BubR1 is required for the mitotic block induced by combretastatin-A4 and a novel cis-restricted ß-lactam analogue in human cancer cells

BubR1 is a well-defined guardian of the mitotic spindle, initiating mitotic arrest in response to the lack of tension and/or chromosome alignment across the mitotic plate. However, the role of BubR1 in combretastatin-induced cell death remains unknown. In this study, we describe the effects of combretastatin A-4 (CA-4) and a synthetic cis-restricted 3,4-diaryl-2-azetidinone (?-lactam) analogue (CA-432) on the modulation and phosphorylation of BubR1 in human cervical cancer-derived cells. We demonstrate that CA-4 and CA-432 depolymerise the microtubular network of human cervical carcinoma-derived cells. Both compounds induced the disassembly of the microtubules and the loss of microtubule tension led to the early phosphorylation of BubR1 and the late cleavage of BubR1. The phosphorylation of BubR1 correlated with the onset of G2M cell cycle arrest whilst the cleavage of BubR1 coincided with apoptosis induced by the combretastatins. The combretastatin-induced apoptosis and the BubR1 cleavage were caspase-dependent. In vitro enzyme digests demonstrated that combretastatin-activated BubR1 is a substrate for caspase-3. Gene silencing of BubR1 with small interfering RNA severely compromised combretastatin-induced G2M cell cycle arrest with a corresponding increase in the formation of polyploid cells in both cervical and breast cancer-derived cells. In summary, BubR1 is required to maintain the G2M arrest and limit the formation of polyploid cells in response to continued combretastatin exposure. Moreover, substitution of the ethylene bridge with 3,4-diaryl-2-azetidinone did not alter the tubulin depolymerising properties or the subsequent mitotic spindle checkpoint response to CA-4 in human cancer cells.     

Clostridium botulinum C2 Toxin Delays Entry into Mitosis and Activation of p34cdc2 Kinase and cdc25-C Phosphatase in HeLa cells

The Clostridium botulinum C2 toxin ADP-ribosylates monomeric actin, thereby inducing disassembly of actin filaments, alteration of focal adhesions, and rounding of cells. After treatment with C2 toxin, cells stop to proliferate but remain viable for about 2 days. In view of reported correlations between the structure of the actin cytoskeleton and cell cycle transition, the effects of C2 toxin on the G(2)/M phase transition of the cell division cycle were studied. Since C2 toxin delayed entry into mitosis in HeLa cells, those enzymes which control entry into mitosis, the cyclin-dependent protein kinase mitosis-promoting factor (MPF) and the phosphatase cdc25-C were examined after treatment of synchronized cells with C2 toxin. MPF is composed of the regulatory cyclin B and the enzymatic p34cdc2 kinase subunits. For its activation at the G2/M border, p34cdc2 needs to be associated with cyclin B and additionally dephosphorylated at Tyr-15 by the specific phosphatase cdc25-C. Treatment of synchronized cells in S or G2 phase with C. botulinum C2 toxin prevented p34cdc2 protein kinase activation by inhibiting its tyrosine dephosphorylation at the G2/M border. Furthermore, the activity of cdc25-C phosphatase was decreased after treatment of cells with C2 toxin. Our results suggest that the prevented activation of the mitotic inducers p34cdc2 kinase and cdc25-C phosphatase represents the final downstream events in the action of C2 toxin resulting in a G(2) phase cell cycle delay in synchronized HeLa cells.     

