Coordinating cell proliferation and differentiation Liang Zhu* and Arthur I Skoultchi† Cell proliferation and differentiation are highly coordinated processes during development. Recent studies have revealed that this coordination may result from dual functions residing in the central regulators of proliferation, allowing them to also regulate differentiation. Studies have also shown that some terminally differentiated cells can be made to divide beyond their normal capacity.
The inverse relationship between proliferation and differentiation
Addresses *Department of Developmental and Molecular Biology, and † Department of Cell Biology, Albert Einstein College of Medicine, Bronx, New York 10461, USA *e-mail: [email protected]
† e-mail: [email protected]
During development of a multicellular organism, cells proliferate for a defined length of time before they begin functional differentiation . The process of differentiation of primitive cells into more specialized cells involves an increasing restriction in proliferative capacity, culminating in cell-cycle exit. Precise regulation of terminal cell division is needed to ensure production of proper numbers of differentiated cells at the appropriate time. Maintenance of cell-cycle arrest in terminally differentiated cells is important for tissue architecture and function.
The decision of cells to differentiate is often made in the G1 phase of the cell cycle. Enforced cell-cycle arrest in G1 achieved by overexpressing dominant acting inhibitors of Cdks has been used to determine whether cell-cycle arrest in G1 per se is sufficient to cause differentiation. This type of experiment is usually performed with cultured cells that have already developed a certain degree of specialization in their transcriptional program. The most-studied differentiation process is skeletal myogenesis . Proliferating myoblasts express MyoD — a member of the myogenic transcription factor family — but they do not differentiate in growth-factor-rich media. Withdrawal of growth factors triggers a coordinated process of cell-cycle exit and myogenic differentiation. In this cell system, forced expression of p21CIP1 and p16INK4a is sufficient to promote cell-cycle withdrawal and expression of certain differentiation markers in the presence of high growth factor concentration . Overexpression of other CKIs such as p57KIP2 and p27CIP1 has also been shown to stimulate the transcriptional activity of MyoD . Conversely, overexpression of cyclin D1 has been shown to inhibit MyoD activity [9,11,12]. Furthermore, overexpression of E2F1 in myoblasts inhibits cell-cycle exit and differentiation upon serum withdrawal , although this effect may depend on supraphysiological levels of E2F1 . Together, these results provide experimental evidence that cell-cycle regulators can indeed affect some aspects of differentiation.
Cell proliferation is governed primarily by the cyclindependent kinases which each consist of a catalytic subunit (Cdk), and a regulatory subunit (cyclin). A key Cdk substrate is the retinoblastoma protein (pRB), which is inactivated by phosphorylation. Inactivation of pRB relieves its repression of E2F transcription factors, which activates the expression of genes whose products promote cell proliferation [2,3]. The enzymatic activities of Cdks are regulated in several ways: by cyclin binding, by Cdk phosphorylation and dephosphorylation, and by binding of Cdk inhibitors (CKIs) [4,5]. Currently, two families of CKIs are known: the INK4 family (p16INK4a, p15INK4b, p18INK4c, and p19INK4d) and the KIP family (p21CIP1, p27KIP1, and p57KIP2) [6,7]. In this review, we discuss how these cell-cycle regulators coordinate cell proliferation and differentiation and maintain terminally differentiated cells in cell-cycle arrest.
The hallmark features of tumor cells are that they have developed a block to normal differentiation and gained unlimited proliferative capacity. Nevertheless, in some tumor cell lines, culture conditions have been identified which force the cells to resume their differentiation programs. In many of these cell systems, forced cell-cycle arrest caused by inhibiting the core cell cycle machinery is sufficient to induce some aspects of differentiation. Several examples include overexpression of p27KIP1 in neuroblastoma cells , p21CIP1 or p27KIP1 in the U937 monocytic leukemia cell , and p19INK4d in 32Dcl3 myeloid cells . In a recent study , it has been shown that expression of p21CIP1 or the combination of p16INK4a and roscovitine, a chemical inhibitor of Cdk2, could induce murine erythroleukemia cells to actually undergo erythroid differentiation including terminal cell division. Conversely, forced expression of cyclin D1 can block myeloid cell differentiation  and overexpression
Current Opinion in Genetics & Development 2001, 10:91–97 0959-437X/01/$ — see front matter © 2001 Elsevier Science Ltd. All rights reserved. Abbreviations Cdk cyclin-dependent kinase CKI Cdk inhibitor HDAC histone deacetylase PCNA proliferating cell nuclear antigen pRB retinoblastoma protein
Studies in many cell types have shown that proliferation and differentiation are inversely correlated processes. With the rapid progress in our understanding of the regulation of the cell cycle, it has become possible to investigate this relationship in greater detail.
