Sample records for altered cell cycle

  1. Alteration of cell cycle progression by Sindbis virus infection

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    Yi, Ruirong; Saito, Kengo [Department of Molecular Virology, Graduate School of Medicine, Chiba University Graduate School of Medicine, 1-8-1 Inohana, Chiba 260-8670 (Japan); Isegawa, Naohisa [Laboratory Animal Center, Graduate School of Medicine, Chiba University Graduate School of Medicine, 1-8-1 Inohana, Chiba 260-8670 (Japan); Shirasawa, Hiroshi, E-mail: [Department of Molecular Virology, Graduate School of Medicine, Chiba University Graduate School of Medicine, 1-8-1 Inohana, Chiba 260-8670 (Japan)


    We examined the impact of Sindbis virus (SINV) infection on cell cycle progression in a cancer cell line, HeLa, and a non-cancerous cell line, Vero. Cell cycle analyses showed that SINV infection is able to alter the cell cycle progression in both HeLa and Vero cells, but differently, especially during the early stage of infection. SINV infection affected the expression of several cell cycle regulators (CDK4, CDK6, cyclin E, p21, cyclin A and cyclin B) in HeLa cells and caused HeLa cells to accumulate in S phase during the early stage of infection. Monitoring SINV replication in HeLa and Vero cells expressing cell cycle indicators revealed that SINV which infected HeLa cells during G{sub 1} phase preferred to proliferate during S/G{sub 2} phase, and the average time interval for viral replication was significantly shorter in both HeLa and Vero cells infected during G{sub 1} phase than in cells infected during S/G{sub 2} phase. - Highlights: • SINV infection was able to alter the cell cycle progression of infected cancer cells. • SINV infection can affect the expression of cell cycle regulators. • SINV infection exhibited a preference for the timing of viral replication among the cell cycle phases.

  2. Magnolol causes alterations in the cell cycle in androgen insensitive human prostate cancer cells in vitro by affecting expression of key cell cycle regulatory proteins. (United States)

    McKeown, Brendan T; McDougall, Luke; Catalli, Adriana; Hurta, Robert A R


    Prostate cancer, one of the most common cancers in the Western world, affects many men worldwide. This study investigated the effects of magnolol, a compound found in the roots and bark of the magnolia tree Magnolia officinalis, on the behavior of 2 androgen insensitive human prostate cancer cell lines, DU145 and PC3, in vitro. Magnolol, in a 24-h exposure at 40 and 80 μM, was found to be cytotoxic to cells. Magnolol also affected cell cycle progression of DU145 and PC3 cells, resulting in alterations to the cell cycle and subsequently decreasing the proportion of cells entering the G2/M-phase of the cell cycle. Magnolol inhibited the expression of cell cycle regulatory proteins including cyclins A, B1, D1, and E, as well as CDK2 and CDK4. Protein expression levels of pRBp107 decreased and pRBp130 protein expression levels increased in response to magnolol exposure, whereas p16(INK4a), p21, and p27 protein expression levels were apparently unchanged post 24-h exposure. Magnolol exposure at 6 h did increase p27 protein expression levels. This study has demonstrated that magnolol can alter the behavior of androgen insensitive human prostate cancer cells in vitro and suggests that magnolol may have potential as a novel anti-prostate cancer agent.

  3. δ-Catenin promotes prostate cancer cell growth and progression by altering cell cycle and survival gene profiles

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    Chen Yan-Hua


    Full Text Available Abstract Background δ-Catenin is a unique member of β-catenin/armadillo domain superfamily proteins and its primary expression is restricted to the brain. However, δ-catenin is upregulated in human prostatic adenocarcinomas, although the effects of δ-catenin overexpression in prostate cancer are unclear. We hypothesized that δ-catenin plays a direct role in prostate cancer progression by altering gene profiles of cell cycle regulation and cell survival. Results We employed gene transfection and small interfering RNA to demonstrate that increased δ-catenin expression promoted, whereas its knockdown suppressed prostate cancer cell viability. δ-Catenin promoted prostate cancer cell colony formation in soft agar as well as tumor xenograft growth in nude mice. Deletion of either the amino-terminal or carboxyl-terminal sequences outside the armadillo domains abolished the tumor promoting effects of δ-catenin. Quantitative RT2 Profiler™ PCR Arrays demonstrated gene alterations involved in cell cycle and survival regulation. δ-Catenin overexpression upregulated cyclin D1 and cdc34, increased phosphorylated histone-H3, and promoted the entry of mitosis. In addition, δ-catenin overexpression resulted in increased expression of cell survival genes Bcl-2 and survivin while reducing the cell cycle inhibitor p21Cip1. Conclusion Taken together, our studies suggest that at least one consequence of an increased expression of δ-catenin in human prostate cancer is the alteration of cell cycle and survival gene profiles, thereby promoting tumor progression.

  4. Rapid alterations of cell cycle control proteins in human T lymphocytes in microgravity

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    Thiel Cora S


    Full Text Available Abstract In our study we aimed to identify rapidly reacting gravity-responsive mechanisms in mammalian cells in order to understand if and how altered gravity is translated into a cellular response. In a combination of experiments using "functional weightlessness" provided by 2D-clinostats and real microgravity provided by several parabolic flight campaigns and compared to in-flight-1g-controls, we identified rapid gravity-responsive reactions inside the cell cycle regulatory machinery of human T lymphocytes. In response to 2D clinorotation, we detected an enhanced expression of p21 Waf1/Cip1 protein within minutes, less cdc25C protein expression and enhanced Ser147-phosphorylation of cyclinB1 after CD3/CD28 stimulation. Additionally, during 2D clinorotation, Tyr-15-phosphorylation occurred later and was shorter than in the 1 g controls. In CD3/CD28-stimulated primary human T cells, mRNA expression of the cell cycle arrest protein p21 increased 4.1-fold after 20s real microgravity in primary CD4+ T cells and 2.9-fold in Jurkat T cells, compared to 1 g in-flight controls after CD3/CD28 stimulation. The histone acetyltransferase (HAT inhibitor curcumin was able to abrogate microgravity-induced p21 mRNA expression, whereas expression was enhanced by a histone deacetylase (HDAC inhibitor. Therefore, we suppose that cell cycle progression in human T lymphocytes requires Earth gravity and that the disturbed expression of cell cycle regulatory proteins could contribute to the breakdown of the human immune system in space.

  5. Menstrual cycle distribution of uterine natural killer cells is altered in heavy menstrual bleeding. (United States)

    Biswas Shivhare, Sourima; Bulmer, Judith N; Innes, Barbara A; Hapangama, Dharani K; Lash, Gendie E


    Heavy menstrual bleeding (HMB) affects 30% of women of reproductive age and significantly interferes with quality of life. Altered endometrial vascular maturation has been reported in HMB and recurrent miscarriage, the latter associated with increased uterine natural killer (uNK) cell numbers. This study compared endometrial leukocyte populations in controls and women with HMB. Formalin-fixed paraffin-embedded endometrial biopsies from controls (without endometrial pathology) and HMB were immunostained for CD14 (macrophages), CD56 (uNK cells), CD83 (dendritic cells), FOXP3 (regulatory T cells/Tregs), CD3 and CD8 (T cells). Leukocyte numbers were analysed as a percentage of total stromal cells in five randomly selected fields of view in the stratum functionalis of each sample. In control women across the menstrual cycle, 2-8% of total stromal cells were CD3(+) cells, 2-4% were CD8(+) T cells and 6-8% were CD14(+) macrophages. Compared with controls, CD3(+) cells were reduced during the mid-secretory phase (4%, P<0.01) and increased in the late secretory phase (12%, P=0.01) in HMB. CD83(+) dendritic cells and FOXP3(+) Tregs were scarce throughout the menstrual cycle in both groups. In controls, 2% of stromal cells in proliferative endometrium were CD56(+) uNK cells, increasing to 17% during the late secretory phase. In HMB, CD56(+) uNK cells were increased in the proliferative (5%, P<0.01) and early secretory (4%, P<0.02) phases, but reduced (10%, P<0.01) in the late secretory phase. This study demonstrates dysregulation of uNK cells in HMB, the functional consequence of which may have an impact on endometrial vascular development and/or endometrial preparation for menstruation.

  6. Mechanical Unloading of Mouse Bone in Microgravity Significantly Alters Cell Cycle Gene Set Expression (United States)

    Blaber, Elizabeth; Dvorochkin, Natalya; Almeida, Eduardo; Kaplan, Warren; Burns, Brnedan


    Spaceflight factors, including microgravity and space radiation, have many detrimental short-term effects on human physiology, including muscle and bone degradation, and immune system dysfunction. The long-term progression of these physiological effects is still poorly understood, and a serious concern for long duration spaceflight missions. We hypothesized that some of the degenerative effects of spaceflight may be caused in part by an inability of stem cells to proliferate and differentiate normally resulting in an impairment of tissue regenerative processes. Furthermore, we hypothesized that long-term bone tissue degeneration in space may be mediated by activation of the p53 signaling network resulting in cell cycle arrest and/or apoptosis in osteoprogenitors. In our analyses we found that spaceflight caused significant bone loss in the weight-bearing bones of mice with a 6.3% reduction in bone volume and 11.9% decrease in bone thickness associated with increased osteoclastic activity. Along with this rapid bone loss we also observed alterations in the cell cycle characterized by an increase in the Cdkn1a/p21 cell cycle arrest molecule independent of Trp53. Overexpression of Cdkn1a/p21 was localized to osteoblasts lining the periosteal surface of the femur and chondrocytes in the head of the femur, suggesting an inhibition of proliferation in two key regenerative cell types of the femur in response to spaceflight. Additionally we found overexpression of several matrix degradation molecules including MMP-1a, 3 and 10, of which MMP-10 was localized to osteocytes within the shaft of the femur. This, in conjunction with 40 nm resolution synchrotron nano-Computed Tomography (nano-CT) observations of an increase in osteocyte lacunae cross-sectional area, perimeter and a decrease in circularity indicates a potential role for osteocytic osteolysis in the observed bone degeneration in spaceflight. To further investigate the genetic response of bone to mechanical

  7. Propionibacterium acnes inhibits FOXM1 and induces cell cycle alterations in human primary prostate cells

    DEFF Research Database (Denmark)

    Sayanjali, Behnam; Christensen, Gitte J M; Al-Zeer, Munir A;


    Propionibacterium acnes has been detected in diseased human prostate tissue, and cell culture experiments suggest that the bacterium can establish a low-grade inflammation. Here, we investigated its impact on human primary prostate epithelial cells. Microarray analysis confirmed the inflammation......-inducing capability of P. acnes but also showed deregulation of genes involved in the cell cycle. qPCR experiments showed that viable P. acnes downregulates a master regulator of cell cycle progression, FOXM1. Flow cytometry experiments revealed that P. acnes increases the number of cells in S-phase. We tested...... the hypothesis that a P. acnes-produced berninamycin-like thiopeptide is responsible for this effect, since it is related to the FOXM1 inhibitor siomycin. The thiopeptide biosynthesis gene cluster was strongly expressed; it is present in subtype IB of P. acnes, but absent from type IA, which is most abundant...

  8. Propionibacterium acnes inhibits FOXM1 and induces cell cycle alterations in human primary prostate cells. (United States)

    Sayanjali, Behnam; Christensen, Gitte J M; Al-Zeer, Munir A; Mollenkopf, Hans-Joachim; Meyer, Thomas F; Brüggemann, Holger


    Propionibacterium acnes has been detected in diseased human prostate tissue, and cell culture experiments suggest that the bacterium can establish a low-grade inflammation. Here, we investigated its impact on human primary prostate epithelial cells. Microarray analysis confirmed the inflammation-inducing capability of P. acnes but also showed deregulation of genes involved in the cell cycle. qPCR experiments showed that viable P. acnes downregulates a master regulator of cell cycle progression, FOXM1. Flow cytometry experiments revealed that P. acnes increases the number of cells in S-phase. We tested the hypothesis that a P. acnes-produced berninamycin-like thiopeptide is responsible for this effect, since it is related to the FOXM1 inhibitor siomycin. The thiopeptide biosynthesis gene cluster was strongly expressed; it is present in subtype IB of P. acnes, but absent from type IA, which is most abundant on human skin. A knock-out mutant lacking the gene encoding the berninamycin-like peptide precursor was unable to downregulate FOXM1 and to halt the cell cycle. Our study reveals a novel host cell-interacting activity of P. acnes.

  9. Aluminum oxide nanoparticles alter cell cycle progression through CCND1 and EGR1 gene expression in human mesenchymal stem cells. (United States)

    Periasamy, Vaiyapuri Subbarayan; Athinarayanan, Jegan; Alshatwi, Ali A


    Aluminum oxide nanoparticles (Al2 O3 -NPs) are important ceramic materials that have been used in a variety of commercial and industrial applications. However, the impact of acute and chronic exposure to Al2 O3 -NPs on the environment and on human health has not been well studied. In this investigation, we evaluated the cytotoxic effects of Al2 O3 -NPs on human mesenchymal stem cells (hMSCs) by using a cell viability assay and observing cellular morphological changes, analyzing cell cycle progression, and monitoring the expression of cell cycle response genes (PCNA, EGR1, E2F1, CCND1, CCNC, CCNG1, and CYCD3). The Al2 O3 -NPs reduced hMSC viability in a dose- and time-dependent manner. Nuclear condensation and fragmentation, chromosomal DNA fragmentation, and cytoplasmic vacuolization were observed in Al2 O3 -NP-exposed cells. The nuclear morphological changes indicated that Al2 O3 -NPs alter cell cycle progression and gene expression. The cell cycle distribution revealed that Al2 O3 -NPs cause cell cycle arrest in the sub-G0-G1 phase, and this is associated with a reduction in the cell population in the G2/M and G0/G1 phases. Moreover, Al2 O3 -NPs induced the upregulation of cell cycle response genes, including EGR1, E2F1, and CCND1. Our results suggested that exposure to Al2 O3 -NPs could cause acute cytotoxic effects in hMSCs through cell cycle regulatory genes.

  10. Genistein inhibits invasive potential of human hepatocellular carcinoma by altering cell cycle, apoptosis, and angiogenesis

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    Yan Gu; Chen-Fang Zhu; Hitoshi Iwamoto; Ji-Sheng Chen


    AIM: To study the in vitro and in vivo inhibitory effects of genistein on invasive potential of Bel 7402 hepatocellular carcinoma (HCC) cells and to explore the underlying mechanism.METHODS: Bel 7402 HCC cells were exposed to genistein. The invasive activity of tumor cells was assayed in transwell cell culture chamber. p125FAK expression and cell cycle were evaluated by a functional assay. Cell apoptosis analysis was performed with TUNEL method. In addition, bilateral subrenal capsule xenograft transplantation of HCC was performed in 10 nude mice.Genistein was injected and the invasion of HCC into the renal parenchyma was observed. Microvessels with immunohistochemical staining were detected.RESULTS: Genistein significantly inhibited the growth of Bel 7402 cells, the inhibitory rate of tumor cells was 26 -42%. The invasive potential of Bel 7402 cells in vitro was significantly inhibited, the inhibitory rate was 11-28%. Genistein caused G2/M cell cycle arrest, S phase decreased significantly. The occurrence of apoptosis in genistein group increased significantly. The expression of p125FAK in 5 μg/mL genistein group (15.26±0.16%)and 10 μg/mL genistein group (12.89±0.36%) was significantly lower than that in the control group (19.75± 1.12%,P<0.05). Tumor growth in genistein-treated nude mice was significantly retarded in comparison to control mice, the inhibitory rate of tumor growth was about 20%. Genistein also significantly inhibited the invasion of Bel 7402 cells into the renal parenchyma of nude mice with xenograft transplant. The positive unit value of microvessels in genistein-treated group (10.422±0.807)was significantly lower than that in control group (22.330 ± 5.696, P< 0.01).CONCLUSION: Genistein can effectively inhibit the invasive potential of Bel 7402 HCC cells by altering cell cycle, apoptosis and angiogenesis, inhibition of focal adhesion kinase may play a significant role in this process.

  11. PLZF mutation alters mouse hematopoietic stem cell function and cell cycle progression. (United States)

    Vincent-Fabert, Christelle; Platet, Nadine; Vandevelde, Amelle; Poplineau, Mathilde; Koubi, Myriam; Finetti, Pascal; Tiberi, Guillaume; Imbert, Anne-Marie; Bertucci, François; Duprez, Estelle


    Hematopoietic stem cells (HSCs) give rise to all blood populations due to their long-term self-renewal and multipotent differentiation capacities. Because they have to persist throughout an organism's life span, HSCs tightly regulate the balance between proliferation and quiescence. Here, we investigated the role of the transcription factor promyelocytic leukemia zinc finger (plzf) in HSC fate using the Zbtb16(lu/lu)mouse model, which harbors a natural spontaneous mutation that inactivates plzf. Regenerative stress revealed that Zbtb16(lu/lu)HSCs had a lineage-skewing potential from lymphopoiesis toward myelopoiesis, an increase in the long-term-HSC pool, and a decreased repopulation potential. Furthermore, oldplzf-mutant HSCs present an amplified aging phenotype, suggesting that plzf controls age-related pathway. We found that Zbtb16(lu/lu)HSCs harbor a transcriptional signature associated with a loss of stemness and cell cycle deregulation. Lastly, cell cycle analyses revealed an important role for plzf in the regulation of the G1-S transition of HSCs. Our study reveals a new role for plzf in regulating HSC function that is linked to cell cycle regulation, and positions plzf as a key player in controlling HSC homeostasis.

  12. Inositol pyrophosphates modulate cell cycle independently of alteration in telomere length. (United States)

    Banfic, Hrvoje; Crljen, Vladiana; Lukinovic-Skudar, Vesna; Dembitz, Vilma; Lalic, Hrvoje; Bedalov, Antonio; Visnjic, Dora


    Synthesis of inositol pyrophosphates through activation of Kcs1 plays an important role in the signalling response required for cell cycle progression after mating pheromone arrest. Overexpression of Kcs1 doubled the level of inositol pyrophosphates when compared to wild type cells and 30 min following the release from α-factor block further increase in inositol pyrophosphates was observed, which resulted that cells overexpressing Kcs1 reached G2/M phase earlier than wild type cells. Similar effect was observed in ipk1Δ cells, which are unable to synthesize IP6-derived inositol pyrophosphates (IP7 and IP8) but will synthesize IP5-derived inositol pyrophosphates (PP-IP4 and (PP)2-IP3). Although ipk1Δ cells have shorter telomeres than wild type cells, overexpression of Kcs1 in both strains have similar effect on cell cycle progression. As it is known that PP-IP4 regulates telomere length through Tel1, inositol polyphosphates, cell cycle and telomere length were determined in tel1Δ cells. The release of the cells from α-factor block and overexpression of Kcs1 in tel1Δ cells produced similar effects on inositol pyrophosphates level and cell cycle progression when compared to wild type cells, although tel1Δ cells possesses shorter telomeres than wild type cells. It can be concluded that telomere length does not affect cell cycle progression, since cells with short telomeres (ipk1Δ and tel1Δ) progress through cell cycle in a similar manner as wild type cells and that overexpression of Kcs1 in cells with either short or normal telomeres will increase S phase progression without affecting telomere length. Furthermore, IP5-derived inositol pyrophosphates can compensate for the loss of IP6-derived inositol pyrophosphates, in modulating S phase progression of the cell cycle.

  13. Hypoxia alters cell cycle regulatory protein expression and induces premature maturation of oligodendrocyte precursor cells.

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    Ravi Shankar Akundi

    Full Text Available BACKGROUND: Periventricular white matter injury (PWMI is a common form of brain injury sustained by preterm infants. A major factor that predisposes to PWMI is hypoxia. Because oligodendrocytes (OLs are responsible for myelination of axons, abnormal OL development or function may affect brain myelination. At present our understanding of the influences of hypoxia on OL development is limited. To examine isolated effects of hypoxia on OLs, we examined the influences of hypoxia on OL development in vitro. METHODOLOGY/FINDINGS: Cultures of oligodendrocyte precursor cells (OPCs were prepared from mixed glial cultures and were 99% pure. OPCs were maintained at 21% O(2 or hypoxia (1% or 4% O(2 for up to 7 days. We observed that 1% O(2 lead to an increase in the proportion of myelin basic protein (MBP-positive OLs after 1 week in culture, and a decrease in the proportion of platelet-derived growth factor receptor alpha (PDGFRalpha-positive cells suggesting premature OL maturation. Increased expression of the cell cycle regulatory proteins p27(Kip1 and phospho-cdc2, which play a role in OL differentiation, was seen as well. CONCLUSIONS: These results show that hypoxia interferes with the normal process of OL differentiation by inducing premature OPC maturation.

  14. Altered Cell Cycle Arrest by Multifunctional Drug-Loaded Enzymatically-Triggered Nanoparticles. (United States)

    Huang, Can; Sun, Ying; Shen, Ming; Zhang, Xiangyu; Gao, Pei; Duan, Yourong


    cRGD-targeting matrix metalloproteinase (MMP)-sensitive nanoparticles [PLGA-PEG1K-cRGD/PLGA-peptide-PEG5K (NPs-cRGD)] were successfully developed. Au-Pt(IV) nanoparticles, PTX, and ADR were encapsulated into NPs-RGD separately. The effects of the drug-loaded nanoparticles on the cell cycle were investigated. Here, we showed that higher cytotoxicity of drug-loaded nanoparticles was related to the cell cycle arrest, compared to that of free drugs. The NPs-cRGD studied here did not disrupt cell cycle progression. The cell cycle of Au-Pt(IV)@NPs-cRGD showed a main S phase arrest in all phases of the cell cycle phase, especially in G0/G1 phase. PTX@NPs-cRGD and ADR@NPs-cRGD showed a higher ratio of G2/M and S phase arrest than the free drugs, respectively. Cells in G0/G1 and S phases of the cell cycle had a higher uptake ratio of NPs-cRGD. A nutrient deprivation or an increase in the requirement of nutrients in tumor cells could promote the uptake of nanoparticles from the microenvironments. In vivo, NPs-cRGD could efficiently accumulate at tumor sites. The inhibition of tumor growth coupled with cell cycle arrest is in line with that in vitro. On the basis of our results, we propose that future studies on nanoparticle action mechanism should consider the cell cycle, which could be different from free drugs. Understanding the actions of cell cycle arrest could affect the application of nanomedicine in the clinic.

  15. T-cell intracellular antigen (TIA-proteins deficiency in murine embryonic fibroblasts alters cell cycle progression and induces autophagy.

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    Carmen Sánchez-Jiménez

    Full Text Available Mice lacking either T-cell intracellular antigen 1 (TIA1 or TIA1 related/like protein (TIAR/TIAL1 show high rates of embryonic lethality, suggesting a relevant role for these proteins during embryonic development. However, intrinsic molecular and cellular consequences of either TIA1 or TIAR deficiency remain poorly defined. By using genome-wide expression profiling approach, we demonstrate that either TIA1 or TIAR inactivation broadly alter normal development-associated signalling pathways in murine embryonic fibroblasts (MEF. Indeed, these analyses highlighted alterations of cytokine-cytokine and ECM-receptor interactions and Wnt, MAPK, TGF-beta dependent signalling pathways. Consistent with these results, TIA1 and TIAR knockout (KO MEF show reduced rates of cell proliferation, cell cycle progression delay and increased cell size. Furthermore, TIA-proteins deficiency also caused metabolic deficiencies, increased ROS levels and DNA damage, promoting a gentle rise of cell death. Concomitantly, high rates of autophagy were detected in both TIA1 and TIAR KO MEF with induction of the formation of autophagosomes, as evidenced by the up-regulation of the LC3B protein, and autolysosomes, measured by colocalization of LC3B and LAMP1, as a survival mechanism attempt. Taken together, these observations support that TIA proteins orchestrate a transcriptome programme to activate specific developmental decisions. This program is likely to contribute to mouse physiology starting at early stages of the embryonic development. TIA1/TIAR might function as cell sensors to maintain homeostasis and promote adaptation/survival responses to developmental stress.

  16. Methoxychlor inhibits growth of antral follicles by altering cell cycle regulators. (United States)

    Gupta, Rupesh K; Meachum, Sharon; Hernández-Ochoa, Isabel; Peretz, Jackye; Yao, Humphrey H; Flaws, Jodi A


    Methoxychlor (MXC) reduces fertility in female rodents, decreases antral follicle numbers, and increases atresia through oxidative stress pathways. MXC also inhibits antral follicle growth in vitro. The mechanism by which MXC inhibits growth of follicles is unknown. The growth of follicles is controlled, in part, by cell cycle regulators. Thus, we tested the hypothesis that MXC inhibits follicle growth by reducing the levels of selected cell cycle regulators. Further, we tested whether co-treatment with an antioxidant, N-acetyl cysteine (NAC), prevents the MXC-induced reduction in cell cycle regulators. For in vivo studies, adult cycling CD-1 mice were dosed with MXC or vehicle for 20 days. Treated ovaries were subjected to immunohistochemistry for proliferating cell nuclear antigen (PCNA) staining. For in vitro studies, antral follicles isolated from adult cycling CD-1 mouse ovaries were cultured with vehicle, MXC, and/or NAC for 48, 72 and 96 h. Levels of cyclin D2 (Ccnd2) and cyclin dependent kinase 4 (Cdk4) were measured using in vivo and in vitro samples. The results indicate that MXC decreased PCNA staining, and Ccnd2 and Cdk4 levels compared to controls. NAC co-treatment restored follicle growth and expression of Ccnd2 and Cdk4. Collectively, these data indicate that MXC exposure reduces the levels of Ccnd2 and Cdk4 in follicles, and that protection from oxidative stress restores Ccnd2 and Cdk4 levels. Therefore, MXC-induced oxidative stress may decrease the levels of cell cycle regulators, which in turn, results in inhibition of the growth of antral follicles.

  17. Altered susceptibility to infection by Sinorhizobium meliloti and Nectria haematococca in alfalfa roots with altered cell cycle. (United States)

    Woo, H-H; Hirsch, A M; Hawes, M C


    Most infections of plant roots are initiated in the region of elongation; the mechanism for this tissue-specific localization pattern is unknown. In alfalfa expressing PsUGT1 antisense mRNA under the control of the cauliflower mosaic virus (CaMV) 35S promoter, the cell cycle in roots is completed in 48 h instead of 24 h, and border cell number is decreased by more than 99%. These plants were found to exhibit increased root-tip infection by a fungal pathogen and reduced nodule formation by a bacterial symbiont. Thus, the frequency of infection in the region of elongation by Nectria haematocca was unaffected, but infection of the root tip was increased by more than 90%; early stages of Sinorhizobium meliloti infection and nodule morphology were normal, but the frequency of nodulation was fourfold lower than in wild-type roots.

  18. Cytoskeleton disorder and cell cycle arrest may be associated with the alteration of protein CEP135 by microgravity (United States)

    Hang, Xiaoming; Sun, Yeqing; Wu, Di; Li, Yixiao; Liu, Zhiyuan

    In the past decades, alterations in the morphology, cytoskeleton and cell cycle have been observed in cells in vitro under microgravity conditions. But the underlying mechanisms are not absolutely identified yet. Our previous study on proteomic and microRNA expression profiles of zebrafish embryos exposed to simulated-microgravity has demonstrated a serial of microgravity-sensitive molecules. Centrosomal protein of 135 kDa (CEP135) was found down-regulated, but the mRNA expression level of it was up-regulated in zebrafish embryos after simulated-microgravity. However, the functional study on CEP135 is very limited and it has not been cloned in zebrafish till now. In this study, we try to determine whether the cytoskeleton disorder and cell cycle arrest is associated with the alteration of CEP135 by microgravity. Full-length cDNA of cep135 gene was firstly cloned from mitosis phase of ZF4. The sequence was analyzed and the phylogenetic tree was constructed based on the similarity to other species. Zebrafish embryonic cell line ZF4 were exposed to simulated microgravity for 24 and 48 hours, using a rotary cell culture system (RCCS) designed by NASA. Quantitative analysis by western blot showed that CEP135 expression level was significantly decreased two times after 24 hour simulated microgravity. Cell cycle detection by flow cytometer indicated ZF4 cells were blocked in G1 phase after 24 and 48 hour simulated microgravity. Moreover, double immunostained ZF4 cells with anti-tubulin and anti-CEP135antibodies demonstrated simulated microgravity could lead to cytoskeleton disorder and CEP135 abnormality. Further investigations are currently being carried out to determine whether knockdown and over-expression of CEP135 will modulate cytoskeleton and cell cycle. In vitro data in combination within vivo results might, at least in part, explain the dramatic effects of microgravity. Key Words: microgravity; CEP135; Cytoskeleton disorder; G1 arrest; ZF4 cell line

  19. Alteration of Cell Cycle Mediated by Zinc in Human Bronchial Epithelial Cells In Vitro (United States)

    Zinc (Zn2+), a ubiquitous ambient air contaminant, presents an oxidant challenge to the human lung and is linked to adverse human health effects. To further elucidate the adaptive and apoptotic cellular responses of human airway cells to Zn2+, we performed pilot studies to examin...

  20. Cell cycle-dependent alteration in NAC1 nuclear body dynamics and morphology (United States)

    Wu, Pei-Hsun; Hung, Shen-Hsiu; Ren, Tina; Shih, Ie-Ming; Tseng, Yiider


    NAC1, a BTB/POZ family member, has been suggested to participate in maintaining the stemness of embryonic stem cells and has been implicated in the pathogenesis of human cancer. In ovarian cancer, NAC1 upregulation is associated with disease aggressiveness and with the development of chemoresistance. Like other BTB/POZ proteins, NAC1 forms discrete nuclear bodies in non-dividing cells. To investigate the biological role of NAC1 nuclear bodies, we characterized the expression dynamics of NAC1 nuclear bodies during different phases of the cell cycle. Fluorescence recovery after photobleaching assays revealed that NAC1 was rapidly exchanged between the nucleoplasm and NAC1 nuclear bodies in interphase cells. The number of NAC1 bodies significantly increased and their size decreased in the S phase as compared to the G0/G1 and G2 phases. NAC1 nuclear bodies disappeared and NAC1 became diffuse during mitosis. NAC1 nuclear bodies reappeared immediately after completion of mitosis. These results indicate that a cell cycle-dependent regulatory mechanism controls NAC1 body formation in the nucleus and suggest that NAC1 body dynamics are associated with mitosis or cytokinesis.

  1. Alterations in the cell cycle in the cerebellum of hyperbilirubinemic Gunn rat: a possible link with apoptosis?

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    María Celeste Robert

    Full Text Available Severe hyperbilirubinemia causes neurological damage both in humans and rodents. The hyperbilirubinemic Gunn rat shows a marked cerebellar hypoplasia. More recently bilirubin ability to arrest the cell cycle progression in vascular smooth muscle, tumour cells, and, more importantly, cultured neurons has been demonstrated. However, the involvement of cell cycle perturbation in the development of cerebellar hypoplasia was never investigated before. We explored the effect of sustained spontaneous hyperbilirubinemia on cell cycle progression and apoptosis in whole cerebella dissected from 9 day old Gunn rat by Real Time PCR, Western blot and FACS analysis. The cerebellum of the hyperbilirubinemic Gunn rats exhibits an increased cell cycle arrest in the late G0/G1 phase (p < 0.001, characterized by a decrease in the protein expression of cyclin D1 (15%, p < 0.05, cyclin A/A1 (20 and 30%, p < 0.05 and 0.01, respectively and cyclin dependent kinases2 (25%, p < 0.001. This was associated with a marked increase in the 18 kDa fragment of cyclin E (67%, p < 0.001 which amplifies the apoptotic pathway. In line with this was the increase of the cleaved form of Poly (ADP-ribose polymerase (54%, p < 0.01 and active Caspase3 (two fold, p < 0.01. These data indicate that the characteristic cerebellar alteration in this developing brain structure of the hyperbilirubinemic Gunn rat may be partly due to cell cycle perturbation and apoptosis related to the high bilirubin concentration in cerebellar tissue mainly affecting granular cells. These two phenomena might be intimately connected.

  2. Demethylation and alterations in the expression level of the cell cycle-related genes as possible mechanisms in arsenic trioxide-induced cell cycle arrest in human breast cancer cells. (United States)

    Moghaddaskho, Farima; Eyvani, Haniyeh; Ghadami, Mohsen; Tavakkoly-Bazzaz, Javad; Alimoghaddam, Kamran; Ghavamzadeh, Ardeshir; Ghaffari, Seyed H


    Arsenic trioxide (As2O3) has been used clinically as an anti-tumor agent. Its mechanisms are mostly considered to be the induction of apoptosis and cell cycle arrest. However, the detailed molecular mechanisms of its anti-cancer action through cell cycle arrest are poorly known. Furthermore, As2O3 has been shown to be a potential DNA methylation inhibitor, inducing DNA hypomethylation. We hypothesize that As2O3 may affect the expression of cell cycle regulatory genes by interfering with DNA methylation patterns. To explore this, we examined promoter methylation status of 24 cell cycle genes in breast cancer cell lines and in a normal breast tissue sample by methylation-specific polymerase chain reaction and/or restriction enzyme-based methods. Gene expression level and cell cycle distribution were quantified by real-time polymerase chain reaction and flow cytometric analyses, respectively. Our methylation analysis indicates that only promoters of RBL1 (p107), RASSF1A, and cyclin D2 were aberrantly methylated in studied breast cancer cell lines. As2O3 induced CpG island demethylation in promoter regions of these genes and restores their expression correlated with DNA methyltransferase inhibition. As2O3 also induced alterations in messenger RNA expression of several cell cycle-related genes independent of demethylation. Flow cytometric analysis revealed that the cell cycle arrest induced by As2O3 varied depending on cell lines, MCF-7 at G1 phase and both MDA-MB-231 and MDA-MB-468 cells at G2/M phase. These changes at transcriptional level of the cell cycle genes by the molecular mechanisms dependent and independent of demethylation are likely to represent the mechanisms of cell cycle redistribution in breast cancer cells, in response to As2O3 treatment.

  3. Melanogenesis stimulation in B16-F10 melanoma cells induces cell cycle alterations, increased ROS levels and a differential expression of proteins as revealed by proteomic analysis

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    Cunha, Elizabeth S.; Kawahara, Rebeca [Departamento de Bioquimica e Biologia Molecular, Setor de Ciencias Biologicas, Universidade Federal do Parana, P.O. Box 19046, CEP 81531-990, Curitiba, PR (Brazil); Kadowaki, Marina K. [Universidade Estadual do Oeste do Parana, Cascavel, PR (Brazil); Amstalden, Hudson G.; Noleto, Guilhermina R.; Cadena, Silvia Maria S.C.; Winnischofer, Sheila M.B. [Departamento de Bioquimica e Biologia Molecular, Setor de Ciencias Biologicas, Universidade Federal do Parana, P.O. Box 19046, CEP 81531-990, Curitiba, PR (Brazil); Martinez, Glaucia R., E-mail: [Departamento de Bioquimica e Biologia Molecular, Setor de Ciencias Biologicas, Universidade Federal do Parana, P.O. Box 19046, CEP 81531-990, Curitiba, PR (Brazil)


    Considering that stimulation of melanogenesis may lead to alterations of cellular responses, besides melanin production, our main goal was to study the cellular effects of melanogenesis stimulation of B16-F10 melanoma cells. Our results show increased levels of the reactive oxygen species after 15 h of melanogenesis stimulation. Following 48 h of melanogenesis stimulation, proliferation was inhibited (by induction of cell cycle arrest in the G1 phase) and the expression levels of p21 mRNA were increased. In addition, melanogenesis stimulation did not induce cellular senescence. Proteomic analysis demonstrated the involvement of proteins from other pathways besides those related to the cell cycle, including protein disulfide isomerase A3, heat-shock protein 70, and fructose biphosphate aldolase A (all up-regulated), and lactate dehydrogenase (down-regulated). In RT-qPCR experiments, the levels of pyruvate kinase M2 mRNA dropped, whereas the levels of ATP synthase (beta-F1) mRNA increased. These data indicate that melanogenesis stimulation of B16-F10 cells leads to alterations in metabolism and cell cycle progression that may contribute to an induction of cell quiescence, which may provide a mechanism of resistance against cellular injury promoted by melanin synthesis. -- Highlights: Black-Right-Pointing-Pointer Melanogenesis stimulation by L-tyrosine+NH{sub 4}Cl in B16-F10 melanoma cells increases ROS levels. Black-Right-Pointing-Pointer Melanogenesis inhibits cell proliferation, and induced cell cycle arrest in the G1 phase. Black-Right-Pointing-Pointer Proteomic analysis showed alterations in proteins of the cell cycle and glucose metabolism. Black-Right-Pointing-Pointer RT-qPCR analysis confirmed alterations of metabolic targets after melanogenesis stimulation.

  4. Human-chimpanzee differences in a FZD8 enhancer alter cell-cycle dynamics in the developing neocortex. (United States)

    Boyd, J Lomax; Skove, Stephanie L; Rouanet, Jeremy P; Pilaz, Louis-Jan; Bepler, Tristan; Gordân, Raluca; Wray, Gregory A; Silver, Debra L


    The human neocortex differs from that of other great apes in several notable regards, including altered cell cycle, prolonged corticogenesis, and increased size [1-5]. Although these evolutionary changes most likely contributed to the origin of distinctively human cognitive faculties, their genetic basis remains almost entirely unknown. Highly conserved non-coding regions showing rapid sequence changes along the human lineage are candidate loci for the development and evolution of uniquely human traits. Several studies have identified human-accelerated enhancers [6-14], but none have linked an expression difference to a specific organismal trait. Here we report the discovery of a human-accelerated regulatory enhancer (HARE5) of FZD8, a receptor of the Wnt pathway implicated in brain development and size [15, 16]. Using transgenic mice, we demonstrate dramatic differences in human and chimpanzee HARE5 activity, with human HARE5 driving early and robust expression at the onset of corticogenesis. Similar to HARE5 activity, FZD8 is expressed in neural progenitors of the developing neocortex [17-19]. Chromosome conformation capture assays reveal that HARE5 physically and specifically contacts the core Fzd8 promoter in the mouse embryonic neocortex. To assess the phenotypic consequences of HARE5 activity, we generated transgenic mice in which Fzd8 expression is under control of orthologous enhancers (Pt-HARE5::Fzd8 and Hs-HARE5::Fzd8). In comparison to Pt-HARE5::Fzd8, Hs-HARE5::Fzd8 mice showed marked acceleration of neural progenitor cell cycle and increased brain size. Changes in HARE5 function unique to humans thus alter the cell-cycle dynamics of a critical population of stem cells during corticogenesis and may underlie some distinctive anatomical features of the human brain.

  5. Phosphorylation states of cell cycle and DNA repair proteins can be altered by the nsSNPs

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    Savas Sevtap


    Full Text Available Abstract Background Phosphorylation is a reversible post-translational modification that affects the intrinsic properties of proteins, such as structure and function. Non-synonymous single nucleotide polymorphisms (nsSNPs result in the substitution of the encoded amino acids and thus are likely to alter the phosphorylation motifs in the proteins. Methods In this study, we used the web-based NetPhos tool to predict candidate nsSNPs that either introduce or remove putative phosphorylation sites in proteins that act in DNA repair and cell cycle pathways. Results Our results demonstrated that a total of 15 nsSNPs (16.9% were likely to alter the putative phosphorylation patterns of 14 proteins. Three of these SNPs (CDKN1A-S31R, OGG1-S326C, and XRCC3-T241M have already found to be associated with altered cancer risk. We believe that this set of nsSNPs constitutes an excellent resource for further molecular and genetic analyses. Conclusion The novel systematic approach used in this study will accelerate the understanding of how naturally occurring human SNPs may alter protein function through the modification of phosphorylation mechanisms and contribute to disease susceptibility.

  6. Altered cell cycle gene expression and apoptosis in post-implantation dog parthenotes.

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    Jung Eun Park

    Full Text Available Mature oocytes can be parthenogenetically activated by a variety of methods and the resulting embryos are valuable for studies of the respective roles of paternal and maternal genomes in early mammalian development. In the present study, we report the first successful development of parthenogenetic canine embryos to the post-implantation stage. Nine out of ten embryo transfer recipients became pregnant and successful in utero development of canine parthenotes was confirmed. For further evaluation of these parthenotes, their fetal development was compared with artificially inseminated controls and differentially expressed genes (DEGs were compared using ACP RT-PCR, histological analysis and immunohistochemistry. We found formation of the limb-bud and no obvious differences in histological appearance of the canine parthenote recovered before degeneration occurred; however canine parthenotes were developmentally delayed with different cell cycle regulating-, mitochondria-related and apoptosis-related gene expression patterns compared with controls. In conclusion, our protocols were suitable for activating canine oocytes artificially and supported early fetal development. We demonstrated that the developmental abnormalities in canine parthenotes may result from defective regulation of apoptosis and aberrant gene expression patterns, and provided evidence that canine parthenotes can be a useful tool for screening and for comparative studies of imprinted genes.

  7. Altered cell cycle-related gene expression in brain and lymphocytes from a transgenic mouse model of Alzheimer's disease [amyloid precursor protein/presenilin 1 (PS1)]. (United States)

    Esteras, Noemí; Bartolomé, Fernando; Alquézar, Carolina; Antequera, Desireé; Muñoz, Úrsula; Carro, Eva; Martín-Requero, Ángeles


    Cumulative evidence indicates that aberrant re-expression of many cell cycle-related proteins and inappropriate neuronal cell cycle control are critical events in Alzheimer's disease (AD) pathogenesis. Evidence of cell cycle activation in post-mitotic neurons has also been observed in murine models of AD, despite the fact that most of these mice do not show massive loss of neuronal bodies. Dysfunction of the cell cycle appears to affect cells other than neurons, as peripheral cells, such as lymphocytes and fibroblasts from patients with AD, show an altered response to mitogenic stimulation. We sought to determine whether cell cycle disturbances are present simultaneously in both brain and peripheral cells from the amyloid precursor protein (APP)/presenilin 1 (PS1) mouse model of AD, in order to validate the use of peripheral cells from patients not only to study cell cycle abnormalities as a pathogenic feature of AD, but also as a means to test novel therapeutic approaches. By using cell cycle pathway-specific RT(2)Profiler™ PCR Arrays, we detected changes in a number of cell cycle-related genes in brain as well as in lymphocytes from APP/PS1 mice. Moreover, we found enhanced 5'-bromo-2'-deoxyuridine incorporation into DNA in lymphocytes from APP/PS1 mice, and increased expression of the cell proliferation marker proliferating cell nuclear antigen (PCNA), and the cyclin-dependent kinase (CDK) inhibitor Cdkn2a, as detected by immunohistochemistry in cortical neurons of the APP/PS1 mice. Taken together, the cell cycle-related changes in brain and blood cells reported here support the mitosis failure hypothesis in AD and validate the use of peripheral cells as surrogate tissue to study the molecular basis of AD pathogenesis.

