JNCI Journal of the National Cancer Institute

JNCI Journal of the National Cancer Institute 1999 91(18):1587-1588; doi:10.1093/jnci/91.18.1587
© 1999 by Oxford University Press

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Journal of the National Cancer Institute, Vol. 91, No. 18, 1587-1588, September 15, 1999
© 1999 Oxford University Press

CORRESPONDENCE

Re: Modification of Clinical Presentation of Prostate Tumors by a Novel Genetic Variant in CYP3A4

Yuichi Ando, Tomonori Tateishi, Yoshitaka Sekido, Toshimichi Yamamoto, Tetsuo Satoh, Yoshinori Hasegawa, Shinichi Kobayashi, Yoshinao Katsumata, Kaoru Shimokata, Hidehiko Saito

Affiliations of authors: Y. Ando, Y. Hasegawa, H. Saito, First Department of Internal Medicine, Nagoya University School of Medicine, Japan; T. Tateishi, S. Kobayashi, Department of Pharmacology, St. Marianna University School of Medicine, Kawasaki, Japan; Y. Sekido, K. Shimokata (Department of Preventive Clinical Medicine), T. Yamamoto, Y. Katsumata (Department of Legal Medicine and Bioethics, Postgraduate School of Medicine), Nagoya University; T. Satoh, The Biomedical Research Institute, HAB Discussion Group, Chiba, Japan.

Correspondence to: Yuichi Ando, M.D., First Department of Internal Medicine, Nagoya University School of Medicine, 65 Tsurumai, Showa-ku, Nagoya 466-8550, Japan (e-mail: yando{at}tsuru.med.nagoya-u.ac.jp).

In the recent study on a new variant of the cytochrome P450 3A4 (CYP3A4) gene by Rebbeck et al. (1), the authors did not provide data that demonstrate an alteration of CYP3A4 function as a consequence of the polymorphism. They suggested that the variant allele might alter disposition of the androgenic substrates of CYP3A4 as a result of decreased enzymatic activity. Worse clinical presentation of prostate cancer (1) and a decreased risk for treatment-related leukemia (2) were reported to be associated with the variant allele, the former probably due to increased bioavailability of testosterone and the latter probably due to reduced production of leukemogenic metabolites of anticancer drugs. Thus, we investigated the possible relationship between CYP3A4 genotypes and nifedipine oxidation activity, a prototype reaction of the encoded enzyme, by use of a human liver microsome system in vitro.

Fifteen liver samples from Caucasian transplant donors were obtained from the National Disease Research Interchange (Philadelphia, PA) through the Biomedical Research Institute, Human and Animal Bridge Discussion Group (Chiba, Japan). The nifedipine oxidation activity and the expression levels of CYP3A4 protein of the samples have been reported elsewhere (3). A variant sequence of the CYP3A4 gene was distinguished from wild-type by a nested polymerase chain reaction-restriction fragment length polymorphism assay by the use of genomic DNA prepared from the liver samples (Fig. 1, A). The institutional review board of St. Marianna University School of Medicine has approved the study. The genotyping analysis revealed one homozygote and four heterozygotes for the variant. The remaining 10 subjects were homozygous for the wild-type allele. No apparent relationships between the genotypes and the activity or amount of CYP3A4 were found (Fig. 1, B).

View larger version (88K): [in this window] [in a new window]   Fig. 1. A) Representative patterns of MboII restriction fragment length polymorphisms (RFLPs) of the CYP3A4 gene. The first-step polymerase chain reaction (PCR) amplification of a 592-base-pair (bp) fragment (nucleotide -570 to +22) was performed by the use of previously described methods (1,2). The second set of PCR amplifications was carried out by the use of the nested primers designed to amplify a 168-bp segment. The mismatched forward and the reverse primer was 5'-GGACAGCCATAGAGACAAGGGGA-3' (-290 to -312; underlining indicates artificial mismatched site) and 5'-CACTCACTGACCTCCTTTGAGTTCA-3' (-145 to -169), respectively. The forward primer was designed to introduce a MboII (Takara Shuzo Co., Ltd., Otsu, Japan) restriction site in wild-type allele (GAAGA, -287 to -291), not in the variant one carrying an A to G transition at -289 from the initial site of the transcription (4). The 1000-fold diluted product of the first PCR was subjected to nested PCR in a volume of 50 µL containing 0.2 mM of each deoxynucleoside triphosphate, 50 mM KCl, 10 mM Tris-HCl (pH 8.3), 1.5 mM MgCl2, 0.5 M of each primer, and 1.3 U of Taq polymerase (Takara Shuzu Co., Ltd.). PCR conditions were 94 °C for 5 minutes followed by 25 cycles of 94 °C for 30 seconds, 60 °C for 40 seconds, and 72 °C for 40 seconds (PCR Thermal Cycler MP; Takara Shuzo Co., Ltd.,). A 2-µL PCR product was digested with 6 U of MboII for 1 hour at 37 °C. Restriction fragments were analyzed by 4% agarose gel electrophoresis and ethidium bromide staining. DNA from wild-type homozygotes (W/W) was digested into 134- and 34-bp fragments; DNA from homozygotes for the variant allele (V/V) gave an undigested 168-bp fragment and DNA from the heterozygous (W/V) genotype gave all three fragments. To corroborate genotyping results of the variant, another mismatched forward primer 5'-GGACAGCCATAGAGACAAGGCCA-3' (-290 to -312; underlining indicates artificial mismatched site) was designed to amplify a 168-bp segment introducing a ScrF I (New England Biolabs, Inc., Beverly, MA) site in variant allele (CCNGG, -288 to -292), that is not in wild-type. The set of PCR amplifications was identical with that for MboII RFLP described above. Digestion of PCR products with ScrF I gave 146- and 22-bp fragments from the variant allele or an undigested 168-bp from wild-type. The heterozygous genotype gave all three fragments. B) Nifedipine oxidation activity and cytochrome P450 3A4 (CYP3A4) protein level by each genotype. Nifedipine oxidation activity was measured according to the method of Guengerich et al. (5). CYP3A4 protein levels were obtained by western blot analysis. Circles indicate subjects given dexamethasone or phenytoin in their last hospitalization. The median nifedipine oxidation activity of the five subjects carrying the variant allele was 2080 pmol/minute/mg protein (interquartile range, 1430-2430 pmol/minute/mg protein; range, 1120-5190), which did not differ statistically significantly from that of the wild-type subjects (1255 pmol/minute/mg protein; interquartile range, 675-1785; range, 100-3440; two-sided P = .11, Mann-Whitney U test). Furthermore, the difference in the CYP3A4 protein levels was not statistically significant (two-sided P = .18, Mann-Whitney U test), i.e., the median was 30.8 pmol/mg protein (interquartile range, 18.9-45.3 pmol/mg protein; range, 5.5-110.9) in the subjects homozygous for the wild-type allele and 64.6 pmol/mg (interquartile range, 35.9-75.5 pmol/mg protein; range, 15.4-165.5) in those who were heterozygous or homozygous for the variant allele.

