Current Topics in Developmental Biology, Volume 04

CURRENT TOPICS IN

DEVELOPMENTAL BIOLOGY VOLUME 4

ADVISORY BOARD

VINCENT G. ALLFREY

DAME HONOR B. FELL, F.R.S.

JEAN BRACHET

JOHN C. KENDREW, F.R.S.

SEYMOUR S. COHEN

S. SPIEGELMAN

BERNARD D. DAVIS

HEWSON W. SWIFT

JAMES D. EBERT

E. N. WILLMER, F.R.S.

MAC V. EDDS, JR.

ETIENNE WOLFF

CONTRIBUTORS

JACK GORSKI

C . SHYAMALA

CLIFFORD GROBSTEIN

PATRICIA G. SPEAR

MAX HAMBURGH

D. TOFT

ALEXKEYNAN

LEONARD WARREN

JACK E. LILIEN

DAVID YAFFE

BERNARD ROIZMAN

CURRENT TOPICS

IN

DEVELOPMENTAL B I O L O G Y EDITED BY

A. A. MOSCONA IIEPAl~TMENT OF BIOLOGY THE UNIVERSITY OF CHICAGO

CHICAGO, ILLINOIS

ALBERT0 MONROY ISTITUTO DI ANATOMIA COMPAHATA UNNERSITA DI PALERMO PALERMO, ITALY

VOLUME 4

1969

ACADEMIC PRESS New York

London

COPYRIGHT @ 1969, BY ACADEMIC PRESS, INC. ALL RIGHTS RESERVED NO PART OF THIS BOOK M A Y BE PRODUCED IN ANY FORM, BY PHOTOSTAT, MICROFILM, RETRIEVAL SYSTEM, OR ANY OTHER MEANS,

WITHOUT WRITTEN PERMISSION FROM

THE PUBLISHERS.

ACADEMIC PRESS, INC.

111 Fifth Avenue, New York, New York 10003

United Kingdom Edition published by ACADEMIC PRESS, INC. (LONDON) LTD. Berkeley Square House, London W l X BBA

LIBRARY OF CONGRESS CATALOG CARDNUMBER:66-28604

PRINTED IN THE UNITED STATES OF AMERICA

LIST OF CONTRIBUTORS Numbers in parmtheses indicate the pages on which the authors’ contributions begin.

GORSKI,Department of Physiology and Biophysics, University of Illinois, Urbanu, Illinois (149) GROBSTEIN, School of Medicine, University of California, Sun CLIFFORD Diego, La Jolla, California (xv) MAXHAMBURGH, Department of Biology, City College of New York, and Department of Anatomy, Albert Einstein College of Medicine, New York, New York (109) ALEX KEYNAN, Department of Microbiological Chemistry, Hebrew University-Hadassah Medical School, Jerusalem, Zsrml ( 1 ) JACK E. LEIEN,* Department of Therapeutic Research, School of Medicine, University of Pennsylvania, Phikulelphia, Pennsyluania ( 169) BERNARD ROIZMAN,Department of Microbiology, The University of Chicago, Chicago, Illinois (79) G. S m A M m A , t Department of Physiology and Biophysics, University of Illinois, Urbana, Illinois (149) PATRICIAG. SPEAR,Department of Microbiology, The Unioersity of Chicago, Chicago, Illinois (79) D. TOFT,$Department of Physiology and Biophysics, University of Illinois, Urbana, Illinois (149) LEONARD WARREN,Department of Therapeutic Research, School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania ( 197) DAVID YAFFE, Department of CeU Biology, The Weizmann Institute of Science, Rekouot, lsrael (37) JACK

0 Present address: Departnient of Zoology, University of Wisconsin, Madison, Wisconsin. t Present address: Ceiicer Research Genetics Laboratory, University of California, Berkeley, California. t Present address: Department of Obstetrics and Gynecology, Vanderbilt University, Nashville, Tennessee.

V

This Page Intentionally Left Blank

PREFACE The Editors wish to thank the contributors to Volume 4 for their cooperation in meeting the specific aims of Current Topics in Deueloy mental Biology. We also thank the members of the Advisory Board and the staff of Academic Press for their continuous efforts to increasc thc usefulness of this publication and to maintain its high standards. A. MONROY A. A. MOSCONA

Septenilier, 1969

vii

This Page Intentionally Left Blank

... . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . .. . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . .

vii

... .. .. . . .. .. ............ .. .. .. .........

xiii

List of Contributors Preface

,

Contents of Previous Volumes

,

,

,

V

Genetics and Genesis

CLIFFORD GROBSTEIN. . . . . . . . . , , , . . . , . . . . . . . . . . . . .

~UTER

xv

1. The Outgrowing Bacterial Endospore

ALEX KEYNAN

.

.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . The Difference between Spores and Vegetative Cells . . . . . . . . . . . . . . Processes Involved in the Transformation of Spores into Vegetable Cells The Overall Program of Biochemical Events during Outgrowth . . . . . . The First Molecular Events during Outgrowth . .. .. . . . . . . .. .. . . . The Pattern of Protein Synthesis during Outgrowth . . . . . . . . . . . . . . . . . Cell Wall Formation ............... .... .. .. .. .. ...... .. ... .. DNA Synthesis and Chromosomal Replication . . . . . . . . . . . . . . . . . . . . Conditions That Influence and Direct the Differentiational Events in the Outgoing Endospore .. . . . . . . .. .. . . . . . . . . . . . . . . . . . ... . . . . .. X. The Outgrowing Bacterial Endospore as a System for the Study of Cellular Differentiation ........................................... XI. Summary .................................................... References ...................................................

I. 11. 111. IV. V. VI. MI. VIII. IX.

CHAPTER

..

..

..

.

2.

.

.

.

..

2 4 8 13 15 21 25 27 30 31 33 33

Cellular Aspects of Muscle Differentiation in Vitro

DAVIDYAFFE

...

.. . . . . . . . . .. .. . . . . . . .. . . .. .. .. .. . . . . . . . . . . . . . . . I. Introduction 11. Differentiation of Primary Cultures . . . . . . . . . .. .. . . . . . . . . . . . . . .

.

ix

. ..

37 39

CONTENTS

X

. . .

........................................... ...................................................

I11 Myogenic Cell Lines IV Induction of DNA Synthesis and Mitosis in Nuclei within Muscle Fibers V Comments References ...................................................

CHAPTER

.

3

45 60

70 75

Macromolecular Biosynthesis in Animal Cells Infected with Cytolytic Viruses

BERNARD ROIZMANAND

PATRICIAG

. SPEAR

.

1 Introduction .................................................. 79 I1 The Inhibition of Host Macromolecular Synthesis ................... 82 111 Why Is Host Macromolecular Synthesis Inhibited by Viruses? ........ 102 105 References ...................................................

. .

CHAPTER

4

.

The Role of Thyroid and Growth Hormones in Neurogenesis

MAX HAMBURGH

. . . .

I I1 I11 IV

Introduction .................................................. The Role of Thyroid Hormone in Neurogenesis .................... The Effect of Growth Hormone on Neurogenesis ................... Remarks References ...................................................

.....................................................

CHAPTER

109 110 139 143 144

5. Interrelationships of Nuclear and Cytoplasmic Estrogen Receptors JACK

. .

I I1 I11. IV. V VI VII

. .

.

GORSIU.G. SHYAMALA.AND D . TOFT

149 Introduction .................................................. Estrogen Receptors ............................................ 150 Subcellular Location of Estrogen-Binding Agent .................. 151 155 Binding of Estrogens to the Cytosol .............................. 158 Cell-Free Binding of Estrogen ................................... Nuclear-Cytosol Interrelationships ............................... 157 184 Conclusions and Speculations ................................... 188 References ...................................................

CONTENTS CZIAPTER

6. Toward a Molecular Explanation for Specific Cell Adhesion JACK

. .

I I1 I11. IV.

E . LILIEN

Introduction .................................................. Sponge Cells as a Model for Specific Cell Adhesion ................. Studies on Vertebrate Cells ..................................... Concluding Remarks ........................................... References ...................................................

CHAPTER

xi

169 170 175 192 193

7 . The Biological Significance of Turnover of the Surface Membrane of Animal Cells

LEONARD WARREN I. Introduction .................................................. I1. Composition of the Surface Membrane ............................ I11. The Dividing and Nondividing Cell .............................. IV. Synthesis and Turnover Rates of the Surface Membrane during the Cell Cycle ....................................................... V. Differential Turnover of Components of the Surface Membrane ...... VI. Synthetic and Degradative Aspects of Turnover .................... VII. Turnover and Repair .......................................... VIII . Speculation on the Malignant Cell ............................... IX . Conclusions .................................................. References ................................................... Author Index Subject Index

197 198 199 207 209 210 212 212 219 220

.......................................................

223

......................................................

232

This Page Intentionally Left Blank

CONTENTS OF PREVIOUS VOLUMES Volume 1 REMARKS Joshua Lederberg OF MESSENGER RNA IN EARLYEMBRYOGENESIS AND ON “MASKED”FORMS IN OTHER DIFFERENTIATING SYSTEMS A. S . Spirin THE TRANSCRIPTION OF GENETIC INFORMATION IN THE SPIRALIAN EMBRYO I. R. Collier SOMEGENETICAND BIOCHEMICAL ASPECTS OF THE REGULATORY PROGRAM FOR SLIMEMOLDDEVELOPMENT Maurice Sussman THE MOLECULARBASISOF DIFFERENTIATION IN EARLY DEVELOPMENT OF AMPHZBIANEMBRYOS H . Tiedemnn THECULTURE OF FREE PLANTCELLSAND ITS SIGNIFICANCE FOR EMBRYOLOGY AND MORPHOGENESIS F . C . Steward, Ann E . Kent, and Marion 0 . Mapes GENETICAND VARIEGATION MOSAICSIN THE EYE OF Drosophih Hans loachim Becker BIOCHEMICAL CONTROL OF ERYTHROID CELLDEVELOPMENT Eugene Goldwasser DEVELOPMENT OF MAMMALIAN ERYTHROID CELLS Paul A. Marks and John S . Kovach GENETIC ASPECXSOF SKINAND LIMBDEVELOPMENT P. F. Goetinck AUTHOR INDEX-SUBJECT INDEX

Volume 2

THE CONTROL OF PRomw SYNTHESIS IN EMBRYONIC DEVELOPMENT AND DIFFERENTIATION Paul R. Gross xiii

xiv

CONTENTS O F PREVIOUS VOLUMES

THE GENESFOR RIBOSOMALRNA AND THEIR TRANSACTION DURING AMPHIBIANDEVELOPMENT Donald D . Brown RIBOSOMEAND ENZYME CHANGES DURING MATURATION AND GERMINATION OF CASTOR BEANSEED E r m o Marrd CONTACT AND SHORT-RANGE INTERACTIONS AFFECTING GROWTH OF ANIMAL CELLSIN CULTURE Michael Stoker AN ANALYSISOF THE MECHANISM OF NEOPLASTIC CELLTRANSFORMATION BY POLYOMA Vmus, HYDROCARBONS, AND X-IRRADIATION Leo Sachs DIFFERENTIATION OF CONNECTIVE TISSUES Frank K. Thorp and Albert Dorfman THEIGA ANTIBODYSYSTEM Mary Ann South, Max D. Cooper, Richard Hong, and Robert A. Good TERATOCARCINOMA: MODELFOR A DEVELOPMENTAL CONCEPT OF CANCER G. Barry Pierce CELLULAR AND SUBCELLULAR EVENTS IN WOLFFIAN LENSREGENERATION Tuneo Yamada AUTHORINDEX-SUBJECT INDEX Volume 3 SYNTHESIS OF MACROMOLECULES AND MORPHOGENESIS IN ACETABULARIA 1. Brachet BIOCHEMICALSTUDIESOF MALE GAMETOGENESIS IN LILIACEOUS PLANTS Herbert Stern and Yasuo Hotta SPECIFICINTERACTIONS BETWEEN TISSUES DURING ORGANOGENESIS Etienne Wolfl LOW-RESISTANCE JUNCTIONS BETWEEN CELLS IN EMBRYOS AND TISSUE CULTURE

Edwin J. Furshpan and David D . Potter COMPUTER ANALYSISOF CELLULAR INTERACTIONS F . Heinmets CELLAGGREGATION AND DIFFERENTIATION IN Dictyostelium Gunther GeTisch HORMONE-DEPENDENT DIFFERENTIATION OF MAMMARY GLANDin Vitro Roger W. Turkington AUTHORINDEX-SUBJECTINDEX

GENETICS AND GENESIS Uliford Grobstein SCHOOL O F MEDICINE

UNIVERSITY O F CALIFORNIA, SAN DIEGO LA JOLLA, CALIFORNIA

The Editors of Current Topics in Deoelopmental Biology, in their Preface to Volume 1, stated their objective to be “useful not only as a source of topical information and interdisciplinary exchange of views but . . . also [to] aid in evaluating concepts and in crystallizing guidelines in the field of developmental biology . . . with emphasis on regulatory mechanisms at the molecular, biochemical, cellular, and histological levels.” With the tables of contents of the first four volumes before me I could not resist checking on how well the Editors have been doing. I find that the first 33 articles fall approximately into the projected editoral pattern with respect to levels of organization. With the reservations which such classification requires, I place 16 of the 33 articles in the molecular category, 7 in the cell category, 8 in the supercellular or histological category, and only 2 in the organismal category. That is certainly reasonably “on” the selected target. With respect to disciplines, the authors include embryologists, pharmacologists, biophysicists, microbiologists, cell biologists, biochemists, geneticists, oncologists, pathologists, internists, pediatricians, plant physidogists, and virologists-with no particular concentration in any one of these areas. Interestingly, one third of the authors are in research instibutes, one third in medical schools, only one third in university departments, and none in liberal arts colleges. If these data reflect the field rather than the Editors’ predilections (my guess is that they reflect both), they suggest that developmental biology is strongly interdisciplinary, is increasingly supported for its high theoretical and medical interest, and is doing poorly in training young people specifically to enter the field. I am satsfied that this is a reasonable characterization of developmental biology, whether or not it is justified by the small sample of 33 articles in four volumes. Perhaps the matter should be put the other way: I believe the Editors are succeeding substantially in reflecting the state of the field. xv

xvi

GENETICS AND GENESIS

If this is so, the four volumes indicate that developmental biology today is very heavily involved with nucleic acid metabolism and protein synthesis, because information on this single topic makes up by far the largest proportion of the contained material. This will not be surprising to anyone who knows the history of biology in the past two decades. Cell bialogy, genetics, virology, microbiology, and biochemistry coalesced in their focus on the mechanisms of nucleic acid and protein turnover, and it became clear that diflerentiation and development would have to be fitted into the picture. The four volumes indicate that the process is well under way but that the emerging conception is still patchy and unresolved, like a developing photographic print as the image just begins to appear. I shall take advantage of this state of conceptual imminence, and the Editors’ desire to “aid in evaluating concepts and in crystallizing guidelines,” to comment on what seems to me to be the need for a little sharper focus in the conceptual image itself. We are nearly all now reasonably persuaded that replication, transcription, and translation, as generally defined, represent a basic outflow track for genetic information transmitted between generations in the form of nucleic acid sequence. We are further persuaded, and evidence is presented in these volumes, that the outflow probably can be regulated and controlled at all three levels, and that the general phenomenon of regulation is essential to provide differential operation of the genome, which we recognize to be necessary for both function and development. Indeed, one of the conceptual problems that has arisen is how to restate our notions of heredity, function, and development so as to reveal their common foundation, and yet to display their differing orbits of relevance. It is probably not necessary to remind biologists of the last third of the twentieth century that embryology and genetics came apart at the turn of the century under the stress of the effort to comprehend continuity between generations. Genetics focused on the transfer of properties, embryology on their apparent origin in the new generation. The two adopted different methodologies and terminologies and hardened into disciplines, frequently seemingly at odds. Today the methodologies are once again becoming similar and the terminologies are rejoining. An important common terminological base is provided by replication, transcription, translation, and regulation. None of these terms is solely genetic, developmental, or functional; rather the phenomena designated are common to all three orbits of relevance. Clearly, however, each orbit requires its own terms to deal with the phenomena beyond these basic four which are

CLIFFORD GROBSTEIN

xvii

additionally involved. It is to the additions required for development that

I want to direct attention. The special orbit of developmental studies is the reconstruction of a total organism, frequently of great complexity, from transmitted genetic information in a single cell or group of cells acting as precursor. “Genetic information” in this sentence may be rewritten as “replicating nucleic acid sequence” with the understanding that the possible involvement of additional forms of information is not excluded. The replicative process i s central to all genetics, and to biological continuity, and has been referred to-when necessary to emphasize this step alone-as transmission genetics. The desirability of this clarifying designation stems from the fact that the scope of the term genetics has been considerably broadened in recent years, as prime interest properly has shifted from foundational transmission genetics to contemporary molecular genetics, in turn seen as the entrke to developmental genetics and development itself. This approach has led to the concept of translational genetics, which is applied usefully to the remaining three fundamental steps, i.e. transcription, translation, and regulation. Transmission genetics has to do with the maintenance, replication, and transmission of the stable hereditary information, translational genetics has to do with the processes controlling the output at a given time of primary gene products-amino acid chains of determined sequence. Important, however, though these mechanisms are, they are not enough to exhaust the developmental process, nor to delineate, sharply or fully, the questions which still need to be asked and answered about developmental controlling mechanisms. We are now aware, for example, that the properties of enzymes are not a simple function of linear amino acid sequence, that certain sequences are more critical than others, and that three-dimensional configuration is essential to aligning the critical sequences into active sites. We also know that many enzymes and structural proteins involve more than one amino acid chain, and that protein interactions with one another and with other macromolecules are the basis for critical relationships in establishing ultrastructure. These complexities are derivative directly from genetically transmitted information, in the sense that many have been shown to “self-assemble“ from genetically determined components. In the process, however, new properties appear4.g. banding patterns based upon register-which arise by transformation of relationships originally specified only in linear sequence. These “new” properties are the sine qua non for still further developmental steps. Such transformational processes without additional genetic information

xviii

GENETICS AND GENESIS

are, therefore, another step-beyond translation-in the metamorphosis of genotype into phenotype. They may be usefully designated metagenesis. Where the transformation involves a single molecular species it is homogeneous metagenesis. Where more than one species is involved, as in membranes, it may be referred to as heterogeneous metagenesis. It is important to note that many metagenetic processes afford either alternative or quantitatively different configurational states which, without requiring new genetic information, have importantly different properties for further developmental progression. Models of such processes include the alternative configurations demonstrated in collagen fibrogenesis and the allosteric postulate for enzyme action. These not only constitute another level for regulation of genetic expression but open a channel for the kind of environmental intervention which provides degrees of freedom between genotype and phenotype. Exciting though it is to be able now to visualize a continuum from bascl sequence to ultrastructure, we should not be excited beyond humility. The fact is that the gap from ultrastructure to a new cell is a major one. Lederberg, in his remarks opening Volume I, came close to “suggesting that a cell will crystallize itself out of the soup when the right components are present.” Perhaps it may, but it is worth recalling that, so far as we are aware, Nature has not seen fit to follow this procedure for a long time now. Development follows the hoary doctrine that it takes a cell to produce a cell. This does not invoke a mysterious process of “organization,” which Lederberg protests, but notes that for the moment the stepby-step conceptual progression of development proceeds from replication to metagenesis and stops. The next phenomenologic step is production of a whole cell, and we do not yet “see through” to this. If mystery is thc other face of ignorance, the organized behavior of the cell is mysterious. So, too, however, was ultrastructure mysterious before we became clearer about protein structure, polymerization, and conformation. We now recognize that order, with new information content, can “emerge” spontaneously. Neogenesis, so essential to developmental recapitulation, can occur. It is not mysterious in the sense of occult, but it does require exquisitely prepared preconditions. What is there about a cell which makes it a sufficient condition for the emergence of another cell? Is it a necessary condition? Is there an essential element, and if so is it describable in terms from replication through metagenesis? Or is there an order-emergent activity which eventually might be labeled cybernogenetic, i.e., giving rise to a new control center for a complex phenomenon? Are centrioles in this category? Is something of this sort operating when

CLIFFORD GROBSTEIN

XiX

Paramecium replicates cortical abnormality or Stentor organizes a new peristomial disc? Having crossed the gap of ignorance with respect to the genesis of new cells we stand again on a small stable conceptual island. Order can emerge in populations of cells, based upon information in their surfaces which we can readily imagine is produced by phenomena ranging through metagenesis, Whether through homogeneous or heterogeneous metagenesis fntercellular ultrastructure arises which locks cells into tissue and organ collectives, In the process cell behaviors are altered and new properties again appear-in both the component cells and the collectives. These are the phenomena of histogenesis (including cytodifferentiation and organogenesis ) whose inner workings remain mysterious though we see ourselves as on the threshold of conceptual understanding. We have not, however, reached the end of developmental progression from genotype to phenotype. Histogenesis leads to collective cellular behaviors and a new series of emergent properties. Nowhere is this more spectacular than in the nervous system. We are all aware of the challenge, and the potential fruits, of comprehension of neurogenesis. Do functionally significant neural patterns self-assemble like kidney tubules or eartilage? Does behavior self-assemble along with neural patterns? We know that some behavior is innate, but surely some is learned. How does experience ( environment) register between genotype and phenotype? It is not surprising that mutation affects behavior; we can expect defective enzymes to lead to behavioral pathogenesis. But does the registration of experience occur between replication and histogenesis? Are there new processes emergent in the incredible complexities and organization of the central nervous system? Shall we perhaps recognize teleogenesis, the origins of purposefulness? And what of egogenesis, the origins of consciousness of self? Are we being mysterious if we suggest that there may be something here in the way of emergence that ignorance yet fails to predict? Would we really have predicted the amino acid sequence of ribonuclease if we had known only its cistronic base sequence? Would we really have predicted its enzymic action if we had only the amino acid sequence and knew nothing of its relationships to substrates? How many similar neogenetic steps lie between ribonuclease and the genesis of choice? How long will it be before we unravel these and acquire the knowledge-as an early teacher of mine used to say-an embryo has without a bachelor’s degree? What I am saying, of course, is that there is much development beyond even translational genetics. Development, along with genetics, has a stake

xx

GENETICS AND GENESIS

in replication, with emphasis particularly on control of its initiation and the occurrence of differential replication. Development is a full partner, indeed is almost indistinguishable from genetics, in approaching the transcriptional and translational mechanisms to which the term epigenetics possibly should be c o h e d . Beyond this, genetics, in the stricter sense, begins to lose interest and physiology begins to gain interest in what development provides. Neogenesis, the appearance of new properties whether structural or functional, is peculiarly the focus of developmental studies. Neogenesis begins with metagenesis at the molecular level and, as I have said, conceivably may extend to egogenesis. At each higher level, as we now see clearly in molecular metagenesis, there is the opportunity for altered combinations and relationships of elements, permitting information to be read in new contexts, and hence to become “new” information. Simultaneously, there is entree for an increasing involvement of non-genetic and environmental influences, hence the higher the level the greater the shared interest between development and physiology. This is especially true since in genesis there frequently are clues to the nature of complex phenomena before their complexity becomes full blown. Nowhere is this more the case than in neurogenesis, long relatively neglected but certain soon to burgeon. Here human development obviously progresses from the onset of integrative systemic control to the initiation of choice and the flicker of awareness. Are these mysterious? Indeed they are. Are they inscrutable? Only to ignorance-and failure to grapple with the steady emergence of new properties with complexity. This is the neogenetic core of development which begins with molecular metagenesis. Thus it lies mostly beyond translational genetics, spanning the gap between it and all the complexity of the functional phenotype. As we fill this gap conceptually-as development does phenomenologically-we shall see mysteries dispell, perhaps even the ultimate mystery of self.

CHAPTER 1

THE OUTGROWING BACTERIAL ENDOSPORE AS A SYSTEM FOR THE STUDY OF CELLULAR DIFFERENTIATION Alex Keynan DEPARTMENT OF MICROBIOLOGICAL CHEMISTRY, HEBREW UNIVERSITY-HADASSAH MEDICAL SCHOOL, JERUSALEM, ISRAEL

.. .. . .... .. .. . . . . .... ...

.. ...

.. . . . I. Introduction . . . .. . 11. The Difference between Spores and Vegetative Cells 111. Processes Involved in the Transformation of Spores into Vegetative Cells . . . . . . . . . . . . . . . . . . . . .. . . . . . . . .. . A. Induction of Developmental Changes in Spore Suspensions and the Succession of Biochemical Events during the Transformation of Spores into Vegetative Cells . . . .. . . . . . . . . . . . . .. . . . . .. . . . . .. . . . . B. The Experimental System for the Study of Outgrowth of Bacterial Endospores . .. . IV. The Overall Program of Biochemical Events during Outgrowth ......................................... .. . . V. The First Molecular Events during Outgrowth A. The Biochemical Potential of the Germinated Spore B. The Problem of the Presence of Messenger RNA in Spores ..................................... C. Sequence and Rate of Synthesis of the Different Kinds of RNA during Outgrowth . . . . . . . .. . .. D. Dependence of Protein Synthesis on Continuous RNA Synthesis in the Outgrowing Spore . . . . . . . . VI. The Pattern of Protein Synthesis during Outgrowth . . A. General .................................... B. The Evidence for Time-Ordered Protein Synthesis in the Outgrowing Spore .. .. .. .. .. .. .. C. The First Proteins Synthesized during Outgrowth .. D. Regdatory Patterns of Protein Synthesis during . .. .. .. . .. .. . . .. outgrowth . . . E. Sequential Enzyme Synthesis during Outgrowth .. .... . . . . . . .. .. .. . ... ... . VII. Cell Wall Formation

.

..... .. .....

... .

..

. ..

.. .. .. .

.....

1

.

..

2 4 8

8

11 13 15 15

17 18

.

20 21 21

.....

21 22

. . .. . .. .

22 24 25

ALEX KEYNAN

VIII. DNA Synthesis and Chromosomal Replication ........ A. Spore DNA ................................ B. Metabolic Activity in DNA at the Time of the Beginning of Outgrowth ...................... C. DNA Replication during Outgrowth ............ D. Chromosome Replication ...................... E. DNA Replication and Differentiation ............ IX. Conditions That Influence and Direct the Differentiational Events in the Outgrowing Endospore .......... X. The Outgrowing Bacterial Endospore as a System for the Study of Cellular Differentiation ................ XI. Summary ....................................... References ......................................

27 27 27 28 29 30 30

31 33 33

1. Introduction

Two interconnected trends, one conceptual and the other experimental, have dominated the study of cellular differentiation in recent years. The conceptual trend searches for a clearer formulation of the problems of cytodifferentiation, and has focused our attention on one major problem of cell differentiaion-the understanding of the mechanisms responsible for different patterns of protein synthesis in cells which harbor identical genomes (Jacob and Monod, 1963; Grobstein, 1963; Sonneborn, 1964; Sussman, 1965b). This approach had led to the adoption of a model which considers cellular differentiation as a process involving control mechanisms, similar to those which have been shown by Monod and Jacob to regulate protein synthesis in bacteria (Jacob, 1986). By applying this model to events which occur during differentiation, we assume that the sequential alterations in the composition, structure, and function of the differentiating cells are brought about by a preprogrammed activation of genes which, as formulated by Lederberg (1966), leads to a “timeordered sequential program of protein synthesis generated from the cell’s informaton” (p. x). Experimentally, cytodifferentiation is approached more and more as a study of the sequence of changes in the macromolecular composition of the differentiating cell. This experimental approach has led to the awareness that differentiation can be viewed as a programmed alteration in the pattern of protein synthesis in cell composition, structure, and function (for references see this series, Volumes 1 and 2). Accepting that one of the main objectives of studies on cell differentiation, at this time, is the understanding of the mechanism responsible for the different patterns of protein synthesis in cells with identical

1. THE

OUTGROWING BACTERIAL ENDOSPORE

3

genomes, we are faced with the need of explaining the fact that differentiation does not involve transient activation of single genes, or of small groups of genes, but time-ordered programs of sequential gene activation leading to rather stable changes in the cell. At present little is known about the nature of such “programs,” but most workers assume that they operate at the level of transcription and translation of the genetic info;mation (Gross et al., 1964); therefore, that “by examining the manner in which the emergence, location, activity and ultimate fate of individual proteins are controlled, by the developmental program, one may hope to learn something about the properties of the program itself’ (Sussman, 1965a, p. 66). This approach focuses on the description of the molecular aspects of “developmental programs” as a central experimental objective for the study of differentiation. Since it is difficult to follow sequential changes in macromolecular composition of differentiating cells in complicated multicellular organisms, many investigators have searched for simple “model systems” for the study of cellular differentiation. A number of such systems are currently explored, most prominently invertebrate embryos, in which the initial stages of development are under intensive investigation. Much work is also being carried out in slime molds and in cultures of mammalian cells undergoing differentiation in uitro (for reviews, see this series, Volumes 1 and 2 ) . In addition, a few microbi‘il unicellular organisms are also being used as models in cellular differentiation. For example, microorganisms that undergo cytodiffereiitiation as part of their normal developmental cycle are Myxobacteria ( Dvorkin, 1966), Paramecium (Sonneborn, 1964), and Volvox (Starr, 1968). This review deals with the spore-forming bacteriu--a group of microorganisms which undergo cytodifferentiation as part of their normal developmental cycle, and which are being used as u model system for the study of this problem. The two differeiitiational events in their life cycle are: sporulation and outgrowth. Sporulation is the formation of a refractile structure in the protoplasm of the parent cell. This refractile structure, the bacterial spore, is an ametabolic, very resistant cell, chemically, structurally, and physiologically very different from the parent cell. Outgrowth, which under appropriate conditions follows germination, is the transformation of the germinated spore into a vegetative cell. While both sporulation and outgrowth have been used successfully for the study of cytodifferentiation, outgrowth has the following advantages as a model system: ( a ) spores can be stored conveniently for long periods, so that genetically identical

4

ALEX KEYNAN

material is available for continuous experimentation, ( b) synchronization of events in the outgrowing cell suspension is easily achieved, ( c ) the time for the completion of the entire process of differentiation is only 2-4 hours, and ( d ) the rate and direction of the process of differentiation can be easily influenced in this system by manipulating its environment. The following is a review of our current knowledge about the process of outgrowth in bacterial spores including a description of the sequential events which occur at the molecular level during outgrowth. No attempt has been made to cover all the literature on the various aspects of the transformation of spores into vegetative cells. Only the work which is considered to be relevant to the subject of cytodifferentiation is reviewed here. It is, of course, by no means certain that the mechanisms responsible for the transformation of a germinating spore into a vegetative cell are identical with the mechanisms responsible for differentiation in multicellular organisms. But in this situation as well, cells harboring the same genome produce different patterns of protein synthesis. The transition from one pattern of protein synthesis to another occurs according to a time-ordered sequential program which leads to a cell with rather stable structural and physiological differences. One can, therefore, assume that what we learn from this system might be of use for the understanding of cytodifferentiation in general. II. The Difference between Spores and Vegetative Cells

The study of differentiation in any cell system must start with a detailed investigation of the composition of both the mother cell and the differentiated cell, in order to determine the exact changes in the molecular characterization of the cell during differentiation. The bacterial endospores differ from vegetative cells in their morphological structure, in much of their chemical composition, and in their physiology. These two forms have been compared extensively in a number of recent reviews and we shall, therefore, give only a very short summary of this topic. (For review see Halvorson, 1965; Vinter, 1967.) Endospores are formed inside the protoplasm of vegetative bacterial cells. When an essential nutrient in the growth medium of an actively multiplying culture of bacteria (Grelet, 1957) is exhausted, the cells cease to multiply, changes occur in their metabolic pattern (Nakata and Halvorson, 1960), and sporulation starts. The first visible morphological change in the cell which is committed to sporulate is the fusion of the

1.

THE OUTGROWING BACTERIAL ENDOSPORE

5

two nuclei of the vegetative cell into one, so-called “nuclear filament” (for review see Robinow, 1960).Then an asymmetric division of the cell occurs, leading to the creation of two compartments inside the original cell wall. In the smaller of these two compartments sequential, structural changes occur leading to the formation of several spore coats which enclose some of the protoplasm and half of the DNA of the original cell, resulting in a structure called a “fore spore.” During spore maturation the specific spore substances are synthesized inside the “fore spore” which

-/\

-

xospo r iu m oat r20-351

FIG. 1. Cross-section of a typical dormant spore. Figures in parentheses indicate approximate percent of total spore weight in each fraction and for several species. The exosporium may not be present in all species. (From Kornberg et al., 1968.)

becomes refractile and heat- and radiation-resistant ( Young and FitzJames, 1959a,b; Fitz-James, 1960; Vinter, 1962, 1963, 1964). The mature spore is released by an autolytic process from its vegetative sporangium; it is rounded and half the size of the mother cell. It has several “spore coats,” made of proteins (Warth et al., 1963) whose sulfur content is five times higher than that of the vegetative cell protein (Vinter, 1959a,b). Between the spore coat and the spore core (which is the spore protoplasm) there is a wide spore cortex which contains a mucopeptide and also calcium and dipicolinic acid ( DPA) (for spore structure see Fig. 1). The spore contains several components which do not occur in the vegetative cell; the most prominent of them is the DPA mentioned above, which accounts for up to 5 to 15% of the dry weight of the spore. Calcium appears in equimolar concentration to DPA, which is an unusually high concentration for this cation. Also, some of the spore

6

ALEX KEYNAN

proteins and some enzymes are different from the vegetative enzymes and proteins. Several spore enzymes have been shown to be identical with vegetative enzymes (Kornberg et al., 1968). During spore formation many of the vegetative cell proteins are broken down and de nouo protein synthesis occurs. In a recent review summing up the evidence from several laboratories for de nouo protein synthesis during spore formation, Halvorson (1965) reached the conclusion, based on turnover and total radioactive labeling experiments, that at least 80% of all spore proteins are synthesized de nouo during sporogenesis. Recently this has also been confirmed by Spudich and Kornberg ( 1968). The appearance of new kinds of protein molecules during spore formation has been shown in n variety of ways. Synthesis of spore-specific surface antigens during sporulation has been demonstrated by immunoelectrophoretic techniques. Spore-specific antigens which could not be demonstrated in vegetative cells could also be isolated from spore lysates (Norris and Wolf, 1961; Baillie and Norris, 1963, 1964; Waites, 1968) . By comparing the behavior of proteins isolated from spores and vegctative cells by acrylamide gel electrophoresis, specific spore and vegetative proteins were shown to exist and to “band in different places on the gel column (Kobayashi et al., 1965). Another approach to the investigation of similarity or diversity of spore and vegetative proteins is to compare spore and vegetative enzymes. Some enzymes seem to be identical in spores and vegetative cells (Falaschi et al., 1965). In other instances spore enzymes, although catalyzing the same reaction as in the vegetative cell, are more heatresistant and sometimes also have different kinetic properties. It has, therefore, been suggested that some of the spore enzymes might bc completely different proteins even though they catalyze the same reaction. More detailed investigations have shown that this conclusion is not necessarily correct. In thf case of glucose dehydrogenase, an enzyme which loses its heat resistdnce and changes its kinetic properties in uiuo during germination, Sadoff demonstrated that a decrease in its molecular size occurs during germination indicating a degradation of this protein into subunits. The subunits are still enzymatically active, but they are much more heat-sensitive and exhibit different enzyme kinetics. In this case, although some of the enzyme properties are different in spores and vegetative cells, the primary structure of the enzyme protein is the same in spores and vegetative cells (Sadoff et al., 1965). In other cases enzymes of identical chemical activity seem to be different in their properties in spore and vegetative cells. This seems to be so in the case of spore

1.

THE OUTGROWING BACTF,RIAL ENDOSPORE

7

catalase and NADH oxidase ( Lawrence and Halvorson, 1954; Sadoff, 1961; Green and Sadoff, 1965). Some enzymes found in spores have not been detected in vegetative cells, such as ribosidase (Powell and Strange, 1956) or glucose dehydrogenase (Bach ‘and Sadoff, 1962). Enzymes which do not exist in the vegetative cells and appear only with the beginning of sporulation have also been described recently by Deutscher et al. (1968). It is obvious from the above summary that there is evidence for the existence of spore-specific proteins, but how many there are and which are, in fact, spore-specific is unknown at present; quantitative evaluation of this question is not yet possible in that we do not know how many of the spore proteins differ in their primary structure from the proteins of vegetative cells. Kornberg et al. (1968) in a recent review state that enzymatic activity in spores and vegetative cells resides in identical proteins, and that the spore-specific proteins are mostly structural proteins. Mandelstam (1968), in whose laboratory this problem has been recently reinvestigated, thinks that some of the spore proteins are also synthesized at a low rate during vegetative growth. However, in examining changes occurring in the protein composition of cells by immunoelectrophoresis, he noted that the “lines” of the vegetative antigens disappeared during sporulation. The differences in the physiological properties of vegetative cells and spores are extreme. The bacterial endospore is one of the most resistant forms of life. It can withstand temperatures above 100°C and is resistant to desiccation and to many bactericidal agents. It is also several times more resistant to radiation than the vegetative cell. No endogenous metabolism can be detected in the resting spore. The respiratory system in spores is also quite different from that in the vegetative cells. The resting spore does not respire. The electron transfer system when investigated immediately after germination or in spore extracts, contains an FMN-dependent soluble DPNH oxidase, but no cytochrome. The vegetative cell, on the other hand, has respiratory particles, which also contain cytochrome in addition to DPNH oxidase (Doi and Halvorson, 1961). The ametabolic, very resistant bacterial spore is an example of the phenomenon of arrested life. Keilin (1959) discussing this phenomenon pointed out that in the normal developmental cycle of many unicellular or multicellular organisms, stages occur in which metabolism is either slowed down or absent altogether and in which the organisms are much more resistant to the environment than in the vegetative stage. He called

8

ALEX XEYNAN

these stages cryptobiotic stages, and the phenomenon itself cryptobiosis (Grossowicz et al., 1961). Some of the main differences between spores and vegetative cells are summarized in Table I. TABLE I PROPERTIES DISTINGUISHING SPORESFROM VEGETATIVE CELLS A. Physical properties typical for spores Refractility Nonpermeability to dyes Resistance to: heat, desiccation, chemicals B. Differences in chemical composition of spores and vegetative cells 5-15% DPA (spores) 5-15% Ca (spores) Some spore proteins differ in immunological and physicochemical properties from vegetative cell proteins Spores contain half the DNA of vegetative cells Spores have five times more cysteine-cystine than vegetative cells, mostly localized in spore wall Spore wall is mostly protein; contains no mucopolysaccharide C. Metabolism and enzymes (general) Endogenous respiration nondetectable in spores Heat-resistant enzymes active in the intact spore Some enzymes only in spores (heat-resistant); others only in vegetative cells Some enzymes are in some way different from similarly acting enzymes in vegetative cells Electron transport systems Spore Vegetative cell Particulate DPNH Soluble (144,000g supernatant) DPNH oxidase Oxidase Flavine Flavoprotein No cytochrome Cytochromes a, b, c DPA- and FMN-stimulated DPA-inhibited

111. Processes Involved in the Transformation of Spores into Vegetative Cells

A. INDUC~ION OF DEVELOPMENTAL CHANCES IN SPORESUSPENSIONS AND THE SUCCESSION OF BIOCHEMICAL EVENTS DURING THE TRANSFORMATION OF SPORESINTO VEGETATIVE CELLS.

It is now believed that three sequential, distinct, and different stages are responsible for the transformation of a bacterial spore into a vege-

2.

THE O U K R O W I N C BACTERIAL ENDOSPORE

9

tative cell: activation, gerniination, and outgrowth. The first twoactivation and germination-are apparently involved in the termination of the “cryptobiotic” state, while the third outgrowth is a typical process of growth and differentiation. It is this outgrowth which we consider to be a model system for the study of cellular differentiation (Keynan and Halvorson, 1965). Following is a short description of these three stages and a summary of the evidence that they involve different biochemical processes (Table 11). Freshly harvested spores are usually reluctant to germinate even under optimal germination conditions and have to be “heat-activated” (by heating them for an hour up to SOOC) or aged (stored for a few weeks or sometimes months). Such activated spores can now be germinated by specific “trigger” substances, which differ for different spore species. L-Alanine is one of the most commonly used germination-inducing substances, but many strains of bacilli need also adenosine in addition to L-alanine for germination, and several strains of Bacillus megaterium can be germinated by glucose alone. When “activated” spores are exposed to “trigger” substances under appropriate conditions, they “germinate” and lose their typical spore properties, such as heat resistance, refractility, and impermeability to dyes. After germination, referred to by some workers as initiation (Murrell, 1961), they also start active respiration and are able to metabolize a variety of substances. The process of germination involves a loss of about 30% of the dry weight of the spore by excretion into the media of some typical spore substances such as DPA, calcium, and mucopeptide. The germinated spores retain all of the original spore structure except for the cortex which is lost partly or completely during germination. The process of spore germination is irreversible and the resulting cell is metabolically active, heat-labile, nonrefractile, and stainable. The germinated spore is still distinct from the vegetative form in structure, protein, and enzyme composition and pattern of metabolism. It will absorb water and swell but no further visible changes will occur in it unless additional nutrients are added to the spore suspension. When nutrients are added the processes of growth start: the germinated spore elongates, begins to synthesize new macromolecules, produces a cell wall, and becomes a typical vegetative cell. The activated spore seems to be identical in its properties with the original spore with the exception that it will now respond to germinationinducing substances. The process of activation, which is reversible and

TABLE I1 DIFFERENCES BETWEEN ACTIVATION,GERMINATIOS, A S D OUTGROWTH General nature Initiators End result of process of process of process Activation

Germination

Outgrowth

Measurement of process

“Activated spore”; ready for germination; still possesses spore properties

No evidence for assumption that process is mediated by metabolism; probably a physical change in stnicture of niacroniolecuIes

Increased gerrnination rate after activation

Chemical inducers (L-alanine, glucose, etc.)

Germinated spore; spore properties irreversibly lost; 30% of dry weight is excreted; retains spore coat and enzymes typical for spores

Enzyme-mediated “trigger reaction” leading to breakdown and excretion; no evidence for synthesis of macromolecules as necessary part of this process

Decrease of OD; loss of heat resistance of spore suspension

Growth mediom

Vegetative cell; full complement of cell enzymes and structure

Synthesis of proteins, enz-ymes, and cell wall

Protein synthesis; cell mass increase

Heat; aging; low PH

h

F

x”

*

z

2:

1.

THE OUTGROWING BACTERIAL ENDOSPORE

11

can be induced also by low pH and reducing agents, does not involve metabolism and probably consists of a change in the configuration of spore macromolecules (Keynan and Evenchik, 1969, in press; Keynan et al., 1964, 1965). The irreversible loss of spore properties occurs during germination where part of the spore substances are hydrolyzed and excreted into the medium; this occurs under conditions in which no RNA or protein synthesis can be demonstrated, and is not blocked by inhibitors of RNA and protein synthesis. These facts suggest that germination involves processes of degradation rather than synthesis and growth (Steinberg et al., 1965). Outgrowth occurs when germinated spores are exposed to a balanced growth medium and, hence, is a process of growth and differentiation. Outgrowth is completely inhibited by inhibitors of RNA or protein synthesis. During outgrowth new macromolecules are formed which were not present in the spore stages. A summary of the processes occurring during transformation of spores into vegetative cells is given in Table I. B. THEEXPERIMENTAL SYSTEMFOR TERIAL ENDOSPORES

THE

STUDY OF OUTGROWTH OF BAC-

For the study of events occurring during outgrowth, spores in suspension are usually heat-activated in distilled water, and then germinated by the addition of a medium which induces germination but not growth. Most spore suspensions used in these experiments complete germination (measured by decrease in turbidity of the suspension or by darkening of the individual spores in the phase microscope) during 5-15 minutes. The suspension is then centrifuged and washed in order to remove the spore exudate. Outgrowth is induced by resuspending the germinated spores in a complete nutrient medium. Using pregerminated spore suspensions for the induction of outgrowth, a high degree of synchrony is achieved; this is determined at the first cell division (Keynan and Issahary, 1968). The time interval between the beginning of outgrowth and the first cell division depends on the medium and on other environmental conditions (Rodenberg et al., 1968) but usually varies between 200 and 300 minutes. Since many investigators have worked with different spore species under dissimilar experimental conditions, there is no agreement on the length of time needed for completion of this differentiation in various systems, and time intervals reported in the literature by different authors cannot be easily compared. Molecular events in this system can be followed by isolation and

12

ALEX KEYNAN

0.3 -

! ! 0 n

0.2

E"

\

0 E

*z a

/Lntroi

A

-

u)

0.1

0

/

-

gL.-r

-

k O '

3 . 0

@Act D a t 0 min o

B

~

o

o

/

30

280k

Act D a t 10 min

00-0-010

d

P

Act D a t 19 mir

o

-

o

~

o

~

o

Control

1.0 3.

r

-0.8- 0E hl

0.6 . 5

OD

n

0.4 0

in c m I

0

10

I 20

I

30 Time (min)

I

I

40

50

FIG.2. The effect of actinomycin D (Act D ) and chloramphenicol (CM) on ( A ) protein synthesis, ( B ) respiration, and ( C ) activity of induced enzymes during germination and outgrowth. Germination of B. cereus spores was induced by adding L-alanine (2.5 mg/ml), adenosine (0.5 mg/ml), and glucose ( 5 mg/ml) to heatactivated spores, suspended in the synthetic medium of Nakata ( 1964). Incorpora-

1.

THE OUTGROWING BACTERIAL ENDOSPORE

13

characterization of pulse-labeled RNA or protein, by the isolation of new enzymes as they appear, by the detection of new structures by microscopic observation, or by the incorporation of isotopes into such structures (Kobayashi et aZ., 1965; Torriani and Levinthal, 1967). IV. The Overall Program of Biochemical Events during Outgrowth

The following is a short summary of the overall events during outgrowth. A detailed description of these events and a discussion of their significance is given in the following chapters. One of the first changes to be noticed when spores germinate is the sudden onset of respiration in the germinated cell. This abrupt appearance of active metabolism, which cannot be demonstrated in uiuo in the dormant spore, is striking, and several workers have considered this to result from de nouo enzyme synthesis during germination. But, there is now clear evidence that the activity of the enzyme systems responsible for respiration in the germinated spore appears suddenly during germination even when RNA and protein synthesis are blocked; therefore, these enzymes preexisted in the resting spore and were activated during germination. There is no evidence of de novo synthesis of enzymes as an obligatory process of germination. The preexisting respiratory system in spores, which is activated during germination, is not identical with the respiratory system in vegetative cells and is of importance in supplying the germinated spore with energy for the first synthetic processes during outgrowth (Steinberg et aZ., 1965). Evidence of this is summarized in Fig. 2 and Table 111. The first event of synthesis of macromolecules which can be observed in the outgrowing spore is RNA synthesis which starts shortly after germination. Protein synthesis lags behind and starts 2-4 minutes after the beginning of RNA synthesis (Higa, 1964; Kobayashi et aZ., 1965; Torriani and Levinthal, 1967). tion of ~-1eucine-14Cinto trichloroacetic acid-insohble material was followed ( A ) and the effect of actinomycin D added at 0, 10, and 19 minutes was observed. The inhibitory effect of chlorampheniml or actinomycin on the second phase of increase in respiratory activity is demonstrated in ( B ) . Ordered transcription during outgrowth is demonstrated in ( C ) . Both a-glucosidase ( M ) and alkaline phosphatase (P)are synthesized in steps which occupy only a fraction of a generation. The effect of chloramphenicol on synthesis of a-glucosidase is illustrated in the lower curve. (Data from Steinberg d al., 1965.)

14

ALEX KEYNAN

In the outgrowing spores it can be seen that different kinds of proteins appear progressively, and a time-ordered sequential appearance of the different enzymes can be demonstrated (Kobayashi et nl., 1965; Torriani and Levinthal, 1967). The cell wall is beginning to be synthesized after 12-15 minutes at the time when the germinated spore swells. At the time when cell wall synthesis begins, the spore starts to elongate. The spore wall remains around the outgrowing cell during swelling and even at the time of TABLE I11 EFFECTOF INHIBITORS OF PROTEIN SYNTHESISON AMINO Acm INCORPORATION AND GLUCOSE OXIDATION BY GEREXINATED sPORESa,b ~~

~

Incubation conditions

Leucine incorporation (pmoles/mg spores)

( pl O,/mg spores/hr )

Control Actinomycin D ( 20 pg/ml) Chloramphenicol(40pg/ml) Puromycin ( 40 pg/ml)

0.39 0.020 0.012 0.028

28.2 27.3 28.8 25.7

QO,

Spores were germinated at 30°C in 0.04 M Tris b d e r (pH 8.3), containing 10-3 M KH,PO,, 0.5 mg adenosine/ml, 2.5 mg L-alanine/ml and 0.5% glucose. Outgrowth conditions: 1 mg spores/ml in Tris-phosphate buffer containing 0.5% glucose, 30°C. Radioactive leucine ( 1 pc/pg/ml) incorporation was followed for 40 minutes, The respiratory activity (QO,) was the initial rate of gliicose oxidation. b Data from Steinberg et al. ( 1965).

elongation. Later on, it is either broken down or, in some species, the cell grows out of it leaving the intact spore coat behind. More of the respiratory system which is activated during germination is synthesized a few minutes after outgrowth begins. This can be seen by comparing the respiration rate in suspensions of outgrowing cells in the presence and absence of chloramphenicol. At 20 minutes after germination, a very significant increase in respiration rate can be noticed in the sample without chloramphenicol as compared with the one to which this antibiotic was added ( Steinberg et al., 1965). The pathway of glucose metabolism changes during outgrowth; although the exact nature of this change is still under discussion, it is quite clear that the hexose monophosphate pathway of glucose metabolism, which accounts for 20% of the glucose used by the germinated spore, is used less and less until it nearly disappears just before the

1.

THE OUTCROWING BACTERIAL ENDOSPORE

15

first cell division. The genninated spore has also no tricarboxylic acid cycle (Blumenthal, 1961; Goldman and Blumenthal, 1964; for review see Sussman and Halvorson, 1966). DNA synthesis starts apparently only 200300 minutes after germination when thc amount of RNA has almost doubled in the genninated spore (Woese and Forro, 1960). Cytochrome appears very late and is sometimes not found during differentiation of the cell up to the first division ( Nakata et al., 1957). V. The First Molecular Events during Outgrowth

POTENTIAL OF A. THEBIOCHEMICAL

THE

GERMINATED SPORE

Thc germinated spore harbors a complex metabolic machinery which, if supplied with the right substrate, is capable of providing the cell with

the necessary energy for the synthesis of new macromolecules. The problem of thc existence of the complete biochemical machinery for protein synthesis in the spores is under current investigation. The fact that protein synthesis starts only several minutes after germination seems to indicate that something is missing in the spore and has to be synthesized, changed, or released before protein synthesis can start ( Steinberg et al., 1965). Investigntions of spore extracts have shown that they contain ribosomes, transfer RNA and activating enzymes. Most investigators have reported that no functional messenger RNA exists in resting spores but this subject is still controversial. In spore extracts very little protein synthesis is apparent even when messenger RNA is added to resting spores and to spores immediately after germination. One has, therefore, to assume that the proteinsynthesizing system is in some way inactive under these conditions (Table I V ) . Whatever this lesion may be, it is rapidly repaired during outgrowth as can be seen by comparing the protein-synthesizing ability of spore extracts before and a few minutes after germination (Kobayashi et al., 1965; Rodenberg et al., 1968). Several hypotheses have been suggested as to the nature of this apparent lesion in the protein-forming system. For instance, it was suggested that the lesion might be due to the fact that spores usually have a low concentration of 70 S ribosomal particles, which were thought to be necessary for protein synthesis. This does not seem a likely explanation for two reasons: smaller particles have been shown to be able to synthesize protein and no great change

16

ALEX KEYNAN

in particle size occurs during the time interval in which the proteinsynthesizing ability is acquired. The rapid activation of protein-synthesizing ability after gennination cannot simply be ascribed to the synthesis of new messenger RNA only, as the addition of messenger RNA will not repair the in vitro system drastically. TABLE IV POLYU STIMULATION OF PHENYLALANINE INCORPORATION INTO CELL-FREE OF SPORES,GERMINATED SPORES,AND VEGETATIVECELLS’, b EXTRACTS

Source of extracts

Phenylalanine incorporated (ppmoles/mg of protein) -Poly u +Poly

u

Dormant spores 3.8 16 3.8 28 Activated spores Germinated spores (20 minutes) 5.8 125 56 761 Vegetative cells 16 949 Vegetative cells ( preincubated) 0 Extracts were prepared by sand grinding in a chilled mortar. Disrupted dormant and heat-activated ( 2 hours at 65°C) spores were extracted with 10-2 M Tris buffer (pH 7.8) containing 0.01 M Mg2+; the reaction mixtures contained the following in pmoles/ml: Tris (pH 7.8), 100; KCI, 40; magnesium acetate, 10; adenosine triphosphate (Na salt), 0.8;0.04 each of guanosine triphosphate, cytidine triphosphate, and uridine triphosphate, Na salts; mercaptoethanol, 4; spermidine, 0.4; 0.04 of each of the 20 L-amino acids minus phenylalanine; ~-phenylalanineJ4C (10 pc/ pmole), 0.2; phosphoenolpyruvate (Na salt), 4; 14 pg of phosphoenolpyruvate kinase; 0.6 mg of B. cereus soluble RNA; 100 pg of poly U; 320 pg of protein extract. The total volume was 0.25 ml. Samples were incubated at 36°C for 60 minutes and the hot trichloroacetic acid insoluble radioactivity determined. b Data from Kobayashi et nl. (1965).

The nature of the lesion responsible for the apparent inactivity of protein synthesis in vitro by spore extracts has been reinvestigated recently by Deutscher et al. ( 1968). These investigations showed that spore extracts contain more nucleases than vegetative extracts and that these nucleases are responsible for the defect in the protein-synthesizing system by destroying both endogenous and added messenger RNA. “Inactive” protein synthesis is also known to exist in some other biological dormant systems, for example, in some kinds of plant seeds (Kornfeld, 1961; Henney and Storck, 1964) and the unfertilized sea urchin egg (Monroy et al., 1965; Salb and Marcus, 1965; Stavy and Gross, 1967 ) .

1. B. THE PROBLEM

17

THE OUTGROWING BACTERIAL ENDOSPORE OF THE

PRESENCE OF MESSENGER RNA

IN

SPORES

In several developmental systems protein synthesis starts by using existing templates of stable RNA which are stored in the cytoplasm before development begins. This is the case in sea urchin eggs, some plant seeds, and some other systems (Gross et al., 1964). In outgrowing spores the initiation of protein synthesis seems to depend on transcriptional events. This is the most simple explanation for the fact that Bacillus cereus spores germinated in the presence of actinomycin D do not incorporate amino acids and their ability to form new proteins is blocked (Higa, 1964; Balassa, 1965; Steinberg et al., 1965; Torriani and Levinthal, 1967). This led most workers to assume that spores have no messenger RNA and new messenger RNA has to be formed before protein synthesis starts. The findings by Doi and Igarashi (1964a) that no messengerlike RNA could be detected by sucrose gradient centrifugation analysis in extracts of resting spores broken up mechanically is additional evidence for this hypothesis. Several workers were unable to detect polysomes in extracts from spores broken up mechanically ( Woese et al., 1960). The findings that amino acids are not incorporated into spore extracts unless supplemented with messenger RNA, seemed also to indicate that spores have no endogenous messenger RNA ( Kobayashi et al., 1965). Although until recently there was a consensus of opinion that messenger RNA is not present in the spore, the new findings by Chambon et al. (1968) call for a revision of this opinion. These workers, using extracts of spores broken up by the much gentler method of Gould and Hitchins (1963), found polysomes in extracts of B. megatetiurn spores. Deutscher et al. (1968) also reported that this extract incorporated amino acids into protein without the addition of messenger RNA to a value of 30% of that obtained in similar extracts from vegetative cells. These findings indicate the existence of some messenger RNA in the resting spore of B. megaterium. The inability by others to demonstrate its presence is explained by their finding in spores an increased amount of nucleases which might degrade it during extraction (Deutscher et al., 1968). The role of this spore messenger RNA in protein synthesis after germination is not clear at this time. Deutscher et al. (1968) state that “it may in fact be a remnant of the templates previously employed during sporulation” ( p. 5125). These findings put even more into focus the problem of the activation of protein synthesis during germination. If mes-

18

ALEX KEYNAN

senger RNA really preexists in resting spores, why then should actinomycin D prevent protein synthesis when added during germination. We have, therefore, to assume that some new RNA has to be formed before protein synthesis can start.

C. SEQUENCE AND RATEOF SYNTHESIS OF DURING OUTGROWTH

THE

DIFFERENT KINDSOF RNA

The overall rate of RNA synthesis changes during outgrowth and depends on the medium. Several workers have shown that the rate of uracil incorporation increases early during outgrowth but then decreases (Kobayashi et al., 1965; Vinter, 1965). It has also been shown that a more stable rate of RNA synthesis is achieved when the medium is supplemented with bases and amino acids (Rodenberg et al., 1968). All classes of RNA are synthesized by the outgrowing spore (Doi and Igarashi, 1964a; Donnelan et al., 1965) but not at the same rate during different time intervals of the outgrowth phase. The sequence of the appearance of different classes of RNA has been investigated by several workers. Armstrong and Sueoka (1968a) (see Fig. 3) published evidence that soluble and ribosomal RNA is formed in their strain of Bacillus subtilis before the onset of messenger RNA synthesis. In their experiments outgrowing spores were pulse-labeled with uridine-:’H bctween 7 and 10 minutes after germination and no messengerlike RNA could be found when extracts of the cells were analyzed by sucrose gradient fractionation. Extracts of outgrowing spores, pulse-labeled and analyzed in the same way at a later time, showed peaks in the 4-16 S region after 10 minutes, indicating the presence of messenger RNA. They concluded, therefore, that ribosomal and transfer RNA synthesis preceded messenger RNA synthesis during outgrowth of these spores. Thrw authors also published evidence that ribosomal RNA wils synthesiztd during some period without ribosomal protein ( Armstrong and Sueokil, 1968b ) , Rates of synthesis of other classes of RNA also vary during outgrowth. Donnelan et al. (1965), using chromatography of extracts of outgrowing spores on methylated albumin kieselgur columns, showed that ribosomal RNA was synthesized faster than transfer RNA. Since Doi and Igarashi (1964b) have shown that transfer RNA exists in spores at a higher concentration than in vegetative cells, the slower synthesis of transfer RNA during outgrowth restores the ratios of transfer RNA to ribosomal RNA which are typical of vegetative cells. At the beginning of outgrowth, labile RNA, defined as the RNA which disappears from the RNA fraction a few minutes after the addition of

3200

-

- 800

a

32001

4320 240

2400

E

01600

160

n I

N

80

800

0 40

0 Fraction number

Fraction number

400 2400 -

300 0

200 a N

n I

I00

0

8

24 32 Fraction number 16

0 40 Fraction number

FIG.3. Sucrose gradient centrifugation of pulse-labeled RNA from outgrowth B. scibtilis spores. Outgrowing spores were pulse-labeled with uridine-3H at ( a ) 7-10; ( b ) 15-18; ( c ) 17-20; and ( d ) 22-25 minutes after induction of germination and centrifuged in 2.5-15% sucrose gradient at 3°C: 39,000 rpm for 5.5 hours ( a and d ) or 7"C, 25,000 rpm for 17 hours ( b and c), X-X, 3'P-labeled B . subtilis RNA marker; 0-0 pulselabeled RNA. (Data from Amstrong and Sueoka, 1968.)