Oncogenes and cell proliferation
of E2F1 blocks M1 myeloblastic leukemia cells from differentiating into macrophages in response to IL-6 . Thus, even in tumor cells it appears that cell-cycle regulators can have strong effects on the differentiation process. Targeted inactivation of cell-cycle regulatory genes in mice has provided a more physiologically relevant approach to assess the importance of cell-cycle exit in cellular differentiation [21•]. The functions of many cell-cycle regulators are apparently redundant because the combined loss of two functionally similar regulators is needed to disrupt cell-cycle control. The resulting disruption of the cell cycle is often accompanied by failure of cellular differentiation in a celltype specific manner. For example, lens fiber cell differentiation is severely impaired in p27KIP1 and p57KIP2 doubly deficient mice ; and failure of myogenesis is observed in p21CIP1 and p57KIP2 doubly null mice . These results demonstrate that certain differentiation decisions are not compatible with continued proliferation. However, studies of other CKI-deficient mice also provide evidence that cell proliferation can be uncoupled from differentiation (see discussion below). Even when differentiation occurs in the presence of inappropriate cell proliferation it may not be perfectly normal. For example, differentiated luteal cells (expressing P450scc) in p27KIP1 null mice were found to undergo active DNA synthesis  but they are functionally impaired such that the mice are infertile. Another frequent consequence of unscheduled cell proliferation in differentiated cells is cell death, resulting in no net increase in the number of differentiated cells [25••,26•]. Other experiments have demonstrated that cell-cycle arrest is not always sufficient for differentiation. Oligodendrocyte differentiation provides a good example in this respect. p27KIP1 protein levels accumulate during the course of oligodendrocyte precusor proliferation and correlates with the onset of differentiation . Precusor cells derived from p27KIP1 null mice undertake one or two extra divisions before initiating differentiation in vitro, indicating that p27KIP1 controls the timing of cell-cycle withdrawal during differentiation. Nevertheless, forced expression of p27KIP1 in these oligodendrocyte precursors did not induce differentiation but it did induce G1 cell-cycle arrest [28,29]. In the Xenopus retina, differentiation of retinoblasts to Müller glial cells can be induced by overexpression of p27Xic1, a homolog of human p27KIP1, yet induction of cellcycle arrest by the amino-terminal cyclin/Cdk binding domain of p27KIP1 cannot induce differentiation, indicating that, in this system, inhibition of cell proliferation per se is not sufficient for differentiation [30••]. Together, these results demonstrate that, in most cases, control of proliferation and differentiation are highly coordinated, although these two processes can certainly be uncoupled in certain instances. It is clear that cell-cycle regulators can have a strong influence on differentiation
decisions. Recent studies discussed in the next section indicate that these differentiation-promoting functions may be separate from their cell cycle regulatory properties.
