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Antioxidant and Xenobiotic-metabolizing Enzyme Gene Expression in Doxorubicin-resistant MCF-7 Breast Cancer Cells¹

Department of Medical Oncology and Therapeutics Research and Division of Biology, City of Hope National Medical Center, Duarte, California 91010

We investigated the expression of the genes for several antixoidant and xenobiotic-detoxifying enzymes in the multidrug-resistant variant of the human breast cancer cell line MCF-7, MCF-7/Dox. MCF-7/Dox is greater than 500-fold resistant to doxorubicin by clonogenic assay. Enzyme activity determinations in the cytoplasmic compartment of MCF-7/Dox revealed a 25-fold increase in glutathione peroxidase level compared to the parent line (mean ± SD, 10 ± 2.8 versus 0.4 ± 0.24 nmol/min/mg; P < 0.005). The activity of the other major hydrogen peroxide-detoxifying enzyme, catalase, was diminished in MCF-7/Dox (2.0 ± 0.4 versus 4.8 ± 1.4 µmol/min/mg; P < 0.025 compared to MCF-7). Superoxide dismutase activity did not differ between the two cell lines. The specific activity of the xenobiotic-detoxifying enzyme DT-diaphorase was 4-fold lower in MCF-7/Dox compared to MCF-7 (DT-diaphorase, 117 ± 45 versus 509 ± 123 nmol/min/mg; P < 0.005). Daunorubicinol-producing carbonyl reductase activity was equal in the two lines. Northern blot analysis demonstrated a 0.9-kilobase band of glutathione peroxidase mRNA in MCF-7/Dox; no glutathione peroxidase mRNA was detected in MCF-7. A 2.4-kilobase catalase and 0.7- and 1.4-kilobase superoxide dismutase mRNAs were detectable in MCF-7/Dox and MCF-7. When normalized to 28S RNA, no difference in the mRNA levels of catalase and superoxide dismutase in MCF-7/Dox and MCF-7 could be determined. DT-diaphorase mRNAs of 1.4 and 2.7 kilobases were found in both MCF-7/Dox and MCF-7 cells. A 1.2-kilobase mRNA homologous to the putative carbonyl reductase cDNA was also easily detectable in both MCF-7 and MCF-7/Dox. The amount of mRNA for both xenobiotic-detoxifying enzymes was decreased 2- to 4-fold in the doxorubicin-resistant cells. Southern blot analysis of PstI- and MspI-restricted genomic DNA revealed no evidence for amplification or rearrangement of the glutathione peroxidase gene. These results indicate that, in addition to the previously described overexpression of anionic glutathione S-transferase in MCF-7/Dox cells, an augmented glutathione peroxidase mRNA level is the major alteration in antioxidant and xenobiotic-detoxifying enzyme expression that could contribute to doxorubicin insensitivity in these multidrug-resistant breast cancer cells.

¹ This study was supported by Grants CA 31788 and CA 33572 from the National Cancer Institute.

² To whom requests for reprints should be addressed, at Department of Medical Oncology and Therapeutics Research, City of Hope National Medical Center, 1500 E. Duarte Road, Duarte, CA 91010.

Received 8/21/89. Revised 11/17/89. Accepted 11/29/89.

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