RESUMEN
Recent evidence suggests an intriguing link between p53 and the Fas pathway. To evaluate this association further, we utilized a recombinant adenoviral vector (AdWTp53) to overexpress wild-type p53 in lung cancer (A549, H23, EKVX and HOP92) and breast cancer (MDA-MB-231 and MCF-7) cell lines and observed an increase in the Fas/CD95/APO-1 protein levels. Furthermore, this increase correlated with the sensitivity of the cell lines to p53-mediated cytotoxicity. To examine the effects of Fas over-expression in cells resistant to p53 over-expression, we constructed AdFas, an adenoviral vector capable of transferring functional human Fas to cancer cells. Interestingly, infection of p53-resistant MCF-7 cells with AdFas sensitized them to p53-mediated apoptosis. These studies indicate that combined over-expression of Fas and wild-type p53 may be an effective cancer gene therapy approach, especially in cells relatively resistant to p53 over-expression.
Asunto(s)
Adenoviridae , Apoptosis/genética , Vectores Genéticos , Proteína p53 Supresora de Tumor/fisiología , Receptor fas/genética , Infecciones por Adenoviridae , Neoplasias de la Mama , Citotoxinas/genética , Cartilla de ADN , ADN de Neoplasias/análisis , Femenino , Expresión Génica , Terapia Genética/métodos , Humanos , Neoplasias Pulmonares , Células Tumorales Cultivadas/química , Células Tumorales Cultivadas/citología , Células Tumorales Cultivadas/virologíaRESUMEN
We constructed an adenoviral vector containing human p16 cDNA in order to evaluate the cytotoxic effects of exogenous p16 expression on cancer cell proliferation and to explore the potential use of p16 in cancer gene therapy. Following infection of human breast (MCF-7, MDA-MB-231, and BT549), osteosarcoma (U-2 OS and Saos-2), cervical (C33a), and lung cancer (H358) cell lines with the recombinant adenovirus Adp16, high levels of p16 expression were observed in all cell lines. Cancer cell lines which were mutant or null for p16 but wild-type for the retinoblastoma gene product (pRb) (MCF-7, MDA-MB-231, BT549 and U-2 OS) were 7-22-fold more sensitive to the cytotoxic effects of Adp16 than to a control virus. In contrast, cancer cell lines which were wild-type for p16 but mutant or null for pRb (Saos-2, C33a and H358) were Asunto(s)
Adenoviridae/genética
, Inhibidor p16 de la Quinasa Dependiente de Ciclina/genética
, Terapia Genética
, Neoplasias/terapia
, Proteínas Proto-Oncogénicas
, Proteína de Retinoblastoma/análisis
, Bromodesoxiuridina/metabolismo
, Ciclo Celular
, Quinasa 4 Dependiente de la Ciclina
, Inhibidor p16 de la Quinasa Dependiente de Ciclina/análisis
, Quinasas Ciclina-Dependientes/metabolismo
, Humanos
, Fosforilación
, Células Tumorales Cultivadas
, Proteína p53 Supresora de Tumor/análisis
RESUMEN
The cytotoxicity of a recombinant adenovirus expressing the wild type tumor suppressor gene p53 (AdWTp53) was studied in two human breast cancer MCF-7 sublines selected for resistance to adriamycin (MCF-Adr) and mitoxantrone (MCF-Mito). Although the levels of wild type p53 protein following infection with AdWTp53 are comparable in all cell lines, the two drug-resistant MCF-7 sublines were 300- and 18-fold more sensitive to killing by AdWTp53 compared with the drug-sensitive parental MCF-7 cell lines. In each cell line, AdWTp53 infection led to cell cycle arrest, and reduction of Cdk2 and cyclin B1-Cdc2 activity. Nucleosomal DNA fragmentation analysis (as a function of apoptosis) following AdWTp53 infection revealed that, while the parental MCF-7 cells failed to undergo apoptosis, both drug-resistant cell lines showed distinct DNA laddering. In MCF-Adr cells, a combination treatment of AdWTp53 and adriamycin was much more toxic than either of the reagents used individually. Finally, exposure of a mixed population of MCF-Adr and CD34+ cells to AdWTp53 selectively prevented MCF-Adr cell colony formation, while there was no inhibition of CFU-GM colony formation from CD34+ cells. These findings suggest that some drug-resistant human breast cancers may be effectively treated with adenovirus expressing wild type p53.
Asunto(s)
Apoptosis , Neoplasias de la Mama/patología , Genes p53 , Terapia Genética/métodos , Adenoviridae , Purgación de la Médula Ósea , Neoplasias de la Mama/terapia , Ciclo Celular/efectos de los fármacos , Ciclo Celular/fisiología , Supervivencia Celular/efectos de los fármacos , Ensayo de Unidades Formadoras de Colonias , Fragmentación del ADN , Doxorrubicina/toxicidad , Resistencia a Múltiples Medicamentos , Resistencia a Antineoplásicos , Femenino , Células Madre Hematopoyéticas/citología , Humanos , Mitoxantrona/toxicidad , Transfección , Proteína p53 Supresora de Tumor/biosíntesisRESUMEN
In order to elucidate the biochemical mechanisms by which the universal cyclin kinase inhibitor p27Kip1 regulates cell cycle progression in human breast cancer cells, a recombinant adenovirus expressing human p27 was constructed (Adp27). Upon infection of human breast cancer cells MDA-MB-231 and MCF-7 with Adp27, a high level of p27 expression was observed, and this resulted in a marked decrease in the proportion of cells in S-phase. In multiple cell lines, comparison of the cytotoxicity of Adp27 with another adenovirus vector expressing the related universal cyclin kinase inhibitor WAF1/Cip1 (AdWAF1), showed Adp27 to be markedly more (up to 56-fold) toxic than AdWAF1. DNA histograms showed Adp27 to cause a G1/S arrest at lower viral doses than AdWAF1. Analysis of cyclin dependent kinase activity following Adp27 infections showed decreased Cdk2 and cyclin B1-Cdc2 activity at lower viral doses when compared with AdWAF1. Adp27 is therefore potentially useful for studies of growth regulation and for gene therapy when growth inhibition is desired.