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1.
Programmed cell death (PCD) or apoptosis is a common form of cellular demise during embryogenesis, tumorigenesis and clonal selection in the immune system. The bcl-2 proto-oncogene has been recently implicated as a potential physiological regulator of the PCD pathway. Gene transfer studies have shown that overexpression of bcl-2 blocks apoptosis mediated by several stimuli in cultured cell lines and promotes the survival of B and T lymphocytes in transgenic mice. However, it remains unclear whether under normal conditions bcl-2 is responsible for controlling cell death. We have investigated the role of bcl-2 in the antimembrane IgM (mIgM)-induced apoptotic death of WEHI-231 B cell lymphoma, a model that mimics clonal deletion of immature B cells by antigen. Signalling of mIgM receptors triggered downregulation of both bcl-2 RNA and protein, and induced apoptosis in WEHI-231 B cells. This effect appeared to be specific since (i) the levels of beta 2-microglobulin and beta-actin RNA remain unchanged and (ii) signalling of the apoptosis-resistant B cell lymphoma line BAL-17 with anti-mu was not associated with downregulation of bcl-2 RNA. However, stable expression of bcl-2 by transfection did not rescue WEHI-231 B cells from apoptosis, yet WEHI-231 cells overexpressing bcl-2 were more resistant to programmed cell death induced by heat-shock.(ABSTRACT TRUNCATED AT 250 WORDS)  相似文献   

2.
It has been demonstrated that the cell lines used for production of biopharmaceuticals are highly susceptible to apoptosis, and that over-expression of the bcl-2 oncogene can protect cells from death. Stress associated with the deprivation of nutrients has been shown to be the main cause of apoptosis in culture. We have extended these studies by investigating the mechanism of cell death under conditions of sub-optimal pH, shear stress and hyperosmolarity, and the protective action of bcl-2 over-expression. At pH 6, there was no clear evidence of protection from cell death. However, at pH 8, the viability of the bcl-2 transfected cells was about 20% higher relative to the control cells. Cultivation of control cells in a flat bottomed bioreactor with a magnetic stirrer bar without a pivot ring resulted in exposure of the cells to a high attrition effect. As a result, cell growth was retarded and a high level of cell death by apoptosis was observed. Under the same conditions, the bcl-2 transfected cell line exhibited a nearly five fold increase in viable cell number. This finding indicates that under apoptosis-suppressed conditions, shear stress can stimulate cell growth. Batch cultivation of both control and bcl-2 transfected cells in 350 and 400 mOsm media resulted in suppression of cell growth, athough the effect was most marked in the control cell line. Adaptation of control cells to 400 mOsm proved to be impossible to achieve. However, the bcl-2 transfected cells exhibited resistance to the osmotic stress resulting in long term adaptation to a high salt environment. Specific productivity of bcl-2 transfected cells grown in high osmolarity medium was 100% higher than that produced by non- adapted bcl-2 transfected cells grown in normal osmolarity medium. These results demonstrate that bcl-2 has a beneficial effect on hybridoma cultivation under a wide range of culture stresses. This revised version was published online in August 2006 with corrections to the Cover Date.  相似文献   

