RESUMO
We investigated the response of SUDHL-1 and L428 cells, derived from t(2;5)-anaplastic large cell lymphoma (ALCL) and Hodgkin's disease (HD), respectively, to recombinant adenoviruses expressing cyclin-dependent kinase inhibitors (CDKIs) p27Kip1 (Adp27), p21Waf1 (Adp21) and p16INK4A (Adp16). Cell cycle analysis of SUDHL-1 cells after 24 h of infection with 200 multiplicity of infection (MOI) of Adp27, Adp21, and Adp16, showed very high levels of cell debris in the subG1 area. The magnitude of cell debris-events was Adp27/Adp21 > Adp16. Cell cycle analysis of L428 cells revealed absence of cell debris and increased G2 phase in all the groups of cells tested as compared to the controls (mock and AdNull). A minimal increase in G1 phase was also evident in cells infected with Adp27 (52%) compared to uninfected cells (43%), AdNull (45%) and to cells infected with Adp21 (37%) and Adp16 (31%). The presence of significant levels of Coxsackie-adenovirus receptor (CAR) on the cell surface of L428 cells excluded the cell membrane-barrier as responsible for the differences in cell observed in response to the recombinant adenovirus-mediated CDKIs expression as compared to SUDHL-1. We also showed that the recombinant adenovirus-mediated cytotoxicity measured as apoptosis was MOI- and vector-dependent in SUDHL-1 cells at lower MOI (100). In conclusion, the therapeutic effect induced by recombinant adenoviruses expressing p27Kip1, p21Waf1 and p16INK4A is cell-dependent in cells derived from selected lymphoid malignancies. Biochemical cellular differences more than cell surface barriers seem to be responsible for differences in response to recombinant adenovirus-mediated expression of cytotoxic genes. Moreover, the cytotoxicity of recombinant adenoviruses expressing p27Kip1, p21Waf1 and p16INK4A may be further explored as a tool for gene therapy of t(2;5)-derived ALCL.
Assuntos
Adenovírus Humanos/genética , Proteínas de Ciclo Celular/biossíntese , Inibidor p16 de Quinase Dependente de Ciclina/biossíntese , Ciclinas/biossíntese , Proteínas do Olho , Regulação Viral da Expressão Gênica , Vetores Genéticos/genética , Doença de Hodgkin/patologia , Lipoproteínas , Linfoma Difuso de Grandes Células B/patologia , Proteínas de Neoplasias/biossíntese , Proteínas do Tecido Nervoso , Proteínas Supressoras de Tumor/biossíntese , Apoptose , Proteínas de Ligação ao Cálcio/biossíntese , Proteínas de Ligação ao Cálcio/genética , Ciclo Celular , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/fisiologia , Inibidor p16 de Quinase Dependente de Ciclina/fisiologia , Inibidor de Quinase Dependente de Ciclina p21 , Inibidor de Quinase Dependente de Ciclina p27 , Ciclinas/genética , Ciclinas/fisiologia , Genes p16 , Terapia Genética , Hipocalcina , Doença de Hodgkin/metabolismo , Doença de Hodgkin/virologia , Humanos , Integrina alfaVbeta3/biossíntese , Integrina alfaVbeta3/genética , Linfoma Difuso de Grandes Células B/metabolismo , Linfoma Difuso de Grandes Células B/virologia , Proteínas de Neoplasias/genética , Fosforilação , Processamento de Proteína Pós-Traducional , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/fisiologia , Recoverina , Proteína do Retinoblastoma/metabolismo , Células Tumorais Cultivadas/metabolismo , Células Tumorais Cultivadas/virologia , Proteínas Supressoras de Tumor/genética , Proteínas Supressoras de Tumor/fisiologiaRESUMO
Anaplastic large cell lymphoma (ALCL) exhibiting the t(2;5) translocation is characterized by the resulting expression of the oncogenic fusion protein nucleophosmin-anaplastic lymphoma kinase (NPM-ALK) gene product. The ALK domain of NPM-ALK contains kinase activity, which is responsible for the autophosphorylation of tyrosine residues of the oncogenic protein and phosphorylation of SH2-protein substrates. Herbimycin A is a general protein tyrosine kinase inhibitor active as an antiproliferative compound against different types of mammalian cells. Herbimycin A inhibited the NPM-ALK-associated autophosphorylating activity in an in vitro cell-free kinase assay. The inhibition was specific when tested against other kinase inhibitors and extended to other cell lines derived from t(2;5)-ALCL. SUDHL-1 cells showed increasing percentage of cells in G(1) after 18 h of incubation with a dose of herbimycin A. NPM-ALK, Akt, and pAkt were down-regulated after 24 h of incubation with herbimycin A. Apoptosis was observed only if the dose of inhibitor was given every 12 h for prolonged time. Our results show that herbimycin A interferes with NPM-ALK and Akt pathways in SUDHL-1 cells. It seems that prolonged inhibition of these biochemical pathways may lead to cell cycle arrest and apoptosis. This study supports the idea of investigating protein kinase inhibitors as therapeutic compounds for t(2;5)-ALCL.
