X Chromosome-linked Inhibitor of Apoptosis Regulates Cell Death Induction by Proapoptotic Receptor Agonists

Proapoptotic receptor agonists cause cellular demise through the activation of the extrinsic and intrinsic apoptotic pathways. Inhibitor of apoptosis (IAP) proteins block apoptosis induced by diverse stimuli. Here, we demonstrate that IAP antagonists in combination with Fas ligand (FasL) or the death receptor 5 (DR5) agonist antibody synergistically stimulate death in cancer cells and inhibit tumor growth. Single-agent activity of IAP antagonists relies on tumor necrosis factor-α signaling. By contrast, blockade of tumor necrosis factor-α does not affect the synergistic activity of IAP antagonists with FasL or DR5 agonist antibody. In most cancer cells, proapoptotic receptor agonist-induced cell death depends on amplifying the apoptotic signal via caspase-8-mediated activation of Bid and subsequent activation of the caspase-9-dependent mitochondrial apoptotic pathway. In the investigated cancer cell lines, induction of apoptosis by FasL or DR5 agonist antibody can be inhibited by knockdown of Bid. However, knockdown of X chromosome-linked IAP (XIAP) or antagonism of XIAP allows FasL or DR5 agonist antibody to induce activation of effector caspases efficiently without the need for mitochondrial amplification of the apoptotic signal and thus rescues the effect of Bid knockdown in these cells.     

Inhibition of Mitochondrial Neural Cell Death Pathways by Protein Transduction of Bcl-2 Family Proteins

Bcl-2 and other closely related members of the Bcl-2 family of proteins inhibit the death of neurons and many other cells in response to a wide variety of pathogenic stimuli. Bcl-2 inhibition of apoptosis is mediated by its binding to pro-apoptotic proteins, e.g., Bax and tBid, inhibition of their oligomerization, and thus inhibition of mitochondrial outer membrane pore formation, through which other pro-apoptotic proteins, e.g., cytochrome c, are released to the cytosol. Bcl-2 also exhibits an indirect antioxidant activity caused by a sub-toxic elevation of mitochondrial production of reactive oxygen species and a compensatory increase in expression of antioxidant gene products. While classic approaches to cytoprotection based on Bcl-2 family gene delivery have significant limitations, cellular protein transduction represents a new and exciting approach utilizing peptides and proteins as drugs with intracellular targets. The mechanism by which proteins with transduction domains are taken up by cells and delivered to their targets is controversial but usually involves endocytosis. The effectiveness of transduced proteins may therefore be limited by their release from endosomes into the cytosol.     

Immunization with a Recombinant Vaccinia Virus That Encodes Nonstructural Proteins of the Hepatitis C Virus Suppresses Viral Protein Levels in Mouse Liver

Chronic hepatitis C, which is caused by infection with the hepatitis C virus (HCV), is a global health problem. Using a mouse model of hepatitis C, we examined the therapeutic effects of a recombinant vaccinia virus (rVV) that encodes an HCV protein. We generated immunocompetent mice that each expressed multiple HCV proteins via a Cre/loxP switching system and established several distinct attenuated rVV strains. The HCV core protein was expressed consistently in the liver after polyinosinic acid–polycytidylic acid injection, and these mice showed chronic hepatitis C-related pathological findings (hepatocyte abnormalities, accumulation of glycogen, steatosis), liver fibrosis, and hepatocellular carcinoma. Immunization with one rVV strain (rVV-N25), which encoded nonstructural HCV proteins, suppressed serum inflammatory cytokine levels and alleviated the symptoms of pathological chronic hepatitis C within 7 days after injection. Furthermore, HCV protein levels in liver tissue also decreased in a CD4 and CD8 T-cell-dependent manner. Consistent with these results, we showed that rVV-N25 immunization induced a robust CD8 T-cell immune response that was specific to the HCV nonstructural protein 2. We also demonstrated that the onset of chronic hepatitis in CN2-29^(+/−)/MxCre^(+/−) mice was mainly attributable to inflammatory cytokines, (tumor necrosis factor) TNF-α and (interleukin) IL-6. Thus, our generated mice model should be useful for further investigation of the immunological processes associated with persistent expression of HCV proteins because these mice had not developed immune tolerance to the HCV antigen. In addition, we propose that rVV-N25 could be developed as an effective therapeutic vaccine.     

