Id Proteins in Cell Cycle Control and Cellular Senescence

Id proteins in cell cycle control and cellular senescence

Zoe Zebedee1 and Eiji Hara*,1,2

1CRC Cell Cycle Group, Paterson Institute for Cancer Research, Christie Hospital NHS Trust, Wilmslow Road, Manchester M20 4BX, UK; 2Department of Biomolecular Sciences, University of Manchester Institute of Science & Technology (UMIST), Manchester M60 1QD, UK

The Id family of helix ± loop ± helix (HLH) proteins are tously expressed (Murre et al., 1989b; Henthorn et thought to aect the balance between cell growth and al., 1990; Zhang et al., 1991; Hu et al., 1992; dierentiation by negatively regulating the function of Skerjanc et al., 1996; Massari and Murre, 2000) basic ± helix ± loop ± helix (bHLH) transcription factors. whereas class B bHLH proteins, which include Although it has been suggested for some time that Id is members such as MyoD, myogenin, NeuroD/BETA2, involved in cell cycle regulation, little is known about the MASH, HAND and TAL, show a tissue-restricted molecular mechanism of this control. Recent studies, pattern of expression. Dimerization is essential for however, have revealed that Id binds to important cell DNA binding and transcriptional activity in vivo cycle regulatory proteins other than bHLH proteins. (Lassar et al., 1991; Massari and Murre, 2000) and in Two such proteins, pRB (retinoblastoma tumour sup- general, class B bHLH proteins form heterodimers pressor protein) family proteins and Ets-family transcrip- with the class A bHLH proteins with few exception, tion factors are known to play key roles in cell cycle although the latter can also operate as homodimers regulation, transformation and tumour suppression. (Shen and Kadesch, 1995). The basic region of each Through the characterization of these pathways we will protein is required for binding to DNA, commonly begin to understand the mechanisms by which Id controls to a region that includes a speci®c sequence motif normal and abnormal cell cycle progression. Oncogene known as the E-box (CANNTG) (Ephrussi et al., (2001) 20, 8317 ± 8325 1985; Kiledjian et al., 1988; Lassar et al., 1989) or the related N-box (CACNAG) (Klambt et al., 1989; Keywords: Id; cell cycle; senescence; Ets; CDKs; Tietze et al., 1992). p16INK4a Id is another category of mammalian HLH protein, which was ®rst identi®ed by Benezra et al. in 1990 during a screen for determination factors Introduction belonging to the bHLH protein family. So far four members of the Id family, Id1 to Id4, have been The basic ± helix ± loop ± helix (bHLH) family of identi®ed in mammalian cells (Benezra et al., 1990; transcription factors have been shown to play a key Christy et al., 1991; Sun et al., 1991; Biggs et al., role in the dierentiation of a number of cell 1992; Ellmeier et al., 1992; Deed et al., 1993; Hara lineages, including B- and T-lymphocytes, muscle et al., 1994; Riechmann et al., 1994; Pagliuca et al., cells, pancreatic b cells, neurons and osteoblasts 1995), apart from its functional homologue, OUT (reviewed in Jan and Jan, 1993; Weintraub, 1993; (Narumi et al., 2000). These proteins do not contain Olson and Klein, 1994: Massari and Murre, 2000). the basic region adjacent to the HLH domain that is This family of proteins generally contains a helix ± essential for speci®c DNA binding in other bHLH loop ± helix (HLH) dimerization domain, consisting of proteins. Id proteins bind to both class-A and class- highly conserved amphipathic helices separated by a B bHLH proteins and inhibit their ability to bind loop of variable length and sequence, and an DNA as homodimeric or heterodimeric complexes. adjacent DNA-binding domain which is rich in basic Because of this activity, these proteins were named amino acids (Murre et al., 1989a; Davis et al., 1990; as Id (inhibitor of DNA binding). In addition to Ellenberger et al., 1994; Ma et al., 1994). There are this ability to inhibit DNA binding, the name Id is two main categories of bHLH proteins. The class A often taken to re¯ect the potential role of these bHLH, also known as the E proteins, such as those proteins in inhibiting dierentiation. For example, encoded by dierentially spliced transcripts from the the expression of Id genes is down-regulated upon E2A (E12, E47 and E2-5/ITF1 proteins), E2-2/ITF2 dierentiation in many cell types (Benezra et al., and HEB/HTF4 genes and Daughterless are ubiqui- 1990; Hara et al., 1991a; Sun et al., 1991; Kreider et al., 1992; Le Jossic et al., 1994; Einarson and Chao, 1995). Conversely, ectopic expression of Id1 inhibits B-cell development (Sun, 1994) and the dierentia- *Correspondence: E Hara, Paterson Institute for Cancer Research, Christie Hospital NHS Trust, Wilmslow Road, Manchester M20 tion of muscle (Jen et al., 1992), myeloid (Kreider et 4BX, UK; E-mail: [email protected] al., 1992), erythroid (Shoji et al., 1994; Lister et al., Id and the cell cycle Z Zebedee and E Hara 8318 1995) and mammary epithelial cells (Desprez et al., Id binding to factors involved in cell cycle regulation 1995, 1998). In many cases, cellular dierentiation is accom- In addition to binding to members of the bHLH panied by cell cycle arrest. It is likely therefore that family, Id proteins have also been shown to associate since Id is able to inhibit dierentiation, this family with other factors involved in cell cycle regulation. A of proteins may also be involved in cell cycle key player regulating the G0 to S phase transition is regulation. Indeed, several