Loss of RCC1, a nuclear DNA-binding protein, uncouples the completion of DNA replication from the activation of cdc2 protein kinase and mitosis.

The temperature-sensitive mutant cell line tsBN2, was derived from the BHK21 cell line and has a point mutation in the RCC1 gene. In tsBN2 cells, the RCC1 protein disappeared after a shift to the non-permissive temperature at any time in the cell cycle. From S phase onwards, once RCC1 function was lost at the non-permissive temperature, p34cdc2 was dephosphorylated and M-phase specific histone H1 kinase was activated. However, in G1 phase, shifting to the non-permissive temperature did not activate p34cdc2 histone H1 kinase. The activation of p34cdc2 histone H1 kinase required protein synthesis in addition to the presence of a complex between p34cdc2 and cyclin B. Upon the loss of RCC1 in S phase of tsBN2 cells and the consequent p34cdc2 histone H1 kinase activation, a normal mitotic cycle is induced, including the formation of a mitotic spindle and subsequent reformation of the interphase-microtubule network. Exit from mitosis was accompanied by the disappearance of cyclin B, and a decrease in p34cdc2 histone H1 kinase activity. The kinetics of p34cdc2 histone H1 kinase activation correlated well with the appearance of premature mitotic cells and was not affected by the presence of a DNA synthesis inhibitor. Thus the normal inhibition of p34cdc2 activation by incompletely replicated DNA is abrogated by the loss of RCC1.     

Biochemical Differences between Staurosporine-Induced Apoptosis and Premature Mitosis

Apoptosis is morphologically related to premature mitosis, an aberrant form of mitosis. Staurosporine, a potent protein kinase inhibitor, induces not only apoptotic cell death in a wide variety of mammalian cells but also premature initiation of mitosis in hamster cells that are arrested in S phase by DNA synthesis inhibitors. Here we report on the biochemical differences between the two phenomena commonly caused by staurosporine. Rat 3Y1 fibroblasts that had been arrested in S phase with hydroxyurea underwent apoptosis by treatment with staurosporine, whereas S-phase-arrested CHO cells initiated mitosis prematurely when similarly treated with a low concentration of staurosporine. Chromosome condensation occurred in both apoptosis (3Y1) and premature mitosis (CHO). However, neither formation of mitotic spindles nor mitosis-specific phosphorylation of MPM-2 antigens was observed in apoptosis of 3Y1 cells, unlike premature mitosis of CHO cells. The p34^cdc2kinase activated in normal and prematurely mitotic cells remained inactive in the apoptotic cells, probably because the active cyclin B/p34^cdc2complex was almost absent in the S-phase-arrested 3Y1 cells. The absence of intracellular activation of p34^cdc2in apoptosis was confirmed by immunohistochemical analyses using a specific antibody raised against Ser⁵⁵-phosphorylated vimentin which is specifically phosphorylated by p34^cdc2during M phase. Furthermore, phosphorylation of histones H1 and H3, which is associated with mitotic chromosome condensation, did not occur in the apoptotic cells. These results indicate that the two phenomena, staurosporine-induced apoptosis and premature mitosis, are different in their requirement for p34^cdc2kinase activation and histone phosphorylation.     

Okadaic acid, a potent inhibitor of type 1 and type 2A protein phosphatases, activates cdc2/H1 kinase and transiently induces a premature mitosis-like state in BHK21 cells.

When BHK21 cells synchronized in early S phase were exposed to okadaic acid (OA), an inhibitor of protein phosphatases 1 and 2A, mitosis specific events such as premature chromosome condensation, the production of MPM-2 antigens, dispersion of nuclear lamins and the appearance of mitotic asters were induced, and then disappeared upon further incubation. These mitosis specific events occurred even in the presence of cycloheximide. Within 1 h of exposure to OA, cdc2/histone H1 kinase activity rose 10-fold compared with untreated controls, but returned to the control level upon further incubation. Using antibodies against either p34cdc2 or cyclin B it was found that p34cdc2 complexed with cyclin B was dephosphorylated after OA treatment concomitant with the activation of cdc2 kinase, and that cyclin B was subsequently degraded concomitant with a decrease in cdc2 kinase activity, as in normal mitosis. In contrast, when cells in G1 phase were treated with OA no increase in cdc2 kinase activity was observed. Moreover when cells in pseudo-metaphase induced by nocodazole were treated with OA, cdc2 kinase was inactivated. These results suggest that OA sensitive protein phosphatases control both the activation and inactivation of the p34cdc2 kinase.     