Expression of cdc2 and p27(KIP1) phosphorylation in mitotic cells of the human retinoblastoma

It was recently demonstrated that a lack of p27(KIP1) degradation resulted in the suppression of cdc2 activity and consequent inhibition of entry into the M-phase. The aim of this study was to examine the distribution of phosphorylated p27(KIP1) on threonine 187 (T187-phospho-p27) and cdc2 in mitotic cells of human retinoblastoma, a malignant retinal neoplasm. Several T187-phospho-p27-immunopositive cells were observed in mitotic retinoblastoma cells, but not in the normal retina. Immunoreactivity for T187-phospho-p27 was located in the prophase and metaphase of mitotic tumor cells. In contrast, tumor cells in the anaphase showed no immunoreactivity for T187-phospho-p27. Nuclear expression of cdc2 was detected in many retinoblastoma cells, including mitotic cells. The immunoreactivity in mitotic cells was located in the prophase, as well as metaphase. In contrast, anaphase cells did not show immunoreactivity. Double staining demonstrated the same localization of T187-phospho-p27 and cdc2 in mitotic cells. These results suggest that p27(KIP1) interacts with cdc2 in the M-phase of human retinoblastoma cells.     

Cell cycle G2 arrest induced by HIV-1 Vpr in fission yeast (Schizosaccharomyces pombe) is independent of cell death and early genes in the DNA damage checkpoint

HIV-1 Vpr induces cell cycle G2 arrest, morphological changes and cell death in human and fission yeast cells. The cellular targets for G2 arrest were expected to be the inhibitory phosphorylation sites of Cdc2, as G2 arrest correlates with hyperphosphorylation and decreased activity of Cdc2 in both human and fission yeast cells. In this study, we present direct evidence of genetic suppression of Vpr-induced G2 arrest by cdc2 mutations. Mutations in cdc2 (cdc2-1w and cdc2-3w) reduce the ability of Vpr to induce G2 arrest. A strain with a mutation changing the Tyr15 of Cdc2 to the non-phosphorylated Phe (Y15F) eliminated Vpr-induced G2 arrest indicating that Tyr15 of Cdc2 is the sole target for induction of G2 arrest by Vpr. Although the G2 arrest induced by DNA damage also proceeds through phosphorylation of Tyr15, the rad1, rad3, rad9 and rad17 mutations, which eliminate the G2 checkpoint for DNA damage, did not block the G2 arrest induced by Vpr. Furthermore, Vpr expression did not alter sensitivity of these rad mutants to UV radiation. Thus, the pathways for the induction of G2 arrest by DNA damage and Vpr are not identical. Interestingly, Vpr still induces cell death and morphological changes in the Y15F Cdc2 strain indicating that G2 arrest is not required for morphological changes and cell death. This conclusion was further supported by the observation that mutations in Vpr, which have lost their ability to induce G2 arrest, retained the ability to kill cells.     

Mitosis in vertebrates: the G2/M and M/A transitions and their associated checkpoints

In this review, I stress the importance of direct data and accurate terminology when formulating and communicating conclusions on how the G2/M and metaphase/anaphase transitions are regulated. I argue that entry into mitosis (i.e., the G2/M transition) is guarded by several checkpoint control pathways that lose their ability to delay or stop further cell cycle progression once the cell becomes committed to divide, which in vertebrates occurs in the late stages of chromosome condensation. After this commitment, progress through mitosis is then mediated by a single Mad/Bub-based checkpoint that delays chromatid separation, and exit from mitosis (i.e., completion of the cell cycle) in the presence of unattached kinetochores. When cells cannot satisfy the mitotic checkpoint, e.g., when in concentrations of spindle poisons that prohibit the stable attachment of all kinetochores, they are delayed in mitosis for many hours. In normal cells, the duration of this delay depends on the organism and ranges from ∼4 h in rodents to ∼22 h in humans. Recent live cell studies reveal that under this condition, many cancer cells (including HeLa and U2OS) die in mitosis by apoptosis within ∼24 h, which implies that biochemical studies on cancer cell populations harvested in mitosis after a prolonged mitotic arrest are contaminated with dead or dying cells.     

