Advances in
CANCER RESEARCH Volume 82
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Advances in
CANCER RESEARCH Volume 82
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George Klein Microbiology and Tumor Biology Center Karolinska Institute Stockholm, Sweden
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Contents
Contributors to Volume 82 ix
Retinoblastoma Protein Partners Erick J. Morris and Nicholas J. Dyson I. An Introduction to pRb-Binding Proteins 2 II. Potential pRb Partners 13 References 47
p53-Dependent Apoptosis Pathways Yan Shen and Eileen White I. II. III. IV. V.
p53 56 Bcl-2 Family 62 Caspase Family 67 IAPs 74 p53-Mediated Apoptosis in Cancer Therapy 75 References 76
von Hippel-Lindau Disease: Clinical and Molecular Perspectives Steven C. Clifford and Eamonn R. Maher I. II. III. IV.
The VHL Gene and VHL Disease 86 The VHL TSG and Sporadic Cancers 94 Functional Analysis of The VHL Tumor Suppressor Gene 95 Conclusion 101 References 101
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Nitric Oxide-Induced Apoptosis in Tumor Cells Victor Umansky and Volker Schirrmacher I. II. III. IV.
Introduction 108 NO and Antimetastatic Resistance 109 Mechanisms of NO-Mediated Apoptosis 116 Concluding Remarks 124 References 125
Detection of Minimal Residual Disease Gottfried Dolken ¨ I. II. III. IV. V. VI.
Introduction 134 Technical Aspects of the Detection of Minimal Residual Disease 135 MRD in Leukemia 143 MRD in Lymphoma 157 MRD in Solid Tumors 165 Concluding Remarks 170 References 172
Modeling Prostate Cancer in the Mouse Diego H. Castrillon and Ronald A. DePinho I. II. III. IV. V. VI.
Introduction 187 Anatomy of the Adult Prostate Gland 188 Embryology and Growth of the Prostate Gland 189 Components of the Epithelial Compartment: Do Prostate Stem Cells Exist? 192 Role of Oncogenes and Tumor Suppressors in Prostate Neoplasia 194 Progress in the Development of CaP Animal Models 197 References 201
Immunity to Oncogenic Human Papillomaviruses Jozsef Konya and Joakim Dillner I. II. III. IV.
Epidemiology of Human Papillomavirus Infection 206 The Antibody Response to HPV Infection 207 Cellular Immunity to HPV Infection 214 Does Cross-Protective Immunity Exist? 226 References 231
Index 239
Contributors
Numbers in parentheses indicate the pages on which the authors’ contributions begin.
Diego H. Castrillon, Department of Adult Oncology, Dana Farber Cancer Institute, and Department of Pathology, Brigham and Women’s Hospital, Harvard Medical School, Boston, Massachusetts 02115 (187) Steven C. Clifford, Section of Medical and Molecular Genetics, Division of Reproductive and Child Health, University of Birmingham, Birmingham B15 2TT, United Kingdom (85) Ronald A. DePinho, Department of Adult Oncology, Dana Farber Cancer Institute, and Department of Medicine (Genetics), Harvard Medical School, Boston, Massachusetts 02115 (187) Joakim Dillner, Laboratory of Tumor Virus Epidemiology, The Microbiology and Tumor Biology Center, Karolinska Institute, S-17177 Stockholm, Sweden (205) ¨ Gottfried Dolken, Department of Hematology and Oncology, Clinic for Internal Medicine C, Errnst-Moritz-Arndt-University Greifswald, D-17487 Greifswald, Germany (133) Nicholas J. Dyson, Laboratory of Molecular Oncology, Massachusetts General Hospital Cancer Center, Charlestown, Massachusetts 02129 (1) Jozsef Konya, Laboratory of Tumor Virus Epidemiology, The Microbiology and Tumor Biology Center, Karolinska Institute, S-17177 Stockholm, Sweden (205) Eamonn R. Maher, Section of Medical and Molecular Genetics, Division of Reproductive and Child Health, University of Birmingham, Birmingham B15 2TT, United Kingdom (85) Erick J. Morris, Laboratory of Molecular Oncology, Massachusetts General Hospital Cancer Center, Charlestown, Massachusetts 02129 (1) Volker Schirrmacher, Division of Cellular Immunology, Tumor Immunology Program, German Cancer Research Center, D-69120 Heidelberg, Germany (107) Yan Shen, Center for Advanced Biotechnology and Medicine, Department of Cell and Developmental Biology, Rutgers University, Piscataway, New Jersey 08854 (55)
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Contributors
Victor Umansky, Division of Cellular Immunology, Tumor Immunology Program, German Cancer Research Center, D-69120 Heidelberg, Germany (107) Eileen White, Howard Hughes Medical Institute, Center for Advanced Biotechnology and Medicine, Department of Molecular Biology and Biochemistry, Department of Cell and Developmental Biology, Cancer Institute of New Jersey, Rutgers University, Piscataway, New Jersey 08854 (55)
Retinoblastoma Protein Partners Erick J. Morris and Nicholas J. Dyson∗ Laboratory of Molecular Oncology, Massachusetts General Hospital Cancer Center, Charlestown, Massachusetts 02129
I. An Introduction to pRb-Binding Proteins A. Clues to Function through Association B. An Introduction to pRb C. A Plethora of pRb-Binding Proteins D. Will the Key pRb-Binding Proteins Please Stand up? E. Future Issues: Identifying the Important Interactors II. Potential pRb Partners A. pRb Is Associated with and Functionally Altered by Kinases and Phosphatases B. Transcriptional Regulators that Associate with pRb C. Other pRb-Associated Proteins References
Studies of the retinoblastoma gene (Rb) have shown that its protein product (pRb) acts to restrict cell proliferation, inhibit apoptosis, and promote cell differentiation. The frequent mutation of the Rb gene, and the functional inactivation of pRb in tumor cells, have spurred interest in the mechanism of pRb action. Recently, much attention has focused on pRb’s role in the regulation of the E2F transcription factor. However, biochemical studies have suggested that E2F is only one of many pRb-targets and, to date, at least 110 cellular proteins have been reported to associate with pRb. The plethora of pRb-binding proteins raises several important questions. How many functions does pRb possess, which of these functions are important for development, and which contribute to tumor suppression? The goal of this review is to summarize the current literature of pRb-associated proteins. C 2001 Academic Press.
∗ Corresponding author. Mailing address: MGH Cancer Center, Building 149, 13th St., Charlestown, MA 02129. Phone: (617) 726-7800; Fax: (617) 726-7808; E-mail: dyson@helix. mgh.harvard.edu
1 Advances in CANCER RESEARCH 0065-230X/01 $35.00
C 2001 by Academic Press. Copyright All rights of reproduction in any form reserved.
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I. AN INTRODUCTION TO pRb-BINDING PROTEINS A. Clues to Function through Association One of the most difficult tasks in biology is to determine the function of a newly identified gene product. A commonly used approach is to identify interacting proteins. It is hoped that such partners may include proteins with known functions or might contain domains that provide important clues. In 1986, it was expected that the isolation of the retinoblastoma tumor suppressor gene would be rapidly followed by a detailed understanding of the biochemistry of its mechanism of action. More than a decade later, many binding partners of pRb have been identified, yet it remains unclear how all of these contribute to the normal function of pRb. Although pRb is a relatively abundant protein, the search for immediate targets of pRb action was hampered by the finding that the pRb found in cell extracts is not in stable complexes with an equally abundant binding partner. Multiple studies examined pRb using immunoprecipitation of extracts of metabolically-labeled cells. However, no coprecipitating proteins have been found consistently using antibodies that recognize epitopes throughout pRb. The absence of a prominent coprecipitating binding partner could occur for several different reasons and the studies described below suggest that multiple factors may underlie this observation: 1. Some pRb complexes are weak and easily disrupted. 2. Many pRb complexes are only found at specific times in the cell cycle so that only a subset of the pRb present in asynchronous cells is actually associated with the target. 3. Some pRb complexes associate with chromatin and are difficult to extract. 4. pRb might have many targets so that no single target would be present at a level comparable to pRb. 5. Some pRb partners are only found in specific cell types. 6. Many pRb targets may be expressed at levels that are much lower than pRb itself. Regardless of the cause, all studies of pRb-associated proteins rely on secondary assays such as western blots, kinase activity, or electrophoretic mobility shift assay (EMSA), which allow small quantities of associated proteins to be detected. The strength of these assays is that they are sensitive and highly specific. Given that pRb has many potential binding partners, a limitation of these assays is that they detect complexes that involve a minor fraction of the total pool of cellular pRb and this may provide a highly skewed view of the state of pRb in the cell. pRb-associated proteins have been identified
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by the testing of candidate proteins and using three random approaches: two-hybrid screens using pRb as bait (Durfee et al., 1993, Durfee et al., 1994; Chen et al., 1996; Sterner et al., 1998; Zheng et al., 2000; Fajas et al., 2000), screens of expression cDNA libraries using recombinant pRb as a probe (Defeo-Jones et al., 1991; Helin et al., 1992; Kaelin et al., 1992; Shan et al., 1992; Buyse et al., 1995; Saijo et al., 1995; Sakai et al., 1995; Numata et al., 1999), and from the analysis of proteins bound to a pRb-affinity column (Kaelin et al., 1991; Qian et al., 1993; Chen et al., 1994). Together, these studies have yielded a large number of potential targets and regulators of pRb.
B. An Introduction to pRb Products of the mammalian Rb tumor suppressor gene family are important regulators of cell cycle, differentiation, and apoptosis. Studies of retinoblastoma patients led to the current Knudson “two-hit” hypothesis for tumorigenesis (Knudson, 1971). Subsequently, Rb was the first tumor suppressor gene to be identified and characterized (Friend et al., 1986). Inactivation of pRb is associated with a significant proportion of human cancers including familial retinoblastoma, osteosarcomas, small-cell lung carcinomas, cervical carcinomas, prostate carcinomas, breast carcinomas, and some forms of leukemias (reviewed in Sellers and Kaelin, 1997). Human pRb contains 928 amino acids. It is a nuclear phosphoprotein with a relatively long half-life (>8 hrs). pRb is synthesized throughout the cell cycle, and its activity is regulated by cell cycle-dependent phosphorylation. pRb is broadly expressed but the levels of the protein are variable between cell types. pRb is a member of a family of proteins, which also includes p107 and p130. These proteins are termed the “pocket proteins” due to the structural nature of these regulators (Livingston et al., 1993). They share structural and functional similarities and have been investigated, in large part, by their ability to bind to the small DNA tumor virus gene products adenovirus E1A (E1A), SV40 T antigen (TAg), and human papilloma virus (HPV) protein E7 (reviewed in Dyson, 1994). These interactions are important for the transforming properties of the viral products and are thought to functionally inactivate pRb. Although there are several ways that protein association might disrupt pRb function, the most widely held view is that the viral oncoproteins prevent pRb from interacting with its normal partners by directly competing for binding sites on pRb or inducing pRb degradation. Mutagenesis of E1A, TAg, and HPV E7 proteins demonstrated that a conserved domain, containing the amino acids LXCXE, is essential for the binding of these proteins to pRb. In contrast, a relatively large portion of pRb is required for the interaction. This domain, termed the “pocket,” contains
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Fig. 1 Cartoon structure of the retinoblastoma (pRb) pocket-protein.
two regions that are essential for viral oncoprotein binding (the A-domain spanning amino acids 379 to 572 and the B-domain spanning amino acids 646 to 772) and are separated by a spacer region (Fig. 1). Many studies of pRb have focused on the pocket domain. The pocket is essential for the growth suppression functions of pRb, and most of the naturally occurring, tumor-derived mutations in Rb affect this region. In addition, most of the known pRb-binding proteins require an intact pocket domain for interaction. However, regions outside of the pRb pocket are also likely to be important. For example, growth suppression by pRb requires sequences C-terminal to the pocket, and the C-terminal domain is required for high affinity interaction with several pRb-binding proteins. Sequences both N-terminal and C-terminal to the pocket are highly conserved between pRb homologs of different species and, perhaps most significantly, several tumorderived mutations of pRb are located N-terminal to the pocket and one C-terminal.
C. A Plethora of pRb-Binding Proteins Currently, there are at least 110 unique, cellular pRb-binding proteins reported in the literature. These interactors fall largely into three groups based on some simple characteristics (Tables I–III). First, there are 15 reported kinases, phosphatases, and kinase regulators that have been found physically-associated with pRb (Table I). Most of these enzymes posttranslationally modify pRb to affect its function. Second, there are 72 reported transcriptional regulators, which fall into three basic functional categories when bound to pRb (Table II): (a) factors which repress transcription, (b) factors which activate transcription, and (c) factors which affect transcription in unknown or unclear ways. Third, and finally, there are 23 reported pRb-interacting proteins which have a variety of miscellaneous functions, and have not yet been shown to directly regulate transcription (Table III). A large number of these proteins directly or indirectly regulate DNA replication, the cell-cycle, and various nuclear processes. The properties of these pRb-binding proteins are discussed in more detail later.
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Table I Kinases, Phosphatases, and Their Regulators, that Functionally Target pRb and Have Been Found Physically Associated with pRb in Mammalian Cell Extracts Interactor
Region of pRb
In Vivoa
Cyclin A
Not mapped
Yes
Cyclin A1 Cyclin D1
Large pocket Small pocket
Yes Yes
Cyclin D2
Small pocket
—
Cyclin D3
Small pocket
—
Cyclin E p25NCK5a cdc2
Not mapped Large pocket Not mapped
— Yes Yes
cdk2
C-domain
—
cdk4 (kinase dead) JNK1
Not mapped
—
C-domain
Yes
Raf
At least small pocket N-domain (89-202)
Yes
C-domain
Yes
Not mapped
Yes
pRb kinase PP1␣
PP1␦
Yes
Reference(s) (Hu et al., 1992; Williams et al., 1992; Hall et al., 1993) (Yang et al., 1999) (Dowdy et al., 1993; Ewen et al., 1993; Hall et al., 1993; Kato et al., 1993) (Dowdy et al., 1993; Ewen et al., 1993; Kato et al., 1993) (Dowdy et al., 1993; Ewen et al., 1993; Kato et al., 1993) (Kelly et al., 1998) (Lee et al., 1997) (Hu et al., 1992; Kitagawa et al., 1992; Williams et al., 1992) (Akiyama et al., 1992; Kelly et al., 1998; Adams et al., 1999) (Kato et al., 1993) (Chauhan et al., 1999; Shim et al., 2000) (Wang et al., 1998) (Sterner et al., 1995, Sterner et al., 1996) (Alberts et al., 1993; Durfee et al., 1993; Ludlow et al., 1993; Tamrakar et al., 1999; Tamrakar and Ludlow, 2000) (Puntoni and Villa-Moruzzi, 1997, Puntoni and Villa-Moruzzi, 1999)
a In vivo interaction has been detected with both endogenous pRb and its respective binding protein in mammalian cell extracts.
D. Will the Key pRb-Binding Proteins Please Stand up? The long list of pRb-binding proteins raises several critical issues. How many functions does pRb-possess? Which of these functions are important for pRb’s role in animal development and how many are relevant to its role as a tumor suppressor? The large number of binding partners for pRb can be viewed in many different ways. This is illustrated by the two extreme viewpoints described below. One viewpoint is that pRb enables many diverse functions to be coordinately regulated during cell cycle progression. Many of the pRb-binding
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Table II pRb-Binding Proteins which Function to Regulate Transcription Interactor
Region of pRb
In Vivoa
Reference(s)
Factors that Are Most Likely Repressed or Function through Repression by Association with pRb: E2F-1 Large pocket Yes (Helin et al., 1992; Kaelin et al., 1992; Shan et al., 1992; Krek et al., 1993; Lees et al., 1993) E2F-2 Large pocket Yes (Ivey-Hoyle et al., 1993; Less et al., 1993) E2F-3a Large pocket Yes (Lees et al., 1993) E2F-3b Not mapped Yes (He et al., 2000; Leone et al., 2000) E2F-4 Large pocket Yes (Ginsberg et al.,1994; Moberg et al., 1996; Li et al., 1997) DP-1 Not mapped Yes (Girling et al., 1993; Bandara et al., 1994; Wu et al., 1995) DP-2 Not mapped Yes (Wu et al., 1995) RBP60 Large pocket — (Arroyo and Raychaudhuri, 1992; Ray et al., 1992) RbAp46 Small pocket Yes (Huang et al., 1991; Qian and Lee, 1995) RbAp48 Small pocket Yes (Qian et al., 1993; Nicolas et al., 2000) HDAC1 Small pocket Yes (Magnaghi-Jaulin et al., 1998; Brehm et al., 1998; Luo et al., 1998; Lai et al., 1999a) HDAC2 Small pocket Yes (Lai et al., 1999a) HDAC3 Small pocket Yes (Lai et al., 1999a) c-Ski B-domain Yes (Tokitou et al., 1999) Sno Not mapped Yes (Tokitou et al., 1999) Sin3a Not mapped Yes (Tokitou et al., 1999) RBP1 Small pocket Yes (Defeo-Jones et al., 1991; Kaelin et al., 1992; Fattaey et al., 1993; Kim et al., 1994; Lai et al., 1999a; Lai et al., 1999b) RBP2 A-domain + spacer — (Defeo-Jones et al., 1991; Fattaey et al., 1993; Kim et al., 1994) Bdp At least C-domain — (Numata et al., 1999) Elf-1 Large pocket Yes (Wang et al., 1993) CtIP At least — (Meloni et al., 1999) small pocket TFIIIB Large pocket Yes (White et al., 1996; Larminie et al., 1997) TBP Not mapped Yes (Larminie et al., 1997) BRF/TFIIB Not mapped Yes (Larminie et al., 1997) UBF C-domain Yes (Kaye et al., 1990; Shan et al., 1992; Cavanaugh et al., 1995; Voit et al., 1997) HBP1 At least — (Lavender et al., 1997; small pocket Tevosian et al., 1997) AHR Not mapped Yes (Ge and Elferink, 1998; Puga et al., 2000) Trip230 Small pocket Yes (Chang et al., 1997) Pax-3 Small pocket — (Wiggan et al., 1998) (continues)
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Table II (continued ) Interactor
Region of pRb
In Vivoa
Pax-5 Pax-6 PHox B4 Chx10 Sp1-I DNMT1 NF-B p50 RBaK p120E4F
Large pocket Not mapped Not mapped Not mapped Small pocket Not mapped Small pocket Not mapped Not mapped B + C-domain
— Yes — — — — Yes Yes — Yes
Reference(s) (Eberhard and Busslinger, 1999) (Cvekl et al., 1999) (Wiggan et al., 1998) (Wiggan et al., 1998) (Wiggan et al., 1998) (Chen et al., 1994) (Robertson et al., 2000) (Tamami et al., 1996) (Skapek et al., 2000) (Fajas et al., 2000)
Factors that Are Most Likely activated by Association with pRb: MyoD Myogenin Myf-5 MRF4 NF-IL6 C/EBP c-Jun JunB JunD Sp1
B + C-domain (605-792) B + C-domain (605-792) B + C-domain (605-792) B + C-domain (605-792) At least small pocket Small pocket B + C-domain (612-767) Not mapped Not mapped Not mapped
Yes
(Gu et al., 1993)
Yes
(Gu et al., 1993)
—
(Gu et al., 1993)
—
(Gu et al., 1993)
Yes
(Chen et al., 1996a)
Yes Yes
(Chen et al., 1996b) (Nead et al., 1998)
Yes Yes Yes
(Nead et al., 1998) (Nead et al., 1998) (Noe et al., 1998)
Factors where the Outcome Is Unknown, Unclear, or Both Activation and Repression Have Been Reported: BRG1
Yes
hBrm1
At least small pocket Small pocket
ATF-1 ATF-2
Not mapped Not mapped
— —
c-myc
Small pocket
—
N-myc PU.1
Small pocket Large pocket
— Yes
AP-2 Id-2
Large pocket At least small pocket Not mapped
Yes Yes
Pur␣
Yes
Yes
(Dunaief et al., 1994; Singh et al., 1995; Strober et al., 1996; Murphy et al., 1999) (Singh et al., 1995; Trouche et al., 1997; Murphy et al., 1999) (Gong et al., 1995) (Kim et al., 1991, Kim et al., 1992; Gong et al., 1995) (Rustgi et al., 1991; Batsche et al., 1994; Adnane and Robbins, 1995) (Rustgi et al., 1991) (Hagemeier et al., 1993; Weintraub et al., 1995; Konishi et al., 1999) (Batsche et al., 1998; Wu and Lee, 1998) (Iavarone et al., 1994; Lasorella et al., 1996; Lasorella et al., 2000) (Johnson et al., 1995) (continues)
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Table II (continued) Interactor Cream-I/ Rbap2 Che-1 TAFII250 TAFII150 TAFII80 RIZ Rim Estrogen receptor PML mdm2 p202 Mi pRb
Region of pRb
In Vivoa
Reference(s)
Not mapped
Yes
(Yan et al., 2000)
Not mapped All regions
Yes —
At least large pocket At least large pocket Not mapped
—
(Fanciulli et al., 2000) (Shao et al., 1995, Shao et al., 1997; Siegert and Robbins, 1999) (Shao et al., 1995)
—
(Shao et al., 1995)
Yes
Not mapped Not mapped
— Yes
(Buyse et al., 1995; Abbondanza et al., 2000) (Fusco et al., 1998) (Abbondanza et al., 2000)
Small pocket C-domain N (1-254) + large pocket Not mapped N (1-300) to C-domain
Yes Yes Yes
(Alcalay et al., 1998; Labbaye et al., 1999) (Xiao et al., 1995; Hsieh et al., 1999) (Choubey and Lengyel, 1995)
— —
(Yavuzer et al., 1995) (Hensey et al., 1994)
a In vivo interaction has been detected with both endogenous pRb and its respective binding protein in mammalian cell extracts.
proteins can be incorporated into a general model in which pRb acts to restrict cell proliferation and to promote differentiation. Almost all of these partners interact with the unphosphorylated form of pRb and not with hyperphosphorylated pRb; as a result, pRb phosphorylation would allow a broad range of activities to be directly coupled to the activity of cyclindependent kinases and cell cycle position. Since pRb interacts not only with transcriptional regulators, but also with a large host of DNA replication control enzymes and other factors, this interpretation places pRb at a pivotal position in the cell cycle with global effects on many aspects of cellular function. Such an interpretation might explain why pRb is such an attractive target for viral oncoproteins and why the functional inactivation of pRb is such a significant event in tumorigenesis. In support of this interpretation, it is noteworthy that 73 of the reported 110 pRb-interactors have been shown to associate with pRb in experiments that examine the endogenous proteins in mammalian cell extracts (see Tables I–III). These types of experiments, which do not depend on interactions between purified proteins or overexpressed proteins, are often regarded as evidence of a genuine interaction. Given the long list of known pRb-binding proteins, one wonders how many different types of processes are regulated by pRb. Even though the first pRb-binding proteins were
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Table III pRb Binding Proteins with Functions that Are Most Likely Distinct from Transcription Binding Protein
Function
Region of pRb
In Vivoa
Reference(s) (Welch and Wang, 1993, Welch and Wang, 1995) (Craven et al., 1995) (Woitach et al., 1998) (Wang et al., 1999) (Aprelikova et al., 1999; Yarden and Brody, 1999)
c-Abl
Tyrosine kinase
C-domain
Yes
Rak Bog Prohibitin BRCA1
Tyrosine kinase Oncogene? Tumor suppressor? Tumor suppressor
Small pocket Not mapped Not mapped Large pocket
Yes Yes Yes Yes
cdk inhibitor cdk inhibitor Metaphase spindle control? Centromere regulation? Nuclear matrix
Small pocket Not mapped Small pocket
Yes — —
Not mapped
—
B + C-domain
—
Nuclear matrix
B + C-domain
—
Nuclear matrix? RNA processing? RNA processing/ apoptosis?
Not mapped Small pocket N-domain (1-300)
— — Yes
Pre-RNA splicing? Unknown Relax DNA supercoiling Replicative DNA polymerase Recombinase Pre-replication complex Chromosomal segregation Heat-shock protein Heat-shock protein
Not mapped
Yes
B + C-domain Small pocket
— Yes
(Kim et al., 1998) (Witte and Scott, 1997) (Durfee et al., 1994; Doostzadeh-Cizeron et al., 1999) (Sakai et al., 1995; Simons et al., 1997) (Saijo et al., 1995) (Bhat et al., 1999)
Not mapped
Yes
(Takemura et al., 1997)
Large pocket N-domain (1-400) Large pocket
Yes Yes
(Fan et al., 1997) (Sterner et al., 1998)
Yes
(Zheng et al., 2000)
N-domain
Yes
Small pocket
Yes
(Nihei et al., 1993; Inoue et al., 1995) (Chen et al., 1996)
1
p21CIP /WAF p57KIP2 H-nuc
1
Mitosin Nuclear lamin A Nuclear lamin C NRP/B P2P-R p84N5
RBQ-1/PACT RBQ-3 Topo-II␣ DNA pol␣ REC2 MCM7 hsHec1p hsc73 hsp75
(Nakanishi et al., 1999) (Nakanishi et al., 1999) (Durfee et al., 1994; Chen et al., 1995) (Zhu et al., 1997) (Mancini et al., 1994; Ozaki et al., 1994) (Shan et al., 1992)
a In vivo interaction has been detected with both endogenous pRb and its respective binding protein in mammalian cell extracts.
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reported almost a decade ago, new pRb-interactors continue to be discovered, and there is no indication that we are at the end of the list. Indeed, even if all of the major functions of pRb have already been identified, it seems likely that the reports of pRb-associated proteins will continue to grow, since many of the known interactors are components of large multisubunit complexes. A different interpretation of this literature is that pRb is simply a regulator of E2F (the best characterized of the pRb-associated factors and the interaction most strongly supported by genetic and biochemical data), and that pRb’s pleiotropic effects on the cell cycle, differentiation and apoptosis reflect the fact that E2F-regulated transcription is critical for each of these processes. According to this interpretation, the only meaningful partners for pRb are the E2F proteins, the repressor complexes that pRb recruits to E2F regulated promoters, and the kinases/phosphatases that regulate pRb activity. In this view, the list of pRb-binding proteins is so long because the sensitivity of assays used to detect protein interactions has enabled complexes to be found that either do not normally form in vivo or are not functionally relevant. Many pRb-protein complexes were first found either using high concentrations of purified proteins in in vitro binding assays or in highly artificial settings in which proteins are overexpressed. A large number of proteins have been tested for pRb-binding because the interaction would fit an attractive model. While most of the protein interactions have been demonstrated by the coimmunoprecipitation of endogenous proteins, these interactions have often been difficult to find and most have not yet been replicated in independent studies from other laboratories. In most cases where the controls are shown, the coprecipitated proteins represent a tiny proportion of the total amounts of pRb and the binding partner. Immunoprecipitation and western blot analysis, EMSA, and kinase assays enable small quantities of proteins to be easily detected. Since pRb is abundant and the assays are very sensitive, it may not be particularly meaningful that small amounts of many different proteins can bind to pRb in cell lysates. A second potential source of artifacts is suggested by the fact that both unphosphorylated pRb, and many of the reported pRb-binding proteins, associate tightly with chromatin. It is important to note that most studies of pRb-associated proteins have not distinguished between proteins that directly contact pRb, and proteins that are only indirectly associated. Immunoprecipitates that are prepared by most standard methods contain substantial amounts of chromatin and few studies have examined the possibility that coprecipitating proteins might be indirectly linked to pRb through protein/DNA intermediates. In addition, several reports have also been based primarily on in vitro binding assays using recombinant proteins that contain only fragments of the wild-type pRb protein. These fragments may expose binding surfaces that do not exist on the surface of the endogenous protein.
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The true picture of pRb function most likely lies between the two extreme views outlined previously. Although there is compelling evidence that E2F is an important target for pRb, it is difficult to believe that all of the studies linking pRb to functions other than E2F-regulation are wrong or irrelevant. Conversely, it is difficult to imagine how one protein could possibly interact with all of the pRb-binding proteins that have been proposed. We note that the vast majority of reports link pRb to transcriptional repression. One simple way to view pRb is as an adapter that tethers large chromatin-regulating complexes to specific DNA elements. Evidence that pRb interacts with several histone deacetylases, methyltransferase, and SWI/SNF complexes suggests that pRb has the potential to recruit different activities to DNA in different settings. While E2F may provide one mechanism to add DNA-binding specificity to pRb, it is reasonable to suppose that other factors could use pRb to target these chromatin-regulating complexes to other sites. It is clear that pRb-recruited complexes can regulate transcriptional initiation, but it seems logical that local changes in chromatin structure could have consequences on other processes including replication, DNA repair and gene silencing.
E. Future Issues: Identifying the Important Interactors Even a cursory glance at the number of pRb binding proteins suggests myriad ways in which pRb might function. Binding proteins have been mapped to regions throughout the entire pRb molecule but, currently, we understand very little about how individual domains of pRb are regulated in vivo and how these domains cooperate to modulate pRb functions. Small changes in pRb structure could translate into large functional outcomes in cellular function. To add further complexity, very little is known about how the pool of pRb is divided among such a large number of potential targets. Given the possibility that pRb might simultaneously bind to several distinct proteins it will be crucial to know what events regulate the assembly of different multisubunit complexes. These issues are very difficult to address, especially when one considers that the binding sites of many pRb-binding proteins have not been mapped on pRb with any degree of precision. Most worryingly, it is unclear how many pRb-binding proteins are even biologically relevant to study. How can one determine which of the pRb-binding proteins are most significant for pRb function? If an interaction is truly important, one would expect either that pRb function should be altered in the absence of the binding partner, or that the function regulated by the binding partner should be altered in Rb−/− cells. If neither of these changes occur, then it is difficult to argue that the interaction is critical. As many pRb-binding proteins also interact with p107 and p130, it is important to know which interactions are
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unique to pRb. In situations where a pRb-binding protein can interact with multiple pRb-family members, it may be necessary to examine cells lacking the entire family of proteins to discover the significance of the interaction. Since many proteins have tissue-specific or cell type specific functions, it may be necessary to study an interaction within a particular tissue by the use of targeted disruption technologies such as the Cre/Lox or FLP/FRT systems. A second criterion that may be useful is to test the effects of specifically eliminating the interaction by targeted mutagenesis. One would expect that subtle mutations that eliminate a key protein/protein interaction, without affecting other binding-surfaces, should specifically alter the function of pRb, or its binding partner. One approach to this problem is to mutagenize the pRb-binding partner so that it no longer interacts with pRb. One potential pitfall of these studies is that if the activity of the protein is altered, it is unclear whether the change is due to the loss of pRb-binding or because the putative binding site is critical for the normal function of the protein. To confirm the specificity of this change, it is necessary to make compensatory mutations that restore the interaction, and to demonstrate restoration of the normal pattern of regulation. Alternatively, this question could be addressed by mutagenesis of pRb. To date, the structure-function analyses of pRb have been relatively crude and fall well short of the ideal situation described above. Most activities of pRb require the pocket domain, and most pRb-associated proteins interact with the pocket. The majority of tumorderived pRb mutations that have been isolated disrupt the pocket domain and eliminate pRb’s tumor-suppressor activity. In many studies, it has been argued that the failure of a pRb-binding partner to interact with pRb containing tumor-derived mutations indicates that this interaction is important in pRb-mediated tumor suppression. However, since the most commonly tested mutations destroy the pocket domain and prevent pRb from interacting with many of its potential targets, this type of correlation is of limited value. Now that a crystal structure of the pocket domain is known (Lee et al., 1998), it should be feasible to map interaction surfaces more precisely and to test the functional consequences of mutations that eliminate pRb’s interaction with specific binding partners, or with subsets of binding proteins. Since many viral pRb-binding proteins contain a conserved LXCXE pRbbinding motif, several studies have used the presence of an LXCXEmotif in candidate cellular pRb-binding protein as evidence that it is a genuine interactor. These types of arguments should be treated cautiously. For example, the -chain of human insulin contains an LXCXE motif (Radulescu and Wendtner, 1992), yet a biologically relevant interaction with pRb is clearly unexpected. While the residues that contact viral LXCXE peptides have been conserved in pRb homologs from plants to animals, it is unclear which of the cellular proteins that might interact with this site are important for pRb function. Indeed, recent studies have shown that mutagenesis of the LXCXE-binding cleft prevents pRb from interacting with
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viral oncoproteins but results in a pRb-molecule that retains many of its cell cycle arrest and growth suppression properties (Dick et al., 2000; Chen and Wang, 2000, Dahiya et al., 2000). Thus, even for the LXCXE-containing proteins, a careful structure function analysis is needed before the significance of this interaction is clear. A third, and perhaps the most important criterion, is that once a key pRb-binding protein has been identified, it should be possible to demonstrate a series of genetic interactions in vivo. Pocket-protein homologs exist in model organisms such as mice, flies, worms, and plants and each of these genetic systems offer tools to test the significance of pRb-binding proteins. Understanding how loss or mutation of a putative pRb-interacting protein affects a pRb-dependent or -deficient phenotype should give results that are highly complementary to the binding studies, as was the case with Rb; E2F1 (Tsai et al., 1998), or Rb; Id2 (Lasorella et al., 2000) doublydeficient mice, Rbf; dE2F1 mutant flies (Du et al., 1996; Du, 2000); and lin35 and lin-53 mutant worms (Lu and Horvitz, 1998; Thomas and Horvitz, 1999; Hsieh et al., 1999). Moreover, insight into pRb function could be greatly aided by conducting unbiased genetic screens for modifiers of pRbdependent or -deficient phenotypes. At present a detailed genetic analysis has not been done for most pRbbinding proteins and the overall significance of the individual interactors is not known. The two exceptions to this are E2F and Id2. Studies showing that mutation of E2F1 can partially rescue the phenotypes of Rb−/− mice (Tsai et al., 1998), and that mutation of dE2F could rescue the lethality of animals with reduced Rbf (Du, 2000), strongly suggest that E2F regulation is a critical aspect of pRb function. In addition, recent evidence demonstrating significant rescue of Rb-deficient mice by loss of Id2 highlight important cell-type specific interactions between pRb and transcriptional regulators during development (Lasorella et al., 2000). However, neither of these studies demonstrated a full rescue of the null mutant phenotype. In addition, in the case for E2F, the interpretation of these results is complicated by the existence of related family members. It remains unclear whether similar types of rescue could be obtained with mutant alleles of other pRb-binding proteins or, alternatively, whether a more extensive elimination of E2F activity, with or without loss of other pRb-binding proteins, could give a more complete rescue of the Rb−/− phenotype.
II. POTENTIAL pRb PARTNERS The following sections review the myriad interactions reported between pRb and a number of unique cellular proteins. These interactions are listed in Tables I, II and III and the characteristics of these complexes are summarized below.
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A. pRb Is Associated with and Functionally Altered by Kinases and Phosphatases pRb is phosphorylated and dephosphorylated in a cell cycle-dependent manner, and it is clear that these changes regulate its activity. pRb has only a low level of phosphorylation in G0- or G1-phase cells. A dramatic increase in pRb phosphorylation is seen as cells enter S-phase. pRb becomes increasingly, heavily phosphorylated during S-phase and G2 before it is abruptly dephosphorylated at metaphase of mitosis. pRb contains up to 16 potential serine/threonine phosphorylation sites for proline-directed kinases. In vitro, pRb can be phosphorylated by many different cyclin-dependent kinases (cdk’s), and it appears that the concerted action of several cdk’s contributes to the appearance of hyperphosphorylated pRb in vivo (reviewed in Mittnacht, 1998). More recent data suggests that pRb is targeted by kinases other than cell cycle-regulating cdks, including Raf-1, JNK/SAPK, and cdk5 (discussed below). Two tyrosine kinases associate with pRb (c-Abl and Rak; see below), but pRb is not overtly tyrosine phosphorylated. An additional G2/M kinase activity has been reported that associates with the amino-terminal domain of pRb but its identity is unknown (Sterner et al., 1995, Sterner et al., 1996).
1. CYCLINS AND CDK’s pRb immunoprecipitates contain small quantities of several kinases and phosphatases that are most likely associated because they act on pRb (Table I). Such immunoprecipitates contain a kinase activity that phosphorylates pRb in vitro (Kitagawa et al., 1992; Hu et al., 1992). Western analysis of pRb immunoprecipitates showed that small quantities of cdc2 (Kitagawa et al., 1992; Hu et al., 1992; Williams et al., 1992), cdk2 (Akiyama et al., 1992; Kelly et al., 1998), cyclin A (Hu et al., 1992; Williams et al., 1992), cyclin A1 (Yang et al., 1999), and cyclin E (Kelly et al., 1998) are associated with pRb in cell extracts. In the case of cdc2, it is unclear whether the cyclin/cdc2 complexes represent enzyme/substrate intermediates or whether pRb contains a specific binding site for cdc2. Phosphorylation of pRb by cyclin/cdk2 depends on a conserved cdk/cyclin binding ZRXL-motif in the C-terminus of pRb (Adams et al., 1999). The presence of ATP perturbs cdk2/cyclin E-pRb binding but not pRb binding to a kinase-dead cdk2 mutant (Kelly et al., 1998). Although it is unclear where the pRb/cyclin A interaction occurs, cyclin E contains a VXCXE motif which has been reported to be essential for pRb binding (Kelly et al., 1998). However, C-terminal fragments of pRb are efficient phosphorylation substrates for both cyclin/cdk2 and cyclin/cdk4
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complexes suggesting that the LXCXE domain is unlikely to be an important determinant in this interaction (Adams et al., 1999). In addition, since cyclin A/cdk2 complexes can also associate with E2F-1 (Krek et al., 1994), it is also possible that at least some of these kinases may be indirectly bound to pRb through other proteins (Bandara et al., 1991). Current evidence indicates that the initial phosphorylation of pRb is likely due to cyclin D-dependent kinases. Cyclins D1, D2 and D3 proteins physically associate with pRb in vitro (Dowdy et al., 1993; Ewen et al., 1993; Kato et al., 1993; Hall et al., 1993) and when coexpressed from baculovirus expression vectors (Dowdy et al., 1993; Kato et al., 1993; Welch and Wang, 1995b). Cyclin D/pRb complexes formed between endogenous proteins have been reported for Cyclin D1 (Dowdy et al., 1993). The interactions between D-type cyclins and pRb have several interesting features. Strong binding appears to involve the intact pocket domain of pRb plus carboxyl-terminal sequences (Ewen et al., 1993). However, although the amino-terminal sequences of each of the D-type cyclins contain an LXCXE motif, mutants in the LXCXE domain of cyclin D1 do not disrupt pRb phosphorylation (Horton et al., 1995; Connell-Crowley et al., 1997), although previous evidence demonstrated reduced pRb-binding in these mutants (Dowdy et al., 1993). Intriguingly, Kato and coworkers found that cyclin D2 and cyclin D3 could bind to pRb in the absence of a kinase partner but a stable complex was not formed when cdk4 was added (Kato et al., 1993). However, stable association between pRb, cdk4 and cyclin D2 or D3 was recovered if a kinase-dead mutant of cdk4 was used. Since pRb is a substrate for cyclin D-dependent kinases, it is possible that D-type cyclins bind to pRb simply to direct the kinase to its substrate. It is also possible that pRb/cyclin D complexes may serve a function in addition to substrate recognition and it has been suggested that pRb may regulate the activity of cyclin D-dependent kinases (Dowdy et al., 1993). Generally, it is thought that pRb inactivation is due to sequential phosphorylation at G1 by cdk4/6 and cdk2, whereby prior cdk4/6 mediated phosphorylation primes pRb for targeting by cdk2 (Ezhevsky et al., 1997; Lundberg and Weinberg, 1998; Harbour et al., 1999).
2. p25nck5a Mammalian neurons express a divergent cdk family member, cdk5, whose activity is induced by binding to its various cognate neuronal-specific activators, including p25nck5a, p35nck5a, p39nck5ai, and p67 (reviewed in Lee et al., 1997b). Together, the cdk5/p25nck5a complex (termed neuronal cdc2like kinase, Nclk) is critically important for proper neuritic process formation and central nervous system development. Purified bovine Nclk has been shown to phosphorylate pRb in vitro (Lee et al., 1997a). Accordingly, p25nck5a contains an LXCXXE motif (all three other cdk5 activators also
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share a similar motif) and has been demonstrated in coimmunoprecipitation assays to bind GST-pRb fusions containing the large pocket of pRb (cdk5 alone was unable to bind pRb). Although cyclin D1 can also bind cdk5, this complex does not form an active kinase and does not phosphorylate pRb. Moreover, binding of cyclin D1 to Nclk reduces the kinase activity of the complex. Currently, it remains to be determined whether pRb is a target of Nclk in vivo and whether these kinase complexes act to regulate pRb function in neurons. In addition, since cdk5 functions in the cytosol to regulate neurofilament dynamics, it is unclear if cdk5 would normally interact with a nuclearly-localized pRb.
3. JNK /SAPK c-Jun N-terminal kinases (JNK), also known as stress-activated protein kinases (SAPK), are members of the MAP kinase family whose activity is rapidly induced in cells that are challenged by a diverse range of toxic and stress-inducing stimuli. These kinases are thought to induce the immediate responses of cells to potentially toxic conditions and regulate both cell survival and apoptosis. By coimmunoprecipitation, pRb was found to associate with JNK1 in both MY5 cells and pRb-reconstituted Saos-2 cells (Chauhan et al., 1999). Moreover, the level of binding of pRb and JNK1 was enhanced in MY5 cells following ␥ -irradiation. JNK1- and pRb immunoprecipitates from control versus ␥ -IR MY5 cells demonstrated enhanced pRb phosphorylation as well as JNK1 kinase activity toward GST-tagged pRb substrates, preferentially at the C-terminal region of pRb at Thr821. Shim and colleagues also demonstrated that this C-terminal portion of pRb could effectively bind JNK1 (Shim et al., 2000). However, pRb directly inhibited JNK1 activity (using GST-c-Jun or -ATF2 as substrate) as well as preferential phosphorylation of JNK1 by SEK1, the JNK1 activating kinase (Shim et al., 2000). Consistent with these results, SEK1/JNK1-mediated apoptosis of Saos-2 cells (caused by UV radiation) was abrogated by overexpression of either full-length or the C-terminal domain of pRb. This effect of pRb was specific in that pRb was unable to inhibit the catalytic activity of either MEKK1 or SEK1 (using myosin basic protein as substrate for SEK1) as well as the related kinases ERK1 and p38 (Shim et al., 2000). The effects of various JNK/SAPK family members may be cell type or condition sensitive since Wang and colleagues have reported that immunoprecipitated p38 from Fas-stimulated Jurkat cells is able to phosphorylate pRb, an effect which is abrogated by cotreatment with the p38 inhibitor, SB203580 (Wang et al., 1999b). Currently, it is unclear if pRb primarily acts as a substrate for or an inhibitor of JNK/SAPK signaling and little is known regarding the role of JNK/SAPK activity on normal pRb function in vivo.
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4. Raf-1 Raf-1, a mitogenic signaling kinase in the MAP kinase cascade, has been reported to physically interact with and phosphorylate pRb (Wang et al., 1998). Raf-1 was shown to bind pRb in vitro (GST pull down and yeast two-hybrid). Moreover, endogenous Raf-1 and pRb were coimmunoprecipitated in vivo from U937 cells (although only 5% of pRb was associated with Raf-1 in asynchronous cells). Raf-1, which integrates tyrosine phosphorylation signaling with Ser/Thr phosphorylation signaling, was shown to associate with pRb in a serum-inducible manner in human HSF8 fibroblasts (althoughRaf-1 was mainly associated withhypophosphorylated pRb).Raf-1 was shown to phosphorylate pRb in vitro, and cotransfection of Raf-1 into Saos-2 cells reversed both pRb-mediated growth suppression and repression of E2F-1 expression. In addition, Raf-1 colocalized with pRb to the nucleus shortly after serum stimulation in HSF8 cells. Further studies are needed to identify a physiologic setting in which Raf-1’s effects on the cell cycle depend on its ability to bind pRb.
5. PHOSPHATASES In vitro assays have implicated serine/threonine phosphatases type 1 (PP-1) in the dephosphorylation of pRb (Alberts et al., 1993; Ludlow, 1993; Puntoni and Villa-Moruzzi, 1997, Puntoni and Villa-Moruzzi, 1999). Using a fragment of pRb as bait in a two-hybrid screen, Durfee and colleagues isolated a variant of PP1 (PP-1␣)(Durfee et al., 1993). Although in vitro binding assays showed that PP-1␣ bound preferentially to unphosphorylated pRb, coimmunoprecipitation experiments revealed that only trace amounts of the protein were bound to pRb in G1-phase cells. Instead, the pRb/PP-1␣ complex was found primarily in M-phase cells during pRb dephosphorylation. In addition to PP-1␣, PP-1␦ activity has been demonstrated to associate with pRb dephosphorylation in HeLa cells, primarily in late M-phase to very early G1 (Puntoni and Villa-Moruzzi, 1997, Puntoni and Villa-Moruzzi, 1999). Analysis of pRb binding, using a PP-1␣-affinity column and phosphospecific pRb antibodies, demonstrated that PP1␣ can bind both hypo- and hyperphosphorylated pRb (Tamrakar et al., 1999). Moreover, select phosphorylated forms of pRb bind to PP-1␣, and the catalytic activity of this phosphatase is not required for pRb binding. Recent evidence demonstrated PP-1␣ binding to the C-domain of pRb and that pRb might actually function as a PP-1␣ inhibitor (Tamrakar and Ludlow, 2000). Further analysis is needed in order to identify the specific amino acid targets of PP-1␦ and to determine why certain phosphatases target particular pRb phospho-residues over others.
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B. Transcriptional Regulators that Associate with pRb Many of the pRb-associated proteins are involved in the regulation of transcription and many genes are directly or indirectly regulated by pRb. pRb association with transcription factors has been reported to increase or decrease the transcriptional activity of these molecules. Moreover, pRb directly and indirectly binds enzymes that affect transcription. For ease of discussion, these interactors are divided into three groups, depending on the reported effect of the interaction on transcription. A general theme runs through many of these interactions: in general, pRb has been found to (a) repress the activity of factors that either promote or are needed for rapid cell proliferation, and (b) enhance the activity of factors that promote differentiation.
1. pRb AS A REPRESSOR OF TRANSCRIPTION In many studies, the interaction of pRb with a target leads to the inhibition of gene expression. pRb has been reported to repress the expression of genes necessary for cell growth, cell death, cell proliferation, and genes that are expressed following T-cell activation. At least four different mechanisms have been suggested whereby pRb represses gene expression: (a) pRb recruits enzymes which directly or indirectly repress transcription, (b) pRb interacts with and inhibits the activity of specific transcriptional activators, (c) pRb interacts and cooperates with specific transcriptional repressors, and (d) pRb regulates gene expression by direct interaction with components of the basal transcriptional machinery, and in this way targets all three mammalian RNA polymerases.
a. E2F, DP, and RBP60 Complexes between pRb and the E2F-transcription factors have been studied intensively (reviewed in Dyson, 1998; Helin and Peters, 1998; Nevins, 1998). E2F/pRb complexes are disrupted by viral oncogenes and the liberation of E2F results in the activation of E2F-dependent transcription. The pRb/E2F complex is cell cycle regulated: E2F associates strongly with hypophosphorylated forms of pRb and has little affinity with hyperphosphorylated pRb. E2F is the collective activity of a heterogenous family of E2F/DP heterodimers. Of the six E2F genes cloned to date, five gene products (E2F-1, E2F-2, E2F-3a, E2F-3b, and E2F-4) have been found in complexes with pRb (Shan et al., 1992; Kaelin et al., 1992; Helin et al., 1992; Ivey-Hoyle et al., 1993; Lees et al., 1993; Krek et al., 1993; Cress et al., 1993; Ginsberg et al., 1994; Moberg et al., 1996; Lee et al., 1997a; He et al., 2000; Leone et al., 2000). These interactions have been found both in vitro and in vivo
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(with endogenous and overexpressed proteins) in multiple cell types. A small binding domain has been identified in E2F-1 that is conserved in five E2F genes (E2F1-5) and missing in E2F-6. This domain is embedded in the transactivation domain of E2F proteins, suggesting that pRb sterically prevents transcriptional activation. E2F binds with low affinity to the small pocket domain of pRb, but with high affinity to fragments containing the pocket domain and the carboxyl-terminus (together termed the large pocket). pRbassociated E2F complexes can contain either DP-1 or DP-2, which potentiate the pRb-binding activity of E2F polypeptides (Girling et al., 1993; Bandara et al., 1994; Wu et al., 1995). The C-terminal region of DP-1 has been shown to bind pRb in vitro in a pocket-independent fashion. An additional protein, RBP60 has been described that binds to pRb in vitro at the large pocket and stabilizes pRb/E2F/DNA complexes in gel-shift assays (Arroyo and Raychaudhuri, 1992; Ray et al., 1992). However, a role for RBP60 in E2F regulation has not yet been shown in cells. Ectopic expression of Rb and E2F genes have antagonistic effects on E2F-dependent transcription and S-phase entry. However, interpretation of the pRb/E2F literature is complicated by a number of issues. First, pRb is only one member of a family of related proteins that regulate E2F activity. Surprisingly, only subtle changes are seen in the cell cycle expression of putative E2F-regulated genes in pRb−/− mouse embryo fibroblasts (Hurford et al., 1997). p107 and p130 are structurally related to pRb; they also regulate E2F activity, and their functions are likely to overlap with pRb (Lee et al., 1996). A second complication is that pRb does not simply silence the transcriptional activation function of E2F. pRb represses transcriptional activation by other promoter elements when tethered to DNA by E2F (Weintraub et al., 1992, Bremner et al., 1995; Sellers et al., 1995; Weintraub et al., 1995). How pRb exerts this effect is still unclear. Recent evidence suggests that pRb can interact directly with elements of the basal transcriptional machinery as well as recruit other transcriptional regulators such as histone deacetylase (HDACs), DNA methyltransferase, and SWI/SNF chromatinregulating complexes. pRb/E2F complexes are detected in E2F EMSA assays prepared from fractions of cells that are primarily in S-phase. This finding is paradoxical since the interaction is detected long after the elevated expression of putative E2F-regulated genes has occurred. One possible explanation is that these bands may represent pRb/E2F complexes that have dissociated from the repression machinery.
b. HDAC Proteins Degrees of acetylation/deacetylation of core histone N-terminal regions are important determinants of gene transcriptional activity and are regulated by histone-acetylase (HAT) and -deacetylase (HDAC) enzymes (reviewed in
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Hassig and Schreiber, 1997b). Generally, acetylation tends to expose chromatin and facilitate transcriptional activation whereas deacetylation acts to repress transcription. There are two classes of HDAC gene products: class I consists of genes similar to the yeast repressor molecule RBP3 and include HDAC1, -2, and -3, whereas class II proteins are yeast HDA1-related and include HDAC4, -5, -6, and -7. HDAC1 and -2 have been found to associate with two large histoneregulating complexes: a) the Sin3a/HDAC complex, which also contains RbAp46 and -48, as well as Sin3a, SAP18, and SAP30 (Zhang et al., 1997), and b) the NuRD complex, which also contains both RbAp46 and -48, as well as Mi2, p70, and p32 (Zhang et al., 1999a). pRb binding has been detected with a number of these proteins suggesting that pRb function might occur within a large multisubunit regulatory network. In 1998, three simultaneous reports demonstrated that HDAC1 physically interacts with pRb to cooperatively repress E2F-mediated gene expression (Brehm et al., 1998; Luo et al., 1998; Magnaghi-Jaulin et al., 1998). The small pocket was found to be necessary and sufficient for HDAC1 binding and transcriptional repression. Further analysis of pRb/HDAC interactions demonstrated that endogenous HDAC1, -2, and -3 bound pRb in vitro and coimmunoprecipitated with endogenous, hypophosphorylated pRb from H1299 cell extracts in vivo (Lai et al., 1999a; Nicolas et al., 2000). Moreover, a HDAC1-competing peptide, containing the LXCXE binding site, prevented pRb-precipitated polynucleosomal deacetylase activity in Jurkat extracts, and HDAC interactions with pRb could not be detected in 293T cells, which express E1A or TAg. p107 and p130 have also been shown to bind HDAC1 (Ferreira et al., 1998). Currently, it is unknown if pocket-proteins bind or function through the activity of HDAC4–7. Interestingly, although HDAC1 and -2 contain IXCXE motifs, HDAC3 does not, yet all three have been reported to be associated with pRb. Recent data suggest that HDACs might be tethered to pRb by other linker proteins, such as RBP1 (discussed below and Lai et al., 1999a).
c. RbAp46, RbAp48, and Sin3a RbAp46 and RbAp48 were the most abundant polypeptides extracted from HeLa cell lysates on an pRb-affinity resin prepared with p56RB (pRb fragment containing large pocket) (Qian et al., 1993). The RbAp48 cDNA was isolated following sequencing of the p56Rb-purified protein (Qian et al., 1993), while RbAp46 was isolated using cross-reactive monoclonal antibodies raised against RbAp48 (Qian and Lee, 1995). RbAp46 and RbAp48 appear to be abundant nuclear proteins that are widely expressed. pRb/ RbAp46 and pRb/RbAp48 complexes were demonstrated in vitro using purified proteins and in vivo using Molt 4 (RbAp48) and HeLa (RbAp46) cell
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extracts (Qian and Lee, 1995; Qian et al., 1993), and the bovine homolog of RbAp48 was found to copurify with histone-deacetylase activity (Taunton et al., 1996). In addition, pRb and RbAp48 are readily coimmunoprecipitated in transiently transfected Saos-2 cells (Nicolas et al., 2000). RbAp46 and -48 are associated primarily with the hypophosphorylated form of pRb and, although neither possess LXCXE motifs, interaction with pRb is competed by wild-type, but not mutant TAg LXCXE peptide sequences. These data suggest that both RbAp46 and -48 might be recruited indirectly to pRb through other pRb-binding factors. Accordingly, incubation of an HDAC1-competing peptide, containing the pRb-binding site, dosedependently decreased RbAp48 binding to GST-pRb in vitro (Nicolas et al., 2000). In addition, endogenous pRb/HDAC1/RbAp48 complexes could be detected in vivo and immunodepletion of RbAp48 decreased pRb-associated HDAC activity (Nicolas et al., 2000). Currently, the exact functions of either RbAp48 or -46 are not clear. Since the pRb/RbAp48 complex can bind E2F-1 directly (Nicolas et al., 2000), RbAp proteins may play a direct role in transcriptional repression. Both, RbAp46 and -48 are found in large chromatin-remodeling complexes including the NuRD/NRD/Mi2 and Sin3a/HDAC complexes (reviewed in Muchardt and Yaniv, 1999) and are likely to directly affect gene expression. It has been proposed that the C. elegans homologs of pRb and RbAp48, lin-35 and lin-53, respectively, can function to repress the synthetic multivulva phenotype and antagonize the Ras signaling pathway through hda-1 (C. elegans HDAC homolog)-dependent mechanisms (Lu and Horvitz, 1998). Both RbAp46 and -48 share approximately 30% amino acid identity with Saccharomyces cerevisiae MSI-1 which acts as a negative regulator of the Ras pathway signaling in yeast (Ruggieri et al., 1989). Sin3a, a Mad-interacting protein required for Mad/Max-dependent transcriptional repression, is part of Sin3a/HDAC large multi-protein complex containing HDAC1, HDAC-2, RbAp48, and RbAp46 (among other proteins) (Hassig et al., 1997a; Zhang et al., 1997). Sin3a-dependent transcriptional repression requires HDAC activity (Hassig et al., 1997a; Zhang et al., 1997), and endogenous Sin3a and pRb were coimmunoprecipitated from CV-1 cells lysates (Tokitou et al., 1999). Currently, it is unclear if Sin3a binds pRb directly or if this interaction is mediated by other proteins such as HDACs, RbAp46 or -48, or other unknown molecules.
d. RBP1, RBP2, and Bdp RBP1 (also termed RBAP2) and RBP2 were isolated by a screen of cDNA expression libraries with labeled fragments of recombinant pRb (Defeo-Jones et al., 1991; Kaelin et al., 1992). The initial isolates of RBP1 and RBP2 were partial cDNAs, and the endogenous proteins (at 200 KD and 195 KD
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respectively) are considerably larger than first suspected (Otterson et al., 1993; Fattaey et al., 1993). RBP1 and RBP2 are nuclear phosphoproteins of unknown function. Both proteins contain LXCXE motifs that mediate in vitro association with pRb. The small pocket is important for both RBP1 and RBP2 binding but only the A-domain and A/B spacer mutants disrupted RBP2 binding (Kim et al., 1994). In vivo association of endogenous pRb and RBP1 occurs but in vivo pRb/RBP2 complexes have yet to be found (Fattaey et al., 1993; Kim et al., 1994; Lai et al., 1999a). RBP1 has been shown to mediate both HDAC-dependent and -independent mechanisms of pRb-mediated transcriptional repression, and RBP1 may function as a linker between pRb and HDACs (Lai et al., 1999a). RBP1 binds to HDAC1, -2, or -3, both when overexpressed and endogenously, although RBP1–HDAC1 complexes are more prevalent (Lai et al., 1999a). Moreover, pRb/RBP1 complexes are not detected in 293T cells (which contain E1A and TAg), although RBP1-HDAC complexes are present (Lai et al., 1999a), suggesting that RBP1 tethers HDACs to pRb. RBP1 overexpression induces growth arrest and inhibits E2F1-dependent transcription. However, RBP1/pRb complexes are present throughout the entire cell cycle (Lai et al., 1999b) and it’s unclear how this interaction is regulated in vivo. The functional relevance of the RBP2 and pRb interaction in unknown. In transcription studies, RBP2 can abrogate pRb-mediated repression of E2F-1 (Kim et al., 1994). Bdp, cloned using a putative tumor suppressor EST to screen a cDNA library, is a nuclear protein which contains a region of homology to sequences in both RBP1 and -2, and is also highly homologous to the mammalian Bright and Drosophila dead ringer DNA binding proteins (Numata et al., 1999). Numata and colleagues demonstrated in vitro Bdp binding to pRb, with an apparent preference for the hypophosphorylated form of pRb (Numata et al., 1999). The C-domain of pRb is sufficient for Bdp binding although it is still unclear if Bdp can bind to the small pocket alone. Currently, it is unclear if Bdp binds pRb in vivo.
e. DNMT1 DNA methylation is an important mechanism for chromatin silencing and is catalyzed by three distinct DNA methyltransferases (DNMT1, 3a, and 3b) (Robertson et al., 2000b). Recently, DNMT1 was found to columncopurify with pRb, E2F1, HDAC1, RbAp46, RbAp48, and BRG1 (although RbAp46/48 and BRG1 were weakly associated) (Robertson et al., 2000a). Further analysis demonstrated that DNMT1 and pRb (large pocket fragment) bound each other both in vitro (GST pull down assay and coimmunoprecipitation) and in vivo (endogenous proteins in HeLa or calf brain nuclear extracts). Moreover, anti-pRb antibodies immunoprecipitate DNMT activity from untransfected NIH3T3 cells. Mapping studies have established that the
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small pocket is sufficient for DNMT binding. Both pRb and DNMT1 function in a cooperative manner as transcriptional repressors, and DNMT1 represses, in part, through both HDAC-recruited activity and de novo methylation of the promoter. Further analysis is needed to identify the aspects of pRb function that are mediated by DNMT1 activity in vivo.
f. c-Ski and Sno c-Ski, a 728-amino acid protooncogene product and member of the ski gene family, has been shown to bind pRb (Tokitou et al., 1999). This gene family is comprised of ski and sno which share homology in their C- and N-terminal domains. pRb and c-Ski interact in vitro (mapped to B-domain) and anti-ski and -sno antibodies coimmunoprecipitated endogenous pRb in HeLa cell lysates (Tokitou et al., 1999). c-Ski was found to associate with the nuclear hormone receptor corepressor (N-CoR), HDAC1, and the smad2 corepressor (Akiyoshi et al., 1999; Nomura et al., 1999) and c-Ski is required for transcriptional repression through Mad and the thyroid hormone receptor-beta (Nomura et al., 1999). Although it is unknown if smad2 interacts with pRb, N-CoR was not found to coimmunoprecipitate with pRb (Tokitou et al., 1999). Antibody microinjection experiments demonstrated that anti-ski and -sno antibodies were able to partially prevent transcriptional repression by a pRb-pocket/Gal4 DNA binding domain fusion protein suggesting that ski proteins might be required for pRb repression activity (Tokitou et al., 1999). The viral transforming oncogene product v-ski, which lacks the C-terminal region of c-ski and acts as a dominant-negative inhibitor of Mad-mediated transcriptional repression (Nomura et al., 1999), was also found to prevent Rb-mediated repression (Tokitou et al., 1999). Since v-Ski also inhibits the binding of pRb to HDAC1 (Tokitou et al., 1999), ski proteins might repress transcription by aiding pRb in the recruitment of HDACs to promoter regions.
g. CtIP The E1A C-terminal interacting protein (CtIP) was identified as a positive interactor in a yeast two-hybrid screen for p130-binding proteins (Meloni et al., 1999). CtIP is an LXCXE motif-containing protein and was found to bind pRb and p130 in yeast two-hybrid (Meloni et al., 1999) and coimmunoprecipitation analysis (Meloni et al., 1999 Dick et al., 2000). Mutation of the LXCXE domain of CtIP disrupts its ability to bind pRb. CtIP, together with CtBP, is part of a transcriptional repressor complex, suggesting that it might act as a corepressor with pocket-proteins (Meloni et al., 1999). Although CtIP functions as a repressor in transient transfection assays (Meloni et al., 1999), it has not yet been shown to be recruited to endogenous promoters by pocket-proteins.
