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Increased expression of unmethylated CDKN2D by 5-aza-2′-deoxycytidine in human lung cancer cells

Abstract

DNA hypermethylation of CpG islands in the promoter region of genes is associated with transcriptional silencing. Treatment with hypo-methylating agents can lead to expression of these silenced genes. However, whether inhibition of DNA methylation influences the expression of unmethylated genes has not been extensively studied. We analysed the methylation status of CDKN2A and CDKN2D in human lung cancer cell lines and demonstrated that the CDKN2A CpG island is methylated, whereas CDKN2D is unmethylated. Treatment of cells with 5-aza-2′-deoxycytidine (5-Aza-CdR), an inhibitor of DNA methyltransferase 1, induced a dose and duration dependent increased expression of both p16INK4a and p19INK4d, the products of CDKN2A and CDKN2D, respectively. These data indicate that global DNA demethylation not only influences the expression of methylated genes but also of unmethylated genes. Histone acetylation is linked to methylation induced transcriptional silencing. Depsipeptide, an inhibitor of histone deacetylase, acts synergistically with 5-Aza-CdR in inducing expression of p16INK4a and p19INK4d. However, when cells were treated with higher concentrations of 5-Aza-CdR and depsipeptide, p16INK4a expression was decreased together with significant suppression of cell growth. Interestingly, p19INK4d expression was enhanced even more by the higher concentrations of 5-Aza-CdR and depsipeptide. Our data suggest that p19INK4d plays a distinct role from other INK4 family members in response to the cytotoxicity induced by inhibition of DNA methylation and histone deacetylation.

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Abbreviations

5-Aza-CdR:

5-aza-2′-deoxycytidine

DNMT:

DNA Methyltransferase

HDAC:

Histone deacetylase

MBP:

Methyl-CpG binding proteins

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Acknowledgements

We thank Drs Yue Xiong and Xin-Hai Pei for kindly providing us information about the putative promoter region of human CDKN2D. We also thank Drs Lawrence Druhan and Melissa Hunter for their technical assistance, and Dr Michael Grever for the gift of depsipeptide. This work is supported in part by grants from the American Cancer Society, Ohio Division, Ohio Cancer Research Associates and by grant P30 CA16058, National Cancer Institute, Bethesda, MD, USA.

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Correspondence to Gregory A Otterson.

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Zhu, WG., Dai, Z., Ding, H. et al. Increased expression of unmethylated CDKN2D by 5-aza-2′-deoxycytidine in human lung cancer cells. Oncogene 20, 7787–7796 (2001). https://doi.org/10.1038/sj.onc.1204970

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