RESUMO
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.
Assuntos
Apoptose/efeitos dos fármacos , Glicoproteínas de Membrana/fisiologia , Proteínas Serina-Treonina Quinases , Proteínas Tirosina Quinases/fisiologia , Linfócitos T/metabolismo , Receptor fas/fisiologia , Trifosfato de Adenosina/metabolismo , Antineoplásicos/farmacologia , Apoptose/genética , Apoptose/fisiologia , Sítios de Ligação , Cromonas/farmacologia , Grupo dos Citocromos c/metabolismo , Doxorrubicina/farmacologia , Inibidores Enzimáticos/farmacologia , Etoposídeo/farmacologia , Proteína Ligante Fas , Humanos , Isoenzimas/metabolismo , Células Jurkat/efeitos dos fármacos , Células Jurkat/metabolismo , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/enzimologia , Morfolinas/farmacologia , Mutagênese Sítio-Dirigida , Proteínas de Neoplasias/biossíntese , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/fisiologia , Fosfatidilinositol 3-Quinases/metabolismo , Inibidores de Fosfoinositídeo-3 Quinase , Fosfolipase C gama , Fosforilação , Processamento de Proteína Pós-Traducional , Proteínas Tirosina Quinases/biossíntese , Proteínas Tirosina Quinases/química , Proteínas Tirosina Quinases/genética , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas c-akt , Proteínas Recombinantes de Fusão/fisiologia , Linfócitos T/efeitos dos fármacos , Transfecção , Fosfolipases Tipo C/metabolismoRESUMO
PPARα and HNF4α are nuclear receptors that control gene transcription by direct binding to specific nucleotide sequences. Using transgenic mice deficient for either PPARα or HNF4α, we show that the expression of the peroxisomal 3-keto-acyl-CoA thiolase B (Thb) is under the dependence of these two transcription factors. Transactivation and gel shift experiments identified a novel PPAR response element within intron 3 of the Thb gene, by which PPARα but not HNF4α transactivates. Intriguingly, we found that HNF4α enhanced PPARα/RXRα transactivation from TB PPRE3 in a DNA-binding independent manner. Coimmunoprecipitation assays supported the hypothesis that HNF4α was physically interacting with RXRα. RT-PCR performed with RNA from liver-specific HNF4α-null mice confirmed the involvement of HNF4α in the PPARα-regulated induction of Thb by Wy14,643. Overall, we conclude that HNF4α enhances the PPARα-mediated activation of Thb gene expression in part through interaction with the obligate PPARα partner, RXRα.