Progesterone Receptor Structure and Protease Activity in Primary Human Endometrial Carcinoma'

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(CANCER RESEARCH 48, I 143-I 147. March 1, 19881 Progesterone Receptor Structure and Protease Activity in Primary Human Endometrial Carcinoma'

Peter D. Feil,2 Christine L. Clarke,3 and Pondichery G. Satyaswaroop Department ofObstetrics and Gynecologyand CancerResearchCenter. TheMilton S. HersheyMedical CenterofThe PennsylvaniaState University, Hershey,PA I 7033

ABSTRACF eliminated the triplet/singlet structure seen on protein blots. Inclusion of a cocktail of protease inhibitors during tumor Monoclonal antibodies were used to investigate progesterone receptor homogenization prevented this loss of structure. Examination structure (isoforms) in 33 primary human endometrial tumors. The monoclonal antibodies recognized on protein blots two progesterone of 33 primary tumors revealed that, when sample preparation receptor proteins with molecular weights of 116,000 and 81,000. TheM, was performed in the presence of the protease inhibitors, the 116,000protein appearedas a triplet, while a single band was found for triplet/singlet structure was present in all PR-positive samples, the M, 81,000 protein. The triplet/singlet structure was found in all regardless of tumor grade. progesterone receptor-positive tumors, regardless of the degree of tumor differentiation. Protease activity, which gave rise to a false-negative MATERIALS AND METhODS pattern on protein blots, was found in approximately one-half of the tumors in which It was investigated. Inclusion of a cocktail of protease Tissue Source and Sample Preparation. Primary tumor samples were Inhibitors during sample preparation resulted in the maintenance of the obtained from patients who were undergoing prescribed surgery for triplet/singlet progesteronereceptor structure. Mixing experiments using endometrial carcinoma. The tumors were transported to the laboratory a progesterone receptor-rich human endometrial carcinoma(EnCa 101), in cold Ham's F-10 medium within 30 mm ofexcision, frozen in liquid which lacks protease activity, and protease-containing primary tumor nitrogen, and pulverized,and the powderswerestored in liquid nitrogen homogenatesindicated that the proteasewas leupeptin sensitive. Inter prior to assay. EnCa 101tumors weregrown in BALB/c -nu/nu athymic estingly, while the proteolytic activity reduced or eliminated the triplet/ mice in the presence of 17@-estradiolpelletsas described (2). Cytosol singlet progesterone receptor structure seen on protein blot analysis, it was prepared by homogenizing (Teflon/glass) the tissue powder in four did not affect progesteronereceptor concentrationmeasuredbyScatchard volumes of ice-cold PEMTG buffer, followed by centrifugation analysis. Sample preparation in the presence of protease inhibitors is (100,000 x g, 1 h). Where indicated, an aliquot of a stock solution therefore a requisite for structural analysis of the progesterone receptor (lOx) of protease inhibitor(s) was added to the PEMTG buffer just in endometrial tumors. prior to homogenization. The final concentration of each protease inhibitor used in the buffer was: 0.5 mM PMSF (Sigma, St. Louis, INTRODUCFION MO); 86 zM leupeptin (Calbiochem, La Jolla, CA); 77 @g/mlaprotinin (Sigma); 1.4 @iMpepstatinA (Sigma); 100 @ig/mlbacitracin(Sigma); Monoclonal antibodies to steroid hormone receptors provide and 25 mM benzamidine (Sigma). The same cytosol sample with or a sensitive tool for characterizing and comparing receptor struc without protease inhibitors was used for both Scatchard (6) and struc ture in endometrial tumors. Such information on receptor tural analysis. structure should complernent steroid binding assays and irn Assay of Progesterone Receptor. The PR binding site concentration prove predictability of tumor response to endocrine therapy. of each sample, prepared in the presence or absence of protease inhib itors, was measured as described previously (7) by Scatchard analysis Our laboratory has recently developed seven rnonoclonal anti (6) using 2 to 20 nM[1,2,6,7-3Hjprogesterone(90Ci/mmol; Amersham, bodies to the human progesterone receptor (1), using as the Arlington Heights, IL) in the presence of 1 @iMcortisol. source of antigen the PR4 from a well-differentiated human The structural form of PR in these samples was determined by endometrial carcinoma (EnCa 101) grown in nude mice in the probing protein blots with the anti-PR monoclonal antibodies (hPRa) presence of estrogen (2). In the EnCa 101 tumor, these mono generated by us (1). The cytosol proteins (200 ag), obtained from clonal antibodies recognize by protein blot analysis 2 PR pro samples prepared in the absence or presence of the protease inhibitors, teins with molecular weights of 116,000 and 81,000. In addi were first separated by 6.5% SDS-PAGE (8) and transferred electro tion, molecular weight isoforms were observed for the 116,000 phoretically (30 V, 16 to 18 h) to nitrocellulose paper according to protein (triplet), while the Mr 81,000 protein appeared as a Towbin (9). All reactions were carried out at room temperature. After single band. Since PR has been detected in well-, moderately, 1-h blocking (10) with Buffer A (10 mt.i Tris-HC1 (pH 7.5):0.15 M NaC1:0.05% Tween 20 (v/v)J, the nitrocellulose paper was reacted (2 and poorly differentiated tumors (3â€”5),we were prornpted to h) with a mixture of monoclonal antibodies enriched from culture ask whether this triplet/singlet structure (isoforms) was asso supernatants by ammonium sulfate (50%)precipitation: hPRa 1 (1:100) ciated with any particular stage of tumor differentiation. Direct + hPRa 2 (1:400) + hPRa 3 (1:800) + hPRa 6 (1:400). Comparison of assessment of this question was precluded by the finding that immunostaining intensity on protein blots using EnCa 101cytosol and many tumors contained a protease activity which, although it serial dilutions of eachantibody was usedto selecteach final dilution. did not affect the binding site concentration, greatly reduced or After probing, the nitrocellulose paper was washed (2 times, 30 mm) with the buffer, reacted with horseradish peroxidase-linked goat anti Received8/12/87; revised11/18/87; accepted12/2/87. mouse IgG (heavy plus light chain specific; Bio-Rad, Richmond, CA), The costsof publicationof this article were defrayedin part by the payment of page charges. This article must therefore be hereby marked advertisementin and the immunoreactive bands revealed using the chromogen 4-chloro accordancewith 18 U.S.C. Section 1734 solely to indicate this fact. 1-naphthol (Bio-Rad) as previously described(1). I This work was supported by National Cancer Institute Grant P01-CA 40011. Protease Activity and Inhibition. Tumor protease activity was assessed 2 To whom requests for reprints should be addressed. by mixing (16 h, 4C) EnCa 101cytosol with cytosol ofprimary tumors 3 Present address: Garvan Institute of Medical Research, St. Vincent's Hospi tal, Darlinghurst, NSW 2010, Australia. without/with the protease inhibitor cocktail prior to structural analysis

