Molecules Involved in Proliferation of Normal and Cancer Cells: Presidential Address1

[CANCER RESEARCH 47. 1488-1491, March 15, 1987] Molecules Involved in Proliferation of Normal and Cancer Cells: Presidential Address1

Arthur B. Pardee Division of Cell Growth and Regulation, Dana-Farber Cancer Institute, Boston, Massachusetts 02115

Overview may permit the escape of cells from growth control. The proposal that a labile protein is necessary for prolifera All definitions of cancer stress the defective regulation of cell tion of normal cells and that its levels are altered in tumorigenic growth and differentiation because such physiological aberra cells led us to examine proteins on two-dimensional gels. A tions underlie the gross derangements of the disease. An im candidate protein which was unstable in normal cells but rela portant goal of basic cancer biology is to provide molecular tively stabilized in tumorigenic cells was indeed identified. It explanations for these defective cellular processes. With recent has been shown to possess a molecular weight of 68,000. This developments in molecular and cellular biology, investigators protein is one of a very small number closely linked to cell have obtained penetrating insights into cell processes and their proliferation during G|. Our work in progress is designed to respective derangements. These are being investigated at four further identify this protein, clone its gene, and study its prop experimental levels: genes and their mRNAs; growth factors erties in normal cells that have been transfected with this gene. and their receptors; biochemical regulation; and cell biology in We are also investigating events following the restriction culture. The results are interrelated and integrated within the point. The rather long time interval (about 2 h) between the framework of the classical cell proliferation cycle. restriction point and the onset of S phase suggests that some Cell proliferation, the creation of two cells from one, is the complex process must occur, one that appears not to require culmination of a vast number of biochemical events which are needed to duplicate all of a cell's components. Among these de novo RNA synthesis. During this time some enzymes needed for DNA synthesis become activated. We have proposed that processes in the cell only a few are regulating pacemakers; the these and other proteins are assembled into a multiprotein rest are controlled although they may be essential for growth. complex which is the actual machinery for synthesis of DNA. It is extremely important to our further understanding of This assembly process may be triggered at the restriction point growth control to discover the regulating events and the mole after an ample supply of protein p68 has been made in the cell. cules involved in them. Current studies demonstrate that there p68 possibly could thus catalyze production of a component are a limited number of such events, located at definite points that "glues" the multienzyme complex together. in the cell cycle. These regulatory processes are deranged in cancer cells; they are the ones on which our research focuses. Within the context of these general thoughts, research from A Restriction Point Protein our laboratory and others has led to several conclusions. A major regulatory control occurs prior to the entry of cells into Information regarding the regulation of cell growth has been S phase. This event appears to regulate not only the onset of summarized frequently. An excellent recent review is in a book DNA synthesis but other events which take place simultane by Baserga (1). The principal guides to my thinking on the ously, such as syntheses of histones and specific enzymes in problem of growth control in normal cells and its derangement volved in DNA synthesis (e.g., thymidine kinase). Cells are in cancer cells are briefly stated at the beginning of this article, committed to complete their DNA duplication about 2 h before and they have been discussed more extensively in several recent the onset of S phase. At this point (the restriction point) they reviews (2, 3). This information and these ideas will not be can go on to make DNA in the absence of external growth presented extensively here. Instead, I will discuss what I con factors. Proliferation is thus controlled by events during d sider to be the central component of this control system, namely rather than by processes occurring during the S, G2, or M the agent responsible for activation of the onset of DNA syn thesis, a prime growth-controlled event in the cell cycle. Dis portions of the cell cycle. These latter cycle stages are