RESUMEN
This chapter describes several methods for recognizing apoptosis in tumor cells following infection with a replication-deficient adenovirus expressing the tumor suppressor gene p53. We include cytotoxicity assays and assays of apoptosis, including DNA-nucleosomal DNA fragmentation (DNA laddering), TUNEL, DAPI staining, analysis of the sub-G1 (subdiploid) population, and degradation of poly(ADP-ribose) polymerase (as assayed by Western blot). Although this is not a comprehensive list of protocols to evaluate apoptosis, we believe that these will cover the majority of conditions of apoptosis that may arise. The chapter also describes the characteristics of each technique, including the advantages and disadvantages of each method.
Asunto(s)
Adenoviridae/genética , Apoptosis/fisiología , Genes p53 , Línea Celular Tumoral , Fragmentación del ADN , ADN Viral , Humanos , Etiquetado Corte-Fin in Situ , Poli(ADP-Ribosa) Polimerasas/metabolismo , Células Tumorales CultivadasRESUMEN
We have developed an approach to cancer gene therapy in which the oncolytic effects of an adenoviral vector have been combined with selective expression of a soluble form of transforming growth factor (TGF)-beta receptor II fused with Fc (sTGFbetaRIIFc). We chose to use adenoviral dl01/07 mutant because it can replicate in all cancer cells regardless of their genetic defects. An oncolytic adenovirus expressing sTGFbetaRIIFc (Ad.sT- betaRFc) was constructed by homologous recombination. Infection of MDA-MB-231 and MCF-7 human breast cancer cells with Ad.sTbetaRFc produced sTGFbetaRIIFc, which was released into the media. The conditioned media containing sTGFbetaRIIFc could bind with TGF-beta 1 and inhibited TGF-beta-dependent transcription in target cells. Infection of MDA-MB-231, MCF-7, and 76NE human breast cancer cells with Ad.sTbetaRFc resulted in high levels of viral replication, comparable to that of a wild-type dl309 virus. Although some viral replication was observed in actively dividing normal human lung fibroblasts, there was no replication in nonproliferating normal cells. Direct injection of Ad.sTbetaRFc into MDA-MB-231 human breast xenograft tumors grown in nude mice resulted in a significant inhibition of tumor growth, causing tumor regression in more than 85% of the animals. These results indicate that it is possible to construct an oncolytic virus expressing sTGFbetaRIIFc in which both viral replication and transgene expression remain intact, and the recombinant adenovirus is oncolytic in a human tumor xenograft model. On the basis of these results we believe that it may be feasible to develop a cancer gene therapy approach using Ad.sTbetaRFc as an antitumor agent.