The importance of DT-diaphorase in mitomycin C resistance in human colon cancer cell lines.
BACKGROUND
Prior studies have suggested the multifactorial nature of mitomycin C (MMC) resistance. However, the relative importance of the different resistance mechanisms is unknown.


METHODS
A panel of colon cancer cell lines with levels of MMC resistance from 2- to 15-fold compared to the parent line HT-29 was produced by repeated MMC exposure. Cell survival was measured using clonogenic assay. Glutathione and related enzymes and DT-diaphorase were measured using biochemical assays. P-glycoprotein expression was measured using flow cytometry. Topoisomerase II activity was measured using the pBR322 DNA relaxation assay.


RESULTS
Multiple drug resistance mechanisms were altered in the resistant cell lines (glutathione reductase, glutathione peroxidase, topoisomerase II). However, the level of DT-diaphorase correlated best with the degree of MMC resistance. The importance of DT-diaphorase was confirmed by using BMY 25282, an MMC analogue which is less dependent on DT-diaphorase for activation. Resistance in the HT-29R54 cell line was 15-fold with MMC compared to 5-fold with BMY 25282. P-glycoprotein-mediated resistance does not appear important in this model.


CONCLUSIONS
Although MMC resistance appears to be multifactorial, the results of this study strongly suggest that DT-diaphorase is the major contributor to MMC resistance under aerobic conditions. Strategies to enhance drug activation may therefore be useful for reversing MMC resistance.
Lambert
P A
PA
Department of Surgery, Eastern Virginia Medical School, Norfolk, Virginia, 23507, USA.
Kang
Y
Y
Greaves
B
B
Perry
R R
RR
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000378:metabolism; D000332:Aerobiosis; D000903:Antibiotics, Antineoplastic / Q000378:metabolism / Q000494:pharmacology*; D002470:Cell Survival / Q000187:drug effects; D003110:Colonic Neoplasms / Q000188:drug therapy* / Q000201:enzymology* / Q000473:pathology; D004250:DNA Topoisomerases, Type II / Q000378:metabolism; D004351:Drug Resistance; D018432:Drug Resistance, Multiple; D005978:Glutathione / Q000378:metabolism; D005979:Glutathione Peroxidase / Q000378:metabolism; D005980:Glutathione Reductase / Q000378:metabolism; D006801:Humans; D016685:Mitomycin / Q000378:metabolism / Q000494:pharmacology*; D008937:Mitomycins; D016660:NAD(P)H Dehydrogenase (Quinone) / Q000378:metabolism*; D010084:Oxidation-Reduction; D014407:Tumor Cells, Cultured; D014410:Tumor Stem Cell Assay
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D000903:Antibiotics, Antineoplastic; D008937:Mitomycins; D016685:Mitomycin; C046941:N(6)-((dimethylamino)methylene)mitomycin C; D005979:Glutathione Peroxidase; D016660:NAD(P)H Dehydrogenase (Quinone); D005980:Glutathione Reductase; D004250:DNA Topoisomerases, Type II; D005978:Glutathione