Combined BCR-ABL inhibition with lentiviral-delivered shRNA and dasatinib augments induction of apoptosis in Philadelphia-positive cells

Myssina, S. et al. (2009) Combined BCR-ABL inhibition with lentiviral-delivered shRNA and dasatinib augments induction of apoptosis in Philadelphia-positive cells. Experimental Hematology, 37(2), pp. 206-214. (doi: 10.1016/j.exphem.2008.10.013)

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<b>Objective</b><br /> This study investigated two approaches, short hairpin RNA (shRNA) and the potent ABL inhibitor, dasatinib, alone and together, to achieve complete inhibition of BCR-ABL activity in Philadelphia-positive (Ph+) cells.<p></p> <b>Materials and Methods</b><br /> shRNA specific for BCR-ABL b3a2 were delivered, by lentiviral transduction or electroporation, to K562 cells, with or without dasatinib. mRNA and protein knockdown were measured by quantitative reverse transcriptase polymerase chain reaction, flow cytometry, and Western blotting. BCR-ABL activity was assessed by intracellular flow cytometry for pCrkL. Cell death and apoptosis were assayed using trypan blue exclusion, Annexin-V, and active caspase-3 staining.<p></p> <b>Results</b><br /> Forty-eight hours after transduction or electroporation of shRNA, BCR-ABL mRNA, and protein were reduced by 75% and >90%, respectively, and sustained for 5 days. Lentiviral delivery and electroporation were equally effective. pCrkL was inhibited in association with cell death. By 5 days after transduction or electroporation, viable cells represented 50% of input, with a 12-fold reduction vs control, which expanded 6-fold. When shRNA, titrated by green fluorescent protein into low and high, was combined with dasatinib (concentration range, 0−10 nM), low shRNA was additive with low dasatinib (0.6 and 1 nM), leading to inhibition of pCrkL, induction of activated caspase-3, expression of Annexin-V, and marked reduction in viable cells.<p></p> <b>Conclusion</b><br /> These results confirm that by lowering BCR-ABL levels with shRNA, complete inhibition of oncoprotein activity can be achieved with a lower concentration of dasatinib, thus providing a rationale for combining these approaches in the setting of high target expression, such as found in advanced phase disease and in the stem cell compartment.<p></p>

Item Type:Articles
Glasgow Author(s) Enlighten ID:Hamilton, Ms Ashley and Holyoake, Professor Tessa and Helgason, Professor Vignir and Mountford, Dr Joanne and Baird, Dr John and Jorgensen, Dr Heather and Serrels, Mr Alan
Authors: Myssina, S., Helgason, G. V., Serrels, A., Jorgensen, H. G., Bhatia, R., Modi, H., Baird, J. W., Mountford, J. C., Hamilton, A., Schemionek, M., Koschmieder, S., Brunton, V. G., and Holyoake, T. L.
College/School:College of Medical Veterinary and Life Sciences > School of Cancer Sciences
College of Medical Veterinary and Life Sciences > School of Psychology & Neuroscience
Journal Name:Experimental Hematology
ISSN (Online):1873-2399
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Project CodeAward NoProject NamePrincipal InvestigatorFunder's NameFunder RefLead Dept
439231Is Bcr-Able expression relevant for the survival of cancer stem cells in chronic myeloid leukaemiaTessa HolyoakeMedical Research Council (MRC)G0600782Institute of Cancer Sciences