3.
Cell lines derived from the hemopoetic lineages are widely used as hosts for the production of biologicals. These cell lines have been demonstrated to undergo high levels of the active death program commonly referred to as apoptosis. The effects of overexpression of the apoptosis suppressor gene bcl-2 on the properties of a Burkitt lymphoma were compared with the control cell line (transfected with a negative control plasmid) under a variety of conditions relevant to cell culture production technology. In stationary batch cultures, there was a clear reduction in both the rate of total cell death and the level of apoptosis during the decline phase of the bcl-2 transfected cell cultures as compared with that of the control cell cultures. Nutrient analysis revealed that the onset of death during the control cell cultures occurred following complete exhaustion of glutamine. However, the bcl-2 transfected cell cultures continued to grow even though glutamine had been exhausted, and a significant decline in viability only occurred when glucose had also been completely exhausted.When cells were cultured in suspension without prior adaptation, the bcl-2 transfected cells grew significantly better, suggesting that the bcl-2 gene protected the cells from apoptosis triggered by either the lack of substrate or the hydrodynamic environment. Fluorescence microscopy revealed that death of the control cells was almost entirely by apoptosis, whereas death was almost exclusively by necrosis in the delayed decline phase of the transfected cell cultures. In both instances, death occurred before total exhaustion of glucose and glutamine.The induction of apoptosis following growth arrest is a major impediment to the development of culture strategies that optimize specific productivity by reducing the growth rate. Results presented here suggest that suppression of apoptosis by bcl-2 under the condition of excess thymidine allows the maintenance of cells in a growth-arrested state for much longer than would otherwise be possible.When cells were transferred to a range of commercial serum-free media, cell growth was, in all cases, much better for the bcl-2 transfected cell line. Moreover, when cells were cultivated in glutamine-free medium, the control cells exhibited a decrease in viable cell number within the first 24 h whereas, for the bcl-2 transfected cell cultures, viable cell number did not exhibit any clear decrease until after 75 h. Clearly, these results indicate that the metabolic engineering approach can be used to alter advantageously the survival and proliferative capacity of cells in cell culture environments. (c) 1996 John Wiley & Sons, Inc.  相似文献   

4.
Vitamin K2 (menaquinone-4: VK2) is a potent inducer for apoptosis in leukemia cells in vitro. HL-60bcl-2 cells, which are derived from a stable transfectant clone of the human bcl-2 gene into the HL-60 leukemia cell line, show 5-fold greater expression of the Bcl-2 protein compared with HL-60neo cells, a control clone transfected with vector alone. VK2 induces apoptosis in HL-60neo cells, whereas HL-60bcl-2 cells are resistant to apoptosis induction by VK2 but show inhibition of cell growth along with an increase of cytoplasmic vacuoles during exposure to VK2. Electron microscopy revealed formation of autophagosomes and autolysosomes in HL-60bcl-2 cells after exposure to VK2. An increase of acid vesicular organelles (AVOs) detected by acridine orange staining for lysosomes as well as conversion of LC3B-I into LC3B-II by immunoblotting and an increased punctuated pattern of cytoplasmic LC3B by fluorescent immunostaining all supported induction of enhanced autophagy in response to VK2 in HL-60bcl-2 cells. However, during shorter exposure to VK2, the formation of autophagosomes was also prominent in HL-60neo cells although nuclear chromatin condensations and nuclear fragments were also observed at the same time. These findings indicated the mixed morphologic features of apoptosis and autophagy. Inhibition of autophagy by either addition of 3-methyladenine, siRNA for Atg7, or Tet-off Atg5 system all resulted in attenuation of VK2-incuded cell death, indicating autophagy-mediated cell death in response to VK2. These data demonstrate that autophagy and apoptosis can be simultaneously induced by VK2. However, autophagy becomes prominent when the cells are protected from rapid apoptotic death by a high expression level of Bcl-2.  相似文献   

5.
6.
The bcl-2 gene has a unique function among mammalian oncogenes as a negative regulator of apoptosis. Its expression pattern in embryonic and adult tissues is consistent with a role in maintaining in vivo survival of specific cell types. The biochemical function of bcl-2 is unknown, but its localization to mitochondrial and microsomal membranes suggests several possibilities, bcl-2 is protective against oxidative stress in mammalian cells and can be replaced by antioxidants in a factor-deprivation model of apoptosis. These results are consistent with a model of apoptotic death involving oxidative stress in a central pathway. The recent discovery of several bcl-2-related genes, some of which also inhibit apoptosis and others that unexpectedly promote apoptosis, has shed new light on several aspects of bcl-2 action. © 1996 Wiley-Liss, Inc.  相似文献   

