Abstract 3003: p73 DNP genotype influences effects of etoposide and 5-aza-2’-deoxycytidine on p73 mRNA isoforms ratios in human cancer cell lines

Abstract

Abstract Background: The p73 gene is a member of the p53 tumor suppressor family. A dinucleotide polymorphism (DNP) in p73 is a G4C14-to-A4T14 linked pair of transitions located in exon 2, between the P1 and P2 promoters, from which TAp73 and DeltaNp73 (DNp73) mRNA isoforms are transcribed, respectively. A p73 dinucleotide polymorphism (DNP) (rs1801173) is a G4C14-to-A4T14 linked pair of transitions located in exon 2 and lies between the P1 and P2 gene promoters. The P1 and P2 p73 gene promoters are the transcription initiation sites for mRNAs encoding TAp73 and deltaNp73 (DNp73) isoforms, respectively. These p73 mRNA isoforms, TAp73 and αNp73, encode protein isoforms which differ in their N-termini. TAp73 isoforms include the full-length N-terminal sequence and are transcriptionally active. αNp73 isoforms lack the N-terminal trans-activation domain and are dominate negative. Recently, we reported the p73 DNP allele was associated with (1) decreased risk [OR = 0.55, 95%CI = 0.31-0.99] for aggressive prostate cancer and (2) increased TAp73/DNp73 protein isoform ratios in ten human cancer cell lines. Etoposide is known to induce p73 gene expression. Others recently reported treatment of T-47D cells with 5-aza-2’-deoxycytidine (5AZA) resulted in increased TAp73/DNp73 mRNA ratios. Hypothesis: We hypothesized that treatment with 5AZA increases the TAp73/DNp73 mRNA ratios in human cell lines and that the presence of the p73 DNP allele potentiates 5AZA-induced increases TAp73/DNp73 cellular ratios. Methods. We treated human cancer cell lines with 50 microM etoposide for 24 hrs and/or 20 microM 5AZA for 48 hrs. RNA was isolated, converted to cDNA and used in TaqMan RT-PCR assays to detect total p73, TAp73 or αNp73 mRNAs. Results: Cell lines wild-type for p73 DNP (DU145, T47-D, MDA-MB-468) had TAp73/DNp73 mRNA ratios that were lower than those heterozygous for p73 DNP (PC-3 and MDA-MB-231) following co-treatment with 5AZA and etoposide or with etoposide alone (p<0.01 for DU145 & T47-D). PC-3 and MDA-MB-231 cell lines had TAp73/DNp73 mRNA ratios following 5AZA treatment (0.8546 and 0.7138, respectively) that were equal to or greater than the three cell lines wild type for p73 DNP: DU145, T47-D and MDA-MB-468 (0.8626, 0.6480, 0.3866, respectively). Conclusion: Of the five cell lines used in this study, cells that harbor a p73 DNP allele had the highest TAp73/DNp73 mRNA ratios following co-treatment with 5AZA and etoposide or with etoposide alone as compared to wild-type cell p73 DNP, which is consistent with our previously reported data. Lastly, our data in this work assessing the effect of 5AZA treatment of T47-D cells on TAp73/DNp73 mRNA ratios is not consistent with a previously reported study. Citation Format: Zachary Connelly, L. Michael Carastro. p73 DNP genotype influences effects of etoposide and 5-aza-2’-deoxycytidine on p73 mRNA isoforms ratios in human cancer cell lines. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3003.