Regulation of Cellular Genes in a Chromosomal Context by the Retinoblastoma Tumor Suppressor Protein

Abstract

The retinoblastoma tumor suppressor gene product (pRb) is involved in controlling cell cycle progression from G 1 into S. pRb functions, in part, by regulating the activities of several transcription factors, making pRb involved in the transcriptional control of cellular genes. Transient-transfection assays have implicated pRb in the transcription of several genes, including c-fos, the interleukin-6 gene, c-myc, cdc-2, c-neu, and the transforming growth factor ␤2 gene. However, these assays place the promoter in an artificial context and exclude the effects of far 5 upstream regions and chromosomal architecture on gene transcription. In these experiments, we have studied the role of pRb in the control of cell cycle-related genes within a chromosomal context and within the context of the G 1 phase of the cell cycle. We have used adenovirus vectors to overexpress pRb in human osteosarcoma cells and breast cells synchronized in early G 1 . By RNase protection assays, we have assayed the effects of this virus-produced pRb on gene expression in these cells. These results indicate that pRb is involved in the transcriptional downregulation of the E2F-1, E2F-2, dihydrofolate reductase, thymidine kinase, c-myc, proliferating-cell nuclear antigen, p107, and p21/Cip1 genes. However, it has no effect on the transcription of the E2F-3, E2F-4, E2F-5, DP-1, DP-2, or p16/Ink4 genes. The results are consistent with the notion that pRb controls the transcription of genes involved in S-phase promotion. They also suggest that pRb negatively regulates the transcription of two of the transcription factors whose activity it also represses, E2F-1 and E2F-2, and that it plays a role in downregulating the immediate-early gene response to serum stimulation.

Molecular characterization of apoptosis induced by CARF silencing in human cancer cells

“…Cell cycle progression requires phosphorylation of RB by cyclin-dependent kinases and their partner cyclins to free E2F proteins and subsequent transactivation of a plethora of cell cycle regulating genes. 6 Hypophosphorylated RB binds to and inhibits the activity of E2F proteins, leading to suppression of a variety of genes including those required for cell cycle progression. Further, RB and E2F proteins have been implicated in regulation of apoptosis, wherein, hypophosphorylated RB binds to E2F1, leading to growth arrest followed by apoptosis.…”

“…5 Regulation of the cell survival and death processes has been largely attributed to p53-dependent and p53-independent pathways involving retinoblastoma (RB), E2F1, p21 WAF1 , Ras-mitogen-activated protein kinases (MAPK) and ataxia telangiectasia mutated (ATM)/ATM-and RAD3-related (ATR) functions. [6][7][8][9] The latter serve as prime mediators of the DNA damage response, instigating apoptosis through Ras-MAPK, RB-E2F1 and ARF-p53-p21 WAF1 or mitotic DNA damage checkpoint mediated by the BRCA1 and CHK1 pathways. [10][11][12][13][14] In the present study, we report that the suppression of CARF induces MC accompanied by activation of the mitochondrial stress and caspase-dependent pathways via induction of DNA damage and disruption of the cell cycle checkpoint regulation, culminating into apoptosis of cancer cells.…”

Antisense-mediated depletion of p300 in human cells leads to premature G 1 exit and up-regulation of c- MYC

“…MSU cells are v-MYC transformed, nontumorigenic, near diploid human fibroblasts with normal morphology. The growth conditions for these two cell lines were described earlier (14,15). AS-1 and AS-2 are two replication defective adenovirus (Ad) vectors that express the 5Ј 1784 or 3905 nucleotides, respectively, of p300 sequences in an AS orientation under the CMV promoter in the E1A region.…”

“…CDK assays were carried out either with ␣-cyclin E or ␣-cyclin A antibodies, as described (19). Procedures for RNA extraction and the details of the probes used for assaying c-MYC, p21, and GAPDH mRNAs are described in a previous report (14). The riboprobe for carbomyl phosphate synthetase, aspartate transcarbomylase and dihydroorotase (CAD) was a gift of P. Farnham, University of Wisconsin.…”

“…MCF10A cells synchronized by serum starvation and infected with appropriate Ad vectors were harvested at different time intervals for FACS analysis or lysed with RIPA buffer (14) for immunoblotting and a kinase lysis buffer for CDK assays (19). CDK assays were carried out either with ␣-cyclin E or ␣-cyclin A antibodies, as described (19).…”