Simvastatin protects neurons from cytotoxicity by up-regulating Bcl-2 mRNA and protein

Statins are most commonly prescribed to reduce hypercholesterolemia; however, recent studies have shown that statins have additional benefits, including neuroprotection. Until now, the mechanism underlying statin-induced neuroprotection has been poorly understood. Recent in vivo studies from our lab reported the novel finding that simvastatin increased expression levels of a gene encoding for a major cell survival protein, bcl-2 [Johnson-Anuna et al., J. Pharmacol. Exp. Ther.312 (2005) 786]. The purpose of the present experiments was to determine if simvastatin could protect neurons from excitotoxicity by altering Bcl-2 levels. Neurons were pre-treated with simvastatin and challenged with a compound known to reduce Bcl-2 levels and induce cell death. Simvastatin pre-treatment resulted in a significant reduction in cytotoxicity (lactate dehydrogenase release and caspase 3 activation) following challenge compared with unchallenged neurons. In addition, chronic simvastatin treatment significantly increased Bcl-2 mRNA and protein levels while challenge resulted in a significant reduction in Bcl-2 protein abundance. G3139, an antisense oligonucleotide directed against Bcl-2, abolished the protective effects of simvastatin and eliminated simvastatin-induced up-regulation of Bcl-2 protein. These findings suggest that neuroprotection by simvastatin is dependent on the drug's previously unexplored and important effect of up-regulating Bcl-2.     

Bcl-2 family member Bcl-G is not a proapoptotic protein

The three major subgroups of the Bcl-2 family, including the prosurvival Bcl-2-like proteins, the proapoptotic Bcl-2 homology (BH)3-only proteins and Bax/Bak proteins, regulate the mitochondrial apoptotic pathway. In addition, some outliers within the Bcl-2 family do not fit into these subgroups. One of them, Bcl-G, has a BH2 and a BH3 region, and was proposed to trigger apoptosis. To investigate the physiological role of Bcl-G, we have inactivated the gene in the mouse and generated monoclonal antibodies to determine its expression. Although two isoforms of Bcl-G exist in human, only one is found in mice. mBcl-G is expressed in a range of epithelial as well as in dendritic cells. Loss of Bcl-G did not appear to affect any of these cell types. mBcl-G only binds weakly to prosurvival members of the Bcl-2 family, and in a manner that is independent of its BH3 domain. To understand what the physiological role of Bcl-G might be, we searched for Bcl-G-binding partners through immunoprecipitation/mass spectroscopy and yeast-two-hybrid screening. Although we did not uncover any Bcl-2 family member in these screens, we found that Bcl-G interacts specifically with proteins of the transport particle protein complex. We conclude that Bcl-G most probably does not function in the classical stress-induced apoptosis pathway, but rather has a role in protein trafficking inside the cell.     

Altered Bcl-2 and Bax expression is associated with cultured first trimester human cytotrophoblasts apoptosis induced by hypoxia

Preeclampsia is associated with placental hypoxia at early gestation. We therefore investigated the effect of hypoxia on the apoptosis of cultured first trimester human cytotrophoblasts and the expression of apoptosis relevant proteins, Bcl-2 and Bax. First trimester human cytotrophoblasts were isolated and cultured under either standard or hypoxic conditions. Cellular apoptosis was monitored by TUNEL and Annexin V binding, and the expression of Bcl-2 and Bax was determined by Western blot analysis. Apoptosis increased significantly in cytotrophoblasts cultured for 24 h under hypoxic conditions in contrast with those cultured under standard conditions, meanwhile expression of Bcl-2 reduced, and that of Bax increased. These changes suggested that hypoxia induced apoptosis in cultured first trimester cytotrophoblasts with altered Bcl-2 and Bax expression. Further study is needed to explore the role of cytotrophoblasts apoptosis in hypoxia-induced maternal and fetal diseases.     

