[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.

Genetically modified adenoviruses against gliomas: from bench to bedside.

Oncolytic or tumor-selective adenoviruses are constructed as novel antiglioma therapies. After infection, the invading genetic adenoviral material is activated within the host cell. E1A and E1B adenoviral proteins are expressed immediately. E1A protein interacts with cell cycle regulatory proteins, such as retinoblastoma (Rb), driving the cell into the S phase and ensuing viral replication. The action of E1A stimulates the cellular p53 tumor suppressor system, which results in growth arrest or apoptosis, and halts adenovirus replication. However, adenoviral E1B interacts with p53 protein, preventing the DNA replication process from being abrogated by the induction of p53-mediated apoptosis. It was subsequently hypothesized that mutant adenoviruses that were unable to express wild-type E1A or E1B proteins could not replicate in normal cells with functional Rb or p53 pathways but instead would replicate and kill glioma cells that had defects in the regulation of these tumor suppressor pathways. Mutant E1B adenoviruses have already entered the clinical setting as an experimental treatment for patients with malignant gliomas. Mutant E1A adenoviruses are now in preclinical development as antiglioma therapy. In this review, the authors describe the mechanisms underlying the production of oncolytic adenoviruses, preclinical and clinical experiences with specific oncolytic adenoviruses, and the possibilities of combining mutant oncolytic adenoviruses with gene therapy or conventional therapies for managing malignant gliomas.

Conditionally replicative adenovirus for gastrointestinal cancers.

The clinical outcome of advanced gastrointestinal (GI) cancers (especially pancreatic and oesophageal cancers) is dismal, despite the advance of conventional therapeutic strategies. Cancer gene therapy is a category of new therapeutics, among which conditionally replicative adenovirus (CRAd) is one promising strategy to overcome existing obstacles of cancer gene therapy. Various CRAds have been developed for GI cancer treatment by taking advantage of the replication biology of adenovirus. Some CRAds have already been tested in clinical trials, but have fallen short of initial expectations. Concerns for clinical applicability include therapeutic potency, replication selectivity and interval end points in clinical trials. In addition, improvement of experimental animal models is needed for a deeper understanding of CRAd biology. Despite these obstacles, CRAds continue to be an exciting area of investigation with great potential for clinical utility. Further virological and oncological research will eventually lead to full realisation of the therapeutic potential of CRAds in the field of GI cancers.

Adenovirus with insertion-mutated E1A selectively propagates in liver cancer cells and destroys tumors in vivo.

The adenovirus E1A proteins are involved in the transcriptional activation of viral and cellular genes needed for controlling cell cycle and virus replication. Undifferentiated embryonic carcinoma cells have the ability to produce an E1A-like activity that can induce the expression of E1A-targeted adenoviral and cellular genes in the absence of the E1A products. Differentiated embryonic carcinoma cells lose the ability to produce the E1A-like activity. In this study, we investigated the E1A-like activity in cancer cells with an adenovirus having a mutated E1a gene. The mutation is generated by the insertion of a large DNA fragment in the E1a gene and interrupts the COOH-terminal region of both the E1A 12S and 13S proteins. The E1a-mutated virus can efficiently replicate in HepG2 and Hep3B liver cancer cells and produce high titers of virus. Replication of the E1a-mutated virus inhibits tumor formation and destroys tumors in vivo. The results obtained in this study imply that cancer cells may produce an E1A-like activity to support the selective replication of mutated virus in cancer cells. In addition, we found that although the E1a-mutated virus could not replicate in Huh1.cl2 liver cells, the viral DNA could amplify in the cells. This result suggests that replication of adenoviral DNA is necessary, but not sufficient, for generating infectious viral progeny and destroying tumor cells.

Thrombin generation by apoptotic vascular smooth muscle cells.

