BAFF regulates B cell survival by downregulating the BH3-only family member Bim via the ERK pathway

The B cell activating factor belonging to the tumor necrosis factor family (BAFF) is required for B cell survival and maturation. The mechanisms by which BAFF mediates B cell survival are less understood. We found that BAFF and a proliferation-inducing ligand (APRIL), which are related, block B cell antigen receptor (BCR)-induced apoptosis upstream of mitochondrial damage, which is consistent with a role for Bcl-2 family proteins. BCR ligation strongly increased expression of the proapoptotic Bcl-2 homology 3-only Bcl-2 protein Bim in both WEHI-231 and splenic B cells, and increases in Bim were reversed by BAFF or APRIL. Small interfering RNA vector-mediated suppression of Bim blocked BCR-induced apoptosis. BAFF also induced Bim phosphorylation and inhibited BCR-induced association of Bim with Bcl-2. BAFF induced delayed but sustained stimulation of extracellular signal-regulated kinase (ERK) and its activators, mitogen-activated protein kinase/ERK activating kinase (MEK) and c-Raf, and MEK inhibitors promoted accumulation and dephosphorylation of Bim. These results suggest that BAFF inhibits BCR-induced death by down-regulating Bim via sustained ERK activation, demonstrating that BAFF directly regulates Bim function. Although transitional immature type 1 (T1) B cell numbers are normal in Bim(-/-) mice, T2 and follicular mature B cells are elevated and marginal zone B cells are reduced. Our results suggest that mature B cell homeostasis is maintained by BAFF-mediated regulation of Bim.     

Chlamydia inhibit host cell apoptosis by degradation of proapoptotic BH3-only proteins

Chlamydia are obligate intracellular bacteria that replicate in a vacuole inside a host cell. Chlamydial infection has been shown to protect the host cell against apoptotic stimuli. This is likely important for the ability of Chlamydia to reproduce in human cells. Here we show that resistance to apoptosis is conveyed by the destruction of the proapoptotic BH3-only proteins Bim/Bod, Puma, and Bad during infection. Apoptotic stimuli were blocked upstream of the mitochondrial activation of Bax/Bak. During infection with both species, Chlamydia trachomatis and Chlamydia pneumoniae, Bim protein gradually disappeared without noticeable changes in Bim mRNA. The disappearance was blocked by inhibitors of the proteasome. Infected cells retained sensitivity to Bim expressed by transfection, indicating functional relevance of the Bim disappearance. Fusion to Bim targeted the green fluorescent protein for destruction during infection. Analysis of truncation mutants showed that a short region of Bim containing the BH3 domain was sufficient for destruction during chlamydial infection. Like Bim, Puma and Bad proteins disappeared during infection. These results reveal a novel way by which microbes can interfere with the host cell's apoptotic machinery, and provide a molecular explanation of the cellular resistance to apoptosis during infection with Chlamydia.     

BH3-only protein Noxa is a mediator of hypoxic cell death induced by hypoxia-inducible factor 1alpha

Hypoxia is a common cause of cell death and is implicated in many disease processes including stroke and chronic degenerative disorders. In response to hypoxia, cells express a variety of genes, which allow adaptation to altered metabolic demands, decreased oxygen demands, and the removal of irreversibly damaged cells. Using polymerase chain reaction-based suppression subtractive hybridization to find genes that are differentially expressed in hypoxia, we identified the BH3-only Bcl-2 family protein Noxa. Noxa is a candidate molecule mediating p53-induced apoptosis. We show that Noxa promoter responds directly to hypoxia via hypoxia-inducible factor (HIF)-1alpha. Suppression of Noxa expression by antisense oligonucleotides rescued cells from hypoxia-induced cell death and decreased infarction volumes in an animal model of ischemia. Further, we show that reactive oxygen species and resultant cytochrome c release participate in Noxa-mediated hypoxic cell death. Altogether, our results show that Noxa is induced by HIF-1alpha and mediates hypoxic cell death.     

