the retinoblastoma gene: a prototypic and multifunctional tumor suppressor

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Experimental Cell Research 264, 2–18 (2001)doi:10.1006/excr.2000.5129, available online at http://www.idealibrary.com on

The Retinoblastoma Gene: A Prototypic and MultifunctionalTumor Suppressor

Lei Zheng and Wen-Hwa Lee1

Department of Molecular Medicine/Institute of Biotechnology, University of Texas Health Science Center at San Antonio,
15355 Lambda Drive, San Antonio, Texas 78245
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Genome instability has been implicated in the gen-eration of multiple somatic mutations that underliecancer. Germline mutation in the retinoblastoma (RB)gene leads to tumor formation in both human andexperimental animal models, and reintroduction ofwild-type RB is able to suppress neoplastic pheno-types. Rb governs the passage of cells through the G1phase-restriction point and this control is lost in mostcancer cells. Rb has also been shown to promote ter-minal differentiation and prevent cell cycle reentry.Recent studies implicate Rb in mitotic progression,faithful chromosome segregation, checkpoint control,and chromatin remodeling, suggesting that Rb mayfunction in the maintenance of genome integrity. It islikely that Rb suppresses tumor formation by virtue ofits multiple biological activities. A single protein ca-pable of performing multiple antioncogenic functionsmay be a common characteristic of other tumor sup-pressors including p53 and BRCA1/2. © 2001 Academic Press

INTRODUCTION

Genetic factors play an important role in tumorigen-esis as evident in hereditary cancer syndromes, partic-ularly childhood tumors. It had long been conceivedthat loss of function of a class of genes, which serve tosuppress tumor formation in normal cells, governs thesusceptibility to these hereditary cancers [1]. The ret-inoblastoma susceptibility gene, mutation of which isresponsible for childhood retinal malignancy, was thefirst human tumor suppressor gene identified [2] andextensively studied as the prototype of this class ofgene. The concept of tumor suppression was substan-tiated by the experimental demonstration that reintro-duction of a wild-type retinoblastoma (RB) allele intocells derived from RB-deficient retinoblastoma andmany other human tumors was able to suppress neo-plastic phenotypes such as anchorage-independentgrowth and tumorigenesis in nude mice [3, 4]. Later,

1 To whom correspondence and reprint requests should be ad-
ressed. Fax: (210) 567-7377. E-mail: [email protected].
20014-4827/01 $35.00Copyright © 2001 by Academic PressAll rights of reproduction in any form reserved.

similar tumor suppression properties were observedwith cancer susceptibility genes identified in other he-reditary cancer diseases, such as p53, WT1, APC,p16INK4a, ARF, PTEN, and others [5–9]. Further obser-vation revealed that loss of exogenously reconstitutedRB reverted cells to the tumorigenic state [10]. It hasalso been demonstrated that reintroduction of RB isable to suppress pituitary tumors, multiple neuroendo-crine neoplasia and lung metastases that develop in Rb1/2 mice [11, 12]. Therefore, inactivation of a tumoruppressor gene represents a threshold event that ini-iates transformation to the neoplastic state.

The identification of growth suppression activity ofumor suppressor genes has revealed potential mech-nisms that govern the gatekeeper event in neoplasticransformation. Deregulation of cell cycle progressions considered to represent the major precipitatingvent that results from inactivation of tumor suppres-or genes. This does not explain, however, why inacti-ation of a single tumor suppressor gene leads to mul-iple genetic alterations that underlie the process ofumorigenesis. Recent studies implicating Rb in gen-ral chromosome metabolism have suggested thataintaining genome integrity is an integral part of Rb

unction. This review revisits mechanisms behind theatekeeper role of Rb in cell cycle regulation and dif-erentiation, present new studies on the role of Rb innsuring proper chromosome functions, and providesuture direction for studies concerning how Rb may benvolved in maintaining genomic stability.

RB AND CELL CYCLE REGULATION

Rb Regulates the G1 Phase of the Cell Cycle

A role for tumor suppressors in cell cycle regulationis exemplified by studies of the gene product of RB.Overexpression of Rb by microinjection into cells ofpurified unphosphorylated Rb protein in early G1phase results in reversible G1 arrest, while injection ofsimilar amounts of Rb in late G1 phase or early S phasehas no effect on DNA synthesis [13]. Similar results are
observed in cells overexpressing Rb through transfec-

3THE RB GENE AND TUMOR SUPPRESSION

tion of an exogenous RB cDNA [14]. These observationsreveal a restriction point that is controlled by Rb dur-ing the cell cycle transition from early G1 phase to lateG1/S phase [15]. Passage through this point commitsnormal cells to DNA synthesis and cell division. Loss ofcontrol of this restriction point as a consequence of lossof function of Rb, however, does not appear to interruptthe cell cycle progression. It is likely that cells withoutproper control of this restriction respond improperly tomitogenic signals, leading to abnormal cell prolifera-tion in the neoplastic state.

Rb–E2F, a Simplified Model for G1 Regulation

How Rb regulates the cell cycle during the G1 phasetransition has been investigated extensively. The iden-tification of the interaction between Rb and E2F-1 pro-vided the first clue [16–18]. The E2F family is a groupof sequence-specific DNA-binding transcription factorsthat have been shown to regulate expression of genesrequired for entry into S phase and for DNA synthesis[19, 20]. The interaction of Rb with E2F, which func-tionally represses transcription, therefore, has beenused as paradigm to demonstrate how Rb restrains cellcycle progression at the G1 phase transition and howRb inhibits cell cycle reentry from quiescence in orderto maintain the terminally differentiated state (Fig. 1).

The studies of E2F regulation provide a compellingmodel for our understanding of the role of Rb in regu-lating G1 phase progression; however, this mechanismis more complicated than first expected. Although thetranscriptional repression activity of Rb appears to beessential for Rb to suppress tumor growth, E2F regu-lation is only one aspect of Rb function, which will bediscussed further in more detail. Other sequence-spe-cific transcription factors must also participate in Rb-mediated transcriptional regulation. Candidates ofsuch transcription factors include HBP1, a sequence-specific high-mobility group transcription repressor[21]; RIZ, an Rb-interacting zinc-finger protein [22];and RbaK, a KRAB-Zinc-Finger family protein thatinteracts with Rb [23]. How these proteins tether Rb tothe specific regulatory elements in Rb-target genes,however, remains to be explored.

Mechanisms of Transcriptional Repression by Rb

Two mechanisms have been suggested to underliethe transcription repression activity of Rb [24]. First,Rb associates directly with the transactivation domainof E2F; therefore, it is proposed that Rb arrests cells inG1 phase by directly inhibiting the transactivation ac-tivity of E2F. Second, Rb binds to DNA by means ofE2F and actively represses transcription. Several linesof evidence support the idea that Rb actively repressestranscription [24]. First, Rb exhibits potent transcrip-
tional repression activity if tethered to DNA. Second,
Rb induces cell cycle arrest when fused to the E2F-1DNA-binding domain. Finally, competitive displace-ment of E2F from promoters blocks transcription re-pression by Rb.

