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Dicer initiates RNA interference by generating small RNAs involved in various silencing pathways. Dicer participates in centromeric silencing, but its role in the epigenetic regulation of other chromatin domains has not been explored. Here we show that Dicer1 deficiency in Mus musculus leads to decreased DNA methylation, concomitant with increased telomere recombination and telomere elongation. These DNA-methylation defects correlate with decreased expression of Dnmt1, Dnmt3a and Dnmt3b DNA methyltransferases (Dnmts), and methylation levels can be recovered by their overexpression. We identify the retinoblastoma-like 2 protein (Rbl2) as responsible for decreased Dnmt expression in Dicer1-null cells, suggesting the existence of Dicer-dependent small RNAs that target Rbl2. We identify the miR-290 cluster as being downregulated in Dicer1-deficient cells and show that it silences Rbl2, thereby controlling Dnmt expression. These results identify a pathway by which miR-290 directly regulates Rbl2-dependent Dnmt expression, indirectly affecting telomere-length homeostasis.
Cyclin D1/cyclin-dependent kinase 2 (Cdk2) complexes are present at high frequency in human breast cancer cell lines, but the significance of this observation is unknown. This report shows that expression of a cyclin D1-Cdk2 fusion protein under the control of the mouse mammary tumor virus (MMTV) promoter results in mammary gland hyperplasia and fibrosis, and mammary tumors. Cell lines isolated from MMTV-cyclin D1-Cdk2 (MMTV-D1K2) tumors exhibit Rb and p130 hyperphosphorylation and up-regulation of the protein products of E2F-dependent genes. These results suggest that cyclin D1/Cdk2 complexes may mediate some of the transforming effects that result from cyclin D1 overexpression in human breast cancers. MMTV-D1K2 cancer cells express the hepatocyte growth factor (HGF) receptor, c-Met. MMTV-D1K2 cancer cells also secrete transforming growth factor beta (TGFbeta), but are relatively resistant to TGFbeta antiproliferative effects. Fibroblasts derived from MMTV-D1K2 tumors secrete factors that stimulate the proliferation of MMTV-D1K2 cancer cells, stimulate c-Met tyrosine phosphorylation, and stimulate the phosphorylation of the downstream signaling intermediates p70(s6k) and Akt on activating sites. Together, these results suggest that deregulation of the Cdk/Rb/E2F axis reprograms mammary epithelial cells to initiate a paracrine loop with tumor-associated fibroblasts involving TGFbeta and HGF, resulting in desmoplasia. The MMTV-D1K2 mice should provide a useful model system for the development of therapeutic approaches to block the stromal desmoplastic reaction that likely plays an important role in the progression of multiple types of human tumors.
It is thought that G(1) cyclin/CDK mediated phosphorylation of pocket proteins from mid G(1) to mitosis is reversed via dephosphorylation in mitosis. We examined the mechanisms involved in the unexpectedly rapid dephosphorylation of the pocket proteins induced via inhibition of cellular protein synthesis by cycloheximide (CHX) as well as direct inhibition of CDKs by flavopiridol. CHX and flavopiridol-induced dephosphorylation of pocket proteins is attributable to inactivation of D-type cyclin/CDKs and G(1)/S CDKs, respectively, which unmasks a phosphatase activity that targets the three pocket proteins apparently throughout the cell cycle. Treatment of cells with phosphatase inhibitors at concentrations selective for PP2A inhibition prevents CHX and flavopiridol-mediated dephosphorylation of pocket proteins in vivo. Also, ectopic expression of SV40 small t antigen, which inhibits PP2A via disruption of trimeric PP2A holoenzymes, delays CHX-induced pocket protein dephosphorylation. Moreover, dephosphorylation of p130 and p107 in cell extracts is inhibited by concentrations of okadaic acid known to inhibit PP2A, but not PP1. Finally, the PP2A catalytic subunit (PP2A/C) specifically interacts with both p130 and p107 in quiescent cells as well as cells progressing throughout the cell cycle. Together, these results demonstrate that the overall phosphorylation state of pocket proteins is determined, at least in part, by a dynamic equilibrium between CDKs and PP2A, or a closely related PP2A-like enzyme. These findings have important implications, as cell cycle or checkpoint-dependent inhibition of CDK activities counteracted by an active PP2A should have imminent effects on the phosphorylation state and activities of pocket proteins.