terminal and necessary for production of a new cell but they appear cussion of our discovery of a possible central regulatory protein much less dependent upon regulatory molecular processes than will be followed by brief comments on the events that lead to are G0 and GÃ¬events. its production and the events that it can subsequently trigger. Some regulation-related events occurring prior to the restric Numerous experiments using inhibitors have demonstrated tion point have been identified. IGF-12 is the only growth factor that protein synthesis is essential for cell cycle transit. The cells required by BALB/c 3T3 fibroblasts in the interval from 2 to 6 must be able not only to synthesize proteins but to make them h prior to S phase. During this same time the ras oncogene is rapidly. This requirement is specific for the GÃ¬period of the activated to produce its message. Studies with inhibitors show cell cycle, the part during which cells prepare for the synthesis that cells cannot initiate S phase without rapid protein synthesis of DNA and the other events in S phase. This particular during this prerestriction point interval. The data strongly sensitivity to protein synthesis inhibitors during ( n led to the idea that an unstable protein with a short half-life (2.5 h) is suggest that normal cells need to make a critical amount of a particular protein that is quite unstable, having a half-life of essential in order to initiate S phase (4-6). During a sabbatical year (1972-1973) in the laboratory of about 2.5 h, in order to pass the restriction point. Various tumorigenic cells behave as if this protein is stabilized or made Dr. Michael Stoker I investigated conditions that differently in excess, suggesting that greater availability of this protein affect the ability of untransformed versus transformed cells to transit the cell cycle (7). Prominent among the conditions that Received 12/2/86: accepted 12/12/86. ' This work was supported by USPHS Research Grant GM24571. preferentially inhibited the growth of nontransformed cells were a The abbreviations used are: IGF-1, insulin-like growth factor 1: p68, a protein those that limit protein synthesis, such as shortages of essential with a molecular weight of 68,000: IK. thymidine kinase. amino acids or low concentration of cycloheximide-related pro- 1488 Downloaded from cancerres.aacrjournals.org on September 26, 2021. © 1987 American Association for Cancer Research. MOLECULES IN NORMAL AND CANCER CELLS tein synthesis inhibitors. These results suggested that an unsta let-derived growth factor, epidermal growth factor, and insulin- ble regulatory protein may be more stable in the transformed like factors (IGF-1) which appear to act sequentially to bring cell. Further work in my laboratory by Drs. Peter Rossow, the cells out of the quiescent state and into S phase of DNA Veronica Riddle, Judith Campisi, and Estela Medrano ex synthesis. This emergence of cells from quiescence involves the tended these findings and provided kinetic data consistent with activation of transcription and protein synthesis (1). This is the increased stability of a regulatory protein in tumor cells (4, interesting from our point of view since rapid protein synthesis 8, 9). At about the same time we demonstrated a similar is essential for the later GÃ¬events, as discussed above. In the phenomenon in yeast, attesting to the generality of labile reg early stages of the G0 to S-phase transition, the cell is put back ulatory proteins in the cancer biology of Gr transition and S into working order after a period of quiescence which has caused phase entry (10). We found that the labile protein of yeast has the gradual disappearance of labile components of the cell's a half-life of only about 10 min in contrast to the 2 to 3 h half- metabolic apparatus. It is well known that a longer time is life reported with mammalian fibroblasts. Popolo and Albergh- required for cells to traverse the Go to S-phase process than is ina (11) and Popolo et al. (12) using two-dimensional gels have required for cycling cells to pass from mitosis to S phase. In identified such a labile protein in yeast which seems to satisfy mouse 3T3 cells the G0 to S-phase time is 12 h, whereas the the criteria that we had proposed for the regulator of the start time from M to S phase is only 6 h. Very likely some of the event. events that transpire during the 6 extra h in the former situation These studies have all pointed to the central role of a labile occur in cycling cells during the prior cycle. protein acting specifically at the onset of DNA synthesis to Events leading more directly to DNA synthesis occur during