The Cell Cycle: Myths and Realities Renato Baserga1

[CANCER RESEARCH 50, 6769-6771, November 1, 1990] Perspectives in Cancer Research

Department of Pathology and the Fels Institute for Cancer Research and Molecular Biology, Temple University School of Medicine, Philadelphia, Pennsylvania 19140

A number of startling discoveries in the past few years have simply overexpression, the regulation of cell growth is affected. given us a picture of the cell cycle that is solidly based on Indeed, even now, despite antioncogenes and yeast genes, one molecular biology and genetics. Growth factors (both stimula cannot disregard protooncogenes in any rational scheme of tory and inhibitory), oncogenes and antioncogenes, the bio animal cell proliferation. The extent of their role may be chemistry of DNA replication, the animal homologues of yeast debatable, but they do have a role. However, their putative cell cycle genes, cyclins, the use of antisense strategies, and growth-regulatory role is based on their relationship to viral many others have opened new vistas to our understanding of transforming genes and not on the fact that they are growth cell cycle controls (1). This perspective, however, is not dedi regulated. cated to a eulogy of these seminal findings; they speak for In the meantime, scores of laboratories (including mine, I themselves. I will, in fact, be the devil's advocate and do the admit) have been picking out, through differential screening of opposite, i.e., have a critical look, not at the findings (they are complementary DNA libraries, an embarrassing number of unarguable), but at some of the conclusions that have been growth-regulated genes. At first, genes the expression of which drawn from these findings. I wish to make myself very clear: increases early in G, (after stimulation of G0 cells) were most there has been a revolution in the past 10 years in our under popular but, more recently, growth-regulated genes at the d-S standing of the cell cycle, a revolution supported by findings boundary have become fashionable. In many instances, the the importance of which cannot be exaggerated. And most of announcement of the discovery of a new growth-regulated gene us have been guarded in our conclusions. But as it happens is accompanied by more or less bold comments about its im often in science, a timid suggestion at the end of a discussion, portant role in the control of cell proliferation. It is time that a carefully worded hypothesis, become transformed in the next this myth be put to rest for two reasons: (a) the expression of paper (often by other authors) into a fact. Thus, a simple some growth-regulated genes and oncogenes is also induced in statement like "gene X may play a role in cell proliferation" situations in which cell proliferation does not occur, or, indeed, becomes in the next paper "gene X is growth regulatory." It is may even be inhibited, for instance, c-fos (5); and (b) more not surprising, therefore, that this transforming capacity of important, some exquisitely growth-regulated genes do not have words has given rise to a number of interpretations of cell cycle any growth-regulatory function. The best example is the thy- events, interpretations that are clearly extrapolations of the midine kinase (TK) gene: growing cells can do without TK; indeed, there are animals whose cells are all TK~ without, facts, and that I have elected, in this perspective, to call myths. It is some of these myths that I would like to discuss here, not obviously, affecting their growth (6). Conclusion: a growth- to pour water on our enthusiasm (which is justified) but for the regulated gene is a growth-regulated gene. To become growth purpose of placing our understanding of the cell cycle back to regulatory it must meet other criteria. its proper perspective, which in simple terms, is: "there are many things that control cell proliferation and they have not all been sorted out." This may seem obvious; yet some of the Myth 2: What's Going On in My Cells Is Universal and Is the comments we have read recently in scientific journals are far Only Truth from following this maxim. Let us look at some of these myths. Probably all of us, at one time or another, commit this sin. Most of us repent quickly; others persist obstinately in their parochial views. The myth takes many forms. For instance, Myth 1: Growth-regulated Genes Are Growth Regulatory some years ago, there was a lot of resistance