Your browser doesn't support javascript.
loading
Show: 20 | 50 | 100
Results 1 - 6 de 6
Filter
1.
J Biol Chem ; 287(7): 5070-8, 2012 Feb 10.
Article in English | MEDLINE | ID: mdl-22184139

ABSTRACT

The ATP-binding cassette transporter ABCB4 is a phosphatidylcholine translocator specifically expressed at the bile canalicular membrane in hepatocytes, highly homologous to the multidrug transporter ABCB1. Variations in the ABCB4 gene sequence cause progressive familial intrahepatic cholestasis type 3. We have shown previously that the I541F mutation, when reproduced either in ABCB1 or in ABCB4, led to retention in the endoplasmic reticulum (ER)/Golgi. Here, Madin-Darby canine kidney cells expressing ABCB1-GFP were used as a model to investigate this mutant. We show that ABCB1-I541F is not properly folded and is more susceptible to in situ protease degradation. It colocalizes and coprecipitates with the ER chaperone calnexin and coprecipitates with the cytosolic chaperone Hsc/Hsp70. Silencing of calnexin or overexpression of Hsp70 have no effect on maturation of the mutant. We also tested potential rescue by chemical and pharmacological chaperones. Thapsigargin and sodium 4-phenyl butyrate were inefficient. Glycerol improved maturation and exit of the mutant from the ER. Cyclosporin A, a competitive substrate for ABCB1, restored maturation, plasma membrane expression, and activity of ABCB1-I541F. Cyclosporin A also improved maturation of ABCB4-I541F in Madin-Darby canine kidney cells. In HepG(2) cells transfected with ABCB4-I541F cDNA, cyclosporin A allowed a significant amount of the mutant protein to reach the membrane of bile canaliculi. These results show that the best strategy to rescue conformation-defective ABCB4 mutants is provided by pharmacological chaperones that specifically target the protein. They identify cyclosporin A as a potential novel therapeutic tool for progressive familial intrahepatic cholestasis type 3 patients.


Subject(s)
ATP Binding Cassette Transporter, Subfamily B, Member 1/metabolism , ATP Binding Cassette Transporter, Subfamily B/metabolism , Calnexin/metabolism , Cryoprotective Agents/pharmacology , Cyclosporine/pharmacology , Enzyme Inhibitors/pharmacology , Glycerol/pharmacology , HSC70 Heat-Shock Proteins/metabolism , Mutation, Missense , ATP Binding Cassette Transporter, Subfamily B/genetics , ATP Binding Cassette Transporter, Subfamily B, Member 1/genetics , Amino Acid Substitution , Animals , Antineoplastic Agents/pharmacology , Calnexin/genetics , Cholestasis/drug therapy , Cholestasis/genetics , Cholestasis/metabolism , Dogs , Endoplasmic Reticulum/genetics , Endoplasmic Reticulum/metabolism , Gene Silencing , HSC70 Heat-Shock Proteins/genetics , Hep G2 Cells , Humans , Phenylbutyrates/pharmacology , Protein Transport/drug effects , Protein Transport/genetics , Thapsigargin/pharmacology
2.
Invest. clín ; 51(4): 479-488, dic. 2010. ilus
Article in Spanish | LILACS | ID: lil-630906

ABSTRACT

The cell response of human HepG2 cells exposed to hypothermia with rewarming was analyzed. Ultrastructural findings in hypothermic stressed cells showed swollen mitochondria, dispersed chromatin, vacuoles and ring-shape nucleolar reorganization. These changes were coupled with significative differences in the induction of Hsp60, inducible Hsp70 and monomeric Hsf1 in all treated samples, but not in Hsc 70 expression. Cellular response to hypothermia could be associated with the synergistic induction of Hsp expression.


En este trabajo se analizó la respuesta celular de células HepG2 expuestas a hipotermia con posterior recuperación. Los hallazgos ultraestructurales en células sometidas a estrés hipotérmico incluyeron mitocondrias edematizadas, núcleos picnóticos, vacuolas y reorganización nucleolar en forma de anillo. Tales cambios están relacionados con diferencias significativas en la inducción de la expresión de Hsp60, Hsp70 inducible y Hsf 1 monomérico en todas las muestras tratadas, pero no de Hsc70. La respuesta celular a la hipotermia puede ser relacionada con la inducción sinergística de las Hsp.


