Bcl-2 inhibits cell death of serum-free mouse embryo cells caused by epidermal growth factor deprivation

SFME cells are brain-derived neural precursor cells that are acutely dependent on epidermal growth factor (EGF) for survival, undergoing apoptosis within 24 h after EGF withdrawal. Because the expression of the protooncogene bcl-2 inhibits apoptosis induced by the withdrawal of interleukins or nerve growth factor in some growth factor-dependent haematopoietic or neuronal cell cultures, we examined the effect of Bcl-2 expression on cell death of SFME cells in the absence of EGF. SFME cells expressing human Bcl-2 showed prolonged survival when deprived of EGF compared to control cells not expressing Bcl-2. A significant fraction of Bcl-2-expressing cells remained viable for 4 days in the absence of EGF and resumed proliferation upon readdition of EGF to the cultures. These results suggest that apoptosis induced by EGF withdrawal in SFME cells may share common mechanisms with other growth factor-related apoptotic systems.     

Detection by the comet assay of apoptosis induced in lymphoid cell lines after growth factor deprivation

Dysregulation of apoptosis contributes to various diseases such as neurodegenerative or aging disorders, autoimmune syndromes or cancers. Numerous experimental paradigms have been explored to characterize molecular and cellular modulators of apoptosis. Similarly, numerous techniques have been described for detecting and/or quantifying accurately cells committed to apoptosis. Besides the conventional techniques, we describe in this report that the comet assay, which detects DNA single- and double-strand breaks in situ, at the cellular level, is relevant for the characterization of apoptotic cells. The comet assay is very sensitive and detects DNA fragmentation occurring in the apoptotic process as early as exposure of phosphatidylserine residues on the outer leaflet. Thus the comet assay can be used for the recognition of apoptosis that follows the death signal caused, for example, by genotoxic stress as well as lack of survival signal as in growth factor deprivation.     

CHOP is involved in neuronal apoptosis induced by neurotrophic factor deprivation

Neurotrophic factors are essential for the survival of neurons. We found that the endoplasmic reticulum (ER) stress-C/EBP homologues protein (CHOP) pathway to be activated during neurotrophic factor deprivation-induced apoptosis in PC12 neuronal cells and in primary cultured neurons, and this apoptosis was suppressed in the neurons from chop(-/-) mice. In addition, we found that CHOP is expressed in the subventricular zone (SVZ) and striatum of the young adult mouse brain. The number of apoptotic cells in the SVZ decreased in chop(-/-) mice. These results indicate that the ER stress-CHOP pathway plays a role in neuronal apoptosis during the development of the brain.     

R-Ras promotes apoptosis caused by growth factor deprivation via a Bcl- 2 suppressible mechanism

The Bcl-2 protein is an important regulator of programmed cell death, but the biochemical mechanism by which this protein prevents apoptosis remains enigmatic. Recently, Bcl-2 has been reported to physically interact with a member of the Ras superfamily of small GTPases, p23-R- Ras. To examine the functional significance of R-Ras for regulation of cell death pathways, the IL-3-dependent cells 32D.3 and FL5.12 were stably transfected with expression plasmids encoding an activated form (38 Glycine-->Valine) of R-Ras protein. R-Ras(38V)-producing 32D.3 and FL5.12 cells experienced increased rates of apoptotic cell death relative to control transfected cells when deprived of IL-3. Analysis of several independent clones of transfected 32D.3 cells revealed a correlation between higher levels of R-Ras protein and faster rates of cell death upon withdrawal of IL-3 from cultures. 32D.3 cells cotransfected with R-Ras(38V) and Bcl-2 exhibited prolonged cell survival in the absence of IL-3, equivalent to 32D.3 cells transfected with Bcl-2 expression plasmids alone. R-Ras(38V) also increased rates of cell death in serum-deprived NIH-3T3 cells, and Bcl-2 again abrogated most of this effect. The ratio of GTP and GDP bound to R- Ras(38V) was not significantly different in control 32D.3 cells vs those that overexpressed Bcl-2, indicating that Bcl-2 does not abrogate R-Ras-mediated effects on cell death by altering R-Ras GDP/GTP regulation. Moreover, purified Bcl-2 protein had no effect on the GTPase activity of recombinant wild-type R-Ras in vitro. When expressed in Sf9 cells using recombinant baculoviruses, R-Ras(38V) bound to and induced activation of Raf-1 kinase irrespective of whether Bcl-2 was coproduced in these cells, suggesting that Bcl-2 does not nullify R-Ras effects by interfering with R-Ras-mediated activation of Raf-1 kinase. Taken together, these findings suggest that R-Ras enhances the activity of a cell death pathway in growth factor-deprived cells and imply that Bcl-2 acts downstream of R-Ras to promote cell survival.     

