Control of cell proliferation by Myc

reviews 5 Futerman, A. H. and Pagano, R. E. (1991) Biochem. J. 280, 295–302 6 Nozue, M. et al. (1988) Int. J. Cancer 42, 734–738 7 Ichikawa, S. et al. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 2703–2707 8 Ichikawa, S. et al. (1996) Proc. Natl. Acad. Sci. U. S. A. 93, 4638–4643 9 Coste, H., Martel, M. B. and Got, R. (1986) Biochim. Biophys. Acta 858, 6–12 10 Trinchera, M., Fabbri, M. and Ghidoni, R. (1991) J. Biol. Chem. 266, 20907–20912 11 Jeckel, D. et al. (1992) J. Cell Biol. 117, 259–267 12 Lannert, H. et al. (1994) FEBS Lett. 342, 91–96 13 Burger, K. N. J., van der Bijl, P. and van Meer, G. (1996) J. Cell Biol. 133, 15–28 14 Futerman, A. H. et al. (1990) J. Biol. Chem. 265, 8650–8657 15 Nakayama, M. et al. (1995) Biosci. Biotechnol. Biochem. 59, 1882–1886 16 Lynch, D. V. et al. (1997) Arch. Biochem. Biophys. 340, 311–316 17 Ichikawa, S., Ozawa, K. and Hirabayashi, Y. Biochem. Mol. Biol. Int. (in press) 18 Tasaka, Y. et al. (1996) EMBO J. 15, 6416–6425 19 Schulte, S. and Stoffel, W. (1993) Proc. Natl. Acad. Sci. U. S. A. 90, 10265–10269

Control of cell proliferation by Myc Caroline Bouchard, Peter Staller and Martin Eilers Myc proteins are key regulators of mammalian cell proliferation. They are transcription factors that activate genes as part of a heterodimeric complex with the protein Max. This review summarizes recent progress in understanding how Myc stimulates cell proliferation and how this might contribute to cellular transformation and tumorigenesis.

20 Stahl, N. et al. (1994) J. Neurosci. Res. 38, 234–242 21 Coetzee, T. et al. (1996) Cell 86, 209–219 22 Bosio, A. et al. (1996) Proc. Natl. Acad. Sci. U. S. A. 93, 13280–13285 23 Kishimoto, Y. (1986) Chem. Phys. Lipids 42, 117–128 24 Hannun, Y. A. (1994) J. Biol. Chem. 269, 3125–3128 25 Kolesnick, R. and Golde, D. W. (1994) Cell 77, 325–328 26 Abe, A., Radin, N. S. and Shayman, J. A. (1996) Biochim. Biophys. Acta 1299, 333–341 27 Ito, M. and Komori, H. (1997) Exp. Med. 15, 1476–1482 28 Lavie, Y. et al. (1996) J. Biol. Chem. 271, 19530–19536 29 Lavie, Y. et al. (1997) J. Biol. Chem. 272, 1682–1687 30 Sando, G. N., Howard, E. J. and Madison, K. C. (1996) J. Biol. Chem. 271, 22044–22051 31 Schwarz, A. and Futerman, A. H. (1997) J. Neurosci. 17, 2929–2938 32 Bird, A. P. (1986) Nature 321, 209–213 33 Hidari, I-P. J. K. et al. (1996) J. Biol. Chem. 271, 14636–14641 34 Fenderson, B. A. et al. (1992) Exp. Cell Res. 198, 362–366 35 Platt, F. M. et al. (1997) J. Biol. Chem. 272, 19365–19372 36 Radin, N. S. (1992) Trends Glycosci. Glycotechnol. 4, 322–325 37 Takamiya, K. et al. (1996) Proc. Natl. Acad. Sci. U. S. A. 93, 10662–10667