  8. Hodgkin and Reed-Sternberg cells harbor alterations in the major tumor suppressor pathways and cell-cycle checkpoints: analyses using tissue microarrays. (United States)

    García, Juan F; Camacho, Francisca I; Morente, Manuel; Fraga, Máximo; Montalbán, Carlos; Alvaro, Tomás; Bellas, Carmen; Castaño, Angel; Díez, Ana; Flores, Teresa; Martin, Carmen; Martinez, Miguel A; Mazorra, Francisco; Menárguez, Javier; Mestre, Maria J; Mollejo, Manuela; Sáez, Ana I; Sánchez, Lydia; Piris, Miguel A


    Tumoral cells in Hodgkin lymphoma (HL) display an increased growth fraction and diminished apoptosis, implying a profound disturbance of the cell cycle and apoptosis regulation. However, limitations of molecular techniques have prevented the analysis of the tumor suppressor pathways and cell-cycle checkpoints. Tissue microarray (TMA) is a powerful tool for analyzing a large number of molecular variables in a large series of tumors, although the feasibility of this technique has not yet been demonstrated in heterogeneous tumors. The expression of 29 genes regulating the cell cycle and apoptosis were analyzed by immunohistochemistry and in situ hybridization in 288 HL biopsies using TMA. The sensitivity of the technique was validated by comparing the results with those obtained in standard tissue sections. The results revealed multiple alterations in different pathways and checkpoints, including G1/S and G2/M transition and apoptosis. Striking findings were the overexpression of cyclin E, CDK2, CDK6, STAT3, Hdm2, Bcl2, Bcl-X(L), survivin, and NF-kappaB proteins. A multiparametric analysis identified proteins associated with increased growth fraction (Hdm2, p53, p21, Rb, cyclins A, B1, D3, and E, CDK2, CDK6, SKP2, Bcl-X(L), survivin, STAT1, and STAT3), and proteins associated with apoptosis (NF-kappaB, STAT1, and RB). The analysis also demonstrated that Epstein-Barr virus (EBV)-positive cases displayed a characteristic profile, confirming the pathogenic role of EBV in HL. Survival probability depends on multiple biologic factors, including overexpression of Bcl2, p53, Bax, Bcl-X(L), MIB1, and apoptotic index. In conclusion, Hodgkin and Reed-Sternberg cells harbor concurrent and overlapping alterations in the major tumor suppressor pathways and cell-cycle checkpoints. This appears to determine the viability of the tumoral cells and the clinical outcome.

  9. R-(+)-perillyl alcohol-induced cell cycle changes, altered actin cytoskeleton, and decreased ras and p34(cdc2) expression in colonic adenocarcinoma SW480 cells. (United States)

    Cerda, S R; Wilkinson, J; Thorgeirsdottir, S; Broitman, S A


    Monoterpenes as S-(-)-perillyl alcohol (PA) have been shown to inhibit the isoprenylation of such growth regulatory proteins as ras. In this study, we investigated the effects of the R-(+) enantiomer of PA on cell cycle, signaling, and cytoskeletal control in the colonic adenocarcinoma cell line SW480, which carries a K-ras mutation. Cell cycle analysis by flow cytometry of SW480 cells treated with 1 mM PA for 24 hours demonstrated an increase in the number of cells in G0/G1 with a decrease in S phase, compared with untreated control cells. These cell cycle changes correlated with an inhibition of protein isoprenylation from (14)C-mevalonate and decreased expression of the cell cycle regulatory kinase p34(cdc2). Additionally, PA-treated cells acquired a flattened morphology with a condensation of cytoskeletal actin spikes to the periphery. This was in contrast to treatment with 15 microM mevinolin (MVN), a direct mevalonate synthesis inhibitor, which imparted to SW480 cells a more rounded and spindly morphology, associated with the depolymerization of actin microfilaments. Together, these data suggest that fluctuations in mevalonate and isoprenoid pools may involve different morphologic phenomenon. Because ras mediated signaling is related to the organization of the actin cytoskeleton, we investigated the effects of PA on the isoprenylation of ras. Although MVN treatment inhibited ras farnesylation, PA treatment decreased the expression of total ras protein. In summary, R-(+)-PA-induced cell signaling events correlated with alterations in the organization of cytoskeletal actin and decreased protein expression of growth regulatory proteins, such as ras and cdc2 kinase. These effects may contribute to the growth inhibitory activity of R-(+)-PA.

  10. Hierridin B Isolated from a Marine Cyanobacterium Alters VDAC1, Mitochondrial Activity, and Cell Cycle Genes on HT-29 Colon Adenocarcinoma Cells

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    Sara Freitas


    Full Text Available Background: Hierridin B was isolated from a marine cyanobacterium Cyanobium sp. strain and induced cytotoxicity selectively in HT-29 adenocarcinoma cells. The underlying molecular mechanism was not yet elucidated. Methods: HT-29 cells were exposed to the IC50 concentration of hierridin B (100.2 μM for 48 h. Non-targeted proteomics was performed using 2D gel electrophoresis and MALDI-TOF/TOF mass spectrometry. The mRNA expression of apoptotic and cell cycle genes were analyzed by real-time PCR. Automated quantification of 160 cytoplasm and mitochondrial parameter was done by fluorescence microscopy using CellProfiler software. Results: Proteomics identified 21 significant different proteins, which belonged to protein folding/synthesis and cell structure amongst others. Increase of VDAC1 protein responsible for formation of mitochondrial channels was confirmed by mRNA expression. A 10-fold decrease of cytoskeleton proteins (STMN1, TBCA provided a link to alterations of the cell cycle. CCNB1 and CCNE mRNA were decreased two-fold, and P21CIP increased 10-fold, indicative of cell cycle arrest. Morphological analysis of mitochondrial parameter confirmed a reduced mitochondrial activity. Conclusion: Hierridin B is a potential anticancer compound that targets mitochondrial activity and function.

  11. ALDH1A1 maintains ovarian cancer stem cell-like properties by altered regulation of cell cycle checkpoint and DNA repair network signaling.

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    Erhong Meng

    Full Text Available OBJECTIVE: Aldehyde dehydrogenase (ALDH expressing cells have been characterized as possessing stem cell-like properties. We evaluated ALDH+ ovarian cancer stem cell-like properties and their role in platinum resistance. METHODS: Isogenic ovarian cancer cell lines for platinum sensitivity (A2780 and platinum resistant (A2780/CP70 as well as ascites from ovarian cancer patients were analyzed for ALDH+ by flow cytometry to determine its association to platinum resistance, recurrence and survival. A stable shRNA knockdown model for ALDH1A1 was utilized to determine its effect on cancer stem cell-like properties, cell cycle checkpoints, and DNA repair mediators. RESULTS: ALDH status directly correlated to platinum resistance in primary ovarian cancer samples obtained from ascites. Patients with ALDHHIGH displayed significantly lower progression free survival than the patients with ALDHLOW cells (9 vs. 3 months, respectively p<0.01. ALDH1A1-knockdown significantly attenuated clonogenic potential, PARP-1 protein levels, and reversed inherent platinum resistance. ALDH1A1-knockdown resulted in dramatic decrease of KLF4 and p21 protein levels thereby leading to S and G2 phase accumulation of cells. Increases in S and G2 cells demonstrated increased expression of replication stress associated Fanconi Anemia DNA repair proteins (FANCD2, FANCJ and replication checkpoint (pS317 Chk1 were affected. ALDH1A1-knockdown induced DNA damage, evidenced by robust induction of γ-H2AX and BAX mediated apoptosis, with significant increases in BRCA1 expression, suggesting ALDH1A1-dependent regulation of cell cycle checkpoints and DNA repair networks in ovarian cancer stem-like cells. CONCLUSION: This data suggests that ovarian cancer cells expressing ALDH1A1 may maintain platinum resistance by altered regulation of cell cycle checkpoint and DNA repair network signaling.

  12. Alteration in cell cycle-related proteins in lymphoblasts from carriers of the c.709-1G>A PGRN mutation associated with FTLD-TDP dementia. (United States)

    Alquezar, Carolina; Esteras, Noemí; Bartolomé, Fernando; Merino, José J; Alzualde, Ainhoa; López de Munain, Adolfo; Martín-Requero, Ángeles


    Frontotemporal lobar degeneration with neuronal inclusions containing TAR DNA binding protein 43 (TDP-43) is associated in most cases with null-mutations in the progranulin gene (PGRN). While the mechanisms by which PGRN haploinsufficiency leads to neurodegeneration remained speculative, increasing evidence support the hypothesis that cell cycle reentry of postmitotic neurons precedes many instances of neuronal death. Based in the mitogenic and neurotrophic activities of PGRN, we hypothesized that PGRN deficit may induce cell cycle disturbances and alterations in neuronal vulnerability. Because cell cycle dysfunction is not restricted to neurons, we studied the influence of PGRN haploinsufficiency, on cell cycle control in peripheral cells from patients suffering from frontotemporal dementia, bearing the PGRN mutation c.709-1G>A. Here we show that progranulin deficit increased cell cycle activity in immortalized lymphocytes. This effect was associated with increased levels of cyclin-dependent kinase 6 (CDK6) and phosphorylation of retinoblastoma protein (pRb), resulting in a G(1)/S regulatory failure. A loss of function of TDP-43 repressing CDK6 expression may result from altered subcellular TDP-43 distribution. The distinct functional features of lymphoblastoid cells from c.709-1 G>A carriers offer an invaluable, noninvasive tool to investigate the etiopathogenesis of frontotemporal lobar degeneration.

  13. In vitro alterations do not reflect a requirement for host cell cycle progression during Plasmodium liver stage infection. (United States)

    Hanson, Kirsten K; March, Sandra; Ng, Shengyong; Bhatia, Sangeeta N; Mota, Maria M


    Prior to invading nonreplicative erythrocytes, Plasmodium parasites undergo their first obligate step in the mammalian host inside hepatocytes, where each sporozoite replicates to generate thousands of merozoites. While normally quiescent, hepatocytes retain proliferative capacity and can readily reenter the cell cycle in response to diverse stimuli. Many intracellular pathogens, including protozoan parasites, manipulate the cell cycle progression of their host cells for their own benefit, but it is not known whether the hepatocyte cell cycle plays a role during Plasmodium liver stage infection. Here, we show that Plasmodium parasites can be observed in mitotic hepatoma cells throughout liver stage development, where they initially reduce the likelihood of mitosis and ultimately lead to significant acquisition of a binucleate phenotype. However, hepatoma cells pharmacologically arrested in S phase still support robust and complete Plasmodium liver stage development, which thus does not require cell cycle progression in the infected cell in vitro. Furthermore, murine hepatocytes remain quiescent throughout in vivo infection with either Plasmodium berghei or Plasmodium yoelii, as do Plasmodium falciparum-infected primary human hepatocytes, demonstrating that the rapid and prodigious growth of liver stage parasites is accomplished independent of host hepatocyte cell cycle progression during natural infection.

  14. Demethoxycurcumin alters gene expression associated with DNA damage, cell cycle and apoptosis in human lung cancer NCI-H460 cells in vitro. (United States)

    Ko, Yang-Ching; Hsu, Shu-Chun; Liu, Hsin-Chung; Hsiao, Yung-Ting; Hsia, Te-Chun; Yang, Su-Tso; Hsu, Wu-Huei; Chung, Jing-Gung


    Lung cancer is the leading cause of cancer-related deaths and new lung cancer cases are continuously emerging around the globe; however, treatment of lung cancer remains unsatisfactory. Demethoxycurcumin (DMC) has been shown to exert cytotoxic effects in human cancer cells via induction of apoptosis. However, the effects of DMC on genetic mechanisms associated with these actions have not been yet elucidated. Human lung cancer NCI-H460 cells were incubated with or without 35 μM of DMC for 24 h and total RNA was extracted for cDNA synthesis labeling and microarray hybridization, followed by fluor-labeled cDNA hybridization on chip. Expression Console software with default Robust Multichip Analysis (RMA) parameters were used for detecting and quantitating the localized concentrations of fluorescent molecules. The GeneGo software was used for investigating key genes involved and their possible interaction pathways. Genes associated with DNA damage and repair, cell-cycle check point and apoptosis could be altered by DMC; in particular, 144 genes were found up-regulated and 179 genes down-regulated in NCI-H460 cells after exposure to DMC. In general, DMC-altered genes may offer information to understand the cytotoxic mechanism of this agent at the genetic level since gene alterations can be useful biomarkers or targets for the diagnosis and treatment of human lung cancer in the future.

  15. α-Phellandrene alters expression of genes associated with DNA damage, cell cycle, and apoptosis in murine leukemia WEHI-3 cells. (United States)

    Lin, Jen-Jyh; Yu, Chien-Chih; Lu, Kung-Wen; Chang, Shu-Jen; Yu, Fu-Shun; Liao, Ching-Lung; Lin, Jaung-Geng; Chung, Jing-Gung


    α-phellandrene (α-PA) is a cyclic monoterpene, present in natural plants such as Schinus molle L. α-PA promotes immune responses in mice in vivo. However, there is no available information on whether α-PA affects gene expression in leukemia cells. The present study determined effects of α-PA on expression levels of genes associated with DNA damage, cell cycle and apoptotic cell death in mouse leukemia WEHI-3 cells. WEHI-3 cells were treated with 10 μM α-PA for 24 h, cells were harvested and total RNA was extracted, and gene expression was analyzed by cDNA microarray. Results indicated that α-PA up-regulated 10 genes 4-fold, 13 by over 3-fold and 175 by over 2-fold; 21 genes were down-regulated by over 4-fold, 26 genes by over 3-fold and expression of 204 genes was altered by at leas 2-fold compared with the untreated control cells. DNA damage-associated genes such as DNA damage-inducer transcript 4 and DNA fragmentation factor were up-regulated by 4-fold and over 2-fold, respectively; cell-cycle check point genes such as cyclin G2 and cyclin-dependent kinases inhibitor 2D and IA (p21) were up-regulated by over 3-fold and over 2-fold, respectively; apoptosis-associated genes such as BCL2/adenovirus EIB interacting protein 3, XIAP-associated factor 1, BCL2 modifying factor, caspase-8 and FADD-like apoptosis regulator were over 2-fold up-regulated. Furthermore, DNA damage-associated gene TATA box binding protein was over 4-fold down-regulated, and D19Ertd652c (DNA segment) over 2-fold down-regulated; cell cycle-associated gene cyclin E2 was over 2-fold down-regulated; apoptosis associated gene growth arrest-specific 5 was over 9-fold down-regulated, Gm5426 (ATP synthase) was over 3-fold down-regulated, and death box polypeptide 33 was over 2-fold down-regulated. Based on these observations, α-PA altered gene expression in WEHI-3 cells in vitro.

  16. Light pulses administered during the circadian dark phase alter expression of cell cycle associated transcripts in mouse brain. (United States)

    Ben-Shlomo, R; Kyriacou, C P


    The circadian mode of cell division has been known for more than a century, but the association between circadian rhythms and mitosis is not yet clear. Synchronization of circadian oscillators with the outside world is achieved because light, or other external temporal cues, have acute effects on the levels of the clock's molecular components. Thus, an important question is whether environmental signals also affect transcription levels of cell machinery genes in a similar manner? In a microarray analysis, we have tested the influence of light pulses on the expression of transcripts in the mouse brain. Light pulses consistently affect transcription levels of genes that are essential and directly control the cell cycle mechanism, as well as levels of genes that are associated with the various cell cycle checkpoints. The changes in the levels and the direction of these changes could possibly lead to cell cycle arrest. We also found consistent changes in transcription levels of genes that are associated with tumorigenesis and are directly implicated with enhanced proliferation and metastasis.

  17. Disrupted cell cycle arrest and reduced proliferation in corneal fibroblasts from GCD2 patients: A potential role for altered autophagy flux

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    Choi, Seung-il; Dadakhujaev, Shorafidinkhuja; Maeng, Yong-Sun; Ahn, So-yeon; Kim, Tae-im [Department of Ophthalmology, Severance Hospital, Yonsei University College of Medicine, Seoul (Korea, Republic of); Corneal Dystrophy Research Institute, Yonsei University College of Medicine, Seoul (Korea, Republic of); Kim, Eung Kweon, E-mail: [Department of Ophthalmology, Severance Hospital, Yonsei University College of Medicine, Seoul (Korea, Republic of); Corneal Dystrophy Research Institute, Yonsei University College of Medicine, Seoul (Korea, Republic of); BK21 Plus Project for Medical Science and Severance Biomedical Science Institute, Yonsei University College of Medicine, Seoul (Korea, Republic of)


    Highlights: • Reduced cell proliferation in granular corneal dystrophy type 2. • Abnormal cell cycle arrest by defective autophagy. • Decreased Cyclin A1, B1, and D1 in Atg7 gene knockout cells. • Increase in p16 and p27 expressions were observed in Atg7 gene knockout cells. - Abstract: This study investigates the role of impaired proliferation, altered cell cycle arrest, and defective autophagy flux of corneal fibroblasts in granular corneal dystrophy type 2 (GCD2) pathogenesis. The proliferation rates of homozygous (HO) GCD2 corneal fibroblasts at 72 h, 96 h, and 120 h were significantly lower (1.102 ± 0.027, 1.397 ± 0.039, and 1.527 ± 0.056, respectively) than those observed for the wild-type (WT) controls (1.441 ± 0.029, 1.758 ± 0.043, and 2.003 ± 0.046, respectively). Flow cytometry indicated a decreased G{sub 1} cell cycle progression and the accumulation of cells in the S and G{sub 2}/M phases in GCD2 cells. These accumulations were associated with decreased levels of Cyclin A1, B1, and E1, and increased expression of p16 and p27. p21 and p53 expression was also significantly lower in GCD2 cells compared to the WT. Interestingly, treatment with the autophagy flux inhibitor, bafilomycin A{sub 1}, resulted in similarly decreased Cyclin A1, B1, D1, and p53 expression in WT fibroblasts. Furthermore, similar findings, including a decrease in Cyclin A1, B1, and D1 and an increase in p16 and p27 expression were observed in autophagy-related 7 (Atg7; known to be essential for autophagy) gene knockout cells. These data provide new insight concerning the role of autophagy in cell cycle arrest and cellular proliferation, uncovering a number of novel therapeutic possibilities for GCD2 treatment.

  18. The pleiotropic effects of fisetin and hesperetin on human acute promyelocytic leukemia cells are mediated through apoptosis, cell cycle arrest, and alterations in signaling networks. (United States)

    Adan, Aysun; Baran, Yusuf


    Fisetin and hesperetin, flavonoids from various plants, have several pharmaceutical activities including antioxidative, anti-inflammatory, and anticancer effects. However, studies elucidating the role and the mechanism(s) of action of fisetin and hesperetin in acute promyelocytic leukemia are absent. In this study, we investigated the mechanism of the antiproliferative and apoptotic actions exerted by fisetin and hesperetin on human HL60 acute promyelocytic leukemia cells. The viability of HL60 cells was evaluated using the MTT assay, apoptosis by annexin V/propidium iodide (PI) staining and cell cycle distribution using flow cytometry, and changes in caspase-3 enzyme activity and mitochondrial transmembrane potential. Moreover, we performed whole-genome microarray gene expression analysis to reveal genes affected by fisetin and hesperetin that can be important for developing of future targeted therapy. Based on data obtained from microarray analysis, we also described biological networks modulated after fisetin and hesperetin treatment by KEGG and IPA analysis. Fisetin and hesperetin treatment showed a concentration- and time-dependent inhibition of proliferation and induced G2/M arrest for both agents and G0/G1 arrest for hesperetin at only the highest concentrations. There was a disruption of mitochondrial membrane potential together with increased caspase-3 activity. Furthermore, fisetin- and hesperetin-triggered apoptosis was confirmed by annexin V/PI analysis. The microarray gene profiling analysis revealed some important biological pathways including mitogen-activated protein kinases (MAPK) and inhibitor of DNA binding (ID) signaling pathways altered by fisetin and hesperetin treatment as well as gave a list of genes modulated ≥2-fold involved in cell proliferation, cell division, and apoptosis. Altogether, data suggested that fisetin and hesperetin have anticancer properties and deserve further investigation.

  19. Alterations in Cell Cycle and Induction of Apoptotic Cell Death in Breast Cancer Cells Treated with α-Mangostin Extracted from Mangosteen Pericarp

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    Hitomi Kurose


    Full Text Available The development of molecularly targeted drugs has greatly advanced cancer therapy, despite these drugs being associated with some serious problems. Recently, increasing attention has been paid to the anticancer effects of natural products. α-Mangostin, a xanthone isolated from the pericarp of mangosteen fruit, has been shown to induce apoptosis in various cancer cell lines and to exhibit antitumor activity in a mouse mammary cancer model. In this study, we investigated the influence of α-mangostin on apoptosis and cell cycle in the human breast cancer cell line MDA-MB231 (carrying a p53 mutation, and HER2, ER, and PgR negative in order to elucidate its anticancer mechanisms. In α-mangostin-treated cells, induction of mitochondria-mediated apoptosis was observed. On cell-cycle analysis, G1-phase arrest, increased p21cip1 expression and decreases in cyclins, cdc(s, CDKs and PCNA were observed. In conclusion, α-mangostin may be useful as a therapeutic agent for breast cancer carrying a p53 mutation and having HER2- and hormone receptor-negative subtypes.

  20. Serum starvation and thymidine double blocking achieved efficient cell cycle synchronization and altered the expression of p27, p53, bcl-2 in canine breast cancer cells. (United States)

    Tong, Jinjin; Sun, Dongdong; Yang, Chao; Wang, Yingxue; Sun, Sichao; Li, Qing; Bao, Jun; Liu, Yun


    Cell synchronization is an approach to obtain cell populations of the same stage, which is a prerequisite to studying the regulation of cell cycle progression in vivo. Serum starvation and thymidine double blocking (TdR) are two important practices in studying cell cycle synchronization. However, their effects on canine cancer cells as well as the regulatory mechanisms by these two methods are poorly understood. In this study, we determined the optimum conditions of serum starvation and TdR and their effects on cell cycle synchronization. We further explored the involvement of PI3K/Akt signaling pathway in the cell cycle synchronization by investigating the expression of three key genes (p27, p53 and bcl-2). Serum starvation resulted in a reversible cell cycle arrest and synchronously progress through G0/G1. The highest percentage of CHMm cells (87.47%) in G0/G1 stage was obtained after 42 h incubation with 0.5% fetal bovine serum (FBS). TdR double blocking could arrest 98.9% of CHMm cells in G1/S phase (0 h of release), and could arrest 93.74% of CHMm cells in S phase after 4h of release. We also found that the p27, p53, bcl-2 genes were most highly expressed in G0/G1 phase. Our current work revealed that serum starvation and TdR methods could achieve sufficient synchronization of CHMm cells. Moreover, the expression of p27, p53 and bcl-2 genes was related to cyclical movements and apoptosis. Our results will provide a new insight into cell cycle regulation and reprogramming of canine cancer cells induced by serum starvation and TdR blocking.

  1. Centrosome structure and function is altered by chloral hydrate and diazepam during the first reproductive cell cycles in sea urchin eggs (United States)

    Schatten, H.; Chakrabarti, A.


    This paper explores the mode of action of the tranquillizers chloral hydrate and diazepam during fertilization and mitosis of the first reproductive cell cycles in sea urchin eggs. Most striking effects of these drugs are the alteration of centrosomal material and the abnormal microtubule configurations during exposure and after recovery from the drugs. This finding is utilized to study the mechanisms of centrosome compaction and decompaction and the dynamic configurational changes of centrosomal material and its interactions with microtubules. When 0.1% chloral hydrate or 350-750 microM diazepam is applied at specific phases during the first cell cycle of sea urchin eggs, expanded centrosomal material compacts at distinct regions and super-compacts into dense spheres while microtubules disassemble. When eggs are treated before pronuclear fusion, centrosomal material aggregates around each of the two pronuclei while microtubules disappear. Upon recovery, atypical asters oftentimes with multiple foci are formed from centrosomal material surrounding the pronuclei which indicates that the drugs have affected centrosomal material and prevent it from functioning normally. Electron microscopy and immunofluorescence studies with antibodies that routinely stain centrosomes in sea urchin eggs (4D2; and Ah-6) depict centrosomal material that is altered when compared to control cells. This centrosomal material is not able to reform normal microtubule patterns upon recovery but will form multiple asters around the two pronuclei. When cells are treated with 0.1% chloral hydrate or 350-750 microM diazepam during mitosis, the bipolar centrosomal material becomes compacted and aggregates into multiple dense spheres while spindle and polar microtubules disassemble. With increased incubation time, the smaller dense centrosome particles aggregate into bigger and fewer spheres. Upon recovery, unusual irregular microtubule configurations are formed from centrosomes that have lost their

  2. Phenethyl isothiocyanate alters the gene expression and the levels of protein associated with cell cycle regulation in human glioblastoma GBM 8401 cells. (United States)

    Chou, Yu-Cheng; Chang, Meng-Ya; Wang, Mei-Jen; Liu, Hsin-Chung; Chang, Shu-Jen; Harnod, Tomor; Hung, Chih-Huang; Lee, Hsu-Tung; Shen, Chiung-Chyi; Chung, Jing-Gung


    Glioblastoma is the most common and aggressive primary brain malignancy. Phenethyl isothiocyanate (PEITC), a member of the isothiocyanate family, can induce apoptosis in many human cancer cells. Our previous study disclosed that PEITC induces apoptosis through the extrinsic pathway, dysfunction of mitochondria, reactive oxygen species (ROS)-induced endoplasmic reticulum (ER) stress, and intrinsic (mitochondrial) pathway in human brain glioblastoma multiforme (GBM) 8401 cells. To the best of our knowledge, we first investigated the effects of PEITC on the genetic levels of GBM 8401 cells in vitro. PEITC may induce G0/G1 cell-cycle arrest through affecting the proteins such as cdk2, cyclin E, and p21 in GBM 8401 cells. Many genes associated with cell-cycle regulation of GBM 8401 cells were changed after PEITC treatment: 48 genes were upregulated and 118 were downregulated. The cell-division cycle protein 20 (CDC20), Budding uninhibited by benzimidazole 1 homolog beta (BUB1B), and cyclin B1 were downregulated, and clusterin was upregulated in GBM 8401 cells treated with PEITC. These changes of gene expression can provide the effects of PEITC on the genetic levels and potential biomarkers for glioblastoma. © 2015 Wiley Periodicals, Inc. Environ Toxicol 32: 176-187, 2017.

  3. Human T-lymphotropic virus type-1 p30 alters cell cycle G2 regulation of T lymphocytes to enhance cell survival

    Directory of Open Access Journals (Sweden)

    Silverman Lee


    Full Text Available Abstract Background Human T-lymphotropic virus type-1 (HTLV-1 causes adult T-cell leukemia/lymphoma and is linked to a number of lymphocyte-mediated disorders. HTLV-1 contains both regulatory and accessory genes in four pX open reading frames. pX ORF-II encodes two proteins, p13 and p30, whose roles are still being defined in the virus life cycle and in HTLV-1 virus-host cell interactions. Proviral clones of HTLV-1 with pX ORF-II mutations diminish the ability of the virus to maintain viral loads in vivo. p30 expressed exogenously differentially modulates CREB and Tax-responsive element-mediated transcription through its interaction with CREB-binding protein/p300 and while acting as a repressor of many genes including Tax, in part by blocking tax/rex RNA nuclear export, selectively enhances key gene pathways involved in T-cell signaling/activation. Results Herein, we analyzed the role of p30 in cell cycle regulation. Jurkat T-cells transduced with a p30 expressing lentivirus vector accumulated in the G2-M phase of cell cycle. We then analyzed key proteins involved in G2-M checkpoint activation. p30 expression in Jurkat T-cells resulted in an increase in phosphorylation at serine 216 of nuclear cell division cycle 25C (Cdc25C, had enhanced checkpoint kinase 1 (Chk1 serine 345 phosphorylation, reduced expression of polo-like kinase 1 (PLK1, diminished phosphorylation of PLK1 at tyrosine 210 and reduced phosphorylation of Cdc25C at serine 198. Finally, primary human lymphocyte derived cell lines immortalized by a HTLV-1 proviral clone defective in p30 expression were more susceptible to camptothecin induced apoptosis. Collectively these data are consistent with a cell survival role of p30 against genotoxic insults to HTLV-1 infected lymphocytes. Conclusion Collectively, our data are the first to indicate that HTLV-1 p30 expression results in activation of the G2-M cell cycle checkpoint, events that would promote early viral spread and T-cell

  4. Flow cytometric detection of micronuclei and cell cycle alterations in fish-derived cells after exposure to three model genotoxic agents: mitomycin C, vincristine sulfate and benzo(a)pyrene. (United States)

    Sánchez, P; Llorente, M T; Castaño, A


    The measurement of cytogenetic alterations in vitro is considered an initial step in the risk assessment procedures for genotoxic agents. The concern about genotoxic pollutants in natural fish population makes the use of fish-derived cells an useful tool for these purposes. The technological improvements in well-established cytogenetic endpoints, such as micronuclei (MN) estimations by means of flow cytometry, have been proposed in the later years using mammalian cells. In this work, we test the capability of flow cytometry to evaluate MN induction and cell cycle alterations in an established fish cell line (RTG-2) using three agent-inductor models at different concentrations and exposure periods. For mitomycin C, an inverse relationship between length of exposure period and concentrations was observed. A dose-response relationship was observed after exposing RTG-2 cells to vincristine sulfate and benzo(a)pyrene. As this study shows, RTG-2 cells respond to clastogenic and aneugenic effects of the tested chemicals through the induction of MN at similar doses to mammalian cells and without the addition of exogenous metabolic activity. The possibility to check cell cycle alterations, in the same sample, gives the opportunity to evaluate early signals of cytotoxicity. The use of flow cytometry improves the assay by means of its speed and objectivity, which makes the assay very useful for genotoxicity assessment of aquatic chemicals.

  5. 一个自闭症家系的B淋巴细胞周期研究%Altered Cell Cycle of B Lymphocytes from an Autistic Family

    Institute of Scientific and Technical Information of China (English)

    申晨; 邹小兵; 邹俊华; 申阿东; 钟南


    目的 了解自闭症儿童的B淋巴细胞的细胞周期是否存在变异,为寻找自闭症的生物标志物并进一步为揭示自闭症的发病机制奠定基础.方法 运用B淋巴细胞建系、淋巴细胞培养、细胞周期检测以及统计学分析的方法,对一个汉族自闭症家系进行研究.该家系由父母及6个子女组成,父母表现型正常,而其中3名子女为自闭症患儿,3名为非自闭症儿童.结果 分析显示,B淋巴细胞的细胞周期在自闭症儿童组(相对于非自闭症儿童组)出现了S期细胞比例的增加和G0/G1期细胞比例的减少,同时代表增殖能力的“S期+G2/M期”的细胞比例也出现显著增加.结论 细胞周期的变化依赖于细胞内调控机制的改变,可能涉及到多种细胞周期蛋白的表达和调控变化,自闭症患儿存在B淋巴细胞细胞周期变异这一现象为进一步揭示自闭症的发病机制提供了线索.此外,如果本研究结果能够在大样本中得到肯定,则可作为自闭症的生物标志物用于疾病辅助诊断而具有良好的临床应用价值.%Objective To discover whether the cell cycle of B lymphocyte from autistic children were different from inartistic children. Methods One family including parents, three autistic children and three non-autistic children was involved in this study. The B lymphoblastoma cell construction, cell culture and cell cycle detection by flow cytometry (FCM) were carried out. Results FCM analysis showed that the percentage of G0/G1 phase cells decreased but that of S phase cells increased in autistic children compared with non-autistic siblings. The cell percentage of "S +G2/M" phase which represents cell proliferation also increased significantly in autistic children. Conclusion Alteration of cell cycle is the result of the intracellular regulating, which may involve variety changes of protein expression and regulation. The phenomenon that autistic children had alerted B lymphocyte cell

  6. Sodium arsenite alters cell cycle and MTHFR, MT1/2, and c-Myc protein levels in MCF-7 cells. (United States)

    Ruiz-Ramos, Ruben; López-Carrillo, Lizbeth; Albores, Arnulfo; Hernández-Ramírez, Raúl U; Cebrian, Mariano E


    There is limited available information on the effects of arsenic on enzymes participating in the folate cycle. Therefore, our aim was to evaluate the effects of sodium arsenite on the protein levels of methylenetetrahydrofolate reductase (MTHFR) and dihydrofolate reductase (DHFR) and its further relationship with the expression MT1/2 and c-myc in MCF-7 cells. Arsenite treatment (0-10 microM) for 4 h decreased MTHFR levels in a concentration-dependent fashion without significant effects on DHFR. The effects on MTHFR were observed at arsenite concentrations not significantly affecting cell viability. We also observed an increase in S-phase recruitment at all concentrations probed. Lower concentrations ( or =5 microM) or longer treatment periods induced apoptosis. Arsenite also induced dose-dependent increases in MT1/2 and c-Myc protein levels. The levels of MTHFR were inversely correlated to MT1/2 and c-Myc overexpression and increased S-phase recruitment. Our findings indicate that breast epithelial cells are responsive to arsenite and suggest that exposure may pose a risk for breast cancer. The reductions in MTHFR protein levels contribute to understand the mechanisms underlying the induction of genes influencing growth regulation, such as c-myc and MT1/2. However, further research is needed to ascertain if the effects here reported following short-time and high-dose exposure are relevant for human populations chronically exposed to low arsenic concentrations.

  7. Treatment with bisphenol A and methoxychlor results in the growth of human breast cancer cells and alteration of the expression of cell cycle-related genes, cyclin D1 and p21, via an estrogen receptor-dependent signaling pathway. (United States)

    Lee, Hye-Rim; Hwang, Kyung-A; Park, Min-Ah; Yi, Bo-Rim; Jeung, Eui-Bae; Choi, Kyung-Chul


    Various endocrine disrupting chemicals (EDCs) are exogenous compounds found in the environment and have the potential to interfere with the endocrine system and hormonal regulation. Among EDCs, bisphenol A (BPA) and 1,1,1-trichloro-2,2-bis(4-methoxyphenol)-ethane [methoxychlor (MXC)] have estrogenic activity resulting in a variety of dysfunctions in the E2-mediated response by binding to estrogen receptors (ERs), causing human health problems such as abnormal reproduction and carcinogenesis. In this study, we investigated the effects of BPA and MXC on cell proliferation facilitated by ER signaling in human breast cancer cells. MCF-7 cells are known to be ERα-positive and to be a highly E2-responsive cancer cell line; these cells are, therefore, a useful in vitro model for detecting estrogenic activity in response to EDCs. We evaluated cancer cell proliferation following BPA and MXC treatment using an MTT assay. We analyzed alterations in the expression of genes associated with the cell cycle in MCF-7 cells by semi-quantitative reverse-transcription PCR following treatment with BPA or MXC compared to EtOH. To determine whether BPA and MXC stimulate cancer cell growth though ER signaling, we co-treated the cells with agonists (propyl pyrazoletriol, PPT; and diarylpropionitrile, DPN) or an antagonist (ICI 182,780) of ER signaling and reduced ERα gene expression via siRNA in MCF-7 cells before treatment with EDCs. These studies confirmed the carcinogenicity of EDCs in vitro. As a result, BPA and MXC induced the cancer cell proliferation by the upregulation of genes that promote the cell cycle and the downregulation of anti-proliferative genes, especially ones affecting the G1/S transition via ERα signaling. These collective results confirm the carcinogenicity of these EDCs in vitro. Further studies are required to determine whether EDCs promote carcinogenesis in vivo.

  8. Cell cycle gene expression under clinorotation (United States)

    Artemenko, Olga


    Cyclins and cyclin-dependent kinase (CDK) are main regulators of the cell cycle of eukaryotes. It's assumes a significant change of their level in cells under microgravity conditions and by other physical factors actions. The clinorotation use enables to determine the influence of gravity on simulated events in the cell during the cell cycle - exit from the state of quiet stage and promotion presynthetic phase (G1) and DNA synthesis phase (S) of the cell cycle. For the clinorotation effect study on cell proliferation activity is the necessary studies of molecular mechanisms of cell cycle regulation and development of plants under altered gravity condition. The activity of cyclin D, which is responsible for the events of the cell cycle in presynthetic phase can be controlled by the action of endogenous as well as exogenous factors, but clinorotation is one of the factors that influence on genes expression that regulate the cell cycle.These data can be used as a model for further research of cyclin - CDK complex for study of molecular mechanisms regulation of growth and proliferation. In this investigation we tried to summarize and analyze known literature and own data we obtained relatively the main regulators of the cell cycle in altered gravity condition.

  9. Cell cycle features of primate embryonic stem cells. (United States)

    Fluckiger, Anne-Catherine; Marcy, Guillaume; Marchand, Mélanie; Négre, Didier; Cosset, François-Loïc; Mitalipov, Shoukhrat; Wolf, Don; Savatier, Pierre; Dehay, Colette


    Using flow cytometry measurements combined with quantitative analysis of cell cycle kinetics, we show that rhesus monkey embryonic stem cells (ESCs) are characterized by an extremely rapid transit through the G1 phase, which accounts for 15% of the total cell cycle duration. Monkey ESCs exhibit a non-phasic expression of cyclin E, which is detected during all phases of the cell cycle, and do not growth-arrest in G1 after gamma-irradiation, reflecting the absence of a G1 checkpoint. Serum deprivation or pharmacological inhibition of mitogen-activated protein kinase kinase (MEK) did not result in any alteration in the cell cycle distribution, indicating that ESC growth does not rely on mitogenic signals transduced by the Ras/Raf/MEK pathway. Taken together, these data indicate that rhesus monkey ESCs, like their murine counterparts, exhibit unusual cell cycle features in which cell cycle control mechanisms operating during the G1 phase are reduced or absent.

  10. MBA-induced differentiation of myeloid leukemic cell lines is associated with altered G1 cell cycle regulators and related genes

    Institute of Scientific and Technical Information of China (English)

    王钦红; 谢毅; 范华骅


    @@The proliferation and differentiation of hematopoietic cells can be regulated by a number of physiological agents including hexamethylene bisacetamide (HMBA). Clinically, HMBA has been used for the treatment of acute myeloid leukemia and myelodysplastic syndrome.1 However, the mechanism of the effect of HMBA on the differentiation of myeloid leukemic cells is largely unkown. Up to now, related reports have not been found. We used HL-60 and U937 cell lines to study the effect of HMBA on the differentiation of myeloid leukemic cells and to explore the possible mechanism.

  11. Furanodiene presents synergistic anti-proliferative activity with paclitaxel via altering cell cycle and integrin signaling in 95-D lung cancer cells. (United States)

    Xu, Wen-Shan; Dang, Yuan-Ye; Chen, Xiu-Ping; Lu, Jin-Jian; Wang, Yi-Tao


    Furanodiene (FUR) is a natural terpenoid isolated from Rhizoma Curcumae, a well-known Chinese medicinal herb that presents anti-proliferative activities in several cancer cell lines. Recently, we found that the combined treatment of FUR with paclitaxel (TAX) showed synergetic anti-proliferative activities in 95-D lung cancer cells. Herein, we showed that FUR reduced the cell numbers distributed in mitosis phase induced by TAX while increased those in G1 phase. The protein levels of cyclin D1, cyclin B1, CDK6 and c-Myc were all down-regulated in the group of combined treatment. The dramatically down-regulated expression of integrin β4, focal adhesion kinase and paxillin might partially contribute to the synergic effect. Though FUR alone obviously induced endoplasmic reticulum stress, this signaling pathway may not contribute to the synergetic anti-proliferative effect as the protein expression of CHOP and BIP was similar in FUR alone and combined treatment group.

  12. The inhibition of PI3K and NFκB promoted curcumin-induced cell cycle arrest at G2/M via altering polyamine metabolism in Bcl-2 overexpressing MCF-7 breast cancer cells. (United States)

    Berrak, Özge; Akkoç, Yunus; Arısan, Elif Damla; Çoker-Gürkan, Ajda; Obakan-Yerlikaya, Pınar; Palavan-Ünsal, Narçin


    Bcl-2 protein has been contributed with number of genes which are involved in oncogenesis. Among the many targets of Bcl-2, NFκB have potential role in induction of cell cycle arrest. Curcumin has potential therapeutic effects against breast cancer through multiple signaling pathways. In this study, we investigated the role of curcumin in induction of cell cycle arrest via regulating of NFκB and polyamine biosynthesis in wt and Bcl-2+ MCF-7 cells. To examine the effect of curcumin on cell cycle regulatory proteins, PI3K/Akt, NFκB pathways and polyamine catabolism, we performed immunoblotting assay. In addition, cell cycle analysis was performed by flow cytometry. The results indicated that curcumin induced cell cycle arrest at G2/M phase by downregulation of cyclin B1 and Cdc2 and inhibited colony formation in MCF-7wt cells. However, Bcl-2 overexpression prevented the inhibition of cell cycle associated proteins after curcumin treatment. The combination of LY294002, PI3K inhibitor, and curcumin induced cell cycle arrest by decreasing CDK4, CDK2 and cyclin E2 in Bcl-2+ MCF-7 cells. Moreover, LY294002 further inhibited the phosphorylation of Akt in Bcl-2+ MCF-7 cells. Curcumin could suppress the nuclear transport of NFκB through decreasing the interaction of P-IκB-NFκB. The combination of wedelolactone, NFκB inhibitor, and curcumin acted different on SSAT expression in wt MCF-7 and Bcl-2+ MCF-7 cells. NFκB inhibition increased the SSAT after curcumin treatment in Bcl-2 overexpressed MCF-7 cells. Inhibition of NFκB activity as well as suppression of ROS generation with NAC resulted in the partial relief of cells from G2/M checkpoint after curcumin treatment in wt MCF-7 cells. In conclusion, the potential role of curcumin in induction of cell cycle arrest is related with NFκB-regulated polyamine biosynthesis.

  13. Genetic alterations in Krebs cycle and its impact on cancer pathogenesis. (United States)

    Sajnani, Karishma; Islam, Farhadul; Smith, Robert Anthony; Gopalan, Vinod; Lam, Alfred King-Yin


    Cancer cells exhibit alterations in many cellular processes, including oxygen sensing and energy metabolism. Glycolysis in non-oxygen condition is the main energy production process in cancer rather than mitochondrial respiration as in benign cells. Genetic and epigenetic alterations of Krebs cycle enzymes favour the shift of cancer cells from oxidative phosphorylation to anaerobic glycolysis. Mutations in genes encoding aconitase, isocitrate dehydrogenase, succinate dehydrogenase, fumarate hydratase, and citrate synthase are noted in many cancers. Abnormalities of Krebs cycle enzymes cause ectopic production of Krebs cycle intermediates (oncometabolites) such as 2-hydroxyglutarate, and citrate. These oncometabolites stabilize hypoxia inducible factor 1 (HIF1), nuclear factor like 2 (Nrf2), inhibit p53 and prolyl hydroxylase 3 (PDH3) activities as well as regulate DNA/histone methylation, which in turn activate cell growth signalling. They also stimulate increased glutaminolysis, glycolysis and production of reactive oxygen species (ROS). Additionally, genetic alterations in Krebs cycle enzymes are involved with increased fatty acid β-oxidations and epithelial mesenchymal transition (EMT) induction. These altered phenomena in cancer could in turn promote carcinogenesis by stimulating cell proliferation and survival. Overall, epigenetic and genetic changes of Krebs cycle enzymes lead to the production of oncometabolite intermediates, which are important driving forces of cancer pathogenesis and progression. Understanding and applying the knowledge of these mechanisms opens new therapeutic options for patients with cancer.

