Wentao Gao

Differential Effects of Endoplasmic Reticulum Stress-induced Autophagy on Cell Survival

Autophagy is a cellular response to adverse environment and stress, but its significance in cell survival is not always clear. Here we show that autophagy could be induced in the mammalian cells by chemicals, such as A23187, tunicamycin, thapsigargin, and brefeldin A, that cause endoplasmic reticulum stress. Endoplasmic reticulum stress-induced autophagy is important for clearing polyubiquitinated protein aggregates and for reducing cellular vacuolization in HCT116 colon cancer cells and DU145 prostate cancer cells, thus mitigating endoplasmic reticulum stress and protecting against cell death. In contrast, autophagy induced by the same chemicals does not confer protection in a normal human colon cell line and in the non-transformed murine embryonic fibroblasts but rather contributes to cell death. Thus the impact of autophagy on cell survival during endoplasmic reticulum stress is likely contingent on the status of cells, which could be explored for tumor-specific therapy.

Autophagy reduces acute ethanol-induced hepatotoxicity and steatosis in mice

BACKGROUND & AIMS Alcohol abuse is a major cause of liver injury. The pathologic features of alcoholic liver disease develop over prolonged periods, yet the cellular defense mechanisms against the detrimental effects of alcohol are not well understood. We investigated whether macroautophagy, an evolutionarily conserved cellular mechanism that is commonly activated in response to stress, could protect liver cells from ethanol toxicity. METHODS Mice were acutely given ethanol by gavage. The effects of ethanol on primary hepatocytes and hepatic cell lines were also studied in vitro. RESULTS Ethanol-induced macroautophagy in the livers of mice and cultured cells required ethanol metabolism, generation of reactive oxygen species, and inhibition of mammalian target of rapamycin signaling. Suppression of macroautophagy with pharmacologic agents or small interfering RNAs significantly increased hepatocyte apoptosis and liver injury; macroautophagy therefore protected cells from the toxic effects of ethanol. Macroautophagy induced by ethanol seemed to be selective for damaged mitochondria and accumulated lipid droplets, but not long-lived proteins, which could account for its protective effects. Increasing macroautophagy pharmacologically reduced hepatotoxicity and steatosis associated with acute ethanol exposure. CONCLUSIONS Macroautophagy protects against ethanol-induced toxicity in livers of mice. Reagents that modify macroautophagy might be developed as therapeutics for patients with alcoholic liver disease.

Protection of Mice and Poultry from Lethal H5N1 Avian Influenza Virus through Adenovirus-Based Immunization

ABSTRACT The recent emergence of highly pathogenic avian influenza virus (HPAI) strains in poultry and their subsequent transmission to humans in Southeast Asia have raised concerns about the potential pandemic spread of lethal disease. In this paper we describe the development and testing of an adenovirus-based influenza A virus vaccine directed against the hemagglutinin (HA) protein of the A/Vietnam/1203/2004 (H5N1) (VN/1203/04) strain isolated during the lethal human outbreak in Vietnam from 2003 to 2005. We expressed different portions of HA from a recombinant replication-incompetent adenoviral vector, achieving vaccine production within 36 days of acquiring the virus sequence. BALB/c mice were immunized with a prime-boost vaccine and exposed to a lethal intranasal dose of VN/1203/04 H5N1 virus 70 days later. Vaccination induced both HA-specific antibodies and cellular immunity likely to provide heterotypic immunity. Mice vaccinated with full-length HA were fully protected from challenge with VN/1203/04. We next evaluated the efficacy of adenovirus-based vaccination in domestic chickens, given the critical role of fowl species in the spread of HPAI worldwide. A single subcutaneous immunization completely protected chickens from an intranasal challenge 21 days later with VN/1203/04, which proved lethal to all control-vaccinated chickens within 2 days. These data indicate that the rapid production and subsequent administration of recombinant adenovirus-based vaccines to both birds and high-risk individuals in the face of an outbreak may serve to control the pandemic spread of lethal avian influenza.

