Reverse-phase microarray assays using phospho-specific antibodies (RPPA) can directly measure levels of phosphorylated protein isoforms. In the current study, lysates from parental and multidrug resistant (MDR) CEM leukemia cells were spotted onto reverse-phase protein microarrays and probed with a panel of phospho-antibodies to ERK, PCK and Akt pathways. In particular, the Akt pathway is considered to play significant roles in leukemia and Akt inhibitor therapy has been proposed as a potential tool in the treatment of this disease. The RPPA data prompted us to investigate deeper this pathway. Here, we found that whereas total Akt1 protein level is higher in parental CEM cells, the activated isoform content, 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 upregulated in MDR-CEM cells. Further exploration of inhibitory therapy in this system was evaluated. The TNF-related apoptosis-inducing ligand, TRAIL, has been shown to selectively kill tumor cells. Herein, we describe that in MDRCEM cells TRAIL responsiveness correlates with a reduced expression of endogenous Akt1, suggesting that the MDR phenotype associated to P-gp sensitizes cells to TRAIL therapy.

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

COCCO, LUCIO ILDEBRANDO;
2011

Abstract

Reverse-phase microarray assays using phospho-specific antibodies (RPPA) can directly measure levels of phosphorylated protein isoforms. In the current study, lysates from parental and multidrug resistant (MDR) CEM leukemia cells were spotted onto reverse-phase protein microarrays and probed with a panel of phospho-antibodies to ERK, PCK and Akt pathways. In particular, the Akt pathway is considered to play significant roles in leukemia and Akt inhibitor therapy has been proposed as a potential tool in the treatment of this disease. The RPPA data prompted us to investigate deeper this pathway. Here, we found that whereas total Akt1 protein level is higher in parental CEM cells, the activated isoform content, 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 upregulated in MDR-CEM cells. Further exploration of inhibitory therapy in this system was evaluated. The TNF-related apoptosis-inducing ligand, TRAIL, has been shown to selectively kill tumor cells. Herein, we describe that in MDRCEM cells TRAIL responsiveness correlates with a reduced expression of endogenous Akt1, suggesting that the MDR phenotype associated to P-gp sensitizes cells to TRAIL therapy.
Maraldi T; Bertacchini J; Benincasa M; Guida M; De Pol A; Liotta LA; Petricoin E; Cocco L; Marmiroli S
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/11585/99545
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