静脉注射41BBLGFP融合表达质粒体内表达及其生物学活性

通过RTPCR方法扩增小鼠41BBLcDNA,以pEGFPN1为载体,构建融合蛋白41BBLGFP重组表达质粒p41BBLGFP.采用基于流体力学原理建立的裸DNA体内转染技术,从小鼠尾静脉快速(15s)注射质粒p41BBLGFP进行体内转染.荧光显微镜观察组织切片,见小鼠肝、脾、肾及肺中均有报告基因GFP表达,尤以肝细胞中荧光最强.进一步用Western印迹和免疫组织化学染色法确定肝细胞表面表达41BBL,用Hsp70H22细胞抗原肽皮下免疫小鼠,同时尾静脉注射质粒p41BBLGFP,检测血清中IL2和IFNγ的分泌.结果显示,质粒注射联合免疫组小鼠血清IL2和IFNγ的浓度分别较生理盐水对照组增加了3倍和4倍;脾细胞对H22细胞的杀伤率则由单独免疫组的45.74%±3.27%增至86.74%±9.36%.结果表明,体内(主要在肝脏)转染质粒p41BBLGFP可以成功表达,表达产物具有41BBL的生物学活性,为进一步研究体内转染41BBL用于基因治疗奠定了基础.     

新型聚乙烯亚胺衍生物体内转染活性的研究

目的:研究新型聚乙烯亚胺衍生物作为基因载体在小鼠体内的转染活性情况。方法:采用荧光素酶质粒DNA作为报告基因,与基因载体复合,对小鼠尾静脉注射,考察组织分布情况;以及对小鼠肌肉注射,应用活体成像技术考察其转染活性。结果:静脉注射后,小鼠肺组织中基因表达量最高;肌肉注射后,可以观察到活性转染结果,表达量高于对照PE(?)25KDa。结论:新型聚乙烯亚胺衍生物在体内可成功输送质粒DNA,但转染效率偏低。     

Lipopolysaccharide recognition protein, MD-2, facilitates cellular uptake of E. coli-derived plasmid DNA in synovium

BACKGROUND: Several cell types are susceptible to transfection in vivo using naked plasmid DNA. The mechanisms involved in mediating in vivo transfection are incompletely known, but evidence suggests that receptor-mediated endocytosis is important for specific types of cells. In this study we tested the hypothesis that residual Escherichia coli lipopolysaccharide (LPS) forms a non-covalent complex with expression plasmid DNA, and host-cell-derived soluble LPS-binding proteins bind to the DNA-LPS complexes in order to facilitate receptor-mediated endocytosis. METHODS: Cells from the murine synovial lining were used as an in vivo model system and in vivo luciferase imaging was used to quantify levels of transgene expression. Using a series of gene-deleted mice, the roles of LPS recognition complex proteins, lipopolysaccharide-binding protein (LBP), CD14 and MD-2, in the process of in vivo transfection were determined. RESULTS: Luciferase expression assays revealed that mice lacking LBP or CD14 had increased luciferase expression (p < 0.023 and < 0.165, respectively), while mice deleted of MD-2 had significant reductions in luciferase expression (p < 0.001). Gene deletion of hyaluronic acid binding protein CD44 was used as a control and had no statistically significant effect on transgene expression in vivo. In muscle tissue, where neither cell surface nor soluble MD-2 is expressed, no MD-2 dependence of plasmid transfection was identified, suggesting the role of MD-2 is tissue or cell type specific. Additionally, depleting mice of macrophages showed that luciferase expression is occurring within fibroblast-like synoviocytes. CONCLUSIONS: Our data support a physical association between LPS and E. coli-derived plasmid DNA, and that in vivo transfection of fibroblast-like synoviocytes is dependent on the soluble form of the LPS-binding protein MD-2.     