Coordinating cell proliferation and differentiation through dual-function cell-cycle regulators Recent studies have revealed the existence of dual-function regulators that can participate in controlling both cell proliferation and differentiation. Certain cell-cycle regulators can directly regulate differentiation and certain differentiation-promoting transcription factors can directly regulate cell proliferation. These dual-function regulators provide attractive mechanisms for coordinating cell proliferation and differentiation. Cdks phosphorylate a number of substrates to promote cell-cycle progression. It is now believed that they also regulate the function of transcription factors that promote differentiation. In muscle-cell differentiation, cyclin-D1dependent kinases can inhibit MyoD activity through phosphorylation [9,11,12]. Physical interaction between MyoD and Cdk4 also inhibits MyoD activity in a phosphorylation-independent manner . Similarly, the kinase inhibitor p57KIP2 can stabilize MyoD by inhibiting cyclin-E/Cdk2-mediated phosphorylation of MyoD  or through direct physical interaction with MyoD [32•]. pRB is best known as a growth suppressor through its ability to repress the E2F transcription factor. pRB can also promote differentiation through cooperation with differentiation-promoting transcription factors such as MyoD and MEF2 [33,34], C/EBPs , and c-jun . As mentioned above, myogenesis can be promoted by ectopic expression of CKIs. This effect of CKIs however is lost in pRB null cells, supporting a role for pRB in myogenesis in addition to cell-cycle arrest . As pRB’s effects on G1 arrest and differentiation can be genetically separated through specific mutations , these two functions of pRB must be mediated by different effectors. How pRB performs these two functions is not known. Recent work shows that the pocket region of pRB, which is well known to bind many proteins, can bind simultaneously both histone deacetylases (HDACs) and hSWI/SNF, two chromatin-modifying activities [39••]. Moreover, phosphorylation of pRB by cyclin-D/Cdk4 abolishes only the pRB–HDAC interaction which relieves repression of only a subset of pRB target genes. As HDACs and hSWI/SNF are intimately involved in the regulation of gene expression, these results may form a framework for pRB to perform multiple, separate functions. MyoD, on the other hand, was initially identified as a differentiation factor, but later work has revealed its function in cell-cycle regulation. MyoD can induce cell-cycle arrest in the absence of myogenic differentiation [40,41]. MyoD transcriptionally activates p21CIP1 expression independently of p53 [42,43] and it can inactivate Cdk4 through physical interaction [44•].
Coordinating cell proliferation and differentiation Zhu and Skoultchi
Recent evidence indicates that certain CKIs may also have functions in both cell cycle regulation and differentiation. In Xenopus, Müller glial cell differentiation can be activated by p27Xic1, as described above. If p27Xic1 acted only as an inhibitor of cyclin-dependent kinases to induce differentiation, other means of arresting the cell cycle in G1 would also be expected to induce differentiation — but this is not the case. The gliogenic function of p27Xic1 is mediated by a portion of the amino-terminal domain near but distinct from the region required to inhibit Cdks [30••]. In this study, it was further shown that glial cell differentiation can be induced by a mutant of human p21CIP1 that cannot inhibit cyclin/Cdk activity. These results strongly suggest that CKIs have differentiation-promoting functions separate from their cell-cycle inhibitory functions. Studies with CKI-deficient mice also suggest roles for CKIs in differentiation independent of their roles in cell-cycle regulation. In p57KIP2 null mouse embryos, abnormalities in muscle, kidney, palate and chondrocyte development were observed in the absence of cell proliferation defects [45–47]. In the developing retina, p57KIP2 seems to play important roles at two different stages, only one of which is related to cell proliferation [26•]. Between embryonic days 14.5 and 16.5, p57KIP2 is upregulated in a subset of retinal progenitor cells and these cells show inappropriate S phase entry in p57KIP2 knockout mice. Between postnatal days two and ten, p57KIP2 is expressed in a subset of amacrine interneurons that are undergoing postmitotic differentiation into subtypes. In p57KIP2-deficient mice, a specific increase in the calbindinexpressing subpopulation of amacrine cells was observed in the absence of inappropriate S-phase entry. These latter observations suggest that p57KIP2 is playing a role in the differentiation of amacrine subtypes even when the cells are in a postmitotic state. In p57KIP2 and p21CIP1 doubly null mice, formation of lung alveoli is impaired, again in the absence of detectable disturbances in cell proliferation or apoptosis . In all these studies with CKI-deficient mice, however, it is not clear whether the observed effects on differentiation resulting from loss of CKIs are dependent upon their inhibitory functions on Cdks or on separate functions of these molecules. It is possible that increases in Cdk activity promoted by the absence of these inhibitors is not sufficient to cause cell-cycle defects but it is nevertheless able to perturb differentiation. The structures of these CKIs can certainly accommodate other functions in addition to inhibition of Cdks. Whereas p21CIP1, p27KIP1, and p57KIP2 all have conserved amino-terminal Cdk-inhibitory domains, their carboxyl termini are different and may interact with other proteins (for p21CIP1, the carboxyl terminus contains the PCNA-interacting domain). Identification of novel functions of CKIs will greatly improve our understanding of the mechanisms that coordinate cell proliferation and differentiation.
