GUINEA PIG MODEL FOR HUMAN NEVI

observations of Pawlowski et al. (24). They observed melanomas in40% of their high-dose group (equivalent to our group 1) after 18months. Many of these tumours metastasized or were able to grow innude mice, confirming their malignancy. The use of depilitory creamto remove hair before DMBA application may explain some differences in nevi and melanoma induction in our system. However, theexact cause of the difference is unclear.

Our model may be modified using the hairless (hr/hr) Hartleyguinea pig (30). Both albino and pigmented hairless guinea pigs areavailable. The obvious advantage of the hairless phenotype is that hairremoval is not required for irradiation protocols and nevi monitoring.However, gross differences occur in other cutaneous carcinogenesisregimes in hairless mice compared with hairy wild-type mice (35). Inaddition, differences in percutaneous absorption (36) and skin changesto UVB (37) between hairless and hairy guinea pigs have been noted.Thus, the dose response of DMBA-induced nevi and augmentation bysolar-simulated light may be very different in hairless guinea pigs.

In conclusion, the frequency of melanocytic nevi is a good indicatorof future development of melanoma and a short-term marker ofadverse reactions to melanoma-inducing sun exposure in humans.Thus, an animal model for sunlight-induced nevi can be used as asurrogate model for sunlight-induced melanoma, allowing such studies as determining the protection of sunscreens against melanomainducing sunlight and defining its action spectrum. Our guinea pigmodel has some of the essential elements required to be a robustanimal model for human nevi.

REFERENCES

I. Parkin, D., Muir, C., Whelan, S., Gao, Y., Ferlay, J.. and Powell, J. (ads.). CancerIncidence in Five Continents. IARC Scientific Publ. Lyon. France: IARC, 1992.

2. Elwood, J. M., and Koh, H. K. Etiology, epidemiology, risk factors, and public healthissues of melanoma. Curr. Opin. Oncol., 6: 179—187,1994.

3. Berwick, M., and Halpern, A. Melanoma epidemiology. Curr. Opin. Oncol.. 9:178—182,1997.

4. Slade, J.. Salopek, T., Marghoob. A., Kopf. A.. and Rigel, D. Risk of developingcutaneous malignant melanoma in atypical-mole syndrome: New York Universityexperience and literature review. In: C. Garbe, S. Schmitz, and C. Orfanos (eds.).Recent Results in Cancer Research 139—SkinCancer, pp. 87—104.Berlin: SpringerVerlag, 1995.

5. Luther, H., Altmeyer, P., Garbe, C., Ellwanger. U.. Jahn, S., Hoffmann, K., andSegerling. M. Increase of melanocytic nevus counts in children during 5 years offollow-up and analysis ofassociated factors. Arch. Dermatol., 132: 1473—1478,1996.

6. Dwyer. T.. Blizzard, L., and Ashbolt, R. Sunburn associated with increased numberof nevi in darker as well as lighter skinned adolescents of northem European descent.Cancer Epidemiol. Biomark. Prey., 4: 825—830,1995.

7. Kelly, J., Rivers, J., MacLennan, R., Harrison, S., Lewis, A., and Tate, B. Sunlight:a major factor associated with the development of melanocytic nevi in Australianschoolchildren. J. Am. Acad. Dermatol., 30: 40—48,1994.

8. Gallagher. R., McLean, D., Yang, C., Coldman, A., Silver, H., Spinelli, J., andBeagrie. M. Anatomic distribution of acquired melanocytic nevi in white children. Acomparison with melanoma: the Vancouver mole study. Arch. Dermatol., 126:466—471. 1990.

9. Friedman, R., Rigel, D., and Heilman, E. The relationship between melanocytic neviand malignant melanoma. Dermatol. Clin. 6: 249—256,1988.

10. Shapiro, R., Duquette, J., Roses, D., Nunes, I., Harris, M., Kamino, H., Wilson, E.,and Rifkin. D. Induction of primary cutaneous melanocytic neoplasms in urokinasetype plasminogen activator (uPA)-deficient and wild-type mice: cellular blue neviinvade but do not progress to malignant melanoma in uPA-deficient animals. CancerRes., 56: 3597—3604,1996.

I I. Chen, S., Thu. H., Wetzel, W., and Philbert, M. Spontaneous melanocytosis intransgenic mice. J. Investig. Dennatol., 106: 1145—1151. 1996.

