Effects of erythropoietin on glucose metabolism.

We purposed to determine the impact of erythropoietin on altering glucose metabolism in the settings of in vitro and in vivo experiments. The acute effect of erythropoietin on lowering blood glucose levels was studied in animal experiments. In [³H]-deoxy-D-glucose isotope studies we measured glucose uptake with insulin and erythropoietin using 3T3-L1 cells cultured under normal or high glucose conditions. Altered activation of Akt and ERK pathways was evaluated in immunoblot analyses. Immunocytochemistry was conducted to determine the glucose transporter 4 translocation to the plasma membrane. Addition of erythropoietin significantly lowered blood glucose levels in vivo in rats. The glucose uptake was markedly increased by erythropoietin treatment (at concentrations 0.15, 0.3, and 0.625 ng/ml) in adipocytes grown in high glucose medium (p<0.05), but it remained unaltered in cells under normal glucose conditions. Significant increase of phosphorylation of ERK and Akt was detected due to erythropoietin (p<0.05). Co-administration of erythropoietin and insulin resulted in higher phosphorylation of Akt and [³H]-deoxy-D-glucose uptake in adipocytes than insulin treatment alone. We found that erythropoietin induced the trafficking of glucose transporter 4 to the plasma membrane. Our data showed that erythropoietin significantly decreased blood glucose levels both in vivo and in vitro, in part, by increasing glucose uptake via the activation of Akt pathway. Preliminary data revealed that adipocytes most likely exhibit a specific receptor for erythropoietin.

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.

Production of homocysteine in serum-starved apoptotic PC12 cells depends on the activation and modification of Ras.

PC12 pheochromocytoma cells expressing a dominant inhibitory mutant of Ha-Ras (M-M17-26) and PC12 cells transfected with normal c-RasH (M-CR3B) have been used to investigate the role of nitrosylation and farnesylation of Ras on the production of homocysteine and the activities of the redox-sensitive transcription factors NF-kappaB and c-Fos. We found that under serum and nerve growth factor withdrawal conditions undifferentiated apoptotic M-CR3B cells accumulated more homocysteine than M-M17-26 cells, and the production of homocysteine decreased in the presence of manumycin and increased in the presence of l-NAME. Furthermore, we have shown that manumycin increased the activity of c-Fos in the M-CR3B cells and decreased the activity of NF-kappaB, while l-NAME decreased the activities of both transcription factors, and accelerated apoptosis of M-CR3B cells. In contrast, in M-M17-26 cells manumycin did not change the activity of c-Fos, nor the activity of NF-kappaB. We conclude that trophic factor withdrawal stimulates Ras, which apparently through the Rac/NADPH oxidase system induces permanent oxidative stress, modulates the activities of NF-kappaB and c-Fos, induces production of homocysteine and accelerates apoptosis. Nitrosylation of Ras is necessary for maintaining the survival of PC12 cells, while farnesylation of Ras stimulates apoptosis under withdrawal conditions.

Sodium salicylate inhibits NF-kappaB and induces apoptosis in PC12 cells.

Sodium salicylate (NaSal) is an effective analgetic and antiinflammatory drug. Beside its well-known inhibitory effect on the cyclooxigenase enzymes, it influences the activity of other signal transduction proteins including nuclear factor kappa B (NF-kappaB) transcription factor. NF-kappaB is found in the cytoplasm bound to an inhibitory protein, inhibitory kappa B (IkappaB). After its phosphorylation, IkappaB is degraded and the released NF-kappaB translocates into the nucleus. Sodium salicylate inhibits the degradation of IkappaB, thus, NF-kappaB activation cannot occur. According to previous observations, the inhibition of this activation can lead to apoptosis. The main goals of this study were to demonstrate that inhibition of NF-kappaB by sodium salicylate decreases the viability of rat phaeochromocytoma PC12 cells and to investigate the nature of cell damage and death. PC12 cells were treated with different concentrations of sodium salicylate (1-20 mM). Higher concentrations (10-20 mM) killed PC12 cells in a dose-dependent manner. The assessments were done by direct cell counting in a Burker chamber and by the WST-1 cytotoxicity assay. We also found a decreased NF-kappaB activity after sodium salicylate treatment by electrophoretic mobility shift assay (EMSA). The cells treated with sodium salicylate were undergoing apoptosis as seen on our records obtained by time-lapse videomicroscopy as well as shown by DNA fragmentation experiments. The decreased DNA binding activity of NF-kappaB indicates that the inhibition of NF-kappaB can play a role in these processes.

