RESUMO
We have studied the posttranslational modifications of the 52-kD protein, an estrogen-regulated autocrine mitogen secreted by several human breast cancer cells in culture (Westley, B., and H. Rochefort, 1980, Cell, 20:353-362). The secreted 52-kD protein was found to be phosphorylated mostly (94%) on high-mannose N-linked oligosaccharide chains, and mannose-6-phosphate signals were identified. The phosphate signal was totally removed by alkaline phosphatase hydrolysis. The secreted 52-kD protein was partly taken up by MCF7 cells via mannose-6-phosphate receptors and processed into 48- and 34-kD protein moieties as with lysosomal hydrolases. By electron microscopy, immunoperoxidase staining revealed most of the reactive proteins in lysosomes. After complete purification by immunoaffinity chromatography, we identified both the secreted 52-kD protein and its processed cellular forms as aspartic and acidic proteinases specifically inhibited by pepstatin. The 52-kD protease is secreted in breast cancer cells under its inactive proenzyme form, which can be autoactivated at acidic pH with a slight decrease of molecular mass. The enzyme of breast cancer cells, when compared with cathepsin D(s) of normal tissue, was found to be similar in molecular weight, enzymatic activities (inhibitors, substrates, specific activities), and immunoreactivity. However, the 52-kD protein and its cellular processed forms of breast cancer cells were totally sensitive to endo-beta-N-acetylglucosaminidase H (Endo H), whereas several cellular cathepsin D(s) of normal tissue were partially Endo H-resistant. This difference, in addition to others concerning tissue distribution, mitogenic activity and hormonal regulation, strongly suggests that the 52-kD cathepsin D-like enzyme of breast cancer cells is different from previously described cathepsin D(s). The 52-kD estrogen-induced lysosomal proteinase may have important functions in facilitating the mammary cancer cells to proliferate, migrate, and metastasize.
Assuntos
Neoplasias da Mama/metabolismo , Proteínas de Neoplasias/metabolismo , Peptídeo Hidrolases , Neoplasias da Mama/ultraestrutura , Linhagem Celular , Feminino , Humanos , Cinética , Manosefosfatos/metabolismo , Microscopia Eletrônica , Peso Molecular , Proteínas de Neoplasias/genética , Fosforilação , Processamento de Proteína Pós-TraducionalRESUMO
It has been proposed that proteases secreted by cancer cells facilitate metastasis by degrading extracellular matrix. Estrogen receptor-positive breast cancer cells secrete a Mr 52,000 pro-cath-D under estrogen stimulation, whereas this protease is produced constitutively by estrogen receptor-negative cancer cells. We report on the degradation in vitro of extracellular matrix by purified Mr 52,000 cathepsin D (cath-D) and by conditioned media prepared from different cell lines. The purified Mr 52,000 pro-cath-D was autoactivated at pH 4.5 into a Mr 51,000 cath-D and found to digest the extracellular matrix of endothelial bovine corneal cells labeled with [3H]proline or [35S]methionine. Culture medium conditioned by estrogen-treated MCF7 cells had a similar effect at pH 4.5 but not at pH 7.4. Matrix degradation was totally inhibited by pepstatin. Other breast cancer cells (BT20, MDA-MB231, T47D cells, etc.) and other cancer cells also secreted a pepstatin-sensitive proteinase able to degrade extracellular matrix. By contrast, the U2 variant of MCF7 cells, which lacks the Mr 52,000 cath-D gene, and the nontumoral epithelial mammary cells secreted a negligible amount of this proteinase. In all conditioned media, the pepstatin-dependent extracellular matrix degrading activity was highly correlated to the Mr 52,000 cath-D concentration measured by immunoenzymatic assay. We conclude that the Mr 52,000 cath-D is the major acidic protease secreted by mammary cancer cells. We suggest that this protease may degrade basement membrane and consequently facilitate tumor invasion when it is released in an acidic microenvironment.
