Expression of the oncogenic NPM-ALK chimeric protein in human lymphoid T-cells inhibits drug-induced, but not Fas-induced apoptosis.
Anaplastic large cell lymphomas (ALCLs) are frequently associated with the t(2;5)(p23;q35) translocation, leading to the expression of NPM-ALK, a fusion protein linking nucleophosmin and anaplastic lymphoma kinase, a receptor tyrosine kinase. In ALCLs, dimerization of NPM-ALK leads to constitutive autophosphorylation and activation of the kinase, necessary for NPM-ALK oncogenicity. To investigate whether NPM-ALK, like other oncogenic tyrosine kinases, can inhibit drug-induced apoptosis, we permanently transfected NPM-ALK into Jurkat T-cells. As in ALCLs, NPM-ALK was expressed as a constitutively kinase-active 80 kDa protein, and could be detected by immunocytochemistry in nucleoli, nuclei and cytoplasm. Doxorubicin-induced apoptosis (assessed by cell morphology and annexin V-FITC binding) was significantly inhibited in two independent NPM-ALK-expressing clones (5.2+/-1.8 and 7.5+/-0.8% apoptosis), compared to control vector-transduced cells (36+/-6.7%). Similar results were observed with etoposide. In contrast, Fas-induced apoptosis was not inhibited. Cytochrome c release into the cytosol was delayed in doxorubicin-, but not anti-Fas-treated transfectant cells, indicating that apoptosis inhibition occurred upstream of mitochondrial events. Using NPM-ALK mutants, we demonstrated that inhibition of drug-induced apoptosis: (1) requires functional kinase activity, (2) does not involve phospholipase C-gamma, essential for NPM-ALK-mediated mitogenicity and (3) appears to be phosphoinositide 3-kinase independent, despite a strong Akt/PKB activation observed in wild type NPM-ALK-expressing cells. These results suggest that the NPM-ALK antiapoptotic and mitogenic pathways are distinct.
Greenland
C
C
CNRS-UPCM, UPR 2163, CHU Purpan, 31059 Toulouse Cedex 03, France.
Touriol
C
C
Chevillard
G
G
Morris
S W
SW
Bai
R
R
Duyster
J
J
Delsol
G
G
Allouche
M
M
D000255:Adenosine Triphosphate / Q000378:metabolism; D000970:Antineoplastic Agents / Q000494:pharmacology; D017209:Apoptosis / Q000187:drug effects* / Q000235:genetics / Q000502:physiology; D001665:Binding Sites; D002867:Chromones / Q000494:pharmacology; D003574:Cytochrome c Group / Q000378:metabolism; D004317:Doxorubicin / Q000494:pharmacology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005047:Etoposide / Q000494:pharmacology; D053222:Fas Ligand Protein; D006801:Humans; D007527:Isoenzymes / Q000378:metabolism; D019169:Jurkat Cells / Q000187:drug effects / Q000378:metabolism; D008562:Membrane Glycoproteins / Q000502:physiology*; D008928:Mitochondria / Q000187:drug effects / Q000201:enzymology; D009025:Morpholines / Q000494:pharmacology; D016297:Mutagenesis, Site-Directed; D009363:Neoplasm Proteins / Q000096:biosynthesis / Q000235:genetics / Q000502:physiology; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism; D000081082:Phosphoinositide-3 Kinase Inhibitors; D051966:Phospholipase C gamma; D010766:Phosphorylation; D011499:Protein Processing, Post-Translational; D017346:Protein Serine-Threonine Kinases; D011505:Protein-Tyrosine Kinases / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000502:physiology*; D011518:Proto-Oncogene Proteins / Q000378:metabolism; D051057:Proto-Oncogene Proteins c-akt; D011993:Recombinant Fusion Proteins / Q000502:physiology; D013601:T-Lymphocytes / Q000187:drug effects / Q000378:metabolism*; D014162:Transfection; D010738:Type C Phospholipases / Q000378:metabolism; D019014:fas Receptor / Q000502:physiology*
D000255:Adenosine Triphosphate; D000970:Antineoplastic Agents; D002867:Chromones; D003574:Cytochrome c Group; D004317:Doxorubicin; D004791:Enzyme Inhibitors; D005047:Etoposide; D053222:Fas Ligand Protein; D007527:Isoenzymes; D008562:Membrane Glycoproteins; D009025:Morpholines; D009363:Neoplasm Proteins; D019869:Phosphatidylinositol 3-Kinases; D000081082:Phosphoinositide-3 Kinase Inhibitors; D051966:Phospholipase C gamma; D017346:Protein Serine-Threonine Kinases; D011505:Protein-Tyrosine Kinases; D011518:Proto-Oncogene Proteins; D051057:Proto-Oncogene Proteins c-akt; D011993:Recombinant Fusion Proteins; D010738:Type C Phospholipases; D019014:fas Receptor; C505661:FASLG protein, human; C085911:2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one; C107023:p80(NPM-ALK) protein; C494918:AKT1 protein, human