RESUMO
Aurintricarboxylic acid (ATA), an endonuclease inhibitor, prevents the death of a variety of cell types in culture. Previously we have shown that ATA, similar to insulin-like growth factor I (IGF-I), protected MCF-7 cells against apoptotic death induced by the protein synthesis inhibitor cycloheximide. Here we show that ATA and a polysulfonated aromatic compound, Evans blue (EB), similar to IGF-I, promote survival and increase proliferation of MCF-7 cells in serum-free culture medium. This may suggest a common signaling pathway shared by the aromatic polyanions and IGF-I. Therefore, the ability of these aromatic compounds to activate the signal transduction pathway of IGF-I was examined. We found that ATA and EB mimicked the IGF-I effect on tyrosine phosphorylation of the IGF-I receptor (IGF-IR) and its major substrates, insulin receptor substrate-1 (IRS-1) and IRS-2; induced the association of these substrates with phosphatidylinositol 3-kinase and Grb2; and activated Akt kinase and p42/p44 mitogen-activated protein kinases. ATA and EB competed for IGF-I binding to the IGF-IR. ATA was found to be selective for the IGF-IR, whereas EB also activated the insulin receptor. Upon fractionation of commercial ATA by size exclusion chromatography, we found that fractions that enhanced the intensity of tyrosyl-phosphorylated IRS-1/IRS-2 also increased the survival of MCF-7 cells in the presence of cycloheximide, whereas fractions devoid of IRS phosphorylation activity had no survival ability. Taken together, these results suggest that the survival/proliferation-promoting effects of ATA and EB in MCF-7 cells are transduced via the IGF-IR signaling pathway.
Assuntos
Apoptose/efeitos dos fármacos , Ácido Aurintricarboxílico/farmacologia , Azul Evans/farmacologia , Fator de Crescimento Insulin-Like I/fisiologia , Proteínas Serina-Treonina Quinases , Transdução de Sinais/efeitos dos fármacos , Células 3T3 , Animais , Ácido Aurintricarboxílico/metabolismo , Divisão Celular/fisiologia , Sobrevivência Celular/fisiologia , Ativação Enzimática , Azul Evans/metabolismo , Humanos , Proteínas Substratos do Receptor de Insulina , Peptídeos e Proteínas de Sinalização Intracelular , Camundongos , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Fosfoproteínas/metabolismo , Fosforilação , Proteínas Proto-Oncogênicas/fisiologia , Proteínas Proto-Oncogênicas c-akt , Receptor IGF Tipo 1/metabolismo , Proteínas Recombinantes , Células Tumorais Cultivadas/patologia , Células Tumorais Cultivadas/fisiologia , Tirosina/metabolismoRESUMO
Previously, we have shown that IGF-1, the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) and aurintricarboxylic acid (ATA) protected MCF-7 cells against death induced by the protein synthesis inhibitor cycloheximide (CHX). We proposed that phosphorylation of a putative cellular proteins(s) may be involved in this survival mechanism. In the present study we investigated the ability of several agents to induce phosphorylation of cellular proteins and correlated this ability to their survival effect. We found that TPA, ATA, and IGF-1 increased the degree of phosphorylation of a 27-kDa protein in a dose- and time-dependent manner in CHX-treated MCF-7 cells. The ED50 values observed were 25 ng/ml, 40 micrograms/ml and 15 ng/ml for TPA, ATA, and IGF-1, respectively. The effect was measured upon 10 min of cell treatment with each agent; it reached maximum at 60 min and thereafter decreased continuously to control levels. The 27-kDa protein was found in the cytosolic fraction as a phosphorylated serine residue. Further characterization with two-dimensional electrophoresis indicated that the 27-kDa phosphorylated serine residue. Further characterization with two-dimensional electrophoresis indicated that the 27-kDa phosphoprotein was resolved into two isoforms with pI 5.7 and 5.9. Such characteristics were observed for the small molecular weight heat shock protein HSP27. Indeed, a single band of 27 kDa was detected immunologically with rabbit polyclonal anti-human HSP27. The inactive phorbol ester alpha TPA, epidermal growth factor (EGF), and 8-bromoadenosine 3'5'-cyclic monophosphate (Br-cAMP) did not increase phosphorylation of the 27-kDa protein. Cell survival was measured by exposure of the CHX-pretreated cells to increasing concentrations of the various agents for 60 min, followed by a further incubation for 48 h in the presence of CHX only. TPA, ATA, and IGF-1 were found to enhance cell survival, whereas alpha-TPA, EGF and Br-cAMP did not. Our results indicate a correlation between phosphorylation of a 27-kDa protein, probably HSP27, and enhanced cell survival, suggesting a role for this phosphoprotein in the survival mechanism.
