Biochemical characterization of a mitomycin C-resistant human bladder cancer cell line

Shivendra V. Singh, Domenic Scalamogna, Hong Xia, Stacy O'Toole, Deodutta Roy, Erling O. Emerson, Vicram Gupta, Howard A. Zaren

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Abstract

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 a 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 β mRNA level was markedly higher in the J82/MMC-2 cell line compared with either J82/WT or 182/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 8.

Original languageEnglish (US)
Pages (from-to)852-857
Number of pages6
JournalInternational Journal of Cancer
Volume65
Issue number6
DOIs
StatePublished - Mar 15 1996

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Mitomycin
Urinary Bladder Neoplasms
Cell Line
NAD(P)H Dehydrogenase (Quinone)
DNA-Directed DNA Polymerase
Messenger RNA
NADPH-Ferrihemoprotein Reductase

ASJC Scopus subject areas

  • Oncology
  • Cancer Research

Cite this

Biochemical characterization of a mitomycin C-resistant human bladder cancer cell line. / Singh, Shivendra V.; Scalamogna, Domenic; Xia, Hong; O'Toole, Stacy; Roy, Deodutta; Emerson, Erling O.; Gupta, Vicram; Zaren, Howard A.

In: International Journal of Cancer, Vol. 65, No. 6, 15.03.1996, p. 852-857.

Research output: Contribution to journalArticle

Singh, Shivendra V. ; Scalamogna, Domenic ; Xia, Hong ; O'Toole, Stacy ; Roy, Deodutta ; Emerson, Erling O. ; Gupta, Vicram ; Zaren, Howard A. / Biochemical characterization of a mitomycin C-resistant human bladder cancer cell line. In: International Journal of Cancer. 1996 ; Vol. 65, No. 6. pp. 852-857.
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abstract = "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 a 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 β mRNA level was markedly higher in the J82/MMC-2 cell line compared with either J82/WT or 182/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 8.",
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