to the concept that the 5'-flanking sequence of the TK gene plays a major role in This is one of the most common and persistent myths. It began several years ago when it was shown that certain pro- the control of TK expression in fibroblasts. The opposition tooncogenes were growth regulated, i.e., that the steady state came mostly from people working with muscle cells, where the mRNA levels of some protooncogenes increased when quies regulation of TK expression is totally different. The point is cent cells (usually fibroblasts or lymphocytes) were stimulated that, in this instance, both parties were right; it just happens to proliferate by growth factors. The first two protooncogenes that fibroblasts and muscle cells have their own particular ways that were shown to be growth regulated were c-myc (2) and c- of regulating TK expression. Jt s (3), but several others (c-fgr, c-myb, c-ets-l, etc.) have been There are many other examples: (a) c-myb clearly plays a added since then (4). role in the proliferation of hemopoietic cells (7), but fibroblasts The extrapolation from growth regulated to growth regula completely ignore it, and even v-myb (with its long terminal tory was, in some respects, justified. After all, protooncogenes repeat) cannot transform fibroblasts; (b) the classical growth- must have something to do with the control of cellular prolif regulated genes of fibroblasts and lymphocytes (c-fos, c-myc, eration, since, when the expression or activity of a protoonco etc.) are not growth regulated in alveolar lung epithelial cells, gene is modified by mutations, translocations, amplification, or or, at least, their mRNA levels are not growth regulated, al though their proteins are (8); (c) HeLa cells, very popular in Received 5/11/90; accepted 8/2/90. ' To whom requests for reprints should be addressed, at Department of many laboratories, are often presented as model cells for cell Pathology, Temple University School of Medicine, 3420 N. Broad Street, Phila cycle regulation of gene expression. Mind you, there is nothing delphia, PA 19140. wrong with the data. I have no doubts that gene expression in 6769

HeLa cells is regulated as advertised. Perhaps, normal cells, more than one growth-regulatory gene, in other words, that T- i.e., cells with growth controls, have different ways of doing antigen and the EIA protein unite, in a single gene product, things ... ; (d) if what's going on in HeLa cells is perhaps more information that, in the normal cell, is divided between two or the exception than the rule, can you imagine making the oocyte more genes. I would be willing, in the meantime, to bestow on a model of cell cycle regulation? A cell already full of all the c-myc the title of (incomplete) growth-regulatory gene. Inciden RNAs and proteins that are necessary for cell growth? A cell tally, \-onc do not count; what we are after here are normal, that, fertilized, can grow up to the stage of gastrula without cellular genes that regulate normal cell growth. synthesizing rRNA? Try to inhibit rRNA synthesis in somatic Where does this leave the oncogenes (besides c-myc)1! As cells: they stop cold; they won't even go through one cycle of already mentioned above, protooncogenes, when suitably mod division. Again, what has been described in oocytes is wonderful ified, can alter the regulation of cell growth. If they only are work, but it applies to oocytes. Its extrapolation to somatic required, why would a mutation (for example) cause them to cells is premature. drive cellular proliferation abnormally? An explanation may be Conclusion: for 30 years I have taught my postdoctoral found in the following illustration: 32D myeloid cells do not fellows and graduate students that what we find in our cells have EGF receptors, are not stimulated by EGF, and have an applies only to our cells, under the conditions we used, at least absolute requirement for interleukin 3. However, if they carry until otherwise proved. It is a maxim that should have more the \-erb-B gene, which is a truncated, permanently activated general currency. EGF receptor, 32D myeloid cells can grow in the absence of interleukin 3 or other growth factors (12). It is as if their cell cycle had been short-circuited. Perhaps, in the case of protoon Myth 3: If Cell Proliferation Is Inhibited by an Antibody or an Antisense RNA to a Certain Gene Product, That Gene Product cogenes, their constitutive activation causes cells to progress is Growth Regulatory through the cell cycle in the absence of those gene products that in normal cells are required for their activation. This seems This statement is either a myth or a truth, depending on how to be the case with viral oncogenes: SV40 T-antigen and aden we define growth regulatory. ovirus EIA are known to activate G.