Several kinases phosphorylate vimentin, the most common intermediate filament protein, in mitosis. Aurora-B and Rho-kinase regulate vimentin filament separation through the cleavage furrow-specific vimentin phosphorylation. Cdk1 also phosphorylates vimentin from prometaphase to metaphase, but its significance has remained unknown. Here we demonstrated a direct interaction between Plk1 and vimentin-Ser55 phosphorylated by Cdk1, an event that led to Plk1 activation and further vimentin phosphorylation. Plk1 phosphorylated vimentin at ∼1 mol phosphate/mol substrate, which partly inhibited its filament forming ability, in vitro. Plk1 induced the phosphorylation of vimentin-Ser82, which was elevated from metaphase and maintained until the end of mitosis. This elevation followed the Cdk1-induced vimentin-Ser55 phosphorylation, and was impaired by Plk1 depletion. Mutational analyses revealed that Plk1-induced vimentin-Ser82 phosphorylation plays an important role in vimentin filaments segregation, coordinately with Rho-kinase and Aurora-B. Taken together, these results indicated a novel mechanism that Cdk1 regulated mitotic vimentin phosphorylation via not only a direct enzyme reaction but also Plk1 recruitment to vimentin.

The study did not demonstrate noninferiority of gefitinib compared with erlotinib in terms of PFS in patients with lung adenocarcinoma according to the predefined criteria.

No abstract

Gemcitabine is one of the most commonly used agents for lung cancer chemotherapy, but the determinants of sensitivity and/or resistance to this agent are not yet fully understood. In this study we used quantitative RT-PCR to examine the expression levels of human equilibrative nucleoside transporter 1 (hENT1) and deoxycytidine kinase (dCK) genes in non-small cell lung cancer (NSCLC) cell lines in relation to sensitivity and resistance to gemcitabine. The basal expression levels of hENT1 were significantly correlated with the IC 50 values for gemcitabine (r = = = =-0.6769, P = = = =0.0005), whereas dCK expression levels were not. In a highly gemcitabinesensitive cell line, NCI-H23, the sensitivity to gemcitabine was inhibited by nitrobenzylmercaptopurine ribonucleoside (NBMPR), an inhibitor of hENT1, without significant modulation of hENT1 expression. These data suggest that hENT1 is associated with gemcitabine sensitivity in lung cancer. We also continuously exposed NCI-H23 cells to gemcitabine and subsequently established the drug-resistant clone H23/GEM-R, which showed a significant decrease of dCK expression; however, hENT1 expression was not altered in the continuously exposed sublines or in the resistant clone. We conclude that increased hENT1 expression is a determinant of gemcitabine sensitivity, while decreased dCK expression is associated with acquired resistance to gemcitabine in NSCLC cells. Thus, hENT1 and dCK might be useful as predictive markers for efficacy of gemcitabine therapy in NSCLC. (Cancer Sci 2004; 95: 753-757) ung cancer is one of the most common malignancies worldwide 1) and remains the leading cause of cancer-related deaths in Western countries.2) Several randomized clinical trials and meta-analyses have demonstrated that survival in patients with advanced stage III or IV non-small cell lung cancer (NSCLC) can be slightly but significantly prolonged with chemotherapy.3, 4) Several new anticancer agents have recently been shown to have encouraging activity against NSCLC, and a recent randomized study provided data supportive of the efficacy and safety of a regimen including gemcitabine. 5)

No abstract

Multidrug-resistance -associated protein, MRP8/ABCC11 (ABCC11), is an efflux pump for nucleotide analogues and 5-fluoro-2 ¶-deoxyuridine 5 ¶-monophosphate (FdUMP). To test whether ABCC11 directly confers 5-fluorouracil (5-FU) resistance, we used the 5-FU -resistant subline PC-6/FU23-26 selected from PC-6 human small-cell lung cancer cells by 5-FU and found that it increases the resistance by f25-fold. The intracellular FdUMP accumulation was reduced in PC-6/FU23-26 cells concomitant with the overexpression of the ABCC11 gene. These findings suggest that ABCC11 confers 5-FU resistance in the sublines by enhancing the efflux for the active metabolite FdUMP. Previously, methotrexate also increased the efflux by ABCC11, and we found crossresistance to methotrexate in PC-6/FU23-26 cells. To confirm our hypothesis, we examined whether decreasing the expression of ABCC11 in PC-6/FU23-26 cells by small interfering RNA altered the cytotoxicity to 5-FU and methotrexate and found that this enhanced 5-FU and methotrexate cytotoxicity in PC-6/FU23-26 cells. These data indicate that expression of the ABCC11 gene is induced by 5-FU, and that ABCC11 is directly involved in 5-FU resistance by the efflux transport of the active metabolite FdUMP. [Mol Cancer Ther 2007;6(1):122 -7]

