RESUMO
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. The Mr 116,000 protein appeared as a triplet, while a single band was found for the Mr 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 progesterone receptor structure. Mixing experiments using a progesterone receptor-rich human endometrial carcinoma (EnCa 101), which lacks protease activity, and protease-containing primary tumor homogenates indicated that the protease was leupeptin sensitive. Interestingly, while the proteolytic activity reduced or eliminated the triplet/singlet progesterone receptor structure seen on protein blot analysis, it did not affect progesterone receptor concentration measured by Scatchard analysis. Sample preparation in the presence of protease inhibitors is therefore a requisite for structural analysis of the progesterone receptor in endometrial tumors.
Assuntos
Peptídeo Hidrolases/análise , Receptores de Progesterona/análise , Neoplasias Uterinas/análise , Feminino , Humanos , Peso MolecularRESUMO
Since interferon inducible 2',5'-oligoadenylate (2,5An) synthetase activity is present in a wide variety of cells and is affected by various hormonal conditions, primary human mammary tumor extracts were examined for the constitutive presence of this enzyme and its possible relationship with the various hormonal receptor levels in these tissues. Further, since 2,5An synthetase has been implicated as a possible factor controlling cell replication, we assayed DNA polymerases in these same tumor extracts to determine any correlation between 2,5An synthetase activity and growth potential. A survey of the soluble extracts from 24 different surgically removed human mammary tumor specimens for 2,5An synthetase activity indicated that this enzyme was indeed present in all extracts but in widely varying amounts of activity (31-2,666 nmol adenosine 5'-phosphate incorporated/mg protein). The 2,5An synthesized in the enzymic reactions ranged in size from di- to hexamers, with trimers being the abundant 2,5An in the majority of tumors. A comparison of the assay results for estrogen and progestin receptors with 2,5An synthesis indicated that high 2,5An synthetase activity was found in both estrogen or progestin positive and negative tumors. Thus, 2,5An synthetase activity was unrelated (r = 0.329 and 0.077, respectively, for estrogen and progestin receptors) to the hormonal receptor content of these tumors. A similar comparison was made between 2,5An synthesis and assay results for the activities of DNA polymerase alpha, regarded as the principal DNA replicating enzyme, and DNA polymerase beta, regarded as the DNA repair enzyme. Although the activity of the polymerases were also quite varied, the majority of tumor extracts demonstrated higher alpha polymerase activity with no parallel difference between the alpha and beta enzymes. There was, however, a weak correlation (r = 0.751) between 2,5An synthetase activity and DNA polymerase alpha activity among the tumors examined. Less of a correlation existed with DNA polymerase beta activity (r = 0.600). These results suggested that the potential of the tumors to synthesize 2,5An was unrelated to their hormonal responsiveness and only weakly related to their growth potential reflected by DNA polymerase alpha activity.
Assuntos
2',5'-Oligoadenilato Sintetase/análise , Neoplasias da Mama/enzimologia , Receptores de Estrogênio/análise , Receptores de Progesterona/análise , Neoplasias da Mama/análise , Neoplasias da Mama/patologia , DNA Polimerase II/análise , Feminino , HumanosRESUMO
Antibody against the progestin receptor was raised in a guinea pig utilizing a partially purified receptor preparation from rabbit uterus. The antibody recognizes the cytosol and nuclear progestin receptor but not glucocorticoid, androgen, or estrogen receptors. In addition, the antibody recognizes the progestin receptor in normal human endometrium as well as that in human breast and endometrial tumor cytosols. Antigen: antibody complex formation between receptor and preimmune or immune serum was judged by shift in sedimentation coefficient on high-salt sucrose gradients, by exclusion from DEAE Affi-Gel Blue columns, and by adsorption with Protein A.
