Studies in NQO2 SNPs and NRH:CB1954 reductase activity in human bladder tumors
Authors: David Jamieson, Kerrie Wilson, Simon Pridgeon, Hing Y. Leung, John Lunec, Alan Boddy
Journal: Cancer Research (2005)

Proc Amer Assoc Cancer Res, Volume 46, 2005

1380 

NQO2 (NRH:Quinone oxidoreductase 2, EC 1.6.99.-) is a homologue of the quinone oxidoreductase, NQO1. Although it has 54% homology with human NQO1, NQO2 appears to have no endogenous enzymatic activity due to the lack of a functional intracellular electron donor. However, NQO2 has potential as a therapeutic target, since the addition of the non-biogenic electron donor dihydronicotinamide riboside (NRH) selectively potentiates the bioactivation of the alkylating agent CB1954. Numerous SNPs in the NQO2 gene have been reported, but it is unknown if any are associated with a decreased enzymatic activity. DNA was extracted from 21 human bladder cancer patients and these were genotyped by RFLP for 7 previously reported variants. Of these, 5 are located in the promoter region (including the A3777G SNP), or in the untranslated exon 1. Of the 2 remaining SNPs, one codes for a silent mutation on exon 5 and the other codes for a non-synonymous mutation on exon 3 (T14055C coding for Phe47Leu). Protein and RNA was extracted from tumour samples of 17 of the patients and assayed for activity and expression. CB1954 reductase activity in nmol/min/mg was determined by the addition of 200μl lysate to a solution containing 100μM CB1954 and 0.5mM NRH in a phosphate buffer and incubation for 80 minutes at 37oC in a final volume of 1ml. Aliquots were removed at 0,5,10,20,30,40,60 and 80 minutes, mixed 1:1 with acetonitrile and the remaining CB1954 concentration determined by HPLC. RNA expression was determined by real time quantitative RT-PCR and expressed as a ratio of HSP90B expression. One sample with a large deletion in the promoter region and no enzyme activity was excluded from further analysis. Two SNPs within the NQO2 gene were associated with differences in CB1954 reductase activity. The 3777G allele was associated with a lower NQO2 activity (median activity AA= 0.4800 versus AG/GG=0.1900, p=0.0474, Mann-Whitney). No enzyme activity was detected in the one variant homozygote. In contrast, no association was detected between the A3777G SNP and expression of NQO2. The 14055C allele was associated with a lower NQO2 activity (median activity TT= 0.4600 versus TC=0.1600, p=0.0392, Mann-Whitney). Three bladder cancer cell lines genotyped for the A3777G and T14055C SNPs also demonstrated a trend of decreased activity associated with the 3777G and 14055C variants. However a broader panel of cell lines of diverse tissue origin failed to demonstrate an association between genotype and activity. There was a close association between the incidence of the two SNPs in the bladder tumour samples (p=0.0008, Fisher’s). Two NQO2 polymorphisms were found to be associated with a decrease in enzymatic activity. The relative contribution of the two variants to decreased CB1954 activation is difficult to ascertain given the linkage in incidence. Further analysis is underway. This work was funded by Protherics PLC and Cancer Research U.K.