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A ribozyme specifically suppresses transformation and tumorigenicity of Ha-ras-oncogene-transformed NIH/3T3 cell
1Department of Microbiology and Immunology, College of Medicine National Cheng Kung University, Tainan, Taiwan, R.O.C.
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 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.
Insights
An anti-ras ribozyme effectively reversed cancer cell transformation by targeting the Ha-ras oncogene. This ribozyme therapy demonstrated tumor regression in vivo, offering a new strategy against Ha-ras-initiated cancers.
Area of Science:
- Molecular Biology
- Cancer Research
- Gene Therapy
Background:
- The Ha-ras oncogene drives cellular transformation and malignancy.
- Targeting oncogenes is a key strategy in cancer therapy.
- Ribozymes offer precise gene-targeting capabilities.
Purpose of the Study:
- To investigate the efficacy of a designed anti-ras ribozyme in reversing the transformed phenotype.
- To evaluate the ribozyme's ability to abrogate Ha-ras oncogene-induced transformation.
- To assess the therapeutic potential of this ribozyme against Ha-ras-driven cancers.
Main Methods:
- Transfection of an anti-ras ribozyme into NIH/3T3-derived 2-12 cells with an inducible Ha-ras oncogene.
- Selection and analysis of ribozyme-expressing clones (ribZ4, ribZ7).
- Assessment of phenotypic characteristics (morphology, growth, colony formation, tumorigenicity) and in vivo tumor regression studies.
Main Results:
- Ribozyme gene expression and basal Ha-ras transgene expression were detected in transfectants.
- Transfected cells exhibited reversed transformed phenotypes, resembling normal NIH/3T3 cells.
- Direct injection of ribozyme DNA into tumors induced tumor regression in mice.
- Enzymatic cleavage of mutant Ha-ras mRNA by the ribozyme in vivo was confirmed.
Conclusions:
- The designed anti-ras ribozyme functions as a site-specific ribonuclease, effectively neutralizing Ha-ras oncogene-induced transformation.
- This ribozyme demonstrates significant potential for developing novel therapeutic strategies against Ha-ras-initiated malignancies.