20

ALEX KEYNAN

actinomycin D, comprises the larger fraction of newly formed RNA. But the relative concentration of this fraction of labile RNA decreases during outgrowth ( Rodenberg et al., 1968). The nature of messenger RNA formed during outgrowth was investigated by Doi and Igarashi (1964a), who demonstrated that the messenger RNA molecules formed during outgrowth are different from those formed during sporulation. They reached this conclusion by showing that these two kinds of RNA did not compete for the same sites of DNA in DNA/RNA hybrid competition experiments. In DNA/RNA hybridization experiments, messenger RNA formed during outgrowth competed effectively with pulse-labeled mRNA from actively growing vegetative cells. We have, therefore, to assume that most of the messenger RNA synthesized during outgrowth seems to be identical with messenger RNA formed during the growth of vegetative cells. There is still no complete consensus of opinion as to the kinds of ribosomal particles which exist in spores. The study of spore ribosomes is complicated by the fact that many methods used for breaking up spores might destroy ribosomal particles, either by mechanical means or by release of dipicolinic acid which chelates metals and therefore might affect ribosomal structure. In spores and in outgrowing germinated spores of B. subtilis, Woese et al. (1960) observed a high percent of an unusual kind of smaller ribosomal particle. It was found that during outgrowth there is a decrease in the amount of the smaller ribosomal particles and an increase in the 70 S particles (Kobayashi and Halvorson, 1968). Several workers were not successful in demonstrating polysomes in spore extracts ( Woese et al., 1960; Chaloupecky, 1964; Fitz-James, 1965). On the other hand, Chambon et al. (1968), although pointing out that the spore ribosome particles were "smaller" in size and quantity than those in vegetative cells, also found polysome-like particles in spores opened by their method. OF PROTEIN SYNTHESIS ON CONTINUOUS RNA SYNTHESIS D. DEPENDENCE IN THE OUTGROWING SPORE

Using actinomycin D which inhibits RNA synthesis nearly instantaneously in the outgrowing spore, it could be shown that protein synthesis during outgrowth is continually dependent on new RNA synthesis. When actinomycin D is added, protein synthesis stops after a delay of a few minutes. This delay is consistent with our information on the half-life of

1.

THE OUTGROWING BACTERIAL ENDOSPORE

21

messenger RNA in these cells which has been shown to be of the order of a few minutes (Levinthal et al., 1962). The relationship between protein and RNA synthesis during outgrowth has been reinvestigated in detail recently ( Rodenberg et al., 1968). These investigators examined the relationship between the rate of protein synthesis in the outgrowing spore and the rate of synthesis of the various classes of RNA. The constant ratio between the rate of protein synthesis and the rate of labile RNA synthesis suggested that the rate of the former depends on the rate of the latter. No rigid dependence of the rate of protein synthesis on the rate of the production of new ribosomes could be shown. VI. The Pattern of Protein Synthesis During Outgrowth

A. GENERAL The study of the pattern of protein synthesis in the outgrowing bacterial spore appeals as a specially productive approach to the study of cell differentiation which is viewed by many workers as a sequential activation of different regions of the genome. Such sequential activation of different gene regions will result in, and can be best measured as, a sequential change in the appearance of different species of protein molecules during development. As no “stable messenger” has been demonstrated in outgrowing spores, protein synthesis is linked to messenger RNA synthesis. Therefore, by following the pattern of protein synthesis we are directly studying transcriptional and translational events ( Rodenberg et al., 1968). B. EVIDENCE FOR TIME-ORDERED PROTEIN SYNTHESISIN THE OUTGROWING SPORE

The existence of a time-ordered sequential synthesis of protein was nated when the sequential appearance of vegetative cell antigens was followed during outgrowth (Norris and Baillie, 1964). More evidence was given by Halvorson (1965) when he labeled outgrowing spores with l e ~ c i n e - ~for H a short time and then, at a later stage, added le~cine-’~C to the same spore preparation. When the protein extracts were separated by chromatography, the differential distribution of label in fractions of these two samples indicated the synthesis of different proteins at different times of outgrowth. The most direct and detailed evidence for sequential synthesis of new kinds of protein has been obtained by employing the method of acryla-

22

ALEX KEYNAN

mide gel electrophoresis after pulse-labeling the proteins of outgrowing spores during different times of outgrowth (Kobayashi et al., 1965; Torriani and Levinthal, 1967). Radioautography of such gels indicates that different vegetative cell proteins appear at different times of outgrowth. With this method it is also easy to identify specific proteins by their location in the gel and to follow their fate as a function of time. In addition, the sequential synthesis of different enzymes during outgrowth described below provides further evidence for time-ordered protein synthesis. The collective evidence on protein synthesis during outgrowth indicates, therefore, that the different kinds of protein molecules are not synthesized simultaneously while the cell is differentiating, but are being synthesized according to a preprogrammed pattern, sequential in time.

C. THE FIRST PROTEINS SYNTHESIZED DURING OUTGROWTH Some proteins are synthesized in germinated spores even in a medium which does not support outgrowth. Such a system in which protein synthesis is limited has been investigated by Torriani and Levinthal (1967) who assume that the building blocks for this kind of protein synthesis are derived from turnover of preexisting spore compounds. By pulse-labeling these cells and studying the “band” pattern of their newly formed proteins, after acrylaniide gel electrophoresis and autoradiography, it became evident that only very fcw kinds of proteins arc synthesized in their system. The same proteins will continue to be synthesized as long as the medium does not support outgrowth (see Fig. 4 ) . It is, therefore, evident that germination itself does not automatically induce the complete transcription of the whole genome but induces only transcription of very few well-defined paits of it. These particular proteins, programmed by the first transcriptional products, have not yet been isolated and studied and their function is still unknown. Judging from the published pictures and data of Torriani and Levinthal (1967) some of these proteins synthesized during such “arrested differentiation” do not appear as prominent bands during protein synthesis of vegetative cells. A high concentration of these proteins, therefore, seems to exist in these cells only during the beginning of differentiation, whereas a relatively smaller amount of at least some of them might also be synthesized at a later stage.

D. REGULATORY PATTERNS OF PROTEIN SYNTHESIS DURING OUTGROWTH When an outgrowing spore in a state of “arrested differentiation,” during which only very few proteins are formed, is transferred to a com-

FIG. 4. Electroplioretic pattern of 1'C-labeled proteins synthesized during a 5minute pulse with 14C-labeled amino acids in outgrowing endospores. ( A ) protein stain of the electrophorograms, ( B ) autoradiogranis. Pulse label was initiated at 0 minutes (column l ) , 5 minutes (column 2 ) , 30 minutes (column 6 ) , 60 minutes (column 8), and in vegetative cells (column 10). (Data from Torriani and Levinthal, 1967.)

24

ALEX KEYNAN

plete medium, the rate of protein synthesis increases rapidly. When such cells are pulse-labeled after transfer to a full medium and their proteins are extracted and separated on acrylamide gel, continuous radioactive background without distinctive bands can be seen in the radioautograms of such gels. This indicates that under these conditions a large number of proteins are being synthesized simultaneously, all at a very similar rate. This pattern of protein synthesis remains constant for some time, but after about 60 minutes the differential rate of synthesis of various individual proteins changes. This becomes evident by the appearance of distinctive bands when examining radioautograms of acrylamide gel of such cell extracts. The pattern of these bands on the acrylamide gel is identical with that of proteins from actively growing vegetative cells. This pattern of protein synthesis remains constant as long as the cells are in the vegetative form. Interpreting these results, the authors point out that “a continuous distribution of label into different proteins may indicate uniform transcription of deoxyribonucleic acid with no control by repression. Thus, if the synthetic process is activated before repressors are made, one could obtain a period of essentially uncontrolled synthesis by all the genes of the cell” (Tomani and Levinthal, 1967) p. 183 (see Fig. 4 ) .

E. SEQUENTIALENZYMESYNTHESIS DURING OUTGROWTH When the activity of various enzymes was followed in outgrowing spores as a function of time, it was found that the rate of synthesis of several of these was not continuous. The synthesis of a specific enzyme occurs only for a short definite time interval during outgrowth and then stops. For instance, in the outgrowing spores of B. cereus strain T, base levels of a-glucosidase activity increase from 10 to 20 minutes after the beginning of germination. In the same cells alkaline phosphatase activity increases from 20 to 25 minutes from zero time. This kind of periodicity of enzyme synthesis was shown to repeat itself over several division cycles during vegetative growth (Kobayashi et al., 1965) and occurred also with L-alanine dehydrogenase and histidase. This ordered increase of enzyme activity could be inhibited by inhibitors of protein synthesis and has therefore to be considered as de nouo synthesis of new protein rather than activation of preexisting dormant enzymes. Histidase and a-glucosidase are inducible enzymes. It was shown also that not only did the base level of these enzymes increase within a short time interval but that induction during outgrowth occurred only during a short time interval which corresponds to the period during

1.

25

THE OUTGROWING BACTERIAL ENDOSPORE

200 -

0.8-

e0 CF

CF

€120-

E

.-

\

0.4-

2

a

80-

.-In

I

0.2 -

i”’i /”.1:* ‘A

e+

A

0-

0

+ ....r x ....

d‘ x

x

In

e0

X

In Q

-2000

0,

E

\

In

c .C

.x.!...?.*‘’ u

1

/

I” P HI &&d..L4:,

40 -

3000

P

\

In c C 3

&-I-@-

,.&P.F x

.q i

I

In a

!?ox-

3

%”PA

160-

:

In

c In .C

UI

>Ad--

0,

0

-1000

eg 0

-

3

0,

4

:

I

60

I

‘0 90

VII. Cell Wall Formation

Most of our knowledge on the molecular events leading to the synthesis of a new cell wall in outgrowing spores comes from Vinter’s laboratory. He measured cell wall synthesis in outgrowing germinated spores by following incorporation of radioactive diaminopimelic acid (DAP) into a fraction insoluble in hot trichloracetic acid. This fraction comprises the newly synthesized cell wall material. In these experiments it was found

26

ALEX KEYNAN

that new cell wall formation could be first detected 20 minutes after the induction of germination. This time period coincides with the late stage of swelling of the germinated spore. The molecular events leading to cell wall synthesis begin earlier during outgrowth, as can be concluded from the following experiments. Actinomycin D will completely inhibit cell wall synthesis when added 10 minutes after the beginning of germination, but a certain rate of cell wall synthesis will continue if it is added after 16 minutes; this indicates that during the time interval of 10 to 16 minutes some of the messenger RNA responsible for later cell wall formation is being synthesized. The enzymes responsible for cell wall formation are apparently formed during the period of 20 to 40 minutes after the beginning of germination. This can be shown by studying the ability of chloramphenicol to inhibit cell wall formation, as a function of time. Chloramphenicol, added 10 minutes after the beginning of germination, inhibits much of cell wall synthesis. Considerably less inhibition is apparent when chloramphenicol is added after 40 minutes. This indicates that after 40 minutes a sufficient amount of stable enzymes has been formed to maintain cell wall synthesis even in the absence of the synthesis of new protein. Vinter has demonstrated that some of the DAP for new cell wall formation might derive internally from a DAP-containing peptide which preexists in the spore and is not completely hydrolyzed and excreted during germination. In outgrowing spores the incorporation of radioactive DAP into a trichloroacetic acid-soluble fraction slightly precedes the incorporation into the hot trichloroacetic acid soluble fraction, indicating the formation of some cell wall intermediates. According to Vinter’s experiments, the sequence of events on thr molecular level leading to cell wall formation in outgrowing spores of B. cereus seems to be as follows: transcription of the genes responsible for cell wall formation occurs between 10 and 18 minutes after the start of germination. The “cell wall messenger” synthesized during this time interval programs the synthesis of enzymes which are directly or indirectly responsible for cell wall formation. The synthesis of these enzymes occurs very actively bctween 20 and 40 minutes after the beginning of germination. At this stage the spore wall still exists and gives a certain degree of osmotic protection to the outgrowing spore. This was apparent when new cell wall synthesis was inhibited by penicillin without resulting in lysis or destruction of the swollen cells at this stage of development. However,

1.

THE OUTGROWING BACTERIAL ENDOSPORE

27

when new cell wall synthesis was inhibited by penicillin at a later stage of development, when the cells start to elongate, most of the cells were killed. This penicillin-induced lysis of cells starts to occur apparently only at the stage of elongation at which time most of the spore wall has already been degraded and does not provide the cell with any more osmotic protection ( Vinter, 1965). VIII. DNA Synthesis and Chromosomal Replication

Several recent investigations are concerned with events occurring with or in DNA during germination and outgrowth. Some investigators have looked for differences between spore DNA and Vegetative DNA and asked themselves whether any “event” can bc observed on the DNA level during germination or at thc beginning of outgrowth. Others have asked when exactly does DNA replication start during outgrowth, and whether the first replication of DNA after spore germination is different from replication that occurs during vegetative cell division. The possible relationship between D N A replication and transcription has also been investigatcd.

A. SPOREDNA The spore has about half the amount of DNA of the vegetative cell (Young and Fitz-James, 1959b), and is genetically complete, as can be shown when DNA from germinated spores is used in transformation experiments (Yoshikawa, 1965). The spore contains a DNA polymerase and other enzymes necessary to synthesize DNA (Falaschi et al., 1965). The physical state of DNA in resting spores might be different from its state in vegetative cells. This conclusion has been reached by the investigation of photochemical reactions occurring in DNA as a result of irradiation with UV. When spores are irradiated with UV, they form certain not yet identified thymine-containing photoproducts instead of the thymine-containing cyclobutane dimers which are formed by vegetative cells under the same conditions. DNA dried in the presence of various salts will also produce different photo products (Donnelan and Setlow, 1965) . B. METABOLICACTIVITY IN DNA AT THE TIMEOF THE BEGINNING OF OUTGROWTH Although the replication of DNA starts relatively late during outgrowth, a short period of DNA synthesis occurs 5-10 minutes after the initiation of germination at the beginning of outgrowth. This is evident

28

ALEX KEYNAN

from incorporation experiments with radioactive DNA precursors (for review see Halvorson et al., 1986). Some controversy exists in the literature about the nature of this early DNA synthesis. Some workers assume that it represents a “repair” mechanism of DNA. Such an active “repair” mechanism has been demonstrated in spores (Donnelan and Setlow, 1965). Recently this problem has been reinvestigated by Steinberg and Halvorson ( 1988a,b) working with a thymine-requiring mutant and bromodeoxy~ridine-~H; these investigators reached the conclusion that the incorporation which occurs at the beginning of outgrowth represents some DNA replication and not just “repair” synthesis. One additional event can be detected in DNA immediately after outgrowth. Stuy ( 1959) showed that germinated spores are more resistant to UV than dormant spores. Stafford and Donnelan (1968) confirmed Stuy’s observation and reinvestigated this question recently. In their experiments 65% of the germinated spores of B. megaterium survived a dose of UV which permitted the survival of only 0.25% of these spores in the dormant state before germination. These investigators correlated this increased resistance of germinated spores with a decrease in the production of thymine-containing photoproducts by this cell when subjected to UV irradiation, This seems to indicate that either the physical properties of the DNA or those of its immediate environment change after germination. C. DNA REPLICATIONDURING OUTGROWTH

From the literature it appears that there is a considerable variation in the time at which DNA synthesis begins in different experiments, even within the same spore species. The factors influencing these variations are not known. The first studies on the timing of DNA replication during outgrowth were those of Fitz-James ( 1955) and Young and Fitz-James ( 1959b) investigating the incorporation of radioactive phosphorus into macromolecules in germinating spores. They showed that incorporation into DNA started only 80 minutes after germination. In other experiments, in which total DNA synthesis was measured, an increase in DNA during outgrowth occurred only after 2 hours. It is evident from these and many other investigations that DNA replication starts when differentiation into a vegetative cell is almost completed. The time of the beginning of DNA replication is given by most recent authors as 80-160 minutes after the start of germination (Woese and

1.

29

THE OUTGROWING BACTERJAL ENDOSPORE

Forro, 1960; Kobayashi et aZ., 1965) (see Fig. 6).The time from the beginning of DNA synthesis until the end of replication was 90 minutes in Wake’s ( 1963) experiments. As mentioned before, the DNA-synthesizing apparatus exists in the spore, but the mechanism controlling the initiation of DNA synthesis is not known. DNA doubling starts roughly after duplication of the RNA content of the cell (Woese and Forro, 1960; Sakakibara et aZ., 1965).

1

2.0 Relat ive

J

DNA Content

2.0

A I

I

100

200

Indole/ methionine Transforming Activity

1.o

Time in Minutes

FIG.6. Top: relative DNA content as a function of time in a germinating B. subtilis spore suspension; bottom: indole/methionine transformation ratios in the DNA lysates obtained at various times from the germinating spore suspension. The indole/methionine ratio for the first sample has been set at 1.0 and subsequent ratios have been normalized to this. (Data from Wake, 1963.)

D. CHROMOSOME REPLICATION The replication of the B . subtilis chromosome during outgrowth occurs in a sequential fashion. This was shown by experiments using marker frequency analysis in DNA transformation studies; the distribution of such transforming markers between old (labeled with heavy isotopes) and newly synthesized (unlabeled) DNA formed during different time

30

ALEX KEYNAN

intervals during outgrowth (Oishi et al., 1964) was examined by separation on cesium chloride density gradients. These authors demonstrated that when the newly synthesized “light” DNA is isolated, it contains increasingly more markers ( as detected in transformation experiments ) as a function of time. The order of gene replication seems to be the same in outgrowing spores as in vegetative cells. This was shown by the same method when a very rich culture medium was used. The chromosomes replicate dichotomously in the sense that some of the new chromosomes start to divide before the division of the parent chromosomes is completed.

E. DNA REPLICATIONA N D DIFFERENTIATION From the description of the events during outgrowth it is evident that differentiation, cxpressed as the sequence of appearance of different protein molecules, enzymes, or structures, is not dependent on chromosomal replication. The independence of differentiational events from DNA replication was demonstrated by Steinberg and Halvorson ( 1968b) who showed that: ( a ) in a thymine auxotroph no thymine addition was necessary for periodic a-glucosidase or histidase synthesis during outgrowth, and ( b ) a DNA inhibitor, 5-fluoro-2-deoxyuridine did not influence enzyme synthesis in these cells. On the other hand, mytomycin inhibited enzyme formation probably by interfering with the template activity of DNA ( Steinberg and Halvorson, 1968b). IX. Conditions That influence and Direct the Differentiational Events in the Outgrowing Endospore

Differentiation in the outgrowing bacterial endospore can be influenced by changing the environment. The most simple case of such an influence is the experiment of Torrinni and Levinthal (1967) in which, by using an incomplete medium, differentiation starts but is interrupted at a stage when only very fcw new proteins are being synthesized. It has been discovered recently that much more drastic modifications of the differentiational events can be induced by a change in the environment. In fact, the whole direction of development of the outgrowing spore can be changed even before outgrowth is completed and before the first dell divides in that the outgrowing spore can be induced to form a new spore instead of developing into a vegetative cell. This has been shown to occur by Vinter and Slepecky (1965) when they diluted the complex medium used to induce outgrowth of B. cereus or B . subtilis

1.

THE OUTGROWING BAClXRIAL ENDOSPOIIE

31

spores shortly after outgrowth started. This kind of sporulation inside an outgrowing spore has been called “microcycle sporulation.” In spores of B. megaterium, Holmes and Levinson (1967) demonstrated that this microcycle sporulation could be induced in germinated spores under controlled nutritional conditions by acetate and also by some other tricarboxylic acid cycle intermediates. Such a “shift down” treatment by diluting the medium does not always lead to spore formation. The stage of outgrowth at which such a system is “shifted down” determines whether the cell will or will not start to differentiate into a spore. Spores do not form if the shift down treatment occurs at a very early phase of development or if the culture has entered the logarithmic growth phase. The best time for the induction by nutritional shift down of secondary spore formation is when DNA starts to replicate, when the outgrowing spore has almost differentiated into a vegetative cell. From these experiments it appears that high nutrient concentrations repress that part of the genome which is responsible for spore formation, whereas depletion of the medium will repress that part of the genome which is responsible for the formation of vegetative cells and derepress the parts of the genome responsible for spore formation; but it also appears that this derepression of the spore genome call occur only at a specific time in the cell cycle. X. The Outgrowing Bacterial Endospore as a System for the Study of Cellular Differentiation

Although much of the information given in this review is still incomplete in its detail and some of it even controversial in its content, it is already possible to evaluate the research potential of the outgrowing spore as a model for the study of cell differentiation. Some of the classical problems of cell differentiation become morc simplified when applied to this model. The problem of whether or not the complete genome is preserved during differentiation does not exist in this system. Differentiation is not unidirectional but cyclic and, therefore, there is no doubt that the genome is prcscrvcd in its completeness during differentiation. Another classic problem is the nature of the substaiicc responsible for the induction of the differentiational changes, the so-called inducer. In this system the “inducer” is simply the nutritional environment. The controls responsible for induction of spore formation are not known, but they are activated whenever an essential nutrient is lacking in the

32

ALEX KEYNAN

medium. A further complication of the study of differentiation in many systems is the fact that it occurs in actively growing cells. In the outgrowing bacterial endospore cell differentiation occurs before the first cell division and can therefore be studied apart from cell division. Therefore, the outgrowing bacterial endospore has several advantages as an experimental model. First, we are dealing with a synchronized single cell suspension; second, it is possible to manipulate the direction and rate of differentiation by changes in the environment, and finally, the fact that differentiation in this system seems to depend on continuous transcription enables us to investigate a situation in which gene action is reflected directly in the sequence of the appearance of new kinds of proteins during development. At this stage most of the information obtained from studying this system is descriptive at the molecular level, i.e., it attempts to describe the developmental program of this cell during differentiation. But some of the information obtained might be relevant to other models which have been suggested for some aspects of cytodifferentiation. The “Jacob-Monod model” of control of protein synthesis assumes the interplay of structural genes which control the structure of proteins (amino acid sequence) and genes which control the quantities of protein products. Applying this model to differentiation one assumes that the control genes are responsible for the sequential “switching on” of the synthesis of new kinds of proteins. In this connection it is interesting to note Mandelstam’s suggestion that small amounts of spore proteins appear also in the vegetative cell. Another aspect of the hypothesis of Jacob and Monod of the “genetic regulatory circuits” that can be applied to this system relates to the fact that there seem to be three sequential periods of regulation. After germination, if no complete medium is available only a very small part of the genome is activated. If complete medium is added all of the “vegetative” parts of the genome seem to become active simultaneously at the same rate until, at a later stage, repressors are formed and appropriate control circuits start to operate. The various descriptions of sequentially timed enzyme syntheses reported in the work of Halvorson and coworkers are also in agreement with a hypothesis of sequential “genetic regulatory circuits” (Jacob and Monod, 1963). Several phenomena connected with this system cannot be applied to this model and need further explanation. One of these is the behavior of DNA during differentiation in the spore-forming bacteria. Before the beginning of spore formation, a kind of fusion occurs between the nuclei

1.

THE OUTGROWING BACTERIAL ENDOSPORE

33

resulting in a DNA “filament” (Robinow, 1960). The nature of this event is not understood. Before the outgrowing spore starts to differentiate, some kind of metabolic change occurs in the DNA (for review see Halvorson et al., 1966); its nature or relation to the processes of differentiation, if any, are not understood either. But Halvorson’s suggestion that this metabolic change in the DNA is, in fact, DNA replication recalls a recent speculation that somatic recombination of replicated genes might explain the formation of a great variety of proteins by the same cell (Edelman and Galy, 1966). Although these phenomena might not be related to differentiation as such, they should at least be noted and their possible significance for differentiational events be considered. XI. Summary

Bacterial endospores are intracellular structures formed during the regular life cycle of several species of bacteria. Endospores differ from vegetative bacterial cells in their morphology, physiological properties, and chemical composition. During germination they lose their typical spore properties, and when suspended in a nutrient medium develop in 1 to 2 hours into typical vegetative cells. This process of “outgrowth” during which a germinated spore turns into a vegetative cell, different in composition, structure, and physiology from the spore, is a process similar to cellular differentiation. Several of the sequential changes occurring at the molecular level during spore outgrowth are described. The advantages of this relatively simple system for the study of cellular differentiation are pointed out. ACKNOWLEDGMENTS

I would like to express my deep gratitude to Mrs. Brigit Wolman, for her unfailing assistance in the writing of this review. I would also like to thank Dr. Amikan Cohen for helping in the preparation of this manuscript and Dr. H. 0. Halvorson, Dr. Y. Mandelstam, and Dr. A. Kornberg, for providing manuscripts prior to their publication. REFERENCES Armstrong, R. L. and Sueoka, N. (1968a). Proc. Natl. Acad. Sciences US. 59, 153. Armstrong, R. L., and Sueoka, N. ( 1968b). PTOC.68th Ann. Meeting Am. SOC. Microbiol. p. 26. Bach, J. A., and Sadoff, H. L. (1962). J. Bacteriol. 83, 699. Baillie, H., and Norris, G. R. (1963).J . Appl. Bacterial. 26, 10. Baillie, H., and Noms, G. R. (1964). J. Bacteriol. 87, 1221. Balassa, G. (1965). “MBcanismes de regulation des activitk cellulaires chez les microorganismes,” p. 565, C.N.S.R., Paris.

34

ALES KEYNAN

Blumenthal, H. J. (1961). In “Spores” (11. 0.Hnlvorson, ed.), Vol. 2, 1’. 120. Burgess, Minneapolis, Minnesota. Chaloupecky, V. ( 1964). Folia Microbiol. 9, 232. Chambon, P., Deutscher, M. P., and Komberg, A. (1968). J. Biol. Chem. (in press), Deutscher, M. P., Chambon, P., and Kornberg, A. (1968). J. Blol. Chem. 243, 5117. Doi, R. H., and Halvorson, H. 0. (1961). J. Bacteriol. 81, 51. Doi, R. H., and Igarashi, R. T. (1964a). Proc. Natl. Acad. Scl. U.S. 52, 755. Doi, R. H., and Igarashi, €3. T. (196413). Nature 203, 1092. Donnelan, J. E., and Setlow, R. B. (1965). Science 149, 308. Donnelan, J. E., Nags, E. H., and Levinson, H. S. (1965). In “Spores” ( L . L. Campbell, and H. 0. Halvorson, eds.), Vol. 3. Am. SOC.Microbiol., Ann Arbor, Michigan. Dvorkin, M. (1966). Ann. Reo. Microbiol. 20, 75. Edelman, G. M., and Galy, J. A. ( 1966). Proc. Natl. Acad. S c i . U.S. 56, 353. Falaschi, A., Spudich, J. A., and Komberg, A. (1965). In “Spores” (L. L. Campbell, and H. 0. Halvorson, eds.), Vol. 3, p. 88. Am. Soc. Microbiol., Ann Arbor, Michigan. Yitz-James, P. C. (1955). Can. J. Blicrobiol. 1, 525. Fitz-James, P. C. (1960). J. Biophys. Biochem. Cytol. 8, 507. Fitz-James, P. C. ( 1965). “Mecanismes de regulation des adivitbs cellulaires chez Ies microorganisms,” p. 529. C.N.R.S., Paris. Goldman, M., and Blumentlial, H. J. (1964). J. Bacteriol. 87, 377. Could, G. W., and Hitchins, A. D. (1963). J. Gen. Microbiol. 33,413. Green, J. H., and Sadoff, H. L. ( 1965). J. Bacteriol. 89, 1499. Grelet, N. (1957). J. Appl. Bacteriol. 20, 315. Grobstein, C. ( 1963). In “Cytodifferentiation and Macromolecular Synthesis” ( M. Locke, ed.), p. 1. Academic Press, New York. Gross, P. R., Malkin, L. I,, and Moyers, W. A. (1964). Proc. Natl. Acad. Sci. U S . 51, 407. Grossowicz, N.,Hestrin, S., and Keynan, A. (1961). “Cryptobiotic Stages in Biological Systems.” Elsevier, Amsterdam. Halvorson, H. 0.(1965). Symp. SOC. Gen. Microbiol. 15, 343. Halvorson, H. O., Vary, J. C., and Steinberg, W. (1966). Ann. Reu. Microbiol. 20, 169. Hanson, R. S., Blicharska, J., Amnud, M., and Szulmajster, J. (1964). Biochem. Biophyv. Res. Commun. 17, 890. Henney, H. R., and Storck, R. (1964). Proc. Natl. Acad. Sci. U S . 51, 1050. Higa, A. ( 1964). Ph.D. Tliesis, Mass. Inst. Techno]., Cambridge, Massachusetts. Holmes, P. K., and Levinson, H. S. ( 1967). J . Bacteriol. 94,434. Jacob, F. (1966). Science 152, 1470. Jacob, F., and Monod, J. (1963). In “Cytodifferentiation and Macromolecular Synthesis” (M. Locke, ed.), p. 30. Academic Press, New York. Keilin, D. (1959). Proc. Roy. Soc. B150, 149. Keynan, A., nnd Evenchik, Z. ( 1969). In “The Bacterial Spore” (G. W. Godd, : ~ n d A. 1).Hitcliing, edq.) Academic Press, New York ( i n press). Keynan, A., and Halvorson, H. 0. (1965). In “Spores” (L. L. Campbell and H. 0. Halvorson, eds.) Vol. 3, p. 174 Am. SOC. Microbiol., Ann Arbor, Michigan. Keynan, A., and Issahary, C. (1968). Unpublished results.

1.

THE OUTGROWING BACTERIAL ENDOSPORE

35

Keynan, A., Evenchik, Z., Halvorson, H. O., and Hastings, J. W. (1964). J. Bacteriol. 88, 313. Keynan, A., Issahary-Brand, G., and Evenchik, Z. ( 1965). In “Spores” (I.. L. Canipbell and H. 0. Halvorson, eds. ), Vol. 3, p. 180. Am. Soc. Microbiol., Ann Arbor, Michigan. Kobayashi, Y., and Halvorson, H. 0. (1968). Arch. Biochem. Biophys. (in press). Kobayashi, Y., Steinberg, W., Higa, A., Halvorson, H. O., and Levinthal, C. (1965). In “Spores” (L. L. Campbell and H. 0.Halvorson, eds.), Vol. 3, p. 200. Am. Soc. Microbiol., Ann Arbor, Michigan. Kornberg, A,, Spudich, J. A., Nelson, D. L., and Deutscher, M. P. (1968). Ann. Rev. Biochem. 37, 51. Kornfeld, J. ( 1961). Ph.D. Thesis, Univ. of Wisconsin, Madison, Wisconsin. Lawrence, N. L., and Halvorson, H. 0. (1954). J. Bacteriol. 68, 334. Lederberg, J. (1966). Current Topics Deuelop. Biol. 1, ix. Levinthal, C., Keynan, A., and Higa, A. (1962). Proc. Natl. Acad. Sci. US.48, 1631. Mandelstam, J. ( 1968). Personal communication. Monroy, A., Maggio, R., and Rinaldi, A. M. (1965). Proc. Natl. Acad. Sci. U.S. 54, 107. Murrell, W. G. (1961). Symp. SOC. Gen. Microbiol. 11, 120. Nakata, D., Matsushiro, A., Konde, K., and Kondolshi, K. (1957). Merl. J. Osaka Univ. 7, 809. Nakata, H. M. (1964). J. Bacteriol. 88, 1522. Nakata, H. M., and Halvorson, H. 0. (1960). J. Bacteriol. 80, 801. Norris, G. R., and Baillie, A. (1964). J. Bacteriol. 88, 264. Norris, G. R., and Wolf, J. (1961). J. Appl. Bacteriol. 24, 42. Oishi, M., Yoshikawa, H., and Sueoka, N. (1964). Nature 204, 1069. Powell, J. F., and Strange, H. E. (1956). Biochern. J. 63, 661. Robinow, C. F. (1960). In “The Bacteria” (I. C. Gimsalus, and R. I. Stanier, eds.), p. 235. Academic Press, New York. Rodenberg, S., Steinberg, W., Piper, J., Nickerson, K., Vary, J. C., Epstein, R., and Halvorson, H. 0. (1968). Submitted for publication. Sadoff, H. L. (1961). In “Spores” (H. 0. Halvorson, ed.) Vol. 2. Burgess, Minneapolis, Minnesota. Sadoff, H. L., Bach, J. A., and Kook, J. W. (1965). In “Spores” (L. L. Campbell and H. 0. Halvorson, eds.), Vol. 3, p. 97. Am. SOC. Microbiol. Ann. Arbor, Michigan. Sakakibara, Y., Saito, H., and Ikeda, Y. (1965). J. Gen. Appl. Microbiol. 11, 243. Salb, J. M., and Marcus, P. I. (1965). Proc. Natl. Acad. Sci. V.S. 54, 1353. Sonnebom, T. M. (1964). Proc. Natl. Acad. Sci. US. 51, 915. Spudich, J. A., and Komberg, A. (1968). J. Biol. Chem. Submitted for publication. Stafford, R. S., and Donnelan, J. E. (1968). Proc. Natl. Acad. Sci. US. 59, 822. Stan, R. C. (1968). Proc. Natl. Acad. Sci. US. 59, 1088. Stavy, L., and Gross, P. R. (1967). Proc. Natl. Acad. Sci. US.57, 735. Steinberg, W., and Halvorson, H. 0. (1968a). J. Bacteriol. 95, 469. Steinberg, W., and Halvorson, H. 0. (196813). J. Bacteriol. 95, 479. Steinberg, W., Halvorson, H. O., Keynan, A., and Weinberg, E. (1965). Nature 208, 710. Stuy, J. (1959). Antonie uon Leeuwenhoek 1. Microbiol. Serol. 22, 337-349.

36

ALEX KEYNAN

Sussman, A. O., and Halvorson, H. 0. (1966). “Spores, Their Dormancy and Germination.” Harper, New York. Sussman, M. (1965a). Brookhawen Symp. Biology 18, 66. Sussman, M. (196513). Ann. Reu. Microbiol. 19, 59. Sussman,M., and Osborn, M. J. (1964). Proc. Natl. Acud. Sci. US. 52, 81. Torriani, A., and Levinthal, C. (1967). J. Bacteriol. 94, 176. Vinter, V. (1959a). Foliu Microbiol. 4, 1. Vinter, V. (1959b). Fdia Microbiol. 4, 216. Vinter, V. (1962). Foliu Microbiol. 7, 115. Vinter, V. ( 1963). Folk MIcrob401. 8, 147. Vinter, V. ( 1964). Folia Microbiol. 9, 58. Vinter, V. (1965). In “Spores” (L. L. CampbelI and H. 0. Halvorson, eds. ), Vol. 3, p. 25. Am. SOC.Microbiol, Ann Arbor, Michigan. Vinter, V. (1967). Folk Microbiol. 12, 89. Vinter, V.,and Slepecky, R. A. (1965). J. Bacteriol. 90, 803. Waits, W. N. (1968). Biochem. J . (in press). Wake, R. G. (1963). Biochem. Biophys. Res. Commun. 13, 67. Warth, A. D., Ohye, D. F., and Murrell, W. G. (1963). J. Cell Bid. 16, 593. Woese, C. R. and Forro, J. R. (1960). J. Bacteriol. 80, 811. Woese, C. R., Langridge, R., and Morowitz, H. J. (1960). J . Buctedol. 79, 777. Wright, B. E., and Anderson, M. L. (1958). In ‘‘Chemical Basis of Development,” (W. D. McElroy and B. Glass, eds.), p. 296. Johns Hopkins Press, Baltimore, Maryland. Yoshikawa, H. (1965). Proc. Nutl. Acad. Sci. U.S. 53, 1476. Young, J. E., and Fitz-James, P. C. (1959a). Nature 183, 372. Young, J. E., and Fitz-James, P. C. (1959b). J. Biophys. Biochem. Cytol. 6, 467.

CHAPTER 2

CELLULAR ASPECTS OF MUSCLE DIFFERENTIATION in Vitro David Yufe DEPARTMENT OF CELL BIOLOGY, THE WEIZMANN INSTITUTE OF SCIENCE, REHOVOT, ISRAEL

I. Introduction . . . . . . . . . . . . . . . , . . . . . . . . . . . . . . . . . . . . 11. Differentiation of Primary Cultures . . . , . . . . . . . . . . . . A. Formation of Multinucleated Muscle Fibers . . . . . . B. Fusion of Myoblasts of Different Species Origin .. . C. I n Vioo “Hybridization” of Muscle Cells . . . . . . . . 111. Myogenic Cell Lines . . . , . .. . . . . . . . . , . . . . . . . . . . . . . A. Effect of Continuous Cell Multiplication on the Retention of Differentiation Potentialities . . . . . . . . B. Cessation of DNA Synthesis at Fusion . . . . . . . . . . . C. Fusion Specificities . .. .. .. .. .. .. .. .. .. .. . . . . . . D. The Inheritance by Single Cells of the Capacity to Differentiate ................................ E. The Effect of Collagen . . . . . . . . . . . . . . . . . . . . . . . . F. Growth Characteristics and Ploidy of Myogenic Cell Lines ...................................... G . Enzymic Manifextations of Differentiation iri Vitro IV. Induction of DNA Synthesis and Mitosis in Nuclei within Muscle Fibers . .. .. .. .. .. .. .. .. . . .. .. . .. .. . V. Comments ...................................... References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

.

37 39 39 41 44 45 45 47 48 49 52 54

57 60

70 75

1. Introduction

The complex interrelationships between cells in the multicellular organism seriously limit the types of questions about differentiation phenomena that can be analyzed in the intact organism. The hetero37

38

DAVID YAFFE

geneity of cell types and the existence of homeostatic regulation mechanisms complicate the interpretation of many observations. Working with simple unicellular organisms as model systems proved to be useful in studying some aspects of cellular differentiation. However, the applicability of such systems is also limited, since they lack one of the most fundamental features of differentiation, i.e., the stability of the differentiated state, manifested in the apparently hereditary perpetuation of differencesbetween cell lines within the same organism. Therefore, the study of some of the main aspects of differentiation necessitates the development of experimental systems in which the inherent properties of isolated and defined cell types can be investigated under controlled conditions. As a result of intensive work on the requirements for the cultivation of somatic cells in uitro many obstacles to the growth of isolated cells have been overcome. While the methods thus developed have contributed to our knowledge of the metabolism of animal and plant cells, considerably less successful have been attempts to elucidate questions related to the nature of differentiation processes. The phenomenon of dedifferentiation, i.e., loss of tissue-specific characteristics after a short period of growth in tissue culture, was so universal as to be almost accepted as a characteristic of cells multiplying in uitro, thus raising doubts about the concept of the stability of differentiation as an inherent property of differentiated cells. However, experiments in recent years have shown that this need not be the case. Retention of differentiated traits in uitro has been achieved in the cultivation of several kinds of cells (Cahn and Cahn, 1966; Coon, 1966; Sato and Yasumura, 1966). Although thesc systems arc still few, they show that in spite of the high interdependency of cells in uiuo, isolated cells can manifest a surprisingly high degree of autonomy in their capacity to differentiate. These observations open thc way to more detailed studies on retention of the capacity for differentiation and its expression at the cellular level. Muscle tissue culture had been included in the repertoire of tissue culturists since the first days of this art (Lake, 1915; Lewis, 1915). Some of the unique properties of this system make it especially attractive for studying various aspects of cell biology; differentiation of muscle tissue is associated with very distinct steps which can be readily analyzed, such as formation of multinucleated fibers, cessation of DNA synthesis, crossstriation, and contractility. While early studies were made on explants of small fragments of muscle tissue, the introduction of techniques for the dissociation of tissue into single cell suspensions by enzymic treatment

2.

MUSCLE DIFFERENTIATION IN VITRO

39

and for the cultivation of cells in monolayers in partially defined media facilitated direct microscopic observations of single living cells, and permitted a more detailed analytical approach ( Moscona, 1952; Konigsberg et al., 1960; Konigsberg, 1961). This article reviews a series of experiments which utilize muscle cell cultures as a model for studying some of the inherent properties of differentiating cells. The following account is intended to familiarize the reader with the experimental system, describe our approach and results, and point out some of the possibilities which this cell system offers for future research. II. Differentiation of Primary Cultures

A. FORMATION OF MULTINUCLEATED MUSCLEFIBERS Monolayer cultures of muscle cells are prepared from embryonic or newborn skeletal muscle. At these stages of development, the tissue consists of mononucleated muscle cell precursors and partially differentiated multinucleated cells; the tissue is dissociated into a suspension of single cells by treatment with trypsin. Most of the fibers are destroyed or lost in this process. After removal of the trypsin, followed by decantation or filtration of the suspension to remove the large tissue debris, the suspension is seeded into tissue culture containers (usually plastic petri dishes), The cells settle down and form a monolayer which consists of a heterogeneous population of mononucleated cells; the majority of these are spindleshaped myoblasts, the mononucleated precursor cells of muscle fibers. The culture also contains fibroblasts and, possibly, other cells from the original muscle. The cells multiply and on day 3-4 of culture, when a confluent cell layer has been formed, the myoblasts begin to aggregate nnd differentiate into thick multinucleated muscle fibers containing hundreds of nuclei. One or two days later, these fibers begin to contract spontaneously and typical cross-striation can be observed (Figs. 1and 2 ) . The mode of formation of these fibers has been a matter of controversy for many years. Most of the earlier workers attributed the multinucleated state to multiplication of nuclei within the growing fibers. However, inorc recent observations, mostly in tissue culture systems, have supported the notion that the multinucleated fibers were formed by fusion of mononucleated myoblasts into ribbonlike syncitia (for reviews, see Betz et al., 1966; Altschul, 1962; Murray, 1960). Even simple observations on the kinetics of fiber formation in uitro made the earlier proposed mechanisms very unlikely; under proper culture conditions one can often

40

DAVID YAFFE

see within a matter of 6 to 12 hours the conversion of a monolayer of mononucleated cells into a network of multinucleated fibers containing hundreds of nuclei. Assuming formation of the fibers by nuclear replication would imply a generation time too short to be plausible. In spite of

FIG. 1. Seventy-hour-old primary rat muscle cell culture fixed at the onset of formation of multinucleated cells. Note aggregation of spindle-shaped myoblasts (arrows) which can be distinguished from the fibroblastic cells.

the rapid formation of the fiber and its increasing content of nuclei, no mitotic figures have been observed within multinucleated cells. Furthermore, the formation of multinucleated fibers could be shown to take place even in the presence of inhibitors of DNA synthesis such as nitrogen mustard and myleran (Bassleer et al., 1963; Konigsberg et al., 1960). More direct evidence that mononucleated cells fuse into multinucleated fibers has been obtained by cinematography (Capers, 1960) and autoradiography ( Stockdale and Holtzer, 1961; Yaffe and Feldman, 1965). Autoradiography of cultures exposed to the tritium-labeled DNA precursor revealed the following features: 1. When myoblasts obtained from rat thigh muscle were exposed to th~rnidine-~H during the first 48 hours of cultivation (before the fonnation of multinucleated fibers), the muscle fibers which subsequently

2.

MUSCLE DIFFERENTIATION IN VITRO

41

formed contained 90-100% thymidine-3H-labeled nuclei. However, when such labeled myoblasts were mixed with unlabeled myoblasts, the resulting fibers contained both labeled and unlabeled nuclei in a random arrangement.

FIG.2. Twelve-day-old rat muscle cell culture consisting of differentiated multinucleated fibers with fibroblastic mononucleated cells in the background.

2. Supply of the labeled nucleotide to cultures during the process of fiber formation, or later on, resulted in the labeling of nuclei within the mononucleated cells but not in the multinucleated fibers. The presence of both labeled and unlabeled nuclei within the same fibers (in the first experiment) indicated that the in oitro formation of multinucleated muscle fibers takes place by fusion of mononucleated cells and not by nuclear replication within the fiber. The lack of incorporation during and after cell fusion (in the second experiment) showed that the nuclei within the fibers are postmitotic and do not synthesize DNA.

B. FUSION OF MYOBLASTS OF DIFFERENT SPECIES ORIGIN The formation of multinucleated fibers via the fusion of mononucleated cells raises questions concerning cell type specificity in the process of

42

DAVID YAFFE

fusion. Experiments performed several years ago ( Holtfreter, 1947; Moscona, 1956) have shown that when cells obtained from different tissues are “co-mingled,” the cells usually sort out and aggregate according to their tissue of origin. Even when cells of different species are mixed together, sorting out into aggregates usually takes place according

FIG.3. Hybrid fibers formed in mixed culture of rat and calf thigh muscle cells. Labeled nuclei are of rat origin. (From Yaffe and Feldman, 1965.)

to tissue type rather than species identity ( Moscona, 1957). These experiments were interpreted as indicating that similarities in tissue-specific cell surface properties can extend across wide taxonomic differences. The fusion property of muscle cells offered another approach for testing the functional similarity of homologous specialized cells. Can inyoblasts from different species fuse with each other? Will fibers having both a cytoplasmic and a nuclear contribution from different species be able to differentiate and function? Will myoblasts fuse with cells from other tissues? To answer these questions, primary cultures of thigh muscle cells were prepared from calf fetuscs, newborn rabbits, and rats. Thc rat cells werc

2.

43

MUSCLE DIFFERENTIATION IN VITRO

labeled by growing them in the presence of th~midine-~H. After 3 days secondary cultures were prepared in which labeled rat cells were mixed with nonlabeled rabbit or calf cells. In both systems differentiation of multinucleated contracting muscle fibers took place. Autoradiographs of cultures of muscle cells produced in cultures of the mixed myoblasts revealed that in the two combinations tested, the muscle fibers contained both labeled and unlabeled nuclei in a pattern indicating that myoblasts of different species origin fused to form hybrid muscle fibers (Fig. 3, Table I). TABLE I FORMATION O F HYBRID MUSCLE FIBERS IN CULTURES OF THIGH MUSCLECELU OF DIFFERENTGENETICORIGIN^ Sources of cells

Thymidine-3Htreated cultures Rat Rat Rat Rat Rat Rat

Untreated cultures

Ralhi t

Calf

Chick

Labeled nuclei in fibers of secondary cultures ( % ) go+ 2b 55 12b 99f 2 37* 9b 9 3 & 6b 51 + 26b

*

From Yaffe and Feldman (1965). The standard deviation is calculated from weighted values according to the number of nuclei in each fiber. a b

In order to test whether thigh myoblasts of mammalian and avian origin will also fuse to form hybrid fibers, rat and chicken cells were mixed under conditions similar to those described above for the ratcalf mixtures. Hybrid cells formed containing nuclei of the two different genetic origins. However, a certain proportion of fibers contained either labeled or unlabeled nuclei, i.e., nuclei either of rat or of chicken origin. This seems to indicate that although rat myoblasts can fuse with chicken myoblasts to form multinucleated fibers, in this situation there is also a tendency toward fusion according to the genetic origin of the cells. No incorporation of labeled nuclei into fibers was observed when myoblasts were mixed with labeled cells of kidney or heart origin, indicating nonparticipation of the latter in muscle fiber formation.*

* Preliminary experiments have suggested the possibility that, in fact, cardiac myoblasts may sometimes fuse into muscle fiber of skeletal origin, but this is restricted to very specific physiological conditions.

44

DAVID YAFFE

These experiments demonstrated that myoblasts derived from as genetically remote organisms as rat, calf, and chicken possess enough similarity in the finer details of differentiation to enable them to fuse and form hybrid cells capable of following an apparently normal course of differentiation within a common cytoplasm. Differentiation of the fiber, following fusion, is associated with various metabolic changes resulting in the formation of the contractile mechanism and appearance of enzymes involved in energy supply for the energetics and biochemistry of contraction (see Section II1,G). Thus, the question arises whether the nuclei of both origins within the hybrid fiber contribute to the differentiation of the cells. If so, how does this affect the synthesis and organization of specialized proteins? Do subunits of structural proteins formed under the direction of the different genomes segregate or form mixed structural elements? C. I n Viuo “HYBRIDIZATION” OF MUSCLECELLS Conclusive evidence that muscle formation in viuo also takes place by cell fusion has recently been produced by Mintz and Baker (1967). Artificial chimeric mice (“allophens”) were formed by aggregating blastomeres dissociated from cleavage stage embryos of different genotypes and reimplanting these combined aggregates into appropriate female hosts for further development. By this procedure these authors obtained adult mice which consisted of a mixture of cells of two genotypes. These allophenic mice were composed of cells originating from two strains of mice, each homozygous for a different allele for an enzyme (isocitric dehydrogenase) . The isozymes could be differentiated by their electrophoretic mobility. Extracts of tissues of genetic F1 hybrids of the parental strains produced on electrophoresis three bands of isozymes: two of the parental type and an intermediate one, the result of the combination of subunits of the two parental isozymes. However, extracts of tissues of the allophenic mice produced only the two parental bands, save for extracts of skeletal muscle tissue which also produced the intermediate band-identical to the pattern produced by the Fl hybrids. Thus, the appearance of a hybrid type of enzyme only in muscle extracts indicates that, unlike in other tissues, in the muscle active nuclei of both genotypes reside within the same muscle cell cytoplasm. Therefore, subunits of the electrophoretically different alleles combine randomly, at status nascendi, to form a hybrid type of isozyme molecules.

2.

MUSCLE DIFFERENTIATION IN VITRO

45

111. Myogenic Cell lines

CELLMULTIPLICATION ON THE RETENTION OF A. EFFECTOF CONTINUOUS DIFFERENTIATION POTENTIALITIES Champy, in a series of articles, has maintained that most of the cells in the body dedifferentiate in tissue cultures. They return, he claims, to a completely indifferent type of cell that no longer shows the imprint of its origin. In explants from late fetal stages he finds that cells of the kidney tubules, of the thyroid, of the parotid and of the submaxillary glands, of the smooth muscle, of the mesenchyme, etc. dedifferentiate into an indifferent embryonic type indistinguishable from each other. This dedifferentiation, he claims, is associated with the phenomena of cell division. The rapidity of dedifferentiation is a function of the rapidity of the celldivision. Furthermore, according to Champy, all cells differentiated for a special function lose or tend to lose during mitosis, their characteristic function. In the animal organism they recover immediately after the telophase, since they are subject to the same functional excitation as before division (Lewis and Lewis, 1917, p. 189).

This phenomenon of dedifferentiation has since been repeatedly observed by many investigators who attempted to grow differentiating cells in culture (Davidson, 1964; Eagle, 1965). Muscle cells in tissue culture manifest a higher capacity to differentiate than other cell types. However, they undergo only a limited number of cell divisions and then fuse into postmitotic multinucleated fibers. Further differentiation of these cells thus takes place in the absence of cell division. Konigsberg has shown that muscle cells can be cloned in tissue culture and still retain the capacity to differentiate ( Konigsberg, 1961). Under these conditions myoblasts multiply and form colonies, each originating from a single cell. However, after several cell divisions these cells also fuse into multinucleated fibers. The capacity of muscle cells to differentiate in vitro may therefore be attributable to their restricted number of cell divisions. Thus, differentiation of these cells may take place on the basis of the utilization of a preexisting stock of informational molecules which were produced in uiuo. Experiments were designed to determine if the fusion and differentiation of myoblasts into postmitotic multinucleated fibers was a rigidly programmed phenomenon which must occur within a definite number of cell divisions. What will be the result of growing the myoblasts under conditions of prolonged multiplication in vitro? Can the capacity to differentiate be retained over extended periods of multiplication? Will the cells continue to grow in the undifferentiated form or will they degenerate? Attempts were made to establish culture conditions which would

46

DAVID YAFFE

promote multiplication of myoblasts and prevent cell fusion. An essential requirement was to start out with cultures which would contain predominantly myoblasts. It was noted that after dispersing primary muscle cell cultures with trypsin, on replating the cells in the standard medium (Medium 199 supplemented with 2% chick embryo extract; 0.5% bovine serum albumin, and 10% horse or fetal calf serum), the myoblasts attached to the plate much more slowly than other cell types present in the suspension. Accordingly young cultures, before the onset of cell fusion, were dispersed with trypsin; after removal of the enzyme, the cells were incubated in growth medium for 30 minutes at which time most of the nonmyoblastic cells had begun to attach to the bottom of the plate. The medium containing the floating cells was collected and placed in dishes which were first coated with a collagen film and seeded with a small number of lethally irradiated cells ( 1 X lo6 irradiated cells per a 60 mm diameter plate). The irradiated cells, which are unable to multiply, served as a feeder layer and thus enabled very dilute populations of myoblasts to survive and continue to multiply. After 3-4 days the cultures consisted of a network of myoblasts with very few nonniyogenic cells. The cultures were repassaged in the above manner at the onset of fusion. In some experiments a carcinogenic agent, methylcholanthrene, was introduced into the culture medium during the first two cell passages. This was done to test whether neoplastic transformation of the cells would facilitate their serial propagation in tissue culture (Earle and Nettleship, 1943; Benvald and Sachs, 1985) and prevent degeneration of the cells ( a phenomenon commonly observed in experiments to maintain cells of primary cultures by serial passaging in uitro (Hayflick, 1965) ) . Several independent experiments for the serial passaging of myoblasts were initiated; most of them resulted in loss of the cells after 4-6 passages by gradual cessation of multiplication. However, two cell lines originating from cultures exposed to methylcholanthrene during the first two passages could be maintained by serial passage without loss of their capacity to replicate and differentiate. One of these lines (designated Lo) has now been maintained in uitro for more than 2 years and the other ( Mdl) for 11 months. The differentiation properties of the two lines are virtually indistinguishable. In the course of the first few passages the cells were cultured on a collagen film and feeder layer for good growth and differentiation; however, after several serial passages they grew readily and differentiated into a very dense network of contracting fibers in the absence of collagen and feeder layer (Figs. 4-6) (Yaffe, 1968). It is not clear whether the exposure of the cells during the first two

2.

MUSCLE DIFFERENTIATION IN VITRO

47

passages to methylcholanthrene had any effect on them. However, in further attempts to establish myogenic cell lines, four lines were finally established, three of which were obtained from cultures not treated with carcinogens (Table I1 ) , It can therefore be stated that whatever the effect of the carcinogen on these cultures, it clearly does not play an essential role in the establishment of myogenic cell lines.

FIG.4. Muscle fiber formation by cells of the line L, fixed on day 8. X 9. (From Yaffe, 1968.)

B. CESSATION OF DNA SYNTHESIS AT FUSION Experiments were performed to test whether the prolonged multiplication of myogenic cells in vitro affects the cessation of DNA synthesis which normally takes place at fusion. Cultures of the line L, were allowed to differentiate. At different stages after the onset of fusion, the cells were then fixed and processed for exposed to a 5-hour pulse of th~midine-~H, autoradiography. In all preparations, th~rnidine-~H was incorporated only into the nuclei of mononucleated cells-in no case were nuclei within multinucleated fibers labeled. Thus, cells after prolonged main-

48

DAVID YAFFE

tenance in a state of continuous multiplication retain their capacity to stop DNA synthesis at fusion.

C. FUSION SPECIFICITIES Another parameter which was considered in relation to possible changes in these cells was fusion specificity. It was shown that fusion of myoblasts

FIG. 5. As Fig. 4 but X 130. Note homogeneity of inononucleated cell type and the paucity of fibroblasts (compare to Figs. 1 arid 2 ) .

in primary cultures is tissue-specific and takes place only between myogenic cells (see Section I1,B). In order to see whether myoblasts of primary cultures will fuse with cells of the established lines, undifferentiated 2-day-old cultures of the line Lo were exposed to a 48-hour pulse of thymidine-8H. The cultures were then trypsinized and the cells were added ( 2 X lo6 cells per plate) to 48-hour primary rat thigh muscle cultures ( 4 X lo6 cells per plate). Two days later, i.e., after formation of inultinucleated fibers, the cultures were fixed and processed for autoradiography and the number of labeled nuclei within the fibers was counted. It was found that whereas in cultures prepared from the

2.

MUSCLE DIFFERENTIATION IN VITRO

49

th~midine-~H-treated Lo cells alone, 98% of the nuclei within the fibers were labeled, the fibers in the mixed cultures contained on the average only 14 2 6% of labeled nuclei. These results indicate that the labeled myoblasts of the La line formed multinucleated fibers with the primary myoblasts.

FIG.6. Cross-striated muscle fibers of line L, fixed on day 12. X 325. (From Yaffe, 1968.)

D. THE INHERITANCE BY SINGLE CELLSOF

THE

CAPACITY TO DIFFER-

ENTIATE

The inheritance of the capacity to differentiate within the population of the myogenic cell line was investigated. Do all cells carry this capacity? Do single cells transmit it to all their progeny? Since the differentiation of these cells is manifested by fusion into multinucleated fibers, differentiation capacity cannot be studied on the single cell level. Therefore, isolated cells were grown under cloning conditions and their potentialities for differentiation were assayed by determining the muscle-forming capacity of colonies derived from single cells. Cultures of the myogenic cell lines Le and Lpl were dispersed by

50

DAVID YAFFE

trypsinization and the cell suspensions seeded at cell densities of 25 to 150 single cells per plate on a feeder layer contained in 60 mm collagencoated dishes. Under these conditions 2040% of the cells multiplied and formed small colonies. It was found that in most experiments all the colonies formed muscle fibers ( Figs. 7-9). Cell suspensions prepared

FIG.7. Muscle-forming colonies derived from single cells of line L,. All colonies contain muscle fibers. X 1.7.

from single clones isolated from such cultures were plated again under cloning conditions. This serial reclonization was repeated several times. In most experiments 100% of the clones produced muscle fibers; only occasionally was a clone obtained which did not form muscle fibers. Some of the few clones which did not form muscle fibers were isolated and the cells were again plated under cloning conditions. One hundred percent of muscle-formipg colonies were obtained in all cases, indicating that even cells of the few clones which for some reason did not form muscle

2.

MUSCLE DIFFERENTIATION IN VITRO

51

FIG.8. ( a ) As Fig. 7 but X 6. Note high homogeneity of colony structure. Compare with colonies formed by cells of another myogenic line, M,, ( b ) .

52

DAVID YAFFE

fibers in these experiments possessed the potentiality to differentiate, as did cells of muscle-forming colonies. Thus, myogenic cells maintained in exponential growth in tissue culture for several months pass on the capacity to differentiate to virtually all their progeny. These experiments

FIG. 9. Colony from Fig. 7 at higher magnification. Note the organization and spatial interrelationships between myoblasts and multinucleated fiber. X 35.

do not exclude the possibility that loss of the capacity to differentiate may be linked with loss of the ability of the cell to multiply and form a colony. However, even if this is so, the high plating efficiency obtained in most experiments demonstrates that the capacity to differentiate is inherited by the mu/ority of progeny. E. THE EFFECTOF COLLAGEN Experiments on cloning chick muscle cells have shown (Konigsberg, 1963) that the culture medium collected from crowded cultures (con-

2.

