Transcriptional silencing of tumor suppressor genes in association with DNA methylation contributes to malignant transformation. However, the specific DNA methyltransferases that initiate this process are unknown. Here we show that a de novo DNA methyltransferase, DNMT3b, substantially contributes to the oncogenic phenotype in a lung cancer model. Normal human bronchial epithelial (NHBE) cells expressing telomerase, SV40 large T antigen, and activated Ras were immortal, formed colonies in soft agar, and expressed DNMT3b. Antisense suppression of DNMT3b prevented soft agar growth. Furthermore, mouse embryo fibroblasts expressing T antigen and Ras formed soft agar colonies and large tumors, but fibroblasts from Dnmt3b À/À mice did not grow in soft agar and were much less tumorigenic in vivo. The tumor suppressor genes, FHIT, TSLC1, and RASSF1A were downregulated in transformed NHBE cells, and antisense DNMT3b treatment resulted in reexpression of FHIT and TSLC1. While expression of TSCL1 correlated with methylation of CpG dinucleotides in its promoter region, the expression of FHIT did not, suggesting that DNMT3b may silence genes by several mechanisms including direct DNA methylation or recruitment of proteins that modify chromatin. Regardless of mechanism, our data indicate that DNMT3b plays an important role in transformation.
The retinoblastoma family proteins pRB, p107, and p130 are phosphorylated and released from E2Fs in the late G 1 phase of the cell cycle. This phosphorylation is thought to contribute to the derepression of E2F-responsive genes and to be mediated, in part, by Cdk4 and Cdk6. Evidence that Cdk4/6 activity is inhibited by p16 INK4A in most pRB(؊) cells suggests that p107 and p130 may be underphosphorylated and remain associated with E2Fs during G 1 -S progression in cells that lack pRB. To examine this, we evaluated the cell cycle-dependent phosphorylation and E2F binding abilities of p107 and p130 in pRB(؊), p16(؉) Saos-2 osteosarcoma cells. p130, but not p107, was phosphorylated and released from E2F-4 in late G 1 and S phase cells, although p130 phosphorylation differed qualitatively in these and other pRB(؊), p16(؉) cells as compared with pRB(؉), p16(؊) cell types. p130 phosphorylation occurred in the absence of cyclin D-Cdk4/6 complexes, coincided with cyclin E-and Cdk2-associated kinase activity, and was prevented by expression of dominant negative Cdk2. Moreover, dominant negative Cdk2 prevented the dissociation of endogenous p130-E2F-4 complexes and inhibited E2F-4-dependent transcription. These findings show that p130 can be phosphorylated and functionally inactivated in a Cdk2-dependent process, and they highlight the involvement of distinct Cdks in the regulation of different pRB family proteins.
The retinoblastoma protein (pRB) is thought to suppress tumorigenesis, in part, through interactions with E2F transcription factors. However, certain low penetrance pRB mutants substantially reduce tumor incidence despite having a minimal ability to bind E2F. These low penetrance mutants retain the ability to induce a senescence-like state, suggesting that they may suppress tumorigenesis through a senescence-associated process. Here, we identify a novel pRB function that is associated with senescence and which is retained by non-E2F binding low penetrance pRB mutants. It was found that pRB and these mutants substantially increased the production of PML nuclear bodies (NBs). In keeping with the role of PML in transcriptional repression, pRB also promoted PML-dependent transcriptional repression by the c-Myc antagonist Mad1. In a series of pRB-p130 chimeric proteins, the ability to increase NB production correlated with the ability to induce a senescence-like phenotype. However, neither NB formation nor PML function were required for pRB to induce the senescencelike response. Together, these observations indicate that a pRB-induced increase in PML NB formation is coordinated with, but separable from, the pRB-induced senescence program. The data further suggest that PML may contribute to an E2F-independent tumor suppressor function of pRB.
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