(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 Cancer ResearchCenter. TheMilton S. HersheyMedical CenterofThe PennsylvaniaState University, Hershey, PA I 7033
ABSTRACF
Monoclonal antibodies were used to investigate progesterone receptor
structure (isoforms) in 33 primary human endometrial tumors. The
monoclonal antibodies recognized on protein blots two progesterone
receptor proteins with molecular weights of 116,000 and 81,000. TheM,
116,000 protein appearedas a triplet, while a single band was found for
the M, 81,000 protein. The triplet/singlet structure was found in all
progesterone receptor-positive tumors, regardless of the degree of tumor
differentiation. Protease activity, which gave rise to a false-negative
pattern on protein blots, was found in approximately
one-half of the
tumors in which It was investigated. Inclusion of a cocktail of protease
Inhibitors
during sample preparation resulted in the maintenance of the
triplet/singlet progesteronereceptor structure. Mixing experiments using
a progesterone receptor-rich human endometrial carcinoma (EnCa 101),
which lacks protease activity, and protease-containing primary tumor
eliminated the triplet/singlet structure seen on protein blots.
Inclusion of a cocktail of protease inhibitors during tumor
homogenization
prevented
this loss of structure.
Examination
of 33 primary tumors revealed that, when sample preparation
was performed in the presence of the protease inhibitors, the
triplet/singlet structure was present in all PR-positive samples,
regardless of tumor grade.
MATERIALS
AND METhODS
Tissue Source and Sample Preparation. Primary tumor samples were
obtained from patients who were undergoing prescribed surgery for
endometrial carcinoma. The tumors were transported to the laboratory
in cold Ham's F-10 medium within 30 mm ofexcision, frozen in liquid
in endometrial tumors.
nitrogen, and pulverized,and the powders were stored in liquid nitrogen
prior to assay. EnCa 101 tumors weregrown in BALB/c -nu/nu athymic
mice in the presence of 17@-estradiolpellets as described (2). Cytosol
was prepared by homogenizing (Teflon/glass) the tissue powder in four
volumes of ice-cold PEMTG buffer, followed by centrifugation
(100,000 x g, 1 h). Where indicated, an aliquot of a stock solution
(lOx) of protease inhibitor(s) was added to the PEMTG buffer just
prior to homogenization. The final concentration of each protease
INTRODUCFION
inhibitor used in the buffer was: 0.5 mM PMSF (Sigma, St. Louis,
MO); 86 zM leupeptin (Calbiochem, La Jolla, CA); 77 @g/mlaprotinin
homogenatesindicated that the protease was leupeptin sensitive. Inter
estingly, while the proteolytic
activity reduced or eliminated the triplet/
singlet progesterone receptor structure seen on protein blot analysis, it
did not affect progesteronereceptor concentrationmeasured by Scatchard
analysis. Sample preparation in the presence of protease inhibitors is
therefore a requisite for structural analysis of the progesterone receptor
(Sigma); 1.4 @iM
pepstatin A (Sigma); 100 @ig/mlbacitracin (Sigma);
Monoclonal antibodies to steroid hormone receptors provide
a sensitive tool for characterizing and comparing receptor struc
ture in endometrial tumors. Such information on receptor
structure
should
complernent
steroid
binding
Assay of Progesterone Receptor. The PR binding site concentration
assays and irn
prove predictability of tumor response to endocrine therapy.
Our laboratory has recently developed seven rnonoclonal anti
bodies to the human progesterone receptor (1), using as the
source of antigen the PR4 from a well-differentiated human
endometrial carcinoma (EnCa 101) grown in nude mice in the
presence
of estrogen
(2). In the EnCa 101 tumor,
these mono
clonal antibodies recognize by protein blot analysis 2 PR pro
teins with molecular weights of 116,000 and 81,000. In addi
tion, molecular weight isoforms were observed for the 116,000
protein (triplet), while the Mr 81,000 protein appeared as a
single band. Since PR has been detected in well-, moderately,
and poorly differentiated tumors (3—5),we were prornpted to
ask whether this triplet/singlet structure (isoforms) was asso
ciated with any particular stage of tumor differentiation. Direct
assessment
of this question
was precluded
by the finding that
Received8/12/87; revised11/18/87; accepted12/2/87.
