Methotrexate resistance in human uroepithelial cells with p53 alterations

Bladder cancers are frequently treated with combination chemotherapy that includes methotrexate (MTX). The development of drug resistance is a common problem in treatment of bladder cancers. We tested if the status of p53 and/or pRb affects the development of MTX resistance in bladder epithelial cell lines.

We used two isogeneic sets of cell lines in which we manipulated the status of p53 and/or pRb by transformation with Human Papillomavirus (HPV) E6 and/or E7 or with a transdominant TP53 mutant (TP53(143)). One series of isogeneic origin was derived from normal human uroepithelial cells (HUC), and the other was derived from a human transitional cell carcinoma (TCC). Cell lines with p53 and/or pRb alterations were cultured for six months while increasing the MTX concentration in each line, as resistance developed.

Two cell lines with both pRb and p53 alterations, alphaE6/E7-HUC and alphaE7-HUCp53mu, acquired the greatest resistance (750 nM) to MTX. One line with p53 loss, E6-TCC#10, acquired intermediate resistance (500 nM), while two lines, alphaE7-HUC and E7-TCC#10, with altered pRb but wildtype p53, showed low levels of MTX resistance (125 nM and 80 nM, respectively). Two clear mechanisms of MTX resistance were identified. All five MTX resistant cell lines showed altered uptake of MTX. In addition, two of five MTX resistant cell lines, both with altered p53, showed dihydrofolate reductase (DHFR) amplification.

p53 alteration increases the risk for development of drug resistance by both DHFR amplification and altered MTX transport in transformed human bladder epithelial cell lines.