 
Moreover, we examined the distribution of the new polymorphism in the Japanese population. DNA samples were prepared from 128 unrelated, healthy volunteers (median age, 22 years; range, 21-40 years) who had given written informed consent for their blood to be used in this study; the study had obtained approval from the Ethics Committee of Nagoya University School of Medicine. Although the frequency of the variant allele is 9.4% in Caucasian individuals (1), the population study revealed that all of the Japanese subjects were homozygous for wild-type.

In our in vitro analysis, we did not find any relationship between the CYP3A4 genotype and the level of nifedipine oxidation activity, which seems inconsistent with the findings presented by Rebbeck et al. (1). The reason for the disagreement is unclear. This may be due to the small sample size in our study. Alternatively, the variant allele may be in linkage disequilibrium with another mutation or gene that more strongly influences prostate carcinogenesis and leukemogenic drug effects. However, CYP3A4 activity is known to exhibit wide interindividual variability due to physiologic factors (e.g., age, food), pathologic conditions (e.g., hepatic diseases), environmental factors (e.g., smoking), and concomitant drug intake (e.g., steroids, anticonvulsants, antifungal agents), aside from any genetic factors (6). We consider the difference in enzymatic activity between the CYP3A4 genotypes, if any, as too small to explain such a wide variation in the observed activity.

It cannot be ruled out that the genetic variant in a 5' regulatory element might be related to a variability in the inducibility of CYP3A4 by such agents as rifampin or anticonvulsants. It is also possible that the induction of CYP3A4 in subjects with the variant allele might be weak compared with those with wild-type, given exposure to the same amount of inducer.

This analysis demonstrates a great difference in the distribution of the variant allele between Caucasian and Japanese populations. Recently, increased attention has been focused on racial variability in drug disposition and sensitivity. The recognized ethnic difference in the distribution of pharmacogenetic polymorphisms should be important in determining drug dosages in different populations. Because the variant allele examined here does not seem to be strongly related to CYP3A4 activity, the ethnic variability in the distribution of the polymorphism would be irrelevant in determining, in different populations, the dosage of drugs that are primarily metabolized by CYP3A4.

REFERENCES

1 Rebbeck TR, Jaffe JM, Walker AH, Wein AJ, Malkowicz SB. Modification of clinical presentation of prostate tumors by a novel genetic variant in CYP3A4. J Natl Cancer Inst 1998;90:1225-9.[Abstract/Free Full Text]cancerlit;98383566

2 Felix CA, Walker AH, Lange BJ, Williams TM, Winick NJ, Cheung NV, et al. Association of CYP3A4 genotype with treatment-related leukemia. Proc Natl Acad Sci U S A 1998;95:13176-81.[Abstract/Free Full Text]cancerlit;99007287

3 Tateishi T, Watanabe M, Moriya H, Yamaguchi S, Sato T, Kobayashi S. No ethnic difference between Caucasian and Japanese hepatic samples in the expression frequency of CYP3A5 and CYP3A7 proteins. Biochem Pharmacol 1999;57:935-9.[CrossRef][Web of Science][Medline]

4 Hashimoto H, Toide K, Kitamura R, Fujita M, Tagawa S, Itoh S, et al. Gene structure of CYP3A4, an adult-specific form of cytochrome P450 in human livers, and its transcriptional control. Eur J Biochem 1993;218:585-95.[Web of Science][Medline]

5 Guengerich FP, Martin MV, Beaune PH, Kremers P, Wolff T, Waxman DJ. Characterization of rat and human liver microsomal cytochrome P-450 forms involved in nifedipine oxidation, a prototype for genetic polymorphism in oxidative drug metabolism. J Biol Chem 1986;261:505-160.[Abstract/Free Full Text]

6 Soons PA, Schellens JH, Breimer DD. Variability in pharmacokinetics and metabolism of nifedipine and other dihydropyridine calcium entry blockers. In: Kalow W, editor. Pharmacogenetics of drug metabolism. New York: Pergamon Press, Inc.; 1992. p. 769-89.

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