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ASIC SCIENCE: GYNECOLOGY

equirements of phosphatidylinositol-3 kinase andammalian target of rapamycin for estrogen-

nduced proliferation in uterine leiomyoma-nd myometrium-derived cell lines

uejun J. Yin, MD, PhD; Gangduo Wang, MD, PhD; Firyal S. Khan-Dawood, PhD

BJECTIVE: This study was undertaken to investigate the effects of7�-estradiol (E2) on G1 cell cycle progression and proliferation interine fibroid and myometrial cells and the roles of phosphatidylino-itol-3 kinase and mammalian target of rapamycin in mediating thesestrogen effects.

TUDY DESIGN: The human uterine smooth muscle–derived cellsUtSM) and uterine leiomyoma–derived cells (UtLM) were treated witharying doses of E2 with or without pretreatment with LY294002, a phos-hatidylinositol-3 kinase inhibitor, or rapamycin, a mammalian target ofapamycin inhibitor. The effects of E2 on cell cycle progression and prolif-ration and the roles of phosphatidylinositol-3 kinase and mammalian tar-

kinase (PI3K)/Akt sioi: 10.1016/j.ajog.2006.09.037

76.e1 American Journal of Obstetrics & Gynecology FEBRUARY 2007

ESULTS: Compared with controls, E2 significantly induced G1 cellycle progression and proliferation in uterine smooth muscle-erived cells and uterine leiomyoma– derived cells. These effects,owever, were significantly blocked when LY294002 or rapamycinas used.

ONCLUSION: E2 significantly induces G1 cell cycle progression andell proliferation in uterine smooth muscle– derived cells and uterineeiomyoma– derived cells, in which phosphatidylinositol-3 kinase and

ammalian target of rapamycin are essentially required.

ey words: 17�-estradiol, mammalian target of rapamycin,

et of rapamycin in E2-induced effects were studied. phosphatidylinositol-3 kinase, proliferation, uterine leiomyomas

ite this article as: Yin XJ, Wang G, Khan-Dawood FS. Requirements of phosphatidylinositol-3 kinase and mammalian target of rapamycin for estrogen-inducedroliferation in uterine leiomyoma- and myometrium-derived cell lines. Am J Obstet Gynecol 2007;196:176.e1-176.e5.

terine leiomyomas, or fibroids, arethe leading cause of hysterectomies

nd the most common type of pelviceoplasm found in women.1 It has be-ome widely accepted that the growth of

leiomyomas is regulated by ovarian ste-roid hormones, particularly estrogenand progesterone. Indeed, estrogen haslong been proposed as the primary pro-moter of fibroid growth.2 However, theeffects of estrogen on fibroid prolifera-tion and growth have not been experi-mentally studied in detail. In addition,the mechanisms through which estrogenexerts its mitogenic effect on fibroids arenot yet clear.

The classical, or genomic, model of es-trogen action involves ligand-mediatedactivation of the nuclear receptors,which interact directly with estrogen re-sponse elements in the promoters oftarget genes and recruit various coactiva-tors to mediate transcriptional reg-ulation. It has been recently dem-onstrated that estrogen also exerts itsmitogenic effect by a nongenomic mech-anism, causing a rapid and transient ac-tivation of distinct intracellular signalingpathways.3 One such estrogen-regulatedpathway is the phosphatidylinositol-3

which has been shown to regulate manycellular processes that are critical for tu-morigenesis.4 The mammalian target ofrapamycin (mTOR), a downstream tar-get of PI3K and Akt, has been recentlyidentified as the central controller of cellgrowth.5 Activated by Akt, mTOR con-trols cell growth by activating p70S6K1, ahighly conserved element in a wide arrayof cellular processes including the mito-genic response to growth factors.6 Stud-ies have shown that PI3K transmits a mi-togenic signal through Akt and mTOR top70S6K1 that is essentially required forG1 cell progression and protein transla-tion in human prostate and ovarian can-cer cells.7,8 The current study was carriedout to test the hypotheses that estrogenincreases G1 cell cycle progression andproliferation in uterine fibroid and myo-metrial cells and that PI3K and mTORplay an important role in mediatingthese estrogen effects. To achieve theseobjectives, we studied the dose- andtime-dependent effects of estrogen on

rom the Department of Pathology (Drsin and Khan-Dawood), and the Maryabb Randolph Cancer Center (Dr Wang),est Virginia University, Morgantown,V. Where the work was done:epartment of Pathology, School ofedicine, West Virginia University,organtown, WV.