Loss of the pro-apoptotic BH3-only Bcl-2 family member Bim inhibits BCR stimulation-induced apoptosis and deletion of autoreactive B cells

During development, the stochastic process assembling the genes encoding antigen receptors invariably generates B and T lymphocytes that can recognize self-antigens. Several mechanisms have evolved to prevent the activation of these cells and the concomitant development of autoimmune disease. One such mechanism is the induction of apoptosis in developing or mature B cells by engagement of the B cell antigen receptor (BCR) in the absence of T cell help. Here we report that B lymphocytes lacking the pro-apoptotic Bcl-2 family member Bim are refractory to apoptosis induced by BCR ligation in vitro. The loss of Bim also inhibited deletion of autoreactive B cells in vivo in two transgenic systems of B cell tolerance. Bim loss prevented deletion of autoreactive B cells induced by soluble self-antigen and promoted accumulation of self-reactive B cells developing in the presence of membrane-bound self-antigen, although their numbers were considerably lower compared with antigen-free mice. Mechanistically, we determined that BCR ligation promoted interaction of Bim with Bcl-2, inhibiting its survival function. These findings demonstrate that Bim is a critical player in BCR-mediated apoptosis and in B lymphocyte deletion.     

Loss of the BH3-only protein Bmf impairs B cell homeostasis and accelerates gamma irradiation-induced thymic lymphoma development

Members of the Bcl-2 protein family play crucial roles in the maintenance of tissue homeostasis by regulating apoptosis in response to developmental cues or exogenous stress. Proapoptotic BH3-only members of the Bcl-2 family are essential for initiation of cell death, and they function by activating the proapoptotic Bcl-2 family members Bax and/or Bak, either directly or indirectly through binding to prosurvival Bcl-2 family members. Bax and Bak then elicit the downstream events in apoptosis signaling. Mammals have at least eight BH3-only proteins and they are activated in a stimulus-specific, as well as a cell type-specific, manner. We have generated mice lacking the BH3-only protein Bcl-2-modifying factor (Bmf) to investigate its role in cell death signaling. Our studies reveal that Bmf is dispensable for embryonic development and certain forms of stress-induced apoptosis, including loss of cell attachment (anoikis) or UV irradiation. Remarkably, loss of Bmf protected lymphocytes against apoptosis induced by glucocorticoids or histone deacetylase inhibition. Moreover, bmf(-/-) mice develop a B cell-restricted lymphadenopathy caused by the abnormal resistance of these cells to a range of apoptotic stimuli. Finally, Bmf-deficiency accelerated the development of gamma irradiation-induced thymic lymphomas. Our results demonstrate that Bmf plays a critical role in apoptosis signaling and can function as a tumor suppressor.     

The proteasome inhibitor bortezomib sensitizes cells to killing by death receptor ligand TRAIL via BH3-only proteins Bik and Bim

Previously, we showed that the proteasome inhibitor bortezomib/Velcade (formerly PS-341) synergizes with the protein tumor necrosis factor alpha-related apoptosis-inducing ligand (TRAIL), a ligand for certain death receptors, to induce apoptosis in cell lines derived from prostate and colon cancers. Because apoptosis is often triggered by BH3-only proteins of the Bcl-2 family, we have explored the hypothesis that bortezomib contributes to the apoptosis by up-regulating their levels. Indeed, bortezomib induced increases of Bik and/or Bim in multiple cell lines but not notably of two other BH3-only proteins (Puma and Bid) nor other family members (Bax, Bak, Bcl-2, and Bcl-xL). The increase in Bik levels seems to reflect inhibition by bortezomib of its proteasome-mediated degradation. Importantly, both Bik and Bim seem central to the proapoptotic function of bortezomib, because mouse embryo fibroblasts in which the genes for both Bik and Bim had been disrupted were refractory to its cytotoxic action. Similarly, the synergy between bortezomib and TRAIL in killing human prostate cancer cells was impaired in cells in which both Bik and Bim were down-regulated by RNA interference. Further evidence that bortezomib acts through the mitochondrial pathway regulated by the Bcl-2 family is that deficiency for APAF-1, which acts downstream of Bcl-2, also blocked its apoptotic effect. These results implicate BH3-only proteins, in particular both Bik and Bim, as important mediators of the antitumor action of bortezomib and establish their role in its enhancement of TRAIL-induced apoptosis.     