  14. Cell proliferation alterations in Chlorella cells under stress conditions

    Energy Technology Data Exchange (ETDEWEB)

    Rioboo, Carmen [Laboratorio de Microbiologia, Facultad de Ciencias, Universidad de A Coruna, Campus da Zapateira s/n, 15008 A Coruna (Spain); O' Connor, Jose Enrique [Laboratorio de Citomica, Unidad Mixta de Investigacion CIPF-UVEG, Centro de Investigacion Principe Felipe, Avda. Autopista del Saler, 16, 46013 Valencia (Spain); Prado, Raquel; Herrero, Concepcion [Laboratorio de Microbiologia, Facultad de Ciencias, Universidad de A Coruna, Campus da Zapateira s/n, 15008 A Coruna (Spain); Cid, Angeles, E-mail: [Laboratorio de Microbiologia, Facultad de Ciencias, Universidad de A Coruna, Campus da Zapateira s/n, 15008 A Coruna (Spain)


    Very little is known about growth and proliferation in relation to the cell cycle regulation of algae. The lack of knowledge is even greater when referring to the potential toxic effects of pollutants on microalgal cell division. To assess the effect of terbutryn, a triazine herbicide, on the proliferation of the freshwater microalga Chlorella vulgaris three flow cytometric approaches were used: (1) in vivo cell division using 5-,6-carboxyfluorescein diacetate succinimidyl ester (CFSE) staining was measured, (2) the growth kinetics were determined by cytometric cell counting and (3) cell viability was evaluated with the membrane-impermeable double-stranded nucleic acid stain propidium iodide (PI). The results obtained in the growth kinetics study using CFSE to identify the microalgal cell progeny were consistent with those determined by cytometric cell counting. In all C. vulgaris cultures, each mother cell had undergone only one round of division through the 96 h of assay and the cell division occurred during the dark period. Cell division of the cultures exposed to the herbicide was asynchronous. Terbutryn altered the normal number of daughter cells (4 autospores) obtained from each mother cell. The number was only two in the cultures treated with 250 nM. The duration of the lag phase after the exposure to terbutryn could be dependent on the existence of a critical cell size to activate cytoplasmic division. Cell size, complexity and fluorescence of chlorophyll a of the microalgal cells presented a marked light/dark (day/night) cycle, except in the non-dividing 500 nM cultures, where terbutryn arrested cell division at the beginning of the cycle. Viability results showed that terbutryn has an algastatic effect in C. vulgaris cells at this concentration. The rapid and precise determination of cell proliferation by CFSE staining has allowed us to develop a model for assessing both the cell cycle of C. vulgaris and the in vivo effects of pollutants on growth and

  15. K+ channels and cell cycle progression in tumor cells

    Directory of Open Access Journals (Sweden)



    Full Text Available K+ ions play a major role in many cellular processes. The deregulation of K+ signaling is associated with a variety of diseases such as hypertension, atherosclerosis, or diabetes. K+ ions are important for setting the membrane potential, the driving force for Ca2+ influx, and regulate volume of growing cells. Moreover, it is increasingly recognized that K+ channels control cell proliferation through a novel signaling mechanisms triggered and modulated independently of ion fluxes. In cancer, aberrant expression, regulation and/or sublocalization of K+ channels can alter the downstream signals that converge on the cell cycle machinery. Various K+ channels are involved in cell cycle progression and are needed only at particular stages of the cell cycle. Consistent with this idea, the expression of Eag1 and HERG channels fluctuate along the cell cycle. Despite of acquired knowledge, our understanding of K+ channels functioning in cancer cells requires further studies. These include identifying the molecular mechanisms controling the cell cycle machinery. By understanding how K+ channels regulate cell cycle progression in cancer cells, we will gain insights into how cancer cells subvert the need for K+ signal and its downstream targets to proliferate.

  16. Metabolic alterations in renal cell carcinoma. (United States)

    Massari, Francesco; Ciccarese, Chiara; Santoni, Matteo; Brunelli, Matteo; Piva, Francesco; Modena, Alessandra; Bimbatti, Davide; Fantinel, Emanuela; Santini, Daniele; Cheng, Liang; Cascinu, Stefano; Montironi, Rodolfo; Tortora, Giampaolo


    Renal cell carcinoma (RCC) is a metabolic disease, being characterized by the dysregulation of metabolic pathways involved in oxygen sensing (VHL/HIF pathway alterations and the subsequent up-regulation of HIF-responsive genes such as VEGF, PDGF, EGF, and glucose transporters GLUT1 and GLUT4, which justify the RCC reliance on aerobic glycolysis), energy sensing (fumarate hydratase-deficient, succinate dehydrogenase-deficient RCC, mutations of HGF/MET pathway resulting in the metabolic Warburg shift marked by RCC increased dependence on aerobic glycolysis and the pentose phosphate shunt, augmented lipogenesis, and reduced AMPK and Krebs cycle activity) and/or nutrient sensing cascade (deregulation of AMPK-TSC1/2-mTOR and PI3K-Akt-mTOR pathways). We analyzed the key metabolic abnormalities underlying RCC carcinogenesis, highlighting those altered pathways that may represent potential targets for the development of more effective therapeutic strategies.

  17. Cell cycle phase regulates glucocorticoid receptor function.

    Directory of Open Access Journals (Sweden)

    Laura Matthews

    Full Text Available The glucocorticoid receptor (GR is a member of the nuclear hormone receptor superfamily of ligand-activated transcription factors. In contrast to many other nuclear receptors, GR is thought to be exclusively cytoplasmic in quiescent cells, and only translocate to the nucleus on ligand binding. We now demonstrate significant nuclear GR in the absence of ligand, which requires nuclear localisation signal 1 (NLS1. Live cell imaging reveals dramatic GR import into the nucleus through interphase and rapid exclusion of the GR from the nucleus at the onset of mitosis, which persists into early G(1. This suggests that the heterogeneity in GR distribution is reflective of cell cycle phase. The impact of cell cycle-driven GR trafficking on a panel of glucocorticoid actions was profiled. In G2/M-enriched cells there was marked prolongation of glucocorticoid-induced ERK activation. This was accompanied by DNA template-specific, ligand-independent GR transactivation. Using chimeric and domain-deleted receptors we demonstrate that this transactivation effect is mediated by the AF1 transactivation domain. AF-1 harbours multiple phosphorylation sites, which are consensus sequences for kinases including CDKs, whose activity changes during the cell cycle. In G2/M there was clear ligand independent induction of GR phosphorylation on residues 203 and 211, both of which are phosphorylated after ligand activation. Ligand-independent transactivation required induction of phospho-S211GR but not S203GR, thereby directly linking cell cycle driven GR modification with altered GR function. Cell cycle phase therefore regulates GR localisation and post-translational modification which selectively impacts GR activity. This suggests that cell cycle phase is an important determinant in the cellular response to Gc, and that mitotic index contributes to tissue Gc sensitivity.

  18. What cycles the cell? -Robust autonomous cell cycle models. (United States)

    Lavi, Orit; Louzoun, Yoram


    The cell cycle is one of the best studied cellular mechanisms at the experimental and theoretical levels. Although most of the important biochemical components and reactions of the cell cycle are probably known, the precise way the cell cycle dynamics are driven is still under debate. This phenomenon is not atypical to many other biological systems where the knowledge of the molecular building blocks and the interactions between them does not lead to a coherent picture of the appropriate dynamics. We here propose a methodology to develop plausible models for the driving mechanisms of embryonic and cancerous cell cycles. We first define a key property of the system (a cyclic behaviour in the case of the embryonic cell cycle) and set mathematical constraints on the types of two variable simplified systems robustly reproducing such a cyclic behaviour. We then expand these robust systems to three variables and reiterate the procedure. At each step, we further limit the type of expanded systems to fit the known microbiology until a detailed description of the system is obtained. This methodology produces mathematical descriptions of the required biological systems that are more robust to changes in the precise function and rate constants. This methodology can be extended to practically any type of subcellular mechanism.

  19. Autoradiography and the Cell Cycle. (United States)

    Jones, C. Weldon


    Outlines the stages of a cell biology "pulse-chase" experiment in which the students apply autoradiography techniques to learn about the concept of the cell cycle. Includes (1) seed germination and plant growth; (2) radioactive labeling and fixation of root tips; (3) feulgen staining of root tips; (4) preparation of autoradiograms; and…

  20. Cell Cycle Progression of Human Cells Cultured in Rotating Bioreactor (United States)

    Parks, Kelsey


    Space flight has been shown to alter the astronauts immune systems. Because immune performance is complex and reflects the influence of multiple organ systems within the host, scientists sought to understand the potential impact of microgravity alone on the cellular mechanisms critical to immunity. Lymphocytes and their differentiated immature form, lymphoblasts, play an important and integral role in the body's defense system. T cells, one of the three major types of lymphocytes, play a central role in cell-mediated immunity. They can be distinguished from other lymphocyte types, such as B cells and natural killer cells by the presence of a special receptor on their cell surface called T cell receptors. Reported studies have shown that spaceflight can affect the expression of cell surface markers. Cell surface markers play an important role in the ability of cells to interact and to pass signals between different cells of the same phenotype and cells of different phenotypes. Recent evidence suggests that cell-cycle regulators are essential for T-cell function. To trigger an effective immune response, lymphocytes must proliferate. The objective of this project is to investigate the changes in growth of human cells cultured in rotating bioreactors and to measure the growth rate and the cell cycle distribution for different human cell types. Human lymphocytes and lymphoblasts will be cultured in a bioreactor to simulate aspects of microgravity. The bioreactor is a cylindrical culture vessel that incorporates the aspects of clinostatic rotation of a solid fluid body around a horizontal axis at a constant speed, and compensates gravity by rotation and places cells within the fluid body into a sustained free-fall. Cell cycle progression and cell proliferation of the lymphocytes will be measured for a number of days. In addition, RNA from the cells will be isolated for expression of genes related in cell cycle regulations.

  1. Evolving Human Alteration of the Carbon Cycle: the Watershed Continuum (United States)

    Kaushal, S.; Delaney Newcomb, K.; Newcomer Johnson, T.; Pennino, M. J.; Smith, R. M.; Beaulieu, J. J.; Belt, K.; Grese, M.; Blomquist, J.; Duan, S.; Findlay, S.; Likens, G.; Mayer, P. M.; Murthy, S.; Utz, R.; Yepsen, M.


    Watersheds experiencing land development are constantly evolving, and their biogeochemical signatures are expected to evolve across both space and time in drainage waters. We investigate how land development influences spatial and temporal evolution of the carbon cycle from small streams to major rivers in the Eastern U.S. Along the watershed continuum, we show that there is spatial evolution in: (1) the amount, chemical form, and bioavailability of carbon; (2) carbon retention/release at the reach scale; and (3) ecosystem metabolism of carbon from headwaters to coastal waters. Over shorter time scales, the interaction between land use and climate variability alters magnitude and frequency of carbon "pulses" in watersheds. Amounts and forms of carbon pulses in agricultural and urban watersheds respond similarly to climate variability due to headwater alteration and loss of ecosystem services to buffer runoff and temperature changes. Over longer time scales, land use change has altered organic carbon concentrations in tidal waters of Chesapeake Bay, and there have been increased bicarbonate alkalinity concentrations in rivers throughout the Eastern U.S. due to human activities. In summary, our analyses indicates that the form and reactivity of carbon have evolved over space and time along the watershed continuum with major implications for downstream ecosystem metabolism, biological oxygen demand, carbon dioxide production, and river alkalinization.

  2. Warming alters coupled carbon and nutrient cycles in experimental streams. (United States)

    Williamson, Tanner J; Cross, Wyatt F; Benstead, Jonathan P; Gíslason, Gísli M; Hood, James M; Huryn, Alexander D; Johnson, Philip W; Welter, Jill R


    Although much effort has been devoted to quantifying how warming alters carbon cycling across diverse ecosystems, less is known about how these changes are linked to the cycling of bioavailable nitrogen and phosphorus. In freshwater ecosystems, benthic biofilms (i.e. thin films of algae, bacteria, fungi, and detrital matter) act as biogeochemical hotspots by controlling important fluxes of energy and material. Understanding how biofilms respond to warming is thus critical for predicting responses of coupled elemental cycles in freshwater systems. We developed biofilm communities in experimental streamside channels along a gradient of mean water temperatures (7.5-23.6 °C), while closely maintaining natural diel and seasonal temperature variation with a common water and propagule source. Both structural (i.e. biomass, stoichiometry, assemblage structure) and functional (i.e. metabolism, N2 -fixation, nutrient uptake) attributes of biofilms were measured on multiple dates to link changes in carbon flow explicitly to the dynamics of nitrogen and phosphorus. Temperature had strong positive effects on biofilm biomass (2.8- to 24-fold variation) and net ecosystem productivity (44- to 317-fold variation), despite extremely low concentrations of limiting dissolved nitrogen. Temperature had surprisingly minimal effects on biofilm stoichiometry: carbon:nitrogen (C:N) ratios were temperature-invariant, while carbon:phosphorus (C:P) ratios declined slightly with increasing temperature. Biofilm communities were dominated by cyanobacteria at all temperatures (>91% of total biovolume) and N2 -fixation rates increased up to 120-fold between the coldest and warmest treatments. Although ammonium-N uptake increased with temperature (2.8- to 6.8-fold variation), the much higher N2 -fixation rates supplied the majority of N to the ecosystem at higher temperatures. Our results demonstrate that temperature can alter how carbon is cycled and coupled to nitrogen and phosphorus. The


    Conazoles are fungicides used as pharmaceuticals and in agriculture. Triadimefon was hepatotoxic and induced follicular cell adenomas in the thyroid gland. In contrast,propiconazole and myclobutanil were hepatotoxic but had no effect on the thyroid gland. It was proposed that tri...

  4. Targeting cell cycle regulators in hematologic malignancies

    Directory of Open Access Journals (Sweden)

    Eiman eAleem


    Full Text Available Hematologic malignancies represent the fourth most frequently diagnosed cancer in economically developed countries. In hematologic malignancies normal hematopoiesis is interrupted by uncontrolled growth of a genetically altered stem or progenitor cell (HSPC that maintains its ability of self-renewal. Cyclin-dependent kinases (CDKs not only regulate the mammalian cell cycle, but also influence other vital cellular processes, such as stem cell renewal, differentiation, transcription, epigenetic regulation, apoptosis, and DNA repair. Chromosomal translocations, amplification, overexpression and altered CDK activities have been described in different types of human cancer, which have made them attractive targets for pharmacological inhibition. Mouse models deficient for one or more CDKs have significantly contributed to our current understanding of the physiological functions of CDKs, as well as their roles in human cancer. The present review focuses on selected cell cycle kinases with recent emerging key functions in hematopoiesis and in hematopoietic malignancies, such as CDK6 and its role in MLL-rearranged leukemia and acute lymphocytic leukemia, CDK1 and its regulator WEE-1 in acute myeloid leukemia, and cyclin C/CDK8/CDK19 complexes in T-cell acute lymphocytic leukemia. The knowledge gained from gene knockout experiments in mice of these kinases is also summarized. An overview of compounds targeting these kinases, which are currently in clinical development in various solid tumors and hematopoietic malignances, is presented. These include the CDK4/CDK6 inhibitors (palbociclib, LEE011, LY2835219, pan-CDK inhibitors that target CDK1 (dinaciclib, flavopiridol, AT7519, TG02, P276-00, terampeprocol and RGB 286638 as well as the WEE-1 kinase inhibitor, MK-1775. The advantage of combination therapy of cell cycle inhibitors with conventional chemotherapeutic agents used in the treatment of AML, such as cytarabine, is discussed.

  5. Structural protein alterations to resistance and endurance cycling exercise training. (United States)

    Parcell, Allen C; Woolstenhulme, Mandy T; Sawyer, Robert D


    The muscle cytoskeleton is necessary for the effective transmission of forces generated by actin-myosin interactions. We have examined the impact of muscle force and exercise volume on the cytoskeleton by measuring desmin and dystrophin content in human skeletal muscle after 12 weeks of progressive resistance or endurance cycle training. Muscle biopsies of the vastus lateralis were obtained before and after training. Desmin and dystrophin content was determined using immunoblotting techniques. After resistance training, desmin content increased 82 +/- 18% (p < 0.05), whereas there was no change in desmin content with endurance cycling. Dystrophin content did not change in either group. One-repetition maximum and VO2max increased (p < 0.05) in the resistance and endurance groups, respectively. These data demonstrate that a high-tension stimulus impacts the cytoskeleton in contrast to high-volume concentric contractions. The tensile loading and eccentric components of resistance training are implicated in desmin alterations. Indeed, the functional improvements resulting from resistance training may be related in part to the mechanical integration provided by the desmin protein.

  6. HSPA6 augments garlic extract-induced inhibition of proliferation, migration, and invasion of bladder cancer EJ cells; Implication for cell cycle dysregulation, signaling pathway alteration, and transcription factor-associated MMP-9 regulation (United States)

    Hwang, Byungdoo; Noh, Dae-Hwa; Park, Sung Lyea; Kim, Won Tae; Park, Sung-Soo; Kim, Wun-Jae; Moon, Sung-Kwon


    Although recent studies have demonstrated the anti-tumor effects of garlic extract (GE), the exact molecular mechanism is still unclear. In this study, we investigated the molecular mechanism associated with the inhibitory action of GE against bladder cancer EJ cell responses. Treatment with GE significantly inhibited proliferation of EJ cells dose-dependently through G2/M-phase cell cycle arrest. This G2/M-phase cell cycle arrest by GE was due to the activation of ATM and CHK2, which appears to inhibit phosphorylation of Cdc25C (Ser216) and Cdc2 (Thr14/Tyr15), this in turn was accompanied by down-regulation of cyclin B1 and up-regulation of p21WAF1. Furthermore, GE treatment was also found to induce phosphorylation of MAPK (ERK1/2, p38MAPK, and JNK) and AKT. In addition, GE impeded the migration and invasion of EJ cells via inhibition of MMP-9 expression followed by decreased binding activities of AP-1, Sp-1, and NF-κB motifs. Based on microarray datasets, we selected Heat shock protein A6 (HSPA6) as the most up-regulated gene responsible for the inhibitory effects of GE. Interestingly, overexpression of HSPA6 gene resulted in an augmentation effect with GE inhibiting proliferation, migration, and invasion of EJ cells. The augmentation effect of HSPA6 was verified by enhancing the induction of G2/M-phase-mediated ATM-CHK2-Cdc25C-p21WAF1-Cdc2 cascade, phosphorylation of MAPK and AKT signaling, and suppression of transcription factor-associated MMP-9 regulation in response to GE in EJ cells. Overall, our novel results indicate that HSPA6 reinforces the GE-mediated inhibitory effects of proliferation, migration, and invasion of EJ cells and may provide a new approach for therapeutic treatment of malignancies. PMID:28187175

  7. "Constructing" the Cell Cycle in 3D (United States)

    Koc, Isil; Turan, Merve


    The cycle of duplication and division, known as the "cell cycle," is the essential mechanism by which all living organisms reproduce. This activity allows students to develop an understanding of the main events that occur during the typical eukaryotic cell cycle mostly in the process of mitotic phase that divides the duplicated genetic material…

  8. Epigenetic dynamics across the cell cycle

    DEFF Research Database (Denmark)

    Kheir, Tony Bou; Lund, Anders H.


    Progression of the mammalian cell cycle depends on correct timing and co-ordination of a series of events, which are managed by the cellular transcriptional machinery and epigenetic mechanisms governing genome accessibility. Epigenetic chromatin modifications are dynamic across the cell cycle...... a correct inheritance of epigenetic chromatin modifications to daughter cells. In this chapter, we summarize the current knowledge on the dynamics of epigenetic chromatin modifications during progression of the cell cycle....

  9. The cyclomodulin cycle inhibiting factor (CIF) alters cullin neddylation dynamics. (United States)

    Toro, Tasha B; Toth, Julia I; Petroski, Matthew D


    The bacterial effector protein cycle inhibiting factor (CIF) converts glutamine 40 of NEDD8 to glutamate (Q40E), causing cytopathic effects and inhibiting cell proliferation. Although these have been attributed to blocking the functions of cullin-RING ubiquitin ligases, how CIF modulates NEDD8-dependent signaling is unclear. Here we use conditional NEDD8-dependent yeast to explore the effects of CIF on cullin neddylation. Although CIF causes cullin deneddylation and the generation of free NEDD8 Q40E, inhibiting the COP9 signalosome (CSN) allows Q40E to form only on NEDD8 attached to cullins. In the presence of the CSN, NEDD8 Q40E is removed from cullins more rapidly than NEDD8, leading to a decrease in steady-state cullin neddylation. As NEDD8 Q40E is competent for cullin conjugation in the absence of functional CSN and with overexpression of the NEDD8 ligase Dcn1, our data are consistent with NEDD8 deamidation causing enhanced deneddylation of cullins by the CSN. This leads to a dramatic change in the extent of activated cullin-RING ubiquitin ligases.

  10. Parenteral nutrition leads to alteration of hepatocyte cell cycle? Gone expression in rats%肠外营养导致大鼠肝细胞周期基因的变化及临床意义

    Institute of Scientific and Technical Information of China (English)

    张献兵; 汪健; 黄顺根; 卢春玉


    目的 本研究利用实时定量PCR芯片技术,检测TPN后肝脏细胞周期基因表达的变化,探讨哪些基因在肝脏损害中发挥作用,为临床预防和治疗PNALD提供资料.方法 选择12只雄性SD大鼠,随机分为TPN组(6只)和生理盐水对照组(6只),7d后应用实时定量PCR芯片检测方法 ,比较两组大鼠间肝脏细胞周期基因的表达.结果 TPN组大鼠肝脏病理表现为肝细胞弥漫的脂肪空泡变性,小叶中央区较周边区更为明显;TPN组大鼠肝脏细胞周期表达上调基因有Atm、Brea1、CAkn1b、Dnajc2、G2a、Nfatc1、Notch2、Pkd1、Ppp2r3a等,表达下调基因有CAc25b、Ccnd1、E2f1、Mcm3、Nek2、Wee1等.结论 TPN组与生理盐水对照组大鼠比较,肝脏细胞周期基因表达存在差异,肝细胞周期基因ATM、BRCA1、Cdkn1b 等表达的改变,提示肝细胞增生减少和凋亡的增加,从而阻止肝细胞的修复,可能TPN与肝脏损伤的发生和发展相关.%Objective Total parenteral nutrition (TPN)-associated liver disease (PNALD) is a well-recognized complication. Despite the severity of this disease, neither the etiology nor the patho-physiology of PNALD is currently understood. The goal of these experiments was to determine changes of cell cycle genes, using real time PCR array. Methods 12 male SD rats were randomized into two groups. The TPN group (N=6) received standard TPN solution through a silastic catheter inserted in the right jugular vein and similarly the control group (N = 6) received the physiologic saline. After 7 days of infusion,we analyze the difference of cell cycle gene expressions between the two groups with real time PCR array. Results The TPN group was found to have large lipid droplets with unclear mar-gins in the perilobular region and smaller lipid droplets in the centrilobular regions. Nine genes were up-regulated in the TPN group: Atm, Brca1, Cdkn1b, Dnajc2, G2a , Nfatc1, Notch2, Pkd1, Ppp2r3a. Seven genes with down-regulated in the TPN

  11. Fission Yeast Cell Cycle Synchronization Methods. (United States)

    Tormos-Pérez, Marta; Pérez-Hidalgo, Livia; Moreno, Sergio


    Fission yeast cells can be synchronized by cell cycle arrest and release or by size selection. Cell cycle arrest synchronization is based on the block and release of temperature-sensitive cell cycle mutants or treatment with drugs. The most widely used approaches are cdc10-129 for G1; hydroxyurea (HU) for early S-phase; cdc25-22 for G2, and nda3-KM311 for mitosis. Cells can also be synchronized by size selection using centrifugal elutriation or a lactose gradient. Here we describe the methods most commonly used to synchronize fission yeast cells.

  12. Molecular mechanisms controlling the cell cycle in embryonic stem cells. (United States)

    Abdelalim, Essam M


    Embryonic stem (ES) cells are originated from the inner cell mass of a blastocyst stage embryo. They can proliferate indefinitely, maintain an undifferentiated state (self-renewal), and differentiate into any cell type (pluripotency). ES cells have an unusual cell cycle structure, consists mainly of S phase cells, a short G1 phase and absence of G1/S checkpoint. Cell division and cell cycle progression are controlled by mechanisms ensuring the accurate transmission of genetic information from generation to generation. Therefore, control of cell cycle is a complicated process, involving several signaling pathways. Although great progress has been made on the molecular mechanisms involved in the regulation of ES cell cycle, many regulatory mechanisms remain unknown. This review summarizes the current knowledge about the molecular mechanisms regulating the cell cycle of ES cells and describes the relationship existing between cell cycle progression and the self-renewal.

  13. Cell cycle and cell signal transduction in marine phytoplankton

    Institute of Scientific and Technical Information of China (English)

    LIU Jingwen; JIAO Nianzhi; CAI Huinong


    As unicellular phytoplankton, the growth of a marine phytoplankton population results directly from the completion of a cell cycle, therefore, cell-environment communication is an important way which involves signal transduction pathways to regulate cell cycle progression and contribute to growth, metabolism and primary production and respond to their surrounding environment in marine phytoplankton. Cyclin-CDK and CaM/Ca2+ are essentially key regulators in control of cell cycle and signal transduction pathway, which has important values on both basic research and applied biotechnology. This paper reviews progress made in this research field, which involves the identification and characterization of cyclins and cell signal transduction system, cell cycle control mechanisms in marine phytoplankton cells, cell cycle proteins as a marker of a terminal event to estimate the growth rate of phytoplankton at the species level, cell cycle-dependent toxin production of toxic algae and cell cycle progression regulated by environmental factors.

  14. Cell-cycle quiescence maintains Caenorhabditis elegans germline stem cells independent of GLP-1/Notch. (United States)

    Seidel, Hannah S; Kimble, Judith


    Many types of adult stem cells exist in a state of cell-cycle quiescence, yet it has remained unclear whether quiescence plays a role in maintaining the stem cell fate. Here we establish the adult germline of Caenorhabditis elegans as a model for facultative stem cell quiescence. We find that mitotically dividing germ cells--including germline stem cells--become quiescent in the absence of food. This quiescence is characterized by a slowing of S phase, a block to M-phase entry, and the ability to re-enter M phase rapidly in response to re-feeding. Further, we demonstrate that cell-cycle quiescence alters the genetic requirements for stem cell maintenance: The signaling pathway required for stem cell maintenance under fed conditions--GLP-1/Notch signaling--becomes dispensable under conditions of quiescence. Thus, cell-cycle quiescence can itself maintain stem cells, independent of the signaling pathway otherwise essential for such maintenance.

  15. Lactobacillus decelerates cervical epithelial cell cycle progression.

    Directory of Open Access Journals (Sweden)

    Katarina Vielfort

    Full Text Available We investigated cell cycle progression in epithelial cervical ME-180 cells during colonization of three different Lactobacillus species utilizing live cell microscopy, bromodeoxyuridine incorporation assays, and flow cytometry. The colonization of these ME-180 cells by L. rhamnosus and L. reuteri, originating from human gastric epithelia and saliva, respectively, was shown to reduce cell cycle progression and to cause host cells to accumulate in the G1 phase of the cell cycle. The G1 phase accumulation in L. rhamnosus-colonized cells was accompanied by the up-regulation and nuclear accumulation of p21. By contrast, the vaginal isolate L. crispatus did not affect cell cycle progression. Furthermore, both the supernatants from the lactic acid-producing L. rhamnosus colonies and lactic acid added to cell culture media were able to reduce the proliferation of ME-180 cells. In this study, we reveal the diversity of the Lactobacillus species to affect host cell cycle progression and demonstrate that L. rhamnosus and L. reuteri exert anti-proliferative effects on human cervical carcinoma cells.

  16. Plant cells in vitro under altered gravity. (United States)

    Klymchuk, D O


    Establishing the role of gravity in plant requires information about how gravity regulates the metabolism of individual cells. Plant cells and tissues in vitro are valuable models for such purpose. Disrupted intercellular relations in such models have allowed to elucidate both the gravity role in non-specialised to gravity plant cells and the correlative relation role of an intact plant organism. The data obtained from non-numerous space and clinostat experiments with plant cells in vitro have demonstrated that their metabolism is sensitive to g-environment. The most experiments have shown a decrease in the biomass production and cell proliferation of spaceflight samples compared with ground controls, although there is study reporting of increased biomass production in an anise suspension culture and D. carota crown gall tissue culture. At the same time, results of experiments with single carrot cells and tomato callus culture demonstrated similarities in differentiation process in microgravity and in ground controls. Noted ultrastructural arrangement in cells, especially mitochondria and plastids, have been related to altered energy load and functions of organelles in microgravity, as well as changes in the lipid peroxidation and the content of malonic dyaldehyde in a haplopappus tissue culture under altered gravity supposed with modification of membrane structural-functional state. This article focuses on growth aspects of the cultured cells in microgravity and under clinostat conditions and considers those aspects that require further analysis.

  17. High-Cycle-Life Lithium Cell (United States)

    Yen, S. P. S.; Carter, B.; Shen, D.; Somoano, R.


    Lithium-anode electrochemical cell offers increased number of charge/ discharge cycles. Cell uses components selected for compatibility with electrolyte solvent: These materials are wettable and chemically stable. Low vapor pressure and high electrochemical stability of solvent improve cell packaging, handling, and safety. Cell operates at modest temperatures - less than 100 degrees C - and is well suited to automotive, communications, and other applications.

  18. Microfluidic Cell Cycle Analysis of Spread Cells by DAPI Staining

    Directory of Open Access Journals (Sweden)

    Jing Sun


    Full Text Available Single-cell cell cycle analysis is an emerging technique that requires detailed exploration of the image analysis process. In this study, we established a microfluidic single-cell cell cycle analysis method that can analyze cells in small numbers and in situ on a microfluidic chip. In addition, factors that influenced the analysis were carefully investigated. U87 or HeLa cells were seeded and attached to microfluidic channels before measurement. Cell nucleic DNA was imaged by 4′-6-diamidino-2-phenylindole (DAPI staining under a fluorescent microscope and subsequently fluorescent intensities of the cell nuclei DNA were converted to depict histograms for cell cycle phases. DAPI concentration, microscopic magnification, exposure time and cell number were examined for optimal cell cycle analysis conditions. The results showed that as few as a few hundred cells could be measured by DAPI staining in the range of 0.4–0.6 μg/mL to depict histograms with typical cell cycle phase distribution. Microscopic magnification during image acquisition, however, could distort the phase distribution. Exposure time did not significantly affect the cell cycle analysis. Furthermore, cell cycle inhibitor rapamycin treatment changed the cell cycle phase distribution as expected. In conclusion, a method for microfluidic single-cell cell cycle analysis of spread cells in situ was developed. Factors such as dye concentration and microscopic magnification had more influence on cell cycle phase distribution. Further studies will focus on detail differentiation of cell cycle phases and the application of such a method for biological meanings.

  19. Nucleosome architecture throughout the cell cycle. (United States)

    Deniz, Özgen; Flores, Oscar; Aldea, Martí; Soler-López, Montserrat; Orozco, Modesto


    Nucleosomes provide additional regulatory mechanisms to transcription and DNA replication by mediating the access of proteins to DNA. During the cell cycle chromatin undergoes several conformational changes, however the functional significance of these changes to cellular processes are largely unexplored. Here, we present the first comprehensive genome-wide study of nucleosome plasticity at single base-pair resolution along the cell cycle in Saccharomyces cerevisiae. We determined nucleosome organization with a specific focus on two regulatory regions: transcription start sites (TSSs) and replication origins (ORIs). During the cell cycle, nucleosomes around TSSs display rearrangements in a cyclic manner. In contrast to gap (G1 and G2) phases, nucleosomes have a fuzzier organization during S and M phases, Moreover, the choreography of nucleosome rearrangements correlate with changes in gene expression during the cell cycle, indicating a strong association between nucleosomes and cell cycle-dependent gene functionality. On the other hand, nucleosomes are more dynamic around ORIs along the cell cycle, albeit with tighter regulation in early firing origins, implying the functional role of nucleosomes on replication origins. Our study provides a dynamic picture of nucleosome organization throughout the cell cycle and highlights the subsequent impact on transcription and replication activity.

  20. Cell cycle activation by plant parasitic nematodes

    NARCIS (Netherlands)

    Goverse, A.; Almeida Engler, de J.; Verhees, J.; Krol, van der S.; Helder, J.; Gheysen, G.


    Sedentary nematodes are important pests of crop plants. They are biotrophic parasites that can induce the (re)differentiation of either differentiated or undifferentiated plant cells into specialized feeding cells. This (re)differentiation includes the reactivation of the cell cycle in specific plan

  1. Cell shape, cytoskeletal mechanics, and cell cycle control in angiogenesis (United States)

    Ingber, D. E.; Prusty, D.; Sun, Z.; Betensky, H.; Wang, N.


    Capillary endothelial cells can be switched between growth and differentiation by altering cell-extracellular matrix interactions and thereby, modulating cell shape. Studies were carried out to determine when cell shape exerts its growth-regulatory influence during cell cycle progression and to explore the role of cytoskeletal structure and mechanics in this control mechanism. When G0-synchronized cells were cultured in basic fibroblast growth factor (FGF)-containing defined medium on dishes coated with increasing densities of fibronectin or a synthetic integrin ligand (RGD-containing peptide), cell spreading, nuclear extension, and DNA synthesis all increased in parallel. To determine the minimum time cells must be adherent and spread on extracellular matrix (ECM) to gain entry into S phase, cells were removed with trypsin or induced to retract using cytochalasin D at different times after plating. Both approaches revealed that cells must remain extended for approximately 12-15 h and hence, most of G1, in order to enter S phase. After this restriction point was passed, normally 'anchorage-dependent' endothelial cells turned on DNA synthesis even when round and in suspension. The importance of actin-containing microfilaments in shape-dependent growth control was confirmed by culturing cells in the presence of cytochalasin D (25-1000 ng ml-1): dose-dependent inhibition of cell spreading, nuclear extension, and DNA synthesis resulted. In contrast, induction of microtubule disassembly using nocodazole had little effect on cell or nuclear spreading and only partially inhibited DNA synthesis. Interestingly, combination of nocodazole with a suboptimal dose of cytochalasin D (100 ng ml-1) resulted in potent inhibition of both spreading and growth, suggesting that microtubules are redundant structural elements which can provide critical load-bearing functions when microfilaments are partially compromised. Similar synergism between nocodazole and cytochalasin D was observed

  2. Lower concentrations of blueberry polyphenolic-rich extract differentially alter HepG2 cell proliferation and expression of genes related to cell-cycle, oxidation and epigenetic machinery (United States)

    In vitro cancer models have been used to study the effect of relatively high concentrations (>200 ug/ml) of phenolic plant extracts upon cell proliferation. In this study we report that the treatment of human hepatocarcinoma HepG2 cells with lower concentrations of blueberry phenolic extract (6.5-10...

  3. Radiofrequency treatment alters cancer cell phenotype (United States)

    Ware, Matthew J.; Tinger, Sophia; Colbert, Kevin L.; Corr, Stuart J.; Rees, Paul; Koshkina, Nadezhda; Curley, Steven; Summers, H. D.; Godin, Biana


    The importance of evaluating physical cues in cancer research is gradually being realized. Assessment of cancer cell physical appearance, or phenotype, may provide information on changes in cellular behavior, including migratory or communicative changes. These characteristics are intrinsically different between malignant and non-malignant cells and change in response to therapy or in the progression of the disease. Here, we report that pancreatic cancer cell phenotype was altered in response to a physical method for cancer therapy, a non-invasive radiofrequency (RF) treatment, which is currently being developed for human trials. We provide a battery of tests to explore these phenotype characteristics. Our data show that cell topography, morphology, motility, adhesion and division change as a result of the treatment. These may have consequences for tissue architecture, for diffusion of anti-cancer therapeutics and cancer cell susceptibility within the tumor. Clear phenotypical differences were observed between cancerous and normal cells in both their untreated states and in their response to RF therapy. We also report, for the first time, a transfer of microsized particles through tunneling nanotubes, which were produced by cancer cells in response to RF therapy. Additionally, we provide evidence that various sub-populations of cancer cells heterogeneously respond to RF treatment.

  4. Forest defoliator pests alter carbon and nitrogen cycles (United States)

    Grüning, Maren; Simon, Judy; Reinhardt, Annett-Barbara; Lamersdorf, Norbert; Thies, Carsten


    Climate change may foster pest epidemics in forests, and thereby the fluxes of elements that are indicators of ecosystem functioning. We examined compounds of carbon (C) and nitrogen (N) in insect faeces, leaf litter, throughfall and analysed the soils of deciduous oak forests (Quercus petraea L.) that were heavily infested by the leaf herbivores winter moth (Operophtera brumata L.) and mottled umber (Erannis defoliaria L.). In infested forests, total net canopy-to-soil fluxes of C and N deriving from insect faeces, leaf litter and throughfall were 30- and 18-fold higher compared with uninfested oak forests, with 4333 kg C ha−1 and 319 kg N ha−1, respectively, during a pest outbreak over 3 years. In infested forests, C and N levels in soil solutions were enhanced and C/N ratios in humus layers were reduced indicating an extended canopy-to-soil element pathway compared with the non-infested forests. In a microcosm incubation experiment, soil treatments with insect faeces showed 16-fold higher fluxes of carbon dioxide and 10-fold higher fluxes of dissolved organic carbon compared with soil treatments without added insect faeces (control). Thus, the deposition of high rates of nitrogen and rapidly decomposable carbon compounds in the course of forest pest epidemics appears to stimulate soil microbial activity (i.e. heterotrophic respiration), and therefore, may represent an important mechanism by which climate change can initiate a carbon cycle feedback. PMID:27853551

  5. Acanthamoeba induces cell-cycle arrest in host cells. (United States)

    Sissons, James; Alsam, Selwa; Jayasekera, Samantha; Kim, Kwang Sik; Stins, Monique; Khan, Naveed Ahmed


    Acanthamoeba can cause fatal granulomatous amoebic encephalitis (GAE) and eye keratitis. However, the pathogenesis and pathophysiology of these emerging diseases remain unclear. In this study, the effects of Acanthamoeba on the host cell cycle using human brain microvascular endothelial cells (HBMEC) and human corneal epithelial cells (HCEC) were determined. Two isolates of Acanthamoeba belonging to the T1 genotype (GAE isolate) and T4 genotype (keratitis isolate) were used, which showed severe cytotoxicity on HBMEC and HCEC, respectively. No tissue specificity was observed in their ability to exhibit binding to the host cells. To determine the effects of Acanthamoeba on the host cell cycle, a cell-cycle-specific gene array was used. This screened for 96 genes specific for host cell-cycle regulation. It was observed that Acanthamoeba inhibited expression of genes encoding cyclins F and G1 and cyclin-dependent kinase 6, which are proteins important for cell-cycle progression. Moreover, upregulation was observed of the expression of genes such as GADD45A and p130 Rb, associated with cell-cycle arrest, indicating cell-cycle inhibition. Next, the effect of Acanthamoeba on retinoblastoma protein (pRb) phosphorylation was determined. pRb is a potent inhibitor of G1-to-S cell-cycle progression; however, its function is inhibited upon phosphorylation, allowing progression into S phase. Western blotting revealed that Acanthamoeba abolished pRb phosphorylation leading to cell-cycle arrest at the G1-to-S transition. Taken together, these studies demonstrated for the first time that Acanthamoeba inhibits the host cell cycle at the transcriptional level, as well as by modulating pRb phosphorylation using host cell-signalling mechanisms. A complete understanding of Acanthamoeba-host cell interactions may help in developing novel strategies to treat Acanthamoeba infections.

  6. Fuel cell hybrid taxi life cycle analysis

    Energy Technology Data Exchange (ETDEWEB)

    Baptista, Patricia, E-mail: [IDMEC-Instituto Superior Tecnico, Universidade Tecnica de Lisboa, Av. Rovisco Pais, 1, 1049-001 Lisboa (Portugal); Ribau, Joao; Bravo, Joao; Silva, Carla [IDMEC-Instituto Superior Tecnico, Universidade Tecnica de Lisboa, Av. Rovisco Pais, 1, 1049-001 Lisboa (Portugal); Adcock, Paul; Kells, Ashley [Intelligent Energy, Charnwood Building, HolywellPark, Ashby Road, Loughborough, LE11 3GR (United Kingdom)


    A small fleet of classic London Taxis (Black cabs) equipped with hydrogen fuel cell power systems is being prepared for demonstration during the 2012 London Olympics. This paper presents a Life Cycle Analysis for these vehicles in terms of energy consumption and CO{sub 2} emissions, focusing on the impacts of alternative vehicle technologies for the Taxi, combining the fuel life cycle (Tank-to-Wheel and Well-to-Tank) and vehicle materials Cradle-to-Grave. An internal combustion engine diesel taxi was used as the reference vehicle for the currently available technology. This is compared to battery and fuel cell vehicle configurations. Accordingly, the following energy pathways are compared: diesel, electricity and hydrogen (derived from natural gas steam reforming). Full Life Cycle Analysis, using the PCO-CENEX drive cycle, (derived from actual London Taxi drive cycles) shows that the fuel cell powered vehicle configurations have lower energy consumption (4.34 MJ/km) and CO{sub 2} emissions (235 g/km) than both the ICE Diesel (9.54 MJ/km and 738 g/km) and the battery electric vehicle (5.81 MJ/km and 269 g/km). - Highlights: > A Life Cycle Analysis of alternative vehicle technologies for the London Taxi was performed. > The hydrogen powered vehicles have the lowest energy consumption and CO{sub 2} emissions results. > A hydrogen powered solution can be a sustainable alternative in a full life cycle framework.

  7. SAMHD1 controls cell cycle status, apoptosis and HIV-1 infection in monocytic THP-1 cells

    Energy Technology Data Exchange (ETDEWEB)

    Bonifati, Serena [Center for Retrovirus Research, Department of Veterinary Biosciences, The Ohio State University, Columbus, OH (United States); Daly, Michele B. [Center for Drug Discovery, Department of Pediatrics, School of Medicine, Emory University, Atlanta, GA (United States); St Gelais, Corine; Kim, Sun Hee [Center for Retrovirus Research, Department of Veterinary Biosciences, The Ohio State University, Columbus, OH (United States); Hollenbaugh, Joseph A.; Shepard, Caitlin [Center for Drug Discovery, Department of Pediatrics, School of Medicine, Emory University, Atlanta, GA (United States); Kennedy, Edward M. [Department of Molecular Genetics and Microbiology, Duke University, Durham, NC (United States); Kim, Dong-Hyun [Department of Pharmacy, School of Pharmacy, Kyung-Hee University, Seoul (Korea, Republic of); Schinazi, Raymond F. [Center for Drug Discovery, Department of Pediatrics, School of Medicine, Emory University, Atlanta, GA (United States); Kim, Baek, E-mail: [Center for Drug Discovery, Department of Pediatrics, School of Medicine, Emory University, Atlanta, GA (United States); Department of Pharmacy, School of Pharmacy, Kyung-Hee University, Seoul (Korea, Republic of); Wu, Li, E-mail: [Center for Retrovirus Research, Department of Veterinary Biosciences, The Ohio State University, Columbus, OH (United States)


    SAMHD1 limits HIV-1 infection in non-dividing myeloid cells by decreasing intracellular dNTP pools. HIV-1 restriction by SAMHD1 in these cells likely prevents activation of antiviral immune responses and modulates viral pathogenesis, thus highlighting a critical role of SAMHD1 in HIV-1 physiopathology. Here, we explored the function of SAMHD1 in regulating cell proliferation, cell cycle progression and apoptosis in monocytic THP-1 cells. Using the CRISPR/Cas9 technology, we generated THP-1 cells with stable SAMHD1 knockout. We found that silencing of SAMHD1 in cycling cells stimulates cell proliferation, redistributes cell cycle population in the G{sub 1}/G{sub 0} phase and reduces apoptosis. These alterations correlated with increased dNTP levels and more efficient HIV-1 infection in dividing SAMHD1 knockout cells relative to control. Our results suggest that SAMHD1, through its dNTPase activity, affects cell proliferation, cell cycle distribution and apoptosis, and emphasize a key role of SAMHD1 in the interplay between cell cycle regulation and HIV-1 infection.