Silencing USP22 by asymmetric structure of interfering RNA inhibits proliferation and induces cell cycle arrest in bladder cancer cells

The ubiquitin specific peptidase 22 (USP22) is a positive regulator of the growth of tumors. However, little is known about the impact of USP22 knockdown on the growth of human bladder cells. In the present study, we designed a series of asymmetric interfering RNAs (aiRNAs) and compared the efficacy of aiRNA and conventional symmetric interfering RNA (siRNA) in the silencing of USP22 expression and the growth of human bladder EJ cells in vitro and in vivo. In comparison with transfection with the USP22-specific siRNA, transfection with 15/21 aiRNA was more potent in down-regulating the USP22 expression and inhibiting EJ cell proliferation in vitro. Furthermore, transfection with 15/21 aiRNA induced higher frequency of EJ cells arrested at the G0/G1 phases, but did not trigger EJ cell apoptosis. Moreover, transfection with either the siRNA or 15/21 aiRNA up-regulated the expression of p53 and p21, but down-regulated the expression of cyclin E and Mdm2 in EJ cells. The up-regulated p53 expression induced by the specific siRNA or aiRNA was abrogated by induction of Mdm2 over-expression. In addition, treatment with the specific siRNA or aiRNA inhibited the growth of implanted human bladder tumors in mice and the aiRNA had more potent anti-tumor activity in vivo. Therefore, our data suggest that knockdown of USP22 expression by the aiRNA may down-regulate the expression of Mdm2 and cyclin E, resulting in the up-regulated expression of p53 and p21 and leading to cell cycling arrest and inhibition of human bladder EJ cell proliferation. Our findings indicate that the USP22-specific aiRNA may be a novel approach for the intervention of human bladder tumors.     

Role of cyclin kinase inhibitor p21 in systemic autoimmunity

The cyclin kinase inhibitor protein p21 affects multiple processes relevant to the immune system, including cell cycle progression, replicative senescence, hemopoietic stem cell quiescence, and apoptosis. Therefore, malfunction of this protein may be a contributor to the pathogenesis of systemic autoimmunity. Here, we report that mixed background p21-deficient 129/Sv x C57BL/6 mice showed increased in vitro and in vivo T cell cycling and activation, moderate hypergammaglobulinemia and, at low penetrance, anti-chromatin autoantibodies. Homeostatic anti-self MHC/peptide ligand-induced proliferation of p21-deficient T cells was also enhanced. However, lymphoid organ enlargement was very mild, presumably due to increased apoptosis of the rapidly dividing cells. Moreover, the older p21-deficient mice had kidney pathology representing a similar, but slightly more advanced, state than that seen in the control mice. The timing and severity of the above serologic, cellular, and histologic manifestations in p21-deficient mice were unaffected by gender. Thus, p21 deficiency significantly enhances T cell activation and homeostatic proliferation, and can induce mild autoimmune manifestations at a low incidence without gender bias, but does not in itself generate the full spectrum of lupus-like disease.     

Thymus-derived inhibitor of lymphocyte proliferation. II. Tissue specificity in vitro and in vivo

A thymus crude factor (TCF) isolated from bovine thymus tissue has been tested for its effects on the proliferation of various murine cells. Specific inhibition in vitro has been found for DNA synthesis in murine T and B lymphocytes which appears not to be based on cytotoxicity. Moreover, TCF, when administered to mice, also interferes with the DNA synthesis in lymphoid tissue in vivo. Our data are suggestive for the presence in TCF of an endogenous 'chalone-like' inhibitor of lymphoid cell proliferation in vitro and in vivo.     