Assuntos
Soros Imunes/análise , Receptores de Progesterona/imunologia , Animais , Complexo Antígeno-Anticorpo/análise , Núcleo Celular/análise , Centrifugação com Gradiente de Concentração , Cromatografia por Troca Iônica , Citosol/análise , Feminino , Cobaias , Coelhos , Útero/análiseRESUMO
Although progesterone receptors have been studied in the uterine cytoplasm of many species, relatively little was known about the nuclear content of these binding proteins. In the present study, a nuclear progesterone receptor was deteced in the guinea pig uterus. The binding of progesterone to the nuclear receptor was hormone and tissue specific. Furthermore, the nuclear localization of the progesterone receptor complex in the uterus was both time and temperature dependent. Since the nuclear receptor was extracted in high salt buffer, the effects of KCl on several physical properties of the cytoplasmic and nuclear binders were studied. In the presence of high salt, cytosol and nuclear receptors were virtually indistinguishable. These proteins were clearly distinguished upon removal of the KCl by rapid dialysis: the nuclear receptor had a slower sedimentation rate, a faster rate of dissociation and a higher binding affinity than did the cytosol receptor for progesterone. We conclude that the cytosol and nuclear receptors for progesterone in the guinea pig uterus are distinct macromolecules. These observations are consistent with the postulate that the cytoplasmic receptor is a precursor of that in the nucleus.
Assuntos
Progesterona/metabolismo , Receptores de Superfície Celular , Útero/metabolismo , Animais , Proteínas de Transporte/metabolismo , Núcleo Celular/metabolismo , Citoplasma/metabolismo , Citosol/metabolismo , Feminino , Cobaias , Cinética , Cloreto de Potássio/farmacologia , Ligação Proteica , Receptores de Superfície Celular/efeitos dos fármacos , Temperatura , Útero/ultraestruturaRESUMO
The guinea pig progestin receptor appears to be unique among mammalian receptors studied to date in that it displays a low binding affinity for some biologically potent 17 alpha-substituted progestinss. [3H]Medroxyprogesterone acetate (MPA) was synthesized and used to investigate this dichotomy between binding affinity and biological activity. The comparison of cytoplasmic and nuclear receptor binding characteristics suggested that progesterone and MPA were bound to the same receptor but with different affinities. Following an intravenous injection of 3H-steroids, the plasma level of progesterone was lower than that of MPA at all time points. Correspondingly, for up to 6 hours following steroid administration, progesterone levels were lower in uterine cytoplasm and higher in nuclei than those of MPA. However, by 24 hours, MPA nuclear levels were higher than those of progesterone, in accordance with plasma levels. We conclude that the potent biological activity of MPA relative to progesterone is due in part to its slower rate of metabolism and longer nuclear retention.
Assuntos
Medroxiprogesterona/metabolismo , Progestinas/metabolismo , Receptores de Droga , Útero/metabolismo , Animais , Sítios de Ligação , Núcleo Celular/metabolismo , Citosol/metabolismo , Feminino , Cobaias , Técnicas In Vitro , Ligantes , Progesterona/sangue , Progesterona/metabolismoRESUMO
Regulation of progesterone receptor (PR) in human endometrial carcinoma was investigated in vivo in a multisite nude mouse tumor experimental system by estrogen administration and withdrawal. The cytosolic PR concentration was low in tumors grown in the absence of 17 beta-estradiol, but increased rapidly upon estrogen administration, reaching a maximal receptor concentration of 1.4-1.6 pmol/mg cytosol protein within 7 days. Protein blot analysis using a monoclonal antibody (hPRa 1) raised against PR from EnCa 101 showed no immunoreactivity in tumors grown in the absence of estrogen. Immunoreactive proteins of mol wt 116,000 and 81,000 were detectable 8 h after estrogen administration and increased in intensity as the cytosolic PR concentration increased. Interestingly, the protein of mol wt 116,000 was composed of mol wt isoforms and was detectable as a doublet 8 h after estrogen administration and finally as a triplet. The effect of estrogen withdrawal on EnCa 101 PR concentration and structure was determined by removal of 17 beta-estradiol pellets (200 pg/ml plasma) from EnCa 101-bearing animals after achievement of maximal tumor PR concentrations. The PR concentration in tumor cytosols decreased in a biphasic manner after estrogen removal, with the initial rapid phase having a half-life of around 2 days. Cytosolic PR was still detectable 21 days after estrogen withdrawal. Protein blot analysis showed that immunoreactive proteins of mol wt 116,000 and 81,000 were also detectable up to that time. Photoaffinity labeling with [3H]R5020 demonstrated that the 81,000 mol wt protein, as well as each of the triplet proteins at mol wt 116,000, was specifically photoaffinity labeled. The 116,000-mol wt protein was detected as a triplet on protein blots until 13 days after estrogen withdrawal, when diminution in the intensity of the highest mol wt triplet protein was noted.