Enhancement of thymidine kinase-mediated killing of malignant glioma by BimS,a BH3-only cell death activator

Herpes simplex virus thymidine kinase (HSV-tk)/gancyclovir (GCV) therapy has the ability to inhibit tumor formation in animal models but the results of clinical trials have been disappointing. To improve the performance of tk/GCV therapy, we tried combination therapy designed to enhance its cytotoxic effects by introducing genes that induce apoptosis of the tumor cells through different pathways. We concentrated our efforts on the use of Bim, a BH3-only member of death activators in the Bcl-2 superfamily, because Bim is not involved in the pathways through which HSV-tk/GCV therapy induces apoptosis in malignant glioma cells. Among three alternative splicing variants, BimEL, BimL, and BimS, BimS lacks the binding domain for the dynein light chain LC8, which negatively regulates the proapoptotic function of BimEL and BimL. All four malignant glioma cell lines, U251, A172, T-430, and U373 underwent cell death after transfer of BimS using an adenovirus vector (AVC2). Intriguingly, combination of AVC2-BimS with AVC2-tk markedly increased the sensitivity of U251 cells to GCV both in vitro and in vivo. In contrast, AVC2-BimL did not induce significant cell death. These results indicated that BimS had the ability to improve the efficiency of HSV-tk/GCV therapy in the treatment of malignant glioma and suggested that the targeting of different proapoptotic pathways may be a useful strategy for the development of an effective gene therapy approach to treatment.     

BH3-only protein mimetic obatoclax sensitizes cholangiocarcinoma cells to Apo2L/TRAIL-induced apoptosis

Human cholangiocarcinomas evade apoptosis by overexpression of Mcl-1. The drug obatoclax (GX15-070) inhibits antiapoptotic members of the Bcl-2 family including Mcl-1. The purpose of this study is to determine if obatoclax sensitizes human cholangiocarcinoma cells to apoptosis. The human cholangiocarcinoma cell lines, KMCH, KMBC, and TFK, were employed for these studies. Protein expression was assessed by immunoblot and protein-protein interactions detected by coprecipitation of the polypeptide of interest with S-tagged Mcl-1. Activation of Bak and Bax was observed by immunocytochemistry with conformation-specific antisera. Obatoclax induced minimal apoptosis alone; however, it increased apoptosis 3- to 13-fold in all three cancer cell lines when combined with Apo2L/tumor necrosis factor-related apoptosis-inducing ligand (TRAIL). Obatoclax did not alter cellular expression of Bid, Bim, Puma, Noxa, Bak, Bax, Mcl-1, or cFLIP. Mcl-1 binding to Bak was readily identified in untreated cells, and this association was disrupted by treating the cells with obatoclax. Additionally, Bim binding to Mcl-1 was markedly decreased by obatoclax treatment. We also identified alterations in Bak and Bax conformation following treatment with obatoclax plus Apo2L/TRAIL but not with either Apo2L/TRAIL or obatoclax alone. In conclusion, obatoclax releases Bak and Bim from Mcl-1 and sensitizes human cholangiocarcinoma cells to Apo2L/TRAIL-induced apoptosis. Obatoclax is a potentially promising adjunctive agent for the treatment of this cancer.     

Synergistic induction of TRAIL-mediated apoptosis by anisomycin in human hepatoma cells via the BH3-only protein Bid and c-Jun/AP-1 signaling pathway

Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) is a member of the TNF super-family, and it has been shown that many human cancer cell lines are refractory to TRAIL-induced cell death. However, the molecular mechanisms underlying resistance are unclear. In the present study, we show that TRAIL-resistance is reversed in human hepatoma cells by anisomycin, which is known to inhibit protein synthesis and induce ribotoxic stress. Synergistic induction of apoptosis in cells treated with anisomycin plus TRAIL was associated with activation of caspases and cleavage of Bid, a pro-apoptotic BH3-only protein. Silencing of Bid expression by small interfering RNA (siRNA) significantly attenuated the loss of mitochondrial membrane potential (MMP, Δψ_m) and significantly increased induction of apoptosis in cells treated with anisomycin and TRAIL, confirming that Bid cleavage is required for the response. In addition, c-Jun/AP-1 was rapidly activated upon stimulation with anisomycin; however, the knockdown of c-Jun/AP-1 expression by c-Jun siRNA markedly reduced anisomycin plus TRAIL-induced loss of MMP and apoptosis. Taken together, the findings show that anisomycin sensitizes TRAIL-mediated hepatoma cell apoptosis via the mitochondria-associated pathway, involving the cleavage of Bid and activation of the c-Jun/AP-1 pathway, indicating that this compound can be used as an anti-tumor agent in combination with TRAIL.     