Although these observations have suggested the im-portance of the Rb–E2F complex in active transcrip-tional repression, other studies have shown that theE2F family of proteins play a positive role in transcrip-tion regulation. Aside from evidence showing that over-expression of E2F activates transcription and inducesS phase entry [25, 26], it has been shown that intro-ducing a dominant-negative mutant of the E2F-bind-ing partner, DP1, inhibited the progression of cells intoS phase [27]. Moreover, embryonic fibroblasts derivedfrom E2F-3 gene knockout mice exhibit delays in Sphase entry [28]. However, it is argued that overex-pression of E2F can competitively replace the Rb–E2Frepressor complex on promoters [24]. Elimination ofE2F sites from many E2F-responsive promoters re-sulted in an increase of transcriptional activity duringthe G0/G1 phases of cell cycle, rather than a decrease

FIG. 1. Rb is a fundamental mediator of growth and differenti-ation signals. A model for involvement of Rb protein in the coordi-nation of cell cycle progression and cellular differentiation in re-sponse to division and differentiation signals. In this model, themitogen-dependent accumulation of D-type cyclins triggers the phos-phorylation of Rb by cdk4,6. Cyclin E/cdk2 collaborates with cyclinD/cdk4,6 to complete Rb phosphorylation. Phosphorylated Rb nolonger interacts with its associated proteins such as E2F. For in-stance, Rb-mediated repression is relieved, and untethered E2Fsactivates genes required for S phase entry. This phosphorylation ofRb is believed to remove the block of G1 phase progression. Simi-larly, cell cycle arrest by anti-mitogenic signals, which is mediatedby cdk inhibitors (CKI), also largely depends on the activity of Rb. Inthe cellular differentiation process, Rb promotes terminal differen-tiation by blocking Id2 or activating C/EBP family proteins andMyoD, which is coordinated with the function of Rb in preventing cellcycle reentry.

in S phase [20]. It is therefore likely that both the

4 ZHENG AND LEE

transactivation activity of E2F and the derepressionresulting from dissociation of the Rb–E2F complex con-tribute to the activation of E2F target gene transcrip-tion. The relative contributions of Rb in repressingtranscription by directly sequestering E2F versus ac-tive repression by the Rb–E2F complex in various bio-logical settings remain to be determined.

Recently, several non-mutually exclusive mecha-nisms have been evoked to explain active transcrip-tional repression by Rb. The association of Rb withhistone deacetylases HDAC1, HDAC2, and HDAC3[29–33] suggests that Rb represses transcription bymeans of histone deacetylation. Interestingly, the firstlink between Rb and histone deacetylation complex isprovided by the identification of Rb-associated pro-teins, RbAp48 and RbAp46 [34]. These two Rb-associ-ated proteins copurified with a histone deacetylasecomplex containing two histone deacetylases, HDAC1and HDAC2 [35]. Rb may function in recruiting such ahistone deacetylase complex to DNA-bound E2F orother transcription factors and alter the chromatinstructure of its target genes into a repression-favoredstatus. Consistent with this scenario, Rb-mediated re-pression is inhibited by histone deacetylase inhibitors[29–31] and also blocked by synthetic mutations thatabolish the interaction of Rb with HDAC1 and HDAC2[33]. The identification of the interaction between Rband HDACs and the dependency of Rb-mediated re-pression on histone deacetylase activities has, there-fore, provided direct evidence to support a role forhistone deacetylase complexes in transcriptional re-pression by Rb.

Rb-mediated transcriptional repression also involvesalteration of higher order chromatin structure, whichis regulated by nucleosome-remodeling complexes inan ATP-dependent manner [36–38]. The best studiedof these complexes is the SWI/SNF complex, initiallyidentified in yeast [39]. Recent studies suggest that theSWI/SNF complex is not only involved in transcrip-tional activation but also in repression, as more genesbecame activated than repressed in yeast strains har-boring SWI2/SNF2 mutation [40]. Rb interacts withBRG1 and Brm, the human homologs of SWI2 andSNF2, respectively [36, 41, 42]. It has also been dem-onstrated that overexpression of Brm can enhance therepression activity of Rb on E2F-1 [36], and BRG1 isrequired for Rb-mediated growth suppression [37, 38].Reciprocally, Rb also appears to be required for growthsuppression induced by overexpression of BRG1 [41].

It needs to be emphasized that histone deacetylaseactivity and chromatin remodeling are not the onlymechanisms for Rb-mediated active transcriptional re-pression. It has been demonstrated that Rb is able torepress transcription through direct contact with thebasal transcription machinery without the require-
ment of histone deacetylase activity [43]. The interac-
tion of Rb with other transcriptional corepressors, suchas RBP1 and CtIP, may also represent alternativemechanisms underlying active transcriptional repres-sion by Rb [44]. It is suggested that RBP1 recruitsHDAC activity to Rb for HDAC-dependent transcrip-tional repression; however, RBP1 may also mediateHDAC-independent repression, the mechanism ofwhich has not, however, been clarified [32]. CtIP waspreviously identified as a protein associated with thebreast cancer susceptibility gene product, BRCA1 [45],and was suggested to participate in the BRCA1-medi-ated corepressor function by recruiting CtBP [46]. Todate, the mechanism by which CtIP–CtBP functions intranscriptional repression remains unclear. Chromatinremodeling coupled with histone deacetylation hasbeen implicated in CtBP-mediated transcription re-pression [47]. Other observations have suggested amechanism of transcription repression by CtBP inde-pendent of histone deacetylase activity [48].

Taken together, multiple mechanisms that mediatethe active role of Rb in transcriptional repression maycontribute to the regulation of E2F target genes atdifferent levels or in response to different signals. It isalso conceivable that multiple transcriptional repres-sion activities are associated with Rb in transcriptionalregulation of different target genes by means of othersequence-specific DNA-binding factors, which, how-ever, remain to be determined.

Further Insights from Structure–Function Studiesof Rb

The majority of tumor-derived Rb mutants are de-fective for multiple biochemical activities, making itdifficult to discern the relative contributions of theseactivities to Rb-mediated tumor suppression. New in-sights into the structural basis for the interactionsbetween Rb and its associated proteins and recentanalysis of a panel of Rb mutations that differentiallyaffect these associations have allowed one to distin-guish different activities of Rb.

Rb contains three distinctive domains: the N-termi-nal region, the central A and B domains separated by alinker region, and the C-terminal region. The struc-tural integrity of the A/B domains is required for theinteraction of Rb with most of its associated proteins[49]. These include a number of proteins that harbor anLXCXE motif [49], which was originally identified to becritical for viral oncoproteins to interact with Rb [50].By contrast, E2F-1 does not contain the LXCXE motifand appears to use a different type of structural motifto bind Rb in the A/B domains [16, 49].

