Bcl-2 protects neuronal cells against taxol-induced apoptosis by inducing multi-nucleation

Taxol-induced peripheral neuropathy is a commonly-occurring side-effect in the treatment of cancer patients with taxoteres or taxanes. Taxol is known to induce apoptosis in a number of tumor cells. This report documents that, similar to proliferating cells, taxol induces apoptosis in NGF-differentiated PC12 cells, as assessed by exogenous FITC-annexin-V binding and nuclear fragmentation. It is shown that PC12 cells that stably overexpress Bcl-2 are protected against the toxic effect of taxol, as evidenced by the XTT assay and by a decreased fraction of propididum iodide positive cells in a dye exclusion test. Also the number of annexin-V-positive cells and the number of fragmented nuclei are lower in the Bcl-2 transfected cells. The effect is similar to the protective effect of Bcl-2 against NGF deprivation in differentiated PC12 cells. Although taxol forced both wild-type and Bcl-2-overexpressing cells into a mitotic state, only in Bcl-2-overexpressing cells did this lead to the appearance of metabolically active, multi-nucleated cells. This suggests that Bcl-2 is able to induce an alternative escape pathway, downstream of the G2/M block, in taxol-treated differentiated PC12 cells.     

A cautionary note on the use of stable transformed cells

Gene transfection and ectopic expression is a widely used method in experimental biology. In the present report, we would like to point out that this approach may, in certain circumstances, lead to a modification of the transfected cell phenotype. Indeed, we observed that after transfection of bcl-2 gene in the neuronal PC12 cell line some of the selected clones have lost their neuronal and catecholaminergic characteristics, i.e. TH expression and ability to grow neurites in response to NGF. Thus, the resistance of some PC12-Bcl-2 clones against neurotoxic insults may not necessarily reflect the potential benefit afforded by Bcl-2 expression. We therefore encouraged authors to verify cell phenotype after stable transfection to avoid misinterpretation of their results.     

依达拉奉对硝普钠诱导的PC12细胞凋亡的保护作用

目的：探讨依达拉奉对硝普钠诱导的PCI2细胞凋亡的影响。方法：体外培养PCI2细胞,并分为依达拉奉对硝普钠保护组（含500μmol／L硝普钠和75μmol／L依达拉奉）、硝普钠诱导组（含500μmol／L硝普钠）和对照组。采用MTT法检测细胞的增殖率：流式细胞术检测细胞的凋亡情况;Western-blot检i受4凋亡抑制蛋白Bcl-2和凋亡促进蛋白Bad的表达。结果：与对照组相比,硝普钠处理的PCI2细胞增殖率显著降低,而细胞凋亡率显著升高,细胞内Bcl-2的表达显著减少,而Bad的表显著增加,差异均具有统计学意义（P〈0．05）;与单纯硝普钠诱导组相比,依达拉奉处理组细的胞增殖率显著增加而细胞凋亡率显著减少,同时Bcl－2的表达显著增加,而Bad的表达明显减少,差异均具有统计学意义（P〈0．05）。结论：依达拉奉对硝普钠诱导的PCI2细胞凋亡具有抑制作用,可能通过增加Bcl-2的表达并降低Bad的表达发挥抗凋亡作用。     

τ in Paired Helical Filaments Is Functionally Distinct from Fetal τ: Assembly Incompetence of Paired Helical Filament-τ

Abstract: During development, many neuronal populations undergo a process of normal, programmed cell death, or apoptosis. Trophic factors regulate this process, but the mechanism by which they suppress apoptosis remains unclear. In the immune system, recent studies have implicated the protooncogene bcl-2 in the lymphocyte survival response to growth factors. To determine whether a similar survival pathway exists in a neuroendocrine cell type, we have expressed bcl-2 in the rat pheochromocytoma PC12 cell line and found that it abrogates the requirement for stimulation by growth factors to survive. bcl-2 expression also substantially delays the onset of injury by the calcium ionophore A23187.     

Gene dosage-dependent effects of bcl-2 expression on cellular survival and redox status

The human oncogene bcl-2 exerts protective functions in numerous models of apoptotic cell death and increased oxidative stress. We investigated the effects of inducible bcl-2 overexpression on cellular survival and redox status in dopaminergic rat pheochromocytoma PC 12 cells. Induction of high-level expression of bcl-2 in PC 12 cells resulted in generation of oxidative stress and cessation of growth by cell cycle arrest. Cell cycle arrest in bcl-2-overexpressing PC 12 cells was prevented by an inhibitor of extracellular signal-related kinase (ERK 1/2) activation. Protective effects of bcl-2 expression against L-DOPA neurotoxicity decreased with increasing amounts of bcl-2. Furthermore, high-level bcl-2 overexpression sensitized cells towards oxidative stress and glutathione depletion. Our data suggest that bcl-2 expression is beneficial only in a limited gene dosage range and that high-level expression of bcl-2 exerts potential deleterious effects.     

