RESUMO
Retinoic acid (RA) induces differentiation of S91 melanoma cells through activation of RA receptor (RAR)gamma without affecting cell viability. The novel RARgamma-agonist CD437 (AHPN), however, also induces concomitant apoptosis through an unknown mechanism which was investigated here. By utilizing DNA microarray analysis, five apoptosis-associated, CD437-induced transcripts (CITs) were identified. Interestingly, all CITs are also regulated by p53 in a DNA damage response, and consistent with this interpretation, CD437 was found to cause DNA adduct-formation. However, p53 is not required for CD437-dependent regulation of CITs. Among this set of genes, induction of p21(WAF1/CIP1) is likely to be responsible for early S-phase growth-arrest of CD437-treated cells, whereas ei24 is a critical mediator of CD437-induced apoptosis in S91 cells. These data suggest an RAR-independent mechanism in which CD437 causes DNA adduct-formation, resulting in induction of a p53-independent DNA damage response, and subsequent growth-arrest and apoptosis. CD437-mediated DNA adduct-formation may also explain its apoptotic effects in other cell types.
Assuntos
Apoptose/efeitos dos fármacos , Melanoma Experimental/patologia , Receptores do Ácido Retinoico/metabolismo , Retinoides/farmacologia , Células 3T3 , Animais , Proteínas Reguladoras de Apoptose , Divisão Celular/efeitos dos fármacos , Inibidor de Quinase Dependente de Ciclina p21 , Ciclinas/genética , Ciclinas/metabolismo , Adutos de DNA/metabolismo , Dano ao DNA/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Melanoma Experimental/metabolismo , Camundongos , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Análise de Sequência com Séries de Oligonucleotídeos , Oligonucleotídeos Antissenso/genética , Especificidade de Órgãos , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Receptores do Ácido Retinoico/genética , Fase S/efeitos dos fármacos , Transfecção , Células Tumorais Cultivadas , Proteína Supressora de Tumor p53/fisiologiaRESUMO
BACKGROUND: Optimal T-cell activation requires not only ligation of the T-cell receptor (TcR) but also delivery of costimulatory signals by various accessory molecules. The interaction of the costimulatory molecule B7.1 (CD80) with its receptor CD28 provides a strong positive signal to T cells. METHODS: The B7.1 gene was transduced into cultured human ovarian, breast, and pancreatic tumor cells by using a retroviral vector. Autologous as well as allogeneic naive T-cells were stimulated with either wild-type or B7.1-transduced tumor cells in a mixed lymphocyte tumor cell culture (MLTC). In addition to cytolytic activity, T-cell proliferation, T-cell subset composition, and the frequencies of TcR variable (V) alpha and beta genes were compared in T cells from both types of MLTC. RESULTS: Introduction of the B7.1 gene into tumor cells was successful in all tumors to a varying degree. Those tumors expressing high levels of B7.1 induced significantly higher levels of T-cell proliferation than wild-type tumor cells. T-cell subset composition did not markedly differ between T cells stimulated with wild-type tumor cells or B7.1-expressing tumor cells. However, T cells stimulated with B7.1-expressing tumor cells showed a significantly increased cytolytic potential. The increased cytotoxic T lymphocyte activity was associated with a higher frequency of specific TcR V alpha and V beta genes. In addition, B7.1 costimulation promoted oligoclonality among the responding T cells. CONCLUSIONS: These data suggest that costimulation through B7.1 promotes T-cell proliferation and cytotoxic activity through clonal expansions of T cells bearing antigen-specific TcR V alpha and V beta genes and through promotion of oligoclonality. The data also suggest that promoting B7.1-mediated costimulation is an important aspect of immune therapies.