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G2 Phase (g2 + phase)
Selected AbstractsMidblastula transition (MBT) of the cell cycles in the yolk and pigment granule-free translucent blastomeres obtained from centrifuged Xenopus embryosDEVELOPMENT GROWTH & DIFFERENTIATION, Issue 5 2005Yasuhiro Iwao We obtained translucent blastomeres free of yolk and pigment granules from Xenopus embryos which had been centrifuged at the beginning of the 8-cell stage with cellular integrity. They divided synchronously regardless of their cell size until they had decreased to 37.5 µm in radius; those smaller than this critical size, however, divided asynchronously with cell cycle times inversely proportional to the square of the cell radius after midblastula transition (MBT). The length of the S phase was determined as the time during which nuclear DNA fluorescence increased in Hoechst-stained blastomeres. When the cell cycle time exceeded 45 min, S and M phases were lengthened; when the cell cycle times exceeded 70 min, the G2 phase appeared; and after cell cycle times became longer than 150 min, the G1 phase appeared. Lengths of G1, S and M phases increased linearly with increasing cell cycle time. Enhanced green fluorescent protein (EGFP)-tagged proliferating cell nuclear antigen (PCNA) expressed in the blastomeres appeared in the S phase nucleus, but suddenly dispersed into the cytoplasm at the M phase. The system developed in this study is useful for examining the cell cycle behavior of the cell cycle-regulating molecules in living Xenopus blastomeres by fluorescence microscopy in real time. [source] Pax6 transcription factor is required for the interkinetic nuclear movement of neuroepithelial cellsGENES TO CELLS, Issue 9 2007Hiroshi Tamai The mammalian cerebral cortex develops from proliferative neuroepithelial cells that exhibit a cell cycle-dependent nuclear movement (interkinetic nuclear migration; INM). Pax6 transcription factor plays pivotal roles in various aspects of corticogenesis. From live observation using cultured cortical slices from the Pax6 mutant rat, we identified the premature descent of S phase cells, the unsteady ascent or descent of G2 phase cells, and ectopic cell division within the basal side of the ventricular zone (VZ). The centrosome normally stayed at the most apical side, apart from the nucleus, in the neuroepithelial cell during the S to G2 phase, while the Pax6 mutant showed unstable movement of the centrosome associated with an abnormal INM. Our results suggest the possibility that Pax6 regulates the INM by stabilizing the centrosome at the apical side. [source] Accumulation of multiple forms of lamin A with down-regulation of FACE-1 suppresses growth in senescent human cellsGENES TO CELLS, Issue 3 2007Ryo Ukekawa 5-Bromodeoxyuridine (BrdU) clearly induces a senescence-like phenomenon in every cell type. Proteome analysis revealed that lamin A and C were most highly increased in the nuclei of HeLa cells upon addition of BrdU. Immunoblot analysis also revealed marked accumulation of nuclear prelamin A. Consistently, farnesylated-proteins converting enzyme 1 (FACE-1) was markedly down-regulated in the same cells. Similar phenomena were also observed in normal human fibroblasts undergoing replicative senescence. Immunochemical analysis confirmed the above results. Lamin A is a major component of lamina and responsible for several genetic diseases. Thus, we ectopically expressed a wild-type, a mature type and a premature type of lamin in HeLa cells. All of these forms similarly inhibited colony formation and delayed cell cycle progression mainly through G2 phase. These results suggest that a change in the amount of lamin A, rather than appearance of its truncated form, is responsible for growth retardation in affected cells. [source] Meiosis induced by inactivation of Pat1 kinase proceeds with aberrant nuclear positioning of centromeres in the fission yeast Schizosaccharomyces pombeGENES TO CELLS, Issue 8 2004Yuji Chikashige Nuclear organization of chromosomes proceeds with significant changes during meiosis. In the fission yeast Schizosaccharomyces pombe, centromeres are clustered at the spindle-pole body (SPB) during the mitotic cell cycle; however, during meiotic prophase telomeres become clustered to the SPB and centromeres dissociate from the SPB. We followed the movement of telomeres, centromeres and sister chromatids in living S. pombe cells that were induced to meiosis by inactivation of Pat1 kinase (a key negative regulator of meiosis). Time-course observation in living cells determined the temporal order of DNA synthesis, telomere clustering, centromere separation and meiotic chromosome