Activation of the Proapoptotic Bcl-2 Protein Bax by a Small Molecule Induces Tumor Cell Apoptosis

The proapoptotic Bcl-2 protein Bax by itself is sufficient to initiate apoptosis in almost all apoptotic paradigms. Thus, compounds that can facilitate disruptive Bax insertion into mitochondrial membranes have potential as cancer therapeutics. In our study, we have identified small-molecule compounds predicted to associate with the Bax hydrophobic groove by a virtual-screen approach. Among these, one lead compound (compound 106) promotes Bax-dependent but not Bak-dependent apoptosis. Importantly, this compound alters Bax protein stability in vitro and promotes the insertion of Bax into mitochondria, leading to Bax-dependent permeabilization of the mitochondrial outer membrane. Furthermore, as a single agent, compound 106 inhibits the growth of transplanted tumors, probably by inducing apoptosis in tumors. Our study has revealed a compound that activates Bax and induces Bax-dependent apoptosis, which may lead to the development of new therapeutic agents for cancer.     

Regulation of Mammalian Target of Rapamycin Complex 1 by Bcl-2 and Bcl-XL Proteins

Mammalian target of rapamycin complex 1 (mTORC1) is a key regulator of cell growth and metabolism. Its activity is controlled by various types of signals, including growth factors, nutrients, and stresses. In this study, we show that changes in expression levels of two antiapoptotic proteins, Bcl-2 and Bcl-X_L, also affect mTORC1 signaling activity. In cells overexpressing Bcl-X_L, mTORC1 activity is increased and becomes less sensitive to growth factor or nutrient conditions. In contrast, reduction in expression levels of the two antiapoptotic proteins inhibits mTORC1 signaling activity. Our results suggest that the effect of Bcl-2 and Bcl-X_L on mTORC1 is mediated by FKBP38, an inhibitor of mTORC1. The two proteins compete with mTORC1 for FKBP38 binding and hence alter mTORC1 activity. This study reveals a novel cross-talk between Bcl-2/X_L and mTORC1 signaling, which is likely to contribute to cancer development.     

Role of Bcl-2 Family Members in Anoxia Induced Cell Death

Anoxia, the condition of oxygen deprivation, induces apoptosis via the intrinsic apoptoticpathway. Cells deficient in both Bax and Bak do not undergo cell death during anoxia.However, the underlying mechanism of anoxia induced cell death is not well defined. Herewe report our latest findings of two critical events that are required to induce cell deathduring anoxia. First, a key member of the Bcl-2 family of pro-survival proteins, Mcl-1,undergoes proteasomal-dependent degradation. The loss of Mcl-1 protein is independentof Bax or Bak indicating this is an early event in the apoptotic cascade. Second, cellsinhibit the mitochondrial electron transport chain to negate the pro-survival function of Bcl-2/Bcl-XL. These observations indicate that loss of pro-survival function is necessary foranoxia induced cell death.     

Regulation of Mammalian Cell Growth and Death by Bacterial Redox Proteins: Relevance to Ecology and Cancer Therapy

Recent evidence indicates that bacterial redox proteins such as cupredoxins andcytochromes, that are normally involved in electron transfer during respiration, can entermammalian cells and induce either apoptosis or inhibition of cell cycle progression. Suchproteins have also been shown to demonstrate a good deal of specificity for entry andinduction of cytotoxic effects in cancer cells, allowing both in vitro cell death and in vivoinhibition of cancer progression. An alteration in the hydrophobicity of the bacterial redoxproteins can lead to a switch from apoptosis to growth arrest and vice versa throughmodulation of the intracellular levels of tumor suppressors. The preferential entry andcytotoxicity of these redox proteins in cancer cells raises interesting questions about thepresence of other bacterial proteins that may affect cell cycle at the G2/M phase, therebypotentially arresting cancer growth. The intracellular localization of the bacterial redoxproteins in nonpathogenic soil bacteria similarly raises questions about their possible rolein allowing various nonpathogenic soil bacteria to defend themselves from environmentalpredators by inducing cytotoxicity when engulfed in large numbers. A new role of theredox proteins in soil bacteria in maintaining an ecological balance among the predatorsand preys is proposed.     

