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P130 and its truncated form mediate p53-induced cell cycle arrest in Rb(-/-) Saos2 cells
Chong-Feng Gao1, Shuo Ren, Jingfei Wang
1Department of Molecular Cellular Oncology, Graduate School, Tokyo Medical and Dental University, 1-5-45, Yushima, Bunkyo-ku, Tokyo, 113-8549, Japan.
Abstract:
In the present study, we investigate the mechanism of how p53 induces growth arrest in Rb-defective Saos2 cells that express temperature-sensitive mutant p53 (ts p53). The activation of p53 at a permissive temperature (32.5 degrees C) induces the cell cycle arrest at both the G1 and G2 stages. The induction of several p53-responsive genes as well as a small form of p130 (S-p130) was detected upon p53 activation. S-p130 retained the functions as a pocket protein and was dominant over p130 at the protein level after 36 h at 32.5 degrees C. A canonical p53 binding site was identified in intron 4 of p130. Furthermore, a novel p53-inducible transcript containing a partial intron 4 sequence downstream of the p53 binding site and exon 5 of p130 was detected by RT-PCR, suggesting S-p130 is induced by p53 at transcriptional level. The results from gel shift assay and immunoprecipitation showed that S-p130 as well as p130 formed complexes with both E2F1 and E2F4 at a permissive temperature. Moreover, the transient expression of E1A (12S) and E2F1 effectively abrogated p53-induced cell cycle arrest. These results strongly suggested that p130 and its truncated form might substitute Rb in mediating p53-induced cell cycle arrest in Rb(-/-) Saos2 cells.
Insights
p53 triggers cell cycle arrest in Rb-defective cells by inducing a small p130 protein (S-p130). This S-p130, along with p130, interacts with E2F proteins, mediating the growth arrest mechanism.
Area of Science:
- Molecular Biology
- Cell Biology
- Cancer Research
Background:
- The tumor suppressor protein p53 plays a critical role in cell cycle regulation.
- Retinoblastoma protein (Rb) is a key regulator of the G1/S transition.
- Rb-defective cancer cells present a unique context for studying p53-mediated growth arrest.
Purpose of the Study:
- To elucidate the mechanism by which p53 induces growth arrest in Rb-defective Saos2 cells.
- To investigate the role of p130 and its potential truncated forms in p53-induced cell cycle regulation.
Main Methods:
- Utilized temperature-sensitive mutant p53 (ts p53) in Rb-defective Saos2 cells.
- Employed RT-PCR to detect novel transcripts and gel shift/immunoprecipitation assays to analyze protein interactions.
- Assessed cell cycle arrest at G1 and G2 stages and the impact of E1A and E2F1 expression.
Main Results:
- p53 activation at permissive temperature induced G1 and G2 cell cycle arrest.
- A small form of p130 (S-p130) was induced, functioning as a pocket protein and dominating over p130.
- S-p130 is transcriptionally induced by p53 and forms complexes with E2F1 and E2F4.
- E1A and E2F1 expression abrogated p53-induced cell cycle arrest.
Conclusions:
- p53 induces cell cycle arrest in Rb-defective cells through the transcriptional induction of S-p130.
- p130 and S-p130 may substitute for Rb function in mediating p53-induced cell cycle arrest.
- The interaction of p130/S-p130 with E2F factors is crucial for this growth arrest mechanism.