segregation. When meiosis was induced by Pat1 inactivation at the G1 phase of mitosis, telomeres clustered to the SPB as per normal meiosis, but in most cells the centromeres remained partially associated with the SPB. When meiosis was initiated at the G2 phase by Pat1 inactivation, both telomeres and centromeres retained their mitotic nuclear positions in the majority of cells. These results indicate that the progression of meiosis induced by Pat1 inactivation is aberrant from normal meiosis in some events. As Pat1 inactivation is often useful to induce S. pombe cells synchronously into meiosis, the temporal order of chromosomal events determined here will provide landmarks for the progression of meiosis downstream the Pat1 inactivation. [source] Combining adenoviral oncolysis with temozolomide improves cell killing of melanoma cellsINTERNATIONAL JOURNAL OF CANCER, Issue 12 2007Christina Quirin Abstract Oncolytic Adenoviruses are emerging agents for treatment of cancer by tumor-restricted virus replication, cell lysis and virus spread. Clinical studies with first generation oncolytic adenoviruses have revealed that an increased potency is warranted in order to achieve therapeutic efficacy. One approach towards this end is to combine adenoviral oncolysis with chemotherapy. Here, a fundamental requirement is that chemotherapy does not interfere with adenovirus replication in cancer cells. We have previously developed a melanoma-targeted oncolytic adenovirus, Ad5/3.2xTyr, which features tyrosinase promoter regulated replication and enhanced cell entry into melanoma cells. In this study, we investigated a combination treatment of melanoma cells with Ad5/3.2xTyr and temozolomide (TMZ), which produces the same active metabolite as Dacarbazine/DTIC, the standard chemotherapy for advanced melanoma. We report that TMZ does not inhibit adenovirus replication in melanoma cells. Additive or synergistic cell killing of melanoma cells, dependent on the cell line used, was observed. Enhanced cell binding was not responsible for synergism of adenoviral oncolysis and TMZ treatment. We rather observed that higher numbers of virus genomes are produced in TMZ-treated cells, which also showed a cell cycle arrest in the G2 phase. Our results have important implications for the clinical implementation of adenoviral oncolysis for treatment of malignant melanoma. It suggests that such studies are feasible in the presence of TMZ or DTIC chemotherapy and recommends the investigation of a viro-chemo combination therapy. © 2007 Wiley-Liss, Inc. [source] Phosphorylation by COP9 Signalosome-Associated CK2 Promotes Degradation of p27 during the G1 Cell Cycle PhaseISRAEL JOURNAL OF CHEMISTRY, Issue 2 2006Xiaohua Huang The cell cycle regulator p27Kip1 (p27) is controlled by 26S proteasome-mediated proteolysis by two different pathways. From the S till the G2 phase of the cell cycle, degradation of p27 takes place in the nucleus and is initiated by CDK2-dependent phosphorylation of threonine 187 with subsequent ubiquitination by the SCFSkp2 ubiquitin ligase. During the G1 cell cycle phase (G1), p27 breakdown is cytosolic and is initiated by nuclear export with subsequent ubiquitination by a RING finger ligase called kip1 ubiquitination complex. Here we show that the COP9 signalosome (CSN) is a regulator of p27 proteolysis during G1. The CSN interacts with p27 and the CSN-associated kinase CK2 phosphorylates p27 at two regions. One is central to the protein (amino acids 101,113), and the other was mapped near to the C-terminus (amino acids 170,189). Elimination of the putative C-terminal phosphorylation sites stabilizes ectopic p27 towards proteasomal degradation and abolishes CSN,p27 binding. Inhibition of CSN-associated kinase activity by curcumin attenuates loss of p27 upon cell cycle re-entry. Similar but not additive effects of the phosphoinositol-3-kinase blocker LY 290042 may point to a common pathway of CSN-associated CK2 and protein kinase B/Akt (Akt) in regulating p27 abundance. Akt is found in Flag pulldowns of lysates obtained from cells permanently expressing Flag-tagged CSN2, indicating that Akt is a novel kinase associated with the CSN. Thus, the CSN seems to regulate p27 proteolysis at G1 downstream of Ras-mediated signal pathways. [source] 2-methoxyestradiol-induced cell death in osteosarcoma cells is preceded by cell cycle arrestJOURNAL OF CELLULAR BIOCHEMISTRY, Issue 5 2008Avudaiappan Maran Abstract 2-Methoxyestradiol (2-ME), a naturally occurring mammalian metabolite of 17,-Estradiol (E2), induces cell death in osteosarcoma cells. To further understand the molecular mechanisms of action, we have investigated cell cycle progression in 2-ME-treated human osteosarcoma (MG63, SaOS-2 and LM8) cells. At 5 µM, 2-ME induced growth arrest by inducing a block in cell cycle; 2-ME-treatment resulted in 2-fold increases in G1 phase cells and a decrease in S phase cells in MG63 and SaOS-2 osteosarcoma cell lines, compared to the appropriate vehicle controls. 