Epidermal growth factor induced apoptosis

Apoptosis, or programmed cell death, plays an important role in the pathogenesis of a number of human diseases, including cancers, autoimmune diseases, and neurodegenerative disorders. Recent evidence suggests that EGF induced signal transduction pathways which govern cell proliferation and cell cycle progression also mediate antiproliferative effects leading to increased apoptosis in cells that express high levels of epidermal growth factor receptors. Treatments designed to increase apoptosis have potential to change the natural progression of cancer and eventually lead to its successful control.     

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.     

Hepatic growth factor (HGF) inhibits cigarette smoke extract induced apoptosis in human bronchial epithelial cells

Low concentrations of cigarette smoke induced DNA damage and repair without leading to apoptosis in human bronchial epithelial cells. Higher concentrations of cigarette smoke, however, could induce either apoptosis or necrosis. The current study demonstrated that 15% cigarette smoke extract (CSE) induced apoptosis as evidenced by DNA content profiling (17.8 ± 2.1% vs 10.2 ± 1.6% of control, p < 0.05), LIVE/DEAD staining (60.2 ± 2.1% viable cells in CSE-treated vs 86.5 ± 2.3% in control cells, p < 0.05), and COMET assay (24.3 ± 0.6% of Apoptotic Index in the cells treated with CSE vs 4.7 ± 0.6% of control, P < 0.05). Hepatocyte growth factor (HGF) significantly blocked the cigarette smoke-induced apoptosis as shown by DNA profiling (10.8 ± 1.5% of CSE + HGF, p < 0.05), LIVE/DEAD staining (78.5 ± 1.2% in CSE + HGF treated cells, p < 0.05), and COMET assay (Apoptotic Index: 10.0 ± 0.8% in CSE + HGF treated cells, P < 0.05). This protective effect of HGF on CSE-induced apoptosis was abolished by PI3K inhibitors, wortmannin and LY294002, and by introduction of the dominant negative AKT into the cells. Furthermore, CSE plus HGF could induce phosphorylation of AKT Thr 308 and the pro-apoptotic protein, BAD. These results suggest that HGF modulates cell survival in response to cigarette smoke exposure through the PI3K/AKT signaling pathway.     

Nonylphenol enhances apoptosis induced by serum deprivation in PC12 cells

Although nonylphenol is well known as an endocrine disrupting chemical, there is little information concerning biological effect of nonylphenol. In this study, we investigated effect of nonylphenol on apoptosis induced by serum deprivation in PC12 cells using TUNEL and DNA fragmentation assays. In addition, changes in contents of proapoptotic factors, Bad and Bax, and antiapoptotic factor, Bcl-2, and enzyme activity of caspase-3 were studied. Below 100 ng/ml of nonylphenol increased TUNEL signals, DNA fragmentation and content of proapoptotic factor, Bad as compared to those by serum deprivation without nonylphenol. Furthermore, addition of nonylphenol enhanced caspase-3 activity and Z-VAD, caspase-3 inhibitor, diminished such effect. These results indicated that below 100 ng/ml of nonylphenol enhanced apoptosis induced by serum deprivation via caspase-3 activation in PC12 cell.     

Vascular endothelial growth factor inhibits programmed cell death of endothelial cells induced by clinorotation

The principal aim of this study was to investigate short- and long-term effects of clinorotation on human endothelial cells (EA hy 926 cell line) using a three-dimensional random positioning machine. Moreover, the impact of vascular endothelial growth factor (VEGF) was addressed. Immediately, within one hour and after four and twenty-four hours an increase of apoptotic cells was detected. VEGF significantly inhibited the amount of apoptotic endothelial cells (EC). VEGF reduced the amount of fas-positive EC. Moreover, after 24 hours, proliferating EC grew in form of three-dimensional multicellular spheroids and also as monolayers. The initially formed spheroids (maximum diameter 3 mm) remained stable up to the 15th day of clinorotation. Some spheroids revealed tubular structures. In addition, a clear increase of extracellular matrix proteins such as osteopontin and fibronectin was measured. The three-dimensional clinostat represents an important tool for cell biological experiments. VEGF significantly attenuated the changes of endothelial cells induced by simulated weightlessness in a cell protective manner.     