MUSCLE DIFFERENTIATION IN VITHO

53

ditioned medium) in the stationary phase is necessary for the development and differentiation of muscle-forming colonies. It was later found that coating the bottom of the culture plate with a collagen film obviates the need for the conditioned medium. The possibility that collagen serves as an inducer for muscle differentiation was therefore raised (Konigsberg and Hauschka, 1965; Hauschka and Konigsberg, 1966). Experiments with rat muscle cultures in our laboratory also indicated a considerable improvement in growth and differentiation (amount of fibers formed) when cultures were grown on collagen-coated plates. This procedure was therefore adopted and all our myogenic lines were isolated and grown in collagen-coated dishes during their first passages. When the role of collagen was later investigated, it was found that all lines could also be grown in uncoated dishes, although this resulted in a decreased degree of differentiation which varied considerably among the different lines. When cells of lines Ls and M,, were plated for cloning in plates not coated with collagen, 100% of the clones were found to produce muscle fibers (Richler and Yaffe, to be published). Since these plates did contain a small number of feeder layer cells (initially 1 X lo6 cells per plate) which could produce some collagen, an attempt was made to clone cells in petri dishes containing neither collagen nor feeder layer cells. Under these conditions the cells grew poorly but eventually formed colonies. These colonies, after several serial passages, were assayed for muscle forming cells under proper cloning conditions. One hundred percent of the clones produced muscle fibers. Although we do not yet know the effect of very prolonged cultivation of the myogenic cells in the absence of collagen, the experiments thus far indicate that coating the dishes with collagen is not necessary for the maintenance of differentiation capacity by the muscle precursor cells. Thus, although collagen undoubtedly has a very distinct effect on the promotion of both growth and differentiation of cells of myogenic lines, it does not seem to act in this system as a specific inducer for muscle differentiation. It appears that collagen enhances in a non-specific way growth and expression of differentiation potentialities of predetermined myoblasts. Promoting effects of collagen were also observed in other differentiating cell systems such as liver (Ehrman and Gey, 1956), pigment, and cartilage cells (Yaffe, unpublished). In cloning experiments (including those with collagen-treated plates) the source of serum was also observed to have an important effect on growth and differentiation of the clones. Sera obtained from different horses vaned considerably in their effect on both plating efficiency and differentiation. There was no obvious relationship between these two

54

DAVID YAFFE

properties; some sera supported growth and differentiation equally well, whereas others lent better support to one or the other. Coon and Cahn (1966) recently reported the fractionation of embryo extract into fractions which differed qualitatively in their effect on the promotion of growth and differentiation in clones of pigment- and cartilage-forming cells. It is also of obvious interest to try to isolate factors responsible for the effect of serum on muscle-forming cells. One might still contend that factors which induce the differentiation of muscle cells are supplied to the cells by one of the medium components. However, the main point to be emphasized is that under the standard conditions which are widely used at present to grow and clone many cell types, only myogenic cells retain the capacity to manifest the specific synthetic pathways of muscle differentiation. Why is it that descendants of muscle cells which have been maintained in culture for more than 600 cell generations continue to form contracting muscle fibers and do not synthesize, say, melanin or hemoglobin?

F. GROWTH CHARACXFZUSTICSAND PLOIDY OF MYOCENICCELL LINES The fact that cell lines in culture retain high differentiation potentialities does not necessarily imply that these cells resemble myoblasts of primary cultures with respect to other parameters, especially as regards their capability to survive culture conditions. Serial passages and serial clonizations were carried out routinely on all the myogenic lines established and maintained in this laboratory. However, repeated attempts to obtain myogenic cell clones from single cells isolated from primary cultures under identical conditions have so far invariably failed due to cessation of multiplication and degeneration of the cells after very few serial passages. All the lines which were successfully established origa s cultures and not inated from serial passages initiated with primary m from single cells. Also, these lines usually went through a period during which generation time increased and pronounced degenerative phenomena occurred. This generally took place between the fourth and eleventh serial passages. This suggests that the evolution of the line may involve some kind of selection and that starting with a large cell population facilitates this process. Cell lines capable of surviving in vitro for a prolonged time have very often been found to undergo changes in chromosome number (Hsu, 1961). Therefore, the chromosomal constitution of the myogenic lines was investigated (Richler and Yaffe, to be published). Results of such ex riments are summarized in Table 11. In all lines the diploid cells are p"

2.

55

MUSCLE DIFFERENTIATION IN VITRO

predominant, and in three cell lines the chromosome number does not vary significantly from that of the primary cultures. This includes the oldest line, L6, maintained in continuous growth in culture for almost 2 years. The lines MS8 and L29 (both of which manifest high differentiation capacity) were found to have a high proportion of polyploid and heteroploid cells. Mb8 had 30% polyploid cells. After repeated cloning TABLE I1 PLOIDY OF MYOCENICCELLLINES

Cell line

MCa

Time cells niaintained in vitro

Diploid cellsb

3 days 18 months 9 months 8 months 10 months 8 months 7 months

96 92 98 70 81 85 91

Methylcholanthrene treatment during the first two cell passages. Number of cells containing 42 chromosomes out of 100 cells counted in metaphase. c First passage of a diploid clone isolated from MS8. d A subline of L, obtained by isolating a polyploid clone. Chromosome numbers range between 60 and 84. a b

this was reduced to 15% (Table 11) which is still significantly higher than the primary cultures. It would seem that there is some kind of inherent chromosomal instability in this line which results in the continuous production of polyploid cells. La, is a subline of L6 obtained by isolation of a polyploid clone. This line is tetraploid and heteroploid with no diploid cells (60-84 chromosomes). In spite of this cultures of this line differentiate into a network of contracting fibers. Thus, the drastic change which occurred in the number of chromosomes of the cells did not interfere with the retention and manifestation of differentiation potentialities by these cells. It is puzzling why the lines are not entirely converted into polyploid cell populations, especially in the case of MS8which has been shown to produce continuously high proportions of new polyploid cells. This suggests that the polyploid cells are also continuously eliminated from the

56

70 DAVlD YAFFE

.c c 0

primary Cultures

2000 2

a2 600

-a 0

.

5001

\

1500 N h

c

W

I000; .

500

z

I00

0

0 0

5-1

2

4 Days o f t e r ploting

n

b M,,

F

4

-2-

25

5

41

50

Day8 a f t e r plating

FIG. 10 ( a ) Enzymic activity in differentiating priniary rat thigh muscle cell culture. Cultures, prepared as usual, were collected at different stages of differentiation. Extracts were assayed for enzymic activity. Each value represents three assays. Blocks

2.

MUSCLE DIFFERENTIATION I N VITRO

57

population. The possibility that the diploid cells have a selective advantage over polyploids should be considered.

G. ENZYMIC MANIFESTATIONS OF DIFFERENTIATION in Vitro The structural proteins which constitute the contracting apparatus are the major and most specific product of the developing muscle. The most direct approach to the study of the process of differentiation on the level of protein synthesis is to follow the kinetics of formation of these proteins. However, the quantitative manipulation of these proteins on a microscale is very difficult compared with the relative ease of quantitative assay of many enzyme proteins. Experiments were therefore carried out to study the correlation between morphological differentiation and the synthesis of specific enzymes in primary thigh muscle cultures as compared to that in myogenic cell lines, attempting to utilize these enzyme activities as biochemical parameters for the study of the synthetic processes which occur during the differentiation of myogenic cell lines. Three enzymes were explored in this study: ( 1 ) creatine phosphokinase (CPK, EC 2.7.3.2); ( 2 ) myokinase (EC 2.7.4.3;adenylate kinase); and ( 3 ) glycogen phosphorylase ( E C 2.4.1.1). These enzymes are involved in the provision of the energy required for the contraction of muscle fibers, and they appear in relatively large amounts during muscle differentiation in vivo (Cosmos, 1966; Kendrick-Jones and Perry, 1967; Reporter et al., 1963). Creatine phosphokinase has also been found to appear in chick muscle cultures ( Reporter et al., 1963). The results of experiments to assay enzyme activity during the differentiation of rat thigh muscle primary cultures are summarized in Fig. 10a (Shainberg et al., to be published). It can be seen that very low levels of all three enzymes are found during the first 2 days of culture, i.e., before fusion starts. However, at onset of fusion, the activity of the enzymes begins to rise and continues to do so at a more or less constant rate. Between days 3 and 7 CPK increases in specific activity more than tenfold. The activity per plate rises up to day 9 reaching a plateau when are m-units activity per mg protein and curves are m-units activity per plate. ( M K ) myokinase; (CPK) creatine phosphokinase; (Ph) glycogen phosphorylase; ( F ) onset of fusion. The values of phosphorylase activity have been multiplied by 20 for convenience of presentation. ( b and c ) Enzymic activity in differentiating cultures of the myogenic cell lines M,, and L,, respectively. "indicates no measurable amount of phosphorylase.

58

DAVID YAFFE

fusion ceases. Very similar behavior is observed with phosphorylase and myokinase. The rise in activity is linked with the process of fusion. When fusion is experimentally delayed, for example, by seeding the cells at low density or by resuspending the culture before fusion and replating, a parallel delay in the onset of elevated enzyme activity is observed. This conclusion is also supported by cytochemical examination. Cultures stained for phosphorylase activity showed staining of multinucleated fibers but not of mononucleated cells. TABLE I11 OF PROTEIN SYNTHESIS ON ENZYMEACTWITYO EFFECTOF INHIBITION

Time (hours)b

CPK Untreated

CH

Phosphorylase Untreated CH

Myokinase Untreated CH

0 4400 27 250 10 490 450 32 300 250 360 250 37 24 440 22 800 400 45 24 430 260 33 680 0 2.8 pg/ml cycloheximide ( C H ) applied to 4-day-old differentiating primary rat thigh muscle cells in culture. Incorporation of leucine-3H into protein was inhibited by 95%. b After addition of the inhibitor. 0 Expressed as m-units activity per plate (one unit of activity represents the amount of enzyme which causes the formation of 1 pmole NADPH per minute at 30°C). Each value represents an average of three assays. Application of inhibitors of protein synthesis like puromycin or cycloheximide at any stage of development stops further elevation in enzyme activity (Table 111);this suggests that the elevation of activity during differentiation represents de nouo synthesis of enzyme and not just activation of preexisting molecules. The experiment with cycloheximide showed also that all three enzymes are relatively stable. Decrease in enzyme activity is not apparent until degeneration of the cycloheximide-treated cells starts. Essentially similar results were obtained when cultures of the myogenic cell lines were examined (Fig. lob, c). Very low levels of activity of CPK and undetectable activity of the phosphorylase were found in young cultures consisting only of mononucleated myoblasts. However, a constantly increasing rise in activity of all three enzymes took place as soon as fusion started. The ratio between phosphorylase and CPK in these cultures remained constant through most of this process

2.

MUSCLE DIFFERENTIATION IN VITRO

59

and resembled that found in primary cultures. The activity of myokinase in these cultures exceeded that of CPK. Also, relatively higher levels of myokinase were found in cultures before fusion. Cytochemical examination of cultures of the La line stained for phosphorylase at various stages of differentiation showed that only multinucleated fibers stained for enzyme activity (Fig. 11).

FIG.11. Cytochemical demonstration of phosphorylase activity in differentiated culture of the line La. Note localization of staining in mukinucleated fibers.

These experiments, although preliminary in nature, indicate the following conclusions: (1) Quantitative increase in activity of at least two out of the three enzymes starts at fusion and takes place in the multinucleated cells. ( 2 ) A similar pattern of enzymic differentiation takes place during the differentiation of cultures of myogenic cell lines after prolonged cultivation in uitro. These results encourage the application of this methodological approach to the study of the regulation of macromolecular synthesis and turnover during differentiation.

60

DAVID YAFFE

IV. Induction of DNA Synthesis and Mitosis in Nuclei within Muscle Fibers

Cessation of DNA synthesis during differentiation is common to many differentiating cell types (e.g., epidermis, erythropoiesis, neural cells). In some systems, this phenomenon is obviously associated with degenerative changes in the nucleus ( epidermis and mammalian red blood cells). It has been pointed out that the formation of multinucleated muscle fibers is associated with a distinct change in the metabolism of DNA. While mononucleated myoblasts synthesize DNA and divide, incorporation of DNA precursors or mitotic figures have not been observed within multinucleated cells, even during the earliest stages of fusion of myoblasts. In fact, it has been shown that myoblasts stop DNA synthesis at least 6-8 hours preceding fusion (Okazaki and- Holtzer, 1966). Autoradiographic experiments have shown also that the formation of multinucleated cells is associated with a pronounced decrease in thc incorporation of labeled RNA precursors (Yaffe and Fuchs, 1867). Experiments were performed to test whether the cessation of DNA synthesis at fusion is irreversible and associated with a change in the structural integrity of the genetic material (Yaffe and Gershon, 1967a,b). Can nuclei within muscle fibers resume DNA synthesis and undergo mitosis? Since oncogenic viruses have been shown to stimulate DNA synthesis in a variety of experimental systems the effect of the oncogenic virus polyonia (PV) on the differentiation of rat muscle cells was studied. Cells of this species are particularly suitable for such a study since rat cells have been shown to undergo malignant transformation by PV without the synthesis of infectious virus particles. It was therefore assumed that rat muscle cells would not sipport PV multiplication and thus facilitate observation of host DNA synthesis, avoiding the complication of cell lysis. Differentiating primary rat thigh muscle cultures were infected with PV at the onset of fusion. In order to measure DNA synthesis in differentiated fibers, duplicate samples of infected and noninfected culture: were exposed to thyn~idine-~H for 6 hours at various times after the introduction of the virus. The cultures were then processed for autoradiography and stained for detailed cytological examination. DNA synthesis was not observed in multinucleated fibers exposed to virus for less than 30 hours, as judged by lack of incorporation of the tritium-labeled thymidine into their nuclei. However, after 30 hours following infection an increasing proportion of the fibers was found to

2.

MUSCLE DIFFERENTIATION IN VITRO

61

synthesize DNA. After 40 hours as many as 15% of the fibers had labeled nuclei. Usually when DNA synthesis was observed in a fiber, all of the nuclei were found to have incorporated the labeled precursor; however, some fibers were found in which only a fraction of the nuclei was labeled. In the latter case the labeled nuclei were clustered in one section of the fiber.

FIG. 12. Early morphological changes caused by PV infection. Cultures in the process of fusion were infected with PV and fixed 50 hours later. Affected fibers are characterized by the presence of swollen, lightly stained nuclei.

Mitotic figures began to appear in multinucleated fibers about 50 hours after infection. After 70 hours about 3% of the fibers contained nuclei in various stages of mitosis. In many such fibers advanced metaphases could be observed at one end of a group of dividing nuclei; distal to these nuclei progressively earlier stages of mitosis were observed which terminated with early prophases and resting nuclei. In other fibers all the nuclei undergoing mitosis were found to be in metaphase (Figs. 12 and 13). In most cases it was difficult to ascertain whether the chromosomes originated from one nucleus or from several nuclei. However, when this was possible the mitotic figures were found to be diploid.

2.

MUSCLE DIFFERENTIATION IN VITRO

63

Division of nuclei within the fibers did not proceed beyond late metaphase. Instead, the fusion of several metaphase figures into large clumps of chromosomes was often observed (Fig. 14). This phenomenon was frequently associated with rounding of the fibers and loss of their

FIG. 14. Late metaphase in a multinucleated fiber. Note the absence of mitotic spindles and clumping of chromosomes from many nuclei; fixed at 72 hours after PV infection. (From Yaffe and Cershon, 1967b.)

elongated form. In cultures fixed 90 hours after infection or later, fibers and rounded cells containing huge abnormal nuclei were frequently found. Judging from the sequence of events these were most probably FIG. 13. Mitosis in a multinucleated muscle fiber. All nuclei are in prometaphase;

86 hours after PV infection. (From Yaffe and Gershon, 1967a.)

64

DAVID YAFFE

the result of the clumping of the dividing nuclei observed in earlier stages. Infectivity tests and methylated albumin kieselguhr (MAK) column chromatography of DNA extracted from infected cultures did not reveal any detectable production of infective particles or synthesis of polyoma DNA in the cultures. The results thus suggest that the fusion of myoblasts is not necessarily associated with an irreversible block of DNA synthesis, and that nuclei within the fibers retain their structural integrity and can be induced to synthesize DNA and undergo mitosis. The fusion of dividing nuclei into abnormal clumps of chromosomes may not be due to aberrations in the chromosomes but rather to the unusual situation of the close proximity of many mitotic apparatuses within the same cytoplasm. Fusion of mitotic figures to form giant nuclei was observed in other cell systems where several nuclei were dividing in a common cytoplasm (Harris et al., 1966). There is also a possibility of interference of the contractile proteins of the specialized muscle cell with the formation of mitotic spindles. Often, chromosomes were found to be arranged in long parallel rows similar to the myofibril arrangement. We have seen that there was no incorporation of th~midine-~H into muscle fibers when exposure to the label was made during the first 30 hours postinfection, while exposure of the cultures at later stages did result in the incorporation of the label into nuclei within fibers. This indicates that the introduction of the virus had induced resting nuclei to resume DNA synthesis. It therefore appears that the effect of PV infection is an induction of DNA synthesis in cells already in a postmitotic state rather than the delay or prevention of the establishment of the mitotic block. However, a conclusion that multinucleated muscle cells are sensitive to direct infection by polyoma might be misleading. At the time of infection the cultures were in the stage of cell fusion, i.e., myoblasts were still joining the multinucleated fibers. It was found that the number of fibers induced by PV to synthesize DNA dropped considerably if infection was delayed by a few days, and almost no response could be found if infection was delayed until fusion ceased. This suggests that the cells susceptible to PV infection are mononucleated myoblasts and that infection of the multinucleated fibers takes place by the subsequent fusion of infected myoblasts into the growing multinucleated fiber. Since mononucleated myoblasts can multiply this assumption is in accordance with the supposition that transformation of cells by oncogenic viruses requires cell division (Todaro and Green, 1966).

2.

MUSCLE DIFFERENTIATION IN VITRO

65

In most of the fibers which were induced to synthesize DNA all the nuclei were synthesizing DNA or undergoing mitosis. The probability that all the myoblasts which participated in the formation of these fibers were infected by PV is very low. This phenomenon leads one, therefore, to assume that the incorporation of a few PV-infected myoblasts into a growing fiber induces DNA synthesis and mitosis in other nuclei present

FIG. 15. Autoradiographic demonstration of incorporation of thymidine-3H into multinucleated fibers formed in cultures prepared from a 1:1 mixture of PV-infected rat myoblasts and untreated bovine embryo myoblasts. All nuclei are labeled.

in the same fiber. This conclusion is strongly supported by the following experiment carried out recently (Yaffe and Gershon, unpublished). One-day-old rat myoblast cultures were infected with PV before the onset of fusion. Twelve hours later the cells were suspended, washed several times, and mixed with myoblasts obtained from bovine embryo thigh muscle. The mixed population was plated in the usual manner and 48-72 hours later, when hybrid fibers had been formed, the cultures were exposed to a 6-hour pulse of th~midine-~H and fixed for autoradiography. It was found that although muscle fibers in cultures of bovine muscle cells alone were not affected by PV infection, in the cultures wntaining both types of myoblasts huge hybrid fibers containing hun-

66

DAVID YAFFE

dreds of labeled nuclei were found (Fig. 15). One day later inany mitotic figures could be observed within such fibers. Unfortunately the large number and great proximity of the mitotic figures within the fibers did not permit conclusive karyotypic examination (Figs. 16 and 17). However, in the autoradiographs many labeled nuclei containing several

FIG. 16. Culture mnditions identical to Fig. 15. Note gradient of progressive stages of mitosis.

nucleoli-very typical of bovine nuclei-could be observed ( Fig. 18). These experiments suggest that the presence of PV-affected rat nuclei within the hybrid fibers resulted in the induction of DNA synthesis and mitosis in the nuclei of bovine origin. Recently, Lee et al. (1988) reported that DNA synthesis in chick muscle fibers could be induced by Row sarcoma virus (RSV) which is an RNA-containing virus. These authors showed that when muscleforming colonies containing myoblasts and differentiating fibers were infected with RSV, fibers incorporating th~midine-~H could be observed 20 hours later. Similar to our observations with PV in many affected fibers all the nuclei were synthesizing DNA. No mitotic figures within

2.

MUSCLE DIFFERENTIATION IN VITRO

67

the fibers were found in these experiments. Unlike the experiments with PV-infected rat muscle cells, fluorescent antibody techniques showed the production of RSV coat antigen in the chick multinucleated muscle cells. Since the effects of RSV on the chick muscle fibers were observed after 20 hours or longer postinfection, the question of whether the RSV af-

FIG.17. As Fig. 16. Hypotonic treatment ruptured the fibers but chromosomal details are accentuated. Most nuclei are in metaphase or late prophase.

fected muscle fiber directly or via the fusion of mononucleated myoblasts remains open (Ebert and Kaighn, 1966). Fogel and Defendi (1967) applied fluorescent antibody techniques to study the appearance of T antigen in PV-infected hamster and rat thigh muscle cultures. This antigen accompanies transformation of cells by PV (but is not related to the production of virus particles ) . Their results are very reminiscent of our results in three main points: ( 1 ) infection of young differentiating cultures resulted in the appearance of T antigen within multinucleated cells in which either large groups of adjacent nuclei or aZZ the nuclei contained T antigen; ( 2 ) the production of viral antigen (which is an indication of virus production) was very rarely

FIG. 18. As in Fig. 15. The light labeling in this branch of the fiber allows the identification of labeled nuclei containing many nucleoli which are assumed to be of calf origin ( arrow )

.

2.

MUSCLE DIFFERENTIATION IN VITRO

69

found in cultures of these species; (3) when cultures of fully formed myotubes were infected by PV no effect could be observed on the fibers whatsoever. Similar results were obtained with SV40-infected human muscle cells. The results thus show that the muscle fiber reacts to oncogenic virus infection as one functional unit. Any explanation of the mechanism by which several PV-infected myoblasts induce the resumption of DNA synthesis and mitosis in other nuclei would at the moment be highly speculative. In experiments on artificial cell fusion with the aid of killed Sendai virus (Harris et al., 1966) cells which synthesize DNA or RNA were hybridized with differentiated cells which usually do not synthesize DNA or RNA. It was found that in all cases the capacity to synthesize the macromolecules was dominant and induced synthesis in the resting nuclei. However, this does not show unequivocally that synthesis of nucleic acid is under positive genetic control. In these experiments the fused cells did not multiply and therefore the nuclei of these cells resided in the original cytoplasm of the donor cells. Under these circumstances one cannot exclude the possibility that genetic control of the synthesis of nucleic acids was negative (i.e., when the regulating genes which control the activity of the structural genes for nucleic acid synthesis are active, the latter are repressed); but in this specific situation the parent cells which synthesized nucleic acids before fusion introduced into the common cytoplasm the enzymes necessary to initiate nucleic acid synthesis ( or residual mRNA which coded for these enzymes). Similarly, when myoblasts affected by PV fuse into a multinucleated muscle fiber DNA synthesis may be "imposed" on the other nuclei in the fiber by factors brought in as a result of fusion with affected myoblasts or by the continued production of such factors (i.e., mRNA for the involved proteins) by the PV-transformed nuclei which were no longer responsive to the mechanism that inhibits DNA synthesis at fusion. It should be mentioned that in both the PV and the RSV systems it was observed that, for the most part, the extent of labeling of individual nuclei within the myotubes was lower than that of nuclei in mononucleated cells. This may imply that DNA synthesis proceeds more slowly in the former cells because of a lower level of DNA synthesizing enzymes. This would be expected if the information for these enzymes is produced only by a small number of nuclei derived from PV-infected myoblasts. Synthesis of viral DNA was not detected in the PV-infected cultures; however, the production of minute amounts of viral DNA which are transmitted intracellularly from PV-infected to uninfected nuclei would not be detectable with the methods employed. Therefore, such an ex-

70

DAVID YAFFE

planation for the almost all-or-none response of muscle fiber, although unlikely, cannot be excluded. Experiments with a different cell system may have some relevance to this question. When SV4O-transformed cells containing T antigen were fused artificially with nontransformed ones, the T antigen appeared also in the nuclei originating from the nontransformed donors. Steplewski et al. (1W) have recently shown that the appearance of T antigen in these nuclei, originating from the nontransformed donor, takes place in the presence of 5-fluorodeoxyuridine, which suppresses DNA synthesis. However, it is dependent on new RNA and protein synthesis. Without the biological function of the T antigen being known, this experiment shows that the synthesis or acquisition by noninfected nuclei of a new protein is induced by the presence of a transformed nucleus, and that this may take place without the production of viral DNA. The resumption of DNA synthesis and mitosis of nuclei within multinucleated fibers has been induced by an oncogenic virus. However, since it has been demonstrated that this activity is not irreversibly blocked in these cells, the possibility that nuclei within muscle fibers can, under nonmalignant but specific physiological conditions, give rise to dividing cells should be considered. The repeated claims that during muscle regeneration in duo mononucleated cells are formed by fragmentation of injured multinucleated muscle fibers should be mentioned here (for reviews see Field, 1961; Betz et al., 1966). The property of myoblasts to fuse into multinucleated cells and the characteristic cessation of DNA synthesis at fusion may offer new experimental approaches to further exploration of the interrelations between malignant and normal cells and throw light on some open questions in carcinogenesis. V. Comments

A main outcome of the experiments reviewed here is the establishment of myogenic cell lines which are able to multiply for very extended periods (apparently indefinitely) in culture and retain their capacity to differentiate. It is presently not clear why some of the attempts to establish myogenic lines resulted in the loss of the cells by degeneration and cessation of multiplication whereas others were successful. This experience is common for many kinds of mammalian or avian cells serially passaged in vitro (Hayflick, 1965; Kuroki et al., 1967; Lithner and Ponten, 1966; Sanford, 1965). In such experiments the cells were either

2.

MUSCLE DIFFERENTIATION IN VITRO

71

lost altogether or a line was established by the appearance of a new type of cell population which differed in its growth characteristics from the original one. The establishment of myogenic cell lines is of special interest with relevance to this phenomenon since it shows that the changes which take place in the growth characteristics during establishment of cell lines may be distinct from the differentiation characteristics of these lines. Although these lines differ in their capacity for growth from populations of freshly isolated myoblasts they nevertheless preserve very similar differentiation properties. Since we do not know the exact nature of the differences between the cell lines and the primary cultures the possibility exists that some of the characteristics that have been studied in the cell lines reflect qualitative or quantitative properties specific to these established lines rather than to normal differentiation; however, the availability of many parameters unique to the differentiation of muscle cells minimizes the likelihood of this happening. The possibility of cloning and thus performing analyses and experiments on homogeneous populations of one kind of cell makes this system valuable and versatile for studying many aspects of cell differentiation. Differentiated primary cultures always contain, in addition to the network of contracting fibers, a population of mononucleated cells which do not participate in fusion. This was attributed to the presence of nonmyogenic cells in the initial cell population derived from the embryonic muscle tissue. However, even in pure populations of cloned muscle cells in which all the cells were supposed to be myoblasts, almost never did all the mononucleated cells become incorporated into fibers. This is clearly seen in the myogenic cell lines; differentiated cultures maintained for weeks always contained mononucleated cells. When these cells were trypsinized and seeded under cloning conditions all the clones formed muscle fibers and no difference could be observed between clones obtained from these residual mononucleated cells and cells obtained from young cultures trypsinized before fiber formation. Even repeated cloning of mononucleated cells from fully differentiated clones did not result in any selection of cells of lower capacity for differentiation. These experiments suggest that an equilibrium exists in these cultures between mononucleated myoblasts and the differentiated fibers. The relative proportion between the amount of mononucleated and multinucleated cells is variable and is influenced by culture conditions. Also, under identical culture conditions, clones produced by different cell lines differ considerably in their mononucleated cell content. These observations may be relevant to a mechanism which regulates

72

DAVID YAFFE

cell populations in viuo; in most tissues there exists throughout adult life a dynamic equilibrium between proliferation of precursor cells and differentiation. This is most clearly exemplified in erythropoietic and epidermal tissue in which the differentiated cells are dead ends which do not divide and proceed into progressive stages of degeneration. However, the tissue is maintained by a continuous regulated multiplication of a stock of precursor cells (stem cells) and production of new differentiating cells. Adult muscle tissue consists predominantly of highly differentiated multinucleated fibers with very few mononucleated cells of unidentified nature. No quantitative replacement of muscle fiber takes place; however, damaged muscle can repair by regeneration and production of new muscle tissue. The cellular origin of this regenerated tissuc is not yet clearly understood. However, most investigations support the notion that mononucleated cells which accompany muscle fibers do multiply and fuse into new fibers (Betz et al., 1966). The observation that mononucleated myoblasts and differentiated fibers are always present together in cultures of myogenic cells suggests that fundamentally a similar situation exists in viuo-some of the mononucleated cells which accompany muscle fiber are indeed muscle precursor cells which participate in replacement and regeneration of muscle tissue. Thus, muscle may not differ qualitatively from tissues which are continuously renewed, such as skin and erythropoietic tissue, but does differ quantitatively in the lifespan of the differentiated cells and in the rate of their replacement. How do precursor cells which multiply continuously retain their capacity to differentiate? The experimental evidence emphasizes the stability of the retention of this capacity. Myogenic cells have been maintained as pure populations in culture for more than 2 years without losing their capacity for differentiation which clearly demonstrates that this capacity is an inherent property, reproduced continuously within these cells and independent of interaction with other cell types, There is no antagonism between cell replication in culture and retention of differentiation potentialities. The mechanism by which the capacity for differentiation is reproduced must be very stable; this is indicated by the fact that regardless of a variety of culture conditions and continuous multiplication, 100% of clones formed by plating single cells differentiated into muscle-forming colonies. In essence, similar results were obtained in experiments with cartilage cells and retina pigment cells (Cahn and Cahn, 1966; Coon, 1966). Experiments on the retention of differentiation capacity in uitro in these two types of cells were made during

2.

MUSCLE DIFFERENTIATION IN VITRO

73

the first passages in tissue culture, i.e., on cells which presumably have not changed or undergone selection in uitro. This shows that retention of differentiation potentialities in vitro in lines of myoblasts undergoing continuous multiplication is not a property which has been fixed during the establishment of such lines but rather a phenomenon of general significance. Since the differentiated multinucleated muscle cells did not divide, they were continuously selected out and the line was maintained only by the precursor cells which did not differentiate beyond the myoblastic level. Thus, the ability to transmit differentiation potentialities to progeny cells is altogether independent of the expression of the traits. The property which is preserved during multiplication of the determined cells is not the differentiated trait itself but the capacity to express this pattern under the right conditions. The cells differ from other cells in their pattern of response to the environment and to signals from other cells. The actual expression of differentiation is flexible and adaptive. Most reported cases of “dedifferentiation” refer, in fact, to the disappearance of manifestations of the differentiation traits (“modulation” according to P. Weiss) but not to a change in the intrinsic capacity of the cells. When cartilage or retina pigment cells, for example, are grown in culture they soon lose their tissue-specific characteristics and acquire a fibroblastic morphology. However, even after many cell divisions these cells still retain a capacity to resume their morphological and biochemical differentiation. As soon as the proper growth conditions are supplied, under identical conditions, fibroblast cells of cartilage origin will form cartilage, and cells of retina pigment origin will become epithelial and pigmented (Cahn and Cahn, 1966; Coon, 1966). Therefore, what condition is perpetuated in precursor cells which preserves their capacity to differentiate? What is the state of activity of the genes which code for muscle-specific proteins? Are they repressed in the same way as other genes which do not participate in differentiation or are they subject to another type of control, tuned for the specific signals which appear at fusion? Can it be that the muscle-specifk genes are active in these cells and produce their mRNA, but the timing of the synthesis of muscle-specific proteins is regulated at the translational level? In many differentiated cell types proteins have been shown to be produced on stable mRNA (De Bellis et al., 1964; Reeder and Bell, 1965; Wilt, 1965; Humphreys et al., 1964; Kafatos and Reich, 1968). The low incorporation of RNA precursors into multinucleated muscle cells and the

74

DAVID YAFFE

relative resistance of muscle cells to the toxic effect of actinomycin D suggests also the existence of stable mRNA in these cells (Yaffe and Feldman, 1964; Yaffe and Fuchs, 1967). At first sight it is tempting to attribute stability of differentiation to the presence in the cell of stablc macromolecules which were formed at earlier stages. However, this cannot apply to the retention of differentiation capacity in continuously multiplying cells, since preformed macromolecules would be diluted out during replication. Furthermore, in most systems, it has been found that stable mRNA appears only in the late stages of differentiation. The existence of stable templates at these stages has an obvious economic advantage for cells which continuously produce large amounts of few proteins. It seems, therefore, more likely that the stable forms of mRNA described in these experiments play a role in the regulation and stabilization of the expression of differentiation traits rather than in the retention of the capacity to differentiate. However, it is also easy to visualize that stable informational macromolecules may participate as components of a complex, self-perpetuating, dynamic system and play a role in stabilizing it during cell division, temporal inhibition of macromolecule synthesis, etc. Jacob and Monod (1963) suggested a model of stable circuits which can maintain specific genes in a continuous state of activity or repression as a modification of the model generally accepted for the regulation of synthesis of adaptive enzymes in bacteria. Such models are appealing for their simplicity and analogy to well-studied regulation mechanisms in microorganisms. However, the greater complexity of phenomena and structures in cells of multicellular organisms as compared to the bacteria suggests also the possibility of new regulation mechanisms which may replace or amplify the regulatory systems suggested by Jacob and Monod. At present too little is known of the molecular events to enable the construction of a model based on experimental data which would explain the stability of cell differentiation. A number of observations may be mentioned here to illustrate the existence of a large variety of mechanisms which may control specific changes in the informational content of a cell: ( a ) the ability of specific genes to replicate and form extra copies. This has been demonstrated to occur in the genes which code for ribosomalRNA in amphibians (Brown and Dawid, 1968); ( b ) the establishment during embryogenesis of replicatioiial or transcriptional differences between chromosomes. A regular loss or inactivation of specific chromosomes during the embryogenesis of some invertebrates was observed at the morphological level many years ago. The phenomenon of inherited

2.

MUSCLE DIFFERENTIATION IN VITRO

75

differences in expression of genetic activity between the two X chromosomes within clones of mammalian somatic cells (De Mars, 1967) has already been pointed out as a possible model for studies of cellular differentiation ( Lederberg, 1966); ( c ) the demonstrated ability of RNA molecules to control their own replication. Thus, replication of RNA viruses is accomplished by the synthesis of a specific replicase. This replicase is coded by the viral RNA and replicates specifically only the viral RNA (Spiegelman and Haruna, 1966). It is not meant to suggest that these phenomena as such play a general role in differentiation. However, their existence even in special situations should be taken into account since an understanding of their underlying mechanisms may give some insight into processes which take place regularly in differentiation. The capability of isolated cells to retain and manifest in uitro their capacity to differentiate, and the possibility to analyze the inheritance of these properties by single cells, make the possible relevance of these phenomena to the mechanism of differentiation more amenable to experimental analysis. The stable retention of the capacity to differentiate demonstrated by cells maintained in uitro in a state of continuous replication and the clear distinction in several experimental systems between the retention of this capacity and its actual expression indicate some of the requirements for a working hypothesis. ACKNOWLEDGMENTS Th'anks are due to Professor M. Feldrnan for his interest and helpful discussions. The participation of Dr. D. Gershon, Dr. G. Yagil, MI. A. Shainberg, and Miss C. Richler in the experiments is gratefully acknowledged, as is the excellent technical assistance of Mrs. S. Neuman, Miss M. Debby, Mr. E. Mor, and Mr. M. Gabbai, and the photographic assistance of Mr. E. Thum. The studies reported in this review were supported in part by the DBlhgation GnCrale B la Recherche Scientifique et Technique, France, and by Grant DRG 1007 from the Damon Runyon Memorial Fund for Cancer Research. REFERENCES Altschul, R. (1962). 2.Zellforsch. Mikroskop. A w t . 56, 425. Bassleer, R., Colignon, P., and Matague-Dhossche, F. (1963). Arch. Biol. (Liege) 74, 79. Benvald, Y., and Sachs, L. (1965). 1. Natl. Cancer Inst. 35, 641. Betz, F. H., Firket, H., and Reznik, M. (1966). Intern. Reu. Cytol. 19, 203. Brown, D. D., and Dawid, I. B. (1968). Science 160, 272. Cahn, R. D., and Cahn, M. B. (1966). Proc. Natl. Acad. Sci. US. 55, 106 Capers, C. R. (1960). I. Biophys. Biochem. Cytol. 7 , 559. Coon, H. G. ( 1966). Proc. Natl. Acad. Sci. U S . 55, 66. Coon, H. G., and Cahn, R. D. (1966). Science 153, 1116.

76

DAVID YAFFE

Cosmos, E. (1966). Deuelop. Biol. 13, 163. Davidson, E. H. (1964). Adoan. Genet. 12, 143. De Bellis, R. H., Gluck, N., and Marks, P. A. (1964). 1. Clin. Inoest. 43, 1329. De Mars, R. (1967). Nutl. Cancer Inst. Monogr. 26, 327. Eagle, H. ( 1965). Science 148, 42. Earle, W. R., and Nettleship, A. (1943). J. Natl. Cancer Inst. 4, 213. Ebert, J. D., and Kaighn, M. E. (1966). In “Major Problems in Developmental Biology” (M. Locke, ed.), p. 29. Academic Press, New York. Ehrman, R. L., and Gey, G. 0. (1956). J. Natl. Cancer Inst. 16, 1375. Field, E. J. (1961). In “Structure and Function of Muscle” (G. H. Bourne, ed.), Vol. 3, p. 139. Academic Press, New York. Fogel, M., and Defendi, V . ( 1967). Proc. Natl. Acad. Sci. U.S.58, 967. Hams, H., Watkins, J, F., Ford, C . E., and Schoefl, G. I. (1966). J. Cell Sci. 1, 1. Hauschka, S. D., and Konigsberg, I. R. (1966). Proc. Natl. Acad. Sci. U S . 55, 119. Hayflick, L. (1965). Exptl. Cell Res. 37, 614. Holtfreter, J. (1947). J. Morphol. 80, 25. Hsu, T. C. (1961). Intern. Rev. Cytol. 12, 64. Humphreys, T., Penman, S., and Bell, E. (1964). Biochern. Biophys. Res. Cornmrrti. 17, 618. Jacob, F., and Monod, J. ( 1963). In “Cytodifferentiation and Macromolecular Synthesis” (M. Locke, ed.), p. 30. Academic Press, New York. Kafatos, F. C., and Reich, 3. (1968). Proc. Natl. Acad. Sci. US. 60, 1458. Kendrick-Jones, J., and Perry, S. V. (1967). Biochem. J. 103, 207. Konigsberg, I. R. (1961). Proc. Natl. Acad. Sci. U.S. 47, 1868. Konigsberg, I. R. ( 1963). Science 140, 1273. Konigsberg, I. R., McElvain, N., Tootle, M., and Hemnann, H. (1960). J. Biophys. Biochem. Cytol. 8, 333. Konigsberg, I. R., and Hauschka, S. D. (1965). In “Reproduction: Molecular, Subcellular and Cellular” ( M . Locke, ed.), p. 243. Academic Press, New York. Kuroki, T., Gob, M., and Sato, H. (1967). Tohoku J. Exptl. Med. 91, 109. Lake, N. C. (1915).J. Physiol. (London) 50, 364. Lederberg, J. (1966). Current Topics Deoelop. Biol. 1, 9. Lee, H. H., Kaighn, M. E., and Ebert, 1. D. (1968). Intern. J. Cancer 3, 126. Lewis, M. R. (1915). Am. J. Physiol. 38, 153. Lewis, W. H., and Lewis, M. R. (1917). Am. J. Anat. 22, 169. Lithner, F., and Ponten, J. (1966). Intern. 1. Cancer 1, 579. Mintz, B., and Baker, W. W. (1967). Proc. Natl. Acad. Sci. U S . 58, 593. Moscona, A. (1952). Exptl. Cell Res. 3, 535. Moscona, A. (1956). Proc. SOC. Exptl. Biol. Med. 92, 410. Moscona, A. (1957). Proc. Natl. Acad. Sci. US.43, 184. Murray, M. R. (1960). In “The Structure and Function of Muscle” (G. H. Bourne, ed.), Vol. 1, pp. 111-136. Academic Press, New York. Okazaki, K., and Holtzer, H. (1966). Proc. Natl. Acad. Sci. U.S. 56, 1484. Reeder, R., and Bell, E. (1965). Science 150, 71. Reporter, M. C., Konigsberg, I. R., and Strehler, B. L. (1963). Exptl. Cell Re.s. 30, 410. Sanford, K. (1965). Intern. Reu. Cytol. 18, 249. Sato, G. H., and Yasumura, Y. (1966). Trans. N.Y. Acad. Sci. 28, 1063.

2.

MUSCLE DIFFERENTIATION M VITRO

77

Shainberg, A., Yagil, G., and Yaffe, D. To be published. Spiegelman, S., and Haruna, I. (1966). Proc. Natl. Acad. Sci. U.S.55, 1539. Steplewski, Z., Knowles, B. B., and Koprowski, H. (1968). Proc. Natl. Acad. Sci. U.S. 59, 769. Stockdale, F. E., and Holtzer, H. (1961). Erptl. Cell Res. 24, 508. Todaro, G. J., and Green, H. (1966). Proc. Natl. Acad. Sci. U.S.55, 302. Wilt, F. H. (1965). J. Mol. Biol. 12, 331. Yaffe, D. (1968). Proc. Natl. Acad. Sci. US. 61, 477. Yaffe, D., and Feldman, M. (1964). Deoelop. Biol. 9, 347. Yaffe, D., and Feldnian, M. (1965). Deoelop. Biol. 11, 300. Yaffe, D., and Fuchs, S. (1967). Develop. B i d . 15, 33. Yaffe, D., and Gershon, D. (1967a). Israel J. Med. Sci. 3, 329. Yaffe, D., and Gershon, D. (196713). Nature 215, 421. Yaffe, D., and Gershon, D. Unpublished.

This Page Intentionally Left Blank

CHAPTER 3

MACROMOLECULAR BIOSYNTHESIS IN ANIMAL

CELLS INFECTED WITH CYTOLYTIC VIRUSES Bernard Roixmun and Patricia G. Spear DEPARTMENT OF MICROBIOLOGY,

THE UNIVERSITY OF CHICAGO, CHICAGO, ILLINOIS

I. Introduction .................................... A. Objectives .................................. B. Historical Background ........................ 11. The Inhibition of Host Macromolecular Synthesis . . . . . A. Experimental Designs ......................... B. The Inhibition of Host Macromolecular Synthesis . . C. The Inhibitor of Host Macromolecular Synthesis ( IHMS) .................................... D. The Mechanisms of Inhibition of Host Macromolecular Synthesis ................................ 111. Why Is Host Macromolecular Synthesis Inhibited by Viruses? ........................................ A. The Source of Genetic Information for the Inhibition of Host Macromolecular Synthesis .............. B, Evolution of the Inhibition of Host Macromolecular Synthesis ................................... C. Evolutionary Basis for the Development of IHMS References ......................................

1.

79 79 81 82 82 84

92 98

102 102 103 103 105

Introduction

A. OBJECTIVES This review deals with the effects of cytolytic viruses on macromolecular biosynthesis of animal cells. One of the earliest manifestations of a 79

80

BERNARD ROIZMAN AND PATFUCIA G. SPEAR

productive infection of animal cells with many viruses is a rapid decline and, ultimately, cessation of synthesis of host macromolecules. The inhibition of the host macromolecular synthesis often precedes the synthesis of viral constituents, assembly of viral progeny, and the irreversible loss of the capacity to synthesize any macromolecules, host or viral. The capacity to inhibit host macromolecular synthesis is not dependent on size-viruses capable of ordering the sequence of 2000 amino acids (many picornaviruses) can be as effective as those carrying 25-fold more information ( poxviruses). This capacity can be a property of DNA viruses (papova, adenoviruses, herpesviruses, and poxviruses ), RNA viruses ( picornaviruses, arboviruses, etc.), viruses that multiply solely in the cytoplasm ( poxviruses, picornaviruses, some myxoviruses, etc. ) , and those that multiply both in the cytoplasm and in the nucleus (adenoviruses, herpesviruses, etc. ). The inhibition of host macromolecular synthesis probably results from the specific effect of one or more viral gene products (for heuristic reasons henceforth designated as inhibitors of host macromolecular synthesis or IHMS) on the synthesis of products of host genes. As a model system for the study of gene interactions in animal cells, virus infection has several advantages i.e., ( a ) prior to infection the cell lacks viral genes or their products, ( b) the introduction of viral genes or their products into the cell is amenable to considerable experimentation, and lastly ( c ) it seems probable at this time that in the foreseeable future all of the gene products of small viruses will be characterized and the structural determinants of function clearly understood. It should be pointed out that the inhibition of host macromolecular synthesis is a frequent, but not the only, sequella of infection. Not all viruses have been reported to affect host metabolism, but both abortive and productive infection nearly always cause some phenotypic modification of the cell. Thus, viruses which are unable to inhibit a particular host may induce the production of interferon; in this instance the host temporarily alters its control of the macromolecules it will make. The cells transformed from normal to neoplastic by oncogenic viruses are another example of infected cells in which host metabolism is altered but not inhibited. The basis for the more lasting phenotypic changes in the transformed cell is less clear. However, no change in host function is as clear and readily measurable as the inhibition of host macromolecular synthesis. This review deals exclusively with animal cells and ignores parallel and, perhaps in its own right, equally instructive work on the effects of bacteriophage on bacterial metabolism. The survey of the literature was com-

3.

CYTOLYTIC VIRUSES

81

pleted in November of 1968. Unlike computerized reviews, the reference list is selective and not exhaustive, and we may have too frequently followed the dictum that what offends our reasoning is perhaps not right. B. HISTORICAL BACKGROUND The inquiry into the effects of viral infection on animal cells barely exceeds a decade and, like most fields of scientific inquiry, it did not originate in a vacuum. The background is of interest. The introduction of tissue culture by Enders et al. (1949) for the study of poliomyelitis viruses was one of the most significant developments in virology for the past two decades; we owe much, if not all we know, about animal viruses today to the rapid acceptance of tissue culture as an essential tool of virology. The immediate consequence was, however, the isolation of several hundred viruses, many, from apparently healthy men and animals. The significance of these unsuspected viruses was uncertain. As the title of a conference sponsored by the New York Academy of Sciences (Huebner, 1957) aptly states, these viruses went “in search of disease” with rather variable results. Lwoffs work (1953) on lysogeny in bacteria became widely disseminated at this time. It is not too surprising that during this period viruses were conjured as obligate intracellular parasites causing little inconvenience to the host. The fact that viruses caused destruction of cells in culture was often thought to be a very useful indication of viral infection but an artifact nonetheless. In this vein, cell injury was thought to be a phenomenon unrelated to virus multiplication ( Ackermann et al., 1954, 1958) even though the evidence linking loss of cell integrity with virus release from infected cells was already published (Lwoff et al., 1955) and at least some of the effects of the T phages on bacterial metabolism were also known. The emphasis of much work presented at numerous meetings and symposia (e.g., Walker et al., 1957) was in fact directed to prevent this artifact and to permit the infected cells to survive indefinitely as models of persistent or latent infections of the multicellular hosts. Consorting with this benevolent view of viruses infecting animal cells were reports that at least early in infection the metabolism of the host showed a net increase (Miroff et al., 1957); Ackermann, 1958; Ackermann et al., 1959; Becker et al., 1959) highly suggestive that host and parasite metabolism went hand in hand. The evidence that viruses alter host metabolism emerged in stages. At first, cells infected with one virus were shown incapable of replicating another (Ledinko and Melnick, 1954; Sabin, 1954; Le Bouvier, 1954). Shortly thereafter it was shown that members of poxvirus (Mishmi and

82

BERNARD ROIZMAN AND PATRICIA G. SPEAR

Bernkopf, 1958; Bernkopf et al., 1959; Brown et al., 1959), myxovirus (Henle et al., 1954; Russell and Morgan, 1959), and adenovirus groups (Rowe et al., 1958; Pereira, 1958; Everett and Ginsberg, 1958) could cause rapid loss of cell function and cell death in the absence of multiplication. Lastly, Salzman et a2. (1959) showed that poliovirus infection in HeLa cells caused a marked inhibition of RNA, DNA, and protein synthesis. These findings were soon extended to cells infected with encephalomyocarditis virus (Martin et al., 1981) and mengovirus (Baltimore and Franklin, 1962). II. The Inhibition of Host Macromolecular Synthesis

A. EXPERIMENTALDESIGNS 1. Requirements For the study of viral inhibition of host synthesis the obvious experimental requirements are that the cells be metabolically active, that they become infected, and that manipulation of the cell should not, alone, account for the alteration in cell metabolism. Experimentally the last requirement is the easiest to control. Most of the experiments described in this review were done on samples containing 106-10E cells grown in continuous cultivation. The number and origin of cells was dictated largely for convenience and for reproducibility. To decrease sampling error and for ease of manipulation many investigators have used cultures of cells growing in suspension. An additional advantage is that the cells in suspension cultures are uniform in shape and size. Alas, many interesting cell lines do not grow in suspension. Experimentally, the requirement for metabolically active cells is met by using cultures of cells increasing exponentially in number. The requirement that all cells be infected is usually met by exposing them to sufficiently high concentrations of virus. If the cells are not clumped or growing in multilayered sheets, i.e., if the conditions of infection are such that every cell has an equal opportunity to become infected, then the amounts of virus required to infect all or most cells can be readily calculated assuming a random distribution of virus infecting the cells. Thus, it may be predicted that approximately 98% of the cells become infected when the average number of infectious virus [e.g. plaque-forming units] (pfu) per cell (this ratio is known as multiplicity of infection) reaches four. Some of the special problems, requirements, and techniques of infection may be summarized as follows: 1. Many experimental designs require synchronously infected cells.

3.

CYTOLYTIC VIRUSES

83

The most common technique to obtain synchronously infected cells is to expose the cells to a very large amount of virus for a short time. Penetration of virus into cells can be synchronized by allowing absorption to take place at 47°C ( McCormick and Penman, 1967). Penetration takes place very rapidly and synchronously while the cells are brought back to the temperature of optimal growth (Huang and Wagner, 1964). Drugs such as puromycin or actidione (Penman and Summers, 1965) have also been used for synchronization of infection. 2. The multiplicity of infection frequently plays a determinant role on the metabolic events and outcome of infection. Thus, at high multiplicities of infection, mumps ( Wacker et al., 1962; Henle et al., 1954) and Newcastle disease virus (Prince and Ginsberg, 1957, Mason and Kaufman, 1960a,b; Rott and Muller, 1965) may be so highly toxic that the cells are no longer capable of. reproducing themselves or of synthesizing v i r u s . Cells infected with vesicular stomatitis virus produce relatively little infectious v i r u s at multiplicities greater than 5 pfu per cell (Huang and Wagner, 1965, Wagner and Huang, 1966). Defective v i r u s is produced in cells infected at relatively high multiplicities with influenza (Von Magnus, 1954; Scholtissek et al., 1966), Rift Valley Fever (Mims, 1956), vesicular stomatitis (Cooper and Bellett, 1959), fowl plaque ( Rott and Schafer, 1960), and with SV40 (Uchida et al., 1966) viruses. Some cells produce viral constituents only after infection at very high multiplicities (Aurelian and Roizman, 1965). Two points should be made. First, a multiplicity of 4 pfu per cell does not mean that every cell is infected with 4 pfu. Assuming random distribution of virus among cells it may be predicted in fact that 56% of the cells will be infected with 4 or more pfu per cell. The second point is that virus particles incapable of replication may have a profound effect on host metabolism. The ratio of particles counted by electron microscopy to pfu is very seldom unity; for many standard virus preparations used in biochemical work it frequently exceeds 100. In effect this means that at a multiplicity of 4 pfu per cell the average number of virions per cell may well exceed 400. 3. The purity of virus used for infection may also play a determinant role on the metabolic events and outcome of infection. For example, crude lysates of cells infected with adenoviruses frequently contain a relatively small structural component which is, by itself, a powerful inhibitor of host macromolecular synthesis ( Levine and Ginsberg, 1967). 4. Mycoplasma (designated once as PPLO or pleuropneumonia-like organisms) are a vexing, not infrequent contaminant of animal cell cultures (Robinson et al., 1956). Mycoplasma affect cell growth and metab-

84

BERNARD ROIZMAN AND PATRICIA G. SPEAR

olism (Pollock et al., 1963;Nardone et al., 1965; Fogh and Fogh, 1965). At least one reported effect of a virus on host cell macromolecules (Randall and Walker, 1963) was later found to be due to mycoplasma (Randall et al., 1965). Mycoplasma contamination in cell cultures is readily monitored, but alas, failure to find them is not meaningful. Few of the authors cited in this review indicate whether they monitor their cultures and virus preparations. 2. Measurement of Macromolecular Synthesis

For purposes relevant to this review, the most widely used method of measuring macromolecular synthesis is by following the incorporation of the appropriate radioactive precursors into DNA, RNA, and protein. However, this method has its pitfalls. It is necessary to consider that drastic changes in precursor pool sizes may occur as a result of infection. In particular, cell permeability to low molecular weight precursors may be considerably altered. Therefore, the specific activity of precursors actually available for macromolecular synthesis may vary widely during the course of infection even under the most controlled conditions. Since information concerning pool sizes is difficult to obtain, too many investigators ignore the problem in experimental design. To obtain information about the inhibition of host macromolecular synthesis it is necessary to differentiate between host and viral products. In some virus-cell systems this may be quite simply achieved. A few examples may be mentioned: (1) Some DNA-containing viruses, such as members of the poxvirus group, replicate completely in the cytoplasm. Therefore, the synthesis of host DNA may be followed independently of viral DNA synthesis by fractionating infected cells into nuclear and cytoplasmic fractions prior to analysis. (2) Other viruses, exemplified by hevesviruses, have DNA’s with guanosine-cytosine contents appreciablv different from those of most animal cells allowing the physical separation of host and viral DNA. (3) The replication of several RNA viruses of the picorna, myxovirus, and arbovirus groups is unaffected by levels of actinomycin D which almost completely suppress host RNA synthesis. Therefore, comparisons of actinomycin-treated and -untreated infected cells may yield information concerning the rate of host RNA synthesis.

B. THEINHIJ~ITION OF HOST MACROMOLECULAR SYNTHESIS I . General Description Some examples of the inhibition of host RNA, DNA, and protein synthesis are shown in Figs. 1-3. The descriptive features of the inhibition of host macromolecular synthesis may be summarized as follows:

3.

85

CYTOLYTIC VIRUSES

1. The interval between the time of exposure and the initiation of the inhibition varies considerably depending on the virus, the macromolecule being studied and the host. In L cells infected with mengovirus, for example, the reduction in host RNA and protein synthesis begins immediately and reaches a maximum about 3 hours after infection (Baltimore et al., 1963a). By contrast the inhibition of host RNA and protein synthesis 30

3

.-c

E

0 0

1

2

3

4

5

6

7

8

9

10

I1

12

Time (hr)

FIG. 1. The pattern of incorporation of thyniidine-3H into DNA of HEp-2 cells infected with herpes simplex virus. The cells were pulse-labeled for 15 minutes at different times after infection. Cellular and viral DNA were separated by isopycnic centrifugation in cesium chloride density gradients. (Sydiskis and Roizman, unpublished data. )

in L cells infected with reovirus begins late in infection coincident with the onset of virus maturation (Gomatos and Tamm, 1963; Kudo and Graham, 1965). 2. The rates of inhibition of DNA, RNA, and protein syntheses in infected cells differ depending on the virus and cells. There is no universal pattern and no evidence that the synthesis of one macromolecule is more readily inhibited than that of another. 3. Some viruses inhibit all macromolecular synthesis in infected cells. Others inhibit the synthesis of one or two of the macromolecules only. In general, the inhibition of host macromoIecular synthesis appears to be a function determined largely but not exclusively by the virus (Section II,B,S). Thus, host RNA, DNA, and protein synthesis are inhibited in HeLa cells infected with poliovirus (Zimmerman et al., 1963; Holland

86

BERNARD ROIZMAN AND PATRICIA G. SPEAR

1962, 1963, 1964a) and in HEp-2 cells infected with herpes simplex virus (Roizman and Roane, 1964; Roizman et al., 1965; Aurelian and Roizman, 1965; Sydiskis and Roizman, 1967). Adenovirus-type 2 inhibits the synthesis of monkey kidney cell RNA and protein but not that of DNA 2.0 1.5

I .o

0.4 0.3 0.2 0.I

0

2.0 1.5 I .o

w 0.4 0.3 n 0.2 0 0.1

2.0 I .5

I .o

0.4 0.3 0.2 0.I

3

9

15

21

3

9

15 21

3

9

15 21

Fraction

FIG.2. The patterns of polyribosomes extracted from HEp-2 cells infected with herpes simplex virus. The polyribosomes were extracted immediately after 15 minutes of incubation with 14C-labeled amino acids and centrifuged in 15 to 30% ( w / w ) sucrose density gradients (Sydiskis and Roizman, unpublished data). The fractions were monitored for both optical density at 260 mp (solid line) and for radioactivity of nascent peptides (dashed line). A, uninfected cells; B, C, D, E, F, and G are profiles obtained from cells I, 2, 4, 5.5, 7, and 9 hours after infection, respectively. (Sydiskis and Roizman, unpublished data.)

( Ledinko, 1966). On the other hand, all strains of Newcastle disease virus inhibit efficiently host protein synthesis, but available data indicate that only a few inhibit host RNA synthesis (Wheelock and Tamm, 1961; Scholtissek and Rott, 1965; Wilson, 1968).

2. Sebctidty of Inhibitory Process Since the inhibition of host macromolecular synthesis is seldom complete, the question arises whether the inhibition is selective, i.e., whether

3.

