Abstract
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-γ, 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.
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Acknowledgements
We are grateful to Georges Cassar from the Service Commun de Cytométrie de l'IFR Purpan for his help with flow cytometry analysis, to Drs Karen Pulford and Bruno Falini for the gift of ALK1 and ALKc mAbs, respectively, and to Dr Michael Cleary for the SU-DHL1 cell line. We also thank Dr Bent Rubin for his advice in transfection, and Dr Olivier Cuvillier for his helpful suggestions in cytochrome c experiments. This work was supported by grant 9937 (M Allouche) from the Association pour la Recherche sur le Cancer, by the ARECA network (Pole protéomique et cancer), by the National Cancer Institute grant CA69129 (SW Morris) and CORE grant CA21765, the American-Lebanese Syrian Associated Charities (ALSAC: SW Morris), St Jude Children's Research Hospital (SW Morris), and by a grant from the Wilhelm-Sander Stiftung (J Duyster).
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Greenland, C., Touriol, C., Chevillard, G. et al. Expression of the oncogenic NPM-ALK chimeric protein in human lymphoid T-cells inhibits drug-induced, but not Fas-induced apoptosis. Oncogene 20, 7386–7397 (2001). https://doi.org/10.1038/sj.onc.1204870
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DOI: https://doi.org/10.1038/sj.onc.1204870
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