Chk2 kinase is required for methylglyoxal-induced G2/M cell-cycle checkpoint arrest: implication of cell-cycle checkpoint regulation in diabetic oxidative stress signaling

Methylglyoxal (MG) is a reactive endogenous metabolite that is produced from the process of degradation of triose-phosphates. Under hyperglycemic conditions the rate of MG formation increases as a result of elevated concentrations of precursors. It has been established that MG elicits oxidative stress signaling, leading to the activation of MAP kinases, p38 MAPK and JNK, yet it remains largely unknown about a role of cell-cycle checkpoint regulation in MG-induced signaling. Here, we show that checkpoint kinases, Chk1 and Chk2, as well as their upstream ATM kinase are phosphorylated and activated following MG treatment of cultured cells. This MG-induced activation of Chk1 and Chk2 were inhibited by either aminoguanidine (AG), an inhibitor of production of advanced glycation end products (AGEs) or N-acetyl-l-cysteine (NAC), an anti-oxidant in dose dependent manners, indicating that oxidative stress via AGEs is involved critically in the activation of Chk1 and Chk2 by MG. Furthermore, it was found that cell-cycle synchronized cells exhibited G(2)/M checkpoint arrest following MG treatment, and that siRNA-mediated knock-down of Chk2, but not Chk1, results in a failure of MG-induced G(2)/M arrest. Thus, the results indicate a critical role for Chk2 in MG-induced G(2)/M cell-cycle checkpoint arrest.     

DTL/CDT2 is essential for both CDT1 regulation and the early G2/M checkpoint

Checkpoint genes maintain genomic stability by arresting cells after DNA damage. Many of these genes also control cell cycle events in unperturbed cells. By conducting a screen for checkpoint genes in zebrafish, we found that dtl/cdt2 is an essential component of the early, radiation-induced G2/M checkpoint. We subsequently found that dtl/cdt2 is required for normal cell cycle control, primarily to prevent rereplication. Both the checkpoint and replication roles are conserved in human DTL. Our data indicate that the rereplication reflects a requirement for DTL in regulating CDT1, a protein required for prereplication complex formation. CDT1 is degraded in S phase to prevent rereplication, and following DNA damage to prevent origin firing. We show that DTL associates with the CUL4-DDB1 E3 ubiquitin ligase and is required for CDT1 down-regulation in unperturbed cells and following DNA damage. The cell cycle defects of Dtl-deficient zebrafish are suppressed by reducing Cdt1 levels. In contrast, the early G2/M checkpoint defect appears to be Cdt1-independent. Thus, DTL promotes genomic stability through two distinct mechanisms. First, it is an essential component of the CUL4-DDB1 complex that controls CDT1 levels, thereby preventing rereplication. Second, it is required for the early G2/M checkpoint.     

SERPINA3 promotes endometrial cancer cells growth by regulating G2/M cell cycle checkpoint and apoptosis

Endometrial carcinoma (EC) is the most common gynecologic cancer worldwide and is one of the leading causes of death in women. Therefore, it is urgent to elucidate the pathological mechanisms of EC. SERPINA3 is a member of the serpin super-family of protease inhibitors. Its aberrant expression has been observed in various tumor cells. However, its clinical significance and biological function in endometrial cancer remains unknown. In the present study, we demonstrated that SERPINA3 expression was significantly up-regulated in EC samples and was closely correlated with lower differentiation, higher stage, positive lymph node or vascular thrombosis and negative estrogen receptor (ER), indicating a poor prognosis. We then demonstrated that SERPINA3 promoted EC cells proliferation by regulating G2/M checkpoint in cell cycle and inhibited cells apoptosis, and we further uncovered that the pro-proliferative effect of SERPINA3 on EC was likely ascribed to the activation of MAPK/ERK1/2 and PI3K/AKT signaling. The results of our study may provide insight into the application of SERPINA3 as a novel predictor of clinical outcomes and a potential therapeutic target of EC.     

