Growth-arrest-specific protein 2 inhibits cell division in Xenopus embryos.

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Tong Zhang

Full Text Available Growth-arrest-specific 2 gene was originally identified in murine fibroblasts under growth arrest conditions. Furthermore, serum stimulation of quiescent, non-dividing cells leads to the down-regulation of gas2 and results in re-entry into the cell cycle. Cytoskeleton rearrangements are critical for cell cycle progression and cell division and the Gas2 protein has been shown to co-localize with actin and microtubules in interphase mammalian cells. Despite these findings, direct evidence supporting a role for Gas2 in the mechanism of cell division has not been reported.To determine whether the Gas2 protein plays a role in cell division, we over-expressed the full-length Gas2 protein and Gas2 truncations containing either the actin-binding CH domain or the tubulin-binding Gas2 domain in Xenopus laevis embryos. We found that both the full-length Gas2 protein and the Gas2 domain, but not the CH domain, inhibited cell division and resulted in multinucleated cells. The observation that Gas2 domain alone can arrest cell division suggests that Gas2 function is mediated by microtubule binding. Gas2 co-localized with microtubules at the cell cortex of Gas2-injected Xenopus embryos using cryo-confocal microscopy and co-sedimented with microtubules in cytoskeleton co-sedimentation assays. To investigate the mechanism of Gas2-induced cell division arrest, we showed, using a wound-induced contractile array assay, that Gas2 stabilized microtubules. Finally, electron microscopy studies demonstrated that Gas2 bundled microtubules into higher-order structures.Our experiments show that Gas2 inhibits cell division in Xenopus embryos. We propose that Gas2 function is mediated by binding and bundling microtubules, leading to cell division arrest.

Effects of low priming dose irradiation on cell cycle arrest of HepG2 cells caused by high dose irradiation

International Nuclear Information System (INIS)

Xia Jingguang; Jin Xiaodong; Chinese Academy of Sciences, Beijing; Li Wenjian; Wang Jufang; Guo Chuanling; Gao Qingxiang

2005-01-01

Human hepatoma cells hepG2 were irradiated twice by 60 Co γ-rays with a priming dose of 5 cGy and a higher dose of 3 Gy performed 4h or 8h after the low dose irradiation. Effects of the priming dose irradiation on cell cycle arrest caused by high dose were examined with flow cytometry. Cells in G 2 /M phase accumulated temporarily after the 5 cGy irradiation, and proliferation of tumor cells was promoted significantly by the low dose irradiation. After the 3 Gy irradiation, G 2 phase arrest occurred, and S phase delayed temporally. In comparison with 3 kGy irradiation only, the priming dose delivered 4h prior to the high dose irradiation facilitated accumulation of hepG2 cells in G 2 /M phase, whereas the priming dose delivered 8h prior to the high dose irradiation helped the cells to overcome G 2 arrest. It was concluded that effects of the priming dose treatment on cell cycle arrest caused by high dose irradiation were dependent on time interval between the two irradiations. (authors)

5-(2-Carboxyethenyl) isatin derivative induces G2/M cell cycle arrest and apoptosis in human leukemia K562 cells

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Zhou, Yao; Zhao, Hong-Ye; Han, Kai-Lin; Yang, Yao; Song, Bin-Bin; Guo, Qian-Nan; Fan, Zhen-Chuan; Zhang, Yong-Min; Teng, Yu-Ou; Yu, Peng

2014-01-01

Highlights: • 5-(2-Carboxyethenyl) isatin derivative (HKL 2H) inhibited K562’s proliferation. • HKL 2H caused the morphology change of G 2 /M phase arrest and typical apoptosis. • HKL 2H induced G2/M cell cycle phase arrest in K562 cells. • HKL 2H induced apoptosis in K562 cells through the mitochondrial pathway. - Abstract: Our previous study successfully identified that the novel isatin derivative (E)-methyl 3-(1-(4-methoxybenzyl)-2,3-dioxoindolin-5-yl) acrylate (HKL 2H) acts as an anticancer agent at an inhibitory concentration (IC 50 ) level of 3 nM. In this study, the molecular mechanism how HKL 2H induces cytotoxic activity in the human chronic myelogenous leukemia K562 cells was investigated. Flow cytometric analysis showed that the cells were arrested in the G 2 /M phase and accumulated subsequently in the sub-G 1 phase in the presence of HKL 2H. HKL 2H treatment down-regulated the expressions of CDK1 and cyclin B but up-regulated the level of phosphorylated CDK1. Annexin-V staining and the classic DNA ladder studies showed that HKL 2H induced the apoptosis of K562 cells. Our study further showed that HKL 2H treatment caused the dissipation of mitochondrial membrane potential, activated caspase-3 and lowered the Bcl-2/Bax ratio in K562 cells, suggesting that the HKL 2H-causing programmed cell death of K562 cells was caused via the mitochondrial apoptotic pathway. Taken together, our data demonstrated that HKL 2H, a 5-(2-carboxyethenyl) isatin derivative, notably induces G 2 /M cell cycle arrest and mitochondrial-mediated apoptosis in K562 cells, indicating that this compound could be a promising anticancer candidate for further investigation

Methyl Sartortuoate Inhibits Colon Cancer Cell Growth by Inducing Apoptosis and G2/M-Phase Arrest.

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Lan, Qiusheng; Li, Shoufeng; Lai, Wei; Xu, Heyang; Zhang, Yang; Zeng, Yujie; Lan, Wenjian; Chu, Zhonghua

2015-08-17

The potential anti-neoplastic activity of terpenoids is of continued interest. In this study, we investigate whether methyl sartortuoate, a terpenoid isolated from soft coral, induced cell cycle arrest and apoptosis in a human colon cancer cell line. Culture studies found that methyl sartortuoate inhibited colon cancer cell (LoVo and RKO) growth and caused apoptotic death in a concentration- and time-dependent manner, by activation of caspase-8, caspase-9, caspase-3, p53 and Bax, and inactivation of B-cell lymphoma 2 (Bcl-2) apoptosis regulating proteins. Methyl sartortuoate treatment led to reduced expression of cdc2 and up-regulated p21 and p53, suggesting that Methyl sartortuoate induced G2-M arrest through modulation of p53/p21/cdc2 pathways. Methyl sartortuoate also up-regulated phospho-JNK and phospho-p38 expression levels. This resulted in cell cycle arrest at the G2-M phase and apoptosis in LoVo and RKO cells. Treatment with the JNK inhibitor SP600125 and the p38 MAPK inhibitor SB203580 prevented methyl sartortuoate-induced apoptosis in LoVo cells. Moreover, methyl sartortuoate also prevented neoplasm growth in NOD-SCID nude mice inoculated with LoVo cells. Taken together, these findings suggest that methyl sartortuoate is capable of leading to activation of caspase-8, -9, -3, increasing p53 and Bax/Bcl-2 ratio apoptosis through MAPK-dependent apoptosis and results in G2-M phase arrest in LoVo and RKO cells. Thus, methyl sartortuoate may be a promising anticancer candidate.

Effects of Chk1 inhibition on the temporal duration of radiation-induced G2 arrest in HeLa cells

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Nahar, Kamrun; Goto, Tatsuaki; Kaida, Atsushi; Deguchi, Shifumi; Miura, Masahiko

2014-01-01

Chk1 inhibitor acts as a potent radiosensitizer in p53-deficient tumor cells by abrogating the G2/M check-point. However, the effects of Chk1 inhibitor on the duration of G2 arrest have not been precisely analyzed. To address this issue, we utilized a cell-cycle visualization system, fluorescent ubiquitination-based cell-cycle indicator (Fucci), to analyze the change in the first green phase duration (FGPD) after irradiation. In the Fucci system, G1 and S/G2/M cells emit red and green fluorescence, respectively; therefore, G2 arrest is reflected by an elongated FGPD. The system also allowed us to differentially analyze cells that received irradiation in the red or green phase. Cells irradiated in the green phase exhibited a significantly elongated FGPD relative to cells irradiated in the red phase. In cells irradiated in either phase, Chk1 inhibitor reduced FGPD almost to control levels. The results of this study provide the first clear information regarding the effects of Chk1 inhibition on radiation-induced G2 arrest, with special focus on the time dimension. (author)

5-(2-Carboxyethenyl) isatin derivative induces G{sub 2}/M cell cycle arrest and apoptosis in human leukemia K562 cells

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Zhou, Yao; Zhao, Hong-Ye; Han, Kai-Lin; Yang, Yao; Song, Bin-Bin; Guo, Qian-Nan [Key Laboratory of Industrial Microbiology, Ministry of Education, College of Biotechnology, Tianjin University of Science and Technology, Tianjin 300457 (China); Tianjin Key Laboratory of Industry Microbiology, College of Biotechnology, Tianjin University of Science and Technology, Tianjin 300457 (China); Fan, Zhen-Chuan [Key Laboratory of Food Nutrition and Safety (Tianjin University of Science and Technology), Ministry of Education, Tianjin 300457 (China); Obesita and Algaegen LLC, College Station, TX 77845 (United States); Zhang, Yong-Min [Université Pierre et Marie Curie-Paris 6, Institut Parisien de Chimie Moléculaire UMR CNRS 8232, 4 Place Jussieu, 75005 Paris (France); Teng, Yu-Ou, E-mail: tyo201485@tust.edu.cn [Key Laboratory of Industrial Microbiology, Ministry of Education, College of Biotechnology, Tianjin University of Science and Technology, Tianjin 300457 (China); Tianjin Key Laboratory of Industry Microbiology, College of Biotechnology, Tianjin University of Science and Technology, Tianjin 300457 (China); Yu, Peng, E-mail: yupeng@tust.edu.cn [Key Laboratory of Industrial Microbiology, Ministry of Education, College of Biotechnology, Tianjin University of Science and Technology, Tianjin 300457 (China); Tianjin Key Laboratory of Industry Microbiology, College of Biotechnology, Tianjin University of Science and Technology, Tianjin 300457 (China)

2014-08-08

Highlights: • 5-(2-Carboxyethenyl) isatin derivative (HKL 2H) inhibited K562’s proliferation. • HKL 2H caused the morphology change of G{sub 2}/M phase arrest and typical apoptosis. • HKL 2H induced G2/M cell cycle phase arrest in K562 cells. • HKL 2H induced apoptosis in K562 cells through the mitochondrial pathway. - Abstract: Our previous study successfully identified that the novel isatin derivative (E)-methyl 3-(1-(4-methoxybenzyl)-2,3-dioxoindolin-5-yl) acrylate (HKL 2H) acts as an anticancer agent at an inhibitory concentration (IC{sub 50}) level of 3 nM. In this study, the molecular mechanism how HKL 2H induces cytotoxic activity in the human chronic myelogenous leukemia K562 cells was investigated. Flow cytometric analysis showed that the cells were arrested in the G{sub 2}/M phase and accumulated subsequently in the sub-G{sub 1} phase in the presence of HKL 2H. HKL 2H treatment down-regulated the expressions of CDK1 and cyclin B but up-regulated the level of phosphorylated CDK1. Annexin-V staining and the classic DNA ladder studies showed that HKL 2H induced the apoptosis of K562 cells. Our study further showed that HKL 2H treatment caused the dissipation of mitochondrial membrane potential, activated caspase-3 and lowered the Bcl-2/Bax ratio in K562 cells, suggesting that the HKL 2H-causing programmed cell death of K562 cells was caused via the mitochondrial apoptotic pathway. Taken together, our data demonstrated that HKL 2H, a 5-(2-carboxyethenyl) isatin derivative, notably induces G{sub 2}/M cell cycle arrest and mitochondrial-mediated apoptosis in K562 cells, indicating that this compound could be a promising anticancer candidate for further investigation.

The inhibition of polo kinase by matrimony maintains G2 arrest in the meiotic cell cycle.

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Youbin Xiang

2007-12-01

Full Text Available Many meiotic systems in female animals include a lengthy arrest in G2 that separates the end of pachytene from nuclear envelope breakdown (NEB. However, the mechanisms by which a meiotic cell can arrest for long periods of time (decades in human females have remained a mystery. The Drosophila Matrimony (Mtrm protein is expressed from the end of pachytene until the completion of meiosis I. Loss-of-function mtrm mutants result in precocious NEB. Coimmunoprecipitation experiments reveal that Mtrm physically interacts with Polo kinase (Polo in vivo, and multidimensional protein identification technology mass spectrometry analysis reveals that Mtrm binds to Polo with an approximate stoichiometry of 1:1. Mutation of a Polo-Box Domain (PBD binding site in Mtrm ablates the function of Mtrm and the physical interaction of Mtrm with Polo. The meiotic defects observed in mtrm/+ heterozygotes are fully suppressed by reducing the dose of polo+, demonstrating that Mtrm acts as an inhibitor of Polo. Mtrm acts as a negative regulator of Polo during the later stages of G2 arrest. Indeed, both the repression of Polo expression until stage 11 and the inactivation of newly synthesized Polo by Mtrm until stage 13 play critical roles in maintaining and properly terminating G2 arrest. Our data suggest a model in which the eventual activation of Cdc25 by an excess of Polo at stage 13 triggers NEB and entry into prometaphase.

Sterigmatocystin-induced DNA damage triggers G2 arrest via an ATM/p53-related pathway in human gastric epithelium GES-1 cells in vitro.

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Donghui Zhang

Full Text Available Sterigmatocystin (ST, which is commonly detected in food and feed commodities, is a mutagenic and carcinogenic mycotoxin that has been recognized as a possible human carcinogen. Our previous study showed that ST can induce G2 phase arrest in GES-1 cells in vitro and that the MAPK and PI3K signaling pathways are involved in the ST-induced G2 arrest. It is now widely accepted that DNA damage plays a critical role in the regulation of cell cycle arrest and apoptosis. In response to DNA damage, a complex signaling network is activated in eukaryotic cells to trigger cell cycle arrest and facilitate DNA repair. To further explore the molecular mechanism through which ST induces G2 arrest, the current study was designed to precisely dissect the role of DNA damage and the DNA damage sensor ataxia telangiectasia-mutated (ATM/p53-dependent pathway in the ST-induced G2 arrest in GES-1 cells. Using the comet assay, we determined that ST induces DNA damage, as evidenced by the formation of DNA comet tails, in GES-1 cells. We also found that ST induces the activation of ATM and its downstream molecules, Chk2 and p53, in GES-1 cells. The ATM pharmacological inhibitor caffeine was found to effectively inhibit the activation of the ATM-dependent pathways and to rescue the ST-induced G2 arrest in GES-1 cells, which indicating its ATM-dependent characteristic. Moreover, the silencing of the p53 expression with siRNA effectively attenuated the ST-induced G2 arrest in GES-1 cells. We also found that ST induces apoptosis in GES-1 cells. Thus, our results show that the ST-induced DNA damage activates the ATM/53-dependent signaling pathway, which contributes to the induction of G2 arrest in GES-1 cells.

Parvovirus B19 NS1 protein induces cell cycle arrest at G2-phase by activating the ATR-CDC25C-CDK1 pathway.

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Peng Xu

2017-03-01

Full Text Available Human parvovirus B19 (B19V infection of primary human erythroid progenitor cells (EPCs arrests infected cells at both late S-phase and G2-phase, which contain 4N DNA. B19V infection induces a DNA damage response (DDR that facilitates viral DNA replication but is dispensable for cell cycle arrest at G2-phase; however, a putative C-terminal transactivation domain (TAD2 within NS1 is responsible for G2-phase arrest. To fully understand the mechanism underlying B19V NS1-induced G2-phase arrest, we established two doxycycline-inducible B19V-permissive UT7/Epo-S1 cell lines that express NS1 or NS1mTAD2, and examined the function of the TAD2 domain during G2-phase arrest. The results confirm that the NS1 TAD2 domain plays a pivotal role in NS1-induced G2-phase arrest. Mechanistically, NS1 transactivated cellular gene expression through the TAD2 domain, which was itself responsible for ATR (ataxia-telangiectasia mutated and Rad3-related activation. Activated ATR phosphorylated CDC25C at serine 216, which in turn inactivated the cyclin B/CDK1 complex without affecting nuclear import of the complex. Importantly, we found that the ATR-CHK1-CDC25C-CDK1 pathway was activated during B19V infection of EPCs, and that ATR activation played an important role in B19V infection-induced G2-phase arrest.

Tributyltin induces G2/M cell cycle arrest via NAD(+)-dependent isocitrate dehydrogenase in human embryonic carcinoma cells.

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Asanagi, Miki; Yamada, Shigeru; Hirata, Naoya; Itagaki, Hiroshi; Kotake, Yaichiro; Sekino, Yuko; Kanda, Yasunari

2016-04-01

Organotin compounds, such as tributyltin (TBT), are well-known endocrine-disrupting chemicals (EDCs). We have recently reported that TBT induces growth arrest in the human embryonic carcinoma cell line NT2/D1 at nanomolar levels by inhibiting NAD(+)-dependent isocitrate dehydrogenase (NAD-IDH), which catalyzes the irreversible conversion of isocitrate to α-ketoglutarate. However, the molecular mechanisms by which NAD-IDH mediates TBT toxicity remain unclear. In the present study, we examined whether TBT at nanomolar levels affects cell cycle progression in NT2/D1 cells. Propidium iodide staining revealed that TBT reduced the ratio of cells in the G1 phase and increased the ratio of cells in the G2/M phase. TBT also reduced cell division cycle 25C (cdc25C) and cyclin B1, which are key regulators of G2/M progression. Furthermore, apigenin, an inhibitor of NAD-IDH, mimicked the effects of TBT. The G2/M arrest induced by TBT was abolished by NAD-IDHα knockdown. Treatment with a cell-permeable α-ketoglutarate analogue recovered the effect of TBT, suggesting the involvement of NAD-IDH. Taken together, our data suggest that TBT at nanomolar levels induced G2/M cell cycle arrest via NAD-IDH in NT2/D1 cells. Thus, cell cycle analysis in embryonic cells could be used to assess cytotoxicity associated with nanomolar level exposure of EDCs.

Effect of caffeine on gamma-ray induced G2 arrest in well-synchronized Chinese hamster ovary cells in vitro

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Masunaga, Shin-ichiro; Keng, P.C.

1996-01-01

G1-rich cells were separated from exponentially growing asynchronous cultured Chinese hamster ovary (CHO-K1) cells by centrifugal elutriation and a Coulter Counter. The G1-rich cells were incubated in medium that contained hydroxyurea (HU) to kill S phase cells and obtain the purest G1/S boundary cells possible. The HU-treated cells were washed, and were again incubated, in medium without HU, to allow these well-synchronized G1/S boundary cells to progress to S and G2/M phases. At various times after release from G1/S boundary, 4 Gy of gamma-ray and/or caffeine was administered to the cells. Eight hours after the removal of HU, cell-cycle analysis was performed with a flow cytometer. G2 arrest induced by gamma-rays was clearly shown when radiation was given earlier than 6.5 hours after HU removal. G2 arrest induced by radiation given 0.5-6.5 hours after HU removal was reduced by caffeine treatment given 6.0-6.5 hours after HU removal. Caffeine released radiation-induced G2 arrest when the radiation was given before the cultured cells entered G2/M phase and when caffeine was applied to the irradiated cells at the time when G1/S boundary cells not treated by radiation or with caffeine entered G2/M phase. Our method of centrifugal elutriation combined with incubation with HU was useful for isolating pure G1/S boundary cells from in vitro asynchronous cultures. (author)

Effect of caffeine on gamma-ray induced G2 arrest in well-synchronized Chinese hamster ovary cells in vitro

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Masunaga, Shin-ichiro [Kyoto Univ., Kumatori, Osaka (Japan). Research Reactor Inst.; Keng, P.C.

1996-11-01

G1-rich cells were separated from exponentially growing asynchronous cultured Chinese hamster ovary (CHO-K1) cells by centrifugal elutriation and a Coulter Counter. The G1-rich cells were incubated in medium that contained hydroxyurea (HU) to kill S phase cells and obtain the purest G1/S boundary cells possible. The HU-treated cells were washed, and were again incubated, in medium without HU, to allow these well-synchronized G1/S boundary cells to progress to S and G2/M phases. At various times after release from G1/S boundary, 4 Gy of gamma-ray and/or caffeine was administered to the cells. Eight hours after the removal of HU, cell-cycle analysis was performed with a flow cytometer. G2 arrest induced by gamma-rays was clearly shown when radiation was given earlier than 6.5 hours after HU removal. G2 arrest induced by radiation given 0.5-6.5 hours after HU removal was reduced by caffeine treatment given 6.0-6.5 hours after HU removal. Caffeine released radiation-induced G2 arrest when the radiation was given before the cultured cells entered G2/M phase and when caffeine was applied to the irradiated cells at the time when G1/S boundary cells not treated by radiation or with caffeine entered G2/M phase. Our method of centrifugal elutriation combined with incubation with HU was useful for isolating pure G1/S boundary cells from in vitro asynchronous cultures. (author)

[Pseudolaric acid B induces G2/M arrest and inhibits invasion and migration in HepG2 hepatoma cells].

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Li, Shuai; Guo, Lianyi

2018-01-01

Objective To investigate the mechanisms of pseudolaric acid B (PAB) blocks cell cycle and inhibits invasion and migration in human hepatoma HepG2 cells. Methods The proliferation effect of PAB on HepG2 cells was evaluated by MTT assay. The effect of PAB on the cell cycle of HepG2 cells was analyzed by flow cytometry. Immunofluorescence cytochemical staining was applied to observe the effect of PAB on the α-tubulin polymerization and expression in HepG2 cells. Transwell TM chamber invasion assay and wound healing assay were performed to detect the influence of PAB on the migration and invasion ability of HepG2 cells. Western blotting was used to determine the expressions of α-tubulin, E-cadherin and MMP-9 in HepG2 cells after treated with PAB. Results PAB inhibited the proliferation of HepG2 cells in a dose-dependent manner and blocked the cell cycle in G2/M phase. PAB significantly changed the polymerization and decreased the expression of α-tubulin. The capacities of invasion and migration of HepG2 cells treated by PAB were significantly depressed. The protein levels of α-tubulin and MMP-9 decreased while the E-cadherin protein level increased. Conclusion PAB can inhibits the proliferation of HepG2 cells by down-regulating the expression of α-tubulin and influencing its polymerization, arresting HepG2 cells in G2/M phase. Meanwhile, PAB also can inhibit the invasion and migration of HepG2 cells by lowering cytoskeleton α-tubulin and MMP-9, and increasing E-cadherin.

Chromosome condensation and radiation-induced G2 arrest studied by the induction of premature chromosome condensation following cell fusion

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Mitchell, J.B.; Bedford, J.S.

1978-01-01

When mitotic and interphase cells are fused together, the chromosomes of the interphase cell sometimes condense prematurely. The phenomenon of premature chromosome condensation (PCC) was utilized in investigating the problem of whether the chromosomes of cells suffering a radiation-induced G 2 delay are capable of condensation. Colcemide-arrested mitotic cells were fused with synchronized G 2 cells, and with irradiated cells suffering a G 2 delay. The frequency of PCC in mitotic X G 2 binucleate cells was determined. This was compared to the PCC frequency in an unirradiated synchronized population rich in G 2 cells after fusion with mitotic cells. Flash-labelling with 3 HTdR and autoradiography allowed S-phase cells to be eliminated. The frequency of G 2 PCCs was not significantly different for the irradiated G 2 -delayed or unirradiated cells. From these results it was concluded that the chromosomes of cells suffering a G 2 arrest are capable of condensation, although the involvement of the condensation process in radiation-induced G 2 delay could not be ruled out. (author)

The effect of modulators of radiation-induced G2 arrest on the repair of radiation-induced DNA damage detectable by neutral filter elution

International Nuclear Information System (INIS)

Rowley, R.; Kort, L.

1988-01-01

The influence of cycloheximide (50 μg/ml), caffeine (5 mM) and cordycepin (0.15 mM) on the repair of the damage detectable in DNA by neutral filter elution was determined. Chinese hamster ovary cells (CHO) were irradiated with X-ray doses of 20, 60 and 100 Gy then allowed to repair without drug treatment or in the presence of each drug for intervals up to 6 h. DNA damage repair proceeded in two phases. The fast component of the repair process (t 1/2 approx. 7 min) was not modified by drug treatment; the slow component (t 1/2 170 min) was unaffected by cycloheximide or cordycepin, but appeared to be inhibited by caffeine. It was concluded that: (a) the lesion which results in radiation-induced G 2 arrest is not the lesion which is detectable by neutral filter elution, and (b) the influence of caffeine on dsb repair is specific to caffeine and is not mediated by a reduction in the duration of G 2 arrest. (author)

Anthocyanins from roselle extract arrest cell cycle G2/M phase transition via ATM/Chk pathway in p53-deficient leukemia HL-60 cells.

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Tsai, Tsung-Chang; Huang, Hui-Pei; Chang, Kai-Ting; Wang, Chau-Jong; Chang, Yun-Ching

2017-04-01

Cell cycle regulation is an important issue in cancer therapy. Delphinidin and cyanidin are two major anthocyanins of the roselle plant (Hibiscus sabdariffa). In the present study, we investigated the effect of Hibiscus anthocyanins (HAs) on cell cycle arrest in human leukemia cell line HL-60 and the analyzed the underlying molecular mechanisms. HAs extracted from roselle calyces (purity 90%) markedly induced G2/M arrest evaluated with flow cytometry analysis. Western blot analyses revealed that HAs (0.1-0.7 mg mL -1 ) induced G2/M arrest via increasing Tyr15 phosphorylation of Cdc2, and inducing Cdk inhibitors p27 and p21. HAs also induced phosphorylation of upstream signals related to G2/M arrest such as phosphorylation of Cdc25C tyrosine phosphatase at Ser216, increasing the binding of pCdc25C with 14-3-3 protein. HAs-induced phosphorylation of Cdc25C could be activated by ATM checkpoint kinases, Chk1, and Chk2. We first time confirmed that ATM-Chk1/2-Cdc25C pathway as a critical mechanism for G2/M arrest in HAs-induced leukemia cell cycle arrest, indicating that this compound could be a promising anticancer candidate or chemopreventive agents for further investigation. © 2016 Wiley Periodicals, Inc. Environ Toxicol 32: 1290-1304, 2017. © 2016 Wiley Periodicals, Inc.

Cell cycle arrest in plants: what distinguishes quiescence, dormancy and differentiated G1?

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Velappan, Yazhini; Signorelli, Santiago; Considine, Michael J

2017-10-17

Quiescence is a fundamental feature of plant life, which enables plasticity, renewal and fidelity of the somatic cell line. Cellular quiescence is defined by arrest in a particular phase of the cell cycle, typically G1 or G2; however, the regulation of quiescence and proliferation can also be considered across wider scales in space and time. As such, quiescence is a defining feature of plant development and phenology, from meristematic stem cell progenitors to terminally differentiated cells, as well as dormant or suppressed seeds and buds. While the physiology of each of these states differs considerably, each is referred to as 'cell cycle arrest' or 'G1 arrest'. Here the physiology and molecular regulation of (1) meristematic quiescence, (2) dormancy and (3) terminal differentiation (cell cycle exit) are considered in order to determine whether and how the molecular decisions guiding these nuclear states are distinct. A brief overview of the canonical cell cycle regulators is provided, and the genetic and genomic, as well as physiological, evidence is considered regarding two primary questions: (1) Are the canonical cell cycle regulators superior or subordinate in the regulation of quiescence? (2) Are these three modes of quiescence governed by distinct molecular controls? Meristematic quiescence, dormancy and terminal differentiation are each predominantly characterized by G1 arrest but regulated distinctly, at a level largely superior to the canonical cell cycle. Meristematic quiescence is intrinsically linked to non-cell-autonomous regulation of meristem cell identity, and particularly through the influence of ubiquitin-dependent proteolysis, in partnership with reactive oxygen species, abscisic acid and auxin. The regulation of terminal differentiation shares analogous features with meristematic quiescence, albeit with specific activators and a greater role for cytokinin signalling. Dormancy meanwhile appears to be regulated at the level of chromatin

Middle infrared radiation induces G2/M cell cycle arrest in A549 lung cancer cells.

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Chang, Hsin-Yi; Shih, Meng-Her; Huang, Hsuan-Cheng; Tsai, Shang-Ru; Juan, Hsueh-Fen; Lee, Si-Chen

2013-01-01

There were studies investigating the effects of broadband infrared radiation (IR) on cancer cell, while the influences of middle-infrared radiation (MIR) are still unknown. In this study, a MIR emitter with emission wavelength band in the 3-5 µm region was developed to irradiate A549 lung adenocarcinoma cells. It was found that MIR exposure inhibited cell proliferation and induced morphological changes by altering the cellular distribution of cytoskeletal components. Using quantitative PCR, we found that MIR promoted the expression levels of ATM (ataxia telangiectasia mutated), ATR (ataxia-telangiectasia and Rad3-related and Rad3-related), TP53 (tumor protein p53), p21 (CDKN1A, cyclin-dependent kinase inhibitor 1A) and GADD45 (growth arrest and DNA-damage inducible), but decreased the expression levels of cyclin B coding genes, CCNB1 and CCNB2, as well as CDK1 (Cyclin-dependent kinase 1). The reduction of protein expression levels of CDC25C, cyclin B1 and the phosphorylation of CDK1 at Thr-161 altogether suggest G(2)/M arrest occurred in A549 cells by MIR. DNA repair foci formation of DNA double-strand breaks (DSB) marker γ-H2AX and sensor 53BP1 was induced by MIR treatment, it implies the MIR induced G(2)/M cell cycle arrest resulted from DSB. This study illustrates a potential role for the use of MIR in lung cancer therapy by initiating DSB and blocking cell cycle progression.

X-ray induction of 6-thioguanine-resistant mutants in division arrested, G0/G1 phase Chinese hamster ovary cells

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O' Neill, J.P.; Flint, K.B.

The cytotoxic and mutagenic effect of X-irradiation was determined with Chinese hamster ovary cells arrested in the G0/G1 phase of the cell cycle through 9 days incubation in serum-free medium. In comparison with exponential phase cultures, the arrested cells showed increased cytotoxicity and mutation induction over the dose range of 50-800 rad. Exponential cultures showed a linear mutant frequency-survival relationship while the arrested cells showed a biphasic linear relationship. A post irradiation holding period 24 h does not result in any change in the mutant frequency. The increased sensitivity of the arrested cells to the mutagenic effects of X-rays appears to be a cell-cycle phase phenomenon. Upon readdition of serum, the arrested cells re-enter the cell cycle in a synchronous manner, reaching S phase at 10-12 h. Cells irradiated at 5 h after serum addition, i.e. in G1, show a similar dose response for mutant frequency, while those irradiated at 10 h or later, i.e. in late G1, S or G2, show lower mutation induction. These observations are consistent with a chromosome interchange mechanism of mutation induction by X-rays, possibly through interactions between repairing regions of the DNA. Irradiation of cells in the G0/G1 phase allow more time for such interactions in the absence of semiconservative DNA replication. (orig.).

MiR-7 triggers cell cycle arrest at the G1/S transition by targeting multiple genes including Skp2 and Psme3.

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Noelia Sanchez

Full Text Available MiR-7 acts as a tumour suppressor in many cancers and abrogates proliferation of CHO cells in culture. In this study we demonstrate that miR-7 targets key regulators of the G1 to S phase transition, including Skp2 and Psme3, to promote increased levels of p27(KIP and temporary growth arrest of CHO cells in the G1 phase. Simultaneously, the down-regulation of DNA repair-specific proteins via miR-7 including Rad54L, and pro-apoptotic regulators such as p53, combined with the up-regulation of anti-apoptotic factors like p-Akt, promoted cell survival while arrested in G1. Thus miR-7 can co-ordinate the levels of multiple genes and proteins to influence G1 to S phase transition and the apoptotic response in order to maintain cellular homeostasis. This work provides further mechanistic insight into the role of miR-7 as a regulator of cell growth in times of cellular stress.

Sulforaphane, a Dietary Isothiocyanate, Induces G2/M Arrest in Cervical Cancer Cells through CyclinB1 Downregulation and GADD45β/CDC2 Association

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Ya-Min Cheng

2016-09-01

Full Text Available Globally, cervical cancer is the most common malignancy affecting women. The main treatment methods for this type of cancer include conization or hysterectomy procedures. Sulforaphane (SFN is a natural, compound-based drug derived from dietary isothiocyanates which has previously been shown to possess potent anti-tumor and chemopreventive effects against several types of cancer. The present study investigated the effects of SFN on anti-proliferation and G2/M phase cell cycle arrest in cervical cancer cell lines (Cx, CxWJ, and HeLa. We found that cytotoxicity is associated with an accumulation of cells in the G2/M phases of the cell-cycle. Treatment with SFN led to cell cycle arrest as well as the down-regulation of Cyclin B1 expression, but not of CDC2 expression. In addition, the effects of GADD45β gene activation in cell cycle arrest increase proportionally with the dose of SFN; however, mitotic delay and the inhibition of proliferation both depend on the dosage of SFN used to treat cancer cells. These results indicate that SFN may delay the development of cancer by arresting cell growth in the G2/M phase via down-regulation of Cyclin B1 gene expression, dissociation of the cyclin B1/CDC2 complex, and up-regulation of GADD45β proteins.

Sinularin Selectively Kills Breast Cancer Cells Showing G2/M Arrest, Apoptosis, and Oxidative DNA Damage

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Hurng-Wern Huang

2018-04-01

Full Text Available The natural compound sinularin, isolated from marine soft corals, is antiproliferative against several cancers, but its possible selective killing effect has rarely been investigated. This study investigates the selective killing potential and mechanisms of sinularin-treated breast cancer cells. In 3-(4,5-dimethylthiazol-2-yl-5-(3-carboxymethoxyphenyl-2-(4-sulfophenyl-2H- tetrazolium, inner salt (MTS assay, sinularin dose-responsively decreased the cell viability of two breast cancer (SKBR3 and MDA-MB-231 cells, but showed less effect on breast normal (M10 cells after a 24 h treatment. According to 7-aminoactinomycin D (7AAD flow cytometry, sinularin dose-responsively induced the G2/M cycle arrest of SKBR3 cells. Sinularin dose-responsively induced apoptosis on SKBR3 cells in terms of a flow cytometry-based annexin V/7AAD assay and pancaspase activity, as well as Western blotting for cleaved forms of poly(ADP-ribose polymerase (PARP, caspases 3, 8, and 9. These caspases and PARP activations were suppressed by N-acetylcysteine (NAC pretreatment. Moreover, sinularin dose-responsively induced oxidative stress and DNA damage according to flow cytometry analyses of reactive oxygen species (ROS, mitochondrial membrane potential (MitoMP, mitochondrial superoxide, and 8-oxo-2′-deoxyguanosine (8-oxodG. In conclusion, sinularin induces selective killing, G2/M arrest, apoptosis, and oxidative DNA damage of breast cancer cells.

Porcine epidemic diarrhea virus through p53-dependent pathway causes cell cycle arrest in the G0/G1 phase.

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Sun, Pei; Wu, Haoyang; Huang, Jiali; Xu, Ying; Yang, Feng; Zhang, Qi; Xu, Xingang

2018-05-22

Porcine epidemic diarrhea virus (PEDV), an enteropathogenic Alphacoronavirus, has caused enormous economic losses in the swine industry. p53 protein exists in a wide variety of animal cells, which is involved in cell cycle regulation, apoptosis, cell differentiation and other biological functions. In this study, we investigated the effects of PEDV infection on the cell cycle of Vero cells and p53 activation. The results demonstrated that PEDV infection induces cell cycle arrest at G0/G1 phase in Vero cells, while UV-inactivated PEDV does not cause cell cycle arrest. PEDV infection up-regulates the levels of p21, cdc2, cdk2, cdk4, Cyclin A protein and down-regulates Cyclin E protein. Further research results showed that inhibition of p53 signaling pathway can reverse the cell cycle arrest in G0/G1 phase induced by PEDV infection and cancel out the up-regulation of p21 and corresponding Cyclin/cdk mentioned above. In addition, PEDV infection of the cells synchronized in various stages of cell cycle showed that viral subgenomic RNA and virus titer were higher in the cells released from G0/G1 phase synchronized cells than that in the cells released from the G1/S phase and G2/M phase synchronized or asynchronous cells after 18 h p.i.. This is the first report to demonstrate that the p53-dependent pathway plays an important role in PEDV induced cell cycle arrest and beneficially contributes to viral infection. Copyright © 2018 Elsevier B.V. All rights reserved.

Salinomycin sensitizes antimitotic drugs-treated cancer cells by increasing apoptosis via the prevention of G2 arrest

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Kim, Ju-Hwa; Yoo, Hye-In; Kang, Han Sung; Ro, Jungsil; Yoon, Sungpil

2012-01-01

Highlights: ► Sal sensitizes antimitotic drugs-treated cancer cells. ► Sal sensitizes them by prevention of G2 arrest and reduced cyclin D1 levels. ► Sal also sensitizes them by increasing DNA damage and reducing p21 level. ► A low concentration of Sal effectively sensitized the cancer cells to antimitotic drugs. -- Abstract: Here, we investigated whether Sal could sensitize cancer cells to antimitotic drugs. We demonstrated that Sal sensitized paclitaxcel (PAC)-, docetaxcel (DOC)-, vinblastin (VIN)-, or colchicine (COL)-treated cancer cell lines, suggesting that Sal has the ability to sensitize the cells to any form of microtubule-targeting drugs. Sensitization to the antimitotic drugs could be achieved with very low concentrations of Sal, suggesting that there is a possibility to minimize Sal toxicity associated with human cancer patient treatments. Sensitization by Sal increased apoptosis, which was observed by C-PARP production. Sal sensitized the cancer cells to antimitotic drugs by preventing G2 arrest, suggesting that Sal contributes to the induction of mitotic catastrophe. Sal generally reduced cyclin D1 levels in PAC-, DOC-, and VIN-treated cells. In addition, Sal treatment increased pH2AX levels and reduced p21 levels in antimitotic drugs-treated cells. These observations suggest that the mechanisms underlying Sal sensitization to DNA-damaging compounds, radiation, and microtubule-targeting drugs are similar. Our data demonstrated that Sal sensitizes cancer cells to antimitotic drugs by increasing apoptosis through the prevention of G2 arrest via conserved Sal-sensitization mechanisms. These results may contribute to the development of Sal-based chemotherapy for cancer patients treated with antimitotic drugs.

Salinomycin sensitizes antimitotic drugs-treated cancer cells by increasing apoptosis via the prevention of G2 arrest

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Kim, Ju-Hwa; Yoo, Hye-In; Kang, Han Sung; Ro, Jungsil [Research Institute, National Cancer Center, Ilsan-gu, Goyang-si, Gyeonggi-do (Korea, Republic of); Yoon, Sungpil, E-mail: yoons@ncc.re.kr [Research Institute, National Cancer Center, Ilsan-gu, Goyang-si, Gyeonggi-do (Korea, Republic of)

2012-02-03

Highlights: Black-Right-Pointing-Pointer Sal sensitizes antimitotic drugs-treated cancer cells. Black-Right-Pointing-Pointer Sal sensitizes them by prevention of G2 arrest and reduced cyclin D1 levels. Black-Right-Pointing-Pointer Sal also sensitizes them by increasing DNA damage and reducing p21 level. Black-Right-Pointing-Pointer A low concentration of Sal effectively sensitized the cancer cells to antimitotic drugs. -- Abstract: Here, we investigated whether Sal could sensitize cancer cells to antimitotic drugs. We demonstrated that Sal sensitized paclitaxcel (PAC)-, docetaxcel (DOC)-, vinblastin (VIN)-, or colchicine (COL)-treated cancer cell lines, suggesting that Sal has the ability to sensitize the cells to any form of microtubule-targeting drugs. Sensitization to the antimitotic drugs could be achieved with very low concentrations of Sal, suggesting that there is a possibility to minimize Sal toxicity associated with human cancer patient treatments. Sensitization by Sal increased apoptosis, which was observed by C-PARP production. Sal sensitized the cancer cells to antimitotic drugs by preventing G2 arrest, suggesting that Sal contributes to the induction of mitotic catastrophe. Sal generally reduced cyclin D1 levels in PAC-, DOC-, and VIN-treated cells. In addition, Sal treatment increased pH2AX levels and reduced p21 levels in antimitotic drugs-treated cells. These observations suggest that the mechanisms underlying Sal sensitization to DNA-damaging compounds, radiation, and microtubule-targeting drugs are similar. Our data demonstrated that Sal sensitizes cancer cells to antimitotic drugs by increasing apoptosis through the prevention of G2 arrest via conserved Sal-sensitization mechanisms. These results may contribute to the development of Sal-based chemotherapy for cancer patients treated with antimitotic drugs.

ERK1/2 signaling plays an important role in topoisomerase II poison-induced G2/M checkpoint activation.

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Kolb, Ryan H; Greer, Patrick M; Cao, Phu T; Cowan, Kenneth H; Yan, Ying

2012-01-01

Topo II poisons, which target topoisomerase II (topo II) to generate enzyme mediated DNA damage, have been commonly used for anti-cancer treatment. While clinical evidence demonstrate a capability of topo II poisons in inducing apoptosis in cancer cells, accumulating evidence also show that topo II poison treatment frequently results in cell cycle arrest in cancer cells, which was associated with subsequent resistance to these treatments. Results in this report indicate that treatment of MCF-7 and T47D breast cancer cells with topo II poisons resulted in an increased phosphorylation of extracellular signal-regulated kinase 1 and 2 (ERK1/2) and an subsequent induction of G2/M cell cycle arrest. Furthermore, inhibition of ERK1/2 activation using specific inhibitors markedly attenuated the topo II poison-induced G2/M arrest and diminished the topo II poison-induced activation of ATR and Chk1 kinases. Moreover, decreased expression of ATR by specific shRNA diminished topo II poison-induced G2/M arrest but had no effect on topo II poison-induced ERK1/2 activation. In contrast, inhibition of ERK1/2 signaling had little, if any, effect on topo II poison-induced ATM activation. In addition, ATM inhibition by either incubation of cells with ATM specific inhibitor or transfection of cells with ATM specific siRNA did not block topo II poison-induced G2/M arrest. Ultimately, inhibition of ERK1/2 signaling greatly enhanced topo II poison-induced apoptosis. These results implicate a critical role for ERK1/2 signaling in the activation of G2/M checkpoint response following topo II poison treatment, which protects cells from topo II poison-induced apoptosis.

Flavokawain derivative FLS induced G2/M arrest and apoptosis on breast cancer MCF-7 cell line

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Ali NM

2016-06-01

Full Text Available Norlaily Mohd Ali,1 M Nadeem Akhtar,2 Huynh Ky,3 Kian Lam Lim,1 Nadiah Abu,4 Seema Zareen,2 Wan Yong Ho,5 Han Kiat Alan-Ong,1 Sheau Wei Tan,6 Noorjahan Banu Alitheen,4 Jamil bin Ismail,2 Swee Keong Yeap,6 Tunku Kamarul7 1Faculty of Medicine and Health Sciences, Universiti Tunku Abdul Rahman, Selangor, 2Department of Industrial Biotechnology, Faculty of Industrial Sciences & Technology, Universiti Malaysia Pahang, Pahang, Malaysia; 3Department of Agriculture Genetics and Breeding, College of Agriculture and Applied Biology, Cantho University, CanTho City, Vietnam; 4Department of Cell and Molecular Biology, Faculty of Biotechnology and Biomolecular Sciences, Universiti Putra Malaysia, 5School of Biomedical Sciences, The University of Nottingham Malaysia Campus, 6Institute of Bioscience, Universiti Putra Malaysia, Selangor, 7Tissue Engineering Group, National Orthopaedic Centre of Excellence for Research and Learning, Department of Orthopaedic Surgery, Faculty of Medicine, University Malaya, Kuala Lumpur, Malaysia Abstract: Known as naturally occurring biologically active compounds, flavokawain A and B are the leading chalcones that possess anticancer properties. Another flavokawain derivative, (E-1-(2'-Hydroxy-4',6'-dimethoxyphenyl-3-(4-methylthiophenylprop-2-ene-1-one (FLS was characterized with 1H-nuclear magnetic resonance, electron-impact mas spectrometry, infrared spectroscopy, and ultraviolet (1H NMR, EI-MS, IR, and UV spectroscopic techniques. FLS cytotoxic efficacy against human cancer cells (MCF-7, MDA-MB-231, and MCF-10A resulted in the reduction of IC50 values in a time- and dose-dependent mode with high specificity on MCF-7 (IC50 of 36 µM at 48 hours against normal breast cell MCF-10A (no IC50 detected up to 180 µM at 72 hours. Light, scanning electron, and fluorescent microscopic analysis of MCF-7 cell treated with 36 µM of FLS displayed cell shrinkage, apoptotic body, and DNA fragmentation. Additionally, induction of G2/M cell

Jaridonin-induced G2/M phase arrest in human esophageal cancer cells is caused by reactive oxygen species-dependent Cdc2-tyr15 phosphorylation via ATM–Chk1/2–Cdc25C pathway

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Ma, Yong-Cheng [Clinical Pharmacology Laboratory, Henan Province People' s Hospital, No. 7, Wei Wu Road, Zhengzhou, Henan (China); Su, Nan [Department of Quality Detection and Management, Henan University of Animal Husbandry and Economy, Zhengzhou, Henan (China); Shi, Xiao-Jing; Zhao, Wen; Ke, Yu [School of Pharmaceutical Sciences, Zhengzhou University, No. 100, Science Avenue, Zhengzhou, Henan (China); Zi, Xiaolin [Department of Urology, University of California, Irvine, Orange, CA (United States); Department of Pharmacology, University of California, Irvine, Orange, CA (United States); Department of Pharmaceutical Sciences, University of California, Irvine, Orange, CA (United States); Zhao, Ning-Min; Qin, Yu-Hua; Zhao, Hong-Wei [Clinical Pharmacology Laboratory, Henan Province People' s Hospital, No. 7, Wei Wu Road, Zhengzhou, Henan (China); Liu, Hong-Min, E-mail: liuhm@zzu.edu.cn [School of Pharmaceutical Sciences, Zhengzhou University, No. 100, Science Avenue, Zhengzhou, Henan (China)

2015-01-15

Jaridonin, a novel diterpenoid from Isodon rubescens, has been shown previously to inhibit proliferation of esophageal squamous cancer cells (ESCC) through G2/M phase cell cycle arrest. However, the involved mechanism is not fully understood. In this study, we found that the cell cycle arrest by Jaridonin was associated with the increased expression of phosphorylation of ATM at Ser1981 and Cdc2 at Tyr15. Jaridonin also resulted in enhanced phosphorylation of Cdc25C via the activation of checkpoint kinases Chk1 and Chk2, as well as in increased phospho-H2A.X (Ser139), which is known to be phosphorylated by ATM in response to DNA damage. Furthermore, Jaridonin-mediated alterations in cell cycle arrest were significantly attenuated in the presence of NAC, implicating the involvement of ROS in Jaridonin's effects. On the other hand, addition of ATM inhibitors reversed Jaridonin-related activation of ATM and Chk1/2 as well as phosphorylation of Cdc25C, Cdc2 and H2A.X and G2/M phase arrest. In conclusion, these findings identified that Jaridonin-induced cell cycle arrest in human esophageal cancer cells is associated with ROS-mediated activation of ATM–Chk1/2–Cdc25C pathway. - Highlights: • Jaridonin induced G2/M phase arrest through induction of redox imbalance. • Jaridonin increased the level of ROS through depleting glutathione in cell. • ATM–Chk1/2–Cdc25C were involved in Jaridonin-induced cell cycle arrest. • Jaridonin selectively inhibited cancer cell viability and cell cycle progression.

β2-adrenoceptor blockage induces G1/S phase arrest and apoptosis in pancreatic cancer cells via Ras/Akt/NFκB pathway

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Zhang Dong

2011-11-01

Full Text Available Abstract Background Smoking and stress, pancreatic cancer (PanCa risk factors, stimulate nitrosamine 4-(methylnitrosamino-1-(3-pyridyl-1-butanone (NNK and catecholamines production respectively. NNK and catecholamine bind the β-adrenoceptors and induce PanCa cell proliferation; and we have previously suggested that β-adrenergic antagonists may suppress proliferation and invasion and stimulate apoptosis in PanCa. To clarify the mechanism of apoptosis induced by β2-adrenergic antagonist, we hypothesize that blockage of the β2-adrenoceptor could induce G1/S phase arrest and apoptosis and Ras may be a key player in PanCa cells. Results The β1 and β2-adrenoceptor proteins were detected on the cell surface of PanCa cells from pancreatic carcinoma specimen samples by immunohistochemistry. The β2-adrenergic antagonist ICI118,551 significantly induced G1/S phase arrest and apoptosis compared with the β1-adrenergic antagonist metoprolol, which was determined by the flow cytometry assay. β2-adrenergic antagonist therapy significantly suppressed the expression of extracellular signal-regulated kinase, Akt, Bcl-2, cyclin D1, and cyclin E and induced the activation of caspase-3, caspase-9 and Bax by Western blotting. Additionally, the β2-adrenergic antagonist reduced the activation of NFκB in vitro cultured PanCa cells. Conclusions The blockage of β2-adrenoceptor markedly induced PanCa cells to arrest at G1/S phase and consequently resulted in cell death, which is possibly due to that the blockage of β2-adrenoceptor inhibited NFκB, extracellular signal-regulated kinase, and Akt pathways. Therefore, their upstream molecule Ras may be a key factor in the β2-adrenoceptor antagonist induced G1/S phase arrest and apoptosis in PanCa cells. The new pathway discovered in this study may provide an effective therapeutic strategy for PanCa.

BDE-47 and BDE-209 inhibit proliferation of Neuro-2a cells via inducing G1-phase arrest.

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Chen, Hongmei; Tang, Xuexi; Zhou, Bin; Xu, Ningning; Zhou, Zhongyuan; Fang, Kuan; Wang, You

2017-03-01

Cell proliferation is closely related to cell cycle which is strictly regulated by genes and regulatory proteins. In the present study, we comparatively analyzed the toxic effects of BDE-47 and BDE-209 on cell proliferation of Neuro-2a cells, and the possible mechanism was discussed. The results indicated that BDE-47 significantly inhibited the cell proliferation and the cell cycle were arrest at G1 phase, while BDE-209 had little effects on either cell proliferation or cell cycle. qRT-PCR and Western blot assay presented that BDE-47 up-regulated the gene expressions of p53 and p21, which down-regulated the expresseion of cyclinD1 and CDK2, and inhibited retinoblastoma protein (pRb) phosphorylation. This process could effectively arrest the cell cycle at G1 phase, which finally caused the inhibition on Neuro-2a cell proliferation. However, BDE-209 was only up-regulated the gene expressions of p53, also suggested to be involved in the inhibition on Neuro-2a cell proliferation. Copyright © 2016. Published by Elsevier B.V.

Tributyltin induces a G2/M cell cycle arrest in human amniotic cells via PP2A inhibition-mediated inactivation of the ERK1/2 cascades.

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Zhang, Yali; Guo, Zonglou; Xu, Lihong

2014-03-01

The molecular mechanisms underlying the cell cycle alterations induced by tributyltin (TBT), a highly toxic environmental contaminant, remain elusive. In this study, cell cycle progression and some key regulators in G2/M phase were investigated in human amniotic cells treated with TBT. Furthermore, protein phosphatase (PP) 2A and the ERK cascades were examined. The results showed that TBT caused a G2/M cell cycle arrest that was accompanied by a decrease in the total cdc25C protein level and an increase in the p-cdc2 level in the nucleus. TBT caused a decrease in PP2A activity and inhibited the ERK cascade by inactivating Raf-1, resulting in the dephosphorylation of MEK1/2, ERK1/2, and c-Myc. Taken together, TBT leads to a G2/M cell cycle arrest in FL cells, an increase in p-cdc2 and a decrease in the levels of total cdc25C protein, which may be caused by the PP2A inhibition-mediated inactivation of the ERK1/2 cascades. Copyright © 2014 Elsevier B.V. All rights reserved.

A novel peptide sansalvamide analogue inhibits pancreatic cancer cell growth through G0/G1 cell-cycle arrest

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Ujiki, Michael B.; Milam, Ben; Ding Xianzhong; Roginsky, Alexandra B.; Salabat, M. Reza; Talamonti, Mark S.; Bell, Richard H.; Gu Wenxin; Silverman, Richard B.; Adrian, Thomas E.

2006-01-01

Patients with pancreatic cancer have little hope for cure because no effective therapies are available. Sansalvamide A is a cyclic depsipeptide produced by a marine fungus. We investigated the effect of a novel sansalvamide A analogue on growth, cell-cycle phases, and induction of apoptosis in human pancreatic cancer cells in vitro. The sansalvamide analogue caused marked time- and concentration-dependent inhibition of DNA synthesis and cell proliferation of two human pancreatic cancer cell lines (AsPC-1 and S2-013). The analogue induced G0/G1 phase cell-cycle arrest and morphological changes suggesting induction of apoptosis. Apoptosis was confirmed by annexin V binding. This novel sansalvamide analogue inhibits growth of pancreatic cancer cells through G0/G1 arrest and induces apoptosis. Sansalvamide analogues may be valuable for the treatment of pancreatic cancer

Serial measurement of neuron specific enolase improves prognostication in cardiac arrest patients treated with hypothermia: A prospective study

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Storm Christian

2012-01-01

Full Text Available Abstract Background Neuron specific enolase (NSE has repeatedly been evaluated for neurological prognostication in patients after cardiac arrest. However, it is unclear whether current guidelines for NSE cutoff levels also apply to cardiac arrest patients treated with hypothermia. Thus, we investigated the prognostic significance of absolute NSE levels and NSE kinetics in cardiac arrest patients treated with hypothermia. Methods In a prospective study of 35 patients resuscitated from cardiac arrest, NSE was measured daily for four days following admission. Outcome was assessed at ICU discharge using the CPC score. All patients received hypothermia treatment for 24 hours at 33°C with a surface cooling device according to current guidelines. Results The cutoff for absolute NSE levels in patients with unfavourable outcome (CPC 3-5 72 hours after cardiac arrest was 57 μg/l with an area under the curve (AUC of 0.82 (sensitivity 47%, specificity 100%. The cutoff level for NSE kinetics in patients with unfavourable outcome (CPC 3-5 was an absolute increase of 7.9 μg/l (AUC 0.78, sensitivity 63%, specificity 100% and a relative increase of 33.1% (AUC 0.803, sensitivity 67%, specificity 100% at 48 hours compared to admission. Conclusion In cardiac arrest patients treated with hypothermia, prognostication of unfavourable outcome by NSE kinetics between admission and 48 hours after resuscitation may be superior to prognostication by absolute NSE levels.

ABT-263 induces G1/G0-phase arrest, apoptosis and autophagy in human esophageal cancer cells in vitro.

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Lin, Qing-Huan; Que, Fu-Chang; Gu, Chun-Ping; Zhong, De-Sheng; Zhou, Dan; Kong, Yi; Yu, Le; Liu, Shu-Wen

2017-12-01

Both the anti- and pro-apoptotic members of the Bcl-2 family are regulated by a conserved Bcl-2 homology (BH3) domain. ABT-263 (Navitoclax), a novel BH3 mimetic and orally bioavailable Bcl-2 family inhibitor with high affinity for Bcl-xL, Bcl-2 and Bcl-w has entered clinical trials for cancer treatment. But the anticancer mechanisms of ABT-263 have not been fully elucidated. In this study we investigated the effects of ABT-263 on human esophageal cancer cells in vitro and to explore its anticancer mechanisms. Treatment with ABT-263 dose-dependently suppressed the viability of 3 human esophageal cancer cells with IC 50 values of 10.7±1.4, 7.1±1.5 and 8.2±1.6 μmol/L, in EC109, HKESC-2 and CaES-17 cells, respectively. ABT-263 (5-20 μmol/L) dose-dependently induced G 1 /G 0 -phase arrest in the 3 cancer cell lines and induced apoptosis evidenced by increased the Annexin V-positive cell population and elevated levels of cleaved caspase 3, cleaved caspase 9 and PARP. We further demonstrated that ABT-263 treatment markedly increased the expression of p21 Waf1/Cip1 and decreased the expression of cyclin D1 and phospho-Rb (retinoblastoma tumor suppressor protein) (Ser780) proteins that contributed to the G 1 /G 0 -phase arrest. Knockdown of p21 Waf1/Cip1 attenuated ABT-263-induced G 1 /G 0 -phase arrest. Moreover, ABT-263 treatment enhanced pro-survival autophagy, shown as the increased LC3-II levels and decreased p62 levels, which counteracted its anticancer activity. Our results suggest that ABT-263 exerts cytostatic and cytotoxic effects on human esophageal cancer cells in vitro and enhances pro-survival autophagy, which counteracts its anticancer activity.

The 20-hydroxyecdysone-induced signalling pathway in G2/M arrest of Plodia interpunctella imaginal wing cells.

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Siaussat, David; Bozzolan, Françoise; Porcheron, Patrick; Debernard, Stéphane

2008-05-01

The mechanisms involved in the control of cellular proliferation by the steroid hormone 20-hydroxyecdysone (20E) in insects are not known. We dissected the 20E signalling pathway responsible for G2/M arrest of imaginal cells from the IAL-PID2 cells of the Indian meal moth Plodia interpunctella. We first used a 5'-3' RACE-based strategy to clone a 4479bp cDNA encoding a putative P. interpunctella HR3 transcription factor named PiHR3. The deduced amino acid sequence of PiHR3 was highly similar to those of HR3 proteins from other lepidopterans, e.g. Manduca sexta and Bombyx mori. Using double-stranded RNA-mediated interference (dsRNAi), we then succeeded in blocking the ability of 20E to induce the expression of PiEcR-B1, PiUSP-2 and PiHR3 genes that encode the P. interpunctella ecdysone receptor B1-isoform, Ultraspiracle-2 isoform, the insect homologue of the vertebrate retinoid X receptor, and the HR3 transcription factor. We showed that inhibiting the 20E induction of PiEcR-B1, PiUSP-2 and PiHR3 mRNAs prevented the decreased expression of B cyclin and consequently the G2/M arrest of IAL-PID2 cells. Using this functional approach, we revealed the participation of EcR, USP and HR3 in a 20E signalling pathway that controls the proliferation of imaginal cells by regulating the expression of B cyclin.

SKLB70326, a novel small-molecule inhibitor of cell-cycle progression, induces G{sub 0}/G{sub 1} phase arrest and apoptosis in human hepatic carcinoma cells

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Han, Yuanyuan; He, Haiyun [State Key Laboratory of Biotherapy and Cancer Center, West China Hospital, West China Medical School, Sichuan University, Chengdu 610041 (China); Peng, Feng [Department of Thoracic Oncology of the Cancer Center and State Key Laboratory of Biotherapy, West China Hospital, West China Medical School, Sichuan University, Chengdu 610041 (China); Liu, Jiyan; Dai, Xiaoyun; Lin, Hongjun; Xu, Youzhi; Zhou, Tian; Mao, Yongqiu; Xie, Gang; Yang, Shengyong; Yu, Luoting; Yang, Li [State Key Laboratory of Biotherapy and Cancer Center, West China Hospital, West China Medical School, Sichuan University, Chengdu 610041 (China); Zhao, Yinglan, E-mail: alancenxb@sina.com [State Key Laboratory of Biotherapy and Cancer Center, West China Hospital, West China Medical School, Sichuan University, Chengdu 610041 (China)

2012-05-18

Highlights: Black-Right-Pointing-Pointer SKLB70326 is a novel compound and has activity of anti-HCC. Black-Right-Pointing-Pointer SKLB70326 induces cell cycle arrest and apoptosis in HepG2 cells. Black-Right-Pointing-Pointer SKLB70326 induces G{sub 0}/G{sub 1} phase arrest via inhibiting the activity of CDK2, CDK4 and CDK6. Black-Right-Pointing-Pointer SKLB70326 induces apoptosis through the intrinsic pathway. -- Abstract: We previously reported the potential of a novel small molecule 3-amino-6-(3-methoxyphenyl)thieno[2.3-b]pyridine-2-carboxamide (SKLB70326) as an anticancer agent. In the present study, we investigated the anticancer effects and possible mechanisms of SKLB70326 in vitro. We found that SKLB70326 treatment significantly inhibited human hepatic carcinoma cell proliferation in vitro, and the HepG2 cell line was the most sensitive to its treatment. The inhibition of cell proliferation correlated with G{sub 0}/G{sub 1} phase arrest, which was followed by apoptotic cell death. The SKLB70326-mediated cell-cycle arrest was associated with the downregulation of cyclin-dependent kinase (CDK) 2, CDK4 and CDK6 but not cyclin D1 or cyclin E. The phosphorylation of the retinoblastoma protein (Rb) was also observed. SKLB70326 treatment induced apoptotic cell death via the activation of PARP, caspase-3, caspase-9 and Bax as well as the downregulation of Bcl-2. The expression levels of p53 and p21 were also induced by SKLB70326 treatment. Moreover, SKLB70326 treatment was well tolerated. In conclusion, SKLB70326, a novel cell-cycle inhibitor, notably inhibits HepG2 cell proliferation through the induction of G{sub 0}/G{sub 1} phase arrest and subsequent apoptosis. Its potential as a candidate anticancer agent warrants further investigation.