12. Millikan, L., Smith, L., and Ochsner, J. Animal models in melanoma. in: H. Maibach(ed), Models in Dermatology, Vol. 1, pp. 22—33.Basel: Karger, 1985

13. Epstein, J. Experimental models for primary melanoma. Photodermatol. Photoimmunol. Photomed., 9: 91—98,1992.

14. Ley, R., Applegate, L., Padilla. S.. and Stuart, T. Ultraviolet radiation-inducedmalignantmelanomain Monodelphisdomestica.Photochem.Photobiol..50: 1—5,1989.

15. Setlow, R., Woodhead, A., and Grist, E. Animal model for ultraviolet radiationinduced melanoma: platyfish-swordtail hybrid. Proc. Natl. Acad. Sci. USA, 86:8922—8926,1989.

16. Sancar, A. Structure and function of DNA photolyase. Biochemistry, 33: 2—9,1994.17. Kato, T., Todo, T., Ayaki, H., Ishizaki, K., Mona, T., Mitra, S., and Ikenaga, M.

Cloning of a marsupial DNA photolyase gene and the lack of related nucleotidesequences in placental animals. Nucleic Acids Res. 22: 4119—4124, 1994.

18. Klein-Szanto,A., Silvers, W., and Mints, B. Ultravioletradiation-inducedmalignantskin melanoma in melanoma-susceptible transgenic mice. Cancer Res., 54: 4569—4572, 1994.

19. Takahashi, M., Iwamoto, T., and Nakashima, I. Proliferation and neoplastic transformation of pigment cells in metallothionein/ret transgenic mice. Pigm. Cell Res., 5:344—347,1992.

20. Husain, Z., Pathak, M., Flotte, T.. and Wick. M. Role of ultraviolet radiation in theinduction of melanocytic tumors in hairless mice following 7,l2-dimethylbenz(a)anthracene application and ultraviolet radiation. Cancer Res.. 51: 4964—4970,1991.

21. Epstein, J.. Epstein, W., and Nakai, T. Production of melanomas from DMBAinduced blue nevi in hairless mice with ultraviolet light. J. NatI. Cancer Inst., 38:19—30,1967.

22. Parrish, J. Responses of skin to visible and ultraviolet radiation. in: L. Goldsmith(ed), Biochemistry and Physiology of the Skin, Vol. 2.. pp. 713—733.New York:Oxford University Press, 1983.

23. Hook, R., Berkelhammer, J., and Oxenhandler, R. Sinclair swine melanoma. Am. J.Pathol., 108: 130—133,1982.

24. Pawlowski, A., Haberman, H., and Menon, I. Skin melanoma induced by 7,12-dimethylbenzanthracene in albino guinea pigs and its similarities to skin melanoma ofhumans.CancerRes.,40: 3652—3660,1980.

25. Pawlowski, A., Haberman, H., and Menon, I. Junctional and compound pigmentednevi induced by 9,l0-dimethyl-l.2-benzanthracene in skin of albino guinea pigs.Cancer Res., 36: 281 3—2821, 1976.

26. Menzies, S. W., Greenoak, G. E., Reeve, V. E., and Gallagher, C. H. UV radiationinduced murine tumors produced in the absence of UV radiation-induced systemictumor immunosuppression. Cancer Res., 51: 2773—2779,1991.

27. Ley, R. Photoreactivation of UV-induced pyrimidine dimers and erythema in themarsupial Monodelphis domestica. Proc. Natl. Aced. Sci. USA, 82: 2409—2411,1985.

28. Ruiter, D., and Brocker, E. Immunohistochemistry in the evaluation of melanocytictumours. Semin. Diagn. Pathol., 10: 76—91,1993.

29. Bacchi, C. E., Bonetti, F., Pea, M., Martignoni, G., and Gown, A. HMB-45. A review.Appl. Immunohistochem.,4: 73—85,1996.

30. Bolognia, J., Murray, M., and Pawelek, J. Hairless pigmented guinea pigs: a newmodel for the study of mammalian pigmentation. Pigm. Cell Res., 3: 150—156,1990.

31. Barnhill, R., and Mihm, M. The histopathology of cutaneous malignant melanoma.Semin. Diagn. Pathol., 10: 47—75,1993.

32. Sutherland, B., and Bennett, P. Human white blood cells contain cyclobutyl pyrimidine dimer photolyase. Proc. NatI. Aced. Sci. USA, 92: 9732—9736,1995.

