Ribozyme-mediated inactivation of mutant K-ras oncogene in a colon cancer cell line.
Mutation of c-K-ras oncogene is an important step in progression of colon cancer. We used a hammerhead ribozyme (KrasRz) against mutated K-ras gene transcripts (codon 12, GTT) to inactivate mutant K-ras function in the colon cancer cell line SW480, harbouring a mutant K-ras gene. The beta-actin promoter-driven KrasRz sequence (pHbeta/KrasRz) was introduced into these cells (SW480/KrasRz), and we evaluated its effects on growth of the colon cancer. The gene expression of angiogenesis-related molecules (vascular endothelial growth factor and thrombospondin) was also estimated in SW480/KrasRz. KrasRz specifically and efficiently cleaved the mutant K-ras mRNA but not wild-type mRNA in vitro. SW480/KrasRz showed decreased growth rate under tissue culture conditions (P< 0.01, Dunnett's test). The xenotransplantability of SW480/KrasRz (XeSW480/KrasRz) was significantly decreased in nude mice (P< 0.05, Fisher's exact test). Tumour volume of the xenografts XeSW480/KrasRz was significantly smaller than that of XeSW480/DisKrasRz (P< 0.01, Dunnett's test). Gene expression of VEGF was suppressed in SW480/KrasRz, while TSP1 gene expression was enhanced. The SW480/KrasRz cells showed apoptosis-related features including nuclear condensation and DNA fragmentation. These results suggested that the hammerhead ribozyme-mediated inactivation of the mutated K-ras mRNA induced growth suppression, apoptosis and alteration of angiogenic factor expression.
Tokunaga
T
T
Department of Pathology, Tokai University School of Medicine, Bohseidai, Isehara, Kanagawa, 259-1193, Japan.
Tsuchida
T
T
Kijima
H
H
Okamoto
K
K
Oshika
Y
Y
Sawa
N
N
Ohnishi
Y
Y
Yamazaki
H
H
Miura
S
S
Ueyama
Y
Y
Nakamura
M
M
D000818:Animals; D003062:Codon; D003110:Colonic Neoplasms / Q000473:pathology*; D004273:DNA, Neoplasm / Q000235:genetics; D016228:Endothelial Growth Factors / Q000096:biosynthesis; D015972:Gene Expression Regulation, Neoplastic; D011905:Genes, ras / Q000235:genetics*; D006801:Humans; D008222:Lymphokines / Q000096:biosynthesis; D051379:Mice; D008819:Mice, Nude; D017354:Point Mutation; D016337:RNA, Catalytic / Q000502:physiology*; D019699:Thrombospondins / Q000096:biosynthesis; D014158:Transcription, Genetic; D014407:Tumor Cells, Cultured; D042461:Vascular Endothelial Growth Factor A; D042442:Vascular Endothelial Growth Factors
D003062:Codon; D004273:DNA, Neoplasm; D016228:Endothelial Growth Factors; D008222:Lymphokines; D016337:RNA, Catalytic; D019699:Thrombospondins; D042461:Vascular Endothelial Growth Factor A; D042442:Vascular Endothelial Growth Factors