lines of evidence suggest the retinoblastoma tumour suppressor protein (pRB) that Id proteins play a role in the G0 to S phase as well as the RB family members, p107 and p130 transition of the cell cycle. The stimulation of (Weinberg, 1995; Mulligan and Jacks, 1998; Vooijsand quiescent ®broblasts with serum or growth factors, and Berns, 1999). The activity of pRB-family proteins for example, induce the expression of Id1, Id2 and are mainly regulated by a series of cyclin-dependent Id3 (Christy et al., 1991; Deed et al., 1993; Hara et kinases (CDKs), including CDK4 and CDK6, acting in al., 1994; Barone et al., 1994). Moreover, inhibiting conjunction with their regulatory partners, cyclins D1, Id protein synthesis by antisense oligonucleotides D2 and D3, and CDK2 in conjunction with cyclins E prevents the re-entry of G0-arrested ®broblasts into and A (reviewed in Sherr, 1994, 1996; Sherr and the cell cycle (Hara et al., 1994; Barone et al., 1994). Roberts, 1999). The current view is that CDK4 and/or This evidence strongly implicates the Id proteins in CDK6 initiate phosphorylation of the C-terminal cell cycle control. However, the role that each Id region of pRB, leading to successive intramolecular member plays in this regulation has been poorly interactions that initially displace histone deacetylase understood. Recent studies have highlighted some of (HDAC) from the pocket domain, blocking active the mechanisms by which Id proteins are able to transcriptional repression by pRB. This facilitates a modulate the cell cycle. In this review, we will discuss second interaction that leads to phosphorylation and possible mechanisms of cell cycle regulation by Id subsequent disruption of the pocket domain by CDK2, based on these studies. preventing pRB from binding to and inactivating E2F transcription factor whose activity is required for entry to S phase (Figure 2) (Harbour et al., 1999). As critical Id expression in cell cycle integrators of mitogenic-signalling pathways, activities of CDKs are regulated at multiple levels, including the Proliferating cells express at least one, or more synthesis and stability of individual components, the commonly multiple, Id genes. In general, expression assembly of functional complexes, post-transcriptional level of Id genes is high in proliferating cells, and is modi®cation and binding to CDK inhibitors (CKIs) low or absent in non-proliferating cells such as (Sherr and Roberts, 1999; Sugimoto et al., 1999). There terminally dierentiated cells with few exceptions are two dierent classes of CKI, the Kip/Cip family (Norton et al., 1998; Norton, 2000). Indeed, Id3 (p21cip1, p27Kip1 and p57kip2) and the INK4 family (HLH462) was originally identi®ed as a delayed early (p16INK4a, p15INK4b, p18INK4c and p19INK4d). In contrast response gene whose expression was induced following to the Kip/Cip family, which inhibits a broad range of mitogenic stimulation in mouse BALB/c 3T3 ®bro- CDKs, the INK4 family protein speci®cally binds to blasts (Christy et al., 1991). The expression of both and inactivates CDK4 and CDK6 (Serrano et al., 1993; Id1 and Id2 transcripts was also observed to rapidly Ruas and Peters, 1998; Sherr and Roberts, 1999). increase (within 1 ± 2 h) following mitogenic stimula- Iavarone et al. (1994) and Lasorella et al. (1996) tion of serum starved primary human ®broblasts have shown that Id2 but not Id1 or Id3 are able to (Hara et al., 1994). Following an initial decline, the bind to pRB and abolish its growth-suppressing expression level of both Id1 and Id2 were further up- activity. In-vitro analysis of the binding of Id2 and regulated as cells progressed through G1 and into the pRB indicated that the binding is dependant on the S phase of the cell cycle, suggesting that both Id1 and HLH domain of Id2, and the pocket domain of pRB Id2 may have bi-functional roles in the G0 to S phase (Lasorella et al., 1996). Growth arrest by pRB was not transition in the cell cycle (Hara et al., 1994). It is antagonized in transfection studies using Id2 lacking interesting to note that Id2 and Id3 are phosphory- the HLH domain. In addition to pRB binding, Id2 has lated by cyclin-dependent kinase 2 (CDK2) in late G1 been shown to associate with p107 and p130 both in- (Hara et al., 1997; Deed et al., 1997), implying that vitro, and in in-vivo transfection experiments (Lasorella phosphorylation of Id2/3 may alter their activity and et al., 1996). Although the HLH region is well perhaps allow Id2/3 to bind to other proteins (Figure conserved between Id family members, only the HLH 1). In addition to cell cycle-linked phosphorylation, Id domain of Id2 was shown to interact with p107 and proteins undergo rapid turnover during the cell cycle p130. Evidence suggesting that Id2 but neither Id1 nor with its levels being regulated by the ubiquitin- Id3 bind to pRB and related proteins implies a distinct proteosome degradation pathway (Bounpheng et al., functional role which may also be underlined by the 1999). Thus Id is regulated at multiple levels, i.e. expression pattern observed for Id2 in cells. The ®rst transcriptional regulation, protein stability and post- increase of Id2 gene expression is observed following translational modi®cation. So far a correlation mitogenic stimulation of quiescent cells. It is possible between Id4 expression and cell growth has not been that Id2 in this circumstance may have in¯uence over identi®ed. the activity of p130-E2F4/5 complexes, which are