Syk is required for p38 activation and G2/M arrest in B cells exposed to oxidative stress

Syk has been demonstrated to play a crucial role in oxidative stress signaling in B cells. In this study, we have investigated the role of Syk in p38 activation and the regulation of cell-cycle progression upon oxidative stress. In B cells, p38 is activated by hydrogen peroxide (H(2)O(2)) stimulation. Syk is required for p38 activation following stimulation with 10-100 microM H(2)O(2), but not with 1 mM H(2)O(2). H(2)O(2)-induced p38 activation is abrogated in phospholipase C-gamma2 (PLC-gamma2)-deficient as well as Syk-deficient cells, suggesting that Syk activates p38 through PLC-gamma2 upon H(2)O(2) stimulation. Although stimulation with 20-100 microM H(2)O(2) induces cellular apoptosis in B cells, pretreatment with SB203580, a p38-specific inhibitor, has no effect on H(2)O(2)-induced apoptosis. Flow cytometric analysis reveals that B cells exposed to 10-20 microM H(2)O(2) exhibit cell-cycle profile of G2/M arrest, and pretreatment with SB203580 inhibits only a little H(2)O(2)-induced G2/M arrest. On the other hand, Syk-deficient cells show no induction of G2/M arrest following H(2)O(2) stimulation. These findings indicate that Syk plays a role in the regulation of cell-cycle progression in G2/M phase via p38-dependent and -independent pathways after oxidative stress.     

Chk1 is an essential kinase that is regulated by Atr and required for the G(2)/M DNA damage checkpoint

Chk1, an evolutionarily conserved protein kinase, has been implicated in cell cycle checkpoint control in lower eukaryotes. By gene disruption, we show that CHK1 deficiency results in a severe proliferation defect and death in embryonic stem (ES) cells, and peri-implantation embryonic lethality in mice. Through analysis of a conditional CHK1-deficient cell line, we demonstrate that ES cells lacking Chk1 have a defective G(2)/M DNA damage checkpoint in response to gamma-irradiation (IR). CHK1 heterozygosity modestly enhances the tumorigenesis phenotype of WNT-1 transgenic mice. We show that in human cells, Chk1 is phosphorylated on serine 345 (S345) in response to UV, IR, and hydroxyurea (HU). Overexpression of wild-type Atr enhances, whereas overexpression of the kinase-defective mutant Atr inhibits S345 phosphorylation of Chk1 induced by UV treatment. Taken together, these data indicate that Chk1 plays an essential role in the mammalian DNA damage checkpoint, embryonic development, and tumor suppression, and that Atr regulates Chk1.     

二烯丙基二硫化物对人胃癌MGC803细胞G2/M期检查点激酶Chk1/2的影响

目的： 观察二烯丙基二硫化物（DADS）对人胃癌MGC803细胞的细胞周期检查点激酶1（Chkl）和细胞周期检查点激酶2(Chk2)的影响。方法：RT-PCR检测MGC803细胞的Chk1和Chk2激酶在mRNA水平的改变;Western blotting 检测Chk1、Chk2的表达和Chk1、Chk2的磷酸化程度,免疫共沉淀检测Chk1、Chk2与细胞分裂周期蛋白25C（Cdc25C）结合情况。结果：RT-PCR检测显示,Chkl和Chk2 mRNA水平在处理组与未处理组之间无明显差异（P>0.05）。Western blotting 检测显示,MGC803细胞分别受30 mg·L-1 DADS刺激1 h和2 h后,与未处理组相比,总Chk1和Chk2蛋白表达在细胞处理前后均无明显改变（P>0.05）;但处理组细胞Chk1磷酸化程度明显增加,并呈时间依赖性（P<0.01）,而Chk2在处理组与未处理组间磷酸化程度无明显差异（P>0.05）。免疫共沉淀分析表明,MGC803细胞中DADS能促进Chk1与Cdc25C结合,而对Chk2与Cdc25C结合无影响。结论：DADS可能是通过激活Chk1引起人胃癌MGC803细胞G2/M期阻滞。     

G proteins in T cell signal transduction.

This review summarises recent data on G protein implication in receptor signalling in T cells. The data show that PPI-specific PLC in T cell membranes is under G protein control. Some evidence indicates that a G protein couples PLC to TCR. Differences are revealed between the effects induced by direct G protein activators, such as GTP gamma S or AlF4-, and TCR ligands, which imply that TCR ligands may trigger some G-protein-independent signals. An analysis of the conflicting results on the action of PTX and CTX, one of the main tools in studying G proteins, has shown that the toxins produce both G protein-dependent and independent effects. The G protein which couples PLC to TCR appears insensitive to both PTX and CTX. Some findings suggest G protein involvement in signalling induced by interleukins; however, in this case the effector molecules implicate often remain unknown. Scarce data on G protein involvement in signalling from differentiation antigens, on direct G protein regulation of ion channels, and on identification of G proteins in T cells, are also discussed.     