Maintenance of cell-cycle arrest in terminally differentiated cells A topic of both fundamental and potential practical importance is the mechanisms that maintain terminally
differentiated cells in cell-cycle arrest. Can these mechanisms be reversed to make phenotypically mature cells divide beyond their normal capacity? If the answer is yes, it could have broad implications for cell-transplantation therapy and possibly even organ regeneration. Much evidence indicates that at least some mature cells are not completely locked in terminal arrest. The evidence comes from experiments in which cell-cycle regulators have been manipulated in differentiated cultured cells or in mice. Skeletal muscle has again served as an important model system for such experiments. Terminally differentiated, multinucleated myotubes are unable to re-enter S phase when growth factors are restored, although they can reexpress certain early G1 genes . Early studies (reviewed in [49•]) showed that expression of viral oncoproteins known to inactivate pRB and its family members, p107 and p130, caused growth-factor-dependent induction of DNA synthesis in terminally differentiated muscle cells, pointing to a role for pRB family proteins in the maintenance of terminal arrest in these cells. Results from inactivation of pRB in mice support these early conclusions. Although mouse embryos deficient in pRB have apparently normal skeletal muscle [50–52], RB–/– myotubes, or RB–/– mouse embryonic fibroblasts driven to differentiate into myocytes by ectopic MyoD expression, respond abnormally to growth-factor stimulation by entering S phase [37,53]. Lack of p107 or p130 does not lead to inappropriate S phase entry, although ectopic expression of p107 or p130 can partially block the response in RB–/– myocytes . The pRB protein family may also control terminal arrest in neuronal cells. Expression of a mutant SV40 T antigen — which can bind to the pRB protein family but not to p300 or p53 — caused inappropriate DNA synthesis, followed by cell death, in fully differentiated neuronal cells in vivo . However, this effect was not observed with a similar adenovirus E1A mutant in vitro in cultured cortical neurons . Together these results suggest that pRB, and possibly other pocket proteins, participate in maintaining terminal arrest in several cell types. The mechanisms by which pRB and other pocket proteins help to keep differentiated cells from cycling remain obscure. Given the importance attached at present to pRBmediated inhibition of E2Fs in cycling cells, one might think that they are logical targets in differentiated cells. In this case, overexpression of E2Fs would be expected to induce cycling in differentiated cells. There are conflicting reports as to whether or not E2F1 is capable of inducing DNA synthesis in differentiated C2 muscle cells [14,56]. A recent report  indicates that overexpression of E2F1 (or E2F4) can induce DNA synthesis in L6-derived myotubes but very high levels of expression are required, resulting in forced nuclear localization of E2F1 which is primarily cytoplasmic in untransfected myotubes. It is not clear at present what the relevant targets of pRB (or other pocket proteins) are in terminally arrested cells. pRB may act in conjunction with transcription factors in differentiated cells to either repress synthesis of proteins needed for cell cycling or to
Oncogenes and cell proliferation
Figure 1 Extracellular signals
? p16, p15, p18, p19
p21, p27, p57
Cyclin-E/Cdk Other proliferation genes
Differentiation-promoting transcription factors
Cell differentiation genes
Current Opinion in Genetics & Development
Extracellular signals are believed to play a major role in a cell’s decision to either proliferate or differentiate, two inversely correlated processes. Cyclin-D-dependent kinases relay proliferative signals by initiating phosphorylation of pRB, which leads to the relief of E2F repression. The positive feedback loop among pRB, E2F, and cyclin-E/Cdk2 facilitates traversal through the restriction point in late G1, after which cells are committed to further cell-cycle progression. As pRB also plays an important role in promoting differentiation, the inactivation of pRB by phosphorylation simultaneously disables the cellular differentiation program. Cells may also rely on an intrinsic clock mechanism that initiates differentiation at a predetermined time. The kinase inhibitor p27KIP1, an important effector of this clock mechanism, inhibits cell proliferation by repressing the activity of cyclin-E/Cdk2. Recent evidence suggests that p27KIP1, and related CKIs, also have separate functions that promote differentiation. Similarly, differentiationpromoting transcription factors can induce the synthesis of CKIs, thereby facilitating terminal arrest in differentiated cells. A central concept of the current model is that previously established regulators of cell proliferation or differentiation may have dual functions. Regulators with functions in both proliferation and differentiation provide a logical mechanism for achieving coordinated regulation of cell proliferation and differentiation.