  8. Altered calcium signaling in cancer cells. (United States)

    Stewart, Teneale A; Yapa, Kunsala T D S; Monteith, Gregory R


    It is the nature of the calcium signal, as determined by the coordinated activity of a suite of calcium channels, pumps, exchangers and binding proteins that ultimately guides a cell's fate. Deregulation of the calcium signal is often deleterious and has been linked to each of the 'cancer hallmarks'. Despite this, we do not yet have a full understanding of the remodeling of the calcium signal associated with cancer. Such an understanding could aid in guiding the development of therapies specifically targeting altered calcium signaling in cancer cells during tumorigenic progression. Findings from some of the studies that have assessed the remodeling of the calcium signal associated with tumorigenesis and/or processes important in invasion and metastasis are presented in this review. The potential of new methodologies is also discussed. This article is part of a Special Issue entitled: Membrane channels and transporters in cancers.

  9. Expression of cell cycle regulator cdk2ap1 suppresses tumor cell phenotype by non-cell autonomous mechanisms


    Zolochevska, Olga; Figueiredo, Marxa L


    We evaluated the effect of expressing the cell cycle regulator cdk2ap1 in epithelial or stromal cell compartments to reduce SCC growth in vitro and in vivo. Cell autonomous and/or non-cell autonomous expression of cdk2ap1 reduced tumor growth and invasion and altered cell cycle, adhesion, invasion, angiogenesis, and apoptotic gene expression, as assessed by several in vitro phenotype assays, quantitative real time PCR, and in vivo molecular imaging using a novel three-way xenograft animal mod...

  10. Differences in the Detection of BrdU/EdU Incorporation Assays Alter the Calculation for G1, S, and G2 Phases of the Cell Cycle in Trypanosomatids. (United States)

    da Silva, Marcelo Santos; Muñoz, Paula Andrea Marin; Armelin, Hugo Aguirre; Elias, Maria Carolina


    Trypanosomatids are the etiologic agents of various infectious diseases in humans. They diverged early during eukaryotic evolution and have attracted attention as peculiar models for evolutionary and comparative studies. Here, we show a meticulous study comparing the incorporation and detection of the thymidine analogs BrdU and EdU in Leishmania amazonensis, Trypanosoma brucei, and Trypanosoma cruzi to monitor their DNA replication. We used BrdU- and EdU-incorporated parasites with the respective standard detection approaches: Indirect Immunofluorescence to detect BrdU after standard denaturation (2 M HCl) and "click" chemistry to detect EdU. We found a discrepancy between these two thymidine analogs due to the poor detection of BrdU, which is reflected on the estimative of the duration of the cell cycle phases G1, S, and G2. To solve this discrepancy, we increase the exposure of incorporated BrdU using different concentrations of HCl. Using a new value for HCl concentration, we re-estimated the phases G1, S, G2+M, and cytokinesis durations, confirming the values found by this approach using EdU. In conclusion, we suggest that the studies using BrdU with standard detection approach, not only in trypanosomatids but also in others cell types, should be reviewed to ensure an accurate estimation of DNA replication monitoring. This article is protected by copyright. All rights reserved.

  11. Microfluidic Cell Cycle Analysis of Spread Cells by DAPI Staining


    Jing Sun; Jiayu Zhang; Haibo Yang; Gongzhuo Wang; Yanzhao Li; Xuxin Zhang; Qidan Chen; Ming-Fei Lang


    Single-cell cell cycle analysis is an emerging technique that requires detailed exploration of the image analysis process. In this study, we established a microfluidic single-cell cell cycle analysis method that can analyze cells in small numbers and in situ on a microfluidic chip. In addition, factors that influenced the analysis were carefully investigated. U87 or HeLa cells were seeded and attached to microfluidic channels before measurement. Cell nucleic DNA was imaged by 4′-6-diamidino-2...

  12. SAFT nickel hydrogen cell cycling status (United States)

    Borthomieu, Yannick; Duquesne, Didier


    An overview of the NiH2 cell development is given. The NiH2 SAFT system is an electrochemical (single or dual) stack (IPV). The stack is mounted in an hydroformed Inconel 718 vessel operating at high pressure, equipped with 'rabbit ears' ceramic brazed electrical feedthroughs. The cell design is described: positive electrode, negative electrode, and stack configuration. Overviews of low earth orbit and geostationary earth orbit cyclings are provided. DPA results are also provided. The cycling and DPA results demonstrate that SAFT NiH2 is characterized by high reliability and very stable performances.

  13. Control of cell cycle and cell growth by molecular chaperones. (United States)

    Aldea, Martí; Garí, Eloi; Colomina, Neus


    Cells adapt their size to both intrinsic and extrinsic demands and, among them, those that stem from growth and proliferation rates are crucial for cell size homeostasis. Here we revisit mechanisms that regulate cell cycle and cell growth in budding yeast. Cyclin Cln3, the most upstream activator of Start, is retained at the endoplasmic reticulum in early G(1) and released by specific chaperones in late G(1) to initiate the cell cycle. On one hand, these chaperones are rate-limiting for release of Cln3 and cell cycle entry and, on the other hand, they are required for key biosynthetic processes. We propose a model whereby the competition for specialized chaperones between growth and cycle machineries could gauge biosynthetic rates and set a critical size threshold at Start.

  14. Tumor cell "dead or alive": caspase and survivin regulate cell death, cell cycle and cell survival. (United States)

    Suzuki, A; Shiraki, K


    Cell death and cell cycle progression are two sides of the same coin, and these two different phenomenons are regulated moderately to maintain the cellular homeostasis. Tumor is one of the disease states produced as a result of the disintegrated regulation and is characterized as cells showing an irreversible progression of cell cycle and a resistance to cell death signaling. Several investigations have been performed for the understanding of cell death or cell cycle, and cell death research has remarkably progressed in these 10 years. Caspase is a nomenclature referring to ICE/CED-3 cysteine proteinase family and plays a central role during cell death. Recently, several investigations raised some possible hypotheses that caspase is also involved in cell cycle regulation. In this issue, therefore, we review the molecular basis of cell death and cell cycle regulated by caspase in tumor, especially hepatocellular carcinoma cells.

  15. Cell alterations induced by a biotherapic for influenza

    Directory of Open Access Journals (Sweden)

    José Nelson Couceiro


    Full Text Available Introduction: Influenza viruses have been responsible for highly contagious acute respiratory illnesses with high mortality, mainly in the elderly, which encourages the development of new drugs for the treatment of human flu. The biotherapics are medicines prepared from biological products, which are not chemically defined. They are compounded following the homeopathic procedures indicated for infectious diseases with known etiology [1]. Aim: The purpose of the present study is to verify cellular alterations induced by a biotherapic prepared from the infectious influenza A virus. Methodology: This biotherapic was prepared for this study in the homeopathic potency of 30X according to the Brazilian Homeopathic Pharmacopeia [2]. The concentration of 10% was not cytotoxic to cells, as verified by neutral red assay. The cellular alterations observed in MDCK cells were analyzed by optical microscopy for the quantification of mitosis, nucleoli and lipid bodies. The mitochondrial activity was assessed by MTT assay and the phosphosfructokinase-1 (PFK-1 enzyme activity was analyzed on the MDCK cells treated for 5, 10 and 30 days. Macrophages J778.G8 were treated with this biotherapic to evaluate the immunostimulatory cytokine release. Results: The cellular alterations observed in MDCK cells were verified by optical microscopy. The number of lipid bodies present in MDCK cells stimulated for 10 days was significantly lower (p <0.05 when compared to controls. The biotherapic significantly increased (p <0.05 the number of mitosis and the mitochondrial activity of MDCK cells stimulated for 10 and 30 days. These changes were confirmed by a significant reduction (p <0.05 on the PFK-1 activity. These results suggest that the biotherapic was able to activate the Krebs cycle and pentose-phosphate metabolism to the generation of amino acids and nucleotides, situations common to cells whose rate of mitosis is increased. The quantification of immunostimulatory


    Energy Technology Data Exchange (ETDEWEB)

    Larry J. Chaney; Mike R. Tharp; Tom W. Wolf; Tim A. Fuller; Joe J. Hartvigson


    A wide variety of conceptual design studies have been conducted that describe ultra-high efficiency fossil power plant cycles. The most promising of these ultra-high efficiency cycles incorporate high temperature fuel cells with a gas turbine. Combining fuel cells with a gas turbine increases overall cycle efficiency while reducing per kilowatt emissions. This study has demonstrated that the unique approach taken to combining a fuel cell and gas turbine has both technical and economic merit. The approach used in this study eliminates most of the gas turbine integration problems associated with hybrid fuel cell turbine systems. By using a micro-turbine, and a non-pressurized fuel cell the total system size (kW) and complexity has been reduced substantially from those presented in other studies, while maintaining over 70% efficiency. The reduced system size can be particularly attractive in the deregulated electrical generation/distribution environment where the market may not demand multi-megawatt central stations systems. The small size also opens up the niche markets to this high efficiency, low emission electrical generation option.

  17. Modeling of SONOS Memory Cell Erase Cycle (United States)

    Phillips, Thomas A.; MacLeod, Todd C.; Ho, Fat H.


    Utilization of Silicon-Oxide-Nitride-Oxide-Silicon (SONOS) nonvolatile semiconductor memories as a flash memory has many advantages. These electrically erasable programmable read-only memories (EEPROMs) utilize low programming voltages, have a high erase/write cycle lifetime, are radiation hardened, and are compatible with high-density scaled CMOS for low power, portable electronics. In this paper, the SONOS memory cell erase cycle was investigated using a nonquasi-static (NQS) MOSFET model. Comparisons were made between the model predictions and experimental data.

  18. Quantitative proteomic analysis of cell cycle of the dinoflagellate Prorocentrum donghaiense (Dinophyceae.

    Directory of Open Access Journals (Sweden)

    Da-Zhi Wang

    Full Text Available Dinoflagellates are the major causative agents of harmful algal blooms in the coastal zone, which has resulted in adverse effects on the marine ecosystem and public health, and has become a global concern. Knowledge of cell cycle regulation in proliferating cells is essential for understanding bloom dynamics, and so this study compared the protein profiles of Prorocentrum donghaiense at different cell cycle phases and identified differentially expressed proteins using 2-D fluorescence difference gel electrophoresis combined with MALDI-TOF-TOF mass spectrometry. The results showed that the synchronized cells of P. donghaiense completed a cell cycle within 24 hours and cell division was phased with the diurnal cycle. Comparison of the protein profiles at four cell cycle phases (G1, S, early and late G2/M showed that 53 protein spots altered significantly in abundance. Among them, 41 were identified to be involved in a variety of biological processes, e.g. cell cycle and division, RNA metabolism, protein and amino acid metabolism, energy and carbon metabolism, oxidation-reduction processes, and ABC transport. The periodic expression of these proteins was critical to maintain the proper order and function of the cell cycle. This study, to our knowledge, for the first time revealed the major biological processes occurring at different cell cycle phases which provided new insights into the mechanisms regulating the cell cycle and growth of dinoflagellates.

  19. Chloroplast Dysfunction Causes Multiple Defects in Cell Cycle Progression in the Arabidopsis crumpled leaf Mutant

    KAUST Repository

    Hudik, Elodie


    The majority of research on cell cycle regulation is focused on the nuclear events that govern the replication and segregation of the genome between the two daughter cells. However, eukaryotic cells contain several compartmentalized organelles with specialized functions, and coordination among these organelles is required for proper cell cycle progression, as evidenced by the isolation of several mutants in which both organelle function and overall plant development were affected. To investigate how chloroplast dysfunction affects the cell cycle, we analyzed the crumpled leaf (crl) mutant of Arabidopsis (Arabidopsis thaliana), which is deficient for a chloroplastic protein and displays particularly severe developmental defects. In the crl mutant, we reveal that cell cycle regulation is altered drastically and that meristematic cells prematurely enter differentiation, leading to reduced plant stature and early endoreduplication in the leaves. This response is due to the repression of several key cell cycle regulators as well as constitutive activation of stress-response genes, among them the cell cycle inhibitor SIAMESE-RELATED5. One unique feature of the crl mutant is that it produces aplastidic cells in several organs, including the root tip. By investigating the consequence of the absence of plastids on cell cycle progression, we showed that nuclear DNA replication occurs in aplastidic cells in the root tip, which opens future research prospects regarding the dialogue between plastids and the nucleus during cell cycle regulation in higher plants.

  20. Chloroplast dysfunction causes multiple defects in cell cycle progression in the Arabidopsis crumpled leaf mutant. (United States)

    Hudik, Elodie; Yoshioka, Yasushi; Domenichini, Séverine; Bourge, Mickaël; Soubigout-Taconnat, Ludivine; Mazubert, Christelle; Yi, Dalong; Bujaldon, Sandrine; Hayashi, Hiroyuki; De Veylder, Lieven; Bergounioux, Catherine; Benhamed, Moussa; Raynaud, Cécile


    The majority of research on cell cycle regulation is focused on the nuclear events that govern the replication and segregation of the genome between the two daughter cells. However, eukaryotic cells contain several compartmentalized organelles with specialized functions, and coordination among these organelles is required for proper cell cycle progression, as evidenced by the isolation of several mutants in which both organelle function and overall plant development were affected. To investigate how chloroplast dysfunction affects the cell cycle, we analyzed the crumpled leaf (crl) mutant of Arabidopsis (Arabidopsis thaliana), which is deficient for a chloroplastic protein and displays particularly severe developmental defects. In the crl mutant, we reveal that cell cycle regulation is altered drastically and that meristematic cells prematurely enter differentiation, leading to reduced plant stature and early endoreduplication in the leaves. This response is due to the repression of several key cell cycle regulators as well as constitutive activation of stress-response genes, among them the cell cycle inhibitor SIAMESE-RELATED5. One unique feature of the crl mutant is that it produces aplastidic cells in several organs, including the root tip. By investigating the consequence of the absence of plastids on cell cycle progression, we showed that nuclear DNA replication occurs in aplastidic cells in the root tip, which opens future research prospects regarding the dialogue between plastids and the nucleus during cell cycle regulation in higher plants.

  1. Cell cycle regulation of hematopoietic stem or progenitor cells. (United States)

    Hao, Sha; Chen, Chen; Cheng, Tao


    The highly regulated process of blood production is achieved through the hierarchical organization of hematopoietic stem cell (HSC) subsets and their progenies, which differ in self-renewal and differentiation potential. Genetic studies in mice have demonstrated that cell cycle is tightly controlled by the complex interplay between extrinsic cues and intrinsic regulatory pathways involved in HSC self-renewal and differentiation. Deregulation of these cellular programs may transform HSCs or hematopoietic progenitor cells (HPCs) into disease-initiating stem cells, and can result in hematopoietic malignancies such as leukemia. While previous studies have shown roles for some cell cycle regulators and related signaling pathways in HSCs and HPCs, a more complete picture regarding the molecular mechanisms underlying cell cycle regulation in HSCs or HPCs is lacking. Based on accumulated studies in this field, the present review introduces the basic components of the cell cycle machinery and discusses their major cellular networks that regulate the dormancy and cell cycle progression of HSCs. Knowledge on this topic would help researchers and clinicians to better understand the pathogenesis of relevant blood disorders and to develop new strategies for therapeutic manipulation of HSCs.

  2. Global analysis of cell cycle gene expression of the legume symbiont Sinorhizobium meliloti. (United States)

    De Nisco, Nicole J; Abo, Ryan P; Wu, C Max; Penterman, Jon; Walker, Graham C


    In α-proteobacteria, strict regulation of cell cycle progression is necessary for the specific cellular differentiation required for adaptation to diverse environmental niches. The symbiotic lifestyle of Sinorhizobium meliloti requires a drastic cellular differentiation that includes genome amplification. To achieve polyploidy, the S. meliloti cell cycle program must be altered to uncouple DNA replication from cell division. In the α-proteobacterium Caulobacter crescentus, cell cycle-regulated transcription plays an important role in the control of cell cycle progression but this has not been demonstrated in other α-proteobacteria. Here we describe a robust method for synchronizing cell growth that enabled global analysis of S. meliloti cell cycle-regulated gene expression. This analysis identified 462 genes with cell cycle-regulated transcripts, including several key cell cycle regulators, and genes involved in motility, attachment, and cell division. Only 28% of the 462 S. meliloti cell cycle-regulated genes were also transcriptionally cell cycle-regulated in C. crescentus. Furthermore, CtrA- and DnaA-binding motif analysis revealed little overlap between the cell cycle-dependent regulons of CtrA and DnaA in S. meliloti and C. crescentus. The predicted S. meliloti cell cycle regulon of CtrA, but not that of DnaA, was strongly conserved in more closely related α-proteobacteria with similar ecological niches as S. meliloti, suggesting that the CtrA cell cycle regulatory network may control functions of central importance to the specific lifestyles of α-proteobacteria.

  3. Cell cycle regulation in human embryonic stem cells: links to adaptation to cell culture. (United States)

    Barta, Tomas; Dolezalova, Dasa; Holubcova, Zuzana; Hampl, Ales


    Cell cycle represents not only a tightly orchestrated mechanism of cell replication and cell division but it also plays an important role in regulation of cell fate decision. Particularly in the context of pluripotent stem cells or multipotent progenitor cells, regulation of cell fate decision is of paramount importance. It has been shown that human embryonic stem cells (hESCs) show unique cell cycle characteristics, such as short doubling time due to abbreviated G1 phase; these properties change with the onset of differentiation. This review summarizes the current understanding of cell cycle regulation in hESCs. We discuss cell cycle properties as well as regulatory machinery governing cell cycle progression of undifferentiated hESCs. Additionally, we provide evidence that long-term culture of hESCs is accompanied by changes in cell cycle properties as well as configuration of several cell cycle regulatory molecules.

  4. A thermodynamic cycle for the solar cell (United States)

    Alicki, Robert; Gelbwaser-Klimovsky, David; Jenkins, Alejandro


    A solar cell is a heat engine, but textbook treatments are not wholly satisfactory from a thermodynamic standpoint, since they present solar cells as directly converting the energy of light into electricity, and the current in the circuit as maintained by an electrostatic potential. We propose a thermodynamic cycle in which the gas of electrons in the p phase serves as the working substance. The interface between the p and n phases acts as a self-oscillating piston that modulates the absorption of heat from the photons so that it may perform a net positive work during a complete cycle of its motion, in accordance with the laws of thermodynamics. We draw a simple hydrodynamical analogy between this model and the ;putt-putt; engine of toy boats, in which the interface between the water's liquid and gas phases serves as the piston. We point out some testable consequences of this model.

  5. The cell cycle as a brake for β-cell regeneration from embryonic stem cells. (United States)

    El-Badawy, Ahmed; El-Badri, Nagwa


    The generation of insulin-producing β cells from stem cells in vitro provides a promising source of cells for cell transplantation therapy in diabetes. However, insulin-producing cells generated from human stem cells show deficiency in many functional characteristics compared with pancreatic β cells. Recent reports have shown molecular ties between the cell cycle and the differentiation mechanism of embryonic stem (ES) cells, assuming that cell fate decisions are controlled by the cell cycle machinery. Both β cells and ES cells possess unique cell cycle machinery yet with significant contrasts. In this review, we compare the cell cycle control mechanisms in both ES cells and β cells, and highlight the fundamental differences between pluripotent cells of embryonic origin and differentiated β cells. Through critical analysis of the differences of the cell cycle between these two cell types, we propose that the cell cycle of ES cells may act as a brake for β-cell regeneration. Based on these differences, we discuss the potential of modulating the cell cycle of ES cells for the large-scale generation of functionally mature β cells in vitro. Further understanding of the factors that modulate the ES cell cycle will lead to new approaches to enhance the production of functional mature insulin-producing cells, and yield a reliable system to generate bona fide β cells in vitro.

  6. Impairment of cell cycle progression by aflatoxin B1 in human cell lines. (United States)

    Ricordy, R; Gensabella, G; Cacci, E; Augusti-Tocco, G


    Aflatoxin B1 is a mycotoxin produced by Aspergillus flavus and Aspergillus parasiticum, which may be present as a food contaminant. It is known to cause acute toxic effects and act as a carcinogenic agent. The carcinogenic action has been related to its ability to form unstable adducts with DNA, which represent possible mutagenic sites. On the other hand, the primary cellular target responsible for its toxic action has not yet been clearly identified. Previous data suggested a possible correlation between cell proliferation and responsiveness to aflatoxin toxicity. These observations led us to investigate the effect of the toxin on cell cycle progression of three human cell lines (HepG2, SK-N-MC and SK-N-SH derived from liver and nervous tissue tumours); they were shown to display different responses to toxin exposure and have different growth kinetics. We performed analysis of the cell cycle, DNA synthesis and expression of p21 and p53 in the presence and absence of the toxin in all cell lines exposed. The results of cell cycle cytofluorometric analysis show significant alterations of cell cycle progression as a result of toxin treatment. In all cell lines exposure to a 24 h toxin treatment causes a dose-dependent accumulation in S phase, however, the ability to recover from impairment to traverse S phase varies in the cell lines under study. SK-N-MC cells appear more prone to resume DNA synthesis when the toxin is removed, while the other two cell lines maintain a significant inhibition of DNA synthesis, as indicated by cytofluorimetry and [(3)H]dTR incorporation. The level of p53 and p21 expression in the three cell lines was examined by western blot analysis and significant differences were detected. The ready resumption of DNA synthesis displayed by SK-N-MC cells could possibly be related to the absence of p53 control of cell cycle progression.

  7. Changes of the cell cycle regulators and cell cycle arrest in cervical cancer cells after cisplatin therapy

    Institute of Scientific and Technical Information of China (English)


    Objective To investigate the changes of the cell cycle regulators ATM,Chk2 and p53 and cell cycle arrest in HeLa cells after cisplatin therapy. Methods The proliferation-inhibiting rates of HeLa cells induced by cisplatin of different concentrations were measured by MTT assays. The mRNA and protein expressions of ATM,Chk2 and p53 of HeLa cells with and without cisplatin were detected by RT-PCR and Western blot,respectively. The cell cycle analysis was conducted by flow cytometric analysis. Results Cisplatin...

  8. Altered lower leg muscle activation patterns in patients with cerebral palsy during cycling on an ergometer

    Directory of Open Access Journals (Sweden)

    Alves-Pinto A


    Full Text Available Ana Alves-Pinto,1,* Tobias Blumenstein,1,* Varvara Turova,1 Renée Lampe1,2 1Research Unit of the Buhl-Strohmaier Foundation for Cerebral Palsy and Paediatric Neuroorthopaedics, Orthopaedic Department, Klinikum rechts der Isar, 2Markus Würth Professorship, Technical University of Munich, Munich, Germany *These authors contributed equally to this work Objective: Cycling on a recumbent ergometer constitutes one of the most popular rehabilitation exercises in cerebral palsy (CP. However, no control is performed on how muscles are being used during training. Given that patients with CP present altered muscular activity patterns during cycling or walking, it is possible that an incorrect pattern of muscle activation is being promoted during rehabilitation cycling. This study investigated patterns of muscular activation during cycling on a recumbent ergometer in patients with CP and whether those patterns are determined by the degree of spasticity and of mobility.Methods: Electromyographic (EMG recordings of lower leg muscle activation during cycling on a recumbent ergometer were performed in 14 adult patients diagnosed with CP and five adult healthy participants. EMG recordings were done with an eight-channel EMG system built in the laboratory. The activity of the following muscles was recorded: Musculus rectus femoris, Musculus biceps femoris, Musculus tibialis anterior, and Musculus gastrocnemius. The degree of muscle spasticity and mobility was assessed using the Modified Ashworth Scale and the Gross Motor Function Classification System, respectively. Muscle activation patterns were described in terms of onset and duration of activation as well as duration of cocontractions.Results: Muscle activation in CP was characterized by earlier onsets, longer periods of activation, a higher occurrence of agonist–antagonist cocontractions, and a more variable cycling tempo in comparison to healthy participants. The degree of altered muscle activation

  9. American cranberry (Vaccinium macrocarpon) extract affects human prostate cancer cell growth via cell cycle arrest by modulating expression of cell cycle regulators. (United States)

    Déziel, Bob; MacPhee, James; Patel, Kunal; Catalli, Adriana; Kulka, Marianna; Neto, Catherine; Gottschall-Pass, Katherine; Hurta, Robert


    Prostate cancer is one of the most common cancers in the world, and its prevalence is expected to increase appreciably in the coming decades. As such, more research is necessary to understand the etiology, progression and possible preventative measures to delay or to stop the development of this disease. Recently, there has been interest in examining the effects of whole extracts from commonly harvested crops on the behaviour and progression of cancer. Here, we describe the effects of whole cranberry extract (WCE) on the behaviour of DU145 human prostate cancer cells in vitro. Following treatment of DU145 human prostate cancer cells with 10, 25 and 50 μg ml⁻¹ of WCE, respectively for 6 h, WCE significantly decreased the cellular viability of DU145 cells. WCE also decreased the proportion of cells in the G2-M phase of the cell cycle and increased the proportion of cells in the G1 phase of the cell cycle following treatment of cells with 25 and 50 μg ml⁻¹ treatment of WCE for 6 h. These alterations in cell cycle were associated with changes in cell cycle regulatory proteins and other cell cycle associated proteins. WCE decreased the expression of CDK4, cyclin A, cyclin B1, cyclin D1 and cyclin E, and increased the expression of p27. Changes in p16(INK4a) and pRBp107 protein expression levels also were evident, however, the changes noted in p16(INK4a) and pRBp107 protein expression levels were not statistically significant. These findings demonstrate that phytochemical extracts from the American cranberry (Vaccinium macrocarpon) can affect the behaviour of human prostate cancer cells in vitro and further support the potential health benefits associated with cranberries.

  10. The cell cycle rallies the transcription cycle: Cdc28/Cdk1 is a cell cycle-regulated transcriptional CDK. (United States)

    Chymkowitch, Pierre; Enserink, Jorrit M


    In the budding yeast Saccharomyces cerevisiae, the cyclin-dependent kinases (CDKs) Kin28, Bur1 and Ctk1 regulate basal transcription by phosphorylating the carboxyl-terminal domain (CTD) of RNA polymerase II. However, very little is known about the involvement of the cell cycle CDK Cdc28 in the transcription process. We have recently shown that, upon cell cycle entry, Cdc28 kinase activity boosts transcription of a subset of genes by directly stimulating the basal transcription machinery. Here, we discuss the biological significance of this finding and give our view of the kinase-dependent role of Cdc28 in regulation of RNA polymerase II.

  11. Spaceflight alters immune cell function and distribution (United States)

    Sonnenfeld, Gerald; Mandel, Adrian D.; Konstantinova, Irina V.; Berry, Wallace D.; Taylor, Gerald R.; Lesniak, A. T.; Fuchs, Boris B.; Rakhmilevich, Alexander L.


    Experiments are described which were performed onboard Cosmos 2044 to determine spaceflight effects on immunologically important cell function and distribution. Results indicate that bone marrow cells from flown and suspended rats exhibited a decreased response to a granulocyte/monocyte colony-stimulating factor compared with the bone marrow cells from control rats. Bone marrow cells showed an increase in the percentage of cells expressing markers for helper T-cells in the myelogenous population and increased percentages of anti-asialo granulocyte/monocyte-1-bearing interleulin-2 receptor bearing pan T- and helper T-cells in the lymphocytic population.

  12. The cell-cycle state of stem cells determines cell fate propensity. (United States)

    Pauklin, Siim; Vallier, Ludovic


    Self-renewal and differentiation of stem cells are fundamentally associated with cell-cycle progression to enable tissue specification, organ homeostasis, and potentially tumorigenesis. However, technical challenges have impaired the study of the molecular interactions coordinating cell fate choice and cell-cycle progression. Here, we bypass these limitations by using the FUCCI reporter system in human pluripotent stem cells and show that their capacity of differentiation varies during the progression of their cell cycle. These mechanisms are governed by the cell-cycle regulators cyclin D1-3 that control differentiation signals such as the TGF-β-Smad2/3 pathway. Conversely, cell-cycle manipulation using a small molecule directs differentiation of hPSCs and provides an approach to generate cell types with a clinical interest. Our results demonstrate that cell fate decisions are tightly associated with the cell-cycle machinery and reveal insights in the mechanisms synchronizing differentiation and proliferation in developing tissues.

  13. AspC-mediated aspartate metabolism coordinates the Escherichia coli cell cycle.

    Directory of Open Access Journals (Sweden)

    Feng Liu

    Full Text Available The fast-growing bacterial cell cycle consists of at least two independent cycles of chromosome replication and cell division. To ensure proper cell cycles and viability, chromosome replication and cell division must be coordinated. It has been suggested that metabolism could affect the Escherichia coli cell cycle, but the idea is still lacking solid evidences.We found that absence of AspC, an aminotransferase that catalyzes synthesis of aspartate, led to generation of small cells with less origins and slow growth. In contrast, excess AspC was found to exert the opposite effect. Further analysis showed that AspC-mediated aspartate metabolism had a specific effect in the cell cycle, as only extra aspartate of the 20 amino acids triggered production of bigger cells with more origins per cell and faster growth. The amount of DnaA protein per cell was found to be changed in response to the availability of AspC. Depletion of (pppGpp by ΔrelAΔspoT led to a slight delay in initiation of replication, but did not change the replication pattern found in the ΔaspC mutant.The results suggest that AspC-mediated metabolism of aspartate coordinates the E. coli cell cycle through altering the amount of the initiator protein DnaA per cell and the division signal UDP-glucose. Furthermore, AspC sequence conservation suggests similar functions in other organisms.

  14. Simvastatin induces cell cycle arrest and inhibits proliferation of bladder cancer cells via PPARγ signalling pathway (United States)

    Wang, Gang; Cao, Rui; Wang, Yongzhi; Qian, Guofeng; Dan, Han C.; Jiang, Wei; Ju, Lingao; Wu, Min; Xiao, Yu; Wang, Xinghuan


    Simvastatin is currently one of the most common drugs for old patients with hyperlipidemia, hypercholesterolemia and atherosclerotic diseases by reducing cholesterol level and anti-lipid properties. Importantly, simvastatin has also been reported to have anti-tumor effect, but the underlying mechanism is largely unknown. We collected several human bladder samples and performed microarray. Data analysis suggested bladder cancer (BCa) was significantly associated with fatty acid/lipid metabolism via PPAR signalling pathway. We observed simvastatin did not trigger BCa cell apoptosis, but reduced cell proliferation in a dose- and time-dependent manner, accompanied by PPARγ-activation. Moreover, flow cytometry analysis indicated that simvastatin induced cell cycle arrest at G0/G1 phase, suggested by downregulation of CDK4/6 and Cyclin D1. Furthermore, simvastatin suppressed BCa cell metastasis by inhibiting EMT and affecting AKT/GSK3β. More importantly, we found that the cell cycle arrest at G0/G1 phase and the alterations of CDK4/6 and Cyclin D1 triggered by simvastatin could be recovered by PPARγ-antagonist (GW9662), whereas the treatment of PPARα-antagonist (GW6471) shown no significant effects on the BCa cells. Taken together, our study for the first time revealed that simvastatin inhibited bladder cancer cell proliferation and induced cell cycle arrest at G1/G0 phase via PPARγ signalling pathway. PMID:27779188

  15. Capacity fade of Sony 18650 cells cycled at elevated temperatures. Part I. Cycling performance (United States)

    Ramadass, P.; Haran, Bala; White, Ralph; Popov, Branko N.

    The capacity fade of Sony 18650 Li-ion cells increases with increase in temperature. After 800 cycles, the cells cycled at RT and 45 °C showed a capacity fade of 30 and 36%, respectively. The cell cycled at 55 °C showed a capacity loss of about 70% after 490 cycles. The rate capability of the cells continues to decrease with cycling. Impedance measurements showed an overall increase in the cell resistance with cycling and temperature. Impedance studies of the electrode materials showed an increased positive electrode resistance when compared to that of the negative electrode for cells cycled at RT and 45 °C. However, cells cycled at 50 and 55 °C exhibit higher negative electrode resistance. The increased capacity fade for the cells cycled at high temperatures can be explained by taking into account the repeated film formation over the surface of anode, which results in increased rate of lithium loss and also in a drastic increase in the negative electrode resistance with cycling.

  16. Tcf3 and cell cycle factors contribute to butyrate resistance in colorectal cancer cells

    Energy Technology Data Exchange (ETDEWEB)

    Chiaro, Christopher, E-mail: [Department of Basic Sciences, The Commonwealth Medical College, 525 Pine Street, Scranton, PA 18509 (United States); Lazarova, Darina L., E-mail: [Department of Basic Sciences, The Commonwealth Medical College, 525 Pine Street, Scranton, PA 18509 (United States); Bordonaro, Michael, E-mail: [Department of Basic Sciences, The Commonwealth Medical College, 525 Pine Street, Scranton, PA 18509 (United States)


    Highlights: Black-Right-Pointing-Pointer We investigate mechanisms responsible for butyrate resistance in colon cancer cells. Black-Right-Pointing-Pointer Tcf3 modulates butyrate's effects on Wnt activity and cell growth in resistant cells. Black-Right-Pointing-Pointer Tcf3 modulation of butyrate's effects differ by cell context. Black-Right-Pointing-Pointer Cell cycle factors are overexpressed in the resistant cells. Black-Right-Pointing-Pointer Reversal of altered gene expression can enhance the anti-cancer effects of butyrate. -- Abstract: Butyrate, a fermentation product of dietary fiber, inhibits clonal growth in colorectal cancer (CRC) cells dependent upon the fold induction of Wnt activity. We have developed a CRC cell line (HCT-R) that, unlike its parental cell line, HCT-116, does not respond to butyrate exposure with hyperactivation of Wnt signaling and suppressed clonal growth. PCR array analyses revealed Wnt pathway-related genes, the expression of which differs between butyrate-sensitive HCT-116 CRC cells and their butyrate-resistant HCT-R cell counterparts. We identified overexpression of Tcf3 as being partially responsible for the butyrate-resistant phenotype, as this DNA-binding protein suppresses the hyperinduction of Wnt activity by butyrate. Consequently, Tcf3 knockdown in HCT-R cells restores their sensitivity to the effects of butyrate on Wnt activity and clonal cell growth. Interestingly, the effects of overexpressed Tcf3 differ between HCT-116 and HCT-R cells; thus, in HCT-116 cells Tcf3 suppresses proliferation without rendering the cells resistant to butyrate. In HCT-R cells, however, the overexpression of Tcf3 inhibits Wnt activity, and the cells are still able to proliferate due to the higher expression levels of cell cycle factors, particularly those driving the G{sub 1} to S transition. Knowledge of the molecular mechanisms determining the variable sensitivity of CRC cells to butyrate may assist in developing approaches that

  17. P27 in cell cycle control and cancer

    DEFF Research Database (Denmark)

    Møller, Michael Boe


    In order to survive, cells need tight control of cell cycle progression. The control mechanisms are often lost in human cancer cells. The cell cycle is driven forward by cyclin-dependent kinases (CDKs). The CDK inhibitors (CKIs) are important regulators of the CDKs. As the name implies, CKIs were...

  18. Tumor-suppressor genes, cell cycle regulatory checkpoints, and the skin

    Directory of Open Access Journals (Sweden)

    Ana Maria Abreu Velez


    Full Text Available The cell cycle (or cell-division cycle is a series of events that take place in a cell, leading to its division and duplication. Cell division requires cell cycle checkpoints (CPs that are used by the cell to both monitor and regulate the progress of the cell cycle. Tumor-suppressor genes (TSGs or antioncogenes are genes that protect the cell from a single event or multiple events leading to cancer. When these genes mutate, the cell can progress to a cancerous state. We aimed to perform a narrative review, based on evaluation of the manuscripts published in MEDLINE-indexed journals using the Medical Subject Headings (MeSH terms "tumor suppressor′s genes," "skin," and "cell cycle regulatory checkpoints." We aimed to review the current concepts regarding TSGs, CPs, and their association with selected cutaneous diseases. It is important to take into account that in some cell cycle disorders, multiple genetic abnormalities may occur simultaneously. These abnormalities may include intrachromosomal insertions, unbalanced division products, recombinations, reciprocal deletions, and/or duplication of the inserted segments or genes; thus, these presentations usually involve several genes. Due to their complexity, these disorders require specialized expertise for proper diagnosis, counseling, personal and family support, and genetic studies. Alterations in the TSGs or CP regulators may occur in many benign skin proliferative disorders, neoplastic processes, and genodermatoses.

  19. Raman spectroscopic study of a genetically altered kidney cell (United States)

    Joshi, Joel; Garcia, Francisco; Centeno, Silvia P.; Joshi, N. V.


    A Raman spectroscopic investigation of a genetically altered Human Embryonic Kidney Cell (HEK293) along with a pathologically normal cell has been carried out by a conventional method. The genetic alteration was carried out with a standard protocol by using a Green Fluorescence Protein (GFP). Raman spectra show that there are dramatic differences between the spectrum obtained from a genetically altered cell and that obtained from a pathologically normal cell. The former shows three broad bands; meanwhile the latter shows several sharp peaks corresponding to the ring vibrational modes of Phen, GFP and DNA. The present analysis provides an indication that the force field near Phen located at 64, 65 and 66 was altered during the genetic transformation. The Raman spectrum could be a direct experimental evidence for substantial modifications triggered due to the expression of specific genes.

  20. Local circadian clock gates cell cycle progression of transient amplifying cells during regenerative hair cycling. (United States)

    Plikus, Maksim V; Vollmers, Christopher; de la Cruz, Damon; Chaix, Amandine; Ramos, Raul; Panda, Satchidananda; Chuong, Cheng-Ming


    Regenerative cycling of hair follicles offers an unique opportunity to explore the role of circadian clock in physiological tissue regeneration. We focused on the role of circadian clock in actively proliferating transient amplifying cells, as opposed to quiescent stem cells. We identified two key sites of peripheral circadian clock activity specific to regenerating anagen hair follicles, namely epithelial matrix and mesenchymal dermal papilla. We showed that peripheral circadian clock in epithelial matrix cells generates prominent daily mitotic rhythm. As a consequence of this mitotic rhythmicity, hairs grow faster in the morning than in the evening. Because cells are the most susceptible to DNA damage during mitosis, this cycle leads to a remarkable time-of-day-dependent sensitivity of growing hair follicles to genotoxic stress. Same doses of γ-radiation caused dramatic hair loss in wild-type mice when administered in the morning, during mitotic peak, compared with the evening, when hair loss is minimal. This diurnal radioprotective effect becomes lost in circadian mutants, consistent with asynchronous mitoses in their hair follicles. Clock coordinates cell cycle progression with genotoxic stress responses by synchronizing Cdc2/Cyclin B-mediated G2/M checkpoint. Our results uncover diurnal mitotic gating as the essential protective mechanism in highly proliferative hair follicles and offer strategies for minimizing or maximizing cytotoxicity of radiation therapies.

  1. Alterations to the Nitrogen Cycle in a Soil Exposed to Trace Veterinary Antibiotics (United States)

    Devries, S. L.; Zhang, P.; Loving, M.; Pons, E.


    Agricultural soils are exposed to veterinary antibiotics when manure fertilizers containing residual active compounds are spread as fertilizers or fertilizer amendments. While there is evidence to suggest that trace antibiotics in soil may significantly alter the microbial community structure or function, a comprehensive assessment of nitrogen cycle dynamics in antibiotic amended soils is currently lacking. Because sustainable fertilizer practices are based on known parameters of soil nitrogen cycle variation, microbial inhibition or delayed activity caused by antibiotics may undermine the ability of modeling tools to make strong fertilizer management recommendations, leading to reduced fertilizer use efficiency and increased inputs of pollutant N species, including N2O and NO3-, to environmental reservoirs. The objective of this study is to assess the impact of six veterinary antibiotics on leachable nitrate concentrations in top soil and the saturated zone. Dose-response and time-series curves were constructed for column and incubation studies conducted on soils treated with sulfamethoxazole, sulfadiazine, sulfamethazine, narasin, gentamicin, and monensin. Dosages ranged from 1-500 ng/L in column studies and 1-500 ng/g in incubation tests and are consistent with concentrations that have been reported in environmental samples. The results to be presented indicate that exposure to veterinary antibiotics, even at concentrations that are well below previously established no-effect limit, may significantly alter the denitrification potential of affected soils.

  2. Nylon wool purification alters the activation of T cells. (United States)

    Wohler, Jillian E; Barnum, Scott R


    Purification of lymphocytes, particularly T cells, is commonly performed using nylon wool. This enrichment method selectively retains B cells and some myeloid cells allowing a significantly more pure T cell population to flow through a nylon wool column. T cells purified in this fashion are assumed to be unaltered and functionally naïve, however some studies have suggested aberrant in vitro T cell responses after nylon wool treatment. We found that nylon wool purification significantly altered T cell proliferation, expression of activation markers and production of cytokines. Our results suggest that nylon wool treatment modifies T cell activation responses and that caution should be used when choosing this purification method.

  3. Cell cycle synchronization reveals greater G2/M-phase accumulation of lung epithelial cells exposed to titanium dioxide nanoparticles. (United States)

    Medina-Reyes, Estefany I; Bucio-López, Laura; Freyre-Fonseca, Verónica; Sánchez-Pérez, Yesennia; García-Cuéllar, Claudia M; Morales-Bárcenas, Rocío; Pedraza-Chaverri, José; Chirino, Yolanda I


    Titanium dioxide has been classified in the 2B group as a possible human carcinogen by the International Agency for Research on Cancer, and amid concerns of its exposure, cell cycle alterations are an important one. However, several studies show inconclusive effects, mainly because it is difficult to compare cell cycle effects caused by TiO2 nanoparticle (NP) exposure between different shapes and sizes of NP, cell culture types, and time of exposure. In addition, cell cycle is frequently analyzed without cell cycle synchronization, which may also mask some effects. We hypothesized that synchronization after TiO2 NP exposure could reveal dissimilar cell cycle progression when compared with unsynchronized cell population. To test our hypothesis, we exposed lung epithelial cells to 1 and 10 μg/cm(2) TiO2 NPs for 7 days and one population was synchronized by serum starvation and inhibition of ribonucleotide reductase using hydroxyurea. Another cell population was exposed to TiO2 NPs under the same experimental conditions, but after treatments, cell cycle was analyzed without synchronization. Our results showed that TiO2 NP-exposed cells without synchronization had no changes in cell cycle distribution; however, cell population synchronized after 1 and 10 μg/cm(2) TiO2 NP treatment showed a 1.5-fold and 1.66-fold increase, respectively, in proliferation. Synchronized cells also reveal a faster capability of TiO2 NP-exposed cells to increase cell population in the G2/M phase in the following 9 h after synchronization. We conclude that synchronization discloses a greater percentage of cells in the G2/M phase and higher proliferation than TiO2 NP-synchronized cells.

  4. Cell-cycle times and the tumour control probability. (United States)

    Maler, Adrian; Lutscher, Frithjof


    Mechanistic dynamic cell population models for the tumour control probability (TCP) to date have used a simplistic representation of the cell cycle: either an exponential cell-cycle time distribution (Zaider & Minerbo, 2000, Tumour control probability: a formulation applicable to any temporal protocol of dose delivery. Phys. Med. Biol., 45, 279-293) or a two-compartment model (Dawson & Hillen, 2006, Derivation of the tumour control probability (TCP) from a cell cycle model. Comput. Math. Methods Med., 7, 121-142; Hillen, de Vries, Gong & Yurtseven, 2009, From cell population models to tumour control probability: including cell cycle effects. Acta Oncol. (submitted)). Neither of these simplifications captures realistic cell-cycle time distributions, which are rather narrowly peaked around the mean. We investigate how including such distributions affects predictions of the TCP. At first, we revisit the so-called 'active-quiescent' model that splits the cell cycle into two compartments and explore how an assumption of compartmental independence influences the predicted TCP. Then, we formulate a deterministic age-structured model and a corresponding branching process. We find that under realistic cell-cycle time distributions, lower treatment intensities are sufficient to obtain the same TCP as in the aforementioned models with simplified cell cycles, as long as the treatment is constant in time. For fractionated treatment, the situation reverses such that under realistic cell-cycle time distributions, the model requires more intense treatment to obtain the same TCP.