4 The abbreviations used are: PR, progesterone receptor, ER, estrogen receptor, by protein blot. The ability of an individual compound in the cocktail EGTA, ethyleneglycolbis(ft-aminoethylether)-N,N,N',N'-tetraacetic acid; R5020, to effectivelyinhibit proteolytic activitywasalso determined. An aliquot 17,21-dimethyl-19-nor-4,9-pregnadiene-3,20-dione;SDS,sodiumdodecylsulfate; ofone of the six proteaseinhibitors (lOx solution) was mixed (5 to 10 PAGE, polyacrylamidegelelectrophoresishPRa, humanprogesteronereceptor antibody;PEMTG buffer, 50 mti phosphate:l0mr@isodiummolybdate:lOmr@s mm, 4'C) with an aliquot of EnCa 101 cytosol (200 @gprotein), EGTA:l2 mM thioglycerol:lO%glycerol(v/v), pH 7.1; PMSF, phenylmethylsul followedby addition ofprimary tumor cytosol (100 @igprotein)to each fonyl fluoride. tube. Following incubation (16 h, 4C), sample buffer [187 mM Tris 1143 Downloaded from cancerres.aacrjournals.org on October 2, 2021. © 1988 American Association for Cancer Research. PR STRUCFURE IN HUMAN ENDOMETRIAL CARCINOMA

