Subject(s)
Apoptosis/physiology , Calcium/deficiency , Cell Nucleus/metabolism , Glycoproteins/physiology , Molecular Chaperones/physiology , Prostate/cytology , Anoikis/drug effects , Anoikis/physiology , Apoptosis/drug effects , Calcium/antagonists & inhibitors , Calcium/pharmacology , Caspase 3 , Caspase 9 , Caspase Inhibitors , Caspases/metabolism , Cell Line, Transformed , Cell Proliferation/drug effects , Clusterin , Cytoplasm/metabolism , DNA Fragmentation/drug effects , Egtazic Acid/analogs & derivatives , Egtazic Acid/pharmacology , Epithelial Cells/drug effects , Epithelial Cells/metabolism , Glycoproteins/genetics , Glycoproteins/metabolism , Humans , Male , Molecular Chaperones/genetics , Molecular Chaperones/metabolism , Poly(ADP-ribose) Polymerases/metabolism , Prostate/metabolism , Protein Isoforms/genetics , Protein Isoforms/metabolism , Protein Isoforms/physiology , Protein Transport/drug effects , TransfectionABSTRACT
Expression of the castration-induced clusterin protein is incompatible with the survival of human prostate cancer cells in tissues and in cell culture. To investigate the fate of human prostate epithelial cells, when engineered to maintain expression of clusterin protein, we have used an IRES-hyg vector and hygromycin selection. PC-3 prostate tumour cells were substantially more sensitive to clusterin expression than nonmalignant PNT1a cells, showing multiple phenotypic changes including cell cycle arrest and increased apoptosis. The results strengthen the hypothesis that clusterin expression is proapoptotic. Expression of exogenous clusterin in both cell types resulted in its relocation from the cytoplasm and a nuclear accumulation of the protein, as was also seen in the same cells when apoptosis was induced by etoposide treatment. To survive clusterin expression, the PC-3 tumour cells developed apoptosis-inhibitory properties. This could have significance for the resistance of prostate cancers to chemo/radiotherapy, where clusterin overexpression is observed.
Subject(s)
Apoptosis , Glycoproteins/metabolism , Molecular Chaperones/metabolism , Prostate/metabolism , Prostatic Neoplasms/metabolism , Cell Cycle , Cell Line, Transformed , Cell Proliferation , Clone Cells , Clusterin , Epithelial Cells/metabolism , Humans , Male , Neoplasms, Hormone-Dependent/metabolism , Neoplasms, Hormone-Dependent/pathology , Prostate/cytology , Prostatic Neoplasms/pathology , Transfection , Tumor Cells, CulturedABSTRACT
Clusterin gene expression is potently induced in experimental models in which apoptosis is activated, such as rat prostate involution following castration. Nevertheless, its precise physiological role has not yet been established, and both anti-apoptotic and pro-apoptotic functions have been suggested for this gene. Clusterin expression level depends on cell proliferation state, and we recently showed that its over-expression inhibited cell cycle progression of SV40-immortalized human prostate epithelial cells PNT2 and PNT1a. Here we studied clusterin expression in PNT1a cells subjected to serum-starvation with the aim of defining clusterin early molecular changes following apoptosis induction. Under serum-starvation conditions, decreased growth rate, slow rounding-up of cells, cell detachment, and formation of apoptotic bodies indicative of anoikis (detachment-induced apoptosis) were preceded by significant downregulation of 70 kDa clusterin precursor and upregulation of 45-40 kDa isoforms. On the 8th day of serum-free culturing, only the higher molecular weight protein-band of about 45 kDa was clearly induced and accumulated in detached cells and apoptotic bodies in which PARP was activated. Anoikis was preceded by induction and transloction of a 45-kDa clusterin isoform to the nucleus. Thus, nuclear targeting of a specific 45-kDa isoform of clusterin appeared to be an early and specific molecular signal triggering anoikis-death. Considering also that clusterin is downregulated during prostate cancer onset and progression, and that its upregulation has inhibited DNA synthesis and cell cycle progression of immortalized human prostate epithelial cells, we suggest that clusterin might be a new anti-oncogene in the prostate.