Bacterial magnetic particles (BMPs)-PEI as a novel and efficient non-viral gene delivery system

BACKGROUND: Non-viral methods of gene delivery, especially using polyethylenimine (PEI), have been widely used in gene therapy or DNA vaccination. However, the PEI system has its own drawbacks, which limits its applications. METHODS: We have developed a novel non-viral delivery system based on PEI coated on the surface of bacterial magnetic nanoparticles (BMPs). The ability of BMPs-PEI complexes to bind DNA was determined by retardation of plasmid DNA in agarose gel electrophoresis. The transfection efficiency of BMPs-PEI/DNA complexes into eukaryotic cells was determined by flow cytometric analysis. The MTT assay was invited to investigate the cytotoxicity of BMPs-PEI/DNA complexes. The expression efficiency in vivo of BMPs-PEI bound to the plasmid pCMVbeta encoding beta-galactosidase was evaluated intramuscularly inoculated into mice. The immune responses of in vivo delivery of BMPs-PEI bound plasmid pcD-VP1 were determined by MTT assay for T cell proliferation and ELISA for detecting total IgG antibodies. RESULTS: BMPs-PEI complexes could bind DNA and provide protection from DNase degradation. The transfection efficiency of BMPs-PEI/DNA complexes was higher than that in PEI/DNA complexes. Interestingly, in contrast to PEI, the BMPs-PEI complex was less cytotoxic to cells in vitro. We further demonstrated that the BMPs-PEI system can deliver an exogenous gene to animals and allow it to be expressed in vivo. Such expression resulted in higher levels of humoral and cellular immune responses against the target antigen compared to controls. CONCLUSIONS: We have developed a novel BMPs-PEI gene delivery system with a high transfection efficiency and low toxicity, which presents an attractive strategy for gene therapy and DNA vaccination.     

Genetic immunization with lung-targeting macroaggregated polyethyleneimine-albumin conjugates elicits combined systemic and mucosal immune responses

Genetic immunization is a novel form of vaccination in which transgenes are delivered into the host to produce the foreign protein within host cells. Although systemic immune responses have been relatively easy to induce by genetic immunization, the induction of regional and mucosal immunity has often been more challenging. To address the problem of eliciting mucosal immunity in the lung, we utilized macroaggregated albumin to target plasmid DNA to the lung. Macroaggregated albumin is trapped in the lung after i. v. injection, and it is routinely used in radiolabeled form as an imaging modality to evaluate pulmonary blood flow. To couple DNA to this targeting agent, polyethyleneimine (a polycation that binds DNA and enhances transfection) was conjugated to serum albumin, and the conjugate was aggregated by heating to produce particles of 25-100 microm. The resulting particles bound plasmid DNA avidly, and when injected i.v. in mice, the particles distributed in the peripheral lung tissue in the alveolar interstitium. Particle-bound luciferase plasmid transfected a variety of cell lines in vitro, and after i.v. injection, gene expression was detected exclusively in the lung. Using human growth hormone as the encoded foreign Ag for immunization, i.v. injection of the particle-bound plasmid elicited both pulmonary mucosal and systemic immune responses, whereas naked DNA injected either i.v. or i.m. elicited only systemic responses. Thus, particle-bound plasmid DNA may have utility for genetic immunization by intravascular delivery to the lung and potentially to other organs and tissues.     

Tumor-targeting magnetic lipoplex delivery of short hairpin RNA suppresses IGF-1R overexpression of lung adenocarcinoma A549 cells in vitro and in vivo