HC1(pH 6.8):5% 2-mercaptoethanol:3% SDS:lO% glycerolJwas added several of the primary tumors, regardless of tumor grade (Fig. to all samples(1:2, v/v) which were then heated(90'C, 2 mm) prior to 1, Lanes 1, 3, 6, 7, and 9). Other results, however, were per SDS-PAGE and protein blot analysis as described above. plexing, since there was often variable staining intensity be tween samples with similar PR concentrations (Fig. 1; compare Lanes 1 versus 3 and 7 versus 9). In addition, little or no RESULTS immunoreaction was detected in several tumors known to be PR concentration and structure (isoforms) were appraised in PR positive by Scatchard analysis (Table 1; Fig. 1, Lanes 2, 4, 33 samples of human endornetrial carcinoma. Each primary 5, and 8). A procedural modification was made in an attempt tumor was classified by the criteria of the Federation Interna to determine the PR StrUCtUreinthese samples. Increasing the tional ofGynecologists and Obstetricians according to whether protein load by 50% did not result in the detection of the it was well (Grade I), moderately (Grade II), or poorly (Grade triplet/singlet structure for these protein-blot-negative tumors III) differentiated. The PR concentration tended to be higher (not shown). An explanation was sought for this discordance. in Grade I (465 to 4770 fmol/mg protein, n = 10) than in We have previously demonstrated in the EnCa 101 tumor Grade II (0 to 1095 frnol/mg protein, n = 11), or Grade III (0 that both the Mr 116,000 and 81,000 PR proteins could be to 2735 fmol/rng protein, n = 12) tumors (Table 1). specifically photoaffinity labeled with [3HJR5020 (2). Analysis PR structure was next investigated in this series of primary by SDS-PAGE ofa photoaffinity-labeled primary tumor cytosol tumors with differing grades of differentiation. Preliminary (protein blot negative) confirmed the absence ofthe Mr 116,000 studies indicated that the use of a panel of the monoclonal and 81,000 PR proteins in this sample (not shown). Several antibodies (hPRa 1+2+3+6) increased the sensitivity of the specifically photolabeled low-molecular-weight proteins (Mr protein blot assay to detect PR structure by 2-fold, from 200 to <50,000) were observed, however, which suggested that proteo 100 fmol/mg cytosol protein. hPRa 1 and 3 recognize both Mr lytic activity in the primary tumors could be responsible for this 116,000 and 81,000 PR proteins, while hPRa 2 and 6 recognize discrepancy between binding and structural analysis. predominantly the Mr I 16,000 protein (1). When protein blots To investigate the presence of protease activity, tumor pow were probed with this mixture, the triplet/singlet structure seen ders from specimens with sufficient sample remaining were previously with EnCa 101 samples (1) was also observed in divided, and cytosols were prepared in PEMTG buffer without

Table 1 Progesteronereceptorconcentrationandproteinblotanalysisofhumanendometrialcarcinomas All tumors wereobtained from postmenopausalpatients exceptNos. 4, 9, 11, 20, 22, and 23 whichwereobtainedfrom pre- or perimenopausalpatients. inhibitorsPRAssayedwithout proteaseinhibitorsAssayed with protean Myometrial blotTumor(fmol/mgconcentrationProtein blotbPR concentrationK,,Protein invasionc protein)(nM)(triplet/singlet)Gradeprotein)Kd (nM)(triplet/singlet)(fmol/mg (%) I123352.5+100247706.1+<30314101.5+15853.1+33420702.7+18803.9+40S625(465r3.4(1.3)â€”(â€”.)4902.7+90626102.6â€”10716552.9.1.25815352.1+12052.9+<20921453.3+28153.4+0109001.3+9153.2+40Grade