The Functional Differences between Pro-survival and Pro-apoptotic B Cell Lymphoma 2 (Bcl-2) Proteins Depend on Structural Differences in Their Bcl-2 Homology 3 (BH3) Domains

Bcl-2 homology 3 (BH3) domains are short sequence motifs that mediate nearly all protein-protein interactions between B cell lymphoma 2 (Bcl-2) family proteins in the intrinsic apoptotic cell death pathway. These sequences are found on both pro-survival and pro-apoptotic members, although their primary function is believed to be associated with induction of cell death. Here, we identify critical features of the BH3 domains of pro-survival proteins that distinguish them functionally from their pro-apoptotic counterparts. Biochemical and x-ray crystallographic studies demonstrate that these differences reduce the capacity of most pro-survival proteins to form high affinity “BH3-in-groove” complexes that are critical for cell death induction. Switching these residues for the corresponding residues in Bcl-2 homologous antagonist/killer (Bak) increases the binding affinity of isolated BH3 domains for pro-survival proteins; however, their exchange in the context of the parental protein causes rapid proteasomal degradation due to protein destabilization. This is supported by further x-ray crystallographic studies that capture elements of this destabilization in one pro-survival protein, Bcl-w. In pro-apoptotic Bak, we demonstrate that the corresponding distinguishing residues are important for its cell-killing capacity and antagonism by pro-survival proteins.     

Pro-apoptotic Bax is the major and Bak an auxiliary effector in cytokine deprivation-induced mast cell apoptosis

The process of apoptosis in immune cells like mast cells is essential to regain homeostasis after an inflammatory response. The intrinsic pathway of apoptosis is ultimately controlled by the pro-apoptotic Bcl-2 family members Bax and Bak, which upon activation oligomerize to cause increased permeabilization of the mitochondria outer membrane leading to cell death. We examined the role of Bax and Bak in cytokine deprivation-induced apoptosis in mast cells using connective tissue-like mast cells and mucosal-like mast cells derived from bax^−/−, bak^−/− and bax^−/−bak^−/− mice. Although both Bax and Bak were expressed at readily detectable protein levels, we found a major role for Bax in mediating mast cell apoptosis induced by cytokine deprivation. We analyzed cell viability by propidium iodide exclusion and flow cytometry after deprivation of vital cytokines for each mast cell population. Upon cytokine withdrawal, bak^−/− mast cells died at a similar rate as wild type, whereas bax^−/− and bax^−/−bak^−/− mast cells were partially or completely resistant to apoptosis, respectively. The total resistance seen in bax^−/−bak^−/− mast cells is comparable with mast cells deficient of both pro-apoptotic Bim and Puma or mast cells overexpressing anti-apoptotic Bcl-2. These results show that Bax has a predominant and Bak a minor role in cytokine deprivation-induced apoptosis in both connective tissue-like and mucosal-like mast cells.     

RNA干扰抑制SiHaB2细胞中Bcl-2基因的表达

Bcl-2基因作为细胞凋亡的一个潜在抑制剂调节细胞的死亡,抑制恶性肿瘤细胞中Bcl-2基因的表达可促进肿瘤细胞的凋亡。采用RNA干扰技术,合成了含有21个核苷酸的小双链干扰RNA（siRNA．Bcl-2）,并构建了含有19个核苷酸基因的质粒载体（pSilencer2．1-U6-Bcl-2）,把合成的siRNA．Bcl-2和pSilencer2．1-U．Bcl-2分别转导入Bcl-2高表达的细胞株SiHaB2中,通过Western印迹检测,免疫荧光法检测及DNA梯（ladder）检测,可观察到在导入siRNA．Bcl-2和pSilencer2．1-U．Bcl-2的SiHaB2细胞被培养72h后,可以明显抑制Bcl-2蛋白的表达。     

Bcl-2 overexpression in CHO cells improves polyethylenimine-mediated gene transfection