33. Kusewitt, D., Applegate, L., and Ley, R. Ultraviolet radiation-induced skin tumors ina South American opossum (Monodelphis domestica). Vet. Pathol., 28: 55-65, 1991.

34. Voulot, C., and Laviolette, P. Les tyrosinases des parties pigmentees de l'oeil chezquatre ezpeces de Rongeurs. C. R. Aced. Sci. Hebd. Seances. Acad. Sci. 0., 283:79—81,1976.

35. Poland, A., Palen, D., and Glover, E. Tumour promotion by TCDD in skin of HRS/Jhairless mice. Nature (Lond.), 300: 271—273,1982.

36. Hisoire, G.. and Bucks, D. An unexpected finding in percutaneous absorption ohserved between haired and hairless guinea pig skin. J. Pharm. Sci., 86: 398—400,1997.

37. Horio, T., Miyauchi, H., and Asada, Y. The hairless guinea pig as an experimentalanimal for photodermatology. Photodermatol. Photoimmunol. Photomed.. 8: 69—72,1991.

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[CANCERRESEARCH58, 5367-5373, December 1, 1998]

studies on ER expression failed to provide clear insights into itspotential role in tumor progression. Moreover, most ovarian cancersremain insensitive to antiestrogen therapy (10).

The recent characterization of ER-@ opens a new way for understanding the physiopathology of estrogen-regulated tissues (11). TheER-@3 is homologous to ER-a and has a similar affinity for the

l7@3-estradio1(12). The question on the respective role of the two ERgene expressions is of particular interest, because a number of estrogen target tissues, including breast, endometria, prostate, testis, andovary, were shown to coexpress ER-a and -/3 (12—14).In normalovaries, ER-@ seems to be the predominant species expressed in rats(13) and humans (12, 14—16)

ER-a and -13are members of the superfamily of nuclear receptors,which transduce hormone signals. ER-ct acts as a ligand-activatedtranscription factor for mediating the principal effects of estrogens ineither normal or cancerous target cells. ER-a expression is involved inbreast tumor progression and in the development of a hormonedependent phenotype of breast cancer (17). A great deal of information regarding the mechanism of ER-a action and the clinical outcomehas been obtained, thus enabling us to use ER-a status as a marker ofprognosis and prediction of hormone sensitivity in breast cancer forover 2 decades (18).

ER-a expression has also been extensively studied in ovariancancer in an attempt to correlate genotype to phenotype. AlthoughER-a is expressed in 40—60%of ovarian cancers, no clear relationship between ER expression and tumor histology, age, or outcome hasbeen noted in epithelial tumors (for review see Ref. 10). Interestingly,a higher ER cytosolic concentration has been observed in cancer ascompared with cysts using LBA, which theoretically recognizes bothER forms (5—9).In contrast, a present report has shown a lower levelof ER-V mRNA expression in neoplastic ovarian tissues as comparedwith normal tissue (16). Comparative analysis of both ER forms is,thus, needed to more accurately study variations in ER expressionbetween normal or benign tumors and cancers.

To study the potential involvement of the two ERs in ovariancarcinogenesis, we analyzed the relative expression of ER-a and -j3mRNAs in normal ovaries and in ovarian cancer cell lines, then inbenign and malignant ovarian epithelial human tumor samples. Forthis aim, we developed a competitive PCR assay based on coamplification of the two ER forms and assessed the relative levels of ER-aand -/3 mRNAs in human ovarian samples that may reflect tumorprogression.

Received 7/13/98; accepted 10/5/98.Thecostsof publicationof thisarticleweredefrayedin partby the paymentof page

charges. ‘l'hisarticle must therefore be hereby marked advertisement in accordance with18US.C. Section1734solelyto indicatethisfact.

I Supported by the “Center Hospitalier Universitaire de Montpellier.― the “LigueNationaIc de Recherche Contre le Cancer,―and the “InstitutNational de la Sante et de IaRechercheMédicale.―

2 To whom requests for reprints should be addressed, at Laboratoire de Biologic

Cdllulaire, HôpitalArnaud de Villeneuve, 271. Av G. Giraud, 34295 Montpellier Cedex5, France.

3 The abbreviations used are: ER, estrogen receptor; RT-PCR, reverse transcription

PcR; 1SH,in situ hybridization;GAPDH,glyceraldehyde-3-phosphatedehydrogenase;QIH, quantitative in situ hybridization; LBA, ligand binding assay; AU, arbitrary unit(s).