2-ME-treatment induced a threefold increase in the G2 phase in LM8 osteosarcoma cells. The results demonstrated steroid specificity, as the tumorigenic metabolite, 16,-hydroxyestradiol (16-OHE), did not have any effect on cell cycle progression in osteosarcoma cells. The cell cycle arrest coincided with an increase in expression of the cell cycle markers p21, p27 and p53 proteins in 2-ME-treated osteosarcoma cells. Also, MG63 cells, transiently transfected with cDNA for a ,loss of function mutant' RNA-dependent protein kinase (PKR) protein, were resistant to 2-ME-induced cell cycle arrest. These results suggest that 2-ME works in concert with factors regulating cell cycle progression, and cell cycle arrest precedes cell death in 2-ME-treated osteosarcoma cells. J. Cell. Biochem. 104: 1937,1945, 2008. © 2008 Wiley-Liss, Inc. [source] Cell cycle checkpoints and their impact on anticancer therapeutic strategiesJOURNAL OF CELLULAR BIOCHEMISTRY, Issue 2 2004Alan Eastman Abstract Cells contain numerous pathways designed to protect them from the genomic instability or toxicity that can result when their DNA is damaged. The p53 tumor suppressor is particularly important for regulating passage through G1 phase of the cell cycle, while other checkpoint regulators are important for arrest in S and G2 phase. Tumor cells often exhibit defects in these checkpoint proteins, which can lead to hypersensitivity; proteins in this class include ataxia,telangiectasia mutatated (ATM), Meiotic recanbination 11 (Mre11), Nijmegen breakage syndrome 1 (Nbs 1), breast cancer susceptibility genes 1 and 2 (BRCA1), and (BRCA2). Consequently, tumors should be assessed for these specific defects, and specific therapy prescribed that has high probability of inducing response. Tumors defective in p53 are frequently considered resistant to apoptosis, yet this defect also provides an opportunity for targeted therapy. When their DNA is damaged, p53-defective tumor cells preferentially arrest in S or G2 phase where they are susceptible to checkpoint inhibitors such as caffeine and UCN-01. These inhibitors preferentially abrogate cell cycle arrest in p53-defective cells, driving them through a lethal mitosis. Wild type p53 can prevent abrogation of arrest by elevating levels of p21waf1 and decreasing levels of cyclins A and B. During tumorigenesis, tumor cells frequently loose checkpoint controls and this facilitates the development of the tumor. However, these defects also represent an Achilles heel that can be targeted to improve current therapeutic strategies. © 2003 Wiley-Liss, Inc. [source] Pro-inflammatory cytokine production from normal human fibroblasts is induced by Tannerella forsythia detaching factorJOURNAL OF PERIODONTAL RESEARCH, Issue 2 2008N. Tomi Background and Objective:,Tannerella forsythia is a periodontal pathogen. Recently, we have reported that the cytopathic component of T. forsythia contains two distinct factors. One arrests the cell cycle at the G2 phase and the other, named forsythia detaching factor, detaches adhesion-dependent immortalized human cells. In this study, we investigated the biological function of forsythia detaching factor using human normal fibroblasts. Material and Methods:, A recombinant forsythia detaching factor, reported previously, was used. TIG-3 cells, cultured in the absence or presence of forsythia detaching factor, were lysed and the supernatant was analyzed by western blotting with polyclonal forsythia detaching factor antibodies. The cells were subsequently fractionated to isolate the cytoplasmic, mitochondrial and remaining fractions. In order to measure the activity of mitochondria using nicotinamide adenine dinucleotide-linked reductase, the water-soluble tetrazolium method was used. The mitochondrial oxidative membrane potential was estimated by measuring the oxidization-dependent fluorogenic conversion of dihydrotetramethylrosamine using flow cytometry. The concentration of interleukin-8 in the culture supernatant was assayed using a Human IL-8 ELISA kit. Results:, Forsythia detaching factor-treated cells detached from the substratum and aggregated from 3 to 24 h. Then, the detached cells resumed adhesion and proliferated after 48 h. The western blot analysis revealed that most forsythia detaching factor trans -located into the mitochondrial fraction. Forsythia detaching factor