Delay In Mitosis Research Articles

The Rho GTP exchange factor Lfc promotes spindle assembly in early mitosis

Rho GTPases regulate reorganization of actin and microtubule cytoskeletal structures during both interphase and mitosis. The timing and subcellular compartment in which Rho GTPases are activated is controlled by the large family of Rho GTP exchange factors (RhoGEFs). Here, we show that the microtubule-associated RhoGEF Lfc is required for the formation of the mitotic spindle during prophase/prometaphase. The inability of cells to assemble a functioning spindle after Lfc inhibition resulted in a delay in mitosis and an accumulation of prometaphase cells. Inhibition of Lfc's primary target Rho GTPase during prophase/prometaphase, or expression of a catalytically inactive mutant of Lfc, also prevented normal spindle assembly and resulted in delays in mitotic progression. Coinjection of constitutively active Rho GTPase rescued the spindle defects caused by Lfc inhibition, suggesting the requirement of RhoGTP in regulating spindle assembly. Lastly, we implicate mDia1 as an important effector of Lfc signaling. These findings demonstrate a role for Lfc, Rho, and mDia1 during mitosis.

The Drosophila Grp/Chk1 DNA Damage Checkpoint Controls Entry into Anaphase

It is well established that DNA damage induces checkpoint-mediated interphase arrest in higher eukaryotes, but recent studies demonstrate that DNA damage delays entry into anaphase as well. Damaged DNA in syncytial and gastrulating Drosophila embryos delays the metaphase/anaphase transition . In human cultured cells, DNA damage also induces a delay in mitosis . However, the mechanism by which DNA damage delays the anaphase onset is controversial. Some studies implicate a DNA damage checkpoint , whereas other studies invoke a spindle checkpoint . To resolve this issue, we compared the effects of random DNA breaks induced by X-irradiation to site-specific I-CreI endonuclease-induced chromosome breaks on cell-cycle progression in wild-type and checkpoint-defective Drosophila neuroblasts. We found that both the BubR1 spindle checkpoint pathway and the Grp/Chk1 DNA damage checkpoint pathway are involved in delaying the metaphase/anaphase transition after extensive X-irradiation-induced DNA damage, whereas Grp/Chk1, but not BubR1, is required to delay anaphase onset in the presence of I-CreI-induced double-strand breaks. On the basis of these results, we propose that DNA damage in nonkinetochore regions produces a Grp/Chk1 DNA-damage-checkpoint-mediated delay in the metaphase/anaphase transition.

The APC Subunit Doc1 Promotes Recognition of the Substrate Destruction Box

Accurate chromosome segregation during mitosis requires the coordinated destruction of the mitotic regulators securin and cyclins. The anaphase-promoting complex (APC) is a multisubunit ubiquitin-protein ligase that catalyzes the polyubiquitination of these and other proteins and thereby promotes their destruction. How the APC recognizes its substrates is not well understood. In mitosis, the APC activator Cdc20 binds to the APC and is thought to recruit substrates by interacting with a conserved target protein motif called the destruction box. A related protein, called Cdh1, performs a similar function during G1. Recent evidence, however, suggests that the core APC subunit Doc1 also contributes to substrate recognition. To better understand the mechanism by which Doc1 promotes substrate binding to the APC, we generated a series of point mutations in Doc1 and analyzed their effects on the processivity of substrate ubiquitination. Mutations that reduce Doc1 function fall into two classes that define spatially and functionally distinct regions of the protein. One region, which includes the carboxy terminus, anchors Doc1 to the APC but does not influence substrate recognition. The other region, located on the opposite face of Doc1, is required for Doc1 to enhance substrate binding to the APC. Importantly, stimulation of binding by Doc1 also requires that the substrate contain an intact destruction box. Cells carrying DOC1 mutations that eliminate substrate recognition delay in mitosis with high levels of APC substrates. Doc1 contributes to recognition of the substrate destruction box by the APC. This function of Doc1 is necessary for efficient substrate proteolysis in vivo.

