Inactivation of XPF Sensitizes Cancer Cells to Gemcitabine

Figure 1

XPF is required for the cellular resistance to gemcitabine. Impact of XPF on gemcitabine sensitivity was examined by clonogenic survival assay. Cells were seeded one day before the gemcitabine treatment in 12-well plates. The indicated concentrations of gemcitabine were added and the cells were exposed to gemcitabine throughout the 5~7 days of incubation. After fixing, the cells were stained with Crystal violet. Surviving fraction was calculated by dividing number of cells with gemcitabine by number of cells without gemcitabine. Three independent experiments were performed and averages of surviving fraction are plotted. The error bars show standard deviations. (a) XPF-deficient UV41 cells are sensitive to gemcitabine and the endonuclease activity of XPF is required for the cellular resistance to gemcitabine. UV41 with vector alone (closed diamond) are sensitive to gemcitabine compared to the UV41 reconstituted with human XPF gene (closed square). Reconstituted UV41 with human XPF gene (closed square) restored resistance to gemcitabine while the endonuclease-deficient XPF (XPF-DA) (closed triangle) failed to restore the gemcitabine resistance. The difference in the gemcitabine sensitivity at each concentration is statistically significant with P<0.01. (b) Nucleotide excision repair (NER) contributes to the cellular resistance to gemcitabine. A NER deficient UV135 showed a moderate sensitivity to gemcitabine compared to the sensitivity in UV41. The differences in the gemcitabine sensitivity at 6.4 μM between AA8 and UV41, AA8 and UV135, and UV41 and UV135 are statistically significant with P<0.05. (c) Suppression of XPF in pancreatic cancer cell line BxPC3 sensitizes cells to gemcitabine. The expression of XPF was suppressed by siRNA in pancreatic cancer cell line BxPC3 and the cellular sensitivity to gemcitabine was examined. The difference in the gemcitabine sensitivity between siControl- and siXPF-treated cells is statistically significant with P<0.05. The western blots showed the suppression of XPF. The expression of XPF was significantly reduced (more than 95%) with the siRNA treatment. GADPH was used as a protein loading control.

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