FIGURE 1.

Level of cisplatin-DNA adducts quantitatively correlates with dual phosphorylation status of SAPK/JNK. A, human fibroblast cells (GM637) were exposed to increasing concentrations of cDDP. 4 and 24 h after continuous cDDP treatment, DNA was isolated, and the level of 1,2-GG intrastrand cross-links was determined by Southwestern analysis as described under “Experimental Procedures.” B, GM637 cells were exposed to increasing concentrations of cisplatin. After incubation periods of 6 (left panel) and 16 h (right panel), cells were harvested, and the dual phosphorylation status of SAPK/JNK (p-JNK) was analyzed by Western blot analysis. In addition, Ser-139 phosphorylation of histone H2AX (γH2AX) was determined. As a loading control, protein expression of ERK2 was monitored. Con, untreated cells. C, GM637 cells were left untreated (Con) or were exposed to different concentrations of cisplatin for 16 h. Afterward, fresh medium was added, and ROS levels were determined by FACS as described under “Experimental Procedures.” D, Balb/c mouse fibroblasts were treated with different concentrations of cDDP for 24 h. DNA platination was measured by ICP-MS as described under “Experimental Procedures.” In parallel, the dual phosphorylation status of SAPK/JNK (p-JNK) was analyzed. After densitometric analysis of the autoradiography, the relative JNK phosphorylation level observed with the highest dose of cisplatin was set to 1.0. E, Balb/c mouse fibroblasts were exposed to different concentrations of cisplatin for 16 h. Afterward, fresh medium was added, and the level of ROS was measured by FACS. F, human fibroblast cells (GM637) were transfected with platinated pEGFP plasmid DNA (cDDP) or non-platinated plasmid (Con). 16 h after transfection, cells were harvested, and the phosphorylation status of SAPK/JNK (p-JNK) and H2AX (γH2AX) was analyzed by Western blot analysis.