Enhanced oncolytic adenoviral production by downregulation of death-domain associated protein and overexpression of precursor terminal protein.

Adequate viral replication in tumor cells is the key to improving the anti-cancer effects of oncolytic adenovirus therapy. In this study, we introduced short hairpin RNAs against death-domain associated protein (Daxx), a repressor of adenoviral replication, and precursor terminal protein (pTP), an initiator of adenoviral genome replication, into adenoviral constructs to determine their contributions to viral replication. Both Daxx downregulation and pTP overexpression increased viral production in variety of human cancer cell lines, and the enhanced production of virus progeny resulted in more cell lysis in vitro, and tumor regression in vivo. We confirmed that increased virus production by Daxx silencing, or pTP overexpression, occurred using different mechanisms by analyzing levels of adenoviral protein expression and virus production. Specifically, Daxx downregulation promoted both virus replication and oncolysis in a consecutive manner by optimizing IVa2-based packaging efficiency, while pTP overexpression by increasing both infectious and total virus particles but their contribution to increased viral production may have been damaged to some extent by their another contribution to apoptosis and autophagy. Therefore, introducing both Daxx shRNA and pTP in virotherapy may be a suitable strategy to increase apoptotic tumor-cell death and to overcome poor viral replication, leading to meaningful reductions in tumor growth in vivo.

Adenovirus 5 E1A Interacts with E4orf3 To Regulate Viral Chromatin Organization.

Human adenovirus expresses several early proteins that control various aspects of the viral replication program, including an orchestrated expression of viral genes. Two of the earliest viral transcriptional units activated after viral genome entry into the host cell nucleus are the E1 and E4 units, which each express a variety of proteins. Chief among these are the E1A proteins that function to reprogram the host cell and activate transcription of all other viral genes. The E4 gene encodes multiple proteins, including E4orf3, which functions to disrupt cellular antiviral defenses, including the DNA damage response pathway and activation of antiviral genes. Here we report that E1A directly interacts with E4orf3 via the conserved N terminus of E1A to regulate the expression of viral genes. We show that E4orf3 indiscriminately drives high nucleosomal density of viral genomes, which is restrictive to viral gene expression and which E1A overcomes via a direct interaction with E4orf3. We also show that during infection E1A colocalizes with E4orf3 to nuclear tracks that are associated with heterochromatin formation. The inability of E1A to interact with E4orf3 has a significant negative impact on overall viral replication, the ability of the virus to reprogram the host cell, and the levels of viral gene expression. Together these results show that E1A and E4orf3 work together to fine-tune the viral replication program during the course of infection and highlight a novel mechanism that regulates viral gene expression.IMPORTANCE To successfully replicate, human adenovirus needs to carry out a rapid yet ordered transcriptional program that executes and drives viral replication. Early in infection, the viral E1A proteins are the key activators and regulators of viral transcription. Here we report, for the first time, that E1A works together with E4orf3 to perfect the viral transcriptional program and identify a novel mechanism by which the virus can adjust viral gene expression by modifying its genome's nucleosomal organization via cooperation between E1A and E4orf3.

ME1 Regulates NADPH Homeostasis to Promote Gastric Cancer Growth and Metastasis.

Genomic alterations of tumor suppressorsoften encompass collateral protein-coding genes that create therapeutic vulnerability to further inhibition of their paralogs. Here, we report that malic enzyme 2 (ME2) is frequently hemizygously codeleted with SMAD4 in gastric cancer. Its isoenzyme ME1 was upregulated to replenish the intracellular reducing equivalent NADPH and to maintain redox homeostasis. Knockdown of ME1 significantly depleted NADPH, induced high levels of reactive oxygen species (ROS), and ultimately cell apoptosis under oxidative stress conditions, such as glucose starvation and anoikis, in ME2-underexpressed cells. Moreover, ME1 promoted tumor growth, lung metastasis, and peritoneal dissemination of gastric cancer in vivo Intratumoral injection of ME1 siRNA significantly suppressed tumor growth in cell lines and patient-derived xenograft-based models. Mechanistically, ME1 was transcriptionally upregulated by ROS in an ETV4-dependent manner. Overexpression of ME1 was associated with shorter overall and disease-free survival in gastric cancer. Altogether, our results shed light on crucial roles of ME1-mediated production of NADPH in gastric cancer growth and metastasis.Significance: These findings reveal the role of malic enzyme in growth and metastasis.Graphical Abstract: http://cancerres.aacrjournals.org/content/canres/78/8/1972/F1.large.jpg Cancer Res; 78(8); 1972-85. ©2018 AACR.