Crystallographic analysis of Rb structure revealsmultiple protein-binding interfaces in the A/B domains[51], which raises the possibility that Rb can simulta-
neously bind several proteins, such as E2F-1 and


HDACs, through the E2F-binding site and the LXCXE-motif-binding site, respectively. Moreover, BRG1,which contains an LXCXE-like motif, appears to bindto Rb at a site distinct from the LXCXE-motif-bindingsite [33]. This allows Rb, along with both HDACs andBRG1/Brm, to form a complex that binds to E2F andactively represses transcription [37]. In addition, pro-teins such as H-nuc and HDAC3, having neither anLXCXE motif nor an E2F-like motif, bind to the A/Bdomains [33, 52]. H-nuc binds to Rb via its C-terminalTPR repeats, revealing another novel Rb-binding motif[53].

Most of tumor-derived Rb mutants, however, aredefective for binding to either LXCXE or E2F-like mo-tifs. To understand the precise role of E2F regulationin tumor suppression, several Rb mutants derived ei-ther from low-penetrant, mutant alleles or from syn-thetic Rb mutations in the conserved residues of theE2F-binding region were analyzed for their biologicalactivities [54–56]. These mutants, whose productsfailed to interact with E2F-1, have no effect on E2F-1-mediated transcription repression but retain the abil-ity to coactivate specific transcription and promote dif-ferentiation. Interestingly, RBR661W (arginine totryptophane substitution at residue 661) and RBD480(residue 480 deleted) mutants were able to suppresscolony formation when reintroduced into Rb-deficientcells, suggesting that E2F regulation is only one aspectof Rb function and E2F binding is dispensable for thegrowth suppression by Rb [54, 55].

Crystallographic analysis has identified the residuespotentially critical for Rb to bind proteins at theLXCXE motif [51]. Subsequent mutations of these res-idues were found to abolish the interaction of Rb withLXCXE motif proteins such as HDAC1 and HDAC2and block Rb activity in tumor growth suppression[33]. These mutations, however, did not affect the in-teraction of Rb with BRG1, HDAC3, and E2F-1 andhad no influence on Rb in blocking E2F activity [33,52]. Human SWI/SNF (hSWI/SNF) activity was alsodispensable for Rb-mediated repression of E2F trans-activation activity, since Rb can effectively block E2Factivity in hSWI/SNF-deficient cells. These results fur-ther suggest that multiple pathways underlie themechanism of growth suppression by Rb.

Mutational analysis of the C-terminus of Rb alsosuggests a contribution of this region to Rb-mediatedgrowth suppression [56]. This region of Rb has anintrinsic nonspecific DNA-binding activity [57]. In ad-dition, c-abl tyrosine kinase and MDM2 bind to thisregion [58, 59]. The c-abl–Rb interaction, which blocksthe tyrosine kinase activity of c-abl, is suggested to beimportant for Rb-mediated growth suppression [60,61], but direct evidence remains to be established tosupport such a notion. The biological relevance of the
Rb–MDM2 interaction was enlightened by a recent
report showing that Rb, in a trimeric complex withMDM2 and p53, prevents the MDM2-mediated degra-dation of p53 [62]. This study provides a functional linkbetween two tumor suppressors, p53 and Rb, and sug-gests a model in which Rb blocks the activity of MDM2in the degradation of p53 and thereby facilitates p53-mediated apoptosis. However, loss of Rb function isknown to trigger apoptosis through both p53-depen-dent and p53-independent pathways [63], suggestingan inhibitory role of Rb in apoptosis. Therefore, it re-mains unclear how these two opposing activities con-tribute to the biological function of Rb.

Although the A/B domains and the C-terminal regionof Rb mediate the interaction with most of its associ-ated proteins and fulfill most of the activities associ-ated with Rb in vitro [49], there is evidence suggestingthat the N-terminal region of Rb is also important forits tumor suppression function. For instance, trans-genic expression of N-terminally deleted human Rb isnot able to rescue Rb2/2 mice completely from embry-onic lethality [64]. Survival of embryos is prolonged,but embryos displayed defects in the terminal differ-entiation of erythrocytes, neurons, and skeletal musclesimilar to those observed in Rb2/2 mouse embryos[65–67]. Moreover, expression of N-terminally deletedhuman Rb fails to prevent pituitary tumor formed inRb1/2 mice but delays tumor formation or progres-sion, whereas expression of wild-type human Rb hadcomplete complementary capability [68, 69]. These re-sults strongly suggest that the N-terminal region of Rbis crucial for the functional integrity of the entire Rbprotein and may provide an underlying explanation forhuman low-penetrant retinoblastoma families withmutations in N-terminal region of RB.

It remains to be explored how the N-terminus of Rbcontributes to the tumor suppression function of Rb.One likely explanation is that the N-terminal regionprovides an integral determinant for the proper con-formation of the full-length Rb protein. Another non-exclusive explanation is that the N-terminal regionmay mediate distinct activities, which coordinate withother functions of Rb in tumor suppression. Consistentwith this hypothesis, several proteins interact primar-ily with the Rb N-terminus, including an 84-kDa nu-clear matrix protein [70], a 70-kDa heat shock cognateprotein [71], a kinase active in G2/M phases [72, 73],and replication licensing factor MCM7 [74]. However,the biological relevance of these interactions to Rbremains unknown.

The presence of a BRCT domain in the N-terminalregion of Rb has shed some light on the potential func-tion of this region. The BRCT domain was originallyidentified in the C-terminal region of BRCA1 [75].Later, this domain was found in a number of proteinsparticipating in cellular response to DNA damage and
was suggested to be involved in protein–protein inter-

6 ZHENG AND LEE

actions [75]. Interestingly, deletion of exon 4 in RB(RBDex4) causes a low-penetrant mutant allele, whichspecifically disrupts the BRCT domain. This deletionhas no effect on activating transcription, promotingdifferentiation, and suppressing colony formation oftumor cells although a negative impact on these activ-ities may be below detection [54, 55]. How this deletioncauses cancers is still an enigma. It will be worthwhileto identify cellular proteins that bind to this BRCTdomain and to examine whether this domain in Rb isalso involved in the DNA damage response.

