- Email: [email protected]
Specific progesterone receptors in human breast cancer
497
SPECIFIC PROGESTERONE RECEPTORS IN HUMAN BREAST CANCER
K. B. Horwitz and W. L. McGuire
Department of Medicine University of Texas Health Science Center 7703 Floyd Curl Drive San Antonio, Texas 78284 btinuscriptreceived: U/22/74 ABSTRACT We have identified a specific progesterone receptor in 11 of 33 human breast cancer cytosols. Since progesterone itself binds to glucocorticoid receptor, to corticosteroidbinding globulin (CBG), and to nonspecific components as well as to its own receptor, we have used a synthetic progestin, R5020 (17,21-dimethyl-19-nor-4,9-pregnadiene-3, 20-dione), whose binding specificity is restricted to progesterone receptor. Bound R5020 sediments at 8 S in sucrose gradients; binding is competed by excess unlabeled R5020 or progesterone. The receptor is distinct from glucocorticoid receptor and CBG as determined by cop petition studies using dexamethasone and hydrocortisone. The dissociation constant for R5020 obtained by Scatchard analysis of dextrsncoated charcoal assays is approximately 2 x 10-9 M.
INTRODUCTION Steroid hormones exert their major effects on target cells through binding to cytoplasmic receptor proteins. The presence of such receptors in neoplastic tissue is evidence that the cells retain at least part of the normal mechanism by which the endocrine system influences cellular activity. Successful application of endocrine therapy in breast cancer is probably dependent upon retention of this regulatory machinery in neoplastic cells. Accordingly, tumors containing estrogen receptors are more likely to respond to hormonal manipulation than tumors without them (1). Nonetheless, the fact that many estrogen receptor-positive tumors fail to respond as predicted suggests that additional markers may prove useful in identification of responsive tumors. Demonstration of a specific receptor for progesterone in
Volume 25, Nwnber 4
S
-S?D=OSDI
April,
1975
mammary tumors may serve as such a marker, since progesterone is involved in regulating the growth and function of normal manmary tissue. Although progesterone binding has been documented
in the reproduc-
tive tracts of several species (2,3,4), in rat and human tissues the identification (5,6).
of specific progesterone receptors has been difficult
Progesterone binds to its own receptor, but also to cytoplasmic
glucocorticoid receptors, and to sites which are probably contaminants from plasma, such as corticosteroid binding globulin (CBG). tion, progesterone specific binding Recently,
In addi-
is sequestered by low-affinity, high-capacity non-
sites.
the progestational
nor-4,9-pregnadiene-3,20-dione)
compound R5020 (17,21-dimethyl-19-
has been found to bind to a progesterone
receptor in the immature rat and mouse uterus (7).
The receptor sedi-
ments with this progestin in the 8 S region of sucrose gradients.
In
the present studies, we have used R5020 to identify a probable progesterone receptor in human breast cancer tissue.
The human progesterone
receptor also migrates in the 8 S region and is distinct from glucocorticoid receptor and CBG.
MATERIALS AND METHODS Human mammary carcinoma biopsies were quick frozen in liquid nitrogen, shipped to San Antonio in dry ice, and stored at -7O'C in a Revco freezer (Revco, Inc., West Columbia, S.C.). At assay, the tissue was crushed with a Thermovac frozen tissue pulverizer, the powder was weighed and then, after thawing to 4OC, homogenized in two volumes of buffer with three lo-second bursts of a Polytron PT lo-ST homogenizer set at low speed. The homogenization buffer, prepared immediately before use, was S x 10-3 sodium phosphate, pH 7.4 at 4"C, containing 10-3 M thioglycerol and 10% glycerol (8). The cytosol was harvested from the cellular debris by centrifugation for 50 minutes at 40,000 rpm (105,000 g), 4'C, with a Beckman 75 Ti rotor. Protein concentration was measured by the method of Lowry -et al. (9).
