DNA damage activates a cell-cycle checkpoint that prevents mitosis while DNA repair is under way. The protein Chk1 enforces this checkpoint by phosphorylating the mitotic inducer Cdc25. Phosphorylation of Cdc25 by Chk1 creates a binding site in Cdc25 for 14-3-3 proteins, but it is not known how 14-3-3 proteins regulate Cdc25. Rad24 is a 14-3-3 protein that is important in the DNA-damage checkpoint in fission yeast. Here we show that Rad24 controls the intracellular distribution of Cdc25. Elimination of Rad24 causes nuclear accumulation of Cdc25. Activation of the DNA-damage checkpoint causes the net nuclear export of Cdc25 by a process that requires Chk1, Rad24 and nuclear-export machinery. Mutation of a putative nuclear-export signal in Rad24 impairs the nuclear exclusion of Rad24, the damage-induced nuclear export of Cdc25 and the damage checkpoint. Thus, Rad24 appears to function as an attachable nuclear-export signal that enhances the nuclear export of Cdc25 in response to DNA damage.

Cdc2, the kinase that induces mitosis, is regulated by checkpoints that couple mitosis to the completion of DNA replication and repair. The repair checkpoint kinase Chk1 regulates Cdc25, a phosphatase that activates Cdc2. Effectors of the replication checkpoint evoked by hydroxyurea (HU) are unknown. Treatment of fission yeast with HU stimulated the kinase Cds1, which appears to phosphorylate the kinase Wee1, an inhibitor of Cdc2. The protein kinase Cds1 was also required for a large HU-induced increase in the amount of Mik1, a second inhibitor of Cdc2. HU-induced arrest of cell division was abolished in cds1 chk1 cells. Thus, Cds1 and Chk1 appear to jointly enforce the replication checkpoint.

Cdc2, a catalytic subunit of cyclin-dependent kinases, is required for both the G 1 -to-S and G 2 -to-M transitions in the fission yeast Schizosaccharomyces pombe. Cdc13, a B-type cyclin, is required for the M-phase induction function of Cdc2. Two additional B-type cyclins, Cig1 and Cig2, have been identified in S. pombe, but none of the B-type cyclins are individually required for the onset of S. We report that Cdc13 is important for DNA replication in a strain lacking Cig2. Unlike ⌬cdc13 cells, double-mutant ⌬cdc13 ⌬cig2 cells are defective in undergoing multiple rounds of DNA replication. The conclusion that Cig2 promotes S is further supported by the finding that Cig2 protein and Cig2-associated kinase activity appear soon after the completion of M and peak during S, as well as the observation that S is delayed in ⌬cig2 cells as they recover from a G 1 arrest induced by nitrogen starvation. These studies indicate that Cig2 is the primary S-phase-promoting cyclin in S. pombe but that Cdc13 can effectively substitute for Cig2 in ⌬cig2 cells. These observations also suggest that the gradual increase in the activity of Cdc2-Cdc13 kinase can be sufficient for the correct temporal ordering of S and M phases in ⌬cig2 cells.A key aim of cell cycle studies is to understand how orderly cell cycle progression is regulated. One productive investigative approach has been to identify fission yeast mutants that have uncoupled the DNA replication (S) and nuclear division (M) phases (32). In the fission yeast Schizosaccharomyces pombe, a single cyclin-dependent kinase complex, consisting of the Cdc2 catalytic subunit bound to the Cdc13 cyclin B subunit, is essential for the induction of mitosis (4, 28). Like B-type cyclins of animal cells, Cdc13 accumulates during interphase and is rapidly degraded upon exit from M. Interestingly, ⌬cdc13 cells undergo multiple rounds of S without intervening M phases (18). This leads to the formation of gigantic cells that each have a huge nucleus. Studies have also shown that high overexpression of Cdc2 and Cdc13 can force the induction of mitosis in G 1 cells (18). These observations have led to the hypothesis that the presence of Cdc2-Cdc13 complex defines a cell as being in G 2 and effectively gives the cell no choice other than to initiate M. The acquisition of the opportunity to undergo DNA replication depends on the destruction of Cdc13 that normally occurs at the end of M.Cdc2 is required for both the G 1 -to-S and G 2 -to-M transitions, whereas Cdc13 is essential only for the onset of M (33). Since cyclin-dependent kinases are inactive unless bound to a cyclin, there must be at least one cyclin that is involved in promoting the onset of S in S. pombe. Genes encoding five additional cyclin-related proteins have been identified in S. pombe. Two of the genes, mcs2 ϩ and pch1 ϩ , encode cyclin C-like proteins having essential functions that do not appear to be directly related to cell cycle control (14,26). A third gene, puc1 ϩ , encodes an unusual type of cyclin that has no ascribed fu...

BackgroundMultiCellular Tumor Spheroid (MCTS) mimics the organization of a tumor and is considered as an invaluable model to study cancer cell biology and to evaluate new antiproliferative drugs. Here we report how the characteristics of MCTS in association with new technological developments can be used to explore the regionalization and the activation of cell cycle checkpoints in 3D.MethodsCell cycle and proliferation parameters were investigated in Capan-2 spheroids by immunofluorescence staining, EdU incorporation and using cells engineered to express Fucci-red and -green reporters.ResultsWe describe in details the changes in proliferation and cell cycle parameters during spheroid growth and regionalization. We report the kinetics and regionalized aspects of cell cycle arrest in response to checkpoint activation induced by EGF starvation, lovastatin treatment and etoposide-induced DNA damage.ConclusionOur data present the power and the limitation of spheroids made of genetically modified cells to explore cell cycle checkpoints. This study paves the way for the investigation of molecular aspects and dynamic studies of the response to novel antiproliferative agents in 3D models.