suppressed the nicotinamide adenine dinucleotide-linked reductase activity in a dose-dependent manner and consequently increased the mitochondrial oxidative membrane potential. The production of interleukin-8 was reinforced in forsythia detaching factor-treated cells at 72 h through an increase of the mitochondrial oxidative membrane potential. Conclusion:, The forsythia detaching factor might be involved in the virulence of T. forsythia through induction of the pro-inflammatory cytokine interleukin-8. [source] The effect of the ionic products of Bioglass® dissolution on human osteoblasts growth cycle in vitroJOURNAL OF TISSUE ENGINEERING AND REGENERATIVE MEDICINE, Issue 4 2007Jun-Ying Sun Abstract In this study, in order to observe the effect of Bioglass® and its ionic products on human esteoblasts growth cycle in vitro, the ionic products of Bioglass have been introduced to a cell culture medium by dissolving Bioglass particles in Dulbecco's modified Eagle's medium (DMEM) at 37 °C for 24 h; this was used as the experimental medium, while DMEM without Bioglass modification was used as the control medium. Human osteoblasts isolated from trabecular bone were treated by the two media and the timing of the osteoblast growth cycle was examined. Cell growth curves were derived after 7 days. Also, human osteoblasts were treated for 1,6 days by the two media, and the G1, S, G2 phase percentages of osteoblasts were recorded by flow cytometry every day, resulting in the cell proliferation activity index: SPF (S-phase fraction) and PI (proliferation index). The difference in cell growth was shown after the second day of culture (p < 0.01), and cell growth in the experimental groups was greater than in control groups. The SPF and PI of the experimental groups were also higher than the control groups in 2 days of culture (p < 0.05 and p < 0.01), which indicates that the growth cycle of the human osteoblasts in experimental medium is about 2 days. In conclusion, Bioglass can promote osteoblast proliferation, reducing the human osteoblast growth cycle to pass through G1 and S phase and then enter G2 phase quickly. Copyright © 2007 John Wiley & Sons, Ltd. [source] Increased wild-type p53-induced phosphatase 1 (Wip1 or PPM1D) expression correlated with downregulation of checkpoint kinase 2 in human gastric carcinomaPATHOLOGY INTERNATIONAL, Issue 9 2007Takeichi Fuku Phosphorylation of checkpoint kinase 2 (Chk2) at Thr68 (pChk2) induced by DNA double-strand breaks is required for inhibition of cell cycle progression in the G2 phase. The purpose of the present paper was to investigate the expression of wild-type p53-induced phosphatase 1 (Wip1 or PPM1D), a negative regulator of Chk2, to better understand its role in human gastric cancer. In non-neoplastic gastric mucosa, most epithelial cells exhibited Wip1-positive and pChk2-negative immunoreactivity, whereas an inverse pattern of protein expression was detected at the surface of the foveolar epithelium. In tumor tissues, 74% of 53 gastric cancers had intense Wip1 immunoreactivity and close correlation with both tumor size (P = 0.0497) and Chk2 dephosphorylation (P = 0.0213). In MKN-74 gastric cancer cells, ionizing radiation (IR)-induced Wip1 upregulation was detected at protein levels, but the Chk2-mediated cell cycle regulatory mechanism was disrupted. In addition, protease inhibitor Z-Leu-Leu-Leu (ZLLL) effectively upregulated Wip1 levels in the presence or absence of IR, suggesting that Wip1 expression can be modulated post-transcriptionally. Understanding the Wip1-mediated signaling pathway in gastric cancer may provide useful information for the development of new chemo- and radiotherapies. [source] Hypericin-mediated Photocytotoxic Effect on HT-29 Adenocarcinoma Cells Is Reduced by Light Fractionation with Longer Dark Pause Between Two Unequal Light DosesPHOTOCHEMISTRY & PHOTOBIOLOGY, Issue 6 2005Veronika Sa ABSTRACT The present study demonstrates the in vitro effect of hypericin-mediated PDT with fractionated light delivery. Cells were photosensitized with unequal light fractions separated by dark intervals (1 or 6 h). We compared the changes in viability, cell number, survival, apoptosis and cell cycle on HT-29 cells irradiated with a single light dose (12 J/cm2) to the fractionated light delivery (1 + 11 J/cm2) 24 and 48 h after photodynamic treatment. We found that a fractionated light regime with a longer dark period resulted in a decrease of hypericin cytotoxicity. Both cell number and survival were higher after light sensitization with a 6-h dark interval. DNA fragmentation occurred after a single light-dose application, but in contrast no apoptotic DNA formation was