Assuntos
Neoplasias da Mama/metabolismo , Carcinoma/metabolismo , Cicloeximida/farmacologia , Proteínas de Choque Térmico/metabolismo , Inibidores da Síntese de Proteínas/farmacologia , 8-Bromo Monofosfato de Adenosina Cíclica/farmacologia , Ácido Aurintricarboxílico/farmacologia , Neoplasias da Mama/patologia , Carcinoma/patologia , Sobrevivência Celular , Citosol/química , Fator de Crescimento Epidérmico/farmacologia , Proteínas de Choque Térmico/química , Humanos , Fator de Crescimento Insulin-Like I/farmacologia , Ponto Isoelétrico , Mitógenos/farmacologia , Peso Molecular , Fosfoproteínas/análise , Fosforilação/efeitos dos fármacos , Fosfosserina/análise , Acetato de Tetradecanoilforbol/farmacologia , Fatores de Tempo , Células Tumorais CultivadasRESUMO
In the present study we investigated the ability of several diverse agents to inhibit MDA-231 cell death induced by two different protein synthesis inhibitors, cycloheximide (CHX) and ricin. Cell death was evaluated by several techniques: trypan blue staining, determination of the released lactic dehydrogenase, transmission electron microscopy, and DNA fragmentation. Results from DNA gel electrophoresis and electron microscopy suggest a mechanism of death by apoptosis which terminates in necrosis. Approximately 60% of cell death was induced either by a continuous exposure to 30 micrograms/ml CHX for 48 hr or by a 1-hr exposure to 250 pg/ml ricin followed by a subsequent incubation of 48 hr in the absence of the drug. Cell survival, in the protein synthesis-inhibited cells, was enhanced by the following diverse agents: the growth factors EGF (20 ng/ml) and IGF-1 (20 ng/ml), the protein kinase C activator 12-0-tetradecanoyl-phorbol-13-acetate (5 ng/ml), the protein kinase A activator 8-bromoadenosine 3':5'-cyclic monophosphate (650 micrograms/ml), the nuclease inhibitor aurintricarboxylic acid (100 micrograms/ml), and fetal bovine serum (5%). The survival agents that stimulated protein synthesis in the control untreated cells had no effect on the CHX-inhibited protein synthesis, which indicates that new protein synthesis is not required for cell survival. The same survival agents attenuated the continuous decrease in protein synthesis in the ricin-exposed cells; therefore, the involvement of new protein synthesis in the survival mechanism could not be excluded. The protein kinase C inhibitor staurosporine blocked, in a dose-dependent manner, the survival effect of 12-0-tetradecanoyl-phorbol-13-acetate and EGF, but not that of aurintricarboxyclic acid or fetal bovine serum, in the protein synthesis-inhibited cells. These results provide evidence for several distinctive pathways, the activation of which inhibits MDA-231 cell death induced by protein synthesis inhibitors. Some of these pathways involved activation of protein kinases, probably protein kinase C.
Assuntos
Apoptose/efeitos dos fármacos , Inibidores da Síntese de Proteínas/farmacologia , Alcaloides/farmacologia , Neoplasias da Mama/patologia , Cicloeximida/farmacologia , DNA/metabolismo , Fator de Crescimento Epidérmico/farmacologia , Feminino , Humanos , Biossíntese de Proteínas , Ricina/farmacologia , Estaurosporina , Acetato de Tetradecanoilforbol/farmacologia , Células Tumorais CultivadasRESUMO
Previously we have shown that IGF-I protected MCF-7 cells against death induced by the protein synthesis inhibitor cycloheximide (CHX). In the present study we investigated the ability of protein kinase C activator 12-0-tetradecanoyl-phorbol-13-acetate (TPA), the protein kinase A activator 8-bromoadenosine 3'5'-cyclic monophosphate (Br-cAMP), and the enzyme inhibitor aurintricarboxylic acid (ATA) to protect MCF-7 cells against death, due to a continuous presence of CHX. Cell death was evaluated after 48 h of incubation by several techniques (trypan blue staining, release of lactic dehydrogenase, cellular ATP content, transmission electron microscopy, and DNA fragmentation). Apoptosis which terminates in necrosis, characterized this mode of cell death. TPA and ATA at optimal concentrations of 40 ng/ml and 100 micrograms/ml, respectively, reduced cell death to the control level (without CHX), while Br-cAMP at an optimal concentration of 650 micrograms/ml reduced cell death only partially. IGF-1, TPA, and ATA, which stimulated protein synthesis in the control MCF-7 cells, had no effect on protein synthesis in the CHX-treated cells, indicating that the survival effect is not due to new protein synthesis. The protein kinase C inhibitor staurosporine blocked the survival effect of TPA and IGF-1 in a dose-dependent manner, however did not affect the survival effect of ATA. The tyrosine kinase inhibitor genistein blocked the survival effect of IGF-1, but not that of TPA and ATA. Our results provide evidence for several distinctive pathways, the activation of which protects MCF-7 cells against death, due to protein synthesis inhibition.