Disruption of HOX-PBX Interaction; a Potential Therapeutic Target in Leukemia.

Most cases of acute myeloid leukemia (AML) are closely associated with gene rearrangements. Appraisal of these translocations and analysis of mouse models of leukemia has revealed that several members of the homeodomain containing family of transcription factors are implicated in the pathogenesis of leukemia. Overexpression of HOXA9 in murine models leads to the development of AML. This study focuses on the role of a subset of the HOX genes and their potential as a target for therapeutic intervention. We have designed a synthetic peptide, HXP4, that disrupts the interaction between HOX and PBX leading to growth inhibition of leukemic cells. An in vitro HOX-induced AML model of leukemia was utilised to determine the efficacy of HXP4 as a therapeutic agent. Using this immortalised cell line overexpressing HOXA9 (imHOXA9), we tested the efficacy of HXP4 in vitro. Cells were treated with HXP4 for four days and analysed. All results are expressed relative to untreated control cells. Following a 60μM dose of HXP4, no viable cells were detected as determined by trypan blue staining, suggesting that HXP4 was cytotoxic. Following treatment with a lower dose of 6μM HXP4, and re-suspension in drug-free medium for a further 6 days, cell regrowth was observed, suggesting a cytostatic effect. RT-PCR was performed to identify potential downstream targets of HXP4. Qualitative analysis showed other HOX family members to be unaffected by treatment with either HXP4 dose. A more detailed study was performed using quantitative RT-PCR with imHOXA9. Cells were treated with either 60μM HXP4, 3μM etoposide, or a combination of the two agents (H+Et) and harvested after 1, 2, and 4 hours. In general, no significant change in gene expression was observed in other HOX family members. However, HOXA1 was upregulated 3-fold when treated with HXP4, and HOXA2 was downregulated 2-fold in HXP4 and H+Et treated cells. The reasons for this are as yet unclear. HXP4 also downregulated N-RAS 3.5-fold at two hours. However, complete loss of N-RAS expression following H+Et treatment suggests that HXP4 may be more effective in combination with etoposide. CDC25 expression was slightly downregulated in HXP4-treated cells. The normal function of CDC25 is to activate CDC2 kinase in the nucleus, however in the absence of CDC25, CDC2 remains inactive leading to a delay in mitosis, supporting the proposed cytostatic mode of HXP4 action. For reasons as yet unclear, CD34 expression was upregulated 4-fold and 6-fold in HXP4 and H+Et treated cells respectively. These preliminary results suggest that HXP4 is a cytostatic agent at relatively low concentrations, with a reversible antiproliferative effect. Downstream genes regulated by disrupting the HOX-PBX interaction with HXP4 have been identified by RT-PCR, but microarray analysis will provide a more comprehensive screen for target genes. In vivo experiments are currently in progress. In conclusion blocking the interaction between HOX and PBX may represent a therapeutic strategy in leukemia treatment.

The dietary isothiocyanate sulforaphane targets pathways of apoptosis, cell cycle arrest, and oxidative stress in human pancreatic cancer cells and inhibits tumor growth in severe combined immunodeficient mice

Anticancer effects of the dietary isothiocyanate sulforaphane were investigated in the human pancreatic cancer cell lines MIA PaCa-2 and PANC-1. Sulforaphane-treated cells accumulated in metaphase as determined by flow cytometry [4C DNA content, cyclin A(-), cyclin B1(+), and phospho-histone H3 (Ser(10))(+)]. In addition, treated cells showed nuclear apoptotic morphology that coincided with an activation of caspase-8, loss of mitochondrial membrane potential, and loss of plasma membrane integrity. The initial detection of caspase-3 cleavage occurring in G(2)-M arrest was independent of a change in phospho-cdc2 (Tyr(15)) protein; consequently, sulforaphane treatment combined with UCN-01 had no significant impact on cellular toxicity. Incubations at higher sulforaphane doses (>10 micromol/L) resulted in cleavage of caspase-3 in the G(1) subpopulation, suggesting that the induction of apoptosis and the sulforaphane-induced mitosis delay at the lower dose are independently regulated. Cellular toxicity in MIA PaCa-2, and to a greater extent in PANC-1, was positively correlated with a decrease in cellular glutathione levels, whereas sustained increases in glutathione observed in MIA PaCa-2 cells or the simultaneous incubation with N-acetyl-L-cysteine in PANC-1 cells were associated with resistance to sulforaphane-induced apoptosis. Daily sulforaphane i.p. injections (375 micromol/kg/d for 3 weeks) in severe combined immunodeficient mice with PANC-1 s.c. tumors resulted in a decrease of mean tumor volume by 40% compared with vehicle-treated controls. Our findings suggest that, in addition to the known effects on cancer prevention, sulforaphane may have activity in established pancreatic cancer.

