95% reduction in K-ras p21 protein synthesis in the clones expressing the 
antisense RNA, whereas H460a cells and sense K- ras clones showed unchanged 
levels of K-ras p21 protein. Total p21 detected with a pan-ras monoclonal 
antibody showed only a slight decrease in the antisense clones, suggesting 
other ras genes were not affected. To confirm this, expression of ras genes 
was measured by cDNA PCR. The cDNA synthesized from the total RNA was 
subjected to PCR amplification using amplimers corresponding to the 5 ’-end of 
the first exon and the 3 ’-end of the second exon. Because the antisense RNA 
was generated from the second and third exons of K-ras. PCR-ampl ified cDNA 
represented the level of endogenous K- ras mRNA. N- and H- ras -specific 
oligonucleotides were used to determine expression of their respective genes. A 
118-bp segment of endogenous was co-ampl ified in the same reaction mixture 
as an internal PCR control. 
Cells expressing antisense RNA showed complete inhibition of K- ras mRNA 
synthesis. There was no change in H- ras or N- ras expression in either 
antisense or sense transfectants. Antisense transfectants showed a 3-fold 
reduction in growth rate compared to sense transfectants and parental H460a 
cells but continued to grow in culture. Expression of antisense K-ras RNA 
reduced the growth rate of H460a tumors in nu/nu mice. Tumorigenicity of cell 
lines expressing antisense RNA was assessed by subcutaneous injection of 10' 
cells in nu/nu mice. Unmodified H460a cells formed tumors in all mice in 15 
days. No tumors developed in mice injected with H460a antisense cells during 
120 days of observation, whereas H460a cells transfected with Apr-l-neo sense 
plasmid formed tumors similar to those formed by unmodified H460a cells. These 
experiments show that in H460a cells engineered to synthesize antisense K- ras 
RNA, the levels of K-ras mRNA and K-ras p21 protein were dramatically reduced. 
Our studies show that a construct can be made that distinguishes among members 
of the family. Previous studies with ras AS oligonucleotides showed 
inhibition of total p21 expression which led to cell death”, whereas our data 
show that antisense RNA generated from the genomic DNA of the K- ras gene can 
specifically inhibit K- ras expression. Inhibition of K- ras reduced the growth 
rate of H460a cells but did not alter cell viability or continued growth in 
culture, which suggests that redundancy in p21 expression may compensate for 
absence of expression by one member of this family so that functions essential 
for maintenance of cell viability are preserved. When antisense K- ras was 
transfected into H322a cells, which are homozygous for wtK-ras. the cells were 
unchanged in viability or rate of growth. These studies provide evidence that 
reduction in the expression of a single mutant gene product can reduce tumor 
cell proliferation and tumorigenicity. 
2. 3. 1.2 Gene construct 
The retroviral vector construct contains the AS-K- ras fragment with its p-actin 
promoter inserted into the LNSX vector”’**. The orientation of the insert is such 
that the transcription of the AS-K-ras is driven by the p-actin promoter 
downstream from the insert. A minor modification eliminating the SV40 promoter 
may also be used. 
2. 3. 1.3 Packaging 
Because recombination events may lead to the production of a replication- 
competent virus, a safe and efficient amphotropic packaging cell line is 
necessary for transfer of exogenous genes into human cancer cells. We used a 
packaging cell line constructed so the qaq - pol and env genes are separated on two 
different plasmids**. For this protocol retroviral supernates from the GP+envAml2 
packaging cell line will be used. This packaging cell line has received prior 
approval for use in human gene therapy clinical trials. The presence of 
functioning retroviral genes in the packaging cell line will be monitored by an 
assay for reverse transcriptase production and by immunoprecipitation of env 
protein by metabolic labeling and inmunoprecipitation with anti - env antiserum**. 
Continued absence of infectious virus will be determined from transfection- 
infection experiments. A neo -containinq vector will be transfected into 
GP+envAM12 cells; colonies will be selected with G418. The supernate will be used 
to infect NIH 3T3 cells. Selection with G418 will be done after one month to 
ensure the survival of rare recombinants that do not have the neo gene but 
subsequently infect neo -positive cells. Supernate from the infected NIH 3T3 cells 
should not be infectious. These secondary supernates will be used to infect naive 
NIH 3T3 cells. Lack of infectivity will indicate absence of replication competent 
virus. Previous human studies have used the GP+envAml2 producer cell line. We 
will use this cell because of the extensive experience with its use and prior 
approval for human use. Supernate from the packaging cells will be produced in 
Recombinant DNA Research, Volume 19 [281] 
