Transcriptional regulation of cytochrome P4503A4 gene expression: effects of inherited mutations in the 5'-flanking region.
1. Understanding the genetic basis of interindividual variability in drug disposition and response is a fundamental focus for rational and individualized drug treatment. Cytochrome P4503A4 (CYP3A4) has a central role in human drug metabolism and polymorphic variation has been reported in this gene. 2. This study reports the in vitro functional analysis of inherited mutations in the 5' flanking region of the CYP3A4 gene using reporter constructs in which the 1141 bp proximal promoter region from the mutant alleles was inserted between a single copy of the CYP3A4 300 bp core distal enhancer (XREM) sequence and the cDNA for human secretory alkaline phosphatase. 3. Reporter constructs were co-transfected with an hPXR expression vector into human liver and intestinal cells in culture and xenobiotic modulation of CYP3A4 promoter activity determined by chemiluminescent secretory alkaline phosphatase assay. DNA-protein interactions were next examined using electrophoretic mobility shift assays. 4. The results demonstrated that inherited mutations in the CYP3A4 gene proximal promoter region could cause significant up-regulation of in vitro transcriptional activation by CYP3A4 xenobiotic inducers. In addition, the magnitude of the effect appeared to be dependent on the cell type used in the functional assays, possibly due to the differing availability of specific intracellular transcription factors or their activating ligands.
Hamzeiy
H
H
Molecular Toxicology Group, School of Biomedical and Molecular Sciences, University of Surrey, Guilford GU2 7XH, UK.
Bombail
V
V
Plant
N
N
Gibson
G
G
Goldfarb
P
P
D024506:5' Flanking Region / Q000235:genetics*; D000469:Alkaline Phosphatase / Q000378:metabolism; D000483:Alleles; D000904:Antibiotics, Antitubercular / Q000378:metabolism; D045744:Cell Line, Tumor; D002467:Cell Nucleus / Q000235:genetics; D051544:Cytochrome P-450 CYP3A; D003577:Cytochrome P-450 Enzyme System / Q000096:biosynthesis*; D004247:DNA / Q000096:biosynthesis / Q000235:genetics; D024202:Electrophoretic Mobility Shift Assay; D015971:Gene Expression Regulation, Enzymologic / Q000235:genetics*; D017930:Genes, Reporter / Q000235:genetics; D006801:Humans; D007527:Isoenzymes / Q000235:genetics; D009154:Mutation / Q000235:genetics*; D017510:Protein Folding; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D012293:Rifampin / Q000378:metabolism
D000904:Antibiotics, Antitubercular; D007527:Isoenzymes; D011994:Recombinant Proteins; D004247:DNA; D003577:Cytochrome P-450 Enzyme System; C104464:CYP3A protein, human; D051544:Cytochrome P-450 CYP3A; C510163:CYP3A4 protein, human; D000469:Alkaline Phosphatase; D012293:Rifampin