Drosophila myb is required for the G2/M transition and maintenance of diploidy

The myb proto-oncogenes are thought to have a role in the cell division cycle. We have examined this possibility by genetic analysis in Drosophila melanogaster, which possesses a single myb gene. We have described previously two temperature-sensitive, recessive lethal mutants in Drosophila myb (Dm myb). The phenotypes of these mutants revealed a requirement for myb in diverse cellular lineages throughout the course of Drosophila development. We now report a cellular explanation for these findings by showing that Dm myb is required for both mitosis and prevention of endoreduplication in wing cells. Myb apparently acts at or near the time of the G₂/M transition. The two mutant alleles of Dm myb produce the same cellular phenotype, although the responsible mutations are located in different functional domains of the gene product. The mutant phenotype can be partially suppressed by ectopic expression of either cdc2 or string, two genes that are known to promote the transition from G₂ to M. We conclude that Dm myb is required for completion of cell division and may serve two independent functions: promotion of mitosis, on the one hand, and prevention of endoreduplication when cells are arrested in G₂, on the other.     

STE11 is a protein kinase required for cell-type-specific transcription and signal transduction in yeast

The STE11 gene of Saccharomyces cerevisiae is one of several genes required for mating between two haploid cell types of this yeast. Its product is required for response to a signal that causes arrest of the mitotic cell cycle in the G1 phase and induction of mating-type-specific genes. The nucleotide sequence of the STE11 gene was determined. The predicted amino acid sequence shows homology to the protein kinase family. We demonstrate that the STE11 product has kinase catalytic activity and that this activity is required for its in vivo functions.     

C. elegans mre-11 is required for meiotic recombination and DNA repair but is dispensable for the meiotic G(2) DNA damage checkpoint

We investigated the roles of Caenorhabditis elegans MRE-11 in multiple cellular processes required to maintain genome integrity. Although yeast Mre11 is known to promote genome stability through several diverse pathways, inviability of vertebrate cells that lack Mre11 has hindered elucidation of the in vivo roles of this conserved protein in metazoan biology. Worms homozygous for an mre-11 null mutation are viable, allowing us to demonstrate in vivo requirements for MRE-11 in meiotic recombination and DNA repair. In mre-11 mutants, meiotic crossovers are not detected, and oocyte chromosomes lack chiasmata but appear otherwise intact. gamma-irradiation of mre-11 mutant germ cells during meiotic prophase eliminates progeny survivorship and induces chromosome fragmentation and other cytologically visible abnormalities, indicating a defect in repair of radiation-induced chromosome damage. Whereas mre-11 mutant germ cells are repair-deficient, they retain function of the meiotic G(2) DNA damage checkpoint that triggers germ cell apoptosis in response to ionizing radiation. Although mre-11/mre-11 animals derived from heterozygous parents are viable and produce many embryos, there is a marked drop both in the number and survivorship of embryos produced by succeeding generations. This progressive loss of fecundity and viability indicates that MRE-11 performs a function essential for maintaining reproductive capacity in the species.     

The eaeB gene of enteropathogenic Escherichia coli is necessary for signal transduction in epithelial cells.

An enteropathogenic Escherichia coli mutant carrying an internal deletion in the eaeB gene (UMD864) was unable to activate epithelial cell signals, including tyrosine phosphorylation, cytoskeletal rearrangements, and the release of inositol phosphates, indicating that the eaeB locus encodes a product that is involved in stimulating signals in epithelial cells.     

二烯丙基二硫诱导肿瘤细胞G2/M 期阻滞的分子机制

二烯丙基二硫（diallyl disulfide,DADS) 对乳腺癌、肺癌、膀胱癌、结肠癌、胃癌、白血病等许多肿瘤均有明显的抑制作用,其作用与G2/M 期阻滞有关。DADS阻滞G2/M 期的分子机制包括抑制p34cdc2的活性,激活MAPK信号通路,增加p21WAF1/cip1蛋白表达与ROS生成等。     

G2 checkpoint control and G2 chromosomal radiosensitivity in cancer survivors and their families

Significant inter-individual variation in G(2) chromosomal radiosensitivity, measured as radiation-induced chromatid-type aberrations in the subsequent metaphase, has been reported in peripheral blood lymphocytes of both healthy individuals and a range of cancer patients. One possible explanation for this variation is that it is driven, at least in part, by the efficiency of G(2)-M checkpoint control. The hypothesis tested in the current analysis is that increased G(2) chromosomal radiosensitivity is facilitated by a less efficient G(2)-M checkpoint. The study groups comprised 23 childhood and adolescent cancer survivors, their 23 partners and 38 of their offspring (Group 1) and 29 childhood and young adult cancer survivors (Group 2). Following exposure to 0.5 Gy of 300 kV X-rays, lymphocyte cultures were assessed for both G(2) checkpoint delay and G(2) chromosomal radiosensitivity. In Group 1, the extent of G(2) checkpoint delay was measured by mitotic inhibition. No statistically significant differences in G(2) checkpoint delay were observed between the cancer survivors (P = 0.660) or offspring (P = 0.171) and the partner control group nor was there any significant relationship between G(2) checkpoint delay and G(2) chromosomal radiosensitivity in the cancer survivors (P = 0.751), the partners (P = 0.634), the offspring (P = 0.824) or Group 1 taken as a whole (P = 0.379). For Group 2, G(2) checkpoint delay was assessed with an assay utilising premature chromosome condensation to distinguish cell cycle stage. No significant relationship between G(2) checkpoint delay and G(2) chromosomal radiosensitivity was found (P = 0.284). Thus, this study does not support a relationship between G(2)-M checkpoint efficiency and variation in G(2) chromosomal radiosensitivity.     

