Fig. 3.

Cytotoxic effect of NADPS is primarily attributable to targeting DHFR. (A) Western blot analysis of DHFR and other dehydrogenases (ALDH1a, GAPDH, and IMPDH). Protein levels were determined using antibodies specific to each dehydrogenase after treating C85 cells with 10 μM NADP-S for 0, 4, 6, 15, and 24 hours; α-tubulin was used as a loading control. (B) Trypan blue exclusion assay was used to determine cell viability of CCRF-CEM/R cells, containing a 5-fold increase of DHFR levels and CCRF-CEM parenteral cells after NADPS treatment for 96 hours. The cells were grown in RPMI-1640 medium supplemented with 10% dialyzed FBS. After harvesting cells, cell viability was determined using the Vi-CELL Series Cell Viability Analyzer. (C) MTS assay, as described in Materials and Methods, was performed to determine cytotoxic effect of NADPS in parental DG44 cells, a hamster ovary cell line with no DHFR expression and in DG44- (DHFR-EGFP), a subline of DG44 cells stably transfected with a DHFR-EGFP fusion construct. Parental DG44 cells were grown in RPMI 1640 medium that was supplemented with hypoxanthine and thymidine to allow these cells to grow in the absence of DHFR (▪, red). DG44-(DHFR-EGFP) cells were grown in RPMI 1640 media with (▴, blue) and without (▾, black) hypoxanthine and thymidine supplementation. Two thousand cells per well were plated, and cells were incubated with NADPS for 96 hours. The percentage cell survival was determined using Softmax Pro software, and GraphPad Prism 4 software was used to determine ED₅₀ values using a sigmoidal dose-response curve fit.