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h. TFIIIB, TBP, and BRF/TFIIB pRb represses RNA polymerase III (polIII) activity in in vitro transcription assays and in transient transfection assays (White et al., 1996). Similar results have been described for p107 and p130 (Sutcliffe et al., 1999). Amino acids 379–928 of pRb provide this activity and mutations in the pocket domain of pRb inhibit the effect. Several lines of evidence suggest that pRb represses polIII by physically interacting with TFIIIB (Larminie et al., 1997). pRb inactivation of polIII transcription could be overcome by the addition of purified TFIIIB. A fraction of pRb was found to copurify with TFIIIB and two components of TFIIIB, TBP/TFIIB and BRF, were coimmunoprecipitated with pRb both in vitro and endogenously from partially fractionated HeLa cell extracts in vivo (Larminie et al., 1997). Perhaps most importantly, the level of TFIIIB activity was elevated in extracts of primary cells taken from Rb−/− embryos compared with wild-type cells (White et al., 1996). Both TFIIIB and TFIIIC2 expression levels are deregulated in SV40-transformed cells raising the possibility that TFIIIC2 might also be regulated by pRb (Larminie et al., 1999). In addition to polIII, pRb represses transcription mediated by the TATA-binding protein, TBP (when tethered to an RNA polymerase II (polII)-regulated promoter) (De Luca et al., 1998). Since TBP is important for transcription through all three RNA polymerases, repression through TBP might be an important mechanism for global transcriptional repression by pRb.
i. UBF pRb accumulates in the nucleoli of differentiated U937 cells and associates with UBF, an auxiliary transcription factor that stimulates transcription of rDNA genes through RNA polymerase I (polI). UBF was one of the cDNA clones isolated by Shan and coworkers from a screen of expression libraries with recombinant pRb protein (p56Rb) (Shan et al., 1992). Cavanaugh and colleagues found that this interaction regulates UBF activation of transcription by polI (Cavanaugh et al., 1995). Using a UBF-responsive in vitro transcription system, large pocket fragments of pRb repressed rDNA transcription by reducing preinitiation complex formation (but not transcriptional elongation) (Voit et al., 1997). Subsequent experiments mapped the pRbbinding site and transcriptional inhibition activity to the C-domain of pRb (Voit et al., 1997). pRb does not prevent the interaction of UBF with either polI or TIF-IB but prevents UBF binding to DNA.
j. HBP1 The HMG-box transcription factor, HBP1, is able to bind to pRb and p130 (Tevosian et al., 1997; Lavender et al., 1997). HBP1 is a member of the high mobility group (HMG) class of transcription factors and was
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identified in a yeast two-hybrid screen that used p130 as bait (Tevosian et al., 1997; Lavender et al., 1997). HBP1 was found to coimmunoprecipitate with pRb when these proteins were coexpressed in C33A or U2OS cells by transient transfection. HBP1 contains two sequences that are similar to the pRb-binding sites of E1A (LXCXE and IXCXE), and these motifs form high and low affinity sites that are important for binding to the small pocket domain of pRb. The functional relationship between HBP1 and pRb is not entirely clear. HBP1 expression levels increase during differentiation, and overexpression of HBP1 can induce growth arrest (Tevosian et al., 1997). In contrast, other studies have found that overexpression of HBP1 can also result in oncogenic transformation (Lavender et al., 1997). Gal4-HBP1 fusion experiments demonstrated that HBP1 contains a strong activation domain that is inhibited by pRb, p107, and p130 in transient-transfection reporter experiments in U2OS cells (Lavender et al., 1997). In transfection assays, HBP1 has been shown to repress both the rat N-MYC promoter (Tevosian et al., 1997) and p21WAF1/CIP1 promoter (Gartel et al., 1998). Moreover, repression of the N-MYC promoter was reduced by mutation of the LXCXE and IXCXE sequences of HBP1. Shih and colleagues suggested that the ratio of pRb/HBP1 within complexes might regulate cell cycle exit and differentiation, possibly through myoD signaling (Shih et al., 1998).
k. Pax-3, Pax-5, Pax-6, PHox, B4, Chx10 Initial screens for p130 binding proteins identified a series of homeodomain proteins as candidate interactors (Wiggan et al., 1998). Using yeast twohybrid and in vitro binding assay with GST-fusions, all three pocket-proteins bound Pax-3, B4, and Chx10 (Wiggan et al., 1998). Moreover, ethidium bromide and DNAseI pretreatment did not perturb these interactions, suggesting that association did not occur indirectly through a DNA intermediate. Immunoprecipitation of either endogenous p107 or p130 in C33A cells demonstrated binding to overexpressed Pax-3 suggesting that these interactions might occur in vivo. Using GST-Chx10 or GST-PHox, these interactions were found to favor hypophosphorylated pRb and mapping studies demonstrated that the pRb small pocket was sufficient for binding. Pax-5 was also found to bind unphosphorylated pRb (deletions and mutations in the large pocket and C-domain reduced binding) and p107 (but not p130), and also to associate with TBP in vitro and in vivo (Eberhard and Busslinger, 1999). Pax-6 binds pRb and endogenous complexes have been detected in lens nuclear extracts (Cvekl et al., 1999). Using reporter constructs containing homeodomain binding promoters, all three pocket proteins were found to repress Pax3-mediated transcriptional activation (Wiggan et al., 1998) suggesting the pRb/homeodomain interactions might function as transcriptional repressors.
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l. AHR The aryl hydrocarbon receptor (AHR) is a ligand-activated nuclear receptor that binds the teratogen and carcinogen ligand, dioxin/TCDD, resulting in changes in gene expression, proliferation, differentiation, and apoptosis in mammalian cells. AHR contains an LXCXE motif and has been shown to bind hypophosphorylated pRb by yeast two-hybrid, in vitro binding assay, and coimmunoprecipitation of endogenous proteins (after IP with antibodies to both proteins) (Ge and Elferink, 1998; Puga et al., 2000). pRb and AHR bind in vivo preferentially after ligand treatment and nuclear transformation/translocation (Puga et al., 2000), although ligand treatment is not required for pRb/AHR binding (Ge and Elferink, 1998). AHR also acts synergistically with pRb to cause E2F1-mediated repression and cell cycle inhibition (Puga et al., 2000). Recently, AHR and BRG-1 have been reported to cooperate with pRb to induce cell cycle arrest (Strobeck et al., 2000).
m. Trip230 Trip230 was initially identified as a positive pocket-protein interactor in a yeast two-hybrid screen using p56Rb as bait (Durfee et al., 1993). Analysis of the Trip230 protein amino acid sequence demonstrated homology in the C-terminal region to Trip11, a protein fragment which interacts with thyroid hormone receptor (TR) in a ligand-dependent fashion (Chang et al., 1997). Using anti-TR antibodies, Chang and coworkers demonstrated that Trip230 associates both with pRb and TR in vivo in coimmunoprecipitation of Wr2E3 cell lysates; association with Trip230 was thyroid hormone (T3) ligand-dependent (Chang et al., 1997). pRb binding to Trip230 was mapped at the small pocket of pRb. In the presence of ligand, Trip230 enhanced TR-dependent transcription. Moreover, cotransfection of pRb repressed Trip230-mediated cooperativity; pRb mutants were ineffective. These data suggest that pRb acts as a transcriptional repressor of TR-regulated gene expression through a Trip230-dependent mechanism.
n. NF-B p50 Nuclear factor-B (NF-B) p50 is a member of the NF-B/Rel family of transcription factors which share homology in their N-terminal Rel homology domain. Endogenous pRb and NF-B p50 were coimmunoprecipitated from Jurkat whole cell extracts and pRb bound NF-B p50 in vitro at the Rel homology domain (Tamami et al., 1996). Although pRb stimulates NF-B p50 DNA binding activity, it represses NF-B p50 transcriptional activation activity (Tamami et al., 1996). The pRb/NF-B p50 transcriptional activity was more sensitive to chymotrypsin digestion, arguing for an inactive conformation of NF-B p50 in the presence of pRb. The pRb/NF-B interaction
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might be biologically relevant during apoptotic signaling. For instance, both NF-B and pRb can suppress E2F1-mediated apoptosis (Phillips et al., 1997, Phillips et al., 1999). E2F1 is hypothesized to kill by blocking NF-B activity (composed of p65/p50 homodimers) through inhibition of IKK and down regulation of TRAF2 protein levels (Phillips et al., 1999). However, since pRb directly inhibits NFB activity, it will be important to determine how apoptotic signaling through NFB occurs in cells expressing wild-type pRb.
o. Elf-1 The association between pRb and Elf-1 has many parallels with pRb/E2F. pRb binds to the transcriptional activation domain of this lymphoid-specific Ets-related transcription factor and represses its activity (Wang et al., 1993). Elf-1 binds preferentially to the hypophosphorylated form of pRb, and complexes formed by endogenous pRb and Elf-1 proteins are disrupted as cells enter S-phase. It has been suggested that this interaction may be important for the coordination of lymphokine production with cell cycle progression in activated T-cells.
p. PU.1 pRb has been proposed to repress the activity of the transcriptional activator, PU.1, by binding to its activation domain (Hagemeier et al., 1993). In vitro, pRb prevents PU.1 from binding to TFIID or to TBP (Weintraub et al., 1995). The pocket domain of pRb has limited sequence similarity with TBP and TFIID (Hagemeier et al., 1993), raising the possibility that pRb may interfere with the activity of many transcription factors by competing for binding with components of the basal transcriptional machinery (Hagemeier et al., 1993; Weintraub et al., 1995). pRb binding to PU.1 is disrupted by the dorsal mesodermal patterning factor, goosecoid protein, which binds PU.1 and negatively regulates erythropoietic differentiation during embryogenesis (Konishi et al., 1999).
q. RBaK RBaK was initially identified as a pocket-protein interactor in a yeast twohybrid screen with p56Rb as bait (Durfee et al., 1993). The cDNA of RBaK predicted a putative transcriptional regulator with Kruppel-type zinc fingers and a KRAB repressor motif (Skapek et al., 2000). RBaK was found to be a 80 kD nuclear protein, which interacted with pRb in vitro (GST pull down). It is unknown if pRb and RBaK interact directly in vivo. RBaK overexpression was found to suppress S-phase progression as well as E2F-dependent activation of E2F1 promoter activity in 10T1/2 fibroblasts (approximately 70% repression activity compared to pRb). Currently, it is unclear if pRb is required for RBaK-mediated repression activity.
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r. p120E4F
Human p120E4F is a ubiquitously expressed GLI-Kruppel-related zinc finger phosphoprotein which has been recently reported to bind pRb (Fajas et al., 2000). p120E4F is a low abundance protein which represses the adenoviral E4 promoter. p120E4F was identified initially as a pRb-binding protein through a yeast two-hybrid screen using pRb as bait (Fajas et al., 2000). Subsequently, pRb was demonstrated to associate with p120E4F both in vitro by GST pull down assay and in vivo with endogenous proteins in U2OS and MCF7 cell lysates (p120E4F did not bind p107 or p130). Mapping studies demonstrated that p120E4F was able to bind pRb at the large pocket with the B-domain and C-terminal domain being sufficient for binding (weak binding to the B-domain alone was detected). Cotransfection and reporter experiments demonstrated that pRb cooperated with p120E4F to efficiently repress an E4F-responsive element and stimulated p120E4F consensus binding. Moreover, overexpression p120E4F is able to induce growth arrest in mouse embryonic fibroblasts in a pRb-dependent manner. The role of p120E4F in normal cell cycle control has yet to be determined.
2. pRb AS AN ACTIVATOR OF TRANSCRIPTION In contrast with the studies mentioned above, there are also a number of studies in which the overexpression of pRb is correlated with an increase in gene expression. These reports are far fewer than those linking pRb to transcription repression, and the mechanisms behind these effects are not well understood. In a few examples, transcriptional activation has been correlated with complex formation with pRb however in several cases the activation appears to be indirect.
a. MyoD, Myogenin Several lines of evidence have suggested a role for pRb in muscle differentiation. pRb cooperates with MyoD (an activator of muscle specific gene expression and myogenic conversion) to promote muscle differentiation when they are both expressed in Saos-2 cells after transfection (Gu et al., 1993). Moreover, myoD induces pRb expression (Martelli et al., 1994) and pRb expression is required for proper muscle gene expression (Novitch et al., 1996). In addition, when myoblasts are formed from pRb−/− cells in vitro, the cell nuclei of differentiated cells are able to re-enter the cell cycle in response to serum stimulation, suggesting that pRb is required for muscle cells to remain quiescent (Schneider et al., 1994). The analysis of transgenic mice in which the homozygous mutation of Rb is partially rescued by an Rb minigene, revealed multiple defects in skeletal muscle differentiation in the absence of normal levels of pRb (Zacksenhaus et al., 1996).
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These defects suggest that pRb is required for myoblasts to exit the cell cycle, for the maintenance of myoblasts, and for the formation of myofibers from myotubes. Gu and coworkers (Gu et al., 1993) found MyoD/pRb and myogenin/pRb complexes in extracts of differentiated C2 myotube extracts and demonstrated that in vitro association between these proteins was mediated by the basic helix-loop-helix region (bHLH) of MyoD and the C-terminal half (amino acids 605–928) of pRb. pRb/MyoD complexes were competed by a TAg peptide. Other myogenic bHLH factors, including myogenin, Myf-5, and MRF4 were reported to bind pRb similarly (Gu et al., 1993). The effects of pRb on MyoD function are not entirely clear and there appears to be a complicated series of interactions between pRb, cell cycle regulators, and muscle-specific transcriptional activators during normal myogenesis. In vitro, GST-pRb was found to inhibit the DNA binding activity of MyoD homodimers, and to promote the formation of MyoD/E2-2 heterodimers; the addition of anti-pRb antibodies to C2 myotube extracts diminished the DNA binding activity (Gu et al., 1993). However, early evidence also pointed to cooperative transactivation of specific promoters and pRb has been found to enhance the MyoD transactivation of an E-box reporter containing MyoD binding sites (Novitch et al., 1999). MyoD has been found to require pRb in order to activate the MEF2 promoter. MEF2 also plays a critical role in the regulation of skeletal muscle-specific genes and Novitch et al., found that pRb enhances MyoD-dependent transcription by enhancing the ability of MyoD to stimulate the MEF2 transactivation domain, a phenomenon which is defective in pRb-deficient cells (Novitch et al., 1999). How pRb achieves this effect is unclear. At least three studies fail to confirm the in vivo pRb/MyoD interaction reported by Gu and colleagues (Halevy et al., 1995; Zhang et al., 1999; Li et al., 2000) and a recent study found that the expression of MyoD failed to rescue the cell-cycle arrest defect in myotubes caused by expression of a dominant-negative pRb fusion protein (Li et al., 2000). Thus, even if the pRb/MyoD interaction occurs, it is likely other pRb targets are important during myogenesis and interact in a complex fashion to regulate differentiation and cell cycle exit. MyoD has also been found to interact with cdk4 and to inhibit cdk4-mediated inactivation of pRb (Zhang et al., 1999).
b. NF-IL6, C/EBP NF-IL6 and C/EBP are members of the family of CCAAT/enhancer-binding proteins that act to promote and maintain differentiation of hematopoietic cells (NF-IL6) and adipocytes (C/EBP). Association of pRb and NF-IL6 was detected in U937 cells when these cells were stimulated to differentiate into monocytes and macrophages (Chen et al., 1996a). The interaction was not
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readily apparent in undifferentiated U937 cells. In vitro association was demonstrated for three naturally occurring forms of NF-IL6. This interaction required two domains of full-length NF-IL6 and sequences within the pocket domain of pRb. pRb appears to modulate the activity of NF-IL6 by altering its DNA-binding activity; the DNA-binding activity of GST-NF-IL6 was stimulated by 10–20 fold by addition of a pRb fragment, p56Rb (Shan et al., 1992). In transient expression experiments, the coexpression of pRb increased the NF-IL6 activation of a reporter construct from 5 fold to greater than 18 fold. A pocket mutant form of pRb had no effect on either DNAbinding or transcriptional activation by NF-IL6, indicating that this effect is a specific property of the wild-type protein. Chen and coworkers found that Rb−/− embryonic lung fibroblasts differed from wild-type in that they failed to differentiate spontaneously into adipocytes when density arrested and could not be induced to undergo adipocyte differentiation in response to hormonal signals (Chen et al., 1996b). The reexpression of pRb in Rb−/− cells restored these properties. pRb binds in vitro to GST fusion proteins containing either C/EBP␣, C/EBP␦ or the transcriptionally-active (LAP) form of C/EBP. Binding to C/EBP was disrupted by mutations in either half of the pocket domain of pRb. The pRbbinding motif on C/EBP is not known; however, it has been suggested that the motif D/E(X3)DLF present in C/EBP␣, C/EBP, C/EBP␦, and NF-IL6 may be involved (Chen et al., 1996b). This sequence is found in the pRb-binding site of E2F-1 and is conserved between E2F family members. The interaction between pRb and C/EBP has many similarities with pRb/NF-IL6 complexes. Physical interaction between pRb and C/EBP␣ or C/EBP was found once cells were induced to differentiate into fibroblasts, but no interaction was seen in the uninduced cells. pRb enhanced the DNA binding activity of C/EBP (but pRb could not be found stably associated with C/EBP when DNA-binding activity was monitored by EMSA). Furthermore, in transient transfection assays, C/EBP stimulated the expression of a reporter construct by 10 fold in the absence of pRb, but this activation was raised to 40 fold when pRb was also coexpressed. Thus, it appears that pRb acts cooperatively with both NF-IL6 and C/EBP, possibly to facilitate differentiation. However, it is still unclear if pRb/NF-IL6 or pRb/C/EBP interactions are required for a proper differentiation program to occur.
c. Sp1, Sp1-I, and Sp3 High levels of expression of pRb have been shown to elevate Sp1 (Kim et al., 1992; Udvadia et al., 1993) and Sp3 (Udvadia et al., 1995)-dependent transcription. Thus far, no direct physical interaction between pRb and Sp3 has been found, and only one report of pRb binding to Sp1 has been published where endogenous Sp1 and pRb were coimmunoprecipitated in
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K1 cell nuclear extracts (Noe et al., 1998). Moreover, the evidence for a direct pRb/Sp1 interaction is questionable since Sp1 was reported to interact with both hypophosphorylated and hyperphosphorylated pRb throughout all phases of the cell cycle and two other reports failed to detect any pRb/Sp1 complex formation (Chen et al., 1994; Shao et al., 1995). One alternative hypothesis is that Sp1 is regulated by pRb indirectly through a negative regulator (Chen et al., 1994). The Sp1-I protein, an inhibitor of Sp1 DNA binding activity to a c-jun Sp1 site, was purified using an pRb affinity column (Chen et al., 1994). These data suggest a mechanism whereby pRb acts to sequester Sp1-I from Sp1, indirectly activating Sp1-mediated transcription.
d. c-Jun, JunD, and JunB c-Jun, a member of the AP-1 family of transcriptional activators, has been shown to bind pRb both in vitro and in vivo (Nead et al., 1998). Coimmunoprecipitations from synchronized HaCaT keratinocytes demonstrated that both active (phosphorylated) and inactive forms of c-Jun associated preferentially with hypophosphorylated pRb during G1. Moreover, these pRb/c-Jun complexes were present in differentiated but not asynchronously growing primary human keratinocytes. Using various pRb–GST fusions, as well as pRb-deletion and mutant constructs, c-Jun binding to pRb was mapped to both the B pocket region and part of the C-terminal domain of pRb and the c-Jun leucine zipper. Both JunD and JunB were also able to bind pRb but it is unclear if they associate in a similar manner as c-Jun. Using transient transfection-reporter assays, pRb bound and enhanced c-Jun-mediated induction of the collagenase gene promoter containing a single AP-1 site (Nead et al., 1998). Currently, it is unclear if pRb is important for the normal cellular function of AP-1 family members or whether these factors serve to regulate pRb activities in vivo.
3. ADDITIONAL TRANSCRIPTION FACTORS THAT BIND pRb a. hBrm1 and BRG1 Based on studies conducted in yeast, hBrm1 and BRG1, the human homologs of yeast SWI2/SNF2, are expected to have pleiotropic effects on gene expression. Both hBrm1 and BRG1 contain LXCXE motifs, associate with pRb in vitro, and antibodies raised against either protein have been shown to coprecipitate pRb from cell lysates (Dunaief et al., 1994; Singh et al., 1995). In addition, all three pocket-proteins have been reported to interact with both hBrm1 and BRG1 in yeast two-hybrid assays (Strober et al., 1996). pRb and hBrm1/BRG1 form a complex that can induce or repress gene transcription. Singh and colleagues showed that pRb cooperated with hBrm to potentiate transcriptional activation mediated by the
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glucocorticoid receptor. This activity was missing when cell lines deficient for pRb (C33A or Saos-2) or hBrm (SW-13) were used but could be reconstituted by the reexpression of either pRb or hBrm1. Dunaief and colleagues found that expression of hBRG1 caused growth arrest in SW-13 cells which was overcome by the coexpression of E1A. Mutants of hBRG1 that were defective for the pRb-interaction had greatly reduced growth arrest activity but it is notable that these mutants remove a substantial portion of the wild-type protein. These experiments suggest that pRb functionally synergizes with hBrm1 and BRG1, potentially through a direct physical interaction. In agreement with this, hBrm1 and BRG1 were found to cooperate with pRb to repress E2F1 and c-fos transcription, respectively (Trouche et al., 1997; Murphy et al., 1999). hBrm1/pRb/E2F1 appear to form complexes both in vitro and in vivo (Trouche et al., 1997). Recently, pRb/BRG1/HDAC1 trimolecular complexes have been found in transiently-transfected C33A cells, and it has been suggested that a specific subset of E2F-regulated genes might be regulated by pRb/BRG1 complexes (Zhang et al., 2000). Currently it is unclear how SWI/SNF complexes and pRb cooperate in transcriptional repression.
b. ATF-1 and ATF-2 The overexpression of pRb has differential effects on the activation properties of the ATF-1 and ATF-2 transcription factors. Cotransfection of a pRb expression vector activates the expression of the transforming growth factor (TGF)-1 and TGF-2 genes (Kim et al., 1991, Kim et al., 1992; Gong et al., 1995). pRb expression stimulates the activity of the TGF-2 by 12-fold, an effect that requires an ATF-binding site in the promoter. This site is a high affinity binding site for ATF-2, and a Gal4-ATF-2 fusion protein was also found to support pRb-mediated transcriptional activation (Kim et al., 1992). However, pRb inhibits the actions of ATF-1 on TGF- expression (Gong et al., 1995). Although ATF-2 was retained on a GST-pRb affinity column, it remains unclear whether pRb acts directly or indirectly on ATF-2 and association between endogenous ATF-2 and pRb has yet to be reported. Although an ATF-1 and pRb interaction has been reported (Gong et al., 1995), the binding regions have not been mapped. Currently, it is unclear how pRb causes differential effects on ATF-mediated transcription.
c. p202 p202, an interferon-inducible nuclear phosphoprotein of 52 kD, is an LXCXE-containing protein that associates with pRb (Choubey and Lengyel, 1995). In vitro pRb/p202 association required two domains of p202. At least two domains of pRb appeared to contribute to the interaction since both amino acid regions 1–254 and 379–928 were sufficient for binding.
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Complexes formed in vivo were detected following interferon treatment of murine AKR-2B cells that had been transfected with an pRb-expression plasmid (Choubey and Lengyel, 1995). p202 has been shown to interact with c-fos and c-jun and to inhibit NF-B and AP-1 activities, although it is unknown if p202’s regulation of these genes is affected by pRb (Min et al., 1996). p202 can inhibit transcriptional activation mediated by E2F1/DP1 (Choubey et al., 1996) and E2F4/DP1 (Choubey and Gutterman, 1997) in a pRb-independent manner, by directly binding to E2F1 or -4 in vitro and in vivo (independent of the presence of pocket-proteins). At present, it seems likely that the p202/pRb interaction is indirectly mediated by E2F proteins.
d. Mi Mi is a bHLH-leucine zipper protein that is encoded by the microphthalmia gene and activates melanocyte-specific gene expression. Expression of E1A was found to repress the expression of several melanocyte specific genes and this effect was relieved by ectopic expression of Mi (Yavuzer et al., 1995). Mi was found to bind to GST-pRb in vitro. It was suggested that by analogy with pRb/MyoD, pRb might act to stimulate the activity of Mi. However, the effects of pRb on Mi-dependent transcription have yet to be reported, and complexes between endogenous Mi and pRb proteins have not yet been found.
e. Id2 Id proteins are naturally-occurring dominant negative forms of bHLHleucine zipper transcription factors that inhibit cellular differentiation in several systems. Id2 was initially reported to reduce the growth suppression activities of pRb on Saos-2 cells without affecting the phosphorylation status of pRb (Iavarone et al., 1994). In vitro binding assays showed that pRb bound to the helix-loop-helix domain of Id2, suggesting that Id2 might act to directly inhibit pRb. No pRb association was seen when two family members, Id1 and Id3, were tested (Lasorella et al., 1996). Id2 was also reported to bind to p107 and p130 in vitro and to prevent them from arresting Saos-2 cells in G1 (Lasorella et al., 1996). Further analysis of Id2 binding demonstrated exclusive association of Id2 to the hypophosphorylated forms of pocket-proteins, with pRb/ and p107/Id2 binding increasing as cells progress through S-phase upon serum stimulation (Lasorella et al., 2000). A recent study demonstrated an interaction with pRb and Id2 in which the loss of Id2 was found to rescue the embryonic lethality in pRb null mice in a dose-dependent fashion (Lasorella et al., 2000). Remarkably, in these animals the loss of Id2 suppressed abnormal cell proliferation and apoptosis caused by lack of pRb, and both cells of the nervous system
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and haematopoietic systems appeared largely normal. However, Id2−/−, pRb−/− mice still did not survive past birth due to incomplete skeletal muscle development, suggesting that other important pRb targets exist in some cell types. Id2 expression correlated highly with N-myc amplification in tumor cells, and myc function was shown to be largely Id2-dependent (Lasorella et al., 2000). Specifically, the ability of myc to bypass a pRb-dependent cell cycle arrest was shown to be dependent on Id2. Further studies are necessary to show that the genetic interaction between pRb and Id2 is due to the physical interactions between their products. Nevertheless, these results suggest that Id2 may be a key partner for pRb during development and that the loss of this interaction may be an important determinant during neoplasia.
f. Pur␣ Pur␣ is a transcriptional activator that binds both single-stranded and double-stranded DNA, and has a higher affinity for single stranded DNA containing repeats of the motif GGN (where N is not a G) (Bergemann et al., 1992b; Bergemann and Johnson, 1992a). Hypophosphorylated pRb was found to bind to Pur␣ in vitro when both proteins were cotransfected in CV-1 cells (Johnson et al., 1995). In addition, endogenous pRb was found in immune complexes prepared from extracts of monkey CV-1 cells with a panel of monoclonal antibodies to Pur␣. Binding of Pur␣ to pRb was competed by a TAg peptide. Pur␣ lacks an LXCXE motif and the binding sequences have been localized to the carboxyl-terminus of Pur␣ but have not been defined in detail. Although the effect of pRb on Pur␣-mediated transactivation has yet to be reported, pRb was found to reduce the DNAbinding activity of Pur␣ suggesting that pRb may inhibit Pur␣ functions. However, recent evidence suggests that Pur␣ can interact directly with E2F1 and suppress its transactivational activity (Darbinian et al., 1999) raising the possibility that the Pur␣/pRb interaction might be mediated through E2F proteins.
g. mdm2 and p53 mdm2 has been studied extensively for its negative regulation of p53 via degradation by the ubiquitin-proteosome pathway. Xiao and colleagues found that a minor fraction of mdm2 and pRb are physically associated in U20S and U937 cells (Xiao et al., 1995). In binding assays, the in vitro association of these two proteins was mediated by the carboxyl-terminus of pRb (amino acids 792–928). pRb has been found to inhibit the ability of mdm2 to direct the degradation of p53 (Hsieh et al., 1999). Accordingly, coexpression of pRb was found to block the negative inhibition of mdm2 on p53-induced apoptosis in Saos-2 and H1299 cells (Hsieh et al., 1999). Both of these effects of pRb required its C-terminal domain. In these experiments,
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pRb had no effect on p53-induced apoptosis or on p53-dependent transactivation in the absence of mdm2. Interestingly, pRb prevented mdm2 from inhibiting p53-mediated repression of gene expression. Hsieh and colleagues further demonstrated that pRb, p53, and mdm2 form a trimolecular complex, whereby hypophosphorylated pRb interacts indirectly with p53 through direct binding to mdm2. No direct interaction could be detected between pRb and p53, in vitro or in vivo, in the absence of mdm2. Phosphorylation of pRb by cyclin E/cdk2 was found to reduce its binding to p53/mdm2. pRb binding was not sufficient to dissociate mdm2 from p53 but instead stabilized a complex in which the apoptotic activity of p53 remained intact. The biological significance of this interaction is still unclear but DNA damage of U2OS, MCF-7, and RKO cells (which all express wild-type p53 and pRb) resulted in more endogenous pRb immunocomplexed with p53 suggesting that this trimolecular complex might regulate DNA damage responses in vivo. p53 and mdm2 have been reported to associate with both E2F-1 and DP-1 (Martin et al., 1995; O’Connor et al., 1995; Sorensen et al., 1996) and to regulate E2F-dependent transcription (Martin et al., 1995; O’Connor et al., 1995; Xiao et al., 1995). However, in vitro association between pRb and mdm2 was seen with mutants of pRb that fail to bind to E2F (Xiao et al., 1995), and regulation of E2F activity by mdm2 and p53 occurs in the absence of pRb (Martin et al., 1995; O’Connor et al., 1995). It is difficult therefore to reconcile the reported interactions between pRb, E2F, DP-1, mdm2 and p53 into a simple model and, currently it remains unclear whether the primary function of the pRb/mdm2 interaction is to regulate the activity of pRb, or mdm2, or p53 or E2F.
h. c-myc and N-myc pRb has been proposed to regulate the activities of c-myc and N-myc by binding to their activation domains (Rustgi et al., 1991). pRb has been reported to activate c-myc activity (Adnane and Robbins, 1995), repress c-myc activity (Rustgi et al., 1991), and to regulate c-myc in opposite ways in the same cell type (Batsche et al., 1994). However, in most cases, pRb has not been found to bind c-myc in vivo or have any effect on myc activity (Beijersbergen et al., 1994; Cziepluch et al., 1993; Gu et al., 1994). Unlike the pRb/E2F interactions, no myc/pRb complexes have been found between the endogenous proteins and the in vivo significance of the in vitro complexes is dubious. p107/c-myc complexes have been found in cell lysates and p107 can regulate the transcriptional activity of myc raising the possibility that other pRb-family members might be more relevant for myc function (Hoang et al., 1995; Beijersbergen et al., 1994; Gu et al., 1994).
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i. AP-2 AP-2 is a 52-kD nuclear transcriptional regulator of histone and Ecadherin genes, possibly through its interaction with pRb (Wu and Lee, 1998; Batsche et al., 1998). AP-2 has both transcriptional activation and repression activities. It was shown to bind pRb at the large pocket and C-domain deletions significantly reduced AP-2 binding. AP-2 interacts with pRb both in vitro and in vivo (after overexpression in transient transfection assays (Wu and Lee, 1998), and endogenously in MDCK and HaCat cell lysates (Batsche et al., 1998). Although pRb inhibited transactivation of the H3core promoter by AP-2 (Wu and Lee, 1998), it stimulated transcription of the Ecadherin promoter through AP-2 (an effect also induced by c-myc through AP-2; Batsche et al., 1998). Further investigation is needed to fully understand how pRb acts to differentially control transcription through AP-2.
j. TAFs Shao and coworkers have suggested that pRb stimulation of Sp1 transcription may be mediated by the TATA-binding protein-associated factor, TAFII250 (Shao et al., 1995). pRb stimulation of Sp1 was partially abrogated in ts13 cells when these cells were maintained at the non-permissive temperature (since they contained a temperature-sensitive mutation in the TAFII250 gene). TAFII250 was found to bind to a GST-pRb fusion protein in vitro and to a Gal4-pRb fusion protein in vivo following cotransfection with an epitope-tagged TAFII250 expression plasmid. pRb also bound (albeit weakly) to other TAFs including TAFII150 and TAFII80, but not TAFII110 (Shao et al., 1995). Unlike most pRb-binding proteins, TAFII250 interacts with the C706F mutant form of pRb and binds pRb independently at two sites, the N-terminus and the large pocket (Shao et al., 1997). The large pocket of pRb inhibits TAFII250 kinase activity. TAFII250-kinase activity is thought to be required for cyclin A and cdc2 gene transcription (O’Brien and Tjian, 1998), raising the possibility that pRb might repress transcription at some TAFII250-dependent promoters (Siegert and Robbins, 1999).
k. RIZ and Estrogen Receptor The pRb-interacting zinc finger protein (RIZ) was identified as a putative pRb-binding protein by screening of a rat cDNA expression library with p56Rb (Buyse et al., 1995). The RIZ cDNA encodes a nuclear protein with three zinc-finger motifs, an LXCXE motif, E1A-cr1 and -cr2 related regions, a GTPase domain, an SH3 domain, and a proline rich domain with several SH3 binding motifs (Buyse et al., 1995). pRb associated with RIZ both in vitro and in vivo (when endogenously immunoprecipitated with anti-RIZ antibodies from HT1080 cell extracts). Endogenous RIZ/pRb binding was also detected in vivo by coimmunoprecipitation in MCF-7 cells (Abbondanza
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et al., 2000). RIZ is able to facilitate estrogen inducibility at a promoter containing an incomplete estrogen responsive element (Medici et al., 1999). RIZ and the estrogen receptor (ER) also coimmunoprecipitated in vivo. Interestingly, although pRb did not bind the estrogen receptor under normal conditions, treatment with estrogen ligand facilitated an ER/pRb and ER/RIZ interaction and diminished the RIZ/pRb interaction (Abbondanza et al., 2000). Treatment with anti-estrogen ligand eliminated both RIZ- and pRb/ER interactions. These interactions were detected in lysates from primary cells (rat uterus) and under physiological levels of estrogen ligand. Further studies are needed in order to identify changes in gene expression that are regulated by these complexes.
l. Che-1 Che-1, initially identified by its ability to interact with the hRPB11 po1II subunit in a yeast two-hybrid screen, is a 62-kD human protein containing a leucine zipper and three nuclear receptor binding consensus motifs (Fanciulli et al., 2000). Che-1, which shares some homology with TAg, was found to bind pRb both in vitro (through GST-fusions and yeast two-hybrid) and in vivo (after overexpression in COS7 cells and endogenously in U2OS cells) (Fanciulli et al., 2000). Che-1 overexpression in Saos-2 cells was able to inhibit the effects of pRb on the E2F1-mediated transactivation of a DHFRluciferase reporter construct as well as the repression of E2F1-Gal4 at a Gal4 responsive promoter. Although Che-1 reduced the growth suppressive activity of pRb in colony-forming experiments with Saos-2 cells, it is unclear how Che-1 might normally function to regulate pRb in vivo.
m. Cream-1/Rbap2 Cream-1 (or Rbap2) is a 120 kD nuclear protein of unknown function with homology to the TFII-I transcriptional regulator (Yan et al., 2000). It was initially isolated from a cDNA expression screen for pRb-interacting proteins (Shan et al., 1992) and later found to associate with pRb (at the C-domain) both in vitro and in vivo (Yan et al., 2000). Cream-1 appears to be identical to the muscle-enhancer-binding protein, Mus-TRD1, and Cream1-Gal4 DNA binding domain fusions are strong activators in yeast, suggesting that Cream-1 might be a transcriptional regulator.
n. PML PML is a nuclear phosphoprotein of unknown function but has been implicated in growth arrest as a transcriptional coregulator. Chromosomal translocations of the PML gene and the retinoic acid receptor-␣ (RAR␣) gene are found in over 95% of acute promyelocytic leukemia patients. PML locates to the nuclear matrix in PML bodies and has been colocalized with
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pRb (Alcalay et al., 1998). pRb was coimmunoprecipitated with PML (and vice versa) in U937 cells which highly overexpressed PML under control of an inducible promoter (Alcalay et al., 1998). Similar results were seen in pRband PML transiently transfected C33A cells. Moreover, endogenous pRb and PML coimmunoprecipitated in human differentiated erythroid hematopoietic progenitor cells suggesting that this interaction likely occurs in vivo (Labbaye et al., 1999). PML bound the small pocket of pRb in vitro, and the exon-21 and -22 pRb pocket mutants and the pRb C-domain fragment all failed to bind to PML (Alcalay et al., 1998). PML is a pRb-independent growth suppressor and overexpression of PML did not affect pRb-mediated repression or activation of E2F1 or Sp1 promoters, respectively (Alcalay et al., 1998). However, PML did suppress the pRb-dependent activation of the glucocorticoid receptor (GR) promoter suggesting that the PML/pRb complex might function during transcriptional regulation in vivo. Interestingly, although the PML–RAR␣ fusion is missing the C-terminal pRb-interaction domain of PML, it still retained pRb binding capacity through an unknown mechanism (Alcalay et al., 1998) and overexpression of the PML–RAR␣ fusion disperses PML bodies and alters pRb localization. Recent evidence suggests that oncogenic ras might contribute to PML and pRb association although details of this interaction are unclear (Ferbeyre et al., 2000).
o. Rim Rim was identified in a yeast two-hybrid screen for Max-interacting proteins (Fusco et al., 1998). Rim is a ubiquitously expressed human protein with two leucine zipper motifs, four putative nuclear localization signals, an E1A/CtBP binding motif, and an LXCXE domain. Subsequent two-hybrid screens identified all three pocket-proteins as Rim interactors. Deletional mutagenesis of Rim identified the LXCXE domain required for pocket protein interaction in yeast two-hybrid assays. Overexpressed Rim fragments were coimmunoprecipitated with pRb in 293 cells. These results are surprising given that these cells express high levels of E1A that disrupts pRb/LXCXE complexes. Currently, there is no known function ascribed to Rim.
C. Other pRb-Associated Proteins 1. KINASES THAT ARE REGULATED BY pRb a. c-Abl pRb binds to the ATP-binding lobe of c-Abl and inhibits the activity of this kinase (Welch and Wang, 1993, Welch and Wang, 1995b). The interaction is readily detected in cell lines in which c-Abl is highly expressed.
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When endogenous proteins were examined, approximately 5% of c-Abl was estimated to coimmunoprecipitate with pRb. The c-Abl binding site of pRb has been mapped by in vitro binding assays. This binding site is C-terminal to the pocket domain and lies between amino acids 768 and 869 of pRb (Welch and Wang, 1993, Welch and Wang, 1995b). Mutational analysis of pRb indicated that the c-Abl-binding site is distinct from both the E2F and cyclin D binding sites. Consistent with this, trimolecular complexes could be found in cell lysates that contained pRb/c-Abl/E2F and pRb/c-Abl/cyclin D2 complexes, and these complexes assembled in vitro using baculovirus expressed proteins (Welch and Wang, 1995b). The functional significance of the pRb/c-Abl complex is not fully understood. At one level, pRb may act to restrict the kinase activity of c-Abl in G1, and phosphorylation of pRb may be one of the signals enabling the activation of c-Abl tyrosine kinase during S-phase (Welch and Wang, 1993). A second connection between c-Abl and pRb is that c-Abl can cause cell cycle arrest when overexpressed (Sawyers et al., 1994). This cytostatic activity of c-Abl requires pRb because the effect is lost in Rb−/− fibroblasts (Wen et al., 1996). In addition to these connections, c-Abl could be a target for growth suppression by pRb. The overexpression of wild-type c-Abl or a kinase defective mutant of c-Abl can prevent growth suppression of Saos-2 cells by pRb and this may be due, in part, to titration of pRb (Welch and Wang, 1995a). Although c-Abl does not bind to the pocket domain, it is clear that the short C-terminal domain of pRb that interacts with c-Abl is important for pRb’s growth suppression activity. Not only is this region required for pRb to suppress the growth of Saos-2 cells, but the expression of a short fragment of pRb that binds to c-Abl was found to prevent suppression by the full length protein (Welch and Wang, 1995b). However it remains to be proven that c-Abl is the critical target of this domain. In addition to growth suppression, c-Abl overexpression leads to apoptosis, which can be partially abrogated by coexpression of pRb or the pRb C-domain (Theis and Roemer, 1998).
b. Rak Rak is an src-related, nuclear, tyrosine kinase that was detected in immune complexes prepared from SK-BR cells using anti-sera to a Rak peptide (Craven et al., 1995). pRb/Rak complexes were detected in lysates of serum starved or hydroxyurea-treated cells but not in nocodozole-treated cells suggesting that Rak binds preferentially to hypophosphorylated forms of pRb. Although Rak is structurally similar to c-Abl, Rak and c-Abl appear to interact with pRb in different ways. In vitro binding assays suggested that the binding domains may include the SH3-domain of Rak and the small pocket domain of pRb. The functional significance of this interaction has yet to be tested.
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2. HEATSHOCK PROTEINS Two heatshock proteins have been reported to associate with pRb and suggested to fold pRb into an active conformation.
a. hsc73 pRb immunoprecipitates have been reported to contain the 73 kD heat shock cognate protein, hsc73 (Nihei et al., 1993), although only a minor proportion of pRb was found in the complex. The hsc73 binding site was mapped to the N-terminal domain of pRb using an in vitro binding assay; a GST fusion protein containing amino acids 1–514 of pRb was the smallest fragment that retained binding activity (Inoue et al., 1995). Hsc73 binds preferentially to the unphosphorylated form of pRb and complex formation was unaffected by the addition of E1A. Hsc73/pRb complexes were found to be disrupted by the addition of ATP. Although the biological significance of these observations are not entirely clear, it is possible that hsp73 might act as a “molecular stabilizer” of nonphosphorylated pRb (Inoue et al., 1995).
b. hsp75 Hsp75 (Chen et al., 1996) was isolated in a two-hybrid screen using a fusion containing the carboxyl-terminal pRb56 fragment. Hsp75 contains an LXCXE motif that mediates interaction with the pocket domain of pRb and is required for high affinity interaction. Hsp75 is ubiquitously expressed and normally located in the cytoplasm, but relocates to the nucleus following heat shock. pRb and hsp75 could not be coimmunoprecipitated from extracts of asynchronous cells. However, pRb is distributed throughout the cytoplasm when the nuclear envelope breaks down and pRb/hsp75 complexes were found in extracts of M-phase cells. In addition, pRb/hsp75 complexes were detected following heatshock treatment during the period when hsp75 is found in the nucleus. Using partial tryptic cleavage of a GST-pRb fusion protein as an assay, Chen and colleagues demonstrated that hsp75 folded GSTpRb into a conformation resistant to cleavage (Chen et al., 1996). Whether hsp75 is the only pRb-binding protein able to refold pRb has not yet been tested.
3. NUCLEAR MATRIX ASSOCIATION The unphosphorylated forms of pRb found in G1-phase cells are resistant to extraction by low salt/detergent extraction buffers (Templeton et al., 1991; Mittnacht and Weinberg, 1991; Templeton, 1992). Conversely, mutant forms of pRb and phosphorylated pRb are readily extracted by the same conditions suggesting that this nuclear tethering may be important for pRb function. A significant proportion of pRb remains bound to the nuclear matrix following extraction with salt, detergent, and nuclease treatment,
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suggesting that pRb might bind directly to a component of the matrix (Mancini et al., 1994).
a. Lamin A /C The nuclear lamina contains three kinds of lamins (A, B, and C). Lamin A and C are alternatively processed products of a single gene and both have been found to associate with pRb. Lamin C was isolated from the screen of cDNA expression libraries with p56Rb (Shan et al., 1992). The respective binding sites have been mapped by in vitro binding assays to a carboxylterminal fragment of pRb (amino acids 611–928) and amino acids 247–355 of Lamin A (Mancini et al., 1994; Ozaki et al., 1994). A subset of pRb is found at the nuclear lamina and the functional significance of this interaction is unclear. Recent studies have shown that pRb accumulated in G1 phase in Lamin A/C-associated structures, and that these perinucleolar foci mark the initial sites of DNA synthesis when primary cells enter S phase (Kennedy et al., 2000).
b. p84N5 p84 is encoded by a cDNA that was isolated in a two-hybrid screen using the amino-terminal 300 amino acids of pRb as a bait (Durfee et al., 1994). The pRb/p84 interaction is cell cycle regulated. GST-p84 fusion proteins bind preferentially to unphosphorylated forms of pRb in cell lysates. Whereas p84 is present throughout the cell cycle, p84 could only be coprecipitated with pRb from CV-1 cell extracts when extracts from G1-phase cells were used. p84 is a component of the nuclear matrix and immunostaining experiments suggest that a high proportion of p84 is colocalized with centers of RNA processing. In addition, p84 contains regions of homology to the death domain of pro-apoptotic regulators, such as the tumor necrosis factor receptor-1, and overexpression or microinjection of p84 resulted in apoptosis (Doostzadeh-Cizeron et al., 1999). Coincubation of purified p84 with GST-pRb before microinjection reduced p84-induced apoptosis. Potentially, the activation of p84 may contribute to the elevated levels of apoptosis when pRb is inactivated. However, this model has yet to be tested.
c. H-Nuc H-Nuc (Chen et al., 1995) was originally isolated in a two-hybrid screen of Durfee and coworkers (Durfee et al., 1994) that used the carboxyl-terminal p56Rb as bait for interacting proteins. H-Nuc is homologous to S. pombe nuc-2 and A. nidulans BimA, two genes that have essential functions during mitosis. pRb binds to the TPR region of H-Nuc, a region comprised of ten repeats of a 34-amino acid motif that is highly conserved in nuc-2 and BimA. The pocket domain of pRb is required for binding to H-Nuc and a GSTH-Nuc fusion protein bound to the unphosphorylated form of pRb.
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Although H-Nuc binds to pRb in yeast and in vitro, efforts to coimmunoprecipitate H-Nuc and pRb complexes from mammalian cells have been unsuccessful (Chen et al., 1995).
d. NRP/B Nuclear restricted protein/brain (NRP/B) is a 67 kD nuclear matrix protein which was originally identified from single-pass sequencing of human brain cDNAs, a strategy employed to identify unique gene products involved in neural development (Kim et al., 1998). NRP/B expression is highly enriched in brain neurons and is associated with the nuclear matrix in areas of the nucleoplasm, peripheral heterochromatin, and nucleolus. NRP/B expression was induced during differentiation of neuroblastoma cells and overexpression of this protein was sufficient to induce a differentiative phenotype in this cell type. In addition, treatment of either PC12 cells or primary hippocampal neurons with antisense oligonucleotides to NRP/B abrogated differentiation of these cells. Immunoprecipitated pRb from SH-SY5Y neuroblastoma cells was shown to associate with recombinant NRP/B in Far Western blotting experiments. However, reduced pRb/NRP/B binding was observed in differentiated cells relative to undifferentiated cells and it is unknown whether this interaction has any functional significance during neuronal development.
4. CELL CYCLE AND DNA REGULATING ENZYMES a. Topo-II␣ DNA topoisomerases are important enzymes which control the topological state of the DNA helical structure and relax DNA supercoiling (reviewed in Wang, 1996). Topo-II␣ activity is important during G2/M and catalyzes an ATP-dependent DNA double-strand break/religation reaction critical for proper chromosomal replication, recombination, segregation, and repair. Using Rh1 cell lysates, topo-II␣ was shown to coimmunoprecipitate primarily with hypophosphorylated pRb (Bhat et al., 1999). Moreover, this association was not perturbed in the presence of ethidium bromide or lysate pretreatment with DNaseI, suggesting that the interaction was not through a DNA intermediate. Utilizing various GST–pRb fusions and lysates of various cell types, topo-II␣ was shown to associate primarily with the large pocket of pRb and also required the A/B spacer region. Wild-type pRb, but not mutant C706F, was able to inhibit topo-II activity in DNA decatenation assays from lysates of transiently-transfected C33A cells as well as in purified, reconstitution assays. These data suggest that phosphorylation of pRb may serve as a switch to release negative inhibition on topo-II function during G2/M. Currently, however, it is unclear if pRb functions to regulate topo-II␣ during the cell cycle in vivo.
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b. DNA polymerase-␣ pRb has been reported to bind to the major replicative DNA polymerase, DNA polymerase-␣ (pol␣) (Takemura et al., 1997). pRb was immunoprecipitated from Raji cell nuclear extracts using anti-pol␣ antibodies, and pol␣ was adsorbed to a pRb-affinity column. Immunopurified calf thymus pol␣ and human pol␣ (HeLa cell) activity was stimulated with recombinant pRb. Moreover, dephosphorylation of pRb with PP2A reduced pol␣ activity, and pRb phosphorylation by cdk2/cyclin E enhanced pRb’s effects on pol␣ activity. These results are unusual since they suggest that the phosphorylation of pRb might stimulate its activity. At present it is unclear if the activity of pol␣ is regulated by pRb in vivo during cell cycle progression.
c. REC2 Recombinase Overexpressed eukaryotic REC2 recombinase, a homolog of the E. coli RecA protein, which has been shown to be important for both homologous recombination as well as DNA damage recombination repair, was found to interact with hypophosphorylated pRb by both immunoprecipitation and in vitro binding assay (Fan et al., 1997). Although REC2 does not contain an LXCXE motif, it was able to bind the small pocket alone but interacted most strongly with the large pocket of pRb. Various mutations and deletions within the large pocket (including C706F, exon22, exon 21, and spacer deletion) either strongly reduced or diminished REC2 binding and a recombinase-deficient REC2 allele was unable to bind pRb. Overexpression of pRb was able to inhibit REC2 overexpression-induced apoptosis in HuH-7 cells. pRb regulation of recombination has not yet been reported.
d. Mitosin Mitosin (or CENP-F) was isolated by screening cDNA expression libraries with purified p56RB (Shan et al., 1992). Mitosin is a large nuclear protein that is expressed during the S, G2, and M-phases of the cell cycle (Zhu et al., 1995). It undergoes dramatic changes in subcellular localization during M-phase and is found at the kinetochore/centromere (early prophase), the spindle (metaphase), and the midbody (telophase) (Zhu et al., 1995; Zhu et al., 1997; Zhu, 1999). Overexpression of mutant forms of mitosin causes an accumulation of G2/M-phase cells. The pRb-binding site has been mapped to a short sequence near the carboxyl-terminus of mitosin that lacks any close sequence homology with other pRb-binding proteins. It remains to be determined whether mitosin/pRb complexes exist in vivo, and the biological significance of this interaction is unresolved.
e. HsHec1p HsHec1p, a coiled-coil protein required for mitosis, was originally identified through a yeast two-hybrid approach with pRb as bait and was shown
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to interact with pRb through its IXCXE motif (Zheng et al., 2000). In yeast, the hsHec1p interaction with an Gal4-pRb fusion was mapped to the large pocket of pRb. By coimmunoprecipitation in T24 cells, hsHec1p interacted preferentially with hypophosphorylated pRb and specifically during G2/Mphase. HsHec1p, pRb, and hSMC1 (human structural maintenance of chromosomes protein-1) were shown to form a complex which might serve to regulate proper chromosomal segregation (Zheng et al., 1999, Zheng et al., 2000). Further studies are needed to determine whether the chromosomal abnormalities seen in pRb-deficient cells result from deregulated hsHec1p function.
f. MCM7 Using a two-hybrid screen with amino acids 1–400 of the N-terminus of pRb, Sterner and colleagues identified two interactors whose cDNAs encoded the DNA initiation factor, microchromosome maintenance protein-7 (MCM7) (Sterner et al., 1998). GST–MCM7 fusions were found to bind GST-N-terminal fusions of pRb, p107, and p130, as well as full length pRb in vitro. Binding assays with short N-terminal deletion mutants of pRb did not perturb MCM7 binding, suggesting that MCM7 interacts with a large surface of the pRb amino terminus. Endogenous MCM7 bound pRb in vivo in coimmunoprecipitation experiments with human ML-1 cells. The use of a Xenopus cell free DNA replication system demonstrated that human N-terminal pRb (which binds Xenopus MCM7) was able to abolish DNA replication and that this effect was competed by addition of a Cterminal portion of MCM7 (which binds the N-terminus of pRb). N-terminal pRb did not interfere with nuclear assembly, transport, or cdk2 activity in these assays. Since MCM7 is a licensing factor of a pre-replication complex formed on chromatin, these data suggest that pRb might function to suppress pre-replication initiation at origins of replication through interactions with MCM proteins, including MCM7. In support of this model, recent studies show that pRb colocalizes with MCM proteins in perinucleolar foci in G1 and early S phase (Kennedy et al., 2000).
g. BRCA1 Germ line transmission of BRCA1 mutant alleles enhances the susceptibility to developing breast and ovarian cancers and loss of heterozygosity often occurs in these tumors. pRb was identified as an interactor of the BRCT (BRCA1 C-terminus) domain of the BRCA1 tumor suppressor during an expression screen for BRCA1-interacting proteins (Yarden and Brody, 1999). pRb/BRCA1 binding occurs through the large pocket of pRb, preferentially with the hypophosphorylated form (Aprelikova et al., 1999) and pRb was colocalized with BRCA1 by immunostaining (Yarden and Brody, 1999). The
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BRCT motif has intrinsic transcriptional activity and is present in a number of DNA repair, DNA replication, and cell cycle checkpoint proteins, including pRb (Yamane et al., 2000). The BRCT domain of BRCA1 was found to interact with RbAp46, RbAp48, HDAC1, HDAC2, and pRb (Yarden and Brody, 1999). Currently, it is unclear if the pRb/BRCA1 interaction occurs indirectly through other proteins in vivo. pRb-deficient cells are resistant to growth suppression by BRCA1 overexpression and HPV E7 abrogates this effect in cells wild-type for Rb (Aprelikova et al., 1999). Moreover, mutations in the pRb-binding domain of BRCA1 abolish the growth suppressive effects of this interactor (Aprelikova et al., 1999). In other studies, it has also been shown that BRCA1 is an E2F1responsive gene and is repressed by pRb (Wang et al., 2000). Both studies raise the possibility that the functions of pRb and BRCA1 may be linked, however further studies are needed to determine whether the roles of these proteins are intimately connected.
h. p21CIP1/WAF1 and p57KIP2
The cdk inhibitors, p21CIP1/WAF1 and p57KIP2, but not p27KIP1, have been reported to bind pRb in vitro (although this has not been shown with full length pRb). Further studies demonstrated that endogenous p21 was coimmunoprecipitated with endogenous pRb in vivo in nuclear extracts from MJ90 human fibroblasts. Mapping studies demonstrated that the N-terminal portion (1–71) of p21 interacted mainly with the small pocket of pRb (weak interaction occurred with the C-domain). Further studies with baculovirus expressed proteins, in Sf9 cells, demonstrated that pRb/p21 binding could be disrupted by phosphorylation with excess quantities of cyclin/cdk complexes. Currently, it is unresolved if the p21/pRb or p57/pRb interaction is mediated through a cyclin/cdk intermediate and whether this interaction is important for pRb function.
i. pRb pRb has been reported to self-associate in vitro with purified proteins as well as through yeast two-hybrid analysis with pRb fragments (Hensey et al., 1994). Initial experiments noted that recombinant pRb, but not N-terminal truncated pRb (p56Rb), could form oligomers on nondenaturing gels. Further analysis by yeast two-hybrid interaction noted that an N-terminal pRb fragment (amino acids 1–300) fused to the Gal4 DNA binding domain could interact with a C-terminal pRb fragment (amino acids 301–928) fused to the Gal4-transactivation domain; C-terminal domains did not interact with each other but it is unclear if N-terminal domains could self-interact. Electron microscopy and immunogold staining with anti-pRb antibodies noted the presence of linearly extended filament structures with full-length pRb
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but not p56Rb. The authors suggest that the N-terminal globular domain of pRb interacted somewhere within amino acids 300–928 of pRb in repeated fashion to form long filament structures. However, these experiments do not rule out the possibility that the Ab245 anti-pRb antibody itself caused some degree of artifactual self-association. Moreover, since the initial observation, no report has demonstrated pRb oligomerization in vivo.
5. PROTEINS OF UNKNOWN OR UNCLEAR FUNCTION a. Bog The B5T-overexpressed gene protein (Bog) has no known function and is overexpressed in various transformed liver epithelial cell lines and human liver tumors. This protein contains an LXCXE motif which has been shown to associate with all three pocket proteins by yeast two-hybrid and coimmunoprecipitation for pRb (Woitach et al., 1998). Endogenous Bog/pRb association in B5T cells coimmunoprecipitated with both anti-bog and antipRb antibodies, suggesting that this interaction is likely to be real and to occur in vivo. Overexpressed Bog is able to displace E2F1 from pRb, overcome TGF1-induced growth arrest, and cause transformation of rat liver epithelial cells leading to hepatoblastomas in nude mice. Bog is overexpressed in a variety of human tumor cell lines, but it is not yet clear if Bog or Bog/pRb interactions contribute to tumorigenesis.
b. P2P-R P2P-R is a member of highly basic proteins that are associated with heterogeneous nuclear ribonucleoprotein (hnRNP) particles (Witte and Scott, 1997). A GST fusion protein containing a portion of P2P-R was found to precipitate pRb from cell lysates. This interaction was most likely mediated by the pocket domain of pRb as it was competed by E1A. It is not known if endogenous P2P-R and pRb proteins interact and the functional significance has not been investigated.
c. Prohibitin Prohibitin (Pro1) is a 30 kD intracellular protein which was initially described by its ability to negatively regulate cell cycle progression through unknown mechanisms (Nuell et al., 1991). Initially utilizing a two-hybrid approach to identify p130 binding proteins, Wang and colleagues demonstrated a positive interaction with Pro1 and all three pocket proteins (Wang et al., 1999a). Subsequent studies demonstrated that in vitro synthesized Pro1 was able to interact with GST-pRb (but not a pRb pocket mutant) and endogenous Pro1 and pRb coimmunoprecipitated from various cell lysates. Pro1 mutants defective for pRb binding were also defective in negative growth control. pRb binding appeared to occur through amino acids 74–116 of Pro1, a region without a LXCXE sequence but 45% identical to RBP1.
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Interestingly, although Pro1 did not affect pRb binding to E2Fs, cotransfection of Pro1 was able to prevent the activation of an E2-CAT reporter construct by E2F1-5. Further characterization demonstrated that this effect might be mediated directly by Pro1 binding to E2Fs (Wang et al., 1999b).
d. RBQ1/PACT and RBQ3 RBQ1 and RBQ3 are unrelated proteins initially identified by screening cDNA expression libraries for pRb interactors (Sakai et al., 1995; Saijo et al., 1995). RBQ1 and RBQ3 bind hypophosphorylated pRb in vitro, and deletions in the B-domain of pRb abolished binding to RBQ3; A-domain deletions and pocket mutations did not perturb binding. RBQ1 is a truncated form of p250PACT, a protein originally identified from an expression screen for p53-interacting proteins (Simons et al., 1997). PACT is a nuclear protein whose endogenous form interacts with both endogenous p53 and pRb in vivo, although a very small proportion of PACT was coimmunoprecipitated with pRb (Simons et al., 1997). Although the function of PACT is unknown, it interferes with p53 DNA binding and is also associated with small nuclear ribonucleoproteins suggesting a role in pre-RNA splicing. The physiologic relevance of pRb and RBQ binding remains unclear.