Assuntos
Estradiol/farmacologia , Receptores de Progesterona/metabolismo , Neoplasias Uterinas/metabolismo , Animais , Linhagem Celular , Citosol/metabolismo , Implantes de Medicamento , Feminino , Humanos , Cinética , Camundongos , Camundongos Nus , Peso Molecular , Transplante de Neoplasias , Receptores de Progesterona/efeitos dos fármacos , Receptores de Progesterona/isolamento & purificação , Transplante Heterólogo , Neoplasias Uterinas/patologiaRESUMO
Monoclonal antibodies to the human progesterone receptor (PR) were used to detect changes in PR form during the menstrual cycle. In proliferative phase samples, two PR proteins with mol wt (Mr) of 116,000 and 81,000 were detected on protein blots probed with anti-PR monoclonal antibodies. The 116,000 Mr protein was comprised of triplet isoforms, while the 81,000 Mr protein was a singlet. By contrast, protein blot analysis of secretory phase samples revealed a doublet isoform at 116,000 Mr and an 81,000 Mr singlet. Organ culture of human endometrium was used to mimic these changes in PR form in vitro. Although the triplet to doublet conversion was not realized in organ culture, time- and dose-related changes in PR form were achieved which were similar to the in vivo state. Furthermore, these changes preceded the progestin-mediated induction of the enzyme estradiol dehydrogenase in parallel cultures, demonstrating that this is a useful system in which to study the relationship between receptor modulation and initiation and maintenance of biological response.
Assuntos
Endométrio/metabolismo , Ciclo Menstrual , Progestinas/fisiologia , Receptores de Progesterona/metabolismo , Anticorpos Monoclonais , Núcleo Celular/metabolismo , Citosol/metabolismo , Estradiol Desidrogenases/metabolismo , Feminino , Humanos , Immunoblotting , Medroxiprogesterona/análogos & derivados , Medroxiprogesterona/farmacologia , Acetato de Medroxiprogesterona , Peso Molecular , Técnicas de Cultura de Órgãos , Receptores de Progesterona/efeitos dos fármacosRESUMO
The action of sex steroids on the growth and differentiation of target tissues requires the presence of specific intracellular receptors. We compared the distribution of cells containing estrogen receptor (ER) and/or progesterone receptor (PR) in rabbit uterus by immunohistochemistry using monoclonal antibodies directed against these receptors. Initial experiments using serial cryostat sections surprisingly revealed the intensity of staining for ER to be inversely proportional to that of PR, as follows: ER, luminal and glandular epithelium greater than myometrium greater than stroma; PR, stroma greater than myometrium greater than glands greater than luminal epithelium. Localization was strictly confined to the nuclei of target cells. Single and dual immunofluorescent labeling of ER and PR in cryostat sections was accomplished using fluorochromes with differing emission spectra. Individual fields of dual labeled sections were examined for red [phycoerythrin (ER)] and green [fluorescein (PR)] fluorescence, with the same distribution as noted by single antibody immunohistochemistry. Myometrial nuclei displayed fluorescence of equivalent relative intensity for both antibodies. Further, sequential exposure photomicrography (exposure first in the spectrum of phycoerythrin emission, followed by exposure in the spectrum of fluorescein emission) revealed the presence of occasional stromal cells staining only for PR and some luminal cells staining only for ER. This differential distribution of ER and PR within various cell populations of rabbit is a novel observation and challenges current concepts of receptor regulation. Dual immunofluorescent localization of both ER and PR within individual cells provides a unique perspective from which to investigate the interactive influences of these sex steroid receptors at the cellular level.
