Figure 2: H460a cells were infected with LNSX-AS-K-ras by incubating 10* cells with 0.5 ml of viral stock 
(6X10* CFU/ml ) produced by the packaging cell line GP+enyAml2 in the presence of ^/ml of 
polybrene. Infection was done daily for 1 to 7 days. Two days later cells were plated in equal 
numbers into selective media containing 20Qfjg/ml G416. Control H460a cells were plated at equal 
cell numbers to determine baseline colony forming efficiency. The infection efficiency was 
measured by determining the percent of the unselected colony number formed by the G418 selected 
colonies. 
The tumorigenicity of the transduced H460a cells was studied in an orthotopic 
lung cancer model. Intratracheal inoculation of H460a cells in irradiated 
(350cGy) nu/nu mice resulted in the growth of endobronchial tumors with 
mediastinal extension in >80X of mice after 4 weeks. The H460a-AS-LNSX, H460a- 
LNSX, and H460a cells (lO’/mouse) were injected endotracheal ly in 0.1 ml of 
medium and the mediastinal block was harvested after 4 weeks. Mice were assessed 
for tumor growth without knowledge of the treatment group. Seven of 9 mice 
inoculated with H460a-LNSX (mean volume 12.5 ± 2.2 SE nm’) and 12 of 14 mice 
inoculated with H460a parental cells (mean volume 39.9 ± 4.25 SE mm’) had tumors. 
Only 3 of 17 mice receiving H460a-AS-LNSX cells had tumors (mean volume 2.95 ± 
1.25 mu’). We conclude that 1) retroviral gene transduction can be used to 
express anti-sense constructs in human tumor cells at levels that mediate a 
biologic effect; 2) AS-mediated inhibition of activated K-ras expression 
effectively inhibits proliferation and tumorigenicity of human cancer cells. 
Expression of the AS-LNSX expression in the H460a cells has been stable up to 6 
months. 
In a second experiment, H460a cells (lO’/nwuse) were injected into the right 
mainstem bronchus via a tracheostomy incision. Three days later mice were 
inoculated endobronchial ly with 0.15 ml of retroviral supernate or control medium 
for 3 days. Tumor growth was suppressed in the group receiving supernate from 
LNSX-AS producer cells (Table 1). Histologic light microscopic study of 3 
specimens from the LNSX-AS group did not reveal any residual H460a cells. 
Table 1: Endotracheal inoculation of retroviral supernate for treatment of established (3 day) H460a human lung 
cancers growing endobronchial ly in nu/nu mice. The H460a cells were injected at 10' per mouse. The titer of the 
retroviral supernate was 5X10* CFU/ml. Mice were injected with 0.15ml of supernate daily for 3 days. 
i^oifucer (Utils ttPU/dose Ha. of tuBwrs/ Itesft tmde Itoluae ♦ $£ 
Mo, of Mice <X) 
H460a 
0 
7/8 
(87. 5X) 
42.3 ± 7.9 
LNSX 
7.5X10’ 
7/8 
(87. 5X) 
44.9 + 10.1 
LNSX-AS 
7.5X10' 
1/6 
(16. 7X) 
7.7* 
Volume of the single tumor that occurred in this group 
2.3.2 Restoration of expression of wtp53 gene product 
2.3.2. 1 Preliminary studies with plasmid DNA 
The eM gene is the most conmonly altered gene yet described in human cancers. To 
Recombinant DNA Research, Volume 19 
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