  5. Effect of p27KIP1 on cell cycle and apoptosis in gastric cancer cells

    Institute of Scientific and Technical Information of China (English)

    Jian-Yong Zheng; Wei-Zhong Wang; Kai-Zong Li; Wen-Xian Guan; Wei Yan


    AIM: To elucidate the effect of p27KIP1 on cell cycle and apoptosis regulation in gastric carcinoma cells.METHODS: The whole length of p27KIP1 cDNA was transfected into human gastric cancer cell line SCG7901by lipofectamine. Expression of p27KIP1 protein or mRNA was analyzed by Western blot and RNA dot blotting,respectively. Effect of p27KIP1 on cell growth was observed by MTT assay and anchorage-independent growth in soft agar. Tumorigenicity in nude mice was used to assess the in vivo biological effect of p27KIP1. Flow cytometry,TUNEL, and electron microscopy were used to assess the effect of p27KIP1 on cell cycle and apoptosis.RESULTS: Expression of p27KIP1 protein or mRNA increased evidently in SCG7901 cells transfected with p27KIP1. The cell growth was reduced by 31% at 48 h after induction with zinc determined by cell viability assay. The alteration of cell malignant phenotype was evidently indicated by the loss of anchorage-independent growth ability in soft agar. The tumorigenicity in nude mice was reduced evidently (0.55±0.14 cm vs 1.36±0.13crn, P<0.01). p27KIP1 overexpression caused cell arrest with 36% increase (from 33.7% to 69.3%,P<0.01) in G1 population. Prolonged p27KIP1 expression induced apoptotic cell death reflected by pre-G1 peak in the histogram of FACS, which was also confirmed by TUNEL assay and electron microscopy.CONCLUSION: p27KIP1 can prolong cell cycle in G1phase and lead to apoptosis. p27KIP1 may be a good candidate for cancer gene therapy.

  6. Butachlor, a suspected carcinogen, alters growth and transformation characteristics of mouse liver cells. (United States)

    Ou, Y H; Chung, P C; Chang, Y C; Ngo, F Q; Hsu, K Y; Chen, F D


    Butachlor is a widely used herbicide in Asia and South America. Previous investigations have indicated that it is a suspected carcinogen. To understand more about the biological effects of butachlor on cultured cells and the mechanism(s) of its carcinogenicity, we studied the alteration of the growth characteristics that was induced by butachlor in normal mouse liver cells (BNL CL2). This study demonstrates that butachlor decreases the population-doubling time of BNL CL2 cells, suggesting that it stimulates cell proliferation. To support this finding, a thymidine incorporation assay was conducted and a similar result that butachlor stimulates cell proliferation was elucidated. In addition, we show that butachlor increases the saturation density of the BNL CL2 cells. When combined with the tumor initiator N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), butachlor transforms cells efficiently, as demonstrated by loss of contact inhibition. These findings indicate that butachlor alters the growth characteristics of BNL CL2 cells and suggest that butachlor may induce malignant transformation through stimulation of cell proliferation, alteration of cell cycle regulation, and suppression of cell density-dependent inhibition of proliferation.

  7. Mitochondrial dynamics and the cell cycle

    Directory of Open Access Journals (Sweden)

    Penny M.A. Kianian


    Full Text Available Nuclear-mitochondrial (NM communication impacts many aspects of plant development including vigor, sterility and viability. Dynamic changes in mitochondrial number, shape, size, and cellular location takes place during the cell cycle possibly impacting the process itself and leading to distribution of this organelle into daughter cells. The genes that underlie these changes are beginning to be identified in model plants such as Arabidopsis. In animals disruption of the drp1 gene, a homolog to the plant drp3A and drp3B, delays mitochondrial division. This mutation results in increased aneuploidy due to chromosome mis-segregation. It remains to be discovered if a similar outcome is observed in plants. Alloplasmic lines provide an opportunity to understand the communication between the cytoplasmic organelles and the nucleus. Examples of studies in these lines, especially from the extensive collection in wheat, point to the role of mitochondria in chromosome movement, pollen fertility and other aspects of development. Genes involved in NM interaction also are believed to play a critical role in evolution of species and interspecific cross incompatibilities.

  8. Changes of the cell cycle regulators and cell cycle arrest in cervical cancer cells after cisplatin therapy

    Institute of Scientific and Technical Information of China (English)

    Ke-xiu Zhu; Ya-li Cao; Bin Li; Jia Wang; Xiao-bing Han


    Objective To investigate the changes of the cell cycle regulators ATM, Chk2 and p53 and cell cycle arrest in HeLa cells after cisplatin therapy. Methods The proliferation-inhibiting rates of HeLa cells induced by eisplatin of different concentrations were measured by MTT assays. The mRNA and protein expressions of ATM, Chk2 and p53 of HeLa cells with and withont cisplatin were detected by RT-PCR and Western blot, respectively. The cell cycle analysis was conducted by flow cytometric analysis. Results Cisplatin inhibited the proliferation of HeLa cells in a dose- and time-dependent manner. The mRNA and protein expressions of ATM, Chk2 and p53 were increased in HeLa cells treated with cisplatin. The cell cycle was arrested in G2/M phase in HeLa cells treated with cisplatin. Conclusion Activation of ATM, Chk2 and p53 might be critical in determining whether cells survive or undergo apoptesis. Targeting ATM, Chk2 and p53 pathway might he a promising strategy for reversing chemoresistance to clsplatin in cervical cancer.

  9. Growth inhibitory effect of 4-phenyl butyric acid on human gastric cancer cells is associated with cell cycle arrest

    Institute of Scientific and Technical Information of China (English)

    Long-Zhu Li; Hong-Xia Deng; Wen-Zhu Lou; Xue-Yan Sun; Meng-Wan Song; Jing Tao; Bing-Xiu Xiao; Jun-Ming Guo


    AIM: To investigate the growth effects of 4-phenyl butyric acid (PBA) on human gastric carcinoma cells and their mechanisms. METHODS: Moderately-differentiated human gastric carcinoma SGC-7901 and lowly-differentiated MGC-803 cells were treated with 5, 10, 20, 40, and 60 μmol/L PBA for 1-4 d. Cell proliferation was detected using the MTT colorimetric assay. Cell cycle distributions were examined using flow cytometry. RESULTS: The proliferation of gastric carcinoma cells was inhibited by PBA in a dose- and time-dependent fashion. Flow cytometry showed that SGC-7901 cells treated with low concentrations of PBA were arrested at the G0/G1 phase, whereas cells treated with high concentrations of PBA were arrested at the G2/M phase. Although MGC-803 cells treated with low concentrations of PBA were also arrested at the G0/G1 phase, cells treated with high concentrations of PBA were arrested at the S phase. CONCLUSION: The growth inhibitory effect of PBA on gastric cancer cells is associated with alteration of the cell cycle. For moderately-differentiated gastric cancer cells, the cell cycle was arrested at the G0/G1 and G2/M phases. For lowly-differentiated gastric cancer cells, the cell cycle was arrested at the G0/G1 and S phases.

  10. Microsporidia infection impacts the host cell's cycle and reduces host cell apoptosis (United States)

    Higes, Mariano; Sagastume, Soledad; Juarranz, Ángeles; Dias-Almeida, Joyce; Budge, Giles E.; Meana, Aránzazu; Boonham, Neil


    Intracellular parasites can alter the cellular machinery of host cells to create a safe haven for their survival. In this regard, microsporidia are obligate intracellular fungal parasites with extremely reduced genomes and hence, they are strongly dependent on their host for energy and resources. To date, there are few studies into host cell manipulation by microsporidia, most of which have focused on morphological aspects. The microsporidia Nosema apis and Nosema ceranae are worldwide parasites of honey bees, infecting their ventricular epithelial cells. In this work, quantitative gene expression and histology were studied to investigate how these two parasites manipulate their host’s cells at the molecular level. Both these microsporidia provoke infection-induced regulation of genes involved in apoptosis and the cell cycle. The up-regulation of buffy (which encodes a pro-survival protein) and BIRC5 (belonging to the Inhibitor Apoptosis protein family) was observed after infection, shedding light on the pathways that these pathogens use to inhibit host cell apoptosis. Curiously, different routes related to cell cycle were modified after infection by each microsporidia. In the case of N. apis, cyclin B1, dacapo and E2F2 were up-regulated, whereas only cyclin E was up-regulated by N. ceranae, in both cases promoting the G1/S phase transition. This is the first report describing molecular pathways related to parasite-host interactions that are probably intended to ensure the parasite’s survival within the cell. PMID:28152065

  11. Overexpression of AQP3 Modifies the Cell Cycle and the Proliferation Rate of Mammalian Cells in Culture. (United States)

    Galán-Cobo, Ana; Ramírez-Lorca, Reposo; Serna, Ana; Echevarría, Miriam


    Abnormal AQP3 overexpression in tumor cells of different origins has been reported and a role for this enhanced AQP3 expression in cell proliferation and tumor processess has been indicated. To further understand the role AQP3 plays in cell proliferation we explore the effect that stable over expression of AQP3 produces over the proliferation rate and cell cycle of mammalian cells. The cell cycle was analyzed by flow cytometry with propidium iodide (PI) and the cell proliferation rate measured through cell counting and BrdU staining. Cells with overexpression of AQP3 (AQP3-o) showed higher proliferation rate and larger percentage of cells in phases S and G2/M, than wild type cells (wt). Evaluation of the cell response against arresting the cell cycle with Nocodazole showed that AQP3-o exhibited a less modified cell cycle pattern and lower Annexin V specific staining than wt, consistently with a higher resistance to apoptosis of AQP3-overexpressing cells. The cell volume and complexity were also larger in AQP3-o compared to wt cells. After transcriptomic analysis, RT-qPCR was performed to highlight key molecules implicated in cell proliferation which expression may be altered by overexpression of AQP3 and the comparative analysis between both type of cells showed significant changes in the expression of Zeb2, Jun, JunB, NF-kβ, Cxcl9, Cxcl10, TNF, and TNF receptors. We conclude that the role of AQP3 in cell proliferation seems to be connected to increments in the cell cycle turnover and changes in the expression levels of relevant genes for this process. Larger expression of AQP3 may confer to the cell a more tumor like phenotype and contributes to explain the presence of this protein in many different tumors.

  12. Calcium signaling in plant cells in altered gravity (United States)

    Kordyum, E. L.


    Changes in the intracellular Ca 2+ concentration in altered gravity (microgravity and clinostating) evidence that Ca 2+ signaling can play a fundamental role in biological effects of microgravity. Calcium as a second messenger is known to play a crucial role in stimulus - response coupling for many plant cellular signaling pathways. Its messenger functions are realized by transient changes in the cytosolic ion concentration induced by a variety of internal and external stimuli such as light, hormones, temperature, anoxia, salinity, and gravity. Although the first data on the changes in the calcium balance in plant cells under the influence of altered gravity have appeared in 80 th, a review highlighting the performed research and the possible significance of such Ca 2+ changes in the structural and metabolic rearrangements of plant cells in altered gravity is still lacking. In this paper, an attempt was made to summarize the available experimental results and to consider some hypotheses in this field of research. It is proposed to distinguish between cell gravisensing and cell graviperception; the former is related to cell structure and metabolism stability in the gravitational field and their changes in microgravity (cells not specialized to gravity perception), the latter is related to active use of a gravitational stimulus by cells presumebly specialized to gravity perception for realization of normal space orientation, growth, and vital activity (gravitropism, gravitaxis) in plants. The main experimental data concerning both redistribution of free Ca 2+ ions in plant cell organelles and the cell wall, and an increase in the intracellular Ca 2+ concentration under the influence of altered gravity are presented. Based on the gravitational decompensation hypothesis, the consequence of events occurring in gravisensing cells not specialized to gravity perception under altered gravity are considered in the following order: changes in the cytoplasmic membrane surface

  13. Culture adaptation alters transcriptional hierarchies among single human embryonic stem cells reflecting altered patterns of differentiation. (United States)

    Gokhale, Paul J; Au-Young, Janice K; Dadi, SriVidya; Keys, David N; Harrison, Neil J; Jones, Mark; Soneji, Shamit; Enver, Tariq; Sherlock, Jon K; Andrews, Peter W


    We have used single cell transcriptome analysis to re-examine the substates of early passage, karyotypically Normal, and late passage, karyotypically Abnormal ('Culture Adapted') human embryonic stem cells characterized by differential expression of the cell surface marker antigen, SSEA3. The results confirmed that culture adaptation is associated with alterations to the dynamics of the SSEA3(+) and SSEA3(-) substates of these cells, with SSEA3(-) Adapted cells remaining within the stem cell compartment whereas the SSEA3(-) Normal cells appear to have differentiated. However, the single cell data reveal that these substates are characterized by further heterogeneity that changes on culture adaptation. Notably the Adapted population includes cells with a transcriptome substate suggestive of a shift to a more naïve-like phenotype in contrast to the cells of the Normal population. Further, a subset of the Normal SSEA3(+) cells expresses genes typical of endoderm differentiation, despite also expressing the undifferentiated stem cell genes, POU5F1 (OCT4) and NANOG, whereas such apparently lineage-primed cells are absent from the Adapted population. These results suggest that the selective growth advantage gained by genetically variant, culture adapted human embryonic stem cells may derive in part from a changed substate structure that influences their propensity for differentiation.

  14. Aurkb/PP1-mediated resetting of Oct4 during the cell cycle determines the identity of embryonic stem cells. (United States)

    Shin, Jihoon; Kim, Tae Wan; Kim, Hyunsoo; Kim, Hye Ji; Suh, Min Young; Lee, Sangho; Lee, Han-Teo; Kwak, Sojung; Lee, Sang-Eun; Lee, Jong-Hyuk; Jang, Hyonchol; Cho, Eun-Jung; Youn, Hong-Duk


    Pluripotency transcription programs by core transcription factors (CTFs) might be reset during M/G1 transition to maintain the pluripotency of embryonic stem cells (ESCs). However, little is known about how CTFs are governed during cell cycle progression. Here, we demonstrate that the regulation of Oct4 by Aurora kinase b (Aurkb)/protein phosphatase 1 (PP1) during the cell cycle is important for resetting Oct4 to pluripotency and cell cycle genes in determining the identity of ESCs. Aurkb phosphorylates Oct4(S229) during G2/M phase, leading to the dissociation of Oct4 from chromatin, whereas PP1 binds Oct4 and dephosphorylates Oct4(S229) during M/G1 transition, which resets Oct4-driven transcription for pluripotency and the cell cycle. Aurkb phosphor-mimetic and PP1 binding-deficient mutations in Oct4 alter the cell cycle, effect the loss of pluripotency in ESCs, and decrease the efficiency of somatic cell reprogramming. Our findings provide evidence that the cell cycle is linked directly to pluripotency programs in ESCs.

  15. Cell cycle controls stress response and longevity in C. elegans (United States)

    Dottermusch, Matthias; Lakner, Theresa; Peyman, Tobias; Klein, Marinella; Walz, Gerd; Neumann-Haefelin, Elke


    Recent studies have revealed a variety of genes and mechanisms that influence the rate of aging progression. In this study, we identified cell cycle factors as potent regulators of health and longevity in C. elegans. Focusing on the cyclin-dependent kinase 2 (cdk-2) and cyclin E (cye-1), we show that inhibition of cell cycle genes leads to tolerance towards environmental stress and longevity. The reproductive system is known as a key regulator of longevity in C. elegans. We uncovered the gonad as the central organ mediating the effects of cell cycle inhibition on lifespan. In particular, the proliferating germ cells were essential for conferring longevity. Steroid hormone signaling and the FOXO transcription factor DAF-16 were required for longevity associated with cell cycle inhibition. Furthermore, we discovered that SKN-1 (ortholog of mammalian Nrf proteins) activates protective gene expression and induces longevity when cell cycle genes are inactivated. We conclude that both, germline absence and inhibition through impairment of cell cycle machinery results in longevity through similar pathways. In addition, our studies suggest further roles of cell cycle genes beyond cell cycle progression and support the recently described connection of SKN-1/Nrf to signals deriving from the germline. PMID:27668945

  16. Influence of cell cycle on responses of MCF-7 cells to benzo[a]pyrene

    Directory of Open Access Journals (Sweden)

    Giddings Ian


    Full Text Available Abstract Background Benzo[a]pyrene (BaP is a widespread environmental genotoxic carcinogen that damages DNA by forming adducts. This damage along with activation of the aryl hydrocarbon receptor (AHR induces complex transcriptional responses in cells. To investigate whether human cells are more susceptible to BaP in a particular phase of the cell cycle, synchronised breast carcinoma MCF-7 cells were exposed to BaP. Cell cycle progression was analysed by flow cytometry, DNA adduct formation was assessed by 32P-postlabeling analysis, microarrays of 44K human genome-wide oligos and RT-PCR were used to detect gene expression (mRNA changes and Western blotting was performed to determine the expression of some proteins, including cytochrome P450 (CYP 1A1 and CYP1B1, which are involved in BaP metabolism. Results Following BaP exposure, cells evaded G1 arrest and accumulated in S-phase. Higher levels of DNA damage occurred in S- and G2/M- compared with G0/G1-enriched cultures. Genes that were found to have altered expression included those involved in xenobiotic metabolism, apoptosis, cell cycle regulation and DNA repair. Gene ontology and pathway analysis showed the involvement of various signalling pathways in response to BaP exposure, such as the Catenin/Wnt pathway in G1, the ERK pathway in G1 and S, the Nrf2 pathway in S and G2/M and the Akt pathway in G2/M. An important finding was that higher levels of DNA damage in S- and G2/M-enriched cultures correlated with higher levels of CYP1A1 and CYP1B1 mRNA and proteins. Moreover, exposure of synchronised MCF-7 cells to BaP-7,8-diol-9,10-epoxide (BPDE, the ultimate carcinogenic metabolite of BaP, did not result in significant changes in DNA adduct levels at different phases of the cell cycle. Conclusions This study characterised the complex gene response to BaP in MCF-7 cells and revealed a strong correlation between the varying efficiency of BaP metabolism and DNA damage in different phases of the cell

  17. Mammalian cryptochromes impinge on cell cycle progression in a circadian clock-independent manner. (United States)

    Destici, Eugin; Oklejewicz, Małgorzata; Saito, Shoko; van der Horst, Gijsbertus T J


    By gating cell cycle progression to specific times of the day, the intracellular circadian clock is thought to reduce the exposure of replicating cells to potentially hazardous environmental and endogenous genotoxic compounds. Although core clock gene defects that eradicate circadian rhythmicity can cause an altered in vivo genotoxic stress response and aberrant proliferation rate, it remains to be determined to what extent these cell cycle related phenotypes are due to a cell-autonomous lack of circadian oscillations. We investigated the DNA damage sensitivity and proliferative capacity of cultured primary Cry1(-/- )|Cry2(-/-) fibroblasts. Contrasting previous in vivo studies, we show that the absence of CRY proteins does not affect the cell-autonomous DNA damage response upon exposure of primary cells in vitro to genotoxic agents, but causes cells to proliferate faster. By comparing primary wild-type, Cry1(-/-) |Cry2(-/-), Cry1(+/-)|Cry2(-/-) and Cry1(-/-)|Cry2(+/-) fibroblasts, we provide evidence that CRY proteins influence cell cycle progression in a cell-autonomous, but circadian clock-independent manner and that the accelerated cell cycle progression of Cry-deficient cells is caused by global dysregulation of Bmal1-dependent gene expression. These results suggest that the inconsistency between in vivo and in vitro observations might be attributed to systemic circadian control rather than a direct cell-autonomous control.

  18. Lectin from Agaricus Bisporus Suppresses Akt Phosphorylation and Arrests Cell Cycle Progression in Primary Human Retinal Pigment Epithelial Cells

    Directory of Open Access Journals (Sweden)

    Y. H. Cheung


    Full Text Available Anomalous retinal pigment epithelial (RPE cells have been implicated in the development of retinal diseases. Lectin from the edible mushroom Agaricus bisporus (ABL was found to inhibit growth of RPE cells. To elucidate the mechanism through which ABL inhibits RPE cell proliferation, we investigated the changes in cell proliferation-related signaling pathways and cell cycle distribution patterns. Primary human RPE cells were grown with or without the lectin (ABL supplement (20ug or 90ug/ml for three days. Phosphorylation statuses of Akt, Jnk and p38 as well as p53 expression level were investigated by Western blotting. Cellular distributions in various cell cycle phases were investigated using flow cytometry. After ABL treatment (90ug/ml, Akt was found to be hypo-phosphorylated while the expression levels of p53, phosphorylated-Jnk and phosphorylated-p38 were not altered. The amount of cells present at S phase was reduced. Our results showed that ABL hypo-phosphorylated Akt and this observation is in line with the finding that ABL could attenuate cell proliferation. As the level of p53 was not significantly altered by ABL, this suggested that the mechanism in which ABL arrested cell proliferation was independent of Akt-mediated MDM2 activation but was possibly mediated by altering G1 to S phase transition.

  19. Characteristics and Behavior of Cycled Aged Lithium Ion Cells (United States)


    service cycle and provide the cornerstone for safety analysis. 18650 Cells with representative chemistry of cells contained in current Army procured...their relevance to this effort warrants inclusion. 1-3 EXPERIMENTAL Representative 18650 cells were cycled at different rates and environmental...conditions. The 18650 chemistry used in this effort is a LiCoO2 lithium ion electrochemical cell. The bulk of this effort was conducted with 1.5 Amp-hr

  20. Cell cycle-dependent gene networks relevant to cancer

    Institute of Scientific and Technical Information of China (English)


    The analysis of sophisticated interplays between cell cycle-dependent genes in a disease condition is one of the largely unexplored areas in modern tumor biology research. Many cell cycle-dependent genes are either oncogenes or suppressor genes, or are closely asso- ciated with the transition of a cell cycle. However, it is unclear how the complicated relationships between these cell cycle-dependent genes are, especially in cancers. Here, we sought to identify significant expression relationships between cell cycle-dependent genes by analyzing a HeLa microarray dataset using a local alignment algorithm and constructed a gene transcriptional network specific to the cancer by assembling these newly identified gene-gene relationships. We further characterized this global network by partitioning the whole network into several cell cycle phase-specific sub-networks. All generated networks exhibited the power-law node-degree dis- tribution, and the average clustering coefficients of these networks were remarkably higher than those of pure scale-free networks, indi- cating a property of hierarchical modularity. Based on the known protein-protein interactions and Gene Ontology annotation data, the proteins encoded by cell cycle-dependent interacting genes tended to share the same biological functions or to be involved in the same biological processes, rather than interacting by physical means. Finally, we identified the hub genes related to cancer based on the topo- logical importance that maintain the basic structure of cell cycle-dependent gene networks.

  1. Metabolic alterations in cancer cells and therapeutic implications

    Institute of Scientific and Technical Information of China (English)

    Naima Hammoudi; Kausar Begam Riaz Ahmed; Celia Garcia-Prieto; Peng Huang


    Cancer metabolism has emerged as an important area of research in recent years. Elucidation of the metabolic differences between cancer and normal cells and the underlying mechanisms will not only advance our understanding of fundamental cancer cell biology but also provide an important basis for the development of new therapeutic strategies and novel compounds to selectively eliminate cancer cells by targeting their unique metabolism. This article reviews several important metabolic alterations in cancer cells, with an emphasis on increased aerobic glycolysis (the Warburg effect) and glutamine addiction, and discusses the mechanisms that may contribute to such metabolic changes. In addition, metabolic alterations in cancer stem cells, mitochondrial metabolism and its influence on drug sensitivity, and potential therapeutic strategies and agents that target cancer metabolism are also discussed.

  2. Amygdala Kindling Alters Estrus Cycle and Ovarian Morphology in the Rat. (United States)

    Pan, Juan; Zhang, Lingwu; Wang, Feng; Liu, Dan; Li, P Andy; Sun, Tao


    The objective of this study is to explore the effects of amygdala kindling on estrus cycle and ovarian morphology. Thirty-five female rats at the age of 8 weeks were randomly designated to electrode kindled, sham-kindled, and normal controls. Kindled rats were implanted with kindling electrodes in the left basolateral amygdala and kindled by brief suprathreshold stimulations with a bipolar electrode. Estrous cycles were daily monitored through vaginal smears. Electrographic and behavioral seizures were recorded and ovarian morphology was evaluated by light and electron microscopies. Our results showed that the kindled rats lost their ovarian periodicity displayed significant ovarian enlargement. H&E staining revealed increased number of growing follicles and total follicles, as well as polycysts in the ovaries of the kindled animals compared to sham and control animals. Ultrastructural study detected numerous apoptotic granulosa cells in growing follicles and thecal cell hyperplasia with secretary granules in the thecal cells in the kindled rats. The results suggest that amygdala kindling is a risk factor for the development of polycystic ovary syndrome.

  3. Chloroplast Dysfunction Causes Multiple Defects in Cell Cycle Progression in the Arabidopsis crumpled leaf Mutant1[C][W (United States)

    Hudik, Elodie; Yoshioka, Yasushi; Domenichini, Séverine; Bourge, Mickaël; Soubigout-Taconnat, Ludivine; Mazubert, Christelle; Yi, Dalong; Bujaldon, Sandrine; Hayashi, Hiroyuki; De Veylder, Lieven; Bergounioux, Catherine; Benhamed, Moussa; Raynaud, Cécile


    The majority of research on cell cycle regulation is focused on the nuclear events that govern the replication and segregation of the genome between the two daughter cells. However, eukaryotic cells contain several compartmentalized organelles with specialized functions, and coordination among these organelles is required for proper cell cycle progression, as evidenced by the isolation of several mutants in which both organelle function and overall plant development were affected. To investigate how chloroplast dysfunction affects the cell cycle, we analyzed the crumpled leaf (crl) mutant of Arabidopsis (Arabidopsis thaliana), which is deficient for a chloroplastic protein and displays particularly severe developmental defects. In the crl mutant, we reveal that cell cycle regulation is altered drastically and that meristematic cells prematurely enter differentiation, leading to reduced plant stature and early endoreduplication in the leaves. This response is due to the repression of several key cell cycle regulators as well as constitutive activation of stress-response genes, among them the cell cycle inhibitor SIAMESE-RELATED5. One unique feature of the crl mutant is that it produces aplastidic cells in several organs, including the root tip. By investigating the consequence of the absence of plastids on cell cycle progression, we showed that nuclear DNA replication occurs in aplastidic cells in the root tip, which opens future research prospects regarding the dialogue between plastids and the nucleus during cell cycle regulation in higher plants. PMID:25037213

  4. Linking genomic reorganization to tumor initiation via the giant cell cycle (United States)

    Niu, N; Zhang, J; Zhang, N; Mercado-Uribe, I; Tao, F; Han, Z; Pathak, S; Multani, A S; Kuang, J; Yao, J; Bast, R C; Sood, A K; Hung, M-C; Liu, J


    To investigate the mechanisms underlying our recent paradoxical finding that mitotically incapacitated and genomically unstable polyploid giant cancer cells (PGCCs) are capable of tumor initiation, we labeled ovarian cancer cells with α-tubulin fused to green fluorescent protein, histone-2B fused to red fluorescent protein and FUCCI (fluorescent ubiquitination cell cycle indicator), and tracked the spatial and time-dependent change in spindle and chromosomal dynamics of PGCCs using live-cell fluorescence time-lapse recording. We found that single-dose (500 nm) treatment with paclitaxel paradoxically initiated endoreplication to form PGCCs after massive cell death. The resulting PGCCs continued self-renewal via endoreplication and further divided by nuclear budding or fragmentation; the small daughter nuclei then acquired cytoplasm, split off from the giant mother cells and acquired competency in mitosis. FUCCI showed that PGCCs divided via truncated endoreplication cell cycle (endocycle or endomitosis). Confocal microscopy showed that PGCCs had pronounced nuclear fragmentation and lacked expression of key mitotic proteins. PGCC-derived daughter cells were capable of long-term proliferation and acquired numerous new genome/chromosome alterations demonstrated by spectral karyotyping. These data prompt us to conceptualize a giant cell cycle composed of four distinct but overlapping phases, initiation, self-renewal, termination and stability. The giant cell cycle may represent a fundamental cellular mechanism to initiate genomic reorganization to generate new tumor-initiating cells in response to chemotherapy-induced stress and contributes to disease relapse. PMID:27991913

  5. A Method to Design Synthetic Cell-Cycle Networks

    Institute of Scientific and Technical Information of China (English)

    MIAO Ke-Ke


    The interactions among proteins, DNA and RNA in an organism form elaborate cell-cycle networks which govern cell growth and proliferation. Understanding the common structure of ce11-cycle networks will be of great benefit to science research. Here, inspired by the importance of the cell-cycle regulatory network of yeast which has been studied intensively, we focus on small networks with 11 nodes, equivalent to that of the cell-cycle regulatory network used by Li et al. [Proc. Natl. Acad. Sci. USA 101(2004)4781] Using a Boolean model, we study the correlation between structure and function, and a possible common structure. It is found that cascade-like networks with a great number of interactions between nodes are stable. Based on these findings, we are able to construct synthetic networks that have the same functions as the cell-cycle regulatory network.

  6. Responses of genes involved in cell cycle control to diverse DNA damaging chemicals in human lung adenocarcinoma A549 cells

    Directory of Open Access Journals (Sweden)

    Gooderham Nigel J


    Full Text Available Abstract Background Many anticancer agents and carcinogens are DNA damaging chemicals and exposure to such chemicals results in the deregulation of cell cycle progression. The molecular mechanisms of DNA damage-induced cell cycle alteration are not well understood. We have studied the effects of etoposide (an anticancer agent, cryptolepine (CLP, a cytotoxic alkaloid, benzo [a]pyrene (BaP, a carcinogenic polycyclic aromatic hydrocarbon and 2-amino-1-methyl-6-phenylimidazo [4,5-b]pyridine (PhIP, a cooked-meat derived carcinogen on the expression of cell cycle regulatory genes to understand the molecular mechanisms of the cell cycle disturbance. Results A549 cells were treated with DMSO or chemicals for up to 72 h and periodically sampled for cell cycle analysis, mRNA and protein expression. DMSO treated cells showed a dominant G1 peak in cell cycle at all times examined. Etoposide and CLP both induced G2/M phase arrest yet the former altered the expression of genes functioning at multiple phases, whilst the latter was more effective in inhibiting the expression of genes in G2-M transition. Both etoposide and CLP induced an accumulation of p53 protein and upregulation of p53 transcriptional target genes. Neither BaP nor PhIP had substantial phase-specific cell cycle effect, however, they induced distinctive changes in gene expression. BaP upregulated the expression of CYP1B1 at 6–24 h and downregulated many cell cycle regulatory genes at 48–72 h. By contrast, PhIP increased the expression of many cell cycle regulatory genes. Changes in the expression of key mRNAs were confirmed at protein level. Conclusion Our experiments show that DNA damaging agents with different mechanisms of action induced distinctive changes in the expression pattern of a panel of cell cycle regulatory genes. We suggest that examining the genomic response to chemical exposure provides an exceptional opportunity to understand the molecular mechanism involved in cellular

  7. Variety in intracellular diffusion during the cell cycle

    DEFF Research Database (Denmark)

    Selhuber-Unkel, C.; Yde, P.; Berg-Sørensen, Kirstine;


    During the cell cycle, the organization of the cytoskeletal network undergoes dramatic changes. In order to reveal possible changes of the viscoelastic properties in the intracellular space during the cell cycle we investigated the diffusion of endogenous lipid granules within the fission yeast...... Schizosaccharomyces Pombe using optical tweezers. The cell cycle was divided into interphase and mitotic cell division, and the mitotic cell division was further subdivided in its stages. During all stages of the cell cycle, the granules predominantly underwent subdiffusive motion, characterized by an exponent...... a that is also linked to the viscoelastic moduli of the cytoplasm. The exponent a was significantly smaller during interphase than during any stage of the mitotic cell division, signifying that the cytoplasm was more elastic during interphase than during division. We found no significant differences...

  8. Changes in oscillatory dynamics in the cell cycle of early Xenopus laevis embryos.

    Directory of Open Access Journals (Sweden)

    Tony Y-C Tsai


    Full Text Available During the early development of Xenopus laevis embryos, the first mitotic cell cycle is long (∼85 min and the subsequent 11 cycles are short (∼30 min and clock-like. Here we address the question of how the Cdk1 cell cycle oscillator changes between these two modes of operation. We found that the change can be attributed to an alteration in the balance between Wee1/Myt1 and Cdc25. The change in balance converts a circuit that acts like a positive-plus-negative feedback oscillator, with spikes of Cdk1 activation, to one that acts like a negative-feedback-only oscillator, with a shorter period and smoothly varying Cdk1 activity. Shortening the first cycle, by treating embryos with the Wee1A/Myt1 inhibitor PD0166285, resulted in a dramatic reduction in embryo viability, and restoring the length of the first cycle in inhibitor-treated embryos with low doses of cycloheximide partially rescued viability. Computations with an experimentally parameterized mathematical model show that modest changes in the Wee1/Cdc25 ratio can account for the observed qualitative changes in the cell cycle. The high ratio in the first cycle allows the period to be long and tunable, and decreasing the ratio in the subsequent cycles allows the oscillator to run at a maximal speed. Thus, the embryo rewires its feedback regulation to meet two different developmental requirements during early development.

  9. Cell cycle networks link gene expression dysregulation, mutation, and brain maldevelopment in autistic toddlers. (United States)

    Pramparo, Tiziano; Lombardo, Michael V; Campbell, Kathleen; Barnes, Cynthia Carter; Marinero, Steven; Solso, Stephanie; Young, Julia; Mayo, Maisi; Dale, Anders; Ahrens-Barbeau, Clelia; Murray, Sarah S; Lopez, Linda; Lewis, Nathan; Pierce, Karen; Courchesne, Eric


    Genetic mechanisms underlying abnormal early neural development in toddlers with Autism Spectrum Disorder (ASD) remain uncertain due to the impossibility of direct brain gene expression measurement during critical periods of early development. Recent findings from a multi-tissue study demonstrated high expression of many of the same gene networks between blood and brain tissues, in particular with cell cycle functions. We explored relationships between blood gene expression and total brain volume (TBV) in 142 ASD and control male toddlers. In control toddlers, TBV variation significantly correlated with cell cycle and protein folding gene networks, potentially impacting neuron number and synapse development. In ASD toddlers, their correlations with brain size were lost as a result of considerable changes in network organization, while cell adhesion gene networks significantly correlated with TBV variation. Cell cycle networks detected in blood are highly preserved in the human brain and are upregulated during prenatal states of development. Overall, alterations were more pronounced in bigger brains. We identified 23 candidate genes for brain maldevelopment linked to 32 genes frequently mutated in ASD. The integrated network includes genes that are dysregulated in leukocyte and/or postmortem brain tissue of ASD subjects and belong to signaling pathways regulating cell cycle G1/S and G2/M phase transition. Finally, analyses of the CHD8 subnetwork and altered transcript levels from an independent study of CHD8 suppression further confirmed the central role of genes regulating neurogenesis and cell adhesion processes in ASD brain maldevelopment.

  10. Effects of berberine on proliferation, cell cycle distribution and apoptosis of human breast cancer T47D and MCF7 cell lines

    Directory of Open Access Journals (Sweden)

    Elmira Barzegar


    Conclusion: Berberine alone and in combination with doxorubicin inhibited cell proliferation, induced apoptosis and altered cell cycle distribution of breast cancer cells. Therefore, berberine showed to be a good candidate for further studies as a new anticancer drug in the treatment of human breast cancer.

  11. Gadd45a inhibits cell migration and invasion by altering the global RNA expression. (United States)

    Shan, Zhanhai; Li, Guiyuan; Zhan, Qimin; Li, Dan


    Gadd45a, the first well-defined p53 downstream gene, can be induced by multiple DNA-damaging agents, which plays important roles in the control of cell cycle checkpoint, DNA repair process and signaling transduction. Our previous findings suggested that Gadd45a maintains cell-cell adhesion and cell contact inhibition. However, little is known about how Gadd45a participates in the suppression of malignancy in human cancer cells. To examine the functions of Gadd45a in cell invasion and metastasis, we performed the adhesion, wound-healing and transwell assays in Gadd45a (+/+) and Gadd45a (-/-) MEF cell lines. We found the adhesion, migration and invasive abilities were much higher in Gadd45a deficient cells. We furthermore applied high-throughput cDNA microarray analysis and bioinformatics analysis to analyze the mechanisms of Gadd45a gene in invasion and metastasis. Compared with the Gadd45a wild type cells, the Gadd45a deficient cells showed a wide range of transcripts alterations. The altered gene pathways were predicted by the MAS software, which indicated focal adhesion,cell communication,ECM-receptor interaction as the three main pathways. Real-time PCR was employed to validate the differentially expressed genes. Interestingly, we figured out that the deregulations of these genes are caused neither by genomic aberrations nor methylation status. These findings provided a novel insight that Gadd45a may involve in tumor progression by regulating related genes expressions.

  12. Effects of bile acids on proliferation and ultrastructural alteration of pancreatic cancer cell lines

    Institute of Scientific and Technical Information of China (English)

    Zheng Wu; Yi Lüi; Bo Wang; Chang Liu; Zuo-Ren Wang,


    AIM: Pancreatic cancer in the head is frequently accompanied by jaundice and high bile acid level in serum. This study focused on the direct effects of bile acids on proliferation and ultrastructural alteration of pancreatic cancer.METHODS: Pancreatic cancer cell lines PANC-1, MIA PaCa2 and PGHAM-1 were explored in this study. The cell lines were cultured in media supplemented with certain bile acids,CA, DCA, LCA, TCDC, TDCA and GCA. Their influence on cell growth was measured with MTT assay after 72 h of incubation. Cell cycles of PANC-1 cells in 40 μM of bile acids media were analyzed by flow cytometry. Ultrastructural alteration of PANC-1 cells induced by DCA was observed using scanning and transmission electron microscope (SEM and TEM).RESULTS: At various concentrations of bile acids and incubation time, no enhanced effects of bile acids on cell proliferation were observed. Significant inhibitory effects were obtained in almost all media with bile acids. DCA and CA increased the percentage of G0+G1 phase cells, while GCA and TDCA elevated the S phase cell number. After 48 h of incubation in DCA medium, PANC-1 cells showed some structural damages such as loss of their microvilli and vacuolization of organelles in cytoplasm.CONCLUSION: Bile acids can reduce proliferation of pancreatic cancer cells due to their direct cytotoxicity. This result implies that elevation of bile acids in jaundiced serum may inhibit pancreatic cancer progression.

  13. DNA fragmentation and cell cycle arrest: a hallmark of apoptosis induced by Ruta graveolens in human colon cancer cells. (United States)

    Arora, Shagun; Tandon, Simran


    In the present study, we investigated the anti-cancer effect of various potencies of Ruta graveolens (Ruta) on COLO-205 cell line, as evidenced by cytotoxicity, migration, clonogenecity, morphological and biochemical changes and modification in the levels of genes associated with apoptosis and cell cycle. On treatment of COLO-205 cells maximal effects were seen with mother tincture (MT) and 30C potencies, wherein decrease in cell viability along with reduced clonogenecity and migration capabilities were noted. In addition morphological and biochemical alterations such as nuclear changes (fragmented nuclei with condensed chromatin) and DNA ladder-like pattern (increased amount of fragmented DNA) in COLO-205 cells indicating apoptotic related cell death were seen. The expression of apoptosis and cell-cycle related regulatory genes assessed by reverse transcriptase-PCR revealed an up-regulation of caspase 9, caspase-3, Bax, p21 and p27 expression and down-regulation of Bcl-2 expression in treated cells. The mode of cell death was suggestive of intrinsic apoptotic pathway along with cell cycle arrest at the G2/M of the cell cycle. Our findings indicate that phytochemicals present in Ruta showed potential for natural therapeutic product development for colon carcinoma.

  14. Connecting the nucleolus to the cell cycle and human disease. (United States)

    Tsai, Robert Y L; Pederson, Thoru


    Long known as the center of ribosome synthesis, the nucleolus is connected to cell cycle regulation in more subtle ways. One is a surveillance system that reacts promptly when rRNA synthesis or processing is impaired, halting cell cycle progression. Conversely, the nucleolus also acts as a first-responder to growth-related stress signals. Here we review emerging concepts on how these "infraribosomal" links between the nucleolus and cell cycle progression operate in both forward and reverse gears. We offer perspectives on how new cancer therapeutic designs that target this infraribosomal mode of cell growth control may shape future clinical progress.

  15. Fisetin and hesperetin induced apoptosis and cell cycle arrest in chronic myeloid leukemia cells accompanied by modulation of cellular signaling. (United States)

    Adan, Aysun; Baran, Yusuf


    Fisetin and hesperetin, naturally occurring flavonoids, have been reported as novel antioxidants with chemopreventive/chemotherapeutic potential against various types of cancer. However, their mechanism of action in CML is still unknown. This particular study aims to evaluate the therapeutic potentials of fisetin and hesperetin and their effects on cell proliferation, apoptosis, and cell cycle progression in human K562 CML cells. The results indicated that fisetin and hesperetin inhibited cell proliferation and triggered programmed cell death in these cells. The latter was confırmed by mitochondrial membrane depolarization and an increase in caspase-3 activation. In addition to that, we have detected S and G2/M cell cycle arrests and G0/G1 arrest upon fisetin and hesperetin treatment, respectively. To identify the altered genes and genetic networks in response to fisetin and hesperetin, whole-genome microarray analysis was performed. The microarray gene profiling analysis revealed some important signaling pathways including JAK/STAT pathway, KIT receptor signaling, and growth hormone receptor signaling that were altered upon fisetin and hesperetin treatment. Moreover, microarray data suggested potential candidate genes for targeted CML therapy. Fisetin and hesperetin significantly modulated the expression of genes involved in cell proliferation and division, apoptosis, cell cycle regulation, and other significant cellular processes such as replication, transcription, and translation. In conclusion, our results suggest that fisetin and hesperetin as potential natural agents for CML therapy.

  16. The Cell Cycle: An Activity Using Paper Plates to Represent Time Spent in Phases of the Cell Cycle (United States)

    Scherer, Yvette D.


    In this activity, students are given the opportunity to combine skills in math and geometry for a biology lesson in the cell cycle. Students utilize the data they collect and analyze from an online onion-root-tip activity to create a paper-plate time clock representing a 24-hour cell cycle. By dividing the paper plate into appropriate phases of…

  17. The cell cycle regulated transcriptome of Trypanosoma brucei.

    Directory of Open Access Journals (Sweden)

    Stuart K Archer

    Full Text Available Progression of the eukaryotic cell cycle requires the regulation of hundreds of genes to ensure that they are expressed at the required times. Integral to cell cycle progression in yeast and animal cells are temporally controlled, progressive waves of transcription mediated by cell cycle-regulated transcription factors. However, in the kinetoplastids, a group of early-branching eukaryotes including many important pathogens, transcriptional regulation is almost completely absent, raising questions about the extent of cell-cycle regulation in these organisms and the mechanisms whereby regulation is achieved. Here, we analyse gene expression over the Trypanosoma brucei cell cycle, measuring changes in mRNA abundance on a transcriptome-wide scale. We developed a "double-cut" elutriation procedure to select unperturbed, highly synchronous cell populations from log-phase cultures, and compared this to synchronization by starvation. Transcriptome profiling over the cell cycle revealed the regulation of at least 430 genes. While only a minority were homologous to known cell cycle regulated transcripts in yeast or human, their functions correlated with the cellular processes occurring at the time of peak expression. We searched for potential target sites of RNA-binding proteins in these transcripts, which might earmark them for selective degradation or stabilization. Over-represented sequence motifs were found in several co-regulated transcript groups and were conserved in other kinetoplastids. Furthermore, we found evidence for cell-cycle regulation of a flagellar protein regulon with a highly conserved sequence motif, bearing similarity to consensus PUF-protein binding motifs. RNA sequence motifs that are functional in cell-cycle regulation were more widespread than previously expected and conserved within kinetoplastids. These findings highlight the central importance of post-transcriptional regulation in the proliferation of parasitic kinetoplastids.

