The antiproliferative action of the retinoblastoma tumor suppressor protein, RB, is disrupted in the majority of human cancers. Disruption of RB activity occurs through several disparate mechanisms, including viral oncoprotein binding, deregulated RB phosphorylation, and mutation of the RB gene. Here we report disruption of RB-signaling in tumor cells through loss of a critical cooperating factor. We have previously reported that C33A cells fail to undergo cell cycle inhibition in the presence of constitutively active RB (PSM-RB). To determine how C33A cells evade RB-mediated arrest, cell fusion experiments were performed with RB-sensitive cells. The resulting fusions were arrested by PSM-RB, indicating that C33A cells lack a factor required for RB-mediated cell cycle inhibition. C33A cells are deficient in BRG-1, a SWI͞SNF family member known to stimulate RB activity. Consistent with BRG-1 deficiency underlying resistance to RB-mediated arrest, we identified two other BRG-1-deficient cell lines (SW13 and PANC-1) and demonstrate that these tumor lines are also resistant to cell cycle inhibition by PSM-RB and p16ink4a, which activates endogenous RB. In cell lines lacking BRG-1, we noted a profound defect in RB-mediated repression of the cyclin A promoter. This deficiency in RB-mediated transcriptional repression and cell cycle inhibition was rescued through ectopic coexpression of BRG-1. We also demonstrate that 3T3-derived cells, which inducibly express a dominant-negative BRG-1, arrest by PSM-RB and p16ink4a in the absence of dominant-negative BRG-1 expression; however, cell cycle arrest was abrogated on induction of dominant-negative BRG-1. These findings demonstrate that BRG-1 loss renders cells resistant to RB-mediated cell cycle progression, and that disruption of RB signaling through loss of cooperating factors occurs in cancer cells.cyclins ͉ Cdk ͉ SWI͞SNF T he retinoblastoma tumor suppressor protein (RB) is a critical regulator of cell cycle progression that is functionally inactivated in the majority of human tumors (1-8). RB functions as a protein-binding protein, binding to greater than 50 identified cellular proteins. However, the requirement of these proteins for RB-mediated cell cycle inhibition is largely unknown. Overall, RB-assembled protein complexes lead to the repression of transcription, and this function of RB is critical for cell cycle regulation. The principal target of RB is believed to be the E2F family of transcriptional activators (6, 9-11). E2F controls the expression of numerous genes directly involved in cell cycle progression or in metabolic processes coupled to DNA replication (6, 9-11). RB binding converts E2F from a transcriptional activator to a repressor through a mechanism that involves the recruitment of histone deacetylases (12, 13). RB also mediates the repression of other gene products, such as cyclin A, through complicated mechanisms that are not clearly understood (14).In response to mitogenic signaling, RB is phosphorylated in mid-G 1 by Cdk4͞cyclin D complexes (1-4)....

The retinoblastoma tumor suppressor protein (RB) is a potent inhibitor of cell proliferation. RB is expressed throughout the cell cycle, but its antiproliferative activity is neutralized by phosphorylation during the G 1 /S transition. RB plays an essential role in the G 1 arrest induced by a variety of growth inhibitory signals. In this report, RB is shown to also be required for an intra-S-phase response to DNA damage. Treatment with cisplatin, etoposide, or mitomycin C inhibited S-phase progression in Rb ؉/؉ but not in Rb ؊/؊ mouse embryo fibroblasts. Dephosphorylation of RB in S-phase cells temporally preceded the inhibition of DNA synthesis. This S-phase dephosphorylation of RB and subsequent inhibition of DNA replication was observed in p21Cip1 -deficient cells. The induction of the RB-dependent intra-S-phase arrest persisted for days and correlated with a protection against DNA damage-induced cell death. These results demonstrate that RB plays a protective role in response to genotoxic stress by inhibiting cell cycle progression in G 1 and in S phase.

Although RB inhibits the G 1 -S transition, the mechanism through which RB prevents cell cycle advancement remains unidentified. To delineate the mechanism(s) utilized by RB to exert its anti-proliferative activity, constitutively active RB proteins (which cannot be inactivated by phosphorylation) or p16ink4a (which prevents RB inactivation) were utilized. Both proteins inhibited the G 1 -S transition, whereas wildtype RB did not. We show that active RB acts to attenuate cyclin A promoter activity, and that overexpression of cyclin E reverses RB-mediated repression of the cyclin A promoter. Although cyclin A is an E2F-regulated gene, and it has been long hypothesized that RB mediates cell cycle advancement through binding to E2F and attenuating its transactivation potential, cyclin E does not reverse dominant negative E2F-mediated repression of the cyclin A promoter. Although active RB repressed both cyclin A and two other paradigm E2F-regulated promoters, only cyclin A transcription was restored upon co-expression of cyclin E. Additionally, we show that RB but not dominant negative E2F regulates the cyclin A promoter through the CCRE element. These data identify cyclin A as a downstream target of RBmediated arrest. Consistent with this idea, ectopic expression of cyclin A reversed RB-mediated G 1 arrest. The findings presented suggest a pathway wherein cyclin A is a downstream target of RB, and cyclin E functions to antagonize this aspect of RB-mediated G 1 -S inhibition.The retinoblastoma tumor suppressor protein (RB), 1 is functionally inactivated in over 60% of human tumors (1-3). The role of RB as a tumor suppressor has been well established, and it is known that RB can inhibit cellular proliferation by halting cell cycle progression (4 -6). RB carries out this growth inhibition through its ability to assemble and modulate a host of multiprotein complexes (5,7,8). At least four distinct proteinbinding domains of RB have been identified and extensively characterized, including: the A/B pocket, the large A/B pocket, the C-pocket, and the N-terminal domain (5, 7, 9). The large A/B pocket is the minimal growth suppressing region of RB and is required to bind the E2F family of transcription factors (10, 11).Binding of RB to proteins such as E2F is regulated by cyclindependent kinase (Cdk)-mediated phosphorylation (4, 5, 12). The full-length RB protein contains 16 consensus Cdk-phosphorylation sites, and phosphorylation at specific sites inhibits the binding of RB to cellular proteins, thereby disrupting the anti-proliferative activity of RB (13-19). Not surprisingly, therefore, overexpression of proteins which cause excessive or deregulated phosphorylation of RB is a common event in human tumors (2,3,20). For example, amplification of Cdk4 and/or its regulatory partner, cyclin D1, are frequently observed in human tumors. In either case, excessive Cdk4/cyclin D kinase activity results in deregulated phosphorylation and inactivation of RB. Similarly, loss of the tumor suppressor p16ink4a, which acts to attenuate C...