Oncogene Id and the cell cycle Z Zebedee and E Hara 8319

Figure 1 Level of expression of a variety of proteins involved in regulation of the cell cycle. Expression levels representing both Id1 and Id2 are shown

Figure 2 Regulation of G1 to S transition by pRb and E2F. Following mitogenic stimulation, Cyclins D and E in complex with CDK4/6 or CDK2 respectively, sequentially phosphorylate pRb thus releasing it from its transcriptionally repressive complex with E2F facilitating S phase progression. The roles of the CDK inhibitors of the INK4 and Cip/Kip family are indicated. Interactions between a number of components of this pathway and Id family proteins are also shown

believed to be inhibitory complexes for cell growth in (Nevins, 1998; Harbour and Dean, 2000; Humbert et G0 phase (Gaubatz et al., 2000; Bruce et al., 2000; al., 2000). Disruption of this complex by Id2 would Hansen et al., 2001). The second peak of Id2 release the E2F1-3 from pRB/p107 and facilitate the expression occurs at late G1, where E2F1-3 are actively gene expression required for progression of cells to S repressed by interaction with pRB and/or p107 phase. These models, however, can not be applied to

Oncogene Id and the cell cycle Z Zebedee and E Hara 8320 Id1 and Id3. It is therefore clear that Id1 and Id3 through a conserved 15 amino acids domain on the regulate the cell cycle through dierent mechanisms. exterior of the bHLH region (Zhang et al., 1999). Recently, Lasorella et al. (2000) reported the genetic Thus, MyoD acts as a CDK inhibitor through two interaction between Rb and Id2 during development independent mechanisms, ®rstly by directly binding to using intercrossed Rb and Id2 mutant mice. In CDK4 and secondly by inducing p21Cip1 gene addition, Lasorella et al. (2000) discovered that Id2 is expression. a transcriptional target of both N-myc and c-myc, Id 1 ± 3, have also been shown to bind to other suggesting that Myc transcription factors increase factors which control the response of cells to mitogenic expression of Id2 to bypass the pRB block and drive stimulation, such as ternary complex factors (TCFs) progression of G1 to S phase transition. But if Id2 is (Yates et al., 1999). These factors, which are a sub- simply restraining RB, then why is the RB null rescued family of ETS domain proteins and include factors by loss of Id2? There must be another component such as Elk-1, SAP-1 and SAP2, are involved in the which could be the mechanism described below, i.e. regulation of expression of immediate early genes such that loss of RB leads to too much Id2 which sequesters as c-fos and egr-1 following mitogenic stimulation or as E proteins and other factors leading to increased CDK a response to stress. Binding of Id to the ETS DNA activity. binding domain of these factors, disrupts the binding In addition to binding directly to pRB in order to of SRF and TCF complexes to the serum responsible dissociate it from the E2F/DP1 complex, Id proteins element (SRE) thus inhibiting transcription of TCF may aect the activity of pRB in a dierent manner. responsive genes. Interestingly, Id1 is under the It has been suggested that the ubiquitous bHLH transcriptional control of EGR-1 (Tournay and protein E2A has the ability to block G1 to S phase Benezra, 1996), suggesting a negative feed-back loop transition at least through the transcriptional activa- which may regulate the response of cells to mitogenic tion of the p21Cip1 gene (Peverali et al., 1994; Prabhu stimulation by setting the threshold level for Id et al., 1997). Id1 and possibly other members of the Id expression. In addition, Id1 has been shown to bind family, are able