eceived April 13, 2006; revised August 8,006; accepted September 25, 2006.

eprints: Firyal S. Khan-Dawood, PhD,epartment of Pathology, School of Medicine,est Virginia University, Morgantown, WV

6506-9203; fikhan@hsc.wvu.edu.

his study was supported in part by WVUealth Sciences Center Internal Grant, Officef Research and Graduate Education.

002-9378/$32.002007 Mosby, Inc. All rights reserved.

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www.AJOG.org Basic Science: Gynecology Research

n uterine fibroid and myometrial cells.ith the addition of the specific inhibi-

ors, critical roles of PI3K and mTOR instrogen-induced cellular responsesere demonstrated.

ATERIALS AND METHODSell culture and treatmenthe human uterine smooth muscle–erived cells (UtSM) and leiomyoma-erived cells (UtLM) (a kind gift fromr. Darlene Dixon of National Institutef Environmental Health Sciences)9

ere maintained at 37°C in a 5% CO2

ncubator in Dulbecco’s modified Ea-le’s medium (DMEM, Invitrogen,arlsbad, CA) supplemented with 100/mL of penicillin, 100 �g/mL of strep-

omycin, and 10% fetal bovine serumFBS, Hyclone Laboratories, Logan,T), verified by the manufacturer to

ontain less than 8.5 pg/mL of 17�-estra-iol (E2). The cells were incubated in 96-ell plates (103 cells per well, for prolif-

ration assay) or 6-well plates (5 � 104

ells per well, for cell cycle assay) in phe-ol red-free DMEM (Invitrogen) sup-lemented with 10% FBS and allowed tolate at 37°C for 24 hours. The cells werehen rinsed with sterile phosphate buff-red solution (PBS) and refed with the

FIGURE 1Effects of E2 on cell proliferation

1 2 3 4 5Cel

l pro

lifer

atio

n (%

of c

ontr

ol o

n da

y 1)

0

100

200

300

400

500

600

*

*

*

UtSM cells

A

ffects of E2 on cell proliferation in A, UtSM aontrol (0.03% ethanol) or E2 at concentrations fupplemented with 10% FBS for 5 days. Data shoP � .05, significantly different between each E

ame medium containing 10% FBS, fol- w

owed by treatment with 10�10 to 10�6

ol/L of E2 (Sigma-Aldrich, St. Louis,O) or vehicle control (0.03% ethanol)

or 6, 12, or 24 hours, or up to 5 consec-tive days. In some cultures, the cellsere treated with dimethyl sulfoxide

DMSO, 0.1%), the PI3K inhibitor LY94002 (10 �mol/L, Cell Signaling, Bev-rly, MA), or the mTOR inhibitor rapa-ycin (10 ng/mL, Cell Signaling) for 2

ours before E2 treatment. The dosagesf inhibitors were selected based on arevious dose-response study (data nothown).

ell proliferation assayell proliferation was determined eachay following the treatment using theellTiter 96 aqueous 1 solution cell pro-

iferation assay (Promega, Madison,I). Briefly, after addition of 10 �L of

he CellTiter 96 aqueous 1 solution re-gent into each well and incubation at7°C in a 5% CO2 incubator for 2 hours,he absorbance at 490 nm was recordedy using a microplate spectrophotome-er reader (SpectraMax 250; Molecularevices Co, Sunnyvale, CA).

ell cycle analysisfter the treatment, cells were washed

e (day)1 2 3 4 5

0

100

200

300

400

500

600Control10-10 M

10-9 M

10-8 M

10-7 M

10-6 M

*

*

**

UtLM cells

B

B, UtLM cells. Cells were treated with vehicle10�10 to 10�6 mol/L in phenol red-free DMEMare means � SE of 3 independent experiments.

reated group and controls.