5367

Differential Expression of Estrogen Receptor-a and 43 Messenger RNAs as aPotential Marker of Ovarian Carcinogenesis1

Pascal Pujol,2 Jean-Marc Rey, Phifippe Nirde, Pascal Roger, Marguerite Gastaldi, François Laffargue,Henri Rochefort, and Thierry MaudelondeServices tie Biologie Cellulaire (P. P., J-M. It, H. R., T. M.J ci de Gynécologie-Obstetrique (F. LI, Centre Hospitalier Universitaire de Mon:pellier. Hdpital Arnaud deVileneuve. 34295 Montpellier; Unite JNSERM 148 (P. P., J-M. R., P. R., H. R., 1'. MI et Unite INSERM 439 (P. N.J 34095 Montpellier; and Service de Biologic Cellulaire,Facultd tie Mtdecine de Ia Timone, 13385 Marseille (M. G.J. France

ABSTRACT

Although estrogen receptor (ER)-a is expressed in both benign andm2lIgn@ntovarian tumors, the role of ER in ovarian carcinogenesis ofepithellal tumors is still unknown. In view of the recent characterizationofER.@3,a second form ofER that seems to be highly expressed in ovaries,we reexamined this issue by studying the relative expression of ER-a and-p in humanovariantumorprogression.

We developed a competitive PCR assay based on coamplification of thetwo ERa In target nucleotide sequences displaying a high homology (exons

3 and 4). Coampilfication experiments with varying amounts of plasmidscontaining ER.a and 43 cDNAs showed that this assay was reliable fordiscriminating as little as a 2-fold difference In the Initial ER-a:ER.fieDNAratio.The relativeexpressionof ER-a comparedwith ER-VmRNAs was StUdIed In human ovarian cancer cell lines (n = 5) and innormal ovaries (n 6), then In human benign and malignant tumorsam@ Including ovarian cysts (n = 24), borderline tumors (n = 3), andcancers (n = 10). In normal ovaries, ER-fl mRNA was the predominantER form, whereas in ovarian cancer cell lines ER-a mRNA was markedlyIncreased as compared with ER43. In benign and borderline tumors,ER..fi mRNA was detected in 78% of tumors, whereas ER-a mRNA wasdetected In 29%. In ovarian carcinomas, both ER-a and 43 mRNAs wereexpressed In 80% oftumors. The ER.a:ER43 mRNA ratio was >1 in onlyone cyst sample (4%). In contrast, the ER-a:ER-fl mRNA ratio wasmarkedly Increased in ovarian cancers because 60% showed an ER-a:ER43 inRNA >1. In situ hybridization experiments showed overlappingtissular distribution of ER-N and -a expression in cancers and cysts, witha main localization in the epithellum and only a low level of expression indramal cell&

In summary, we found an increase in the ER-a:ER-@ mRNA ratio inovarian carcinomas as compared with normal ovaries and cysts. Thesedata suggest that overexpression of ER-a relative to ER-fl mRNA may bea marker of ovarian carcinogenesis.

INTRODUCTION

Ovarian cancer is the second most common gynecological cancerand the second cause of death from gynecological cancers in westerncounti•ies(1). Ovarian carcinogenesis mechanisms has not yet beenfully elucidated (2, 3), and available molecular markers for clinicalassessment of ovarian tumor progression are, thus, still missing.

Ovaries represent both a main producer and target tissue of estrogens. There are experimental and clinical studies suggesting a role ofestrogen in ovarian carcinogenesis (4). ER3 is expressed in bothbenign and malignant human ovarian epitheial tumors (5—9),but

MATERIALS AND METHODS

Patients. Six normal ovaries, 24 ovarian cysts, 3 borderline tumors, and 10ovarian cancer specimens were obtained from patients after surgical therapy inour institution in 1992 and 1993 (Department of Gynecology, UniversityHospital of Montpellier). The tissues were harvested in the operating room.

One partof the tissue was snap-frozenin liquid nitrogenand stored in ourtumor bank, whereas the other part was used for histological examination. The

histological subtypes of ovarian tumors included: serous cysts (n = 16),mucinous cysts (n = 6), endometrioid cysts (n = 2), serous borderline tumors(n 3), serous carcinomas (n 8), and mucinous carcinomas (n 2).

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ER.a AND .@ EXPRESSION IN OVARIAN TUMORS

ISH

ER@D3 ER*3 ERxF4 ERDR2-@,------.@-@

ER@CDNA@@ I 2 I 3 I@

The integrity of extracted RNA was controlled by agarose gel analysis throughthe rRNA fraction.

