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Modulation of protein expression in endometrial adenocarcinoma cells by in vitro exposure to estradiol and progesterone
Modulation of protein expression in endometrial adenocarcinoma cells by in vitro exposure to estradiol and progesterone T. U’.
J. O’BRIEN HERNANDEZ
I’. J. JERNSTROM 1). A. SEYMOUR (:.
I’.
MORROW
J.
A.
SYKES
Los .4 ngy1e.c. California Protein maps of in vitro hormone-modulated adenocarcinoma cells from an endometrial tumor are described. It is noted that mapping of tumor tissues by means of the two-dimensional analysis described by O’Farrell and associate9 of 35S-methionine-labeled proteins can provide a characteristic map for each tumor and that the methionine-containing proteins of tumor cells can be independently modulated by the in vitro additions of estradiol and progesterone. Such protein modulation could be indicative of hormonal therapeutic responsiveness of individual endometrial adenocarcinomas and other hormone receptor-positive tumors. Tumor maps may further provide data for the identification and isolation of new tumor markers which might be correlated with the radiotherapeutic and chemotherapeutic sensitivity of the malignancy. (AM. J. OBSTET. GYNECOL. 139:67, 1981.)
EVA I. u A TI ON of tumor responsiveness to endocrine manipulation has been carried out with some success by hormone receptor analysis. Patients with receptorpositive breast tumors have been shown to have a better prognosis than those with tumors which are borderline or negative. ‘-’ Evidence for responsiveness of endometrial adenocarcinoma to progestin therapy has also been documented” and suggests that receptor-positive tumors are responsive to progestin therapy. Receptor positivity. therefore, would appear to be a basic reFrom the Department of Obstetrics and Gynecology, Univrrsit~ of Southern California School of Medicine, and the Southern Calijonia Cancer Center, California Hospital Medical Center. Supported by Robert E. und May R. Wright Foundation Grant No. 55 and National Institutes of Health Grant No. 2RlK4 20749-04. Receir~ed for publication Accepted August
May
21, 1980.
26, 1980.
Reprint requrcts: Dr. 7‘. J. O’Brien, Room L903, 1240 N. M&ion Rood, California 90033. 0002~9378/X1/010067+06$00.60/0~
1981The
Women’s Hospital, Los Angeles,
C. V.Mosby
Co.
quirement for hormone responsiveness of a target organ or tissue; however, receptor positivity may not always result in responsiveness due to the possibility of defective binding of the hormone or defective mechanisms in processing the hormone receptor complex. It is, therefore, appropriate that estrogen or progestin efficacy be evaluated by analyzing the direct responsiveness of tumors or tissues to individual hormones. The present approach has been to develop “tumol maps” of %-labeled proteins from the tumor cells separated by the technique of O’Farrell and associates’ of two-dimensional analysis after an appropriate in vitro exposure to hormone and ‘%-methionine. Such maps provide a characteristic display of proteitls for each tumor, indicating its metabolic and histologic make-up. It is reasonable to assume that the presence or absence of individual proteins could predispose the tumor to therapeutic responsiveness or resistance. Protein markers which might indicate such sensitivity or resistance would have significant value in the selection of therapy for individual cases. The present study provides evidence that cells from a hormone receptor67
68
O’Brien
et al.
Fig. 1. ~~‘ell-diffe~entirt~d lin and eositl. X 100.)
Fig. 2. Solid inflammatol-v
area bill\.
adenoc-ar~i!loma
of poorly dift‘erentiated (Hematosyliu and eosin.
ot the endotnett~ium
adenocarcinoma X-400.)
with
with
papillat-v
microcysts
f~atutc\.
and
I flcttiatc)\\
inhltr;~tiotl
h\
Protein
Fig. 3. Solid area of carcinoma with numerous
lipid-laden
expression
histioqtes.
in endometrial
(Hematoxylin
adenocarcinoma
69
and eosin.
x -lOO.)