discovered as the cellular homologue of the transforming oncogene of several chicken retroviruses. The c-myc gene is transcribed in a strictly proliferationdependent manner in several cell types (for a detailed review, see Ref. 1). Growth-factor-independent expression of c-myc occurs in tumour cells as a result of either lesions in the c-myc gene itself or mutations in signalling pathways that regulate c-myc expression. In cultured cells, constitutive expression of c-myc induces proliferation even in the complete absence of growth factors. Under these conditions, proliferation is limited only by the concomitant induction of apoptosis by c-myc. Conversely, inhibition of Myc function by antisense approaches or overexpression either of dominant–negative alleles of Myc or of Mad proteins (which antagonize Myc function) severely impairs the growth-factor-induced proliferation of cells in culture2; fibroblasts, in which both alleles of c-myc are inactivated by homologous recombination are viable but have a greatly diminished rate of proliferation, with a marked delay in passage through both the G1 and the G2 phases of the cell cycle3. Knockout animals in which c-myc has been disrupted die early in embryogenesis4. Taken together, these data show that Myc is an important regulator of cell proliferation in vivo, although it might not be essential for cell-cycle progression per se. In vertebrates, c-myc is part of a small gene family with four closely related members (c-, N-, L- and s-myc); a fifth gene, B-myc, is also similar to other myc genes but lacks several domains believed to be crucial for the functions of Myc proteins. c-, N- and L-myc all transform primary rat fibroblasts in cooperation with an activated allele of ras, demonstrating that crucial functions of the encoded proteins are conserved5. Surprisingly, this is also true for the recently discovered Drosophila homologue of Myc;

The authors are in the Institute for Molecular Biology and Tumour Research, University of Marburg, Germany. E-mail: bouchard@ imt.uni-marburg.de

One way in which quiescent mammalian cells differ from their proliferating counterparts is that they do not transcribe a number of genes required for cell proliferation, such as those that encode cyclins, cyclindependent kinases (CDKs), replication factors and some enzymes of nucleotide metabolism. Cellular proliferation is clearly controlled at least in part at the level of gene transcription, and activation of transcription factors is a crucial step in the transition from quiescence to proliferation. It has long been known that one such transcription factor is encoded by the c-myc gene, which was

202

Copyright © 1998 Elsevier Science Ltd. All rights reserved. 0962-8924/98/$19.00 PII: S0962-8924(98)01251-3

trends in CELL BIOLOGY (Vol. 8) May 1998

reviews TAD thus, the function of Myc proteins is much more highly conserved evolutionarily than previously anticipated6,7. However, several experiments demonstrate that the physiological functions of the N- and L-myc genes might differ from that of c-myc8; for example, L-myc-deficient mice are viable and do not show obvious compensatory changes in the expression pattern of other myc genes9. This review focuses on the c-myc gene itself and its product, Myc protein, and summarizes recent progress in understanding how Myc transduces signals from the environment into changes in the proliferative programme of a cell.

** * 1

Bin1 p107 TBP α-tubulin TRAP-1

*

45 63

129 143

I

II

A

240 262

NLS B HLH LZ

*

320 328 355 368

410 439

amino acids

MAX YY1 MIZ-1

FIGURE 1 Structural domains of c-Myc. The domains are indicated at the top: a transcriptional activation domain (TAD) with the Myc-box I (I) and Myc-box II (II), an acidic region (A), a nuclear-localization signal (NLS), a basic region (B), a helix–loop–helix motif (HLH) and a leucine zipper domain (LZ). The locations of the major in vivo phosphorylation sites are indicated by an asterisk (*). Black bars represent the domains of c-Myc involved in the interaction with other proteins.

