Ugh these murine Bcr/Abl ALL cells contain an identical transgene
Ugh these murine Bcr/Abl ALL cells contain an identical transgene, they also exhibited different sensitivity to this drug.PHA-739358 induces apoptosis and leads to an accumulation of cells with 4N DNA contentThe ability of PHA-739358 to induce apoptosis was measured by Annexin V/PI staining in Pt2 and UCSF02 cells treated with increasing concentrations of the drug for 48 hours. As demonstrated in BIM-22493 site Figure 2A, PHA-739358 induced apoptosis both in Pt2 and UCSF02 cells. Since inhibition of Aurora kinases causes endoreduplication and polyploidy [28], we assessed DNA content at different time points in Ph-positive BLQ1 and Ph-negative US6 cells treated with PHA-739358. Mutations and deletions of p53 are rare in ALL and of the samples examined here, only US6 had defective p53 function (by p53 deletion; not shown). In agreement with previous findings using Aurora kinase inhibitors (PHA-739358, VX-680, AZD1152) in other types of cancer cells [24,28-30], PHA-739358 caused accumulation of BLQ1 and US6 cells with more than or equal to 4 N DNA content as early as 16 hours (Figure 2B). Moreover, 1 M PHA-739358 generated polyploid (4 N DNA) cells and produced a significant reduction PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/27321907 in viability, as assessed by the percentage of cells in the sub-G1 DNA content.PHA-739358 targets both Bcr/Abl and Aurora kinase activitiesResultsPHA-739358 reduces viability of acute lymphoblastic leukemia cells including those with the Bcr/Abl T315I mutationTo determine the impact of the Bcr/Abl status on the efficacy of PHA-739358, we treated human ALL cells including BLQ1, Pt2 (Bcr/Abl+, T315I mutation), UCSF02, TXL2 (Bcr/Abl+, wild type), US7, US7R (non-Bcr/Abl) and mouse 8093 and Bin2 cells (Bcr/Abl+, wild type) with increasing concentrations of PHA-739358 for 72 hours. In Phase I-II clinical trials, a Cmax of 4? M/h was observed for CML patients harboring the T315I mutation when PHA-739358 was administered at 330 mg/m2/day [27]. Therefore, we used clinically relevant and achievable concentrations of up to 5 M PHA-739358 in our experiments. As shown in Figure 1, increasing concentrations of PHA-739358 caused a cytotoxic effect on all the leukemia cells tested as measured by the decreased viability of thePHA-739358 was reported to inhibit both Bcr/Abl kinase and Aurora kinase in vitro [31], whereas dasatinib targets Bcr/Abl and Src-family kinases [32]. To examine this in human Ph-positive ALL cells, the effect of PHA-739358 on the activity of Bcr/Abl was determined by examining the phosphorylation of overall tyrosine, of Crkl and of Stat5. A concentration of 1 M PHA-739358 blocked the generation of total phosphotyrosine significantly in both T315I Bcr/Abl BLQ1 and wild type Bcr/Abl UCSF02 cells (Figure 3A). As shown in Figure 3A, increasing concentrations of PHA-739358 decreased the phosphorylation status of Crkl. Stat5 phosphorylation was completely inhibited even at 1 M PHA-739358. Treatment with 100 nM dasatinib (concentration chosen based on the maximum plasma concentration range [33]) 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 (data not shown). 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 [34] using Phpositive BLQ1 and Ph-negative US6 cells.
