DNA glycosylases with dual ss/dsDNA substrate specificity are downregulated in Melphalan-resistant cells.

<p>Levels of DNA glycosylases (A), relative UDG activities (B) and genomic 8-oxodG-content in sensitive and resistant cells prior and after exposure to 50 µM Melphalan. Western blot analysis revealed downregulation of UNG2, MPG and NEIL1 (dual ss/dsDNA-specific glycosylases) in the Melphalan-resistant cells compared to sensitive cells while this was not observed for the dsDNA-specific glycosylases TDG, MBD4 and OGG1, the latter barely above the detection level in the cells (A). In agreement with UNG2 protein levels, activity assays showed that uracil excision activity was significantly higher in sensitive cells against [³H]dUMP-containing ss- and dsDNA substrates. Moreover, high dose Melphalan mediated an apparent increase in UDG activity in both cell lines, although this was only significant (P<0.05) for the resistant cells. Addition of the UNG-specific inhibitor Ugi to the reactions demonstrated that the observed reduced uracil-excision activity in the resistant cell line was mainly accounted for by reduction of UNG-activity. Each bar constitutes an average from four independent experiments, each run in triplicate, with standard deviations as demonstrated. P-values were calculated using unpaired Student t-test (B). A twofold increased steady state level of genomic 8-oxodG was found in the resistant compared to the sensitive cells. Each bar constitutes and average from three independent experiments with standard deviations as indicated (C).