The clinical significance of Cyclin B1 and Wee1 expression innon-small-cell lung cancer

Cyclin B1 has an important role in the G2-M phase transition of the cell cycle. Wee1 delays mitosis by suppressing the activity of the Cyclin B1/cdc2 complex. The objective of the present study was to elucidate the clinicopathological and prognostic significance of Cyclin B1 and Wee1 expression in non-small-cell lung cancer (NSCLC). An immunohistochemical assessment of Cyclin B1 and Wee1 expression was performed in 79 patients with NSCLC. The expression of Cyclin B1 was correlated with differentiation (P = 0.0423) and vascular invasion (P = 0.001). Patients with overexpression of Cyclin B1 had higher mean values for both the Ki-67 proliferative index (Ki-Index) (P <0.0001) and proliferating cell nuclear antigen labeling index (PCNA-LI) (P <0.0001), and a poorer prognosis (P = 0.0068). Patients lacking expression of Wee1 had a higher recurrence rate (P = 0.0084) and a poorer prognosis (P = 0.0457), and tended to have higher Ki-Index and PCNA-LI values. Multivariate analysis suggested that both Cyclin B1 (P = 0.0244) and Wee1 (P = 0.0444) expression were significant prognostic factors. These findings suggest that Cyclin B1 expression could be a significant prognostic parameter in NSCLC. The loss of Wee1 expression may have a potential role in promoting tumor progression and may be a significant prognostic indicator in NSCLC.

Control of mitotic entry after DNA damage in Drosophila.

In the presence of DNA damage, cells delay the entry into mitosis, presumably to allow time for repair. Methods to detect the delay of mitosis in a multicellular model organism, Drosophila melanogaster, are described here. These include the collection of embryos and larvae, irradiation with x-rays to damage DNA, and fixing and staining of tissues with an antibody to phosphorylated histone H3 to measure the mitotic index. These methods should be useful in identifying potential mutants that are unable to regulate mitosis following DNA damage.

Gamma-tubulin plays an essential role in the coordination of mitotic events.

Recent data from multiple organisms indicate that gamma-tubulin has essential, but incompletely defined, functions in addition to nucleating microtubule assembly. To investigate these functions, we examined the phenotype of mipAD159, a cold-sensitive allele of the gamma-tubulin gene of Aspergillus nidulans. Immunofluorescence microscopy of synchronized material revealed that at a restrictive temperature mipAD159 does not inhibit mitotic spindle formation. Anaphase A was inhibited in many nuclei, however, and after a slight delay in mitosis (approximately 6% of the cell cycle period), most nuclei reentered interphase without dividing. In vivo observations of chromosomes at a restrictive temperature revealed that mipAD159 caused a failure of the coordination of late mitotic events (anaphase A, anaphase B, and chromosomal disjunction) and nuclei reentered interphase quickly even though mitosis was not completed successfully. Time-lapse microscopy also revealed that transient mitotic spindle abnormalities, in particular bent spindles, were more prevalent in mipAD159 strains than in controls. In experiments in which microtubules were depolymerized with benomyl, mipAD159 nuclei exited mitosis significantly more quickly (as judged by chromosomal condensation) than nuclei in a control strain. These data reveal that gamma-tubulin has an essential role in the coordination of late mitotic events, and a microtubule-independent function in mitotic checkpoint control.

