Modulation of the ribonucleotide reductase-antimetabolite drug interaction in cancer cell lines

Abstract

RRM1 is a determinant of gemcitabine efficacy in cancer patients. However, the precision of predicting tumor response based on RRM1 levels is not optimal. We used gene-specific overexpression and RNA interference to assess RRM1's impact on different classes of cytotoxic agents, on drug-drug interactions, and the modulating impact of other molecular and cellular parameters. RRM1 was the dominant determinant of gemcitabine efficacy in various cancer cell lines. RRM1 also impacted the efficacy of other antimetabolite agents. It did not disrupt the interaction of two cytotoxic agents when combined. Cell lines with truncation, deletion, and null status of p53 were resistant to gemcitabine without apparent relationship to RRM1 levels. Pemetrexed and carboplatin sensitivity did not appear to be related to p53 mutation status. The impact of p53 mutations in patients treated with gemcitabine should be studied in prospective clinical trials to develop a model with improved precision of predicting drug efficacy.

Figure 1

Modification of RRM1 expression by stable transfection with RRM1 and shRRM1 expression vectors in cell lines H23, MCF7, and HCT. Wt, wild-type cell lines, R1, clones of cell lines transfected with RRM1; Ct, clones transfected with an out-of-frame RRM1 vector; shR1, clones transfected with a small hair-pin RRM1 vector; shCt, clones transfected with a random control small hair-pin vector. (a) RRM1 protein (red) and mRNA (green) expression in stably transfected clones of H23, MCF7, and HCT8. (b) Western blots of H23, MCF7, and HCT8 clones. (c) Cytotoxicity of MCF7 clones following gemcitabine treatment for 6 days. Each point is the mean of at least three independent experiments. The dashed line indicates the 50% survival fraction.

Figure 2

Knock-down of RRM1 and ERCC1 expression in three NSCLC cell lines by RNA interference and impact on gemcitabine efficacy. (a) Western blots showing that RRM1-specific siRNA reduced RRM1 protein expression to undetectable levels, while random control and ERCC1-specific siRNAs did not affect RRM1 expression. Likewise, ERCC1-specific siRNA reduced ERCC1 protein expression to undetectable levels, while random control and RRM1-specific siRNAs did not affect ERCC1 expression. (b) IC50 values of gemcitabine cytotoxicity in cell lines A549, H292, and H460. Wt, wild-type cell lines; si-control, cell lines transfected with nonspecific siRNA; si-ERCC1; cell lines transfected with ERCC1-specific siRNA; si-RRM1, cell lines transfected with RRM1-specific siRNA.

Figure 3

Western blots of knock-down of TP53 expression in four NSCLC cell lines by RNA interference. TP53-specific siRNA reduced TP53 protein expression to undetectable levels in A549 and H292 and greater then 10-fold in H23 and H460, while random control siRNA did not affect TP53 or RRM1 expression.

Figure 4

Western blots of cell line H358. There is no detectable TP53 expression in wild-type cells; while transfected cells clearly show TP53 expression.