Phosphorylation of Rb by Cyclin-Dependent Kinases

As discussed earlier, Rb governs cell cycle passagethrough the restriction point during G1 phase. Whencells are stimulated by growth factors to enter the cellcycle from G0, they generally require continuous mito-genic stimulation to be driven to the restriction point,after which mitogens can be withdrawn and cells willenter S phase and complete the cycle in their absence.Conversely, antiproliferative compounds, such astransforming growth factor-b, can only arrest the pro-liferation of cells that are progressing through G1phase but have not yet reached the restriction point[15]. In yeast, the role of cyclin-dependent kinase in thecontrol of the G1 restriction point has been well estab-lished [76]. cdc2 and Cdc28, the only member of cyclin-dependent kinase family present in Schizosaccharomy-ces pombe and Saccharomyces cerevisiae, respectively,regulate both G1/S and G2/M transition by partneringwith different cyclins [77]. In mammalian cells, multi-ple Cdk kinases are involved. The G1/S transition ismainly attributed to Cdk2, 4, and 6, and the G2/Mtransition is mainly attributed to mammalian cdc2[15]. While Cdk kinases are stable during the cell cycle,their activities are regulated by their cyclin partners,which are expressed in a cell-cycle-dependent manner.In mammalian cells, two types of cyclins, cyclins D andE, partnering with cdk4/6 and cdk2, respectively, areinvolved in regulation of G1 passage and G1/S transi-tion. In addition, cyclin A, partnering with cdk2, isinvolved in S phase progression and the transitionduring later S and G2/M phase. Induction of cyclin B,partnering with cdc2, controls M phase entry, and itsdegradation contributes to the metaphase to anaphasetransition.

The cellular Rb protein is modulated by phosphory-lation in a cell-cycle-dependent manner [78–80]. Hy-pophosphorylated forms of Rb predominate in early G1phase and reappear during M phase, while hyperphos-phorylated forms of Rb are present from late G1 phasethroughout S, G2, and M phase [78, 81]. The inductionof D-type cyclins coincides with the initial phosphory-lation of Rb. The mitogen-dependent accumulation of
D-type cyclins triggers the phosphorylation of Rb by
cdk4 and cdk6 [82, 83] (Fig. 1). This phosphorylation ofRb is believed to remove the block to G1 phase progres-sion. Phosphorylated Rb no longer interacts with itsassociated proteins, such as E2Fs and HDACs [19, 24].For instance, Rb-mediated repression is relieved, anduntethered E2Fs therefore activates the transcriptionof genes required for S phase entry and DNA synthesis.Meanwhile, E2Fs induce the expression of cyclin E andcyclin A, and in turn, cyclin E/cdk2 collaborates withcyclin D/cdk4,6 to complete Rb phosphorylation [84–86].

Different sites on Rb appear to be phosphorylatedpreferentially by specific cyclin/cdk kinases, and phos-phorylation of specific sites may modulate distinct bio-chemical activities. For example, binding of E2F,LxCxE-motif-containing proteins, and c-abl is specifi-cally regulated by distinct sets of phorphorylation sites[61, 87]. A recent study has suggested that phosphor-ylation of the C-terminal region of Rb by cyclinD–Cdk4/6 initiates a folding of the C-terminal region tointeract intramolecularly with the central A/B domainsand displaces HDACs from the A/B domains. Such astructural modification facilitates the phosphorylationof the A/B domains by cyclin E–cdk2 and subsequentlydisrupts the structures of the A/B domain [88]. Byanalyzing transcriptional repression activity of theRb–E2F complexes, this study provides compelling ev-idence for understanding the necessity of successivephosphorylation of Rb by cyclin D–cdk4/6 and cyclinE–cdk2. However, further investigation will be neces-sary for a more comprehensive understanding of howsuccessive phosphorylation of Rb modulates biologicalactivities other than E2F regulation and, particularly,how distinctive phosphorylation of Rb and subsequentstructural modification of Rb determine the multipleroles of Rb in various biological settings.

Modulation of Rb activity by different cdk kinaseshas been proposed to allow differential regulation of Rbtarget genes involved in cell cycle regulation. This ideais supported by a study showing that Rb forms twodistinct complexes at different points of cell cycle, onewith both HDAC and hSWI/SNF and another with onlyhSWI/SNF [37]. The HDAC–Rb–hSWI/SNF complexappears to function in the repression of E2F targetgenes such as cyclin E, dihydrofolate reductase, andthymidine kinase, which are normally activated duringG1 phase. The Rb–hSWI/SNF complex appears tofunction in the repression of other E2F target genessuch as cyclin A and cdc2, which are normally acti-vated during S phase. It has been suggested that phos-phorylation of Rb by cyclin D–cdk4/6 inhibits Rb bind-ing to HDAC, but not to BRG1, and thereby specificallydisrupt the HDAC–Rb–hSWI/SNF complex, leading toelevated cyclin E expression. The Rb–hSWI/SNF com-plex is not disrupted until Rb is subsequently phos-
phorylated by cyclinE–cdk2, allowing the induction of


cyclin A and cdc2. Such a model suggests that twodistinct transcriptional repression activities associatedwith Rb are successively inactivated by phosphoryla-tion, thereby maintaining the sequential order of cy-clin/cdk activity during the cell cycle. Execution ofthese orderly events, according to this model, relies ontargeting E2F-dependent gene-specific transcription.How these two transcriptional repressor complexesdistinguish their target genes has not been clarified.More complicated mechanisms must be involved toensure targeting of specific cyclin/cdk gene promoterssuccessively and, therefore, remain to be explored.

Finally, it has not yet been addressed how phosphor-ylation modulates the structure and function of theN-terminal region, given that there are six potentialphosphoacceptor sites localized in the N-terminal re-gion. Further exploration on this topic may help clari-fying the tumor-suppressive activity that requires theN-terminal region.

Rb Conducts Cell Cycle Orchestration beyond the G1Phase

The presence of the Rb–hSWI/SNF complex in Sphase and continued repression of the cyclin A andcdc2 genes suggests a role for Rb in the remote controlof S phase exit and subsequent M phase entry. Theseobservations are consistent with another study show-ing that Rb/E2F-mediated G1 regulation coordinateswith the mechanism that controls G2/M transition[89]. It has been shown that, in S phase, cyclin A/cdk2phosphorylates Cdh1, the substrate-specific activatorof the anaphase-promoting complex (APC), therebyblocking the activity of this ubiquitin ligase in degrad-ing cyclin B. Conversely, Rb–E2F-mediated repressionof cyclin A expression leads to the activation of APC,which degrades cyclin B, presumably creating a low-cyclin-B environment for the later induction of cyclin Bduring M phase entry. It can be predicted that furtherphosphorylation of Rb by cyclin E/cdk2 would relievethe repression of cyclin A expression, leading to accu-mulation of cyclin B and cdc2, which allows movementinto M phase. Therefore, Rb not only plays a role incontrolling the cell cycle passage through the G1 re-striction point, but also coordinates the regulatory ma-chinery in G1 with that in S, G2, and M phase byorchestrating a cascade of cyclin induction and cdkactivation.