Assuntos
Neoplasias da Mama/enzimologia , Catepsina D/metabolismo , Matriz Extracelular/metabolismo , Colágeno/metabolismo , Precursores Enzimáticos/metabolismo , Espaço Extracelular/enzimologia , Humanos , Concentração de Íons de Hidrogênio , Técnicas In Vitro , Pepstatinas/farmacologia , Proteoglicanas/metabolismo , Células Tumorais CultivadasRESUMO
The Mr 52,000 glycoprotein is regulated by estrogen and released by breast cancer cells in culture (B. Westley and H. Rochefort, Cell, 20: 352-362, 1980). This rare protein was partially purified from 25 liters of medium conditioned by MCF7 cells and injected into Biozzi's selected mice. The spleen lymphocytes of one immunized mouse was fused with the murine myeloma P3-X63-Ag8-653. Sixteen hybridomas producing monoclonal antibodies to the Mr 52,000 protein were isolated, and seven of them were cloned and purified. The seven monoclonal antibodies were all of the immunoglobulin G1 isotype, and their dissociation constants ranged from 0.35 to 2.3 nM. The antibodies specifically recognized the secreted Mr 52,000 protein as evidenced by double immunoprecipitation and by immunoblotting after electrophoretic separation and transfer. Double-determinant immunoradiometric assay indicated that the seven purified monoclonal antibodies recognized three distinct regions of the Mr 52,000 protein, and it was used to assay the Mr 52,000 protein in biological fluids. These antibodies did not react with the external plasma membrane of MCF7 cells, as shown by immunofluorescence analysis. By contrast, the cytoplasm of MCF7 cells (but not T47D and RBA cells) was stained by the peroxidase-immunoperoxidase complex after plasma membrane permeation, indicating that the protein is secreted by exocytosis rather than shed from the plasma membrane.
Assuntos
Anticorpos Monoclonais , Neoplasias da Mama/análise , Proteínas de Neoplasias/análise , Animais , Linhagem Celular , Exocitose , Imunofluorescência , Humanos , Técnicas Imunoenzimáticas , CamundongosRESUMO
In human mammary cancer cells, pro-cathepsin D (pro-Cath-D) is induced by estrogens and 50% of it is secreted. To determine whether its secretion is characteristic of mammary cells or transformed cells, we compared its production, processing, and glycosylation in primary cultures of normal mammary epithelial cells to those found in breast cancer cell lines. The cytosolic concentration of total cathepsin D (precursor and mature enzyme) measured by enzyme-linked immunosorbent assay was 8 times higher in cancer cells. Its mRNA level estimated by Northern blot analysis was 8 to 50 times higher and its secretion was 30 times higher in cancer cells. Using pulse-chase labeling, the cellular processing of pro-Cath-D was altered in hormone-dependent and -independent breast cancer cells in comparison to normal cells. This alteration resulted in a lower accumulation of mature enzyme, while the secretion and cytoplasmic accumulation of pro-Cath-D was greater in breast cancer cells than in normal cells. NH4Cl increased secretion of the proenzyme in normal cells but not in cancer cells. The secreted proenzyme was markedly heterogeneous and had a more acidic pI in MCF7 cells than in normal mammary cells. These acidic forms disappeared following endo-beta-N-acetylglucosaminidase H treatment indicating that the structural difference between pro-Cath-D of normal and of cancer mammary cells was located on high mannose or hybrid N-linked oligosaccharides. This difference may be responsible for the altered routing of the pro-Cath-D in breast cancer cells.
Assuntos
Neoplasias da Mama/enzimologia , Catepsina D/genética , Precursores Enzimáticos/genética , Processamento de Proteína Pós-Traducional , Mama/enzimologia , Catepsina D/metabolismo , Linhagem Celular , Citosol/enzimologia , Precursores Enzimáticos/metabolismo , Feminino , Glicosilação , Humanos , Peso Molecular , RNA Mensageiro/análise , RNA Mensageiro/genética , Valores de Referência , Transcrição GênicaRESUMO
In an attempt to find estrogen-specific responses in breast cancer, we have established primary cell culture from metastatic pleural effusions of breast cancer and have analyzed the proteins labeled by [35S]methionine and released into the culture medium using sodium dodecyl sulfate-polyacrylamide gel electrophoresis. We show that the synthesis of a Mr 52,000 glycoprotein which is released by metastatic breast cancer cells in primary cultures is stimulated by estradiol in four of six patients. This protein is similar to the Mr 52,000 protein of MCF7 cells on the basis of its mobility in one- and two-dimensional gel electrophoresis [the molecular weight of this protein was originally found to be 46,000; it is closer to 52,000 using labeled proteins from New England Nuclear as molecular weight markers], its immunoprecipitation by antisera raised against the Mr 52,000 protein, and its binding to concanavalin A. We conclude that, similar to some breast cancer cell lines, some metastatic breast cancers synthesize a Mr 52,000 glycoprotein which is regulated by estrogens and exported from the cells into the medium. This study also shows that some primary cultures established from metastatic breast cancer remain responsive to estradiol in vitro for the synthesis of specific proteins. More clinical studies are needed to prove the interest of the Mr 52,000 secreted protein as an additional marker of the hormone responsiveness of breast cancer.
