ABSTRACT
Perturbations in cell-extracellular matrix (ECM) interactions are a consistent feature of mammary tumors and cells in culture. We have utilized MCF-10ATG3B human breast epithelial cells to examine whether the organochlorine Kepone induces alterations in cell adhesion molecules important to cell-cell and cell-ECM interactions. Kepone effects on the levels and association of proteins involved in adherens junctions or desmosomes were examined using immunoblot analysis and immunoprecipitation. MCF-10ATG3B cells cultured on an ECM of Matrigel form lattice-like structures that are disrupted with 0.1 and 1 microM Kepone. E-cadherin protein levels decreased significantly by approximately 23% and approximately 69% following treatment with 0.1 and 1.0 microM Kepone, respectively, relative to solvent-treated cells. Desmoglein and alpha- and gamma-catenin levels did not vary significantly with Kepone. Beta-catenin protein levels decreased significantly by approximately 37%, 36% and 53% at 0.01, 0.1 and 1.0 microM Kepone, respectively. E-cadherin-gamma-catenin association was disrupted with 0.1 and 1.0 microM Kepone. Thus, Kepone disrupts cellular architecture, specifically E-cadherin-gamma-catenin containing adherens junctions, which may ultimately affect cellular phenotype.
Subject(s)
Adherens Junctions/drug effects , Breast/drug effects , Chlordecone/pharmacology , Pesticide Residues/pharmacology , Trans-Activators , Adherens Junctions/ultrastructure , Breast/cytology , Breast/metabolism , Breast Neoplasms/chemically induced , Cadherins/metabolism , Cell Adhesion Molecules/metabolism , Cells, Cultured/drug effects , Cells, Cultured/metabolism , Cells, Cultured/ultrastructure , Chlordecone/adverse effects , Collagen , Cytoskeletal Proteins/metabolism , Desmogleins , Desmoplakins , Desmosomes/drug effects , Desmosomes/ultrastructure , Drug Combinations , Epithelial Cells/drug effects , Epithelial Cells/metabolism , Epithelial Cells/ultrastructure , Extracellular Matrix/physiology , Female , Humans , Laminin , Pesticide Residues/adverse effects , Proteoglycans , alpha Catenin , beta Catenin , gamma CateninABSTRACT
BACKGROUND: Proliferative breast disease (PBD) may increase a woman's risk of developing breast cancer, perhaps by decreasing cellular sensitivity to apoptosis. To determine whether resistance to apoptosis develops during PBD, we investigated apoptosis initiated through the Fas pathway in a series of cell lines that recapitulates the morphologic changes of PBD in nude/beige mice. METHODS: The series of cell lines used was MCF-10A cells (parental preneoplastic human breast epithelial cells), MCF-10AT cells (transformed with T(24) Ha-ras), and MCF-10ATG3B cells (derivative cells that progress to carcinoma). Fas-mediated apoptosis, induced when a Fas monoclonal antibody bound to and activated the Fas receptor on these cells, was assessed morphologically and by flow cytometry. Levels of proteins involved in Fas-mediated apoptosis and cleavage of poly(adenosine diphosphate-ribose) polymerase (PARP), an end product of caspase activation, were determined by immunoblotting. Bcl-2 and Bax heterodimerization was examined by coimmunoprecipitation. All statistical tests were two-sided. RESULTS: Sensitivity to Fas-mediated apoptosis decreased with the tumorigenic potential of cells: MCF-10A cells were extremely susceptible, MCF-10AT cells were less susceptible, and MCF-10ATG3B cells were resistant. The percentage of apoptotic cells declined, from 24% to 8% to 6%, respectively. All lines produced Fas ligand (FasL) and had comparable levels of Fas receptor, FasL, Fas-associated death-domain protein, and caspases 3 and 6. Levels of caspase 8 were similar in MCF-10A and MCF-10AT cells but about 30% lower in MCF-10ATG3B cells (P>.01 but <.05). Levels of caspase 10 were about 20% lower in MCF-10AT cells (P>.005 but <.01) and about 59% lower in MCF-10ATG3B cells than in MCF-10A cells (P>.01 but <.05). PARP cleavage was detected in MCF-10A and MCF-10AT cells but not in MCF-10ATG3B cells. Levels of Bax, Bid, and Bak proteins were similar in all lines, but levels of Bcl-2 were lower in MCF-10AT and MCF-10ATG3B cells than in MCF-A cells, and Bcl-2-Bax heterodimerization progressively declined in the series. CONCLUSION: Resistance to Fas-mediated apoptosis appears to develop progressively in the MCF-10AT cell series.
