Adenoviral-mediated p53 transgene expression sensitizes both wild-type and null p53 prostate cancer cells in vitro to radiation.
OBJECTIVE
The effect of adenoviral-mediated p53 transgene expression on the radiation response of two human prostate cancer cell lines, the p53(wild-type) LNCaP and p53(null) PC3 lines, was examined. The objective was to determine if this vector sensitizes cells to radiation independently of their p53 status.


METHODS
A recombinant adenovirus-5 vector (RPR/INGN 201, Introgen Therapeutics, Houston, TX) containing a CMV promoter and wild-type p53-cDNA (Ad5-p53) was used to facilitate p53 transgene expression. A multiplicity of infection (MOI) of 10-40 viral particles per cell was used, based on Ad5/CMV/lacz infection and staining for the beta-galactosidase reporter gene product. Clonogenic assays were performed to evaluate the degree of sensitization to radiation of viral-transduced cells compared with irradiated nontransduced controls. The relative efficacy of these treatments to induce apoptotic cell death was determined using the TUNEL assay.


RESULTS
The delivery of Ad5-p53 (10 MOI) reduced control plating efficiency from 36.5% to 0.86% in the LNCaP cell line and from 75.1% to 4.1% in the PC3 cell line. After correcting for the effect of Ad5-p53 on plating efficiency, the surviving fraction after 2 Gy (SF2) of gamma-irradiation was reduced over 2.5-fold, from 0.187 to 0.072, with transgene p53 expression in the LNCaP cell line. Surviving fraction after 4 Gy (SF4) was reduced over 4.5-fold, from 0.014 to 0.003, after Ad5-p53 treatment. In the PC3 cell line, Ad5-p53 (40 MOI) reduced SF2 over 1.9-fold from 0.708 to 0.367, and SF4 over 6-fold from 0.335 to 0.056. In both the LNCaP and PC3 cell lines, the combination of Ad5-p53 plus radiation (2 Gy) resulted in supra-additive apoptosis (approximately 20% for LNCaP and approximately 15% for PC3 at 50 MOI), above that seen from the addition of the controls; control vector Ad5-pA plus RT (0.15% for LNCaP and 1.44% for PC3), Ad5-p53 alone (28.6% for LNCaP and 21.7% for PC3), RT alone (0% for LNCaP and 0.23% for PC3), or Ad5-pA alone (0.1% for LNCaP and 0.29% for PC3).


CONCLUSIONS
The clonogenic survival and apoptosis data demonstrate that p53 transgene expression sensitizes human prostate adenocarcinoma cells in vitro to irradiation. As this effect was observed in both the p53(wild-type) LNCaP and p53(null) PC3 lines, radiosensitization was independent of p53 status.
Colletier
P J
PJ
Department of Radiation Oncology, The University of Texas M. D. Anderson Cancer Center, Houston, TX, USA. Apollack@notes.mdacc.tmc.edu
Ashoori
F
F
Cowen
D
D
Meyn
R E
RE
Tofilon
P
P
Meistrich
M E
ME
Pollack
A
A
D000256:Adenoviridae / Q000235:genetics; D017209:Apoptosis / Q000235:genetics*; D002470:Cell Survival / Q000235:genetics; D015972:Gene Expression Regulation, Neoplastic / Q000235:genetics*; D017930:Genes, Reporter; D016158:Genes, p53 / Q000235:genetics*; D005822:Genetic Vectors / Q000502:physiology; D006801:Humans; D008297:Male; D011471:Prostatic Neoplasms / Q000235:genetics* / Q000503:physiopathology / Q000532:radiotherapy*; D011836:Radiation Tolerance / Q000235:genetics*; D011853:Radiobiology; D014162:Transfection; D019076:Transgenes / Q000235:genetics*; D014407:Tumor Cells, Cultured / Q000528:radiation effects; D001616:beta-Galactosidase / Q000235:genetics
D001616:beta-Galactosidase