Induction and intracellular regulation of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) mediated apotosis in human malignant glioma cells.

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) preferentially triggers apoptosis in tumor cells versus normal cells, thus providing a therapeutic potential. In this study, we examined a large panel of human malignant glioma cell lines and primary cultures of normal human astrocytes for their sensitivity to TRAIL. Of 13 glioma cell lines, 3 were sensitive (80-100% death), 4 were partially resistant (30-79% death), and 6 were resistant (< 30% death). Normal astrocytes were also resistant. TRAIL-induced cell death was characterized by activation of caspase-8 and -3, poly(ADP-ribose) polymerase cleavage, and DNA fragmentation. Decoy receptor (DcR1 and DcR2) expression was limited in the glioma cell lines and did not correlate with TRAIL sensitivity. Both sensitive and resistant cell lines expressed TRAIL death receptor (DR5), adapter protein Fas-associated death domain (FADD), and caspase-8; but resistant cell lines expressed 2-fold higher levels of the apoptosis inhibitor phosphoprotein enriched in diabetes/phosphoprotein enriched in astrocytes-15 kDa (PED/PEA-15). In contrast, cellular FADD-like IL-1beta-converting enzyme-like inhibitory protein (cFLIP) expression was similar in sensitive and resistant cells. Transfection of sense PED/PEA-15 cDNA in sensitive cells resulted in cell resistance, whereas transfection of antisense in resistant cells rendered them sensitive. Inhibition of protein kinase C (PKC) activity restored TRAIL sensitivity in resistant cells, suggesting that PED/ PEA-15 function might be dependent on PKC-mediated phosphorylation. In summary, TRAIL induces apoptosis in > 50% of glioma cell lines, and this killing occurs through activation of the DR pathway. This caspase-8-induced apoptotic cascade is regulated by intracellular PED/PEA-15, but not by cFLIP or decoy receptors. This pathway may be exploitable for glioma and possibly for other cancer therapies.

PED/PEA-15: an anti-apoptotic molecule that regulates FAS/TNFR1-induced apoptosis.

PED/PEA-15 is a recently cloned 15 kDa protein possessing a death effector domain (DED). In MCF-7 and HeLa cells, a fivefold overexpression of PED/PEA-15 blocked FasL and TNFalpha apoptotic effects. This effect of PED overexpression was blocked by inhibition of PKC activity. In MCF-7 and HeLa cell lysates, PED/PEA-15 co-precipitated with both FADD and FLICE. PED/PEA-15-FLICE association was inhibited by overexpression of the wild-type but not of a DED-deletion mutant of FADD. Simultaneous overexpression of PED/PEA-15 with FADD and FLICE inhibited FADD-FLICE co-precipitation by threefold. Based on cleavage of the FLICE substrate PARP, this inhibitory effect was paralleled by a threefold decline in FLICE activation in response to TNF-alpha. TNFalpha, in turn, reduces PED association with the endogenous FADD and FLICE of the cells. Thus, PED/PEA-15 is an endogenous protein inhibiting FAS and TNFR1-mediated apoptosis. At least in part, this function may involve displacement of FADD-FLICE binding through the death effector domain of PED/PEA-15.

Protein kinase C (PKC)-alpha activation inhibits PKC-zeta and mediates the action of PED/PEA-15 on glucose transport in the L6 skeletal muscle cells.

Overexpression of the PED/PEA-15 protein in muscle and adipose cells increases glucose transport and impairs further insulin induction. Like glucose transport, protein kinase C (PKC)-alpha and -beta are also constitutively activated and are not further stimulatable by insulin in L6 skeletal muscle cells overexpressing PED (L6(PED)). PKC-zeta features no basal change but completely loses insulin sensitivity in L6(PED). In these cells, blockage of PKC-alpha and -beta additively returns 2-deoxy-D-glucose (2-DG) uptake to the levels of cells expressing only endogenous PED (L6(WT)). Blockage of PKC-alpha and -beta also restores insulin activation of PKC-zeta in L6(PED) cells, with that of PKC-alpha sixfold more effective than PKC-beta. Similar effects on 2-DG uptake and PKC-zeta were also achieved by 50-fold overexpression of PKC-zeta in L6(PED). In L6(WT), fivefold overexpression of PKC-alpha or -beta increases basal 2-DG uptake and impairs further insulin induction with no effect on insulin receptor or insulin receptor substrate phosphorylation. In these cells, overexpression of PKC-alpha blocks insulin induction of PKC-zeta activity. PKC-beta is 10-fold less effective than PKC-alpha in inhibiting PKC-zeta stimulation. Expression of the dominant-negative K(281)-->W PKC-zeta mutant simultaneously inhibits insulin activation of PKC-zeta and 2-DG uptake in the L6(WT) cells. We conclude that activation of classic PKCs, mainly PKC-alpha, inhibits PKC-zeta and may mediate the action of PED on glucose uptake in L6 skeletal muscle cells.