Vascular endothelial growth factor inhibits maturation of dendritic cells induced by lipopolysaccharide,but not by proinflammatory cytokines

Purpose: Dendritic cells (DCs) play an important role in the hosts immunosurveillance against cancer. It has been shown that the function of DCs is impaired and their population decreased in a cancer-bearing host. In the present study, we investigated the mechanism of down-regulation of DCs in a cancer-bearing host. Methods: We evaluated the relationship between DC infiltration and production of vascular endothelial growth factor (VEGF) in carcinoma tissue by immunohistochemistry. Furthermore, functional and phenotypical alterations of DCs were evaluated when monocyte-derived, mature DCs were treated with VEGF in vitro. Monocyte-derived DCs were generated in a culture of monocyte with interleukin 4 (IL-4) and granulocyte-macrophage colony-stimulating factor, and the maturation of DCs was induced by either lipopolysaccharide (LPS) or a proinflammatory cytokine cocktail: tumor-necrosis factor , prostaglandin E₂, IL-6, and IL-1. Results: A significant inverse correlation was found between the density of DCs and the quantity of VEGF production in gastric carcinoma tissue (r=–0.39, p<0.05). In LPS-induced maturation, the ability of mature DCs to stimulate allogenic T cells and produce IL-12 (p70 heterodimer) was suppressed by the addition of VEGF in a dose-dependent manner. A lesser expression of costimulatory molecules (CD80 and CD86) was seen in DCs treated with exogenous VEGF than in DCs not treated with VEGF. The population of dead DCs (early and late apoptosis) treated with VEGF increased more than that without VEGF treatment, using the annexin V and propidium iodide evaluation in DCs matured by LPS. In contrast, in DCs matured by the proinflammatory cytokine cocktail, the down-regulation of costimulatory molecules and induction of DC apoptosis was not seen. Conclusions: These findings show that the inhibition of DC maturation by VEGF differs depending on the maturation status of the DCs.Abbreviations APC antigen-presenting cells - DC dendritic cells - ELISA enzyme-linked immunosorbent assay - FACS fluorescence-activated cell sorter - FCS fetal calf serum - FITC fluorescein isothiocyanate - GM-CSF granulocyte-macrophage colony-stimulating factor - HLA human leukocyte antigen - IL interleukin - LPS lipopolysaccharide - mAb monoclonal antibody - MHC major histocompatibility complex - PBS phosphate-buffered saline - PCNA proliferative cell nuclear antigen - PE phycoerythrin - PG prostaglandin - PI propidium iodide - TNF tumor-necrosis factor - VEGF vascular endothelial growth factor This work was supported by grants from the Ministry of Education, Culture, Sports, Science and Technology in Japan.     

Apoptosis of vascular endothelial cells by fibroblast growth factor deprivation

Survival and proliferation of many types of vascular endothelial cells are influenced by fibroblast growth factor (FGF)1. Removal of FGF from the medium of human umbilical vein endothelial cells (HUVEC) in culture resulted in death of the cells. Here we show that the death caused by deprivation of FGF is active death or apoptosis, and the process of apoptosis can be inhibited by cycloheximide, an inhibitor of protein synthesis. The present study shows apoptosis occurs in endothelial cells in culture. The process of active death of vascular endothelial cells is inhibited by growth factor. This mechanism may be important for the regulation of vascular organization through the degeneration of vessels.     

Hepatocyte growth factor inhibits apoptosis by the profibrotic factor angiotensin II via extracellular signal-regulated kinase 1/2 in endothelial cells and tissue explants

Hepatocyte growth factor (HGF), an endogenous tissue repair factor, attenuates apoptosis in many primary cell types, but the mechanism is not completely understood. Our laboratory demonstrated that angiotensin (Ang) II activates the intrinsic apoptotic pathway in primary endothelial cells (ECs) via reduction of the antiapoptotic protein Bcl-x(L). Ang II decreased Bcl-x(L) mRNA half-life by reducing its binding to nucleolin, a protein that normally binds a 3' AU-rich region and stabilizes Bcl-x(L) mRNA. We hypothesized HGF may block apoptosis induced by Ang II. We used primary EC and ex vivo cultures of rat lung tissue to investigate HGF inhibition of Ang II-induced apoptosis. Our data indicated HGF abrogated Ang II-induced apoptosis by inhibiting cytochrome c release, caspase-3 activation, and DNA fragmentation. RNA-immunoprecipitation experiments demonstrated that HGF stabilized Bcl-x(L) mRNA by increasing nucleolin binding to the 3'-untranslated region that was associated with cytoplasmic localization of nucleolin. Cytoplasmic localization of nucleolin and Bcl-x(L) mRNA stabilization required HGF activation of extracellular signal-regulated kinase (ERK)1/2, but not phosphatidylinositol 3-kinase. HGF also blocked Ang II-induced caspase-3 activation and lactate dehydrogenase release in tissue explants in an ERK-dependent manner.     