  18. Cell cycle regulation by glucosamine in human pulmonary epithelial cells. (United States)

    Chuang, Kun-Han; Lu, Chih-Shen; Kou, Yu Ru; Wu, Yuh-Lin


    Airway epithelial cells play an important role against intruding pathogens. Glucosamine, a commonly used supplemental compound, has recently begun to be regarded as a potential anti-inflammatory molecule. This study aimed to uncover how glucosamine impacts on cellular proliferation in human alveolar epithelial cells (A549) and bronchial epithelial cells (HBECs). With trypan blue-exclusion assay, we observed that glucosamine (10, 20, 50 mM) caused a decrease in cell number at 24 and 48 h; with a flow cytometric analysis, we also noted an enhanced cell accumulation within the G(0)/G(1) phase at 24 h and induction of late apoptosis at 24 and 48 h by glucosamine (10, 20, 50 mM) in A549 cells and HBECs. Examination of phosphorylation in retinoblastoma (Rb) protein, we found an inhibitory effect by glucosamine at 20 and 50 mM. Glucosamine at 50 mM was demonstrated to elevate both the mRNA and protein expression of p53 and heme oxygenase-1 (HO-1), but also caused a reduction in p21 protein expression. In addition, glucosamine attenuated p21 protein stability via the proteasomal proteolytic pathway, as well as inducing p21 nuclear accumulation. Altogether, our results suggest that a high dose of glucosamine may inhibit cell proliferation through apoptosis and disturb cell cycle progression with a halt at G(0)/G(1) phase, and that this occurs, at least in part, by a reduction in Rb phosphorylation together with modulation of p21, p53 and HO-1 expression, and nuclear p21 accumulation.

  19. Nylon Wool Purification Alters the Activation of T Cells (United States)

    Wohler, Jillian E.; Barnum, Scott R.


    Purification of lymphocytes, particularly T cells, is commonly performed using nylon wool. This enrichment method selectively retains B cells and some myeloid cells allowing a significantly more pure T cell population to flow through a nylon wool column. T cells purified in this fashion are assumed to be unaltered and functionally naïve, however some studies have suggested aberrant in vitro T cell responses after nylon wool treatment. We found that nylon wool purification significantly altered T cell proliferation, expression of activation markers and production of cytokines. Our results suggest that nylon wool treatment modifies T cell activation responses and that caution should be used when choosing this purification method. PMID:18952296

  20. Glioblastoma Stem Cells Respond to Differentiation Cues but Fail to Undergo Commitment and Terminal Cell-Cycle Arrest

    Directory of Open Access Journals (Sweden)

    Helena Carén


    Full Text Available Glioblastoma (GBM is an aggressive brain tumor whose growth is driven by stem cell-like cells. BMP signaling triggers cell-cycle exit and differentiation of GBM stem cells (GSCs and, therefore, might have therapeutic value. However, the epigenetic mechanisms that accompany differentiation remain poorly defined. It is also unclear whether cell-cycle arrest is terminal. Here we find only a subset of GSC cultures exhibit astrocyte differentiation in response to BMP. Although overtly differentiated non-cycling astrocytes are generated, they remain vulnerable to cell-cycle re-entry and fail to appropriately reconfigure DNA methylation patterns. Chromatin accessibility mapping identified loci that failed to alter in response to BMP and these were enriched in SOX transcription factor-binding motifs. SOX transcription factors, therefore, may limit differentiation commitment. A similar propensity for cell-cycle re-entry and de-differentiation was observed in GSC-derived oligodendrocyte-like cells. These findings highlight significant obstacles to BMP-induced differentiation as therapy for GBM.

  1. Cholesterol biosynthesis and homeostasis in regulation of the cell cycle.

    Directory of Open Access Journals (Sweden)

    Pushpendra Singh

    Full Text Available The cell cycle is a ubiquitous, multi-step process that is essential for growth and proliferation of cells. The role of membrane lipids in cell cycle regulation is not explored well, although a large number of cytoplasmic and nuclear regulators have been identified. We focus in this work on the role of membrane cholesterol in cell cycle regulation. In particular, we have explored the stringency of the requirement of cholesterol in the regulation of cell cycle progression. For this purpose, we utilized distal and proximal inhibitors of cholesterol biosynthesis, and monitored their effect on cell cycle progression. We show that cholesterol content increases in S phase and inhibition of cholesterol biosynthesis results in cell cycle arrest in G1 phase under certain conditions. Interestingly, G1 arrest mediated by cholesterol biosynthesis inhibitors could be reversed upon metabolic replenishment of cholesterol. Importantly, our results show that the requirement of cholesterol for G1 to S transition is absolute, and even immediate biosynthetic precursors of cholesterol, differing with cholesterol merely in a double bond, could not replace cholesterol for reversing the cell cycle arrest. These results are useful in the context of diseases, such as cancer and Alzheimer's disease, that are associated with impaired cholesterol biosynthesis and homeostasis.

  2. Cell cycling and patterned cell proliferation in the wing primordium of Drosophila.



    The pattern of cell proliferation in the Drosophila imaginal wing primordium is spatially and temporally heterogeneous. Direct visualization of cells in S, G2, and mitosis phases of the cell cycle reveals several features invariant throughout development. The fraction of cells in the disc in the different cell cycle stages is constant, the majority remaining in G1. Cells in the different phases of the cell cycle mainly appear in small synchronic clusters that are nonclonally derived but resul...

  3. Disconnected circadian and cell cycles in a tumor-driven cell line


    Pendergast, Julie S.; Yeom, Mijung; Bryan A. Reyes; Ohmiya, Yoshihiro; Yamazaki, Shin


    Cell division occurs at a specific time of day in numerous species, suggesting that the circadian and cell cycles are coupled in vivo. By measuring the cell cycle rhythm in real-time, we recently showed that the circadian and cell cycles are not coupled in immortalized fibroblasts, resulting in a rapid rate of cell division even though the circadian rhythm is normal in these cells. Here we report that tumor-driven Lewis lung carcinoma (LLC) cells have perfectly temperature compensated circadi...

  4. Effects of Coptidis Rhizoma on Cell Cycle, DNA Damage, and Apoptosis in L929 Murine Fibroblast Cells

    Institute of Scientific and Technical Information of China (English)

    Yan-fei Huang; Man-man Gu; Jing Xu; Chun-yang Han; Teng-fei Liu; Cui-yan Liu


    Objective Coptidis Rhizoma(CR), a widely used traditional Chinese herbal medicine, is commonly believed to be non-toxic. However, little is known about its cytotoxicity and relevant mechanisms at cellular and genetic levels. The present study was conducted to explore the cytotoxicity of CR and its mechanisms related to cell cycle arrest, DNA damage, cell apoptosis, and mitochondrial membrane potential in L929 murine fibroblast cells. Methods The cells were cultured and treated with different concentration of CR aqueous extract for 24 h. Cell viability was determined by CCK-8 method, morphological changes, and mitochondrial membrane potential were observed with an inverted microscope, cell cycle and cell apoptosis were examined by flow cytometry and DNA damages were detected by comet assay. Results Our results showed that cell viability was significantly decreased in a dose-dependent manner when concentration was higher than 0.2 mg/m L. A concentration above 1 mg/mL altered the cells morphology. Each DNA damage indicator score increased in the groups with the concentration of above 0.1 mg/mL. Cells at G2/M phase, cell apoptosis and mitochondrial membrane potential changed in the 2 mg/m L group. Conclusion Overall, our study suggests that CR at a high dosage exhibits cytotoxicity on L929 cells, which is likely to be the consequences of cell cycle arrest, DNA damage, cell apoptosis and mitochondrial membrane potential reduction.

  5. Cell cycle-dependent induction of autophagy, mitophagy and reticulophagy. (United States)

    Tasdemir, Ezgi; Maiuri, M Chiara; Tajeddine, Nicolas; Vitale, Ilio; Criollo, Alfredo; Vicencio, José Miguel; Hickman, John A; Geneste, Olivier; Kroemer, Guido


    When added to cells, a variety of autophagy inducers that operate through distinct mechanisms and target different organelles for autophagic destruction (mitochondria in mitophagy, endoplasmic reticulum in reticulophagy) rarely induce autophagic vacuolization in more than 50% or the cells. Here we show that this heterogeneity may be explained by cell cycle-specific effects. The BH3 mimetic ABT737, lithium, rapamycin, tunicamycin or nutrient depletion stereotypically induce autophagy preferentially in the G(1) and S phases of the cell cycle, as determined by simultaneous monitoring of cell cycle markers and the cytoplasmic aggregation of GFP-LC3 in autophagic vacuoles. These results point to a hitherto neglected crosstalk between autophagic vacuolization and cell cycle regulation.

  6. Brucella abortus Cell Cycle and Infection Are Coordinated. (United States)

    De Bolle, Xavier; Crosson, Sean; Matroule, Jean-Yves; Letesson, Jean-Jacques


    Brucellae are facultative intracellular pathogens. The recent development of methods and genetically engineered strains allowed the description of cell-cycle progression of Brucella abortus, including unipolar growth and the ordered initiation of chromosomal replication. B. abortus cell-cycle progression is coordinated with intracellular trafficking in the endosomal compartments. Bacteria are first blocked at the G1 stage, growth and chromosome replication being resumed shortly before reaching the intracellular proliferation compartment. The control mechanisms of cell cycle are similar to those reported for the bacterium Caulobacter crescentus, and they are crucial for survival in the host cell. The development of single-cell analyses could also be applied to other bacterial pathogens to investigate their cell-cycle progression during infection.

  7. MFSD2A is a novel lung tumor suppressor gene modulating cell cycle and matrix attachment

    Directory of Open Access Journals (Sweden)

    Shames David S


    Full Text Available Abstract Background MFSD2A (major facilitator superfamily domain containing 2 gene maps on chromosome 1p34 within a linkage disequilibrium block containing genetic elements associated with progression of lung cancer. Results Here we show that MFSD2A expression is strongly downregulated in non-small cell lung cancer cell lines of different histotypes and in primary lung adenocarcinomas. Experimental modulation of MFSD2A in lung cancer cells is associated with alteration of mRNA levels of genes involved in cell cycle control and interaction with the extracellular matrix. Exogenous expression of MFSD2A in lung cancer cells induced a G1 block, impaired adhesion and migration in vitro, and significantly reduced tumor colony number in vitro (4- to 27-fold, P in vivo (~3-fold, P Conclusion Together these data suggest that MFSD2A is a novel lung cancer tumor suppressor gene that regulates cell cycle progression and matrix attachment.

  8. Identification of G1-regulated genes in normally cycling human cells.

    Directory of Open Access Journals (Sweden)

    Maroun J Beyrouthy

    Full Text Available BACKGROUND: Obtaining synchronous cell populations is essential for cell-cycle studies. Methods such as serum withdrawal or use of drugs which block cells at specific points in the cell cycle alter cellular events upon re-entry into the cell cycle. Regulatory events occurring in early G1 phase of a new cell cycle could have been overlooked. METHODOLOGY AND FINDINGS: We used a robotic mitotic shake-off apparatus to select cells in late mitosis for genome-wide gene expression studies. Two separate microarray experiments were conducted, one which involved isolation of RNA hourly for several hours from synchronous cell populations, and one experiment which examined gene activity every 15 minutes from late telophase of mitosis into G1 phase. To verify synchrony of the cell populations under study, we utilized methods including BrdU uptake, FACS, and microarray analyses of histone gene activity. We also examined stress response gene activity. Our analysis enabled identification of 200 early G1-regulated genes, many of which currently have unknown functions. We also confirmed the expression of a set of genes candidates (fos, atf3 and tceb by qPCR to further validate the newly identified genes. CONCLUSION AND SIGNIFICANCE: Genome-scale expression analyses of the first two hours of G1 in naturally cycling cells enabled the discovery of a unique set of G1-regulated genes, many of which currently have unknown functions, in cells progressing normally through the cell division cycle. This group of genes may contain future targets for drug development and treatment of human disease.

  9. Disconnected circadian and cell cycles in a tumor-driven cell line. (United States)

    Pendergast, Julie S; Yeom, Mijung; Reyes, Bryan A; Ohmiya, Yoshihiro; Yamazaki, Shin


    Cell division occurs at a specific time of day in numerous species, suggesting that the circadian and cell cycles are coupled in vivo. By measuring the cell cycle rhythm in real-time, we recently showed that the circadian and cell cycles are not coupled in immortalized fibroblasts, resulting in a rapid rate of cell division even though the circadian rhythm is normal in these cells. Here we report that tumor-driven Lewis lung carcinoma (LLC) cells have perfectly temperature compensated circadian clocks, but the periods of their cell cycle gene expression rhythms are temperature-dependent, suggesting that their circadian and cell cycles are not connected. These data support our hypothesis that decoupling of the circadian and cell cycles may underlie aberrant cell division in tumor cells.

  10. Soil fungal:bacterial ratios are linked to altered carbon cycling

    Directory of Open Access Journals (Sweden)

    Ashish A. Malik


    Full Text Available Despite several lines of observational evidence, there is a lack of consensus on whether higher fungal:bacterial (F:B ratios directly cause higher soil carbon (C storage. We employed RNA sequencing, protein profiling and isotope tracer techniques to evaluate whether differing F:B ratios are associated with differences in C storage. A mesocosm 13C labeled foliar litter decomposition experiment was performed in two soils that were similar in their physico-chemical properties but differed in microbial community structure, specifically their F:B ratio (determined by PLFA analyses, RNA sequencing and protein profiling; all three corroborating each other. Following litter addition, we observed a consistent increase in abundance of fungal phyla; and greater increases in the fungal dominated soil; implicating the role of fungi in litter decomposition. Litter derived 13C in respired CO2 was consistently lower, and residual 13C in bulk SOM was higher in high F:B soil demonstrating greater C storage potential in the fungal:bacterial dominated soil. We conclude that in this soil system, the increased abundance of fungi in both soils and the altered C cycling patterns in the fungal:bacterial dominated soils highlight the significant role of fungi in litter decomposition and indicate that F:B ratios are linked to higher C storage potential.

  11. Cell Cycle Related Differentiation of Bone Marrow Cells into Lung Cells

    Energy Technology Data Exchange (ETDEWEB)

    Dooner, Mark; Aliotta, Jason M.; Pimental, Jeffrey; Dooner, Gerri J.; Abedi, Mehrdad; Colvin, Gerald; Liu, Qin; Weier, Heinz-Ulli; Dooner, Mark S.; Quesenberry, Peter J.


    Green-fluorescent protein (GFP) labeled marrow cells transplanted into lethally irradiated mice can be detected in the lungs of transplanted mice and have been shown to express lung specific proteins while lacking the expression of hematopoietic markers. We have studied marrow cells induced to transit cell cycle by exposure to IL-3, IL-6, IL-11 and steel factor at different times of culture corresponding to different phases of cell cycle. We have found that marrow cells at the G1/S interface have a 3-fold increase in cells which assume a lung phenotype and that this increase is no longer seen in late S/G2. These cells have been characterized as GFP{sup +} CD45{sup -} and GFP{sup +} cytokeratin{sup +}. Thus marrow cells with the capacity to convert into cells with a lung phenotype after transplantation show a reversible increase with cytokine induced cell cycle transit. Previous studies have shown the phenotype of bone marrow stem cells fluctuates reversibly as these cells traverse cell cycle, leading to a continuum model of stem cell regulation. The present studies indicate that marrow stem cell production of nonhematopoietic cells also fluctuates on a continuum.

  12. Side population sorting separates subfractions of cycling and non-cycling intestinal stem cells

    Directory of Open Access Journals (Sweden)

    Richard J. von Furstenberg


    Full Text Available We report here that side population (SP sorting allows for the simultaneous isolation of two intestinal stem cell (ISC subsets from wild-type (WT mice which are phenotypically different and represent cycling and non-cycling pools of cells. Following 5-ethynyl-2′-deoxyuridine (EdU injection, in the upper side population (USP the percentage of EdU+ was 36% showing this fraction to be highly proliferative. In the lower side population (LSP, only 0.4% of cells were EdU+, indicating this fraction to be predominantly non-cycling. Using Lgr5-EGFP mice, we show that Lgr5-EGFPhi cells, representing actively cycling ISCs, are essentially exclusive to the USP. In contrast, using histone 2B-YFP mice, SP analysis revealed YFP label retaining cells (LRCs in both the USP and the LSP. Correspondingly, evaluation of the SP fractions for mRNA markers by qRT-PCR showed that the USP was enriched in transcripts associated with both quiescent and active ISCs. In contrast, the LSP expressed mRNA markers of quiescent ISCs while being de-enriched for those of the active ISC. Both the USP and LSP are capable of generating enteroids in culture which include the four intestinal lineages. We conclude that sorting of USP and LSP fractions represents a novel isolation of cycling and non-cycling ISCs from WT mice.

  13. Estrogen receptor alpha is cell cycle-regulated and regulates the cell cycle in a ligand-dependent fashion. (United States)

    JavanMoghadam, Sonia; Weihua, Zhang; Hunt, Kelly K; Keyomarsi, Khandan


    Estrogen receptor alpha (ERα) has been implicated in several cell cycle regulatory events and is an important predictive marker of disease outcome in breast cancer patients. Here, we aimed to elucidate the mechanism through which ERα influences proliferation in breast cancer cells. Our results show that ERα protein is cell cycle-regulated in human breast cancer cells and that the presence of 17-β-estradiol (E2) in the culture medium shortened the cell cycle significantly (by 4.5 hours, P fashion. These results provide the rationale for an effective treatment strategy that includes a cell cycle inhibitor in combination with a drug that lowers estrogen levels, such as an aromatase inhibitor, and an antiestrogen that does not result in the degradation of ERα, such as tamoxifen.

  14. Impact of the cell division cycle on gene circuits (United States)

    Bierbaum, Veronika; Klumpp, Stefan


    In growing cells, protein synthesis and cell growth are typically not synchronous, and, thus, protein concentrations vary over the cell division cycle. We have developed a theoretical description of genetic regulatory systems in bacteria that explicitly considers the cell division cycle to investigate its impact on gene expression. We calculate the cell-to-cell variations arising from cells being at different stages in the division cycle for unregulated genes and for basic regulatory mechanisms. These variations contribute to the extrinsic noise observed in single-cell experiments, and are most significant for proteins with short lifetimes. Negative autoregulation buffers against variation of protein concentration over the division cycle, but the effect is found to be relatively weak. Stronger buffering is achieved by an increased protein lifetime. Positive autoregulation can strongly amplify such variation if the parameters are set to values that lead to resonance-like behaviour. For cooperative positive autoregulation, the concentration variation over the division cycle diminishes the parameter region of bistability and modulates the switching times between the two stable states. The same effects are seen for a two-gene mutual-repression toggle switch. By contrast, an oscillatory circuit, the repressilator, is only weakly affected by the division cycle.

  15. Discrete gene replication events drive coupling between the cell cycle and circadian clocks. (United States)

    Paijmans, Joris; Bosman, Mark; Ten Wolde, Pieter Rein; Lubensky, David K


    Many organisms possess both a cell cycle to control DNA replication and a circadian clock to anticipate changes between day and night. In some cases, these two rhythmic systems are known to be coupled by specific, cross-regulatory interactions. Here, we use mathematical modeling to show that, additionally, the cell cycle generically influences circadian clocks in a nonspecific fashion: The regular, discrete jumps in gene-copy number arising from DNA replication during the cell cycle cause a periodic driving of the circadian clock, which can dramatically alter its behavior and impair its function. A clock built on negative transcriptional feedback either phase-locks to the cell cycle, so that the clock period tracks the cell division time, or exhibits erratic behavior. We argue that the cyanobacterium Synechococcus elongatus has evolved two features that protect its clock from such disturbances, both of which are needed to fully insulate it from the cell cycle and give it its observed robustness: a phosphorylation-based protein modification oscillator, together with its accompanying push-pull read-out circuit that responds primarily to the ratios of different phosphoform concentrations, makes the clock less susceptible to perturbations in protein synthesis; the presence of multiple, asynchronously replicating copies of the same chromosome diminishes the effect of replicating any single copy of a gene.

  16. Cell-Cycle Inhibition by Helicobacter pylori L-Asparaginase (United States)

    Scotti, Claudia; Sommi, Patrizia; Pasquetto, Maria Valentina; Cappelletti, Donata; Stivala, Simona; Mignosi, Paola; Savio, Monica; Chiarelli, Laurent Roberto; Valentini, Giovanna; Bolanos-Garcia, Victor M.; Merrell, Douglas Scott; Franchini, Silvia; Verona, Maria Luisa; Bolis, Cristina; Solcia, Enrico; Manca, Rachele; Franciotta, Diego; Casasco, Andrea; Filipazzi, Paola; Zardini, Elisabetta; Vannini, Vanio


    Helicobacter pylori (H. pylori) is a major human pathogen causing chronic gastritis, peptic ulcer, gastric cancer, and mucosa-associated lymphoid tissue lymphoma. One of the mechanisms whereby it induces damage depends on its interference with proliferation of host tissues. We here describe the discovery of a novel bacterial factor able to inhibit the cell-cycle of exposed cells, both of gastric and non-gastric origin. An integrated approach was adopted to isolate and characterise the molecule from the bacterial culture filtrate produced in a protein-free medium: size-exclusion chromatography, non-reducing gel electrophoresis, mass spectrometry, mutant analysis, recombinant protein expression and enzymatic assays. L-asparaginase was identified as the factor responsible for cell-cycle inhibition of fibroblasts and gastric cell lines. Its effect on cell-cycle was confirmed by inhibitors, a knockout strain and the action of recombinant L-asparaginase on cell lines. Interference with cell-cycle in vitro depended on cell genotype and was related to the expression levels of the concurrent enzyme asparagine synthetase. Bacterial subcellular distribution of L-asparaginase was also analysed along with its immunogenicity. H. pylori L-asparaginase is a novel antigen that functions as a cell-cycle inhibitor of fibroblasts and gastric cell lines. We give evidence supporting a role in the pathogenesis of H. pylori-related diseases and discuss its potential diagnostic application. PMID:21085483

  17. Studies on regulation of the cell cycle in fission yeast.

    Directory of Open Access Journals (Sweden)

    Miroslava Požgajová


    Full Text Available All living organisms including plants and animals are composed of millions of cells. These cells perform different functions for the organism although they possess the same chromosomes and carry the same genetic information. Thus, to be able to understand multicellular organism we need to understand the life cycle of individual cells from which the organism comprises. The cell cycle is the life cycle of a single cell in the plant or animal body. It involves series of events in which components of the cell doubles and afterwards equally segregate into daughter cells. Such process ensures growth of the organism, and specialized reductional cell division which leads to production of gamets, assures sexual reproduction. Cell cycle is divided in the G1, S, G2 and M phase. Two gap-phases (G1 and G2 separate S phase (or synthesis and M phase which stays either for mitosis or meiosis. Essential for normal life progression and reproduction is correct chromosome segregation during mitosis and meiosis. Defects in the division program lead to aneuploidy, which in turn leads to birth defects, miscarriages or cancer. Even thou, researchers invented much about the regulation of the cell cycle, there is still long way to understand the complexity of the regulatory machineries that ensure proper segregation of chromosomes. In this paper we would like to describe techniques and materials we use for our studies on chromosome segregation in the model organism Schizosaccharomyces pombe.

  18. Cell-cycle inhibition by Helicobacter pylori L-asparaginase.

    Directory of Open Access Journals (Sweden)

    Claudia Scotti

    Full Text Available Helicobacter pylori (H. pylori is a major human pathogen causing chronic gastritis, peptic ulcer, gastric cancer, and mucosa-associated lymphoid tissue lymphoma. One of the mechanisms whereby it induces damage depends on its interference with proliferation of host tissues. We here describe the discovery of a novel bacterial factor able to inhibit the cell-cycle of exposed cells, both of gastric and non-gastric origin. An integrated approach was adopted to isolate and characterise the molecule from the bacterial culture filtrate produced in a protein-free medium: size-exclusion chromatography, non-reducing gel electrophoresis, mass spectrometry, mutant analysis, recombinant protein expression and enzymatic assays. L-asparaginase was identified as the factor responsible for cell-cycle inhibition of fibroblasts and gastric cell lines. Its effect on cell-cycle was confirmed by inhibitors, a knockout strain and the action of recombinant L-asparaginase on cell lines. Interference with cell-cycle in vitro depended on cell genotype and was related to the expression levels of the concurrent enzyme asparagine synthetase. Bacterial subcellular distribution of L-asparaginase was also analysed along with its immunogenicity. H. pylori L-asparaginase is a novel antigen that functions as a cell-cycle inhibitor of fibroblasts and gastric cell lines. We give evidence supporting a role in the pathogenesis of H. pylori-related diseases and discuss its potential diagnostic application.

  19. The impact of altered task mechanics on timing and duration of eccentric bi-articular muscle contractions during cycling. (United States)

    Connick, Mark J; Li, François-Xavier


    In order to understand muscle adaptations to altered task mechanics during cycling, this study investigated the impact of altered seat height and cadence on timing and duration of gastrocnemius (GAST), biceps femoris (BF) and vastus lateralis (VL) eccentric contractions and muscle activation patterns, and cycling economy. Ten male cyclists completed 9 × 5 min of cycling at 3 seat heights and 3 cadences. Three-dimensional leg kinematics and muscle activation patterns were recorded to estimate timing of eccentric muscle contractions. Onset, offset and duration of eccentric contractions and, onset, offset and duration of muscle activation were calculated, along with cycling economy. Duration of GAST and VL eccentric contractions decreased with increasing seat height due to earlier offset of eccentric muscle contractions. Duration of BF eccentric contractions significantly increased with seat height due to a later eccentric contraction offset. Offset of GAST and BF muscle activation occurred earlier with increasing cadence. Cycling economy was significantly affected by cadence but not seat height. The results suggest that as a consequence of altered seat height, proprioceptive feedback is used to fine-tune the timing of bi-articular eccentric muscle contractions. These results may have implications for seat height self-selection.

  20. Intercellular Coupling of the Cell Cycle and Circadian Clock in Adult Stem Cell Culture. (United States)

    Matsu-Ura, Toru; Dovzhenok, Andrey; Aihara, Eitaro; Rood, Jill; Le, Hung; Ren, Yan; Rosselot, Andrew E; Zhang, Tongli; Lee, Choogon; Obrietan, Karl; Montrose, Marshall H; Lim, Sookkyung; Moore, Sean R; Hong, Christian I


    Circadian clock-gated cell division cycles are observed from cyanobacteria to mammals via intracellular molecular connections between these two oscillators. Here we demonstrate WNT-mediated intercellular coupling between the cell cycle and circadian clock in 3D murine intestinal organoids (enteroids). The circadian clock gates a population of cells with heterogeneous cell-cycle times that emerge as 12-hr synchronized cell division cycles. Remarkably, we observe reduced-amplitude oscillations of circadian rhythms in intestinal stem cells and progenitor cells, indicating an intercellular signal arising from differentiated cells governing circadian clock-dependent synchronized cell division cycles. Stochastic simulations and experimental validations reveal Paneth cell-secreted WNT as the key intercellular coupling component linking the circadian clock and cell cycle in enteroids.

  1. PKCeta enhances cell cycle progression, the expression of G1 cyclins and p21 in MCF-7 cells. (United States)

    Fima, E; Shtutman, M; Libros, P; Missel, A; Shahaf, G; Kahana, G; Livneh, E


    Protein kinase C encodes a family of enzymes implicated in cellular differentiation, growth control and tumor promotion. However, not much is known with respect to the molecular mechanisms that link protein kinase C to cell cycle control. Here we report that the expression of PKCeta in MCF-7 cells, under the control of a tetracycline-responsive inducible promoter, enhanced cell growth and affected the cell cycle at several points. The induced expression of another PKC isoform, PKCdelta, in MCF-7 cells had opposite effects and inhibited their growth. PKCeta expression activated cellular pathways in these cells that resulted in the increased expression of the G1 phase cyclins, cyclin D and cyclin E. Expression of the cyclin-dependent kinase inhibitor p21(WAF1) was also specifically elevated in PKCeta expressing cells, but its overall effects were not inhibitory. Although, the protein levels of the cyclin-dependent kinase inhibitor p27(KIP1) were not altered by the induced expression of PKCeta, the cyclin E associated Cdk2 kinase activity was in correlation with the p27(KIP1) bound to the cyclin E complex and not by p21(WAF1) binding. PKCeta expression enhanced the removal of p27(KIP1) from this complex, and its re-association with the cyclin D/Cdk4 complex. Reduced binding of p27(KIP1) to the cyclin D/Cdk4 complex at early time points of the cell cycle also enhanced the activity of this complex, while at later time points the decrease in bound p21(WAF1) correlated with its increased activity in PKCeta-expressing cells. Thus, PKCeta induces altered expression of several cell cycle functions, which may contribute to its ability to affect cell growth.

  2. Understanding and altering cell tropism of vesicular stomatitis virus (United States)

    Hastie, Eric; Cataldi, Marcela; Marriott, Ian; Grdzelishvili, Valery Z.


    Vesicular stomatitis virus (VSV) is a prototypic nonsegmented negative-strand RNA virus. VSV’s broad cell tropism makes it a popular model virus for many basic research applications. In addition, a lack of preexisting human immunity against VSV, inherent oncotropism and other features make VSV a widely used platform for vaccine and oncolytic vectors. However, VSV’s neurotropism that can result in viral encephalitis in experimental animals needs to be addressed for the use of the virus as a safe vector. Therefore, it is very important to understand the determinants of VSV tropism and develop strategies to alter it. VSV glycoprotein (G) and matrix (M) protein play major roles in its cell tropism. VSV G protein is responsible for VSV broad cell tropism and is often used for pseudotyping other viruses. VSV M affects cell tropism via evasion of antiviral responses, and M mutants can be used to limit cell tropism to cell types defective in interferon signaling. In addition, other VSV proteins and host proteins may function as determinants of VSV cell tropism. Various approaches have been successfully used to alter VSV tropism to benefit basic research and clinically relevant applications. PMID:23796410

  3. Regulation of cell cycle by the anaphase spindle midzone

    Directory of Open Access Journals (Sweden)

    Sluder Greenfield


    Full Text Available Abstract Background A number of proteins accumulate in the spindle midzone and midbody of dividing animal cells. Besides proteins essential for cytokinesis, there are also components essential for interphase functions, suggesting that the spindle midzone and/or midbody may play a role in regulating the following cell cycle. Results We microsurgically severed NRK epithelial cells during anaphase or telophase, such that the spindle midzone/midbody was associated with only one of the daughter cells. Time-lapse recording of cells severed during early anaphase indicated that the cell with midzone underwent cytokinesis-like cortical contractions and progressed normally through the interphase, whereas the cell without midzone showed no cortical contraction and an arrest or substantial delay in the progression of interphase. Similar microsurgery during telophase showed a normal progression of interphase for both daughter cells with or without the midbody. Microsurgery of anaphase cells treated with cytochalasin D or nocodazole indicated that interphase progression was independent of cortical ingression but dependent on microtubules. Conclusions We conclude that the mitotic spindle is involved in not only the separation of chromosomes but also the regulation of cell cycle. The process may involve activation of components in the spindle midzone that are required for the cell cycle, and/or degradation of components that are required for cytokinesis but may interfere with the cell cycle.

  4. Two cell cycle blocks caused by iron chelation of neuroblastoma cells: separating cell cycle events associated with each block. (United States)

    Siriwardana, Gamini; Seligman, Paul A


    Studies have presented evidence that besides the well described S phase block, treatment of cancer cell lines with the iron chelator deferrioxamine (DFO) also results in an earlier block in G1 phase. In this article, measurements of cell cycle regulatory proteins define this block at a very specific point in G1. DFO treatment results in markedly decreased cyclin A protein levels. Cyclin E levels that accumulate in early to mid-G1 are increased in cells treated with DFO as compared to the resting cells. The DFO S phase block is shown after cells are arrested at G1/S by (aphidicolin) then released into DFO. The same S phase block occurs with DFO treatment of a neuroblastoma cell line relatively resistant to the G1 DFO block. These experiments clearly differentiate the S phase DFO block from the earlier block pinpointed to a point in mid-G1, before G1/S when cyclin E protein increases but before increased cyclin A synthesis. Apoptosis was observed in cells inhibited by DFO at both cell cycle arrest points.

  5. The circadian clock and cell cycle: interconnected biological circuits. (United States)

    Masri, Selma; Cervantes, Marlene; Sassone-Corsi, Paolo


    The circadian clock governs biological timekeeping on a systemic level, helping to regulate and maintain physiological processes, including endocrine and metabolic pathways with a periodicity of 24-hours. Disruption within the circadian clock machinery has been linked to numerous pathological conditions, including cancer, suggesting that clock-dependent regulation of the cell cycle is an essential control mechanism. This review will highlight recent advances on the 'gating' controls of the circadian clock at various checkpoints of the cell cycle and also how the cell cycle can influence biological rhythms. The reciprocal influence that the circadian clock and cell cycle exert on each other suggests that these intertwined biological circuits are essential and multiple regulatory/control steps have been instated to ensure proper timekeeping.

  6. Technoeconomy of different solid oxide fuel cell based hybrid cycle

    DEFF Research Database (Denmark)

    Rokni, Masoud


    Gas turbine, steam turbine and heat engine (Stirling engine) is used as bottoming cycle for a solid oxide fuel cell plant to compare different plants efficiencies, CO2 emissionsand plants cost in terms of $/kW. Each plant is then integrated with biomass gasification and finally six plants...... configurations are compared with each other. Technoeconomy is used when calculating the cost if the plants. It is found that when a solid oxide fuel cell plant is combined with a gas turbine cycle then the plant efficiency will be the highest one while if a biomass gasification plant is integrated...... with these hybrid cycles then integrated biomass gasification with solid oxide fuel cell and steam cycle will have the highest plant efficiency. The cost of solid oxide fuel cell with steam plant is found to be the lowest one with a value of about 1030$/kW....

  7. Cellular Clocks : Coupled Circadian Dispatch and Cell Division Cycles

    NARCIS (Netherlands)

    Merrow, Martha; Roenneberg, Till


    Gating of cell division by the circadian clock is well known, yet its mechanism is little understood. Genetically tractable model systems have led to new hypotheses and questions concerning the coupling of these two cellular cycles.

  8. Genome-wide examination of myoblast cell cycle withdrawal duringdifferentiation

    Energy Technology Data Exchange (ETDEWEB)

    Shen, Xun; Collier, John Michael; Hlaing, Myint; Zhang, Leanne; Delshad, Elizabeth H.; Bristow, James; Bernstein, Harold S.


    Skeletal and cardiac myocytes cease division within weeks of birth. Although skeletal muscle retains limited capacity for regeneration through recruitment of satellite cells, resident populations of adult myocardial stem cells have not been identified. Because cell cycle withdrawal accompanies myocyte differentiation, we hypothesized that C2C12 cells, a mouse myoblast cell line previously used to characterize myocyte differentiation, also would provide a model for studying cell cycle withdrawal during differentiation. C2C12 cells were differentiated in culture medium containing horse serum and harvested at various time points to characterize the expression profiles of known cell cycle and myogenic regulatory factors by immunoblot analysis. BrdU incorporation decreased dramatically in confluent cultures 48 hr after addition of horse serum, as cells started to form myotubes. This finding was preceded by up-regulation of MyoD, followed by myogenin, and activation of Bcl-2. Cyclin D1 was expressed in proliferating cultures and became undetectable in cultures containing 40 percent fused myotubes, as levels of p21(WAF1/Cip1) increased and alpha-actin became detectable. Because C2C12 myoblasts withdraw from the cell cycle during myocyte differentiation following a course that recapitulates this process in vivo, we performed a genome-wide screen to identify other gene products involved in this process. Using microarrays containing approximately 10,000 minimally redundant mouse sequences that map to the UniGene database of the National Center for Biotechnology Information, we compared gene expression profiles between proliferating, differentiating, and differentiated C2C12 cells and verified candidate genes demonstrating differential expression by RT-PCR. Cluster analysis of differentially expressed genes revealed groups of gene products involved in cell cycle withdrawal, muscle differentiation, and apoptosis. In addition, we identified several genes, including DDAH2 and Ly

  9. Dynamic Pax6 expression during the neurogenic cell cycle influences proliferation and cell fate choices of retinal progenitors

    Directory of Open Access Journals (Sweden)

    Yang Xian-Jie


    Full Text Available Abstract Background The paired homeobox protein Pax6 is essential for proliferation and pluripotency of retinal progenitors. However, temporal changes in Pax6 protein expression associated with the generation of various retinal neurons have not been characterized with regard to the cell cycle. Here, we examine the dynamic changes of Pax6 expression among chicken retinal progenitors as they progress through the neurogenic cell cycle, and determine the effects of altered Pax6 levels on retinogenesis. Results We provide evidence that during the preneurogenic to neurogenic transition, Pax6 protein levels in proliferating progenitor cells are down-regulated. Neurogenic retinal progenitors retain a relatively low level of Pax6 protein, whereas postmitotic neurons either elevate or extinguish Pax6 expression in a cell type-specific manner. Cell imaging and cell cycle analyses show that neurogenic progenitors in the S phase of the cell cycle contain low levels of Pax6 protein, whereas a subset of progenitors exhibits divergent levels of Pax6 protein upon entering the G2 phase of the cell cycle. We also show that M phase cells contain varied levels of Pax6, and some correlate with the onset of early neuronal marker expression, forecasting cell cycle exit and cell fate commitment. Furthermore, either elevating or knocking down Pax6 attenuates cell proliferation and results in increased cell death. Reducing Pax6 decreases retinal ganglion cell genesis and enhances cone photoreceptor and amacrine interneuron production, whereas elevating Pax6 suppresses cone photoreceptor and amacrine cell fates. Conclusion These studies demonstrate for the first time quantitative changes in Pax6 protein expression during the preneurogenic to neurogenic transition and during the neurogenic cell cycle. The results indicate that Pax6 protein levels are stringently controlled in proliferating progenitors. Maintaining a relatively low Pax6 protein level is necessary for S phase

  10. Auxin Transport and Ribosome Biogenesis Mutant/Reporter Lines to Study Plant Cell Growth and Proliferation under Altered Gravity (United States)

    Valbuena, Miguel A.; Manzano, Ana I.; van Loon, Jack JWA.; Saez-Vasquez, Julio; Carnero-Diaz, Eugenie; Herranz, Raul; Medina, F. J.


    We tested different Arabidopsis thaliana strains to check their availability for space use in the International Space Station (ISS). We used mutants and reporter gene strains affecting factors of cell proliferation and cell growth, to check variations induced by an altered gravity vector. Seedlings were grown either in a Random Positioning Machine (RPM), under simulated microgravity (μg), or in a Large Diameter Centrifuge (LDC), under hypergravity (2g). A combination of the two devices (μgRPM+LDC) was also used. Under all gravity alterations, seedling roots were longer than in control 1g conditions, while the levels of the nucleolar protein nucleolin were depleted. Alterations in the pattern of expression of PIN2, an auxin transporter, and of cyclin B1, a cell cycle regulator, were shown. All these alterations are compatible with previous space data, so the use of these strains will be useful in the next experiments in ISS, under real microgravity.

  11. Altered cytoskeletal structures in transformed cells exhibiting obviously metastatic capabilities

    Institute of Scientific and Technical Information of China (English)



    Cytoskeletal changes in transformed cells (LM-51) eshibiting obviously metastatic capabilities were investigated by utilization of double-fluorescent labelling through combinations of:(1) tubulin indirect immunofluorescence plus Rhodamine-phalloidin staining of F-actins;(2) indirect immunofluorescent staining with α-actinin polyclonal-and vinculin monoclonal antibodies.The LM-51 cells which showed metastatic index of >50% were derived from lung metastasis in nude mice after subcutaneous inoculation of human highly metastatic tumor DNA transfected NIH3T3 cell transformants.The parent NIH3T3 cells exhibited well-organized microtubules,prominent stress fibers and adhesion plaques while their transformants showed remarkable cytoskeletal alterations:(1)reduced microtubules but increased MTOC fluorescence;(2)disrupted stress fibers and fewer adhesion plaques with their protein components redistributed in the cytoplasm;(3)Factin-and α-actinin/vinculin aggregates appeared in the cytoplasm.These aggregates were dot-like,varied in size(0.1-0.4μm) and number,located near the ventral surface of the cells.TPA-induced actin/vinculin bodies were studied too.Indications that actin and α-actinin/vinculin redistribution might be important alterations involved in the expression of metastatic capabilities of LM-51 transformed cells were discussed.

  12. Calcium signaling and T-type calcium channels in cancer cell cycling

    Institute of Scientific and Technical Information of China (English)

    James T Taylor; Xiang-Bin Zeng; Jonathan E Pottle; Kevin Lee; Alun R Wang; Stephenie G Yi; Jennifer A S Scruggs; Suresh S Sikka; Ming Li


    Regulation of intracellular calcium is an important signaling mechanism for cell proliferation in both normal and cancerous cells. In normal epithelial cells,free calcium concentration is essential for cells to enter and accomplish the S phase and the M phase of the cell cycle. In contrast, cancerous cells can pass these phases of the cell cycle with much lower cytoplasmic free calcium concentrations, indicating an alternative mechanism has developed for fulfilling the intracellular calcium requirement for an increased rate of DNA synthesis and mitosis of fast replicating cancerous cells. The detailed mechanism underlying the altered calcium loading pathway remains unclear;however, there is a growing body of evidence that suggests the T-type Ca2+ channel is abnormally expressed in cancerous cells and that blockade of these channels may reduce cell proliferation in addition to inducing apoptosis. Recent studies also show that the expression of T-type Ca2+ channels in breast cancer cells is proliferation state dependent, i.e. the channels are expressed at higher levels during the fast-replication period, and once the cells are in a non-proliferation state, expression of this channel isminimal. Therefore, selectively blocking calcium entry into cancerous cells may be a valuable approach for preventing tumor growth. Since T-type Ca2+ channels are not expressed in epithelial cells, selective T-type Ca2+ channel blockers may be useful in the treatment of certain types of cancers.

  13. Alteration of forest succession and carbon cycling under elevated CO2. (United States)

    Miller, Adam D; Dietze, Michael C; DeLucia, Evan H; Anderson-Teixeira, Kristina J


    Regenerating forests influence the global carbon (C) cycle, and understanding how climate change will affect patterns of regeneration and C storage is necessary to predict the rate of atmospheric carbon dioxide (CO2 ) increase in future decades. While experimental elevation of CO2 has revealed that young forests respond with increased productivity, there remains considerable uncertainty as to how the long-term dynamics of forest regrowth are shaped by elevated CO2 (eCO2 ). Here, we use the mechanistic size- and age- structured Ecosystem Demography model to investigate the effects of CO2 enrichment on forest regeneration, using data from the Duke Forest Free-Air Carbon dioxide Enrichment (FACE) experiment, a forest chronosequence, and an eddy-covariance tower for model parameterization and evaluation. We find that the dynamics of forest regeneration are accelerated, and stands consistently hit a variety of developmental benchmarks earlier under eCO2 . Because responses to eCO2 varied by plant functional type, successional pathways, and mature forest composition differed under eCO2 , with mid- and late-successional hardwood functional types experiencing greater increases in biomass compared to early-successional functional types and the pine canopy. Over the simulation period, eCO2 led to an increase in total ecosystem C storage of 9.7 Mg C ha(-1) . Model predictions of mature forest biomass and ecosystem-atmosphere exchange of CO2 and H2 O were sensitive to assumptions about nitrogen limitation; both the magnitude and persistence of the ecosystem response to eCO2 were reduced under N limitation. In summary, our simulations demonstrate that eCO2 can result in a general acceleration of forest regeneration while altering the course of successional change and having a lasting impact on forest ecosystems.

