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1.
Leuk Res ; 38(1): 131-7, 2014 Jan.
Article in English | MEDLINE | ID: mdl-24268349

ABSTRACT

Regulation of ABCB1 (P-glycoprotein/Pgp) in AML was investigated. In a historical cohort with Pgp and transcriptional regulator expression profiling data available (n=141), FOXO1 correlated with Pgp protein expression. This was confirmed in an independent cohort (n=204). Down-regulation (siRNA) or hyperactivation (nicotinamide) of FOXO1 led to corresponding changes in Pgp. Low FOXO1 expression correlated with FLT3-ITDs (p<0.001) and siRNA inhibition of FLT3-ITD up-regulated FOXO1. As FOXO1 is a key growth regulator, it may underpin biological differences between Pgp-positive clones (low WBC and primary resistant disease) and clones with a FLT3-ITD (associated with a high WBC and early relapse).


Subject(s)
ATP Binding Cassette Transporter, Subfamily B, Member 1/genetics , Forkhead Transcription Factors/genetics , Leukemia, Myeloid/genetics , fms-Like Tyrosine Kinase 3/genetics , ATP Binding Cassette Transporter, Subfamily B, Member 1/metabolism , Acute Disease , Adult , Blotting, Western , Cell Line, Tumor , Flow Cytometry , Forkhead Box Protein O1 , Forkhead Transcription Factors/metabolism , Gene Expression Regulation, Leukemic , HL-60 Cells , Humans , Leukemia, Myeloid/metabolism , Leukemia, Myeloid/pathology , Leukocyte Count , Mutation , Oligonucleotide Array Sequence Analysis , Prognosis , RNA Interference , Reverse Transcriptase Polymerase Chain Reaction , Tandem Repeat Sequences , Transcriptome , fms-Like Tyrosine Kinase 3/metabolism
2.
Clin Cancer Res ; 15(23): 7291-8, 2009 Dec 01.
Article in English | MEDLINE | ID: mdl-19934300

ABSTRACT

PURPOSE: Acute myeloid leukemia cells with an internal tandem duplication mutation of FLT3 (FLT3-ITD) have effective DNA repair mechanisms on exposure to drugs. Despite this, the phenotype is not associated with primary resistant disease. We show defects in the response of mutant FLT3 AML cells to the S-phase drug clofarabine that could account for the apparent contradiction. EXPERIMENTAL DESIGN: We studied responses of AML cells to clofarabine in vitro. RESULTS: When treated with a short pulse of clofarabine, FLT3-ITD-harboring MOLM-13 and MV4.11 cells undergo similar damage levels (gammaH2AX foci) to wild-type cells but have a better repair capability than wild-type cells. However, whereas the wild-type cells undergo rapid S-phase arrest, the S-phase checkpoint fails in mutant cells. Cell cycle arrest in response to DNA damage in S phase is effected via loss of the transcriptional regulator cdc25A. This loss is reduced or absent in clofarabine-treated FLT3 mutant cells. Furthermore, cdc25A message levels are maintained by the FLT3-ITD, such that message is reduced by 87.5% on exposure to FLT3 small interfering RNA. Primary FLT3-ITD samples from untreated patients also display impaired cell cycle arrest and show enhanced sensitivity on prolonged treatment with clofarabine compared with wild-type samples. CONCLUSION: There is a reversal of phenotype in mutant FLT3 cells dependent on the length of exposure to clofarabine. Efficient DNA repair may render the cells resistant to a short pulse of the drug, but a failure of cell cycle checkpoint(s) in S phase renders the cells sensitive to prolonged exposure.


Subject(s)
Adenine Nucleotides/pharmacology , Antineoplastic Agents/pharmacology , Arabinonucleosides/pharmacology , Leukemia, Myeloid, Acute/drug therapy , Leukemia, Myeloid, Acute/genetics , S Phase/drug effects , Tandem Repeat Sequences , fms-Like Tyrosine Kinase 3/genetics , Apoptosis , Cell Cycle , Cell Line, Tumor , Clofarabine , DNA Repair , Gene Expression Regulation, Leukemic , Histones/metabolism , Humans , Leukemia, Myeloid, Acute/pathology , Mutation , Phenotype
3.
Br J Haematol ; 147(5): 653-61, 2009 Dec.
Article in English | MEDLINE | ID: mdl-19775300