_1S@@ 8i— cDNAsynthesiswasperformedwith1 @goftotalRNAina25-p.lfinal. . 1560volumecontaining10mMDDT,50mMTris-HC1(pH8.3),75mMKC1,3________________ mMMgCl2, 300 msi dNTP, and 2.72 mr@irandom hexamer oligonucleotides

@-@-@--@ _I_5@@ 8 I@j5j;•(Promega,Lyon,France).Sampleswereheatedat65°Cfor5 mmandrapidlychilled on ice before adding 40 IU of RNase inhibitor and 200 LUof M-MTV

-- reverse transcriptase (Life Technologies, Inc.). The final mixture was then-------. incubated for 1 h at 37°C and heated for 5 mm at 90°C. Reverse transcriptase

--.--.--.-.-. efficiency was controlled by GAPDH housekeeping gene amplification, as

ERacDN@@ I 2 1 3T

-@- -.@---@@

ERaD3 ERa9/ saas ERuR2

described previously (19).

Competitive PCR. To competitively amplify the specific ER-a and -(3gene fragments, the 5'-end oligonucleotide was identical for both PCRs (Fig.1). To further minimize the differences in reaction efficiency between the two

gene amplifications, the target nucleotide sequence was chosen in exons 3 and4 displaying 71% homology (11, 20). The 5' sense oligonucleotide was

5'-AAGAGCTGCCAGGCCTGCC-3' (ERxF4, located at nucleotides 702-

720 and 454—472 of the ER-a and -(3 sequences, respectively). The 3'antisense oligonucleotide for ER-a was 5'-TFGGCAGCTCTCAT

GTCTCC-3' (ERaR2, located at nucleotides 850—869), amplifying a l68-bpPCR product, whereas the 3' antisense oligonucleotide for ER-(3 was 5'-GCGCACTGGGGCGGCTGATCA-3' (ER(3Rl, located at nucleotides 701-721), generating a 267-bp specific fragment.

The reversetranscriptase(1 @tl)productwas amplifiedby PCR in a 25-idfinal volume containing 20 nmi Tris-HC1 (pH 8.55), 16 mM (NH4)2SO4,2.5mM MgCl2, 10% DMSO, 50 pM of each dNTP (with 10 pmol of oligonucleotides ERxF4, ERaR2, and ER(3Rl), and 0.125 unit of ‘FaqDNA polymerase(Bioprobe, Montreuil, France). After an initial 2-mm denaturation step at 94°C,PCRs were carried out on a DNA thermocycler (Perkin-Elmer Corp., Courtaboeuf, France) under the following conditions: 1-mm denaturation at 94°C,1-mm annealing at 62°C,and a 2-mm extension at 72°C.The samples under

went a final extension step for 7 mm at 72°C.Plasmids (pSG5) containing thewild-type full-length cDNA of ER-a and -(3 genes were used as DNA templates in the coamplification reaction. Each amplification product (12 @d)was

ER@: 267@

,.ERa: I6$bp

Fig. I. Strategy used for competitive PCR of ER-a and -@3cDNAs. Schematicrepresentation of ER-a and -13eDNA organization: vertical bars indicate positions ofexonhintron boundaries; horizontal bars indicate locations of primers used for synthesis ofISH probes and for competitive PCR. PCRs using sense primer(within exon 3) andantisense primer (within exon 4) for ER-(3 and -a generated 267 bp- and 168 bp-specificfragments. respectively.

Cell Lines. The following humanovariancancercell lines wereusedin thisstudy: BG1, SKOV3, PEO4, PEO14, and NIH:OVCAR3 (OVCAR3). BG1

(obtained from Dr. C. E. Welander, Emory University, Atlanta, GA) andSKOV3 (obtained from the American Type Culture Collection) were culturedin DMEM supplemented with 10% FCS. PEO4 and PEO14 (obtained from Dr.

Langdon, Hospital of Edinburgh, S.P. Edinburgh, United Kingdom) andOVCAR3 (obtained from the American Type Culture Collection) were cultivated in RPMI 1640, 10% FCS, supplemented with either 10 @.tg/mlinsulin(PEO4)or 2 mML-glutamine(PEO14andOVCAR3).Cells were harvestedat75% confluence.

