Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UNIPROT:P10415 (Bcl-2)
 33,771 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Programmed cell death (PCD) has been observed in a wide variety of cell types in response to physiologic signals or types of stress. How these stimuli trigger PCD, and whether there is a common PCD signal transduction pathway, is not clear. As more genes are described that may participate in or regulate PCD, an assay system in which gene products can easily be introduced and/or modulated would be of great value. To avoid the generation and screening of multiple individual stable cell transfectants, a simple transient transfection death assay has been developed. 2B4.11, a murine T cell hybridoma, was transfected by electroporation with a constitutively active beta-galactosidase reporter gene and the cells were incubated in culture medium or with a PCD-inducing stimulus. The amount of beta-galactosidase activity remaining in the intact cells at the end of the culture period represented only viable transfected cells. Bcl-2 was chosen to examine whether this system would be useful to study the effect of transiently transfected genes since it blocks PCD in a number of experimental systems. Consistent with data obtained using stable transfectants, transient expression of Bcl-2 in 2B4.11 completely protected cells from glucocorticoid- and cytotoxic agent-induced PCD. This protection from death was confirmed at the individual cell level by the transient co-expression of a class I Ld surface antigen and flow cytometric analysis. Some of the advantages of the transient transfection death assay described here are; (1) the simple and sensitive beta-galactosidase assay, (2) the rapidity of the assay, (3) the ability to perform conventional viability assays to monitor treatment-induced cytotoxicity, (4) multiple gene products can be tested alone, and in combination, (5) antisense or dominant negative approaches can be used, and (6) the adaptability of this assay system to other cell types, transfection techniques, or reporter and expression vectors. The transient transfection death assay should make it easier to identify and order important steps in the PCD signal transduction pathways.
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PMID:A simple assay for examining the effect of transiently expressed genes on programmed cell death. 789 44

Interactions of the Bcl-2 protein with itself and other members of the Bcl-2 family, including Bcl-X-L, Bcl-X-S, Mcl-1, and Bax, were explored with a yeast two-hybrid system. Fusion proteins were created by linking Bcl-2 family proteins to a LexA DNA-binding domain or a B42 trans-activation domain. Protein-protein interactions were examined by expression of these fusion proteins in Saccharomyces cerevisiae having a lacZ (beta-galactosidase) gene under control of a LexA-dependent operator. This approach gave evidence for Bcl-2 protein homodimerization. Bcl-2 also interacted with Bcl-X-L and Mcl-1 and with the dominant inhibitors Bax and Bcl-X-S. Bcl-X-L displayed the same pattern of combinatorial interactions with Bcl-2 family proteins as Bcl-2. Use of deletion mutants of Bcl-2 suggested that Bcl-2 homodimerization involves interactions between two distinct regions within the Bcl-2 protein, since a LexA protein containing Bcl-2 amino acids 83-218 mediated functional interactions with a B42 fusion protein containing Bcl-2 amino acids 1-81 but did not complement a B42 fusion protein containing Bcl-2 amino acids 83-218. In contrast to LexA/Bcl-2 fusion proteins, expression of a LexA/Bax protein was lethal to yeast. This cytotoxicity could be abrogated by B42 fusion proteins containing Bcl-2, Bcl-X-L, or Mcl-1 but not those containing Bcl-X-S (an alternatively spliced form of Bcl-X that lacks a well-conserved 63-amino acid region). The findings suggest a model whereby Bax and Bcl-X-S differentially regulate Bcl-2 function, and indicate that requirements for Bcl-2/Bax heterodimerization may be different from those for Bcl-2/Bcl-2 homodimerization.
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PMID:Interactions among members of the Bcl-2 protein family analyzed with a yeast two-hybrid system. 793 47

Bcl-2 is an oncogene associated with prevention of apoptosis in a variety of cell types. Bcl-2 expression in B lymphoid cells prolongs antibody production, in vitro and in vivo. A line of transgenic mice (B6) has been developed that expresses human Bcl-2 in the B cells of SWR/SJL mice. B6 transgenic, nontransgenic littermates, and BALB/c mice were immunized with beta-galactosidase (B-gal) or sheep red blood cells (SRBC). The number of spleen cells recovered from immunized B6 mice was 3-4 times greater than syngeneic, nontransgenic littermates or BALB/c mice. Spleen cells from B-gal or SRBC immune B6, SWR/SJL, and BALB/c mice were fused with P3 myeloma cells to produce hybridomas. Forty-eight percent of the wells plated with fused B6 spleen cells produced B-gal-specific antibodies compared to 14% from BALB/c and 12% from SWR/SJL. Antibody-specific wells were subcloned, resulting in enhanced recovery of antigen-specific subclones with B6-derived fusions compared to controls. In the SRBC fusions, 17% of the wells plated with fused B6 spleen cells produced SRBC-specific antibodies compared to 6% for BALB/c and SWR/SJL spleens. After subcloning, B6-derived clones produced 8% positive subclones compared to 9.5% from SWR/SJL and 3.5% from BALB/c. Comparison of the isotype distribution of subclones showed a higher ratio of IgG antibodies compared to IgM from B6 mice in the B-gal fusions. IgA antibodies were recovered only from B6 mice. These data indicate that B6 transgenic mice that overexpress Bcl-2 in their B cells may be superior to other mouse strains for production of antigen-specific hybridomas.
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PMID:Evaluation of Bcl-2/B cell transgenic mice (B6) for hybridoma production. 891 86