VEGF-B inhibits apoptosis via VEGFR-1-mediated suppression of the expression of BH3-only protein genes in mice and rats

Despite its early discovery and high sequence homology to the other VEGF family members, the biological functions of VEGF-B remain poorly understood. We revealed here a novel function for VEGF-B as a potent inhibitor of apoptosis. Using gene expression profiling of mouse primary aortic smooth muscle cells, and confirming the results by real-time PCR using mouse and rat cell lines, we showed that VEGF-B inhibited the expression of genes encoding the proapoptotic BH3-only proteins and other apoptosis- and cell death-related proteins, including p53 and members of the caspase family, via activation of VEGFR-1. Consistent with this, VEGF-B treatment rescued neurons from apoptosis in the retina and brain in mouse models of ocular neurodegenerative disorders and stroke, respectively. Interestingly, VEGF-B treatment at the dose effective for neuronal survival did not cause retinal neovascularization, suggesting that VEGF-B is the first member of the VEGF family that has a potent antiapoptotic effect while lacking a general angiogenic activity. These findings indicate that VEGF-B may potentially offer a new therapeutic option for the treatment of neurodegenerative diseases.     

The pro‐apoptotic BH3‐only protein Bim regulates cell cycle progression of hematopoietic progenitors during megakaryopoiesis

Summary. Background: The pro‐apoptotic BH3‐only protein Bim is recognized as a pivotal regulator of apoptosis induced by the depletion of cytokines. In the present study, we examined the role of Bim in megakaryopoiesis. Methods: Megakaryocyte (MK) progenitors obtained from bim knockout (KO) mice were analyzed in vitro for liability to apoptosis after the depletion of cytokines, ability to differentiate into MKs and proliferation/cell cycle progression in response to thrombopoietin (TPO). The production of platelets in vitro was evaluated by assaying the formation of proplatelets in MKs. Megakaryopoiesis in vivo was observed in a mouse model of thrombocytopenia induced by injecting fluorouracil (5‐FU). Results: Bim‐deficient CD34‐/c‐kit+/Sca‐1+/Lineage‐ stem cells and MKs were highly resistant to apoptosis induced by cytokine depletion, suggesting that Bim is involved in the apoptotic process in both stem cells and MKs. As bim KO mice exhibited splenomegaly and thrombocytopenia, splenectomized mice were used for experiments in vivo. Platelet recovery after 5‐FU‐induced thrombocytopenia was significantly delayed in bim KO mice. Corresponding with this, numbers of MKs in the recovery phase bone marrow were significantly reduced in bim KO mice. Culture of c‐kit+/Lineage‐ progenitors with TPO revealed that Bim‐deficient cells poorly proliferate and differentiate into CD41+ cells in comparison with wild‐type (WT) cells. However, once differentiated into MKs, these cells matured normally. Furthermore, cell cycle analyses demonstrated that transition from the G1 to the S phase was delayed in Bim‐deficient stem cells. Conclusions: In the present study, we demonstrated that Bim plays a pivotal role in the regulation of cell cycle progression in hepatopoietic progenitors during megakaryopiesis.     

极端热环境心肌细胞损伤乳鼠心肌细胞凋亡与Bim及caspase-3蛋白的表达

背景:目前有关极端气候条件下疾病发生规律、机制及防治措施的研究,国内只有零星报道,均为病理生理表现的研究,尚未深入到分子机制的研究.目的:观察前凋亡蛋白Bim及caspase-3在极端热环境致乳鼠心肌细胞损伤中的作用.方法:体外培养1～3 d龄SD大鼠心肌细胞,建立心肌细胞热环境损伤模型(42℃,60 min).观察热处珲后恢复37℃孵育0,4,8,12,16,24 h时心肌细胞损伤情况.倒置显微镜下观测各组心肌细胞搏动频率和节律;MTS/PMS法检测细胞存活率;全自动生化分析仪测定心肌细胞培养液乳酸脱氢酶活性;Annexin V-FITC/PI双染流式细胞术检测细胞凋亡率;Western blot检测Bim及caspase-3蛋白表达.结果与结论:体外培养乳鼠心肌细胞热处理后即刻(0 h)搏动频率略有增加,并可见异常节律,此后随时间的延长心肌细胞搏动频率逐渐减慢,存活率下降(P<0.05),乳酸脱氧酶活性升高(P<0.05);恢复37℃孵育8 h时前凋亡蛋白Bim显著升高(P<0.05):caspase-3从恢复37℃孵育时表达即明显升高(P<0.05).因此,提示极端热环境使心肌细胞发生了损伤,且以凋亡为主要损伤形式,前凋亡蛋白Bim与caspase-3可能参与了极端热环境致乳鼠心肌细胞凋亡的过程.     

