Variations in Ribonucleotide Reductase subunit expression induce gemcitabine resistance in a cell cycle dependent manner in Suit-2 pancreatic cancer cells
Session type: Oral
Gemcitabine has cytostatic and cytotoxic effects. Inhibition of Ribonucleotide Reductase (RRM) pausing the cell cycle in G1, while incorporation of fluorinated nucleotides in DNA causes damage and cell death. Resistant pancreatic cancer cell lines were developed by exposure to increasing gemcitabine concentrations. RRM1 levels have been associated with gemcitabine resistance, but we've identified several resistant cell lines with lower expression. The p53R2 subunit is a p53-regulated homologue of RRM2. Knocking down RRM1 will reduce gemcitabine sensitivity in sensitive but not resistant cells (Suit-2R). Resistance to the S-phase dependent cytotoxicity from low RRM expression will induce p53R2 expression, in place of other RRM2 isoforms. Chk1 is a check point kinase that arrests the cycle to facilitate repair and expression will highlight G1 arrest.
Suit-2 and Suit-2R cells were transfected with RRM1 siRNA, having been previously shown to produce a successful knockdown after 24hrs. The following day varying doses of gemcitabine were added for 48hrs. An MTS assay was performed and IC50 calculated. Western analyses of RRM2, p53R2 and Chk1 were performed on cell lysates.
The initial Suit-2 and Suit-2R assays demonstrated a gemcitabine IC50 of 17nm and 83nm, but the knockdown assay demonstrated a decrease in IC50 of both cell lines; 11nm and 25nm respectively. Suit-2R Western analysis depicted the expected reduction in RRM1, no significant change to RRM2 but a significant increase in p53R2 and Chk1.
Reduced RRM1 slows growth in both Suit-2 and Suit-2R cell lines, but is insufficent to protect cells from the S-phase specific cytotoxicity of gemcitabine. This may be due to the increase in p53R2 which was not seen in the acquired resistance to gemcitabine associated with reduction in RRM. Increasing Chk1 levels validates this theory by halting progression to S-phase. This suggests a p53-mediated sensitivity to gemcitabine that acquired resistance overcomes.