Protocol THS 94-002 
REVISED 3/28/94 
Page 6 
lines were chosen for this study: cell line H358, which has a homozygous deletion of 
the p53 gene, cell line H322, which has a point mutation of the p53 gene at codon 
248 (G to T), and cell line H460. which has a wild-type p53 gene. The cells were 
treated with Ad5CMV-p53 and Ad5/RSV/GL2 at 10 FFU/cell. Triplicate sets of the 
viral-infected and mock-infected cells were counted every day for 6 days. Growth 
rates of the Ad5CMV-p53-infected H358 and H322 cells were inhibited 79% and 72%, 
respectively, compared to that of the mock-infected cells (Fig. 6. A and B, Appendix 
D). The growth rate of the Ad5CMV-p53-treated H460 cells was inhibited by 28% of 
that of the mock-infected H460 cells (Fig. 6C, Appendix D). Cells infected with the 
control virus Ad5/RSV/GL2 had growth rates similar to those of the mock-infected 
cells. When the Ad5CMV-p53-infected cells were harvested at 24 h after infection 
and replated in cell culture dishes, only 14% of the treated H358 and H322 cell 
populations reattached and survived, whereas the mock-infected cells had a 
replating efficiency of more than 95%. This suggested that the viability of the 
Ad5CMV-p53-infected H358 and H322 cells was greatly reduced. 
Inhibition of tumorigenicity mediated by Ad5CMV-p53. To examine whether the 
Ad5CMV-p53 virus can inhibit tumorigenicity of human NSCLC cells, nude mice were 
injected s.c. with H358 cells to induce tumor. Each mouse received one injection of 
2x10® cells that had been infected with Ad5CMV-p53 or control vector Ad5/RSV/GL2 
at 10 PFU/cell for 24 h. H358 cells treated with medium alone were used as mock- 
infected controls. Tumors, first palpable at postinjection day 14. were induced only 
by the mock- or control virus-infected cells (Table I); mice that received AdSCMV- 
p53-treated cells did not develop tumors (Table I). The tumors at the end of a 6- 
week period were 4-10 mm in diameter. This study was initiated with five mice per 
group; one mouse each in the Ad5CMV-p53 or Ad5/RSV/GL2 group failed to 
complete the study. The early deaths were presumably due to infection, since the 
mice that received Injections were kept in a regular animal room after they were 
taken out from a barrier area for radiation treatment. 
Table I. Effect of Ad5CMV-p53 on tumorigenicity In H358 in nude mice* 
Treatment No. of Tumors/ Mean Volume 
No. of Mice (%) (mm® ± SD) 
Medium 4/5 (80) 37 ± 12 
Ad5/RSV/GL2 3/4 (75) 30 ± 14 
Ad5CMV-p53 0/4 (0) — 
• The treated H358 cells were injected s.c. at 2 x 10® cells/mouse. 
Tumor sizes were determined at the end of a 6-week period. 
Recombinant 
The efficacy, of Ad5CMV-p53 in inhibiting tumorigenicity was further evaluated in the 
mouse model of orthotopic human lung cancer[27j. Since H358 and H322 cells did 
not produce tumors in this model, cell line H226Br was used. This cell line has an 
origin of squamous lung cancer and metastasized from lung to brain. It has a’ point 
mutation (ATC to GTC) at exon 7, codon 254, of the p53 gene and is tumorigenic in 
mice. The irradiated nude mice were inoculated with 2x10® H226Br cells/mouse by 
intratracheal instillation. Three days after inoculation, each of the mice (8-10 mice 
per group) were treated with 0.1 ml of either Ad5CMV-p53 or Ad5/RSV/GL2 or 
vehicle (PBS) by intratracheal Instillation once a day for two days. The virus dosage 
used was 5x10® PFU/mouse. Tumor formation was evaluated at the end of a 6- 
week period by dissecting the lung and mediastinum tissues and measuring the 
tumor size. The procedure Is depicted in Figure 7, Appendix D. The representative 
samples of dissection are demonstrated in Figure 8, Appendix D. The detected 
DNA Research, Volume 19 [5051 