Overexpression of the proto-oncogene bcl-2 has been shown to protect a variety of cell types from oxidative and non-oxidative injury, blocking apoptotic and necrotic types of cell death. Retroviral vectors were used to stably overexpress bcl-2 in primary murine astrocyte cultures with more than 95% efficiency. Compared to beta-galactosidase-expressing and uninfected control cells, bcl-2 overexpressing astrocytes suffered < 40% injury after 24 h glucose deprivation, while controls were essentially completely injured. After exposure to 0.2 mM hydrogen peroxide, the bcl-2 overexpressing astrocytes suffered < 40% the injury seen in controls. In contrast, when the cultures were injured by combined oxygen-glucose deprivation, no difference in the extent or time course of injury was found between cells overexpressing bcl-2 and those expressing beta-galactosidase. To investigate one possible mechanism of bcl-2 protection, we measured the levels of glutathione and three antioxidant enzymes. Astrocytes overexpressing bcl-2 had elevated glutathione levels (130-200%), increased superoxide dismutase (170%) and glutathione peroxidase (140%) activities compared with beta-galactosidase-expressing controls. Bcl-2 overexpressing astrocytes suffered less lipid peroxidation after glucose deprivation, as assessed by cis-parinaric acid fluorescence, and demonstrated more rapid removal of hydrogen peroxide from the medium. When glutathione levels were decreased 80% by pretreatment with buthionine sulfoximine, the extent of protection from glucose deprivation of bcl-2 overexpressing cells was decreased by about half. Increased antioxidant defence contributes to protection from glucose deprivation in bcl-2 overexpressing astrocytes.
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PMID:Potentiation of murine astrocyte antioxidant defence by bcl-2: protection in part reflects elevated glutathione levels. 974 79

Apoptosis has been identified as a mechanism of pancreatic islet beta-cell death in autoimmune diabetes. Proinflammatory cytokines are candidate mediators of beta-cell death in autoimmune diabetes, and these cytokines can induce beta-cell death by apoptosis. In the present study, we examined whether transfection of human islet beta-cells with an anti-apoptotic gene, bcl-2, can prevent cytokine-induced beta-cell destruction. Human islet beta-cells were transfected by a replication-defective herpes simplex virus (HSV) amplicon vector that expressed the bcl-2 gene (HSVbcl-2) and, as a control, the same HSV vector that expressed a beta-galactosidase reporter gene (HSVlac). Two-color immunohistochemical staining revealed that 95+/-3% of beta-cells transfected with HSVbcl-2 expressed Bcl-2 protein compared with 14+/-3% of beta-cells transfected with HSVlac and 19+/-4% of nontransfected beta-cells. The bcl-2-transfected beta-cells were fully protected from impaired insulin secretion and destruction resulting from incubation for 5 days with the cytokine combination of interleukin (IL)-1beta, tumor necrosis factor (TNF)-alpha, and interferon (IFN)-gamma. In addition, the bcl-2-transfected islet cells were significantly protected from cytokine-induced lipid peroxidation and DNA fragmentation. These results demonstrate that cytokine-induced beta-cell dysfunction and death involve mechanisms subject to regulation by an anti-apoptotic protein, Bcl-2. Therefore, bcl-2 gene therapy has the potential to protect human beta-cells in pancreatic islets, or islet grafts, from immune-mediated damage in type 1 diabetes.
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PMID:Transfection of human pancreatic islets with an anti-apoptotic gene (bcl-2) protects beta-cells from cytokine-induced destruction. 1034 8