Low cdc27 and high securin expression predict short survival for breast cancer patients

Cell cycle regulators cdc27 and securin participate in control of the mitotic checkpoint and survey the mitotic spindle to maintain chromosomal integrity. This is achieved by their functions in metaphase–anaphase transition, DNA damage repair, enhancement of mitotic arrest and apoptosis. We report on the roles of cdc27 and securin in aneuploidy and prognosis of breast cancer. The study comprises 429 breast cancer patients with up to 22 years of follow‐up. DNA content was determined by image cytometry, and immunopositivity for cdc27 and securin was based on tissue microarrays. An inverse association between cdc27 and securin expression was observed in both image cytometric and immunohistochemical analyses. Low cdc27 and high securin expression identified patients with significant difference in disease outcome. Cdc27 and securin immunoexpression identified patients at risk of early cancer death within five years from diagnosis. In multivariate analysis, the combination of cdc27 and securin immunohistochemistry was the strongest predictor of cancer death after lymph node status. We demonstrate, for the first time in human breast cancer, the prognostic value of cdc27 and securin immunohistochemistry. Cdc27 and securin appear promising biomarkers for applications in predicting disease progression, prognostication of individual patients and potential in anti‐mitotic drug development.     

Phosphorylation of adenovirus E1A proteins by the p34cdc2 protein kinase.

Adenovirus early region 1A (E1A) products are phosphorylated nuclear oncoproteins which appear to derive transforming activity largely through interactions with cellular proteins including the tumor suppressor p105/Rb-1 and cyclin A (p60cycA), a regulatory subunit associated with p34cdc2 and the related protein kinase p33cdk2. We have identified several sites of phosphorylation on E1A proteins previously and showed that phosphorylation at Ser-89 alters electrophoretic mobility significantly and affects E1A-mediated transforming activity to some extent. We now report that both Ser-89 and Ser-219, the major E1A phosphorylation site, were phosphorylated in vitro by p34cdc2 purified from HeLa cells. We also found that E1A proteins seemed to be phosphorylated at the highest levels in vivo in mitotic cells which express maximal levels of p34cdc2 kinase activity. Thus, in addition to forming complexes with p60cycA, a regulator of p34cdc2 and related kinases, and p105/Rb-1 which exhibits cell cycle-dependent phosphorylation, E1A proteins seem to be substrates for p34cdc2. These data suggested that a link could exist between phosphorylation, cell cycle progression, and the regulation of transforming activity of E1A proteins.     

Pleotropic effects of genistein on MCF-7 breast cancer cells

Soy isoflavone, genistein has been shown to induce growth inhibition, cell cycle arrest and apoptosis in cultured cancer cell lines derived from head and neck, breast, lung, and prostate cancers and showed antitumor activity against tumors in multiple animal models. In the present study we show that genistein inhibits the growth of MCF-7 breast cancer cell line in a dose dependent manner. The genistein induced growth inhibition is accompanied by the reduction in the number of mitotic cells and overexpression of cyclin dependent kinase inhibitor p21WAF1 leading to cell cycle arrest. In addition, the telomeric area was significantly reduced in genistein treated MCF-7 cells. Analysis of multiple genes involving the apoptotic pathway reveals inhibition of Akt activity without affecting the steady state levels of Akt protein expression and the down regulation of proapoptotic gene BAD expression. From these results, we conclude that genistein-induced inhibition of cell division is partly mediated by decreased telomere length, reduced mitosis and inhibition of Akt activation, leading to induction of apoptosis.     

Fission yeast genes involved in coupling mitosis to completion of DNA replication.

We have isolated fission yeast mutants that enter mitosis when DNA replication is blocked with hydroxyurea. The mutants define eight linkage groups, three of which consist of alleles of the rad1, rad3, and rad17 genes. Recently, these fission yeast genes have been shown to be required for radiation-induced cell cycle arrest, as is the budding yeast RAD9 gene. The other five genes are called hus (hydroxyurea sensitive) 1-5. We propose that these genes participate in an intracellular signal transduction pathway that monitors the completion of DNA replication and transmits information to the mitotic control protein cdc2. Mutations that bypass the requirement for cdc25 (an activator of the mitotic regulator cdc2) also uncouple mitosis from DNA replication. However, mitosis is blocked by inhibitors of DNA replication in strains in which the cdc25 gene has been deleted, indicating that although cdc25 influences the coupling of mitosis to the completion of DNA replication, it is not essential for this control.     