Regulation of transcription by Myc Myc proteins are transcription factors of the helix– loop–helix/leucine zipper family (Fig. 1) that activate transcription as obligate heterodimers with a partner phenotype of the transformed cells and for the control protein, Max (for a detailed review, see Ref. 1). The of proliferation by Myc. For example, one such reMyc–Max complex binds to E-box DNA elements with pressed gene encodes the transcription factor c/EBP-␣, the core sequence CAC(G/A)TG (Fig. 2). Dimerizwhich induces cell-cycle arrest and commits cells to ation with Max and binding to DNA are required for adipocyte differentiation23. Of course, Myc might all biological effects of Myc. In contrast to Myc, Max can also form homodimers or heterodimers with a simply induce the synthesis of a repressor protein, family of proteins termed Mad, Mxi-1 and Mnt and the repression might be an indirect consequence (Rox)10–13 (Fig. 2). Heterodimeric complexes of Max of transcriptional activation by Myc. However, a number of observations argue against this idea: for and Mad are transcriptional repressors at least in part example, mutations in Myc have been identified because they recruit histone deacetylases through a that affect transactivation and transrepression difprotein termed Sin3 (e.g. Refs 14–16). Mad proteins ferentially24. More likely, therefore, at least some accumulate during differentiation in several cell types; for example, induced differentiation of U937 cells is genes are repressed by Myc through complex foraccompanied by a switch from Myc–Max to Mad–Max mation with partner proteins distinct from Max. For heterodimers17. Thus, the opposing biochemical propexample, Myc forms complexes with YY-1 and with TFII-I, two proteins that activate transcription from erties and patterns of expression of Myc–Max cominitiator elements25; binding of Myc to YY-1 precludes plexes and Mad–Max complexes might explain how proliferation-dependent regulation of E-box elements is achieved. Max Target genes of the Myc–Max complex Max c-Myc Max have been isolated by a number of difc-Myc ferent approaches, including differenMad tial screening methods, immunoprecipiMad3 Heterodimerization Mad4 tation of Myc–Max complexes and, with other factors Mxi-1 most frequently, by educated guesses. A Homodimerization Mnt (Rox) list of candidate genes was compiled reMax–Max Heterodimerization cently18: it contains a number of genes Max–Mad required for cell proliferation, and at least Max–Mad3 -CAC(G/A)TGYY-1 TFII-1 Miz-1 two genes, ornithine decarboxylase19 and Max–Mad4 Cdc25A20, whose overexpression in culMax–Mxi-1 ture partially mimics the transforming Max–Mnt (Rox) properties of Myc. Taken together, these results leave little doubt that Myc exerts Sin3 at least part of its effect on cell proliferation by activating the transcription of a set of crucial target genes. However, Activation of transcription Repression a number of observations suggest that e.g. proTα, ODC, Cdc25A, of transcription some of the essential target genes by CAD, eIF-4E which Myc influences proliferation have FIGURE 2 not yet been identified (see below). A second open issue stems from the The Myc network of proteins. Myc–Max heterodimers are transcriptional activators that bind to the finding that cells transformed by Myc CAC(G/A)TG motif, found in genes including alpha prothymosin (proT␣), ornithine decarboxylase are characterized by the loss of expression (ODC) and Cdc25A phosphatase. Max can also form homodimers or heterodimers with Mad, Mad3, of a number of genes (e.g. Refs 21 and 22). Mad4, Mxi-1 and Mnt (Rox). These complexes are transcriptional repressors that recruit histone The identity of the repressed genes sug- deacetylases through the Sin3 protein. In addition to the formation of heterodimers with Max, Myc gests that repression is important for the protein might interact with and inhibit transactivation by YY-1, TFII-1 and Miz-1. trends in CELL BIOLOGY (Vol. 8) May 1998

203

reviews Cyclin D1 Cdk4

pRb

p107

E2F

Myc

Cyclin E

? Cyclin E

Cdk2

Bin-1

Cdc25A Cyclin E

Cyclin E

Cdk2

Cdk2

p27 Cdk2 P P

P FIGURE 3

Control of G1-phase progression by Myc. The figure presents a working model as to how Myc might be integrated in the control of G1 cyclin-dependent kinase (CDK) activation. Activation of Myc promotes the formation of binary active cyclin-E–Cdk2 complexes from ternary inactive cyclin-E–Cdk2–p27 complexes by controlling p27 metabolism and degradation and by activating Cdc25A phosphatase activity. Cyclin E synthesis might be downstream of pRb phosphorylation by cyclin-D–Cdk4 complexes55,56. The evidence supporting a role for p107 as a regulator of Myc is discussed in the text.