G2M arrest and apoptosis in murine T lymphoma cells following exposure to 212Bi alpha particle irradiation

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Palayoor, S.T.; Humm, J.L.; Macklis, R.M.

1993-01-01

Asynchronous exponentially growing EL4 murine T lymphoma cells were exposed either to high LET α-radiation from 212 Bi-DTPA or to γ-radiation from a 137 Cs source. Radiation-induced cell cycle perturbation was studied by flow cytometry. Alpha irradiation, like γ, transiently arrested cells in the G2M phase in a dose-dependent manner. The maximum percentages of cells accumulated in G2M 18 h after α- and γ-irradiation were comparable, though the dose-response relationships differed. The ''RBE'' value for G2M block for α- versus γ-radiation was approx. 4. (author)

Piperine causes G1 phase cell cycle arrest and apoptosis in melanoma cells through checkpoint kinase-1 activation.

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Neel M Fofaria

Full Text Available In this study, we determined the cytotoxic effects of piperine, a major constituent of black and long pepper in melanoma cells. Piperine treatment inhibited the growth of SK MEL 28 and B16 F0 cells in a dose and time-dependent manner. The growth inhibitory effects of piperine were mediated by cell cycle arrest of both the cell lines in G1 phase. The G1 arrest by piperine correlated with the down-regulation of cyclin D1 and induction of p21. Furthermore, this growth arrest by piperine treatment was associated with DNA damage as indicated by phosphorylation of H2AX at Ser139, activation of ataxia telangiectasia and rad3-related protein (ATR and checkpoint kinase 1 (Chk1. Pretreatment with AZD 7762, a Chk1 inhibitor not only abrogated the activation of Chk1 but also piperine mediated G1 arrest. Similarly, transfection of cells with Chk1 siRNA completely protected the cells from G1 arrest induced by piperine. Piperine treatment caused down-regulation of E2F1 and phosphorylation of retinoblastoma protein (Rb. Apoptosis induced by piperine was associated with down-regulation of XIAP, Bid (full length and cleavage of Caspase-3 and PARP. Furthermore, our results showed that piperine treatment generated ROS in melanoma cells. Blocking ROS by tiron protected the cells from piperine mediated cell cycle arrest and apoptosis. These results suggest that piperine mediated ROS played a critical role in inducing DNA damage and activation of Chk1 leading to G1 cell cycle arrest and apoptosis.

Increased expression of cyclin B1 mRNA coincides with diminished G2-phase arrest in irradiated HeLa cells treated with staurosporine or caffeine

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Bernhard, E.J.; Maity, A.; McKenna, W.G.; Muschel, R.J.

1994-01-01

The irradiation of cells results in delayed progression through the G 2 phase of the cell cycle. Treatment of irradiated HeLa cells with caffeine greatly reduces the G 2 -phase delay, while caffeine does not alter progression of cells through the cell cycle in unirradiated cells. In this report we demonstrate that treatment of HeLa cells with the kinase inhibitor staurosporine, but not with the inhibitor H7, also results in a reduction of the G 2 -phase arrest after irradiation. Cell cycle progression in unirradiated cells is unaffected by 4.4 nM (2ng/ml) staurosporine, which releases the radiation-induced G 2 -phase arrest. In HeLa cells, the G 2 -phase delay after irradiation in S phase is accompanied by decreased expression of cyclin B1 mRNA. Coincident with the reduction in G 2 -phase delay, we observed an increase in cyclin B1 mRNA accumulation in irradiated, staurosporine-treated cells compared to cells treated with irradiation alone. Caffeine treatment of irradiated HeLa cells also resulted in an elevation in the levels of cyclin B1 message. These results support the hypothesis that diminished cyclin B1 mRNA levels influence G 2 -phase arrest to some degree. The findings that both staurosporine and caffeine treatments reverse the depression in cyclin B1 expression suggest that these two compounds may act on a common pathway of cell cycle control in response to radiation injury. 33 refs., 6 figs

Cyclin G2 is a centrosome-associated nucleocytoplasmic shuttling protein that influences microtubule stability and induces a p53-dependent cell cycle arrest

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Arachchige Don, Aruni S.; Dallapiazza, Robert F.; Bennin, David A.; Brake, Tiffany; Cowan, Colleen E.; Horne, Mary C.

2006-01-01

Cyclin G2 is an atypical cyclin that associates with active protein phosphatase 2A. Cyclin G2 gene expression correlates with cell cycle inhibition; it is significantly upregulated in response to DNA damage and diverse growth inhibitory stimuli, but repressed by mitogenic signals. Ectopic expression of cyclin G2 promotes cell cycle arrest, cyclin dependent kinase 2 inhibition and the formation of aberrant nuclei [Bennin, D. A., Don, A. S., Brake, T., McKenzie, J. L., Rosenbaum, H., Ortiz, L., DePaoli-Roach, A. A., and Horne, M. C. (2002). Cyclin G2 associates with protein phosphatase 2A catalytic and regulatory B' subunits in active complexes and induces nuclear aberrations and a G 1 /S-phase cell cycle arrest. J Biol Chem 277, 27449-67]. Here we report that endogenous cyclin G2 copurifies with centrosomes and microtubules (MT) and that ectopic G2 expression alters microtubule stability. We find exogenous and endogenous cyclin G2 present at microtubule organizing centers (MTOCs) where it colocalizes with centrosomal markers in a variety of cell lines. We previously reported that cyclin G2 forms complexes with active protein phosphatase 2A (PP2A) and colocalizes with PP2A in a detergent-resistant compartment. We now show that cyclin G2 and PP2A colocalize at MTOCs in transfected cells and that the endogenous proteins copurify with isolated centrosomes. Displacement of the endogenous centrosomal scaffolding protein AKAP450 that anchors PP2A at the centrosome resulted in the depletion of centrosomal cyclin G2. We find that ectopic expression of cyclin G2 induces microtubule bundling and resistance to depolymerization, inhibition of polymer regrowth from MTOCs and a p53-dependent cell cycle arrest. Furthermore, we determined that a 100 amino acid carboxy-terminal region of cyclin G2 is sufficient to both direct GFP localization to centrosomes and induce cell cycle inhibition. Colocalization of endogenous cyclin G2 with only one of two GFP-centrin-tagged centrioles, the

Oxygen-Glucose Deprivation Induces G2/M Cell Cycle Arrest in Brain Pericytes Associated with ERK Inactivation.

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Wei, Wenjie; Yu, Zhiyuan; Xie, Minjie; Wang, Wei; Luo, Xiang

2017-01-01

Growing evidence has revealed that brain pericytes are multifunctional and contribute to the pathogenesis of a number of neurological disorders. However, the role of pericytes in cerebral ischemia, and especially the pathophysiological alterations in pericytes, remains unclear. In the present study, our aim was to determine whether the proliferation of pericytes is affected by cerebral ischemia and, if so, to identify the underlying mechanism(s). Cultured brain pericytes subjected to oxygen-glucose deprivation (OGD) were used as our model of cerebral ischemia; the protein expression levels of cyclin D1, cyclin E, cdk4, and cyclin B1 were determined by Western blot analysis, and cell cycle analysis was assessed by flow cytometry. The OGD treatment reduced the brain pericyte proliferation by causing G2/M phase arrest and downregulating the protein levels of cyclin D1, cyclin E, cdk4, and cyclin B1. Further studies demonstrated a simultaneous decrease in the activity of extracellular regulated protein kinases (ERK), suggesting a critical role of the ERK signaling cascade in the inhibition of OGD-induced pericyte proliferation. We suggest that OGD inhibition of the proliferation of brain pericytes is associated with the inactivation of the ERK signaling pathway, which arrests them in the G2/M phase.

Lamprey Prohibitin2 Arrest G2/M Phase Transition of HeLa Cells through Down-regulating Expression and Phosphorylation Level of Cell Cycle Proteins.

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Shi, Ying; Guo, Sicheng; Wang, Ying; Liu, Xin; Li, Qingwei; Li, Tiesong

2018-03-02

Prohibitin 2(PHB2) is a member of the SFPH trans-membrane family proteins. It is a highly conserved and functionally diverse protein that plays an important role in preserving the structure and function of the mitochondria. In this study, the lamprey PHB2 gene was expressed in HeLa cells to investigate its effect on cell proliferation. The effect of Lm-PHB2 on the proliferation of HeLa cells was determined by treating the cells with pure Lm-PHB2 protein followed by MTT assay. Using the synchronization method with APC-BrdU and PI double staining revealed rLm-PHB2 treatment induced the decrease of both S phase and G0/G1 phase and then increase of G2/M phase. Similarly, cells transfected with pEGFP-N1-Lm-PHB2 also exhibited remarkable reduction in proliferation. Western blot and quantitative real-time PCR(qRT-PCR) assays suggested that Lm-PHB2 caused cell cycle arrest in HeLa cells through inhibition of CDC25C and CCNB1 expression. According to our western blot analysis, Lm-PHB2 was also found to reduce the expression level of Wee1 and PLK1 and the phosphorylation level of CCNB1, CDC25C and CDK1 in HeLa cells. Lamprey prohibitin 2 could arrest G2/M phase transition of HeLa cells through down-regulating expression and phosphorylation level of cell cycle proteins.

Asperlin induces G2/M arrest through ROS generation and ATM pathway in human cervical carcinoma cells

International Nuclear Information System (INIS)

He, Long; Nan, Mei-Hua; Oh, Hyun Cheol; Kim, Young Ho; Jang, Jae Hyuk; Erikson, Raymond Leo; Ahn, Jong Seog; Kim, Bo Yeon

2011-01-01

Highlights: → A new anti-cancer effect of an antibiotics, asperlin, is exploited. → Asperlin induced human cervical cancer cell apoptosis through ROS generation. → Asperlin activated DNA-damage related ATM protein and cell cycle associated proteins. → Asperlin could be developed as a new anti-cancer therapeutics. -- Abstract: We exploited the biological activity of an antibiotic agent asperlin isolated from Aspergillus nidulans against human cervical carcinoma cells. We found that asperlin dramatically increased reactive oxygen species (ROS) generation accompanied by a significant reduction in cell proliferation. Cleavage of caspase-3 and PARP and reduction of Bcl-2 could also be detected after asperlin treatment to the cells. An anti-oxidant N-acetyl-L-cysteine (NAC), however, blocked all the apoptotic effects of asperlin. The involvement of oxidative stress in asperlin induced apoptosis could be supported by the findings that ROS- and DNA damage-associated G2/M phase arrest and ATM phosphorylation were increased by asperlin. In addition, expression and phosphorylation of cell cycle proteins as well as G2/M phase arrest in response to asperlin were significantly blocked by NAC or an ATM inhibitor KU-55933 pretreatment. Collectively, our study proved for the first time that asperlin could be developed as a potential anti-cancer therapeutics through ROS generation in HeLa cells.

Action of caffeine on x-irradiated HeLa cells. III. enhancement of x-ray-induced killing during G2 arrest

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Busse, P.M.; Bose, S.K.; Jones, R.W.; Tolmach, L.J.

1978-01-01

The ability of caffeine to enhance the expression of potentially lethal x-ray damage in HeLa S3 cells was examined as a function of the age of the cells in the generation cycle. Synchronous populations were irradiated at different times after mitotic collection and treated for various intervals with 1 mM caffeiene, which causes negligible killing of unirradiated cells. The response was thereby determined as a function of cell age at both the time of irradiation and the time of exposure to caffeine. The amount of cell killing depends strongly on when in the cycle caffeine is present and only weakly on when the cells are irradiated. If cells are irradiated in early G 1 , caffeine treatment enhances killing for 2 to 3 hr. No additional enhancement is observed until 16 to 17 hr postcollection, corresponding to G 2 ; here they enter a second period of much greater sensitivity. Similarly, fluorodeoxyuridine resynchronized cells irradiated during S and treated with caffeine suffer no enhanced killing until they pass into this sensitive phase in G 2 , approximately 7 hr after release from the fluorodeoxyuridine block. The sensitive period appears to coincide with G 2 arrest. The rate and extent of killing during this period are dependent upon the x-ray dose and the caffeine concentration. In the absence of caffeine, cells irradiated in G 1 lose sensitivity to caffeine in about 9 hr; they do so faster in G 2 . It is concluded that the potentially lethal x-ray damage expressed on treatment with caffeine is retained for many hours in the presence of caffeine and is maximally manifested by G 2 -arrested cells

Cordyceps militaris Fraction induces apoptosis and G2/M Arrest via c-Jun N-Terminal kinase signaling pathway in oral squamous carcinoma KB Cells.

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Xie, Wangshi; Zhang, Zhang; Song, Liyan; Huang, Chunhua; Guo, Zhongyi; Hu, Xianjing; Bi, Sixue; Yu, Rongmin

2018-01-01

Cordyceps militaris fraction (CMF) has been shown to possess in vitro antitumor activity against human chronic myeloid leukemia K562 cells in our previous research. The in vitro inhibitory activities of CMF on the growth of KB cells were evaluated by viability assay. The apoptotic and cell cycle influences of CMF were detected by 4',6-diamidino-2-phenylindole staining and flow cytometry assay. The expression of different apoptosis-associated proteins and cell cycle regulatory proteins was examined by Western blot assay. The nuclear localization of c-Jun was observed by fluorescence staining. The objective of this study was to investigate the antiproliferative effect of CMF as well as the mechanism underlying the apoptosis and cell cycle arrest it induces in KB cells. CMF suppressed KB cells' proliferation in a dose- and time-dependent manner. Flow cytometric analysis indicated that CMF induced G2/M cell cycle arrest and apoptosis. Western blot analysis revealed that CMF induced caspase-3, caspase-9, and PARP cleavages, and increased the Bax/Bcl-2 ratio. CMF also led to increased expression of p21, decreased expression of cyclin B1, mitotic phosphatase cdc25c, and mitotic kinase cdc2, as well as unchanged expression of p53. In addition, CMF stimulated c-Jun N-terminal kinases (JNK) protein phosphorylations, resulting in upregulated expression of c-Jun and nuclear localization of c-Jun. Pretreatment with JNK inhibitor SP600125 suppressed CMF-induced apoptosis and G2/M arrest. CMF is capable of modulating c-Jun caspase and Bcl-2 family proteins through JNK-dependent apoptosis, which results in G2/M phase arrest in KB cells. CMF could be developed as a promising candidate for the new antitumor agents. CMF exhibited strong anticancer activity against oral squamous carcinoma KB cellsCMF inhibited KB cells' proliferation via induction of apoptosis and G2/M cell cycle arrestCMF activated JNK signaling pathway and promoted the nuclear localization of c-JunCMF regulated the

Genistein induces G2/M cell cycle arrest and apoptosis via ATM/p53-dependent pathway in human colon cancer cells.

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Zhang, Zhiyu; Wang, Chong-Zhi; Du, Guang-Jian; Qi, Lian-Wen; Calway, Tyler; He, Tong-Chuan; Du, Wei; Yuan, Chun-Su

2013-07-01

Soybean isoflavones have been used as a potential preventive agent in anticancer research for many years. Genistein is one of the most active flavonoids in soybeans. Accumulating evidence suggests that genistein alters a variety of biological processes in estrogen-related malignancies, such as breast and prostate cancers. However, the molecular mechanism of genistein in the prevention of human colon cancer remains unclear. Here we attempted to elucidate the anticarcinogenic mechanism of genistein in human colon cancer cells. First we evaluated the growth inhibitory effect of genistein and two other isoflavones, daidzein and biochanin A, on HCT-116 and SW-480 human colon cancer cells. In addition, flow cyto-metry was performed to observe the morphological changes in HCT-116/SW-480 cells undergoing apoptosis or cell cycle arrest, which had been visualized using Annexin V-FITC and/or propidium iodide staining. Real-time PCR and western blot analyses were also employed to study the changes in expression of several important genes associated with cell cycle regulation. Our data showed that genistein, daidzein and biochanin A exhibited growth inhibitory effects on HCT-116/SW-480 colon cancer cells and promoted apoptosis. Genistein showed a significantly greater effect than the other two compounds, in a time- and dose-dependent manner. In addition, genistein caused cell cycle arrest in the G2/M phase, which was accompanied by activation of ATM/p53, p21waf1/cip1 and GADD45α as well as downregulation of cdc2 and cdc25A demonstrated by q-PCR and immunoblotting assay. Interestingly, genistein induced G2/M cell cycle arrest in a p53-dependent manner. These findings exemplify that isoflavones, especially genistein, could promote colon cancer cell growth inhibition and facilitate apoptosis and cell cycle arrest in the G2/M phase. The ATM/p53-p21 cross-regulatory network may play a crucial role in mediating the anticarcinogenic activities of genistein in colon cancer.

Identification of herpesvirus proteins that contribute to G1/S arrest.

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Paladino, Patrick; Marcon, Edyta; Greenblatt, Jack; Frappier, Lori

2014-04-01

Lytic infection by herpesviruses induces cell cycle arrest at the G1/S transition. This appears to be a function of multiple herpesvirus proteins, but only a minority of herpesvirus proteins have been examined for cell cycle effects. To gain a more comprehensive understanding of the viral proteins that contribute to G1/S arrest, we screened a library of over 200 proteins from herpes simplex virus type 1, human cytomegalovirus, and Epstein-Barr virus (EBV) for effects on the G1/S interface, using HeLa fluorescent, ubiquitination-based cell cycle indicator (Fucci) cells in which G1/S can be detected colorimetrically. Proteins from each virus were identified that induce accumulation of G1/S cells, predominantly tegument, early, and capsid proteins. The identification of several capsid proteins in this screen suggests that incoming viral capsids may function to modulate cellular processes. The cell cycle effects of selected EBV proteins were further verified and examined for effects on p53 and p21 as regulators of the G1/S transition. Two EBV replication proteins (BORF2 and BMRF1) were found to induce p53 but not p21, while a previously uncharacterized tegument protein (BGLF2) was found to induce p21 protein levels in a p53-independent manner. Proteomic analyses of BGLF2-interacting proteins identified interactions with the NIMA-related protein kinase (NEK9) and GEM-interacting protein (GMIP). Silencing of either NEK9 or GMIP induced p21 without affecting p53 and abrogated the ability of BGLF2 to further induce p21. Collectively, these results suggest multiple viral proteins contribute to G1/S arrest, including BGLF2, which induces p21 levels likely by interfering with the functions of NEK9 and GMIP. Most people are infected with multiple herpesviruses, whose proteins alter the infected cells in several ways. During lytic infection, the viral proteins block cell proliferation just before the cellular DNA replicates. We used a novel screening method to identify proteins

Chromosomal instability and the abrogated G2/M arrest in x-irradiated myelodysplastic syndrome cells

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Ban, S.; Sudo, H.; Saegusa, K.; Sagara, M.; Imai, T.; Kimura, A.

2003-01-01

A preliminary epidemiological study demonstrated that myelodysplastic syndrome (MDS) has an excess relative risk per sievert of 13 in atomic bomb survivors in Hiroshima. MDS is the only other radiogenic blood disease apart from leukemia. Clinically, MDS involves dysplastic hematopoiesis and an increased risk of leukemic transformation. Because it is uncertain whether MDS pathogenesis affects lymphoid progenitor cells as well as myeloid progenitor cells, we investigated the karyotypes of bone marrow cells and the micronucleus (MN) frequency in peripheral T lymphocytes of twenty- three atomic bomb survivors with MDS and five normal individuals. Aneuploidy was observed in 10 of 23 patients. Chromosome aberrations were observed in 3 of 12 patients with mild symptoms, and six of 11 patients of severe symptoms. The spontaneous- and X-ray-induced-MN frequencies were significantly higher in MDS patients than in normal individuals. Interestingly, radiation sensitivity increased along with the severity of MDS clinical subtypes. Because many of the patients in this study had not been exposed to chemo- or radiation- therapy, their unusual radiosensitivity may be related to their chromosomal or genomic instability. Immortalized lymphoid cell lines were established from B-lymphocytes infected with Epstein-Barr virus in vitro. The abrogation of radiation-induced-G2/M arrest was observed in 10 of 12 MDS-B lymphoid cell lines, but not in the normal B lymphoid cell lines. Our data suggest that the control of chromosomal stability is impaired in pluripotent stem cells of MDS patients, and that the abrogated G2/M arrest may be involved in the pathophysiology of disease progression and the high radiation sensitivity of patients

Increased expression of cyclin B1 mRNA coincides with diminished G{sub 2}-phase arrest in irradiated HeLa cells treated with staurosporine or caffeine

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Bernhard, E.J.; Maity, A.; McKenna, W.G.; Muschel, R.J. [Univ. of Pennsylvania School of Medicine, Philadelphia, PA (United States)

1994-12-01

The irradiation of cells results in delayed progression through the G{sub 2} phase of the cell cycle. Treatment of irradiated HeLa cells with caffeine greatly reduces the G{sub 2}-phase delay, while caffeine does not alter progression of cells through the cell cycle in unirradiated cells. In this report we demonstrate that treatment of HeLa cells with the kinase inhibitor staurosporine, but not with the inhibitor H7, also results in a reduction of the G{sub 2}-phase arrest after irradiation. Cell cycle progression in unirradiated cells is unaffected by 4.4 nM (2ng/ml) staurosporine, which releases the radiation-induced G{sub 2}-phase arrest. In HeLa cells, the G{sub 2}-phase delay after irradiation in S phase is accompanied by decreased expression of cyclin B1 mRNA. Coincident with the reduction in G{sub 2}-phase delay, we observed an increase in cyclin B1 mRNA accumulation in irradiated, staurosporine-treated cells compared to cells treated with irradiation alone. Caffeine treatment of irradiated HeLa cells also resulted in an elevation in the levels of cyclin B1 message. These results support the hypothesis that diminished cyclin B1 mRNA levels influence G{sub 2}-phase arrest to some degree. The findings that both staurosporine and caffeine treatments reverse the depression in cyclin B1 expression suggest that these two compounds may act on a common pathway of cell cycle control in response to radiation injury. 33 refs., 6 figs.

(4-Methoxyphenyl)(3,4,5-trimethoxyphenyl)methanone inhibits tubulin polymerization, induces G2/M arrest, and triggers apoptosis in human leukemia HL-60 cells

International Nuclear Information System (INIS)

Magalhães, Hemerson I.F.; Wilke, Diego V.; Bezerra, Daniel P.; Cavalcanti, Bruno C.; Rotta, Rodrigo; Lima, Dênis P. de; Beatriz, Adilson; Moraes, Manoel O.; Diniz-Filho, Jairo; Pessoa, Claudia

2013-01-01

(4-Methoxyphenyl)(3,4,5-trimethoxyphenyl)methanone (PHT) is a known cytotoxic compound belonging to the phenstatin family. However, the exact mechanism of action of PHT-induced cell death remains to be determined. The aim of this study was to investigate the mechanisms underlying PHT-induced cytotoxicity. We found that PHT displayed potent cytotoxicity in different tumor cell lines, showing IC 50 values in the nanomolar range. Cell cycle arrest in G 2 /M phase along with the augmented metaphase cells was found. Cells treated with PHT also showed typical hallmarks of apoptosis such as cell shrinkage, chromatin condensation, phosphatidylserine exposure, increase of the caspase 3/7 and 8 activation, loss of mitochondrial membrane potential, and internucleosomal DNA fragmentation without affecting membrane integrity. Studies conducted with isolated tubulin and docking models confirmed that PHT binds to the colchicine site and interferes in the polymerization of microtubules. These results demonstrated that PHT inhibits tubulin polymerization, arrests cancer cells in G 2 /M phase of the cell cycle, and induces their apoptosis, exhibiting promising anticancer therapeutic potential. - Highlights: • PHT inhibits tubulin polymerization. • PHT arrests cancer cells in G 2 /M phase of the cell cycle. • PHT induces caspase-dependent apoptosis

Histone Modification Is Involved in Okadaic Acid (OA Induced DNA Damage Response and G2-M Transition Arrest in Maize.

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Hao Zhang

Full Text Available Histone modifications are involved in regulation of chromatin structure. To investigate the relationship between chromatin modification and cell cycle regulation during plant cell proliferation, Okadaic acid (OA, a specific inhibitor of serine/threonine protein phosphatase, was applied in this study. The results showed that OA caused the cell cycle arrest at preprophase, leading to seedling growth inhibition. Western blotting assay revealed that the spatial distribution of phosphorylation of Ser10 histone H3 tails (H3S10ph signals was altered under OA treatment. Reactive oxygen species (ROS was found to be at higher levels and TdT-mediated dUTP nick end labeling (TUNEL assay displayed DNA breaks happened at the chromatin after treatment with OA, companied with an increase in the acetylation of histone H4 at lysine 5 (H4K5ac level. From these observations, we speculated that the alteration of the spatial distribution of H3S10ph and the level of H4K5ac was involved in the procedure that OA induced DNA breaks and G2-M arrested by the accumulation of ROS, and that the histone H3S10ph and H4K5ac might facilitate DNA repair by their association with the chromatin decondensation.

Asperlin induces G{sub 2}/M arrest through ROS generation and ATM pathway in human cervical carcinoma cells

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He, Long; Nan, Mei-Hua [Chemical Biology Research Center, Korea Research Institute of Bioscience and Biotechnology (KRIBB), 30 Yeongudanji-ro, Ochang-eup, Cheongwon-gun, Chungbuk 363-883 (Korea, Republic of); Oh, Hyun Cheol [College of Medical and Life Sciences, Silla University, 100 Silladaehak-gil, Sasang-gu, Busan 617-736 (Korea, Republic of); Kim, Young Ho [College of Pharmacy, ChungNam National University, Yuseong, Daejeon, 305-764 (Korea, Republic of); Jang, Jae Hyuk [Chemical Biology Research Center, Korea Research Institute of Bioscience and Biotechnology (KRIBB), 30 Yeongudanji-ro, Ochang-eup, Cheongwon-gun, Chungbuk 363-883 (Korea, Republic of); Erikson, Raymond Leo [Department of Molecular and Cellular Biology, Harvard University, Cambridge, MA 02138 (United States); Ahn, Jong Seog, E-mail: jsahn@kribb.re.kr [Chemical Biology Research Center, Korea Research Institute of Bioscience and Biotechnology (KRIBB), 30 Yeongudanji-ro, Ochang-eup, Cheongwon-gun, Chungbuk 363-883 (Korea, Republic of); Kim, Bo Yeon, E-mail: bykim@kribb.re.kr [Chemical Biology Research Center, Korea Research Institute of Bioscience and Biotechnology (KRIBB), 30 Yeongudanji-ro, Ochang-eup, Cheongwon-gun, Chungbuk 363-883 (Korea, Republic of); World Class Institute, KRIBB, 30 Yeongudanji-ro, Ochang-eup, Cheongwon-gun, Chungbuk 363-883 (Korea, Republic of)

2011-06-10

Highlights: {yields} A new anti-cancer effect of an antibiotics, asperlin, is exploited. {yields} Asperlin induced human cervical cancer cell apoptosis through ROS generation. {yields} Asperlin activated DNA-damage related ATM protein and cell cycle associated proteins. {yields} Asperlin could be developed as a new anti-cancer therapeutics. -- Abstract: We exploited the biological activity of an antibiotic agent asperlin isolated from Aspergillus nidulans against human cervical carcinoma cells. We found that asperlin dramatically increased reactive oxygen species (ROS) generation accompanied by a significant reduction in cell proliferation. Cleavage of caspase-3 and PARP and reduction of Bcl-2 could also be detected after asperlin treatment to the cells. An anti-oxidant N-acetyl-L-cysteine (NAC), however, blocked all the apoptotic effects of asperlin. The involvement of oxidative stress in asperlin induced apoptosis could be supported by the findings that ROS- and DNA damage-associated G2/M phase arrest and ATM phosphorylation were increased by asperlin. In addition, expression and phosphorylation of cell cycle proteins as well as G2/M phase arrest in response to asperlin were significantly blocked by NAC or an ATM inhibitor KU-55933 pretreatment. Collectively, our study proved for the first time that asperlin could be developed as a potential anti-cancer therapeutics through ROS generation in HeLa cells.

NF-κB and JNK mediated apoptosis and G0/G1 arrest of HeLa cells induced by rubiarbonol G, an arborinane-type triterpenoid from Rubia yunnanensis.

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Zeng, Guang-Zhi; Wang, Zhe; Zhao, Li-Mei; Fan, Jun-Ting; Tan, Ning-Hua

2018-06-28

Rubia yunnanensis is a medicinal plant mainly grown in Yunnan province in Southwest China, and its root named "Xiaohongshen" has been used as a herb in Yunnan for the treatment of cancers. Three major types of chemical components, Rubiaceae-type cyclopeptides, quinones, and triterpenoids, were identified from R. yunnanensis, in which some of compounds including rubiarbonol G (RG), a unique arboriane-type triterpenoid, showed cytotoxicity on cancer cells. But the cytotoxic mechanism of RG has not been reported. To investigate the cytotoxic mechanism of RG on cancer cells. RG was evaluated its cytotoxicity on 7 cancer cell lines by the SRB assay, and detected the effect on apoptosis and cell cycle arrest by Annexin V-FITC/PI apoptosis assay and DNA contents analysis. The expression and activity of apoptosis and cell cycle related proteins were also investigated by western blot and caspase activity assay. Furthermore, the effect of RG on NF-κB signaling was also tested by luciferase assay, western blot, and immunofluorescence staining. RG showed potent cytotoxicity on 7 human cancer cell lines, whose activity was attributed to apoptosis induction and G 0 /G 1 arrest in HeLa cells. Results from the mechanism study showed that RG promoted the activation of ERK1/2 and JNK pathway in MAPK family, which in turn increased the expression of p53, thereby triggering the G 0 /G 1 arrest through p53/p21/cyclin D1 signaling. Moreover, RG-mediated JNK activation down-regulated the expression of the anti-apoptotic protein Bcl-2, which caused the release of cytochrome c to the cytosol and activated the cleavage of caspase cascade and poly(ADP-ribose) polymerase, thereby inducing apoptosis in HeLa cells. In addition, RG was also found to inhibit the activation of NF-κB signaling by down-regulating the expression and attenuating the translocation to nucleus of NF-κB p65, by which the down-stream p53, cyclin D1, Bcl-2, and caspases were regulated, thereby triggering apoptosis and G

Cisplatin resistance in non-small cell lung cancer cells is associated with an abrogation of cisplatin-induced G2/M cell cycle arrest.

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Navin Sarin

Full Text Available The efficacy of cisplatin-based chemotherapy in cancer is limited by the occurrence of innate and acquired drug resistance. In order to better understand the mechanisms underlying acquired cisplatin resistance, we have compared the adenocarcinoma-derived non-small cell lung cancer (NSCLC cell line A549 and its cisplatin-resistant sub-line A549rCDDP2000 with regard to cisplatin resistance mechanisms including cellular platinum accumulation, DNA-adduct formation, cell cycle alterations, apoptosis induction and activation of key players of DNA damage response. In A549rCDDP2000 cells, a cisplatin-induced G2/M cell cycle arrest was lacking and apoptosis was reduced compared to A549 cells, although equitoxic cisplatin concentrations resulted in comparable platinum-DNA adduct levels. These differences were accompanied by changes in the expression of proteins involved in DNA damage response. In A549 cells, cisplatin exposure led to a significantly higher expression of genes coding for proteins mediating G2/M arrest and apoptosis (mouse double minute 2 homolog (MDM2, xeroderma pigmentosum complementation group C (XPC, stress inducible protein (SIP and p21 compared to resistant cells. This was underlined by significantly higher protein levels of phosphorylated Ataxia telangiectasia mutated (pAtm and p53 in A549 cells compared to their respective untreated control. The results were compiled in a preliminary model of resistance-associated signaling alterations. In conclusion, these findings suggest that acquired resistance of NSCLC cells against cisplatin is the consequence of altered signaling leading to reduced G2/M cell cycle arrest and apoptosis.

Specific Consideration on Superior Performance and Evaluation Methods of Polymer-housed Surge Arresters

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Ishizaki, Yoshihiro; Kobayashi, Misao; Suzuki, Hironori; Futagami, Koichi

It is very suitable to select the polymer materials for the housings of surge arresters (SAs), because the polymer materials are generally soft and light weight. Therefore, many kinds of polymer-housed SAs using various polymer materials have been developed, and expanding into many countries. Considering these backgrounds, the JEC technical report (JEC-TR) 23002-2008; polymer-housed surge arrester(1) has been established based on the existent relevant standards of arresters, such as JEC-2371-2003; Insulator-housed surge arresters(2) and IEC 60099-4 Edition 2.2, Metal-oxide surge arresters (MOSAs) without gaps for a.c. systems(3) in order to introduce the technology and provide a common guide for testing of polymer-housed SAs. According as the JEC-TR, the various new applications of the polymer-housed SAs, which are caused by superior advantages such as compact, light weight, safe failure mode, anti-seismic performance, anti-pollution performance and cost efficiency design, have been realized recently in Japan. Therefore, this paper gives specific consideration on the superior performance of the polymer-housed SAs and the evaluation methods of the polymer-housed SAs, because there are some issues in the existent standards to be solved.

(4-Methoxyphenyl)(3,4,5-trimethoxyphenyl)methanone inhibits tubulin polymerization, induces G{sub 2}/M arrest, and triggers apoptosis in human leukemia HL-60 cells

Energy Technology Data Exchange (ETDEWEB)

Magalhães, Hemerson I.F. [Departamento de Fisiologia e Farmacologia, Faculdade de Medicina, Universidade Federal do Ceará, Fortaleza, Ceará (Brazil); Centro de Ciências da Saúde, Departamento de Ciências Farmacêuticas, Universidade Federal da Paraíba, João Pessoa, Paraíba (Brazil); Wilke, Diego V. [Departamento de Fisiologia e Farmacologia, Faculdade de Medicina, Universidade Federal do Ceará, Fortaleza, Ceará (Brazil); Bezerra, Daniel P., E-mail: danielpbezerra@gmail.com [Centro de Pesquisa Gonçalo Moniz, Fundação Oswaldo Cruz, Salvador, Bahia (Brazil); Cavalcanti, Bruno C. [Departamento de Fisiologia e Farmacologia, Faculdade de Medicina, Universidade Federal do Ceará, Fortaleza, Ceará (Brazil); Rotta, Rodrigo; Lima, Dênis P. de; Beatriz, Adilson [Centro de Ciências Exatas e Tecnológicas (Laboratório LP4), Universidade Federal do Mato Grosso do Sul, Campo Grande, Mato Grosso do Sul (Brazil); Moraes, Manoel O.; Diniz-Filho, Jairo [Departamento de Fisiologia e Farmacologia, Faculdade de Medicina, Universidade Federal do Ceará, Fortaleza, Ceará (Brazil); Pessoa, Claudia, E-mail: c_pessoa@yahoo.com [Departamento de Fisiologia e Farmacologia, Faculdade de Medicina, Universidade Federal do Ceará, Fortaleza, Ceará (Brazil)

2013-10-01

(4-Methoxyphenyl)(3,4,5-trimethoxyphenyl)methanone (PHT) is a known cytotoxic compound belonging to the phenstatin family. However, the exact mechanism of action of PHT-induced cell death remains to be determined. The aim of this study was to investigate the mechanisms underlying PHT-induced cytotoxicity. We found that PHT displayed potent cytotoxicity in different tumor cell lines, showing IC{sub 50} values in the nanomolar range. Cell cycle arrest in G{sub 2}/M phase along with the augmented metaphase cells was found. Cells treated with PHT also showed typical hallmarks of apoptosis such as cell shrinkage, chromatin condensation, phosphatidylserine exposure, increase of the caspase 3/7 and 8 activation, loss of mitochondrial membrane potential, and internucleosomal DNA fragmentation without affecting membrane integrity. Studies conducted with isolated tubulin and docking models confirmed that PHT binds to the colchicine site and interferes in the polymerization of microtubules. These results demonstrated that PHT inhibits tubulin polymerization, arrests cancer cells in G{sub 2}/M phase of the cell cycle, and induces their apoptosis, exhibiting promising anticancer therapeutic potential. - Highlights: • PHT inhibits tubulin polymerization. • PHT arrests cancer cells in G{sub 2}/M phase of the cell cycle. • PHT induces caspase-dependent apoptosis.

Alkali-soluble polysaccharide, isolated from Lentinus edodes, induces apoptosis and G2/M cell cycle arrest in H22 cells through microtubule depolymerization.

Science.gov (United States)

You, Ru-Xu; Liu, Jin-Yu; Li, Shi-Jun; Wang, Liu; Wang, Kai-Ping; Zhang, Yu

2014-12-01

The aim of the study was to evaluate the pro-apoptotic effects of polysaccharides derived from Lentinus edodes and further elucidated the mechanisms of this action. Our results demonstrated that marked morphological changes of apoptosis were observed after treatment of L. edodes polysaccharides [Lentinan (LTN)]. Moreover, LTN-induced cell apoptosis was characterized by a rapid stimulation of reactive oxygen species production, the loss of mitochondrial membrane potential and an increase in intracellular concentration of Ca(2+) . In addition, the results of the haematoxylin and eosin and TUNEL assay further confirmed that LTN-induced apoptosis in vivo. Furthermore, flow cytometry analysis showed that LTN could arrest the cell cycle at G2/M phase, and immunofluorescence showed LTN caused disruption of microtubule. These results suggest that disruption of cellular microtubule network, arrest of the cell cycle at G2/M phase and induction of apoptosis may be one of the possible mechanisms of anti-tumour effect of LTN. Copyright © 2014 John Wiley & Sons, Ltd.

A methoxyflavanone derivative from the Asian medicinal herb (Perilla frutescens) induces p53-mediated G2/M cell cycle arrest and apoptosis in A549 human lung adenocarcinoma.

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Abd El-Hafeez, Amer Ali; Fujimura, Takashi; Kamei, Rikiya; Hirakawa, Noriko; Baba, Kenji; Ono, Kazuhisa; Kawamoto, Seiji

2017-07-14

Perilla frutescens is an Asian dietary herb consumed as an essential seasoning in Japanese cuisine as well as used for a Chinese medicine. Here, we report that a newly found methoxyflavanone derivative from P. frutescens (Perilla-derived methoxyflavanone, PDMF; 8-hydroxy-5,7-dimethoxyflavanone) shows carcinostatic activity on human lung adenocarcinoma, A549. We found that treatment with PDMF significantly inhibited cell proliferation and decreased viability through induction of G 2 /M cell cycle arrest and apoptosis. The PDMF stimulation induces phosphorylation of tumor suppressor p53 on Ser15, and increases its protein amount in conjunction with up-regulation of downstream cyclin-dependent kinase inhibitor p21 Cip1/Waf1 and proapoptotic caspases, caspase-9 and caspase-3. We also found that small interfering RNA knockdown of p53 completely abolished the PDMF-induced G 2 /M cell cycle arrest, and substantially abrogated its proapoptotic potency. These results suggest that PDMF represents a useful tumor-preventive phytochemical that triggers p53-driven G 2 /M cell cycle arrest and apoptosis.

GADD45a Regulates Olaquindox-Induced DNA Damage and S-Phase Arrest in Human Hepatoma G2 Cells via JNK/p38 Pathways

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Daowen Li

2017-01-01

Full Text Available Olaquindox, a quinoxaline 1,4-dioxide derivative, is widely used as a feed additive in many countries. The potential genotoxicity of olaquindox, hence, is of concern. However, the proper mechanism of toxicity was unclear. The aim of the present study was to investigate the effect of growth arrest and DNA damage 45 alpha (GADD45a on olaquindox-induced DNA damage and cell cycle arrest in HepG2 cells. The results showed that olaquindox could induce reactive oxygen species (ROS-mediated DNA damage and S-phase arrest, where increases of GADD45a, cyclin A, Cdk 2, p21 and p53 protein expression, decrease of cyclin D1 and the activation of phosphorylation-c-Jun N-terminal kinases (p-JNK, phosphorylation-p38 (p-p38 and phosphorylation-extracellular signal-regulated kinases (p-ERK were involved. However, GADD45a knockdown cells treated with olaquindox could significantly decrease cell viability, exacerbate DNA damage and increase S-phase arrest, associated with the marked activation of p-JNK, p-p38, but not p-ERK. Furthermore, SP600125 and SB203580 aggravated olaquindox-induced DNA damage and S-phase arrest, suppressed the expression of GADD45a. Taken together, these findings revealed that GADD45a played a protective role in olaquindox treatment and JNK/p38 pathways may partly contribute to GADD45a regulated olaquindox-induced DNA damage and S-phase arrest. Our findings increase the understanding on the molecular mechanisms of olaquindox.

Synchronization and Arrest of the Budding Yeast Cell Cycle Using Chemical and Genetic Methods.

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Rosebrock, Adam P

2017-01-03

The cell cycle of budding yeast can be arrested at specific positions by different genetic and chemical methods. These arrests enable study of cell cycle phase-specific phenotypes that would be missed during examination of asynchronous cultures. Some methods for arrest are reversible, with kinetics that enable release of cells back into a synchronous cycling state. Benefits of chemical and genetic methods include scalability across a large range of culture sizes from a few milliliters to many liters, ease of execution, the absence of specific equipment requirements, and synchronization and release of the entire culture. Of note, cell growth and division are decoupled during arrest and block-release experiments. Cells will continue transcription, translation, and accumulation of protein while arrested. If allowed to reenter the cell cycle, cells will do so as a population of mixed, larger-than-normal cells. Despite this important caveat, many aspects of budding yeast physiology are accessible using these simple chemical and genetic tools. Described here are methods for the block and release of cells in G 1 phase and at the M/G 1 transition using α-factor mating pheromone and the temperature-sensitive cdc15-2 allele, respectively, in addition to methods for arresting the cell cycle in early S phase and at G 2 /M by using hydroxyurea and nocodazole, respectively. © 2017 Cold Spring Harbor Laboratory Press.

Small Molecule TH-39 Potentially Targets Hec1/Nek2 Interaction and Exhibits Antitumor Efficacy in K562 Cells via G0/G1 Cell Cycle Arrest and Apoptosis Induction.

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Zhu, Yongxia; Wei, Wei; Ye, Tinghong; Liu, Zhihao; Liu, Li; Luo, Yong; Zhang, Lidan; Gao, Chao; Wang, Ningyu; Yu, Luoting

2016-01-01

Cancer is still a major public health issue worldwide, and new therapeutics with anti-tumor activity are still urgently needed. The anti-tumor activity of TH-39, which shows potent anti-proliferative activity against K562 cells with an IC50 of 0.78 µM, was investigated using immunoblot, co-immunoprecipitation, the MTT assay, and flow cytometry. Mechanistically, TH-39 may disrupt the interaction between Hec1 and Nek2 in K562 cells. Moreover, TH-39 inhibited cell proliferation in a concentration- and time-dependent manner by influencing the morphology of K562 cells and inducing G0/G1 phase arrest. G0/G1 phase arrest was associated with down-regulation of CDK2-cyclin E complex and CDK4/6-cyclin D complex activities. Furthermore, TH-39 also induced cell apoptosis, which was associated with activation of caspase-3, down-regulation of Bcl-2 expression and up-regulation of Bax. TH-39 could also decrease mitochondrial membrane potential (Δψm) and increase reactive oxygen species (ROS) accumulation in K562 cells. The results indicated that TH-39 might induce apoptosis via the ROS-mitochondrial apoptotic pathway. This study highlights the potential therapeutic efficacy of the anti-cancer compound TH-39 in treatment-resistant chronic myeloid leukemia. © 2016 The Author(s) Published by S. Karger AG, Basel.

Vitamin K2 and cotylenin A synergistically induce monocytic differentiation and growth arrest along with the suppression of c-MYC expression and induction of cyclin G2 expression in human leukemia HL-60 cells.

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Maniwa, Yasuhisa; Kasukabe, Takashi; Kumakura, Shunichi

2015-08-01

Although all-trans retinoic acid (ATRA) is a standard and effective drug used for differentiation therapy in acute promyelocytic leukemia, ATRA-resistant leukemia cells ultimately emerge during this treatment. Therefore, the development of new drugs or effective combination therapy is urgently needed. We demonstrate that the combined treatment of vitamin K2 and cotylenin A synergistically induced monocytic differentiation in HL-60 cells. This combined treatment also synergistically induced NBT-reducing activity and non-specific esterase-positive cells as well as morphological changes to monocyte/macrophage-like cells. Vitamin K2 and cotylenin A cooperatively inhibited the proliferation of HL-60 cells in short-term and long-term cultures. This treatment also induced growth arrest at the G1 phase. Although 5 µg/ml cotylenin A or 5 µM vitamin K2 alone reduced c-MYC gene expression in HL-60 cells to approximately 45% or 80% that of control cells, respectively, the combined treatment almost completely suppressed c-MYC gene expression. We also demonstrated that the combined treatment of vitamin K2 and cotylenin A synergistically induced the expression of cyclin G2, which had a positive effect on the promotion and maintenance of cell cycle arrest. These results suggest that the combination of vitamin K2 and cotylenin A has therapeutic value in the treatment of acute myeloid leukemia.

Sulforaphane Induces Cell Death Through G2/M Phase Arrest and Triggers Apoptosis in HCT 116 Human Colon Cancer Cells.

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Liu, Kuo-Ching; Shih, Ting-Ying; Kuo, Chao-Lin; Ma, Yi-Shih; Yang, Jiun-Long; Wu, Ping-Ping; Huang, Yi-Ping; Lai, Kuang-Chi; Chung, Jing-Gung

2016-01-01

Sulforaphane (SFN), an isothiocyanate, exists exclusively in cruciferous vegetables, and has been shown to possess potent antitumor and chemopreventive activity. However, there is no available information that shows SFN affecting human colon cancer HCT 116 cells. In the present study, we found that SFN induced cell morphological changes, which were photographed by contrast-phase microscopy, and decreased viability. SFN also induced G2/M phase arrest and cell apoptosis in HCT 116 cells, which were measured with flow cytometric assays. Western blotting indicated that SFN increased Cyclin A, cdk 2, Cyclin B and WEE1, but decreased Cdc 25C, cdk1 protein expressions that led to G2/M phase arrest. Apoptotic cell death was also confirmed by Annexin V/PI and DAPI staining and DNA gel electrophoresis in HCT 116 cells after exposure to SFN. The flow cytometric assay also showed that SFN induced the generation of reactive oxygen species (ROS) and Ca[Formula: see text] and decreased mitochondria membrane potential and increased caspase-8, -9 and -3 activities in HCT 116 cell. Western blotting also showed that SFN induced the release of cytochrome c, and AIF, which was confirmed by confocal microscopy examination. SFN induced ER stress-associated protein expression. Based on those observations, we suggest that SFN may be used as a novel anticancer agent for the treatment of human colon cancer in the future.

Defining the interactions and role of DCAF1/VPRBP in the DDB1-cullin4A E3 ubiquitin ligase complex engaged by HIV-1 Vpr to induce a G2 cell cycle arrest.

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Francine C A Gérard

Full Text Available HIV viral protein R (Vpr induces a cell cycle arrest at the G2/M phase by activating the ATR DNA damage/replication stress signalling pathway through engagement of the DDB1-CUL4A-DCAF1 E3 ubiquitin ligase via a direct binding to the substrate specificity receptor DCAF1. Since no high resolution structures of the DDB1-DCAF1-Vpr substrate recognition module currently exist, we used a mutagenesis approach to better define motifs in DCAF1 that are crucial for Vpr and DDB1 binding. Herein, we show that the minimal domain of DCAF1 that retained the ability to bind Vpr and DDB1 was mapped to residues 1041 to 1393 (DCAF1 WD. Mutagenic analyses identified an α-helical H-box motif and F/YxxF/Y motifs located in the N-terminal domain of DCAF1 WD that are involved in exclusive binding to DDB1. While we could not identify elements specifically involved in Vpr binding, overall, the mutagenesis data suggest that the predicted β-propeller conformation of DCAF1 is likely to be critical for Vpr association. Importantly, we provide evidence that binding of Vpr to DCAF1 appears to modulate the formation of a DDB1/DCAF1 complex. Lastly, we show that expression of DCAF1 WD in the absence of endogenous DCAF1 was not sufficient to enable Vpr-mediated G2 arrest activity. Overall, our results reveal that Vpr and DDB1 binding on DCAF1 can be genetically separated and further suggest that DCAF1 contains determinants in addition to the Vpr and DDB1 minimal binding domain, which are required for Vpr to enable the induction of a G2 arrest.

Apoptosis Induction by Polygonum minus is related to antioxidant capacity, alterations in expression of apoptotic-related genes, and S-phase cell cycle arrest in HepG2 cell line.

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Mohd Ghazali, Mohd Alfazari; Al-Naqeb, Ghanya; Krishnan Selvarajan, Kesavanarayanan; Hazizul Hasan, Mizaton; Adam, Aishah

2014-01-01

Polygonum minus (Polygonaceae) is a medicinal herb distributed throughout eastern Asia. The present study investigated antiproliferative effect of P. minus and its possible mechanisms. Four extracts (petroleum ether, methanol, ethyl acetate, and water) were prepared by cold maceration. Extracts were subjected to phytochemical screening, antioxidant, and antiproliferative assays; the most bioactive was fractionated using vacuum liquid chromatography into seven fractions (F1-F7). Antioxidant activity was measured via total phenolic content (TPC), 2,2-diphenyl-1-picrylhydrazyl (DPPH), and ferric reducing antioxidant power (FRAP) assays. Antiproliferative activity was evaluated using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Most active fraction was tested for apoptosis induction and cell cycle arrest in HepG2 cells using flow cytometry and confocal microscopy. Apoptotic-related gene expression was studied by RT-PCR. Ethyl acetate extract was bioactive in initial assays. Its fraction, F7, exhibited highest antioxidant capacity (TPC; 113.16 ± 6.2 mg GAE/g extract, DPPH; EC50: 30.5 ± 3.2 μg/mL, FRAP; 1169 ± 20.3 μmol Fe (II)/mg extract) and selective antiproliferative effect (IC50: 25.75 ± 1.5 μg/mL). F7 induced apoptosis in concentration- and time-dependent manner and caused cell cycle arrest at S-phase. Upregulation of proapoptotic genes (Bax, p53, and caspase-3) and downregulation of antiapoptotic gene, Bcl-2, were observed. In conclusion, F7 was antiproliferative to HepG2 cells by inducing apoptosis, cell cycle arrest, and via antioxidative effects.

Transcription arrest by a G quadruplex forming-trinucleotide repeat sequence from the human c-myb gene.

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Broxson, Christopher; Beckett, Joshua; Tornaletti, Silvia

2011-05-17

Non canonical DNA structures correspond to genomic regions particularly susceptible to genetic instability. The transcription process facilitates formation of these structures and plays a major role in generating the instability associated with these genomic sites. However, little is known about how non canonical structures are processed when encountered by an elongating RNA polymerase. Here we have studied the behavior of T7 RNA polymerase (T7RNAP) when encountering a G quadruplex forming-(GGA)(4) repeat located in the human c-myb proto-oncogene. To make direct correlations between formation of the structure and effects on transcription, we have taken advantage of the ability of the T7 polymerase to transcribe single-stranded substrates and of G4 DNA to form in single-stranded G-rich sequences in the presence of potassium ions. Under physiological KCl concentrations, we found that T7 RNAP transcription was arrested at two sites that mapped to the c-myb (GGA)(4) repeat sequence. The extent of arrest did not change with time, indicating that the c-myb repeat represented an absolute block and not a transient pause to T7 RNAP. Consistent with G4 DNA formation, arrest was not observed in the absence of KCl or in the presence of LiCl. Furthermore, mutations in the c-myb (GGA)(4) repeat, expected to prevent transition to G4, also eliminated the transcription block. We show T7 RNAP arrest at the c-myb repeat in double-stranded DNA under conditions mimicking the cellular concentration of biomolecules and potassium ions, suggesting that the G4 structure formed in the c-myb repeat may represent a transcription roadblock in vivo. Our results support a mechanism of transcription-coupled DNA repair initiated by arrest of transcription at G4 structures.

Isorhapontigenin (ISO) inhibited cell transformation by inducing G0/G1 phase arrest via increasing MKP-1 mRNA Stability.

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Gao, Guangxun; Chen, Liang; Li, Jingxia; Zhang, Dongyun; Fang, Yong; Huang, Haishan; Chen, Xiequn; Huang, Chuanshu

2014-05-15

The cancer chemopreventive property of Chinese herb new isolate isorhapontigenin (ISO) and mechanisms underlying its activity have never been explored. Here we demonstrated that ISO treatment with various concentrations for 3 weeks could dramatically inhibit TPA/EGF-induced cell transformation of Cl41 cells in Soft Agar assay, whereas co-incubation of cells with ISO at the same concentrations could elicit G0/G1 cell-cycle arrest without redundant cytotoxic effects on non-transformed cells. Further studies showed that ISO treatment resulted in cyclin D1 downregulation in dose- and time-dependent manner. Our results indicated that ISO regulated cyclin D1 at transcription level via targeting JNK/C-Jun/AP-1 activation. Moreover, we found that ISO-inhibited JNK/C-Jun/AP-1 activation was mediated by both upregulation of MKP-1 expression through increasing its mRNA stability and deactivating MKK7. Most importantly, MKP-1 knockdown could attenuate ISO-mediated suppression of JNK/C-Jun activation and cyclin D1 expression, as well as G0/G1 cell cycle arrest and cell transformation inhibition, while ectopic expression of FLAG-cyclin D1 T286A mutant also reversed ISO-induced G0/G1 cell-cycle arrest and inhibition of cell transformation. Our results demonstrated that ISO is a promising chemopreventive agent via upregulating mkp-1 mRNA stability, which is distinct from its cancer therapeutic effect with downregulation of XIAP and cyclin D1 expression.

The SecA2 pathway of Mycobacterium tuberculosis exports effectors that work in concert to arrest phagosome and autophagosome maturation.

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Zulauf, Katelyn E; Sullivan, Jonathan Tabb; Braunstein, Miriam

2018-04-30

To subvert host defenses, Mycobacterium tuberculosis (Mtb) avoids being delivered to degradative phagolysosomes in macrophages by arresting the normal host process of phagosome maturation. Phagosome maturation arrest by Mtb involves multiple effectors and much remains unknown about this important aspect of Mtb pathogenesis. The SecA2 dependent protein export system is required for phagosome maturation arrest and consequently growth of Mtb in macrophages. To better understand the role of the SecA2 pathway in phagosome maturation arrest, we identified two effectors exported by SecA2 that contribute to this process: the phosphatase SapM and the kinase PknG. Then, utilizing the secA2 mutant of Mtb as a platform to study effector functions, we identified specific steps in phagosome maturation inhibited by SapM and/or PknG. By identifying a histidine residue that is essential for SapM phosphatase activity, we confirmed for the first time that the phosphatase activity of SapM is required for its effects on phagosome maturation in macrophages. We further demonstrated that SecA2 export of SapM and PknG contributes to the ability of Mtb to replicate in macrophages. Finally, we extended our understanding of the SecA2 pathway, SapM, and PknG by revealing that their contribution goes beyond preventing Mtb delivery to mature phagolysosomes and includes inhibiting Mtb delivery to autophagolysosomes. Together, our results revealed SapM and PknG to be two effectors exported by the SecA2 pathway of Mtb with distinct as well as cumulative effects on phagosome and autophagosome maturation. Our results further reveal that Mtb must have additional mechanisms of limiting acidification of the phagosome, beyond inhibiting recruitment of the V-ATPase proton pump to the phagosome, and they indicate differences between effects of Mtb on phagosome and autophagosome maturation.

A novel muscarinic antagonist R2HBJJ inhibits non-small cell lung cancer cell growth and arrests the cell cycle in G0/G1.

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Nan Hua

Full Text Available Lung cancers express the cholinergic autocrine loop, which facilitates the progression of cancer cells. The antagonists of mAChRs have been demonstrated to depress the growth of small cell lung cancers (SCLCs. In this study we intended to investigate the growth inhibitory effect of R2HBJJ, a novel muscarinic antagonist, on non-small cell lung cancer (NSCLC cells and the possible mechanisms. The competitive binding assay revealed that R2HBJJ had a high affinity to M3 and M1 AChRs. R2HBJJ presented a strong anticholinergic activity on carbachol-induced contraction of guinea-pig trachea. R2HBJJ markedly suppressed the growth of NSCLC cells, such as H1299, H460 and H157. In H1299 cells, both R2HBJJ and its leading compound R2-PHC displayed significant anti-proliferative activity as M3 receptor antagonist darifenacin. Exogenous replenish of ACh could attenuate R2HBJJ-induced growth inhibition. Silencing M3 receptor or ChAT by specific-siRNAs resulted in a growth inhibition of 55.5% and 37.9% on H1299 cells 96 h post transfection, respectively. Further studies revealed that treatment with R2HBJJ arrested the cell cycle in G0/G1 by down-regulation of cyclin D1-CDK4/6-Rb. Therefore, the current study reveals that NSCLC cells express an autocrine and paracrine cholinergic system which stimulates the growth of NSCLC cells. R2HBJJ, as a novel mAChRs antagonist, can block the local cholinergic loop by antagonizing predominantly M3 receptors and inhibit NSCLC cell growth, which suggest that M3 receptor antagonist might be a potential chemotherapeutic regimen for NSCLC.

3-(3-Hydroxy-4-methoxyphenyl)-4-(3,4,5-trimethoxyphenyl)-1,2,5-selenadiazole (G-1103), a novel combretastatin A-4 analog, induces G2/M arrest and apoptosis by disrupting tubulin polymerization in human cervical HeLa cells and fibrosarcoma HT-1080 cells.

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Zuo, Daiying; Guo, Dandan; Jiang, Xuewei; Guan, Qi; Qi, Huan; Xu, Jingwen; Li, Zengqiang; Yang, Fushan; Zhang, Weige; Wu, Yingliang

2015-02-05

Microtubule is a popular target for anticancer drugs. In this study, we describe the effect 3-(3-hydroxy-4-methoxyphenyl)-4-(3,4,5-trimethoxyphenyl)-1,2,5-selenadiazole (G-1103), a newly synthesized analog of combretastatin A-4 (CA-4), showing a strong time- and dose-dependent anti-proliferative effect on human cervical cancer HeLa cells and human fibrosarcoma HT-1080 cells. We demonstrated that the growth inhibitory effects of G-1103 in HeLa and HT-1080 cells were associated with microtubule depolymerization and proved that G-1103 acted as microtubule destabilizing agent. Furthermore, cell cycle analysis revealed that G-1103 treatment resulted in cell cycle arrest at the G2/M phase in a time-dependent manner with subsequent apoptosis induction. Western blot analysis revealed that down-regulation of cdc25c and up-regulation of cyclin B1 was related with G2/M arrest in HeLa and HT-1080 cells treatment with G-1103. In addition, G-1103 induced HeLa cell apoptosis by up-regulating cleaved caspase-3, Fas, cleaved caspase-8 expression, which indicated that G-1103 induced HeLa cell apoptosis was mainly associated with death receptor pathway. However, G-1103 induced HT-1080 cell apoptosis by up-regulating cleaved caspase-3, Fas, cleaved caspase-8, Bax and cleaved caspase-9 expression and down-regulating anti-apoptotic protein Bcl-2 expression, which indicated that G-1103 induced HT-1080 cell apoptosis was associated with both mitochondrial and death receptor pathway. Taken together, all the data demonstrated that G-1103 exhibited its antitumor activity through disrupting the microtubule assembly, causing cell cycle arrest and consequently inducing apoptosis in HeLa and HT-1080 cells. Therefore, the novel compound G-1103 is a promising microtubule inhibitor that has great potentials for therapeutic treatment of various malignancies. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

Ziyuglycoside I Inhibits the Proliferation of MDA-MB-231 Breast Carcinoma Cells through Inducing p53-Mediated G2/M Cell Cycle Arrest and Intrinsic/Extrinsic Apoptosis.

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Zhu, Xue; Wang, Ke; Zhang, Kai; Zhang, Ting; Yin, Yongxiang; Xu, Fei

2016-11-22

Due to the aggressive clinical behavior, poor outcome, and lack of effective specific targeted therapies, triple-negative breast cancer (TNBC) has currently been recognized as one of the most malignant types of tumors. In the present study, we investigated the cytotoxic effect of ziyuglycoside I, one of the major components extracted from Chinese anti-tumor herbal Radix Sanguisorbae , on the TNBC cell line MDA-MB-231. The underlying molecular mechanism of the cytotoxic effect ziyuglycoside I on MDA-MB-231 cells was investigated with cell viability assay, flow cytometric analysis and Western blot. Compared to normal mammary gland Hs 578Bst cells, treatment of ziyuglycoside I resulted in a significant growth inhibitory effect on MDA-MB-231 cells. Ziyuglycoside I induced the G2/M phase arrest and apoptosis of MDA-MB-231 cells in a dose-dependent manner. These effects were found to be partially mediated through the up-regulation of p53 and p21 WAF1 , elevated Bax/Bcl-2 ratio, and the activation of both intrinsic (mitochondrial-initiated) and extrinsic (Fas/FasL-initiated) apoptotic pathways. Furthermore, the p53 specific siRNA attenuated these effects. Our study suggested that ziyuglycoside I-triggered MDA-MB-231 cell cycle arrest and apoptosis were probably mediated by p53. This suggests that ziyuglycoside I might be a potential drug candidate for treating TNBC.