RNA Extraction and Reverse Transcription. Total RNAs were preparedfrom ovarian cancer and ovarian cyst samples by TRizol extraction accordingto the manufacturer's instructions (Life Technologies, Inc., Eraguy, France).

n cycles

M 17 20 23 26 29 32

@...... d

— ER@

a

b

C

Numberof cycle

a.@

I

.@ *001

e@00l

0I@@ 20 :@@

Numberof cycle

ERat .@

IER@

Fig. 2. Coamplification of ER-a and -@ as afunction of the number of cycles. a, plasmids contaming ER-a or -@ cDNAs. Each plasmid (20 pg)was coamplified for 17, 20, 23. 26, 29, and 32cycles. M. molecular weight marker. b. BGI ovarian cancer cell line. c, Human ovarian cancer samplc. d.f, Quantification of PCR products. U, ER-a;0. ER-B.

Number of cycle

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1 23 4 5 67 8 9 1011I.fl-..-@--

ER.a AND .@ EXPRESSIONIN OVARIANTUMORS

MgCl2,50 @.LMdNTP (with 5 pmol of oligonucleotides), and 0.125 unit of TaqDNA polymerase (Bioprobe). Thirty-six rounds of PCR amplification werecarried out with a 30-s denaturation step at 94°C,30 s of annealing at 56°C,and a 1-mm extension at 72°C.RT-PCR products were eluted from a 0.8%agarose gel using QiAquick gel extraction (QIAgen, Courtaboeuf, France).

The PCR products were then subcloned in the PGEMT plasmid (Promega,Charbonnières,France). Digoxigenin-labeled antisense and sense RNA probeswere synthesized with the Dig labeling transcription kit, according to themanufacturer's instructions, using 11 and SP6 RNA polymerase (Boehringer

0 -ERa Mannheim, Mannheim, Germany). Adjacent 5—7 @tmof frozen ovarian tissue

I -ERR sections were collected on Silane-prepTM slides (Sigma Diagnostics, St.F Louis, MO), and ISH was performed as described (22, 23). The antisense and

sense probes were added to the tissue at a final 40-ng/@.d concentration in 20,.d of hybridization buffer. For each sample, nonspecific binding controlsincluded slides without probe, sections hybridized with sense oligonucleotideprobe, and slides treated with RNase A before hybridization. mRNA integritywas controlled by amplification of the GAPDH housekeeping gene on adjacenttissue sections, as described above.

ISH signal was quantifiedwith an image analyzer(SAMBA TITN;Unilog,Grenoble, France) adapted to a Leitz DMRB light microscope (Leica GmbH,Wetzlar, Germany) and a 3-CCD DXC-950P color video camera (Sony Cor

poration, Tokyo, Japan) connected to a microcomputer. The results wereexpressed by a QIH score given in AU (the percentage of stained sur

face X mean absorbance). The background QIH score of the negative control(sense probe) in an adjacent section was subtracted from the total QLH score toobtain specific staining.

Statistical Analysis. The y@test was used to compare frequencies of ER-a-and -(3-positivetumors between patient subgroups. Nonparametric ER-a:ER-(3ratio values were compared using the Kruskal-Wallis test.

RESULTS

Optimization of a Competitive PCR Assay for ER-a and -13

Linearity. Amplification of increasing known amounts of plasmidcontaining full-length ER-a or -(3 cDNAs was performed to optimizea competitive PCR assay for the two ERs. The linear PCR amplification phase was determined for equal amounts of plasmid containingER-a and -(3 template cDNAs (Fig. 2a), then for reverse transcriptaseproducts from the ovarian cancer BG1 cell line (Fig. 2b) and from anovarian tissue sample (serous carcinoma; Fig. 2c). PCR products werequantified by measuring the fluorescence intensity as described in“Materialsand Methods.―Coamplification of ER-a and -(3 was linearbetween 20 and 26 cycles for plasmids (Fig. 2d) and 23—29cycles for

a

ER@

ERa

llutmdiERf3(P&PRa

b

2 10 50 2 10 50 2 10 50 - 50

50 5050 10 10 10 2 2 2 50 -

@ 1000

I @ffli]I@@L1IHI

run on a 5% acrylamide gel and stained with ethidium bromide. Two mdcpendent RT-PCR reactions were performed for each tumor sample.

PCR Product Quantification. The fluorescence intensity of the sampleproducts and the standard was quantified by digital image processing ofagarose gel bands on an Argus 100 biological imaging workstation(Hamamatsu Photomcs, Hamamatsu, Japan) with a high-performance CCDcamera (Cohu, San Diego, CA), as previously reported (21). After backgroundsubtraction,the net fluorescencewas plotted as a functionof the standardamount.