ith PBS (pH 7.4), fixed with 70% etha- 2

FEBRUARY 2007 America

ol at �20°C overnight. The cells werehen washed with PBS, incubated in.1% Triton X-100/PBS containing 0.2g/mL RNase A (Sigma-Aldrich) on ice

or 30 minutes, followed by incubationith a propidium iodide solution (Sigma-ldrich, 200 �g/mL in 0.1% Triton-100/PBS) on ice for 30 minutes. Cellycle distribution of cells was then deter-ined by flow cytometry with the use ofFACSCalibur (Becton-Dickinson, Saniego, CA) and the CellQuest Pro soft-are (Becton-Dickinson).

tatistical analysisata are compiled from 3 independent

xperiments performed in triplicate andxpressed as means � SE. The signifi-ance of the interaction among the dif-erent treatment groups for the differentarameters at each time point was as-essed using an analysis of varianceANOVA). The significance of differ-nce between individual groups was an-lyzed using the Tukey-Kramer post hocest. For all analyses, the criterion of sig-ificance was set at P � .05.

ESULTS2, at concentrations ranging from0�10 to 10�6 mol/L, significantly in-reased proliferation of UtSM (FigureA) and UtLM (Figure 1B) cells in aose-dependent manner at 2 to 4 daysfter the treatment. On day 5, prolifera-ion in the E2-treated UtSM cells, but notontrols, was stopped or decreased ashown by a declination in the prolifera-ion curves for these cells. Meanwhile, alightly decreased proliferation wasbserved in either the E2-treated (�8-5%) or the non-E2-treated (�13-35%)tLM cells compared with the respectivetSM cells from day 2 to day 4 (Figure

). As shown in Figure 2, the E2-inducedell proliferation in both UtSM (FigureA-D) and UtLM (Figure 2E-H) cellsas significantly blocked by LY294002r rapamycin, with the maximum effectsccurred at 3-5 days after the treatment.he 2 inhibitors were also found to sig-ificantly decrease cell proliferation, in aimilar manner, in both non-E2–treatedtSM (Figure 2B-D) and UtLM (Figure

Tim

ndromwn2-t

F-H) cells at the same time.

n Journal of Obstetrics & Gynecology 176.e2

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Research Basic Science: Gynecology www.AJOG.org

1

On the other hand, E2 significantly de-reased the percentage of the cell popu-ation at G0/G1 phase (Figure 3A and B),ut increased those at G2/M phase inoth UtSM and UtLM cells (Figure 3Cnd D). These responses were dose- andime-dependent, with no markedhanges appearing at 6 hours (data nothown) but significant effects occurring

ainly at higher E2 doses and at 12 hoursr later after the treatment. When the2-treated cells were pretreated withY294002 or rapamycin, however, theeduction in the percentage of the cellopulation at G0/G1 phase in both cellypes was totally restored at 12 or 24ours after the treatment (Figure 3A and). In contrast, the E2-induced increase

n the cell population at G2/M phase intSM and UtLM cells was completelylocked (Figure 3C and D). Treatmentith LY294002 or rapamycin alone sig-ificantly increased the percentage of0/G1 cell population (Figure 3A and

FIGURE 2Effects of PI3K and mTOR inhibito

Cel

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n (%

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ontr

ol o

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0

100

200

300

400

500DMSO + E2LY294002 + E2Rapamycin + E2

0 10-1010-9 10-8 10-7 10-6

BA

0

100

200

300

400

500DMSO + E2LY294002 + E2Rapamycin + E2

0 1

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valuation of the effects of LY294002 and rapamreated with DMSO (0.1%), LY294002 (10 �mor E2 at concentrations from 10�10 to 10�6 moays. Data shown are means � SE of 3 indepe

), but decreased that of the cells at d

76.e3 American Journal of Obstetrics & Gynecol

2/M phase (Figure 3C and D) in bothtSM and UtLM cells.

OMMENThe current study demonstrates that E2

ignificantly induces G1 cell cycle pro-ression and proliferation in both uter-ne fibroid and myometrial cells. Com-ared with findings from the previoustudies with the ELT 3 rat leiomyomaells,10 our current study provides directvidence that E2 increases proliferationn fibroid cells and support the notionhat estrogen acts as a promoter of fi-roid growth.2 On the other hand, theurrent study shows that E2 also in-reased the proliferation in human uter-ne myometrial cells. This indicates that2 or E2-primed milieu may drive theormal myometrial cells into an in-reased growth potential, while stimu-ating the proliferation of tumor cells.