detected with a 6-h dark pause. After fractionation the percentage of cells in the G1 phase of the cell cycle was increased, while the proportion of cells in the G2 phase decreased as compared to a single light-dose application, i.e. both percentage of cells in the G1 and G2 phase of the cell cycle were near control levels. We presume that the longer dark interval after the irradiation of cells by first light dose makes them resistant to the effect of the second illumination. These findings confirm that the light application scheme together with other photodynamic protocol components is crucial for the photocytotoxicity of hypericin. [source] High temperature causes arrest of cell cycle in G2 phase in BmN cells derived from the silkworm, Bombyx moriPHYSIOLOGICAL ENTOMOLOGY, Issue 3 2007TAKASHI KIUCHI Abstract The influence of temperature on the insect cell line, BmN, derived from the silkworm, Bombyx mori is investigated. These cells proliferate at an accelerated pace as the temperature increases from 22 to 30 °C, but the growth rate slows at 34 °C, and proliferation stops at 38 °C. At high temperatures, abnormal cellular morphology is observed. Cells treated at 38 °C have cytoplasmic bilateral protrusions and they gradually aggregate and float in the medium. BmN cells without proliferation at 38 °C are viable but have reduced DNA synthesis. At high temperatures, the cell cycle of BmN cells halts at the G2 phase. After heat treatment of the larvae, an accumulation of larval haemocytes with high DNA content is found, which suggests that the cell cycle arrest at G2 also occurs in the silkworm at high temperatures. [source] Tobacco BY-2 cells expressing fission yeast cdc25 bypass a G2/M block on the cell cycleTHE PLANT JOURNAL, Issue 2 2005Craig B. Orchard Summary The mitotic inducer gene from Schizosaccharomyces pombe, Spcdc25, was used as a tool to investigate regulation of G2/M in higher plants using the BY-2 (Nicotiana tabacum) cell line as a model. Spcdc25 -expressing BY-2 cells exhibited a reduced mitotic cell size through a shortening of the G2 phase. The cells often formed isodiametric double files both in BY-2 cells and in cell suspensions derived from 35S::Spcdc25 tobacco plants. In Spcdc25 -expressing cells, the tobacco cyclin-dependent kinase, NtCDKB1, showed high activity in early S phase, S/G2 and early M phase, whereas in empty vector cells CDKB1 activity was transiently high in early S phase but thereafter remained lower. Spcdc25- expressing cells also bypassed a block on G2/M imposed by the cytokinin biosynthetic inhibitor lovastatin (LVS). Surprisingly, cytokinins were at remarkably low levels in Spcdc25 -expressing cells compared with the empty vector, explaining why these cells retained mitotic competence despite the presence of LVS. In conclusion, synchronised Spcdc25 -expressing BY-2 cells divided prematurely at a small cell size, and they exhibited premature, but sustained, CDKB1 activity even though endogenous cytokinins were virtually undetectable. [source] The role of auxin-binding protein 1 in the expansion of tobacco leaf cellsTHE PLANT JOURNAL, Issue 6 2001Jin-Gui Chen Summary Tobacco leaf was used to investigate the mechanism of action of auxin-binding protein 1 (ABP1). The distributions of free auxin, ABP1, percentage of leaf nuclei in G2 and the amount of auxin-inducible growth were each determined in control tobacco leaves and leaves over-expressing Arabidopsis ABP1. These parameters were compared with growth of tobacco leaves, measured both spatially and temporally throughout the entire expansion phase. Within a defined window of leaf development, juvenile leaf cells that inducibly expressed Arabidopsis ABP1 prematurely advanced nuclei to the G2 phase. The ABP1-induced increase in cell expansion occured before the advance to the G2 phase, indicating that the ABP1-induced G2 phase advance is an indirect effect of cell expansion. The level of ABP1 was highest at the position of maximum cell expansion, maximum auxin-inducible growth and where the free auxin level was the lowest. In contrast, the position of maximum cell division correlated with higher auxin levels and lower ABP1 levels. Consistent with the correlations observed in leaves, tobacco cells (BY-2) in culture displayed two dose-dependent responses to auxin. At a low auxin concentration, cells expanded, while at a relatively higher concentration, cells divided and incorporated [3H]-thymidine. Antisense suppression of ABP1 in these cells dramatically reduced cell expansion with negligible effect on cell division. Taken together, the data suggest that ABP1 acts at a relatively low level of auxin to mediate cell expansion, whereas high