ISH. The cellulardistributionof ER-a and -(3mRNAs was analyzedwitha nonisotopic ISH procedureusing digoxigenin-labeledprobe. Specific ofER-a and -(3 probes were chosen in the region of ERs displaying a lowhomology (exons 1 and 2 corresponding to the A/B domain ofthe protein; Fig.1). Probespecificity was checkedusing the GeneJockeyprogramon a Macintosh ilCi and by screening all nucleotide sequences stored in GenBank forhomology. ER-a and -(3cDNA templates were synthesized by RT-PCR. TotalRNA(1 gLg)from the BG1 celiline was reverse transcribed as described above.The sense and antisense primers for amplification of a 328-hp ER-a-specificfragment were 5'-CCCTACTGCATCAGATCCAAGG-3' (ERaD1, nucleotides 330-351) and 5'-CFGCAGGAAAGGCGACAGC-3' (ERaR1, nucleotides 640—658),respectively. The sense and antisense primers for amplification of a 268-bp-specific ER-(3 fragment were 5'-GTGATGAATFA

CAGCATFCCCAOC-3' (ER(3Dl, nucleotides 16-40) and 5'-GAACCTGGACCAGTAACAGG-3' (ERf3R1, nucleotides 265—284), respectively. Reverse transcriptase product (1 ,.tl) was amplified by PCR in a 25-@l finalvolume containing 20 mM Tris-HCI (pH 8.55), 16 mM (NH4)2S04, 2.5 mM

Fig. 3. Coamplification of ER-a and -(3 as a function of the initial quantity of eDNAtemplate. a, coamplification of different amounts of plasmids containing ER-a or -f3cDNAs. b, quantification of corresponding PCR products.

a CM 1 2 3 4 5 67

‘U

‘UFig. 4. Coampliulcationof increasing amounts ofER-aor ER-@cDNAs.a, PCRproductswithincreased amounts of ER-a plasmid and a constantamount of ER-H plasmid (10 pg). b. PCR productswith increased amounts of ER-@ plasmid and aconstant amount of ER-a plasmid (10 pg). c and d,quantification of final PCR products.

ER@ (pg)

ERt@(pg)

b d°0

‘U

‘U

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ER-a AND .@ EXPRESSION IN OVARIAN TUMORS

amount of ER-a plasmid when the quantity of ER(3 plasmid remainedconstant (Fig. ‘Ia).Inversely, for a constant amount of ER-a plasmid,the competitive PCR was done with increasing quantity of ER-(3plasmid (Fig. 4b). The logs of ratio between the fluorescence intensities were plotted as a function of the increasing amount of eitherER-a (Fig. 4c) or ER-(3 (Fig. 4d) plasmids, and the curve fitting wascomputed by linear regression. The PCR product quantificationgraphs showed that the assay can discriminate the ER-a:ER-(3 expression ratio in the order of 1:8 to 8:1.

RT-PCRAnalysisof OvarianCancerCell Linesand HumanTissue Samples

In an attempt to study the potential involvement of the two ERs inovarian carcinogenesis, we assessed the relative levels of ER-a and -(3mRNA expression in normal ovaries and in human ovarian cancer celllines, then in benign, borderline, and malignant ovarian tumors thatmay reflect tumor progression. Six normal ovaries, 5 human ovariancancer cell lines, 24 human ovarian cysts, 3 serous borderline tumors,and 10 cancer samples were analyzed.

Fig. 5 shows the relative expression of ER-a and ER-(3 genes inovarian cancer cell lines and in normal ovaries. The mRNA integritywas controlled by amplification of the GAPDH housekeeping gene(Fig. 5b). In ovarian cancer tumors and cell lines, ER-a was thepredominant ER form, whereas in normal ovaries, ER-(3 was the mainexpressed form of ER. All ovarian cell lines analyzed displayed adetectable ER-a mRNA expression by our method (Fig. 5a), althoughPEO14 and OVCAR3 have a low level of ER-a expression, consistentwith their ER-negative phenotype observed at the protein level. Ascontrols, the ER-a-positive ZR-75 and ER-a-negative MDA-MB-231breast cancer cell lines are shown for the same experiment. Bothbreast cancer cell lines expressed a detectable level of ER-(3 mRNA.