Loss of the proapoptotic protein, Bim, breaks B cell anergy

Although B cells that respond with high avidity to self-antigen are eliminated early in their development, many autoreactive B cells escape elimination and are tolerized later in their lives via anergy. Anergic B cells are unresponsive to antigen and die prematurely. It has been suggested that the proapoptotic protein, Bim, controls the fate of anergic B cells. To test this idea, mice lacking Bim were crossed with mice that express soluble hen egg lysozyme and whose B cells bear receptors specific for the protein. In Bim+/+ mice these B cells are anergic and die rapidly. If the mice lack Bim, however, the B cells live longer, are more mature, respond to antigen, and secrete anti-hen egg lysozyme antibodies. This break of tolerance is not due to expression of endogenous B cell receptors, nor is it dependent on T cells. Rather, it appears to be due to a reduced requirement for the cytokine BAFF. Normal B cells require BAFF both for differentiation and survival. Bim-/- B cells, on the other hand, require BAFF only for differentiation. Therefore, autoreactive B cells are allowed to survive if they lack Bim and thus accumulate sufficient signals from differentiating factors to drive their maturation and production of autoantibodies.     

CD27 cooperates with the pre-T cell receptor in the regulation of murine T cell development

CD27 is a lymphocyte-specific member of the TNF receptor family and has a TNF-related transmembrane ligand, CD70. The CD27/CD70 receptor-ligand pair cooperates with the TCR in the regulation of the peripheral T cell response. The study presented here reveals that CD27 may play a similar role in thymic pre-T cell development. We have previously cloned the cDNA encoding murine CD27, prepared specific mAbs and observed that murine CD27 is expressed on virtually all thymocytes, with the exception of a subpopulation of CD4-8- precursor T cells. It is shown here that induction of murine CD27 expression occurs at the transition from the CD4-8-25+ to the CD4-8-25- precursor T cell stage and is regulated by the pre-TCR. Therefore, we investigated whether CD27 contributes to pre-TCR-mediated thymocyte development. Pre-TCR function was mimicked by the induction of CD3 signaling in thymocytes of recombination activating gene (RAG)-deficient mice. This in vivo anti- CD3 epsilon mAb treatment induces an about fifty fold numerical expansion of CD4-8-25+ thymocytes and their differentiation to the CD4+8+25- stage. Co-injection of anti-CD27 mAb inhibited the CD3- mediated expansion and differentiation of the CD4-8-25+ precursor population. Also, injection of anti-CD27 mAb in TCR alpha-/- mutant mice led to a reduction in the absolute number of CD4+8+25- thymocytes. We present evidence that in these in vivo systems, anti-CD27 mAb inhibits CD27-ligand interaction. Therefore, we conclude that CD27 may contribute to normal murine T cell development by synergizing with the pre-TCR-mediated signal.     

细胞凋亡在帕金森病发病机制中的作用

目的研究帕金森病(PD)病因中细胞凋亡的发生及作用机制。方法应用6－OHDA毁损大鼠纹状体,以制备PD大鼠模型,应用酪氨酸羟化酶(TH)和DNA原位末端标记免疫组化双标染色检测黑质内多巴胺(DA)神经元细胞凋亡的发生及其变化规律。结果成功复制出符合临床特点的PD大鼠模型,其黑质内DA神经元的丢失是以细胞凋亡为主要形式,且于术后2周及1月为最明显,术后2个月仍然存在细胞凋亡情况。结论黑质内DA神经元细胞凋亡的发生将使其数目减少,从而导致帕金森病的发生,其机制可能与纹状体区病变有关。     

Essential role of survivin, an inhibitor of apoptosis protein, in T cell development, maturation, and homeostasis

Survivin is an inhibitor of apoptosis protein that also functions during mitosis. It is expressed in all common tumors and tissues with proliferating cells, including thymus. To examine its role in apoptosis and proliferation, we generated two T cell-specific survivin-deficient mouse lines with deletion occurring at different developmental stages. Analysis of early deleting survivin mice showed arrest at the pre-T cell receptor proliferating checkpoint. Loss of survivin at a later stage resulted in normal thymic development, but peripheral T cells were immature and significantly reduced in number. In contrast to in vitro studies, loss of survivin does not lead to increased apoptosis. However, newborn thymocyte homeostatic and mitogen-induced proliferation of survivin-deficient T cells were greatly impaired. These data suggest that survivin is not essential for T cell apoptosis but is crucial for T cell maturation and proliferation, and survivin-mediated homeostatic expansion is an important physiological process of T cell development.     

