Related Experiment Videos
Somatic genetic analysis of cyclic AMP action: selection of unresponsive mutants
Abstract:
Dibutyryl cyclic AMP and theophylline kill S49.1 mouse lymphoma tissue culture cells. When cells are grown in soft agar with these drugs, the few clones that survive are resistant to cytolysis. The rate of mutation to resistance is 1-3 times 10-7/cell/generation in both diploid and tetraploid cells. The incidence of mutants is increased by treatment with a chemical mutagen, ICR 191. The mutation is consistently associated with greatly reduced or absent cytoplasmic cyclic AMP binding protein. These results suggest that a somatic mutation leads to a defect of the protein kinase regulatory subunit and that activity of this kinase is required for induction of cell death by cyclic AMP.
Insights
Dibutyryl cyclic AMP and theophylline induce cell death in lymphoma cells. Mutations conferring resistance suggest a defect in the cyclic AMP binding protein, essential for this cell death pathway.
Area of Science:
- Cell Biology
- Molecular Biology
- Cancer Research
Background:
- S49.1 mouse lymphoma cells are sensitive to dibutyryl cyclic AMP and theophylline.
- Cyclic AMP (cAMP) plays a role in cellular regulation and apoptosis.
- Understanding resistance mechanisms to cAMP-induced cell death is crucial for cancer therapy.
Purpose of the Study:
- To investigate the mechanism of resistance to cAMP-induced cytolysis in S49.1 mouse lymphoma cells.
- To determine the mutation rate and genetic basis of resistance.
- To identify the molecular target affected by the mutation.
Main Methods:
- Culturing S49.1 cells in soft agar with cytotoxic drugs.
- Measuring mutation rates to drug resistance.
- Treating cells with a chemical mutagen (ICR 191) to assess mutagenicity.
- Analyzing cytoplasmic cyclic AMP binding protein levels in resistant mutants.
Main Results:
- A low rate of mutation (1-3 x 10^-7/cell/generation) to resistance was observed.
- Chemical mutagenesis increased the incidence of resistant mutants.
- Resistant mutants consistently showed reduced or absent cytoplasmic cAMP binding protein.
- The mutation appears to affect the regulatory subunit of protein kinase.
Conclusions:
- Somatic mutation leading to a defective cAMP binding protein confers resistance to cAMP-induced cell death.
- Protein kinase activity is essential for mediating cAMP-induced apoptosis in these cells.
- This study identifies a specific molecular defect underlying drug resistance in lymphoma cells.