Parafibromin inhibits cancer cell growth and causes G1 phase arrest

International Nuclear Information System (INIS)

Zhang Chun; Kong Dong; Tan, M.-H.; Pappas, Donald L.; Wang, P.-F.; Chen, Jindong; Farber, Leslie; Zhang Nian; Koo, H.-M.; Weinreich, Michael; Williams, Bart O.; Teh, B.T.

2006-01-01

The HRPT2 (hereditary hyperparathyroidism type 2) tumor suppressor gene encodes a ubiquitously expressed 531 amino acid protein termed parafibromin. Inactivation of parafibromin predisposes one to the development of HPT-JT syndrome. To date, the role of parafibromin in tumorigenesis is largely unknown. Here, we report that parafibromin is a nuclear protein that possesses anti-proliferative properties. We show that overexpression of parafibromin inhibits colony formation and cellular proliferation, and induces cell cycle arrest in the G1 phase. Moreover, HPT-JT syndrome-derived mutations in HRPT2 behave in a dominant-negative manner by abolishing the ability of parafibromin to suppress cell proliferation. These findings suggest that parafibromin has a critical role in cell growth, and mutations in HRPT2 can directly inhibit this role

Abnormal G1 arrest in the cell lines from LEC strain rats after X-irradiation

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Hayashi, M.; Uehara, K.; Kirisawa, R.; Endoh, D.; Arai, S.; Okui, T.

1997-01-01

The effect of X-irradiation of cell lines from LEC and WKAH strain rats on a progression o cell cycle was investigated. When WKAH rat ells were exposed to 5 Gy of X-rays and their cell cycle distribution was determined by a flow cytometer, the proportion of S-phase cells decrease and that of G2/M-phase cells in creased at 8 hr post-irradiation. At 18 and 24 hr post-irradiation, approximately 80% of the cells appeared in the G1 phase. On the contrary, the proportion of S-phase cells increased and that of G1-phase cells decreased in LEC rats during 8-24 hr post-irradiation, compared with that at 0 hr post-irradiation. Thus, radiation-induced delay in the progression from the G1 phase to S phase (G1 arrest) was observed inWKAH rat cells but not in LEC rat cells. In the case of WKAH rat cells, the intensities of the bands of p53 protein increased at 1 and 2 hr after X-irradiation at 5 Gy, compared with those of un-irradiated cells and at 0 hr post-irradiation. In contrast, the intensities of the bands were faint and did not significantly increase in LEC rat ells during 0-6 hr incubation after X-irradiation. Present results suggested that the radioresistant DNA synthesis in LEC rat cells is thought to be due to the abnormal G1 arrest following X-irradiation

A class of DNA-binding peptides from wheat bud causes growth inhibition, G2 cell cycle arrest and apoptosis induction in HeLa cells

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Elgjo Kjell

2009-07-01

Full Text Available Abstract Background Deproteinized DNA from eukaryotic and prokaryotic cells still contains a low-molecular weight peptidic fraction which can be dissociated by alkalinization of the medium. This fraction inhibits RNA transcription and tumor cell growth. Removal from DNA of normal cells causes amplification of DNA template activity. This effect is lower or absent in several cancer cell lines. Likewise, the amount of active peptides in cancer cell DNA extracts is lower than in DNA preparation of the corresponding normal cells. Such evidence, and their ubiquitous presence, suggests that they are a regulatory, conserved factor involved in the control of normal cell growth and gene expression. Results We report that peptides extracted from wheat bud chromatin induce growth inhibition, G2 arrest and caspase-dependent apoptosis in HeLa cells. The growth rate is decreased in cells treated during the S phase only and it is accompanied by DNA damage and DNA synthesis inhibition. In G2 cells, this treatment induces inactivation of the CDK1-cyclin B1 complex and an increase of active chk1 kinase expression. Conclusion The data indicate that the chromatin peptidic pool inhibits HeLa cell growth by causing defective DNA replication which, in turn, arrests cell cycle progression to mitosis via G2 checkpoint pathway activation.

Radiosensitization of NSCLC cells by EGFR inhibition is the result of an enhanced p53-dependent G1 arrest

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Kriegs, Malte; Gurtner, Kristin; Can, Yildiz; Brammer, Ingo; Rieckmann, Thorsten; Oertel, Reinhard; Wysocki, Marek; Dorniok, Franziska; Gal, Andreas; Grob, Tobias J.; Laban, Simon; Kasten-Pisula, Ulla; Petersen, Cordula; Baumann, Michael; Krause, Mechthild; Dikomey, Ekkehard

2015-01-01

Purpose: How EGF receptor (EGFR) inhibition induces cellular radiosensitization and with that increase in tumor control is still a matter of discussion. Since EGFR predominantly regulates cell cycle and proliferation, we studied whether a G1-arrest caused by EGFR inhibition may contribute to these effects. Materials and methods: We analyzed human non-small cell lung cancer (NSCLC) cell lines either wild type (wt) or mutated in p53 (A549, H460, vs. H1299, H3122) and HCT116 cells (p21 wt and negative). EGFR was inhibited by BIBX1382BS, erlotinib or cetuximab; p21 was knocked down by siRNA. Functional endpoints analyzed were cell signaling, proliferation, G1-arrest, cell survival as well as tumor control using an A549 tumor model. Results: When combined with IR, EGFR inhibition enhances the radiation-induced permanent G1 arrest, though solely in cells with intact p53/p21 signaling. This increase in G1-arrest was always associated with enhanced cellular radiosensitivity. Strikingly, this effect was abrogated when cells were re-stimulated, suggesting the initiation of dormancy. In line with this, only a small non-significant increase in tumor control was observed for A549 tumors treated with fractionated RT and EGFR inhibition. Conclusion: For NSCLC cells increase in radiosensitivity by EGFR inhibition results from enhanced G1-arrest. However, this effect does not lead to improved tumor control because cells can be released from this arrest by re-stimulation

Cucurbitacin B inhibits proliferation, induces G2/M cycle arrest and autophagy without affecting apoptosis but enhances MTT reduction in PC12 cells

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Chuanhong Wu

2016-03-01

Full Text Available In the present study, the effect of cucurbitacin B (a natural product with anti-cancer effect was studied on PC12 cells. It significantly reduced the cell number, changed cell morphology and inhibited colony formation while MTT results showed increased cell viability. Cucurbitacin B treatment increased activity of succinode hydrogenase. No alteration in the integrity of mem-brane, the release of lactic dehydrogenase, the mitochondrial membrane potential, and the expression of apoptotic proteins suggested that cucurbitacin B did not induce apoptosis. The cell cycle was remarkably arrested at G2/M phase. Furthermore, cucurbitacin B induced autophagy as evidence by accumulation of autophagic vacuoles and the increase of LC3II. In addition, cucurbitacin B up-regulated the expression of p-beclin-1, p-ULK1, p-Wee1, p21 and down-regulated p-mTOR, p-p70S6K, CDC25C, CDK1, Cyclin B1. In conclusion, cucurbitacin B inhibited PC12 proliferation but caused MTT pitfall. Cucurbitacin B induced G2/M cell cycle arrest, autophagy, but not the apoptosis in PC12 cells.

Differential sensitivity of p53+ and p53- cells to caffeine-induced radiosensitization and override of G2 delay

International Nuclear Information System (INIS)

Powell, S.N.; DeFrank, J.S.; Connell, P.; Eogan, M.; Preffer, F.; Dombkowski, D.; Tang, W.; Friend, S.H.

1995-01-01

Purpose: Most drug discovery efforts have focused on finding new DNA damaging agents to kill tumor cells preferentially. An alternative approach is to find ways to increase tumor specific killing by modifying tumor specific responses to that damage. We asked whether cells lacking the G1/S arrest in response to X-rays are more sensitive to X-ray damage when treated with agents that override G2/M arrest. Materials and Methods: Mouse embryonic fibroblasts genetically matched to be (+/+) or (-/-) p53 and rat embryonic fibroblasts (REF) made (+) or (-) for wild-type p53 function by transfection were irradiated with and without caffeine, a known checkpoint inhibitor. Caffeine treatment was maintained for 24 hours from 1 hour prior to irradiation. Cell survival following ionizing radiation was measured by clonogenic assay. For cell-cycle analysis, cells were in exponential asynchronous growth at the time of irradiation. The proportion of cells in G1, S and G2/M phases of the cell cycle were recorded immediately before and following irradiation and subsequently at 3,6,9,12,24 and 48 hours following irradiation. Results: Caffeine was found to cause radiosensitzation at low dose (0.5mM) in (-/-) cells but not in (+/+) cells. The sensitization enhancement ratio (SER) was 1.45 at 0.1 survival and 1.56 at 0.01 survival. At this dose of caffeine, this SER reflected therapeutic gain as there was no detectable effect on (+/+) cells. At 1mM caffeine, sensitization of (-/-) cells was 1.77, but (+/+) cells now also showed sensitization (SER=1.25). In (-/-) cells at 0.1mM caffeine the SER was 1.5 at 0.01 survival. The transfected REF cells (functionally null for p53) also exhibited caffeine-induced radiosensitization at both 0.5 and 2mM caffeine with a SER 1.45 for 2mM at 0.1 survival. No significant sensitization could be demonstrated for REF cells at the same doses of caffeine. The REF cells, with wild-type p53, transfected with pCMVneo alone showed no change in radiosensitivity or

Cell cycle arrest induced by radiation

International Nuclear Information System (INIS)

Okaichi, Yasuo; Matsumoto, Hideki; Ohnishi, Takeo

1994-01-01

It is known that various chemical reactions, such as cell cycle arrest, DNA repair and cell killing, can occur within the cells when exposed to ionizing radiation and ultraviolet radiation. Thus protein dynamics involved in such chemical reactions has received considerable attention. In this article, cell cycle regulation is first discussed in terms of the G2/M-phase and the G1/S-phase. Then, radiation-induced cell cycle arrest is reviewed. Cell cycle regulation mechanism involved in the G2 arrest, which is well known to occur when exposed to radiation, has recently been investigated using yeasts. In addition, recent study has yielded a noticeable finding that the G1 arrest can occur with intracellular accumulation of p53 product following ionization radiation. p53 is also shown to play an extremely important role in both DNA repair and cell killing due to DNA damage. Studies on the role of genes in protein groups induced by radiation will hold promise for the elucidation of cell cycle mechanism. (N.K.) 57 refs

4β-Hydroxywithanolide E from Physalis peruviana (golden berry inhibits growth of human lung cancer cells through DNA damage, apoptosis and G2/M arrest

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Guo Zong-Lun

2010-02-01

Full Text Available Abstract Background The crude extract of the fruit bearing plant, Physalis peruviana (golden berry, demonstrated anti-hepatoma and anti-inflammatory activities. However, the cellular mechanism involved in this process is still unknown. Methods Herein, we isolated the main pure compound, 4β-Hydroxywithanolide (4βHWE derived from golden berries, and investigated its antiproliferative effect on a human lung cancer cell line (H1299 using survival, cell cycle, and apoptosis analyses. An alkaline comet-nuclear extract (NE assay was used to evaluate the DNA damage due to the drug. Results It was shown that DNA damage was significantly induced by 1, 5, and 10 μg/mL 4βHWE for 2 h in a dose-dependent manner (p p 50 of 4βHWE in H1299 cells for 24 and 48 h were 0.6 and 0.71 μg/mL, respectively, suggesting it could be a potential therapeutic agent against lung cancer. In a flow cytometric analysis, 4βHWE produced cell cycle perturbation in the form of sub-G1 accumulation and slight arrest at the G2/M phase with 1 μg/mL for 12 and 24 h, respectively. Using flow cytometric and annexin V/propidium iodide immunofluorescence double-staining techniques, these phenomena were proven to be apoptosis and complete G2/M arrest for H1299 cells treated with 5 μg/mL for 24 h. Conclusions In this study, we demonstrated that golden berry-derived 4βHWE is a potential DNA-damaging and chemotherapeutic agent against lung cancer.

Inhibition of PI3K by ZSTK474 suppressed tumor growth not via apoptosis but G0/G1 arrest

International Nuclear Information System (INIS)

Dan, Shingo; Yoshimi, Hisashi; Okamura, Mutsumi; Mukai, Yumiko; Yamori, Takao

2009-01-01

Phosphoinositide 3-kinase (PI3K) is a potential target in cancer therapy. Inhibition of PI3K is believed to induce apoptosis. We recently developed a novel PI3K inhibitor ZSTK474 with antitumor efficacy. In this study, we have examined the underlying mode of action by which ZSTK474 exerts its antitumor efficacy. In vivo, ZSTK474 effectively inhibited the growth of human cancer xenografts. In parallel, ZSTK474 treatment suppressed the expression of phospho-Akt, suggesting effective PI3K inhibition, and also suppressed the expression of nuclear cyclin D1 and Ki67, both of which are hallmarks of proliferation. However, ZSTK474 treatment did not increase TUNEL-positive apoptotic cells. In vitro, ZSTK474 induced marked G 0 /G 1 arrest, but did not increase the subdiploid cells or activate caspase, both of which are hallmarks of apoptosis. These results clearly indicated that inhibition of PI3K by ZSTK474 did not induce apoptosis but rather induced strong G 0 /G 1 arrest, which might cause its efficacy in tumor cells.

Blocking CHK1 Expression Induces Apoptosis and Abrogates the G2 Checkpoint Mechanism

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Yan Luo

2001-01-01

Full Text Available Checkpoint kinase 1 (Chki is a checkpoint gene that is activated after DNA damage. It phosphorylates and inactivates the Cdc2 activating phosphatase Cdc25C. This in turn inactivates Cdc2, which leads to G2/M arrest. We report that blocking Chki expression by antisense or ribozymes in mammalian cells induces apoptosis and interferes with the G2/M arrest induced by adriamycin. The Chki inhibitor UCN-01 also blocks the G2 arrest after DNA damage and renders cells more susceptible to adriamycin. These results indicate that Chki is an essential gene for the checkpoint mechanism during normal cell proliferation as well as in the DNA damage response.

Circulatory Arrest, Brain Arrest and Death Determination

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Sam David Shemie

2018-03-01

Full Text Available Technological advances, particularly in the capacity to support, replace or transplant failing organs, continue to challenge and refine our understanding of human death. Given the ability to reanimate organs before and after death, both inside and outside of the body, through reinstitution of oxygenated circulation, concepts related to death of organs (e.g. cardiac death are no longer valid. This paper advances the rationale for a single conceptual determination of death related to permanent brain arrest, resulting from primary brain injury or secondary to circulatory arrest. The clinical characteristics of brain arrest are the permanent loss of capacity for consciousness and loss of all brainstem functions. In the setting of circulatory arrest, death occurs after the arrest of circulation to the brain rather than death of the heart. Correspondingly, any intervention that resumes oxygenated circulation to the brain after circulatory arrest would invalidate the determination of death.

Studies with GFP-Vpr fusion proteins: induction of apoptosis but ablation of cell-cycle arrest despite nuclear membrane or nuclear localization

International Nuclear Information System (INIS)

Waldhuber, Megan G.; Bateson, Michael; Tan, Judith; Greenway, Alison L.; McPhee, Dale A.

2003-01-01

The human immunodeficiency virus type 1 (HIV-1) Vpr protein is known to arrest the cell cycle in G 2 /M and induce apoptosis following arrest. The functions of Vpr relative to its location in the cell remain unresolved. We now demonstrate that the location and function of Vpr are dependent on the makeup of fusion proteins and that the functions of G 2 /M arrest and apoptosis are separable. Using green fluorescence protein mutants (EGFP or EYFP), we found that fusion at either the N- or C-terminus compromised the ability of Vpr to arrest cell cycling, relative to that of His-Vpr or wild-type protein. Additionally, utilizing the ability to specifically identify cells expressing the fusion proteins, we confirm that Vpr can induce apoptosis, but appears to be independent of cell-cycle arrest in G 2 /M. Both N- and C-terminal Vpr/EYFP fusion proteins induced apoptosis but caused minimal G 2 /M arrest. These studies with Vpr fusion proteins indicate that the functions of Vpr leading to G 2 /M arrest and apoptosis are separable and that fusion of Vpr to EGFP or EYFP affected the localization of the protein. Our findings suggest that nuclear membrane localization and nuclear import and export are strongly governed by modification of the N-terminus of Vpr

1-(2,6-Dihydroxy-4-methoxyphenyl-2-(4-hydroxyphenyl Ethanone-Induced Cell Cycle Arrest in G1/G0 in HT-29 Cells Human Colon Adenocarcinoma Cells

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Ma Ma Lay

2014-01-01

Full Text Available 1-(2,6-Dihydroxy-4-methoxyphenyl-2-(4-hydroxyphenyl ethanone (DMHE was isolated from the ethyl acetate fraction of Phaleria macrocarpa (Scheff. Boerl fruits and the structure confirmed by GC-MS (gas chromatography-mass spectrometry and NMR (nuclear magnetic resonance analysis. This compound was tested on the HT-29 human colon adenocarcinoma cell line using MTT (method of transcriptional and translational cell proliferation assay. The results of MTT assay showed that DMHE exhibited good cytotoxic effect on HT-29 cells in a dose- and time-dependent manner but no cytotoxic effect on the MRC-5 cell line after 72 h incubation. Morphological features of apoptotic cells upon treatment by DMHE, e.g., cell shrinkage and membrane blebbing, were examined by an inverted and phase microscope. Other features, such as chromatin condension and nuclear fragmentation were studied using acridine orange and propidium iodide staining under the fluorescence microscope. Future evidence of apoptosis/necrosis was provided by result fromannexin V-FITC/PI (fluorescein-isothiocyanate/propidium iodide staining revealed the percentage of early apoptotic, late apoptotic, necrotic and live cells in a dose- and time-dependent manner using flow cytometry. Cell cycle analysis showed G0/G1 arrest in a time-dependent manner. A western blot analysis indicated that cell death might be associated with the up-regulation of the pro-apoptotic proteins Bax PUMA. However, the anit-apotptic proteins Bcl-2, Bcl-xL, and Mcl-1 were also found to increase in a time-dependent manner. The expression of the pro-apoptotic protein Bak was not observed.

4β-Hydroxywithanolide E from Physalis peruviana (golden berry) inhibits growth of human lung cancer cells through DNA damage, apoptosis and G2/M arrest

International Nuclear Information System (INIS)

Yen, Ching-Yu; Guo, Zong-Lun; Cheng, Yu-Shan; Liu, Yin-Chang; Lan, Yu-Hsuan; Chang, Yu-Ching; Ko, Ying-Chin; Chang, Hsueh-Wei; Wu, Yang-Chang; Chiu, Chien-Chih; Chang, Fang-Rong; Chen, Jeff Yi-Fu; Hwang, Chi-Ching; Hseu, You-Cheng; Yang, Hsin-Ling; Lee, Alan Yueh-Luen; Tsai, Ming-Tz

2010-01-01

The crude extract of the fruit bearing plant, Physalis peruviana (golden berry), demonstrated anti-hepatoma and anti-inflammatory activities. However, the cellular mechanism involved in this process is still unknown. Herein, we isolated the main pure compound, 4β-Hydroxywithanolide (4βHWE) derived from golden berries, and investigated its antiproliferative effect on a human lung cancer cell line (H1299) using survival, cell cycle, and apoptosis analyses. An alkaline comet-nuclear extract (NE) assay was used to evaluate the DNA damage due to the drug. It was shown that DNA damage was significantly induced by 1, 5, and 10 μg/mL 4βHWE for 2 h in a dose-dependent manner (p < 0.005). A trypan blue exclusion assay showed that the proliferation of cells was inhibited by 4βHWE in both dose- and time-dependent manners (p < 0.05 and 0.001 for 24 and 48 h, respectively). The half maximal inhibitory concentrations (IC 50 ) of 4βHWE in H1299 cells for 24 and 48 h were 0.6 and 0.71 μg/mL, respectively, suggesting it could be a potential therapeutic agent against lung cancer. In a flow cytometric analysis, 4βHWE produced cell cycle perturbation in the form of sub-G 1 accumulation and slight arrest at the G 2 /M phase with 1 μg/mL for 12 and 24 h, respectively. Using flow cytometric and annexin V/propidium iodide immunofluorescence double-staining techniques, these phenomena were proven to be apoptosis and complete G 2 /M arrest for H1299 cells treated with 5 μg/mL for 24 h. In this study, we demonstrated that golden berry-derived 4βHWE is a potential DNA-damaging and chemotherapeutic agent against lung cancer

4β-Hydroxywithanolide E from Physalis peruviana (golden berry) inhibits growth of human lung cancer cells through DNA damage, apoptosis and G2/M arrest

Science.gov (United States)

2010-01-01

Background The crude extract of the fruit bearing plant, Physalis peruviana (golden berry), demonstrated anti-hepatoma and anti-inflammatory activities. However, the cellular mechanism involved in this process is still unknown. Methods Herein, we isolated the main pure compound, 4β-Hydroxywithanolide (4βHWE) derived from golden berries, and investigated its antiproliferative effect on a human lung cancer cell line (H1299) using survival, cell cycle, and apoptosis analyses. An alkaline comet-nuclear extract (NE) assay was used to evaluate the DNA damage due to the drug. Results It was shown that DNA damage was significantly induced by 1, 5, and 10 μg/mL 4βHWE for 2 h in a dose-dependent manner (p < 0.005). A trypan blue exclusion assay showed that the proliferation of cells was inhibited by 4βHWE in both dose- and time-dependent manners (p < 0.05 and 0.001 for 24 and 48 h, respectively). The half maximal inhibitory concentrations (IC50) of 4βHWE in H1299 cells for 24 and 48 h were 0.6 and 0.71 μg/mL, respectively, suggesting it could be a potential therapeutic agent against lung cancer. In a flow cytometric analysis, 4βHWE produced cell cycle perturbation in the form of sub-G1 accumulation and slight arrest at the G2/M phase with 1 μg/mL for 12 and 24 h, respectively. Using flow cytometric and annexin V/propidium iodide immunofluorescence double-staining techniques, these phenomena were proven to be apoptosis and complete G2/M arrest for H1299 cells treated with 5 μg/mL for 24 h. Conclusions In this study, we demonstrated that golden berry-derived 4βHWE is a potential DNA-damaging and chemotherapeutic agent against lung cancer. PMID:20167063

Human cytochrome c enters murine J774 cells and causes G1 and G2/M cell cycle arrest and induction of apoptosis

International Nuclear Information System (INIS)

Hiraoka, Yoshinori; Granja, Ana Teresa; Fialho, Arsenio M.; Schlarb-Ridley, Beatrix G.; Das Gupta, Tapas K.; Chakrabarty, Ananda M.; Yamada, Tohru

2005-01-01

Cytochrome c is well known as a carrier of electrons during respiration. Current evidence indicates that cytochrome c also functions as a major component of apoptosomes to induce apoptosis in eukaryotic cells as well as an antioxidant. More recently, a prokaryotic cytochrome c, cytochrome c 551 from Pseudomonas aeruginosa, has been shown to enter in mammalian cells such as the murine macrophage-like J774 cells and causes inhibition of cell cycle progression. Much less is known about such functions by mammalian cytochromes c, particularly the human cytochrome c. We now report that similar to P. aeruginosa cytochrome c 551 , the purified human cytochrome c protein can enter J774 cells and induce cell cycle arrest at the G 1 to S phase, as well as at the G 2 /M phase at higher concentrations. Unlike P. aeruginosa cytochrome c 551 which had no effect on the induction of apoptosis, human cytochrome c induces significant apoptosis and cell death in J774 cells, presumably through inhibition of the cell cycle at the G 2 /M phase. When incubated with human breast cancer MCF-7 and normal mammary epithelial cell line MCF-10A1 cells, human cytochrome c entered in both types of cells but induced cell death only in the normal MCF-10A1 cells. The ability of human cytochrome c to enter J774 cells was greatly reduced at 4 deg. C, suggesting energy requirement in the entry process

G2 arrest and apoptosis of cultured Raji cells by continuous low dose rate beta irradiation therapy with 188Re-perrhenate

International Nuclear Information System (INIS)

Yim, S. J.; Kim, E. H.; Lee, T. S.; Woo, K. S.; Jeong, W. S.; Choi, C. W.; Yim, S. M.

2001-01-01

Beta emitting radionuclide therapy gives exponentially decreasing radiation dose rate and results in cell death presumably by apoptosis. We observed changes in DNA content and apoptosis in relatively low dose rate beta irradiation. Raji cells were cultured and incubated with 188Re-perrhenate (3.7MBq, or 370MBq/ml) for 4 hours to give irradiation dose of 0.4, 4, or 40 Gy. After changing the culture media, cells were cultured for 2,4,8,16, and 24 hours. The cells were stained with Trypan blue, Annexin-V and Propidium Iodide (PI) to observe cell viability, cell membrane alternation by apoptosis and changes in DNA content respectively. Flowcytometry was done for Annexin-V and PI to quantitate apoptosis and necrosis in the irradiated cells. DAPI(4,6-diamidino-2-phenylindole) stain was also done to observe the damage in the nucleus. Cell viability decreased with an increasing radiation dose. Cells irradiated in 40 Gy showed early uptake of both Annexin-V and PI suggesting cell death by necrosis. Cells irradiated in 0.4 Gy showed delayed uptake of Annexin-V only, and later on PI uptake suggesting cell death mainly by apoptosis. The cells irradiated in 0.4 Gy showed G2 arrest in 16 hours after irradiation, but the cells irradiated in 40 Gy showed early DNA fragmentation within 2 hours after irradiation. In DAPI stain, early nucleus damage was observed in the cells irradiated in 40 Gy. On the other hand, slowly increasing apoptotic bodies were observed in the cells irradiated in 0.4 Gy. These results suggest that continuous low-dose irradiation induces G2 arrest and progressive apoptosis in cells while continuous high-dose irradiation induces rapid necrosis. Therefore, we expect therapeutic effect by continuous low-dose rate irradiation with beta emitting radiopharmaceuticals

Dux4 induces cell cycle arrest at G1 phase through upregulation of p21 expression

International Nuclear Information System (INIS)

Xu, Hongliang; Wang, Zhaoxia; Jin, Suqin; Hao, Hongjun; Zheng, Lemin; Zhou, Boda; Zhang, Wei; Lv, He; Yuan, Yun

2014-01-01

Highlights: • Dux4 induced TE671 cell proliferation defect and G1 phase arrest. • Dux4 upregulated p21 expression without activating p53. • Silencing p21 rescued Dux4 mediated proliferation defect and cell cycle arrest. • Sp1 binding site was required for Dux4-induced p21 promoter activation. - Abstract: It has been implicated that Dux4 plays crucial roles in development of facioscapulohumeral dystrophy. But the underlying myopathic mechanisms and related down-stream events of this retrogene were far from clear. Here, we reported that overexpression of Dux4 in a cell model TE671 reduced cell proliferation rate, and increased G1 phase accumulation. We also determined the impact of Dux4 on p53/p21 signal pathway, which controls the checkpoint in cell cycle progression. Overexpression of Dux4 increased p21 mRNA and protein level, while expression of p53, phospho-p53 remained unchanged. Silencing p21 rescued Dux4 mediated proliferation defect and cell cycle arrest. Furthermore, we demonstrated that enhanced Dux4 expression increased p21 promoter activity and elevated expression of Sp1 transcription factor. Mutation of Sp1 binding site decreased dux4 induced p21 promoter activation. Chromatin immunoprecipitation (ChIP) assays confirmed the Dux4-induced binding of Sp1 to p21 promoter in vivo. These results suggest that Dux4 might induce proliferation inhibition and G1 phase arrest through upregulation of p21

Dux4 induces cell cycle arrest at G1 phase through upregulation of p21 expression

Energy Technology Data Exchange (ETDEWEB)

Xu, Hongliang; Wang, Zhaoxia; Jin, Suqin; Hao, Hongjun [Department of Neurology, Peking University First Hospital, Beijing 100034 (China); Zheng, Lemin [The Institute of Cardiovascular Sciences, Peking University Health Science Center, Key Laboratory of Molecular Cardiovascular Sciences of Education Ministry, Key Laboratory of Cardiovascular Molecular Biology and Regulatory Peptides of Health Ministry, Beijing 100191 (China); Zhou, Boda [The Department of Cardiology, Peking University Third Hospital, Beijing 100191 (China); Zhang, Wei; Lv, He [Department of Neurology, Peking University First Hospital, Beijing 100034 (China); Yuan, Yun, E-mail: yuanyun2002@sohu.com [Department of Neurology, Peking University First Hospital, Beijing 100034 (China)

2014-03-28

Highlights: • Dux4 induced TE671 cell proliferation defect and G1 phase arrest. • Dux4 upregulated p21 expression without activating p53. • Silencing p21 rescued Dux4 mediated proliferation defect and cell cycle arrest. • Sp1 binding site was required for Dux4-induced p21 promoter activation. - Abstract: It has been implicated that Dux4 plays crucial roles in development of facioscapulohumeral dystrophy. But the underlying myopathic mechanisms and related down-stream events of this retrogene were far from clear. Here, we reported that overexpression of Dux4 in a cell model TE671 reduced cell proliferation rate, and increased G1 phase accumulation. We also determined the impact of Dux4 on p53/p21 signal pathway, which controls the checkpoint in cell cycle progression. Overexpression of Dux4 increased p21 mRNA and protein level, while expression of p53, phospho-p53 remained unchanged. Silencing p21 rescued Dux4 mediated proliferation defect and cell cycle arrest. Furthermore, we demonstrated that enhanced Dux4 expression increased p21 promoter activity and elevated expression of Sp1 transcription factor. Mutation of Sp1 binding site decreased dux4 induced p21 promoter activation. Chromatin immunoprecipitation (ChIP) assays confirmed the Dux4-induced binding of Sp1 to p21 promoter in vivo. These results suggest that Dux4 might induce proliferation inhibition and G1 phase arrest through upregulation of p21.

Exposure of Human Lung Cancer Cells to 8-Chloro-Adenosine Induces G2/M Arrest and Mitotic Catastrophe

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Hong-Yu Zhang

2004-11-01

Full Text Available 8-Chloro-adenosine (8-CI-Ado is a potent chemotherapeutic agent whose cytotoxicity in a variety of tumor cell lines has been widely investigated. However, the molecular mechanisms are uncertain. In this study, we found that exposure of human lung cancer cell lines A549 (p53-wt and H1299 (p53-depleted to 8-CI-Ado induced cell arrest in the G2/M phase, which was accompanied by accumulation of binucleated and polymorphonucleated cells resulting from aberrant mitosis and failed cytokinesis. Western blotting showed the loss of phosphorylated forms of Cdc2 and Cdc25C that allowed progression into mitosis. Furthermore, the increase in Ser10-phosphorylated histone H3-positive cells revealed by fluorescence-activated cell sorting suggested that the agent-targeted cells were able to exit the G2 phase and enter the M phase. Immunocytochemistry showed that microtubule and microfilament arrays were changed in exposed cells, indicating that the dynamic instability of microtubules and microfilaments was lost, which may correlate with mitotic dividing failure. Aberrant mitosis resulted in mitotic catastrophe followed by varying degrees of apoptosis, depending on the cell lines. Thus, 8-CI-Ado appears to exert its cytotoxicity toward cells in culture by inducing mitotic catastrophe.

Pleiotropic role of growth arrest-specific gene 6 in atherosclerosis

NARCIS (Netherlands)

Tjwa, Marc; Moons, Lieve; Lutgens, Esther

2009-01-01

Growth arrest-specific gene 6 (Gas6) belongs to the family of vitamin K-dependent coagulation proteins, but in contrast to its other members, has only a limited role in hemostasis. Instead, Gas6 plays a prominent role in conditions of injury, inflammation and repair. Gas6 amplifies the activation of

Fisetin induces G2/M phase cell cycle arrest by inactivating cdc25C-cdc2 via ATM-Chk1/2 activation in human endometrial cancer cells

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Zhan-Ying Wang

2015-06-01

Full Text Available Endometrial cancer is one of the most prevalent gynaecological malignancies where, currently available therapeutic options remain limited. Recently phytochemicals are exploited for their efficiency in cancer therapy. The present study investigates the anti-proliferative effect of fisetin, a flavonoid on human endometrial cancer cells (KLE and Hec1 A. Fisetin (20-100 µM effectively reduced the viability of Hec1 A and KLE cells and potentially altered the cell population at G2/M stage. Expression levels of the cell cycle proteins (cyclin B1, p-Cdc2, p-Cdc25C, p-Chk1, Chk2, p-ATM, cyclin B1, H2AX, p21 and p27 were analyzed. Fisetin suppressed cyclin B1 expression and caused inactiva-tion of Cdc25C and Cdc2 by increasing their phosphorylation levels and further activated ATM, Chk1 and Chk2. Increased levels of p21 and p27 were observed as well. These results suggest that fisetin induced G2/M cell cycle arrest via inactivating Cdc25c and Cdc2 through activation of ATM, Chk1 and Chk2.

The G2/M DNA damage checkpoint inhibits mitosis through Tyr15 phosphorylation of p34cdc2 in Aspergillus nidulans.

Science.gov (United States)

Ye, X S; Fincher, R R; Tang, A; Osmani, S A

1997-01-02

It is possible to cause G2 arrest in Aspergillus nidulans by inactivating either p34cdc2 or NIMA. We therefore investigated the negative control of these two mitosis-promoting kinases after DNA damage. DNA damage caused rapid Tyr15 phosphorylation of p34cdc2 and transient cell cycle arrest but had little effect on the activity of NIMA. Dividing cells deficient in Tyr15 phosphorylation of p34cdc2 were sensitive to both MMS and UV irradiation and entered lethal premature mitosis with damaged DNA. However, non-dividing quiescent conidiospores of the Tyr15 mutant strain were not sensitive to DNA damage. The UV and MMS sensitivity of cells unable to tyrosine phosphorylate p34cdc2 is therefore caused by defects in DNA damage checkpoint regulation over mitosis. Both the nimA5 and nimT23 temperature-sensitive mutations cause an arrest in G2 at 42 degrees C. Addition of MMS to nimT23 G2-arrested cells caused a marked delay in their entry into mitosis upon downshift to 32 degrees C and this delay was correlated with a long delay in the dephosphorylation and activation of p34cdc2. Addition of MMS to nimA5 G2-arrested cells caused inactivation of the H1 kinase activity of p34cdc2 due to an increase in its Tyr15 phosphorylation level and delayed entry into mitosis upon return to 32 degrees C. However, if Tyr15 phosphorylation of p34cdc2 was prevented then its H1 kinase activity was not inactivated upon MMS addition to nimA5 G2-arrested cells and they rapidly progressed into a lethal mitosis upon release to 32 degrees C. Thus, Tyr15 phosphorylation of p34cdc2 in G2 arrests initiation of mitosis after DNA damage in A. nidulans.

Curcumin induces G2/M arrest, apoptosis, NF-κB inhibition, and expression of differentiation genes in thyroid carcinoma cells.

Science.gov (United States)

Schwertheim, Suzan; Wein, Frederik; Lennartz, Klaus; Worm, Karl; Schmid, Kurt Werner; Sheu-Grabellus, Sien-Yi

2017-07-01

The therapy of unresectable advanced thyroid carcinomas shows unfavorable outcome. Constitutive nuclear factor-κB (NF-κB) activation in thyroid carcinomas frequently contributes to therapeutic resistance; the radioiodine therapy often fails due to the loss of differentiated functions in advanced thyroid carcinomas. Curcumin is known for its anticancer properties in a series of cancers, but only few studies have focused on thyroid cancer. Our aim was to evaluate curcumin's molecular mechanisms and to estimate if curcumin could be a new therapeutic option in advanced thyroid cancer. Human thyroid cancer cell lines TPC-1 (papillary), FTC-133 (follicular), and BHT-101 (anaplastic) were treated with curcumin. Using real-time PCR analysis, we investigated microRNA (miRNA) and mRNA expression levels. Cell cycle, Annexin V/PI staining, and caspase-3 activity analysis were performed to detect apoptosis. NF-κB p65 activity and cell proliferation were analyzed using appropriate ELISA-based colorimetric assay kits. Treatment with 50 μM curcumin significantly increased the mRNA expression of the differentiation genes thyroglobulin (TG) and sodium iodide symporter (NIS) in all three cell lines and induced inhibition of cell proliferation, apoptosis, and decrease of NF-κB p65 activity. The miRNA expression analyses showed a significant deregulation of miRNA-200c, -21, -let7c, -26a, and -125b, known to regulate cell differentiation and tumor progression. Curcumin arrested cell growth at the G2/M phase. Curcumin increases the expression of redifferentiation markers and induces G2/M arrest, apoptosis, and downregulation of NF-κB activity in thyroid carcinoma cells. Thus, curcumin appears to be a promising agent to overcome resistance to the conventional cancer therapy.

Resistance to bleomycin in cancer cell lines is characterized by prolonged doubling time, reduced DNA damage and evasion of G2/M arrest and apoptosis.

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Qi Wang

Full Text Available To establish, characterize and elucidate potential mechanisms of acquired bleomycin (BLM resistance using human cancer cell lines. Seven BLM-resistant cell lines were established by exposure to escalating BLM concentrations over a period of 16-24 months. IC50 values and cell doubling times were quantified using a real time cytotoxicity assay. COMET and γ-H2AX assays, cell cycle analysis, and apoptosis assessment further investigated the mechanisms of BLM resistance in these cell lines.Compared with parental cell lines, real time cytotoxicity assays revealed 7 to 49 fold increases in IC50 and a mean doubling time increase of 147 % (range 64 %-352% in BLM-resistant sub-clones (p<0.05 for both. Higher maintenance BLM concentrations were associated with higher IC50 and increased doubling times (p<0.05. Significantly reduced DNA damage (COMET and γ-H2AX assays, G2/M arrest, and apoptosis (p<0.05 for each set of comparison following high-dose acute BLM exposure was observed in resistant sub-clones, compared with their BLM-sensitive parental counterparts. Three weeks of BLM-free culturing resulted in a partial return to BLM sensitivity in 3/7 BLM-resistant sub-clones (p<0.05.Bleomycin resistance may be associated with reduced DNA damage after bleomycin exposure, resulting in reduced G2/M arrest, and reduced apoptosis.

Induction of G1 and G2/M cell cycle arrests by the dietary compound 3,3'-diindolylmethane in HT-29 human colon cancer cells

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Choi Hyun

2009-05-01

Full Text Available Abstract Background 3,3'-Diindolylmethane (DIM, an indole derivative produced in the stomach after the consumption of broccoli and other cruciferous vegetables, has been demonstrated to exert anti-cancer effects in both in vivo and in vitro models. We have previously determined that DIM (0 – 30 μmol/L inhibited the growth of HT-29 human colon cancer cells in a concentration-dependent fashion. In this study, we evaluated the effects of DIM on cell cycle progression in HT-29 cells. Methods HT-29 cells were cultured with various concentrations of DIM (0 – 30 μmol/L and the DNA was stained with propidium iodide, followed by flow cytometric analysis. [3H]Thymidine incorporation assays, Western blot analyses, immunoprecipitation and in vitro kinase assays for cyclin-dependent kinase (CDK and cell division cycle (CDC2 were conducted. Results The percentages of cells in the G1 and G2/M phases were dose-dependently increased and the percentages of cells in S phase were reduced within 12 h in DIM-treated cells. DIM also reduced DNA synthesis in a dose-dependent fashion. DIM markedly reduced CDK2 activity and the levels of phosphorylated retinoblastoma proteins (Rb and E2F-1, and also increased the levels of hypophosphorylated Rb. DIM reduced the protein levels of cyclin A, D1, and CDK4. DIM also increased the protein levels of CDK inhibitors, p21CIP1/WAF1 and p27KIPI. In addition, DIM reduced the activity of CDC2 and the levels of CDC25C phosphatase and cyclin B1. Conclusion Here, we have demonstrated that DIM induces G1 and G2/M phase cell cycle arrest in HT-29 cells, and this effect may be mediated by reduced CDK activity.

Induction of G1 and G2/M cell cycle arrests by the dietary compound 3,3'-diindolylmethane in HT-29 human colon cancer cells.

Science.gov (United States)

Choi, Hyun Ju; Lim, Do Young; Park, Jung Han Yoon

2009-05-29

3,3'-Diindolylmethane (DIM), an indole derivative produced in the stomach after the consumption of broccoli and other cruciferous vegetables, has been demonstrated to exert anti-cancer effects in both in vivo and in vitro models. We have previously determined that DIM (0 - 30 micromol/L) inhibited the growth of HT-29 human colon cancer cells in a concentration-dependent fashion. In this study, we evaluated the effects of DIM on cell cycle progression in HT-29 cells. HT-29 cells were cultured with various concentrations of DIM (0 - 30 micromol/L) and the DNA was stained with propidium iodide, followed by flow cytometric analysis. [3H]Thymidine incorporation assays, Western blot analyses, immunoprecipitation and in vitro kinase assays for cyclin-dependent kinase (CDK) and cell division cycle (CDC)2 were conducted. The percentages of cells in the G1 and G2/M phases were dose-dependently increased and the percentages of cells in S phase were reduced within 12 h in DIM-treated cells. DIM also reduced DNA synthesis in a dose-dependent fashion. DIM markedly reduced CDK2 activity and the levels of phosphorylated retinoblastoma proteins (Rb) and E2F-1, and also increased the levels of hypophosphorylated Rb. DIM reduced the protein levels of cyclin A, D1, and CDK4. DIM also increased the protein levels of CDK inhibitors, p21CIP1/WAF1 and p27KIPI. In addition, DIM reduced the activity of CDC2 and the levels of CDC25C phosphatase and cyclin B1. Here, we have demonstrated that DIM induces G1 and G2/M phase cell cycle arrest in HT-29 cells, and this effect may be mediated by reduced CDK activity.

[Association study between 834+7G/A and +1332C/T polymorphisms in the growth arrest specific 6 gene and risk of severe preeclampsia in Chinese population].

Science.gov (United States)

Ye, Liyan; Guan, Linbo; Fan, Ping; Liu, Xinghui; Liu, Rui; Chen, Jinxin; Zhu, Yue; Wei, Xin; Liu, Yu; Bai, Huai

2017-02-10

To investigate the relationship between polymorphisms of the growth arrest specific 6 (GAS6) gene and severe preeclampsia in a South West Han Chinese population. Blood samples from 167 patients with severe preeclampsia and 312 normal pregnant women as controls from Han Chinese in Chengdu area were analyzed by polymerase chain reaction-restriction fragment length polymorphisms. C and T allele frequencies for +1332C/T site were 85.63% and 14.37% in the patient group, respectively, and 78.04% and 21.96% in control group, respectively. The TT genotype and variant T allelic frequencies of the +1332C/T polymorphism were significantly lower in patients with severe preeclampsia than in the control group (both Ppreeclampsia was 0.602 (95%CI: 0.401-0.904) in carriers for the variant T allele (χ 2 =6.045, P=0.014). G and A allele frequencies for 834+7G/A site were 72.75% and 27.25% in case group, respectively, and 74.36% and 25.64% in control group, respectively. The genotype and allele frequencies of the 834+7G/A polymorphism in patients with severe preeclampsia and controls showed no significant differences (both P>0.05). In addition, there was no significant association between the polymorphisms and blood pressure levels in the patient or control groups. The variant GAS6+1332 T allele is associated with a decreased risk for severe preeclampsia in a South West Han Chinese population. On the other hand, the 834+7G/A polymorphism has no effect on the severe preeclampsia.

Carbon ions of different linear energy transfer (LET values induce apoptosis & G2 cell cycle arrest in radio-resistant melanoma cells

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Žakula Jelena

2016-01-01

Full Text Available Background & objectives: The main goal when treating malignancies with radiation is to deprive tumour cells of their reproductive potential. One approach is to induce tumour cell apoptosis. This study was conducted to evaluate the ability of carbon ions ( [12] C to induce apoptosis and cell cycle arrest in human HTB140 melanoma cells. Methods: In this in vitro study, human melanoma HTB140 cells were irradiated with the 62 MeV/n carbon ( [12] C ion beam, having two different linear energy transfer (LET values: 197 and 382 keV/μm. The dose range was 2 to 16 Gy. Cell viability was estimated by the sulforhodamine B assay seven days after irradiation. The cell cycle and apoptosis were evaluated 48 h after irradiation using flow cytometry. At the same time point, protein and gene expression of apoptotic regulators were estimated using the Western blot and q-PCR methods, respectively. Results: Cell viability experiments indicated strong anti-tumour effects of [12] C ions. The analysis of cell cycle showed that [12] C ions blocked HTB140 cells in G2 phase and induced the dose dependent increase of apoptosis. The maximum value of 21.8 per cent was attained after irradiation with LET of 197 keV/μm at the dose level of 16 Gy. Pro-apoptotic effects of [12] C ions were confirmed by changes of key apoptotic molecules: the p53, Bax, Bcl-2, poly ADP ribose polymerase (PARP as well as nuclear factor kappa B (NFκB. At the level of protein expression, the results indicated significant increases of p53, NFκB and Bax/Bcl-2 ratio and PARP cleavage. The Bax/Bcl-2 mRNA ratio was also increased, while no change was detected in the level of NFκB mRNA. Interpretation & conclusions: The present results indicated that anti-tumour effects of [12] C ions in human melanoma HTB140 cells were accomplished through induction of the mitochondrial apoptotic pathway as well as G2 arrest.

Inhibition of G1-phase arrest induced by ionizing radiation in hematopoietic cells by overexpression of genes involved in the G1/S-phase transition

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Epperly, M.; Berry, L.; Halloran, A.; Greenberger, J.S.

1995-01-01

D-type cyclins and cyclin-dependent kinase (cdk-4) are likely involved in regulating passage of cells through the G 1 phase of the cell cycle. A decrease in the proportion of cells in G 1 , a relatively radiation-sensitive phase of the cell cycle, should result in increased resistance to ionizing radiation; however, the effect of such overexpression on X-ray-induced G 1 -phase arrest is not known. Radiation survival curves were obtained at a dose rate of either 8 cGy/min or 1 Gy/min for subclones of the IL-3-dependent hematopoietic progenitor cell line 32D cl 3 expressing transgenes for either cyclin-D1, D2 or D3 or cdk-4. We compared the results to those with overexpression of the transgene for Bcl-2, whose expression enhances radiation survival and delays apoptosis. Cells overexpressing transgenes for each D-type cyclin or Bcl-2 had an increased number of cells in S phase compared to parent line 32D cl 3; however, overexpression of cdk-4 had no effect on cell cycle distribution. Cell death resulting from withdrawal of IL-3 was not affected by overexpression of D2, cdk-4 or Bcl-2. Flow cytometry 24 h after 5 Gy irradiation demonstrated that overexpression of each G 1 -phase regulatory transgene decreased the proportion of cells at the G 1 /S-phase border. Western analysis revealed induction of cyclin-D protein levels by irradiation, but no change in the D O , but a significant increase in the rvec n for cyclin-D or cdk-4 transgene-overexpressing clones at 1 Gy/min (P 1 /S-phase arrest. 31 refs., 4 figs., 4 tabs

Ponatinib promotes a G1 cell-cycle arrest of merlin/NF2-deficient human schwann cells.

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Petrilli, Alejandra M; Garcia, Jeanine; Bott, Marga; Klingeman Plati, Stephani; Dinh, Christine T; Bracho, Olena R; Yan, Denise; Zou, Bing; Mittal, Rahul; Telischi, Fred F; Liu, Xue-Zhong; Chang, Long-Sheng; Welling, D Bradley; Copik, Alicja J; Fernández-Valle, Cristina

2017-05-09

Neurofibromatosis type 2 (NF2) is a genetic syndrome that predisposes individuals to multiple benign tumors of the central and peripheral nervous systems, including vestibular schwannomas. Currently, there are no FDA approved drug therapies for NF2. Loss of function of merlin encoded by the NF2 tumor suppressor gene leads to activation of multiple mitogenic signaling cascades, including platelet-derived growth factor receptor (PDGFR) and SRC in Schwann cells. The goal of this study was to determine whether ponatinib, an FDA-approved ABL/SRC inhibitor, reduced proliferation and/or survival of merlin-deficient human Schwann cells (HSC). Merlin-deficient HSC had higher levels of phosphorylated PDGFRα/β, and SRC than merlin-expressing HSC. A similar phosphorylation pattern was observed in phospho-protein arrays of human vestibular schwannoma samples compared to normal HSC. Ponatinib reduced merlin-deficient HSC viability in a dose-dependent manner by decreasing phosphorylation of PDGFRα/β, AKT, p70S6K, MEK1/2, ERK1/2 and STAT3. These changes were associated with decreased cyclin D1 and increased p27Kip1levels, leading to a G1 cell-cycle arrest as assessed by Western blotting and flow cytometry. Ponatinib did not modulate ABL, SRC, focal adhesion kinase (FAK), or paxillin phosphorylation levels. These results suggest that ponatinib is a potential therapeutic agent for NF2-associated schwannomas and warrants further in vivo investigation.

Proteasome activity is important for replication recovery, CHK1 phosphorylation and prevention of G2 arrest after low-dose formaldehyde

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Ortega-Atienza, Sara; Green, Samantha E.; Zhitkovich, Anatoly, E-mail: anatoly_zhitkovich@brown.edu

2015-07-15

Formaldehyde (FA) is a human carcinogen with numerous sources of environmental and occupational exposures. This reactive aldehyde is also produced endogenously during metabolism of drugs and other processes. DNA–protein crosslinks (DPCs) are considered to be the main genotoxic lesions for FA. Accumulating evidence suggests that DPC repair in high eukaryotes involves proteolysis of crosslinked proteins. Here, we examined a role of the main cellular proteolytic machinery proteasomes in toxic responses of human lung cells to low FA doses. We found that transient inhibition of proteasome activity increased cytotoxicity and diminished clonogenic viability of FA-treated cells. Proteasome inactivation exacerbated suppressive effects of FA on DNA replication and increased the levels of the genotoxic stress marker γ-H2AX in normal human cells. A transient loss of proteasome activity in FA-exposed cells also caused delayed perturbations of cell cycle, which included G2 arrest and a depletion of S-phase populations at FA doses that had no effects in control cells. Proteasome activity diminished p53-Ser15 phosphorylation but was important for FA-induced CHK1 phosphorylation, which is a biochemical marker of DPC proteolysis in replicating cells. Unlike FA, proteasome inhibition had no effect on cell survival and CHK1 phosphorylation by the non-DPC replication stressor hydroxyurea. Overall, we obtained evidence for the importance of proteasomes in protection of human cells against biologically relevant doses of FA. Biochemically, our findings indicate the involvement of proteasomes in proteolytic repair of DPC, which removes replication blockage by these highly bulky lesions. - Highlights: • Proteasome inhibition enhances cytotoxicity of low-dose FA in human lung cells. • Active proteasomes diminish replication-inhibiting effects of FA. • Proteasome activity prevents delayed G2 arrest in FA-treated cells. • Proteasome inhibition exacerbates replication stress by FA in

Betulinic Acid Inhibits Growth of Cultured Vascular Smooth Muscle Cells In Vitro by Inducing G1 Arrest and Apoptosis

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Raja Kumar Vadivelu

2012-01-01

Full Text Available Betulinic acid is a widely available plant-derived triterpene which is reported to possess selective cytotoxic activity against cancer cells of neuroectodermal origin and leukemia. However, the potential of betulinic acid as an antiproliferative and cytotoxic agent on vascular smooth muscle (VSMC is still unclear. This study was carried out to demonstrate the antiproliferative and cytotoxic effect of betulinic acid on VSMCs using 3-[4,5-dimethylthizol-2-yl]-2,5-diphenyltetrazolium bromide (MTT assay, flow cytometry cell cycle assay, BrdU proliferation assay, acridine orange/propidium iodide staining, and comet assay. Result from MTT and BrdU assays indicated that betulinic acid was able to inhibit the growth and proliferation of VSMCs in a dose-dependent manner with IC50 of 3.8 μg/mL significantly (P<0.05. Nevertheless, betulinic acid exhibited G1 cell cycle arrest in flow cytometry cell cycle profiling and low level of DNA damage against VSMC in acridine orange/propidium iodide and comet assay after 24 h of treatment. In conclusion, betulinic acid induced G1 cell cycle arrest and dose-dependent DNA damage on VSMC.

Repair of U/G and U/A in DNA by UNG2-associated repair complexes takes place predominantly by short-patch repair both in proliferating and growth-arrested cells

DEFF Research Database (Denmark)

Akbari, Mansour; Otterlei, Marit; Pena Diaz, Javier

2004-01-01

Nuclear uracil-DNA glycosylase UNG2 has an established role in repair of U/A pairs resulting from misincorporation of dUMP during replication. In antigen-stimulated B-lymphocytes UNG2 removes uracil from U/G mispairs as part of somatic hypermutation and class switch recombination processes. Using......, PCNA and DNA ligase, the latter detected as activity. Short-patch repair was the predominant mechanism both in extracts and UNG2-ARC from proliferating and less BER-proficient growth-arrested cells. Repair of U/G mispairs and U/A pairs was completely inhibited by neutralizing UNG...

AZD2014 Radiosensitizes Oral Squamous Cell Carcinoma by Inhibiting AKT/mTOR Axis and Inducing G1/G2/M Cell Cycle Arrest.

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Chih-Chia Yu

Full Text Available Oral squamous cell carcinoma (OSCC is one of the most common malignant neoplasms in Taiwan. Activation of the mTOR signaling pathway has been linked to decreased radiation responsiveness in human oral cancer, thus it limits efficacy of radiotherapy. To address this question, we investigated the effect of AZD2014, a novel small molecular ATP-competitive inhibitor of mTORC1 and mTORC2 kinase, as a radiosensitizer in primary OSCC and OSCC-derived cell line models.We isolated primary tumor cells from OSCC tissues and cell lines. AZD2014 was administered with and without ionizing radiation. The radiosensitizing effect of AZD2014 were then assessed using cell viability assays, clonogenic survival assays, and cell cycle analyses. Western blotting was used to detect protein expression.Combination treatment with AZD2014 and irradiation resulted in significant reduction in OSCC cell line and primary OSCC cell colony formation due to the enhanced inhibition of AKT and both mTORC1 and mTORC2 activity. Pre-treatment with AZD2014 in irradiated oral cancer cells induced tumor cell cycle arrest at the G1 and G2/M phases, which led to disruption of cyclin D1-CDK4 and cyclin B1-CDC2 complexes. Moreover, AZD2014 synergized with radiation to promote both apoptosis and autophagy by increasing caspase-3 and LC3 in primary OSCC cells.These findings suggest that in irradiated OSCC cells, co-treatment with AZD2014, which targets mTORC1 and mTORC2 blockade, is an effective radiosensitizing strategy for oral squamous cell carcinoma.

Live-Cell Imaging Visualizes Frequent Mitotic Skipping During Senescence-Like Growth Arrest in Mammary Carcinoma Cells Exposed to Ionizing Radiation

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Suzuki, Masatoshi, E-mail: msuzuki@nagasaki-u.ac.jp [Department of Radiation Medical Sciences, Atomic Bomb Disease Institute, Nagasaki University Graduate School of Biomedical Sciences, Nagasaki (Japan); Yamauchi, Motohiro; Oka, Yasuyoshi; Suzuki, Keiji; Yamashita, Shunichi [Department of Radiation Medical Sciences, Atomic Bomb Disease Institute, Nagasaki University Graduate School of Biomedical Sciences, Nagasaki (Japan)

2012-06-01

Purpose: Senescence-like growth arrest in human solid carcinomas is now recognized as the major outcome of radiotherapy. This study was designed to analyze cell cycle during the process of senescence-like growth arrest in mammary carcinoma cells exposed to X-rays. Methods and Materials: Fluorescent ubiquitination-based cell cycle indicators were introduced into the human mammary carcinoma cell line MCF-7. Cell cycle was sequentially monitored by live-cell imaging for up to 5 days after exposure to 10 Gy of X-rays. Results: Live-cell imaging revealed that cell cycle transition from G2 to G1 phase without mitosis, so-called mitotic skipping, was observed in 17.1% and 69.8% of G1- and G2-irradiated cells, respectively. Entry to G1 phase was confirmed by the nuclear accumulation of mKO{sub 2}-hCdt1 as well as cyclin E, which was inversely correlated to the accumulation of G2-specific markers such as mAG-hGeminin and CENP-F. More than 90% of cells skipping mitosis were persistently arrested in G1 phase and showed positive staining for the senescent biochemical marker, which is senescence-associated ss-galactosidase, indicating induction of senescence-like growth arrest accompanied by mitotic skipping. While G2 irradiation with higher doses of X-rays induced mitotic skipping in approximately 80% of cells, transduction of short hairpin RNA (shRNA) for p53 significantly suppressed mitotic skipping, suggesting that ionizing radiation-induced mitotic skipping is associated with p53 function. Conclusions: The present study found the pathway of senescence-like growth arrest in G1 phase without mitotic entry following G2-irradiation.

Live-Cell Imaging Visualizes Frequent Mitotic Skipping During Senescence-Like Growth Arrest in Mammary Carcinoma Cells Exposed to Ionizing Radiation

International Nuclear Information System (INIS)

Suzuki, Masatoshi; Yamauchi, Motohiro; Oka, Yasuyoshi; Suzuki, Keiji; Yamashita, Shunichi

2012-01-01

Purpose: Senescence-like growth arrest in human solid carcinomas is now recognized as the major outcome of radiotherapy. This study was designed to analyze cell cycle during the process of senescence-like growth arrest in mammary carcinoma cells exposed to X-rays. Methods and Materials: Fluorescent ubiquitination-based cell cycle indicators were introduced into the human mammary carcinoma cell line MCF-7. Cell cycle was sequentially monitored by live-cell imaging for up to 5 days after exposure to 10 Gy of X-rays. Results: Live-cell imaging revealed that cell cycle transition from G2 to G1 phase without mitosis, so-called mitotic skipping, was observed in 17.1% and 69.8% of G1- and G2-irradiated cells, respectively. Entry to G1 phase was confirmed by the nuclear accumulation of mKO 2 -hCdt1 as well as cyclin E, which was inversely correlated to the accumulation of G2-specific markers such as mAG-hGeminin and CENP-F. More than 90% of cells skipping mitosis were persistently arrested in G1 phase and showed positive staining for the senescent biochemical marker, which is senescence-associated ß-galactosidase, indicating induction of senescence-like growth arrest accompanied by mitotic skipping. While G2 irradiation with higher doses of X-rays induced mitotic skipping in approximately 80% of cells, transduction of short hairpin RNA (shRNA) for p53 significantly suppressed mitotic skipping, suggesting that ionizing radiation-induced mitotic skipping is associated with p53 function. Conclusions: The present study found the pathway of senescence-like growth arrest in G1 phase without mitotic entry following G2-irradiation.

N-(4-Hydroxyphenyl) retinamide potentiated paclitaxel for cell cycle arrest and apoptosis in glioblastoma C6 and RG2 cells

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Janardhanan, Rajiv; Butler, Jonathan T.; Banik, Naren L.; Ray, Swapan K.

2009-01-01

Glioblastoma grows aggressively due to its ability to maintain abnormally high potentials for cell proliferation. The present study examines the synergistic actions of N-(4-hdroxyphenyl) retinamide (4-HPR) and paclitaxel (PTX) to control the growth of rat glioblastoma C6 and RG2 cell lines. 4-HPR induced astrocytic differentiation was accompanied by increased expression of the tight junction protein e-cadherin and sustained down regulation of Id2 (member of inhibitor of differentiation family), catalytic subunit of rat telomerase reverse transcriptase (rTERT), and proliferating cell nuclear antigen (PCNA). Flow cytometric analysis showed that the microtubule stabilizer PTX caused cell cycle deregulation due to G2/M arrest. This in turn could alter the fate of kinetochore-spindletube dynamics thereby halting cell cycle progression. An interesting observation was induction of G1/S arrest by combination of 4-HPR and PTX, altering the G2/M arrest induced by PTX alone. This was further ratified by the upregulation of tumor suppressor protein retinoblastoma, which repressed the expression of the key signaling moieties to induce G1/S arrest. Collectively, combination of 4-HPR and PTX diminished the survival factors (e.g., rTERT, PCNA, and Bcl-2) to make glioblastoma cells highly prone to apoptosis with activation of cysteine proteases (e.g., calpain, cathepsins, caspase-8, caspase-3) in two glioblastoma cell lines. Hence, combination 4-HPR and PTX can be considered as an effective therapeutic strategy for controlling the growth of heterogeneous glioblastoma cell populations. PMID:19285047

Persea declinata (Bl. Kosterm Bark Crude Extract Induces Apoptosis in MCF-7 Cells via G0/G1 Cell Cycle Arrest, Bcl-2/Bax/Bcl-xl Signaling Pathways, and ROS Generation

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Putri Narrima

2014-01-01

Full Text Available Persea declinata (Bl. Kosterm is a member of the Lauraceae family, widely distributed in Southeast Asia. It is from the same genus with avocado (Persea americana Mill, which is widely consumed as food and for medicinal purposes. In the present study, we examined the anticancer properties of Persea declinata (Bl. Kosterm bark methanolic crude extract (PDM. PDM exhibited a potent antiproliferative effect in MCF-7 human breast cancer cells, with an IC50 value of 16.68 µg/mL after 48 h of treatment. We observed that PDM caused cell cycle arrest and subsequent apoptosis in MCF-7 cells, as exhibited by increased population at G0/G1 phase, higher lactate dehydrogenase (LDH release, and DNA fragmentation. Mechanistic studies showed that PDM caused significant elevation in ROS production, leading to perturbation of mitochondrial membrane potential, cell permeability, and activation of caspases-3/7. On the other hand, real-time PCR and Western blot analysis showed that PDM treatment increased the expression of the proapoptotic molecule, Bax, but decreased the expression of prosurvival proteins, Bcl-2 and Bcl-xL, in a dose-dependent manner. These findings imply that PDM could inhibit proliferation in MCF-7 cells via cell cycle arrest and apoptosis induction, indicating its potential as a therapeutic agent worthy of further development.