regulate the progression of cells through the cycle. Such a the latter part of the G0 to S-phase transition approximately 6 protein, however, had not actually been identified. The afore h prior to DNA synthesis. This period corresponds to the M to mentioned kinetic and inhibitor studies had yielded the follow S-phase part of the cycle in proliferating cells. The production ing three major properties which were used to seek for and of adequate amounts of labile protein(s) including p68 occurs identify such a protein: (a) the regulatory protein should be during the first 4 h of this G, period. Synthesis of these produced in GÃ¬ofthe cell cycle; (b) in normal cells this protein protein(s) requires the presence of growth factors (14, 15) and should be unstable with a half-life of 2-3 h; and (c) the protein is blocked by mild inhibition of protein synthesis. Experiments should be altered in transformed cells so that its apparent are currently in progress to determine which growth factors are stability is increased. most responsible for progression through G|. It has been found Dr. Robert Croy, working in my laboratory, then set out to that epidermal growth factor seems to be essential for early GÃ¬ find a protein that satisfied all of these criteria (13). Using the events. However, IGF-1 is the principal growth factor required technique of two-dimensional gel electrophoresis he observed by mouse cells during late (Â»,.since within about 6 h of S phase at least 1000 spots. Among these about a dozen satisfied the these cells can progress when only IGF-1 is supplied (9, 16). criterion of being made during d, but not by resting cells. Relatively little else is known about specific events during Pulse-chase experiments revealed that most of these proteins this 6-h pre-DNA synthetic period except that during this time in normal cells had half-lives of longer than a few h; only one the ras oncogene is activated (17) and appears to be essential of the dozen Gi-related proteins was unstable. Furthermore, for cell progression, as shown with microinjected antibody (18). this protein alone was made in large amounts in transformed Also, except for the production of the p68 protein, differences cells, where it was stable for at least 8 h. This protein has been during this period between normal and cancer cells remain called p68 because of its molecular weight of 68,000. It has an largely unknown. isoelectric point of 6.3. We consider p68 to be of special interest because its proper Events That Follow the Restriction Point ties coincide so well with the conditions we defined, and because it is synthesized so differently by normal cells as compared to The cell's metabolic requirement(s) change dramatically at a variety of transformed cells. Few other proteins have been about 2 h before the onset of S phase (8, 15, 19). After this identified that satisfy even one of these criteria. Enzyme activ time, serum and growth factors are not required to complete ities and messenger RNAs have been reported that differ in the cycle, and neither is rapid protein synthesis (8). We have normal versus transformed cells or that are made specifically in designated this point of commitment to begin S phase as the the pre-S phase part of the cell cycle. However, since none of restriction point (7). We consider it as a key regulating event these proteins satisfy the three criteria outlined above, they are in the cell cycle, because after cells come within about 2 h of probably not regulatory proteins. It is more likely that they are the onset of S phase, their progression through the remainder the consequences of regulatory events (e.g., housekeeping en of the cycle and into the next G\ becomes essentially automatic zymes needed for processes such as nucleotide synthesis). and uncontrolled by extracellular factors. Although it is possible Our current work is aimed at testing the role of p68 in growth to stop cells in this latter part of the cycle (e.g., with inhibitors regulation. In order to accomplish this goal we have purified of DNA synthesis such as hydroxyurea), no major physiological this very rare protein and commenced production of monoclo controls have yet been reported for cell lines in culture such as nal antibodies. Our ultimate goal is to clone the gene for p68 we have been studying. and make antibodies to the purified protein. These tools will It is remarkable that approximately 2 h transpire between the enable us to examine its role in cell cycle progression, its loss of serum dependence and the onset of S phase. What location in the cell, and its properties. happens during this time? We have been investigating this question using two different approaches. One is to study the Events Leading to the Synthesis of p68 and the Subsequent production of enzymes that are known to be needed for DNA Onset of S Phase synthesis, and the other is to investigate the assembly of these enzymes into a multienzyme complex which functions to syn Cell proliferation is primarily regulated by extracellular fac thesize DNA. tors. Prominent among these are growth factors such as plate For many years it has been known that histones and certain 1489

