myc and E1A oncogenes alter the responses of PC12 cells to nerve growth factor and block differentiation

PC12 rat pheochromocytoma cells respond to nerve growth factor (NGF) by neuronal differentiation and partial growth arrest. Mouse c-myc and adenovirus E1A genes were introduced into PC12 cells to study the influence of these nuclear oncogenes on neuronal differentiation. Expression of myc or E1A blocked morphological differentiation and caused NGF to stimulate rather than inhibit cell proliferation. NGF binding to cell surface receptors and ornithine decarboxylase induction were similar in myc- and E1A-expressing clones compared with wild-type PC12 cells, suggesting that changes in the cellular response to NGF were at a post-receptor level. These results illustrate that NGF can promote either growth or differentiation of PC12 cells, and that myc or E1A alter the phenotypic responses to growth factors and hormones.     

Role of Ras in signal transduction from the nerve growth factor receptor: relationship to protein kinase C, calcium and cyclic AMP.

A dominant inhibitory ras mutant (Ha-ras Asn-17) has been used to investigate the role of Ras in nerve growth factor (NGF)-mediated signal transduction in PC12 cells. Expression of Ha-Ras Asn-17 blocks neuronal differentiation of these cells in response to NGF treatment. The Ha-Ras Asn-17 block was bypassed by treatment with NGF plus dibutyryl cAMP or NGF plus the Ca2+ ionophore ionomycin, but not by NGF plus 12-O-tetradecanoyl phorbol acetate (TPA). Direct stimulation of the cAMP or Ca2+ pathways thus appeared to act synergistically with a Ras-independent NGF signaling pathway. This Ras-independent pathway was also distinct from protein kinase C, since its activity was not affected by protein kinase C down-regulation. It thus appears that NGF stimulation generates a Ras-independent intracellular signal that contributes to neuronal differentiation independently of the cAMP, Ca2+ or protein kinase C second messenger systems. Since TPA did not bypass the Ha-Ras Asn-17 block to differentiation, protein kinase C also did not appear to be sufficient for Ras-dependent pathways mediating NGF-induced differentiation. Down-regulation experiments further indicated that protein kinase C was not required for NGF induction of early response genes via either Ras-dependent or Ras-independent pathways. Moreover, the formation of inositol phosphates and mobilization of intracellular calcium in response to NGF was not inhibited in PC12 cells expressing the Ha-Ras Asn-17 protein. Therefore, although calcium was able to bypass the Ha-Ras Asn-17 block to PC12 differentiation, Ras activity was not required for activation of phospholipase C in response to NGF. It thus appears that both Ras-dependent and Ras-independent signaling pathways contribute to NGF-induced PC12 cell differentiation independently of the cAMP, calcium and protein kinase C second messenger systems.     

Regulation of growth by a nerve growth factor-like protein which modulates paracrine interactions between a neoplastic epithelial cell line and stromal cells of the human prostate.

Nerve growth factor-like substance(s) were identified in both conditioned media of a human prostatic tumor epithelial cell line (TSU-pr1) and a human prostatic stromal cell line (HPS) by Western blot analysis and bioassay of neurite outgrowth of PC12 cells. Nerve growth factor-beta (NGF) immunofluorescence was also localized to secretory vesicles in the cytoplasm of both the TSU-pr1 and HPS cells. Western blot of the TSU-pr1 and HPS cell-secreted protein identified an Mr 65,000 major protein which immunoreacted with murine NGF antibody. NGF Western blot of HPS cell-secreted protein also identified an Mr 42,000 minor band under reduced and nonreduced conditions and an Mr 61,000 minor band under reduced conditions. The secreted protein from the TSU-pr1 cells (50 micrograms/ml) and HPS (50 micrograms/ml), as well as murine NGF (50 ng/ml) or human recombinant NGF (50 ng/ml), stimulated neurite outgrowth from PC12 cells. This neurite outgrowth activity was partially inhibited by treatment with NGF antibody. Neither the serum containing growth medium nor bovine serum albumin (50 micrograms/ml) stimulated neurite outgrowth. The NGF-like secretory protein appeared to play a role in the paracrine regulation of prostatic growth between TSU-pr1 cells and HPS cells. The relative growth of TSU-pr1 cells, as indicated by [3H]thymidine incorporation, in response to HPS secretory protein was stimulated 2.8-fold in a dose-dependent manner. In the converse interaction, the relative growth of HPS cells in response to TSU-pr1 secretory protein was stimulated 1.8-fold in a dose-dependent manner. Immunoneutralization of TSU-pr1 and HPS secretory protein was performed with antibody against NGF, acidic fibroblast growth factor, and basic fibroblast growth factor. Removal of the NGF-like protein from the maximal stimulatory dose of TSU-pr1 secretory protein (100 micrograms/ml) with NGF antibody reduced HPS proliferation to 52% of maximal levels, and immunoneutralization of the NGF-like protein in the maximal stimulatory dose of HPS secretory protein (20 micrograms/ml) also reduced TSU-pr1 proliferation to 16% of maximal levels. Addition of normal rabbit serum or prior immunoprecipitation of either TSU-pr1 or HPS secretory protein with antibody against acidic fibroblast growth factor and basic fibroblast growth factor did not inhibit the proliferation of either cell type. These results suggest that TSU-pr1 tumor cells and HPS cells secrete NGF-like protein(s) which modulate their paracrine interactive growth in vitro.     