Transforming growth factor beta (TGF-beta) induces cell cycle arrest of most nontransformed epithelial cell lines. In contrast, many human carcinomas are refractory to the growth-inhibitory effect of TGF-beta. TGF-beta overexpression inhibits tumorigenesis, and abolition of TGF-beta signaling accelerates tumorigenesis, suggesting that TGF-beta acts as a tumor suppressor in mouse models of cancer. A screen to identify agents that potentiate TGF-beta-induced growth arrest demonstrated that the potential anticancer agent rapamycin cooperated with TGF-beta to induce growth arrest in multiple cell lines. Rapamycin also augmented the ability of TGF-beta to inhibit the proliferation of E2F1-, c-Myc-, and (V12)H-Ras-transformed cells, even though these cells were insensitive to TGF-beta-mediated growth arrest in the absence of rapamycin. Rapamycin potentiation of TGF-beta-induced growth arrest could not be explained by increases in TGF-beta receptor levels or rapamycin-induced dissociation of FKBP12 from the TGF-beta type I receptor. Significantly, TGF-beta and rapamycin cooperated to induce growth inhibition of human carcinoma cells that are resistant to TGF-beta-induced growth arrest, and arrest correlated with a suppression of Cdk2 kinase activity. Inhibition of Cdk2 activity was associated with increased binding of p21 and p27 to Cdk2 and decreased phosphorylation of Cdk2 on Thr(160). Increased p21 and p27 binding to Cdk2 was accompanied by decreased p130, p107, and E2F4 binding to Cdk2. Together, these results indicate that rapamycin and TGF-beta cooperate to inhibit the proliferation of nontransformed cells and cancer cells by acting in concert to inhibit Cdk2 activity.
Crk-associated substrate (p130(Cas), Cas) is a docking protein first recognized as having elevated phosphotyrosine content in mammalian cells transformed by v-Src and v-Crk oncoproteins. Subsequent studies have implicated Cas in the control of normal cell behavior through its roles in integrin-mediated signal transduction and organization of the actin cytoskeleton at sites of cell adhesion. In this study, we sought to gain new insight into normal Cas function by identifying previously unrecognized interacting proteins. A yeast two-hybrid screen using the C-terminal region of Cas as a bait identified the Src homology 3 (SH3) domain of the mouse "nephrocystin" protein-orthologous to a human protein whose loss of function leads to the cystic kidney disease familial juvenile nephronophthisis. The putative full-length mouse and partial canine nephrocystin sequences were deduced from cDNA clones. Additional studies using epitope-tagged mouse nephrocystin indicated that nephrocystin and Cas can interact in mammalian cells and revealed that both proteins prominently localize at or near sites of cell-cell contact in polarized Madin-Darby canine kidney epithelial cells. Our findings provide novel insight into the normal cellular activities regulated by both Cas and nephrocystin, and raise the possibility that these proteins have a related function in polarized epithelial cells.
Copyright 2000 Academic Press.
Binding of the CXC chemokine, melanoma growth stimulatory activity (MGSA), to the class II IL-8 receptor on cells which overexpress this G-protein coupled receptor results in enhanced phosphorylation on serine residues. In experiments described herein, it is demonstrated that MGSA also enhances the tyrosine phosphorylation of two endogenously tyrosine phosphorylated proteins approximately 130 and 70 kDa in size. MGSA treatment (5 nM) of the clonally selected, stably transfected placental cell line, 3ASubE P-3, which overexpresses the class II IL-8 receptor, results in the maximal tyrosine phosphorylation of the 130 kDa protein before 2 min. This enhanced phosphorylation of the 130 kDa protein returns to basal level after a 5 min treatment. Based upon cell fractionation studies, the 130 kDa protein is concentrated in the membrane fraction of the cells. The 70 kDa protein which also shows tyrosine phosphorylation is predominantly cytosolic. The identity of the 130 kDa tyrosine phosphorylated protein was determined by immunoprecipitation and Western blot analyses. In these experiments, the 130 kDa tyrosine phosphorylated protein was shown to immunoprecipitate with antibody to the cas antigen (crk-associated substrate) and with antibody to the p130 tyrosine phosphorylated protein described as undergoing tyrosine phosphorylation in src transformed cells. The data suggest that MGSA binding to the class II IL-8 receptor is associated with tyrosine phosphorylation of p130/cas. The data also suggest that p130 and the cas antigen are the same protein.