Assuntos
Neoplasias da Mama/metabolismo , Inibidores da Síntese de Proteínas/farmacologia , Células Tumorais Cultivadas/efeitos dos fármacos , Células Tumorais Cultivadas/patologia , 8-Bromo Monofosfato de Adenosina Cíclica/farmacologia , Ácido Aurintricarboxílico/farmacologia , Neoplasias da Mama/patologia , Sobrevivência Celular/efeitos dos fármacos , Cicloeximida/farmacologia , Dano ao DNA , Genisteína , Humanos , Fator de Crescimento Insulin-Like I/farmacologia , Isoflavonas/farmacologia , Microscopia Eletrônica , Acetato de Tetradecanoilforbol/farmacologiaRESUMO
The involvement of growth factors in cell survival in the presence of anticancer drugs was investigated. Cell death was induced in the human breast cancer cell line MCF-7, by the structurally and mechanistically unrelated chemotherapeutic drugs puromycin, actinomycin D, 5-fluorouracil, cisplatin, and adriamycin. The effect of insulin-like growth factor-1 (IGF-1), epidermal growth factor (EGF), and insulin on cell death was evaluated by two different methods: (1) trypan blue dye exclusion test and (2) lactic dehydrogenase release into the culture medium. IGF-1 inhibited cell death induced by each of the diverse drugs in a concentration-dependent manner reaching a maximal effect at 40 ng/ml. Insulin mimicked the effect of IGF-1 only at supraphysiological concentration with an optimal effect at 10,000 ng/ml. EGF had no effect on cell death up to 100 ng/ml. Our finding that IGF-1 specifically enhanced MCF-7 cell survival in the presence of different anticancer drugs suggests the involvement of growth factors in the mechanism of drug resistance.
Assuntos
Antineoplásicos/farmacologia , Fator de Crescimento Insulin-Like I/farmacologia , Neoplasias da Mama/patologia , Morte Celular/efeitos dos fármacos , Resistência a Medicamentos , Fator de Crescimento Epidérmico/farmacologia , Humanos , Insulina/farmacologia , Células Tumorais CultivadasRESUMO
The ability of epidermal growth factor (EGF), insulin-like growth factor-1 (IGF-1), insulin, 12-O-tetradecanoylphorbol-13-acetate (TPA), and aurintricarboxylic acid (ATA) to protect the human breast cancer cell line MDA-231 from death induced by the anticancer drug adriamycin was investigated. Cell death was induced in the MDA-231 cells either by a short-time exposure to a high dose of adriamycin (2 micrograms.ml-1.1h-1) and further culturing in the absence of the drug, or by continuous exposure to a low dose of adriamycin (0.3 micrograms/ml). Cell death was evaluated after 48 h of incubation by several techniques (trypan blue dye exclusion, lactic dehydrogenase activity, cellular ATP content, transmission electron microscopy, and DNA fragmentation). EGF, TPA, and ATA, each at an optimal concentration of 20 ng/ml, 5 ng/ml, and 100 micrograms/ml respectively, substantially enhanced survival of cells exposed either to a high or low dose of adriamycin. Neither IGF-1 nor insulin, each at concentrations of 20 ng/ml, had an effect on cell survival. The three survival factors enhanced protein synthesis in the untreated cells and attenuated the continuous decrease in protein synthesis in the adriamycin-treated cells. Moreover, the three survival factors protected the MDA-231 cells from death in the absence of protein synthesis (cycloheximide 30 micrograms/ml). These results suggest that EGF, TPA, and ATA promote survival of adriamycin pretreated cells by at least two mechanisms: enhancement of protein synthesis and by a protein synthesis independent process, probably a posttranslational modification effect.
