Reverse-phase protein microarrays (RPPA) as a diagnosticand therapeutic guide in multidrug resistant leukemia

Abstract. Reverse-phase microarray assays using phosphospecificantibodies (RPPA) can directly measure levels ofphosphorylated protein isoforms. In the current study, lysatesfrom parental and multidrug resistant (MDR) CEM leukemiacells were spotted onto reverse-phase protein microarraysand probed with a panel of phospho-antibodies to ERK, PCKand Akt pathways. In particular, the Akt pathway is consideredto play significant roles in leukemia and Akt inhibitor therapyhas been proposed as a potential tool in the treatment of thisdisease. The RPPA data prompted us to investigate deeperthis pathway. Here, we found that whereas total Akt1 proteinlevel is higher in parental CEM cells, the activated isoformcontent, p-Akt1, increases in doxorubicin-selected CEM cells(MDR-CEM). This was backed up by Western blot analysis,confirming that Akt1 activity/phosphorylation may be upregulatedin MDR-CEM cells. Further exploration of inhibitorytherapy in this system was evaluated. The TNF-relatedapoptosis-inducing ligand, TRAIL, has been shown toselectively kill tumor cells. Herein, we describe that in MDRCEMcells TRAIL responsiveness correlates with a reducedexpression of endogenous Akt1, suggesting that the MDRphenotype associated to P-gp sensitizes cells to TRAIL therapy.