Leptin induces mitogenic effect on human choriocarcinoma cell line (JAr) via MAP kinase activation in a glucose-dependent fashion.

Leptin and glucose effect on cell growth has been investigated in the JAr human choriocarcinoma cells. When JAr cells were cultured in the presence of 6m M glucose (LG), proliferation and thymidine incorporation were induced by serum but not by leptin. At variance, at 25m M glucose (HG), proliferation and thymidine incorporation were stimulated by leptin and serum to a comparable extent. HG culturing also enhanced leptin-stimulated insulin receptor substrate 1 (IRS1) and MAPK phosphorylation. Blockage of MAPK activity with PD98059 caused an inhibition of glucose- and leptin-dependent thymidine incorporation. At variance with HG conditions no effects were observed in cells cultured in 6m M glucose upon treatment with PD98059. Neither glucose nor leptin determined a modification in leptin receptors total content. In this study, we provide evidence that in placental cells, leptin, similarly to that observed with insulin, stimulates cell proliferation by inducing the IRS1/MAPK pathway in a glucose-dependent fashion.

Molecular mechanisms involved in GnRH analogue-related apoptosis for uterine leiomyomas.

GnRH agonist therapy is known to reduce uterine leiomyoma volume, although the molecular mechanisms responsible for this effect remain poorly understood. In this study, we have investigated the molecular mechanisms involved in the anti-proliferative effect of a GnRH agonist, leuprolide acetate (LA), in uterine leiomyomas obtained from six patients treated with LA for 3 months before surgery (group B), compared with tumours from six untreated patients (group A). To this end, we have evaluated the expression and the activity of molecules involved in the regulation of cell survival and proliferation. In group B, the total activity of PI3K was reduced by 60% compared with control samples. Furthermore, LA caused a reduction of PKB activation of approximately 50%, measured as serine 473 phosphorylation. In parallel with PKB reduction in LA samples, we observed a 60% reduction in the phosphorylation of its substrate BAD. While Bcl-xL/BAD association was not significantly modified in LA-treated leiomyomas, BAD/14.3.3 interaction was reduced, due to a 50% decreased 14.3.3 expression. In addition, LA was able to reduce the expression of the antiapoptotic proteins FLIP and PED/PEA15 by 70 and 50% respectively, compared with control samples. We next evaluated the activation of MAP kinases in leiomyomas. Activation of p42 and p44 MAP kinase isoforms was increased by 30% in group B. However, the phosphorylation of the transcription factor Elk1 was not increased in a similar fashion in LA-treated leiomyomas compared with group A. Thus, these data suggest that LA reduction of leiomyoma volume is mediated at least in part by a decreased activation of the PI3K/PKB survival pathway and by the suppression of antiapoptotic factors.

Insulin receptor substrate-2 phosphorylation is necessary for protein kinase C zeta activation by insulin in L6hIR cells.

We have investigated glycogen synthase (GS) activation in L6hIR cells expressing a peptide corresponding to the kinase regulatory loop binding domain of insulin receptor substrate-2 (IRS-2) (KRLB). In several clones of these cells (B2, F4), insulin-dependent binding of the KRLB to insulin receptors was accompanied by a block of IRS-2, but not IRS-1, phosphorylation, and insulin receptor binding. GS activation by insulin was also inhibited by >70% in these cells (p < 0.001). The impairment of GS activation was paralleled by a similarly sized inhibition of glycogen synthase kinase 3 alpha (GSK3 alpha) and GSK3 beta inactivation by insulin with no change in protein phosphatase 1 activity. PDK1 (a phosphatidylinositol trisphosphate-dependent kinase) and Akt/protein kinase B (PKB) activation by insulin showed no difference in B2, F4, and in control L6hIR cells. At variance, insulin did not activate PKC zeta in B2 and F4 cells. In L6hIR, inhibition of PKC zeta activity by either a PKC zeta antisense or a dominant negative mutant also reduced by 75% insulin inactivation of GSK3 alpha and -beta (p < 0.001) and insulin stimulation of GS (p < 0.002), similar to Akt/PKB inhibition. In L6hIR, insulin induced protein kinase C zeta (PKC zeta) co-precipitation with GSK3 alpha and beta. PKC zeta also phosphorylated GSK3 alpha and -beta. Alone, these events did not significantly affect GSK3 alpha and -beta activities. Inhibition of PKC zeta activity, however, reduced Akt/PKB phosphorylation of the key serine sites on GSK3 alpha and -beta by >80% (p < 0.001) and prevented full GSK3 inactivation by insulin. Thus, IRS-2, not IRS-1, signals insulin activation of GS in the L6hIR skeletal muscle cells. In these cells, insulin inhibition of GSK3 alpha and -beta requires dual phosphorylation by both Akt/PKB and PKC zeta.