Prostaglandin E1 transported into cells blocks the apoptotic signals induced by nerve growth factor deprivation

Neuronal apoptosis in rat pheochromocytoma PC12 cells, which was confirmed by TUNEL (terminal transferase-mediated dUTP-biotin nick end-labeling) staining and detection of chromatin condensation, appeared within 8 h after nerve growth factor (NGF) deprivation. Prostaglandin (PG) E1 (10(-7)-10(6) M) reduced the incidence of apoptotic cell death in PC12 cells. The genes encoding PG transporter specific to prostaglandins such as PGE2 or PGF2alpha were expressed in the cell lines as shown by RT-PCR. Bromcresol green, an inhibitor of PG transporter, reversed the antiapoptotic effect of PGE1. Moreover, treatment of PC12 cells with an antisense oligonucleotide corresponding to PG transporter cDNA also blocked the inhibitory effects of PGE1 on apoptotic cell death. In addition, PGE1 counteracted the increased activities of stress-activated protein kinase/cJun N-terminal kinase within 1-2 h after NGF deprivation in PC12 cells. These results indicated that the antiapoptotic effect of PGE1 in NGF-deprived PC12 cells was achieved by inhibitory signals following uptake into neurons through the PG transporter.     

Caspase activation independent of cell death is required for proper cell dispersal and correct morphology in PC12 cells

Caspase activation is indispensable for the proper execution of apoptosis. However, to date, little is known about other possible physiologic functions for this class of enzymes in addition to their well-defined role in apoptosis. In this report, we described an action of caspase-3 involving cell dispersion that is independent of cell death. Using an in vitro neuronal model system consisting of PC12 cells, we observed a transient activation of caspase-3 both in situ and by Western blot analysis that was evident at 1 h following plating, was maximal by 3 h, and was attenuated by 24 h. Preincubation of PC12 cells with either the caspase-3 inhibitor, DEVD, or antisense caspase-3 oligonucleotides caused cells to be more rounded in appearance and led to a failure of cells to disperse properly. Additional experiments demonstrated a possible target for caspase cleavage to be the cytoskeletal protein, tau. These data suggest a requirement for caspase activation and subsequent disassembly of the cytoskeleton during cell dispersion and represent a novel role for caspases that may allow for proper migration of neurons to target locations during development.     

Nitric oxide-induced carbonylation of Bcl-2, GAPDH and ANT precedes apoptotic events in insulin-secreting RINm5F cells

Generation of high levels of nitric oxide (NO) following induction of NOS2 by interleukin-1 beta (IL-1beta) triggers beta cell apoptosis in insulin-secreting RINm5F cells. Mitochondrial and nuclear events such as downregulation of the antiapoptotic protein Bcl-2, activation of the pore responsible for the permeability transition (PT) and DNA fragmentation are involved in the process. We report in the present paper that exposure of insulin-producing RINm5F cells to NO donors and to IL-1beta leads to oxidative carbonylation of both Bcl-2 and the adenine nucleotide translocator (ANT) component of the mitochondrial PT pore. When the effect of endogenous generation of high concentrations of NO following exposure of cells to IL-1beta was studied, carbonylation of Bcl-2 preceded downregulation of the protein. Overexpression of Mn-SOD decreases substantially the extent of Bcl-2 carbonylation in SIN-1-exposed cells. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) inhibition, carbonylation and translocation from cytoplasm to nucleus and DNA fragmentation were also induced by DETA/NO exposure. DETA/NO-induced carbonylation of Bcl-2 and ANT proteins takes place 6 h before apoptotic release of histone-associated DNA to cytoplasm. Time course studies also reveal a close parallel between GAPDH translocation to nucleus and carbonylation. Inhibitors of lipooxidation end products formation such as piridoxamine (PM) and aminoguanidine (AG) block NO-triggered carbonylation of Bcl-2, ANT and GAPDH, prevent NO-induced GAPDH enzyme inhibition and nuclear translocation and DNA fragmentation. Our results support the notion that the oxidative carbonylation of proteins plays a role in the control of NO-induced apoptosis.     