Wogonin induced G1 cell cycle arrest by regulating Wnt/β-catenin signaling pathway and inactivating CDK8 in human colorectal cancer carcinoma cells

International Nuclear Information System (INIS)

He, Licheng; Lu, Na; Dai, Qinsheng; Zhao, Yue; Zhao, Li; Wang, Hu; Li, Zhiyu; You, Qidong; Guo, Qinglong

2013-01-01

Highlights: • Wogonin inhibited HCT116 cells growth and arrested at G1 phase of the cell cycle. • Wogonin down-regulated the canonical Wnt/β-catenin signaling pathway. • Wogonin interfered in the combination of β-catenin and TCF/Lef. • Wogonin limited the kinase activity of CDK8. - Abstract: Wogonin, a naturally occurring mono-flavonoid, has been reported to have tumor therapeutic potential and good selectivity both in vitro and in vivo. Herein, we investigated the anti-proliferation effects and associated mechanisms of wogonin in human colorectal cancer in vitro. The flow-cytometric analysis showed that wogonin induced a G1 phase cell cycle arrest in HCT116 cells in a concentration- and time-dependent manner. Meanwhile, the cell cycle-related proteins, such as cyclin A, E, D1, and CDK2, 4 were down-regulated in wogonin-induced G1 cell cycle arrest. Furthermore, we showed that the anti-proliferation and G1 arrest effect of wogonin on HCT116 cells was associated with deregulation of Wnt/β-catenin signaling pathway. Wogonin-treated cells showed decreased intracellular levels of Wnt proteins, and activated degradation complex to phosphorylated and targeted β-catenin for proteasomal degradation. Wogonin inhibited β-catenin-mediated transcription by interfering in the transcriptional activity of TCF/Lef, and repressing the kinase activity of CDK8 which has been considered as an oncogene involving in the development of colorectal cancers. Moreover, CDK8 siRNA-transfected HCT116 cells showed similar results to wogonin treated cells. Thus, our data suggested that wogonin induced anti-proliferation and G1 arrest via Wnt/β-catenin signaling pathway and it can be developed as a therapeutic agent against human colorectal cancer

Prolyl oligopeptidase inhibition-induced growth arrest of human gastric cancer cells

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Suzuki, Kanayo [Laboratory of Cell Biology, Osaka University of Pharmaceutical Sciences, 4-20-1 Nasahara, Takatsuki, Osaka 569-1094 (Japan); Sakaguchi, Minoru, E-mail: sakaguti@gly.oups.ac.jp [Laboratory of Cell Biology, Osaka University of Pharmaceutical Sciences, 4-20-1 Nasahara, Takatsuki, Osaka 569-1094 (Japan); Tanaka, Satoshi [Laboratory of Cell Biology, Osaka University of Pharmaceutical Sciences, 4-20-1 Nasahara, Takatsuki, Osaka 569-1094 (Japan); Yoshimoto, Tadashi [Department of Life Science, Setsunan University, 17-8 Ikeda-Nakamachi, Neyagawa, Osaka 572-8508 (Japan); Takaoka, Masanori [Laboratory of Cell Biology, Osaka University of Pharmaceutical Sciences, 4-20-1 Nasahara, Takatsuki, Osaka 569-1094 (Japan)

2014-01-03

Highlights: •We examined the effects of prolyl oligopeptidase (POP) inhibition on p53 null gastric cancer cell growth. •POP inhibition-induced cell growth suppression was associated with an increase in a quiescent G{sub 0} state. •POP might regulate the exit from and/or reentry into the cell cycle. -- Abstract: Prolyl oligopeptidase (POP) is a serine endopeptidase that hydrolyzes post-proline peptide bonds in peptides that are <30 amino acids in length. We recently reported that POP inhibition suppressed the growth of human neuroblastoma cells. The growth suppression was associated with pronounced G{sub 0}/G{sub 1} cell cycle arrest and increased levels of the CDK inhibitor p27{sup kip1} and the tumor suppressor p53. In this study, we investigated the mechanism of POP inhibition-induced cell growth arrest using a human gastric cancer cell line, KATO III cells, which had a p53 gene deletion. POP specific inhibitors, 3-((4-[2-(E)-styrylphenoxy]butanoyl)-L-4-hydroxyprolyl)-thiazolidine (SUAM-14746) and benzyloxycarbonyl-thioprolyl-thioprolinal, or RNAi-mediated POP knockdown inhibited the growth of KATO III cells irrespective of their p53 status. SUAM-14746-induced growth inhibition was associated with G{sub 0}/G{sub 1} cell cycle phase arrest and increased levels of p27{sup kip1} in the nuclei and the pRb2/p130 protein expression. Moreover, SUAM-14746-mediated cell cycle arrest of KATO III cells was associated with an increase in the quiescent G{sub 0} state, defined by low level staining for the proliferation marker, Ki-67. These results indicate that POP may be a positive regulator of cell cycle progression by regulating the exit from and/or reentry into the cell cycle by KATO III cells.

Xylopine Induces Oxidative Stress and Causes G2/M Phase Arrest, Triggering Caspase-Mediated Apoptosis by p53-Independent Pathway in HCT116 Cells

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Luciano de Souza Santos

2017-01-01

Full Text Available Xylopine is an aporphine alkaloid that has cytotoxic activity to cancer cells. In this study, the underlying mechanism of xylopine cytotoxicity was assessed in human colon carcinoma HCT116 cells. Xylopine displayed potent cytotoxicity in different cancer cell lines in monolayer cultures and in a 3D model of cancer multicellular spheroids formed from HCT116 cells. Typical morphology of apoptosis, cell cycle arrest in the G2/M phase, increased internucleosomal DNA fragmentation, loss of the mitochondrial transmembrane potential, and increased phosphatidylserine externalization and caspase-3 activation were observed in xylopine-treated HCT116 cells. Moreover, pretreatment with a caspase-3 inhibitor (Z-DEVD-FMK, but not with a p53 inhibitor (cyclic pifithrin-α, reduced xylopine-induced apoptosis, indicating induction of caspase-mediated apoptosis by the p53-independent pathway. Treatment with xylopine also caused an increase in the production of reactive oxygen/nitrogen species (ROS/RNS, including hydrogen peroxide and nitric oxide, but not superoxide anion, and reduced glutathione levels were decreased in xylopine-treated HCT116 cells. Application of the antioxidant N-acetylcysteine reduced the ROS levels and xylopine-induced apoptosis, indicating activation of ROS-mediated apoptosis pathway. In conclusion, xylopine has potent cytotoxicity to different cancer cell lines and is able to induce oxidative stress and G2/M phase arrest, triggering caspase-mediated apoptosis by the p53-independent pathway in HCT116 cells.

A Subpopulation of the K562 Cells Are Killed by Curcumin Treatment after G2/M Arrest and Mitotic Catastrophe.

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Macario Martinez-Castillo

Full Text Available Curcumin is extensively investigated as a good chemo-preventive agent in the development of many cancers and particularly in leukemia, including treatment of chronic myelogenous leukemia and it has been proposed as an adjuvant for leukemia therapies. Human chronic myeloid leukemia cells (K562, were treated with 20 μM of curcumin, and we found that a subpopulation of these cells were arrested and accumulate in the G2/M phase of the cell cycle. Characterization of this cell subpopulation showed that the arrested cells presented nuclear morphology changes resembling those described for mitotic catastrophe. Mitotic cells displayed abnormal chromatin organization, collapse of the mitotic spindle and abnormal chromosome segregation. Then, these cells died in an apoptosis dependent manner and showed diminution in the protein levels of BCL-2 and XIAP. Moreover, our results shown that a transient activation of the nuclear factor κB (NFκB occurred early in these cells, but decreased after 6 h of the treatment, explaining in part the diminution of the anti-apoptotic proteins. Additionally, P73 was translocated to the cell nuclei, because the expression of the C/EBPα, a cognate repressor of the P73 gene, was decreased, suggesting that apoptosis is trigger by elevation of P73 protein levels acting in concert with the diminution of the two anti-apoptotic molecules. In summary, curcumin treatment might produce a P73-dependent apoptotic cell death in chronic myelogenous leukemia cells (K562, which was triggered by mitotic catastrophe, due to sustained BAX and survivin expression and impairment of the anti-apoptotic proteins BCL-2 and XIAP.

Proteasome-mediated degradation of cell division cycle 25C and cyclin-dependent kinase 1 in phenethyl isothiocyanate-induced G2-M-phase cell cycle arrest in PC-3 human prostate cancer cells.

Science.gov (United States)

Xiao, Dong; Johnson, Candace S; Trump, Donald L; Singh, Shivendra V

2004-05-01

Phenethyl isothiocyanate (PEITC), a constituent of many cruciferous vegetables, offers significant protection against cancer in animals induced by a variety of carcinogens. The present study demonstrates that PEITC suppresses proliferation of PC-3 cells in a dose-dependent manner by causing G(2)-M-phase cell cycle arrest and apoptosis. Interestingly, phenyl isothiocyanate (PITC), which is a structural analogue of PEITC but lacks the -CH(2) spacers that link the aromatic ring to the -N=C=S group, neither inhibited PC-3 cell viability nor caused cell cycle arrest or apoptosis. These results indicated that even a subtle change in isothiocyanate (ITC) structure could have a significant impact on its biological activity. The PEITC-induced cell cycle arrest was associated with a >80% reduction in the protein levels of cyclin-dependent kinase 1 (Cdk1) and cell division cycle 25C (Cdc25C; 24 h after treatment with 10 micro M PEITC), which led to an accumulation of Tyr(15) phosphorylated (inactive) Cdk1. On the other hand, PITC treatment neither reduced protein levels of Cdk1 or Cdc25C nor affected Cdk1 phosphorylation. The PEITC-induced decline in Cdk1 and Cdc25C protein levels and cell cycle arrest were significantly blocked on pretreatment of PC-3 cells with proteasome inhibitor lactacystin. A 24 h exposure of PC-3 cells to 10 micro M PEITC, but not PITC, resulted in about 56% and 44% decrease in the levels of antiapoptotic proteins Bcl-2 and Bcl-X(L), respectively. However, ectopic expression of Bcl-2 failed to alter sensitivity of PC-3 cells to growth inhibition or apoptosis induction by PEITC. Treatment of cells with PEITC, but not PITC, also resulted in cleavage of procaspase-3, procaspase-9, and procaspase-8. Moreover, the PEITC-induced apoptosis was significantly attenuated in the presence of general caspase inhibitor and specific inhibitors of caspase-8 and caspase-9. In conclusion, our data indicate that PEITC-induced cell cycle arrest in PC-3 cells is likely due

The APC/C Coordinates Retinal Differentiation with G1 Arrest through the Nek2-Dependent Modulation of Wingless Signaling.

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Martins, Torcato; Meghini, Francesco; Florio, Francesca; Kimata, Yuu

2017-01-09

The cell cycle is coordinated with differentiation during animal development. Here we report a cell-cycle-independent developmental role for a master cell-cycle regulator, the anaphase-promoting complex or cyclosome (APC/C), in the regulation of cell fate through modulation of Wingless (Wg) signaling. The APC/C controls both cell-cycle progression and postmitotic processes through ubiquitin-dependent proteolysis. Through an RNAi screen in the developing Drosophila eye, we found that partial APC/C inactivation severely inhibits retinal differentiation independently of cell-cycle defects. The differentiation inhibition coincides with hyperactivation of Wg signaling caused by the accumulation of a Wg modulator, Drosophila Nek2 (dNek2). The APC/C degrades dNek2 upon synchronous G1 arrest prior to differentiation, which allows retinal differentiation through local suppression of Wg signaling. We also provide evidence that decapentaplegic signaling may posttranslationally regulate this APC/C function. Thus, the APC/C coordinates cell-fate determination with the cell cycle through the modulation of developmental signaling pathways. Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.

Proteomic analysis of the response to cell cycle arrests in human myeloid leukemia cells.

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Ly, Tony; Endo, Aki; Lamond, Angus I

2015-01-02

Previously, we analyzed protein abundance changes across a 'minimally perturbed' cell cycle by using centrifugal elutriation to differentially enrich distinct cell cycle phases in human NB4 cells (Ly et al., 2014). In this study, we compare data from elutriated cells with NB4 cells arrested at comparable phases using serum starvation, hydroxyurea, or RO-3306. While elutriated and arrested cells have similar patterns of DNA content and cyclin expression, a large fraction of the proteome changes detected in arrested cells are found to reflect arrest-specific responses (i.e., starvation, DNA damage, CDK1 inhibition), rather than physiological cell cycle regulation. For example, we show most cells arrested in G2 by CDK1 inhibition express abnormally high levels of replication and origin licensing factors and are likely poised for genome re-replication. The protein data are available in the Encyclopedia of Proteome Dynamics (

Growth arrest specific protein (GAS) 6

DEFF Research Database (Denmark)

Haase, T N; Rasmussen, Morten; Jaksch, C A M

2013-01-01

using RNA microarray and quantitative PCR. The role of a differentially expressed gene, growth arrest specific protein 6 (GAS6), was evaluated in vitro using neonatal rat islets. Results The mRNA level of Gas6, known to be mitogenic in other tissues, was reduced in LP offspring. The mRNA content of Mafa...... was increased in LP offspring suggesting an early maturation of beta cells. When applied in vitro, GAS6 increased proliferation of neonatal pancreatic beta cells, while reducing glucose-stimulated insulin secretion without changing the total insulin content of the islets. In addition, GAS6 decreased the m......RNA content of Mafa. Conclusions/interpretation We propose a role for GAS6 in the regulation of pancreatic beta cells in the critical period around the time of birth. Our results support the hypothesis that the reduced beta cell mass seen in LP offspring is caused by a change in the intra-uterine environment...

The chalcone flavokawain B induces G2/M cell-cycle arrest and apoptosis in human oral carcinoma HSC-3 cells through the intracellular ROS generation and downregulation of the Akt/p38 MAPK signaling pathway.

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Hseu, You-Cheng; Lee, Meng-Shiou; Wu, Chi-Rei; Cho, Hsin-Ju; Lin, Kai-Yuan; Lai, Guan-Hua; Wang, Sheng-Yang; Kuo, Yueh-Hsiung; Kumar, K J Senthil; Yang, Hsin-Ling

2012-03-07

Chalcones have been described to represent cancer chemopreventive food components that are rich in fruits and vegetables. In this study, we examined the anti-oral cancer effect of flavokawain B (FKB), a naturally occurring chalcone isolated from Alpinia pricei (shell gingers), and revealed its molecular mechanism of action. Treatment of human oral carcinoma (HSC-3) cells with FKB (1.25-10 μg/mL; 4.4-35.2 μM) inhibited cell viability and caused G(2)/M arrest through reductions in cyclin A/B1, Cdc2, and Cdc25C levels. Moreover, FKB treatment resulted in the induction of apoptosis, which was associated with DNA fragmentation, mitochondria dysfunction, cytochrome c and AIF release, caspase-3 and caspase-9 activation, and Bcl-2/Bax dysregulation. Furthermore, increased Fas activity and procaspase-8, procaspase-4, and procaspase-12 cleavages were accompanied by death receptor and ER-stress, indicating the involvement of mitochondria, death-receptor, and ER-stress signaling pathways. FKB induces apoptosis through ROS generation as evidenced by the upregulation of oxidative-stress markers HO-1/Nrf2. This mechanism was further confirmed by the finding that the antioxidant N-acetylcysteine (NAC) significantly blocked ROS generation and consequently inhibited FKB-induced apoptosis. Moreover, FKB downregulated the phosphorylation of Akt and p38 MAPK, while their inhibitors LY294002 and SB203580, respectively, induced G(2)/M arrest and apoptosis. The profound reduction in cell number was observed in combination treatment with FKB and Akt/p38 MAPK inhibitors, indicating that the disruption of Akt and p38 MAPK cascades plays a functional role in FKB-induced G(2)/M arrest and apoptosis in HSC-3 cells.

Growth arrest specific gene 2 in tilapia (Oreochromis niloticus): molecular characterization and functional analysis under low-temperature stress.

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Yang, ChangGeng; Wu, Fan; Lu, Xing; Jiang, Ming; Liu, Wei; Yu, Lijuan; Tian, Juan; Wen, Hua

2017-07-17

Growth arrest specific 2 (gas2) gene is a component of the microfilament system that plays a major role in the cell cycle, regulation of microfilaments, and cell morphology during apoptotic processes. However, little information is available on fish gas2. In this study, the tilapia (Oreochromis niloticus) gas2 gene was cloned and characterized for the first time. The open reading frame was 1020 bp, encoding 340 amino acids; the 5'-untranslated region (UTR) was 140 bp and the 3'-UTR was 70 bp, with a poly (A) tail. The highest promoter activity occurred in the regulatory region (-3000 to -2400 bp). The Gas2-GFP fusion protein was distributed within the cytoplasm. Quantitative reverse transcription-polymerase chain reaction and western blot analyses revealed that gas2 gene expression levels in the liver, muscle, and brain were clearly affected by low temperature stress. The results of gas2 RNAi showed decreased expression of the gas2 and P53 genes. These results suggest that the tilapia gas2 gene may be involved in low temperature stress-induced apoptosis.

Cdk2 is required for p53-independent G2/M checkpoint control.

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Jon H Chung

2010-02-01

Full Text Available The activation of phase-specific cyclin-dependent kinases (Cdks is associated with ordered cell cycle transitions. Among the mammalian Cdks, only Cdk1 is essential for somatic cell proliferation. Cdk1 can apparently substitute for Cdk2, Cdk4, and Cdk6, which are individually dispensable in mice. It is unclear if all functions of non-essential Cdks are fully redundant with Cdk1. Using a genetic approach, we show that Cdk2, the S-phase Cdk, uniquely controls the G(2/M checkpoint that prevents cells with damaged DNA from initiating mitosis. CDK2-nullizygous human cells exposed to ionizing radiation failed to exclude Cdk1 from the nucleus and exhibited a marked defect in G(2/M arrest that was unmasked by the disruption of P53. The DNA replication licensing protein Cdc6, which is normally stabilized by Cdk2, was physically associated with the checkpoint regulator ATR and was required for efficient ATR-Chk1-Cdc25A signaling. These findings demonstrate that Cdk2 maintains a balance of S-phase regulatory proteins and thereby coordinates subsequent p53-independent G(2/M checkpoint activation.

G2 checkpoint abrogator abates the antagonistic interaction between antimicrotubule drugs and radiation therapy

International Nuclear Information System (INIS)

Sui Meihua; Zhang Hongfang; Di Xiaoyun; Chang Jinjia; Shen Youqing; Fan Weimin

2012-01-01

Background and purpose: We previously demonstrated that radiation may arrest tumor cells at G2 phase, which in turn prevents the cytotoxicity of antimicrotubule drugs and results in antagonistic interaction between these two modalities. Herein we tested whether G2 abrogators would attenuate the above antagonistic interaction and improve the therapeutic efficacy of combination therapy between radiation and antimicrotubule drugs. Materials and methods: Breast cancer BCap37 and epidermoid carcinoma KB cell lines were administered with radiation, UCN-01 (a model drug of G2 abrogator), paclitaxel or vincristine, alone or in combinations. The antitumor activities of single and combined treatments were analyzed by a series of cytotoxic, apoptotic, cell cycle, morphological and biochemical assays. Results: UCN-01 significantly enhanced the cytotoxicity of radiation, antimitotic drugs, and their combined treatments in vitro. Further investigations demonstrated that UCN-01 attenuated radiation-induced G2 arrest, and subsequently repressed the inhibitory effect of radiation on drug-induced mitotic arrest and apoptosis. Conclusions: This is the first report demonstrating that G2 checkpoint abrogation represses the inhibitory effect of radiation on antimicrotubule drugs, which may be implicated in cancer combination therapy. Considering that G2 abrogators are under extensive evaluation for cancer treatment, our findings provide valuable information for this class of promising compounds.

Arctigenin, a natural lignan compound, induces G0/G1 cell cycle arrest and apoptosis in human glioma cells.

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Maimaitili, Aisha; Shu, Zunhua; Cheng, Xiaojiang; Kaheerman, Kadeer; Sikandeer, Alifu; Li, Weimin

2017-02-01

The aim of the current study was to investigate the anticancer potential of arctigenin, a natural lignan compound, in malignant gliomas. The U87MG and T98G human glioma cell lines were treated with various concentrations of arctigenin for 48 h and the effects of arctigenin on the aggressive phenotypes of glioma cells were assessed. The results demonstrated that arctigenin dose-dependently inhibited the growth of U87MG and T98G cells, as determined using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide and bromodeoxyuridine incorporation assays. Arctigenin exposure also induced a 60-75% reduction in colony formation compared with vehicle-treated control cells. However, arctigenin was not observed to affect the invasiveness of glioma cells. Arctigenin significantly increased the proportion of cells in the G 0 /G 1 phase and reduced the number of cells in the S phase, as compared with the control group (Parctigenin increased the expression levels of p21, retinoblastoma and p53 proteins, and significantly decreased the expression levels of cyclin D1 and cyclin-dependent kinase 4 proteins. Additionally, arctigenin was able to induce apoptosis in glioma cells, coupled with increased expression levels of cleaved caspase-3 and the pro-apoptotic BCL2-associated X protein. Furthermore, arctigenin-induced apoptosis was significantly suppressed by the pretreatment of cells with Z-DEVD-FMK, a caspase-3 inhibitor. In conclusion, the results suggest that arctigenin is able to inhibit cell proliferation and may induce apoptosis and cell cycle arrest at the G 0 /G 1 phase in glioma cells. These results warrant further investigation of the anticancer effects of arctigenin in animal models of gliomas.

Pyrogallol, ROS generator inhibits As4.1 juxtaglomerular cells via cell cycle arrest of G2 phase and apoptosis

International Nuclear Information System (INIS)

Park, Woo Hyun; Han, Yong Hwan; Kim, Suhn Hee; Kim, Sung Zoo

2007-01-01

Pyrogallol as a catechin compound has been employed as an O 2 · - generator and often used to investigate the role of ROS in the biological system. Here, we investigated the in vitro effect of pyrogallol on cell growth, cell cycle and apoptosis in As4.1 juxtaglomerular cells. Dose-dependent inhibition of cell growth was observed with IC 50 of about 60 μM for 48 h using MTT assay. Pyrogallol (100 μM) did not alter intracellular H 2 O 2 level and catalase activity, but increased the intracellular O 2 · - level and decreased SOD activity in As4.1 cells. DNA flow cytometric analysis indicated that 50 and 100 μM pyrogallol significantly increased G2 phase cells as compared with those of pyrogallol-untreated cells. Also, pyrogallol induced apoptosis as evidenced by flow cytometric detection of sub-G1 DNA content, annexin V binding assay and DAPI staining. This apoptosis process was accompanied with the loss of mitochondrial transmembrane potential (ΔΨ m ), Bcl-2 decrease, caspase-3 activation and PARP cleavage. Pan caspase inhibitor (Z-VAD) could significantly rescue As4.1 cells from pyrogallol-induced cell death. But, the inhibitors of caspase-3, caspase-8, and caspase-9 did not prevent apoptotic events in pyrogallol-treated As4.1 cells. Taken together, we have demonstrated that an ROS inducer, pyrogallol inhibits the growth of As4.1 JG cells via cell cycle arrest and apoptosis, and suggest that the compound exhibits an anti-proliferative efficacy on these cells

4beta-Hydroxywithanolide E from Physalis peruviana (golden berry) inhibits growth of human lung cancer cells through DNA damage, apoptosis and G2/M arrest.

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Yen, Ching-Yu; Chiu, Chien-Chih; Chang, Fang-Rong; Chen, Jeff Yi-Fu; Hwang, Chi-Ching; Hseu, You-Cheng; Yang, Hsin-Ling; Lee, Alan Yueh-Luen; Tsai, Ming-Tz; Guo, Zong-Lun; Cheng, Yu-Shan; Liu, Yin-Chang; Lan, Yu-Hsuan; Chang, Yu-Ching; Ko, Ying-Chin; Chang, Hsueh-Wei; Wu, Yang-Chang

2010-02-18

The crude extract of the fruit bearing plant, Physalis peruviana (golden berry), demonstrated anti-hepatoma and anti-inflammatory activities. However, the cellular mechanism involved in this process is still unknown. Herein, we isolated the main pure compound, 4beta-Hydroxywithanolide (4betaHWE) derived from golden berries, and investigated its antiproliferative effect on a human lung cancer cell line (H1299) using survival, cell cycle, and apoptosis analyses. An alkaline comet-nuclear extract (NE) assay was used to evaluate the DNA damage due to the drug. It was shown that DNA damage was significantly induced by 1, 5, and 10 microg/mL 4betaHWE for 2 h in a dose-dependent manner (p < 0.005). A trypan blue exclusion assay showed that the proliferation of cells was inhibited by 4betaHWE in both dose- and time-dependent manners (p < 0.05 and 0.001 for 24 and 48 h, respectively). The half maximal inhibitory concentrations (IC50) of 4betaHWE in H1299 cells for 24 and 48 h were 0.6 and 0.71 microg/mL, respectively, suggesting it could be a potential therapeutic agent against lung cancer. In a flow cytometric analysis, 4betaHWE produced cell cycle perturbation in the form of sub-G1 accumulation and slight arrest at the G2/M phase with 1 microg/mL for 12 and 24 h, respectively. Using flow cytometric and annexin V/propidium iodide immunofluorescence double-staining techniques, these phenomena were proven to be apoptosis and complete G2/M arrest for H1299 cells treated with 5 microg/mL for 24 h. In this study, we demonstrated that golden berry-derived 4betaHWE is a potential DNA-damaging and chemotherapeutic agent against lung cancer.

Sensitization of Tumor to 212Pb Radioimmunotherapy by Gemcitabine Involves Initial Abrogation of G2 Arrest and Blocked DNA Damage Repair by Interference With Rad51

International Nuclear Information System (INIS)

Yong, Kwon Joong; Milenic, Diane E.; Baidoo, Kwamena E.; Brechbiel, Martin W.

2013-01-01

Purpose: To elucidate the mechanism of the therapeutic efficacy of targeted α-particle radiation therapy using 212 Pb-TCMC-trastuzumab together with gemcitabine for treatment of disseminated peritoneal cancers. Methods and Materials: Mice bearing human colon cancer LS-174T intraperitoneal xenografts were pretreated with gemcitabine, followed by 212 Pb-TCMC-trastuzumab and compared with controls. Results: Treatment with 212 Pb-TCMC-trastuzumab increased the apoptotic rate in the S-phase-arrested tumors induced by gemcitabine at earlier time points (6 to 24 hours). 212 Pb-TCMC-trastuzumab after gemcitabine pretreatment abrogated G2/M arrest at the same time points, which may be associated with the inhibition of Chk1 phosphorylation and, in turn, cell cycle perturbation, resulting in apoptosis. 212 Pb-TCMC-trastuzumab treatment after gemcitabine pretreatment caused depression of DNA synthesis, DNA double-strand breaks, accumulation of unrepaired DNA, and down-regulation of Rad51 protein, indicating that DNA damage repair was blocked. In addition, modification in the chromatin structure of p21 may be associated with transcriptionally repressed chromatin states, indicating that the open structure was delayed at earlier time points. Conclusion: These findings suggest that the cell-killing efficacy of 212 Pb-TCMC-trastuzumab after gemcitabine pretreatment may be associated with abrogation of the G2/M checkpoint, inhibition of DNA damage repair, and chromatin remodeling

Oleanolic acid induces mitochondrial-dependent apoptosis and G0/G1 phase arrest in gallbladder cancer cells

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Li HF

2015-06-01

Full Text Available Huai-Feng Li,1–3,* Xu-An Wang,1–3,* Shan-Shan Xiang,1–3,* Yun-Ping Hu,1–3 Lin Jiang,1–3 Yi-Jun Shu,1–3 Mao-Lan Li,1–3 Xiang-Song Wu,1–3 Fei Zhang,1–3 Yuan-Yuan Ye,1–3 Hao Weng,1–3 Run-Fa Bao,1–3 Yang Cao,1–3 Wei Lu,1–3 Qian Dong,1–3 Ying-Bin Liu1–3 1Department of General Surgery, 2Laboratory of General Surgery, 3Institute of Biliary Tract Disease, Xinhua Hospital, Affiliated to Shanghai Jiao Tong University, School of Medicine, Shanghai, People’s Republic of China *These authors contributed equally to this work Abstract: Oleanolic acid (OA, a naturally occurring triterpenoid, exhibits potential antitumor activity in many tumor cell lines. Gallbladder carcinoma is the most common malignancy of the biliary tract, and is a highly aggressive tumor with an extremely poor prognosis. Unfortunately, the effects of OA on gallbladder carcinoma are unknown. In this study, we investigated the effects of OA on gallbladder cancer cells and the underlying mechanism. The results showed that OA inhibits proliferation of gallbladder cancer cells in a dose-dependent and time-dependent manner on MTT and colony formation assay. A flow cytometry assay revealed apoptosis and G0/G1 phase arrest in GBC-SD and NOZ cells. Western blot analysis and a mitochondrial membrane potential assay demonstrated that OA functions through the mitochondrial apoptosis pathway. Moreover, this drug inhibited tumor growth in nude mice carrying subcutaneous NOZ tumor xenografts. These data suggest that OA inhibits proliferation of gallbladder cancer cells by regulating apoptosis and the cell cycle process. Thus, OA may be a promising drug for adjuvant chemotherapy in gallbladder carcinoma. Keywords: oleanolic acid, gallbladder carcinoma, apoptosis, cell cycle arrest, mitochondrial pathway

Hexamethoxylated Monocarbonyl Analogues of Curcumin Cause G2/M Cell Cycle Arrest in NCI-H460 Cells via Michael Acceptor-Dependent Redox Intervention.

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Li, Yan; Zhang, Li-Ping; Dai, Fang; Yan, Wen-Jing; Wang, Hai-Bo; Tu, Zhi-Shan; Zhou, Bo

2015-09-09

Curcumin, derived from the dietary spice turmeric, holds promise for cancer prevention. This prompts much interest in investigating the action mechanisms of curcumin and its analogues. Two symmetrical hexamethoxy-diarylpentadienones (1 and 2) as cucumin analogues were reported to possess significantly enhanced cytotoxicity compared with the parent molecule. However, the detailed mechanisms remain unclear. In this study, compounds 1 and 2 were identified as the G2/M cell cycle arrest agents to mediate the cytotoxicity toward NCI-H460 cells via Michael acceptor-dependent redox intervention. Compared with curcumin, they could more easily induce a burst of reactive oxygen species (ROS) and collapse of the redox buffering system. One possible reason is that they could more effectively target intracellular TrxR to convert this antioxidant enzyme into a ROS promoter. Additionally, they caused up-regulation of p53 and p21 and down-regulation of redox-sensitive Cdc25C along with cyclin B1/Cdk1 in a Michael acceptor- and ROS-dependent fashion. Interestingly, in comparison with compound 2, compound 1 displayed a relatively weak ability to generate ROS but increased cell cycle arrest activity and cytotoxicity probably due to its Michael acceptor-dependent microtubule-destabilizing effect and greater GST-inhibitory activity, as well as its enhanced cellular uptake. This work provides useful information for understanding Michael acceptor-dependent and redox-mediated cytotoxic mechanisms of curcumin and its active analogues.

G2 Checkpoint Responses in Arabidopsis

Energy Technology Data Exchange (ETDEWEB)

Britt, Anne [Univ. of California, Davis, CA (United States)

2013-03-18

This project focused on the mechanism and biological significance of the G2 arrest response to replication stress in plants. We employed both forward and reverse genetic approaches to identify genes required for this response. A total of 3 different postdocs, 5 undergraduates, and 2 graduate students participated in the project. We identified several genes required for damage response in plants, including homologs of genes previously identified in animals (ATM and ATR), novel, a plant-specific genes (SOG1) and a gene known in animals but previously thought to be missing from the Arabidopsis genome (ATRIP). We characterized the transcriptome of gamma-irradiated plants, and found that plants, unlike animals, express a robust transcriptional response to damage, involving genes that regulate the cell cycle and DNA metabolism. This response requires both ATM and the transcription factor SOG1. We found that both ATM and ATR play a role in meiosis in plants. We also found that plants have a cell-type-specific programmed cell death response to ionizing radiation and UV light, and that this response requires ATR, ATM, and SOG1. These results were published in a series of 5 papers.

Microvesicles derived from human Wharton's Jelly mesenchymal stem cells ameliorate ischemia-reperfusion-induced renal fibrosis by releasing from G2/M cell cycle arrest.

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Chen, Wenxia; Yan, Yongbin; Song, Chundong; Ding, Ying; Du, Tao

2017-12-14

Studies have demonstrated that microvesicles (MVs) derived from human Wharton's Jelly mesenchymal stromal cells (hWJMSCs) could ameliorate renal ischemia/reperfusion injury (IRI); however, the underlying mechanisms were not clear yet. Here, MVs were isolated and injected intravenously into rats immediately after ischemia of the left kidney, and Erk1/2 activator hepatocyte growth factor (HGF) or inhibitor U0126 was administrated. Tubular cell proliferation and apoptosis were identified by Ki67 or terminal-deoxynucleotidyl transferase-mediated nick end labeling immunostaining. Masson's tri-chrome straining and alpha-smooth muscle actin staining were used for assessing renal fibrosis. The mRNA or protein expression in the kidney was measured by quantitative reverse transcription-PCR or Western blot, respectively. The total collagen concentration was also determined. In vitro , NRK-52E cells that treated with MVs under hypoxia injury and with HGF or U0126 administration were used, and cell cycle analysis was performed. The effects of hWJMSC-MVs on enhancing the proliferation and mitigating the apoptosis of renal cells, abrogating IRI-induced fibrosis, improving renal function, decreasing collagen deposition, and altering the expression levels of epithelial-mesenchymal transition and cell cycle-related proteins in IRI rats were found. In vitro experiment showed that hWJMSC-MVs could induce G2/M cell cycle arrest and decrease the expression of collagen deposition-related proteins in NRK-52E cells after 24 or 48 h. However, U0126 treatment reversed these effects. In conclusion, MVs derived from hWJMSCs ameliorate IR-induced renal fibrosis by inducing G2/M cell cycle arrest via Erk1/2 signaling. © 2017 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.

The novel anthraquinone derivative IMP1338 induces death of human cancer cells by p53-independent S and G2/M cell cycle arrest.

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Choi, Hyun Kyung; Ryu, Hwani; Son, A-Rang; Seo, Bitna; Hwang, Sang-Gu; Song, Jie-Young; Ahn, Jiyeon

2016-04-01

To identify novel small molecules that induce selective cancer cell death, we screened a chemical library containing 1040 compounds in HT29 colon cancer and CCD18-Co normal colon cells, using a phenotypic cell-based viability assay system with the Cell Counting Kit-8 (CCK-8). We discovered a novel anthraquinone derivative, N-(4-[{(9,10-dioxo-9,10-dihydro-1-anthracenyl)sulfonyl}amino]phenyl)-N-methylacetamide (IMP1338), which was cytotoxic against the human colon cancer cells tested. The MTT cell viability assay showed that treatment with IMP1338 selectively inhibited HCT116, HCT116 p53(-/-), HT29, and A549 cancer cell proliferation compared to that of Beas2B normal epithelial cells. To elucidate the cellular mechanism underlying the cytotoxicity of IMP1338, we examined the effect of IMP1338 on the cell cycle distribution and death of cancer cells. IMP1338 treatment significantly arrested the cell cycle at S and G2/M phases by DNA damage and led to apoptotic cell death, which was determined using FACS analysis with Annexin V/PI double staining. Furthermore, IMP1338 increased caspase-3 cleavage in wild-type p53, p53 knockout HCT116, and HT29 cells as determined using immunoblotting. In addition, IMP1338 markedly induced the phosphorylation of histone H2AX and Chk1 in both cell lines while the combination of 5-fluorouracil (5-FU) and radiation inhibited the viability of HCT116, HCT116 p53(-/-), and HT29 cells compared to 5-FU or radiation alone. Our findings indicated that IMP1338 induced p53-independent cell death through S and G2/M phase arrest as well as DNA damage. These results provide a basis for future investigations assessing the promising anticancer properties of IMP1338. Copyright © 2016. Published by Elsevier Masson SAS.

Oridonin nanosuspension was more effective than free oridonin on G2/M cell cycle arrest and apoptosis in the human pancreatic cancer PANC-1 cell line

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Qi XL

2012-04-01

Full Text Available Xiaoli Qi1, Dianrui Zhang2, Xia Xu1, Feifei Feng2, Guijie Ren1, Qianqian Chu1, Qiang Zhang3, Keli Tian11Department of Biochemistry and Molecular Biology, Shandong University School of Medicine, Jinan, 2Department of Pharmaceutics, College of Pharmacy, Shandong University, Jinan, 3State Key Laboratory of Natural and Biomimetic Drugs, School of Pharmaceutical Sciences, Peking University, Beijing, People's Republic of ChinaAbstract: Oridonin, a diterpenoid isolated from Rabdosia rubescencs, has been reported to have antitumor effects. However, low solubility has limited its clinical applications. Preparation of drugs in the form of nanosuspensions is an extensively utilized protocol. In this study, we investigated the anticancer activity of oridonin and oridonin nanosuspension on human pancreatic carcinoma PANC-1 cells. 3-(4,5-dimethylthiazol-2-yl-2,5-diphenyltetrazolium bromide assay was performed to investigate the effect of oridonin on cell growth. Propidium iodide and Hoechst 33342 staining were used to detect morphologic changes. The percentage of apoptosis and cell cycle progression was determined by flow cytometric method staining with propidium iodide. Annexin V-fluorescein isothiocyanate (FITC/PI staining was used to evaluate cell apoptosis by flow cytometry. Caspase-3 activity was measured by spectrophotometry. The apoptotic and cell cycle protein expression were determined by Western blot analysis. Both oridonin and oridonin nanosuspension induced apoptosis and G2/M phase cell cycle arrest, and the latter had a more significant cytotoxic effect. The ratio of Bcl-2/Bax protein expression was decreased and caspase-3 activity was stimulated. The expression of cyclin B1 and p-cdc2 (T161 was suppressed. Our results showed that oridonin nanosuspension was more effective than free oridonin on G2/M cell cycle arrest and apoptosis in the human pancreatic cancer PANC-1 cell line.Keywords: cyclin B1, cdc2, caspase-3, Bcl-2, Bax

Enhanced efficacy of combined 213Bi-DTPA-F3 and paclitaxel therapy of peritoneal carcinomatosis is mediated by enhanced induction of apoptosis and G2/M phase arrest

International Nuclear Information System (INIS)

Vallon, Mario; Seidl, Christof; Blechert, Birgit; Li, Zhoulei; Gaertner, Florian C.; Senekowitsch-Schmidtke, Reingard; Essler, Markus; Gilbertz, Klaus-Peter; Baumgart, Anja; Aichler, Michaela; Feuchtinger, Annette; Walch, Axel K.; Bruchertseifer, Frank; Morgenstern, Alfred

2012-01-01

Targeted therapy with α-particle emitting radionuclides is a promising new option in cancer therapy. Stable conjugates of the vascular tumour-homing peptide F3 with the α-emitter 213 Bi specifically target tumour cells. The aim of our study was to determine efficacy of combined 213 Bi-diethylenetriaminepentaacetic acid (DTPA)-F3 and paclitaxel treatment compared to treatment with either 213 Bi-DTPA-F3 or paclitaxel both in vitro and in vivo. Cytotoxicity of treatment with 213 Bi-DTPA-F3 and paclitaxel, alone or in combination, was assayed towards OVCAR-3 cells using the alamarBlue assay, the clonogenic assay and flow cytometric analyses of the mode of cell death and cell cycle arrest. Therapeutic efficacy of the different treatment options was assayed after repeated treatment of mice bearing intraperitoneal OVCAR-3 xenograft tumours. Therapy monitoring was performed by bioluminescence imaging and histopathologic analysis. Treatment of OVCAR-3 cells in vitro with combined 213 Bi-DTPA-F3 and paclitaxel resulted in enhanced cytotoxicity, induction of apoptosis and G2/M phase arrest compared to treatment with either 213 Bi-DTPA-F3 or paclitaxel. Accordingly, i.p. xenograft OVCAR-3 tumours showed the best response following repeated (six times) combined therapy with 213 Bi-DTPA-F3 (1.85 MBq) and paclitaxel (120 μg) as demonstrated by bioluminescence imaging and histopathologic investigation of tumour spread on the mesentery of the small and large intestine. Moreover, mean survival of xenograft mice that received combined therapy with 213 Bi-DTPA-F3 and paclitaxel was significantly superior to mice treated with either 213 Bi-DTPA-F3 or paclitaxel alone. Combined treatment with 213 Bi-DTPA-F3 and paclitaxel significantly increased mean survival of mice with peritoneal carcinomatosis of ovarian origin, thus favouring future therapeutic application. (orig.)

Distinct transcriptional networks in quiescent myoblasts: a role for Wnt signaling in reversible vs. irreversible arrest.

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Sindhu Subramaniam

Full Text Available Most cells in adult mammals are non-dividing: differentiated cells exit the cell cycle permanently, but stem cells exist in a state of reversible arrest called quiescence. In damaged skeletal muscle, quiescent satellite stem cells re-enter the cell cycle, proliferate and subsequently execute divergent programs to regenerate both post-mitotic myofibers and quiescent stem cells. The molecular basis for these alternative programs of arrest is poorly understood. In this study, we used an established myogenic culture model (C2C12 myoblasts to generate cells in alternative states of arrest and investigate their global transcriptional profiles. Using cDNA microarrays, we compared G0 myoblasts with post-mitotic myotubes. Our findings define the transcriptional program of quiescent myoblasts in culture and establish that distinct gene expression profiles, especially of tumour suppressor genes and inhibitors of differentiation characterize reversible arrest, distinguishing this state from irreversibly arrested myotubes. We also reveal the existence of a tissue-specific quiescence program by comparing G0 C2C12 myoblasts to isogenic G0 fibroblasts (10T1/2. Intriguingly, in myoblasts but not fibroblasts, quiescence is associated with a signature of Wnt pathway genes. We provide evidence that different levels of signaling via the canonical Wnt pathway characterize distinct cellular states (proliferation vs. quiescence vs. differentiation. Moderate induction of Wnt signaling in quiescence is associated with critical properties such as clonogenic self-renewal. Exogenous Wnt treatment subverts the quiescence program and negatively affects clonogenicity. Finally, we identify two new quiescence-induced regulators of canonical Wnt signaling, Rgs2 and Dkk3, whose induction in G0 is required for clonogenic self-renewal. These results support the concept that active signal-mediated regulation of quiescence contributes to stem cell properties, and have implications for

Distinct transcriptional networks in quiescent myoblasts: a role for Wnt signaling in reversible vs. irreversible arrest.

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Subramaniam, Sindhu; Sreenivas, Prethish; Cheedipudi, Sirisha; Reddy, Vatrapu Rami; Shashidhara, Lingadahalli Subrahmanya; Chilukoti, Ravi Kumar; Mylavarapu, Madhavi; Dhawan, Jyotsna

2014-01-01

Most cells in adult mammals are non-dividing: differentiated cells exit the cell cycle permanently, but stem cells exist in a state of reversible arrest called quiescence. In damaged skeletal muscle, quiescent satellite stem cells re-enter the cell cycle, proliferate and subsequently execute divergent programs to regenerate both post-mitotic myofibers and quiescent stem cells. The molecular basis for these alternative programs of arrest is poorly understood. In this study, we used an established myogenic culture model (C2C12 myoblasts) to generate cells in alternative states of arrest and investigate their global transcriptional profiles. Using cDNA microarrays, we compared G0 myoblasts with post-mitotic myotubes. Our findings define the transcriptional program of quiescent myoblasts in culture and establish that distinct gene expression profiles, especially of tumour suppressor genes and inhibitors of differentiation characterize reversible arrest, distinguishing this state from irreversibly arrested myotubes. We also reveal the existence of a tissue-specific quiescence program by comparing G0 C2C12 myoblasts to isogenic G0 fibroblasts (10T1/2). Intriguingly, in myoblasts but not fibroblasts, quiescence is associated with a signature of Wnt pathway genes. We provide evidence that different levels of signaling via the canonical Wnt pathway characterize distinct cellular states (proliferation vs. quiescence vs. differentiation). Moderate induction of Wnt signaling in quiescence is associated with critical properties such as clonogenic self-renewal. Exogenous Wnt treatment subverts the quiescence program and negatively affects clonogenicity. Finally, we identify two new quiescence-induced regulators of canonical Wnt signaling, Rgs2 and Dkk3, whose induction in G0 is required for clonogenic self-renewal. These results support the concept that active signal-mediated regulation of quiescence contributes to stem cell properties, and have implications for pathological

Sensitization of Tumor to {sup 212}Pb Radioimmunotherapy by Gemcitabine Involves Initial Abrogation of G2 Arrest and Blocked DNA Damage Repair by Interference With Rad51

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Yong, Kwon Joong; Milenic, Diane E.; Baidoo, Kwamena E. [Radioimmune and Inorganic Chemistry Section, Radiation Oncology Branch, National Cancer Institute, National Institutes of Health, Bethesda, Maryland (United States); Brechbiel, Martin W., E-mail: martinwb@mail.nih.gov [Radioimmune and Inorganic Chemistry Section, Radiation Oncology Branch, National Cancer Institute, National Institutes of Health, Bethesda, Maryland (United States)

2013-03-15

Purpose: To elucidate the mechanism of the therapeutic efficacy of targeted α-particle radiation therapy using {sup 212}Pb-TCMC-trastuzumab together with gemcitabine for treatment of disseminated peritoneal cancers. Methods and Materials: Mice bearing human colon cancer LS-174T intraperitoneal xenografts were pretreated with gemcitabine, followed by {sup 212}Pb-TCMC-trastuzumab and compared with controls. Results: Treatment with {sup 212}Pb-TCMC-trastuzumab increased the apoptotic rate in the S-phase-arrested tumors induced by gemcitabine at earlier time points (6 to 24 hours). {sup 212}Pb-TCMC-trastuzumab after gemcitabine pretreatment abrogated G2/M arrest at the same time points, which may be associated with the inhibition of Chk1 phosphorylation and, in turn, cell cycle perturbation, resulting in apoptosis. {sup 212}Pb-TCMC-trastuzumab treatment after gemcitabine pretreatment caused depression of DNA synthesis, DNA double-strand breaks, accumulation of unrepaired DNA, and down-regulation of Rad51 protein, indicating that DNA damage repair was blocked. In addition, modification in the chromatin structure of p21 may be associated with transcriptionally repressed chromatin states, indicating that the open structure was delayed at earlier time points. Conclusion: These findings suggest that the cell-killing efficacy of {sup 212}Pb-TCMC-trastuzumab after gemcitabine pretreatment may be associated with abrogation of the G2/M checkpoint, inhibition of DNA damage repair, and chromatin remodeling.

Enhanced efficacy of combined {sup 213}Bi-DTPA-F3 and paclitaxel therapy of peritoneal carcinomatosis is mediated by enhanced induction of apoptosis and G2/M phase arrest

Energy Technology Data Exchange (ETDEWEB)

Vallon, Mario; Seidl, Christof; Blechert, Birgit; Li, Zhoulei; Gaertner, Florian C.; Senekowitsch-Schmidtke, Reingard; Essler, Markus [Technische Universitaet Muenchen, Department of Nuclear Medicine, Munich (Germany); Gilbertz, Klaus-Peter [German Armed Forces, Institute of Radiobiology, Munich (Germany); Baumgart, Anja [Technische Universitaet Muenchen, III. Medical Department, Munich (Germany); Aichler, Michaela; Feuchtinger, Annette; Walch, Axel K. [Helmholtz Zentrum Muenchen, Institute of Pathology, Neuherberg (Germany); Bruchertseifer, Frank; Morgenstern, Alfred [Institute for Transuranium Elements, European Commission, Joint Research Centre, Karlsruhe (Germany)

2012-12-15

Targeted therapy with {alpha}-particle emitting radionuclides is a promising new option in cancer therapy. Stable conjugates of the vascular tumour-homing peptide F3 with the {alpha}-emitter {sup 213}Bi specifically target tumour cells. The aim of our study was to determine efficacy of combined {sup 213}Bi-diethylenetriaminepentaacetic acid (DTPA)-F3 and paclitaxel treatment compared to treatment with either {sup 213}Bi-DTPA-F3 or paclitaxel both in vitro and in vivo. Cytotoxicity of treatment with {sup 213}Bi-DTPA-F3 and paclitaxel, alone or in combination, was assayed towards OVCAR-3 cells using the alamarBlue assay, the clonogenic assay and flow cytometric analyses of the mode of cell death and cell cycle arrest. Therapeutic efficacy of the different treatment options was assayed after repeated treatment of mice bearing intraperitoneal OVCAR-3 xenograft tumours. Therapy monitoring was performed by bioluminescence imaging and histopathologic analysis. Treatment of OVCAR-3 cells in vitro with combined {sup 213}Bi-DTPA-F3 and paclitaxel resulted in enhanced cytotoxicity, induction of apoptosis and G2/M phase arrest compared to treatment with either {sup 213}Bi-DTPA-F3 or paclitaxel. Accordingly, i.p. xenograft OVCAR-3 tumours showed the best response following repeated (six times) combined therapy with {sup 213}Bi-DTPA-F3 (1.85 MBq) and paclitaxel (120 {mu}g) as demonstrated by bioluminescence imaging and histopathologic investigation of tumour spread on the mesentery of the small and large intestine. Moreover, mean survival of xenograft mice that received combined therapy with {sup 213}Bi-DTPA-F3 and paclitaxel was significantly superior to mice treated with either {sup 213}Bi-DTPA-F3 or paclitaxel alone. Combined treatment with {sup 213}Bi-DTPA-F3 and paclitaxel significantly increased mean survival of mice with peritoneal carcinomatosis of ovarian origin, thus favouring future therapeutic application. (orig.)

The ubiquitin peptidase UCHL1 induces G0/G1 cell cycle arrest and apoptosis through stabilizing p53 and is frequently silenced in breast cancer.

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Tingxiu Xiang

Full Text Available Breast cancer (BrCa is a complex disease driven by aberrant gene alterations and environmental factors. Recent studies reveal that abnormal epigenetic gene regulation also plays an important role in its pathogenesis. Ubiquitin carboxyl- terminal esterase L1 (UCHL1 is a tumor suppressor silenced by promoter methylation in multiple cancers, but its role and alterations in breast tumorigenesis remain unclear.We found that UCHL1 was frequently downregulated or silenced in breast cancer cell lines and tumor tissues, but readily expressed in normal breast tissues and mammary epithelial cells. Promoter methylation of UCHL1 was detected in 9 of 10 breast cancer cell lines (90% and 53 of 66 (80% primary tumors, but rarely in normal breast tissues, which was statistically correlated with advanced clinical stage and progesterone receptor status. Pharmacologic demethylation reactivated UCHL1 expression along with concomitant promoter demethylation. Ectopic expression of UCHL1 significantly suppressed the colony formation and proliferation of breast tumor cells, through inducing G0/G1 cell cycle arrest and apoptosis. Subcellular localization study showed that UCHL1 increased cytoplasmic abundance of p53. We further found that UCHL1 induced p53 accumulation and reduced MDM2 protein level, and subsequently upregulated the expression of p21, as well as cleavage of caspase3 and PARP, but not in catalytic mutant UCHL1 C90S-expressed cells.UCHL1 exerts its tumor suppressive functions by inducing G0/G1cell cycle arrest and apoptosis in breast tumorigenesis, requiring its deubiquitinase activity. Its frequent silencing by promoter CpG methylation may serve as a potential tumor marker for breast cancer.

Cell cycle arrest in the jewel wasp Nasonia vitripennis in larval diapause.

Science.gov (United States)

Shimizu, Yuta; Mukai, Ayumu; Goto, Shin G

2018-04-01

Insects enter diapause to synchronise their life cycle with biotic and abiotic environmental conditions favourable for their development, reproduction, and survival. One of the most noticeable characteristics of diapause is the blockage of ontogeny. Although this blockage should occur with the cessation of cellular proliferation, i.e. cell cycle arrest, it was confirmed only in a few insect species and information on the molecular pathways involved in cell cycle arrest is limited. In the present study, we investigated developmental and cell cycle arrest in diapause larvae of the jewel wasp Nasonia vitripennis. Developmental and cell cycle arrest occur in the early fourth instar larval stage of N. vitripennis under short days. By entering diapause, the S fraction of the cell cycle disappears and approximately 80% and 20% of cells arrest their cell cycle in the G0/G1 and G2 phases, respectively. We further investigated expression of cell cycle regulatory genes and some housekeeping genes to dissect molecular mechanisms underlying the cell cycle arrest. Copyright © 2016 Elsevier Ltd. All rights reserved.

Menadione induces G2/M arrest in gastric cancer cells by down-regulation of CDC25C and proteasome mediated degradation of CDK1 and cyclin B1

OpenAIRE

Lee, Min Ho; Cho, Yoonjung; Kim, Do Hyun; Woo, Hyun Jun; Yang, Ji Yeong; Kwon, Hye Jin; Yeon, Min Ji; Park, Min; Kim, Sa-Hyun; Moon, Cheol; Tharmalingam, Nagendran; Kim, Tae Ue; Kim, Jong-Bae

2016-01-01

Menadione (vitamin K3) has been reported to induce apoptotic cell death and growth inhibition in various types of cancer cells. However, involvement of menadione in cell cycle control has not been considered in gastric cancer cells yet. In the current study, we have investigated whether menadione is involved in the cell cycle regulation and suppression of growth in gastric cancer cells. In the cell cycle analysis, we found that menadione induced G2/M cell cycle arrest in AGS cells. To elucida...

Prometaphase arrest-dependent phosphorylation of Bcl-2 family proteins and activation of mitochondrial apoptotic pathway are associated with 17α-estradiol-induced apoptosis in human Jurkat T cells.

Science.gov (United States)

Han, Cho Rong; Jun, Do Youn; Kim, Yoon Hee; Lee, Ji Young; Kim, Young Ho

2013-10-01

In Jurkat T cell clone (JT/Neo), G2/M arrest, apoptotic sub-G1 peak, mitochondrial membrane potential (Δψm) loss, and TUNEL-positive DNA fragmentation were induced following exposure to 17α-estradiol (17α-E2), whereas none of these events (except for G2/M arrest) were induced in Jurkat cells overexpressing Bcl-2 (JT/Bcl-2). Under these conditions, phosphorylation at Thr161 and dephosphorylation at Tyr15 of Cdk1, upregulation of cyclin B1 level, histone H1 phosphorylation, Cdc25C phosphorylation at Thr-48, Bcl-2 phosphorylation at Thr-56 and Ser-70, Mcl-1 phosphorylation, and Bim phosphorylation were detected in the presence of Bcl-2 overexpression. However, the 17α-E2-induced upregulation of Bak levels, activation of Bak, activation of caspase-3, and PARP degradation were abrogated by Bcl-2 overexpression. In the presence of the G1/S blocking agent hydroxyurea, 17α-E2 failed to induce G2/M arrest and all apoptotic events including Cdk1 activation and phosphorylation of Bcl-2, Mcl-1 and Bim. The 17α-E2-induced phosphorylation of Bcl-2 family proteins and mitochondrial apoptotic events were suppressed by a Cdk1 inhibitor but not by aurora A and aurora B kinase inhibitors. Immunofluorescence microscopic analysis showed that an aberrant bipolar microtubule array, incomplete chromosome congression at the metaphase plate, and prometaphase arrest, which was reversible, were the underlying factors for 17α-E2-induced mitotic arrest. The in vitro microtubule polymerization assay showed that 17α-E2 could directly inhibit microtubule formation. These results show that the apoptogenic activity of 17α-E2 was due to the impaired mitotic spindle assembly causing prometaphase arrest and prolonged Cdk1 activation, the phosphorylation of Bcl-2, Mcl-1 and Bim, and the activation of Bak and mitochondria-dependent caspase cascade. Copyright © 2013 Elsevier B.V. All rights reserved.

Loss of p53 induces M-phase retardation following G2 DNA damage checkpoint abrogation.

Science.gov (United States)

Minemoto, Yuzuru; Uchida, Sanae; Ohtsubo, Motoaki; Shimura, Mari; Sasagawa, Toshiyuki; Hirata, Masato; Nakagama, Hitoshi; Ishizaka, Yukihito; Yamashita, Katsumi

2003-04-01

Most cell lines that lack functional p53 protein are arrested in the G2 phase of the cell cycle due to DNA damage. When the G2 checkpoint is abrogated, these cells are forced into mitotic catastrophe. A549 lung adenocarcinoma cells, in which p53 was eliminated with the HPV16 E6 gene, exhibited efficient arrest in the G2 phase when treated with adriamycin. Administration of caffeine to G2-arrested cells induced a drastic change in cell phenotype, the nature of which depended on the status of p53. Flow cytometric and microscopic observations revealed that cells that either contained or lacked p53 resumed their cell cycles and entered mitosis upon caffeine treatment. However, transit to the M phase was slower in p53-negative cells than in p53-positive cells. Consistent with these observations, CDK1 activity was maintained at high levels, along with stable cyclin B1, in p53-negative cells. The addition of butyrolactone I, which is an inhibitor of CDK1 and CDK2, to the p53-negative cells reduced the floating round cell population and induced the disappearance of cyclin B1. These results suggest a relationship between the p53 pathway and the ubiquitin-mediated degradation of mitotic cyclins and possible cross-talk between the G2-DNA damage checkpoint and the mitotic checkpoint.

An engineering interpretation of pop-in arrest and tearing arrest in terms of static crack arrest, Ksub(Ia)

International Nuclear Information System (INIS)

Witt, F.J.