DNA Damage during Mitosis in Human Cells Delays the Metaphase/Anaphase Transition via the Spindle-Assembly Checkpoint

Background: DNA damage during mitosis triggers an ATM kinase-mediated cell cycle checkpoint pathway in yeast and fly embryos that delays progression through division. Recent data suggest that this is also true for mammals. Here we used laser microsurgery and inhibitors of topoisomerase IIα to break DNA in various mammalian cells after they became committed to mitosis. We then followed the fate of these cells and emphasized the timing of mitotic progression, spindle structure, and chromosome behavior.Results: We find that DNA breaks generated during late prophase do not impede entry into prometaphase. If the damage is minor, cells complete mitosis on time. However, more significant damage substantially delays exit from mitosis in many cell types. In human (HeLa, CFPAC-1, and hTERT-RPE) cells, this delay occurs during metaphase, after the formation of a bipolar spindle and the destruction of cyclin A, and it is not dependent on a functional p53 pathway. Pretreating cells with ATM kinase inhibitors does not abrogate the metaphase delay due to chromosome damage. Immunofluorescence studies reveal that cells blocked in metaphase by chromosome damage contain one or more Mad2-positive kinetochores, and the block is rapidly overridden when the cells are microinjected with a dominant-negative construct of Mad2 (Mad2ΔC).Conclusions: We conclude that the delay in mitosis induced by DNA damage is not due to an ATM-mediated DNA damage checkpoint pathway. Rather, the damage leads to defects in kinetochore attachment and function that, in turn, maintain the intrinsic Mad-2-based spindle assembly checkpoint.

Preventive medicine: biomonitoring and chemoprevention

The aim of chemoprevention is to delay or prevent the development of pathological conditions, or to correct abnormal regulatory mechanisms and in some cases even reverse the process. For this intervention to succeed, biomarkers must be found that characterize impaired health status in a phase when impairment is still reversible. Genotoxicological parameters may function as biomarkers of this kind, such as inhibition or delay of mitosis, inhibition of apoptosis, increase in the number of chromosomal aberrations, decrease in the capacity of DNA repairing enzymes, or parameters characterizing immunological status (eg. decreased NK activity). With the help of early signs, impending negative changes or illness (which could not be prevented without effective chemoprevention) can be predicted in apparently healthy individuals. Thus, the first and most important step in chemoprevention is risk characterization. Risk characterization is a complex concept, which includes risk analysis, risk assessment and risk management. Chemoprevention is a part of the latter process. Biomarkers, which can be studied mainly by up-to-date molecular biological methods, are used in discovering risk factors and also in the assessment of caused biological effects. Besides avoiding risk factors, it is very important to strengthen the protective mechanisms, to promote the metabolism of toxic substances, and to repair damage (ward off denaturation of macromolecules caused by free oxygen radicals with antioxidants for example). Chemopreventive agents are therefore diverse in their targets. Most of them have antioxidant properties, such as plant-derived substances, like glycosides, flavonoids, various vitamins, carotinoids, and some trace elements such as selenium. Another group of chemopreventive agents inhibit cell proliferation, or induce programmed cell death (apoptosis). Another group influence terminal differentiation, or inhibit angiogenesis. Some chemopreventive agents affect the metabolism or detoxification of xenobiotics, or boost the functions of the immune system. In many cases these effects are mediated by the rate of methylation of DNA molecules.

Finding degradation at the checkpoint

![Graphic][1] Chfr can ubiquitinate itself. When the mitotic apparatus is disturbed by agents such as nocodazole, mitotic checkpoints delay mitosis to avert disaster. Defects in the anaphase-onset checkpoint are rarely seen in cancers, but defects in the G2/M (prophase) checkpoint are

Mutation in the beta-tubulin signature motif suppresses microtubule GTPase activity and dynamics, and slows mitosis.

