Protocol HNS 94-001 
October 4, 1994 
Page 6 
PCR analysis on the DNA samples prepared from the Ad5CMV-p53-treated H358 
cells failed to detect the viral DNA after post-infection 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 HNSCC cell growth. Five human HNSCC cell 
lines were chosen for this study: cell line Tul 38 and Tul 77, which both have 
homozygous mutations of the p53 gene, and cell lines MDA 686, 886 and 1986 
which have wild-type p53 genes. Additionally, fibroblasts derived from stromal tissue 
culture outgrowth of patients afflicted with head and neck cancer were also assessed. 
The cells were treated with Ad5CMV-p53 and dl312 ( replication defective adenovirus 
control) at 50 PFU/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 
HNSCC cells were significantly inhibited compared to that of the mock-infected cells 
and dl31 2 infected cells. (Fig. 6., Appendix D). Unlike other epithelial cancer cell lines 
previously tested, within 72 hours post-infection, no viable cell colonies can be 
obtained in reculturing transduced cell culture dishes. Twenty-four hours after 
infection, an apparent morphologic change occurred with portions of the cell 
population rounding up and their outer membranes forming blebs. Cells infected with 
dl312, (replication-defective adenovirus), demonstrated normal growth characteristics 
with no histomorphologic abnormalities. Ad5CMV-p53 had no effect on cell growth or 
morphology in non-malignant fibroblasts derived from cancer patients. (Fig. 6, 
Appendix D) 
Inhibition of tumorigeniclty mediated by Ad5CMV-p53. To examine 
whether the Ad5CMV-p53 virus can inhibit tumorigenicity of human HNSCC cells, the 
effect of AdCMV-p53 on established subcutaneous tumor nodules was determined 
in nude mice in a defined pathogen-free environment. Briefly, following induction of 
acepromazine anesthesia, three separate s.c. flaps were elevated on each animal and 
5 x 10 6 cells in 150pl nude mice were injected s.c. After four days, palpable tumors 
were noted in each pocket site ( 6mm 3 or greater in size) and animals were 
reanesthetized, and the flaps were re-elevated for the delivery of 100pJ of a) 
Ad5CMVp53 (1 0 8 P.F.U.) in the right anterior flap; b) replication defective adenovirus, 
dl31 2, (1 0 8 P.F.U.) in the right posterior flap and c) transport medium alone in the left 
posterior flap. Animals were observed daily and sacrificed on day 20 following 
experimental interventions. 
Seven animals were tested for each cell line. One animal in the Tu-177 group 
died following the second flap surgery and delivery of the therapeutic interventions, 
presumably due to profound anesthesia and subsequent mutilation by cage mates. 
Necropsy revealed no evidence of metastasis or systemic effects. Figure 7, 
Appendix D shows representative Tu-138 (left) and Tu-177 recipients (right). Sizable 
tumors are apparent on both posterior flaps of the animals (i.e., the sites that did not 
receive Ad5CMV-p53). The lack of tumor progression is significant in the right 
anterior flaps of the animals, which received Ad5CMV-p53 in both cell lines tested. 
That Tu-177 cells have a slower growth rate has previously been established in these 
animals (unpublished data). Two animals in the Tu-177 group had complete clinical 
and pathologic regression of their established subcutaneous tumor nodule. Two 
animals in the Tu-138 group were killed early because they were experiencing rapid 
growth and ulceration of the control tumor sites. All surgical sites had developed 
lesions of at least 6 mm 3 before intervention. The tumor volumes on necropsy are 
shown in Table 1. 
Recombinant DNA Research, Volume 20 
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