It was observed, however, UtLM cells

on E2-induced cell proliferation

A

Concentration (M)

1010-9 10-8 10-7 10-6 0 10-1010-9 10-8 10-7

C

G

* * **

**** * * * * *

***

**

** * * * *

*****

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***

Day 3 Day 4

in on E2-induced cell proliferation in UtSM (to, or rapamycin (10 ng/mL) for 2 hours, followedin phenol red-free DMEM supplemented with 1nt experiments. *P � .05, significantly differen

id not show increased responses to E2 p

ogy FEBRUARY 2007

ith respect to proliferation comparedirh UtSM cells. In fact, the E2-treatedtLM cells showed slightly decreased

�8-35%) proliferation compared withhe UtSM cells (Figure 1). Because UtLMells also exhibited a similarly lower�13-35%) proliferation pattern rela-ive to the UtSM cells in the absence of2, the slight difference in proliferationetween the 2 cell types in response to E2ould be ignored. A similar observationas also been reported previously in therimary cultures of human leiomyomaells and adjacent myometrial tissues,oth in the absence and presence of E2.11

hese findings are a bit incongruousith the fact of in vivo tumor growth and

he estrogen hypothesis of fibroid devel-pment.2 A most likely explanation forhis discrepancy is that the formationnd/or development of fibroids mightot be caused only by increased growthotential, but could also be resulted fromhe other factors such as decreased apo-

-6 0 10-1010-9 10-8 10-7 10-6

D

H

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**

*

*

*

*

*** *

*

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anel) and UtLM (lower panel) cells. Cells weretreatment with vehicle control (0.03% ethanol)FBS for A, E, 2, B, F, 3, C, G, 4, or D, H, 5,

om DMSO � E2 group.

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www.AJOG.org Basic Science: Gynecology Research

oughs et al12 has revealed an increasedate of proliferation, but a decreased ratef apoptosis in leiomyomas relative toge-matched myometrium in the Ekerat model. Interestingly, the authorsound that the normal myometrium oferipubertal animals proliferated at itsreatest rate on the day of proestrus, theime at which serum E2 concentrationeaked. Apoptosis, on the other hand,as maximal when E2 and progesterone

evels were both low. Another study byukuhara et al13 showed that secretedrizzled-related protein 1 (SFRP1), a

odulator of Wnt signaling exerting an-

FIGURE 3Effects of PI3K and mTOR inhibito

12 24

G0/

G1

phas

e ce

lls (

%)

0

20

40

60

80

100

12 24

G2/

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%)

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5

10

15

20

A

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UtSM cells

C

ffects of E2 on cell cycle distribution and effecell cycle progression in A, C, UtSM and B, D,0.03% ethanol) or E2 at concentrations fromupplemented with 10% FBS for 12 or 24 hourMSO (0.1%), LY294002 (10 �mol/L), or rapeans � SE of 3 independent experiments. *P

05, significantly different from 10�8 mol/L E2

iapoptotic effects, was highly expressed e

n leiomyomas compared with normalyometrium. The expression of SFRP1

n leiomyomas was strongest in the lateollicular phase (high-estrogenic milieu)f the menstrual cycle and could be in-uced in vitro by the treatment of cul-ured leiomyoma cells with E2, but notrogesterone.13 These observations sug-est that both increased growth potentialnd decreased apoptotic responses in fi-roid tumors could be driven by a high-strogenic environment in vivo and thatpossible defect in the regulation of ap-ptosis may exist in these tumors, butot in the normal myometrium. Inter-

on E2-induced G1 cell cycle

12 240

20

40

60

80

100Control

10-9

M

10-8

M

10-7 M

LY294002 + 10-8 M E2Rapamycin + 10-8 M E2LY294002Rapamycin

ime (h)12 24

0

5

10

15

20

*

* *

* *

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##

*

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UtLM cells

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D

f LY294002 and rapamycin on E2-induced G1M cells. Cells were treated with vehicle control�9 to 10�7 mol/L in phenol red-free DMEMefore E2 treatment, the cells were treated with

ycin (10 ng/mL) for 2 hours. Data shown are.05, significantly different from controls. #P �ne group.

