Assessment of the relationship between genotypic status of a DT-diaphorase point mutation and enzymatic activity

Abstract

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

Expression of the prodrug-activating enzyme DT-diaphorase via Ad5 delivery to human colon carcinoma cells in vitro

“…BE cells infected with Ad5.DTD 609T exhibited no major increases in MMC sensitivity, consistent with previous observations of impaired catalytic activity and protein instability of the DTD 609T isoform. 18,19 As shown in Figure 5C, exogenous expression of DTD was confirmed in cells infected with Ad5.DTD 609C by assessing DTD activities in cells that were infected in parallel with the MMC sensitivity studies described above.…”

“…-gal or Ad5.DTD A single-base transition corresponding to nucleotide 609 of DTD cDNA ( C -to -T ) has been shown to result in impaired DTD activity and decreased protein stability. 18,19 HT29 cells ( homozygous for the wild -type DTD allele ) displayed mean ± SEM (n = 5) DTD activity of 1060 ± 215 nmol / min / mg protein, whereas BE cells (homozygous for the variant DTD allele ) contained very low to undetectable DTD activity ( less than 20 nmol /min /mg protein ). Consistent with the fact that the variant allele promotes decreased DTD protein stability, 19 HT29 cells display Western blot reactivity whereas BE cells express very low to undetectable levels of DTD protein.…”

“…Similar levels of 609T isoform expression were observed following Ad5.DTD 609T infection of BE cells at 10 and 100 pfu/cell ( Fig 3 ). The lower molecular weight band ( 0.5-1 kDa smaller than wild -type DTD ) observed in Figure 3A may be a result of proteolytic processing or chemical degradation of the 609T isoform 19 and was included in the densitometric analysis.…”

“…Ten milliliters of growth medium was added to each dish and, after 48 hours, cells were harvested by scraping. Total cell protein extracts were prepared, protein concentrations determined, and Western blot and densitometry analyses carried out as described previously, 19 with the exception that blots were probed for integrin V expression ( using a 1:1000 dilution of antihuman integrin V ) as a loading control and protease inhibitors were not used.…”

“…15,18 Individuals homozygous for this mutation exhibit little or no DTD activity and heterozygotes have approximately half the DTD activity observed in wild -type cells. 18,19 Homozygous mutants have a much reduced sensitivity to MMC under aerobic exposure conditions in vitro. 10,15 To reduce the problem of heterogeneity and to enhance DTD activity in tumor cells, recombinant adenoviral vectors were constructed, which express DTD minigenes for both wild -type and mutant ( C -to -T base change ) DTD under the control of the cytomegalovirus (CMV ) promoter.…”