Liposomal magnetofection potentiates gene transfection by applying a magnetic field to concentrate magnetic lipoplexes onto target cells. Magnetic lipoplexes are self-assembling ternary complexes of cationic lipids with plasmid DNA associated with superparamagnetic iron oxide nanoparticles (SPIONs). Type1insulin-like growth factor receptor (IGF-1R), an important oncogene, is frequently overexpressed in lung cancer and mediates cancer cell proliferation and tumor growth. In this study, we evaluated the transfection efficiency (percentage of transfected cells) and therapeutic potential (potency of IGF-1R knockdown) of liposomal magnetofection of plasmids expressing GFP and shRNAs targeting IGF-1R (pGFPshIGF-1Rs) in A549 cells and in tumor-bearing mice as compared to lipofection using Lipofectamine 2000. Liposomal magnetofection provided a threefold improvement in transgene expression over lipofection and transfected up to 64.1% of A549 cells in vitro. In vitro, IGF-1R specific-shRNA transfected by lipofection inhibited IGF-1R protein by 56.1 ± 6% and by liposomal magnetofection by 85.1 ± 3%. In vivo delivery efficiency of the pGFPshIGF-1R plasmid into the tumor was significantly higher in the liposomal magnetofection group than in the lipofection group. In vivo IGF-1R specific-shRNA by lipofection inhibited IGF-1R protein by an average of 43.8 ± 5.3%; that by liposomal magnetofection inhibited IGF-1R protein by 43.4 ± 5.7%, 56.3 ± 9.6%, and 72.2 ± 6.8%, at 24, 48, and 72 h, respectively, after pGFPshIGF-1R injection. Our findings indicate that liposomal magnetofection may be a promising method that allows the targeting of gene therapy to lung cancer.     

Concerted effect of transforming growth factor-beta, cyclin inhibitor p21, and c-myc on smooth muscle cell proliferation

Increased aortic smooth muscle cell (SMC) proliferation is a key event in the pathogenesis of atherosclerosis. Transforming growth factor-beta (TGF-beta) is one of the potent inhibitors of SMC proliferation. The purpose of this study was 1) to explore the effect of TGF-beta inhibition on proliferation of SMC and expression of growth regulatory molecules like p21 and c-myc and 2) to determine whether restoration of cell cycle regulatory molecules normalizes the altered proliferation. To test the role of TGF-beta in SMC proliferation, using antisense plasmid DNA, we inhibited TGF-beta gene from aortic SMC, which resulted in a significant increase (P < 0.03) in proliferation (studied by quantifying new DNA synthesis with [(3)H]thymidine uptake assay). In TGF-beta-altered SMC (TASMC), the mRNA expression (studied by RT-PCR) of c-myc was increased whereas that of the cyclin inhibitor p21 was completely inhibited. Using p21 sense plasmid DNA, we transfected p21 gene in TASMC, which restored p21 mRNA and protein expression and decreased proliferation (P < 0.002) in TASMC. Similar treatment with c-myc antisense oligonucleotides significantly (P < 0.001) decreased the proliferation of TASMC. TASMC also exhibited alteration in morphological changes in SMC but returned to normal with treatment of p21 and TGF-beta sense plasmid DNA. Two-dimensional gel electrophoresis analysis of SMC and TASMC demonstrated differential expression of proteins relevant to cellular proliferation and atherosclerosis. This study uniquely analyzes the effect of TGF-beta at the molecular level on proliferation of SMC and on cell cycle regulatory molecules, implicating their potential role in the pathogenesis of atherosclerosis.     

Magnolol suppresses proliferation of cultured human colon and liver cancer cells by inhibiting DNA synthesis and activating apoptosis

Magnolol, a hydroxylated biphenyl compound isolated from the Chinese herb Hou p'u of Magnolia officinalis, has been reported to have anti-cancer activity. In the present study, magnolol at very low concentrations of 3-10 microM inhibited DNA synthesis and decreased cell number in cultured human cancer cells (COLO-205 and Hep-G2) in a dose-dependent manner, but not in human untransformed cells such as keratinocytes, fibroblasts, and human umbilical vein endothelial cells (HUVEC). Magnolol was not cytotoxic at these concentrations and this indicates that it may have an inhibitory effect on cell proliferation in the subcultured cancer cell lines. [(3)H] thymidine incorporation and flow cytometry analyses revealed that magnolol treatment decreased DNA synthesis and arrested the cells at the G0/G1 phase of the cell cycle. Moreover, the magnolol-induced cell cycle arrest occurred when the cyclin-CDK system was inhibited, just as p21 protein expression was augmented. When magnolol concentration was increased to 100 microM, apoptosis was observed in COLO-205 and Hep-G2 cells, but not in cultured human fibroblasts and HUVEC. COLO-205 cells implanted subcutaneously in nude mice formed solid tumors; subsequent daily i.p.-injections of magnolol led to profound regression of these tumors of up to 85%. In these tumors, an increase in the expression of p21 protein level and the occurrence of apoptosis were observed. These findings demonstrate for the first time that magnolol can inhibit the proliferation of tumor cells in vitro and in vivo.     