High pressure sensitizes murine erythroleukemia cells to caffeine-induced premature mitosis

Murine erythroleukemia (MEL) cells were exposed to a high pressure of 80 MPa or aphidicolin (APH), DNA polymerase inhibitor. The effects of caffeine on cell cycle were examined using these cells. During the culture of 80 MPa-treated MEL cells at atmospheric pressure, the cells arrested in the G2 phase, and cyclin B and hyperphosphorylated p34(cdc2) were accumulated. Namely, maturation promoting factor (MPF) composed of p34(cdc2) and cyclin B was inactive. However, upon exposure to caffeine, these cells entered prematurely into mitosis by activating MPF. Caffeine-induced premature mitosis was suppressed by butyrolactone I and orthovanadate. On the other hand, APH-treated MEL cells, which were not exposed to 80 MPa, were not so sensitive to caffeine-induced premature mitosis despite cyclin B accumulation. In this case, dephosphorylation of p34(cdc2) was not induced by caffeine. Interestingly, caffeine-induced premature mitosis in the 80 MPa-treated cells was also suppressed by APH. These results suggest that the premature mitosis of 80 MPa-treated MEL cells by caffeine is induced by active MPF, and that APH-sensitive molecules such as DNA polymerase may also play an important role in the checkpoint that controls the transition from G2 to M phase.     

抑制plk1表达导致胃癌细胞cyclinB/cdc2复合体活性升高

目的观察抑制polo样激酶基因1(plkl)表达对胃癌细胞株MKN45的cyclinB／cde2复合体(MPF)及细胞周期的影响。方法化学合成小片断干扰RNA(siRNA)阻断MKN45细胞plkl基因的表达;实时定量PCR及Western印迹检测干扰前后plkl mRNA及蛋白质表达的变化;倒置显微镜观察细胞形态的变化;流式细胞仪检测MKN45细胞周期分布的变化;Western印迹检测cyclinB与cdc2表达水平的变化,激酶活性分析检测cdc2激酶活性变化。结果经靶向plkl的siRNA作用后,plkl siRNA mRNA及蛋白质表达明显下降;较多MKN45细胞变圆及呈现G_2期细胞DNA含量(P<0．05);cyclinB平均表达水平在48及72h,较对照siRNA组升高了82．38％和32．89％(P<0．05),cdc2表达水平无明显变化,但其平均活性在48及72h较对照siRNA组升高了29．34％和80．33％(P<0．05)。结论 PLKl与其下游的cyclinB／cdc2复合体活性之间在MKN45细胞有丝分裂的前后期可能存在着不同的调控机制,PLKl在MKN45细胞有丝分裂的启动及退出方面均发挥关键作用。     

The contribution of cytolethal distending toxin to bacterial pathogenesis

Cytolethal distending toxin (CDT) is a bacterial toxin that initiates a eukaryotic cell cycle block at the G2 stage prior to mitosis. CDT is produced by a number of bacterial pathogens including: Campylobacter species, Escherichia coli, Salmonella enterica serovar Typhi, Shigella dystenteriae, enterohepatic Helicobacter species, Actinobacillus actinomycetemcomitans (the cause of aggressive periodontitis), and Haemophilus ducreyi (the cause of chancroid). The functional toxin is composed of three proteins; CdtB potentiates a cascade leading to cell cycle block, and CdtA and CdtC function as dimeric subunits, which bind CdtB and delivers it to the mammalian cell interior. Once inside the cell, CdtB enters the nucleus and exhibits a DNase I-like activity that results in DNA double-strand breaks. The eukaryotic cell responds to the DNA double-strand breaks by initiating a regulatory cascade that results in cell cycle arrest, cellular distension, and cell death. Mutations in CdtABC that cause any of the three subunits to lose function prevent the bacterial cell from inducing cytotoxicity. The result of CDT activity can differ somewhat depending on the eukaryotic cell types affected. Epithelial cells, endothelial cells, and keratinocytes undergo G2 cell cycle arrest, cellular distension, and death; fibroblasts undergo G1 and G2 arrest, cellular distension, and death; and immune cells undergo G2 arrest followed by apoptosis. CDT contributes to pathogenesis by inhibiting both cellular and humoral immunity via apoptosis of immune response cells, and by generating necrosis of epithelial-type cells and fibroblasts involved in the repair of lesions produced by pathogens resulting in slow healing and production of disease symptoms. Thus, CDT may function as a virulence factor in pathogens that produce the toxin.     

