Activation of p21WAF1/Cip1 Transcription through Sp1 Sites by Histone Deacetylase Inhibitor Apicidin

Abstract

We previously reported that apicidin, a novel histone deacetylase inhibitor, inhibited the proliferation of tumor cells via induction of p21 WAF1/Cip1 . In this study, we determined the molecular mechanisms by which apicidin induced the p21 WAF1/Cip1 gene expression in HeLa cells. Apicidin induced p21 WAF1/Cip1 mRNA independent of the de novo protein synthesis and activated the p21 WAF1/Cip1 promoter through Sp1-3 site located at ؊82 and ؊77 relative to the transcription start site. This transcriptional activation appears to be mediated by protein kinase C (PKC), because calphostin C, a PKC inhibitor, significantly attenuated the activation of p21 WAF1/Cip1 promoter via Sp1 sites, which was accompanied by a marked suppression of p21 WAF1/Cip1 mRNA and protein expression induced by apicidin. Consistent with the transcriptional activation of p21 WAF1/Cip1 promoter by apicidin, apicidin treatment led to the translocation of PKC⑀ from cytosolic to particulate fraction, which was reversed by pretreatment with calphostin C, indicating the involvement of PKC in the transcriptional activation of p21 WAF1/Cip1 via Sp1 sites by apicidin. However, the PKC-mediated transcriptional activation of p21 WAF1/Cip1 by apicidin appears to be independent of the histone hyperacetylation, because apicidin-induced histone hyperacetylation was not affected by calphostin C. Furthermore, a PKC activator, phorbol 12,13-dibutyrate, alone induced the transcriptional activation of p21 WAF1/Cip1 promoter, p21 WAF1/Cip1 mRNA, and protein expression without induction of the histone hyperacetylation, suggesting that the transcriptional activation of p21 WAF1/Cip1 by apicidin might have been mediated by a mechanism other than chromatin remodeling through the histone hyperacetylation. Taken together, these results suggest that the PKC signaling pathway plays a pivotal role in the transcriptional activation of the p21 WAF1/Cip1 gene by apicidin.

HDAC4 represses p21WAF1/Cip1 expression in human cancer cells through a Sp1-dependent, p53-independent mechanism

“…HDAC4 represses p21 WAF1/Cip1 in cancer cells in vitro in a Sp1-dependent manner Although most reports demonstrated that HDAC inhibitors induced p21 WAF1/Cip1 expression in an Sp1/Sp3-dependent manner Sowa et al, 1997Sowa et al, , 1999Han et al, 2001;Wilson et al, 2006), a role for p53 in HDAC-associated p21 WAF1/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).…”

“…Epigenetic suppression of p21 WAF1/Cip1 is directly linked to HDAC activity, as inhibition of HDAC activities in cancer cells induces transcriptional reactivation of p21 WAF1/Cip1 through hyperacetylation of histones H3 and H4 in the proximal promoter region Richon et al, 2000;Sawa et al, 2004). In other studies, p21 Waf1/Cip1 induction by HDAC inhibitors was mediated through Sp1 sites through Sp1 family transcription factors, Sp1 and/or Sp3 Sowa et al, 1997Sowa et al, , 1999Han et al, 2001;Wilson et al, 2006). To date, the most common model for p21 WAF1/Cip1 repression in cancer cells is the recruitment of HDACs by Sp1/-Sp3 transcription factors in the proximal promoter region encompassing the Sp1/Sp3 binding sites, which in turn repress p21 WAF1/Cip1 transcription by deacetylating histones H3 and H4.…”

“…Among the genes that are consistently upregulated in cancer cells treated with HDAC inhibitors is the cell cycle mediator p21 WAF1/Cip1 Sowa et al, 1997;Archer et al, 1998;Kim et al, 1999;Saito et al, 1999;Sambucetti et al, 1999;Richon et al, 2000;Han et al, 2001;Rocchi et al, 2005). p21 WAF1/Cip1 arrests cell growth by inhibiting cyclin/Cdk complexes (Xiong et al, 1993) and can act as tumor suppressor gene by negatively regulating the cell cycle.…”

Expression of p21WAF1/Cip1 through Sp1 sites by histone deacetylase inhibitor apicidin requires PI 3-kinase–PKCε signaling pathway

“…In addition, it has been shown that the transcriptional activation of p21 WAF1/Cip1 by apicidin is dependent on protein kinase C (PKCs) as indicated by studies of the PKC inhibitor (Han et al, 2001). However, which of the PKC isoforms was involved in p21 WAF1/Cip1 gene expression by apicidin or upstream signaling events that leads to PKC activation are not yet clear.…”

“…Recent evidence suggests that the induction of p21 WAF1/Cip1 by these stimuli could be regulated by various signal transduction pathways that mediate cell growth, differentiation, and stress response (Hu et al, 1999;Lee et al, 2000;Mitsuuchi et al, 2000). Also, it has been demonstrated that p21 WAF1/Cip1 induction by HDAC inhibitors was mediated via Sp1 sites through Sp1 family transcription factors, Sp1 and/or Sp3 (Nakano et al, 1997;Sowa et al, 1999;Huang et al, 2000;Han et al, 2001). However, the detailed signal transduction pathways involved in p21 WAF1/Cip1 gene regulation by HDAC inhibitors still remain poorly understood.…”