Prevalence of Neutralizing Antibodies to Adenoviral Serotypes 5 and 35 in the Adult Populations of The Gambia, South Africa, and the United States

ABSTRACT One of the major limitations of the use of adenoviruses as gene therapy vectors is the existence of preformed immunity in various populations. Recent studies have linked failure of adenoviral gene therapy trials to the presence of antiadenoviral neutralizing antibodies (NAb). Understanding the distribution and specificity of such antibodies will assist in the design of successful recombinant adenoviral gene therapies and vaccines. To assess the prevalence of NAb to adenovirus serotypes 5 and 35 (Ad5 and Ad35), we analyzed serum samples from adult immunocompetent individuals living in The Gambia, South Africa, and the United States by using a neutralization assay. Serum samples were incubated with A549 lung carcinoma cells and adenoviruses encoding enhanced green or yellow fluorescent proteins; results were analyzed by fluorescence microscopy and flow cytometry. Using this technique, we found a high prevalence of NAb against Ad5 in Gambian, South African, and U.S. subjects at both low and high titers. Conversely, all subjects displayed a low prevalence of NAb to Ad35; when present, anti-Ad35 NAb were seen at low titers. Because of the ability of adenoviruses to elicit systemic and mucosal immune responses, Ad35 with its low NAb prevalence appears to be an attractive candidate vector for gene therapy applications.

Effects of a SARS-associated coronavirus vaccine in monkeys

Summary The causative agent of severe acute respiratory syndrome (SARS) has been identified as a new type of coronavirus. Here, we have investigated the ability of adenoviral delivery of codon-optimised SARS-CoV strain Urbani structural antigens spike protein S1 fragment, membrane protein, and nucleocapsid protein to induce virus-specific broad immunity in rhesus macaques. We immunised rhesus macaques intramuscularly with a combination of the three Ad5-SARS-CoV vectors or a control vector and gave a booster vaccination on day 28. The vaccinated animals all had antibody responses against spike protein S1 fragment and T-cell responses against the nucleocapsid protein. All vaccinated animals showed strong neutralising antibody responses to SARS-CoV infection in vitro. These results show that an adenoviral-based vaccine can induce strong SARS-CoV-specific immune responses in the monkey, and hold promise for development of a protective vaccine against the SARS causal agent.

Autophagy in the liver

A great part of our current understanding of mammalian macroautophagy is derived from studies of the liver. The term “autophagy” was introduced by Christian de Duve in part based on ultrastructural changes in rat liver following glucagon injection. Subsequent morphological, biochemical, and kinetics studies of autophagy in the liver defined the basic process of autophagosome formation, maturation, and degradation and the regulation of autophagy by hormones, phosphoinositide 3‐kinases, and mammalian target of rapamycin. It is now clear that macroautophagy in the liver is important for the balance of energy and nutrients for basic cell functions, the removal of misfolded proteins resulting from genetic mutations or pathophysiological stimulations, and the turnover of major subcellular organelles such as mitochondria, endoplasmic reticulum, and peroxisomes under both normal and pathophysiological conditions. Disturbance of autophagy function in the liver could thus have a major impact on liver physiology and liver disease. (HEPATOLOGY 2008.)

UpGene: Application of a Web-Based DNA Codon Optimization Algorithm

Although DNA codon optimization is a standard molecular biology strategy to overcome poor gene expression, to date no public software exists to facilitate this process. Among the uses of codon optimization, human immunodeficiency virus (HIV) vaccine development represents one of the most difficult challenges. A key obstacle to an effective DNA‐based vaccine is the low‐level expression of HIV genes in mammalian cells, which is due primarily to the instability of HIV mRNAs resulting from AU‐rich elements and rare codon usage. In this report we describe the development of a DNA optimization algorithm integrated with a PCR primer design program to redesign specific coding sequences for maximal gene expression. Using this algorithm combination, together with PCR‐based gene assembly, we have successfully optimized gene sequences for simian immunodeficiency virus (SIV) strain mac239 structural antigenic proteins gag and env, resulting in high‐level gene expression in eukaryotic cells. Our findings demonstrate that our user‐friendly algorithm is a valuable tool for DNA‐based HIV vaccine development. Moreover, it can be used to optimize any other genes of interest and is freely available online at http://www.vectorcore.pitt.edu/upgene.html.