ACKNOWLEDGMENTS We thank past and present members of the Laboratory of Molecular Oncology, particularly F. Dick, O. Stevaux, and B. Kennedy, as well as W. Kaelin, Jr. and D. Park for helpful discussion and comments on the manuscript. An updated catalog of pRb-binding proteins will be linked to the following URL, http://www.mgh.harvard.edu/dept/cancercenter/dyson.html.
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p53-Dependent Apoptosis Pathways Yan Shen2,4 and Eileen White1–5,∗ 1 Howard Hughes Medical Institute, 2Center for Advanced Biotechnology and Medicine, 3Department of Molecular Biology and Biochemistry, 4Department of Cell and Developmental Biology, 5Cancer Institute of New Jersey & Rutgers University, Piscataway, New Jersey 08854
I. p53 A. p53 Is a Tumor Suppressor B. p53 Is a Transcription Regulator C. Regulation of p53 Activation II. Bcl-2 Family A. The Bcl-2 Family Members B. The Structure of Bcl-2 Related Proteins C. Interactions between Bcl-2 Family Members D. Regulation of Mitochondrial Function by Bcl-2 Family Members E. Bax in p53-Dependent Apoptosis F. Antiapoptotic Bcl-2 Family Members Block Bax Activity Downstream of p53 Death Signaling III. Caspase Family A. The Caspase Cascade B. Caspase-9 C. Caspase-3 D. Caspase-6 and -7 E. Caspase-8 F. E1B 19K Inhibits Caspase-9 Activation G. Viral Caspase Inhibitors IV. IAPs A. IAP Family Members B. Structure–Function of IAPs C. Inhibitors of IAPs V. p53-Mediated Apoptosis in Cancer Therapy References
The p53 tumor suppressor limits cellular proliferation by inducing cell cycle arrest and apoptosis in response to cellular stresses such as DNA damage, hypoxia, and oncogene activation. Many apoptosis-related genes that are transcriptionally regulated by ∗ To whom correspondence should be addressed; Phone: (732) 235-5329; Fax: (732) 235-5795; E-mail:
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55 Advances in CANCER RESEARCH 0065-230X/01 $35.00
C 2001 by Academic Press. Copyright All rights of reproduction in any form reserved.
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p53 have been identified. These are candidates for implementing p53 effector functions. In response to oncogene activation, p53 mediates apoptosis through a linear pathway involving bax transactivation, Bax translocation from the cytosol to membranes, cytochrome c release from mitochondria, and caspase-9 activation, followed by the activation of caspase-3, -6, and -7. p53-mediated apoptosis can be blocked at multiple death checkpoints, by inhibiting p53 activity directly, by Bcl-2 family members regulating mitochondrial function, by E1B 19K blocking caspase-9 activation, and by caspase inhibitors. Understanding the mechanisms by which p53 induces apoptosis, and the reasons why cell death is bypassed in transformed cells, is of fundamental importance in cancer research, and has great implications in the design of anticancer therapeutics. C 2001 Academic Press.
Apoptosis, or programmed cell death, is an important cellular process that secures normal growth and homeostatsis, and has been implicated as a means for eliminating abnormal, damaged, cancerous cells. Apoptosis is a cell suicide program that normally responds to stresses such as growth factor deprivation, hormone or cytokine exposure, DNA damage, cell cycle dysfunction, and oncogene activation. Tumorigenesis can occur when apoptosis is inhibited by genetic mutations. Characteristic morphological events that typify apoptosis include chromosomal DNA condensation, DNA fragmentation in a laddering pattern, cytosolic boiling, cell membrane blebbing, and cell shrinkage followed by engulfment by neighboring cells (Wyllie, 1980). All of these events represent molecular occurrences critical to the proper execution of the cell death program. At the molecular level, apoptosis is highly regulated by evolutionarily conserved pathways. Some conserved genes that regulate apoptosis are members of the Bcl-2 (B-cell lymphoma/leukemia) family, Apaf-1 like adapter proteins, caspases, and the inhibitors of apoptosis (IAPs). On one hand, apoptosis is an efficient self-protective mechanism for multicellular organisms to eliminate damaged cells. On the other hand, apoptosis eliminates surplus cells during development and tissue remodeling. Aberrant regulation of apoptosis has been linked to various diseases including cancer, AIDS, Alzheimer’s disease, and rheumatoid arthritis, and may also result in serious defects in embryonic development. Thus, understanding the regulation of apoptosis will have great medical implications.
I. p53 A. p53 Is a Tumor Suppressor Tumor suppressor genes can function by promoting apoptosis, thereby inhibiting oncogenic transformation. Loss of function of tumor suppressor
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genes in vivo thus leads to a tumor-prone state. This has been most elegantly demonstrated for the p53 tumor suppressor gene. p53 is mutant in about half of human cancers across a wide spectrum of tissues, suggesting that the loss of function of p53 renders cells more susceptible to tumorigenic transformation. Indeed, the incidence of spontaneous tumors increases dramatically in p53-deficient mice (Donehower et al., 1992), and in man (reviewed by Akashi and Koeffler, 1998; Hainaut, 2000). Homozygous p53-deficient animals die during the first six months of life, mainly of lymphomas, while the heterozygous animals display a wider spectrum of tumor formation, where sarcomas and lymphomas are the most common. The p53-deficient mice are also extremely sensitive to radiation, viral infection, and carcinogen-induced tumorigenesis (Harvey et al., 1993a; Kemp et al., 1994). Humans heterozygous for p53 mutations are strongly predisposed to a variety of cancers with early onset (Akashi and Koeffler, 1998). These studies confirm the role of p53 as a tumor suppressor in man, which can be recapitulated in animal models. The high incidence of tumors in p53-deficient animals partially results from a lack of p53-induced apoptosis. Thus, tumorous cells can replicate and the population of aberrant cells increases. Indeed, a high incidence of chromosome aberrations are observed in the somatic cells of the p53-deficient mice (Harvey et al., 1993b). p53-deficient mice show decreased apoptosis in hematopoietic cells, hippocampal granule cells, and tunica vasculosa lentis cells in response to apoptosis-inducing agents (Dausset et al., 2000; Lotem and Sachs, 1993; Reichel et al., 1998; Sakhi et al., 1996). The importance of p53 in conveying oncogene-induced apoptosis is also illustrated in cells deficient in p53. Adenovirus E1A oncogene expression in primary cells containing wild-type p53 normally results in an immediate burst of proliferation, but is followed by apoptosis, resulting in abortive transformation (Rao et al., 1992). In contrast, dominant interfering mutants of p53 can cooperate with E1A to transform cells by blocking an E1A-mediated apoptosis. Moreover, E1A expression in primary cells deficient in p53 results in a successful cellular transformation due to the lack of apoptosis (Lowe et al., 1994). Coexpressing E1A and a dominant interfering temperature-sensitive p53 mutant (p53val(135)) is sufficient to transform cells at the restrictive temperature, when p53 is in the mutant conformation. In contrast, cells undergo apoptosis at the permissive temperature, when p53 reverts to the wild-type conformation and is functional (Debbas and White, 1993). Additionally, expression of p53 in cell lines derived from osteosarcoma, colon tumor, and myeloid leukemia that are devoid of endogenous p53, results in apoptosis (Baker et al., 1990; Shaw et al., 1992; Yonish-Rouach et al., 1991). Thus, overcoming p53dependent apoptosis is crucial for successful oncogenic transformation. p53 also promotes cell cycle arrest. For example, p53 can arrest the cell cycle in osteosarcoma cells and in glioblastoma tumor cell lines that
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conditionally express p53 (Diller et al., 1990; Mercer et al., 1990). Similar conditional expression of a temperature-sensitive p53 mutant inhibits transformation by inhibiting cell proliferation (Michalovitz et al., 1990). Therefore, p53 possesses two distinct mechanisms as a tumor suppressor. Firstly, p53 initiates a program that results in cell death; and secondly, p53 prevents aberrant cells from progressing through the cell cycle. Both are effective means for preventing tumor progression. The decision between promoting cell cycle arrest versus apoptosis partially relies on the p53 protein levels. High levels of p53 protein are correlated with apoptosis and low levels with cell cycle arrest in Saos2 and H1299 cells that express p53 conditionally (Chen et al., 1996). The decision to induce cell cycle arrest or apoptosis is also related to the cellular context and environment. DNA damage heightens the apoptotic response to p53 without altering p53 protein levels in cells (Chen et al., 1996). Furthermore, blocking p53dependent apoptosis downstream of p53 reveals cell cycle arrest (Debbas and White, 1993). This suggests that pathways to induce cell cycle arrest and apoptosis are simultaneously activated, but apoptosis is dominant to cell cycle arrest in cells competent to die.
B. p53 Is a Transcription Regulator The most well-characterized biochemical function of p53 is as a regulator of transcription (Fig. 1, see color plate). The amino-terminus of p53 contains an acidic transactivation domain. This domain allows p53 to recruit the basal transcription machinery including the TATA box binding proteins (TBP) and TBP-associated factors (TAFs), both of which are components of the general transcription factor TFIID (Chang et al., 1995; Chen et al., 1993; Liu et al., 1993; Lu, 1995; Thut et al., 1995; Truant et al., 1993). The transactivation domain is responsible for both sequence-specific transactivation (SST) and transcriptional repression of target genes. The transactivation domain is followed by a poly-proline (PP) domain, which is required for p53-mediated apoptosis, as the p53 PP deletion mutants are defective in mediating apoptosis (Sakamuro et al., 1997; Venot et al., 1998; Walker and Levine, 1996; Zhu et al., 1999). The PP deletion mutants are also defective for transcriptional repression but not activation, suggesting a functional relationship between transcriptional repression and apoptosis. The PP domain is followed by a sequence-specific DNA-binding domain, where the majority of p53 mutations in human tumors cluster, suggesting that p53 mediates most of its tumor suppressive effects through DNA binding and transcriptional mechanisms (Cho et al., 1994; Farmer et al., 1992; Hollstein et al., 1991; Zambetti et al., 1992). At the carboxy terminus of p53 there is a tetramerization domain required for oligomerization and DNA binding. Also, the
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basic carboxy-terminal tail of p53 is a negative regulatory region. Posttranslational modifications of the carboxy-terminal tail allosterically promote efficient sequence-specific DNA-binding by p53 (Hupp et al., 1992; Jayaraman ¨ and Prives, 1995; Sturzbecher et al., 1992). These domains act in synergy to detect abnormalities of cellular function and mediate cellular responses. Among the genes that are transcriptionally upregulated by p53, there are those involved in cell cycle regulation, such as p21WAF1 and 14-3-3 (El-Deiry et al., 1993; Hermeking et al., 1997). Accumulating evidence suggests that p53 mediates apoptosis by a mechanism independent from that of cell cycle arrest (Guillouf et al., 1995; Polyak et al., 1996; Wagner et al., 1994). A number of p53-regulated, apoptosis-related genes have been identified. These include Bcl-2 family members that are important regulators of apoptosis. Bax, bak, and noxa are proapoptotic Bcl-2 family members that are upregulated by p53 (Miyashita et al., 1994b; Miyashita and Reed, 1995; Oda et al., 2000; Pearson et al., 2000; Pohl et al., 1999). They may function downstream of p53 and convey p53-related toxicity. Fas, DR5/KILLER, and TRID are genes that belong to the tumor necrosis factor receptor (TNFR) family of death or decoy receptors. They are important regulators of apoptosis in the immune system, and are among the transactivation targets of p53, suggesting that p53 may play a role in death receptor-induced apoptosis (Owen-Schaub et al., 1995; Sheikh et al., 1999; Wu et al., 1997b). Another class of p53 transactivation targets includes many genes involved in signal transduction regulation, such as IGF-BP3, PTGF-beta, and PERP, which have all been implicated in promoting apoptosis, suggesting that p53 may alter the cellular contexts by manipulating signal transduction pathways (Attardi et al., 2000; Buckbinder et al., 1995; Tan et al., 2000). Based on the identity of another group of p53 inducible genes (PIGs), it has been suggested that some p53 target gene products regulate the production of reactive oxygen species that may also contribute to p53-mediated apoptosis (Polyak et al., 1997). As more p53 target genes are identified through constantly improved screening techniques, it is becoming apparent that the spectrum of p53 target gene expression and their contribution to apoptosis vary among different cell types, and in response to different stimuli (Zhao et al., 2000). The significance of these gene targets in apoptosis needs to be carefully evaluated. Although p53 is mostly known as a transcriptional activator, it also represses gene transcription. For example, p53 represses the expression of the antiapoptotic Bcl-2 family member Bcl-2 (Miyashita et al., 1994a,b). Other genes that are repressed by p53 include c-fos, cyclin A, map-4, and interleukin 6 (Desdouets et al., 1996; Kley et al., 1992; Santhanam et al., 1991; Zhang et al., 1998). Recent DNA array studies have also exemplified that in response to irradiation, p53 can upregulate genes involved in inhibiting cell cycle progression and genes involved in DNA repair, while repressing
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genes involved in cell division (Zhao et al., 2000). By activating some genes while repressing others, p53 may provide a potent ability to dramatically alter cellular functions. p53-mediated apoptosis can also occur in the absence of protein or RNA synthesis (Bissonnette et al., 1997; Caelles et al., 1994), demonstrating that SST-independent mechanisms may also contribute to p53-mediated apoptosis. A truncated p53 mutant that lacks a major portion of the DNA-binding domain, and is defective in activating p53-responsive promoters, can induce extensive apoptosis, and is able to suppress transformation of rat fibroblast (Haupt et al., 1995). Moreover, a human p53 protein carrying mutations in residues 22 and 23 also triggers apoptosis in HeLa cells, despite failing to induce significant activation of relevant p53 target promoters (Haupt et al., 1995). These data suggest that p53 may also induce apoptosis through SSTindependent pathways (Chen et al., 1996). The contribution of these transcriptionally independent apoptotic mechanisms to p53 tumor suppressor function remains to be determined.
C. Regulation of p53 Activation As a central regulator of many cellular processes, the activity of p53 is tightly regulated by multiple mechanisms. Regulation of p53 occurs mostly at the level of protein stability. DNA damage, hypoxia, and oncogene activation cause an increase in the half-life of p53, and result in an accumulation of the p53 protein. The most important regulator of p53 stability is the oncogene product Mdm2 (Momand et al., 2000; Yap et al., 1999). Mdm2 interacts with the amino-terminal transactivation domain of p53, and promotes its ubiquitin-dependent degradation (Haupt et al., 1997; Kubbutat et al., 1997; Oliner et al., 1993). Mdm2 itself is transcriptionally upregulated by p53 (Barak et al., 1993). When p53 is activated by DNA damage, for example, Mdm2 is upregulated, which promotes p53 degradation, thus forming a negative feedback loop to modulate p53 protein levels. Aside from regulating p53 protein levels, Mdm2 also directly inhibits p53 transactivation activity through interaction with the transactivation domain of p53 (Thut et al., 1997). The negative regulation of p53 by Mdm2 is also demonstrated in that a p53 null background rescues the lethality in Mdm2-deficient mice (Jones et al., 1995; Montes de Oca Luna et al., 1995), suggesting that Mdm2 functions to inhibit p53 in vivo. Oncogene expression promotes the accumulation and activation of p53. Levels of p53 are extremely low in untransformed cells, due to rapid turnover. Expression of adenovirus oncogene E1A, as well as cellular oncogenes c-Myc, E2F-1, and Ras, leads to the stabilization of p53 protein (Chiou et al., 1994b; Hermeking and Eick, 1994; Lowe and Ruley, 1993; Sabbatini et al., 1995).
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Accumulation of p53 occurs in part because these oncogene products can inactivate Rb, which leads to the release and activation of E2Fs, and E2Fs induce p19ARF transcription. p19ARF sequesters Mdm2 through direct interaction (de Stanchina et al., 1998; Palmero et al., 1998; Pomerantz et al., 1998; Zindy et al., 1998). Inactivation of Mdm2 relieves ubiquitin-mediated degradation of p53, and results in p53 accumulation. The increase in p53 protein levels by E1A also results from the inactivation of p300, which directly blocks p53-dependent Mdm2 transcription, and thus directly alleviates Mdm2-mediated degradation and inactivation of p53 (Thomas and White, 1998). DNA damage can induce the accumulation of p53 protein by interfering with the signal transduction pathways. Posttranslational modification by extensive protein phosphorylation affects p53 protein stability. Phosphorylation of p53 occurs on amino-terminal serine residues 15, 20, and 37 by protein kinases such as PI3K, ataxia telangiectasia mutated protein (ATM), Ataxia- and Rad-related kinase (ATR), checkpoint kinases Chk1 and Chk2, DNA-dependent protein kinase (DNA-PK), c-Jun amino-terminal kinase (JNK), casein kinase 1 (CK1), and CDK activating kinase (CAK) (Caspari, 2000; Jimenez et al., 1999; Meek, 1999). Phosphorylation of these residues reduces Mdm2 binding to p53, and removes Mdm2-mediated p53 degradation, thus stabilizing p53 (Shieh et al., 1997; Siliciano et al., 1997). The activity of p53 can be regulated by posttranslational modification without changes in protein levels. Phosphorylation of the p53 amino terminus directly alleviates Mdm2 inhibition. Other posttranslational modifications such as dephosphorylation and acetylation also rigorously adjust p53 activity. Dephosphorylation of serine 376 by irradiation enables p53 to interact with 14-3-3, which increases the ability of p53 to bind DNA (Waterman et al., 1998). Acetylation of the carboxy-terminal tail alters the conformation of p53, and disrupts the interaction between the carboxy terminus of p53 and the core DNA binding domain, thus promoting p53 binding to DNA (Gu and Roeder, 1997; Sakaguchi et al., 1998). While multiple mechanisms exist for activating p53, so too are there multiple mechanisms for inactivating p53. In human tumors, the most common mechanism for inactivating p53 is through mutations in the p53 gene. Lossof-function mutations in p53 have been found in about half of all human cancers. The oncogenic mutation “hot spots” are found within the sequencespecific DNA-binding domain, suggesting that SST-dependent mechanisms are important for p53 tumor suppression function. Also, Mdm2 amplification is associated with p53 inactivation and tumor induction in sarcomas (Oliner et al., 1992). Besides p53 protein levels and protein conformation, the subcellular localization of p53 also contributes to the regulation of p53 activation. In some breast cancer cells, p53 is localized to the cytoplasm but not the nuclei. Although p53 was wild-type, it was not functional (Moll et al.,
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1992). These multiple mechanisms for regulating p53 activity emphasize the importance of ablating p53 function during tumorigenesis. Oncoproteins encoded by DNA tumor viruses can inhibit the function of p53. The adenovirus E1B 55-kDa protein (Sarnow et al., 1982; Yew and Berk, 1992) and the SV-40 T antigen (Lane and Crawford, 1979; Linzer and Levine, 1979) bind to p53 and sequester it in an inactive complex. The E6 protein of the high-risk human papillomaviruses promotes the ubiquitination and degradation of p53 (Lechner et al., 1992; Scheffner et al., 1990; Werness et al., 1990). These viruses prevent p53 function to allow viral replication and tumorigenesis.
II. Bcl-2 FAMILY A. The Bcl-2 Family Members Multiple lines of evidence suggest that Bcl-2 family members are involved in p53-mediated apoptosis. Some Bcl-2 family members like bax, bak, and noxa are transactivationally upregulated by p53 (Ho et al., 1999; Miyashita et al., 1994b; Miyashita and Reed, 1995; Oda et al., 2000; Pearson et al., 2000), while bcl-2 is transcriptionally suppressed by p53 (Miyashita and Reed, 1995). Bcl-2 was originally identified as a gene translocated in human follicular B cell lymphoma, which caused overexpression of the Bcl-2 protein. Overexpression of Bcl-2 blocks apoptosis of mammalian cells triggered by a number of different stimuli, such as factor deprivation, irradiation, E1A, c-Myc, p53, and anticancer drugs (Alnemri et al., 1992; Chiou et al., 1994a; Rao et al., 1992; Sentman et al., 1991; Wagner et al., 1993). Bcl-2 is the founding member of a multigene family that includes many mammalian, invertebrate, and viral homologs (Fig. 2, see color plate). Bcl-2 family members come in two functional categories, those that inhibit apoptosis and those that promote apoptosis. The antiapoptotic members include mammalian Bcl-2, Bcl-xL (Boise et al., 1993), Bcl-w (Gibson et al., 1996), A1/Bfl-1 (D’Sa-Eipper et al., 1996; Lin et al., 1993), Mcl-1 (Reynolds et al., 1994), adenovirus E1B19K (Chiou et al., 1994b), and C. elegans CED-9 (Hengartner et al., 1992). The proapoptotic members include Bax (Oltvai et al., 1993), Bcl-xS, Nbk/Bik (Boyd et al., 1995; Han et al., 1996b), Bak (Chittenden et al., 1995; Kiefer et al., 1995), Bad (Yang et al., 1995), Bid (Wang et al., 1996), and Noxa (Oda et al., 2000). Four conserved regions within Bcl-2 related proteins have been identified that have been designated Bcl-2 homology (BH) domains BH1, BH2, BH3, and BH4. The BH1 and BH2 regions are important for the antiapoptotic function of Bcl-2 and Bcl-xL, and for their interaction with Bax. The BH3
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domain is sufficient for proapoptotic Bcl-2 members to interact with the antiapoptotic Bcl-2 members. Furthermore, BH3 is required for the killing activity of the proapoptotic Bcl-2 members. The subgroup of BH3-only Bcl-2 family members are nevertheless potent apoptosis-promoting proteins. The BH4 domain of Bcl-2 has been shown to be necessary for Bcl-2 to heterodimerize with Bax, and to inhibit apoptosis (Hirotani et al., 1999; Huang et al., 1998).
B. The Structure of Bcl-2 Related Proteins The three-dimensional structure of Bcl-xL has been determined by X-ray and NMR analysis (Muchmore et al., 1996). Bcl-xL consists of two central, hydrophobic ␣-helices, which are surrounded by amphipathic helices. The three functionally important Bcl-2 homology regions (BH1, BH2, and BH3) are in close spatial proximity and form an elongated hydrophobic cleft into which the BAK BH3 binds, as revealed from the structure of Bcl-xL-BAK BH3 peptide complex (Sattler et al., 1997). The arrangement of the ␣-helices in Bcl-xL is reminiscent of the membrane translocation domain of bacterial toxins, in particular, diphtheria toxin and the colicins, suggesting that Bcl-xL may also generate pores in membranes. The structure of Bid, a BH3-domain-only proapoptotic Bcl-2 family member, has also been determined. Bid also contains two central hydrophobic helices, similar to that of the Bcl-xL, although the sequence homology between the two is limited to only the 16-residue BH3 domain (Chou et al., 1999; McDonnell et al., 1999). Bid is a proximal substrate of caspase-8 in the Fas and TNF-␣ apoptotic-signaling pathways. Full-length Bid localizes to the cytosol; however, after cleavage by caspase-8, the carboxy-terminal fragment of Bid (tBid) translocates to mitochondria. It is speculated that Bid cleavage by caspase-8 induces the exposure of its BH3 domain, and causes a significant change in the protein surface hydrophobicity, which promotes the translocation of tBid from cytosol to the mitochondrial membranes (Chou et al., 1999; McDonnell et al., 1999). tBid will then further activate Bax by interacting with Bax, which induces a conformational change that activates the Bax killing function (Perez and White, 2000). Based on sequence and secondary structure alignments, the proapoptotic members of the Bcl-2 family are proposed to be grouped into two main structural categories. One group has their BH3 domain buried, and therefore needs a posttranslational modification for activation. The other group has their BH3 domains exposed constantly, and would be constitutively active (McDonnell et al., 1999). This may provide us with a mechanism by which the activities of Bcl-2 family members are regulated.
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C. Interactions between Bcl-2 Family Members Interaction between Bcl-2 family members is one of the most important activities of Bcl-2 family proteins. Homodimerization appears to be important for some Bcl-2 family members to assume proper conformation, subcellular localization, and function. For example, enforced dimerization of Bax results in translocation of the Bax dimers from cytosol to the mitochondria (Gross et al., 1998). Detergent buffer conditions induce Bax dimerization and a conformational change that exposes the amino terminus of the protein (Hsu et al., 1997). One model holds that under normal circumstances, the amino terminus of Bax is concealed to keep the protein in a closed conformation and inactive. Upon receiving an activation stimulus, such as interacting with tBid, Bax undergoes a conformational change. The amino terminus of Bax unfolds and the BH3 is exposed, which then enables Bax to function (Perez and White, 2000). The conformational changes of Bax are accompanied by changes in its oligomerization status, which may represent all or part of the membrane pore-forming activity of Bax (Desagher et al., 1999; Gross et al., 1999; Hsu and Youle, 1997). Thus, interactions between the Bcl-2 family members play an important role in the regulation of the protein function. Heterodimerization between Bcl-2 family members can be antagonistic, such that antiapoptotic members associate with proapoptotic family members and inhibit their functions. The determining factor for cell viability may be the ratio of the protein levels. Antagonistic interactions have been reported between Bcl-2 and Bax, Bid, Bak, Bcl-xS, and Bad; between Bcl-xL and Bcl-xS and Bak; and between E1B19K and Bax, Bak, or Nbk/Bik. The structural basis for these interactions may lie on the hydrophobic and electrostatic interactions between the hydrophobic clefts on antiapoptotic Bcl-2 family members and the BH3 domains of the proapoptotic Bcl-2 family members. Consistent with the structural analysis, heterodimerization between Bcl-2 family members is heavily conformation dependent. E1B 19K, for example, does not interact with Bax when Bax is in an inactive, closed conformation. In fact, E1B 19K only binds Bax when its amino terminal is exposed by a cellular death stimulus. The 19K–Bax interaction then antagonizes the proapoptotic function of Bax (Perez and White, 2000). Thus, interactions between Bcl-2 family members contribute significantly to their biochemical function in the regulation of apoptosis.
D. Regulation of Mitochondrial Function by Bcl-2 Family Members The structural resemblance between Bcl-2 family members and bacterial toxins raised the possibility that Bcl-2 family members may form pores in
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membranes. In vitro experiments using purified proteins demonstrated that Bcl-xL, Bcl-2, and Bax were all able to form channels in synthetic lipid membranes (Antonsson et al., 1997, 2000; Minn et al., 1997; Schendel et al., 1997). These channels are usually pH sensitive and voltage dependent, allowing ions and small molecules to pass with limited selectivity. These pore-forming activities appear to be consistent with the cellular localization of Bcl-2 family members and their ability to regulate mitochondrial functions. Bcl-2 family members can be found both in the cytosol and in membranebound forms. Bcl-2 is normally localized to the outer mitochondrial membranes, and endoplasmic reticulum, as well as nuclear membranes. Bcl-xL and Bax can be found either in cytosol or in membrane-bound forms. Accumulating evidence indicates that the subcellular localization of proapoptotic Bcl-2 family members changes during apoptosis. Bax translocation from the cytosol to mitochondrial membranes has been reported in response to a variety of death stimuli (Gilmore et al., 2000; Khaled et al., 1999; Nomura et al., 1999; Tremblais et al., 1999). Similar translocation is also observed for Bak, a Bax-like proapoptotic Bcl-2 family member (Griffiths et al., 1999). Thus, the subcellular localization, protein conformation, and the oligomerization status together regulate the activities of Bcl-2 family members. Some proapoptotic Bcl-2 family members like Bax and Bak have also been shown to associate with the adenine nucleotide translocator (ANT) and the voltage dependent anion selective channel (VDAC), both of which are components of the permeability transition pore (PTP) on mitochondria. This suggests that other mitochondrial proteins may assist Bcl-2 family members in the regulation mitochondrial events (for a review, see Gross et al., 1999). The permeability of the mitochondrial membrane has been correlated with the apoptotic process. It has been proposed that the proapoptotic Bcl-2 members such as Bax, Bid, and Bak disrupt the normal mitochondrial function and cause the loss of mitochondrial membrane potential during apoptosis. Recent studies further identified molecules that are released from mitochondria, which are normally confined to the intermembrane space. Some of these molecules play important roles in activating the caspase cascade, such as cytochrome c (Liu et al., 1996) and Smac/DIABLO (Du et al., 2000; Verhagen et al., 2000). In p53-mediated apoptosis, cytochrome c is released from mitochondria, which is efficiently blocked by both Bcl-2 and E1B 19K at an early stage of p53 activation, possibly by antagonizing Bax activity (Henry et al., 2001). Both Bcl-2 and E1B 19K failed to block cytochrome c release after a prolonged p53 stimulus (Henry et al., 2001). The fact that the mitochondrial events are highly regulated by Bcl-2 family members is indicative of the important role of mitochondria in apoptosis. Thus, the mitochondria are an important cell death checkpoint, where strict regulations are required to control apoptosis.
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E. Bax in p53-Dependent Apoptosis The observation that Bax is able to induce apoptosis in a p53-deficient background suggests that Bax functions downstream of p53. Bax functions as one of the effectors of p53 to promote apoptosis. Upregulated Bax protein levels are observed in BRK cells after p53 activation (Han et al., 1996a). Bax changes conformation in response to a p53 death signal, which causes the exposure of the Bax amino terminus. Furthermore, depletion of Bax from cell extracts prepared form p53-expressing cells blocks cytochrome c release from isolated mitochondria in vitro, suggesting that Bax may be required for cytochrome c release downstream of p53 stimulus (Schuler et al., 2000). Indeed, primary fibroblasts from Bax-deficient mice expressing the E1A oncogene, a setting where apoptosis is dependent on endogenous p53, are resistant to chemotherapy-induced apoptosis (McCarthy et al., 1997). Bax-deficient neurons (-/-) exhibit significant protection against excitotoxins or DNA damage, both of which trigger p53-dependent cell death in neurons containing at least one copy of the Bax gene (Xiang et al., 1998). These observations suggest that Bax functions downstream of p53 and conveys p53 toxicity. However, Bax is not the only player downstream of p53-mediated apoptosis. p53-induced Bax accumulation by itself is not sufficient to mediate apoptosis (Sakamuro et al., 1997). Consistent with the involvement of other factors besides Bax in p53-mediated apoptosis, the ability of bax-/- E1Amouse embryo fibroblasts (MEFs) to resist apoptosis is less robust than that of MEFs lacking p53 (McCurrach et al., 1997). Some other proapoptotic Bcl-2 family members, such as Bak and Bad, share certain functionality with Bax and may partially substitute Bax function in Bax-deficient cells. A newly identified BH3-only member of Bcl-2 family, Noxa, has also been implicated in p53-mediated apoptosis by functioning at the mitochondria (Oda et al., 2000). These observations suggest that some proapoptotic Bcl-2 family members function downstream of p53 death signaling by pushing cells through the mitochondria death checkpoint.
F. Antiapoptotic Bcl-2 Family Members Block Bax Activity Downstream of p53 Death Signaling Antiapoptotic Bcl-2 family members are able to block p53-mediated apoptosis in many cases. High levels of Bcl-2 are found in many p53-intensitive tumor cell types, suggesting that Bcl-2 may block p53-dependent apoptosis in these cells. Bcl-2 has also been shown to modulate p53 function by altering p53 subcellular trafficking during cell cycles (Marchenko et al., 2000; Ryan et al., 1994). Indeed, both Bcl-2 and the adenovirus E1B 19K protein block
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E1A-induced, p53-mediated apoptosis in BRK cells (Chiou et al., 1994a; Debbas and White, 1993; Rao et al., 1992; Henry et al., 2001). The E1B 19K protein inhibits apoptosis completely when expressed in BRK cells transformed by E1A plus the temperature-sensitive p53val(135) mutant, when p53 assumes the wild-type conformation. The 19K protein does not interact with p53, nor are the levels or localization of p53 affected. Therefore, E1B 19K functions on the components downstream of p53 or pathways that bypass p53. E1B 19K prevents neither the p53-induced Bax upregulation nor the conformational change in Bax. E1B 19K has the ability to directly interact with and antagonize Bax (Han et al., 1996a, 1998a; Perez and White, 2000). Transient interaction between E1B 19K and Bax is detected at the early stage of p53 activation (Henry et al., 2001). These observations indicate that mitochondria are an important death checkpoint in p53-mediated apoptosis, requiring both pro- and antiapoptotic Bcl-2 family members to regulate synergistically. E1B 19K inhibits Bax by at least by interacting with Bax directly.
III. CASPASE FAMILY A. The Caspase Cascade The caspase family is another evolutionarily conserved gene family that is involved in p53-mediated apoptosis. Caspases are cysteine proteases that specifically cleave substrates with an aspartic acid in the P1 position (Alnemri et al., 1996). More than 14 caspase family members have been identified so far, with many represented in humans, and several are conserved in flies and worms (Fig. 3, see color plate). Caspases are constitutively presented in most cells as inactive single chain proenzymes. They are activated to fully functional proteases through a primary proteolytic cleavage event that divides the proenzyme into large and small subunits, and a second cleavage event that removes the amino-terminal prodomain from the large subunit. The subunits assemble into a tetramer with two active sites (Rotonda et al., 1996; Walker et al., 1994) (Fig. 3). Since proteolytic cleavage generates mature caspases, these enzymes can be activated by being cleavage substrates of other caspases; thus they can function in an activation cascade (Enari et al., 1996). Caspases with large prodomains that may interact with death signaling molecules are usually referred to as the “initiator” or “regulatory” caspases. Caspase-8 and caspase-9 are representatives of this category. These upstream caspases are activated upon interacting with their adapter proteins in response to signals from the cell surface death receptors or from internal stress signals such as cytosolic cytochrome c. Two general types of
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interaction domains have been identified in the prodomains of initiator caspases. Procaspases-8 and -10 each contain two tandem death effector domains (DEDs) (Boldin et al., 1996; Muzio et al., 1996), while procaspases-1, -2, -4, -5, and -9 contain caspase recruitment domains (CARDs). Despite low amino acid sequence homology, CARDs and DEDs have similar threedimensional structures. CARDs and DEDs are composed of six closely packed, amphipathic, antiparallel ␣-helices, a structure that is also found in the death domains (DD) of Fas receptor and FADD protein (Huang et al., 1996). Procaspases bind to adapter proteins containing similar domains. The adapter proteins either directly aggregate or aggregate through interacting with other proteins upon receiving a death stimulus. Caspase recruitment results in the formation of protein complexes where procaspases are in close proximity to each other, and they autocatalyze and activate themselves. Since the activation of “initiator” caspases may be amplified by downstream caspases and result in dramatic changes in cell physiology, this step is usually tightly regulated, and functions as an important cell death checkpoint. Caspases with short prodomains such as caspase-3, -6, and -7 are referred to as “effector” caspases. Effector caspases lack protein interaction motifs, and are activated predominantly through the action of upstream caspases. They function at the distal end of the caspase cascade, and implement cellular destruction by cleaving important cellular proteins during apoptosis. Caspases have conserved sequences for substrate binding and catalysis. They recognize and cleave their substrates after specific tetrapeptide sequences that contain an aspartic acid at the P1 position. Each tetrapeptide sequence bears substrate specificity to different caspases. For example, caspase-3 prefers the sequence DEVD as its cleavage site, while caspase-8 prefers IETD. Many important cellular proteins are cleaved by caspases, which contributes to various aspects of apoptosis (Cryns and Yuan, 1998). Caspase activation is detected in E1A-induced, p53-mediated apoptosis in BRK cells. The p53-responding caspase profile includes caspase-9, -3, -6, and -7 (Henry et al., 2001). Activation of caspase-9 and caspase-3 also occurs when p53 is overexpressed in p53-null Saos-2 cells (Schuler et al., 2000). Furthermore, p53-dependent apoptosis in both cases is blocked by zVADfmk, a broad-spectrum caspase inhibitor (Sabbatini et al., 1997; Schuler et al., 2000). This suggests that the apoptotic pathways downstream of p53 may converge on caspase activation. However, the mechanism by which p53 activates the caspase cascade, and whether these caspase activities are required for p53-mediated apoptosis, are yet to be determined.
B. Caspase-9 Based on knowledge of evolutionarily conserved pathways, and recent biochemistry discoveries, the caspase-9 activation pathway has been deciphered.
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This is a pathway of fundamental importance in apoptosis, conserved from worms to humans. About a decade ago, ced-3, ced-4, and ced-9 were identified by genetic analysis as central players in programmed cell death in C. elegans (Hengartner et al., 1992; Yuan and Horvitz, 1990). The Ced-4 gene product has the ability to oligomerize, and recruit Ced-3, a caspase family member, through interactions via CARDs on both proteins, and promotes Ced-3 proteolytic activation (Yang et al., 1998; Yuan et al., 1993). Ced-9, a Bcl-2 homologue, can form a multimeric protein complex with Ced-4 and Ced-3 in vivo, which leads to inhibition of Ced-3 activation (Wu et al., 1997a). Thus, the activation of Ced-3 is the death checkpoint that decides the fate of the worm cells, which is regulated by the adapter protein Ced-4 and the Bcl-2 homologue Ced-9. Interestingly, a conserved pathway to the C. elegans Ced-3, Ced-4, and Ced-9 pathway has been identified in mammalian cells. This involves the Bcl-2 family members, Apaf-1 (Zou et al., 1997), and caspase-9 (Li et al., 1997). Although cytochrome c has not been shown to play a role in the C. elegans cell death pathway, it has been suggested to function as a cofactor in mammalian systems to assist in the activation of caspase-9 (Liu et al., 1996). Apaf-1 is a mammalian homolog of Ced-4. It contains a CARD and a Ced-4 homology domain at its amino terminus, and 12 WD40 repeats at its carboxy terminus. Apaf-1 assumes an inactive conformation in nonapoptotic cells where its carboxy-terminal tail inhibits the activity of its amino terminus (Hu et al., 1998b). Upon interacting with dATP and cytochrome c, Apaf-1 changes conformation to expose its amino terminus, and self-aggregates through the Ced-4 homology domains (Hu et al., 1998b). Oligomerized Apaf-1 recruits multiple caspase-9 molecules into a large protein complex, designated the apoptosome (Cain et al., 1999; Rodriguez and Lazebnik, 1999; Srinivasula et al., 1998). The interaction between Apaf-1 and caspase-9 is carried out through CARDs on both proteins, similar to that of Ced-4/Ced-3 interaction. Caspase-9 is then processed and activated in apoptosomes (Zou et al., 1999). Caspase-3 is also found in the apoptosome, where it is cleaved and activated by caspase-9. Ordering the cytochrome c-initiated caspase cascade reveals a possible hierarchical activation of caspases-2, -3, -6, -7, -8, and -10 all in a caspase-9-dependent manner (Slee et al., 1999). Cytochrome c release from mitochondria is observed in E1A-induced, p53mediated apoptosis. Cytochrome c release may explain the activation of caspase-9 downstream of the p53 death stimulus. Furthermore, Apaf-1 has been purified from 293 cells as a cofactor required to facilitate E1A-induced apoptosis in a cell-free system (Fearnhead et al., 1998), suggesting that the Apaf-1/caspase-9 activation may be required to mediate p53-dependent apoptosis, and may function as a death checkpoint. Other evidence for the involvement of Apaf-1 and caspase-9 downstream of p53 comes from studies with mutant mice. Caspase-9 and Apaf-1 deficient
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mice mostly die prenatally. Their phenotypes include severe craniofacial malformations and brain overgrowth, suggesting that both Apaf-1 and caspase-9 functions are crucial during embryo development, especially brain development (Cecconi et al., 1998; Hakem et al., 1998; Kuida et al., 1998; Yoshida et al., 1998). Caspase-9 or Apaf-1 null MEFs exhibit a reduced response to various apoptotic stimuli, including UV irradiation and ␥ -irradiation, which are known stimuli for the activation of p53 (Hakem et al., 1998; Yoshida et al., 1998). Deficiency in either Apaf-1 or caspase-9 also substitutes for p53 loss in promoting oncogenic transformation by Myc in MEFs. This suggests that Apaf-1 and caspase-9 may function in the same pathway as p53 in mediating Myc-induced cell death (Soengas et al., 1999).
C. Caspase-3 Caspase-3 is a general executioner of apoptosis that functions in many apoptotic pathways. Caspase-3 can be activated by both caspase-8 and caspase-9 in vivo, depending on the death signal and/or the cell type. Among substrates of caspase-3 are cellular proteins having important functions. Cleavage of these substrates significantly alters cell physiology toward apoptosis. These substrates include basic protein synthesis machinery such as polypeptide chain initiation factor eIF4GI and eukaryotic initiation factor 2-alpha, which may affect the protein synthesis process (Bushell et al., 2000; Marissen et al., 2000). The cell cycle regulator p21WAF1 is also cleaved by caspase-3 in certain cell types, which may promote apoptosis over growth arrest in these cells (Jin et al., 2000). Many molecules in the signal transduction pathways are cleavage targets of caspase-3, which may have significant impact on cellular context. These substrates include protein kinase C (PKC) -mu, -zeta, and -theta (Datta et al., 1997; Fasulo et al., 2000; Frutos et al., 1999), the serine/threonine kinase AKT/PKB (Bachelder et al., 1999), ATM (Hotti et al., 2000), beta-catenin (Steinhusen et al., 2000), GDP dissociation inhibitor D4-GDI (Rho-GDI 2) (Essmann et al., 2000; Krieser and Eastman, 1999), p21-activated protein kinase gamma-PAK (Walter et al., 1998), and focal adhesion kinase (Cicala et al., 2000). Caspase-3 may also affect the morphology of cells by cleaving cytoskeleton-related proteins such as actin, actin-capping protein ␣-adducin (van De Water et al., 2000), and the Ste20related kinase SLK (Sabourin et al., 2000). Cleavage of scaffold attachment factor A (SAF-A) by caspase-3 may contribute to the breakdown of the nuclear matrix during apoptosis (Kipp et al., 2000). Antiapoptotic Bcl-2 family members such as Bcl-2 and Bcl-xL can also be cleaved by caspase-3 at the amino terminus and have their BH4 removed. The cleaved Bcl-2 and Bcl-xL possess sequences resembling those of the proapoptotic family members, and they promote apoptosis (Fujita et al., 1998; Kirsch et al., 1999). Caspase-3
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has also been implicated in playing a role in Alzheimer’s disease, since the Alzheimer-associated presenilins are also a caspase-3 substrate (Kim et al., 1997; Vito et al., 1997). Caspase-3 dependent cleavage and inactivation of the inhibitor of caspase-activated-deoxyribonuclease (ICAD) removes ICAD from the CAD–ICAD complex, which releases and activates CAD. CAD functions to cause chromosomal DNA fragmentation during apoptosis, generating the DNA laddering pattern, which is usually considered a hallmark of apoptosis (Enari et al., 1998; Liu et al., 1998; Sakahira et al., 1998). Nucleoside-induced poly(ADP-ribose) polymerase (PARP), which functions in DNA repair, is also a caspase-3 substrate; however, whether it plays a role in apoptosis is not clear (Smulson et al., 2000). PARP cleavage is recognized as a marker for caspase-3 activation. Caspase-3 deficient mice have defects similar to caspase-9 or Apaf-1 deficient mice, indicating that caspase-3 may be involved in the same cell death pathway as caspase-9 (Kuida et al., 1996). Indeed, caspase 9-deficient thymocytes show resistance to a subset of apoptotic stimuli, including absence of caspase 3-like cleavage and delayed DNA fragmentation, confirming that caspase-3 is a necessary downstream component of the caspase-9-mediated apoptosis pathways (Kuida et al., 1998). Caspase-3-deficient neurons exhibit a remarkable delay in apoptosis and a dramatic decrease in TUNEL-positive cells in response to p53 activation, suggesting that p53-mediated apoptosis is executed through caspase-3 activation (Cregan et al., 1999). In fact, activation of p53 induces caspase-3 activation, PARP cleavage, and nuclear DNA fragmentation in many cases (Bachelder et al., 1999; Debbas and White, 1993; Geske et al., 2000; Ho et al., 1999; Hietanen, 2000; Sabbatini et al., 1997; Schuler et al., 2000; Shaw et al., 1992; Henry et al., 2001). Thus, caspase-3 may function as a distal machinery that carries out p53-mediated cellular destruction.
D. Caspase-6 and -7 Like caspase-3, caspase-6 and -7 are effector caspases that are usually activated by initiator caspases, and they cleave cellular target proteins. These three caspases can compensate for one another to some extent, since apoptosis is not ablated by knocking out each one of them. However, these caspases are not redundant. Each has a somewhat overlapping, yet distinct substrate profile, and plays different roles during mouse development. Caspase-6 deficient mice have mild defects, and mice appear to develop normally (reviewed in Zheng and Flavell, 2000). Caspase-7 deficient mice, however, die extremely early during gestation (Zheng and Flavell, 2000). Since lamins are common substrates for both caspase-6 and -7, and lamins are cleaved during p53-mediated apoptosis, it is very likely that capsase-6 and/or -7 are
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activated by p53. They may function in synergy with caspase-3 in p53 death pathways. Lamin cleavage by caspases is known to facilitate dismantling of the nucleus during apoptosis (Rao et al., 1996); therefore, a redundant cleavage mechanism would be expected.
E. Caspase-8 Caspase-8 is an “initiator” caspase that is activated by death receptors such as tumor necrosis factor receptor 1 (TNFR1) and Fas. Multiple members of the TNFR family and their decoy receptors, such as fas, DR5/KILLER, and TRID, are transactivation targets of p53 (Owen-Schaub et al., 1995; Sheikh et al., 1999; Wu et al., 1997b). Fas and DR5/KILLER trimerize upon interacting with their ligands, Fas ligand (FasL) and the TNF-related, apoptosisinducing ligand (TRAIL), respectively, thereby recruiting adapter proteins into a protein complex usually referred to as the death-inducing signaling complex (DISC) (Kischkel et al., 1995). FADD is one of the adapter proteins that is important for Fas signaling, while its role in DR5 signaling remains to be determined (Chinnaiyan et al., 1995; Kuang et al., 2000). Caspase-8 is recruited to the DISC by adapter proteins through interactions between their mutual DEDs, and caspase-8 then autoprocesses and becomes activated (Muzio et al., 1996). TRID is an antagonistic decoy receptor that has an extracellular region similar to that of TNFRs, but lacks the cytoplasmic DD. TRID protects TRAIL-induced apoptosis by competing with TRAIL receptors DR4 and DR5 for binding to TRAIL. Caspase-8 and FADD are important for mouse development. Deficiency in caspase- 8, as well as FADD, results in impaired formation of cardiac muscles and defects in response to death receptor-mediated apoptosis (Zheng and Flavell, 2000). However, the significance of death receptor-mediated apoptosis downstream of p53 remains to be established.
F. E1B 19K Inhibits Caspase-9 Activation Adenovirus E1B 19K functions downstream of E1A-induced, p53mediated apoptosis (Debbas and White, 1993; Henry et al., 2001). It potently inhibits caspase-9 activation even after cytochrome c release from mitochondria (Henry et al., 2001). E1B 19K does not associate with caspase-9 directly. However, 19K may interfere with the normal apoptosome function, providing a mechanism for 19K action downstream of cytochrome c release at the caspase-9 checkpoint. This did not rule out the possibility that 19K may target other Apaf-1-like proteins in p53-mediated apoptosis. E1B 19K has also been shown to be able to antagonize Ced-4 function and prevent Ced-4-induced caspase activation (Han et al., 1998b), suggesting
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that 19K may have the activity to block other Apaf-1-like proteins. E1B 19K expression, however, does not protect apoptosis after the activation of effector caspases in vitro (Henry et al., 2001). PARP and lamin cleavage, nuclear DNA condensation, and DNA laddering proceed normally after effector caspases are activated by p53, regardless of 19K expression. So far, E1B 19K has been shown to function only upstream of caspase-9 activation. After this death checkpoint, cells become irreversibly committed to apoptosis. A question of debate is whether mammalian Bcl-2 family members interact with Apaf-1. Thus far, Bcl-xL and Boo/Diva have been shown to interact with Apaf-1 by some (Hu et al., 1998a; Inohara et al., 1998; Pan et al., 1998; Song et al., 1999), but not by others (Moriishi et al., 1999). Purified Bcl-xL, however, does not inhibit Apaf-1 function in vitro (Newmeyer et al., 2000), and no evidence was found for an interaction between Bcl-2 and Apaf-1 (Moriishi et al., 1999). Thus, different Bcl-2 family members may have different activites. Some Bcl-2 family members such as Ced-9 and E1B 19K may be able to interact with and inhibit Apaf-1 or Apaf-1-like proteins, while others such as Bcl-2 may not. E1B 19K is more active than Bcl-2 in blocking p53-mediated apoptosis, which is likely due to a gain of function over Bcl-2. Bcl-2 may have evolved more specialized functions that are restricted to the level of protecting mitochondria. Alternatively, there may be other mammalian Bcl-2 family members that antagonize Apaf-1-like activities. Aven, a protein with no significant homology to Bcl-2 family members, interacts with both Bcl-xL and Apaf-1, thereby preventing Apaf-1 self-aggregation and caspase-9 activation (Chau et al., 2000). This finding suggests that proteins other than Bcl-2 family members may function in synergy with Bcl-2 family members to function at the caspase-9 activation death checkpoint.
G. Viral Caspase Inhibitors Many DNA viruses possess inhibitory mechanisms to prevent the premature death of infected host cells by encoding caspase inhibitors. These include CrmA, p35, IAPs, and viral-FLICE-inhibitory proteins (vFLIPs) (Clem and Miller, 1994; Irmler et al., 1997; Kataoka et al., 1998; Liston et al., 1996; Roy et al., 1997; Thome et al., 1997). Cowpox CrmA binds and inhibits caspase-1 and -8 efficiently, but has little inhibitory activity toward downstream caspases such as caspase-3, -6, and -7 (Gagliardini et al., 1994; Miura et al., 1995; Ray et al., 1992; Tewari and Dixit, 1995; Tewari et al., 1995; Zhou et al., 1997). Baculovirus encodes caspase inhibitory functions in p35 and IAPs (Birnbaum et al., 1994; Clem and Miller, 1994). p35 can be cleaved by certain caspases, and then binds to and inhibits these caspases as a suicide substrate (Ahmad et al., 1997; Beidler et al., 1995; Bump et al., 1995;
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Rabizadeh et al., 1993; Xue and Horvitz, 1995). IAPs have been found to directly interact with and inhibit activated caspases (Crook et al., 1993; Seshagiri and Miller, 1997; Uren et al., 1996). They are also found in the apoptosome, where they inhibit the activation of caspase-9 (Deveraux and Reed, 1999; Du et al., 2000). vFLIPs are presented in several ␥ -herpesviruses and human molluscipoxvirus. They interact with the DED of FADD and/or caspase-8 and inhibit caspase-8 recruitment and activation (Bertin et al., 1997; Thome et al., 1997; Wang et al., 1997). These caspase inhibitors are important for viruses to combat the immune response of the host in order to achieve productive infection and latency.
IV. IAPs A. IAP Family Members The baculovirus IAP family members are caspase inhibitors, with homologs also found in human cells. These human IAP homologs include c-IAP-1, c-IAP-2, XIAP, survivin, NAIP, and BRUCE (reviewed in Deveraux and Reed, 1999) (Fig. 4, see color plate). Mammalian IAPs, especially XIAP, directly and specifically interact with the active forms of caspase-3 and caspase-7, thereby potently inhibiting their activities (Roy et al., 1997). IAPs are also reported to interact with the proform of caspase-9 and prevent its activation (Huang et al., 2000, Deveraux et al., 1999). c-IAPs are transcription targets of NF-B, and are also identified in association with the TNFR2 complex through interaction with TNFR-associated factors (TRAFs) (Rothe et al., 1995; Wang et al., 1998). These findings suggest that IAPs may also be involved in signal transduction pathways under the regulation of TNFRs. IAP homologs are identified in a wide variety of species including yeast, C. elegans, Drosophila melanogaster, insect cells that are hosts to baculovirus, chicken, mice, rat, pigs, and man. The involvement of many of these IAP family members in apoptosis is yet to be established.
B. Structure–Function of IAPs All IAP family members contain one or more copies of the characteristic baculovirus IAP repeat (BIR) motif (Fig. 4). This is a highly conserved putative zinc coordination domain that is sufficient and required for IAP antiapoptotic function (Takahashi et al., 1998). The BIR motifs mediate oligomerization of IAPs, and are required for the interaction of c-IAPs with TRAFs (Hozak et al., 2000; Rothe et al., 1995). Most IAP family members
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also have a RING domain at the carboxy terminus. The requirement of the RING domain for the antiapoptotic function of IAPs appears to depend on the cellular context. The RING domain is implicated in facilitating the ubiquitinization and proteasome-dependent degradation of c-IAP-1 during apoptosis, and the XIAP mutant that lacks the RING domain provides superior stability and inhibition of apoptosis compared to the wild-type XIAP (Yang et al., 2000). Consistent with the ubiquitinization of IAPs, BRUCE actually contains a ubiquitin-conjugating (UBC) domain at its carboxy end, strongly suggesting that the ubiquitin–proteasome pathway may function in regulating IAPs and apoptosis (Hauser et al., 1998). The c-IAPs also contain CARDs that interact with caspases. IAP family represents another group of apoptotic checkpoint regulators that control the caspase activity.
C. Inhibitors of IAPs IAPs also have their own inhibitors. The IAP interacting protein Smac/ DIABLO directly blocks IAP activity in the apoptosome, which greatly elevates the ability of Apaf-1 to activate caspase-9 (Du et al., 2000; Verhagen et al., 2000). Smac/DIABLO is normally a mitochondrial protein, but is released into the cytosol when cells undergo apoptosis. In Drosophila, IAP activity is negatively regulated by Reaper, Hid, and Grim, which are potent proapoptotic proteins (Goyal et al., 2000; Vucic et al., 1998; Wang et al., 1999). IAPs can also be inactivated by being specifically cleaved by caspases, or being degraded through the ubiquitin pathway during apoptosis (Deveraux et al., 1999). Thus, inactivation of IAPs also appears to be an important regulatory step during the apoptotic process.
V. p53-MEDIATED APOPTOSIS IN CANCER THERAPY It is widely recognized that dysfunction in apoptotic regulation frequently occurs in cancers. One strategy to combat cancer is to restore the normal apoptosis pathways in these genetically altered cells, and to drive the cancer cells to die by apoptosis. Multiple cell death checkpoints appear to be involved in p53-mediated apoptosis, such as the regulation of mitochondrial function and the regulation of caspase-9 activation (Fig. 5, see color plate). Although a great deal of research has been done on the general apoptosis machinery and the involvement of p53 in cell death, the downstream mechanism underlying p53-dependent apoptosis still needs to be established. The functionality of the multiple p53-transactivated and suppressed gene products in apoptosis pathways is yet to be clearly defined. To understand why
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cell death checkpoints are bypassed in cancer cells, and how to reactivate them, may provide potent new strategies in cancer treatment.
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von Hippel-Lindau Disease: Clinical and Molecular Perspectives Steven C. Clifford1 and Eamonn R. Maher2,∗ 1
UK Medical Research Council Research Fellow and 2Professor of Medical Genetics, Section of Medical and Molecular Genetics, Division of Reproductive and Child Health, University of Birmingham Birmingham, B15 2TT, United Kingdom
I. The VHL Gene and VHL Disease A. Background and Clinical Features of VHL Disease B. Isolation of the VHL Gene: Genomic Structure, Expression Pattern, and Gene Products C. Germline Mutation of the VHL TSG in VHL Disease D. Diagnosis of VHL Disease E. Surveillance in VHL Disease F. Genotype–Phenotype Correlations and Modifier Effects in VHL Disease II. The VHL TSG and Sporadic Cancers III. Functional Analysis of the VHL Tumor Suppressor Gene A. Tumor Suppressor Activity of the Major pVHL Gene Products B. pVHL-Dependent Target Gene Regulation and Its Role in Angiogenesis C. pVHL-Associated Proteins: pVHL as the Recognition Component of a Ubiquitin Ligase Complex D. pVHL Has Multiple Functions: Further Intracellular Roles E. The Molecular Basis of Tumorigenesis and Genotype–Phenotype Relationships in VHL Disease IV. Conclusion References
Von Hippel-Lindau (VHL) disease (MIM 193300) is the most common cause of familial clear cell renal cell carcinoma (RCC). VHL disease results from germline mutations in the VHL tumor suppressor gene and is characterized by variable expression and the development of benign and malignant neoplasms in multiple organs. The clinical ∗ To whom correspondence should be addressed at Section of Medical and Molecular Genetics, Division of Reproductive and Child Health, University of Birmingham, The Medical School, Edgbaston, Birmingham B15 2TT, UK. Telephone: +44 121 627 2630; FAX: +44 121 627 2618; Email:
[email protected]
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management of VHL disease is challenging and requires a coordinated multidisciplinary approach. However, early detection of VHL tumors by annual surveillance has improved the prognosis for VHL gene carriers. Complex genotype–phenotype correlations for the major manifestations of VHL disease result from allelic heterogeneity and suggest that the VHL gene product has multiple and tissue-specific functions. Recent studies suggest that the VHL protein represents the adaptor unit of an Skp1-Cdc53/Cul1-F-box (SCF)like protein complex which targets specific proteins for ubiquitinylation and proteolysis. Tumors from VHL patients and sporadic tumors with VHL gene inactivation (e.g., most clear cell RCC) are hypervascular and overexpress hypoxia-inducible mRNAs such as vascular epithelial growth factor (VEGF). Recently, pVHL has been shown to regulate proteolysis of the transcription factors HIF-1 and HIF-2 (EPAS). Thus absence or inactivation of pVHL leads to constitutive HIF-1 and HIF-2 expression, which activates transcription of VEGF and other hypoxia-inducible mRNAs. Evidence for further pVHL functions including roles in fibronectin metabolism and cell cycle regulation has also been reported, but it is unclear whether these functions are mediated via pVHL-targeted proteolysis or other mechanisms. Clinical and laboratory studies of VHL disease have provided a paradigm for demonstrating the importance of familial cancer syndromes in elucidating mechanisms of tumorigenesis in familial and sporadic cancer. C 2001 Academic Press.
I. THE VHL GENE AND VHL DISEASE A. Background and Clinical Features of VHL Disease von Hippel-Lindau disease (VHL) is an autosomal dominant familial cancer syndrome, caused by mutation of the VHL tumor suppressor gene (TSG), and with an approximate incidence of 1/36,000 live births (Maher et al., 1990a; Latif et al., 1993). The age at onset and initial presentation of VHL disease is variable. The three major features of VHL disease are predisposition to retinal and central nervous system (CNS) hemangioblastomas (HABs) and clear cell renal cell carcinoma (RCC). The age-dependent penetrance for each of these tumors differs, but overall the risk for each tumor has been predicted to be in excess of 70% by age 60 years (Maher et al., 1990a). However, mean age at symptomatic diagnosis varies (24.5 years for retinal angioma, 29 years for cerebellar HAB, and 44 years in RCC). Because of the earlier onset of retinal angioma and cerebellar HAB, these complications appear more frequent than RCC in cross-sectional studies and are the most common first manifestations of the disease. VHL disease may present in childhood or old age, but most patients present in the second and third decades, and penetrance is almost complete by age 60 years. Increasingly, familial cases are diagnosed presymptomatically following DNA-based predictive testing. These individuals are offered regular surveillance to enable early diagnosis of complications (see later).