IIII3101.1+<30l2a985NDâ€•+60l3a1095ND+65140â€”95154351.2+9516675(510)1.0(0.9)â€”(â€”)6450.7+40175002.5+<25188751.7+<20190â€”0â€”100204256.5â€”3653.2+0219151.4â€”13002.0+60Grade

III220â€”90230â€”90240â€”30250â€”0â€”7526415(275)3.4(3.5)â€”(â€”)2853.1+902727352.4+90280â€”0â€”90299601.9+85301103.4â€”1053.2+85311201.5+1203.6+33328050.9+8652.3+<25332651.143652.2+0

a PR concentration (fmol/mg protein) and the equilibrium dissociation constant (K, nM) were obtained from Scatchard analysis except for Samples 12 and 13 which were obtained by single-point analysis due to limited sample size. Three samples (Nos. 5, 16, and 26) were assayedrepeatedly with the values shown in parentheses. a Protein blot analysis was usedto determine the presence(+) or absence(â€”)ofthe triplet/singlet PR structure. C The percentage of myometrial invasion was obtained from pathology reports. I ND, not determined. 1144

EnCa101 GRADE I GRADE U GRADE ID

Fig. 2. Protein blot analysis of progesterone receptor structure in human endometrial tumor samples prepared in the presence or absenceof protean EnCa inhibitors. Tumor cytosols were prepared in PEMTG buffer in the absence(â€”) 101 GRADE I GRADE II GRADE III or presence(+) of the protean inhibitorcocktail,and the proteins(200 @g)were separated by 6.5% SDS-PAGE, transferred to nitrocellulose, probed with a Fig. 1. Protein blot analysis of progesterone receptor structure in primary mixture of monoclonalantibodies(hPRa 1+2+3+6) in Buffer A, and washed humanendometrialcarcinoma.PR concentration(fmol/mgcytosolprotein)was with Buffer A, and the immunoreactive bands were revealed with 4-chloro-l- determinedin eachtumor cytosolby Scatchardanalysis.For structuralanalysis, naphtholasdescribedinâ€œMaterialsandMethods.â€•Thesamplesarereferredto thecytoplasmicproteins(200@g)were separatedby 6.5% SDS-PAGE, transferred by their tumor number shown in Table I, with EnCa 101 (50 @sg)shownfor to nitrocellulose, probed with a mixture of monoclonal antibodies (hPRa reference(EnCa101):Grade I tumors,Nos. 3 (LaneA), 5 (Lane B), 10 (Lane C); 1+2+3+6) in Buffer A:l M NaCl, and washedwith Buffer A:l M NaCI, and the Grade II tumors, Nos. 16 (Lane D), 19 (Lane E), 20 (Lane I); and Grade III immunoreactivebandswere revealedwith 4-chloro-I-naphthol as describedin tumors, Nos. 26 (Lane 6), 30 (Lane II), 31 (Lane I), 33 (Lane J). Molecular â€œMaterialsandMethods.â€•Thesamples are referred to by their tumor number weight(MW@standards(Bio-Rad)are shownon theleft (DF, dyefront). shown in Table 1, with EnCa 101 (200 @g)shownfor reference (EnCa 101): Grade I tumors, Nos. 4 (Lane 1), 5 (Lane 2), 7 (Lane 3), 6 (Lane 4); Grade II tumors,Nos. 16 (Lane 5), 17 (Lane 6), 18 (Lane 7); and Grade III tumors,Nos. 26 (Lane 8) and 29 (Lane 9). Molecular weight(MW) standards(Bio-Rad) are shownon the left (DF, dye front). The light bandsin the EnCa 101 sampleare MW prestained standards (Diversified Biotech, Newton Centre, MA) used as lane markers. 116K-@'@ or with a cocktail of protease inhibitors. The protease inhibitor p. cocktail consisted of PMSF, leupeptin, aprotinin, pepstatin A, bacitracin, and benzamidine. The PR binding site concentration 81K-@- was not significantly different in any ofthe 17 samples prepared in the presence or absence of the protease cocktail (Table 1). I â€¢ Except for the PR-negative tumors, all samples tested exhibited enhancement of PR structure following preparation in the @ presence of the inhibitors, as judged by protein blot analysis @*â€˜ (Fig. 2). This was most strikingly evident in six tumor samples (Fig. 2, Lanes B, D, F, G, H, and J) which, although PR positive by binding analysis, were previously