Transient gene expression (TGE) in Chinese hamster ovary (CHO) cells with polyethylenimine (PEI) as a transfection reagent has been considered as an attractive method to produce recombinant proteins rapidly for pre-clinical studies. A high level of transfection efficiency, which is required for high-level TGE in CHO cells, can be achieved by increasing the PEI concentration. However, PEI induces cytotoxicity in a dose-dependent manner. To overcome this problem, Bcl-2 protein, an anti-apoptotic protein, was overexpressed in CHO cells (DG44). At a ratio of PEI to DNA (an N/P ratio) of 10, there were no significant differences in transfection efficiency and cell viability between Bcl-2 overexpressing and non-overexpressing cells. The transfection efficiency and cell viability were 2–11% and 83–92%, respectively. However, there were significant differences (P < 0.05) in the transfection efficiency and cell viability between them at a higher N/P ratio. At an N/P ratio of 40, the transfection efficiency and cell viability of Bcl-2 non-overexpressing cells were 24–38% and 35–40%, respectively, while those of Bcl-2 overexpressing cells were 48–53% and 43–56%, respectively. Furthermore, compared with Bcl-2 non-overexpressing cells, more DNAs entered the Bcl-2 overexpressing cells, resulting in a higher rate of TGE per cell. PE-Annexin V apoptosis revealed that Bcl-2 overexpression suppressed PEI-induced apoptotic cell death at high N/P ratios. Taken together, Bcl-2 overexpression in CHO cells suppresses apoptotic cell death during PEI-mediated transient transfection, resulting in enhanced transfection efficiency and TGE.     

Mitochondrial permeability transition mediates apoptosis induced by N-methyl(R)salsolinol,an endogenous neurotoxin,and is inhibited by Bcl-2 and rasagiline,N-propargyl-1(R)-aminoindan

The role of mitochondrial permeability transition (PT) in apoptosis induced by an endogenous neurotoxin, N-methyl(R)salsolinol [NM(R)Sal], was studied by use of dopaminergic neuroblastoma SH-SY5Y cells. NM(R)Sal reduced mitochondrial membrane potential, DeltaPsim, in the early phase of apoptosis, which was not suppressed by a pan-caspase inhibitor, but was antagonized by Bcl-2 and cyclosporin A, suggesting the involvement of the PT in NM(R)Sal-induced loss of DeltaPsim. NM(R)Sal-induced apoptosis was completely inhibited not only by Bcl-2 and a pan-caspase inhibitor, but also by cyclosporin A, suggesting the essential role of the PT in NM(R)Sal-induced apoptosis. In mitochondria isolated from rat liver, NM(R)Sal induced swelling and reduced DeltaPsim, which was inhibited by cyclosporin A and Bcl-2 overexpression. These results indicate that NM(R)Sal induced the PT by direct action on the mitochondria. Rasagiline, N-propargyl-1(R)-aminoindan, which is a now under a clinical trial for Parkinson's disease, suppressed the DeltaPsim reduction, release of cytochrome c, and apoptosis induced by NM(R)Sal in SH-SY5Y cells. Rasagiline also inhibited the NM(R)Sal-induced loss of DeltaPsim and swelling in the isolated mitochondria, proving that rasagiline directly targets the mitochondria also. Altogether, mitochondrial PT plays a key role both in NM(R)Sal-induced cell death and the neuroprotective effect of rasagiline.     

Transfection-enforced Bcl-2 overexpression and an anti-Parkinson drug, rasagiline, prevent nuclear accumulation of glyceraldehyde-3-phosphate dehydrogenase induced by an endogenous dopaminergic neurotoxin, N-methyl(R)salsolinol