The costsof publicationof this article were defrayedin part by the payment
of page charges. This article must therefore be hereby marked advertisementin
accordancewith 18 U.S.C. Section 1734 solely to indicate this fact.
2 To
work
whom
3 Present
was
supported
requests
address:
for
Garvan
by
National
reprints
Cancer
should
Institute
of
be
Medical
Institute
Grant
P01-CA
St.
Vincent's
abbreviations
used
are:
PR,
progesterone
receptor,
ER,
estrogen
Arlington Heights, IL) in the presence of 1 @iM
cortisol.
The structural form of PR in these samples was determined by
probing protein blots with the anti-PR monoclonal antibodies (hPRa)
generated by us (1). The cytosol proteins (200 ag), obtained from
samples prepared in the absence or presence of the protease inhibitors,
were first separated by 6.5% SDS-PAGE
(8) and transferred
electro
phoretically (30 V, 16 to 18 h) to nitrocellulose paper according to
Towbin (9). All reactions were carried out at room temperature. After
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
h) with a mixture of monoclonal antibodies enriched from culture
supernatants by ammonium sulfate (50%)precipitation:
hPRa 1 (1:100)
+ hPRa 2 (1:400) + hPRa 3 (1:800) + hPRa 6 (1:400). Comparison
of
serial dilutions of each antibody was used to selecteach final dilution.
After probing, the nitrocellulose paper was washed (2 times, 30 mm)
with the buffer, reacted with horseradish peroxidase-linked goat anti
mouse IgG (heavy plus light chain specific; Bio-Rad, Richmond, CA),
and the immunoreactive bands revealed using the chromogen 4-chloro
1-naphthol (Bio-Rad) as previously described(1).
by mixing (16 h, 4C) EnCa 101 cytosol with cytosol ofprimary tumors
Hospi
without/with the protease inhibitor cocktail prior to structural analysis
tal, Darlinghurst, NSW 2010, Australia.
4 The
(6) using 2 to 20 nM[1,2,6,7-3Hjprogesterone(90Ci/mmol; Amersham,
Protease Activity and Inhibition. Tumor protease activity was assessed
40011.
addressed.
Research,
of each sample, prepared in the presence or absence of protease inhib
itors, was measured as described previously (7) by Scatchard analysis
immunostaining intensity on protein blots using EnCa 101 cytosol and
many tumors contained a protease activity which, although it
did not affect the binding site concentration, greatly reduced or
I This
and 25 mM benzamidine (Sigma). The same cytosol sample with or
without protease inhibitors was used for both Scatchard (6) and struc
tural analysis.
receptor,
EGTA, ethyleneglycolbis(ft-aminoethylether)-N,N,N',N'-tetraacetic acid; R5020,
by protein blot. The ability of an individual compound in the cocktail
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, polyacrylamidegel electrophoresishPRa, humanprogesteronereceptor
antibody;PEMTG buffer, 50 mti phosphate:l0 mr@isodiummolybdate:lOmr@s
EGTA:l2 mM thioglycerol:lO%glycerol(v/v), pH 7.1; PMSF, phenylmethylsul
fonyl fluoride.
mm, 4'C) with an aliquot of EnCa 101 cytosol (200 @gprotein),
followedby addition ofprimary tumor cytosol (100 @ig
protein) to each
tube. Following incubation (16 h, 4C), sample buffer [187 mM Tris
1143
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PR STRUCFURE
IN HUMAN
HC1(pH 6.8):5% 2-mercaptoethanol:3% SDS:lO% glycerolJwas added
to all samples(1:2, v/v) which were then heated(90'C, 2 mm) prior to
ENDOMETRIAL
CARCINOMA
several of the primary tumors, regardless of tumor grade (Fig.
