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F16. Depletion of DNA Methyltransferase (DNMT)1, and/or DNMT3b Mediates Growth Arrest and Apoptosis in Cultured Thoracic Malignancies
Edmund S. Kassis, Ming Zhao, Julie A. Hong, G. Aaron Chen, Dao M. Nguyen, David S. Schrump; Bethesda, MD

Objective: Elevated levels of DNMT1 and/or 3b facilitate malignant transformation via chromatin remodeling mechanisms. The present study was undertaken to examine if antisense-mediated inhibition of DNMT expression could mediate growth arrest in cultured lung and esophageal cancer, and malignant pleural mesothelioma (MPM) cells, as a prelude to evaluation of these agents in thoracic oncology patients.
Methods: A549 and CALU-6 lung cancer cells, SKGT5 and BIC esophageal adenocarcinoma cells, H2373 and H2052 MPM cells, and normal human bronchial epithelial (NHBE) cells were transfected with phosphorothioate-modified antisense oligos (ASOs) specifically targeting DNMT1 or DNMT3b, or mismatch oligos using lipofectamine techniques. Quantitative RT-PCR, western blot, trypan blue exclusion, and ApoBrdU techniques were used to evaluate DNMT expression, proliferation, and apoptosis following ASO transfections. Gene expression profiles were assessed by long-oligo arrays techniques.
Results: Under exposure conditions 200-fold lower than achieved in phase I clinical studies, ASOs mediated specific, dose-dependent depletion of DNMT1 and DNMT3b, resulting in pronounced (>80%) inhibition of proliferation of all cancer cell lines; these effects were not observed following ASO transfection of NHBE cells. Depletion of DNMT1 and /or DNMT3b coincided with dramatic, caspase- dependent, p53-independent apoptosis in 4 of the 6 cancer lines. The antiproliferative effects of the ASOs were not attributable to induction of RASSF1A or p16 tumor suppressor genes, and did not coincide with demethylation of genes encoding cancer testis antigens. p21 expression was induced in all of the cancer lines following DNMT1 and/or DNMT3b depletion; however, p21 expression levels did not directly coincide with sensitivity to ASO-mediated apoptosis. Micro-array analysis of DNMT-depleted lung cancer cells revealed pronounced induction of numerous genes mediating response to genotoxic stress. Interestingly, gene expression profiles following DNMT1, DNMT3b, or combined DNMT1/3b knockdown were remarkably similar, yet distinctly different from expression profiles mediated by low dose deoxyazacytidine.
Conclusions: ASOs targeting DNMT1 and DNMT3b induce genomic stress, and mediate potent growth inhibition in cultured lung and esophageal cancer and MPM cells. These findings support further evaluation of DNMT knockdown strategies for the treatment of thoracic malignancies.

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