Protocol HNS 94-001 
October 4, 1994 
Page 5 
(H226b) showed increased tumorigenicity when transfected with AS -p53. The 
H226b cells expressing AS-p53grow significantly more rapidly in nu/nu mice than the 
cells transfected with the control plasmid. This indicates that elimination of the wtp53 
gene product enhances features of the malignant phenotype. 
These studies showed that wtp53 is dominant and can suppress the malignant 
phenotype in cells with mutant or deleted p53. The presence of the mutant p53 
confers transforming potential to the gene product, which can be suppressed by AS- 
p53. Thus, in cancer cells both the absence of wtp53 and the presence of certain 
p53 mutations may enhance the malignant phenotype. 
2.3.1 .2 Generation of recombinant p53 adenovirus. 
The p53 expression cassette (Figure 1 , Appendix D), which contains human 
cytomegalovirus (CMV) promoter 21 , wild-type p53 cDNA, and SV40 early 
polyadenylation signal, was inserted between the Xba I and Cla I sites of pXCJL.1 , a 
plasmid kindly 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 PCR analysis of the 
DNA samples prepared from the cell culture supernatants was described 
elsewhere 24 . The wild-type sequence of the p53 cDNA in the Ad5CMV-p53 virus was 
confirmed by dideoxy DNA sequencing on the CsCI-gradient-purified viral DNA. 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), Ad5CMV-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 25 . Single viral clones were propagated in 293 cells. 
The culture medium of the 293 cells showing the completed cytopathic effect was 
collected and centrifuged at 1000 x gfor 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 viral 
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 protein In HNSCC 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 
HNSCC cell lines Tu138 and Tul77 that were infected by Ad5CMV-p53 at an MOI of 
50 PFU/cell. When Tul38 or Tu177 cells were infected at the same MOI, the level of 
expression of the exogenous wild-type p53 gene was at least four times higher than 
that of the endogenous mutated protein in Tu138 and 14 times higher than that of 
the endogenous wild-type protein in MDA 1986 cells (data not shown). 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 post-infection day 3, 
sharply decreased after day 5, and lasted for at least 15 days (Fig. 5, Appendix D). 
[650] 
Recombinant DNA Research, Volume 20 
