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Aromatase and breast cancer - W39R, an inactive protein

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					European Journal of Endocrinology (2002) 146 583–589                                                                              ISSN 0804-4643


EXPERIMENTAL STUDY

Aromatase and breast cancer: W39R, an inactive protein
                                        ´
C Nativelle-Serpentini, S Lambard, G E Seralini and P Sourdaine
                                         ´                                                       ´                  ´
Institut de Biochemie et Biologie Appliquee, EA 2608, Laboratoire de Biochimie et de Biologie Moleculaire, Universite de Caen, Esplanade de la Paix,
14032 Caen Cedex, France
                                            ´
(Correspondence should be addressed to G E Seralini; Email: bioch.bio.mol@ibba.unicaen.fr)




                             Abstract
                             Background: Aromatase (CYP19) catalyzes the conversion of androgens into estrogens. It is in
                             particular involved in development, reproduction and breast cancer. One of its polymorphisms,
                             W39R localized in the N-terminal region of CYP19, significantly decreases breast cancer risk
                             among Japanese women and was chosen for this study. In this work, we studied the structure –
                             function relationships between W39R polymorphism and CYP19 enzyme activity.
                             Objective: To examine the kinetic properties of the mutant W39R recombinant protein in transfected
                             human cells devoid of steroidogenic activity.
                             Methods: Expression vectors for the wild-type or the mutated R39 aromatase were transiently trans-
                             fected into E293 human embryonal kidney cells. The conversions of androstenedione to estrone and
                             of testosterone and nortestosterone to 17b-estradiol were assayed by RIA. Expression of recombinant
                             cDNAs was analyzed by semi-quantitative RT-PCR and immunoblotting.
                             Results: W39R recombinant protein was devoid of aromatase activity whatever the substrate used.
                             This absence of activity was not due to the lack of expression of the recombinant enzyme since the
                             mRNA and protein were detected.
                             Conclusion: Our present in vitro study shows that the R39 mutant is unable to synthesize estrogens.
                             This work provides a novel observation, being consistent with the fact that Japanese women with the
                             variant allele arg have significantly lower risk of developing a breast tumor.

                              European Journal of Endocrinology 146 583–589




Introduction                                                                cancer (8). However, recently, Miyoshi et al. (9) have
                                                                            identified two new polymorphisms. The first one, also
The human aromatase (CYP19), in concert with the                            described by Kristensen et al. (10), is a tetranucleotide
ubiquitous flavoprotein NADPH-cytochrome P450                                repeat in intron 4 which could be associated with
reductase, is a key enzymatic complex that catalyzes                        breast cancer susceptibility. The second polymorphism,
the conversion of androgens into estrogens. It is now                       chosen for our study, is localized in the N-terminal
well established that estrogens are involved in various                     region of the CYP19 gene among Japanese women.
physiological functions but that they also promote the                      Miyoshi et al. (9) observed that this single homozygous
growth of breast cancer cells (1, 2). This enzymatic                        or heterozygous amino acid substitution from trypto-
complex belongs to the class of mammalian endo-                             phan to arginine at codon 39 (W39R), significantly
plasmic reticulum cytochrome P450, anchored with                            decreased the breast cancer risk. The same authors
the N-terminal membrane domain extending from                               speculated that this polymorphism may reduce the
A20 to W39 (3, 4) (Throughout, the conventional                             activity of CYP19 enzyme, resulting in the lower pro-
single letter abbreviations for amino acids are used:                       duction of estrogens. In this work, we tested this
A, alanine; C, cysteine; E, glutamic acid; Q, glutamine;                    hypothesis, i.e. the relationship between W39R poly-
R, arginine; W, tryptophan.) Moreover, Amarneh and                          morphism and CYP19 enzyme activity. Therefore in
Simpson (5) have reported that the deletion of the                          the present work, the W39R mutant was generated
first 53 residues of the human aromatase protein                             in order to determine its potential impact on the
abolishes its activity. In the last decade, several point                   structure – function relationships of aromatase. A
mutations of CYP19 have been associated with a loss                         mammalian cell expression plasmid, pCMV-HA, was
of the aromatase function (for review see (6)). On the                      mutated or not, and transiently expressed in E293
other hand, one polymorphism, R264C, in the coding                          human embryonic kidney cells in order to determine
area of the gene was observed (7) but without affecting                     the kinetic properties of the recombinant proteins.
activity and without being correlated with breast                           E293 cells were used because they are an appropriate