Cyclin-dependent kinase and Cks/Suc1 interact with the proteasome in yeast to control proteolysis of M-phase targets

Cell cycle-specific proteolysis is critical for proper execution of mitosis in all eukaryotes. Ubiquitination and subsequent proteolysis of the mitotic regulators Clb2 and Pds1 depend on the cyclosome/APC and the 26S proteasome. We report here that components of the cell cycle machinery in yeast, specifically the cell cycle regulatory cyclin-dependent kinase Cdc28 and a conserved associated protein Cks1/Suc1, interact genetically, physically, and functionally with components of the 26S proteasome. A mutation in Cdc28 (cdc28-1N) that interferes with Cks1 binding, or inactivation of Cks1 itself, confers stabilization of Clb2, the principal mitotic B-type cyclin in budding yeast. Surprisingly, Clb2-ubiquitination in vivo and in vitro is not affected by mutations in cks1, indicating that Cks1 is not essential for cyclosome/APC activity. However, mutant Cks1 proteins no longer physically interact with the proteasome, suggesting that Cks1 is required for some aspect of proteasome function during M-phase-specific proteolysis. We further provide evidence that Cks1 function is required for degradation of the anaphase inhibitor Pds1. Stabilization of Pds1 is partially responsible for the metaphase arrest phenotype of cks1 mutants because deletion of PDS1 partially relieves the metaphase block in these mutants.     

Hypoxia-inducible factor-1alpha induces cell cycle arrest of endothelial cells

BACKGROUND: Hypoxia can induce tissue injury, including apoptosis of endothelial cells. However, little is known about the effects of hypoxia on endothelial cell function. We assessed the effects of hypoxia inducible factor (HIF)-1alpha on the functional characteristics of endothelial cells, particularly on cell cycle regulators, by cationic liposome-mediated transfection of HIF-1alpha-expression vector into the cells. RESULTS: Transfection of the HIF-1alpha gene in endothelial cells resulted in (a) reduced proliferation and detachment of the cells; (b) up-regulation of intracellular p21waf1/cip1 and down-regulation of bcl-2; (c) reduced activities of cyclin-dependent kinase (CDK)-4 and CDK-6; (d) cell cycle arrest at G0/G1 phase; and (e) apoptosis of the cells. CONCLUSIONS: HIF-1alpha can induce cell cycle arrest, resulting in the reduced proliferation and apoptosis of endothelial cells, and the hypoxia-induced cell death may be involved by suppression of anti-apoptotic molecule, bcl-2.     

Premature chromosome condensation revisited: a novel chemical approach permits efficient cytogenetic analysis of cancers

Chemical induction of premature chromosome condensation (PCC) was investigated and optimized to be able to analyze the chromosomal constitution of cancer cells independent of mitosis and with minimal culture artifacts. A potent protein phosphatase inhibitor, calyculin A, was used to induce PCC in normal diploid cells, in several established human tumor cell lines, and in cells isolated from freshly dissected adenomatous polyps of a patient with hereditary colorectal cancer. In parallel, mitotic arrest was pursued by use of Colcemid. In cell lines, a difference of up to 10-fold was found between frequency of cells with PCC induced by calyculin A (PCC index) and the mitotic index after treatment with Colcemid. In the fresh tumor specimens, Colcemid failed to result in metaphase formation, whereas a regimen of 80 nM calyculin A for 75 min, after only 2 days of culturing, resulted in a PCC index of 2-5%. pq-COBRA-FISH (COmbined Binary RAtio labeling-fluorescence in situ hybridization) was used for a detailed analysis of four cell lines treated with calyculin A, which proved that PCC spreads are amenable to molecular karyotyping, and a comparison between PCC spreads and metaphases from mitotic arrest revealed no discrepancies in karyotypes. pq-COBRA-FISH on PCC spreads from fresh colon tumor samples revealed only numerical and no structural abnormalities. Calyculin A-induced PCC combined with multicolor FISH gives a new opportunity for analysis of the chromosomal constitution of G(1) and G(2) cancer cells and may find application in the study of the role of chromosome instability in cancer development.     