central dosage of 3.000 mg hours. Three weeks later a total abdominal hyscerectom\ with bilateral salpingooophorec tom! 11as carried out. The patient had an uneventful recovery and was placed on a regimen of megestrol acetate. 40 mg twice daily. The patient has been free of‘ disease for the past 9 months. ,1t operation a portion of the tumor was taken for Iiistologic examination and part was saved to1 Ilc)I.lnone-hintIjng studies. The pathologist reported that the rissue \zas grossly the equivalent of I ml ot fornl~~liIl-l,atl~e~~, matlogan~ red. soft. irregular endometrial c klrcttirigs. ~Ii~I-osc,opic~ill~, sections showed sparse amounts of malignant endometrial glandular elements in fragmented papillarv and solid form. The former was well diffcwntiated akl the latter was poorly differentiated. Foci of’ papillar-v carcinoma had minimal nondescript sLrom;~ (I;ig. 1) in contrast Lo the solid areas (Figs. 9 and S) I\ llerriti microcystic spaces and numerous vacuaiated tell5 ac~conipanied inllamniatoi~y cells. Slost ot the fiwrller- had snlali. cenrral to slightly ec.cent!ic nuclei surI-oundetl b\ foam\. cytoplasm most consistent with lipidladc,n hi~tioc.! tes. ‘I‘he p.irt of the tumor specimen for hormonebinding stutlics and mapping ~vas collected directly inro ice-cold t)host’haLe-buff’ered saline. The specimen was slicccl into Z1mm cubes. and 100 mg (about one rhird) was transferred to storage buffer (50 m&l tromethamine, p!I 7:4, 1 mM dithiothreitol, and SO’% glycerol) and stored in liquid nitrogen for steroid receptor analysis. ‘l% rrmaining tissue W;IS transferred to 5 ml ot
methionine-deficient medium (iYeymouth’s medium containing 20c% fetal calf serum, 1 PM methionine, 50 w1Lgirnl of Kanamycin, 15 ICiml of penicillin, and 15 p~g/ml of streptomycin) and incubated at room temperature for 30 minutes in the presence of 0.1% Collagenase II (Worthington). The dispersed cells were harvested by centrifugation at HO0 x g for 10 minutes and resuspended in 2.0 ml of methionine-deficient medium. Alicluots (0.5 ml) were then placed in individual culture tubes containing 200 p(:i of‘ ““S-methioiline (approximately 800 (~i/mniole, I-\niershaln Corporation). Individual tubes were then classified as control (no additives except :i pl of 704 ;~lwtlol), esrradiol (.i ~1 of estl-adiol in 70% alcohol, final concenLration 10 -TM). progesterone (5 I*] of progrsLerone in 70% alcohol. tinal concentration IOF% progeswrone). and estradiol pl11s progesterone (5 pl of‘ a mixture of estradiol and progesteroLle in 70’1 alcohol, final concentration IO-“M of each steroid). .I’he culture tubes were incubated overnighr at 37” C on a rocker. Sulfur%-labeled cells were harvested 1~~.centrifLlgarion followed by renio~al of the ““S medium. The cell\ were resuspended and washed twice with 10 nil of’ complete M’eymouth’s medium. The cells were then washrd with 0.35N mannitol in clistilled water containing 0.0 IM tromethamine, pH 7.4. to remove salts. The cells were lysed in 50 ~1 of. buffer (!?.SM urea, 2%, NI’,O, 2% ampholytes, and 5% Irlel-captoethanol) by resuspension in plastic pipettes for .S minutes at 4” Ct. The ‘3 lysate \vas assayed ti)r radioactivitk I by separating aliquots of 2 ~1 into 0.j ml of 0. 1% bovine serum albumin and 0.5
70 O’Brien et al
Fig. 4B.
Two-dimensional
FOI- legend
analysis
SW
of “%
Fig.
4A
proteins.