Human Rad9 is required for the activation of S-phase checkpoint and the maintenance of chromosomal stability

In response to DNA damage or replication block, cells activate a battery of checkpoint signaling cascades to control cell cycle progression and elicit DNA repair in order to maintain genomic stability and integrity. Identified as a homolog of its fission yeast counterpart, human Rad9 was proposed to form a Rad9-Hus1-Rad1 protein complex to mediate checkpoint signals. However, the precise function of Rad9 in the process of checkpoint activation is not fully understood. Using the RNA interference technique, we investigated the role of Rad9 in the genotoxic stress-induced activation of S-phase checkpoint and the maintenance of chromosomal stability. We found that Rad9 knockdown reduced the phosphorylation of Rad17, Chk1 and Smc1 in response to DNA replication block and certain types of DNA damage. Immunofluorescence studies showed that the removal of Rad9 disrupted the foci formation of phosphorylated Chk1, but not ATR. Moreover, Rad9 knockdown resulted in radioresistant DNA synthesis and reduced cell viability under replication stress. Finally, removal of Rad9 by RNAi led to increased accumulation of spontaneous chromosomal aberrations. Taken together, these results suggest a critical and specific role of Rad9 in the activation of S-phase checkpoint and the maintenance of chromosome stability.     

Cooperative effects of genes controlling the G(2)/M checkpoint

It is believed that multiple effectors independently control the checkpoints permitting transitions between cell cycle phases. However, this has not been rigorously demonstrated in mammalian cells. The p53-induced genes p21 and 14-3-3sigma are each required for the G(2) arrest and allow a specific test of this fundamental tenet. We generated human cells deficient in both p21 and 14-3-3sigma and determined whether the double knockout was more sensitive to DNA damage than either single knockout. p21(-/-) 14-3-3sigma(-/-) cells were significantly more sensitive to DNA damage or to the exogenous expression of p53 than cells lacking only p21 or only 14-3-3sigma. Thus, p21 and 14-3-3sigma play distinct but complementary roles in the G(2)/M checkpoint, and help explain why genes at the nodal points of growth arrest pathways, like p53, are the targets of mutation in cancer cells.     

PU.1 and Spi-B are required for normal B cell receptor-mediated signal transduction

PU.1 and Spi-B have previously been implicated in the regulation of genes encoding B cell receptor (BCR) signaling components. Spi-B-/- B lymphocytes respond poorly to BCR stimulation; PU.1-/- mice, however, lack B cells, precluding an analysis of BCR responses. We now show that PU.1+/- Spi-B-/- B cells exhibit more extensive defects than Spi-B-/- B cells, indicating that both PU.1 and Spi-B are required for normal BCR signaling. Strikingly, BCR cross-linking results in substantially reduced protein tyrosine phosphorylation in mutant B cells. Further analysis shows that Igalpha is phosphorylated and syk is recruited and becomes phosphorylated but that BLNK and PLCgamma phosphorylation are defective in mutant cells. Our data support the existence of a novel component coupling syk to downstream targets.     

Biochemical signal transduction of mechanical strain in osteoblast-like cells.

The responses to mechanical loading of two types of osteoblast-like cells and skin fibroblasts were investigated using two new devices for applying defined and homogeneous strains to cells. The results indicate that only periostal (bone surface) osteoblasts are sensitive to strains within the physiological range and that a specific strain mechanism is responsible. Osteoblasts derived from the haversian system and skin fibroblasts do not respond except at higher, unphysiological strains. The mechanism is located in the cytoskeleton and activates the membrane phospholipase C within milliseconds and may react to distension of a strain sensitive protein. Activation of phospholipase C can account for only some of the observed responses of bone to mechanical loading such as stimulation of cell division, increase in collagen and collagenase production. Application of over 10,000 mu strains results in a de-differentiation of the osteoblasts and a change in cell morphology to become fibroblast-like.