Enhancement of radiation sensitivity with BH3I-1 in non-small cell lung cancer

BACKGROUND: Anti-apoptotic proteins, such as Bcl-2 and Bcl-xL, are frequently over-expressed in human malignancies, and this is correlated with resistance to chemotherapeutic drugs and gamma- radiation. Recently identified small organic molecules capable of inhibiting Bcl-2 and/or Bcl-xL function, may enhance radiation sensitivity of cancer cells in which they are over expressed. We examined whether specific blockade of the BH3-domain binding to Bcl-xL could sensitize cancer cells to gamma- radiation. METHODS: Human non-small-cell lung cancer H460 cells with wild-type p53 and H1792 cells with mutant p53 were exposed to various doses of radiation and/or BH3I-1 and for different points of time to BH3I-1 treatment. XTT and clonogenic survival assays were used to evaluate the growth-inhibitory effects of the antagonist BH3I-1, ionizing radiation or both. Western blot analysis was used to examine the cellular effect of the expression of Bcl-xL, Bax, and p53. Apoptosis and cell cycle distribution were analyzed by confocal microscopy with Hoechst 33258 staining and cytochrome c, and flow cytometry, respectively. RESULTS: BH3I-1 appeared to induce a dose- and time-dependent apoptosis in H460 and H1792 cells, regardless of p53 status. After 2 days of BH3I-1 treatment, the cells that remained attached were exposed to ionizing radiation. Followed by clonogenic assay, BH3I-1 treatment enhanced the radiation sensitivity of H1792 surviving cells with mutant p53, but not in H460 cells with wild-type p53. A transient time-dependent cell cycle blockade at G2-M phase was identified for H1792 cells without subsequent modification of cell cycle distribution. CONCLUSION: These findings suggest a potential role for the small molecule inhibitor as a novel radiation sensitizer in non-small cell lung cancer.     

Bruton's tyrosine kinase cooperates with the B cell linker protein SLP-65 as a tumor suppressor in Pre-B cells

Expression of the pre-B cell receptor (pre-BCR) leads to activation of the adaptor molecule SLP-65 and the cytoplasmic kinase Btk. Mice deficient for one of these signaling proteins have an incomplete block in B cell development at the stage of large cycling pre-BCR+CD43+ pre-B cells. Our recent findings of defective SLP-65 expression in approximately 50% of childhood pre-B acute lymphoblastic leukemias and spontaneous pre-B cell lymphoma development in SLP-65-/- mice demonstrate that SLP-65 acts as a tumor suppressor. To investigate cooperation between Btk and SLP-65, we characterized the pre-B cell compartment in single and double mutant mice, and found that the two proteins have a synergistic role in the developmental progression of large cycling into small resting pre-B cells. We show that Btk/SLP-65 double mutant mice have a dramatically increased pre-B cell tumor incidence ( approximately 75% at 16 wk of age), as compared with SLP-65 single deficient mice (<10%). These findings demonstrate that Btk cooperates with SLP-65 as a tumor suppressor in pre-B cells. Furthermore, transgenic low-level expression of a constitutive active form of Btk, the E41K-Y223F mutant, prevented tumor formation in Btk/SLP-65 double mutant mice, indicating that constitutive active Btk can substitute for SLP-65 as a tumor suppressor.     

Expression of HCV E1 protein in baculovirus-infected cells: effects on cell viability and apoptosis induction

The molecular mechanisms of pathogenesis in hepatitis C virus (HCV) infection are not yet understood. Recently, we reported that the expression of the envelope protein E1 is toxic for Escherichia coli cells. The toxicity is related to the ability of C-terminal transmembrane (TM) domain of E1 to modify membrane permeability. In this study we expressed the E1 protein, complete (a.a. 192-383) or deleted (a.a. 192-340) of the TM region, fused to the C-terminus of glutathione-S-transferase by two recombinant baculoviruses. Infection of Sf9 insect cells by E1 baculovirus induced a rapid decrease in cell viability in the first 18-24 h postinfection. Premature cytopathic changes and low level of E1 protein expression were also reported. The analysis of DNA isolated from cells revealed a typical internucleosomal ladder pattern characteristic of apoptosis. The DNA degradation was first detected at 18 h postinfection by ethidium bromide gel electrophoresis and was confirmed by TUNEL assay. The results indicated that the C-terminal domain of E1 is essential for apoptosis induction as neither cell death nor DNA degradation were observed following infection with the recombinant baculovirus expressing the C-terminal-deleted E1. These findings support the hypothesis that the TM domain of E1 may play a role in viral pathogenesis.