Genotype-phenotype relationships in studies of a polymorphism in NAD(P)H

The NAD(P)H:quinone oxidoreductase 1 (NQO1) genotype-phenotype relationship was examined in individuals with a polymorphism in NQO1. The polymorphism comprises a C to T base change at position 609 of the human NQO1 cDNA (C609T) and codes for a proline to serine substitution in the amino acid structure of the NQO1 protein. Genotyping was performed by polymerase chain reaction-restriction fragment length polymorphism analysis of genomic DNA. Phenotyping was performed using enzyme activity assays and/or immunoblotting of human tumor cell lines and of saliva and bone marrow samples from healthy donors. Phenotyping of uninvolved lung and lung tumors from archived biopsy material was performed by immunohistochemistry. NQO1 activity and protein could be detected in wild-type (C/C) human tumor cells (HT-29) under conditions where NQO1 protein could not be detected in cells (BE) homozygous for the C609T change (T/T). Trace levels of NQO1 protein could be detected in BE cells; however, when immunoblots were subjected to chemiluminescence detection for prolonged periods. In saliva samples from 11 individuals carrying the homozygous C609T change (T/T), no NQO1 protein could be detected even after prolonged chemiluminescence detection. The amount of NQO1 protein present in saliva was quantified and found to be significantly less in heterozygous individuals (C/T) than in wild-type individuals (C/C). In bone marrow stromal cultures, both NQO1 activity and protein could be detected in heterozygotes (C/T) and in wild-type (C/C) samples. In a bone marrow stromal culture from an individual genotyped as T/T at position 609, no NQO1 protein or activity could be detected. NQO1 is elevated in non-small cell lung cancers and could be readily observed as intense immunostaining throughout lung adenocarcinomas genotyped as C/C but no immunostaining could be detected in adenocarcinomas genotyped as T/T at position 609. NQO1 is expressed in normal human lung but is localized to respiratory epithelium and to vascular endothelium. In normal lung tissue from individuals genotyped as T/T, no or faint immunostaining for NQO1 could be detected in either respiratory epithelium or vascular endothelium. These results demonstrate that tissues from individuals homozygous for the C609T change have no detectable or, at best, only trace amounts of NQO1 protein and are devoid of NQO1 activity.