A more direct role for Rb in the cell cycle beyond G1phase and independent of G1 cyclin control has alsobeen implicated. It has been shown that phosphoryla-tion-site-mutated Rb not only has the capability ofarresting cell at G1 phase, but is also able to inhibit Sphase progression. Such an inhibitory effect cannot bebypassed through overexpression of G1 cyclins such as
cyclin E, suggesting a distinct role of Rb in S phase of
the cell cycle [90]. Taken together, these studies sug-gest that Rb orchestrates the progression of the entirecell cycle. Based on this model, inactivation of Rb willresult in the uncoupling of G1/S phase and G2/M phaseprogression, leading to additional defects besides im-proper growth control, particularly improper timing ofDNA synthesis and chromosome segregation.

Rb, a Signal Mediator of Cell Cycle Control

It is not surprising that cell cycle activation by manytypes of mitogenic stimuli requires relief of Rb-medi-ated cell cycle inhibition. Similarly, cell cycle arrest byantiproliferative signals largely depends on Rb activ-ity. Rb also mediates cell cycle regulation in responseto nonmitogenic signals [49]. Moreover, negative regu-latory machinery intrinsic to the cell cycle itself existsand also functions through Rb (Fig. 1). This negativeregulatory machinery is considered to comprise the cdkinhibitors [15]. Two major classes of cdk inhibitorshave been defined. One is INK4 (inhibitors of cdk4)proteins that specifically target the cyclin-D-dependentkinases, such as cdk4 and cdk6. To date, four INK4proteins have been identified, including p16INK4a,p15INK4b, p18INK4c, and p19INK4d. The ability ofINK4 proteins to arrest the cell cycle in G1 phasedepends on the presence of functional Rb, implyingthat by inhibiting cyclin-D-dependent kinases, Rb re-mains hypophosphorylated and able to block G1 pro-gression [91–93]. Among INK4 proteins, loss of func-tion of p16INK4a occurs frequently in human cancer[94] and is considered to represent an additional meansthrough which the Rb pathway is disabled in cancercells.

Another class of cdk inhibitors is the Cip/Kip familyproteins, which include p21Cip1/WAF1, p27Kip1, andp57Kip2 [95]. Recent studies suggest that the Cip/Kipproteins function mainly to inhibit cdk2, rather thanfunction as general cdk inhibitors. Phosphorylation ofRb by cyclin D/cdk4,6 may even be facilitated by itsassociation with a Cip/Kip protein. Mitogen-dependentaccumulation of cyclin-D-dependent kinase sequestersCip/Kip proteins and subsequent Rb phosphorylation,thereby facilitating cyclin E–cdk2 activation. This isconsidered to complement the induction of cyclin E byRb–E2F-mediated transcription regulation [96]. Oncecyclin E-cdk2 is activated, it phosphorylates p27, lead-ing to the degradation of p27, which contributes to theirreversibility of passage through the restriction point.Therefore, Rb and p27, which is a haploinsufficienttumor suppressor [97], are functionally linked togetherby coordinating the passage of G1 phase cell cycle.Interestingly, like genetic inactivation of RB, haploin-sufficency of p27 also predisposes mice to pituitaryadenomas.

Recent studies showing that Rb is essential for G1/S

R[tas

8 ZHENG AND LEE

arrest in response to DNA damage have provided apotential link between Rb and p21. p21 is inducedtranscriptionally upon treatment of cells with a varietyof DNA-damaging agents and is believed to mediateG1/S arrest in DNA-damage-induced checkpoint con-trol [98, 99]. Interestingly, ionizing irradiation inhibitsthe specific phosphorylation of Rb by cdk2 in a p21-dependent manner [100], suggesting that Rb is a com-ponent of the p21-induced checkpoint control pathway.Therefore, feedback control of the cell cycle in responseto DNA damage signals also functions through theactivity of Rb. Thus, Rb appears to be a central medi-ator for cell growth and division signals.

RB AND DIFFERENTIATION

Rb, a Mediator of Differentiation Signals

Rb knockout mice exhibit developmental defects inneurogenesis and erythropoiesis [65, 66, 101]. It hasbeen demonstrated that these developmental defectsare not merely caused by ectopic mitosis and increasedapoptosis present in Rb-deficient embryos, but moresignificantly caused by loss of function in differentia-tion [102]. Rb-deficient embryos have abnormal neuro-nal differentiation as displayed by decreased or absentexpression of a group of neuronal differentiation mak-ers such as p75 NGFR, bII-tubulin, and trkA in thetrigeminal ganglia and dorsal root ganglia. Further-more, primary cultures of neuronal cells from theseregions of Rb-deficient embryos show decreased out-growth of axons compared with similar cultures fromwild-type embryos. Thus, these studies provide in vivoevidence suggesting that Rb is critical for cellular dif-ferentiation and embryonic development. By mediat-ing both differentiation and cell division signals, Rbmay function in coupling cell growth control with in-duction of differentiation during the developmentalprocess.

Rb Controls Terminal Differentiation Mediated byC/EBP Family Proteins

Rb has been shown to promote adipocyte differenti-ation by enhancing the DNA-binding and transactiva-tion activity of C/EBPb [103]. This study, showing thatRb is essential for adipose differentiation, provides di-rect evidence to support a role for Rb in cell differen-tiation. By similar approaches, a recent study alsosuggests an essential role for Rb in adipogenesis andreveals that C/EBPa may also mediate the activity of

b, although the precise mechanism is not known104]. NF-IL6, another C/EBP family transcription fac-or, which is important for the initiation of differenti-tion of leukocytes, was also activated by Rb [105]. In
um, these studies have revealed the C/EBP pathway
as a potential mechanism underlying the role of Rb indifferentiation (Fig. 1).

Rb Promotes Erythrocyte and Neuron Differentiationthrough Id2

Not only does Rb play a positive role in promotingterminal differentiation by C/EBP family proteins, butit also functions in removing those differentiationblocks that are set at certain stages of developmentalprocess, thereby maintaining the proper timing of dif-ferentiation (Fig. 1). A recent study suggests that un-restricted activation of Id2 underlies the defects indifferentiation of erythrocytes and neuronal cells ofRb-knockout mice [106]. Id2, which is required tomaintain the timing of differentiation in many pro-cesses of mammalian development and also serves as apositive regulator of cell cycle progression, is a target ofRb [107]. The Id family proteins are known to inhibitdifferentiation through sequestration of basic helix–loop–helix (bHLH) transcription factors. Specific inter-action between Rb and Id2 is proposed to sequesterId2, thereby relieving the inhibition of bHLH factors byId2 [108]. The defects in neurogenesis and erythropoi-esis caused by loss of Rb in the developing Rb2/2mouse embryo appear to correspond to the conse-quences of uncontrolled Id2 activity, as suggested bythe observation that a null mutation of Id2 both res-cues defects and prolongs the life span of Rb2/2 em-bryo to birth [106]. Conversely, overexpression of Id2can disrupt the antiproliferative effects of Rb andp16INK4a. Similarly, activation of Id2 transcription byMyc, presumably leading to the presence of excessiveamount of Id2, is able to bypass the inhibitory effect ofRb [106]. These results, therefore, suggest that thetumor suppression function of Rb could be compro-mised by oncogenic activation of the Myc–Id2 tran-scriptional pathway, implicating a link between Rb-mediated cell differentiation and tumor suppression.

