Ras mutations are frequent in thyroid tumors, the most common endocrine malignancy. The ability of Ras to transform thyroid cells is thought to rely on its mitogenic activity. Unexpectedly, acute expression of activated Ras in normal rat thyroid cells induced a DNA damage response, followed by apoptosis. Notably, a subpopulation of cells evaded apoptosis and emerged with features of transformation, including the loss of epithelial morphology, dedifferentiation, and the acquisition of hormone-and anchorage-independent proliferation. Strikingly, the surviving cells showed marked chromosomal instability. Acutely, Ras stimulated replication stress as evidenced by the induction of ataxia telangiectasia mutated and Rad3-related protein kinase (ATR) activity (Chk1 phosphorylation) and of ;H2A.X, a marker of DNA damage. Despite the activation of a checkpoint, cells continued through mitosis in the face of DNA damage, resulting in an increase in cells harboring micronuclei, an indication of defects in chromosome segregation and other forms of chromosome damage. Cells that survived exposure to Ras continued to exhibit replication stress (ATR activation) but no longer exhibited ;H2A.X or full activation of p53. When rechallenged with Ras or DNA-damaging agents, the surviving cells were more resistant to apoptosis than parental cells. These data show that acute expression of activated Ras is sufficient to induce chromosomal instability in the absence of other signals, and suggest that Ras-induced chromosomal instability arises as a consequence of defects in the processing of DNA damage. Hence, abrogation of the DNA damage response may constitute a novel mechanism for Ras transformation. (Cancer Res 2006; 66(21): 10505-12)

Overexpression of protein kinase C ␦ (PKC␦) stimulates apoptosis in a wide variety of cell types through a mechanism that is incompletely understood. PKC␦-deficient cells are impaired in their response to DNA damage-induced apoptosis, suggesting that PKC␦ is required to mount an appropriate apoptotic response under conditions of stress. The mechanism through which it does so remains elusive. In addition to effects on cell survival, PKC␦ elicits pleiotropic effects on cellular proliferation. We now provide the first evidence that the ability of PKC␦ to stimulate apoptosis is intimately linked to its ability to stimulate G 1 phase cell cycle progression. Using an adenoviral-based expression system to express PKC␣, -␦, and -⑀ in epithelial cells, we demonstrate that a modest increase in PKC␦ activity selectively stimulates quiescent cells to initiate G 1 phase cell cycle progression. Rather than completing the cell cycle, PKC␦-infected cells arrest in S phase, an event that triggers caspasedependent apoptotic cell death. Apoptosis was preceded by the activation of cell cycle checkpoints, culminating in the phosphorylation of Chk-1 and p53. Strikingly, blockade of S phase entry using the phosphatidylinositol 3-kinase inhibitor LY294002 prevented checkpoint activation and apoptosis. In contrast, inhibitors of mitogen-activated protein kinase cascades failed to prevent apoptosis. These findings demonstrate that the biological effects of PKC␦ can be extended to include positive regulation of G 1 phase cell cycle progression. Importantly, they reveal the existence of a novel, cell cycle-dependent mechanism through which PKC␦ stimulates cell death. Protein kinase C (PKC)3 is a family of serine/threonine protein kinases composed of three subclasses consisting of the classical (␣, ␤I, ␤II, and ␥), novel (␦, ⑀, , and ), and atypical ( and /) PKC isoforms. PKC isoforms differ in their requirement for calcium and their responsiveness to the lipid second messenger diacylglycerol. The classical (calcium-dependent) and novel (calcium-independent) isoforms are responsive to diacylglycerol, whereas the atypical isoforms are diacylglycerol-insensitive (reviewed in Ref. 1). Most cells express multiple PKC isozymes that require distinct cofactors and exhibit unique intracellular localizations. Pharmacological inhibitors have been used to assess the roles of individual PKC isozymes. Much of what is known regarding the biological roles of PKC␦ has been derived from studies using the PKC␦-selective inhibitor rottlerin, the specificity of which is in question. The isolation of cell lines overexpressing or lacking individual isozymes has been used to decipher the physiological roles of select PKC isozymes. A major limitation of this approach is that it does not readily distinguish between the effects of PKC activation versus the subsequent down-regulation of PKC expression. We opted to use adenoviruses to drive selective, modest increases in the activities of PKC␣, -␦, and -⑀, a powerful approach that has provided substantial insight into...

Genetic evidence indicates that Ras plays a critical role in the initiation and progression of human thyroid tumors. Paradoxically, acute expression of activated Ras in normal rat thyroid cells induced deregulated cell cycle progression and apoptosis. We investigated whether cell cycle progression was required for Ras-stimulated apoptosis. Ras increased CDK-2 activity following its introduction into quiescent cells. Apoptotic cells exhibited a sustained increase in CDK-2 activity, accompanied by the loss of CDK-2-associated p27. Blockade of Ras-induced CDK-2 activity and S phase entry via overexpression of p27 inhibited apoptosis. Inactivation of the retinoblastoma protein in quiescent cells through expression of HPV-E7 stimulated cell cycle progression and apoptosis, indicating that deregulated cell cycle progression is sufficient to induce apoptosis. Ras failed to induce G 1 phase growth arrest in normal rat thyroid cells. Rather, Ras-expressing thyroid cells progressed into S and G 2 phases and evoked a checkpoint response characterized by the activation of ATR. Ras-stimulated ATR activity, as evidenced by Chk1 and p53 phosphorylation, was blocked by p27, suggesting that cell cycle progression triggers checkpoint activation, likely as a consequence of replication stress. These data reveal that Ras is capable of inducing a DNA damage response with characteristics similar to those reported in precancerous lesions. Our findings also suggest that the frequent mutational activation of Ras in thyroid tumors reflects the ability of Ras-expressing cells to bypass checkpoints and evade apoptosis rather than to simply increase proliferative potential.

Thyroid cell proliferation is regulated by the concerted action of TSH/cAMP and serum growth factors. The specific contributions of cAMP-dependent vs. -independent signals to cell cycle progression are not well understood. We examined the molecular basis for the synergistic effects of TSH and serum on G1/S phase cell cycle progression in rat thyroid cells. Although strictly required for thyroid cell proliferation, TSH failed to stimulate G1 phase cell cycle progression. Together with serum, TSH increased the number of cycling cells. TSH enhanced the effects of serum on retinoblastoma protein hyperphosphorylation, cyclin-dependent kinase 2 activity, and cyclin A expression. Most notably, TSH and serum elicited strikingly different effects on p27 localization. TSH stimulated the nuclear accumulation of p27, whereas serum induced its nuclear export. Unexpectedly, TSH enhanced the depletion of nuclear p27 in serum-treated cells. Furthermore, only combined treatment with TSH and serum led to rapamycin-sensitive p27 turnover. Together, TSH and serum stimulated p70S6K activity that remained high through S phase. These data suggest that TSH regulates cell cycle progression, in part, by increasing the number of cycling cells through p70S6K-mediated effects on the localization of p27.