Abstract Malignant tumors and cell lines are characterized by increased expression of DNMT1, DNMT3a and DNMT3b. While evidence suggests that DNMT1, DNMT3a and DNMT3b co-operate in methylation and gene silencing during carcinogenesis, very little is known regarding gene targets for DNMT3a and DNMT3b and their impact on the development of adenocarcinoma. We developed an in vitro model using human telomerase/cyclin dependent kinase 4-immortalized human bronchial epithelial cell lines (HBECs) to identify key molecular changes that drive transformation and clonal outgrowth of preneoplastic lung epithelial cells during exposure to tobacco carcinogens. Carcinogen exposure (0.5mM MNU and 0.05µM BPDE) for 12 weeks induced transformation of HBEC2 cells and transformation efficiency was associated with DNA repair capacity. A significant increase in DNMT1 protein was observed during carcinogen exposure while moderate increases in DNMT3a and DNMT3b were observed in transformed cells. Increased DNMT3a and DNMT3b levels have been reported in several cancers. We observed 5-20 fold increased expression of DNMT3a and DNMT3b in tumor derived lung cancer cell lines. Therefore we tested the hypothesis that over expressing DNMT3a and DNMT3b would accelerate transformation by targeting specific genes for methylation and promote full malignancy in carcinogen-treated HBECs. Over expression of DNMT3a or DNMT3b did not affect DNA repair capacity and was not sufficient to drive spontaneous transformation as evidenced by inability to form colonies on soft agar. Transformation efficiency was increased 2-2.5 fold in the two HBEC2 cell lines over expressing DNMT3b after 12 weeks of carcinogen treatment relative to treated parental HBEC2. In addition, after 8 weeks, colony formation was evident in the DNMT3b over expressing, but not in parental HBECs indicating that increased expression of this de novo DNMT accelerates transformation. Over expressing DNMT3a had no effect on carcinogen-induced transformation. Current studies are evaluating global re-programming of the epigenome in transformed clones using the Illumina 450k promoter array and the effect of over expression of DNMT3b on stem cell markers. This study was supported by R01 ES008801 to S.A.B. and 1F32 CA157082 to I.T. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1049. doi:1538-7445.AM2012-1049