γ-H2AX, induced by cisplatin treatment, is also strongly enhanced by NPRL2.

H1229 cells treated with an empty vector and IC20 dose of cisplatin (3.0 µM), NPRL2, or NPRL2 and IC20 dose of cisplatin were harvested at 24, 48, and 72 h after treatment. (A) Western blot shows remarkably enhanced expression of γ-H2AX in cells treated with NPRL2 and cisplatin compared with cells treated with empty vector and cisplatin. (B) γ-H2AX expression was examined at 48 h after treatment by immunofluorescence staining and flow cytometric analysis. γ-H2AX expression was detected in cells treated with empty vector and cisplatin or NPRL2 but not in cells treated with empty vector only, and this expression was dramatically enhanced in cells treated with NPRL2 and cisplatin. Magnification: ×800. Mean fluorescein isothiocyanate (FITC) intensity was also examined at 24, 48, and 72 h after treatment. At these three time points, γ-H2AX expression in cells treated with NPRL2 and cisplatin was significantly higher than that in cells in all other treatments (P<0.02 or P<0.0005). Bars, SDs of the mean in two individual experiments. (C) γ-H2AX expression was analyzed by immunofluorescence staining in an orthotopic model of H322 pleural dissemination, as described in the Materials and Methods section. The pleural tumor cells from the mice treated with LacZ and cisplatin or NPRL2 were weakly stained; in contrast, γ-H2AX was hyperexpressed in cells treated with NPRL2+ cisplatin. Magnification: ×400.