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B. Isolation of the VHL Gene: Genomic Structure, Expression Pattern, and Gene Products The VHL TSG was isolated by a positional cloning strategy in 1993, 5 years after it was mapped to chromosome 3p25 by family linkage studies (Latif et al., 1993; Seizinger et al., 1988). Although no patients with VHL disease and major chromosomal rearrangements have been described, the isolation of the VHL gene was facilitated by analysis of a small subgroup of patients with submicroscopic deletions (50–250 kb) (Yao et al., 1993; Richards et al., 1994). The VHL gene was identified finally by the detection of germline deletions and intragenic mutations in VHL patients and intragenic mutations in sporadic RCC cell lines (Latif et al., 1993). The VHL gene encodes a 4.7 kb mRNA which is widely expressed in both fetal and adult tissues, such that expression of the VHL transcript and protein is not restricted to organs in which VHL tumors occur, with expression of the VHL protein (pVHL) predominantly (though not exclusively) localized to the cytoplasm of cells in vivo (Latif et al., 1993; Los et al., 1996; Corless et al., 1997). The pattern of VHL mRNA expression in the fetal kidney was considered to be consistent with a role in normal renal tubular development and differentiation (Richards et al., 1996). Development of a mouse model of VHL disease indicated that VHL expression is critical for normal extra-embryonic vascular development. While mice heterozygous for VHL (+/−) appeared phenotypically normal, homozygous VHL (−/−) mice died in utero at 10.5 to 12.5 days of gestation. This resulted from placental dysgenesis caused by a failure of embryonic vasculogenesis of the placenta and development of hemorrhagic lesions in the placenta (Gnarra et al., 1997). The VHL coding sequence is organized in three exons and encodes two proteins. Two alternatively spliced VHL transcripts have been detected, reflecting the presence (isoform I) or absence (isoform II) of exon 2. No endogenous isoform II-associated protein product has been reported to date, and the identification of VHL patients with germline deletions of exon 2, resulting in the expression of isoform II only from the mutant allele, suggests that isoform II does not encode a functional gene product. Moreover, stable overexpression of isoform II produces a nonfunctional protein (Clifford et al., in preparation). The long 3′ untranslated region (UTR) has been characterized recently (Renbaum et al., 1996), and the 5′ UTR and its flanking sequence were defined by Kuzmin et al. (1995). A minimal promoter region of 106 bp has been defined, and we have recently obtained evidence for the presence of functional SP1 and AP2 binding sites within the minimal promoter (Kuzmin et al., 1995; Zatyka et al., manuscript in preparation).
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The full-length 213-amino acid VHL protein (pVHL30) migrates with an apparent molecular weight of ∼28–30 kDa. A second major VHL gene product, with an apparent molecular weight of ∼18–19 kDa (pVHL19), arises by internal translation initiation from the codon 54 methionine, producing a 160-amino acid protein. Evolutionary conservation of the VHL sequence is very strong over most of the sequence included in pVHL19, but the first 53 amino acids included in pVHL30 are less conserved (Woodward et al., 2000a). Thus, the N-terminal sequence of pVHL30 contains eight copies of a GXEEX acidic repeat motif in human and higher primates, but only three copies are present in the marmoset and one copy in rodent VHL genes. However, cross-species analysis of 5′ VHL coding regions suggests that this part of the gene is undergoing a stabilizing evolutionary selection, and hence is of some functional significance (Woodward et al., 2000a). The primary sequence of pVHL shows minimal homology to any known protein.
C. Germline Mutation of the VHL TSG in VHL Disease Following the identification of the VHL gene, germline mutations have been identified in >500 kindreds (Crossey et al., 1994; Richards et al., 1995; Chen et al., 1995a; Zbar et al., 1996; Maher et al., 1996), and a VHL mutation database is maintained at http://www.umd.necker.fr (see Beroud et al., 1998). VHL mutations are extremely heterogeneous and are distributed widely throughout the coding sequence, with the exception that no intragenic mutations have been detected 5′ to the translation initiation site for pVHL19. Germline VHL mutations may be divided into three groups: (a) large deletions, which account for ∼40% of all mutations, (b) intragenic missense mutations (∼30%), and (c) protein truncating mutations (nonsense, frameshift insertions and deletions, splice site mutations) (∼30%). VHL codons which are mutated are more likely to be conserved or semiconserved on an evolutionary basis (e.g., between human/C. elegans) than those residues which are not mutated (Woodward et al., 2000a). Furthermore, VHL missense mutations do show some clustering at specific regions across the protein, which may be relevant to pVHL structure or functional domains (see later). Molecular genetic analysis of the complete VHL coding region by direct sequencing and large deletion detection (e.g., quantitative Southern blot and fluoresence in situ hybridization (FISH) analyses) has been reported to detect muations in up to 100% of cases (Stolle et al., 1998; Pack et al., 1999). VHL patients without detectable mutations may be mosaic (Sgambati et al., 2000). The detection of germline VHL gene mutations enables a definite diagnosis in patients who do not satisfy clinical criteria for VHL disease (see below) and provides an opportunity for carrier testing of at-risk relatives. A few
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recurrent mutations (e.g., C694T, C712T, G713A, T505C) have been identified in multiple VHL kindreds. Most of these represent multiple de novo mutations secondary to hypermutable sequences (e.g., CpG dinucleotides, small repeats) (Richards et al., 1995). However, the T505C mutation common in southwest Germany and in American kindreds with German origin has been demonstrated to result from a founder mutation (Brauch et al., 1995). Germline missense mutations are of particular interest in VHL disease because of their relevance to genotype–phenotype correlations (see later).
D. Diagnosis of VHL Disease Conventional clinical diagnostic criteria for VHL disease requires the presence of a typical VHL tumor with a positive family history. However, isolated cases without a family history can only be identified clinically when two tumors (e.g., two HABs or a HAB and a visceral tumor) have developed. Consequently, diagnosis of VHL disease is often delayed greatly in de novo cases. The new mutation rate in VHL disease was estimated at ∼2–4 × 10−6/gene/generation (Maher et al., 1991). A diagnosis of VHL disease should be considered in all cases of retinal and CNS HABs, but also in patients with familial, multicentric, or young onset pheochromocytoma and RCC. Molecular genetic analysis provides a method by which an early diagnosis of VHL disease can be made in patients who do not satisfy the current clinically based diagnostic criteria. Thus mutation analysis has revealed that ∼50% of patients with apparently isolated familial pheochromocytoma or bilateral pheochromocytoma have germline VHL gene mutations (Woodward et al., 1997). In addition, ∼4% of patients with an apparently isolated CNS HAB have a germline VHL gene mutation even if family history and screening for subclinical features of VHL disease are negative (Hes et al., 2000). Estimates of the risk of VHL disease in patients presenting with apparently isolated retinal angioma are available (Webster et al., 2000). For patients with familial RCC, in addition to VHL disease, a diagnosis of familial papillary RCC and familial clear cell RCC which is not allelic with VHL disease should also be considered (Teh et al., 1997; Schmidt et al., 1997; Woodward et al., 2000b). At-risk children in VHL families with a known VHL mutation are usually considered for predictive testing from age 5 years.
1. THE EYE IN VHL DISEASE Retinal angiomas are the most common presenting feature of VHL disease and are multiple in many cases. Approximately 68% of cases have retinal involvement with mean 1.85 lesions. Webster et al. (1999) estimated that
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the cumulative risk of visual loss by age 50 years was 35% in gene carriers and 55% in those with retinal angiomas. Histologically, retinal angiomas are benign HABs. Untreated retinal angiomas enlarge progressively and may produce retinal detachment and hemorrhage, resulting in visual impairment. The early detection of these tumors enables treatment by laser therapy or cryotherapy and reduces the risk of visual loss.
2. THE CENTRAL NERVOUS SYSTEM IN VHL DISEASE Within the CNS, the cerebellum is the most frequent site of HAB, followed by the spinal cord and brain stem. The incidence of supratentorial lesions is small. Approximately 30% of all patients with cerebellar HAB have VHL disease, and the mean age at diagnosis of those with VHL disease is considerably younger than in sporadic cases (Maher et al., 1990b). HABs are benign, and the results of surgery for single peripherally located cerebellar lesions are often excellent. However, the treatment of multiple CNS HABs and the management of brain stem and spinal tumors may be hazardous and result in significant morbidity. The future prospects for effective anti-vascular endothelial growth factor (VEGF) therapy may offer a medical approach to the treatment of inoperable CNS and retinal HABs.
3. THE KIDNEY IN VHL DISEASE The major renal manifestations of VHL disease are renal cysts and clear cell RCC. For many years, RCC was overlooked as a major feature of VHL disease until it emerged as the leading cause of death (Maher et al., 1990a). Recognition of the high risk of RCC has led to a policy of annual renal imaging in adult VHL patients and at-risk relatives. Although computer tomography has been considered to be the most sensitive method for following renal lesions, regular surveillance is usually performed by magnetic resonance imaging (MRI) or ultrasound scans to avoid radiation exposure. Most small solid renal tumors enlarge slowly (mean <2 cm/year) (Choyke et al., 1992), and the risk of distant metastasis from a solid lesion <3 cm appears to be very remote. Thus, small solid lesions are followed up until they reach 3 cm in size. At that stage, conservative nephron sparing surgery is performed in order to maintain renal function for as long as possible. Follow up of VHL patients managed by such a nephron-sparing approach suggests that although the risk of local recurrence (from new primary tumors) is high, the risk of distant metastasis is low (Steinbach et al., 1995). In contrast, 25% of VHL patients with a RCC >3 cm (treated by nephron-sparing surgery or nephrectomy) developed metastatic disease (Walther et al., 1999). Renal transplantation is an option for a VHL patient in end-stage renal failure, and experience so far suggests that immunosuppression does not affect adversely the underlying course of VHL disease.
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Renal cystic disease in VHL does not usually compromise renal function, so no treatment is required. However, RCC may develop within a cyst, and complex cysts are followed carefully, as they can develop into solid lesions (Choyke et al., 1992). The epithelium lining the cyst wall is frequently atypical or shows evidence of carcinoma in situ. Careful examination of the renal parenchyma may also demonstrate microfoci of clear cell RCC in VHL disease (Walther et al., 1995).
4. ADRENAL GLAND IN VHL DISEASE Pheochromocytoma is an important complication of VHL disease, which demonstrates marked interfamilial variability (Richard et al., 1992). In many families, pheochromocytoma is absent or rare but in some, pheochromocytoma is the most frequent manifestation. These variations result from allelic heterogeneity (see later). In most families, pheochromocytoma in VHL disease is also associated with a significant risk of HAB and RCC. However, certain mutations (e.g., Tyr98His founder mutation) are associated with haemangioblastoma and pheochromocytoma susceptibility with a very low risk of RCC. Furthermore, certain families with apparently isolated familial pheochromocytoma have germline VHL mutations which have not been found in VHL disease families, suggesting that such mutations may specifically cause pheochromocytomas only (Crossey et al., 1995; Neumann et al., 1995; see later). Pheochromocytomas in VHL disease may be extra-adrenal, and <5% are malignant. Compared to sporadic cases, onset of pheochromocytoma is earlier in VHL patients (∼20 years), and patients and at-risk relatives are screened from age 10 years.
5. PANCREAS IN VHL DISEASE Although multiple cysts are the most frequent pancreatic manifestation, these are rarely of clinical significance, as impairment of pancreatic function is uncommon. More significant is the risk of pancreatic tumors which occur in 5–10% of cases and are usually nonsecretory islet cell tumors. A high frequency of that malignancy has been reported in VHL-associated islet cell tumors, and early surgical intervention is generally recommended (Binkovitz et al., 1990). Recently, it has been suggested that surgical management can be individualized according to the size of the pancreatic tumor such that tumors <1 cm can be monitored, while those >3 cm should be resected (Libutti et al., 1998).
6. OTHER ORGAN INVOLVEMENT IN VHL DISEASE Endolymphatic sac tumors can be detected by MRI in up to 11% of cases (Manski et al., 1997), although only a minority are symptomatic, in which
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case they present with hearing loss, tinnitus, and vertigo. Epididymal cysts are very frequent in males with VHL disease and can, if bilateral, impair fertility. However, epididymal cysts are not infrequent in the general population, and their presence in an at-risk relative does not necessarily indicate carrier status.
E. Surveillance in VHL Disease The widespread recognition that surveillance and presymptomatic diagnosis of VHL tumors reduces morbidity and mortality in VHL disease has improved prognosis in VHL disease. Targeting of surveillance to gene carriers only (following molecular genetic analysis) improves the cost effectiveness of screening. Full details of screening regimens are described elsewhere (Hodgson and Maher, 1999). The effective management of VHL patients and families requires a multidisciplinary approach. Although specific complications may require expert investigations and treatment from organ-based specialists (e.g., ophthalmologists, neurosurgeons, urologists, etc.), it is essential that responsibility for the overall coordination of family ascertainment and screening is assumed. In particular, strenuous efforts should be made to identify all at-risk relatives and offer them screening to detect subclinical disease.
F. Genotype–Phenotype Correlations and Modifier Effects in VHL Disease Complex genotype–phenotype associations are a notable feature of VHL disease. This is particularly apparent for pheochromocytoma which shows wide interfamilial differences in frequency. Thus, large deletions and truncating mutations typically predispose to HABs and RCC but not pheochromocytomas (Crossey et al., 1994; Maher et al., 1996; Zbar et al., 1996). This clinical phenotype is designated Type 1 in the NCI classification of VHL disease. Missense mutations may produce a type 1 phenotype, but can also cause a Type 2 phenotype (pheochromocytoma present) and variants thereof. Thus, in type 2A phenotype there is susceptibility to HAB and pheochromocytoma but rarely RCC; in type 2B phenotype HAB, RCC, and pheochromocytoma occur; and type 2C is characterized by a pheochromocytoma-only phenotype (Brauch et al., 1995; Neumann et al., 1995; Woodward et al., 1997). Genotype-phenotype associations in VHL disease are summarized in Fig. 1. From a clinical viewpoint, the genotype–phenotype correlations may be helpful in management. Thus, for patients with deletions and truncating mutations, the overall risk of pheochromocytoma by age 50 years
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Fig. 1 Genotype–phenotype relationships in VHL disease. Mutations are described using standard amino acid abbreviations. , deletion; RCC, renal cell carcinoma; HAB, hemangioblastoma; PHE, pheochromocytoma.
is 5%, but is 10-fold higher in patients with missense mutations. Nevertheless, missense mutations are heterogeneous, and there are relatively few instances in which there is extensive clinical information available from multiple kindreds for a specific mutation (e.g., T505C and G713A are associated with type 2A and 2B phenotypes, respectively). Patients presenting with a familial pheochromocytoma-only history (type 2C) may have mutations that are associated with other phenotypes (e.g., G713A, suggesting that gene carriers are also at risk for HAB and RCC) or missense mutations which have not been reported in other subtypes of VHL disease (suggesting that they may indeed cause pheochromocytoma specifically). These complex correlations suggest that the VHL gene product has multiple and tissue-specific functions. Furthermore, the elucidation of the molecular basis of the genotype–phenotype correlations should provide important insights into the relationship between specific pVHL functions and tumorigenesis. In addition to phenotypic variability caused by allelic heterogeneity, there is also evidence that genetic modifiers may influence the phenotypic expression of VHL disease (Webster et al., 1998). Thus individuals with ocular HABs were found to have a significantly increased incidence of cerebellar HAB and RCC (hazard ratios 2.3 and 4.0, respectively) compared to those without retinal involvement. Relative-pair analysis revealed a significantly positive correlation with regard to the number of ocular tumors in individuals of 1/2 degree relatedness, which was not apparent in more distantly related family members or in those unrelated individuals with the same mutation (Webster
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et al., 1999). These findings suggested that the development of VHL tumors is determined at an early age and is influenced by genetic modifier effects that act at multiple sites. However, the molecular basis of the putative modifiers has not been elucidated.
II. THE VHL TSG AND SPORADIC CANCERS Statistical analysis of the age at onset of cerebellar HAB and RCC in VHL disease and sporadic cases was consistent with a one-hit and two-hit model as for familial and sporadic retinoblastoma (Maher et al., 1990b). Following the identification of the VHL TSG, Prowse et al. (1997) examined tumors from VHL patients for VHL gene mutation, deletion, and hypermethylation. Evidence of somatic VHL gene inactivation was found in each tumor type investigated (HAB, RCC, pheochromocytoma, and pancreatic cancer), suggesting that a common two-stage mechanism of tumorigenesis occurs. Studies of very early lesions (small carcinomas from renal cysts) have demonstrated VHL allele loss (Lubensky et al., 1996). Familial forms of RCC (including VHL disease) account for only about 2% of all adult renal cancers. RCC in VHL disease is of the clear cell variety, and about 80% of all adult kidney cancers are classified as clear cell RCC. Although chromosome 3p deletions are frequent in sporadic RCC, there is much evidence that several TSGs map to chromosome 3p (reviewed by Kok et al., 1997). The identification of the VHL TSG enabled investigation of the hypothesis that VHL inactivation was implicated in the pathogenesis of sporadic RCC. Somatic VHL mutations and allele loss occur in up to 60% of clear cell RCC tumors and cell lines (Foster et al., 1994; Gnarra et al., 1994; Clifford et al., 1998). Furthermore, transcriptional silencing by promoter hypermethylation occurs in RCC cell lines and ∼15% of sporadic primary clear cell RCC (Herman et al., 1994; Clifford et al., 1998). These findings are compatible with VHL inactivation, representing an early, frequent, and possibly necessary step in clear cell renal carcinogenesis. Recently, Brauch et al. (2000) reported an association between somatic VHL mutation/hypermethylation and tumor stage in sporadic clear cell RCC. In addition to the involvement of the VHL gene in sporadic clear cell RCC, there is genetic and functional evidence for further chromosome 3p TSGs at 3p12-p21 (see Kok et al., 1997). Analysis of clear cell RCC of known VHL mutation status for chromosome 3p allele loss has suggested that inactivation of TSG(s) in 3p12-p21 occurs in clear cell RCC with and without VHL inactivation, and so VHL inactivation alone is probably not sufficient for tumorigenesis (van den Berg et al., 1997; Clifford et al., 1998).
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Somatic VHL inactivation is also frequent in sporadic HABs (Kanno et al., 1994). However, somatic VHL mutations are uncommon in sporadic pheochromocytoma (Eng et al., 1995). Those that have been detected are missense mutations, suggesting that specific mutations are required to instigate a pheochromocytoma, analogous to the missense mutations predominantly associated with the development of pheochromocytoma in type 2 VHL disease. Although chromosome 3p allele loss is a feature of many human cancers, tumor types which display frequent chromosome 3 allele loss but which do not occur in VHL disease (e.g., lung, ovarian, head and neck, and breast cancers) do not demonstrate somatic VHL gene mutations.
III. FUNCTIONAL ANALYSIS OF THE VHL TUMOR SUPPRESSOR GENE A. Tumor Suppressor Activity of the Major pVHL Gene Products The tumor suppressor activity of pVHL30 was confirmed experimentally by reintroduction of wild-type pVHL30 into VHL (−/−) RCC cells, causing a reduction in tumor formation in vivo in nude mice. Notably, reintroduction of pVHL30 variants containing disease-causing mutations did not have equivalent tumor suppressive effects (Chen et al., 1995b; Iliopoulos et al., 1995). Furthermore, pVHL19 is biologically active and retains the ability to suppress RCC formation in nude mice, as well as other biochemical functions (Iliopoulos et al., 1998; Schoenfeld et al., 1998; Blankenship et al., 1999). The primary sequence of pVHL shows minimal homology to any known protein and hence, several strategies have been taken to elucidate possible pVHL functions.
B. pVHL-Dependent Target Gene Regulation and Its Role in Angiogenesis VHL disease tumors (e.g., RCC, HAB) are typically highly vascular in nature, which led to the investigation of pVHL as a candidate mediator of gene regulation during neovascularization. pVHL was demonstrated to play a key role in the regulation of a number of hypoxia-inducible mRNAs, including VEGF, glucose transporter-1 (GLUT-1), and platelet-derived growth factor (PDGF), which may underlie the angiogenic phenotype of VHL tumors. mRNAs for these genes are constitutively upregulated in VHL (−/−)
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cells, with their oxygen-dependent regulation restored upon reintroduction of wild-type pVHL (Siemeister et al., 1996; Gnarra et al., 1996; Iliopoulos et al., 1996; Levy et al., 1996). Furthermore, a role for pVHL in vasculogenesis is supported by mouse models of VHL disease, which have demonstrated that VHL expression is critical for normal extra-embryonic vascular development during embryogenesis (Gnarra et al., 1997). Other genes negatively regulated by pVHL include transforming growth factor-␣ (Knebelmann et al., 1998) and the transmembrane carbonic anhydrases, CA9 and CA12, which have been postulated to play a role in regulation of pH in the extracellular tumor microenvironment (Ivanov et al., 1998). Insights into the mechanisms of pVHL target gene regulation were provided by Maxwell et al. (1999), who demonstrated that pVHL plays a critical role in the regulation of the hypoxia-inducible transcription factors HIF-1 and HIF-2. HIF-1 and HIF-2 transcription factors play a key role in the cellular response to hypoxia (oxygen sensing) and the regulation of genes involved in energy metabolism, angiogenesis, and apoptosis (e.g., GLUT-1 and VEGF, previously shown to be regulated by pVHL—see above). HIF-1␣ and HIF-2␣ subunits are normally degraded by the proteasome, but are stabilized by hypoxia, and pVHL targets these HIF subunits for oxygen-dependent proteolysis. pVHL and HIF alpha-subunits coimmunoprecipitate, and pVHL is present in the hypoxic HIF-1 DNA-binding complex (see Fig. 2). Accordingly, constitutively high HIF-1␣ and HIF-2␣ levels are observed with pVHL inactivation, with oxygen-dependent instability restored by reexpression of pVHL, paralleling the pattern of expression observed for pVHL-regulated mRNAs (see above). Thus, constitutive HIF-1 activation is linked to pVHL target gene regulation and the angiogenic phenotype of VHL-associated tumors. In addition to this transcriptional basis for pVHL-mediated target gene regulation, a number of groups have reported that mRNA stabilization may also underly the overexpression of pVHL-dependent hypoxia-inducible target genes (e.g., VEGF, GLUT1, TGF␣) in the absence of pVHL (Gnarra et al., 1996; Levy et al., 1996; Knebelmann et al., 1997). Furthermore, Levy et al. (1996) visualized the components of a constitutively elevated hypoxia-inducible RNA–protein complex in a cell line lacking wild-type pVHL, whose presence correlated with the constitutive stabilization of VEGF mRNA levels. These studies indicate that target gene regulation by pVHL may be pleiotropic, with both transcriptional and posttranscriptional effects.
C. pVHL-Associated Proteins: pVHL as the Recognition Component of a Ubiquitin Ligase Complex Several groups have sought to identify proteins which interact with pVHL. In addition to its association with HIF-1␣/2␣ subunits (see earlier), pVHL
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interacts with two proteins, elongins B and C, which bind to a region of the C-terminal third of the VHL protein that is frequently altered by VHLassociated mutations (Duan et al., 1995; Kibel et al., 1995). Elongin B and C, when bound to elongin A, generate a transcriptional elongation complex called Elongin or SIII; however, there is little evidence that VHL-mediated tumor suppression is exerted by inhibiting transcriptional elongation. Subsequently, a further protein, Cul-2, a member of the cullin protein family, was shown to associate with pVHL, elongin B, and elongin C to form the pVHL/elongin C/elongin B/Cul-2 (VCBC) complex (Pause et al., 1997; Lonergan et al., 1998). Folding and assembly of pVHL into a complex with elongin B and C is directly mediated by the chaperonin TRiC/CCT (Feldman et al., 1999). Identification of the VCBC complex led to new insights into putative pVHL function, based on homologies noted between complex components and other known protein complexes. Elongin C and Cul-2 show significant sequence homology to the yeast proteins Skp1 and Cdc53, respectively. These proteins are members of an SCF (Skp1-Cdc53/Cull-F-box) complex, which forms part of a family of E3 ubiquitin ligases. SCF complexes regulate multiple cellular processes (e.g., cell cycle, signaling, and development) in a diversity of eukaryotic organisms from yeast to humans (Tyers and Rottapel, 1999). Thus, it was proposed that the VCBC complex might play a role in targeting cellular proteins for ubiquitinization and proteolytic degradation. Under this model pVHL is predicted to act as an adaptor protein, analogous to the F-box protein in SCF complexes, to recruit specific protein targets to the core ubiquitination complex. Three critical pieces of evidence have recently been reported to substantiate this role for pVHL: Firstly, the VCBC-associated protein, Rbx-1, was identified as an essential general component of SCF complexes (Kamura et al., 1999). Secondly, solving of the crystal structure for the pVHL/elonginB/ elonginC complex provided important insights into pVHL function. pVHL has two domains: an ∼100-residue amino-terminal domain rich in -sheet (the -domain) and a smaller carboxy-terminal ␣-helical domain (the ␣domain). A large portion of the ␣-domain surface interacts with elongin C and significantly, a large proportion of disease-causing VHL mutations map to the ␣-domain and its residues that contact elongin C. A structural analogy was also noted between the F-box protein in SCF complexes and the elongin C binding site in pVHL. Elongin B binds pVHL indirectly through elongin C. The majority of other mutations map to the -domain, specifically to a patch not implicated in elongin C binding (Stebbins et al., 1999). This indicates that the presence of two intact and distinct macromolecular binding sites may be required for pVHL function, and furthers the intriguing possibility that the -domain may be involved in targeting proteolytic substrates to the general ubiquitylation complex which is bound via the ␣domain. Finally, two independent studies have now demonstrated that the
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VCBC complex does indeed promote ubiquitin ligase activity, identifying pVHL as a component of an SCF-like E3 ubiquitin-protein ligase complex. Importantly, promotion of ligase activity was only associated with wild-type pVHL and not disease-causing pVHL mutants (Lisztwan et al., 1999; Iwai et al., 1999). On the basis of their observed pVHL-dependent proteolysis (Maxwell et al., 1999), HIF-1␣ subunits provide the most striking candidate target for VCBC-targeted ubiquitin ligase activity. Indeed, Cockman et al. (2000) recently provided compelling evidence that pVHL regulates HIF-1␣ proteolysis by acting as the recognition component of a ubiquitin ligase complex, with substrate recognition mediated through the pVHL -domain. They showed that extracts from VHL-deficient renal carcinoma cells have a defect in HIF-1␣ ubiquitylation activity, which is restored by exogenous pVHL. Furthermore, the pVHL/HIF-1␣ interaction was disrupted by tumor-associated mutations in the -domain of pVHL, and loss of interaction was associated with defective HIF-1␣ ubiquitylation and regulation. These results define a mechanism by which pVHL mutation may result in the upregulation of hypoxia-inducible target genes and an enhanced tumor angiogenesis phenotype. Regulation of HIF-1␣ subunits by VCBC targeted ubiquitylation and oxygen-dependent proteasomal degradation is summarized in Fig. 2. With the demonstration of both VCBC-targeted ubiquitin ligase activity and HIF-1␣ as a specific pVHL -domain target substrate, the search is now underway for further VCBC target protein substrates. Initial reports by Iwai et al. (1999) have identified two putative VCBC targets, specifically, proteins of 100 and 220 kDa that associate with the VCBC complex and are
Fig. 2 Oxygen-dependent regulation of HIF1␣ subunits by the VCBC complex. VHL acts as an adaptor molecule, binding the general ubiquitylation machinery (VCBC complex, RBX-1, E2 enzyme) through its ␣-domain. HIF-1␣ subunits are targeted for proteasomal degradation by binding to the pVHL -domain. Proteolytic degradation of HIF-1␣ is inhibited at low oxygen tensions. EB, elongin B; EC, elongin C; ubq, ubiquitin. See text for further details.
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ubiquitylated in a wild-type pVHL-dependent fashion, but fail to associate with VCBC complexes containing disease-causing pVHL mutants. To date, these proteins are unidentified. However, these experiments specifically raise the issues that: (i) the VCBC complex may target a number of other proteins for degradation, and (ii) common mechanisms of regulation may underlie multiple pVHL functions (see below).
D. pVHL Has Multiple Functions: Further Intracellular Roles Several lines of evidence have emerged to suggest that pVHL is a multifunctional protein. Firstly, although some studies have reported that the pVHL tumor suppressive effect occurs without affecting the growth rate and cell cycle profile of cells in culture (Iliopoulos et al., 1996), VHL-dependent cell cycle effects have been reported. VHL (−/−) RCC cells fail to exit the cell cycle upon serum withdrawal, whereas reintroduction of wild-type pVHL into these cells restores their ability to exit the cell cycle and enter G0/quiescence in low serum (Pause et al., 1998). Thus, defects in cell cycle control may play a role in VHL-mediated tumorigenesis, consistent with a gatekeeper function for pVHL in the kidney. Secondly, pVHL has been reported to play a role in extracellular fibronectin matrix (EFM) assembly (Ohh et al., 1998). Fibronectin coimmunoprecipitates with wild-type pVHL but not tumor-derived pVHL mutants. Moreover, EFM formation/assembly is grossly defective in VHL (−/−) human RCC and VHL (−/−) mouse embryo fibroblasts, whereas this is corrected in the wild-type pVHL restored/VHL (+/+) counterparts, respectively. These findings strongly suggest that pVHL may have multiple intracellular roles that contribute to its tumor suppressor activity. The multiple pVHL functions described may help to define further targets for VCBC-targeted ubiquitin ligase activity and help to further illuminate pVHL function. For instance, the effects of pVHL on fibronectin and EFM assembly could potentially be mediated by VCBC ubiquitin ligase activity (fibronectin is comparably sized to the p220 species identified by Iwai et al., 1999) or by HIF-1␣-directed gene expression. Similarly, HIF-1␣-mediated target gene regulation or other unidentified VCBC target proteins may be involved in the regulation of hypoxia-inducible mRNA stability (e.g., targeting mRNA stabilizing RNA-binding proteins), or pathways governing cell cycle exit under serum-starved conditions. Thus, the demonstration of whether any function ascribed to pVHL is dependent upon (or occurs independently of) VCBC-targeted ubiquitin ligase activity is of prime importance to the understanding of pVHL function. Known pVHL functions are summarized in Fig. 3.
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Fig. 3 Summary of pVHL functions elucidated to date. +VCBC = regulated by pVHLtargeted ubiquitylation/proteasomal degradation; ??? = significance unknown/mechanism of regulation unknown; pointed arrows = positive regulation; blunted arrows = inhibitory function; HRE, hypoxia response element; HIF-1, hypoxia inducible factor 1. See text for further details.
E. The Molecular Basis of Tumorigenesis and Genotype–Phenotype Relationships in VHL Disease It is hoped that the recent insights gained into pVHL function will now begin to illuminate the molecular basis of VHL tumorigenesis and genotype–phenotype relationships in VHL disease. In addition to the multiple functions defined biochemically, genotype–phenotype associations and the related mutation-dependent tumor risks further suggest that pVHL has multiple and tissue-specific functions. The striking associations between specific pVHL missense mutations and each disease phenotype provide a powerful tool for the molecular analysis of pVHL function, its disruption in VHL tumorigenesis, and the basis of VHL genotype–phenotype relationships. Key questions include whether (i) all pVHL functions are lost uniformly in VHL tumorigenesis, (ii) if loss is nonuniform, does loss of specific pVHL functions correlate with specific disease phenotypes, (iii) gains of pVHL function(s) are associated with any disease phenotype, and (iv) retention of specific pVHL function(s) are associated with any given phenotype. Structural analysis of the VCBC complex (Stebbins et al., 1999) has provided some initial observations in this respect, noting that type 1 mutations (RCC + HAB) show a strong disposition toward -domain hydrophobic core mutations, which would be predicted to cause complete disruption to
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the pVHL structure. In contrast, type 2A/2B mutations (HAB ± RCC + PC) show a trend against hydrophobic core mutations, causing mostly local effects, suggesting that type 2 mutations have a strong bias against total loss of function. This is consistent with the rarity of gross deletion mutations in type 2 cases (Chen et al., 1995a; Maher et al., 1996). Moreover, experimental observation of protein binding patterns for pVHL missense mutants associated with each of the disease phenotypes do suggest a trend toward retention of pVHL-associated protein binding in type 2 cases, in contrast to more disruptive effects for type 1 mutants (Clifford et al., in preparation). Thus, based on initial structural and experimental evidence, development of pheochromocytoma in VHL disease may be associated with a partial retention of pVHL function, perhaps because residual pVHL function is necessary for the viability of certain cell types. Further evidence to support the differential effects of pVHL mutants is provided by the observations that (i) different type 2 mutants, which are not predicted to disrupt the hydrophobic core, map to different pVHL protein binding domains (Stebbins et al., 1999), raising the possibility of disparate protein-binding effects, and (ii) differential tumor risks are associated with-type 2A, 2B, and 2C mutants.
IV. CONCLUSION VHL disease is a complex disorder relevant to many clinical specialities. Although the identification of the VHL TSG did not provide immediate insights into the biochemical basis of VHL disease, it has (a) improved the management of VHL families by allowing molecular genetic diagnosis and (b) provided insights into the molecular pathogenesis of sporadic clear cell RCC. Recently, clues to pVHL function have been elucidated, and pVHL has been implicated in the regulation of tumor angiogenesis. Future research should further elucidate pVHL functions and their relevance to genotype– phenotype correlations in VHL disease. Furthermore, there is the prospect of a rational approach for developing effective medical treatments for VHL tumors, as knowledge of pVHL function increases (e.g., VEGF antagonists).
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Nitric Oxide-Induced Apoptosis in Tumor Cells Victor Umansky∗ and Volker Schirrmacher Division of Cellular Immunology, Tumor Immunology Program German Cancer Research Center, D-69120 Heidelberg, Germany
I. Introduction II. NO and Antimetastatic Resistance A. NO and Antimetastatic Resistance Mediated by Macrophages B. NO and Antimetastatic Resistance Mediated by Endothelial Cells III. Mechanisms of NO-Mediated Apoptosis A. Death Receptors B. The p53 Response C. Mitochondrial Control D. The Bcl-2 Family E. Caspase Activation IV. Concluding Remarks References
Nitric oxide (NO), an important molecule involved in neurotransmission, vascular homeostasis, immune regulation, and host defense, is generated from a guanido nitrogen of L-arginine by the family of NO synthase enzymes. Large amounts of NO produced for relatively long periods of time (days to weeks) by inducible NO synthase in macrophages and vascular endothelial cells after challenge with lipopolysaccharide or cytokines (such as interferons, tumor necrosis factor-␣, and interleukin-1), are cytotoxic for various pathogenes and tumor cells. This cytotoxic effect against tumor cells was found to be associated with apoptosis (programmed cell death). The mechanism of NO-mediated apoptosis involves accumulation of the tumor suppressor protein p53, damage of different mitochondrial functions, alterations in the expression of members of the Bcl-2 family, activation of the caspase cascade, and DNA fragmentation. Depending on the amount, duration, and the site of NO production, this molecule may not only mediate apoptosis in target cells but also protect cells from apoptotis induced by other apoptotic stimuli. In this review, we will concentrate on the current knowledge about the role of NO as an effector of apoptosis in tumor cells and discuss the mechanisms of NO-mediated apoptosis.
∗ Address reprint requests to Dr. Victor Umansky, Division of Cellular Immunology, Tumor Immunology Program, German Cancer Research Center, D-69120 Heidelberg, Germany; Tel.: 49-6221-423757; Fax: 49-6221-423702; E-mail: V. Umansky @dkfz-heidelberg.de
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I. INTRODUCTION The small gaseous molecule nitric oxide (NO) is generated from a guanido nitrogen of L-arginine by the family of NO synthase (NOS) enzymes. Despite its short half-life (usually a matter of seconds) and rapid oxidation to the stable, inactive end-products, nitrite and nitrate, NO has been reported as a potent biological modulator for a number of physiological functions, including vasodilation, neurotransmission, and natural defense of the immune system (Bredt et al., 1990; Moncada et al., 1991; Nathan, 1992). Following the identification of NO as endothelial-derived relaxing factor (Ignarro et al., 1987; Palmer et al., 1987), different cell types (such as macrophages, endothelial cells, neurons, fibroblasts, hepatocytes, epithelial and smooth muscle cells) have been shown to produce this free radical (Schmidt and Walter, 1994). At least three distinct isoforms of the NOS encoded by three distinct genes have been isolated (Knowles and Moncada, 1994; Nathan and Xie, 1994; Morris and Billiar, 1994). Two of them, endothelial NOS and neuronal NOS, are expressed constitutively and require intracellular Ca2+ and calmodulin for its activation. The other isoform, inducible NOS (iNOS), is usually induced in the body by bacterial products and/or by some inflammatory cytokines such as interferons, interleukin (IL)-1, and tumor necrosis factor (TNF)-␣. iNOS activity can also be upregulated by certain viruses (Umansky et al., 1996; Saura et al., 1999). The fourth isoform of NOS has recently been found in the mitochondrial inner membrane (Ghafourifar and Richter, 1997; Giulivi et al., 1998). Mitochondrial NOS is constitutively expressed, is Ca2+-dependent, and exerts control over mitochondrial respiration and mitochondrial transmembrane potential (m). A low level of NO synthesized by constitutive NOS for short periods of time acts as a neurotransmitter and as a regulator of blood pressure and platelet aggregation (Moncada et al., 1991; Schmidt and Walter, 1994; ¨ Forstermann, 2000). In contrast, large amounts of NO produced by iNOS in macrophages and endothelial cells after challenge with lipopolysaccharide or cytokines are effective against various microbes (Nussler and Billiar, 1993; MacMicking et al., 1997), parasites (Green et al., 1991; Diefenbach et al., 1998), and viruses (Karupiah et al., 1993; Saura et al., 1999). Moreover, cytokine-activated macrophages and vascular endothelial cells were reported to kill tumor cells in vitro via NO production (Stuehr and Nathan, 1989; Li et al., 1991a,b). Consistent with these experimental data, iNOS knockout mice were susceptible to infections and showed poor macrophage killer function against microorganisms and tumor cells (MacMicking et al., 1995). The expression of iNOS was also reported in human macrophages from patients with different infectious, autoimmune, and inflammatory dis¨ eases (Nicholson et al., 1996; MacMicking et al., 1997; Kronke et al., 1998).
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It was demonstrated that NO-mediated cytotoxicity involved the inhibition of mitochondrial respiration and DNA synthesis in cell targets, including tumor cells (Stuehr and Nathan, 1989; Moncada et al., 1991; Kwon et al., 1991; Kurose et al., 1993). Moreover, this cytotoxic effect was found to be associated with apoptosis (programmed cell death) in normal (Albina et al., 1993; Sarih et al., 1993; Messmer et al., 1994; Fehsel et al., 1995) and tumor cells (Xie et al., 1993; Cui et al., 1994; Geng et al., 1996; Umansky et al., 1997). Apoptosis results from the action of a genetically encoded suicide program occurring during development and differentiation, in tumor cell deletion, and in response to different stimuli such as TNF, CD95 (Fas/APO-1) ligand, TNF-related apoptosis-inducing ligand (TRAIL) (APO-2 ligand), and shortage of growth factors or certain metabolities (Thompson, 1995). Apoptosis involves an initial commitment phase followed by an execution phase characterized by the activation of a cascade of cytoplasmic cystein proteases (caspases) (Henkart, 1996) and by structural changes including externalization of phosphatidylserine, cell shrinkage, condensation of nuclear chromatin, DNA fragmentation, plasma membrane blebbing, and the breakdown of the cell into small fragments (apoptotic bodies) that are then phagocytosed (Earnshaw, 1995). Aberrant cell survival resulting from inhibition of apoptosis is known to contribute to tumor progression (Williams and Smith, 1993; Krammer et al., 1998), and cancer cells would gain a selective growth advantage by blocking apoptosis (Reed, 1997; Dong et al., 1994). In particular, matrix-independent survival of metastatic carcinoma cells during extravasation may depend upon high resistance to apoptosis, since detachment of epithelial cells from the extracellular matrix induces programmed cell death (Frisch and Francis, 1994). It is important to note that depending on the site, magnitude, and duration of NOS acitivity, NO may not only mediate apoptotic cell death but also protect from apoptosis induced by other agents. This review will concentrate on the current knowledge about the role of NO as an effector of apoptosis in tumor cells.
II. NO AND ANTIMETASTATIC RESISTANCE Tumor cell–host cell interactions play a decisive role for tumor development or regression in various types of cancer. Numerous data from experimental tumor systems and clinical observations have resulted in the conclusion that the majority of tumor cells die rapidly in the circulation, and only a few of them can survive and proliferate to form distant metastases (Fidler, 1990; Hart and Saini, 1992; Nicolson, 1993; Schirrmacher et al., 1996). Both primary tumor lesions and metastases are infiltrated
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by different host cells. Tumor microenvironment includes not only organ parenchymal cells but also T lymphocytes with the potential of mediating specific antitumor immune reactions and antigen nonspecific host cells like natural killers, fibroblasts, granulocytes, endothelial cells, and macrophages (Whitworth et al., 1990; Belloni and Tressler, 1990; Shirrmacher, 1992). The last cell type constitutes a large proportion of stroma cells, and together with endothelial cells can produce in situ cytotoxic amounts of NO after appropriate activation (Thomsen and Miles, 1998; Xie and Fidler, 1998). In addition to cytotoxicity, NO can modulate several steps in the metastatic ¨ process, including suppression of platelet aggregation (Forstermann, 2000), downregulation of expression of adhesion molecules such as VCAM and ICAM (De Caterina et al., 1995), and inhibition of angiogenesis (Sakkoula et al., 1994).
A. NO and Antimetastatic Resistance Mediated by Macrophages 1. NO PRODUCTION DURING TUMOR METASTASIS To study NO production by macrophages infiltrating metastatic lesions, the ESb/ESb-MP mouse lymphoma model was used (Schirrmacher et al., 1982, 1995). ESb cells represent a spontaneous highly metastatic (liver as main site) variant of the chemically induced T cell lymphoma L5178 Y (Eb) of DBA/2 mice. A plastic-adherent variant, ESb-MP, retaining most of its ESb-derived antigenic and biochemical characteristics, has reduced growth capacity in vivo and metastasizes at a lower rate than parental ESb cells. To detect and quantify spontaneous lymphoma metastases at the single cell level, ESbL cells, a subline of ESb lymphoma cells, were transduced with ¨ the bacterial lacZ gene coding for -galactosidase (Kruger et al., 1994a). After intradermal inoculation in immunocompetent syngeneic mice, this lymphoma variant showed a characteristic three-phase kinetics of primary tumor growth and liver metastasis including an initial expansion phase, a plateau ¨ phase, and a final expansion phase leading to the death of the animals (Kruger et al., 1994b). Liver macrophages (Kupffer cells) were isolated during ESbL-lacZ lymphoma metastasis using a new method which provides high cell viability (>93%) and allows the direct ex vivo examination of separated cells without further in vitro culture (Rocha et al., 1996). A correlation between an increased NO production by Kupffer cells and the arrest of tumor growth and metastasis at the plateau phase was demonstrated (Umansky et al., 1995). This increase in NO synthesis may be due to upregulation of iNOS activity by various cytokines, the possible source of which in our model could be
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host T lymphocytes. In support for this assumption, SCID mice, which lack both T and B lymphocytes, and nude (nu/nu) mice, which lack only T lymphocytes, demonstrated no increase in NO production by Kupffer cells and spleen macrophages in response to application of either live or irradiated lymphoma cells. Kupffer cell cytotoxicity mediated by NO has also been reported in a rat hepatoma model in vitro (Kurose et al., 1993; Aono et al., 1994). This cytotoxicity was abrogated by the treatment with the specific iNOS inhibitor N-monomethyl-L-arginine (NMMA). The regression of murine sarcoma liver metastasis in vivo has been reported to correlate with the upregulation of iNOS expression and NO Synthesis within the tumor lesions after treatment with a macrophage activator encapsulated in liposomes (Xie et al., 1995a). application of the iNOS inhibitor prevented NO production and apoptosis in the tumor cells. Interestingly, direct transfection of iNOS into highly metastatic murine melanoma cells caused partial apoptosis of these cells associated with suppression of their tumorigenicity and metastatic potential (Xie et al., 1995b). Tumor cells producing large amounts of NO stimulated not only autocytolysis but also destruction of bystander tumor cells (Xie et al., 1997a). On the other hand, highly metastatic cells may evade NO-mediated apoptosis by development of protective mechanisms. For example, NO production by Kupffer cells was found to be substantially inhibited at the final expansion phase of ESbL-lacZ lymphoma metastasis (Umansky et al., 1995). The downregulation of iNOS activity may be caused by the release of soluble tumor derived factors (Murata et al., 1994). It has been found that overproduction of acidic phosphoprotein osteopontin by metastatic tumor cells suppressed the cytokine-mediated iNOS activation and tumoricidal activity of macrophages (Denhardt and Chambers, 1994; Feng et. al., 1995). In addition, metastatic cells are able to develop different defense mechanisms ¨ to scavenge or to detoxify NO (Kronke et al., 1997; Inai et al., 1996).
2. ROLE OF NF-B IN THE REGULATION OF NO PRODUCTION In has been shown that the transcription of iNOS in stimulated macrophages is predominantly activated by NF-B (Xie et al., 1994; Liang et al., 1999). This transcription factor regulates the expression of various genes encoding cytokines, cell surface proteins, and other genes regulating the immune response and apoptosis (Bauerle and Henkel, 1994). In unstimulated cells, NF-B is presented in the cytoplasm in an inactive form, complexed with its inhibitory subunit IB. Inflammatory cytokines, TNF-␣, LPS, UVirradiation, or reactive oxygen species (ROS) induce the activity of IB kinases, which allows the degradation of the phosphorylated IB protein and
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the translocation of NF-B into the nucleus, where it binds to DNA enhancer motifs (Stancovski and Baltimore, 1997). Newcastle disease virus (NDV) has also been found to activate NF-B in rat macrophages (Fisher et al., 1994; Bauerle and Henkel, 1994). This virus has received much attention because of its nonspecific immune stimulating potential and its various antitumor activities in tumor mouse models and in cancer patients (Ahlert et al., 1997; Schirrmacher et al., 1998). Using mouse spleen macrophages, we demonstrated that treatment with NDV in vitro and in vivo induced NF-B activation and NO synthesis in these cells (Umansky et al., 1996). This activation of NF-B and NO production was completely inhibited by the antioxidant butylated hydroxyanisole (BHA) and therefore required the participation of ROS. It has been proposed that the stimulation of certain kinases and proteases in the process of NF-B activation is regulated by the intracellular redox state (Westendorp et al., 1995; Cahill et al., 1996). Moreover, NDV-induced NF-B activation and NO synthesis were blocked by an inhibitor of protein tyrosine kinase (genistein) and protein kinase A (H-89) but not by an inhibitor of protein kinase C (staurosporin), which suggests that signaling requirements of both NF-B activation and NO production in NDV-treated macrophages are similar (Umansky et al., 1996). As to the role of the produced NO in the modulation of NF-B activity, there still exists no clear picture. NO was reported to activate the DNAbinding activity of NF-B in T cells (Lander et al., 1995), whereas it inhibited this transcription factor in other cell types (Peng et al., 1995; Matthews et al., 1996). To clarify these apparent contradictions, we incubated endothelial TC10 cells with the NO donor and tested its effect on NF-B activation (Umansky et al., 1998). It was found that at low concentrations, NO increased the DNA-binding activity of NF-B prestimulated with low amounts of TNF-␣. In contrast, high concentrations of NO inhibited this activation. The following mechanism of interaction between NF-B and iNOS in endothelial cells and macrophages could thus be proposed: The activation of NF-B by cytokines and other stimuli results in the induction of iNOS expression. This leads to an increased NO synthesis, which in turn costimulates NF-B by a self-amplifying mechanism. High amounts of generated NO can not only induce apoptosis in neighboring tumor cells but also prevent the NF-B activation followed by reduced iNOS transcription and NO production in macrophages or endothelial cells. The inhibition of the NF-B activation was proposed to result either from a direct S-nitrosylation of cysteine 62 in the p50 NF-B subunit (Matthews et al., 1996) or from stabilization of the inhibitory IB-␣ protein (Peng et al., 1995). Importantly, NO did not affect the viability of endothelial cells at concentrations causing the inhibition of DNA-binding activity of NF-B, thus allowing the establishment of an autoregulatory loop (Umansky et al., 1998).
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3. NO IN IMMUNOTHERAPY OF METASTASIS In situ activated host macrophages, which are able to produce cytotoxic amounts of NO, were also shown to play an important role in a highly effective adoptive cellular immunotherapy (ADI) of ESb T lymphoma in DBA/2 (H-2d, Mlsa) mice. The therapy protocol consists of a single intravenous injection of anti-tumor-immune spleen lymphocytes from ESb-tumor resistant, MHC identical but superantigen different B10.D2 (H-2d, Mlsb) mice into tumor-bearing DBA/2 mice. This results in rejection of primary tumors (1.5 cm in diameter) from the skin and eradication of liver metastases (Schirrmacher et al., 1991, 1995). This immunotherapy requires only relatively few specific T cells and involves synergistic interactions between CD4 and CD8 tumor-immune donor T cells and host Kupffer cells at the site of ¨ ¨ liver metastases (Schirrmacher et al., 1995; Muerk oster et al., 1999). It was found that Kupffer cells ex vivo isolated at different time points after adoptive transfer of immune spleen cells produced high amounts of NO (Umansky et al., 1995). Furthermore, the depletion of Kupffer cells with liposomes containing chlodronate in tumor-bearing mice during ADI led to ¨ ¨ nearly complete prevention of the immunotherapeutic effect (Muerk oster et al., 1999). This treatment with chlodronate selectively affected Kupffer cells but not other liver cells (e.g., lymphocytes, dendritic, or endothelial cells) and did not result in cytokine release (Rocha et al., 1995; Van Rooijen and Sanders, 1997). After immunohistological staining of livers from ADI-treated mice, iNOSpositive host macrophages were observed at early time points (days 5 and 8 after cell transfer) in close association with apoptotic cells in areas of ¨ ¨ metastases (Muerk oster et al., 2000). Moreover, in vivo experiments, in which iNOS activity was suppressed by constant infusion of the inhibitor N-(3-aminomethylbenzyl)acetamidin (1400W) immediately after ADI treatment, showed a marked reduction of the survival of mice. These findings suggest a cytotoxic role of NO in the regression of liver metastasis (which was completely eradicated at day 12 after ADI). It has been reported that the secretion of NO by macrophages and dendritic cells in vitro is mediated by interaction between CD40 expressed on these cells and CD40 ligand (CD40L) on the surface of T lymphocytes (Tian et al., 1995; Lu et al., 1996). In our therapy model, iNOS-positive macrophages coexpressed the CD40 molecule. Furthermore, the in vivo blockade of CD40L with respective monoclonal antibodies led to a significant reduction of both CD40 and iNOS expression in macrophages and to a considerable inhibition of the immunotherapeutic effect, supporting thereby the involvement of CD40–CD40L interaction in ¨ ¨ the iNOS induction during ADI (Muerk oster et al., 2000). Cytotoxic and regulatory fuctions of NO, including the suppression of T cell proliferation, the inhibition of Thl cytokine secretion, and the
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downregulation of the MHC class II expression (Kolb and Kolb-Bachofen, 1998; Bogdan, 1998, 2000), may also be operative at a later stage of ADI, when local T cell responses should be terminated, and the liver has to be cleared from undesired transferred immune cells. At these time points (in particular, at day 20 after ADI), iNOS expressing cells were detected around periportal veins in close proximity to donor CD8 and CD4 T cells undergoing ¨ ¨ apoptosis (Muerk oster et al., 2000). After day 20, the total numbers of CD8 and CD4 liver-infiltrating T lymphocytes were found to decrease markedly ¨ ¨ (Muerk oster et al., 1998). In contrast, in livers of mice treated with the iNOS-specific inhibitor 1400W at later time points after ADI, the numbers of CD8 and CD4 T cells remained at the high level that correlated with the reduced survival of animals. Similar results were observed when antiCD40L antibodies were injected into ADI-treated mice. Since at these later time points the metastases were already eradicated, the animals died most likely from graft versus host (GvH) disease caused by donor T cells which were not eliminated from the liver via host versus graft (HvG) reactivity and which preserved their proliferative potential. Therefore, CD40–CD40L interactions leading to iNOS induction in Kupffer cells could contribute to the destruction of liver metastases at early time points after ADI and to elimination of the infiltrating allogeneic T cells at a later stage.
B. NO and Antimetastatic Resistance Mediated by Endothelial Cells Interactions between tumor and endothelial cells are not limited only to the promotion of invasion and metastasis through the formation of new capillary vessels or upregulation of adhesion molecule expression which leads to increased tumor cell binding (Belloni and Tressler, 1990; Folkman, 1995). Activated endothelial cells are also able to induce tumor cell death via secretion of cytotoxic factors including NO. It is known that endothelial cells produce NO in vitro and in vivo both by constitutive and inducible mech¨ anisms (Schmidt and Walter, 1994; Moncada, 1997; Forstermann, 2000). The endothelial isoform of constitutive NOS plays an important role in the stimulation of angiogenesis, blood flow, and platelet aggregation (Ziche et al., 1994; Fukumura and Jain, 1998), whereas large amounts of NO produced by iNOS in cytokine-activated vascular endothelial cells are capable of lysing murine and human tumor cells in vitro (Li et al., 1991a, b; Geng et al., 1996). To study whether a similar mechanism may function in vivo, NO production by ex vivo isolated liver endothelial cells was determined during the different phases of ESbL-lacZ lymphoma metastasis (Rocha et al., 1995). A dramatic increase in NO synthesis in liver endothelial cells was found to
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correlate with the arrest of primary tumor growth and a low plateau of liver metastasis. However, when the growth of primary tumor and liver metastasis started again at the final expansion phase, NO production by endothelial cells significantly decreased, reaching the basal level observed in normal mice. Metastatic cells were reported to produce some factors like transforming growth factor-beta (TGF-) or osteopontin, which could downregulate the iNOS activity in both endothelial cells and macrophages (Vodovotz et al., 1993; Denhardt and Chambers, 1994; Feng et al., 1995). In this way, tumor cells appear to be able to defend themselves against being killed by NO produced by cytokine-activated host endothelial cells and/or macrophages. The upregulation of iNOS activity in endothelial cells during tumor growth suppression at the plateau phase may be induced by cytokines such as interferon ␥ (IFN-␥ ) or TNF-␣ produced by activated cells of the tumor microenvironment (macrophages and/or lymphocytes) (Estrada et al., 1992; Bordeling and Murphy, 1995). To investigate this possible regulatory mechanism in the mouse ESbL-lacZ lymphoma model, selective elimination of Kupffer cells was performed by injection of chlodronate entrapped in liposomes (Rocha et al., 1995). Importantly, liver endothelial cells were not functionally affected by this treatment (Boggers et al., 1991; Van Rooijen, and Sanders, 1997). It was found that the depletion of Kupffer cells in lymphoma-bearing DBA/2 mice caused no changes in the high level of NO production by liver endothelial cells during the arrest of tumor growth and metastasis, suggesting that this production is completely independent from Kupffer cells. The role of T lymphocytes in the stimulation of NO synthesis in endothelial cells was studied using different protocols of tumor cell inoculation into immunocompetent or immunocompromised mice. NO production was considerably increased only in liver endothelial cells of tumor cell-injected immunocompetent DBA/2 mice, regardless of the site of lymphoma cell inoculation, whereas injected immunocompromised animals (sublethally ␥ -irradiated, nude or SCID mice) did not show any induction of NO synthesis. The nature and mechanisms of the inductive signals transmitted to the iNOS of liver endothelial cells after tumor cell vaccination at distant sites are still unknown. The systemic effect observed in immunocompetent syngeneic mice might be explained by migration of activated CD4 T lymphocytes from the draining lymph nodes of the vaccination site to the liver where they could produce, among others, IFN-␥ or TNF-␣, powerful inducers of iNOS activity. A central role of CD4 T cells in the induction of NO synthesis by host cells in the tumor microenvironment associated with a strong antitumor immune responses has been recently demonstrated in mouse tumor models (Hung et al., 1998; Nishimura et al., 1999). The inducible endothelial NO response thus plays an important role in the suppression of lymphoma metastasis in the liver and seems to require mature T lymphocytes and a T cell dependent antitumor immune response.
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In another set of experiments, we demonstrated that endothelial cells isolated ex vivo from livers of ESbL-lacZ lymphoma-bearing mice could induce apoptosis in coincubated ESbL-lacZ target cells. Since it was technically difficult to get ex vivo sufficient amounts of liver endothelial cells to study in detail this effect, we decided to use the well-characterized bovine endothelial cell line (BEC) as a source of effector cells (Umansky et al., 1997). An important advantage of BEC is their fast and strong adherence to plastic, which permits the separation of nonadherent lymphoma target cells after coincubation. It was found that treatment with human TNF-␣ induced BEC iNOS activation and NO production, which could be completely blocked with the iNOS inhibitor NMMA. Coculture of activated BEC with lymphoma cells caused apoptosis in the latter cells, which was considerably reduced after pretreatment of endothelial cells with NMMA. A similar apoptotic effect was also observed after incubation of tumor cells with NO donors such as DETA / NONOate (NOC-18) or glycerol trinitrate (GTN). Interestingly, nonactivated BEC were not able to produce NO and showed a substantially lower level of apoptotic cell death. This might mean that part of the apoptotic effect of endothelial cells against lymphoma cells is mediated not only by NO, but also by other agent(s) as observed in some cases of macrophageinduced tumor cytotoxicity (Mateo et al., 1996; Lavnikova et al., 1997). Besides BEC, TNF-␣ was reported to stimulate iNOS also in human vascular endothelial cells, followed by NO-induced apoptosis in human leukemic cells in vitro (Geng et al., 1996). It might, therefore, be suggested that appropriately activated vascular endothelial cells are able to induce apoptosis in circulating metastasizing cells via NO and thereby contribute to prevention of the extravasation phase of metastasis.
III. MECHANISMS OF NO-MEDIATED APOPTOSIS A. Death Receptors It is known that members of the TNF receptor superfamily play a critical role in the development of apoptosis in various cell types (Nagata, 1997; Krammer et al., 1998). CD95L and TNF-␣ were shown to induce apoptosis by binding to their respective death domain-containing receptors, CD95 and TNFR-1. Binding of CD95L or agonistic antibodies to CD95 leads to a signal transduction cascade initiated by death receptor associated molecules such as FADD/MORT1 (Chinnaiyan et al., 1996) and FADD associated FLICE/MACH (caspase-8), a chimeric molecule containing a death effector domain and a proteolytic ICE-like domain (Boldin et al., 1996). This leads
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to proteolytic processing of caspases into active proteases. Caspase-8 is the first in a cascade of caspases activated by CD95 (Medema et al., 1997). The CD95 system is critical for growth control of T cells. Elimination of peripheral T cells involves the induction of a suicide of fratricide mechanism triggered by CD95 ligand/receptor interaction (Dhein et al., 1995; Ju et al., 1995; Peter and Krammer, 1998). Triggering of CD95 ligand/receptor interaction in lymphoid and nonlymphoid cells may also be caused by cytotoxic drugs or viral proteins (Friesen et al., 1996; Chlichlia et al., 1997). Another member of the TNF superfamily, named TRAIL/APO-2L, has been identified based on the homology of its extracellular domain with CD95L and TNF (Wiley et al., 1995). TRAIL induces rapid apoptosis in a variety of transformed cell lines, but does not appear to be cytotoxic to normal cells in vitro (Walczak et al., 1999). It has been recently reported that human monocytes rapidly expressed TRAIL, but not CD95L or TNF, after activation with IFN-␥ or -␣ and acquire the ability to kill tumor cells through apoptosis (Griffith et al., 1999). The involvement of the CD95/CD95L system in NO-induced apoptosis in human neoplastic lymphoid cells was studied using CD95-sensitive Jurkat cells (APO-S clone) and their CD95-resistant subclone (APO-R), characterized by lack of the CD95 receptor on the cell surface (Peter et al., 1995). APO-S cells were found to be much more susceptible to apoptosis induced by NO donor glycerol trinitrate (GTN) than APO-R cells (Chlichlia et al., 1998). Moreover, NO triggered apoptosis in freshly isolated human leukemic lymphocytes which were also sensitive to anti-CD95 treatment. These findings were confirmed using an in vitro model mimicking a relevant in vivo situation and based on coculture of APO-S target cells with TNF-activated bovine endothelial cells generating NO as effectors. A significant level of lymphoid cell apoptosis was observed which was completely abrogated by the iNOS inhibitor NMMA. Incubation of APO-S cells with the NO donor resulted in a strong increase in the expression of CD95L and TRAIL mRNA. Although CD95L mRNA expression was transient and reduced after prolonged (up to 24 h) incubation of the cells with NO, TRAIL expression was stable for the same time period. Unlike CD95L, whose transcripts are predominantly restricted to stimulated T cells and sites of immune privilege, TRAIL expression is detected in many normal human tissues (Wiley et al., 1995). It was proposed that TRAILmediated apoptosis may play a role in antileukemic growth control in APO-S Jurkat cells, although TRAIL had only 25% of the activity of CD95L in inducing apoptosis (Jeremias et al., 1998). However, the requirement of the CD95/CD95L system in the mechanism of NO-induced apoptosis is not strict. Some neoplastic lymphoid cells resistant to CD95-mediated apoptosis (e.g., BL60 cells) were shown to be sensitive to treatment with NO (Chlichlia et al., 1998). Furthermore, NO was able
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to trigger apoptosis in human breast cancer cell lines derived from primary tumor (BT-20) or from metastases (MCF-7) independently of CD95/CD95L interaction (Umansky et al., 2000a). Thus, in contrast to APO-S Jurkat leukemia cells, NO failed to induce mRNA expression of CD95L in these breast cancer cells. Moreover, BT-20 and MCF-7 cells showed a strong expression of the mRNA of Fas-associated phosphatase-1 (FAP-1). The FAP-1 protein is associated with a negative regulatory domain of CD95 and can thereby inhibit CD95-mediated apoptosis (Sato et al., 1995). Abrogation of this association has been reported to restore CD95-mediated cell death in a colon cancer cell line (Yanagisawa et al., 1997). In addition, FAP-1 expression in some tumor cell lines was found to correlate with their resistance to apoptosis induced by CD95 (Ungefroren et al., 1998).
