Abstract
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 G1 phase growth arrest in normal rat thyroid cells. Rather, Ras-expressing thyroid cells progressed into S and G2 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.
Highlights
Point mutations in H, K, and N-Ras are frequent in human thyroid tumors (9 –13)
In contrast to the induction of G1 phase growth arrest observed in primary human diploid or murine embryonic fibroblasts [18], retroviral-mediated expression of activated Ras stimulates sustained proliferation in primary human thyrocytes, cells that normally fail to proliferate in vitro [19, 20]
Inducible expression of activated Ras in rat thyroid PC-CL3 cells [22] or infection of Wistar rat thyroid cells with an adenovirus expressing activated Ras [23] stimulated cell cycle progression as expected and induced apoptosis, a result difficult to reconcile with the high frequency of Ras mutations in human thyroid tumors
Summary
Cell Culture and Adenoviruses—Wistar rat thyroid cells were cultured in Coon’s modified Ham’s F-12 medium supplemented with bovine thyroid-stimulating hormone (1 milliunit/ ml), insulin (10 g/ml), calf serum (5%), and transferrin (5 g/ml) as previously described [29]. RasV12 (or RasL61) was infected at 5000 particles/cell (unless otherwise noted), and the other Ras mutants were infected at doses that resulted in equal levels of Ras expression. Determined by counting the number of DAPI-positive nuclei that reacted with the active caspase-3 antibody. When analyzed in detached cells, pooled cells from four 100-mm plates were used to precipitate CDK-2 (sc-163G; Santa Cruz) from equal amounts of cell protein as in total and adherent cells. The number of BrdUrd-positive cells is expressed as the percentage of the total nuclei (mean Ϯ S.E.). FACS Analysis for DNA Content—Detached and adherent cells were collected, fixed, treated with 200 units/ml RNase, stained with 0.1 mg/ml propidium iodide, and subjected to FACS analysis as described in Ref. 30
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