negative by protein blot analysis. Sample preparation in the presence of the inhibitors resulted in preservation of the triplet Mr 116,000 and 81,000 PR proteins. Low-molecular-weight proteins were also observed 1 2 3 4 5 6 7 8 9 10 in cytosols of several tumors prepared in the presence of inhib Fig. 3. Effect of various protease inhibitors on the protease activity present in itors. These proteins could be receptor-related breakdown prod a primary human endometrialcarcinoma.Cytosolsfrom the EnCa 101 tumor (200 gig) and a primary endometrial tumor (No. 5; 100 @@g)weremixed (16 h, ucts, since they were not observed with PR-negative tumors. 4C) with or without proteaseinhibitor(s).The proteinswere then separatedby Immunostaining of a Mr 200,000 protein(s) (Fig. 2) was non 6.5% SDS-PAGE, transferredto nitrocellulose,probedwith a mixtureof mono specific since addition of 1 M NaCl to buffer A during the clonal antibodies (hPRa 1+2+3+6) in Buffer A, and washedwith Buffer A, and the immunoreactive complexes were detected with 4-chloro-1-naphthol as de probing and washing steps eliminated this band (compare Figs. scribed in â€œMaterialsandMethods.â€•Sampleswere composed of the following: 1 and 2). EnCa101cytosol+ tumorcytosol+ proteaseinhibitorcocktail(Lane1);EnCa 101cytosol+ tumorcytosol(Lane2); tumorcytosol+ proteaseinhibitorcocktail Based on the present and previous studies, the EnCa 101 (Lane 3); EnCa 101 cytosol + proteaseinhibitor cocktail(Lane 4); and EnCa 101 tumor appeared to contain little or no protease activity to affect cytosol + tumor cytosol + protease inhibitor-benzamidine (Lane 5), bacitracin its PR structure. Therefore, EnCa 101 cytosolic PR was used (Lane 6), pepstatin A (Lane 7), aprotinin (Lane 8), leupeptin (Lane 9), or PMSF (Lane JU) at the final concentration given in TMMaterialsand Methods.â€•The as a substrate to test for proteolytic activity in the primary positionof the 2 PR proteinswith molecularweights(MW) of 116,000and tumors. Three separate mixing experiments with EnCa 101 81,000 is indicated on the left. cytosol and protease-positive primary tumor cytosol were car ned out. Protein blot analysis indicated that EnCa 101 PR was cocktail of protease inhibitors was used in these studies, the degraded by protease(s) present in the primary tumor (Fig. 3, inhibitory capacity of each individual reagent was tested sepa Lane 2). The proteolysis could be inhibited by incubation of the rately. Protein blot analysis indicated that leupeptin was the cytosols with the protease cocktail (Fig. 3, Lane 1). Since a most effective agent in the cocktail in preventing protease 1145 Downloaded from cancerres.aacrjournals.org on October 2, 2021. © 1988 American Association for Cancer Research. PR STRUCI1JRE IN HUMAN ENDOMETRIAL CARCINOMA activity (Fig. 3, Lane 9). Irnrnunostaining of low-molecular more, EnCa 101 does not possess an inhibitor of the protease weight proteins (Fig. 3) has previously been attributed to sec activity, as evidenced by the lack of effect on protease activity ondary antibody (goat anti-mouse) recognition of mouse serum present in primary tumors in mixing experiments. The EnCa components which contaminate EnCa 101 cytosols (1 1). 