7.
TB/C3 hybridoma cells were transected with either pEF-MClneopA or pEF bcl2-MClneopA vectors to produce a control cell line (TB/C3 pEF) and a cell line that overexpresses the "antiapoptotic" human bcl-2 protein (TB/C3 bcl2). Flow cytometry analysis of intracellular bcl-2 protein levels enabled near on-line monitoring of the stability of bcl-2 expression in the absence of drug selection. It was possible to maintain spontaneous selection of cells with the overexpression of bcl-2 protein during semicontinuous cultures at very low dilution rates, where cells were subjected to the selective conditions of nutrient limitation and high toxic metabolite concentrations. Interestingly, cells that overexpressed bcl-2 were adapted to suspension culture conditions significantly faster than control cells. Dual fluorescence staining with acridine orange and propidium iodide allowed for discrimination between viable, apoptotic, secondary necrotic, and necrotic cells, respectively. Compared with the usual trypan blue method of establishing culture viability, dual staining demonstrated that under stressful conditions a significant proportion of cells that excluded trypan blue were also undergoing cell death through apoptosis. In batch cultures the overexpression of bcl-2 more than doubled the membrane intact (MI) cell productive period (the integral of Ml cell density with respect to culture time) and increased the monoclonal antibody (mAb) production by approximately 40% when compared with the control cell line. The overexpression of bcl-2 protein also significantly extended the cell integrity and viability by the suppression of apoptosis in conditions of hypoxia, hyperoxia, glutamine deprivation, glucose deprivation, and serum limitation. The suppression of apoptosis in anaerobic conditions suggests that bcl-2 exerts its antiapoptotic activity by a mechanism that does not involve an oxidative reactive pathway. In conditions of excess thymidine, which suppressed cell proliferation, Ml cell density and specific mAb productivity were further enhanced by the overexpression of bcl-2, which suggests the possibility of accomplishing a controlled proliferation in immortalized cell lines without invoking cell death. Cell size and intracellular mAb were increased for TB/C3 bcl2 cells compared with TB/C3 pEF control cells when analyzed by flow cytometry. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 54: 1-16, 1997.  相似文献   

8.
Expression of c-myc and macromolecular synthesis have been associated with physiological cell death. We have studied their requirement for the death of factor (interleukin-3)-dependent cells (FDC-P1) bearing an inducible bcl-2 expression construct. FDC-P1 cells expressing bcl-2 turned off expression of c-myc when deprived of interleukin-3 but remained viable as long as bcl-2 was maintained. A subsequent decline in Bcl-2 allowed the cells to undergo apoptosis directly from G0, in the absence of detectable c-myc expression. Thus c-myc expression may lead to apoptosis in some cases but is not directly involved in the mechanism of physiological cell death that can be controlled by Bcl-2. The macromolecular synthesis inhibitors actinomycin D and cycloheximide triggered rapid cell death of FDC-P1 cells in the presence of interleukin-3, but the cells could be protected by Bcl-2. Thus, the cell death machinery can exist in a quiescent state and can be activated by mechanisms that do not require synthesis of RNA or protein.  相似文献   

9.
The p53 tumor suppressor gene is thought to be required for the induction of programmed cell death (apoptosis) initiated by DNA damage. We show here, however, that the human promyelocytic leukemia cell line HL-60, which is known to be deficient in p53 because of large deletions in the p53 gene, can be induced to undergo apoptosis following X-irradiation. We demonstrate that the decision to undergo apoptosis in this cell line appears to be made at a G2 checkpoint. In addition, we characterize an HL-60 variant, HCW-2, which is radioresistant. HCW-2 cells display DNA damage induction and repair capabilities identical to those of the parental HL-60 cell line. Thus, the difference between the two cell lines appears to be that X-irradiation induces apoptosis in HL-60, but not in HCW-2, cells. Paradoxically, HCW-2 cells display high levels of expression of bax, which enhances apoptosis, and no longer express bcl-2, which blocks apoptosis. HCW-2 cells' resistance to apoptosis may be due to the acquisition of expression of bcl-XL, a bcl-2-related inhibitor of apoptosis. In summary, apoptosis can be induced in X-irradiated HL-60 cells by a p53-independent mechanism at a G2 checkpoint, despite the presence of endogenous bcl-2. The resistance shown by HCW-2 cells suggests that bcl-XL can block this process.  相似文献   

