RESUMEN
Replication-deficient E1-/E3-deleted adenoviral vectors are commonly used to introduce transgenes into cells in vitro and have been used for certain kinds of gene therapy protocols in vivo. We have demonstrated that transduction of cells using these vectors can induce p53 expression in cells containing a wild-type p53 gene. This response is different from p53 induction observed after DNA damage because the time course of induction is slower and because it occurs in cells with an attenuated DNA damage response. However, this vector-induced p53 is transcriptionally active and, therefore, p53 function is not inactivated by viral proteins. The mechanism of induction appears to be an increased rate of protein translation because immunoprecipitation analyses demonstrated increased levels of 35S-labeled p53 protein, even after a short 15-min labeling time. Induction of p53 by adenoviral vectors may have various effects on transduced cells, including apoptosis and altered chemotherapy chemosensitivity. Therefore, the influence of the vector might confound the impact of any particular gene used in a gene therapy application.
Asunto(s)
Adenoviridae/genética , Terapia Genética , Proteínas Nucleares , Proteína p53 Supresora de Tumor/biosíntesis , Replicación Viral , Daño del ADN , Vectores Genéticos , Humanos , Fosfatidilinositol 3-Quinasas/fisiología , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Proto-Oncogénicas c-mdm2 , Células Tumorales CultivadasRESUMEN
Susceptibility of a tumor cell to undergo chemotherapy-induced apoptosis appears to be dependent upon the balance of proapoptotic and survival factors that are expressed within any given cell. We have chosen to evaluate how expression of several of these proteins influences chemosensitivity of a panel of 10 pediatric tumor cell lines chosen from three tumor histiotypes: neuroblastoma, rhabdomyosarcoma, and pediatric glial tumors. The proteins evaluated were p53 and six members of the Bax/Bcl-2 family: three proapoptotic proteins (Bax, Bak, and Bcl-xS) and three survival factors (Bcl-2, Bcl-xL, and Mcl-1). We investigated whether there was any relationship between endogenous expression of these proteins and chemosensitivity (or resistance) to three chemotherapeutic agents that directly damage DNA (doxorubicin, actinomycin D, and topotecan) and a mitotic spindle poison (vincristine). Even though exogenous overexpression of wild-type p53 has been associated with a chemosensitive phenotype in several model systems we demonstrated no such relationship in these studies. In addition, expression levels of Bcl-2, Bcl-xL, Bcl-xS, Bak, or Mcl-1 did not correlate with sensitivity or resistance to the four drugs. However, there was a statistically significant correlation between endogenous levels of Bax protein and sensitivity to both doxorubicin and actinomycin D. We conclude that even though many proteins such as p53 and Bcl-2 have been shown to influence drug response when exogenously overexpressed in model systems, in unmodified cell lines endogenous protein levels may not generate the same results. We have demonstrated that endogenous Bax expression was the only protein found to be associated with chemosensitivity across the three different tumor histiotypes and propose that analysis of Bax may be a more useful prognostic indicator for tumor response to therapy than either p53 or Bcl-2.
Asunto(s)
Glioma/tratamiento farmacológico , Neuroblastoma/tratamiento farmacológico , Proteínas Nucleares , Proteínas Proto-Oncogénicas c-bcl-2/análisis , Proteínas Proto-Oncogénicas/análisis , Rabdomiosarcoma/tratamiento farmacológico , Proteína p53 Supresora de Tumor/análisis , Animales , Apoptosis/efectos de los fármacos , Niño , Glioma/química , Humanos , Mutación , Neuroblastoma/química , Proteínas Proto-Oncogénicas c-mdm2 , Conejos , Rabdomiosarcoma/química , Células Tumorales Cultivadas , Proteína X Asociada a bcl-2RESUMEN
The p53 tumor suppressor gene is deleted or mutated in over 50% of human tumors. Mutations frequently extend the half-life of the p53 protein; and a high level of nuclear p53 expression, detected by immunohistochemistry, has been used to predict the p53 status of tumors. We compared the sensitivity and reactivity of five frequently used, commercially available monoclonal antibodies (1801, DO1, DO7, BP53.12 and 421) in immunoblot and immunofluorescence assays, and found that results differed among the antibodies. Comparison of immunoblot analysis of denatured nuclear and cytoplasmic p53 protein were consistent with antibodies DO1, DO7 and BP53.12, each of which generated a strong specific signal in both cell fractions. However, in situ analysis demonstrated that although all antibodies recognized nuclear p53, only BP53.12 and 421 recognized p53 protein in the cytoplasm. In addition, 1801 produced a signal in p53-negative tumor cell lines. Differences in situ among the antibodies were probably due to the accessibility of their respective epitopes and suggested that nuclear and cytoplasmic p53 either have different three-dimensional conformations or are bound to different proteins. A third p53 protein conformation was also suggested by the observation that only two of the five antibodies (BP53.12 and DO7) detected induced levels of p53 in situ following exposure to ionizing radiation. In summary, except for the fact that DO7 does not recognize cytoplasmic p53 in situ, we found it to be the most specific, versatile, and reliable antibody. We conclude that the p53 antibody of choice depends upon the specific goal of a study and the method used to detect this protein.