Induction of CDK inhibitor p21 gene as a new therapeutic strategy against pulmonary fibrosis

Alveolar epithelial cells are known to be present at the primary site of lung damage in pulmonary fibrosis. Apoptosis has been implicated as being involved in epithelial cell damage and pulmonary fibrosis. Because the cyclin-dependent kinase inhibitor p21 induces G1 arrest and DNA repair and because it also prevents apoptosis in some cells, we hypothesized that p21 gene transfer may attenuate bleomycin-induced pulmonary fibrosis in mice, the pathogenesis of which likely involves epithelial cell apoptosis. Human p21 protein was expressed in mouse alveolar epithelial cells at 1-7 days in vitro and was detected predominantly in lung epithelial cells at 1-7 days in vivo after adenoviral transfer of the human p21 gene. Inflammatory cell infiltration and fibrosis had already begun at 7 days in this model. Adenoviral transfer of the human p21 gene at 7 days after intratracheal instillation of bleomycin led to a decrease in the number of apoptotic cells, lung inflammation, and fibrosis at 14 days. Therefore, the forced expression of p21 exerted both anti-apoptotic and anti-fibrotic effects, which would facilitate the ultimate goal of treatment for pulmonary fibrosis.     

Ultrasound／Microbubble Enhances Foreign Gene Expression in ECV304 Cells and Murine Myocardium

The success of gene therapy is largely dependent onthe development of vectors or vehicles that can selectivelyand efficiently deliver a therapeutic gene to cells or targetissues with minimal toxicity. Viruses are efficient transducing vectors. However, the safety concerns regardingthe use of virus vector in human make nonviral deliverysystem an attractive focus. Nonviral vectors are particularly suitable with respect to the simplicity of use, possibility of large-scale production and lack of s…     

Pigment epithelium-derived factor reduces the PDGF-induced migration and proliferation of human aortic smooth muscle cells through PPARγ activation

Our previous study demonstrated that pigment epithelium-derived factor (PEDF) plays an important role in the proliferation and migration of human aortic smooth muscle cells (HASMCs). In the present study, we examined whether PEDF inhibited platelet-derived growth factor (PDGF)-stimulated HASMC migration and proliferation. PEDF dose-dependently reduced PDGF-induced HASMC migration and proliferation in vitro and also arrested cell cycle progression in the G0/G1 phase, and this was associated with decreased expression of cyclin D1, cyclin E, CDK2, CDK4, and p21(Cip1) and increased expression of the cyclin-dependent kinase inhibitor p27(Kip1). The antiproliferative and antimigratory effects of PEDF were partially blocked by the PPARγ antagonist GW9662, but not by the PPARα antagonist MK886. In in vivo studies, the femoral artery of C57BL/6 mice was endothelial-denuded and the mice injected intravenously with PEDF or vehicle. After 2 weeks, both the neointima/media area ratio and cell proliferation (proliferating cell nuclear antigen-positive cells) in the neointima were significantly reduced and again these effects were partially reversed by GW9662 pretreatment. Our data show that PEDF increases PPARγ activation, preventing entry of HASMCs into the cell cycle in vitro and reducing the neointimal area and cell proliferation in the neointima in vivo. Thus, PEDF may represent a safe and effective novel target for the prevention and treatment of vascular proliferative diseases.     

