5.

Effects of H₂O₂ and TSA on p21^WAF1, acH3, acH4 and HDAC1 expression and on the p21^WAF1 promoter status in HCT116 p53⁻/⁻ cells. (A) H₂O₂ and TSA pre-treatment induce p21^WAF1 mRNA expression after 6 hrs. (B) Whereas H₂O₂ induces both, p21^WAF1 and HDAC1 protein expression, TSA alone and H₂O₂+ TSA have no effect on HDAC1 expression. (C) There was a significant decrease (*P<0.05) in the activity of HDACs 6 hrs after H₂O₂ or TSA addition, but TSA pre-treatment resulted in prolonged reduction of the HDACs activity. (D) Western Blotting showed that there was a decrease in the total acH3 and acH4 levels after TSA pre-treatment. (E, F) ChIP experiment using immunoprecipitation with Ac-H4 (E) and HDAC1 (F) antibodies in control cells (C), and 6 hrs and 24 hrs after treatment with TSA (200 ng/ml, 6 hrs, T), and with H₂O₂ (30 mM, 3 min, H), and TSA and H₂O₂ (T + H), respectively, showing that there is only a slight increase in acH4 around the p21^WAF1-Sp1 promoter region after H₂O₂ treatment. Pre-treatment with TSA slightly reinforces the acH4 on this region. Furthermore, the pre-treatment do not significantly affect HDAC1 binding at the Sp1 promoter site.