-S boundary genes (like By now there are quite a few reports in the literature that TK, PCNA, etc.) in the absence of cellular products that are microinjected antibodies, or antisense RNA, or antisense oli- normally required by serum-stimulated cells; e.g., the products godeoxynucleotides can block cellular proliferation if targeted of early growth-regulated genes (13). to an appropriate gene product. A sample of such gene products includes c-ras, c-myc, c-fos, cdc2, c-myb, and the proliferating cell nuclear antigen. Let us consider PCNA2 (9). The question Myth 4: The Time of Appearance in the Cell Cycle of a mRNA is, "Is PCNA growth regulatory?" The answer is yes and maybe, or a Protein Is an Indication of the Time in the Cell Cycle at depending on our definitions. PCNA is a cofactor of DNA Which That Gene Product Is Required polymerase 5, which is necessary for cellular DNA synthesis. It For instance, since ribonucleotide reducÃaseactivity reaches is fair to say that PCNA (and c-fos, and c-myc, etc.) is necessary a peak at 50 h after partial hepatectomy, that must be the time for cellular proliferation and, in this respect, it is growth regu when ribonucleotide reducÃaseis mostly needed. Unfortunately latory. However, under this broad definition of growth regula for the myth, ribonucleotide reducÃase is needed for DNA tory, many other gene products would be growth regulatory (for synthesis, and, by 50 h after parlial hepaleclomy, DNA synthe- instance, all the genes necessary for the synthesis of nonessen- sis in the regenerating liver has ceased (6). tial amino acids), indeed, all kinds of things, like ATP and This is a harmless myth, although il may mislead us in our anything else that is essential for the life processes of the cell. search for a funclion. Il is especially misleading in ihe case of Lower the intracellular concentration of ATP, and the cell will mRNA levels. Somelimes, il is proposed lhal cell cycle pro not divide. There is nothing wrong in calling growth regulatory gression requires Ihe orderly expression of differenl genes, using so many genes and molecules, except that the informational mRNA levels as Ihe indicalors of such orderly progression. content of the word then becomes almost trivial. There may or ihere may noi be an orderly sequence of evenls, I suggest the following distinction: when we show that an bul we cannol support it on ihe basis of mRNA levels. Take, antibody, or an antisense RNA, or an antisense oligodeoxynu- for inslance, c-fos and c-myc. Al Ihe slarling gale of Gu, c-fos cleotide targeted to an appropriate gene product blocks cell beals c-myc by aboul 1 h; i.e., c-fos mRNA levels increase l h cycle progression, we should say that particular gene is required earlier lhan c-myc mRNA levels. The finding has no heurislic for cellular proliferation. We should reserve the term "growth conlenl: (a) because cycloheximide experimenls unequivocally regulatory" only to those genes that actually induce cellular show lhat the c-myc gene is a primary responder thai does not proliferation in quiescent cells. Unfortunately, under this defi need the c-fos protein or any other de novo synthesized prolein nition, the best examples are not cellular genes. Microinjection for aclivalion and (b) because whal we see on Northern blots is of SV40 T-antigen or adenovirus EIA protein cause quiescent only what is delectable on Northern blots. Who can say that cells to enter DNA synthesis or divide (6), without any other there is no c-myc mRNA in the first 20 min after a mitogenic manipulation (growth factors, for instance). At present, there slimulus? All we can say is lhal it's not deteclable under ihe is no single cellular gene that, microinjected or transfected into condilions used. It's possible thai Ihere is enough lo make quiescent cells, causes them to divide. Cyclin does that in sufficienl myc prolein for whalever its function is. The point is oocytes (10) but, as mentioned above, that is a special case not lhal ihe Â¡nlensilyof a band on a Northern blol depends on applicable to somatic cells. The closest thing to the SV40 T- several faclors, one of which is exquisilely biological, i.e., Ihe antigen is c-myc, when microinjected into quiescent 3T3 cells, number of mRNA copies per cell al any given lime. it induces DNA synthesis (11) but only if insulin-like growth Conclusion: sometimes there is a correlation between time of factor 1 is also added. It suggests that somatic cells may need appearance of a gene product and its function in the cell cycle, 2The abbreviations used are: PCNA, proliferating cell nuclear antigen; EOF. but it is not a strict correlation and, in some cases, it can even epidermal growth factor. be misleading. 6770

