Protocol THS 94—002 
revised 3/28/94 
Page 4 
obstmcting the airway and that have failed or are unabie to receive external beam 
radiotherapy will be considered for this protocol. Existing therapies for this condition offer 
only short-term palliation. Most patients have recurred despite external beam radiotherapy. It 
may be possible to insert a brachytherapy catheter arxJ administer additional radiotherapy. 
Patients receiving this treatment have a median survival of 6 months[20]. Patients failing 
brachytherapy would also be eligible to receive gene therapy. Tumor can be removed from 
the airway with the laser or biopsy forceps. This can be done in conjunction with injection of 
the adenovirus construct thus decreasing the volume that must be injected. Patients with 
unresectable local-regional tumors who have failed surgery or radiation therapy have a poor 
prognosis. Chemotherapy is only palliative and the median survival is less than 6 months. 
Patients with malignant pleural effusions are not curable and are usually treated 
symptomatically with sclerosis. The administration of the adenovirus constructs would not 
preclude the patient from receiving other palliative therapy if the tumor progresses. 
2.3 Structure and characteristics of the biological system 
2.3.1 Restoration of expression of wtp53 gene product 
2.3. 1.1 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 ceil 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 wtp53 expressed in lung cancer cells with mutant or deleted p53 and the 
effects of reducing wildtype and mutant p53 expression (1 1]. 
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 antisense p53 (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 wlp53 and the presence of certain 
p53 mutations may enhance the malignant phenotype. 
ecombinant DNA Research, Volume 19 
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