1983-01-01

When fracture toughness specimens are tested under displacement controlled conditions, they are often observed to exhibit unstable cleavage fracture followed by arrest of the cleavage mode wherein a significant load remains on the specimen (pop-in arrest). This behavior carries over into the ductile tearing regime wherein tearing may occur rapidly identified by load reduction and then proceeds at a discernible less rate (tearing arrest). Both these behaviors represent an initiation condition followed by an arrest condition. In this paper it is demonstrated that from either of the arrest conditions an arrest value may be determined which, for available experimental data, is shown to be an engineering estimate for the static crack arrest toughness, Ksub(Ia). A data analysis procedure is outlined and Ksub(Ic) and Ksub(Ia) estimates from sixty-eight 1/2, 1 and 2 in. thick compact specimens from two steels (A533 Grade B Class 1 and AISI 1018) tested between -40 deg F and 200 deg F are summarized. The crack arrest estimates are seen to compare favorably with Ksub(Ia) results obtained by other investigators using 2 in. thick specimens. Also it is demonstrated that when failure is by fully ductile tearing, the crack arrest toughness is at least equal to the estimate for Ksub(Ic) for the specimen. (author)

Deficiency of G1 regulators P53, P21Cip1 and/or pRb decreases hepatocyte sensitivity to TGFβ cell cycle arrest

Directory of Open Access Journals (Sweden)

Harrison David J

2007-11-01

Full Text Available Abstract Background TGFβ is critical to control hepatocyte proliferation by inducing G1-growth arrest through multiple pathways leading to inhibition of E2F transcription activity. The retinoblastoma protein pRb is a key controller of E2F activity and G1/S transition which can be inhibited in viral hepatitis. It is not known whether the impairment of pRb would alter the growth inhibitory potential of TGFβ in disease. We asked how Rb-deficiency would affect responses to TGFβ-induced cell cycle arrest. Results Primary hepatocytes isolated from Rb-floxed mice were infected with an adenovirus expressing CRE-recombinase to delete the Rb gene. In control cells treatment with TGFβ prevented cells to enter S phase via decreased cMYC activity, activation of P16INK4A and P21Cip and reduction of E2F activity. In Rb-null hepatocytes, cMYC activity decreased slightly but P16INK4A was not activated and the great majority of cells continued cycling. Rb is therefore central to TGFβ-induced cell cycle arrest in hepatocytes. However some Rb-null hepatocytes remained sensitive to TGFβ-induced cell cycle arrest. As these hepatocytes expressed very high levels of P21Cip1 and P53 we investigated whether these proteins regulate pRb-independent signaling to cell cycle arrest by evaluating the consequences of disruption of p53 and p21Cip1. Hepatocytes deficient in p53 or p21Cip1 showed diminished growth inhibition by TGFβ. Double deficiency had a similar impact showing that in cells containing functional pRb; P21Cip and P53 work through the same pathway to regulate G1/S in response to TGFβ. In Rb-deficient cells however, p53 but not p21Cip deficiency had an additive effect highlighting a pRb-independent-P53-dependent effector pathway of inhibition of E2F activity. Conclusion The present results show that otherwise genetically normal hepatocytes with disabled p53, p21Cip1 or Rb genes respond less well to the antiproliferative effects of TGFβ. As the function of

Zerumbone-loaded nanostructured lipid carrier induces G2/M cell cycle arrest and apoptosis via mitochondrial pathway in a human lymphoblastic leukemia cell line

Directory of Open Access Journals (Sweden)

Rahman HS

2014-01-01

Full Text Available Heshu Sulaiman Rahman,1–3 Abdullah Rasedee,1,2 Ahmad Bustamam Abdul,2,4 Nazariah Allaudin Zeenathul,1,2 Hemn Hassan Othman,1,3 Swee Keong Yeap,2 Chee Wun How,2 Wan Abd Ghani Wan Nor Hafiza4,51Faculty of Veterinary Medicine, 2Institute of Bioscience, Universiti Putra Malaysia, Selangor, Malaysia; 3Faculty of Veterinary Medicine, University of Sulaimanyah, Sulaimanyah City, Kurdistan Region, Northern Iraq; 4Faculty of Medicine and Health Science, Universiti Putra Malaysia, Selangor, Malaysia; 5College of Medical Laboratory Technology, Institute for Medical Research, Kuala Lumpur, MalaysiaAbstract: This investigation evaluated the antileukemia properties of a zerumbone (ZER-loaded nanostructured lipid carrier (NLC prepared by hot high-pressure homogenization techniques in an acute human lymphoblastic leukemia (Jurkat cell line in vitro. The apoptogenic effect of the ZER-NLC on Jurkat cells was determined by fluorescent and electron microscopy, Annexin V-fluorescein isothiocyanate, Tdt-mediated dUTP nick-end labeling assay, cell cycle analysis, and caspase activity. An MTT (3-(4,5-dimethylthiazol-2-yl-2,5 diphenyltetrazolium bromide assay showed that ZER-NLC did not have adverse effects on normal human peripheral blood mononuclear cells. ZER-NLC arrested the Jurkat cells at G2/M phase with inactivation of cyclin B1 protein. The study also showed that the antiproliferative effect of ZER-NLC on Jurkat cells is through the intrinsic apoptotic pathway via activation of caspase-3 and caspase-9, release of cytochrome c from the mitochondria into the cytosol, and subsequent cleavage of poly (adenosine diphosphate-ribose polymerase (PARP. These findings show that the ZER-NLC is a potentially useful treatment for acute lymphoblastic leukemia in humans.Keywords: zerumbone-loaded nanostructured lipid carrier, cell cycle arrest, apoptosis, mitochondrial pathway

Nuclear interaction of Smac/DIABLO with Survivin at G2/M arrest prompts docetaxel-induced apoptosis in DU145 prostate cancer cells

International Nuclear Information System (INIS)

Kim, Ji Young; Chung, Jin-Yong; Lee, Seung Gee; Kim, Yoon-Jae; Park, Ji-Eun; Yoo, Ki Soo; Yoo, Young Hyun; Park, Young Chul; Kim, Byeong Gee; Kim, Jong-Min

2006-01-01

Smac/DIABLO is released by mitochondria in response to apoptotic stimuli and is thought to antagonize the function of inhibitors of apoptosis proteins. Recently, it has been shown that, like XIAP, Survivin can potentially interact with Smac/DIABLO. However, the precise mechanisms and cellular location of their action have not been determined. We report for the first time that Smac/DIABLO translocates to the nucleus and is colocalized with Survivin at mitotic spindles during apoptosis resulting from G2/M arrest due to docetaxel treatment of DU145 prostate cancer cells. Our data demonstrate that the nuclear interaction of Smac/DIABLO with Survivin is an important step for suppressing the anti-apoptotic function of Survivin in Doc-induced apoptosis. This suggests that the balance between cellular Smac/DIABLO and Survivin levels could be critical for cellular destiny in taxane-treated cancer cells

Two Chitotriose-Specific Lectins Show Anti-Angiogenesis, Induces Caspase-9-Mediated Apoptosis and Early Arrest of Pancreatic Tumor Cell Cycle.

Directory of Open Access Journals (Sweden)

Ruby Singh

Full Text Available The antiproliferative activity of two chito-specific agglutinins purified from Benincasa hispida (BhL and Datura innoxia (DiL9 of different plant family origin was investigated on various cancer cell lines. Both lectins showed chitotriose specificity, by inhibiting lectin hemagglutinating activity. On further studies, it was revealed that these agglutinins caused remarkable concentration-dependent antiproliferative effect on human pancreatic cancerous cells but not on the normal human umbilical vein endothelial cells even at higher doses determined using MTT assay. The GI50 values were approximately 8.4 μg ml(-1 (0.247 μM and 142 μg ml(-1 (14.8 μM for BhL and DiL9, respectively, against PANC-1 cells. The growth inhibitory effect of these lectins on pancreatic cancer cells were shown to be a consequence of lectin cell surface binding and triggering G0/G1 arrest, mitochondrial membrane depolarization, sustained increase of the intracellular calcium release and the apoptotic signal is amplified by activation of caspases executing cell death. Interestingly, these lectins also showed anti-angiogenic activity by disrupting the endothelial tubulogenesis. Therefore, we report for the first time two chito-specific lectins specifically binding to tumor glycans; they can be considered to be a class of molecules with antitumor activity against pancreatic cancer cells mediated through caspase dependent mitochondrial apoptotic pathway.

Inhibition of Rac1 activity induces G1/S phase arrest through the GSK3/cyclin D1 pathway in human cancer cells.

Science.gov (United States)

Liu, Linna; Zhang, Hongmei; Shi, Lei; Zhang, Wenjuan; Yuan, Juanli; Chen, Xiang; Liu, Juanjuan; Zhang, Yan; Wang, Zhipeng

2014-10-01

Rac1 has been shown to regulate the cell cycle in cancer cells. Yet, the related mechanism remains unclear. Thus, the present study aimed to investigate the mechanism involved in the regulation of G1/S phase transition by Rac1 in cancer cells. Inhibition of Rac1 by inhibitor NSC23766 induced G1/S phase arrest and inhibited the proliferation of A431, SW480 and U2-OS cells. Suppression of GSK3 by shRNA partially rescued G1/S phase arrest and inhibition of proliferation. Incubation of cells with NSC23766 reduced p-AKT and inactivated p-GSK3α and p-GSK3β, increased p-cyclin D1 expression and decreased the level of cyclin D1 protein. Consequently, cyclin D1 targeting transcriptional factor E2F1 expression, which promotes G1 to S phase transition, was also reduced. In contrast, constitutive active Rac1 resulted in increased p-AKT and inactivated p-GSK3α and p-GSK3β, decreased p-cyclin D1 expression and enhanced levels of cyclin D1 and E2F1 expression. Moreover, suppression of GSK3 did not alter p-AKT or Rac1 activity, but decreased p-cyclin D1 and increased total cyclin D1 protein. However, neither Rac1 nor GSK3 inhibition altered cyclin D1 at the RNA level. Moreover, after inhibition of Rac1 or GSK3 following proteasome inhibitor MG132 treatment, cyclin D1 expression at the protein level remained constant, indicating that Rac1 and GSK3 may regulate cyclin D1 turnover through phosphorylation and degradation. Therefore, our findings suggest that inhibition of Rac1 induces cell cycle G1/S arrest in cancer cells by regulation of the GSK3/cyclin D1 pathway.

Radiosensitization by inhibiting survivin in human hepatoma HepG2 cells to high-LET radiation

International Nuclear Information System (INIS)

Jin Xiaodong; Li Qiang; Wu Qingfeng; Li Ping; Gong Li; Hao Jifang; Dai Zhongying; Matsumoto, Yoshitaka; Furusawa, Yoshiya

2011-01-01

In this study, whether survivin plays a direct role in mediating high-linear energy transfer (LET) radiation resistance in human hepatoma cells was investigated. Small interfering RNA (siRNA) targeting survivin mRNA was designed and transfected into human hepatoma HepG2 cells. Real-time polymerase chain reaction (PCR) and western blotting analyses revealed that survivin expression in HepG2 cells decreased at both transcriptional and post-transcriptional levels after treatment with survivin-specific siRNA. Caspase-3 activity was determined with a microplate reader assay as well. Following exposure to high-LET carbon ions, a reduced clonogenic survival effect, increased apoptotic rates and caspase-3 activity were observed in the cells treated with the siRNA compared to those untreated with the siRNA. The cells with transfection of the survivin-specific siRNA also increased the level of G 2 /M arrest. These results suggest that survivin definitely plays a role in mediating the resistance of HepG2 cells to high-LET radiation and depressing survivin expression might be useful to improve the therapeutic efficacy of heavy ions for radioresistant solid tumors. (author)

Berberine induces p53-dependent cell cycle arrest and apoptosis of human osteosarcoma cells by inflicting DNA damage

International Nuclear Information System (INIS)

Liu Zhaojian; Liu Qiao; Xu Bing; Wu Jingjing; Guo Chun; Zhu Faliang; Yang Qiaozi; Gao Guimin; Gong Yaoqin; Shao Changshun

2009-01-01

Alkaloid berberine is widely used for the treatment of diarrhea and other diseases. Many laboratory studies showed that it exhibits anti-proliferative activity against a wide spectrum of cancer cells in culture. In this report we studied the mechanisms underlying the inhibitory effects of berberine on human osteosarcoma cells and on normal osteoblasts. The inhibition was largely attributed to cell cycle arrest at G1 and G2/M, and to a less extent, to apoptosis. The G1 arrest was dependent on p53, as G1 arrest was abolished in p53-deficient osteosarcoma cells. The induction of G1 arrest and apoptosis was accompanied by a p53-dependent up-regulation of p21 and pro-apoptotic genes. However, the G2/M arrest could be induced by berberine regardless of the status of p53. Interestingly, DNA double-strand breaks, as measured by the phosphorylation of H2AX, were remarkably accumulated in berberine-treated cells in a dose-dependent manner. Thus, one major mechanism by which berberine exerts its growth-inhibitory effect is to inflict genomic lesions on cells, which in turn trigger the activation of p53 and the p53-dependent cellular responses including cell cycle arrest and apoptosis

Berberine induces p53-dependent cell cycle arrest and apoptosis of human osteosarcoma cells by inflicting DNA damage

Energy Technology Data Exchange (ETDEWEB)

Liu Zhaojian; Liu Qiao; Xu Bing; Wu Jingjing [Key Laboratory of Experimental Teratology of Ministry of Education and Institute of Molecular Medicine and Genetics, Shandong University School of Medicine, Jinan, Shandong 250012 (China); Guo Chun; Zhu Faliang [Institute of Immunology, Shandong University School of Medicine, Jinan, Shandong 250012 (China); Yang Qiaozi [Department of Genetics, Rutgers University, Piscataway, NJ 08854 (United States); Gao Guimin [Key Laboratory of Experimental Teratology of Ministry of Education and Institute of Molecular Medicine and Genetics, Shandong University School of Medicine, Jinan, Shandong 250012 (China); Gong Yaoqin [Key Laboratory of Experimental Teratology of Ministry of Education and Institute of Molecular Medicine and Genetics, Shandong University School of Medicine, Jinan, Shandong 250012 (China)], E-mail: yxg8@sdu.edu.cn; Shao Changshun [Key Laboratory of Experimental Teratology of Ministry of Education and Institute of Molecular Medicine and Genetics, Shandong University School of Medicine, Jinan, Shandong 250012 (China); Department of Genetics, Rutgers University, Piscataway, NJ 08854 (United States)], E-mail: shao@biology.rutgers.edu

2009-03-09

Alkaloid berberine is widely used for the treatment of diarrhea and other diseases. Many laboratory studies showed that it exhibits anti-proliferative activity against a wide spectrum of cancer cells in culture. In this report we studied the mechanisms underlying the inhibitory effects of berberine on human osteosarcoma cells and on normal osteoblasts. The inhibition was largely attributed to cell cycle arrest at G1 and G2/M, and to a less extent, to apoptosis. The G1 arrest was dependent on p53, as G1 arrest was abolished in p53-deficient osteosarcoma cells. The induction of G1 arrest and apoptosis was accompanied by a p53-dependent up-regulation of p21 and pro-apoptotic genes. However, the G2/M arrest could be induced by berberine regardless of the status of p53. Interestingly, DNA double-strand breaks, as measured by the phosphorylation of H2AX, were remarkably accumulated in berberine-treated cells in a dose-dependent manner. Thus, one major mechanism by which berberine exerts its growth-inhibitory effect is to inflict genomic lesions on cells, which in turn trigger the activation of p53 and the p53-dependent cellular responses including cell cycle arrest and apoptosis.

TEAD4-YAP interaction regulates tumoral growth by controlling cell-cycle arrest at the G1 phase

International Nuclear Information System (INIS)

Takeuchi, Shin; Kasamatsu, Atsushi; Yamatoji, Masanobu; Nakashima, Dai; Endo-Sakamoto, Yosuke; Koide, Nao; Takahara, Toshikazu; Shimizu, Toshihiro; Iyoda, Manabu; Ogawara, Katsunori; Shiiba, Masashi; Tanzawa, Hideki; Uzawa, Katsuhiro

2017-01-01

TEA domain transcription factor 4 (TEAD4), which has critical functions in the process of embryonic development, is expressed in various cancers. However, the important role of TEAD4 in human oral squamous cell carcinomas (OSCCs) remain unclear. Here we investigated the TEAD4 expression level and the functional mechanism in OSCC using quantitative reverse transcriptase-polymerase chain reaction, Western blot analysis, and immunohistochemistry. Furthermore, TEAD4 knockdown model was used to evaluate cellular proliferation, cell-cycle analysis, and the interaction between TEAD4 and Yes-associated protein (YAP) which was reported to be a transcription coactivator of cellular proliferation. In the current study, we found that TEAD4 expression increased significantly in vitro and in vivo and correlated with tumoral size in OSCC patients. TEAD4 knockdown OSCC cells showed decreased cellular proliferation resulting from cell-cycle arrest in the G1 phase by down-regulation of cyclins, cyclin-dependent kinases (CDKs), and up-regulation of CDK inhibitors. We also found that the TEAD4-YAP complex in the nuclei may be related closely to transcriptions of G1 arrest-related genes. Taken together, we concluded that TEAD4 might play an important role in tumoral growth and have potential to be a therapeutic target in OSCCs. - Highlights: • TEAD4 contributes to tumor progression in OSCCs. • TEAD4 knockdown results in cell-cycle arrest at the G1phase in OSCC cells. • In TEAD4 knockdown cells, the amount of YAP in the nucleus decreases. • Activation of the TEAD4-YAP complex is an important factor in OSCC tumor growth. • TEAD4 might be a critical biomarker and a therapeutic target for OSCCs.

Mutation analysis of the negative regulator cyclin G2 in gastric cancer

African Journals Online (AJOL)

Jane

2011-10-24

Oct 24, 2011 ... Key words: Cyclin G2, gastric cancer, negative regulator, mutation screen. INTRODUCTION ... cerebellum, thymus, spleen, prostate, kidney and the immune ..... and B cell antigen receptor-mediated cell cycle arrest. J. Biol.

TGEV nucleocapsid protein induces cell cycle arrest and apoptosis through activation of p53 signaling

International Nuclear Information System (INIS)

Ding, Li; Huang, Yong; Du, Qian; Dong, Feng; Zhao, Xiaomin; Zhang, Wenlong; Xu, Xingang; Tong, Dewen

2014-01-01

Highlights: • TGEV N protein reduces cell viability by inducing cell cycle arrest and apoptosis. • TGEV N protein induces cell cycle arrest and apoptosis by regulating p53 signaling. • TGEV N protein plays important roles in TGEV-induced cell cycle arrest and apoptosis. - Abstract: Our previous studies showed that TGEV infection could induce cell cycle arrest and apoptosis via activation of p53 signaling in cultured host cells. However, it is unclear which viral gene causes these effects. In this study, we investigated the effects of TGEV nucleocapsid (N) protein on PK-15 cells. We found that TGEV N protein suppressed cell proliferation by causing cell cycle arrest at the S and G2/M phases and apoptosis. Characterization of various cellular proteins that are involved in regulating cell cycle progression demonstrated that the expression of N gene resulted in an accumulation of p53 and p21, which suppressed cyclin B1, cdc2 and cdk2 expression. Moreover, the expression of TGEV N gene promoted translocation of Bax to mitochondria, which in turn caused the release of cytochrome c, followed by activation of caspase-3, resulting in cell apoptosis in the transfected PK-15 cells following cell cycle arrest. Further studies showed that p53 inhibitor attenuated TGEV N protein induced cell cycle arrest at S and G2/M phases and apoptosis through reversing the expression changes of cdc2, cdk2 and cyclin B1 and the translocation changes of Bax and cytochrome c induced by TGEV N protein. Taken together, these results demonstrated that TGEV N protein might play an important role in TGEV infection-induced p53 activation and cell cycle arrest at the S and G2/M phases and apoptosis occurrence

Induction of G2/M arrest and apoptosis through mitochondria pathway by a dimer sesquiterpene lactone from Smallanthus sonchifolius in HeLa cells.

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Kitai, Yurika; Zhang, Xia; Hayashida, Yushi; Kakehi, Yoshiyuki; Tamura, Hirotoshi

2017-07-01

Dimer sesquiterpene lactones (SLs), uvedafolin and enhydrofolin, against four monomer SLs isolated from yacon, Smallanthus sonchifolius, leaf were the most cytotoxic substances on HeLa cells (IC 50 values 2.96-3.17 μM at 24 hours). However, the cytotoxic mechanism of dimer SL has not been elucidated yet. Therefore, in this study, we clarified the in vitro cytotoxic mechanism of uvedafolin on the HeLa cells, and evaluated the cytotoxicity against NIH/3T3 cells which were used as normal cells. In consequence, the dimer SLs had low toxicity for the NIH/3T3 cells (IC 50 4.81-4.98 μM at 24 hours) and then the uvedafolin mediated cell cycle arrest at the G 2 /M phase and induced apoptosis on the HeLa cells evidenced by appearance of a subG1 peak. Uvedafolin induced apoptosis was attributed to caspase-9 and caspase-3/7 activities. An effectively induced apoptosis pathway was demonstrated from mitochondria membrane potential change and cytochrome c release to cytosol. These results reveal that uvedafolin induced apoptosis via the mitochondria pathway. The present results indicate the potential of uvedafolin as a leading compound of new anticancer agents. Copyright © 2016. Published by Elsevier B.V.

Rare sugar D-allose induces specific up-regulation of TXNIP and subsequent G1 cell cycle arrest in hepatocellular carcinoma cells by stabilization of p27kip1.

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Yamaguchi, Fuminori; Takata, Maki; Kamitori, Kazuyo; Nonaka, Machiko; Dong, Youyi; Sui, Li; Tokuda, Masaaki

2008-02-01

'Rare sugars' are defined as monosaccharides that exist in nature but are only present in limited quantities. The development of mass production method of rare sugars revealed some interesting physiological effects of these on animal cells, but the mechanisms have not been well studied. We examined the effect of D-allose on the proliferation of cancer cells and the underlying molecular mechanism of the action. The HuH-7 hepatocellular carcinoma cells were treated with various monosaccharides for 48 h and D-allose was shown to inhibit cell growth by 40% in a dose-dependent manner. D-allose induced G1 cell cycle arrest but not apoptosis. The microarray analysis revealed that D-allose significantly up-regulated thioredoxin interacting protein (TXNIP) gene expression, which is often suppressed in tumor cells and western blot analysis confirmed its increase at protein level. The overexpression of TXNIP also induced G1 cell cycle arrest. Analysis of cell cycle regulatory genes showed p27kip1, a key regulator of G1/S cell cycle transition, to be increased at the protein but not the transcriptional level. Protein interaction between TXNIP and jab1, and p27kip1 and jab1, was observed, suggesting stabilization of p27kip1 protein by the competitive inhibition of jab1-mediated nuclear export of p27kip1 by TXNIP. In addition, increased interaction and nuclear localization of TXNIP and p27kip1 were apparent after D-allose treatment. Our findings surprisingly suggest that D-allose, a simple monosaccharide, may act as a novel anticancer agent via unique TXNIP induction and p27kip1 protein stabilization.

CpG oligodeoxynucleotide induces apoptosis and cell cycle arrest in A20 lymphoma cells via TLR9-mediated pathways.

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Qi, Xu-Feng; Zheng, Li; Kim, Cheol-Su; Lee, Kyu-Jae; Kim, Dong-Heui; Cai, Dong-Qing; Qin, Jun-Wen; Yu, Yan-Hong; Wu, Zheng; Kim, Soo-Ki

2013-07-01

Recent studies have suggested that the anti-cancer activity of CpG-oligodeoxynucleotides (CpG-ODNs) is owing to their immunomodulatory effects in tumor-bearing host. The purpose of this study is to investigate the directly cytotoxic activity of KSK-CpG, a novel CpG-ODN with an alternative CpG motif, against A20 and EL4 lymphoma cells in comparison with previously used murine CpG motif (1826-CpG). To evaluate the potential cytotoxic effects of KSK-CpG on lymphoma cells, cell viability assay, confocal microscopy, flow cytometry, DNA fragmentation, Western blotting, and reverse transcription-polymerase chain reaction (RT-PCR) analysis were used. We found that KSK-CpG induced direct cytotoxicity in A20 lymphoma cells, but not in EL4 lymphoma cells, at least in part via TLR9-mediated pathways. Apoptotic cell death was demonstrated to play an important role in CpG-ODNs-induced cytotoxicity. In addition, both mitochondrial membrane potential decrease and G1-phase arrest were involved in KSK-CpG-induced apoptosis in A20 cells. The activities of apoptotic molecules such as caspase-3, PARP, and Bax were increased, but the activation of p27 Kip1 and ERK were decreased in KSK-CpG-treated A20 cells. Furthermore, autocrine IFN-γ partially contributed to apoptotic cell death in KSK-CpG-treated A20 cells. Collectively, our findings suggest that KSK-CpG induces apoptotic cell death in A20 lymphoma cells at least in part by inducing G1-phase arrest and autocrine IFN-γ via increasing TLR9 expression, without the need for immune system of tumor-bearing host. This new understanding supports the development of TLR9-targeted therapy with CpG-ODN as a direct therapeutic agent for treating B lymphoma. Copyright © 2013 Elsevier Ltd. All rights reserved.

Smad4 sensitizes colorectal cancer to 5-fluorouracil through cell cycle arrest by inhibiting the PI3K/Akt/CDC2/survivin cascade.

Science.gov (United States)

Zhang, Binhao; Leng, Chao; Wu, Chao; Zhang, Zhanguo; Dou, Lei; Luo, Xin; Zhang, Bixiang; Chen, Xiaoping

2016-03-01

5-Fluorouracil (5-FU), a cell cycle-specific antimetabolite, is one of the most commonly used chemotherapeutic agents for colorectal cancer (CRC). Yet, resistance to 5-FU-based chemotherapy is still an obstacle to the treatment of this malignancy. Mutation or loss of Smad4 in CRC is pivotal for chemoresistance. However, the mechanism by which Smad4 regulates the chemosensitivity of CRC remains unclear. In the present study, we investigated the role of Smad4 in the chemosensitivity of CRC to 5-FU, and whether Smad4-regulated cell cycle arrest is involved in 5-FU chemoresistance. We used Smad4-expressing CT26 and Smad4-null SW620 cell lines as experimental models, by knockdown or transgenic overexpression. Cells or tumors were treated with 5-FU to determine chemosensitivity by cell growth, tumorigenicity assay and a mouse model. Cell cycle distribution was examined with flow cytometric analysis, and cell cycle-related proteins were examined by western blotting. Smad4 deficiency in CT26 and SW620 cells induced chemoresistance to 5-FU both in vitro and in vivo. Smad4 deficiency attenuated G1 or G2 cell cycle arrest by activating the PI3K/Akt/CDC2/survivin pathway. The PI3K inhibitor, LY294002, reversed the activation of the Akt/CDC2/survivin cascade in the Smad4-deficient cells, while it had little effect on cells with high Smad4 expression. In conclusion, we discovered a novel mechanism mediated by Smad4 to trigger 5-FU chemosensitivity through cell cycle arrest by inhibiting the PI3K/Akt/CDC2/survivin cascade. The present study also implies that LY294002 has potential therapeutic value to reverse the chemosensitivity of CRC with low Smad4 expression.

The G2/M DNA damage checkpoint inhibits mitosis through Tyr15 phosphorylation of p34cdc2 in Aspergillus nidulans.

OpenAIRE

Ye, X S; Fincher, R R; Tang, A; Osmani, S A

1997-01-01

It is possible to cause G2 arrest in Aspergillus nidulans by inactivating either p34cdc2 or NIMA. We therefore investigated the negative control of these two mitosis-promoting kinases after DNA damage. DNA damage caused rapid Tyr15 phosphorylation of p34cdc2 and transient cell cycle arrest but had little effect on the activity of NIMA. Dividing cells deficient in Tyr15 phosphorylation of p34cdc2 were sensitive to both MMS and UV irradiation and entered lethal premature mitosis with damaged DN...

Curcumin analog WZ35 induced cell death via ROS-dependent ER stress and G2/M cell cycle arrest in human prostate cancer cells

International Nuclear Information System (INIS)

Zhang, Xiuhua; Chen, Minxiao; Zou, Peng; Kanchana, Karvannan; Weng, Qiaoyou; Chen, Wenbo; Zhong, Peng; Ji, Jiansong; Zhou, Huiping; He, Langchong; Liang, Guang

2015-01-01

Prostate cancer is the most commonly diagnosed malignancy among men. The Discovery of new agents for the treatment of prostate cancer is urgently needed. Compound WZ35, a novel analog of the natural product curcumin, exhibited good anti-prostate cancer activity, with an IC 50 of 2.2 μM in PC-3 cells. However, the underlying mechanism of WZ35 against prostate cancer cells is still unclear. Human prostate cancer PC-3 cells and DU145 cells were treated with WZ35 for further proliferation, apoptosis, cell cycle, and mechanism analyses. NAC and CHOP siRNA were used to validate the role of ROS and ER stress, respectively, in the anti-cancer actions of WZ35. Our results show that WZ35 exhibited much higher cell growth inhibition than curcumin by inducing ER stress-dependent cell apoptosis in human prostate cells. The reduction of CHOP expression by siRNA partially abrogated WZ35-induced cell apoptosis. WZ35 also dose-dependently induced cell cycle arrest in the G2/M phase. Furthermore, we found that WZ35 treatment for 30 min significantly induced reactive oxygen species (ROS) production in PC-3 cells. Co-treatment with the ROS scavenger NAC completely abrogated the induction of WZ35 on cell apoptosis, ER stress activation, and cell cycle arrest, indicating an upstream role of ROS generation in mediating the anti-cancer effect of WZ35. Taken together, this work presents the novel anticancer candidate WZ35 for the treatment of prostate cancer, and importantly, reveals that increased ROS generation might be an effective strategy in human prostate cancer treatment. The online version of this article (doi:10.1186/s12885-015-1851-3) contains supplementary material, which is available to authorized users

The inhibition of activated hepatic stellate cells proliferation by arctigenin through G0/G1 phase cell cycle arrest: persistent p27(Kip1) induction by interfering with PI3K/Akt/FOXO3a signaling pathway.

Science.gov (United States)

Li, Ao; Wang, Jun; Wu, Mingjun; Zhang, Xiaoxun; Zhang, Hongzhi

2015-01-15

Proliferation of hepatic stellate cells (HSCs) is vital for the development of fibrosis during liver injury. In this study, we describe that arctigenin (ATG), a major bioactive component of Fructus Arctii, exhibited selective cytotoxic activity via inhibiting platelet-derived growth factor-BB (PDGF-BB)-activated HSCs proliferation and arrested cell cycle at G0/G1 phase, which could not be observed in normal human hepatocytes in vitro. The cyclin-dependent kinase (CDK) 4/6 activities could be strongly inhibited by ATG through down-regulation of cyclin D1 and CDK4/6 expression in early G1 phase arrest. In the ATG-treated HSCs, the expression level of p27(Kip1) and the formation of CDK2-p27(Kip1) complex were also increased. p27(Kip1) silencing significantly attenuated the effect of ATG, including cell cycle arrest and suppression of proliferation in activated HSCs. We also found that ATG suppressed PDGF-BB-induced phosphorylation of Akt and its downstream transcription factor Forkhead box O 3a (FOXO3a), decreased binding of FOXO3a to 14-3-3 protein, and stimulated nuclear translocation of FOXO3a in activated HSCs. Furthermore, knockdown of FOXO3a expression by FOXO3a siRNA attenuated ATG-induced up-regulation of p27(Kip1) in activated HSCs. All the above findings suggested that ATG could increase the levels of p27(Kip1) protein through inhibition of Akt and improvement of FOXO3a activity, in turn inhibited the CDK2 kinase activity, and eventually caused an overall inhibition of HSCs proliferation. Copyright © 2014 Elsevier B.V. All rights reserved.

SUN1 silencing inhibits cell growth through G0/G1 phase arrest in lung adenocarcinoma

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Huang W

2017-06-01

Full Text Available Weiyi Huang,* Haihua Huang,* Lei Wang, Jiong Hu, Weifeng Song Department of Oncology, The First People’s Hospital Affiliated to Shanghai Jiaotong University, Shanghai, People’s Republic of China *These authors contributed equally to this work Purpose: Cytoskeleton is critical for carcinoma cell proliferation, migration, and invasion. Sad-1 and UNC-84 domain containing 1 (SUN1 is one of the core linkers of nucleoskeleton and cytoskeleton. However, the functions of SUN1 in lung adenocarcinoma are largely unknown.Methods: In this study, we first transduced the lentivirus delivering the short hairpin RNA (shRNA against SUN1 to lung adenocarcinoma cells (A549 and 95D cells with high efficiency. After lentivirus infection, quantitative real-time polymerase chain reaction and Western blotting were used to detect the expressions of SUN1 mRNA and protein. The cell proliferation and colony formation were detected by MTT assay and colony formation assay, respectively. The cell distribution in the cell cycle was analyzed by flow cytometry.Results: Both mRNA and protein levels of SUN1 were significantly decreased in A549 and 95D cells after lentivirus infection, as indicated by quantitative real-time polymerase chain reaction and Western blot. Next, we found that cell proliferation and colony formation were markedly reduced in SUN1 silenced cells. Moreover, suppression of SUN1 led to cell cycle arrest at G0/G1 phase. Furthermore, Cyclin D1, CDK6, and CDK2 expressions were obviously reduced in A549 cells after SUN1 silencing.Conclusion: These results suggest that SUN1 plays an essential role in proliferation of lung adenocarcinoma cells in vitro and may be used as a potential therapeutic target for the treatment of lung adenocarcinoma in the future. Keywords: SUN1, lung cancer, proliferation

Low Oxygen Tension Enhances Expression of Myogenic Genes When Human Myoblasts Are Activated from G0 Arrest

DEFF Research Database (Denmark)

Sellathurai, Jeeva; Nielsen, Joachim; Hejbøl, Eva Kildall

2016-01-01

-PCR, immunocytochemistry and western blot. RESULTS AND CONCLUSIONS: We found an increase in proliferation rate of myoblasts when activated at a low oxygen tension (1% O2) compared to 21% O2. In addition, the gene expression studies showed up regulation of the myogenesis related genes PAX3, PAX7, MYOD, MYOG (myogenin), MET......, NCAM, DES (desmin), MEF2A, MEF2C and CDH15 (M-cadherin), however, the fraction of DES and MYOD positive cells was not increased by low oxygen tension, indicating that 1% O2 may not have a functional effect on the myogenic response. Furthermore, the expression of genes involved in the TGFβ, Notch...... and Wnt signaling pathways were also up regulated in low oxygen tension. The differences in gene expression were most pronounced at day one after activation from G0-arrest, thus the initial activation of myoblasts seemed most sensitive to changes in oxygen tension. Protein expression of HES1 and β...

Effects of DNA double-strand and single-strand breaks on intrachromosomal recombination events in cell-cycle-arrested yeast cells

International Nuclear Information System (INIS)

Galli, A.; Schiestl, R.H.

1998-01-01

Intrachromosomal recombination between repeated elements can result in deletion (DEL recombination) events. We investigated the inducibility of such intrachromosomal recombination events at different stages of the cell cycle and the nature of the primary DNA lesions capable of initiating these events. Two genetic systems were constructed in Saccharomyces cerevisiae that select for DEL recombination events between duplicated alleles of CDC28 and TUB2. We determined effects of double-strand breaks (DSBs) and single-strand breaks (SSBs) between the duplicated alleles on DEL recombination when induced in dividing cells or cells arrested in G1 or G2. Site-specific DSBs and SSBs were produced by overexpression of the I-Sce I endonuclease and the gene II protein (gIIp), respectively. I-Sce I-induced DSBs caused an increase in DEL recombination frequencies in both dividing and cell-cycle-arrested cells, indicating that G1- and G2-arrested cells are capable of completing DSB repair. In contrast, gIIp-induced SSBs caused an increase in DEL recombination frequency only in dividing cells. To further examine these phenomena we used both γ-irradiation, inducing DSBs as its most relevant lesion, and UV, inducing other forms of DNA damage. UV irradiation did not increase DEL recombination frequencies in G1 or G2, whereas γ-rays increased DEL recombination frequencies in both phases. Both forms of radiation, however, induced DEL recombination in dividing cells. The results suggest that DSBsbut not SSBs induce DEL recombination, probably via the single-strand annealing pathway. Further, DSBs in dividing cells may result from the replication of a UV or SSB-damaged template. Alternatively, UV induced events may occur by replication slippage after DNA polymerase pausing in front of the damage. (author)

Change of cell cycle arrest of tumor cell lines after 60Co γ-irradiation

International Nuclear Information System (INIS)

Tang Yi; Liu Wenli; Zhou Jianfeng; Gao Qinglei; Wu Jianhong

2003-01-01

Objective: To observe the cell cycle arrest changes in peripheral blood mononuclear cells (PBMNCs) of normal persons and several kinds of tumor cell lines after 60 Co γ-irradiation. Methods: PBMNCs of normal persons, HL-60, K562, SiHA and 113 tumor cell lines were irradiated with 60 Co γ-rays at the absorbed doses of 6, 10,15 Gy. Cell cycles changes were checked 6, 12, 24, 48 and 60 h after the irradiation. Results: A stasis state was observed in normal person PBMNCs, 95 percents of which were in G 1 phase, and they still remained stasis after the irradiation. Except the 113 cell line manifesting G 1 phase arrest, all other tumor cell lines showed G 2 /M phase arrest after irradiation. The radiation sensitivity of HL-60 was higher than that of SiHA cell line. Conclusion: Different cell lines have different cell cycle arrest reaction to radiation and their radiation sensitivity are also different

Iso-suillin isolated from Suillus luteus, induces G1 phase arrest and apoptosis in human hepatoma SMMC-7721 cells.

Science.gov (United States)

Jia, Zhi-Qiang; Chen, Ying; Yan, Yong-Xin; Zhao, Jun-Xia

2014-01-01

Iso-suillin, a natural product isolated from Suillus luteus, has been shown to inhibit the growth of some cancer cell lines. However, the molecular mechanisms of action of this compound are poorly understood. The purpose of this study was to investigate how iso-suillin inhibits proliferation and induces apoptosis in a human hepatoma cell line (SMMC-7721). We demonstrated the effects of iso-suillin on cell proliferation and apoptosis in SMMC-7721 cells, with no apparent toxicity in normal human lymphocytes, using colony formation assays and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) analysis. Western blotting was used to examine the expression of G1 phase-regulated and apoptosis-associated protein levels in iso-suillin treated SMMC-7721 cells. The results indicated that iso-suillin significantly decreased viability, induced G1 phase arrest and triggered apoptosis in SMMC-7721cells. Taken together, these results suggest the potential of iso-suillin as a candidate for liver cancer treatment.

The SFP1 gene product of Saccharomyces cerevisiae regulates G2/M transitions during the mitotic cell cycle and DNA-damage response

International Nuclear Information System (INIS)

Xu, Z.; Norris, D.

1998-01-01

In eukaryotic cells, checkpoint pathways arrest cell-cycle progression if a particular event has failed to complete appropriately or if an important intracellular structure is defective or damaged. Saccharomyces cerevisiae strains that lack the SFP1 gene fail to arrest at the G2 DNA-damage checkpoint in response to genomic injury, but maintain their ability to arrest at the replication and spindle-assembly checkpoints. sfp1D mutants are characterized by a premature entrance into mitosis during a normal (undamaged) cell cycle, while strains that overexpress Sfp1p exhibit delays in G2. Sfp1p therefore acts as a repressor of the G2/M transition, both in the normal cell cycle and in the G2 checkpoint pathway. Sfp1 is a nuclear protein with two Cys2His2 zinc-finger domains commonly found in transcription factors. We propose that Sfp1p regulates the expression of gene products involved in the G2/M transition during the mitotic cell cycle and the DNA-damage response. In support of this model, overexpression of Sfp1p induces the expression of the PDS1 gene, which is known to encode a protein that regulates the G2 checkpoint. (author)

Predicting Arrest in a Sample of Youth Perinatally Exposed to HIV: The Intersection of HIV and Key Contextual Factors.

Science.gov (United States)

Elkington, Katherine S; Peters, Zachary; Choi, C Jean; Bucek, Amelia; Leu, Cheng-Shiun; Abrams, Elaine J; Mellins, Claude A

2017-11-22

We examined the role of youth HIV status and other key factors on past-year arrest in perinatally HIV-exposed but uninfected (PHIV-) and perinatally HIV-infected (PHIV+) youth using data from a multi-site study of psychosocial behaviors in PHIV-exposed urban youth (N = 340; 61% PHIV+; 51% female; ages 9-16 at baseline). Youth and caregivers were administered 5 interviews, spanning approximately 7.5 years. Using longitudinal logistic mixed-effect models, we explored the association between past year arrest, internal [e.g., substance use disorder (SUD)] and external (e.g., neighborhood arrest rates) contextual factors, and social-regulation processes (e.g., in-school/work). Arrest rates increased from 2.6 to 19.7% across follow-ups; there were no differences in arrest over time by HIV status. In the final model, odds of arrest were greater for youth who were male, with SUD, ≥ 18 years old, with high levels of city stress, and neither in school nor employed. PHIV-exposed, urban youth have much higher rates of arrest than national samples. Lack of differences in arrest by HIV status suggests key contextual factors are more important in promoting arrest.

Metoclopramide-induced cardiac arrest

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Martha M. Rumore

2011-11-01

Full Text Available The authors report a case of cardiac arrest in a patient receiving intravenous (IV metoclopramide and review the pertinent literature. A 62-year-old morbidly obese female admitted for a gastric sleeve procedure, developed cardiac arrest within one minute of receiving metoclopramide 10 mg via slow intravenous (IV injection. Bradycardia at 4 beats/min immediately appeared, progressing rapidly to asystole. Chest compressions restored vital function. Electrocardiogram (ECG revealed ST depression indicative of myocardial injury. Following intubation, the patient was transferred to the intensive care unit. Various cardiac dysrrhythmias including supraventricular tachycardia (SVT associated with hypertension and atrial fibrillation occurred. Following IV esmolol and metoprolol, the patient reverted to normal sinus rhythm. Repeat ECGs revealed ST depression resolution without pre-admission changes. Metoclopramide is a non-specific dopamine receptor antagonist. Seven cases of cardiac arrest and one of sinus arrest with metoclopramide were found in the literature. The metoclopramide prescribing information does not list precautions or adverse drug reactions (ADRs related to cardiac arrest. The reaction is not dose related but may relate to the IV administration route. Coronary artery disease was the sole risk factor identified. According to Naranjo, the association was possible. Other reports of cardiac arrest, severe bradycardia, and SVT were reviewed. In one case, five separate IV doses of 10 mg metoclopramide were immediately followed by asystole repeatedly. The mechanism(s underlying metoclopramide’s cardiac arrest-inducing effects is unknown. Structural similarities to procainamide may play a role. In view of eight previous cases of cardiac arrest from metoclopramide having been reported, further elucidation of this ADR and patient monitoring is needed. Our report should alert clinicians to monitor patients and remain diligent in surveillance and

Prophase I arrest and progression to metaphase I in mouse oocytes: comparison of resumption of meiosis and recovery from G2-arrest in somatic cells

Czech Academy of Sciences Publication Activity Database

Šolc, Petr; Schultz, R. M.; Motlík, Jan

2010-01-01

Roč. 16, č. 9 (2010), s. 654-664 ISSN 1360-9947 R&D Projects: GA ČR(CZ) GC301/09/J036; GA MŠk ME08030 Grant - others:NIH(US) HD22681 Institutional research plan: CEZ:AV0Z50450515 Keywords : resumption of meiosis * prophase I arrest * oocyte Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 3.506, year: 2010

Folate deprivation induces cell cycle arrest at G0/G1 phase and apoptosis in hippocampal neuron cells through down-regulation of IGF-1 signaling pathway.

Science.gov (United States)

Yang, Yang; Li, Xi; Sun, Qinwei; He, Bin; Jia, Yimin; Cai, Demin; Zhao, Ruqian

2016-10-01

Folate deficiency contributes to impaired adult hippocampal neurogenesis, yet the mechanisms remain unclear. Here we use HT-22 hippocampal neuron cells as model to investigate the effect of folate deprivation (FD) on cell proliferation and apoptosis, and to elucidate the underlying mechanism. FD caused cell cycle arrest at G0/G1 phase and increased the rate of apoptosis, which was associated with disrupted expression of folate transport and methyl transfer genes. FOLR1 and SLC46A1 were (Pmethyl transfer pathway and hypermethylation of IGF-1 gene promoter. Copyright © 2016 Elsevier Ltd. All rights reserved.

Witnessed arrest, but not delayed bystander cardiopulmonary resuscitation improves prehospital cardiac arrest survival.

Science.gov (United States)

Vukmir, R B

2004-05-01

This study correlated the effect of witnessing a cardiac arrest and instituting bystander CPR (ByCPR), as a secondary end point in a study evaluating the effect of bicarbonate on survival. This prospective, randomised, double blinded clinical intervention trial enrolled 874 prehospital cardiopulmonary arrest patients encountered in a prehospital urban, suburban, and rural regional emergency medical service (EMS) area. This group underwent conventional advanced cardiac life support intervention followed by empiric early administration of sodium bicarbonate (1 mEq/l), monitoring conventional resuscitation parameters. Survival was measured as presence of vital signs on emergency department (ED) arrival. Data were analysed using chi(2) with Pearson correlation and odds ratio where appropriate. The overall survival rate was 13.9% (110 of 792) of prehospital cardiac arrest patients. The mean (SD) time until provision of bystander cardiopulmonary resuscitation (ByCPR) by laymen was 2.08 (2.77) minutes, and basic life support (BLS) by emergency medical technicians was 6.62 (5.73) minutes. There was improved survival noted with witnessed cardiac arrest-a 2.2-fold increase in survival, 18.9% (76 of 402) versus 8.6% (27 of 315) compared with unwitnessed arrests (ptwo minutes (p = 0.3752). Survival after prehospital cardiac arrest is more likely when witnessed, but not necessarily when ByCPR was performed by laymen.

Diacerein retards cell growth of chondrosarcoma cells at the G2/M cell cycle checkpoint via cyclin B1/CDK1 and CDK2 downregulation

International Nuclear Information System (INIS)

Lohberger, Birgit; Leithner, Andreas; Stuendl, Nicole; Kaltenegger, Heike; Kullich, Werner; Steinecker-Frohnwieser, Bibiane

2015-01-01

Chondrosarcoma is characterized for its lack of response to conventional cytotoxic chemotherapy, propensity for developing lung metastases, and low rates of survival. Research within the field of development and expansion of new treatment options for unresectable or metastatic diseases is of particular priority. Diacerein, a symptomatic slow acting drug in osteoarthritis (SYSADOA), implicates a therapeutic benefit for the treatment of chondrosarcoma by an antitumor activity. After treatment with diacerein the growth behaviour of the cells was analyzed with the xCELLigence system and MTS assay. Cell cycle was examined using flow cytometric analysis, RT-PCR, and western blot analysis of specific checkpoint regulators. The status for phosophorylation of mitogen-activated protein kinases (MAPKs) was analyzed with a proteome profiler assay. In addition, the possible impact of diacerein on apoptosis was investigated using cleaved caspase 3 and Annexin V/PI flow cytometric analysis. Diacerein decreased the cell viability and the cell proliferation in two different chondrosarcoma cell lines in a dose dependent manner. Flow cytometric analysis showed a classical G2/M arrest. mRNA and protein analysis revealed that diacerein induced a down-regulation of the cyclin B1-CDK1 complex and a reduction in CDK2 expression. Furthermore, diacerein treatment increased the phosphorylation of p38α and p38β MAPKs, and Akt1, Akt2, and Akt 3 in SW-1353, whereas in Cal-78 the opposite effect has been demonstrated. These observations accordingly to our cell cycle flow cytometric analysis and protein expression data may explain the G2/M phase arrest. In addition, no apoptotic induction after diacerein treatment, neither in the Cal-78 nor in the SW-1353 cell line was observed. Our results demonstrate for the first time that the SYSADOA diacerein decreased the viability of human chondrosarcoma cells and induces G2/M cell cycle arrest by CDK1/cyclin B1 down-regulation

The Growth Suppressing Effects of Girinimbine on Hepg2 Involve Induction of Apoptosis and Cell Cycle Arrest

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Tang Sook Wah

2011-08-01

Full Text Available Murraya koenigii is an edible herb widely used in folk medicine. Here we report that girinimbine, a carbazole alkaloid isolated from this plant, inhibited the growth and induced apoptosis in human hepatocellular carcinoma, HepG2 cells. The MTT and LDH assay results showed that girinimbine decreased cell viability and increased cytotoxicity in a dose-and time-dependent manner selectively. Girinimbine-treated HepG2 cells showed typical morphological features of apoptosis, as observed from normal inverted microscopy and Hoechst 33342 assay. Furthermore, girinimbine treatment resulted in DNA fragmentation and elevated levels of caspase-3 in HepG2 cells. Girinimbine treatment also displayed a time-dependent accumulation of the Sub-G0/G1 peak (hypodiploid and caused G0/G1-phase arrest. Together, these results demonstrated for the first time that girinimbine could effectively induce programmed cell death in HepG2 cells and suggests the importance of conducting further investigations in preclinical human hepatocellular carcinoma models, especially on in vivo efficacy, to promote girinimbine for use as an anticancer agent against hepatocellular carcinoma.

Distinct mechanisms act in concert to mediate cell cycle arrest.

Science.gov (United States)

Toettcher, Jared E; Loewer, Alexander; Ostheimer, Gerard J; Yaffe, Michael B; Tidor, Bruce; Lahav, Galit

2009-01-20

In response to DNA damage, cells arrest at specific stages in the cell cycle. This arrest must fulfill at least 3 requirements: it must be activated promptly; it must be sustained as long as damage is present to prevent loss of genomic information; and after the arrest, cells must re-enter into the appropriate cell cycle phase to ensure proper ploidy. Multiple molecular mechanisms capable of arresting the cell cycle have been identified in mammalian cells; however, it is unknown whether each mechanism meets all 3 requirements or whether they act together to confer specific functions to the arrest. To address this question, we integrated mathematical models describing the cell cycle and the DNA damage signaling networks and tested the contributions of each mechanism to cell cycle arrest and re-entry. Predictions from this model were then tested with quantitative experiments to identify the combined action of arrest mechanisms in irradiated cells. We find that different arrest mechanisms serve indispensable roles in the proper cellular response to DNA damage over time: p53-independent cyclin inactivation confers immediate arrest, whereas p53-dependent cyclin downregulation allows this arrest to be sustained. Additionally, p21-mediated inhibition of cyclin-dependent kinase activity is indispensable for preventing improper cell cycle re-entry and endoreduplication. This work shows that in a complex signaling network, seemingly redundant mechanisms, acting in a concerted fashion, can achieve a specific cellular outcome.

"House Arrest" or "Developmental Arrest"? A Study of Youth Under House Arrest.

Science.gov (United States)

Chamiel, Elad; Walsh, Sophie D

2018-06-01

Studies have examined the potential benefits and risks of alternative forms of detention, such as house arrest, for adults but, despite its growing use, little research has examined the implications of house arrest for juveniles. The current research examined the experience of 14 adolescents under house arrest. Six main themes were identified in the narratives of the participants: the experience of detention, daily schedule and utilization of time, emotions and self-reflection, relationships with peers, relation to parents and supervisor(s), and contact with professionals. Findings emphasized the potential developmental dangers of house arrest at the critical stage of adolescence. Yet, analysis also showed that the period of house arrest has the potential to be a period of positive changes, and can be used for successful rehabilitation.

[Ability of typical greenery shrubs of Beijing to adsorb and arrest PM2.5 ].

Science.gov (United States)

Liang, Dan; Wang, Bin; Wang, Yun-qi; Zhang, Hui-lan; Yang, Song-nan; Li, Ang

2014-09-01

Four typical types of green shrubs of Beijing (Euonymus japonicus, Buxus microphylla, Berberis thunbergii cv. atropurpurea, Taxus cuspidate cv. nana) were selected to study their capacities in adsorbing and arresting PM2.5 using both field observations and air chamber simulations. Concurrently, in order to analyze the pollution characteristics of Beijing in winter and spring, the PM2.5 concentrations of December 2012 to May 2013 were collected. Experimental results showed that: From the gas chamber experiments, the ability to adsorb and arrest PM2.5 was in the order of Berberis thunbergii cv. atropurpurea > Buxus microphylla > Taxus cuspidate cv. nana > Euonymus japonicus, mainly due to the differences in leaf characteristics; Outside measurement results showed that the ability to adsorb and arrest PM2.5 was ranked as Buxus microphylla > Berberis thunbergii cv. atropurpurea > Taxus cuspidate cv. nana > Euonymus japonicus. Chamber simulation and outdoor observation showed that Buxus microphylla and Berberis thunbergii cv. atropurpurea had strong ability to adsorb and arrest PM2.5; Meanwhile, the slight differences between the chamber simulation and outdoor observation results might be related to plant structure. Compared to tree species, the planting condition of shrub species was loose, and it greened quickly; By analyzing the Beijing PM2.5 concentration values in winter and spring, it was found that the PM2.5 concentration was particularly high in the winter of Beijing, and evergreen shrubs maintained the ability to adsorb and arrest PM2.5.

An inhibitor of polyamine synthesis arrests cells at an earlier stage of G1 than does calcium deprivation.

OpenAIRE

Cheetham, B F

1983-01-01

Methylglyoxal bis(guanylhydrazone) completely inhibits the induction of thymidine kinase after serum stimulation of quiescent fibroblasts only if added within 3 h after serum, whereas calcium deprivation blocks this induction up to 12 h after serum stimulation. Experiments in which one of these blocks was imposed as the other was released confirmed that cells blocked by methylglyoxal bis(guanylhydrazone) are arrested at an earlier stage in G1 than cells blocked by calcium deprivation.

Galiellalactone induces cell cycle arrest and apoptosis through the ATM/ATR pathway in prostate cancer cells.

Science.gov (United States)

García, Víctor; Lara-Chica, Maribel; Cantarero, Irene; Sterner, Olov; Calzado, Marco A; Muñoz, Eduardo

2016-01-26

Galiellalactone (GL) is a fungal metabolite that presents antitumor activities on prostate cancer in vitro and in vivo. In this study we show that GL induced cell cycle arrest in G2/M phase, caspase-dependent apoptosis and also affected the microtubule organization and migration ability in DU145 cells. GL did not induce double strand DNA break but activated the ATR and ATM-mediated DNA damage response (DDR) inducing CHK1, H2AX phosphorylation (fH2AX) and CDC25C downregulation. Inhibition of the ATM/ATR activation with caffeine reverted GL-induced G2/M cell cycle arrest, apoptosis and DNA damage measured by fH2AX. In contrast, UCN-01, a CHK1 inhibitor, prevented GL-induced cell cycle arrest but enhanced apoptosis in DU145 cells. Furthermore, we found that GL did not increase the levels of intracellular ROS, but the antioxidant N-acetylcysteine (NAC) completely prevented the effects of GL on fH2AX, G2/M cell cycle arrest and apoptosis. In contrast to NAC, other antioxidants such as ambroxol and EGCG did not interfere with the activity of GL on cell cycle. GL significantly suppressed DU145 xenograft growth in vivo and induced the expression of fH2AX in the tumors. These findings identify for the first time that GL activates DDR in prostate cancer.

2-Methoxy-4-vinylphenol can induce cell cycle arrest by blocking the hyper-phosphorylation of retinoblastoma protein in benzo[a]pyrene-treated NIH3T3 cells

International Nuclear Information System (INIS)

Jeong, Jin Boo; Jeong, Hyung Jin

2010-01-01

Research highlights: → 2M4VP activated the expression of p21 and p15 protein, and down-regulated the expression of cyclin D1 and cyclin E. → 2M4VP inhibited hyper-phosphorylation of Rb protein. → 2M4VP induced cell cycle arrest from G1 to S. → 2M4VP inhibited hyper-proliferation of the cells in BaP-treated cells. → 2M4VP induces growth arrest of BaP-treated cells by blocking hyper-phosphorylation of Rb via regulating the expression of cell cycle-related proteins. -- Abstract: Benzo[a]pyrene (BaP) is an environment carcinogen that can enhance cell proliferation by disturbing the signal transduction pathways in cell cycle regulation. In this study, the effects of 2M4VP on cell proliferation, cell cycle and cell cycle regulatory proteins were studied in BaP-treated NIH 3T3 cells to establish the molecular mechanisms of 2M4VP as anti-proliferative agents. 2M4VP exerted a dose-dependent inhibitory effect on cell growth correlated with a G1 arrest. Analysis of G1 cell cycle regulators expression revealed 2M4VP increased expression of CDK inhibitor, p21Waf1/Cip1 and p15 INK4b, decreased expression of cyclin D1 and cyclin E, and inhibited kinase activities of CDK4 and CDK2. However, 2M4VP did not affect the expression of CDK4 and CDK2. Also, 2M4VP inhibited the hyper-phosphorylation of Rb induced by BaP. Our results suggest that 2M4VP induce growth arrest of BaP-treated NIH 3T3 cells by blocking the hyper-phosphorylation of Rb via regulating the expression of cell cycle-related proteins.

2-Methoxy-4-vinylphenol can induce cell cycle arrest by blocking the hyper-phosphorylation of retinoblastoma protein in benzo[a]pyrene-treated NIH3T3 cells

Energy Technology Data Exchange (ETDEWEB)

Jeong, Jin Boo [Bioresource Sciences, Andong National University, Andong 760749 (Korea, Republic of); Jeong, Hyung Jin, E-mail: jhj@andong.ac.kr [Bioresource Sciences, Andong National University, Andong 760749 (Korea, Republic of)

2010-10-01

Research highlights: {yields} 2M4VP activated the expression of p21 and p15 protein, and down-regulated the expression of cyclin D1 and cyclin E. {yields} 2M4VP inhibited hyper-phosphorylation of Rb protein. {yields} 2M4VP induced cell cycle arrest from G1 to S. {yields} 2M4VP inhibited hyper-proliferation of the cells in BaP-treated cells. {yields} 2M4VP induces growth arrest of BaP-treated cells by blocking hyper-phosphorylation of Rb via regulating the expression of cell cycle-related proteins. -- Abstract: Benzo[a]pyrene (BaP) is an environment carcinogen that can enhance cell proliferation by disturbing the signal transduction pathways in cell cycle regulation. In this study, the effects of 2M4VP on cell proliferation, cell cycle and cell cycle regulatory proteins were studied in BaP-treated NIH 3T3 cells to establish the molecular mechanisms of 2M4VP as anti-proliferative agents. 2M4VP exerted a dose-dependent inhibitory effect on cell growth correlated with a G1 arrest. Analysis of G1 cell cycle regulators expression revealed 2M4VP increased expression of CDK inhibitor, p21Waf1/Cip1 and p15 INK4b, decreased expression of cyclin D1 and cyclin E, and inhibited kinase activities of CDK4 and CDK2. However, 2M4VP did not affect the expression of CDK4 and CDK2. Also, 2M4VP inhibited the hyper-phosphorylation of Rb induced by BaP. Our results suggest that 2M4VP induce growth arrest of BaP-treated NIH 3T3 cells by blocking the hyper-phosphorylation of Rb via regulating the expression of cell cycle-related proteins.

Methylselenol, a selenium metabolite, induces cell cycle arrest in G1 phase and apoptosis via the extracellular-regulated kinase 1/2 pathway and other cancer signaling genes.

Science.gov (United States)

Zeng, Huawei; Wu, Min; Botnen, James H

2009-09-01

Methylselenol has been hypothesized to be a critical selenium (Se) metabolite for anticancer activity in vivo, and our previous study demonstrated that submicromolar methylselenol generated by incubating methionase with seleno-l-methionine inhibits the migration and invasive potential of HT1080 tumor cells. However, little is known about the association between cancer signal pathways and methylselenol's inhibition of tumor cell invasion. In this study, we demonstrated that methylselenol exposure inhibited cell growth and we used a cancer signal pathway-specific array containing 15 different signal transduction pathways involved in oncogenesis to study the effect of methylselenol on cellular signaling. Using real-time RT-PCR, we confirmed that cellular mRNA levels of cyclin-dependent kinase inhibitor 1C (CDKN1C), heme oxygenase 1, platelet/endothelial cell adhesion molecule, and PPARgamma genes were upregulated to 2.8- to 5.7-fold of the control. BCL2-related protein A1, hedgehog interacting protein, and p53 target zinc finger protein genes were downregulated to 26-52% of the control, because of methylselenol exposure. These genes are directly related to the regulation of cell cycle and apoptosis. Methylselenol increased apoptotic cells up to 3.4-fold of the control and inhibited the extracellular-regulated kinase 1/2 (ERK1/2) signaling and cellular myelocytomatosis oncogene (c-Myc) expression. Taken together, our studies identify 7 novel methylselenol responsive genes and demonstrate that methylselenol inhibits ERK1/2 pathway activation and c-Myc expression. The regulation of these genes is likely to play a key role in G1 cell cycle arrest and apoptosis, which may contribute to the inhibition of tumor cell invasion.

Modulation of radiation-induced apoptosis and G2/M block in murine T-lymphoma cells

International Nuclear Information System (INIS)

Palayoor, S.T.; Macklis, R.M.; Bump, E.A.; Coleman, C.N.

1995-01-01

Radiation-induced apoptosis in lymphocyte-derived cell lines is characterized by endonucleolytic cleavage of cellular DNA within hours after radiation exposure. We have studied this phenomenon qualitatively (DNA gel electrophoresis) and quantitatively (diphenylamine reagent assay) in murine EL4 T-lymphoma cells exposed to 137 Cs γ irradiation. Fragmentation was discernible within 18-24 h after exposure. It increased with time and dose and reached a plateau after 8 Gy of γ radiation. We studied the effect of several pharmacological agents on the radiation-induced G 2 /M block and DNA fragmentation. The agents which reduced the radiation-induced G 2 /M-phase arrest (caffeine, theobromine, theophylline and 2-aminopurine) enhanced the degree of DNA fragmentation at 24 h. In contrast, the agents which sustained the radiation-induced G 2 /M-phase arrest (TPA, DBcAMP, IBMX and 3-aminobenzamide) inhibited the DNA fragmentation at 24 h. These studies on EL4 lymphoma cells are consistent with the hypothesis that cells with radiation-induced genetic damage are eliminated by apoptosis subsequent to a G 2 /M block. Furthermore, it may be possible to modulate the process of radiation-induced apoptosis in lymphoma cells with pharmacological agents that modify the radiation-induced G 2 /M block, and to use this effect in the treatment of patients with malignant disease. 59 refs., 7 figs

Natriuretic peptide receptor A inhibition suppresses gastric cancer development through reactive oxygen species-mediated G2/M cell cycle arrest and cell death.