Timeless links replication termination to mitotic kinase activation

The mechanisms that coordinate the termination of DNA replication with progression through mitosis are not completely understood. The human Timeless protein (Tim) associates with S phase replication checkpoint proteins Claspin and Tipin, and plays an important role in maintaining replication fork stability at physical barriers, like centromeres, telomeres and ribosomal DNA repeats, as well as at termination sites. We show here that human Tim can be isolated in a complex with mitotic entry kinases CDK1, Auroras A and B, and Polo-like kinase (Plk1). Plk1 bound Tim directly and colocalized with Tim at a subset of mitotic structures in M phase. Tim depletion caused multiple mitotic defects, including the loss of sister-chromatid cohesion, loss of mitotic spindle architecture, and a failure to exit mitosis. Tim depletion caused a delay in mitotic kinase activity in vivo and in vitro, as well as a reduction in global histone H3 S10 phosphorylation during G2/M phase. Tim was also required for the recruitment of Plk1 to centromeric DNA and formation of catenated DNA structures at human centromere alpha satellite repeats. Taken together, these findings suggest that Tim coordinates mitotic kinase activation with termination of DNA replication.     

Depletion of Chk1 leads to premature activation of Cdc2-cyclin B and mitotic catastrophe

Mitotic catastrophe occurs as a result of the uncoupling of the onset of mitosis from the completion of DNA replication, but precisely how the ensuing lethality is regulated or what signals are involved is largely unknown. We demonstrate here the essential role of the ATM/ATR-p53 pathway in mitotic catastrophe from premature mitosis. Chk1 deficiency resulted in a premature onset of mitosis because of abnormal activation of cyclin B-Cdc2 and led to the activation of caspases 3 and 9 triggered by cytoplasmic release of cytochrome c. This deficiency was associated with foci formation by the phosphorylated histone, H2AX (gammaH2AX), specifically at S phase. Ectopic expression of Cdc2AF, a mutant that cannot be phosphorylated at inhibitory sites, also induced premature mitosis and foci formation by gammaH2AX at S phase in both embryonic stem cells and HCT116 cells. Depletion of ATM and ATR protected against cell death from premature mitosis. p53-deficient cells were highly resistant to lethality from premature mitosis as well. Our results therefore suggest that ATM/ATR-p53 is required for mitotic catastrophe that eliminates cells escaping Chk1-dependent mitotic regulation. Loss of this function might be important in mammalian tumorigenesis.     

Haspin inhibition delays cell cycle progression through interphase in cancer cells

Haspin (Haploid Germ Cell-Specific Nuclear Protein Kinase) is a serine/threonine kinase pertinent to normal mitosis progression and mitotic phosphorylation of histone H3 at threonine 3 in mammalian cells. Different classes of small molecule inhibitors of haspin have been developed and utilized to investigate its mitotic functions. We report herein that applying haspin inhibitor CHR-6494 or 5-ITu at the G1/S boundary could delay mitotic entry in synchronized HeLa and U2OS cells, respectively, following an extended G2 or the S phase. Moreover, late application of haspin inhibitors at S/G2 boundary is sufficient to delay mitotic onset in both cell lines, thereby, indicating a direct effect of haspin on G2/M transition. A prolonged interphase duration is also observed with knockdown of haspin expression in synchronized and asynchronous cells. These results suggest that haspin can regulate cell cycle progression at multiple stages at both interphase and mitosis.     

A novel protein with similarities to Rb binding protein 2 compensates for loss of Chk1 function and affects histone modification in fission yeast

The conserved protein kinase Chk1 mediates cell cycle progression and consequently the ability of cells to survive when exposed to DNA damaging agents. Cells deficient in Chk1 are hypersensitive to such agents and enter mitosis in the presence of damaged DNA, whereas checkpoint-proficient cells delay mitotic entry to permit time for DNA repair. In a search for proteins that can improve the survival of Chk1-deficient cells exposed to DNA damage, we identified fission yeast Msc1, which is homologous to a mammalian protein that binds to the tumor suppressor Rb (RBP2). Msc1 and RBP2 each possess three PHD fingers, domains commonly found in proteins that influence the structure of chromatin. Msc1 is chromatin associated and coprecipitates a histone deacetylase activity, a property that requires the PHD fingers. Cells lacking Msc1 have a dramatically altered histone acetylation pattern, exhibit a 20-fold increase in global acetylation of histone H3 tails, and are readily killed by trichostatin A, an inhibitor of histone deacetylases. We postulate that Msc1 plays an important role in regulating chromatin structure and that this function modulates the cellular response to DNA damage.     