Subject(s)
Active Transport, Cell Nucleus/physiology , Apoptosis/physiology , Glycoproteins/genetics , Molecular Chaperones/genetics , Simian virus 40/genetics , Cell Division , Cell Line, Tumor , Cell Nucleus/metabolism , Cell Transformation, Viral , Clusterin , Culture Media, Serum-Free , Epithelial Cells/metabolism , Epithelial Cells/pathology , Gene Expression Regulation, Neoplastic , Glycoproteins/metabolism , Humans , Keratins/metabolism , Kinetics , Male , Molecular Chaperones/metabolism , Prostatic Neoplasms/metabolism , Prostatic Neoplasms/pathology , Protein Isoforms/genetics , Protein Isoforms/metabolismABSTRACT
In 3T3 cells temperatures higher than physiological stimulated amino acid transport activity in a dose-dependent manner up to 44 degrees C. However, the temperature increase did not induce widespread transport increase of all other nutrients tested. The activities of both amino acid transport systems A and ASC were enhanced within a few minutes following cell exposure to increased temperature. The maintenance of this effect required continuous exposure of the cells to hyperthermia. Kinetic analysis indicated that the stimulation of the activity of transport System A occurred through a mechanism affecting Vmax rather than Km. The continuous presence of cycloheximide did not prevent the transport changes induced by hyperthermia. These results suggest that the increased amino acid uptake reflects an activation or relocation of existing amino acid transport proteins. During the hyperthermic treatment, the content of ninhydrin-positive substances (NPS), mostly amino acids, increased within the cells and the accumulation of these compatible osmolytes was parallelled by an increase in cell volume. The withdrawal of amino acids from the culture medium immediately before and during the shock phase counteracted the increase and reduced the NPS content but did not prevent the increase in amino acid transport, the cell swelling and the induction of the heat shock response.
Subject(s)
Hot Temperature , beta-Alanine/analogs & derivatives , 3T3 Cells , Amino Acids/metabolism , Amino Acids/pharmacokinetics , Animals , Biological Transport , Blotting, Northern , Cycloheximide/pharmacology , Dose-Response Relationship, Drug , Glutamine/metabolism , HSP70 Heat-Shock Proteins/metabolism , Kinetics , Mice , Ninhydrin/metabolism , Proline/metabolism , Protein Binding , Protein Synthesis Inhibitors/pharmacology , Stress, Physiological , Temperature , Time Factors , beta-Alanine/pharmacokineticsABSTRACT
When porcine endothelial cells were exposed to hypertonicity, both the level of ATA2 (amino acid transporter 2) mRNA and activity of amino acid transport System A increased transiently, peaking after about 6 and 9 h, respectively. Cycloheximide, like actinomycin D, prevented both responses, showing that an earlier step also involves protein synthesis. Withdrawal of hypertonicity after 6 h increased the rate of down regulation. These findings confirm that ATA2 is a major isoform of System A and show that changes in the expression of ATA2 mRNA precede both the induction and subsequent down regulation of transport activity.
Subject(s)
Amino Acid Transport System A , Amino Acids/metabolism , Carrier Proteins/metabolism , Endothelium, Vascular/metabolism , Membrane Proteins/metabolism , RNA, Messenger/metabolism , Water-Electrolyte Balance/physiology , beta-Alanine/analogs & derivatives , Animals , Biological Transport/physiology , Carrier Proteins/genetics , Cells, Cultured , Cycloheximide/pharmacology , Dactinomycin/pharmacology , Endothelium, Vascular/cytology , Endothelium, Vascular/drug effects , Hypertonic Solutions/pharmacology , Membrane Proteins/genetics , Nucleic Acid Synthesis Inhibitors/pharmacology , Protein Synthesis Inhibitors/pharmacology , Swine , Transfection , Water-Electrolyte Balance/drug effects , beta-Alanine/pharmacokineticsABSTRACT
We studied the responses to hypertonicity of cultured endothelial cells from swine pulmonary arteries. In 0.5 osmol/kgH(2)O medium, initial cell shrinkage was followed by a regulatory volume increase (RVI), complete after 1 h, concomitant with an increase in cellular K(+) content. Then the activity of amino acid transport System A increased, accompanied by an accumulation of ninhydrin-positive solutes (NPS), reaching a peak at approximately 6 h. The subsequent decline in System A activity was paralleled by an induction of the betaine-GABA transporter (BGT-1), detected as increases of BGT-1 mRNA and of transport activity, which peaked at approximately 24 h. Inhibitors of transcription or translation prevented induction of both transport activities. The increased expression of BGT-1, which involved activation of "tonicity-responsive enhancer," was inhibited by 5 mM extracellular betaine. Cellular K(+) concentration gradually declined after the accumulation of NPS and during the induction of BGT-1. This very effective adaptation to hypertonicity suggests it has a physiological role.
