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Assessment of the relationship between genotypic status of a DT-diaphorase point mutation and enzymatic activity
V Misra1, A Grondin, H J Klamut
1Department of Medical Biophysics, University of Toronto and Division of Experimental Therapeutics, Ontario Cancer Institute, 610 University Avenue, Toronto, Ontario, M5G 2M9, Canada.
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.
Insights
Genetic variations in DT-diaphorase (DTD) affect its activity. A specific gene substitution leads to reduced protein levels and enzyme function, impacting drug activation and detoxification.
Area of Science:
- Biochemistry
- Pharmacogenetics
- Molecular Biology
Background:
- DT-diaphorase (DTD) is a crucial enzyme for activating chemotherapeutic prodrugs and detoxifying carcinogens.
- A common C to T nucleotide substitution at position 609 in DTD cDNA is linked to reduced protein stability and enzyme activity.
Purpose of the Study:
- To investigate the correlation between the allelic status of the 609 C to T substitution in DT-diaphorase and its enzymatic activity.
- To understand the molecular basis for undetectable enzymatic activity in a specific heterozygous strain.
Main Methods:
- PCR-RFLP assay was used to determine the genotypic status of the DTD polymorphism in 45 human skin fibroblast strains.
- Allele-specific RT-PCR-RFLP was employed to analyze mRNA expression patterns.
- Western blot analysis was performed to assess DT-diaphorase protein levels.
Main Results:
- Enzymatic activity varied with genotypic status, with wild-type homozygotes showing approximately 2-fold higher activity than heterozygotes.
- A unique heterozygous strain (3437T) exhibited undetectable enzymatic activity due to exclusive expression from the variant allele.
- Homozygous variant strains displayed significantly lower DT-diaphorase protein levels, confirming the substitution's role in protein instability.
Conclusions:
- Genotypic status serves as a reliable indicator of DT-diaphorase enzymatic activity.
- The 609 C to T substitution impacts DTD function through reduced protein expression and altered mRNA expression patterns.
- Understanding these genetic variations is crucial for predicting individual responses to DTD-dependent therapies and detoxification processes.