Subject(s)
Adaptor Proteins, Signal Transducing , Apoptosis , Breast Neoplasms/metabolism , Breast Neoplasms/pathology , Animals , BH3 Interacting Domain Death Agonist Protein , Carrier Proteins/biosynthesis , Carrier Proteins/metabolism , Caspase 10 , Caspase 3 , Caspase 6 , Caspase 8 , Caspase 9 , Caspases/biosynthesis , Cell Line, Transformed , Cell Lineage , Dimerization , Fas Ligand Protein , Fas-Associated Death Domain Protein , Female , Flow Cytometry , Humans , Immunoblotting , Membrane Glycoproteins/metabolism , Membrane Proteins/metabolism , Mice , Mice, Nude , Poly(ADP-ribose) Polymerases/metabolism , Precipitin Tests , Protein Binding , Proto-Oncogene Proteins/metabolism , Proto-Oncogene Proteins c-bcl-2/metabolism , Time Factors , Tubulin/metabolism , Tumor Cells, Cultured , bcl-2 Homologous Antagonist-Killer Protein , bcl-2-Associated X Protein , fas Receptor/metabolismABSTRACT
In the peripheral olfactory organ, continual olfactory receptor neuron (ORN) turnover exposes neighboring cells to potentially damaging cellular debris such as free radicals. These, in turn, may be inactivated by binding directly onto glutathione (GSH) or by enzymatic conjugation with glutathione S-transferase (GST). In this study, we have investigated GSH and GST during retrograde degeneration and regeneration of the olfactory nerve in rainbow trout. In these fish, prolonged ORN physiological activity and structural integrity following transection of the olfactory nerve may be mediated by GSH and GST. In the olfactory mucosa, early changes following nerve lesion and prior to ORN degeneration included a shift of intense GSH labeling from the dendrites and perikarya of a subpopulation of ORN, and from melanophores, to olfactory nerve fascicles. GSH levels were unchanged, but GST activity decreased by 33% and GST-immunoreactivity (GST-IR) in nerve fascicles diminished slightly. When the process of massive degeneration terminated and ORN were largely absent, GSH levels and GST activity decreased further, GSH labeling was confined to melanophores, and GST-IR was absent. As ORN repopulated the olfactory mucosa, GST-IR was widespread. The combination of increased GST activity (92% of preoperative values) and low GSH levels suggests GSH utilization for GST conjugation reactions. These changes imply that GSH provides protection from cellular debris associated with ORN degeneration. Recovery of GST activity and widespread GST-IR during regeneration indicates modulation of neuroprotective, developmental, and/or physiological processes by GST.
Subject(s)
Glutathione Transferase/metabolism , Glutathione/metabolism , Nerve Degeneration , Nerve Regeneration , Olfactory Mucosa/metabolism , Olfactory Nerve/physiology , Animals , Axons/physiology , Blotting, Western , Denervation , Hemocyanins/pharmacokinetics , Immunohistochemistry , Oncorhynchus mykiss , Tissue DistributionABSTRACT
In the fish olfactory system, glutathione S-transferases (GST) which detoxify electrophilic substances and participate in reactions of lipophilic compounds, may be active in the biotransformation of odorants and xenobiotics. In this study GST activity in the rainbow trout olfactory mucosa was high (477.6 +/- 218 nmol/min per mg protein). The GST pi class was demonstrated by Western immunoblot analysis and localized by immunofluorescence to the dendritic and perinuclear regions of olfactory receptor neurons; areas previously shown to contain elevated glutathione. The presence of GST and glutathione in fish olfactory receptor neurons suggests that these cells utilize the glutathione pathway.
