METHOTREXATE RESISTANCE IN HUMAN UROEPITHELIAL CELLS WITH P53 ALTERATIONS

Purpose: Bladder cancers are frequently treated with combination chemo therapy that includes methotrexate (MTX). The development of drug resi stance is a common problem in treatment of bladder cancers. We tested if the status of p53 and/or pRb affects the development of MTX resista nce in bladder epithelial cell lines. Materials and Methods: We used t wo isogeneic sets of cell lines in which we manipulated the status of p53 and/or pRb by transformation with Human Papillomavirus (HPV) E6 an d/or E7 or with a transdominant TP53 mutant (Tp53(143)). One series of isogeneic origin was derived from normal human uroepithelial cells (H UC), and the other was derived from a human transitional cell carcinom a (TCC). Cell lines with p53 and/or pRb alterations were cultured for six months while increasing the MTX concentration in each line, as res istance developed. Results: Two cell lines with both pRb and p53 alter ations, alpha E6/E7-HUC and alpha E7-HUCp53mu, acquired the greatest r esistance (750 nM) to MTX. One line with p53 loss, EG-TCC#10, acquired intermediate resistance (500 nM), while two lines, alpha E7-HUC and E 7-TCC#10, with altered pRb but wildtype p53, showed low levels of MTX resistance (125 nM and 80 nM, respectively). Two clear mechanisms of M TX resistance were identified. All five MTX resistant cell lines showe d altered uptake of MTX. In addition, two of five MTX resistant cell l ines, both with altered p53, showed dihydrofolate reductase (DHFR) amp lification. Conclusions: p53 alteration increases the risk for develop ment of drug resistance by both DHFR amplification and altered MTX tra nsport in transformed human bladder epithelial cell lines.