Barham H M, Inglis R, Chinje E C, Stratford I J
MRC Radiobiology Unit, Chilton, Didcot, Oxon, UK.
Br J Cancer. 1996 Oct;74(8):1188-93. doi: 10.1038/bjc.1996.515.
As part of an 'enzyme-directed' approach to bioreductive drug development, we have measured the activity of NADH: cytochrome b5 reductase (B5R) in human cancer cell lines in order to assess the role of this enzyme in activating bioreductive drugs, and thus in influencing the cytotoxicity of these compounds. At present, there is no validated assay reported in the literature for measuring the activity of B5R in tumour cells, and current measurements have assumed that the enzyme activity can be measured either as the NADH-dependent reduction of cytochrome c or as the non-dicoumarol-inhibitable activity in the DT-diaphorase assay. Using p-hydroxymercuribenzoate (pHMB) as an inhibitor of B5R, we have quantified the contribution of B5R to the NADH-dependent reduction of cytochrome c and to the overall reduction of cytochrome c in the DT-diaphorase assay. In the former we found that residual uninhibited activity remained in the presence of pHMB, in some cases accounting for up to 60% of the total reduction of cytochrome c. Thus, simply measuring the NADH-dependent reduction of cytochrome c consistently overestimated B5R activity. We also found that the non-dicoumarol-inhibitable activity in the DT-diaphorase assay underestimated B5R activity, especially in cell lines with high DT-diaphorase activity. Therefore, we have developed a spectrophotometric assay for measuring B5R activity as the pHMB-inhibitable NADH-dependent reduction of cytochrome c. This has been used to measure the B5R activity of a panel of 22 human tumour cell lines, in which we found 7-fold and 3-fold variations in activity expressed per cell or per mg protein respectively.
作为生物还原药物开发“酶导向”方法的一部分，我们测定了人癌细胞系中NADH：细胞色素b5还原酶（B5R）的活性，以评估该酶在激活生物还原药物中的作用，进而影响这些化合物的细胞毒性。目前，文献中尚未报道用于测量肿瘤细胞中B5R活性的有效测定方法，当前的测量方法假定该酶活性可以通过细胞色素c的NADH依赖性还原或DT-黄递酶测定中的非双香豆素抑制活性来测量。使用对羟基汞苯甲酸（pHMB）作为B5R的抑制剂，我们在DT-黄递酶测定中量化了B5R对细胞色素c的NADH依赖性还原以及细胞色素c总体还原的贡献。在前者中，我们发现在pHMB存在下仍保留有残余的未抑制活性，在某些情况下占细胞色素c总还原量的60%。因此，简单地测量细胞色素c的NADH依赖性还原一直高估了B5R活性。我们还发现DT-黄递酶测定中的非双香豆素抑制活性低估了B5R活性，尤其是在具有高DT-黄递酶活性的细胞系中。因此，我们开发了一种分光光度测定法，用于测量B5R活性，即作为pHMB可抑制的细胞色素c的NADH依赖性还原。这已用于测量一组22种人肿瘤细胞系的B5R活性，我们发现每细胞或每毫克蛋白质表达的活性分别有7倍和3倍的变化。
