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A polymorphism in NAD(P)H:quinone oxidoreductase (NQO1): relationship of a homozygous mutation at position 609 of the NQO1 cDNA to NQO1 activity Sir -NQO1 has attracted considerable attention owing to its ability to deactivate a broad range of xenobiotics while activating certain anti-tumour quinones (Ross et al., 1994). A number of recent reports have highlighted the occurrence and potential significance of a point mutation in the NQO1 gene which is associated with a loss of NQO1 activity in both normal and tumour tissue (Traver et al., 1992; Eickelmann et al., 1994a,b;Rosvold et al., 1995;Kolesar et al., 1995). The mutation is a C to T point mutation at position 609 of the NQO1 cDNA which codes for a proline to serine substitution in the amino acid sequence of the protein (Traver et al., 1992). The mutation was originally characterised in the BE human colon adenocarcinoma cell line by SSCP analysis and sequencing (Traver et al., 1992) and subsequently in the H596 human non-small-cell lung cancer cell line by a polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) technique (1995) have recently examined the relationship between the heterozygous mutation at position 609 and NQO1 activity. These data demonstrate a wide range of NQO1 activity in IC to T heterozygotes and suggests a significant role for post-transcriptional modification in determination of NQO1 activity.In the paper by Kuehl et al. (1995), the BE cell line is described as heterozygous for the 9C-T point mutation with very low NQO1 activity. An additional fibroblastoid cell line, the 3701T line, was reported as homozygous for the mutation with very low NQO1 activity. Since these results differed, with respect to the BE cell line, from the original report of Traver et al. (1992) al., 1995). Because of the somewhat nonspecific nature of both DCPIP as a substrate and dicoumarol as an inhibitor (Ross et al., 1993), it is possible that the extremely low rates of dicoumarol-inhibitable DCPIP reduction obtained in BE, H596 or 3701T cells may reflect the presence of reductases other than NQO1. Our joint findings confirm previous results that the BE cells are homozygous for the 609C-T mutation and that the presence of the homozygous mutation is associated with a loss of NQO1 protein activity (Traver et al., 1992. The same conclusion was reached by Eickelmann et al. (1994) who reported that a cell line and three human kidney carcinoma samples without detectable NQO1 activity were all homozygous for the IC to T mutation.An alternatively spliced form of NQO1 lacking exon 4 and the quinone binding site has recently been reported (Gasdaska et al., 1995). This form of NQO1 has minimal enzyme activity with traditional model substrates for NQO1 but retains immunoreactivity and can be detected using a polyclonal antibody to NQO1 (Gasdaska et al., 1995). We have also examined BE and H596 cells for NQO1 protein using the same polyclonal antibody used by Gasdaska and colleagues and were unable to detect NQO1 protein. We have recently sugg...

DT-diaphorase, a cytosolic reductase, has been implicated as an activator of chemotherapeutic prodrugs and a detoxifier of certain potentially carcinogenic xenobiotics. A common C to T nucleotide 609 substitution in DT-diaphorase cDNA has been associated with protein instability and reduced catalytic activity. The degree to which the allelic status of the substitution correlates with enzymatic activity was assessed in 45 normal human skin fibroblast strains using a PCR-RFLP assay. Included in this study was the 3437T strain, which is unique in that it is heterozygous for the polymorphism yet contains undetectable enzymatic activity. An allele-specific RT-PCR-RFLP technique attributed this phenomenon to exclusive DT-diaphorase mRNA expression from the variant allele. Overlap in activities was observed between individual strains homozygous for the wild-type allele and heterozygotes, but the former group displayed enzymatic activity that was on average 2-fold higher. Western blot analysis of the two strains in this panel that are homozygous for the variant allele revealed that they express relatively low amounts of DT-diaphorase protein, consistent with the role of the substitution in protein instability. This work confirms that genotypic status is a reliable initial estimate of DT-diaphorase activity. © 2000 Cancer Research Campaign