CYTOLYTIC VIRUSES

87

the virus discriminates between the synthesis of one host protein and another or of one RNA molecule and another. Most of the available information on this question concerns host RNA synthesis. Early studies by Fenwick (1963), Homma and Graham ( 1963), and Tobey ( 1964) showed

.-cE

> 4-

c a

0

0

0 0

G

X

Distance ( m m )

FIG. 3. The synthesis of cytoplasmic RNA in HEp-2 cells infected with herpes simplex virus. The infected HEp-2 cells were incubated for 30 minutes in medium containing 10 &/ml uridine-3H and then for an additional 2 hours in medium containing 10-4 M unlabeled uridine. The cytoplasmic RNA from the infected cultures and an uninfected control culture ( 0 hour) was extracted with phenol and electrophoresed on a 10 cm polyacrylamide gel consisting of two 5 cm segments 2.5% ( A ) and 5.5% ( B ) , respectively. The gels were monitored for optical density at 260 mp (solid line) and radioactivity (dashed line). (Wagner and Roizman, 1969.)

that in cells infected with picornaviruses messenger, ribosomal, and transfer RNA are inhibited to approximately the same extent. More recent studies by Darnel1 et al. (1967) showed that in HeLa cells infected with poliovirus the synthesis of ribosomal precursor RNA was inhibited more rapidly than that of mRNA. They showed moreover that ( a ) the synthesis of 45 S ribosomal RNA precursor and its conversion in the nucleus to 32 S precursor and 16 S ribosomal RNA were depressed and ( b ) whereas the small ribosomal subunit containing preformed 16 S ribosomal RNA

88

BERNARD ROIZMAN AND PATRICIA G. SPEAR

was assembled and released into the cytoplasm without appreciable delay, the assembly of the large ribosomal subunit containing 28 S ribosomal RNA was blocked. Recent studies on HEp-2 cells infected with herpes simplex virus (Wagner and Roizman, 1969) showed that ribosomal RNA synthesis was inhibited more rapidly and to a greater extent than 4 S RNA. At present it is not clear whether the selectively higher level of 4 S RNA synthesis is due to the synthesis of viral transfer RNA (Subak-Sharpe et al., 1966) or whether it represents selective sparing of synthesis of certain host transfer RNA species. Studies of nuclear RNA synthesis indicated that the syntheses of both 18 S and 28 S RNA were reduced to the same extent. Analysis of ribosomal RNA synthesis in infected cells showed that the 45 S ribosomal precursor RNA was made at a reduced rate and methylated. However, the ribosomal precursor was degraded; it was not processed into ribosomal RNA. In these respects the inhibition of ribosomal RNA synthesis in herpesvirus-infected cells differs from that seen in HeLa cells infected with poliovirus (Darnel1 et al., 1967). 3. The Specificity of the Inhibitory Process

In this review we have defined specificity as the capacity of a virus to differentiate in its inhibition between the synthesis of its own macromolecules and the synthesis of heterologous macromolecules, either of the host or of another virus, regardless of the mechanisms involved. The question arises as to how specific is the inhibition of macromolecular synthesis mediated by viruses infecting animal cells. The capacity of a virus to discriminate between its own and heterologous macromolecular synthesis is difficult to measure since there is no good way of determining to what extent viral synthesis may be affected by its own products. A pragmatic test is to compare viral yields from cells infected at different multiplicities of infection, the assumption being that, if the virus cannot efficiently differentiate between its own and host macromolecular synthesis, increasing the multiplicity of infection should decrease the virus yield. The capacity to discriminate between the synthesis of its own macromolecules and those of another virus is easiest to measure. In general many viruses interfere with the synthesis of another superinfecting virus as evidenced, simply enough, by the inability of the superinfecting virus to multiply. Among the exceptions are related viruses introduced into the cell in close succession (Roizman, 1963; Cords and Holland, 1964) and occasionally a virus which cannot inhibit heterologous macromolecular synthesis, either that of another virus or that of the host (Holmes and

3.

CYTOLYTIC VIRUSES

89

Choppin, 1966). The lack of interference between related viruses argues for a degree of specificity in the inhibitory process. In general the inhibitory processes initiated by the small picornaviruses appear to be among the most specific in that they readily differentiate among homologous and heterologous ( virus and host) macromolecular synthesis. At the other extreme are adenoviruses. Purified structural components of the adenovirion and crude adenovirus preparations (Bello and Ginsberg, 1967; Levine and Ginsberg, 1967, 1968) have an effect on both homologous and heterologous macromolecular synthesis. In consequence, ( a ) host macromolecular synthesis is inhibited, ( b) heterologous viruses are inhibited, and ( c ) under certain conditions even homologous, i.e., viral macromolecular synthesis, is inhibited. Many viruses cannot be readily classified. Into this category fall viruses like SV5 which inhibit macromolecular metabolism only in rare hosts (Holmes and Choppin, 1966) and others like the reoviruses (Gomatos and Tamm, 1963; Kudo and Graham, 1965) which inhibit only very late in infection. Of particular interest are two different virus groups, i.e., a group exemplified by vesicular stomatitis and members of the myxovirus group. These viruses appear to differentiate between viral and host macromolecular synthesis at low multiplicities of infection; at high multiplicities of infection the yield of virus is diminished. Whether the diminished yield of virus is due to impairment of viral macromolecular metabolism or excessive injury to the cell is not clear. The problems are in part best illustrated with vesicular stomatitis in that at high multiplicities of infection the cell yields defective v i r u s which lacks a full complement of RNA and cannot multiply by itself (Huang et al., 1966). The defective particle shares with the infectious v i r u s the capacity to inhibit host macromolecular metabolism and to diminish infectious virus yields from cells infected at high multiplicities. However, the defective particle has the added capacity to interfere even at low multiplicities with the formation of infectious virus (Hackett, 1964; Huang et al., 1966; Wagner and Huang, 1966). The mechanisms of interference at low multiplicities are probably differentfrom the indiscriminant inhibition of both viral and host metabolism at high multiplicities of infection since the former is abolished by UV irradiation of the particles whereas the latter is not (Huang and Wagner, 1960).

4. Inhibition of Synthesis of Host Macromolecules: Interdependence The question has often arisen whether host DNA, RNA, and protein synthesis are each inhibited by a specific gene product or whether the cessation of synthesis of one macromolecule is simply the consequence of

90

BERNARD ROIZMAN AND PATRICIA G. SPEAR

the inhibition of the synthesis of another macromolecule. Systematic studies specifically designed to discriminate between a single versus multiple inhibitory gene products have not been done, and available data bearing on this question are fragmentary. The evidence suggesting that different gene products and possibly entirely different mechanisms of inhibition are involved consists of three observations concerning RNA and protein synthesis, i.e., ( a ) in several cell-virus systems the inhibition of host protein synthesis precedes that of RNA synthesis (Zimmerman et al., 1963; Bablanian et al., 1965, McCormick and Penman, 1967; Brown et al., 1968), ( b ) the rate of inhibition of protein synthesis is more rapid than the estimated decay of host mRNA (half-life 35 hours), and ( c ) in poliovirus-infected cells host polyribosomes disaggregate more rapidly than in cells treated with actinomycin D (Penman et al., 1963; Dalgarno et al., 1967). A priori it would be expected that inhibition of synthesis of one macromolecule should, after a time lag, result in cessation of synthesis of the other two. The interpretation of the evidence cited above hinges on the significance of the difference in the rates of inhibition of various macromolecules caused by virus infection and by defined chemical inhibitors such as actinomycin D. It seems to us that the specific questions we wish to have answered cannot be resolved by comparing rates of inhibition of macromolecules for two reasons. First, in any one cell line the extent to which DNA, RNA, and protein synthesis can continue when one of these macromolecules ceases to be made may hinge on the site where, and the mechanism by which, it is inhibited. Second, our understanding of the “natural” interdependence between DNA, RNA, and protein synthesis in animal cells is limited and based solely on the use of several drugs. These drugs may or may not affect specifically the synthesis of only one macromolecule but certainly we do not have drugs to cover every single possible way by which the synthesis of the macromolecule might be inhibited. The evidence suggesting that only one gene product is responsible for the inhibition of host macromolecular synthesis consists of two observations. (1)Studies on the biologic properties of adenovirus structural components suggest that a single protein constituent may be responsible for the inhibition of host RNA, DNA, and protein synthesis (Levine and Ginsberg, 1967, 1968). (2) As indicated in the next section, the inhibition of host macromolecular synthesis may vary from very rapid to slow or absent depending on the host and conditions of infection. It is of interest to note that when a change in pattern or rate of inhibition occurs it seldom affects just one macromolecule; usually changes in patterns of

3.

CYTOLYTIC VIRUSES

91

inhibition of RNA and protein or of all three macromolecules run in parallel. Thus, mengovirus was found to inhibit rapidly and early in infection the synthesis of both RNA and protein in Novikoff rat hepatoma stain NISI 63 but not in stain NISI 67 (Plagemann and Swim, 1966). The same virus efficiently inhibits RNA and protein synthesis in L cells; in HEp-2 cells the inhibition of both is delayed and reduced ( McCormick and Penman, 1967). Similar parallel variations in the inhibition of RNA and protein synthesis were seen in HeLa, human embryonic lung cells, and ERK cells infected with poliovirus (Bablanian et al., 1965). Lastly, in contrast to that of HEp-2 cells, the inhibition of dog kidney celI DNA, RNA, and protein synthesis was dependent on the multiplicity of infection to approximately the same extent ( Aurelian and Roizman, 1965).

5. Host Dependence of the Inhibitory Process There are several reports which illustrate quite clearly that the occurrence, the time, and the rate of inhibition of host macromolecular synthesis may, in part, be determined by the host. Three examples, all dealing with mengovirus, may be cited as follows: 1. Mengovirus grows equally well in both L and HeLa cells. In L cells the inhibition of host RNA synthesis by mengovirus is rapid and occurs soon after infection ( Franklin and Baltimore, 1962). McCormick and Penman (1967) confirmed these observations on the effect of the virus on L cells and showed that the inhibition of host RNA synthesis in HeLa cells by mengovirus is less severe and takes place late in infection. The rate of inhibition of RNA and protein synthesis in HeLa cells by mengovirus parallels that seen in HeLa cells infected with poliovirus (Zimmerman et al. 1963; McCormick and Penman, 1967). 2. Levy (1964) found that the inhibition of host RNA synthesis in mengovirus-infected L cells was delayed by several hours if the cell monolayer cultures were fully grown and cell growth had ceased. 3. In the first of two reports, Plagemann and Swim (1966) reported differences in the time and extent of inhibition of host RNA and protein synthesis by mengovirus in two strains of Novikoff rat hepatoma propagated in vitro. The parent strain, NISI 63, grows in a medium containing 20% horse serum. From NISI 63 they isolated a strain designated as No. 67 which grows in a medium containing 5% calf serum and 2 mum1 of pancreatic autolysate. Mengovirus inhibits RNA, DNA, and protein synthesis of cell strain 63 rapidly and early in infection. In strain NISI 67 the inhibition occurs very late in infection and coincides with loss of cell

92

BERNARD ROIZMAN AND PATRICIA G. SPEAR

viability. In the second report Plagemann (1968) showed that the delay in the inhibition of host macromolecular synthesis by mengovirus in NISI 67 cells might be characteristic of cells selected to grow in medium containing 5% calf serum and 2 mg/ml of pancreatic autolysate. Thus, a strain of L cells, selected on the basis of its ability to grow in that medium and designated as L 67, responded to mengovirus infection in a manner very similar to that of NISI 67 cells. Moreover, NISI 67 cells grown for 20 passages in medium containing 20% horse serum responded to infection in a manner similar to that of NISI 63 cells. The evidence cited above that the time and pattern of inhibition of host macromolecular synthesis can be in part determined by the host may explain the early results of Ackermann ( 1958), Ackermann et al. ( 1959), Maasab et al. (1957), and Becker et al. (1959) who reported an increase in the rates of synthesis of macromolecules in cells infected with poliovirus. C. THEINHIBITOR OF HOSTMACROMOLECULAR SYNTHESIS(IHMS ) 1. The Nature, Source, and Time of Synthesis of IHMS

The data summarized in the preceding sections show that IHMS might be expected to vary considerably. Thus ( a ) the ability to differentiate between viral and heterologous macromolecular synthesis ( the specificity of IHMS) is determined largely by the virus, and ( b ) the ability to inhibit the host varies depending on the virus, host species, and metabolic condition of the host. As might be expected, the time of synthesis, the source, and possibly, even the nature of IHMS may vary depending on the virus. It seems desirable to summarize the available data according to the v i r u s group. a. Adenouiruses. Available evidence (Ginsberg et al., 1967) indicates that adenovirus IHMS is a structural component of the virus and that it is made relatively late in infection after the onset of viral DNA and protein synthesis (about 8-10 hours after infection). As shown schematically in Fig. 4, the surface components of adenoviruses are a hexon or antigen A, a penton or antigen B, and a fiber or antigen C (Ginsberg et al., 1968). Pereira (1960) showed that HeLa cells exposed before infection to the fiber antigen produced by adenovirus types 1, 2, 4, 5, and 6 failed to replicate adenovirus type 5, poliovirus type 1, and vaccinia virus. The hexon antigen was ineffective. The inhibition was not mediated by interferon, nor did the fiber antigen affect virus adsorption. Levine and Ginsberg (1967) con6rmed the finding of Pereira and showed, moreover, that

3.

93

CYTOLYTIC VIRUSES

the fiber antigen combines irreversibly with KB cells and blocks the synthesis of host RNA, DNA, and protein. b. Picomuiruses. Available evidence suggests that picornavirus IHMS is a protein, is made early after infection and, moreover, that it is not a structural component of the virus. The experimental evidence is based on two kinds of data. Hexon (Antigen A )

T

/

Penton base Penton (Antigen B)

Fiber (Antigen C )

0 0

I I

FIG. 4. Adenovirus antigens and their relationship to the virion according to Russell et al (1967).

First, photoinactivated ( Holland, 1964a,b) and UV-light-irradiated virus (Franklin and Baltimore, 1962; Penman and Summers, 1965) do not inhibit host macromolecular biosynthesis. These data are taken to mean that IHMS is not a structural component of the virus, and moreover, that a functional viral genome is required. Second, in several studies the inhibition of host macromolecular synthesis was prevented or significantly diminished by chemical inhibitors of macromolecular synthesis or by amino acid analogs added during an after infection. Thus Baltimore et al. ( 1963a) reported that the inhibition of host RNA synthesis in L cells infected with mengovirus was reduced by p-fluorophenylalanine and abolished by puromycin. Puromycin ( Holland and Peterson, 1964; Darnel1 et al., 1967) and actidione (Willems and Penman, 1966) were similarly effective in HeLa cells infected with poliovirus. Furthermore, p-fluorophenylalanine in relatively small amounts ( 20 pg/ml) was reported by Verwoerd and Hausen (1963) to prevent the inhibition of host RNA synthesis in L cells infected with ME virus. On the other hand, incubation of poliovirus-infected cells at 40°C (Fenwick, 1963) or exposure to concentrations of guanidine which prevent viral RNA synthesis (Bablanian et al., 1965; Summers et al., 1965; Penman and Summers, 1965) failed to prevent the inhibition of host protein synthesis. The inter-

94

BERNARD ROIZMAN AND PATRICIA C. SPEAR

pretation of these data is that the inhibition requires a protein synthesized early in infection, i.e., before viral RNA is replicated. It should be pointed out that while it is most likely that the picorna IHMS is a protein made in the cell early in infection, the data taken one by one are not entirely convincing for two reasons. First, the failure of inactivated virus to inhibit the host is not very meaningful since exposure to heat, UV, or to other inactivating agents causes alteration in structure as evidenced by a change in immunologic specificity (Roizman et al., 1959) and loss of the capacity to absorb to cells (Katagiri et al., 1968). Second, in many experiments the concentrations of chemical inhibitors and of amino acid analogs required to abolish the inhibition of the host were frequently (Baltimore et d.,1963a; Farnham, 1965) far in excess of the amount required to abolish viral protein and RNA synthesis (Baltimore and Franklin, 1963; Eggers et aZ., 1963). c. Herpesuiruses. The inhibition of host macromolecular synthesis in cells infected with herpesviruses takes place during the first 3 hours after infection before the onset of viral DNA synthesis and before the bulk of viral proteins are made (Roizman, 1969). Reports concerning the nature of herpesvirus IHMS, that is, whether it is a structural component of the virus or a protein made after infection, are contradictory. Ben-Porat and Kaplan ( 1965) demonstrated that puromycin (20 pg/ml) decreased the rate of inhibition of host DNA synthesis in rabbit kidney cells infected with pseudorabies virus, a member of the herpesvirus group. On the other hand, Newton (1968) reported that UV-irradiated herpes simplex virus was effective in blocking host DNA synthesis and, moreover, the effect of the v i r u s could not be abolished by chemical inhibitors of protein synthesis. Herpes simplex virus loses its capacity to disaggregate HEp-2 cell polyribosomes and inhibit protein synthesis following UV-light irradiation (Sydiskis and Roizman, 1967). However, whereas actinomycin D, pfluorophenylalanine and 6-azauridine prevented the disaggregation of host polyribosomes in dog kidney cells abortively infected at relatively high (lo00 pfu per cell) multiplicities of infection, the drugs were ineffective in HEp-2 cells infected at low multiplicities of infection (Sydiskis and Roizman, 1967). d. Poxuiruses. There is little information on the nature and source of poxvirus IHMS. Inhibition of HeLa cell DNA synthesis by heated (60°C at 15 minutes) or by UV-light-irradiated vaccinia virus, a member of the poxvirus group, is similar to that obtained with untreated virus (Jungwirth and Launer, 1968; Hanafusa, 1960a,b, 1961,1962). Treatment of infected cells with fluorodeoxyuridine prevents the synthesis of viral DNA

3.

CYTOLYTIC VIRUSES

95

but does not diminish the inhibition of cell DNA synthesis (Jungwirth and Launer, 1968). Ultraviolet light-inactivated purified vaccinia virus also inhibits host protein synthesis; heated or detergent-treated virus is ineffective ( Moss, 1968). The inhibition, moreover, is multiplicity dependent ( Moss, 1968). The inhibition of protein synthesis also takes place early in infection and is not affected by actinomycin D (Shatkin, 1963; Moss, 1968), cycloheximide (Moss, 1968) or by interferon (Joklik and Merigan, 1966). Inhibition of host RNA synthesis on the other hand becomes apparent only beginning 3 4 hours after infection (Becker and Joklik, 1964; Salzman et al., 1964). The inhibition of DNA and protein synthesis is probably brought about by a constituent of the virus. The nature of this constituent is unknown. e . Myxoviruses, arbouiruses, and vesicular stomatitis virus. There is a paucity of data concerning myxovirus, arbovirus, and vesicular stomatitis virus IHMS. Studies on cells infected with Newcastle’s disease virus, a member of the myxovirus group, showed that inhibition of protein synthesis takes place between 5 and 9 hours after infection, and that 6azauridine prevents the shut-off when added at the time of infection, whereas cycloheximide and puromycin prevent the shut-off when added as late as 3.5 hours after infection (Bolognesi and Wilson, 1966). The ability to inhibit host RNA synthesis, as indicated in Section I,B,l, does not appear to be a universal property of all Newcastle disease virus strains ( Wilson, 1968). The inhibition of host RNA synthesis is also prevented by cycloheximide or by 6-azauridine. These data led to the conclusion that in infection with Newcastle disease virus both RNA and protein synthesis must precede inhibition of the host. It is not clear whether Newcastle disease IHMS is a structural component of the virus. The undiminished capacity of UV-irradiated complete or of defective vesicular stomatitis virus to inhibit host RNA synthesis (Wagner and Huang, 1966; Huang and Wagner, 1966) suggests that IHMS is a structural component of the virus.

2. I H M S and Cell Death Frequently during the last stages in the reproduction of animal viruses all macromolecular metabolism ceases; the cells become distorted in shape and lose their structural integrity. Some viruses alter the adhesiveness of cells to glass; others cause cells to interact differently among themselves leading to the formation of cell clumps, polykaryocytes, etc. The process is usually irreversible. For the purposes of this review cell death may be defined as cessation of energy production and of cell macromolecular

96

BERNARD ROIZMAN AND PATRICIA G. SPEAR

synthesis. The question arises whether IHMS is alone responsible for the death of the cell. There is no apparent answer applicable to all viruses and several points should be made. 1. Selective cessation of synthesis of one or another host macromolecule occurs throughout the reproductive cycle of the cell and also in cells in cultures reaching a critical density. Cell death is not a necessary consequence of selective inhibition lasting several hours. 2, Many picornaviruses cause loss of cell integrity and loss of adhesiveness to glass surfaces much faster than do inhibitors like actinomycin D, puromycin, and mitomycin C. We may deduce from this that cell death caused by viruses and drugs is not a consequence of an identical arrest or alteration of a biologic process. 3. Cell death usually occurs at the end of the reproductive cycle. When inhibition of host macromolecular synthesis occurs late in infection concurrently with inhibition of viral synthesis, it could be argued that the two events-cessation of cell macromolecular synthesis and immediate death of the cell-are interconnected. It is more difficult to establish a causal connection between the inhibiting process occurring early in infection and the cell death occurring some 12-15 hours later. D. THEMECHANISMS OF INHIBITION OF HOSTMACROMOLECULAR SYNTHESIS

1. General Consklerations

The data presented in the preceding sections have shown that the IHMS produced by different viruses vary with respect to specificity, biosynthetic requirements, and the time at which they become effective. There appears to be not one but possibly two or more evolutionary pathways by which viruses affect their hosts. Thus, the IHMS of some viruses, exemplified by picornaviruses, is probably not a structural component of the virion. The characteristics of this IHMS is that it is made early in infection, that it requires protein synthesis only to be effective, and that it is generally highly specific in terms of its ability to differentiate between host and viral macromolecular synthesis. The prediction is that increasing the multiplicity of infection should, to a limited extent, synchronize the inhibition of the host without affecting the yield of the viral progeny. On the other hand, the IHMS of viruses exemplified by adenovirus is a structural protein of the virion. At relatively low multiplicities of infection the inhibition of the host concurs with the onset of synthesis of structural proteins. Characteristically, the biosynthesis of IHMS requires both

3.

CYTOLYTIC VIRUSES

97

protein and nucleic acid synthesis (Ginsberg et al., 1967; Wilson, 1968) and, moreover, the IHMS is incapable of differentiating between viral and host macromolecular synthesis. We may predict that exposure of sensitive cells to concentrated extracts of virus containing IHMS should accelerate the inhibition and cause a greatly reduced viral yield. These predictions generally hold well for adenoviruses (Levine and Ginsberg, 1967, 1968) and perhaps a little less well for poxviruses (Moss, 1968), myxoviruses (Von Magnus, 1954; Scholtissek et al., 1966; Rott and Schafer, 1960), and vesicular stomatitis virus (Wagner and Huang, 1966; Huang et al., 1966; Huang and Wagner, 1966). The inhibition of protein synthesis by purified pox virions is multiplicity dependent, but it does not appear to affect viral protein synthesis (Moss, 1968).In particular, with myxoviruses and vesicular stomatitis virus high multiplicities of infection result in the formation of defective virions lacking a full complement of RNA. The defective particle is made in cells infected at both high and low multiplicities of infection and therefore it is not entirely clear whether or not the defectiveness is a consequence of an unselective reduction in macromolecular metabolism. However, the decrease in infectious (complete) virus yield from cells infected at high multiplicities of infection might be due to successful competition for the RNA polymerase by the fractured fragment of RNA in the defective particles. 2. The Inhibition of RNA Spthesis

Virtually all available data were obtained on cells infected with picornaviruses and concern mechanisms which are probably not the ones actually responsible for the inhibition of host RNA synthesis. To begin with, the inhibition of RNA synthesis is real enough and not an artifact since it cannot be accounted for by a more rapid breakdown of host RNA in infected cells (Martin et al., 1961; Franklin and Baltimore, 1962) or by changes in the rate of entry of labeling precursors into the cellular pools (Martin et al., 1961; Martin and Work, 1962). Second, it seems likely that the inhibition of RNA synthesis is not due to breakdown of host DNA (Holland, 1962; Franklin and Baltimore, 1962). The inhibition of RNA synthesis coincides with a decrease in the activity of host DNA-dependent RNA, polymerase (aggregate enzyme) (Holland, 1962; Baltimore and Franklin, 1962). The enzyme activity, however, is bound tightly to DNA and it is not entirely clear whether the decrease in activity is due to inactivation of enzyme or of template. In in uitro tests deproteinized DNA and DNA-protein complexes extracted from infected cells could not be differentiated with respect to priming ability from cor-

9s

BERNARD ROIZhlAN AND PATRICIA G. SPEAR

responding preparations extracted from uninfected cells ( Holland, 1962). However, we are not sure that the in uitro experiments measure the same kind of priming activity as that measured in uioo. The meaning of the in vitro experiments is unclear. 3. Inhibition of DNA Synthesis

There is general agreement that the inhibition of host DNA synthesis is not the consequence of degradation of the DNA or competition of virus and host for enzymes and precursors (Kitt and Dubbs, 1962; Ben-Porat and Kaplan, 1965).Available data suggest that two different mechanisms may be involved. One mechanism may be exemplified by adenoviruses. Levine and Ginsberg (1968)have shown that the adenovirus fiber antigen binds to host DNA. The fiber antigen is by itself inhibitory. The suggestion is that the inhibition of host DNA synthesis following exposure of cells to fiber antigen might be due to the formation of DNA-fiber complexes. Similarly Ackermann and Wahl (1956)found that the omission of arginine from the medium prevented the inhibition of HeLa cell DNA synthesis (somewhat reduced by the absence of arginine) without affecting the yield of poliovirus. They suggested that the inhibition of the host DNA synthesis might be due to an arginine-rich protein, possibly a histone, binding to the DNA. The same laboratory reported a stimulation in the biosynthesis of histones in HeLa cells infected with poliovirus (Sokol et al., 1965). A second mechanism which might be operative in cells infected with some viruses is the inhibition of synthesis of proteins required for cell DNA synthesis. The evidence that the decline in host DNA synthesis is a consequence of the inhibition of protein synthesis is less compelling; it is based on the observation that in cells infected with poliovirus, mengovirus, and a number of other viruses the inhibition of DNA synthesis follows that of protein synthesis. As we noted elsewhere in the text (Section II,B,4) the delay may not be very meaningful. Two additional points, however, should be made here concerning the special relationship between DNA and protein synthesis. First, unless special efforts are made, cell cultures tend to grow asynchronously, and a constant fraction of cells is replicating DNA at any one time. Most of the studies cited in this review were done on asynchronously growing cells. In the reproductive cycle of animal cells the interval of DNA synthesis ( S ) is sandwiched between the two cycles of protein synthesis; the first (GI) is required for DNA synthesis, whereas the second ( G 2 ) is required for the subsequent cell division. On the basis of these considerations if the decline in DNA

3.

CYTOLYTIC VlRUSES

99

synthesis is a consequence of the inhibition of protein synthesis, it would be expected that the inhibition of DNA synthesis would be immediately effective for cells entering into G1 but not for those already in the S phase. It might be calculated that for completely asynchronous cultures of cells dividing every 18-24 hours the complete inhibition of DNA synthesis may take 10 hours. This is somewhat longer than the observed 3-6 hours. It should be noted however that these calculations are based on “complete” asynchrony. In practice any manipulation of cells such as transfers, changes of media, etc. tends to partially synchronize growth. The effects of partial synchronization particularly of cells arrested in G1 prior to infection might be to decrease the time required for complete inhibition of DNA synthesis to considerably less than the predicted 10 hours. Perhaps a far more compelling link between the inhibition of host protein and DNA synthesis would be evidence that the virus inhibits selectively the synthesis of proteins in GIbut not those in Gz.Evidence of this type is not available for any v i r u s infecting animal cells. Recently however, it has been shown by Lin (1968) that in L cells infected with the agent of meningopneumonitis, the inhibition of host protein synthesis is selective, affects primarily proteins of the GI phase, and satisfactorily accounts for the inhibition of L cell DNA synthesis.

4. The Inhibition of Prolein Synthesis The inhibition of protein synthesis coincides with, and is probably due to, disaggregation of polyribosomes (Penman et al., 1963, 1964; Summers et al., 1965; Joklik and Merigan, 1966; Sydiskis and Roizman, 1966; Dalgarno et al., 1967). The mechanism of disaggregation of host polyribosomes is unknown, but it does not appear to be due to a loss of the capacity of preexisting ribosomes to participate in protein synthesis (Kerr et al., 1962; Kerr, 1963; Baltimore et al., 196313; Summers et d.,1964). Willems and Penman (1966) showed that host polyribosomes from cells infected with poliovirus and treated with actidione remained intact both with respect to number and size. When actidione was removed polyribosomes decreased in number but not in size. The extent of disaggregation was proportional to the duration of exposure to the drug. Willems and Penman concluded that the polyribosomes were modified in some fashion so that they disaggregated once the drug was withdrawn. While the authors probably could not detect the loss of a few nucleotides from host messenger RNA, the data excluded random scission of the messenger RNA. It is noteworthy, however, that in HeLa cells infected with vaccinia virus host polyribosomes decreased both in amount and in size (Moss, 1968).

100

BERNARD ROIZMAN AND PATRICIA G. SPEAR

Some clues concerning the mechanism of inhibition of host protein synthesis emerge from three observations. First, in some cell-virus systems the concentrations of chemical inhibitors required to prevent the inhibition of the host are far in excess of that required to inhibit viral macromolecular synthesis. Thus, Baltimore et al. (1963a) reported that the inhibition of host RNA synthesis in L cells infected with mengovirus was decreased by 500 pg of p-fluorophenylalanine per milliliter of medium or 200 pg of puromycin per milliliter of medium. Second, as cited in Section II,C,7 actinomycin D, p-fluorophenylalanine, and 6-azauridine prevented polyribosome disaggregation in dog kidney cells abortively infected at high multiplicity with herpes simplex virus but not in HEp-2 cells productively infected at relatively low multiplicities with the same virus (Sydiskis and Roizman, 1967). The difference in the effectiveness of the drugs was not due to inherent differences between HEp-2 and DK cells since actinomycin D was ineffective in preventing the disaggregation of host polyribosomes in DK cells infected with a herpes virus mutant capable of growing in these cells. The inhibition of host macromolecular synthesis in abortively infected cells appears to be more sensitive to drugs than that in productively infected cells. Present data suggest that wild strains of herpes simplex virus make altered or defective proteins in the nonpermissive DK cells (Spring et aZ., 1968). The third observation is that host protein synthesis is inhibited in infected cells treated with interferon at a concentration sufficient to prevent v i r u s multiplication. Interferon probably prevents the synthesis of viral proteins (Levy, 1964; Joklik and Merigan, 1966). One hypothesis which may account for these observations may be stated as follows. ( a ) In productive infection of permissive cells the inhibitor of protein synthesis functions catalytically and is highly efficient. ( b ) Even trace amounts of this substance made in the presence of chemical inhibitors or of interferon are sufficient to inhibit the host. ( c ) In abortively infected cells the inhibitor of protein synthesis is less efficient and more inhibitor molecules are necessary to shut off host protein synthesis; hence any interference by drugs with either the synthesis of the inhibitor or its function tends to prevent the inhibition of the host.

5. The Discrimination between Host and Viral Macromolecular Biosynthesis One of the most striking hdings reported in this review is that the IHMS of some viruses readily differentiate between viral and cellular macromolecular synthesis. The differentiation must necessarily be based

3.

CYTOLYTIC VIRUSES

101

on some inherent differences in viral and cellular biosynthesis. There are three generalized hypotheses which may account for some of the specificity, i.e., (a) corresponding viral and cellular macromolecules differ in some higher order structure, ( b ) the synthesis of viral and host macromolecules follows different pathways, and ( c ) viral and host macromolecules are synthesized in different compartments of the cell. Examples supporting these arguments are that ( 1 ) replicating cellular DNA in vivo forms a highly structured DNA-protein complex whereas viral DNA probably does not, (2) the biosynthetic pathway for the synthesis of RNA by RNA viruses is different from that of the host RNA, and ( 3 ) the biosynthesis of picorna and pox viruses takes place entirely in the cytoplasm. If we accept these arguments as the basis for the differentiation of viral and cellular macromolecular synthesis two additional points should be made. First, both viral and cellular protein synthesis occur in the cytoplasm. Viral protein synthesis involves participation of cellular ribosomes and at least some cellular enzymes. If indeed the macromolecules and structures involved in viral and cellular protein synthesis do not differ among themselves to an extent greater than do host polyribosomes making different peptides, it would necessarily follow that the cessation of host protein svnthesis is a consequence of the inhibition of the synthesis of some other 'macromolecule. Evidence suggesting that there must be differences between macromolecules and structures involved in host and viral protein svnthesis emerges from studies of the mod(. of action of interferon. Interferon is a product of the cell made in response to selected classes of macromolecules foreign to the cell (Lampson et al., 1967; Field et a l , 1967; Tytell et al., 1967). Purified interferon has no demonstrable effect on cell RNA or protein synthesis (Baron et al., 1966; Levy and Merigan, 1966 ). Infected cells treated with interferon fail to make viral constituents possibly by preventing the translation of viral messenger RNA (Marcus and Salb, 1966; Joklik and Merigan, 1966, Sonnabend et al., 1967; Carter and Levy, 1967, 1968). If, as currently suggested by Marcus and Salb ( 1966), Levy and Carter (1968), and Carter and Levy (1968), ribosomes treated with interferon differentiate between viral and cellular messenger RNA, several conclusions necessarily follow, i.e. : ( a ) there is a difference between corresponding cellular and viral RNA involved in protein synthesis; this difference may be the basis for cellular exclusion of viral protein synthesis and vice versa. ( b ) In noninterferon-treated cells host components of the protein-synthesizing machinery do not discriminate between viral and cellular RNA. ( c ) Last, interferon imposes a

102

BERNARD ROIZMAN AND PATRICIA G. SPEAR

restriction on the cell, and from the temporary nature of the restriction it follows that it is not in the best interests of the cell or organism to restrict its translation of RNA to molecules bearing a specific cognitive structure. The second comment concerning the discrimination between the host and the viral macromolecular biosynthesis stems from the conclusion that in order to inhibit the host without inhibiting viral macromolecular synthesis the IHMS must react with one or more of the host components involved in the biosynthesis of host macromolecules. To emphasize, in this instance the differentiation of self from nonself involves a subsequent reaction of IHMS with nonself. It is entirely conceivable, and there is in fact evidence, that a virus could develop in the course of evolution an enzyme which has a greater affinity for viral nucleic acids than for cellular nucleic acids. It is more difficult to visualize the evolutionary formulation of a macromolecule with greater affinity for host templates than its own particularly in view of the fact that many animal viruses multiply and effectively inhibit host macromolecular synthesis in a wide variety of species throughout the animal kingdom. Perhaps the most important consideration is that while the cell has sufficient genetic storage capacity to accommodate the distinguishing features of many different viruses which would be recognized as nonself, most small RNA viruses do not have similar capacity for all the different kinds of cells they infect. The differentiation between self and nonself most probably involves an invariable feature of animal cell protein synthesis. The nucleotide sequences specifying initiation and possibly termination of protein synthesis seem to us to be the most invariant among pathways, macromolecules, and structures involved in protein synthesis. 111. Why Is Host Macromolecular Synthesis Inhibited By Viruses?

A. THE SOURCEOF GENETICINFORMATION FOR MACROMOLECULAR SYNTHESIS

THE

INHIBITION OF HOST

At the time of writing there has been no direct evidence that the structural information of IHMS is specified by the virus. For our purposes direct evidence would consist of the in vitro synthesis of IHMS starting with a viral template as a source of structural information. Since we do not know the nature of IHMS or how it functions the point is moot. The argument that the source of genetic information is viral stems from two observations, i.e., ( a ) in some instances the IHMS is a structural component of the virus, and ( b ) when prevailing evidence suggests that the

3.

CYTOLYTIC VIRUSES

103

IHMS is not a structural component of the virion, the inhibition of the host may be prevented by UV irradiation of the virus. The argument that IHMS is determined by the cell rather than the virus is based on two observations on cells infected with viruses whose IHMS is probably not a structural component of the virion. Those observations are that ( a ) interferon, which prevents the synthesis of viral components, does not prevent the inhibition of L cell metabolism by vaccinia (Joklik and Merigan, 1966) or by mengovirus (Levy, 1984), and ( b ) as cited in Section II,C,l the amounts of chemical inhibitors required to prevent the inhibition of the host have been in some instances far in excess of the amounts required to inhibit the synthesis of viral components. As we have indicated ( Section II,D,4) one trivial but rather appealing explanation for these findings is that even trace amounts of IHMS made in the presence of chemical inhibitors are sufficient to inhibit host functions.

B. EVOLUTION OF THE INHIBITION OF HOSTMACR~MOLECULAR SYNTHESIS This review dealt mostly with picorna-, pox-, herpes-, adeno-, and myxoviruses. The viruses comprising these groups share in common the property of inhibiting host macromolecular synthesis at some point of their reproductive cycle, but differ in that the IHMS of picomaviruses and possibly of herpesviruses are made early in infection and do not appear to be structural components of the virion whereas the IHMS of adenoviruses, and possibly of myxoviruses are made relatively late in infection and are structural components of the virus. Analysis of the nature, synthesis, and mode of action of IHMS of different viruses does not shed light on their evolutionary relationship. It has been suggested that viruses may have originated from a component of the cell; a possible prototype of the inhibitory mechanisms developed by viruses is the mechanism developed by the cell which is mediated by interferon. Unfortunately, there is presently insufficient data to compare, from the point of view of an evolutionary relationship, the inhibition of the host by viruses with the inhibition of viral macromolecular synthesis in cells treated with interferon.

C. EVOLUTIONARY BASISFOR THE DEVELOPMENT OF IHMS This section concerns not so much thc nature of IHMS but rather the fact that viruses cause the inhibition of host functions. It is indeed not very clear why viruses have acquired, retained, and express the capacity to inhibit the host. Two aspects of the problem deserve consideration. From the point of view of the cell it could be argued that the inhibition of host functions and cell death are desirable consequences of viral infec-

104

BERNARD ROIZMAN AND PATRICIA G. SPEAR

tion. The argument is based on considerations of the function of animal cells in the artificial in vitro environment of the cell culture and in the whole animal. I n culture cells act as single entities competing independently for survival. In the animal they are dependent components of a complex multicellular organism, readily expendable if they constitute a threat to the life of the animal. In evolution the selective pressure operates only at the level of the entire animal. From this point of view it could be argued that the ease with which viruses appear to inhibit and kill the cell may in fact be very desirable for the organism as a whole for prolonged survival of the infected cell is undesirable. This line of argument is supported to some extent by the observations cited in Section II,B,5 that the effectiveness of inhibition is dependent on the host. The argument is weakened, however, by the observation that similar inhibition occurs in unicellular organisms like bacteria. From the point of view of the virus one pertinent observation would seem to suggest that inhibition of host macromolecular synthesis may be a prerequisite for virus multiplication. This conclusion is based on the observation that herpes simplex virus strain MPdk- multiplies and effectively inhibits cells of human derivation but does not produce infectious progeny or effectively inhibit canine kidney cells probably because some proteins specified in canine cells malfunction ( Aurelian and Roizman, 1965; Sydiskis and Roizman, 1967; Spring et al., 1968). The significant finding is that in canine cells viral DNA and proteins are synthesized only in cells infected at a multiplicity sufficiently high to inhibit the host. At low multiplicities of infection the cell makes interferon only. These observations would seem to indicate that ( a ) host response to infection and inhibition of host macromolecular synthesis are competing processes initiated simultaneously on infection, and ( b ) at low multiplicities of infection the cells attain the upper hand only because the amount of effective inhibitor specified by the virus is insufficient to inhibit the host in time to prevent it from making interferon ( Aurelian and Roizman, 1965; Sydiskis and Roizman, 1967). It would be of interest to determine whether the competing processes uncovered in that investigation are a general property of all animal cells infected with viruses capable of inducing interferon. If this were the case it could follow that viruses which elicit the synthesis of interferon have, in the course of evolution, acquired the capacity to inhibit host functions. ACKNOWLEDGMENTS We would like to acknowledge our indebtedness to Mrs. Norma Coleman and Mrs. Deirdre Kondor for assistance in the preparation of the manuscript and to numerous

3.

CYTOLYTIC VIRUSES

105

colleagues for many useful discussions. The published and unpublished data from our laboratory cited here were accumulated with the aid of grants from the American Cancer Society, the National Science Foundation, the United States Public Health Service, and the Whitehall Foundation. REFERENCES Ackemiann, W. W. (1958).Bacteriol. Rev. 22,223. Ackermann, W. W., and Wahl, D. (1956).J . Bacteriol. 92, 1051. Ackermann, W. W., Rabson, A., and Kurtz, H. (1954).J. Erptl. Med. 100,437. Ackermann, W.W., Payne, F. E., and Kurtz, H. (1958).J. Immunol. 81, 1. Ackermann, W. W., Loh, P. C., and Payne, F. E. (1959).Virology 7, 170. Aurelian, L., and Roizman, B. (1965).J. Mol. B i d . 11, 539. Bablanian, R., Eggers, H. J,, and Tamm, I. (1965).Virology 26, 100. Baltimore, D., and Franklin, R. M. (1962).Proc. Nutl. Acud. Sci. U.S. 48, 1383. Baltimore, D.,and Franklin, R. M. (1963).Biochim. Biophys. Acta 76, 431. Baltimore, D., Franklin, R. M., and Callender, J. ( 1963a).Biochim. Biophys. Acta 76,425. Baltimore, D., Eggers, H. J., and Tamm, I. (1963b).Biochim. Biophys. Acta 76, 644. Baron, S., Merigan T. C., and McKerlie, M. L. (1966).Proc. Exptl. B i d . Med. 121, 50. Becker, Y.,and Joklik, W. K. (1964).Proc. Nutl. Acad. Sci. US.51, 577. Becker, Y.,Grossowim, N., and Bernkopf, H. (1959).Bull. Res. Council Israel Sect. E 8,NO. 1-2. Bello, L. J., and Ginsberg, H. S. (1967).J . Virol. 1, 543. Ben-Porat, T., and Kaplan, A. S . (1965). Virology 25, 22. Bernkopf, H., Mishmi, M., and Rosin, A. (1959).J. Immunol. 83, 635. Bolognesi, D. P., and Wilson, D. E. (1966).J . Bacteriol. 91, 1896. Brown, A., Mayyasi, S. A., and Officer, J. E. (1959).J. Infect. Diseases 104, 193. Brown, F., Martin, S. J., and Underwood, B. (1966).Biochim. Biophys. Acta 129, 166. Carter, W. A., and Levy, H. B. (1967).Science 155, 1254. Carter, W. A., and Levy, H. B. (1968).Biochim. Biophys. Acta 155, 437. Cooper, P. D., and Bellett, A. J. D. (1959).I. Gen. Microbiol. 21, 485. Cords, C. E., and Holland, J. J. (1964).Virology 22, 226. Dalgarno, L., Cox, R. A., and Martin, E. M. (1967).Biochim. Biophys. Actu 138, 316. Damell, J. E., Girard, M., Baltimore, D., Summers, D. F., and Maizel, J. V. (1967).In “The Molecular Biology of Viruses” (J. Colter, ed.), pp. 375-402.Academic Press, New York. Eggers, H. J., Baltimore, D., and Tamm, I. (1963).Virology 21, 281. Enders, J. F.,Weller, T. H., and Robbins, F. C. (1949).Science 109,55. Everett, S. F., and Ginsberg, H. S. (1958).Virology 6, 770. Farnham, A. E. (1965).Virobgy 27, 73. Fenwick, M. L. (1963).Virology 19, 241. Field, A. K.,Tytell, A. A., Lampson, G. P., and Hillelnau, M. R. (1967).Proc. Natl. Acad. Sci. U.S. 58, 1004. Fogh, J., and Fogh, H. (1965).Proc. Soc. Exptl. B i d . Med. 119, 233. Franklin, R. M.,and Baltimore, D. (1962).Cold Spring Harbor Symp. Quunt. Biol. 27, 175.

106

BERNARD ROIZMAN AND PATRICIA

C.

SPEAR

Ginsberg, H. S., Pereira, H. G., Valentine, R. C., and Wilcox, W. C. (1966).Virology 28, 782. Ginsberg, H. S., Bello, L. J., and Levine, A. J. (1967).In “Molecular Biology of Viruses” (J. Colter, ed.), pp. 547-572.Academic Press, New York. Comatos, P. J., and Tamm, I. (1963).Biochim. Biophys. Acta 72, 651. Hackett, A. J. (1964).Virology 24, 51. Hanafusa, H. (1960a).Biken’s J. 3, 313. Hanafusa, H. (1960b).Biken’s J. 3, 191. Hanafusa, H. (1961). Biken’s 3. 4, 97. Hanafusa, H. (1962).Cold Spring Harbor Symp. (Iuant. B i d . 27, 209. Henle, G., Deinhardt, F., and Girardi, A. (1954).Proc. SOC. Exptl. B i d . Med. 87, 386. Holland, J. J. (1962).Biochem. Biophys. Res. Commun. 9, 556. Holland, J. J. (1963).Proc. Natl. Acad. Sci. U S . 49, 23. Holland, J. J. (1964a).Bacteriol. Reo. 28, 3. Holland, J. J. (1964b).J. Mol. Biol. 8, 574. Holland, J. J., and Peterson, J. A. (1964).J. Mol. B i d . 8, 556. Holmes, K. V., and Choppin, P. W. (1966).J. Exptl. Med. 124, 501. Homma, M., and Graham, A. F. (1963).J. Cell Comp. Physiol. 62,179. Huang, A. S., and Wagner, R. R. (1964).Proc. SOC. Erptl. Bid. Med. 116, 863. Huang, A. S.,and Wagner, R. R. (1965).Proc. Natl. Acad. Sci. U S . 54, 1579. Huang, A. S., and Wagner, R. R. (1966).Virology 30, 173. Huang, A. S., Greenawalt, J. W., and Wagner, R. R. (1966).Virology 30, 161. Huebner, R. J. (1957).Ann. N.Y. Acad. Sci. 67, 209. Joklik, W.K.,and Merigan, T. C. (1966). Proc. Natl. Acad. Sci. U.S.56, 558. Jungwirth, C., and Launer, J. (1968).I. Virol. 2, 401. Katagiri, S., Hinuma, Y., and Ishida, N. (1968).Virology 34, 797. Ken, I. M. (1963).Ph.D. Thesis, Univ. of London. Ken, I. M., Martin, E. M., Hamilton, M. G., and Work, T. S. (1962).Cold Spring Harbor Symp. Quunt. Biol. 27, 259. Kitt, S., and Dubbs, D. R. (1962).Virology 18, 286. Kudo, H., and Graham, A. F. (1965).J. Bacterfol. 90,936. Lampson, G. P., Tytell, A. A., Field, A. K., Nemes, M. M., and Hillernan, M. R. (1967).Proc. Natl. Acad. Sci. US. 58, 782. Le Bouvier, G. L. (1954).Nature 174, 849. Ledinko, N. ( 1966). Virology 28,679. Ledinko, N., and Melnick, J. L. (1954).J. Exptl. Med. 100, 247. Levine, A. J., and Ginsberg, H. S. ( 1967).J. Virol. 1, 747. Levine, A. J., and Ginsberg, H. S. (1968).J. Virol. 2,430. Levy, H. B. (1964).Virology 22, 575. Levy, H. B., and Carter, W. A. (1968).J. Mol. Bid. 31, 561. Levy, H. B., and Merigan, T. C. (1966).Proc. Soc. Exptl. B i d . Med. 121, 53. Lin, H. (1968).J. Bacteriol. 96,2054. Lwoff, A. ( 1953).Bacteriol. Rev. 17,269. Lwoff, A.,Dulbecco, R., Vogt, M., and Lwoff, M. (1955).Virology 1, 128. McCormick, W., and Penman, S. (1967).Virology 31, 135. Maasab, H.F.,Loh, P. C., and Ackermann, W. W. (1957).J. Exptl. Med. 106, 641. Marcus, P. I., and Salb, J. M. (1966).Virology 30, 502.

3.

CYTOLYTIC VIRUSES

107

Martin, E. M., and Work, T. S . (1962). Biochem. J. 83, 574. Martin, E. M., Malec, J., Sved, S., and Work, T. S. (1961).Biochem. J. 80, 585. Mason, E. J., and Kaufman, N. (1960a). Am. J. Pathol. 37, 231. Mason, E. J., and Kaufman, N. (1960b). J. Infect. Diseases 107, 245. Mims, C. A. (1956). J. Erptl. Pathol. 37, 129. Miroff, G., Cornaker, W. E., and Fischer, R. G. (1957). J. Biol. Chem. 228, 255. Mishmi, M., and Bernkopf, J. (1958). J. Immunol. 81, 460. Moss, B. (1968). J. Virol. 2, 1028. Nardone, R. M., Todd, J., Gonzalez, P., and Gaffney E. V. (1965). Science 149, 1100. Newton, A. (1968). PTOC.1st Intern. COngT. Virol. Helsinki, Finland p. 253. Karger, Basel. Penman, S., and Summers, D. F. (1965). Virology 27, 614. Penman, S., Schemer, K., Becker, Y., and Darnell, J. E. (1963). Proc. Natl. Acud. Sci. US.49, 654. Penman, S., Becker, Y.,and Darnell, J. E. (1964). J. Mol. Biol. 8, 541. Pereira, H. G. (1958). Virobgy 6, 601. Pereira, H. G. (1960). Virobgy 11, 590. Plagemann, P. G. W. (1968). J. Virol. 2, 461. Plagemann, P. G. W., and Swim, H. E. (1966). J. Bacteriol. 91, 2317. Pollock, M. E., Treadwell, P. E. and Kenny, G. E. (1963). Erptl. Cell Res. 31, 321. Prince, A. M., and Ginsberg, H. S. (1957). J. Immunol. 79,94. Randall, C . C., and Walker, B. M. (1963). J. Bacteriol. 86, 138. Randall, C. C., Gafford, L. G., Gentry, G. A., and Lawson, L. A. (1965). Science 149, 1098. Robinson, L. B., Wichelhausen, R. H., and Roiunan, B. (1956). Science 124, 1147. Roizman, B. (1963). PTOC. Natl. Acud. Sci. U.S.49, 165. Roizman, B. (1969). In “Biochemistry of Viruses” (J. Levy, ed.), 11. 415. Marcel Dekker. Roizman, B., and Roane, P. R., Jr. (1964). Virology 22, 262. Roizman, B., Mayer, M. M., and Roane, P. R. Jr. (1959). 1. Zmmunol. 82, 19. Roizman, B., Borman, G. S., and Kamali-Rousta, M. (1965). Nature 206, 1374. Rott, R., and Muller, G. (1965). Arch. Ges. Virusforschung 17, 139. Rott, R., and Schafer, W. (1960). 2. Natuflorscli. 15b, 691. Rowe, W. P., Hartley, J. W., Roizman, B., and Levy, H. B. (1958). J. Erptl. Med. 108. 713. Russell, P. K., and Morgan, H. R. (1959). J. Inject. Diseases 104, 38. Russell, W. C., Hayashi, K., Sanderson, P. J., and Pereira, H. G. ( 1967). 1. Gen. Virol. 1, 495. Sabin, A. B. ( 1954). Science 120,357. Salzman, N. P., Lockart, R. Z., Jr. and Sebring, E. D. (1959). Virology 9, 244. S a h a n , N. P., Shatkin, A. J., and Sebring, E. D. (1964). J. Mol. Biol. 8, 405. Scholtissek, C., and Rott, R. (1965). Nature 206, 729. Scholtissek, C., Drzeniek, R., and Rott, R. (1966). Virology 30, 313. Shatkin, A. J. ( 1963). Nature 199, 357. Sokol, F., Cox, D. C., Dinka, S., and Ackermann, W. W. (1965). Proc. SOC. Erptl. Biol. Med. 119, 1015. Sonnabend, J. A., Martin, E. M., Mew, E., and Fantes, K. H. (1967). J. Gen. Viral. 1: 41.

108

BERNARD ROIZMAN AND PATRICIA G . SPEAR

Spring, S. B., Roizman, B., and Schwartz, J. (1968). J. Virol. 2, 384. Subak-Sharpe, H., Shepherd, W. M., and Hay, J. (1986). Cold Spring Harbor Symp. Quant. Biol. 31, 583. Summers, D. F., McElvain, N. F., Thoren, M. M., and Levintow, L. (1984). Biochem. Blophys. Res. Commun. 15, 290. Summers, D. F., Maize], J. V., and Darnell, J. E. (1965). Proc. Natl. Acad. Sci. U.S. 54, 505. Sydiskis, R. J., and Roizman, B. (1966). Science 153, 76. Sydiskis, R. J., and Roizman, B. (1967). Virology 32, 678. Tobey, R. A. (1964). Virology 23, 10. Tytell, A. A,, Lampson, G. P., Field, A. K., and Hilleman, M. R. (1967). Proc. Natl. Acad. Sci. US. 58, 1719. Uchida, S., Watanabe, S., and Kato, M. (1966). Virology 28, 135. Verwoerd, D. W., and Hausen, P. ( 1963). Virology 21, 628. Von Magnus, P. ( 1954). Virus Res. 2, 59. Wacker, W. B., Lipton, M. M., and Steigman, A. J. (1962). J . Immunol. 89, 525. Wagner, E. K., and Roizman, B. (1969). J . Virol. 4, 36. Wagner, R. R., and Huang, A. S. (1966). Virology 28, 1. Walker, D. L., Hanson, R. P., and Evans, A. S. (1957). Proc. Symp. Latency Masking Viral Rickettsia1 Infections. Burgess, Minneapolis, Minnesota. Wheelock, E. F., and Tamm, I. (1961). J. Exptl. Med. 114, 617. Willems, M., and Penman, S. (1966). Virology 30, 355. Wilson, D. E. (1968). J . Virol. 2, 1. Zimmerman, E. F., Heeter, M., and Darnell, J. E. (1963). Virology 19, 400.

CHAPTER 4

THE ROLE OF THYROID AND

GROWTH HORMONES IN NEUROGENESIS Max Hamburgh DEPARTMENT OF BIOLOGY,

CITY COLLEGE OF NEW YORK, AND DEPARTMENT OF ANATOMY, ALBERT EINSTEIN COLLEGE OF MEDICINE, NEW YORK, NEW YORK

I. Introduction ..................................... The Role of Thyroid Hormone in Neurogenesis . . . . . . . . A. Effects of Thyroid Hormone on Mammalian Neurogenesis ..................................... B. Theories of Mechanisms of Action of Thyroid Hormone on Processes of Neural Differentiation ...... 111. The Effect of Growth Hormone on Neurogenesis . . . . . . A. Observations ................................ B. Interpretations ............................... IV. Remarks ........................................ References ...................................... 11.

I.

109 110 110

129 139 139 140 143 144

Introduction

The action of hormones in development has been studied at three levels: (1) The “fundamentalist approach” aims to demonstrate that hormones influence and participate in the control of differentiation of various target structures; it relies on procedures that involve either removal of the source of the hormone, or administration of excess hormone during fetal life and subsequent observation of the effects of such treatments on postnatal stages. ( 2 ) The “analytical approach in fetal endocrinology focuses on critical periods during embryogenesis in an attempt to identify the specific developmental processes that are subject to hormonal interference during the maturation of selected embryonic targets. (3) The third ap109

110

M A X HAMBURGH

proach attempts to interpret hormone action in terms of ultimate cellular mechanisms; it aims to fit the effect of hormones on target structures with the “central doctrine of experimental biology” according to which differentiation reflects selective protein synthesis. Hormones, it has been asserted, may intervene in these processes through differential repression or derepression of genes that code for different proteins (Villee, 1969; Tata, 1965,1966, 1969). The notion that the thyroid influences neural development dates back centuries to the observations that in mentally retarded human cretinoid babies the thyroid fails to develop normally. The implications of this were not recognized for a long time. Experimental embryologists were for many years preoccupied with Speeman’s organizer, even though the discovery of Gudernatsch (1912, 1914) that amphibian metamorphosis is initiated by thyroid hormone drew attention to the significance of hormones in development ( Eayrs, 1964a; Kollros, 1968 ) . Neuroembryologists, under the leadership of Hamburger (1928, 1934, 1939, 1958), LeviMontalcini and Levi (1942, 1943), Detweiler (1923, 1924), and Detweiler and Lewis (1925) focused their attention almost exclusively on the mechanism by which peripheral structures signal to the developing nerve centers. Thus, only 15 years ago it was stated that “there is very little direct evidence to prove that hormones act upon the nervous system” ( Reach, 19.52) . II. The Role of Thyroid Hormone in Neurogenerir

A. EFFECTSOF THYROID HORMONE ON MAMMALIAN NEUROGENESIS 1. General Speculations about the role of the thyroid in mammalian neurogenesis have been much influenced by the hypothesis that in mammals there may be a critical period of development during which rapid changes in the morphology and physiology of prospective nerve and glia cells take place, analogous to changes in amphibian metamorphosis. Whether the presumed analogy between this critical period in mammalian development and the more dramatic thyroid-mediated metamorphic changes in amphibians is meaningful or merely superficial (Myant, 1966; 1969) has prompted many to study the effect of thyroid hormone on the developing mammalian central nervous system. The consequences of experimental thyroid deficiency during the prenatal or neonatal period have their counterpart in the human cretinoid baby. Cretinism is characterized by various degrees of mental retarda-

4.

ROLE OF HORMONES IN NEUROGENESIS

111

tion. The mental disturbance in hypothyroid babies becomes noticeable within 6 months after birth; it seems to be the result of prenatal damage to the developing brain as is evident also from the fact that the mental symptoms of cretinism can be reversed if thyroid hormone treatment is begun at an early age (Brown et al., 1939; Smith et al., 1949; Andersen, 1969). Neurological and mental disturbances are the most important lesions in severe congenital hypothyroidism ( Andersen, 1966; Money and Lewis, 1964). The electroencephalogram (EEG) in very young hypothyroid children may show a slow alpha rhythm. A recently reported retardation in the development of the normal sleep pattern in EEG's of newborn and older babies indicates that the neural connection between brain stems and cerebral cortex may have been retarded (Schultz et al., 1967). (For a review, see Andersen, 1969.) The credit for launching a modern and a systematic study of the influence of thyroid hormone on the developing mammalian nervous system belongs to J. T. Eayrs. His investigations were based entirely upon studies with the laboratory rat which is a particularly suitable animal for studies of hormonal effects on nervous development because its brain is relatively immature and undergoes after birth maturational events which in many other mammals occur during intrauterine life. In the rat deprivation of thyroid can be achieved early in the newborn by surgical removal of the gland, or by a single injection of lS1I to the newborn rat, or by feeding goitrogens such as thiouracil to the suckling mother who will pass it through the milk to the newborn.

