These mutations render the p21 protein gene product 
constitutively active by keeping the protein in the GTP-bound 
state. We began investigations on alterations of expression of 
p21 22 . A homozygous mutation at codon 61 was detected in 
the H460a large-cell undifferentiated NSCLC cell line clone; a 
normal glutamine residue (CAA) was substituted by histidine 
(CAT) using hybridization with specific oligonucleotide probes. 
Direct PCR DNA sequencing confirmed this. An antisense K-ras 
RNA construct selectively blocked the production of mutant p21 
so the contribution of the mutated p21 protein to the malignant 
phenotype could be studied. A recombinant plasmid clone was 
constructed using a normal wildtype 2-Kb K-ras genomic DNA 
segment carrying second and third exons with flanking intron 
sequences subcloned into an Apr-1 -neo expression vector in 
antisense orientation. The intron sequence used has a low 
degree of homology with other ras genomic sequences; it 
allowed specific inhibition of K-ras while preserving H-ras and N- 
ras expression. For example, the 241 bases flanking exon 2 
and the 240 bases flanking exon 3 have no significant homology 
between K-ras and H-ras sequences. Previous studies of 
uptake of ras antisense oligonucleotides by cancer cells resulted 
in cell death instead of regulated growth, probably because 
functioning p21 is necessary for cell viability, and the 
oligonucleotides unselectively blocked p21 expression 23 . 
Unselective blockade of oncogene expression can therefore be 
harmful to both normal and cancer cells. An additional novel 
feature of this construct was the use of a /?-actin promoter that 
can constitutively direct synthesis of RNA in a human tumor cell. 
The 2-Kb DNA insert was stably integrated into H460a cells, as 
shown by Southern hybridization. Northern blot analysis 
detected expression of antisense RNA. Western blot analysis 
showed a 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-amplified 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 p53 was 
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Recombinant DNA Research, Volume 16 