Molecular dissection of TrkA signal transduction pathways mediating differentiation in human neuroblastoma cells

Activation of the neurotrophin receptor TrkA by its ligand nerve growth factor (NGF) initiates a cascade of signaling events leading to neuronal differentiation in vitro and might play an important role in the differentiation of favorable neuroblastomas (NB) in vivo. To study TrkA signal transduction pathways and their effects on differentiation in NB, we stably expressed wild-type TrkA and five different TrkA mutants in the NGF unresponsive human NB cell line SH-SY5Y. Resulting clones were characterized by TrkA mRNA and protein expression, and by autophosphorylation of the receptor. Introduction of wild-type TrkA restored NGF responsiveness of SH-SY5Y cells, as demonstrated by morphological differentiation, activation of mitogen-activated protein kinases (MAPK) and induction of immediate-early genes. Expression of TrkA in the absence of NGF resulted in growth inhibition of transfectants compared to parental cells, whereas NGF-treatment increased their proliferation rate. Analysis of downstream signal transduction pathways indicated that NGF-induced differentiation was dependent on TrkA kinase activity. Our data suggest that several redundant pathways are present further downstream, but activation of the RAS/MAPK signaling pathway seems to be of major importance for NGF mediated differentiation of NB cells. Our results also show that the signaling effector SH2-B is a substrate of NGF-mediated Trk signaling in NB, whereas it is not activated by NGF in rat pheochromocytoma PC12 cells. This might explain the differences we observed in TrkA signaling between neuroblastoma and PC12 cells. Further insight into TrkA signaling may suggest new options for the treatment of NB.     

Nerve growth factor induces rapid accumulation of the GTP-bound form of p21ras in rat pheochromocytoma PC12 cells.

The ras gene product (p21) is thought to transduce signals from various growth and differentiation factors. p21 is a GTP-binding protein, and its activity is regulated by the bound GDP/GTP ratio. We analysed p21-bound nucleotides in cell lysates of rat pheochromocytoma cell line PC12 cells stimulated with various factors. Nerve growth factors (NGF) rapidly increased the relative amount of active p21-GTP complex to as much as 20% of the total amount of p21 within 2 min. The amount of p21-GTP then declined to 8% after 10 min, and this level was sustained for at least 2 h. Epidermal growth factor (EGF) also stimulated a rapid accumulation of p21-GTP to the same extent as seen with NGF, but the amount of p21-GTP declined to 5% after 10 min and gradually returned to the basal level within 60 min. In contrast, basic fibroblast growth factor, interleukin 6 and dibutyryl cAMP, which induce neuronal differentiation of PC12 cells, did not stimulate the accumulation of p21-GTP at any time point examined. Phorbol 12-myristate 13-acetate also had no effect. Interestingly, the protein kinase inhibitor K-252a specifically suppressed the NGF-induced accumulation of p21-GTP, but did not suppress the EGF-induced response. These results strongly suggest that an active p21-GTP complex transduces the differentiation signal from NGF. It may also be suggested that the process of activating p21 is mediated by a K-252a-sensitive protein kinase(s).     