Assuntos
Receptores de Estrogênio/análise , Receptores de Progesterona/análise , Útero/citologia , Animais , Estradiol/sangue , Feminino , Imunofluorescência , Progesterona/sangue , Coelhos , Radioimunoensaio , Maturidade SexualRESUMO
Progesterone receptor (PR) from a human endometrial carcinoma (EnCa 101) grown in nude mice consists of two hormone-binding proteins with mol wt around 116,000 and 85,000. To generate monoclonal antibodies against this receptor, PR was partially purified from EnCa 101 and used to immunize Robertsonian mice. Immune mouse spleens were fused with HL-1 Friendly myeloma-653 cells, and hybridomas were screened by solid phase dot-blot assay and double antibody precipitation. Seven stable hybridomas were obtained, designated hPRa 1-7. Subisotyping revealed that hPRa 1 and 6 were immunoglobulin G2b, while the remainder were immunoglobulin G1. Ultracentrifugation in high salt sucrose gradients showed that six of the seven antibodies effected a shift of [3H]progestin-labeled PR from EnCa 101; only hPRa 4 was ineffective in this regard. Protein blots of EnCa 101 cytosols and DEAE eluates revealed that hPRa 1, 3, 4, 5, and 7 recognized both PR proteins equally. hPRa 2 recognized principally the 116,000 mol wt PR protein; it recognized the lower mol wt PR protein very poorly if at all, whereas hPRa 6 recognized only the 116,000 mol wt protein. Interestingly, the latter was consistently detected as a closely migrating triplet. Immunolocalization of PR by hPRa 1-7 in tissue sections was confined to nuclei of target tissues and varied in intensity: hPRa 7 greater than 3 = 5 greater than 6 = 2 greater than 1 greater than 4. In proliferative phase uterus, the intensity of staining was ranked: endometrial gland nuclei (3+) greater than myometrial cell nuclei (2-3+) greater than endometrial stromal cell nuclei (0-1+). Thus, seven monoclonal antibodies directed against human PR have been prepared, and their suitability for the study of PR by biochemical and immunohistochemical techniques has been demonstrated.
Assuntos
Anticorpos Monoclonais/imunologia , Receptores de Progesterona/imunologia , Animais , Anticorpos Monoclonais/biossíntese , Especificidade de Anticorpos , Centrifugação com Gradiente de Concentração , Eletroforese em Gel de Poliacrilamida , Feminino , Histocitoquímica , Humanos , Hibridomas/imunologia , Imunoensaio , Técnicas de Imunoadsorção , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Peso Molecular , Receptores de Progesterona/análise , Neoplasias Uterinas/análise , Útero/análiseRESUMO
The uptake of androgens into the nuclei of caput epididymis, ventral prostate, seminal vesicle and testis was studied by recirculating physiological and pharmacological concentrations of [3H]testosterone in an artificial medium through the lower half (hemicorpus) of castrated or hypophysectomized rats. The accumulation of dihydrotestosterone in accessory sex organ nuclei was saturable, inhibited by perfusion of excess testosterone or cyproterone acetate, and associated with binding to 3S salt-extractable molecules. In castrated preparations the mean saturation levels (pmol/mg DNA) were different in the three organs: seminal vesicle, 2.8; ventral prostate, 1.8; caput epididymis, 0.9. The saturation level was significantly lower in ventral prostate of hypophysectomized rats (1.2) treated with testosterone to regenerate the accessory sex organs. Testosterone was the major nuclear androgen in the testes of mature hypophysectomized preparations perfused with testosterone. Although there was a large amount of nonspecific accumulation, testosterone binding to 3S molecules was shown by sucrose gradient centrifugation. Binding of dihydrotestosterone to 3S molecules in testicular nuclei was also demonstrated. The ratio of dihydrotestosterone to testosterone was different in immature and mature testicular nuclei and was altered by treatments known to affect testicular 5 alpha-reductase activity. The results suggest that in rat accessory sex organs and immature testis the major active androgen is dihydrotestosterone, whereas in mature testis it is testosterone. The shift in the predominant nuclear androgen in the testis from dihydrotestosterone to testosterone is most simply explained by the maturational change in 5 alpha-reductase activity.
