Abstract
Cisplatin is a commonly used antitumor agent in the treatment of various human cancers, with nephrotoxicity as a major side effect. Cisplatin causes the loss of cell-cell contacts of renal proximal tubular epithelial cells prior to the onset of apoptosis. We studied the involvement of protein kinase C in these events in the renal epithelial cell line LLC-PK1. Cisplatin caused apoptosis in LLC-PK1 cells, which was directly related to the activation of caspase-3 and DNA fragmentation. Apoptosis was almost completely inhibited by the protein kinase C inhibitors bisindolylmaleimide (Bis) I and Gö6983 [2-[1-(3-dimethylaminopropyl)-5-methoxyindol-3-yl]-3-(1H-indol-3-yl) maleimide], but not by Gö6976 [12-(2-cyanoethyl)-6,7,12,13-tetrahydro-13-methyl-5-oxo-5H-indolo(2,3-a)pyrrolo(3,4-c)-carbazole]. Also, in primary cultured rat renal proximal tubular cells, inhibition of protein kinase C (PKC) inhibited apoptosis. Cisplatin also caused the early loss of cell-cell adhesions, which was associated with the altered localization of the adherens junction-associated protein β-catenin in association with PKC-mediated phosphorylation of the actincapping protein adducin. These events preceded and were independent of caspase activation. β-Catenin did not dissociate from E-cadherin. Cisplatin-induced loss of cell-cell contacts was associated with the increased formation of F-actin stress fibers, which was inhibited by Bis I and Gö6983 as well as dominant-negative PKC-ϵ. Also, the loss of cell-cell adhesions by cisplatin was prevented by Bis I and Gö6983. Activation of protein kinase C with phorbol esters promoted cisplatin-induced loss of cell-cell adhesions as well as apoptosis. In conclusion, the combined data fit a model whereby protein kinase C mediates the cisplatin-induced loss of cellular interactions. Such a loss of these interactions has a role in the onset of apoptosis.
Footnotes
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This work was supported by Grants 902-21-217 and 911-02-022 of the Netherlands Organization for Scientific Research. B. van de Water was supported by a fellowship from the Netherlands Royal Academy for Arts and Sciences.
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doi:10.1124/jpet.104.072678.
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ABBREVIATIONS: PTC, proximal tubular epithelial cells; ECM, extracellular matrix; RPTE, renal proximal tubular epithelial; PKC, protein kinase C; DMEM, Dulbecco's modified Eagle's medium; FBS, fetal bovine serum; BSA, bovine serum albumin; AMC, 7-amino-4-methylcoumarin; Bis I, bisindolylmaleimide I; Gö6976, 12-(2-cyanoethyl)-6,7,12,13-tetrahydro-13-methyl-5-oxo-5H-indolo(2,3-a)pyrrolo(3,4-c)-carbazole; PDBu, phorbol 12,13-dibutyrate; Ac-DEVD-AMC, acetyl-Asp-Glu-Val-Asp-AMC; DN, dominant-negative; LDH, lactate dehydrogenase; 4FD, fluorescein-labeled 4-kDa dextran; PBS, phosphate-buffered saline; RT, room temperature; MSB, microtubule stabilization buffer; PAGE, polyacrylamide gel electrophoresis; PDI, protein disulfide isomerase; TBP, blocking buffer; CLSM, confocallaser scanning microscopy; Gö6983, 2-[1-(3-dimethylaminopropyl)-5-methoxyindol-3-yl]-3-(1H-indol-3-yl) maleimide.
- Received June 16, 2004.
- Accepted September 17, 2004.
- The American Society for Pharmacology and Experimental Therapeutics
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