Abstract
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 theEscherichia coli (E. coli) lac operator/repressor system, and displays a transformed phenotype after isopropyl-β-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.
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Abbreviations
- IPTG :
-
isopropyl β-d-thiogalactoside
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Chang, MY., Won, SJ. & Liu, HS. A ribozyme specifically suppresses transformation and tumorigenicity of Ha-ras-oncogene-transformed NIH/3T3 cell lines. J Cancer Res Clin Oncol 123, 91–99 (1997). https://doi.org/10.1007/BF01269886
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DOI: https://doi.org/10.1007/BF01269886