Class Note for BME 510 at UA 2
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432 80 Mitelman F 2000 Recurrent chromosome aberra ons in cancer Mum Res 462 2477253 81 Elliott B andJay39n M 2002 Doublers and breaks and ansloca ons in cancer C511 Mo Life Sci 59 3737385 82 SachsR K eta 19971gtroximity effectsinthe produc on of chromosome aherran39ons by ionizing radiation 1m IRadi39at Biol 711719 TRENDS in Cell Biology Vol12 No9 September 2002 1397143 leukaemia Hum Genet 10057535 83 Savage J R andPapworth D G 1973 The relationship of radia onrinduced dicen ic yield to chromosome annnumber Mum Res 19 84 Lukasova E eta 1997Localisan39on and distance between ABLandBCR genes in interphasenuclei of honemarrow cells of control donors andpa ents with chronic myeloid 85 Neves H eta 1 999 The nuclear topography of ABL BCR PML and RARalplrra genes evidence for geneproximity in speci c phases of the cell cycle and stages ofhematopoie c differen a on Blood93 119771207 86 Nikiforova M N eta 2000Proximity of chromosomal loci that parn39cipatein radja onr induced rearrangements in human cells Science 290 1 3 e141 Signalling cell cycle and pluripotency in embryonic stem cells Tom BurdonAustin Smith and Pierre Savatier Pluripotent mouse embryonic stem ES cells can be expanded in large numbers in Vitro owing to a process of symmetrical selfrenewal Selfrenewal entails proliferation with a concomitant suppression of differentiation Here we describe how the cytokine leukaemia inhibitory factor LIF sustains selfrenewal through activation ofthetranscription factorSTAT3and how two othersignals extracellularsignalrelated kinase ERK and phosphatidylinositol30H kinase PI3K can influence differentiation and propagation respectively We relate these observations to the unusual cellcycle properties of ES cellsand speculate on the role ofthe cell cycle in maintaining pluripotency Tom Burden Deparlmenl of Gene Expressron and Developmenl Roslrn Inslrlule Roslrn Mrdlolhran UK EH25 9P5 Austin Smith Cenlrelor Genome Research Unrversrly ol Edrnburgh Krng s Burldrngs Wesl Marns Road Edrnburgh UK EH93JQ e marlauslrnsmrlh edacuk Pierre Savatier Ecole Normale Superreure deLyon Laboralorre de Brologre Molecularre el Cellularre CNRS UMR 5665 INRA LA913 46 allee d llaIIE 69364Ly0n Cedex07 France Mouse embryonic stem ES cells are the in vitro counterparts of an in Vivopopulation of cells known as the epiblast that are speci c to the early embryo 1 3 Epiblast cells are pluripotent which means that an individual cell can give rise to all cell types of the foetus ES cells retain the developmental identity and potential of the epiblast even after prolonged culture This has been shown conclusively by their complete integration into a developing embryo after being reintroduced into the blastocyst 4 ES cells can efficiently colonize the germ line resulting in chimaeric animals These produce functional gametes which allows ES cells to be used as vehicles for introducing sophisticated genetic modifications into mice 5 ES cells can also undergo multilineage differentiation in vitro and produce a range of well differentiated progeny 67 Currently there is considerable interest in the prospect of exploiting this potential in analogous human pluripotent cells 8 to generate specific differentiated types of cell for drug development for therapies based on cell replacement and for delivering gene therapies Les s attention has been paid to the unusual proliferative properties of ES cells 9 11 ES cells are derived without the intervention of any immortalizin g agent do not undergo either crisis or senescence and retain a diploid karyotype They proliferate without apparent limit 12 and can readily be propagated hll pIc hlrendsmm 0962 3924023 clonally They can multiply in the absence of serum and are not subject to contact inhibition or anchorage dependence In fact there is no known means of inducing cell cycle arrest and quiescence in ES cells Apart from the normal kary otype these are features that are typical of transformed cells and indeed ES cells are tumorigenic In contrast to their behaviour when introduced into the early embryo they produce teratocarcinomas when injected into adult mice Thus ES cells can be considered as conditional tumour cells Embryonic stem cells undergo symmetrical self renewal that is they produce two identical stem cell daughters when they divide Self renewal entails the suppression of differentiation during proliferation Here we review current data on the regulation of ES cell self renewal by signalling networks and discus s the relation ship between cell cycle control and the retention of pluripotency Cytokinedependent