Marcel Mongay Garcia - Academia.edu (original) (raw)
Papers by Marcel Mongay Garcia
Cancer research, 1983
In an attempt to find estrogen-specific responses in breast cancer, we have established primary c... more 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...
Cancer research, 1981
5-Androstene-3 beta, 17 beta-diol (ADIOL) has previously been shown to have a high affinity for t... more 5-Androstene-3 beta, 17 beta-diol (ADIOL) has previously been shown to have a high affinity for the estrogen receptor and to translocate it to the nucleus in vitro and in vivo. This compound and related C19 delta 5-steroids of adrenal origin have now been examined for their ability to induce the synthesis in MCF7 cells of an estrogen-dependent secreted glycoprotein (M.W. 46,000). Concentrations required for half-maximum induction were 2 nM for ADIOL and 500 nM for dehydroepiandrosterone (DHEA). Dehydroepiandrosterone sulfate showed weak inducing ability at concentrations of 1 microM or greater. The induction by ADIOL was unaffected by the presence of an aromatase inhibitor, and 5 alpha-androstane-3 beta, 17 beta-diol, which cannot be aromatized, also induced the M.W. 46,000 protein at low concentrations. When cells were exposed to 10 nM [3H]ADIOL, the cytosol and nuclear fractions contained [3H]ADIOL resistant to charcoal adsorption. The bound [3H]ADIOL in the cytosol and nucleus wa...
Cancer research, 1986
We have recently shown that the integrity of the polyamine pathway is essential but not sufficien... more We have recently shown that the integrity of the polyamine pathway is essential but not sufficient for expression of the mitogenic effect of estradiol (E2) in the N-nitrosomethylurea-induced rat mammary tumor grown in vitro in the soft agar clonogenic assay. To elucidate the mechanism of E2 action in this system, we tested whether E2 may stimulate tumor growth through induction of secretory growth factor(s), as already proposed for human breast cancer cell lines in culture. Furthermore, we investigated the potential interaction between such "autocrine" control of tumor growth by E2 and the polyamine pathway. Conditioned medium obtained from E2-treated tumors (E2-CM) but not from control tumors (C-CM) consistently stimulated colony formation when added to N-nitrosomethylurea-mammary tumors plated in soft agar under serum-free media conditions. Such growth-promoting effects of the E2-CM was found to increase with increasing protein concentrations of the medium and was abolis...
Cancer research, 1989
The Mr 52,000 cathepsin D is the precursor of a lysosomal protease secreted in excess by breast c... more The Mr 52,000 cathepsin D is the precursor of a lysosomal protease secreted in excess by breast cancer cells. This protease can degrade extracellular matrices and proteoglycans and is induced by estrogens in estrogen receptor-positive breast cancer cell lines. In a 4- to 6-yr retrospective cohort study, the concentration of the total cathepsin D (precursor plus intermediate and mature chains) was assayed in cytosols of primary tumors from 242 pre/perimenopausal and 154 postmenopausal breast cancer patients in a solid-phase immunoassay using two specific monoclonal antibodies. Patients were initially divided into groups with low, intermediate, or high concentrations of cathepsin D corresponding to the quartiles of the overall distribution. Using these groupings, the level of Mr 52,000 cathepsin D was not significantly associated with the recognized prognostic factors of age, lymph node involvement, tumor size, and/or grade of anaplasia. A significant association was found between cat...
Cancer research, 1978
To improve our understanding of the effects of androgens on the induction and growth of mammary t... more To improve our understanding of the effects of androgens on the induction and growth of mammary tumors, the interaction of androgen on the estrogen receptor (ER) has been evaluated in the 7,12-dimethylbenz(a)anthracene-induced rat mammary tumor. In vitro competitive experiments have shown that androgens interact on ER with a low affinity but have a good stereospecificity for C,9 steroids bearing a 170-and/or 3/3-hydroxyl group. The administration of one dose (-20 mg) of 5a-dihydrotestosterone to 1-day ovariectomized rats induced ER nuclear translocation in 92% of the mammary tumors. In 60% of these tumors, this translocation was followed by a signifi cant stimulation of the rate of [3H]leucine incorporation into cytosoluble proteins by 5a-dihydrotestosterone or estradici. When injected together, 5a-dihydrotestosterone did not antagonize the effect of estradiol. We conclude that one very high dose of 5a-dihydrotestosterone that stimulates leucine incorporation into pro teins is also able to occupy the cytosol ER in mammary tumors and to translocate it in vivo into the nucleus. We suggest that a direct interaction of androgens with the ER located in mammary tumors is responsible, as in rat uteri, for the stimulating effect of very high doses of androgens. We cannot ascertain that the same mechanism is involved in the androgen-induced regression of the mammary tu mors.