auxin levels stimulate cell division via an unidentified receptor. [source] Damage-induced reactivation of cohesin in postreplicative DNA repairBIOESSAYS, Issue 1 2008Alexander R. Ball Jr Cohesin establishes sister-chromatid cohesion during S phase to ensure proper chromosome segregation in mitosis. It also facilitates postreplicative homologous recombination repair of DNA double-strand breaks by promoting local pairing of damaged and intact sister chromatids. In G2 phase, cohesin that is not bound to chromatin is inactivated, but its reactivation can occur in response to DNA damage. Recent papers by Koshland's and Sjögren's groups describe the critical role of the known cohesin cofactor Eco1 (Ctf7) and ATR checkpoint kinase in damage-induced reactivation of cohesin, revealing an intricate mechanism that regulates sister-chromatid pairing to maintain genome integrity.1,2 BioEssays 30:5,9, 2008. © 2007 Wiley Periodicals, Inc. [source] Block to DNA replication in meiotic maturation: a unified view for a robust arrest of cell cycle in oocytes and somatic cellsBIOESSAYS, Issue 4 2003Yumiko Kubota Under certain conditions, the cell cycle can be arrested for a long period of time. Vertebrate oocytes are arrested at G2 phase, while somatic cells arrest at G0 phase. In both cells, nuclei have lost the ability to initiate DNA synthesis. In a pair of recently published papers,1,2 Méchali and colleagues and Coué and colleagues have clarified how frog oocytes prevent untimely DNA synthesis during the long G2 arrest. Intriguingly, they found only Cdc6 is responsible for the inability of immature oocytes to replicate DNA. Cdc6 is a key component for replication licensing, and for G0 cells to re-enter the proliferative stage. Strikingly similar strategies for preventing the untimely replication in both cells suggest that the suppression of replication licensing is a universal mechanism for securing the prolonged arrest of the cell cycle. BioEssays 25:313,316, 2003. © 2003 Wiley Periodicals, Inc. [source] Effect of ultraviolet (UV) A, UVB or ionizing radiation on the cell cycle of human melanoma cellsBRITISH JOURNAL OF DERMATOLOGY, Issue 5 2007M. Placzek Summary Background, One important component of the cellular response to irradiation is the activation of cell cycle checkpoints. It is known that both ultraviolet (UV) radiation and ionizing radiation (IR) can activate checkpoints at transitions from G1 to S phase, from G2 phase to mitosis and during DNA replication. Objectives, To evaluate the effects of irradiation with different wavelengths on cell cycle alterations. Methods, p53-deficient IPC-298 melanoma cells were irradiated with 10 J cm,2 UVA, 40 mJ cm,2 UVB, or with 7·5 Gy IR. Cell cycle effects were then determined by DNA/5-bromodeoxyuridine dual-parameter flow cytometry. Results, IPC-298 cells irradiated in G1 with UVA were not arrested at the G1/S transition, but at the G2/M transition. Despite p53 deficiency, the cells showed a G1 arrest after UVB exposure. Furthermore, IR did not affect G1 or S phase, but induced G2 phase arrest. Hence, the effects of UVA, but not of UVB, on the cell cycle in p53-deficient melanoma cells are comparable with those of IR. Conclusions, UVA and IR induce radical-mediated strand breaks and DNA lesions, and UVB essentially induces thymine dimers that lead to excision repair-related strand breaks. Different cell cycle effects may be a consequence of different types of DNA damage. The results showed that UVB-irradiated p53-deficient cells are arrested in G1. Irradiation with the solar radiation component UVB can therefore result in a beneficial retardation of tumour promotion in human skin carrying p53-mutated cell clones. [source] Oxaliplatin induces mitotic catastrophe and apoptosis in esophageal cancer cellsCANCER SCIENCE, Issue 1 2008Chew Yee Ngan The platinum-based chemotherapeutic agent oxaliplatin displays a wide range of antitumor activities. However, the underlying molecular responses to oxaliplatin in esophageal cancer remain largely unknown. In the present study, we investigated the effect of oxaliplatin on two esophageal cancer cell lines, squamous cell carcinoma (TE3) and adenocarcinoma (TE7). Following cell-cycle arrest at G2 phase after oxaliplatin treatment, TE3 cells died via apoptosis and TE7 cells died via mitotic catastrophe. Survivin was inhibited more in TE7 cells compared with TE3 cells, but inhibition of survivin using small interfering RNA induced mitotic catastrophe in both cell lines. Further investigations indicated that survivin promoter activity was also inhibited by oxaliplatin. Among mitotic catastrophe-associated proteins, 14,3-3, was decreased in TE7 cells; no