MTH-68/H oncolytic viral treatment in human high-grade gliomas.

Application of virus therapy to treat human neoplasms has over a three decade history. MTH-68/H, a live attenuated oncolytic viral strain of the Newcastle disease virus, is one of the viruses used in the treatment of different malignancies. Here we report on the administration of MTH-68/H to patients with glioblastoma multiforme, the most common and most aggressive neuroectodermal neoplasm with a poor prognosis, averaging six months to a year. Four cases of advanced high-grade glioma were treated with MTH-68/H after the conventional modalities of anti-neoplastic therapies had failed. This treatment resulted in survival rates of 5-9 years, with each patient still living today. Against all odds, each patient resumed a lifestyle that resembles their previous daily routines and enjoys a good quality of life, Each of these patients has regularly received MTH-68/H as their sole form of onco-therapy for a number of years now without interruption.

Nuclear translocation of p90Rsk and phosphorylation of CREB is induced by ionomycin in a Ras-independent manner in PC12 cells.

In the present study we examined the possible role of p90Rsk in pathways leading to neuronal differentiation of PC12 cells induced by nerve growth factor (NGF) and the calcium ionophore ionomycin. PC12-M17 cells, expressing a dominant inhibitory Ras protein, do not undergo neuronal differentiation in response to NGF like wild-type PC12 cells, but exhibit neurite outgrowth when treated with NGF in combination with ionomycin. However, the blockade of Ras in these cells results in failure of activation of mitogen-activated protein kinase (MAPK)/extracellular signal regulation kinase (ERK) (MEK) and ERK activation as well, therefore kinases other than those of the ERK pathway might play a role in the induction of neuronal differentiation in this case. Here we show that p90Rsk translocates to the nucleus in response to ionomycin in both wild-type PC12 and PC12-M17 cells, and this spatial distribution is followed by increased phosphorylation of the cAMP response element binding protein (CREB). Since CREB is believed to be the transcription factor that can integrate Ca2+, growth factor and cAMP-induced signals, we suggest that p90Rsk may be one of the kinases which is able to replace ERKs under certain circumstances, thereby participating in Ras-independent neuronal differentiation induced by NGF plus ionomycin.

Nerve growth factor in combination with second messenger analogues causes neuronal differentiation of PC12 cells expressing a dominant inhibitory Ras protein without inducing activation of extracellular signal-regulated kinases.

In the present work, nerve growth factor (NGF) was used in combination with the calcium ionophore, ionomycin or dibutyryl cyclic AMP (dbcAMP), to study the connection between neuronal differentiation and extracellular signal-regulated kinase (ERK) activation of PC12 rat pheochromocytoma cells expressing a dominant negative, Ha-Ras Asn17 protein. Due to the block of endogenous Ras activity, neurite outgrowth in response to NGF is completely inhibited in these cells. However, this blockade can be bypassed by combined treatment with NGF plus ionomycin or NGF plus dbcAMP. The mitogen-activated protein kinase (MAPK) /ERK kinase inhibitor, PD98059, proved to be insufficient in inhibiting the neurite outgrowth under these conditions. Moreover, although both long-term ERK activation and nuclear translocation of ERKs are believed to be key events in neuronal differentiation, neither detectable ERK phosphorylation, nor nuclear translocation of these enzymes, occurred upon combination treatments in our experimental system. However, the neuritogenesis induced by either the combination of NGF/ionomycin or NGF/dbcAMP was inhibited by the Trk inhibitor, K252a. Ras-independent pathways, originating from the NGF receptor, can thus synergize with second messenger analogues bypassing the ERK cascade but leading to the same biological result--neurite formation.

