DNA methylation and DNMT in ACC cell lines

Objectives:To investgate the promoter methylation of tumor suppressor genes(P16, RASSF1A and E-cadherin) in salivary adenoid cystic carcinoma(ACC) and discuss the role of DNA methyltransferase(DNMT1 and DNMT3b ) in gene hypermethylation.Methods:The genomic DNA was extracted from the adenoid cystic carcinoma cell line ACC-2 and high-rate metastasis cell line ACC-M. Promoter hypermethylation of P16, RASSF1A and E-cadherin were analysed by methylation-specific PCR(MSP). Meanwhile, total RNA and total protein were also isolated from the cells, 2 ug of total RNA was reverse-transcribed to cDNA and cDNA was amplified with primer specific for DNMT1 and DNMT3b respectively. The detection of DNMT1 and DNMT3b proteins was carried out using Western-Blot. GAPDH was used in both RT-PCR and Western-Blot as a inner control. The images from RT-PCR and Western-Blot were semiquantitatively analysed using Totallab 2.01 software.Results:The promoters of P16 and E-cadherin gene were methylated in ACC-2 and ACC-M cells, while the hypermethylation of RASSF1A was not found. The expression of DNMT1 and DNMT3b was detected both in ACC-2 and ACC-M cell lines on mRNA and protein levels and the amounts of DNMT1 and DNMT3b in ACC-2 cell line were1.25 and 1.17 times more than that in ACC-M cell line respectively. In ACC-M and ACC-2 cell lines the amounts of DNMT1 was 1.25 and 1.34 times more than that of DNMT3b respectively. Conclusions:Inactivation of P16 and E-cadherin resulting from promoter hypermethylation may be associated with the tumorigenesis of ACC. Both DNMT1 and DNMT3b play a role in gene methylation but DNMT1 may be more important in methylation maintaince than DNMT3b.