stimulate transcription of genes encoding other cell-cycle brakes (e.g. CKIs), or both. A recent study suggested that pRB can also inhibit DNA replication in cycling cells by targeting MCM7 (minichromosome maintenance 7), a component of the DNA replication initiation complex .
compartments of mice in which one or two CKI genes have been inactivated. Using BrdU incorporation as a marker of S phase, Zhang et al.  found that mouse embryos lacking both p27KIP1 and p57KIP2 had dramatically increased levels of ectopic DNA synthesis in normally postmitotic lens fiber cells. Likewise, although embryos deficient for both p21CIP1 and p57KIP2 are severely impaired in muscle differentiation, Zhang et al.  found that residual myotubes incorporate BrdU, which is never seen in wild-type or the single mutant embryos. Of course, studies of BrdU incorporation do not indicate whether such highly differentiated cells can actually undergo cell division. However, Zindy et al. [25••] have shown by staining for phosphorylated histone H3 — a marker for G2 and M phases — and by observing BrdU-positive mitotic figures and cell doublets, that bona fide central nervous system neurons in mice deficient for p19INK4d and p27KIP1 not only enter S phase but also progress through G2 and M and undergo cytokinesis. Similarly, hair cells and supporting cells in the body of Corti in postnatal and adult p27KIP1 null mice were found to undergo DNA synthesis and cell division, whereas these cells normally cease dividing during embryogenesis [59•,60•]. It is therefore clear that at least some terminally differentiated cells retain the capacity for cell division and that pRB family proteins and CKIs are important regulators of terminal arrest. Are CKIs working here simply to activate pRB and other pocket proteins? The much higher number of lens fiber cells exhibiting ectopic DNA synthesis in p27KIP1 and p57KIP2 doubly deficient mutants compared with RB–/– lenses  suggests that pRB is not the only target of these CKIs. A recent study by Mal et al. [61••] also reveals that the ability of adenovirus E1A to activate DNA synthesis in differentiated muscle cells is not simply based on targeting pRB; E1A also needs to bind to and inactivate p21CIP1, indicating that both p21CIP1 and pRB are required to prevent S-phase re-entry in these cells. CKIs play a major role both in determining the number of terminal cell divisions and in maintenance of the final arrest. If their functions can be reversed, what is the limit to the number of terminally differentiated cells that can be produced? Recently, Matushansky et al.  demonstrated that the final cell divisions of cultured erythroid cells committed to terminal differentiation could be greatly extended by ectopic expression of both Cdk2 and a CKIresistant mutant of Cdk4. Mature, nucleated red cell number was increased greater than 100-fold without affecting hemoglobin levels. Interestingly, a CKI-resistant mutant of Cdk6 could not substitute for the mutant Cdk4, suggesting a functional distinction between these two highly related Cdks in differentiated hematopoietic cells.