S
TREOXDI
Progesterone-1,2,6,7-3H (81 Ci/mmol) obtained from New England Nuclear, or R5020 (17,21-dimethyl-19-nor-4,9-pregnadiene-3,20-dione-6, 7-3H; 51.4 Ci/mmol) obtained from Roussel-Uclaf were added in 2 ul ethanol to 250 ~1 cytosol and incubated for 4 hours at 4°C. Parallel samples were preincubated for 15 minutes with excess unlabeled hormone (progesterone, R5020, estradiol-178, hydrocortisone [118,17a,21-trihydroxy-4-pregnene-3,20-dione] or dexamethasone [9-fluoro-118,17ct,21trihydroxy-16a-methyl-l,4-pregnadiene-3,2O-dione] added in 1 IJ.LL ethanol. Pellets were prepared from a 1 ml suspension of dextran-coated charcoal (0.25% Norit A, 0.0025% Dextran in 0.01 M Tris-HCl, pH 8.0 at 4°C) by a 10 minute centrifugation at 3200 rpm (2000 g). The charged cytosol was transferred onto the pellet, mixed, and incubated 15 minutes After recentrifugation for 10 at 4'C to adsorb unbound radioactivity. minutes at 3200 rpm, a 200 ul aliquot of the supematant was layered onto a 5 to 20 percent sucrose gradient prepared in the homogenization buffer. Bovine serum albumin-14C (lo), 1500 cpm/lO ~1 buffer was added Gradients were centrifuged in to each gradient as an internal marker. a Beckman SW56 rotor at 50,000 rpm (246,000 g,,) for 17 hours. Four drop fractions were collected and counted in 5 ml modified Bray's solution (11). To analyze saturation kinetics, 200 ul cytosol prepared as above was incubated with increasing quantities of the 3H-hormone added in Parallel incubations contained excess non50 ul homogenization buffer. radioactive hormone. After 16 hours of incubation at 4"C, 0.5 ml of the charcoal suspension was added, and the mixture was vigorously shaken for 30 minutes in an Eberbach shaker. The charcoal was sedimented with a 10 minute, 3200 rpm centrifugation, and an aliquot of the supematant was counted in 5 ml Bray's solution. The data was analyzed according to Scatchard (12), after subtraction of nonspecific binding calculated from the preparations competed with nonradioactive hormone.
RESULTS We first sought to determine whether saturable, specific binding could be demonstrated in human breast cancer cytosol using 3H-progesterone.
The sucrose gradient pattern of a representative tumor, F6, is
seen in Figure 1A.
The protein-bound hormone migrated to the 4 S
region, making it difficult to distinguish binding of progesterone to its own receptor from binding to CBG, glucocorticoid receptor, or nonspecific components.
We estimated the relative contribution of these
by studying the binding
of 3H-progesterone in the presence of excess
nonradioactive
(Figure IA, Table 1).
steroids
Unlabeled progesterone
j. A
3H -R5020 DEX
R-5020 -PG
TOP FRACTION NO
FRACTION NO.
TUMOR F6
TUMOR F6
Figure 1: Sucrose density gradient centrifugation of F6 tumor cytosol (9.5 mg/ml protein) incubated with 16 pmoles/ml 3R-hormone, in the presence or absence of 1600 pmoles/ml unlabeled competitor: PG, progesterone; DEX, dexamethasone; F, hydrocortisone; 3H-PG or 3a-~5020, no competitor. Arrow indicates 4.6 S sedimentation of A -- 3H-progesterone binding; B -- 3Hbovine serum albumin-l4C. R5020 binding.
TABLE
1
Percent Inhibition of 38-125020 or 3H-Progesterone Binding in Human Mammary Tumor Cytosols by Unlabeled Competitor
Competitor
Tumor F6 3H-Progesterone* 3~-~5020* 85 4s 8s 45
Progesterone R5020 Dexamethasone Hydrocortisone Estradiol
-__ --__
91 30 14 57 18
93 95 15 0 36
69 62 12 0 27
Tumor U5 3H-Progesterone* 3~-~50204 8S 4s 8s 4s
95 96 16 0 26
94 81 27 43 20
93 -0 0 7
89 _0 0 13
Tumor cytosolwas preincubated with 1600 pmoles/ml unlabeled hormone before addition of 16 pmoles/ml 3H-hormone (*) or preincubated with 100 pmoles/ml unlabeled hormone before addition of 2 pmoles/ml 3H-hormone Competition was determined from percent depression of the area (?. under the peaks seen in sucrose gradients. F6 cytosol was 9.5 mglml protein, U5 cytosol was 11.3 mg/ml protein.
competes efficiently
(91%) so that generalized nonspecific binding can
not account for the 4 S peak.