p27Kip1 and p130 cooperate to regulate hematopoietic cell proliferation in vivo

To investigate the potential functional cooperation between p27Kip1 and p130 in vivo, we generated mice deficient for both p27Kip1 and p130. In p27Kip1-/-; p130-/- mice, the cellularity of the spleens but not the thymi is significantly increased compared with that of their p27Kip1-/- counterparts, affecting the lymphoid, erythroid, and myeloid compartments. In vivo cell proliferation is significantly augmented in the B and T cells, monocytes, macrophages, and erythroid progenitors in the spleens of p27Kip1-/-; p130-/- animals. Immunoprecipitation and immunodepletion studies indicate that p130 can compensate for the absence of p27Kip1 in binding to and repressing CDK2 and is the predominant CDK-inhibitor associated with the inactive CDK2 in the p27Kip1-/- splenocytes. The finding that the p27Kip1-/-; p130-/- splenic B cells are hypersensitive to mitogenic stimulations in vitro lends support to the concept that the hyperproliferation of splenocytes is not a result of the influence of their microenvironment. In summary, our findings provide genetic and molecular evidence to show that p130 is a bona fide cyclin-dependent kinase inhibitor and cooperates with p27Kip1 to regulate hematopoietic cell proliferation in vivo.     

Enhancement of dendrimer-mediated transfection using synthetic lung surfactant exosurf neonatal in vitro.

Pulmonary surfactants enhance adenovirus-mediated gene transfer but inhibit cationic liposome-mediated transfection in lung epithelial cells in vitro. This study examines the effect of the synthetic lung surfactant Exosurf on dendrimer-mediated transfection in eukaryotic cells. Exosurf significantly enhanced dendrimer-luciferase plasmid transfection in a number of cell lines and was very effective in primary cells. Luciferase expression increased up to 40-fold in primary normal human bronchial/tracheal epithelial cells (NHBE). FACScan analysis demonstrated that the transfection rate of the human T cell leukemia Jurkat cell line has significantly improved from 10 to 90% of cells at 24 h after transfection. Analysis of the components of Exosurf revealed that the nonionic surfactant tyloxapol was responsible for the enhancement of dendrimer-mediated gene transfer. The tyloxapol effect was due to increased cell membrane porosity and DNA uptake. Our results demonstrate that Exosurf and its component, tyloxapol, constitute a powerful enhancer for dendrimer-mediated gene transfer in vitro.     

On the role of APC-activation for in vitro versus in vivo T cell priming

Professional antigen-presenting cells take up antigens for processing and presentation in association with MHC class I and II molecules. When APCs receive the right stimuli, they undergo a maturation process and migrate to secondary lymphoid organs to trigger T cell activation. In this study, we compared side-by-side in vivo and in vitro activation of T cells. Transgenic CD8(+) T cells specific for the p33 epitope, derived from the lymphocytic choriomeningitis virus glycoprotein, were labeled with CFSE and injected into syngeneic mice or alternatively, co-cultured in vitro with APCs. The p33 epitope was delivered as free peptide or genetically fused to virus-like particles. Whereas proliferation of specific T cells was comparable in both systems, the production of IFN-gamma and the expression of CD25 showed important differences. Induction of effector function and expression of activation markers were strongly enhanced in vitro by both the free peptide and VLPs. Surprisingly, addition of CpG-containing immune-stimulating DNA for activation of APCs dramatically increased effector T cell differentiation in vitro, whereas no enhancement could be observed in vitro. Thus, activation of professional APCs was mandatory for induction of effector CD8(+) T cell responses in vivo, while this step was largely dispensable in vitro.     

