p53 acetylation and downstream target gene activation in LNCaP prostate cancer cells

摘要

To improve the therapeutic response of prostate cancer to anti-androgens, we have compared the effects of two histone deacetylase (HDAC) inhibitors CG-1521 (7-phenyl-2,4,6-hepta-trienoic hydroxamic acid) and Trichostatin (TSA). While both drugs block histone deacetylation, they also stabilize acetylation of p53. CG-1521 stabilizes the acetylation of p53 at Lys373 (Ac-Lys373 p53), inducing p53-dependent increase in p21 levels, cell cycle arrest, and cell death. In contrast TSA induces transient acetylation of p53 at Lys382 (Ac-Lys382 p53), inducing p21 and cell cycle arrest, but not cell death. Gene array analysis on LNCaP cells treated with CG-1521 and TSA, and subsequent Real Time PCR (RT-PCR) (Sybrgreen) analysis, confirms that the two HDAC inhibitors induce substantially different subsets of genes regulating cell cycle, cell proliferation, cell death and transcription. Co-immunoprecipitation analysis using anti-Ac-Lys373 p53 and anti-Ac-Lys282 p53 columns, shows that the two HDAC inhibitors initiate the formation of different transcriptional complexes. Ac-Lys373 p53 forms complexes containing p300, Baf60, Foxa1, Foxm1 and the androgen receptor (AR), which is itself acetylated. Ac-Lys382 p53 forms complexes with CBP and Brm-1 but not with p300, Baf60, AR or the forkhead transcription factors. Pol II and TFIIH are associated with the complexes formed after treatment with both CG-1521 or TSA, suggesting that the complexes associated with Ac-Lys373 p53 and Ac-Lys382 p53 are both capable of productively initiating transcription. Chromatin immunoprecipitation analysis on two p53 response elements in the p21 promoter, a p53 target gene, indicates that complexes formed on Ac-Lys373 p53 and Ac-Lys382 p53 can bind to and stimulate transcription from the two p53REs.