2.3.2 Restoration of expression of wt p53 gene product 
2.3.2. 1 Preliminary studies with plasmid DNA 
The p53 gene is the most commonly altered gene yet described 
in human cancers. To 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 
p53 protein as shown by the presence of high levels of 
endogenous p53 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) cDNA with a /?-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. 16 
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 wtp53 can 
suppress transformation in cancer cells expressing a mutant p53 
or 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 wtp53 (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 wtp53 gene product 
enhances features of the malignant phenotype. 
Our studies showed that wtp53 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 wtp53 and the 
presence of certain p53 mutations may enhance the malignant 
phenotype. 
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Recombinant DNA Research, Volume 16 