2. Evidence from Behavioral Studies Eayrs and Lishman (1955) and Eayrs (1964b) first tested the effect of deficiency as well as excess of thyroid hormone during the period of postnatal maturation of young rats on the appearance of several automatic and innate responses. Their results clearly suggested that deprivation of thyroid hormone at or soon after birth caused retardation of neural mechanisms responsible for mediation of automatic behavior, while excess thyroid hormone caused an accelerated emergence of this behavior. Hamburgh and Vicari (1957) first reported precocious eye opening, precocious startle, and other auditory responses in hyperthyroid mice; this was confirmed for rats by Schapiro and Norman (1967) and Schapiro (1968), who also reported that young adult rats treated with thyroxine since birth learned to avoid an electric shock faster than did controls. Lasting damage can be imposed on the maturing nervous system by early exposure of thc! central nervous system (CNS) to excess thyroxine (Eayrs,

112

M A X HAMBURGH

1964b, 1969, Khamsi and Eayrs, 1966). Despite the initial precocity associated with hyperthyroidism, adult rats made hyperthyroid at birth perform less well than littermate controls when confronted with the Hebb-Williams closed field test which is a most sensitive index of impairment of cerebral function (Eayrs, 1964b, 1969). Considerably more attention has been devoted to the effect of thyroid deficiency on the developing nervous system. Eayrs and Lishman (1955) showed that cretinous rats ran a simple T maze more slowly and with greater error score than their normal littermates. Adult rats thyroidectomized at birth made significantly more errors than controls in learning a simple escape-avoidance task in response to a conditioned auditory stimulus suggesting a diminished sensitivity to environmental change in the cretinoid rat ( Eayrs and Levine, 1962). Using the Hebb-Williams closed field test, Eayrs (1961) showed that the severity of impairment of the capacity for adaptive behavior was inversely proportional to the interval between birth and the time when thyroidectomy was performed; thyroidectomy effectively impaired achievement only when it was performed between birth and 10 days of postnatal age. Replacement therapy in animals thyroidectomized at birth proved effective only when begun during the first 10 days of postnatal age (Eayrs, 1953, 1959, 1966, 1969). Using a water escape response test devised by Essman and Jarvik (1961), Hamburgh et al. (1964) and Essman et al. (1968) reported that with few exceptions hypothyroid rats failed to “learn” even in the four trials offered. Unlike some of the automatic responses tested by Eayrs and Lishman (1955) and Eayrs and Levine (1962) the negative performance on the tests devised by Hamburgh et al. (1964) and Essman et al. (1968) did not improve with increasing age. Evidence that changes in behavior induced in rats thyroidectomized at birth is also reflected in the electroencephalogram was provided by studies of Bradley et al. (1960). That a developmental effect exerted by the hormone is responsible for the changes in EEG is further suggested by experiments in which the EEG of mature rats that had been thyroidectomized at 24 days of age was measured and found to be no different from that of controls. On the other hand, the EEG of young adult cretinous rats that had been given thyroid hormone from the 24th day of age was intermediate between that of normal and neonatally thyroidectomized animals. All these observations point to an important role of the thyroid hormone during a critical period of CNS development, during which changes in

4.

ROLE OF HORMONES IN NEUROGENESIS

113

the level of this hormone can cause either transitory acceleration or delay of some aspects of neural maturation. In the rat, this critical period extends over the first 10-14 days of life, and thereafter neither thyroidectomy nor excess thyroxine can change the course of normal brain development; nor can thyroid medication initiated after day 14 to the neonatally thyroidectomized rat reverse some of the damage to the maturing brain caused by the deprivation of thyroid hormone during that critical period.

3. Eljidence from Neurohistological Studies Considerable study has been directed toward the nature of the defect underlying the behavioral deficiencies in cretinoid rats ( Eayrs, 1955, 1960, 1964a, 1966, 1969). The present evidence suggests that thyroid hormone is neither available to nor required by the rat fetus during uterine life (Hamburgh et al., 1962). Therefore, the influence of thyroid hormone must be exerted during the postnatal period, The histogenesis of the cerebral cortex is initiated by migration of neuroblasts before they come to occupy their definitive places in the cortical laminae in which they later differentiate into their adult configurations and connection. In the rat these preliminary events, i.e., proliferation, and migration, are reaching their conclusion at birth. Thyroid hormone must therefore influence subsequent postnatal processes of the differentiation sequence. Studies of the structure and architecture of the brain of hypothyroid rats have revealed a reduced rate of growth both of perikarya and axons resulting in a cortex in which the cell population appears more packed, the density of the axonal neuropil is diminished, and the basal dendrites associated with each neuron are shorter and less branched (Eayrs and Taylor, 1951; Eayrs, 1955).The development of the axonal network is particularly impaired in the region occupied by specific afferent plexuses (layer 4 ) . Eayrs (1966, 1969) suggested that the interference with axonal and dendritic branching results in a drastic reduction in the probability of axodendritic interactions which could be responsible for the deficit in the behavior of the cretinoid rat (see Figs. 1 and 2 ) . Evidence that the development of the nerve terminals is affected by presence or absence of thyroid hormone comes also from the following biochemical data. Balazs et al. (1968, 1969) noted that in the cerebral cortex of neonatally thyroidectomized rats the activity of succinic dehydrogenase and glutamate decarboxylase which are associated with synaptosomal mitochondria and nerve terminals are decreased, while the activity of lactate dehydrogenase, an enzyme associated with cell sap, and of

114

MAX HAMBURGH

glutamate dehydrogenase, an enzyme associated with mitochondria of nerve cell perikarya, were less affected by thyroid deprivation. Perhaps the most noticeable morphological sign of neural differentiation is the formation of a myelin sheath around the axon. However, the evidence concerning the effect of thyroid hormone on myelinization is A Normal

Cretin

Difference

I 3

10

2

5

I

FIG.1. Effect of neonatal thyroidectomy on growth of cortical neuropil. ( A ) Axonal component density of axons (axons/mm2/104) in successive cortical laminae (solid) and difference between normal and cretinous rats (dotted). ( B ) Dendritic component number of dendrites of successive distance from center of perikaryon. XX normal; 0cretinous; abscissa = 18 p intervals. (From Eayn 1969.)

somewhat contradictory. It has been reported that in the hypothyroid animal myelinogenesis of the developing brain is retarded and that the total amount of myelin in some tracts is reduced (Barnett, 1948). Precocious myelinization was reported (Schapiro, 1968) in infant rats that had received thyroxine for 4 days before sacrifice. Confirmation that defects of myelinization are one of the consequences of thyroid deficiency

4.

ROLE OF HORMONES IN NEUROGENESIS

115

comes from experiments by Balazs et al. (1969) who found that the lipid content of the brain was reduced in rats thyroidectomized at birth. The constituents most affected were the cerebrosides. On the other hand, A

Behavior

B

Dendrit ic field

Axo-dend rit ic interact ion

FIG.2. Correlation between behavior scores, rate of decay of the dendritic field, and probability of axo-dendritic interaction in ( A ) rats thyroidectomized at the ages shown (days) and ( B ) rats thyroidectomized at birth and given thyroid hormone from the ages shown. The data indicate the mean differences between measures taken from thyroidectomized animals and littermate controls. ( From Eayrs, 1969.)

Myant and Cole (1966) and Myant (1969) found that thyroxine injected into immature rats during the period of active myelinization had 7u) significant effect on the ratio of myelin to nonmyelin phospholipids in the whole brain, although there was an increase in the total amount of brain

116

M A X HAMBURGH

phospholipids. These authors also failed to find any effect of thyroxine on the relative rates of incorporation of szP into each of the four classes of phospholipids. Myant (1966) concluded that treatment of newborn rats with thyroxine in dosages high enough to bring about precocious maturity, such as earlier eye opening, had no selective effect on increasing the rate of deposition of myelin phospholipids. Hamburgh ( 1966) tested the effect of excess thyroxine added to tissue culture medium in which cerebella of newborn rats were developing. The most consistent effect observed was the accelerated appearance of myelin in thyroxine-supplemented cultures (Fig. 3). In the same study, he also observed a much more rapid disappearance of newly formed myelin in cerebellar culture maintained in thyroxine-supplemented medium as compared with those maintained without hormone. Possibly the thyroxine may exert its effect on myelinogenesis by a one step triggering reaction rather than by influencing a sequence of synthetic reactions of myelin precursors. Normally cerebellar cultures obtained from newborn rats begin to myelinate by day 12-14. However, if explants are incubated at 29 to 30°C,myelin does not form. If, after 14 days the cultures are shifted from 29 to 35"C,myelin will appear in less than 24 hours (Hamburgh, unpublished) indicating that all the precursors must have been synthesized and only a final step necessary to form the visible myelin sheath is temperature-sensitive. Within certain limits this temperature effect can be mimicked by thyroxine. Adding thyroxine to the medium will enable cerebellar cultures that are incubated at 29" to 30°C to form some myelin at such low temperatures (Hamburgh, 1966 ) . Attempts to establish a positive correlation between damage to cerebral architecture due to thyroid deficiency and behavioral changes have not been altogether successful. It has been stated by Eayrs (1969) that neonatally thyroidectomized rats given thyroxine replacement therapy will recover normal neurohistological architecture without a corresponding amelioration of all the behavioral deficiencies consequent upon lack of thyroid hormone during maturation. This suggests that the developmental anomalies underlying the persistent functional deficiencies are due to biochemical errors the detection of which will require more subtle methods. 4. Evidence from Metabolic Studies It has been reported that excess thyroxine accelerates metabolic maturation of brain tissue, as measured by rates of 0 2 consumption of

4.

ROLE OF HORMONES IN NEUROGENESIS

117

glucose by brain slices (Reiss et al., 1956; Hoexter, 1954; Fazekas et al., 1951). Fazekas et al. (1951) demonstrated a small but significant elevation of the rate of respiration (0,consumption) in whole brains of

7 8 9 10 II 12 13 14 15 16 17 18 19 20 21 Days after explantation FIG. 3. Emergence of myelin in cultures of cerebella with and without thyroxine treatment obtained from newborn rats. The cultures were maintained at 36°C. Control cultures were grown in standard medium. Experimental cultures were grown in medium supplemented with thyroxine. The number of cultures in each group is given in parantheses. 0 = control cultures; 0 = experimental cultures. (From Hamburgh, 1966. )

thyroxine-treated young rats that lasted until adult levels were reached at about 30 days of age. The respiration of hyperthyroid young rats at any stage resembled that of 2 to 3 day older controls. Hamburgh et al. (1964) tested the effect of administration of triiodo-

118

M A X HAMBURGH

thyronine starting at birth and continuing throughout the period of weaning on the respiration of three brain areas: the cerebral cortex, the cerebellum, and the medulla oblongata. They reported an increase in 0 2 consumption of cerebral cortex and cerebellum but not of medulla oblongata that was significant only during the first week of postnatal age. The period during which the maturing brain responds with increased respiratory rate to the availability of excessive amounts of thyroxine is probably very limited. This might explain why thyroxine administration to the newborn animal did not cause the expected increase in 0 2 consumption of the brain (Schapiro, 1968). Using a more critical test Ghittoni and Gomez (1964) confirmed that in brain slices of hypothyroid rats there was a significant decrease in 0 2 uptake between day 12 and 20 of postnatal life, while at 30 to 40 days of age the differences between hypothyroid rats and controls were no longer significant. The O2 uptake of brain slices of hypothyroid rats was not stimulated by potassium at high concentrations which enhance respiration and glycolysis. The authors postulated the failure of brain cells of hypothyroid rats to respond to the high concentration of potassium might be related to delay in mitochondrial maturation (Ghittoni and Gomez, 1964). According to Gomez and de Guglielmone (1967)neonatal thyroidectomy also depresses the rate of conversion of glucose into amino acids, and produces a temporary depression in cortical levels of glutamate, glutamine, gamma aminobutyric acid, and a permanent decrease in aspartate. The very limited effects which thyroid withdrawal or excess thyroxine exert on the respiratory metabolism of the developing nervous system contrast with the well-known metabolic actions which thyroxine exerts on a variety of adult tissues, and makes it unlikely that energy metabolism is the key %mechanismthrough which this hormone influences growth and differentiation of nerve cells and nerve cell circuits.

5. Evidence from Studies on Enzyme Activities Evidence that the calorigenic effect of thyroxine may be secondary to itsreffect on synthesis of enzyme proteins was provided by Weiss and Sokoloff (1983).The administration of puromycin, a drug which blocks protein synthesis, reverses also the hypermetabolism induced in rats by prior administration of thyroxine, and restores the 0 2 consumption of thyroxine-treated rats to the level of normal controls. The proposition that the metabolic alterations observed in the maturing mammalian brain as a consequence of manipulation with thyroid hormone may be secondary to the effect of the hormone on the synthesis of

4.

119

ROLE OF HORMONES IN NEUROGENESIS

specific enzymes and other proteins was also tested by Hamburgh (1955) and Hamburgh and Flexner (1957). They demonstrated that in the cerebral cortex the development of succinic dehydrogenase and cholinesterase was irreversibly impaired when thyroid hormone was withheld from the newborn rat (Figs. 4 and 5), but such treatment was without effect on the increase of aldolase and cytochrome oxidase in the maturing rat brain. Hormone therapy in thyroidectomized young rats, when started on the 10th day of postnatal life, led to a normal level of succinic dehydrogenase activity in young adults (25 days of age), but such therapy I

3

I

1

I

I

I

1.0 -

0.

1

-

I

I

I

I

I

120

M A X HAMBURGH

pretation is the existence of a lag period between hormone manipulation and the observable end effect. The efficacy of thyroid replacement therapy in bringing enzyme concentration to normal levels depends on whether it is instituted before or after the critical period of enzyme synthesis. This implies that in the absence of thyroid hormone during this critical period some irreversible changes may occur in the developing cerebral cortex. The reduction in the activity of succinate dehydrogenase in cerebral cortex of hypothyroid rats first reported by Hamburgh and Flexner ( 1957) has hence been confirmed (Garcia Argiz et al., 1967; Pasquini et al., 1967).

Glutamate dehydrogenase in the brain, another mitochondria1 enzyme, has been found to be less affected by thyroidectomy (Balazs et al., 1968, 1969) than succinic dehydrogenase (see Table I and Fig. 4 ) . About 75% of glutamate dehydrogenase activity is contained in mitochondria of cell bodies of nerve and glia cells, whereas about 75% of succinic dehydrogenase is located in the synapstosomal mitochondria (Salganicoff and De Robertis, 1965; Balazs et al., 1966). These observations suggested to Balazs et aZ. (1988, 1969) that the mitochondria of the nerve terminals

4. ROLE

OF HORMONES IN NEUROGENESIS

121

are probably more sensitive to thyroxine or lack of it than those of the cell bodies. The demonstration of a selective effect of thyroid hormones on enzymes associated with the synaptosomal fractions has since been extended to glutamate decarboxylase ( Balazs et al., 1968, 1969). Gee1 and Timiras (1967a) have shown that thyroid hormone deficiency induced at birth by radioiodine treatment is accompanied by a 15% Thyroidectomi zed

k 0.28

2 0.28

Group I

Thyroid deficient animal with therapy

k 0.31

? 0.40

Nr7

N=3

,up 2

Group 3

FIG. 6. Effect of replacement therapy on succinic dehydrogenase activity of animals thyroidectomized at birth. In Group 1 hormone therapy was started at the 10th day and continued for 15 days; in Group 2 therapy was started at the 15th day and continued for 15 days in six animals, for 50 days in one animal, and for 60 days in one animal; in Group 3 therapy was started at the 20th day and continued for 15 days. Over each bar, the height of which represents the mean, is placed the standard error of the mean, and N = the number of animals in the group. Note that animals for this series came from a different strain derived from a different dealer than those used for determinations represented in Fig. 5. The base values of the controls differ correspondingly by 1 unit. ( Froin Hamburgh and Flexner, 1957.)

decrease in total cholinesterase (CHE) and a 26% decrease in acetylcholinesterase (ACHE ) concentration in cerebral cortex and hypothalamus. When enzyme activity was expressed per unit DNA or per cell, the reduction in CHE and ACHE was 26 and 35%, respectively. The

122

MAX HAMBURGH

TABLE I SOMEENZYMIC ACTIVITIES IN THE CEREBRAL CORTEX OF RATS THYROIDECTOMIZED AT BIRTH^ Glutamate decarboxylase Age ( days 1

Normal

Thyroidectomized

Alanine aminotrazferase Normal

Thyroidectomized

( I ) ( pmoles/g/min )

14 24 ( a )

(b) 32 46

24 ( b )

0.387 (2) 0.662 f 0.0053 (10) 0.657 f 0.0295 (5) 0.797 2 0.0170 (4) 0.767 f.0.0243 (4)

0.330 (2) 0.557 f 0.0115' -

(4)

0.548 f.0.01351

-

(9) 0.670 k 0.0218"

-

-

-

(4) 2.20 f 0.93 0.700 & 0.0245 (4) (3) (11) (pmoles/pg atom DNA-P/min) 0.291 f 0.0142 0.220 f 0.0065' -

Age

Normal

-

-

Thyroidectomized

Glutamate dehydrogenase Normal

( I ) ( pmoles/g/min)

Thyroidectomized

-

-

-

116 -t 3.1

110 f.4.6 (9)

21.1 f.0.87 (3) 20.9 f 0.59 (5)

20.8 2 1.40 (4) 21.4 2 0.67

21.7 t 1.73 (2) (4) (11) ( pmoles/ug atom DNA-P/min ) 51.4 f 1.86 44.4 t 2.219 16.1 2 0.54

20.3 f 1.28 (4)

-

113

-

109

(2)

24 ( b )

2.28 f 0.174 (3)

-

(5)

32 46

-

-

14 24 ( a )

(b)

1.16 (2) 2.30 & 0.95 (3)

-

Lactate dehydrogenase ( days 1

1.18 (2) 2.35 & 0.133

-

(9)

-

14.6 & 0.404

-

( a ) Female rats radiothyroidectomizecl at birth were used together with littermate controls at the age indicated; series ( b ) at 24 days of age contained both sexes. The enzymic activities were determined upon the homogenates of cerebral gray matter and are expressed as pmoles/gm wet wt/min (f.SEM; number of animals is in parentheses). The values underlined mean that the difference between the experimental and the control group is significant; the p values were
4.

ROLE O F HORMONES IN NEUROCENESIS

123

administration of thyroxine prevented these changes. Similarly Schapiro (1968) reported an increase in acetyl cholinesterase activity in cerebral cortex and hypothalamus in 13-day-old rats that had received exogenous thyroxine on postnatal days 2,3,4, and 5. The enzyme level of the medulla oblongata and cerebellum was unaffected by hormone treatment. Enzymic changes in line with those in the mammalian CNS have aIso been reported for the amphibian brain (Pesetsky, 1965a,b,c, 1966b, 1968a, 1969a), during metamorphosis when brain maturation is thyroid dependent, the administration of thyroxine to Rana pipiens larvae resulted in increased activity of NADPH ( TPNH ) diaphorase, glucose &phosphate dehydrogenase and thiamine pyrophosphatase ( TPPase) activity ( Figs. 7-10).

NADPH (TPNH) diaphorase activity in a cross-section through Fk. 7. ( I @ ) . U larva. Incubation was the hindbrain of a normal, untreated stage VIII R U ~ pipiens for 55 minutes. Note intensely black “pigment” granules. X40. (From Pesetsky, 1965c. ) FIG.8. (right). NADPH (TPNH) diaphorase activity in the hindbrain of a similar larva treated with L-thyroxine, 222 ~g/liter,for 6 days. This titer stimulated hind limb deve!opment to stage XI. The section was incubated “back-to-back” with the tissue shown in Fig. 7. Enzyme activity is increased throughout the section but is especially intense in the ependyma. Staining is most conspicuous in cells to either side of the median su!cus. Note “pigment” granules in the midventral ependymal cells beneath the median sulcus. X 4 0 . (From Pesetsky 1965c.)

124

MAX HAMBURGH

Conversely, the inhibition of metamorphosis by thyroidectomy in premetamorphosizing tadpoles is accompanied by a reduction in activity of these enzymes. These changes in enzyme activity in the tadpole brain are most marked in the ependymal layer throughout the hind brain. Since ependymal elements are believed by some to represent the major neuroglial population in the amphibian brain, Pesetsky (1969b) interprets his observations to mean that glial elements may be the primary target of thyroxine action in the maturing amphibian brain. This view agrees with the observations ( Pesetsky, 1968a) that amphibian ependymal cells have a high level of carbonic anhydrase activity, and that the activities of this enzyme vary markedly in response to thyroid manipulation. Since neuroglial elements contain levels of anhydrase activity far higher than neurons (Giacobini, 1964), Pesetsky’s hypothesis and its applicability on the mammalian CNS merit serious attention. 6. Eoidence from Studies of Protein Synthesis

The stimulatory effects of thyroxine on certain brain enzymes are paralleled by the effects of this hormone on overall protein synthesis, as measured by amino acid incorporation (Sokoloff and Klee, 1963; Klee and Sokoloff, 1964). From considerations presented above, one is led to assume that thyroxine stimulates selectively the synthesis of some brain enzymes while exerting no influence on others. Nevertheless, the spectrum of proteins affected by thyroxine must be sufficiently wide to be recognizable when total protein synthesis of the developing brain is measured. Gelber et al. (1964) measured the effect of thyroxine on D, L-leucineI4C incorporation into cell-free preparations obtained from adult and infant rat brains (Table 11). They reported that in preparations from the infant brain thyroxine stimulated the amino acid incorporation into protein when hydroxybutyrate is the oxidizable substrate. No hormone effect on incorporation was noted in cell-free preparations obtained from adult brains. In order to determine which of the cell fractions were involved mitochondria, microsomes, and cell sap were isolated from both mature and immature brain and mixed together in all possible combinations (Sokoloff and Klee, 1963).It could clearly be shown that thyroxine stimulation of amino acid incorporation depended on the source of the mitochondria1 fraction that was present in the incubating system (Fig. 11).Replacement of the adult brain mitochondrial fraction by infant brain mitochondria resulted in thyroxine stimulation of amino acid incorporation by a cell-free system

4. HOLE OF HORMONES IN NEUROGENESIS

126

MAX HAMBURGH

that consisted otherwise of components obtained from adult brain tissue. Replacement of the infant brain mitochondria with adult brain mitochondria in an otherwise “infant system” abolished thyroxine stimulation (Fig. 12). For a review see also Sokoloff (1967). Supplementing these findings with those reported by Balazs et al. (1968, 1969) it is conceivable that one of the specific sites at which hormonal influence is exerted in nerve cells or neuroblasts are the mitoTABLE I1 EFFECTSOF 6.5 x b’f L-THYROXINE ON D,L-hUCINE-1-14C INCORPORATION I N T O PROTEININ CELL-FREEFREPAFUTIONS FROM ADULT AND INFANT RATLIVER AND BRAIN@

Tissue

Animal age

Protein nitrogen content of homogL-Thyroxine enate effect (mg/ Control L-Thyroxine % flask) (cpm/mg) (cpm/mg) Acpm/mg

Liver

Adult (3) Infant ( 2 )

1.86 1.61

38.7 31.6

61.3 44.6

4-22.6 -I-13.0

Brain

Adult (7) Infant ( 6 )

1.12 1.13

11.7

8.9 50.2

- 2.8b

44.2

+ 6.0b

+57 +42 -21 + 14

0 From data of Gelber et ol. ( 1984). The data presented are the means of the values obtained in the numbers of experiments indicated in parentheses. The homogenates were prepared as previously described. Adult rats were 40-50 days old; infant rats were 15-16 days old. b Denotes statistically significant effect ( p < 0.05) as determined by method of paired comparison.

chondria, particularly those inhabiting the synaptosomes. It is difficult at present to reconcile these results with recent observations by Mitskevich and Moscovkin (1969) who examined by electron microscopy brains of hypothyroid rats and found a delay in the attachment of cytoplasmic free ribosomes to the membranes of the endoplasmic reticulum. Possibly the disturbance of mitochondria1 maturation is more lasting than the retardation on ribosomal maturation exerted by lack of thyroid. If the above observations are confirmed it might be well worthwhile to repeat the experiments of Sokoloff and Klee (1963) and test the incorporation capacity of cell-free brain preparation obtained from very young hypothyroid animals.

4.

127

ROLE OF HORMONES IN NEUROGENESIS

There is additional evidence that thyroxine stimulates protein biosynthesis in vivo as well as in vitro (Geel and Timiras, 1967b, 1969; Geel et al., 1967). Following the injection of uniformly labeled ~-1eucine-I~C in proportion to body weight, it was found that the specific activity of cerebral protein-bound leucine was reduced in hypothyroid rats, indicating a

1

Miiochondrio Microsomes Cell sap

I

I

-* I

A

I

I I

I I

A

I

I

A

I rnrnature

A A A

1 A A

A A I A A I

Adult*

FIG. 11. Role of mitochondria in the differences in the rates of amino acid incorporation into protein and on the thyroxine effects on amino acid incorporation in adult and infant brain preparations. The incubation conditions and assay procedures are the same as those described in Table 11 except that the cell fractions were derived from the tissues as indicated in the figure. The thyroxine concentration was 6.5 X 10-5 M. (I) indicates that the homogenate fraction was obtained from hmature brain, and ( A ) indicates that it was obtained from adult brain. The asten& indicates at least two components derived from sources. (From Klee and Sokoloff, 1964).

decrease in protein synthesis. This deficiency in protein synthesis could be prevented by thyroxine medication. Whether the influence of thyroid hormone is exerted on the level of transcription or translation is not clear. There is evidence for both. Gee1 and Timiras (1969) reported that neonatal hypothyroidism resulted in a significant decrease in cerebral cortical nuclear and cytoplasmic RNA content when expressed per unit wet weight or per unit DNA. These

128

M A X HAMBURGH

authors reported that the reduced RNA content in brains of thyroidectomized rats could not be accounted for by a depression of RNA synthesis. Measurements of the incorporation of orotic acidJ4C into the cerebral cortex of normal and hypothyroid rats revealed that by this criterion the rate of synthesis of RNA was not altered. Therefore, Gee1 and Timiras (1969) proposed that the rate of “utilization” or breakdown of cerebral RNA, rather than its rate of synthesis, may differ between controls and

80

- 20

1

t

Mitochondria Microsomes Cell sap

--

L L L

A L L

L A L

Liver*

L L A

A A A

L A A

A L A

A A L

Adult brain*

FIG. 12. Role of mitochondria in the difference in effect5 of thyroxine on amino acid incorporation into protein in adult brain and liver preparations. The incubation conditions and assay procedures were the same as those described in Table I1 except that the cell fractions were derived from the tissues as indicated in the figure. The thyroxine concentration was 8.5 X 10-6 M. ( A ) , and the asterisk are as in Fig, 11. L indicates the fraction is obtained from liver. (From Klee and Sokoloff, 1984).

hypothyroid rats. They concluded that the reduced RNA content observed in the brains of hypothyroid rats suggests an increased RNA turnover with no change in net synthesis. Balazs et al. (1969) were led to identical conclusions on the basis of similar observations. They found that the rate of amino acid incorporation into cerebral protein was decreased in neonatally thyroidectomized rats, while the synthesis of RNA (measured as incorporation of orotic acid-14C) was not changed by thyroidectomy during infancy; they concluded that thyroid hormone probably in-

4.

ROLE OF HORMONES IN NEUROGENESIS

129

terferes with translation rather than with transcription in the developing nervous tissue.

B. THEORIES OF MECHANISMS OF AC~IONOF THYROID HORMONE ON PROCESSES OF

NEURAL DIFFERENTIATION

One of the favorite objects for investigations into the mechanisms of thyroid hormone action in development is the Mauthner ( M ) cell situated in the hindbrain of amphibian tadpoles at the entrance of the vestibular nerve. In the larval brain, a pair of M cells can easily be distinguished from other neurons by their large size. After metamorphosis they undergo regression and lose volume until they can no longer be distinguished from the surrounding cells. Like other metamorphic events, the regression of these neurons is under thyroid control. Weiss and Rosetti (1951) reported that while M cells involute under the influence of thyroid hormone other cells in the medulla oblongata adjacent to M neurons increase in size as a result of thyroid treatment. It would appear from their observations that different neurons in the same locality, i.e., the M cells and the non-M cells can respond in different ways to local applications of thyroid hormone, resulting in what Weiss and Rosetti (1951) called changes in “opposite signs.” Regression of M cells is coupled to a burst of mitotic activity in the adjacent neural epithelium leading to an increase in the neighboring non-M cell population. These results and interpretations have since been disputed by Pesetsky and Kollros (1956) and Pesetsky (1962) who reported enlargement of both M cells and non-M cells subsequent to local implantation of thyroid pellets into the hindbrain of R a m pipiells larvae. Pesetsky and Kollros (1956) and Pesetsky (1962, 1966a) advanced an alternative hypothesis to account for the regression of M cells during metamorphosis. They proposed that during the latter portion of larval life the enlargement of M cells may be initially accelerated by thyroxine, the levels of which go up during metamorphosis. Mauthner’s cells may become so dependent on this hormone that the sharp drop in levels of circulating hormone which takes place after metamorphosis may result in their subsequent shrinkage. All workers agree that the administration of exogenous thyroxine stimulates ependymal mitoses. Since ependymal cells represent the major portion of the glial population, Pesetsky (1969b) suggested that mitotic stimulation might be limited to neuroglia and therefore that glia cells might be the major targets of thyroid hormone in the developing nervous system. Stimulation by thyroxine treatment of mitotic activity of neuroblasts in the lumbosacral spinal cord of R a m pipiens larvae was reported by

130

MAX HAMBURGH

Reynolds (1962, 1966). She found that the spinal cord of thyroxinetreated larvae failed to conform to the normal pattern of mitosis, according to which cells preparing for mitosis migrate to the ependymal area and showed instead a marked increase of mitotic figures adjacent to the cpendymal layer in thc spinal cord. In the mammalian brain a direct effect on proliferation of neuronal elements has been demonstrated by Legrand et al. (1961) and Legrand (1963, 1967a,b) and independently by Hamburgh et al. (1964). In the cerebellar cortex of rats one of the most striking effects of thyroid deficiency initiated during the late fetal period or shortly after birth is the persistence of the external granular zone long after this layer of cells has disappeared in control littermates. The postnatal development of the rat cerebellum during the first 3 weeks is characterized by extensive migration of cells from this transitory external zone to form the layer of granular cells of the cerebellum. This external zone is thickest at about 8 days after birth and subsequently diminishes progressively. It disappears about the 21st day at which time the surface of the cerebellum has acquired the characteristic aspect of the adult (Fig. 13). In hypothyroid rats, the external superficial granular layer of the cerebellar cortex is maintained in the “fetal” condition as late as the 28th day of age (Figs. 13, 14, 15a and b). After that date, the cerebellar cortex of hypothyroid rats begins to assume the normal achitecture even though the thyroid hormone may not be made available to the rat (Legrand et al., 1961; Legrand, 1963,1967a,b; Hamburgh et al., 1964). A lasting consequence of this delayed migration of cells from the external granular zone is a hypoplasia of the dendritic spread of the Purkinje cells ( Fig. 14). Golgi-Cox preparations by Legrand (1967a, b, 1969) have clearly revealed that this hypoplasia persists long after the rest of the cerebellar architecture has finally assumed its normal histological organization in such hypothyroid rats. Thyroxine treatment initiated before the end of the second postnatal week corrects all these defects, but a delay in replacement therapy beyond this day will not reverse the hypoplasia. Hamburgh and Burkart ( 1969), by means of radioautography, have shown that the fetal external granular layer that persists in hypothyroid rats after day 21, when it has completely disappeared in controls consists of cells that will actively take up tritiated thymidine, and therefore are presumably engaged in DNA synthesis preparatory to mitosis (Figs. 16 and 17). On the basis of these observations, Hamburgh (1968) and Hamburgh and Burkart (1969) hypothesized that thyroxine may push cells into

4.

ROLE OF HORMONES IN NEUROGENESIS

131

FIG. 13. Development of the arborization of the dendritic tree of Purkinje cells of a normal rat, 14 days old. Golgi-Cox preparation. X330. (From Legrand, 1967b).

132

MAX HAMBURGH

FIG. 14. Development of the arborization of the dendritic tree of Purkinje cells of a hypothyroid (propylthiouracil treated) rat, 14 days old. Treatment started at birth. Golgi-Cox preparation. X 330. (From Legrand, 1967b).

4.

ROLE OF HORMONES IN NEUROGENESIS

133

FIG. 15a. Section through cerebellum of a 21-day-old normal rat. The external granular layer has disappeared, the molecular layer is practically cell-free, and the typical architecture of the adult cerebellum is established. X 120. (From Hamburgh et al., 1964.)

134

MAX HAMBURGH

Section through cerebellum of a 21-day-old hypothyroid rat. The exFIG. 1%. ternal granular layer is still present as a multicellular layer. Extensive cell proliferation and migration of cells through the molecular layer is still visible. X 120. (From Hamburgh et a]., 1964.)

FIGS,16 ( t o p ) and 17 (bottom). Radioautographs of a cerebellum of a 21-dayold normal (Fig. 16) and hypothyroid (Fig. 17) rat sacrificed 6 hours after an injection of 1 kCi/gm body weight of tritiated thymidine and exposed for 2 weeks. X440. Note high concentration of grains in Fig. 17. (From Hamburgh and Burkart,

1969.)

136

MAX HAMBURGH

the “differentiative” phase by taking them out of the Proliferative or mitotic phase. By acting as a timer the optimal amount of thyroxine may turn off the proliferative phase, and thus allow the differentiative phase to begin. Excess thyroxine may turn off the proliferative phase too soon, while thyroid deficiency prolongs it, and thus delays the onset of differentiation. The wrong timing may then lead to both permanent as well as to transitory abnormalities. It is conceivable that the differentiation of the Purkinje network depends on some inductive influence exerted by the granule cells during their migration past the row of Purkinje neurons prior to taking their proper place in the internal granular layer. The possibility that an error in the passing of maturational processes of the developing nervous system may lead to permanent defects in the CNS was also advanced by Eayrs (1969) on the basis of behavioral studies of rats made hyperthyroid during the neonatal period. The notion that the effect exerted by thyroid hormone on the developing brain may be mediated by the hormone’s interference with the timing of the proliferative phase is further supported by studies of Zamenhof et al. (1969). They treated chick eggs with the goitrogen thiourea on days 10-13 of incubation, and found that the experimental animals on hatching displayed larger brain weights (39% heavier) with a 10% increase of total DNA. On the other hand, thyroxine introduced into 9-day-old chick eggs depressed the amount of DNA ( 5 4 % ) and presumably cell division. These differences in the total DNA of the whole brain are admittedly small. Possibly much greater differences could have been revealed by examining specific areas of the brain in which maturational periods coincide with the critical period of thyroid sensitivity. For the rat, such sensitive periods for brain maturation have been identified by Eayrs and Taylor (1951) and Eayrs and Horn (1955) and by Hamburgh and Flexner (1957). Gee1 and Timiras (1969) reported a significant increase in total DNA content of the cerebral cortex of rats thyroidectomized at birth, but failed to find any comparable effect when only the diencephalon of thyroidectomized rats was analyzed. This is exactly what one would expect for it attests to the regional specificity of thyroid hormone on brain development. The hypothesis that thyroxine may direct cells from the prolifera t‘we phase into the “differentiative” phase is attractive, particularly in view of the idea that growth and differentiation are mutually exclusive. One might speculate that the increased duration of the proliferative period might affect the timing of RNA synthesis and thus delay the production of important enzymes. Pasquini et a2. (1967) followed changes in nucleic acid content during postnatal development of the cerebral cortex and

4. ROLE

OF HORMONES IN NEUROGENESIS

137

cerebellum in normal rats and in rats thyroidectomized at birth. They noted that neonatal thyroidectomy results in substantial decreases in RNA and protein in both tissues, accompanied by a significant increase in the DNA content of the cerebral cortex and some increase in the cerebellum. Similar findings were reported by others (Geel and Timiras, 1967b, 1969; Geel et aZ., 1967) (Table 111). Speculations about the role of thyroid hormone in the differentiation and development of the mammalian CNS have been influenced by observations on amphibian metamorphosis which depends on thyroxine (Myant, 1969). Possibly the major process directly initiated by thyroxine in the sequence of metamorphic events in the tadpole is the “resorptive action” or “self-digestion” of larval structures to make way for the adult structures ( Etkin, 1955, 1964; Frieden, 1968; Kaltenbach, 1968; Weber, 1963, 1967). Tata (1965, 1966, 1969) has shown that the immediate consequence of thyroxine stimulation in metamorphosis is the synthesis of RNA in metamorphosizing tissue, presumably required for the formation or activation of lytic enzymes for the resorption of larval structures. The suggestion that thyroxine may influence mammalian CNS maturation also by stimulating degenerative changes is worthy of further testing ( Beaudoin, 1955, 1956; Race, 1961; Reynolds, 1962). Beaudoin ( 1955) first showed that in the developing lateral motor column of Rana pipiens larvae many of the cells present at the outset of development do not survive beyond the midlarval period. At the midlarval stage, when thyroid hormone secretion causes rapid growth of the hind limb, over one half of the motor cells of the lateral column are lost, while the surviving cells increase in size. Race (1961) showed that hypophysectomized tadpoles (whose limbs had been amputated), raised in solution of DL-thyroxine, lost one half of the lateral motor cells; he reasoned that thyroxine might hasten the degeneration of those motor cells which have lost or failed to establish peripheral connections. Observations of the effects of hormone on nervous tissue growth in &TO reported by Hamburgh (1966, 1968) also demonstrated resorptive effects of thyroxine. Explants of cerebella from newborn rats and mice maintained in culture according to procedures of Bornstein and Murray (1958) and Murray ( 1965) continued differentiation into neurons and myelinated axons in vitro. Addition of thyroxine to the culture medium hastened the breakdown of those myelin sheath that had already formed and resulted in rapid disappearance of myelin. In unsupplemented control medium myelin persisted for some time, even if the medium was not renewed for as long as 7-9 days. All these results, as well as the demon-

TABLE I11 EFFECTOF NEONATAL HYPOTHYRODIISAI, THYROXINE, A N D GROWTHHOFMOSETHERAPY ON NUCLEIC ACID CONTENT OF CEREBRALCORTICAL FRACTIONS AT 25 DAYS OF AGE* Group

RNAa

Normal

Nuclear Cytoplasmic

Hypothyroidb Hypothyroidb T4 Earlyd

Nuclear Cytoplasmic Nuclear Cytoplasmic

Hypothyroidb T4 Late

Nuclear Cytoplasmic

0.621 k 0.015 1.319k 0.022 0.5982 0.014 1.210k 0.017 p 0.613 2 0.027 1.3372 0.031 0.616& 0.017 1.271-+ 0.052

DNA=

RNA/DNAa

( pg/mg wet tissue)

( pg/mg wet tissue)

0.934 f 0.028

< 0.01

1.074-C 0.033 p 0.969 f 0.059 0.957 f 0.025

< 0.01

0.672 f 0.009 1.420f 0.068 0.569f 0.010 p 1.165 f 0.036 p 0.652 f 0.095 1.422f 0.080 0.673f 0.013 1.3222 0.067

< 0.001 < 0.01

Hypothyroidb Nuclear 0.594 f 0.017 1.058& 0.026 p < 0.01 0.5852 0.018 p < 0.001 and GHe Cytoplasmic 1.191k 0.017 p < 0.01 1.131 f 0.039 p < 0.01 5 Values are means & SEM. p Values refer to significant differences between experimental groups and normal controls. Pregnant mothers were maintained on a low iodine diet and female offspring were injected with 100 pCi 1311 at 1 day of age. 0 pg L-Thyroxine/100 gm body weight daily from 15th day of age. d rg L-Thyroxine/lOO gm body weight daily from 6th day of age. e 50 pg Bovine growth hormone/100 gm body weight daily from 6th day of age. * From Gee1 and Timiras (1969).

*

5

x

Fg m

4.

ROLE OF HORMONES I N NEUROGENESIS

139

stration by Weiss and Rossetti (1951) that M cells atrophy under the influence of thyroid hormone, warrant further testing of the hypothesis that thyroxine influences the maturing CNS through control of the neuronal cell population effected by two contrasting mechanisms, namely, the “phasing of proliferation,” and the “resorption of excess cell numbers.” 111. The Effect of Growth Hormone on Neurogenerir

A. OBSERVATIONS That growth hormone may have a direct effect on brain development was first suggested by Zamenhof (1941, 1942). Tadpoles of different age groups responded to intraperitoneal injections of growth hormone preparations with a 6 1 2 8 % increase in the cell population of the cerebral hemispheres. A similar increase (86.5%) in number of cells per volume of cortex was noted in rats born to mothers that had been given subcutaneous injections of growth hormone during pregnancy. In addition to the increase in cell number the offspring from growth hormone-treated mothers showed a 36% increase in weight of cerebral hemispheres, a 21% increase in the thickness of the cerebral cortex, and an increase in volume and cell density of the cortex. Since these early experiments were done with impure growth hormone preparation available at the time Zamenhof et al. (1966) repeated them recently using more refined methods and preparations. Pregnant rats were injected daily with highly purified bovine pituitary growth hormone from the seventh until the 20th day of pregnancy. After delivery total DNA in brains from control and experimental groups was determined and found to be 24% higher in the brains of experimental animals; this suggested an increase in the total number of brain cells in rats exposed to an excess of growth hormone during fetal life. Similarly, chick embryos treated on days 8-13 of incubation with bovine growth hormone showed a 4 7 % increase in brain DNA over that of controls (Zamenhof et al., 1969). Further scrutiny of these results (Zamenhof et al., 1969) has raised some doubts whether the effects of growth hormone were in fact due to a specific hormonal action on the dividing neuroblasts or whether the added hormone simply increased the protein pool available to the fetus. Different results were obtained by Clendinnen and Eayrs (1961) who treated pregnant rats with purified growth hormone and studied the newborn rats up to 140 days after birth. In their experience treatment with growth hormone did not result in an increase in the relative number of

140

MAX HAMBURGH

brain cells, but promoted a hypertrophy rather of the cortical cells associated with a hypertrophy of the dendritic field. They suggested that this hypertrophy, by increasing the probability of axo-dendritic interactions, might enhance learning ability and demonstrated that rats treated with growth hormone during fetal life committed significantly fewer errors than the controls when tested for learning capacity after attaining maturity (85 days of postnatal age). Block and Essman (1985) also found that offspringfrom mothers injected with growth hormone exhibited at maturity a significantly better performance in conditioned avoidance tests. B. INTERPRETATIONS Assuming for a moment that the prenatal administration of purified growth hormone can modify cerebral development as claimed by Zamenhof et al. ( 1988), Clendinnen and Eayrs ( 1981), and Block and Essman (1985) one is faced with the situation that, as Clendinnen and Eayrs (1961) put it, “reactions which depend for their integration upon subcortical structures are relatively little influenced by the experimental treatment, while cortically mediated behavior is significantly affected.” This is the opposite of what would be expected if the growth hormone exerted a direct and immediate effect on the nervous system at the time of its administration to the fetus, since in the rat the development of subcortical structures is completed before cortical matur at‘ion commences. Results of Clendinnen and Eayrs (1981) raise the problem of how an antecedent availability of excess of growth hormone influences processes of cortical differentiation that begin long after the administration of the hormone. Conceivably, growth hormone given prenatally influences cerebral development through a delayed action effect by stimulating neuroblast mitosis. Zamenhof et al. (1986) assumed that the increase in cell number is due to extension of the proliferation period, and that the cells most sensitive to the hormone are neuroblasts which normally have a short proliferation rather than glia cells which continue to proliferate after birth. The consequence of an enlarged population of neuroblasts in the brain would not become apparent in the rat until after birth when cell proliferation in the cortex of the rat has ceased and fiber outgrowth is starting. The main objection to a direct effect of growth hormone on cerebral maturation during intrauterine life is the uncertainty that maternal pituitary hormone can pass through the placenta (Gitlin et al., 1985; Laron et al., 1988). The divergent reports on modifications of cerebral development in mammalian embryos by growth hormone administration suggest

4.

ROLE OF HORMONES IN NEUROGENESIS

141

that either these effects of the hormone are indirect, nonspeciiic, and mediated through influences on the mother, or that a small amount of the hormone can cross the placenta to the fetus. There is evidence to support both views. Excess growth hormone administered to the pregnant mother might merely add to the maternal supply of nutrients, and protein deprivation before and during pregancy can indeed result in a significant decrease in neonatal brain weight and brain DNA (Zamenhof et al., 1969). In a comparative study between two groups of pregnant rats, one maintained throughout gestation on a 8% protein-containing diet, the other on a 27% protein diet, Zamenhof et al. (1968) could show that the brain weight of newborn offspring was reduced by 23% and total brain DNA by 10% in the low protein group. On the other hand, injection of 300 mg of bovine growth hormone into chick embryos increased neonatal brain DNA (Zamenhof et al., 1969), and it seems unlikely that the addition of 300 mg of this hormone protein would significantly enrich the large nutritional depot in the chicken egg. An alternative possibility that merits further study is that growth hormone may influence brain maturation through a mechanism of action shared with other hormones. Biological systems sometimes can utilize alternative pathways to assure “important” end results. Thus, Gomez et at. (1966) reported that rats thyroidectomized at birth and treated daily with growth hormone recovered their normal weight and their righting reflex; furthermore, O2 consumption of brain tissue, which was severely depressed by thyroid deprivation during postnatal maturation, returned to normal levels following growth hormone treatment. Hamburgh et al. (1964) and Hamburgh (1968) reported that the impairment of the thermoregulatory mechanisms typical of hypothyroid rats could be partially reversed by growth hormone replacement therapy so that hypothyroid rats given this hormone from birth were able to maintain an almost normal (35°C)internal body temperature upon reaching maturity, while hypothyroid rats were unable to adjust their internal temperature above 2830°C (Fig. 18). These observations suggest that the action of thyroid hormone upon some aspects of cerebral development can be mimicked by growth hormone. This is not restating that growth and thyroid hormone act synergistically in development, or that the effect of one hormone is mediated through its action on the other. The whole spectrum of developmental defects of hypothyroidism cannot be reversed by growth hormone replacement therapy started at birth as would be expected if the lack of thyroxine

142

MAX HAMBURGH

inhibited postnatal development of the brain primarily by shutting off growth hormone supply. The idea that growth hormone and thyroxine influence thc development of the central nervous system through a synergistic collaboration is

FIG. 18. Effect of chemical thyroidectomy and somatotrophin replacement therapy on internal temperature control. Croup 1 represents controls; in Group 2 thyroid deprivation was started prior to birth; Group 3 consists of animals deprived of thyroid hormone and simultaneously given somatotrophin replacement therapy; in Group 4 thyroid deprivation was started on day 10 of postnatal life. (From Hamburgh, 1968. )

rendered unlikely by results of Diamond (1968). He compared the brain growth in rats hypophysectomized at 6 days with their littermate controls and with hypophysectomized animals given thyroxine replacement therapy, and concluded that absence or presence of growth hormone at that time did not influence cortical development. Independence of the growing

4.

ROLE OF HORMONES IN NEUROGENESIS

143

brain in the postnatal rat from growth hormone has also been suggested by Walker et al. (1950). Thus, in the postnatal rat, growth hormone may function merely as one of several control mechanisms serving to diminish the need for thyroxine in subsequent development and thus limit the critical period of thyroid sensitivity. IV. Remarks

The question remains whether those changes in growth or differentiation that are brought about by hormonal interferences during fetal or early postnatal life represent responses to the direct action of the hormone or are due to indirect effects. Removal or addition of any hormone during ontogeny not only disturbs the remaining endocrine balance but initiates diverse metabolic changes in the mother, the embryo, or both; it is therefore difficult to determine with confidence whether the developmental changes that follow hormone manipulation are not mediated through effects on blood supply, nutritional state, metabolic rate, etc., rather than through direct intervention of the hormone with differentiation of the target cell. Tissue culture has provided a welcome tool to resolve some of these doubts, and it is generally agreed that in those cases where the in uiuo effect of a hormone on developing tissue can be continued in uitro a direct action is indicated. Unfortunately the available tissue culture methods do not always lend themselves well to the study of the effects of hormones on neurogenesis. Successful culturing of nervous tissue usually requires the presence in the culture medium of plasma, serum, or chick embryo extract and cannot be carried out successfully in any known synthetic medium. It is therefore not possible to control adequately or exclude hormones from the culture medium in which embryonic nervous tissue is to be grown, Tests for the effects of horniones on neurogenesis in uitro rely, therefore, heavily on experiments in which excess of hormone is made available to the developing neuroblasts. Studies in this direction have been started (Hamburgh, 1966),but the information that can be obtained in this manner is limited. The availability of a hormone above the optimum amount may not have any effect on development; yet this does not mean that requirements for the hormone do not exist. Fetal endocrinologists interested in the effects of hormones on the differentiation of nervous tissue may have to design synthetic media which will support nerve tissue in culture or employ serum or plasma obtained from animals from which the circu-

144

MAX HAMBURCH

lating hormone, whose effect is to be tested, has been excluded. Until that task is achieved hormone research on the developing nervous system in viuo will have to take into account possible effects of the endocrine imbalance on other physiological mechanisms that may conceivably be the primary targets of alteration in the hormonal environment of embryos. There is always an element of “arbitrariness” in the selection for comment from the large variety of papers that deal with the problems discussed here. The selection usually leans heavily towards the special interests of the reviewer, and the present case is no exception. Other hormones than the ones considered here play a most important role in neurogenesis. For example, evidence has recently been presented by Moscona ( 1989) and Moscona and Piddington (1968,1987) that certain corticosteroids precociously induce the accumulation of glutamine synthetase in the developing neural retina and enhance the differentiation of this tissue in uiuo and in culture; and Granich and Timiras (1989) have presented evidence that cortisol influences maturation of brain lipid patterns in young chicks. The role of sex hormones in neurogenesis has attracted considerable attention in recent years, but has been extensively reviewed elsewhere (Barraclough, 1967; Harris, 1964; Levine, 1966, 1967; Levine and Mullins, 1968).The only rationale that guided the author in the restriction of discussion to those hormones that were selected for review is that they conveniently illustrate to him different levels of information available at the time of writing, and point to areas in pressing need for exploration. ACKNOWLEDGMENT This paper is dedicated to Dr. L. B. Flexner, who suggested the problem of “Hormones in Neurogenesis” to me and in this way caused me many a sleepless night. REFERENCES Andersen, H. J. (1966).Med. Sn’. 7,210. hdersen, H. J. (1969).In “Proceedings of an International Symposium on Hormones in Development” ( M . Hamburgh and E. J. W. Barrington, eds.). Natl. Found., New York, in press. Balazs, R., Dahl, D., and Harwood, J. R. (1966).I. Neurochem. 13, 897. Balazs, R., Kovacs, S., Teichgraber, P., Cocks, W. A,, and Eayrs, J. T. (1968).J. Neurocbem. 15, 1335. Balazs, R., Cocks, W. A., Eayrs, J. T., and Kovacs, S. ( 1969). In “Proceedings of an International Symposium on Hormones in Development” (M. Hamburgh and E. J. W. Barrington, Eds.), Natl. Found., New York, in press. Barnett, R. J. ( 1948). Ph.D. Thesis. Yale University, School of Medicine. Barraclough, C. A. ( 1967). In “Neuroendocrinology” (L. Martini and W. F. Ganong, eds.), Vol. 2, p. 81. Academic Press, New York. Beach, F. A. (1952).Ciba Found. C o h q . Endocn’nol. 3, 209.

4.

ROLE OF HORMONES IN NEUROGENESIS

145

Beaudoin, A. R. (1955). Anat. Record 121, 81. Beaudoin, A. R. (1956). Anat. Record 125, 247. Block, J. B., and Essman, W. B. (1965). Nature 205, 1136 Bornstein, M. B., and Murray, M. R. (1958). J . Biophys. Biochem. Cytol. 4, 499. Bradley, P. B., Eayrs, J. T., and Schumalbach, K. (1960). Ekctroencephalog. Clin. Neurophysiol. 12, 467. Brown, A. W., Bronstein, I. P., and Kraines, R. (1939). Am. J. Diseases Children 57, 517. Clendinnen, B. G., and Eayrs, J. T. (1961). J. Endocrinol. 22, 183. Detwiler, S. R. (1923). J . Exptl. Zool. 37, 339. Detwiler, S. R. (1924). Am. J. Anat. 33, 407. Detwiler, S. R., and Lewis, R. W. (1925). J. Comp. Neurol. 39, 291. Diamond, M. C. (1968). Brain Res. 7, 407. Eayrs, J. T. (1953). Brit. 1. Animal Behauiour 1, 144. Eayrs, J. T. (1955). Acta Anat. 25, 160. Eayrs, J. T. (1959). Brit. J. Animal Behauiour 7, 1. Eayrs, J. T. (1960). Brit. Med. Bull. 16, 122. Eayrs, J. T. (1961). J. Endocrinol. 22, 409. Eayrs, J. T. (1964a) Arch. Biol. (Liege) 75, 529. Eayrs, J. T. ( 1964b). Animal Behauiour 12, 195. Eayrs, J. T. (1966). In “Scientific Basis of Medicine Annual Reviews” (J. P. Ross, ed.), p. 317. Athlone Press, London. Eayrs, J.T. (1969). In “Proceedings of an International Symposium on Hormones in Development” ( M. Hamburgh, and E. J. W. Barrington, eds. ). Natl. Found., New York, in press. Eayrs, J. T., and Horn, G. (1955). Anat. Record 121, 53. Eayrs, J. T., and Levine, S. (1962). J. Endocrinol. 25, 505. Eayrs, J. T., and Lishman, W. A. (1955). Brit. J. Animal Behauiour 3, 17. Eayrs, J. T., and Taylor, S. H. ( 1951). J. Anat. 85, 350. Essman, W. B., and Jarvik, M. E. (1961). Psychol. Rept. 8, 58. Essman, W. B., Mendoza, L. A., and Hamburgh, M. (1968). Psychol. Rept. 23, 795. Etkin, W. (1955). In “Analysis of Development” (B. H. Willier, P. A. Weiss, and V. Hamburger, eds. ), p. 631. Saunders, Philadelphia, Pennsylvania, Etkin, W. (1964). In “Physiology of Amphibian” (J. A. Moore, ed.), p. 427. Academic Press, New York. Fazekas, J. F., Graves, F. B., and Alman, R. W. (1951). Endocrinology 48, 169. Frieden, E. (1968). I n “Metamorphosis” (W. Etkin and L. I. Gilbert, eds.), p. 349. Appleton, New York. Garcia Argiz, C. A., Pasquini, J. M., Kaplim, B., and Gomez, C. J. (1967). Brain Res. 6, 635. Geel, S . E., and Timiras, P. S. (1967a). Endocrinology 80, 1069. Geel, S. E., and Timiras, P. S. (1967b). Brain Res. 4, 135. Geel, S. E., and Timiras, P. S. ( 1969). In “Proceedings of an International Symposium on Hormones in Development” (M. Hamburgh, and E. J. W. Barrington, eds.). Natl. Found., New York, in press. Geel, S. E., Valcana, T., and Timiras, P. S. (1967). Brain Res. 4, 143. Gelber, S., Campbell, P. L., Deibler G. E., and Sokoloff, L. (1964). J. Neurochem. 11, 221.

146

MAX HAMBURGH

Ghittoni, N. E., and Gomez, C. J. (1964).Life Sci. 3, 979. Giacobini. E. ( 1964). I n “Morphological and Biochemical Correlates of Neural Activity” (M. M. Cohen and R. S. Snider, e&.), p. 15.Hoeber, New York. Gitlin, D., Kumate, J., and Morales, C. (1965).J . Clin. Endocrinol. 25, 1599. Gomez, C. J., Ghittoni, N. E., and Dellacha, J. M. (1966).Life Sci. 5, 243. Gomez, C. J., and Ramirez de Guglielmone, A. E. (1967).J. Neurochem. 14, 1119. Granich, M., and Timiras, P. S. (1969).In “Proceedings of an International Symposium on Hormones in Development” (M. Hamburgh and E. J. W. Barrington, eds.), Natl. Found., New York, in press. Gudematsch, J. F. ( 1912).Arch. Entwicklungsmech. Org. 35, 457. Gudematsch, J. F. (1914).Am. J . Anat. 15, 431. Hamburger, V. (1928).Arch. Entwicklungsmech. Org. 114, 272. Hamburger, V. (1934).J. Erptl. Zool. 68, 449. Hamburger, V. (1939).Physiol. Zool. 12, 268. Hamburger, V. (1958).Am. J . Anat. 102, 365. Hamburgh, M. (1955).Anat. Record 121, 304. Hamburgh, M. (1966).Develop. Biol. 13, 15. Hamburgh, M. (1968). Gen. Comp. Endocrinol. 10, 198. Hamburgh, M., and Burkart, J. (1969).In “Proceedings of an International Syniposium on Hormones in Development” (M. Hamburgh and E. J. W. Barrington, eds.). Natl. Found., New York, in press. Hamburgh, M., and Flexner, L. B. (1957).J. Neurochem. 1, 279. Hamburgh, M., and Vicari, E. (1957).Anat. Record 127, 302. Hamburgh, M., and Sobel, E., Koblin, R., and Rhinestone, A. (1962).Anat. Record

144,219.

Hamburgh, M., Lynn, E., and Weiss, E. P. (1964).Anat. Record 150, 147. Harris, G. W. (1964). Endocrinology 75, 627. Hoexter, F. M. (1954).Endocrinology 54, 1. Kaltenbach, J. C. (1988).I n “Metamorphosis” (W. Etkin and L. I. Gilbert, eds.), p. 399. Appleton, New York. Khamsi, F., and Eayrs, J. T. ( 1968).Growth 30, 143. Klee, C. B., and Sokoloff, L. (1964).J. Neurochem. 11, 709. Kollros, J. J. (1968).Ciba Found. Symp. Growth Nervous System p. 179. Laron, Z., Pertzelan, A., Mannheimer, S., Goldman, J., and Guttman, S. (1966).Acla Endocrinol. 53, 687. Legrand, J. (1963).Arch Anat. Microscop. Morphol. Erptl. 52, 205. Legrand, J. (1967a).Arch. Anat. Microscop. Morphol. Erptl. 96, 205. Legrand, J. (1967b).Arch. Anat. Microscop. Morphol. Erptl. 56, 291. Legrand, J. (1969).I n “Proceedings of an International Symposium on Hormones in Development” ( M. Hamburgh and E. J. W. Barrington, eds. ), Natl. Found., New York, in press. Legrand, J., Kriegel, A,, and Jost, A. (1961).Arch. d’Atwt. Microscup. Morphol. 50,

506.

Levi-Montalcini, R., and Levi, G. (1942).Arch. B i d . 53, 537. Levi-Montalcini, R., and Levi, G. (1943).Arch. B i d . 54, 189. hvine, S. (1966).Sci. Am. 214, 84. bvine, S. ( 1967) I n “Symposium on Reproduction” (K. LissQk, ed.). Akadkmiai Kiad6, Budapest. Levine, S. and Mullins, R. F. (1966). Science 152, 1585.

4.

