PDF《Oncogene (2009) 28, 243–256》

Sowa et al., 1997;

Archer et al., 1998;

Kim et al., 1999;

Sambucetti et al., 1999;

Han et al., 2000;

Huang and Pardee, 2000;

Richon et al., 2000;

Sawa et al., 2004;

Rocchi et al., 2005) and inhibit class I and class II HDACs equally well. To identify the speci?c HDACs that participate in regulating p21WAF1/Cip1 , we transfected IGROV-1 cells with siRNAs directed against seven class I and II HDACs (HDAC1 through HDAC7) (Supplementary Figure S1A and B) and examined p21WAF1/Cip1 by real-time polymerase chain reaction (RTCPCR) and western blot analysis. Both HDAC1 and HDAC4 silencing (HDAC4 siRNA no. 1) signi?cantly increased p21WAF1/Cip1 gene expression both at mRNA and protein level (Figures 1a and b). However, the combined silencing of HDAC1 and HDAC4 did not show synergy on p21WAF1/Cip1 induction compared with individual siRNA suggesting that speci?c HDAC4 inhibition is suf?cient to increase p21WAF1/Cip1 gene expression (Supplementary Figure S2). To exclude a non-speci?c effect of HDAC4 siRNA used, we transfected IGROV-1 cells with two siRNAs whose sequences did not overlap with those of the effective HDAC4 siRNA and evaluated p21WAF1/Cip1 mRNA and protein levels by RTCPCR and western blot analysis (Figures 1c and d). Transfection with siRNA HDAC4 no. 2, which effectively reduced HDAC4 abundance, also induced p21WAF1/Cip1 expres- sion. In contrast, siRNA HDAC4 no.

3 which silenced HDAC4 expression less ef?ciently was also less ef?cient in increasing p21WAF1/Cip1 expression. In the subsequent experiment, siRNA HDAC4 no.

2 was used because it did not affect expression levels of the other HDACs (Supplementary Figure S3A and B), suggesting that speci?c HDAC4 silencing is involved in the induction of p21WAF1/Cip1 expression. HDAC4 silencing also induced p21WAF1/Cip1 expression in human cervical cancer HeLa cells (Figure 1e), glioblastoma U87-MG cells (Figure 1f) and breast cancer MCF7 cells (Figure 1g), suggesting that the effect was not cell type speci?c. HDAC4 represses p21WAF1/Cip1 in cancer cells in vitro in a Sp1-dependent manner Although most reports demonstrated that HDAC inhibitors induced p21WAF1/Cip1 expression in an Sp1/Sp3-dependent manner (Nakano et al., 1997;

Sowa et al., 1997, 1999;

Han et al., 2001;

Wilson et al., 2006), a role for p53 in HDAC-associated p21WAF1/Cip1 expression has also been reported in several human cancer cell lines (Lagger et al., 2003;

Luo et al., 2004;

Roy et al., 2005;

Zhao et al., 2006). To determine the role of p53 and Sp1/Sp3 transcrip- tion factors in the activation of p21WAF1/Cip1 promoter activity after HDAC4 silencing, we used a series of p21WAF1/Cip1 promoter constructs (Figure 2a). IGROV-1 cells were transfected with a full-length human p21WAF1/Cip1 promoter luciferase reporter construct (p21-WT) and a p21WAF1/Cip1 promoter luciferase reporter construct that was mutated in the ?rst (p21-p53 mut1) or second (p21- p53 mut2) p53-binding site. Neither mutation decreased the HDAC4 inhibition-induced increase in the relative luciferase activity of the full-length p21WAF1/Cip1 promo- ter. Similarly, the p21-101 vector, which lacks the two p53-binding sites but contains four Sp1Cbinding sites important for induction of p21WAF1/Cip1 by HDAC inhibitors (Nakano et al., 1997;

Sowa et al., 1997;

Wilson et al., 2006), was strongly activated by HDAC4 inhibition (Figure 2b). This ?nding suggests that the Sp1 elements, not the p53-binding sites, are required for the transcriptional activation of p21WAF1/Cip1 promoter in- duced by HDAC4 silencing. In addition, these results demonstrated that the basal level of promoter activity was partially inhibited when p53 response elements were HDAC4 represses p21WAF1/Cip1 expression in human cancer cells D Mottet et al