Assuntos
Ácido Aurintricarboxílico/farmacologia , Neoplasias da Mama/patologia , Sobrevivência Celular/efeitos dos fármacos , Doxorrubicina/farmacologia , Fator de Crescimento Epidérmico/farmacologia , Acetato de Tetradecanoilforbol/farmacologia , Trifosfato de Adenosina/metabolismo , Apoptose , Cicloeximida/farmacologia , DNA de Neoplasias/metabolismo , Doxorrubicina/administração & dosagem , Humanos , Fator de Crescimento Insulin-Like I/farmacologia , L-Lactato Desidrogenase/metabolismo , Microscopia Eletrônica , Biossíntese de Proteínas , Células Tumorais CultivadasRESUMO
In the present study, we investigated the ability of epidermal growth factor (EGF), insulin-like growth factor-1 (IGF-1), and insulin to protect the human breast cancer cell line MDA-231 from death induced by the antitumor drug actinomycin D (ACT-D). ACT-D is an inhibitor of RNA and protein synthesis, and its cytotoxicity may result due to continuous depletion in some vital protein molecules. Cell death was induced in the MDA-231 cells by either continuous exposure to a low dose of ACT-D (0.2 microgram/ml), or by a short-time exposure to a high dose of ACT-D (2 micrograms/ml) and further culturing in the absence of the drug. Cell death was evaluated by the trypan blue dye exclusion test, the release of lactic dehydrogenase into the culture medium, and the depletion in the cellular ATP content. EGF and IGF-1, each at an optimal concentration of 20 ng/ml, enhanced substantially survival of cells exposed either to a low or a high dose of ACT-D. The combination of EGF (10 ng/ml) and IGF-1 (10 ng/ml) had an additive survival effect, which proposes that each of the growth factors enhanced survival by a distinct pathway. Insulin up to 40 ng/ml had no effect on cell survival. Pretreatment of the cells for 1 to 5 h with EGF and IGF-1 protected cells from the cytotoxic effect of ACT-D. Exposure of the cells to 2 micrograms/ml of ACT-D for 1 h resulted in a drastic inhibition in uridine incorporation and only in a slight inhibition in leucine incorporation.(ABSTRACT TRUNCATED AT 250 WORDS)
Assuntos
Dactinomicina/antagonistas & inibidores , Fator de Crescimento Epidérmico/fisiologia , Fator de Crescimento Insulin-Like I/fisiologia , Neoplasias da Mama/patologia , Neoplasias da Mama/ultraestrutura , Morte Celular/efeitos dos fármacos , Morte Celular/fisiologia , Dactinomicina/farmacologia , Resistência a Medicamentos , Humanos , Fatores de Tempo , Células Tumorais CultivadasRESUMO
Prolonged exposure of cells to the potent protein synthesis inhibitor cycloheximide (CHX) terminates in cell death. In the present study we investigated the effect of epidermal growth factor (EGF), insulin-like growth factor-1 (IGF-1), and insulin on cell death induced by CHX in the human cancerous cell lines MDA-231 and MCF-7 (breast), KB (oral epidermoid), HEP-2 (larynx epidermoid), and SW-480 (colon), and correlated this effect to the inhibition rate of protein synthesis. Cell death was evaluated by measuring either dead cells by trypan blue dye exclusion test or by the release of lactic dehydrogenase into the culture medium. CHX was shown to induce cell death in a concentration (1 to 60 micrograms/ml) and time (24 to 72 h)-dependent manner in each of the five cell lines. EGF at physiologic concentrations (2 to 40 ng/ml) reduced cell death close to control level (without CHX) in the cell lines HEP-2, KB, MDA-231, and SW-480, but had almost no effect on cell death in the MCF-7 cells. IGF-1 at physiologic concentrations (2 to 40 ng/ml) reduced cell death nearly to control level in the MCF-7 cells, but had only a partial effect in the other four cell lines. Insulin at supraphysiologic concentration (10,000 ng/ml) mimicked the effect of IGF-1 in each of the cell lines. CHX at concentrations that induced about 60% cell death, inhibited about 90% of protein synthesis as measured by [3H]leucine incorporation. Protein synthesis remained inhibited although cell viability was preserved by EGF or IGF-1. These results indicated that the mechanism by which EGF or IGF-1 preserve cell viability does not require new protein synthesis and may be mediated via a posttranslational modification effect.
Assuntos
Sobrevivência Celular/efeitos dos fármacos , Fator de Crescimento Epidérmico/farmacologia , Fator de Crescimento Insulin-Like I/farmacologia , Células Tumorais Cultivadas/efeitos dos fármacos , Neoplasias da Mama , Neoplasias do Colo , Cicloeximida/antagonistas & inibidores , Humanos , Leucina/metabolismo , Neoplasias Bucais , Biossíntese de ProteínasRESUMO
Prolonged exposure of cells to the potent protein synthesis inhibitor cycloheximide terminates in cell death. In the present study we investigated the effect of insulinlike growth factor-1, insulin, and epidermal growth factor on cell death induced by cycloheximide in the confluent MCF-7 cells, and correlated this effect to the inhibition rate of protein synthesis. Cell death was evaluated by measuring either dead cells by the trypan blue dye exclusion test or by the release of lactic dehydrogenase into the culture medium. After 48 h incubation, cycloheximide (10 to 50 micrograms/ml) was shown to induce cell death in a concentration-dependent manner. Insulinlike growth factor-1, at physiologic concentrations (0.2 to 5 ng/ml), reduced this cell death. Insulin at supraphysiologic concentrations (1 to 10 micrograms/ml) mimicked the effect of insulinlike growth factor-1, whereas epidermal growth factor (10 to 50 ng/ml) had no effect. More than 90% of protein synthesis measured by [3H]leucine incorporation was inhibited by 10 to 50 micrograms/ml cycloheximide. Insulinlike growth factor-1 and insulin at the concentrations that reduced cell death to control level, had no effect on the protein synthesis inhibition rate induced by cycloheximide. These results indicate that inhibition of cell death by insulinlike growth factor-1 does not depend on protein synthesis and may be mediated via a posttranslational modification effect.