Oncogene, 2001
Cathepsin-D, a lysosomal aspartyl proteinase, is highly secreted by breast cancer cells and its o... more Cathepsin-D, a lysosomal aspartyl proteinase, is highly secreted by breast cancer cells and its over-expression by transfection stimulates cancer cell proliferation. The mechanism by which this protease aects proliferation remains, however, unknown. In order to determine whether proteolytic activity is necessary, we abolished its enzymatic activity using site-directed mutagenesis followed by stable transfection in 3Y1-Ad12 cancer cells. Substitution of the aspartic acid residue 231 by an asparagine residue in its catalytic site abrogated the cathepsin-D proteolytic activity but did not aect its expression level, processing or secretion. However, like wild-type cathepsin-D, this mutated catalytically-inactive cathepsin-D retained its capacity to stimulate proliferation of cells embedded in Matrigel or collagen I matrices, colony formation in soft agar and tumor growth in athymic nude mice. Addition on the mocktransfected cells, of either conditioned media containing the wild-type or the mutated pro-cathepsin-D, or of the puri®ed mutated pro-cathepsin-D, partially mimicked the mitogenic activity of the transfected cathepsin-D, indicating a role of the secreted pro-enzyme. Moreover, addition of two anti-cathepsin-D antibodies on the cathepsin-D transfected cells inhibited their proliferation, suggesting an action of the secreted pro-cathepsin-D via an autocrine loop. A synthetic peptide containing the 27-44 residue moiety of the cathepsin-D pro-fragment was, however, not mitogenic suggesting that a receptor for the pro-fragment was not involved. Furthermore, the cathepsin-D mitogenicity was not blocked by inhibiting the interaction of pro-cathepsin-D with the mannose-6phosphate receptors. Our results altogether demonstrate that a mutated cathepsin-D devoid of catalytic activity is still mitogenic and suggest that it is acting extracellularly by triggering directly or indirectly a yet unidenti®ed cell surface receptor. Oncogene (2001) 20, 6920 ± 6929.
Proceedings of the National Academy of Sciences, 1992
Breast cancers containing estrogen receptors are responsive to antiestrogen treatment and have a ... more Breast cancers containing estrogen receptors are responsive to antiestrogen treatment and have a better prognosis than estrogen receptor-negative tumors. The loss of estrogen and progesterone receptors appears to be associated with a progression to less-differentiated tumors. We transfected the human estrogen receptor into the estrogen receptor-negative metastatic breast cancer cell line MDA-MB-231 in an attempt to restore their sensitivity to antiestrogens. Two stable sublines of MDA-MB-231 cells (HC1 and HE5) expressing functional estrogen receptors were studied for their ability to grow and invade in vitro and to metastasize in athymic nude mice. The number and size of lung metastases developed by these two sublines in ovariectomized nude mice was not markedly altered by tamoxifen but was inhibited 3-fold by estradiol. Estradiol also significantly inhibited in vitro cell proliferation of these sublines and their invasiveness in Matrigel, a reconstituted basement membrane, whereas...
Molecular Endocrinology, 1988
Two \gt11 libraries containing complementary DNAs from human breast cancer MCF7 cells were screen... more Two \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-receptornegative 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. (Molecular Endocrinology 2: 186-192, 1988)
CANCER AND METASTASIS REVIEW, 1990
Cathepsin D is an acidic lysosomal protease present in all cells. In estrogen receptor positive a... more Cathepsin D is an acidic lysosomal protease present in all cells. In estrogen receptor positive and negative breast cancer cell lines, the mRNA coding for pro-cathepsin D is overexpressed and sorting and maturation of the pro-enzyme are altered, via possibly saturation of the Man-6-P/IGF-II receptor, leading to accumulation of the active proteinase in large endosomes and to secretion of the precursor (52K protein). In MCF7 cells, the cathepsin D mRNA is induced directly and transcriptionally by estrogens and indirectly by growth factors. In patients, there is a significant correlation between high cathepsin D concentrations in the cytosol of primary breast cancer and development of metastasis. This marker is independent of other prognostic factors and appears to be particularly useful in axillary node-negative tumors. Transfection of a human cDNA cathepsin D expression vector under the control of SV40 promoter increases the metastatic potential of 3YA1-Adl2 rat tumorigenic cells when intravenously injected into nude mice. The mechanism of cathepsin D-induced metastasis is currently unknown. These results indicate that overexpression of cathepsin D might facilitate breast cancer metastasis, suggesting new possible therapeutic approaches.