The 30% competition by R5020 shms
that
only 30% of this binding is to progesterone receptor; hydrocortisone competition shows that 57% is to either CBG or glucocorticoid receptor. The latter possibility is excluded by the minimal competition of dexamethasone, which binds to glucocorticoid
receptor but not to CBG (13).
Since competition studies evaluate progesterone receptor levels only indirectly, we measured the binding of radioactive R5020 directly. Cytosol from the same tumor as above (F6), incubated with 3H-R5020, showed the sucrose gradient pattern seen in Figure 1B. bound R5020 had two components, sedimenting of the gradient.
The protein-
in the S S and 4 S regions
Either excess cold R5020 or excess cold progesterone
completely inhibited binding to the 8 S peak.
Hydrocortisone
compete at all and dexamethasone
(15%).
estradiolcompetes
only minimally
did not
Since excess
only weakly, R5020 is not binding to estrogen receptor.
When unlabeled R5020 is used as a competitor of 3H-estradiol binding, the displacement of 8 S estrogen receptor is less than 10% (data not shown).
Thus,
R5020 is apparently binding to a specific 8 S progesterone
receptor distinguishable
from CBG, glucocorticoid receptor, and estrogen
receptor; a considerable
portion of the 4 S binding also appears due to
the progesterone
receptor.
Eleven of thirty-three breast tumor cytosols
surveyed with 3H-R5020 likewise bound the ligand to receptor in the 8 S region of the gradient. The Scatchard plot of the specific binding obtained by incubating increasing concentrations 2.
of 3H-R5020 with F6 cytosol is shown in Figure
The specific binding sites approached saturation; the extrapolated
number of binding sites was 228 fmolesfmg protein.
The dissociation
502
0
02
04 B
TUMOR
06
Figure 2: Scatchard plots of F6 tumor cytosol (2.25 mg/ml protein) incubated with 0.5 to 20 pmoles/ml 3H-hormone. Nonspecific binding has been subtracted as indicated in methods. (0) 3H-R5020; (0) 3H-progesterone.
08
00-9Ml
F6
L
constant
(Kd) of R5020 binding to the protein was estimated to be
1.9 x 10-9 M.
3H-progesterone binding, also shown, was not saturable
at the concentrations R5020
used.
is especially useful for receptor studies because, in con-
trast to progesterone,
it does not bind to CBG.
However, one would
anticipate that in occasional tumors 3H-progesterone itself could be used to demonstrate progesterone showed considerable
In fact, tumor U5, which
8 S binding with 3H-R5020, did have a distinct 8 S
peak with 3H-progesterone and dexamethasone
receptor.
(Figure 3).
Competition by hydrocortisone
(Table 1) showed that this tumor also had somewhat
lower CBG levels than F6.
The US cytosol Kd with 3H-progesterone was
2.8 x 10-9 M, and the number of binding sites was approximately 400 fmoles/mg protein (Figure 3H).
FRACTION
TUMOR U5
NO.
B PROGESTERONE (IO-‘M)
TUMOR U5
Figure 3: A -- Sucrose density gradient centrifugation of U5 tumor cytosol (11.3 mglml protein) incubated with 16 pmolesfml 3Hsteroid. Arrow indicates 4.6 S sedimentation of bovine serum albumin-14C. (Ir)3H-ES020 (53,000 cpm/pm), (0) 3H-progesterone (85,000 cpm/pm), (A,) 3H-progesteronewith lOO-fold excess unlabeled progesterone. B -- Scatchard plot of U5 tumr cytosol (2.9 mg/ml protein) incubated with 0.1 to 20 pmoles/ml 3H-progesterone. Nonspecific binding has been subtracted as indicated in methods.