An endogenous dopaminergic neurotoxin, N-methyl(R)salsolinol, was found to induce apoptosis in human dopaminergic SH-SY5Y cells by step-wise activation of apoptotic cascade; collapse in mitochondrial membrane potential, DeltaPsim, activation of caspases, and fragmentation of DNA. Recently, accumulation of gylceraldehyde-3-phosphate dehydrogenase (GAPDH) in nuclei was proposed to play an important role in apoptosis. In this paper, involvement of GAPDH in apoptosis induced by N-methyl(R)salsolinol was studied. The isoquinoline reduced DeltaPsim within 3 h, as detected by a fluorescence indicator, JC-1, then after 16 h incubation, GAPDH accumulated in nuclei by detection with immunostaining. To clarify the role of GAPDH in apoptotic process, a stable cell line of Bcl-2 overexpressed SH-SY5Y cells was established. Overexpression of Bcl-2 prevented the decline in DeltaPsim and also apoptotic DNA damage induced by N-methyl(R)salsolinol. In Bcl-2 transfected cells, nuclear translocation of GAPDH was also completely suppressed. In addition, a novel antiparkinsonian drug, rasagiline, prevented nuclear accumulation of GAPDH induced by N-methyl(R)salsolinol in control cells. These results suggest that GAPDH may accumulate in nuclei as a consequence of signal transduction, which is antagonized by anti-apoptotic Bcl-2 protein family and rasagiline. The results are discussed in concern to intracellular mechanism underlying anti-apoptotic function of rasagiline analogues.     

Detection of Bcl-2 family member Bcl-G in mouse tissues using new monoclonal antibodies

Bcl-G is an evolutionarily conserved member of the Bcl-2 family of proteins that has been implicated in regulating apoptosis and cancer. We have generated monoclonal antibodies that specifically recognise mouse Bcl-G and have used these reagents to analyse its tissue distribution and subcellular localisation using western blotting, immunohistochemistry and immunofluorescence. We found that Bcl-G predominantly resides in the cytoplasm and is present in a wide range of mouse tissues, including the spleen, thymus, lung, intestine and testis. Immunohistochemical analyses revealed that Bcl-G is expressed highly in mature spermatids in the testis, CD8⁺ conventional dendritic cells (DCs) in hematopoietic tissues and diverse epithelial cell types, including those lining the gastrointestinal and respiratory tracts. The Bcl-G monoclonal antibodies represent new tools for studying this protein, using a variety of techniques, including immunoprecipitation and flow cytometry.     

p21 promotes ceramide-induced apoptosis and antagonizes the antideath effect of Bcl-2 in human hepatocarcinoma cells

p21, a potent cyclin-dependent kinase inhibitor, has been known to induce cell cycle arrest in response to DNA-damaging agents. Although p21 has been reported to play an important role in the regulation of apoptosis, the postulated role for p21 in apoptosis is still controversial. Previously, we reported that p21 was induced in a p53-independent manner during ceramide-induced apoptosis in human hepatocarcinoma cell lines. In the present study, we investigated the precise role of p21 in ceramide-induced apoptosis in human hepatocarcinoma cells by using a tetracycline-inducible expression system. Overexpression of p21 by itself did not induce apoptosis in p53-deficient Hep3B cells. However, Hep3B/p21 cells were more sensitive to ceramide-induced apoptosis. In these cells, p21 overexpression did not result in G1 arrest. The expression level of Bax was increased in Hep3B/p21 cells treated with ceramide and its expression was more accelerated under the p21-overexpressed condition compared to that of the p21-repressed condition. Overexpression of Bax induced apoptosis in Hep3B cells. On the other hand, the levels of p21 and Bax protein were increased by ceramide in another hepatocarcinoma cell line, SK-Hep-1, while the Bcl-2 protein level was not changed. Overexpression of Bcl-2 not only suppressed apoptosis but also completely prevented induction of p21 and Bax caused by ceramide in SK-Hep-1 cells. Furthermore, overexpression of p21 antagonized the death-protective function of Bcl-2 and upregulated expression of Bax protein. These results suggest that p21 promotes ceramide-induced apoptosis by enhancing the expression of Bax, thereby modulating the molecular ratio of Bcl-2:Bax in human hepatocarcinoma cells.     