Rb Controls Muscle Differentiation

Although Id2–Rb double-knockout mouse embryoshave minimal defects in neurogenesis and hematopoi-esis, they die at birth from severe reduction of muscletissue [106]. This observation suggests that Rb is es-sential for muscle differentiation that is mediated bythe MyoD family of basic helix–loop–helix proteins[109]. It has been shown that Rb augments the activityof MyoD, an early muscle transcription factor. More-over, MyoD-mediated transcriptional activation of cer-tain myogenic genes requires the presence of a func-tional Rb [110]. Other studies suggest Rb relieves aHBP1-imposed myogenic differentiation block, therebyactivating the expression of members of the MyoD fam-ily, including myogenin and MyoD itself [111]. There-
fore, the essential role of Rb in muscle differentiation

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Loss of Differentiation and Carcinogenesis

A recent study provides a direct link between a de-ficiency in cellular differentiation and carcinogenesis[112]. It was demonstrated that loss of Rb in miceresults in delayed melanotroph maturation as Rb2/2ells display impaired innervation. Failure to establishppropriate dopaminergic growth inhibitory nerve con-acts was suggested as the initial event of melanotrophalignant transformation, which in turn, promotes ab-

rrant cellular proliferation and eventually leads to theevelopment of melanotroph tumors of the pituitary.herefore, the requirement for Rb in the maintenancef a differentiated state for melanotrophs plays a keyole in the suppression of pituitary tumor carcinogen-sis.

b, a Mediator of Hormone Response

In addition to integrating differentiation and cellivision signals, Rb plays a role in integrating hor-onal signals that regulate general metabolic and cat-

bolic pathways. Rb interacts with Trip230, a thyroideceptor (TR) coactivator [113], thereby inhibiting thectivity of Trip230 in enhancing the transactivationctivity of TR in a thyroid-hormone-dependent manner113, 114]. This result suggests that Rb negativelyegulates the thyroid hormone signaling pathway.

Rb appears also to positively regulate the glucocor-icoid hormone signal pathway. It has been shown thatb enhances the transactivation activity of glucocorti-

oid receptor, suggesting a role of Rb in glucocorticoidormone response [115]. It remains, however, unclearhether these activities of Rb are involved in tumor

uppression.

b Activates Transcription Factors

The studies described above suggest that Rb acti-ates a group of transcription factor involved in cellu-ar differentiation. Such activity has also been ob-erved in studies showing that Rb cooperates with-jun to activate transcription [116]. This activity of Rb

also appears to be important for its function in tumorsuppression [55]. How Rb activates transcription is notcompletely understood. It was shown that Rb can en-hance the DNA-binding activity of C/EBP, but is notpresent in the DNA-binding complex, implying that Rbmay chaperone C/EBP to activate its transcriptionalactivity [103]. Such a chaperone-like activity of Rb mayexplain other functions in which Rb apparently plays a
positive regulatory role.
Rb-Related Proteins p107 and p130 in Cell CycleRegulation and Differentiation

p107 and p130 are proteins homologous to Rb withinthe A/B domains, which also bind to viral oncoproteinsand E2F [117]. Like Rb, p107 and p130 can inactivateE2F and inhibit the E2F-responsive promoter [118],recruit HDAC and CtIP/CtBP corepressors that ac-tively repress transcription [44, 119], and mediategrowth arrest [120–122]. There are also significantdifferences between Rb, p107, and p130. The spacerregion between the A/B domains in Rb is dispensablefor Rb function, whereas this region is conserved be-tween 107 and p130 and mediate their interactionswith cyclin E– and cyclin A–cdk2 complexes and theirinhibitory effect on these kinases [123–126]. Moreover,Rb, p107, and p130 preferentially bind to differentmembers of E2F family proteins during cell growth andterminal differentiation, suggesting that they have dis-tinct roles in cell cycle regulation and differentiation[19, 20]. Most distinctively, mice heterozygous for Rbmutations develop tumor [65, 66], whereas mice withp107 or p130 mutations do not [127, 128].

Nonetheless, Rb, p107, and p130 have partially over-lapping functions, particularly between p107 and p130[111]. Although mice homozygous for either p107 orp130 mutations are normal and fertile, mice homozy-gous for both mutations die perinatally, suggestingthat p107 and p130 can functionally substitute for eachother [127, 128]. Therefore, all these studies suggestthat Rb and Rb-related proteins have distinct but par-tially overlapping roles in tumor suppression and de-velopment.

RB: GENOME INTEGRITY AND CANCERSUSCEPTIBILITY

The roles of Rb in cell cycle regulation and differen-tiation are well established, and these roles can explainhow Rb suppresses tumor growth, but do not com-pletely explain why cancer susceptibility results fromloss of Rb function. In particular, why the inactivationof Rb leads to multiple genetic alterations that predis-pose cells for the process of tumorigenesis remainsunsolved. As discussed earlier, the susceptibility of thegenome to multiple genetic alterations can be attrib-uted to a loss of function in maintaining genome sta-bility. Such functions are known to rely on activitiesinvolved in DNA metabolism, chromosome structuralmaintenance and dynamic movement, and cell cyclecheckpoint control. Recent studies have implicated arole for Rb in these processes, thereby providing in-sights into the relationship between inactivation of Rb
and genome instability.

Ac itot

10 ZHENG AND LEE

Rb Modulates Chromosome Segregation

The structural proteins required for the reorganiza-tion of chromosomes after replication and proteins as-sociated with kinetochore assembly are two majorgroups of proteins that play an essential role in chro-mosome segregation [129–131]. Rb is associated withthese two groups of proteins via interactions with mi-tosin (aka CENP-F), a structural component of thekinetochore [132], and Hec1, a conserved regulator ofmultiple mitotic events [133–136] (Fig. 2).

During the chromosome reorganization process,linkage of duplicated DNA molecules, termed sisterchromatid cohesion, is established, and paired sisterchromatids undergo condensation [137–139]. Sisterchromatid cohesion and chromosome condensation arerequired for proper spindle attachments and chromo-some movements. Moreover, the separation of pairedchromatids is triggered by the dissolution of sisterchromatid cohesion at the metaphase–anaphase tran-sition. All of these steps must be precisely regulated toensure the faithful transmission of chromosomes. Hec1interacts with the SMC (structural maintenance ofchromosomes) family of proteins, which are chromo-some structural proteins essential for establishment of

FIG. 2. Rb regulates chromosome segregation and M phase progfaithful chromosome segregation, and M phase progression by assoc

mong them, Hec1, a recently identified protein, is a conserved reguhromatin reconfiguration, and ubiquitin-dependent proteolysis of m

sister chromatid cohesion and chromosome condensa-

tion (Fig. 2). Studies showing that chromosome segre-gation in either mammalian cells or yeast cells is se-verely disturbed when Hec1 is inactivated by eithermicroinjection of specific anti-Hec1 antibodies or byintroducing genetic mutations suggest that Hec1 isessential for chromosome segregation [133, 136, 140].This essential function of Hec1 appears to be mediatedin part by its interaction with SMC proteins [136].Such an interaction is important for the stable bindingof SMC1 proteins to DNA with high potential to formsecondary structures, which is a characteristic of SMC-associated chromatin regions in vivo [139].