.A IotaI
oi
“0 /iI of’ :=S lyxate of each sample was plw~i Ott :I .i’i; ac~rylamide disk gel containing I .!I%- amphoh tch, pH 3 to 10. ‘I‘he sample was overlqcd with IO ~1 of iysis buffer
dilured
I : 1 and
focused
at 400
V fi)l-
.G,!$ hour5.
lCI( sulfuric acid and I$? ethanolamine usul :I5 tht% c,iecti-ode solutions. Aiier- focuaiilg. the gels \\Cl-t’ c‘xtrudeci trorn the tubes and ~)lawd in sodiunl dodeu I sulfate f)uffer (0.0(i.SM tromethatnine. pH 6.X. L’.L’.i% sodium dodecyl sulfate. 3% lliei-~aptoerhanol. 2nd lO!p 2 horll-s with one change of glycerol) with shaking tar buff’er. ‘I‘he sodium dodecvl sul~‘a~e-ecluilil)l att’d gels were then placed on a polyacrylamide gI-adient slab gel ( lOc/, to IS%) and run at 20 mamp until the tl-;l&ng dye (hromophenol blue) entered the lower- buffer. ‘The gels were stained bvith Coomassir Blue (0. 1’; in 3% 11.ichloroacetic acid), destained (in 5% acetic acid). :tnd dried. All two-dimensional procedures w\?cre cascntiall\ those of O’Farrell and associates.” .4utoradiog1-;tms ivere ohtained by exposing Ihe gels lo Y-IX\ film I \;R2. with
Protein
expression
in endometrial
adenocarcinoma
71
t
Fig. 4C. For
Kodak) finveloped in tures were grams with Copv Film (irack 3 at
legend see Fig. 1A.
approximately 2 weeks. X-ray film was deautomatic developer and positive picmade hy photography of the autoradioa 35 mm camera and Kodak High Contrast followed by printing on Kodahromide the original sizr. Receptor analysis. The tissue was removed from licfuitl nitrogen storage (not longer than 1 week), thawed. and homogenized in 10 volumes of buffer (50 mM tromethnmine, pH 7.4, 5 mM dithiothreitol 1 mM ethylenetliaminetetra-acetic acid, and 10% glycerol) in 5-second bursts five times with a Tekmar Tissumizer. The homogenate was centrifuged at 2,000 x g for 20 minutes and the supernatant was recentrifuged for 1 hour at 100,000 x g. The cytosol was treated with dextran-coated charcoal for 2 minutes and recentrifuged at 2,000 x g for 10 minutes: the supernatant was diluted to 1 mg of protein per milliliter according to the Bradford’ assay, and 0.2 ml (0.2 mg of protein) ;III
Fig. 4D.
For
legend see Fig. 4A.
was assayed for estradiol and progesterone receptors in a 200 ~1 assay overnight at 3” C. Assays f’or progesterone receptor were carried out in 10. ?, and 0.5 nM “H-R5020 (New England Nuclear) with duplicate tubes containing l(K)-fold excess of unlabeled K5020. A single-point estradiol receptor assay was carried out at 10 nM “H-estradiol (tetra lahle, Amersham) with a duplicate tube containing lO&fotd excess of diethylstilhestrol. Unbound steroid was removed by dextran-coated charcoal (5 minutes at 4” C), and the supernatants of a centrifugation at 2,000 x g (10 minutes) were counted in 20 ml of Aquasol (New England Nuclear) on a Beckman scintillation counter.
Results Progesterone and estradiot receptor analysis of a portion of the tumor specimen showed a positive receptor population for both hormones. The progesterone receptor concentration, based on a three-point
72
O’Brien
analysis.
et al.
was
fourrtl
while
the estratliol
tein,
based
tlie
in
on
\.itro
to I~ave
177
receptor ;I single-poitlt
hoI-mane-niod~il;ttetl
in Fig.
4. (~hal-;tc~tei~isti~~iil~
other
oi~gaiiisms I” most
tion
the
and
ot’the
vitro
pH
.i ;tnd
5
maps
it i5 observed
the
c.rc.eptiotib
1R),
!\~hTlY
prrsent
In fx.1.
spots
tit\-.
‘I‘he
(FIK.
IWO
in;ited
IIlL\\.
3 and
other
the
spots
Nos.