interaction between YY-1 and TATA-binding protein and TFIIB, providing a potential mechanism for inhibition26. More recently, two-hybrid cloning identified a zinc-finger protein, Miz-1, that associates with Myc27. Miz-1 binds to and transactivates the promoters of at least two genes that are repressed by Myc in vivo. Complex formation with Myc inhibits transactivation by Miz-1. This inhibition depends on protein association and on the integrity of a small N-terminal domain of Miz-1 that has been termed the POZ domain; in other proteins, this domain strongly represses transcription. These data suggest a model in which association with Myc activates the function of a latent repressive domain in Miz-1. In cultured cells, Miz-1 exerts a potent growth-arrest function, which is alleviated by Myc. Thus, Myc might control proliferation both by activating genes, when complexed to Max, and by inhibiting transcription factors such as Miz-1, TFII-1 and YY-1. CDKs as effectors of Myc The earliest response to activation of Myc in resting cells is a rapid induction of cyclin-E–Cdk2 kinase activity; other CDKs lag behind28. Microinjection experiments show that inhibition of Cdk2 kinase activity blocks all downstream responses to Myc in the cell cycle, most notably induction of transcription of the gene encoding cyclin A29. Expression of Cdk2 is unaltered in response to Myc and there is only a moderate increase in the amount of transcripts encoding cyclin E, suggesting that Myc acts primarily at a step after cyclin E synthesis. This is supported by the observation that cells expressing a dominant–negative allele of myc arrest with high levels of cyclin E and Cdk2, yet the complex lacks detectable kinase activity30.

204

One potential target gene of Myc implicated in this activation is Cdc25A, a phosphatase that removes two inhibitory phosphates from Cdk2. As mentioned above, expression of Cdc25A is stimulated by Myc via an E-box in the first intron of the cdc25A gene20. In vivo, however, activation of cyclin-E–Cdk2 kinase by Myc correlates more closely with loss of the CDK inhibitor p27Kip1 from cyclin-E–Cdk2 complexes28 rather than with dephosphorylation by Cdc25A, and a cell-cycle block imposed by ectopic expression of p27 is partially antagonized by ectopic expression of Myc31. These data suggest that some aspect of p27 metabolism and degradation is controlled by Myc, but the crucial proteins involved are still unknown. Another possible mechanism is through dissociation of p27 from Cdk2 complexes, which is brought about by proteins that compete for the same binding site on Cdk2 complexes – several proteins (including Cdc25A and E2F2, encoded by another potential target gene of Myc32) are now known to use similar motifs to bind to CDK complexes33 (Fig. 3). One complication is that the process has elements of feedback control: for example, p27 is phosphorylated by cyclin-E–Cdk2 and cyclin-A–Cdk2, and this facilitates its dissociation from Cdk2 complexes34 and is required for subsequent degradation35,57. Also, transcription of the gene encoding cyclin A is stimulated by cyclin-E–Cdk2 kinase, and this might further enhance p27 turnover29. Therefore, minor changes (for example, the increase in the expression of cyclin E) could be amplified by a positive-feedback system to yield large changes in cyclin-E–Cdk2 kinase activity36. As a consequence of these alterations, the normal order of events in the G1 phase of the cell cycle is disrupted in Myc-transformed cells37. In exponentially growing Myc-transformed cells, activation of transcription of the genes encoding cyclin E and cyclin A, activation of their associated kinases and degradation of p27 all occur immediately upon exit from mitosis. Hyperphosphorylation of the retinoblastoma protein (pRb) and expression of the gene encoding cyclin E have been linked to passage through the restriction point, after which cells are committed for another round of DNA replication. If this is true, then Myctransformed cells become committed to another round of proliferation immediately after exit from mitosis and lack a pre-restriction phase of the cell cycle. This type of alteration in the cell cycle could explain why Myc-transformed cells proliferate even in the complete absence of growth factors. These data also raise the issue of why transformed cells still initiate DNA replication at the same size as nontransformed cells, despite having almost complete deregulation of CDK activity and E2F-dependent transcription. Control of Myc function Myc activity is controlled at multiple levels by signaltransduction cascades. One such level is c-myc transcription, which is strongly induced by growth factors. Both transcription initiation and elongation at the end of the first intron are regulated. Several observations implicate the Ras–Raf signalling cascade in c-myc promoter regulation. In response to colony-stimulating factor 1 (CSF-1), activation of c-myc expression requires trends in CELL BIOLOGY (Vol. 8) May 1998