Zebrafish lacking Alzheimer presenilin enhancer 2 (Pen-2) demonstrate excessive p53-dependent apoptosis and neuronal loss

Gamma-secretase cleavage, mediated by a complex of presenilin, presenilin enhancer (Pen-2), nicastrin, and Aph-1, is the final proteolytic step in generating amyloid beta protein found in brains of Alzheimer's disease patients and Notch intracellular domain critical for proper neuronal development. Here, we employ the zebrafish model to study the role of Pen-2 in neuronal survival. We found that (i) knockdown of Pen-2 using antisense morpholino led to a reduction of islet-1 positive neurons, (ii) Notch signaling was reduced in embryos lacking Pen-2 or other gamma-secretase components, (iii) neuronal loss in Pen-2 knockdown embryos is not as a result of a lack of neuronal precursor cells or cell proliferation, (iv) absence of Pen-2 caused massive apoptosis in the whole animal, which could be suppressed by simultaneous knockdown of the tumor suppressor p53, (v) loss of islet-1 or acetylated tubulin positive neurons in Pen-2 knockdown embryos could be partially rescued by knockdown of p53. Our results demonstrate that knockdown of Pen-2 directly induces a p53-dependent apoptotic pathway that contributes to neuronal loss and suggest that Pen-2 plays an important role in promoting neuronal cell survival and protecting from apoptosis in vivo.     

Reduction in TrkA-immunoreactive neurons is not associated with an overexpression of galaninergic fibers within the nucleus basalis in Down's syndrome

Down's syndrome (DS) individuals develop neuropathological features similar to Alzheimer's disease (AD), including degeneration of cholinergic basal forebrain (CBF) neurons. In AD a reduction in CBF/trkA-containing neurons has been suggested to trigger a hyperexpression of galaninergic fibers within the nucleus basalis subfield of the basal forebrain. The present study examined the interrelationship between reductions in CBF/trkA-containing neurons and the overexpression of galaninergic fibers within the nucleus basalis in DS. Within the nucleus basalis stereologic evaluation revealed a 46% reduction in the number of trkA-immunopositive neurons, whereas optical density measurements displayed a nonsignificant 18% reduction in neuronal trkA immunoreactivity in DS as compared with age-matched controls. Western blot analysis also showed a significant reduction in cortical trkA protein levels in DS. A semiquantitative examination of galaninergic fibers in the nucleus basalis revealed only a modest hypertrophy of galaninergic fibers within the nucleus basalis in DS. The present findings indicate a significant reduction in trkA within the nucleus basalis and cortex with only a moderate hypertrophy of galaninergic fibers in DS. These observations suggest that DS may not be an exact genetic model for investigation of changes in the AD basal forebrain.     

Phosphorylated PP2A (tyrosine 307) is associated with Alzheimer neurofibrillary pathology

Down-regulation of protein phosphatase 2A (PP2A) is thought to play a critical role in tau hyperphosphorylation in Alzheimer's disease (AD). In vitro phosphorylation of PP2A catalytic subunit at Y307 efficiently inactivates PP2A. A specific antibody against phosphorylated (p) PP2A (Y307) (PP2Ac-Yp307) was used to investigate possible PP2A down-regulation by known pathophysiological changes associated with AD, such as Abeta accumulation and oestrogen deficiency. Immunohistochemistry and immunofluorescence confocal microscopy showed an aberrant accumulation of PP2Ac-Yp307 in neurons that bear pretangles or tangles in the susceptible brain regions, such as the entorhinal cortical cortex and the hippocampus. Experimentally, increased PP2Ac-Yp307 was observed in mouse N2a neuroblastoma cells that stably express the human amyloid precursor protein with Swedish mutation (APPswe) compared with wild-type, and in the brains of transgenic APPswe/ presenilin (PS1, A246E) mice, which corresponded to the increased tau phosphorylation. Treating N2a cells with Abeta25-35 mimicked the changes of PP2Ac-Yp307 and tau phosphorylation in N2a APPswe cells. Knockout of oestrogen receptor (ER) alpha or ERbeta gave similar changes of PP2Ac-Yp307 level and tau phosphorylation in the mouse brain. Taken together, these findings suggest that increased PP2A phosphorylation (Y307) can be mediated by Abeta deposition or oestrogen deficiency in the AD brain, and consequently compromise dephosphorylation of abnormally hyperphosphorylated tau, and lead to neurofibrillary tangle formation.     

