Silencing KLB expression decreases HCC cell growth via cell cycle arrest Having

To examine this in human Ph positive ALL cells, the effect of PHA 739358 on the activity of BcrAbl was determined by examining the phosphorylation of overall tyrosine, ABT-737 構造 of Crkl and of Stat5. A concentration of 1 uM PHA 739358 blocked the gener ation of total phosphotyrosine significantly in both T315I BcrAbl BLQ1 and wild type BcrAbl UCSF02 cells. As shown in Figure 3A, increasing concentra tions of PHA 739358 decreased the phosphorylation status of Crkl. Stat5 phosphorylation was completely inhibited even at 1 uM PHA 739358. Treatment with 100 nM dasa tinib also induced a distinct inhibition in phosphotyosine, p Crkl, p Stat5 and p Src in UCSF02 cells. However, as expected, there was no effect of dasatinib in BLQ1 cells harboring the T315I mutation. Similar results were also obtained with cell cycle analysis.<br><br> We also evaluated the effect of PHA 739358 on Aurora B kinase activity, by measuring inhibition of phosphorylation of its substrate histone H3 at position Ser10 using Ph positive BLQ1 and Ph negative US6 cells. As shown in Figure 3B, 24 hours of treatment with 1 uM PHA 739358 caused an obvious オーダー AEB071 reduction of p histone H3 to 0. 1% compared to 1. 6% and 1. 4% in untreated BLQ1 and US6 cells respectively. ALL cells resume proliferation after short term PHA 739358 treatment As mentioned above, in the presence of stroma, 1 uM PHA 739358 treatment for 3 days left 50% of the Pt2 and UCSF02 cells in an apparently viable state. In the study by Gontarewicz et al. they observed that PHA 739358 significantly inhibited the proliferation of K562 cells in vivo during 10 days of treatment.<br><br> However, when the application supplier AG-014699 of the drug was terminated, K562 cells started to proliferate again. We therefore examined the effect of short term treat ment of PHA 739358, followed by no treatment. Pt2 and UCSF02 cells were exposed to 1 uM of PHA 739358 for 3 days in the presence of stroma, after which drug was removed. As shown in Figure 4A, after re moval of PHA 739358 on day 3, viability of both Pt2 and UCSF02 cultures increased gradually. By day 16, cells began to proliferate again and the viability of the cells reached a level similar to that of the control culture. However, such cells remained sensitive to re treatment with PHA 739358, and BcrAbl exhibited a sensitivity similar to that displayed by the orignal non drug treated cells.<br><br> This indicates that the ALL cells had not acquired genetic re sistance to this Aurora kinase inhibitor. Combination treatment significantly increases effect of PHA 739358 To investigate the possibility of increasing the effect of PHA 739358 on cell cycle inhibition, we tested it in combination with a second drug that also affects cell cycle. Farnesyltransferase inhibitors inhibit farne sylation of mitotic proteins CENP E and CENP F while Aurora kinases inhibitors will inhibit the phosphoryl ation of CENP E. We therefore treated Pt2 and UCSF02 with 500 nM or 1 uM of the FTI Lonafarnib alone or together with 1 uM PHA 739358 for 3 days. As shown in Figure 4B, exposure of Pt2 or UCSF02 to 500 nM or 1 uM FTI alone resulted in min imal toxicity as judged by viability, but consistent with its inhibition of cell cycle, did prevent cell proliferation.