Cancer research, Jan 15, 1988
The Mr 52,000 cathepsin-D-like protease induced by estrogens in MCF7 human breast cells was assay... more The Mr 52,000 cathepsin-D-like protease induced by estrogens in MCF7 human breast cells was assayed in 182 primary breast cancer cytosols prepared for receptor assays from pre- and post-menopausal patients. Using two solid-phase sandwich immunoenzymatic assays, we quantified the total Mr 52,000 cathepsin D (52K-cath-D) (the Mr 52,000 precursor protein and its Mr 48,000 and 34,000 processed forms) and the Mr 52,000 precursor alone. The value of total 52K-cath-D varied between 3 and 165 pmol/mg protein and the proportion of the precursor varied from 0 to 28% of total 52K-cath-D. There was no correlation between the concentrations of 52K-cath-D and estrogen receptor, but the estrogen receptor status (greater than or less than 10 fmol/mg protein) was correlated to the 52K-cath-D status (greater than or less than 15 pmol/mg protein) according to the chi 2 test (P less than 0.001). The correlation with progesterone receptor concentrations and status was low (r = 0.43) and absent, respecti...
Enzyme and Protein, 1996
Cathepsin D (Cath D) overexpression in breast cancer cells is associated with increased risk of m... more Cathepsin D (Cath D) overexpression in breast cancer cells is associated with increased risk of metastasis in patients according to several clinical studies. The amino acid sequence of Cath D in two breast cancer cell lines was normal, but glycosylation appears to be different with more acidic isoforms. Transfection of a human cDNA Cath D expression vector increases the metastatic potential of 3Y1-Ad12 embryonic rat tumorigenic cells when intravenously injected into nude mide. The mechanism of Cath-D-induced metastasis seems to require maturation of the proenzyme, mostly in large acidic compartments identified as phagosomes. Cath D is mitogenic in different cell types, and different substrates (growth inhibitors, precursors of growth factors, etc.) are proposed to mediate this activity.
Astrophysics and Space Science Library
We determined the stellar parameters for thirteen Galactic early-O type stars by modeling spectra... more We determined the stellar parameters for thirteen Galactic early-O type stars by modeling spectra from the Far Ultraviolet Spectroscopic Explorer (FUSE) (905-1187A) and from IUE and HST/STIS (1150-3250A) with line-blanketed, hydrodynamic, spherical non-LTE synthetic spectra, computed with the WMbasic code (Pauldrach et al. 2003). We find effective temperatures significantly lower (by 15-20%) than typically assigned to these spectral types (Bianchi & Garcia 2002; Bianchi et al. 2003; Garcia & Bianchi 2003). The stellar luminosities are also lower. The result is of great importance for the energy balance of HII regions and to understand massive star evolution.
International Journal of Radiation Applications and Instrumentation. Part B. Nuclear Medicine and Biology, 1987
We have studied estrogen-regulated proteins in an attempt to understand the mechanism by which es... more 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 I ysosomes via mannose-6-phosphate receptor. The protease is mitogen ic 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 ccl I lines, whi le in some ant iestrogen resistant variants, it becomes inducible by tamoxifen, contrary to the wild type MCF7 cells. Cloning of its cDNA in Xgtll has a I lowed to show that the mRNA is rapidly induced by estrogens and to sequence the protein and compare it to that of the norma I human kidney cathepsin-D. We conclude that the estrogen-induced cathepsin-D like protease may have important autocrine and/or paracrine functions in stimulating the growth and invasion of hormone-dependent and independent breast cancers and may be useful as a tissue marker to predict high risk mastopathies and breast cancer invasiveness. In addition to other estrogen regulated growth factors, the precursor of I ysosoma I proteases has the potential to stimulate the pro1 iferation and invasiveness of breast cancer cells as long as they are secreted in excess rather than being routed to lysosomes.
Enzyme and Protein, 1996
Cathepsin D (Cath D) overexpression in breast cancer cells is associated with increased risk of m... more Cathepsin D (Cath D) overexpression in breast cancer cells is associated with increased risk of metastasis in patients according to several clinical studies. The amino acid sequence of Cath D in two breast cancer cell lines was normal, but glycosylation appears to be different with more acidic isoforms. Transfection of a human cDNA Cath D expression vector increases the metastatic potential of 3Y1-Ad12 embryonic rat tumorigenic cells when intravenously injected into nude mide. The mechanism of Cath-D-induced metastasis seems to require maturation of the proenzyme, mostly in large acidic compartments identified as phagosomes. Cath D is mitogenic in different cell types, and different substrates (growth inhibitors, precursors of growth factors, etc.) are proposed to mediate this activity.