evident changes were observed for aurora kinases. Oxaliplatin-induced apoptosis in the TE3 cells was caspase dependent. However, downregulation of Bad, Bid, Puma, and Noxa, lack of cytochrome c release, and limited loss of mitochondrial membrane potential in early phase indicated possible initiation by pathways other than the mitochondrial pathway. Mechanistic studies showed that downregulation of survivin by oxaliplatin in TE7 cells was partially due to the proteasome-mediated protein degradation pathway and partially due to the downregulation of Sp1 transcription factor. Similar results were obtained for another gastric adenocarcinoma cell line, MKN45, in which survivin was previously shown to be inhibited by oxaliplatin. These data indicate that survivin may be a key target for oxaliplatin. The ability of oxaliplatin to induce different modes of cell death may contribute to its efficacy in esophageal cancer. (Cancer Sci 2008; 99: 129,139) [source] Effects of PKC , on early genome transcription activation in mouse 1-cell stage fertilized eggsCELL BIOCHEMISTRY AND FUNCTION, Issue 6 2007Bing-zhi Yu Abstract Effects of PKC , on the activation of embryonic transcription in 1-cell stage fertilized mouse eggs were explored. The effects of PKC antagonist calphostin C and PKC , specific inhibitor on the activation of embryonic early transcription were observed by Western blotting and cell immunofluorescence. PKC activity increased gradually from G1 phase to late G2 phase in mouse 1-cell stage fertilized eggs, and reached a maximum in G2 stage. Calphostin C inhibited PKC activity by about 47% in 1-cell stage fertilized eggs. Calphostin C inhibited early transcription in 1-cell stage fertilized eggs (p,<,0.01). PKC ,-Thr410 in G2 were about 27% and 110% higher than those in G1 phase of 1-cell stage fertilized eggs and MII oocytes, respectively. PKC , specific inhibitor can also inhibit early transcription in 1-cell stage fertilized eggs (p,<,0.05). The results suggest that PKC , participates in early transcription activation in mouse 1-cell stage fertilized eggs. Copyright © 2006 John Wiley & Sons, Ltd. [source] Activation of extracellular signal-regulated kinase (ERK) in G2 phase delays mitotic entry through p21CIP1CELL PROLIFERATION, Issue 4 2006S. Dangi In contrast, the role of extracellular signal-regulated kinase during G2 phase and mitosis (M phase) is largely undefined. Previous studies have suggested that inhibition of basal extracellular signal-regulated kinase activity delays G2 - and M-phase progression. In the current investigation, we have examined the consequence of activating the extracellular signal-regulated kinase pathway during G2 phase on subsequent progression through mitosis. Using synchronized HeLa cells, we show that activation of the extracellular signal-regulated kinase pathway with phorbol 12-myristate 13-acetate or epidermal growth factor during G2 phase causes a rapid cell cycle arrest in G2 as measured by flow cytometry, mitotic indices and cyclin B1 expression. This G2 -phase arrest was reversed by pre-treatment with bisindolylmaleimide or U0126, which are selective inhibitors of protein kinase C proteins or the extracellular signal-regulated kinase activators, MEK1/2, respectively. The extracellular signal-regulated kinase-mediated delay in M-phase entry appeared to involve de novo synthesis of the cyclin-dependent kinase inhibitor, p21CIP1, during G2 through a p53-independent mechanism. To establish a function for the increased expression of p21CIP1 and delayed cell cycle progression, we show that extracellular signal-regulated kinase activation in G2 -phase cells results in an increased number of cells containing chromosome aberrations characteristic of genomic instability. The presence of chromosome aberrations following extracellular signal-regulated kinase activation during G2 -phase was further augmented in cells lacking p21CIP1. These findings suggest that p21CIP1 mediated inhibition of cell cycle progression during G2/M phase protects against inappropriate activation of signalling pathways, which may cause excessive chromosome damage and be detrimental to cell survival. [source] Role and regulation of human XRCC4-like factor/cernunnos,JOURNAL OF CELLULAR BIOCHEMISTRY, Issue 5 2008Kirsten Dahm Abstract In mammalian cells, non-homologous end joining (NHEJ) is the major double strand break (DSB) repair mechanism during the G1 phase of the cell cycle. It also contributes to DSB repair during the S and G2 phases. Ku heterodimer, DNA PKcs, XRCC4 and DNA Ligase IV constitute the core NHEJ machinery, which joins directly ligatable ends. XRCC4-like factor/Cernunnos (XLF/Cer) is a recently