The tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) can trigger growth inhibition, epithelial-mesenchymal transition (EMT) -like cell scattering, and migration of hepatoma cells HepG2 in a protein kinase C-A (PKC-A) -dependent manner. Saikosaponin a, an ingredient of antitumorigenic Chinese herb Sho-Saiko-to, inhibited cell growth but did not induce EMT-like cell scattering and cell migration of HepG2. Saikosaponin a and TPA induced transient (for 30 minutes) and sustained (until 6 hours) phosphorylation of extracellular signal-regulated kinase (ERK), respectively. Generation of the reactive oxygen species (ROS) was induced by TPA, but not saikosaponin a, for 3 hours. As expected, scavengers of ROS, such as superoxide dismutase, catalase, and mannitol, and the thiol-containing antioxidant N-acetylcystein dramatically suppressed the TPA-triggered cell migration but not growth inhibition of HepG2. The generation of ROS induced by TPA was PKC, but not ERK, dependent. On the other hand, scavengers of ROS and N-acetylcystein also prevented PKC activation and ERK phosphorylation induced by TPA. On the transcriptional level, TPA can induce gene expression of integrins A 5 , A 6 , and B 1 and reduce gene expression of E-cahedrin in a PKC-and ROS-dependent manner. In conclusion, ROS play a central role in mediating TPA-triggered sustained PKC and ERK signaling for regulation of gene expression of integrins and E-cahedrin that are responsible for EMT and migration of HepG2. (Mol Cancer Res 2006;4(10):747 -58)
Mature vaccinia virus enters cells through either fluid-phase endocytosis/macropinocytosis or plasma membrane fusion. This may explain the wide range of host cell susceptibilities to vaccinia virus entry; however, it is not known how vaccinia virus chooses between these two pathways and which viral envelope proteins determine such processes. By screening several recombinant viruses and different strains, we found that mature virions containing the vaccinia virus A25 and A26 proteins entered HeLa cells preferentially through a bafilomycin-sensitive entry pathway, whereas virions lacking these two proteins entered through a bafilomycin-resistant pathway. To investigate whether the A25 and A26 proteins contribute to entry pathway specificity, two mutant vaccinia viruses, WR⌬A25L and WR⌬A26L, were subsequently generated from the wild-type WR strain. In contrast to the WR strain, both the WR⌬A25L and WR⌬A26L viruses became resistant to bafilomycin, suggesting that the removal of the A25 and A26 proteins bypassed the low-pH endosomal requirement for mature virion entry. Indeed, WR⌬A25L and WR⌬A26L virus infections of HeLa, CHO-K1, and L cells immediately triggered cell-to-cell fusion at a neutral pH at 1 to 2 h postinfection (p.i.), providing direct evidence that viral fusion machinery is readily activated after the removal of the A25 and A26 proteins to allow virus entry through the plasma membrane. In summary, our data support a model that on vaccinia mature virions, the viral A25 and A26 proteins are low-pH-sensitive fusion suppressors whose inactivation during the endocytic route results in viral and cell membrane fusion. Our results also suggest that during virion morphogenesis, the incorporation of the A25 and A26 proteins into mature virions may help restrain viral fusion activity until the time of infections.Vaccinia virus has a wide host range and infects many cell lines in cultures and animal species (14). It belongs to the genus Orthopoxvirus of the family Poxviridae and replicates in the cytoplasm of infected cells. Several unique features of vaccinia virus help to maximize its ability to transmit virus infections in different host cells. First, vaccinia virus produces several forms of infectious particles, including mature virus (MV), intracellular wrapped virus (WV), and extracellular enveloped virus (EEV) particles, that are suited for inter-or intrahost cell transmission and dissemination (8). Second, vaccinia virus MV attaches to cell surface components that are commonly expressed on cells. MV contains at least four attachment proteins, of which viral H3, A27, and D8 bind to cell surface glycosaminoglycans (GAGs) (7,18,24) and A26 binds to the extracellular matrix protein laminin (6). Third, MV enters cells through more than one route, since both endocytosis (9, 31) and plasma membrane fusion (1,3,4,10,12,25,39) were reported previously. The endocytosis of vaccinia virus MV is dependent on low pH (4.5 to 5.0) and sensitive to chemicals such as NaF, cytochalasin B (31), as well as bafilomycin A (BFLA),...
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