q 2002 Society of the European Journal of Endocrinology                                                          Online version via http://www.eje.org
584     C Nativelle-Serpentini and others                                    EUROPEAN JOURNAL OF ENDOCRINOLOGY (2002) 146


cell line to study recombinant steroidogenic enzyme        E293 cell culture and transient transfection
(11) since they are naturally devoid of aromatase,         assay
5a-reductase and 17b-dehydrogenase enzyme activi-
                                                           The stable reductase-transfected human embryonal
ties (12). In order to investigate if this polymorphism
                                                           kidney cell line E293 (a gift from Dr Van Luu-The,
at codon 39 affects the expression of aromatase or its
                                                                       ´
                                                           CHUL, Quebec, Canada) was maintained at 37 8C (5%
cellular localization, we have performed a semi-quanti-
                                                           CO2, 95% air) in EMEM phenol red-free medium
tative RT-PCR analysis of mRNA from transfected E293
                                                           containing 2 mmol/l glutamine, 100 U/ml antibiotics
cells and a CYP19 protein analysis in microsomal frac-
                                                           (penicillin, streptomycin and Fungizone), 10% heat-
tions by Western blot. According to the aromatase
                                                           inactivated fetal calf serum (supreme serum), and 1%
sequence, one possible structural modification induced
                                                           non-essential amino acids (BioWhittaker, Gagny,
by R39 could result from its ionic interaction with the
                                                           France). Forty-eight hours prior to transfection, cells
i+3 residue, E42, leading to a modification of the hinge
                                                           were grown up to 50% confluence on 24-well cell
region (13). In order to check this hypothesis, we have
                                                           culture plates, washed with medium and supplemented
performed the double mutation W39R-E42A. In such a
                                                           with 500 ml EMEM-free serum. Cell transfection was as
protein, alanine will be unable to form an electrostatic
                                                           reported by Boussif et al. (15). Briefly, 2 mg plasmid and
interaction with R39 and, thus, could restore the
                                                           54 nmol polyethylenimine (PEI) aqueous solution (pH
aromatase activity.
                                                           7) were diluted separately with 50 ml NaCl
   In this study, we have shown that the W39R recom-
                                                           (150 mmol/l), incubated 10 min at room temperature
binant protein is devoid of aromatase activity whatever
                                                           and mixed together. After a further 10 min incubation,
the substrate used and that the double mutation
                                                           PEI – DNA mix was added to each well. Then, after 3-4 h
W39R-E42A is unable to restore the activity.
                                                           incubation at 37 8C, cells were supplemented with
                                                           500 ml EMEM-10% serum. Aromatase activity ‘in cell’