ROLE OF HORMONES IN NEUROGENESIS

147

Mitskevich, M. S., and Moscovkin, G. N. (1969). In “Proceedings of an International Symposium on Hormones in Development” (M. Hamburgh and E. J. W. Barrington, eds.). Natl. Found., New York, in press. Money, J, and Lewis, V. (1964). Ciba Found. Study Group 18. Moscona, A. A. ( 1969). In “Proceedings of an International Symposium on Hormones in Development” (M. Hamburgh and E. J. W. Barrington, eds.). Natl. Found., New York, in press. Moscona, A. A., and Piddington, R. (1966). Biochim. Biophys. Acta 121, 409. Moscona, A. A., and Piddington, R. ( 1967). Science 158, 496. Murray, M. R. ( 1965). In “Biology of Cells and Tissues in Culture” (E. N. Wiumer, ed.), Vol. 2, p. 373. Academic Press, New York. Myant, N. B. ( 1966). B i d . Neonatorum 9, 148. Myant, N. B. ( 1969). In “Proceedings of an International Symposium on Hormones in Development” (M. Hamburgh and E. J. W. Barrington, eds.). Natl. Foiind., New York, in press. Myant, N. B., and Cole, L. A. (1966). J. Neurochem. 13, 1299. Pasquini, J. M., Kapllin, B., Garcia Argiz, C. A., and Gomez, C. J. (1967). Bruin Res. 6, 621. Pesetsky, I. ( 1962). Gen. Comp. Endocrinol. 2, 229. Pesetsky, I. (1965a). Anat. Record 151, 399. Pesetsky, I. (196513). Anat. Record 151, 471. Pesetsky, I. ( 1965c). Gen. Comp. Endocrinol. 5, 411. Pesetsky, I. (1966a). Z. Zellforsch. 75, 138. Pesetsky. I. ( 1966b). Anat. Record 154, 401. Pesetsky, I. (1968a). Anat. Record. 160, 408. Pesetsky, I. (1969a). Gen. Comp. Endocrinol. Suppl. 2, 238. Pesetsky, I. (1969b). In “Proceedings of an International Symposium on Hormones in Development” (M. Hamburgh and E. J. W. Barrington, eds.). Natl. Found., New York, in press. Pesetsky, I. and Kollros, J. J. (1956). Exptl. Cell Re.s. 20, 477. Race, J,, Jr. ( 1961). Gen. Comp. Endocsinol. 1, 322. Reiss, J., Reiss, M., and Wyatt, A. (1956). Proc. Soc. Erptl. B i d . Med. 93, 19. Reynolds, W. A. ( 1962). Thesis. University Microfilms, Inc., Ann Arbor, Michigan. Reynolds, W. A. (1966). Gen. Comp. Endocrinol. 6, 453. Salganicoff, L., and De Robertis, E. (1965). 1. Neurochem. 12, 287. Schapiro, S. (1968). Gen. Comp. Endocsinol. 10, 214. Schapiro, S., and Norman, R. (1967). Science 155, 1270. Schultz, M. A., Schultz, F. J., and Parmele, A. H. (1967). Mschr. Kinderheilk. 115, 284. Smith, D. W., Blizzard, R. M., and Wilkins, L. (1949). Pediatrics 9, 1011. Sokoloff, L. (1967). Am. 1. Diseases Children 114, 498. Sokoloff, L., and Klee, C. B. (1963). Assoc. Res. Nerve Mental Disease Publ. 43, 371. Tata, J. R. ( 1965). Nature 207, 378. Tata, J. R. (1966). Deuelop. B i d . 13, 77. Tata, J. R. (1969). In “Proceedings of an International Symposium on Hormones in Development” (M. Hamburgh and E. J. W. Bamngton, eds. ). Natl. Found,, New York, in press. Villee, C. A. (1969). In “Proceedings of an International Symposium on Hormones

148

MAX HAMBURCH

in Development” (M. Hamburgh and E. J. W. Barrington, eds.). Natl. Found., New York, in press. Walker, D. G., Simpson, M. E., Asling, C. W., and Evans, H. M. (1950). Anat. Record 106,539. Weber, R. (1963). Ciba Found., Symp. Lysosomes, p. 282. Weber, R. ( 1967). In “Biochemistry of Animal Development” (R. Weber, ed.), p. 227. Academic Press, New York. Weiss, P., and Rossetti, F. (1951). Proc. Natl. Acad. Sci. US.37, 540. Weiss, W. P., and Sokoloff, L. (1963). Science 140, 1324. Zamenhof, S. (1941). Growth 5, 123. Zamenhof, S. (1942). Physiol. 2001.15, 281. Zamenhof, S., Mosley, J., and Schuller, E. (1966). Science 152, 1396. Zamenhof, S., Van Marthens, E., and Margolis, F. L. (1968). Science 160, 322. Zamenhof, S., Van Marthens, E., and Bursztyn, H. (1969). In “Proceedings of an International Symposium of Hormones in Development” ( M . Hamburgh and E. J. W. Barrington, eds. ). Natl. Found., New York, in press.

CHAPTER 5

INTERRELATIONSHIPS OF NUCLEAR AND CYTOPLASMIC ESTROGEN RECEPTORS* Jack Gorski, G. Shyamah,+ and U . Toft] DEPARTMENT OF PHYSIOLOGY AND BIOPHYSICS, UNIVERSITY OF ILLINOIS, URBANA, ILLINOIS

1. 11. 111. IV. V. VI. VII.

Introduction ............................................ Estrogen Receptors ...................................... Subcellular Location of Estrogen-Binding Agent . . . . . . . . . . . . . . Binding of Estrogens to the Cytosol ........................ Cell-Free Binding of Estrogen ............................. Nuclear-Cytosol Interrelationships ......................... Conclusions and Speculations ............................. References .............................................

1.

149 150 151 155 156 157 164 166

introduction

An essential feature in the evolution of multicellular organisms was the development of systems of intercellular communication represented by the nervous and endocrine systems. These systems differ probably only in degree from communication systems involved in embryological development and certain other cell-tocell or tissue-to-tissue interactions. It would appear that in most of these cases, chemical compounds moving from one cell to another cell serve as the means of communication. In order to understand such communication systems one first needs to examine the transmitting system, what the chemical messenger is, how it is synthesized, and how it is secreted. Only in certain endocrines is such information available to the extent that the general outlines of these processes can be discussed. In addition, to fully understand any communi-

*

Supported by grant A-MB327 from the National Institutes of Health.

t Present address: Cancer Research Genetics Laboratory, University of California, Berkeley, California. $ Present address: Department of Obstetrics and Gynecology, Vanderbilt University, Nashville, Tennessee.

149

150

JACK CORSKI, G . SHYAMALA, AND D. TOFT

cation system it is necessary to describe the receiving system in the target cells. In essence, such a system appears to need a molecular sensory or detection unit coupled to a means of amplification. Pharmacologists and endocrinologists have for many years referred to such sensory or detection molecules as “receptors.” Since the time at which the concept of receptors was postulated, numerous attempts have been made to characterize these theoretical receptor molecules and their amplification systems. No receptor molecule has as yet been isolated in pure form or characterized; hence we do not know very much about these systems in multicellular organisms. On the other hand, we may have a model for such sensory or detection molecules in the regulatory mechanisms operating in bacteria. While, with certain exceptions, bacteria are not concerned with communicating directly with each other, they must communicate with their environments which, in turn, often contain products of other cells. This type of communication in bacteria appears to be involved in feedback regulation of enzyme activity and in induced synthesis of enzymes. In both of these cases, a molecular model has evolved according to which allosteric proteins act as sensory or detection units as well as amplifiers. These models have been well described in the case of feedback regulation of aspartyl transcarbamylase activity ( Gerhart and Pardee, 1962; Gerhart and Schachman, 1965) and repression and derepression of gene expression on the Lac operon (Gilbert and Muller-Hill, 1967). Asparty1 transcarbainylase appears to bc a protein which has binding sites for its feedback regulator (sensory unit) and for its substrate (amplification system). The Lac operon repressor has binding sites for an inducer (sensory unit) and for a specific sequence of nucleotides in the DNA of the Lac operator locus (amplification system). In both cases interaction of small molecules with the sensory unit brings about modifications in the activity of the protein, probably by mechanisms discussed in great detail by Koshland and Neet (1968). It would not be surprising if the molecular sensory or detection systems of multicellular organisms evolved from such simpler systems, but this remains to be proven. With regard to this possibility, we will discuss, in this paper, the preliminary characterization of an estrogen-binding protein from the rat uterus and a theoretical model of its mechanism of amplification. II. Estrogen Receptors

Estrogenic hormones are associated with the control of growth and differentiation of the female reproductive tract. These hormones bring

5.

ESTROGEN RECEPTORS

151

this about apparcntly by regulating niacromolecular syntheses ( Mueller et al., 1958; Gorski et al., 1965). Within minutes after the administration of these hormones, changes in a number of metabolic activities can be detected. In trying to find out how estrogen brings about both the early and the later responses, a number of investigators have tried to elucidate the first step in the hormone's action: the initial interaction of the hormone with receptor molecules in the cells of the target tissue. Attempts to identify steroid hormone receptors go back a number of years. With the increasing availability of radioactive carbon-labeled steroids in the late 1940's and the early 195O's, a great deal of work was carried out on steroid uptake and metabolism. No marked differences between target and nontarget tissues were detected in uptake of 14C steroids. The liver appeared to be the principal site of metabolism of the steroids and, as a result, most of the studies on steroid hormone-tissue interactions were carried out in this tissue. Such studies have importance in the understanding of steroid metabolism, but do not contribute significantly to clarification of the function of hormones in their respective target tissues. In the late 1950's Glascock and Hoekstra (1959) and Jensen and Jacobson (1960) utilized tritium-labeled estrogens of high specific activity to show that there was a marked difference in the ability of the female reproductive tract to take up estrogen as compared to other tissues. Jensen and Jacobson (1962) showed that estrogen was not metabolized in the uterus of the immature rat. They also showed that the differential uptake in the uterus was seen only at physiological doses of estrogen, whereas at higher doses there was little difference between various tissues. These studies raised many questions concerning the mechanism of estrogen action. The most important of these was whether the binding of estrogen is directly related to the primary tissue response and hence actually involves the receptor; this question has not yet been answered and cannot be until we have a complete description in molecular terms of the primary action of estrogen. However, considerable evidence has accumulated, which indicates that the estrogen-binding agent of the uterus has the attributes of a physiological receptor. 111.

Subcellular Location of Estrogen-Binding Agent

Soinc of the earlier work done in our laboratory dealt with the subcellular location of the bound estrogen in the rat uterus after in vivo administration of 17P-e~tradiol-~H. The estrogen-binding agent is dis-

152

JACK GORSKI, G. SHYAMALA, A N b D. TOFT

tributed in crude nuclear-myofibrillar (800g X 10 minutes), mitochondrial (15,OOOg X 15 minutes), microsomal (105,OOOg X 60 minutes supernatant) fractions as shown in Fig. 1. Approximately half of the estrogen was bound to the nuclear-myofibrillar fraction of the uterus (Noteboom and Gorski, 1965). Shyamala and Gorski (1967, 1969) using an improved method of fractionation which 0.01pg injected 6o

r

1.6 I

0.1pg injected 4.0 X 10'4pg

1-5p9

n n lnnlllInnll

2.0 x 10-4po

8 x 10-6pg

d

U

FIG. 1. The distribution of radioactive estradiol in subcellular fractions of rat uteri. Uteri from five immature rats were pooled for each determination. At 0.01 pg injected dose, approximately 0.30% of the injected activity was recovered in the uterus and at 0.1 pg, 0.07% was recovered. (Noteboom and Gorski, 1965.)

results in relatively clean nuclei (Table I), found that most of the particulate bound estrogen was in the nuclei and furthermore that it was associated with a crude chromatin fraction. King et al. (1965) had previously suggested that chromatin was involved in binding estrogens to a mammary tumor; estrogen bound to chromatin prepared from calf endometrium was described by Maurer and Chalkley (1967). The chromatin preparation in all of these studies is relatively crude and con-

5.

INCORPORATION OF

Preparation

153

ESTROGEN RECEPTORS

TABLE I ESTRADIOL-3H INTO HOMOCENATE AND NUCLEAR FRACTIONS OF IMMATURE RAT UTERUS~ Specific activity

Bound estradiol (cpm/uteriu)

DNA

Protein

( Pghterus)

( pg/uterus)

(cpm/Pg DNA)

(cpm/pg protein)

5082 800

219 72

2448 228

23.2 11.1

2.07 3.50

559

47

100

11.8

5.59

Homogenate Nuclei Purified chromatin

The 17fi-estradiol-:iH (0.05 &rat) was injected intraperitoneally 3 hours prior to killing; 15 rats were used in this study. (From Shyamala and Gorski, 1967.) 5

tains basic and nonbasic proteins as well as RNA and DNA. Treatment of the nuclear fractions with degradative enzymes, as shown in Fig. 2 (Noteboom and Gorski, 1965) indicates that proteolytic enzymes reduced the amount of bound estrogen, whereas nucleases had no such effect. Treatment of the chromatin preparation with the same enzymes gave similar results (Shyamala and Gorski, 1967, 1969). These data suggested 1000

-

Bound estrodiol-H3 0

0-

Q

-

._

Control

I3 DNose

Cone of DNA

_-

A

RNase

8

Control

Trips in

DNase OL

0

15

30

45

60

0 Minutes

15

30

45

60

FIG.2. Effect of enzyme degradation on the release of estradiol-3H from the nuclear-myofibrillar (N-M) fraction of the rat uterus. Each point represents the mean value of two determinations on the equivalent of one half of a uterus. The lower curves on the right side of the figure show the amount of DNA remaining in the N-M fraction after incubation with the enzyme. Incubations with 500 pg of RNase or DNase were at 31°C. Incubation with 5 pg trypsin was at 10°C. (Noteboom and Gorski, 1965.)

154

JACK GORSKI, G. SHYAMALA, AND D. TOFT

that the estrogen bound to the nuclear fraction was associated with a protein and not directly with nucleic acid. In receptor theory an important consideration is the specificity of the interaction. When various unlabeled steroids or nonsteroid estrogens are injected into the rat prior to injection of 17fl-e~tradiol-~H(Fig. 3), only physiologically active compounds like diethylstilbestrol ( DES ) are found to interfere with the uptake of labeled 17P-e~tradiol-~H (Note-

7Z% 0)-

Ex

cg 10.0

.-

?;-

0

.o g TIL ow

2% u)

0)w

-5

5.0

% .:

.o c

lLi.E! +

- 0

5u'ce

0

0

0 Hours

FIG.3. Effect of 2-hour pretreatment with testosterone, cortisol, diethylstibestrol, and 17a-estradiol on the incorporation of 17p-estradiol-sH into the nuclear-myofibrillar (N-M) fraction of the rat uterus. The amount of hormone injected as the pretreatment was 1.0 pg, and the amount of estradiol-SH injected was 0.01 pg. Each point represents the mean of values from four individual uteri expressed as pmoles x 10-8 per N-M fraction of one uterus. Estradiol was extracted with ethanol, ethanolchloroform ( l:Z),and ether. (Noteboom and Gorski, 1965.)

boom and Gorski, 1965). Preinjection of 17a-estradiol, a stereoisomer of 17P-estradiol and a very weak estrogen had only a slight effect on the As shown by the reciprocal plot of Fig. 4, uptake of the 17P-e~tradiol-~H. the effect of DES appears to be due to competition with 17P-estradiol for the same binding site. These data demonstrated that the binding agent in the nuclear fraction was stereospecific for estrogens and inactivated by proteolytic enzymes; it was also inactivated by high temperature and other protein-denaturing agents, thus suggesting that the binding agent was a protein.

5.

ESTROGEN RECEPTORS

155

IV. Binding of Estrogens to the Cytosol

After in viuo injection of e~tradiol-~H the cytosol fraction (105,OOOg X 60 minutes supernatant fraction) contains approximately 30% of the estrogen. By means of sucrose density gradient fractionation it is possible to separate a specific estrogen-binding agent from the bulk of the cytosol proteins, as shown in Fig. 5 (Toft and Gorski, 1966). The only fraction

FIG. 4. Inliibition of the incorporation of estradiol-311 by diethylstilbestrol ( DES ) in the nuclear-myofibrillar fraction of the rat uterus. Each point represents the mean values of determinations of individual uteri. The brackets indicate standard errors. Various levels of the estrogens were injected ip simultaneously, uteri removed after 3 hours, and the estradiol extracted as described in Fig. 3. Radioactivity extracted from the uterus was converted to (ES) per uterus by using the specific activity of the estradiol-3H of 125 pCi/pg. (Elnlected)refers to the concentration of injected estradiol-3H. ( Noteboom and Gorski, 1965.)

which binds estrogen at low physiological doses of this hormone is one which has an estimated sedimentation constant of 9 S. A second peak ( 4 6 S ) of bound estrogen is detectable, but only at higher doses of injected hormone. Further studies in viuo showed that only the 9 S peak reflected the effects of estrogen competitors and that nontarget tissues such as duodenum, liver, and blood serum did not contain the 9 S material. Treatment of the cytosol fraction with various enzymes and denaturing agents again indicated that the 9 S material was protein.

156

JACK GORSKI, G. SHYAMALA, AND D. TOFT

2

4

Bottom

6 8 10 12 14 16 18 20 2 4 6 8 10 12 14 16 18 20 Fraction number

TOP

FIG.5. Density gradient patterns of the soluble fraction 3.5 hours after injection of various amounts of estradiol-3H. Each soluble fraction is from five uteri homogenized in 0.5 ml of medium (Tris, MgCI,, KCl); 0.2 ml was layered on 5 to 20% sucrose gradient and centrifuged for 9.5 hours at 39,000 revolutions per minute, 10°C. Estradiol injections: ( A ) 0.05 pg/rat; (B)0.1 pg; ( C ) 0.15 pg; (D)0.2 pg. Counting efficiency, 13-14%. Sedimentation is from right to left. (Toft and Gorski, 1966.)

V. Cell-Free Binding of Estrogen When small amounts of estradiol-3H were added directly to the cytosol preparation obtained from immature rat uteri and the cytosol then fractionated on sucrose density gradients, the results shown in Fig. 6 were obtained (Toft et al., 1967). Only the binding to the 9 S peak is apparent with smaller concentrations of estrogen while at higher concentrations of estrogen, binding also occurred in the 4-6 S region of the gradient. Figure 7 shows the saturation curves for the 9 S and 4-6 S peaks when various levels of estrogen were added to the cytosol preparation. These same data are expressed in Fig. 8 by means of a Scatchard (1949) plot. This plot enables one to estimate the dissociation constant (7 X 10-lo M) for 17P-estradiol and the total number of binding sites (20,OOO per cell), This latter figure is considerably higher than the estimate of 2500 molecules of estrogen per cell after in vivo estrogen administration (Noteboom and Gorski, 1965).

5.

157

ESTROGEN RECEPTORS

9.5 s

0

I

0

I 4

Bottom

I 0

I

12 16 Fraction number

I

I

20

J

24

TOP

FIG.6. Density gradient patterns of uterine soluble fraction after the addition of increasing concentrations of 17p-estradiol-3H. One-tenth milliliter of soluble fraction containing the amount of estradiol designated was layered on 5 to 20% sucrose gradients and centrifuged for 13 hours at 200,000 g, 3°C. Counting efficiency was 19%. (Toft et al., 1967.) Further attempts to purify the estrogen-binding agent have not been successful but are continuing. VI. Nuclear-Cytosol Interrelationships

When whole rat uteri are incubated in tissue culture medium at 37°C in the presence of e~tradiol-~H the steroid is taken up by the tissue; within 30 minutes the distribution of the label between the particulate

158

JACK COHSKI, C. SIIYAMALA, AND D. TOFT

(principally, the nuclear fraction) and the cytosol fractions is similar to that found after in uiuo administration of the labeled steroid (Fig. 9 ) ( Shyamah and Gorski, 1967, 1969). With a similar treatment of tissue at O”C, estradioL3H is taken up to some extent by the cytosol but there is very little uptake by the nuclear fraction. These observations suggested the possibility that movement of estrogen into the nucleus may be a two-step reaction which could be studied experimentally. Figure 10 shows the results of first exposing uteri to estradioPH at 0°C for 1minute, removing the medium containing the estrogen, and then incubating the tissue at 37°C. The labeled estrogen is able to bind to the 9 S cytosol receptor during the first 0°C incubation and then, during incubation at 37°C the estrogen moves into the nucleus.

7

14 28 TOTAL ESTROGEN (lo-’ pmoles)

71

FIG.7. Titration plot from the density gradient analysis of estradiol binding in the uterine soluble fraction. The sucrose gradient techniques were as described in “methods,” using 0.1 ml samples of the soluble fraction. The amount of estradiol-SH in the 9 S region, 3-8 S region, and free estradiol were determined and plotted versus total estradiol concentration. For these determinations, the radioactivity was measured and converted to pmoles of estradiol. The counting efficiency was 19% and the specific activity of estradiol was 140 fiCi/fig. (Toft et al., 1987.)

5.

159

ESTROGEN RECEPTORS

I

0

Amount bound (iO-'"M)

FIG.8. Determination of the dissociation constants and the number of binding sites. Using sucrose gradient analysis, measurements were made of the amount of estradiol bound to the 9 S component and the amount of "free" estradiol (total - 9 S bound estradiol). These values are adjusted to correspond to a protein concentration of 1 mg/ml. The actual protein concentration was 8.6/ml. The relationship used is: bound/free = 1 / K binding sites - bound where K = l/slope and binding sites = X-intercept. (Toft et d.,1967).

x

7 . /Particulate

ooc

Time of incubation (min)

FIG.9. The distribution of radioactive estradiol between the cytosol and particulate fraction, In each group two uteri from immature rats were incubated in 2 ml Eagle's medium containing 0.005 pg 17P-estradiol-3H at 0 or 37°C for various times as indicated. (Shyamala and Gorski, 1967, 1969.)

JACK GORSKI, G. SHYAMALA, AND D. TOIT

160

Previously, we had been unable to demonstrate specific binding when estrogen was added directly to cell-free preparations of purified uterine nuclei at either 0 or 37°C. In addition, chromatin (Shyamala and Gorski, 1967, 1969) extracts of nuclei in 0.3 M KC1-0.2 M POa, pH 7.4, [which had been shown to contain estrogen bound to a 5 S macromolecule after either in uiuo injection or whole tissue in uitro incubation [DeSombre et al., 1967)], did not bind estrogen. All of these observations suggested the possibility that initially the receptor was entirely in the cytosol. The estrogen-receptor interaction then results in the whole complex moving into the nucleus as shown in the model in Fig. 11.

*

x

Particulate

c 0

P

-4

-3 0

t

0

4-

-

'c

0

41

Cytosol

$

0

0

I

I

I

I

I

10 20 30 40 50 Time of 37OC incubation (min)

60

FIG.10. The release of radioactive estradiol from cytosol into particulate fraction upon incubation at 37°C. In each group two uteri were incubated at 0°C for 1 minute in 2 ml of Eagle's medium containing 0.02 pg 17fJ-estradiol-~H.The uteri were then transferred to fresh medium without any estradiol and incubated at 37°C for various times as indicated. (Shyamala and Gorski, 1967, 1969.)

This model was first tested by looking at what happens to the 9 S peak in the cytosol. Simultaneously with its appearance in the nucleus, the estrogen disappears from the 9 S region of the sucrose gradient, as shown in Fig. 12 (Shyamala and Gorski, 1967, 1969). What happens to the 9 S binding protein itself under these conditions? Figure 13 indicates that the 9 S binding protein in the cytosol loses its ability to bind estrogen when estradioPH is added directly to the cytosol fractions after various incubations of the whole tissue. If we assume that the 9 S protein had just released the estrogen to a nuclear receptor and that it had itself remained in the cytosol, it should have been free to bind

5.

161

ESTROGEN RECEPTORS Uterine cell

I

+;

Nucleus

I

Receptor

Estrogen

FIG. 11. Hypothetical model for estrogen interaction with uterine cell. (Gonki et al., 1968.)

875 0 +

-

750 -

3

.5 E

625-

&0. 500v)

2 3753

u”

250I25

-

OO

h 4 ;

Bottom

Ib 1; Id I k Fraction number

;1

2;

4

2; TOP

FIG.12. Sucrose density gradient patterns of uterine cytosol. In each group four uteri were incubated at 0°C for 1 minute, in 2 ml Eagle’s medium containing 0.05 kg 17~-estradiol-3H.The uteri were then transferred to fresh medium without any estradiol and treated as follows: G-1 had no 37°C incubation, in G 2 the uteri were incubated at 37°C for 15 minutes, and in G-3 the uteri were incubated at 37°C for 1 hour. Two-tenths milliliter samples of cytosol from each group were layered on 5 to 20% sucrose gradients and centrifuged at 45,000 rpm for 15 hours at 4°C. Counting efficiency was 19-2076, Data are adjusted to represent one uterus. (Shyamala and Gorski, 1967, 1969. )

162

TACK GORSKI, G. SHYAMALA, A N D D. TOFT

more estrogen under the cell-free conditions. It should be noted that at 37°C incubation of the cytosol without estrogen has no effect of the capacity of the 9 S protein to bind estrogen. Figure 14 presents the reciprocal plots of binding of estrogen in the particulate (nuclear fraction after incubations of whole uteri with different levels of estrogen (Shyamala and Gorski, 1969). The K,,, derived from these data for the whole process of estradiol

0 Bottom

I

5

I

10

I

15

Fraction number

I

20

i i TOP

FIG. 13. Sucrose density gradient patterns of uterine cytosol. Treatment of the uteri were as follows: in G-1 four uteri were incubated at 37°C for 1 hour in 2 ml Eagle’s medium, in C-2 four uteri were incubated at 0°C for 1 minute in 2 ml Eagle’s medium containing 0.05 pg 17fl-estradiol-sH, in C-3 four uteri were incubated at 0°C for 1 minute in 2 in1 Eagle’s medium containing 0.05 pg 17P-estradiol-3H and then transferred to fresh medium without estrogen and incubated for 1 hour at 37°C. In each case 0.005 wg 17fl-estradiol-3Hwas added directly to 0.4 ml of cytosol. Twotenths milliliter samples were layered on 5 to 20% sucrose gradients and centrifuged at 48,000 revolutions per minute for 14 hours at 4°C. Counting efficiency was 1920%. Data are adjusted to represent one uterus. (Shyamala and Gorski, 1967, 1969.)

5.

163

ESTROGEN RECEPTORS

movement into the nucleus is similar to the dissociation constant previously derived for the cytosol alone. Because the intracellular concentrations of estrogen may vary from that of the media this K , is only a crude estimate. The estimates of affinity (dissociation constants and Michaelis constants ) obtained in the above experiments were derived under different conditions and therefore are not directly comparable. However, these constants are closely related and the similarity of the values is in accord with a model in which only one receptor is involved. It is also interesting that the estimates of the total number of binding sites K,,

No. of nuclear bindinq sites = 8.3 x 10-13 moles/uterus

No. of nuclear binding sites; 1.0 x 10-13 moler/uterus

K m (Cytosol receptor complex)= 7.0 X 10-10M

K,

- ESTRADIOL-

G

= 0.62 x 10-9 M

K m = 9.1 X 10-10 M

H3

(Cytosol receptor complex)= 2 x 10-9 M

E S T R I O L - H3

0

1.0

G X

X

In

A’ 0 . 4

z

v)

w

-

\

0.2

-f

I

2

4

6

I / E mpmoles X lo3

2

a

I

4

.

I

6

I / E mpmoles X 10

FIG. 14. Determination of the dissociation constants and the number of binding sites in the particulate fraction. In each group two uteri were incubated at 0°C for 1 minute in 2 ml of Eagle’s medium containing known amounts of estradiol-3H or e~tri01-3H.The uteri were then transferred to fresh medium without any estrogen and incubated at 37°C for 1 hour. Radioactivity extracted from the uterus was converted to ( E S ) per uterus, and E refers to the concentration of absorbed estradiol-3H or est1iol-3H. The particulate fraction was prepared as described in “Methods.” (Shyamala and Gorski, 1967, 1969.)

164

JACK GORSKI, G. SHYAMALA, AND D. TOFT

derived from these calculations are less subject to error and are also similar. Again, these observations would fit a model in which all the receptor is initially in the cytoplasm and is able to move into the nucleus only under the influence of estrogen. Jensen et at. ( 1968), have recently made similar conclusions from a rather different line of evidence. VII. Conclusions and Speculations

The model of estrogen interaction with uterine cells discussed here is clearly speculative and needs more proof. In particular, evidence is needed that the receptor molecule actually moves into the nucleus after interacting with estrogen molecules. However, should future evidence further substantiate this model, it would then partly explain the primary action of estrogen. The initial sensory or detection system in the uterus is the estrogen-binding site on the cytosol receptor protein. This site is analogous to the allosteric or feedback control site in aspartate transcarbamylase mentioned in the introduction to this paper. The amplification system involves a conformational transition of the protein due to estrogen-binding which, in turn, permits the protein to move into or on the nucleus. The observation that the 9 S receptor molecule can be dissociated into a smaller molecule ( 4 5 S) with high salt suggests that interactions between protein subunits may be involved in the conformational changes (Erdos, 1968). Prescott ( 1963), Goldstein ( 1963), and others have presented evidence indicating that the cytoplasm has an effect on nuclear function and that this effect may involve the transfer of proteins from cytoplasm to nucleus. It would certainly be of interest if this transfer step were a controlling process in eukaryotic cell regulation. Once this protein is in the nucleus it might regulate gene expression at any of a number of sites ranging from a direct interaction with DNA to control of nuclear membrane transfer of RNA to the cytoplasm. Assuming that a hormone effects a transfer of a protein from the cytoplasm into the nucleus, let us speculate further that the changes in the cytosol also may be of considerable importance. If, for example, the cytoplasmic receptor proteins are associated with an intracellular membrane, one might expect various alterations in metabolic activities in the cytoplasm immediately after the interaction of estrogen with the receptor. Protein synthesis and lipid synthesis are associated with these membrane fractions, Some of the enzymes associated with glycolysis appear to be present partially in membrane-bound as well as in soluble forms. If these membranes are made up of closely interacting proteins and lipids, the change in conformation and solubilization of one such membrane protein

5.

ESTROGEN RECEPTORS

165

might have a great effect on the whole membrane and its various activities. The diverse responses to estrogen which are known to occur within minutes after hormone administration might best be explained by such a mechanism. The complex events which must be involved in gene expression and nuclear function in animal cells are difficult to visualize as being responsible for some of the cellular responses which occur within minutes after estrogen administration. Rapid changes via gene expression occur in bacterial systems, where translation closely follows transcription and where very few copies of a particular messenger RNA (mRNA) are present at any one time. On the other hand, regulation of gene expression in animal cells involves changing the rate of RNA synthesis, the transport of the finished mRNA to the cytoplasm, and its accumulation. Penman et al. (1968) recently reported a lag of 15 minutes before newly synthesized mRNA could be detected in polysomes. Furthermore, because of the large numbers of RNA and protein molecules of any one type in animal cells, it is necessary to discuss accumulation of these macromolecules in terms of modifying the pool size of any particular RNA and again of its particular protein product. Berlin and Schimke (1965)have discussed the problem of changes in accumulation of protein in terms of both synthesis and turnover. Proteins which have a rapid turnover will show rapid changes in accumulation when protein synthesis is increased. Proteins which have a slow turnover will come to a new steady state accumulation at a slower rate. The same kind of analysis holds true with RNA and is particularly important when one is comparing control of bacterial messenger RNA, where turnover is a matter of seconds and minutes, to animal cell RNA where turnover appears to be much longer, i.e., hours or perhaps even days. It is possible that increased synthesis of an RNA having a rapid turnover and therefore a relatively small pool size might bring about a rapid modification of metabolic activity. Our only evidence for early involvement of gene expression with estrogen action comes from studies using inhibitors of RNA synthesis (Ui and Mueller, 1963).Studies indicating early estrogen effects on precursor incorporation into crude RNA fractions do not provide answers to the problem raised above. The varied responses of the uterus to estrogen occurring at longer periods of time are more likely to be due to regulation of gene expression and would require some interaction, direct or indirect, with the nucleus. In our speculative model of estrogen action, the estrogen interacts with a cytoplasmic receptor protein to modify its conformation. This results

166

JACK GORSKI, G . SHYAMALA, AND D. TOFT

in the protein moving from the cytoplasm and therefore modifying some cytoplasmic function. The receptor enters the nucleus where it now has an effect on the apparatus involved in gene expression. We thus have the possibility of both a cytoplasmic and a nuclear event resulting from the interaction of estrogen with one type of receptor molecule. Whether the above model of the initial interaction of one particular cell regulator will have any general applications, particularly in developmental biology, is problematical. Estrogens are still small, relatively simple molecules which are not dissimilar to the regulators discussed by Monod et al. ( 1963), in their classical paper on allosteric proteins. However many of the regulators of animal cells, including inducing factors and hormones, are complex macromolecules which have a much greater information potential and therefore more possibilities as to mechanism of action. It is of interest that macromolecular effectors have not been described in bacterial systems and subsequently have not been discussed in terms of molecular models of cellular regulation. In any case, it seems probable that models of the mechanism of action of protein and small molecule hormones will lay the foundation for the understanding of intercellular and developmental control systems in higher organisms. REFERENCES Berlin, C., and Schimke, R. (1965).Mol. Pharmacol. 1, 149. DeSombre, E., Hurst, D., Kawashima, T., Jungblut, P. W., and Jensen, E. V. (1967). Federation Proc. 26, 536. Erdos, T. (1968).Biochem. Biophys. Res. Commun. 32, 338. Gerhart, J., and Pardee, A. B. (1962).J . Biol. Chem. 237, 891. Gerhart, J., and Schachman, H. K. ( 1965). Biochemistry 4, 1054. Gilbert, W.,and Muller-Hill, B. (1967).Proc. Natl. Acad. Sci. U.S. 58, 2415. Glascock, R. F., and Hoekstra, W. G. (1959).Biochem. J. 72, 673-682. Coldstein, L. (1963).Syrnp. Intern. SOC. Cell Biol. 2, 129. Gorski, J., Noteboom, W. D., and Nicolette, J. (1965).J . Cellular Comp. Physiol. 66, Suppl. 1, 91. Gorski, J., Toft, D., Shyamala, G., Smith, D., and Notides, A. (1968).Recent Progr. Hormone Res. 24, 45. Jensen, E. V., and Jacobson, H. I. ( 1960).I n “Biological Activities of Steroids in Relation to Cancer” (G. Pincus and E. P. Vollmer, eds.), pp. 161-178.Academic Press, New York. Jensen, E. V., and Jacobson, H. I. (1962).Recent Progr. Hormone Res. 18,387-414. Jensen, E. V., Suzuki, T., Kawashima, T., Stermpf, W., Jungblut, P., and DeSombre, E. (1968).Proc. Natl. Acad. Sci. US.59, 632. King, R. J. B., Cordon, J,, and Martin, L. (1965).Biochem. J. 97, 28P. Koshland, D.E., and Neet, K. E. (1968).Ann. Reo. Biochem. 37, 359. Maurer, H.R., and Chalkley, G. R. (1967).J . Mol. Biol. 27, 431-441.

5.

ESTROGEN RECEPTORS

167

Monod, J., Changeux, J. P., and Jacob, F. (1963). J . Mol. Biol. 6, 306. Mueller, G . C., Herranen, A., and Jervell, D. (1958). Recent Progr. Honnone Res. 8, 95. Noteboom, W. D., and Gorski, J. (1965). Arch. Biochim. Biophys. 111, 559-568. Penman, S., Vesco, C., and Penman, M. (1968). J. Mol. Bid. 34, 49. Prescott, D. M. (1963). Symp. Intern. SOC. Cell Bfol. 2, 111. Scatchard, G. (1949). Ann. N.Y. Acad. Scl. 51, 660. Shyamala, G., and Gorski, J. (1967). J . Cell Biol. 35, 125A (1967). Shyamala, G., and Gorski, J. (1969). J. B i d . Chem. (in press). Toft, D., and Gorski, J. (1966). Proc. Natl. Acad. Sci. US. 55, 1574. Toft, D., Shyamala, G., and Gorski, J. (1967). Proc. Natl. Acad. Sci. U S . 57, 1740. Ui, H., and Mueller, G. C. (1963). Proc. Natl. Acad. Sci. US.50, 256.

This Page Intentionally Left Blank

CHAPTER 6

TOWARD A MOLECULAR EXPLANATION FOR SPECIFIC CELL ADHESION Jack E . LiZien* DEPARTMENT OF THERAPEUTIC

RESEARCH,

SCHOOL OF MEDICINE,

UNIVERSITY OF

PENNSYLVANIA,

PHILADELPHIA,

PENNSYLVANIA

I. Introduction ..................................... 11. Sponge Cells as a Model for Specific Cell Adhesion .... A. Immunological Approaches to Specific Adhesion of Sponge Cells ................................ B. Macromolecular Preparations of Cellular Origin Which Promote Cell Aggregation ............... C. Characterization of the Aggregation-Promoting Materials .................................... 111. Studies on Vertebrate Cells ........................ A. Tissue Disaggregation and the Specificity of Reaggregation ................................. B. Techniques of Measuring Cell Adhesion and Its Specificity ................................... C. Immunological Approaches to Specific Adhesion of Vertebrate Cells .............................. D. Inhibitors of Cell Reaggregation ................ E. The Onset of Specificity F. Macromolecular Preparations Which Promote Cell Aggregation ................................. IV. Concluding Remarks .............................. References ......................................

.......................

169 170 171 171 174 175 175 176 179 180 185 187 192 193

1. Introduction

Morphogenetic development in embryonic systems involves the movement of cells until they attain relatively stable positions and become organized in characteristic and functional architectural frameworks.

* Present address: Department of Zoology, University of Wisconsin, Madison, Wisconsin. 169

170

JACK E. LILIEN

During this period individual cells and tissues continually change their relative positions and mutual associations. Understanding the mechanisms which mediate these changes and endow cells with the selective affinities manifested during embryonic morphogenesis is a major goal of developmental biology. Several hypotheses have been advanced to explain the mechanisms underlying morphogenetic phenomena. They have been based largely on studies of the reaggregation of living cells disaggregated from sponges, amphibian embryos, and avian embryos. Few attempts have been made to “dissect” the reaggregating systems into their component parts and to study the mechanisms involved at a molecular level. Such studies require the use of an experimental system which is amenable to controlled manipulation and is free of extrinsic factors which might affect the behavior of the cells. The reaggregation of dissociated cells in suspension cultures maintained under controlled centripetal forces ( Moscona, 1961a) meets these requirements. Under these conditions dispersed cells will readhere in the absence of added nutrients, such as serum or embryo extract which might obscure or modify the inherent adhesive properties of the cells, and subsequently establish histotypic associations forming functional tissues or organs ( Moscona, 1961a, 1962a,b). The process of cell aggregation and tissue reconstruction from cell suspensions has generally been accepted as an experimental manifestation of the “normal” morphogenetic propensities of the cells, and as such has been used as a tool in studies on the mechanisms of morphogenesis and selective cell adhesion. This article briefly reviews some of the work directed at understanding the nature of chemical and molecular mechanisms involved in the formation of specific associations between embryonic cells. II. Sponge Cells as a Model for Specific Cell Adhesion

In 1907, Wilson demonstrated that sponge tissue could be mechanically dispersed into single cells by pressing through a fine silk cloth and that the cells would aggregate and readhere under appropriate conditions to eventually reconstruct miniature sponges. The readhesion of aggregating sponge cells was subsequently shown to be preferentially species specific (Wilson, 1907, 1910; Galtsoff, 19W), i.e., in mixtures of cells from two species each species tends to adhere to homologous cells; in some cases the cells of each species remain separate throughout the processes of reaggregation. These properties make sponge cells an ideal model system for studying the molecular basis of selective cell adhesion.

6.

SPECIFIC CELL ADHESION

171

A. IMMUNOLOGICAL TO SPECIFIC ADHESION OF SPONGE CELLS APPROACHES Prompted by the hypothesis of Tyler (1947) and Weiss (1947) that specific cell adhesion might be a result of antigen-antibody-like combinations at the cell periphery, Spiegel (1954a) prepared antisera against cell suspensions from two species of sponges. These antisera were found to be specific in their ability to inhibit cell reaggregation, that is, antiseruni against species A inhibited only the reaggregation of A cells but not C cells, and vice versa. MacLennan (1963) prepared antisera against six species of sponge tissue. These antisera agglutinated homologous cells and could be neutralized by water soluble glycopeptides extracted from the homologous tissue; these glycopeptides were found to be qualitatively similar, although they were immunologically species specific. These studies demonstrated that sponges contain species-specific antigenic determinants. Although antibodies to these specific antigens inhibit reaggregation or cause agglutination of homologous cells, this does not necessarily mean that such antigenic determinants are actually involved in specific cell adhesion. It is possible that the demonstrated antigens are only spatially related to the specific adhesive components. Indeed, working with alloantigens on the surface of thymus cells, Boyse et al. (1968) have recently shown that niasking of some antigenic sites may be caused by attachment of antibody to neighboring antigens.

PREPARATIONS OF CELLULAR ORIGINWHICHPROB. MACROMOLECULAR MOTE CELLAGGREGATION

By far the most productive studies on the mechanisms of specific adhesion of cells have been attempts to isolate the molecular components involved. These studies were initiated on vertebrate embryonic cells (Moscona, 196%) and continued on sponge cells (Moscona, 1962b). The integrity of the adhesive mechanism of sponge cells after mechanical dissociation, i.e., dispersion of the cells by pressing fragments of sponge tissue through a fine sieve, is demonstrated by their ability to form histotypic adhesions at suboptimal temperature ( Humphreys, 1963; Moscona, 1963a). This finding led to the attempted isolation of the components involved in selective adhesion from the surface of such mechanically dissociated sponge cells. Mechanically dissociated sponge cells when washed in cold Ca2+- and Mg2+-freesea water (CMFSW) are referred to as “chemically dissociated

172

JACK E. LLLIEN

cells.” These cells no longer have the ability to reaggregate typically at suboptimal temperature and remain dispersed or form small, loose clumps. Addition of the supernatant fluid in which the cells were washed (hereafter referred to as “factor”) restores the ability to readhere at reduced temperature ( Humphreys, 1963; Moscona, 1963a). This factor will also cause the adhesion of chemically dissociated cells ( CMFSW-treated ) fixed in formalin (Moscona, 1963a, 1968) which shows that the effect of the factor is not dependent on the metabolic activities of the cells and is mediated through the cell periphery. Enhanced adhesion in the presence of factor is accompanied by a concomitant loss of activity from the medium (Moscona, 1968) indicating that the active factor becomes associated with the cells. Activity of the factor is dependent, on the presence of Ca2+, as is reaggregation in the absence of factor (Humphreys, 1963; Margoliash et aZ., 1965). Materials which promote adhesion of homologous sponge cells have been obtained from several sponge species (Humphreys, 1963; Moscona, 1963a; Gasic and Galanti, 1966; Galanti and Gasic, 1967). Not all sponge species studied have yielded totally specific aggregation-promoting materials. Only in cases where sorting is absolute (i-e., two species of cells form separate aggregates when “co-mingled”) are the factors totally specific ( Humphreys, 1969). Do the specificities exhibited by reaggregating sponge cells and their factors mimic cellular affinities in situ? Recent experiments ( Moscona, 1968) have demonstrated that heterospecific grafts are consistently rejected while homospecifk grafts are completely accepted and integrated, further supporting the concept that the reaggregation of dispersed cell populations is an expression of in situ tissue and cellular afEnities. These findings on the reaggregation of sponge cells and their specific factors can be interpreted in the following way: specific adhesive components are removed from the cells in a functional state by washing them in CMFSW. The cells can no longer readhere without prior synthesis of the missing components. Addition of factor replaces the missing constituent(~)enabling the cells to again readhere in the absence of biosynthetic activity. The factor then functions between cells binding them together (Humphreys, 1963, 19f35a; Moscona, 1963a, 1968). This interpretation rests on the assumption that the isolated factor reconstructs adhesions which are the same as those binding cells together in situ. The

6.

SPECIFIC CELL ADHESION

173

evidence thus far presented is compatible with this assumption but does not conclusively prove it.

As a further test of this assumption antiserum was prepared against concentrated and partially purified factor ( Moscona, 1968). The antiserum specifically inactivated the factor against which it was prepared (it did not inactivate factor from another species of sponge). When tested for its ability to agglutinate homologous and heterologous cells obtained by mechanical dissociation, agglutination was observed only with homologous cells. Chemically dissociated cells were also agglutinated but to a lesser degree ( Moscona, 1968). These experiments further demonstrate the specificity of components in the supernatant solution (including the active components); moreover they show that components at the surface of cells obtained by mechanical dissociation are partially removed after CMFSW treatment, and are antigenically indistinguishable from the functional components in the active supernatant solution. Although all the data thus far described are compatible with the interpretation rendered above, other alternatives have been suggested. Based on the early observations of Galtsoff (1929) that some species of sponges contain materials which are lethal to other species, Curtis (1967) has speculated that factor preparations may contain such materials; thus the specacity exhibited by the factors is a lethal effect on heterospecific cells. Two possibilities were then suggested for enhanced reaggregation in the presence of factor: ( a ) nonspecific repair of damage resulting from washing the cells in CMFSW, and ( b ) inactivation of a hypothetical inhibitor of cell aggregation liberated during the washing procedure (Curtis and Greaves, 1965). Humphreys ( 1967) investigated the possibility of heterospecific lethality of factors in cultures containing two species of sponge cells aggregating in the presence of factor from one of the species; as expected, aggregation of this species was promoted at reduced temperature. When the temperature was raised to physiological level the other cell type reaggregated normally without showing any detrimental effects of the heterospecific factor. Recently Moscona ( 1968) has shown that mechanically dissociated sponge cells also respond to the aggregation enhancing effect of the specific factor; this clearly eliminates the possibility that the effect of the factor results from artifacts intro.. duced by CMFSW treatment.

174

JACK E. LILIEN

C. CHARACTERIZATION OF THE ACCREGATION-PROMOTING MATERIALS

The activity of the supernatant solution can be sedimented at 100,OOO

g for 90 minutes (Humphreys, 1963; 196513; Margoliash et al., 1965); preliminary tests suggested that it moves as a single band on sucrose density gradient with a sedimentation constant of 90 to 100 S indicating a molecular weight of greater than 5 X 10" (Humphreys, 1965b). Margoliash et al. (1965) have performed chemical analysis on a preparation after purification by repeated sedimentation of the active component, and found it to contain predominantly carbohydrate and protein in approximately equal proportions; the lipid content was below 1 mole per mole of protein and less than 5% nucleic acids were detected. On the basis of its behavior in the ultracentrifuge and its appearance in the electron microscope the preparation appeared to be homogeneous. These analyses suggested that the activity of the factor resided in glycoprotein particles, the most basic unit being approximately 20 A in diameter with an estimated molecular weight of 15,000 to 20,000. More recent analysis (Humphreys, 1969) on a preparation purified 1530 times, in which most of the physical material sediments on sucrose density gradient coincident with activity, was found to be composed of 70% carbohydrate. This was further substantiated by its high density in cesium chloride (1.5-1.55). The particle appears to be a single macromolecule as evidenced by its integrity in ionic detergent (0.5% sodium dodecyl sulfate) ( Humphreys, 1969). Proteolytic enzymes have been reported to destroy activity of sponge aggregation-promoting materials (Gasic and Galanti, 1966;Galanti and Gasic, 1967; Moscona, 1968; Humphreys, 1969). The body of evidence indicates that carbohydrate and protein, most likely covalently linked and organized into a particulate, are involved in the adhesive process of sponge cells, and that specificity resides in such macromolecules. The chemical purity of these preparations has not been established, and thus the definitive chemical compositions of the active molecules in the preparations remains to be determined in detail and correlated with biological activity. The specificity with which sponge cells readhere and the functional demonstration of macromolecules which play a role in this phenomenon have provided developmental biologists with a model through which some insight and understanding of the mechanisms involved in the histotypic readhesion of vertebrate cells may be gained.

6.

SPECIFIC CELL ADHESION

175

111. Studies on Vertebrate Cells

A. TISSUE DISAGGREGATION AND THE SPECIFICITY OF REAGGREGATION Studies directed at elucidating the mechanisms of cell adhesion must of necessity deal with the problems of tissue disaggregation. This problem is less obvious in dealing with sponge or amphibian tissues, from which suspensions of viable cells can be obtained by mechanical dissociation or changes in pH. Since the introduction of trypsin as an agent for the dissociation of tissues into viable cells (Moscona, 1952) various proteolytic enzymes have been employed in the dissociation of avian or mammalian tissue ( Rinaldini, 1958; Moscona, 1962b) ; however, trypsin remains generally used. Ethylenediaminetetracetic acid ( EDTA ) has also been used to dissociate avian tissue (Curtis, 1963) in the hope that it would damage the cells less than enzymes. Recently it has been shown that significant amounts of carbohydrate and protein are liberated on dissociation with EDTA (Kemp et al., 1967). This, and other evidence to be cited later on, make questionable the claim that EDTA is less injurious than trypsin. That the action of trypsin is due to its proteolytic activity and not some other effect has been demonstrated by using trypsin inhibitors (Moscona, 1963b) and trypsin inactivated with diisopropyhorophosphate (Easty and Mutolo, 1960). The need for proteolytic enzymes suggests that proteinaceous materials are involved in intercellular adhesion and may possibly be involved in the contact specificities of cells manifested during development and in in uitro reaggregation. The ability of embryonic avian cells to aggregate, to adhere selectively to each other, and to form groupings structured in a manner characteristic of the tissues of origin has been amply reviewed recently (Moscona, 1960, 1962b, 1965; Trinkaus, 1965); therefore, only a brief comparison of the behavior of sponge and vertebrate cells will be presented here. Embryonic vertebrate cells ( mammalian and avian) when “co-mingled” in suspension do not sort out preferentially according to species specificities, as do sponge cells, but according to tissue specificity. In mixed suspensions of embryonic mouse and chick cells, sorting occurs preferentially on a tissue-spec8c basis ( Moscona, 1957, 1962a). Furthermore, embryonic vertebrate cells do not manifest the same degree of specificity as reaggregating sponge cells. In suspensions containing cells from two different tissues composite reaggregates are formed which comprise cells of both tissue types; within such composite aggregates the

176

JACK E. LILLEN

cells sort out into tissues which are arranged relative to each other in a manner representative of their in situ relationships. ( Holtfreter, 1939; Moscona, 1956, 1960, 1962a, 1965; Steinberg, 1963). Although the basic mechanisms underlying the adhesive specificities manifested by reaggregating sponge cells and vertebrate cells may be similar, it is obvious from this brief consideration that the study of vertebrate cells is complicated by their inherent properties and the techniques used.

B. TECHNIQUES OF MEASURING CELLADHESIONAND ITS SPECIFICITY The reaggregation in rotation culture of embryonic avian cells dissociated with trypsin consists of a series of events which culminate in the formation of aggregates of cells with a characteristic histological organization. Depending on conditions, the number of aggregates can vary from one to many. Two characteristics of aggregating cells are usually mcasured: the ability of cells in a population to adhere to one another, and the ability of different kinds of cells in a mixed population to sort out and form selective adhesions. In dealing with a cell suspension of a single tissue type, regardless of the techniques employed, the goal has been to measure the ability of these cells to adhere to one another. Using aggregation by rotation, adhesiveness was first correlated with aggregate size after 16-24 hours of culture. The size of the aggregates was shown to decrease progressively at increased speeds of rotation and at lower temperatures of incubation (Moscona, 196la,b, 1962a). These studies led to the conclusion that the size distribution and number of aggregates produced by a given cell population under a given set of circumstances was determined by a balance between the adhesiveness of the cells, the shear forces exerted on them, and the medium composition. Aggregate size has also been shown to decrease when cells derived from progressively older tissues are used; this has been interpreted as indicating a decline with advancing differentiation or specialization of the cells in their capacity to restitute normal adhesive mechanisms following dissociation ( Moscona, 1962a). Kuroda (1963) has also shown that cells maintained in monolayer cultures for increasing periods of time progressively lost both the ability to reaggregate and to differentiate; this indicated a correlation between developmental competence and the ability of cells to aggregate. In a study comparing the ability of normal and the mutant talpid3 embryonic chick wing bud cells to reaggregate, Ede and Agerbak (1968) concluded that the adhesiveness of cells was inversely related to the size of the resulting aggregates. This was based on an analysis of the histological

6.

SPECIFIC CELL ADHESION

177

appearance of the aggregates and the rate of reaggregation during the first hour in rotation culture. Cells derived from the mutant tissue reaggregated at a more rapid rate (judged by the number of single cells counted on a hemocytometer remaining in suspension at 1 hour) during the initial hour of culture, yet formed more numerous, smaller aggregates than the comparable normal cells. Histologically the chrondrioblastic cells found in the center of the aggregates were more tightly packed and the exterior epithelial cells were more flattened in the mutant cell reaggregates. These results serve to emphasize that reaggregate size is most likely determined by a combination of factors, and alone is not an adequate measure of the adhesiveness of the individual cells in a population. Recognizing the need for a more direct means of assessing cell adhesion, Ball (1965, 1966) examined the kinetics of the initial phases of reaggregation. The actual technique was to count with a Coulter counter the number of single cells and cell clusters present in the population of aggregating cells at various times after the onset of rotation culture. Using embryonic chick retina cells of several ages dissociated with crystalline trypsin and a serum containing medium he found that reaggregation proceeded with no detectable lag and at a rate that was characteristic for the cells of each age. The initial rate of aggregation measured as the incorporation of single cells into clusters decreases with increasing embryonic age (Ball, 1965) in agreement with results obtained in earlier studies using aggregate size as a measure of cell adhesiveness. Some exceptions to this were noted; in the presence of embryo extract the rate of aggregation is depressed while aggregate size is increased ( Ball, 1965). These results indicate that the composition of the culture medium may significantly affect the rates and patterns of reaggregation. Curtis and Greaves (1965) have used a hemocytometer to count single cells and cell clusters at various times during the process of cell adhesion on a reciprocating shaker. The number of single cells remaining in suspension served as an index of the degree to which cell adhesion had progressed. Using cells dissociated with EDTA these workers observed a lag phase of approximately 2 hours prior to detectable cell-clumping in serum-containing medium. In the absence of serum no such lag was observed. These results will be further commented on in Section 111, D. As will be seen, this technique may lead to an impression of extensive reaggregation when, in fact, no structurally intact aggregates are formed. Steinberg and Granger (1966) have measured the rate of reaggregation by rotation of embryonic chick cells in serum-containing medium by filtering the reaggregating suspension on a millipore filter at various times

178

JACK E. LILIEN

after the onset of rotation, staining the cells and clusters on the filter, and counting the number of each cluster size present. These data were reported as the average cluster size at each time period measured. When cells dissociated with trypsin were used, a lag phase of approximately 40 minutes was observed; when the cells were dissociated with chelating agents no such lag was observed. A change in turbidity associated with the progressive reaggregation of cells has also been used to measure the effects of various additives on the rate of readhesion and reaggregation (Jones, 1966; Knight et al., 1966; Kemp et al., 1967; Cunningham and Hirst, 1967). Reaggregation was shown to proceed with no detectable lag phase using cells dissociated with EDTA or trypsin in medium with or without serum (Kemp et al., 1967). The question of whether there is a lag phase prior to the onset of cell adhesion probably depends largely on the techniques used to dissociate the tissues and the culture medium used. Since, in each of the above experiments the methods of measurement and the techniques of culture are different, it is difficult to make valid comparisons. The techniques used by Steinberg and Granger ( 1966) and Kemp et al. (1967) indicate the average rate at which reaggregation is progressing, whereas those used by Ball (1965) and Curtis and Greaves (1965) give the rate at which single cells are incorporated into clusters or the rate at which clusters of a particular size are formed. The interest in these methods lies not only in the existence of a latent period but in their effectiveness for measuring the effects of various exogenous or cellular constituents on reaggregation. The significance of a lag period, if it exists, is difficult to assess without the additional knowledge of whether the adhesions formed by the cells in the initial phases of aggregation have, under all conditions, the same degree and type of specificity as is seen in the later phases when the reaggregating cells become histologically organized. The question of the lag phase will be commented on further in Section 111, E. The specificity of aggregation, or the ability of different cell types to sort in mixed suspensions of cells, has been difficult to measure quantitatively. It can only be assessed in terms of the histological organization of cells in aggregates formed by two or more cell types “co-mingled” and reaggregated. Steinberg ( 1963, 1964 ) has compared the behavior of embryonic chick cells from several tissues with regard to the positions which they assume within composite aggregates and has attempted to explain specific sorting out of the cells on this basis. In most cases an internal mass composed of one tissue type is surrounded by the second tissue type. Study of various combinations yielded a hierarchy of cell

6.

SPECIFIC

CELL ADHESION

179

types with regard to the position they assume. If cell type A assumes a position external to B, and if B is external to C, then A will be external to C. The data led Steinberg to propose that sorting out of cells results from differences in adhesive strength or “work of adhesion.” The “work of adhesion” of the internal tissue, A, being greater than that of the external tissue, B, heterotypic adhesions were predicted to be of intermediate value. Recently, Roth and Weston (1967) have reported a more quantitative method of measuring selective cell adhesion. Unlabeled cell aggregates of two tissue types are cultured with a labeled cell suspension of the same or different tissue types. Differences in the number of labeled cells adhering to each aggregate type is then a quantitative means of comparing the preferential adhesiveness of single cells. C. IMMUNOLOGICAL APPROACHESTO SPECIFICADHESIONOF VERTEBRATE CELLS The high degree of specificity manifested by antibodies has prompted many workers to utilize immunological techniques to demonstrate tissue, organ, and species specific antigenic determinants. In an extension of his previous work on sponge cells, Spiegel (1954b) utilized antisera to study the antigenic nature of the specific adhesive mechanism of amphibian cells. Antiserum prepared against R a m pipiens gastrula completely inhibited the reaggregation of dissociated R. pipiens gastrula cells but had no effect on cells dissociated from gastrula of Triton alpestis. Moscona and Moscona (1962) prepared antiserum in rabbits against suspensions of living embryonic chick liver cells dissociated with trypsin and found that the heat-inactivated antiserum inhibited the reaggregation of liver cells but not of neural retina cells. In another study, Lilien ( 1966; unpublished) prepared antisera against homogenates of embryonic chick liver, neural retina, and heart ventricle. Each antiserum was able to inhibit the reaggregation of all three cell types. Cross-absorption of each antiserum with the two heterologous tissue homogenates rendered the antisera specific; inhibition of aggregation occurred only when homologous cells and antiserum were combined. The cells remained viable during the entire period of the experiment as evidenced by their ability to readhere and reaggregate when the cells were washed. Subsequent to cross-absorption the antisera showed no precipitin lines in agar when tested against the soluble portion of each tissue homogenate indicating the insolubility of the antigenic determinant. These studies suggest the existence of insoluble, tissue-specific, anti-

180

JACK E. LILIEN

genic components, presumably at the cell periphery. They do not, however demonstrate that these components are actually involved in adhesive mechanisms. As in the discussion on sponge cells inhibition could result if antibody is specifically adsorbed near the attachment mechanism causing masking of the adhesive components. The failure of specific antisera to agglutinate cells may result from several conditions. (1)Antibody excess is known to inhibit agglutination and could be the case in these studies as no detailed studies of concentration dependence were made. ( 2 ) Enfolding of the cell surface might geometrically prevent antibody from combining at the periphery of two cells. (3) Since these cells are metabolically active the secretion or turnover of cell surface constituents might cause a continual shedding of the antibody from the cell periphery. This would prevent reaggregation since adhesive sites would be continually blocked while agglutination could not occur, because antibody would not remain firmly attached at its specific site. D. INHIBITORS OF CELLREAGGREGATION 1. Temperature

It was initially observed that reduced temperature inhibited the reaggregation of trypsin-dissociated embryonic cells measured as a reduction in aggregate size (Moscona, 1961b). This led to the suggestion that biosynthesis of specific macromolecules was necessary for reaggregation (Moscona, 1962a, l W a , 1965). Steinberg ( 1962) confirmed the basic observation that reduced temperature inhibited reaggregation, again based on aggregate size, but interpreted this as being due to arrest of pseudopodial movement through which cell contacts presumably were made. Curtis and Greaves (1965) have also investigated the effects of rcduced temperature on the reaggregation of embryonic chick cells using EDTA as the dissociating agent and “flocculation kinetics” as a model of aggregation, presuming that both phenomena are identical. In the presence of serum (33%) at 38°C a lag period of several hours was observed prior to any adhesion, while at 3°C adhesion was severely inhibited. However, in the absence of serum no lag period was observed and the cells clumped at either temperature. These authors isolated an inhibitor of reaggregation from various types of sera, and suggest that the absence of cell aggregation observed by others at low temperature was due to an inability of the cells to inactivate or destroy this inhibitor at the suboptimal temperature.

