The molecular basis of polymorphic debrisoquin hydroxylation was studied in 223 Swedish white subjects, 187 extensive metabolizers and 36 poor metabolizers phenotyped with debrisoquin and desipramine. Restriction fragment length polymorphism (RFLP) analysis of the CYP2D6 gene revealed that 52% of unrelated poor metabolizers were homozygous for Xba I 29 kb fragment, and only 8% had two mutant alleles detected with RFLP. Allele-specific polymerase chain reaction (PCR)-based DNA amplification, however, revealed that all but one of the poor metabolizers had two mutant alleles of the CYP2D6A or CYP2D6B type or both. Extensive metabolizers who were heterozygous for wild-type and CYP2D6B genes had metabolic ratios for debrisoquin and desipramine that were higher than those of subjects who were homozygous for the wild-type gene. The 16 + 9 kb Xba I RFLP pattern was associated with the poor metabolizer phenotype and CYP2D6B mutations. Three extremely rapid metabolizers of debrisoquin had a 44 kb Xba I fragment that did not carry either CYP2D6A or CYP2D6B mutations. In conclusion, in the Swedish population studied, allele-specific PCR amplification allowed prediction of the debrisoquin hydroxylation phenotype with 99% accuracy.
We have previously shown that confluent growth of the human hepatoma cell line Huh7 substantially induces the CYP3A4 mRNA, protein, and activity levels. Here, the mechanisms behind were investigated, and a transcriptome analysis revealed significant up-regulation of liver-specific functions, whereas pathways related to proliferation and cell cycle were downregulated in the confluent cells. Reporter analysis revealed that the CYP3A4 gene was transcriptionally activated during confluence in a process involving pregnane X receptor (PXR). PXR expression was increased, and PXR protein accumulated in the nuclei during confluent growth. The PXR ligand rifampicin further increased the expression of CYP3A4, and siRNAmediated knock-down of PXR in confluent cells resulted in decreased CYP3A4 expression. Cyclin-dependent kinase 2 (CDK2), a known modulator of the cell cycle and a negative regulator of PXR, was more highly expressed in proliferating control cells. Trypsinization of the confluent cells and replating them subconfluent resulted in a decrease in CYP3A4 and PXR expression back to levels observed in subconfluent control cells, whereas the CDK2 levels increased. Knock-down of CDK2 in proliferating control cells increased the CYP3A4 and PXR protein levels. Moreover, the CDK inhibitor roscovitine stimulated the expression of CYP3A4. A phosphorylationdeficient mutation (S350A) in the PXR protein significantly induced the CYP3A4 transcription. In conclusion, the data strongly suggest that the increased CYP3A4 expression in confluent Huh7 cells is caused by the endogenous induction of PXR as a result of cell-cell contact inhibited proliferation and subsequent decreased CDK2 activities, indicating an endogenous, non-ligand-dependent regulation of PXR and CYP3A4, possibly of physiologic and pharmacological significance.
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