DNA-related enzymes are synthesized at the onset of S phase. synthesis as well. Its catalysis of DNA synthesis does not require Therefore, importantly, S phase is not synonymous simply with addition of template DNA. We have calculated reputase to have DNA synthesis. Rather a set of more or less tightly coupled a molecular weight of about 5 million which is approximately events occur at the same time (20, 21). For example, histone the size of a ribosome. synthesis commences with and is tightly coupled to DNA Of particular interest for this article, we found that the synthesis, primarily through changes in the stability of histone complex is present in S-phase cells but absent from cells in GÂ¡ mRNAs (22): if DNA synthesis is arrested by an inhibitor, (25). Various reputase enzymes may or may not be present histone mRNA decays very rapidly and histone synthesis then during d; also, those found then are principally located in the comes to a halt, providing another example of control through cytoplasm. We propose therefore that assembly of the reputase lability. complex occurs during the 2-h period between the restriction A different mechanism is involved in turning on the produc point and the onset of S phase and that the complex, once tion of certain enzymes that are involved in DNA synthesis. assembled, permits DNA synthesis. It is of great interest to Several of these enzymes increase dramatically at the onset of learn what mechanism is responsible for the assembly of such DNA synthesis. TK is one such enzyme which has been studied a complex. Is it the final synthesis of some glue molecule which in our laboratory (20). The activity of this enzyme increases at provides nucleation for reputase assembly? Is it controlled by least 40-fold during S phase. The enzyme is not obligatory for spontaneous intermolecular forces (much as the classical work DNA synthesis (as shown with thymidine kinase-negative mu of Dr. Nomura has shown for ribosome assembly)? And, finally, tants), nor is DNA synthesis essential for its appearance since what relation does this multienzyme complex have to the nu DNA synthesis inhibitors (such as hydroxyurea) do not block clear matrix, to which several enzymes, including DNA polym increases in TK activity. erase-a, are at least in part bound? Our most recent studies have centered on regulation of the Some of our recent work to determine answers to the above mRNA for thymidine kinase.3 This increase in TK mRNA, queries has shown that the activation of thymidylate synthase, which occurs at the beginning of S phase, is partly due to an a component of this reputase, occurs an h or so before the onset enhanced transcriptional rate. We have reported enhanced tran of S phase and does not require protein synthesis (21 ). This scription as a mechanism for increasing levels of TK mRNA. result is consistent with activation of this enzyme being related However, there also appears to be a strong component of to its assembly into the complex. Furthermore, studies by us stabilization of this mRNA or its precursor, so that the amount and others have shown that the activity of thymidylate synthase of TK mRNA is dependent on both its increased rate of pro in intact cells is strongly inhibited by drugs that directly inhibit duction and a decreased rate of degradation. Other mechanisms ribonucleotide reducÃase,DNA polymerase-a, or DNA lopoi- such as more efficient nuclear processing or increased mRNA somerase II (27). Our explanation for ihis indirect inhibition, transport may also contribute to the accumulation of this mes which does not occur in extracts, is that it is caused by allosleric sage during S phase. inleractions between thymidylate synthase and the olher, inhib An important consequence of studies on DNA synthetic ited enzymes within the reputase complex. All of these data enzymes is the opportunity to investigate cell cycle regulation suggest an important role in DNA synthesis for multienzyme by working backwards from a measurable event turned on at complexes and therefore, between assembly of the complex and the beginning of S phase. In particular, the appearance