Increased histidine decarboxylase expression during in vitro monocyte maturation; a possible role of endogenously synthesised histamine in monocyte/macrophage differentiation.

OBJECTIVE: In this study the expression of histidine decarboxylase (HDC), the pivotal enzyme in histamine formation and the effect of endogenously produced histamine on differentiation antigens was examined during in vitro differentiation of human monocytes. MATERIAL AND TREATMENT: Human elutriated monocytes from healthy volunteers were incubated with macrophage colony stimulating factor (M-CSF) and the expression of HDC was followed at both mRNA and protein levels. To study the possible function of histamine we followed the expression of some cell surface markers (CD14, CD16, CD91, CD49d and CD11c) relevant for phagocytic differentiation upon incubation in the presence of different histamine inhibitors, an HDC inhibitor: S(+)-alpha-fluoromethyl-histidine HCl, (alphaFMH), a compound that disturbs the interaction of histamine with intracellular cyp450 moieties: N,N-diethyl-2-[4-(phenylmethyl) phenoxy]-ethanamine HCI, (DPPE); and H1 and H2 receptor antagonists, Triprolidine and Cimetidine. RESULTS: During in vitro culture of elutriated human monocytes, in the presence of M-CSF, the gene expression and biosynthesis of HDC was considerably increased. The various antihistamine agents decreased the expression of the cell surface markers examined in this study. CONCLUSIONS: These data support the elevation of HDC expression during human monocytic differentiation and the possibility that monocyte-derived histamine is partially involved in regulation of M-CSF induced in vitro human monocyte/macrophage phagocytic differentiation.

Inhibition of effects of endogenously synthesized histamine disturbs in vitro human dendritic cell differentiation.

Histamine, a principal mediator in various physiological and pathological cell functions is synthesized from L-histidine exclusively by histidine decarboxylase, an enzyme, which is expressed in many tissues of mammalian organism. Histamine plays a role in various cellular functions, including cell differentiation. The aim of this study was to determine the presence and to characterize the role of the endogenously produced histamine during in vitro dendritic cell (DC) differentiation induced by interleukin-4 (IL-4) and granulocyte-monocyte colony stimulating factor (GM-CSF). The changes in intracellular histamine content, biosynthesis and gene expression of histidine decarboxylase were investigated during this process. One also studied how histamine receptor antagonists and a histamine synthesis blocker influence the expression of differentiation antigens on the DC during in vitro maturation. During in vitro differentiation parallel culture incubations were performed by adding H1 receptor antagonist triprolidine, H2 receptor antagonist tiotidine, the tamoxifene derivate DPPE which blocks the intracellular binding of histamine, and an irreversible blocker of histidine decarboxylase, alpha-fluoromethyl histamine (alpha-FMH). The results show simultaneous increase in both histidine decarboxylase level and histamine content during differentiation of elutriated monocytes toward DC. Both blockade of de novo histamine production (by alpha-FMH) and inhibition of histamine binding (by H1 and H2 receptor antagonists, triprolidine and tiotidine, respectively) markedly decreased CD40 expression and that of CD45 from the 3rd day of treatment. DPPE by disturbing intracellular interaction of histamine with cytochrome P-450 moieties was able to decrease the expression of CD45, CD86, HLA-DR, CD33, CD40 and CD11c. Based on the data it is suggested that endogenous histamine is actively synthesized during cytokine-induced in vitro DC differentiation. The functional relevance and autocrine and paracrine action of endogenously produced histamine is supported by the data showing that inhibition of histamine synthesis by HDC, blocking of histamine binding by both 'extracellular' histamine receptors (by specific antagonists, triprolidine and tiotidine) and 'intracellular' antagonists (DPPE) disturb the differentiation of DC. This conclusion is supported by the fact, that by the inhibition of histamine acting in an autocrine/paracrine way, the expression pattern of differentiation markers on DC is markedly changed.

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 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.

Multi-color analysis of monocyte and dendritic cell precursor heterogeneity in whole blood.