Identification and Characterization of Maize and Barley Lsi2-Like Silicon Efflux Transporters Reveals a Distinct Silicon Uptake System from That in Rice

Silicon (Si) uptake has been extensively examined in rice (Oryza sativa), but it is poorly understood in other gramineous crops. We identified Low Silicon Rice 2 (Lsi2)-like Si efflux transporters from two important gramineous crops: maize (Zea mays) and barley (Hordeum vulgare). Both maize and barley Lsi2 expressed in Xenopus laevis oocytes showed Si efflux transport activity. Furthermore, barley Lsi2 was able to recover Si uptake in a rice mutant defective in Si efflux. Maize and barley Lsi2 were only expressed in the roots. Expression of maize and barley Lsi2 was downregulated in response to exogenously applied Si. Moreover, there was a significant positive correlation between the ability of roots to absorb Si and the expression levels of Lsi2 in eight barley cultivars, suggesting that Lsi2 is a key Si transporter in barley. Immunostaining showed that maize and barley Lsi2 localized only at the endodermis, with no polarity. Protein gel blot analysis indicated that maize and barley Lsi2 localized on the plasma membrane. The unique features of maize and barley Si influx and efflux transporters, including their cell-type specificity and the lack of polarity of their localization in Lsi2, indicate that these crops have a different Si uptake system from that in rice.     

The Herpes Simplex Virus Type 1 UL17 Gene Encodes Virion Tegument Proteins That Are Required for Cleavage and Packaging of Viral DNA

Previous studies have suggested that the U_L17 gene of herpes simplex virus type 1 (HSV-1) is essential for virus replication. In this study, viral mutants incorporating either a lacZ expression cassette in place of 1,490 bp of the 2,109-bp U_L17 open reading frame [HSV-1(ΔU_L17)] or a DNA oligomer containing an in-frame stop codon inserted 778 bp from the 5′ end of the U_L17 open reading frame [HSV-1(U_L17-stop)] were plaque purified on engineered cell lines containing the U_L17 gene. A virus derived from HSV-1(U_L17-stop) but containing a restored U_L17 gene was also constructed and was designated HSV-1(U_L17-restored). The latter virus formed plaques and cleaved genomic viral DNA in a manner indistinguishable from wild-type virus. Neither HSV-1(ΔU_L17) nor HSV-1(U_L17-stop) formed plaques or produced infectious progeny when propagated on noncomplementing Vero cells. Furthermore, genomic end-specific restriction fragments were not detected in DNA purified from noncomplementing cells infected with HSV-1(ΔU_L17) or HSV-1(U_L17-stop), whereas end-specific fragments were readily detected when the viruses were propagated on complementing cells. Electron micrographs of thin sections of cells infected with HSV-1(ΔU_L17) or HSV-1(U_L17-stop) illustrated that empty capsids accumulated in the nuclei of Vero cells, whereas DNA-containing capsids accumulated in the nuclei of complementing cells and enveloped virions were found in the cytoplasm and extracellular space. Additionally, protein profiles of capsids purified from cells infected with HSV-1(ΔU_L17) compared to wild-type virus show no detectable differences. These data indicate that the U_L17 gene is essential for virus replication and is required for cleavage and packaging of viral DNA. To characterize the U_L17 gene product, an anti-U_L17 rabbit polyclonal antiserum was produced. The antiserum reacted strongly with a major protein of apparent M_r 77,000 and weakly with a protein of apparent M_r 72,000 in wild-type infected cell lysates and in virions. Bands of similar sizes were also detected in electrophoretically separated tegument fractions of virions and light particles and yielded tryptic peptides of masses characteristic of the predicted U_L17 protein. We therefore conclude that the U_L17 gene products are associated with the virion tegument and note that they are the first tegument-associated proteins shown to be required for cleavage and packaging of viral DNA.     

Characterization of CELO Virus Proteins That Modulate the pRb/E2F Pathway

The avian adenovirus CELO can, like the human adenoviruses, transform several mammalian cell types, yet it lacks sequence homology with the transforming, early regions of human adenoviruses. In an attempt to identify how CELO virus activates the E2F-dependent gene expression important for S phase in the host cell, we have identified two CELO virus open reading frames that cooperate in activating an E2F-inducible reporter system. The encoded proteins, GAM-1 and Orf22, were both found to interact with the retinoblastoma protein (pRb), with Orf22 binding to the pocket domain of pRb, similar to other DNA tumor virus proteins, and GAM-1 interacting with pRb regions outside the pocket domain. The motif in Orf22 responsible for the pRb interaction is essential for Orf22-mediated E2F activation, yet it is remarkably unlike the E1A LxCxD and may represent a novel form of pRb-binding peptide.     

