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Somatic genetic analysis of cyclic AMP action: characterization of unresponsive mutants
Abstract:
N-6,O-2'-dibutyryl adenosine 3',5'-monophosphate kills cultured mouse lymphosarcoma cells, but not resistant mutants derived by a single-step clonal selection. Resistant clones lack the cyclic AMP binding proteins present in wild type, cyclic AMP sensitive clones. Both endogenous cyclic AMP, accumulated in response to isoproterenol or cholera toxin, and exogenous dibutyryl cyclic AMP induce cyclic AMP phosphodiesterase, slow growth, and eventually kill wild type cells. In the resistant mutants, however, the endogenous and exogenous cyclic nucleotides appear to be completely inactive. These results indicate that an intracellular receptor for cyclic AMP, previously shown to be associated with a cyclic AMP-dependent protein kinase, mediates cyclic AMP's regulation of growth and phosphodiesterase synthesis.
Insights
N-6,O-2'-dibutyryl adenosine 3',5'-monophosphate effectively kills mouse lymphosarcoma cells by targeting cyclic AMP (cAMP) pathways. Resistant cells lacking cAMP binding proteins are unaffected, indicating a crucial role for these proteins in cAMP-mediated cell death.
Area of Science:
- Cell Biology
- Molecular Biology
- Biochemistry
Background:
- Cyclic adenosine monophosphate (cAMP) is a crucial second messenger involved in various cellular processes.
- Understanding the mechanisms of cAMP signaling is vital for comprehending cell growth regulation and drug resistance.
Purpose of the Study:
- To investigate the role of cyclic AMP (cAMP) binding proteins in mediating the cytotoxic effects of N-6,O-2 '-dibutyryl adenosine 3 ',5 '-monophosphate (dibutyryl cAMP) on mouse lymphosarcoma cells.
- To elucidate the mechanism by which resistant cell mutants evade cAMP-induced cell death.
Main Methods:
- Utilized N-6,O-2 '-dibutyryl adenosine 3 ',5 '-monophosphate (dibutyryl cAMP) to treat wild-type and resistant mouse lymphosarcoma cell lines.
- Performed clonal selection to derive resistant mutants.
- Assessed the presence of cyclic AMP binding proteins in wild-type and resistant cells.
- Evaluated the cellular response to both endogenous and exogenous cyclic nucleotides.
Main Results:
- N-6,O-2 '-dibutyryl adenosine 3 ',5 '-monophosphate (dibutyryl cAMP) demonstrated cytotoxicity against wild-type lymphosarcoma cells.
- Resistant mutants, selected through single-step clonal selection, were insensitive to dibutyryl cAMP.
- Resistant clones lacked the cyclic AMP binding proteins found in sensitive wild-type cells.
- Both endogenous and exogenous cyclic AMP failed to elicit responses in resistant mutants, while they induced phosphodiesterase synthesis, growth inhibition, and cell death in wild-type cells.
Conclusions:
- Intracellular cyclic AMP (cAMP) binding proteins are essential mediators of cAMP's regulatory effects on cell growth and phosphodiesterase synthesis.
- The absence of functional cAMP binding proteins confers resistance to cAMP-mediated cytotoxicity.
- A specific intracellular receptor for cAMP, linked to cAMP-dependent protein kinase, plays a pivotal role in mediating these cellular responses.