to inhibit E2A mediated expression of to Ets1 and Ets2, regulating expression of the p16INK4a the p21Cip1 gene (Prabhu et al., 1997). p21Cip1 binds to gene (Ohtani et al., 2001). This pathway will be further and inactivates CDKs and enhances the activity of discussed below. pRB, thereby blocking G1 to S phase transition. Therefore, it is possible that Id proteins mediate G1 Id and cellular senescence to S phase transition through the downregulation of p21Cip1 gene expression by heterodimerization with In contrast to germ line cells and certain stem cells, E2A. Although it is still not clear whether E2A most somatic cells permanently stop dividing after a functions as a homodimer or heterodimer in the ®nite number of cell divisions in culture and enter a regulation of p21Cip1 transcription, it has been state termed cellular or replicative senescence. These reported that overexpression of MyoD also induces cells are irreversibly arrested in the G1 phase of the p21Cip1 gene expression (Halevy et al., 1995; Parker et cell cycle and are no longer able to grow despite al., 1995). Therefore, it is likely that E2A hetero- remaining viable and metabolically active for long dimerizes with MyoD in order to regulate p21Cip1 gene periods of time, thereby distinguishing, senescence expression at least in myogenic dierentiation and from quiescence or programmed cell death (apoptosis) perhaps other class B bHLH proteins in other (Campisi et al., 1996). The ®nite replicative lifespan of lineages. Id proteins have been shown to antagonize cells was ®rst discovered 40 years ago by Hay¯ick and the activity of the class A and class B heterodimers Moorhead (1961), and cellular senescence is often which could therefore lead to the downregulation of termed the `Hay¯ick limit'. Most tumours contain p21 Cip1 and activation of the CDKs in preparation cells that appear to have bypassed this limit and for RB phosphorylation and cell cycle transit. It has evaded senescence. Immortality, or even an extended been reported that there are two CDK phosphoryla- replicative lifespan, greatly increases the susceptibility tion sites, Ser-48 and Ser-154, within the N-terminal to malignant progression because it permits the domain of E2A. Ser-154 is preferentially phosphory- extensive cell divisions needed to acquire successive lated by cyclinD1-CDK4, whereas Ser-48 is preferen- mutations. In this sense, cellular senescence could act tially phosphorylated by cyclinA-CDK2 or cyclinE- as a barrier to cancer and play an important role in CDK2. Although the growth suppressive activity of tumour suppression (Figure 3). It could also be a E2A is negatively regulated by phosphorylation of fundamental factor involved in the ageing process. A Ser-154 by cyclinD1-CDK4, phosphorylation of these number of hypotheses have been proposed to explain sites are not required for inhibition of E2A transcrip- the mechanisms of cellular senescence, and they can tional activity by CDKs (Chu and Kohtz, 2001). be grouped into two broad categories. One set These results suggest that the growth suppressor and proposes that the loss of proliferative potential is transcriptional activator functions of E2A may be due to random accumulation of damage or stress, for distinct. It is therefore interesting to examine whether example through inappropriate culture conditions or not this can be applied to the regulation of p21Cip1 (Sherr and DePinho, 2000; Tang et al., 2001; Mathon expression by E12/E47. It is also interesting to note et al., 2001). The other set proposes that genetically that MyoD directly binds to and inhibits CDK4 programmed processes, such as those observed in

