Related Experiment Videos
Isolation of variants resistant to methylglyoxal bis(guanylhydrazone) from adenovirus-transformed rat cells
Abstract:
It is presumed that proteins encoded by viral oncogenes interact with proteins encoded by cellular genes to bring about the transformed phenotype. To demonstrate the existence of such cellular genes we attempted to isolate mutants with a nontransformed phenotype from an adenovirus-transformed rat cell line (F4) which contains multiple copies of the transforming E1 region. F4 cells were mutagenized with ethyl methanesulfonate and variants resistant to the anticancer drug methylglyoxal bis(guanylhydrazone) were selected. The proportion of such variants was about one in 10(6) and increased 5-fold after mutagenesis. Two variant clones (G1 and G2) were isolated and characterized: they were 5-fold more resistant to methylglyoxal bis(guanylhydrazone); they had a stable phenotype; they showed decreased drug uptake; they had a reduced ability to grow in soft agar, low serum, and nude mice; there was no detectable change in the restriction pattern of integrated viral genes or in the expression of the E1a and E1b proteins. These properties suggest that selection for methylglyoxal bis(guanylhydrazone) resistance may result in the isolation of variants with phenotypic characteristics of nontransformed cells. It was likely that these variants were altered in a cellular function required for the maintenance of the transformed phenotype.
Insights
Researchers isolated non-transformed cell variants from adenovirus-transformed cells by selecting for resistance to methylglyoxal bis(guanylhydrazone). This suggests cellular genes are crucial for maintaining the transformed phenotype.
Area of Science:
- Molecular Biology
- Virology
- Cancer Research
Background:
- Viral oncogenes are believed to interact with cellular genes to cause cell transformation.
- Adenovirus-induced transformation involves the E1 region of the viral genome.
- Identifying cellular genes involved in transformation is key to understanding cancer development.
Purpose of the Study:
- To demonstrate the existence of cellular genes that maintain the transformed phenotype.
- To isolate and characterize mutants with a non-transformed phenotype from an adenovirus-transformed cell line.
- To investigate the role of cellular functions in sustaining viral oncogenesis.
Main Methods:
- Mutagenesis of an adenovirus-transformed rat cell line (F4) using ethyl methanesulfonate.
- Selection of variants resistant to the anticancer drug methylglyoxal bis(guanylhydrazone).
- Phenotypic characterization of selected resistant clones, including drug resistance, growth properties, and viral gene expression analysis.
Main Results:
- Two variant clones (G1 and G2) exhibited stable resistance to methylglyoxal bis(guanylhydrazone) and reduced drug uptake.
- These variants showed decreased ability to grow in soft agar, low serum conditions, and in nude mice.
- No detectable changes were observed in the integrated viral genes or the expression of E1a and E1b viral proteins.
Conclusions:
- Selection for methylglyoxal bis(guanylhydrazone) resistance can isolate cells with non-transformed characteristics.
- These findings suggest alterations in cellular functions required for maintaining the transformed state.
- Cellular genes play a critical role in the maintenance of the transformed phenotype induced by adenovirus.