稳定表达流感病毒基质蛋白2的哺乳动物细胞系的建立

本研究构建了稳定表达甲型流感病毒基质蛋白2(M2)的哺乳动物细胞系。应用PCR方法扩增A/PR/8/34(H1N1)株流感病毒M2基因,将其克隆至真核表达载体pcDNA5/FRT(pDF)上,构建出pDF-M2重组质粒。将鉴定正确的pDF-M2与表达Flp重组酶的pOG44质粒共转染Flp-In-CHO细胞,通过体内同源重组使目的基因整合到宿主细胞染色体上。筛选具有Hygromycin B抗性的重组细胞株命名为CHO-M2。以间接免疫荧光法(IFA)和Western blot法检测M2的表达,共获得15株高表达M2蛋白的重组细胞株。这些重组细胞株在连续培养10代后,PCR方法仍可检测到M2基因的存在,IFA也检测到蛋白的稳定表达。本研究成功获得了稳定表达甲型流感病毒M2的哺乳动物细胞系,为M2蛋白的功能研究和非复制型流感病毒疫苗的研制提供了新的工具。     

The Molecular Biology of Mammalian SIRT Proteins: SIRT2 Functions on Cell Cycle Regulation

Sir2, an NAD+-dependent protein deacetylase, extends the lifespan in diverse species from yeast to flies. Mammals have 7 homologues of Sir2, SIRT1-7, which affect aging and metabolism and which are potential targets for pharmacologic intervention. We identified SIRT2, which preferentially deacetylates tubulin and histone H4, as a down-regulated protein in gliomas due to its epigenetic aberration. We herein discuss the role of SIRT2 in the mitotic checkpoint function and show that it may be as a potential target of anti-cancer drugs.     

Molecular Cloning and Characterization of a cDNA Clone That Encodes a Cdc2 Homolog from Nicotiana tabacum

We have isolated a cDNA clone (cdc2Nt1) that encodes a homologof p34^cdc2/CDC28 kinase from tobacco (Nicotiana tabacum). Thecdc2Ntl protein showed extensive similarity to other homologsof Cdc2 from plants. Complementation studies showed that thecdc2Ntl gene was able to overcome cell cycle arrest at boththe G1/S and the G2/M transitions of cdc28^ts mutants of buddingyeast, demonstrating that the cdc2Ntl protein was able to replacethe Cdc28 kinase at both the G1/S and the G2/M transitions.Analysis of gene expression demonstrated that the cdc2Ntl genewas transcribed constitutively throughout the cell cycle butthat it was preferentially expressed in actively dividing tobaccoBY-2 cells. (Received July 13, 1995; Accepted February 15, 1996)     

Functional Characterization of a Wheat NHX Antiporter Gene TaNHX2 That Encodes a K+/H+ Exchanger

The subcellular localization of a wheat NHX antiporter, TaNHX2, was studied in Arabidopsis protoplasts, and its function was evaluated using Saccharomyces cerevisiae as a heterologous expression system. Fluorescence patterns of TaNHX2-GFP fusion protein in Arabidopsis cells indicated that TaNHX2 localized at endomembranes. TaNHX2 has significant sequence homology to NHX sodium exchangers from Arabidopsis, is abundant in roots and leaves and is induced by salt or dehydration treatments. Western blot analysis showed that TaNHX2 could be expressed in transgenic yeast cells. Expressed TaNHX2 protein suppressed the salt sensitivity of a yeast mutant strain by increasing its K⁺ content when exposed to salt stress. TaNHX2 also increased the tolerance of the strain to potassium stress. However, the expression of TaNHX2 did not affect the sodium concentration in transgenic cells. Western blot analysis for tonoplast proteins indicated that the TaNHX2 protein localized at the tonoplast of transgenic yeast cells. The tonoplast vesicles from transgenic yeast cells displayed enhanced K⁺/H⁺ exchange activity but very little Na^+/H⁺ exchange compared with controls transformed with the empty vector; Na⁺/H⁺ exchange was not detected with concentrations of less than 37.5 mM Na⁺ in the reaction medium. Our data suggest that TaNHX2 is a endomembrane-bound protein and may primarily function as a K⁺/H⁺ antiporter, which is involved in cellular pH regulation and potassium nutrition under normal conditions. Under saline conditions, the protein mediates resistance to salt stress through the intracellular compartmentalization of potassium to regulate cellular pH and K⁺ homeostasis.     

Genomic Organization of the Mammalian SLC14a2 Urea Transporter Genes

Urea transporters encoded by the UT-A gene play fundamental roles in the kidney and possibly other tissues. Knowledge of the genomic organization of the mouse, rat and human UT-A genes has enabled the engineering of transgenic and knockout animals and these have helped refine our understanding of the role of UT-A proteins. This review summarizes the published work that has accrued on the structure and regulation of these genes. It also documents a novel cDNA, human UT-A3, which has enabled a major refinement of the human UT-A gene structure. This and other information contained in this review should prove useful for future comparative genomic analysis, studies addressing gene regulation and for the engineering of transgenic and knockout animal strains.