reviews BOX 1 – MYC AND APOPTOSIS

binding of transcription factors of the Ets family to a joint Ets/E2F site in the c-myc promoter38. Activation of conditional Raf-ER alleles results in rapid phosphorylation of the Ets-2 protein39 and induction of c-myc promoter activity. Finally, dominant– negative alleles of Raf, when expressed from an inducible promoter, block the serum induction of the c-myc gene40. Taken together, these data strongly suggest that the Ras–Raf pathway plays a role in c-myc induction. However, injection of dominant–negative alleles of Ras does not block induction of the c-myc promoter in response to addition of platelet-derived growth factor (PDGF), suggesting that other signaltransduction pathways might contribute to c-myc induction41. It has also been suggested that the c-myc promoter is controlled by the E2F transcription factor, and E2F proteins can activate the promoter in transient-transfection assays42. However, expression of c-myc is not regulated in a cell-cycle-dependent manner; also, more-moderate expression of E2F from an adenoviral vector does not induce c-myc expression43, and c-myc expression is not elevated in cells lacking either pRb, p107 and/or p130, pocket proteins that negatively regulate E2F function44. Taken together, these data suggest that endogenous E2F does not contribute significantly to the transcriptional regulation of the c-myc gene. A second level of control is through proteins that interact with the N-terminus of Myc. Evidence suggesting that such mechanisms exist comes from sequence analysis of viral myc genes and of c-myc genes isolated from lymphoma cells. Both accumulate a similar set of mutations around a conserved domain (so-called Myc-box I) in the N-terminus of Myc. One protein that interacts with this domain is Bin-1, which represses transactivation and transformation by Myc and appears to be mutated in a number of breast cancers45. Bin-1 is similar to a yeast protein that controls cell-cycle exit in response to nitrogen starvation and could transmit negative regulatory signals to Myc. A second protein that can interact with Myc-box I in the N-terminus of Myc in vivo is p10746,47, although a stoichiometric association of endogenous p107 and Myc proteins has not yet been demonstrated. Ectopically expressed p107 negatively controls transactivation and transformation by Myc and exerts a cell-cycle arrest that can be relieved by Myc. Repression of transactivation by p107 is released by coexpression of Cdk4, suggesting that phosphorylation of p107 by D1–Cdk4 kinases relieves repression48. The findings suggest that D-type kinases act upstream of Myc in a manner that is analogous to the control of E2F function by D-type kinases through phosphorylation of the pocket proteins pRb and p107. This model could explain the strong overlap in function between D-type cyclins and Myc during mitogenic stimulation (Fig. 3). The most recent addition to the group of proteins that interact with the N-terminus of Myc is TRAP-1, a member of the ATM family of lipid kinases (M. Cole, pers. commun.). TRAP-1 interacts with Myc-box II, a domain that is crucial for transformation by Myc and is implicated in both transrepression and transactivation by Myc. Depletion of TRAP-1 by antisense trends in CELL BIOLOGY (Vol. 8) May 1998