The topoisomerase II inhibitor VM-26 induces marked changes in histone H1 kinase activity, histones H1 and H3 phosphorylation, and chromosome condensation in G2 phase and mitotic BHK cells

We have examined the effects of topoisomerase inhibitors on the phosphorylation of histones in chromatin during the G2 and the M phases of the cell cycle. Throughout the G2 phase of BHK cells, addition of the topoisomerase II inhibitor VM-26 prevented histone H1 phosphorylation, accompanied by the inhibition of intracellular histone H1 kinase activity. However, VM-26 had no inhibitory effect on the activity of the kinase in vitro, suggesting an indirect influence on histone H1 kinase activity. Entry into mitosis was also prevented, as monitored by the absence of nuclear lamina depolymerization, chromosome condensation, and histone H3 phosphorylation. In contrast, the topoisomerase I inhibitor, camptothecin, inhibited histone H1 phosphorylation and entry into mitosis only when applied at early G2. In cells that were arrested in mitosis, VM-26 induced dephosphorylation of histones H1 and H3, DNA breaks, and partial chromosome decondensation. These changes in chromatin parameters probably reverse the process of chromosome condensation, unfolding condensed regions to permit the repair of strand breaks in the DNA that were induced by VM- 26. The involvement of growth-associated histone H1 kinase in these processes raises the possibility that the cell detects breaks in the DNA through their effects on the state of DNA supercoiling in constrained domains or loops. It would appear that histone H1 kinase and topoisomerase II work coordinately in both chromosome condensation and decondensation, and that this process participates in the VM-26- induced G2 arrest of the cell.     

Phosphorylation-induced rearrangement of the histone H3 NH2-terminal domain during mitotic chromosome condensation

The NH2-terminal domain (N-tail) of histone H3 has been implicated in chromatin compaction and its phosphorylation at Ser10 is tightly correlated with mitotic chromosome condensation. We have developed one mAb that specifically recognizes histone H3 N-tails phosphorylated at Ser10 (H3P Ab) and another that recognizes phosphorylated and unphosphorylated H3 N-tails equally well (H3 Ab). Immunocytochemistry with the H3P Ab shows that Ser10 phosphorylation begins in early prophase, peaks before metaphase, and decreases during anaphase and telophase. Unexpectedly, the H3 Ab shows stronger immunofluorescence in mitosis than interphase, indicating that the H3 N-tail is more accessible in condensed mitotic chromatin than in decondensed interphase chromatin. In vivo ultraviolet laser cross-linking indicates that the H3 N-tail is bound to DNA in interphase cells and that binding is reduced in mitotic cells. Treatment of mitotic cells with the protein kinase inhibitor staurosporine causes histone H3 dephosphorylation and chromosome decondensation. It also decreases the accessibility of the H3 N-tail to H3 Ab and increases the binding of the N-tail to DNA. These results indicate that a phosphorylation-dependent weakening of the association between the H3 N-tail and DNA plays a role in mitotic chromosome condensation.     

Role of inhibitory CDC2 phosphorylation in radiation-induced G2 arrest in human cells

The activity of the mitosis-promoting kinase CDC2-cyclin B is normally suppressed in S phase and G2 by inhibitory phosphorylation at Thr14 and Tyr15. This work explores the possibility that these phosphorylations are responsible for the G2 arrest that occurs in human cells after DNA damage. HeLa cell lines were established in which CDC2AF, a mutant that cannot be phosphorylated at Thr14 and Tyr15, was expressed from a tetracycline-repressible promoter. Expression of CDC2AF did not induce mitotic events in cells arrested at the beginning of S phase with DNA synthesis inhibitors, but induced low levels of premature chromatin condensation in cells progressing through S phase and G2. Expression of CDC2AF greatly reduced the G2 delay that resulted when cells were X- irradiated in S phase. However, a significant G2 delay was still observed and was accompanied by high CDC2-associated kinase activity. Expression of wild-type CDC2, or the related kinase CDK2AF, had no effect on the radiation-induced delay. Thus, inhibitory phosphorylation of CDC2, as well as additional undefined mechanisms, delay mitosis after DNA damage.     

Mutations of p34cdc2 phosphorylation sites induce premature mitotic events in HeLa cells: evidence for a double block to p34cdc2 kinase activation in vertebrates.