activation of STAT3 drives ES cell selfrenewal The propagation of mouse ES cells is dependent on the presence of leukaemia inhibitory factor LIF or related cytokines that can activate signal transduction from cell surface receptors 13 15 LIF can be provided by a feeder layer of embryonic fibroblasts 1617 andor as a recombinant protein LIF engages a heterodimeric receptor complex consisting of two related cytokine receptors LIF receptor LIFR and gp130 18 This complex activates associated Janus associated J AK tyrosine kinases that phosphorylate the receptor chains The phosphorylated tyrosines then act as docking sites for proteins containing Src homology 2 SH2 domains that might themselves be phosphorylated by the JAKs Fig l The signal transducer and activator of transcription STAT family of transcription factors Seelronl maller 2002 ElsevierScience Ltd All righlsreserved PH 30962 39240202352 s TRENDSii GelBiology Vol12 No9 September 2002 433 Estrad o ES ce se frenewa TRENDS in ca BDagy Fig1 LIF dependenl aclivalion ol STATS blocks ES cell dillerenlialion and promoles sell renewal Leukaemia inhibilorylaclorLlF slabilizes Ihe associalion ol LIFR and gplSO cylokine receplors Resullanl aclivalion ol receplor associaled JAK kinasescauseslhe recruilmenl lyrosine phosphorylalion and dimerizalion ol STATS The STATS dimerslhenlranslocalelolhe nucleus where lheyconlrollhe lranscriplion ol genes regulaling sell renewal The Importance ol STATS isconlirmed bylhe demonslralion lhal acondilionally regulaled lorm ol Ihe lranscriplion laclor STATSERcan when aclivaled subslilule lor LIF addilion boxed inserl Abbrevialion ER ligand binding and dimerizalion domain ol Ihe oeslrogen receplor bind receptor phosphotyrosines and are key substrates for J AKs Phosphorylation of STATS promotes their dimerization through reciprocal interactions between an SH2 domain and phosphotyrosine This triggers their translocation to the nucleus and their binding to target sites on DNA In ES cells LIF predominantly activates STAT3 1 9 Recruitment and activation of STAT3 is essential for self renewal ofES cells 1920 and expression of an inhibitory STAT3 mutant in ES cells forces differentiation 1921 Studies using a chimaeric STAT3 molecule that can be activated directly by estradiol Fig 1 indicate that STAT3 activation is not only necessary but might be sufficient to block differentiation 22 Activation of this chimaeric molecule sustains ES cell self renewal without the addition of LIF It should be noted however that these experiments were carried out at moderate to high densities of cells in the presence of serum which might provide additional signals that support ES cell viability and or proliferation 22 ERKsantagonize ES cell selfrenewal Signalling downstream ofgp130 is not limited to activation of STAT3 but includes stimulation of the Rasmitogen activated protein kinase MAPK pathway The ERK MAPKs p42 and p44 regulate many different cellular responses in somatic cells and have particularly well documented roles in proliferation and differentiation In its simplest hll KIMIE hlrendSEom 7 Grb2 l SHPQMBmO HAS Raf l MEK 7 PD98059 UO126 ERK i l l D fferent at on Se fren ewa MKP3 TRENDS in ca BDagy Fig 2 Reduclion in RasMEKERK signalling promoles ES cell sell renewal Binding ollhe adaplors Grb2 or SHP 2couples cell surlace receplorslolhe RasRalMEKERK signalling palhway lnlerlerence wdh lhis palhway by mulalion ol Grb20r ShpZ by inhibilinglhe aclivalion ol MEKs wdh Ihe small molecule inhibilors PD98059and U0126 or by dephosphorylaling ERKs by milogen aclivaled prolein kinase phosphalase S MKP S limilslhe dillerenlialion ol ES cellsand promolessell renewal form the ERK pathway is engaged through the recruitment of a complex containing the Grb2 adaptor and S05 guanine nucleotide exchange factor to activated receptors Localization of Sos at the membrane promotes activation of Ras This initiates a cascade of tran sphophorylations involving Raf and MAPK kinase MEK kinases that culminates in activation of ERK 23 Fig 2 Active ERKs phosphorylate cytoplasmic targets and also undergo nuclear translocation which enables them to modulate the activities of transcriptional regulators such as Elk Ets Myc and the serum response factor SRF Receptor recruitment of the Grb2 Sos complex can be indirect In the context of LIFR gp130 akey intermediate in this recruitment is the protein tyrosine phosphatase SHP 2 Tyrosine phosphorylation of SHP 2 generates binding sites for Grb2 SHP 2 also associates with the scaffold protein Grb2 associated binder protein 1 Gab1 This protein recruits the lipid kinase PI3K and by binding the resulting phospholipid products through