The interaction between Rb and Hec1 occurs specif-ically in G2/M phase [133, 135]. It has been demon-strated that the fidelity of chromosome segregation inyeast cells is enhanced upon induced expression ofexogenous human Rb, and this activity requires thespecific interaction between Rb and Hec1 [135]. More-over, the DNA secondary-structure-binding activity ofSMC1 protein partially purified from these cells is alsoimproved, implicating a role for Rb in chromosomestructural maintenance. Rb, however, does not appearto directly participate in the DNA-binding activity ofSMC1, but appears to function as a chaperone through

sion. Rb is proposed to participate in mitotic chromatin remodeling,ion with protein phosphatase 1a (PP1a), mitosin, H-nuc, and Hec1.r of multiple mitotic events including kinetochore functions, mitoticic cyclins. Rb has also been implicated in the control of mitotic exit.

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modulating the activity of Hec1.


Kinetochore proteins are responsible for attachmentand movement of chromosomes along the microtubulesof the mitotic spindle. Rb specifically interacts withmitosin [132], which is proposed, based on its proteinstructure, to function in the kinetochore assembly.Hec1 is also localized to the centromeric regions, im-plicating a role of Hec1 in kinetochore function [133]. Itis possible that Rb, through interacting with mitosinand Hec1, modulates chromosome segregation by in-fluencing kinetochore activities (Fig. 2).

Rb Regulates Cell Cycle at G2/M Phase

The structural dynamics of chromosomes must becoordinated with the cell cycle regulatory machinery toexecute the precise order of mitotic events. Ubquitin-dependent proteolysis appears to be a major regulatorymechanism modulating M phase progression as well asother mitotic events. The APC complex has been dem-onstrated to be essential for the degradation of mitoticcyclins. Furthermore, it controls the onset of sisterchromatid separation and the metaphase–anaphasetransition by degradation of specific regulators of chro-mosome transmission such as Pds1, Cut2, and Ase1[141]. Rb was found to associate with H-nuc, the hu-man homolog of S. pombe nuc2 and S. cerevisiae Cdc27,an integral component of APC complex [53] (Fig. 2).

The association between Rb and Hec1 may also in-fluence the G2/M progression, since Hec1 directly in-teracts with 26S proteasome subunits that are shownto function in the control of degradation of mitoticcyclins during G2/M transition in yeast [142]. Hec1 canmodulate the activity of 26S proteasome subunit invitro, and consistently, ectopic expression of Hec1 inmammalian cells inhibits the degradation of mitoticcyclins such as cyclin A [134] (Fig. 2). Hec1 itself isphosphorylated by Nek2, a G2/M specific kinase, andthis phosphorylation is essential for cell survival, high-fidelity chromosome segregation, and G2/M progres-sion [143] (Fig. 2).

Although the manner in which Rb regulates H-nucand Hec1 to control mitotic cyclin degradation remainsunclear, several lines of evidence suggest a role for Rbin cell cycle transition during G2/M phases. First, asdiscussed earlier, phosphorylation of Rb in G1/Sphases has a coordinate effect on mitotic cyclin induc-tion and degradation in G2/M phases [89]. Second,overexpression of Rb in S phase arrests cells at G2phase [144]. Third, when treated with microtubule-destabilizing agents, cells lacking functional Rb do notfinish mitosis properly, but exit M phase and undergoa new cycle of DNA replication, leading to hyperploidy[145, 146] (Fig. 2). Finally, p53-mediated G2/M arrestin response to DNA damage requires the presence offunctional Rb [147]. Taken together, these results sug-
gest that, without functional Rb, mitotic division can-
not be completed in a controlled order, resulting inimproper chromosome segregation.

A role for Rb in G2/M phases is supported by studiesshowing the presence of hypophosphorylated Rb, thefunctional form of Rb, in these cell cycle stages. It hasbeen shown that Rb is dephosphorylated during G2/Mphases, and a protein phosphatase activity is sug-gested to be responsible [78, 148, 149]. Consistent withthis idea, Rb was found to interact with protein phos-phatase 1a specifically during G2/M phases [148].Whether protein phosphatase 1a is responsible for Rbdephosphorylation, however, remains to be explored.Alternatively, instead of being a modulator of Rb, pro-tein phosphatase 1a could be a target of Rb. The yeasthomolog of protein phosphatase 1a has been shown tobe essential for kinetochore function, execution of mi-totic kinetochore/spindle checkpoint, and faithful chro-mosome segregation [150, 151]. A regulatory role in theactivity of protein phosphatase 1a mediated by Rbwould be consistent with other potential activities ofRb in G2/M progression.

Rb Ensures Proper Timing of DNA Synthesis

Rb has been suggested to inhibit new DNA synthe-sis, that is, rereplication, before the completion of chro-mosome segregation and cytokinesis [145, 146]. Otherstudies suggest that functional Rb is required for in-hibiting reinitiation of DNA synthesis, that is, en-doreplication, in cell cycle progression beyond S phase.This activity is important to prevent initiation of DNAsynthesis during DNA-damage-induced G1/S andG2/M arrest [152]. Furthermore, a role for Rb in regu-lating initiation of DNA replication is suggested by itsbinding to a replication licensing factor MCM7 [74].Alternatively, Rb may control the initiation of DNAreplication indirectly through transcription regulationof genes involved in DNA replication. Such an activityin preventing DNA synthesis is necessary for properexecution of checkpoint controls and ensures the main-tenance of correct genome contents when cells reenterthe cell cycle. Together with the evidence showing thatRb is required for DNA-damage-induced G1/S andG2/M arrest, these results have revealed a potentialfunction for Rb in DNA-damage-induced checkpointcontrols.

Rb and Global Genomic Fluidity

An important feature of Rb function as revealed byrecent studies is associated with chromatin remodel-ing. Rb has been implicated in mitotic chromatin reor-ganization including the establishment of sister chro-matid cohesion and chromosome condensation asdiscussed earlier. Consistent with a role in chromatindynamics, Rb is found to physically associate with to-
poisomerase II, which is involved in multiple chroma-

12 ZHENG AND LEE

tin DNA metabolic processes including DNA replica-tion and chromosome segregation [153]. Top2, theyeast topoisomerase II, is essential for remodelingchromatin structure in favor of establishing sisterchromatid cohesion and chromosome condensation[154]. Therefore, Rb appears to participate in the pro-cess that keeps the chromatin structures in an orderlyfashion.