Pemetrexed (MTA) is a multitargeted antifolate with promising clinical activity in lung cancer. We exposed the small cell lung cancer cell line PC6 to stepwise-increasing pemetrexed concentrations of 0.4, 1.6, and 4.0 lM, and established three pemetrexed-resistant lung cancer cell lines: PC6 ⁄ MTA-0.4, PC6 ⁄ MTA-1.6, and PC6 ⁄ MTA-4.0 cells. To investigate the mechanisms of acquired resistance to pemetrexed, we measured the expression levels of the thymidylate synthase (TS), reduced folate carrier (RFC), and folylpolygamma-glutamate synthetase (FPGS) genes. TS gene expression was significantly increased in PC6 ⁄ MTA-1.6 and PC6 ⁄ MTA-4.0 cells relative to parental cells in a pemetrexed dose-dependent manner. In contrast, the levels of RFC gene expression in PC6 ⁄ MTA-0.4 cells and FPGS in PC6 ⁄ MTA-1.6 cells were significantly decreased, whereas the levels of both genes were restored in PC6 ⁄ MTA-4.0 cells. Knockdown of TS expression using siRNA enhanced pemetrexed cytotoxicity in PC6 ⁄ MTA-4.0 cells. The expression level of the TS gene was significantly correlated with the concentration of pemetrexed for 50% cell survival (IC 50 ) in 11 non-small cell lung cancer cell lines. These results suggest that the alteration of molecular pharmacological factors in relation with pemetrexed resistance is dose-dependent, and that up-regulation of the expression of the TS gene may have an important role in the acquired resistance to pemetrexed. In addition, TS may be a predictive marker for pemetrexed sensitivity in lung cancer. (Cancer Sci 2010; 101: 161-166) P emetrexed is an MTA that targets the folate-dependent enzymes TS, DHFR, GARFT, and AICARFT, all of which are involved in the de novo biosynthesis of thymidine and purine nucleotides.(1) Pemetrexed is transported intracellularly, predominantly via the RFC, where it is metabolized to polyglutamated forms. Pemetrexed was found to be one of the best substrates for mammalian FPGS, and it is believed that polyglutamation and the polyglutamated metabolites play important roles in determining both the selectivity and antitumor activity of this agent.(2,3) The polyglutamated metabolites of pemetrexed are most active against TS, followed by DHFR, GARFT and AI-CARFT, and natural folate competes with this inhibition in all cases.(4) Therefore, the primary mechanism of the action of pemetrexed is inhibition of TS, which results in a decrease in the available thymidine necessary for DNA synthesis. (2,4) Pemetrexed is a single agent that is currently approved for second-line treatment of advanced-stage NSCLC.(5,6) In chemotherapy-naïve patients with advanced NSCLC, double combinations of platinum compounds with gemcitabine, vinorelbine, paclitaxel, and docetaxel are standard regimens. A recent phase III trial found that cisplatin ⁄ pemetrexed provides equivalent efficacy with significantly fewer side effects and more convenient administration than cisplatin ⁄ gemcitabine in advanced NSCLC.(7) Preclinical examinations found that the combination of pemetrexed with gemcitabine or...

We examined the expression levels of the multidrug resistance protein 5 (ABCC5) gene in non -small cell lung cancer (NSCLC) cell lines to clarify the relationship with the sensitivity to gemcitabine. The expression levels of ABCC5 were inversely correlated with gemcitabine sensitivity significantly (r = 0.628; P < 0.01) in 17 NSCLC cells, whereas the expression of ABCC5 in the gemcitabine-resistant NSCLC cell line H23/GEM-R was the same as that in parental NCI-H23 cells. Treatment with the ABCC5 inhibitor zaprinast altered the sensitivity to gemcitabine in ABCC5-expressing NSCLC cells. In addition, decreasing the expression of ABCC5 by small interfering RNA altered the cytotoxicity to gemcitabine. These results indicate that modulation of ABCC5 activity could be used to increase the gemcitabine sensitivity in NSCLC. Previously, we found a decreased expression of deoxycytidine kinase in H23/GEM-R cells, and further investigation in this study showed an increased expression of ribonucleotide reductase subunit 1 in H23/GEM-R cells. We therefore also examined the effect of modifying the expression of both genes on gemcitabine resistance. We found that using small interfering RNA to decrease the expression of ribonucleotide reductase subunit 1 resulted in a decreased resistance to gemcitabine in H23/GEM-R cells. Furthermore, pretreatment with pemetrexed resulted in an increased deoxycytidine kinase expression concomitant with the alteration of the resistance to gemcitabine in H23/GEM-R cells. The determinants for sensitivity and the acquired resistance in gemcitabine are quite different; nonetheless, modification of these factors may increase the efficacy of gemcitabine in the treatment of NSCLC.