of TK regulation of Ihe cell cycle al onsel of DNA synthesis. mRNA provides a tool for studying growth control. It will evidently be much easier to study the mRNA regulation of a On the Dynamic State of Regulatory Processes single gene, which is dependent upon transcriptional activation, stabilization, and processing, than to study the yet incompletely One generalization that arises from these studies is the im understood and multicomponent process of DNA synthesis. By portance of dynamic stales in regulalion. This dynamic state is investigating the molecular events which control the production a reflection of the age-old principle of Ying and Yang, in which of an enzyme such as TK, we will learn about molecules all the world's processes are considered lo be balanced between produced during d that regulate various coordinated events of active and passive components. Again and again, at every level S phase. This knowledge will thereby permit us to understand of cell biology and biochemistry, one observes lhat molecules more completely molecular biology of growth control. such as mRNAs, proteins, and phosphorylated compounds are Our second approach to post-restriction point events is to in a dynamic state, often with very rapid turnover rale. Futile investigate a multienzyme complex that is required for DNA cycles have been reported in which a compound can continually synthesis. Many data over the past decade, starting with work be synthesized and broken down, or phosphorylaled and de- by Seki and Muller (23) and Baril's laboratory (see Ref. 24), phosphorylaled, etc. (28). At first glance these processes appear have suggested that DNA is made in eukaryotic cells by a to be wasteful of metabolic energy. However, they can be multienzyme complex rather than by soluble enzymes. Dr. G. justified upon realizing lhat they permil rapid adjust ments of a P. V. Reddy and I have extended this concept to include not syslem lo altered conditions and therefore are vital for the only the enzymes directly involved in DNA synthesis, such as maintenance of light control of importanl processes. DNA polymerase-a, topoisomerases, primase, etc., but also According lo this reasoning, a substance subject to both rapid enzymes of DNA precursor synthesis including ribonucleotide synthesis and degradation exists in a steady state, which in this reducÃase,thymidine kinase, thymidylate synthase, and others dynamic system is highly dependenl upon both inflow and (25). Our data indicate that all of these proteins are parts of a outflow rales, and so ils amounl changes dramalically owing to larger complex which we have called a reputase (26). With Dr. small changes in either of those rates. An analogy that comes H. Noguchi, we have managed to purify such a complex nearly close to home is Ihe dynamic state of one's checking account, 10-fold, although it is quite unstable. This complex carries with which can fluctuate widely depending upon the amount of input it nascent DNA and several of the enzymes included in DNA and output. Similarly in cell cycle regulation, the dynamic slale 3 D. L. Coppock, J. A. Lewis, and A. B. Pardee. Dual control of thymidine of Ihe p68 prolein in unlransformed cells could allow rapid kinase mRNA during the cell cycle, submitted for publication. responses lo growth-conlrolling subslances depending upon 1490 Downloaded from cancerres.aacrjournals.org on September 26, 2021. © 1987 American Association for Cancer Research. MOLECULES IN NORMAL AND CANCER CELLS the Gi-to-S transition in Saccharomyces cerevisiae. Proc. Nati. Acad. Sci. events which govern its production, such as rates of protein USA, 81:120-124, 1984. synthesis and availability of growth factors, and also on other 12. Popolo, L., Vai, M., and Alberghina, L. Identification of a glycoprotein events that determine its stability. Stabilization of this protein involved in cell cycle progression in yeast. J. Biol. Chem., 261: 3479-3482, 1986. in tumor cells would therefore increase the steady state level of 13. Croy, R. G., and Pardee, A.B. Enhanced synthesis and stabilization of M, p68 and result in relaxation of growth control. Given this 68,000 protein in transformed BALB/C-3T3 cells: candidate for restriction point control of cell growth. Proc. Nati. Acad. Sci. USA, 80: 4699-4703, apparent power to modulate concentrations of regulatory con 1983. stituents, it seems apparent that the dynamic state will prove 14. Campisi, J., Morreo, G., and Pardee, A. B. Kinetics of GÃ¬transit following to be a major principle for biology of regulation at every level. brief starvation for serum factors. Exp. Cell Res., Â¡52:459-466, 1984. 