Assuntos
Neoplasias da Mama/metabolismo , Estradiol/farmacologia , Glicoproteínas/biossíntese , Proteínas de Neoplasias/biossíntese , Células Cultivadas , Epitélio/fisiologia , Feminino , Glicoproteínas/metabolismo , Humanos , Peso Molecular , Proteínas de Neoplasias/metabolismoRESUMO
We have studied the regulation by estradiol of the mannose-6-phosphate (Man-6-P)/insulin-like growth factor-II (IGF-II) receptor concentration in different breast cancer cell lines. The mRNA level was assayed by Northern blot using the H5.1 cDNA probe. The protein level was assayed by Western ligand blot, by binding saturation with [125I]procathepsin-D on total membrane preparations, and by immunoprecipitation of 35S-labeled proteins. In three estrogen receptor-positive cell lines (MCF7, T47D, and ZR75-1), estradiol specifically decreased the steady state level of the Man-6-P/IGF-II receptor protein and mRNA. Moreover, in different cell lines and in primary culture of normal mammary cells, the secretion of procathepsin-D was inversely correlated with the level of Man-6-P/IGF-II receptor protein and mRNA. We conclude that estradiol down-regulates the Man-6-P/IGF-II receptor in breast cancer cells. Since two of its ligands, procathepsin-D and IGF-II, are induced by estrogen, we propose that the Man-6-P/IGF-II receptor becomes saturated after estrogen treatment. This model might explain the previously described estrogen-induced secretion of procathepsin-D and other lysosomal proenzymes routed by the same transport system.
Assuntos
Catepsina D/biossíntese , Precursores Enzimáticos/metabolismo , Estradiol/farmacologia , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Lisossomos/enzimologia , Receptores de Superfície Celular/genética , Neoplasias da Mama , Linhagem Celular , Relação Dose-Resposta a Droga , Indução Enzimática , Feminino , Humanos , Fator de Crescimento Insulin-Like II/metabolismo , Cinética , Lisossomos/efeitos dos fármacos , Manosefosfatos/metabolismo , RNA Mensageiro/efeitos dos fármacos , RNA Mensageiro/genética , Receptor IGF Tipo 2 , Receptores de Somatomedina , Transcrição Gênica/efeitos dos fármacosRESUMO
Two lambda gt11 libraries containing complementary DNAs from human breast cancer MCF7 cells were screened by expression with monoclonal antibodies to the secreted 52K protein and with a 36-mer oligonucleotide derived from the N-terminal amino acid sequence of the secreted 52K protein. Four overlapping clones were sequenced, and found to be extensively homologous to the cathepsin D of normal human kidney, except for 5-point mutations resulting in one amino acid change (Ala to Val) in the profragment of cathepsin D. Northern blot analysis showed the 2.2 kilobase (kb) cathepsin D mRNA to be induced by estradiol in MCF7 cells and produced constitutively at high levels in the estrogen-receptor-negative BT20 cell line. A simple restriction pattern consistent with the restriction map of cathepsin D cDNA was obtained in Southern blot analysis of MCF7 cell DNA. In situ hybridization of the 52K-9 cDNA probe on normal lymphocytes assigned the 52K cathepsin D gene at the extremity of the short arm of chromosome 11, in the p15 band, close to the H-ras gene and in the region whose deletion increases the risk of invasive breast cancer. We conclude that the estrogen induced 52K protein has the same sequence as normal pro-cathepsin D and we propose that the 52K protein correspond to the only pro-cathepsin D expressed in MCF7 cells.