Subject(s)
Humans , Cold Temperature , Carcinoma, Hepatocellular/pathology , /biosynthesis , DNA-Binding Proteins/biosynthesis , Gene Expression Regulation, Neoplastic , /biosynthesis , Liver Neoplasms/pathology , Neoplasm Proteins/biosynthesis , Transcription Factors/biosynthesis , Cell Line, Tumor/metabolism , Cell Line, Tumor/ultrastructure , /genetics , Cold Temperature/adverse effects , DNA-Binding Proteins/genetics , /genetics , Mitochondria/ultrastructure , Neoplasm Proteins/genetics , Rewarming , Temperature , Transcription Factors/genetics
3.
Invest Clin ; 51(4): 479-88, 2010 Dec.
Article in English | MEDLINE | ID: mdl-21361146

ABSTRACT

The cell response of human HepG2 cells exposed to hypothermia with rewarming was analyzed. Ultrastructural findings in hypothermic stressed cells showed swollen mitochondria, dispersed chromatin, vacuoles and ring-shape nucleolar reorganization. These changes were coupled with significative differences in the induction of Hsp60, inducible Hsp70 and monomeric Hsfl in all treated samples, but not in Hsc 70 expression. Cellular response to hypothermia could be associated with the synergistic induction of Hsp expression.


Subject(s)
Carcinoma, Hepatocellular/pathology , Chaperonin 60/biosynthesis , Cold Temperature , DNA-Binding Proteins/biosynthesis , Gene Expression Regulation, Neoplastic , HSP72 Heat-Shock Proteins/biosynthesis , Liver Neoplasms/pathology , Neoplasm Proteins/biosynthesis , Transcription Factors/biosynthesis , Cell Line, Tumor/metabolism , Cell Line, Tumor/ultrastructure , Chaperonin 60/genetics , Cold Temperature/adverse effects , DNA-Binding Proteins/genetics , HSP72 Heat-Shock Proteins/genetics , Heat Shock Transcription Factors , Humans , Mitochondria/ultrastructure , Neoplasm Proteins/genetics , Rewarming , Temperature , Transcription Factors/genetics
4.
J Toxicol Pathol ; 23(4): 235-43, 2010 Dec.
Article in English | MEDLINE | ID: mdl-22272033

ABSTRACT

In order to evaluate the effects of hyperthermia on adriamycin cardiomyopathy and its relationship with heat shock protein induction and myosin accumulation, female Sprague-Dawley rats (21-24 days) were randomized into four groups: the control, adriamycin, temperature and temperature-adriamycin groups. Adriamycin was injected i.v. at a dose of 27 mg/Kg (0.1 ml). The rats were exposed to a temperature of 45ºC for 35 min, followed by a recovery (1 h) at room temperature prior to adriamycin treatment. Body weight was recorded weekly. The thickness of the ventricular wall and percentage of cellular damage were biometrically and ultrastructurally evaluated, respectively. Heat shock protein 25 and myosin accumulation were determined through Western blot analysis. The determinations were carried out monthly until the third month after treatment. At eight and twelve weeks after treatment, the thickness of the ventricular wall seemed to decrease in the adriamycin-treated rats in relation to the other groups. An electron microscopic analysis of the adriamycin group's left ventricular wall samples, showed more sarcomeric changes and loss of myofibrils than the control, temperature and temperature-adriamycin groups. At 24 hours after treatment with adriamycin, higher levels of heat shock protein 25 and myosin were observed (week 0) in the temperature-adriamycin group than in the control and adriamycin groups (4, 8 and 12 weeks). Hyperthermia was confirmed by a multivariate approach to induce heat shock protein 25 and myosin, which would strengthen cardiac-sarcomeric myosin arrangement.