Assuntos
Neoplasias da Mama/patologia , Cicloeximida/farmacologia , Fator de Crescimento Insulin-Like I/farmacologia , Neoplasias da Mama/metabolismo , Morte Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Fator de Crescimento Epidérmico/farmacologia , Humanos , Insulina/farmacologia , L-Lactato Desidrogenase/metabolismo , Leucina/metabolismo , Modelos Biológicos , Proteínas/metabolismo , Trítio , Células Tumorais Cultivadas/efeitos dos fármacos , Células Tumorais Cultivadas/patologiaRESUMO
Continuous exposure of cells to cycloheximide (CHM) terminates in cell death. This may result from CHM's inhibition of protein synthesis. In the present study we investigated the effect of serum and insulin on cell death induced by CHM in the human breast cancer cell line MCF-7, and correlated this effect to the inhibition of protein synthesis. Cell death was evaluated by measuring either dead cells by the trypan blue dye exclusion test or by the release of lactic dehydrogenase into the culture medium. CHM (0.1 to 50 micrograms/ml) was shown to induce cell death in a time- and concentration-dependent manner. Including either fetal bovine serum or insulin in the culture medium inhibited this cell death in a concentration-dependent manner. Protein synthesis as measured by [3H]leucine incorporation was inhibited by the increasing concentration of CHM. However, fetal bovine serum and insulin did not alter the protein synthesis inhibition rate induced by CHM. These results indicate that inhibition of protein synthesis is not enough for cell death to proceed. Insulin or factors present in serum may stabilize some crucial cell proteins (key enzymes, cytoskeletal or membrane components) which are vital for cell life.
Assuntos
Proteínas Sanguíneas/farmacologia , Neoplasias da Mama/patologia , Cicloeximida/farmacologia , Insulina/farmacologia , Neoplasias da Mama/enzimologia , Neoplasias da Mama/metabolismo , Morte Celular/efeitos dos fármacos , Citotoxinas/antagonistas & inibidores , Relação Dose-Resposta a Droga , Humanos , L-Lactato Desidrogenase/metabolismo , Leucina/metabolismo , Proteínas/metabolismo , Fatores de Tempo , Trítio , Azul Tripano , Células Tumorais Cultivadas/enzimologia , Células Tumorais Cultivadas/metabolismo , Células Tumorais Cultivadas/patologiaRESUMO
OBJECTIVE: To determine the effects of single dose tamoxifen on plasma estrogen (E)-binding equivalents and endometrial estradiol (E2) and progesterone (P) receptors. DESIGN: Controlled clinical study. SETTING: Normal human volunteers were studied in an academic research environment. PATIENTS: Premenopausal and postmenopausal women with histologically normal endometrium undergoing curettage or hysterectomy were selected. INTERVENTIONS: Tamoxifen was administered orally; blood and endometrial samples were collected 4 to 96 hours after tamoxifen administration. MAIN OUTCOME MEASURES: Plasma E-binding equivalents, endometrial cytosolic and nuclear E2 and P receptors. RESULTS: (1) Plasma E-binding equivalents increased eightfold at 4 to 24 hours of tamoxifen administration and declined exponentially thereafter, reaching control levels at 73 to 96 hours. Plasma E-binding equivalents were not affected by endogenous E2 levels. (2) Endometrial total E2 and P receptor levels increased in all women 2.9 to 19.2-fold after tamoxifen. (3) Tamoxifen resulted in an increase in the fraction of the E2 receptor measured in the nuclear extract 2.1 to 7.5-fold in midcycle, secretory, and menopausal endometria but not in proliferative endometrium. CONCLUSIONS: (1) Tamoxifen has an E2 agonistic effect on histologically normal human endometrium. (2) Irrespective of the total level of the endometrial E2 receptor, the nuclear capacity of that receptor in vivo is limited (approximately 75% to 80% of the total level).
Assuntos
Endométrio/efeitos dos fármacos , Estradiol/fisiologia , Estrogênios/metabolismo , Menopausa , Tamoxifeno/farmacologia , Adulto , Núcleo Celular/metabolismo , Citosol/metabolismo , Endométrio/ultraestrutura , Feminino , Humanos , Pessoa de Meia-Idade , Receptores de Estradiol/metabolismo , Receptores de Progesterona/metabolismo , Valores de Referência , Distribuição TecidualRESUMO
UNLABELLED: The effect of castration on the incorporation of [35S]methionine into secreted proteins in 7,12-dimethylbenz(a)anthracene (DMBA)-induced rat mammary tumors, was investigated. Biopsy specimens were obtained from 19 tumors, 0, 24, 48, 72, and 96 h after castration. In 14 tumors, castration induced an increase in the incorporation (mean of 5-fold), reaching the maximal level after 24 h (3 tumors), 48 h (7 tumors), 72 h (3 tumors), and after 96 h (1 tumor). In three tumors castration did not alter the incorporation rate, while in two tumors incorporation declined immediately after castration. One-dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis of the labeled secreted protein showed that castration did not decrease or increase significantly the incorporation of [35S]methionine into any of the major labeled proteins. CONCLUSION: tumor regression following hormonal deprivation is apparently preceded by an increased synthesis of secreted proteins. However, no qualitative differences in any major labeled proteins could be observed.