Assuntos
Androgênios/metabolismo , Genitália Masculina/metabolismo , Animais , Núcleo Celular/metabolismo , Epididimo/metabolismo , Hipofisectomia , Masculino , Orquiectomia , Perfusão , Próstata/metabolismo , Ratos , Glândulas Seminais/metabolismo , Testículo/metabolismoRESUMO
PIP: The use of medroxyprogesterone acetate (MPA) was incorporated into a nuclear receptor assay for progestin receptor in human endometrium. The assay was developed because MPA is a better ligand than progesterone since it does not bind to corticosteroid-binding globulin and gives greater kinetic stability to the nuclear complex. The MPA nuclear receptor complex for malignant endometrium dissociated at a faster rate than did the complex obtained from normal endometrium, an alteration in binding kinetics which could not be explained by instability of the receptors from malignant endometrium. Factors, including radiation therapy, plasma proteins, endogenous steroids, receptor degradation, tissue heterogeneity, and limited sample size, which influence the interpretation of receptor assay were systematically evaluated. In spite of these controls, it would be premature to conclude that the clinical observations indicate altered receptor from malignant tissue. Further studies are required on endometrial carcinoma which is free of normal tissue fragments. Clinically, nuclear receptor levels were highest in normal endometrium but decreased in samples of malignant endometrium as tumors became more anaplastic. The lowest nuclear binding activity was detected in samples of metastatic endometrial tissue (carcinoma). Hopefully, this nuclear receptor assay (which uses MPA because its dissociation was slower than progesterone) will provide data for correlating clinical response to therapy.^ieng
Assuntos
Núcleo Celular/metabolismo , Endométrio/metabolismo , Receptores de Progesterona/metabolismo , Doenças Uterinas/metabolismo , Ligação Competitiva , Feminino , Humanos , Cinética , Medroxiprogesterona/metabolismo , Progesterona/metabolismoRESUMO
Estrone and estradiol concentrations in breast tumor tissue are an order of magnitude higher than circulating plasma levels in postmenopausal women with breast cancer. Local production of estrogen in the neoplastic tissue is one of several possible explanations for this plasma/tissue gradient. This study evaluated breast tumor estrogen production via the estrone sulfate to estrone (sulfatase) pathway and compared this with the androstenedione to estrone (aromatase) system in human and rodent mammary tumors. Estrogen production from estrone sulfate was related linearly with time and tissue concentrations, exhibited an apparent Km of 20 microM, and produced a linear Eadie-Hofstee kinetic plot consistent with a single class of enzymatic sites. Measurement of sulfatase in 35 human breast tumors using enzyme saturating conditions revealed estrone production ranging from 0.8-125 mumol/g protein . h. The corresponding range in host mammary tumors was 3.5-7.1 mumol/g protein . h. In human breast tumors, sulfatase activity did not correlate with the levels of estrogen receptor or progesterone receptor. Comparison of sulfatase with aromatase activity in human tumors at physiological levels of substrate revealed estrone formation via sulfatase of 2.8 pmol estrone produced/g protein . h, while aromatase produced only 0.27 pmol/g protein . h. In rat mammary tumors, sulfatase activity was similar to that in human tumors, whereas aromatase activity could not be detected, even with a highly sensitive assay. Thus, estrone sulfatase appears to be the enzyme primarily responsible for intratissue estrone production in hormone-dependent breast carcinomas.