Oncogene Id and the cell cycle Z Zebedee and E Hara 8321

Figure 3 Model of cellular senescence. Human cells has at least two dierent barriers to cancer. One is the Hay¯ick limit which is induced by a variety of stress. The other is the telomere limit which is induced by telomere shortening. PDL: Population doubling level dierentiation, result in cellular senescence (Shay and (such as mitogenic stress), than by telomere shortening. Wright, 2000). Although much evidence suggests that This is because accumulation of p16INK4A is also cellular senescence in human cells is genetically observed in rodent senescent cells where we cannot controlled by a cell-division counting mechanism observe detectable telomere shortening (Palmero et al., called telomere shortening, recent reports strongly 1997; Zindy et al., 1997). In addition, Serrano et al. suggest that it can also be induced by physiological (1997) have shown that aberrant growth signals provided stress (Artandi and DePinho, 2000; Wright and Shay, by oncogenic Ras induce p16INK4A gene expression 2000; Sherr and DePinho, 2000; Lundberg et al., without telomere shortening in both human and rodent 2000). Both types of mechanisms will likely play a role cells. The introduction of oncogenic Ras into normal in cellular senescence. primary cells results in the induction of various In both human and rodent cells, the pRB and p53 antiproliferative proteins, including the p16INK4A and tumour suppressor proteins are crucial gatekeepers of p53 tumour suppressors; the accompanying cell cycle senescence (Campisi et al., 1996; Carnero et al., 2000). As arrest resembles cellular senescence and is termed mentioned earlier, the activities of RB are highly `premature senescence'. Normal cells must therefore regulated by CDK4, CDK6 and CDK2. The loss of have a sensor that detects aberrant growth signals such growth potential is accompanied by elevated expression as oncogene activation, and that may induce p16INK4A. of p16INK4a in human senescent cells (Hara et al., 1996a; In rodent cells, inactivation of either the p16 INK4A/pRB Alcorta et al., 1996; Loughran et al., 1996; Rezniko et pathway or p53 pathway seems to be enough to override al., 1996) . Overexpression of p16INK4A causes activation both replicative senescence and premature senescence of pRB, resulting in a senescent-like growth arrest (Lundberg et al., 2000). In human cells, however, (McConnell et al., 1998; Kato et al., 1998). Furthermore, inactivation of both pathways is required (Shay et al., the p16INK4A gene, a recognized tumour suppressor gene, 1991; Hara et al., 1991b). Targeted inactivation of all of is frequently inactivated by a variety of mechanisms in a the pRB family genes (pRB, p107 and p130) immorta- wide range of human cancers. So how is p16INK4A lizes mouse cells despite the presence of high levels of expression induced in senescence? Although we can not p53, suggesting that pRB family proteins play a role completely exclude the connection between p16INK4A downstream of the p53-pathway in mouse cell senescence expression and telomere shortening, p16INK4A expression (Sage et al., 2000; Dannenberg et al., 2000; Peeper et al., is more likely to be regulated by physiological stress 2001). In human cell senescence on the other hand, p53