Science.gov (United States)

Li, Zheng; Wang, Ji-Wei; Wang, Wei-Zhi; Zhi, Xiao-Fei; Zhang, Qun; Li, Bo-Wen; Wang, Lin-Jun; Xie, Kun-Ling; Tao, Jin-Qiu; Tang, Jie; Wei, Song; Zhu, Yi; Xu, Hao; Zhang, Dian-Cai; Yang, Li; Xu, Ze-Kuan

2016-10-01

Natriuretic peptide receptor A (NPRA), the major receptor for atrial natriuretic peptide (ANP), has been implicated in tumorigenesis; however, the role of ANP-NPRA signaling in the development of gastric cancer remains unclear. Immunohistochemical analyses indicated that NPRA expression was positively associated with gastric tumor size and cancer stage. NPRA inhibition by shRNA induced G2/M cell cycle arrest, cell death, and autophagy in gastric cancer cells, due to accumulation of reactive oxygen species (ROS). Either genetic or pharmacologic inhibition of autophagy led to caspase-dependent cell death. Therefore, autophagy induced by NPRA silencing may represent a cytoprotective mechanism. ROS accumulation activated c-Jun N-terminal kinase (JNK) and AMP-activated protein kinase (AMPK). ROS-mediated activation of JNK inhibited cell proliferation by disturbing cell cycle and decreased cell viability. In addition, AMPK activation promoted autophagy in NPRA-downregulated cancer cells. Overall, our results indicate that the inhibition of NPRA suppresses gastric cancer development and targeting NPRA may represent a promising strategy for the treatment of gastric cancer. Copyright © 2016 Elsevier Inc. All rights reserved.

P53-dependent upregulation of neutral sphingomyelinase-2: role in doxorubicin-induced growth arrest.

Science.gov (United States)

Shamseddine, A A; Clarke, C J; Carroll, B; Airola, M V; Mohammed, S; Rella, A; Obeid, L M; Hannun, Y A

2015-10-29

Neutral sphingomyelinase-2 (nSMase2) is a ceramide-generating enzyme that has been implicated in growth arrest, apoptosis and exosome secretion. Although previous studies have reported transcriptional upregulation of nSMase2 in response to daunorubicin, through Sp1 and Sp3 transcription factors, the role of the DNA damage pathway in regulating nSMase2 remains unclear. In this study, we show that doxorubicin induces a dose-dependent induction of nSMase2 mRNA and protein with concomitant increases in nSMase activity and ceramide levels. Upregulation of nSMase2 was dependent on ATR, Chk1 and p53, thus placing it downstream of the DNA damage pathway. Moreover, overexpression of p53 was sufficient to transcriptionally induce nSMase2, without the need for DNA damage. DNA-binding mutants as well as acetylation mutants of p53 were unable to induce nSMase2, suggesting a role of nSMase2 in growth arrest. Moreover, knockdown of nSMase2 prevented doxorubicin-induced growth arrest. Finally, p53-induced nSMase2 upregulation appears to occur via a novel transcription start site upstream of exon 3. These results identify nSMase2 as a novel p53 target gene, regulated by the DNA damage pathway to induce cell growth arrest.

Socioeconomic factors associated with outcome after cardiac arrest in patients under the age of 65.

Science.gov (United States)

Uray, Thomas; Mayr, Florian B; Fitzgibbon, James; Rittenberger, Jon C; Callaway, Clifton W; Drabek, Tomas; Fabio, Anthony; Angus, Derek C; Kochanek, Patrick M; Dezfulian, Cameron

2015-08-01

In a prior study of seven North American cities Pittsburgh had the highest crude rate of cardiac arrest deaths in patients 18 to 64 years of age, particularly in neighborhoods with lower socioeconomic status (SES). We hypothesized that lower SES, associated poor health behaviors (e.g., illicit drug use) and pre-existing comorbid conditions (grouped as socioeconomic factors [SE factors]) could affect the type and severity of cardiac arrest, thus outcomes. We retrospectively identified patients aged 18 to 64 years treated for in-hospital (IHCA) and out-of hospital arrest (OHCA) at two Pittsburgh hospitals between January 2010 and July 2012. We abstracted data on baseline demographics and arrest characteristics like place of residence, insurance and employment status. Favorable cerebral performance category [CPC] (1 or 2) was our primary outcome. We examined the associations between SE factors, cardiac arrest variables and outcome as well as post-resuscitation care. Among 415 subjects who met inclusion criteria, unfavorable CPC were more common in patients who were unemployed, had a history of drug abuse or hypertension. In OHCA, favorable CPC was more often associated with presentation with ventricular fibrillation/tachycardia (OR 3.53, 95% CI 1.43-8.74, p = 0.006) and less often associated with non-cardiovascular arrest etiology (OR 0.22, 95% CI 0.08-0.62, p = 0.004). We found strong associations between specific SE factors and arrest factors associated with outcome in OHCA patients only. Significant differences in post-resuscitation care existed based on injury severity, not on SES. SE factors strongly influence type and severity of OHCA but not IHCA resulting in an association with outcomes. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

An inhibitor of polyamine synthesis arrests cells at an earlier stage of G1 than does calcium deprivation.

Science.gov (United States)

Cheetham, B F

1983-01-01

Methylglyoxal bis(guanylhydrazone) completely inhibits the induction of thymidine kinase after serum stimulation of quiescent fibroblasts only if added within 3 h after serum, whereas calcium deprivation blocks this induction up to 12 h after serum stimulation. Experiments in which one of these blocks was imposed as the other was released confirmed that cells blocked by methylglyoxal bis(guanylhydrazone) are arrested at an earlier stage in G1 than cells blocked by calcium deprivation. PMID:6843551

Matrine induced G0/G1 arrest and apoptosis in human acute T-cell lymphoblastic leukemia (T-ALL cells

Directory of Open Access Journals (Sweden)

Aslı Tetik Vardarlı

2018-05-01

Full Text Available Matrine, a natural product extracted from the root of Sophora flavescens, is a promising alternative drug in different types of cancer. Here, we aimed to investigate the therapeutic effects and underlying molecular mechanisms of matrine on human acute lymphoblastic leukemia (ALL cell line, CCRF-CEM. Cell viability and IC50 values were determined by WST-1 cell cytotoxicity assay. Cell cycle distribution and apoptosis rates were analyzed by flow cytometry. Expression patterns of 44 selected miRNAs and 44 RNAs were analyzed by quantitative reverse transcription polymerase chain reaction (qRT-PCR using the Applied Biosystems 7500 Fast Real-Time PCR System. Matrine inhibited cell viability and induced apoptosis of CCRF-CEM cells in a dose-dependent manner. Cell cycle analysis demonstrated that matrine-treated CCRF-CEM cells significantly accumulated in the G0/G1 phase compared with the untreated control cells. hsa-miR-376b-3p (-37.09 fold, p = 0.008 and hsa-miR-106b-3p (-16.67 fold, p = 0.028 expressions were decreased, whereas IL6 (95.47 fold, p = 0.000011 and CDKN1A (140.03 fold, p = 0.000159 expressions were increased after matrine treatment. Our results suggest that the downregulation of hsa-miR-106b-3p leads to the upregulation of target p21 gene, CDKN1A, and plays a critical role in the cell cycle progression by arresting matrine-treated cells in the G0/G1 phase.

PRR11 regulates late-S to G2/M phase progression and induces premature chromatin condensation (PCC)

Energy Technology Data Exchange (ETDEWEB)

Zhang, Chundong; Zhang, Ying; Li, Yi; Zhu, Huifang; Wang, Yitao; Cai, Wei [Department of Biochemistry and Molecular Biology, Chongqing Medical University, Chongqing 400016 (China); Molecular Medicine and Cancer Research Center, Chongqing Medical University, Chongqing 400016 (China); Zhu, Jiang [Molecular Medicine and Cancer Research Center, Chongqing Medical University, Chongqing 400016 (China); Ozaki, Toshinori [Laboratory of DNA Damage Signaling, Chiba Cancer Center Research Institute, 666-2 Nitona, Chuohku, Chiba 260-8717 (Japan); Bu, Youquan, E-mail: buyqcn@aliyun.com [Department of Biochemistry and Molecular Biology, Chongqing Medical University, Chongqing 400016 (China); Molecular Medicine and Cancer Research Center, Chongqing Medical University, Chongqing 400016 (China)

2015-03-13

Recently, we have demonstrated that proline-rich protein 11 (PRR11) is a novel tumor-related gene product likely implicated in the regulation of cell cycle progression as well as lung cancer development. However, its precise role in cell cycle progression remains unclear. In the present study, we have further investigated the expression pattern and functional implication of PRR11 during cell cycle in detail in human lung carcinoma-derived H1299 cells. According to our immunofluorescence study, PRR11 was expressed largely in cytoplasm, the amount of PRR11 started to increase in the late S phase, and was retained until just before mitotic telophase. Consistent with those observations, siRNA-mediated knockdown of PRR11 caused a significant cell cycle arrest in the late S phase. Intriguingly, the treatment with dNTPs further augmented PRR11 silencing-mediated S phase arrest. Moreover, knockdown of PRR11 also resulted in a remarkable retardation of G2/M progression, and PRR11-knockdown cells subsequently underwent G2 phase cell cycle arrest accompanied by obvious mitotic defects such as multipolar spindles and multiple nuclei. In addition, forced expression of PRR11 promoted the premature Chromatin condensation (PCC), and then proliferation of PRR11-expressing cells was massively attenuated and induced apoptosis. Taken together, our current observations strongly suggest that PRR11, which is strictly regulated during cell cycle progression, plays a pivotal role in the regulation of accurate cell cycle progression through the late S phase to mitosis. - Highlights: • PRR11 started to increase in the late S phase and was retained until just before mitotic telophase. • PRR11-knockdown caused a significant cell cycle arrest in the late S phase and G2 phase. • The treatment with dNTPs further augmented PRR11 silencing-mediated S phase arrest. • PRR11-knockdown led to multipolar spindles and multiple nuclei. • Forced expression of PRR11 promoted the PCC and inhibited

PRR11 regulates late-S to G2/M phase progression and induces premature chromatin condensation (PCC)

International Nuclear Information System (INIS)

Zhang, Chundong; Zhang, Ying; Li, Yi; Zhu, Huifang; Wang, Yitao; Cai, Wei; Zhu, Jiang; Ozaki, Toshinori; Bu, Youquan

2015-01-01

Recently, we have demonstrated that proline-rich protein 11 (PRR11) is a novel tumor-related gene product likely implicated in the regulation of cell cycle progression as well as lung cancer development. However, its precise role in cell cycle progression remains unclear. In the present study, we have further investigated the expression pattern and functional implication of PRR11 during cell cycle in detail in human lung carcinoma-derived H1299 cells. According to our immunofluorescence study, PRR11 was expressed largely in cytoplasm, the amount of PRR11 started to increase in the late S phase, and was retained until just before mitotic telophase. Consistent with those observations, siRNA-mediated knockdown of PRR11 caused a significant cell cycle arrest in the late S phase. Intriguingly, the treatment with dNTPs further augmented PRR11 silencing-mediated S phase arrest. Moreover, knockdown of PRR11 also resulted in a remarkable retardation of G2/M progression, and PRR11-knockdown cells subsequently underwent G2 phase cell cycle arrest accompanied by obvious mitotic defects such as multipolar spindles and multiple nuclei. In addition, forced expression of PRR11 promoted the premature Chromatin condensation (PCC), and then proliferation of PRR11-expressing cells was massively attenuated and induced apoptosis. Taken together, our current observations strongly suggest that PRR11, which is strictly regulated during cell cycle progression, plays a pivotal role in the regulation of accurate cell cycle progression through the late S phase to mitosis. - Highlights: • PRR11 started to increase in the late S phase and was retained until just before mitotic telophase. • PRR11-knockdown caused a significant cell cycle arrest in the late S phase and G2 phase. • The treatment with dNTPs further augmented PRR11 silencing-mediated S phase arrest. • PRR11-knockdown led to multipolar spindles and multiple nuclei. • Forced expression of PRR11 promoted the PCC and inhibited

Benzylidene derivatives of andrographolide inhibit growth of breast and colon cancer cells in vitro by inducing G1 arrest and apoptosis

Science.gov (United States)

Jada, S R; Matthews, C; Saad, M S; Hamzah, A S; Lajis, N H; Stevens, M F G; Stanslas, J

2008-01-01

Background and purpose: Andrographolide, the major phytoconstituent of Andrographis paniculata, was previously shown by us to have activity against breast cancer. This led to synthesis of new andrographolide analogues to find compounds with better activity than the parent compound. Selected benzylidene derivatives were investigated for their mechanisms of action by studying their effects on the cell cycle progression and cell death. Experimental approach: Microculture tetrazolium, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) and sulphorhodamine B (SRB) assays were utilized in assessing the in vitro growth inhibition and cytotoxicity of compounds. Flow cytometry was used to analyse the cell cycle distribution of control and treated cells. CDK1 and CDK4 levels were determined by western blotting. Apoptotic cell death was assessed by fluorescence microscopy and flow cytometry. Key results: Compounds, in nanomolar to micromolar concentrations, exhibited growth inhibition and cytotoxicity in MCF-7 (breast) and HCT-116 (colon) cancer cells. In the NCI screen, 3,19-(2-bromobenzylidene) andrographolide (SRJ09) and 3,19-(3-chloro-4-fluorobenzylidene) andrographolide (SRJ23) showed greater cytotoxic potency and selectivity than andrographolide. SRJ09 and SRJ23 induced G1 arrest and apoptosis in MCF-7 and HCT-116 cells, respectively. SRJ09 downregulated CDK4 but not CDK1 level in MCF-7 cells. Apoptosis induced by SRJ09 and SRJ23 in HCT-116 cells was confirmed by annexin V-FITC/PI flow cytometry analysis. Conclusion and implications: The new benzylidene derivatives of andrographolide are potential anticancer agents. SRJ09 emerged as the lead compound in this study, exhibiting anticancer activity by downregulating CDK4 to promote a G1 phase cell cycle arrest, coupled with induction of apoptosis. PMID:18806812

Modulation of radiation-induced apoptosis and G{sub 2}/M block in murine T-lymphoma cells

Energy Technology Data Exchange (ETDEWEB)

Palayoor, S.T.; Macklis, R.M.; Bump, E.A.; Coleman, C.N. [Harvard Medical School, Boston, MA (United States)

1995-03-01

Radiation-induced apoptosis in lymphocyte-derived cell lines is characterized by endonucleolytic cleavage of cellular DNA within hours after radiation exposure. We have studied this phenomenon qualitatively (DNA gel electrophoresis) and quantitatively (diphenylamine reagent assay) in murine EL4 T-lymphoma cells exposed to {sup 137}Cs {gamma} irradiation. Fragmentation was discernible within 18-24 h after exposure. It increased with time and dose and reached a plateau after 8 Gy of {gamma} radiation. We studied the effect of several pharmacological agents on the radiation-induced G{sub 2}/M block and DNA fragmentation. The agents which reduced the radiation-induced G{sub 2}/M-phase arrest (caffeine, theobromine, theophylline and 2-aminopurine) enhanced the degree of DNA fragmentation at 24 h. In contrast, the agents which sustained the radiation-induced G{sub 2}/M-phase arrest (TPA, DBcAMP, IBMX and 3-aminobenzamide) inhibited the DNA fragmentation at 24 h. These studies on EL4 lymphoma cells are consistent with the hypothesis that cells with radiation-induced genetic damage are eliminated by apoptosis subsequent to a G{sub 2}/M block. Furthermore, it may be possible to modulate the process of radiation-induced apoptosis in lymphoma cells with pharmacological agents that modify the radiation-induced G{sub 2}/M block, and to use this effect in the treatment of patients with malignant disease. 59 refs., 7 figs.

Sulforaphane induces cell cycle arrest by protecting RB-E2F-1 complex in epithelial ovarian cancer cells

Directory of Open Access Journals (Sweden)

Morris Robert

2010-03-01

Full Text Available Abstract Background Sulforaphane (SFN, an isothiocyanate phytochemical present predominantly in cruciferous vegetables such as brussels sprout and broccoli, is considered a promising chemo-preventive agent against cancer. In-vitro exposure to SFN appears to result in the induction of apoptosis and cell-cycle arrest in a variety of tumor types. However, the molecular mechanisms leading to the inhibition of cell cycle progression by SFN are poorly understood in epithelial ovarian cancer cells (EOC. The aim of this study is to understand the signaling mechanisms through which SFN influences the cell growth and proliferation in EOC. Results SFN at concentrations of 5 - 20 μM induced a dose-dependent suppression of growth in cell lines MDAH 2774 and SkOV-3 with an IC50 of ~8 μM after a 3 day exposure. Combination treatment with chemotherapeutic agent, paclitaxel, resulted in additive growth suppression. SFN at ~8 μM decreased growth by 40% and 20% on day 1 in MDAH 2774 and SkOV-3, respectively. Cells treated with cytotoxic concentrations of SFN have reduced cell migration and increased apoptotic cell death via an increase in Bak/Bcl-2 ratio and cleavage of procaspase-9 and poly (ADP-ribose-polymerase (PARP. Gene expression profile analysis of cell cycle regulated proteins demonstrated increased levels of tumor suppressor retinoblastoma protein (RB and decreased levels of E2F-1 transcription factor. SFN treatment resulted in G1 cell cycle arrest through down modulation of RB phosphorylation and by protecting the RB-E2F-1 complex. Conclusions SFN induces growth arrest and apoptosis in EOC cells. Inhibition of retinoblastoma (RB phosphorylation and reduction in levels of free E2F-1 appear to play an important role in EOC growth arrest.

Study of nuclear heat application systems for arresting CO2 emission

International Nuclear Information System (INIS)

Fumizawa, Motoo; Inaba, Yoshitomo; Hishida, Makoto; Ogata, Kan; Yamada, Seiya.

1996-11-01

The objective of the paper is to investigate the systems for arresting CO 2 emission and for the effective utilization of fossil fuel. We studied the fossil fuel reforming systems to decrease the CO 2 emission rate per unit amount of heat generation by fossil fuel. Feed materials for reforming system were natural gas, crude oil, oil sand, oil shale and coal. Products by reforming were hydrogen, methane, methanol and gasoline. We examined CO 2 emission ratio of ten systems with different feed material and product. The CO 2 emission ratio was the ratio of CO 2 emission rate per unit amount of heat generation between the products and the feed materials, and was the important index. As the results, the CO 2 emission ratio for the coal to methane reforming system using steam gasifier had the lowest value of 51%. It means that the CO 2 emission rate of the product from the coal to methane reforming system was 51% of the emission rate of the feed material, that is, the system is very effective to arrest the CO 2 emission. The CO 2 emission ratio increases in the following order: the reforming systems from coal to methanol, heavy oil to hydrogen and natural gas to hydrogen. It was clarified that the system of coal to methane reforming was very effective for arresting CO 2 emission compared to the other systems, moreover the nuclear heat using rate and thermal efficiency of the plant of the system were the highest. (author)

Detection of FMD virus type specific IgG1, IgG2 and IgA antibodies in milk and serum of buffaloes vaccinated with oil adjuvanted polyvalent FMD vaccine

Directory of Open Access Journals (Sweden)

R. Sharma

2010-02-01

Full Text Available The present investigation was carried out on 15 randomly selected milch buffaloes divided into three groups on the basis of lactation at an organized farm, to study the foot and mouth disease virus type specific antibodies in milk and serum following FMD vaccination. Milk and serum samples collected before vaccination i.e. 0 day and on 7, 14, 28, 42 and 56 days post vaccination, were analyzed for the detection of FMD virus specific IgG1, IgG2 and IgA antibody response by indirect double antibody sandwich ELISA. Significant FMD virus type specific antibody titres (IgG1, IgG2 and IgA were detected in milk and serum of buffaloes on different days post vaccination, though the levels of antibodies were lower in milk as compared to serum. FMD virus type specific IgG1 was found to be the predominant subclass as compared to IgG2 and IgA both in milk and serum of vaccinated buffaloes. Milk and serum IgG1, IgG2 and IgA antibody titres were positively correlated with values of regression coefficient (R as 0.506, 0.434 and 0.396, respectively.

Galactosylated poly(ε-caprolactone) membrane promoted liver-specific functions of HepG2 cells in vitro

Energy Technology Data Exchange (ETDEWEB)

Zhang, Yan, E-mail: zhang_yan@ecust.edu.cn [The Key Laboratory for Ultrafine Materials of Ministry of Education, School of Materials Science and Engineering, East China University of Science and Technology, Shanghai 200237 (China); Zhang, Yi [The Key Laboratory for Ultrafine Materials of Ministry of Education, School of Materials Science and Engineering, East China University of Science and Technology, Shanghai 200237 (China); Chen, Min; Zhou, Yan [The State Key Laboratory of Bioreactor Engineering, School of Bioengineering, East China University of Science and Technology, Shanghai, 200237 (China); Lang, Meidong, E-mail: mdlang@ecust.edu.cn [The Key Laboratory for Ultrafine Materials of Ministry of Education, School of Materials Science and Engineering, East China University of Science and Technology, Shanghai 200237 (China)

2014-08-01

The lack of pendant functional groups on the PCL backbone has been a great challenge for surface bioactivation of poly(ε-caprolactone) (PCL). In the present study, covalently galactosylated PCL (GPCL) was developed through coupling between the amino-functionalized PCL (NPCL) and the lactobionic acid (LA) and its potential application in maintenance of physiological functions of HepG2 cells was further evaluated. The structure and properties of GPCL were explored by {sup 1}H NMR, FT-IR, GPC and DSC. Moreover, the incorporation of galactose ligands onto GPCL membranes not only promoted higher wettability, but also radically changed surface morphology in comparison with PCL and NPCL according to the contact angle measurement and atomic force microscopy. When HepG2 cells were seeded onto these membranes, the cells on GPCL membranes showed more pronounced cell adhesion and tended to form aggregates during the initial adhesion stage and then progressively grew into multi-layer structures compared to those without galactose ligands by the observation with fluorescence microscope and scanning electron microscopy. Furthermore, live–dead assay and functional tests demonstrated that HepG2 cells on GPCL membranes had superior viability and maintained better liver-specific functions. Collectively, GPCL has great potential for hepatic tissue engineering scaffolds. - Graphical abstract: The specific recognition between the galactose ligands on the galactosylated poly(ε-caprolactone) membrane and the ASGPR on the HepG2 cell surface. The galactosylated poly(ε-caprolactone) membranes improved the cell-matrix interaction. The galactosylated functionalized PCL scaffold is a potential candidate for liver tissue engineering. - Highlights: • The specific recognition between the galactose ligands on the galactosylated poly(ε-caprolactone) membrane and the ASGPR on the HepG2 cell surface. • The galactosylated poly(ε-caprolactone) membranes improved the cell-matrix interaction. �

Glycoprotein 5 of porcine reproductive and respiratory syndrome virus strain SD16 inhibits viral replication and causes G2/M cell cycle arrest, but does not induce cellular apoptosis in Marc-145 cells

International Nuclear Information System (INIS)

Mu, Yang; Li, Liangliang; Zhang, Beibei; Huang, Baicheng; Gao, Jiming

2015-01-01

Cell apoptosis is common after infection with porcine reproductive and respiratory syndrome virus (PRRSV). PRRSV GP5 has been reported to induce cell apoptosis. To further understand the role of GP5 in PRRSV induced cell apoptosis, we established Marc-145 cell lines stably expressing full-length GP5, GP5 Δ84-96 (aa 84-96 deletion), and GP5 Δ97-119 (aa 97-119 deletion). Cell proliferation, cell cycle progression, cell apoptosis and virus replication in these cell lines were evaluated. Neither truncated nor full-length GP5 induced cell apoptosis in Marc-145 cells. However, GP5 Δ97-119 , but not full-length or GP5 Δ84-96 , induced a cell cycle arrest at the G2/M phase resulting in a reduction in the growth of Marc-145 cells. Additionally, GP5 Δ84-96 inhibited the replication of PRRSV in Marc-145 cells through induction of IFN-β. These findings suggest that PRRSV GP5 is not responsible for inducing cell apoptosis in Marc-145 cells under these experimental conditions; however it has other important roles in virus/host cell biology. - Highlights: • Marc-145 cell lines stable expression PRRSV GP5 or truncated GP5 were constructed. • GP5 Δ97-119 expression in Marc-145 cell induced cell cycle arrest at G2/M phase. • Expression of GP5 and truncated GP5 could not induce Marc-145 cells apoptosis. • PRRSV replication in Marc-145-GP5 Δ84-96 was significantly inhibited

Monoclonal antibody Zt/g4 targeting RON receptor tyrosine kinase enhances chemosensitivity of bladder cancer cells to Epirubicin by promoting G1/S arrest and apoptosis.

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Chen, Jun-Feng; Yu, Bi-Xia; Yu, Rui; Ma, Liang; Lv, Xiu-Yi; Cheng, Yue; Ma, Qi

2017-02-01

Epirubicin (EPI) is one of the most used intravesical chemotherapy agents after transurethral resection to non-muscle invasive bladder tumors (NMIBC) to prevent cancer recurrence and progression. However, even after resection of bladder tumors and intravesical chemotherapy, half of them will recur and progress. RON is a membrane tyrosine kinase receptor usually overexpressed in bladder cancer cells and associated with poor pathological features. This study aims to investigate the effects of anti-RON monoclonal antibody Zt/g4 on the chemosensitivity of bladder cells to EPI. After Zt/g4 treatment, cell cytotoxicity was significantly increased and cell invasion was markedly suppressed in EPI-treated bladder cancer cells. Further investigation indicated that combing Zt/g4 with EPI promoted cell G1/S-phase arrest and apoptosis, which are the potential mechanisms that RON signaling inhibition enhances chemosensitivity of EPI. Thus, combing antibody-based RON targeted therapy enhances the therapeutic effects of intravesical chemotherapy, which provides new strategy for further improvement of NMIBC patient outcomes.

Mechanism for G2 phase-specific nuclear export of the kinetochore protein CENP-F.

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Loftus, Kyle M; Cui, Heying; Coutavas, Elias; King, David S; Ceravolo, Amanda; Pereiras, Dylan; Solmaz, Sozanne R

2017-08-03

Centromere protein F (CENP-F) is a component of the kinetochore and a regulator of cell cycle progression. CENP-F recruits the dynein transport machinery and orchestrates several cell cycle-specific transport events, including transport of the nucleus, mitochondria and chromosomes. A key regulatory step for several of these functions is likely the G2 phase-specific export of CENP-F from the nucleus to the cytosol, where the cytoplasmic dynein transport machinery resides; however, the molecular mechanism of this process is elusive. Here, we have identified 3 phosphorylation sites within the bipartite classical nuclear localization signal (cNLS) of CENP-F. These sites are specific for cyclin-dependent kinase 1 (Cdk1), which is active in G2 phase. Phosphomimetic mutations of these residues strongly diminish the interaction of the CENP-F cNLS with its nuclear transport receptor karyopherin α. These mutations also diminish nuclear localization of the CENP-F cNLS in cells. Notably, the cNLS is phosphorylated in the -1 position, which is important to orient the adjacent major motif for binding into its pocket on karyopherin α. We propose that localization of CENP-F is regulated by a cNLS, and a nuclear export pathway, resulting in nuclear localization during most of interphase. In G2 phase, the cNLS is weakened by phosphorylation through Cdk1, likely resulting in nuclear export of CENP-F via the still active nuclear export pathway. Once CENP-F resides in the cytosol, it can engage in pathways that are important for cell cycle progression, kinetochore assembly and the faithful segregation of chromosomes into daughter cells.

The role of the RACK1 ortholog Cpc2p in modulating pheromone-induced cell cycle arrest in fission yeast.

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Magdalena Mos

Full Text Available The detection and amplification of extracellular signals requires the involvement of multiple protein components. In mammalian cells the receptor of activated C kinase (RACK1 is an important scaffolding protein for signal transduction networks. Further, it also performs a critical function in regulating the cell cycle by modulating the G1/S transition. Many eukaryotic cells express RACK1 orthologs, with one example being Cpc2p in the fission yeast Schizosaccharomyces pombe. In contrast to RACK1, Cpc2p has been described to positively regulate, at the ribosomal level, cells entry into M phase. In addition, Cpc2p controls the stress response pathways through an interaction with Msa2p, and sexual development by modulating Ran1p/Pat1p. Here we describe investigations into the role, which Cpc2p performs in controlling the G protein-mediated mating response pathway. Despite structural similarity to Gβ-like subunits, Cpc2p appears not to function at the G protein level. However, upon pheromone stimulation, cells overexpressing Cpc2p display substantial cell morphology defects, disorientation of septum formation and a significantly protracted G1 arrest. Cpc2p has the potential to function at multiple positions within the pheromone response pathway. We provide a mechanistic interpretation of this novel data by linking Cpc2p function, during the mating response, with its previous described interactions with Ran1p/Pat1p. We suggest that overexpressing Cpc2p prolongs the stimulated state of pheromone-induced cells by increasing ste11 gene expression. These data indicate that Cpc2p regulates the pheromone-induced cell cycle arrest in fission yeast by delaying cells entry into S phase.

NBPF1, a tumor suppressor candidate in neuroblastoma, exerts growth inhibitory effects by inducing a G1 cell cycle arrest

International Nuclear Information System (INIS)

Andries, Vanessa; Vandepoele, Karl; Staes, Katrien; Berx, Geert; Bogaert, Pieter; Van Isterdael, Gert; Ginneberge, Daisy; Parthoens, Eef; Vandenbussche, Jonathan; Gevaert, Kris; Roy, Frans van

2015-01-01

NBPF1 (Neuroblastoma Breakpoint Family, member 1) was originally identified in a neuroblastoma patient on the basis of its disruption by a chromosomal translocation t(1;17)(p36.2;q11.2). Considering this genetic defect and the frequent genomic alterations of the NBPF1 locus in several cancer types, we hypothesized that NBPF1 is a tumor suppressor. Decreased expression of NBPF1 in neuroblastoma cell lines with loss of 1p36 heterozygosity and the marked decrease of anchorage-independent clonal growth of DLD1 colorectal carcinoma cells with induced NBPF1 expression further suggest that NBPF1 functions as tumor suppressor. However, little is known about the mechanisms involved. Expression of NBPF was analyzed in human skin and human cervix by immunohistochemistry. The effects of NBPF1 on the cell cycle were evaluated by flow cytometry. We investigated by real-time quantitative RT-PCR the expression profile of a panel of genes important in cell cycle regulation. Protein levels of CDKN1A-encoded p21 CIP1/WAF1 were determined by western blotting and the importance of p53 was shown by immunofluorescence and by a loss-of-function approach. LC-MS/MS analysis was used to investigate the proteome of DLD1 colon cancer cells with induced NBPF1 expression. Possible biological interactions between the differentially regulated proteins were investigated with the Ingenuity Pathway Analysis tool. We show that NBPF is expressed in the non-proliferative suprabasal layers of squamous stratified epithelia of human skin and cervix. Forced expression of NBPF1 in HEK293T cells resulted in a G1 cell cycle arrest that was accompanied by upregulation of the cyclin-dependent kinase inhibitor p21 CIP1/WAF1 in a p53-dependent manner. Additionally, forced expression of NBPF1 in two p53-mutant neuroblastoma cell lines also resulted in a G1 cell cycle arrest and CDKN1A upregulation. However, CDKN1A upregulation by NBPF1 was not observed in the DLD1 cells, which demonstrates that NBPF1 exerts cell-specific

Structure of human ubiquitin-conjugating enzyme E2 G2 (UBE2G2/UBC7)

International Nuclear Information System (INIS)

Arai, Ryoichi; Yoshikawa, Seiko; Murayama, Kazutaka; Imai, Yuzuru; Takahashi, Ryosuke; Shirouzu, Mikako; Yokoyama, Shigeyuki

2006-01-01

The crystal structure of human UBE2G2/UBC7 was solved at 2.56 Å resolution. The superimposition of UBE2G2 on UbcH7 in a c-Cbl–UbcH7–ZAP70 ternary complex suggested that the two loop regions of UBE2G2 interact with the RING domain in a similar way as UbcH7. The human ubiquitin-conjugating enzyme E2 G2 (UBE2G2/UBC7) is involved in protein degradation, including a process known as endoplasmic reticulum-associated degradation (ERAD). The crystal structure of human UBE2G2/UBC7 was solved at 2.56 Å resolution. The UBE2G2 structure comprises a single domain consisting of an antiparallel β-sheet with four strands, five α-helices and two 3 10 -helices. Structural comparison of human UBE2G2 with yeast Ubc7 indicated that the overall structures are similar except for the long loop region and the C-terminal helix. Superimposition of UBE2G2 on UbcH7 in a c-Cbl–UbcH7–ZAP70 ternary complex suggested that the two loop regions of UBE2G2 interact with the RING domain in a similar way to UbcH7. In addition, the extra loop region of UBE2G2 may interact with the RING domain or its neighbouring region and may be involved in the binding specificity and stability

Runway Arrested Landing Site (RALS)

Data.gov (United States)

Federal Laboratory Consortium — The Runway Arrested Landing Site includes an underground complex located on a Mod 2, Mod 3, and Mod 3+ arresting gear and are located under the runway and accurately...

Effects of gamma-radiation on cell growth, cycle arrest, death, and superoxide dismutase expression by DU 145 human prostate cancer cells

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Vucic V.

2006-01-01

Full Text Available Gamma-irradiation (gamma-IR is extensively used in the treatment of hormone-resistant prostate carcinoma. The objective of the present study was to investigate the effects of 60Co gamma-IR on the growth, cell cycle arrest and cell death of the human prostate cancer cell line DU 145. The viability of DU 145 cells was measured by the Trypan blue exclusion assay and the 3(4,5-dimethylthiazol-2-yl-2,5,diphenyltetrazolium bromide test. Bromodeoxyuridine incorporation was used for the determination of cell proliferation. Cell cycle arrest and cell death were analyzed by flow cytometry. Superoxide dismutase (SOD, specifically CuZnSOD and MnSOD protein expression, after 10 Gy gamma-IR, was determined by Western immunoblotting analysis. gamma-IR treatment had a significant (P < 0.001 antiproliferative and cytotoxic effect on DU 145 cells. Both effects were time and dose dependent. Also, the dose of gamma-IR which inhibited DNA synthesis and cell proliferation by 50% was 9.7 Gy. Furthermore, gamma-IR induced cell cycle arrest in the G2/M phase and the percentage of cells in the G2/M phase was increased from 15% (control to 49% (IR cells, with a nonsignificant induction of apoptosis. Treatment with 10 Gy gamma-IR for 24, 48, and 72 h stimulated CuZnSOD and MnSOD protein expression in a time-dependent manner, approximately by 3- to 3.5-fold. These data suggest that CuZnSOD and MnSOD enzymes may play an important role in the gamma-IR-induced changes in DU 145 cell growth, cell cycle arrest and cell death.

Evidence for a second 'Prereplicative G2' repair mechanism, specific for γ-induced damage, in wild-type schizosaccharomyces pombe

International Nuclear Information System (INIS)

Gentner, N.E.; Atomic Energy of Canada Ltd., Chalk River, Ontario. Chalk River Nuclear Labs.)

1977-01-01

The major part of the substantial γ-resistance of wild-type Schizosaccharomyces pombe appears to be due to prereplicative recombinational repair mechanisms. The existence of a second 'prereplicative G2' repair pathway, specific for γ-induced damage, has now been deduced from studies of the effect of the repair inhibitor caffeine on γ-irradiated G1 phase and G2 phase cells. Only G2 cells are additionally inactivated on exposure to caffeine after γ-irradiation. This shows that both known caffeine-sensitive γ-repair processes (Genter and Werner, Molec. gen. Genet. 145, 1-5 [1976]) are dependent on the presence of a duplicated genome (2c) at the time of radiation exposure. Pathway I is the known 'prereplicative G2' repair process (Fabre, Radiation Res. 56, 528-539 [1973]) which is involved in both UV- and γ-repair, and which requires post-irradiation protein synthesis for activity. Pathway II represents a second distinct 'prereplicative G2' repair mechanism; it differs from the first in that it is specific for repair of γ-induced damage and appears to be constitutive. (orig.) [de

The Forkhead Transcription Factor FOXP2 Is Required for Regulation of p21WAF1/CIP1 in 143B Osteosarcoma Cell Growth Arrest.

Science.gov (United States)

Gascoyne, Duncan M; Spearman, Hayley; Lyne, Linden; Puliyadi, Rathi; Perez-Alcantara, Marta; Coulton, Les; Fisher, Simon E; Croucher, Peter I; Banham, Alison H

2015-01-01

Mutations of the forkhead transcription factor FOXP2 gene have been implicated in inherited speech-and-language disorders, and specific Foxp2 expression patterns in neuronal populations and neuronal phenotypes arising from Foxp2 disruption have been described. However, molecular functions of FOXP2 are not completely understood. Here we report a requirement for FOXP2 in growth arrest of the osteosarcoma cell line 143B. We observed endogenous expression of this transcription factor both transiently in normally developing murine osteoblasts and constitutively in human SAOS-2 osteosarcoma cells blocked in early osteoblast development. Critically, we demonstrate that in 143B osteosarcoma cells with minimal endogenous expression, FOXP2 induced by growth arrest is required for up-regulation of p21WAF1/CIP1. Upon growth factor withdrawal, FOXP2 induction occurs rapidly and precedes p21WAF1/CIP1 activation. Additionally, FOXP2 expression could be induced by MAPK pathway inhibition in growth-arrested 143B cells, but not in traditional cell line models of osteoblast differentiation (MG-63, C2C12, MC3T3-E1). Our data are consistent with a model in which transient upregulation of Foxp2 in pre-osteoblast mesenchymal cells regulates a p21-dependent growth arrest checkpoint, which may have implications for normal mesenchymal and osteosarcoma biology.

Gender-Specific Effect of -102G>A Polymorphism in Insulin Induced Gene 2 on Obesity in Chinese Children

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Fang-Hong Liu

2015-01-01

Full Text Available Background. Insulin induced gene 2 (INSIG2 encodes a protein that has a biological effect on regulation of adipocyte metabolism and body weight. This study aimed to investigate the association of INSIG2 gene -102G>A polymorphism with obesity related phenotypes in Chinese children and test gender-specific effects. Methods. The 2,030 independent individuals aged from 7 to 18 years, including 705 obese cases and 1,325 nonobese controls, were recruited from local schools. We measured the obesity-related phenotypes and detected the serum lipids. We genotype -102G>A polymorphism by using the matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS. Results. In all individuals, we found that the GG/GA genotype of INSIG2 -102G>A polymorphism was associated with risk of severe obesity (OR = 1.62, 95% CI: 1.11–2.36, and P=0.012 under the dominant model. The association with severe obesity existed only in boys (OR = 1.91, 95% CI: 1.15–3.17, P=0.012. The GG/GA genotype of -102G>A polymorphism was also associated with higher waist circumference (β=2.61 cm, P=0.031 in boys. No similar association was found in girls. The polymorphism was not associated with other obesity-related phenotypes, neither in all individuals nor in gender-specific population. Conclusions. This study identified a gender-specific effect of INSIG2 -102G>A polymorphism on risk of severe obesity and waist circumference in Chinese boys.

Age-specific prognostication after out-of-hospital cardiac arrest - The ethical dilemma between 'life-sustaining treatment' and 'the right to die' in the elderly.

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Sulzgruber, Patrick; Sterz, Fritz; Poppe, Michael; Schober, Andreas; Lobmeyr, Elisabeth; Datler, Philip; Keferböck, Markus; Zeiner, Sebastian; Nürnberger, Alexander; Hubner, Pia; Stratil, Peter; Wallmueller, Christian; Weiser, Christoph; Warenits, Alexandra-Maria; van Tulder, Raphael; Zajicek, Andreas; Buchinger, Angelika; Testori, Christoph

2017-03-01

While prognostic values on survival after out-of-hospital cardiac arrest have been well investigated, less attention has been paid to their age-specific relevance. Therefore, we aimed to identify suitable age-specific early prognostication in elderly patients suffering out-of-hospital cardiac arrest in order to reduce the burden of unnecessary treatment and harm. In a prospective population-based observational trial on individuals suffering out-of-hospital cardiac arrest, a total of 2223 patients receiving resuscitation attempts by the local emergency medical service in Vienna, Austria, were enrolled. Patients were stratified according to age as follows: young and middle-aged individuals (85 years). There was an increasing rate of 30-day mortality (+21.8%, p 85-year-olds. Frailty was directly associated with mortality (HR 1.22, 95% CI 1.01-1.51, p = 0.049), showing a 30-day survival of 5.6% and a favourable neurological outcome of 1.1% among elderly individuals. An initially shockable electrocardiogram proved to be a suitable tool for risk assessment and decision making in order to predict a successful outcome in elderly victims of out-of-hospital cardiac arrest. However, the outcomes of elderly patients seemed to be exceptionally poor in frail individuals and need to be considered in order to reduce unnecessary treatment decisions.

Growth Arrest-Specific 6 (Gas6) and TAM Receptors in Mouse Platelets.

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Uras, Fikriye; Küçük, Burhanettin; Bingöl Özakpınar, Özlem; Demir, Ahmet Muzaffer

2015-03-05

Growth arrest-specific 6 (Gas6) is a newly discovered vitamin K-dependent protein, which is a ligand for TAM receptors [Tyro3 (Sky), Axl, and Mer] from the tyrosine kinase family. Gas6 knockout mice were resistant to venous and arterial thrombosis. There are contradictory reports on the presence of Gas6 and its receptors in mouse platelets. The objective of this study was to investigate whether Gas6 and its receptors were present in mouse platelets or not. Specific pathogen-free BALB/c male and female mice of 8-10 weeks old and 25-30 g in weight were anesthetized under light ether anesthesia and blood samples were taken from their hearts. RNAs were isolated from isolated platelets, and then mRNAs encoding Gas6 and TAM receptors were detected by reverse transcription-polymerase chain reaction (RT-PCR). Protein concentrations of Gas6 and TAM receptors in platelets were measured by ELISA, but not those of Mer, because of the absence of any commercial ELISA kit for mouse specimens. RT-PCR results indicated the presence of mRNAs encoding Gas6 and Mer in mouse platelets. However, although RT-PCR reactions were performed at various temperatures and cycles, we could not detect the presence of mRNAs encoding Axl and Tyro3 (Sky). Receptor protein levels of Axl and Tyro3 were below the detection limits of the ELISA method. We found the presence of mRNAs encoding Gas6 and the receptor Mer in mouse platelets, but not Axl and Tyro3. Gas6, Axl, and Tyro3 protein levels were below the detection limits of the ELISA. The presence of mRNA is not obvious evidence of protein expression in platelets that have no nucleus or DNA. Further studies are required to clarify the presence of Gas6/TAM receptors in platelets using real-time PCR and more sensitive immunological methods, and future studies on mechanisms will indicate whether the Gas6/TAM pathway is a strategy for treatment of disorders.

Association of Ambient Fine Particles With Out-of-Hospital Cardiac Arrests in New York City

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Silverman, Robert A.; Ito, Kazuhiko; Freese, John; Kaufman, Brad J.; De Claro, Danilynn; Braun, James; Prezant, David J.

2010-01-01

Cardiovascular morbidity has been associated with particulate matter (PM) air pollution, although the relation between pollutants and sudden death from cardiac arrest has not been established. This study examined associations between out-of-hospital cardiac arrests and fine PM (of aerodynamic diameter ≤2.5 μm, or PM2.5), ozone, nitrogen dioxide, sulfur dioxide, and carbon monoxide in New York City. The authors analyzed 8,216 out-of-hospital cardiac arrests of primary cardiac etiology during the years 2002–2006. Time-series and case-crossover analyses were conducted, controlling for season, day-of-week, same-day, and delayed/apparent temperature. An increased risk of cardiac arrest in time-series (relative risk (RR) = 1.06, 95% confidence interval (CI): 1.02, 1.10) and case-crossover (RR = 1.04, 95% CI: 0.99, 1.08) analysis for a PM2.5 increase of 10 μg/m3 in the average of 0- and 1-day lags was found. The association was significant in the warm season (RR = 1.09, 95% CI: 1.03, 1.15) but not the cold season (RR = 1.01, 95% CI: 0.95, 1.07). Associations of cardiac arrest with other pollutants were weaker. These findings, consistent with studies implicating acute cardiovascular effects of PM, support a link between PM2.5 and out-of-hospital cardiac arrests. Since few individuals survive an arrest, air pollution control may help prevent future cardiovascular mortality. PMID:20729350

Effect of caffeine on γ-ray induced G2 delay in ataxia telangiectasia

International Nuclear Information System (INIS)

Bates, P.R.; Lavin, M.F.

1985-01-01

Exposure of normal control and ataxia-telangiectasia (A-T) lymphoblastoid cell lines to ionizing radiation gives rise to an increase in the proportion of G2 phase cells. The size and extent of the G2 phase block is greater in A-T cells than in normal cells. Caffeine has a similar overall effect in control and A-T cell lines in reducing the G2 arrest observed after ionizing radiation. While the proportion of cells accumulated in G2 in A-T cells is considerably greater than in controls, addition of caffeine at the time of maximal G2 block brings about a return of G2 phase cell numbers to unirradiated values in 3 hours in both cell types. In normal control cells the caffeine-mediated decrease in G2 cells is reflected by an increase in mitotic cells. These mitotic cells have a higher frequency of chromosome aberrations compared to cells harvested in the absence of caffeine. Similarly in A-T cells addition of caffeine to irradiated cultures, delayed in G2 phase, increased the number of mitotic cells and the frequency of chromosome aberrations. (author)

Indirect semiquantitative determination of p34cdc2 levels in G1 and G2 cells of the carbohydrate-starved root meristems in Vicia faba var. minor

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Justyna Polit

2014-01-01

Full Text Available In eukaryotes, the 34kDa kinase (p34 encoded by the cdc2 gene is a key regulator of both the onset of DNA synthesis (G1 to S phase transition and the onset of mitosis (G1 to M phase transition. Using mouse anti-human PSTAIRE and FITClabelled goat antibodies, indirect semiquantitative determination of p34cdc2 levels was performed in meristematic cells from the control (intact and excised, carbohydrate-starved main roots of Vicia faba var. minor. No evident differences in the intensity of fluorescence was found either between the G1 and G2 cells or between the control cells and the cells arrested at both Principal Control Points by carbohydrate starvation. It seems thus, that the cell cycle block induced in meristematic cells of V. faba var. minor is not correlated with the absolute level of the key cell cycle enzyme responsible for phosphory-lution of cellular proteins, but primarily with the altered activity of p34cdc2.

Indole-3-carbinol induces G1 cell cycle arrest and apoptosis through aryl hydrocarbon receptor in THP-1 monocytic cell line.

Science.gov (United States)

Mohammadi, Saeed; Seyedhosseini, Fakhri Sadat; Behnampour, Nasser; Yazdani, Yaghoub

2017-10-01

The role of aryl hydrocarbon receptor (AhR) in carcinogenesis has been studied recently. Indole-3-carbinol (I3C) is an AhR agonist and a potential anticancer agent. Here, we investigated the effects of I3C on cell cycle progression and apoptosis through activation of AhR on THP-1 acute myeloid leukemia (AML) cell line. MTT viability assay was used to measure the cytotoxic effects of I3C on THP-1 cells. Apoptosis and cell cycle assays were investigated using flow cytometry. Real time RT-PCR was conducted to measure the alterations in the expression of AhR gene, key genes associated with AhR activation (IL1β and CYP1A1) and major genes involved in cell cycle regulation and apoptosis including P27, P21, CDK2, P53, BCL2 and FasR. Our findings revealed that I3C inhibits the proliferation of THP-1 cells in a dose- and time-dependent manner with minimal toxicity over normal monocytes. The AhR target genes (CYP1A1, IL1β) were overexpressed upon I3C treatment (p cycle arrest was also observed using flow cytometry. G1-acting cell cycle genes (P21, P27 and P53) were overexpressed (p cycle arrest in a dose- and time-dependent manner. Therefore, AhR could be targeted as a novel treatment possibility in AML.

Inhibition of DNA methyltransferase induces G2 cell cycle arrest and apoptosis in human colorectal cancer cells via inhibition of JAK2/STAT3/STAT5 signalling.

Science.gov (United States)

Xiong, Hua; Chen, Zhao-Fei; Liang, Qin-Chuan; Du, Wan; Chen, Hui-Min; Su, Wen-Yu; Chen, Guo-Qiang; Han, Ze-Guang; Fang, Jing-Yuan

2009-09-01

DNA methyltransferase inhibitors (MTIs) have recently emerged as promising chemotherapeutic or preventive agents for cancer, despite their poorly characterized mechanisms of action. The present study shows that DNA methylation is integral to the regulation of SH2-containing protein tyrosine phosphatase 1 (SHP1) expression, but not for regulation of suppressors of cytokine signalling (SOCS)1 or SOCS3 in colorectal cancer (CRC) cells. SHP1 expression correlates with down-regulation of Janus kinase/signal transducers and activators of transcription (JAK2/STAT3/STAT5) signalling, which is mediated in part by tyrosine dephosphorylation events and modulation of the proteasome pathway. Up-regulation of SHP1 expression was achieved using a DNA MTI, 5-aza-2'-deoxycytidine (5-aza-dc), which also generated significant down-regulation of JAK2/STAT3/STAT5 signalling. We demonstrate that 5-aza-dc suppresses growth of CRC cells, and induces G2 cell cycle arrest and apoptosis through regulation of downstream targets of JAK2/STAT3/STAT5 signalling including Bcl-2, p16(ink4a), p21(waf1/cip1) and p27(kip1). Although 5-aza-dc did not significantly inhibit cell invasion, 5-aza-dc did down-regulate expression of focal adhesion kinase and vascular endothelial growth factor in CRC cells. Our results demonstrate that 5-aza-dc can induce SHP1 expression and inhibit JAK2/STAT3/STAT5 signalling. This study represents the first evidence towards establishing a mechanistic link between inhibition of JAK2/STAT3/STAT5 signalling and the anticancer action of 5-aza-dc in CRC cells that may lead to the use of MTIs as a therapeutic intervention for human colorectal cancer.

Multi-functional roles of a soldier-specific volatile as a worker arrestant, primer pheromone and an antimicrobial agent in a termite.

Science.gov (United States)

Mitaka, Yuki; Mori, Naoki; Matsuura, Kenji

2017-07-26

Division of labour in eusocial insects is characterized by efficient communication systems based on pheromones. Among such insects, termites have evolved specialized sterile defenders, called soldiers. Because they are incapable of feeding themselves, it has been suggested that soldiers are sustained by workers and emit the pheromone arresting workers. However, such a soldier pheromone has not been identified in any termite species, and the details of the soldier-worker interaction remain to be explored. Here, we identified a soldier-specific volatile sesquiterpene as a worker arrestant, which also acts as a primer pheromone regulating soldier differentiation and fungistatic agent in a termite Reticulitermes speratus Chemical analyses revealed that (-)- β -elemene is the major component of soldier extract, and its authentic standard exhibited arrestant activity to workers and inhibited the differentiation from workers to soldiers. This compound also showed fungistatic activity against entomopathogenic fungi. These suggest that (-)- β -elemene secreted by soldiers acts not only as a worker arrestant but also as one component of inhibitory primer pheromone and an anti-pathogenic agent. Our study provides novel evidence supporting the multi-functionality of termite soldier pheromone and provides new insights into the role of soldiers and the evolutionary mechanisms of pheromone compounds. © 2017 The Author(s).

Discrimination, arrest history, and major depressive disorder in the U.S. Black population.

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Anglin, Deidre M; Lighty, Quenesha; Yang, Lawrence H; Greenspoon, Michelle; Miles, Rashun J; Slonim, Tzachi; Isaac, Kathleen; Brown, Monique J

2014-09-30

Everyday discrimination contributes negatively to depressive symptomatology among Blacks in the US and being arrested could add to this depression. Using data from the National Survey on American Life, the present study determined the association between an arrest history and major depressive disorder (MDD), while accounting for discrimination among African Americans, US-born Afro-Caribbeans and first-generation Black immigrants. Findings from logistic regression analyses adjusted for discrimination suggested an arrest history is associated with 12-month MDD (Adjusted OR=1.47; 95% CI=1.02-2.10) and lifetime MDD (Adjusted OR=1.56 CI=1.17-2.09). Accounting for drug and alcohol dependence attenuated the association between arrest history and 12-month MDD, but not lifetime MDD. The associations between arrest history and both 12-month and lifetime MDD, and discrimination and lifetime MDD varied by ethnic/immigrant group. Specifically, while the association between arrest history and MDD (both 12-month and lifetime) was strongest among US-born Afro-Caribbeans, evidence consistent with the immigrant paradox, the association between discrimination and lifetime MDD was particularly relevant for first-generation Black immigrants, suggesting discrimination may hinder the protection of first-generation status. Mental health prevention and treatment programs should target the stress associated with being arrested and experiencing discrimination among US Blacks. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

p27Kip1 Is Required to Mediate a G1 Cell Cycle Arrest Downstream of ATM following Genotoxic Stress.

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Erica K Cassimere

Full Text Available The DNA damage response (DDR is a coordinated signaling network that ensures the maintenance of genome stability under DNA damaging stress. In response to DNA lesions, activation of the DDR leads to the establishment of cell cycle checkpoints that delay cell-cycle progression and allow repair of the defects. The tumor suppressor p27Kip1 is a cyclin-CDK inhibitor that plays an important role in regulating quiescence in a variety of tissues. Several studies have suggested that p27Kip1 also plays a role in the maintenance of genomic integrity. Here we demonstrate that p27Kip1 is essential for the establishment of a G1 checkpoint arrest after DNA damage. We also uncovered that ATM phosphorylates p27Kip1 on a previously uncharacterized residue (Ser-140, which leads to its stabilization after induction of DNA double-strand breaks. Inhibition of this stabilization by replacing endogenous p27Kip1 with a Ser-140 phospho-mutant (S140A significantly sensitized cells to IR treatments. Our findings reveal a novel role for p27Kip1 in the DNA damage response pathway and suggest that part of its tumor suppressing functions relies in its ability to mediate a G1 arrest after the induction of DNA double strand breaks.

Chikusetsusaponin IVa methyl ester induces cell cycle arrest by the inhibition of nuclear translocation of β-catenin in HCT116 cells

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Lee, Kyung-Mi [Natural Products Research Institute, College of Pharmacy, Seoul National University, Seoul (Korea, Republic of); Yun, Ji Ho [Natural Products Research Center, Korea Institute of Science and Technology, Gangneung, 210-340 (Korea, Republic of); Lee, Dong Hwa [Department of Food Science and Nutrition, Andong National University, Andong 760-749 (Korea, Republic of); Park, Young Gyun [Natural Products Research Center, Korea Institute of Science and Technology, Gangneung, 210-340 (Korea, Republic of); Son, Kun Ho [Department of Food Science and Nutrition, Andong National University, Andong 760-749 (Korea, Republic of); Nho, Chu Won, E-mail: cwnho@kist.re.kr [Natural Products Research Center, Korea Institute of Science and Technology, Gangneung, 210-340 (Korea, Republic of); Kim, Yeong Shik, E-mail: kims@snu.ac.kr [Natural Products Research Institute, College of Pharmacy, Seoul National University, Seoul (Korea, Republic of)

2015-04-17

We demonstrate that chikusetsusaponin IVa methyl ester (CME), a triterpenoid saponin from the root of Achyranthes japonica, has an anticancer activity. We investigate its molecular mechanism in depth in HCT116 cells. CME reduces the amount of β-catenin in nucleus and inhibits the binding of β-catenin to specific DNA sequences (TCF binding elements, TBE) in target gene promoters. Thus, CME appears to decrease the expression of cell cycle regulatory proteins such as Cyclin D1, as a representative target for β-catenin, as well as CDK2 and CDK4. As a result of the decrease of the cell cycle regulatory proteins, CME inhibits cell proliferation by arresting the cell cycle at the G0/G1 phase. Therefore, we suggest that CME as a novel Wnt/β-catenin inhibitor can be a putative agent for the treatment of colorectal cancers. - Highlights: • CME inhibits cell proliferation in HCT116 cells. • CME increases cell cycle arrest at G0/G1 phase and apoptosis. • CME attenuates cyclin D1 and regulates cell cycle regulatory proteins. • CME inhibits β-catenin translocation to nucleus.

Chikusetsusaponin IVa methyl ester induces cell cycle arrest by the inhibition of nuclear translocation of β-catenin in HCT116 cells

International Nuclear Information System (INIS)

Lee, Kyung-Mi; Yun, Ji Ho; Lee, Dong Hwa; Park, Young Gyun; Son, Kun Ho; Nho, Chu Won; Kim, Yeong Shik

2015-01-01

We demonstrate that chikusetsusaponin IVa methyl ester (CME), a triterpenoid saponin from the root of Achyranthes japonica, has an anticancer activity. We investigate its molecular mechanism in depth in HCT116 cells. CME reduces the amount of β-catenin in nucleus and inhibits the binding of β-catenin to specific DNA sequences (TCF binding elements, TBE) in target gene promoters. Thus, CME appears to decrease the expression of cell cycle regulatory proteins such as Cyclin D1, as a representative target for β-catenin, as well as CDK2 and CDK4. As a result of the decrease of the cell cycle regulatory proteins, CME inhibits cell proliferation by arresting the cell cycle at the G0/G1 phase. Therefore, we suggest that CME as a novel Wnt/β-catenin inhibitor can be a putative agent for the treatment of colorectal cancers. - Highlights: • CME inhibits cell proliferation in HCT116 cells. • CME increases cell cycle arrest at G0/G1 phase and apoptosis. • CME attenuates cyclin D1 and regulates cell cycle regulatory proteins. • CME inhibits β-catenin translocation to nucleus

Effects on g2/m phase cell cycle distribution and aneuploidy formation of exposure to a 60 Hz electromagnetic field in combination with ionizing radiation or hydrogen peroxide in l132 nontumorigenic human lung epithelial cells.

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Jin, Hee; Yoon, Hye Eun; Lee, Jae-Seon; Kim, Jae-Kyung; Myung, Sung Ho; Lee, Yun-Sil

2015-03-01

The aim of the present study was to assess whether exposure to the combination of an extremely low frequency magnetic field (ELF-MF; 60 Hz, 1 mT or 2 mT) with a stress factor, such as ionizing radiation (IR) or H2O2, results in genomic instability in non-tumorigenic human lung epithelial L132 cells. To this end, the percentages of G2/M-arrested cells and aneuploid cells were examined. Exposure to 0.5 Gy IR or 0.05 mM H2O2 for 9 h resulted in the highest levels of aneuploidy; however, no cells were observed in the subG1 phase, which indicated the absence of apoptotic cell death. Exposure to an ELF-MF alone (1 mT or 2 mT) did not affect the percentages of G2/M-arrested cells, aneuploid cells, or the populations of cells in the subG1 phase. Moreover, when cells were exposed to a 1 mT or 2 mT ELF-MF in combination with IR (0.5 Gy) or H2O2 (0.05 mM), the ELF-MF did not further increase the percentages of G2/M-arrested cells or aneuploid cells. These results suggest that ELF-MFs alone do not induce either G2/M arrest or aneuploidy, even when administered in combination with different stressors.

A miR-21 inhibitor enhances apoptosis and reduces G2-M accumulation induced by ionizing radiation in human glioblastoma U251 cells

International Nuclear Information System (INIS)

Li, Yi; Li, Qiang; Asai, Akio; Kawamoto, Keiji; Zhao Shiguang; Zhen Yunbo; Teng Lei

2011-01-01

MicroRNAs (miRNAs) are small noncoding RNAs that take part in diverse biological processes by suppressing target gene expression. Elevated expression of miR-21 has been reported in many types of human cancers. Radiotherapy is a standard adjuvant treatment for patients with glioblastoma. However, the resistance of glioblastoma cells to radiation limits the success of this treatment. In this study, we found that miR-21 expression was upregulated in response to ionizing radiation (IR) in U251 cells, which suggested that miR-21 could be involved in the response of U251 cells to radiation. We showed that a miR-21 inhibitor enhanced IR-induced glioblastoma cell growth arrest and increased the level of apoptosis, which was probably caused by abrogation of the G 2 -M arrest induced by IR. Further research demonstrated that the miR-21 inhibitor induced the upregulation of Cdc25A. Taken together, these findings suggest that miR-21 inhibitor can increase IR-induced growth arrest and apoptosis in U251 glioblastoma cells, at least in part by abrogating G 2 -M arrest, and that Cdc25A is a potential target of miR-21. (author)

Characterization of mitochondrial injury after cardiac arrest (COMICA).

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Donnino, Michael W; Liu, Xiaowen; Andersen, Lars W; Rittenberger, Jon C; Abella, Benjamin S; Gaieski, David F; Ornato, Joseph P; Gazmuri, Raúl J; Grossestreuer, Anne V; Cocchi, Michael N; Abbate, Antonio; Uber, Amy; Clore, John; Peberdy, Mary Anne; Callaway, Clifton W

2017-04-01

Mitochondrial injury post-cardiac arrest has been described in pre-clinical settings but the extent to which this injury occurs in humans remains largely unknown. We hypothesized that increased levels of mitochondrial biomarkers would be associated with mortality and neurological morbidity in post-cardiac arrest subjects. We performed a prospective multicenter study of post-cardiac arrest subjects. Inclusion criteria were comatose adults who suffered an out-of-hospital cardiac arrest. Mitochondrial biomarkers were measured at 0, 12, 24, 36 and 48h after return of spontaneous circulation as well as in healthy controls. Out of 111 subjects enrolled, 102 had evaluable samples at 0h. Cardiac arrest subjects had higher baseline cytochrome c levels compared to controls (2.18ng/mL [0.74, 7.74] vs. 0.16ng/mL [0.03, 0.91], p<0.001), and subjects who died had higher 0h cytochrome c levels compared to survivors (3.66ng/mL [1.40, 14.9] vs. 1.27ng/mL [0.16, 2.37], p<0.001). There were significantly higher Ribonuclease P (RNaseP) (3.3 [1.2, 5.7] vs. 1.2 [0.8, 1.2], p<0.001) and Beta-2microglobulin (B2M) (12.0 [1.0, 22.9], vs. 0.6 [0.6, 1.3], p<0.001) levels in cardiac arrest subjects at baseline compared to the control subjects. There were no differences between survivors and non-survivors for mitochondrial DNA, nuclear DNA, or cell free DNA. Cytochrome c was increased in post- cardiac arrest subjects compared to controls, and in post-cardiac arrest non-survivors compared to survivors. Nuclear DNA and cell free DNA was increased in plasma of post-cardiac arrest subjects. There were no differences in mitochondrial DNA, nuclear DNA, or cell free DNA between survivors and non-survivors. Mitochondrial injury markers showed mixed results in the post-cardiac arrest period. Future research needs to investigate these differences. Copyright © 2017 Elsevier B.V. All rights reserved.