6.

SPECIFIC CELL ADHESION

181

To test this suggestion Moscona and Moscona (1966a) repeated the earlier work on temperature sensitivity of aggregation of trypsin-dissociated cells using serum-free medium and again found marked inhibition of aggregate size at 4°C. The possibility of a difference in temperature sensitivity of aggregation between trypsin-and EDTA-dissociated cells was investigated by Ball (1966) using the number of single cells remaining in suspension after 60 minutes and 24 hours of rotation culture as the measure of reaggregation. With trypsin-dissociated cells, cultured in the absence of serum at 3"C, there was marked inhibition of aggregation as compared to control cultures at 37°C. With EDTA-dissociated cells there was extensive cell adhesion at 3°C in the absence of serum. However, typical reaggregates were not formed at either 3°C or 37°C when EDTA was used as the dissociating agent; only loosely organized clusters of cells were observed. Moscona and Moscona (1967) further compared dissociation with trypsin and EDTA (using the dissociation method of Curtis) with respect to the viability of the cells, their capacity to form aggregates in serum-free medium, and their ability to undergo enzyme induction. The degree of monocellularity of suspensions prepared with EDTA was poor and many of the cells were nonviable; the aggregates formed by these cells after 24 hours of culture consisted of loosely adherent clusters of cells quite different from the compact tissuelike aggregates formed by trypsin-dissociated cells under the same conditions. The authors point out that much of the seeming discrepancies in the above results are largely semantic, due to different definitions of reaggregation ( Moscona and Moscona, 1967). The process of histogenetic reaggregation involves not only the formation of initial adhesions and clusters but the subsequent development and differentiation of the cells into groups having a definite limiting boundary and tissuelike organization. It is probable from the studies just described that the flocculation observed by Curtis and Greaves (1965) cannot be compared with the histogenetic aggregates studies by other workers.

2. Inhibitors of Protein Synthesis The effects of puromycin (Moscona and Moscona, 1963; 1966b; Richmond et al., 1968), actinomycin D (Moscona and Moscona, 1963; Richmond et al., 1968), and cycloheximide (Richmond et aZ., 1968) on aggregate size have been studied on embryonic chick cells dissociated with trypsin. All of these compounds inhibit aggregate size and organization of aggregates both in the presence (Moscona and Moscona, 1963; Richmond et al., 1968) and absence of serum in the culture medium

182

JACK E. LILIEN

(Moscona and Moscona, 196613). In the presence of puromycin cell clustering proceeds for approximately 60 minutes; in the presence of actinomycin D reaggregation proceeds for approxinately 4 hours before inhibition takes place; therefore, the aggregates are significantly larger than those formed in the presence of puromycin or cycloheximide (Moscona and Moscona, 1963). The effect of puromycin was reversible even after 6 hours (Moscona and Moscona, 1963). Lilien (1968) investigated the effect of inhibition of protein synthesis with cycloheximide on cell aggregation by measuring the rate of decline in the number of single cells dissociated with trypsin in serum-free medium. No apparent effect of the inhibitor was observed for the first 2 hours after the onset of rotation although, as noted previously, aggregate size and organization were ultimately inhibited. Kemp et al. (1967) using a different method, observed that puromycin also does not affect the initial rate of cluster formation ( 0 3 0 minutes,) although aggregation beyond this point is inhibited. Curtis and Greaves (1965)reported that inhibitors of protein synthesis did not affect the reaggregation of cells dissociated with EDTA in serumfree medium. Kemp et al. ( 1967) using the dissociation methods of Curtis have found that in the absence of serum puromycin causes a marked inhibition in the formation of aggregates although initial cell clusters were formed. Glaeser et al. (1968) have compared the dissociation and reaggregation of embryonic chick cells prepared with EDTA and trypsin. Their dissociation procedure differed from that of Curtis in that instead of 0.002 M, 0.05 M EDTA was used. Although the degree of monocellularity and the ability to incorporate labeled precursors was much less than with cells dissociated with trypsin, compact reaggregates were formed resembling those formed by trypsin-dissociated cells. The use of a higher concentration of EDTA may have resulted in less mechanical damage thus allowing for better cell function. Puromycin and cycloheximide, at concentrations which inhibited reaggregation (judged by reaggregate size) and blocked the incorporation of labeled precursors in cells dissociated with trypsin, had little effect on aggregate size or the incorporation of labeled precursors in cells dissociated with EDTA. The formation of reaggregates can be correlated in all cases with the ability of the cells to incorporate labeled precursors (Richmond et al., 1968). Thus EDTA-dissociated cells which appear to be insensitive to inhibitors of macromolecular synthesis are still able to incorporate labeled precursors. This insensitivity may be related to the reduced sensitivity

6.

SPECIFIC CELL ADHESION

183

of these cells to suboptimal temperatures. As yet, no explanation for this difference in effect is available. The process of reaggregation can be operationally separated into two phases: the initial adhesion of single cells to each other, which appears to be insensitive to inhibitors of macromolecular synthesis, and the ensuing formation of large clusters and histogenetic aggregates which is sensitive to these inhibitors. It is not surprising that inhibitors of macromolecular synthesis do not affect the formation of initial adhesions while reduced temperature does. Reduced temperature may affect the rate at which adhesions are formed independent of any affect on metabolic activity. The term “regeneration” has been used in connection with the possible biogenesis of macromolecular constituents modified or destroyed by dissociation with trypsin and essential for cell aggregation. This term may imply that once such components are replaced, aggregation should ensue regardless of metabolic or biosynthetic activity. Moscona and Moscona (1963) found that addition of puromycin to cultures 6 hours after the beginning of aggregation halted the process. Kemp et al. ( 1967) found that the addition of puromycin 2 hours after the onset of reaggregation, a time supposedly sufficient for “regeneration” to occur, still caused a marked inhibition. Cycloheximide has also been shown to inhibit reaggregation when added at 2, 4, 6, and 8 hours after onset (Lilien, unpublished). It has been suggested that such inhibition is not directly related to the biosynthesis of components actually involved in specific cell attachment (Kemp et al., 1967). Indeed, Giudice ( 1965) suggested that puromycin inhibits oxygen consumption and reaggregation in sea urchin embryo cells, and Kemp et al. (1967) found that dinitrophenol inhibited the formation of aggregates of vertebrate embryonic cells. The above notion rests on the assumption that surface constituents once repaired or replaced are stable. Recent studies on the turnover of cell surface membranes (Warren and Glick, 1968; also see Chapter 7, this volume) demonstrated that under culture conditions in which cells do not divide there is rapid metabolic turnover of surface membranes. Turnover was also shown to be linked to degradative processes as evidenced by the ability of puromycin to increase the half-life of isotope incorporated into the surface membrane. It could be suggested then that components of the attachment mechanism will also undergo metabolic turnover and may be constantly replaced. Another consideration is the possible effect of degradative enzymes

184

JACK E. LILIEN

released from cells damaged during the dissociation procedure ( DeDuve, 1964, Weiss, 1967). The presence of such enzymes may have a considerable effect on the attachment mechanism, particlarly when inhibitors of macromolecular synthesis are present. Degradation would occur without concomitant replacement. 3. Inhibition by Periodate and Amino Sugars

Periodate reacts with molecules containing adjacent cis hydroxyl groups and therefore may destroy carbohydrate residues. Moscona (1962a, 1964) reported that periodate inhibited to some extent the reaggregation of trypsin-dissociated embryonic chick cells, thus possibly indicating a role of carbohydrate residues at the cell surface in the normal progression of this process. Glaeser et al. (1968) confirmed this and also found that uptake of labeled precursors was inhibited. In contrast, the aggregation of cells dissociated with EDTA appeared to be insensitive to inhibition with periodatc and the incorporation of labeled precursors was not hampered (Glaeser et aZ., 1968). On this basis Glaeser et al. (1968) have considered that periodate may exert its effect by interfering with cellular metabolism rather than by altering carbohydrate residues on the cell surface. The insensitivity of cells dissociated with EDTA to both periodate and to inhibitors of protein synthesis might suggest that the refractiveness of these cells may represent a change in permeability caused by treatment with EDTA. Garber ( 1963) has reported that glucosamine inhibits reaggregate size of embryonic chick neural retina cells and has suggested that thc sugar may react competitively at the cell periphery; however, the possibility of a toxic effect was not ruled out. This observation has been repeated and extended to both trypsin- and EDTA-dissociated cells from embryonic chick neural retina and liver and to a variety of other amino sugars (Glaeser et al., 1968). Regardless of cell type or dissociation procedure all the tested amino sugars inhibited both the incorporation of labeled precursors and reaggregate size. Thus, under the conditions examined, there appears to be no specificity to inhibition of cell aggregation by amino sugars and the effect may be a metabolic one rather than one spec& for the cell surface. The direction of the work described in this section has been to determine whether the specific readhesion of dissociated embryonic cells is directly dependent upon metabolic processes related to the biosynthesis of specsc macromolecular constituents involved in cell adhesion. These studies have yielded considerable information showing that normal reat-

6.

SPECIFIC CELL ADHESION

185

tachment of cells requires metabolic activities; however, since only a functional test (reaggregation) is used to assess the effect of inhibitors, the results may be interpreted in two ways: ( a ) inhibition of protein synthesis affects the formation of macromolecules that are directly involved in specific cell adhesion and thereby retards cell reaggregation, and ( b ) processes not directly related to the actual mechanism of cell adhesion are affected and indirectly retard cell reaggregation. A third suggestion was raised by Curtis and Greaves (1965) on the basis of their findings with cells dissociated with EDTA; blocking protein synthesis retards cell aggregation by preventing the degradation by the cells of inhibitors of aggregation present in serum. This is unlikely since reaggregation is retarded by both puromycin and cycloheximide in serum free media.

E. THE ONSETOF SPECIFITY Most workers have agreed that in the initial phase of cell reaggregation there is a period of nonspecificity during which adhesion between heterotypic cells is as likely as between homotypic cells (Moscona and Moscona, 1952; Weiss and James, 1955; Trinkaus and Groves, 1955; Moscona, 1965). No definitive experimental evidence exists as to the duration of this period; however, it has been estimated to last for up to 24 hours. Using the previously described system of collecting cells on aggregates for quantitatively determining the specificity of adhesion, Roth (1968) found that as soon as adhesions between preformed aggregates and dissociated cells formed they were specific; for example, more single liver cells were found adhering to liver cell aggregates than to retina cell aggregates, in a mixture containing all of these, and vice versa. This suggested that a nonspecific phase did not exist, at least when one of the adhering surfaces was intact, such as that of a preformed aggregate. This conclusion differed from the earlier observation on the behavior of freshly dissociated single cells which suggested that the initial clusters and aggregates of heterotypic cells consisted of randomly associated cell types, and that up to 24 hours may be required for all the cells in the aggregates to become fully sorted into discrete, type-specific groupings ( Moscona, 1965). Since adhesions between dissociated cells and preformed aggregates may not be the same as adhesions between freshly dissociated single cells, Roth tested the effect of the presence of heterotypic cells on homotypic adhesion between cells and aggregates. To accomplish this a

186

JACK E. LlLIEN

suspension of embryonic chick liver cells labeled with tritiated thymidine was mixed with a suspension of nonlabeled embryonic chick retina cells; aggregates of liver cells and of retina cells of equal sizes were also included in the mixture. A suspension of liver cells alone with retina and liver cell aggregates served as a control. Compared with control values, the presence of retina cells in the mixture not only caused a decrease in the number of liver cells adhering to the liver aggregate, but also caused an increase in the number of liver cells adhering to the retina aggregates. Similar results were obtained in the reciprocal situation. When this experiment was repeated with cells which had been circulated in culture for 4 hours prior to combination and addition of aggregates, no such interference was observed. The simplest interpretation of these results is that nonspecific adhesions between heterotypic cells are, in fact, common in the early phases of aggregation; they are most likely to occur prior to 4 hours of culture, and may physically interfere with homotypic adhesions between cells and aggregates of the same histotype; when such an adhesion is formed a heterotypic cell may be carried along. In the previous section on inhibitors, the sequence of aggregation was operationally divided into an initial phase of cluster formation which is insensitive to inhibition of macromolecular synthesis and the subsequent formation of tightly packed reaggregates which is sensitive to this inhibition. It is interesting that the period of insensitivity to metabolic inhibition roughly corresponds to the period of nonspecificity of cell adhesion suggested by the above experiments. Although it is tempting to speculate that the onset of contact specificity and the onset of sensitivity to inhibitors of protein synthesis are reflections of the same phenomenon, the critical experiments remain to be done. The above experiments serve to emphasize the need for caution in the interpretation of kinetic and inhibitor studies on cell aggregation when all measurements are made during the first 4 hours of aggregation. The absence of a lag phase d x i n g the initial phases of cell adhesion observed in kinetic studies and the insensitivity of this phase to inhibitors of protein synthesis have been suggested as evidence that surface components involved in cell adhesion are not removed or destroyed by trypsin treatment (Kemp et al., 1967). More relevant to the argument that biogenesis of macromolecules is necessary for normal aggregation is the demonstration that a lag period of 2 to 4 hours exists before specific adhesions ensue, and that inhibition of protein synthesis prevents the onset of specific adhesion (Roth, 1968). In fact, the initial nonspecific adhesion

6.

SPECIFIC CELL ADHESION

187

or flocculation of cells may have little relevance to the actual mechanism involved in histogenetic cell aggregation.

F. MACROMOLECULAR PREPARATIONS WHICHPROMOTE AGGREGATION The preceding discussion has emphasized the need for the direct demonstration and isolation of components involved in specific adhesion as a first step towards understanding the process at the molecular level. The first such attempt was made by Moscona (1962a). Reasoning that such materials may be synthesized and released into the culture medium under conditions which prevent the readhesion of cells, he obtained a cell-free supernatant from embryonic chick neural retina cells which increased the reaggregate size of freshly dissociated homologous cells. The supernatant solution was obtained by swirling the cells in flasks at 100 rpm (cells do not appreciably adhere at this speed) for 2 hours at 37°C in serum-free medium. The supernatant solution manifested cell type specificity, and activity could not be obtained from cells maintained at 4°C. However, difficulties in obtaining large quantities of consistently active supernatants delayed the extension of this work and prompted a search for other methods. It has recently been reported (Kuroda, 1968) that a supernatant solution similarly prepared from embryonic chick liver cells, but in medium containing serum and embryo extract, promotes reaggregate size of freshly dissociated liver cells. However, no data on the cell specificity of the preparation was given. Roth (1968) also reported the enhancement of cell adhesion by a medium with serum after incubation with various cell types, but found no significant tissue specificity, A serum constituent bound to various types of embryonic chick cells in monolayer culture and subsequently released in serum-free medium has also been reported to promote cell reaggregation (Lilien, 1968). It is likely that a wide variety of cells are able to “activate” serum constituents causing them to promote cell adhesion; the relevance of these serum-dependent effects to the actual mechanism of specific cell adhesion is doubtful.

1. Preparation and Activity of a Specific Cell Aggregation-Enhancing Material Several important points emerge from the above studies: (1) Cells cultured under conditions that are conducive to rapid reaggregation do not liberate into the medium materials capable of promoting the size of reaggregates. ( 2 ) Cells prevented from reaggregating by low temper-

188

JACK E. LILIEN

ature also do not liberate such materials. ( 3 ) It is necessary to exclude serum from the culture medium in studies directed at obtaining (or at testing) specific aggregation-promoting cellular products. From these points a procedure has been developed that makes it possible to obtain a supernatant solution from cultured cells which promotes the size of reaggregates and the rate of reaggregation of homologous cells (Lilien and Moscona, 1967; Lilien, 1968). The supernatant solution, which is active in promoting the aggregation of embryonic chick neural retina cells, was obtained from monolayer cultures of 10-day embryonic chick neural retina cells. After 48 hours of culture in medium containing serum, monolayers of neural retina cells were washed extensively and incubated for an additional 24 hours in medium without serum; the medium collected after 24 hours is referred to as R1. Fresh, serum-free medium is added for an additional 24 hours and collected as R2. R1 and R2 are cleared of cells and cell debris by centrifugation and are dialyzed extensively against Tyrode’s solution. Activity is assayed at concentrations ranging from 10 to 100% (v/v) R1 or R2 in Tyrode’s solution on freshly dissociated cells from embryonic chick retina, heart, liver, and limb bud. Both R1 and R2 enhance the reaggregate size of retina cells by more than three times; they have no effect on any other cell types. The presence of serum (horse or fetal bovine) in the assay medium completely masks the effect of both R1 and R2. Both supematant solutions are active over a range of concentrations from 25 to 80% (v/v) showing no gradation in response. The most consistent results are obtained with 50% R1 and 75% R2. R1 contains approximately 0.1 mg/ml protein and R2, 0.01 mg/ml, yet they are active over a similar range of concentrations. This suggests that R1 contains a large amount of proteinaceous material not invoIved in the promotion of reaggregation. Although it is not known why R1 is not active at high concentrations (above 6085%), the similarity in activity of R1 and R2, the large difference in protein concentration, and the ability of serum to inhibit activity suggest that an inhibitor may be present in R1. The inhibitory effect of serum and possibly of a component in R1 may manifest itself by either competing for sites on the cell or by binding directly with the active component. The accumulation of activity in R1 by the monolayered cells does not occur at reduced temperature and is prevented by inhibiting protein synthesis with cycloheximide.

6.

SPECIFIC CELL ADHESION

189

2. Effectsof Cell Dissociation Procedure and the Time of Addition of the Aggregation-Promoting Factor on Cell Aggregation Retina cells obtained by mechanical dissociation either from monolayer cultures maintained for 24 hours in serum-free medium or from whole tissue do not respond to added R1. These experiments suggest that a modification of the cell by trypsin is necessary for an effect of R1. Furthermore, they imply that cells maintained in rotation culture for a period sufficient for the repair of the tryptic damage may not respond to R1. This is the case, since the addition of R1 to trypsin-dissociated cells 2 hours after the onset of aggregation results in no activity. The 2-hour period after which R1 no longer promotes an increase in the size of aggregates corresponds in time to the period of insensitivity to inhibitors of protein synthesis and is similar to the period of nonspecificity. These may be three indications of the same phenomenon, i.e., the replacement or repair of macromolecular constituents involved in specific cell adhesion. 3. The Interaction of the Aggregation-Promoting Factor and Responding

Cells Enhanced aggregation in the presence of R1 involves two main phases: association of the active component with the test cells and its functional utilization. These phases were separated on the following basis. Reduced temperature (4"C), or the presence of cycloheximide in the culture medium prevented enhancement of aggregate size by R1. Cells in R1 maintained at 4°C and then shifted to 37°C showed a typical response. When cultures containing R1 were maintained at 4°C for 24 hours and the medium was removed and assayed on freshly dissociated cells, it showed no effect. These experiments demonstrated that the active component in R1 cannot function in the absence of metabolic or biosynthetic activity but that it does become associated with the cells under these conditions. Similar experiments utilizing, in place of retina cells, embryonic liver cells which do not respond to R1, have shown that activity can be recovered from the culture medium after incubation at 4°C with R1; this indicates that the association of the active component in R1 with test cells is specific. The specificity of the association between the active component in R1 and test cells suggests the existence of specific attachment sites and lends particular significance to this phase. The experiments described previously which indicated that tryptic modilkation of the cell surface may be requisite to the activity of €31 further suggest

190

JACK E. LILIEN

that the proposed attachment sites are masked by surface constituents (not necessarily those involved in specific adhesion) and that these are removed by trypsin. 4. Localization of the Aggregation-Promoting Factor at the Cell Periphery The association of the active component in R1 with test cells was investigated to determine if it was localized internally or at the cell periphery. Antiserum was prepared against supernatant solution R2 (R2 was used because it required less protein to give a positive result and was therefore judged purer than R1), This antiserum inhibited the reaggregation of retinal cells at a concentration of 20% (v/v), and affected a slight amount of enhancement at a concentration of 5% (v/v) (see Section 111,c). It had no effect on the reaggregation of liver cells. If a component( s ) in R1 were localized at the cell periphery antiserum should agglutinate retina cells preincubated with R1 at 4°C in a manner directly analogous to hemagglutination of sensitized red blood cells. Prior to the addition of the antiserum cycloheximide was added to the cultures to prevent aggregation due to endogenous synthetic activity. The results (Table 1) indicate that a component( s ) in R1 is taken up at the cell periphery where it is available for combination with antibody. It is most significant that freshly trypsinized cells showed some agglutination. Taken together, the results of the experiments on the association of the active component in R1 with test cells and results utilizing antibody directed against R2 are most easily interpreted as indicating that the active factor attaches at the cell periphery where it is available for combination with antibody. The requirement for metabolic processes by the trypsin-dissociated cells in order to respond to R1 distinguishes this situation from specific TABLE I THEAGGLUTINATION OF RETINACELLS BY ANTISERUMAGAINSTR2a Reincubation in R1

No Yes No Yes

Serum Normal serum Normal serum Anti-R2 Anti-R2

Result

0 0 +1 +4

Test cells were preincubated at 4°C in the presence or absence of R1 for 24 hours. Ten percent serum was added and a further incubation at 37°C was carried out. Agglutination was recorded on a scale from 0 to +4. a

6. SPECIFIC CELL

ADHESION

191

cellular adhesion of sponge cells obtained by nonenzymic dissociation. Two explanations can be offered for this requirement: ( a ) the active component in R1 stimulates endogenous synthesis of adhesive materials thus promoting aggregation, or ( b ) the active factor contains the normal adhesive constituents of the cell surface, and these are actually utilized in the formation of specific cell adhesions. Biosynthetic activity may be required to replace or otherwise repair secondary attachment sites at the cell surface damaged by enzymic dissociation. At present no clear distinction between these hypotheses can be made although the specificity of the effect, the attachment to the cell surface and the immunologic cross-reactivity with constituents normally present at the cell surface clearly favor the second hypothesis.

5. Age-Dependent Studies on the Promotion

of Cell Aggregation

The problem of adhesive specificity of cells during development is one of continuous change in cell affinities. The studies thus far discussed deal almost exclusively with one developmental age and do not reflect the dynamic character of morphogenetic phenomena. Studies comparing the reaggregation of 7-, lo-, and 14-day embryonic retina cells have shown a characteristic decline in both aggregate size (Moscona, 1962a) and the rate of aggregation (Ball, 1965) with increasing embryonic age. For this reason studies concerning the effect of R1 on 7-, lo-, and 14-day embryonic retina cells were conducted. An assessment of whether it was possible to obtain from cells of these different ages preparations with aggregation-enhancing activity similar to that of R1 was also made ( Lilien, 1966 unpublished). Supernatant solutions from monolayer cultures of 7-, lo-, and 1Cday retina cells were collected in the exact manner as R1 and assayed at various concentrations on freshly dissociated 10-day retina cells. Cells from each of the three ages yielded active supernatant solution when tested on freshly dissociated 10-day retina cells. It was not possible to determine quantitative differences in the activity of these preparations owing to the qualitative nature of the assay and the possible existence of inhibitory proteins, as discussed above. These experiments do suggest that no qualitative changes in the active component in R1 occur during this period of development. The response of 14-day retina cells to R1 (from 10-day retina monolayers) is similar to that of 10-day cells, although more R1 is required. This might suggest that the afEinity of the active component in R1 for these cells is reduced, indicating possibly a qualitative change in attach-

192

JACK E. LILIEN

ment sites. Seven-day retina cells do not respond to R1 even though they do produce an active supernatant. The extremely rapid rate of reaggregation of these cells (Ball, 1965) may preclude the possibility of R1 showing any demonstrable activity; that is, if for every collision between cells a positive union is formed in the absence of R1, the possibility of enhanced adhesion by R1 is immediately excluded. Although still preliminary, these experiments do suggest that some changes in the attachment mechanisms of cells may occur during the development of a single tissue. Identification of these changes will be important for future studies. IV. Concluding Remarks

The studies summarized here provide convincing evidence that specific cellular adhesion in sponge and vertebrate cells is mediated by specific macromolecular constituents at the cell periphery as postulated some time ago (Moscona, 1960, 1962a, 1963a, 1968). The preparation of such constituents opens to experimentation their molecular nature, organization at the cell periphery and role in morphogenesis. Although the data thus far available are incomplete some general outlines of the process involved in specific cell adhesion have become apparent. The aggregation of embryonic vertebrate cells can be separated into two phases: the adhesion of single cells to one another immediately following tryptic dissociation, and the subsequent adhesion of these clusters into tightly packed aggregates with a definite histological structure. The initial adhesion of single embryonic cells freshly dissociated by trypsin is nonspecific and insensitive to inhibitors of macromolecular synthesis. This phase in the sequence of reaggregation most probably reflects the modification or destruction of cell surface components during tissue dissociation by the enzyme. Its duration is dependent in principle on the time required to repair such modification. Where enzymic dissociation is unnecessary, as is the case with sponge tissue, reaggregat'ion cannot be divided into comparable phases; here specific adhesion may proceed in the absence of metabolic activities. The second phase in the reaggregation of vertebrate cells is sensitive to inhibition of macromolecular synthesis. It is not clear whether inhibition results primarily from interference with the biosynthesis of specific adhesive components altering the normal turnover of these components or with secondary processes not directly related to specific attachment.

6.

SPECIFIC CELL ADHESION

193

The mechanism of specific cellular attachment has been most completely elucidated in the sexually agglutinative yeast Hamenula wingei. A mating factor isolated froin one mating type has been shown to act as a specific agglutinin for cells of the opposite mating type (Taylor, 1964; Taylor and Orton, 1967; Brock, 1965). Recently a second substance has been isolated from the agglutinable strain which specifically inhibits agglutination in a manner resembling an antigen-antibody reaction (Crandall and Brock, 1968). It was concluded that these macromolecules reacted by virtue of complementary molecular configurations. Evidence that the specific cell aggregation-promoting materials prepared from sponges and from embryonic retina cells attach specifically at the cell periphery in the absence of metabolic activity is also compatible with a mechanism of cell recognition based on molecular complementarity.

REFERENCES Ball, W. D. (1965). Ph.D. dissertation. The Univ. of Chicago, Illinois. Ball, W. D. (1966). Nature 210, 1075. Boyse, E. A., Old, L. J,, and Stockert, E. (1968). Proc. Natl. Acad. Sn’. U.S. 60, 886. Brock, T. D. (1965). Proc. Natl. Acad. Sci. U.S. 54, 1104. Crandall, M. A., and Brock, T. D. (1968). Science 161, 473. Cunningham, I., and Hirst, J. H. R. (1967). Experientia 23, 693. Curtis, A. S. G. ( 1963). Nature 200, 1235. Curtis, A. S. G. (1967). “The Cell Surface: Its Molecular Role in Morphogenesis.” Logos Press, London. Curtis, A. S. G., and Greaves, M. F. (1965). J . Ernbryol. Exptl. Morphol. 13, 309. DeDuve, C. (1964). In “Lysosomes” ( A . V. S. deReuck, M. P. Cameron, and J. Knight, eds. ), p. 1. Little, Brown, Boston, Massachusetts. Easty, G. D., and Mutolo, V. (1960). Exptl. Cell Res. 21, 374. Ede, D. A., and Agerbak, G. S. (1968). J. Embryol. Exptl. Morphol. 20, 81. Galanti, N. L., and Gasic, G. J. ( 1967). Biologica 40, 23. Galtsoff, P. S. (1925). J. Exptl. Zool. 42, 183. Galtsoff, P. S. (1929). Biol. Bull. 45, 153. Carber, B. (1983). Deoelop. Biol. 7, 630. Gasic, G. J., and Galanti, N. L. (1966). Science 151, 204. Giudice, G. (1965). Der;elop. Biol. 12, 233. Claeser, R. M., Richmond, J. E., and Todd, P. W. (1968). Exptl. Cell Res. 52, 71. Holtfreter, J. ( 1939). Arch Entwicklungsmech. Organ. 139, 110. Humphreys, T. (1963). Deoelop. B i d . 8, 27. Humphreys, T. (1965a). 1. Exptl. 2002.160, 235. Humphreys, T. ( 1965b). Exptl. Cell Res. 40, 539. Humphreys, T. (1967). In “The Specificity of Cell Surfaces” (B. D. Davis and L. Warren, etls. ), 1). 195 Prentice-Hall, Englewood Cliffs, New Jersey.

194

JACK E. LILIEN

Humphreys, T. ( 1969). In “Porifera: Their Biology and Experimental Significance” ( W. G. Fry, ed.) Academic Press, New York (in press). Jones, B. M. (1966).Nature 212, 362. Kemp, R. B., Jones, B. M., Cunningham, I,, and James, M. C. M. (1967).I. Cell Sci. 2, 323. Knight, V. A., Jones, B. M., and Jones, P. C. T. (1966).Nature 210, 1008. Kuroda, Y. ( 1963). Exptl. Cell Res. 30, 446. Kuroda, Y. (1968).Exptl. Cell Res. 49,626. Lilien, J. E. (1968).Deoelop. Biol. 17, 657. Lilien, J. E.,and Moscona, A. A. (1967).Science 157, 70. MacLennan, A. P. (1963).Biochem. 1. 89, 99P. Margoliash, E., Schenck, J. R., Hargie, M. P., Burokas, B., Richter, W. R., Barlow, R. H., and Moscona, A. A. (1965).Biochem. Biophys. Res. Commun. 20, 383. Moscona, A. A. ( 1952).Exptl. Cell Res. 3, 536. Moscona, A. A. (1956).Proc. Soc. Exptl. Bfol. Med. 92,410. Moscona, A. A. (1957).Proc. Natl. Acad. S c i . U.S . 43, 184. Moscona, A. A. ( 1960).In “Developing Cell Systems and Their Control” ( D. Rudnik, ed.), p. 45. Ronald Press, New York. Moscona, A. A. (1961a).Exptl. Cell. Res. 22, 544. Moscona, A. A. (1961b).Nature 190, 408. Moscona, A. A. (1962a).1. Cellular Comp. Physiol. 80, Suppl. 1, 65. Moscona, A. A. (1962b).Intern. Reo. Exptl. Pathol. 1, 371. Moscona, A. A. (1963a).Proc. Natl. Acad. Sci. U. S . 49, 742. Moscona, A. A. (1963b).Nature 199, 379. Moscona, A. A. (1964).In “Biological Interactions in Normal and Neoplastic Growth” M. J. Brennan, and W. L. Simpson, eds.), p. 113. Little, Brown, Boston, Massachusetts. Moscona, A. A. ( 1965).In “Cells and Tissues in Culture” (E. N. Willmer, ed.), Vol. I. p. 489. Academic Press, New York. Moscona, A. A. (1968).Deuelop. Biol. 18,250. Moscona, A. A., and Moscona, H. (1952).I. Anat. 86,287. Moscona, A. A., and Moscona, M. H. (1962).Anut. Record 142, 319. Moscona, A. A., and Moscona, M. H. (1966a).Exptl. Cell Res. 41,697. Moscona, A. A., and Moscona, M. H. (1967).Exptl. Cell Res. 45,239, Moscona, M. H.,and Moscona, A. A. (1963).Science 142, 1070. Moscona, M. H.,and Moscona, A. A. (1966b).Exptl. Cell Res. 41, 703. Richmond, J. E.,Glaeser, R. M., and Todd, P. W. (1968).Exptl. Cell Res. 52, 43. Rinaldini, J. M. L. (1958).Intern. Reo. Cytol. 7, 587. Roth, S. A. (1968).Deoelop. Blol. 18, 602. Roth, S. A., and Weston, J. A. (1967).Proc. Natl. Acad. Sci. U. S . 58, 974. Spiegel, M. (1954a).Bid. Bull. 107, 130. Spiegel, M. (1954b).Bid. Bull. 107, 149. Steinberg, M. S. (1962).Exptl. Cell Res. 28, 1. Steinberg. M. S. (1963).Science 141, 401. Steinberg, M. S. ( 1964).In “Cellular Membranes in Development” (M. Locke, ed.), p. 321. Academic Press, New York. Steinberg, M. S., and Granger, E. (1966).Am. Zool. 8,579. Taylor, N. W.( 1964).I. Bactsn’ol. 87, 863.

6.

SPECIFIC CELL ADHESION

195

Taylor, N. W., and Orton, W. L. ( 1967). Arch. Biochem. Biophys. 120,602. Trinkaus, J, P. (1965).I n “Organogenesis” (R.L. DeHaan and H. Ursprung, eds.), p. 55. Holt, New York. Trinkaus, J. P., and Groves, P. W. (1955).Proc. Natl. Acad. Sci. U. S . 41,787. Tyler, A. ( 1947).Growth 10,7. Warren, L., and Glick, M. C. (1968).J. Cell Biol. 37,729. Weiss, L. (1967).“The Cell Periphery, Metastasis, and Other Contact Phenomena.” North-Holland Publ. Amsterdam. Weiss, P. (1947).Yale J. Biol. Med. 19, 235. Weiss, P. and James, R. (1955).1. Erptl. Zool. 121,449. Wilson, H.V. ( 1907). 1. Exptl. Zool. 5,245. Wilson, H. V. (1910).Bull. Bur. Fisheries 30, 1.

This Page Intentionally Left Blank

CHAPTER 7

THE BIOLOGICAL SIGNIFICANCE OF TURNOVER OF THE SURFACE MEMBRANE OF ANIMAL CELLS Leonard Warren DEPARTMENT OF THERAPEUTIC RESEARCH,

MJ3DICINE, PENNSYLVANIA, PHILADELPHIA, PENNSYLVANIA

SCHOOL OF

UNIVERSITY O F

I. Introduction ..................................... 197 11. Composition of the Surface Membrane . . . . . . . . . . . . . . . 198

.................

199

brane during the Cell Cycle ....................... of Components of the Surface Membrane ...................................... VI. Synthetic and Degradative Aspects of Turnover . . . . . . . VII. Turnover and Repair ............................. VIII. Speculation on the Malignant Cell . . . . . . . . . . . . . . . . . . IX. Conclusions ..................................... References ......................................

207

111. The Dividing and Nondividing Cell

IV. Synthesis and Turnover Rates of the Surface MemV. Differential Turnover

1.

209 210 212 212 219 220

Introduction

It should be stated at the outset of this highly speculative chapter that it is written by an “outsider” looking at certain biological problems without the intimate knowledge and insights of a researcher totally involved in these problems. One of the things that has prompted this writing is the disparity between the mass of published observations and the few viable integrative ideas with which one is confronted. Perhaps this chapter will be of heuristic value. The modern view of “the dynamic state of body constituents” was firmly established by Schoenheimer in a monograph bearing that title 197

198

LEONARD WARREN

( 1942). It was convincingly argued that the structural and functional molecules of an organism are in a state of flux. In this chapter we will examine the flux that occurs, in the components of the surface membrane of animal cells, insofar as they are known and we will speculate on the possible relevance of this phenomenon to cellular behavior. Most of the data that will be discussed is our own, either published (Warren and Glick, 1968) or unpublished. At the present time there is just enough information to excite the imagination as to its possible general significance and not enough of it to crush even wild speculation. During turnover of the surface membrane some membrane leaves the structure and at the same time an equivalent amount is replaced so that no net change occurs in the amount of membrane present. This is to be distinguished from situations in which there is a net increase in surface membrane as in cell multiplication or cell enlargement. Experimentally, turnover is difficult to examine and the data frequently even more difficult to interpret. Indeed, the immediate interpretation of our data to be discussed while reasonable and likely, is by no means unique. Interpretation is obscured by our lack of knowledge concerning pool sizes of simple and complex membrane precursors, of possible exchanges between pools of precursors and membranes, and of the fate and degree of recycling of rejected membrane, to name but a few factors. The rate at which an isotopic precursor ( e.g. 14C-labeled amino acids or glucosamine-14C) enters and leaves the membrane is dependent upon these processes. We have studied cultured cells in defined medium. How do these differ from cells in tissues? It is obvious that turnover data in whole tissues can be even more difficult to interpret than that of individual cells in culture. II. Composition of the Surface Membrane

Our studies on the turnover of the surface membrane of the L cell and other cells are based upon our ability to isolate this structure. We have devised a method by which we “toughen” the surface membrane with a reagent, fluoroscein mercuric acetate ( FMA), that combines with sulfhydryl groups in a hypotonic solution. The cells swell and the surface structure rises from the underlying cytoplasm. At this point the cells are homogenized. The surface membranes are obtained as empty bags with a hole through which the cytoplasm and nucleus were expelled (Warren et al., 1968).The bag comprises 4.7% of the total protein of the cell. It consists of approximately 58% protein, 38% lipid, 3% carbohydrate, and 1.4% RNA. The carbohydrates found in the membrane are sialic acid, N-acetylhexosamine, fucose, mannose, galactose, and possibly xylose. In

7.

SURFACE MEMBRANE OF ANIMAL CELLS

199

the electron microscope the membranes display a characteristic trilaminar appearance. 4

111. The Dividing and Nondividing Cell

The work to be described in this section was done with the L cell, a mouse fibroblast grown on a glass surface or in suspension in Eagles’ medium with 10% bovine fetal serum. In this medium cells double in number approximately every 21 hours. The cells were counted and their size distributions were determined with a Coulter counter. Two types of experiments were done to establish the rates of turnover. These will be briefly outlined here. For details, previous reports should be reviewed ( Warren and Glick, 1968). In the first, the rate of incorporation of isotope into surface membranes was determined. L cells were grown in the presence of a 14C-labeled precursor such as D-glucose, Lleucine, L-valine, or D-glucosamine, a precursor of the N-acetylhexosamines and sialic acids of the cellular glycoproteins. After a certain length of time the cells were harvested, washed twice with physiological saline solution, and the surface membranes were removed and isolated as whole ghosts by the FMA method (Warren et al., 1966; Warren and Glick, 1968). The protein content of the membrane was determined by the method of Lowry et al. (1951) and the radioactivity was measured. Results are expressed as counts per minute per microgram of membrane protein. Cells in two states were tested, dividing and nondividing. Cells could be prevented from dividing by elevating the cell number to 1 to 2 X lo8 per milliliter, in fresh medium. For unknown reasons the cells did not divide in most but not all experiments, nor did they change size significantly under these conditions. When pulsed with tritiated thymidine they incorporated about 8% of the amount of radioactivity that dividing cells incorporated. The cells were viable as tested with Trypan blue. In Fig. 1 it can be seen that the rate of rise of the specific activity of 14Cfrom glucose-14Cin the surface membrane is approximately the same, whether or not the cells are dividing. The rise in specific activity of the dividing cell is expected as one cell becomes two and the equivalent of one cell must be synthesized per generation. From the data one cannot tell whether there is turnover or replacement of the membrane in the dividing cell. Quite unexpectedly, in the nondividing cell an equally rapid rise in 14Cspecific activity is observed. Since the size and number of the cells are not changing and the mass of the membrane remains constant, the incorporation can only be explained by turnover within the membrane where newly synthesized, labeled material goes into the membrane and

200

LEONARD WARREN

0

1

2

3

4

5

6

7

Time (hr)

FIG. 1. Incorporation of glucose-14C into surface membranes and cell particulate fractions of growing and nongrowing L cells. A. Nongrowing cells: L cells in plateau phase were resuspended in 100 ml of fresh medium which contained glucose-1% (21,700 counts per minute per pmole). The cell number was adjusted to 2.2 X 106 per ml. The spinner flask was gassed with 5% CO, in air and was incubated at 37°C. Samples of 25 ml were removed every 1.75 hours at which time a cell count and size estimation were made. No significant change in either of these parameters was observed during the experiment, Cell samples (25 ml ) were washed twice with saline and were processed by the FMA method, B. Growing cells: L cells which were growing logarithmically were centrifuged and resuspended in 300 ml of fresh medium containing glucose-14C (28,700 counts per minute per m o l e ) in a spinner flask. The cell number was 5.13 X 105 at the beginning of the experiment and increased 20% without change in cell size during a &hour period at 37°C. Samples (100 ml) were removed every 2 hours, washed with saline, and processed by the FMA method. The numbers on the graphs beside the points on the membrane curve (0-0) refer to the percent of the total counts of the membrane that were extractable into surface membrane; A-A cell particulates. It ch1oroform:methanol. 0-0 should be noted that the specific activity of the glucose in the medium used in experiment B (28,700 counts per minute per m o l e glucose) was 32% higher than in experiment A (21,700 counts per minute per m o l e glucose). A corresponding correction should be made in comparing the curves of experiments A and B.

7.

201

SURFACE MEMBRANE OF ANIMAL CELLS

an equivalent amount of unlabeled membrane is removed. The same pattern of labeling is seen with a general precursor (D-glucose, Fig. l), protein precursors (L-leucine, L-valine), (Table I), or a carbohydrate precursor (D-glucosamine). With all I4C substrates the rate of rise of specific activity in the surface membrane of nongrowing cells is 80100% that of growing cells. This observation has been made on a variety of tissue culture cells and on chick fibroblasts grown in suspension culture or on a glass surface (unpublished work with Drs. C. Buck and V. Defendi). The spec& activities of the membranes of the cells in the two states (dividing or nondividing) rise essentially at the same rate over several days or generation times until a maximum labeling is attained (Warren and Glick, 1968). In the second type of turnover experiment the removal of I4C-labeled material from prelabeled membrane was studied. L cells were heavily labeled by growing them for several days in the presence of the 14Csubstrates listed above. The cells were washed and the surface membranes of a portion of the cells were obtained to determine the initial specific activity. The remaining cells were placed in fresh medium without isotope and allowed to grow or were suspended at high concentration to prevent growth. On successive days thereafter a portion of the cells were processed for surface membranes and the remainder were allowed to continue in culture in nonradioactive medium. Specific activities of the surface membranes of each of these samples were determined. COMPARISONSOF

THE

INCORPORATE

TABLE I ABILITY OF DIVIDINGAND NONDWIDING L CELLSTO L-VALINE-14C INTO SURFACE MEMBRANE+

Vessel

1. 2. 3. 4.

Control, growing cells Control, nongrowing cells Cells vinblastine sulfate Cells thymidine

+ +

Specific activity of surface membrane (cpm/pg protein) 25.6 20.4 20.8 24.4

4

Two spinner flasks were set up, each with 500 ml of fresh medium containing 400,000 cells per milliliter. To one flask was added vinblastine sulfate, 0.1 pg per milliliter; to the second flask was added thymidine to a concentration of 10-2 M. The h k s were incubated at 37°C for 21 hours. ~-Valine-ldC(67,000 counts per minute per milliliter of medium) was then added to these vessels as well as to two other control flasks. The first mitained L cells which were rapidly dividing and a second contained cells in high concentration. In 24 hours of culture in radioactive medim, the cells of vessel 1 had doubled in number while those in vessel 2 had increased only 6%. There was no increase in cell number in vessels 3 and 4. The size of cells in vessels 3 and 4 had increased markedly. The cells were processed after 24 hours incubation in radioactive medium. a

202

LEONARD WARREN

In the growing cell, if there is no turnover, i.e., no replacement of labeled membrane with newly synthesized nonradioactive membrane, then the specific activity of the surface membrane should fall by a predicted value of 50% as one old labeled cell forms the equivalent of a new cell from nonradioactive precursors. If the specific activity of the surface membrane falls to a level lower than predicted ( < 50%) as cells double in number, the fall beyond 50% is considered to be caused by turnover of membrane components. It can be seen in Fig. 2 that the observed fall in specific activity closely follows, but is slightly below, the predicted, over several generations. The same result has been obtained using several 14C precursors of proteins and carbohydrates of the cell membrane. In all of our experiments the specific activities of the membrane were, on the average, 7.1% lower than that predicted by dilution due to increase of cell number. This would suggest that there is relatively little turnover of the surface membrane in the dividing cell; this cell replaces 7.1% of its surface membrane per generation. The apparent small turnover of the surface membrane of dividing cells does not preclude the possibility that there are certain components in the membrane, small in amount, that are rapidly turning over. Our methods measure the overall turnover and the turnover of minor components might be masked. In the nondividing cell, where the mass is not changing, one would expect no decrease in specific activity if there is no turnover. In Fig. 3 it may be seen that the specific activity does in fact fall by 40 to 50% in the equivalent of one generation time which suggests a rapid turnover. This is consistent with the results of the incorporation experiment described above (Fig. 1). Similar results have been obtained using cells labeled with valine-14C and gluc~samine-~~C. The most reasonable interpretation of the data so briefly described is that the cell, when it is not dividing, may make as much or almost as much membrane material as when it is dividing. It can be calculated that it is making 50-100% of a cell's component of surface membrane in the equivalent of a generation time when not dividing. Newly formed membrane is incorporated into the surface structure of the dividing cell and most of it stays there, while in the stationary state it goes in but an equivalent amount must be rejected. The fate of the rejected membrane is not known at the present time. It could pass into the cell or be pushed into the medium, or it may or may not be completely degraded, reutilized, or metabolized. The extent of recycling of material is unknown but it is

7.

SURFACE MEMBRANE OF ANIMAL CELLS

203

probably small. If there is recycling, the turnover rate would be greater than we have observed. Kruse et al. (1967) have shown that the level of protein synthesis in the Jensen sarcoma is similar at different rates of growth and that at slower growth rates the products of protein synthesis are secreted into the medium. This could be the material rejected in the course of membrane turnover. The results described above also are consistent with those of Herrmann and Marchok (1963). They found that actively growing chick embryo explants, pulsed with a 14C-labeled amino acid retained virtually all of the radioactivity when allowed to grow further in nonradioactive medium. Protein breakdown did not appear to take place. However, when the growth rate of explants was reduced or completely inhibited by an amino acid analog or by eliminating nutrients in the medium following the radioactive pulse, significant amounts of radioactivity were lost from the explant. In the turnover experiments described above a particulate fraction was obtained from the same cells from which surface membranes were derived. This fraction contained the mitochondria, endoplasmic reticulum, and nuclei (i.e., intracellular membranes). It was found that, in general, the pattern of labeling of this fraction closely followed that of the surface membrane. This suggests that the overall turnover rates of all of the membrane systems of the cell are similar to each other in the dividing and stationary phase. The extent to which pinocytosis and phagocytosis play a part in the removal of surface membrane is not really known. We have compared the rate of uptake of glucoseJ4C into the surface membranes of L cells which were incubated in the presence or absence of styrene beads and other particles. There was barely a significant difference in the uptake of isotope by the membranes over a 5-hour period which would suggest that phagocytosis is not an important factor in our turnover studies (Warren and Glick unpublished). On the other hand, Christiansen and Marshall (1965) have estimated that during optimal growth, the ameba Chaos chaos may remove 4 M O % of its surface in 1 hour by phagocytosis, and Chapman-Andresen (1962) has reported a turnover of 12% per hour for amebas recovering from pinocytosis. Amebas appear to have a large pool of surface membrane precursors and can replace their surface structure in 35 minutes (Nachmias, 1966). Little is known about the size of the pool of surface membrane precursors in the L cell, although from our data a preliminary rough estimate suggests that there is almost no

204

LEONARD WARREN

internal pool in the dividing cell and a pool of about 10-15% of that which is on the surface of the nondividing L cell (unpublished). Ray et al. (1968) have recently provided evidence that in rat liver a pool of surface membrane precursors exists that continues to be incorporated into the surface membrane after protein synthesis is inhibited by cycloheximide. 70-’

Counts i n membrane extracled into chloroform: methanol (%)

30.6

20.6

29.6

30.0

*

*

,I

*

60 I

.?0. 5 0 -

Glucose-

14

C

5 .-W

40-

c

z

30-

W

2e

20-

W

I 10 -

0 0

A

,

I

10

20

30

40

50

60

70

80

90

100

110 120

T i m e (hr)

49 41

17 V

9 I

B

0

2

I

Days

FIG.2A and B. See facing page for legend.

3

7.

205

SURFACE MEMBRANE OF ANIMAL CELLS

W

n e

5E

0

C

2

I

3

Days

FIG.2. Decay in 14C specific activity of the surface membrane of L cells labeled with ( A ) glucose-l4C, ( B ) 1%-labeled L-valine and L-leucine, and ( C ) D-glucosamine-14C and subsequently grown in nonradioactive medium. A. L cells were grown in glucose-14C (specific activity = 56,400 counts per minute per pmole). The cells were centrifuged and washed twice with saline (0.16 M NaCl), a portion was taken for processing to obtain surface membranes and cell particulates, and the remainder was placed in 100 ml of fresh nonradioactive medium and allowed to grow with a TD of approximately 18-20 hours. At the times indicated on the graph, half the culture was removed and processed. An equal volume of fresh nonradioactive medium was added back and the cells continued to grow. The cells appeared healthy as observed under the phase contrast microscope and remained constant in size. The predicted curve was calculated on the basis of increase in cell number, i.e., if the cells doubled in number in nonradioactive medium the predicted specific activity would be 50% of the initial specific activity. B. The design of this experiment is the same as that described in A. Cells were labeled with ~-valine-l4Cand ~-1eucine-14C(specific activity of each amino acid was 546,000 counts per minute per pmole). The L cells grew in nonradioactive medium as shown by the doubling time on the abscissa. The cells appeared healthy as observed in the phase contrast microscope and remained constant in size. 0-0 surface membrane, counts per minute per pg protein, predicted and observed; - - surface membrane, counts per minute per cell ghost X 10-4, predicted and observed. C. The design of this experiment is the same as that described in A. Cells were labeled with 1%-labeled Dglucosamine-HC1 (1.2 X 106 counts per minute per milliliter). The L cells grew rapidly in nonradioactive medium as shown by the doubling time on the abscissa. The cells appeared healthy as observed in the phase contrast microscope and remained constant in size. 0-0 surface membrane, counts per minute per pg protein, predicted and observed; - - - surface membrane, counts per minute per cell ghost X 10-4, predicted and observed.

-

1

19 18 17 16

15

:

h

l4

5

0

13

v

.E 12 P) +

g

II

4 0 .2

0

8

0

% 7 p 6

3

5

4

r

3 (extracted with ethanol)

2

I

4 4 4 20,2 19.3 24.2 ICounts in membrane extracted by ch1oroform:methanol (%) 1 -

I

2

I

1

I

I

3

Time (days)

FIG.3. Decay of specific activity of nongrowing, 14C-labeled L cells. L cells in logarithmic phase were grown in the presence of glucose-14C (specific activity = 15,800 counts per minute per pmole glucose) for 3 days. The cells were harvested, washed twice with saline, and then resuspended in 300 ml nonradioactive medium, The cell count was 2.05 X 100 per milliliter. A sample was removed before resuspension and each day thereafter for 3 days. Cell number increased to only 2.34 X 106 per milliliter on the third day. There was no significant change in cell size during the experiment. At the times indicated one sample was removed to obtain surface membranes and cell particulates. A second sample was centrifuged, the medium was removed, and the pellet was washed and extracted with 4 ml of 30% ethanol. The ethanol extraction was repeated twice. The cells after this treatment appeared remarkably intact. Protein and 14C contents of the cell pellets were determined. The glucose content of the medium on day 3 was 45% that of fresh medium. Values for the percent of counts extracted from the membrane by chlorofomxmethanol were Obtained.

7.

SURFACE MEMBRANE OF ANIMAL CELLS

207

IV. Synthesis and Turnover Rates of the Surface Membrane During the Cell Cycle

It is believed that cells in tissue culture in the plateau phase are in a G1 phase of the cell cycle ( Baserga, 1965), and it is not unreasonable to assume that the nondividing cells in our experiments are also in the GI phase. Our results indicate that the cell's ability to make the surface membrane is not impaired greatly if at all in this phase (Table I, Fig. 1).We have also blocked cell division by excess thymidine which arrests cells at the end of G1 or in early S phase and by vinblastine which stops the completion of mitosis. In both of these phases biosynthesis of membranes proceeds. As seen in Table I incorporation of L-valine into surface membranes of nongrowing cells is approximately 80% that of growing cells. Cells prevented from proliferating by thymidine or vinblastine are equally capable of incorporating ~-valine-'~C into the membrane as calculated on a count per minute per microgram protein basis. However, cells inhibited from dividing by these two agents are enlarged and their surface membrane is correspondingly enlarged as measured by the protein content per cell ghost. Isotope was added after the cells had enlarged. Thus, when the incorporation data of Table I are recalculated on a per cell basis the total amount of synthesis of membrane per cell is greater than that of the smaller growing and nongrowing controls. It would appear that the larger nondividing cell arrested in G1-S interphase or M phase is capable of a greater amount of synthesis and turnover of surface membrane. In order to determine more precisely the cell's ability to make the surface membrane at various times during the cell cycle, Dr. E. Gerner and I have measured the rates of incorporation of isotopic precursors into the surface membrane of KB cells in synchronous culture. KB cells were synchronized by two 20-hour treatments with thymidine (Bello, 1988). After transferring the cells to the usual growth medium (time 0), the cells were pulsed at various times for l-hour periods with D-glucosamine-14C or ~-1eucineJ~C. At the end of incubation with isotope the cells were harvested, washed twice with physiological saline, and then subjected to the FMA procedure to obtain surface membranes, cell particulates, and soluble protein fractions of the cell. In Fig. 4 can be seen the results of experiments in which ~-1eucineJ~C and D-glu~osamine-~~C were used as precursors. The cell can make a surface membrane at all stages of the cell cycle but there appears to be a burst of synthesis just after the cell divides in the GI period. From other experiments we have been led to believe that there is little or no elevation in synthetic rate during the actual division period ( M ) . Elevation in rate seen here is most

208

LEONARD WARREN

CI

- 250

0 x -

E u) -

\ W

0

.-Y-o o

- 200

W

a 2.0

v)

-

0 ?

I .o

0

3

6

9

1

I

I

I

I

12

15

18

21

24

1150

A

Time ( h r ) FIG.4. Synthesis of surface membranes and other cell components throughout the cell cycle. KB cells were synchronized by the double-thymidine block method as described by Bello (1968). At time 0 cells were suspended in fresh medium and

probably due to those cells that have already divided and are, in fact, in the GI phase. It would seem then that the cell surface is made throughout the cell cycle with an extra burst of synthesis in the GI phase just after cell division. If cells are prevented from completing the cell cycle, turnover commences at the point of arrest wherever it may be. In the normal course of division of a tissue culture cell during which there is little membrane turnover, there is probably an accumulation of membrane precursor either internally as a pool awaiting incorporation or, more probably, there is actual incorporation into the membrane. If this is so, the membrane should increase in size, either by an enlargement of the entire cell or by the development of microvilli and folds on the cell surface. This questioii is being investigated at the present time. A t mitosis two relatively small

7.

6.0

h

209

SURFACE MEMBRANE OF ANIMAL CELLS

G lucosa mine -I4C

5 .O

07

a

\

E

a

,“ 4.0 .-c0)

v

X

-

c

0

h 3.0

250

h

+ .5

W

0

._ c 0 0

>-E

2.0

200

1.0

150

0 ._ c

.-

0

0)

Q

07 0

f

0 0

3

6

100 9

12

15

18

21

24

B

Time (hr)

aliquots were pulsed with ( A ) leucine-14C or ( B ) glucosamine-14C for 1 hour at the times indicated on the graphs. The cells were then processed for surface membranes, cell particulates, and soluble proteins by the method as previously described (Warren and Click, 1968). Cells were counted with a hemocytometer.

cells are formed from one large cell upon division, and our interpretation is that an increased synthetic rate in the GI phase just after division rapidly brings the cells to ‘normal” size. In other words, by the time the division process is taking place most of two cell’s worth of membrane is present and the “deficit” is made with haste just after division. V. Differential Turnover of Components of the Surface Membrane

In all of our experiments on turnover we have found that the rates of incorporation and release of isotope from the surface membrane followed a similar pattern whether a 14C-labeledprecursor for proteins (L-valine and L-leucine ) carbohydrates (D-glucosamine) or a general precursor such as D-glucose was used. Further, in most experiments the lipids of the isolated membranes were completely removed by extraction with chloro-

210

LEONARD WARREN

form-methanol and the percent of the total radioactivity of the membrane found to be lipid soluble was determined. We found that in a given experiment using the above listed isotopic precursors the proportion of total radioactivity in the lipid was constant, within experimental limits. If there was a differential turnover between the lipid and the rest of the membrane one might expect the percent of lipid soluble counts of the membrane to change progressively in a regular fashion. The constancy suggests that grossly there is little difference between the turnover rates of the lipid and protein or carbohydrate components of the membrane and they all turn over in tandem. However, these experiments are not definitive since the data measure overall values. It will be necessary to measure the turnover rates of specific chemical entities of all classes of compounds in the membrane before more precise statements can be made on differential turnover of the individual components. Omura et al. (1967)have found that the lipids of smooth and rough endoplasmic reticulum (ER) of rat liver turn over 10 to 30% faster than does the protein. Differences between these results and ours may not only be explained on a technical basis but also might possibly be accounted for by differences in the types of membrane (surface us. E R ) and differences in the cell environment ( culture us. tissue). VI. Synthetic and Degradative Aspects of Turnover

It was shown several years ago that the release of isotopic macromolecular material from liver slices was inhibited by metabolic inhibitors (Simpson, 1952) and by amino acid analog (Steinberg and Vaughan, 1956) as if inhibition of protein synthesis inhibited degradation and release of cellular material. We have carried out experiments based on these observations to see if inhibition in a variety of ways affected both incorporation into, and release of isotope from, the surface membrane (Fig. 5 ) . We measured the degree of inhibition of glucoseJ4C incorporation into the surface membrane by puromycin, KCN, dinitrophenol (DNP), and by omitting either glutamine or valine, leucine, and isoleucine from the medium. The control was a nondividing cell. It was found, as expected, that there were varying degrees of inhibition of isotope incorporation into cells deprived of essential amino acids or in contact with inhibitors. A corresponding experiment was done in which the conditions in the vessels were identical except that the removal of isotope from cell membranes of labeled cells was observed. The cells used had been heavily labeled with glucose-14C and then washed. A sample was removed to

7.

211

SVRFACE MEMBRANE OF ANIMAL CELLS

obtain an initial specific activity of radioactivity in the membrane, and the remaining cells were divided among six vessels in nonradioactive medium, a control, and five others, each with an inhibitor or lacking amino acids as described above. In this experiment we were able to measure the degree of inhibition of removal of isotope from the surface membrane-the degradative aspect of turnover. In Fig. 5 it can be seen that if a certain treatment inhibited incorporation 50%, then removal of isotope was also decreased by 50%. This would suggest that the synthetic Puromycin.

Puromycin Cell particulates

Surfoce membrane

NO ValI leuc, iso~euc.