10.
Bcl-2 is the founding member of a family of proteins that influence apoptosis. During kidney development bcl-2 not only acts as a survival factor, but may also impact cell adhesive mechanisms and by extension branching morphogenesis. The interrelationship between cell adhesion, migration and apoptosis, important during development, is poorly understood. Here we examined the impact lack of bcl-2, an inhibitor of apoptosis, has on ureteric bud (UB) cell adhesion, migration, and branching morphogenesis. Bcl-2 -/- UB cells demonstrated increased cell migration, increased cell invasion and decreased adhesion to vitronectin and fibronectin compared with wild-type cells. Bcl-2 +/+ UB cells readily branched in collagen gel and Matrigel while bcl-2 -/- UB cells did not undergo significant branching in either matrix. Re-expression of bcl-2 in bcl-2 -/- UB cells restored their ability to undergo branching morphogenesis in Matrigel. Consistent with our in vitro data, we show that in the absence of bcl-2, embryonic kidneys undergo decreased UB branching. We observed decreased numbers of UB branch points, UB branch tips and a decreased distance to the first UB branch point in the absence of bcl-2. The alterations in bcl-2 -/- UB cell adhesion and migration was also associated with a significant alteration in expression of a number of extracellular matrix proteins. Bcl-2 -/- UB cells exhibited increased fibronectin expression and decreased thrombospondin-1 and osteopontin expression. Taken together, these data suggest that bcl-2 is required for the proper regulation of cell adhesive and migratory mechanisms, perhaps through modulation of the cellular microenvironment.  相似文献   

11.
Divalent cations, including Zinc and Manganese ions, are important modulators of cell activation. We investigated the ability of these two divalent cations to modulate apoptosis in human Burkitt lymphoma B cells line (Ramos). We found that Zinc (from 10 to 50 microM) inhibited Manganese-induced caspase-3 activation and apoptosis of Ramos cells. Higher concentration of Zinc (50 to 100 microM) did not prevent Manganese-mediated apoptosis but rather increased cell death among Ramos cells. This Zinc-mediated cell death was associated with apoptotic features such as cell shrinkage, the presence of phosphatidylserine residues on the outer leaflet of the cells, chromatin condensation, DNA fragmentation and decrease of mitochondrial transmembrane potential. Zinc-mediated apoptosis was associated with caspase-9 and caspase-3 activation as revealed by the appearance of active p35 fragment of caspase-9 and p19 and p17 of caspase-3 as well as in vivo cleavage of PARP and of a cell-permeable fluorogenic caspase-3 substrate (Phiphilux-G(1)D(2)). Both Zinc-mediated apoptosis and caspase-3 activation were prevented by the cell-permeable, broad-spectrum inhibitor of caspases (zVAD-fmk) or overexpression of bcl-2. In addition, we show that Zinc-induced loss of transmembrane mitochondrial potential is a caspase-independent event, since it is not modified by the presence of zVAD-fmk, which is inhibited by overexpression of bcl-2. These results indicate that depending on its concentration, Zinc can exert opposite effects on caspase-3 activation and apoptosis in human B lymphoma cells: concentrations below 50 microM inhibit caspase-3 activation and apoptosis whereas higher concentrations of Zinc activate a death pathway associated with apoptotic-like features and caspase-3 activation.  相似文献   

12.
It is well known that BCL-2 protects against cell death by both apoptosis and necrosis. The culture of bcl-2-transfected normal fibroblasts showed a shorter life span by about 12 population doubling levels compared to that of vector transfectants (64 vs 76 population doubling levels, respectively). An MTT assay revealed that BCL-2-overexpressing cells (HCA2/bcl-2) showed more severe growth suppression due to hydrogen peroxide or doxorubicin treatment than vector control cells (HCA2/vector). We observed a significant number of dead cells in the HCA2/bcl-2 culture, but not in the HCA2/vector culture. Other BCL-2 family proteins with both antiapoptotic and proapoptotic activity and other apoptosis-related factors were maintained at similar levels, indicating that overexpression of BCL-2 is the major reason that normal fibroblasts are sensitized to cell death. A broad caspase inhibitor (z-Val-Ala-Asp-fmk) and inhibitors of specific caspases (acetyl-Asp-Glu-Val-Asp-CHO, acetyl-Ile-Glu-Thr-Asp-CHO, and acetyl-Leu-Glu-His-Asp-CHO) suppressed cell death of HCA2/bcl-2 effectively, suggesting involvement of caspase 3-, 8-, and 9-dependent pathways in cell death and that the form of death is apoptosis. Unexpectedly, involvement of active MEK in cell death was shown by the use of its inhibitor, suggesting that crosstalk between BCL-2 and the MAP kinase cascade regulates death as well as life span.  相似文献   