B. The p53 Response The p53 tumor suppressor gene is a key target for mutation in many types of human cancer (Mowat, 1998; Brown and Wouters, 1999). A main biological function of the p53 protein is positive regulation of apoptosis in response to signals such as genomic damage and aberrant activation of certain oncogenes (Lakin and Jackson, 1999). In many cell types, stressrelated activation of p53 associated with a rapid increase in its levels induces cell-cycle arrrest, which can provide a sufficient time window for DNA repair. When DNA damage is excessive, the cell may undergo apoptosis mediated by p53. The apoptotic activity of p53 has been demonstrated to be crucial for tumor growth suppression in vitro and in vivo (Mowat, 1998; Brown and Wouters, 1999). Evidence for the involvement of accumulation of the p53 gene products in NO-mediated apoptosis was initially provided for normal cells such as RAW 264.7 macrophages (Messmer et al., 1994), pancreatic RINm5F cells (Ankakrona et al., 1994), and murine thymocytes (Fehsel et al., 1995). Upregulation of iNOS activity in macrophages or treatment of these cells with NO donors caused p53 accumulation, which preceded DNA fragmentation at the late apoptotic phase, while inhibition of NO synthesis suppressed p53 ¨ ¨ accumulation (Messmer and Brune, 1996; Brune et al., 1999). Moreover, after incubation with the NO donor S-nitrosoglutathione, macrophages stably transfected with plasmids encoding p53 antisense RNA showed lower p53 levels and significantly reduced DNA fragmentation, suggesting thereby an importance of p53 expression during NO-induced apoptosis. It has recently been reported that NO-mediated accumulation of p53 involves inhibition of the proteasome (Glockzin et al., 1999). The latter molecular complex is responsible for the degradation of many short-lived proteins (including p53) that regulate cell proliferation and cell death.
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The role of wild-type p53 in NO-mediated apoptosis was also demonstrated in tumor systems. In murine melanoma cells, incubation with cytokines such as IFN-␥ and IL-1␣ stimulated iNOS expression and NO production, which correlated with increased expression of wild-type p53 at the mRNA and protein levels (Xie et al., 1997b). Moreover, this effect on p53 expression could be reversed by inhibiting iNOS activity. A similar induction of p53 expression in melanoma cells was achieved using NO donors and was concentration and time dependent (Xie and Fidler, 1998). In another study, human colon carcinoma cells containing wild-type p53 were more sensitive to NO-mediated apoptosis than cells without p53 or with mutated p53 (Ho et al., 1996). In addition to wild-type p53, NO was found to induce the expression of the p21/ WAF1/CIP1 protein, which may eventually promote apoptosis in these cells. The upregulation of p21/ WAF1/CIP1 expression was also demonstrated in human pancreatic carcinoma cell lines in the process of NO-mediated apoptosis (Gansauge et al., 1998). It is necessary to note that in cancer cells with mutated p53, NO may not induce apoptosis but rather promote tumor growth associated with increased neovascularization, contributing thereby to cancer progression (Ambs et al., 1998). However, p53 negative cells (such as U937 cells) have recently been reported to undergo apoptosis after NO exposure. This observation substantiates the hypothesis for the existence of p53-independent signaling pathways during ¨ ¨ et al., 2000). NO-mediated apoptosis (Brockhaus and Brune, 1998; Brune
C. Mitochondrial Control Recent studies performed in various cellular systems, including cell-free extracts, clearly indicated a crucial role for mitochondrial damage in the effector phase of apoptosis in mammalian cells induced by different agents, including NO (Kroemer et al., 1997; Van der Heiden et al., 1997; Scaffidi et al., 1998; Brown and Borutaite, 1998). This damage includes the early disruption of mitochondrial transmembrane potential m, the generation of ROS, the opening of permeability transition (PT) pores, and the release of 15-kDa protein cytochrome c release from mitochondria (Marchetti et al., 1996; Susin et al., 1997; Li et al., 1997; Kluck et al., 1997; Kuida et al., 1998). m results from the asymmetric distribution of ions on both sides of the inner mitochondrial membrane, giving rise to a gradient which is essential for mitochondrial function. Functional experiments show that the mechanism of the early m loss involves the PT pores, dynamic multiprotein complexes formed at the contact site between the inner and the outer mitochondrial membranes (Kroemer et al., 1997). Cytochrome c, identified as apoptotic protease activating factor 2 (Apaf 2) is required for the formation of the complex between Apaf 1 and caspase-9 (Apaf 3). The latter becomes
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activated under such conditions and in turn activates caspase-3, which leads to DNA fragmentation and apoptosis (Li et al., 1997). Cytochrome c is normally present on the outer surface of the inner mitochondrial membrane and shuttles electrons between complexes III and IV of the respiratory chain (Brown and Borutaite, 1998). The decrease of m due to the opening of PT pores followed by the depolarization of the inner mitochondrial membrane and massive ROS production was demonstrated in normal thymocytes undergoing apoptosis mediated by NO (Hortelano et al., 1997). To study whether this mechanism is operative also in tumor cells, we treated APO-S Jurkat leukemic cells with the NO donor and observed a time-dependent increase in the number of cells with low m (Ushmorov et al., 1999). The role of PT in the NO-induced apoptosis in our model was elucidated with the help of bongkrekic acid (BA) which is known to be a specific inhibitor of PT, affecting the molecular conformation of the adenine nucleotide translocator (a protein participating in the formation of PT pores) and was shown to suppress dexamethasoneinduced apoptosis in mouse thymocytes (Marchetti et al., 1996). However, only a limited inhibitory effect on NO-induced apoptosis in APO-S Jurkat cells was observed, and only a moderate decrease in the number of cells with low m was observed, suggesting that PT is possibly not a major facotor causing NO-induced reduction of m and apoptosis in Jurkat cells (Ushmorov et al., 1999). Upon NO treatment, an intensive and rapid cytochrome c release into the cytosol of Jurkat cells was found. To clarify the mechanism of this release, we studied the function of respiratory chain complexes and the content of cardiolipin. This major mitochondrial lipid is necessary for the activity of respiratory complexes (Hatch, 1998) and plays a crucial role in cytochrome c attachment to the inner mitochondrial membrane (Choi and Swanson, 1995; Salamon and Tollin, 1996). We found that exposure to NO could significantly inhibit the activity of all complexes of the mitochondrial electron transport chain. These findings are in agreement with publications reporting on NO-mediated inhibiton of cytochrome c oxidase (complex IV) in different cell types via reversible binding to its heme moiety (Takehara et al., 1996; Clementi et al., 1998; Richter et al., 1999). This downregulation of complex IV activity resulted in the upregulation of ROS synthesis, followed by the formation of the strong oxidant peroxynitrite anion (ONOO−) Which can induce irreversible inhibition of complexes I and III but not complex IV (Casina and Radi, 1996). The alternative mechanism of a direct effect of NO on these complexes is also possible in Jurkat leukemic cells, since a long-term exposure to NO in vitro has been recently shown to block complex I activity in murine macrophages, due to S-nitrosylation of this enzyme (Brown and Cooper, 1994; Clementi et al., 1998; Richter et al., 1999).
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After exposure to NO, inhibition of complex IV activity was found to correlate with a significant time-dependent accumulation of Jurkat cells with low cardiolipin concentration (Ushmorov et al., 1999). Moreover, a potent inhibitor of lipid peroxidation, trolox (a vitamin E analog) significantly inhibited NO-induced apoptotic cell death and restored complex IV activity. In addition, trolox considerably reduced the number of NO-treated Jurkat cells with low cardiolipin content. A similar protective effect of trolox has also been reported for NO-exposed rat astrocytes (Heales et al., 1994). To study further the role of mitochondrial lipid cardiolipin in the resistance of Jurkat leukemia cells to NO-mediated apoptosis, we sorted the cells with high cardiolipin concentration (Cardiolipinhigh) which survived after exposure to NO donors for 24 h (Umansky et al., 2000b). These cells were substantially less sensitive to NO-induced apoptosis than unsorted parental Jurkat cells and maintained the same low level of sensitivity during long-term culture. Elevated cardiolipin concentration has recently been reported to be involved in the resistance of hepatocytes to some apoptotic stimuli (Lieser et al., 1998). However, in Cardiolipinhigh Jurkat cells, the increased cardiolipin content quickly returned back to the level observed in parental cells and remained unchanged during the whole period of culture, suggesting that the stimulation of cardiolipin synthesis is associated with Jurkat cell survival after NO treatment but does not play a crucial role in this process. In contrast to cardiolipin, the content of glutathione in Cardiolipinhigh Jurkat cells was significantly and constantly higher than in parental NOsensitive cells (Umansky et al., 2000b). Glutathione as a physiological oxidant plays an important role in maintaining intracellular redox balance ¨ and in the cellular defense against oxidative stress (Droge et al., 1994). It has been reported that glutathione can suppress apoptosis mediated by different agents, including NO, by inhibition of the mitochondrial damage (Clementi et al., 1998; Ghibelli et al., 1998). A depletion of intracellular glutathione has been demonstrated in various apoptotic systems, including exposure of the cells to NO (Van den Dobbelsteen et al., 1996; Clementi et al., 1998; Ghibelli et al., 1998) and has been considered as an early apoptotic event (Macho et al., 1997). In contrast, increased glutathione concentration may downregulate NO-induced apoptosis in smooth muscle cells (Zhao et al., 1997). An antiapoptotic effect is also provided by the inhibition of glutathione efflux via bcl-xL gene overexpression (Bojes et al., 1997) or via pretreatment with methionine (Ghibelli et al., 1998). In our experiments, glutathione depletion in Cardiolipinhigh NO-resistant cells with buthionine-sulfoximine (BSO) resulted in the stimulation of NOmediated apoptosis, whereas the exposure of parental NO-sensitive cells to the glutathione precursor N-acetylcysteine caused a substantial
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suppression of apoptotic cell death. These data suggest an important role of glutathione in the protection against NO-induced apoptosis in human leukemia cells.
D. The Bcl-2 Family Bcl-2 belongs to a growing family of proteins that can either block (Bcl-2, Bcl-xL, etc.) or promote (Bax, Bad, Bak, etc.) apoptosis. Bcl-2-related proteins are integrated in the outer mitochondrial, outer nuclear, and endoplasmic reticular membranes with the help of carboxy-terminal membrane anchor (Hockenbery et al., 1993; Kluck et al., 1997). It was demonstrated that transfection of Bcl-2 specifically prevented normal and tumor cells from apoptosis evoked by NO, which was produced endogenously after exposure to inflammatory cytokines or derived from NO donors (Xie et al., 1996, 1997b; Messmer et al., 1996a; Albine et al., 1996; Melkova et al., 1997). In contrast, expression of the proapoptotic protein Bax increased during NOmediated apoptosis, at least in macrophages and mesangial cells (Messmer et al., 1996a). Mechanisms of Bcl-2 related antiapoptotic effects at the mitochondrial level are linked with (i) inhibition of PT and stabilization of m (Marchetti et al., 1996); (ii) prevention of caspase activation and cleavage of poly (ADP-ribose) polymerase cleavage (Melkova et al., 1997); (iii) regulation of proton flux (Shimizu et al., 1998); and (iv) blocking of the proapoptotic effect of Bax protein (Antonsson et al., 1997). In addition, Bcl-2 can not only prevent the release of cytochrome c from mitochondria by inhibition of lipid peroxidation (in particular, cardiolipin) (Hockenberry et al., 1993; Tyurina et al., 1997) but can also interfere with cytochrome c already released into the cytosol (Zhivotovski et al., 1998). On the other hand, p53 accumulation remained unchanged in Bcl-2 transfected cells, which led to the conclusion that Bcl-2 acts downstream of p53 but upstream to cytochrome ¨ et al., 1999). c release (Brune In our experiments, we used Jurkat leukemia cells transfected with Bcl-2 and found that Bcl-2 overexpression resulted in a complete resistance to apoptosis in cells treated with NO donors (GTN or DETA / NONOate) even at high concentrations. Furthermore, Bcl-2 was able to block the damage of mitochondrial functions observed after exposure to NO donors. It normalized the content of cardiolipin and ROS production, m and respiratory complex activities, and prevented cytochrome c release and lipid peroxidation in mitochondria (Ushmorov et al., 1999). From our data on the effect of BA, an inhibitor of mitochondrial PT, and of trolox, an inhibitor of lipid peroxidation, we suggest that Bcl-2-induced suppression of lipid peroxidation plays a more important role in protection against NO-mediated apoptosis in human leukemia cells than the inhibition of PT.
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NO has been reported to induce apoptosis also by direct downregulation of Bcl-2 expression in target cells. Thus, treatment of murine melanoma cells with inflammatory cytokines or with NO donors led to a considerable reduction of Bcl-2 expression followed by apoptosis (Xie et al., 1996). This effect was reversed by inhibiting NO synthesis. However, short-term NO production at low concentrations has been demonstrated to suppress apoptosis in B lymphocytes (Mannick et al., 1994; Genaro et al., 1995) and in endothelial cells (Suschek et al., 1999). This protection was due to the increased expression of Bcl-2 at the mRNA and protein levels. It appears that the influence of NO on Bcl-2 expression is dose and time dependent: prolonged exposure to high-level NO results in Bcl-2 suppression, whereas short incubation with trace-level NO had the reverse effect.
E. Caspase Activation It is assumed that the activation of a cascade of cytoplasmic cysteine proteases (caspases) that specifically cleave a number of cellular substrates is essential for apoptosis, regardless of the initial death signal (Henkart, 1996; Salvesen and Dixit, 1997). Currently, the caspase family consists of 12 members which share several amino acid residues important for substrate binding and catalysis. Caspases are expressed as inactive precursors that are activated by proteolytic processing. According to the model (Cryns and Yuan, 1998), two classes of caspases, initiators and effectors, are involved in apoptosis. Apoptotic agents stimulate initiator caspases such as caspase-2, -8, and -9. This stimulation is autocatalytic and requires the binding of various specific cofactors. Activated initiator caspases process effector caspases (like caspase-3, -6, and -7) which in turn cause cell collapse by cleaving a certain set of substrates. Each initiator caspase seems to be activated only in response to particular apoptotic signals. For example, caspase-8 (FLICE) was shown to be the most upstream in the CD95 signaling pathway (Boldin et al., 1996; Medema et al., 1997). Upon triggering of CD95 receptor, the active subunits of caspase-8 are released into the cytosol, where they may stimulate effector caspases. Caspase-9, another initiator caspase, is known to be activated in the presence of other factors like Apaf 1 and ATP by cytochrome c released from mitochondria (Li et al., 1997; Kuida et al., 1998; Fearnhead et al., 1998). This leads, in turn, to cleavage and activation of caspase-3 followed by DNA fragmentation and apoptosis. It should be noted that caspase activation requires sufficient amount of ATP and could be significantly reduced by ATP depletion (Leist et al., 1997a). In this case, when energy levels are rapidly compromised, cells triggered to undergo apoptosis may instead die by necrosis.
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A series of studies demonstrated that endogenously produced or exogenously supplied NO activates caspases in connection with apoptosis in various normal (Messmer et al., 1996b; Leist et al., 1997b; Melkova et al., ¨ et al., 1999) and tumor cells (Yabuki et al., 1997; Sandau et al., 1998; Brune 1997; Chlichlia et al., 1998; Ushmorov et al., 1999; Umansky et al., 2000a). Importantly, NO-induced caspase-8 activation in human leukemia cells and caspase-3, -6, and -9 activation in human breast cancer cells was completely blocked by zVAD, a broad-spectrum caspase inhibitor. This enzyme inhibition was associated with complete inhibition of NO-mediated apoptosis in these cells (Chlichlia et al., 1998; Umansky et al., 2000a). Cytochrome c accumulation in the cytosol of NO-treated Jurkat leukemia cells was observed before caspase activation (Ushmorov et al., 1999). This suggests that NO-mediated cytochrome c release causes the activation of the caspase cascade followed by apoptosis, as was already shown for CD95 and other apoptotic death signals (Yang et al., 1997; Scaffidi et al., 1998). It has recently been reported by several groups that NO is able not only to increase but also to inhibit caspase activity by S-nitrosylation or oxidation of the catalytically reactive cysteine moiety (Dimmeler et al., 1997; Mohr et al., 1997; Li et al., 1999). However, most of these studies have been performed in cell extracts or with purified proteins. Under cellular conditions (normal hepatocytes) (Li et al., 1999), the concentration of NO suppressing TNF-mediated apoptosis via reduction of caspase activity was much lower than in experiments showing NO-mediated caspase activation. Again, as in the case of Bcl-2, the effect of NO seems to be strictly dependent on its concentration, on the time of NO exposure, and eventually on the type of target cells.
IV. CONCLUDING REMARKS A large body of evidence shows that NO either produced by activated host cells or delivered exogenously by NO donors induces apoptotic cell death in murine and human tumor cells in vitro and in vivo. High concentrations of NO can be produced for a long time in localized “hot spots” by activated iNOS expressed in macrophages within the tumors, endothelial cells in the tumor microvasculature, or in peripheral blood monocytes. The mechanism of NO-mediated apoptosis in tumor cells involves accumulation of the tumor suppressor protein p53, mitochondrial damage (including cytochrome c release, a downregulation of respiratory complex activity, lipid degradation, and glutathione depletion), alterations in the expression of members of Bcl-2 family, followed by activation of the caspase cascade and DNA fragmentation. Thus, stimulation of iNOS in host cells of the tumor
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microenvironment with appropriate inflammatory cytokines could be of importance for effective treatment of cancer patients by induction of apoptosis in tumor lesions and in circulating tumor cells. In addition, NO donors might be promising new agents in therapeutic protocols. However, NO produced in the tumor-bearing host can act as a doubleedged sword against the tumor or the host, depending on the circumstances. For example, NO is able not only to mediate apoptosis but also to protect target cells from apoptosis induced by other agents or to exert regulatory functions. Moreover, NO produced by tumor cells is often conducive to tumor progression and metastasis and is thus detrimental to the host. It appears that the mode of NO effect is dependent on the type of target cells as well as on the magnitude and duration of NO production. Prolonged exposure to high-level NO may result in apoptotic cell death, whereas short incubation with low-level NO may cause a cell protective effect.
ACKNOWLEDGMENTS The authors thank the members of our research group, collaborators, and colleagues who have contributed to the research described in this review. This work was supported in part by Grant No. 10-0980-Schi2 from the Dr. Mildred Scheel Stiftung and by the D. Hopp Stiftung.
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Detection of Minimal Residual Disease Gottfried Dolken ¨ Department of Hematology and Oncology, Clinic for Internal Medicine C Errnst-Moritz-Arndt-University Greifswald D-17487 Greifswald, Germany
I. Introduction II. Technical Aspects of the Detection of Minimal Residual Disease A. Detection of MRD by Morphological and Immunological Techniques B. Detection of MRD by Cytogenetic and Molecular Techniques C. Detection of MRD by in Vitro Amplification of Specific DNA and RNA Sequences III. MRD in Leukemia A. Philadelphia Chromosome (Ph1 )-Positive Chronic Myelogenous Leukemia B. Acute Lymphoblastic Leukemia C. Acute Myeloblastic Leukemia (AML) IV. MRD in Lymphoma A. t(14;18)-Positive Follicular Lymphoma B. Mantle Cell Lymphoma (MCL) V. MRD in Solid Tumors A. Colorectal Carcinoma VI. Concluding Remarks References
A high percentage of patients with leukemia, lymphoma, and solid tumors achieve a complete clinical remission after initial treatment, but the majority of these patients will finally relapse from residual tumor cells detectable in clinical remission only by the most sensitive methods. The in vitro amplification of tumor-specific DNA or RNA sequences by polymerase chain reaction (PCR) allows identification of a few neoplastic cells in 104 to 106 normal cells. Depending on the underlying malignant disease and therapeutic treatment, the presence of residual tumor cells in an individual patient may herald relapse, but a long-term stable situation or slowly vanishing tumor cells are also possible. Molecular monitoring of residual leukemia and lymphoma cells by quantitative PCR techniques has provided important information about the effectiveness of treatment and the risk of recurrent disease as shown by minimal residual disease (MRD) analysis in patients with various malignant diseases. Such diseases include childhood acute lymphoblastic leukemia, after induction therapy; acute promyelocytic leukemia, during and after chemotherapy; and chronic myelogenous leukemia, during treatment with ␣-interferon and after allogeneic bone marrow transplantation. Evaluation of the
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predictive value of the detection of MRD has to take into account its evolution and course, the pathogenesis, biology, and natural course of the underlying malignant disease, the molecular genetic lesion, and finally, the type of treatment. Quantification of minimal residual cells by the recently developed real-time quantitative PCR technique will surely have a major impact on our therapeutic strategies for patients with leukemia, lymphomas, and solid tumors. Based on quantitative PCR data, the terms molecular remission and molecular relapse have to be exactly defined and validated in prospective clinical trials to assess the biological and clinical significance of MRD in various types of malignancies. C 2001 Academic Press.
I. INTRODUCTION The detection of malignant cells by morphological, immunological, cytogenetic, and molecular techniques is not only important for an accurate staging at diagnosis but also for monitoring response to therapeutic interventions. Although a high percentage of patients with leukemia, lymphoma, and solid tumors achieve a complete clinical remission after initial treatment, the majority of these patients will finally relapse and die from recurrent disease. It is generally believed that these relapses are due to minimal residual tumor cells persisting undetectable by standard diagnostic procedures. The detection of chromosomal translocations by cytogenetics followed by the identification of the genes involved, and their breakpoints, have given new insights into the pathogenesis of malignant tumors and provided the basis for the development of very sensitive molecular techniques to detect minimal residual tumor cells (Rabbitts, 1994; Look, 1997). The in vitro amplification of DNA or RNA by polymerase chain reaction (PCR) allows identification of a few neoplastic cells in 104 to 106 normal cells. When in complete clinical remission—defined by the absence of malignant disease detectable by clinical, radiological, standard cytological, and histological means—malignant cells can only be found by very sensitive molecular or immunological techniques, the situation is called minimal residual disease (MRD). MRD describes the lowest level of disease detectable in patients in complete clinical remission, by the most sensitive methods available. With regard to the high probability of relapse in most tumor patients and the high incidence of MRD as detected by PCR, one might assume that all patients with MRD should receive additional treatment to eradicate these minimal numbers of tumor cells. Unfortunately, this is not justified. Depending on the malignant disease and therapeutic treatment, the presence of residual tumor cells in an individual patient may herald relapse, but it could also indicate a long-term stable situation or slowly vanishing tumor cells. Therefore, the terms molecular remission and molecular relapse have been introduced. By applying these terms, it is necessary to define and state the sensitivity of the detection method in any single assay. This can easily be
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accomplished in the case of quantitative PCR assays, since only these tests fulfill most of the essential criteria. The evaluation of MRD has been complicated by the finding that by very sensitive molecular techniques so-called primary chromosomal aberrations as well as clone-specific chromosomal rearrangements thought to be specific for malignant cells have been found in healthy individuals as well as in patients in long-term remission. The evaluation of the predictive value of the detection of MRD in a single patient has to take into account its evolution and course, the pathogenesis, biology and natural course of the underlying malignant disease, the molecular genetic lesion, and finally, the type of treatment.
II. TECHNICAL ASPECTS OF THE DETECTION OF MINIMAL RESIDUAL DISEASE A. Detection of MRD by Morphological and Immunological Techniques Depending on the malignant disease, a variety of techniques have been used to detect MRD (Table I). In leukemia and lymphoma, the detection limit of morphological (cytological, cytochemical, and histological) techniques is 5% malignant cells; in the case of solid tumors, it may be lower. By standard immunophenotypic analysis, 1–5% malignant cells can be detected, but it is possible to increase the specificity and sensitivity to 0.1–0.01% by using combinations of monoclonal antibodies and multicolor flow cytometry. Tumor cells stained with monoclonal antibodies can be detected at a high sensitivity in the presence of large numbers of normal cells, but unfortunately all immunological techniques are hampered by the fact that most monoclonal antibodies used are not truly tumor specific, with only a few exceptions (e.g., antibodies specific for new tumor-associated fusion proteins). Therefore, the identification of tumor cells is mainly based on the detection of differentiation antigens or antigen combinations that are preferentially found on tumor cells but rarely on surrounding normal cells, since normal circulating blood cells or bone marrow cells do not regularly express mRNAs and proteins typically found in epithelial cells (e.g., cytokeratins or tyrosinase) (for review, see Ghossein et al., 1999).
B. Detection of MRD by Cytogenetic and Molecular Techniques Using standard karyotypic analysis, chromosomal abnormalities have been found in 50–70% of hematological malignancies (Rowley and Testa, 1982;
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Table I Sensitivity of Different Techniques Used to Detect MRD
Method Cytological, cytochemical, or histological analysis Immunophenotyping by flow cytometry (FACS) Immunophenotyping on cytospins, immunohistochemistry Southern blot analysis Standard cytogenetics
Sensitivity (%) 5
1–5 (0.1) 1–5 (0.01)
1
Positive cells in total cells 5 in 100
Standard clinical procedure, low sensitivity
1–5 in 100 (to 1,000) 1–5 in 100 (to 10,000)
Lack of tumor-specific antigens and antibodies Lack of tumor-specific antigens and antibodies
1 in 100
Time consuming, laborious, low sensitivity Labor intensive, high quality metaphases Interphase FISH: false positive results, labor intensive, no need for metaphases
5–10
5–10 in 100
5–10 1–3 0.01–0.0001
5–10 in 100 1–3 in 100 1 in 104 to 106
Fluorescence in situ hybridization (FISH) —S-FISH —D-FISH Polymerase chain reaction (PCR)
PCR combined with stochastic analysis
0.00001
Features
1 in 107
mRNA: total amount of RNA used for cDNA synthesis, and final PCR is important for sensitivity DNA: 1–10 g DNA can be tested; for ≥ 2 g DNA, 2-step PCR is recommended; false-positive results due to contamination or carry-over are a serious problem
Yunis et al., 1982). The sensitivity is low (5%), and suitable metaphases must be present in adequate numbers. Fluorescence in situ hybridization (FISH) can be carried out on interphase nuclei, with a sensitivity of 5–10%. Doublecolor FISH (D-FISH) on interphase nuclei and hypermetaphase FISH seem to be about 10 times more sensitive. The most sensitive techniques for the detection of tumor cells are based on the finding of nonrandom chromosomal translocations associated with well-defined malignancies, identification of the genes adjacent to the chromosomal breakpoints, and nucleotide sequence analysis of these genes and their mRNA expression. The molecular consequence of these translocations, new tumor-specific fusion genes and mRNA transcripts, can be used to diagnose clinically important subtypes of leukemia and lymphoma as well as to specifically detect minimal residual tumor cells (Rabbitts, 1998).
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The second type of clonal genetic rearrangements used for the detection of MRD are intrachromosomal rearrangements of immune receptor genes of B and T lymphocytes that can serve as clone-specific markers in lymphoid leukemias and lymphomas (Arnold et al., 1983; Cleary et al., 1984; Bertness et al., 1985). Since most B and T cell malignancies originate from immune cells that have already undergone their physiologic intrachromosomal gene recombinations that occur during B and T cell differentiation, all cells belonging to this malignant clone carry the same receptor gene rearrangement as a unique clonal marker. Nonrandom chromosomal translocations and clonal receptor gene rearrangements can be detected by DNA restriction fragment length polymorphism (RFLP) analysis combined with Southern blot hybridization using radiolabeled DNA probes. To be detectable by this technique, at least 1% of the cells under investigation must be clonal cells (Cleary et al., 1984). The analysis of peripheral blood and bone marrow cells from patients with leukemia and lymphomas has shown the presence of minimal residual neoplastic cells in the majority of patients in complete clinical remission (Arnold et al., 1983). The use of this technique in clinical studies on large numbers of patient samples is hampered by low sensitivity, it is a time-consuming laborious procedure, and there is the restriction that only a limited number of tests can be carried out simultaneously.
C. Detection of MRD by in Vitro Amplification of Specific DNA and RNA Sequences Development of PCR has solved most of these problems (Saiki et al., 1985). This technique has revolutionized basic and clinical research after further major improvements have been introduced: r automatization of thermal cycling r use of heat-resistant Taq DNA polymerase cloned from the hot springs bacterium Thermus aquaticus (Saiki et al., 1988) r reamplification of a small aliquot of a first-round PCR with a second set of internal primers by so-called two-step nested PCR techniques (Mullis and Faloona, 1987) r use of dUTP and uracil-N-glycosylase (UNG) to control contamination due to carryover of previously amplified DNA (Longo et al., 1990) r hot start techniques to improve the specificity (Chou et al., 1992; Birch et al., 1996) r and the very recent development of real-time quantitative PCR (Heid et al., 1996; Gibson et al., 1996) In the past, quantitative PCR techniques have not been used as often, because they were thought to be only semiquantitative or because they are very laborious with a high risk of false-positive results due to contamination
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or carryover or previously amplified DNA. These quantitative PCR techniques have been (1) limiting dilution assays combined with single round or ¨ two-step PCR (Sykes et al., 1992; Brisco et al., 1994; Dolken et al., 1996), (2) competitive PCR techniques based on the observation that the amount of added standard increases as the native signal decreases (Gilliland et al., 1990), and (3) noncompetitive PCR techniques that use the quantification of two products synthesized in parallel under conditions when the native signal is not altered by the standard (Freeman et al., 1999). Real-time quantitative PCR is based on a kinetic cycle by cycle quantification using target gene-specific fluorogenic probes (Holland et al., 1991; Lee et al., 1993; Heid et al., 1996; Gibson et al., 1996). To utilize all advantages of this technique, an integrated system for thermal cycling, real-time fluorescence detection in closed tubes, and subsequent computerized analysis is necessary. Double-labeled fluorogenic probes (TaqManTM) are oligonucleotides with a fluorescent 5′ -reporter dye and a 3′ -quencher dye. If the probe is intact and flexible, the reporter fluorescence is quenched by the 3′ -quencher dye due to fluorescence energy transfer. When during PCR amplification, probes specifically anneal to the homologous target sequences between the two primer sites, any bound fluorogenic probe is hydrolyzed by the the 5′ -nuclease activity of Taq DNA polymerase, and the 5′ -reporter dye is released, yielding an unquenched signal. After cleavage, the shortened probe dissociates from the complementary strand, and DNA synthesis can continue without any interference. This process is repeated during all cycles, resulting in an increase of free reporter molecules proportional to the numbers of target sequences amplified. The increase in fluorescence intensity can be followed in real-time, based on the normalized fluorescence signal (Fig. 1A). The system generates an amplification plot: product synthesized −−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−→ Fig. 1 (A) Detection of the t(8;21)/AML1-ETO fusion transcript by real-time quantitative PCR. The amplified AML1-ETO cDNA fragment was cloned, and defined copy numbers based on a spectrophotometric determination of the DNA content of a plasmid preparation were amplified by PCR in the presence of a double labeled fluorogenic probe. The increase in fluorescence intensity was followed up in real-time based upon the normalized fluorescence signal. The background is determined during the first 3–15 cycles. When after further amplification the fluorescence intensity measured in some reaction vials has reached a value above background, these time points or fractional cycle numbers (CT = threshold cycle) are determined for the various samples with increasing copy numbers of AML1-ETO. (B) Determination of a standard curve for a quantitative analysis of unknown samples. The fractional cycle numbers (CT = threshold cycle) determined by the system are proportional to the initial number of target copies in the different samples. This relation is expressed as CT = A log (N)+B, in which N is the initial copy number, A the slope of the curve, and B the Y-intercept or the CT of one initial copy. The standard curve shown has been corrected with regard to the initially spectrophotometrically determined DNA content by a stochastic analysis using limiting dilution assays of the AML1-ETO plasmid DNA in combination with real-time PCR. AML1-ETO mRNA transcripts are then quantitatively determined in patient samples using this standard curve and a limited number of positive controls tested in each separate experiment.
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versus cycle number (time). Subsequently, for any positive sample, the time point (cycle number) when the fluorescence intensity has reached a detectable value above the background determined during the first 3–15 cycles is calculated by the computer. This time point is a fractional cycle number (CT = threshold cycle) and is proportional to the initial number of target copies in the sample (Fig. 1B). The amplification has to be carefully optimized with regard to Mg++ concentration, annealing temperature, and primer and probe sequences, as well as their concentrations, to achieve an amplification efficiency as close as possible to 100%. When this is the case, a perfect correlation is found between initial copy numbers (5–10 up to 105) and the CT values (Fig. 1B). These assays are highly reproducible, the risk of contamination is greatly reduced, since there is no need for pipetting amplified products, and the sensitivity of single-round real-time PCR is at least as high as that of two-step nested PCR, since both techniques seem to detect even a ¨ single copy, based on stochastic analyses (Dolken et al., 1998). When DNA is used as the target, the simultaneous amplification of the gene of interest and a control gene, e.g., -actin or wild-type K-ras, provides information about the maximum sensitivity of the assay, expressed in copy numbers of the control gene or total numbers of cells analyzed in a single PCR reaction. In the case of mRNA targets, it is not so easy to establish a correlation between mRNA copy numbers and cell numbers. Therefore, “relative” quantifications (Freeman et al., 1999) of the mRNA transcript of interest in relation to the mRNA of a housekeeping gene, like porphobilinogen deaminase (PBGD) (Finke et al., 1993a; Mensink et al., 1998) or ABL (van Rhee et al., 1995; Hochhaus et al., 1996a), are carried out. When samples should be analyzed for MRD at the highest available sensitivity, control genes expressed at a much higher rate than the gene of interest, and genes for which highly homologous pseudogenes exist (e.g., GAPDH or ALD), should be avoided (Finke et al., 1993a). If pseudogenes are present, control reactions have to be carried out after DNase digestion, which could reduce the overall sensitivity. The sensitivity of most PCR assays carried out with genomic DNA is one positive cell in 105–6 normal cells, and it can reach one in 107 normal cells if ¨ stochastic analyses are included (Liu et al., 1994; Dolken et al., 1996). Using real-time quantitative PCR, the inhibitory effects of DNA and RNA/cDNA preparations can be quantitatively assessed for the first time: purified high molecular weight DNA in amounts ≥ 2 g—corresponding to about 280,000 cells—in 50 l reaction volume of a single-round PCR start to increasingly ¨ inhibit PCR amplification (Dolken et al., 1998). If the estimation of the DNA content of a DNA preparation isolated by centrifugation methods—not by spooling—is based on OD measurements, quite large errors can be introduced due to degradation of DNA; in worst cases, deviations by a factor of 10–100 have been observed. By applying real-time quantitative PCR, it
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is possible now to determine very accurately the total amount of genomic DNA used in a single assay, which corresponds to a certain number of cells and in turn indicates the sensitivity of the single assay. In RT-PCR experiments, cDNA synthesized in the presence of high KCl concentrations (>50 mM) is able to strongly inhibit single-round PCR amplification when ≥5% of the cDNA mixture is added to the final PCR reaction ¨ (Schuler et al., 1998, 2000). When 1 g total RNA isolated from 105 cells is transcribed into cDNA, and 5–10% of this cDNA mixture is subjected to PCR amplification in a single assay, the resulting highest sensitivity of a single RT-PCR reaction is one positive cell in 0.5–1 × 104 normal cells. This sensitivity can be increased only by repetitive testing combined with a stochastic analysis or by extracting more RNA from a larger number of cells and using specific antisense primers for cDNA synthesis. The sensitivity of the PCR technique is one of the most important factors when evaluating MRD in serial samples of single patients as well as large groups of patients in clinical studies, aiming at a comparative analysis with results of other trials. PCR is an extremely sensitive method, but there are many technical limitations that could lead to false-negative and false-positive results (Table II). Based on recent developments, at least some technical problems have already Table II Detection of MRD: Limitations of the PCR Technology with Regard to Target Cells and Target Molecules, DNA, or mRNA I. False-positive results due to —Carry-over of previously amplified DNA (DNA, RNA) —Contamination with foreign cells due to invasive procedures during the process of sample collection (DNA, RNA) —Sample contamination with foreign cells or nucleic acids in the laboratory (DNA, RNA) —Illegitimate transcription (RNA) —Pseudogenes (RNA) II. False-negative results due to —Problems associated with sample collection due to an intermittent shedding of cells into the blood stream or a patchy distribution of leukemia or lymphoma cell infiltrates in the bone marrow (DNA, RNA) —Degradation of nucleic acids during purification and storage (DNA, RNA) —Variable PCR sensitivity (DNA, RNA) —Polymorphic sites at primer binding sites (DNA, RNA) —Inhibitors of amplification (DNA, RNA) —Technical errors (DNA, RNA) —Tumor cell heterogeneity, oligoclonality, subclones (DNA, RNA) —Clonal evolution (DNA, RNA) —Variations in transcription rates (RNA) —Downregulation of the transcription of target genes (RNA) —Heterogeneity of fusion transcripts (RNA)
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been eliminated or can be controlled, such as carryover of previously amplified DNA and the presence of inhibitors of amplification. But many difficulties and limitations still exist and have to be controlled—most of them are discussed later in this review, when the results of MRD detection in various diseases with specific DNA and RT-PCR assays are described. For the detection of MRD, micrometastasis, or occult tumor cells in leukemia, lymphoma, and solid tumors, PCR amplification assays for tumor-specific chromosomal aberrations, clonal rearrangements, tumor cell-specific gene mutations, or deletions and tissue-specific mRNA expression have been extensively used (Table III). By far the most studied chromosomal translocations are t(9;22) in patients with Philadelphia chromosome-positive chronic myelogenous leukemia (CML) and t(14;18) in patients with follicular lymphoma. In solid tumors, most experience has been gathered with RT-PCR for CEA
Table III DNA-PCR and RT-PCR Targets for the Detection of MRD in Leukemia and Lymphoma as Well as for the Detection of Micrometastasis or Occult Tumor Cells in Patients with Solid Tumors I. Tumor-specific chromosomal aberrations (fusion genes) Chronic myelogenous leukemia (CML) t(9;22)(q34.1;q11.2) Acute lymphoblastic leukemia (ALL) t(9;22)(q34.1;q11.2) t(1;19)(p23;p13) t(17;19)(q23;p13) Acute myeloblastic leukemia (AML) t(8;21)(q22;q22) inv(16)(p13q22) t(16;16)(p13;q23) t(15;17)(q22;q12) Follicular lymphoma (FL) t(14;18)(q32;q21) Mantle cell lymphoma (MCL) t(11;14)(q13;q32) Ewing’s sarcoma (ES) and primitive t(11;22)(q24;q12) neuroectodermal tumor (PNET) t(21;22)(q22;q12) II. Clonal rearrangements B-cell leukemia/lymphoma T-cell leukemia/lymphoma III. Tumor-specific gene mutations Gastrointestinal carcinomas
BCR-ABL BCR-ABL E2A-PBX1 E2A/HFL AML1-ETO CBF-MYH11
RT-PCR RT-PCR RT-PCR RT-PCR RT-PCR RT-PCR
PML-RARa BCL-2/IgH BCL-1/IgH EWS/FLI1 EWS/ERG
RT-PCR DNA-PCR DNA-PCR RT-PCR RT-PCR
IgH-CDR-3 rearrangements TCR-rearrangements
DNA-PCR DNA-PCR
K-ras mutations p53 mutations, deletions
DNA-PCR
IV. Tissue-specific gene expression (mRNA) Gastrointestinal carcinomas, Carcinoembryonic antigen (CEA), breast cancer cytokeratin 20 Hepatocellular carcinoma ␣1-fetoprotein (AFP), albumin Melanoma Tyrosinase Prostate carcinoma Prostate-specific antigen (PSA)
RT-PCR RT-PCR RT-PCR RT-PCR
Note. Most of the proteins encoded by the fusion genes listed here, and the so-called tissue-specific genes, can also be detected by monoclonal antibodies.
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and cytokeratin 20 as epithelial cell markers, and K-ras mutations or p53 mutations/deletions as tumor-specific genetic alterations in gastrointestinal cancer.
III. MRD IN LEUKEMIA A. Philadelphia Chromosome (Ph1 )-Positive Chronic Myelogenous Leukemia The Philadelphia chromosome translocation (t(9;22)(q34.1;q11.21) can be found in about 95% of patients with CML (Nowell and Hungerford, 1960; Rowley, 1973). Reciprocal translocation of the long arms of chromosome 9 and 22 results in a new fusion gene consisting of 5′ -BCR (22q11) and 3′ -ABL (9q34) sequences (de Klein et al., 1982; Heisterkamp et al., 1985; Shtivelman et al., 1985). The breakpoints in the ABL gene usually occur in the first ABL intron of about 200 kb (Fig. 2). The 5′ part of ABL is translocated into the 5.8-kb major breakpoint cluster region (M-bcr) of the BCR gene on chromosome 22 between exons b2 and b4. The resulting chimeric BCR– ABL genes are transcribed into 8.5-kb mRNAs, showing either a b2a2 or a b3a2 junction (Stam et al., 1985; Shtivelman et al., 1986). This hybrid mRNA is translated into a fusion protein of 210 kDa, p210BCR-ABL. Usually one, but also both types of mRNA can be expressed in the same patient, due to alternative splicing. About 30% of adult patients with Ph1-positive acute lymphoblastic leukemia (ALL) show a BCR–ABL joining at the mRNA level, similar to that in CML (de Klein et al., 1986). In the remaining cases, the breakpoints are located in the first intron of the BCR gene within the minor breakpoint cluster region (m-bcr) (Fainstein et al., 1987). This leads to a fusion mRNA of 7.0 kb; the first BCR exon (e1) is spliced to the second ABL exon (a2), resulting in an e1a2 joining and a hybrid protein of 190-kDa, p190BCR-ABL (Kurzrock et al., 1987). Rare fusion mRNAs are those with an e19a2 junction (p230BCR-ABL) resulting from a breakpoint in -bcr and associated with neutrophilic CML (Saglio et al., 1990; Pane et al., 1996), b2a3 or b3a3 (p203BCR-ABL) (Van der Plas et al., 1991), e1a3 (Soekarman et al., 1990), e2a2 (Leibundgut et al., 1999), e6a2 (Hochhaus et al., 1996b), e13a2 and e8a2 (How et al., 1999). Some fusion transcripts may be generated by alternative splicing; others are due to different locations of breakpoints. All fusion mRNAs can be detected by RT-PCR using different sets of primers (Fig. 2). Cytogenetic analysis is the standard diagnostic procedure for CML. The Ph1 chromosome can be found in 90% of patients, and in a further 5%, the BCR–ABL translocation can be detected by Southern or Northern blot hybridization or by RT-PCR. In the past, standard cytogenetic analysis has been
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Fig. 2 Schematic representation of the BCR gene on chromosome 22, the ABL gene on chromosome 9, and their heterogeneous BCR–ABL fusion transcripts due to the Philadelphia chromosome translocation t(9;22). The horizontal lines indicate the genomic DNA sequences, the boxes represent the positions of the exons(a = ABL exons; e, b, or c = BCR exons). The breakpoint regions are indicated by arrows. The breakpoints in the ABL gene are located in a large region of about 250 kb 5′ of exon a2. Three breakpoint cluster regions have been identified in the BCR gene. The minor breakpoint cluster region (m-bcr) is located within the first large intron (65 kb) of the BCR gene and associated with Ph1-positive ALL, the transcripts mainly code for a protein p190BCR-ABL. The major breakpoint cluster region (M-bcr) is located more 3′ between bcr exons e13 and e15 (b2 and b4). The resulting b2a2 and b3a2 mRNAs and the encoded protein p210BCR-ABL are mainly found in Ph1-positive CML. In about 10%, exon a2 is not expressed, leading to b2a3, b3a3, and e1a3 mRNA transcripts. Different locations of breakpoints or alternative splicing may also lead to e6a2, e8a2, e13a2, and e18a2 fusion transcripts. A rare product is p230BCR-ABL which results from breakpoints between exons e19 and e20 (-bcr). This type of transcript (e19a2) seems to be associated with a more benign course of CML with higher neutrophilic count and an initial thrombocytosis. The sizes and distances are not to scale.
used for monitoring CML patients following various kinds of treatments, to establish remission criteria. But the procedure is laborious, time consuming, and of low sensitivity due to the limited number of metaphases (20–25) investigated per sample (Talpaz et al., 1986). Interphase FISH using one fluorescent probe (S-FISH) on peripheral blood cells can be used for monitoring, but it needs at least about 10% Ph1-positive cells because of false-positive find¨ ings (Muhlmann et al., 1998). Interphase FISH using double-color probes
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(D-FISH) is easy to perform on a large number of peripheral blood cells (500–5000 cells), and cytogenetic responses can easily be assessed with a false-positive rate in the range of 0.1–2.0% (Dewald et al., 1998; Buno et al., 1998). Hypermetaphase FISH allows analysis of up to 500 cells in metaphase, but it cannot be applied to peripheral blood samples and very often only insufficient numbers of metaphases can be obtained with bone marrow samples from patients treated with interferon-␣ (Seong et al., 1995, 1997). Because of its high sensitivity and easy and rapid performance, RT-PCR has been extensively used for monitoring MRD in CML. Conventional chemotherapy with hydroxyurea or busulfan rarely leads to hematological remissions or a suppression of the Ph1 clone, and the patients will inevitably progress to blast crisis. Interferon-␣ was the first biological agent that produced cytogenetic remissions in patients with CML. Hematological remissions and major cytogenetic responses (< 35 % Ph1 metaphases in bone marrow samples) were observed in 70–80% of the patients, and there were complete cytogenetic responses (CCR) in 15–20% (Talpaz et al., 1991). Only very few patients in CCR were found to be also negative by standard qualitative one-step or two-step RT-PCR (Lee et al., 1989; Opalka et al., 1991; Dhingra et al., 1992; Kurzrock et al., 1998). These assays indicate only the presence or absence of BCR–ABL transcripts. By applying quantitative PCR analyses, it has been shown that BCR/ABL transcript levels decrease in peripheral blood leukocytes and bone marrow mononuclear cells of interferon-␣ responders only, and that they are rarely if ever completely eliminated (Hochhaus et al., 1996a, 2000). The levels of BCR–ABL mRNA transcripts vary to a large extent even in complete cytogenetic remission (Malinge et al., 1992; Hochhaus et al., 1996a), but low levels of residual transcripts found in serial tests correlate with a high probability of remaining in remission (Hochhaus et al., 2000). Increases in transcript levels before hematological relapse do not necessarily reflect increasing cell numbers, but they may also represent increasing steady-state levels in malignant cells, heralding disease progression toward blast crisis (Gaiger et al., 1995). These results suggest that quantitative molecular monitoring of leukemic cells might be helpful to individualize the therapeutic strategy for patients on interferon-␣ treatment (Cross, 1998; Faderl et al., 1999; Goldman et al., 1999; Lion, 1999). Many studies have been carried out to prospectively analyze the kinetics of disappearance and reappearance of Ph1-positive cells after allogeneic bone marrow transplantation (BMT). There is convincing evidence that persistent negative results obtained by a qualitative two-step nested PCR indicate a high probability for maintaining a stable remission, since < 5% relapses were found in these patients (Roth et al., 1992; Miyamura et al., 1993; Radich et al., 1995). The presence of Ph1-positive cells detectable by
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two-step nested PCR during the first 3–6 months after allogeneic BMT was not associated with a higher rate of recurrence, compared to negative patients (Hughes et al., 1991; Radich et al., 1995). In a study on 346 CML patients, PCR-positivity during a period of 6–12 months after allogeneic related BMT was associated with a high risk of relapse (42%), whereas the risk in negative patients was only 3% (Radich et al., 1995). The predictive value of persistent PCR-positivity for relapse was almost lost when patients in clinical remission remained PCR-positive for more than 36 months. In comparison, the risk of relapse in patients being PCR-positive 6–12 months after allogeneic unrelated BMT was only 18%. A high rate of PCR-positivity (80%) was found 6–24 months after T-cell depleted allogeneic BMT, in correlation with a high rate of relapse. Only 26–30% of the patients transplanted with unmanipulated allogeneic bone marrow were found to be PCR-positive, and the relapse rate was significantly lower (Pichert et al., 1995). These results suggest that residual malignant cells in CML patients transplanted with manipulated or untreated allografts can be eliminated by a graft-versus-leukemia response (GvL) due to competent T cells. Quantitative competitive RT-PCR techniques for the detection of BCR– ABL mRNA have been introduced by Lion et al. (1992) and Thompson et al. (1992). In a large clinical study, it has been shown that BCR–ABL transcripts increase in number during progression from complete cytogenetic remission to cytogenetic relapse and finally hematological relapse (Cross et al., 1993; Cross, 1998). Serial quantitative monitoring of residual disease after allogeneic BMT seems to provide more information than qualitative PCR, since undetectable, decreasing, or low levels of BCR–ABL transcripts (< 50/g RNA) correlated with a low probability of relapse (1/69 patients) after allogeneic BMT (Lin et al., 1996). Increasing or persistent high numbers of BCR–ABL transcripts (> 50/g RNA) correlated with a high incidence of relapse (21/29) and were detected several months prior to cytogenetic and clinical relapse. A shorter BCR–ABL transcript doubling time before relapse seems to indicate the development of a more aggressive disease to be recognized at clinical relapse later. In conclusion, the kinetics of decreasing or increasing BCR–ABL transcripts are important for predicting relapse or continuing remission. After allogeneic BMT, the donor immune system plays an important role in controlling recipient Ph1-positive cells, either leading to an eradication or keeping them in a dormant state (Uhr et al., 1997). The combined analysis of T-cell chimerism and MRD after T-cell depleted allogeneic BMT showed that mixed T-cell chimerism detected after a median follow-up of 12 months was associated with a high rate of residual Ph1positive cells (18/22) and relapse (9/22), whereas only 3/10 patients with full donor chimerism had residual Ph1-positive cells and only one patient relapsed (Mackinnon et al., 1994). Relapses after allogeneic BMT can be successfully
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treated with donor lymphocyte infusions in 60–80% of cases (Kolb et al., 1990; van Rhee et al., 1994). These effects are mediated by T cells reacting against CD34-positive CML progenitor cells (Smit et al., 1998). The analysis of mixed chimerism of CD34-positive progenitor cells and peripheral T-cells has shown that changes toward donor phenotype provide the earliest molecular evidence of a subsequent molecular response (Baurmann et al., 1998; Gardiner et al., 1998). In addition, the molecular criteria “BCR-ABL/ABL mRNA ratio >0.02% or >100 BCR–ABL transcripts /g RNA in two sequential samples obtained later than four months after BMT” (Hochhaus et al., 1996a; Lin et al., 1996) have been used to define molecular relapse based on quantitative PCR results. The detection of molecular relapse in CML after allogeneic BMT seems to be important, since donor lymphocyte infusions are much more effective when given as early as possible (van Rhee et al., 1994; Raanani et al., 1997). In a recent study, a characteristic evolution pattern has been described for relapsing patients: persisting p210BCR-ABL positivity, increasing mixed chimerism in myeloid cells, p190BCR-ABL positivity, and finally, cytogenetic relapse (Serrano et al., 2000). In two patients successfully treated with donor lymphocyte infusions, the exact inverse pattern was observed. In CML patients, the treatment with interferon-␣, allogeneic BMT, donor lymphocyte infusions, and most recently, tyrosine kinase inhibitors (Druker et al., 1996) should be monitored at the molecular level by quantitative PCR. Based on these data, it seems to be possible in the near future to individualize treatment strategies. The kinetics are important not merely as qualitative data, since a possible “cure” from CML as a malignant disease does not necessarily mean “PCR-negativity” (Faderl et al., 1999). Real-time quantitative PCR combined with a standardized purification procedure of RNA seems to be the method of choice for molecular monitoring (Mensink et al., 1998; Emig et al., 1999; Preudhomme et al., 1999; Branford et al., 1999). But there are still some limitations due to false-negative and false-positive PCR results. Primitive CML progenitors as well as nonproliferating Ph1positive cells do not necessarily express BCR–ABL mRNA or the encoded protein (Bedi et al., 1993; Keating et al., 1994; Chomel et al., 2000). Such cells cannot be detected by RT-PCR, but they could possibly be identified by FISH if present in appropriate numbers or by amplification of long targets of human DNA (5–20 kb) by applying long-template DNA–PCR (Waller et al., 1999). Furthermore, the detection of residual leukemic cells by RT-PCR may be complicated by the heterogeneity of BCR–ABL mRNA transcripts (Fig. 2) due to variable breakpoints and alternative splicing, since a b3a2positive progenitor cells may variably express b2a2 and e1a2 as well as b3a2 (Shtivelman et al., 1986; van Rhee et al., 1996). False-positive results due to the detection of BCR–ABL mRNA transcripts as in healthy individuals are
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rather unlikely, since these results were obtained by PCR techniques 10–100 times more sensitive than standard 2-step nested PCR (Biernaux et al., 1995; Bose et al., 1998).
B. Acute Lymphoblastic Leukemia 1. STANDARD RISK ALL Hematological remission in patients with acute leukemia is defined as less than 5% morphologically identifiable leukemic blasts in the bone marrow. Theoretically, this could still mean a total leukemic cell burden of up to 1010 leukemic cells. Therefore, all therapeutic strategies aim at reducing the remaining residual leukemic cells as far as possible to achieve a cure. The detection and quantification of minimal residual leukemic cells during and after chemotherapy for acute leukemia may be a helpful guide for controlling established chemotherapeutic protocols and for developing new strategies. Acute lymphoblastic leukemia of childhood is a disease very sensitive to chemotherapy. The treatment strategies have been elaborated so well that virtually all patients achieve a remission, and about 70% of all children with ALL are eventually cured (Brisco et al., 1994). Treatment results in adult ALL are not as good; the overall cure rate is in the range of 50% (Hoelzer, 1994). In patients with standard risk B-cell lineage ALL—characterized by the absence of chromosomal aberrations associated with a high risk, such as t(9;22), t(1;19), t(17;19) or t(4;11)—the detection of MRD has been greatly facilitated by the presence of clonal rearrangements of the variable, diversity, and junctional genes (V, D, J) of the immunoglobulin heavy chain (IgH) locus. By using PCR primers complementary to highly conserved family-specific regions, leukemia-specific clonal rearrangements can be amplified in a first step and then sequenced, and clone-specific oligonucleotide primers or probes can be chosen to detect leukemia-specific IgH-CDR3 (V–D–J) rearrangements by PCR in subsequent samples obtained from the same patient (Yamada et al., 1989; Billadeau et al., 1991). The sensitivity of the leukemia-specific PCR with one clone-specific primer—usually chosen from the V–N–D–N–J region—is one positive cell in 104–6 normal cells. A major limitation of this technique is due to the fact that further gene rearrangements may occur in ALL cells of the pre-B-phenotype, generating leukemic subclones (Wright et al., 1987; Raghavachar et al., 1987; Yamada et al., 1990; Rovera et al., 1991; Bird et al., 1991). In some cases, these subclones may not be detected by the primary clone-specific PCR, leading to false-negative results. Most cases of apparent biclonality or oligoclonality have been shown to be due to secondary rearrangements, i.e., VH gene replacement (Rovera et al., 1991; Wasserman et al., 1992a; Kitchingman, 1993; Steward et al., 1994).
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These changes alter the VH–N–DH sequence, leaving the DH–N–JH sequence mostly unaffected, which has to be kept in mind when choosing allele-specific primers or probes. T-cell receptor (TCR)-␥ , -␦ and - genes are rearranged in most cases of T-ALL and can be used to follow up the malignant clone. These rearrangements are not restricted to T-cell neoplasms, but have also been found in B-ALL (50–70%) and in B-NHL (Waldmann, 1987). The TCR-␦ and TCR-␥ gene rearrangements seem to be very suitable targets: almost all children with T-ALL and about 80% of those with common ALL show such rearrangements (Yokota et al., 1991a; Trainor et al., 1991). To be detectable by PCR, the sample must contain at least 5–10% clonal cells. The sensitivity of PCR assays with clone-specific probes ranges from 1 positive cell in 103 to 106 cells. Unfortunately, secondary rearrangements of IgH and TCR genes in leukemic cells have been found in 30–90% of B- and T-ALL, due to either further somatic rearrangements because of continuing recombinase activity or the outgrowth of minor subclones already present before primary treatment and selected thereby (Biondi et al., 1992a; Baruchel et al., 1995; Beishuizen et al., 1994). Whenever possible, detection of MRD in ALL is carried out by following at least two marker genes to avoid false-negative results, since in clinical trials at least one rearranged IgH, TCR-␥ , or TCR-␦ allele was found to be unchanged, by a comparative analysis of leukemic cells from first presentation and relapse (Beishuizen et al., 1994; van Dongen et al., 1998). In 12–26% of patients with T-ALL, the TAL-1 and SIL loci on chromosome 1 are involved in a specific recombination event leading to a submicroscopical deletion of about 90 kb, the so-called TAL-1 deletion (Brown et al., 1990; Jonsson et al., 1991). This deletion seems to be generated by the VDJrecombinase using sequences resembling heptamer–nonamer recombination signal sequences of Ig and TCR genes. Almost the entire SIL locus including all SIL exons is deleted, and the intact coding region of TAL-1 is placed under the control of the SIL gene promotor. PCR analysis combined with sequencing shows that the SIL–TAL-1 fusion region has a clone-specific sequence with randomly inserted N-nucleotides, representing an ideal target for MRD analysis (Breit et al., 1993). Initially, MRD was solely tested by standard nonquantitative PCR techniques. Patients with high or low tumor burden could not be identified and were simply classified as “PCR positive.” Kinetics of increments or decrements in residual leukemic cells could not be detected, but this was possible after the introduction of quantitative PCR techniques. Studies in pediatric patients provided the most promising results, and insights into the clinical significance of residual disease were determined at different time points after blocks of conventional therapy. In most patients with ALL, the eradication of leukemic cells seems to be a slow process, taking several months.