Enteropathogenic Campylobacter jejuni, C. coli and C. lari are currently the most common causes o... more Enteropathogenic Campylobacter jejuni, C. coli and C. lari are currently the most common causes of acute infectious diarrhoeal illness in the UK. Many domestic animals, including pigs, act as natural reservoirs of these organisms and infection may occur through the ingestion of contaminated food-stu¡s. C. jejuni and C. coli, isolated from the livers of bacon pigs, were examined at subspecies level by multilocus enzyme electrophoresis (MEE) typing with seven enzymic loci. Polymorphological variation was highest with indophenol oxidase, isocitrate dehydrogenase and l-phenylalanyl-l-leucine peptidase giving 5, 5 and 4 alleles at these loci, respectively. The 35 Campylobacter isolates examined in this study (12 C. jejuni and 23 C. coli) represented 30 unique electrophoretic types (ETs). Of these ETs, 8 unique types were detected for the 12 C. jejuni isolates and 19 unique ETs were detected for the 23 C. coli isolates. In addition, 3 types (ETs 2, 5, 10) were shared in common among C. jejuni and C. coli. The average number of alleles per enzyme locus was 3.28. The mean genetic diversity, i.e. arithmetic average over all loci assayed, including monomorphic values, was 0.5573 and 0.5350 for C. jejuni and C. coli, respectively. Alleles were shared by C. jejuni and C. coli, suggesting an exchange of genetic material between the species. MEE analyses of isolates showed that there was a wide range of subspecies types within both C. jejuni and C. coli in porcine livers. In certain cases, up to four phenotypically di¡erent strains of C. coli were isolated from one liver, indicating multiple infections.
Astrophysics and Space Science Library
We determined the stellar parameters for thirteen Galactic early-O type stars by modeling spectra... more We determined the stellar parameters for thirteen Galactic early-O type stars by modeling spectra from the Far Ultraviolet Spectroscopic Explorer (FUSE) (905-1187A) and from IUE and HST/STIS (1150-3250A) with line-blanketed, hydrodynamic, spherical non-LTE synthetic spectra, computed with the WMbasic code (Pauldrach et al. 2003). We find effective temperatures significantly lower (by 15-20%) than typically assigned to these spectral types (Bianchi & Garcia 2002; Bianchi et al. 2003; Garcia & Bianchi 2003). The stellar luminosities are also lower. The result is of great importance for the energy balance of HII regions and to understand massive star evolution.
International Journal of Radiation Applications and Instrumentation. Part B. Nuclear Medicine and Biology, 1987
We have studied estrogen-regulated proteins in an attempt to understand the mechanism by which es... more 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 I ysosomes via mannose-6-phosphate receptor. The protease is mitogen ic 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 ccl I lines, whi le in some ant iestrogen resistant variants, it becomes inducible by tamoxifen, contrary to the wild type MCF7 cells. Cloning of its cDNA in Xgtll has a I lowed to show that the mRNA is rapidly induced by estrogens and to sequence the protein and compare it to that of the norma I human kidney cathepsin-D. We conclude that the estrogen-induced cathepsin-D like protease may have important autocrine and/or paracrine functions in stimulating the growth and invasion of hormone-dependent and independent breast cancers and may be useful as a tissue marker to predict high risk mastopathies and breast cancer invasiveness. In addition to other estrogen regulated growth factors, the precursor of I ysosoma I proteases has the potential to stimulate the pro1 iferation and invasiveness of breast cancer cells as long as they are secreted in excess rather than being routed to lysosomes.
Journal of Steroid Biochemistry, 1989
Pro-cathepsin D is overexpressed in breast cancer cells compared to normal mammary epithelial cel... more 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.
Enteropathogenic Campylobacter jejuni, C. coli and C. lari are currently the most common causes o... more Enteropathogenic Campylobacter jejuni, C. coli and C. lari are currently the most common causes of acute infectious diarrhoeal illness in the UK. Many domestic animals, including pigs, act as natural reservoirs of these organisms and infection may occur through the ingestion of contaminated food-stu¡s. C. jejuni and C. coli, isolated from the livers of bacon pigs, were examined at subspecies level by multilocus enzyme electrophoresis (MEE) typing with seven enzymic loci. Polymorphological variation was highest with indophenol oxidase, isocitrate dehydrogenase and l-phenylalanyl-l-leucine peptidase giving 5, 5 and 4 alleles at these loci, respectively. The 35 Campylobacter isolates examined in this study (12 C. jejuni and 23 C. coli) represented 30 unique electrophoretic types (ETs). Of these ETs, 8 unique types were detected for the 12 C. jejuni isolates and 19 unique ETs were detected for the 23 C. coli isolates. In addition, 3 types (ETs 2, 5, 10) were shared in common among C. jejuni and C. coli. The average number of alleles per enzyme locus was 3.28. The mean genetic diversity, i.e. arithmetic average over all loci assayed, including monomorphic values, was 0.5573 and 0.5350 for C. jejuni and C. coli, respectively. Alleles were shared by C. jejuni and C. coli, suggesting an exchange of genetic material between the species. MEE analyses of isolates showed that there was a wide range of subspecies types within both C. jejuni and C. coli in porcine livers. In certain cases, up to four phenotypically di¡erent strains of C. coli were isolated from one liver, indicating multiple infections.