Selective regulation of CD8 effector T cell migration by the p110 gamma isoform of phosphatidylinositol 3-kinase

Chemokine-mediated T cell migration is essential to an optimal immune response. The p110gamma isoform of PI3K is activated by G protein-coupled receptors and regulates neutrophil and macrophage chemotaxis. We used p110gamma-deficient mice to examine the role of p110gamma in CD8 T cell migration and activation in response to viral challenge. Naive CD8 T cell migration in response to CCL21 in vitro and trafficking into secondary lymphoid organs in vivo was unaffected by the loss of p110gamma. Furthermore, loss of p110gamma did not affect CD8 T cell proliferation and effector cell differentiation in vitro in response to anti-CD3 stimulation or in vivo in response to vaccinia virus (VV) challenge. However, there was reduced migration of p110gamma knockout (p110gamma(-/-)) CD8 effector T cells into the peritoneum following i.p. challenge with VV. The role of p110gamma in CD8 effector T cell migration was intrinsic to T cells, as p110gamma(-/-) CD8 effector T cells exhibited impaired migration into the inflamed peritoneum following secondary transfer into wild-type recipients. In addition, p110gamma(-/-) CD8 effector T cells exhibited impaired migration in vitro in response to inflammatory chemoattractants. Although wild-type mice efficiently cleared VV at high viral doses, infection of p110gamma knockout mice resulted in visible illness and death less than a week after infection. Thus, p110gamma is dispensable for constitutive migration of naive CD8 T cells and subsequent activation and differentiation into effector CD8 T cells, but plays a central role in the migration of effector CD8 T cells into inflammatory sites.     

Lack of tumor necrosis factor alpha induces impaired proliferation of hepatitis B virus-specific cytotoxic T lymphocytes

Recent studies have shown that tumor necrosis factor alpha (TNF-alpha) plays critical roles in not only viral clearance but also lymphoid tissue development and stem cell differentiation. In this study, we attempted to induce hepatitis B virus (HBV)-specific cytotoxic T lymphocytes (CTLs) by immunization of TNF-alpha knockout (TNF-alpha(-/-)) mice with HBsAg-encoding plasmid DNA. An immunization with the HBV plasmid failed to induce CTL responses in TNF-alpha(-/-) mice, although CTLs were readily induced in wild-type mice by the same protocol. Weak CTL responses were produced in TNF-alpha(-/-) mice after two sessions of immunization with the HBV plasmid; however, TNF-alpha was required to maintain the responses of these CTL lines to in vitro stimulation and, even then, the responses were lost after 3 weeks. Interestingly, a limiting dilution of a CTL line showed that HBV-specific CTL clones with high specific cytotoxicity were present in TNF-alpha(-/-) mice, but these clones again failed to proliferate for more than 3 weeks. Furthermore, since exogenously added TNF-alpha enhanced the proliferation of a TNF-alpha(-/-) clone but suppressed that of a TNF-alpha(+/+) clone in vitro, TNF-alpha also has a direct effect on the proliferation of CTLs. In conclusion, TNF-alpha is essential rather than important for the proliferation of HBV-specific CTLs both in vivo and in vitro and this effect is not only due to the activation of dendritic cells but is also induced by the direct effect on CTLs.     

Regulation of apoptosis and cell cycle arrest by Zac1, a novel zinc finger protein expressed in the pituitary gland and the brain.

The proliferation rate of a cell population reflects a balance between cell division, cell cycle arrest, differentiation and apoptosis. The regulation of these processes is central to development and tissue homeostasis, whereas dysregulation may lead to overt pathological outcomes, notably cancer and neurodegenerative disorders. We report here the cloning of a novel zinc finger protein which regulates apoptosis and cell cycle arrest and was accordingly named Zac1. In vitro Zac1 inhibited proliferation of tumor cells, as evidenced by measuring colony formation, growth rate and cloning in soft agar. In vivo Zac1 abrogated tumor formation in nude mice. The antiproliferative activity of Zac1 was due to induction of extensive apoptosis and of G1 arrest, which proceeded independently of retinoblastoma protein and of regulation of p21(WAF1/Cip1), p27Kip1, p57Kip2 and p16INK4a expression. Zac1-mediated apoptosis was unrelated to cell cycle phase and G1 arrest was independent of apoptosis, indicating separate control of apoptosis and cell cycle arrest. Zac1 is thus the first gene besides p53 which concurrently induces apoptosis and cell cycle arrest.