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A novel cell-based assay for the evaluation of anti-ras compounds
D C Jenkins1, J N Stables, J Wilkinson
1Department of Cell Biology, Wellcome Research Laboratories, Beckenham, Kent, UK.
Abstract:
In order to identify drugs active against mutated ras oncogenes we have developed an in vitro assay employing two clones of the human fibrosarcoma cell-line, HT1080 which carries an N-ras gene mutated at codon 61. Clone, HT1080scc2, retains the transformed phenotype of the parental line, whilst the other, HT1081c, is a morphologically flat, non-tumourigenic, revertant with under-representation of the chromosome carrying the transforming N-ras allele. The clear implication of mutant ras in maintaining the transformed nature of HT1080scc2 was confirmed when these cells were microinjected with the pan ras neutralising antibody Y13-259, which resulted in the morphological detransformation of these cells to a phenotype resembling that of the HT10801c clone. A number of known anti-cancer drugs with modes of action unrelated to ras function were found to be equipotent against both clones. However, when compounds chosen on the grounds of their potential selective cytotoxic or differentiating activity were tested some interesting results were obtained. Thus 8-bromo cAMP affected some morphological detransformation of HT1080scc2 cells and reduced their colony forming potential. The IMP-dehydrogenase inhibitors, tiazafurin and mycophenolic acid also flattened the morphology of the transformed clone. Fumagillin, an antibiotic reported to exhibit selective activity against ras transformed cells showed very marked and selective cytostatic effects against HT1080scc2 cells with IC50 values as low as 1 x 10(-11) M.
Insights
Researchers developed an in vitro assay to find drugs targeting mutated ras oncogenes. Fumagillin showed potent, selective cytostatic effects against N-ras mutated fibrosarcoma cells.
Area of Science:
- Oncology
- Molecular Biology
- Drug Discovery
Background:
- Mutated ras oncogenes are key drivers in various cancers.
- Developing targeted therapies for ras-driven cancers remains a challenge.
- HT1080 fibrosarcoma cell line with mutated N-ras provides a model for drug screening.
Purpose of the Study:
- To establish an in vitro assay for identifying drugs active against mutated ras oncogenes.
- To screen compounds for selective activity against ras-transformed cells.
- To evaluate the efficacy of specific agents like 8-bromo cAMP, IMP-dehydrogenase inhibitors, and fumagillin.
Main Methods:
- Utilized two clones of HT1080 fibrosarcoma cells: HT1080scc2 (transformed, mutated N-ras) and HT1081c (revertant, non-tumorigenic).
- Employed microinjection with a pan ras neutralizing antibody (Y13-259) to confirm ras dependency.
- Assessed drug effects on cell morphology, colony formation, and cytostatic activity (IC50 values).
Main Results:
- The pan ras neutralizing antibody Y13-259 induced morphological detransformation in HT1080scc2 cells.
- 8-bromo cAMP and IMP-dehydrogenase inhibitors (tiazofurin, mycophenolic acid) showed some morphological effects.
- Fumagillin exhibited highly potent and selective cytostatic effects against HT1080scc2 cells, with IC50 values as low as 1 x 10(-11) M.
Conclusions:
- The developed in vitro assay is effective for identifying drugs targeting mutated ras oncogenes.
- Fumagillin demonstrates significant selective anti-cancer activity against N-ras mutated fibrosarcoma cells.
- This study highlights potential therapeutic strategies for cancers driven by ras mutations.