Cancer research, 1983
In an attempt to find estrogen-specific responses in breast cancer, we have established primary c... more 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...
Cancer research, 1981
5-Androstene-3 beta, 17 beta-diol (ADIOL) has previously been shown to have a high affinity for t... more 5-Androstene-3 beta, 17 beta-diol (ADIOL) has previously been shown to have a high affinity for the estrogen receptor and to translocate it to the nucleus in vitro and in vivo. This compound and related C19 delta 5-steroids of adrenal origin have now been examined for their ability to induce the synthesis in MCF7 cells of an estrogen-dependent secreted glycoprotein (M.W. 46,000). Concentrations required for half-maximum induction were 2 nM for ADIOL and 500 nM for dehydroepiandrosterone (DHEA). Dehydroepiandrosterone sulfate showed weak inducing ability at concentrations of 1 microM or greater. The induction by ADIOL was unaffected by the presence of an aromatase inhibitor, and 5 alpha-androstane-3 beta, 17 beta-diol, which cannot be aromatized, also induced the M.W. 46,000 protein at low concentrations. When cells were exposed to 10 nM [3H]ADIOL, the cytosol and nuclear fractions contained [3H]ADIOL resistant to charcoal adsorption. The bound [3H]ADIOL in the cytosol and nucleus wa...
Cancer research, 1986
We have recently shown that the integrity of the polyamine pathway is essential but not sufficien... more We have recently shown that the integrity of the polyamine pathway is essential but not sufficient for expression of the mitogenic effect of estradiol (E2) in the N-nitrosomethylurea-induced rat mammary tumor grown in vitro in the soft agar clonogenic assay. To elucidate the mechanism of E2 action in this system, we tested whether E2 may stimulate tumor growth through induction of secretory growth factor(s), as already proposed for human breast cancer cell lines in culture. Furthermore, we investigated the potential interaction between such "autocrine" control of tumor growth by E2 and the polyamine pathway. Conditioned medium obtained from E2-treated tumors (E2-CM) but not from control tumors (C-CM) consistently stimulated colony formation when added to N-nitrosomethylurea-mammary tumors plated in soft agar under serum-free media conditions. Such growth-promoting effects of the E2-CM was found to increase with increasing protein concentrations of the medium and was abolis...
Cancer research, 1989
The Mr 52,000 cathepsin D is the precursor of a lysosomal protease secreted in excess by breast c... more The Mr 52,000 cathepsin D is the precursor of a lysosomal protease secreted in excess by breast cancer cells. This protease can degrade extracellular matrices and proteoglycans and is induced by estrogens in estrogen receptor-positive breast cancer cell lines. In a 4- to 6-yr retrospective cohort study, the concentration of the total cathepsin D (precursor plus intermediate and mature chains) was assayed in cytosols of primary tumors from 242 pre/perimenopausal and 154 postmenopausal breast cancer patients in a solid-phase immunoassay using two specific monoclonal antibodies. Patients were initially divided into groups with low, intermediate, or high concentrations of cathepsin D corresponding to the quartiles of the overall distribution. Using these groupings, the level of Mr 52,000 cathepsin D was not significantly associated with the recognized prognostic factors of age, lymph node involvement, tumor size, and/or grade of anaplasia. A significant association was found between cat...
Cancer research, 1978
To improve our understanding of the effects of androgens on the induction and growth of mammary t... more To improve our understanding of the effects of androgens on the induction and growth of mammary tumors, the interaction of androgen on the estrogen receptor (ER) has been evaluated in the 7,12-dimethylbenz(a)anthracene-induced rat mammary tumor. In vitro competitive experiments have shown that androgens interact on ER with a low affinity but have a good stereospecificity for C,9 steroids bearing a 170-and/or 3/3-hydroxyl group. The administration of one dose (-20 mg) of 5a-dihydrotestosterone to 1-day ovariectomized rats induced ER nuclear translocation in 92% of the mammary tumors. In 60% of these tumors, this translocation was followed by a signifi cant stimulation of the rate of [3H]leucine incorporation into cytosoluble proteins by 5a-dihydrotestosterone or estradici. When injected together, 5a-dihydrotestosterone did not antagonize the effect of estradiol. We conclude that one very high dose of 5a-dihydrotestosterone that stimulates leucine incorporation into pro teins is also able to occupy the cytosol ER in mammary tumors and to translocate it in vivo into the nucleus. We suggest that a direct interaction of androgens with the ER located in mammary tumors is responsible, as in rat uteri, for the stimulating effect of very high doses of androgens. We cannot ascertain that the same mechanism is involved in the androgen-induced regression of the mammary tu mors.
