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Colin J. Wilde - One of the best experts on this subject based on the ideXlab platform.
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influence of microenvironment on mammary epithelial cell survival in primary culture
Journal of Cellular Physiology, 1999Co-Authors: David R Blatchford, Lynda H. Quarrie, Elizabeth Tonner, Corinna Mccarthy, David J Flint, Colin J. WildeAbstract:Mammary epithelial cells cultured on Engelbreth-Holm-Swarm (EHS) matrix form multicellular structures termed mammospheres, in which cells and matrix become arranged around a central luminal space. In the presence of lactogenic hormones, cells within mammospheres become polarized, form tight intercellular junctions, and secrete milk proteins vectorially into the luminal space. This study examined the mechanism of lumen formation. Histological examination of developing mammospheres showed that cavitation was associated spatially and temporally with the appearance of fragmented nuclear material in apoptotic bodies, and with the presence of cells positively labeled by terminal deoxynucleotide transferase-mediated deoxyuridine nick end-labeling (TUNEL). Analysis of [32P]-deoxynucleotide end-labeled genomic DNA by electrophoresis and autoradiography showed DNA Laddering indicative of apoptosis. A transient increase in Laddering coincided with both lumen formation and the presence of TUNEL-positive cells. Lumen formation, DNA Laddering, and detection of TUNEL-positive cells were all accelerated when matrix composition was altered. They were also impaired coordinately when caspase inhibitor was present during the first two days of culture. Therefore, lumen formation in mammosphere cultures is due to selective apoptosis of centrally located cells. Mammosphere cavitation was accompanied by redistribution of matrix constituents to the mammosphere periphery. Western blotting and Western ligand blotting of culture medium showed that lumen formation was also associated with a transient increase in insulin-like growth factor binding protein-5 (IGFBP5), a factor implicated in mammary apoptosis in vivo. We propose that epithelial cell survival during mammosphere development is induced selectively through stabilization by basement membrane constituents, which may act directly on the epithelial cell or confer protection against autocrine apoptotic factors. J. Cell. Physiol. 181:304–311, 1999. © 1999 Wiley-Liss, Inc.
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Programmed cell death during mammary tissue involution induced by weaning, litter removal, and milk stasis
Journal of cellular physiology, 1996Co-Authors: Lynda H. Quarrie, Caroline V. P. Addey, Colin J. WildeAbstract:Programmed cell death in mammary tissue was studied during natural weaning in lactating mice and after litter removal or milk stasis. All treatments stimulated mammary apoptosis, indicating that this process is an integral part of the tissue's involution after lactation. Induction of apoptosis was slower in natural weaning than after litter removal but occurred earlier when mice were concurrently pregnant during natural weaning. Ipsilateral induction of apoptosis by milk stasis in teat-sealed glands indicates that cell death is under local (i.e., intramammary) as well as endocrine regulation. Apoptosis detected by DNA Laddering was associated with changes in expression of p53 and bax, two genes implicated in the regulation of cell death, and was accompanied by structural degeneration characteristic of mammary involution. Reciprocal changes in stromelysin mRNA, and that of its inhibitor TIMP-2, suggested that this structural reorganisation was the result of coordinated changes in gene expression favouring proteolysis of the extracellular matrix. © 1996 Wiley-Liss, Inc.
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Apoptosis in lactating and involuting mouse mammary tissue demonstrated by nick-end DNA labelling
Cell and Tissue Research, 1995Co-Authors: Lynda H. Quarrie, Caroline V. P. Addey, Colin J. WildeAbstract:Mammary involution after cessation of milk removal is associated with extensive loss of secretory epithelial cells. Ultrastructural changes and the appearance of oligonucleosomal DNA Laddering in ethidium bromide-stained gels indicates that cell loss during involution occurs by apoptosis. In this study, a technique for nick end-labelling of genomic DNA with radiolabelled deoxynucleotide has been used to monitor the induction of programmed cell death in mice after litter removal at peak lactation. This technique proved more sensitive than conventional ethidium bromide staining, and results suggested that apoptosis was induced rapidly by milk stasis, before extensive tissue re-modelling had begun. Oligonucleosomal DNA Laddering on agarose gels was detected within 24 h of milk stasis, and increased progressively for at least 4 days. Nick-end labelling also detected Laddering before litter removal, suggesting that programmed cell death is a normal feature of the lactating tissue. The DNA end-labelling technique was also adapted for in situ visualisation of apoptotic cells in tissue sections. By this criterion, apoptotic cells were identified in both the secretory epithelium lining the alveoli of the gland and, increasingly with prolonged milk stasis, amongst those sloughed into the alveolar lumen. The results demonstrate the utility of these techniques for study of mammary cell death and suggest that, whilst apoptosis is rapidly induced by milk stasis, it is also a normal physiological event in the lactating mammary gland.