                                                           was evaluated 48 h post-transfection. Transfection effi-
                                                           ciency was evaluated by ELISA of the wild-type and
Materials and methods                                      several mutated proteins without showing differences
                                                           in recombinant aromatase expressions (16).
pCMV-human aromatase cDNA construction
(pCMV-HA)
                                                           Aromatase activity ‘in cell’
A 2920 bp fragment of human aromatase cDNA
EcoR1-EcoR1 was subcloned into the pCMV EcoRI              The conversion of androstenedione to estrone and of
site (14). Orientation was then checked by sequencing.     testosterone and nortestosterone to 17b-estradiol was
The pCMV-HA was purified from the transformed               measured in E293 cell supernatants by RIA according
JM109 bacterial strain by using the Qiagen Plasmid         to Auvray et al. (14). Briefly, transfected cells were
Mega kit (Qiagen, Courtaboeuf, France). The length,        washed with EMEM and incubated with increasing
the concentration and the purity of the plasmid-cDNA       concentrations of androstenedione (0 –800 nmol/l) or
construction were verified by 1% agarose electrophor-       200 nmol/l testosterone or 20 nmol/l nortestosterone
esis and ethidium bromide staining.                        for 45 min at 37 8C under a 5% CO2 atmosphere. After
                                                           incubation, cells were placed for 5 min on ice and
                                                           medium was extracted by 10 volumes diethyl ether.
                                                           Anti-estrone and anti-estradiol rabbit antibodies (Ref
                                                                                                    `
                                                           PF1247 and Ref 1287, P.A.R.I.S., Compiegne, France)
Site-directed mutagenesis                                  were prepared according to the manufacturer’s instruc-
The introduction of specific base changes in the aroma-     tion. The extraction efficiency was 80^5% and the
tase cDNA was accomplished by using the Quickchange        sensitivity of this RIA was 10 pg/ml. Intra- and inter-
site directed mutagenesis kit from Stratagene (France).    assay coefficients of variations were approximately 3
Oligonucleotide primers used for the mutagenesis           and 7% respectively. Results are the mean of at least
experiments were synthesized at Eurobio (Les Ulis,         triplicate experiments^S.D.
France). Sequences of oligonucleotides were 50 -CCT
TTT TCT CTT GGT GAG GAA TTA TG-30 and 50 -CAT
                                                           RNA extraction and semi-quantitative RT-PCR
AAT TCC TCA CCA AGA GAA AAA GG-30 for W39R
and 50 -CTT GGT GAG GAA TTA TGC GGG CAC ATC                Total cellular RNA was isolated from E293 cells, alone
C-30 and 50 -GGA TGT GCC CGC ATA ATT CCT CAC               or transfected with pCMV-HA (used as positive control)
CAA G-30 for W39R-E42A. All mutated cDNAs were             or pCMV-R39 and pCMV-R39-A42, using the TRIzol
sequenced by using the ABI Prism BigDye Terminator         reagent (Gibco BRL, Cergy Pontoise, France). Total
Cycle Sequencing Ready Reaction from Biosystems            RNA (3 mg) was treated for 10 min at 37 8C with 2
(Warrington, UK) to confirm that there were no other        units RNAse-free DNAse (Promega, Charbonnieres,   `
mutations except those designed.                           France) and reverse transcribed with 200 IU M-MLV