The prognosis for patients with esophageal cancer remains poor, prompting the search for new treatment strategies. Overexpression of E2F-1 has been shown to induce apoptosis in several cancer cell types. In the present study, the effect of adenovirus-mediated E2F-1 overexpression on human esophageal cancer cell lines Yes-4 and Yes-6 was evaluated. Cells were treated by mock infection, infection with an adenoviral vector expressing beta-galactosidase (Ad5CMV-LacZ), or E2F-1 (Ad5CMVE2F-1). Western blot analysis confirmed marked overexpression of E2F-1 in Ad5CMVE2F-1-infected cells. Overexpression of E2F-1 resulted in marked growth inhibition and rapid loss of cell viability due to apoptosis, although Yes-6 cells were somewhat more resistant to E2F-1-mediated growth inhibition than Yes-4 cells. Cell cycle analysis revealed that overexpression of E2F-1 led to G2 arrest, followed by apoptotic cell death. p53 expression remained undetectable in both cell lines after E2F-1 overexpression. The apoptosis inhibitor proteins of the Bcl-2 gene family, Bcl-2, Mcl-1, and BcI-XL, decreased at 48 h after infection in Yes-4 cells, but remained unchanged in Yes-6 cells. Levels of retinoblastoma gene product (pRb) declined at 48 h after E2F-1 infection in Yes-4 cells, at which apoptosis predominated, whereas pRb expression remained constant in Yes-6 cells. Expression of p14ARF did not change after E2F-1 infection in either cell line. Involvement of caspase 3 and caspase 6 in E2F-1-mediated apoptosis was demonstrated by cleavage of caspase 3/CPP32 and poly-ADP-ribose polymerase, as well as fragmentation of the caspase 6 substrate, lamin B. These results indicate that the sensitivity of esophageal cancer cells to E2F-1-mediated apoptosis may be related to differential expression of Bcl-2 family member proteins and suggest that the adenovirus-mediated E2F-1 gene therapy may be a promising treatment strategy for the treatment of this disease.
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PMID:Caspase activation and changes in Bcl-2 family member protein expression associated with E2F-1-mediated apoptosis in human esophageal cancer cells. 1077 92

The tumor suppressor protein p53 participates in normal cell differentiation as well as induction of programmed cell death. The authors investigated the effect of p53 overexpression on spermatogenesis by transferring p53 gene into the rat testes. Replication-deficient recombinant adenovirus vectors were constructed to include cytomegalovirus (CMV) promoter driving wild-type p53 (Ad-CMV-p53) or beta-galactosidase (Ad-CMV-beta-gal). Virus was delivered to cells of the tubules by slow retrograde injection through the rete testis. At 0, 4, 7, and 14 days, testes were removed, weighed, and analyzed histopathologically, including immunohistochemistry for p53, Bcl-2, Bax, and interleukin-1beta converting enzyme (ICE). Testicular weight was decreased in Ad-CMV-p53 group at 14 days after injection, while no change occurred in phosphate-buffered saline-injected controls or Ad-CMV-beta-gal-infected testes. Beyond 4 days, cell degradation in tubules interfered with immunohistochemical observation in the Ad-CMV-p53 group. At 4 days, p53 was expressed mostly in spermatocytes. Bax showed greater expression in the p53 group than in the control or Ad-CMV-beta-gal group. ICE, expressed mostly in spermatids, was more abundant in the p53 group than in controls. Overall, p53 overexpression in the testis impaired spermatogenesis.
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PMID:Adenovirus-mediated p53 gene transfer to rat testis impairs spermatogenesis. 1133 49

Although overexpression of E2F-1 can induce apoptosis in a variety of tumor cell lines, the mechanisms by which E2F-1 induces apoptosis remain ambiguous. In this study, we examine the ability of E2F-1 to induce apoptosis in colon cancer and the molecular mechanisms underlying E2F-1-mediated apoptosis. HT-29 and SW-620 colon adenocarcinoma cells (both mutant p53) were treated by mock infection or adenoviral vectors Ad5CMV (empty vector), Ad5CMVLacZ (beta-galactosidase), and Ad5CMVE2F-1 (E2F-1) at multiplicity of infection of 100. Western blot analysis confirmed marked overexpression of E2F-1 in both cell lines. By 5 days after infection, E2F-1 overexpression resulted in >25-fold reduction in cell growth and >90% loss of cell viability in both cell lines. Cell cycle analysis of Ad-E2F-1-infected cells revealed an increase in G(2)/M and sub-G(1) populations. By in situ terminal deoxynucleotidyl transferase (Tdt)-mediated nick end labeling analysis, evidence of apoptosis was observed including internucleosomal DNA fragmentation and the formation of apoptotic bodies. In addition, caspase-3 and poly(ADP-ribose) polymerase apoptotic fragments were detected by 48 h after treatment with Ad-E2F-1. Of mechanistic importance, overexpression of E2F-1 caused a G(2)/M arrest followed by increased levels of c-Myc and p14(ARF) proteins. Additionally, expression of the antiapoptotic Bcl-2 family member Mcl-1 was down-regulated in E2F-1-overexpressing cells. In conclusion, E2F-1 overexpression initiates apoptosis and suppresses growth in HT-29 and SW620 colon adenocarcinoma cells. Overexpression of E2F-1 triggers apoptosis and is associated with up-regulation of c-Myc and p14(ARF) proteins and down-regulation of Mcl-1. Therefore, E2F-1 is a potentially active gene therapy agent for the treatment of colon cancer.
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PMID:E2F-1 up-regulates c-Myc and p14(ARF) and induces apoptosis in colon cancer cells. 1170 81