Assuntos
Neoplasias da Mama/genética , Catepsina D/genética , Mapeamento Cromossômico , Cromossomos Humanos Par 11 , Clonagem Molecular , Humanos , Células Tumorais CultivadasRESUMO
High levels of cytosolic cathepsin D expression have been associated with poor prognosis in breast cancer node-negative patients. In this work, we provide evidence that three cell lines established from human metastatic melanomas--IIB-MEL-J, IIB-MEL-LES, and IIB-MEL-IAN--express high levels of procathepsin D mRNA. IIB-MEL-J cells secreted into the conditioned media about 30% of the newly synthesized protein, which was active at acidic pH. Melanoma tumors arising in nude mice after injection of the three different cell lines expressed high levels of procathepsin D mRNA. Moreover, 13 human metastatic melanomas expressed variable levels of procathepsin D mRNA. To study the possible association between cathepsin D expression and melanoma development, samples corresponding to 10 primary tumors, 11 metastatic melanomas, 10 dysplastic nevi, 27 nevocellular nevi, and normal melanocytes were studied by immunohistochemistry for cathepsin D-specific staining. We found that cathepsin D was expressed in all of the dysplastic nevi and primary and metastatic melanomas tested but in only 18% of nevocellular nevi (five of 27), whereas normal melanocytes showed no cathepsin D expression. The overall data indicate that cathepsin D is expressed at a high level by melanoma cells, and because of its expression in preneoplastic lesions, it may be associated with melanoma development.
Assuntos
Catepsina D/análise , Síndrome do Nevo Displásico/metabolismo , Melanoma/secundário , Catepsina D/genética , Catepsina D/metabolismo , Meios de Cultivo Condicionados , Expressão Gênica , Humanos , Imuno-Histoquímica , Melanoma/química , Melanoma/genética , RNA Mensageiro/análise , Células Tumorais CultivadasRESUMO
A 52K glycoprotein is secreted by human breast cancer cells in culture after estrogen stimulation. Using monoclonal antibodies, we have quantitated and characterized the corresponding proteins of the cell compartment. Using pulse-chase experiments, we have shown that about 40% of the 52K protein is secreted, the majority being successively processed into a 48K and a 34K protein. This last protein is very stable. The processing is inhibited by lysosomotropic agents and leupeptin, suggesting that it occurs in acidic vesicles, such as lysosomes or endosomes. Estradiol increased the intracellular level of immunoreactive 52K related proteins by 4-fold. Its effect is, however, more obvious in the medium, since there is a constitutive level in the cell. The stimulatory effects of estradiol on [3H]mannose and [35S]methionine incorporation into these proteins were similar and the endoglycosydase H sensitivity of the proteins was not altered, suggesting that estradiol did not modulate the glycosylation step. Antiestrogens did not stimulate synthesis and glycosylation of the 52K related proteins. Estradiol also increased the stability of the 52K precursor as well as that of total proteins. We conclude that the secreted 52K protein is the precursor of two cellular proteins of 48K and 34K. Estradiol stimulates both the intracellular accumulation of these proteins and the secretion of the precursor.
Assuntos
Neoplasias da Mama/fisiopatologia , Estradiol/farmacologia , Proteínas de Neoplasias/fisiologia , Cloreto de Amônio/farmacologia , Anticorpos Monoclonais , Neoplasias da Mama/metabolismo , Compartimento Celular , Antagonistas de Estrogênios/farmacologia , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Glicosilação , Humanos , Leupeptinas/farmacologia , Peso Molecular , Monensin/farmacologia , Proteínas de Neoplasias/metabolismo , Processamento de Proteína Pós-Traducional/efeitos dos fármacosRESUMO
The growth of MCF 7 human breast cancer cells is stimulated in vitro by estradiol (E2) and we have previously shown that estrogen-regulated glycoproteins released into the culture medium can partly mimic this effect. In this paper, we evaluate the mitogenic activity of the 52 K glycoprotein, which is a major E2-stimulated protein released by MCF 7 cells. The 52 K protein was purified 600-fold by affinity chromatography on Concanavalin A and an anti-52 K monoclonal antibody Sepharose columns. The 99% purified 52 K protein fraction stimulated the growth of estrogen-deprived MCF 7 cells. A mean 1.7-fold increase was obtained with nanomolar concentrations of seven different preparations of 52 K protein. This stimulation represented 40% of the mitogenic effect of E2. Both the 52 K protein and E2 induced microvilli at the cell surface but the effect of the 52 K protein occurred earlier. Other putative growth factors which are also stimulated by E2 and observed by [35S]cysteine labeling did not comigrate with the purified 52 K protein. Finally, the labeled 52 K protein was found to enter MCF 7 cells and to be processed into an immunoreactive 34 K protein. These data indicate that the E2-regulated 52 K glycoprotein is an autocrine mitogen on MCF 7 cells in culture and support the hypothesis that estrogens stimulate the growth of mammary cancer via this (and possibly other) secreted protein(s) acting as autocrine (and paracrine?) growth factors.