In vertebrates, entry into mitosis is accompanied by dephosphorylation of p34cdc2 kinase on threonine 14 (Thr14) and tyrosine 15 (Tyr15). To examine the role of these residues in controlling p34cdc2 kinase activation, and hence the onset of mitosis, we replaced Thr14 and/or Tyr15 by non-phosphorylatable residues and transfected wild-type and mutant chicken p34cdc2 cDNAs into HeLa cells. While expression of wild-type p34cdc2 did not interfere with normal cell cycle progression, p34cdc2 carrying mutations at both Thr14 and Tyr15 displayed increased histone H1 kinase activity and rapidly induced premature mitotic events, including chromosome condensation and lamina disassembly. No phenotype was observed in response to mutation of only Thr14, and although single-site mutation at Tyr15 did induce premature mitotic events, effects were partial and their onset was delayed. These results identify both Thr14 and Tyr15 as sites of negative regulation of vertebrate p34cdc2 kinase, and they suggest that dephosphorylation of p34cdc2 represents the rate-limiting step controlling entry of vertebrate cells into mitosis.     

Cell cycle-specific activity of type I and type II cyclic adenosine 3':5'-monophosphate-dependent protein kinases in Chinese hamster ovary cells.

Types I and II cyclic adenosine 3':5'-monophosphate (cAMP)-dependent protein kinases have been studied during the cell cycle of Chinese hamster ovary cells. Chinese hamster ovary cells were synchronized by selective detachment of mitotic cells from monolayer cultures. Protein kinases were separated by DEAE-cellulose chromatography and were similar to the types of cAMP-dependent protein kinases studied in skeletal muscle and in heart extracts. The total amount of protein kinases activity per cell was substantial, both in mitosis and at the G1/S boundary. During mitosis, the relatively high activity of protein kinase was due to a predominance of type I protein kinase. During early G1, the activity of type I protein kinase decreased and there was little detectable type II activity. A rapid increase in the activity of type II was evident at the G1/S boundary. The administration of puromycin (50 mug/ml) from 1 to 5 hours after selective detachment of mitotic cells abolished the activity of type II cAMP-dependent protein kinase seen at the G1/S border, but had no observable effect on the activity of type I protein kinase. The data presented demonstrate cell cycle-specific activity patterns of type I and type II protein kinase Type I protein kinase activity is high in mitosis and is constant throughout the cell cycle. Increased type II protein kinase activity seems to be related to the initiation of DNA synthesis in S phase. The data suggest a translational control of type II cAMP-dependent protein kinase activity.     

DNA replication stress in CHK1-depleted tumour cells triggers premature (S-phase) mitosis through inappropriate activation of Aurora kinase B

The disruption of DNA replication in cells triggers checkpoint responses that slow-down S-phase progression and protect replication fork integrity. These checkpoints are also determinants of cell fate and can help maintain cell viability or trigger cell death pathways. CHK1 has a pivotal role in such S-phase responses. It helps maintain fork integrity during replication stress and protects cells from several catastrophic fates including premature mitosis, premature chromosome condensation and apoptosis. Here we investigated the role of CHK1 in protecting cancer cells from premature mitosis and apoptosis. We show that premature mitosis (characterized by the induction of histone H3 phosphorylation, aberrant chromatin condensation, and persistent RPA foci in arrested S-phase cells) is induced in p53-deficient tumour cells depleted of CHK1 when DNA synthesis is disrupted. These events are accompanied by an activation of Aurora kinase B in S-phase cells that is essential for histone H3 Ser10 phosphorylation. Histone H3 phosphorylation precedes the induction of apoptosis in p53−/− tumour cell lines but does not appear to be required for this fate as an Aurora kinase inhibitor suppresses phosphorylation of both Aurora B and histone H3 but has little effect on cell death. In contrast, only a small fraction of p53+/+ tumour cells shows this premature mitotic response, although they undergo a more rapid and robust apoptotic response. Taken together, our results suggest a novel role for CHK1 in the control of Aurora B activation during DNA replication stress and support the idea that premature mitosis is a distinct cell fate triggered by the disruption of DNA replication when CHK1 function is suppressed.     

