DNA Promoter Methylation-dependent Transcription of the Double C2-like Domain Beta (DOC2B) Gene Regulates Tumor Growth in Human Cervical Cancer

Kabekkodu, Shama Prasada and Bhat, Samatha and Raghu, Radhakrishnan and Aithal, Abhijit and Mascarenhas, Roshan and Pandey, Deeksha and Rai, Lavanya and Kushtagi, Pralhad and Mundyat, Gopinath Puthiya and Satyamoorthy, K (2014) DNA Promoter Methylation-dependent Transcription of the Double C2-like Domain Beta (DOC2B) Gene Regulates Tumor Growth in Human Cervical Cancer. Journal of Biological Chemistry, 289 (15). pp. 10637-10649.

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Abstract

Double C2 like Domain Beta (DOC2B) gene encodes for a calcium binding protein which is involved in neurotransmitter release, sorting and exocytosis. We have identified promoter region of DOC2B gene as hypermethylated in pre-malignant, malignant cervical tissues and cervical cancer cell lines by methylation sensitive dimethyl sulfoxide polymerase chain reaction (MS-DMSO-PCR) and bisulfite genome sequencing (BGS); while, it was unmethylated in normal cervical tissues (P<0.05). The promoter hypermethylation was inversely associated with mRNA expression in SiHa, CaSki and HeLa cells and treatment with demethylating agent 5-aza-2–deoxycytidine restored DOC2B expression. The region -630 to +25bp of DOC2B gene showed robust promoter activity by luciferase reporter assay and was inhibited by in vitro artificial methylation with Sss1 methylaseprior to transient transfections. The overexpression of DOC2B gene in SiHa cells when compared to controls, showed significantly reduced colony formation, cell proliferation and doubling time, induced cell cycle arrest and repressed cell migration and invasion (P<0.05). Ectopic expression of DOC2B resulted in anoikis mediated cell death and repressed tumor growth in a nude mice xenograft model (P<0.05). DOC2B expressing cells showed a significant increase in intracellular calcium level (P<0.05), impaired AKT1 and ERK1/2 signaling and induced actin cytoskeleton remodeling. Our results show that promoter hypermethylation and silencing of DOC2B gene is an early and frequent event during cervical carcinogenesis and whose reduced expression due to DNA promoter methylation may lead to selective cervical tumor growth.

Item Type: Article
Uncontrolled Keywords: Calcium, cell cycle, cell growth, cell invasion, cell motility, DNA promoter methylation, Human Papillomavirus, DOC2B, actin remodeling, AKT, ERK, cervical cancer.
Subjects: Life Sciences > MLSC Manipal
Medicine > KMC Mangalore > Obstetrics & Gynaecology
Medicine > KMC Manipal > Obstetrics & Gynaecology
Depositing User: KMCMLR User
Date Deposited: 02 Jul 2014 06:42
Last Modified: 24 Oct 2016 08:56
URI: http://eprints.manipal.edu/id/eprint/140002

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