Jeng-Woei Lee

Retardation of cell growth by avian reovirus p17 through the activation of p53 pathway

Abstract The second open reading frame of avian reovirus S1 gene segment encodes a 17kDa non-structural protein, named p17. The biological role of p17 is fully unknown so far. Using trypan blue dye exclusion and MTT assay, we demonstrated that the ectopic expression of p17 results in the reduction of viable cell number and cell proliferation rate of Vero, BHK, 293, and HeLa cells. Measurement of LDH activity and DNA fragmentation analysis revealed that p17 expression did not cause cell death or apoptosis. These data indicated that the p17 possessed the growth retardation function. Semi-quantitative RT-PCR and Western blotting revealed that p17-expressing cells induced the expression of CDK inhibitor p21cip1/waf1 in a time- and dose-dependent manner, but the transcripts of CDK inhibitor p15INK4b, p16INK4a, or p27kip were not altered. In the presence of p17, the p53 protein level and p53-driven reporter activity were elevated significantly. Dominant negative p53 alleviated the p21 accumulation, p53 activation, and growth inhibition effect induced by p17. Taken together, these studies revealed a possible intrinsic function of p17 in growth regulation through the activation of p53 and p21cip1/waf1.

Modulation of p53 by mitogen-activated protein kinase pathways and protein kinase C δ during avian reovirus S1133-induced apoptosis

ARV S1133 infection caused apoptosis in vivo and in vitro; however, the intracellular signaling pathways have not been fully delineated. We have previously demonstrated that ARV S1133 activates proapoptotic signaling from Src to p53, and further investigated how ARV S1133 modulates p53. We found that ARV S1133 forms syncytia and induces apoptosis in CEF, DF1 and Vero cells with different kinetics. Enhancement of p53 phosphorylation and DNA-binding capacity to bax and bad promoters was found in this study to increase bax and bad expression in ARV S1133-infected cells. ARV S1133 activates PKC delta and p38 and JNK/SAPK pathways, and inhibition of Ras, p38, JNK/SAPK and PKC delta works efficiently against apoptosis. Suppression of p38, JNK/SAPK and PKC delta selectively abolished ARV S1133-mediated p53 phosphorylation; moreover, inhibition of Src did not affect ARV S1133-induced p38 and JNK/SAPK activation, whereas blocking of Ras resulted in a reduction in the activities of p38 and JNK/SAPK.

Upregulated claudin-1 expression confers resistance to cell death of nasopharyngeal carcinoma cells.

Accumulating evidence reveals that aberrant expression of claudins manifests in various tumors; however, their biological functions are poorly understood. Here, we report on the elevated expression of claudin‐1 in nasopharyngeal carcinoma (NPC) cell lines under serum deprivation or fluorouracil (5‐FU) treatment. Interestingly, an increase in expression of claudin‐1 considerably reduced apoptosis rather than enhancing cell proliferation. However, claudin‐1 expression and activity were unaffected by external stimuli or Akt and NF‐κB activation. Notably, predominant cytoplasmic and nuclear localization of claudin‐1 in NPC cells reflected the aforementioned feature. On the other hand, loss of epithelial morphology and E‐cadherin expression was associated with serum withdrawal in NPC cells. Interestingly, restoration of E‐cadherin inhibited the protein elevation and antiapoptotic activity of claudin‐1. In conclusion, our data demonstrate the regulation and novel biological function of claudin‐1 and indicate the important role of claudin‐1 in NPC tumorigenesis.

TNF-α mediates pseudorabies virus-induced apoptosis via the activation of p38 MAPK and JNK/SAPK signaling

PRV infection causes apoptosis in vitro and in vivo. However, the significance of PRV-induced apoptosis and its signaling pathways is still unknown. This work investigates the role of MAPK pathways in mediating PRV-induced apoptosis. Flow cytometry, apoptosis ELISA and western blotting using antibodies against cleaved caspase-3, -6 and PARP demonstrated that PRV induces apoptosis in a time- and dose-dependent manner. p38 and JNK/SAPK inhibitors significantly protected cells from PRV-induced apoptosis. Inhibitor treatment did not affect Us3a gene transcription and progeny virus production. Western blotting revealed that PRV activates p38 and JNK/SAPK signaling. Inhibition of NF-kappaB had no effect on PRV-mediated apoptosis. Non-replicative PRV failed to activate p38 and JNK/SAPK or induce apoptosis. PRV infection increases TNF-alpha transcription, translation and secretion, as well as TNF-alpha receptor expression. Inhibition of p38 and JNK/SAPK reduced PRV-induced TNF-alpha up-regulation. Neutralization assay confirmed that TNF-alpha is a key mediator involved in PRV-induced apoptosis.