Deregulated expression of Myc not only promotes cell proliferation but also active cell death – apoptosis1; this ability is thought to act as a safeguard protecting the organism from tumours that might otherwise arise as a consequence of a single mutation at a myc gene locus. In order to proliferate, cells require not only growth-promoting signals but also survival signals, which can be provided by factors such as insulin-like growth factors (IGFs) and transmitted by a phosphoinositide-3-kinase-dependent signalling cascade2,3. When survival signals are missing, for example when cells are forced to proliferate by ectopic expression of Myc, they activate a death pathway that involves the CD95 receptor–ligand interaction4. So, how does Myc promote apoptosis? One model is that apoptosis occurs due to an inherent conflict that arises when proliferation is induced by Myc alone and other signals that promote proliferation are missing. The model predicts that inhibition of Myc-induced proliferation should relieve the conflict and thus inhibit Myc-induced apoptosis. Support for this view comes from the observation that antisense-mediated inhibition of Cdc25A expression inhibited Myc-induced apoptosis in one cell line5. However, a number of related studies concluded that Myc-induced proliferation and apoptosis are at least partially independent from each other. For example, injection of high amounts of cyclindependent kinase (CDK) inhibitors blocked Myc-induced cell-cycle progression but not apoptosis in a closely related set of experiments6. In myeloid cells, activation of ornithine decarboxylase (ODC) activity is required for both Myc-induced apoptosis and proliferation; ectopic expression of ODC is sufficient to give apoptosis but not proliferation7. Recent observations from Kari Alitalo’s laboratory offer an explanation as to how Myc promotes apoptosis in response to the cytokine tumour necrosis factor alpha (TNF-␣)8. Cytokines such as TNF-␣ induce both pro-apoptotic and anti-apoptotic signals in their target cells, and the balance of signals determines the fate of the cell. Alitalo and coworkers report that the signal-transduction pathways that mediate survival signals upon binding of TNF-␣ are inhibited by activation of Myc. For example, activation of NF-␬B, which has been implicated in cell survival, is inefficient in Myc-transformed cells; re-expression of NF-␬B restores cell survival. Although the molecular mechanisms underlying the observation are unresolved, the findings suggest that a Myc-induced imbalance in cellular signalling underlies its ability to promote apoptosis. Whether this also applies to death in the absence of survival factors remains to be determined.

References 1 2 3 4 5 6 7 8

Evan, G. I. et al. (1992) Cell 69, 119–128 Kauffmann-Zeh, A. et al. (1997) Nature 385, 544–548 Kennedy, S. G. et al. (1997) Genes Dev. 11, 701–713 Hueber, A. O. et al. (1997) Science 278, 1305–1309 Galaktionov, K., Chen, X. and Beach, D. (1996) Nature 382, 511–517 Rudolph, B. et al. (1996) EMBO J. 15, 3065–3076 Packham, G., Porter, C. W. and Cleveland, J. L. (1996) Oncogene 13, 461–469 Klefstrom, J. et al. (1997) EMBO J. 16, 7382–7392

techniques abolishes transformation by Myc, suggesting that TRAP-1 carries out a downstream effector function of Myc protein rather than acting upstream to control Myc function (M. Cole, pers. commun.). Oncogene cooperation and Myc While Myc can be sufficient to induce mitogenesis in established cell lines, it fails to transform primary cells. Similarly, while activated alleles of ras transform established cell lines, Ras alone is insufficient to transform primary rat embryo fibroblasts. Myc and Ras need to cooperate in the transformation of such cells. Several proposals have been put forward to explain this behaviour. One is that activation of conditional alleles of Myc induces only a partial activation of CDK

