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Spontaneous or carcinogen-mediated amplification of a mutated ras gene promotes neoplastic transformation
V Sorrentino1, M D McKinney, V Drozdoff
1Molecular Biology Program, Memorial Sloan-Kettering Cancer Center, New York, NY 10021.
Abstract:
Mouse C3H10T1/2 cells were co-transfected with a plasmid containing the KiSV DNA and a plasmid carrying sequences coding for resistance to mycophenolic acid. Only 30% of the transfected colonies had a transformed phenotype, i.e. highly refractile rounded cells. The remaining colonies had varied morphologies with flat or slightly elongated cells. Analysis of p21 ras protein indicated that higher levels of the protein were expressed in cells with the more transformed phenotype. Tumors formed by a poorly tumorigenic clone were found to have undergone in vivo amplification of the transfected KiSV sequence. Transformed variants of this clone were also isolated in vitro. Treatment with 5-azacytidine resulted in an increase of about 10 fold in the formation of transformed variants. All transformed cells isolated, either spontaneous or 5-azacytidine induced, were tumorigenic in nude mice. The neoplastic conversion of these cells was accompanied by amplification of the transfected K-ras sequences. The data reported here indicate that a chemical agent that modifies DNA structure can enhance the frequency of amplification events in cellular DNA and, by affecting ras copy number, promote cell transformation.
Insights
Chemicals altering DNA structure can increase gene amplification, promoting cell transformation. This study shows 5-azacytidine enhances K-ras gene amplification and neoplastic conversion in mouse cells.
Area of Science:
- Molecular Biology
- Cell Biology
- Cancer Research
Background:
- Cell transformation is a key step in cancer development.
- Ras gene amplification is implicated in neoplastic progression.
Purpose of the Study:
- To investigate the role of K-ras gene amplification in cell transformation.
- To determine if DNA-modifying agents can influence K-ras amplification and transformation frequency.
Main Methods:
- Co-transfection of mouse C3H10T1/2 cells with KiSV DNA and a resistance gene.
- Phenotypic analysis of transfected colonies.
- Western blot analysis of p21 ras protein levels.
- In vivo and in vitro isolation of transformed variants.
- Treatment with 5-azacytidine.
- Tumorigenicity assays in nude mice.
Main Results:
- Only 30% of transfected cells showed a transformed phenotype.
- Higher p21 ras protein levels correlated with a more transformed phenotype.
- In vivo K-ras sequence amplification occurred in poorly tumorigenic clones.
- 5-azacytidine treatment increased transformed variant formation tenfold.
- All isolated transformed cells were tumorigenic.
- Neoplastic conversion was associated with K-ras sequence amplification.
Conclusions:
- DNA-modifying agents like 5-azacytidine can enhance gene amplification events.
- Increased ras copy number promotes cell transformation and neoplastic conversion.
- This highlights a mechanism linking DNA structural changes to cancer progression.