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)....

Truncating mutations and homozygous deletions in the hSNF5/INI1/BAF47 subunit of human SWI/SNF complexes occur in most malignant rhabdoid tumors and some other malignancies. How loss of hSNF5 contributes to tumorigenesis remains unknown. Because the SWI/ SNF subunit BRG1 is required for RB-mediated cell cycle arrest, we hypothesized that hSNF5 deficiency disrupts RB signaling. Here we demonstrate that unlike BRG1, hSNF5 deficient cells retain functional RB since ectopic expression of either p16ink4a or a constitutively active form of RB (PSM -RB) led to cell cycle arrest. To determine how hSNF5 loss might contribute to tumorigenesis, we used a retrovirus to introduce hSNF5 into multiple deficient cell lines. In all cases, re-expression inhibited colony formation and induced cell cycle arrest characterized by a flattened morphology. Flow cytometry revealed that these cells accumulated in G 0 /G 1 . Importantly, arrested cells exhibited strong induction of p16ink4a, hypophosphorylated RB, and down-regulation of cyclin A, suggesting that hSNF5 signals upstream of RB to induce growth arrest. Co-expression of SV40 T/t abolished hSNF5-induced G 1 arrest and activation of RB. Likewise, HPV-16 E7 was sufficient to partially overcome cell cycle arrest. These results suggest that hSNF5 loss is not equivalent to BRG1/ BRM loss in human tumor cell lines. Furthermore, hSNF5-induced cell cycle arrest of deficient cells is mediated in part through activation of p16ink4a expression. These findings provide insight into mechanisms of hSNF5-mediated tumor suppression.

Mammalian cells express two homologs of the SWI2 subunit of the SWI/SNF chromatin-remodeling complex called BRG1 and BRM. Whether the SWI/SNF complexes formed by these two subunits perform identical or dierent functions remains an important question. In this report, we show concomitant downregulation of BRG1 and BRM in six human tumor cell lines. This down-regulation occurs at the level of mRNA abundance. We tested whether BRM could aect aberrant cellular functions attributed to BRG1 in tumor cell lines. By transient transfection, we found that BRM can restore RB-mediated cell cycle arrest, induce expression of CD44 protein and suppress Cyclin A expression. Therefore, BRM may be consistently downregulated with BRG1 during neoplastic progression because they share some redundant functions. However, assorted tissues from BRM null/BRG1-positive mice lack CD44 expression, suggesting that BRM-containing SWI/SNF complexes regulate expression of this gene under physiological conditions. Our studies further de®ne the mechanism by which chromatin-remodeling complexes participate in RB-mediated cell cycle arrest and provide additional novel evidence that the functions of SWI/SNF complexes containing BRG1 or BRM are not completely interchangeable.

Aberrant regulation of CD44, a transmembrane glycoprotein, has been implicated in the growth and metastasis of numerous tumors. Although both CD44 overexpression and loss have been implicated in tumor progression, the mechanism of CD44 down-regulation in these tumor types is not known. By immunoblot and reverse transcription-polymerase chain reaction analysis we determined that a cervical carcinoma cell line, C33A, lacks CD44 expression. To determine how CD44 is down-regulated in C33A cells, we utilized cell fusions of C33A cells with a CD44-expressing cell line (SAOS-2). We found that SAOS-2 fusion restored CD44 expression in C33A cells, suggesting that a trans-acting factor present in SAOS-2 cells promotes CD44 production. C33A cells are BRG-1-deficient, and we found that CD44 was absent in another BRG-1-deficient tumor cell line, indicating that loss of BRG-1 may be a general mechanism by which cells lose CD44. Reintroduction of BRG-1 into these cells restored CD44 expression. Furthermore, disruption of BRG-1 function through the use of dominant-negative BRG-1 demonstrated the requirement of BRG-1 in CD44 regulation. Finally, we show that Cyclin E overexpression resulted in the attenuation of CD44 stimulation, which is consistent with previous observations that Cyclin E can abrogate BRG-1 action. Taken together, these results suggest that BRG-1 is a critical regulator of CD44 expression, thus implicating SWI/SNF components in the regulation of cellular adhesion and metastasis.The CD44 family of transmembrane glycoproteins has been implicated in cell-cell and cell-matrix adhesion, cell growth, and metastasis (1-3). A number of different CD44 proteins are produced through alternative RNA splicing, and these proteins are extensively modified. Many tumors express higher than normal levels of total CD44 protein as well as splice variants that do not occur in normal cells (1,3, 4). How CD44 expression is regulated in normal cells and in tumors is poorly understood.A role for CD44 in tumor progression has been documented in numerous clinical and experimental studies (1, 2). Ectopic expression of some forms of CD44 can enhance metastasis and tumor growth both in vitro and in vivo (5-7). It is believed that CD44 expression in some tumors increases as the tumor becomes more proliferative and invasive (1). These findings suggest that CD44 might be regulated by environmental or genetic factors that have been shown to contribute to cancer progression. The expression of activated oncogenes like v-Ras, v-Src, and v-Fos, which promote transformation and invasion, have been reported to induce CD44 expression (8 -10). In addition, the epidermal growth factor receptor has also been shown to stimulate CD44 (10,11).In contrast to studies that correlate CD44 overexpression with cancer progression, a significant number of reports also indicate that loss of CD44 expression can contribute to tumorigenesis (12). Specifically, it has been shown that loss of CD44 in cervical carcinomas, neuroblastomas, prostate carcinomas, melanomas, an...

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...