Wen-Ling Shih

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.

Suppression of hepatitis B virus x protein-mediated tumorigenic effects by ursolic Acid.

This study investigated the potential effects of natural products ursolic acid (UA) and oleanolic acid (OA) against HBx-mediated tumorigenic activities in vitro and in vivo. HBx transactivated Sp-1 and Smad 3/4 in Huh7 and FL83B hepatocytes and induced cell migration of Huh7 and HepG2. HBx also induced MMP-3 secretion in Huh7 and acted against TGF-β-induced apoptosis in Hep3B. UA almost completely blocked the HBx-mediated effects, while OA had a partial inhibitive effect. Utilization of specific MAPK inhibitors and immunoblotting demonstrated that UA selectively activated MAPK signaling in certain tested cells. Preintraperitoneal injection of UA fully prevented the tumor growth of HBV-containing 2.2.15 cells, while OA-treated mice had smaller tumors than the control group. Our results suggested that UA possesses a hepatoprotective ability and illustrated the evident effects against HBx-mediated tumorigenic activities without toxicity in a mouse model.

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.

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.

Bovine ephemeral fever virus-induced apoptosis requires virus gene expression and activation of Fas and mitochondrial signaling pathway

Although induction of apoptosis by bovine ephemeral fever virus (BEFV) in several cell lines has been previously demonstrated by our laboratory, less information is available on the process of BEFV-induced apoptosis in terms of cellular pathways and specific proteins involved. In order to determine the step in viral life cycle at which apoptosis of infected cells is triggered, chemical and physical agents were used to block viral infection. Treatment of BHK-21 infected cells with ammonium chloride (NH4Cl) or cells infected with UV-inactivated BEFV was seen to abrogate virus apoptosis induction, suggesting that virus uncoating and gene expression are required for the induction of apoptosis. Using soluble death receptors Fc:Fas chimera to block Fas signaling, BEFV-induced apoptosis was inhibited in cells. BEFV infection of BHK-21 cells results in the Fas-dependent activation of caspase 8 and cleavage of Bid. This initiated the dissipation of the membrane potential and the release of cytochrome c but not AIF or Smac/DIABLO from mitochondrial into cytoplasm leading to activation of caspase 9. Combined activation of the death receptor and mitochondrial pathways results in activation of the downstream effecter caspase 3 leading to cleavage of PARP. Fas-mediated BEFV-induced apoptosis could be suppressed by the overexpression of Bcl-2 or by treatment with caspase inhibitors and soluble death receptors Fc:Fas chimera. Taken together, this study provided first evidence demonstrating that BEFV-induced apoptosis requires viral gene expression and occurs through the activation of Fas and mitochondrion-mediated caspase-dependent pathways.

AMF/PGI transactivates the MMP-3 gene through the activation of Src-RhoA-phosphatidylinositol 3-kinase signaling to induce hepatoma cell migration

We have previously shown that AMF/PGI induces hepatoma cell migration through the induction of MMP-3. This work investigates how AMF/PGI activates the MMP-3 gene. We demonstrated that AMF/PGI transactivates the MMP-3 gene promoter through AP-1. The transactivation and induction of cell migration effect of AMF/PGI directly correlates with its enzymatic activity. Various analyses showed that AMF/PGI stimulated the Src-RhoA-PI3-kinase signaling pathway, and these three signaling molecules could form a complex. Our results demonstrate a new mechanism of AMF/PGI-induced cell migration and a link between Src-RhoA-PI3-kinase, AP-1, MMP-3 and hepatoma cell migration.

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.