Aromatase and breast cancer - W39R, an inactive protein

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





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