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Supplementary Figures 1-6 from Suppression of Apoptosis by PIF1 Helicase in Human Tumor Cells

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posted on 2023-03-30, 20:24 authored by Mary E. Gagou, Anil Ganesh, Ruth Thompson, Geraldine Phear, Cyril Sanders, Mark Meuth
Supplementary Figures 1-6 from Suppression of Apoptosis by PIF1 Helicase in Human Tumor Cells

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ARTICLE ABSTRACT

Defining the processes that sustain telomere maintenance is critical to our understanding of cancer and longevity. PIF1 is a nonprocessive 5′->3′ human DNA helicase that exhibits broad substrate specificity. In vitro studies have implicated PIF1 in maintaining telomeres and processing stalled DNA replication forks, but disruption of the murine Pif1 gene did not yield any apparent phenotype. In this study, we evaluated the function of the PIF1 gene in human cells by using siRNA knockdown strategies to gauge its role in the response to DNA replication stress. We found that PIF1 depletion reduced the survival of both p53-deficient and p53-proficient human tumor cells by triggering apoptosis. In contrast, nonmalignant cells were unaffected by PIF1 depletion. Apoptosis induction in tumor cells was augmented by cotreatment with replication inhibitors (thymidine, hydroxyurea, or gemcitabine). When sensitive PIF1-depleted cells were released from a thymidine-induced S-phase arrest, there remained a subpopulation of cells that failed to enter S-phase. This cell subpopulation displayed an increase in levels of cyclin E and p21, as well as a deficiency in S-phase checkpoint markers that were induced with thymidine in PIF1 expressing cells. Specifically, CHK1 activation was suppressed and we detected no consistent changes in ATM S1981 autophosphorylation, γH2AX induction, or RPA hyperphosphorylation. Death in PIF1-depleted cells was detected in late G1/early S-phase and was dependent on caspase-3 activity. Taken together, our findings suggest roles for PIF1 in S-phase entry and progression that are essential to protect human tumor cells from apoptosis. Cancer Res; 71(14); 4998–5008. ©2011 AACR.

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