Object: The authors sought to determine whether combining p53 gene transfer with radiation therapy would enhance the therapeutic killing of p53 wild-type glioma cells. It has been shown in several reports that adenovirus-mediated delivery of the p53 gene into p53 mutant gliomas results in dramatic apoptosis, but has little effect on gliomas containing wild-type p53 alleles. Therefore, p53 gene therapy alone may not be a clinically effective treatment for gliomas because most gliomas are composed of both p53 mutant and wild-type cell populations. One potential approach to overcome this problem is to exploit the role p53 plays as an important determinant in the cellular response to ionizing radiation.
Methods: In vitro experiments were performed using the glioma cell line U87MG, which contains wild-type p53. Comparisons were made to the glioma cell line U251MG, which contains a mutant p53 allele. Monolayer cultures were infected with an adenovirus containing wild-type p53 (Ad5CMV-p53), a control vector (dl312), or Dulbecco's modified Eagle's medium (DMEM). Two days later, cultures were irradiated and colony-forming efficiency was determined. Transfection with p53 had only a minor effect on the plating efficiency of nonirradiated U87MG cells, reducing the plating efficiency from 0.23 +/- 0.01 in DMEM to 0.22 +/- 0.04 after addition of Ad5CMV-p53. However, p53 transfection significantly enhanced the radiosensitivity of these cells. The dose enhancement factor at a surviving fraction of 0.10 was 1.5, and the surviving fraction at 2 Gy was reduced from 0.61 in untransfected controls to 0.38 in p53-transfected cells. Transfection of the viral vector control (dl312) had no effect on U87MG radiosensitivity. In comparison, transfection of Ad5CMV-p53 into the p53 mutant cell line U251 MG resulted in a significant decrease in the surviving fraction of these cells compared with controls, and no radiosensitization was detected. To determine whether Ad5CMV-p53-mediated radiosensitization of U87MG cells involved an increase in the propensity of these cells to undergo apoptosis, flow cytometric analysis of terminal deoxynucleotidyl transferase-mediated biotinylated-deoxyuridinetriphosphate nick-end labeling-stained cells was performed. Whereas the amount of radiation-induced apoptosis in uninfected and dl312-infected control cells was relatively small (2.1 +/- 0.05% and 3.7 +/- 0.5%, respectively), the combination of Ad5CMV-p53 infection and radiation treatment significantly increased the apoptotic frequency (18.6 +/- 1.4%). To determine whether infection with Ad5CMV-p53 resulted in increased expression of functional exogenous p53 protein, Western blot analysis of p53 was performed on U87MG cells that were exposed to 9 Gy of radiation 2 days after exposure to Ad5CMV-p53, dl312, or DMEM. Infection with Ad5CMV-p53 alone increased p53 levels compared with DMEM- or dl312-treated cells. Irradiation of AdSCMV-p53-infected cells resulted in a further increase in p53 that reached a maximum at 2 hours postirradiation. To determine whether exogenous p53 provided by Ad5CMV-p53 had transactivating activity, U87MG cells were treated as described earlier and p21 messenger RNA levels were determined. Infection of U87MG cells with Ad5CMV-p53 only resulted in an increase in p21 compared with DMEM- and dl312-treated cells. Irradiation of AdSCMV-p53-infected cells resulted in an additional time-dependent increase in p21 expression.
Conclusions: These data indicate that adenovirus-mediated delivery of p53 may enhance the radioresponse of brain tumor cells containing wild-type p53 and that this radiosensitization may involve converting from a clonogenic to the more sensitive apoptotic form of cell death. Although the mechanism underlying this enhanced apoptotic susceptibility is unknown, the AdSCMV-p53-infected cells have a higher level of p53 protein, which increases further after irradiation, and this exogenous p53 is transcriptionally active. (ABSTRACT TRUNCATE