Respective roles of carbamylcholine and cyclic adenosine monophosphate in their synergistic regulation of cell cycle in thyroid primary cultures

The stimulation of thyroid cell proliferation by TSH through cAMP depends o n permissive comitogenic factors, generally the insulin-like growth factors and insulin. In dog thyroid primary cultures, the use of the phosphodieste rase-resistant analog of cAMP (Bu)(2)cAMP instead of TSH allowed to unveil a potent comitogenic activity of carbamylcholine, which can substitute for insulin and was shown to mimic insulin action on cell cycle regulatory prot eins. Like insulin, carbamylcholine induced the accumulation of cyclin D3 a nd overcame the repression by cAMP of this protein, which was shown 1) to b e essential for cell cycle progression by means of microinjections of a neu tralizing antibody; and 2) to be rate limiting for the cAMP-dependent assem bly of cyclin D3-cdk4 complexes, their nuclear translocation and the phosph orylation of pRb. Relative to insulin, carbamylcholine offers the significa nt experimental advantage that its signaling cascades can be immediately de activated by the muscarinic antagonist atropine. In the presence of carbamy lcholine, the elimination of (Bu)(2)cAMP blocked within 2 h the entry of ce lls into DNA synthesis phase, but the addition of atropine still permitted the entry of cells in S phase. These data support our view that the progres sion in G1 phase stimulated by cAMP consists of at least two essential acti ons that are clearly dissociated: in a first stage, depending on the suppor tive activity of an agent that stimulates the required cyclin D3 accumulati on, cAMP induces the assembly and nuclear translocation of cyclin D3-cdk4 c omplexes, and then cAMP can exert alone the last crucial control that deter mines the cell commitment toward DNA replication.