DISCUSSION We have demonstrated in human breast tumors the presence of a specific progesterone binder, probably a receptor, sediment&g at 8 S in sucrose gradients and distinct from receptors for glucocorticoids. Similar receptors have previously been described in reproductive tract tissues of several species (2,3,4,14) including man (15). However, som
in
tissues the binding of progesterone has been difficult to demon-
strate, perhaps because of the presence of large amounts of CBG competing for the steroid. The synthetic progestin I?5020was first employed by Philibert and Raynaud (7), who were able to identify a progestin receptor in immature
rat uteri under conditions where progesterone itself bound poorly. the present study, R5020 permitted the demonstration
In
of progestin re-
ceptors in human mammary tumor cytosols even when progesterone bound primarily to other macromolecules. (16) measured progesterone binding
In an alternative approach, Terenius in aliquots of human mammary tumor
cytosol using 3H-progesterone with excess unlabeled hydrocortisone
to
eliminate CBG binding of the progesterone. The concept underlying endocrine therapy for breast cancer is that some tumors have retained the ability to respond to hormone signals. Currently, the presence of estrogen receptors is used to identify such tumors.
Unfortunately,
40% of these estrogen receptor-positive
fail to respond to hormone additional measurement
therapy or endocrine ablation (1).
of progesterone
tumors The
receptors in such tumors may pro-
vide further indication that the endocrine regulatory complex is complete, thus offering a more sensitive method for distinguishing sive from nonresponsive
respon-
carcinomas.
ACKNOWLEDGMENTS Tumor specimens were obtained from Wilford Hall Air Force Hospital, San Antonio, Texas (W. Kemmerer, M.D. and J. McCulloch, M.D.); Bexar County Hospital, San Antonio, Texas (A. Cruz, M.D.); The Methodist Hospital, San Antonio, Texas (E. Gregory, M.D.); The Alton Ochsner Medical Foundation, New Orleans, Louisiana (A. Segaloff, M.D.); The Henry Ford Hospital, Detroit, Michigan (R. Talley, M.D.); University Hospitals, Cleveland, Ohio (0. Pearson, M.D. and C. Hubay, M.D.); University Hospitals, Madison, Wisconsin (F. Ansfield, M.D.); Duke University Medical Center, Durham, North Carolina (W. Shingleton, M.D. and L. Stocks, M.D.). We thank Doctors D. Philibert and J.-P. Raynaud for providing the R.5020. This work has been aided by the USPHS CA-11378, CB-23862, the American Cancer Society BC-23D, and the Robert A. Welch Foundation.
REFERENCES 1.
McGuire, W.L., Pearson, O.H. and Segaloff, A. In ESTROGEN RECEPTOR IN HUMAN BREAST CANCER (W.L. McGuire, P.P. Carbone and E.P. Vollmer, eds.). In Press (1975).
2.
Leavitt, W.W., Toft, D.O., Strott, C.A. and O'Malley, B.W. ENDOCRINOLOGY 94, 1041 (1974).
3. Milgrom, E., Thi, L., Atger, M. and Baulieu, E.-E. J. BIOL. CHEM. 248, 6366 (1973). 4.
Faber, L.E., Sandmann, M.L. and Stavely, H.E. 74 (1973).
5.
Feil, P.D., Glasser, S.R., Toft, D.O. and O'Malley, B.W. ENDOCRINOLOGY 91, 738 (1972).
6.
Verma, U. and Laumas, K.R. (1973).
7.
Fhilibert, D. and Raynaud, J.-P.
8.
Faber, L.E., Sandmann, M.L. and Stavely, H.E. (1973).
9.
Lowry, O.H., Rosebrough, N.J., Farr, A.L. and Randall, R.J. J. BIOL. CHEM. 193, 265 (1951).
10. Rice, R.H. and Means, G.E.
ENDOCRINOLOGY 93,
BIOCHIM. ET BIOPHYS. ACTA 317, 403
STEROIDS 22, 89 (1973). FED. PROC. 22, 229
3. BIOL. CHEM. 246, 831 (1971).
ANAL. BIOCHEM. 1, 279 (1960).
11.
Bray, G.A.
12.
Scatchard, G.
13.
Rousseau, G.G., Baxter, J.D. and Tomkins, G.M. 99 (1972).
14.
Philibert, D. and Raynaud, J.-P. ENDOCRINOLOGY 94, 627 (1974).
15.
Kontula, K., Janne, O., Luukkainen, T. and Vihko, R. BIOPHYS. ACTA 328, 145 (1973).
16. Terenius, L.
ANN. N.Y. ACAD. SCI. 51, 660 (1949).
ELJROP.J. CANCER 9, 291 (1973).