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PCR negativity is usually reached late during treatment, or soon thereafter (Yamada et al., 1990; Yokota et al., 1991a; Neale et al., 1991). The kinetics of leukemia cell reduction during the first 3 months of conventional therapy permits identification of two MRD-based risk groups of patients with a low and a high risk of relapse (Wasserman et al., 1992b; Brisco et al., 1994; van Dongen et al., 1998). For the low-risk patients, therapy may be reduced, whereas patients in the high risk group might benefit from more intensive therapy, including allogeneic BMT. After induction therapy, the number of leukemic cells was found to vary to a great extent between >10−2 and <10−6. High residual disease at this point, i.e, ≥ 10−2 residual leukemia cells, indicated a high risk of relapse during further therapy (Brisco et al., 1994; Cave et al., 1998; van Dongen et al., 1998). Each 10-fold higher level of residual leukemic cells (≤ 10−4 to ≥ 10−2) correlated with a 2-fold higher relapse rate (van Dongen et al., 1998). The level of MRD after induction therapy can be regarded as an indicator of the in vivo drug sensitivity of leukemic cells. In patients responding to further therapy, each treatment block reduced the frequency as well as the degree of MRD. Most children exhibit PCR-positivity at the end of the induction therapy, if the sensitivity of the PCR technique is high enough (e.g., ≤ 5 × 10−6) (Roberts et al., 1997). In two large recent studies on 441 children, about 40–50% were still positive after induction therapy or at the beginning of consolidation therapy. The sensitivity of the PCR techniques used for the detection of IgH and TCR rearrangements was ≤ 5 × 10−5 (Cave et al., 1998) and 10−4 to 10−5 (van Dongen et al., 1998), respectively. About 45% of the patients PCR-positive at this point relapsed later; PCR-negative patients had a very low risk of relapse (< 10%). Relapses in previously PCR-negative patients are thought to be due to the emergence of chemotherapy-resistant subclones with altered receptor gene rearrangements. In addition, PCR assays carried out on bone marrow cells are about 10 times more sensitive than tests using peripheral blood cells (van Rhee et al., 1995; Brisco et al., 1997a; van Dongen et al., 1998). At any later time points, even low numbers of residual cells correlate with poor outcome. The critical values were found to be ≥ 10−3 leukemic cells, using a quantitative competitive PCR technique (Cave et al., 1998), or ≥ 10−4 by applying semiquantitative PCR (van Dongen et al., 1998). In B-lineage ALL, about 30% of the bone marrow samples are still PCR positive during maintenance therapy, but at the end of this treatment, most patients become negative. MRD still detectable after treatment is rare, but is associated with a high rate of relapse (Potter et al., 1993; Nizet et al., 1993). In T-ALL, the presence of MRD at the beginning of the maintenance therapy indicates a poor outcome (Dibenedetto et al., 1997). Reappearance of clonal cells and, especially, increasing levels over several months seem to predict hematological relapse. All studies agree that an analysis of MRD evolution rather than the simple qualitative detection of leukemic cells provides prognostic
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information regarding the clinical outcome (Yamada et al., 1990; Yokota et al., 1991b; Neale et al., 1991; Biondi et al., 1992a; Potter et al., 1993; Nizet et al., 1993, 2000; Cave et al., 1994; Roberts et al., 1997). Surprisingly, persisting low numbers of clonal cells have been observed in a few patients in long-term remission (Yamada et al., 1990; Biondi et al., 1992a; Roberts et al., 1997; Ito and Miyamura, 1994; Cave et al., 1998). These findings suggest that a cure from ALL does not necessarily require the elimination of all clonal cells (leukemic cells?), since small numbers of normal precursor cells could still survive the treatment of leukemia (Roberts et al., 1997). In summary, the presence or absence of MRD, including the quantitative sequential analysis of residual leukemic cells, was found to be an important independent prognostic factor in childhood ALL irrespective of age, white blood cell count at presentation, immunophenotype, risk, or treatment group (Cave et al., 1998; van Dongen et al., 1998). The results of two large clinical studies suggest that the quantitative detection of MRD by PCR at the end of induction therapy allows identification of two risk groups for which a new treatment stratification should be introduced to improve the results. To further optimize the therapeutic strategy for individual patients, additional clinical trials are necessary that include the use of standardized PCR assays based on the technology of real-time quantitative PCR ( Pongers-Willemse et al., 1998, 1999).
2. PHILADELPHIA CHROMOSOME (Ph1 )-POSITIVE ALL The Ph1 chromosome or BCR–ABL mRNA transcripts have been found in 3–5% of pediatric patients with ALL (Crist et al., 1990a) and in 12–43% of adults (Maurer et al., 1991; Radich et al., 1994). About 90% of children and 70% of adults have breakpoints in m-bcr and express p190BCR–ABL (Suryanarayan et al., 1991; Maurer et al., 1991). Ph1-positive ALL is a very aggressive leukemia and has an extremely poor prognosis when treated with conventional or even intensified chemotherapy for ALL only. The event-free survival rates are 25–30% in children and 5–20% in adults (Crist et al., 1990a; Preti et al., 1994). It seems to be almost impossible to eradicate Ph1positive cells by conventional chemotherapy, since almost all patients in clinical and cytogenetic remission after induction therapy are still PCR-positive at a > 10−3 level, which is associated with a high risk of relapse in standard risk childhood ALL (Brisco et al., 1997b). Therefore, autologous BMT has been used in trying to improve the outcome, but almost all autografts have been found to contain leukemic cells, and minimal residual leukemic cells were detected in the majority of the patients after transplantation followed by relapse (Martin et al., 1994; Mitterbauer et al., 1995; Preudhomme et al., 1997). Patients that became PCR-negative after transplantation with
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a purged PCR-negative autograft did not relapse or did so very late, whereas PCR-positive patients relapsed quite soon. Somewhat surprising is that in two autografted patients in remission for 2 and 6 years, residual Ph1-positive cells were found at a low level (< 0.1% BCR–ABL/ABL mRNA transcripts) (Mitterbauer et al., 1999). Allogeneic BMT has been introduced as first-line therapy after induction therapy (Forman et al., 1987; Snyder et al., 1999; Arico et al., 2000). Relapse rates are in the range of 12–35%, and disease-free survival rates are 35–65% after allogeneic related and about 50% after allogeneic unrelated BMT. Persistence of low numbers of residual cells during the first 3 months after allogeneic unrelated BMT does not indicate a high risk of relapse: 4/7 PCR-positive patients were found to be negative 9 months later (Sierra et al., 1997). In comparison, PCR-positivity after allogeneic related transplants correlates with clinical relapse in the near future (Miyamura et al., 1992). At 3 years after BMT, a relapse rate of 52% was observed in 20 patients PCR-positive until day 100 after BMT, whereas in 14 patients that had already become PCR-negative before day 100, a relapse rate of only 17% was found (Radich et al., 1997). Relapses occurred in 90% of the patients expressing p190BCR–ABL, but in only 12% expressing p210BCR–ABL and in 50% that were positive for both. These findings suggest that leukemias with m-bcr breakpoints have a worse prognosis than those with a breakpoint in M-bcr; this is supported by observations in transgenic mice that the more aggressive leukemia type is associated with p190BCR–ABL (Kelliher et al., 1991). Negative PCR assays do not always indicate cure from the disease; a change to positivity eventually occurs within less than 3 months, shortly followed by relapse and then death, demonstrating the very aggressive nature of Ph1-positive ALL (Miyamura et al., 1992). Prognostic information might be obtained by quantitative PCR assays carried out in short-term intervals of 1–2 months on bone marrow cells rather than peripheral blood cells (van Rhee et al., 1995). The median time interval from the first positive PCR assay (molecular relapse) to clinical relapse is about 100 days (Radich et al., 1997). This time window is large enough to start therapeutic interventions, since donor lymphocyte infusions have been successfully used in patients with Ph1-positive ALL (Mitterbauer et al., 1999). Molecular monitoring by PCR seems to be an important tool and a guide to start therapy after relapse as early as possible, since patients transplanted after hematological relapse or in advanced refractory disease have a very poor prognosis even after allogeneic unrelated BMT (Sierra et al., 1997; Radich et al., 1997).
3. t(1;19)- AND t(17;19)-POSITIVE ALL The t(1;19)(q23;p13.3) translocation can be detected cytogenetically in about 5% of ALL, mainly in pre-B ALL (Williams et al., 1984). This subtype
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associated with a poor prognosis can be successfully treated with intensified chemotherapy (Crist et al., 1990b). The translocation t(1;19) results in a fusion gene that codes for a novel chimeric mRNA consisting of the transcription activating domain of the basic helix-loop-helix transcription factor E2A from chromosome 19 and the DNA-binding homeodomain of the transcription factor PBX1 from chromosome 1 (Nourse et al., 1990; Kamps et al., 1990; Hunger et al., 1991). This fusion mRNA and most—but not all—of its variants can be detected by RT-PCR, suggesting that there might be a gene other than PBX1 involved in t(1;19) variant translocations (Privitera et al., 1992; Izraeli et al., 1992). PCR analyses for MRD have shown that a substantial proportion of children achieve a molecular remission, and persistently negative PCR assays indicate a low risk of relapse (Devaraj et al., 1995; Privitera et al., 1996; Lanza et al., 1996; Hunger et al., 1998). Qualitative detection of MRD by PCR at the end of consolidation therapy does not seem to have predictive value for relapse and should not be taken as a reason to change therapy in these patients, since minimal residual E2A-PBX1 mRNA positive cells have been observed in patients in clinical remission for several years without any signs of recurrent disease (Izraeli et al., 1993; Privitera et al., 1996). To achieve the highest percentage of positive results in follow-up studies, PCR assays on bone marrow cells as well as peripheral blood cells were found to be necessary (Hunger et al., 1998). Quantitative PCR analyses of bone marrow and peripheral blood cells carried out in short-term intervals might provide prognostic information. The t(17;19)(q22;p13.3) translocation can be envisaged as a variant of the t(1;19) translocation leading to the E2A–HLF fusion gene (Inaba et al., 1992; Hunger et al., 1992, 1994). This results in chimeric transcripts containing the amino-terminal E2A sequence fused to the DNA-binding and dimerization domain of the hepatic leukemia factor HLF. RT-PCR has been used to monitor MRD in this subtype of ALL with a very poor prognosis. Only three patients have been studied: they were positive at diagnosis and in all subsequent samples, irrespective whether they were in complete clinical remission or in relapse. Two patients remained positive after allogeneic BMT, and all three patients died from relapse (Devaraj et al., 1994).
C. Acute Myeloblastic Leukemia (AML) 1. t(8;21)-POSITIVE AML The translocation t(8;21)(q22;q22.3) is generated by a reciprocal translocation of the long arms of chromosomes 8 and 21, resulting in a new fusion gene, AML1–ETO (Miyoshi et al., 1991; Erickson et al., 1992). This
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translocation or the AML1–ETO fusion transcript have been found in approximately 10% of adult patients with AML, especially the subtype M2 (Nucifora et al., 1993a; Chang et al., 1993; Grimwade et al., 1998). The presence of this translocation is associated with a high remission rate and prolonged disease-free survival in patients treated with standard induction therapy followed by consolidation treatment consisting of at least three cycles of high-dose cytarabine (Byrd et al., 1999). Despite the relatively good prognosis, relapses are the major cause of treatment failures. Several groups have shown by standard RT-PCR that AML1–ETO transcripts can be found in patients in long-term remission after chemotherapy (Chang et al., 1993; Nucifora et al., 1993b; Maruyama et al., 1994; Kusec et al., 1994). These results have been confirmed by quantitative PCR analysis (Tobal and Liu Yin, 1996; Miyamoto et al., 1996; Tobal et al., 2000). Some groups did not find AML1–ETO transcripts in patients in long-term remission, which could be explained at least in part by a low sensitivity of the PCR technique used or by differences in treatment modalities (Satake et al., 1995; Morschhauser et al., 2000). In a study on 18 patients in complete remission (lasting 12–150 months) after conventional chemotherapy (n = 14) or autologous peripheral blood stem cell transplantation (PBSCT) (n = 4), all patients were found to be positive by PCR (Miyamoto et al., 1996). AML1–ETO mRNA-positive multipotent hematopoietic progenitors were cultured from the bone marrow of these patients, and their clonal origin could be demonstrated by X-chromosomal inactivation patterns of PGK in four female patients. The number of AML1– ETO mRNA-positive progenitor cells decreased with the duration of remission. AML1–ETO mRNA-positive progenitor cells could not be detected in four PCR-negative patients after allogeneic BMT (Miyamoto et al., 1996). The efficacy of eradication of AML1–ETO mRNA-positive cells seems to be higher after allogeneic BMT, presumably due to alloreactive T-cells, but PCR-positive patients in long-term remission can also be found after this treatment (Jurlander et al., 1996; Tobal and Liu Yin, 1996). Taken together, these results suggest that a complete elimination of AML1–ETO mRNApositive cells is not necessary for achieving a long-term remission. The clonogenic progenitor cell of t(8;21)-positive AML apparently originates from a t(8;21)-positive nonleukemic multipotent progenitor cell, since the presence of the AML1–ETO fusion transcript by itself does not seem to be sufficient to render these cells fully malignant and, therefore, the detection of AML– ETO mRNA does not necessarily identify a leukemic cell (Miyamoto et al., 1996; Saunders et al., 1997). Molecular monitoring by quantitative PCR seems to provide prognostic information. Induction chemotherapy leads to a decrease of leukemic cells by 2–3 logs, and a further reduction has been observed under successive cycles of chemotherapy (Miyamoto et al., 1995; Tobal and Liu Yin, 1996; ¨ Marcucci et al., 1998; Schuler et al., 1998). Serial analyses by competitive
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or real-time quantitative PCR seem to be useful to identify patients at risk for relapse, since increases in transcripts (molecular relapse) can be found 2–4 months before clinical relapse (Tobal and Liu Yin, 1996; Marcucci et al., ¨ et al., 2000; Tobal et al., 2000), providing 1998; Krauter et al., 1999; Schuler an opportunity for early therapeutic interventions to prevent hematological relapse.
2. inv(16)-POSITIVE AML The pericentric inversion of chromosome 16 [inv(16)(p13q22)] and the translocation t(16;16)(p13;q22) are associated with acute myelomonocytic leukemia with increased eosinophils (AML M4 Eo), but are not restricted to this subtype (Le Beau et al., 1983). Four forms of new chimeric transcripts, mainly type A, are generated by a fusion between the core binding factor ß gene (CBFB or CBFß) on chromosome 16q22 and the smooth muscle myosin heavy chain gene (MYH11) on chromosome 16p13 (Liu et al., 1993; Claxton et al., 1994). Patients with inv(16)-positive AML have a favorable prognosis, but relapses are the main cause of treatment failures. In some studies on patients in long-term remission after chemotherapy, CBFB-MYH11 mRNA transcripts have been detected (Hebert et al., 1994; Tobal et al., 1995a; Evans et al., 1997); in others, negative PCR results have been found (Claxton et al., 1994; Costello et al., 1997). The different results seem to be at least in part due to a different sensitivity of the PCR techniques used. In a study on 10 patients, mainly negative results were observed by PCR at a sensitivity of 10−4; negative PCR results obtained 8 months after the start of chemotherapy indicated a low risk of relapse (Costello et al., 1997). A complete elimination of CBFB-MYH11 mRNA positive cells has been observed after allogeneic BMT (Laczika et al., 1998; Elmaagacli et al., 1998).
3. ACUTE PROMYELOCYTIC LEUKEMIA (APL) Acute promyelocytic leukemia (or FAB-M3, according to the French– American–British Association) accounts for 10–15% of de novo AML in young adults (Grimwade et al., 1998), and it is associated with the t(15;17) (q22;q21) translocation in the majority of cases (Rowley et al., 1977). This type of AML with hypergranular blasts is characterized by some features that need specific therapeutic interventions: r r r r r
life-threatening coagulopathy a high chemotherapeutic sensitivity to anthracyclins a differentiation response to all-trans-retinoic acid (ATRA) a relatively low risk of relapse no overall benefit from first-line autologous or allogeneic BMT for most patients
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The t(15;17) translocation disrupts the PML gene at three breakpoint clusters on chromosome 15q22 and the retinoic acid receptor-␣ gene (RAR␣) within the 2 intron on chromosome 17q21. This translocation leads to a new PML–RAR␣ fusion transcript that interferes with differentiation and inhibits apoptosis in myeloid cells (de The et al., 1990, 1991). The two major breakpoint regions within the PML gene are bcr3 within intron 3, occurring in about 30% of the cases and leading to “short” PML–RAR␣ transcripts, and bcr1 within intron 6, occurring in about 60% and leading to “long” transcripts. The remaining breakpoints are found at variable positions within bcr2 located in exon 6, causing “variable” transcripts (Grimwade et al., 1996a). Different locations of breakpoints, alternative splicing, and the use of alternative RAR␣ polyadenylation sites generate heterogeneous PML– RAR␣ transcripts of different sizes (de The et al., 1991; Pandolfi et al., 1992; Biondi et al., 1992b). Patients with the PML–RAR␣ type of transcript, and presumably also those with the variant translocations t(5;17)(q32;q21) [NPM–RAR␣ (Redner et al., 1996)] and t(11;17)(q13;q21) [NuMA–RAR␣ (Wells et al., 1997)], benefit from combination treatment of ATRA and chemotherapy. On the other hand, patients with t(11;17)(q23;q21)-positive APL carrying the PZLF–RAR␣ transcript (Chen et al., 1993) are ATRA resistant. Therefore it is important to detect the PML–RAR␣-rearrangement in any case by at least one method: cytogenetics, FISH, Southern or Northern blot analysis, or RT-PCR, since in about 15% of the cases, the typical translocation t(15;17) can be missed by cytogenetics due to either variant complex aberrations (< 10%) or technical problems (Burnett et al., 1999). Patients with t(15;17) or PML–RAR␣ mRNA have a favorable prognosis (Grimwade et al., 1998), since the combination of chemotherapy and extended courses of ATRA has been introduced (Burnett et al., 1999). Therefore it is the goal of MRD studies to identify the small number of patients that have a high risk of relapse and might benefit from more intensive treatment. The majority of patients treated with chemotherapy and ATRA will become PCR-negative by applying conventional PCR assays with a sensitivity of 10−4. The persistence of PML–RAR␣ transcripts is highly predictive of relapse (Grimwade et al., 1996b; Mandelli et al., 1997; Burnett et al., 1999). The majority of those patients that will later relapse are PCR-negative at the end of combination therapy. This can be explained by the low sensitivity of RT-PCR for the detection of PML–RAR␣ mRNA, presumably due to its low expression (Seale et al., 1996). If PCR analyses are carried out on bone marrow cells in at least 3-monthly intervals, the majority of patients that will relapse later could be prospectively identified (Diverio et al., 1998). The median time interval from molecular relapse to hematological relapse is about 3 months. Early diagnosis of molecular relapse and early treatment before hematological relapse could help to improve the overall treatment results, since < 50% of the patients suffering from hematological relapse are going
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to survive for more than 2 years (Burnett et al., 1999). Determination of the kinetics of disappearance of PML–RAR␣ mRNA and the reciprocal transcript RAR␣–PML mRNA (Chang et al., 1992; Grimwade et al., 1996b)— resulting in a 10 × higher sensitivity—have shown that positive PCR results after the end of induction therapy indicate a high risk of relapse, and the delayed disappearance of t(15;17)-associated transcripts turned out to be an independent prognostic factor in APL (Burnett et al., 1999). When RT-PCR techniques with increased sensitivity were used in studies on MRD, both types of transcripts were also found in long-term remission (Tobal et al., 1995b; Tobal and Liu Yin, 1998). Molecular monitoring in the setting of autologous BMT suggests that transplantation of PCR-negative bone marrow is likely to result in prolonged clinical and molecular remission, but PCR-positivity before transplant is associated with a high risk of relapse (Meloni et al., 1997). On the other hand, an autograft contaminated with a few PML–RAR␣-mRNA-positive cells does not necessarily exclude a secondary long-term remission with negative PCR results (Sanz et al., 1998), as has been found in inv(16)-positive AML ¨ et al., 2000). Us(Grimwade et al., 1997) and t(8;21)-positive AML (Schuler ing standard PCR techniques, negative results have been found in patients in long-term remission after autologous BMT. The presence of PCR-detectable transcripts 3 months after BMT seems to indicate a high risk of relapse (Meloni et al., 1997; Roman et al., 1997). Future studies should take advantage of the very sensitive real-time quantitative PCR technology to monitor patients with APL at the molecular level during induction therapy and follow-up (Cassinat et al., 2000). It would be of special interest to evaluate the efficacy of prolonged therapy with ATRA after autologous BMT (Tallman et al., 1997).
IV. MRD IN LYMPHOMA A. t(14;18)-Positive Follicular Lymphoma The most frequent primary chromosomal aberration in human B-cell lymphomas is the t(14;18)(q32;q21) translocation, which has been detected by cytogenetics in about 90% of patients with follicular lymphoma and in 20% of patients with diffuse large cell lymphoma (Fukuhara et al., 1979; Yunis et al., 1982; Levine et al., 1985). This translocation involves the J region of the IgH gene locus on chromosome 14q32 and the BCL-2 gene on chromosome 18q21 (Tsujimoto et al., 1984; Cleary and Sklar, 1985; Bakhshi et al., 1985). The chromosomal breakpoints are located 5′ or 3′ to the BCL-2 gene, leaving intact the coding part of this gene. The association of the BCL-2 gene
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with the heavy chain locus results in chimeric transcripts and a constitutive expression of the translocated BCL-2 gene, inhibiting apoptosis (Seto et al., 1988; Nunez et al., 1990; Korsmeyer, 1992). Despite the mature B-cell phenotype of follicular lymphoma, the t(14;18) translocation occurs at an early step of pre B-cell differentiation, when the D and J gene segments of the IgH locus are being rearranged (Bakhshi et al., 1987). In about 50–60% of follicular lymphomas, the breakpoints are clustered within the 3′ -untranslated part of the third BCL-2 exon within the so-called major breakpoint region (MBR) (Tsujimoto et al., 1985). In 10– 20% of the cases, the breakpoints are located more than 20 kb downstream from MBR within the minor cluster region (mcr) (Cleary et al., 1986), and in a few cases they have been found 5′ of BCL-2 exon 1 in the variant cluster region (vcr) (Tsujimoto et al., 1987). Since the known t(14;18) translocations are structurally quite uniform, they represent ideal DNA target sequences for PCR analysis with defined BCL-2 and JH-consensus primers (Lee et al., 1987; Crescenzi et al., 1988; Ngan et al., 1989). Different breakpoints within the breakpoint cluster regions of the BCL-2 gene and the J segments, as well as the addition of N-nucleotides comparable to IgH-VDJ recombinations, lead to patient- and clone-specific t(14;18) translocations. Clone-specific translocation fragments amplified by PCR can be identified by their size, as determined by gel electrophoresis eventually combined with Southern blot hybridization and ultimately by nucleotide sequence analysis, which allows establishment ¨ of clone-specific PCR assays (Dolken et al., 1996). The nucleotide sequence of clone-specific t(14;18) translocations in lymphoma patients was found to ¨ be stable for at least about 10 years (Finke et al., 1993b; Dolken and Hirt, ¨ 1997; Hirt and Dolken, 2000). Different PCR techniques are capable of detecting one t(14;18)-positive cell in 105 to 106 normal cells and even one in 107 normal cells when stochastic experiments are performed (Table I). Therefore, in vitro amplification has been extensively utilized to detect t(14;18)-positive cells in patients with follicular lymphoma, to carry out molecular staging before therapy, as well as molecular monitoring to detect and follow up the evolution of MRD in clinical remission. In patients with low-stage follicular lymphoma (stage I, II) at initial presentation, several investigators have found t(14;18)-positive cells (occult lymphoma cells) in the bone marrow or circulating in the peripheral blood, with no morphological evidence for bone marrow infiltration or leukemic generalization (Berinstein et al., 1993a,b; Yuan et al., 1993; Lambrechts et al., 1993). After successful treatment of localized follicular lymphoma by radiotherapy, circulating t(14;18)-positive cells carrying the same translocation as the lymphoma cells within the primary biopsy have ¨ been found in long-term remission (Finke et al., 1993b; Dolken and Hirt, 1997). These circulating t(14;18)-positive cells were quantitatively determined by limiting dilution assays combined with a two-step nested PCR
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as well as real-time quantitative PCR and were found to be stable within one order of magnitude in the case patients remaining in long-term remission, whereas logarithmically increasing cell numbers were found in relapsing ¨ ¨ ¨ patients C Dolken and Hirt, 1997; Dolken et al., 1998; Hirt and Dolken, 2000). With regard to the excellent long-term results of radiotherapy alone for patients with localized, low-stage disease, the finding of occult lymphoma cells has not been used to change the standard therapy, i.e., the “local” therapy concept. By PCR analysis, t(14;18)-positive cells have been found in the bone marrow of most patients with advanced disease at presentation but also in patients in clinical remission after chemotherapy without any morphological evidence for bone marrow infiltration (Gribben et al., 1991a; Lambrechts et al., 1992, 1994). Although molecular responses in follicular lymphomas have been observed after conventional chemotherapy in a substantial number of patients, correlating with durable remissions (Lopez et al., 1998), long-term follow-up studies suggest that conventional chemotherapy does not eradicate minimal residual t(14;18)-positive cells in all patients, as detected by PCR. In the majority of patients with previously advanced disease, circulating t(14;18)-positive cells bearing the same translocation as the initial lymphoma cell clone were also detected in clinical remission (Price et al., 1991a; Lambrechts et al., 1992, 1994). A recent very promising study shows that chemotherapy in combination with anti-CD20 chimeric antibody (Rituximab) can induce durable clinical and molecular remissions (Czuczman et al., 1999). Long-term clinical and molecular follow up in a randomized trial is necessary to ascertain the clinical relevance of these preliminary data. Myeloablative chemoradiotherapy followed by transplantation of autologous bone marrow cells or autologous peripheral blood stem cells has been increasingly used to treat patients with recurrent follicular lymphoma (Gribben et al., 1991b; Johnson et al., 1994; Leonard et al., 1998; Freedman et al., 1999). As outlined above, these patients are at high risk to be reinfused with occult lymphoma cells, detectable by PCR in morphologically normal bone marrow or peripheral blood stem cell harvests. Therefore, purging techniques have been introduced to eliminate contaminating B-lymphoma cells by the use of 3 to 5 B-cell-specific mouse monoclonal antibodies in combination with complement-mediated lysis (Gribben et al., 1991b; Freedman et al., 1999) or immunomagnetic beads (Kiesel et al., 1987; Gribben et al., 1992). Unfortunately, until now no clinical studies have been successfully carried out that compare the reinfusion of purged versus unpurged hematopoietic stem cell products, presumably because of the large number of patients necessary. No investigation has proven until now that lymphoma cells present in an autograft contribute to relapse (Bachier et al., 1999), but there is clinical evidence that purging may be effective (Gribben et al., 1991b). Patients transplanted with bone marrow preparations that still contained PCR-detectable
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lymphoma cells after immunological purging—purging was ineffective in about 50% of the patients—had an increased incidence of relapse compared to those that received a PCR-negative purged bone marrow. In this study, the most important prognostic indicator in predicting relapse was the inability to purge residual lymphoma cells. This finding is remarkable with regard to the observation that the majority of relapsing patients do so at the sites of prior disease, which has been confirmed in all following studies. In a recent paper, results were presented of a long-term follow up of patients treated with autologous BMT for relapsed follicular lymphoma (Freedman et al., 1999). The overall survival rates and the disease-free survival rates for all patients were 66 and 42%, respectively. Patients transplanted with a purged PCR-negative bone marrow had a significantly higher probability of freedom from relapse (80% after 8 years) than those transplanted with a purged but still PCR-positive bone marrow (20% after 8 years). Unfortunately, no quantitative PCR analyses were carried out. In two subsequent studies from another group, the proportion of patients transplanted with a purged PCR-negative bone marrow was much lower (about 25%), but the clinical outcome was quite similar (Johnson et al., 1994; Apostolidis et al., 2000). The differences in purging efficiency of bone marrow harvests was attributed to differences in the sensitivity of the PCR methods. Surprisingly, the PCR status of the purged reinfused bone marrow did not have any influence on survival or freedom from relapse (Apostolidis et al., 2000). The prognostic value of PCR analyses of MRD after autologous BMT is difficult to assess. The main reason seems to be the use of PCR techniques with varying sensitivities. In a large study, 77 of 134 patients who had a recent PCR-negative bone marrow sample—either because they never had detectable lymphoma cells post-ABMT (n = 58) or because they were positive only during the first 2 years (n = 19)—did not relapse, but 25 of 35 patients with positive PCR tests on all bone marrow samples had recurrent disease (Gribben et al., 1993). The persistence or reappearance of t(14;18)positive cells after autologous BMT seems to indicate a high risk of relapse. Eight relapses have been observed in the 22 remaining patients who had t(14;18)-positive cells in bone marrow samples, detectable by PCR for varying periods of time. All 33 patients that finally relapsed had PCR-detectable residual lymphoma cells in the bone marrow before clinical relapse. These results have been confirmed by using a comparable PCR technique: patients remaining PCR-negative after autologous BMT have a low risk of relapse (4/23 = 17%), whereas continuously PCR-positive patients have a high risk of relapse (12/13 = 92%) (Apostolidis et al., 2000). Several groups have found that patients in long-term remission after autologous BMT for relapsed follicular lymphoma still had PCR-detectable circulating t(14;18)-positive cells carrying the same translocation as the primary lymphoma (Johnson ¨ et al., 1994; Colombat et al., 1994; Hirt and Dolken, 2000). Based on these
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findings, it may be concluded that t(14;18)-positive lymphoma cells need not be completely eradicated to achieve a long-term remission or even a “cure.” Since new therapeutic options are available to treat patients with residual lymphoma cells, who are at high risk for relapse, reliable quantitative PCR analyses are necessary to identify those patients that might benefit from further treatment. The therapeutic efficacy of tumor cell vaccination with genetically engineered cells (Schultze et al., 1995), vaccination with idiotypic peptides derived from lymphoma cells with GM-CSF (Kwak et al., 1992; Bendandi et al., 1999), or dendritic cells (Hsu et al., 1996), or the antitumor activity of unconjugated monoclonal antibodies like the humanized anti-CD20 (Rituximab) (Shan et al., 1998; Czuczman et al., 1999) should be controlled not only by clinical evaluation but also at the molecular level by real-time quantitative PCR. This has already been done in a study on fol¨ licular lymphoma patients after autologous BMT (Hirt and Dolken, 2000). The interpretation of PCR results in MRD studies has been complicated by the findings that B cells carrying t(14;18) translocations have been detected by very sensitive two-step PCR techniques in benign hyperplastic lymphoid lymph nodes and tonsils (Limpens et al., 1991; Aster et al., 1992), in peripheral blood lymphocytes of healthy individuals (Limpens et al., 1995; Ji ¨ et al., 1995; Dolken et al., 1996), and in spleens of patients who died from diseases other than malignant lymphomas (Liu et al., 1994). The t(14;18) translocations found in healthy blood donors are typical BCL-2-MBR/JH rearrangements, as shown by nucleotide sequence analysis; they cannot be distinguished from those found in follicular lymphoma. Not only patients with follicular lymphoma (Price et al., 1991b), but also healthy individuals were found to carry more than one t(14;18)-positive cell clone, based on size and nucleotide sequence analysis of the amplified DNA fragments ¨ (Limpens et al., 1995; Ji et al., 1995; Dolken et al., 1996). Therefore is it important to know the size of the t(14;18)-DNA fragment amplified from the malignant lymphoma cell clone as well as its nucleotide sequence. In healthy individuals, t(14;18)-positive cell clones have been found to persist for at least 5 years, suggesting that t(14;18)-positive B cell clones can be long-lived and immortalized like cells carrying Epstein–Barr virus in healthy seropositive individuals. These results support the idea that the occurrence of the t(14;18) translocation is not restricted to malignant follicular lymphoma cells and that the presence of a t(14;18) translocation does not identify a lymphoma cell per se. Several findings suggest that the deregulated expression of BCL-2 itself is not sufficient for the development of B-cell lymphomas. Gene transfer experiments and studies with transgenic mice carrying a BCL-2/IgH-minigene have shown that additional events are necessary to develop malignant B-cell lymphomas (e.g., a translocation of c-myc; McDonnell et al., 1989; McDonnell and Korsmeyer, 1991). The incidence of t(14;18)-positive cells in healthy
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individuals is high, about 50% carry circulating B cells with a rearrangement within the MBR region of the BCL-2 gene. Since MBR translocations can be detected by PCR in only 40–60% of cytogenetically t(14;18)-positive follicular lymphomas, it seems to be justified to assume that almost all healthy individuals carry at least one t(14;18)-positive B-cell clone once during their lifetime. Based on these findings, it has been suggested (Fig. 3) that in healthy individuals “pre-pre-lymphoma cells” carrying the t(14;18) translocation as the first and only genetic aberration are stochastically generated, presumably by recombination-activating gene (RAG)-mediated transposition (Agrawal et al., 1998; Hiom et al., 1998). These positive cells can be detected by PCR in the peripheral blood or in lymphatic organs if the sensitivity of the assay is ¨ high enough (Dolken et al., 1996). These t(14;18)-positive cells are long-lived and presumably immortalized due to the constitutive expression of the BCL2 protein. The proliferation of t(14;18)-positive B cells may be positively and negatively controlled by various mechanisms, including antigen stimulation and antiidiotypic antibodies. During clonal expansion, additional cytogenetic or molecular changes may occur in single t(14;18)-positive cells now designated “pre-lymphoma cells.” Further accumulating genetic changes leads to malignant follicular lymphoma cells which may progress to highly malignant lymphomas, a process called transformation or progression. Successful treatment of t(14;18)-positive follicular lymphoma by radiotheraphy or myeloablative therapy may reduce the total burden of lymphoma cells to minimal residual numbers that could possibly be controlled or even eliminated by the immune system. Minimal residual B lymphoma cells can be kept under immunological control by the idiotype–antiidiotype network, as has been shown in the case of murine BCL1-lymphoma (Uhr et al., 1997). Alternatively, malignant lymphoma cells may be eradicated by therapeutic intervention, but t(14;18)-positive pre- or pre-pre-lymphoma cells persist for the rest of a person’s life. This hypothesis could explain the positive PCR results of patients in long-term remission after successful radiotherapy for lowstage follicular lymphoma (Finke et al., 1993b), after chemotherapy (Price et al., 1991a), and after high-dose chemotherapy combined with autologous BMT (Johnson et al., 1994). It may be very important for our understanding of the evolution of follicular lymphoma to identify additional molecular changes that determine the malignant phenotype of t(14;18)-positive follicular lymphoma cells in comparison to t(14;18)-positive B cells found in healthy individuals. The sensitivity of PCR techniques and the reliability of PCR results from different laboratories in Europe and North America have been investigated in a collaborative study (Johnson et al., 1999). By single-round and twostep nested PCR, 100 positive cells could be safely detected in a single assay, but below this cell number, only the two-step nested PCR was able to produce reliable results. The false-positive rate was about 30%, reported from
Fig. 3 Multistep evolution of t(14;18)-positive malignant follicular lymphomas.
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9/20 laboratories. The authors came to the conclusion that there is urgent need to improve accuracy and reproducibility of PCR results. Standardized protocols for DNA isolation in conjunction with real-time quantitative PCR might solve most of the problems. The sensitivity of real-time quantitative PCR using 50 rounds of amplification is about the same as that of nested PCR with at least 55 cycles of amplification, since both techniques are able ¨ to detect one positive cell per assay (Dolken et al., 1998; Luthra et al., 1998; Mandingers et al., 1998; Johnson et al., 1999). Real-time quantitative PCR employing quantitative determination of the t(14;18) translocation and a control gene as a reference on any sample seems to be the method of choice to determine the quality of the isolated DNA and the sensitivity of every single PCR assay. This will provide the information necessary to calculate and to adjust the sensitivity of PCR results to a defined detection limit for a comparative analysis of different samples obtained from one patient at different ¨ times as well as of results from different laboratories (Dolken et al., 1998).
B. Mantle Cell Lymphoma (MCL) Mantle cell lymphoma is a B-cell lymphoma characterized by a pan Bpositive (CD19, CD20, CD22, CD24), CD5-positive, cyclin D1-positive, but CD23-negative phenotype (Campo et al., 1999) and genetically by the t(11;14)(q13;q32) translocation that fuses the bcl-1 locus with the IgH locus (Van den Berghe et al., 1979; Erikson et al., 1984). The molecular consequence of this translocation is a constant overexpression of the cyclin D1 (PRAD-1/CCDN1) gene (Rosenberg et al., 1991; Withers et al., 1991; Rimokh et al., 1993), which probably leads to a deregulation of the cell cycle due to an interaction with the tumor suppressor retinoblastoma protein (Rb). The t(11;14) translocation can be detected in 70–100% of patients with MCL. The breakpoints on chromosome 11 are scattered over 100 kb of genomic DNA. In 30–50% of MCL, the breakpoints on chromosome 11 have been detected within the major translocation cluster (MTC) (Williams et al., 1991), representing a suitable DNA target for PCR amplification (Williams et al., 1993; Rimokh et al., 1994). About 80% of patients with MCL can be monitored for residual lymphoma cells by PCR using as targets either the BCL-1/IgH translocation or the CDR-3 region of the IgH locus (Andersen et al., 1997). Mantle cell lymphoma is a disease of the elderly, is usually very aggressive, and is almost incurable (Weisenburger and Armitage, 1996). Response to conventional chemotherapy is poor, and patients have a median survival of 3 to 4 years (Fisher et al., 1995). The lack of efficacy of conventional treatment has been documented by quantitative PCR: molecular remissions are rare, and MCL cells persist in the majority of patients at a high level (around
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1%) in the bone marrow and peripheral blood (Jacquy et al., 1999). Therefore, aggressive chemotherapy protocols including autologous BMT have been used as part of a frontline treatment for younger patients whenever possible. At the time of transplantation, all patients still had PCR-detectable lymphoma cells in their bone marrow, irrespective of previous histological involvement (Andersen et al., 1997). In several studies, PCR-negative autografts were obtained in only about 10% of the patients, even after immunological purging. After BMT with untreated as well as purged autografts, the majority of patients show persistent PCR-positivity (Andersen et al., 1997; Corradini et al., 1997; Jacquy et al., 1999). Reinfusion of a PCR-positive bone marrow or persistent PCR-positive results after transplant are associated with a high probability of relapse. Until now, there has been no evidence for long-term remissions after autologous BMT (Freedman et al., 1998). A few patients were transplanted with allogeneic blood stem cells with quite promising results (Khouri et al., 1999). The transplant-related mortality was quite high (6/16), but the overall survival and failure-from-diseaseprogression was 55% at 3 years for all patients. All patients had MRD detectable by PCR at transplant, about 50% (7/16) were still positive after BMT, and four patients achieved molecular remission 4 months later. One patient who was PCR-positive after BMT became negative later; another was borderline positive/negative. These results suggest that there might be a graft-versus-lymphoma (GvL) effect (Jones et al., 1991; Mandingers et al., 1998) in MCL patients after allogeneic BMT. Because molecular remissions were achieved concomitant with GvHD, PCR-positivity detected early after transplant was converted to PCR-negativity, and the only relapse observed occurred in a patient who failed to engraft. In three other patients, clinical and molecular remissions were reported after allogeneic BMT (Andersen et al., 1997; Corradini et al., 1996). Sensitive and reliable quantitative PCR techniques for the detection of residual lymphoma cells (Luthra et al., 1999; Gerard et al., 1998; Olsson et al., 1999) will be very helpful for developing innovative therapeutic strategies for MCL patients, and are urgently needed. Low-dose, nonmyeloablative conditioning regimens in the setting of allogeneic BMT seem to be very promising to treat patients older than 60 years of age in first remission.
V. MRD IN SOLID TUMORS A. Colorectal Carcinoma Solid epithelial cancers are the most common malignancies worldwide. Mortality among patients with solid tumors mainly results from early
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dissemination of neoplastic cells undetectable by conventional cytology and histopathology. The identification of occult tumor cells or micrometastatic disease at secondary sites seems to be essential for individual management of cancer patients after surgical resection of the primary tumor. Sensitive immunological and molecular techniques have been developed in the past 15 years to detect micrometastasis in patients with solid tumors. Mainly, regional lymph nodes, peripheral blood, and bone marrow have been the sites analyzed for the presence of occult tumor cells. In patients with proven micrometastatic disease, systemic adjuvant treatment using conventional chemotherapy or innovative therapeutic regimens and strategies should be evaluated in prospective clinical trials aiming at prevention of metastatic relapse, since recurrent metastatic disease cannot be successfully treated in most solid cancers. Colorectal cancer (CRC) is one of the leading causes of cancer deaths worldwide. Research on micrometastasis as an important risk factor for metastatic disease seems to be by far most advanced in CRC, therefore this tumor entity is included in this review as a model for solid tumors (Ghossein et al., 1999; Pantel et al., 1999). In CRC, the stage at primary surgery correlates very well with prognosis (Dukes and Bussey, 1958). In patients with localized disease, the single most important prognostic factor is the presence or absence of nodal metastasis at the time of resection of the primary tumor (Deans et al., 1992). Stage II patients showing histologically no metastatic involvement of locoregional lymph nodes have a 5-year survival rate of about 80%, whereas patients with lymph node metastasis (stage III) have a 40–50% survival rate (Dukes and Bussey, 1958; Hermanek, 1995). Surgical resection followed by adjuvant chemotherapy is the standard practice in stage III patients, since it reduces the risk of recurrent disease and the rate of death. Patients with stage II tumors are treated with surgery only; adjuvant therapy is reserved for patients in experimental clinical trials (Moertel et al., 1995; O’Connell et al., 1997). To be on the safe side, a minimum of 12 lymph nodes should be examined by the pathologist and found to be free of tumor cells, until the patient can be considered as being in stage II without lymph node metastasis (Fielding et al., 1991). Since 20– 30% of stage II patients are going to die from metastatic disease within 5 years after primary surgery (Ovaska et al., 1990; Kune et al., 1990; Cohen et al., 1991), the question arises whether these patients can be identified by the detection of micrometastasis in regional lymph nodes and selected for adjuvant therapeutic trials to prevent recurrent disease. Table IV summarizes molecular and clinical results of the most relevant studies that looked for occult tumor cells in regional lymph nodes of patients with CRC stage I/II in relation to recurrent disease. PCR amplification at the DNA level was used for the detection of mutated K-ras or p53 present in
Table IV Detection of Micrometastasis in Regional Lymph Nodes of Patients with Colorectal Cancer (CRC) in Relation to Recurrent Disease
Reference
Stage (n)
Method
Target genes
Number of patients
Pos. ln/total ln studied
%# pos. pat.
No. ln studied/ patient
Follow-up (years)
Relapse rate (%)
Recurrent disease
Cancer-related deaths
Significance (∗ )
263/581
52 (37/71)
8.2
5
27/37 ln+ 0/34 ln−
not stated
yes
2.5
I (21) II (59) I+II (38) I+II (12)
not stated
no
7/14 ln+ 1/12 ln− 6/14 ln+ 1/36 ln−
yes
Hayashi et al. (1995)
I (39) II (32)
PCR (DNA)
K-ras p53
71
Nakamori et al. (1997) Liefers et al. (1998) Greenson et al. (1994)
I (5) II (12) II (26)
PCR (DNA)
K-ras p53 CEA
17 26
36/192
54 (14/26)
7.4
5–6
II (31)
2/9 ln+ 0/8 ln− not stated
CK
50
33/568
28 (14/50)
11.3
5
II (14)
not stated
II (50)
2-step RT-PCR (mRNA) immunohistochemical (protein)
53 (9/17)
13
Note. #, percentage of patients that had micrometastasis detected in at least one lymph node; ln = lymph node; ∗ statistical significance of a comparison, negative versus positive lymph nodes and risk of relapse.
yes
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tumor cells. K-ras mutations at codons 12, 13, or 61 occur early during carcinoma development, are stable throughout the course of the disease, and can be detected in about 40–50% of colorectal cancers (Bos, 1989; Vogelstein et al., 1988; Fearon et al., 1987). Mutations or deletions of p53 have been found in 75–80% of CRC (Levine et al., 1991; Ohue et al., 1994). For the detection of the epithelial cell-specific and more or less tumorspecific mRNAs of cytokeratin 19 (CK19), cytokeratin 20 (CK20) (Burchill et al., 1995; Gunn et al., 1996), and the carcinoembryonic antigen (CEA) (Neumaier et al., 1995; Mori et al., 1995), RT-PCR was used. In a retrospective study on CRC patients in stage I/II, Hayashi et al. (1994, 1995) used a PCR technique for an allele-specific amplification of mutations in K-ras (codons 12, 13, 61) or p53 (exons 5–8). Somatic mutations in at least one of these genes were detected in 71/120 (59.2%) primary tumors. Histologically tumor-free regional lymph nodes from 71 patients were analyzed by PCR: lymph nodes of 37 patients were positive by PCR, and within 5 years after primary surgery, 27 relapses were observed in this group. No relapses were observed in patients without micrometastasis. Forty-nine percent (184/450) lymph nodes resected from patients with recurrent tumors were positive by PCR, but only 79/581 (13.6%) lymph nodes from patients without recurrence. In every patient with either a local relapse or distant metastasis, PCR-positive lymph nodes were found, but in only 23% of patients without recurrent disease. The differences between the two groups of patients were statistically significant. Nakamori et al. (1997) analyzed regional lymph nodes from 17 patients using the same PCR technique. Two out of nine patients with PCR-positive lymph nodes had recurrent disease within 2 years after primary resection, but none of the 8 patients with PCRnegative lymph nodes. These differences were not statistically significant. In the study of Liefers et al. (1998), micrometastasis in regional lymph nodes, as detected by CEA, mRNA specific RT-PCR turned out to be an important prognostic factor. The adjusted 5-year survival rate decreased from 91% in patients with PCR-negative lymph nodes to 50% in patients with micrometastasis. Despite the fact that only seven lymph nodes were analyzed per patient (and there were some doubts regarding the specificity of CEART-PCR; Ghossein, 1998; Bostick et al., 1998; Ko et al., 1998), this study clearly identified patients with stage II disease who might benefit from adjuvant therapy based on the detection of CEA mRNA in regional lymph nodes. Two studies should be mentioned that used conventional staining of lymph node sections and immunohistochemistry to identify tumor cells. Greenson et al. (1994) studied 50 patients with CRC stage II and analyzed 568 lymph nodes by staining with a cytokeratin-specific monoclonal antibody. Tumor cells were detected in 33 lymph nodes from 14 patients; 6 of them (42.8%)
Detection of Minimal Residual Disease
169
died within 66 months, from recurrent disease. No lymph node involvement was found in 36 patients, and only 1 died from recurrent carcinoma. In a case control study on 44 patients, immunohistochemical staining of tumor cells in lymph nodes by cytokeratin-specific antibodies did not identify patients at risk for relapse (Nakanishi et al., 1999). Comparable to studies using RT-PCR for CEA or cytokeratins, immunohistochemically identifiable CKpositive cells may not represent tumor cells in all instances, and especially not tumor cells that are still able to divide and grow. Furthermore, in most studies, only one or a few sections of the lymph nodes—not the entire lymph node—were analyzed by immunohistochemistry. At present, the prognostic significance of micrometastasis detected by immunological methods is still a somewhat controversial matter. Two-step nested RT-PCR techniques used for the detection of CK19, CK20, or CEA are very sensitive, but they might produce false-positive results due to “illegitimate” transcription, contamination of the tissue samples with a few normal epithelial cells, or due to the amplification of a pseudogene resulting from contaminating DNA, as in the case of CK-19 (Schoenfeld et al., 1994; Krismann et al., 1995; Burchill et al., 1995; Gunn et al., 1996; Ghossein, 1998; Bostick et al., 1998). These studies have shown that CK19 mRNA is not only expressed in epithelial cells but also to a variable but easy detectable extent in peripheral blood cells and lymph nodes, which argues against its usefulness as a tumor cell marker detectable by RT-PCR. K-ras or p53 gene mutations can be successfully detected in 70–80% of CRC, but at present, there is no evidence that they can be found in nonneoplastic cells. Therefore, PCR amplification techniques using these targets seem to be the assays of choice due to their high specificity, but normal tissue should be frequently tested at the same high sensitivity as possible metastatic tissue samples. It has been argued that these assays do not necessarily detect viable tumor cells, since they are able to detect a piece of mutated DNA coming from degraded carcinoma cells and being transported within lymph vessels (Yamamoto et al., 1997). On the other hand, these assays did provide information for stage I/II patients about a low or high risk for recurrent disease after primary surgery (Hayashi et al., 1995). By a quantitative evaluation of micrometastatic disease in lymph nodes by real-time quantitative PCR, it will be possible to detect occult tumor cells more reproducibly, reliably, and rapidly. By applying this technique, it may be possible in the near future to immediately use PCR results to choose therapeutic strategies for individual ¨ patients (Schuler et al., 1999; Miyake et al., 2000). Prospective studies on large numbers of patients have to be carried out until molecular diagnostics becomes routine procedure in this situtation. The molecular genetic as well as histochemical findings should be confirmed until randomized adjuvant clinical trials can be initiated.
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VI. CONCLUDING REMARKS Molecular monitoring of residual malignant cells by quantitative PCR techniques in patients with leukemia and lymphoma during and after therapy provided important information about the effectiveness of treatment. Sequential quantitative determinations of residual malignant cells in bone marrow and peripheral blood are of prospective value, as has been shown by MRD analysis in patients with various malignant diseases, such diseases include childhood ALL at several time points after the induction therapy (van Dongen et al., 1998; Cave et al., 1998); APL during and after chemotherapy (Diverio et al., 1998); CML during treatment with ␣-interferon or after allogeneic BMT (Lin et al., 1996; Hochhaus et al., 1996a; Mensink et al., 1998); and after radiotherapy for localized follicular lymphoma or autologous BMT ¨ ¨ for relapsed lymphoma (Dolken and Hirt, 1997; Hirt and Dolken, 2000). The quantification of minimal residual cells by real-time quantitative PCR will presumably have a major impact on our therapeutic strategies to treat patients with leukemia, lymphomas, and solid tumors. By following the decline of leukemia or lymphoma cells during first-line therapy, new drugs, drug combinations, and schedules can be developed, and existing ones can be optimized. The kinetics of disappearance of tumor cells will provide information about the efficacy of the treatment, including an assessment of chemotherapy resistence or sensitivity to be confirmed later by the results of long-term molecular monitoring and clinical follow up. Some novel therapeutic strategies might have a great chance to be effective preferentially in MRD, such as adoptive immunotherapy, vaccination with genetically engineered tumor cells or idiotypes, and monoclonal antibody based therapies. These new therapeutic approaches should be controlled at the molecular level; the goal may be molecular remission, since in many diseases this correlates with a high chance for maintaining stable clinical and hematological remission or to being cured. Very promising results have been obtained in a vaccination study using B-cell lymphoma-specific idiotypes in combination with GM-CSF, since conversions to PCR-negativity and sustained molecular remissions have been observed after this treatment in PCR-positive patients with follicular lymphoma in (1.) clinical remission after chemotherapy (Bendandi et al., 1999). In addition, it seems to be very important to recognize a molecular relapse in patients with leukemia as soon as possible to start treatment early, since low numbers of tumor cells might be killed more efficiently than large numbers at hematological relapse, usually detected several months later. Further clinical studies incorporating quantitative PCR assays have to be carried out to be able to use the terms molecular remission and molecular relapse, since these are evolutionary terms that have to be defined for every malignant disease by sequential quantitative PCR analyses
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and not just single assays or numbers of malignant cells. The combination of chemotherapy with monoclonal antibody to CD20 leading to negative PCR results in low grade follicular lymphomas might change our view about MRD in patients with this disease. At present, it is not known whether the absence of circulating t(14;18)-positive B cells (lymphoma cells?) after this treatment will be translated into an increased cure rate. The T-cell dosage in CML patients after allogeneic BMT is another case in point—to give enough T cells to induce the GvL effect to reduce Ph1-positive myeloid progenitor cells seems to be important, but life-threatening GvHD should be avoided. Furthermore, in autologous blood stem cell transplantation or BMT, the efficacy of different purging procedures of hematopoietic stem cell harvests can be efficiently controlled. One of the most fascinating findings in MRD studies on patients with leukemia and lymphoma was the observation that the persistence of translocation-carrying cells is compatible with remaining in long-term remission, as has been shown in t(14;18)-positive lymphoma, t(8;21)-positive AML, and inv(16)-positive AML. Therefore, it might not be necessary to achieve a “complete molecular remission” defined by negative PCR results at a certain sensitivity. Stable low levels of PCR-positive cells seem to be compatible with continuing remission in many malignant diseases; increasing levels seem to announce or indicate recurrent disease. In patients with t(8;21)-positive AML or t(14;18)-positive lymphoma in long-term remission, translocation-specific transcripts or DNA fragments have been detected in presumably nonmalignant pre-leukemic or pre-lymphoma cells. These clonal cells detected after successful chemotherapy may be precursor cells of the malignant disease insofar as they lack at least some or all additional genetic changes required for a full malignant phenotype. The survival of these cells can be explained on the basis of a multistep evolution of most hematological malignancies and differences in the susceptibility of cells in different stages of progression to a malignant phenotype to the killing effects of chemotherapy. Successful chemotherapy might eradicate the fully malignant clones, but their ancestors, the “premalignant clones,” may survive, detectable by PCR. These results are also compatible with the findings in healthy individuals of cells carrying so-called primary chromosomal aberrations thought to be specific for certain types of leukemia and lymphoma. These transloca¨ ¨ tions [t(14;18), t(9;22), t(8;14) (Muller et al., 1995), t(2;5) (Trumper et al., 1998)] are believed to belong to the primary events in tumorigenesis, but are by themselves not able to generate the full malignant phenotype, since further progression by acquisition of additional genetic changes seems to be necessary. Alternatively, tumor cells might survive in long-term remission in a state of dormancy for prolonged periods of time (Uhr et al., 1997). In solid tumors, the metastatic process in most tumors is quite inefficient, since most of the circulating tumor cells are going to be killed (Weiss, 1990).
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Despite these findings, micrometastatic disease detected by PCR in regional lymph nodes has been correlated with an increased incidence of recurrent disease in CRC (Liefers et al., 1998; Hayashi et al., 1995). If these results can be confirmed in large prospective clinical trials using standardized PCR techniques, the detection of occult tumor cells will form the basis for innovative adjuvant treatment protocols to be analyzed in randomized clinical trials. Real-time quantitative PCR is a very sensitive and reproducible technique that will help to detect micrometastatic disease in CRC in the most reliable way necessary for large clinical studies. Using this technique, it may be possible to avoid unnecessary chemotherapy or surgery, to start adjuvant therapy earlier, to control its efficacy, and to decide about the duration of the treatment based on molecular monitoring. Finally, this strategy may be applied to other malignant solid tumors to improve the overall treatment results. Molecular diagnosis and staging as well as molecular monitoring of MRD in patients with hematopoietic malignancies and solid tumors have the potential to aid clinicians in making therapeutic decisions, since a highly sensitive evaluation cannot be performed by current conventional methods. Therefore, there is urgent need for optimized and standardized protocols for PCR analysis. Quantitative PCR assays based on real-time technology are believed to provide solutions to many problems associated with the use of the standard PCR technique. The tumor cell-specific PCR assays have to be evaluated in prospective clinical trials to assess the biological and clinical significance of MRD in various malignancies. If the results of quantitative PCR assays for the detection of micrometastasis in solid tumors or MRD in hematological malignancies are found to reliably predict recurrent disease, they will surely have a major impact on future treatment strategies.
ACKNOWLEDGMENTS The research work of the author and his coworkers has been supported by grants from ¨ Deutsche Forschungsgemeinschaft and Deutsche Krebshilfe, Dr. Mildred Scheel Stiftung fur Krebsforschung. Dr. Thomas Kiefer is gratefully acknowledged for his critical reading of the manuscript.
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Modeling Prostate Cancer in the Mouse Diego H. Castrillon1,2 and Ronald A. DePinho1,3 1
Department of Adult Oncology, Dana Farber Cancer Institute, 2Department of Pathology, Brigham and Women’s Hospital, 3Department of Medicine (Genetics), Harvard Medical School, Boston, Massachusetts 02115
I. Introduction II. Anatomy of the Adult Prostate Gland A. General Features B. Anatomy of the Mouse Prostate C. Anatomy of the Human Prostate III. Embryology and Growth of the Prostate Gland A. Embryology B. Signaling Pathways Involved in Prostate Development C. Relevance of Prostate Development Signaling Pathways to Prostate Tumorigenesis IV. Components of the Epithelial Compartment: Do Prostate Stem Cells Exist? A. Prostatic Epithelium Cell Types B. Evidence for Prostatic Stem Cells V. Role of Oncogenes and Tumor Suppressors in Prostate Neoplasia A. Difficulties in Studying Genetic Alterations in CaP B. RAS Activating Mutations C. Amplification of c-MYC D. Inactivation of MXI1 E. Inactivation of PTEN F. Potential Roles for Other Oncogenes and Tumor Suppressor Genes VI. Progress in the development of CaP Animal Models A. General Comments B. Approaches for the Generation of Mouse CaP Models C. Future Prospects References
I. INTRODUCTION Prostate cancer (CaP) is a major worldwide health problem. In the United States alone, an estimated 185,000 cases were diagnosed in 1998, and nearly 40,000 men died of the disease (Landis et al., 1998). Although many prostate cancers eventually metastasize, the majority remain localized and clinically 187 Advances in CANCER RESEARCH 0065-230X/01 $35.00
C 2001 by Academic Press. Copyright All rights of reproduction in any form reserved.
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silent. Despite years of intensive study, the nature of the genetic lesions responsible for tumor initiation and progression in the prostate remain largely unknown. Most oncogenes and tumor suppressor genes, such as RB1 and P53, are infrequently altered in CaP. Moreover, while cytogenetic and molecular data attest to the existence of multiple prostate tumor suppressor genes (Verma et al., 1999), many of these genes remain unidentified, and the isolation of familial predisposition loci (Smith et al., 1996) has proved challenging. Clearly, alternative experimental systems are required to advance these critical initiatives. Mouse model systems of prostate cancer are being constructed and refined and have already yielded useful insights into mechanisms regulating growth and development of the prostate. Due to rapid advances in the field of mouse genetics, it is anticipated that the mouse will become an effective model system for the identification and study of genes and regulatory pathways involved in the genesis, progression, and maintenance of human CaP.
II. ANATOMY OF THE ADULT PROSTATE GLAND A. General Features The prostate is an accessory reproductive gland that in many species produces the bulk of seminal fluid. Unlike other accessory reproductive glands such as the seminal vesicles and bulbourethral (Cowper’s) glands, a distinct prostate gland is present in all mammalian orders, including monotremes and rodents (Price, 1963). In both mouse and human, the prostate is located at the base of the urinary bladder where it surrounds and is intimately associated with the urethra. Although the prostate in diverse mammalian species consists of a relatively small number of separate ductal units, these ductal units vary in their arrangement and precise relationship to each other, making precise anatomic comparisons among species difficult.
B. Anatomy of the Mouse Prostate In the mouse, the prostate consists of three anatomically discrete paired lobes known as the anterior, dorsolateral, and ventral lobes (Fig. 1, see color plate). The anterior lobe is also known as the coagulating gland because of its role in coagulating seminal fluid. The dorsolateral lobe can be divided into dorsal and lateral lobes, based on the anatomic origin of the ducts in the urethra (Sugimura et al., 1986). The location of origin of the prostatic ducts in the urethra would appear to represent the best basis for determining
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homology of prostatic lobes or anatomic regions (Price, 1963); however, as discussed below, such anatomic homology is no guarantee of functional homology.
C. Anatomy of the Human Prostate In man, the prostate is compact and lacks discrete lobes. The prostatic primary ducts (approximately 16–32 in humans) originate independently from the urethra, successively branching peripherally to form separate (i.e., noncommunicating) ductal units (McNeal, 1988). All of the ductal units are surrounded by a common dense stroma consisting of collagenous fibers, fibroblasts, and smooth muscle, and most of the gland is enveloped in a thin capsule of connective tissue. Three anatomic regions—the peripheral, central, and transitional zones—have been described based on histologic appearance, point of origin of the associated prostatic ducts in the urethra, and association with specific disease processes (Fig. 2, see color plate) (McNeal, 1988). Although anatomic comparisons can be made between the mouse and human prostates (for example, the mouse dorsolateral lobe roughly corresponds to the human peripheral zone), such interpretations are subjective, and there are significant differences in homologous lobes in secretory function and response to hormones even in species as closely related as the ¨ mouse and rat (reviewed in Aumuller and Seitz, 1990; McNeal, 1988; Price, 1963).
III. EMBRYOLOGY AND GROWTH OF THE PROSTATE GLAND A. Embryology Much of what is known of prostate development has been learned from studies in the mouse and rat because of the increased opportunities for experimental manipulation, although several observations indicate that prostate development in man is fundamentally similar. The formation of the urorectal septum divides the primitive cloaca into the rectum and urogenital sinus (UGS). The UGS, initially a simple epithelium with surrounding mesenchyme, gives rise to the urethra, most of the urinary bladder, and prostate. At approximately 10 weeks of human fetal development (17 days in the mouse) prostatic buds first develop as outpouchings of the UGS, and subsequently undergo differentiation with hierarchical branching and ductal outgrowth into the surrounding mesenchyme. This process is strictly dependent on
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testicular androgens: UGS from a female fetus will form prostatic tissue if exposed to androgens. Conversely, males with mutations in the androgen receptor (AR) or 5␣-reductase (which converts testosterone to the more potent dihydrotestosterone) do not undergo prostatic development (reviewed in Cunha et al., 1987).