  14. Simulation of the Prokaryotic Cell Cycle at

    Directory of Open Access Journals (Sweden)

    Arieh Zaritsky


    Full Text Available The bacterial Cell Cycle is presented using the Simulation program CCSim, which employs four parameters related to time (inter-division τ, replication C, division D and size (mass at replication initiation Mi, sufficient to describe and compare bacterial cells under various conditions. The parameter values can be altered and the effects of the alterations can be seen. CCSim is easy to use and presents the kinetics of cell growth in a digestible format. Replicating chromosomes and growing bacillary cells are coupled to parameters that affect the cell cycle and animated. It serves as an educational tool to teach bacteriology and to compare experimental observations with the model best describing cell growth thus improve our understanding of regulatory mechanisms. Examples are displayed of transitions between known physiological states that are consistent with experimental results, including one that explains strange observations. The program predicts enhanced division frequencies after a period of slow replication under thymine limitation if a minimal distance (Eclipse exists between successive replisomes, as observed. Missing features and ways to improve, extend and refine CCSim are proposed, for both single cells and random populations under steady-states of exponential growth and during well-defined transitions. Future improvements and extensions are proposed for single cells and populations.

  15. Simulation of the Prokaryotic Cell Cycle at

    Directory of Open Access Journals (Sweden)

    Charles E. Helmstetter


    Full Text Available The bacterial Cell Cycle is presented using the Simulation program CCSim, which employs four parameters related to time (inter-division τ, replication C, division D and size (mass at replication initiation Mi, sufficient to describe and compare bacterial cells under various conditions. The parameter values can be altered and the effects of the alterations can be seen. CCSim is easy to use and presents the kinetics of cell growth in a digestible format. Replicating chromosomes and growing bacillary cells are coupled to parameters that affect the cell cycle and animated. It serves as an educational tool to teach bacteriology and to compare experimental observations with the model best describing cell growth thus improve our understanding of regulatory mechanisms. Examples are displayed of transitions between known physiological states that are consistent with experimental results, including one that explains strange observations. The program predicts enhanced division frequencies after a period of slow replication under thymine limitation if a minimal distance (Eclipse exists between successive replisomes, as observed. Missing features and ways to improve, extend and refine CCSim are proposed, for both single cells and random populations under steady-states of exponential growth and during well-defined transitions. Future improvements and extensions are proposed for single cells and populations.

  16. Eukaryotic checkpoints are absent in the cell division cycle of Entamoeba histolytica

    Indian Academy of Sciences (India)

    Sulagna Banerjee; Suchismita Das; Anuradha Lohia


    Fidelity in transmission of genetic characters is ensured by the faithful duplication of the genome, followed by equal segregation of the genetic material in the progeny. Thus, alternation of DNA duplication (S-phase) and chromosome segregation during the M-phase are hallmarks of most well studied eukaryotes. Several rounds of genome reduplication before chromosome segregation upsets this cycle and leads to polyploidy. Polyploidy is often witnessed in cells prior to differentiation, in embryonic cells or in diseases such as cancer. Studies on the protozoan parasite, Entamoeba histolytica suggest that in its proliferative phase, this organism may accumulate polyploid cells. It has also been shown that although this organism contains sequence homologs of genes which are known to control the cell cycle of most eukaryotes, these genes may be structurally altered and their equivalent function yet to be demonstrated in amoeba. The available information suggests that surveillance mechanisms or ‘checkpoints’ which are known to regulate the eukaryotic cell cycle may be absent or altered in E. histolytica.

  17. The timing of T cell priming and cycling

    Directory of Open Access Journals (Sweden)

    Reinhard eObst


    Full Text Available The proliferation of specific lymphocytes is the central tenet of the clonal selection paradigm. Antigen recognition by T cells triggers a series of events that produces expanded clones of differentiated effector cells. TCR signaling events are detectable within seconds and minutes and are likely to continue for hours and days in vivo. Here, I review the work done on the importance of TCR signals in the later part of the expansion phase of the primary T cell response, primarily regarding the regulation of the cell cycle in CD4+ and CD8+ cells. The results suggest a degree of programming by early signals for effector differentiation, particularly in the CD8+ T cell compartment, with optimal expansion supported by persistent antigen presentation later on. Differences to CD4+ T cell expansion and new avenues towards a molecular understanding of cell cycle regulation in lymphocytes are discussed.

  18. ARTD1 regulates cyclin E expression and consequently cell-cycle re-entry and G1/S progression in T24 bladder carcinoma cells. (United States)

    Léger, Karolin; Hopp, Ann-Katrin; Fey, Monika; Hottiger, Michael O


    ADP-ribosylation is involved in a variety of biological processes, many of which are chromatin-dependent and linked to important functions during the cell cycle. However, any study on ADP-ribosylation and the cell cycle faces the problem that synchronization with chemical agents or by serum starvation and subsequent growth factor addition already activates ADP-ribosylation by itself. Here, we investigated the functional contribution of ARTD1 in cell cycle re-entry and G1/S cell cycle progression using T24 urinary bladder carcinoma cells, which synchronously re-enter the cell cycle after splitting without any additional stimuli. In synchronized cells, ARTD1 knockdown, but not inhibition of its enzymatic activity, caused specific down-regulation of cyclin E during cell cycle re-entry and G1/S progression through alterations of the chromatin composition and histone acetylation, but not of other E2F-1 target genes. Although Cdk2 formed a functional complex with the residual cyclin E, p27(Kip 1) protein levels increased in G1 upon ARTD1 knockdown most likely due to inappropriate cyclin E-Cdk2-induced phosphorylation-dependent degradation, leading to decelerated G1/S progression. These results provide evidence that ARTD1 regulates cell cycle re-entry and G1/S progression via cyclin E expression and p27(Kip 1) stability independently of its enzymatic activity, uncovering a novel cell cycle regulatory mechanism.

  19. NONO couples the circadian clock to the cell cycle. (United States)

    Kowalska, Elzbieta; Ripperger, Juergen A; Hoegger, Dominik C; Bruegger, Pascal; Buch, Thorsten; Birchler, Thomas; Mueller, Anke; Albrecht, Urs; Contaldo, Claudio; Brown, Steven A


    Mammalian circadian clocks restrict cell proliferation to defined time windows, but the mechanism and consequences of this interrelationship are not fully understood. Previously we identified the multifunctional nuclear protein NONO as a partner of circadian PERIOD (PER) proteins. Here we show that it also conveys circadian gating to the cell cycle, a connection surprisingly important for wound healing in mice. Specifically, although fibroblasts from NONO-deficient mice showed approximately normal circadian cycles, they displayed elevated cell doubling and lower cellular senescence. At a molecular level, NONO bound to the p16-Ink4A cell cycle checkpoint gene and potentiated its circadian activation in a PER protein-dependent fashion. Loss of either NONO or PER abolished this activation and circadian expression of p16-Ink4A and eliminated circadian cell cycle gating. In vivo, lack of NONO resulted in defective wound repair. Because wound healing defects were also seen in multiple circadian clock-deficient mouse lines, our results therefore suggest that coupling of the cell cycle to the circadian clock via NONO may be useful to segregate in temporal fashion cell proliferation from tissue organization.

  20. Distinct regulation of cell cycle and survival in lymphocytes from patients with Alzheimer's disease and amyotrophic lateral sclerosis. (United States)

    Bartolomé, Fernando; Muñoz, Ursula; Esteras, Noemí; Esteban, Jesús; Bermejo-Pareja, Félix; Martín-Requero, Angeles


    Alterations in cell cycle progression seem to be associated with neuronal death in Alzheimer's disease (AD) and amyotrophic lateral sclerosis (ALS). We previously reported disturbances in the control of cell survival/death fate in immortalized lymphocytes from AD patients. These cell cycle dysfunction and impaired apoptosis were considered systemic manifestations of AD disease. The purpose of this study was to evaluate whether these abnormalities are characteristic of AD, or they may be seen in other neurodegenerative disorders such ALS. Our results indicate that alterations in signaling molecules, Akt and ERK1/2, and in the cyclin-dependent kinase complex inhibitors (CDKis) p21(Cip1) and p27(Kip1) are detectable in lymphoblasts from AD patients, but not in ALS patients, suggesting that these variables may be considered for the development of biomarkers of AD. However, lymphocytes from ALS patients do not represent a useful model to study cell cycle-related events associated with neurodegeneration of motoneurons.

  1. Effects of valproic acid and pioglitazone on cell cycle progression and proliferation of T-cell acute lymphoblastic leukemia Jurkat cells

    Directory of Open Access Journals (Sweden)

    Marie Saghaeian Jazi


    Full Text Available Objective(s: T-cell acute lymphoblastic leukemia (T-ALL is an aggressive hematologic malignant tumor. Administration of chemical compounds influencing apoptosis and T cell development has been discussed as promising novel therapeutic strategies. Valproic acid (VPA as a recently emerged anti-neoplastic histone deacetylase (HDAC inhibitor and pioglitazone (PGZ as a high-affinity peroxisome proliferator-activated receptor-gamma (PPARγ agonist have been shown to induce apoptosis and cell cycle arrest in different studies. Here, we aimed to investigate the underlying molecular mechanisms involved in anti-proliferative effects of these compounds on human Jurkat cells. Materials and Methods: Treated cells were evaluated for cell cycle progression and apoptosis using flowcytometry and MTT viability assay. Real-time RT-PCR was carried out to measure the alterations in key genes associated with cell death and cell cycle arrest. Results: Our findings illustrated that both VPA and PGZ can inhibit Jurkat E6.1 cells in vitro after   24 hr; however, PGZ 400 μM presents the most anti-proliferative effect. Interestingly, treated cells have been arrested in G2/M with deregulated cell division cycle 25A (Cdc25A phosphatase and cyclin-dependent kinase inhibitor 1B (CDKN1B or p27 expression. Expression of cyclin D1 gene was inhibited when DNA synthesis entry was declined. Cell cycle deregulation in PGZ and VPA-exposed cells generated an increase in the proportion of aneuploid cell population, which has not reported before. Conclusion: These findings define that anti-proliferative effects of PGZ and VPA on Jurkat cell line are mediated by cell cycle deregulation. Thus, we suggest PGZ and VPA may relieve potential therapeutic application against apoptosis-resistant malignancies.

  2. Circadian gating of the cell cycle revealed in single cyanobacterial cells. (United States)

    Yang, Qiong; Pando, Bernardo F; Dong, Guogang; Golden, Susan S; van Oudenaarden, Alexander


    Although major progress has been made in uncovering the machinery that underlies individual biological clocks, much less is known about how multiple clocks coordinate their oscillations. We simultaneously tracked cell division events and circadian phases of individual cells of the cyanobacterium Synechococcus elongatus and fit the data to a model to determine when cell cycle progression slows as a function of circadian and cell cycle phases. We infer that cell cycle progression in cyanobacteria slows during a specific circadian interval but is uniform across cell cycle phases. Our model is applicable to the quantification of the coupling between biological oscillators in other organisms.

  3. Palladium Nanoparticles Induce Disturbances in Cell Cycle Entry and Progression of Peripheral Blood Mononuclear Cells: Paramount Role of Ions

    Directory of Open Access Journals (Sweden)

    Claudia Petrarca


    Full Text Available There is concern about the possible toxicity of palladium nanoparticles (Pd-NP, as they are released in the environment through many applications. We previously studied the toxicity of Pd-NP at high concentrations; here we address the possible toxicity of Pd-NP at low, subtoxic doses. In particular, we have exposed normal human PBMC entering into the first in vitro mitotic division to Pd-NP and to Pd(IV ions to evaluate ROS generation and cell cycle progression. We have measured a statistically significant increase of intracellular ROS in Pd(IV exposed cells, but not in Pd-NP exposed cells. TEM revealed accumulation of lipid droplets and autophagic and mitophagic vacuoles, which appeared more conspicuous in cells exposed to Pd(IV ions than to Pd-NP. Pd-NP were visible in the cytoplasm of Pd-NP exposed cells. Pd-NP addition was associated with a significant increase of cells within the G0/G1-phase and a significant reduction in GS- and G2/M-phases. Cells exposed to Pd(IV ions showed a significant amplification of these cell cycle alterations. These results suggest that ions, per se or released by NPs, are the true inducers of Pd toxicity. It will be essential to verify whether the observed disturbance represents a temporary response or might result in permanent alterations.

  4. Nanosecond pulsed electric fields and the cell cycle (United States)

    Mahlke, Megan A.

    Exposure to nanosecond pulsed electrical fields (nsPEFs) can cause poration of external and internal cell membranes, DNA damage, and disassociation of cytoskeletal components, all of which are capable of disrupting a cell's ability to replicate. The phase of the cell cycle at the time of exposure is linked to differential sensitivities to nsPEFs across cell lines, as DNA structure, membrane elasticity, and cytoskeletal structure change dramatically during the cell cycle. Additionally, nsPEFs are capable of activating cell cycle checkpoints, which could lead to apoptosis or slow population growth. NsPEFs are emerging as a method for treating tumors via apoptotic induction; therefore, investigating the relevance of nsPEFs and the cell cycle could translate into improved efficacy in tumor treatment. Populations of Jurkat and Chinese Hamster Ovary (CHO) cells were examined post-exposure (10 ns pulse trains at 150kV/cm) by analysis of DNA content via propidium iodide staining and flow cytometric analysis at various time points (1, 6, and 12h post-exposure) to determine population distribution in cell cycle phases. Additionally, CHO and Jurkat cells were synchronized in G1/S and G2/M phases, pulsed, and analyzed to evaluate the role of cell cycle phase in survival of nsPEFs. CHO populations appeared similar to sham populations post-nsPEFs but exhibited arrest in the G1 phase at 6h after exposure. Jurkat cells exhibited increased cell death after nsPEFs compared to CHO cells but did not exhibit checkpoint arrest at any observed time point. The G1/S phase checkpoint is partially controlled by the action of p53; the lack of an active p53 response in Jurkat cells could contribute to their ability to pass this checkpoint and resist cell cycle arrest. Both cell lines exhibited increased sensitivity to nsPEFs in G2/M phase. Live imaging of CHO cells after nsPEF exposure supports the theory of G1/S phase arrest, as a reduced number of cells undergo mitosis within 24 h when

  5. Metabolic monosaccharides altered cell responses to anticancer drugs. (United States)

    Chen, Long; Liang, Jun F


    Metabolic glycoengineering has been used to manipulate the glycochemistry of cell surfaces and thus the cell/cell interaction, cell adhesion, and cell migration. However, potential application of glycoengineering in pharmaceutical sciences has not been studied until recently. Here, we reported that Ac(4)ManNAc, an analog of N-acetyl-D-mannosamine (ManNAc), could affect cell responses to anticancer drugs. Although cells from different tissues and organs responded to Ac(4)ManNAc treatment differently, treated cells with increased sialic acid contents showed dramatically reduced sensitivity (up to 130 times) to anti-cancer drugs as tested on various drugs with distinct chemical structures and acting mechanisms. Neither increased P-glycoprotein activity nor decreased drug uptake was observed during the course of Ac(4)ManNAc treatment. However, greatly altered intracellular drug distributions were observed. Most intracellular daunorubicin was found in the perinuclear region, but not the expected nuclei in the Ac(4)ManNAc treated cells. Since sialoglycoproteins and gangliosides were synthesized in the Golgi, intracellular glycans affected intracellular signal transduction and drug distributions seem to be the main reason for Ac(4)ManNAc affected cell sensitivity to anticancer drugs. It was interesting to find that although Ac(4)ManNAc treated breast cancer cells (MDA-MB-231) maintained the same sensitivity to 5-Fluorouracil, the IC(50) value of 5-Fluorouracil to the same Ac(4)ManNAc treated normal cells (MCF-10A) was increased by more than 20 times. Thus, this Ac(4)ManNAc treatment enlarged drug response difference between normal and tumor cells provides a unique opportunity to further improve the selectivity and therapeutic efficiency of anticancer drugs.

  6. How the cell cycle impacts chromatin architecture and influences cell fate

    Directory of Open Access Journals (Sweden)

    Yiqin eMa


    Full Text Available Since the earliest observations of cells undergoing mitosis, it has been clear that there is an intimate relationship between the cell cycle and nuclear chromatin architecture. The nuclear envelope and chromatin undergo robust assembly and disassembly during the cell cycle, and transcriptional and post-transcriptional regulation of histone biogenesis and chromatin modification is controlled in a cell cycle-dependent manner. Chromatin binding proteins and chromatin modifications in turn influence the expression of critical cell cycle regulators, the accessibility of origins for DNA replication, DNA repair, and cell fate. In this review we aim to provide an integrated discussion of how the cell cycle machinery impacts nuclear architecture and vice-versa. We highlight recent advances in understanding cell cycle-dependent histone biogenesis and histone modification deposition, how cell cycle regulators control histone modifier activities, the contribution of chromatin modifications to origin firing for DNA replication, and newly identified roles for nucleoporins in regulating cell cycle gene expression, gene expression memory and differentiation. We close with a discussion of how cell cycle status may impact chromatin to influence cell fate decisions, under normal contexts of differentiation as well as in instances of cell fate re-programming.

  7. Cell cycles and proliferation patterns in Haematococcus pluvialis (United States)

    Zhang, Chunhui; Liu, Jianguo; Zhang, Litao


    Most studies on Haematococcus pluvialis have been focused on cell growth and astaxanthin accumulation; far less attention has been paid to cell cycles and proliferation patterns. The purpose of this study was to clarify cell cycles and proliferation patterns in H. pluvialis microscopically using a camera and video recorder system. The complicated life history of H. pluvialis can be divided into two stages: the motile stage and the non-motile stage. All the cells can be classified into forms as follows: motile cell, non-motile cell, zoospore and aplanospore. The main cell proliferation, both in the motile phase and non-motile phase in H. pluvialis, is by asexual reproduction. Under normal growth conditions, a motile cell usually produces two, sometimes four, and exceptionally eight zoospores. Under unfavorable conditions, the motile cell loses its flagella and transforms into a non-motile cell, and the non-motile cell usually produces 2, 4 or 8 aplanospores, and occasionally 20-32 aplanospores, which further develop into non-motile cells. Under suitable conditions, the non-motile cell is also able to release zoospores. The larger non-motile cells produce more than 16 zoospores, and the smaller ones produce 4 or 8 zoospores. Vegetative reproduction is by direct cell division in the motile phase and by occasional cell budding in the non-motile phase. There is, as yet, no convincing direct evidence for sexual reproduction.

  8. The cell cycle-regulated genes of Schizosaccharomyces pombe.

    Directory of Open Access Journals (Sweden)

    Anna Oliva


    Full Text Available Many genes are regulated as an innate part of the eukaryotic cell cycle, and a complex transcriptional network helps enable the cyclic behavior of dividing cells. This transcriptional network has been studied in Saccharomyces cerevisiae (budding yeast and elsewhere. To provide more perspective on these regulatory mechanisms, we have used microarrays to measure gene expression through the cell cycle of Schizosaccharomyces pombe (fission yeast. The 750 genes with the most significant oscillations were identified and analyzed. There were two broad waves of cell cycle transcription, one in early/mid G2 phase, and the other near the G2/M transition. The early/mid G2 wave included many genes involved in ribosome biogenesis, possibly explaining the cell cycle oscillation in protein synthesis in S. pombe. The G2/M wave included at least three distinctly regulated clusters of genes: one large cluster including mitosis, mitotic exit, and cell separation functions, one small cluster dedicated to DNA replication, and another small cluster dedicated to cytokinesis and division. S. pombe cell cycle genes have relatively long, complex promoters containing groups of multiple DNA sequence motifs, often of two, three, or more different kinds. Many of the genes, transcription factors, and regulatory mechanisms are conserved between S. pombe and S. cerevisiae. Finally, we found preliminary evidence for a nearly genome-wide oscillation in gene expression: 2,000 or more genes undergo slight oscillations in expression as a function of the cell cycle, although whether this is adaptive, or incidental to other events in the cell, such as chromatin condensation, we do not know.

  9. Genetic alterations in head and neck squamous cell carcinomas

    Directory of Open Access Journals (Sweden)

    Nagai M.A.


    Full Text Available The genetic alterations observed in head and neck cancer are mainly due to oncogene activation (gain of function mutations and tumor suppressor gene inactivation (loss of function mutations, leading to deregulation of cell proliferation and death. These genetic alterations include gene amplification and overexpression of oncogenes such as myc, erbB-2, EGFR and cyclinD1 and mutations, deletions and hypermethylation leading to p16 and TP53 tumor suppressor gene inactivation. In addition, loss of heterozygosity in several chromosomal regions is frequently observed, suggesting that other tumor suppressor genes not yet identified could be involved in the tumorigenic process of head and neck cancers. The exact temporal sequence of the genetic alterations during head and neck squamous cell carcinoma (HNSCC development and progression has not yet been defined and their diagnostic or prognostic significance is controversial. Advances in the understanding of the molecular basis of head and neck cancer should help in the identification of new markers that could be used for the diagnosis, prognosis and treatment of the disease.

  10. Plant Characteristics of an Integrated Solid Oxide Fuel Cell Cycle and a Steam Cycle

    DEFF Research Database (Denmark)

    Rokni, Masoud


    Plant characteristics of a system containing a solid oxide fuel cell (SOFC) cycle on the top of a Rankine cycle were investigated. Natural gas (NG) was used as the fuel for the plant. A desulfurization reactor removes the sulfur content in the fuel, while a pre-reformer broke down the heavier...... hydrocarbons in an adiabatic steam reformer (ASR). The pre-treated fuel then entered to the anode side of the SOFC. The remaining fuels after the SOFC stacks entered a catalytic burner for further combusting. The burned gases from the burner were then used to produce steam for the Rankine cycle in a heat...... recovery steam generator (HRSG). The remaining energy of the off-gases was recycled back to the topping cycle for further utilization. Several parameter studies were carried out to investigate the sensitivity of the suggested plant. It was shown that the operation temperature of the desulfurization unit...

  11. Cycle life characteristics of Li-TiS2 cells (United States)

    Deligiannis, Frank; Shen, D.; Huang, C. K.; Surampudi, S.


    The development of lithium ambient temperature rechargeable cells is discussed. During the development process, we hope to gain a greater understanding of the materials and the properties of the Li-TiS2 cell and its components. The design will meet the requirements of 100 Wh/Kg and 1000 cycles, at 50 percent depth-of-discharge, by 1995.

  12. Organo-mineral complexation alters carbon and nitrogen cycling in stream microbial assemblages (United States)

    Hunter, William Ross; Wanek, Wolfgang; Prommer, Judith; Mooshammer, Maria; Battin, Tom


    of OM and may, consequently, alter the carbon and nitrogen cycling dynamics within aquatic ecosystems.

  13. Arecoline decreases interleukin-6 production and induces apoptosis and cell cycle arrest in human basal cell carcinoma cells

    Energy Technology Data Exchange (ETDEWEB)

    Huang, Li-Wen [Department of Medical Laboratory Science and Biotechnology, Kaohsiung Medical University, Kaohsiung 80708, Taiwan (China); Hsieh, Bau-Shan; Cheng, Hsiao-Ling [Department of Biochemistry, Faculty of Medicine, College of Medicine, Kaohsiung Medical University, Kaohsiung 80708, Taiwan (China); Hu, Yu-Chen [Graduate Institute of Medicine, College of Medicine, Kaohsiung Medical University, Kaohsiung 80708, Taiwan (China); Chang, Wen-Tsan [Graduate Institute of Medicine, College of Medicine, Kaohsiung Medical University, Kaohsiung 80708, Taiwan (China); Division of Hepatobiliarypancreatic Surgery, Department of Surgery, Kaohsiung Medical University Hospital, Kaohsiung 80708, Taiwan (China); Chang, Kee-Lung, E-mail: [Department of Biochemistry, Faculty of Medicine, College of Medicine, Kaohsiung Medical University, Kaohsiung 80708, Taiwan (China); Graduate Institute of Medicine, College of Medicine, Kaohsiung Medical University, Kaohsiung 80708, Taiwan (China)


    Arecoline, the most abundant areca alkaloid, has been reported to decrease interleukin-6 (IL-6) levels in epithelial cancer cells. Since IL-6 overexpression contributes to the tumorigenic potency of basal cell carcinoma (BCC), this study was designed to investigate whether arecoline altered IL-6 expression and its downstream regulation of apoptosis and the cell cycle in cultured BCC-1/KMC cells. BCC-1/KMC cells and a human keratinocyte cell line, HaCaT, were treated with arecoline at concentrations ranging from 10 to 100 μg/ml, then IL-6 production and expression of apoptosis- and cell cycle progress-related factors were examined. After 24 h exposure, arecoline inhibited BCC-1/KMC cell growth and decreased IL-6 production in terms of mRNA expression and protein secretion, but had no effect on HaCaT cells. Analysis of DNA fragmentation and chromatin condensation showed that arecoline induced apoptosis of BCC-1/KMC cells in a dose-dependent manner, activated caspase-3, and decreased expression of the anti-apoptotic protein Bcl-2. In addition, arecoline induced progressive and sustained accumulation of BCC-1/KMC cells in G2/M phase as a result of reducing checkpoint Cdc2 activity by decreasing Cdc25C phosphatase levels and increasing p53 levels. Furthermore, subcutaneous injection of arecoline led to decreased BCC-1/KMC tumor growth in BALB/c mice by inducing apoptosis. This study demonstrates that arecoline has potential for preventing BCC tumorigenesis by reducing levels of the tumor cell survival factor IL-6, increasing levels of the tumor suppressor factor p53, and eliciting cell cycle arrest, followed by apoptosis. Highlights: ► Arecoline has potential to prevent against basal cell carcinoma tumorigenesis. ► It has more effectiveness on BCC as compared with a human keratinocyte cell line. ► Mechanisms involved including reducing tumor cells’ survival factor IL-6, ► Decreasing Cdc25C phosphatase, enhancing tumor suppressor factor p53, ► Eliciting G2/M

  14. Altered entrainment to the day/night cycle attenuates the daily rise in circulating corticosterone in the mouse. (United States)

    Sollars, Patricia J; Weiser, Michael J; Kudwa, Andrea E; Bramley, Jayne R; Ogilvie, Malcolm D; Spencer, Robert L; Handa, Robert J; Pickard, Gary E


    The suprachiasmatic nucleus (SCN) is a circadian oscillator entrained to the day/night cycle via input from the retina. Serotonin (5-HT) afferents to the SCN modulate retinal signals via activation of 5-HT1B receptors, decreasing responsiveness to light. Consequently, 5-HT1B receptor knockout (KO) mice entrain to the day/night cycle with delayed activity onsets. Since circulating corticosterone levels exhibit a robust daily rhythm peaking around activity onset, we asked whether delayed entrainment of activity onsets affects rhythmic corticosterone secretion. Wheel-running activity and plasma corticosterone were monitored in mice housed under several different lighting regimens. Both duration of the light:dark cycle (T cycle) and the duration of light within that cycle was altered. 5-HT1B KO mice that entrained to a 9.5L:13.5D (short day in a T = 23 h) cycle with activity onsets delayed more than 4 h after light offset exhibited a corticosterone rhythm in phase with activity rhythms but reduced 50% in amplitude compared to animals that initiated daily activity entrainment suggested that the endogenous rhythm of adrenal responsiveness to ACTH remained aligned with SCN-driven behavioral activity. Circadian clock gene expression in the adrenal cortex of these same animals suggested that the adrenal circadian clock was also aligned with SCN-driven behavior. Under T cycles entrainment to short day (winter-like) conditions, manifest as long delays in activity onset after light offset, severely reduces the amplitude of the diurnal rhythm of plasma corticosterone. Such a pronounced reduction in the glucocorticoid rhythm may alter rhythmic gene expression in the central nervous system and in peripheral organs contributing to an array of potential pathophysiologies.

  15. CD20(+) B Cell Depletion Alters T Cell Homing

    NARCIS (Netherlands)

    Kap, Yolanda S.; van Driel, Nikki; Laman, Jon D.; Tak, Paul P.; 't Hart, Bert A.


    Depleting mAbs against the pan B cell marker CD20 are remarkably effective in the treatment of autoimmune-mediated inflammatory disorders, but the underlying mechanisms are poorly defined. The primary objective of this study was to find a mechanistic explanation for the remarkable clinical effect of

  16. Characteristics of nobiletin-mediated alteration of gene expression in cultured cell lines

    Energy Technology Data Exchange (ETDEWEB)

    Nemoto, Kiyomitsu, E-mail: [Department of Molecular Toxicology, School of Pharmaceutical Sciences, University of Shizuoka, 52-1 Yada, Suruga-ku, Shizuoka 422-8526 (Japan); Ikeda, Ayaka; Yoshida, Chiaki; Kimura, Junko; Mori, Junki [Department of Molecular Toxicology, School of Pharmaceutical Sciences, University of Shizuoka, 52-1 Yada, Suruga-ku, Shizuoka 422-8526 (Japan); Fujiwara, Hironori [Department of Anti-Dementia Functional Food Development, Research Center of Supercritical Fluid Technology, Graduate School of Engineering, Tohoku University, 6-6-7 Aoba, Aramaki, Aoba-ku, Sendai 980-8579 (Japan); Yokosuka, Akihito; Mimaki, Yoshihiro [Department of Medicinal Pharmacognosy, School of Pharmacy, Tokyo University of Pharmacy and Life Sciences, 1432-1 Horinouchi, Hachioji 192-0392 (Japan); Ohizumi, Yasushi [Department of Molecular Toxicology, School of Pharmaceutical Sciences, University of Shizuoka, 52-1 Yada, Suruga-ku, Shizuoka 422-8526 (Japan); Department of Anti-Dementia Functional Food Development, Research Center of Supercritical Fluid Technology, Graduate School of Engineering, Tohoku University, 6-6-7 Aoba, Aramaki, Aoba-ku, Sendai 980-8579 (Japan); Laboratory of Kampo Medicines, Yokohama College of Pharmacy, 601 Matano-cho, Totsuka-ku, Yokohama 245-0066 (Japan); Degawa, Masakuni [Department of Molecular Toxicology, School of Pharmaceutical Sciences, University of Shizuoka, 52-1 Yada, Suruga-ku, Shizuoka 422-8526 (Japan)


    Highlights: ► Nobiletin-mediated alterations of gene expression were examined with DNA microarrays. ► Three organ-derived cell lines were treated with 100 μM nobiletin for 24 h. ► In all cell lines, 3 endoplasmic reticulum stress-responsive genes were up-regulated. ► Some cell cycle-regulating and oxidative stress-promoting genes were down-regulated. ► These alterations may contribute to nobiletin-mediated biological effects. -- Abstract: Nobiletin, a polymethoxylated flavonoid that is highly contained in the peels of citrus fruits, exerts a wide variety of beneficial effects, including anti-proliferative effects in cancer cells, repressive effects in hyperlipidemia and hyperglycemia, and ameliorative effects in dementia at in vitro and in vivo levels. In the present study, to further understand the mechanisms of these actions of nobiletin, the nobiletin-mediated alterations of gene expression in three organ-derived cell lines – 3Y1 rat fibroblasts, HuH-7 human hepatocarcinoma cells, and SK-N-SH human neuroblastoma cells – were first examined with DNA microarrays. In all three cell lines, treatments with nobiletin (100 μM) for 24 h resulted in more than 200% increases in the expression levels of five genes, including the endoplasmic reticulum stress-responsive genes Ddit3, Trib3, and Asns, and in less than 50% decreases in the expression levels of seven genes, including the cell cycle-regulating genes Ccna2, Ccne2, and E2f8 and the oxidative stress-promoting gene Txnip. It was also confirmed that in each nobiletin-treated cell line, the levels of the DDIT3 (DNA-damage-inducible transcript 3, also known as CHOP and GADD153) and ASNS (asparagine synthetase) proteins were increased, while the level of the TXNIP (thioredoxin-interacting protein, also known as VDUP1 and TBP-2) protein was decreased. All these findings suggest that nobiletin exerts a wide variety of biological effects, at least partly, through induction of endoplasmic reticulum stress and

  17. Notch3 overexpression causes arrest of cell cycle progression by inducing Cdh1 expression in human breast cancer cells. (United States)

    Chen, Chun-Fa; Dou, Xiao-Wei; Liang, Yuan-Ke; Lin, Hao-Yu; Bai, Jing-Wen; Zhang, Xi-Xun; Wei, Xiao-Long; Li, Yao-Chen; Zhang, Guo-Jun


    Uncontrolled cell proliferation, genomic instability and cancer are closely related to the abnormal activation of the cell cycle. Therefore, blocking the cell cycle of cancer cells has become one of the key goals for treating malignancies. Unfortunately, the factors affecting cell cycle progression remain largely unknown. In this study, we have explored the effects of Notch3 on the cell cycle in breast cancer cell lines by 3 methods: overexpressing the intra-cellular domain of Notch3 (N3ICD), knocking-down Notch3 by RNA interference, and using X-ray radiation exposure. The results revealed that overexpression of Notch3 arrested the cell cycle at the G0/G1 phase, and inhibited the proliferation and colony-formation rate in the breast cancer cell line, MDA-MB-231. Furthermore, overexpressing N3ICD upregulated Cdh1 expression and resulted in p27(Kip) accumulation by accelerating Skp2 degradation. Conversely, silencing of Notch3 in the breast cancer cell line, MCF-7, caused a decrease in expression levels of Cdh1 and p27(Kip) at both the protein and mRNA levels, while the expression of Skp2 only increased at the protein level. Correspondingly, there was an increase in the percentage of cells in the G0/G1 phase and an elevated proliferative ability and colony-formation rate, which may be caused by alterations of the Cdh1/Skp2/p27 axis. These results were also supported by exposing MDA-MB-231 cells or MCF-7 treated with siN3 to X-irradiation at various doses. Overall, our data showed that overexpression of N3ICD upregulated the expression of Cdh1 and caused p27(Kip) accumulation by accelerating Skp2 degradation, which in turn led to cell cycle arrest at the G0/G1 phase, in the context of proliferating breast cancer cell lines. These findings help to illuminate the precision therapy targeted to cell cycle progression, required for cancer treatment.

  18. Effects of ultraviolet irradiation on the cell cycle. (United States)

    Bolognia, J L; Sodi, S A; Chakraborty, A K; Fargnoli, M C; Pawelek, J M


    Cultured mouse Cloudman melanoma cells, EMT6 breast carcinoma cells, and 3T3 fibroblasts all accumulated in the G2/M phase of the cell cycle when exposed to UVB radiation. The effects of UVB were maximal at 20-30 mJ/cm2 for all three cell lines, and could be observed by flow cytometry as early as 12 hr post irradiation. It has been known since the mid-1970s that MSH receptor binding activity is highest on Cloudman melanoma cells when they are in the G2/M phase of their cycle. Here we show that either UVB irradiation or synchronization of Cloudman cells with colchicine results in a stimulation of MSH binding within 24 hr following treatment, a time when both treatments have resulted in accumulation of cells in the G2/M phase of the cycle. Furthermore, the two treatments performed together on the melanoma cells stimulated MSH receptor activity to the same extent as either treatment performed separately, suggesting that each may be influencing MSH receptor activity solely through a G2/M accumulation of cells. Together, these results raise the possibility that an increase in the number of cells in the G2 phase of the cell cycle is a generalized cellular response to injury, such as UV irradiation. However, in the case of pigment cells this response includes a mechanism for increasing melanin formation, i.e., increased MSH receptor activity. Should this be the case, similar G2/M "injury responses" of other cell types might be expected, consistent with their differentiated phenotypes.

  19. Alterations induced in Escherichia Coli cells by gamma radiation

    Energy Technology Data Exchange (ETDEWEB)

    Kappke, J.; Schelin, H.R.; Paschuk, S.A.; Denyak, V.; Silva, E.R. da [Federal University of Technology of Parana (CPGEI/UTFPR), Curitiba, PR (Brazil)]. E-mails:;;; Jesus, E.F.O. de; Lopes, R.T. [Universidade Federal do Rio de Janeiro (UFRJ), RJ (Brazil). Coordenacao dos Programas de Pos-graduacao de Engenharia (COPPE). Lab. de Instrumentacao Nuclear]. E-mails:;; Carlin, N.; Toledo, E.S. [Universidade de Sao Paulo (USP), SP (Brazil). Inst. de Fisica]. E-mail:


    Modifications occurred in Escherichia coli cells exposed to gamma radiation ({sup 60}Co source) were investigated. The irradiations were done at the LIN-COPPE laboratory of the UFRJ and the analysis at the Biology Department of the UTFPR. The E. coli cells were irradiated with 30, 60, 90, 120, 150, 180, 210, 240, 300, 480, 600 e 750 Gy doses. The samples were analyzed with Gram-stain, biochemical tests in EPM, MIO and Lysine Broth, Simmons Cytrate Medium and Rhamnose Broth, antibiogram and isolation of auxotrophic mutants. It was observed that for the received doses the E. coli did not show morphological alterations in the tests. Some E. Coli cells showed to be able to deaminade the L-tryptophan or they changed their sensibility for amoxillin and cephaloonine after the irradiation. The existence of aauxotrophic mutants after irradiation was also verified. (author)

  20. Altered B cell receptor signaling in human systemic lupus erythematosus (United States)

    Jenks, Scott A.; Sanz, Iñaki


    Regulation of B cell receptor signaling is essential for the development of specific immunity while retaining tolerance to self. Systemic lupus erythematosus (SLE) is characterized by a loss of B cell tolerance and the production of anti-self antibodies. Accompanying this break down in tolerance are alterations in B cell receptor signal transduction including elevated induced calcium responses and increased protein phosphorylation. Specific pathways that negatively regulate B cell signaling have been shown to be impaired in some SLE patients. These patients have reduced levels of the kinase Lyn in lipid raft microdomains and this reduction is inversely correlated with increased CD45 in lipid rafts. Function and expression of the inhibitory immunoglobulin receptor FcγRIIB is also reduced in Lupus IgM- CD27+ memory cells. Because the relative contribution of different memory and transitional B cell subsets can be abnormal in SLE patients, we believe studies targeted to well defined B cell subsets will be necessary to further our understanding of signaling abnormalities in SLE. Intracellular flow cytometric analysis of signaling is a useful approach to accomplish this goal. PMID:18723129

  1. The circadian mutation PER2(S662G) is linked to cell cycle progression and tumorigenesis. (United States)

    Gu, X; Xing, L; Shi, G; Liu, Z; Wang, X; Qu, Z; Wu, X; Dong, Z; Gao, X; Liu, G; Yang, L; Xu, Y


    Circadian oscillation and cell cycle progression are the two most essential rhythmic events present in almost all organisms. Circadian rhythms keep track of time and provide temporal regulation with a period of about 24 h. The cell cycle is optimized for growth and division, but not for time keeping. Circadian gated cell divisions are observed in nearly all organisms. However, the implications of this coupling to the physiology of mammals are unknown. A mutation (S662G) in the clock protein PERIOD2 (PER2) is responsible for familial advanced sleep phase syndrome in which sleep onset occurs in the early evening and wakefulness occurs in the early morning. Here, we provide evidence that the PER2(S662) mutation leads to enhanced resistance to X-ray-induced apoptosis and increased E1A- and RAS-mediated oncogenic transformation. Accordingly, the PER2(S662) mutation affects tumorigenesis in cancer-sensitized p53(R172H/+) mice. Finally, analyzing the clock-controlled cell cycle genes p21, c-Myc, Cyclin D1 and p27, we found that the relative phases between p21 and Cyclin D expression profiles have been changed significantly in these Per2 allele mutant mouse embryonic fibroblasts. This key role of the Per2-mediated phase alteration of p21 provides what we believe to be a novel mechanism in understanding cell cycle progression, its plasticity and its resistance to interference.

  2. - a comprehensive multi-organism online database of cell-cycle experiments

    DEFF Research Database (Denmark)

    Gauthier, Nicholas Paul; Larsen, Malene Erup; Wernersson, Rasmus


    .org, for viewing and downloading these data. The user interface facilitates searches for genes of interest as well as downloads of genome-wide results. Individual genes are displayed with graphs of expression profiles throughout the cell cycle from all available experiments. These expression profiles...

  3. A cell cycle and nutritional checkpoint controlling bacterial surface adhesion.

    Directory of Open Access Journals (Sweden)

    Aretha Fiebig


    Full Text Available In natural environments, bacteria often adhere to surfaces where they form complex multicellular communities. Surface adherence is determined by the biochemical composition of the cell envelope. We describe a novel regulatory mechanism by which the bacterium, Caulobacter crescentus, integrates cell cycle and nutritional signals to control development of an adhesive envelope structure known as the holdfast. Specifically, we have discovered a 68-residue protein inhibitor of holdfast development (HfiA that directly targets a conserved glycolipid glycosyltransferase required for holdfast production (HfsJ. Multiple cell cycle regulators associate with the hfiA and hfsJ promoters and control their expression, temporally constraining holdfast development to the late stages of G1. HfiA further functions as part of a 'nutritional override' system that decouples holdfast development from the cell cycle in response to nutritional cues. This control mechanism can limit surface adhesion in nutritionally sub-optimal environments without affecting cell cycle progression. We conclude that post-translational regulation of cell envelope enzymes by small proteins like HfiA may provide a general means to modulate the surface properties of bacterial cells.

  4. MiR-107 and MiR-185 can induce cell cycle arrest in human non small cell lung cancer cell lines.

    Directory of Open Access Journals (Sweden)

    Yukari Takahashi

    Full Text Available BACKGROUND: MicroRNAs (miRNAs are short single stranded noncoding RNAs that suppress gene expression through either translational repression or degradation of target mRNAs. The annealing between messenger RNAs and 5' seed region of miRNAs is believed to be essential for the specific suppression of target gene expression. One miRNA can have several hundred different targets in a cell. Rapidly accumulating evidence suggests that many miRNAs are involved in cell cycle regulation and consequentially play critical roles in carcinogenesis. METHODOLOGY/PRINCIPAL FINDINGS: Introduction of synthetic miR-107 or miR-185 suppressed growth of the human non-small cell lung cancer cell lines. Flow cytometry analysis revealed these miRNAs induce a G1 cell cycle arrest in H1299 cells and the suppression of cell cycle progression is stronger than that by Let-7 miRNA. By the gene expression analyses with oligonucleotide microarrays, we find hundreds of genes are affected by transfection of these miRNAs. Using miRNA-target prediction analyses and the array data, we listed up a set of likely targets of miR-107 and miR-185 for G1 cell cycle arrest and validate a subset of them using real-time RT-PCR and immunoblotting for CDK6. CONCLUSIONS/SIGNIFICANCE: We identified new cell cycle regulating miRNAs, miR-107 and miR-185, localized in frequently altered chromosomal regions in human lung cancers. Especially for miR-107, a large number of down-regulated genes are annotated with the gene ontology term 'cell cycle'. Our results suggest that these miRNAs may contribute to regulate cell cycle in human malignant tumors.