J. MOL. BIOL. 67,
BIOCHIM. ET
SPECIFIC PROGESTERONE RECEPTORS IN HUMAN BREAST CANCER
K. B. Horwitz and W. L. McGuire
Department of Medicine University of Texas Health Science Center 7703 Floyd Curl Drive San Antonio, Texas 78284 btinuscriptreceived: U/22/74 ABSTRACT We have identified a specific progesterone receptor in 11 of 33 human breast cancer cytosols. Since progesterone itself binds to glucocorticoid receptor, to corticosteroidbinding globulin (CBG), and to nonspecific components as well as to its own receptor, we have used a synthetic progestin, R5020 (17,21-dimethyl-19-nor-4,9-pregnadiene-3, 20-dione), whose binding specificity is restricted to progesterone receptor. Bound R5020 sediments at 8 S in sucrose gradients; binding is competed by excess unlabeled R5020 or progesterone. The receptor is distinct from glucocorticoid receptor and CBG as determined by cop petition studies using dexamethasone and hydrocortisone. The dissociation constant for R5020 obtained by Scatchard analysis of dextrsncoated charcoal assays is approximately 2 x 10-9 M.
INTRODUCTION Steroid hormones exert their major effects on target cells through binding to cytoplasmic receptor proteins. The presence of such receptors in neoplastic tissue is evidence that the cells retain at least part of the normal mechanism by which the endocrine system influences cellular activity. Successful application of endocrine therapy in breast cancer is probably dependent upon retention of this regulatory machinery in neoplastic cells. Accordingly, tumors containing estrogen receptors are more likely to respond to hormonal manipulation than tumors without them (1). Nonetheless, the fact that many estrogen receptor-positive tumors fail to respond as predicted suggests that additional markers may prove useful in identification of responsive tumors. Demonstration of a specific receptor for progesterone in
Volume 25, Nwnber 4
S
-S?D=OSDI
April,
1975
mammary tumors may serve as such a marker, since progesterone is involved in regulating the growth and function of normal manmary tissue. Although progesterone binding has been documented
in the reproduc-
tive tracts of several species (2,3,4), in rat and human tissues the identification (5,6).
of specific progesterone receptors has been difficult
Progesterone binds to its own receptor, but also to cytoplasmic
glucocorticoid receptors, and to sites which are probably contaminants from plasma, such as corticosteroid binding globulin (CBG). tion, progesterone specific binding Recently,
In addi-
is sequestered by low-affinity, high-capacity non-
sites.
the progestational
nor-4,9-pregnadiene-3,20-dione)
compound R5020 (17,21-dimethyl-19-
has been found to bind to a progesterone
receptor in the immature rat and mouse uterus (7).
The receptor sedi-
ments with this progestin in the 8 S region of sucrose gradients.
In
the present studies, we have used R5020 to identify a probable progesterone receptor in human breast cancer tissue.
The human progesterone
receptor also migrates in the 8 S region and is distinct from glucocorticoid receptor and CBG.
MATERIALS AND METHODS Human mammary carcinoma biopsies were quick frozen in liquid nitrogen, shipped to San Antonio in dry ice, and stored at -7O'C in a Revco freezer (Revco, Inc., West Columbia, S.C.). At assay, the tissue was crushed with a Thermovac frozen tissue pulverizer, the powder was weighed and then, after thawing to 4OC, homogenized in two volumes of buffer with three lo-second bursts of a Polytron PT lo-ST homogenizer set at low speed. The homogenization buffer, prepared immediately before use, was S x 10-3 sodium phosphate, pH 7.4 at 4"C, containing 10-3 M thioglycerol and 10% glycerol (8). The cytosol was harvested from the cellular debris by centrifugation for 50 minutes at 40,000 rpm (105,000 g), 4'C, with a Beckman 75 Ti rotor. Protein concentration was measured by the method of Lowry -et al. (9).