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Local Control of Mammary Apoptosis by Milk Stasis
Intercellular Signalling in the Mammary Gland, 1995Co-Authors: Lynda H. Quarrie, Caroline V. P. Addey, Colin J. WildeAbstract:Mammary involution after lactation is associated with extensive loss of secretory epithelial cells. Laddering of genomic DNA (Wyllie et al., 1980) and altered gene expression indicate that this cell loss occurs by apoptosis. Neither the mechanism nor the regulation of mammary cell death is well-characterised, but both circulating mammogenic hormones and locally-active factors may be involved. Apoptosis induced in lactating mouse mammary gland by litter removal was prevented by prolactin treatment (Sheffield and Kotolski, 1992), whereas non-milking of one gland of lactating goats increased DNA Laddering in that gland (Quarrie et al., 1994). In this study, we present evidence for local induction of apoptosis in glands of lactating mice after 24 h of milk stasis.
Wilson S Colucci - One of the best experts on this subject based on the ideXlab platform.
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inhibition of copper zinc superoxide dismutase induces cell growth hypertrophic phenotype and apoptosis in neonatal rat cardiac myocytes in vitro
Circulation Research, 1999Co-Authors: Deborah A Siwik, John D Tzortzis, David R Pimental, Donny L F Chang, Patrick J Pagano, Krishna Singh, Douglas B Sawyer, Wilson S ColucciAbstract:Abstract—Oxidative stress has been implicated in the pathophysiology of myocardial failure. We tested the hypothesis that inhibition of endogenous antioxidant enzymes can regulate the phenotype of cardiac myocytes. Neonatal rat ventricular myocytes in vitro were exposed to diethyldithiocarbamic acid (DDC), an inhibitor of cytosolic (Cu, Zn) and extracellular superoxide dismutase (SOD). DDC inhibited SOD activity and increased intracellular superoxide in a concentration-dependent manner. A low concentration (1 μmol/L) of DDC stimulated myocyte growth, as demonstrated by increases in protein synthesis, cellular protein, prepro–atrial natriuretic peptide, and c-fos mRNAs and decreased sarcoplasmic reticulum Ca2+ATPase mRNA. These actions were all inhibited by the superoxide scavenger Tiron (4,5-dihydroxy-1,3-benzene disulfonic acid). Higher concentrations of DDC (100 μmol/L) stimulated myocyte apoptosis, as evidenced by DNA Laddering, characteristic nuclear morphology, in situ terminal deoxynucleotidyl trans...
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norepinephrine stimulates apoptosis in adult rat ventricular myocytes by activation of the β adrenergic pathway
Circulation, 1998Co-Authors: Catherine Communal, Krishna Singh, David R Pimentel, Wilson S ColucciAbstract:Background—Myocardial sympathetic activity is increased in heart failure. We tested the hypothesis that norepinephrine (NE) stimulates apoptosis in adult rat ventricular myocytes in vitro. Methods and Results—Myocytes were exposed to NE alone (10 μmol/L), NE+propranolol (2 μmol/L), NE+prazosin (0.1 μmol/L), or isoproterenol (ISO, 10 μmol/L) for 24 hours. NE and ISO decreased the number of viable myocytes by ≈35%. This effect was completely blocked by the β-adrenergic antagonist propranolol but was not affected by the α1-adrenergic antagonist prazosin. NE increased DNA Laddering on agarose gel electrophoresis and increased the percentage of cells that were stained by terminal deoxynucleotidyl transferase–mediated nick end-labeling from 5.8±1.0% to 21.0±2.3% (P<0.01; n=4). NE likewise increased the percentage of apoptotic cells with hypodiploid DNA content as assessed by flow cytometry from 7.8±0.7% to 16.7±2.2% (P<0.01; n=6), and this effect was abolished by propranolol but not prazosin. ISO and forskolin ...