We introduced a threonine-to-glycine point mutation at position 143 in the "tubulin signature motif" 140Gly-Gly-Gly-Thr-Gly-Ser-Gly146 of Saccharomyces cerevisiae beta-tubulin. In an electron diffraction model of the tubulin dimer, this sequence comes close to the phosphates of a guanine nucleotide bound in the beta-tubulin exchangeable E site. Both the GTP-binding affinity and the microtubule (MT)-dependent GTPase activity of tubulin isolated from haploid tub2-T143G mutant cells were reduced by at least 15-fold, compared to tubulin isolated from control wild-type cells. The growing and shortening dynamics of MTs assembled from alphabeta:Thr143Gly-mutated dimers were also strongly suppressed, compared to control MTs. The in vitro properties of the mutated MTs (slower growing and more stable) are consistent with the effects of the tub2-T143G mutation in haploid cells. The average length of MT spindles in large-budded mutant cells was only 3.7 +/- 0.2 microm, approximately half of the size of MT arrays in large-budded wild-type cells (average length = 7.1 +/- 0.4 microm), suggesting that there is a delay in mitosis in the mutant cells. There was also a higher proportion of large-budded cells with unsegregated nuclei in mutant cultures (30% versus 12% for wild-type cells), again suggesting such a delay. The results show that beta:Thr143 of the tubulin signature motif plays an important role in GTP binding and hydrolysis by the beta-tubulin E site and support the idea that tubulins belong to a family of proteins within the GTPase superfamily that are structurally distinct from the classic GTPases, such as EF-Tu and p21(ras). The data also suggest that MT dynamics are critical for MT function in yeast cells and that spindle MT assembly and disassembly could be coordinated with other cell-cycle events by regulating beta-tubulin GTPase activity.

Induction of a mitosis delay and cell lysis by high-level secretion of mouse alpha-amylase from Saccharomyces cerevisiae.

Some foreign proteins are produced in yeast in a cell cycle-dependent manner, but the cause of the cell cycle dependency is unknown. In this study, we found that Saccharomyces cerevisiae cells secreting high levels of mouse alpha-amylase have elongated buds and are delayed in cell cycle completion in mitosis. The delayed cell mitosis suggests that critical events during exit from mitosis might be disturbed. We found that the activities of PP2A (protein phosphatase 2A) and MPF (maturation-promoting factor) were reduced in alpha-amylase-oversecreting cells and that these cells showed a reduced level of assembly checkpoint protein Cdc55, compared to the accumulation in wild-type cells. MPF inactivation is due to inhibitory phosphorylation on Cdc28, as a cdc28 mutant which lacks an inhibitory phosphorylation site on Cdc28 prevents MPF inactivation and prevents the defective bud morphology induced by overproduction of alpha-amylase. Our data also suggest that high levels of alpha-amylase may downregulate PPH22, leading to cell lysis. In conclusion, overproduction of heterologous alpha-amylase in S. cerevisiae results in a negative regulation of PP2A, which causes mitotic delay and leads to cell lysis.

Activating the DNA damage checkpoint in a developmental context.

Studies in unicellular systems have established that DNA damage by irradiation invokes a checkpoint that acts to stall cell division. During metazoan development, the modulation of cell division by checkpoints must occur in the context of gastrulation, differential gene expression and changes in cell cycle regulation. To understand the effects of checkpoint activation in a developmental context, we examined the effect of X-rays on post-blastoderm embryos of Drosophila melanogaster. In Drosophila, DNA damage was previously found to delay anaphase chromosome separation during cleavage cycles that lack a G2 phase. In post-blastoderm cycles that included a G2 phase, we found that irradiation delayed the entry into mitosis. Gastrulation and the developmental program of string (Cdc25) gene expression, which normally regulates the timing of mitosis, occurred normally after irradiation. The radiation-induced delay of mitosis accompanied the exclusion of mitotic cyclins from the nucleus. Furthermore, a mutant form of the mitotic kinase Cdk1 that cannot be inhibited by phosphorylation drove a mitotic cyclin into the nucleus and overcame the delay of mitosis induced by irradiation. Developmental changes in the cell cycle, for example, the introduction of a G2 phase, dictate the response to checkpoint activation, for example, delaying mitosis instead of or in addition to delaying anaphase. This unprecedented finding suggests that different mechanisms are used at different points during metazoan development to stall cell division in response to checkpoint activation. The delay of mitosis in post-blastoderm embryos is due primarily to inhibitory phosphorylation of Cdk1, whereas nuclear exclusion of a cyclin-Cdk1 complex might play a secondary role. Delaying cell division has little effect on gastrulation and developmentally regulated string gene expression, supporting the view that development generally dictates cell proliferation and not vice versa.