Capicua suppresses hepatocellular carcinoma progression by controlling the ETV4-MMP1 axis.

Hepatocellular carcinoma (HCC) is developed by multiple steps accompanying progressive alterations of gene expression, which leads to increased cell proliferation and malignancy. Although environmental factors and intracellular signaling pathways that are critical for HCC progression have been identified, gene expression changes and the related genetic factors contributing to HCC pathogenesis are still insufficiently understood. In this study, we identify a transcriptional repressor, Capicua (CIC), as a suppressor of HCC progression and a potential therapeutic target. Expression of CIC is posttranscriptionally reduced in HCC cells. CIC levels are correlated with survival rates in patients with HCC. CIC overexpression suppresses HCC cell proliferation and invasion, whereas loss of CIC exerts opposite effects in vivo as well as in vitro. Levels of polyoma enhancer activator 3 (PEA3) group genes, the best-known CIC target genes, are correlated with lethality in patients with HCC. Among the PEA3 group genes, ETS translocation variant 4 (ETV4) is the most significantly up-regulated in CIC-deficient HCC cells, consequently promoting HCC progression. Furthermore, it induces expression of matrix metalloproteinase 1 (MMP1), the MMP gene highly relevant to HCC progression, in HCC cells; and knockdown of MMP1 completely blocks the CIC deficiency-induced HCC cell proliferation and invasion. CONCLUSION: Our study demonstrates that the CIC-ETV4-MMP1 axis is a regulatory module controlling HCC progression. (Hepatology 2018;67:2287-2301).

Activation of the orphan receptor GPR55 by lysophosphatidylinositol promotes metastasis in triple-negative breast cancer.

The orphan G protein-coupled receptor GPR55 has been directly or indirectly related to basic alterations that drive malignant growth: uncontrolled cancer cell proliferation, sustained angiogenesis, and cancer cell adhesion and migration. However, little is known about the involvement of this receptor in metastasis. Here, we show that elevated GPR55 expression in human tumors is associated with the aggressive basal/triple-negative breast cancer population, higher probability to develop metastases, and therefore poor patient prognosis. Activation of GPR55 by its proposed endogenous ligand lysophosphatidylinositol confers pro-invasive features on breast cancer cells both in vitro and in vivo. Specifically, this effect is elicited by coupling to Gq/11 heterotrimeric proteins and the subsequent activation, through ERK, of the transcription factor ETV4/PEA3. Together, these data show that GPR55 promotes breast cancer metastasis, and supports the notion that this orphan receptor may constitute a new therapeutic target and potential biomarker in the highly aggressive triple-negative subtype.

Competitive Inhibition of Lysine Acetyltransferase 2B by a Small Motif of the Adenoviral Oncoprotein E1A.

The adenovirus early region 1A (E1A) oncoprotein hijacks host cells via direct interactions with many key cellular proteins, such as KAT2B, also known as PCAF (p300/CBP associated factor). E1A binds the histone acetyltransferase (HAT) domain of KAT2B to repress its transcriptional activation. However, the molecular mechanism by which E1A inhibits the HAT activity is not known. Here we demonstrate that a short and relatively conserved N-terminal motif (cNM) in the intrinsically disordered E1A protein is crucial for KAT2B interaction, and inhibits its HAT activity through a direct competition with acetyl-CoA, but not its substrate histone H3. Molecular modeling together with a series of mutagenesis experiments suggests that the major helix of E1A cNM binds to a surface of the acetyl-CoA pocket of the KAT2B HAT domain. Moreover, transient expression of the cNM peptide is sufficient to inhibit KAT2B-specific H3 acetylation H3K14ac in vivo Together, our data define an essential motif cNM in N-terminal E1A as an acetyl-CoA entry blocker that directly associates with the entrance of acetyl-CoA binding pocket to block the HAT domain access to its cofactor.