The retinoblastoma tumor suppressor protein (RB) is a negative regulator of the cell cycle that inhibits both G 1 and S-phase progression. While RB-mediated G 1 inhibition has been extensively studied, the mechanism utilized for S-phase inhibition is unknown. To delineate the mechanism through which RB inhibits DNA replication, we generated cells which inducibly express a constitutively active allele of RB (PSM-RB). We show that RB-mediated S-phase inhibition does not inhibit the chromatin binding function of MCM2 or RPA, suggesting that RB does not regulate the prereplication complex or disrupt early initiation events. However, activation of RB in S-phase cells disrupts the chromatin tethering of PCNA, a requisite component of the DNA replication machinery. The action of RB was S phase specific and did not inhibit the DNA damage-mediated association of PCNA with chromatin. We also show that RB-mediated PCNA inhibition was dependent on downregulation of CDK2 activity, which was achieved through the downregulation of cyclin A. Importantly, restoration of cyclin-dependent kinase 2 (CDK2)-cyclin A and thus PCNA activity partially restored S-phase progression in the presence of active RB. Therefore, the data presented identify RB-mediated regulation of PCNA activity via CDK2 attenuation as a mechanism through which RB regulates S-phase progression. Together, these findings identify a novel pathway of RB-mediated replication inhibition.The retinoblastoma tumor suppressor protein RB is a critical negative regulator of cell cycle progression (1, 24, 54, 6 2, 63). Extensive analysis has shown that RB functions as a protein-binding protein, assembling multiprotein complexes that regulate gene transcription (24,62,63). For example, RB binds to the E2F transcription factor and recruits histone deacetylase through an independent protein-binding module to repress specific E2F target genes (4, 42). Similarly, RB interacts with Brg-1 to mediate transcriptional repression of discrete target genes (57, 65). Naturally occurring mutant alleles of RB found in tumors invariably disrupt RB-mediated transcriptional repression function, lending credence to the hypothesis that this function is required to block inappropriate proliferation.Cell cycle progression is a highly ordered process driven by the activity of cyclin-dependent kinase (CDK)-cyclin complexes. Mitogenic signaling interfaces with the cell cycle machinery through the activation of D-type cyclins and their associated catalytic subunits (CDK4 or CDK6) (1, 52, 54). The CDK-cyclin D complexes initiate the phosphorylation of RB in mid-G 1 , with complete RB hyperphosphorylation mediated by CDK2-associated kinase activity (43). RB is the critical substrate for cyclin D-associated kinase activity, as cyclin D and CDK4 are not required in the absence of functional RB (38,40). In contrast, CDK2-cyclin E and -cyclin A complexes have additional substrates, as RB-deficient cells are readily arrested by attenuation of CDK2 activity (46). CDK-mediated phosphorylation of RB disrupts i...

Phosphorylation/inactivation of RB is typically required for cell cycle progression. However, we have identi®ed a tumor cell line, C33A, which progresses through the cell cycle in the presence of an active allele of RB (PSM-RB). To determine how C33A cells evade RB-mediated arrest, we compared RB signaling to downstream eectors in this resistant cell line to that of the RBsensitive SAOS-2 cell line. Although introduction of PSM-RB repressed E2F-mediated transcription in both C33A and SAOS-2 cells, PSM-RB failed to repress Cyclin A promoter activity in C33A. Ectopic expression of PSM-RB in SAOS-2 cells resulted in a decrease in both Cyclin A and Cdk2 protein levels without aecting Cyclin E or Cdk4. In contrast, over-expression of PSM-RB in C33A cells did not alter endogenous Cyclin A, Cyclin E, or Cdk2 protein levels or impact Cdk2 kinase activity, indicating that signaling from RB to downstream targets is abrogated in this cell line. The importance of Cdk2 activity was demonstrated by p27Kip1, which attenuated Cdk2 activity and inhibited cell cycle progression in C33A cells. Since RB signaling to Cdk2 is disrupted in these tumor cells, we coexpressed two proteins that cooperate with RB in transcriptional repression, AHR and BRG-1, in an attempt to correct this signaling dysfunction. Coexpression of AHR/BRG-1 with PSM-RB attenuated Cyclin A and Cdk2 expression as well as Cdk2-associated kinase activity, resulting in cell cycle inhibition of C33A cells. Importantly, ectopic expression of Cyclin A was able to reverse the arrest mediated by co-expression of AHR/BRG-1 with PSM-RB. These results indicate that down-regulation of Cdk2 activity is requisite for RB-mediated cell cycle arrest. Thus, this study reveals a new mechanism through which tumor cells evade anti-proliferative signals, and provides insight into how RB-signaling is mediated.