Regulation of Mitotic Inhibitor Mik1 Helps to Enforce the DNA Damage Checkpoint

The protein kinase Chk1 enforces the DNA damage checkpoint. This checkpoint delays mitosis until damaged DNA is repaired. Chk1 regulates the activity and localization of Cdc25, the tyrosine phosphatase that activates the cdk Cdc2. Here we report that Mik1, a tyrosine kinase that inhibits Cdc2, is positively regulated by the DNA damage checkpoint. Mik1 is required for checkpoint response in strains that lack Cdc25. Long-term DNA damage checkpoint arrest fails in Deltamik1 cells. DNA damage increases Mik1 abundance in a Chk1-dependent manner. Ubiquitinated Mik1 accumulates in a proteasome mutant, which indicates that Mik1 normally has a short half-life. Thus, the DNA damage checkpoint might regulate Mik1 degradation. Mik1 protein and mRNA oscillate during the unperturbed cell cycle, with peak amounts detected around S phase. These data indicate that regulation of Mik1 abundance helps to couple mitotic onset to the completion of DNA replication and repair. Coordinated negative regulation of Cdc25 and positive regulation of Mik1 ensure the effective operation of the DNA damage checkpoint.

Fission yeast orb6, a ser/thr protein kinase related to mammalian rho kinase and myotonic dystrophy kinase, is required for maintenance of cell polarity and coordinates cell morphogenesis with the cell cycle.

The molecular mechanisms that coordinate cell morphogenesis with the cell cycle remain largely unknown. We have investigated this process in fission yeast where changes in polarized cell growth are coupled with cell cycle progression. The orb6 gene is required during interphase to maintain cell polarity and encodes a serine/threonine protein kinase, belonging to the myotonic dystrophy kinase/cot1/warts family. A decrease in Orb6 protein levels leads to loss of polarized cell shape and to mitotic advance, whereas an increase in Orb6 levels maintains polarized growth and delays mitosis by affecting the p34(cdc2) mitotic kinase. Thus the Orb6 protein kinase coordinates maintenance of cell polarity during interphase with the onset of mitosis. orb6 interacts genetically with orb2, which encodes the Pak1/Shk1 protein kinase, a component of the Ras1 and Cdc42-dependent signaling pathway. Our results suggest that Orb6 may act downstream of Pak1/Shk1, forming part of a pathway coordinating cell morphogenesis with progression through the cell cycle.

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.

Xe-p9, a Xenopus Suc1/Cks homolog, has multiple essential roles in cell cycle control.

The small Suc1/Cks protein is a ubiquitous subunit of Cdk/cyclin complexes, but its precise function has remained unclear. We have isolated a Xenopus homolog, Xe-p9, of the Suc1/Cks protein by virtue of its ability to rescue a fission yeast mutant that enters mitosis prematurely. To assess its functional role in cell cycle control, we have both overexpressed p9 in Xenopus egg extracts and immunodepleted the protein from these extracts. We found that addition of recombinant His6-p9 to egg extracts results in a pronounced delay of mitosis that can be attributed to an inhibition of the tyrosine dephosphorylation of the inactive Cdc2/cyclin B complex. In immunodepletion studies, we observed that the consequences of removing p9 from egg extracts depend on the stage of the cell cycle. Specifically, in the case of interphase extracts, the removal of p9 abolishes the entry into mitosis as a result of a failure in the activation of the Cdc2/cyclin B complex by tyrosine dephosphorylation. Furthermore, mitotic extracts lacking p9 fail to exit mitosis because of a defect in the destruction of cyclin B. Collectively, these results indicate that p9 has multiple essential roles in the cell cycle by governing the interaction of the Cdc2/cyclin B complex with both positive and negative regulators.