Assuntos
Aromatase/metabolismo , Neoplasias da Mama/enzimologia , Estrogênios/biossíntese , Oxirredutases/metabolismo , Sulfatases/metabolismo , Animais , Feminino , Humanos , Neoplasias Mamárias Experimentais/enzimologia , Ratos , Receptores de Estrogênio/isolamento & purificação , Receptores de Progesterona/isolamento & purificação , Especificidade por SubstratoRESUMO
Human breast carcinomas contain aromatase, the enzyme necessary for the conversion of androgens to estrogens. If present in sufficient amounts, aromatase could catalyze the synthesis of estrogens from plasma steroid precursors and produce high breast cancer tissue concentrations. To determine the biological importance of tumor aromatase, we validated a specific and highly sensitive 3H-labeled water release assay for aromatase and used this to quantitate the amount of estrogen synthesized in vitro in breast tumors. As proof of assay validity, the [3H] water release assay detected 22.7 +/- 0.09 (+/- SEM) pmol/g . h estrogen formed vs. 24.7 pmol/g . h with the direct product isolation assay. Of 61 human breast tumors studied, 48 contained measurable aromatase activity, ranging from 5-70.5 pmol estrone formed/g . h. Three aromatase inhibitors (aminoglutethimide, testololactone, and 4-hydroxyandrostenedione) blocked this activity at concentrations similar to those affecting aromatase activity in other tissues. If biologically important, the estrogen formed locally from aromatase would be expected to stimulate production of the progesterone receptor. Under these circumstances, a positive correlation of progesterone receptor and local estrogen production should be found. In contrast, no significant correlation between aromatase activity and progesterone receptor level was observed (r = -0.27; P = NS). In addition, no correlation between estrogen receptor content and aromatase activity was detected. Finally, the amount of aromatase activity present in most tumors was insufficient to produce biologically meaningful saturation of estrogen receptors. These observations suggested that aromatase, while present in the majority of breast cancer tissues, may only be biologically important in those few tumors with very high aromatase activity.
Assuntos
Aromatase/metabolismo , Neoplasias da Mama/enzimologia , Oxirredutases/metabolismo , Aminoglutetimida/farmacologia , Androstenodiona/análogos & derivados , Androstenodiona/farmacologia , Inibidores da Aromatase , Neoplasias da Mama/metabolismo , Estrona/biossíntese , Feminino , Humanos , Receptores de Estrogênio/metabolismo , Receptores de Progesterona/metabolismo , Testolactona/farmacologiaRESUMO
One-third of the cases of breast cancer in postmenopausal women are hormone-dependent and the lesions regress upon treatment with antiestrogens or inhibition of estrogen biosynthesis. In these patients, estrogens are synthesized in extraglandular tissues from adrenal precursors and re-enter plasma to produce estrone levels of 52 +/- 6.5 pg/ml (mean +/- SEM) and estradiol concentrations of 13.1 +/- 0.7 pg/ml. However, the fact that the levels of estrogen in breast tumor tissue are an order of magnitude higher than plasma levels suggested the possibility of in situ estrogen production. To address this possibility, we measured several enzymes involved in estradiol biosynthesis in human tumors. Forty-eight of 61 tumors contained aromatase (estrogen synthetase) activity ranging from 5-80 pg/gm protein per hour. By comparison, the levels of estrone sulfatase were 10(6) higher, ranging from 0.8-125 micrograms/gm protein per hour. Because the sulfatase enzyme was of lower affinity (i.e., Km = 27 microM) than that of aromatase (i.e., 0.027 microM), the amount of estrogen formed under conditions of similar substrate concentrations was compared and found to be 10-fold higher via the sulfatase enzyme. In 41 additional tumors, the 17 beta-hydroxysteroid dehydrogenase enzyme, catalyzing the conversion of estrone to estradiol, was uniformly present. To test the biologic relevance of the estrone sulfate to estrone to estradiol pathway, estrogen-dependent nitrosomethylurea rat mammary tumors were grown in soft agar in the presence of estrone sulfate. Concentrations of estrone sulfate of 10(-6) microM significantly (p less than 0.01) stimulated colony formation in this system in which 75.5-98.6% of estrone sulfate was converted to estrone and 0.2 to 6% to estradiol. These data support the hypothesis that mammary carcinomas can synthesize estradiol in situ from circulating estrogen precursor and that local conversion is biologically important. On the basis of comparative data, the estrone sulfate to estrone to estradiol pathway is quantitatively more important than that involving androstenedione to estrone to estradiol.