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
RESULTS
Lanes 1 versus 3 and 7 versus 9). In addition, little or no
immunoreaction was detected in several tumors known to be
tween samples with similar PR concentrations
PR concentration and structure (isoforms) were appraised in
33 samples of human endornetrial carcinoma. Each primary
tumor was classified by the criteria of the Federation Interna
tional ofGynecologists and Obstetricians according to whether
it was well (Grade I), moderately (Grade II), or poorly (Grade
III) differentiated. The PR concentration tended to be higher
in Grade I (465 to 4770 fmol/mg protein, n = 10) than in
Grade II (0 to 1095 frnol/mg protein, n = 11), or Grade III (0
to 2735 fmol/rng protein, n = 12) tumors (Table 1).
PR structure was next investigated
in this series of primary
tumors with differing grades of differentiation. Preliminary
studies indicated that the use of a panel of the monoclonal
antibodies (hPRa 1+2+3+6) increased the sensitivity of the
protein blot assay to detect PR structure by 2-fold, from 200 to
100 fmol/mg cytosol protein. hPRa 1 and 3 recognize both Mr
1 16,000 and 81,000 PR proteins, while hPRa 2 and 6 recognize
predominantly the Mr I 16,000 protein (1). When protein blots
were probed with this mixture,
the triplet/singlet
structure
seen
previously with EnCa 101 samples (1) was also observed in
(Fig. 1; compare
PR positive by Scatchard analysis (Table 1; Fig. 1, Lanes 2, 4,
5, and 8). A procedural modification was made in an attempt
to determine the PR StrUCtUrein these samples. Increasing the
protein load by 50% did not result in the detection of the
triplet/singlet structure for these protein-blot-negative tumors
(not shown). An explanation was sought for this discordance.
We have previously demonstrated in the EnCa 101 tumor
that both the Mr 116,000 and 81,000 PR proteins could be
specifically photoaffinity labeled with [3HJR5020 (2). Analysis
by SDS-PAGE ofa photoaffinity-labeled primary tumor cytosol
(protein blot negative) confirmed the absence ofthe Mr 116,000
and 81,000 PR proteins in this sample (not shown). Several
specifically photolabeled low-molecular-weight proteins (Mr
<50,000) were observed, however, which suggested that proteo
lytic activity in the primary tumors could be responsible
for this
discrepancy between binding and structural analysis.
To investigate the presence of protease activity, tumor pow
ders from specimens with sufficient sample remaining were
divided, and cytosols were prepared in PEMTG buffer without
Table 1 Progesterone
receptorconcentrationandproteinblotanalysisofhuman endometrialcarcinomas
All tumors wereobtained from postmenopausalpatients except Nos. 4, 9, 11, 20, 22, and 23 whichwereobtained from pre- or perimenopausalpatients.
inhibitorsPR
Assayedwithout proteaseinhibitorsAssayed
with protean
Myometrial
blotTumor(fmol/mgconcentrationProtein
protein)(nM)(triplet/singlet)Grade
protein)Kd
concentrationK,,Protein
blotbPR
invasionc
(%)
(nM)(triplet/singlet)(fmol/mg
I123352.5+100247706.1+<30314101.5+15853.1+33420702.7+18803.9+40S625(465r3.4(1.3)—(—.)4902.7+90626102.6—10716552.9.1.25815352.1+12052.9+<20921
IIII3101.1+<30l2a985ND―+60l3a1095ND+65140—95154351.2+9516675(510)1.0(0.9)—(—)6450.7+40175002.5+<25188751.7+<20190—0—100204256.5—3653
III220—90230—90240—30250—0—7526415(275)3.4(3.5)—(—)2853.1+902727352.4+90280—0—90299601.9+85301103.4—1053.2+85311201.5+1203.6
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(—)of the triplet/singlet PR structure.
C The
I ND,
percentage
of
myometrial
invasion
was
obtained
from
pathology
reports.
not determined.
