Histone deacetylase inhibitor, Trichostatin A, activates p21WAF1/CIP1 expression through downregulation of c-myc and release of the repression of c-myc from the promoter in human cervical cancer cells

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Abstract

Histone deacetylase (HDAC) inhibitors have shown promise in clinical cancer therapy and to consistently induce p21WAF1/CIP1 expression in a p53-independent manner and via increased acetylation of the chromatin at the Sp1 sites in the p21WAF1/CIP1 promoter region. However, the exact mechanism by which HDAC inhibitors induce p21WAF1/CIP1 remains unclear. In this study, we observed that Trichostatin A (TSA), a HDAC inhibitor, induced strikingly p21WAF1/CIP1 expression in human cervical cancer (HeLa) cells, and this induction correlated with downregulation of c-myc expression. Coincident with this observation, knock down of c-myc with a c-myc specific small interfering RNA dramatically induced expression of p21WAF1/CIP1 in these cancer cells. These data suggest that c-myc may play a critical role in repression of p21WAF1/CIP1 expression in HeLa cells. More importantly, using chromatin immunoprecipitation assay, we observed for the first time that c-myc bound to the endogenous p21WAF1/CIP1 promoter in untreated HeLa cells, but not in TSA-treated cells. Taken together, TSA induced c-myc downregulation and release from the endogenous p21WAF1/CIP1 promoter contributes, at least partially, to transcriptional activation of the p21WAF1/CIP1 in HeLa cells.

Section snippets

Materials and methods

Cell line and reagents. Human cervical carcinoma cells (HeLa) were maintained in Dulbecco’s modified Eagle’s medium (DMEM), supplemented with 10% fetal bovine serum (Hyclone), 1% penicillin, and streptomycin (Invitrogen). Trichostatin A (Sigma) was dissolved in dimethyl sulphoxide (DMSO).

Reverse transcription and polymerase chain reaction. Total cellular RNA was isolated with TRIzol reagent (Invitrogen) and DNAs were removed with DNA-free kit (Ambion) according to the manufacturer’s

TSA induces morphological changes of HeLa cells

HeLa is an epithelia-like cervical carcinoma cell line which contains human papillomavirus type 18. HeLa cells are normally oval or polygonal, with few cells showing elongation (Fig. 1A). However, treatment with 100 nM TSA for 16 h resulted in characteristic elongated cells with filamentous protrusions (Fig. 1B). These changes in morphology resemble cells that have undergone replicative senescence, cell cycle arrest, and/or apoptosis [19], [7].

TSA-induced p21WAF1/CIP1 expression correlates with downregulation of c-myc in HeLa cells

The induction of p21WAF1/CIP1 is one of the common

Discussion

In the current study, we found that HeLa cells appeared to undergo morphological changes after TSA treatment for 16 h, which were consistent with morphology of the cells that have undergone replicative senescence, cell arrest, and/or apoptosis [19], [7]. Thus, TSA is highly effective in suppressing the growth of HeLa cells. Expression of p21WAF1/CIP1, a cyclin-dependent kinase inhibitor which has an important role in arresting the cell cycle in the G1 phase, is likely to account for this effect.

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