Assuntos
Aromatase/metabolismo , Neoplasias da Mama/metabolismo , Estrogênios/biossíntese , Sulfatases/metabolismo , 17-Hidroxiesteroide Desidrogenases/análise , Adrenalectomia , Aminoglutetimida/uso terapêutico , Androstenodiona/análogos & derivados , Androstenodiona/farmacologia , Animais , Estradiol/análise , Estrona/análogos & derivados , Estrona/análise , Feminino , Humanos , Hidrocortisona/uso terapêutico , Neoplasias Mamárias Experimentais/metabolismo , Neoplasias Hormônio-Dependentes/metabolismo , Ratos , Testolactona/análogos & derivados , Testolactona/farmacologiaRESUMO
In this study, we assessed the rate of estradiol degradation via the 17 beta-hydroxysteroid dehydrogenase (HSD) enzyme in breast tumors from postmenopausal women. We initially studied the effects of time, level of enzyme activity, amount of tissue assayed, and substrate concentration on the linearity of conversion of estradiol to estrone in breast tumor homogenates. The reaction was demonstrated to be linear when less than 15% conversion of estradiol to estrone occurred over 30 min with homogenates produced from 2.5 mg of tissue. Detailed kinetic experiments demonstrated the presence of two classes of enzyme activity, one with high affinity and the other with low affinity. In 83% of the tumors examined, the high affinity form was present and had a median Km of 0.62 microM and Vmax of 82 nmol/g protein/h. In 29 tumors, HSD activity could be precisely quantified and correlated with clinical parameters. No statistically significant correlation of enzyme activity with estrogen receptor (r2 = 0.06) or progesterone receptor (r2 = 0.006) or with patient age could be detected (r2 = 0.001). In 12 additional tumors, activity exceeded 15% conversion of estradiol to estrone at 30 min and precise quantitation was not possible. The average content of progesterone receptor was similar for these 12 tumors as for the 19 with lower HSD activity. However, estrogen receptor content and patient age were lower in the group with high HSD activity. The finding of a high affinity form of HSD in this study provides support for the biological importance of this enzyme in breast cancer tissues.
Assuntos
17-Hidroxiesteroide Desidrogenases/metabolismo , Neoplasias da Mama/enzimologia , Estradiol/biossíntese , Estrona/biossíntese , Humanos , CinéticaRESUMO
Medroxyprogesterone acetate (MPA)3, a potent synthetic progestin, was employed as the 3H-ligand to investigate the status of the rabbit uterine progestin receptor under various hormonal conditions. MPA dissociated slowly from the cytosol and nuclear progestin receptor obtained from ovariectomized, estrogen-primed rabbits. This slow rate of dissociation permitted a partial characterization of the receptors in pregnant and immature, unprimed rabbit uteri. High affinity progestin binding to 6--7 S and 4 S macromolecular species was detected in cytosol and nuclear extracts, respectively, from these animals. In contrast to the 3 peaks of cytosol receptor activity from estrogen-primed animals which were eluted stepwise from DEAE-cellulose columns, predominantly 2 peaks were obtained with cytosols from pregnant and immature, unprimed uteri. Further studies are required to determine to what extent progestin action is modified through interaction of these binding components with themselves and other cellular proteins.