5 ;iiid
01 ~lx!atl\ lxxillccd. tirap (Fig. 41)).
tliosc
iri the
control
or gi-eatI\-
the
noted,
all
OllC
cstr~idiol
01’
(Fig.
spots Nos.
were 3 and
wduc-rd
4
in quan-
progesterone
map
cmnpletel~
elim-
almost 6 wt’rc
ot
in all in
maps.
map
‘1\‘;16 in tile
go”’ tradiol
esamitl,~-
nlost
f’o111-
cnllallc-cd
I ws
On
orceptions
othcl~
beet1 points
co~nn~on
ol
as fill-
have
that art’
#watly
In Al1
tutnot.,
4.4).
01‘
No.
of iirc
isoelectric
use
alm2nt sf)ot
this
\
esccption
where
anti
with
1).
entirely
I(:),
(Fig.
a1.e (‘0111111011 to AI iioteci
(Nos.
eithei-
r:iilgc proteins
conditions. nl;i;ol~
m;rps
which
have
of al1 fot~i-
rtw
wc‘rc
for tissue
proteins
“:‘S-mcthioniric-labeled
of pro-
Protein
;Icl~no~;i1-cinom~i
tii;tnv
tnapp’C1,~-
of‘ proteirl
I l-l I inolesimg ;issay.
5ho~vn
betweet
f’t~~ole.s/tn~
w;is
either-
conipletel~
111 tl1e },rogeste~~olle tlie obvious spots
plus
es-
resetnldfxl
nlal).
Comment ‘l‘lie
searc.h
fi)r
sclec.1 ion of’ therap
ti~nioi. . and
iiiarkers tumor
useful monitoring
for
ciiagnohis. is a diwrse
REFERENCES hIcGuiw, 12’. I.., Horx
6.
7.
8. 9.
IO.
11.
Steroid Receptors and the ManLippman. M.. editors: agement of Cancer. Florida, 1979, CRC Pr-es. p. l9!i. O’Farrell. P. %., Goodman. I-1. hi., and O’Fa1rel1, P. H.: High resolution two-dirncnsional electrophorcsi\ of basit as well as acidic pwteins, Cell 12: 1133. 1977. Bradford. M.: A rapid ;mtl wnsitivc methcjd tar the cfuantitation of microgram quantities of ploteilr rttililing the principle of protein-dve binding. Anal. Hioc-hem. 72:248. 1976. O’Farrell, I’. H.: High rcsoiution two-rlirrlensiollal clectrophoresis of proteins. J. Biol. (:hem. 250:~~1)1)7, 11175. Alton. T. H., and Lodish. H. F.: Tr-anslational control of protein synthesis during the early stages ot’ dit‘f’erentiation of’ the slime mold dictvostelium discoideum. C:ell 12:301. 1977. O’Farrell, P. H.. and O‘Farrell. 1’. /..: -rMo-dilllellsii)llai pol!acx-ylamide gel electrophoretic frac-tionariw. hlethads Cell Biol. 16:407, 1977. Garrels, J. I.: Two-dimensional gel electrophorcsis and computer analysis of proteins synthesized 117 clonal cell lilies. J. Biol. Chem. 254:796l. 1979.
J. O’BRIEN HERNANDEZ
I’. J. JERNSTROM 1). A. SEYMOUR (:.
I’.
MORROW
J.
A.
SYKES
Los .4 ngy1e.c. California Protein maps of in vitro hormone-modulated adenocarcinoma cells from an endometrial tumor are described. It is noted that mapping of tumor tissues by means of the two-dimensional analysis described by O’Farrell and associate9 of 35S-methionine-labeled proteins can provide a characteristic map for each tumor and that the methionine-containing proteins of tumor cells can be independently modulated by the in vitro additions of estradiol and progesterone. Such protein modulation could be indicative of hormonal therapeutic responsiveness of individual endometrial adenocarcinomas and other hormone receptor-positive tumors. Tumor maps may further provide data for the identification and isolation of new tumor markers which might be correlated with the radiotherapeutic and chemotherapeutic sensitivity of the malignancy. (AM. J. OBSTET. GYNECOL. 139:67, 1981.)