13.
The productivity of recombinant mammalian cell lines growth in batch culture is often limited by the rapidity with which cells die on entry into the decline phase (the period of culture after the maximum cell density has been reached and where cell viability begins to fall). We examined the decline phase characteristics of the NSO myeloma cell line with a view to modulating the cell death that ensues. Examination of nuclear morphology during culture revealed that the onset of the decline phase was marked by a time-dependent increase in the percentage of cells that exhibited condensed and fragmented nuclei. Furthermore, these changes coincided with a fall in DNA integrity. High molecular weight DNA appeared to be degraded into oligonucleosomal fragments. Taken together, these observations indicated that NSO cells die by the process of apoptosis. The protein encoded by the bcl-2 gene has been shown to counter apoptosis induced by a large variety of stimuli and in a number of different cell types, but is not expressed in NSO cells. We examined whether overexpression of this protein could prevent/delay the onset of cell death seen during batch culture and also in response to serum limitation. Bcl-2 failed to affect the decline phase characteristics and serum dependence of NSO cells. In our search to explain these findings, we found that the NSO cell line expresses bax and also a high level of another Bcl-2 related protein, Bcl-x(L). Given that Bcl-X(L) is a sequence and functional homologue of Bcl-2, it is possible that Bcl-2 is redundant in the NSO cell background. These data therefore indicate that cells such as NSO, which are used in biotechnologically important processes such as generation of hybridomas and expression of recombinant proteins, may express only a subset of genes important in apoptotic regulation. Modulation of the death characteristics of such cells will need to take account of the expression profile of such genes and their regulatory interactions. (c) 1996 John Wiley & Sons, Inc.  相似文献   

14.
15.
Apoptosis which is also a called programmed cell death plays an important role during development, homeostasis and in many diseases such as cancer. Apoptosis is a genetically encoded cell death program defined by characteristic morphological and biochemical features. It is well recognized as a distinct pathologic mechanism in tumours responding to anticancer therapies. Many genes play an important role in this process. We evaluated an expression of the tumour supressor gene p53 and proteins p21 and bcl-2 in non-small cell lung cancer. We examined resected tumour tissues from 30 patients who received neoadjuvant chemotherapy. As a control we assessed tissues from patients treated without chemotherapy. Histological slides of the resected tumours were evaluated by TUNEL, in situ hybridisation and with immunoperoxidase staining procedure. The results were documented by photography. We examined the level of extinction using cytophotometry. In conclusion, preoperative chemotherapy induces apoptosis in cancer cells. The level of p53 correlates with the acceleration of TUNEL reaction. The loss of bcl-2 expression correlated with an increased apoptotic cell death. There was an increased p21 protein expression in the examined cancer tissues after chemotherapy.  相似文献   

16.
《Autophagy》2013,9(5):629-640
Vitamin K2 (menaquinone-4: VK2) is a potent inducer for apoptosis in leukemia cells in vitro. HL-60bcl-2 cells, which are derived from a stable transfectant clone of the human bcl-2 gene into the HL-60 leukemia cell line, show 5-fold greater expression of the Bcl-2 protein compared with HL-60neo cells, a control clone transfected with vector alone. VK2 induces apoptosis in HL-60neo cells, whereas HL-60bcl-2 cells are resistant to apoptosis induction by VK2 but show inhibition of cell growth along with an increase of cytoplasmic vacuoles during exposure to VK2. Electron microscopy revealed formation of autophagosomes and autolysosomes in HL-60bcl-2 cells after exposure to VK2. An increase of acid vesicular organelles (AVOs) detected by acridine orange staining for lysosomes as well as conversion of LC3B-I into LC3B-II by immunonoblotting and an increased punctuated pattern of cytoplasmic LC3B by fluorescent immunostaining all supported induction of enhanced autophagy in response to VK2 in HL-60bcl-2 cells. However, during shorter exposure to VK2, the formation of autophagosomes was also prominent in HL-60neo cells although nuclear chromatin condensations and nuclear fragments were also observed at the same time. These findings indicated the mixed morphologic features of apoptosis and autophagy. Inhibition of autophagy by either addition of 3-methyladenine, siRNA for Atg7, or Tet-off Atg5 system all resulted in attenuation of VK2-incuded cell death, indicating autophagy-mediated cell death in response to VK2. These data demonstrate that autophagy and apoptosis can be simultaneously induced by VK2. However, autophagy becomes prominent when the cells are protected from rapid apoptotic death by a high expression level of Bcl-2.  相似文献   