ABSTRACT

FLT3-internal tandem duplication (ITD) mutations are heterogeneous with regards to length and proportion of DNA harbouring the mutation and the expression level of FLT3 also varies widely, however very little is known about the biological effects of these variables. We studied FLT3-associated biological parameters in 322 acute myeloid leukaemia samples to establish their importance. Expression of total FLT3 transcripts was shown to be significantly higher in the FLT3-ITD cohort (n = 121) compared to the wild-type cohort (P = 0.004). Whilst phosphorylated signal transducer and activator of transcription 5 (phospho-STAT5) was not confined to FLT3-ITD samples, within the FLT3-ITD group phosphorylation correlated with adjusted FLT3-ITD levels assessed by determining the total transcripts and proportion of FLT3-ITD within a sample. Expression of the STAT5 downstream target Bcl-xl (an isoform of BCL2L1) was strongly correlated with FLT3 total and adjusted FLT3-ITD levels in FLT3-ITD samples (P < 0.001), however there was no association between Bcl-xl and phospho-STAT5 levels suggesting that STAT5 is not the sole regulator of Bcl-xl in FLT3-ITD cells. We further stratified our cohort by the presence/absence of a cytoplasmic nucleophosmin NPMc+ mutation. Samples co-expressing NPMc+ had longer FLT3-ITD mutations (P = 0.01) and there was a high occurrence of NPMc+ in samples that had >1 FLT3-ITD mutation. Phospho-STAT5 levels were reduced in the FLT3-ITD/NPMc+ group (P = 0.04) suggesting that NPMc+ may oppose the FLT3-ITD-dependent activation of STAT5.


Subject(s)
Leukemia, Myeloid, Acute/genetics , Mutation , Nuclear Proteins/genetics , STAT5 Transcription Factor/metabolism , fms-Like Tyrosine Kinase 3/genetics , DNA Mutational Analysis/methods , Gene Duplication , Humans , Leukemia, Myeloid, Acute/metabolism , Middle Aged , Neoplasm Proteins/metabolism , Nucleophosmin , Phosphorylation , Polymerase Chain Reaction/methods , Tandem Repeat Sequences/genetics , bcl-X Protein/metabolism , fms-Like Tyrosine Kinase 3/biosynthesis
5.
Exp Hematol ; 35(12): 1793-800, 2007 Dec.
Article in English | MEDLINE | ID: mdl-17923246

ABSTRACT

OBJECTIVE: P-glycoprotein (pgp) is a membrane transporter encoded by the multidrug resistance (MDR1, ABCB1) gene. Pgp is a poor prognostic factor in elderly patients with acute myeloid leukemia (AML). In addition to its role in drug efflux, pgp has been implicated in cellular cholesterol homeostasis. We investigated the effects of exogenous cholesterol removal on pgp expression and function. METHODS: KG1a drug-naïve, primitive leukemia cells were cultured in serum-free medium with or without the addition of low-density lipoprotein (LDL) cholesterol. After 72 hours, pgp expression and function was assessed by flow cytometry and total cholesterol content of the KG1a cells was determined by the Amplex Red cholesterol assay. The addition of clinically available cholesterol-lowering agents, 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors to KG1a cells was also assessed. RESULTS: There was a 39% (SEM = 8.3%; p = 0.03) decrease in pgp protein expression after 3 days of serum-free culture. The decrease was also observed at the message and functional levels. In the presence of low-density lipoprotein cholesterol, pgp expression was restored to 86% of the basal value. Addition of a HMG-CoA reductase inhibitor to KG1a cells resulted in an additional 26% (lovastatin, p = 0.03) and 16% (pravastatin, p = 0.05) reduction in pgp, respectively. Furthermore, toxicity of the pgp substrate drug daunorubicin was enhanced following lovastatin preculture (p = 0.04). CONCLUSION: LDL cholesterol contributes to pgp expression and chemoresistance in primitive leukemia cells. Use of HMG-CoA reductase inhibitors may be of clinical value in lowering pgp expression in AML.


Subject(s)
ATP Binding Cassette Transporter, Subfamily B, Member 1/metabolism , Cholesterol, LDL/metabolism , Down-Regulation , Hydroxymethylglutaryl-CoA Reductase Inhibitors/pharmacology , Leukemia, Experimental/metabolism , Animals , Base Sequence , Cell Line, Tumor , DNA Primers , Immunophenotyping , Leukemia, Experimental/pathology
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