EVA I. u A TI ON of tumor responsiveness to endocrine manipulation has been carried out with some success by hormone receptor analysis. Patients with receptorpositive breast tumors have been shown to have a better prognosis than those with tumors which are borderline or negative. ‘-’ Evidence for responsiveness of endometrial adenocarcinoma to progestin therapy has also been documented” and suggests that receptor-positive tumors are responsive to progestin therapy. Receptor positivity. therefore, would appear to be a basic reFrom the Department of Obstetrics and Gynecology, Univrrsit~ of Southern California School of Medicine, and the Southern Calijonia Cancer Center, California Hospital Medical Center. Supported by Robert E. und May R. Wright Foundation Grant No. 55 and National Institutes of Health Grant No. 2RlK4 20749-04. Receir~ed for publication Accepted August
May
21, 1980.
26, 1980.
Reprint requrcts: Dr. 7‘. J. O’Brien, Room L903, 1240 N. M&ion Rood, California 90033. 0002~9378/X1/010067+06$00.60/0~
1981The
Women’s Hospital, Los Angeles,
C. V.Mosby
Co.
quirement for hormone responsiveness of a target organ or tissue; however, receptor positivity may not always result in responsiveness due to the possibility of defective binding of the hormone or defective mechanisms in processing the hormone receptor complex. It is, therefore, appropriate that estrogen or progestin efficacy be evaluated by analyzing the direct responsiveness of tumors or tissues to individual hormones. The present approach has been to develop “tumol maps” of %-labeled proteins from the tumor cells separated by the technique of O’Farrell and associates’ of two-dimensional analysis after an appropriate in vitro exposure to hormone and ‘%-methionine. Such maps provide a characteristic display of proteitls for each tumor, indicating its metabolic and histologic make-up. It is reasonable to assume that the presence or absence of individual proteins could predispose the tumor to therapeutic responsiveness or resistance. Protein markers which might indicate such sensitivity or resistance would have significant value in the selection of therapy for individual cases. The present study provides evidence that cells from a hormone receptor67
68
O’Brien
et al.
Fig. 1. ~~‘ell-diffe~entirt~d lin and eositl. X 100.)
Fig. 2. Solid inflammatol-v
area bill\.
adenoc-ar~i!loma
of poorly dift‘erentiated (Hematosyliu and eosin.
ot the endotnett~ium
adenocarcinoma X-400.)
with
with
papillat-v
microcysts
f~atutc\.
and
I flcttiatc)\\
inhltr;~tiotl
h\
Protein
Fig. 3. Solid area of carcinoma with numerous
lipid-laden
expression
histioqtes.
in endometrial
(Hematoxylin
adenocarcinoma
69
and eosin.
x -lOO.)
central dosage of 3.000 mg hours. Three weeks later a total abdominal hyscerectom\ with bilateral salpingooophorec tom! 11as carried out. The patient had an uneventful recovery and was placed on a regimen of megestrol acetate. 40 mg twice daily. The patient has been free of‘ disease for the past 9 months. ,1t operation a portion of the tumor was taken for Iiistologic examination and part was saved to1 Ilc)I.lnone-hintIjng studies. The pathologist reported that the rissue \zas grossly the equivalent of I ml ot fornl~~liIl-l,atl~e~~, matlogan~ red. soft. irregular endometrial c klrcttirigs. ~Ii~I-osc,opic~ill~, sections showed sparse amounts of malignant endometrial glandular elements in fragmented papillarv and solid form. The former was well diffcwntiated akl the latter was poorly differentiated. Foci of’ papillar-v carcinoma had minimal nondescript sLrom;~ (I;ig. 1) in contrast Lo the solid areas (Figs. 9 and S) I\ llerriti microcystic