Oncogene Id and the cell cycle Z Zebedee and E Hara 8322 and pRB have overlapping, but distinct roles. Although transduction pathway as critical for Ras-induced it is still not clear whether or not senescence occurs in senescence (Lin et al., 1998; Zhu et al., 1998). Ohtani vivo, it is clear that senescence is the ®nal common state et al. (2001) have shown that p16INK4A expression is of a cell provoked by a variety of distinct physiological controlled by a balance between a positive factor, Ets1/ stimuli/stress. 2, and a negative factor, Id1. Normally, the Ets1/2 Loss of Id function has been linked to replicative transcription factor is phosphorylated and activated by senescence (Hara et al., 1994) and recent studies MAP kinase at a certain point in the cell cycle. suggest that Id, especially Id1, is implicated in the However, Id1 expression is also induced by EGR1 in regulation of cell senescence and immortalization response to MAP kinase signalling (Tournay and (Hara et al., 1996b; Alani et al., 1999; Nickolo et Benezra, 1996). Id1 therefore, counterbalances this al., 2000). Id1, for example, has been shown to activity by binding to and inactivating Ets1/2 function. cooperate with a pRB binding mutant of SV40 large However, oncogenic Ras/MAP kinase signalling con- T antigen to reactivate DNA synthesis in senescent stitutively phosphorylates and activates Ets1/2. This human ®broblasts (Hara et al., 1996b). The role of Id1 aberrant activation overrides the steady state and has also been shown to extend the replicative lifespan results in the induction of p16INK4A which causes of human keratinocytes (Alani et al., 1999; Nickolo et premature senescence. In normal replicative senescence, al., 2000). Alani et al. (1999) suggested that the ectopic which is provoked by cumulative cell divisions, expression of Id1, 2 and 3 extended the life-span of the expression levels of Ets1 increase whilst Id1 levels primary human keratinocytes, but Id1 had the most decline. Thus, Ets1 induces p16INK4A expression, aided signi®cant eect and immortalized them. Although by the concomitant down regulation of Id1 (Figure 4). Nickolo et al. (2000) failed to immortalize the In this way, the balance between Ets1/2 and Id1 seems primary human keratinocytes by overexpression of to act as a sensor that detects aberrant growth signals Id1, they also observed that overexpression of Id1 (mitogenic stress/oncogenic stress). These ideas are extended the lifespan of human keratinocytes. Both consistent with recent reports that mouse embryo reports showed increased level of phosphorylated, ®broblasts (MEFs) derived from Id1-de®cient mice inactive pRB in Id1 expressing cells (Alani et al., express high levels of p16INK4a (Lyden et al., 1999). It is 1999; Nickolo et al., 2000). Nickolo et al. (2000) clear, however, that it is important to determine also reported that the level of p16INK4a expression whether additional factors are involved in the regula- decreased following overexpression of Id1, underlining tion of p16INK4A. Indeed, it has been reported that bmi- the suggestion that Id1 regulates pRB activity through 1, JunB, 14-3-3s and LMP1 also regulate p16INK4A the regulation of p16INK4a expression. expression (Jacobs et al., 1999; Passegue and Wagner, 2000; Dellambra et al., 2000; Yang et al., 2000). Moreover, Id3 expression is induced by Ras-ERK A molecular sensor induces p16INK4A gene expression in MAP kinase cascade (Bain et al., 2001). It is therefore response to oncogenic mutation likely that the activity of Ets1/2 is regulated by several dierent factors. Understanding the mechanisms of Previous experiments with Ras mutants, which are p16INK4A regulation might help us to ®nd a way of unable to activate one or another eector pathway of controlling its expression and inducing senescence in Ras signalling, have identi®ed the MAP kinase signal cancer.

Figure 4 Model for p16INK4a induction: opposing eects of Ets and Id1. (a) Young cells express relatively low levels of p16INK4a because signalling via the predominant Ets protein, Ets2, is counterbalanced by Id1. (b) Oncogenic Ras over-rides this steady state via the phosphorylation of Ets1/2, resulting in induction of p16INK4a and premature senescence. (c) In senescent cells, signalling via the Ras/Raf/MEK pathway is attenuated and Ets2 levels are low. Here, the up-regulation of p16INK4a depends on the increased expression of Ets1, in the absence of any interference by Id1

Oncogene Id and the cell cycle Z Zebedee and E Hara 8323 Final comments regulation. Additionally, the dierences of binding speci®cities as well as regulation by phosphorylation In addition to the pRB- and Ets-family proteins, three also underline the dierences in function of each additional non-bHLH proteins, MIDA1 (Shoji et al., member. Further work is needed to de®ne pathways 1995; Inoue et al., 2000), Pax transcription factors which control expression of the Id proteins and to (Roberts et al., 2001) and adenovirus E1A protein identify target genes whose expression is regulated by (Nakajima et al., 1998) also interact with Id proteins. Id. Therefore, Id is likely to have dierent functions depending on the binding target. Much work remains to be done in order to fully understand the mechanisms Acknowledgements of action of each Id family protein in cell cycle control. We thank Drs G Peters, TJ Huot, N Ohtani, M Sugimoto, It is clear from evidence of overlapping expression I Palmero, M Serrano, J Campisi and N Jones for valuable during cell cycle progression, that each Id family discussion. This work was supported by United Kingdom member is responsible for dierent eects on growth Cancer Research Campaign.

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