Activation of PI 3-kinase/Akt/NF-κB and Stat3 signaling by avian reovirus S1133 in the early stages of infection results in an inflammatory response and delayed apoptosis.

Avian reovirus (ARV) strain S1133 causes apoptosis in host cells in the middle to late stages of infection. This study investigated the early-stage biological response and intracellular signaling in ARV S1133-infected Vero and chicken cells. Treatment with conditioned medium from ARV S1133-infected cells increased the chemotactic activity of U937 cells. Neutralizing antibodies against IL-1beta and IL-6 showed that both cytokines contribute to viral-induced inflammation but neither affect cell survival. Inhibition of Akt, NF-kappaB, and Stat3 released the chemotactic activity and anti-apoptotic effect elicited by ARV S1133. ARV S1133 activated PI 3-kinase-dependent Akt/NF-kappaB and p70 S6 kinase, as well as Stat3; however, p70 S6 kinase was not involved in ARV S1133-mediated effects. DF1 cells over-expressing constitutively active PI 3-kinase and Stat3 showed association with enhancement of anti-apoptotic activity. In conclusion, in the early stages of ARV S1133 infection, activation of cell survival signals contributes to virus-induced inflammation and anti-apoptotic response.

A Novel l-Amino Acid Oxidase from Trichoderma harzianum ETS 323 Associated with Antagonism of Rhizoctonia solani

Trichoderma spp. are used as biocontrol agents against phytopathogens such as Rhizoctonia solani, but their biocontrol mechanisms are poorly understood. A novel L-amino oxidase (Th-LAAO) was identified from the extracellular proteins of Trichoderma harzianum ETS 323. Here, we show a FAD-binding glycoprotein with the best substrate specificity constant for L-phenylalanine. Although the amino acid sequence of Th-LAAO revealed limited homology (16-24%) to other LAAO members, a highly conserved FAD-binding motif was identified in the N-terminus. Th-LAAO was shown to be a homodimeric protein, but the monomeric form was predominant when grown in the presence of deactivated Rhizoctonia solani. Furthermore, in vitro assays demonstrated that Th-LAAO had an antagonistic effect against Rhizoctonia solani and a stimulatory one on hyphal density and sporulation in T. harzianum ETS 323. These findings further our understanding of T. harzianum as a biocontrol agent and provide insight into the biological function of l-amino acid oxidase.

Identification of antibacterial mechanism of l‐amino acid oxidase derived from Trichoderma harzianum ETS 323

Although l‐amino oxidase (LAAO; EC 1.4.3.2) has been reported to be a potent antibacterial agent, the mechanism responsible for its antibacterial activity has not been identified. The present study aimed to identify the mechanism responsible for the antibacterial activity of Th‐LAAO, an LAAO recently isolated from the extracellular proteins of Trichoderma harzianum ETS 323, at the same time as elucidating the nature of this enzyme. The results obtained indicate that the enzyme activity and structure of Th‐LAAO are stable at pH 6–8 and less stable at both pH 4–5.5 and pH 9. At pH 7.0, the optimum temperature for Th‐LAAO was found to be 40 °C, comprising the temperature at which enzymatic activity is greatest, with enzymatic activity deceasing with further increases in temperature as a result of thermal denaturation of the enzyme, leading to partial denaturation at 50 °C. The results obtained by confocal microscopy and flow cytometry indicate that Th‐LAAO interacts with bacteria to cause membrane permeabilization, and this interaction may be promoted by the amphipathic sequence in Th‐LAAO and other cytotoxic LAAOs located at the N‐terminus. The findings of increased exogenous H2O2 production and reactive oxidative species accumulation in Th‐LAAO‐treated bacteria indicate that reactive oxidative species accumulation may trigger forms of cell damage, including lipid peroxidation and DNA strand breakage that results in bacterial growth inhibition. Taken together, the results indicate that the processes of bacterial interaction, membrane permeabilization and H2O2 production are involved in the mechanism responsible for the antibacterial activity of Th‐LAAO.