S
TREOXDI
Progesterone-1,2,6,7-3H (81 Ci/mmol) obtained from New England Nuclear, or R5020 (17,21-dimethyl-19-nor-4,9-pregnadiene-3,20-dione-6, 7-3H; 51.4 Ci/mmol) obtained from Roussel-Uclaf were added in 2 ul ethanol to 250 ~1 cytosol and incubated for 4 hours at 4°C. Parallel samples were preincubated for 15 minutes with excess unlabeled hormone (progesterone, R5020, estradiol-178, hydrocortisone [118,17a,21-trihydroxy-4-pregnene-3,20-dione] or dexamethasone [9-fluoro-118,17ct,21trihydroxy-16a-methyl-l,4-pregnadiene-3,2O-dione] added in 1 IJ.LL ethanol. Pellets were prepared from a 1 ml suspension of dextran-coated charcoal (0.25% Norit A, 0.0025% Dextran in 0.01 M Tris-HCl, pH 8.0 at 4°C) by a 10 minute centrifugation at 3200 rpm (2000 g). The charged cytosol was transferred onto the pellet, mixed, and incubated 15 minutes After recentrifugation for 10 at 4'C to adsorb unbound radioactivity. minutes at 3200 rpm, a 200 ul aliquot of the supematant was layered onto a 5 to 20 percent sucrose gradient prepared in the homogenization buffer. Bovine serum albumin-14C (lo), 1500 cpm/lO ~1 buffer was added Gradients were centrifuged in to each gradient as an internal marker. a Beckman SW56 rotor at 50,000 rpm (246,000 g,,) for 17 hours. Four drop fractions were collected and counted in 5 ml modified Bray's solution (11). To analyze saturation kinetics, 200 ul cytosol prepared as above was incubated with increasing quantities of the 3H-hormone added in Parallel incubations contained excess non50 ul homogenization buffer. radioactive hormone. After 16 hours of incubation at 4"C, 0.5 ml of the charcoal suspension was added, and the mixture was vigorously shaken for 30 minutes in an Eberbach shaker. The charcoal was sedimented with a 10 minute, 3200 rpm centrifugation, and an aliquot of the supematant was counted in 5 ml Bray's solution. The data was analyzed according to Scatchard (12), after subtraction of nonspecific binding calculated from the preparations competed with nonradioactive hormone.
RESULTS We first sought to determine whether saturable, specific binding could be demonstrated in human breast cancer cytosol using 3H-progesterone.
The sucrose gradient pattern of a representative tumor, F6, is
seen in Figure 1A.
The protein-bound hormone migrated to the 4 S
region, making it difficult to distinguish binding of progesterone to its own receptor from binding to CBG, glucocorticoid receptor, or nonspecific components.
We estimated the relative contribution of these
by studying the binding
of 3H-progesterone in the presence of excess
nonradioactive
(Figure IA, Table 1).
steroids
Unlabeled progesterone
j. A
3H -R5020 DEX
R-5020 -PG
TOP FRACTION NO
FRACTION NO.
TUMOR F6
TUMOR F6
Figure 1: Sucrose density gradient centrifugation of F6 tumor cytosol (9.5 mg/ml protein) incubated with 16 pmoles/ml 3R-hormone, in the presence or absence of 1600 pmoles/ml unlabeled competitor: PG, progesterone; DEX, dexamethasone; F, hydrocortisone; 3H-PG or 3a-~5020, no competitor. Arrow indicates 4.6 S sedimentation of A -- 3H-progesterone binding; B -- 3Hbovine serum albumin-l4C. R5020 binding.
TABLE
1
Percent Inhibition of 38-125020 or 3H-Progesterone Binding in Human Mammary Tumor Cytosols by Unlabeled Competitor
Competitor
Tumor F6 3H-Progesterone* 3~-~5020* 85 4s 8s 45
Progesterone R5020 Dexamethasone Hydrocortisone Estradiol
-__ --__
91 30 14 57 18
93 95 15 0 36
69 62 12 0 27
Tumor U5 3H-Progesterone* 3~-~50204 8S 4s 8s 4s
95 96 16 0 26
94 81 27 43 20
93 -0 0 7
89 _0 0 13
Tumor cytosolwas preincubated with 1600 pmoles/ml unlabeled hormone before addition of 16 pmoles/ml 3H-hormone (*) or preincubated with 100 pmoles/ml unlabeled hormone before addition of 2 pmoles/ml 3H-hormone Competition was determined from percent depression of the area (?. under the peaks seen in sucrose gradients. F6 cytosol was 9.5 mglml protein, U5 cytosol was 11.3 mg/ml protein.
competes efficiently
(91%) so that generalized nonspecific binding can
not account for the 4 S peak.