Reactive oxygen species mediates the apoptosis induced by transforming growth factor beta(2) in human lens epithelial cells

Transforming growth factor beta(2) (TGF-beta(2)), a growth regulator of human lens epithelial cells (HLECs), also regulates the death of these cells. Dose-response analysis showed that the TGF-beta(2) concentration needed to induce HLECs death (100 pg/ml) was 10 times that needed to inhibit growth in these cells (10 pg/ml). TGF-beta(2)-induced apoptosis in HLECs was preceded by an induction of reactive oxygen species (ROS) and a decrease in glutathione in the intracellular content, indicating that this factor induces oxidative stress in HLECs. Studies performed to analyze the levels of c-fos mRNA, a gene whose expression is modulated by the redox state, demonstrated that only high, apoptotic concentrations of TGF-beta(2) (100 pg/ml) produced an increase in the mRNA levels of this gene, the level of induction being similar to that found when cells were incubated in the presence of hydrogen peroxide. Finally, the cell death induced by TGF-beta(2) in HLECs was partially blocked by radical scavengers, which decreased the percentage of apoptotic cells, whereas these agents did not modify the growth-inhibitory effect elicited by TGF-beta(2) in these cells. The results presented in this paper provide evidence for the involvement of an oxidative process in the apoptosis elicited by TGF-beta(2) in HLECs.     

Maltol inhibits apoptosis of human neuroblastoma cells induced by hydrogen peroxide

To analyze the effect of Maltol on the apoptosis of Human Neuroblastoma Cells (SH-SY5Y) treated by free radical which was generated from Hydrogen Peroxide (H2O2), flow cytometry analysis on Phosphatidylserine (PS) inverting percentage was applied to determine the apoptosis. MTT (3-(4,5-dimethythiazol-2-yl)-2,5-diphenyl-tetrazolium bromide) assay was employed to analyze the cell viability. DNA electrophoresis was used to detect DNA fragmentation. Moreover intracellular calcium of concentration ([Ca2+]i) was measured by fluorescence emission. Flow cytometry analysis on the function of mitochondria and Western blot analysis of NF-kappaB. The results showed that the pretreatment with maltol for 2 hours could prevent the H2O2-induced apoptosis. Maltol could reduce the inverting percentage of PS, DNA fragmentation and [Ca2+]i, and enhance the cellular function of mitochondria. NF-kappaB activated by H2O2 is reduced. The experiments suggest that maltol could effectively inhibit the apoptosis induced by H2O2. As a novel anti-oxidant, maltol is a new promising drug in protecting the neurological cells from the damage by free radical.     

GRP75经由Raf/Mek/Erk1/2信号转导通路抑制缺糖诱导的细胞凋亡

本文旨在探讨促存活信号通路Raf/Mek/Erk1/2是否参与了葡萄糖调节蛋白75(glucose-regulated protein75,GRP75)对缺糖诱导的细胞凋亡的抑制作用。GRP75过表达的PC12细胞给予Raf/Mek/Erk1/2通路抑制剂U0126预处理之后,无糖培养6、12和24h,同时以DMSO预处理的GRP75过表达PC12细胞组为对照。Western blot检测Erk1/2的磷酸化和表达水平,MTT实验检测细胞存活率,Hoechst 33258染色观察凋亡细胞核的形态学改变,流式细胞仪检测细胞亚二倍体峰,免疫荧光检测细胞色素c(cytochrome c,Cytc)向胞浆的弥散情况。结果显示:U0126在没有影响Erk1/2表达水平的前提下,阻断了GRP75对Erk1/2磷酸化水平的维持;U0126处理组的凋亡率明显高于对照组;U0126处理组Cytc从线粒体向胞浆释放的时间明显早于对照组,同时Cytc向胞浆的弥散程度大于对照组。以上结果提示,U0126通过抑制Erk1/2磷酸化,阻断了缺糖状态下GRP75对Cytc释放和细胞凋亡的抑制作用,这表明GRP75是通过Raf/Mek/Er...     

