study this gene, a cell culture model system of cell lines varying in p53 
expression was established. The H322a lung adenocarcinoma cell line expresses the 
mutant protein as shown by the presence of high levels of endogenous mRNA 
and phosphorylated protein. We showed that the H322a cell line has a G:T 
transversion at codon 248 (Arg to Leu) with absence of the wildtype allele. The 
H358a cell line has a homozygous p53 deletion. The H460a and H226b cell lines are 
homozygous for the wildtype p53 . Expression vectors for sense (S- p53 ) and 
antisense p53 (AS- p53 1 cDNA with a f-actin promoter were constructed to study the 
effect of wt p53 expressed in lung cancer cells with mutant or deleted p53 and the 
effects of reducing wildtype and mutant p53 expression. “ 
Stable transfectants of p53 mutant cells (H322a) or deleted p53 (H358) expressing 
S- p53 could not be rescued. Failure to isolate colonies expressing sense p53 RNA 
in cells with homozygous mutant or deleted alleles shows that wt p53 can suppress 
transformation in cancer cells expressing a mutant eM o'" having a homozygous p53 
deletion. 
In general, transfection with AS- p53 reduced colony formation (10-fold) by cells 
with endogenous mutant p53 . This indicates that expression of mutant p53 
contributes to the transformed phenotype. As expected, cells with wt p53 (H226b) 
showed increased tumorigenicity when transfected with AS- p53 . The H226b cells 
expressing AS- p53 grow significantly more rapidly in nu/nu mice than the cells 
transfected with the control plasmid. This indicates that elimination of the 
wt p53 gene product enhances features of the malignant phenotype. 
Our studies showed that wt p53 is dominant and can suppress the malignant 
phenotype in cells with mutant or deleted p53 . The presence of the mutant p53 
confers transforming potential to the gene product, which can be suppressed by 
AS- p53 . Thus, in cancer cells both the absence of wt p53 and the presence of 
certain p53 mutations may enhance the malignant phenotype. 
2. 3. 2. 2 Gene construct 
The retroviral vector construct contains p53 cDNA with its f-actin promoter 
inserted into the LNSX vector”'”. Preclinical studies were performed with this 
vector. Subsequently a new vector was developed which does not have the SV40 
promoter. This is designated LNp53B and will be used in the clinical trials. 
2. 3. 2. 3 Packaging 
See section 2. 3. 1.3. 
2. 3. 2. 4 Preclinical studies 
The LNSX- p53 and the DC- p53 were transduced into H322a (mutant p53 ) . H358a 
(deleted £53), and H460a (wt £^). H322a cells that underwent one cycle of 
infection with the wt p53 construct but without G418 selection had an over 3-fold 
reduction in proliferation compared to cells that received either the unmodified 
vector or no treatment. Two cycles of transduction without G418 selection 
resulted in a 5-fold reduction in proliferation (Fig. 3). A similar result was 
observed for the H358a cell line when transduced with LNSX- p53 . The proliferation 
of the H460a cell line which has a wildtype p53 was not altered by transduction 
with any of the p53 retroviral constructs (Fig. 4). Thus, retroviral mediated 
gene transfer of wt p53 into human lung cancer cells with deleted or mutated £M 
significantly reduces the proliferation of those cells. The expression of the 
mutated £M protein is uniform in cultured cell lines as detected by 
immunohi stochemi stry. In fresh lung tumors that express high levels of p53 
protein, expression is detected in >90% of cells. 
A critical question is the ability of the retroviral constructs to transduce 
established tumor cells in vivo . This question was addressed by injecting H460a 
(10’) cells in the mouse right mainstem bronchus followed 3 days later by lavage 
with LNSX retroviral supernate (10* CFU in 0.1 ml). LNSX was used so that the neo 
gene could be used as a marker for transduction. It was necessary to recover 
tumor cells for analysis so that the AS construct was not used. Tumors were 
harvested and the presence of the neo gene was assessed by Southern 
hybridization. The neo gene was detected in the DNA from the H460a cells 
indicating successful transduction of the retrovirus 30 days after lavage. 
Although this data is encouraging, the model has limitations. Direct injection of 
endobronchial tumor is not possible in this model. Other sites of direct 
injection do not accurately simulate the milieu of endobronchial lung cancer. 
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Recombinant DNA Research, Volume 19 