The 30% competition by R5020 shms
that
only 30% of this binding is to progesterone receptor; hydrocortisone competition shows that 57% is to either CBG or glucocorticoid receptor. The latter possibility is excluded by the minimal competition of dexamethasone, which binds to glucocorticoid
receptor but not to CBG (13).
Since competition studies evaluate progesterone receptor levels only indirectly, we measured the binding of radioactive R5020 directly. Cytosol from the same tumor as above (F6), incubated with 3H-R5020, showed the sucrose gradient pattern seen in Figure 1B. bound R5020 had two components, sedimenting of the gradient.
The protein-
in the S S and 4 S regions
Either excess cold R5020 or excess cold progesterone
completely inhibited binding to the 8 S peak.
Hydrocortisone
compete at all and dexamethasone
(15%).
estradiolcompetes
only minimally
did not
Since excess
only weakly, R5020 is not binding to estrogen receptor.
When unlabeled R5020 is used as a competitor of 3H-estradiol binding, the displacement of 8 S estrogen receptor is less than 10% (data not shown).
Thus,
R5020 is apparently binding to a specific 8 S progesterone
receptor distinguishable
from CBG, glucocorticoid receptor, and estrogen
receptor; a considerable
portion of the 4 S binding also appears due to
the progesterone
receptor.
Eleven of thirty-three breast tumor cytosols
surveyed with 3H-R5020 likewise bound the ligand to receptor in the 8 S region of the gradient. The Scatchard plot of the specific binding obtained by incubating increasing concentrations 2.
of 3H-R5020 with F6 cytosol is shown in Figure
The specific binding sites approached saturation; the extrapolated
number of binding sites was 228 fmolesfmg protein.
The dissociation
502
0
02
04 B
TUMOR
06
Figure 2: Scatchard plots of F6 tumor cytosol (2.25 mg/ml protein) incubated with 0.5 to 20 pmoles/ml 3H-hormone. Nonspecific binding has been subtracted as indicated in methods. (0) 3H-R5020; (0) 3H-progesterone.
08
00-9Ml
F6
L
constant
(Kd) of R5020 binding to the protein was estimated to be
1.9 x 10-9 M.
3H-progesterone binding, also shown, was not saturable
at the concentrations R5020
used.
is especially useful for receptor studies because, in con-
trast to progesterone,
it does not bind to CBG.
However, one would
anticipate that in occasional tumors 3H-progesterone itself could be used to demonstrate progesterone showed considerable
In fact, tumor U5, which
8 S binding with 3H-R5020, did have a distinct 8 S
peak with 3H-progesterone and dexamethasone
receptor.
(Figure 3).
Competition by hydrocortisone
(Table 1) showed that this tumor also had somewhat
lower CBG levels than F6.
The US cytosol Kd with 3H-progesterone was
2.8 x 10-9 M, and the number of binding sites was approximately 400 fmoles/mg protein (Figure 3H).
FRACTION
TUMOR U5
NO.
B PROGESTERONE (IO-‘M)
TUMOR U5
Figure 3: A -- Sucrose density gradient centrifugation of U5 tumor cytosol (11.3 mglml protein) incubated with 16 pmolesfml 3Hsteroid. Arrow indicates 4.6 S sedimentation of bovine serum albumin-14C. (Ir)3H-ES020 (53,000 cpm/pm), (0) 3H-progesterone (85,000 cpm/pm), (A,) 3H-progesteronewith lOO-fold excess unlabeled progesterone. B -- Scatchard plot of U5 tumr cytosol (2.9 mg/ml protein) incubated with 0.1 to 20 pmoles/ml 3H-progesterone. Nonspecific binding has been subtracted as indicated in methods.