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EUROPEAN JOURNAL OF ENDOCRINOLOGY (2002) 146                                         Aromatase and breast cancer     585

RT (Promega) and 0.2 mg oligo dT primer (12 – 18           Subcellular fractionation and Western
bases), in RT buffer (Promega) containing 500 mmol/l       analysis
dNTPs and 24 IU Rnasin for 1 h at 37 8C, in a total
                                                           Transfected cells from confluent T75 tissue culture
volume of 10 ml. PCR amplification of 5 ml of reversed
                                                           flasks were scraped into 20 mmol/l Tris –HCl (pH
transcribed sample was carried out in a final volume
                                                           7.5), 20% glycerol, 15 mmol/l b-mercapto-ethanol,
of 50 ml PCR buffer supplemented with 200 mmol/l
                                                           50 mmol/l NaCl, 5 mmol/l EDTA, 1 mmol/l dithio-
dNTP, 50 pmol of each primer, 1.5 mmol/l MgCl2 and
                                                           threitol containing 4 mg/ml polymethylsulfonyl
1.5 IU Taq DNA polymerase from Promega. Amplifica-
                                                           fluoride, and a mixture of antiproteases (benzamidine,
tion was performed on a Stratagene Robocycler
                                                           aprotinin, leupeptin, pepstatin A and antipain at
through 30 PCR cycles with the profile: 94 8C (1 min),
                                                           1 mg/ml each). Then cell lysates were centrifuged at
60 8C (1 min) and 72 8C (2 min) and an extra cycle of
                                                           20 000 g for 15 min at 4 8C and the supernatant was
72 8C (5 min). The sequences of sense and antisense pri-
                                                           transferred to a fresh tube and centrifuged at
mers were 50 -AAA GGA AAT CCA GAC TGTT ATT GGT
                                                           100 000 g for 1 h at 4 8C to obtain the microsomal
GAG-30 (exon IX, 1116 – 1143) and 50 -GTA TCT TCT
                                                           fraction. Each fraction was mixed with an equal
GTA TGC TCT CAA CAC ACT GTG-30 (exon X, 1511 –
                                                           volume of twofold concentrated Laemmli sample
1540) respectively. The resulting PCR product of
                                                           buffer (17) and denatured by heating in boiling water
424 bp was resolved on a 1.5% agarose gel and visual-
                                                           for 2 min. Proteins (100 mg) were resolved on a 10%
ized by ethidium bromide staining.
                                                           SDS-PAGE using Tris base (0.25 mol/l), glycine
   The internal control (GAPDH) was amplified for 23
                                                           (1.92 mol/l), SDS (1%) running buffer and then electro-
cycles, in the conditions described above. The sequences
                                                           transferred onto PVDF membrane (Appligen, Illkirch,
of sense and antisense primers used to detect the PCR
                                                           France). Proteins were detected by immunoblotting
identification of the GAPDH mRNAs were 50 -TGA
                                                           with the P450 aromatase antiserum (1:1000) pro-
ACG GGA AGC TCA CTG GCA TGG CTT T-30
                                                           duced in our laboratory (18), followed by an incubation
(731– 758) and 50 -GTG TGG TGG GGG ACT GAG TGT
                                                           with horseradish peroxidase-linked anti-rabbit IgG at
GGC AGG GAC-30 (1132 –1162) respectively. The result-
                                                           1:3000 (BioRad, Ivry sur Seine, France) and addition
ing PCR product of 431 bp was resolved on a 1.5%
                                                           of Sigma Fast 3,30 -diaminobenzidine (Tablet Sets;
agarose gel and visualized by ethidium bromide staining.
                                                           Sigma, Saint Quentin Fallavier, France).
   Twelve microliter aliquots of the PCR reaction were
size separated on 1.5% agarose equilibrated in
0.04 mol/l Tris –acetate, 0.001 mol/l EDTA. Gels were      Results
stained with ethidium bromide (1 mg/ml), photographed
using Polaroid film under UV light and analyzed using an    The aim of our study was to examine the enzyme
AGFA Snap-Scan 1200p Scanner, Adobe Photoshop              kinetic properties of the mutants W39R and
(version 4) software and the NIH image computer pro-       W39R-E42A in transfected E293 cells and to determine
gram (http://rsb.info.nih.gov/nih-image).                  the importance of these residues in protein activity.




                                                                       Figure 1 Aromatase activity and mutations. (A)
                                                                       Enzymatic activity of pCMV-HA (upper curve), and
                                                                       of R39 and R39-A42 (lower two curves)
                                                                       transfected in E293 cells, incubated for 45 min with
                                                                       increasing doses of androstenedione
                                                                       (0– 400 nmol/l). Results are expressed as pg
                                                                       estrone (E1) formed/min per mg protein and are
                                                                       the mean of triplicate values^S.E.M. Insert:
                                                                       apparent Km value determination of the wild-type
                                                                       enzyme by a Lineweaver– Burk plot (results are
                                                                       representative of one from three experiments
                                                                       showing similar profiles). (B, C) Enzymatic activity
                                                                       of pCMV, pCMV-HA, R39 and R39-A42
                                                                       transfected E293 cells, incubated for 45 min with
                                                                       200 nmol/l testosterone (B) or nortestosterone (C).
                                                                       Results are expressed as pg estradiol (E2)
                                                                       formed/min per mg protein and are the mean of
                                                                       triplicate values^S.E.M.


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586     C Nativelle-Serpentini and others                                     EUROPEAN JOURNAL OF ENDOCRINOLOGY (2002) 146