PIAS3绿色荧光蛋白表达载体构建及其对胶质瘤U251细胞增殖和凋亡的影响

目的：构建PIAS3绿色荧光蛋白真核表达载体,探讨外源性PIAS3对胶质瘤细胞U251周期和凋亡的影响。方法：利用RT-PCR扩增人全长PIAS3编码序列,并将其克隆到带有绿色荧光蛋白的真核表达载体pEGFP-N1中,得到重组质粒pEGFP-N1-PIAS3。应用脂质体法转染体外培养的人胶质瘤U251细胞株。荧光显微镜观察蛋白表达,用RT-PCR和Westernblot检测PIAS3的表达,Annexin V2FITC和PI双染流式细胞术检测细胞凋亡,流式细胞术检测细胞增殖周期变化。结果：转染PIAS3基因的细胞株外源目的基因和蛋白表达增强,瞬时转染48 h后,光镜下可见部分细胞变圆,部分细胞脱落死亡;对照组和未转染组无明显变化。流式细胞仪分析显示,pEGFP-N1-PIAS3转染组凋亡细胞增多,细胞凋亡率显著高于未转染组和pEGFP-N1转染组,P=0.0073;U251细胞转染pEGFP-N1-PIAS3后细胞周期阻滞于S期,S期细胞比例增高,P=0.025,G2期细胞比例降低。结论：外源性PIAS3使U251阻滞在S期,诱导胶质瘤细胞凋亡。     

Myc drives apoptosis in PC12 cells in the absence of Max.

A conditionally active chimeric form of the c-Myc protein fused to the ligand-binding domain of the estrogen receptor (MycER) was expressed in PC12 cells. Induction of Myc activity resulted in a threefold increase in apoptosis after 5 days when cells were maintained in 1% serum. The effect of Myc overexpression was dependent on its DNA-binding domain but not on its heterodimeric binding protein Max, which is absent in PC12 cells. Preincubation of the c-Myc overexpressing cells with either NGF or bFGF, but not EGF, prevented the Myc-mediated increase in apoptosis, although the signaling pathways used by NGF and bFGF to block cell death differed. NGF-mediated rescue was mediated by the phosphatidylinositol 3'-OH (P13) kinase/Akt pathway while rescue by bFGF was not affected by P13 kinase inhibitors. These results show that Myc can induce apoptosis in PC12 cells in a Max-independent manner and that alternate signaling pathways exist to mediate cell survival.     

Nerve growth factor induces survival and differentiation through two distinct signaling cascades in PC12 cells.

Nerve growth factor induces differentiation and survival of rat PC12 pheochromocytoma cells. The activation of the erk cascade has been implicated in transducing the multitude of signals induced by NGF. In order to explore the role of this signaling cascade in NGF mediated survival, differentiation and proliferation, we generated recombinant adenoviruses which express the intermediates of the erk cascade in their wild type, dominant negative and constitutively activated forms. We show that differentiation of PC12 cells requires activity of the ras/erk pathway, whereas inhibition of this pathway had no effect on survival or proliferation. Constitutively active forms of ras, raf and mek induced PC12 cell differentiation, while dominant interfering forms inhibited differentiation. Survival of PC12 cells in serum-free medium did not require activity of the ras/erk pathway. Instead, PI3 Kinase signaling was necessary for PC12 cell survival. Interestingly, constitutively activated versions of raf and mek were able to promote survival, but again this was dependent on activation of PI3 Kinase. Therefore, at least two distinct signaling pathways are required in PC12 cells for mediation of NGF functions.     

一种新型小分子化合物增强PC3细胞的辐射敏感性

目的 检测小分子化合物8-氧-8H-苊并[1,2-b]吡咯-9-腈的氨基取代衍生物（S1）对前列腺癌细胞系PC3辐射敏感性的作用。方法 采用流式细胞术分别检测不同剂量X射线和不同浓度S1化合物以及两者联合作用后,PC3细胞凋亡率的变化;采用Western blot方法检测S1作用后不同时间Bcl-2蛋白在PC3细胞中的表达变化。结果 X射线诱导PC3细胞凋亡具有剂量依赖特性,2Gy、5Gy X 射线照射未发现明显的凋亡,10Gy、20Gy照射组有明显的凋亡产生 (P<0.01);S1化合物能够显著诱导PC3细胞凋亡,并具有剂量依赖特性,其中5μmol/L和10μmol/L组凋亡率明显高于对照组（P<0.01）;S1作用后2~12h PC3细胞中Bcl-2蛋白表达逐渐减低;S1作用后,分别给予PC3细胞2Gy和8Gy X射线照射,细胞凋亡率明显高于单独照射组和单独S1作用组（P<0.01）。结论 S1能够增强PC3细胞对X射线的敏感性,可望为临床提高前列腺癌放疗效果,降低辐射损伤提供新的治疗方案。     

Ras controls coupling of growth factor receptors and protein kinase C in the membrane to Raf-1 and B-Raf protein serine kinases in the cytosol.

