Abstract
The p53 tumor suppressor protein regulates the transcription of regulatory genes involved in cell cycle arrest and apoptosis. We reported previously that overexpression of p202, an interferon-inducible negative regulator of cell growth, negatively regulates the transcriptional activity of p53. Now we identify the gene encoding p202 as one whose mRNA and protein expression decrease in cells following the expression of wild-type, but not mutant, p53. Furthermore, the levels of p202 also decrease after exposure of cells to ultra violet light, which correlate with increase in the levels of p53. We report that the sequence-specific DNA binding of p53 to the 5'-regulatory region of the 202 gene contributes to the transcriptional repression of the 202 gene. Interestingly, overexpression of p202 in cells induced to undergo p53-dependent apoptosis significantly delays this process, indicating that the negative regulation of the 202 gene by wild-type p53 is important to potentiate apoptosis.
Highlights
The protein, p202, is an interferon (IFN)1-inducible phosphoprotein (52 kDa) whose ectopic expression in a variety of cell types retards proliferation [1,2,3,4]
We report that the sequence-specific DNA binding of p53 to the 5-regulatory region of the 202 gene contributes to the transcriptional repression of the 202 gene
Expression of Wild-type p53 Results in a Decrease in p202 Levels—To test if the expression of wild-type p53 regulates the expression of p202, we generated a stable cell line of murine AKR-2B fibroblasts
Summary
Cell Cultures, Antibodies, and Reagents—Murine AKR-2B cells To express the temperature-sensitive mutant of p53, murine AKR-2B fibroblasts were cotransfected with plasmid pCMV53Val135 and pCMV (in 10:1 ratio) and the transfected cells were selected in G418 (500 g/ml). To initiate apoptosis in the pooled transfected cells, cells were cultured without Zeocin and were shifted to the permissive temperature (32.5 °C) for the indicated times. Gel Electrophoretic Mobility Shift Assays—Nuclear extracts were prepared from murine AKR-2B cells treated with UV-C (5 mJ/m2) for 24 h or untreated cells as described previously [36]. Luciferase Assays—For reporter assays, subconfluent cells in six-well plates were transfected with the reporter plasmids 202-luc (5 g) and pRL-TK (0.5 g) along with indicated amounts of protein expression plasmid using the calcium phosphate transfection system (Life Technologies, Inc.), as suggested by the supplier. The luciferase activity in control vector-transfected cells is shown as 1
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