Myth 5: Cell Cycle Progression Is Simply Regulated by the cellular proliferation and, in doing so, we miss not only the Degree of Phosphorylation of Certain Key Proteins differences but also the commonalities that could serve as a clue to the identification of the fundamental mechanism(s). It This myth has received a lot of publicity by the fact that two is possible (but not yet demonstrated) that somatic cells may very important genes, the p34cdc2gene (14) and the retinoblas- need two or more gene products for growth regulation and toma (RB) gene, are regulated in that way. In both cases, many others for growth (the genes I would like to define as amounts of RNA and protein are essentially constant through genes required for cell cycle progression). Personally, I picture out the cell cycle; what varies is the degree of phosphorylation of the protein. There is no question in anybody's mind that the cell cycle as having three distinct critical steps: Step 1, the transition from G() to G, (c-wyc?); Step 2, the transition from these two genes are very important. The evidence that the G! to S (c-mybl); and Step 3, the transition from S to mitosis p34cdc2gene is required from the transition from S to M is (p34"k2, cyclins, c-mos), each of them requiring several gene especially convincing (14, 15); it seems that the binding of its products. Clearly, my view of the cell cycle includes Gn, an product to other key proteins depends on its degree of phos arbitrary decision, which is hotly contested these days by some phorylation. However, it phosphorylates also at the G,-S colleagues, who would like to exclude the G0 state from the boundary, and the evidence that it is also needed there is poor: definition of the cell cycle, also an arbitrary decision. The indeed, the same antibody to the p34cdc2gene product that, difference between growth-regulated genes and cell cycle-regu microinjected, inhibits S to M transition has no effect on the lated genes, advocated these days by several groups, seems to GI to S transition. The RB gene almost certainly plays a role me an unnecessary separation of two processes that are too in retinoblastoma and certain osteosarcomas. There is no evi closely intertwined to be separated. Again, we should not take dence, however, that it plays any role in the control of cell cycle HeLa cells as models of cellular behavior. But to return to my progression in, for instance, fibroblasts or lymphocytes, al three critical steps: it is, of course, just an opinion. Now, to though, even in these cells, the degree of phosphorylation varies avoid the creation of another myth, I have to promise (and the with the cell cycle. This latter finding is by no means evidence readers must remember) not to say in the next paper: the cell that the RB gene controls cell proliferation in fibroblasts or cycle is regulated by three critical steps .... lymphocytes. Indeed, Bookstein et al. (16), in a seminal paper, have shown that, in cells carrying a mutant RB gene, the References introduction of a normal one abolishes their tumorigencity 1. Pardee, A. B. G, events and regulation of cell proliferation. Science (Wash without altering their growth characteristics in culture (inciden ington, DC). 246: 603-608. 1989. 1. Kelly. K.. Cochran. B. H.. Stiles. C. D., and Leder, P. Cell-specific regulation tally, this takes care of another myth, that transformation and of the c-myb gene by lymphocyte mitogens and platelet-derived growth factor. cell proliferation are regulated by the same genes. There are Cell. 35: 603-610, 1983. things in common between the two, but there also differences). 3. Greenberg, M. E.. and Ziff. E. B. Stimulation of 3T3 cells induces transcrip tion of the c-fos proto-oncogene. Nature (Lond.), 311: 433-438, 1984. Once could actually be impertinent and remind the reader of 4. Studzinski, G. P. Oncogenes, growth and the cell cycle: an overview. Cell what a professor of biochemistry used to tell his students: that Tissue Kinet., 22: 405-424. 