Abstract B56: Human PIF1 helicase depletion sensitizes RAS-transformed human fibroblasts to gemcitabine and suppresses DNA replication fork progression and initiation efficiency.

Pif1 helicase is implicated in a wide range of DNA transactions that are required for orderly replication and genome stability in lower and higher eukaryotes. While mouse pif1-/- knockouts show no phenotype and PIF1 is dispensable in non-cancerous human cells, Pif1 knockdowns sensitize many tumor cell lines to replication inhibitors, slow S-phase progression, and trigger apoptosis. Here we investigated the effects of PIF1 depletion on a non-tumor cell line, MRC5-SV2, and HRAS-transformed derivatives. We show that PIF1 depletion reduces the growth and viability of RAS-transformed MRC5-SV2 cells relative to parental and increases sensitivity to the therapeutic DNA replication inhibitor gemcitabine. Since previous work suggested that PIF1 depletion affected S-phase entry and transition in cultured tumor cells, we used single fiber analysis of pulse-labeled DNA to investigate the potential role of PIF1 in these events. Here we show that PIF1 depletion triggers replication stress characterized by slower fork rates and increased fork arrest during normal cycling conditions in non-tumor MRC5-SV2 cells. These effects were enhanced in the RAS-transformed derivative. Impaired fork movement upon PIF1-depletion did not augment DNA double-stranded break formation or trigger DNA damage responses. However PIF1 depletion affected resumption of DNA synthesis after prolonged replication inhibitor exposure, accompanied by diminished new origin firing and S-phase entry. Taken together, our data establish a novel functional role for human PIF1 in DNA replication that becomes critical for cell growth during oncogenic stress. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):B56. Citation Format: Mary E. Gagou, Anil Ganesh, Mark Meuth. Human PIF1 helicase depletion sensitizes RAS-transformed human fibroblasts to gemcitabine and suppresses DNA replication fork progression and initiation efficiency. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr B56.