Cdc18+ regulates initiation of DNA replication in Schizosaccharomyces pombe.

In the fission yeast Schizosaccharomyces pombe the cdc18'+gene is required both for initiation of DNA replication and for coupling mitosis to the completion of S phase. Cells lacking Cdc18 fail to enter S phase but still undergo nuclear division. Expression of cdc18+ is sufficient to drive a G1-arrested cdc10ts mutant into the S phase of the cell cycle, indicating that cdc18+ represents a critical link between passage through START and the initiation of DNA replication. Here we show that Cdcl8 is a highly unstable protein that is expressed only once per cell cycle at the boundary between GI and S phase. De novo synthesis of Cdc18 is required before, but not after, the initiation of DNA replication, indicating that Cdc18 function is not necessary once the initiation event has occurred. Overproduction of the protein results in an accumulation of cells with DNA content of greater than 2C and delays mitosis, suggesting that Cdc18 is sufficient to cause reinitiation of DNA replication within a given cell cycle. Our data indicate that the synthesis of Cdc18 protein is a critical rate-limiting step in the initiation of DNA replication during each cell cycle. The extreme lability of the protein may contribute to the prevention of reinitiation.

Transforming growth factor β decreases the rate of proliferation of rat vascular smooth muscle cells by extending the G2 phase of the cell cycle and delays the rise in cyclic AMP before entry into M phase

Transforming growth factor beta 1 (TGF-beta 1) decreased the rate of proliferation of rat aortic vascular smooth muscle cells (VSMCs) stimulated with serum showing a maximal effect at > 5 ng/ml (200 pM). However, it did not reduce the proportion of cells which passed through S phase (> 90%) and entry into S phase was delayed by less than 3 h. The proportion of cells passing through M phase (> 90%) was also unaffected, but entry into mitosis was delayed by approx. 24 h. This increase in cell cycle time was therefore due mainly to an increase in the G2 to mitotic metaphase period. Addition of TGF-beta 1 late in G1 or late in S phase failed to delay the onset of mitosis, but the presence of TGF-beta 1 between 0 and 12 h after the addition of serum to quiescent cells was sufficient to cause the maximal delay in mitosis of approx. 24 h. The role of cyclic AMP in the mechanism of the TGF-beta 1 effects on the cell cycle was examined. Entry into mitosis was preceded by a transient 2-fold increase in cyclic AMP concentration and TGF-beta 1 delayed both this increase in cyclic AMP and entry into mitosis to the same extent. Addition of forskolin or 8-(4-chlorophenylthio)-cyclic AMP to cells 30 h after stimulation with serum completely reversed the increase in duration of G2 in the presence of TGF-beta 1, suggesting that the rise in cyclic AMP levels which precedes mitosis might trigger entry of the VSMCs into M phase. Addition of forskolin late in S phase (26 h after stimulation with serum) advanced the entry of the cells into M phase and they divided prematurely. This effect was unaffected by the addition of cycloheximide with the forskolin; however, the effect of forskolin on cell division was completely inhibited when cycloheximide was added late in G1. TGF-beta 1 prevented the loss of smooth-muscle-specific myosin heavy chain (SM-MHC), which occurs in primary VSMC cultures in the presence or absence of serum, and the cells proliferated while maintaining a differentiated phenotype. However, TGF-beta 1 did not cause re-differentiation of subcultured VSMCs which contained very low amounts of SM-MHC and the effect of TGF-beta 1 in extending the G2 phase of the cell cycle is exerted independently of its effect on differentiation.