17.
18.
Protein phosphorylation plays an indispensable role in cellular regulation of mitosis, metabolism, differentiation, and death. We previously reported that the protein phosphatase inhibitor okadaic acid (OKA) induces apoptosis in renal epithelial cells in culture. In the present study, we examined the role of phosphotidylinositol 3 (PI3) kinase signaling in okadaic acid-induced apoptosis by pre-treating normal rat kidney renal epithelial cells expressing human bcl-2 with the PI3 kinase inhibitors, LY294002 and wortmannin, followed by apoptosis-inducing concentrations of okadaic acid. Given the reported cell survival activity of PI3 kinase signaling mostly attributed to Akt kinase activation, we hypothesized that inhibition of PI3 kinase would enhance okadaic-induced apoptosis. Surprisingly, our data show that pretreatment with LY294002, but not wortmannin, attenuated okadaic acid-induced apoptosis. In contrast, to LY294002, wortmannin enhanced apoptosis. Interestingly, we also found that LY294002 treatment increased bcl-2 protein levels in normal rat kidney epithelial cells expressing bcl-2 (NRK-bcl-2). In untreated cells, bcl-2 appeared to be mainly perinuclear, coincident with the nuclear membrane, or in the cytosol. In OKA treated cells that were pre-treated with Ly294002, bcl-2 was highly co-localized with mitochondria, but in cells treated with okadaic acid alone, bcl-2 was associated with fragmented chromatin. In this model, it appears that LY294002 may exert anti-apoptotic effects by a previously unreported treatment related increase in bcl-2. Although it is widely accepted that bcl-2 protein can inhibit apoptosis, we propose that the subcellular location of bcl-2 is an important determinant in whether bcl-2 effectively inhibits apoptosis.  相似文献   

19.
Malignant glioma is resistant to the induction of apoptosis, resulting in a subsequent failure of chemotherapy in clinical treatment strategies. Downregulation of bcl-2 and bcl-xl expression in glioblastoma cells can induce apoptosis. BH3-only proteins, which include Bmf, are essential initiators of stress-induced cell death and apoptosis. Whether PS-341 regulates expression of BH3-only proteins in glioblastoma cells during the procedure of apoptosis is unclear. This study was designed to investigate the effects of PS-341 on glioma cell death and its possible signaling pathway. Our results demonstrate that Bmf is upregulated by PS-341 in A172 and T98G cells, and Bmf has a crucial role in PS-341-mediated cell death. In addition, we found that expression of Bmf is regulated by JNK phosphorylation.  相似文献   

20.
The function of the proteasome has been linked to various pathologies, including cancer and neurodegeneration. Proteasomal inhibition can lead to death in a variety of cell types, however the manner in which this occurs is unclear, and may depend on the particular cell type. In this work we have extended previous findings pertaining to the effects of pharmacological proteasomal inhibitors on PC12 cells, by examining in more detail the induced death pathway. We find that cell death is apoptotic by ultrastructural criteria. Caspase 9 and 3 are processed, cytochrome c is released from the mitochondria and a dominant negative form of caspase 9 prevents death. Furthermore, Bax undergoes a conformational change and is translocated to the mitochondria in a caspase-independent fashion. Total cell levels of Bax however do not change, whereas levels of the BH3-only protein Bim increase with proteasomal inhibition. Transient overexpression of bcl-xL or, to a lesser extent, of bcl-2, significantly decreased apoptotic death and prevented Bax conformational change. We conclude that death elicited by proteasomal inhibition of PC12 cells follows a classical “intrinsic” pathway. Significantly, antiapoptotic bcl-2 family members prevent apoptosis by inhibiting Bax conformational change. Increased levels of Bim may contribute to cell death in this model.  相似文献   

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