Conclusions Many studies have demonstrated inappropriate DNA synthesis in diverse, highly differentiated cellular
Significant progress in the study of the cell cycle has allowed a deeper understanding of the inverse relationship
Coordinating cell proliferation and differentiation Zhu and Skoultchi
between cellular differentiation and cell proliferation. The ability to manipulate cell-cycle regulation through various techniques, particularly in vivo analysis with genetically engineered mice, has revealed that the two processes can be dissociated to some degree. Recent studies have also provided an important insight into a mechanism for coordinating cell proliferation and differentiation. Regulators at the center of both processes appear to have dual functions, allowing them to promote cell-cycle exit as well as cell differentiation (see Figure 1). Further characterization of the mechanisms utilized by these regulators will play a major part in the construction of a more detailed network connecting cell-cycle regulation with differentiation. Although much remains to be learned about the precise mechanisms that maintain terminally differentiated cells in proliferative quiescence, it is now clear that the capacity for further, perhaps extensive, cell division exists in many terminally differentiated cell types and their immediate precursors. Unlocking this potential in a cell type specific way, might someday lead to methods for regenerating differentiated cells to replace counterparts lost by degeneration or injury. A major obstacle to be overcome in this regard is apoptosis in terminally differentiated cells induced to re-enter the cell cycle. Equally important, safeguards will be needed to prevent the generation of cells that escape proliferation control to form tumors. Certain species have the ability to regenerate lost tissue; perhaps we can learn also.
Update Since the submission of the first draft of this review, a paper of special interest has been published [63•].
Acknowledgements The authors’ laboratories are supported by National Institutes of Health grants (L Zhu and AI Skoultchi), and grants from US Army and the American Cancer Society (L Zhu). L Zhu is a Leukemia & Lymphoma Society Scholar.
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38. Sellers WR, Novitch BG, Miyake S, Heith A, Otterson GA, Kaye FJ, Lassar AB, Kaelin WGJ, Sellers WR: Stable binding to E2F is not required for the retinoblastoma protein to activate transcription, promote differentiation, and suppress tumor cell growth. Genes Dev 1998, 12:95-106. 39. Zhang HS, Gavin M, Dahiya A, Postigo AA, Ma D, Luo RX, •• Harbour JW, Dean DC: Exit from G1 and S phase of the cell cycle is regulated by repressor complexes containing HDAC-RbhSWI/SNF and Rb-hSWI/SNF. Cell 2000, 101:79-89. This paper provides a mechanistic basis for differential regulation of different sets of genes by pRB. It is shown that an HDAC–pRB–hSWI/SNF complex and a separate pRB–hSWI/SNF complex are required for repression of different genes involved in controlling different stages of the cell cycle. Furthermore, phosphorylation of pRB by cyclin-D/Cdk4 selectively inhibits its interaction with HDAC. The results might also help explain how pRB can perform dual functions in cell proliferation and differentiation. 40. Sorrentino V, Pepperkok R, Davis RL, Ansorge W, Philipson L: Cell proliferation inhibited by MyoD1 independently of myogenic differentiaiton. Nature 1990, 345:813-815.
Takahashi K, Nakayama K, Nakayama K: Mice lacking a CDK inhibitor, p57Kip2, exhibit skeletal abnormalities and growth retardation. J Biochem 2000, 127:73-83.
51. Jacks T, Fazeli A, Schmitt E, Bronson R, Goodell M, Weinberg R: Effects of an Rb mutation in the mouse. Nature 1992, 359:295-300. 52. Lee EY-HP, Chang C-Y, Hu N, Wang Y-CJ, Lai C-C, Herrup K, Lee W-H, Bradley A: Mice deficient for Rb are nonviable and show defects in neurogenesis and haematopoiesis. Nature 1992, 359:288-294. 53. Schneider JW, Gu W, Zhu L, Mahdavi V, Nadal-Ginard B: Reversal of terminal differentiation mediated by p107 in Rb-//- muscle cells. Science 1994, 264:1467-1471. 54. Feddersen RM, Clark HB, Yunis WS, Orr HT: In vivo viability of postmitotic Purkinje neurons requires pRb family member function. Mol Cell Neurosci 1995, 6:153-167. 55. Slack RS, El-Bizri H, Wong J, Belliveau DJ, Miller FD: A critical temporal requirement for the retinoblastoma protein family during neuronal determination. J Cell Biol 1998, 140:1497-1509. 56. Pajalunga D, Tognozzi D, Tiainen M, D’Angelo M, Ferrantelli F, Helin K, Sacchi A, Crescenzi M: E2F activates late-G1 events but cannot replace E1A in inducing S phase in terminally differentiated skeletal muscle cells. Cell Growth Differ 1999, 18:5054-5062. 57.