stingly, it was observed in our current d

FEBRUARY 2007 America

tudy that most E2-treated UtSM cellsut not UtLM cells, showed a substantiveecrease in proliferation on day 5 com-ared with day 4 (Figure 1). The reason

or this declined proliferation is not fullynderstood. It is possible, however, that

hese cells might be undergoing aarked apoptosis on day 5 that de-

reased proliferation. Because a possibleefect in the regulation of apoptosis mayxist in leiomyomas,12,13 UtLM cells didot show such an apoptotic response,ut instead, a continuously increasedroliferation at the mean time. The facthat UtSM cells and UtLM cells respondimilarly to estrogen with respect to pro-iferation as indicated in our currenttudy further suggests that disregulationf apoptotic responses may be a crucialechanism underlying the disruption of

issue homeostasis and development ofterine leiomyomas.Previous studies have shown that E2

nduces cell proliferation in target tissuesy stimulating progression through the1 phase of the cell cycle, as shown by

ntiestrogens that are able to arrest thestrogen-dependent cancer cells in the0/G1 phases.14 In human fibroids dur-

ng all phases of the menstrual cycle, cy-lin D1 expression was elevated in com-arison with adjacent myometriumhere cyclin D1 was barely detected.15

he rate of this increase appeared to beycle-dependent, with the highest eleva-ion observed during the estrogen-dom-nated proliferative phase. During

enopause, however, cyclin D1 levels ineiomyomas were decreased and similaro that in myometrium. These resultsuggest that estrogen may play a role inhe induction of key regulators that areesponsible for accelerating cell progres-ion through the G1 phase. Corroborat-ng these earlier findings, the currenttudy provides direct evidence that E2nduces G1 cell cycle progression accom-anied by increased proliferation interine fibroid and myometrial cells.ithin the first 24 hours after treatment,

2 dose dependently decreased the cellopulation at G0/G1 phase but increasedhose G2/M phase in both cell types, al-hough had no significant effect on cellumber-based proliferation index on

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n Journal of Obstetrics & Gynecology 176.e4

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Research Basic Science: Gynecology www.AJOG.org

1

f G2/M cells determined did not pro-ortionally reflect respective cell prolif-ration, the results suggest that induc-ion of G1 cell cycle progression by E2ccurred before and was the cause of cellumber increases determined later.As an important part of our current

tudy, we investigated the roles of PI3Knd mTOR in mediating the effects of E2n G1 cell cycle progression and prolif-ration in UtSM and UtLM cells. Previ-us studies have shown that the activa-ion of Akt is greater in human uterinebroid samples than correspondingyometrium, especially in the estrogen-

ominated proliferative phase of theenstrual cycle.15 These studies gave the

rst clue showing that the PI3K/Akt sig-aling is implicated in fibroid develop-ent and that estrogen may play a role in

ctivation of the PI3K/Akt signaling inbroids. In the current study, we showed

hat the E2-induced G1 cell cycle pro-ression and proliferation were effi-iently blocked when the activity of PI3Kr mTOR in cells was inhibited by theirpecific inhibitors, revealing essentialoles of PI3K and mTOR in mediatinghese estrogen effects. These are believedo be the first data to indicate that PI3Knd mTOR are essentially required forhe E2-induced G1 cell cycle progressionnd proliferation in human uterine fi-roid and myometrial cells. Interest-

ngly, treatment with either LY294002 orapamycin alone significantly inhibitedell proliferation, accompanied by G1rrest, in UtSM and UtLM cells. This in-icates that PI3K and mTOR, which areonstitutively expressed in both UtSMnd UtLM cells, are required for G1 cellycle progression and proliferation in

hese cells. n

76.e5 American Journal of Obstetrics & Gynecol

In conclusion, our current study pro-ides evidence indicating an induction of1 cell cycle progression and prolifera-

