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Biomedical subjects

F Capony

Publications and source records attributed to F Capony.

At least 37 records · Page 2Linked to original sources
PubMed1989

Overexpression and hormonal regulation of pro-cathepsin D in mammary and endometrial cancer.

Pro-cathepsin D is overexpressed in breast cancer cells compared to normal mammary epithelial cells. Moreover, its processing and maturation are altered resulting in increased secretion. In estrogen-responsive breast cancer cell lines such as MCF7 cells and ZR75-1 cells, the 2.2-kb cathepsin D mRNA is accumulated specifically by estrogens and growth factors. Estrogen regulation is mostly transcriptional, while growth factors stabilize the mRNA and act indirectly. In estrogen-receptor-negative cell lines, there is a constitutive high production of cathepsin D mRNA. Moreover in uterine cells, progesterone is the inducer rather than estrogen, indicating the complexity of regulation by steroids, depending on the tissue. The increased production of cathepsin D appears to be correlated with the aggressiveness of the tumour, as shown by retrospective clinical studies, suggesting a role in mammary carcinogenesis.

Breast↗
PubMed1989

[Pro-cathepsin D can activate in vitro pro-cathepsin B secreted by ovarian cancers].

A precursor form of cathepsin B (Mr 45-47 kd) was purified from ascitic fluids of patients with ovarian adenocarcinomas. Following pepsin activation, this precursor produced a 33 kd cathepsin B-like proteinase closely related to lysosomal cathepsin B. A similar activation was found using the 52 kd pro-cathepsin D secreted by the MCF7 human breast cancer cells. This activation was a time, dose and pH dependent process. These results suggest that the 52 kd pro-cathepsin D may be involved in the early steps of the "metastatic cascade", activating pro-cathepsin B in an acidic environment.

Adenocarcinoma↗
PubMed1988

In vitro degradation of extracellular matrix with Mr 52,000 cathepsin D secreted by breast cancer cells.

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.

Breast Neoplasms↗
PubMed1988

Structure, function, regulation and clinical significance of the 52K pro-cathepsin D secreted by breast cancer cells.

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)

Biomarkers, Tumor↗
PubMed1988

Cloning and sequencing of the 52K cathepsin D complementary deoxyribonucleic acid of MCF7 breast cancer cells and mapping on chromosome 11.

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.

Breast Neoplasms↗
PubMed1987

Estrogen-induced lysosomal proteases secreted by breast cancer cells: a role in carcinogenesis?

In an attempt to understand the mechanism by which estrogens stimulate cell proliferation and mammary carcinogenesis, metastatic human breast cancer cell lines (MCF7, ZR75-1) were found to secrete a 52,000 dalton (52K) protein under estrogen stimulation. Following its purification to homogeneity, the 52K protein was identified as a secreted procathepsin-D-like aspartyl protease bearing mannose-6-phosphate signals. This precursor displays an in vitro autocrine mitogenic activity on estrogen-deprived MCF7 cells and is able to degrade basement membrane and proteoglycans following its autoactivation. The total protease (52K + 48K and 34K) was detected and assayed by monoclonal antibodies and was found to be highly concentrated in proliferative and cystic mastopathies. In breast cancer, its cytosolic concentration appears to be correlated more to tumor invasiveness than to hormone responsiveness. The mRNA of the 52K protease accumulates rapidly following estradiol treatment, as was shown by Northern blot analysis with cloned cDNA. The 52K cathepsin-D-like protease is the first example of a lysosomal protease induced by estrogens in cancer cells. Results obtained using different approaches suggest that two cysteinyl cathepsins are also related to cell transformation and invasiveness. It has been proposed that cathepsin-B is involved in breast cancer and metastatic melanoma, and its regulation by estrogen has been shown in the rat uterus. Cathepsin-L corresponds to the major excreted protein (MEP) whose synthesis and secretion are markedly increased by transformation of NIH 3T3 cells with Ki ras and are regulated by several growth factors. In addition to secreted autocrine growth factors and to other proteases (plasminogen activator, collagenase), lysosomal cathepsins may therefore play an important role in the process of tumor growth and invasion as long as their precursor is secreted abundantly.

Breast Neoplasms↗
PubMed1987

The estrogen-regulated 52K-cathepsin-D in breast cancer: from biology to clinical applications.

We have studied estrogen-regulated proteins in an attempt to understand the mechanism by which estrogens stimulate cell proliferation and mammary carcinogenesis. In estrogen receptor positive human breast cancer cell lines (MCF7, ZR75-1) estrogens specifically increase the production into the culture medium of a 52,000 daltons (52K) glycoprotein. Several high affinity monoclonal antibodies to the partially purified secretory 52K protein have allowed to purify to homogeneity this protein and its cellular processed products. The 52K protein has been identified as the secreted precursor of a cathepsin-D like protease bearing mannose-6-phosphate signals and routed to lysosomes via mannose-6-phosphate receptor. The protease is mitogenic in vitro on estrogen deprived MCF7 cells and is able to degrade basement membrane and proteoglycans following its activation. The cellular related proteins, as detected by immunohistochemistry and immunoassay are more concentrated in proliferative mammary ducts than in resting ducts and their concentration in breast cancer cytosol appears to be more correlated with lymph nodes invasion and disease free survival (with S. Thorpe, Copenhagen) than with the estrogen receptor (RE) level. The protein is also produced constitutively by RE-negative cell lines, while in some antiestrogen resistant variants, it becomes inducible by tamoxifen, contrary to the wild type MCF7 cells. Cloning of its cDNA in lambda gt11 has allowed to show that the mRNA is rapidly induced by estrogens and to sequence the protein and compare it to that of the normal human kidney cathepsin-D.(ABSTRACT TRUNCATED AT 250 WORDS)

Breast Neoplasms↗
PubMed1987

Phosphorylation, glycosylation, and proteolytic activity of the 52-kD estrogen-induced protein secreted by MCF7 cells.