Assuntos
Neoplasias da Mama/ultraestrutura , Proteínas de Neoplasias/farmacologia , Anticorpos Monoclonais , Divisão Celular/efeitos dos fármacos , Linhagem Celular , Cromatografia de Afinidade , Relação Dose-Resposta a Droga , Eletroforese em Gel de Poliacrilamida , Estradiol/farmacologia , Feminino , Fluorometria , Humanos , Microscopia Eletrônica de Varredura , Peso Molecular , Propriedades de SuperfícieRESUMO
Ovarian cancers are highly invasive. In a first attempt to define the hormones and factors involved in the control of tumor invasion and metastasis, we have used the human ovarian cancer cell line BG-1 which contains both estrogen and progesterone receptors. Protein synthesis and secretion was assayed by [35S]methionine incorporation and polyacrylamide gel electrophoresis followed by fluorography. Three responses to estradiol were found: 1) procathepsin D secretion was increased, whereas the corresponding intracellular proteins were not significantly affected; 2) an abundant but nonidentified 120-kilodalton (kDa) estrogen-induced secreted glycoprotein, different from CA125, was detected for the first time; and 3) the number of cells as determined by DNA assay was markedly stimulated, reaching a higher level of confluency. The antiestrogen OH-tamoxifen was weakly agonist at low concentrations to stimulate cell growth but was a pure antagonist on the 120-kDa protein. The steroid specificity of these responses strongly suggests that they are mediated by the estrogen receptor. We conclude that cathepsin D secretion is specifically stimulated by estrogen in this ovarian cancer cell line as it is in estrogen receptor-positive breast cancer cells. Both cathepsin D and a newly described 120-kDa secreted glycoprotein are potential markers of hormone responsiveness and/or aggressiveness which deserve to be further studied in clinical ovarian cancers.
Assuntos
Catepsina D/metabolismo , Precursores Enzimáticos/metabolismo , Estradiol/farmacologia , Neoplasias Ovarianas/metabolismo , Proteínas/metabolismo , Divisão Celular/efeitos dos fármacos , Di-Hidrotestosterona/farmacologia , Eletroforese em Gel de Poliacrilamida , Antagonistas de Estrogênios/farmacologia , Feminino , Humanos , Peso Molecular , Neoplasias Ovarianas/patologia , Testes de Precipitina , Proteínas/química , Tamoxifeno/análogos & derivados , Tamoxifeno/farmacologia , Células Tumorais CultivadasRESUMO
An estrogen regulated glycoprotein of molecular weight 52,000 is released by metastatic human breast cancer cells in culture. In order to detect this protein directly in human tissues, several high affinity monoclonal antibodies were produced against the 52,000 mol wt protein. Frozen sections of human breast cancer samples were stained by the peroxidase-anti-peroxidase method using these antibodies. In 20 of 25 samples, specific immunoperoxidase staining was observed in the cytoplasm of epithelial cells with six monoclonal antibodies to the 52,000 mol wt protein. The 5 samples that were not stained contained no detectable estrogen receptor. Epithelial cells were not stained in 6 normal mammary glands collected during reduction mammoplasties and in 9 normal uteri, whether tissues were collected during the follicular or luteal phase. The demonstration that the 52,000 mol wt estrogen regulated protein is present in the cytoplasm of some primary breast cancers but absent in normal mammary tissue and uterus indicates its possible use as a tumor marker.