Caspase-3 activation is an early event and initiates apoptotic damage in a human leukemia cell line

Many apoptotic pathways culminate in the activation of caspase cascades usually triggered by the apical caspases-8 or -9. We describe a paradigm where apoptosis is initiated by the effector caspase-3. Diethylmaleate (DEM)-induced apoptotic damage in Jurkat cells was blocked by the anti-apoptotic protein Bcl-2, whereas, a peptide inhibitor of caspase-3 but not caspase-9 blocked DEM-induced mitochondrial damage. Isogenic Jurkat cell lines deficient for caspase-8 or the adaptor FADD (Fas associated death domain) were not protected from DEM-induced apoptosis. Caspase-3 activation preceded that of caspase-9 and initial processing of caspase-3 was regulated independent of caspase-9 and Bcl-2. However, inhibitors of caspase-9 or caspase-6 regulated caspase-3 later in the pathway. We explored the mechanism by which caspase-3 processing is regulated in this system. DEM triggered a loss of Erk-1/2 phosphorylation and XIAP (X-linked inhibitor of apoptosis protein) expression. The phorbol ester PMA activated a MEK-dependent pathway to block caspase-3 processing and cell death. Constitutively active MEK-1 (CA-MEK) upregulated XIAP expression and exogenous XIAP inhibited DEM-induced apoptotic damage. Thus, we describe a pathway where caspase-3 functions to initiate apoptotic damage and caspase-9 and caspase-6 amplify the apoptotic cascade. Further, we show that MEK may regulate caspase-3 activation via the regulation of XIAP expression in these cells.     

Compromised proteasome degradation elevates neuronal nitric oxide synthase levels and induces apoptotic cell death

The significance of impairment of proteasome activity in PC12 cells was examined in connection with nitrative/nitrosative stress and apoptotic cell death. Treatment of differentiated PC12 cells with MG132, a proteasome inhibitor, elicited a dose- and time-dependent increase in neuronal nitric oxide synthase (nNOS) protein levels, decreased cell viability, and increased cytotoxicity. Viability and cytotoxicity were ameliorated by L-NAME (a broad NOS inhibitor). Nitric oxide/peroxynitrite formation was increased upon treatment of PC12 cells with MG132 and decreased upon treatment with the combination of MG132 and 7-NI (a specific inhibitor of nNOS). The decreases in cell viability appeared to be effected by an activation of JNK and its effect on mitochondrial Bcl-x_L phosphorylation. These effects are strengthened by the activation of caspase-9 along with increased caspase-3 activity upon treatment of PC12 cells with MG132. These results suggest that impairment of proteasome activity and consequent increases in nNOS levels lead to a nitrative stress that involves the coordinated response of JNK cytosolic signaling and mitochondrion-driven apoptotic pathways.     

Molecular mechanisms of programmed cell death

Programmed cell death is currently under active investigation. A recent meeting focused on the molecular machinery of programmed cell death and on its role in the pathogenesis of human diseases.     

细胞色素c的释放是多巴胺诱导PC12细胞凋亡的重要环节

通过末端脱氧核苷酸转移酶介导dUTP缺口翻译法和DNA凝胶电泳观察多巴胺(DA)对PC12细胞凋亡的诱导作用, 并经蛋白质印迹法检测胞浆细胞色素c、Bcl-2和Bax蛋白以及活化型半胱氨酸蛋白酶3(caspase-3)水平. 结果表明, 在DA诱导PC12细胞凋亡的过程中, 可见PC12细胞中活化型caspase-3蛋白表达, 胞浆中细胞色素c水平明显增高, 同时Bcl-2蛋白水平下降, 而Bax蛋白水平明显增加. 环孢菌素A预处理对细胞色素c释放和caspase-3激活有明显的抑制作用, 而对Bcl-2和Bax蛋白影响不明显. 结果提示, Bcl-2和Bax蛋白、细胞色素c以及caspase-3可能参与DA诱导PC12细胞凋亡, 线粒体细胞色素c向胞浆释放可能是其中的中心环节.     

Neuroprotective effects of tetramethylpyrazine on hydrogen peroxide-induced apoptosis in PC12 cells

In the present study, we investigated the effects of tetramethylpyrazine (TMP) on hydrogen peroxide (H2O2)-induced apoptosis in PC12 cells. The apoptosis in H2O2-induced PC12 cells was accompanied by a decrease in Bcl-2/Bax protein ratio, release of cytochrome c to cytosol and the activation of caspase-3. TMP not only suppressed the down-regulation of Bcl-2, up-regulation of Bax and the release of mitochondrial cytochrome c to cytosol, but also attenuated caspase-3 activation and eventually protected against H2O2-induced apoptosis. These results indicated that TMP blocked H2O2-induced apoptosis by the regulation of Bcl-2 family members, suppression of cytochrome c release, and caspase cascade activation in PC12 cells.     