B. Signaling Pathways Involved in Prostate Development A series of now classic tissue recombination experiments in the mouse established the importance of mesenchymal–epithelial interactions in prostatic development. These experiments utilized combinations of UGS mesenchyme and epithelium from wild-type and testicular feminization mice with AR gene mutations, and established several important principles: 1. The initial steps in prostate development (epithelial budding and ductal branching) require the presence of AR only in the mesenchyme, demonstrating an essential role for UGS mesenchyme in the induction of prostatic epithelial differentiation. 2. Normal epithelial function, as determined by secretion of prostatespecific proteins, requires AR in the epithelium. 3. The epithelium in turn is necessary for normal mesenchymal differentiation into smooth muscle (Cunha and Lung, 1978). Intriguingly, mesenchyme derived from distinct regions of the UGS is capable of inducing lobe-specific differentiation (Takeda et al., 1990). The generation of tissue recombinants will likely continue to be an important experimental strategy in the analysis of transgenic or knockout mice with defects in prostate development. Relatively little is known about the signaling cascade mediating these mesenchymal–epithelial interactions. The secreted glycoprotein Sonic hedgehog is expressed in the UGS epithelium coincident with the formation of the primary prostatic ducts, consistent with a role as a paracrine factor involved in the initiation of prostate development, and blockade of Shh function interferes with prostatic ductal growth and morphogenesis (Podlasek et al., 1999). Growth factors including FGF 7 and FGF 10 are expressed in the UGS mesenchyme and may also function as paracrine epithelial regulators. However, these growth factors appear not to be regulated by androgens in vivo, suggesting that other growth factors are also involved (Sugimura et al., 1996; Thomson et al., 1997). The homeobox gene Nkx3.1 has emerged as an important marker of prostate embryonic development. Although Nkx3.1 is transiently expressed
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in several nonurogenital tissues during embryonic and postnatal development; its expression in adults is highly restricted, being expressed only in the prostate (all three lobes in the mouse) and the bulbourethral gland, and not in any adult female tissues (Bhatia-Gaur et al., 1999; Bieberich et al., 1996; Sciavolino et al., 1997). In the embryonic UGS, Nkx3.1 expression is confined to the epithelium and demarcates regions where prostatic buds will arise, predating their emergence by 2 days (Bhatia-Gaur et al., 1999); Nkx3.1 is thus the earliest known marker of prostatic epithelial differentiation. Importantly, Nkx3.1 expression is highly regulated by androgens, with mRNA levels decreasing 10- to 30-fold within 3 days of castration (Bieberich et al., 1996; Sciavolino et al., 1997), and its expression in the human prostate cell line LNCaP is markedly increased upon androgen stimulation (He et al., 1997). AR signaling thus appears to be directly required for maintenance of Nkx3.1 expression. AR signaling must also be required for initiation of Nkx3.1 expression (since Nkx3.1 is not expressed in the female UGS), but this is likely indirect and mediated by the UGS mesenchyme (Bhatia-Gaur et al., 1999). Shh is a candidate mediator of this indirect regulation, as it has been shown to induce Nkx3.1 expression during somite formation (Kos et al., 1998). Mice with targeted disruption of the Nkx3.1 locus had abnormal ductal morphogenesis with fewer prostatic duct tips than wild-type controls, although the overall size and weight of prostatic lobes were normal. In adults, prostatic epithelium exhibited altered production of secretory proteins. Since Nkx3.1 is not essential for prostatic differentiation, other factors must be involved (Bhatia-Gaur et al., 1999). One recently identified example is the p53 homolog p63 (see next section): no prostatic duct formation or epithelial budding occurs in p63-deficient mice (Signoretti et al., 2000).
C. Relevance of Prostate Development Signaling Pathways to Prostate Tumorigenesis Further elucidation of molecular signaling pathways controlling prostate growth and development is an important goal, since such pathways are likely to be involved in prostate tumorigenesis. AR signaling illustrates this point, as it is necessary for prostate development and function (Cunha et al., 1987) as well as CaP initiation and maintenance. Nkx3.1 has also been implicated in prostate tumorigenesis. Nkx3.1 loss in the mouse leads to a preneoplastic phenotype of age-dependent prostatic hyperplasia and dysplasia. The human ortholog maps to 8p21, a chromosomal region that undergoes frequent loss of heterozygosity (LOH) in prostate cancer. Coding region mutations have not yet been identified in human prostate cancers (Voeller et al., 1997), but other mechanisms of gene inactivation may be operating, and the locus
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is haploinsufficient in the mouse, as Nkx3.1 heterozygotes exhibit some degree of age-dependent prostate hyperplasia and dysplasia (Bhatia-Gaur et al., 1999). That Nkx3.1 is the 8p21 tumor suppressor gene remains an intriguing hypothesis that warrants further study, and the work to date illustrates the utility of the mouse for dissecting signaling pathways involved in both prostate development and tumorigenesis.
IV. COMPONENTS OF THE EPITHELIAL COMPARTMENT: DO PROSTATE STEM CELLS EXIST? A. Prostatic Epithelium Cell Types Prostate glands contain three distinct cell types: secretory, basal, and neuroendocrine (Fig. 3, see color plate). The luminal secretory layer is responsible for the synthesis of products such as prostate-specific antigen (PSA) in man and probasin in rodents. The basal cell layer lies below the secretory cells, rests on the basement membrane, and expresses a different group of markers, including high molecular weight keratins (HWMK). The prostate (like the gastrointestinal tract and lung) also contains scattered but widely distributed neuroendocrine cells. These cells are not readily distinguished with routine histochemical stains, but are highlighted through the use of specific immunohistochemical markers such as chromogranin and synaptophysin (McNeal, 1988). Neuroendocrine cells contain cytoplasmic dense core neurosecretory granules with bioactive products such as serotonin and assorted neuropeptides. The physiologic role of neuroendocrine cells in the prostate remains mysterious, but it is speculated that they regulate prostatic cell growth, differentiation, and/or secretory processes (reviewed in Abrahamsson, 1999).
B. Evidence for Prostatic Stem Cells Several observations suggest that the prostate, like the skin, colon, and hematopoietic lineage, contains a multipotent stem cell compartment that replenishes secretory cells, and perhaps neuroendocrine cells as well (De Marzo et al., 1998). These putative prostatic stem cells most likely represent a subset of cells within the basal layer. Following androgen withdrawal by castration, the prostate involutes and the great majority of prostatic secretory cells (but not basal cells) undergoes apoptosis (Kyprianou and Isaacs, 1988). Yet, upon readministration of androgens, the epithelium regenerates and the prostate returns to normal size and function. This process can be repeated many times,
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revealing the existence of a reserve population of androgen-independent but androgen-sensitive stem cells that can be stimulated to divide and differentiate. In radiolabeling experiments as well as immunohistochemistry using cell proliferation markers such as PCNA and Ki-67, the basal cell layer appears to be the predominant proliferative compartment in the normal prostate. Furthermore, immunohistochemical studies have demonstrated in normal prostate glands occasional cells expressing HMWK + PSA or HMWK + chromogranin (Bonkhoff et al., 1994). The presence of such double-positive cells suggests that the three prostatic epithelial cell types are interrelated, and is consistent with the existence of prostatic stem cells. p63 is expressed in basal cells in a variety of human and mouse tissues, including the skin, cervix, and prostate (Yang et al., 1998), and remarkably, p63-deficient mice exhibit prostate agenesis (Signoretti et al., 2000), among other severe abnormalities in epithelial development in tissues where p63 is expressed (Yang et al., 1999). Further investigation will be required to determine if p63 is required for stem cell function per se or for later steps in prostatic epithelial development or maturation. Another marker—prostate stem cell antigen (PSCA)—may also be useful for further lineage analyses. PSCA is a cell surface marker whose expression is most prominent in the bladder and prostate. In the prostate, PSCA transcripts have been localized by mRNA in situ hybridization to a subset of basal cells, suggesting a possible role in stem cell maintenance or differentiation (Reiter et al., 1998). The majority of human CaP exhibit predominantly secretory differentiation, as evidenced by their selective expression of secretory markers such as PSA (McNeal, 1988) and lack of expression of the basal cell markers p63 and HWMK (Signoretti et al., 2000). Interestingly, at least 10% of prostatic adenocarcinomas contain scattered cells exhibiting neuroendocrine differentiation, typically identified by immunohistochemistry. Such neuroendocrine differentiation has been associated with androgen resistance and worse prognosis, but not all studies agree. Since neuroendocrine cells are androgen independent, it is speculated that neuroendocrine differentiation is a mechanism for the development of androgen resistance in CaP (Abrahamsson, 1999). Lesions exhibiting basal cell differentiation have also been described in the prostate. These appear to be essentially benign basal cell hyperplasias that are unrelated to prostatic intraepithelial neoplasia (PIN) or CaP, although exceedingly rare basaloid carcinomas have been identified (Epstein, 1995). The existence of prostatic stem cells is of profound importance for understanding prostate development, homeostasis, and pathology. It also has fundamental implications for the development of mouse CaP models, as discussed below. Though the preponderance of evidence favors the existence of prostatic stem cells as a component of the basal layer, such cells remain to be definitively identified, isolated, and characterized.
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V. ROLE OF ONCOGENES AND TUMOR SUPPRESSORS IN PROSTATE NEOPLASIA A. Difficulties in Studying Genetic Alterations in CaP Many oncogenes and tumor suppressor loci have been evaluated for genetic alterations in CaP. Studies have in many cases come to greatly different conclusions as to the frequency and role of particular genetic alterations in CaP. It is also remarkable that no gene has been found to be mutated in greater than 10% of cases even for well-characterized tumor suppressor loci that undergo frequent (>50%) LOH in CaP. Several features of CaP make genetic analyses especially difficult and potentially unreliable: 1. The tumors are relatively small (often less then 1 or 2 cm), limiting the amount of tissue available for research. 2. The tumors are difficult and in many cases impossible to appreciate grossly. 3. The tumors are irregularly infiltrative, and even in involved areas, tumor cells are often admixed with an abundance of stroma and normal glands. 4. The distinction between malignant and benign cells (i.e., invasive carcinoma vs atrophy, a secondary change due to aging or antiandrogen treatment) can be extremely subtle, requiring consultation with a pathologist. These features may lead to significant underestimation of mutation frequencies. Fortunately, tissue microdissection provides a means to circumvent these problems (Simone et al., 1998) and should improve the yield and accuracy of cancer gene discovery efforts. In the subsections that follow, we will not attempt to review exhaustively previous studies that have catalogued CaP-associated genetic changes, but rather aim to discuss those genetic alterations that are either reasonably well established or might be especially relevant to the development of mouse CaP models.
B. RAS Activating Mutations Activating mutations in H-, K-, and N-RAS have been found in CaP in 0 to 25% of cases, depending on the study (Moyret-Lalle et al., 1995; Shiraishi et al., 1998), and most authors have concluded such mutations are rare. However, these studies, although small, have identified activating mutations in H-, K-, or N-RAS in at least 5% of primary tumors in Western males, compared with a uniformly higher incidence of 20–30% in Japanese men (Gumerlock et al., 1991; Konishi et al., 1997; Shiraishi et al., 1998; Watanabe et al., 1994). Therefore, RAS mutations appear to be present in
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at least 5% of CaP, and when present, likely contribute to human prostate tumorigenesis. Notably, ras has been shown to contribute to the formation of prostate tumors in a reconstituted mouse organ model system (Thompson et al., 1989).
C. Amplification of c-MYC Comparative genomic hybridization studies have documented gain of chromosome 8q in CaP, and fluorescence in situ hybridization (FISH) has demonstrated low-level amplification of c-MYC, which maps to 8q24, in more than 20% of advanced CaP. c-MYC amplification correlated with overexpression of c-MYC protein (Jenkins et al., 1997). The cell-surface marker PSCA, originally discovered on the basis of its overexpression in CaP and discussed above, also maps to 8q24. In some cases evaluated by dual-probe FISH, c-MYC but not PSCA was overrepresented, indicating that PSCA lies outside the minimal amplicon and therefore is probably not the biological target of amplification (Reiter et al., 2000). Although it remains to be demonstrated formally that c-MYC amplification has a causal role in CaP, MYC has been shown to cooperate with RAS in the above-mentioned reconstituted mouse organ model system (Thompson et al., 1989). The role of c-MYC in CaP clearly warrants further investigation.
D. Inactivation of MXI1 Other evidence pointing to the importance of MYC in CaP comes from analysis of mice with mutations in Mxi1. Mxi1 is a member of the Mad(Mxi1) family of proteins that function as active transcriptional repressors and potent antagonists of the myc oncoprotein (Schreiber–Agus and DePinho, 1998); Mxi1 loss is therefore functionally equivalent to myc overexpression. Mxi1 mice consistently showed a number of age-related changes in the prostate, including significant dysplasia and hyperplasia, seen as foci of enlarged and complex glandular structures. Proliferation of the prostatic epithelium, as measured by mitotic indices and Ki67 immunostaining, was markedly higher in Mxi1 prostates than in wild-type controls, indicating that the prostatic hyperplasia observed histologically in a permissive genetic background is the result of aberrant growth control due to loss of Mxi1 function (Schreiber–Agus et al., 1998). This phenotype bears a striking resemblance to PIN, the in situ precursor lesion to invasive carcinoma (Epstein, 1995). These observations are additional evidence that misregulation of MYC can contribute to prostate tumorigenesis. However, studies evaluating the frequency of MXI1 mutations in human CaP have yielded conflicting results (e.g., Eagle et al., 1995; Kuczyk et al., 1998).
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E. Inactivation of PTEN The PTEN locus maps to 10q23, a region of frequent chromosome loss in CaP (>50% of cases, by a variety of techniques), particularly in higher-stage tumors. Studies evaluating PTEN mutational status in CaP have confirmed the high incidence of LOH at the locus, but have identified a relatively low incidence of PTEN mutations. A recently published metaanalysis of such studies found that in 271 prostate tumors of all stages analyzed to date, a total of 26 PTEN mutations were identified (10%), 16 of which were homozygous deletions, and 10 of which were point mutations. In comparison to CaP, PTEN mutations were identified in 45% of endometrial adenocarcinomas and 24% of malignant gliomas, tumors that frequently undergo 10q23 loss (Ali et al., 1999). Complete loss of PTEN protein expression occurs in 20% of CaP cases evaluated by immunostaining, and loss of expression correlated with higher Gleason scores (McMenamin et al., 1999). PTEN haploinsufficiency may account in part for the lack of second allele mutations in some tumors, since a noticeable increase in phosphorylation of the downstream kinase AKT is observed in Pten +/− embryonic stem cells (Sun et al., 1999), and Pten +/− mice develop hyperplastic lesions in multiple organs early in development (Di Cristofano et al., 1998). Targeted gene disruption of Pten results in embryonic lethality in homozygous mice, but heterozygotes develop preneoplastic and frankly malignant lesions at multiple sites, demonstrating that Pten is a potent tumor suppressor. In the prostate, these mice develop epithelial hyperplasia and dysplasia, consistent with a role for Pten as an important regulator of prostatic epithelial growth in vivo (Di Cristofano et al., 1998; Podsypanina et al., 1999). Identification of invasive CaPs in Pten +/− mice may require extended longitudinal studies. Additional details of the PTEN pathway are provided in Fig. 4 (see color plate). It will be instructive to analyze PI3K and other components of this pathway, including the AKT kinases, forkhead transcription factors AFX, FKHR1, and FKHRL, and the forkhead-responsive gene targets for genetic alterations in CaP. Though PTEN is likely a 10q23 prostate tumor suppressor locus, the possibility exists of additional cooperating loci near the 10q23 region, and has been suggested by some studies (Robertson et al., 1999). One candidate for such a cooperating locus is MXI1, located at 10q25 (Schreiber–Agus et al., 1998).
F. Potential Roles for Other Oncogenes and Tumor Suppressor Genes Studies of P53 have yielded variable results, but mutations appear to be present in perhaps 20% of advanced cases (e.g., Macera et al., 1999).
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Immunohistochemical analyses have found accumulation of the protein (an indirect marker of missense mutations) in CaP cells; in several studies, p53 overexpression correlated with higher tumor grade and stage (MacGrogan and Bookstein, 1997). P53 mutations thus appear to occur as a relatively late event in CaP progression. Other classic tumor suppressor genes, such as RB1 and INK4A, have not been strongly implicated in human CaP, and significant prostatic lesions have not been reported in mouse models bearing homozygous mutations in these loci. Amplification of the Her2/neu oncogene, a frequent event in breast carcinomas, does occur in CaP, but studies have come to widely ranging conclusions as to its frequency and utility as a prognostic marker. The CDK4 inhibitor p27 kip1 is frequently downregulated in PIN and CaP and has utility as a prognostic marker (Macri and Loda, 1998). Mutations are rare, and downregulation occurs through other mechanisms, including ubiquitin-mediated proteolysis. The p27kip1 chromosomal region 12p12–13 is frequently lost in CaP (Kibel et al., 1998), and the mouse p27kip1 gene is haploinsufficient for tumor suppression in multiple tissues (Fero et al., 1998), with heterozygous mice exhibiting hyperplasia in multiple organs, including the prostate (Cordon-Cardo et al., 1998). The loss of a single allele may thus be sufficient to promote prostate tumorigenesis. In light of recent data linking Myc and Mxi1 transcriptional regulation to ubiquitin-mediated proteolysis (O’Hagan et al., 2000), it is tempting to speculate that c-MYC amplification and p27kip1 downregulation in CaP are mechanistically related.
VI. PROGRESS IN THE DEVELOPMENT OF CaP ANIMAL MODELS A. General Comments Mouse models provide unique opportunities to generate and test hypotheses in vivo, which can then be further tested and verified in humans. The fact that no mouse model of CaP can recapitulate all aspects of the human disease should be no disincentive to generate, characterize, and refine such models. For example, there is evidence that in man, expression of PSA (a secreted protein of the kallikrein protease family) confers a selective advantage and a more invasive phenotype to prostate cancer cells, apparently due to digestion of the extracellular matrix or other substrates (Webber et al., 1995). However, there is no ortholog of human PSA in the mouse. Most mouse models would therefore not recapitulate this potentially relevant molecular trait of human CaP. On the other hand, a mouse model might afford an opportunity
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to study the importance of PSA expression in CaP progression through (for example) the generation of transgenic mice expressing human PSA in the prostate, a feat that has been accomplished (Wei et al., 1997). Every mouse CaP model will lead to unique insights, and it will be advantageous to have a diversity of models available. For a complete list of mouse CaP models generated to date, the reader is referred to a recent review (Sharma and Schreiber–Agus, 1999).
B. Approaches for the Generation of Mouse CaP Models Mouse CaP models, that is, mice that develop PIN or invasive prostatic carcinoma, can be generated by a number of approaches, and combinations of these approaches will likely become an important experimental strategy in the future. Several conventional knockout mouse strains exhibit prostatic hyperplasia and/or dysplasia (Pten, Nkx3.1, and Mxi1, discussed in the preceding section), either as heterozygotes or homozygotes. Consistent with the need for two or more cooperating mutations in most experimental systems of tumorigenesis, these single gene knockouts to date have not resulted in frankly invasive carcinomas. Pten exemplifies another limitation of this approach. PIN is observed in Pten heterozygotes, but embryonic lethality precludes analysis of homozygous Pten loss (Di Cristofano et al., 1998; Podsypanina et al., 1999), which might result in a more severe phenotype with decreased latency. Even in heterozygotes, early mortality from tumors at other locations might preclude long-term studies of progression in the prostate. The generation of conditional (“floxed”) alleles using the Cre/loxP system should be useful for the analysis of mutations that are homozygous lethal. Intronic loxP sites would be introduced into the gene to be studied, and Cre recombinase would be directed to prostatic epithelium using a prostatespecific promoter (see below). Subsequent Cre mediated deletion would result in a prostatic epithelium-specific knockout. Transgenic mice specifically expressing Cre in prostatic epithelium have recently been reported (Maddison et al., 2000). A variation of this approach permitting temporal as well as spatial control would be the use of Cre fused to a mutant human estrogen receptor (ERT) which binds tamoxifen but not endogenous estrogens (Brocard et al., 1997). Cre-ERT is induced following administration of tamoxifen at the desired developmental stage. Such transient induction might also be advantageous, given anecdotal evidence that Cre can be cytotoxic. However, there is a caveat to this approach, and careful controls would be required. Although ERT does not bind to and is not effectively induced by endogenous estrogens, tamoxifen per se does have potent biological effects
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in vivo, acting as an agonist or antagonist of naturally occurring estrogen receptors. The prostate is highly hormone sensitive, and several studies have documented that even a single neonatal administration of tamoxifen or estrogen has potent, long-term effects on the rodent prostate, including decreased weight, ductal branching and AR expression, and increased inflammation (Singh and Handelsman, 1999; Stoker et al., 1999). Inducible systems permit the expression of dominantly acting oncoproteins in somatic tissues, and are useful for studies of factors involved in tumor initiation and progression. A system that has been employed successfully in an inducible mouse model of melanoma makes use of two separate transgenic elements, one consisting of the reverse tetracycline transactivator (rtTA) under the control of a tissue-specific promoter and another consisting of a dominant oncogene (in the melanoma model, H-Ras V12G) driven by a minimal promoter containing multimerized tet-operons (designated Tet– Ras). Transcriptional activation of the oncogene is induced by administration of doxycycline in the drinking water, and is rapidly reversed by withdrawal of doxycycline (Chin et al., 1999). This approach should be applicable to the study of any oncoprotein with a known or suspected role in CaP, such as MYC and RAS, among others. Several strategies for the generation of mouse CaP models depend on the ability to direct the expression of transgenes in prostatic epithelium. Promoter fragments derived from the rat probasin (rPB) gene have proven reliable and are being used widely for this purpose. The rPB gene encodes a secreted protein of unknown function but with similarity to odorant-binding proteins; expression is limited to the prostate and is highest in the dorsolateral lobe. The promoter contains multiple androgen response elements; rPB expression is highly androgen dependent and is dramatically decreased following castration. Transgene expression has been studied using minimal (426-bp) and long (12-kb) rPB promoter constructs linked to a reporter gene. Not all founder lines expressed the transgene, but expression was prostate specific in expressing lines. The level of expression was highly dependent on the site of integration, with different transgenic lines exhibiting marked variation (two to three logs) in expression levels in individual prostate lobes, even with the 12-kb promoter (Yan et al., 1997). Cointegration of a short promoter construct with a matrix attachment region (MAR) resulted in restriction of transgene expression to the dorsolateral lobe; variation in expression levels was not eliminated (Greenberg et al., 1994). Promoter sequences from the human PSA gene have also been used to direct the expression of transgenes in the mouse prostate. In an early study, a small (650-bp) PSA promoter fragment was fused to an activated RAS allele. Transgenic animals developed salivary gland tumors due to missexpression of the construct in salivary gland; expression was undetectable in the prostate. This misexpression was due to absence of essential nearby cis-elements, since
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subsequent studies using larger PSA promoter constructs resulted in prostatespecific expression. A 6-kb PSA promoter fragment targeted expression of the LacZ gene to the prostate, limited to the lateral lobe (Cleutjens et al., 1997), and a 14-kb human genomic DNA fragment encompassing the PSA coding region and flanking regulatory sequences mediated androgen-dependent prostate-specific expression of PSA in transgenic mice (Wei et al., 1997). It is remarkable that transcriptional regulation in the prostate, including the function of both cis-acting control elements and trans-acting transcriptional factors, is conserved to such a degree in the mouse and human. The first transgenic models of CaP used minimal or long rPB promoter constructs linked to SV40 T antigen (Greenberg et al., 1995; Kasper et al., 1998). In these models, 100% of males developed prostate tumors, beginning as multifocal hyperplasia by 10 weeks of age that progressed to severe diffuse hyperplasia and invasive cancer. Tumors grew to large size and were always palpable well before death from tumor burden or urinary obstruction. No primary neoplasms were detected at other sites or in females, consistent with prostate-specific expression of the transgenes. In one model, tumors were largely androgen independent, and metastases were observed in 100% of mice by 28 weeks, whereas in a second model, tumors were androgen dependent, and metastases were not observed (Gingrich et al., 1996). These differences in biological behavior likely relate to the use of different T antigen constructs. In the metastatic, androgen-independent CaP model, both small and large T antigens are expressed, whereas only large T antigen is expressed in the nonmetastatic, androgen-dependent model (Kasper et al., 1998). The activation of additional targets by small T antigen is likely responsible for the more aggressive phenotype. These groundbreaking studies demonstrated the general utility of rPB promoter constructs and established the feasibility of creating mouse CaP models. It is notable that the rPB/T antigen models developed highly aggressive tumors, even though rPB is expressed in a compartment (the secretory cells) that is ostensibly terminally differentiated. Presumably, T antigen forces reentry into the cell cycle, although leaky expression in a stem cell or intermediate compartment is also possible. Oncoproteins with less potent or less pleiotropic effects on the cell cycle, however, might not force such a cell cycle reentry, and it is possible that the transgene-expressing secretory cells would be eliminated through physiologic mechanisms (i.e., sloughing, cell death) before a phenotype became manifest or before the accumulation of additional random genetic events led to an observable phenotype. It may be desirable to direct expression of transgenes to stem cells, should such an approach become technically feasible. However, because we know little about prostate normal growth or tumor progression, this is a theoretical consideration, and the fact remains that the rPB-based tumor models faithfully mimic human CaP in having a predominantly secretory phenotype.
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C. Future Prospects Ultimately, the availability of a wide assortment of transgenic and knockout mice will permit the generation of mouse models useful for exploring the contribution of individual genetic lesions to the neoplastic phenotype, including androgen dependence, abnormal growth, invasion, and metastasis. These mouse model systems will also be useful for the identification and isolation of novel genes important in prostate tumorigenesis. Feasible approaches are many and include the identification of genetic modifiers of mouse prostate tumorigenesis present in laboratory strains, the existence of which is suggested by prior studies (Thompson et al., 1989); insertional mutagenesis based on proviral integration by expression of the avian leukosis virus (ALV) receptor in the prostate, thereby rendering it susceptible to efficient ALV infection and proviral integration (Federspiel et al., 1994); and genome-wide scans using DNA microarrays enabling more focused genetic screens and validation efforts. It is expected that work done in the mouse and human will synergize and accelerate our understanding of the genetic basis of CaP.
ACKNOWLEDGMENTS We thank William Sellers, Massimo Loda, and David Berman for helpful comments on the manuscript. We also thank Michael Shen and the CSH Laboratory Press for permission to reproduce Fig. 1. RAD is an American Cancer Society Research Professor and is supported by NIH grants 9R01 CA86379-08 and 5U01 CA84313-02. DHC was a Damon Runyan–Walter Winchell Foundation Fellow and is supported by NIH grant K08 CA84044-03, a Cap Cure Award, and a Massachusetts Public Health Service Prostate Cancer grant.
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Immunity to Oncogenic Human Papillomaviruses Jozsef Konya and Joakim Dillner1 Laboratory of Tumor Virus Epidemiology, The Microbiology and Tumor Biology Center, Karolinska Institute, S-17177 Stockholm, Sweden
I. Epidemiology of Human Papillomavirus Infection A. The Natural History of Human Papillomavirus (HPV) Infection Is Dynamic II. The Antibody Response to HPV Infection A. The Capsid Antibody Response Is a Marker of HPV Exposure B. Sensitivity, Specificity, and Stability over Time/Natural History and Inter- and Intralaboratory Variability C. Antibodies of Different Isotypes D. Use of HPV Antibody Detection in Epidemiological Studies III. Cellular Immunity to HPV Infection A. Histological Studies B. Role of Cytokines in the Antipapillomaviral Responses C. HLA Class I Association of Cervical Neoplasia D. HLA Class II Association of HPV-Related Cervical Diseases E. HPV-Associated Lesions in Immunosuppressed Patients F. Cell-Mediated Immunity (CMI) in Immunocompetent Women with HPV-Associated Diseases G. Cytotoxic T Cells IV. Does Cross-Protective Immunity Exist? A. Possible Explanations for the Observed Antagonism B. Cooccurrence of HPV Types in Cervical Samples C. The Dynamics of the HPV Infection: Implications for Estimating the Effects of Vaccination References
The establishment of human papillomavirus (HPV) infection as a major cause of several human cancer forms, notably cervical cancer, has spurred development of prophylactic and/or therapeutic HPV vaccines for prevention of cervical neoplasia. Knowledge of the immunity to HPV forms the basis for such endeavors. Method: A literature review of humoral and cellular immunity to HPV. The overview on human leukocyte antigen (HLA) and cervical cancer was expanded to a formal metaanalysis, where relevant articles were located by Medline search and citation analysis and graded by preassigned
1
To whom correspondence should be addressed at the present address: Department of ¨ Sweden. Medical Microbiology, MAS University Hospital, Lund University, S-20502 Malmo,
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C 2001 by Academic Press. Copyright All rights of reproduction in any form reserved.
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quality criteria on study design. Results: The antibody response to the HPV particle is dominated by a neutralizing antibody response to a typespecific, conformationally dependent immunodominant epitope. Vaccines based on viral particles lacking the viral genome (virus-like particles, VLPs) have been highly successful in preventing and treating HPV infection in several animal model systems. In humans, the serum antibody response to VLPs is stable over time, also after the HPV infection has been cleared, resulting in HPV serology being used as a marker of cumulative HPV exposure in spite of the fact that a significant proportion of HPV-exposed subjects fail to seroconvert. More than 90% of HPV infections will clear spontaneously. The factors that determine whether an HPV infection is cleared or persists and increases the risk for cancer are not known, but cellular immunity is implicated. Several HLA class II haplotypes are associated with cervical cancer: DQw3 increases and DR13 decreases the risk for cervical cancer in general (odds ratios (OR) and 95% confidence intervals (CI): 1.25(1.15–1.37) and 0.69 (0.56–0.85), respectively); DR15 increases the risk for HPV16-carrying cancer (OR: 1.47; CI: 1.20–1.81); and DR7 may be either protective or increase the risk. Most cervical cancers have downregulated the expression of at least one HLA class I antigen, whereas class II expression is increased in infected epithelium. A Th2 cytokine profile is associated with progression to cervical cancer. HPV-antigen-specific proliferative responses have been detected in many studies, although it is not entirely clear whether these responses are HPV type specific or may be cross-reactive between HPV types. Specific cytotoxic T lymphocyte (CTL) responses were originally reported in only a minority of infected subjects, typically cancer patients, but with advancing technology, specific CTLs can be stimulated from about half of the women with HPV-carrying disease. In animal model systems, CTL responses can mediate clearance. Conclusion: The antibody response to HPV is a mediator of type-specific protective immunity, which forms the basis for prophylactic vaccine candidates. The cellular immunity to HPV is implicated as an important factor in cervical carcinogenesis, but the main targets and types of responses that mediate HPV clearance are not established. C 2001 Academic Press.
I. EPIDEMIOLOGY OF HUMAN PAPILLOMAVIRUS INFECTION A. The Natural History of Human Papillomavirus (HPV) Infection Is Dynamic The HPV infection is characterized by a very high rate of acquisition. Numerous longitudinal studies have established that each change of sexual partner involves a substantial risk for HPV infection. In Sweden, the risk for seroconversion to the major oncogenic HPV type, HPV16, increases linearly by about 4% for each life-time sexual partner up to a plateau of about 32% among women with on average eight lifetime sexual partners (Dillner et al., 1996). A longitudinal cohort study of teenage girls found that none of the girls without sexual experience was or became seropositive for HPV16 or 33, whereas among girls who had had five or more partners, 54% were positive ¨ et al., 1994, 1996). at some point in time (Andersson–Ellstrom
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Fortunately, HPV infection is also characterized by a very high rate of spontaneous clearance. The cohort studies of HPV DNA positive women have been quite consistent in their estimates of a 70% clearance rate on a 12-month follow-up (Hildesheim et al., 1994; Evander et al., 1995; Ho et al., 1998). After 18 months, > 80% of infections have cleared (Ho et al., 1998). It is women with persistent type-specific positivity that are at increased risk to develop invasive cervical cancer (Wallin et al., 1999). The dynamic nature of the infection makes point prevalence studies of HPV DNA with measurements at only one point in time hard to interpret. There are several examples of how such studies have provided misleading comparisons of the total HPV exposure in different populations, possibly because of incidences that peak at different ages in different populations (Kjaer et al., 1993; Reeves et al., 1994). The HPV DNA point prevalence thus measures recent exposures and a subset of old exposures that have become persistent. The determinants of which infections become persistent are not well known and may be different in different populations.
II. THE ANTIBODY RESPONSE TO HPV INFECTION A. The Capsid Antibody Response Is a Marker of HPV Exposure A serological assay based on HPV16 capsids (also denoted virus-like particles, VLPs) that correlates with type-specific detection of HPV infection, as determined by detection of the viral genome, was established in 1994 (Kirnbauer et al., 1994). Several years of research were required to validate these assays, for several reasons: 1. Early methods for HPV DNA detection were inaccurate, to the extent that the misclassification made early studies of HPV epidemiology seriously flawed (Franco, 1991). 2. If we also count partially characterized HPV types, there are over 130 different types, many of which are not malignancy associated and not sexually transmitted. Serological cross-reactions are hard to predict based on DNA homology. 3. Because most HPV infections are rapidly cleared spontaneously, many people testing negative for HPV DNA may have had a previous infection. 4. Seroconversions can be delayed many months after the detection of ¨ et al., 1995a,b; Carter et al., viral DNA in a subset of patients (Wikstrom 1996). Thus, many people with a recently acquired HPV infection may not yet have seroconverted.
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5. Testing for the HPV genome of samples taken from the uterine cervix will not detect infections at other body sites. In spite of these major theoretical difficulties, serology using viral capsids has, for several HPV types, shown an amazing concordance with detection of viral DNA at the cervix. In the original report, serum IgG antibodies against HPV16 capsids of a wild-type HPV16 strain were found in 59% of women testing positive for cervical HPV16 DNA, whereas only 6% of women negative for cervical HPV DNA or positive for the benign HPV types 6 and 11 had these antibodies (Kirnbauer et al., 1994). HPV16 capsids prepared using the originally cloned HPV16 isolate had a serological reactivity devoid of HPV type-specificity. A single amino acid change in the new, wild-type HPV16 isolate enabled the formation of the apparently immunodominant and type-restricted serologically reactive epitope (Kirnbauer et al., 1994). The vast majority of the antibody response in human serum against intact HPV16 capsids can be entirely blocked by a single neutralizing monoclonal antibody, designated V5 (Wang et al., 1997). This apparently immunodominant and type-specific epitope is conformation dependent and only present on assembled capsids, not on monomers of the major capsid protein. This property of the major type-specific epitope forms the basis for two of the methods that can be used to confirm type specificity of an HPV antibody response: (i) The HPV capsid can be disrupted, usually by treatment with high pH carbonate buffer, to destroy the type-specific epitope. Type-specific serological reactivity will be lost by capsid disruption, whereas cross-reactive antibody responses will remain unaffected (Heino et al., 1995). (ii) The HPV16 capsid can be blocked with the V5 monoclonal antibody. Type-specific serological reactivity will be lost, whereas cross-reactive antibodies will remain unaffected (Wang et al., 1997). An alternative method for assaying type-specific antibodies is based on the fact that type-specific antibodies are usually present in higher titers than cross-reactive antibodies. By assigning a “cut-off” value that classifies lowtitered reactivity as negative, type-specific results can be obtained without a negative control or confirmatory assays (Wideroff et al., 1995).
B. Sensitivity, Specificity, and Stability over Time/Natural History and Inter- and Intralaboratory Variability Sensitivity of assays is measured using panels of serum samples obtained from individuals with a documented infection with the virus in question, as measured using detection of the viral genome. State-of-the-art detection of
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viral DNA is not entirely straightforward, with misclassification attributable to inability to distinguish between some of the many viral genotypes, to contamination in polymerase chain reaction (PCR) assays, and to inadequate sampling being the most common problems. Whereas there is good to excellent agreement between laboratories for certain assays, such as the PCR– ELISA system based on the general primers GP5+/GP6+, there is poor agreement between different HPV DNA detection assays (Jacobs et al., 1999). In general, it can be said that studies of the sensitivity of HPV capsid serology that has used state-of-the-art HPV DNA detection methodology has found a sensitivity of 50% or greater (Kirnbauer et al., 1994; Carter et al., 1996; ¨ Wideroff et al., 1995, 1996; Kjellberg et al., 1999; Andersson–Ellstrom et al., 1994, 1996). In a large population-based study that used nested PCR technology, the sensitivity was found to be 65–75% (Kjellberg et al., 1999). Two covariates of HPV seropositivity have been described that may result from misclassification, or may be biological phenomena: viral load and persistence. Clearly detectable presence of HPV DNA is more commonly associated with HPV seropositivity than is weakly detectable presence of HPV DNA (Viscidi et al., 1997). Possibly, a large infection may produce more viral protein that may more effectively induce an antibody response. Alternatively, weakly detectable presence of HPV DNA may more commonly be misclassified and not true infections. Persistent presence of HPV DNA in samples taken at two different occasions from the same woman is more commonly associated with seropositivity than is transient presence of HPV DNA that was not detectable in a second sample taken from the same woman (Wideroff et al., 1995). Possibly, transient infections may not be present long enough in the body to evoke an antibody response. Alternatively, HPV DNA detections that could not be repeated in a second sample may have been misclassified or may have reflected presence of the viral genome that never resulted in an infection. The HPV virion is a stable molecule that is resistant to desiccation and will retain viability extracellularly for at least 1 week (Roden et al., 1997). Specificity is assayed by comparing serum samples taken from women infected with the same HPV type, women infected with other HPV types, and women not exposed to HPV. Comparisons with women infected with other types of HPV are confounded by the fact that the different genital oncogenic types are transmitted similarly and that high-risk group women presently infected with a certain HPV type may have previously had infections with other HPV types. All studies of type specificity of the HPV capsid serology has found a strong type-restricted component, and in a large populationbased study performed in a population with a modest amount of life-time sexual partners, there was no covariation with presence of other HPV types,
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indicating type specificity (Kjellberg et al., 1999). Type specificity of HPV capsid-based assays is also supported by a very large amount of experimental studies on immunological cross-reactivity of monoclonal antibodies against HPV capsids. Whereas disrupted or partially disrupted viruses expose epitopes that are broadly cross-reactive or even group specific (Dillner et al., 1991; Jenson et al., 1980), conformationally dependent epitopes on intact capsids have generally been HPV type specific (Cowsert et al., 1987; Christensen et al., 1996a). The exception is HPV6 and 11, that have been shown to contain shared epitopes and type-specific epitopes on intact capsids (Christensen et al., 1994,1996b). Specificity of HPV capsid serology is also indicated by the fact that panels of serum samples taken from subjects with no or little sexual experience have very low seroprevalences. Monogamous women have, in many studies, been found to have low seroprevalences (between 2 and 7%) (Kjellberg et al., ¨ et al., 1994,1996; Wideroff 1999; Dillner et al., 1996; Andersson–Ellstrom et al., 1996; Viscidi et al., 1997; Carter et al., 1996). Adult virginal women have so far not been found to have any HPV seropositivity, albeit the total ¨ et al., number of virginal women tested is not very large (Andersson–Ellstrom 1994, 1996). Large-scale surveys of seroprevalences among children younger than 13 years of age found seroprevalences on the order of 2% (af Geijerstam et al., 1999; Mund et al., 1997). Although there is consensus that oncogenic genital HPVs are mainly sexually transmitted, there exists controversial data regarding whether nonsexual transmission exists. As the data on this issue have been extensively reviewed (Dillner et al., 1999), it is not repeated here. Suffice it to say that the specificity of HPV capsid serology for sexually transmitted HPV infections is at least 98% and that the specificity may be even higher if some nonsexually transmitted infections have indeed occurred among the control groups of sexually inexperienced subjects. The natural history of the HPV serum antibody response is well known. Seroconversions against the HPV16 capsids are seen concomitantly with ¨ or within a few months following acquisition of HPV16 DNA (Wikstrom ¨ et al., 1994,1996). et al., 1995a, b; Carter et al., 1996; Andersson–Ellstrom In large-scale follow-up studies, the antibody levels have been stable over time (Carter et al., 1996; af Geijerstam et al., 1998a; Shah et al., 1997), even after more than a decade of follow-up (Shah et al., 1997). This is also well in line with the fact that IgG seropositivity to oncogenic genital HPVs is strongly correlated with the life-time number of sexual partners (Carter et al., 1996; Wideroff et al., 1996; Dillner et al., 1996; Viscidi et al., 1997; Olsen et al., 1997; Wang et al., 2000) (a correlate of the life-time cumulative HPV exposure), but not correlated with the recent number of partners (Olsen et al., 1997; Wang et al., 2000), which would have been correlated if seropositivity
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had had more limited stability over time. By comparison, presence of cervical HPV DNA is commonly transient (i.e., not stable over time), and presence of cervical HPV DNA correlates better with the recent number of partners than with the life-time number of partners (Olsen et al., 1997; Hildesheim et al., 1993). The distinction that “seropositivity = lifetime exposure,” whereas “HPV DNA = recent exposure,” is of course not detectable in studies of adolescent women, where life-time and recent number of partners are closely related variables and where HPV DNA detection and HPV seropositivity are usually strongly correlated (Karlsson et al., 1995). The biological and assay variability of HPV serology has been formally quantified in large-scale studies. The variability observed when retesting the same serum sample was similar to the variability seen when serial samples taken after more than 2 years of follow-up were tested, indicating limited biological variability over time (af Geijerstam et al., 1998a). Persistence of HPV antibody levels has been reported even after 15 years of follow-up (Shah et al., 1997). Formal studies of variability among different laboratories has found good agreement, even when assay protocols were not specifically standardized (Strickler et al., 1997) (Table I).
C. Antibodies of Different Isotypes The major isotypes of serum antibodies against HPV capsids are IgG1 and IgA (Wang et al., 2000). Other IgG subclasses are only occasionally detected (Wang et al., 2000). The serum IgA response is also HPV type specific, as demonstrated by its correlation with presence of type-specific HPV DNA (Wang et al., 2000). In contrast to serum IgG, however, serum IgA correlates with the recent number of sexual partners and with the life-time number of partners, mostly among young women (Wang et al., 2000), suggesting that the IgA response is not as biologically stable over time as is the IgG response. Serum IgA may be a marker of recent or active infection, which could also be used in addition to IgG measurements to improve the sensitivity of HPV capsid serology (Wang et al., 2000). Secretory IgA antibodies to HPV capsids are detectable in cervical mucus (Wang et al., 1996). Local IgG has also been found in cervical secretions (Wang et al., 1996). The local IgA has been demonstrated to be HPV type specific (Wang et al., 1996; Bontkes et al., 1999). Specific IgM responses have been difficult to demonstrate in seroepidemiological studies. The typical time-course of the antibody response after experimental inoculation is a rapid IgM and IgA response, followed by rapid disappearance of IgM, maintenance or slow decline of IgA, and appearance of stable IgG levels, but it is not known if this also applies to the natural HPV infection.
Table I Basic Characteristics of the Serum Antibody Response to HPV and Its Use in HPV Seroepidemiology Basic Current Knowledge
Characteristic Biological properties Epitopes recognized Type-specificity
Time-course of induction Biological stability over time
Sensitivity, using detection of cervical HPV DNA as gold standard Specificity, as determined by analysis of unexposed control groups Assay formats
Major isotypes Major applications of HPV seroepidemiology
HPV antibodies in human serum known to be neutralizing. The reactivity of the vast majority of antibodies in human sera can be blocked by a single type-specific and neutralizing monoclonal antibody, implying the existence of an immunodominant type-specific site on the intact HPV particle. Disrupted and partially denatured particles expose epitopes with limited HPV type-specificity. Reactivity of human sera with intact HPV found to be highly type-specific in epidemiological studies, with the exception of HPV6 and 11 which are substantially cross-reactive. Induction of antibodies may be delayed several months after the first detection of HPV DNA. In follow-up studies of patients with condylomas, very late seroconversions (years) have been documented. In large-scale follow-up studies up to 5 years, no significant change compared to random fluctuation noted in antibody levels. Persistence of serum antibody response documented for >15 years. Seroprevalences strongly related to life-time number of sexual partners, but not to number of recent partners. Varies by exact serological assay format and quality of HPV DNA detection methods used for comparison. Most validation studies report about 50% sensitivity. About 98%. The possibility exists that the specificity may be higher, if some seropositive subjects in the control groups of children and virginal women have indeed been infected (i.e., if nonsexual transmission exists). Three major assay formats are extensively used:(a) Direct ELISA using disrupted particles as control antigen, (b) direct ELISA using no control antigen, but high cut-off values, (c) two-site ELISA, also called capture or sandwich ELISA. An intermethod comparison between different laboratories found fair agreement (Kappa: 0.4–0.6). The IgG response is dominated by IgG1. IgG response forms basis for seroepidemiology. IgA response also abundant, but seems less stable over time than the IgG response. Due to its stability over time, it can be used as a marker of life-time cumulative HPV exposure. Not recommended for diagnosis in the individual woman, due to low sensitivity, but useful in comparisons of groups. Major uses: –Studies of trends over time in HPV infection. –Studies of the risk for cancer among HPV exposed individuals. –Studies of the HPV-associated risk of cancer at noncervical sites. –Ecological studies.
Note. For references, see text or the review by Dillner (1999).
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D. Use of HPV Antibody Detection in Epidemiological Studies Since it is the IgG response that is stable over time, it is the IgG seropositivity that is used in epidemiological studies of HPV exposure. As discussed above, the natural history of HPV infection with a high rate of acquisition and a high rate of spontaneous clearance has made it difficult to assess the total amount of HPV exposure in ecological studies based on HPV DNA detection (Kjaer et al., 1993). Indeed, a cervical cancer high-risk population on Greenland has lower HPV DNA prevalences than a lower cervical cancer risk population in Denmark (Kjaer et al., 1993). However, if HPV serology is used, it is found that the population of Greenland does indeed have a higher level of cumulative HPV exposure than the population in Denmark (Nonnenmacher et al., 1996). HPV seroprevalences have in general correlated well with cervical cancer incidences. As an example, the prevalences of HPV16 seropositivity among healthy population-based control subjects in Colombia and Spain were 22 and 3%, respectively (Nonnenmacher et al., 1995), and the incidence of cervical cancer is correspondingly 8-fold higher in Colombia than in Spain. Thus, HPV serology has enabled more informative ecological studies of the importance of the amount of HPV exposure for explaining regional variations in cancer incidence. There are also striking differences in HPV seroprevalences over calendar time. Very low HPV16 seroprevalences (2%) are reported in a populationbased survey from rural Finland in 1968 (Lehtinen et al., 1996; Dillner et al., 1998) and from southern Norway in 1973 (Dillner et al., 1997), whereas present-day HPV16 seroprevalences in these populations are approaching 20% (Olsen et al., 1996; Kibur et al., 2000). A study of HPV16 seroprevalences in a population-based sample of pregnant women in Stockholm, Sweden, between 1969 and 1989 found a 50% increase from 1969 to 1983, but stable seroprevalences during the 1980s (af Geijersstam et al., 1998b). The seroprevalences of herpes simplex virus (HSV) type 2 had been assayed in the same samples and showed a strikingly similar trend, indicating that seroprevalences of common sexually transmitted diseases (STDs) such as HPV and HSV-2 reflect the overall rate of sexual contacts in the population (af Geijersstam et al., 1998b). Also, a survey of seroprevalences and agespecific attack rates that was performed in Helsinki, Finland, found stable seroprevalences and attack rates during the 1980s (Kibur et al., 2000). Thus, antibody-based studies have found clear evidence of a major increase in the total exposure to infection with the major oncogenic HPV type, HPV16, during the 1960s and 1970s, but the epidemic appears to have been stable during the 1980s.
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III. CELLULAR IMMUNITY TO HPV INFECTION A. Histological Studies The first evidence of antipapillomaviral cellular immunity was obtained by means of histology, which revealed that most skin warts have an inflammation with infiltrating mononuclear cells (Tagami et al., 1974). The inflammatory components have been extensively studied in skin warts (Aiba et al., 1986; Chardonnet et al., 1986), genital warts (Bishop et al., 1990; Coleman et al., 1993, 1994), and in laryngeal papillomas (Viac et al., 1987, 1992). The lymphocyte infiltrate has different characteristics in different dermal and mucosal compartments. In the stroma, there is a delayed-type hypersensitivity (DTH) reaction (the majority of the infiltrating lymphocytes are CD4+), whereas in the epithelium, about equal amounts of CD8+ and CD4+ T lymphocytes are found (Aiba et al., 1986; Coleman et al., 1994; Viac et al., 1992). An intense stromal infiltration in the mucosal papillomas is associated with ICAM-1 and major histocompatibility complex (MHC) class II (HLA-DR) expression on the keratinocyte surface (Viac et al., 1987, 1992). In a significant proportion of cervical intraepithelial neoplasia (CIN) lesions, HLA-DR is upregulated on the keratinocytes (Cromme et al., 1993; Glew et al., 1993a). These changes are seen not only in CIN, but in various inflammatory skin diseases and are likely to be mediated by the proinflammatory cytokine interferon (IFN)-␥ and in part also by tumor necrosis factor (TNF)-␣ (Barker et al., 1990; Coleman et al., 1993; Majewski et al., 1991). In the keratinocytes, as in many nonimmune cell types, the inducible expression of the MHC class II antigens varies for the different alleles (Glew et al., 1992a; Glimcher and Kara, 1992). In inflamed warts, or upon IFN-␥ treatment, the keratinocytes are induced to express HLA-DR, but not DP or DQ, antigens on the cell surface (Aiba et al., 1986; Basham et al., 1985; Bishop et al., 1990; Drijkoningen et al., 1988). Even after the malignant transformation of the keratinocytes, the expression of HLA-DR antigens significantly correlates with the extent of lymphocyte infiltration, while that of the DP or DQ antigens is less frequent, is unrelated to the inflammatory component, and is probably due to the concomitant genomic alterations (Hilders et al. 1993; Markey et al., 1990). The HLA-DR upregulation is inversely associated with clinical [F´ed´eration Internationale de Gyn´ecologie et d’obst´etrique (FIGO)] stage of incident cervical cancer cases (van Driel et al., 1996). In summary, HLA-DR is the MHC class II antigen that is most likely to be involved in the antipapillomaviral immunity, although one cannot exclude the possibility that professional antigenpresenting cells could activate CD4+ T lymphocytes through DP or DQ restriction.
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Most infiltrating T lymphocytes carry HLA-DR antigens (a late activation marker), but only few of them express the early activation antigen CD25 (Bishop et al., 1990; Coleman et al., 1994) suggesting that the T-cell activation occurs in distant places, plausibly in the regional lymph nodes. The Langerhans cells (LCs) form the afferent arm between the local keratinocytes and the regional lymph node. The LCs are the largest population of tissue dendritic cells (DCs) and are specialized for professional antigen presentation and activation of precursor T lymphocytes, even for naive precursor cells (Croft, 1994). In the epithelial layers, the primary DC function is to take up the antigen. Reaching the draining lymph node, DCs change to be highly potent antigen-presenting cells with enhanced expression of MHC and costimulatory molecules The HPV-induced CIN (Morelli et al., 1993) and skin warts (Chardonnet et al., 1986) are characterized by a depletion of LCs. However, this depletion appears uniform in the regressing and the progressing warts (Bishop et al., 1990; Coleman et al., 1994), suggesting that the LC density does not affect directly the amplitude of the immune response. Histological studies have concluded that the regressing but not the persistent/progressing warts show clinical and histological signs of immune rejection, i.e., the regressing warts show an enhanced immune activation compared to the nonregressing ones. All types of infiltrating mononuclear cells are found in greater numbers and have a higher level of activation.
B. Role of Cytokines in the Antipapillomaviral Responses The type of T-lymphocyte stimulation by the antigenic stimulus seems pivotal for the outcome of HPV-infection. The regression-associated histological changes (see above) are known to be mediated by Th1 type cytokines. Increased level of serum interleukin (IL)-2 was found to predict a favorable outcome for HPV16- or HPV18-associated genital lesions (Stellato et al., 1997). Also, a pathogenic role for Th2 responses is indicated by the finding that the peripheral mononuclear cells of high grade CIN patients have a decreased IL-2, IFN-␥ , and elevated IL-4, IL10 production in response to mitogenic stimuli (Clerici et al., 1997). Locally, there is also an increased tendency of Th2 cytokine production of the T lymphocytes infiltrating the squamaus intraepithelial lesions (SIL) (al Saleh et al., 1998), while in invasive cancer, both types of T-helper activity may be suppressed (de Gruijl et al., 1999a). Under physiological conditions, the epithelial cells themselves also communicate through cytokines. The keratinocyte IL-1␣ induces autocrine and paracrine proliferation (Ristow, 1987) and induces the further production
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of IL-1␣, IL-6, IL-8, and GM-CSF (Kondo, 1999). IL-1␣ and GM-CSF synergistically enhance the antigen presentation function of the LCs (Heufler et al., 1988). The proposed physiological functions of IL-6 and IL-8 are to induce keratinocyte proliferation and chemotaxis, respectively, and thereby to promote wound healing (Grossman et al., 1989; Michel et al., 1992). Keratinocytes also produce TNF-␣ which has pleiotropic effects in the epithelium: it is a negative autoregulator of keratinocyte proliferation (Symington, 1989), and enhances the migration of the LC/DC into the regional lymph nodes (Cumberbatch and Kimber, 1992). Keratinocytes secrete the antiinflammatory IL-10, which downregulates expression of chemokine receptors, costimulatory molecules of LC/DCs, and inhibits migration to the regional lymph node (Cumberbatch and Kimber, 1992; Kawamura and Furue, 1995). IL-1␣ and IL-6 are autocrine growth factors for neoplastic cells of keratinocyte origin (Castrilli et al., 1997; Eustace et al., 1993; Iglesias et al., 1995). Enhanced secretion of IL-6 by HPV16-transformed epithelial cells was demonstrated both in vitro (Bryan et al., 1995; Malejczyk et al., 1991) and in vivo (Tartour et al., 1994). Since IL-6 acts on the lymphocytes as a Th type 2 cytokine, its overproduction might promote the growth of HPVtransformed cells (Clerici et al., 1998). After transformation by the virus, the infected cells tend to gain resistance to the negative autoregulator TNF-␣. The underlying mechanisms for this resistance can be the upregulation of the epidermal growth factor receptor ligand, amphiregulin (Woodworth et al., 1995) or the secretion of soluble TNF-␣ receptors (Malejczyk et al., 1996). Increased serum levels of TNF-␣ receptors and increased serum TNF-␣ inhibitory activity have been found in patients with condyloma acuminata or invasive cervical cancer (Malejczyk et al., 1997).
C. HLA Class I Association of Cervical Neoplasia On the keratinocyte surface in unaffected skin and mucous membranes, there is a steady-state level expression of human leukocyte antigen (HLA) class I antigens. With more severe levels of cervical neoplasia, the HLA class I antigens tend to be downregulated or even missing (Cromme et al., 1993; Glew et al., 1993a); 30–75% of cervical cancers have downregulated the expression of at least one HLA class I antigen (Connor and Stern, 1990; Hilders et al., 1993). In one study, about one quarter of cervical cancers was found to retain the normal level of the HLA-A and -B antigens on the cell surface and about one sixth did not express any of these antigens (Keating et al., 1995). The remaining cervical cancers showed allele-specific loss of 1–3 HLA-antigens. The HLA antigens most frequently downregulated are A2, A3, A9 group, B5 group, B7, B8, and B44. Significant proportions
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of these allele-specific losses occur when TAP expression is normal (Keating et al., 1995), suggesting that these HLA antigens are involved in presenting dominant epitopes of the rejection antigens. The HLA-B12 serospecificity (this includes B44 as well) was reported to be associated with cervical cancer more than 20 years ago (Koenig et al., 1976). Recently, a disease association of A2 (Montoya et al., 1998) and B44 (Bontkes et al., 1998b) has been suggested, and an association with HLA-B7 has been established (Duggan Keen et al., 1996). The most likely underlying mechanism is the allele-specific downregulation of these antigens during cervical carcinogenesis. Downregulation of HLA-B7 on cervical cancer cells is associated with worse survival compared to normal expression of this antigen (Duggan Keen et al., 1996). The existence of HPV 16 variants with E6 mutations affecting HLA-A2 and -B7 binding motifs suggests that lack of CD8-restricted epitopes may enable an immune escape (Ellis et al., 1995; Yamada et al., 1995).
D. HLA Class II Association of HPV-Related Cervical Diseases A large number of human studies have focused on the association of HLA class II and CIN or cervical cancer. However, a number of limitations of these studies results in a less than straightforward interpretation: 1. There are many alleles for each HLA locus, and most studies are limited in size. Thus, the low number of observations for the individual alleles results in a limited statistical power, complicated by the problem of multiple comparisons. 2. Most studies have not had an epidemiologically valid study design. In many cases, the study base has not been defined, and there are examples of studies where the controls have been knowingly drawn from populations different from the cases. 3. A susceptibility factor may not be comparable in populations with different exposures. Susceptibility factors, by definition, confer increased risk of disease in the case of exposure to an environmental risk factor. For cervical cancer, it is assumed that certain HLA haplotypes may increase the risk of disease in the case of exposure to an oncogenic HPV infection. The prevalence of HPV infection may be very different in high-risk and lowrisk populations (Dillner et al., 1997; Nonnenmacher et al., 1995) and may furthermore vary with calendar time (af Geijersstam et al., 1998a). 4. Some HLA molecules, e.g., HLA-DQ, are composed of two variable polypeptides, both of which are probably involved in antigen presentation. Searching for associations with alleles coding for only one of these polypeptides will attenuate the risks and may lead to variable results, if the
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frequencies of the alleles coding for the other complexed polypeptide is different in the different populations. 5. Due to the linkage disequilibrium in the HLA region, one might detect a positive association not only with the allele involved in the disease but also with a closely linked one. To enable an assessment of the status of evidence, we performed a metaanalysis as follows: Studies with the words “HLA” and “papillomavirus” or “cervical cancer/neoplasia” in the title or abstract were located by Medline searches. Studies that cited the ones located from Medline were identified using the Science Citation Index. The HLA haplotypes that were reported to have a significant association (corrected p < 0.05) in at least one of the above studies were subjected to systematic review. The reviewed studies provided data of HLA frequencies in three different ways: (i) proportion of persons carrying a certain HLA entity, (ii) allele frequency calculated from the number of persons (n) in the study groups, and (iii) allele frequency calculated from the cumulative number of alleles (2n) in the study group. The disease risk of an HLA allele will vary slightly depending of which of the above methods is used to calculate the allele frequency. We recalculated the point estimates and confidence intervals according to method (ii) The data given according to method (i) were included into the systematic analysis as if they had been obtained by method (ii) The point estimates (odds ratios) were calculated with the program EpiInfo 6.3, and the beta values and the corresponding variances were also calculated (beta, the logistic regression coefficient is the natural logarithmic value of the odds ratio). A weighted average of beta values with the weight assigned to each beta proportional to the inverse of its variance was calculated and converted into an average odds ratio. Studies that derived the controls from populations other than the cases, or did not specify the source of the controls, were not considered informative and were not included in the metaanalysis. Albeit population-based studies and/or cohort studies are preferable, studies using hospital-based controls or local blood donor controls were considered eligible for metaanalysis. It should be noted that when several stratifications (HPV types, disease stages, HLA haplotypes) are introduced, the statistical analyses will have less power. Therefore, we restricted our analysis to stratifications by (i) HPV-associated cervical disease, (ii) HPV 16-associated cervical disease, and (iii) HPV16negative disease. HPV 16 was the only virus sufficiently prevalent in cervical disease to make type-restricted metaanalysis meaningful. We also calculated susceptibility risk of the HLA types for HPV16-associated cervical disease compared to the HPV16-negative disease. Since the latter calculations are not affected by healthy controls, we therefore included studies with inappropriate control selection.