Microtubule dependency of p34cdc2 inactivation and mitotic exit in mammalian cells

The protein kinase inhibitor 2-aminopurine induces checkpoint override and mitotic exit in BHK cells which have been arrested in mitosis by inhibitors of microtubule function (Andreassen, P. R., and R. L. Margolis. 1991. J. Cell Sci. 100:299-310). Mitotic exit is monitored by loss of MPM-2 antigen, by the reformation of nuclei, and by the extinction of p34cdc2-dependent H1 kinase activity. 2-AP-induced inactivation of p34cdc2 and mitotic exit depend on the assembly state of microtubules. During mitotic arrest generated by the microtubule assembly inhibitor nocodazole, the rate of mitotic exit induced by 2-AP decreases proportionally with increasing nocodazole concentrations. At nocodazole concentrations of 0.12 microgram/ml or greater, 2-AP induces no apparent exit through 75 min of treatment. In contrast, 2-AP brings about a rapid exit (t1/2 = 20 min) from mitotic arrest by taxol, a drug which causes inappropriate overassembly of microtubules. In control mitotic cells, p34cdc2 localizes to kinetochores, centrosomes, and spindle microtubules. We find that efficient exit from mitosis occurs under conditions where p34cdc2 remains associated with centrosomal microtubules, suggesting it must be present on these microtubules in order to be inactivated. Mitotic slippage, the natural reentry of cells into G1 during prolonged mitotic block, is also microtubule dependent. At high nocodazole concentrations slippage is prevented and mitotic arrest approaches 100%. We conclude that essential components of the machinery for exit from mitosis are present on the mitotic spindle, and that normal mitotic exit thereby may be regulated by the microtubule assembly state.     

Chromatin-bound histone 1 kinase activity in synchronized HeLa S3 cells

The chromatin-bound H1 kinase activity of HeLa S3 cells that had been synchronized with 2.7 mM thymidine for 24 h has been followed during their progression into mitosis. They were arrested at this stage of the cell cycle by adding 0.13 microM nocodazole 8 h after the removal of thymidine. The kinase was partially purified by extracting chromatin proteins with 0.4 M NaCl and fractionation with ammonium sulfate (17.5-35%), a procedure in which a significant amount of in vivo histone 1 phosphorylating activity was retained. H1 kinase activity increased as the cells entered mitosis, rising to a maximum level sevenfold higher than interphase as the mitotic index reached about 50%. A rapid decrease in activity followed this maximum approximately 2 h after cells started to accumulate in mitosis. At this time, the mitotic index was still increasing, although at a lower rate than during the increase of the kinase activity. Other protein kinase activities measured by using core histones, casein, and protamine as substrates remained fairly constant at a comparatively low level. HeLa H1 kinase activity was further distinguished from several known protein kinase activities by the lack of stimulation or inhibition with known modulators of protein phosphorylating activities.     

Premature initiation of mitosis in yeast lacking RCC1 or an interacting GTPase.

A fission yeast mutant is described in which the onset of mitosis is uncoupled from the completion of DNA replication. pim1 (premature initiation of mitosis) cells can undergo mitotic chromosome condensation and mitotic spindle formation without completion of S phase and without the cdc25 mitotic inducer. The M phase kinase is required for pim1-induced mitosis and becomes activated. pim1 encodes a homolog of the human RCC1 nuclear protein. pim1 mutants are fully rescued by overexpression of spi1, a newly identified essential gene whose predicted product shares 81% identity with human TC4. spi1 and TC4 define a new subclass within the "ras-like" GTPase superfamily that is structurally distinct from the ras, rho, or sec4 families. Diploid yeast that carry one wild-type and one disrupted copy of spi1 have multiple satellite nuclei, and mitotic haploidization occurs at very high frequency. spi1 appears to interact with pim1 in the maintenance of a coordinated cell cycle.     

Effect of inhibitors serine/threonine protein kinases and protein phosphatases on mitosis progression of synchronized tobacco by-2 cells

In order to investigate the role of various serine/ threonine protein kinases and protein phosphatases in the regulation of mitosis progression in plant cells the influence of cyclin-dependent (olomoucine) and Ca2+ -calmodulin-dependent (W7) protein kinases inhibitors, as well as protein kinase C inhibitors (H7 and staurosporine) and protein phosphatases inhibitor (okadaic acid) on mitosis progression in synchronized tobacco BY-2 cells has been studied. It was found that BY-2 culture treatment with inhibitors of cyclin dependent protein kinases and protein kinase C causes prophase delay, reduces the mitotic index and displaces of mitotic peak as compare with control cells. Inhibition of Ca2+ -calmodulin dependent protein kinases enhances the cell entry into prophase and delays their exit from mitosis. Meanwhile inhibition of serine/threonine protein phosphatases insignificantly enhances of synchronized BY-2 cells entering into all phases of mitosis.     

