The regulation of cell proliferation and survival is essential for normal function. Thyroid stimulating hormone (TSH) regulates thyroid cell survival via cyclic AMP (cAMP), and protein kinase A (PKA) activity is an important determinant of the survival of thyroid cells (2). Given the important role of cAMP Response Element Binding protein (CREB) in mediating the transcriptional effects of PKA, we investigated the role of CREB family members in thyroid cell survival. Inhibition of CREB function using a dominant negative CREB mutant (A-CREB) induced apoptosis in rat thyroid cells. Expression of A-CREB inhibited mitogenesis, in part due to delayed cell cycle transit. Protracted S phase cell cycle progression in A-CREB-expressing cells was sufficient to activate a DNA damage checkpoint response. Entry into S phase was required for apoptosis as the overexpression of p27 prevented cell cycle progression and rescued apoptosis in A-CREB-expressing cells. These data reveal a novel mechanism through which interference with CREB abrogates cell survival through checkpoint activation secondary to cell cycle delay. In the course of these studies we found that insulin increased the sensitivity of A-CREB-expressing cells to apoptosis. To determine whether insulin increases the susceptibility of thyroid cells to apoptosis in the absence of CREB inhibition, its effects on survival were examined. Surprisingly, cells treated with TSH/insulin or with insulin alone underwent caspase-mediated apoptosis. Activation of PI3K and mTOR by insulin was essential for the induction of apoptosis. Serum also protected TSH/insulin-treated cells from apoptosis, though not through the modulation of PI3K or mTOR activity. Cell cycle progression in the presence of TSH/insulin was delayed compared to that stimulated by TSH/serum. Blockade of cell cycle progression and mTOR activity, or acceleration of cell cycle progression in the presence of mTOR activity was sufficient to block apoptosis. In contrast, blockade of cell cycle progression in the presence of mTOR activity did not prevent apoptosis. Our working model is that insulin uncouples cell growth from cell proliferation, possibly depleting cellular energy reserves, and enhancing sensitivity to apoptosis. The ability of insulin or CREB inhibition to stimulate apoptosis was conserved in two rat thyroid cell lines, but was not observed in rat fibroblasts. These results suggest that thyroid cells may be particularly sensitive to apoptosis induced by aberrant proliferation
