Abstract 2984: Association of the Uracil DNA glycosylase (UNG) promoter in human lung cancer cells with the transcription factor family E2F is lost after pemetrexed treatment
Authors: Lachelle D. Weeks, Lili Liu, Stanton L. Gerson
Journal: Cancer Research (2010)

Abstract We have previously reported that the base excision repair (BER) protein, uracil DNA glycosylase (UNG) is induced in response to DNA damage caused by fludarabine, a nucleoside analog and pemetrexed, an inhibitor of dihidrofolate reductase (DHFR). To understand the mechanism underlying this induction, we sought to identify transcriptional activators of the UNG gene that bind the UNG promoter region after treatment with DNA damaging agents. Members of the E2F transcription factor family have been reported to bind to the UNG promoter resulting in a cell cycle dependent regulation of mRNA expression in untreated colon cancer cells. Treatment of a human lung cancer cell line (H460) with pemetrexed (100-400uM) causes dUTP incorporation in DNA, S-phase arrest, double strand breaks and apoptosis initiation. S-phase arrest, strand break accumulation, and apoptosis are all observed following 24 hours of treatment with 200uM pemetrexed and increase with longer drug treatment times. However, UDG levels are only increased in H460 extracts from cells treated with 200uM pemetrexed for longer than 48 hours. To investigate whether induction of UDG after pemetrexed treatment occurs via activation of the UNG promoter by E2F, we performed chromatin immunoprecipitation (ChIP) assays using antibodies specific to E2F-1 and E2F-4. Results of these studies show that the UNG promoter is immunoprecipitated with both E2F-1 and E2F-4 in untreated cells, but not in cells treated with pemetrexed (200uM). Furthermore, western blot analysis of E2F-1 and UNG protein levels in H460 cells treated with 200uM pemetrexed for 0-72h illustrates a decline in total E2F-1 protein in cells treated beyond 12h, and an increase in total UDG levels. Additionally, the UNG promoter was not immunoprecipitated by E2F transcription factors in extrtacts from H460 cells treated with the DHFR inhibitor, methotrexate (12.5uM), but was immunoprecipitated with E2F-1 and E2F-4 in H460 extracts from cells treated with the crosslinking agent, cisplatin (12.5uM). These data thus suggest that the transcription factor family E2F, which is responsible for cell cycle regulation of UNG protein levels, is not responsible for induction of UNG protein following pemetrexed treatment. The UDG promoter region contains consensus sites for several other transcription factors including c-myc, AP-1 (c-Jun) and Sp-1; the binding of these transcription factors with the UNG promoter of pemetrexed treated cells have also been evaluated in this study. Together, these data illustrate, for the first time, a disparate mechanism of UNG gene activation and regulation of gene expression in untreated cells and cells treated with DHFR inhibitors, methotrexate and pemetrexed. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2984.