spaces and numerous vacuaiated tell5 ac~conipanied inllamniatoi~y cells. Slost ot the fiwrller- had snlali. cenrral to slightly ec.cent!ic nuclei surI-oundetl b\ foam\. cytoplasm most consistent with lipidladc,n hi~tioc.! tes. ‘I‘he p.irt of the tumor specimen for hormonebinding stutlics and mapping ~vas collected directly inro ice-cold t)host’haLe-buff’ered saline. The specimen was slicccl into Z1mm cubes. and 100 mg (about one rhird) was transferred to storage buffer (50 m&l tromethamine, p!I 7:4, 1 mM dithiothreitol, and SO’% glycerol) and stored in liquid nitrogen for steroid receptor analysis. ‘l% rrmaining tissue W;IS transferred to 5 ml ot
methionine-deficient medium (iYeymouth’s medium containing 20c% fetal calf serum, 1 PM methionine, 50 w1Lgirnl of Kanamycin, 15 ICiml of penicillin, and 15 p~g/ml of streptomycin) and incubated at room temperature for 30 minutes in the presence of 0.1% Collagenase II (Worthington). The dispersed cells were harvested by centrifugation at HO0 x g for 10 minutes and resuspended in 2.0 ml of methionine-deficient medium. Alicluots (0.5 ml) were then placed in individual culture tubes containing 200 p(:i of‘ ““S-methioiline (approximately 800 (~i/mniole, I-\niershaln Corporation). Individual tubes were then classified as control (no additives except :i pl of 704 ;~lwtlol), esrradiol (.i ~1 of estl-adiol in 70% alcohol, final concenLration 10 -TM). progesterone (5 I*] of progrsLerone in 70% alcohol. tinal concentration IOF% progeswrone). and estradiol pl11s progesterone (5 pl of‘ a mixture of estradiol and progesteroLle in 70’1 alcohol, final concentration IO-“M of each steroid). .I’he culture tubes were incubated overnighr at 37” C on a rocker. Sulfur%-labeled cells were harvested 1~~.centrifLlgarion followed by renio~al of the ““S medium. The cell\ were resuspended and washed twice with 10 nil of’ complete M’eymouth’s medium. The cells were then washrd with 0.35N mannitol in clistilled water containing 0.0 IM tromethamine, pH 7.4. to remove salts. The cells were lysed in 50 ~1 of. buffer (!?.SM urea, 2%, NI’,O, 2% ampholytes, and 5% Irlel-captoethanol) by resuspension in plastic pipettes for .S minutes at 4” Ct. The ‘3 lysate \vas assayed ti)r radioactivitk I by separating aliquots of 2 ~1 into 0.j ml of 0. 1% bovine serum albumin and 0.5
70 O’Brien et al
Fig. 4B.
Two-dimensional
FOI- legend
analysis
SW
of “%
Fig.
4A
proteins.
.A IotaI
oi
“0 /iI of’ :=S lyxate of each sample was plw~i Ott :I .i’i; ac~rylamide disk gel containing I .!I%- amphoh tch, pH 3 to 10. ‘I‘he sample was overlqcd with IO ~1 of iysis buffer
dilured
I : 1 and
focused
at 400
V fi)l-
.G,!$ hour5.
lCI( sulfuric acid and I$? ethanolamine usul :I5 tht% c,iecti-ode solutions. Aiier- focuaiilg. the gels \\Cl-t’ c‘xtrudeci trorn the tubes and ~)lawd in sodiunl dodeu I sulfate f)uffer (0.0(i.SM tromethatnine. pH 6.X. L’.L’.i% sodium dodecyl sulfate. 3% lliei-~aptoerhanol. 2nd lO!p 2 horll-s with one change of glycerol) with shaking tar buff’er. ‘I‘he sodium dodecvl sul~‘a~e-ecluilil)l att’d gels were then placed on a polyacrylamide gI-adient slab gel ( lOc/, to IS%) and run at 20 mamp until the tl-;l&ng dye (hromophenol blue) entered the lower- buffer. ‘The gels were stained bvith Coomassir Blue (0. 1’; in 3% 11.ichloroacetic acid), destained (in 5% acetic acid). :tnd dried. All two-dimensional procedures w\?cre cascntiall\ those of O’Farrell and associates.” .4utoradiog1-;tms ivere ohtained by exposing Ihe gels lo Y-IX\ film I \;R2. with
Protein
expression
in endometrial
adenocarcinoma
71
t
Fig. 4C. For
Kodak) finveloped in tures were grams with Copv Film (irack 3 at
legend see Fig. 1A.