Oncogene, 2001
Cathepsin-D, a lysosomal aspartyl proteinase, is highly secreted by breast cancer cells and its o... more Cathepsin-D, a lysosomal aspartyl proteinase, is highly secreted by breast cancer cells and its over-expression by transfection stimulates cancer cell proliferation. The mechanism by which this protease aects proliferation remains, however, unknown. In order to determine whether proteolytic activity is necessary, we abolished its enzymatic activity using site-directed mutagenesis followed by stable transfection in 3Y1-Ad12 cancer cells. Substitution of the aspartic acid residue 231 by an asparagine residue in its catalytic site abrogated the cathepsin-D proteolytic activity but did not aect its expression level, processing or secretion. However, like wild-type cathepsin-D, this mutated catalytically-inactive cathepsin-D retained its capacity to stimulate proliferation of cells embedded in Matrigel or collagen I matrices, colony formation in soft agar and tumor growth in athymic nude mice. Addition on the mocktransfected cells, of either conditioned media containing the wild-type or the mutated pro-cathepsin-D, or of the puri®ed mutated pro-cathepsin-D, partially mimicked the mitogenic activity of the transfected cathepsin-D, indicating a role of the secreted pro-enzyme. Moreover, addition of two anti-cathepsin-D antibodies on the cathepsin-D transfected cells inhibited their proliferation, suggesting an action of the secreted pro-cathepsin-D via an autocrine loop. A synthetic peptide containing the 27-44 residue moiety of the cathepsin-D pro-fragment was, however, not mitogenic suggesting that a receptor for the pro-fragment was not involved. Furthermore, the cathepsin-D mitogenicity was not blocked by inhibiting the interaction of pro-cathepsin-D with the mannose-6phosphate receptors. Our results altogether demonstrate that a mutated cathepsin-D devoid of catalytic activity is still mitogenic and suggest that it is acting extracellularly by triggering directly or indirectly a yet unidenti®ed cell surface receptor. Oncogene (2001) 20, 6920 ± 6929.
Proceedings of the National Academy of Sciences, 1992
Breast cancers containing estrogen receptors are responsive to antiestrogen treatment and have a ... more Breast cancers containing estrogen receptors are responsive to antiestrogen treatment and have a better prognosis than estrogen receptor-negative tumors. The loss of estrogen and progesterone receptors appears to be associated with a progression to less-differentiated tumors. We transfected the human estrogen receptor into the estrogen receptor-negative metastatic breast cancer cell line MDA-MB-231 in an attempt to restore their sensitivity to antiestrogens. Two stable sublines of MDA-MB-231 cells (HC1 and HE5) expressing functional estrogen receptors were studied for their ability to grow and invade in vitro and to metastasize in athymic nude mice. The number and size of lung metastases developed by these two sublines in ovariectomized nude mice was not markedly altered by tamoxifen but was inhibited 3-fold by estradiol. Estradiol also significantly inhibited in vitro cell proliferation of these sublines and their invasiveness in Matrigel, a reconstituted basement membrane, whereas...
Molecular Endocrinology, 1988
Two \gt11 libraries containing complementary DNAs from human breast cancer MCF7 cells were screen... more Two \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-receptornegative 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. (Molecular Endocrinology 2: 186-192, 1988)
CANCER AND METASTASIS REVIEW, 1990
Cathepsin D is an acidic lysosomal protease present in all cells. In estrogen receptor positive a... more Cathepsin D is an acidic lysosomal protease present in all cells. In estrogen receptor positive and negative breast cancer cell lines, the mRNA coding for pro-cathepsin D is overexpressed and sorting and maturation of the pro-enzyme are altered, via possibly saturation of the Man-6-P/IGF-II receptor, leading to accumulation of the active proteinase in large endosomes and to secretion of the precursor (52K protein). In MCF7 cells, the cathepsin D mRNA is induced directly and transcriptionally by estrogens and indirectly by growth factors. In patients, there is a significant correlation between high cathepsin D concentrations in the cytosol of primary breast cancer and development of metastasis. This marker is independent of other prognostic factors and appears to be particularly useful in axillary node-negative tumors. Transfection of a human cDNA cathepsin D expression vector under the control of SV40 promoter increases the metastatic potential of 3YA1-Adl2 rat tumorigenic cells when intravenously injected into nude mice. The mechanism of cathepsin D-induced metastasis is currently unknown. These results indicate that overexpression of cathepsin D might facilitate breast cancer metastasis, suggesting new possible therapeutic approaches.