Free radical-dependent nuclear localization of Bcl-2 in the central nervous system of aged rats is not associated with Bcl-2-mediated protection from apoptosis

We have previously reported that Bcl-2 is up-regulated in the CNS of aged F344 rats as a consequence of oxidative stress. In addition to increased levels of expression, we now report that there is a subcellular redistribution of Bcl-2 in the CNS of aged F344 rats. Using western blotting, we found Bcl-2 predominantly located in the cytosol of young rats. However, in aged rats Bcl-2 was found primarily in the nucleus. This distribution, in the hippocampus and cerebellum, was reversed by treatment with the nitrone spin trap N-tert-butyl-alpha-phenylnitrone (PBN). Paradoxically, PBN treatment in young rats had the opposite effect, changing Bcl-2 from predominantly cytosolic to nuclear. We also detected an increase in Bax in aged hippocampal samples (both nuclear and cytosolic), which was reversed by treatment with PBN. The distribution of Bcl-2 and Bax in the cytosol of aged rats dramatically decreased the Bcl-2/Bax ratio, a probable indicator of neuronal vulnerability, which was restored upon treatment with PBN. In order to assess the effect of nuclear association of Bcl-2 we used PC12 cells stably transfected with a Bcl-2 construct to which we added the nuclear localization sequence of the SV40 large T antigen to the N-terminus which resulted in nuclear targeting of Bcl-2. Measurement of cell death using lactate dehydrogenase assays showed that, contrary to wild-type Bcl-2, Bcl-2 localized to the nucleus was not effective in protecting cells from treatment with 250 microm H2O2. These results suggest that nuclear localization of Bcl-2 observed in the aged CNS may not reflect a protective mechanism against oxidative stress, a major component of age-associated CNS impairments.     

Cytoskeleton inhibitors combined with TRAIL induce apoptosis in HeLa carcinoma cells overexpressing antiapoptotic protein Bcl-2

TRAIL (Apo2L), a cytokine from the family of tumor necrosis factors (TNF), causes apoptosis in various types of tumor cells but is not toxic for normal cells. Recombinant TRAIL obtained using an original method stimulates the release of cytochrome c from mitochondria into the cytoplasm and apoptosis in HeLa carcinoma cells. Expression of oncoprotein Bcl-2 in these cells blocks both processes. The microtubule inhibitors taxol, nocodazole, and colcemid, as well as an inhibitor of actin microfilaments cytochalasin D, enhance the action of TRAIL and allow it to overcome protection caused by overexpression of Bcl-2. This effect is not associated with enhancement of early steps of TRAIL-dependent apoptosis leading to activation of caspase-8 and Bid protein. The inactivation of Bcl-2 also does not define the effect of cytoskeleton inhibitors. It is supposed that destruction of cytoskeleton alters the mechanism of the TRAIL- (or TNF)-dependent cytochrome c release from mitochondria by making it resistant to Bcl-2. The combined use of cytoskeleton inhibitors, which are antitumor drugs, with the recombinant TRAIL preparations may be efficient in therapy of tumors resistant to traditional chemotherapy.     

4-HPR-mediated leukemia cell cytotoxicity is triggered by ceramide-induced mitochondrial oxidative stress and is regulated downstream by Bcl-2