Same-Sex and Race-Based Disparities in Statutory Rape Arrests.

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Chaffin, Mark; Chenoweth, Stephanie; Letourneau, Elizabeth J

2016-01-01

This study tests a liberation hypothesis for statutory rape incidents, specifically that there may be same-sex and race/ethnicity arrest disparities among statutory rape incidents and that these will be greater among statutory rape than among forcible sex crime incidents. 26,726 reported incidents of statutory rape as defined under state statutes and 96,474 forcible sex crime incidents were extracted from National Incident-Based Reporting System data sets. Arrest outcomes were tested using multilevel modeling. Same-sex statutory rape pairings were rare but had much higher arrest odds. A victim-offender romantic relationship amplified arrest odds for same-sex pairings, but damped arrest odds for male-on-female pairings. Same-sex disparities were larger among statutory than among forcible incidents. Female-on-male incidents had uniformly lower arrest odds. Race/ethnicity effects were smaller than gender effects and more complexly patterned. The findings support the liberation hypothesis for same-sex statutory rape arrest disparities, particularly among same-sex romantic pairings. Support for race/ethnicity-based arrest disparities was limited and mixed. © The Author(s) 2014.

Sudden Cardiac Arrest during Participation in Competitive Sports.

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Landry, Cameron H; Allan, Katherine S; Connelly, Kim A; Cunningham, Kris; Morrison, Laurie J; Dorian, Paul

2017-11-16

The incidence of sudden cardiac arrest during participation in sports activities remains unknown. Preparticipation screening programs aimed at preventing sudden cardiac arrest during sports activities are thought to be able to identify at-risk athletes; however, the efficacy of these programs remains controversial. We sought to identify all sudden cardiac arrests that occurred during participation in sports activities within a specific region of Canada and to determine their causes. In this retrospective study, we used the Rescu Epistry cardiac arrest database (which contains records of every cardiac arrest attended by paramedics in the network region) to identify all out-of-hospital cardiac arrests that occurred from 2009 through 2014 in persons 12 to 45 years of age during participation in a sport. Cases were adjudicated as sudden cardiac arrest (i.e., having a cardiac cause) or as an event resulting from a noncardiac cause, on the basis of records from multiple sources, including ambulance call reports, autopsy reports, in-hospital data, and records of direct interviews with patients or family members. Over the course of 18.5 million person-years of observation, 74 sudden cardiac arrests occurred during participation in a sport; of these, 16 occurred during competitive sports and 58 occurred during noncompetitive sports. The incidence of sudden cardiac arrest during competitive sports was 0.76 cases per 100,000 athlete-years, with 43.8% of the athletes surviving until they were discharged from the hospital. Among the competitive athletes, two deaths were attributed to hypertrophic cardiomyopathy and none to arrhythmogenic right ventricular cardiomyopathy. Three cases of sudden cardiac arrest that occurred during participation in competitive sports were determined to have been potentially identifiable if the athletes had undergone preparticipation screening. In our study involving persons who had out-of-hospital cardiac arrest, the incidence of sudden cardiac

Menadione induces G2/M arrest in gastric cancer cells by down-regulation of CDC25C and proteasome mediated degradation of CDK1 and cyclin B1

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Lee, Min Ho; Cho, Yoonjung; Kim, Do Hyun; Woo, Hyun Jun; Yang, Ji Yeong; Kwon, Hye Jin; Yeon, Min Ji; Park, Min; Kim, Sa-Hyun; Moon, Cheol; Tharmalingam, Nagendran; Kim, Tae Ue; Kim, Jong-Bae

2016-01-01

Menadione (vitamin K3) has been reported to induce apoptotic cell death and growth inhibition in various types of cancer cells. However, involvement of menadione in cell cycle control has not been considered in gastric cancer cells yet. In the current study, we have investigated whether menadione is involved in the cell cycle regulation and suppression of growth in gastric cancer cells. In the cell cycle analysis, we found that menadione induced G2/M cell cycle arrest in AGS cells. To elucidate the underlying mechanism, we investigated the cell cycle regulatory molecules involved in the G2/M cell cycle transition. After 24 h of menadione treatment, the protein level of CDK1, CDC25C and cyclin B1 in AGS cells was decreased in a menadione dose-dependent manner. In the time course experiment, the protein level of CDC25C decreased in 6 h, and CDK1and cyclin B1 protein levels began to decrease after 18 h of menadione treatment. We found that mRNA level of CDC25C decreased by menadione treatment in 6 h. Menadione did not have an influence on mRNA level of CDK1 and cyclin B1 though the protein levels were decreased. However, the decreased protein levels of CDK1 and cyclin B1 were recovered by inhibition of proteasome. Collectively, these results suggest that menadione inhibits growth of gastric cancer cells by reducing expression of CDC25C and promoting proteasome mediated degradation of CDK1 and cyclin B1 thereby blocking transition of the cell cycle from G2 phase to M phase. PMID:28077999

Opposing roles of C/EBPbeta and AP-1 in the control of fibroblast proliferation and growth arrest-specific gene expression

DEFF Research Database (Denmark)

Gagliardi, Mark; Maynard, Scott; Miyake, Tetsuaki

2003-01-01

in the levels of AP-1 proteins. Therefore, C/EBPbeta is a negative regulator of AP-1 expression and activity in CEF. The expression of cyclin D1 and cell proliferation were stimulated by the dominant negative mutant of C/EBPbeta but not in the presence of TAM67, a dominant negative mutant of c-Jun and AP-1. CEF......Chicken embryo fibroblasts (CEF) express several growth arrest-specific (GAS) gene products in G0. In contact-inhibited cells, the expression of the most abundant of these proteins, the p20K lipocalin, is activated at the transcriptional level by C/EBPbeta. In this report, we describe the role of C....../EBPbeta in CEF proliferation. We show that the expression of a dominant negative mutant of C/EBPbeta (designated Delta184-C/EBPbeta) completely inhibited p20K expression at confluence and stimulated the proliferation of CEF without inducing transformation. Mouse embryo fibroblasts nullizygous for C/EBPbeta had...

High-dose irradiation induces cell cycle arrest, apoptosis, and developmental defects during Drosophila oogenesis.

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Hee Jin Shim

Full Text Available Ionizing radiation (IR treatment induces a DNA damage response, including cell cycle arrest, DNA repair, and apoptosis in metazoan somatic cells. Because little has been reported in germline cells, we performed a temporal analysis of the DNA damage response utilizing Drosophila oogenesis as a model system. Oogenesis in the adult Drosophila female begins with the generation of 16-cell cyst by four mitotic divisions of a cystoblast derived from the germline stem cells. We found that high-dose irradiation induced S and G2 arrests in these mitotically dividing germline cells in a grp/Chk1- and mnk/Chk2-dependent manner. However, the upstream kinase mei-41, Drosophila ATR ortholog, was required for the S-phase checkpoint but not for the G2 arrest. As in somatic cells, mnk/Chk2 and dp53 were required for the major cell death observed in early oogenesis when oocyte selection and meiotic recombination occurs. Similar to the unscheduled DNA double-strand breaks (DSBs generated from defective repair during meiotic recombination, IR-induced DSBs produced developmental defects affecting the spherical morphology of meiotic chromosomes and dorsal-ventral patterning. Moreover, various morphological abnormalities in the ovary were detected after irradiation. Most of the IR-induced defects observed in oogenesis were reversible and were restored between 24 and 96 h after irradiation. These defects in oogenesis severely reduced daily egg production and the hatch rate of the embryos of irradiated female. In summary, irradiated germline cells induced DSBs, cell cycle arrest, apoptosis, and developmental defects resulting in reduction of egg production and defective embryogenesis.

Characterization of Mitochondrial Injury after Cardiac Arrest (COMICA)

Science.gov (United States)

Donnino, Michael W.; Liu, Xiaowen; Andersen, Lars W.; Rittenberger, Jon C.; Abella, Benjamin S.; Gaieski, David F.; Ornato, Joseph P.; Gazmuri, Raúl J.; Grossestreur, Anne V.; Cocchi, Michaen N.; Abbate, Antonio; Uber, Amy; Clore, John; Peberdy, Mary Anne; Callaway, Clifton

2017-01-01

Introduction Mitochondrial injury post-cardiac arrest has been described in pre-clinical settings but the extent to which this injury occurs in humans remains largely unknown. We hypothesized that increased levels of mitochondrial biomarkers would be associated with mortality and neurological morbidity in post-cardiac arrest subjects. Methods We performed a prospective multicenter study of post-cardiac arrest subjects. Inclusion criteria were comatose adults who suffered an out-of-hospital cardiac arrest. Mitochondrial biomarkers were measured at 0, 12, 24, 36 and 48 hours after return of spontaneous circulation as well as in healthy controls. Results Out of 111 subjects enrolled, 102 had evaluable samples at 0 hours. Cardiac arrest subjects had higher baseline cytochrome c levels compared to controls (2.18 ng/mL [0.74, 7.74] vs. 0.16 ng/mL [0.03, 0.91], p<0.001), and subjects who died had higher 0 hours cytochrome c levels compared to survivors (3.66 ng/mL [1.40, 14.9] vs. 1.27 ng/mL [0.16, 2.37], p<0.001). There were significantly higher RNAase P (3.3 [1.2, 5.7] vs. 1.2 [0.8, 1.2], p<0.001) and B2M (12.0 [1.0, 22.9], vs. 0.6 [0.6, 1.3], p<0.001) levels in cardiac arrest subjects at baseline compared to the control subjects. There were no differences between survivors and non-survivors for mitochondrial DNA, nuclear DNA, or cell free DNA. Conclusions Cytochrome C was increased in post-cardiac arrest subjects compared to controls, and in post-cardiac arrest non-survivors compared to survivors. Nuclear DNA and cell free DNA was increased in plasma of post-cardiac arrest subjects. There were no differences in mitochondrial DNA, nuclear DNA, or cell free DNA between survivors and non-survivors. Mitochondrial injury markers showed mixed results in post-arrest period. Future research needs to investigate these differences. PMID:28126408

Induction of cell cycle arrest at G1 and S phases and cAMP-dependent differentiation in C6 glioma by low concentration of cycloheximide

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Zhang Samuel S

2010-12-01

Full Text Available Abstract Background Differentiation therapy has been shown effective in treatment of several types of cancer cells and may prove to be effective in treatment of glioblastoma multiforme, the most common and most aggressive primary brain tumor. Although extensively used as a reagent to inhibit protein synthesis in mammalian cells, whether cycloheximide treatment leads to glioma cell differentiation has not been reported. Methods C6 glioma cell was treated with or without cycloheximide at low concentrations (0.5-1 μg/ml for 1, 2 and 3 days. Cell proliferation rate was assessed by direct cell counting and colony formation assays. Apoptosis was assessed by Hoechst 33258 staining and FACS analysis. Changes in several cell cycle regulators such as Cyclins D1 and E, PCNA and Ki67, and several apoptosis-related regulators such as p53, p-JNK, p-AKT, and PARP were determined by Western blot analysis. C6 glioma differentiation was determined by morphological characterization, immunostaining and Western blot analysis on upregulation of GFAP and o p-STAT3 expression, and upregulation of intracellular cAMP. Results Treatment of C6 cell with low concentration of cycloheximide inhibited cell proliferation and depleted cells at both G2 and M phases, suggesting blockade at G1 and S phases. While no cell death was observed, cells underwent profound morphological transformation that indicated cell differentiation. Western blotting and immunostaining analyses further indicated that changes in expression of several cell cycle regulators and the differentiation marker GFAP were accompanied with cycloheximide-induced cell cycle arrest and cell differentiation. Increase in intracellular cAMP, a known promoter for C6 cell differentiation, was found to be elevated and required for cycloheximide-promoted C6 cell differentiation. Conclusion Our results suggest that partial inhibition of protein synthesis in C6 glioma by low concentration of cycloheximide induces cell cycle

Synthesis of an anthraquinone derivative (DHAQC) and its effect on induction of G2/M arrest and apoptosis in breast cancer MCF-7 cell line.

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Yeap, SweeKeong; Akhtar, Muhammad Nadeem; Lim, Kian Lam; Abu, Nadiah; Ho, Wan Yong; Zareen, Seema; Roohani, Kiarash; Ky, Huynh; Tan, Sheau Wei; Lajis, Nordin; Alitheen, Noorjahan Banu

2015-01-01

Anthraquinones are an important class of naturally occurring biologically active compounds. In this study, anthraquinone derivative 1,3-dihydroxy-9,10-anthraquinone-2- carboxylic acid (DHAQC) (2) was synthesized with 32% yield through the Friedel-Crafts condensation reaction. The mechanisms of cytotoxicity of DHAQC (2) in human breast cancer MCF-7 cells were further investigated. Results from the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay showed that DHAQC (2) exhibited potential cytotoxicity and selectivity in the MCF-7 cell line, comparable with the naturally occurring anthraquinone damnacanthal. DHAQC (2) showed a slightly higher IC50 (inhibitory concentration with 50% cell viability) value in the MCF-7 cell line compared to damnacanthal, but it is more selective in terms of the ratio of IC50 on MCF-7 cells and normal MCF-10A cells. (selective index for DHAQC (2) was 2.3 and 1.7 for damnacanthal). The flow cytometry cell cycle analysis on the MCF-7 cell line treated with the IC50 dose of DHAQC (2) for 48 hours showed that DHAQC (2) arrested MCF-7 cell line at the G2/M phase in association with an inhibited expression of PLK1 genes. Western blot analysis also indicated that the DHAQC (2) increased BAX, p53, and cytochrome c levels in MCF-7 cells, which subsequently activated apoptosis as observed in annexin V/propidium iodide and cell cycle analyses. These results indicate that DHAQC (2) is a synthetic, cytotoxic, and selective anthraquinone, which is less toxic than the natural product damnacanthal, and which demonstrates potential in the induction of apoptosis in the breast cancer MCF-7 cell line.

Calotropin from Asclepias curasavica induces cell cycle arrest and apoptosis in cisplatin-resistant lung cancer cells.

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Mo, En-Pan; Zhang, Rong-Rong; Xu, Jun; Zhang, Huan; Wang, Xiao-Xiong; Tan, Qiu-Tong; Liu, Fang-Lan; Jiang, Ren-Wang; Cai, Shao-Hui

2016-09-16

Calotropin (M11), an active compound isolated from Asclepias curasavica L., was found to exert strong inhibitory and pro-apoptotic activity specifically against cisplatin-induced resistant non-small cell lung cancer (NSCLC) cells (A549/CDDP). Molecular mechanism study revealed that M11 induced cell cycle arrest at the G2/M phase through down-regulating cyclins, CDK1, CDK2 and up-regulating p53 and p21. Furthermore, M11 accelerated apoptosis through the mitochondrial apoptotic pathway which was accompanied by increase Bax/Bcl-2 ratio, decrease in mitochondrial membrane potential, increase in reactive oxygen species production, activations of caspases 3 and 9 as well as cleavage of poly ADP-ribose polymerase (PARP). The activation and phosphorylation of JNK was also found to be involved in M11-induced apoptosis, and SP610025 (specific JNK inhibitor) partially prevented apoptosis induced by M11. In contrast, all of the effects that M11 induce cell cycle arrest and apoptosis in A549/CDDP cells were not significant in A549 cells. Drugs with higher sensitivity against resistant tumor cells than the parent cells are rather rare. Results of this study supported the potential application of M11 on the non-small lung cancer (NSCLC) with cisplatin resistance. Copyright © 2016 Elsevier Inc. All rights reserved.

Dendrobium candidum inhibits MCF-7 cells proliferation by inducing cell cycle arrest at G2/M phase and regulating key biomarkers

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Sun J

2015-12-01

<0.05. The general apoptosis biomarker, Bcl-2, was significantly decreased and the Bax was significantly increased compared to the control group (P<0.05. In contrast to that in MCF-7, D. candidum does not affect cell proliferation at any concentration and any time points in normal breast epithelial cells, MCF10A cells. Conclusion: D. candidum could decrease the cell viability of MCF-7 cells by inducing cell cycle arrest at the G2/M phase and regulating the key biomarkers in breast cancer cells. Keywords: breast cancer, D. candidum, proliferation, biomarker, inhibition

The transcription factor Swi4 is target for PKA regulation of cell size at the G1 to S transition in Saccharomyces cerevisiae.

Science.gov (United States)

Amigoni, Loredana; Colombo, Sonia; Belotti, Fiorella; Alberghina, Lilia; Martegani, Enzo

2015-08-03

To investigate the specific target of PKA in the regulation of cell cycle progression and cell size we developed a new approach using the yeast strain GG104 bearing a deletion in adenylate cyclase gene and permeable to cAMP ( cyr1Δ, pde2Δ, msn2Δ, msn4Δ). In this strain the PKA activity is absent and can be activated by addition of cAMP in the medium, without any other change of the growth conditions. In the present work we show that the activation of PKA by exogenous cAMP in the GG104 strain exponentially growing in glucose medium caused a marked increase of cell size and perturbation of cell cycle with a transient arrest of cells in G1, followed by an accumulation of cells in G2/M phase with a minimal change in the growth rate. Deletion of CLN1 gene, but not of CLN2, abolished the transient G1 phase arrest. Consistently we found that PKA activation caused a transcriptional repression of CLN1 gene. Transcription of CLN1 is controlled by SBF and MBF dual-regulated promoter. We found that also the deletion of SWI4 gene abolished the transient G1 arrest suggesting that Swi4 is a target responsible for PKA modulation of G1/S phase transition. We generated a SWI4 allele mutated in the consensus site for PKA (Swi4(S159A)) and we found that expression of Swi4(S159A) protein in the GG104-Swi4Δ strain did not restore the transient G1 arrest induced by PKA activation, suggesting that Swi4 phosphorylation by PKA regulates CLN1 gene expression and G1/S phase transition.

Gene-specific characterization of human histone H2B by electron capture dissociation.

Science.gov (United States)

Siuti, Nertila; Roth, Michael J; Mizzen, Craig A; Kelleher, Neil L; Pesavento, James J

2006-02-01

The basis set of protein forms expressed by human cells from the H2B gene family was determined by Top Down Mass Spectrometry. Using Electron Capture Dissociation for MS/MS of H2B isoforms, direct evidence for the expression of unmodified H2B.Q, H2B.A, H2B.K/T, H2B.J, H2B.E, H2B.B, H2B.F, and monoacetylated H2B.A was obtained from asynchronous HeLa cells. H2B.A was the most abundant form, with the overall expression profile not changing significantly in cells arrested in mitosis by colchicine or during mid-S, mid-G2, G2/M, and mid-G1 phases of the cell cycle. Modest hyperacetylation of H2B family members was observed after sodium butyrate treatment.

Prolonged early G1 arrest by selective CDK4/CDK6 inhibition sensitizes myeloma cells to cytotoxic killing through cell cycle–coupled loss of IRF4

Science.gov (United States)

Huang, Xiangao; Di Liberto, Maurizio; Jayabalan, David; Liang, Jun; Ely, Scott; Bretz, Jamieson; Shaffer, Arthur L.; Louie, Tracey; Chen, Isan; Randolph, Sophia; Hahn, William C.; Staudt, Louis M.; Niesvizky, Ruben; Moore, Malcolm A. S.

2012-01-01

Dysregulation of cyclin-dependent kinase 4 (CDK4) and CDK6 by gain of function or loss of inhibition is common in human cancer, including multiple myeloma, but success in targeting CDK with broad-spectrum inhibitors has been modest. By selective and reversible inhibition of CDK4/CDK6, we have developed a strategy to both inhibit proliferation and enhance cytotoxic killing of cancer cells. We show that induction of prolonged early-G1 arrest (pG1) by CDK4/CDK6 inhibition halts gene expression in early-G1 and prevents expression of genes programmed for other cell-cycle phases. Removal of the early-G1 block leads to S-phase synchronization (pG1-S) but fails to completely restore scheduled gene expression. Consequently, the IRF4 protein required to protect myeloma cells from apoptosis is markedly reduced in pG1 and further in pG1-S in response to cytotoxic agents, such as the proteasome inhibitor bortezomib. The coordinated loss of IRF4 and gain of Bim sensitize myeloma tumor cells to bortezomib-induced apoptosis in pG1 in the absence of Noxa and more profoundly in pG1-S in cooperation with Noxa in vitro. Induction of pG1 and pG1-S by reversible CDK4/CDK6 inhibition further augments tumor-specific bortezomib killing in myeloma xenografts. Reversible inhibition of CDK4/CDK6 in sequential combination therapy thus represents a novel mechanism-based cancer therapy. PMID:22718837

Prolonged early G(1) arrest by selective CDK4/CDK6 inhibition sensitizes myeloma cells to cytotoxic killing through cell cycle-coupled loss of IRF4.

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Huang, Xiangao; Di Liberto, Maurizio; Jayabalan, David; Liang, Jun; Ely, Scott; Bretz, Jamieson; Shaffer, Arthur L; Louie, Tracey; Chen, Isan; Randolph, Sophia; Hahn, William C; Staudt, Louis M; Niesvizky, Ruben; Moore, Malcolm A S; Chen-Kiang, Selina

2012-08-02

Dysregulation of cyclin-dependent kinase 4 (CDK4) and CDK6 by gain of function or loss of inhibition is common in human cancer, including multiple myeloma, but success in targeting CDK with broad-spectrum inhibitors has been modest. By selective and reversible inhibition of CDK4/CDK6, we have developed a strategy to both inhibit proliferation and enhance cytotoxic killing of cancer cells. We show that induction of prolonged early-G(1) arrest (pG1) by CDK4/CDK6 inhibition halts gene expression in early-G(1) and prevents expression of genes programmed for other cell-cycle phases. Removal of the early-G(1) block leads to S-phase synchronization (pG1-S) but fails to completely restore scheduled gene expression. Consequently, the IRF4 protein required to protect myeloma cells from apoptosis is markedly reduced in pG1 and further in pG1-S in response to cytotoxic agents, such as the proteasome inhibitor bortezomib. The coordinated loss of IRF4 and gain of Bim sensitize myeloma tumor cells to bortezomib-induced apoptosis in pG1 in the absence of Noxa and more profoundly in pG1-S in cooperation with Noxa in vitro. Induction of pG1 and pG1-S by reversible CDK4/CDK6 inhibition further augments tumor-specific bortezomib killing in myeloma xenografts. Reversible inhibition of CDK4/CDK6 in sequential combination therapy thus represents a novel mechanism-based cancer therapy.

An Ingenol Derived from Euphorbia kansui Induces Hepatocyte Cytotoxicity by Triggering G0/G1 Cell Cycle Arrest and Regulating the Mitochondrial Apoptosis Pathway in Vitro

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Xiaojing Yan

2016-06-01

Full Text Available Natural product lingenol, a purified diterpenoid compound derived from the root of Euphorbia kansui, exerts serious hepatotoxicity; however, the molecular mechanisms remain to be defined. In the present study, cell counting Kit-8 (CCK-8, inverted phase contrast microscope and flow cytometry were used to demonstrate that lingenol significantly inhibited L-O2 cells proliferation, and induced cell cycle arrest and apoptosis. Moreover, the results investigated that lingenol markedly disrupted mitochondrial functions by high content screening (HCS. In addition, the up-regulation of cytochrome c, AIF and Apaf-1 and activation of caspases were found in L-O2 cells detected by Western blotting and ELISA assay, which was required for lingenol activation of cytochrome c-mediated caspase cascades and AIF-mediated DNA damage. Mechanistic investigations revealed that lingenol significantly down-regulated the Bcl-2/Bax ratio and enhanced the reactive oxygen species (ROS in L-O2 cells. These data collectively indicated that lingenol modulation of ROS and Bcl-2/Bax ratio led to cell cycle arrest and mitochondrial-mediated apoptosis in L-O2 cells in vitro. All of these results will be helpful to reveal the hepatotoxicity mechanism of Euphorbia kansui and to effectively guide safer and better clinical application of this herb.

Baicalein induces G1 arrest in oral cancer cells by enhancing the degradation of cyclin D1 and activating AhR to decrease Rb phosphorylation

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Cheng, Ya-Hsin, E-mail: yhcheng@mail.cmu.edu.tw [Department of Physiology, School of Medicine, China Medical University, Taichung 40402, Taiwan, ROC (China); Li, Lih-Ann; Lin, Pinpin; Cheng, Li-Chuan [Division of Environmental Health and Occupational Medicine, National Health Research Institutes, Zhunan, Miaoli 35053, Taiwan, ROC (China); Hung, Chein-Hui [Graduate Institute of Clinical Medicine Sciences, Chang Gung University, Puizi City, Chiayi 613, Taiwan, ROC (China); Chang, Nai Wen [Department of Biochemistry, School of Medicine, China Medical University, Taichung, Taiwan, ROC (China); Lin, Chingju [Department of Physiology, School of Medicine, China Medical University, Taichung 40402, Taiwan, ROC (China)

2012-09-15

Baicalein is a flavonoid, known to have anti-inflammatory and anti-cancer effects. As an aryl hydrocarbon receptor (AhR) ligand, baicalein at high concentrations blocks AhR-mediated dioxin toxicity. Because AhR had been reported to play a role in regulating the cell cycle, we suspected that the anti-cancer effect of baicalein is associated with AhR. This study investigated the molecular mechanism involved in the anti-cancer effect of baicalein in oral cancer cells HSC-3, including whether such effect would be AhR-mediated. Results revealed that baicalein inhibited cell proliferation and increased AhR activity in a dose-dependent manner. Cell cycle was arrested at the G1 phase and the expression of CDK4, cyclin D1, and phosphorylated retinoblastoma (pRb) was decreased. When the AhR was suppressed by siRNA, the reduction of pRb was partially reversed, accompanied by a decrease of cell population at G1 phase and an increase at S phase, while the reduction of cyclin D1 and CDK4 did not change. This finding suggests that the baicalein activation of AhR is indeed associated with the reduction of pRb, but is independent of the reduction of cyclin D1 and CDK4. When cells were pre-treated with LiCl, the inhibitor of GSK-3β, the decrease of cyclin D1 was blocked and the reduction of pRb was recovered. The data indicates that in HSC-3 the reduction of pRb is both mediated by baicalein through activation of AhR and facilitation of cyclin D1 degradation, which causes cell cycle arrest at the G1 phase, and results in the inhibition of cell proliferation. -- Highlights: ► Baicalein causes the G1 phase arrest by decreasing Rb phosphorylation. ► Baicalein modulates AhR-mediated cell proliferation. ► Both AhR activation and cyclin D1 degradation results in hypophosphorylation of Rb. ► Baicalein facilitates cyclin D1 degradation by signalling the GSK-3β pathway.

A case of thyroid storm with cardiac arrest

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Nakashima Y

2014-05-01

Full Text Available Yutaka Nakashima,1 Tsuneaki Kenzaka,2 Masanobu Okayama,3 Eiji Kajii31Department for Support of Rural Medicine, Yamaguchi Grand Medical Center, 2Division of General Medicine, Center for Community Medicine, Jichi Medical University School of Medicine, Shimotsuke, Japan; 3Division of Community and Family Medicine, Center for Community Medicine, Jichi Medical University School of Medicine, Shimotsuke, JapanAbstract: A 23-year-old man became unconscious while jogging. He immediately received basic life support from a bystander and was transported to our hospital. On arrival, his spontaneous circulation had returned from a state of ventricular fibrillation and pulseless electrical activity. Following admission, hyperthyroidism led to a suspicion of thyroid storm, which was then diagnosed as a possible cause of the cardiac arrest. Although hyperthyroidism-induced cardiac arrest including ventricular fibrillation is rare, it should be considered when diagnosing the cause of treatable cardiac arrest.Keywords: hyperthyroidism, ventricular fibrillation, treatable cardiac arrest, cardiac arrest, cardiopulmonary arrest

Maturation arrest of human oocytes at germinal vesicle stage

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Zhi Qin Chen

2010-01-01

Full Text Available Maturation arrest of human oocytes may occur at various stages of the cell cycle. A total failure of human oocytes to complete meiosis is rarely observed during assisted conception cycles. We describe here a case of infertile couples for whom all oocytes repeatedly failed to mature at germinal vesicle (GV stage during in vitro fertilization/Intra cytoplasmic sperm injection (IVF/ICSI. The patient underwent controlled ovarian stimulation followed by oocyte retrieval and IVF/ICSI. The oocytes were stripped off cumulus cells prior to the ICSI procedure and their maturity status was defined. The oocyte maturation was repeatedly arrested at the GV. Oocyte maturation arrest may be the cause of infertility in this couple. The recognition of oocyte maturation arrest as a specific medical condition may contribute to the characterization of the currently known as "oocyte factor." The cellular and genetic mechanisms causing oocyte maturation arrest should be the subject for further investigation.

The effect of ultraviolet light on arrested human diploid cell populations

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Kantor, G.J.; Warner, C.; Hull, D.R.

1977-01-01

The results of the experiments to determine an effect of UV (254 nm) on human diploid fibroblasts (HDF) arrested with respect to division by using 0.5% fetal calf serum in the culture medium are reported. A fraction of cells from irradiated arrested populations, maintained in the arrested state post-irradiation, was lost from the populations. The extent of cell loss was fluence-dependent and cell strain specific. A Xeroderma pigmentosum cell strain was more sensitive to UV than were normal HDF. No difference in sensitivity were observed when arrested populations established from normal HDF populations of various in vitro ages were used. The length of the pre-irradiation arrested period affected the sensitivity of normal HDF, which appeared more resistant at longer arrested periods, but not the sensitivity of arrested Xeroderma populations. These results suggest that DNA repair processes play a role in maintaining irradiated cells in the arrested state. The suggestion is made that the lethal event caused by UV is an effect on transcription leading to an inhibition of required protein synthesis. (author)

Huaier Aqueous Extract Induces Hepatocellular Carcinoma Cells Arrest in S Phase via JNK Signaling Pathway

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Chengshuo Zhang

2015-01-01

Full Text Available Huaier aqueous extract, the main active constituent of Huaier proteoglycan, has antihepatocarcinoma activity in experimental and clinical settings. However, the potential and associated antihepatoma mechanisms of Huaier extract are not yet fully understood. Therefore, in this study, we aimed to elucidate the inhibitory proliferation effect of Huaier extract on apoptosis and cycle of HepG2 and Bel-7402 cells. Our data demonstrated that incubation with Huaier extract resulted in a marked decrease in cell viability dose-dependently. Flow cytometric analysis showed that a 48 h treatment of Huaier extract caused cell apoptosis. Typical apoptotic nucleus alterations were observed with fluorescence microscope after Hoechst staining. Immunoblot analysis further demonstrated that Huaier extract activated caspase 3 and PARP. Additionally, Huaier extract inhibited the activity of p-ERK, p-p38, and p-JNK in terms of MAPK. Furthermore, Huaier extract induced HCC cells arrest in S phase and decreased the cycle related protein expression of β-catenin and cyclin D1. Studies with JNK specific inhibitor, SP600125, showed that Huaier extract induced S phase arrest and decreased β-catenin and cyclin D1 expression via JNK signaling pathway. In conclusion, we verify that Huaier extract causes cell apoptosis and induces hepatocellular carcinoma cells arrest in S phase via JNK pathway, which advances our understanding on the molecular mechanisms of Huaier extract in hepatocarcinoma management.

Casticin impairs cell growth and induces cell apoptosis via cell cycle arrest in human oral cancer SCC-4 cells.

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Chou, Guan-Ling; Peng, Shu-Fen; Liao, Ching-Lung; Ho, Heng-Chien; Lu, Kung-Wen; Lien, Jin-Cherng; Fan, Ming-Jen; La, Kuang-Chi; Chung, Jing-Gung

2018-02-01

Casticin, a polymethoxyflavone, present in natural plants, has been shown to have biological activities including anti-cancer activities. Herein, we investigated the anti-oral cancer activity of casticin on SCC-4 cells in vitro. Viable cells, cell cycle distribution, apoptotic cell death, reactive oxygen species (ROS) production, and Ca 2+ production, levels of ΔΨ m and caspase activity were measured by flow cytometric assay. Cell apoptosis associated protein expressions were examined by Western blotting and confocal laser microscopy. Results indicated that casticin induced cell morphological changes, DNA condensation and damage, decreased the total viable cells, induced G 2 /M phase arrest in SCC-4 cells. Casticin promoted ROS and Ca 2+ productions, decreases the levels of ΔΨ m , promoted caspase-3, -8, and -9 activities in SCC-4 cells. Western blotting assay demonstrated that casticin affect protein level associated with G2/M phase arrest and apoptosis. Confocal laser microscopy also confirmed that casticin increased the translocation of AIF and cytochrome c in SCC-4 cells. In conclusion, casticin decreased cell number through G 2 /M phase arrest and the induction of cell apoptosis through caspase- and mitochondria-dependent pathways in SCC-4 cells. © 2017 Wiley Periodicals, Inc.

Effect of Irradiation on Apoptosis, Cell Cycle Arrest and Calcified Nodule Formation of Rat Calvarial Osteoblast

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Lee, Young Mi; Choi, Hang Moon; Heo, Min Suk; Lee, Sam Sun; Choi, Soon Chul; Park, Tae Won

2000-01-01

The study was aimed to detect the induction of apoptosis, cell cycle arrest and calcified nodule formation after irradiation on primarily cultured osteoblasts. Using rat calvarial osteoblasts, the effects of irradiation on apoptosis, cell cycle arrest, and calcified nodule formation were studied. The single irradiation of 10, 20 Gy was done with 5.38 Gy/min dose rate using the 137 Cs cell irradiator at 4th and 14th day of culture. Apoptosis induction and cell cycle arrest were assayed by the flow cytometry at 1, 2, 3, and 4 days after irradiation. The formation of calcified nodules was observed by alizarin red staining at 1, 3, 10, 14 days after irradiation at 4th day of culture, and at 1, 4, 5 days after irradiation at 14th day of culture. Apoptosis was not induced by 10 or 20 Gy independent of irradiation and culture period. Irradiation did not induced G1 arrest in post-irradiated osteoblasts. After irradiation at 4th-day of culture, G2 arrest was induced but it was not statistically significant after irradiation at 14th-day of culture. In the case of irradiated cells at 4th day of culture, calcified nodules were not formed and at 14th-day of culture after irradiation, calcified nodule formation did not affected. Taken together, these results suggest that irradiation at the dose of 10-20 Gy would not affect apoptosis induction of osteoblasts. Cell cycle and calcified nodule formation were influenced by the level of differentiation of osteblasts.

Growth Arrest-Specific Protein 6 is Hepatoprotective Against Ischemia/Reperfusion Injury

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Llacuna, Laura; Bárcena, Cristina; Bellido-Martín, Lola; Fernández, Laura; Stefanovic, Milica; Marí, Montserrat; García-Ruiz, Carmen; Fernández-Checa, José C.; de Frutos, Pablo García; Morales, Albert

2010-01-01

Growth arrest-specific gene 6 (GAS6) promotes growth and cell survival during tissue repair and development in different organs, including the liver. However, the specific role of GAS6 in liver ischemia/reperfusion (I/R) injury has not been previously addressed. Here, we report an early increase in serum GAS6 levels following I/R exposure. Moreover, unlike wild type mice, Gas6-/- mice were highly sensitive to partial hepatic I/R, with 90% of mice dying within 12 hours of reperfusion due to massive hepatocellular injury. I/R induced early hepatic AKT phosphorylation in wild type but not in Gas6-/- mice, without significant changes in JNK phosphorylation or nuclear NF-κB translocation, whereas hepatic IL-1β and TNF mRNA levels were higher in Gas6-/- mice compared to wild type mice. In line with the in vivo data, in vitro studies indicated that GAS6 induced AKT phosphorylation in primary mouse hepatocytes protecting them from hypoxia-induced cell death, while GAS6 diminished lipopolysaccharide (LPS)-induced cytokine expression (IL-1β and TNF) in murine macrophages. Finally, in vivo recombinant GAS6 treatment not only rescued GAS6 knockout mice from I/R-induced severe liver damage, but also attenuated hepatic damage in wild type mice following I/R. In conclusion, our data uncover GAS6 as a new player in liver I/R injury, emerging as a potential therapeutic target to reduce post-ischemic hepatic damage. PMID:20730776

Acute dose and low dose-rate irradiation of carcinoma cells expressing human papillomavirus E6 and E7 oncoproteins - the significance of p53, Rb and G1 arrest status

International Nuclear Information System (INIS)

DeWeese, Theodore L.; Walsh, Jonathan C.; Dillehay, Larry E.; Shao, Y.; Kessis, Theodore D.; Cho, Kathleen R.; Nelson, William G.

1995-01-01

Purpose: The development of carcinomas in a number of sites including the cervix, vulva and anus have been associated with cellular infection by human papillomaviruses (HPV), including HPV 16 and HPV 18. The mechanism by which these viruses contribute to tumor development or progression seems in part to be related to the integration of the viral genome into the host cells DNA, and the binding of p53 protein by the HPV E6 oncoprotein as well as the binding of the retinoblastoma (Rb) protein and Rb-like proteins by the HPV E7 oncoprotein. These interactions lead to loss of p53 and Rb function including loss of the G 1 cell cycle checkpoint. Although it is believed that both p53 and Rb play a role in the radiosensitivity of the cell, whether alteration in either protein enhances or diminishes cellular radiation response is not clear from the literature. Because HPV-associated tumors such as cervical cancer are often treated with acute dose and/or low dose-rate radiation, we set out to evaluate the radiation response of several carcinoma cell sublines expressing either oncogenic E6 or E7 to both types of radiation, and to determine if p53/Rb dependent G 1 arrest is an important determinant of cell fate after irradiation. Materials and Methods: We have previously developed a series of RKO colorectal carcinoma cell sublines expressing both low-risk (HPV 11) and high-risk (HPV 16) E6 and E7 genes. p53-dependent G 1 arrest is intact in RKO parental cells and cells expressing low-risk E6 proteins, while the G 1 arrest is abrogated in cells expressing high-risk E6 or E7. Clonogenic survival was assessed after exposure to acute dose (1 Gy/min) and low dose-rate (0.25 Gy/hour) radiation. The radiobiologic parameters α, β and the surviving fraction at 2 Gy (SF2) were determined. SDS-PAGE/immunoblotting was carried out to assess both p53 and p21 WAF1/CIP1 levels after exposure to radiation. Flow cytometry was performed before and after exposure to low dose-rate radiation to

F-box protein FBXO31 is a dedicated checkpoint protein to facilitate cell cycle arrest through activation of regulators in radiation induced DNA damage

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Santra, Manas Kumar

2017-01-01

In response to radiation-induced DNA damage, eukaryotic cells initiate a complex signalling pathway, termed the DNA damage response (DDR), which coordinates cell cycle arrest with DNA repair. Previous study showed that induction of G1 arrest in response to radiation induced DNA damage is minimally a two-step process: a fast p53-independent initiation of G1 arrest mediated by cyclin D1 proteolysis and a slower maintenance of arrest resulting from increased p53 stability. We elucidated the molecular mechanism of slow and fast response of radiation induced DDR. We showed that FBXO31, a member of F-box family proteins, plays important role in DDR induced by ionizing radiation. We show that FBXO31 is responsible for promoting MDM2 degradation following radiation. FBXO31 interacts with and directs the degradation of MDM2 in ATM dependent phosphorylation of MDM2. FBXO31-mediated loss of MDM2 leads to elevated levels of p53, resulting in growth arrest. In cells depleted of FBXO31, MDM2 is not degraded and p53 levels do not increase following genotoxic stress. Thus, FBXO31 is essential for the classic robust increase in p53 levels following DNA damage

Master curve based correlation between static initiation toughness KIC and crack arrest toughness KIa

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Wallin, K.; Rintamaa, R.

1999-01-01

Historically the ASME reference curve concept assumes a constant relation between static fracture toughness initiation toughness and crack arrest toughness. In reality, this is not the case. Experimental results show that the difference between K IC and K Ia is material specific. For some materials there is a big difference while for others they nearly coincide. So far, however, no systematic study regarding a possible correlation between the two parameters has been performed. The recent Master curve method, developed for brittle fracture initiation estimation, has enabled a consistent analysis of fracture initiation toughness data. The Master curve method has been modified to be able to describe also crack arrest toughness. Here, this modified 'crack arrest master curve' is further validated and used to develop a simple, but yet (for safety assessment purpose) adequately accurate correlation between the two fracture toughness parameters. The correlation enables the estimation of crack arrest toughness from small Charpy-sized static fracture toughness tests. The correlation is valid for low Nickel steels ≤ (1.2% Ni). If a more accurate description of the crack arrest toughness is required, it can either be measured experimentally or estimated from instrumented Charpy-V crack arrest load information. (orig.)

N-Methyl-N'-nitro-N-nitrosoguanidine-induced senescence-like growth arrest in colon cancer cells is associated with loss of adenomatous polyposis coli protein, microtubule organization, and telomeric DNA

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Narayan Satya

2004-01-01

Full Text Available Abstract Background Cellular senescence is a state in which mammalian cells enter into an irreversible growth arrest and altered biological functions. The senescence response in mammalian cells can be elicited by DNA-damaging agents. In the present study we report that the DNA-damaging agent N-methyl-N'-nitro-N-nitrosoguanidine (MNNG is able to induce senescence in the HCT-116 colon cancer cell line. Results Cells treated with lower concentrations of MNNG (0–25 microM for 50 h showed a dose-dependent increase in G2/M phase arrest and apoptosis; however, cells treated with higher concentrations of MNNG (50–100 microM showed a senescence-like G0/G1 phase arrest which was confirmed by increased expression of β-galactosidase, a senescence induced marker. The G2/M phase arrest and apoptosis were found to be associated with increased levels of p53 protein, but the senescence-like G0/G1 phase arrest was dissociated with p53 protein levels, since the p53 protein levels decreased in senescence-like arrested cells. We further, determined whether the decreased level of p53 was a transcriptional or a translational phenomenon. The results revealed that the decreased level of p53 protein in senescence-like arrested cells was a transcriptional phenomenon since p53 mRNA levels simultaneously decreased after treatment with higher concentrations of MNNG. We also examined the effect of MNNG treatment on other cell cycle-related proteins such as p21, p27, cyclin B1, Cdc2, c-Myc and max. The expression levels of these proteins were increased in cells treated with lower concentrations of MNNG, which supported the G2/M phase arrest. However, cells treated with higher concentrations of MNNG showed decreased levels of these proteins, and hence, may not play a role in cell cycle arrest. We then examined a possible association of the expression of APC protein and telomeric DNA signals with cellular senescence in MNNG-treated cells. We found that protein and m

Application of Food-specific IgG Antibody Detection in Allergy Dermatosis

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Yine Hu

2015-01-01

Full Text Available The application of food-specific IgG antibody detection in allergy dermatoses was explored. 181 patients with allergy dermatoses were diagnosed from January to September 2014 and 20 healthy subjects were selected. Fourteen kinds of food-specific IgG antibodies were detected by ELISA method among all the subjects. The positive rates of IgG antibody of the patient group and the healthy group were respectively 65.2% and 5.0%. The positive rates of IgG antibody of egg, milk, shrimp and crab took a large proportion in three groups of patients with three kinds of allergy dermatoses of urticaria, eczema and allergic dermatitis, the proportion of which was respectively 70.2%, 77.8% and 71.7%. Among urticaria and allergic dermatitis patients with positive antibody, the positive rate of children was significantly higher than that of adults (p0.05. Allergy dermatoses are closely related to food-specific IgG antibodies, and the allergy dermatoses patients have a high incidence rate of food intolerance; detecting IgG antibody in the serum of patients is of great significance for the diagnosis and treatment of allergy dermatoses.

SOX4 inhibits GBM cell growth and induces G0/G1 cell cycle arrest through Akt-p53 axis.

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Zhang, Jing; Jiang, Huawei; Shao, Jiaofang; Mao, Ruifang; Liu, Jie; Ma, Yingying; Fang, Xuefeng; Zhao, Na; Zheng, Shu; Lin, Biaoyang

2014-11-01

SOX4 is a transcription factor required for tissue development and differentiation in vertebrates. Overexpression of SOX4 has been reported in many cancers including glioblastoma multiforme (GBM), however, the underlying mechanism of actions has not been studied. In this study, we investigated the role of SOX4 in GBM. Kaplan-Meier analysis was performed to assess the association between SOX4 expression levels and survival times in primary GBM samples. Cre/lox P system was used to generate gain or loss of SOX4 in GBM cells, and microarray analysis uncovered the regulation network of SOX4 in GBM cells. High SOX4 expression was significantly associated with good prognosis of primary GBMs. SOX4 inhibited the growth of GBM cell line LN229, A172G and U87MG, partly via the activation of p53-p21 signaling and down-regulation of phosphorylated AKT1. Gene expression profiling and subsequent gene ontology analysis showed that SOX4 influenced several key pathways including the Wnt/ beta-catenin and TGF-beta signaling pathways. Our study found that SOX4 acts as a tumor suppressor in GBM cells by induce cell cycle arrest and inhibiting cell growth.

A Novel Polysaccharide Conjugate from Bullacta exarata Induces G1-Phase Arrest and Apoptosis in Human Hepatocellular Carcinoma HepG2 Cells.

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Liao, Ningbo; Sun, Liang; Chen, Jiang; Zhong, Jianjun; Zhang, Yanjun; Zhang, Ronghua

2017-03-01

Bullacta exarata has been consumed in Asia, not only as a part of the normal diet, but also as a traditional Chinese medicine with liver- and kidney-benefitting functions. Several scientific investigations involving extraction of biomolecules from this mollusk and pharmacological studies on their biological activities have been carried out. However, little is known regarding the antitumor properties of polysaccharides from B. exarata , hence the polysaccharides from B. exarata have been investigated here. One polysaccharide conjugate BEPS-IA was isolated and purified from B. exarata . It mainly consisted of mannose and glucose in a molar ratio of 1:2, with an average molecular weight of 127 kDa. Thirteen general amino acids were identified to be components of the protein-bound polysaccharide. Methylation and NMR studies revealed that BEPS-IA is a heteropolysaccharide consisting of 1,4-linked-α-d-Glc, 1,6-linked-α-d-Man, 1,3,6-linked-α-d-Man, and 1-linked-α-d-Man residue, in a molar ratio of 6:1:1:1. In order to test the antitumor activity of BEPS-IA, we investigated its effect against the growth of human hepatocellular carcinoma cells HepG2 in vitro. The result showed that BEPS-IA dose-dependently exhibited an effective HepG2 cells growth inhibition with an IC 50 of 112.4 μg/mL. Flow cytometry analysis showed that BEPS-IA increased the populations of both apoptotic sub-G1 and G1 phase. The result obtained from TUNEL assay corroborated apoptosis which was shown in flow cytometry. Western blot analysis suggested that BEPS-IA induced apoptosis and growth inhibition were associated with up-regulation of p53, p21 and Bax, down-regulation of Bcl-2. These findings suggest that BEPS-IA may serve as a potential novel dietary agent for hepatocellular carcinoma.

A Novel Polysaccharide Conjugate from Bullacta exarata Induces G1-Phase Arrest and Apoptosis in Human Hepatocellular Carcinoma HepG2 Cells

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Ningbo Liao

2017-03-01

Full Text Available Bullacta exarata has been consumed in Asia, not only as a part of the normal diet, but also as a traditional Chinese medicine with liver- and kidney-benefitting functions. Several scientific investigations involving extraction of biomolecules from this mollusk and pharmacological studies on their biological activities have been carried out. However, little is known regarding the antitumor properties of polysaccharides from B. exarata, hence the polysaccharides from B. exarata have been investigated here. One polysaccharide conjugate BEPS-IA was isolated and purified from B. exarata. It mainly consisted of mannose and glucose in a molar ratio of 1:2, with an average molecular weight of 127 kDa. Thirteen general amino acids were identified to be components of the protein-bound polysaccharide. Methylation and NMR studies revealed that BEPS-IA is a heteropolysaccharide consisting of 1,4-linked-α-d-Glc, 1,6-linked-α-d-Man, 1,3,6-linked-α-d-Man, and 1-linked-α-d-Man residue, in a molar ratio of 6:1:1:1. In order to test the antitumor activity of BEPS-IA, we investigated its effect against the growth of human hepatocellular carcinoma cells HepG2 in vitro. The result showed that BEPS-IA dose-dependently exhibited an effective HepG2 cells growth inhibition with an IC50 of 112.4 μg/mL. Flow cytometry analysis showed that BEPS-IA increased the populations of both apoptotic sub-G1 and G1 phase. The result obtained from TUNEL assay corroborated apoptosis which was shown in flow cytometry. Western blot analysis suggested that BEPS-IA induced apoptosis and growth inhibition were associated with up-regulation of p53, p21 and Bax, down-regulation of Bcl-2. These findings suggest that BEPS-IA may serve as a potential novel dietary agent for hepatocellular carcinoma.

Cytokinetically quiescent (G0/G1) human multiple myeloma cells are susceptible to simultaneous inhibition of Chk1 and MEK1/2.

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Pei, Xin-Yan; Dai, Yun; Youssefian, Leena E; Chen, Shuang; Bodie, Wesley W; Takabatake, Yukie; Felthousen, Jessica; Almenara, Jorge A; Kramer, Lora B; Dent, Paul; Grant, Steven

2011-11-10

Effects of Chk1 and MEK1/2 inhibition were investigated in cytokinetically quiescent multiple myeloma (MM) and primary CD138(+) cells. Coexposure to the Chk1 and MEK1/2 inhibitors AZD7762 and selumetinib (AZD6244) robustly induced apoptosis in various MM cells and CD138(+) primary samples, but spared normal CD138(-) and CD34(+) cells. Furthermore, Chk1/MEK1/2 inhibitor treatment of asynchronized cells induced G(0)/G(1) arrest and increased apoptosis in all cell-cycle phases, including G(0)/G(1). To determine whether this regimen is active against quiescent G(0)/G(1) MM cells, cells were cultured in low-serum medium to enrich the G(0)/G(1) population. G(0)/G(1)-enriched cells exhibited diminished sensitivity to conventional agents (eg, Taxol and VP-16) but significantly increased susceptibility to Chk1 ± MEK1/2 inhibitors or Chk1 shRNA knock-down. These events were associated with increased γH2A.X expression/foci formation and Bim up-regulation, whereas Bim shRNA knock-down markedly attenuated lethality. Immunofluorescent analysis of G(0)/G(1)-enriched or primary MM cells demonstrated colocalization of activated caspase-3 and the quiescent (G(0)) marker statin, a nuclear envelope protein. Finally, Chk1/MEK1/2 inhibition increased cell death in the Hoechst-positive (Hst(+)), low pyronin Y (PY)-staining (2N Hst(+)/PY(-)) G(0) population and in sorted small side-population (SSP) MM cells. These findings provide evidence that cytokinetically quiescent MM cells are highly susceptible to simultaneous Chk1 and MEK1/2 inhibition.

Evaluation of cysticercus-specific IgG (total and subclasses and IgE antibody responses in cerebrospinal fluid samples from patients with neurocysticercosis showing intrathecal production of specific IgG antibodies

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Lisandra Akemi Suzuki

Full Text Available In the present study, an enzyme-linked immunosorbent assay (ELISA standardized with vesicular fluid of Taenia solium cysticerci was used to screen for IgG (total and subclasses and IgE antibodies in cerebrospinal fluid (CSF samples from patients with neurocysticercosis showing intrathecal production of specific IgG antibodies and patients with other neurological disorders. The following results were obtained: IgG-ELISA: 100% sensitivity (median of the ELISA absorbances (MEA=1.17 and 100% specificity; IgG1-ELISA: 72.7% sensitivity (MEA=0.49 and 100% specificity; IgG2-ELISA: 81.8% sensitivity (MEA=0.46 and 100% specificity; IgG3-ELISA: 63.6% sensitivity (MEA=0.12 and 100% specificity; IgG4-ELISA: 90.9% sensitivity (MEA=0.85 and 100% specificity; IgE-ELISA 93.8% sensitivity (MEA=0.60 and 100% specificity. There were no significant differences between the sensitivities and specificities in the detection of IgG-ELISA and IgE-ELISA, although in CSF samples from patients with neurocysticercosis the MEA of the IgG-ELISA was significantly higher than that of the IgE-ELISA. The sensitivity and MEA values of the IgG4-ELISA were higher than the corresponding values for the other IgG subclasses. Future studies should address the contribution of IgG4 and IgE antibodies to the physiopathology of neurocysticercosis.

Inhibition of the oncogenic fusion protein EWS-FLI1 causes G2-M cell cycle arrest and enhanced vincristine sensitivity in Ewing's sarcoma.

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Zöllner, Stefan K; Selvanathan, Saravana P; Graham, Garrett T; Commins, Ryan M T; Hong, Sung Hyeok; Moseley, Eric; Parks, Sydney; Haladyna, Jessica N; Erkizan, Hayriye V; Dirksen, Uta; Hogarty, Michael D; Üren, Aykut; Toretsky, Jeffrey A

2017-10-03

Ewing's sarcoma (ES) is a rare and highly malignant cancer that grows in the bones or surrounding tissues mostly affecting adolescents and young adults. A chimeric fusion between the RNA binding protein EWS and the ETS family transcription factor FLI1 (EWS-FLI1), which is generated from a chromosomal translocation, is implicated in driving most ES cases by modulation of transcription and alternative splicing. The small-molecule YK-4-279 inhibits EWS-FLI1 function and induces apoptosis in ES cells. We aimed to identify both the underlying mechanism of the drug and potential combination therapies that might enhance its antitumor activity. We tested 69 anticancer drugs in combination with YK-4-279 and found that vinca alkaloids exhibited synergy with YK-4-279 in five ES cell lines. The combination of YK-4-279 and vincristine reduced tumor burden and increased survival in mice bearing ES xenografts. We determined that independent drug-induced events converged to cause this synergistic therapeutic effect. YK-4-279 rapidly induced G 2 -M arrest, increased the abundance of cyclin B1, and decreased EWS-FLI1-mediated generation of microtubule-associated proteins, which rendered cells more susceptible to microtubule depolymerization by vincristine. YK-4-279 reduced the expression of the EWS-FLI1 target gene encoding the ubiquitin ligase UBE2C, which, in part, contributed to the increase in cyclin B1. YK-4-279 also increased the abundance of proapoptotic isoforms of MCL1 and BCL2, presumably through inhibition of alternative splicing by EWS-FLI1, thus promoting cell death in response to vincristine. Thus, a combination of vincristine and YK-4-279 might be therapeutically effective in ES patients. Copyright © 2017 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.

Cells bearing chromosome aberrations lacking one telomere are selectively blocked at the G2/M checkpoint

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Rodriguez, Pilar [Unitat de Biologia Cel.lular, Departament de Biologia Cel.lular, Fisiologia i Immunologia, Universitat Autonoma de Barcelona, 08193 Bellaterra (Spain); Barquinero, Joan Francesc [Unitat d' Antropologia Biologica, Departament de Biologia Animal, Biologia Vegetal i Ecologia, Universitat Autonoma de Barcelona, 08193 Bellaterra (Spain); Duran, Assumpta [Unitat de Biologia Cel.lular, Departament de Biologia Cel.lular, Fisiologia i Immunologia, Universitat Autonoma de Barcelona, 08193 Bellaterra (Spain); Caballin, Maria Rosa [Unitat d' Antropologia Biologica, Departament de Biologia Animal, Biologia Vegetal i Ecologia, Universitat Autonoma de Barcelona, 08193 Bellaterra (Spain); Ribas, Montserrat [Servei de Radiofisica i Radioproteccio de l' Hospital de la Santa Creu i Sant Pau, 08025 Barcelona (Spain); Barrios, Leonardo, E-mail: Lleonard.Barrios@uab.cat [Unitat de Biologia Cel.lular, Departament de Biologia Cel.lular, Fisiologia i Immunologia, Universitat Autonoma de Barcelona, 08193 Bellaterra (Spain)

2009-11-02

Cell cycle checkpoints are part of the cellular mechanisms to maintain genomic integrity. After ionizing radiation exposure, the cells can show delay or arrest in their progression through the cell cycle, as well as an activation of the DNA repair machinery in order to reduce the damage. The G2/M checkpoint prevents G2 cells entering mitosis until the DNA damage has been reduced. The present study evaluates which G0 radiation-induced chromosome aberrations are negatively selected in the G2/M checkpoint. For this purpose, peripheral blood samples were irradiated at 1 and 3 Gy of {gamma}-rays, and lymphocytes were cultured for 48 h. Calyculin-A and Colcemid were used to analyze, in the same slide, cells in G2 and M. Chromosome spreads were consecutively analyzed by solid stain, pancentromeric and pantelomeric FISH and mFISH. The results show that the frequency of incomplete chromosome elements, those lacking a telomeric signal at one end, decreases abruptly from G2 to M. This indicates that cells with incomplete chromosome elements can progress from G0 to G2, but at the G2/M checkpoint suffer a strong negative selection.

Cells bearing chromosome aberrations lacking one telomere are selectively blocked at the G2/M checkpoint

International Nuclear Information System (INIS)

Rodriguez, Pilar; Barquinero, Joan Francesc; Duran, Assumpta; Caballin, Maria Rosa; Ribas, Montserrat; Barrios, Leonardo

2009-01-01

Cell cycle checkpoints are part of the cellular mechanisms to maintain genomic integrity. After ionizing radiation exposure, the cells can show delay or arrest in their progression through the cell cycle, as well as an activation of the DNA repair machinery in order to reduce the damage. The G2/M checkpoint prevents G2 cells entering mitosis until the DNA damage has been reduced. The present study evaluates which G0 radiation-induced chromosome aberrations are negatively selected in the G2/M checkpoint. For this purpose, peripheral blood samples were irradiated at 1 and 3 Gy of γ-rays, and lymphocytes were cultured for 48 h. Calyculin-A and Colcemid were used to analyze, in the same slide, cells in G2 and M. Chromosome spreads were consecutively analyzed by solid stain, pancentromeric and pantelomeric FISH and mFISH. The results show that the frequency of incomplete chromosome elements, those lacking a telomeric signal at one end, decreases abruptly from G2 to M. This indicates that cells with incomplete chromosome elements can progress from G0 to G2, but at the G2/M checkpoint suffer a strong negative selection.

Cdk5 phosphorylates non-genotoxically overexpressed p53 following inhibition of PP2A to induce cell cycle arrest/apoptosis and inhibits tumor progression

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Kumari Ratna

2010-07-01

Full Text Available Abstract Background p53 is the most studied tumor suppressor and its overexpression may or may not cause cell death depending upon the genetic background of the cells. p53 is degraded by human papillomavirus (HPV E6 protein in cervical carcinoma. Several stress activated kinases are known to phosphorylate p53 and, among them cyclin dependent kinase 5 (Cdk5 is one of the kinase studied in neuronal cell system. Recently, the involvement of Cdk5 in phosphorylating p53 has been shown in certain cancer types. Phosphorylation at specific serine residues in p53 is essential for it to cause cell growth inhibition. Activation of p53 under non stress conditions is poorly understood. Therefore, the activation of p53 and detection of upstream kinases that phosphorylate non-genotoxically overexpressed p53 will be of therapeutic importance for cancer treatment. Results To determine the non-genotoxic effect of p53; Tet-On system was utilized and p53 inducible HPV-positive HeLa cells were developed. p53 overexpression in HPV-positive cells did not induce cell cycle arrest or apoptosis. However, we demonstrate that overexpressed p53 can be activated to upregulate p21 and Bax which causes G2 arrest and apoptosis, by inhibiting protein phosphatase 2A. Additionally, we report that the upstream kinase cyclin dependent kinase 5 interacts with p53 to phosphorylate it at Serine20 and Serine46 residues thereby promoting its recruitment on p21 and bax promoters. Upregulation and translocation of Bax causes apoptosis through intrinsic mitochondrial pathway. Interestingly, overexpressed activated p53 specifically inhibits cell-growth and causes regression in vivo tumor growth as well. Conclusion Present study details the mechanism of activation of p53 and puts forth the possibility of p53 gene therapy to work in HPV positive cervical carcinoma.

ICT, Policy, Politics, and Democracy: An Integrated Framework for G2G Implementation

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Iliana Mizinova

2006-12-01

Full Text Available This research approaches the issue of G2G digitization using an integrated policy dynamics model. The essence of the contradictions in the G2G integration discourse is followed by a description of two policy paradigms that are then incorporated into an integrated or synthetic framework to evaluate the specifics of the G2G implementation in DHS and HUD. Speculations are made about the implications of this study for the democratic principles of government rule.