Effects of Adenovirus Type 5 E1A Isoforms on Viral Replication in Arrested Human Cells.

Human adenovirus has evolved to infect and replicate in terminally differentiated human epithelial cells, predominantly those within the airway, the gut, or the eye. To overcome the block to viral DNA replication present in these cells, the virus expresses the Early 1A proteins (E1A). These immediate early proteins drive cells into S-phase and induce expression of all other viral early genes. During infection, several E1A isoforms are expressed with proteins of 289, 243, 217, 171, and 55 residues being present for human adenovirus type 5. Here we examine the contribution that the two largest E1A isoforms make to the viral life cycle in growth-arrested normal human fibroblasts. Viruses that express E1A289R were found to replicate better than those that do not express this isoform. Importantly, induction of several viral genes was delayed in a virus expressing E1A243R, with several viral structural proteins undetectable by western blot. We also highlight the changes in E1A isoforms detected during the course of viral infection. Furthermore, we show that viral DNA replication occurs more efficiently, leading to higher number of viral genomes in cells infected with viruses that express E1A289R. Finally, induction of S-phase specific genes differs between viruses expressing different E1A isoforms, with those having E1A289R leading to, generally, earlier activation of these genes. Overall, we provide an overview of adenovirus replication using modern molecular biology approaches and further insights into the contribution that E1A isoforms make to the life cycle of human adenovirus in arrested human fibroblasts.

A positive role of DBC1 in PEA3-mediated progression of estrogen receptor-negative breast cancer.

Deleted in Breast Cancer 1 (DBC1), a negative regulator of deacetylase SIRT1, has been shown to act as an estrogen receptor α (ER) coactivator that has a key role in ER transcription complex assembly and estrogen-dependent breast cancer cell proliferation. However, little is known about its physiological role and mechanism of action in ER-negative breast cancer cells. Here we report that DBC1 functions as a coactivator for the oncogenic ETS transcription factor PEA3 to promote ER-negative breast cancer progression. DBC1 is required for the expression of PEA3 target genes and for recruitment of PEA3 and RNA polymerase II to PEA3 target promoters. We also demonstrated that acetylation of PEA3 stimulates its DNA binding and association with DBC1 by disrupting the intramolecular interaction of PEA3. The molecular mechanism underlying DBC1 function in PEA3-mediated transcription involves inhibition of SIRT1 interaction with PEA3 and of SIRT1-mediated deacetylation of PEA3. Moreover, DBC1 depletion inhibited the tumorigenic properties of ER-negative breast cancer cells in vitro and in vivo. Importantly, increased DBC1 expression correlated with shorter relapse-free survival of ER-negative breast cancer patients. Our results firmly established DBC1 as a critical coactivator of PEA3 and as a key player in PEA3-mediated breast cancer progression.

Oncolytic adenovirus-induced autophagy: tumor-suppressive effect and molecular basis.