We have previously reported that, in leukemia cells, the cytotoxicity of the anticancer agent N-(4-hydroxyphenyl)retinamide (4-HPR) is mediated by mitochondria-derived reactive oxygen species (ROS) and cardiolipin peroxidation. Here, we have analyzed at greater depth the 4-HPR-triggered molecular events, demonstrating that 4-HPR induces an early (15 min) increase in ceramide levels by sphingomyelin hydrolysis and later (from 1 h) by de novo synthesis. Using specific inhibitors of both pathways, we demonstrate that ceramide accumulation is responsible for early ROS generation, which act as apoptotic signalling intermediates leading to conformational activation of Bak and Bax, loss of mitochondrial membrane potential (ΔΨm), mitochondrial membrane permeabilization (MMP) and cell death. Enforced expression of Bcl-2 has no effect on 4-HPR-induced oxidative stress, but notably prevents mitochondrial alterations and apoptosis, indicating that Bcl-2 functions by regulating events downstream of ROS generation. In conclusion, our study delineates for the fist time the sequence and timing of the principal events induced by 4-HPR in leukemia cells and points to the potential use of modulators of ceramide metabolism as enhancers in 4-HPR-based therapies.     

4-HPR-mediated leukemia cell cytotoxicity is triggered by ceramide-induced mitochondrial oxidative stress and is regulated downstream by Bcl-2

We have previously reported that, in leukemia cells, the cytotoxicity of the anticancer agent N-(4-hydroxyphenyl)retinamide (4-HPR) is mediated by mitochondria-derived reactive oxygen species (ROS) and cardiolipin peroxidation. Here, we have analyzed at greater depth the 4-HPR-triggered molecular events, demonstrating that 4-HPR induces an early (15 min) increase in ceramide levels by sphingomyelin hydrolysis and later (from 1 h) by de novo synthesis. Using specific inhibitors of both pathways, we demonstrate that ceramide accumulation is responsible for early ROS generation, which act as apoptotic signalling intermediates leading to conformational activation of Bak and Bax, loss of mitochondrial membrane potential (ΔΨm), mitochondrial membrane permeabilization (MMP) and cell death. Enforced expression of Bcl-2 has no effect on 4-HPR-induced oxidative stress, but notably prevents mitochondrial alterations and apoptosis, indicating that Bcl-2 functions by regulating events downstream of ROS generation. In conclusion, our study delineates for the fist time the sequence and timing of the principal events induced by 4-HPR in leukemia cells and points to the potential use of modulators of ceramide metabolism as enhancers in 4-HPR-based therapies.     

Bcl-2 down modulation in WEHI-3B/CTRES cells resistant to Cholera Toxin （CT）-induced apoptosis

The very different effects of Cholera Toxin （CT） on cell growth and proliferation may depend on the type of ganglioside receptors in cell membranes and different signal transduction mechanisms triggered, but other functions related to the drug resistance mechanisms can not be excluded. The effect of CT treatment on the ＂in vitro＂ clonogenicity, the Population Doubling Time （PDT）, apoptosis, PKA activation and Bax and Bcl-2 expression was evaluated in WEHI-3B cell line and its CT-resistant subclone （WEHI-3B/CTRES）. In WEHI-3B parental cells the dramatic accumulation of cAMP induced by CT correlated well with PKA activation, increased PDT value, inhibition of clonogenicity and apoptosis. H-89 treatment inhibited PKA activation by CT but did not protect the cells from apoptosis and growth inhibition. In WEHI-3B/CTRES no significant CT-dependent accumulation of cAMP occurred with any increase of PKA activity and PDT. In CT resistant cells （WEHI-3B/CTRES）, Bcl-2 expression was down regulated by both CT or drug treatment （eg, ciprofloxacin, CPX） although these cells were protected from CT-dependent apoptosis but not from drug-induced apoptosis. Differently from other cell models described, down regulation of Bcl-2 is proved to be independent on cAMP accumulation and PKA activation. Our observations support the implication of cAMP dependent kinase （PKA） in the inhibition of WEHI-3B cells growth and suggest that, in WEHI-3B/CTRES, Bcl-2 expression could be modulated by CT in the absence of cAMP accumulation. Also in consideration of many contradictory data reported in literature, our cell models （of one sensitive parental cell strain and two clones with different uncrossed specific resistance to CT and CPX） provides a new and interesting tool for better investigating the relationship between the CT signal transduction mechanisms and Bcl-2 expression and function.