its N terminal pleckstrin homology PH domain stabilizes the association of the SHP 2 Gab1 Grb2 complex at the membrane and potentiates coupling to Ras 2A In ES cells eliminating the SHP 2 binding site from a chimaeric gp130 receptor blocks coupling to the Ras pathway but enhances the self renewal response 25 This effect is partly due to the elimination of a negative feedback effect on JAK activity 26 TRENDS in Cell Biology Vol12 No9 September 2002 ES ce surv va p27 l ES ce cyc e progress on TRENDSn cal Boagy Fig 3 PISK dependenl signals promole ES cell survival and cell cycle progression Localizalion ol phosphalidylinosilol 3 OH kinase Pl3Kal lhe plasma membranelhrough a receplorassocialed adaplor prolein such as Gab1increaseslhe amounls ol 3 phosphorylaled inosilol lipidsPldlns345P These moleculescolocalize Wilh lhe phospholipid binding kinases PDK1 and PKB and promole lhe aclivalion ol PKB by PDK1 ln ES cells polenlialion ol PISK signalling by mulalion ollhe PISK anlagonisl PTENa3 phosphale lipid phosphalase enhances ES cell survivaland increases prolileralion rale by suppressingaclivily ollhe cell cycle inhibilor p27K D The reporl ol a novel lipid phosphalase s SHIP expressed in ES cellsand haemalopoielic slem cellsislanlalizing however a role lorlhis molecule in slem cell signalling hasyel lo be eslablished which is independent of Ras But speci c attenuation of ERK signalling either by pharmacological inhibition of MEK activity or by forced expression of ERK phosphatases also facilitates self renewal by reducing differentiation 25 and T Burdon unpublished Notably inhibition of ERK does not replace the requirement for activation of STAT3 but rather enhances the actions of STAT3 although it is not clear whether this effect is direct or indirect The indications that ERK activation has a pro differentiation effect and is antagonistic to ES cell self renewal are corroborated further by genetic disruption of either Grb2 27 or Shp2 28 which results in impaired differentiation Fig 2 Reintroducing either a Grb2 Sos chimaera or an activated form of Ras into Grb2 ES cells restores normal differentiation 27 ERK activity re ects the input not only of gp130 cytokines but also of ligands that stimulate Ras through receptor tyrosine kinases and other cell surface receptors The overall balance of con icting activation of STAT3 and ERKs might determine the efficiency of ES self renewal 29 PI3K signalling in ES cell propagation An increased amount of 3 phosphorylated phosphoinositides is frequently associated with growth factor and cytokine signalling pathways This increase occurs through receptor mediated translocation of PI3K to the cell membrane The PI3K products phosphatidylinositol 34 bisphosphate hllpIEhIrEndSEom PtdIns34P2 and pho sphatidylinositol 345 trisphosphate PtdIns345P3 are ligands or the PH domains of several signal transducers including the serinethreonine kinases pho sphoinositide dependent kinase 1 PDK1 and protein kinase B PKBAkt Coordinate localization of these lipid bound kinases at the membrane facilitates PDK1 mediated phosphorylation of PKB which then modulates the activity of key regulators of apoptosis the cell cycle and cellular metabolism in various types of cell 30 The PI3K signalling pathway is also linked to cellular transformation The tumour suppressor PTEN is a lipid phosphatase that functions as a negative regulator of the PI3K pathway by removing the phosphate from the 3 position of3 phosphoinositides The PI3K signalling pathway has been implicated in ES cellpropagation through studies on Pten ES cells These cells have both enhanced viability and an increased rate of cell proliferation 31 Improved cell survival is correlated with elevated amounts of PtdIns345P3 enhanced phosphorylation of PKB and inactivation of the pro apoptotic protein B ad An accelerated transit through G1 seems to be caused by an increase in the rate of degradation of the p27Kipl inhibitor of cell cycle progression Fig 3 Notably the increase in the rate of cell division is more marked in Pten ES cells than in Pten fibroblasts which suggests that PI3K dependent signals could be relatively more signi cant in regulating the cell cycle of ES cells than in regulating that of fibroblasts The PI3K dependent signals that in uence the proliferation and survival of ES cells have not been defined Although PKB might seem to be a likely candidate ES cells lacking the up stream activator PDK1 are viable with no reported proliferation defect 32 They show negligible activation of PKB and also fail to activate other targets of PDK1 including p90 Rsk and p70 S6 kinase These results raise the possibility that PI3K can