Assuntos
Neoplasias Mamárias Experimentais/metabolismo , Proteínas de Neoplasias/biossíntese , 9,10-Dimetil-1,2-benzantraceno , Animais , Feminino , Peso Molecular , Proteínas de Neoplasias/química , Ovariectomia , Ratos , Fatores de TempoRESUMO
The susceptibility of the progesterone receptor, liganded either by the antiprogestin RU 486 or by the progestin ORG 2058, to chymotrypsin and trypsin degradation was investigated. The nuclear fraction was isolated from T47D cells previously exposed either to 0.1 microM [3H]RU 486 or to 0.1 microM [3H]ORG 2058. The proteolytic digestion was performed on the micrococcal nuclease hydrolysate. The molecular weights of the receptor fragments were calculated, in high salt buffer, from the sedimentation coefficients determined on a sucrose gradient and from the Stokes radii estimated by gel filtration on an Agarose A-0.5 m column. Micrococcal nuclease solubilized receptor forms with molecular weights of 80,000 and 75,000 for the antiprogestin- or progestin-liganded receptor, respectively. Chymotrypsin degraded these receptor forms to fragments with molecular weights of 23,000 either for the antiprogestin- or progestin-liganded receptor. Similar molecular weights of 23,000 were calculated for the progesterone receptor liganded either by the antiprogestin RU 436 or the progestin ORG 2058 following trypsin cleavage. We conclude that the degradation pattern of the progesterone receptor liganded either by the antiprogestin RU 486 or the progestin ORG 2058 following chymotrypsin or trypsin digestion seems to be similar.
Assuntos
Núcleo Celular/metabolismo , Quimotripsina/metabolismo , Mifepristona/metabolismo , Pregnenodionas/metabolismo , Receptores de Progesterona/metabolismo , Tripsina/metabolismo , Linhagem Celular , Centrifugação com Gradiente de Concentração , Fenômenos Químicos , Físico-Química , Nuclease do Micrococo/metabolismo , Peso Molecular , Fragmentos de Peptídeos/metabolismo , Congêneres da Progesterona/metabolismoRESUMO
Total progesterone receptor levels (TRP) were measured in various tissues and segments of normal human uteri in basal conditions (late proliferative phase, control group, n = 6) and after short term estrogen administration (test group, n = 8). Similar trends of distribution were observed in both groups in basal endometrial TRP levels along the longitudinal axis, i.e. the highest levels were found at the height of the body and the lowest at the height of the internal os. Differences were found in the trends of distribution of the TRP levels between the two groups along the transverse axis of the uterus at the height of the fundus. In the control group TRP levels in the functional endometrium were higher than those in the basal endometrium and myometrium. In the test group, however, the trend was reversed. Since the RP is estradiol induced and dependent, these results indicate the possibility of a selective estrogen effect on various segments or tissues in the human uterus.
Assuntos
Estrogênios/farmacologia , Receptores de Progesterona/análise , Útero/análise , Adulto , DNA/análise , Endométrio/análise , Estradiol/farmacologia , Estrogênios/administração & dosagem , Feminino , Humanos , Pessoa de Meia-Idade , Miométrio/análise , Fatores de TempoRESUMO
Estradiol receptor (RE2) levels were measured in normal (n = 22) and pathological (n = 14) human endometria and in various segments and tissues of the same uteri (n = 8). The highest total estradiol levels (TRE2) were found in midcycle normal cyclic endometria and the lowest in the secretory phase of normal cyclic endometria and in normal postmenopausal endometria (the TRE2 levels in the latter two groups were within similar ranges). The highest nuclear RE2 levels were found in proliferative-phase normal cyclic endometria. TRE2 levels in pathological postmenopausal endometria were high, in the range of those found in proliferative normal cyclic endometria, but the nuclear RE2 levels were low as compared with those in normal proliferative endometria. Examination of the distribution of the TRE2 levels in various segments and tissues of normal proliferative uteri revealed similar trends in all cases as follows: the highest levels were measured in the functional endometrium (fundus) and the lowest in the myometrium (fundus). Basal endometrial TRE2 levels decreased along the longitudinal axis from fundus to isthmus. In 4 cases, TRE2 levels in leiomyoma tissues were higher than those in the corresponding myometrium. These results indicate the possibility of a differing regulating mechanism of the RE2 in various segments or tissues of the human uterus.
