Effects of Purvalanol A on Imatinib-Sensitive and -Insensitive Chronic Myeloid Leukaemia Cell Lines

Phoomvuthisarn, Panrawee
Effects of Purvalanol A on Imatinib-Sensitive and -Insensitive Chronic Myeloid Leukaemia Cell Lines. PhD thesis, University of Liverpool.

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Objectives: This project aimed to investigate effects of purvalanol A on imatinib-sensitive and -insensitive chronic myeloid leukaemia (CML) cell lines, and normal white blood cells. Methods: Imatinib-sensitive (LAMA-84) and –insensitive (KCL-22) CML cell lines and human neutrophils and PBMCs were incubated with imatinib, and purvalanol A. Cell apoptosis was measured using flow cytometry and the expression of proteins was measured by Western blotting. Mcl-1 mRNA expression was quantified using real-time PCR. Results: The CDK2 inhibitor, purvalanol A, induced cell death in both CML cell lines. In the KCL-22 cell line, purvalanol A treatment resulted in a rapid (≤1 h) decrease in levels of the anti-apoptotic protein, Mcl-1. This decrease in Mcl-1 was mediated by a decrease in mRNA and a marked acceleration in the rate of turnover of the protein (half-life decreased from ~3 h in control cells to ~1.5 h in drug-treated cells). Purvalanol A also induced cell death in LAMA-84 cell line, although it was not as potent and cell death occurred in the absence of a decrease in Mcl-1, suggesting different signalling pathways in these two lines and hence different capacity to alter Mcl-1 stability. Furthermore, purvalanol A also increased Mcl-1 turnover rate and phosphorylated p38-MAPK activity in neutrophils and in KCL-22 cells. In addition, the p38 MAPK inhibitor, BIRB796, decreased apoptosis and prevented the decrease in Mcl-1 expression induced by purvalanol A in both CML cell lines and neutrophils. Conclusions: The development of drug resistance in patients with CML results in a continuing need to develop new ways to treat patients that become refractory to tyrosine kinase inhibitors. The results of this study indicate that purvalanol A might provide an adjunct to TKIs in the therapy of CML. This study shows that the effects of purvalanol A on induction of cell death in KCL-22 cell line are independent of the effects of this drug on cell cycle kinetics, but instead point to a direct or indirect effect on post-translational modifications of Mcl-1 that enhance its turnover rate.

Item Type: Thesis (PhD)
Additional Information: Date: 2015-09 (completed)
Subjects: ?? Q1 ??
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Depositing User: Symplectic Admin
Date Deposited: 15 Jan 2016 14:16
Last Modified: 17 Dec 2022 01:39
DOI: 10.17638/02036859
URI: https://livrepository.liverpool.ac.uk/id/eprint/2036859