Study of pp60v-src protein kinase activity in synchronized chicken embryo fibroblasts infected with Rous sarcoma virus

Chicken embryo fibroblast (CEF) cultures, synchronized by the addition of serum to stationary cells, were exposed to Schmidt-Ruppin strain of Rous Sarcoma Virus (SR-RSV) and the appearance of pp60v-src protein kinase activity was examined through the cell cycle. In cells infected either at the beginning or at the end of G1, the onset of pp60v-src protein kinase activity was coincidental, closely following mitosis, with a delay between the infection of cells with SR-RSV and the appearance of protein kinase activity of about 20 and 16 h, respectively. In cells infected during the S phase this delay was 16 h, as observed for late G1 cells. These experiments show that the activity of pp60v-src protein kinase, which cannot be detected before the first mitosis following infection does not depend on G1. The aphidicolin prevented protein kinase activity if added before or at the beginning of S phase, but not if added later, which is presumably related to the inhibition of S phase, required for provirus integration. The use of colcemid, which suppresses cell division, did not inhibit but delayed the appearance of protein kinase activity. These results show that the synthesis of an active oncogene product, such as pp60v-src protein kinase, depends on both S phase and mitosis.     

Aurora kinase is required for chromosome segregation in tobacco BY-2 cells

Post-translational modifications of core histone tails play crucial roles in chromatin structure and function. Although phosphorylation of Ser10 and Ser28 (H3S10ph and H3S28ph) of histone H3 is ubiquitous among eukaryotes, the phosphorylation mechanism during the cell cycle remains unclear. In the present study, H3S10ph and H3S28ph in tobacco BY-2 cells were observed in the pericentromeric regions during mitosis. Moreover, the Aurora kinase inhibitor Hesperadin inhibited the kinase activity of Arabidopsis thaliana Aurora kinase 3 (AtAUR3) in phosphorylating both Ser10 and Ser28 of histone H3 in vitro. Consistently, Hesperadin inhibited both H3S10ph and H3S28ph during mitosis in BY-2 cells. These results indicate that plant Aurora kinases phosphorylate not only Ser10, but also Ser28 of histone H3 in vivo. Hesperadin treatment increased the ratio of metaphase cells, while the ratio of anaphase/telophase cells decreased, although the mitotic index was not affected in Hesperadin-treated cells. These results suggest that Hesperadin induces delayed transition from metaphase to anaphase, and early exit from mitosis after chromosome segregation. In addition, micronuclei were observed frequently and lagging chromosomes, caused by the delay and failure of sister chromatid separation, were observed at anaphase and telophase in Hesperadin-treated BY-2 cells. The data obtained here suggest that plant Aurora kinases and H3S10ph/H3S28ph may have a role in chromosome segregation and metaphase/anaphase transition.     

Chromosome condensation induced by fostriecin does not require p34cdc2 kinase activity and histone H1 hyperphosphorylation, but is associated with enhanced histone H2A and H3 phosphorylation.

Chromosome condensation at mitosis correlates with the activation of p34cdc2 kinase, the hyperphosphorylation of histone H1 and the phosphorylation of histone H3. Chromosome condensation can also be induced by treating interphase cells with the protein phosphatase 1 and 2A inhibitors okadaic acid and fostriecin. Mouse mammary tumour FT210 cells grow normally at 32 degrees C, but at 39 degrees C they lose p34cdc2 kinase activity and arrest in G2 because of a temperature-sensitive lesion in the cdc2 gene. The treatment of these G2-arrested FT210 cells with fostriecin or okadaic acid resulted in full chromosome condensation in the absence of p34cdc2 kinase activity or histone H1 hyperphosphorylation. However, phosphorylation of histones H2A and H3 was strongly stimulated, partly through inhibition of histone H2A and H3 phosphatases, and cyclins A and B were degraded. The cells were unable to complete mitosis and divide. In the presence of the protein kinase inhibitor starosporine, the addition of fostriecin did not induce histone phosphorylation and chromosome condensation. The results show that chromosome condensation can take place without either the histone H1 hyperphosphorylation or the p34cdc2 kinase activity normally associated with mitosis, although it requires a staurosporine-sensitive protein kinase activity. The results further suggest that protein phosphatases 1 and 2A may be important in regulating chromosome condensation by restricting the level of histone phosphorylation during interphase, thereby preventing premature chromosome condensation.