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  • Original Paper
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Histone deacetylase inhibitors activate INK4d gene through Sp1 site in its promoter

Abstract

Histone deacetylase (HDAC) inhibitors are known to arrest human tumor cells at the G1 phase of the cell cycle and activate the cyclin-dependent kinase inhibitor, p21WAF1/Cip1. However, several studies have suggested the existence of a p21WAF1/Cip1-independent molecular pathway. We report here that HDAC inhibitors activate a member of the INK4 family, the INK4d gene, causing G1 phase arrest, in the human T cell leukemia cell line, Jurkat. One of the major Trichostatin A (TSA)-responsive elements is a specific Sp1 binding site in the INK4d promoter. Electrophoretic mobility-shift assay revealed that Sp1 and Sp3 can specifically interact with this Sp1 binding site. Furthermore, using chromatin immunoprecipitation assay, we demonstrated that HDAC2 was present in the INK4d proximal promoter region in the absence, but not the presence, of TSA. Taken together, these results suggest that treatment with TSA transcriptionally activates INK4d by releasing HDAC2 from the histone–DNA complex at the INK4d promoter. Using a p21WAF1/Cip1-deleted human colorectal carcinoma cell line, HCT116 p21 (−/−), we show that upregulation of p19INK4d by TSA is associated with inhibition of cell proliferation. Moreover, mouse embryo fibroblasts lacking Ink4d were resistant to the growth inhibitory effects of TSA as compared to their wild-type counterpart. Our findings suggest that p19INK4d in addition to p21WAF1/Cip1 is an important molecular target of HDAC inhibitors inducing growth arrest.

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Abbreviations

HDAC:

histone deacetylase

TSA:

Trichostatin A

EMSA:

electrophoretic mobility-shift assay

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Acknowledgements

We gratefully acknowledge Dr Frank L Graham, McMaster University for the human p19 expression vector (pMH4p19) and its control vector (pMH4). We also thank Rose Mathew (SJCRH), Dr Yoshihiro Sowa (Kyoto Prefectural University of Medicine) and Dr Gwyn T Williams (Keele University) for technical advice and helpful discussions. This work was supported in part by NCI grant CA71907 (MFR), Core grant CA 21765 of St Jude Children's Research Hospital (SJCRH) and the American Lebanese Syrian Associated Charities (ALSAC), the Ministry of Education, Science, Sports and Culture of Japan and a grant (H11-Seikatsu-018) for Research on Environmental Health from the Ministry of Health, Labor and Welfare of Japan.

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Correspondence to Toshiyuki Sakai.

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Yokota, T., Matsuzaki, Y., Miyazawa, K. et al. Histone deacetylase inhibitors activate INK4d gene through Sp1 site in its promoter. Oncogene 23, 5340–5349 (2004). https://doi.org/10.1038/sj.onc.1207689

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