Assuntos
Receptores de Estradiol/análise , Útero/análise , Adulto , Feminino , Humanos , Pessoa de Meia-Idade , Doenças Uterinas/patologiaRESUMO
Total estradiol and progesterone receptor levels and DNA concentrations were measured in endometria of four women with nonuniform postovulatory delayed maturation of the epithelium. All four women underwent curettage on Days 21 to 23 of their cycle. The results in these women were compared with results obtained from a control group of women with histologically normal cyclic endometrium. In three women with corpus luteum defect, total estradiol and progesterone receptor levels and DNA concentrations were in the range of midcycle or early secretory normal (control) endometria. In one patient with a normally functioning corpus luteum, the above parameters were similar to those found in the late secretory phase of controls. Summation of the results of the present study along with those previously reported in endometria of women, both with uniform and nonuniform delayed maturation, indicate the possibility of endometrial postacceptorial progesterone defect, which may either be secondary to corpus luteum defect or a result of primary endometrial defect.
Assuntos
DNA/metabolismo , Endométrio/metabolismo , Fase Luteal , Receptores de Estradiol/metabolismo , Receptores de Estrogênio/metabolismo , Receptores de Progesterona/metabolismo , Adulto , Endométrio/patologia , Endométrio/fisiopatologia , Epitélio/fisiopatologia , Feminino , Humanos , Pessoa de Meia-IdadeRESUMO
The assumption that a different conformational form was induced in the nuclear estrogen receptor following binding by antiestrogens compared to estrogens was studied by analysing the proteolytic fragments of the receptor following limited digestion with chymotrypsin and trypsin. Nuclei were isolated from MCF-7 cells previously exposed to [3H] 4-OHTAM. The proteolytic digestion was performed either on the micrococcal nuclease hydrolysate or on intact nuclei. The molecular weights (Mr) were calculated from the sedimentation coefficients (S) determined on a sucrose gradient and from the Stokes radii (Rs) estimated by gel filtration. Digestion of the nuclei with micrococcal nuclease solubilized a receptor form of Mr = 155,000. This receptor form was degraded by chymotrypsin to a receptor of Mr = 63,000 which could not be further dissociated by 0.4 M KCl and 3 M urea. A similar receptor molecule was released by chymotrypsin from intact nuclei. Digestion of the micrococcal nuclease hydrolysate with trypsin degraded the receptor to a form of a Mr = 67,000 which could not be further dissociated by 0.4 M KCl and 3 M urea. Digestion of intact nuclei with trypsin followed by micrococcal nuclease, solubilized a receptor form of Mr = 80,000 which could be further dissociated with 0.4 M KCl and 3 M urea to a receptor form of Mr = 67,000. This trypsin degraded receptor form seems to be similar in Mr to the chymotrypsin degraded form. On the other hand different receptor fragments of Mr = 33,000 and Mr = 60,000 were excised by chymotrypsin and trypsin respectively from the estradiol ligated estrogen receptor. (Geier et al., J. steroid Biochem. 26 [1987] 35-40.) These results support the assumption of a different conformational form for the antiestrogen ligated receptor, compared to the estrogen ligated receptor since they were differentially susceptible to proteolytic degradation by chymotrypsin.
Assuntos
Núcleo Celular/metabolismo , Antagonistas de Estrogênios/metabolismo , Receptores de Estrogênio/metabolismo , Tamoxifeno/análogos & derivados , Animais , Linhagem Celular , Neoplasias Mamárias Experimentais/metabolismo , Camundongos , Conformação Proteica , Receptores de Estrogênio/isolamento & purificação , Tamoxifeno/metabolismoRESUMO
We assessed the hypothesis that due to variations in the conformation of the progesterone receptor induced by the antiprogestin RU38486 compared to the progestin ORG 2058, differences may result in the association of the receptor with some of the chromatin components. The physical properties of the receptor-bound chromatin fragments released by micrococcal nuclease digestion were characterized by sucrose gradient sedimentation and by gel filtration on Agarose A-1.5m or Agarose A-5m columns. The nuclear fraction was isolated from T47D cells previously exposed to 0.1 microM [3H]RU38486 or 0.1 microM [3H]ORG 2058. Micrococcal nuclease digestion solubilized two receptor forms sedimenting at 4.4 S and 6.3 S for the antiprogestin bound receptor and only one receptor at 4.4 S for the progestin ligated receptor. High-salt buffer dissociated either the antiprogestin or the progestin-bound receptor to smaller receptor forms sedimenting at 3.5 S. Chemical cross-linking with the cross-linker 2-iminothiolane of the micrococcal nuclease solubilized receptor forms resulted in 6.7-S and 4.4-S forms sedimenting on 0.4 M KCl gradients for the antiprogestin and progestin ligated receptors, respectively. Stokes radii of 7.3 nm and 6.4 nm were determined by gel filtration in 0.4 M KCl for the 6.7-S and the 4.4-S receptor forms, respectively. Using the sedimentation coefficient and the Stokes radius, molecular weights of 202,000 and 116,000 were calculated for the antiprogestin and progestin ligated receptors. We conclude that the micrococcal nuclease solubilized antiprogestin ligated receptor is associated with additional or different chromatin components compared to the progestin bound receptor.