Oncogenic transformation confers a selective susceptibility to the combined suppression of the proteasome and autophagy

The proteasome and the autophagy systems are two evolutionarily conserved mechanisms for degrading intracellular materials. They are functionally coupled and suppression of the proteasome promotes autophagy. Although suppression of the proteasome leads to cell death, suppression of autophagy can be either prodeath or prosurvival. To understand the underlining mechanism of this dichotomy and its potential clinical implications, we treated various transformed and nontransformed human cells with proteasome inhibitors. We found that whether autophagy served a prosurvival role in this scenario was contingent on the cellular oncogenic status. Thus, autophagy suppression enhanced apoptosis induced by proteasome inhibitors in transformed cells, but not in nontransformed cells. Oncogenic transformation enhanced the ability of cells to initiate autophagy in response to stress, reflecting a stronger dependence of transformed cells on autophagy for survival. Indeed, a combined use of bortezomib, the only Food and Drug Administration–approved proteasome inhibitor for clinical use, and chloroquine, which inhibits autophagy by disturbing lysosomal functions, suppressed tumor growth more significantly than either agent alone in a xenograft model. These findings indicate that suppression of both intracellular degradation systems could constitute a novel strategy for enhanced cancer control in a tumor-specific way. [Mol Cancer Ther 2009;8(7):2036–45]

Induction of macroautophagy by exogenously introduced calcium.

Macroautophagy can be activated by a broad range of agents and cellular manipulations. In performing cellular transfection using the calcium phosphate method, we noticed that the calcium phosphate precipitates (CPP) could induce LC3 punctation. Because of the wide use of this transfection method in mammalian cells and the potential significance of calcium in autophagy induction, we investigated whether CPP could specifically induce macroautophagy. We found that CPP-induced LC3 punctation was dependent on calcium and could be neutralized by extracelluar or intracellular calcium chelator. The punctation was not due to nonspecific aggregation of LC3 since it depended on the amino acid residue Glycine120, which is specifically required for LC3 to conjugate to phosphatidylethanolamine (PE). Consistently, there was also a significant increase of the PE-conjugated form of LC3. Electron microscopy confirmed the accumulation of typical autophagosomes following CPP treatment. Flux analysis indicated that CPP induced but did not inhibit autophagic degradation. Finally CPP-induced autophagy depended on the classical macroautophagy machinery including Beclin 1, the class III phosphoinositide-3 kinase and Atg5. Our studies thus indicate that exogenously introduced calcium in the form of CPP could specifically induce macroautophagy, which may have the practical significance in the use of this agent for introducing genes into cells, and for studying the mechanism of autophagy as a model system.

Human adenovirus type 35: nucleotide sequence and vector development

In this report, we describe the complete 34 794 base pair genomic sequence of the human adenovirus serotype 35 (Ad35) Holden strain. The viral genome exhibits a compact organization similar to other adenoviral serotypes, with overlapping genes on both strands. In all, 47 open reading frames (ORFs) were identified, including early (E1, 2, 3, 4) and late (L1, 2, 3, 4, 5) regions conserved among the adenoviridae family. In addition, 14 ORFs were identified that do not encode known adenoviral genes. Comparison of the predicted translational products of the conserved genes with those of other adenoviruses revealed that Ad35 has high homology to Ad7, Ad3, Ad21, Ad17, and simian Ads25. Based on the complete Ad35 DNA sequence, E3-, E1-, and E1/E3-deleted Ad35-based vector systems were developed. An HEK293-derived cell line was established for the propagation of the E1-deleted Ad35 vector, avoiding the emergence of replication-competent adenovirus. Moreover, production of the E1-deleted recombinant Ad35 vector was achieved by transient transduction of a plasmid encoding the Ad35 E1B gene in HEK293 cells. Testing showed that the Ad35-based vector efficiently infects both human and rhesus macaque dendritic cells. Our novel Ad35-based vectors and their corresponding packaging cell lines will provide a versatile and powerful system for DNA-based vaccine development and gene therapy applications.