Involvement of C-terminal truncation mutation of kinesin-5 in resistance to kinesin-5 inhibitor.

Cultured cells easily develop resistance to kinesin-5 inhibitors (K5Is) often by overexpressing a related motor protein, kinesin-12/KIF15, or by acquiring mutations in the N-terminal motor domain of kinesin-5/KIF11 itself. We aimed to identify novel mechanisms responsible for resistance to S-trityl L-cysteine (STLC), one of the K5Is, using human osteosarcoma cell lines. Among six lines examined, U-2OS and HOS survived chronic STLC treatment and gave rise to resistant cells with IC50s at least 10-fold higher than those of the respective parental lines. Depletion of KIF15 largely eliminated the acquired K5I resistance in both cases, consistent with the proposed notion that KIF15 is indispensable for it. In contrast to the KIF11-independent property of the cells derived from HOS, those derived from U-2OS still required KIF11 for their growth and, intriguingly, expressed a C-terminal truncated variant of KIF11 resulting from a frame shift mutation (S1017fs). All of the isolated clones harbored the same mutation, suggesting its clonal expansion in the cell population due to the growth advantage during chronic STLC treatment. Transgenic expression of KIF11S1017fs in the parental U-2OS cells, as well as in HeLa cells, conferred a moderate but reproducible STLC resistance, probably owing to STLC-resistant localization of the mutant KIF11 on mitotic spindle. Our observations indicate that both KIF15 and the C-terminal-truncated KIF11 contributes to the STLC resistance of the U-2OS derived cells

Changes in the Chemical and Physical Soil Conditions of a Marginal Land Planted with Three Strata Forage System under Three Years of Grazing

This study was conducted to evaluate the changes in the chemical and physical conditions of marginal planted with three strata forage system (TSFS) during three years of grazing period. A-3.2ha of marginal land was divided into eight plots (each plot was 40m×100m), then four plots were planted with TSFS and other four plots were planted with native grasses (Control:C). Eight head of male Bali cattle (average body weight 150kg) grazed continuously in the experimental land (1 cattle/plot). The experiment was carried out for three consecutive years (1992-1995). Results of this study indicated that at the end of the third year, the physical chemical conditions of the soil planted with TSTF were better than the C. Sand percentage in the soil of the TSFS was less than that of the C (71.81 versus 77.86%). Total-N, total-P and total-K contents of the TSFS were higher than those of the C (0.27 versus 0.20%, 224.07 versus 114.34ppm, 6.89 versus 5.79ppm). Similarly, top soil of the TSFS was thicker than that of the C (17.86 versus 9.53cm), and water run off the TSFS was slower than that of the C (19.88 versus 34.88mm/h). However, soil pH, P Bray and infiltration rate were not singificantly different (P>0.05) between the TSFS and the C(6.81 versus 6.78; 16.43 versus 17.01ppm, and 1.55 versus 1.40mm/h, respectively). Results also revealed that the experimental period affected soil pH, total-N, infiltaration rate, top soil, and water run off in the TSFS and the C, but the effects among the periods were not persistent. Analysis also showed that there was no interaction between the planting system (TSFS and C) and the growing periods

NAD(P)H:Quinone Oxidoreductase-1 Expression Sensitizes Malignant Melanoma Cells to the HSP90 Inhibitor 17-AAG

<div><p>The KEAP1-NRF2 pathway regulates cellular redox homeostasis by transcriptional induction of genes associated with antioxidant synthesis and detoxification in response to oxidative stress. Previously, we reported that KEAP1 mutation elicits constitutive NRF2 activation and resistance to cisplatin (CDDP) and dacarbazine (DTIC) in human melanomas. The present study was conducted to clarify whether an HSP90 inhibitor, 17-AAG, efficiently eliminates melanoma with KEAP1 mutation, as the NRF2 target gene, NQO1, is a key enzyme in 17-AAG bioactivation. In melanoma and non-small cell lung carcinoma cell lines with or without KEAP1 mutations, NQO1 expression and 17-AAG sensitivity are inversely correlated. NQO1 is highly expressed in normal melanocytes and in several melanoma cell lines despite the presence of wild-type KEAP1, and the NQO1 expression is dependent on NRF2 activation. Because either CDDP or DTIC produces reactive oxygen species that activate NRF2, we determined whether these agents would sensitize NQO1-low melanoma cells to 17-AAG. Synergistic cytotoxicity of the 17-AAG and CDDP combination was detected in four out of five NQO1-low cell lines, but not in the cell line with KEAP1 mutation. These data indicate that 17-AAG could be a potential chemotherapeutic agent for melanoma with KEAP1 mutation or NQO1 expression.</p></div

Combination treatment with 17-AAG and CDDP or DTIC.

<p>Combination treatment with 17-AAG and CDDP or DTIC.</p