in uence ES cell grth through apathway that is not dependent on PDK1PKB The PKB related protein serum and glucocorticoid induced kinase 1 SGK might ful l this role 3334 Alternatively the PI3KPDK1 pathway might have a supportive but dispensable role in self renewal Unique signalling adaptors in ES cells Embryonic stem cells express a variant of SH2 containing inositol 5 phosphatase SHIP that lacks the SH2 domain 35 This enzyme normally removes 5 phosphates from the lipid products of PI3K and in some systems it inhibits the activation of downstream signals such as PKB The variant expressed in ES cells is reported to bind the adaptor protein Grb2 but remains unphosphorylated and does not associate with the docking protein Shc 35 Embryonic stem cells also speci cally express large amounts of a variant Gabl molecule This protein lacks the N terminal PH domain which TRENDS GelBiology Vol12 No9 September 2002 435 a M togens b SHP2 4 gp130 HSTATB w LIF l S ph ase TRENDSn ca BIDagy Fig 4 Models ol cell cycle control In dIllerenlIaled cellsand embryonic stem EScells a The RBEZF and Myc palhwayslunclion In parallelioconlrol accumulation ol cyclIn ECDKZand enlry Into 8 phase In dIllerenlIaled cells 7 Hypothetical palhwayslorregulalinglhe accumulation ol cyclIn ECDKZ In ES cells The broken lIne IndIcalesihalcyclIn DCDK4 orcyclIn DCDKG kinase activity seemslo be dispensable In activating RB results in attenuated coupling to the RasERK cascade T B urdon andA Smith unpublished The presence of these novel isoforms indicates that the signalling circuitry in ES cells is likely to differ from that in other cell types although the functional signi cance of these molecules is yet to be determined Cellcycle control differs in differentiated cells and ES cells Proliferation of differentiated mammalian cells is controlled primarily by regulating the progression through G1 phase and entry into S phase The retinoblastoma RB protein and its relatives p107 and p130 are essential components in the control of the G1S transition Fig 4 The activity ofRB is regulated by phosphorylation hypophosphorylated G1 specific RB inhibits the expression of genes that are required for entry into S phase by sequestering the E2F family of transcription factors During progression through G1 RB is phosphorylated sequentially by complexes of cyclins and cyclin dependent kinases CDKs Phosphorylation by cyclin DCDK4 or cyclin DCDK6 induces apartial hll KIMIE hlrendSEom release of E2F which is sufficient to activate transcription of the cyclin B and the cdc25A genes The cdc25A phosphatase removes inhibitory phosphates from CDK2 and the resulting cyclin ECDK2 complex then completes RB phosphorylation leading to full release of E2F activation of target genes and entry into S phase 36 38 A second pathway involves the c mycproto oncogene which directly stimulates transcription of the genes that encode cyclin E and cdc25A to generate cyclin ECDK2 kinase 38 Fig 4 The Myc and the RB E2F pathways are now thought to be two parallel and cooperative G1S control pathways that converge on cyclin ECDK2 kinase the activity of which determines entry into S phase 38 Mutations in either or both pathways are frequently encountered in cancers 3940 re ecting their fundamental role in controlling the cell cycle The tumour suppressors p 16M la and p27 1 are inhibitors of cyclin DCDK4 or cyclin DCDK6 and cyclin ECDK2 respectively They are activated in response to various growth inhibitory signals including senescence contact inhibition and terminal differentiation 41 Embryonic stem cells have a short G1 phase of roughly 15 h during which hypophosphorylated RB is virtually undetectable 9 Thus RB is likely to be rephosphorylated immediately after mitosis in TRENDS in Cell Biology Vol12 No9 September 2002 ES cells in contrast to differentiated types of cell An important issue therefore is whether ES cells are subject to G1 regulation by RB or other members of the RB family or whether RB is functionally inactivated by constitutive hyperphosphorylation In addition to RB ES cells express p107 42 but not p130 43 B ut much evidence supports the notion that the RB pathway does not regulate the ES cell cycle First ES cells are refractory to the growth inhibitory activity of p 1 61quot la 10 Resistance to growth inhibition mediated by p16i 1k la is a common feature of cancer cells in which the RB pathway is disrupted 4445 Withdrawal of LIF and subsequent differentiation is accompanied by sensitization to growth inhibition mediated by p16ink l which indicates that RB control of G1 is imposed during differentiation 10 Second inactivating disruptions in all three genes ofthe RB