Modulation of RhoA GTPase Activity Sensitizes Human Cervix Carcinoma Cells to γ-Radiation by Attenuating DNA Repair Pathways

Figure 3

Inhibition of RhoA activity by C3 toxin expression strongly affects DNA damage response, including global and specific DNA repair mechanisms in HeLa cells, following γ-radiation. (a) Dendritic morphology of HeLa cells (HeLa + C3 images) associated with decreased RhoA-GTP levels (on the right), 24 h after transfection with a plasmid for C3 toxin expression. Images on the right are insets from those on the left, 200x. (b) Estimates of DNA damage and repair efficiency (by olive tail moment, or OTM, measurements from comet assays) in HeLa cell expressing the C3 toxin, following γ-radiation. (c) Immunoblotting analysis of the effects of γ-radiation (15 Gy) on phosphorylated Chk1/Chk2 and histone H2AX levels in HeLa cells expressing the C3 toxin (using α-Tubulin as a loading control). (d) and (e) Assays for GFP-based detection of homologous recombination (HR, using HeLa-DR-GFP) or nonhomologous end joining (NHEJ, using HeLa-EJ5-GFP) after DNA damage induced by I-SceI restriction enzyme expression. (d) Phase contrast (left) and green fluorescence (right) images of cells transfected with a plasmid for I-SceI expression (I-SceI), or with an empty vector (EV), showing the appearance of GFP-positive cells indicative of HR (HeLa-EJ5-GFP) or NHEJ (HeLa-DR-GFP), 72 h after transfection. (e) Quantification of HR and NHEJ assays, with (EV + C3 and I-SceI + C3 groups) or without (EV and I-SceI groups) concomitant C3 toxin expression. Graphs (with mean ± SD values) and immunoblots are representative of three independent experiments. , , and , between treated and untreated conditions (by ANOVA).

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