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.

Breast Neoplasms↗
PubMed1986

Purification and first characterization of the secreted and cellular 52-kDa proteins regulated by estrogens in human-breast cancer cells.

An estrogen-regulated 52-kDa glycoprotein secreted by MCF7 breast cancer cells was first purified from serum-free conditioned medium by concanavalin-A--Sepharose (ConA--Sepharose). The 13% pure protein was then used to obtain monoclonal antibodies to the 52-kDa protein [Garcia et al. (1985) Cancer Res. 45, 709-716]. Using ConA--Sepharose and monoclonal antibody affinity chromatographies, the secreted 52-kDa protein was finally purified to homogeneity as verified by silver staining of sodium dodecyl sulfate/polyacrylamide gel electrophoresis (SDS-PAGE) and one single N-terminal amino acid. The purification factor was approximately 1400 and the yield 40%. The same two-step procedure, applied to MCF7 cell extracts, yielded four immunologically related proteins of 52 kDa, 48 kDa, 34 kDa and 17 kDa, which were purified 1250-fold with a yield of 30%. These components were further separated by high-performance liquid chromatography gel filtration under denaturing conditions. The final products were homogeneous on the basis of silver-stained SDS-PAGE and gel filtration. However, isoelectrofocusing showed that the pI of the secreted 52-kDa protein and the cellular 34-kDa protein varied from 5.5 to 6.5. Amino acid analysis of the secreted and the related cellular 34-kDa protein is given. Western immunoblotting, pulse chase studies and post-translational studies indicate that the 52-kDa protein is the precursors of a lysosomal enzyme which is partially secreted and partially processed into smaller cellular forms.

Amino Acids↗
PubMed1986

The 52-kDa estrogen-induced protein secreted by MCF7 cells is a lysosomal acidic protease.

An estrogen-induced 52-kDa glycoprotein secreted by human breast cancer cells and able to autostimulate the growth of MCF7 cells has been purified, using monoclonal antibodies, and characterized. The protein contains mannose 6-phosphate signals on its N-linked high-mannose chains, suggesting that it is a lysosomal enzyme. Both the secreted 52-kDa protein and its processed cellular forms (52-, 48- and 34-kDa) were identified as carboxyl proteinases having an optimal activity at pH 3.5 and being specifically inhibited by pepstatin. This protease is characterized by its inducibility by estrogens and its high concentration in proliferative benign and malignant mammary tissue, when detected by immunohistochemistry. The estrogen-induced secretion of this protease may help to understand how estrogens stimulate mammary tumor growth and/or invasion.

Antibodies, Monoclonal↗
PubMed1986

Autocrine growth stimulation of the MCF 7 breast cancer cells by the estrogen-regulated 52 K protein.

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.

Antibodies, Monoclonal↗
PubMed1986

Processing and estrogen regulation of the 52-kilodalton protein inside MCF7 breast cancer cells.

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.

Ammonium Chloride↗
PubMed1985

Estradiol increases the production of alpha 1-antichymotrypsin in MCF7 and T47D human breast cancer cell lines.

alpha 1-Antichymotrypsin (Achy) is an antiprotease of the acute inflammation phase, which is also released by MCF7 human breast cancer cells in culture. Using a fluorimetric assay with the synthetic substrate L-Seryl-L-Tyrosyl-2-N-naphthylamide, we have shown that a medium conditioned by MCF7 cells treated by estradiol inhibits the activity of alpha-chymotrypsin. This inhibition increased when physiological concentrations of estradiol were added to the cells for 2 days. It was due to an increased production of Achy and not to a direct effect of estradiol on alpha-chymotrypsin activity as shown by double immunoprecipitation with an antiserum against human alpha 1-antichymotrypsin. An increased accumulation by estradiol of an antigen located in the cytoplasm of MCF7 cells, which was revealed by immunoperoxidase staining with antibodies to Achy, also indicated that estradiol increased the production of Achy in these cells. Similar immunostaining was observed in a breast cancer tissue. Most of the estrogen regulated 60-68 kDa protein secreted by T47D cells (Chalbos et al. (1982) J. Clin. Endocrinol. Metab. 55, 276-283) was also specifically immunoprecipitated by the antibodies to Achy. Thus, alpha 1-antichymotrypsin is the first protein to be identified which is induced by estradiol and secreted by breast cancer cells.

Breast Neoplasms↗
PubMed1985

Effect of tunicamycin and endoglycosidase H and F on the estrogen regulated 52000-Mr protein secreted by breast cancer cells.

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.

Acetylglucosaminidase↗