Cancer research, Jan 15, 1988
The Mr 52,000 cathepsin-D-like protease induced by estrogens in MCF7 human breast cells was assay... more The Mr 52,000 cathepsin-D-like protease induced by estrogens in MCF7 human breast cells was assayed in 182 primary breast cancer cytosols prepared for receptor assays from pre- and post-menopausal patients. Using two solid-phase sandwich immunoenzymatic assays, we quantified the total Mr 52,000 cathepsin D (52K-cath-D) (the Mr 52,000 precursor protein and its Mr 48,000 and 34,000 processed forms) and the Mr 52,000 precursor alone. The value of total 52K-cath-D varied between 3 and 165 pmol/mg protein and the proportion of the precursor varied from 0 to 28% of total 52K-cath-D. There was no correlation between the concentrations of 52K-cath-D and estrogen receptor, but the estrogen receptor status (greater than or less than 10 fmol/mg protein) was correlated to the 52K-cath-D status (greater than or less than 15 pmol/mg protein) according to the chi 2 test (P less than 0.001). The correlation with progesterone receptor concentrations and status was low (r = 0.43) and absent, respecti...
Enzyme and Protein, 1996
Cathepsin D (Cath D) overexpression in breast cancer cells is associated with increased risk of m... more Cathepsin D (Cath D) overexpression in breast cancer cells is associated with increased risk of metastasis in patients according to several clinical studies. The amino acid sequence of Cath D in two breast cancer cell lines was normal, but glycosylation appears to be different with more acidic isoforms. Transfection of a human cDNA Cath D expression vector increases the metastatic potential of 3Y1-Ad12 embryonic rat tumorigenic cells when intravenously injected into nude mide. The mechanism of Cath-D-induced metastasis seems to require maturation of the proenzyme, mostly in large acidic compartments identified as phagosomes. Cath D is mitogenic in different cell types, and different substrates (growth inhibitors, precursors of growth factors, etc.) are proposed to mediate this activity.
Astrophysics and Space Science Library
We determined the stellar parameters for thirteen Galactic early-O type stars by modeling spectra... more We determined the stellar parameters for thirteen Galactic early-O type stars by modeling spectra from the Far Ultraviolet Spectroscopic Explorer (FUSE) (905-1187A) and from IUE and HST/STIS (1150-3250A) with line-blanketed, hydrodynamic, spherical non-LTE synthetic spectra, computed with the WMbasic code (Pauldrach et al. 2003). We find effective temperatures significantly lower (by 15-20%) than typically assigned to these spectral types (Bianchi & Garcia 2002; Bianchi et al. 2003; Garcia & Bianchi 2003). The stellar luminosities are also lower. The result is of great importance for the energy balance of HII regions and to understand massive star evolution.
International Journal of Radiation Applications and Instrumentation. Part B. Nuclear Medicine and Biology, 1987
We have studied estrogen-regulated proteins in an attempt to understand the mechanism by which es... more 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 I ysosomes via mannose-6-phosphate receptor. The protease is mitogen ic 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 ccl I lines, whi le in some ant iestrogen resistant variants, it becomes inducible by tamoxifen, contrary to the wild type MCF7 cells. Cloning of its cDNA in Xgtll has a I lowed to show that the mRNA is rapidly induced by estrogens and to sequence the protein and compare it to that of the norma I human kidney cathepsin-D. We conclude that the estrogen-induced cathepsin-D like protease may have important autocrine and/or paracrine functions in stimulating the growth and invasion of hormone-dependent and independent breast cancers and may be useful as a tissue marker to predict high risk mastopathies and breast cancer invasiveness. In addition to other estrogen regulated growth factors, the precursor of I ysosoma I proteases has the potential to stimulate the pro1 iferation and invasiveness of breast cancer cells as long as they are secreted in excess rather than being routed to lysosomes.
Enzyme and Protein, 1996
Cathepsin D (Cath D) overexpression in breast cancer cells is associated with increased risk of m... more Cathepsin D (Cath D) overexpression in breast cancer cells is associated with increased risk of metastasis in patients according to several clinical studies. The amino acid sequence of Cath D in two breast cancer cell lines was normal, but glycosylation appears to be different with more acidic isoforms. Transfection of a human cDNA Cath D expression vector increases the metastatic potential of 3Y1-Ad12 embryonic rat tumorigenic cells when intravenously injected into nude mide. The mechanism of Cath-D-induced metastasis seems to require maturation of the proenzyme, mostly in large acidic compartments identified as phagosomes. Cath D is mitogenic in different cell types, and different substrates (growth inhibitors, precursors of growth factors, etc.) are proposed to mediate this activity.
