Protocol THS 9A-002 
REVISED 3/28/94 
Page 5 
2.3.1. 2 Generation of recombinant p53 adenovirus. 
The p53 expression cassette (Figure 1, Appendix D), which contains human 
cytomegaiovirus (CMV) promoter[21], wiid-type p53 cDNA, and SV40 early 
polyadenylation signai, was inserted between the Xba i and Cla i sites of pXCJLl, a 
piasmid kindiy provided by Dr. Frank L Graham of McMaster University, Hamilton, 
Ontario, Canada. The p53 shuttle vector (pEC53) and the recombinant plasmid 
pJM17(22] were cotransfected into 293 cells[23] by liposome-mediated transfection 
with DOTAP (Boehringer Mannheim Corp., Indianapolis, IN)[24]. The transfected 
cells were maintained in medium until the onset of the cytopathic effect. 
Identification of newly generated p53 recombinant adenoviruses (Ad5CMV-p53) with 
PGR analysis of the DMA samples prepared from the ceil culture supernatants was 
described elsewhere [24]. The wild-type sequence of the p53 cDNA in the AdSCMV- 
p53 virus was confirmed by dideoxy DMA sequencing on the CsCI-gradient-purified 
viral DMA. The control virus Ad5/RSV/GL2, generated in a similar manner, has a 
structure similar to that of Ad5CMV-p53 except a Rous sarcoma viral promoter and 
luciferase cDNA were used in its expression cassette. The recombinant adenovirus 
that carries a E. coli b-galactosidase gene (LacZ), Ad5GMV-LacZ, also has a 
structure similar to that of Ad5CMV-p53, and was kindly provided by Dr. Frank L 
Graham. Structural analysis of the vectors is shown in Fig. 2, Appendix D. 
2.3.1. 3 Viral stock, titer, and infection. Individual clones of the Ad5CMV-p53, Ad5/RSV/GL2, 
and Ad5CMV-LacZ viruses were obtained by plaque-purification according to the 
method of Graham and Prevec[25j. Single viral clones were propagated in 293 cells. 
The culture medium of the 293 ceils showing the completed cytopathic effect was 
collected and centrifuged at 1000 x g for 10 min. The pooled supernatants were 
aliquoted and stored at -20 °C as viral stocks. The viral titers were determined by 
plaque assays[25). Infections of the cell lines were carried out by addition of the 
virai solutions (0.5 ml per 60-mm dish) to cell monolayers and incubation at room 
temperature for 30 min with brief agitation every 5 min. This was followed by the 
addition of culture medium and the return of the infected cells to the 37 °C 
incubator. 
2.3. 1.4 Preclinical studies 
Expression of exogenous p53 In human lung cancer cells. To obtain a high level 
expression of p53, the human CMV promoter[21 ] was used to drive the p53 cDNA 
carried by Ad5CMV-p53. As shown by Immunostaining and Western blot in Figures 3 
and 4 (Appendix D), a high level of expression of exogenous p53 was achieved in 
the H358 cells that were Infected by Ad5CMV-p53 at an MOI of 30 PFU/cell. When 
H322 or H460 cells were infected at the same MOI, the level of expression of the 
exogenous p53 gene was three times higher than that of the endogenous mutated 
protein In H322 and 14 times higher than that of the endogenous wild-type protein in 
H460 cells. The time course of the expression of the exogenous p53 after a single 
infection of 10 PFU/cell was studied In H358 cells. The protein expression peaked at 
postinfection day 3, sharply decreased after day 5, and lasted for at least 15 days 
(Fig. 5, Appendix D). PCR analysis on the DNA samples prepared from the Ad5CMV- 
p53-treated H358 cells failed to detect the viral DNA after postinfection day 15 (data 
not shown). The decrease in expression of the exogenous p53 probably resulted 
from the cellular attenuation on the CMV promoter or degradation of the viral DNA in 
the treated cell population [26]. This Is a critical point with respect to safety of the . 
vector. Transient p53 expression is sufficient for mediating apoptosis. However, 
normal cells taking up the vector will express the exogenous p53 for only a short 
time. 
Effect of exogenous p53 on lung cancer cell growth. Three human NSCLC cell 
Recombinant DNA Research, Volume 19 