approximately 2 weeks. X-ray film was deautomatic developer and positive picmade hy photography of the autoradioa 35 mm camera and Kodak High Contrast followed by printing on Kodahromide the original sizr. Receptor analysis. The tissue was removed from licfuitl nitrogen storage (not longer than 1 week), thawed. and homogenized in 10 volumes of buffer (50 mM tromethnmine, pH 7.4, 5 mM dithiothreitol 1 mM ethylenetliaminetetra-acetic acid, and 10% glycerol) in 5-second bursts five times with a Tekmar Tissumizer. The homogenate was centrifuged at 2,000 x g for 20 minutes and the supernatant was recentrifuged for 1 hour at 100,000 x g. The cytosol was treated with dextran-coated charcoal for 2 minutes and recentrifuged at 2,000 x g for 10 minutes: the supernatant was diluted to 1 mg of protein per milliliter according to the Bradford’ assay, and 0.2 ml (0.2 mg of protein) ;III
Fig. 4D.
For
legend see Fig. 4A.
was assayed for estradiol and progesterone receptors in a 200 ~1 assay overnight at 3” C. Assays f’or progesterone receptor were carried out in 10. ?, and 0.5 nM “H-R5020 (New England Nuclear) with duplicate tubes containing l(K)-fold excess of unlabeled K5020. A single-point estradiol receptor assay was carried out at 10 nM “H-estradiol (tetra lahle, Amersham) with a duplicate tube containing lO&fotd excess of diethylstilhestrol. Unbound steroid was removed by dextran-coated charcoal (5 minutes at 4” C), and the supernatants of a centrifugation at 2,000 x g (10 minutes) were counted in 20 ml of Aquasol (New England Nuclear) on a Beckman scintillation counter.
Results Progesterone and estradiot receptor analysis of a portion of the tumor specimen showed a positive receptor population for both hormones. The progesterone receptor concentration, based on a three-point
72
O’Brien
analysis.
et al.
was
fourrtl
while
the estratliol
tein,
based
tlie
in
on
\.itro
to I~ave
177
receptor ;I single-poitlt
hoI-mane-niod~il;ttetl
in Fig.
4. (~hal-;tc~tei~isti~~iil~
other
oi~gaiiisms I” most
tion
the
and
ot’the
vitro
pH
.i ;tnd
5
maps
it i5 observed
the
c.rc.eptiotib
1R),
!\~hTlY
prrsent
In fx.1.
spots
tit\-.
‘I‘he
(FIK.
IWO
in;ited
IIlL\\.
3 and
other
the
spots
Nos.
5 ;iiid
01 ~lx!atl\ lxxillccd. tirap (Fig. 41)).
tliosc
iri the
control
or gi-eatI\-
the
noted,
all
OllC
cstr~idiol
01’
(Fig.
spots Nos.
were 3 and
wduc-rd
4
in quan-
progesterone
map
cmnpletel~
elim-
almost 6 wt’rc
ot
in all in
maps.
map
‘1\‘;16 in tile
go”’ tradiol
esamitl,~-
nlost
f’o111-
cnllallc-cd
I ws
On
orceptions
othcl~
beet1 points
co~nn~on
ol
as fill-
have
that art’
#watly
In Al1
tutnot.,
4.4).
01‘
No.
of iirc
isoelectric
use
alm2nt sf)ot
this
\
esccption
where
anti
with
1).
entirely
I(:),
(Fig.
a1.e (‘0111111011 to AI iioteci
(Nos.
eithei-
r:iilgc proteins
conditions. nl;i;ol~
m;rps
which
have
of al1 fot~i-
rtw
wc‘rc
for tissue
proteins
“:‘S-mcthioniric-labeled
of pro-
Protein
;Icl~no~;i1-cinom~i
tii;tnv
tnapp’C1,~-
of‘ proteirl
I l-l I inolesimg ;issay.
5ho~vn
betweet
f’t~~ole.s/tn~
w;is
either-
conipletel~
111 tl1e },rogeste~~olle tlie obvious spots
plus
es-
resetnldfxl
nlal).
Comment ‘l‘lie
searc.h
fi)r
sclec.1 ion of’ therap
ti~nioi. . and
iiiarkers tumor
useful monitoring
for
ciiagnohis. is a diwrse
REFERENCES hIcGuiw, 12’. I.., Horx
6.
7.
8. 9.
IO.
11.
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