DISCUSSION We have demonstrated in human breast tumors the presence of a specific progesterone binder, probably a receptor, sediment&g at 8 S in sucrose gradients and distinct from receptors for glucocorticoids. Similar receptors have previously been described in reproductive tract tissues of several species (2,3,4,14) including man (15). However, som
in
tissues the binding of progesterone has been difficult to demon-
strate, perhaps because of the presence of large amounts of CBG competing for the steroid. The synthetic progestin I?5020was first employed by Philibert and Raynaud (7), who were able to identify a progestin receptor in immature
rat uteri under conditions where progesterone itself bound poorly. the present study, R5020 permitted the demonstration
In
of progestin re-
ceptors in human mammary tumor cytosols even when progesterone bound primarily to other macromolecules. (16) measured progesterone binding
In an alternative approach, Terenius in aliquots of human mammary tumor
cytosol using 3H-progesterone with excess unlabeled hydrocortisone
to
eliminate CBG binding of the progesterone. The concept underlying endocrine therapy for breast cancer is that some tumors have retained the ability to respond to hormone signals. Currently, the presence of estrogen receptors is used to identify such tumors.
Unfortunately,
40% of these estrogen receptor-positive
fail to respond to hormone additional measurement
therapy or endocrine ablation (1).
of progesterone
tumors The
receptors in such tumors may pro-
vide further indication that the endocrine regulatory complex is complete, thus offering a more sensitive method for distinguishing sive from nonresponsive
respon-
carcinomas.
ACKNOWLEDGMENTS Tumor specimens were obtained from Wilford Hall Air Force Hospital, San Antonio, Texas (W. Kemmerer, M.D. and J. McCulloch, M.D.); Bexar County Hospital, San Antonio, Texas (A. Cruz, M.D.); The Methodist Hospital, San Antonio, Texas (E. Gregory, M.D.); The Alton Ochsner Medical Foundation, New Orleans, Louisiana (A. Segaloff, M.D.); The Henry Ford Hospital, Detroit, Michigan (R. Talley, M.D.); University Hospitals, Cleveland, Ohio (0. Pearson, M.D. and C. Hubay, M.D.); University Hospitals, Madison, Wisconsin (F. Ansfield, M.D.); Duke University Medical Center, Durham, North Carolina (W. Shingleton, M.D. and L. Stocks, M.D.). We thank Doctors D. Philibert and J.-P. Raynaud for providing the R.5020. This work has been aided by the USPHS CA-11378, CB-23862, the American Cancer Society BC-23D, and the Robert A. Welch Foundation.
REFERENCES 1.
McGuire, W.L., Pearson, O.H. and Segaloff, A. In ESTROGEN RECEPTOR IN HUMAN BREAST CANCER (W.L. McGuire, P.P. Carbone and E.P. Vollmer, eds.). In Press (1975).
2.
Leavitt, W.W., Toft, D.O., Strott, C.A. and O'Malley, B.W. ENDOCRINOLOGY 94, 1041 (1974).
3. Milgrom, E., Thi, L., Atger, M. and Baulieu, E.-E. J. BIOL. CHEM. 248, 6366 (1973). 4.
Faber, L.E., Sandmann, M.L. and Stavely, H.E. 74 (1973).
5.
Feil, P.D., Glasser, S.R., Toft, D.O. and O'Malley, B.W. ENDOCRINOLOGY 91, 738 (1972).
6.
Verma, U. and Laumas, K.R. (1973).
7.
Fhilibert, D. and Raynaud, J.-P.
8.
Faber, L.E., Sandmann, M.L. and Stavely, H.E. (1973).
9.
Lowry, O.H., Rosebrough, N.J., Farr, A.L. and Randall, R.J. J. BIOL. CHEM. 193, 265 (1951).
10. Rice, R.H. and Means, G.E.
ENDOCRINOLOGY 93,
BIOCHIM. ET BIOPHYS. ACTA 317, 403
STEROIDS 22, 89 (1973). FED. PROC. 22, 229
3. BIOL. CHEM. 246, 831 (1971).
ANAL. BIOCHEM. 1, 279 (1960).
11.
Bray, G.A.
12.
Scatchard, G.
13.
Rousseau, G.G., Baxter, J.D. and Tomkins, G.M. 99 (1972).
14.
Philibert, D. and Raynaud, J.-P. ENDOCRINOLOGY 94, 627 (1974).
15.
Kontula, K., Janne, O., Luukkainen, T. and Vihko, R. BIOPHYS. ACTA 328, 145 (1973).
16. Terenius, L.
ANN. N.Y. ACAD. SCI. 51, 660 (1949).
ELJROP.J. CANCER 9, 291 (1973).
J. MOL. BIOL. 67,
BIOCHIM. ET