Enhancement of radiosensitivity of wild-type p53 human glioma cells by adenovirus-mediated delivery of the p53 gene.
OBJECTIVE
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
Lang
F F
FF
Department of Neurosurgery, The University of Texas, M.D. Anderson Cancer Center, Houston 77030, USA.
Yung
W K
WK
Raju
U
U
Libunao
F
F
Terry
N H
NH
Tofilon
P J
PJ
D000256:Adenoviridae / Q000235:genetics*; D000483:Alleles; D017209:Apoptosis / Q000235:genetics / Q000528:radiation effects; D015153:Blotting, Western; D001932:Brain Neoplasms / Q000235:genetics* / Q000532:radiotherapy; D002455:Cell Division / Q000235:genetics; D002470:Cell Survival; D004396:Coloring Agents; D003470:Culture Media; D004307:Dose-Response Relationship, Radiation; D005434:Flow Cytometry; D015972:Gene Expression Regulation, Neoplastic / Q000528:radiation effects; D018014:Gene Transfer Techniques; D016158:Genes, p53 / Q000528:radiation effects*; D005822:Genetic Vectors; D005910:Glioma / Q000235:genetics* / Q000532:radiotherapy; D006801:Humans; D009154:Mutation / Q000235:genetics; D014411:Neoplastic Stem Cells / Q000528:radiation effects; D016283:Proto-Oncogene Proteins p21(ras) / Q000032:analysis / Q000235:genetics / Q000528:radiation effects; D012333:RNA, Messenger / Q000032:analysis / Q000235:genetics / Q000528:radiation effects; D011836:Radiation Tolerance; D011879:Radiotherapy Dosage; D014158:Transcription, Genetic / Q000235:genetics / Q000528:radiation effects; D014162:Transfection / Q000235:genetics; D014407:Tumor Cells, Cultured; D016159:Tumor Suppressor Protein p53 / Q000032:analysis / Q000235:genetics / Q000528:radiation effects
D004396:Coloring Agents; D003470:Culture Media; D012333:RNA, Messenger; D016159:Tumor Suppressor Protein p53; C501009:HRAS protein, human; D016283:Proto-Oncogene Proteins p21(ras)