Oncoprotein 18/stathmin (Op18/stathmin) plays a crucial role in maintaining cell biological characteristics by regulating microtubule dynamics, especially entry into mitosis; phosphorylated Op18/stathmin promotes microtubule polymerization to form the mitotic spindle, which is essential for chromosome segregation and cell division. Cdc2 is a critical kinase in starting M phase events in cell-cycle progression and is a positive regulator of the cell cycle. Latent membrane protein 1 (LMP1) is an Epstein-Barr virus (EBV)-encoded oncogenic protein that is able to induce carcinogenesis via various signaling pathways. This study focused on regulation by LMP1 of Op18/stathmin signaling in nasopharyngeal carcinoma (NPC) cells and showed that LMP1 regulates Op18/stathmin signaling through cdc2 mediation, LMP1 upregulates cdc2 kinase activity, and Op18/stathmin phosphorylation promotes the interaction of cdc2 with Op18/stathmin and microtubule polymerization during mitosis, and inhibition of LMP1 expression attenuates the interaction of cdc2 and Op18/stathmin and promotes microtubule depolymerization. These results reveal a new pathway via which LMP1 regulates Op18/stathmin signaling by cdc2 mediation; this new signaling pathway not only perfects the LMP1 regulation network but also elucidates the molecular mechanism of LMP1 that leads to carcinogenesis. ' 2008 Wiley-Liss, Inc.Key words: Op18; LMP1; cdc2; NPC; signaling pathway Epstein-Barr virus (EBV) is closely associated with nasopharyngeal carcinoma (NPC) and latently infects the majority of the world's population. EBV infection is characterized mainly by expression of type II latent proteins, including latent membrane proteins (1, 2) and Epstein-Barr nuclear antigen-1 encoded by EBV in NPCs. Latent membrane protein 1 (LMP1) is an EBVencoded oncogenic protein that is able to induce carcinogenesis, which includes promoting the cellular transformation of B lymphocytes and epithelial cells and of immortalized cells into neoplastic cells.

Purvalanol A is a highly selective inhibitor of Cdc2 [also known as cyclin-dependent kinase 1 (CDK1)]. Taxol is an anti-tumor chemotherapeutic drug which is widely used clinically. In this study, the CDK1 inhibitor, purvalanol A was applied to explore the relevance of Cdc2 signaling and taxol sensitivity through analyses, such as cellular proliferation and apoptosis assays, ELISA, western blot analysis and immunoprecipitation. We demonstrated that purvalanol A effectively enhanced the taxol-induced apoptosis of NCI-H1299 cells, as well as its inhibitory effects on cellular proliferation and colony formation. In combination, purvalanol A and taxol mainly decreased the expression of oncoprotein 18 (Op18)/stathmin and phosphorylation at Ser16 and Ser38, while purvalanol A alone inhibited the phosphorylation of Op18/stathmin at all 4 serine sites. Co-treatment with purvalanol A and taxol weakened the expression of Bcl-2 and activated the extrinsic cell death pathway through the activation of caspase-3 and caspase-8. Further experiments indicated that Cdc2 kinase activities, including the expression of Cdc2 and the level of phospho-Cdc2 (Thr161) were significantly higher in taxol-resistant NCI-H1299 cells compared with the relatively sensitive CNE1 cells before and following treatment with taxol. These findings suggest that Cdc2 is positively associatd with the development of taxol resistance. The Cdc2 inhibitor, purvalanol A, enhanced the cytotoxic effects of taxol through Op18/stathmin. Our findings may prove to be useful in clinical practice, as they may provide a treatment strategy with which to to reduce the doses of taxol applied clinically, thus alleviating the side-effects.

NCI-H1299 cells are evidently resistant to taxol-induced cellular apoptosis, inhibition of cellular proliferation and wound recovery, as well as cell migration and invasion interference, which are closely associated with the changes of microtubule dynamics. High expression of Op18/stathmin is perhaps a crucial determinant of taxol-resistant development in NCI-H1299 cells.