1989. proteins can be divided into two classes, the ones that are 5. Morgan, J. I., Cohen. D. R.. Hempstead, J. L., and Curran. T. Mapping patterns of c-fos expression in the central nervous system after seizure. phosphorylated and those that have not yet been studied. Even Science (Washington. DC). 237: 192-197, 1987. conceding that some proteins are selectively phosphorylated 6. Baserga, R. The Biology of Cell Reproduction. 251 pp. Cambridge. MA: Harvard University Press, 1985. during the cell cycle, there are many that do so, including DNA 7. Gewirtz, A. M., and Calabretta, B. A c-myb antisense oligodeoxynucleotide polymerase a, RNA polymerase I and RNA polymerase II. inhibits normal human hematopoiesis in vitro. Science (Washington, DC), Clearly, if one uses phosphorylation as the sole criterion, there 242: 1303-1306, 1988. 8. Clement, A., Campisi, J.. Farmer, S. R.. and Brody. J. S. Constitutive are many candidates for a controlling role in the cell cycle. This expression of growth-related mRNAs in proliferating and non-proliferating caution is shared by Horowitz et al. (17) who clearly state their lung epithelial cells in primary culture: evidence for growth-dependent trans- doubts that the RB gene may not be critical for growth regula lational control. Proc. Nati. Acad. Sci. USA, 87: 318-322, 1990. 9. Jaskulski. D., deRiel, J. K.. Mercer. W. E., Calabretta, B., and Baserga, R. tion in many of the cell types in which it is expressed. Their Inhibition of cellular proliferation by antisense oligodeoxynucleotides to concluding statement is a model of caution and salutary skep PCNA cyclin. Science (Washington, DC). 240: 1544-1546, 1988. ticism: "Since the regulatory pathways that constrain cell pro Murray. A. M., and Kirschner. M. W. Dominoes and clocks: the union of two views of the cell cycle. Science (Washington, DC), 246: 614-621, 1989. liferation are poorly understood, we are still many years away Kaczmarek, L., Hyland, J. K., Watt, R., Rosenberg, M.. and Baserga, R. from directly addressing these possibilities." Microinjected c-myb as a competence factor. Science (Washington, DC), The same comments, of course, apply to any other posttrans- 228: 1313-1315, 1985. 12. Pierce, J. H., Ruggiero, M., Fleming, T. P., DiFiore. P. P., Greenberger, J. lational modification of proteins; per se, it is not proof that S., Varticovski, L., Schlessinger, J., Rovera, G., and Aaronson. S. A. Signal protein modifications control cell cycle progression. The only transduction through the EGF receptor transfected in lL-3-dependent he- matopoietic cells. Science (Washington. DC). 239: 628-631. 1988. valid criteria remain those discussed under Myth 3. 13. Liu. H. T.. Baserga. R.. and Mercer, W. E. Adenovirus type 2 activates cell cycle-dependent genes that are subset of those activated by serum. Mol. Cell. Biol.. J.-2936-2942, 1985. Concluding Remarks 14. Draetta, G.. Pianica-Worms. H., Morrison, D., Drunker. B.. Roberts, T., and Beach, D. Human cdc2 protein kinase is a major cell-cycle regulated tyrosine kinase substrate. Nature (Lond.), 336: 738-744, 1988. I would like to propose that we reserve the term of growth- 15. Nurse, P. Universal control mechanism regulating onset of M-phase. Nature regulatory genes only to those that, like the SV40 T-antigen or (Lond.), 344: 503-508. 1990. the adenovirus EIA protein, can induce cellular proliferation 16. Bookstein, R., Shew. J. Y., Chen, P. L., Scully. P., and Lee, W. H. Suppres sion of tumorigenicity of human prostate carcinoma cells by replacing a in quiescent cells. Cyclin, therefore, is growth regulatory in mutated RB gene. Science (Washington, DC), 343: 712-715, 1990. oocytes (10). But this brings us promptly to the second recom 17. Horowitz, J. M., Park. S. H.. Bogenmann, E., Cheng, J. C.. Yandell, D. W., Kaye, F. J.. Minna, J. D., Dryja, T. P.. and Weinberg, R. A. Frequent mendation, that different cells may utilize different genes. Too inactivation of the retinoblastoma anti-oncogene is restricted to a subset of often, investigators forget this diversity in the regulation of human tumor cells. Proc. Nati. Acad. Sci. USA. 87: 2775-2779. 1990.

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Renato Baserga

Cancer Res 1990;50:6769-6771.

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