Presenilin 2 (PS2) is a polytopic membrane protein that is mutated in some cases of familial Alzheimer's disease (AD). The normal functions of PS2 and its pathogenic role in AD remain unclear. We investigated the biological role of this protein in neurons, using adenovirus-mediated transduction of the PS2 gene into rat primary cortical neurons. Immunocytochemical analyses demonstrated increased PS2 immunoreactivity in most neurons infected with recombinant adenoviruses expressing PS2. Neurons infected with wild-type or mutant (N141I) PS2-expressing adenoviruses showed a significant increase in basal cell death, compared with those infected with control beta-galactosidase-expressing adenovirus. Moreover, PS2 overexpression markedly increased neuronal susceptibility to staurosporine-induced apoptosis. Mutant PS2 was more effective in enhancing apoptosis than its wild-type counterpart. Staurosporine-induced death was significantly inhibited by a specific caspase 3 inhibitor. Western analyses revealed that Bcl-2 protein expression was specifically down-regulated in neurons overexpressing PS2, which temporally corresponded to the accumulation of C- and N-terminal fragments of PS2. Additionally, expression of mutant, but not wild-type PS2, increased the production of beta-amyloid protein (Abeta) 42. These data collectively suggest that the pro-apoptotic effect of PS2 is mediated by down-regulation of Bcl-2. PS2 mutations may increase the susceptibility of neurons to apoptotic stimuli by perturbing the regulation of cell death.
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PMID:Pro-apoptotic effect of presenilin 2 (PS2) overexpression is associated with down-regulation of Bcl-2 in cultured neurons. 1175 57

Pancreatic cancer is often resistant to conventional chemotherapy. In this study, we examined the role of adenovirus-mediated overexpression of E2F-1 in inducing apoptosis and increasing the sensitivity of pancreatic cancer cells to chemotherapeutic agents. MIA PaCa-2 pancreatic head exocrine adenocarcinoma cells (mutant p53) were treated by mock infection or adenoviruses expressing beta-galactosidase or E2F-1 (Ad-E2F-1) alone or in combination with sublethal concentrations of each chemotherapeutic drug. Cell growth and viability were assessed at selected time points. Apoptosis was evaluated by flow cytometry, characteristic changes in cell morphology and poly (ADP-ribose) polymerase (PARP) cleavage. Western blot analysis was used to examine the expression of E2F-1 and Bcl-2 family member proteins and PARP cleavage. Western blot analysis revealed marked overexpression of E2F-1 at a multiplicity of infection (MOI) of 20 and 70. By 3 days after infection, Ad-E2F-1 treatment at an MOI of 70 resulted in approximately a 20-fold reduction in cell growth and 60% reduction in cell viability as compared to mock-infected cells. Cell cycle analysis, PARP cleavage and changes in cell morphology supported apoptosis as the mechanism of cell death in response to E2F-1. In order to test the efficacy of treatment with a combination of gene therapy and chemotherapy, we utilized concentrations of Ad-E2F-1 which reduced viability to 50% in combination with each chemotherapeutic agent. Cotreatment of the cells with E2F-1 virus and roscovitine (ROS) or etoposide resulted in an additive effect on cell growth inhibition and induction of apoptosis. Interestingly, 5-fluorouracil did not cooperate with Ad-E2F-1 in the mediation of tumor death or inhibition of cell growth. Immunoblotting for Bcl-2 family members revealed no significant changes in the expression levels of Bcl-2, Bcl X(L), Bax or Bak following gene or 'chemogene' therapy with E2F-1. However, a Bax cleavage product was noted which was substantially increased by cotreatment with ROS or etoposide. E2F-1 overexpression initiates apoptosis and suppresses growth in pancreatic MIA PaCa-2 cells in vitro. E2F-1-mediated apoptosis was not associated with significant changes in the expression of Bcl-2 family member proteins in these pancreatic cancer cells. ROS and etoposide, when combined with E2F-1 overexpression, induce apoptosis in an additive manner. This chemogene combination may provide a potentially useful therapeutic strategy for advanced pancreatic cancer.
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PMID:E2F-1 gene therapy induces apoptosis and increases chemosensitivity in human pancreatic carcinoma cells. 1206 45


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