Arginine-specific ADP-ribosytransferases 1 (ART1) is able to modify the arginine of specific proteins by mono-ADP-ribosylation. We previously reported that the expression of ART1 in human colon adenocarcinoma tissues was higher than in adjacent tissues. Herein, we primarily revealed that ART1 could regulate the epithelial-mesenchymal transition (EMT) and, therefore, the development of colon carcinoma. In CT26 cells, which overexpressed ART1 by lentiviral transfection, the following were promoted: alterations of spindle-like non-polarization, expression of EMT inducers and mesenchymal markers, migration, invasion and adhesion. However, epithelial marker expression was decreased. Correspondingly, knockdown of ART1 in CT26 cells had the opposite effects. The effect of ART1 on EMT and carcinoma metastasis was also verified in a liver metastasis model of BALB/c mice. To further explore the molecular mechanism of ART1 in EMT, CT26 cells were treated with several specific inhibitors and gene silencing. Our data suggest that ART1 could regulate EMT by regulating the RhoA/ROCK1/AKT/β-catenin pathway and its downstream factors (snail1, vimentin, N-cadherin and E-cadherin) and that it therefore plays an important role in the progression of colon carcinoma.

The MAPKs are activated by a variety of cellular stimuli to participate in a series of signaling cascades and mediate diverse intracellular responses. One potential target of the MAPKs is Op18/ stathmin, a molecule that acts as an integrator of diverse cell signaling pathways and regulates the dynamics of microtubules, which are involved in modulating a variety of cellular processes, including cell cycle progression and cell growth. Our study focused on the regulation of the MAPK-mediated Op18/stathmin signaling pathway, which is triggered by the Epstein-Barr virusencoded latent membrane protein 1 (LMP1) oncogene in nasopharyngeal carcinoma cells. The results showed that the activity of MAPK, which was induced by LMP1, varied with cell cycle progression; LMP1 upregulated phosphorylation of ERK during the G 1 /S phase, but negatively regulated phosphorylation of ERK during the G 2 /M phase. We found that the regulation of Op18/ stathmin signaling by LMP1 was mainly mediated through ERK. The inhibition of LMP1 expression attenuated the interaction of ERK with Op18/stathmin and promoted microtubule depolymerization. These findings indicate the existence of a new cell cycle-associated signaling pathway in which LMP1 regulates ERK-mediated Op18/stathmin signaling. (Cancer Sci 2012; 103: 993-999) T he Epstein-Barr virus (EBV) is a prototype gamma herpes virus. Infection with EBV is mainly characterized by the expression of type II latent proteins, including latent membrane protein (LMP) 1 and LMP2 in nasopharyngeal carcinoma (NPC).(1-3) LMP1, an EBV-encoded oncogene, abnormally activates the nuclear factor-jB, activator protein-1, and signal transducer and activator of transcription signaling pathways by phosphorylating epidermal growth factor receptor, Jun N-terminal kinase (JNK), Janus-activated kinase, and other signaling molecules in NPC cells.(1-4) However, the full complement of LMP1-regulated proteins has not been thoroughly elucidated. Recently, we identified phosphorylation sites on 25 new components of the LMP1 signaling pathway, including oncoprotein 18 (Op18)/stathmin, heat shock protein 27, annexin I, and several kinases. A novel target of LMP1, Op18/stathmin is a highly conserved small cytosolic phosphoprotein. Op18/stathmin that is overexpressed in tumors, including leukemia and breast carcinomas, (6) regulates microtubule dynamics. During the cell cycle, Op18/stathmin integrates different signals to regulate microtubule polymerization and depolymerization, and its activation adapts to the phase of the cell cycle.(7) Recently, we showed that LMP1 accelerates the cell cycle progression by cdc2-mediated Op18/stathmin phosphorylation during the G 2 / M phase.(8) A dynamic microtubule equilibrium is crucial to a series of biological features, including cell morphology stabilization, substance transportation, cell division and proliferation, and cell migration and invasion.(9) The level of Op18/stathmin expression also correlates with pathologic features and outcomes in the clinic. (10) Numerous kinases,...