Role of phospholipase C-gamma in NGF-stimulated differentiation and gene induction.

The PC12 phaeochromocytoma cell line provides a useful model to study nerve growth factor-induced neuronal differentiation. The central signaling route of this process is mediated by the Ras-dependent extracellular signal-regulated kinase cascade. However, Ras-independent pathways are also stimulated by nerve growth factor and may contribute to differentiation signaling. One mediator for Ras-independent signal transduction in PC12 cells is phospholipase C-gamma that generates the second messengers diacylglycerol and inositol-trisphosphate. To probe the possible involvement of this enzyme in nerve growth factor-promoted differentiation, we used the phospholipase C inhibitor U73122 and the inositol-trisphosphate-receptor inhibitor Xestospongin C. Our results show that both chemicals block nerve growth factor-promoted neurite outgrowth, but the blockage of phospholipase C does not inhibit nerve growth factor-induced expression of c-fos, zif268 and transin genes. In addition, induction of these genes by nerve growth factor plus dibutyryl-cAMP is comparable in wild-type PC12 cells as well as in cells in which both Ras- and phospholipase C-gamma-mediated pathways are inhibited. The phospholipase C-gamma pathway thus belongs to those nerve growth factor receptor-originated signaling routes that contribute to the biological response of PC12 cells to nerve growth factor, but its gene activating potential does not have a major role in its neuritogenic effect.

Antiestrogenic effect of opioid peptides in rat uterus.

The effects of a single injection or continuous infusion of opioid peptide, [D-Met(2),pro(5)]enkephalinamide (ENK) on the hormone binding and transcriptional properties of estrogen receptors were investigated in estradiol (E(2)) treated rat uterus. The level of estrogen- (ER) and progesterone receptor (PR) proteins, the hormone binding of E(2) receptors and the effects of single injection of ENK with or without naltrexone (NAL) on the E(2)-induced changes in the level of Fos and Jun proteins and the binding of AP-1 proteins to DNA were studied. The receptor proteins levels were determined by Western blots and the binding of AP-1 to DNA by electrophoretic mobility shift assay. Both the ER and PR protein concentrations and the [3H]Estradiol binding to the high affinity nuclear receptors decreased after ENK treatment during the first two days. At 72 h the PR concentration decreased further, while no significant changes were found in the level of ER, however, at this time the former competitive E(2) binding turned into positive cooperativity. The E(2)-induced increase in the level of Fos proteins and the binding of AP-1 proteins to DNA was inhibited by a single injection of ENK. We conclude that the endogenous opioid peptides may interact with E(2) in the gene regulation of rat uterus.

Regulation of activator protein-1-DNA binding activity by opioid peptides in estrogen-sensitive cells of rat hypothalamus and uterus.

The present studies demonstrate, for the first time, that the binding of activator protein-1 (AP-1)-DNA in rat uterus and the estrogen-sensitive areas of the hypothalamus, as measured by electrophoretic mobility shift assay, is increased 2 h after intraperitoneal injection of [D-Met(2),Pro(5)]enkephalinamide. The effect was prevented by the opiate antagonist naltrexone given 30 min before the administration of [D-Met(2),Pro(5)]enkephalinamide, suggesting the involvement of opioid peptide receptors in the observed effects. The present findings support the role of opioid peptides in the regulation of transcription in estrogen-sensitive cells.

Beneficial treatment of patients with advanced cancer using a Newcastle disease virus vaccine (MTH-68/H).

Newcastle Disease Virus Vaccine (MTH-68/H) was administered to patients suffering from advanced neoplastic diseases after non-efficient tumor-destructive treatment. Case reports of selected patients suggest promising effects of this treatment. A prospectively-randomized clinical study (phase III; in accordance with Good Clinical Practice, GCP) was proposed to confirm these results and is currently under consideration.

Induction of apoptosis by a Newcastle disease virus vaccine (MTH-68/H) in PC12 rat phaeochromocytoma cells.

The attenuated Newcastle Disease Virus (NDV) vaccine MTH-68/H has been found to cause regression of various tumors including certain types of human neoplasms (See Table 1 and References 86-88). The mechanism of its oncolytic action is poorly understood, but it appears to affect specific signaling pathways in the target cell. We studied the cellular effects of NDV employing PC12 rat phaeochromocytoma cells, a widely used model system to analyze differentiation, proliferation and apoptosis. The MTH-68/H vaccine was found to be cytotoxic on PC12 cells. It caused internucleosomal DNA fragmentation, the most characteristic feature of programmed cell death (PCD). A brief exposure (30 min) of P12 cells to the virus was sufficient to produce a full-blown apoptotic response. Major mitogen-activated protein kinase pathways (including the stress inducible c-Jun N-terminal kinase pathway and p38 pathway) or mechanisms regulated by reactive oxygen species appear to have no role in virus-induced cell death. The PCD-inducing effect of MTH-68/H could not be prevented by simultaneous treatment of the P12 cells with growth factors or second messenger analogs stimulating protein kinase C or Ca(++)-mediated pathways. In contrast, treatment with a cyclic AMP analog partially protected the them from virus-induced apoptosis. These experimental results suggests that MTH-68/H might disrupt a growth factor-stimulated survival pathway and that direct stimulation of protein kinase A-catalyzed phosphorylation events bypass this NDV-induced block.

Anisomycin uses multiple mechanisms to stimulate mitogen-activated protein kinases and gene expression and to inhibit neuronal differentiation in PC12 phaeochromocytoma cells.

Treatment of PC12 cells with nerve growth factor (NGF) stimulates extracellular signal-regulated kinases (ERKs), as well as stress-activated c-Jun N-terminal kinases (JNKs) and p38 kinase, and induces neuronal differentiation. While the pivotal role of ERKs in NGF-induced morphological differentiation is well established, the contribution of JNK- and p38-pathways is less clear. The role of the JNK- and p38-pathway in PC12 cells was analysed by using anisomycin, a protein synthesis inhibitor that activates JNKs and p38. Non-toxic concentrations of anisomycin were found to stimulate these enzyme activities as well as the expression of the early response genes c-jun, c-fos and zif268, and to inhibit NGF-induced neurite formation. These effects of anisomycin appear to be mediated by the generation of reactive oxygen species (ROS), which in turn act through both TrkA/Ras-dependent and -independent signalling pathways. In addition, cross-talk between the p38- and ERK-pathways appears to play a role in the action of anisomycin.

Anisomycin affects both pro- and antiapoptotic mechanisms in PC12 cells.

Survival and differentiation of PC12 cells depend on the proper balance between the activities of several mitogen-activated protein kinase (MAPK) pathways. We have previously shown that low, nontoxic doses of anisomycin stimulated these MAPKs as well as the expression of several early-response genes and inhibited NGF-induced neurite formation. In the present work we show that protein synthesis-inhibiting concentrations of anisomycin, in contrast, cause apoptosis of PC12 cells. To try to characterize the apoptosis-inducing mechanisms of anisomycin we compared the signaling effects of subinhibitory and inhibitory drug concentrations. Anisomycin in a nontoxic dosis activates the same MAPK pathways and early-response genes as in protein synthesis inhibiting concentrations. In contrast, while the subinhibitory anisomycin treatment stimulates Akt and induces Bcl-2, two antiapoptotic proteins, the translation-inhibiting concentration of the drug prevents these survival-promoting biochemical events. Anisomycin thus triggers both pro- and antiapoptotic processes in PC12 cells; stimulation of stress-responsive MAPK cascades is not sufficient to mediate apoptotic signaling: the inhibition of key antiapoptotic proteins appears to be more important for PC12 cell death by anisomycin treatment.