Changes in activation balance of different protein kinase C (PKC) isoenzymes have been linked to cancer development. The current study investigated the effect of different PKC inhibitors on cellular contacts in cultured high-grade urinary bladder carcinoma cells (5637 and T24). Exposure of the cells to isoenzyme-specific PKC inhibitors yielded variable results: Go6976, an inhibitor of PKC␣ and PKC isoenzymes, induced rapid clustering of cultured carcinoma cells and formation of an increased number of desmosomes and adherens junctions. Safingol, a PKC␣ inhibitor, had similar but less pronounced effects. In contrast, a PKC␦ inhibitor, rottlerin, had an opposite effect on cell clustering and caused dissociation of cell junctions. A broad-spectrum PKC inhibitor bisindolylmaleimide I did not have any apparent effect on the morphology of the cultures or on the number of cell junctions. Additional studies with Go6976 demonstrated that inhibition of PKC␣ and  isoenzymes induced translocation of 1-integrin from the cell-matrix junctions and that 4-integrin was translocated to face the culture substratum. Go6976 was also highly effective in inhibiting migration of carcinoma cells and inhibited invasion through artificial basement membrane. Our results on urinary bladder carcinoma cells emphasize that Go6976 is a potential anticancer drug due to its effects on cell-cell and cell-matrix junctions, migration, and invasion. Furthermore, the results may be explained by changes in PKC activation balance promoted by inhibition of PKC␣/.
Hypoxia causes an inflammatory reaction in RPE cells that is potentiated by pre-treatment with the Toll-like receptor-activating agent, LPS. The secretion of VEGF from these cells is regulated directly by hypoxia and is not mediated by inflammation.
We first studied expression of neurofibromin by immunohistochemistry in scars obtained from operations involving areas of healing wounds. The results demonstrated increased immunoreactivity for neurofibromin in the fibroblastic cell population of the lesions when compared with fibroblasts of apparently healthy perilesional skin, or those of intact control skin. Furthermore, dermal fibroblasts of 19 and 34 wk-old fetuses displayed a clearly detectable immunosignal for neurofibromin. In vitro studies were designed to investigate the potential effects of selected growth factors--known to be operative in wound healing--on neurofibromin mRNA steady-state levels in cultured fibroblasts. Northern transfer analyses revealed that different isoforms of platelet derived growth factor (PDGF) exerted selective effects on the neurofibromin mRNA levels: PDGF isoform AB elevated neurofibromin mRNA levels in a concentration-dependent manner when concentrations of 0.1, 1, 10, and 30 ng per ml were used. The maximal upregulatory effect of PDGF BB was reached at a concentration of 1 ng per ml. In contrast, PDGF AA did not alter the steady-state levels of neurofibromin mRNA. As estimated by RNase protection assay, transforming growth factor-beta1 (TGF-beta1) upregulated neurofibromin gene expression when concentrations of 0.5 and 5 ng per ml were used. Reverse transcription followed by polymerase chain reaction did not detect apparent alterations in the ratio of type I/type II neurofibromin isoforms in PDGF- or TGF-beta1-treated cultures. Taken together, our results suggest that expression of tumor suppressor protein neurofibromin is upregulated in response to skin injury, and that this upregulation can be mediated through PDGF and TGF-beta.
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