Cells were transfected with either the wild-type plasmid   for R39-A42 in comparison with mRNA coding for
(pCMV-HA) or with the mutated plasmids (W39R and           the wild-type enzyme. The Western blot analysis of
W39R-E42A) or with pCMV plasmid, used as control.          the wild-type protein and the recombinant R39 and
Forty-eight hours after transfection, cells were incu-     R39-A42 proteins on subcellular fractions, illustrated
bated for 45 min with increasing concentrations of         in Fig. 3, reveals that the proteins are seen only in
androstenedione (0-800 nmol/l) or with 200 nmol/l          the microsomal fraction and not in the cytosol.
testosterone or nortestosterone. As illustrated in Fig.
1, the mutation of W39R and the double mutation
induced a total activity loss with androstenedione         Discussion
(Fig. 1A). With 200 nmol/l testosterone as substrate,
both mutations decreased strongly the aromatase            Recent studies of 204 Japanese women have shown
activity (activities below 6 and 3% relative to the        that the single amino acid substitution in aromatase
wild-type for R39 and R39-A42 respectively) (Fig. 1B).     of tryptophan by arginine at codon 39 was significantly
In the same manner, a lower activity with 200 nmol/l       associated with a decrease in breast cancer risk (9).
nortestosterone was observed for the R39 and               Those authors speculated that this polymorphism
R39-A42 (41 and 27% of the wild-type respectively)         may reduce the activity of the CYP19 enzyme, conse-
(Fig. 1C). Moreover, Fig. 1A shows a typical result of     quently resulting in a lower production of estrogens.
kinetic analysis of the wild-type aromatase. From          We tested this hypothesis.
these results, the apparent Km value of the wild-type         In our study, aromatase activity was measured ‘in
aromatase expressed in E293 was 76:6^14:1 nmol=l           cell’ and allowed us to determine an apparent Km
for androstenedione. To study the effects of the point     value for the wild-type protein for androstenedione
mutation T/A on the CYP19 expression, aromatase            (76 nmol/l) in the same range as values previously
mRNA from the E293 transfected cells was analyzed          reported by others (19). However, different estimates
by RT-PCR. As illustrated in Fig. 2A, the corresponding    of apparent Km and Vm values can be obtained from
424 bp RT-PCR product of aromatase mRNA was                microsomal and intact cell methods. According to
absent in untransfected E293 cells and present in          Zhou et al. (20), similar Km values were obtained with
E293 cells transfected with pCMV-HA, pCMV-HA-              both approaches but Vm values were lower by using
W39R and pCMV-HA-W39R-E42A, and in granulosa               microsomes. The authors suggested instability of the
cells used as a positive control. As illustrated in        microsomal preparation. Moreover, Kadohama et al.
Fig. 2C, the semi-quantitative RT-PCR analysis of          (21) have suggested that the cytochrome P450 reduc-
CYP19 mRNA levels, by using GAPDH mRNA as                  tase/aromatase ratio may be involved in differences in
standard (Fig. 2B), showed a 34% decrease in mRNA          the two protocols. In such a way, we have used stable
coding for R39 and a 56% decrease in mRNA coding           reductase transfected E293 cells. Our results show




                                                               Figure 2 (A) RT-PCR amplification of aromatase mRNA
                                                               followed by each negative control without reverse
                                                               transcriptase from granulosa cells (lanes 1 and 2),
                                                               untransfected cells (3 and 4), transfected cells with R39 (5
                                                               and 6), with R39-A42 (7 and 8) and with pCMV-HA (9 and
                                                               10). The size and position of the expected 424 bp transcript
                                                               is indicated on the right. (B) RT-PCR amplification of
                                                               GAPDH mRNA and negative controls. The size and position
                                                               of the expected 431 bp transcript is indicated on the right.
                                                               (C) Semi-quantification by RT-PCR of P450 aromatase
                                                               mRNA levels in E293 cells transfected with 2 mg, pCMV-HA
                                                               (W39), R39 and R39-A42; the respective P450
                                                               aromatase/GAPDH ratios are shown. Each bar is the mean
                                                               of three separate values^S.E.M. *P , 0.01, **P , 0.05.