Differing sensitivity of tumor cells to apoptosis induced by iron deprivation in vitro

We studied the sensitivity of tumor cells to the induction of apoptosis by iron deprivation. Iron deprivation was achieved by the employment of a defined iron-deficient culture medium. Mouse 38C13 cells and human Raji cells die within 48 and 96 h of incubation in iron-deficient medium, respectively. On the contrary, mouse EL4 cells and human HeLa cells are completely resistant to the induction of death under the same experimental arrangement. Deoxyribonucleic acid fragmentation analysis by agarose gel electrophoresis as well as flow cytometric analysis after propidium iodide staining detected in 38C13 and Raji cells, but not in EL4 and HeLa cells, changes characteristic to apoptosis. The 38C13 cells, sensitive to iron deprivation, also displayed a similar degree of sensitivity to apoptosis induction by thiol deprivation (achieved by 2-mercaptoethanol withdrawal from the culture medium) as well as by rotenone (50 nM), hydroxyurea (50 microM), methotrexate (20 nM), and doxorubicin (100 nM). Raji cells shared with 38C13 cells a sensitivity to rotenone, methotrexate, doxorubicin, and, to a certain degree, to hydroxyurea. However, Raji cells were completely resistant to thiol deprivation. EI4 and HeLa cells, resistant to iron deprivation, also displayed a greater degree of resistance to most of the other apoptotic stimuli than did their sensitive counterparts. We conclude that some tumor cells in vitro are sensitive to apoptosis induction by iron deprivation, while other tumor cells are resistant. All the tumors found to be sensitive to iron deprivation in this study (four cell lines) are of hematopoietic origin. The mechanism of resistance to apoptosis induction by iron deprivation differs from the mechanism of resistance to thiol deprivation.     

Effect of neural stem cells on apoptosis of PC12 cells induced by serum deprivation

Neural stem cells (NSCs) have a bright application prospect to be used to treat neurodegenerative diseases due to their capacity to give rise to the appropriate cell types when they are grafted. At present, however, the function of NSCs after transplantation is not quite ensured, whether to replace the degenerative cells or to secrete nutrient factors. On the other hand, pheochromocytoma cell line 12 (PC12) cells have been widely used for investigating Parkinson's disease (PD) since their apoptosis is similar to that of dopaminergic neuron cells. Therefore, the possible cytoprotective effects of NSCs on the apoptosis of PC12 cells induced by serum deprivation were investigated in this paper. PC12 cells were cocultured with NSCs in DMEM/F12 medium free of serum, and their morphologies, viabilities, and survival were observed with an inverted microscope and assessed with a CCK-8 assay. In addition, the concentrations of glial derived neurotrophic factor (GDNF) in different medium were detected with a GDNF Elisa kit, and the mechanism of NSC's protective effect on PC12 cell apoptosis induced by serum deprivation was analyzed. The results showed that (1) PC12 cell apoptosis induced by serum deprivation increased with time, and only about 44.25% PC12 cells survived after 72 h; (2) NSCs culture medium protected against PC12 cell apoptosis insignificantly; (3) NSCs' supernatant and NSCs mildly prevented PC12 cells from apoptosis; (4) the amount of GDNF secreted by NSCs increased after the coculture with the apoptotic PC12 cells induced by serum deprivation. It can be concluded that there exists clear interaction between NSCs and apoptotic PC12 cells, and that GDNF secretion from NSCs is one of the important mechanisms to prevent the apoptosis of PC12 cells.     

Thymidine phosphorylase inhibits apoptosis induced by cisplatin

An angiogenic factor, platelet-derived endothelial cell growth factor/thymidine phosphorylase (PD-ECGF/TP), stimulates the chemotaxis of endothelial cells and confers resistance to apoptosis induced by hypoxia. 2-Deoxy-D-ribose, a degradation product of thymidine generated by TP, partially prevents hypoxia-induced apoptosis. TP is expressed at higher levels in tumor tissues compared to the adjacent non-neoplastic tissues in a variety of human carcinomas. High expression of TP is associated with an unfavorable prognosis. To investigate the effect of TP on cisplatin-induced apoptosis, human leukemia Jurkat cells were transfected with wild-type or mutant (L148R) TP cDNA. TP inhibited a number of steps in the cisplatin-induced apoptotic pathway, activation of caspases 3 and 9 and mitochondrial cytochrome c release. These findings suggest a mechanism by which TP confers resistance to apoptosis induced by cisplatin. Moreover, mutant TP that has no enzymatic activity also suppressed cisplatin-induced apoptosis. These findings indicate that TP has cytoprotective functions against cytotoxic agents which are independent of its enzymatic activity.