Dennis W Choi - One of the best experts on this subject based on the ideXlab platform.
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staurosporine induced neuronal apoptosis
Experimental Neurology, 1995Co-Authors: Byoung Joo Gwag, Joseph A Demaro, Stefano L Sensi, Cynthia A Csernansky, Lorella M T Canzoniero, Dennis W ChoiAbstract:Staurosporine, a nonselective protein kinase inhibitor, has been shown to induce apoptosis in several different nonneuronal cell types. We tested the hypothesis that staurosporine would also induce apoptosis in central neurons. Exposure of murine cortical cell cultures to 30-100 nM staurosporine induced concentration-dependent selective neuronal degeneration over the following day; at higher concentrations, staurosporine damaged glial cells as well. Staurosporine-induced neuronal death was accompanied by cell body shrinkage, chromatin condensation, and DNA Laddering. In contrast, NMDA-induced neuronal death was accompanied by acute cell body swelling without DNA Laddering. Staurosporine-induced neuronal death, unlike excitotoxic death, was markedly attenuated by the protein synthesis inhibitor cycloheximide; this protective effect was not reversed by a glutathione synthesis inhibitor, buthionine sulfoximine. Interestingly, the glial cell death induced by 1 μM staurosporine was markedly potentiated by cycloheximide. Staurosporine-induced neuronal death was not accompanied by an increase in intracellular free Ca2+ and was attenuated by 30 mM K+; this protective effect of high K+ was blocked by nimodipine or Co2+. Present data suggest that staurosporine can induce apoptosis in cultured cortical neurons and that this apoptosis can be blocked by raising intracellular Ca2+ or by blocking protein synthesis. Staurosporine exposure may be useful as a model for studying central neuronal apoptosis in vitro.
Lynda H. Quarrie - One of the best experts on this subject based on the ideXlab platform.
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influence of microenvironment on mammary epithelial cell survival in primary culture
Journal of Cellular Physiology, 1999Co-Authors: David R Blatchford, Lynda H. Quarrie, Elizabeth Tonner, Corinna Mccarthy, David J Flint, Colin J. WildeAbstract:Mammary epithelial cells cultured on Engelbreth-Holm-Swarm (EHS) matrix form multicellular structures termed mammospheres, in which cells and matrix become arranged around a central luminal space. In the presence of lactogenic hormones, cells within mammospheres become polarized, form tight intercellular junctions, and secrete milk proteins vectorially into the luminal space. This study examined the mechanism of lumen formation. Histological examination of developing mammospheres showed that cavitation was associated spatially and temporally with the appearance of fragmented nuclear material in apoptotic bodies, and with the presence of cells positively labeled by terminal deoxynucleotide transferase-mediated deoxyuridine nick end-labeling (TUNEL). Analysis of [32P]-deoxynucleotide end-labeled genomic DNA by electrophoresis and autoradiography showed DNA Laddering indicative of apoptosis. A transient increase in Laddering coincided with both lumen formation and the presence of TUNEL-positive cells. Lumen formation, DNA Laddering, and detection of TUNEL-positive cells were all accelerated when matrix composition was altered. They were also impaired coordinately when caspase inhibitor was present during the first two days of culture. Therefore, lumen formation in mammosphere cultures is due to selective apoptosis of centrally located cells. Mammosphere cavitation was accompanied by redistribution of matrix constituents to the mammosphere periphery. Western blotting and Western ligand blotting of culture medium showed that lumen formation was also associated with a transient increase in insulin-like growth factor binding protein-5 (IGFBP5), a factor implicated in mammary apoptosis in vivo. We propose that epithelial cell survival during mammosphere development is induced selectively through stabilization by basement membrane constituents, which may act directly on the epithelial cell or confer protection against autocrine apoptotic factors. J. Cell. Physiol. 181:304–311, 1999. © 1999 Wiley-Liss, Inc.