1144
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PR STRUCFURE
IN HUMAN
ENDOMETRIAL
CARCINOMA
EnCa
101
EnCa
101
GRADE I
GRADE II
GRADE III
Fig. 1. Protein blot analysis of progesterone receptor structure in primary
humanendometrialcarcinoma.PR concentration(fmol/mg cytosolprotein)was
determinedin eachtumor cytosolby Scatchardanalysis.For structuralanalysis,
thecytoplasmicproteins(200@g)were separatedby 6.5% SDS-PAGE, transferred
to nitrocellulose, probed with a mixture of monoclonal antibodies (hPRa
1+2+3+6) in Buffer A:l M NaCl, and washedwith Buffer A:l M NaCI, and the
immunoreactivebandswere revealedwith 4-chloro-I-naphthol as describedin
“Materials
and Methods.―The samples are referred to by their tumor number
shown in Table 1, with EnCa 101 (200 @g)
shown for 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
prestained standards (Diversified Biotech, Newton Centre, MA) used as lane
markers.
@
GRADE U
GRADE ID
Fig. 2. Protein blot analysis of progesterone receptor structure in human
endometrial tumor samples prepared in the presence or absence of protean
inhibitors. Tumor cytosols were prepared in PEMTG buffer in the absence(—)
or presence(+) of the protean inhibitorcocktail,and the proteins(200 @g)
were
separated by 6.5% SDS-PAGE, transferred to nitrocellulose, probed with a
mixture of monoclonalantibodies(hPRa 1+2+3+6) in Buffer A, and washed
with Buffer A, and the immunoreactive bands were revealed with 4-chloro-lnaphtholas describedin “Materials
and Methods.―
The samplesare referredto
by their tumor number shown in Table I, with EnCa 101 (50 @sg)
shown for
reference(EnCa 101):Grade I tumors,Nos. 3 (LaneA), 5 (Lane B), 10 (Lane C);
Grade II tumors, Nos. 16 (Lane D), 19 (Lane E), 20 (Lane I); and Grade III
tumors, Nos. 26 (Lane 6), 30 (Lane II), 31 (Lane I), 33 (Lane J). Molecular
weight(MW@standards(Bio-Rad) are shownon the left (DF, dye front).
MW
116K-@'@
p.
or with a cocktail of protease inhibitors. The protease inhibitor
cocktail consisted of PMSF, leupeptin, aprotinin, pepstatin A,
bacitracin, and benzamidine. The PR binding site concentration
was not significantly different in any ofthe 17 samples prepared
in the presence or absence of the protease cocktail (Table 1).
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
(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
GRADE I
81K-@-
I
•
@*‘
1 2 3 4 5
proteins were also observed
6 7
8 9 10
in cytosols of several tumors prepared in the presence of inhib
itors. These proteins could be receptor-related breakdown prod
ucts, since they were not observed with PR-negative tumors.
Immunostaining of a Mr 200,000 protein(s) (Fig. 2) was non
specific since addition of 1 M NaCl to buffer A during the
probing and washing steps eliminated this band (compare Figs.
Fig. 3. Effect of various protease inhibitors on the protease activity present in
a primary human endometrialcarcinoma.Cytosolsfrom the EnCa 101 tumor
(200 gig) and a primary endometrial tumor (No. 5; 100 @@g)
were mixed (16 h,
4C) with or without proteaseinhibitor(s).The proteinswere then separatedby
6.5% SDS-PAGE, transferredto nitrocellulose,probedwith a mixture of mono
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
scribed in “Materials
and Methods.―
Samples were composed of the following:
1 and 2).
