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Expression and function of drug transporters in primitive CML cells

Jordanides, Niove E. (2008) Expression and function of drug transporters in primitive CML cells. PhD thesis, University of Glasgow.

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Abstract

Chronic myeloid leukaemia (CML) is a stem cell (SC) disorder initiated by the reciprocal translocation between chromosome 9 and 22, giving rise to the Philadelphia (Ph) chromosome and the resulting expression of the oncogenic fusion protein BCR-ABL. The current first line of treatment is imatinib mesylate (IM), a tyrosine kinase inhibitor (TKI) that competes with ATP to block ABL kinase activity, which in turn prevents tyrosine phosphorylation of downstream molecules and selectively induces apoptosis of BCR-ABL cells. However, despite excellent cytogenetic responses, only a minority of patients achieve complete molecular response (CMR). We have previously identified a population of quiescent (q) Ph+ SC found in chronic phase (CP) CML that are relatively insensitive to IM and other TKIs and which may be responsible for the molecular persistence of this disease. This population may be insensitive because TKIs do not reach therapeutic concentrations within the cell. Such resistance to classical chemotherapeutic drugs, the phenomenon of multidrug resistance (MDR), is mediated by ABC transporters. In this study we have investigated whether CML SC express the clinically relevant ABC transporters and determine their interaction with TKIs. In addition, we determined whether the inhibition of these transporters increased the efficacy of TKI against CML SC. Using CML CD34+ cells isolated from newly diagnosed patients, normal CD34+ cells and cell lines transduced with specific transporters as controls, the relative expression of drug transporters were determined in CML CD34+ cells and intracellular staining confirmed protein expression. The interaction of drug transporters with TKIs was assessed using a combination of substrate displacement assays and radiolabelled assays. The effect of transporter inhibitors 3 with TKIs on the growth and differentiation of q34+ and more mature CD34+ cells from CML patients in CP were assessed with regard to cell division, apoptosis and BCR-ABL kinase activity. When compared to normal CD34+ cells, CML CD34+ cells over-expressed ABCG2 mRNA. In contrast MDR1 expression was reduced in CML CD34+ cells and MRP1 was detected at similar expression levels in both populations. All three drug transporters were expressed at the protein levels in CML CD34+ cells. It was determined that at therapeutic concentrations (5μM) IM and nilotinib both inhibited ABCG2 and MDR1 and nilotinib also inhibited MRP1. Neither drug was a substrate for any of the transporters. In contrast, dasatinib was shown to be a substrate for ABCG2 and MRP1, but had no effect on MDR1. Therefore activity and concentration of dasatinib but not IM or nilotinib may be altered by the activity of these proteins. In keeping with their inhibitory activity, neither IM nor nilotinib demonstrated significantly increased efficacy when combined with specific ABC transporter inhibitors (FTC or PSC 833). Surprisingly, although dasatinib was a substrate for ABCG2 and MRP1, dasatinib did not further increase apoptosis, or reduce the qSC population. Therefore, although MDR1, MRP1 and ABCG2 were found to be expressed and functional in CML CD34+ cells and to interact with TKI, the co-treatment of TKIs with drug transporter inhibitors did not further increase apoptosis, reduce BCRABL kinase activity or reduce the qSC population. Therefore, modulation of individual transporter activity is unlikely to reverse the resistance of this population of cells to TKI and will not improve the clinical response to these drugs.

Item Type: Thesis (PhD)
Qualification Level: Doctoral
Subjects: Q Science > QP Physiology
Colleges/Schools: College of Medical Veterinary and Life Sciences > Institute of Cancer Sciences
Supervisor's Name: Holyoake, Prof. T. and Mountford, Dr. J.
Date of Award: 2008
Depositing User: Miss Fiona Riggans
Unique ID: glathesis:2008-604
Copyright: Copyright of this thesis is held by the author.
Date Deposited: 24 Feb 2009
Last Modified: 10 Dec 2012 13:20
URI: http://theses.gla.ac.uk/id/eprint/604

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