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1.
Mol Cancer Ther ; 9(3): 661-72, 2010 Mar.
Article in English | MEDLINE | ID: mdl-20159992

ABSTRACT

Aurora kinases play an essential role in orchestrating chromosome alignment, segregation, and cytokinesis during mitotic progression and both aurora-A and B are frequently overexpressed in a variety of human malignancies. In this study, we report the effects of AZD1152-HQPA, a highly selective inhibitor of aurora-B kinase, in acute myeloid leukemia (AML) cell lines and primary samples. We show that AZD1152-HQPA inhibits the phosphorylation of Histone H3 (pHH3) on serine 10 resulting in polyploid cells, apoptosis, and loss of viability in a panel of AML cell lines. We also show that AZD1152-HQPA sensitivity in our cell lines is irrespective of p53 status and the FLT3-ITD-expressing MOLM-13 and MV4-11 cell lines are particularly sensitive to AZD1152-HQPA. Internal tandem duplications (ITD) within the FLT3 tyrosine kinase receptor are found in approximately 25% of AML patients and are associated with a poor prognosis. Here, we report that AZD1152-HQPA directly targets phosphorylated FLT3 along with inhibiting its downstream target phospho-signal transducer and activator of transcription 5 (STAT5) in the FLT3-ITD cell lines. We show pHH3 expression in primary AML blasts and its inhibition by AZD1152-HQPA at low doses in all of our primary samples tested. AZD1152-HQPA inhibits the clonogenic potential of primary AML samples, with FLT3-ITD samples being the most sensitive (P = 0.029). FLT3-ITD primary samples are also more sensitive to pHH3 inhibition (P = 0.022) and are particularly sensitive to pSTAT5 downregulation after treatment with AZD1152-HQPA compared with FLT3 wild-type samples (P = 0.007). We conclude that mutant FLT3 is a secondary target of AZD1152-HQPA and that FLT3-ITD primary samples are particularly sensitive to the drug.


Subject(s)
Leukemia, Myeloid, Acute/drug therapy , Leukemia, Myeloid, Acute/genetics , Quinazolines/administration & dosage , Quinazolines/pharmacology , fms-Like Tyrosine Kinase 3/genetics , Antineoplastic Agents/administration & dosage , Antineoplastic Agents/pharmacology , Apoptosis/drug effects , Aurora Kinase B , Aurora Kinases , Cell Survival/drug effects , Drug Delivery Systems , Drug Evaluation, Preclinical , Gene Expression Regulation, Leukemic/drug effects , Histones/genetics , Histones/metabolism , Humans , Leukemia, Myeloid, Acute/pathology , Mutagenesis, Insertional/physiology , Phosphorylation/drug effects , Protein Kinase Inhibitors/administration & dosage , Protein Kinase Inhibitors/pharmacology , Protein Serine-Threonine Kinases/antagonists & inhibitors , Tandem Repeat Sequences/genetics , Tumor Cells, Cultured , U937 Cells , fms-Like Tyrosine Kinase 3/metabolism
2.
Exp Hematol ; 33(1): 62-72, 2005 Jan.
Article in English | MEDLINE | ID: mdl-15661399

ABSTRACT

OBJECTIVE: Ceramide, an intermediate of apoptosis induction in response to chemotherapy, can be detoxified by glycosylation at the cytoplasmic surface of the Golgi membrane. P-glycoprotein (p-gp) might augment ceramide glycosylation by translocating glucosylceramide (GC) across the Golgi membrane. We aimed to show that glucosylceramide synthase (GCS) activity is linked to p-gp expression and resistance to ceramide-induced apoptosis in acute myeloid leukemia (AML). METHODS: Apoptosis and cell-cycle analysis were measured using propidium iodide staining and flow cytometry. Fluorescent microscopy assessed p-gp expression in, and rhodamine 123 uptake by, the Golgi. P-gp interaction with GC was assessed by modulation of rhodamine accumulation. The GCS activity assay was based upon the transfer of UDP-(3)H-glucose to C8-ceramide to form radiolabeled GC, by rate-limiting cell-derived GCS. TLC and fluorimetry were used to measure the metabolites of fluorescent ceramide. Cell viability was measured using 7-amino-actinomycin D staining and flow cytometry with an internal standard for cell enumeration. RESULTS: P-gp(+) cell lines (KG1a, TF-1) were resistant to C8-ceramide-induced apoptosis compared to p-gp(-) cell lines (HL-60, U937). P-gp inhibitors GF120918 and cyclosporin A enhanced ceramide-induced apoptosis in the p-gp expressing cells. P-gp expression was identified in the Golgi of these cells. Pgp's efflux function in TF-1 but not KG1a cells was inhibited by glucosylceramide. In the presence of p-gp inhibitors, R123 accumulation in the Golgi of TF-1 cells was lost, and GCS activity and lactosylceramide formation were downregulated. Intact cells were necessary for the involvement of p-gp in the regulation of GCS activity. CONCLUSION: Our data suggests that ceramide induces apoptosis in AML cells and that p-gp confers resistance to ceramide-induced apoptosis, with modulation of the ceramide-glucosylceramide pathway making a marked contribution to this resistance in TF-1 cells.


Subject(s)
ATP Binding Cassette Transporter, Subfamily B, Member 1/physiology , Apoptosis , Ceramides/physiology , Glucosyltransferases/metabolism , Leukemia, Myeloid/pathology , ATP Binding Cassette Transporter, Subfamily B, Member 1/genetics , Acute Disease , Cell Cycle , Cell Line, Tumor , Cell Survival , Ceramides/metabolism , Glucosylceramides/biosynthesis , Glucosylceramides/metabolism , Golgi Apparatus/metabolism , Humans
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