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Programmed cell death during mammary tissue involution induced by weaning, litter removal, and milk stasis
Journal of cellular physiology, 1996Co-Authors: Lynda H. Quarrie, Caroline V. P. Addey, Colin J. WildeAbstract:Programmed cell death in mammary tissue was studied during natural weaning in lactating mice and after litter removal or milk stasis. All treatments stimulated mammary apoptosis, indicating that this process is an integral part of the tissue's involution after lactation. Induction of apoptosis was slower in natural weaning than after litter removal but occurred earlier when mice were concurrently pregnant during natural weaning. Ipsilateral induction of apoptosis by milk stasis in teat-sealed glands indicates that cell death is under local (i.e., intramammary) as well as endocrine regulation. Apoptosis detected by DNA Laddering was associated with changes in expression of p53 and bax, two genes implicated in the regulation of cell death, and was accompanied by structural degeneration characteristic of mammary involution. Reciprocal changes in stromelysin mRNA, and that of its inhibitor TIMP-2, suggested that this structural reorganisation was the result of coordinated changes in gene expression favouring proteolysis of the extracellular matrix. © 1996 Wiley-Liss, Inc.
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Apoptosis in lactating and involuting mouse mammary tissue demonstrated by nick-end DNA labelling
Cell and Tissue Research, 1995Co-Authors: Lynda H. Quarrie, Caroline V. P. Addey, Colin J. WildeAbstract:Mammary involution after cessation of milk removal is associated with extensive loss of secretory epithelial cells. Ultrastructural changes and the appearance of oligonucleosomal DNA Laddering in ethidium bromide-stained gels indicates that cell loss during involution occurs by apoptosis. In this study, a technique for nick end-labelling of genomic DNA with radiolabelled deoxynucleotide has been used to monitor the induction of programmed cell death in mice after litter removal at peak lactation. This technique proved more sensitive than conventional ethidium bromide staining, and results suggested that apoptosis was induced rapidly by milk stasis, before extensive tissue re-modelling had begun. Oligonucleosomal DNA Laddering on agarose gels was detected within 24 h of milk stasis, and increased progressively for at least 4 days. Nick-end labelling also detected Laddering before litter removal, suggesting that programmed cell death is a normal feature of the lactating tissue. The DNA end-labelling technique was also adapted for in situ visualisation of apoptotic cells in tissue sections. By this criterion, apoptotic cells were identified in both the secretory epithelium lining the alveoli of the gland and, increasingly with prolonged milk stasis, amongst those sloughed into the alveolar lumen. The results demonstrate the utility of these techniques for study of mammary cell death and suggest that, whilst apoptosis is rapidly induced by milk stasis, it is also a normal physiological event in the lactating mammary gland.
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Local Control of Mammary Apoptosis by Milk Stasis
Intercellular Signalling in the Mammary Gland, 1995Co-Authors: Lynda H. Quarrie, Caroline V. P. Addey, Colin J. WildeAbstract:Mammary involution after lactation is associated with extensive loss of secretory epithelial cells. Laddering of genomic DNA (Wyllie et al., 1980) and altered gene expression indicate that this cell loss occurs by apoptosis. Neither the mechanism nor the regulation of mammary cell death is well-characterised, but both circulating mammogenic hormones and locally-active factors may be involved. Apoptosis induced in lactating mouse mammary gland by litter removal was prevented by prolactin treatment (Sheffield and Kotolski, 1992), whereas non-milking of one gland of lactating goats increased DNA Laddering in that gland (Quarrie et al., 1994). In this study, we present evidence for local induction of apoptosis in glands of lactating mice after 24 h of milk stasis.
Douglas Yee - One of the best experts on this subject based on the ideXlab platform.
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Strain-specific differences in formation of apoptotic DNA ladders in MCF-7 breast cancer cells
Cancer letters, 1999Co-Authors: Jennifer L. Gooch, Douglas YeeAbstract:We tested the ability of seven MCF-7 strains to undergo DNA fragmentation, as measured by DNA Laddering, following doxorubicin-induced apoptosis. Four strains were found to undergo DNA Laddering while three were not. All strains were inhibited by doxorubicin, although sensitivity differed. Finally, we show by detection of sub-G1 DNA that doxorubicin induced the same fold increase in apoptosis in MCF-7-ATCC, which did not ladder, and MCF-7-MG, which did ladder. Therefore, detection of DNA ladders is not an accurate indicator of apoptosis in MCF-7 cells as fragmentation of DNA appears to vary between strains.