The cancer-germline antigen SSX2 causes cell cycle arrest and DNA damage in cancer cells

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Greve, Katrine Buch Vidén; Lindgreen, Jonas; Terp, Mikkel Green

2011-01-01

The SSX family of cancer and germline antigens is mainly expressed in the germ cells of healthy individuals as well as wide range of cancers and is therefore potential targets for immunotherapy. However, little is known about the role of SSX proteins in tumorigenesis and normal cell function. Here......, we show that SSX2 is involved in regulation of cancer cell growth. We found that ectopic expression of SSX2 in melanoma and colon cancer cells strongly reduced cell growth and induced apoptosis in vitro. Importantly, in a xenograft mouse model, the growth of tumors derived from SSX2 overexpressing...... melanoma cells was severely reduced compared to those derived from the isogenic parental cell line. Cell cycle analysis showed that SSX2 caused an accumulation of cells arrested in G1. Consistent with this, we observed a marked decrease in cells expressing the proliferation marker Ki67 and concomitantly...

A novel anti-alpha-fetoprotein single-chain variable fragment displays anti-tumor effects in HepG2 cells as a single agent or in combination with paclitaxel.

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Ji, Xiaonan; Shen, Yanli; Sun, Hao; Gao, Xiangdong

2016-08-01

Human hepatocellular carcinoma (HCC) has a high rate of tumor recurrence and metastasis, resulting in shortened survival time. The function of alpha-fetoprotein (AFP) as a regulatory factor in the growth of HCC cells has been well defined. The aim of this study was to investigate the use of a novel AFP-specific single-chain variable fragment that blocked AFP and inhibited HCC cell growth. The results indicated that the anti-AFP single-chain variable fragment (scFv) induced growth inhibition of AFP-expressing HCC cell lines in vitro through induction of G1 cell cycle arrest and apoptosis. The mechanism of apoptosis probably involved with blocking AFP internalization and regulation of the PTEN/PI3K/Akt signaling network. Moreover, the anti-AFP-scFv also effectively sensitized the HepG2 cells to paclitaxel (PTX) at a lower concentration. The combination effect of PTX and anti-AFP-scFv displayed a synergistic effect on HepG2 cells both in vitro and in vivo. Our results demonstrated that targeting AFP by specific antibodies has potential immunotherapeutic efficacy in human HCC.

PPARγ-independent induction of growth arrest and apoptosis in prostate and bladder carcinoma

International Nuclear Information System (INIS)

Chaffer, Christine L; Thomas, David M; Thompson, Erik W; Williams, Elizabeth D

2006-01-01

Although PPARγ antagonists have shown considerable pre-clinical efficacy, recent studies suggest PPARγ ligands induce PPARγ-independent effects. There is a need to better define such effects to permit rational utilization of these agents. We have studied the effects of a range of endogenous and synthetic PPARγ ligands on proliferation, growth arrest (FACS analysis) and apoptosis (caspase-3/7 activation and DNA fragmentation) in multiple prostate carcinoma cell lines (DU145, PC-3 and LNCaP) and in a series of cell lines modelling metastatic transitional cell carcinoma of the bladder (TSU-Pr1, TSU-Pr1-B1 and TSU-Pr1-B2). 15-deoxy-prostaglandin J 2 (15dPGJ2), troglitazone (TGZ) and to a lesser extent ciglitazone exhibited inhibitory effects on cell number; the selective PPARγ antagonist GW9662 did not reverse these effects. Rosiglitazone and pioglitazone had no effect on proliferation. In addition, TGZ induced G0/G1 growth arrest whilst 15dPGJ2 induced apoptosis. Troglitazone and 15dPGJ2 inhibit growth of prostate and bladder carcinoma cell lines through different mechanisms and the effects of both agents are PPARγ-independent

Crystal structures of the human G3BP1 NTF2-like domain visualize FxFG Nup Repeat Specificity

DEFF Research Database (Denmark)

Vognsen, Tina Reinholdt; Möller, Ingvar Rúnar; Kristensen, Ole

2013-01-01

Ras GTPase Activating Protein SH3 Domain Binding Protein (G3BP) is a potential anti-cancer drug target implicated in several cellular functions. We have used protein crystallography to solve crystal structures of the human G3BP1 NTF2-like domain both alone and in complex with an FxFG Nup repeat...... peptide. Despite high structural similarity, the FxFG binding site is located between two alpha helices in the G3BP1 NTF2-like domain and not at the dimer interface as observed for nuclear transport factor 2. ITC studies showed specificity towards the FxFG motif but not FG and GLFG motifs. The unliganded...

Endosulfan inhibiting the meiosis process via depressing expressions of regulatory factors and causing cell cycle arrest in spermatogenic cells.

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Guo, Fang-Zi; Zhang, Lian-Shuang; Wei, Jia-Liu; Ren, Li-Hua; Zhang, Jin; Jing, Li; Yang, Man; Wang, Ji; Sun, Zhi-Wei; Zhou, Xian-Qing

2016-10-01

Endosulfan is a persistent organic pollutant and widely used in agriculture as a pesticide. It is present in air, water, and soil worldwide; therefore, it is a health risk affecting especially the reproductive system. The aim of this study was to evaluate the toxicity of endosulfan in the reproductive system. To investigate the effect of endosulfan on meiosis process, 32 rats were divided into four groups, treated with 0, 1, 5, and 10 mg/kg/day endosulfan, respectively, and sacrificed after the 21 days of treatments. Results show that endosulfan caused the reductions in sperm concentration and motility rate, which resulted into an increased in sperm abnormality rate; further, endosulfan induced downregulation of spermatogenesis- and oogenesis-specific basic helix-loop-helix transcription factor (Sohlh1) which controls the switch on meiosis in mammals, as well cyclin A1, cyclin-dependent kinases 1 (CDK1), and cyclin-dependent kinases 2 (CDK2). In vitro, endosulfan induced G2/M phase arrest in the spermatogenic cell cycle and caused proliferation inhibition. Moreover, endosulfan induced oxidative stress and DNA damage in vivo and vitro. The results suggested that endosulfan could inhibit the start of meiosis by downregulating the expression of Sohlh1 and induce G2/M phase arrest of cell cycle by decreasing the expression of cyclin A1, CDK1, and CDK2 via oxidative damage, which inhibits the meiosis process, and therefore decrease the amount of sperm.

The production of reactive oxygen species and the mitochondrial membrane potential are modulated during onion oil-induced cell cycle arrest and apoptosis in A549 cells.

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Wu, Xin-jiang; Stahl, Thorsten; Hu, Ying; Kassie, Fekadu; Mersch-Sundermann, Volker

2006-03-01

Protective effects of Allium vegetables against cancers have been shown extensively in experimental animals and epidemiologic studies. We investigated cell proliferation and the induction of apoptosis by onion oil extracted from Allium cepa, a widely consumed Allium vegetable, in human lung cancer A549 cells. GC/MS analysis suggested that propyl sulfides but not allyl sulfides are major sulfur-containing constituents of onion oil. Onion oil at 12.5 mg/L significantly induced apoptosis (13% increase of apoptotic cells) as indicated by sub-G1 DNA content. It also caused cell cycle arrest at the G2/M phase; 25 mg/L onion oil increased the percentage of G2/M cells almost 6-fold compared with the dimethyl sulfoxide control. The action of onion oil may occur via a reactive oxygen species-dependent pathway because cell cycle arrest and apoptosis were blocked by the antioxidants N-acetylcysteine and exogenous glutathione. Marked collapse of the mitochondrial membrane potential suggested that dysfunction of the mitochondria may be involved in the oxidative burst and apoptosis induced by onion oil. Expression of phospho-cdc2 and phospho-cyclin B1 were downregulated by onion oil, perhaps accounting for the G2/M arrest. Overall, these results suggest that onion oil may exert chemopreventive action by inducing cell cycle arrest and apoptosis in tumor cells.

2-Aminopurine overrides multiple cell cycle checkpoints in BHK cells.

OpenAIRE

Andreassen, P R; Margolis, R L

1992-01-01

BHK cells blocked at any of several points in the cell cycle override their drug-induced arrest and proceed in the cycle when exposed concurrently to the protein kinase inhibitor 2-aminopurine (2-AP). For cells arrested at various points in interphase, 2-AP-induced cell cycle progression is made evident by arrival of the drug-treated cell population in mitosis. Cells that have escaped from mimosine G1 arrest, from hydroxyurea or aphidicolin S-phase arrest, or from VM-26-induced G2 arrest subs...

Kaposi sarcoma herpes virus latency associated nuclear antigen protein release the G2/M cell cycle blocks by modulating ATM/ATR mediated checkpoint pathway.

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Amit Kumar

Full Text Available The Kaposi's sarcoma-associated herpesvirus infects the human population and maintains latency stage of viral life cycle in a variety of cell types including cells of epithelial, mesenchymal and endothelial origin. The establishment of latent infection by KSHV requires the expression of an unique repertoire of genes among which latency associated nuclear antigen (LANA plays a critical role in the replication of the viral genome. LANA regulates the transcription of a number of viral and cellular genes essential for the survival of the virus in the host cell. The present study demonstrates the disruption of the host G2/M cell cycle checkpoint regulation as an associated function of LANA. DNA profile of LANA expressing human B-cells demonstrated the ability of this nuclear antigen in relieving the drug (Nocodazole induced G2/M checkpoint arrest. Caffeine suppressed nocodazole induced G2/M arrest indicating involvement of the ATM/ATR. Notably, we have also shown the direct interaction of LANA with Chk2, the ATM/ATR signalling effector and is responsible for the release of the G2/M cell cycle block.

Novel Epstein-Barr virus-like particles incorporating gH/gL-EBNA1 or gB-LMP2 induce high neutralizing antibody titers and EBV-specific T-cell responses in immunized mice.

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Perez, Elizabeth M; Foley, Joslyn; Tison, Timelia; Silva, Rute; Ogembo, Javier Gordon

2017-03-21

Previous Epstein-Barr virus (EBV) prophylactic vaccines based on the major surface glycoprotein gp350/220 as an immunogen have failed to block viral infection in humans, suggesting a need to target other viral envelope glycoproteins. In this study, we reasoned that incorporating gH/gL or gB, critical glycoproteins for viral fusion and entry, on the surface of a virus-like particle (VLP) would be more immunogenic than gp350/220 for generating effective neutralizing antibodies to prevent viral infection of both epithelial and B cell lines. To boost the humoral response and trigger cell-mediated immunity, EBV nuclear antigen 1 (EBNA1) and latent membrane protein 2 (LMP2), intracellular latency proteins expressed in all EBV-infected cells, were also included as critical components of the polyvalent EBV VLP. gH/gL-EBNA1 and gB-LMP2 VLPs were efficiently produced in Chinese hamster ovary cells, an FDA-approved vehicle for mass-production of biologics. Immunization with gH/gL-EBNA1 and gB-LMP2 VLPs without adjuvant generated both high neutralizing antibody titers in vitro and EBV-specific T-cell responses in BALB/c mice. These data demonstrate that will be invaluable not only in preventing EBV infection, but importantly, in preventing and treating the 200,000 cases of EBV-associated cancers that occur globally every year.

Pharmacologic ATM but not ATR kinase inhibition abrogates p21-dependent G1 arrest and promotes gastrointestinal syndrome after total body irradiation.

Science.gov (United States)

Vendetti, Frank P; Leibowitz, Brian J; Barnes, Jennifer; Schamus, Sandy; Kiesel, Brian F; Abberbock, Shira; Conrads, Thomas; Clump, David Andy; Cadogan, Elaine; O'Connor, Mark J; Yu, Jian; Beumer, Jan H; Bakkenist, Christopher J

2017-02-01

We show that ATM kinase inhibition using AZ31 prior to 9 or 9.25 Gy total body irradiation (TBI) reduced median time to moribund in mice to 8 days. ATR kinase inhibition using AZD6738 prior to TBI did not reduce median time to moribund. The striking finding associated with ATM inhibition prior to TBI was increased crypt loss within the intestine epithelium. ATM inhibition reduced upregulation of p21, an inhibitor of cyclin-dependent kinases, and blocked G1 arrest after TBI thereby increasing the number of S phase cells in crypts in wild-type but not Cdkn1a(p21 CIP/WAF1 )-/- mice. In contrast, ATR inhibition increased upregulation of p21 after TBI. Thus, ATM activity is essential for p21-dependent arrest while ATR inhibition may potentiate arrest in crypt cells after TBI. Nevertheless, ATM inhibition reduced median time to moribund in Cdkn1a(p21 CIP/WAF1 )-/- mice after TBI. ATM inhibition also increased cell death in crypts at 4 h in Cdkn1a(p21 CIP/WAF1 )-/-, earlier than at 24 h in wild-type mice after TBI. In contrast, ATR inhibition decreased cell death in crypts in Cdkn1a(p21 CIP/WAF1 )-/- mice at 4 h after TBI. We conclude that ATM activity is essential for p21-dependent and p21-independent mechanisms that radioprotect intestinal crypts and that ATM inhibition promotes GI syndrome after TBI.

Major life events as potential triggers of sudden cardiac arrest.

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Wicks, April F; Lumley, Thomas; Lemaitre, Rozenn N; Sotoodehnia, Nona; Rea, Thomas D; McKnight, Barbara; Strogatz, David S; Bovbjerg, Viktor E; Siscovick, David S

2012-05-01

We investigated the risk of sudden cardiac arrest in association with the recent loss of, or separation from, a family member or friend. Our case-crossover study included 490 apparently healthy married residents of King County, Washington, who suffered sudden cardiac arrest between 1988 and 2005. We compared exposure to spouse-reported family/friend events occurring ≤ 1 month before sudden cardiac arrest with events occurring in the previous 5 months. We evaluated potential effect modification by habitual vigorous physical activity. Recent family/friend events were associated with a higher risk of sudden cardiac arrest (odds ratio [OR] = 1.6; 95% confidence interval [CI] = 1.1-2.4). ORs for cases with and without habitual vigorous physical activity were 1.1 (0.6-2.2) and 2.0 (1.2-3.1), respectively (interaction P = 0.02). These results suggest family/friend events may trigger sudden cardiac arrest and raise the hypothesis that habitual vigorous physical activity may lower susceptibility to these potential triggers.

HIV-1 Vif's Capacity To Manipulate the Cell Cycle Is Species Specific.

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Evans, Edward L; Becker, Jordan T; Fricke, Stephanie L; Patel, Kishan; Sherer, Nathan M

2018-04-01

Cells derived from mice and other rodents exhibit profound blocks to HIV-1 virion production, reflecting species-specific incompatibilities between viral Tat and Rev proteins and essential host factors cyclin T1 (CCNT1) and exportin-1 (XPO1, also known as CRM1), respectively. To determine if mouse cell blocks other than CCNT1 and XPO1 affect HIV's postintegration stages, we studied HIV-1 NL4-3 gene expression in mouse NIH 3T3 cells modified to constitutively express HIV-1-compatible versions of CCNT1 and XPO1 (3T3.CX cells). 3T3.CX cells supported both Rev-independent and Rev-dependent viral gene expression and produced relatively robust levels of virus particles, confirming that CCNT1 and XPO1 represent the predominant blocks to these stages. Unexpectedly, however, 3T3.CX cells were remarkably resistant to virus-induced cytopathic effects observed in human cell lines, which we mapped to the viral protein Vif and its apparent species-specific capacity to induce G 2 /M cell cycle arrest. Vif was able to mediate rapid degradation of human APOBEC3G and the PPP2R5D regulatory B56 subunit of the PP2A phosphatase holoenzyme in mouse cells, thus demonstrating that Vif NL4-3 's modulation of the cell cycle can be functionally uncoupled from some of its other defined roles in CUL5-dependent protein degradation. Vif was also unable to induce G 2 /M cell cycle arrest in other nonhuman cell types, including cells derived from nonhuman primates, leading us to propose that one or more human-specific cofactors underpin Vif's ability to modulate the cell cycle. IMPORTANCE Cells derived from mice and other rodents exhibit profound blocks to HIV-1 replication, thus hindering the development of a low-cost small-animal model for studying HIV/AIDS. Here, we engineered otherwise-nonpermissive mouse cells to express HIV-1-compatible versions of two species-specific host dependency factors, cyclin T1 (CCNT1) and exportin-1 (XPO1) (3T3.CX cells). We show that 3T3.CX cells rescue HIV-1

Inhibition of autophagy enhances DNA damage-induced apoptosis by disrupting CHK1-dependent S phase arrest

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Liou, Jong-Shian; Wu, Yi-Chen; Yen, Wen-Yen; Tang, Yu-Shuan [Institute of Cellular and Organismic Biology, Academia Sinica, Taipei 115, Taiwan, ROC (China); Kakadiya, Rajesh B.; Su, Tsann-Long [Institute of Biomedical Sciences, Academia Sinica, Taipei 115, Taiwan, ROC (China); Yih, Ling-Huei, E-mail: lhyih@gate.sinica.edu.tw [Institute of Cellular and Organismic Biology, Academia Sinica, Taipei 115, Taiwan, ROC (China)

2014-08-01

DNA damage has been shown to induce autophagy, but the role of autophagy in the DNA damage response and cell fate is not fully understood. BO-1012, a bifunctional alkylating derivative of 3a-aza-cyclopenta[a]indene, is a potent DNA interstrand cross-linking agent with anticancer activity. In this study, BO-1012 was found to reduce DNA synthesis, inhibit S phase progression, and induce phosphorylation of histone H2AX on serine 139 (γH2AX) exclusively in S phase cells. Both CHK1 and CHK2 were phosphorylated in response to BO-1012 treatment, but only depletion of CHK1, but not CHK2, impaired BO-1012-induced S phase arrest and facilitated the entry of γH2AX-positive cells into G2 phase. CHK1 depletion also significantly enhanced BO-1012-induced cell death and apoptosis. These results indicate that BO-1012-induced S phase arrest is a CHK1-dependent pro-survival response. BO-1012 also resulted in marked induction of acidic vesicular organelle (AVO) formation and microtubule-associated protein 1 light chain 3 (LC3) processing and redistribution, features characteristic of autophagy. Depletion of ATG7 or co-treatment of cells with BO-1012 and either 3-methyladenine or bafilomycin A1, two inhibitors of autophagy, not only reduced CHK1 phosphorylation and disrupted S phase arrest, but also increased cleavage of caspase-9 and PARP, and cell death. These results suggest that cells initiate S phase arrest and autophagy as pro-survival responses to BO-1012-induced DNA damage, and that suppression of autophagy enhances BO-1012-induced apoptosis via disruption of CHK1-dependent S phase arrest. - Highlights: • Autophagy inhibitors enhanced the cytotoxicity of a DNA alkylating agent, BO-1012. • BO-1012-induced S phase arrest was a CHK1-dependent pro-survival response. • Autophagy inhibition enhanced BO-1012 cytotoxicity via disrupting the S phase arrest.

RBP-J-interacting and tubulin-associated protein induces apoptosis and cell cycle arrest in human hepatocellular carcinoma by activating the p53–Fbxw7 pathway

International Nuclear Information System (INIS)

Wang, Haihe; Yang, Zhanchun; Liu, Chunbo; Huang, Shishun; Wang, Hongzhi; Chen, Yingli; Chen, Guofu

2014-01-01

Highlights: • RITA overexpression increased protein expression of p53 and Fbxw7 and downregulated the expression of cyclin D1, cyclin E, CDK2, Hes-1 and NF-κB p65. • RITA can significantly inhibit the in vitro growth of SMMC7721 and HepG2 cells. • RITA exerts tumor-suppressive effects in hepatocarcinogenesis through induction of G0/G1 cell cycle arrest and apoptosis and suggest a therapeutic application of RITA in HCC. - Abstract: Aberrant Notch signaling is observed in human hepatocellular carcinoma (HCC) and has been associated with the modulation of cell growth. However, the role of Notch signaling in HCC and its underlying mechanism remain elusive. RBP-J-interacting and tubulin-associated (RITA) mediates the nuclear export of RBP-J to tubulin fibers and downregulates Notch-mediated transcription. In this study, we found that RITA overexpression increased protein expression of p53 and Fbxw7 and downregulated the expression of cyclin D1, cyclin E, CDK2, Hes-1 and NF-κB p65. These changes led to growth inhibition and induced G0/G1 cell cycle arrest and apoptosis in SMMC7721 and HepG2 cells. Our findings indicate that RITA exerts tumor-suppressive effects in hepatocarcinogenesis through induction of G0/G1 cell cycle arrest and apoptosis and suggest a therapeutic application of RITA in HCC

Cell cycle arrest and the evolution of chronic kidney disease from acute kidney injury.

Science.gov (United States)

Canaud, Guillaume; Bonventre, Joseph V

2015-04-01

For several decades, acute kidney injury (AKI) was generally considered a reversible process leading to complete kidney recovery if the individual survived the acute illness. Recent evidence from epidemiologic studies and animal models, however, have highlighted that AKI can lead to the development of fibrosis and facilitate the progression of chronic renal failure. When kidney injury is mild and baseline function is normal, the repair process can be adaptive with few long-term consequences. When the injury is more severe, repeated, or to a kidney with underlying disease, the repair can be maladaptive and epithelial cell cycle arrest may play an important role in the development of fibrosis. Indeed, during the maladaptive repair after a renal insult, many tubular cells that are undergoing cell division spend a prolonged period in the G2/M phase of the cell cycle. These tubular cells recruit intracellular pathways leading to the synthesis and the secretion of profibrotic factors, which then act in a paracrine fashion on interstitial pericytes/fibroblasts to accelerate proliferation of these cells and production of interstitial matrix. Thus, the tubule cells assume a senescent secretory phenotype. Characteristic features of these cells may represent new biomarkers of fibrosis progression and the G2/M-arrested cells may represent a new therapeutic target to prevent, delay or arrest progression of chronic kidney disease. Here, we summarize recent advances in our understanding of the biology of the cell cycle and how cell cycle arrest links AKI to chronic kidney disease. © The Author 2014. Published by Oxford University Press on behalf of ERA-EDTA. All rights reserved.

Impact of DNA demethylation of the G0S2 gene on the transcription of G0S2 in squamous lung cancer cell lines with or without nuclear receptor agonists

International Nuclear Information System (INIS)

Kusakabe, Masashi; Watanabe, Kousuke; Emoto, Noriko; Aki, Naomi; Kage, Hidenori; Nagase, Takahide; Nakajima, Jun; Yatomi, Yutaka; Ohishi, Nobuya; Takai, Daiya

2009-01-01

We recently identified that DNA methylation of the G0S2 gene was significantly more frequent in squamous lung cancer than in non-squamous lung cancer. However, the significance of G0S2 methylation levels on cancer cells is not yet known. We investigated the effect of G0S2 methylation levels on cell growth, mRNA expression, and chromatin structure using squamous lung cancer cell lines and normal human bronchial epithelial cells. DNA methylation and mRNA expression of G0S2 were inversely correlated, and in one of the squamous lung cancer cell lines, LC-1 sq, G0S2 was completely methylated and suppressed. Overexpression of G0S2 in LC-1 sq did not show growth arrest or apoptosis. The G0S2 gene has been reported to be a target gene of all-trans retinoic acid and peroxisome proliferator-activated receptor agonists. We treated LC-1 sq with 5-Aza-2'-deoxycytidine, Trichostatin A, all-trans retinoic acid, Wy 14643, or Pioglitazone either alone or in combination. Only 5-Aza-2'-deoxycytidine restored mRNA expression of G0S2. Chromatin immunoprecipitation revealed that histone H3 lysine 9 was methylated regardless of DNA methylation or mRNA expression. In summary, mRNA expression of G0S2 was regulated mainly by DNA methylation in squamous lung cancer cell lines. When the G0S2 gene was methylated, nuclear receptor agonists could not restore mRNA expression of G0S2 and did not show any additive effect on mRNA expression of G0S2 even after the treatment with 5-Aza-2'-deoxycytidine.

Enhancement of Cisplatin-Mediated Apoptosis in Ovarian Cancer Cells through Potentiating G2/M Arrest and p21 Upregulation by Combinatorial Epigallocatechin Gallate and Sulforaphane

Directory of Open Access Journals (Sweden)

Huaping Chen

2013-01-01

Full Text Available Advanced-stage ovarian cancer is characterized by high mortality due to development of resistance to conventional chemotherapy. Novel compounds that can enhance the efficacy of conventional chemotherapy in ovarian cancer may overcome this drug resistance. Consumption of green tea (epigallocatechin gallate, EGCG and cruciferous vegetables (sulforaphane, SFN is inversely associated with occurrence of ovarian cancer and has anticancer effects through targeting multiple molecules in cancer cells. However, the effects of EGCG and SFN combinational treatment on ovarian cancer cells and on efficacy of cisplatin to these cells are unknown. In this study, EGCG or SFN was used to treat both cisplatin-sensitive (A2780 and cisplatin-resistant (A2780/CP20 ovarian cancer cells alone or in combination with cisplatin. We found that EGCG and SFN combinational treatment can reduce cell viability of both ovarian cancer cell lines time- and dose-dependently. Furthermore, EGCG and SFN combinational treatment can enhance cisplatin-induced apoptosis and G2/M phase arrest, thereby enhancing the efficacy of cisplatin on both cisplatin-sensitive and cisplatin-resistant ovarian cancer cells. EGCG and SFN combinational treatment upregulated p21 expression induced by cisplatin in cisplatin-sensitive ovarian cancer cells, while p27 expression was not regulated by these treatments. Collectively, these studies provide novel approaches to overcoming cisplatin chemotherapy resistance in ovarian cancer.

Location of cardiac arrest and impact of pre-arrest chronic disease and medication use on survival

DEFF Research Database (Denmark)

Granfeldt, Asger; Wissenberg, Mads; Hansen, Steen Møller

2017-01-01

location and a higher mortality can be explained by differences in chronic diseases and medication. METHODS: We identified 27,771 out-of-hospital cardiac arrest patients ≥18 years old from the Danish Cardiac Arrest Registry (2001-2012). Using National Registries, we identified pre-arrest chronic disease......INTRODUCTION: Cardiac arrest in a private location is associated with a higher mortality when compared to public location. Past studies have not accounted for pre-arrest factors such as chronic disease and medication. AIM: To investigate whether the association between cardiac arrest in a private...

Current Pharmacological Advances in the Treatment of Cardiac Arrest

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Andry Papastylianou

2012-01-01

Full Text Available Cardiac arrest is defined as the sudden cessation of spontaneous ventilation and circulation. Within 15 seconds of cardiac arrest, the patient loses consciousness, electroencephalogram becomes flat after 30 seconds, pupils dilate fully after 60 seconds, and cerebral damage takes place within 90–300 seconds. It is essential to act immediately as irreversible damage can occur in a short time. Cardiopulmonary resuscitation (CPR is an attempt to restore spontaneous circulation through a broad range of interventions which are early defibrillation, high-quality and uninterrupted chest compressions, advanced airway interventions, and pharmacological interventions. Drugs should be considered only after initial shocks have been delivered (when indicated and chest compressions and ventilation have been started. During cardiopulmonary resuscitation, no specific drug therapy has been shown to improve survival to hospital discharge after cardiac arrest, and only few drugs have a proven benefit for short-term survival. This paper reviews current pharmacological treatment of cardiac arrest. There are three groups of drugs relevant to the management of cardiac arrest: vasopressors, antiarrhythmics, and other drugs such as sodium bicarbonate, calcium, magnesium, atropine, fibrinolytic drugs, and corticosteroids.

Epidemiology and Outcomes After In-Hospital Cardiac Arrest After Pediatric Cardiac Surgery

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Gupta, Punkaj; Jacobs, Jeffrey P.; Pasquali, Sara K.; Hill, Kevin D.; Gaynor, J. William; O’Brien, Sean M.; He, Max; Sheng, Shubin; Schexnayder, Stephen M.; Berg, Robert A.; Nadkarni, Vinay M.; Imamura, Michiaki; Jacobs, Marshall L.

2014-01-01

Background Multicenter data regarding cardiac arrest in children undergoing heart operations are limited. We describe epidemiology and outcomes associated with postoperative cardiac arrest in a large multiinstitutional cohort. Methods Patients younger than 18 years in the Society of Thoracic Surgeons Congenital Heart Surgery Database (2007 through 2012) were included. Patient factors, operative characteristics, and outcomes were described for patients with and without postoperative cardiac arrest. Multivariable models were used to evaluate the association of center volume with cardiac arrest rate and mortality after cardiac arrest, adjusting for patient and procedural factors. Results Of 70,270 patients (97 centers), 1,843 (2.6%) had postoperative cardiac arrest. Younger age, lower weight, and presence of preoperative morbidities (all p < 0.0001) were associated with cardiac arrest. Arrest rate increased with procedural complexity across common benchmark operations, ranging from 0.7% (ventricular septal defect repair) to 12.7% (Norwood operation). Cardiac arrest was associated with significant mortality risk across procedures, ranging from 15.4% to 62.3% (all p < 0.0001). In multivariable analysis, arrest rate was not associated with center volume (odds ratio, 1.06; 95% confidence interval, 0.71 to 1.57 in low- versus high-volume centers). However, mortality after cardiac arrest was higher in low-volume centers (odds ratio, 2.00; 95% confidence interval, 1.52 to 2.63). This association was present for both high- and low-complexity operations. Conclusions Cardiac arrest carries a significant mortality risk across the stratum of procedural complexity. Although arrest rates are not associated with center volume, lower-volume centers have increased mortality after cardiac arrest. Further study of mechanisms to prevent cardiac arrest and to reduce mortality in those with an arrest is warranted. PMID:25443018

Linalool Induces Cell Cycle Arrest and Apoptosis in Leukemia Cells and Cervical Cancer Cells through CDKIs

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Mei-Yin Chang

2015-11-01

Full Text Available Plantaginaceae, a popular traditional Chinese medicine, has long been used for treating various diseases from common cold to cancer. Linalool is one of the biologically active compounds that can be isolated from Plantaginaceae. Most of the commonly used cytotoxic anticancer drugs have been shown to induce apoptosis in susceptible tumor cells. However, the signaling pathway for apoptosis remains undefined. In this study, the cytotoxic effect of linalool on human cancer cell lines was investigated. Water-soluble tetrazolium salts (WST-1 based colorimetric cellular cytotoxicity assay, was used to test the cytotoxic ability of linalool against U937 and HeLa cells, and flow cytometry (FCM and genechip analysis were used to investigate the possible mechanism of apoptosis. These results demonstrated that linalool exhibited a good cytotoxic effect on U937 and HeLa cells, with the IC50 value of 2.59 and 11.02 μM, respectively, compared with 5-FU with values of 4.86 and 12.31 μM, respectively. After treating U937 cells with linalool for 6 h, we found an increased sub-G1 peak and a dose-dependent phenomenon, whereby these cells were arrested at the G0/G1 phase. Furthermore, by using genechip analysis, we observed that linalool can promote p53, p21, p27, p16, and p18 gene expression. Therefore, this study verified that linalool can arrest the cell cycle of U937 cells at the G0/G1 phase and can arrest the cell cycle of HeLa cells at the G2/M phase. Its mechanism facilitates the expression of the cyclin-dependent kinases inhibitors (CDKIs p53, p21, p27, p16, and p18, as well as the non-expression of cyclin-dependent kinases (CDKs activity.

Measuring the performance of G2G services in Iran

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Zarei, Behrouz; Safdari, Maryam

To highlight the growth of e-government and the importance of its services it is essential to evaluate the performance of the service delivery to customers. Research indicates that traditional performance indexes are not suitable for this evaluation; moreover, it is noticeable that the e-government services are intangible and invisible. Among different e-government services, measurement of quality government to government (G2G) services has been less attractive for researchers while crucial for government policy-makers. This calls for a better understanding of the specific needs of users of these services in order to provide appropriate type and level of services that meets those needs. In this paper, the performance of the G2G services is measured in the Iranian context. For this purpose, SERVQUAL, which is a well-known method for assessing service quality, is employed. This study proposes and tests a five-factor of SERVQUAL instrument to explain user satisfaction and gap analysis, between expectations and perceptions of its customers, consisting thirty ministries and main governmental organizations. Based on a Chi-square test, factor analysis, gap analysis and correlations, it is concluded the gap between expectations and perceptions of G2G customers is significant and customer satisfaction of G2G services is at low level.

N,N'-di-(m-methylphenyi)-3,6-dimethyl-1,4-dihydro-1,2,4,5-tetrazine-1,4-dicarboamide (ZGDHu-1) suppresses the proliferation of PANC-1 pancreatic cancer cells via apoptosis and G2/M cell cycle arrest.

Science.gov (United States)

Chen, Su-Feng; Xia, Jun; Lv, Ya-Ping; Liu, Jin-Lin; Li, Wan-Xiang; Yu, Xi-Ping; Hu, Wei-Xiao; Zhou, Yong-Lie

2015-04-01

Pancreatic cancer is one of the human gastrointestinal malignancies with a high mortality and poor prognosis. Approximately eighty percent of patients are diagnosed with unresectable or metastatic disease. Thus, development of novel chemicals in the treatment of pancreatic cancer is imperative. This study aimed to investigate the anticancer effects of N,N'-di-(m-methylphenyi)-3,6-dimethyl-1,4-dihydro-1,2,4,5-tetrazine-1,4-dicarboamide (ZGDHu-1), a new tetrazine derivative, on the PANC-1 pancreatic cancer cell line and clarify the underlying molecular mechanism. Using an MTT assay, we found that ZGDHu-1 significantly suppressed the proliferation of PANC-1 cells in a time- and dose-dependent manner. Moreover, according to the morphological and flow cytometric analysis, the results indicated that ZGDHu-1 induced PANC-1 cell apoptosis and G2/M cell cycle arrest in a dose-dependent manner. In the western blot analysis, expression of the pro-apoptotic Bax gene was upregulated while the anti-apoptotic Bcl-2 gene was downregulated following treatment with ZGDHu-1. ZGDHu-1 also activated pro-caspase-3 and PARP and increased the expression of NF-κB inhibitor IκB. Furthermore, the expression levels of G2/M regulatory molecules such as cyclin B1 and cdc2 were decreased while that of Chk1 was increased. These results suggested that ZGDHu-1 suppressed the proliferation of pancreatic cancer cells, rendering it a potential therapeutic drug for the treatment of pancreatic cancer.

Nek1 silencing slows down DNA repair and blocks DNA damage-induced cell cycle arrest.

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Pelegrini, Alessandra Luíza; Moura, Dinara Jaqueline; Brenner, Bethânia Luise; Ledur, Pitia Flores; Maques, Gabriela Porto; Henriques, João Antônio Pegas; Saffi, Jenifer; Lenz, Guido

2010-09-01

Never in mitosis A (NIMA)-related kinases (Nek) are evolutionarily conserved proteins structurally related to the Aspergillus nidulans mitotic regulator NIMA. Nek1 is one of the 11 isoforms of the Neks identified in mammals. Different lines of evidence suggest the participation of Nek1 in response to DNA damage, which is also supported by the interaction of this kinase with proteins involved in DNA repair pathways and cell cycle regulation. In this report, we show that cells with Nek1 knockdown (KD) through stable RNA interference present a delay in DNA repair when treated with methyl-methanesulfonate (MMS), hydrogen peroxide (H(2)O(2)) and cisplatin (CPT). In particular, interstrand cross links induced by CPT take much longer to be resolved in Nek1 KD cells when compared to wild-type (WT) cells. In KD cells, phosphorylation of Chk1 in response to CPT was strongly reduced. While WT cells accumulate in G(2)/M after DNA damage with MMS and H(2)O(2), Nek1 KD cells do not arrest, suggesting that G(2)/M arrest induced by the DNA damage requires Nek1. Surprisingly, CPT-treated Nek1 KD cells arrest with a 4N DNA content similar to WT cells. This deregulation in cell cycle control in Nek1 KD cells leads to an increased sensitivity to genotoxic agents when compared to WT cells. These results suggest that Nek1 is involved in the beginning of the cellular response to genotoxic stress and plays an important role in preventing cell death induced by DNA damage.

Serum IgG2 and tissue IgG2 plasma cell elevation in orbital IgG4-related disease (IgG4-RD): Potential use in IgG4-RD assessment.

Science.gov (United States)

Chan, Anita S Y; Mudhar, Hardeep; Shen, Sunny Yu; Lang, Stephanie S; Fernando, Malee; Hilmy, Maryam Hazly; Guppy, Naomi Jayne; Rennie, Ian; Dunkley, Lisa; Al Jajeh, Issam

2017-11-01

To determine the role of serum and tissue IgG2 in orbital biopsies with the histological features of IgG4-related disease (IgG4-RD) in comparison with non-IgG4-related orbital inflammatory disorders (OID), including autoimmune disorders. This is an international (Sheffield, UK, and Singapore) collaborative, retrospective case review of 69 patients (38 from Singapore National Eye Centre and 31 from Royal Hallamshire Hospital, Sheffield) with orbital inflammatory biopsies between 2002 and 2016. Clinical information and histology were reviewed and cases were classified into three groups: Group 1: IgG4-RD orbital inflammation (n=43); Group 2: idiopathic OID (n=12) and Group 3: autoimmune OID (n=14). Serum IgG1, IgG2, IgG3 and IgG4 levels were collated where available and immunohistochemistry (IHC) for tissue IgG2 plasma cells was performed. Dual IHC showed IgG2 plasma cells as a distinct population from IgG4 plasma cells. Significant (twofold) serum IgG2 elevation was noted among IgG4-RD (group 1), idiopathic (group 2) and autoimmune OID (group 3). Similarly, significant elevation of tissue IgG2 plasma cells was also seen among IgG4-RD (group 1), idiopathic and autoimmune OID (groups 2 and 3). Significant elevations of serum IgG2 and tissue IgG2 plasma cells are present in orbital IgG4-RD in comparison with non-IgG4 orbital inflammation (idiopathic and autoimmune OID), suggesting that IgG2 may play a role in IgG4-RD. A serum IgG2 cut-off >5.3 g/L was found to be 80% sensitive and 91.7% specific for orbital IgG4-RD, with an accuracy of 0.90. Tissue IgG2 and IgG4 subclass reporting may provide additional insight regarding the 'IgG4-RD' pathogenesis. © Article author(s) (or their employer(s) unless otherwise stated in the text of the article) 2017. All rights reserved. No commercial use is permitted unless otherwise expressly granted.

Optimizing Neurologically Intact Survival from Sudden Cardiac Arrest: A Call to Action

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Jeffrey M. Goodloe

2014-11-01

Full Text Available The U.S. national out-of-hospital and in-hospital cardiac arrest survival rates, although improving recently, have remained suboptimal despite the collective efforts of individuals, communities, and professional societies. Only until very recently, and still with inconsistency, has focus been placed specifically on survival with pre-arrest neurologic function. The reality of current approaches to sudden cardiac arrest is that they are often lacking an integrative, multi-disciplinary approach, and without deserved funding and outcome analysis. In this manuscript, a multidisciplinary group of authors propose practice, process, technology, and policy initiatives to improve cardiac arrest survival with a focus on neurologic function. [West J Emerg Med. 2014;15(7:-0.

2-Hexadecynoic acid inhibits plasmodial FAS-II enzymes and arrests erythrocytic and liver stage Plasmodium infections.

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Tasdemir, Deniz; Sanabria, David; Lauinger, Ina L; Tarun, Alice; Herman, Rob; Perozzo, Remo; Zloh, Mire; Kappe, Stefan H; Brun, Reto; Carballeira, Néstor M

2010-11-01

Acetylenic fatty acids are known to display several biological activities, but their antimalarial activity has remained unexplored. In this study, we synthesized the 2-, 5-, 6-, and 9-hexadecynoic acids (HDAs) and evaluated their in vitro activity against erythrocytic (blood) stages of Plasmodium falciparum and liver stages of Plasmodium yoelii infections. Since the type II fatty acid biosynthesis pathway (PfFAS-II) has recently been shown to be indispensable for liver stage malaria parasites, the inhibitory potential of the HDAs against multiple P. falciparum FAS-II (PfFAS-II) elongation enzymes was also evaluated. The highest antiplasmodial activity against blood stages of P. falciparum was displayed by 5-HDA (IC(50) value 6.6 μg/ml), whereas the 2-HDA was the only acid arresting the growth of liver stage P. yoelii infection, in both flow cytometric assay (IC(50) value 2-HDA 15.3 μg/ml, control drug atovaquone 2.5 ng/ml) and immunofluorescence analysis (IC(50) 2-HDA 4.88 μg/ml, control drug atovaquone 0.37 ng/ml). 2-HDA showed the best inhibitory activity against the PfFAS-II enzymes PfFabI and PfFabZ with IC(50) values of 0.38 and 0.58 μg/ml (IC(50) control drugs 14 and 30 ng/ml), respectively. Enzyme kinetics and molecular modeling studies revealed valuable insights into the binding mechanism of 2-HDA on the target enzymes. All HDAs showed in vitro activity against Trypanosoma brucei rhodesiense (IC(50) values 3.7-31.7 μg/ml), Trypanosoma cruzi (only 2-HDA, IC(50) 20.2 μg/ml), and Leishmania donovani (IC(50) values 4.1-13.4 μg/ml) with generally low or no significant toxicity on mammalian cells. This is the first study to indicate therapeutic potential of HDAs against various parasitic protozoa. It also points out that the malarial liver stage growth inhibitory effect of the 2-HDA may be promoted via PfFAS-II enzymes. The lack of cytotoxicity, lipophilic nature, and calculated pharmacokinetic properties suggests that 2-HDA could be a useful compound to

2-Hexadecynoic Acid Inhibits Plasmodial FAS-II Enzymes and Arrest Erythrocytic and Liver Stage Plasmodium Infections

Science.gov (United States)

Tasdemir, Deniz; Sanabria, David; Lauinger, Ina L.; Tarun, Alice; Herman, Rob; Perozzo, Remo; Zloh, Mire; Kappe, Stefan H.; Brun, Reto; Carballeira, Néstor M.

2010-01-01

Acetylenic fatty acids are known to display several biological activities, but their antimalarial activity has remained unexplored. In this study, we synthesized the 2-, 5-, 6-, and 9-hexadecynoic acids (HDAs) and evaluated their in vitro activity against erythrocytic (blood) stages of Plasmodium falciparum and liver stages of P. yoelii infections. Since the type II fatty acid biosynthesis pathway (PfFAS-II) has recently been shown to be indispensable for liver stage malaria parasites, the inhibitory potential of the HDAs against multiple P. falciparum FAS-II (PfFAS-II) elongation enzymes was also evaluated. The highest antiplasmodial activity against blood stages of P. falciparum was displayed by 5-HDA (IC50 value 6.6. μg/ml), whereas the 2-HDA was the only acid arresting the growth of liver stage P. yoelii infection, in both flow cytometric assay (IC50 value 2-HDA 15.3 μg/ml, control drug atovaquone 2.5 ng/ml) and immunofluorescense analysis (IC50 2-HDA 4.88 μg/ml, control drug atovaquone 0.37 ng/ml). 2-HDA showed the best inhibitory against the PfFAS-II enzymes PfFabI and PfFabZ with IC50 values of 0.38 and 0.58 μg/ml (IC50 control drugs 14 and 30 ng/ml) respectively. Enzyme kinetics and molecular modeling studies revealed valuable insights into the binding mechanism of 2-HDA on the target enzymes. All HDAs showed in vitro activity against Trypanosoma brucei rhodesiense (IC50 values 3.7–31.7 μg/ml), Trypanosoma cruzi (only 2-HDA, IC50 20.2 μg/ml), and Leishmania donovani (IC50 values 4.1–13.4 μg/ml) with generally low or no significant toxicity on mammalian cells. This is the first study to indicate therapeutic potential of HDAs against various parasitic protozoa. It also points out that the malarial liver stage growth inhibitory effect of the 2-HDA may be promoted via PfFAS-II enzymes. The lack of cytotoxicity, lipophilic nature and calculated pharmacokinetic properties suggest that 2-HDA could be a useful compound to study the interaction of fatty

A phthalide derivative isolated from endophytic fungi Pestalotiopsis photiniae induces G1 cell cycle arrest and apoptosis in human HeLa cells

International Nuclear Information System (INIS)

Chen, C.; Yang, R.L.

2013-01-01

MP [4-(3′,3′-dimethylallyloxy)-5-methyl-6-methoxyphthalide] was obtained from liquid culture of Pestalotiopsis photiniae isolated from the Chinese Podocarpaceae plant Podocarpus macrophyllus. MP significantly inhibited the proliferation of HeLa tumor cell lines. After treatment with MP, characteristic apoptotic features such as DNA fragmentation and chromatin condensation were observed in DAPI-stained HeLa cells. Flow cytometry showed that MP induced G1 cell cycle arrest and apoptosis in a dose-dependent manner. Western blotting and real-time reverse transcription-polymerase chain reaction were used to investigate protein and mRNA expression. MP caused significant cell cycle arrest by upregulating the cyclin-dependent kinase inhibitor p27 KIP1 protein and p21 CIP1 mRNA levels in HeLa cells. The expression of p73 protein was increased after treatment with various MP concentrations. mRNA expression of the cell cycle-related genes, p21 CIP1 , p16 INK4a and Gadd45α, was significantly upregulated and mRNA levels demonstrated significantly increased translation of p73, JunB, FKHR, and Bim. The results indicate that MP may be a potential treatment for cervical cancer

A phthalide derivative isolated from endophytic fungi Pestalotiopsis photiniae induces G1 cell cycle arrest and apoptosis in human HeLa cells

Energy Technology Data Exchange (ETDEWEB)

Chen, C. [College of Life Science, Hebei University, Baoding (China); Yang, R.L. [Key Laboratory of Microbial Diversity Research and Application of Hebei Province, Baoding, China, Key Laboratory of Microbial Diversity Research and Application of Hebei Province, Baoding (China)

2013-07-30

MP [4-(3′,3′-dimethylallyloxy)-5-methyl-6-methoxyphthalide] was obtained from liquid culture of Pestalotiopsis photiniae isolated from the Chinese Podocarpaceae plant Podocarpus macrophyllus. MP significantly inhibited the proliferation of HeLa tumor cell lines. After treatment with MP, characteristic apoptotic features such as DNA fragmentation and chromatin condensation were observed in DAPI-stained HeLa cells. Flow cytometry showed that MP induced G1 cell cycle arrest and apoptosis in a dose-dependent manner. Western blotting and real-time reverse transcription-polymerase chain reaction were used to investigate protein and mRNA expression. MP caused significant cell cycle arrest by upregulating the cyclin-dependent kinase inhibitor p27{sup KIP1} protein and p21{sup CIP1} mRNA levels in HeLa cells. The expression of p73 protein was increased after treatment with various MP concentrations. mRNA expression of the cell cycle-related genes, p21{sup CIP1}, p16{sup INK4a} and Gadd45α, was significantly upregulated and mRNA levels demonstrated significantly increased translation of p73, JunB, FKHR, and Bim. The results indicate that MP may be a potential treatment for cervical cancer.

Selumetinib suppresses cell proliferation, migration and trigger apoptosis, G1 arrest in triple-negative breast cancer cells.

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Zhou, Yan; Lin, Shuchen; Tseng, Kuo-Fu; Han, Kun; Wang, Yaling; Gan, Zhi-Hua; Min, Da-Liu; Hu, Hai-Yan

2016-10-21

Triple-negative breast cancer (TNBC) has aggressive progression with poor prognosis and ineffective treatments. Selumetinib is an allosteric, ATP-noncompetitive inhibitor of MEK1/2, which has benn known as effective antineoplastic drugs for several malignant tumors. We hypothesized that Selumetinib might be potential drug for TNBC and explore the mechanism. After treated with Selumetinib, the viability and mobility of HCC1937 and MDA-MB-231 were detected by MTT, tunnel, wound-healing assay, transwell assay and FCM methods. MiR array was used to analysis the change of miRs. We predicted and verified CUL1 is the target of miR-302a using Luciferase reporter assay. We also silenced the CUL1 by siRNA, to clarify whether CUL1 take part in the cell proliferation, migration and regulated its substrate TIMP1 and TRAF2. Moreover, after transfection, the antagomir of miR-302a and CUL1 over-expressed plasmid into HCC1937 and MDA-MB-231 cell accompanied with the Selumetinib treatment, we detected the proliferation and migration again. Selumetinib reduce the proliferation, migration, triggered apoptosis and G1 arrest in TNBC cell lines. In this process, the miR-302a was up-regulated and inhibited the CUL1 expression. The later negatively regulated the TIMP1 and TRAF2. As soon as we knockdown miR-302a and over-expression CUL1 in TNBC cells, the cytotoxicity of Selumetinib was reversed. MiR-302a targeted regulated the CUL1 expression and mediated the Selumetinib-induced cytotoxicity of triple-negative breast cancer.

Crisis management during anaesthesia: cardiac arrest.

Science.gov (United States)

Runciman, W B; Morris, R W; Watterson, L M; Williamson, J A; Paix, A D

2005-06-01

Cardiac arrest attributable to anaesthesia occurs at the rate of between 0.5 and 1 case per 10 000 cases, tends to have a different profile to that of cardiac arrest occurring elsewhere, and has an in-hospital mortality of 20%. However, as individual practitioners encounter cardiac arrest rarely, the rapidity with which the diagnosis is made and the consistency of appropriate management varies considerably. To examine the role of a previously described core algorithm "COVER ABCD-A SWIFT CHECK", supplemented by a sub-algorithm for cardiac arrest, in the management of cardiac arrest occurring in association with anaesthesia. The potential performance of this structured approach for each the relevant incidents among the first 4000 reported to the Australian Incident Monitoring Study (AIMS) was compared with the actual management as reported by the anaesthetists involved. There were 129 reports of cardiac arrest associated with anaesthesia among the first 4000 AIMS incident reports. Identified aetiological factors were grouped into five categories: (1) anaesthetic technique (11 cases with this category alone; 32 with this and one or more of the other categories, representing 25% of all 129 cardiac arrests); (2) drug related (16; 32, 25%); (3) associated with surgical procedure (9; 29, 22%); (4) associated with pre-existing medical or surgical disease (30; 82, 64%); (5) unknown (8; 14, 11%). The "real life" presentation and management of cardiac arrest in association with anaesthesia differs substantially from that detailed in general published guidelines. Cardiac rhythms at the time were sinus bradycardia (23%); asystole (22%); tachycardia/ventricular tachycardia/ventricular fibrillation (14%); and normal (7%), with a further third unknown. Details of treatment were recorded in 110 reports; modalities employed included cardiac compression (72%); adrenaline (61%); 100% oxygen (58%); atropine (38%); intravenous fluids (25%), and electrical defibrillation (17%). There

False-negative type-specific glycoprotein G antibody responses in STI clinic patients with recurrent HSV-1 or HSV-2 DNA positive genital herpes, The Netherlands.

Science.gov (United States)

van Rooijen, Martijn S; Roest, Wim; Hansen, Gino; Kwa, David; de Vries, Henry J C

2016-06-01

Herpes simplex virus (HSV) type-discriminating antibody tests (glycoprotein G (gG) directed) are used to identify naïve persons and differentiate acute infections from recurrences. We studied test characteristics of three commercially available antibody tests in patients with recurrent (established by viral PCR tests) herpes simplex virus type 1 (HSV-1) or herpes simplex virus type 2 (HSV-2) genital herpes episodes. Serum samples (at minimum 3 months after t=0) were examined for the presence of gG-1-specific or gG-2-specific antibodies using the HerpeSelect 1 and 2 Immunoblot IgG, the HerpeSelect 1 and 2 enzyme linked immunoassays IgG and the LIAISON HSV-1 and HSV-2 IgG indirect chemiluminescence immunoassays. The immunoblot was HSV-1 positive in 70.6% (95% CI 44.0% to 89.7%), the LIAISON in 88.2% (95% CI 63.5% to 98.5%) and the ELISA in 82.4% (95% CI 56.6% to 96.2%) of the 17 patients with a recurrent HSV-1 episode. From 33 patients with a recurrent HSV-2 episode, the immunoblot was HSV-2 positive in 84.8% (95% CI 68.1% to 94.9%), the LIAISON in 69.7% (95% CI 51.3% to 84.4%) and the ELISA in 84.8% (95% CI 68.1% to 94.9%). Among 15/17 (88.2%; 95% CI 63.5% to 98.5%) patients with HSV-1 and 30/33 (90.1%; 95% CI 75.7% to 98.1%) patients with HSV-2, HSV-1 or HSV-2 antibodies, respectively, were detected in at least one of the three antibody tests. Commercial type-specific gG HSV-1 or HSV-2 antibody assays were false negative in 12-30% of patients with recurrent HSV-1 or HSV-2 DNA positive genital lesions. The clinical and epidemiological use of type-specific HSV serology can be hampered by false-negative results, especially if based on a single test. Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://www.bmj.com/company/products-services/rights-and-licensing/

Inhibition of mTORC2 Induces Cell-Cycle Arrest and Enhances the Cytotoxicity of Doxorubicin by Suppressing MDR1 Expression in HCC Cells.

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Chen, Bryan Wei; Chen, Wei; Liang, Hui; Liu, Hao; Liang, Chao; Zhi, Xiao; Hu, Li-Qiang; Yu, Xia-Zhen; Wei, Tao; Ma, Tao; Xue, Fei; Zheng, Lei; Zhao, Bin; Feng, Xin-Hua; Bai, Xue-Li; Liang, Ting-Bo

2015-08-01

mTOR is aberrantly activated in hepatocellular carcinoma (HCC) and plays pivotal roles in tumorigenesis and chemoresistance. Rapamycin has been reported to exert antitumor activity in HCC and sensitizes HCC cells to cytotoxic agents. However, due to feedback activation of AKT after mTOR complex 1 (mTORC1) inhibition, simultaneous targeting of mTORC1/2 may be more effective. In this study, we examined the interaction between the dual mTORC1/2 inhibitor OSI-027 and doxorubicin in vitro and in vivo. OSI-027 was found to reduce phosphorylation of both mTORC1 and mTORC2 substrates, including 4E-BP1, p70S6K, and AKT (Ser473), and inhibit HCC cell proliferation. Similar to OSI-027 treatment, knockdown of mTORC2 induced G0-G1 phase cell-cycle arrest. In contrast, rapamycin or knockdown of mTORC1 increased phosphorylation of AKT (Ser473), yet had little antiproliferative effect. Notably, OSI-027 synergized with doxorubicin for the antiproliferative efficacy in a manner dependent of MDR1 expression in HCC cells. The synergistic antitumor effect of OSI-027 and doxorubicin was also observed in a HCC xenograft mouse model. Moreover, AKT was required for OSI-027-induced cell-cycle arrest and downregulation of MDR1. Our findings provide a rationale for dual mTORC1/mTORC2 inhibitors, such as OSI-027, as monotherapy or in combination with cytotoxic agents to treat HCC. Mol Cancer Ther; 14(8); 1805-15. ©2015 AACR. ©2015 American Association for Cancer Research.

NFBD1/MDC1 participates in the regulation of G2/M transition in mammalian cells

International Nuclear Information System (INIS)

Bu, Youquan; Suenaga, Yusuke; Okoshi, Rintaro; Sang, Meixiang; Kubo, Natsumi; Song, Fangzhou; Nakagawara, Akira; Ozaki, Toshinori

2010-01-01

NFBD1/MDC1 is a large nuclear protein involved in the early cellular response to DNA damage. Upon DNA damage, NFBD1 has an ability to facilitate the efficient DNA repair. In the present study, we have found that, in addition to DNA damage response, NFBD1 plays a critical role in the regulation of G2/M transition. Expression study using synchronized HeLa cells demonstrated that, like the mitotic kinase Plk1, NFBD1 expression level is maximal in G2/M-phase of the cell cycle. siRNA-mediated knockdown of NFBD1 resulted in G2/M arrest as well as simultaneous apoptosis in association with a significant increase in the amounts of γH2AX and pro-apoptotic p73. Since a remarkable down-regulation of mitotic phospho-histone H3 was detectable in NFBD1-knocked down cells, it is likely that knocking down of NFBD1 inhibits G2/M transition. Taken together, our present findings suggest that NFBD1 has a pivotal role in the regulation of proper mitotic entry.

DNA Damage during G2 Phase Does Not Affect Cell Cycle Progression of the Green Alga Scenedesmus quadricauda

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Vítová, Milada; Bišová, Kateřina; Zachleder, Vilém

2011-01-01

DNA damage is a threat to genomic integrity in all living organisms. Plants and green algae are particularly susceptible to DNA damage especially that caused by UV light, due to their light dependency for photosynthesis. For survival of a plant, and other eukaryotic cells, it is essential for an organism to continuously check the integrity of its genetic material and, when damaged, to repair it immediately. Cells therefore utilize a DNA damage response pathway that is responsible for sensing, reacting to and repairing damaged DNA. We have studied the effect of 5-fluorodeoxyuridine, zeocin, caffeine and combinations of these on the cell cycle of the green alga Scenedesmus quadricauda. The cells delayed S phase and underwent a permanent G2 phase block if DNA metabolism was affected prior to S phase; the G2 phase block imposed by zeocin was partially abolished by caffeine. No cell cycle block was observed if the treatment with zeocin occurred in G2 phase and the cells divided normally. CDKA and CDKB kinases regulate mitosis in S. quadricauda; their kinase activities were inhibited by Wee1. CDKA, CDKB protein levels were stabilized in the presence of zeocin. In contrast, the protein level of Wee1 was unaffected by DNA perturbing treatments. Wee1 therefore does not appear to be involved in the DNA damage response in S. quadricauda. Our results imply a specific reaction to DNA damage in S. quadricauda, with no cell cycle arrest, after experiencing DNA damage during G2 phase. PMID:21603605

DNA damage during G2 phase does not affect cell cycle progression of the green alga Scenedesmus quadricauda.

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Monika Hlavová

Full Text Available DNA damage is a threat to genomic integrity in all living organisms. Plants and green algae are particularly susceptible to DNA damage especially that caused by UV light, due to their light dependency for photosynthesis. For survival of a plant, and other eukaryotic cells, it is essential for an organism to continuously check the integrity of its genetic material and, when damaged, to repair it immediately. Cells therefore utilize a DNA damage response pathway that is responsible for sensing, reacting to and repairing damaged DNA. We have studied the effect of 5-fluorodeoxyuridine, zeocin, caffeine and combinations of these on the cell cycle of the green alga Scenedesmus quadricauda. The cells delayed S phase and underwent a permanent G2 phase block if DNA metabolism was affected prior to S phase; the G2 phase block imposed by zeocin was partially abolished by caffeine. No cell cycle block was observed if the treatment with zeocin occurred in G2 phase and the cells divided normally. CDKA and CDKB kinases regulate mitosis in S. quadricauda; their kinase activities were inhibited by Wee1. CDKA, CDKB protein levels were stabilized in the presence of zeocin. In contrast, the protein level of Wee1 was unaffected by DNA perturbing treatments. Wee1 therefore does not appear to be involved in the DNA damage response in S. quadricauda. Our results imply a specific reaction to DNA damage in S. quadricauda, with no cell cycle arrest, after experiencing DNA damage during G2 phase.

Growth arrest-specific protein 6 is hepatoprotective against murine ischemia/reperfusion injury.

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Llacuna, Laura; Bárcena, Cristina; Bellido-Martín, Lola; Fernández, Laura; Stefanovic, Milica; Marí, Montserrat; García-Ruiz, Carmen; Fernández-Checa, José C; García de Frutos, Pablo; Morales, Albert

2010-10-01

Growth arrest-specific gene 6 (GAS6) promotes growth and cell survival during tissue repair and development in different organs, including the liver. However, the specific role of GAS6 in liver ischemia/reperfusion (I/R) injury has not been previously addressed. Here we report an early increase in serum GAS6 levels after I/R exposure. Moreover, unlike wild-type (WT) mice, Gas6(-/-) mice were highly sensitive to partial hepatic I/R, with 90% of the mice dying within 12 hours of reperfusion because of massive hepatocellular injury. I/R induced early hepatic protein kinase B (AKT) phosphorylation in WT mice but not in Gas6(-/-) mice without significant changes in c-Jun N-terminal kinase phosphorylation or nuclear factor kappa B translocation, whereas hepatic interleukin-1β (IL-1β) and tumor necrosis factor (TNF) messenger RNA levels were higher in Gas6(-/-) mice versus WT mice. In line with the in vivo data, in vitro studies indicated that GAS6 induced AKT phosphorylation in primary mouse hepatocytes and thus protected them from hypoxia-induced cell death, whereas GAS6 diminished lipopolysaccharide-induced cytokine expression (IL-1β and TNF) in murine macrophages. Finally, recombinant GAS6 treatment in vivo not only rescued GAS6 knockout mice from severe I/R-induced liver damage but also attenuated hepatic damage in WT mice after I/R. Our data have revealed GAS6 to be a new player in liver I/R injury that is emerging as a potential therapeutic target for reducing postischemic hepatic damage.

An evidence on G2/M arrest, DNA damage and caspase mediated apoptotic effect of biosynthesized gold nanoparticles on human cervical carcinoma cells (HeLa)

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