Enteropathogenic Campylobacter jejuni, C. coli and C. lari are currently the most common causes o... more Enteropathogenic Campylobacter jejuni, C. coli and C. lari are currently the most common causes of acute infectious diarrhoeal illness in the UK. Many domestic animals, including pigs, act as natural reservoirs of these organisms and infection may occur through the ingestion of contaminated food-stu¡s. C. jejuni and C. coli, isolated from the livers of bacon pigs, were examined at subspecies level by multilocus enzyme electrophoresis (MEE) typing with seven enzymic loci. Polymorphological variation was highest with indophenol oxidase, isocitrate dehydrogenase and l-phenylalanyl-l-leucine peptidase giving 5, 5 and 4 alleles at these loci, respectively. The 35 Campylobacter isolates examined in this study (12 C. jejuni and 23 C. coli) represented 30 unique electrophoretic types (ETs). Of these ETs, 8 unique types were detected for the 12 C. jejuni isolates and 19 unique ETs were detected for the 23 C. coli isolates. In addition, 3 types (ETs 2, 5, 10) were shared in common among C. jejuni and C. coli. The average number of alleles per enzyme locus was 3.28. The mean genetic diversity, i.e. arithmetic average over all loci assayed, including monomorphic values, was 0.5573 and 0.5350 for C. jejuni and C. coli, respectively. Alleles were shared by C. jejuni and C. coli, suggesting an exchange of genetic material between the species. MEE analyses of isolates showed that there was a wide range of subspecies types within both C. jejuni and C. coli in porcine livers. In certain cases, up to four phenotypically di¡erent strains of C. coli were isolated from one liver, indicating multiple infections.
Astrophysics and Space Science Library
We determined the stellar parameters for thirteen Galactic early-O type stars by modeling spectra... more We determined the stellar parameters for thirteen Galactic early-O type stars by modeling spectra from the Far Ultraviolet Spectroscopic Explorer (FUSE) (905-1187A) and from IUE and HST/STIS (1150-3250A) with line-blanketed, hydrodynamic, spherical non-LTE synthetic spectra, computed with the WMbasic code (Pauldrach et al. 2003). We find effective temperatures significantly lower (by 15-20%) than typically assigned to these spectral types (Bianchi & Garcia 2002; Bianchi et al. 2003; Garcia & Bianchi 2003). The stellar luminosities are also lower. The result is of great importance for the energy balance of HII regions and to understand massive star evolution.
International Journal of Radiation Applications and Instrumentation. Part B. Nuclear Medicine and Biology, 1987
We have studied estrogen-regulated proteins in an attempt to understand the mechanism by which es... more 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 I ysosomes via mannose-6-phosphate receptor. The protease is mitogen ic 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 ccl I lines, whi le in some ant iestrogen resistant variants, it becomes inducible by tamoxifen, contrary to the wild type MCF7 cells. Cloning of its cDNA in Xgtll has a I lowed to show that the mRNA is rapidly induced by estrogens and to sequence the protein and compare it to that of the norma I human kidney cathepsin-D. We conclude that the estrogen-induced cathepsin-D like protease may have important autocrine and/or paracrine functions in stimulating the growth and invasion of hormone-dependent and independent breast cancers and may be useful as a tissue marker to predict high risk mastopathies and breast cancer invasiveness. In addition to other estrogen regulated growth factors, the precursor of I ysosoma I proteases has the potential to stimulate the pro1 iferation and invasiveness of breast cancer cells as long as they are secreted in excess rather than being routed to lysosomes.
Journal of Steroid Biochemistry, 1989
Pro-cathepsin D is overexpressed in breast cancer cells compared to normal mammary epithelial cel... more 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.
Enteropathogenic Campylobacter jejuni, C. coli and C. lari are currently the most common causes o... more Enteropathogenic Campylobacter jejuni, C. coli and C. lari are currently the most common causes of acute infectious diarrhoeal illness in the UK. Many domestic animals, including pigs, act as natural reservoirs of these organisms and infection may occur through the ingestion of contaminated food-stu¡s. C. jejuni and C. coli, isolated from the livers of bacon pigs, were examined at subspecies level by multilocus enzyme electrophoresis (MEE) typing with seven enzymic loci. Polymorphological variation was highest with indophenol oxidase, isocitrate dehydrogenase and l-phenylalanyl-l-leucine peptidase giving 5, 5 and 4 alleles at these loci, respectively. The 35 Campylobacter isolates examined in this study (12 C. jejuni and 23 C. coli) represented 30 unique electrophoretic types (ETs). Of these ETs, 8 unique types were detected for the 12 C. jejuni isolates and 19 unique ETs were detected for the 23 C. coli isolates. In addition, 3 types (ETs 2, 5, 10) were shared in common among C. jejuni and C. coli. The average number of alleles per enzyme locus was 3.28. The mean genetic diversity, i.e. arithmetic average over all loci assayed, including monomorphic values, was 0.5573 and 0.5350 for C. jejuni and C. coli, respectively. Alleles were shared by C. jejuni and C. coli, suggesting an exchange of genetic material between the species. MEE analyses of isolates showed that there was a wide range of subspecies types within both C. jejuni and C. coli in porcine livers. In certain cases, up to four phenotypically di¡erent strains of C. coli were isolated from one liver, indicating multiple infections.