discovered interaction partner of XRCC4. Current evidence suggests the following model for the role of XLF/Cer in NHEJ: after DSB induction, the XRCC4-DNA Ligase IV complex promotes efficient accumulation of XLF/Cer at DNA damage sites via constitutive interaction of the XRCC4 and XLF/Cer head domains and dependent on components of the DNA PK complex. Ku alone can stabilise the association of XLF/Cer with DNA ends. XLF/Cer stimulates ligation of complementary and non-complementary DNA ends by XRCC4-DNA Ligase IV. This activity involves the carboxy-terminal DNA binding region of XLF/Cer and could occur via different, non-exclusive modes: (i) enhancement of the stability of the XRCC4-DNA Ligase IV complex on DNA ends by XLF/Cer, (ii) modulation of the efficiency and/or specificity of DNA Ligase IV by binding of XLF/Cer to the XRCC4-DNA Ligase IV complex, (iii) promotion of the alignment of blunt or other non-complementary DNA ends by XLF/Cer for ligation. XLF/Cer promotes the preservation of 3, overhangs, restricts nucleotide loss and thereby promotes accuracy of DSB joining by XRCC4-DNA Ligase IV during NHEJ and V(D)J recombination. J. Cell. Biochem. 104: 1534,1540, 2008. © 2008 Wiley-Liss, Inc. [source] A role for protein kinase CK2 in plant development: evidence obtained using a dominant-negative mutantTHE PLANT JOURNAL, Issue 1 2008Jordi Moreno-Romero Summary Protein kinase CK2 is an evolutionary conserved Ser/Thr phosphotransferase composed of two distinct subunits, , (catalytic) and , (regulatory), that combine to form a tetrameric complex. Plant genomes contain multiple genes for each subunit, the expression of which gives rise to different active holoenzymes. In order to study the effects of loss of function of CK2 on plant development, we have undertaken a dominant-negative mutant approach. We generated an inactive catalytic subunit by site-directed mutagenesis of an essential lysine residue. The mutated open reading frame was cloned downstream of an inducible promoter, and stably transformed Arabidopsis thaliana plants and tobacco BY2 cells were isolated. Continuous expression of the CK2 kinase-inactive subunit did not prevent seed germination, but seedlings exhibited a strong phenotype, affecting chloroplast development, cotyledon expansion, and root and shoot growth. Prolonged induction of the transgene was lethal. Moreover, dark-germinated seedlings exhibited an apparent de-etiolated phenotype that was not caused by disruption of the light-signalling pathways. Short-term induction of the CK2 kinase-inactive subunit allowed plant survival, but root growth and lateral root formation were significantly affected. The expression pattern of CYCB1;1::GFP in the root meristems of mutant plants demonstrated an important decrease of mitotic activity, and expression of the CK2 kinase-inactive subunit in stably transformed BY2 cells provoked perturbation of the G1/S and G2 phases of the cell cycle. Our results are consistent with a model in which CK2 plays a key role in cell division and cell expansion, with compelling effects on Arabidopsis development. [source] Heat shock-induced arrests in different cell cycle phases of rat C6-glioma cells are attenuated in heat shock-primed thermotolerant cellsCELL PROLIFERATION, Issue 3 2000N. M. Kühl The response kinetics of rat C6 glioma cells to heat shock was investigated by means of flow cytometric DNA measurements and western blot analysis of HSP levels. The results showed that the effects on cell cycle progression are dependent on the cell cycle phase at which heat shock is applied, leading to either G1 or G2/M arrest in randomly proliferating cells. When synchronous cultures were stressed during G0 they were arrested with G1 DNA content and showed prolongation of S and G2 phases after release from the block. In proliferating cells, HSC70 and HSP68 were induced during the recovery and reached maximum levels just before cells were released from the cell cycle blocks. Hyperthermic pretreatment induced thermotolerance both in asynchronous and synchronous cultures as evidenced by the reduced arrest of cell cycle progression after the second heat shock. Thermotolerance development was independent of the cell cycle phase. Pre-treated cells already had high HSP levels and did not further increase the amount of HSP after the second treatment. However, as in unprimed cells, HSP reduction coincided with the release from the cell cycle blocks. These results imply that the cell cycle machinery can be rendered thermotolerant by heat shock pretreatment and supports the assumption that HSP70 family members might be involved in thermotolerance development. [source] | |||||||||||||||||||||||||