Biochemical characterization of a mitomycin C-resistant human bladder cancer cell line.
This study describes characteristics of a mitomycin C (MMC)-resistant human bladder cancer cell line, J82/MMC-2, which was established by repeated in vitro exposures of a 6-fold MMC-resistant variant (J82/MMC) to 18 nM MMC. A 9.6-fold higher concentration of MMC was required to kill 50% of the J82/MMC-2 sub-line compared with parental cells (J82/WT). NADPH cytochrome P450 reductase and DT-diaphorase activities were significantly lower in J82/MMC-2 cells compared with J82/WT, suggesting that reduced sensitivity of J82/MMC-2 cells to MMC resulted from impaired drug activation. Consistent with this hypothesis, the formation of MMC-alkylating metabolites was significantly lower in J82/MMC-2 cells compared with J82/WT. Furthermore, DT-diaphorase activity in J82/MMC-2 cells was significantly lower compared with the 6-fold MMC-resistant variant. Glutathione (GSH) levels were comparable in all 3 cell lines. Although GSH transferase (GST) activity was significantly higher in the J82/MMC-2 cells compared with J82/WT, this enzyme activity did not differ between 6- and 9.6-fold MMC-resistant variants. Whereas DNA polymerase alpha mRNA expression was comparable in these cell lines, levels of DNA ligase I mRNA were slightly lower in both MMC-resistant variants relative to J82/WT. However, the DNA polymerase beta mRNA level was markedly higher in the J82/MMC-2 cell line compared with either J82/WT or J82/MMC. Thus, emergence of a higher level of resistance to MMC in J82/MMC-2 cells compared with J82/MMC may be attributed to (i) impaired drug activation through further reduction in DT-diaphorase activity and (ii) enhanced DNA repair through over-expression of DNA polymerase beta.
Singh
S V
SV
Mercy Cancer Institute, Mercy Hospital, Pittsburgh, Pennsylvania 15219, USA.
Scalamogna
D
D
Xia
H
H
O'Toole
S
S
Roy
D
D
Emerson
E O
EO
Gupta
V
V
Zaren
H A
HA
D000903:Antibiotics, Antineoplastic / Q000493:pharmacokinetics / Q000494:pharmacology*; D001711:Biotransformation; D002470:Cell Survival / Q000187:drug effects; D004256:DNA Polymerase I / Q000378:metabolism; D019008:Drug Resistance, Neoplasm; D004354:Drug Screening Assays, Antitumor; D005978:Glutathione / Q000378:metabolism; D005982:Glutathione Transferase / Q000378:metabolism; D006801:Humans; D016685:Mitomycin / Q000493:pharmacokinetics / Q000494:pharmacology*; D012333:RNA, Messenger / Q000378:metabolism; D014407:Tumor Cells, Cultured; D001749:Urinary Bladder Neoplasms / Q000188:drug therapy* / Q000378:metabolism / Q000473:pathology*
D000903:Antibiotics, Antineoplastic; D012333:RNA, Messenger; D016685:Mitomycin; D005982:Glutathione Transferase; D004256:DNA Polymerase I; D005978:Glutathione