Avian reovirus S1133-induced DNA damage signaling and subsequent apoptosis in cultured cells and in chickens

In this study, intracellular signaling in ARV S1133-mediated apoptosis was investigated. A microarray was used to examine the gene expression profiles of cells upon ARV S1133 infection and ARV-encoded pro-apoptotic protein σC overexpression. The analysis indicated that in the set of DNA-damage-responsive genes, DDIT-3 and GADD45α were both upregulated by viral infection and σC overexpression. Further investigation demonstrated that both treatments caused DNA breaks, which increased the expression and/or phosphorylation of DNA damage response proteins. ROS and lipid peroxidation levels were increased, and ARV S1133 and σC caused apoptosis mediated by DNA damage signaling. ROS scavenger NAC, caffeine and an ATM-specific inhibitor significantly reduced ARV S1133- and σC-induced DNA breaks, DDIT-3 and GADD45α expression, H2AX phosphorylation, and apoptosis. Overexpression of DDIT-3 and GADD45α enhanced the oxidative stress and apoptosis induced by ARV S1133 and σC. In conclusion, our results demonstrate the involvement of the DNA-damage-signaling pathway in ARV S1133- and σC-induced apoptosis.

Monocyte Chemotactic Protein 1 (MCP-1) Modulates Pro-Survival Signaling to Promote Progression of Head and Neck Squamous Cell Carcinoma

Background Monocyte chemotactic protein-1 (MCP-1) recruits monocytes and macrophages to inflammation sites, and inflammatory infiltration correlates with the progression of head and neck squamous cell carcinoma (HNSCC). This study aims to determine whether MCP-1 expression is related to HNSCC malignancy and patient survival. We also investigated the relationship between MCP-1 expression and the phosphorylation state of the pro-survival pathway factors Akt, ERK, and STAT3. Methods Expression of MCP-1 and related proteins in HNSCC cell lines was investigated using western blotting. HNSCC patients (34) without distant metastasis at diagnosis were recruited for tissue specimen evaluation of MCP-1 expression and clinical outcomes. The relationship between MCP-1 expression and survival was evaluated using the Cox proportional hazard model with stepwise selection. Results High-grade HNSCC cell lines were found to have higher levels of active Akt, ERK, and/or STAT3 than did lower grade cell lines under serum-free condition. OCSL, the most malignant cell line, had the highest level of endogenous MCP-1. Administration of exogenous recombinant MCP-1 increased phosphorylation of Akt, ERK, and STAT3 in a dose- and time-dependent manner and increased cellular resistance to serum starvation. Inhibition of Akt, ERK, or STAT3 reduced cell growth and caused cell death. Long-term survival of HNSCC patients was negatively associated with the histological intensity of MCP-1, implicating MCP-1 as a potential prognostic marker for HNSCC. Conclusions These results suggest that overexpressed MCP-1 in cancer cells may promote HNSCC progression through upregulating pro-survival signaling pathways. High cellular MCP-1 expression is related to poor overall survival rate in HNSCC patients.

PI 3-kinase/Akt and STAT3 are required for the prevention of TGF-β-induced Hep3B cell apoptosis by autocrine motility factor/phosphoglucose isomerase

We established Hep3B cells stably-expressing wild-type and mutated AMF/PGI with differing enzymatic activities in order to investigate how AMF/PGI affects TGF-beta-induced apoptosis, and demonstrated that AMF/PGI against TGF-beta-induced apoptosis was correlated with its enzymatic activity. AMF/PGI did not alter TGF-beta-receptor expression nor affect TGF-beta-induced PAI-1 gene promoter or Smad3/4 activity. AMF/PGI induced PI 3-kinase activity, IRS and Akt phosphorylation, which can further regulate BAD phosphorylation. Constitutively-active p110 enhanced AMF/PGI-mediated anti-apoptosis activity, and dominant negative Akt alleviated anti-TGF-beta-induced apoptosis. We also demonstrated that STAT3 is a weak anti-apoptotic agent but has an increased anti-apoptotic effect in cooperation with PI 3-kinase/Akt.