/

I

.I_

t / 0

A

/

_I .I

.No

vol, I

20 40 60 80 100/0 20 40 60 80 Inhibition of isotope incorporotion (%I Inhibition of isotope incorporation (%)

100

B

FIG. 5. Effect of inhibitors on the incorporation of isotope into, and loss of isotope from, the surface membrane and cell particulates. A. Incorporation of isotope. Six flasks were set up, the first containing 2.5 X 100 cells per milliliter (25 ml). This served as a control with nongrowing cells. The remaining five flasks each contained 100 ml of medium with 5 X lo5 cells per milliliter: flask 1, control; flask 2, 35 pg puromycin per milliliter; flask 3, 10-3 M KCN; flask 4, 10-4 M dinitrophenol; flask 5, glutamine omitted from medium; flask 6, valine, leucine, and isoleucine omitted from medium. Flasks were incubated for 2 hours at 37°C. Glucose-14C was added (specific activity 10,500 counts per minute per pmole glucose) and the flasks were then incubated for 23.5 hours. Cells were harvested and processed for membranes by the FMA method. Cell growth did not occur in any of the vessels nor were there significant changes in cell sizes. B. Loss of isotope. L cells were labeled by growing them in the presence of glucose-14C overnight. The cells were harvested and washed twice with saline. One portion of the cells (3.4 X 107) was processed for surface membranes. The remainder was divided among six flasks as described under ( A ) . The medium in this experiment contained no isotope. Volumes of the medium, cell counts, and inhibitor concentrations were the same as in ( A ) . The cells were incubated for 23.5 hours and then processed for surface membranes. The only difference between this experiment and that of ( A ) was that there was no preincubation with inhibitors for 2 hours. Cell growth and change of cell size were not significant.

212

LEONARD WARREN

and degradative aspects of turnover are coupled: if synthesis is inhibited a corresponding degree of inhibition of degradation occurs. How this coupling is effected is completely unknown. Perhaps a balance between synthetic and degradative processes is an important factor in various pathological conditions and in the process of death itself. VII. Turnover and Repair

In the previous sections evidence was presented for the constant replacement of the surface membrane which proceeds at a rapid rate when the cell is not dividing and at a slower pace in the dividing cell. This process can be viewed as a mechanism of repair in that surface structures, if damaged, would be replaced in the normal processes of turnover. In preliminary experiments we have measured the rate of incorporation of 14C-labeledL-valine, L-leucine, and D-glucosamine into the surface membranes of two groups of chick fibroblasts, controls and cells treated in a medium containing 0.25% trypsin for 10 to 20 minutes. This enzyme is commonly used for removing cells from glass surfaces and from one another in a tissue, and it acts by cleaving and removing surface proteins, i.e., it damages the cell surface. It was found that the rates of rise of 14C specific activity in the membranes of both control and trypsin-treated cells were approximately the same immediately and 24 hours after trypsin treatment. Evidence was not found for a burst of synthesis for repair of damaged parts. Rather, it appears that the cell would have replaced the surface structure whether or not it was damaged. This of course does not rule out the possibility that there are specific repair mechanisms but it would seem that in this instance, the cell, instead of specifically repairing what was damaged, replaced the whole unit with a new one (C. Buck, L. Warren, and M. C. Glick, unpublished results). The rate of repair is then presumably linked to the turnover rate of the surface membrane and one would predict that repair of the surface of a nondividing cell is considerably more rapid than that of a dividing cell. Although it is difficult to comprehend why there should be rapid turnover at all, one of its consequences would be the replacement of defective parts and these might not be uncommon at the surface of a cell in a hostile environment containing degradative enzymes such as proteinases, phospholipases, and glycosidases. VIII. Speculation on the Malignant Cell

The work discussed in previous sections has led us to a consideration of the malignant cell and to the question of how this cell possibly differs

7.

SURFACE MEMBRANE OF ANIMAL CELLS

213

from a normal cell with regard to the metabolic turnover and the properties of the surface membrane. Many workers feel that the surface of a malignant cell is altered (Weiss, 1967), and in this section an hypothesis will be presented which relates the alteration of surface membrane to the peculiar behavior of the malignant cell. Although the hypothesis is grounded on little data, it does have the merit of being testable and provides a basis for devising experiments. The hypothesis rests on the assumption that the surface membrane of a malignant cell has a higher rate of turnover than that of a nonmalignant cell. The basis of the postulated enhanced turnover rate could be the production of an “imperfect” membrane protein brought about by genetic or other alterations. The critical carcinogenic event would be the production of altered membrane proteins. As a result of this alteration an “unstable” membrane might be produced in which the proteins would not be as tightly associated as usual. The membrane might then continually shed various or all components, necessitating replacement and this would constitute turnover. Changeux et a2. (1967) have discussed the far reaching consequences of altering some of the proteins of a membrane. A change in one type of protein affects the behavior of all of them in a highly integrated membrane complex. Enhanced turnover in the nondividing state might also be a consequence of malfunctioning of control mechanisms within malignant cells (Reid, 1965; Pitot and Cho, 1965). In this instance a normal reduction of membrane synthesis would not take place when cell division ceases. We suggest that cells in tissue culture are, from the point of view of turnover, highly malignant for they are turning over rapidly when not dividing and are synthesizing almost as much structural material in this state as when they are dividing. I n tissue culture there appears to be little control over the rate of synthesis of the membrane. We postulate that a normal, nondividing cell in a tissue is largely “shut off,” and that when not dividing its biosynthetic functions are operating at only a small fraction of the rate that it is when it is dividing, with a relatively low rate of turnover. Here the rate of synthesis is geared to the needs of the cell. If cells with a high turnover capacity, as measured under tissue culture conditions, are injected into a host, the host may be able to depress the rate of synthesis and turnover of the surface membrane and the cell is controlled; if not, a tumor results. Perhaps the cell in culture or the malignant cell remains operating in some phase of the cell cycle (GI, S , Gz, M ) even if it is not dividing. However, the normal cell in a tissue after dividing would be capable of

214

LEONARD WARREN

shifting to a state (perhaps G o ) where synthesis can be reduced and the cell then remains in this state with a low rate of turnover. One of the consequences of increased turnover might be a decreased adhesiveness between malignant cells or a decreased anchorage to glass or plastic surfaces in tissue culture. This is believed to be a characteristic of the malignant cell (Coman, 1944, 1961; McCutcheon et al., 1948; Tjernberg and Zajicek, 1965). If we postulate that cells adhere to one another through intercellular proteins anchored in the cell surface, and that the surface structure is constantly giving way as the surface material turns over and is shed, then the links between malignant cells could be relatively weakened. The resulting accumulation of rejected surface material around the cell might account for some of the observations that have been made on the malignant cell. It has been reported that malignant cells have a greater negative surface charge density than do corresponding cells as shown by electrophoresis of living cells ( Forrester et al., 1964). Sialic acid, a component of the surface membrane, is responsible for much of this charge ( Ruhenstroth-Bauer, 1965). Other workers have not found enhanced surface charge in malignant cells (Vassar, 1963). Our view is that increased charge at the surface is brought about by the piling up of varying amounts of rejected surface material and that this frequently, although not necessarily, contains sialic acid, a component of surface membranes. The building up of such a layer in a culture of cells or in a tissue may, of course, depend not only on an increased rate of production but also on the rapidity with which it can be removed. In this regard the inherent adhesive or “sticky” quality of the material (Coman, 1961) is a very important factor. This property may be altered in the malignant cell so that it would not be as readily removed from the surface of the cell. Histochemical methods have also shown that virus-transformed tissue culture cells contain large amounts of sialic acid-containing material (Defendi and Gasic, 1963) and acid mucopolysaccharide (MartinezPaloma and Brailovsky, 1968; Morgan, 1968) at their cell surface. Martinez-Paloma and Brailovsky (1968) have measured the thickness of a layer stainable with ruthenium red on the surface of hamster embryo cells and their counterparts transformed by Adenovirus ( A d ) 12 or SV40 virus. While the mean thickness of the outer layer (outer leaflet of membrane plus material staining with ruthenium red) was 130 A in the normal cell, it was 330 A for the Ad 12-transformed cells and 230 A for the SV40 transformant, Much of the material staining with ruthenium red can be removed with hyaluronidase (Morgan, 1968) indicating that it is an acid mucopolysaccharide. Whether the stain binds to surface membrane mate-

7.

SURFACE MEMBRANE OF ANIMAL CELLS

215

rial other than acid niucopolysaccharide is not known. The surface material found external to the cell membrane trilaminar structure may be only one component of the cell surface structure, i.e., a selection process may take place or there may be an overproduction of one component relative to others. The formation of a capsulelike structure, if only 100 A in thickness might be able to counter the host’s immunological defense system. Since it is external to the real cell surface and could have antigenic activity, it might serve as a first line of defense against humoral antibodies as well as the host cellular defense system. The exaggerated surface coat of a malignant cell might be comparable to the capsule of a bacteria which is frequently an effective defense against the host macrophages. Further, the malignant cell’s “capsule” might also be of survival value in the formation of metastases in sights that are foreign to the cell since the cell would be providing its own immediate physical environment. Little is known about the specific mechanisms that control and regulate cell growth, i.e., by what means cells in culture or in tissues stop dividing or how under certain circumstances they begin to divide. The concept of “contact inhibition” of motion ( Abercrombie and Heaysman, 1954) which has broadened to encompass contact inhibition of growth, metabolism, and biosynthesis (Levine et al., 1965) has been a dominating force in our thinking in recent years. Investigations have shown that in addition to a contact factor, control may reside in growth factors or nutritional agents and inhibitors (Todaro et al., 1965; Rubin, 1966; Holley and Kiernan, 1968; Beierle, 1968). In transformed cells which are much less susceptible to contact inhibition, the requirement for the growth factor is reduced (Holley and Kiernan, 19S8). Whether control is by contact, or by humoral mechanisms, or both it is undoubtedly mediated through the surface membrane, and may ultimately operate through a common control apparatus at that site. Control of cellular metabolism from the cell surface is seen in the action of colicines. Very few molecules of this material attached to a bacterial cell surface have a profound inhibitory effect on the metabolism of the microorganism (Jacob et at., 1952; Nomura, 1964;Luria, 1964). In considering the mechanisms by which cells control one another in regulated growth it may be worth examining the factor of intercellular distance. Is it possible that the intimacy of physical contact is a critical factor in determining whether cells can shut each other down? If so, it may be that the accumulation of material on the surface of a malignant cell, as previously discussed, might separate cells beyond a critical dis-

216

LEONARD WARREN

tance at which they are functionally and morphologically isolated, Thus, in a normal tissue most cells are closely apposed, and the resting rate of synthesis and turnover is at a low level while in malignancy many cells are separated from one another. This might be demonstrated with the electron microscope. However, the testing of this idea would be difficult because of possible changes in intercellular distances during the processing of material for the electron microscope ( e.g., dehydration). Complications could arise because even in a tumor many cells could be resting, or cell separation for release from “control” might only be necessary over part of the cell surface. It is of interest that House and Stoker (1966) have shown that there are spaces between cultured BHK21 cells that have been transformed spontaneously or by SV40 virus which are not seen in the untransformed cell. Perhaps there is rejected surface membrane material in these spaces. A possible correlation between changes in intercellular distances and enhancement of biosynthetic activity can be seen in the regenerating liver of the rat. In the first 6 hours after excision of most of a rat’s liver prior to an enormous burst of cell division, characteristic changes occur at the cell surface of the remaining hepatocytes. There is a simplification of the surface structure with loss of sinusoidal villi, loss of contact between intercellular surfaces, and “progressive separation of cell surfaces and disengagement of the interdigitations” ( p. 187). However, morphological evidence for the formation of new substances at the cell surface could not be found (Lane and Becker, 1966). There also appears to be an increased stickiness of the cell surface as well as a rapid rise in the electrophoretic mobility of liver cells (Eisenberg et al., 1962; Chaudhuri and Lieberman, 1965) reminiscent of the malignant cell and possibly associated with the accumulation of glycoprotein (rejected surface membrane?) at the surface of the cells. One can only speculate on the mechanism by which cells that are very closely apposed could shut each other down. It is possible that there is mediation by soluble or surface-associated factors such as those observed by Holley and Kiernan (1968) or by Beierle (1968). Perhaps under normal conditions there is a buildup in concentration of this type of factor in the very small intercellular space between closely apposed normal cells, and that these factors can be carried away much more rapidly when this space enlarges. We have also considered the possibility that cells which appose each other very closely stimulate each other to produce agents that inhibit one another without necessitating the passage of material between the cells.

7.

SURFACE MEMBRANE OF ANIMAL CELLS

217

In the past year, Dr. M. C. Glick and I, have shown that preparations of surface membranes of L cells can incorporate amino acids into proteinlike products (Glick and Warren, 1968, 1969). We believe that synthesis may take place in a population of ribosomelike particles close to or just under the surface membrane, and that these might be especially responsive to environmental changes such as the close approach of another cell. Although there is no real evidence to support the idea, one could visualize the production of proteins or polypeptides or small peptides at the cell surface which upon passing inward partially inhibit various key systems. This would be an amputation system leading to inhibition of macromolecular synthesis and mitosis. Control in this instance would be mediated by a constant supply of inhibitors derived in part from the cell surface. A recently reported trypsin-sensitive inhibitor of protein synthesis found in microsomal membranes (Salb and Marcus, 1966; Scornick et al., 1967) could conceivably be such a substance. It might originate at the cell surface and keep the cells in a metabolically quiescent state. In the regenerating rat liver its synthesis would cease and inhibition of general synthesis of the cell would end. The liver cell would commence synthesis for division. An hypothesis could also be constructed on the basis of the synthesis of activators at the cell surface which would be formed only when cells are separated from one another. Serum factors, previously discussed, might mimic the action of these activators. To test the hypothesis of enhanced turnover rates of the cell surface of malignant cells, Dr. V. Defendi and I have been comparing the rates of incorporation of 14C-labeledamino acids into the surface membranes of “normal” tissue culture cells and their SV40 or polyoma virus-transformed counterparts. We have found that the incorporation rate of 14Clabeled glucose or amino acids is approximately twice as high in the transformed cell lines W18Va2 and W126Va4 as into their diploid, nontransformed counterparts W138 and W126, respectively, both when the cells are in logarithmic and plateau phases of growth. On the other hand, the polyoma-transformed cell, P138, incorporates at no faster a rate than does C 13, the cell line from which it was derived. Interpretation of experiments using transformed cell lines is not simple because in the process of transformation only a few cells of a large population are in fact transformed and new lines are derived from these. It is quite possible that the cells from which the new lines are derived are quite atypical in a heterogeneous population even before transformation and so the “control” may not be a valid one. Further, the behavior of cells in culture is not directly comparable to the behavior of normal and

218

LEONARD WARREN

malignant cells in a tissue environment. Although a tissue culture cell and its transformed counterpart may have the same incorporation and turnover rate in culture, when injected into an animal the transformed cell might maintain its relatively high rate of turnover while the rate of the control cells might be reduced by the host to “normal” tissue levels. Therefore, the differential in turnover rates between normal and malignant cells which we postulate, would in some instances be seen only in the tumor in viuo but not in culture. The comparison of most relevance would be the turnover rates of the surface membranes of malignant cells and their controls in a tissue. Experiments with Dr. C. Buck that bear on this comparison have recently been carried out. We have consistently found that embryonic chick fibroblasts and their RSV-transformed counterparts in tissue culture incorporate radioactive leucine or glucosamine into the surface membrane and cell particulate matter at the same rate. This is so whether the cells are in the log or plateau phases of growth. On the other hand, when a comparison is made of the rate of incorporation of isotopic precursors into isolated, whole sarcomatous nodules, produced by infecting chorioallantoic membrane with Rous sarcoma virus, with the incorporation into nonnodular areas of the same chorioallantoic membrane or into chorioallantoic membrane of eggs not infected with virus, it is found that the sarcomatous nodules have twice the 14Cspecific activity as the controls. In these studies isotope incorporation into the whole cell was measured; surface membranes were not isolated. If these results are codrmed in future studies in which incorporation of the isotope into the surface membranes of specific types of cells of the chorioallantoic membrane is measured, they will not only lend some support to the hypothesis proposed above but will also suggest that comparisons of normal and transformed malignant cells should be made in vioo rather than in culture. As stated before, it is quite possible that “normal” cells in culture are equivalent to malignant cells and are not suitable as controls. Stated differently, “normal” and malignant cells may tend to approximate one another, in some respects, in culture. There are a number of reports in the literature which indicate that cells in various experimental situations tend to either carry on specialized function or produce DNA and divide (Holtzer et al., 1960; Stockdale and Holtzer, 1961; Stockdale et d.,1963). Several exceptions to this statement, however, are now known (Cahn and Lasher, 1967). It is known that the conditions of culture may determine whether a cell can do both and perhaps the distinction resides in the biosynthetic potential of a cell

7.

SURFACE MEMBRANE OF ANIMAL CELLS

219

under given conditions, i.e., those with limited or reduced potential can do one or the other while those with higher biosynthetic and metabolic capacity can manage both. Our work with the L cell (and with the primary chick fibroblast) in culture suggests that it synthesizes as much cellular material ( surface membrane, particulates ) when it is not dividing as when it divides. The nondividing cell is, in this respect, equivalent to a dividing cell and under the standard conditions of culture may not be capable of doing more than synthesizing cellular material. Thus, specialized, differentiated functioi is lost. This could conceivably be the basis of the commonly observed loss of specialized function of the cell in tissue culture and of the malignant cell which resembles the cell in culture. IX. Conclusions

On the basis of what is admittedly incomplete data on the turnover of surface membranes in tissue culture cells, we have attempted to construct an hypothesis that affords some explanation of certain aspects of cellular behavior, both social and metabolic. We begin with the notion that the cell in culture synthesizes almost as much surface membrane material in the dividing and nondividing state. When dividing, the new material goes into the making of a new cell, with a small amount of turnover; when not dividing the material goes into the cell structure and an equivalent amount is rejected and here there is a high rate of turnover. If the surface structure is damaged it is replaced in the course of turnover at the same rate that it would have been if it had not been damaged. We have presented an oversimplified hypothesis of the basis of the manifestation of malignancy, in which many complications have been ignored. Nevertheless it may have some validity and provide a testable, rational basis for further experimentation. The difference in turnover rates of the surface membrane of dividing and nondividing cells could be reflected in differences in virus receptivity, antigenic specificity, adhesiveness, permeability, and electrical coupling ( Lowenstein, 1967). The quality of the surface membrane of a dividing and nondividing cell may be different. Further, it is possible that alteration of the rate of turnover of the surface and other components of the cell in response to environmental conditions, especially in development, may be very important. Turnover may also serve as the vehicle of change in the specific nature of a membrane. Kalckar (1965) has discussed the possible metabolic mechanisms by which galactose and other sugars might be lacking at the

220

LEONARD WARREN

surface of malignant cells leading to changes of function and antigenic specificity. Through turnover new, different membrane precursors, formed in the absence of specific sugars, might be incorporated leading to the formation of an altered membrane. Indeed one wonders how, normally, the properties of the membrane are maintained in the face of rapid turnover in culture. It seems unlikely that membranes are rather static supports to which special factors or enzymes are added or removed. Dallner et al. (1966) have shown that in the rat, at birth, certain enzymes rapidly increase in the endoplasmic reticulum of liver cells. It is quite possible that these enzymes ( glucose-6-phosphatase,ATPase, NADH cytochrome c reductase, and others) are not just formed and then hung on a rather static preexisting membrane but rather are formed, incorporated into loose subunit associations, and are then incorporated into the membrane during the normal course of membrane turnover in nondividing cells and into new membrane in dividing cells. The rates of incorporation of an enzyme are probably determined by the rate of its formation, its availability at the site of membrane precursor assembly, the number of molecules of enzymc usually going into the precursor complex, the size of the precursor complex pool, and the rate of membrane turnover. If the enzyme (or antigen) is not present a t the time of precursor assembly the membrane can still be made. Underlying this view is the unproven assumption that there can be considerable latitude in the composition of a precursor complex that can still be aggregated into a functional membrane. We are constantly looking at pictures of the cell and its membranes that are fixed in time and space and this static impression cannot help but influence our thinking. Certainly visual evidence of turnover is rare and its firm interpretation as turnover is virtually impossible. This review may be of use for it emphasizes the dynamic nature of membrane systems. The turnover discussed is a fundamental cellular process that surely must have a profound impact on our consideration of physiological and pathological phenomena. ACKNOWLEDGMENTS This paper was written with the support of United States Public Health Service grant No. 1-ROlHD 005201 and the American Cancer Society grant No. T-333. We wish to thank Dr. L. Bello for kindly providing KB cells and assisting us in synchronizing the culture. REFERENCES Abercrombie, M., and Heaysman, 1. E. M. (1954). Exptl. Cell Res. 6, 293. Baserga, R. (1965).Cancer Res. 25, 581.

7.

SURFACE MEMBRANE OF ANIMAL CELLS

221

Beierle, J. W. ( 1968). Science 161, 798. Bello, L. (1968). Biochim. Biophys. Acta 157, 8. Cahn, R. D., and Lasher, R. (1967). Proc. Natl. Acad. Sci. U S . 58. Changeux, J. P., Thiery, J., Tung, Y., and Kittel, C. ( 1967). Proc. Natl. Acad. Sci. us. 57, 334. Chapman-Andresen, C. (1962). Compt. Rend. Lab. Carlsberg 33, 73. Chaudhuri, S., and Lieberman, I. (1965). Biochem. Biophys. Res. Commun. 20, 303. Christiansen, R. G., and Marshall, J. M. (1965). J. Cell Biol. 25, 443. Coman, D. R. (1944). Cancer Res. 4, 625. Coman, D. R. (1961). Cancer Res. 21, 1436. Dallner, G., Siekevitz, P., and Palade, G. E. (1966). J. Cell Biol. 30, 97. Defendi, V., and Gasic, G. (1963). J. Cellular Comp. Physiol. 62, 23. Eisenberg, S., Ben-Or, S., and Doljanski, F. (1962). Exptl. Cell Res. 26, 451. Forrester, J. H., Ambrose, E. J., and Stoker, M. G. P. (1964). Nature 201, 945. Click, M. C., and Warren, L. ( 1968). Federation Proc. 27, 299. Click, M. C., and Warren, L. (1969). Proc. Natl. Acad. Sci. U.S. 63, 563. Henmann, H., and Marchok, A. (1963). Deuelop. Biol. 7 , 207. Holley, R. W., and Kiernan, J. A. (1968). Proc. Natl. Acad. Sci. U.S. 60, 300. Holtzer, H., Abbot, J., Lash, J., and Holtzer, S. (1960). Proc. Natl. Acad. Sn’. U.S. 12, 1533. House, W., and Stoker, M. G. P. (1966). J. Cell Sci. 1, 169. Jacob, F., Siminovitch, L., and Wollman, E. L. (1952). Ann. Inst. Pasteur 83, 295. Kalckar, H. M. ( 1965). Science 150, 305. Kruse, P. F., Jr., Miedema, E., and Carter, H. C. (1967). Biochemistry 6, 949. Lane, B. P., and Becker, F. F. (1966). Am. J. Pathol. 48, 183. Levine, M., Becker, Y., Boone, C. W., and Eagle, H. (1965). Proc. Natl. Acad. Sci. U S . 53, 350. Lowenstein, W. (1967). Deoelop. Biol. 15, 503. Lowly, 0. H., Rosebrough, N. J., Farr, A. L., and Randall, R. J. ( 1951). J . Biol. Chem. 193, 265. Luria, S. E. (1964). Ann. Inst. Pasteur 107, 67. McCutcheon, M., Coman, D. R., and Fontaine, B. M. (1948). Cancer 1, 460. Martinez-Palomo, A,, and Brailovsky, C. (1968). Virology 34, 379. Morgan, H. R. (1968). J. Virol. 2, 1133. Nachmias, V. T. (1966). Exptl. Cell Res. 43, 583. Nomura, M. (1964). Proc. Natl. Acad. Sci. U S . 52, 1514. Omura, T., Siekevitz, P., and Palade, G. E. (1967). J. Biol. Chem. 242, 2389. Pitot, H. C., and Cho, Y. S. (1965). Progr. Elrptl. Tumor Res. 7 , 158. Ray, T. K., Lieberman, I., and Lansing, A. I. (1968). Biochem. Biophys. Res. Commun. 31, 54. Reid, E. (1965). “Biochemical Approaches to Cancer,” p. 138. Permagon, Oxford. Rubin, H. (1966). Exptl. Cell Res. 41, 138. Ruhenstroth-Bauer, G. ( 1965). In “Cell Electrophoresis” (E. J. Ambrose, ed.), p. 66. Churchill, London. Salb, J. M., and Marcus, P. I. (1966). 1. Cell Biol. 31, 98A. Schoenheimer, R. (1942). “The Dynamic State of Body Constituents.” Harvard Univ. Press, Cambridge, Massachusetts. Scomick, 0. A., Hoagland, M. B., Pfefferkorn, L. C.,and Bishop, E. A. (1967). J. Biol. Chem. 242, 131.

222

LEONARD WARREN

Simpson, M. V. (1952).1. Biol. Chem. 201, 143. Steinberg, D.,and Vaughan, M. (1956).Arch. Biochem. Biophys. 65, 93. Stockdale, F. E.,Abbot, J., Holtzer, S., and Holtzer, H. (1963).Deuelop. Biol. 7, 293. Stockdale, F. E., and Holtzer, H. (1961).Exptl. Cell Res. 24, 508. Tjernberg, B., and Zajicek, J. (1965).A d a Cytol. 9,197. Todaro, G. J., Lazar, G. K., and Green, H. (1965).J. CeUukr Comp. Physiol. 66, 325. Vassar, P. S. (1963).Lab. Znuest. 12, 1072. Warren, L., and Glick, M. C. (1968).J. Cell Biol. 37, 729. Warren, L.,Glick, M. C., and Nass, M. K. (1966).J. CeU Physiol. 68, 269. Weiss, L. (1967).“The Cell Periphery, Metastasis and Other Contact Phenomena.” North Holland Publ., Amsterdam.

AUTHOR INDEX Numbers in italics refer to the pages on which the complete references are listed. A Abbot, J., 218, 221, 222 Abercrombie, M., 215, 220 Ackermann, W. W., 81, 92, 98, 105, 106 Agerbak, G . S., 176, 193 Alman,R. W., 117, 145 Altschul, R., 39, 75 Ambrose, E. J., 214, 221 Andersen, H. J., 111, 144 Anderson, M. L., 25, 36 Armstrong, R. L., 18, 19, 33 h a u d , M., 34 Asling, C. W., 143, 148 Aurelian, L., 83, 86, 91, 104, 105

B Bablanian, R., 90, 91, 93, 105 Bach, J. A., 6, 7, 33, 35 Baillie, A., 21, 35 Baillie, H., 8, 33 Baker, W. W., 44, 76 Balassa, G., 17, 33 Balazs, R., 113, 115, 120, 121, 122, 126, 128, 144 Ball, W. D., 177, 178, 181, 191,192,193 Baltimore, D., 82, 85, 87, 88, 91, 93, 94, 97, 99, 100, 105 Barlow, R. H., 172, 174, 194 Barnett, R. J., 114, 144 Baron, S., 101, 105 Barraclough, C. A., 144 Baserga, R., 207, 220 Bassleer, R., 40, 75 Beach, F. A., 110, 144 Beaudoin, A. R., 137, 145 Becker, F. F., 216, 221 Becker, Y., 81, 90, 92, 95, 99, 105, 107, 215, 221 Beierle, J. W., 215, 216, 221 Bell, E., 73, 76 Bellett, A. J. D., 83, 105 Bello, L., 207, 2.08, 221 Bello, L. J., 89, 92, 97, 105, 106 Ben-Or, S., 216, 221

Ben-Porat,. T.,. 94.. 98., 105 Berlin, C. M., 165, 166 Bernkopf, H., 81, 82, 92,105 Bernkopf, J., 82, 107 Berwald, Y., 46, 75 Betz, F. H., 39,70, 72, 75 Bishop, E. A., 217, 221 Blicharska, J., 34 Blizzard, R. M., 111, 147 Block, J. B., 140, 145 Blumenthal, H. J., 15, 34 Bolognesi, D. P., 95, 105 Boone, C. W., 215, 221 Borman, G. S., 86, 107 Bornstein, M. B., 137, 145 Boyse, E. A., 171, 193 Bradley, P. B., 112, 145 Brailovsky, C., 214, 221 Block, J. B., 140, 145 Bronstein, I. P., 111, 145 Brown, A., 82, 105 Brown, A. W., 111, 145 Brown, D. D., 74, 75 Brown, F., 90, 105 Burkart, J., 130, 135, 146 Burokas, B., 172, 174, 194 Bursztyn, H., 136, 139, 141,147

C Cahn, M. B., 38,72,73, 75 Cahn, R. D., 38, 54, 72, 73, 75, 218, 221 Callender, J., 85, 93, 94, 100,105 Campbell, P. L., 124, 126, 145 Capers, C. R., 40, 75 Carter, H. C., 203, 221 Carter, W. A., 101,105,106 Chalkley, C . R., 152, 166 Chaloupecky, V., 20, 34 Chambon, P., 7, 16, 17, 20, 34 Changeux, J. P., 167, 213, 221 Chapman-Andresen, C., 203, 221 Chaudhuri, S., 216, 221 Cho, Y. S., 213, 221 Choppin, P. W., 88, 89, 106 Christiansen, R. G., 203, 221

223

224

AUTHOR INDEX

Clendinnen, B. G., 139, 140, 145 Cocks, W. A., 113, 115, 120, 121. 122, 128, 128, 144 Cole, L. A., 115, 147 Colignon, P., 40, 75 Coman, D. R., 214, 221 Coon, H. G., 38, 54, 72, 73, 75 Cooper, P. D., 83, 105 Cords, C. E., 88, 105 Cornatzer, W. E., 81, 107 Cosmos, E., 57, 76 Cox, D. C., 98, 107 Cox, R. A,, 90, 99, 105 Crandall, M. A., 193 Cunningham, I., 175, 178, 182, 183, 186, 193, 194 Curtis, A. S. G., 173, 175, 177, 178, 180, 181, 182, 185, 193

D Dahl, D., 120, 144 Dalgarno, L., 90, 99, 105 Dallner, G., 220, 221 Darnell, J. E., 85, 87, 88, 90,91, 93, 99, 105, 107, 108 Davidson, E. H., 45, 76 Dawid, I. B., 74, 75 De Bellis, R. H., 73, 76 DeDuve, C., 184, 193 Defendi, V., 67, 76, 214, 221 Deibler, G. E., 124, 128, 145 Deinhardt, F., 82, 83, 106 Dellacha, J. M., 141,146 De Mars, R., 75, 76 De Robertis, E., 120, 147 DeSombre, E., 160, 164, 166 Detwiler, S. R., 110, 145 Deutscher, M. P., 5, 6, 7, 16, 17, 20, 34, 35 Diamond, M. C., 142,145 Dinka, S., 98, 107 Doi, R. H., 7, 17, 18.20, 34 Doljanski, F., 216, 221 Donnelan, J. E., 18, 27, 28, 34, 35 Drzeniek, R., 83, 97, 107 Dubbs, D. R., 98, 106 Dulbecco. R., 81. 106 Dvorkin, .M.; 3, 34

E Eagle, H., 45, 76, 215, 221 Earle, W. R., 46, 76 Easty, G. D., 175,193 Eayrs, 'J. T., 110, 111, 112, 113, 114, 115, 116, 120, 121, 122, 126, 128, 136, 139, 140, 144, 145, 146 Ebert, J. D., 66, 67, 76 Ede, D. A., 176, 193 Edelman, G. M., 33, 34 Eggers, H. J., 90, 91, 93, 94, 99, 105 Ehrman, R. L., 53, 76 Eisenberg, S., 216, 221 Enders, J. F., 81, 105 Epstein, R., 11, 15, 18, 20,21, 35 Erdos, T., 164, 166 Essman, W. B., 112, 140, 145 Etkin, W., 137, 145 Evans, A. S., 81, 82, 108 Evans, H. M., 143, 148 Evenchik, Z., 11, 34, 35 Everett, S. F., 82, 105

F Falaschi, A., 6, 27, 34 Fantes, K. H., 101, 107 Farnham, A. E., 94, 105 Farr, A. L., 199, 221 Fazekas, J. F., 117, 145 Feldman, M., 40, 42, 43, 74, 77 Fenwick, M. L., 87, 93,105 Field, A. K., 101, 105, 106, 108 Field, E. J., 70, 76 Firket, H., 39, 70, 72, 75 Fischer, R. G., 81, 107 Fitz-James, P. C., 5, 20, 25, 27, 28, 34, 36 Flexner, L. B., 119, 120, 121, 136, 146' Fogel, M., 67, 76 Fogh, H., 84, 105 Fogh, J., 84, 105 Fontaine, B. M., 214, 221 Ford, C . E., 64, 69, 76 Forrester, J. H., 214, 221 Forro, 1. R., 15, 28, 29, 36 Franklin, R. M., 82, 85, 91, 93, 94, 97, 100, 105

22s

AUTHOR INDEX

Frieden, E., 137, 145 Fuchs, S., 60, 74, 77

G Gaffney, E. V., 84, 107 Gafford, L. G., 84, 107 Galanti, N. L., 172, 174,193 Galtsoff, P. S., 170, 173, 193 Galy, J. A., 33, 34 Garber, B., 184, 193 Garcia Argiz, C. A., 120, 136, 145, 147 Gasic, G., 214, 221 Gasic, G. J., 172, 174, 193 Geel, S. E., 121, 127, 128, 136, 137, 138, 145 Gelber, S., 124, 126, 145 Gentry, G. A., 84, 107 Gerhart, J., 150, 166 Gershon, D., 53, 60, 63, 65, 77 Gey, G. O., 53, 76 Ghittoni, I%. E., 118, 141,145, 146 Giacobini, E., 124, 146 Gilbert, W., 150, 166 Ginsberg, H. S., 82, 83, 89, 90, 92, 97, 98, 105, 106, 107 Girard, M., 87, 88, 93, 105 Girardi, A., 82, 83, 106 Gitlin, D., 140, 146 Giudice, G., 183, 193 Glaeser, R. M., 181, 182, 184, 193, 194 Glascock, R. F., 151, 166 Click, M. C., 183, 195, 198, 199, 201, 209, 217, 221, 222 Gluck, N., 73, 76 Goldman, J., 140, 146 Goldman, M., 15, 34 Goldstein, L., 164, 166 Gomatos, P. J., 85, 89, 106 Gomez, C. J., 118, 120, 136, 141, 145, 146, 147 Gonzalez, P., 84, 107 Gordon, J., 152, 166 Gorski, J., 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 166, 167 Goto, M., 70, 76 Gould, G. W., 17, 34

Graham, A. F., 85, 87, 89,106 Granger, E., 177, 178, 194 Granich, M., 144, 146 Graves, F. B., 117, 145 Greaves, M. F., 173, 177, 178, 180, 181, 182, 185, 193 Green, H., 64,77, 215, 222 Green, J. H., 7, 34 Greenawdt, J. W., 89, 97,106 Grelet, N., 4, 34 Grobstein, C., 2, 34 Gross, P. R., 3, 16, 17, 34, 36 Crossowicz, N., 8, 34, 81, 92, 105 Groves, P. W., 185, 195 Gudematsch, J. F., 110, 146 Guttman, S., 140, 146

H Hackett, A. J., 89, 106 Halvorson, H. O., 4, 6, 7, 9, 11, 13, 14, 15, 16, 17, 18, 20, 21, 22, 24, 25, 28, 30, 33, 34, 35, 36 Hamburger, V., 110, 146 Hamburgh, M., 111, 112, 113, 116, 117, 119, 120, 121, 130, 133, 134, 135, 136, 137, 141,142,143,145,146 Hamilton, M. G., 99, 106 Hanafusa, H., 94, 106 Hanson, R. P., 81, 82, 108 Hanson, R. S., 34 Hargie, M. P., 172, 174, 194 Harris, G. W., 144, 146 Harris, H., 64, 69, 76 Hartley, J. W., 82, 107 Haruna, I., 75, 77 Hanvood, J. R., 120, 144 Hastings, J. W., 11, 35 Hauschka, S. D., 53, 76 Hausen, P., 93, 108 Hay, J., 88, 108 Hayashi, K., 93, 107 Hayflick, L., 46, 70, 76 Heaysman, J. E. M., 215,220 Heeter, M., 85, 90, 91, 108 Henle, G., 82, 83, 106 Henney, H. R., 16, 34 Hermann, H., 203, 221 Herranen, A., 151, 167

226

AUTHOR INDEX

Herrmann, H., 39, 40, 76, 203, 221 Hestrin, S., 8, 34 Higa, A., 6, 13, 14, 15, 16, 17, 18, 21, 22, 24, 25, 28, 34, 35 Hilleman, M. R., 101,105,106,108 Hinuma, Y., 94, 106 Hirst, J. H . R., 178, 193 Hitchins, A. D., 17, 34 Hoagland, M. B., 217, 221 Hoekstra, W. G., 151, 166 Hoexter, F. M., 117, 146 Holland, J. J., 86, 88, 93, 97, 98, 105, 106 Holley, R. W., 215,216, 221 Holmes, K. V., 88, 89, 106 Holmes, P. K., 31, 34 Holtfreter, J., 42, 76, 176, 193 Holker, H., 40, 60, 76, 77, 218, 221, 222 Holker, S., 218, 221, 222 Homma, M., 87, 106 Horn, G., 136, 145 House, W., 216, 221 Hsu, T. C., 54, 76 Huang, A. S., 83, 89,95,97, 106, 108 Huebner, R. J., 81, 106 Humphreys, T., 73, 76, 171, 172, 173, 174, 193, 194 Hurst, D., 160, 166

I Igarashi, R. T., 17, 18, 20, 34 Ikeda, Y., 29, 35 Ishida, N., 94, 106 Issahary, C., 11, 34 Issahary-Brand, G., 11, 35

J Jacob, F., 2, 32, 34, 74, 76, 167, 215, 221 Jacobson, H. I., 151, 166 James, M. C. M., 175, 178, 182, 183, 186, 194 James, R., 185, 195 Jarvik, M. E., 112, 145 Jensen, E. V., 151, 180, 164,166 Jervell, D., 151, 167 Joklik, W. K., 95, 99, 100, 101, 103, 105, 106

Jones, B. M., 175, 178, 182, 183, 186, 194 Jones, P. C. T., 178, 194 Jost, A., 130, 146 Jungblut, P. W., 160, 164, 166 Jungwirth, C., 94, 95, 106

K Kafatos, F. C., 73, 76 Kaighn, M. E., 66,67, 76 Kalckar, H. M., 219, 221 Kaltenbach, J. C., 137, 146 Kamali-Rousta, M., 86, 107 Kaplan, A. S., 94, 98, 105 KaplGn, B., 120, 136, 145, 147 Katagiri, S., 94, 106 Kato, M., 83, 108 Kaufman, N., 83, 107 Kawashima, T., 180, 164,166 Keilin, D., 7, 34 Kemp, R. B., 175, 178, 182, 183, 186, 194 Kendrick-Jones, J., 57, 76 Kenny, G. E., 84, 107 Kerr, I. M., 99, 106 Keynan, A., 8, 9, 11, 13, 14, 15, 17, 21, 25, 34, 35, 36 Khamsi, F., 112, 146 Kieman, J. A., 215, 216, 221 King, R. J. B., 152, 166 Kitt, S., 98, 106 Kittel, C., 213, 221 Klee, C. B., 124, 126, 127, 128, 146, 147 Knight, V. A., 178, 194 Knowles, B. B., 70, 77 Kobayashi, Y., 6, 13, 14, 15, 16, 17, 18, 20, 22, 24, 25,28, 35 Koblin, R., 113, 146 Kollros, J. J., 124, 127, 128, 129, 146, 147 Konde, K., 15, 35 Kondolshi, K., 15, 35 Konigsberg, I. R., 39, 40, 45, 58, 53, 57, 76 Kook, J. W., 6, 35 Koprowski, H., 70, 77

227

AUTHOR INDEX

1

Lewis, W. H., 45, 76 Lieberman, I., 204, 216, 221 Lilien, J. E., 182, 187, 188,194 Lin, H., 99, 106 Lipton, M. M., 83, 108 Lishman, W. A., 111, 112,145 Lithner, F., 70, 76 Lockart, R. Z., Jr., 82, 107 Loh, P. C., 81, 92, 105, 106 Lowenstein, W., 219, 221 Lowry, 0. H., 199, 221 Luria, S. E., 215, 221 Lwoff, A., 81, 106 Lwoff, M., 81, 106 LYM, E., 112, 117, 130, 133, 134, 141, 146

Lake, N. C., 38, 76 Lampson, G. P., 101, 105, 106, 108 Lane, B. P., 216, 221 Langridge, R., 17, 20, 36 Lansing, A. I., 204,221 Laron, Z., 140, 146 Lash, J., 218, 221 Lasher, R., 218, 221 Launer, J., 94,95, 106 Lawrence, N. L., 7, 35 Lawson, L. A., 84, 107 Lazar, G. K., 215, 222 Le Bouvier, G. L., 81, 106 Lederberg, J., 2, 35, 75, 76 Ledinko, N., 81, 86, 106 Lee, H . H., 66, 76 Legrand, J., 130, 131, 132, 146 Levi, G., 110, 146 Levi-Montalcini, R., 110, 146 Levine, A. J., 83, 89, 90, 92, 97, 98, 106 Levine, M., 215, 221 Levine, S., 112, 144, 145, 146 Levinson, H . S., 18, 31, 34 Levinthd, C., 6, 13, 14, 15, 16, 17, 18, 21, 22, 23, 24,25, 28, 30, 35, 36 Levintow, L., 99, 108 Levy, H. B., 82, 91, 100, 101, 103, 105, 106, 107 Lewis, M. R., 38,45,76 Lewis, R. W., 110, 145 Lewis, V., 111, 147

Maasab, H. F., 92, 106 McCormick, W., 83,90, 91, 106 McCutcheon, M., 214, 221 McElvain, N., 39, 40, 76 McElvain, N . F., 99, 108 McKerlie, M. L., 101, 105 MacLennan, A. P., 171, 194 Maggio, R., 16, 35 Maizel, J. V., 87, 88, 93, 99, 105, 108 Malec, J., 82, 97,107 Malkin, L. I., 3, 17, 34 Mandelstam, J., 7, 35 Mannheimer, S., 140, 146 Marcliok, A., 203, 221 Marcus, P. I., 16, 35, 101, 106, 217, 221 Margoliash, E., 172, 174, 194 Margolis, F. L., 141, 148 Marks, P. A., 73, 76 Marshall. J. M., 203, 221 Martin, E. M., 82, 90, 97, 99, 101, 105, 106, 107 Martin, L., 152, 166 Martin, S. J., 90, 105 Martinez-Palomo, A., 214, 221 Mason, E. J., 83,107 Matague-Dhossche, F., 40, 75 Matsushiro, A., 15, 35 Maurer, H. R., 152, 166 Mayer, M. M., 94, 107 Mayyasi, S. A., 82, 105

Kornberg, A., 5, 6, 7, 16, 17, 20, 27, 34, 35 Kornfeld, J., 16, 25, 35 Koshland, D. E., 150, 166 Kovacs, S., 113, 115, 120, 121, 122, 126, 128, 144 Kraines, R., 111, 145 Kriegel, A., 130, 146 Kruse, P. F., Jr., 203, 221 Kudo, H., 85, 89, 106 Kumate, J., 140, 146 Kuroda, Y.,176, 187, 188,194 Kuroki, T., 70, 76 Kurtz, H., 81, 105

M

228

AUTHOR INDEX

Mecs, E., 101, 107 Melnick, J. L., 81, 106 Mendoza, L. A., 112, 145 Merigan, T. C . , 95, 99, 100, 101, 103, 105, 106 Miedema, E., 203, 221 Mims, C . A., 83,107 Mink, B., 44, 76 Miroff, G., 81, 107 Mishmi, M., 82, 95, 97, 99, 105, 107 Mitskevich, M. S . , 126, 147 Money, J., 111, 147 Monod, J., 2, 32, 34, 74, 76, 165 Monroy, A., 16, 35 Morales, C., 140, 146 Morgan, H. R., 82,107, 214,221 Morowitz, H. J., 17,20, 36 Moscona, A. A., 35, 39, 42, 76, 144, 147, 170, 171, 172, 173, 174, 175, 176, 179, 180, 181, 182, 183, 184, 185, 187, 188, 191, 192, 194 Moscona, H., 185, 194 Moscona, M. H., 179,181, 182,183,194 Moscovkin, G. N., 126, 147 Mosley, J., 139, 140, 148 Moss, B., 95, 97,99, 107 Moyers, W. A., 3, 17, 34 Mueller, G. C., 151, 165, 167 Muller, G., 83, 107 Muller-Hill, B., 150, 166 Mullins, R. F., 144, 146 Murray, M. R., 39, 76,137,145,147 Murrell, W. G., 5, 9, 35, 36 Mutolo, V., 175, 193 Myant, N. B., 110, 115, 116, 137, 147

N Nachmias, V. T., 203, 221 Nags, E. H., 18, 34 Nakata, D., 15, 35 Nakata, H. M., 4, 12, 35 Nardone, R. M., 84, 107 Nass, M. K., 198,199,222 Neet, K. E., 150, 166 Nelson, D. L., 5, 6, 7 , 35 Nemes, M. M., 101,106 Nettleship, A., 48, 76 Newton, A., 94, 107 Nickerson, K.,11, 15, 18,20, 21, 35

Nicolette, J., 151, 166 Nomura, M., 215, 221 Norman, R., 111, 147 Norris, G. R., 6, 21, 33, 35 Noteboom, W. D., 151, 152, 153. 154, 155, 156, 166, 167 Notides, A,, 161, 166

0 Ofker, J. E., 82, 105 Ohye, D. F., 5, 36 Oishi, M . , 30, 35 Okado, A., 106 Okazaki, K., 60, 76 Old, L. J., 171, 193 Omura, T., 210, 221 Orton, W. L., 193, 195 Osborn, M. J., 25, 36

P Palade, G. E., 210, 220, 221 Pardee, A. B., 150, 166 Parmele, A. H., 111, 147 Pasquini, J. M., 120, 136, 145,147 Payne, F. E., 81, 92, 105 Penman, M., 165, 167 Penman, S., 73, 76, 83, 90, 91, 93, 99, 106,107, 108, 165, 167 Pereira, H. G., 82, 92, 93, 106, 107 Perry, S . V.,57, 76 Pertzelan, A., 140, 146 Pesetsky, I., 123, 124, 125, 129, 147 Peterson, J. A., 93, 106 Pfefferkorn, L. C . , 217, 221 Piddington, R., 144, 147 Piper, J,, 11, 15, 18, 20, 21,35 Pitot, H. C., 213, 221 Plagemann, P. G. W., 91, 92, 107 Pollock, M. E., 84, 107 Ponten, J., 70, 76 Powell, J. F., 7, 35 Prescott, D. M., 164, 167 Prince, A. M., 83, 107

R Rabson, A., 81, 105 Race, J., Jr.. 137, 147 Ramirez de Guglielmone, A. E., 118, 146

AUTHOR INDEX

229

Sata, G. H., 38, 76 Sato, H., 70, 76 Scatchard, G., 167 Schachman, H. K., 150, 166 Schafer, W., 83, 97, 107 Schapiro, S., 111, 114, 118, 123, 147 Schenck, J. R., 172, 174, 194 Scherrer, K., 90,99, 107 Schimke, R., 165, 166 Schoefl, G. I., 64, 69, 76 Schoenheimer, R., 198, 221 Scholtissek, C., 83, 86, 97, 107 Schuller, E., 139, 140, 148 Schultz, F. J., 111, 147 Schultz, M. A., 111, 147 Schumalbach, K., 112, 145 Schwartz, J., 100, 104, 108 Scornick, 0. A., 217, 221 Sebring, E. D., 82, 95,107 Setlow, R. B., 27,28,34 Shainberg, A., 57, 77 Shatkin, A. J., 95, 107 Shepherd, W. M., 88, 108 Shyaniala, G., 152, 153, 156, 157, 158, 159, 180, 161, 162, 163, 166, 167 Siekevitz, P.,210, 220, 221 Siminovitch, L., 215, 221 Simpson, M. E., 143, 148 Simpson, M. V., 210, 222 Slepecky, R. A., 30, 36 Smith, D., 161, 166 Smith, D. W., 111, 147 Sobel, E., 113, 146 Sokol, F., 98, 107 Sokoloff, L., 118, 124, 126, 127, 128, 145, 146, 147, 148 Sonnabend, J. A., 101, 107 S Sonneborn, T. M., 2, 3, 35 Spiegel, M., 171, 179, 194 Sabin, A. B., 81, 107 Spiegelman, S., 75, 77 Sachs, L., 46, 75 Spring, S. B., 100, 104, 108 Sadoff, H. L., 6, 7, 33, 34, 35 Spudich, J. A., 5, 6, 7, 27, 34, 35 Saito, H., 29, 35 Stafford, R. S.,28, 35 Sakakibara, Y.,29, 35 Starr, R. C., 3, 35 Salb, J. M., 16, 35, 101, 106, 217, 221 Stavy, L., 16, 35 Salganicoff, L., 120, 147 Steigman, A. J., 83, 108 Salzman, N. P., 82, 95,107 Steinberg, D., 210, 222 Sanderson, P. J., 93, 107 Steinberg, M. S., 176, 177, 178, 180, 194 Sanford, K., 70, 76 Steinberg, W., 6, 11, 13, 14, 15, 16, 17,

Randall, C. C., 84,107 Randall, R. J., 199, 221 Ray, T. K., 204, 221 Reeder, R., 73, 76 Reich, J., 73, 76 Reid, E., 213, 221 Reiss, J., 117, 147' Reiss, M., 117, 147 Reporter, M. C . , 57, 76 Revivi, C., 53, 54, 77 Reynolds, W. A., 130, 137, 147 Reznik, M., 39, 70,72, 75 Rhinestone, A., 113, 146 Richmond, J. E., 181, 182, 184, 193, 194 Richter, W. R., 172, 174, 194 Rinaldi, A. M., 16, 35 Rinaldini, J. M. L., 175, 194 Rome, P. R., Jr., 86, 94, 107 Robbins, F. C., 81, 105 Robinow, C. F., 5, 33, 35 Robinson, L. B., 83. I07 Rodenberg, S., 11, 15, 18, 20, 21, 35 Roizman, B., 82, 83, 86, 87, 88, 91, 94, 99, 100, 104, 105, 107, 108 Rosebrough, N. J,, 199, 221 Rosin, A., 82, 105 Rossetti, F., 139, 148 Roth, S. A., 179, 185, 186, 187, 194 Rott, R., 83, 86, 97, 107 Rowe, W. P., 82,107 Rnbin, H., 215, 221 Ruhenstroth-Bauer, G., 214. 221 Russell, P. K., 82, 107 Russell, W. C., 93, 107

230

AUTHOR INDEX

18, 20, 21, 22, 24, 25, 28, 30, 33, 34, 35, 36 Steplewski, Z.,70, 77 Stermpf, W., 184, 166 Stockdale, F. E., 40,77, 218,222 Stocked, E.,171, 193 Stoker, M. G. P., 214,216,221 Storck, R., 16, 34 Strange, R. E., 7, 35 Strehler, B. L., 57, 76 Stuy, J., 28, 35 Subak-Sharpe, H.,88, 108 Sueoka, N.,18,19,30,33, 35 Summers, D. F., 83, 87, 88,93, 99, 105,

Tung, Y., 213, 221 Tyler, A., 171, 195 Tytell, A. A., 101,lOS,IO6,108

U Uchida, S., 83, 108 Ui, H.,165, 167 Underwood, B., 90, 105

V Valcana, T., 127, 137, 145 Valentine, R. C., 92,106 Van Marthens, E., 136, 139, 141, 148 Vary, J. C., 11, 15, 18,20, 21, 33,34, 35 Vassar, P. S., 214,222 107, 108 Vaughan, M., 210, 222 Sussman, A. O., 15,36 Verwoerd, D.W., 93,108 Sussman, M., 2, 3, 25, 36 Vesco, C.,165, 167 Suzuki, T., 184, I66 Vicari, E., 111, 146 Sved, S., 82,97, 107 Villee, C. A., 110, 147 Swim, H. E.,91,107 Vinter, V., 4,5, 18,27,30,36 Sydiskis, R. J., 86, 94, 99, 100, 104, 108 Vogt, M., 81, 106 Szulmajster, J., 34 Von Magnus, P., 83,97,108

T W Tamm, I., 85, 86, 89, 90,91,93, 94,99. Wacker, W. B., 83, 108 105, 106, 108 Wagner, E. R., 87, 88, 108 T a b , J. R., 110,137,147 Wagner, R. R., 83, 89, 95, 97, 106, 108 Taylor, N.W., 193,194 Wahl, D., 98, 105 Taylor, S. H., 113, 136,145 Waites, W. N., 6, 36 Teichgraber, P.,113, 120, 121, 122, 126, Wake, R. G., 29,36 144 Walker, B. M., 84,I07 Thiery, J., 213, 221 Walker, D. G.,143, 148 Thoren, M. M., 99,108 Walker, D.L., 81,82,108 Timiras, P. S., 121, 127, 128, 136, 137, Warren, L., 183, 195, 198, 199, 201, 138, 144,145, 146 209, 217, 221, 222 Tjernberg, B., 214, 222 Warth, A. D., 5,36 Tobey, R. A., 87, 108 Watanabe, S., 83, 108 Todaro, G . J., 64,77, 215,222 Watkins, J. F., 64,69,76 Todd, J,, 84, 107 Weber, R., 137, 147 Todd, P. W., 181, 182, 184, 193, 194 Weinberg, E.,11, 13, 14, 15, 17, 25, 35 Toft, D., 155, 156, 157, 158, 159, 161, Weiss, E. P., 112, 117, 130, 133, 134, 166, 167 141, 146 Tootle, M., 39,40, 76 Weiss, L., 184,195, 213,222 Torriani, A., 13, 14, 17, 22, 23, 24, 30, Weiss, P.,139,148, 171,185,195 36 Weiss, W. P., 118,148 Treadwell, P.E.,84,107 Weller, T. H.. 81, 105 Trinkaus, J. P., 175,185,195 Weston, J. A., 179,194

231

AUTHOR INDEX

Wheelock, E. F., 86, 108 Wichelhausen, R. H., 88, 94,107 wilcox, w. c., 92,106 Wilkins, L., 111, 147 WiUems, M., 93,99,108 Wilson, D. E., 86,95,97,105,108 Wilson, H. V., 170, 195 Wilt, F. H., 73, 77 Woese, C. R., 15, 17, 20, 28,29, 36 Wolf, J., 6, 35 Wollman, E. L., 215, 221 Work, T. S., 82, 97, 99,106, 107 Wright, B. E., 25, 36 Wyatt, A., 117, 147

Y Yaffe, D., 40, 42, 43, 46, 47, 49, 53, 54, 57, 60, 63, 65, 74, 77 Yagil, G., 57, 77 Yasumura, Y., 38, 76 Yoshikawa, H., 27, 30,35,36’ Young, J. E., 5, 25,27, 28, 36

2 Zajicek, J., 214, 222 Zamenhof, S., 136, 139, 140, 141, 148 Zimmerman, E. F., 85, 90, 91, 108

A Adenoviruses, macromolecular biosynthesis in animal cells infected by, 92-93 Arboviruses macromolecular biosynthesis in animal cells infected by, 95

B Bacterial endospore (outgrowing), 138 activation, germination, and outgrowth processes of, 10 biochemical events in, 13-15 cell wall formation in, 25-27 differential events in, 30-31 DNA synthesis and chromosomal replication in, 27-30 experimental system for study of, I l 13 first molecular events in, 15-21 biochemical potential of germinated spore, 15-16 presence of mRNA in spores in, 17-18 protein synthesis, 20-21 RNA synthesis in, 18.20 protein synthesis in, 21-25 enzyme synthesis and, 24-25 first proteins synthesized, 22 regulatory patterns, 2 2 2 4 time-ordered, 21-22 spores and vegetative cells compared, 4-8 transformation processes, 8-13 as system for study of cellular differentiation, 31-33

protein-synthesis inhibitors, 181184 temperature, 180-181 macromolecular preparations which promote aggregation, 187-192 age-dependent studies, 191-192 aggregation-enhancing material, 187-191 measurement techniques for, 176-179 molecular explanation for, 189-195 onset of specificity and, 185-187 in sponge cells, 170-174 tissue disaggregation and the specificity of reaggregation, 175-176 in vertebrate cells, 175-192 Cells, surface membrane of, see Surface membrane of animal cells Cytolytic-virus infected animal cells, macromolecular biosynthesis in, 79108 Cytosol, estrogen binding to, 155-156

E Endospore, of bacteria, see Bacterial endospore Estrogen receptors, nuclear and cytoplasmic, 14CL187 cell-free binding of estrogen, 156-157 estrogen binding to cytosol, 155-156 nuclear-cytosol interrelationships, 157164 subcellular location of estrogen binding agent, 151-154

0 Growth hormone in neurogenesis, 139143

C

H

Cell adhesion immunological approaches to, 179. 180 inhibitors of cell reaggregation and, 180-185 periodate and amino sugars, 184185

Herpesvirus, macromolecular biosynthesis in animal cells infected by, 94

M Macromolecular biosynthesis in animal cells infected with cytolytic viruses, 79-108

232

233

SUBJECT INDEX

historical background, 81-82 host and viral types, 100-102 inhibition of host macromolecular synthesis, 82-102 inhibitor of ( IHMS), 92-96 specificity of, 88-89 mechanisms of, 96-102 DNA synthesis inhibition of, 9899 protein synthesis inhibition, 99-100 RNA synthesis inhibition of, 9798 v i r u s inhibition of, 102-104 Malignant cells, surface membrane studies on, 212-219 Muscle differentiation in uitro, 37-77 enzymic manifestations of, 57-59 induction of DNA synthesis and mitosis in, 60-70 myogenic cell lines, 45-59 cessation of DNA synthesis and, 4748 collagen effects, 5 2 5 4 continuous cell multiplication and, 45-47 fusion specificities, 4t?49 growth characteristics, 54-57 of primary cultures, 39-44 formation of multinucleated muscle fibers, 39-41 fusion of myoblasts in, 41-44 “hybridization” of muscle cells in, 44 Myxoviruses, macromolecular biosynthesis in animal cells infected by, 95

N Neurogenesis, thyroid and growth hormones in, 109-148

P Picomaviruses, macromolecular biosynthesis in animal cells infected by, 93-94 Poxviruses, macromolecular biosynthesis in animal cells infected by, 9495

5 Sponge cells, cell-adhesion studies on, 170-174 Spores, compared to vegetative cells, 48 Surface membrane of animal cells biological significance of turnover on, 197-222 composition of, 198-199 differential turnover of components of, 209-210 of dividing and nondividing cell, 199206 of malignant cell, 212-219 synthesis and turnover rates of, 207209 synthetic and degradative aspects of turnover in, 210-212 turnover and repair in, 212

T Thyroid hormone in neurogenesis, 110139 mammalian neurogenesis, 110-129 mechanisms of, 129-139

V Vegetative cells, spores compared to, 48 Vesicular stomatitis virus, macromolecular biosynthesis in animal cells infected by, 95

This Page Intentionally Left Blank