Recombinant DNA Advisory Committee - 12/1-2/94 
Dr. Zhang presented data demonstrating the lack of any growth effect on a normal human fibroblast cell line 
whereas, tumor cells were killed. p53 gene expression in the normal fibroblasts lasted between 5 and 7 days. 
He showed additional data demonstrating apoptosis. Addressing the issue of p53 mutations in the vector 
stocks, Dr. Zhang showed data derived from in vitro transformation assays on NIH3T3 cells. No transformed 
fod were observed indicating the lack of oncogenic effect of the adenovirus vector. The likelihood of any 
oncogenic effect is slight because gene expression is transient. 
Dr. Roth said that mutant p53 alone cannot transform normal cells; transformation occurs in cooperation with 
other oncogenes. No evidence of NIH3T3 transformation has been observed at high MOI compared to the 
positive transformation observed with the Abelson retrovirus. Transgenic murine experiments suggest that the 
dominant negative effect of mutant p53 is very weak. 
Dr. Roth stated that the protein molecule which appears to mediate the "bystander" effect ranges between 30 
to 100 kilodaltons. This protein causes tumor cell killing through a mechanism that is consistent with 
apoptosis. This protein is inactivated by trypsin digestion, heating, acetonitrile, and 0.1% sodium dodecyl 
sulfate. De novo protein synthesis is required for the "bystander" effect to occur, as suggested by 
cycloheximide inhibition. Progress is ongoing in an attempt to isolate this protein. 
Dr. Par km an said that the biological significance of p53 mutations is the critical issue. He inquired whether 
the primary reviewers of this protocol are comfortable with the results of the transformation assays using 
replication competent adenovirus. Dr. Miller said that retrovirus transformation can be observed by focus 
formation in the NIH3T3 cells 2 weeks post-infection; similar assays cannot be performed with the 
adenoviruses since expression is transient. Dr. Samulski suggested that "hot spot" p53 mutations should be 
monitored using a properly validated PCR assay. Dr. Roth stated that PCR sensitivity for detecting a 
particular pS3 exon mutation or a single strand conformational polymorphism assay for point mutations is 
approximately 10%. Dr. Miller asked if animal models are available to test for p53 mutations. Dr. Roth 
responded that such experiments had been conducted in mice but cotton rat experiments were not conducted. 
Dr. Glorioso suggested that a mutant p53 adenovirus vector should be constructed to use as a transformation 
assay control. Dr. Roth said that other p53 mutant vectors have been shown not to cause cell transformation. 
p53 requires oncogenes to transform most normal cells, e.g., K-ras. 
Dr. Parkman said that a validated and sensitive transforming assay is preferable to a biochemical assay for the 
detection of p53 mutations. The important factor is the biological significance of these mutations. A 
transformation assay could be constructed using helper oncogenes to assay mutant p53 oncogenic activity. Dr. 
Samulski agreed that cell transformation requires multiple events; a system to detect p53 oncogenic mutations 
has to be set up in cooperation with other oncogenes. Dr. Glorioso said such a biological system to assess the 
risk is necessary, te., to test the vector stocks in a cooperation type of transformation assay. Dr. Miller 
commented that constructing an adenovirus with an oncogenic p53 is a risky experiment. Mr. Capron 
suggested an alternative strategy for providing a biological containment, i.e., tumor-specific promotor which 
could limit mutant p53 expression to target cells. Dr. Miller said that the albumin promoter elicits some 
degree of specificity for liver cells; however, development of a tumor specific promotor would be difficult. Dr. 
Samulski agreed with Mr. Capron that development of such a vector would be preferable. Dr. Roth 
responded that a cancer specific promoter is a complex issue and should not distract the present proposal. 
Committee Motion 
A motion was made by Dr. Haselkorn and seconded by Dr. Brinckerhoff to approve the protocol contingent 
on the submission of a validated assay which can detect potentially oncogenic p53 mutations in the vector 
stock. Dr. Haselkorn expressed concern that the construction of an oncogenic p53 adenovirus would be more 
hazardous than the theoretical risk of the proposed study. 
Dr. Saha asked about the frequency of p53 mutations in the general population. Dr. Roth responded that 
hereditary p53 mutations are rare. Li-Fraumeni syndrome represents a rare predisposition for the 
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Recombinant DNA Research, Volume 20 