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EUROPEAN JOURNAL OF ENDOCRINOLOGY (2002) 146                                               Aromatase and breast cancer   587

that mutation of residue 39 abolishes or reduces con-             and, thus, could restore the aromatase activity. In the
siderably the protein activity, whatever the substrate            present study, the double mutant R39-A42 was
tested, and consequently suggest that this position               devoid of activity whatever the substrate tested. This
appears to be important for aromatase activity. Slight            lack of activity may result from a lower expression
differences according to the substrates is in agreement           rate of the double mutant, since corresponding
with our previous results. According to multiple                  mRNA levels were 56% lower than these of the wild-
sequence analysis, W39 is well conserved in most                  type. Another hypothesis to explain this lack of activity
P450 aromatase enzymes (human (22), horse (23),                   may be that the mutation W39R could prevent the
pig (24) and rabbit (25)) except in rat (26), as well as          interaction between the cytochrome P450 aromatase
in other P450s involved in steroidogenesis (human                 and the reductase. For instance, Lee-Robichaud et al.
P450scc       (27),    steroid    17a-hydroxylase/lyase           (34) have described the importance of arginine residues
(CYP17A) (28)) or other P450s such as P450 2E1                    which are involved in protein – protein interaction
(29) and P450 2C2 (30). Tryptophan is a hydrophobic               between cytochrome b5 and human CYP17 and conse-
amino acid, in contrast to arginine, which is a posi-             quently affect the lyase activity of the enzyme required
tively charged residue, and consequently could be                 for androgen formation. Moreover, Muller-Enoch &
involved in an ionic interaction with E42 according               Gruler (35) have recently reported that the N-terminal
to structural models (31). Moreover, several ionic                binding domain of cytochrome P450 enzymes deter-
interactions between arginine and glutamic acid have              mines the complexation process of the binary P450
been described in P450 cytochromes which could                    reductase system. We also hypothesized that R39 may
favor secondary structures of the protein. Carani et al.          form a salt-bridge with another glutamic acid residue
(32) have identified a homozygous mutation, R365Q                  located far away in the sequence, as has been described
on the CYP19 gene, which is associated with a very                in CYP19 between R9 and E296 (36), and conse-
low activity of the enzyme. According to the computer             quently could prevent the interaction between P450
model of Graham-Lorence et al. (31), R365 may form a              aromatase and NADPH reductase. Further studies will
salt-bridge with E362 and may play a major role in the            be necessary to explore this hypothesis.
stabilization of the general structure of the aromatase              Genetic polymorphism of the gene CYP19 may be
protein. Moreover, glutamic acid is likely to play a              involved in mechanisms other than in the protein
role in aromatase structure and function by affecting             structure, such as mRNA stability, modulation of tran-
enzyme activity and modulating substrate binding                  scription or post-translational regulation of expression.
affinities (33). We speculated that this W39R mutation             Recently, Kristensen et al. (37) have observed that the
could destabilize the structure of aromatase by forming           C/T polymorphism in exon 10 of the CYP19 gene is
a salt-bridge with E42, leading to its inactivity. Thus, to       associated with variations in mRNA levels. They
check this possibility, we analyzed the double-mutant             reported that, according to the computer model of
pCMV-HA-R39-A42. In such a case, alanine will be                  Walter et al. (38), the C/T change may influence
unable to form an electrostatic interaction with R39              mRNA stability. In addition, it was shown that the
                                                                  introduction of a polar residue in the hydrophobic
                                                                  anchor domain of cytochromes P450 2E1 and 2B4
                                                                  changes their cellular location from membrane to cyto-
                                                                  sol (39). Because a point mutation could modify the
                                                                  expression of the protein (37), or its cell trafficking
                                                                  (39), we have further analyzed this mutant at the
                                                                  RNA and subcellular levels. To study the effects of the
                                                                  single nucleotide mutation T/A on CYP19 gene
                                                                  expression, aromatase mRNA from the E293 trans-
                                                                  fected cells were analyzed by RT-PCR. The results
                                                                  show that the corresponding product of aromatase
                                                                  mRNA was detected in E293 cells transfected with
                                                                  R39. However, the semi-quantitative RT-PCR analy-
                                                                  sis of mRNA levels have shown that they were sig-
                                                                  nificantly affected by the mutation. In fact, a 34%
                                                                  decrease in HA-R39 mRNA levels was observed in com-
Figure 3 Detection of aromatase proteins. E293 cells used for     parison with HA-W39 mRNA levels. The new mRNA
enzymatic assays were harvested and centrifuged; 100 mg           structures may affect their half-lives but this hypothesis
proteins from microsomal (M) and cytosolic (C) fractions were     needs further investigation.
resolved on SDS 10% polyacrylamide gels. The proteins were           In order to determine whether the enzyme localiza-
electroblotted onto a PVDF membrane and probed with rabbit
anti-P450 aromatase antiserum. The size and position of the       tion was modified by the W39R mutation, an immuno-
expected 53 kDa aromatase protein is shown on the right and the   blot analysis was performed on subcellular fractions. As
molecular mass markers (MW, kDa) are on the left.                 illustrated in Fig. 3, aromatase was detected only in the