Gill RM, Hamel PA: Subcellular compartmentalization of E2F family members is required for maintenance of the postmitotic state in terminally differentiated muscle. J Cell Biol 2000, 148:1187-1201.
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Coordinating cell proliferation and differentiation Zhu and Skoultchi
59. Lowenheim H, Furness DN, Kil J, Zinn C, Gultig K, Fero ML, Frost D, • Gummer AW, Roberts JM, Rubel EW et al.: Gene disruption of p27(Kip1) allows cell proliferation in the postnatal and adult organ of corti. Proc Natl Acad Sci USA 1999, 96:4084-4088. The highly differentiated hair and supporting cells of the organ of Corti in the developing inner ear undergo terminal arrest during embryogenesis and have never been observed to divide postnatally in mammals. Together with [60•] this paper shows that loss of p27KIP1 results in proliferation of these cells in adult mice. Hearing loss often results from hair-cell degeneration. These studies suggest that it may be possible to find ways to regenerate these cells. 60. Chen P, Segil N: p27(Kip1) links cell proliferation to • morphogenesis in the developing organ of Corti. Development 1999, 126:1581-1590. See annotation [59•]. 61. Mal A, Chattopadhyay D, Ghosh MK, Poon RY, Hunter T, Harter ML: •• p21 and retinoblastoma protein control the absence of DNA replication in terminally differentiated muscle cells. J Cell Biol 2000, 149:281-292. Several different types of studies have indicated pRB’s role in the block to reentry into S phase in terminally differentiated muscle cells. p21CIP1 is induced during muscle differentiation and as it can activate pRB, it seems logical that p21CIP1 is acting through pRB to prevent cycling in these cells. However, this study indicates there is more to the story. Adenovirus E1A, which has been known for years to bind to and inactivate pRB and to induce DNA synthesis in
differentiated muscle cells, is shown here to also bind p21CIP1 and prevent it from inhibiting cyclin E/Cdk2 kinase activity. Moreover, a mutant E1A (E1A.dl236), which binds pRB but not p21CIP1, fails to induce DNA synthesis in myotubes. Therefore, both pRB and p21CIP1 are needed to prevent re-entry into S phase in muscle cells. p21CIP1 may be acting both directly to inhibit DNA replication through its PCNA-binding function, as well as indirectly by preventing cyclinE/Cdk2 complexes from phosphorylating proteins involved in replication. 62. Matushansky I, Radparvar F, Skoultchi AI: Manipulating the onset of cell cycle withdrawal in differentiated erythroid cells with cyclindependent kinases and inhibitors. Blood 2000, 96:2755-2764. 63. Lasorella A, Noseda M, Beyna M, Iavarone A: Id2 is a retinoblastoma • protein target and mediates signalling by Myc oncoproteins. Nature 2000, 407:592-598. Id proteins inhibit differentiation through direct interaction with and dominant negative interference of basic helix-loop-helix differentiation promoting transcription factors. One member of this family, Id-2, has been implicated as a dual function regulator because in addition to its differentiation inhibiting functions, when overexpressed, it can also oppose the antiproliferative actions of the pRB pocket proteins through direct physical interaction. This recent paper now provides a genetic connection between Id-2 and pRB by showing that loss of Id-2 can rescue the neuronal and hematopoietic differentiation defects present in RB–/– mouse embryos. It also provides biochemical and genetic evidence strongly implicating the proliferation-promoting function of Id-2 in the formation of neuroblastomas with N-Myc amplification.