Muscarinic activation of mitogen-activated protein kinase in PC12 cells

Muscarinic acetylcholine receptors (mAChRs) activate many downstream signaling pathways, some of which can lead to mitogen-activated protein kinase (MAPK) phosphorylation and activation. MAPKs play roles in regulating cell growth, differentiation, and synaptic plasticity. Here, the activation of MAPK was examined in PC12 cells endogenously expressing mAChRs. Western blot analysis using a phosphospecific MAPK antibody revealed a dose-dependent and atropine-sensitive increase in MAPK phosphorylation in cells stimulated with carbachol (CCh). The maximal response occurred after 5 min and was rapidly reduced to baseline. To investigate the receptors responsible for CCh activation of MAPK in PC12 cells, the mAChR subtypes present were determined using RT-PCR and immunoprecipitation. RT-PCR was used to amplify fragments of the appropriate sizes for m1, m4, and m5, and the identities of the bands were confirmed with restriction digests. Immunoprecipitation using subtype-specific antibodies showed that approximately 95% of the expressed receptors were m4, whereas the remaining approximately 5% were m1 and m5. A highly specific m1 toxin completely blocked MAPK phosphorylation in response to CCh stimulation. The mAChR-induced MAPK activation was abolished by protein kinase C down-regulation and partially inhibited by pertussis toxin. Although m1 represents a small proportion of the total mAChR population, pharmacological evidence suggests that m1 is responsible for MAPK activation in PC12 cells.     

Inhibition by epigallocatechin gallate of CoCl2-induced apoptosis in rat PC12 cells

Epigallocatechin-3-gallate (EGCG) is a major constituent of green tea polyphenols. This study was aimed to investigate the possible mechanisms of EGCG-mediated inhibition against apoptosis in rat pheochromocytoma PC12 cells by exposure to CoCl(2). Exposure to CoCl(2) caused the generation of ROS and induced cell death with appearance of apoptotic morphology and DNA fragmentation. However, EGCG rescued the loss of viability in the cells exposed to CoCl(2) and led the reduction of DNA fragmentation and sub-G(1) fraction of cell cycle. Also, EGCG attenuated the CoCl(2)-induced disruption of mitochondrial membrane potential (DeltaPsim), release of cytochrome c from the mitochondria to cytosol and abolished the CoCl(2)-stimulated activities of the caspase cascades, caspase-9 and caspase-3. In addition, EGCG ameliorated the increase in the Bax to Bcl-2 ratio, a marker of apoptosis proceeding, induced by CoCl(2) treatment. Taken together, the present results suggest that EGCG inhibit the CoCl(2)-induced apoptosis of PC12 cells through the mitochondria-mediated apoptosis pathway involved in modulating the Bcl-2 family.     

Propofol protects against hydrogen peroxide-induced injury in cardiac H9c2 cells via Akt activation and Bcl-2 up-regulation

Propofol is a widely used intravenous anesthetic agent with antioxidant properties secondary to its phenol based chemical structure. Treatment with propofol has been found to attenuate oxidative stress and prevent ischemia/reperfusion injury in rat heart. Here, we report that propofol protects cardiac H9c2 cells from hydrogen peroxide (H₂O₂)-induced injury by triggering the activation of Akt and a parallel up-regulation of Bcl-2. We show that pretreatment with propofol significantly protects against H₂O₂-induced injury. We further demonstrate that propofol activates the PI3K-Akt signaling pathway. The protective effect of propofol on H₂O₂-induced injury is reversed by PI3K inhibitor wortmannin, which effectively suppresses propofol-induced activation of Akt, up-regulation of Bcl-2, and protection from apoptosis. Collectively, our results reveal a new mechanism by which propofol inhibits H₂O₂-induced injury in cardiac H9c2 cells, supporting a potential application of propofol as a preemptive cardioprotectant in clinical settings such as coronary bypass surgery.     

Enhancing Effect of Manganese on L-DOPA-Induced Apoptosis in PC12 Cells

L-DOPA and manganese both induce oxidative stress-mediated apoptosis in catecholaminergic PC12 cells. In this study, exposure of PC12 cells to 0.2 mM MnCl2 or 10-20 microM L-DOPA neither affected cell viability, determined by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, nor induced apoptosis, tested by flow cytometry, fluorescence microscopy, and the TUNEL technique. L-DOPA (50 microM) induced decreases in both cell viability and apoptosis. When 0.2 mM MnCl2 was associated with 10, 20, or 50 microM L-DOPA, a concentration-dependent decrease in cell viability was observed. Apoptotic cell death also occurred. In addition, manganese inhibited L-DOPA effects on dopamine (DA) metabolism (i.e., increases in DA and its acidic metabolite levels in both cell lysate and incubation medium). The antioxidant N-acetyl-L-cysteine significantly inhibited decreases in cell viability, apoptosis, and changes in DA metabolism induced by the manganese association with L-DOPA. An increase in autoxidation of L-DOPA and of newly formed DA is suggested as a mechanism of manganese action. These data show that agents that induce oxidative stress-mediated apoptosis in catecholaminergic cells may act synergistically.