205

reviews activity in the absence of serum growth factors even in established cell lines28. Addition of serum growth factors or complementation with an activated allele of ras leads to a synergistic induction of CDK activity28,49. For example, growth factors (and Ras activity) are required for expression of cyclin D1 and its associated kinase activity50 and potentially to activate the Cdc25A phosphatase51. Thus, cooperation between Ras and Myc occurs at the level of CDK activity even in established cell lines. If there is a threshold level of CDK activity that is required for proliferation and if this threshold differs between primary and established cells, oncogene cooperation could simply result from the fact that different oncogenes control different steps in the assembly of active cyclin–CDK complexes. A slightly different model is suggested by the finding that ectopic expression of EJ Ras induces a cell-cycle arrest in primary mouse embryo fibroblasts (but not in established cells) and that cooperating nuclear oncogenes are required to overcome this arrest52,53. The arrest involves induction of the CDK inhibitor p21 by a pathway that depends on p53 activity at least in some primary cell types52. Thus, cells might have a safeguard against malfunction of the ras gene, and oncogene cooperation might result from the disruption of this arrest programme by nuclear oncogenes. Both models explain oncogene cooperation in terms of cyclin–CDK activity. Clearly, however, other forms of oncogene cooperation exist in vivo, such as loss of apoptotic programmes (see Box 1), induction of angiogenesis, loss of interaction with neighbouring cells and/or induction of telomerase activity: and both Myc and Ras might function in these pathways. For example, ectopic expression of N-Myc downregulates the expression of an integrin in neuroblastoma cells and might therefore promote metastatic behaviour (e.g. Ref. 22). Finally, even transient activation of Myc promotes the accumulation of genetic damage in cells54. Thus, there might be multiple effector pathways by which deregulated expression of Myc contributes to tumorigenesis in vivo. Concluding remarks A wealth of biological data supports the notion that Myc proteins are central regulators of cell-cycle progression. It has become clear that activation of Cdk2 kinase activity and antagonizing the function of CDK inhibitors such as p27 is one mechanism by which Myc promotes mitogenesis; however, the details of this reaction are still not understood. Similarly, our knowledge about the factors that control Myc function is still too limited to allow a precise view as to how transactivation and potentially transrepression by Myc are regulated. Finally, one of the most pressing questions is whether the ability of Myc to promote proliferation underlies its widespread deregulation in human tumours. Clearly, the signal-transduction pathway that is defined by Myc protein stills holds many mysteries. Acknowledgements We sincerely apologize to all our colleagues whose work we could not cite owing to space constraints.

206

References 1 Henriksson, M. and Lüscher, B. (1996) Adv. Cancer Res. 68, 109–182 2 Roussel, M. F. et al. (1996) Mol. Cell. Biol. 16, 2796–2801 3 Mateyak, M. K. et al. (1997) Cell Growth Differ. 8, 1039–1048