Interestingly, the transcription repression activity ofRb also features chromatin remodeling. The chromatinremodeling activity in transcription repression is totighten the association between histones and chroma-tin DNA, thereby stabilizing the nucleosome struc-tures, or to counteract the activity that disrupts theregular arrays of nucleosomes. This activity is similarto sister chromatid cohesion and chromosome conden-sation and keeps nucleosome structures in an unre-laxed orderly fashion. Although chromatin is decon-densed beyond M phase, it still maintains the orderlystructure of nucleosome arrays before replication andimmediately after replication and in DNA regions thatare not actively transcribed. Such an orderly chroma-tin structure is important to prevent the loss of geneticmaterial.

It has been conceived that genomic DNA must retaina degree of fluidity for the processes of DNA replicationand gene transcription. Apparently, such genome flu-idity must be properly controlled to keep the genomestable. How genome fluidity is controlled has not yetbeen studied. Interestingly, chromatin was found ingeneral to be more relaxed in Rb-deficient cells than innormal cells [155]. Therefore, it would be intriguing toexamine whether Rb plays a role in the control ofglobal genome fluidity.

RB: MORE THAN A GATEKEEPER

Rb is considered a gatekeeper that controls a thresh-old event leading to malignant transformation [156].Gatekeepers are expected to directly control cellgrowth. Other tumor suppressor genes, such as p53,APC, and WT1, share similar properties with Rb. Themutational inactivation or deletion of a tumor suppres-sor gene initiates transformation to the neoplastic sta-tus, whereas reintroduction of the wild-type tumorsuppressor gene into tumor cells suppresses tumorgrowth. Therefore, bona fide tumor suppressor genesconstitute a subgroup of cancer susceptibility genes.Genetic inactivation of this class of genes not onlyincreases the susceptibility to develop cancer, but alsorepresents a threshold event in tumorigenesis.

Another group of cancer susceptibility genes, exem-plified by MSH2, a gene involved in DNA mismatchrepair, does not encode tumor suppression activities.Functional inactivation of this group of genes destabi-
lizes the genome such that mutations in other genes
can accumulate more frequently; therefore, thesegenes appear to function as caretakers in the mainte-nance of genome stability [156]. The initiation of ma-lignant transformation, thus, relies on the subsequentmutational inactivation of gatekeepers or activation ofoncogene.

Apparently, bona fide tumor suppressors includingRb, p53, APC, WT1, etc., must be also responsible forthe cancer susceptibility. Loss of a gatekeeper functiondoes not readily cause multiple genetic alterations.Rather, loss of a caretaker function appears to be nec-essary for this process, therefore, suggesting that thesetumor suppressors also have caretaker activity inmaintaining genome integrity. As discussed earlier,recent studies do support a role of Rb in the mainte-nance of genome stability, that is, a caretaker functionfor Rb. In addition, p53 has also been proposed to be aguardian of the genome [157]. Other tumor suppres-sors may also exhibit caretaker functions, which re-mains to be explored.

Thus, tumor suppressor genes can be categorizedinto three classes. The first class of genes, representedby tumor suppressors such as Rb and p53, has bothcaretaker and gatekeeper functions. The second classof genes, represented by MSH2, has only caretakerfunction. It is possible that there is a third class oftumor suppressors that have only gatekeeper function.Loss of function of these genes would not increase thecancer susceptibility because they do not have care-taker function in maintaining genome stability. How-ever, loss of function of this class of genes, if it occursdue to somatic mutations, will facilitate transforma-tion to the neoplastic state. This class of tumor sup-pressors may be composed of those genes that aremutated in later developmental stage of cancers.Germline mutations in these genes, however, wouldnot increase the cancer incidence in human carriers orexperimental mouse carriers. A potential example ofthis class of genes is p130, an Rb-related gene. Micecarrying germline mutations of p130 do not have in-creased incidence of developing tumors; however, so-matic mutations of p130 are documented in a variety ofhuman cancers [158]. p130 appears to have gatekeeperfunction since reintroducing p130 to these cancer cellscan suppress neoplastic phenotypes [159].

An entire process of tumorigenesis initiated by he-reditary mutations of cancer susceptibility genesshould include events that cause loss of both caretakerand gatekeeper functions. It has not been carefullyexamined whether BRCA1 or BRCA2 has the capabil-ity of suppressing tumor growth, a gatekeeper func-tion. Unlike MSH2, BRCA1 and BRCA2 are not evolu-tionarily conserved proteins that function as basiccomponents of the DNA repair machinery. Rather,they share certain properties with Rb, including tran-
scription regulation of genes involved in the control of


cell cycle progression, differentiation and apoptosis.Interestingly, it has been shown that growth arrestinduced by overexpression of BRCA1 requires func-tional Rb [160], suggesting that BRCA1 may suppresstumor growth through an Rb-mediated cell cycle regu-latory pathway.

Although multiple genetic defects underlie the pro-cess of tumorigenesis, reconstitution of a single tumorsuppressor gene is able to suppress tumor formation.The success of tumor suppression by Rb in experimen-tal studies has provoked great expectation for cancergene therapy. However, it remains unanswered whyreconstitution of a single RB gene can suppress tumorformation even though multiple genetic defects haveoccurred. Nevertheless, this finding indicates that Rband other tumor suppressors such as p53, by servingmultiple functions in tumor suppression, can funda-mentally change the course of carcinogenesis, in whichloss of genome integrity, growth inhibition, and differ-entiation all exist. Similarly, an effective cancer treat-ment can only be achieved by those cancer therapiesthat are designed to challenge the cancer cells from allthese different angles or to change the microenviron-ment in which the cancer cells with unstable genomesevolve. By contrast, if a therapeutic strategy is mainlytargeted to an activity that controls cell growth, it canbe predicted that tumor cells are likely to become re-sistant to such treatment as a result of underlyinggenome instability and compensatory selection for mu-tations that inactivate other functions in cell growthcontrol. Therefore, understanding the precise functionof Rb and other tumor suppressor genes, particularlyin maintaining genome stability, is one of the majorchallenges for future research.

We are indebted to Drs. Nickolas Ting, Tom Boyer, and Paul Hastyfor their critical reading of this manuscript and their constructivecomments. We also thank Drs. Phang-Lang Chen and Yumay Chenfor many inspiring discussion. We apologize to our colleagues whosework was not cited because of space limitation. This work wassupported by NIH grants (EY05758 and CA58318) and Alice P.McDermott endowment funds to W.-H.L.

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eceived December 4, 2000ublished online February 8, 2001

the retinoblastoma gene: a prototypic and multifunctional tumor suppressor

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