Puromycin aminonucleoside and adriamycin disturb cytoskeletal and extracellular matrix protein organization, but not protein synthesis of cultured glomerular epithelial cells.
Puromycin aminonucleoside (PA) and Adriamycin (ADR) cause glomerular proteinuria associated with degenerative alterations of glomerular visceral epithelial cells (GVEC) and detachment from the glomerular basement membrane when administered to rats. This in vitro study was performed to define, in detail, the quantitative and qualitative changes of a number of adhesion-associated proteins (cytoskeletal, extracellular matrix and integrin proteins) upon exposure to PA and ADR. By immunofluorescence we observed: (1) dose- and incubation-time-dependent filament pattern changes and decreased staining of the cytoskeletal proteins actin, vimentin, keratin, and beta-tubulin; (2) an altered distribution, and decreased expression of the extracellular matrix proteins laminin and heparan sulfate and (3) a loss of the beta 1-integrin focal adhesions upon exposure to PA and ADR. Using an ELISA, a concentration-dependent decrease was found (a 50% reduction with 50 micrograms/ml PA for 48 h and with 2 micrograms/ml ADR for 24 h) in the production of cytoskeletal and extracellular matrix proteins per cell. These general effects were suggestive of a disturbance of protein synthesis but, by metabolic labelling studies, no reduction in overall protein synthesis was found. Using two-dimensional PAGE on 35S-methionine steady-state labeled cells, no changes were found in intracellular protein patterns of PA- and ADR-treated cells (pH 5-7.5, MW 110-20 kD). We hypothesize that exposure of GVEC in vitro to PA and ADR might result, directly or indirectly, in perturbation of the macromolecular organization of cytoskeletal and extracellular matrix proteins with loss of GVEC adhesion.
Coers
W
W
Department of Pathology, University of Groningen, The Netherlands.
Huitema
S
S
van der Horst
M L
ML
Weening
J J
JJ
D000818:Animals; D002448:Cell Adhesion; D002455:Cell Division; D002470:Cell Survival; D002478:Cells, Cultured; D003598:Cytoskeletal Proteins / Q000378:metabolism*; D004317:Doxorubicin / Q000494:pharmacology*; D015180:Electrophoresis, Gel, Two-Dimensional; D004847:Epithelial Cells; D004848:Epithelium / Q000378:metabolism; D016326:Extracellular Matrix Proteins / Q000378:metabolism*; D005455:Fluorescent Antibody Technique; D007678:Kidney Glomerulus / Q000166:cytology / Q000378:metabolism*; D014176:Protein Biosynthesis; D011692:Puromycin Aminonucleoside / Q000494:pharmacology*; D051381:Rats; D017207:Rats, Sprague-Dawley
D003598:Cytoskeletal Proteins; D016326:Extracellular Matrix Proteins; D011692:Puromycin Aminonucleoside; D004317:Doxorubicin