ion by E2 in cultured human uterine fi-roid and myometrial cells and an essen-ial requirement of PI3K and itsownstream mTOR signaling for these2 effects. It should be noted, however,

he roles of PI3K and mTOR in mediat-ng these E2 effects as revealed in the cur-ent study is solely based on the cellularesponses to their pharmaceutical inhib-tors. Nevertheless, a potential involve-

ent of the PI3K/Akt/mTOR/p70S6K1ignaling transduction pathway in theevelopment of fibroids and its role inediating estrogen effects on human

terine fibroid and myometrial cells areikely. A better understanding of thisathway will provide more valuable in-

ormation needed for designing effectivegents for the therapy of fibroid tu-ors. f

CKNOWLEDGMENTSe are grateful to Dr Darlene Dixon (National

nstitute of Environmental Health Sciences) forenerously providing the UtSM and UtLM cells.

EFERENCES. Wilcox LS, Koonin LM, Pokras R, Strauss LT,ia Z, Peterson HB. Hysterectomy in the Unitedtates, 1988-1990. Obstet Gynecol 1994;83:49-55.. Flake GP, Andersen J, Dixon D. Etiology andathogenesis of uterine leiomyomas: a review.nviron Health Perspect. 2003;111:1037-54.. Falkenstein E, Tillmann HC, Christ M, Feuring, Wehling M. Multiple actions of steroid

ormones—a focus on rapid, nongenomic ef-ects. Pharmacol Rev 2000;52:513-56.. Luo J, Manning BD, Cantley LC. Targetinghe PI3K-Akt pathway in human cancer: ratio-

ale and promise. Cancer Cell 2003;4:257-62. 2

ogy FEBRUARY 2007

. Hay N, Sonenberg N. Upstream and down-tream of mTOR. Genes Dev 2004;18:926-45.. Grewe M, Gansauge F, Schmid RM, Adler G,eufferlein T. Regulation of cell growth and cy-lin D1 expression by the constitutively activeRAP-p70s6K pathway in human pancreaticancer cells. Cancer Res 1999;59:3581-7.. Gao N, Zhang Z, Jiang BH, Shi X. Role ofI3K/AKT/mTOR signaling in the cell cycle pro-ression of human prostate cancer. Biochemiophys Res Commun 2003;310:1124-32.. Gao N, Flynn DC, Zhang Z, et al. G1 cell cyclerogression and the expression of G1 cyclinsre regulated by PI3K/AKT/mTOR/p70S6K1ignaling in human ovarian cancer cells. Am Jhysiol Cell Physiol 2004;287:C281-91.. Carney SA, Tahara H, Swartz CD, et al. Im-ortalization of human uterine leiomyoma andyometrial cell lines after induction of telomer-

se activity: molecular and phenotypic charac-eristics. Lab Invest 2002;82:719-28.0. Howe SR, Gottardis MM, Everitt JI, Walker. Estrogen stimulation and tamoxifen inhibitionf leiomyoma cell growth in vitro and in vivo.ndocrinology 1995;136:4996-5003.1. Loy CJ, Evelyn S, Lim FK, Liu MH, Yong EL.rowth dynamics of human leiomyoma cellsnd inhibitory effects of the peroxisome prolif-rator-activated receptor-gamma ligand, piogli-azone. Mol Hum Reprod 2005;11:561-6.2. Burroughs KD, Fuchs-Young R, Davis B,alker CL. Altered hormonal responsiveness of

roliferation and apoptosis during myometrialaturation and the development of uterine

eiomyomas in the rat. Biol Reprod 2000;3:1322-30.3. Fukuhara K, Kariya M, Kita M, et al. Se-reted frizzled related protein 1 is overex-ressed in uterine leiomyomas, associated withhigh estrogenic environment and unrelated toroliferative activity. J Clin Endocrinol Metab002;87:1729-36.4. Wakeling AE, Dukes M, Bowler J. A potentpecific pure antiestrogen with clinical potential.ancer Res 1991;51:3867-73.5. Kovacs KA, Lengyel F, Kornyei JL, et al.ifferential expression of Akt/protein kinase B,cl-2 and Bax proteins in human leiomyomand myometrium. J Steroid Biochem Mol Biol

003;87:233-40.