A ribozyme specifically suppresses transformation and tumorigenicity of Ha-ras-oncogene-transformed NIH/3T3 cell lines.
In this study, the efficacy of an anti-ras ribozyme in reversing a transformed phenotype was investigated. A murine NIH/3T3-derived cell line, designated 2-12, contains an inducible Ha-ras oncogene, which is regulated by the Escherichia coli (E. coli) lac operator/repressor system, and displays a transformed phenotype after isopropyl-beta-D-thiogalactoside induction. To reverse the transformed characteristics, the ribozyme, which specifically targets the Ha-ras oncogene at the codon 12 mutation site (GGC to GUC), was transfected into 2-12 cells. Two (ribZ4 and ribZ7) clones were subsequently selected and analyzed for their transforming features. Our results show that, in the transfectants, ribozyme gene expression was detected, and the target Ha-ras transgene was expressed at basal levels. Their phenotypic responses, including morphology, cell growth rate, colony-formation efficiency and tumorigenicity in mice with severe combined immunodeficiency were more similar to those of NIH/3T3 than 2-12 transformed cells. Directly injecting the ribozyme DNA into tumors induced by transformed 2-12 cells in BALB/c mice also caused tumor regression. The enzymatic cleavage products of the ribozyme acting on mutant Ha-ras mRNA in vivo were detected by primer-extension analysis. These results indicate that the ribozyme were designed exhibits a site-specific ribonuclease function that effectively abrogates Ha-ras-oncogene-induced transformation, and this unique anti-Ha-ras property should shed light on the development of strategies against the Ha-ras-oncogene-initiated malignancy.
Chang
M Y
MY
Department of Microbiology and Immunology, College of Medicine National Cheng Kung University, Tainan, Taiwan, R.O.C.
Won
S J
SJ
Liu
H S
HS
D016475:3T3 Cells; D000818:Animals; D000970:Antineoplastic Agents; D001483:Base Sequence; D002455:Cell Division; D002471:Cell Transformation, Neoplastic / Q000187:drug effects* / Q000235:genetics / Q000473:pathology; D015870:Gene Expression; D011905:Genes, ras; D008297:Male; D051379:Mice; D008807:Mice, Inbred BALB C; D016513:Mice, SCID; D008969:Molecular Sequence Data; D016283:Proto-Oncogene Proteins p21(ras) / Q000235:genetics*; D016337:RNA, Catalytic / Q000494:pharmacology*
D000970:Antineoplastic Agents; D016337:RNA, Catalytic; D016283:Proto-Oncogene Proteins p21(ras)