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588     C Nativelle-Serpentini and others                                                         EUROPEAN JOURNAL OF ENDOCRINOLOGY (2002) 146


microsomal fraction and not in the cytosol as was                              increase the risk of breast cancer. Pharmacogenetics 1998 8
observed for P450 2E1 and 2B4 (39). However, the                               43 –48.
                                                                          11   Dufort I, Soucy P, Labrie F & Luu-The V. Molecular cloning of
mutants are completely inactive whatever the                                   human type 3 3 alpha-hydroxysteroid dehydrogenase that differs
substrate used. This inactivity may partly result from                         from 20 alpha-hydroxysteroid dehydrogenase by seven amino
a decrease in the protein expression but also from a                           acids. Biochemical and Biophysical Research Communications 1996
structural modification of the enzyme.                                          228 474 –479.
                                                                          12   Corbin CJ, Trant JM, Walters KW & Conley AJ. Changes in
   In conclusion, our present study provides a novel                           testosterone metabolism associated with the evolution of
observation, being consistent with the fact that                               placental and gonadal isozymes of porcine aromatase cytochrome
Japanese women with the variant allele arg have a                              P450. Endocrinology 1999 140 5202–5220.
significantly lower risk than the non-carrier of                           13   Yamazaki S, Sato K, Suhara K, Sakaguchi M, Mihara K & Omura
developing a breast tumor, as hypothesized by Miyoshi                          T. Importance of the proline-rich region following signal-anchor
                                                                               sequence in the formation of correct conformation of
et al. (9). However, the lack of activity of the mutated                       microsomal cytochrome P-450s. Journal of Biochemistry 1993
W39R protein raises the question of the consequences                           114 652 –657.
of such an homozygous mutation in the normal post-                        14   Auvray P, Sourdaine P & Seralini GE. PAAn-1b and PAAn-E: two
natal female physiology. In this context, further in                           phosphorothioate antisense oligodeoxynucleotides inhibit human
                                                                               aromatase gene expression. Biochemical and Biophysical Research
vivo studies concerning their aromatase status (activity,                      Communications 1998 253 1– 9.
expression) and estrogen levels would be interesting.                     15   Boussif O, Lezoualc’h F, Zanta MA, Mergny MD, Scherman D,
                                                                               Demeneix B et al. A versatile vector for gene and oligonucleotide
                                                                               transfer into cells in culture and in vivo: polyethylenimine.
                                                                               PNAS 1995 92 7297–7301.
Acknowledgements                                                          16                                                           ´
                                                                               Auvray P, Nativelle C, Bureau R, Dallemagne P, Seralini GE &
                                                                               Sourdaine P. Study of substrate specificity of human aromatase by
This work was supported by Agro-biological industries                          site directed mutagenesis. European Journal of Biochemistry (In Press).
(AGROBIO) Committee, Fonds FEDER (European                                17   Laemmli UK. Cleavage of structural proteins during the
                                                                               assembly of the head of bacteriophage T4. Nature 1970 227
Funds for Development and Research) and the Ligue                              680– 685.
                                 ´
Nationale Contre le Cancer (Comite de la Manche et                        18   Almadhidi J, Seralini GE, Fresnel J, Silberzahn P & Gaillard JL.
du Calvados).                                                                  Immunohistochemical localization of cytochrome P450 aroma-
                                                                               tase in equine gonads. Journal of Histochemistry and Cytochemistry
                                                                               1995 43 571 –577.
                                                                          19   Zhou DJ, Korzekwa KR, Poulos T & Chen SA. A site-directed
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