Abstract 2214: Dynamic transcriptome analysis of DNA damage pathways under hypoxia

Abstract

Abstract During the outgrowth of tumor, gradients of hypoxic regions develop due to reduced oxygen diffusion. Intratumoral hypoxia is a prognostic factor associated with decreased disease-free survival and increased resistance to radiotherapy and chemotherapy in different solid cancers. We have previously shown that chronic hypoxia suppresses DNA damage repair pathways partly through decreased protein translation efficiency. The RNA levels of repair pathway genes have also been shown to be downregulated in hypoxia; however, most of the RNA studies do not distinguish between RNA synthesis and degradation. Here, we use novel metabolic labeling of newly synthesized RNA to investigate the dynamic changes in transcription rates of DNA damage repair pathways under hypoxia. 4-thiouridine (4sU), a uridine analogue, was added to the media of DU145 cells (prostate cancer cell line) that have been cultured in 21% or 0.2% oxygen concentrations for 72 hours. Total RNA was extracted and 4sU-tagged RNA was biotinylated and separated from untagged pre-existing RNA using streptavidin coated magnetic beads. Real-time RT-PCR was used to quantify the expression of repair pathways in three RNA subsets (total, pre-existing, and newly synthesized). Parallel experiments were performed in RNA harvested from the cytoplasmic and nuclear factions of cells. We found that with 30-minute 4sU incubation, most 4sU-tagged RNA is still in the nucleus, and is subjected to little degradation, thus reflecting the average transcription rate. The percentage of newly synthesized RNA is lower in hypoxic than in normoxic cells. Importantly, the transcript levels of homologous recombination (HR) repair pathway regulators, RAD51, BRCA1 and BRCA2, are decreased in both total and newly-synthesized RNA of hypoxic cells. Our results demonstrate that in addition to translational regulation, hypoxia may also downregulate repair pathways through suppression of transcription. This 4sU assay provides opportunities for further analysis of differential RNA synthesis and decay of DNA damage repair pathways between hypoxia and normoxia. Citation Format: Winnie W. Lo, Gaetano Zafarana, Robert G. Bristow. Dynamic transcriptome analysis of DNA damage pathways under hypoxia. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2214. doi:10.1158/1538-7445.AM2015-2214