family p107 p130 Rb triple knockout TKO cells do not seem to compromise the proliferation of ES cells but do reduce differentiation in teratocarcinomas 4647 This indicates further that RB dependence is acquired only as ES cells undergo differentiation Last ES cells share striking similarities in proliferative behaviour with TKO embryonic fibroblasts B oth TKO MEFs and ES cells fail to arrest in G1 at confluency 4647 In normal fibroblasts this phenomenon is accompanied by increased amounts of p27kll 1 decreased amounts of cyclin D1 and an accumulation of hypophosphorylated RB which leads to G1 arrest 48 ES cells and TKO MEFs also escape replicative senescence and are immortal 4647 In other cells replicative senescence and G1 arrest are associated with an accumulation of hypophosphorylated RB which is caused by inhibition of CDK kinases mediated by p16 48 and p21Cipl 49 B oth ES cells and TKO MEFs fail to arrest in G1 after DNA damage but they do arrest at the RB independent G2M checkpoint 46475051 ES cells like TKO MEFs therefore escape from contact inhibition are immortal and lack the G1 checkpoint Together these data strongly support the notion that ES cells are not controlled by RB in G1 Cyclin expression and function during 61 in ES cells What mechanism underlies the functional inactivation of RB in ES cells In certain tumour cells that do not have a mutation in the RB gene RB protein is hyperphosphorylated by constitutive expression of cyclin DCDK4 cyclinCDK6 andor cyclin ECDK2 kinases 405253 Cyclin D1 and cyclin D3 are present in low amounts in ES cells whereas cyclin D2 is not expressed CDK4 associated kinase activity is virtually undetectable The low amount of D type cyclins in ES cells re ects the situation in epiblast cells which do not express appreciable quantities of D type cyclins until gastrulation commences 54 hllpIEhIrEndSEom The differentiation of ES or epiblast cells results in robust expression of D type cyclins and appreciable CDK4 associated kinase activity which signifies the adoption of G1 regulatory control 10 Regulation of the basal expression of cyclin D1 differs between ES cells and other cells First the RasERK pathway which is central to transcriptional activation of cyclin D1 expression in somatic cells stimulated by grth factor 55 57 does not contribute to the expression of cyclin D1 in ES cells 58 Second the amount of cyclin D1 protein 59 is dependent on PI3K signalling but this seems to be uncoupled from any specificmitogenic stimulation 5 8 Thus neither PI3K activity nor cyclin D1 expression is downregulated after serum starvation Taken together these data lead us to the conclusion that basal expression of cyclin D1 is disconnected from mitogenic signals transduced by tyrosine kinase receptors in ES cells Constitutive albeit low expression of cyclin D1 could contribute to constitutive pho sphorylation of RB Alternatively the functional significance of cyclin DCDK4 complexes in ES cells might be to sequester p27kipl and prevent this inhibitor acting on cy clin ECDK2 kinase 41 The resistance to p 1 6 1MB implies however that neither function is es sential for ES cell proliferation In differentiated cells enforced expression of cyclin E is sufficient to overcome growth arrest that is mediated by p16 k4 Constitutive cyclin ECDK2 activity in fibroblasts is also as sociated with anchorage independent growth 60 another property shown by ES cells The cyclin B gene is subject to repression by the active form of RB in differentiated cells but as we have discussed above the RB pathway seems to be inoperative in ES cells Consistent with this an active form ofRB is undetectable L Vitelli and P Savatier unpublished and cyclin ECDK2 kinase activity seems to be constitutive in these cells 10 Gp13D signalling and cellcycle control in ES cells The G1S transition thus seems to be driven uniquely by cyclin ECDK2 during ES cell self renewal A currently unresolved issue is whether the apparently constitutive activity of cyclin ECDK2 is an intrinsic property of ES cells or is dependent on gp 130 signalling Withdrawal of LIF induces differentiation of ES cells rather than cell cycle arrest But because cell cycle regulation changes early in differentiation this does not preclude the possibility that STAT3 could direct the expression of key regulators of the mitotic cycle in ES cells and stimulate their entry into S phase STAT3 can in uence G1S transition in some types of differentiated cells In the lymphoid cell line BAF 03 STAT3 activates expression of specific cell cycle regulators including D type cyclins p27k 1 1 c Mycand Pim 1 61 Pim 1 is a serinethreonine kinase that phosphorylates