the £53 gene and are essential for the transforming ability of the oncogene. The wildtype £W 
gene may directly suppress uncontrolled cell growth or indirectly activate genes that suppress 
this growth. Thus, absence or inactivation of wildtype £^ 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'""”; they occur in both NSCLC and SCLC cell lines and fresh tumors”’”. 
Options for specific targeting of oncogenes include inhibition of expression of a dominant gene 
or 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. Initially, a model for 
specific inhibition of K- ras was developed. We chose to work with K- ras because of the 
applicability of the findings to a large number of tumors, because of our previous work with K- 
ras . and because information on the genetic organization and sequence of the ras gene family was 
readily available. Advances in antisense and retroviral gene transfer technology suggested that 
application of these techniques may mediate specific inhibition of oncogene expression. 
Antisense mRNA, which is precisely complementary to the corresponding sense mRNA, inhibits 
translation. The mechanisms for this inhibition have not been completely defined but include 
inhibition of translation by ribosomes, degradation of sense-antisense duplexes by enzymes, and 
failure of export from the nucleus. Thus, specific targeting of a gene in a multigene family 
could occur if it possessed unique sequences in a region amenable to antisense inhibition, such 
as an initiation codon or splice site. 
The working hypothesis that we developed is that reversal of a single altered genetic event 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 our laboratory indicate that reversal of a 
single genetic alteration has profound effects on the growth and tumorigenicity of lung cancer 
cells'*’”. Additional support for this concept comes from a recent study by Soriano and coworkers'' 
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 fvn 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. 
Preliminary data on transfection of an antisense K- ras expression vector indicated that 
inhibition of expression of a single oncogene reduced the growth rate of cancer cells and 
tumorigenicity in nu/nu mice. However, transfected cells retained viability, as did cells with no 
endogenous K- ras mutation that were also transfected with the construct. The wt p53 appears 
dominant over the mutant gene and will select against proliferation when transfected into cells 
with the mutant gene'*’”. Normal expression of the transfected wt p53 does not affect the growth of 
cells with endogenous wt p53 . Thus, such constructs might be taken up by normal cells without 
adverse effects. This protocol focuses regional delivery of the two gene constructs, antisense K- 
ras and wt p53 . to lung cancer cells in patients with unresectable obstructing endobronchial 
cancers. The efficiency of delivery and gene expression will be evaluated both in lung cancer 
cells and in normal cells in vivo . This is of importance for the design of constructs that may 
be useful therapeutically. The effects of these constructs on clinical progression of the cancer 
will be studied. 
These approaches may lead to cancer therapy based on direct alteration of gene expression in 
cancer cells. Current therapy relies on attempts to kill or remove the last cancer cell. However, 
tumor cell dormancy is an established phenomenon making effective killing highly unlikely. 
Although inhibition of expression of some oncogenes may be lethal to the cancer cell, in some 
cases cell replication will slow or cease, thus rendering these cancers clinically dormant. Even 
if absolute specificity is not achieved, single oncogenes may still be important targets, because 
it is likely that adverse effects to normal cells will be minimal. 
2.2 Natural history of locally unresectable NSCLC 
Patients with NSCLC will die of their cancer in 86% of cases. Regional delivery of gene 
constructs to areas at risk for development of cancer has important implications for both 
prevention and therapy. Failure of therapy at the primary tumor site is a significant problem'*’'”. 
Of the 161,000 patients newly diagnosed with lung cancer in 1991, 45,080 will undergo surgical 
resection. Local recurrence as the first site of failure will occur in 9,000 of those patients. 
Recombinant DNA Research, Volume 19 [279] 
