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Combined Wee1 and Rad51 inhibitor treatment decrease repair of double-strand breaks, survival and alters cells cycle of irradiated T-leukemia cells
Authors: Havelek Radim | Ćmielová Jana | Královec Karel | Brůčková Lenka | Vávrová Jiřina | Řezáčová Martina | Bílková Zuzana
Year: 2014
Type of publication: ostatní - článek ve sborníku
Name of source: FEBS Journal Vol. 281, s1
Publisher name: Wiley-Blackwell
Place: Hoboken
Page from-to: 714
Titles:
Language Name Abstract Keywords
eng Combined Wee1 and Rad51 inhibitor treatment decrease repair of double-strand breaks, survival and alters cells cycle of irradiated T-leukemia cells Aim: To evaluate cytotoxic and apoptosis-inducing effects of ionizing radiation, specific inhibitor of Wee1 kinase 681641, novel inhibitor of Rad51 recombinase RI-1 and its combination on Jurkat (p53 deficient) and MOLT-4 (p53 wild-type) leukemic cell lines. We also aim to clarify basic cellular and molecular mechanisms involved in ionizing radiation induced double-strand breaks (DSBs) repair in presence or absence of examined inhibitors. Methods: In this work, we measured the mitochondrial dehydrogenase activity to quantify survival of Jurkat and MOLT-4 cells. Flow cytometry based methods were used to assess the effect of the two inhibitors on cell cycle and apoptosis quantified by means of Annexin V-FITC and propidium iodide dual staining. Immunofluorescence stained gammaH2AX positive cells were detected 24 h after irradiation to semiquantitatively analyse delayed DSBs repair by epi-fluorescence microscopy under low magnification. Visual aspects of persistent gammaH2AX foci with 53BP1 colocalization were captured by high-resolution imaging using confocal microscope. Expressions of selected proteins involved in DNA repair and cell cycle arrest were detected by Western blot. Results: Combining Wee1 681641 and Rad51 RI-1 with ionizing radiation significantly reduced the cell survival for both Jurkat and MOLT-4 cells. Pre-treatment with Wee1 681641 or Rad51 RI-1 inhibitor alone increased the sensitivity of Jurkat cells to the irradiation, however combining both inhibitors together resulted in further enhancement of apoptosis. Jurkat cells pre-treated with inhibitors were positive for gammaH2AX foci 24 h upon irradiation. MOLT-4 cells were less affected by inhibitors application followed by ionizing radiation exposure. Pre-treatment with Rad51 RI-1 had minimal effect on increased apoptosis induction; however Wee1 681641 increased ionizing radiation-induced cell death in MOLT-4. When dosed together, the combination had additive effect on MOLT-4 cell death. Wee1 and Rad51 inhibitor; treatment; double-strand breaks; irradiated; T-leukemia cells

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