Eickelmann P, Schulz W A, Rohde D, Schmitz-Dräger B, Sies H
Institut für Physiologische Chemie I, Heinrich-Heine-Universität Düsseldorf, Germany.
Biol Chem Hoppe Seyler. 1994 Jul;375(7):439-45. doi: 10.1515/bchm3.1994.375.7.439.
The human bladder carcinoma cell line RT112 and the mitomycin C-resistant cell line RT112MMC, derived from RT112 cells, were examined for their expression of NAD(P)H:quinone oxidoreductase (NQOR) and glutathione S-transferases (GSTs). RT112 cells were 40-fold more sensitive towards mitomycin C than RT112MMC cells. The NQOR mRNA level in RT112MMC cells was decreased to 15% as compared to RT112 cells. NQOR enzyme activity was 391 +/- 140 mU/mg protein in RT112 cells, whereas NQOR activity in RT112MMC cells was not measurable. As shown by a fast PCR-based assay and DNA-sequencing, the cell line RT112 is heterozygous, whereas RT112MMC is homozygous for a null allele of the NQOR gene without enzymatic activity. Accordingly, both wild-type and null allele mRNAs were present in RT112 cells, whereas only null allele mRNA was found in RT112MMC. The lack of NQOR enzyme activity in RT112MMC cells was thus associated with loss of heterozygosity at the NQOR locus. By a PCR-RFLP assay, three kidney carcinoma patients without measurable NQOR enzyme activity were shown to be homozygous for the null allele. The PCR assay described here is useful for examination of large numbers of samples. The relative amount of GST-Pi mRNA was decreased by 30% in RT112MMC as compared to RT112, contributing to a diminished level of GST enzyme activity, using CDNB as a substrate, from 95 +/- 62 mU/mg protein in RT112 to 26 +/- 6 mU/mg protein in RT112MMC.
对人膀胱癌细胞系RT112以及源自RT112细胞的丝裂霉素C耐药细胞系RT112MMC进行了烟酰胺腺嘌呤二核苷酸磷酸（NAD(P)H）：醌氧化还原酶（NQOR）和谷胱甘肽S-转移酶（GSTs）表达情况的检测。RT112细胞对丝裂霉素C的敏感性比RT112MMC细胞高40倍。与RT112细胞相比，RT112MMC细胞中NQOR mRNA水平降至15%。RT112细胞中NQOR酶活性为391±140 mU/mg蛋白，而RT112MMC细胞中的NQOR活性无法检测到。基于快速聚合酶链反应（PCR）的检测和DNA测序显示，细胞系RT112是杂合子，而RT112MMC对于无酶活性的NQOR基因无效等位基因是纯合子。因此，RT112细胞中同时存在野生型和无效等位基因mRNA，而在RT112MMC中仅发现无效等位基因mRNA。RT112MMC细胞中NQOR酶活性的缺乏因此与NQOR基因座杂合性的丧失有关。通过PCR-限制性片段长度多态性（RFLP）检测，三名无法检测到NQOR酶活性的肾癌患者被证明对于无效等位基因是纯合子。这里描述的PCR检测对于大量样本的检测很有用。与RT112相比，RT112MMC中谷胱甘肽S-转移酶Pi（GST-Pi）mRNA的相对量减少了30%，这导致以1-氯-2,4-二硝基苯（CDNB）为底物时，GST酶活性水平降低，从RT112中的95±62 mU/mg蛋白降至RT112MMC中的26±6 mU/mg蛋白。