Fig. 6 shows the relative expression of ER-a and -(3 genes in a@ representative subset of ovarian cysts (Fig. 6a) and ovarian cancers

:@ (Fig.6c).ER-f3wasthepredominantERformexpressedinbenignorborderline tumors. ER-f3 mRNA was expressed in 18 of 24 cysts

@ (75%) and in all 3 borderline tumors. ER-a mRNA was detectable at,@ lowlevelsinthe6normalovaries,in5of24cysts(20%),andinthe@ 3 borderline tumors. The ER-a:ER-(3 mRNA ratio was >1 in only one

cyst sample (4%; Fig. 7).In contrast to normal ovaries and benign ovarian tumors, ER-a

mRNA expression as compared with ER-(3 was markedly higher inovarian cancer samples. Eight (80%) ovarian cancers were ER-apositive, and eight (80%) were ER-(3 positive (Fig. 6c). Six (60%)cancers showed an ER-a:ER-(3 mRNA ratio >1 (Fig. 7). The difference in the ER-a:ER-(3 ratio of mRNA (>1 versus 1) betweennormal ovaries, ovarian cysts, borderline tumors, and cancers wasstatistically significant (Kruskal-Wallis test, P <0.01). No correla

M @â€/̃@ @3//@

a ___

b

Fig. 5. Expression of ER-a and 43 mRNAs in ovarian cancer cell lines and normalovaries. a, ER-a and -(3 eDNA coamplifications for the human ovarian cancer cell linesBG1. SKOV3, PEO4, OVCAR3, and PEO14 are shown together with the human breastcancer cell lines MDA-MB-23l and ZR-75, and normal ovaries (N). b, Amplification ofthe housekeeping GAPDH gene in the corresponding samples.

@—

CYSTS

:@@ 2 3 4 5 6 7 8 9 l0:@

: L@

Fig. 6. Expression of ER-a and -(3 mRNAs in ovarian cysts (a) as compared withovarian cancers (c). b and d, GAPDH mRNA expression in the corresponding samples.

CARCINOMAS

M I 2 3 4 5 6 7 8 9 10

cell lines and tumor samples (Fig. 2, e and f, respectively). Allsubsequent analyses were, thus, performed at 26 cycles.

The ER-a:ER-f3 ratio was maintained during the coamplificationreaction for a greater number of cycles than the range of linearamplification (Fig. 2, b and c, 29—32 cycles). This might be explained

by PCR limiting factors (e.g., Taq polymerase, dNTPs, or primerconcentrations) when the plate phase was reached.

Quantification of the ER-a:ER13 Ratio Using CalibratedAmounts of Plasmids. We compared the ER-a:ER-(3 ratio of thecDNA products obtained after amplification with the various knownamounts of ER-a and -(3 plasmid introduced in the mixture. As littleas 2 pg of each plasmid (—0.8attomoles) could be detected. The ER-aand -/3 signal intensity ratio was maintained during the amplificationprocess with regard to the initial various proportions of ER-a and -(3plasmids (Fig. 3, a and b; Lanes 1, 2, 4, 6, 8, and 9). The ER-a:ER-(3ratio remained at around 1 when PCR was performed from 2 pg, 10pg, or 50 pg of each plasmid (1.1, 1.4, and 1.3, respectively), showingthat the relative signal does not depend on the initial quantity of thetwo plasmids (Fig. 3, a and b; Lanes 3, 5 and 7, respectively).

Competitive ER-aIER-(3 PCR was performed with an increasing

1-15

10

5

I

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1)Normal Cysts Borderline

(n=6) (n=24) tumors(n=3)

Cancars(n=1O)

Fig. 7. ER-a:ER-13 mRNA ratio in ovarian tumor progression. a, mean values; P(Kruskal-Wallis test).

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ER-a AND -@ EXPRESSION IN OVARIAN TUMORS

h1p.

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h

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Fig. 8. In situ localization of ER-a and 43 mRNA expression in ovarian cysts and cancers. ISH was performed on adjacent sections of an ovarian serous cyst (a, b, e, and I) andan ovarian serous carcinoma (c, d, g, and h) with ER-a antisense probe (a and c) and ER-V antisense probe (b and d). Hybridization with ER-a (e and g) or ER-(3 (land h) sense probesare shown as controls.

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1996;56:3270-3275. Cancer Res   Mirjam J. A. P. Govers, Denise S. M. L. Termont, John A. Lapré, et al.   HumansSecondary Bile Acids and Thus Inhibits Colonic Cytotoxicity in Calcium in Milk Products Precipitates Intestinal Fatty Acids and

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