PIP: The hypothesis that variations in the conformation of the progesterone receptor induced by the antiprogesterone RU38486 compared to the progestin ORG2058 may cause differences in the association of the receptor with some of the chromatin components was investigated. The approach to analyzing the functional organization of the receptor in the chromatin was to study the receptor-bound fragments released by nuclear digestion. The physical properties of these receptor-bound chromatin fragments were characterized by sucrose gradient sedimentation and gel filtration. Micrococcal nuclease digestion solubilized 2 receptor forms sedimenting at 4.4 S and 6.3 S for the antiprogestin bound receptor and only 1 receptor at 4.4 S for the progestin ligated receptor. High-salt buffer dissociated either the antiprogestin or the progestin-bound receptor to smaller receptor forms sedimenting at 3.5 S. Chemical cross-linking resulted in 6.7 S forms for the antiprogestin receptors and 4.4 S forms for the progestin ligated receptors. Use of the sedimentation coefficient and the Stokes radius yielded molecular weights of 202,000 and 116,000 for the antiprogestin and progestin ligated receptors, respectively. Overall, these kinetic studies were unable to explain the variation in responses of the target cell to agonist or antagonist ligands. It is postulated that the antagonist exerts a subtle, but important, conformational change in the receptor, which alters the receptor's ability to associate with some chromatin components and thus affects the normal events in gene expression.
Assuntos
Cromatina/metabolismo , Estrenos/metabolismo , Pregnenodionas/metabolismo , Receptores de Progesterona/metabolismo , Linhagem Celular , Centrifugação com Gradiente de Concentração , Cromatografia em Gel , Reagentes de Ligações Cruzadas , Imidoésteres , Nuclease do Micrococo/metabolismo , Mifepristona , Peso Molecular , Congêneres da Progesterona , Progestinas/antagonistas & inibidoresRESUMO
Progesterone (P) receptor levels were measured in the cytosol and in the 0.4 M KCl nuclear extract in human cervical tissues with [3H]-R5020 as a ligand and the results compared with those obtained in the myometrium and endometrium of the same uteri. Tissue samples were obtained from 28 women, grouped as follows: group A, 12 premenopausal controls; group B, 7 perimenopausal women who received estrogen 7 to 11 days before operation; and group C, 9 perimenopausal and postmenopausal women who received estrogen as in group B plus P injection 1 to 3 hours before operation. Estrogen administration resulted in a significant rise in total P receptor levels in the cervix, compared with the endometrium and myometrium. P injection after estrogen priming resulted in down-regulation of the P receptor in the cervix to undetectable levels, whereas in the endometrium and myometrium it resulted only in redistribution of the P receptor, with higher nuclear levels and lower cytosol levels. These results indicate the possibility of a different mechanism of regulation of the P receptor in the human cervix, compared with that in the endometrium and myometrium.
Assuntos
Colo do Útero/metabolismo , Estrogênios/farmacologia , Progesterona/farmacologia , Receptores de Progesterona/metabolismo , Adulto , Colo do Útero/efeitos dos fármacos , Endométrio/efeitos dos fármacos , Endométrio/metabolismo , Feminino , Humanos , Pessoa de Meia-Idade , Miométrio/efeitos dos fármacos , Miométrio/metabolismoRESUMO
The proteolytic fragments of the nuclear estrogen receptor in the MCF-7 cell line were characterized following limited digestion with chymotrypsin and trypsin. Nuclei were isolated from cells previously exposed to 10 nM [3H]estradiol. The proteolytic digestion was performed either on the micrococcal nuclease hydrolysate or on intact nuclei. The molecular weights (Mr) were calculated from the sedimentation coefficients determined on a sucrose gradient and from the Stokes radii estimated by gel filtration. Digestion of the nuclei with micrococcal nuclease solubilized a receptor form of Mr = 151,000. This receptor form was degraded by chymotrypsin to a receptor of Mr = 33,000 and by trypsin to a receptor of Mr = 60,000. Digestion of intact nuclei with chymotrypsin solubilized a receptor form of Mr = 62,000 which dissociated in 0.4 M KCl to a receptor of Mr = 32,000. Digestion of intact nuclei with trypsin followed by micrococcal nuclease solubilized a receptor form of Mr = 75,000 which was further dissociated by 0.4 M KCl to a receptor form of Mr = 60,000. The ability of the receptor forms to bind DNA was tested using DNA-cellulose column chromatography. About 40% of the micrococcal nuclease solubilized receptor form, compared to about 7% of the chymotrypsin degraded receptor and to about 13% of the trypsin degraded receptor forms, all bound to the column and could be eluted by high salt concentrated buffer. We conclude that the nuclear estrogen receptor in the MCF-7 cell line can be partially degraded either in the micrococcal nuclease hydrolysate or in intact nuclei by chymotrypsin or trypsin generating protein moieties, probably receptor fragments of Mr = 33,000 and 60,000 respectively. Both fragments retain their estradiol binding domain and it may be hypothesized that the heavier fragment retains its chromatin binding domain.