Thrombin activation requires assembly of a prothrombinase complex of activated coagulation factors on an anionic phospholipid surface, classically provided by activated platelets. We have previously shown that anionic phosphatidylserine is exposed by rat vascular smooth muscle cells (VSMCs) undergoing apoptosis after serum withdrawal. In this study, using a chromogenic assay, we have shown thrombin generation by apoptotic VSMCs expressing c-myc (VSMC-myc) with an area under the thrombin-generation curve (AUC) of 305 +/- 17 nmol x min/L and a peak thrombin (PT) of 154 +/- 9 nmol/L. The thrombin-generating potential of the apoptotic VSMC-myc cells was greater than that of unactivated platelets (P = .003 for AUC; P = .0002 for PT) and similar to calcium-ionophore activated platelets (AUC of 332 +/- 15 nmol x min/L, P = .3; PT of 172 +/- 8 nmol/L, P = .2). Thrombin activation was also seen with apoptotic human VSMCs (AUC of 211 +/- 8 nmol x min/L; PT of 103 +/- 4 nmol/L) and was inhibited by annexin V (P < .0001 for AUC and PT). VSMC-myc cells maintained in serum generated less thrombin than after serum withdrawal (P = .0002 for AUC and PT). VSMCs derived from human coronary atherosclerotic plaques that apoptose even in serum also generated thrombin (AUC of 260 +/- 2 nmol x min/L; PT of 128 +/- 4 nmol/L). We conclude that apoptotic VSMCs possess a significant thrombin-generating capacity secondary to phosphatidylserine exposure. Apoptotic cells within atherosclerotic plaques may allow local thrombin activation, thereby contributing to disease progression.

Transcription factors RFX1/EF-C and ATF-1 associate with the adenovirus E1A-responsive element of the human proliferating cell nuclear antigen promoter.

The proliferating cell nuclear antigen (PCNA) is an adenovirus E1A-inducible factor that is intimately linked to the processes of DNA replication and cell cycle regulation. Previously, we defined a novel cis-acting element, the PCNA E1A-responsive element (PERE), that confers induction by the E1A 243R oncoprotein upon the human PCNA promoter. To better understand the regulation of PCNA expression by E1A 243R, we have identified cellular transcription factors that associate with the PERE. In electrophoretic mobility shift assays, the PERE formed three major complexes (P1, P2 and P3) with proteins in nuclear extracts from HeLa or 293 cells. Formation of complexes P2 and P3, which correlates with PCNA promoter activity in vivo, requires the activating transcription factor (ATF) binding site found within the PERE [Labrie et al. (1993) Mol. Cell. Biol., 13, 1697-1707]. Antibody interference experiments and mobility shift assays performed with in vitro-synthesized protein indicated that the transcription factor ATF-1 is a major component of these complexes. Similar assays demonstrated that the hepatitis B virus enhancer-associated protein RFX1 constitutes a major component of the P1 complex. In addition, we examined the binding of proteins to the minimal E1A-responsive promoter to identify other factors important for transcription from the PCNA promoter. Mobility shift assays revealed that a fragment encompassing the region from -87 to +62 relative to the transcription initiation site forms at least five complexes, EH1-EH5, with HeLa cell nuclear extracts. The transcription factor YY1 associates with the initiator element of the PCNA promoter. The identification of these transcription factors will allow their roles in the activation of PCNA by E1A to be evaluated.

Relief of YY1 transcriptional repression by adenovirus E1A is mediated by E1A-associated protein p300.

YY1 represses transcription when bound upstream of transcriptional initiation sites. This repression can be relieved by adenovirus E1A. Here, we present genetic evidence that the ability of E1A to relieve YY1 repression was impaired by mutations that affect E1A binding to its associated protein p300. This suggests that E1A may modulate the repressor activity of YY1 by binding to p300, which may be physically complexed with YY1. A YY1/p300 protein complex in vivo was demonstrated by several independent approaches, and the YY1-interacting domain was mapped to the carboxy-terminal region of p300, distinct from the E1A-binding site. Unlike E2F/RB, the YY1/p300 complex is not disrupted by E1A. Functional studies using recombinant p300 demonstrated unequivocally that p300 is capable of mediating E1A-induced transcriptional activation through YY1. Taken together, these results reveal, for the first time, a YY1/p300 complex that is targeted by E1A and demonstrate a function for p300 in mediating interactions between YY1 and E1A. Our data thus identify YY1 as a partner protein for p300 and uncover a molecular mechanism for the relief of YY1-mediated repression by E1A.