The G 2 M Arrest Caused by Iodide Is Unrelated to the Effects of Iodide at Adenylate Cyclase

Abstract

Previously, we have shown that iodide was able to inhibit TSH induced thyrocyte proliferation by arresting the cell cycle at G₀G₁ and G₂M, suggesting that iodide may be exerting its effects through more than the TSH-adenylate cyclase-cAMP system. To confirm the effects of iodide on the adenylate cyclase (AC) system, forskolin- and dibutyryl-cyclic-AMP (dBcAMP)-stimulated FRTL5 thyroid cells were exposed to inhibitory concentrations of iodide and the resultant effects on the cell cycle were compared to the effects observed with TSH, using flow cytometric DNA analysis. Forskolin stimulated the proliferation of FRTL5 cells in a dose-dependent manner. Cell numbers rose from baseline by 169 ± 4% to peak at 10 μM forskolin. Interestingly, 100 μM forskolin inhibited cell proliferation, causing cell numbers to fall by approximately 50%. Iodide inhibited forskolin-induced proliferation to baseline levels. However, the pattern of cell cycle perturbation was different to that with TSH-stimulated cells. There were no differences in the proportion of cells in G₀G₁ between forskolin alone and forskolin + NaI, while there was a marked fall in the proportion of cells in S phase, indicating possible partial arrest at G₀G₁. Furthermore, there was a marked accumulation of cells in G₂M over and above that found with TSH + Nal, indicating arrest at G₂M. dBcAMP maximally stimulated cell numbers to rise from baseline by 125% with 1 mM dBcAMP. Again, higher concentrations of the mitogen had an inhibitory effect on proliferation. The addition of NaI inhibited dBcAMP stimulated cell proliferation. However, unlike the effect of NaI on TSH- or forskolin-induced proliferation, there was no G₀G₁ arrest in the cell cycle. With dBcAMP, there were decreases in the proportion of cells in both G₀G₁ and S phases with a marked accumulation of cells in G₂M indicating that the inhibitory effect of iodide is due purely to G₂M cell cycle arrest. In this study, the requirement of AC activation for the mediation of TSH-induced proliferation has been confirmed. Further, the inhibitory effects of iodide are mediated by at least 2 mechanisms, one AC related and the other independent of the AC system. Iodide caused significant inhibition at G₀G₁ and G₂M with cells stimulated by TSH, while in the presence of forskolin, which stimulates the cAMP cascade at the catalytic component of AC, iodide caused little or no cell cycle arrest at G₀G₁, but induced marked cell cycle arrest at G₂M. Further, iodide in the presence of dBcAMP, which acts distal to AC, induced only a G₂M arrest. This indicates that the G₀G₁ arrest caused by iodide is due to effects at or proximal to the catalytic component of AC while the G₂M arrest is unrelated to the effects of iodide at AC.