101 tumor therefore was useful in determining that the protease activity in the primary tumors was most sensitive to leupeptin. The present study has thus identified a subgroup of tumors DISCUSSION which has receptor characteristics common to endometrial tu mors, but which in addition possesses protease activity. Fur Protein blot analysis of 33 primary human endometrial car thermore, the protease activity in most, but not all, of these cinornas has revealed the presence of two PR proteins with primary tumors was associated with aggressive behavior that molecular weights of 116,000 and 81,000 in PR-positive tu included >30% invasion of the myometrium. While the role of mors; PR-negative specimens were devoid of these protein the protease(s) and the triplet/singlet structure in biological bands. Thus, there was excellent agreement between receptor activity and metastatic potential (21) remain to be determined, binding and PR structure in this series of tumors. All PR it is clear that any studies aimed at the structural analysis of positive samples prepared in the presence of the protease inhib PR must include protease inhibitors to prevent receptor deg itors, regardless of tumor grade, displayed the characteristic radation. The availability of monoclonal antibodies (1, 22â€”25) triplet isoform of the M, 116,000 protein and a single band for to PR provides a new dimension to the study of hormone the M, 81,000 protein. Therefore, this band pattern is not action, since they will enable us to further our understanding associated with a particular stage of tumor differentiation. We ofhow PR modulates progestin action in normal and neoplastic have previously observed this triplet/singlet band pattern for endometrium. the progesterone receptor in the EnCa 101 tumor (1). That this triplet/singlet structure is not unique to PR in the nude mouse grown EnCa 101 tumor has thus been demonstrated by the ACKNOWLEDGMENTS current study. Since most of the samples in this study were The authors would like to thank Jeri Miller, Shane Miller, and obtained from postrnenopausal women, the triplet/singlet struc Arunthathy Sivarajah for their excellent technical assistance and Bar ture is obviously present in the absence of progesterone, as bars Hynum for typing this manuscript. noted earlier for the EnCa 101 tumor which is grown in castrated nude mice exposed to estrogen (2). Interestingly, a similar triplet/singlet PR structure is also consistently observed REFERENCES in samples of primary human breast cancer and in normal 1. Clarke, C. L, Zaino, R. J., Feil, P. D., Miller, J. V., Steck,M. E., Ohlsson proliferative phase endometrium.5 Horwitz and coworkers first Wilhelm, B. M., and Satyaswaroop,P.G. 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Distribution of proges cocktail of protease inhibitors during sample homogenization terone receptor, estradiol dehydrogenase,and 20a-dihydroprogesterone de prevented generation of receptor fragments which were unrec hydrogenaseactivities in human endometrial glands and stroma progestin inductionof steroiddehydrogenaseactivitiesin vitro is restrictedto the ognized by the antibody. Endogenous proteases which affect glandular epithelium. Endocrinology, 111: 743â€”749,1982. ER (15â€”17, 19) and PR (15, 17â€”19)characteristics, such as 8. Laemmli, U. K. Cleavageof structuralproteinsduringthe assemblyofthe isoelectric point (16) or sedimentation properties on sucrose headofbacteriophage T4. Nature (Lond.), 227: 680-685, 1970. 9. Towbin,H., Staehelin,T., andGordon,J. Electrophoretictransferofproteins gradients (15, 17, 18), have also been described by other inves from polyacrylamidegelsto nitrocellulosesheets.Proc. Natl. Acad. Sd. tigators. 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1147 Downloaded from cancerres.aacrjournals.org on October 2, 2021. © 1988 American Association for Cancer Research. Progesterone Receptor Structure and Protease Activity in Primary Human Endometrial Carcinoma

Peter D. Feil, Christine L. Clarke and Pondichery G. Satyaswaroop

Cancer Res 1988;48:1143-1147.

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