and activates cdc25A thereby TRENDS GelBiology Vol12 No9 September 2002 potentiatin g the accumulation of active cyclin ECDK2 kinase Myc and Pim 1 combine synergistically to effect interleukin 6 IL 6 dependent proliferation of BAF cells 62 Enforced expression of Myc and Pim 1 is sufficient to overcome cell cycle arrest mediated by IL 6 starvation which indicates that they are essential targets of STAT3 This pathway can drive RB hyperphosphorylation and entry into S phase in the presence of minimal cyclin DCDK4 or cyclin DCDK6 complexes Fig 4b Although the lack of a reported proliferation phenotype in ES cells lacking mycorpim 1 seems to argue against such a mechanism operating in ES cells this lack of phenotype could also be explained by functional redundancy with related members of these gene families Data on transcriptional activity of effectors of G1S transition is necessary to determine whether STAT3 directly stimulates proliferation Fig 4b or whether ES cells cycle autonomously until entering into differentiation It is also possible that LIFR or gp130 signalling could contribute to G1 S transition by recruiting PI3K through SHP 2 and Gab 1 Fig 4b This pathway has been identified in the T47D breast cancer cell line in which IL 6 has been shown to control cell migration by activating MAPK and PI3K via the gp130SHP 2Gab1 pathway 63 In ES cells LIF dependent activation of PI3K seems to sustain cyclin D1 by positively regulating the rate of its synthesis via p70 S6 kinase and by negatively regulating the GSK3 dependent rate of protein degradation 58 Observations from ES cells lacking PTEN indicate that PI3K signalling also promotes degradation of the p27 1 inhibitor 31 These different actions could result in a low amount of p271 1 that is effectively sequestered by cyclin DCDK4 see above thereby ensuring that cyclin ECDK2 remains con stitutively active It should be noted that in the presence of serum and feeders gp130 signalling might not be the only or even the most significant activator of PI3K in Acknowledgements Welhanklan Chambers lorcommenls onlhe manuscrIpIResearch In Iheaulhors laboralorles Is supporled bylhe Blolechnology and Blologlcal Sclences Research CouncIITB ASIhe UK Medlcal Research CouncIIAS and by Assoclallon pour la Recherche conlre e Cancerand ngue Nallonal conlre e CancerPS Cultures ofES cells References 1 Gardner R L andBrook FA 1997 Re ec ons on thebiology of embryonic stem cells Int J Dev Biol 41 2357243 2 Nichols J 20011nnoducing embryonic stem cells Cult Biol 11 R5037R505 3 Smith A 2001 Embryonic stem cells In Stem Cell Biology Nlarshak D R eta eds pp 205723 0 Cold Spring Harbor Laboratory Press 4 Beddjngton R S P and Robertson E J 1989An assessment of the developmental potential of embryonic stem Cells in themidgesta on mouse embryo Development 105 73 37737 5 Bradley A eta 1992 Modifying themouse design and desire Biotechnology 10 5347539 6 Doetschman T C et a 1 985 The in VitIo development of blastiocystederived embryonic stem cell lines fonnation of visceral yolk sac blood islands and myocardium I Embryo EXP Mozplaol 87 2745 h KIMIE hlrendSEom 437 Concluding remarks As we have discussed above ES cells have an unorthodox cell cycle in which the G1 control pathways that operate in other types of cell are reduced or absent Such features are associated with the deregulated proliferation of tumour cells however constitutive replication is also a common aspect of early embryo development in many species This might simply re ect the fundamental requirement of establishing sufficient cell numbers to initiate gastrulation It is possible however that the uncoupling from G1 regulation might also be involved in sustaining the undifferentiated state Active hyp ophosphorylated RB forms complexes with and promotes the activity of differentiation promoting transcription factors such as MyoD myogenin and CEBP 64 67 Efficient hyperphosphorylation and inactivation of RB and its other family members might therefore be important to shield pluripotent cells from activities that induce differentiation The acquisition of G1S regulation which involves activation of RB seems to be an early event in the differentiation of ES cells Another pos sibility is that constitutive transit through G1 could con strain the temp oral opportunity for both chromatin remodelling and the establishment of heritable transcription programs Imposition of G1 control might be necessary for the heritable changes in gene expression that signify cell commitment If this were so then it might explain why ES cells express RB and components of the RasERK pathway 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