No abstract

In primary human umbilical vein endothelial cells (HUVECs), incubation with phorbol-12-myristate-13-acetate (PMA) enhanced basal and bradykinin-stimulated nitric oxide production. In the HUVEC-derived cell line EA.hy 926, PMA and phorbol-12,13-dibutyrate stimulated endothelial nitric oxide synthase (NOS III) mRNA expression in a concentration- and time-dependent manner. Maximal mRNA expression (3.3-fold increase) was observed after 18 hr. NOS III protein and activity were increased to a similar extent. The specific protein kinase C (PKC) inhibitors bisindolylmaleimide I (1 microM), Gö 6976 [12-(2 cyanoethyl)-6,7,12, 13-tetrahydro-13-methyl-5-oxo-5H-indolo[2,3-a]pyrrolo-[3, 4-c]carbazole] (1 microM), Ro-31-8220 [3-[1-[3(amidinothio)propyl-1H-inoyl-3-yl]3-(1-methyl-1H- indoyl-3-yl) maleimide methane sulfonate] (1 microM), and chelerythrine (3 microM) did not change NOS III expression when applied alone, but they all prevented the up-regulation of NOS III mRNA produced by PMA. Of the PKC isoforms expressed in EA.hy 926 cells (alpha, beta I, delta, epsilon, eta, zeta, lambda, and mu), only PKC alpha and PKC epsilon showed changes in protein expression after PMA treatment. Incubation of EA.hy 926 cells with PMA for 2-6 hr resulted in a translocation of PKC alpha and PKC epsilon from the cytosol to the cell membrane, indicating activation of these isoforms. After 24 hr of PMA incubation, both isoforms were down-regulated. The time course of activation and down-regulation of these two PKC isoforms correlated well with the PMA-stimulated increase in NOS III expression. When human endothelial cells (ECV 304 or EA.hy 926) were transiently or stably transfected with a 3.5-kb fragment of the human NOS III promoter driving a luciferase reporter gene, PMA stimulated promoter activity up to 2.5-fold. On the other hand, PMA did not change the stability of the NOS III mRNA. These data indicate that stimulation of PKC alpha, PKC epsilon, or both by active phorbol esters represents an efficacious pathway activating the human NOS III promoter in human endothelium.

No abstract

Sphingolipids were discovered more than a century ago in the brain. Cerebrosides and sphingomyelins were named so because they were first isolated from neural tissue. Although glycosphingolipids and especially those containing sialic acid in their oligosaccharide moiety are particularly abundant in the brain, sphingolipids are ubiquitous cellular membrane components. They form cell- and species-specific profiles at the cell surfaces that characteristically change in development, differentiation, and oncogenic transformation, indicating the significance of these lipid molecules for cell-cell and cell-matrix interactions as well as for cell adhesion, modulation of membrane receptors and signal transduction. This review summarizes sphingolipid metabolism with emphasis on aspects particularly relevant in neural cell types, including neurons, oligodendrocytes and neuroblastoma cells. In addition, the reader is briefly introduced into the methodology of lipid evaluation techniques and also into the putative physiological functions of glycosphingolipids and their metabolites in neural tissue.

The tumor suppressor gene PTEN (phosphatase and tensin homologue deleted from chromosome 10) encodes a dual specific protein and phospholipid phosphatase that affects cell proliferation, apoptosis and migration. In our study, we examined protein expression of PTEN in renal carcinogenesis. PTEN protein levels were examined in 42 clear cell renal cell carcinomas (ccRCC) and oncocytomas as well as in the corresponding normal renal tissue of the same patients using Western blot analysis. Cellular localization was analyzed by immunohistochemistry. PTEN was highly expressed in all investigated normal renal tissue specimens. Immunohistochemical analysis showed an almost exclusive staining of proximal tubulus epithelial cells known to be precursor cells of ccRCC. Within the proximal tubulus cells, PTEN exhibited a membrane predominant immunostaining pattern. In ccRCCs PTEN expression was markedly reduced to an average of less than 10% compared with normal tissue as evidenced by Western blot analysis (p < 0.001). The degree of reduction was similar in highly differentiated (G1) carcinomas and in less differentiated (G2-G4) carcinomas. Key words: PTEN; renal cell carcinoma; oncocytoma; proximal tubulus epithelial cellThe human tumor suppressor gene PTEN (phosphatase and tensin homologue deleted from chromosome 10) located on chromosome 10q23 1 encodes a dual specific protein and phospholipid phosphatase that is involved in regulation of a variety of signal transduction pathways. PTEN inhibits Shc (Src-homology collagen) phosphorylation following epidermal growth factor (EGF) stimulation and therefore blocks the activation of the Ras/MAPkinase pathway. This has been proposed as an explanation for the tumor suppressive effect of PTEN. 2 Another mechanism that involves the protein phosphatase activity of PTEN is dephosphorylation and inactivation of focal adhesion kinase (FAK), thus implying a crucial role of PTEN for the interaction between extracellular matrix and the cytoskeleton. This mechanism may lead to negative regulation of integrin mediated cell spreading, migration and invasion. 3,4 Besides its function as a protein phosphatase, PTEN acts as a phospholipid phosphatase with phosphatidylinositol 3,4,5-triphosphate (PIP3) as a substrate. [5][6][7] One of the direct downstream targets of PIP3, protein kinase B (Akt/PKB), is continually activated by phosphorylation in cells lacking functional PTEN and the elevated levels of activation can in turn be reduced to normal levels by expression of wild-type PTEN. 8 These data suggest that the tumor suppressive properties of PTEN relate in part to its ability to downregulate the Akt/PKB pathway and hence inhibit cell proliferation and facilitate apoptosis. PTEN is frequently deleted or mutated in advanced cancers, thus suggesting a crucial role in tumor development. Loss of PTEN has been implicated in the carcinogenesis in a wide variety of tissues including prostate, bladder, melanoma, breast and brain. 9,10 However, in renal cancer, PTEN has not yet been shown to play a rol...