[Show abstract][Hide abstract] ABSTRACT: hSNF5, the smallest member of the SWI/SNF chromatin remodeling complex, is lost in most malignant rhabdoid tumors (MRT). In MRT cell lines, reexpression of hSNF5 induces G1 cell cycle arrest, elevated p16INK4a, and activated replicative senescence markers, such as beta-galactosidase (beta-Gal) and plasminogen activator inhibitor-1. To compare the replicative senescence caused by hSNF5 in A204 cells to normal cellular senescence, we examined the activation of both p16INK4a and p21CIP/WAF1. Analogous to normal cellular senescence, both p16INK4a and p21CIP/WAF1 were up-regulated following hSNF5 restoration. Furthermore, we found that hSNF5 bound the p16INK4a and p21CIP/WAF1 promoters, suggesting that it directly regulates transcription of these genes. Using p16INK4a RNA interference, we showed its requirement for the replicative senescence caused by hSNF5 but not the growth arrest. Instead, p21CIP/WAF1 remained activated by hSNF5 in the absence of high p16INK4a expression, apparently causing the growth arrest in A204. Interestingly, we also found that, in the absence of p16INK4a, reexpression of hSNF5 also increased protein levels of a second cyclin-dependent kinase (CDK) inhibitor, p18INK4c. However, our data show that lack of hSNF5 does not abrogate cellular responsiveness to DNA damage or growth-inhibitory factors. In summary, our studies suggest that hSNF5 loss may influence the regulation of multiple CDK inhibitors involved in replicative senescence.
[Show abstract][Hide abstract] ABSTRACT: 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 G0/G1. 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 G1 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.
[Show abstract][Hide abstract] ABSTRACT: 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 different functions remains an important question. In this report, we show concomitant down-regulation 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 affect 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 down-regulated 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 define 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.
[Show abstract][Hide abstract] ABSTRACT: The BRG-1 subunit of the SWI-SNF complex is involved in chromatin remodeling and has been implicated in the action of the retinoblastoma tumor suppressor (RB). Given the importance of BRG-1 in RB function, germ line BRG-1 mutations in tumorigenesis may be tantamount to RB inactivation. Therefore, in this study we assessed the behavior of cells harboring discrete BRG-1 alleles for the RB-signaling pathway. Using p16ink4a, an upstream activator of endogenous RB, or a constitutively active RB construct (PSM-RB), we determined that the majority of tumor lines with germ line defects in BRG-1 were sensitive to RB-mediated cell cycle arrest. By contrast, A427 (lung carcinoma) cells were resistant to expression of p16ink4a and PSM-RB. Analysis of the SWI-SNF subunits in the different tumor lines revealed that A427 are deficient for BRG-1 and its homologue, Brm, whereas RB-sensitive cell lines retained Brm expression. Similarly, the RB-resistant SW13 and C33A cell lines were also deficient for both BRG-1/Brm. Reintroduction of either BRG-1 or Brm into A427 or C33A cells restored RB-mediated signaling to cyclin A to cause cell cycle arrest. Consistent with this compensatory role, we observed that Brm could also drive expression of CD44. We also determined that loss of these core SWI-SNF subunits renders SW13 cells resistant to activation of the RB pathway by the chemotherapeutic agent cisplatin, since reintroduction of either BRG-1 or Brm into SW13 cells restored the cisplatin DNA-damage checkpoint. Together, these data demonstrate that Brm can compensate for BRG-1 loss as pertains to RB sensitivity.
Preview · Article · Mar 2002 · Journal of Biological Chemistry
[Show abstract][Hide abstract] ABSTRACT: We have previously described a functional model for identification of human liver tumor suppressor genes in which human chromosome 11 was introduced into rat liver epithelial tumor cell lines via microcell-mediated chromosome transfer, producing microcell hybrid (MCH) cell lines that exhibit suppression of tumorigenicity in vivo. Chromosome deletion mapping studies identified a 950-kb region of 11p11.2-p12 that was retained in all suppressed MCH cell lines, suggesting that this region may harbor one or more genes with liver tumor suppressor function. In this study, we generated a comprehensive transcription map of the 11p11.2-p12 liver tumor suppressor region through examination of 142 expressed sequence tag (EST) markers among a group of suppressed MCH cell lines. Of 142 ESTs examined, 19 were localized within the 11p11.2-p12 liver tumor suppressor region. RT-PCR analysis of gene expression for these 19 ESTs among an index panel of suppressed MCH cell lines (n = 3) identified 11 potential candidate liver tumor suppressor genes. Examination of candidate gene expression among six additional suppressed MCH cell lines reduced the number of potential candidate genes to three (stSG30184, stSG10014, and stSG29748). Northern blot analysis of suppressed MCH cell lines and derived tumor cell lines suggested stSG30184 as the best candidate liver tumor suppressor gene. The 3.7 kb stSG30184 transcript was expressed by all suppressed MCH cell lines, but expression was extinguished coordinately with reexpression of tumorigenicity by these cells, consistent with a tumor suppressor gene. Subsequent characterization of this EST indicates that it is a novel transcript with expression in a broad range of tissue types. Further characterization of the genes identified in this study will provide a greater understanding of their role in the molecular pathogenesis of neoplastic liver disease.
Full-text · Article · Jan 2002 · Genes Chromosomes and Cancer
[Show abstract][Hide abstract] ABSTRACT: The organization of genomic DNA into chromatin aids in the regulation of gene expression by limiting the access of transcriptional binding domains. The SWI/SNF family of chromatin-remodeling complexes, which are conserved from yeast to humans, open the chromatin to facilitate the transcriptional machinery to access their targets. The gene encoding the BAF47/hSNF5 subunit of the complex has been found mutated in both rhabdoid cell lines and in primary rhabdoid tumors. Since the pediatric tumors rhabdomyosarcoma (RMS) and Wilms' tumor (WT) share a similar genetic link with rhabdoid tumors, it was hypothesized that they may also show alterations of the BAF47 gene. Using primary tumors, the BAF47 protein was detected in all WT but less than 75% of the RMS tested. In cell lines, the BAF47 protein was missing in all rhabdoid cell lines and one RMS cell line. Analysis of sample DNA displayed either a mutation or deletion of the BAF47 gene in all samples negative for the protein. Several other subunits of the human SWI/SNF complex, including BRG1 which is the subunit directly interacting with the Rb tumor suppressor gene, were detected in all tumor samples. Alteration of BAF47 may be a genetic marker associated with the poor prognosis seen in all rhabdoid tumors but only some RMS.