5.
Hepatology ; 49(4): 1218-27, 2009 Apr.
Article in English | MEDLINE | ID: mdl-19185004

ABSTRACT

UNLABELLED: Progressive familial intrahepatic cholestasis type 3 (PFIC3) is a rare liver disease characterized by early onset of cholestasis that leads to cirrhosis and liver failure before adulthood. PFIC3 may be improved by chronic administration of ursodeoxycholic acid, although in many cases liver transplantation is the only therapy. The disease is caused by mutations of the adenosine triphosphate (ATP)-binding cassette, sub-family B, member 4 (ABCB4) [multidrug resistance 3 (MDR3)] gene encoding a specific hepatocellular canalicular transporter involved in biliary phosphatidylcholine secretion. Several mutations have been reported; however, the effect of individual mutations has not been investigated. ABCB4 is highly homologous to ATP-binding cassette, sub-family B, member 1 (ABCB1) (MDR1), the multidrug transporter responsible for drug resistance of cancer cells. We have studied the effect of mutation I541F localized to the first nucleotide-binding domain, which is highly conserved between ABCB4 and ABCB1. Plasmids encoding the wild-type human ABCB4 or rat ABCB1-green fluorescing protein (GFP) construct, and corresponding I541F-mutants, were expressed in hepatocellular carcinoma, human (HepG2) and Madin-Darby canine kidney (MDCK) cells. Expression studies showed that ABCB4 was localized at the bile canalicular membrane in HepG2 cells and at the apical surface in MDCK cells, whereas the I541F mutant was intracellular. In MDCK cells, ABCB1-I541F also accumulated intracellularly in compartments, which were identified as the endoplasmic reticulum and cis-Golgi, and remained partially endoH-sensitive. After shifting cells to 27 degrees C, ABCB1-I541F was expressed at the apical cell surface in a mature and active form. Similarly, ABCB4 was significantly trafficked to the membrane of bile canaliculi in HepG2 cells. CONCLUSION: Mutation I541F causes mislocalization of both ABCB4 and ABCB1. Intracellular retention of ABCB4-I541F can explain the disease in PFIC3 patients bearing this mutation. The observation that plasma membrane expression and activity can be rescued by low temperature opens perspectives to develop novel therapies for the treatment of PFIC3.


Subject(s)
ATP Binding Cassette Transporter, Subfamily B, Member 1/genetics , ATP Binding Cassette Transporter, Subfamily B/genetics , Cholestasis, Intrahepatic/genetics , Protein Folding , ATP Binding Cassette Transporter, Subfamily B, Member 1/metabolism , Amino Acid Sequence , Animals , Bile Canaliculi/metabolism , Cell Line, Tumor , Cell Membrane/metabolism , Cold Temperature , Dogs , Endoplasmic Reticulum/metabolism , Female , Gene Expression , Golgi Apparatus/metabolism , Green Fluorescent Proteins , Humans , Molecular Sequence Data , Mutation, Missense , Protein Interaction Domains and Motifs
6.
Tissue Cell ; 37(1): 59-65, 2005 Feb.
Article in English | MEDLINE | ID: mdl-15695177

ABSTRACT

To understand hypothermia as a stress condition we determined the expression and localization of Hsp70 under hyperthermic and hypothermic stress in human hepatoma HepG2 cells. Western blot analysis indicates that there was a statistically significant increase of Hsp70 expression under thermal stresses. Immunohistochemically, the distribution of inducible Hsp70 in stressed cells showed a granular pattern mostly in the cytoplasm. At subcellular level, Hsp70 was localized in the nucleus, vacuoles, cytoskeletal components and dispersed throughout the cytoplasm. Accumulation of Hsp70 in cells under hypothermia could be related to restitution of cell equilibrium modified by this thermal stress condition. The protective effect of hypothermia could be associated with promotion of Hsp expression. We suggest that hypothermia is a stress capable of inducing Hsp70 expression in human HepG2 cells.


Subject(s)
Cell Nucleus/metabolism , Cytoplasm/metabolism , HSP70 Heat-Shock Proteins/biosynthesis , Hypothermia/physiopathology , Blotting, Western , Carcinoma, Hepatocellular , Cell Line, Tumor , Gene Expression , HSP70 Heat-Shock Proteins/metabolism , Humans , Immunoenzyme Techniques , Microscopy, Electron, Transmission
SELECTION OF CITATIONS
SEARCH DETAIL
...