Resistance to TRAIL-induced apoptosis caused by constitutional phosphorylation of Akt and PTEN in acute lymphoblastic leukemia cells.
OBJECTIVE
Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a member of the tumor necrosis factor superfamily, which induces apoptosis in cancer cells but not in normal cells. Akt/protein kinase B, when phosphorylated to its active form, promotes cell survival and blocks apoptosis. The aim of this study was to investigate the role of Akt pathway in acquired TRAIL resistance of acute lymphoblastic leukemia cells.


METHODS
MB-IT and NALM-24 cells that developed resistance to TRAIL, i.e., TRAIL-resistant cells (MB-IT R and NALM-24 R) were established from TRAIL-sensitive acute lymphoblastic leukemia cell lines (MB-IT S and NALM-24 S), respectively, through application of TRAIL and repetitive limiting dilution. Apoptosis was measured by flow cytometry using propidium iodide/Annexin-V fluorescein isothiocyanate staining. TRAIL receptor cell surface expression of MB-IT and NALM-24 were analyzed by flow cytometry. Protein levels were analyzed by Western blot analysis.


RESULTS
The obtained resistant cell lines presented the same pattern of receptor expression as sensitive parent cells, and the internalization of DR5 after TRAIL treatment was similar. Caspase-8/3, FLIP, BID, XIAP were cleaved/downregulated in sensitive cells after treatment with TRAIL, but not in the resistant cells. We also observed that phosphoinositide-3-kinase (PI3K)/Akt pathway was constitutively active in resistant clones, and was not downregulated upon TRAIL treatment. Phosphate and tensin homologue deleted on chromosome 10 (PTEN) level was the same in both sensitive cells and resistant cells, but was quickly downregulated in sensitive cells after TRAIL treatment. Also, resistant cells expressed a high level of phosphorylated inactive form of PTEN than the sensitive cells. Expression levels of PH domain leucine-rich repeat protein phosphatase were slightly higher in sensitive than resistant cells. When resistant cells were treated with LY 294002 (a PI3K inhibitor), the expression level of phosphorylated Akt was distinctly downregulated, and there was induction of apoptosis when these cells were treated with a combination of TRAIL and LY 294002. When MB-IT-sensitive cells were treated with okadaic acid, a phosphatase inhibitor, TRAIL-induced apoptosis was significantly reduced.


CONCLUSIONS
These results suggest that cellular resistance to TRAIL could be developed through phosphorylation (activation) of Akt and phosphorylation (inactivation) of PTEN.
Dida
Francis
F
Department of Pediatrics and Developmental Science, Mie University Graduate School of Medicine, Tsu, Mie, Japan.
Li
Yufeng
Y
Iwao
Atsushi
A
Deguchi
Takao
T
Azuma
Eiichi
E
Komada
Yoshihiro
Y
D015703:Antigens, CD / Q000235:genetics; D017209:Apoptosis / Q000502:physiology*; D045744:Cell Line, Tumor; D019008:Drug Resistance, Neoplasm; D005260:Female; D006801:Humans; D016130:Immunophenotyping; D007621:Karyotyping; D018613:Microscopy, Confocal; D051059:PTEN Phosphohydrolase / Q000235:genetics / Q000378:metabolism*; D010766:Phosphorylation; D054198:Precursor Cell Lymphoblastic Leukemia-Lymphoma / Q000235:genetics / Q000378:metabolism*; D051057:Proto-Oncogene Proteins c-akt / Q000235:genetics / Q000378:metabolism*; D053221:TNF-Related Apoptosis-Inducing Ligand / Q000235:genetics / Q000502:physiology*
D015703:Antigens, CD; D053221:TNF-Related Apoptosis-Inducing Ligand; C505653:TNFSF10 protein, human; D051057:Proto-Oncogene Proteins c-akt; D051059:PTEN Phosphohydrolase; C494929:PTEN protein, human