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Presence of a heterozygous substitution and its relationship to DT-diaphorase activity
B L Kuehl1, J W Paterson, J W Peacock
1Department of Medical Biophysics, University of Toronto, Ontario, Canada.
Abstract:
A point mutation in the mRNA of NADP(H): quinone oxidoreductase 1 (NQO1, DT-diaphorase) is believed to be responsible for reduced enzyme activity in the adenocarcinoma BE cell line. The present study examined nine cultured human non-cancerous fibroblast cell strains, five of which were from members of a single cancer-prone family, which demonstrated widely varying activity levels of DT-diaphorase (41 - 3462 nmol min-1 mg-1 protein), to determine if genetic alteration of the NQO1 or NOQ2 gene was involved in determining enzyme activity. All cell strains expressed NQO1 and NQO2 mRNA as measured by a quantitative polymerase chain reaction amplification technique. No relationship was found between the level of mRNA expressed and the enzyme activity in the cells. Sequencing of the entire complementary DNA from the cell strains revealed only a single base substitution at nucleotide 609 in one allele encoding NQO1 in every cell strain from members of the cancer-prone family, except for one cell strain which expressed only the T at nucleotide 609 in both alleles. Subsequent examination of genomic DNA from 44 individuals revealed that this base substitution is present in approximately 50% of the population. The presence of the T at nucleotide 609 in the NQO1 locus does not appear to be directly causal for altered DT-diaphorase activity.
Insights
Genetic variations in the NADP(H): quinone oxidoreductase 1 (NQO1) gene were investigated for their impact on DT-diaphorase enzyme activity. A common NQO1 single nucleotide polymorphism was found not to be directly causal for altered enzyme levels.
Area of Science:
- Biochemistry
- Genetics
- Molecular Biology
Background:
- NADP(H): quinone oxidoreductase 1 (NQO1), also known as DT-diaphorase, is an enzyme involved in cellular redox homeostasis.
- A specific point mutation in NQO1 mRNA was previously linked to reduced enzyme activity in an adenocarcinoma cell line.
Purpose of the Study:
- To investigate the role of genetic alterations in the NQO1 and NOQ2 genes in determining DT-diaphorase enzyme activity levels.
- To examine a common single nucleotide polymorphism (SNP) in the NQO1 gene and its association with enzyme activity.
Main Methods:
- Quantitative polymerase chain reaction (qPCR) was used to measure NQO1 and NQO2 mRNA expression levels.
- Complementary DNA (cDNA) and genomic DNA sequencing were performed to identify genetic variations.
- DT-diaphorase enzyme activity was measured in cultured human fibroblast cell strains.
Main Results:
- All examined cell strains expressed both NQO1 and NQO2 mRNA, with no correlation found between mRNA levels and enzyme activity.
- A single base substitution at nucleotide 609 in the NQO1 gene was identified in members of a cancer-prone family.
- This NQO1 nucleotide 609 polymorphism was found in approximately 50% of the general population studied.
Conclusions:
- The identified single base substitution in the NQO1 gene at nucleotide 609 is not directly causal for altered DT-diaphorase enzyme activity.
- Genetic variations, beyond the studied SNP, may contribute to the wide range of DT-diaphorase activity observed in human populations.