Assuntos
Neoplasias da Mama/metabolismo , Mama/metabolismo , Estrogênios/fisiologia , Glicoproteínas/análise , Proteínas de Neoplasias/análise , Útero/metabolismo , Anticorpos Monoclonais , Feminino , Histocitoquímica , Humanos , Técnicas Imunoenzimáticas , Peso MolecularRESUMO
Overexpression of cathepsin D in human breast cancers is associated with a higher risk of relapse and metastasis. Also, pro-enzyme routing is altered in several tumoral mammary cell lines, leading to its hypersecretion. MCF7 cells compared to normal kidney carry a C-->T transition at position 224 in the cathepsin D gene which converts Ala to valine in its pro-fragment. Using polymerase chain reaction-single strand conformational polymorphism analysis (PCR-SSCP), the variant T allele frequency was found to be 23-30%, and equally distributed in cancer and normal cells. Six to nine per cent of genotypes were homozygous T/T, 34-41% were heterozygous T/C and 50-59% were homozygous C/C. Moreover, genotypes were identical in 19 out of 20 matched sets of tumoral mammary cells and normal white blood cells from the same patients. Loss of heterozygosity was noted in 1 case. C/T224 transition is thus not due to a somatic event. However, this missense polymorphism might modify procathepsin D secretion and/or maturation in breast cancer cells.
Assuntos
Neoplasias da Mama/genética , Catepsina D/genética , Polimorfismo Genético/genética , Alelos , Sequência de Bases , Neoplasias da Mama/enzimologia , Catepsina D/biossíntese , Feminino , Frequência do Gene , Humanos , Dados de Sequência Molecular , Mutação , Reação em Cadeia da Polimerase , Células Tumorais CultivadasRESUMO
The glycosylation and immunoreactivity of an estrogen regulated glycoprotein secreted by breast cancer cells in culture and defined by its molecular mass (52 000-Mr protein) have been studied indirectly using an inhibitor of glycosylation and specific endoglycosidases. The protein and its deglycosylated forms were immunoprecipitated with specific monoclonal antibodies to the 52 000-Mr protein and analyzed by SDS polyacrylamide gel electrophoresis. The 52 000-Mr protein was intensely labelled by [3H] mannose or [35S] methionine. Tunicamycin treatment of the cells, endoglycosidase H or endoglycosidase F digestion of conditioned media, gave two identical deglycosylated forms of 50 000-Mr and 48 000-Mr which remained immunoreactive. The 48 000-Mr protein, in contrast to the 52 000 and 50 000-Mr proteins, was unable to bind concanavalin A. The 52 000-Mr protein was resolved into five spots of decreasing pI on two-dimensional gels following immunoprecipitation. Endoglycosidase H treatment decreased the molecular weight and reduced the intensity of spots of lower pI, suggesting that the N-glycosylated chains contain acidic molecules. We conclude that: The 52 000-Mr secreted protein contains at least two high mannose or hybrid N-glycosylated chains of approximately 2,000 molecular weight corresponding to 8% of the mass of the 52 000-Mr protein. The two types of monoclonal antibodies (site 1 and 2) raised against the 52 000-Mr glycoprotein are still able to recognize the 48 000-Mr N-deglycosylated form indicating that they do not interact with the N-glycosylated moiety of the molecule.
