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Title: Defining disease risk groups through the quantification of genetic heterogeneity across single leukaemia cells
Author: Karimiani, Ehsan Ghayoor
Awarding Body: University of Manchester
Current Institution: University of Manchester
Date of Award: 2012
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Chronic myeloid leukaemia (CML) is typified by the BCR-ABL fusion gene. Primitive CML cells are less responsive to treatment and have high BCR-ABL mRNA and protein expression. BCR-ABL may also be required for cell adhesion, which may possess increased resistance. Previous studies have analysed bulk cell populations but the significance of BCR-ABL expression heterogeneity at the single cell level is unknown. In this study, the K562 CML cell line was used. Surface-adherent (K562/Adh) and non-adherent (K562/NonAdh) cell populations from standard suspensions were generated through 4 months of passages. Isolated K562/Adh and K562/NonAdh were used for the detection and quantification of BCR-ABL DNA, mRNA and protein levels across single and bulk cell populations, using fluorescent in situ hybridisation (FISH), RT-qPCR, flow cytometry and proximal ligation assay (PLA). Cell viability was measured for both Adh and NonAdh cells grown in the presence of a tyrosine kinase inhibitor (Imatinib) using the Cell Proliferation Kit (XTT).The passage of K562 cells demonstrated a small fraction (5% + 2.1%) of K562/Adh cells adhering to the plastic surface of the culture flask. Genomic BCR-ABL gene amplification was present in K562 cells, but displayed no significant difference between K562/Adh and K562/NonAdh cell types (P=0.8227). RT-qPCR showed up-regulation of BCR-ABL mRNA in K562/Adh cells, compared to K562/NonAdh in single and bulk cells (p<0.0001). In addition, a microfluidic platform was developed for measuring the BCR-ABL fusion gene. The proximal ligation Assay (PLA) and flow cytometry showed higher expression of phospho-BCR-ABL protein in K562/Adh cells than K562/NonAdh (63.42% and 23.1%). Imatinib significantly inhibited the growth of K562/NonAdh; whereas the treatment of K562/Adh had less effect (p<0.005), suggesting resistance of K562/Adh to Imatinib. These results provide some evidence, at both single and bulk cell levels, for the existence of an adherent subpopulation of K562 cells with higher BCR-ABL mRNA and phosphorylated protein which are resistant to Imatinib, suggesting the possibility of a similar subpopulation of cells in CML which may cause clinical resistance.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available