CORRESPONDENCE |
RESPONSE: Re: Modification of Clinical Presentation of Prostate Tumors by a Novel Genetic Variant in CYP3A4
Behnoosh Amirimani,
Amy H. Walker,
Barbara L. Weber,
Timothy R. Rebbeck
Affiliations of authors: B. Amirimani (Departments of Biostatistics and
Epidemiology and Medicine), B. L. Weber (Departments of Medicine and Genetics), A. H.
Walker, T. R. Rebbeck (Department of Biostatistics and Epidemiology), University of
Pennsylvania School of Medicine, Philadelphia.
Correspondence to: Timothy R. Rebbeck, Ph.D.,
Department of Biostatistics and Epidemiology and Cancer Center, University of Pennsylvania
School of Medicine, Philadelphia, PA 19104 (e-mail: trebbeck{at}cceb.med.upenn.edu).
We are pleased to see the work of Ando et al. that addresses the
functional significance of the cytochrome P450-3A4 (CYP3A4) genetic
variant originally reported by our group (1). We would like to
comment on the findings reported in their correspondence and contribute
additional data that complement the findings of those authors.
We have previously reported that the frequency of the CYP3A4 variant allele differs
substantially across ethnic groups (2). We estimated that the frequency of
the variant allele among healthy, unrelated individuals was 53% in African-Americans (n
= 70), 9% in U.S. Caucasians (n = 132), and 0% in Taiwanese (n
= 130). The observation by Ando et al. of no variant alleles in 123 Japanese individuals
provides further evidence that the CYP3A4 variant may be found only rarely, if at all, in Asians.
However, the high frequency of variant alleles in African-Americans, and the significant
differences in allele frequencies between the racial groups studied to date, could in part explain
differences in CYP3A4-associated disease susceptibility or drug metabolism by race.
The results of expression analyses presented by Ando et al. are similar to those recently
performed in our laboratory. To evaluate the functional significance of the CYP3A4 variant, we
generated two eukaryotic reporter constructs (Fig. 1,
A). One construct
contained the wild-type (W) CYP3A4 promoter sequence, and the other contained the variant (V)
CYP3A4 promoter sequence. The expression of luciferase by these promoter constructs was
studied in the hepatoma cell line HepG2 and in the breast cancer cell line MCF7. As shown in Fig.
1
, B, average luciferase expression was 1.4-fold higher for the V
promoter compared with the W promoter in HepG2 cells (
21
= 18.04; P<.0001) and 1.9-fold higher for the V compared with the W
promoter in MCF7 cells (
21 = 33.29; P<.0001). Both sets of experiments were repeated in triplicate nine times. These results suggest
that the CYP3A4-V promoter may be associated with higher expression of CYP3A4 than the
CYP3A4-W promoter.


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Fig. 1. A) Diagram of the reporter-gene construct
used to evaluate the effect of a 1-kilobase (kb)-segment of the CYP3A4
promoter on luciferase expression. The vector used is a pGL3 Enhancer
vector (Promega Corp., Madison, WI) containing an SV40 enhancer
sequence and the wild-type (W) or variant (V) CYP3A4 promoter driving
luciferase expression. Genomic DNA from a homozygous wild-type (W/W)
individual and a homozygous variant (V/V) individual was amplified by
polymerase chain reaction (PCR) by use of a forward primer:
5'-TCCTGTTTCCAGACATGCAG-3' and reverse primer:
5'-TCAGTGAGGCTGTT GGATTG-3'. 100 ng of genomic DNA was used as
template in a 50-µL reaction containing 2.5 U of PWO
Taq polymerase and 5 µL of 10 x PCR buffer without
MgSO4 (Boehringer Mannheim, Indianapolis, IN), 0.2
mM deoxynucleoside triphosphate, 1 mMMgSO4, and 0.5 µM primers. Blunt-ended PCR
products were ligated into the PCR-Blunt vector (Invitrogen, Carlsbad,
CA). All CYP3A4 DNA sequences were confirmed after amplification by use
of an ABI Prism 377 sequencer (The Perkin-Elmer Corp., Foster City,
CA). The W and V constructs were generated by inserting restriction
digested CYP3A4 5'-flanking DNA fragments corresponding to promoter
sequence -14 to -1018, which were digested with XhoI and
KpnI (New England Biolabs, Beverly, MA) into the pGL3-Enhancer
vector (Promega Corp.). *denotes the approximate position of the
CYP3A4 variant (an A to G transition in the nifedipine-specific
element, or NFSE) located -290 bp from the transcription start site
of CYP3A4. B) Luciferase reporter gene expression by CYP3A4
genotype. The expression of luciferase under the influence of either
the wild-type (W) or variant (V) CYP3A4 promoter is reported relative
to that of empty vector. Cell culture and transient transfections were
undertaken by use of HepG2 and MCF-7 cell lines maintained in high
glucose Dulbecco's minimal Eagle medium (Life Technologies, Inc.
[GIBCO BRL], Gaithersburg, MD) supplemented with 10% fetal bovine
serum (Life Technologies, Inc.). Transient transfection was performed
after plating 3 x 105 cells/well in six-well plates plated
the day prior to transfection using Superfect Reagent (Qiagen,
Valencia, CA). In general, the transfection mixes in each well
contained 0.5 µg pCH110 ß-galactosidase (Pharmacia, Piscataway,
NJ) and 1.0 µg carrier plasmid. The cells were washed with 1 x
phosphate buffer solution and covered with fresh medium after 3 hours
incubation at 37 °C and 5% CO2. Nine triplicate
repeat assays for ß-galactosidase and luciferase enzyme activities
were performed after 48 hours post-transfection incubation by use of
the respective enzyme assay kits manufactured by Promega.
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Ando et al. reported no statistically significant differences in nifedipine oxidation or CYP3A4
protein levels between wild-type and variant homozygotes. However, they do report 1.6-fold
higher nifedipine oxidation and a 2.1-fold higher CYP3A4 protein levels in V versus W
genotypes. As suggested by those authors, the lack of a statistically significant difference in these
levels may reflect insufficient statistical power to detect the reported magnitude of effect. Taken
together with our results, however, it appears that CYP3A4-V may be associated with CYP3A4
overexpression compared with CYP3A4-W. While the magnitude of these differences may be
small, it is possible that the downstream effect of these genetic differences on some metabolic
pathways could be physiologically relevant. Additional research will be required to define the
regulatory features of this potential difference and to clarify the biologic mechanism by which
CYP3A4 genotype may explain the observed epidemiologic associations of CYP3A4 with
prostate cancer (1) and childhood leukemias (3).
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Walker AH, Jaffe JM, Gunasegaram S, Cummings SA, Huang
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CYP3A4: ethnic distribution and implications for prostate cancer risk. Hum Mutat 1998;12:289.[Medline]
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Cheung NK, 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]
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