The coexpression analysis of the 55-kDa lipopolysaccharide receptor (CD14) and the Fc gamma-receptor III (CD16) reveals a broad heterogeneity of blood monocytes which in our previous work could be divided into five subpopulations based on correlated differences in expression of the pan-myeloid antigen CD33 and the adhesion antigens CD11a, CD11b and CD56. An even larger complexity of myeloid cells with antigen presenting capacity in peripheral blood is suggested by the description of small populations of immature and mature precursors of dendritic cells which rapidly develop potent costimulatory activity and a dendritic morphology in in vitro culture. The identity of the subsets of cells which have been described based on heterogeneous analytical approaches, however, remains unclear. The goal of this study, therefore, was the correlated analysis of monocyte subpopulations and dendritic cell precursors in a quantitative whole blood assay. This was achieved based on simultaneous expression analysis of the monocyte markers CD14 and CD16 with antigens such as CD33, HLA-DR, the integrin CD11c, and the interleukin-3 receptor alpha-chain (CD123) which in absence of lineage-related antigens have been used for description of dendritic cell precursors. The selected marker panel revealed identity of cells previously described as CD33bright CD14dim dendritic cell precursors with CD11c+lin-HLA-DR+ cells. Dendritic cell precursors considered to be less mature which have been alternatively described as CD33+ CD14dim CD16- cells or CD123hi dendritic cell precursors, however, were shown to differ in phenotype from each other with regard to expression density of CD33 and expression of CD14. In summary, our study revealed a complex heterogeneity of monocytes and dendritic cell precursors in peripheral blood and indicates that a direct comparison of the analytical approaches of different authors is needed to further clarify the ontogeny of human monocytes and dendritic cells.

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.

Different roles for RhoA during neurite initiation, elongation, and regeneration in PC12 cells.

The goal of the present study was to characterize the effects of RhoA at different stages of nerve growth factor (NGF)-induced neuronal differentiation in the PC12 model. This comparative analysis was prompted by previous studies that reported apparently opposite effects for Rho in different models of neuronal differentiation and regeneration. PC12 cells were transfected with activated V14RhoA or dominant negative N19RhoA under the control of either a constitutive or a steroid-regulated promoter. Upon exposure to NGF, V14RhoA cells continued to proliferate and did not extend neurites; however, they remained responsive to NGF, as indicated by the activation of extracellular signal-regulated kinases. This inability to differentiate was reversed by C3 toxin and activation of cyclic AMP signaling, which inactivate RhoA. N19RhoA expression led to an increase in neurite initiation and branching. In contrast, when the RhoA mutants were expressed after NGF priming, only the rate of neurite extension was altered; V14RhoA clones had neurites approximately twice as long, whereas neurites of N19RhoA cells were approximately 50% shorter than those of appropriate controls. The effects of Rho in neurite regeneration mimicked those observed during the initial stages of morphogenesis; activation inhibited, whereas inactivation promoted, neurite outgrowth. Our results indicate that RhoA function changes at different stages of NGF-induced neuronal differentiation and neurite regeneration.

Role of different Ca2+ sources in the superoxide production of human neutrophil granulocytes.

The role of different Ca2+ sources in the activation of the NADPH oxidase was investigated in human neutrophil granulocytes. Selective depletion of the stimulus-responsive intracellular Ca2+ -pool and the consequent opening of the store-dependent Ca2+ channel of the plasma membrane was achieved with thapsigargin, an inhibitor of microsomal Ca2+ -ATPase. Low concentration (10-100 nM) of thapsigargin did not induce any O2*- -production, indicating that elevation of [Ca2+]ic to similar level and probably via similar route as following stimulation of chemotactic receptors, by itself is not sufficient to activate the NADPH oxidase. In significantly higher concentration (1-10 microM) thapsigargin did induce O2*- -generation but this effect was not the result of elevation of [Ca2+]ic. In the absence of external Ca2+ a gradual decrease of the responsive Ca2+ pool was accompanied by a gradual decrease of the rate and duration of the respiratory response stimulated by formyl-methionyl-leucyl-phenylalanin. Maximal extent of receptor-initiated O2*- -production could only be obtained when the intracellular [Ca2+] was higher than the resting level. Under this condition Ca2+ originating from intracellular or external source was equally effective in supporting the biological response.

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.