The effect of the C3435T mutation at exon 26 of the MDR1 gene on the expression levels of MDR1 messenger ribonucleic acid (mRNA) was evaluated by means of real‐time polymerase chain reaction in 51 biopsy specimens of duodenum obtained from 13 healthy Japanese subjects. The mRNA levels of MDR1 were 0.38 ± 0.15, 0.56 ± 0.14, and 1.13 ± 0.42 (mean value ± SE) in the subjects with the homozygote of wild‐type allele (C/C), compound heterozygote with mutant T allele (C/T), and the homozygote of the mutant allele (T/T), respectively, reasonably explaining the lower digoxin serum concentration after administration of a single oral dose to subjects harboring a mutant T allele. Good correlation (r = .797; P < .01) was observed between the mRNA concentrations of MDR1 and CYP3A4 in the individual biopsy specimens. This finding suggested a lower plasma concentration of the substrates for CYP3A4 in subjects harboring the C3435T mutation of the MDR1 gene.Clinical Pharmacology & Therapeutics (2002) 71, 297–303; doi: 10.1067/mcp.2002.122055

The expression levels of mRNAs for MDR1 (P-glycoprotein), multidrug resistance-associated proteins (MRP1, MRP2), and cytochrome P450 3A (CYP3A) in Caco-2 cells were quantitatively compared with those in human duodenal enterocytes, normal colorectal tissues, and colorectal adenocarcinomas. Caco-2 cells (passages 36–88) were kindly supplied by several laboratories in Japan. Human duodenal enterocytes were obtained from five healthy male volunteers. Normal colorectal tissues and colorectal adenocarcinomas were simultaneously obtained from seven patients with primary colorectal adenocarcinoma. MDR1, MRP1, MRP2, and CYP3A mRNA levels were determined by real-time quantitative polymerase chain reactions (PCR). Relative concentrations of mRNAs for target proteins (MDR1, MRP1, MRP2, and CYP3A) and glyceraldehyde-3-phosphate dehydrogenase in Caco-2 cells were 1.00 ± 0.15, 1.02 ± 0.06, 0.94 ± 0.10, and 0.68 ±0.60, respectively, and those in human enterocytes were about 12-, 3-, 7-, and 8000-fold higher than in the Caco-2 cells, respectively. In contrast, MDR1, MRP1, and CYP3A mRNA levels in Caco-2 cells were comparable to those in normal colorectal tissue and colorectal adenocarcinoma.

In the present study, we examined whether polymorphisms in the ATP-binding cassette (ABC) transporter genes, MDR1, MRP1 and MRP2, were associated with their respective mRNA expression levels in duodenal enterocytes of 13 healthy Japanese volunteers. MDR1 genotypes of T-129C, G2677(A,T) and C3435T, MRP1 genotypes of G128C, C218T, G2168A and G3173A, and MRP2 genotypes of C-24T, G1249A, C2302T, C2366T and G4348A were determined by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) or direct sequencing. Mutations T-129C, G2677(A,T) and C3435T of MDR1 gene were found at allele frequencies of 2/26, 16/26 and 12/26, respectively. Mutations G2168A of the MRP1 gene and C-24T of the MRP2 gene were also found at allele frequencies of 1/26 and 6/26, respectively, whereas other mutations were not detected in MRP1 and MRP2 genes. The relative concentrations (mean±S.E.) of MDR1 mRNA to villin mRNA were 0.38±0.15, 0.56±0.14 and 1.13±0.42 in the subjects with C/C3435, C/T3435 and T/T3435, respectively, which supported the lower serum concentrations of digoxin after single oral administration in the subjects with the mutant T-allele at position 3435. Genetic collaboration between positions 3435 and 2677 was suggested, and those in G/G2677, G/(A,T)2677 and T/(A,T)2677 were 0.16±0.05, 1.10±0.40, and 0.63±0.16, respectively (p=0.107). However, there was no remarkable effect of the G2168A of the MRP1 gene or of C-24T of the MRP2 gene on the relative MRP1 or MRP2 mRNA concentrations, respectively.

Prostate cancer progresses from an androgen-dependent to androgen-independent stage after androgen ablation therapy. Mitochondrial DNA plays a role in cell death and metastatic competence. Further, heteroplasmic large-deletion mitochondrial DNA is very common in prostate cancer. To investigate the role of mitochondrial DNA in androgen dependence of prostate cancers, we tested the changes of normal and deleted mitochondrial DNA in accordance with the progression of prostate cancer. We demonstrated that the androgen-independent cell line C4-2, established by inoculation of the androgen-dependent LNCaP cell line into castrated mice, has a greatly reduced amount of normal mitochondrial DNA and an accumulation of large-deletion DNA. Strikingly, the depletion of mitochondrial DNA from androgen-dependent LNCaP resulted in a loss of androgen dependence. Reconstitution of normal mitochondrial DNA to the mitochondrial DNA-depleted clone restored androgen dependence. These results indicate that mitochondrial DNA determines androgen dependence of prostate cancer cell lines. Further, mitochondrial DNA-deficient cells formed tumors in castrated athymic mice, whereas LNCaP did not. The accumulation of large deletion and depletion of mitochondrial DNA may thus play a role in the development of androgen independence, leading to progression of prostate cancers.

Escherichia coliis one of the most common pathogens in urinary tract infections (UTIs), and antibiotic resistance inE. coliis becoming a serious problem in treating UTI. Efflux system overexpression is reported to contribute toE. coliresistance to several antibiotics. This study investigated the correlation of antibiotic susceptibilities with the overexpression of the efflux pump genes such asmarA,yhiU,yhiV, andmdfAand with risk factors for antibiotic resistance inE. coliisolated from UTI patients. We examined the expression level of efflux pump genes using quantitative real-time reverse transcription-PCR (qRT-PCR). We also tested thein vitrosusceptibilities to 12 kinds of antibiotics in 64 clinical strains ofE. coliisolated from UTI patients. By multivariate analyses we revealed significant relationships between the overexpression of (i)marAand MICs of cefepime (FEP) and nalidixic acid (NAL), (ii)yhiVand MICs of minocycline (MIN), and (iii)mdfAand MICs of sitafloxacin (STX). In our investigation of the efflux pump genes, risk factors such as gender and the previous use of fluoroquinolones correlated with the overexpression ofmarA, and indwelling catheter use correlated with the overexpression ofmdfA. In conclusion, we demonstrated that the increased expression of efflux pump genes such asmarAandmdfAcan lead to fluoroquinolone resistance inE. coli. These results contribute to our knowledge of the efflux system and raise the possibility of developing new agents, such as efflux pump inhibitors (EPIs), to antibiotic-resistantE. coli.