Autophagy is a catabolic process that produces energy through lysosomal degradation of intracellular organelles. Autophagy functions as a cytoprotective factor under physiological conditions such as nutrient deprivation, hypoxia, and interruption of growth factors. On the other hand, infection with pathogenic viruses and bacteria also induces autophagy in infected cells. Oncolytic virotherapy with replication-competent viruses is thus a promising strategy to induce tumor-specific cell death. Oncolytic adenoviruses induce autophagy and subsequently contribute to cell death rather than cell survival in tumor cells. We previously developed a telomerase-specific replication-competent oncolytic adenovirus, OBP-301, which induces cell lysis in tumor cells with telomerase activities. OBP-301-mediated cytopathic activity is significantly associated with induction of autophagy biomarkers. In this review, we focus on the tumor-suppressive role and molecular basis of autophagic machinery induced by oncolytic adenoviruses. Addition of tumor-specific promoters and modification of the fiber knob of adenoviruses supports the oncolytic adenovirus-mediated autophagic cell death. Autophagy is cooperatively regulated by the E1-dependent activation pathway, E4-dependent inhibitory pathway, and microRNA-dependent fine-tuning. Thus, future exploration of the functional role and molecular mechanisms underlying oncolytic adenovirus-induced autophagy would provide novel insights and improve the therapeutic potential of oncolytic adenoviruses.

Advances in adenovirus-mediated p53 cancer gene therapy.

INTRODUCTION: The tumor suppressor p53 gene regulates diverse cellular processes, such as cell-cycle arrest, senescence, apoptosis and autophagy, and it is frequently inactivated by genetic alterations in ∼ 50% of all types of human cancers. To restore wild-type p53 function in p53-inactivated tumors, adenovirus-mediated p53 gene therapy has been developed as a promising antitumor strategy in preclinical experiments and clinical studies. AREAS COVERED: This review focuses on the clinical relevance of replication-deficient adenovirus vectors that carry the wild-type p53 gene (Ad-p53; Advexin, Gendicine and SCH-58500) in clinical studies of patients with various cancers and the future perspectives regarding conditionally replicating adenovirus vectors expressing the wild-type p53 gene (CRAd-p53; AdDelta24-p53, SG600-p53, OBP-702) in preclinical experiments. Moreover, the recent advances in our understanding of the molecular basis for the p53-mediated tumor suppression network induced by Ad-p53 and CRAd-p53 vectors and the combination therapies for promoting the therapeutic potential of adenovirus-mediated p53 gene therapy are discussed. EXPERT OPINION: Exploration of the molecular mechanism underlying the p53-mediated tumor suppression network and the effective strategy for enhancing the p53-mediated cell death signaling pathway would provide novel insights into the improvement of clinical outcome in p53-based cancer gene therapy.

FOXO3a-Dependent Mechanism of E1A-Induced Chemosensitization.

Gene therapy trials in human breast, ovarian, and head and neck tumors indicate that adenovirus E1A can sensitize cancer cells to the cytotoxic effects of paclitaxel in vitro and in vivo. Resistance to paclitaxel has been reported to occur in cells expressing low levels of the Forkhead transcription factor FOXO3a. In this article, we report that FOXO3a is critical for E1A-mediated chemosensitization to paclitaxel. RNA interference-mediated knockdown of FOXO3a abolished E1A-induced sensitivity to paclitaxel. Mechanistic investigations indicated that E1A indirectly stabilized FOXO3a by acting at an intermediate step to inhibit a ubiquitin-dependent proteolysis pathway involving the E3 ligase ßTrCP and the FOXO3a inhibitory kinase IKKß. E1A derepressed this inhibitory pathway by stimulating expression of the protein phosphatase 2A (PP2A)/C protein phosphatases, which by binding to the TGF-ß-activated kinase TAK1, inhibited its ability to activate IKKß and, thereby, to suppress ßTrCP-mediated degradation of FOXO3a. Thus, by stimulating PP2A/C expression, E1A triggers a signaling cascade that stabilizes FOXO3a and mediates chemosensitization. Our findings provide a leap forward in understanding paclitaxel chemosensitization by E1A, and offer a mechanistic rational to apply E1A gene therapy as an adjuvant for improving therapeutic outcomes in patients receiving paclitaxel treatment.

Polyomavirus enhancer activator 3 protein promotes breast cancer metastatic progression through Snail-induced epithelial-mesenchymal transition.