Assuntos
Acetilglucosaminidase/farmacologia , Neoplasias da Mama/metabolismo , Estradiol/farmacologia , Glucosamina/análogos & derivados , Glicosídeo Hidrolases/farmacologia , Hexosaminidases/farmacologia , Proteínas de Neoplasias/metabolismo , Tunicamicina/farmacologia , Linhagem Celular , Feminino , Fucose/metabolismo , Humanos , Cinética , Manose/metabolismo , Manosil-Glicoproteína Endo-beta-N-Acetilglucosaminidase , Metionina/metabolismo , Peso Molecular , Proteínas de Neoplasias/biossíntese , Proteínas de Neoplasias/isolamento & purificação , Radioisótopos de Enxofre , TrítioRESUMO
In estrogen-receptor-positive human breast cancer cell lines (MCF7, ZR75-1), estrogens specifically increase the secretion into the culture medium of a 52,000 Da (52K) glycoprotein and stimulate cell proliferation. The 52K protein has been purified to homogeneity using monoclonal antibodies and identified as the secreted precursor of a cathepsin D bearing mannose-6-phosphate signals. The secreted precursor 52K protein is mitogenic in vitro in estrogen-deprived MCF7 cells, can be taken up by these cells via mannose-6-phosphate receptors, and can degrade extracellular matrix and proteoglycans following its auto-activation. The protease is also produced constitutively by ER-negative cell lines, and is inducible by tamoxifen in some antiestrogen-resistant variants. The corresponding cDNA has been cloned using N-terminal sequencing of the protein and monoclonal antibodies. Its complete sequencing indicates a strong homology with pro-cathepsin D of normal tissues. Using a cDNA probe, the regulation of 52K cathepsin D mRNA by estrogens and antiestrogens has been studied and chromosome localization determined by in situ hybridization. Clinical studies using both immunohistochemistry and immunoenzymatic assay of breast cancer cytosol have shown that the concentration of total cellular cathepsin D (52K + 48K + 34K) is related to the proliferation of mammary ducts and to the prognosis of breast cancer. Its cytosolic concentration in primary tumors of postmenopausal patients is correlated slightly with lymph node invasion and significantly with shorter disease-free intervals in a 6-year retrospective study with the Danish Breast Cancer Groups and Finsen Institute (S. Thorpe et al.).(ABSTRACT TRUNCATED AT 250 WORDS)
Assuntos
Neoplasias da Mama/enzimologia , Catepsina D/metabolismo , Precursores Enzimáticos/metabolismo , Biomarcadores Tumorais/análise , Neoplasias da Mama/diagnóstico , Catepsina D/genética , Catepsina D/isolamento & purificação , Linhagem Celular , Transformação Celular Neoplásica , Precursores Enzimáticos/genética , Precursores Enzimáticos/isolamento & purificação , Estrogênios/farmacologia , Feminino , Humanos , Peso Molecular , Prognóstico , Biossíntese de Proteínas , Processamento de Proteína Pós-TraducionalRESUMO
Various analogs of statine, a remarkable amino acid component of the protease inhibitor pepstatine, were synthesized and evaluated as tripeptide derivatives for their activity against cathepsin D and HIV-1 protease.
Assuntos
Aminoácidos/farmacologia , Catepsina D/antagonistas & inibidores , Oligopeptídeos/farmacologia , Inibidores de Proteases/farmacologia , Aminoácidos/química , Inibidores da Protease de HIV/química , Inibidores da Protease de HIV/farmacologia , Humanos , Oligopeptídeos/química , Pepstatinas/química , Inibidores de Proteases/química , Relação Estrutura-Atividade , Células Tumorais CultivadasRESUMO
PIP: The binding of an antiestrogen with the uterine estrogen receptor (R) has been studied directly in vitro using tritiated dimethylstilbestrol (DMS). The affinity of DMS for R as determined at equilibrium was similar to that of estradiol-17beta (E2) (K(D) approximately .3 nN) when taking into account the higher nonspecific binding of DMS. The number of DMS binding sites was constantly found to be inferior to that of E2. The fact that the DMS binding entity specifically bound estrogen and antiestrogen, was destroyed by pronase, displayed an 8S sedimentation constant, and interacted in vitro with DNA, strongly suggested that DMS interacted directly with R. The association of DMS to R was a simple 2nd-order reaction while its dissociation was a 1st-order reaction with 2 slopes. The association and dissociation rate constants of the R-DMS complex were, respectively, slower and higher than those of the R-E2 complex. The rapid dissociation rate of DMS could be responsible for its inability to protect the receptor binding sites against thermo-inactivation. Tritiated DMS was able in vivo to induce the nuclear translocation of the receptor. However, as with other short-acting antiestrogens and contrary to Nafoxidine, the time of nuclear retention of R was short. These results are in agreement with the assumption that the length of the nuclear retention of R is determinant in explaining the weak agonist activity of this compound.^ieng
Assuntos
Receptores de Estrogênio/metabolismo , Estilbenos/metabolismo , Núcleo Celular/metabolismo , Citosol/metabolismo , Estradiol/metabolismo , Estrona/metabolismo , Feminino , Temperatura Alta , Cinética , Nafoxidina/metabolismo , Progesterona/metabolismo , Testosterona/metabolismo , Útero/metabolismoRESUMO
Interactions between estrogen recpetors and triaryl ethylene anti-estrogens (U-11100A, MER 25 and CI 628) were tested on immature rat uteri. The accessible and the total (accessible and occupied) estrogen receptor sites were assayed in the cytosol and nuclear extracts using charcoal adsorption. After in vivo administration of anti-estrogens, the estradiol receptor sites were occupied and subsequently transferred to the nuclear compartment. The nuclear localisation of the receptor induced by the antagonist lasted for several days, during which replenishment of the cytosol receptor occurred. The nuclear receptors transferred by anti-estrogens and labelled in vitro with [3H]estradiol, were similar to the nuclear receptor-estradiol complex formed in vivo as far as their sedimentation constants and extractability from nuclei were concerned. Although these anti-estrogens are capable to translocate the estrogen receptor to the nucleus and to induce the replenishment of the cytosol receptor, the mechanism of their antagonism and of their weak estrogenic activity is still not clear.