A dominant negative mutant of Ras, M17 Ras, was used to study the role of Ras in receptor coupling of Raf-1 and B-Raf protein serine/threonine kinases (PSKs). We found that mutant Ras blocks serum- and 12-O-tetradecanoyl phorbol 13-acetate-induced activation of Raf-1 kinase in NIH3T3 cells and Raf-1 as well as B-Raf PSK stimulation by nerve growth factor (NGF) in PC12 pheochromocytoma cells. Mitogen stimulation of Raf kinase was measured by determination of Raf hyperphosphorylation and activity towards exogenous substrates and both of these events were inhibited in cells expressing M17 Ras. In contrast, tyrosine phosphorylation of a direct substrate of activated tyrosine kinase receptors, phospholipase C-gamma 1 (PLC-gamma 1), was unaffected. These data indicate that tyrosine phosphorylation of PLC-gamma 1 is not sufficient for growth induction in NIH3T3 cells and that Ras mediates signal transfer from activated membrane receptors to Raf kinases in the cytosol. As activated Raf induced differentiation in PC12 cells expressing M17 Ras we conclude that Raf kinase activation may be sufficient to account for this aspect of NGF function.     

Mitogenic effect of neurotrophic factors on human IMR 32 neuroblastoma cells

The effect of growth factors with neurotrophic properties on proliferation of the human IMR 32 neuroblastoma (NB) cell line was studied. A colorimetric proliferation assay and an anchorage-dependent cell culture system were used. Basic fibroblast growth factor (bFGF), nerve growth factor (NGF), neurite-inducing factor (NIF), ciliary neurotrophic factor (CNTF), and a cell-free extract from selected embryonic chick eye tissues (CIPE) were assayed for their capacity to control proliferation. Basic FGF, NGF, and CIPE stimulated proliferation of IMR 32 NB cells in serum-containing culture conditions. The NIF and CNTF had no effect. The concentration of bFGF required to induce half-maximal cell growth was 4.6 +/- 1.8 ng/ml, but the half-maximally effective dose of NGF was 7.5 +/- 2.7 ng/ml. In combination these two growth factors were additive within a small concentration range. In serum-free culture conditions bFGF affected both proliferation and cell differentiation by promoting neurite growth and cell aggregate formation. In contrast, NGF induced cell neurite outgrowth only. These results, in conjunction with the evidence that bFGF-like molecules are present, in IMR 32 NB cells may support the notion that NB cells regulate their proliferation by an autocrine mechanism. Basic FGF and NGF, two distinct neurotrophic factors, appear to be involved in the regulation of NB cell proliferation.     

In vivo and in vitro modulatory effect of human immunodeficiency virus type-1 (HIV-1) Tat protein on protein kinase C activity

Extracellular HIV-1 Tat protein shows a pleiotropic activity on the survival/proliferation of different cell types, which may be relevant to the pathogenesis of the immune suppression as well as of the frequent neoplastic disorders observed during the course of HIV-1 disease. Therefore, we investigated the effect of recombinant Tat on the protein kinase C (PKC) activity in Jurkat CD4(+) T lymphoma cells by using a serine substituted specific PKC peptide substrate, which allowed the evaluation of the whole catalytic activity of both Ca++-dependent and Ca++-independent PKC isoforms. High concentrations of recombinant Tat (1 mu g/ml) induced an early (5 min) stimulation followed by a secondary (30-60 min) inhibition of PKC in whole Jurkat cell homogenates. Immuno-localization experiments showed that recombinant Tat protein was rapidly taken up by Jurkat cells within the first 5 min from the addition in culture, thus suggesting the possibility that the secondary inhibitory phase of Tat on PKC activity in Jurkat cells could be due to a direct interaction between the two proteins. Consistently, PKC immunoprecipitated from Jurkat cells or purified from rat brain was significantly inhibited by the addition of high (0.1-1 mu g) but not low (1-10 ng) doses of Tat in a cell-free in vitro assay. The inhibition of PKC catalytic activity mediated by 1 mu g of Tat was at least partially due to competition among substrates. The present data may help in understanding the opposite effects on the survival/proliferation of different cell types observed in the presence of picomolar (stimulation) vs nanomolar (inhibition) concentrations of recombinant Tat.