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We found three HLA-DR specificities conferring an altered susceptibility for neoplasia upon exposure to HPV. Interestingly, the three DR specificities revealed different susceptibility patterns. HLA-DR13 was protective, regardless of the infecting HPV type. HLA-DR15 conferred an increased susceptibility only when the infecting HPV type was 16. HLA DR-7 had a “Janus face,” being protective upon exposure to a type other than HPV16 and being a susceptibility factor for HPV16-associated cervical neoplasia (Table II). A decreased risk by DR13 for cervical neoplasia was suggested by most studies. In the systematic analysis of the DR13 effect, we included the studies that published data of DR6-positive persons (Glew et al., 1993b; Syrjanen et al., 1996; Wank and Thomssen. 1991). The DR6 serospecificity is encoded by DRB1∗ 13xx and 14xx alleles. From the studies where DRB1∗ 14xx data were available (Apple et al., 1994, 1995; Duggan Keen et al., 1996; Hamsikova et al., 1999; Odunsi et al., 1996; Sanjeevi et al., 1996), we estimated the association of this allele with cervical disease (comprising both CIN and cervical cancer) and calculated a joint odds ratio (OR) of 0.69 (95% confidence interval: 0.50–0.96). Since it was very similar to the joint OR of DRB1∗ 13xx, we decided that the DR6 data were comparable with DR13 estimates. The metaanalysis (Table II) revealed that DR13 is protective against both premalignant and malignant cervical disease, but not specific for HPV type 16, suggesting that the mechanism is independent of HPV, or involves common epitopes shared by the most prevalent HPV types. The studies not included in the metaanalysis because of inappropriate controls did, however, also reveal a protection (joint OR: 0.52, 95% confidence interval: 0.38–0.70) (Allen et al., 1996; Krul et al., 1999; Sastre Garau et al., 1996). For the analysis of the entire DR15 group, we included the studies that published data of DR2 positive women (Glew et al., 1993b; Syrjanen et al., 1996; Wank and Thomssen, 1991). The DR2 serospecificity is encoded by DRB1∗ 15xx and 16xx alleles. The latter alleles do not influence the risk for cervical neoplasia (estimated joint OR: 0.93, 95% CI: 0.60–1,43, based on the studies by Apple et al., 1994, 1995; Duggan Keen et al., 1996; Hamsikova et al., 1999; Odunsi et al., 1996; Sanjeevi et al., 1996). Thus, our use of DR2 data in the metaanalysis may have conservatively biased the disease association of the DR15 specificity. The more stringent stratification (DRB1∗ 1501 or DQB1∗ 0602) was, however, not biased by the DRB1∗ 16xx alleles. Since DRB1∗ 1501 is almost exclusively linked to DQB1∗ 0602 in the investigated populations, and several reports focused only on DQ alleles, we considered the DQB1∗ 0602 data relevant for DRB1∗ 1501 (Table II). It was impossible to differentiate whether the observed effect was due to DRB1∗ 1501 or DQB1∗ 0602 because the number of observations for DQB1∗ 0602 linked to DRB1 alleles other than DRB1∗ 1501 was too small.
Table II HLA-DR Association of HPV-Related Cervical Disease—Cumulative Odds Ratio (95% Confidence Interval) Disease vs Control
HPV16+ve Disease vs Control
HPV16−ve Disease vs Control
HPV16+ve Disease vs HPV16-ve Disease
DR7
1.01 (0.80–1.27) b 2,3,6,8,10,17,18,19,21
1.42 (1.12–1.80) b 2,3,6,8,19
0.61 (0.41–0.92) b 2,3,6,8,19
2.63 (1.67–4.16) b 1,2,3,4,6,8,14,19,20
DR13
0.69 (0.56–0.85) b 2,3,6,8,10,17,18,19,21
0.51 (0.38–0.68) b 2,3,6,8,19
0.53 (0.38–0.75) b 2,3,6,8,19
1.06 (0.68–1.64) b 1,2,3,4,6,8,14,19,20
DR15
1.29 (1.13–1.47) b 2,3,6,8,10,13,17,18,19,21
1.47 (1.20–1.81) b 2,3,6,8,13,19
0.94 (0.74–1.19) b 2,3,6,8,13,19
1.34 (1.01–1.78) b 1,2,3,4,6,8,13,14,19,20
1.15 (0.98–1.34) b 2,3,6,9,11,12,13,16 18,19,21
1.36 (1.10–1.69) b 2,3,6,9,11, 12,13,19
1.02 (0.79–1.31) b 2,3,6,9,11, 12,13,19
1.28 (0.96–1.71) b 1,2,3,4,6,8,9,11,12 13,14,19,20
HLA Class II
a
DRB1∗ 1501
a
Including also the DQB1∗ 0602 data from the reports that did not investigate the HLA-DR antigens. References processed in the metaanalysis: 1 (Allen et al. 1996), 2 (Apple et al., 1995), 3 (Apple et al., 1994), 4 (Bontkes et al., 1998a), 5 (David et al., 1992), 6 (Duggan Keen et al., 1996), 7 (Glew et al., 1992b), 8 (Glew et al., 1993b), 9 (Gregoire et al., 1994), 10 (Hamsikova et al., 1999), 11 (Helland et al., 1994), 12 (Helland et al., 1998), 13 (Hildesheim et al., 1998), 14 (Krul et al., 1999), 15 (Mehal et al., 1994), 16 (Montoya et al., 1998), 17 (Wank and Thomssen, 1991), 18 (Odunsi et al., 1996), 19 (Sanjeevi et al., 1996), 20 (Sastre Garau et al., 1996), 21 (Syrjanen et al., 1996). b
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The DQB1∗ 03xx alleles, which define DQw3 specificity, was the first to be reported to confer an increased risk for cervical cancer (Wank and Thomssen, 1991). For systematic review, we analyzed both the cumulative and the single effects of the DQB1∗ 03xx alleles (Table III). When considering all the alleles coding for DQw3 specificity, we found a moderate but significant disease association which was independent of the infecting HPV type. On the level of the single alleles, both DQB1∗ 0302 and DQB1∗ 0303 proved to be a susceptibility factor upon HPV16 infection. For DQB1∗ 0301, no disease association was confirmed when the infecting HPV type was introduced into the analysis. The different DQB1∗ 03xx alleles (0301, 0302, 0303) can be found in linkage with several DQA1 alleles and plausibly define a group of DQ molecules with heterogeneous antigen-presenting properties. The different proportion of the DQA1–DQB1∗ 03xx allele linkages in different populations may give heterogeneous results if only the DQB1 alleles are considered. For instance, in the European Caucasian populations, from which most study participants were drawn, DQB1∗ 0301 is found in two frequent allele linkages, namely with DQA1∗ 0301/2 and DQA1∗ 0501. On the other hand, DQB1∗ 0302, which revealed a clearcut association with disease and virus type, is predominantly found in linkage with a DQA1 allele, the ∗ DQA1∗ 0301/2 (Doherty et al., 1992; Helland et al., 1998; Ronningen et al., 1990; Sanjeevi et al., 1996). The other positive association was found with the rare DQB1∗ 0303 allele, which in turn is in linkage with DQA1∗ 0201 and DR7 alleles. The fact that DQB1∗ 0303 and DR7 alleles of the same linkage were associated with HPV16-related cervical disease in a similar manner indicates that complete typing of the DR-DQ alleles will be necessary in future studies.
E. HPV-Associated Lesions in Immunosuppressed Patients Epidermodysplasia verruciformis (EV) is a rare, multifactorial skin disease with an autosomal recessive-like genetic predisposition characterized by the presence of EV-associated HPV types and deficiency of several immune functions such as reduced T-cell number involving particularly the CD4+ population, reduced lymphocyte activation by phytohemagglutinin, and possibly altered LC function (Vardy et al., 1990). However, several EV-associated HPV types have also been found in nonmelanoma skin cancers of both immunosuppressed and immunocompetent individuals (Shamanin et al., 1996). A higher rate of HPV-associated lesions among patients with iatrogenic immunosuppression due to organ transplantation indicated that the HPV infected keratinocytes are rejected in the same way as the transplanted
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Table III HLA-DO Association of HPV-Related Cervical Disease—Cumulative Odds Ratio (95% Confidence Interval)
Disease vs.Control
HPV16+ve Disease vs Control
HPV16−ve Disease vs Control
HPV16+ve Disease vs HPV16-ve Disease
DQB1∗ 03xx
1.25 (1,15-1,37) a 2,3,5,6,7,9,10,11,12 13,15,16,17,18,19,21
1.21 (1,02-1,43) a 2,3,6,9,11, 12,13,19
1.17 (1,02-1,37) a 2,3,6,9,11, 12,13,19
1.04 (1,04-1,25) a 1,2,3,6,9,11, 12,13,14,19,20
DQB1∗ 0301
1,22 (1,07-1,39) a 2,3,6,9,10,11,12, 13,16,18,19
1,08 (0,89-1,30) a 2,3,6,9,11, 12,13,19
1,14 (0,84-1,54) a 2,3,6,9,11, 12,13,19
0,90 (0,66-1,25) a 1,2,3,6,9,11, 12,13,19,20
DQB1∗ 0302
1,25 (1,07-1,45) a 2,3,6,9,10,11,12, 13,16,18,19
1,34 (1,06-1,69) a 2,3,6,9,11, 12,13,19
0,92 (0,69-1,24) a 2,3,6,9,11, 12,13,19
1,38 (1,00-1,90) a 1,2,3,6,9,11, 12,13,19,20
DQB1∗ 0303
1,34 (1,02-1,77) a 2,3,6,9,10,11,12, 13,16,18,19
1,60 (1,03-2,47) a 2,3,6,9,11,12, 13,19
1,21 (0,73-1,98) a 2,3,6,9,11,12, 13,19
1,30 (0,71-2,40) a 1,2,3,6,9,11, 12,13,19,20
HLA Class II
a
References processed in the metaanalysis: 1 (Allen et al., 1996), 2 (Apple et al., 1995), 3 (Apple et al., 1994), 4 (Bontkes et al., 1998a), 5 (David et al., 1992), 6 (Duggan Keen et al., 1996), 7 (Glew et al., 1992b), 8 (Glew et al., 1993b), 9 (Gregoire et al., 1994), 10 (Hamsikova et al., 1999), 11 (Helland et al., 1994), 12 (Helland et al., 1998), 13 (Hildesheim et al., 1998), 14 (Krul et al., 1999), 15 (Mehal et al., 1994), 16 (Montoya et al., 1998), 17 (Wank and Thomssen, 1991), 18 (Odunsi et al., 1996), 19 (Sanjeevi et al., 1996), 20 (Sastre Garau et al., 1996), 21 (Syrjanen et al., 1996).
allogeneic tissues. Transplantation patients receiving antirejection therapy have enormously increased incidences of nonmelanoma skin cancers and premalignant keratoses, keratoacanthomas in their sun-exposed skin areas (Euvrard et al., 1997). HPV infection and SILs tend to progress into advanced stages in the genitalia of women after renal transplantation (Petry et al., 1994). Acquired immunodeficiency is associated with an increased risk for HPVassociated, anogenital neoplasia. Cervical cancer is actually an AIDS-defining illness (Maiman et al., 1997). However, both HIV and HPV are sexually transmitted, i.e., infection by one of them is associated with an increased risk for the other, merely by the similar mode of transmission. Nevertheless, several studies have indicated that HIV-associated immunodeficiency indeed contributed to HPV pathogenesis. HIV-related immunosuppression, as determined by CD4+ cell count, is associated with a higher amount papillomavirus DNA in the infected women (Hillemanns et al., 1996;
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Ho et al., 1994; Petry et al., 1994) and with an increased tendency for HPV infections to persist (Vernon et al., 1994). Apart from the increased prevalence of HPV infections, HIV-related immunosuppression is associated with an increased rate of progression to SILs (Six et al., 1998). The increased rates are associated in particular with the advanced clinical stages of HIV infection (Sopracordevole et al., 1996; Spinillo et al., 1993). An increased amount of plasma HIV RNA, which is an indirect indicator of advanced immunodeficiency, is also associated with HPV infection (Palefsky et al., 1999). HIVseropositive women with low CD4+/CD8+ ratio have a reduced number of infiltrating LCs (Barberis et al., 1998). In men with HIV infection, HPV infections tend to persist (Critchlow et al., 1998), and in the anal region, there is an increased amount of papillomaviruses (Friedman et al., 1998) and an increased frequency of anal intraepithelial neoplasias (Palefsky et al., 1998b). A low CD4/CD8 ratio with an underlying anal HPV infection exposes the host to frequent anal condylomas or squamous cell atypia lesions (Melbye et al., 1990). Anal condylomas are more resistant to therapy in HIV-infected than in noninfected men (von Krogh et al., 1995). A proper highly active antiretroviral therapy improving the immune functions of HIV-infected persons results in regression of SILs (Heard et al., 1998). Antibodies to HPV-6 and HPV-16 capsids are readily detected in HIV-seropositive men with anogenital intraepithelial lesions (Hagensee et al., 1997), in concordance with the concept that HPV-specific antibodies are not important for HPV clearance.
F. Cell-Mediated Immunity (CMI) in Immunocompetent Women with HPV-Associated Diseases HPV-associated genital neoplasias have a high incidence in the general population and usually develop in women, who are apparently immunocompetent both clinically and by routine laboratory tests. The HLA associations of the disease strongly support an essential role of T lymphocytes for immune surveillance. Presently, prediction of the targeted antigens is based on the knowledge of the successive appearance of viral proteins during the viral life cycle, which is closely linked to differentiation of the squamous eptihelium. The earlier appearance of a viral protein during the viral cycle renders it a more favorable target of protective immune responses. The E1 and E2 proteins mediate maintenance of the episomal genome and regulation of viral transcription and must therefore be expressed in the basal and suprabasal layers, while the E6, E7, and E5 proteins drive the spinosal layer into excessive proliferation. The E4, L1, and L2 antigens are expressed in nondividing terminally differentiated keratinocytes, and therefore these proteins have not been considered to be rejection antigens. However, these considerations did
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not take into account that the L1 and L2 structural antigens of the virions enter the cell with the infecting particle and can be processed for antigen presentation even before any viral replication has started. There is at least one human viral infection, the cytomegalovirus infection, where cytotoxic T lymphocytes (CTLs) that are specific to structural antigens can act immediately upon virus entry and thereby inhibit the spread of infection within the same individual (Riddell et al., 1991; Wills et al., 1996). This means that studies determining the correlates of viral clearance should include the structural virus proteins as target antigens of T-lymphocyte recognition. Several antigens derived from both the early (E2, E5, E6, E7) and the late (L1) open reading frames of HPV 16 have indeed been found to be immunogenic in T-cell proliferation assays (Altmann et al., 1992; Gill et al., 1998; Lehtinen et al., 1995; Strang et al., 1990). These CMI responses tend to be specific for the infecting HPV type and closely related types (Kadish et al., 1994; Konya et al., 1997; Shepherd et al., 1996). In most cross-sectional studies, the controls were defined as having no history of cervical disease. High proportions of these controls were found to respond to either E7 or L1 epitopes, but the L1-specific responses were somewhat (but not significantly) increased among the CIN patients (Luxton et al., 1996; Shepherd et al., 1996). Several studies reported little or no disease specificity of lymphocyte proliferation specific for E7 antigens (Kadish et al., 1994; Luxton et al., 1996). Even a study that used virginal women as control group found little disease specificity (Nakagawa et al., 1996). Conclusions about the immune surveillance by CMI are difficult to make based on cross-sectional studies for several reasons. (i) In a control group, referred to merely as having no history of cervical disease, we do not know who has been exposed to HPV resulting in immune memory and who has never been infected. HPV serology and sexual behavior questionnaires may help in this regard. (ii) Follow-up of the different disease groups is needed to investigate whether or not the antiHPV CMI will be of benefit and will induce regression. In a follow-up study, positive lymphocyte proliferation to E6 or E7 at one visit indeed predicted regression of the lesion at the next visit (Kadish et al., 1997). Several studies used an assay measuring IL-2 production upon stimulation with papillomaviral antigens. An L1-specific IL-2 production was maintained long after HPV 16 infection, irrespective of whether the virus was cleared at different stages of infection or induced persisting/progressing CIN (de Gruijl et al., 1999b). These IL-2 responses were found in patients lacking both HPV16 seropositivity and previous HPV16 DNA positivity, indicating that the L1 antigen used was not entirely type restricted. The E7-specific IL-2 responsiveness was studied in two different studies. A crosssectional study within a cohort revealed that incident HPV16-positive CIN patients tended to respond less frequently to E7 than women who were
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HPV16-infected in the past, but did not develop CIN. The responding frequencies were inversely correlated also with severity of the disease (Tsukui et al., 1996). The other approach was to measure the E7-specific IL-2 responses in relation to the outcome of CIN (Bontkes et al., 1999; de Gruijl et al., 1998). The E7-specific responses appeared earlier in the HPV16 clearance group than in the persistence group, and their appearance was linked to a period around the viral clearance. In the persistence group, the E7 specific responses developed only after prolonged exposure to the virus. Both research groups reported that patients with invasive cervical cancer were less responsive to E7 than the high-grade CIN patients. In patients with CIN regression, the frequency of responders against the C-terminal domain of E2 was similar to that against E7 peptides, while in the persistence group, the E2-C responses were less frequent (Bontkes et al., 1999). In conclusion, an antigen-specific Th1 type activation appears to promote a favorable outcome of the infection. The patients with persistent/progressing CIN tend to develop IL-2 responsiveness against the most abundant antigens only in a late stage of the infection, when the Th1 type activation might be neutralized by the increased secretion of soluble IL-2 receptor (Hildesheim et al., 1997) or could be suppressed by the overwhelming Th2 type responses (Clerici et al., 1997; de Gruijl et al., 1999a).
G. Cytotoxic T Cells The importance of CTLs, which are the ultimate effector cells of the Th1 type activation, is suggested by the histological findings that demonstrated either CD8+ T lymphocytes infiltrating the infected epithelium or the selective loss of MHC class I antigens in the malignant and the premalignant lesions. CTL stimulations have reported variable results depending on the stimulating protocol used. Oligopeptide epitopes (Ressing et al., 1996) or HPV16+−ve cell lines (Evans et al., 1996) stimulated in vitro CTLs only occasionally from the peripheral blood of HPV16+ve and MHC-matched cervical cancer patients. Viral vectors with recombinant E6 and E7 could readily stimulate CTLs in vitro from 40–60% of HPV16+ve CIN patients (Nakagawa et al., 1997; Nimako et al., 1997) and even from the peripheral and tumor infiltrating lymphocytes of cervical cancer patients (Evans et al., 1997; Peter Stern, personal communication). Although CMI specific to HPV can apparently be detected in different stages of HPV infections, there is little knowledge of which CMI responses at which stage of the infection will mediate the clearance the HPV-infected cells. The determinants of viral clearance will need to be elucidated in prospective studies using reproducible cellular immunology methods with minimal in vitro manipulations.
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IV. DOES CROSS-PROTECTIVE IMMUNITY EXIST? The term interaction refers to the epidemiological phenomenon that the joint effect of two or more different exposures for disease development is more or less than expected than if the exposures are independent. In the HPV field, interaction between different HPV types has been studied with regard to (i) joint effect on cancer development and (ii) infection by other HPV types. If interaction between different HPV types exists, elimination of some HPV types by preventive vaccination might change the occurrence of and/or the pathogenicity of interacting HPV types. As HPV exists as more than 80 types, it is difficult to predict the population biology of the various papillomaviruses and how this may be influenced by changes over time in factors affecting the spread of HPV, e.g., prevention. A first indirect indication of possible antagonism between the different types of HPV was the finding by Evans et al. (1992) that showed a protective effect on cervical cancer by a history of condyloma accuminata. In the HPV seroepidemiology field, the issue was raised by the finding that seropositivity against certain broadly cross-reactive HPV antigens was protective against cervical cancer (Dillner et al., 1994) and by the finding that the cervical cancer risk associated with HPV 16 seropositivity differed substantially between populations at low risk for STDs, with a highly elevated cervical cancer risk (OR = 11.8) among those seropositive for HPV 16 and no excess risk (OR = 1.1) in an STD high-risk population (Dillner et al., 1997). Two possible explanations for the observed reduced risk in the high STD risk population were: (a) interaction with another STD protecting against HPV16 or (b) a saturation of the STD high-risk population with HPV 16 exposures, making it impossible to correctly identify any HPV 16-unexposed subjects. A similar oncogenic risk difference for a serological exposure marker in highand low-risk populations had previously been found in the case of hepatitis B virus (HBV) infection (Szmuness, 1978), with HBsAg giving risks of 5–10 in low-incidence and 30–60 in high-incidence areas. To follow-up on the above-mentioned findings, we have investigated whether interaction of HPV 16 and other HPVs might exist. Two separate seroepidemiological studies have been performed, both of which found statistically significant evidence of a protective role of HPV 6/11 in HPV 16associated cervical carcinogenesis. The paper by Luostarinen et al. (1999) was a nested case-control study in a cohort of 530.000 women who had donated blood samples between 1973 and 1994. During follow-up, 182 women developed invasive cervical cancer, and the serum samples taken at baseline from these women and from matched women who remained healthy during an equal length of follow-up were analyzed for presence of antibodies to capsids of HPV 6, 11, 16, 18, and 33. The cancer risks of each virus per se and
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their joint effects using the jointly unexposed as the reference group were analyzed. The strongest interaction was observed for the combination of HPV 6/11 and 16. There was no observed excess risk (OR = 1.0) for cases positive for both viruses, although the expected joint risk was markedly elevated (OR = 12). Thus, the observed risk indicated an antagonistic interference among these virus types (p = 0.001). As discussed above, a substantial proportion of HPV-exposed women will not seroconvert for HPV. The sensitivity of HPV serology, using detection of viral DNA in cervical samples as the reference, has been estimated at about 50–65% (Kjellberg et al., 1999). To investigate whether the interaction seen might be an effect of misclassification of HPV exposure by the HPV serology, the effects of misclassification were considered by gradually assuming worse levels of sensitivity (proportion of HPV-exposed subjects testing seropositive), specificity (proportion of seropositive subjects that are HPV-exposed), and cross-reactivity (positivity for one virus specifically induced by exposure to the other virus) of the serological test. Misclassification-corrected antagonism was greater than the observed antagonism in all combinations and reached infinity at fairly modest levels of misclassification by specificity and cross-reactivity (Luostarinen et al., 1999). The models assumed nondifferential misclassification. Since all samples were taken from healthy individuals who had not yet developed disease, nondifferential misclassification by case status is a reasonably safe assumption. The study by Silins et al. (1999) describes a case-control study of 218 incident, untreated cancer cases and 219 age-matched controls and focuses on the separate oncogenic risk evaluation of HPV 6, 11, 16, 18, and 33, as well as on possible interaction among the different viruses. Statistically significant antagonistic interaction was observed only for the HPV 6 and 16 combination, showing an expected risk for joint seropositivity of 5.59, but an observed risk of 1.64 (p = 0.018) in a multiplicative model. The relative excess risk due to interaction in an additive model was −2.35, which was also statistically significant. In the paper by Luostarinen et al. (1999), HPV 6 and HPV 11 antibodies were not considered separately, because of the strong cross-reactivity between the capsids of these HPV types. In the paper by Silins et al. (1999), HPV 6 and HPV 11 antibodies were analyzed separately. The responses were, as expected, highly correlated. Albeit the antagonism with HPV 16 was only detected for HPV 6, this might have been attributable to better statistical power (more subjects were positive for HPV 6 than for HPV 11). In an as yet unpublished work exploring possible existence of other interactions in a prospective study of CIN from northern Sweden (Chua et al., 1996), preliminary data show an even more pronounced antagonizing effect of HPV 73 seropositivity against the effects of HPV 16 and Chlamydia trachomatis (unpublished data). There is no a priori reason to suspect that only HPV 6 and 16 would have an interaction, and exploration of possible
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interactions between various HPV types is likely to become an expanding research area in the future.
A. Possible Explanations for the Observed Antagonism As two different HPV types cannot infect the same cell, a molecular interaction mechanism within the infected cell is not a possible explanation. Animal studies show that HPV anticapsid antibodies can prevent infection, but it has been well documented that there is very little cross-neutralizing ability among HPV types at the antibody level (Roden et al., 1996). Furthermore, the interaction is not possibly at the level of preventing infection: The jointly seropositive subjects have indeed been infected with both HPV types (seroconversion is induced by infection). Still, the oncogenic effect of both HPV 6 and HPV 16 infection is less than that of HPV 16 alone. Therefore, the only reasonable explanation that we can think of is that prior infection with HPV 6 does not prevent HPV 16 infection, but prevents it from becoming persistent and causing cancer. The mechanisms that determine whether HPV infection is cleared or becomes persistent are not known, but cellular immunity is implicated. The type specificity of HPV cellular immunity is not well known. Proliferative responses against the E7 antigen seem to be HPV type specific (Kadish et al., 1994), but the HPV early proteins whose expression is required for episomal maintenance (i.e., E1 and E2) are highly conserved among HPV types. The E2 protein can induce proliferative responses (Lehtinen et al., 1995), and a CTL epitope in the E2 protein has been mapped (Konya et al., 1997).
B. Cooccurrence of HPV Types in Cervical Samples Studies on HPV DNA presence usually show up to 10% multiple infections with two or more different HPV types. It is not entirely clear, however, how much of these multiple positivities is attributable to an insufficient specificity of HPV detection and typing methods. One of the largest published studies on HPV DNA cooccurrence among 1425 healthy low-income women in Brazil evaluated 357 women who were HPV-positive at least once. Among 13 more common HPV types, 78 HPV pair combinations were found (Franco et al., 1995). Some HPV types, such as HPV 16 and 53, tended to be more common in combinations, and some, such as HPV 61 and 70, are usually alone. However, as most HPV types are rare infections, and quite commonly unknown HPV types are found, limited statistical power implies some caution in interpretation.
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Regarding HPV-serological methods, cooccurrence of several type-specific antibodies is a common phenomenon, because of the very long persistence of HPV-specific serum antibodies after HPV DNA has been cleared and because of the common mode of transmission of these viruses. Therefore, if studies on cooccurrence, both for HPV DNA and HPV antibodies, are interpreted to show facilitation or protection against infection, they will be biased by the common modes of transmission of these viruses. Studies on HPV DNA occurrence are furthermore complicated by the fact that prior exposure cannot be detected. If a prior infection has resulted in cross-protective immunity facilitating clearance of infections, such prior infection is not likely to still be detectable as cervical HPV DNA. Cooccurrence of HPV DNA of various types is also not very informative, since it is known that, in particular, cellular immunity to HPV develops only slowly, and a large part of currently detectable HPV DNA is likely to reflect either recent infections or a subset of infections that have failed to induce an appropriate cellular immunity and have become persistent. Our hypothesis on the existence of cross-protective immunity could be tested using a cohort study that has followed whether HPV 16 infections become persistent or are cleared, simply by testing whether HPV 6 seropositivity at onset predicts clearance/persistence.
C. The Dynamics of the HPV Infection: Implications for Estimating the Effects of Vaccination The spread of every sexually transmitted agent is dependent on a number of factors that are either intrinsic to the agent itself or the population in which it is spreading. Apart from the fact that behavioral or other changes in the susceptible population may affect the spread of the agent, the population dynamics of other related agents may greatly influence the occurrence of disease. The latter scenario can occur if different agents compete for the same ecological niche or if they influence the susceptibility or pathogenicity of related agents. In “worst case” scenarios, complete removal of predominant pathogenic serotypes can result in increased occurrence of disease, by the resurgence of previously rare serotypes that had been interfered with by the removed serotypes (Lipsitch, 1997). Of course, “best case” scenarios are possible where removal of a minor fraction of only some serotypes can result in large effects on disease prevention, e.g., by herd immunity effects. The resulting effects with regard to disease of influencing the occurrence of a particular serotype will be even more complex to predict if there exists interference influencing not only occurrence of infections, but also their oncogenicity (Table IV). Prediction of HPV population dynamics will be even more difficult, considering the fact that different HPV types are already known to be transmitted
Table IV Population Dynamics of HPV Infection and Its Possible Consequences for HPV Vaccination Phenomenon Herd immunity
Competition between viral types, i.e., attack rates of infection is influenced by infection with other virus types. Interactions in pathogenicity, i.e., probability that infection will result in disease is influenced by infection with other virus types.
Potential Effect on Vaccination
Relevance for HPV?
Protective effect of vaccination greater than that achieved by vaccine efficacy and vaccination coverage: Attempts to eradicate oncogenic HPV could be contemplated also with vaccines with rather low efficacy and limited duration of protection. Vaccination may result in increases of viral types not included in vaccine. Even completely effective vaccines may thus not protect against disease, and may in certain worst case scenarios even increase disease.
Likely to be the case for HPV. Expected herd immunity effects have, however, not been estimated for HPV.
Vaccination may result in previously less pathogenic types, previously interfered with by the types eradicated by the vaccine, becoming more pathogenic.
No evidence that this may occur for HPV. Absence of cross-neutralization and random distribution of multiple infections in cervical samples suggest that it does not occur. Two studies have indeed disclosed an interaction, between HPV16 and HPV6/11. The HPV type specificity of the cellular immunity responsible for viral clearance is not well known.
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somewhat differently. Thus, although infection of both benign and oncogenic HPV types is generally closely related to the number of lifetime sexual partners, low-risk HPV types (6 and 11) have a weaker correlation with sexual activity than oncogenic HPV types (Franco et al., 1995, Silins et al., 2000), suggesting different rates of transmissibility and/or duration of infectivity. Thus, although accurate prediction of vaccination effects may be hard to achieve, it is necessary to realize that HPV is an infectious risk factor and that predictions of disease-preventive effects by preventing various types of HPV infections cannot be made in the simple ways used in the traditional epidemiologic study of noninfectious risk factors. Thus, an improved knowledge of the cellular immunity to HPV and whether it is HPV type specific or not will be required not only for understanding the natural history of HPV infection and design of immunotherapeutics, but also for design and monitoring of prophylactic HPV vaccination efforts and prediction of their effects.
ACKNOWLEDGMENTS J. D. is supported by the Academy of Finland and by the Swedish Medical Research Council. We are indebted to Dr. Matti Lehtinen for having pointed out the population dynamics issue and for continuous discussions on HPV immunity throughout these works.
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Index
A ABL gene schematic representation, 144f ABL intron, 143 Acquired immunodeficiency (AIDS) HPV, 222–223 Activated initiator caspases, 123 Acute lymphoblastic leukemia (ALL), 143 Acute myeloblastic leukemia (AML)1-ETO, 153–154 cDNA fragment detection, 138f–139f Acute promyelocytic leukemia (APL), 155–157 Adenine nucleotide translocator (ANT) Bcl-2, 65 Adenovirus E1B 19K caspase-9, 73–74 Adipocytes, 30 Adrenal gland VHL, 91 AFX, 196 AHR, 6t pRb, 26 AIDS HPV, 222–223 AKT kinase, 196 ALL, 143 Allogeneic blood stem cells transplantation, 165 Allogeneic related BMT, 146 Allogeneic unrelated BMT, 146 All-trans-retinoic acid (ATRA), 156–157 Amino acid proto-oncogene products, 23 AML1-ETO, 153–154 cDNA fragment detection, 138f–139f Androgen receptor (AR) signaling prostate tumorigenesis, 191–192
Angiogenesis pVHL-dependent target gene, 95–96 ANT Bcl-2, 65 Anterior lobe mouse prostate gland, 188 Antibody response HPV epidemiological studies, 213–214 isotypes, 211 sensitivity, specificity, stability, 208–211 serum, 212t Anti-vascular endothelial growth factor therapy HABs, 90 AP-2, 7t pRb, 35–36 APL, 155–157 Apoptosis, 56 caspases, 123 TNF receptor, 116 tumor cells, 109 Apoptotic protease activating factor 1 (Apaf1) Bcl-2, 73–74 Apoptotic protease activating factor 2 (Apaf2), 119–121 AR signaling prostate tumorigenesis, 191–192 Aryl hydrocarbon receptor (AHR) pRb, 26 Assays sensitivity HPV antibody response, 208–209 specificity HPV antibody response, 209–210 ATF-1, 7t pRb, 32
239
240 ATF-2, 7t pRb, 32 ATRA, 156–157 Autologous BMT, 151–152 MCL, 165 molecular monitoring, 157
B B4, 7t pRb, 25 Baculovirus, 74 Baculovirus IAP repeat (BIR), 75 Bad structure, 62 Bak, 62–63 ANT, 65 Basal cells prostate, 192 Bax, 62–63 ANT, 65 p53, 66–67 B-cell lymphoma/leukemia (Bcl-2) Apaf-1, 73–74 characterized, 164 deregulated expression, 161 exon 1, 158 family members, 62–64 interaction between, 64 MBR/JH, 161 mitochondria, 64–65 p53, 59–60 related proteins structure, 62 XL structure, 62 Bcl-2 homology (BH) domains, 62–68 Bcr1, 156 BCR-ABL, 143 levels, 145 mRNA transcripts, 151–152 transcripts remission, 146 BCR gene schematic representation, 144f Bdp, 6t pRb, 21–22 BEC iNOS activation, 116 Benign hyperplastic lymphoid lymph nodes PCR, 161 BH domains, 62–68
Index
BHLH-leucine zipper protein, 33 Bid structure, 62 BIR, 75 Blood stem cells allogeneic transplantation, 165 BMT. See Bone marrow transplantation (BMT) Bog, 9t pRb, 46 Bone marrow transplantation (BMT) allogeneic related, 146 allogeneic unrelated, 146 autologous, 151–152 MCL, 165 molecular monitoring, 157 Ph1-positive cells, 145–146 T-cell depleted allogeneic, 146 Bovine endothelial cell line (BEC) iNOS activation, 116 BRCA1, 9t pRb, 44–45 BRCT (BRCA1 C-terminus) pRb, 44–45 Breast cancer cells apoptosis, 118 caspase inhibitor, 124 BRF/TFIIB, 6t pRb, 24 BRG1, 7t pRb, 31–32 BRUCE p53, 74 BSO NO-mediated apoptosis, 121 BT-20 apoptosis, 118 B5T-overexpressed gene protein (bog), 9t pRb, 46 Buthionine-sulfoximine (BSO) NO-mediated apoptosis, 121
C C-Ab1, 9t pRb, 38–39 CaP. See Prostate cancer (CaP) Capsid antibody HPV, 207–208
241
Index
Cardiolipin NO-mediated apoptosis, 121 CARDs, 68 Caspase, 67–74 cascade, 67–69 classes, 123 Caspase-3, 123 p53, 70–71 Caspase-6 p53, 72 Caspase-7 p53, 72 Caspase-8 FLICE, 123 p53, 68, 72 Caspase-9, 123 E1B 19K, 73–74 p53, 68, 69–70 Caspase recruitment domains (CARDs), 68 CD40 NO production, 113–114 CD95 death receptors, 116–117 Cdc2 pRb, 5t, 14–15 Cdk2 pRb, 5t Cdk4 pRb, 5t CDKs pRb, 14–15 CDNA fragment AML1-ETO detection, 138f–139f CD4T cells NO synthesis, 115 CEA detection, 169 C/EBP, 7t pRb, 30 Cell cycle arrest p53, 57–58 Cell cycle regulating enzymes pRb, 42–47 Cell-mediated immunity (CMI) immunocompetent women HPV-associated disease, 223–228 Cellular immunity HPV infection, 214–225 CENP-F pRb, 43
Central nervous system VHL, 90 Cerebellar HAB VHL, 94 Cerebellar HABs VHL, 90 Cervical neoplasia HLA class I antigens, 216–217 HLA class II antigens, 217–221 C-fos p53, 59 Chc-1, 8t Che-1 pRb, 37 Chemotherapy MCL, 164 t(14; 18), 159 Children ALL, 148 MRD, 150 T-ALL, 149 Chromosome 22 schematic representation, 144f Chromosome 14q32, 157 Chromosome 15q22 PML, 156 Chromosome 17q21 PML, 156 Chromosome 18q21, 157 Chx10, 7t pRb, 25 C-IAP-1 p53, 74 C-IAP-2 p53, 74 C-Jun, 7t pRb, 31 C-Jun N terminal kinase (JNK) pRb, 16–17 CK19 detection, 169 CK20 detection, 169 Clear cell renal cell carcinoma VHL, 86, 90–91, 94 CMI immunocompetent women HPV-associated disease, 223–228
242 CML cytogenetic analysis, 143 Philadelphia chromosome translocation, 143 C-Myc, 7t CaP, 195 p53, 60–61 pRb, 35 Coagulating gland mouse prostate gland, 188 Cognate neuronal-specific activators. See P25nck5a Coiled-coil protein, 43–44 Colorectal cancer (CRC), 165–169 micrometastasis detection, 167f Complex IV downregulation, 120–121 Conventional staining lymph node sections, 168–169 CRC, 165–169 Cream-1/Rbap2, 7t pRb, 37 CrmA, 74 Cross-protective immunity HPV, 226–231 C-Ski, 6t pRb, 23 CtIP, 6t pRb, 23 CTLs HPV, 225 CT values, 140 Cul-2 pVHL, 97 Cyclin A p53, 59 pRb, 5t, 14 Cyclin A1 pRb, 5t Cyclin D1 pRb, 5t Cyclin D2 pRb, 5t Cyclin D3 pRb, 5t Cyclin D-dependent kinases pRb, 15 Cyclin E pRb, 5t, 14
Index
Cyclins pRb, 14–15 Cytochrome c, 119–120 Cytochrome c oxidase (complex IV), 120–121 Cytogenetic analysis CML, 143 Cytogenetics MRD detection, 136t Cytokines HPV infection, 215–216 NF-B activation, 112 Cytological analysis MRD detection, 136t Cytoplasmic cysteine proteases (caspases), 123
D DCs, 215 Death effector domains (DEDs), 68 Delayed-type sensitivity (DTH) HPV, 214 Dendritic cells (DCs), 215 D-FISH CML, 145 MRD detection, 136 DNA MRD detection, 137 PCR, 141t MRD detection, 142t RFLP analysis MRD detection, 137 DNA methylation pRb, 22–23 DNA polymerase-␣, 9t pRb, 42–43 DNA regulating enzymes pRb, 42–47 DNA topoisomerases, 9t pRb, 42–47 DNMT1, 7t pRb, 22–23 Dorsolateral lobe mouse prostate gland, 188 Double-color fluorescence in situ hybridization (D-FISH) MRD detection, 136 Double-color probes, 144 Double-labeled fluorogenic probes, 138
243
Index
DP, 6t pRb, 18–19 DP-1, 6t DP-2, 6t DR5/KILLER, 72 DTH HPV, 214
E E1A p53, 60 E1A C-terminal interacting protein, 23 E2A-HLF fusion gene, 153 ECs NO production, 114 E2F, 6t pRb, 10–11, 18–19 E2F-1, 6t p53, 60 E2F-2, 6t E2F-4, 6t E2F-3a, 6t E2F-3b, 6t Effector caspases, 68, 123 EFM pVHL, 99 Electrophoretic mobility shift assay (EMSA) pRb-binding proteins, 2 Elf-1, 6t pRb, 27 Elongin B pVHL, 97 Elongin C pVHL, 97 EMSA pRb-binding proteins, 2 Endolymphatic sac tumors VHL, 91–92 Endothelial cells (ECs) NO production, 114 Endothelial NOS, 108 Epidermodysplasia verruciformis (EV) HPV, 221 Epididymal cysts VHL, 92 ER pRb, 36–37 Esb cells, 110 ESbL-lacZ lymphoma metastasis, 110–111 Estrogen receptor, 8t
Estrogen receptor (ER) pRb, 36–37 EV HPV, 221 Exon 6, 156 Extracellular fibronectin matrix (EFM) pVHL, 99 Eye VHL, 89–90
F FAB-M3, 155 FACS MRD detection, 136t FADD, 72 death receptors, 116–117 FADD/MORT1 death receptors, 116–117 FAP-1 apoptosis, 118 Fas-associated phosphatase-1 (FAP-1) apoptosis, 118 FISH MRD detection, 136 FKHR1, 196 FLICE caspase-8, 123 FLICE/MACH (caspase-8) death receptors, 116–117 Fluorescence in situ hybridization (FISH) MRD detection, 136, 136t Forkhead transcription factors, 196 Fractional cycle number, 140
G Genetic alterations CaP, 194 Genital neoplasia HPV-associated, 223 Genital warts infiltrating mononuclear cells, 214 GLI-Kruppel-related zinc finger phosphoprotein, 28 Glutathione NO-mediated apoptosis, 121 Glycerol trinitrate (GTN), 116 GM-CSF HPV infection, 215–216
244 Graft-versus-lymphoma (GvL) MCL, 165 GTN, 116 Guanido nitrogen, 108 GvL MCL, 165
H HABs VEGF therapy, 90 VHL, 86 HAT pRb, 19–20 HBP1, 6t pRb, 24–25 HBrm1, 7t pRb, 31–32 HDAC, 6t pRb, 19–20 HDAC1, 6t HDAC2, 6t HDAC3, 6t Heatshock proteins pRb, 39–40 Hemangioblastomas (HABs), 90 VHL, 86 Hematological malignancies karyotypic analysis, 135–136 Hematological remission, 148 Hematopoietic cells, 30 Hepatic leukemia factor (HLF), 153 Her2/neu oncogene CaP, 197 HIF-1␣, 98, 98f High molecular weight keratins (HWMK), 192–193 Histone-acetylase (HAT) pRb, 19–20 Histone-deacetylase (HDAC) pRb, 19–20 HIV HPV, 223 HLA class I antigens cervical neoplasia, 216–217 class II antigens cervical neoplasia, 217–221 HLA-DO HPV cumulative odds ration, 222t
Index
HLA-DR HPV, 214–215, 219 cumulative odds ratio, 220t HLF, 153 HMG-box transcription factor pRb, 24–25 H-Nuc, 9t pRb, 41 Homeodomain proteins, 25 HPV. See Human papillomavirus (HPV) H-RAS CaP, 194–195 H-Ras V12G, 199 Hsc73, 9t pRb, 40 HsHec1p pRb, 43–44 HsHec1p, 9t Hsp75, 9t pRb, 40 Human leukocyte antigen (HLA) class I antigens cervical neoplasia, 216–217 class II antigens cervical neoplasia, 217–221 Human papillomavirus (HPV) associated genital neoplasia, 223 associated lesions immunosuppressed patients, 221–223 capsid-based assays specificity, 210 capsids isotypes, 211 capsid serology sensitivity, 209 cellular immunity, 214–225 cytokines, 215–216 histological studies, 214–215 cross-protective immunity, 226–231 CTLs, 225 seropositivity, 209 transmission, 210 types antagonism, 226–228 cooccurrence, 228–229 vaccination effects, 229–231, 230t Human papillomavirus (HPV) antibody response, 207–213 epidemiological studies, 213–214 isotypes, 211
245
Index
sensitivity, specificity, stability, 208–211 serum, 212t natural history, 206–207 Human papillomavirus (HPV) 6 antagonism, 228 HPV 11 interaction, 227 Human papillomavirus (HPV) 16 antagonism, 228 capsids serological assay, 207–208 HPV 73 antagonism, 227–228 interaction, 226–227 seroconversion, 206–207, 210–211 seropositivity prevalence, 213 HWMK, 192–193
I IAPs, 74–76 family members, 74–75 function, 75 inhibitors, 75–76 p53, 74 structure, 75 ICAD, 71 ICAM, 110 Id2 pRb, 33 Id-2, 7t IgA HPV capsids, 211 IgG HPV, 213 IgG1 HPV capsids, 211 IkB kinases, 111–112 IL-2 HPV, 224 IL-6 HPV infection, 216 p53, 59 IL-8 HPV infection, 216 IL-1␣ HPV infection, 215–216 Immunohistochemistry MRD detection, 136t tumor cell identification, 168–169 Immunological purging, 160
Immunophenotyping by flow cytometry MRD detection, 136t Immunophenotyping on cytospins MRD detection, 136t Immunosuppressed patients HPV-associated lesions, 221–223 Immunotherapy metastasis NO, 113–114 Inducible NOS (iNOS), 108 activity upregulation, 115 NF-B activation, 112 NO production, 113–114 Induction chemotherapy leukemic cells, 154 Induction therapy MRD, 150 Infiltrating mononuclear cells HPV, 214 Inhibitor of caspase-activated-deoxyribonuclease (ICAD), 71 Initiator caspase, 123 INK4A CaP, 197 INOS. See Inducible NOS (iNOS) Interferon inducible nuclear phosphoprotein, 33 Interphase FISH CML, 144 Intrachromosomal rearrangements MRD detection, 137 Intron 6, 156 Inv(16)-positive AML, 155 [inv(16)(p13q22)], 155
J JNK pRb, 16–17 JNK1 pRb, 5t JunB, 7t pRb, 31 JunD, 7t pRb, 31 Jun N terminal kinase (JNK) pRb, 16–17 Jurkat cells, 120
246 Jurkat leukemia cells Bcl-2, 122 NO-mediated apoptosis, 121
K Karyotypic analysis hematological malignancies, 135–136 14-kb human genomic DNA fragment, 200 6-kb PSA promoter fragment, 200 30-kDa intracellular protein, 46–47 67-kDa nuclear matrix protein, 42 52-kDa nuclear transcriptional regulator, 35 Ki-67, 193 immunostaining Mxi1, 195 Kidney VHL, 90–91 Kinase activity assay pRb-binding proteins, 2 Kinase regulators pRb, 4 Kinases pRb, 4, 5t, 14–17, 38–39 Knockout mouse models CaP, 198 Knudson “two-hit” hypothesis tumorigenesis, 3 K-RAS CaP, 194–196 mutations carcinoma development, 168 Kupffer cells, 110 cytotoxicity, 111 NO production, 113–114
L LacZ gene, 200 Lamin A/C pRb, 41 Langerhans cells (LCs), 215 Laryngeal papillomas infiltrating mononuclear cells, 214 LCs, 215 Leukemia complete clinical remission, 133 detection limit, 135 MRD detection, 1425 Liver ECs, 114, 116
Index
Liver macrophages, 110 Long rPB promoter SV40 T antigen, 200 Lymph node sections conventional staining, 168–169 Lymphoma complete clinical remission, 133 detection limit, 135 MRD detection, 1425
M Major breakpoint region (MBR), 158 Malignant cells detection, 134 Malignant disease detection limit, 135 Map-4 p53, 59 MBR, 158 MCF-7 apoptosis, 118 MCM7, 9t pRb, 44 Mdm2, 8t p53, 60 pRb, 34–35 Mesenchymal epithelial interactions prostatic development, 190 Metastasis immunotherapy NO, 113–114 Metastatic cells NO-mediated apoptosis, 111 Metastatic process solid tumors, 171–172 Mi, 8t pRb, 33 Micrometastatic disease identification, 166 Minimal residual disease (MRD), 133 ALL, 148–153 AML, 153–157 colorectal carcinoma, 165–169 description, 134 detection, 133–172 cytogenetic and molecular techniques, 135–137 morphological and immunological techniques, 135
Index
PCR limitations, 141t technical aspects, 135–143 technique sensitivity, 136t in vitro DNA and RNA amplification, 137–143 leukemia, 143–157 lymphoma, 157–165 MCL, 164–165 Philadelphia chromosome (Ph1)-positive CML, 143–148 solid tumors, 165–168 t(14; 18)-positive follicular lymphoma, 157–164 Mitochondria Bcl-2, 64–65 Mitochondrial lipid cardiolipin NO-mediated apoptosis, 121 Mitochondrial NOS, 108 Mitogenic signaling kinase, 17 Mitosin, 9t pRb, 43 Molecular monitoring autologous BMT, 157 CML, 147 MRD, 158 quantitative PCR, 154 residual malignant cells, 170 Molecular relapse, 134, 170 Molecular remission, 134, 170 Molecular staging MRD, 158 Mouse models CaP, 197–200 Mouse prostate gland, 188–189 MRD. See Minimal residual disease (MRD) MRF4, 7t Murine thymocytes NO-mediated apoptosis, 118 Mxi1 CaP, 195 Ki67 immunostaining, 195 Myf-5, 7t MyoD, 7t pRb, 28–29 Myogenin, 7t pRb, 28–29
N NAIP p53, 74
247 NDV, 112 Neuroendocrine cells prostate, 192 Neuronal NOS, 108 Newcastle disease virus (NDV), 112 NF-IL6, 7t pRb, 30 NF-B NO production, 111–112 p50, 7t pRb, 26–27 Nitric oxide (NO). See NO Nkx3.1, 190–191 NMMA, 111, 116 N-monomethyl-L-arginine (NMMA), 111 N-myc, 7t pRb, 35 NO antimetastatic resistance, 109–116 endothelial cells, 114–116 macrophages, 110–114 apoptosis caspase, 124 apoptotic cell death, 109 cytotoxic and regulatory functions, 113–114 induced apoptosis Bcl-2 downregulation, 123 death receptors, 116–118 mediated apoptosis Bcl-2 family, 122–123 caspase activation, 123–124 death receptors, 116–118 mitochondrial control, 119–122 p53, 118–119 p53 response, 118–119 production liver ECs, 114 macrophages, 110 NF-B, 111–112 NF-B activation, 112 NO synthase (NOS), 108 induced, 108, 112–114 Noxa, 62–63 N-RAS CaP, 194–195 NRP/B, 9t pRb, 42 Nuclear factor-B (NF-B) p50 pRb, 7t, 11–112, 26–27 Nuclear lamin A, 9t
248 Nuclear lamin C, 9t Nuclear matrix association pRb, 40–42 Nuclear phosphoprotein, 37 Nuclear restricted protein/brain (NRP/B), 9t pRb, 42 Nucleotide sequence analysis t(14; 18), 158 Nude mice described, 111
O Occult lymphoma cells, 158 PCR, 159 Occult tumor cells identification, 166 MRD detection, 1425 Oncogene expression p53, 60 Osteopontin, 115
P P53 activation regulation, 60–62 Bcl-2, 62–67 CaP, 196–197 carcinoma development, 168 cell cycle arrest, 57–58 mediated apoptosis Bax, 66 Bcl-2, 62–67 cancer therapy, 76 caspase, 67–74 IAPs, 74–76 transcription regulation, 58–60 tumor suppression, 56–58 pRb, 34–35 transcription regulator, 58–60 tumor suppressor, 56–58 P67, 15 P202, 8t pRb, 33 PACT, 9t pRb, 47
Index
Pancreas VHL, 91 Pancreatic RINm5F cells NO-mediated apoptosis, 118 Pax, 6t-7t Pax-3, 6t pRb, 25 Pax-5, 6t pRb, 25 Pax-6, 7t pRb, 25 PBaK, 7t P21CIPI/WAF1 pRb, 9t, 45 PCNA, 193 PCR. See also Real-time quantitative PCR; RT-PCR ALL limitation, 148 analysis t(14;18), 159 assays sensitivity, 140–141 follicular lymphoma reliability, 162–164 improvements, 137–138 MRD, 153 prognosis, 160 MRD detection, 136t and stochastic analysis, 136t quantitative molecular monitoring, 154 residual malignant cells, 170 quantitative assays real-time technology, 172 technical limitations, 141–142, 141t P120E4F, 7t pRb, 28 Pericentric inversion chromosome 16, 155 Permeability transition pore (PTP), 65 Permeability transition (PT) pores, 119–120 Pheochromocytoma VHL, 91 Philadelphia chromosome (Ph1)-positive ALL, 151–152 Phosphatases pRb, 4, 5t Phosphatases type 1 (PP-1) pRb, 17
249
Index
PHox, 7t pRb, 25 PIN, 193 mouse models, 198–200 P27KIP1 CaP, 197 P57KIP2, 9t pRb, 45 PML, 8t pRb, 37–38 P84N5, 9t pRb, 41 P25nck5a pRb, 5t, 15–16 P35nck5a pRb, 15–16 P39nck5a pRb, 15–16 Polymerase chain reaction (PCR). See PCR Poly-proline (PP) domain, 58 PP1␣ pRb, 5t, 17 PP1␦ pRb, 5t PP domain, 58 P2P-R, 9t pRb, 46 PRb, 8t binding proteins, 2–13 assays, 2–3 examples, 4 interactor identification, 11–13 transcription regulation, 6t–8t cell cycle regulating enzymes, 42–47 cyclins and CDKs, 14–15 DNA regulating enzymes, 42–47 E2F, 10–11 heatshock proteins, 39–40 interacting proteins, 4 interacting zinc finger protein, 36 kinases, 14–17, 38–39 nuclear matrix association, 40–42 phosphatases, 17 pocket protein, 3–4, 4f transcriptional regulators, 18–38 activation, 28–31 repression, 18–28 Pre-lymphoma cells, 162 Pre-pre-lymphoma cells, 162
Pro1 pRb, 9t, 46–47 Procaspase-8 p53, 68 Procaspase-10 p53, 68 Programmed cell death, 56 caspases, 123 TNF receptor, 116 tumor cells, 109 Progression malignant lymphomas, 162 Prohibitin (PRO1) pRb, 9t, 46–47 Prostate cancer (CaP), 187–201 animal models, 197–200 c-MyC, 195 genetic alterations, 194 Mxi1, 195 PTEN, 196 RAS activating mutations, 194–195 tumor suppressor genes, 196–197 Prostate gland anatomy, 188–189 embryology, 189–190 signaling pathways, 190–192 Prostate-specific antigen (PSA), 192 Prostatic epithelium cell types, 192 Prostatic intraepithelial neoplasia (PIN), 193 Prostatic primary ducts, 189 Prostatic stem cells, 192–193 Protein retinoblastoma. See PRb PSA, 192 PSLF-RAR ␣ transcript APL, 156 PTEN CaP, 196 PTP, 65 PT pores, 119–120 PU.1, 7t pRb, 27 Pur␣ pRb, 7t, 34 PVHL intracellular roles, 99, 100f ubiquitin ligase complex, 96–99 PVHL30 tumor suppressor activity, 95
250 PVHL-associated proteins, 96–99 PVHL-dependent target gene angiogenesis, 95–96 regulation, 95–96 PVHL gene products tumor suppressor activity, 95 P21WAF1 pRb, 59
Q Quantitative PCR molecular monitoring, 154 residual malignant cells, 170 Quantitative PCR assays real-time technology, 172
R Raf pRb, 5t Raf-1 pRb, 17 RAG-mediated transposition, 162 Rak, 9t pRb, 39 RAR␣ PML, 37–38, 156 RAS activating mutations CaP, 194–195 p53, 60 Rat probasin (rPB), 199 RAW 264.7 macrophages NO-mediated apoptosis, 118 RB1 CaP, 197 RBaK pRb, 27–28 RbAp, 6t RbAp2, 7t pRb, 21–22, 37 RbAp46, 6t pRb, 20–21 RbAp48, 6t pRb, 20–21 RBP, 6t RBP1, 6t pRb, 21–22 RBP2, 6t pRb, 21–22
Index
RBP60, 6t pRb, 18–19 RBQ3, 9t pRb, 47 RBQ1/PACT, 9t pRb, 47 Rbx-1, 97 RCC VHL, 86, 90–91, 94 Real-time fluorescence detection, 138 Real-time quantitative PCR, 138–140, 138f–139f AML, 155 CRC, 172 minimal residual cells, 170 t(14; 18), 164 REC2, 9t recombinase pRb, 43 Recombination-activating gene (RAG)-mediated transposition, 162 Renal cell carcinoma (RCC) VHL, 86, 90–91, 94 Renal cysts VHL, 90–91 Residual malignant cells molecular monitoring, 170 Restriction fragment length polymorphism (RFLP) analysis MRD detection, 137 Retinal angiomas VHL, 89–90 Retinoblastoma. See PRb Retinoic acid receptor-␣ gene (RAR␣) PML, 37–38, 156 Reverse tetracycline transactivator (rtTA), 199 RFLP analysis MRD detection, 137 Rim, 8t pRb, 38 RIZ, 8t pRb, 36–37 RNA PCR limitations, 141t RPB, 199 RPB/T antigen models CaP, 200 RT-PCR, 141
251
Index
AML1-ETO, 154 assays MRD detection, 142 MRD, 145 PSLF-RAR ␣ mRNA, 156 targets MRD detection, 1425 techniques BCR-ABL mRNA detection, 146 two-step nested, 169 RtTA, 199
S SAPK pRb, 16–17 SCID mice described, 111 Secretory cells prostate, 192 Serial analyses AML, 154 Serine/threonine phosphatases type 1 pRb, 17 Serum IgG antibodies HPV16 capsids, 208 Signaling cascade mesenchymal epithelial interactions prostatic development, 190 Signaling pathways prostate gland, 190–192 Sin3a, 6t pRb, 20–21 Skin warts infiltrating mononuclear cells, 214 Smac/DIABLO, 65, 75 Sno, 6t pRb, 23 Solid tumors complete clinical remission, 133 MRD detection, 1425 Sonic hedgehog UGS epithelium, 190 Southern blot analysis MRD detection, 136t Southern blot hybridization MRD detection, 137 t(14; 18), 158 Sp1, 7t pRb, 31
Sp3 pRb, 31 Sp1-I, 7t pRb, 31 Spleens, 161 Sporadic pheochromocytoma VHL, 95 Src-related nuclear tyrosine kinase, 39 Standard risk ALL, 148–151 Stress-activated protein kinases (SAPK). See also C-Jun N terminal kinase pRb, 16–17 Sublethally ␥ -irradiated nude mice, 115 Sublethally ␥ -irradiated SCID mice, 115 Survivin p53, 74 SV40 T antigen long rPB promoter, 200
T T(1; 19) positive ALL, 152–153 T(8; 21) AML1-ETO detection, 138f–139f positive AML, 153–155 T(11; 17) (q13; q21), 156 T(14; 18) positive malignant follicular lymphomas multistep evolution, 163f T(15; 17) translocation PML, 156 T(16; 16) (p13; q22), 155 T(17; 19) positive ALL, 152–153 (q22; p13.3) translocation, 153 TAFB80, 8t TAFB150, 8t TAFs, 36, 58 pRb, 36 Taq DNA polymerase, 138 Target cells PCR limitations, 141t Target molecules PCR limitations, 141t TATA-binding protein-associated factor (TAFs), 36, 58
252 TATA box binding proteins (TBP), 6t, 24, 58 TBP, 6t, 58 pRb, 24 TBP-associated factors (TAFs), 36, 58 T-cell depleted allogeneic BMT, 146 T-cell receptor (TCR), 149 TCR, 149 TFIIB, 6t pRb, 24 TFIID, 58 TFIIIB, 6t pRb, 24 TGF-, 115 Threonine phosphatases type 1 pRb, 17 Threshold cycle, 140 Th1 type cytokines HPV infection, 215 Th2 type cytokines HPV infection, 216 T lymphocytes NO synthesis, 115 TNF-␣ HPV infection, 216 NO production, 116 TNFR apoptosis, 116 TNFR1, 72 Tonsils, 161 Topo-II␣ pRb, 9t, 42 TRAIL/APO-21, 117 Transcriptional regulators p53, 58–60 pRb, 4, 18–38 activation, 28–31 repression, 18–28 Transformation malignant lymphomas, 162 Transforming growth factor-beta (TGF-), 115 Transgenic mouse models CaP, 198 Trip230, 6t pRb, 26 Tumor cell death activated ECs, 114 Tumor cell identification immunohistochemistry, 168–169
Index
Tumor cell vaccination therapeutic efficacy, 161 Tumorigenesis Knudson “two-hit” hypothesis, 3 prostate gland signaling pathways, 191–192 Tumor metastasis NO production, 110–111 Tumor necrosis factor-␣ HPV infection, 216 NO production, 116 Tumor necrosis factor receptor apoptosis, 116 Tumor necrosis factor receptor 1, 72 Tumor suppressor genes CaP, 196–197 p53, 56–58 Two-step nested RT-PCR techniques CRC, 169
U UBF, 6t pRb, 24 Ubiquitin ligase complex pVHL, 96–99 UGS, 189–190 Unmanipulated allogeneic bone marrow, 146 Urogenital sinus (UGS), 189–190 Urorectal septum prostate gland, 189–190
V Vaccination HPV infection, 229–231, 230t Vascular endothelial growth factor (VEGF) therapy HABs, 90 VCAM, 110 VDAC Bcl-2, 65 VEGF therapy HABs, 90 VFLIPs p53, 74 VHL. See Von Hippel-Lindau disease
253
Index
Viral caspase inhibitors p53, 74 Viral-FLICE-inhibitory proteins (vFLIPs) p53, 74 Viral load HPV seropositivity, 209 Viral persistence HPV seropositivity, 209 Von Hippel-Lindau disease (VHL), 85–100. See also PVHL clinical features, 86 diagnosis, 89–92 gene, 87–88 genotype-phenotype correlations, 92–94, 93f molecular basis, 100–101 surveillance, 92 tumorigenesis, 100–101 tumor suppressor gene
functional analysis, 95–101 sporadic cancers, 94–95 tumor suppressor gene germline mutation, 88–89 tumor suppressor gene mutation database, 88 VHL gene, 87–88 VHL TSG sporadic cancers, 94–95 VHL TSG germline mutation, 88–89
W Western blot pRb-binding proteins, 2
X XIAP p53, 74