4 Davis, A. C. et al. (1993) Genes Dev. 7, 671–682 5 Mukherjee, B., Morgenbesser, S. D. and DePinho, R. A. (1992) Genes Dev. 6, 1480–1492 6 Gallant, P. et al. (1996) Science 274, 1523–1527 7 Schreiber-Agus, N. et al. (1997) Proc. Natl. Acad. Sci. U. S. A. 94, 1235–1240 8 Morgenbesser, S. D. et al. (1995) EMBO J. 14, 743–756 9 Hatton, K. S. et al. (1996) Mol. Cell. Biol. 16, 1794–1804 10 Zervos, A. S., Gyuris, J. and Brent, R. (1993) Cell 72, 223–232 11 Ayer, D. E., Kretzner, L. and Eisenman, R. N. (1993) Cell 72, 211–222 12 Hurlin, P. J. et al. (1995) EMBO J. 14, 5646–5659 13 Hurlin, P. J., Queva, C. and Eisenman, R. N. (1997) Genes Dev. 11, 44–58 14 Laherty, C. D. et al. (1997) Cell 89, 349–356 15 Sommer, A. et al. (1997) Curr. Biol. 7, 357–365 16 Alland, L. et al. (1997) Nature 387, 49–55 17 Ayer, D. E. and Eisenman, R. N. (1993) Genes Dev. 7, 2110–2119 18 Grandori, C. and Eisenman, R. N. (1997) Trends Biochem. Sci. 22, 177–181 19 Bello-Fernandez, C., Packham, G. and Cleveland, J. L. (1993) Proc. Natl. Acad. Sci. U. S. A. 90, 7804–7808 20 Galaktionov, K., Chen, X. and Beach, D. (1996) Nature 382, 511–517 21 Penn, L. J. Z. et al. (1990) EMBO J. 9, 1113–1121 22 Judware, R. and Culp, L. A. (1995) Oncogene 11, 2599–2607 23 Freytag, S. O. and Geddes, T. J. (1992) Science 256, 379–382 24 Li, L. et al. (1994) EMBO J. 13, 4070–4079 25 Roy, A. L. et al. (1993) Nature 365, 359–361 26 Shrivastava, A. et al. (1996) Proc. Natl. Acad. Sci. U. S. A. 93, 10638–10641 27 Peukert, K. et al. (1997) EMBO J. 16, 5672–5686 28 Steiner, P. et al. (1995) EMBO J. 14, 4814–4826 29 Rudolph, B. et al. (1996) EMBO J. 15, 3065–3076 30 Berns, K., Hijmans, E. M. and Bernards, R. (1997) Oncogene 15, 1347–1356 31 Vlach, J. et al. (1996) EMBO J. 15, 6595–6604 32 Sears, R., Ohtani, K. and Nevins, J. R. (1997) Mol. Cell. Biol. 17, 5227–5235 33 Saha, P. et al. (1997) Mol. Cell. Biol. 17, 4338–4345 34 Muller, D. et al. (1997) Oncogene 15, 2561–2576 35 Sheaff, R. J. et al. (1997) Genes Dev. 11, 1464–1478 36 Perez-Roger, I. et al. (1997) Oncogene 14, 2373–2381 37 Pusch, O. et al. (1997) Oncogene 15, 649–656 38 Roussel, M. F. et al. (1994) Oncogene 9, 405–415 39 McCarthy, S. A. et al. (1997) Mol. Cell. Biol. 17, 2401–2412 40 Kerkhoff, E. et al. (1998) Oncogene 16, 211–216 41 Barone, M. V. and Courtneidge, S. A. (1995) Nature 378, 509–512 42 Oswald, F. et al. (1994) Oncogene 9, 2029–2036 43 DeGregori, J., Kowalik, T. and Nevins, J. R. (1995) Mol. Cell. Biol. 15, 4215–4224 44 Hurford, R. K., Jr et al. (1997) Genes Dev. 11, 1447–1463 45 Sakamuro, D. et al. (1996) Nat. Genet. 14, 69–77 46 Beijersbergen, R. L. et al. (1994) EMBO J. 13, 4080–4086 47 Gu, W. et al. (1994) Science 264, 251–254 48 Haas, K. et al. (1997) Oncogene 15, 179–192 49 Leone, G. et al. (1997) Nature 387, 422–426 50 Peeper, D. S. et al. (1997) Nature 386, 177–181 51 Galaktionov, K., Jessus, C. and Beach, D. (1995) Genes Dev. 9, 1046–1058 52 Lloyd, A. C. et al. (1997) Genes Dev. 11, 663–677 53 Serrano, M. et al. (1997) Cell 88, 593–602 54 Chernova, O. B. et al. (1998) Mol. Cell. Biol. 18, 536–545 55 Alevizopoulos, K. et al. (1997) EMBO J. 16, 5322–5333 56 Lukas, J. et al. (1997) Genes Dev. 11, 1479–1492 57 Vlach, J. et al. (1997) EMBO J. 16, 5334–5344 trends in CELL BIOLOGY (Vol. 8) May 1998