Polyomavirus enhancer activator 3 protein (Pea3), also known as ETV4, is a member of the Ets-transcription factor family, which promotes metastatic progression in various types of solid cancer. Pea3-driven epithelial-mesenchymal transition (EMT) has been described in lung and ovarian cancers. The mechanisms of Pea3-induced EMT, however, are largely unknown. Here we show that Pea3 overexpression promotes EMT in human breast epithelial cells through transactivation of Snail (SNAI1), an activator of EMT. Pea3 binds to the human Snail promoter through the two proximal Pea3 binding sites and enhances Snail expression. In addition, knockdown of Pea3 in invasive breast cancer cells results in down-regulation of Snail, partial reversal of EMT, and reduced invasiveness in vitro. Moreover, knockdown of Snail partially rescues the phenotype induced by Pea3 overexpression, suggesting that Snail is one of the mediators bridging Pea3 and EMT, and thereby metastatic progression of the cancer cells. In four breast cancer patient cohorts whose microarray and survival data were obtained from the Gene Expression Omnibus database, Pea3 and Snail expression are significantly correlated with each other and with overall survival of breast cancer patients. We further demonstrate that nuclear localization of Pea3 is associated with Snail expression in breast cancer cell lines and is an independent predictor of overall survival in a Chinese breast cancer patient cohort. In conclusion, our results suggest that Pea3 may be an important prognostic marker and a therapeutic target for metastatic progression of human breast cancer.

Adenovirus E1A and E1B-19K proteins protect human hepatoma cells from transforming growth factor beta1-induced apoptosis.

Primary and some transformed hepatocytes undergo apoptosis in response to transforming growth factor beta1 (TGFbeta). We report that infection with species C human adenovirus conferred resistance to TGFbeta-induced apoptosis in human hepatocellular carcinoma cells (Huh-7). Protection against TGFbeta-mediated cell death in adenovirus-infected cells correlated with the maintenance of normal nuclear morphology, lack of pro-caspases 8 and 3 processing, maintenance of the mitochondrial membrane potential, and lack of cellular DNA degradation. The TGFbeta pro-apoptotic signaling pathway was blocked upstream of mitochondria in adenovirus-infected cells. Both the N-terminal sequences of the E1A proteins and the E1B-19K protein were necessary to protect infected cells against TGFbeta-induced apoptosis.

Increased OXPHOS activity precedes rise in glycolytic rate in H-RasV12/E1A transformed fibroblasts that develop a Warburg phenotype.

BACKGROUND: The Warburg phenotype in cancer cells has been long recognized, but there is still limited insight in the consecutive metabolic alterations that characterize its establishment. We obtained better understanding of the coupling between metabolism and malignant transformation by studying mouse embryonic fibroblast-derived cells with loss-of-senescence or H-RasV12/E1A-transformed phenotypes at different stages of oncogenic progression. RESULTS: Spontaneous immortalization or induction of senescence-bypass had only marginal effects on metabolic profiles and viability. In contrast, H-RasV12/E1A transformation initially caused a steep increase in oxygen consumption and superoxide production, accompanied by massive cell death. During prolonged culture in vitro, cell growth rate increased gradually, along with tumor forming potential in in vitro anchorage-independent growth assays and in vivo tumor formation assays in immuno-deficient mice. Notably, glucose-to-lactic acid flux increased with passage number, while cellular oxygen consumption decreased. This conversion in metabolic properties was associated with a change in mitochondrial NAD+/NADH redox, indicative of decreased mitochondrial tricarboxic acid cycle and OXPHOS activity. CONCLUSION: The high rate of oxidative metabolism in newly transformed cells is in marked contrast with the high glycolytic rate in cells in the later tumor stage. In our experimental system, with cells growing under ambient oxygen conditions in nutrient-rich media, the shift towards this Warburg phenotype occurred as a step-wise adaptation process associated with augmented tumorigenic capacity and improved survival characteristics of the transformed cells. We hypothesize that early-transformed cells, which potentially serve as founders for new tumor masses may escape therapies aimed at metabolic inhibition of tumors with a fully developed Warburg phenotype.

