Akman S A, Forrest G, Chu F F, Esworthy R S, Doroshow J H
Department of Medical Oncology and Therapeutics Research, City of Hope National Medical Center, Duarte, California 91010.
Cancer Res. 1990 Mar 1;50(5):1397-402.
We investigated the expression of the genes for several antioxidant 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 less than 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 mumol/min/mg; P less than 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 less than 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.
我们研究了人乳腺癌细胞系MCF-7的多药耐药变体MCF-7/Dox中几种抗氧化酶和外源性物质解毒酶基因的表达情况。通过克隆形成试验，MCF-7/Dox对阿霉素的耐药性比亲代细胞系高500倍以上。对MCF-7/Dox细胞质部分的酶活性测定显示，与亲代细胞系相比，谷胱甘肽过氧化物酶水平增加了25倍（平均值±标准差，10±2.8对0.4±0.24 nmol/分钟/毫克；P<0.005）。另一种主要的过氧化氢解毒酶过氧化氢酶的活性在MCF-7/Dox中降低（2.0±0.4对4.8±1.4 μmol/分钟/毫克；与MCF-7相比P<0.025）。两种细胞系中超氧化物歧化酶的活性没有差异。外源性物质解毒酶DT-黄递酶的比活性在MCF-7/Dox中比MCF-7低4倍（DT-黄递酶，117±45对509±123 nmol/分钟/毫克；P<0.005）。两种细胞系中产生柔红霉素醇的羰基还原酶活性相同。Northern印迹分析显示MCF-7/Dox中有一条0.9千碱基的谷胱甘肽过氧化物酶mRNA条带；在MCF-7中未检测到谷胱甘肽过氧化物酶mRNA。在MCF-7/Dox和MCF-7中可检测到2.4千碱基的过氧化氢酶以及0.7和1.4千碱基的超氧化物歧化酶mRNA。以28S RNA作标准化对照时，无法确定MCF-7/Dox和MCF-7中过氧化氢酶和超氧化物歧化酶mRNA水平的差异。在MCF-7/Dox和MCF-7细胞中均发现了1.4和2.7千碱基的DT-黄递酶mRNA。在MCF-7和MCF-7/Dox中也很容易检测到一条与假定的羰基还原酶cDNA同源的1.2千碱基mRNA。在阿霉素耐药细胞中，两种外源性物质解毒酶的mRNA量减少了2至4倍。对经PstI和MspI酶切的基因组DNA进行Southern印迹分析，未发现谷胱甘肽过氧化物酶基因扩增或重排的证据。这些结果表明，除了先前描述的MCF-7/Dox细胞中阴离子型谷胱甘肽S-转移酶的过表达外，谷胱甘肽过氧化物酶mRNA水平的升高是抗氧化酶和外源性物质解毒酶表达的主要变化，这可能导致这些多药耐药乳腺癌细胞对阿霉素不敏感。