Assuntos
Medroxiprogesterona/metabolismo , Prenhez , Útero/metabolismo , Envelhecimento , Animais , Ligação Competitiva , Castração , Núcleo Celular/metabolismo , Citosol/metabolismo , Dexametasona/farmacologia , Feminino , Cinética , Gravidez , Progesterona/metabolismo , Coelhos , Útero/crescimento & desenvolvimentoAssuntos
Progesterona/metabolismo , Ligação Proteica , Útero/metabolismo , Animais , Sítios de Ligação , Castração , Centrifugação com Gradiente de Concentração , Citoplasma , Estrogênios/farmacologia , Etilmaleimida/farmacologia , Feminino , Glicerol/farmacologia , Cinética , Camundongos , Ovário/fisiologia , Ratos , Receptores de Droga , Soroglobulinas/metabolismo , Sacarose , TrítioAssuntos
Endométrio/efeitos dos fármacos , Glicogênio/biossíntese , Progesterona/farmacologia , Animais , Meios de Cultura , Cicloeximida/farmacologia , Dactinomicina/farmacologia , Dietilestilbestrol/farmacologia , Endométrio/metabolismo , Feminino , Cobaias , Insulina/farmacologia , Medroxiprogesterona/análogos & derivados , Medroxiprogesterona/farmacologia , Técnicas de Cultura de Órgãos , Ovário/fisiologia , Fatores de TempoAssuntos
Dietilestilbestrol/farmacologia , Progesterona/farmacologia , Receptores de Superfície Celular/efeitos dos fármacos , Útero/metabolismo , Animais , Sítios de Ligação , Castração , Cromatografia , Citosol/efeitos dos fármacos , Citosol/metabolismo , Feminino , Cobaias , Meia-Vida , Ovário/fisiologia , Progesterona/metabolismo , Ligação Proteica , Dióxido de Silício , Fatores de Tempo , Trítio , Útero/citologia , Útero/efeitos dos fármacosRESUMO
Photoaffinity labeling with [17 alpha-methyl-3H]promegestone ([ 3H]R5020) is an effective technique for the covalent labeling of the progesterone receptor (PR), which allows monitoring of the steroid receptor complex under denaturing conditions. The present study was initiated to evaluate whether photolabeled PR could be used also as a marker for PR under nondenaturing conditions. Accordingly, the effect of irradiation on each component of the reaction was evaluated separately. When [3H]R5020 alone was irradiated, there was a rapid (less than 5 min), light dependent destruction of [3H]R5020, as evident from increased formation of a more polar tritiated product on TLC and a concomitant decrease in the ability of the irradiated preparation to bind to PR. When rabbit uterine PR was irradiated in the absence of steroid, a gradual decrease in the binding capacity was observed, reaching 70% of the nonirradiated control in 10 min. The optimal irradiation time for covalent [3H]R5020-PR complex formation was determined by irradiation for up to 5 min, and separation of the products by sodium dodecylsulfate (SDS)-polyacrylamide gel electrophoresis. Specific labeling of proteins of Mr 116,000 and 85,000 was observed, with the rate of labeling of the two being similar, and reaching a plateau by 4 min of irradiation. The photolabeling efficiency ranged from 2 to 12%. Sucrose gradient ultracentrifugation of photolabeled PR revealed that both the irradiated sample and the nonirradiated control sedimented to the same position. Subsequent SDS-polyacrylamide gel electrophoresis of the sucrose gradient peak from the photolabeled sample showed the presence of both labeled proteins of Mr 116,000 and 85,000. In addition, photolabeled rabbit uterine PR (Mr 116,000 and 85,000) could be immunoprecipitated with a guinea pig antiserum raised against rabbit uterine PR. Analysis of the photoaffinity labeling procedure in our system revealed that the photodestruction of [3H]R5020 was very rapid. However, maximal labeling with [3H]R5020 was obtainable with minimal photodestruction of PR which suggests that photolabeled receptor can be used as a marker for PR under nondenaturing conditions.