15. Zetterberg, A., and Larsson, O. Kinetic analysis of regulatory events in (,, leading to proliferation or quiescence of Swiss 3T3 cells. Proc. Nati. Acad. Sci. USA, 82:5365-5369, 1985. Acknowledgments 16. Leof, E. B., Wharton, W., Van Wyk, J. J., and Pledger, W. J. Epidermal growth factor (EGF) and somatomedin C regulate G! progression of com I would like to thank Marjorie Rider for preparing the manuscript. petent BALB/c 3T3 cells. Exp. Cell Res., Â¡41:107-115, 1982. 17. Campisi, J., Gray, H. E., Pardee, A. B., Dean, M., and Sonenshein, G. E. Cell-cycle control of c-myc but not c-ras expression is lost following chemical transformation. Cell, 36: 241-247, 1984. References 18. Mulcahy, L. S., Smith, M. R., and Stacey, D. W. Requirement for ras proto oncogene function during serum-stimulated growth of NIH 3T3 cells. Nature (Lond.), 3Â¡3:24I-243, 1985. 1. Baserga. R. The Biology of Cell Reproduction. Cambridge. MA: Harvard 19. Ii-min. H. M. Stimulation by serum of multiplication of stationary chick University Press, 1985. cells. J. Cell. Physiol., 78:161-170, 1971. 2. Pardee, A. B. Principles of cancer biology: biochemistry and cell biology. ///. V. T. DeVita, Jr., S. Hellman. and S. A. Rosenberg (eds.). Cancer Principles 20. Coppock, D. L., and Pardee, A. B. Regulation of thymidine kinase activity and Practice of Oncology, pp. 3-22. Philadelphia: J. B. Lippincott, 1985. in the cell cycle by a labile protein. J. Cell. Physiol., Â¡24:269-274, 1985. 21. Yang, H. C., and Pardee, A. B. Insulin-like growth factor I regulation of 3. Campisi, J., Fingert, H. J., and Pardee. A. B. Basic biology and biochemistry transcription and DNA replicating enzyme induction necessary for DNA of cancer. In: R. C. Knapp and R. S. Berkowitz (eds.), Gynecologic Oncology, synthesis. J. Cell. Physiol., 727:410-416, 1986. pp. 3-46. New York: Macmillan Publishing, 1986. 22. Sittman, D. B., Graves, R. A., and Marzluff, W. F. Histone mRNA concen 4. Rossow, P. W., Riddle. V. G. H., and Pardee, A. B. Synthesis of labile, trations are regulated at the level of transcription and mRNA degradation. serum-dependent protein in early G, controls animal cell growth. Proc. Nati. Proc. Nati. Acad. Sci. USA, 80:1849-1853, 1983. Acad. Sci. USA, 76:4446-4450, 1979. 23. Seki, S., and Mueller, G. C. Dissociation and reconstitution of the DNA 5. Schneiderman, M. H., Dewey, W. C, and Highneid, D. P. Inhibition of replicase system of HeLa cell nuclei. Biochim. Biophys. Acta, 435: 236-250, DNA synthesis in synchronized Chinese hamster cells treated in G, with 1976. cycloheximide. Exp. Cell Res., 67: 147-155, 1971. 24. Vishwanatha, J. K . Coughlin, S. A., Wesolowski-Owen, M., and Barii, E. F. 6. Brooks, R. F. Continuous protein synthesis is required to maintain the A multiprotein form of DNA polymerase a from HeLa cells. J. Biol. Chem., probability of entry into S phase. Cell, 12: 311-317, 1977. 261:6619-6628, 1986. 7. Pardee. A. B. A restriction point for control of normal animal cell prolifera 25. Reddy, G. P. V., and Pardee, A. B. Multienzyme complex for metabolic tion. Proc. Nati. Acad. Sci. USA, 71:1286-1290, 1974. channeling in mammalian DNA replication. Proc. Nati. Acad. Sci. USA, 77.- 8. Campisi, J., Medrano. E. E., Morreo, G., and Pardee. A. B. Restriction point 3312-3316, 1980. control of cell growth by a labile protein: evidence for increased stability in 26. Noguchi, H., Reddy, G. P. V., and Pardee, A. B. Rapid incorporation of transformed cells. Proc. Nati. Acad. Sci. USA, 79:436-440, 1982. label from ribonucleoside diphosphates into DNA by a cell-free high molec 9. Campisi. J., and Pardee, A. B. Post-transcriptional control of the onset of ular weight fraction from animal cell nuclei. Cell, 32: 443-451, 1983. DNA synthesis by an insulin-like growth factor. Mol. Cell. Biol., 4: 1807- 27. Reddy, G. P. V., and Pardee, A. B. Inhibitor evidence for allosteric interaction 1814, 1984. in the reputase multienzyme complex. Nature (Lond.), 303:86-88, 1983. 10. Shilo. B., Riddle, V. G. H.. and Pardee, A. B. Protein turnover and cell cycle 28. Newsholme. E. A., Challiss, R. A. J., and Crabtree, B. Substrate cycles: their initiation in yeast. Exp. Cell. Res., Â¡23:221-227, 1979. role in improving sensitivity in metabolic control. Trends Biochem. Sci., 9: 11. Popolo, L., and Alberghina. L. Identification of a labile protein involved in 277-280, 1984.

1491 Downloaded from cancerres.aacrjournals.org on September 26, 2021. © 1987 American Association for Cancer Research. Molecules Involved in Proliferation of Normal and Cancer Cells: Presidential Address

Arthur B. Pardee

Cancer Res 1987;47:1488-1491.

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