EnCa101cytosol+ tumorcytosol+ proteaseinhibitorcocktail(Lane1); EnCa
Based on the present and previous studies, the EnCa 101
tumor appeared to contain little or no protease activity to affect
its PR structure. Therefore, EnCa 101 cytosolic PR was used
as a substrate to test for proteolytic activity in the primary
tumors. Three separate mixing experiments with EnCa 101
cytosol and protease-positive primary tumor cytosol were car
ned out. Protein blot analysis indicated that EnCa 101 PR was
degraded by protease(s) present in the primary tumor (Fig. 3,
Lane 2). The proteolysis could be inhibited by incubation of the
cytosols with the protease cocktail (Fig. 3, Lane 1). Since a
101cytosol+ tumorcytosol(Lane2); tumorcytosol+ proteaseinhibitorcocktail
(Lane 3); EnCa 101 cytosol + proteaseinhibitor cocktail(Lane 4); and EnCa 101
cytosol + tumor cytosol + protease inhibitor-benzamidine (Lane 5), bacitracin
(Lane 6), pepstatin A (Lane 7), aprotinin (Lane 8), leupeptin (Lane 9), or PMSF
(Lane JU) at the final concentration given in TMMaterialsand Methods.―The
positionof the 2 PR proteinswith molecularweights(MW) of 116,000and
81,000 is indicated on the left.
cocktail of protease inhibitors was used in these studies, the
inhibitory capacity of each individual reagent was tested sepa
rately. Protein blot analysis indicated that leupeptin was the
most effective agent in the cocktail in preventing protease
1145
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PR STRUCI1JRE
IN HUMAN
activity (Fig. 3, Lane 9). Irnrnunostaining of low-molecular
weight proteins (Fig. 3) has previously been attributed to sec
ondary antibody (goat anti-mouse) recognition of mouse serum
components which contaminate EnCa 101 cytosols (1 1).
DISCUSSION
Protein blot analysis of 33 primary human endometrial car
cinornas has revealed the presence of two PR proteins with
molecular weights of 116,000 and 81,000 in PR-positive tu
mors; PR-negative specimens were devoid of these protein
bands. Thus, there was excellent agreement between receptor
binding and PR structure in this series of tumors. All PR
positive samples prepared
in the presence of the protease
inhib
itors, regardless of tumor grade, displayed the characteristic
triplet isoform of the M, 116,000 protein and a single band for
the M, 81,000 protein. Therefore, this band pattern is not
associated with a particular stage of tumor differentiation. We
have previously observed this triplet/singlet band pattern for
the progesterone receptor in the EnCa 101 tumor (1). That this
triplet/singlet
structure
of primary
more, EnCa 101 does not possess an inhibitor of the protease
activity, as evidenced by the lack of effect on protease activity
present in primary tumors in mixing experiments. The EnCa
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
which has receptor characteristics common to endometrial tu
mors, but which in addition possesses protease activity. Fur
thermore, the protease activity in most, but not all, of these
primary
was associated
with aggressive
behavior
that
PR must include protease inhibitors to prevent receptor deg
radation. The availability of monoclonal antibodies (1, 22—25)
to PR provides a new dimension to the study of hormone
action, since they will enable us to further our understanding
ofhow PR modulates progestin action in normal and neoplastic
endometrium.
human
breast
cancer
ACKNOWLEDGMENTS
The authors would like to thank Jeri Miller, Shane Miller, and
Arunthathy Sivarajah for their excellent technical assistance and Bar
bars Hynum for typing this manuscript.
REFERENCES
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That several known PR-positive tumors were protein blot
negative was intriguing. In these specimens, inclusion of a
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prevented generation of receptor fragments which were unrec
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S Unpublished
tumors
included >30% invasion of the myometrium. While the role of
the protease(s) and the triplet/singlet structure in biological
activity and metastatic potential (21) remain to be determined,
it is clear that any studies aimed at the structural analysis of
proliferative phase endometrium.5 Horwitz and coworkers first
described an upshift in the molecular weights of the 2 PR
proteins in T47D human breast cancer cells cultured in the
presence of progestin (12). A similar upshift in the Mr 110,000
gradients
CARCINOMA
is not unique to PR in the nude mouse
grown EnCa 101 tumor has thus been demonstrated by the
current study. Since most of the samples in this study were
obtained from postrnenopausal women, the triplet/singlet struc
ture is obviously present in the absence of progesterone, as
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
in samples
ENDOMETRIAL
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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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