Adenoviral E1A function through Myc.

The study of DNA tumor viruses has been invaluable in uncovering the cellular nodes and pathways that contribute to oncogenesis. Perhaps one of the best-studied oncoproteins encoded by a DNA tumor virus is adenovirus E1A, which modifies the function of key regulatory proteins such as retinoblastoma (Rb) and the chromatin remodeling protein p400. Although the interaction of E1A with Rb has long been known to target regulation of the E2F transcription factors, the downstream target of the E1A-p400 interaction has remained elusive. We have recently reported that a critical downstream link of the E1A-p400 nexus is the oncoprotein transcription factor c-Myc. Through its interaction with p400, E1A stabilizes Myc and promotes formation of Myc-p400 complexes on chromatin, leading to activation of Myc target genes. These findings point to an important role for p400 in Myc function and reveal that E1A drives oncogenesis by tapping into two important transcriptional networks: those of E2F and Myc.

Adenovirus 5 E1A is responsible for increased expression of insulin receptor substrate 4 in established adenovirus 5-transformed cell lines and interacts with IRS components activating the PI3 kinase/Akt signalling pathway.

Using mass spectrometric analysis insulin receptor substrate 4 (IRS-4) has been identified as a novel adenovirus 5 early region 1A (Ad5E1A)-binding protein. IRS-4 interacts with both the transcriptional activation domain (conserved region 3) and the N-terminal region of Ad5E1A13S. Prolonged expression of Ad5E1A13S is required for the observed dramatic increase in the levels of IRS-4 mRNA and protein in Ad5E1-transformed human cell lines. Once expressed, as well as binding to E1A and the insulin receptor, IRS-4 remains tyrosine phosphorylated and constitutively associates with the regulatory p85 subunit of phosphoinositide 3 kinase, resulting in the phosphorylation of Akt (causing activation) and GSK-3beta (causing inhibition). Reducing IRS-4 expression using small interfering RNA (siRNA) in established Ad5E1A-expressing cell lines decreases the activation of Akt and cellular proliferation. During Ad5 infection, IRS-4 is not expressed. However, Ad5E1A associates with IRS-1, increasing Akt and GSK-3beta phosphorylation and tyrosine phosphorylation of IRS-1 itself. We conclude that the association and altered regulation of IRS proteins by Ad5E1A contribute to the adenovirus-transformed phenotype and modulates viral infection in an Akt-dependent manner.

Adenovirus E1A regulates lung epithelial ICAM-1 expression by interacting with transcriptional regulators at its promoter.

We focused on the regulation of inflammatory mediator expression by adenovirus E1A in lung epithelial cells and the role of this viral protein in the pathogenesis of chronic obstructive pulmonary disease (COPD). We previously reported that E1A, a well-known regulator of host genes, increased ICAM-1 expression in human bronchial epithelial (HBE) and A549 cells in response to LPS stimulation. In this report, we clarified the mechanism of this regulation. We found NF-kappaB translocation to the nucleus after LPS stimulation in both E1A-positive and -negative HBE cells. ICAM-1 promoter reporter constructs revealed that a mutation in the proximal NF-kappaB binding site completely inhibited increased transcription, whereas the mutation in a distal site did not. We analyzed the participation of E1A in transcriptional complex formation at this promoter using chromatin immunoprecipitation. In E1A-positive HBE and A549 cells, LPS stimulation increased ICAM-1 promoter immunoprecipitation by NF-kappaB p65 and p300 but not activator protein-1 antibodies with a concomitant increase by the E1A antibody. No increase was found in E1A-negative cells except in HBE cells with p65 antibody. The association of E1A with the increased promoter immunoprecipitation with p300 was also observed after TNF-alpha stimulation of A549 cells. These results suggest that adenovirus E1A regulates the ICAM-1 promoter through its proximal NF-kappaB binding site, most likely by interacting with the transcriptional complex that forms at this site. E1A regulation of the LPS response may play a role in acute exacerbations as a consequence of bacterial infections in COPD.