Assuntos
Estradiol/metabolismo , Antagonistas de Estrogênios , Receptores de Superfície Celular/efeitos dos fármacos , Útero/metabolismo , Animais , Sítios de Ligação , Núcleo Celular/metabolismo , Citosol/metabolismo , Etamoxitrifetol/farmacologia , Feminino , Cinética , Nafoxidina/farmacologia , Nitromifeno/farmacologia , Concentração Osmolar , Cloreto de Potássio/farmacologia , Ligação Proteica , RatosRESUMO
The interaction of tamoxifen (ICI 46,474), a synthetic antiestrogen, with uterine cytosol proteins of immature calf and rat has been studied directly using the tritiated compound labeled with a high specific activity. The binding complexes were measured by the dextrancoated charcoal, protamine sulfate and hydroxyapatite assays. Scatchard plots revealed a single class of high-affinity (KD congruent to 1.7 nM) binding sites, with a binding capacity similar to that of estradiol. Competitive experiments showed the same binding specificity for estrogens and antiestrogens. Sucrose gradient analysis revealed an 8S binding protein which could be partially proteolysed by trypsin into a 4S binding protein. Kinetic studies showed that the association rate of tamoxifen was 5 times lower than that of estradiol and reacted according to a second order kinetics. The first-order kinetics of dissociation was considerably higher than that of estradiol, giving a half-dissociation time of 20--40 min at 0--2 degrees C. In some cases tamoxifen displayed two slopes of dissociation, but the proportion of the slow-dissociating complex was always inferior to that found with estradiol. In contrast to estradiol, the kinetic constants ratio (k-/k+) gave a calculated dissociation constant, similar to that determined in equilibrium conditions (KD), agreeing with a simple reactional scheme. We conclude that the antiestrogen tamoxifen binds directly to the 8S cytosol receptor for estrogens and not to another receptor for the antagonists. In contrast to estradiol, the antagonist is rapidly dissociated from the receptor sites and is unable to protect them against thermal inactivation. The affinity of tamoxifen for its receptor sites as determined directly is surprisingly high when compared to its affinity evaluated indirectly by competitive experiments. It is then suggested that the two ligands either bind on two different sites of the same protein or induce a different conformational change of the same binding site.
Assuntos
Estradiol/metabolismo , Receptores de Estrogênio/metabolismo , Tamoxifeno/metabolismo , Animais , Ligação Competitiva , Bovinos , Feminino , Cinética , Ratos , Útero/metabolismoRESUMO
In the MCF7 human breast cancer cell line, estradiol stimulates the synthesis of a 52 K secretory glycoprotein and has been reported to increase the plasminogen activator (PA) activity in the culture medium. Since one PA isozyme has a molecular weight close to 52 000 daltons under denaturing conditions, we asked whether the 52 K protein was a PA. The PA activity released in serum-free conditioned medium was evaluated by the increase in [125I]casein digestion observed in the presence of plasminogen. The 52 K protein was estimated by analysing the released proteins on SDS-polyacrylamide gel electrophoresis. When the conditioned medium was chromatographed on concanavalin A-Sepharose, the 52 K protein was retained on the gel, but not the PA. The two proteins also appeared different on the basis of their competing efficiency in a radioimmunoassay developed to quantify the 52 K protein. An antiserum against human urokinase failed to immunoprecipitate the 52 K protein. Under our culture conditions estradiol increased 52 K, but not PA, production. These results clearly indicate that the estradiol-regulated 52 K protein is not a plasminogen activator.