Protocol HNS 94-001 
October 4, 1994 
Page 2 
dominant tumor suppressor genes may influence certain characteristics of cells that contribute to the 
malignant phenotype. 
Despite increasing knowledge of the mechanisms involved in oncogene-mediated transformation, 
little progress has occurred in developing therapeutic strategies that specifically target oncogenes 
and their products. Initially, research in this area was focused on dominant oncogenes, as these were 
the first to be characterized. DNA-mediated gene transfer studies showed acquisition of the malignant 
phenotype by normal cells following the transfer of DNA from malignant human tumors. Activated 
oncogenes of the ras family were identified by this technique with transfection of human DNA into 
mouse NIH 3T3 cells. More recently a class of tumor suppressor genes have been identified. Mutation 
or deletion of both copies of a tumor suppressor gene is required to eliminate its function and cause 
the cell to acquire characteristics of the malignant phenotype. 
Tumor Suppressor Gene Mutations in Head and Neck Squamous Cell Carcinoma 
The p53 gene is the most frequently mutated gene yet identified in human cancers. It is mutated in 
over 50% of human HNSCC 3 . The p53 gene encodes a 375-amino-acid phosphoprotein that can 
form complexes with viral proteins such as large-T antigen and E1B 4 . Missense mutations are 
common for the p53 gene and are essential for the transforming ability of the oncogene. The wildtype 
p53 gene may directly suppress uncontrolled cell growth or indirectly activate genes that suppress 
this growth. Thus, absence or inactivation of wildtype p53 may contribute to transformation. However, 
some studies indicate that the presence of mutant p53 may be necessary for full expression of the 
transforming potential of the gene. Mutations of p53 are common in a wide spectrum of tumors 5-8 ; 
they occur in both HNSCC cell lines and fresh tumors 9 . Additionally, they occur in second primary 
cancers that may arise in over 20% of head and neck cancer patients. 10 
An option for specific targeting of tumor suppressor genes is replacement of a deleted or mutated 
tumor suppressor gene. Progress in the understanding of the critical genes involved in tumor 
development and in technology for altering gene expression logically led to our studies of techniques 
for achieving these options. t 
Our working hypothesis Is that overexpression of a wildtype p53 tumor suppressor 
gene in the cancer cell can potentially reverse critical features of the malignant 
phenotype of that cell. This finding has important therapeutic implications. Cancer cells have 
multiple genetic alterations. Therapy directed toward oncogenes will be practical only if therapeutic 
effects occur with targeting of one or two genes. It is unlikely that any therapy targeting oncogenes or 
their products will be absolutely specific for cancer cells. If other genes can compensate for loss of 
normal function by a specific oncogene mediated by an antisense construct, the harmful effects of 
the therapy will be reduced. Studies from Roth et al. indicate that reversal of a single genetic 
alteration has profound effects on the growth and tumorigenicity of carcinoma cells 11 . Additional 
support for this concept comes from a recent study by Soriano and co-workers 12 in which transgenic 
mice were created that lacked a functional c-src proto-oncogene. The resulting developmental defect 
in the mice was osteopetrosis. The ubiquity of c-src, its high degree of conservation among species, 
and its role in mitosis suggest that inactivation would be lethal, but this was not the case; viable mice 
were recovered. A possible explanation is that other closely related nonreceptor tyrosine kinases 
such as yes and fyn can compensate for loss of c-src. Introduction of a single copy of a wildtype tumor 
suppressor gene into normal cells would be unlikely to have adverse effects if it occurred during 
therapy directed at replacing inactivated tumor suppressor genes in cancer cells. To support this, 
work in our laboratory has investigated the effect of exogenous wild-type p53 transduced in non- 
malignant fibroblasts and found that no alteration in cell growth or morphology occurs as compared to 
tumor cells that undergo cell cycle arrest, inhibition of tumorigenicity and apoptosis, (dayman, 
unpublished data Appendix D. Figure 6.) 
Wild-type p53 appears dominant over its mutant gene and will select against proliferation when 
transfected into cells with the mutant gene 11 - 13 . Our experiments have shown that expression of the 
transfected wt p53 does not affect the growth of non-mafignant cells with endogenous wtp53. Thus, 
such constructs might be taken up by normal cells without adverse effects. This protocol wjll study 
Recombinant DNA Research, Volume 20 
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