p53 deficiency and defective mitotic checkpoint in proliferating T lymphocytes increase chromosomal instability through aberrant exit from mitotic arrest

During the proliferation of T cells for successful immune responses against pathogens, the fine regulation of cell cycle is important to the maintenance of T cell homeostasis and the prevention of lymphoproliferative disorders. However, it remains to be elucidated how the cell cycle is controlled at the mitotic phase in proliferating T cells. Here, we show that during the proliferation of primary T cells, the disruption of the mitotic spindle leads to cell-cycle arrest at mitosis and that prolonged mitotic arrest results in not only apoptosis but also the form of chromosomal instability observed in human cancers. It is interesting that in response to spindle damage, the phosphorylation of BubR1, a mitotic checkpoint kinase, was significantly induced in proliferating T cells, and the expression of the dominant-negative mutant of BubR1 compromised mitotic arrest and subsequent apoptosis and thus led to the augmentation of polyploidy formation. We also show that in response to prolonged spindle damage, the expression of p53 but not of p73 was significantly induced. In addition, following sustained mitotic arrest, p53-deficient T cells were found to be more susceptible to polyploidy formation than the wild type. These results suggest that during flourishing immune response, mitotic checkpoint and p53 play important roles in the prevention of chromosomal instability and in the maintenance of the genomic integrity of proliferating T cells.     

Aberrations of cell cycle and cell death in normal development of the chick embryo growth plate.

The epiphyses of femurs from 7.5-15 day chicken embryos were studied by electron microscopy. Several forms of aberrant cell cycles were present: (1) in the perichondrium, polyploid metaphases, segmentating large (giant) cells, and mitotic catastrophe (midway between mitosis and apoptosis) were observed; (2) in the resting zone, premature chromosome condensation was found; (3) in the proliferative zone, approximately 5% of divisions were aberrant, representing most often mitosis restitution from metaphase and more seldom from the anaphase; (4) in all layers, 'dark chondrocytes' representing a premortal form of hypersecretory cells undergoing often a-mitotic nuclear segmentation were present. Many of the aberrations of cell cycle were combined with cell death. These deviations omitting or adapting the cell cycle check-points represent evidently the normal epigenetic mechanisms of development and repair. At the same time, by origin and appearances they seem very close to the loss of the growth control displayed by malignant tumours. This connection is briefly analysed in view of some current concepts of carcinogenesis.     

A mouse cdc25 homolog is differentially and developmentally expressed.

The timing and activation of the p34cdc2 kinase in mammals is associated with dephosphorylation of phosphotyrosine and phosphothreonine residues on the p34cdc2 kinase. For fission yeast, the timing of mitosis is regulated by cyclic accumulation of cdc25, which promotes dephosphorylation of p34cdc2 and concomitant protein kinase activation. We report the identification and characterization of a structural and functional mouse homolog, Cdc25M2, of the cdc25 phosphatase. Cdc25M2 shows high sequence identity to the previously reported human homolog cdc25Hu2. Cdc25M2 can functionally complement for a Schizosaccharomyces pombe cdc25ts mutation, and when expressed in Escherichia coli and purified, Cdc25M2 is an active phosphatase. cdc25M2 mRNA shows variation in expression in different tissues in the mouse embryo and is expressed in a developmental and cell-cycle-dependent fashion. We suggest that the expression and accumulation of the cdc25 mitotic inducer may play a critical role in the regulation of mouse development.