Transduction effect of antisense K-ras on malignant phenotypes in gastric cancer cells.
The antitumoral effects of antisense RNA to K-ras were investigated in gastric cancer cell lines by examining the level of K-ras expression and the tumorigenicity in vitro and in vivo. Polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP), DNA sequencing, and immunoblotting analysis revealed that YCC-1 gastric cancer cells overexpressed wild type K-ras, whereas YCC-2 cells had a homozygous mutation in codon 12 from GGT (glycine) to AGT (serine), while SNU-1 cells had a heterozygous mutation to GAT (asparagine) in the identical position. Both YCC-1 and YCC-2 cells were transduced by LNC-AS/K-ras containing the antisense 2.2 kb genomic K-ras DNA fragment covering exon 2 and exon 3 specific for K-ras. The application of antisense K-ras significantly downregulated the expression of K-ras and had no influence on the expression of either H-ras or N-ras. The antisense-transduced YCC-2 cells grew considerably slower than the control group transduced by LNCX, whereas the growth inhibition of antisense-transduced YCC-1 cells was less prominent than that of transduced YCC-2 cells. In addition, the tumorigenicity of YCC-2 cells transduced by LNC-AS/K-ras was totally lost. Therefore, our results imply that the specific inhibition of K-ras p21 protein can be accomplished by introducing the antisense covering the K-ras- specific region to gastric cancer cells with aberrant K-ras expression, resulting in a reduction of the growth rate and suppression of tumorigenicity.
Song
J J
JJ
Institute for Cancer Research, Yonsei University College of Medicine, Yonsei Cancer Center, Seoul, South Korea.
Lee
H
H
Kim
E
E
Kim
Y S
YS
Yoo
N C
NC
Roh
J K
JK
Kim
B S
BS
Kim
J
J
D000818:Animals; D002455:Cell Division / Q000502:physiology; D015536:Down-Regulation; D015870:Gene Expression; D015972:Gene Expression Regulation, Neoplastic; D011905:Genes, ras / Q000235:genetics*; D015316:Genetic Therapy; D005822:Genetic Vectors; D006801:Humans; D008297:Male; D051379:Mice; D008819:Mice, Nude; D009154:Mutation / Q000235:genetics; D009368:Neoplasm Transplantation; D020320:Oligoribonucleotides, Antisense / Q000235:genetics*; D015689:Oncogene Protein p21(ras) / Q000096:biosynthesis / Q000235:genetics; D010641:Phenotype; D016133:Polymerase Chain Reaction; D018807:Polymorphism, Single-Stranded Conformational; D012190:Retroviridae / Q000235:genetics; D013274:Stomach Neoplasms / Q000235:genetics* / Q000378:metabolism / Q000473:pathology; D014161:Transduction, Genetic; D014407:Tumor Cells, Cultured
D020320:Oligoribonucleotides, Antisense; D015689:Oncogene Protein p21(ras)