[Expression of toll-like receptor 4 in human alveolar epithelial cells and its role in cellular inflammation].

OBJECTIVE: To investigate if type II alveolar epithelial cells express Toll-like receptor 4 (TLR4) and to investigate the role of TLR4 in airway inflammation of chronic obstructive pulmonary diseases (COPD). METHODS: A549, the line of human type II alveolar epithelial cells were cultured and divided into 3 groups: normal control group, E1A(+) group transfected with adenovirus E1A plasmid, E1A(-) group transferred with blank plasmid without adenovirus E1A. Lipopolysaccharide (LPS) of the concentrations of 0, 0.1, 1, and 10 microg/ml, IL-1 beta of the concentrations of 0, and 0.1 ng/ml, and cigarette smoking extract (CSE) of the concentrations of 0, 10%, 20%, and 40% were used to stimulated the A549 cells for 12 and 24 h. Reverse transcription polymerase chain reaction was used to detect the mRNA expression of IL-8 and TLR4. Western blotting was used to detect the protein expression of nuclear factor kappaB (NF-kappaB) subunit P65. RESULTS: Twenty-four hours after the stimulation of 10 microg/ml LPS, 0.1 ng/ml IL-1beta, and 20% CSE, the IL-8 mRNA expression of the E1A(+) group was 2.82, 1.87, and 4.70 respectively, all significantly higher than those of the normal control group (0.95, 0.78, and 1.02 respectively, all P < 0.05) and those of the E1A(-) group (0.97, 0.81, and 1.12 respectively, all P < 0.05). Twelve and twenty-four hours after the stimulation of 10 microg/ml of LPS, the TLR4 mRNA expression of the E1A+ group were 4.52 and 7.99, both significantly higher than those of the normal control group (1.91 and 3.81 respectively, both P < 0.05) and those of the E1A(-) group (2.00 and 3.88 respectively, both P < 0.05). IL-1beta increased the expression of TLR4 mRNA too, but CSE did not change the expression of TLR4 mRNA in all these groups. LPS, IL-1beta, and CSE all increased the expression levels of NF-kappaB subunit P65 protein. CONCLUSIONS: Pulmonary type II epithelial cells express TLR4. LPS and IL-1beta up-regulate the release of IL-8 which may be mediated via the activation of NF-kappaB induced by TLR4.

E1A oncogene enhancement of caspase-2-mediated mitochondrial injury sensitizes cells to macrophage nitric oxide-induced apoptosis.

The adenovirus E1A oncogene induces innate immune rejection of tumors by sensitizing tumor cells to apoptosis in response to injuries, such as those inflicted by macrophage-produced TNF alpha and NO. E1A sensitizes cells to TNF by repressing its activation of NF-kappaB-dependent, antiapoptotic defenses. This suggested the hypothesis that E1A blockade of the NF-kappaB activation response might be the central mechanism of E1A induced cellular sensitivity to other proapoptotic injuries, such as macrophage-produced NO. However, creation of E1A-positive NIH-3T3 mouse cell variants with high-level, NF-kappaB-dependent resistance to TNF did not coselect for resistance to apoptosis induced by either macrophage-NO or chemical-NO, as the hypothesis would predict. E1A expression did block cellular recovery from NO-induced mitochondrial injury and converted the reversible, NO-induced cytostasis response of cells to an apoptotic response. This viral oncogene-induced phenotypic conversion of the cellular injury response of mouse and human cells was mediated by an E1A-related increase in NO-induced activation of caspase-2, an apical initiator of intrinsic apoptosis. Blocking caspase-2 activation or expression eliminated the NO-induced apoptotic response of E1A-positive cells. These results define an NF-kappaB-independent pathway through which the E1A gene of human adenovirus sensitizes mouse and human cells to apoptosis by enhancement of caspase-2-mediated mitochondrial injury.