  5. Viral infections and cell cycle G2/M regulation

    Institute of Scientific and Technical Information of China (English)

    Richard Y.ZHAO; Robert T.ELDER


    Progression of cells from G2 phase of the cell cycle to mitosis is a tightly regulated cellular process that requires activation of the Cdc2 kinase, which determines onset of mitosis in all eukaryotic cells. In both human and fission yeast(Schizosaccharomyces pombe) cells, the activity of Cdc2 is regulated in part by the phosphorylation status of tyrosine 15(Tyr15) on Cdc2, which is phosphorylated by Wee1 kinase during late G2 and is rapidly dephosphorylated by the Cdc25 tyrosine phosphatase to trigger entry into mitosis. These Cdc2 regulators are the downstream targets of two well-characterized G2/M checkpoint pathways which prevent cells from entering mitosis when cellular DNA is damaged or when DNA replication is inhibited. Increasing evidence suggests that Cdc2 is also commonly targeted by viral proteins,which modulate host cell cycle machinery to benefit viral survival or replication. In this review, we describe the effect of viral protein R (Vpr) encoded by human immunodeficiency virus type 1 (HIV-1) on cell cycle G2/M regulation. Based on our current knowledge about this viral effect, we hypothesize that Vpr induces cell cycle G2 arrest through a mechanism that is to some extent different from the classic G2/M checkpoints. One the unique features distinguishing Vpr-induced G2 arrest from the classic checkpoints is the role of phosphatase 2A (PP2A) in Vpr-induced G2 arrest.Interestingly, PP2A is targeted by a number of other viral proteins including SV40 small T antigen, polyomavirus T antigen, HTLV Tax and adenovirus E4orf4. Thus an in-depth understanding of the molecular mechanisms underlying Vpr-induced G2 arrest will provide additional insights into the basic biology of cell cycle G2/M regulation and into the biological significance of this effect during host-pathogen interactions.

  6. A combined gas cooled nuclear reactor and fuel cell cycle (United States)

    Palmer, David J.

    Rising oil costs, global warming, national security concerns, economic concerns and escalating energy demands are forcing the engineering communities to explore methods to address these concerns. It is the intention of this thesis to offer a proposal for a novel design of a combined cycle, an advanced nuclear helium reactor/solid oxide fuel cell (SOFC) plant that will help to mitigate some of the above concerns. Moreover, the adoption of this proposal may help to reinvigorate the Nuclear Power industry while providing a practical method to foster the development of a hydrogen economy. Specifically, this thesis concentrates on the importance of the U.S. Nuclear Navy adopting this novel design for its nuclear electric vessels of the future with discussion on efficiency and thermodynamic performance characteristics related to the combined cycle. Thus, the goals and objectives are to develop an innovative combined cycle that provides a solution to the stated concerns and show that it provides superior performance. In order to show performance, it is necessary to develop a rigorous thermodynamic model and computer program to analyze the SOFC in relation with the overall cycle. A large increase in efficiency over the conventional pressurized water reactor cycle is realized. Both sides of the cycle achieve higher efficiencies at partial loads which is extremely important as most naval vessels operate at partial loads as well as the fact that traditional gas turbines operating alone have poor performance at reduced speeds. Furthermore, each side of the cycle provides important benefits to the other side. The high temperature exhaust from the overall exothermic reaction of the fuel cell provides heat for the reheater allowing for an overall increase in power on the nuclear side of the cycle. Likewise, the high temperature helium exiting the nuclear reactor provides a controllable method to stabilize the fuel cell at an optimal temperature band even during transients helping

  7. Intercellular Variability in Protein Levels from Stochastic Expression and Noisy Cell Cycle Processes. (United States)

    Soltani, Mohammad; Vargas-Garcia, Cesar A; Antunes, Duarte; Singh, Abhyudai


    Inside individual cells, expression of genes is inherently stochastic and manifests as cell-to-cell variability or noise in protein copy numbers. Since proteins half-lives can be comparable to the cell-cycle length, randomness in cell-division times generates additional intercellular variability in protein levels. Moreover, as many mRNA/protein species are expressed at low-copy numbers, errors incurred in partitioning of molecules between two daughter cells are significant. We derive analytical formulas for the total noise in protein levels when the cell-cycle duration follows a general class of probability distributions. Using a novel hybrid approach the total noise is decomposed into components arising from i) stochastic expression; ii) partitioning errors at the time of cell division and iii) random cell-division events. These formulas reveal that random cell-division times not only generate additional extrinsic noise, but also critically affect the mean protein copy numbers and intrinsic noise components. Counter intuitively, in some parameter regimes, noise in protein levels can decrease as cell-division times become more stochastic. Computations are extended to consider genome duplication, where transcription rate is increased at a random point in the cell cycle. We systematically investigate how the timing of genome duplication influences different protein noise components. Intriguingly, results show that noise contribution from stochastic expression is minimized at an optimal genome-duplication time. Our theoretical results motivate new experimental methods for decomposing protein noise levels from synchronized and asynchronized single-cell expression data. Characterizing the contributions of individual noise mechanisms will lead to precise estimates of gene expression parameters and techniques for altering stochasticity to change phenotype of individual cells.

  8. Labeling of lectin receptors during the cell cycle. (United States)

    Garrido, J


    Labeling of lectin receptors during the cell cycle. (Localizabión de receptores para lectinas durante el ciclo celular). Arch. Biol. Med. Exper. 10: 100-104, 1976. The topographic distribution of specific cell surface receptors for concanavalin A and wheat germ agglutinin was studied by ultrastructural labeling in the course of the cell cycle. C12TSV5 cells were synchronized by double thymidine block or mechanical selection (shakeoff). They were labeled by means of lectin-peroxidase techniques while in G1 S, G2 and M phases of the cycle. The results obtained were similar for both lectins employed. Interphase cells (G1 S, G2) present a stlihtly discontinous labeling pattern that is similar to the one observed on unsynchronized cells of the same line. Cells in mitosis, on the contrary, present a highly discontinous distribution of reaction product. This pattern disappears after the cells enters G1 and is not present on mitotic cells fixed in aldehyde prior to labeling.

  9. Computation Molecular Kinetics Model of HZE Induced Cell Cycle Arrest (United States)

    Cucinotta, Francis A.; Ren, Lei


    Cell culture models play an important role in understanding the biological effectiveness of space radiation. High energy and charge (HZE) ions produce prolonged cell cycle arrests at the G1/S and G2/M transition points in the cell cycle. A detailed description of these phenomena is needed to integrate knowledge of the expression of DNA damage in surviving cells, including the determination of relative effectiveness factors between different types of radiation that produce differential types of DNA damage and arrest durations. We have developed a hierarchical kinetics model that tracks the distribution of cells in various cell phase compartments (early G1, late G1, S, G2, and M), however with transition rates that are controlled by rate-limiting steps in the kinetics of cyclin-cdk's interactions with their families of transcription factors and inhibitor molecules. The coupling of damaged DNA molecules to the downstream cyclin-cdk inhibitors is achieved through a description of the DNA-PK and ATM signaling pathways. For HZE irradiations we describe preliminary results, which introduce simulation of the stochastic nature of the number of direct particle traversals per cell in the modulation of cyclin-cdk and cell cycle population kinetics. Comparison of the model to data for fibroblast cells irradiated photons or HZE ions are described.

  10. Metabolic flux prediction in cancer cells with altered substrate uptake. (United States)

    Schwartz, Jean-Marc; Barber, Michael; Soons, Zita


    Proliferating cells, such as cancer cells, are known to have an unusual metabolism, characterized by an increased rate of glycolysis and amino acid metabolism. Our understanding of this phenomenon is limited but could potentially be used in order to develop new therapies. Computational modelling techniques, such as flux balance analysis (FBA), have been used to predict fluxes in various cell types, but remain of limited use to explain the unusual metabolic shifts and altered substrate uptake in human cancer cells. We implemented a new flux prediction method based on elementary modes (EMs) and structural flux (StruF) analysis and tested them against experimentally measured flux data obtained from (13)C-labelling in a cancer cell line. We assessed the quality of predictions using different objective functions along with different techniques in normalizing a metabolic network with more than one substrate input. Results show a good correlation between predicted and experimental values and indicate that the choice of cellular objective critically affects the quality of predictions. In particular, lactate gives an excellent correlation and correctly predicts the high flux through glycolysis, matching the observed characteristics of cancer cells. In contrast with FBA, which requires a priori definition of all uptake rates, often hard to measure, atomic StruFs (aStruFs) are able to predict uptake rates of multiple substrates.

  11. Cell Division, a new open access online forum for and from the cell cycle community

    Directory of Open Access Journals (Sweden)

    Kaldis Philipp


    Full Text Available Abstract Cell Division is a new, open access, peer-reviewed online journal that publishes cutting-edge articles, commentaries and reviews on all exciting aspects of cell cycle control in eukaryotes. A major goal of this new journal is to publish timely and significant studies on the aberrations of the cell cycle network that occur in cancer and other diseases.

  12. Cell "circadian" cycle: new role for mammalian core clock genes. (United States)

    Borgs, Laurence; Beukelaers, Pierre; Vandenbosch, Renaud; Belachew, Shibeshih; Nguyen, Laurent; Malgrange, Brigitte


    In mammals, 24 hours rhythms are organized as a biochemical network of molecular clocks that are operative in all tissues, with the master clock residing in the hypothalamic suprachiasmatic nucleus (SCN). The core pacemakers of these clocks consist of auto-regulatory transcriptional/post-transcriptional feedback loops. Several lines of evidence suggest the existence of a crosstalk between molecules that are responsible for the generation of circadian rhythms and molecules that control the cell cycle progression. In addition, highly specialized cell cycle checkpoints involved in DNA repair after damage seem also, at least in part, mediated by clock proteins. Recent studies have also highlighted a putative connection between clock protein dysfunction and cancer progression. This review discusses the intimate relation that exists between cell cycle progression and components of the circadian machinery.

  13. Circadian clock gene Per2 plays an important role in cell proliferation, apoptosis and cell cycle progression in human oral squamous cell carcinoma. (United States)

    Wang, Qingqing; Ao, Yiran; Yang, Kai; Tang, Hong; Chen, Dan


    Previous studies have shown that the aberrant expression of period circadian clock 2 (Per2) is closely related to the occurrence and development of cancers, but the specific mechanism remains unclear. In the present study, we used shRNA to downregulate Per2 in oral squamous cell carcinoma (OSCC) Tca8113 cells, and then detected the alterations in cell cycle, cell proliferation and apoptosis by flow cytometric analysis and mRNA expression alterations in all the important genes in the cyclin/cyclin-dependent protein kinase (CDK)/cyclin-dependent kinase inhibitor (CKI) cell cycle network by RT-qPCR. We found that in the Tca8113 cells, after Per2 downregulation, the mRNA expression levels of cyclin A2, B1 and D1, CDK4, CDK6 and E2F1 were significantly increased (Pcycle progression and the balance of cell proliferation and apoptosis by regulation of the cyclin/CDK/CKI cell cycle network. Further research on Per2 may provide a new effective molecular target for cancer treatments.

  14. Coordinating cell polarity and cell cycle progression: what can we learn from flies and worms? (United States)

    Noatynska, Anna; Tavernier, Nicolas; Gotta, Monica; Pintard, Lionel


    Spatio-temporal coordination of events during cell division is crucial for animal development. In recent years, emerging data have strengthened the notion that tight coupling of cell cycle progression and cell polarity in dividing cells is crucial for asymmetric cell division and ultimately for metazoan development. Although it is acknowledged that such coupling exists, the molecular mechanisms linking the cell cycle and cell polarity machineries are still under investigation. Key cell cycle regulators control cell polarity, and thus influence cell fate determination and/or differentiation, whereas some factors involved in cell polarity regulate cell cycle timing and proliferation potential. The scope of this review is to discuss the data linking cell polarity and cell cycle progression, and the importance of such coupling for asymmetric cell division. Because studies in model organisms such as Caenorhabditis elegans and Drosophila melanogaster have started to reveal the molecular mechanisms of this coordination, we will concentrate on these two systems. We review examples of molecular mechanisms suggesting a coupling between cell polarity and cell cycle progression.

  15. Vertebrate Cell Cycle Modulates Infection by Protozoan Parasites (United States)

    Dvorak, James A.; Crane, Mark St. J.


    Synchronized HeLa cell populations were exposed to Trypanosoma cruzi or Toxoplasma gondii, obligate intracellular protozoan parasites that cause Chagas' disease and toxoplasmosis, respectively, in humans. The ability of the two parasites to infect HeLa cells increased as the HeLa cells proceeded from the G1 phase to the S phase of their growth cycle and decreased as the cells entered G2-M. Characterization of the S-phase cell surface components responsible for this phenomenon could be beneficial in the development of vaccines against these parasitic diseases.

  16. Comparison of cell cycle components, apoptosis and cytoskeleton-related molecules and therapeutic effects of flavopiridol and geldanamycin on the mouse fibroblast, lung cancer and embryonic stem cells. (United States)

    Aktug, Huseyin; Acikgoz, Eda; Uysal, Aysegul; Oltulu, Fatih; Oktem, Gulperi; Yigitturk, Gurkan; Demir, Kenan; Yavasoglu, Altug; Bozok Cetintas, Vildan


    Similarities and differences in the cell cycle components, apoptosis and cytoskeleton-related molecules among mouse skin fibroblast cells (MSFs), mouse squamous cell lung carcinomas (SqCLCs) and mouse embryonic stem cells (mESCs) are important determinants of the behaviour and differentiation capacity of these cells. To reveal apoptotic pathways and to examine the distribution and the role of cell cycle-cell skeleton comparatively would necessitate tumour biology and stem cell biology to be assessed together in terms of oncogenesis and embryogenesis. The primary objectives of this study are to investigate the effects of flavopiridol, a cell cycle inhibitor, and geldanamycin, a heat shock protein inhibitor on mouse somatic, tumour and embryonic stem cells, by specifically focusing on alterations in cytoskeletal proteins, cell polarity and motility as well as cell cycle regulators. To meet these objectives, expression of several genes, cell cycle analysis and immunofluorescence staining of intracellular cytoskeletal molecules were performed in untreated and flavopiridol- or geldanamycin-treated cell lines. Cytotoxicity assays showed that SqCLCs are more sensitive to flavopiridol than MSFs and mESCs. Keratin-9 and keratin-2 expressions increased dramatically whereas cell cycle regulatory genes decreased significantly in the flavopiridol-treated MSFs. Flavopiridol-treated SqCLCs displayed a slight increase in several cell cytoskeleton regulatory genes as well as cell cycle regulatory genes. However, gene expression profiles of mESCs were not affected after flavopiridol treatment except the Cdc2a. Cytotoxic concentrations of geldanamycin were close to each other for all cell lines. Cdkn1a was the most increased gene in the geldanamycin-treated MSFs. However, expression levels of cell cytoskeleton-associated genes were increased dramatically in the geldanamycin-treated SqCLCs. Our results revealing differences in molecular mechanisms between embryogenesis and

  17. Modelling cell cycle synchronisation in networks of coupled radial glial cells. (United States)

    Barrack, Duncan S; Thul, Rüdiger; Owen, Markus R


    Radial glial cells play a crucial role in the embryonic mammalian brain. Their proliferation is thought to be controlled, in part, by ATP mediated calcium signals. It has been hypothesised that these signals act to locally synchronise cell cycles, so that clusters of cells proliferate together, shedding daughter cells in uniform sheets. In this paper we investigate this cell cycle synchronisation by taking an ordinary differential equation model that couples the dynamics of intracellular calcium and the cell cycle and extend it to populations of cells coupled via extracellular ATP signals. Through bifurcation analysis we show that although ATP mediated calcium release can lead to cell cycle synchronisation, a number of other asynchronous oscillatory solutions including torus solutions dominate the parameter space and cell cycle synchronisation is far from guaranteed. Despite this, numerical results indicate that the transient and not the asymptotic behaviour of the system is important in accounting for cell cycle synchronisation. In particular, quiescent cells can be entrained on to the cell cycle via ATP mediated calcium signals initiated by a driving cell and crucially will cycle in near synchrony with the driving cell for the duration of neurogenesis. This behaviour is highly sensitive to the timing of ATP release, with release at the G1/S phase transition of the cell cycle far more likely to lead to near synchrony than release during mid G1 phase. This result, which suggests that ATP release timing is critical to radial glia cell cycle synchronisation, may help us to understand normal and pathological brain development.

  18. TRPV3 Channel Negatively Regulates Cell Cycle Progression and Safeguards the Pluripotency of Embryonic Stem Cells. (United States)

    Lo, Iek Chi; Chan, Hing Chung; Qi, Zenghua; Ng, Kwun Lam; So, Chun; Tsang, Suk Ying


    Embryonic stem cells (ESCs) have tremendous potential for research and future therapeutic purposes. However, the calcium handling mechanism in ESCs is not fully elucidated. Aims of this study are (1) to investigate if transient receptor potential vanilloid-3 (TRPV3) channels are present in mouse ESCs (mESCs) and their subcellular localization; (2) to investigate the role of TRPV3 in maintaining the characteristics of mESCs. Western blot and immunocytochemistry showed that TRPV3 was present at the endoplasmic reticulum (ER) of mESCs. Calcium imaging showed that, in the absence of extracellular calcium, TRPV3 activators camphor and 6-tert-butyl-m-cresol increased the cytosolic calcium. However, depleting the ER store in advance of activator addition abolished the calcium increase, suggesting that TRPV3 released calcium from the ER. To dissect the functional role of TRPV3, TRPV3 was activated and mESC proliferation was measured by trypan blue exclusion and MTT assays. The results showed that TRPV3 activation led to a decrease in mESC proliferation. Cell cycle analysis revealed that TRPV3 activation increased the percentage of cells in G2 /M phase; consistently, Western blot also revealed a concomitant increase in the expression of inactive form of cyclin-dependent kinase 1, suggesting that TRPV3 activation arrested mESCs at G2 /M phase. TRPV3 activation did not alter the expression of pluripotency markers Oct-4, Klf4 and c-Myc, suggesting that the pluripotency was preserved. Our study is the first study to show the presence of TRPV3 at ER. Our study also reveals the novel role of TRPV3 in controlling the cell cycle and preserving the pluripotency of ESCs.

  19. An apoptotic cell cycle mutant in Saccharomyces cerevisiae

    DEFF Research Database (Denmark)

    Villadsen, Ingrid


    The simple eukaryote Saccharomyces cerevisiae has proved to be a useful organism for elucidating the mechanisms that govern cell cycle progression in eukaryotic cells. The excellent in vivo system permits a cell cycle study using temperature sensitive mutants. In addition, it is possible to study...... many genes and gene products from higher eukaryotes in Saccharomyces cerevisiae because many genes and biological processes are homologous or similar in lower and in higher eukaryotes. The highly developed methods of genetics and molecular biology greatly facilitates studies of higher eukaryotic...... processes.Programmmed cell death with apoptosis plays a major role in development and homeostatis in most, if not all, animal cells. Apoptosis is a morphologically distinct form of death, that requires the activation of a highly regulated suicide program. Saccharomyces cerevisiae provides a new system...

  20. Glucocorticoids play a key role in circadian cell cycle rhythms.

    Directory of Open Access Journals (Sweden)

    Thomas Dickmeis


    Full Text Available Clock output pathways play a pivotal role by relaying timing information from the circadian clock to a diversity of physiological systems. Both cell-autonomous and systemic mechanisms have been implicated as clock outputs; however, the relative importance and interplay between these mechanisms are poorly understood. The cell cycle represents a highly conserved regulatory target of the circadian timing system. Previously, we have demonstrated that in zebrafish, the circadian clock has the capacity to generate daily rhythms of S phase by a cell-autonomous mechanism in vitro. Here, by studying a panel of zebrafish mutants, we reveal that the pituitary-adrenal axis also plays an essential role in establishing these rhythms in the whole animal. Mutants with a reduction or a complete absence of corticotrope pituitary cells show attenuated cell-proliferation rhythms, whereas expression of circadian clock genes is not affected. We show that the corticotrope deficiency is associated with reduced cortisol levels, implicating glucocorticoids as a component of a systemic signaling pathway required for circadian cell cycle rhythmicity. Strikingly, high-amplitude rhythms can be rescued by exposing mutant larvae to a tonic concentration of a glucocorticoid agonist. Our work suggests that cell-autonomous clock mechanisms are not sufficient to establish circadian cell cycle rhythms at the whole-animal level. Instead, they act in concert with a systemic signaling environment of which glucocorticoids are an essential part.

  1. Genetic alterations in B-cell non-Hodgkin's lymphoma

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    Magić Zvonko


    Full Text Available Background. Although the patients with diagnosed B-NHL are classified into the same disease stage on the basis of clinical, histopathological, and immunological parameters, they respond significantly different to the applied treatment. This points out the possibility that within the same group of lymphoma there are different diseases at molecular level. For that reason many studies deal with the detection of gene alterations in lymphomas to provide a better framework for diagnosis and treatment of these hematological malignancies. Aim. To define genetic alterations in the B-NHL with highest possibilities for diagnostic purposes and molecular detection of MRD. Methods. Formalin fixed and paraffin embedded lymph node tissues from 45 patients were examined by different PCR techniques for the presence of IgH and TCR γ gene rearrangement; K-ras and H-ras mutations; c-myc amplification and bcl-2 translocation. There were 34 cases of B-cell non-Hodgkin’s lymphoma (B-NHL, 5 cases of T-cell non-Hodgkin’s lymphoma (T-NHL and 6 cases of chronic lymphadenitis (CL. The mononuclear cell fraction of the peripheral blood of 12 patients with B-NHL was analyzed for the presence of monoclonality at the time of diagnosis and in 3 to 6 months time intervals after an autologous bone marrow transplantation (BMT. Results. The monoclonality of B-lymphocytes, as evidenced by DNA fragment length homogeneity, was detected in 88 % (30/34 of B-NHL, but never in CL, T-NHL, or in normal PBL. Bcl-2 translocation was detected in 7/31 (22.6% B-NHL specimens, c-myc amplification 9/31 (29%, all were more than doubled, K-ras mutations in 1/31 (3.23% and H-ras mutations in 2/31 (6.45% of the examined B-NHL samples. In the case of LC and normal PBL, however, these gene alterations were not detected. All the patients (12 with B-NHL had dominant clone of B-lymphocyte in the peripheral blood at the time of diagnosis while only in 2 of 12 patients MRD was detected 3 or 6 months after

  2. Altered entrainment to the day/night cycle attenuates the daily rise in circulating corticosterone in the mouse.

    Directory of Open Access Journals (Sweden)

    Patricia J Sollars

    Full Text Available The suprachiasmatic nucleus (SCN is a circadian oscillator entrained to the day/night cycle via input from the retina. Serotonin (5-HT afferents to the SCN modulate retinal signals via activation of 5-HT1B receptors, decreasing responsiveness to light. Consequently, 5-HT1B receptor knockout (KO mice entrain to the day/night cycle with delayed activity onsets. Since circulating corticosterone levels exhibit a robust daily rhythm peaking around activity onset, we asked whether delayed entrainment of activity onsets affects rhythmic corticosterone secretion. Wheel-running activity and plasma corticosterone were monitored in mice housed under several different lighting regimens. Both duration of the light:dark cycle (T cycle and the duration of light within that cycle was altered. 5-HT1B KO mice that entrained to a 9.5L:13.5D (short day in a T = 23 h cycle with activity onsets delayed more than 4 h after light offset exhibited a corticosterone rhythm in phase with activity rhythms but reduced 50% in amplitude compared to animals that initiated daily activity <4 h after light offset. Wild type mice in 8L:14D (short day in a T = 22 h conditions with highly delayed activity onsets also exhibited a 50% reduction in peak plasma corticosterone levels. Exogenous adrenocorticotropin (ACTH stimulation in animals exhibiting highly delayed entrainment suggested that the endogenous rhythm of adrenal responsiveness to ACTH remained aligned with SCN-driven behavioral activity. Circadian clock gene expression in the adrenal cortex of these same animals suggested that the adrenal circadian clock was also aligned with SCN-driven behavior. Under T cycles <24 h, altered circadian entrainment to short day (winter-like conditions, manifest as long delays in activity onset after light offset, severely reduces the amplitude of the diurnal rhythm of plasma corticosterone. Such a pronounced reduction in the glucocorticoid rhythm may alter rhythmic gene expression in

  3. Impact of cell cycle delay on micronucleus frequency in TK6 cells. (United States)

    Sobol, Zhanna; Spellman, Richard A; Thiffeault, Catherine; Dobo, Krista L; Schuler, Maik


    Previous studies with TK6 cells have shown that extending the recovery period after pulse treatment allows for greater micronucleus expression for some compounds. This study explores the role of cell cycle delay in micronucleus expression after pulse treatment with three model genotoxins [mitomycin C, etoposide (ETOP), vinblastine]. Cells were treated for 4 hr and allowed to recover for 36 hr with samples removed at various time points during the recovery period and analyzed for cell cycle distribution, apoptosis and micronucleus frequency. Our results show that mitomycin C causes cell cycle delay for 20 hr after pulse treatment and cell cycle perturbation is no longer evident after 36 hr of recovery. The micronucleus frequency of cells sampled at 36 hr is doubled when compared with cells sampled at 20 hr after mitomycin C removal. When cells were treated with indirect acting genotoxins (ETOP, vinblastine), cell cycle perturbation was not observed at the 20 hr time point. Micronucleus frequency after treatment with either ETOP or vinblastine did not differ between the 20 hr and the 36 hr time point. All three compounds induced similar levels of apoptosis ranging from 4.5 to 5.6% with maximum induction occurring at the 36-hr time point. We conclude that TK6 cells exhibit extended cell cycle arrest after exposure to MMC and can go on to express micronuclei, after overcoming cell cycle arrest.

  4. Altered T cell costimulation during chronic hepatitis B infection. (United States)

    Barboza, Luisa; Salmen, Siham; Peterson, Darrell L; Montes, Henry; Colmenares, Melisa; Hernández, Manuel; Berrueta-Carrillo, Leidith E; Berrueta, Lisbeth


    T-cell response to hepatitis B virus (HBV) is vigorous, polyclonal and multi-specific in patients with acute hepatitis who ultimately clear the virus, whereas it is narrow and inefficient in patients with chronic disease, where inappropriate early activation events could account for viral persistence. We investigated the induction of activation receptors and cytokine production in response to HBcAg and crosslinking of CD28 molecules, in CD4+ cells from a group of chronically infected patients (CIP) and naturally immune subjects (NIS). We demonstrated that CD4+ cells from CIP did not increase levels of CD40L and CD69 following stimulation with HBcAg alone or associated to CD28 crosslinking, in contrast to subjects that resolved the infection (p<0.01). Furthermore, CD4+ cells from CIP produced elevated levels of IL-10 in response to HBcAg. These results suggest that a predominant inhibitory environment may be responsible for altered T cell costimulation, representing a pathogenic mechanism for viral persistence.

  5. Ureaplasma parvum infection alters filamin a dynamics in host cells

    Directory of Open Access Journals (Sweden)

    Brown Mary B


    Full Text Available Abstract Background Ureaplasmas are among the most common bacteria isolated from the human urogenital tract. Ureaplasmas can produce asymptomatic infections or disease characterized by an exaggerated inflammatory response. Most investigations have focused on elucidating the pathogenic potential of Ureaplasma species, but little attention has been paid to understanding the mechanisms by which these organisms are capable of establishing asymptomatic infection. Methods We employed differential proteome profiling of bladder tissues from rats experimentally infected with U. parvum in order to identify host cell processes perturbed by colonization with the microbe. Tissues were grouped into four categories: sham inoculated controls, animals that spontaneously cleared infection, asymptomatic urinary tract infection (UTI, and complicated UTI. One protein that was perturbed by infection (filamin A was used to further elucidate the mechanism of U. parvum-induced disruption in human benign prostate cells (BPH-1. BPH-1 cells were evaluated by confocal microscopy, immunoblotting and ELISA. Results Bladder tissue from animals actively colonized with U. parvum displayed significant alterations in actin binding proteins (profilin 1, vinculin, α actinin, and filamin A that regulate both actin polymerization and cell cytoskeletal function pertaining to focal adhesion formation and signal transduction (Fisher's exact test, P U. parvum perturbed the regulation of filamin A. Specifically, infected BPH-1 cells exhibited a significant increase in filamin A phosphorylated at serine2152 (P ≤ 0.01, which correlated with impaired proteolysis of the protein and its normal intracellular distribution. Conclusion Filamin A dynamics were perturbed in both models of infection. Phosphorylation of filamin A occurs in response to various cell signaling cascades that regulate cell motility, differentiation, apoptosis and inflammation. Thus, this phenomenon may be a useful

  6. Visualisation of cell cycle modifications by X-ray irradiation of single HeLa cells using fluorescent ubiquitination-based cell cycle indicators. (United States)

    Kaminaga, K; Noguchi, M; Narita, A; Sakamoto, Y; Kanari, Y; Yokoya, A


    To explore the effects of X-ray irradiation on mammalian cell cycle dynamics, single cells using the fluorescent ubiquitination-based cell cycle indicator (Fucci) technique were tracked. HeLa cells expressing Fucci were used to visualise cell cycle modifications induced by irradiation. After cultured HeLa-Fucci cells were exposed to 5 Gy X-rays, fluorescent cell images were captured every 20 min for 48 h using a fluorescent microscope. Time dependence of the fluorescence intensity of S/G2 cells was analysed to examine the cell cycle dynamics of irradiated and non-irradiated control cells. The results showed that irradiated cells could be divided into two populations: one with similar cell cycle dynamics to that of non-irradiated cells, and another displaying a prolonged G2 phase. Based on these findings, it is proposed in this article that an underlying switch mechanism is involved in cell cycle regulation and the G2/M checkpoint of HeLa cells.

  7. Alterations of proteins in MDCK cells during acute potassium deficiency. (United States)

    Peerapen, Paleerath; Ausakunpipat, Nardtaya; Chanchaem, Prangwalai; Thongboonkerd, Visith


    Chronic K(+) deficiency can cause hypokalemic nephropathy associated with metabolic alkalosis, polyuria, tubular dilatation, and tubulointerstitial injury. However, effects of acute K(+) deficiency on the kidney remained unclear. This study aimed to explore such effects by evaluating changes in levels of proteins in renal tubular cells during acute K(+) deficiency. MDCK cells were cultivated in normal K(+) (NK) (K(+)=5.3 mM), low K(+) (LK) (K(+)=2.5 mM), or K(+) depleted (KD) (K(+)=0 mM) medium for 24 h and then harvested. Cellular proteins were resolved by two-dimensional gel electrophoresis (2-DE) and visualized by SYPRO Ruby staining (5 gels per group). Spot matching and quantitative intensity analysis revealed a total 48 protein spots that had significantly differential levels among the three groups. Among these, 46 and 30 protein spots had differential levels in KD group compared to NK and LK groups, respectively. Comparison between LK and NK groups revealed only 10 protein spots that were differentially expressed. All of these differentially expressed proteins were successfully identified by Q-TOF MS and/or MS/MS analyses. The altered levels of heat shock protein 90 (HSP90), ezrin, lamin A/C, tubulin, chaperonin-containing TCP1 (CCT1), and calpain 1 were confirmed by Western blot analysis. Global protein network analysis showed three main functional networks, including 1) cell growth and proliferation, 2) cell morphology, cellular assembly and organization, and 3) protein folding in which the altered proteins were involved. Further investigations on these networks may lead to better understanding of pathogenic mechanisms of low K(+)-induced renal injury.

  8. Regulation of mitochondrial morphology and cell cycle by microRNA-214 targeting Mitofusin2. (United States)

    Bucha, Sudha; Mukhopadhyay, Debashis; Bhattacharyya, Nitai Pada


    Huntington's disease (HD) is an autosomal dominant neurodegenerative disease caused by the increase in CAG repeats beyond 36 at the exon1 of the gene Huntingtin (HTT). Among the various dysfunctions of biological processes in HD, transcription deregulation due to abnormalities in actions of transcription factors has been considered to be one of the important pathological conditions. In addition, deregulation of microRNA (miRNA) expression has been described in HD. Earlier, expression of microRNA-214 (miR-214) has been shown to increase in HD cell models and target HTT gene; the expression of the later being inversely correlated to that of miR-214. In the present communication, we observed that the expressions of several HTT co-expressed genes are modulated by exogenous expression of miR-214 or by its mutant. Among several HTT co-expressed genes, MFN2 was shown to be the direct target of miR-214. Exogenous expression of miR-214, repressed the expression of MFN2, increased the distribution of fragmented mitochondria and altered the distribution of cells in different phases of cell cycle. In summary, we have shown that increased expression of miR-214 observed in HD cell model could target MFN2, altered mitochondrial morphology and deregulated cell cycle. Inhibition of miR-214 could be a possible target of intervention in HD pathogenesis.

  9. Ecdysone Receptor (EcR Is Involved in the Transcription of Cell Cycle Genes in the Silkworm

    Directory of Open Access Journals (Sweden)

    Wenliang Qian


    Full Text Available EcR (ecdysone receptor-mediated ecdysone signaling pathway contributes to regulate the transcription of genes involved in various processes during insect development. In this work, we detected the expression of EcR gene in silkworm ovary-derived BmN4 cells and found that EcR RNAi result in an alteration of cell shape, indicating that EcR may orchestrate cell cycle progression. EcR RNAi and EcR overexpression analysis revealed that in the cultured BmN4 cells, EcR respectively promoted and suppressed the transcription of E2F-1 and CycE, two genes controlling cell cycle progression. Further examination demonstrated that ecdysone application in BmN4 cells not only changed the transcription of these two cell cycle genes like that under EcR overexpression, but also induced cell cycle arrest at G2/M phase. In vivo analysis confirmed that E2F-1 expression was elevated in silk gland of silkworm larvae after ecdysone application, which is same as its response to ecdysone in BmN4 cells. However, ecdysone also promotes CycE transcription in silk gland, and this is converse with the observation in BmN4 cells. These results provide new insights into understanding the roles of EcR-mediated ecdysone signaling in the regulation of cell cycle.

  10. Arginine starvation in colorectal carcinoma cells: Sensing, impact on translation control and cell cycle distribution. (United States)

    Vynnytska-Myronovska, Bozhena O; Kurlishchuk, Yuliya; Chen, Oleh; Bobak, Yaroslav; Dittfeld, Claudia; Hüther, Melanie; Kunz-Schughart, Leoni A; Stasyk, Oleh V


    Tumor cells rely on a continued exogenous nutrient supply in order to maintain a high proliferative activity. Although a strong dependence of some tumor types on exogenous arginine sources has been reported, the mechanisms of arginine sensing by tumor cells and the impact of changes in arginine availability on translation and cell cycle regulation are not fully understood. The results presented herein state that human colorectal carcinoma cells rapidly exhaust the internal arginine sources in the absence of exogenous arginine and repress global translation by activation of the GCN2-mediated pathway and inhibition of mTOR signaling. Tumor suppressor protein p53 activation and G1/G0 cell cycle arrest support cell survival upon prolonged arginine starvation. Cells with the mutant or deleted TP53 fail to stop cell cycle progression at defined cell cycle checkpoints which appears to be associated with reduced recovery after durable metabolic stress triggered by arginine withdrawal.

  11. The Effect of Spaceflight on Cartilage Cell Cycle and Differentiation (United States)

    Doty, Stephen B.; Stiner, Dalina; Telford, William G.


    In vivo studies have shown that spaceflight results in loss of bone and muscle. In an effort to understand the mechanisms of these changes, cell cultures of cartilage, bone and muscle have been subjected to spaceflight to study the microgravity effects on differentiated cells. However it now seems possible that the cell differentiation process itself may be the event(s) most affected by spaceflight. For example, osteoblast-like cells have been shown to have reduced cellular activity in microgravity due to an underdifferentiated state (Carmeliet, et al, 1997). And reduced human lymphocyte growth in spaceflight was related to increased apoptosis (Lewis, et al, 1998). Which brings us to the question of whether reduced cellular activity in space is due to an effect on the differentiated cell, an effect on the cell cycle and cell proliferation, or an effect on cell death. This question has not been specifically addressed on previous flights and was the question behind die present study.

  12. α-Mangostin Induces Apoptosis and Cell Cycle Arrest in Oral Squamous Cell Carcinoma Cell (United States)

    Kwak, Hyun-Ho; Park, Bong-Soo


    Mangosteen has long been used as a traditional medicine and is known to have antibacterial, antioxidant, and anticancer effects. Although the effects of α-mangostin, a natural compound extracted from the pericarp of mangosteen, have been investigated in many studies, there is limited data on the effects of the compound in human oral squamous cell carcinoma (OSCC). In this study, α-mangostin was assessed as a potential anticancer agent against human OSCC cells. α-Mangostin inhibited cell proliferation and induced cell death in OSCC cells in a dose- and time-dependent manner with little to no effect on normal human PDLF cells. α-Mangostin treatment clearly showed apoptotic evidences such as nuclear fragmentation and accumulation of annexin V and PI-positive cells on OSCC cells. α-Mangostin treatment also caused the collapse of mitochondrial membrane potential and the translocation of cytochrome c from the mitochondria into the cytosol. The expressions of the mitochondria-related proteins were activated by α-mangostin. Treatment with α-mangostin also induced G1 phase arrest and downregulated cell cycle-related proteins (CDK/cyclin). Hence, α-mangostin specifically induces cell death and inhibits proliferation in OSCC cells via the intrinsic apoptosis pathway and cell cycle arrest at the G1 phase, suggesting that α-mangostin may be an effective agent for the treatment of OSCC. PMID:27478478

  13. α-Mangostin Induces Apoptosis and Cell Cycle Arrest in Oral Squamous Cell Carcinoma Cell

    Directory of Open Access Journals (Sweden)

    Hyun-Ho Kwak


    Full Text Available Mangosteen has long been used as a traditional medicine and is known to have antibacterial, antioxidant, and anticancer effects. Although the effects of α-mangostin, a natural compound extracted from the pericarp of mangosteen, have been investigated in many studies, there is limited data on the effects of the compound in human oral squamous cell carcinoma (OSCC. In this study, α-mangostin was assessed as a potential anticancer agent against human OSCC cells. α-Mangostin inhibited cell proliferation and induced cell death in OSCC cells in a dose- and time-dependent manner with little to no effect on normal human PDLF cells. α-Mangostin treatment clearly showed apoptotic evidences such as nuclear fragmentation and accumulation of annexin V and PI-positive cells on OSCC cells. α-Mangostin treatment also caused the collapse of mitochondrial membrane potential and the translocation of cytochrome c from the mitochondria into the cytosol. The expressions of the mitochondria-related proteins were activated by α-mangostin. Treatment with α-mangostin also induced G1 phase arrest and downregulated cell cycle-related proteins (CDK/cyclin. Hence, α-mangostin specifically induces cell death and inhibits proliferation in OSCC cells via the intrinsic apoptosis pathway and cell cycle arrest at the G1 phase, suggesting that α-mangostin may be an effective agent for the treatment of OSCC.

  14. Effects of cell cycle noise on excitable gene circuits

    CERN Document Server

    Veliz-Cuba, Alan; Bennett, Matthew R; Josić, Krešimir; Ott, William


    We assess the impact of cell cycle noise on gene circuit dynamics. For bistable genetic switches and excitable circuits, we find that transitions between metastable states most likely occur just after cell division and that this concentration effect intensifies in the presence of transcriptional delay. We explain this concentration effect with a 3-states stochastic model. For genetic oscillators, we quantify the temporal correlations between daughter cells induced by cell division. Temporal correlations must be captured properly in order to accurately quantify noise sources within gene networks.

  15. Cell cycle-arrested tumor cells exhibit increased sensitivity towards TRAIL-induced apoptosis (United States)

    Ehrhardt, H; Wachter, F; Grunert, M; Jeremias, I


    Resting tumor cells represent a huge challenge during anticancer therapy due to their increased treatment resistance. TNF-related apoptosis-inducing ligand (TRAIL) is a putative future anticancer drug, currently in phases I and II clinical studies. We recently showed that TRAIL is able to target leukemia stem cell surrogates. Here, we tested the ability of TRAIL to target cell cycle-arrested tumor cells. Cell cycle arrest was induced in tumor cell lines and xenografted tumor cells in G0, G1 or G2 using cytotoxic drugs, phase-specific inhibitors or RNA interference against cyclinB and E. Biochemical or molecular arrest at any point of the cell cycle increased TRAIL-induced apoptosis. Accordingly, when cell cycle arrest was disabled by addition of caffeine, the antitumor activity of TRAIL was reduced. Most important for clinical translation, tumor cells from three children with B precursor or T cell acute lymphoblastic leukemia showed increased TRAIL-induced apoptosis upon knockdown of either cyclinB or cyclinE, arresting the cell cycle in G2 or G1, respectively. Taken together and in contrast to most conventional cytotoxic drugs, TRAIL exerts enhanced antitumor activity against cell cycle-arrested tumor cells. Therefore, TRAIL might represent an interesting drug to treat static-tumor disease, for example, during minimal residual disease. PMID:23744361

  16. NONO couples the circadian clock to the cell cycle


    Kowalska, Elzbieta; Ripperger, Juergen A.; Hoegger, Dominik C.; Bruegger, Pascal; Buch, Thorsten; Birchler, Thomas; Mueller, Anke; Albrecht, Urs; Contaldo, Claudio; Steven A Brown


    Mammalian circadian clocks restrict cell proliferation to defined time windows, but the mechanism and consequences of this interrelationship are not fully understood. Previously we identified the multifunctional nuclear protein NONO as a partner of circadian PERIOD (PER) proteins. Here we show that it also conveys circadian gating to the cell cycle, a connection surprisingly important for wound healing in mice. Specifically, although fibroblasts from NONO-deficient mice showed approximately n...

  17. Cell cycle control by a minimal Cdk network.

    Directory of Open Access Journals (Sweden)

    Claude Gérard


    Full Text Available In present-day eukaryotes, the cell division cycle is controlled by a complex network of interacting proteins, including members of the cyclin and cyclin-dependent protein kinase (Cdk families, and the Anaphase Promoting Complex (APC. Successful progression through the cell cycle depends on precise, temporally ordered regulation of the functions of these proteins. In light of this complexity, it is surprising that in fission yeast, a minimal Cdk network consisting of a single cyclin-Cdk fusion protein can control DNA synthesis and mitosis in a manner that is indistinguishable from wild type. To improve our understanding of the cell cycle regulatory network, we built and analysed a mathematical model of the molecular interactions controlling the G1/S and G2/M transitions in these minimal cells. The model accounts for all observed properties of yeast strains operating with the fusion protein. Importantly, coupling the model's predictions with experimental analysis of alternative minimal cells, we uncover an explanation for the unexpected fact that elimination of inhibitory phosphorylation of Cdk is benign in these strains while it strongly affects normal cells. Furthermore, in the strain without inhibitory phosphorylation of the fusion protein, the distribution of cell size at division is unusually broad, an observation that is accounted for by stochastic simulations of the model. Our approach provides novel insights into the organization and quantitative regulation of wild type cell cycle progression. In particular, it leads us to propose a new mechanistic model for the phenomenon of mitotic catastrophe, relying on a combination of unregulated, multi-cyclin-dependent Cdk activities.

  18. Cell-cycle radiation response: Role of intracellular factors (United States)

    Blakely, E.; Chang, P.; Lommel, L.; Bjornstad, K.; Dixon, M.; Tobias, C.; Kumar, K.; Blakely, W. F.

    We have been studying variations of radiosensitivity and endogenous cellular factors during the course of progression through the human and hamster cell cycle. After exposure to low-LET radiations, the most radiosensitive cell stages are mitosis and the G1/S interface. The increased activity of a specific antioxidant enzyme such as superoxide dismutase in G1-phase, and the variations of endogenous thiols during cell division are thought to be intracellular factors of importance to the radiation survival response. These factors may contribute to modifying the age-dependent yield of lesions or more likely, to the efficiency of the repair processes. These molecular factors have been implicated in our cellular measurements of the larger values for the radiobiological oxygen effect late in the cycle compared to earlier cell ages. Low-LET radiation also delays progression through S phase which may allow more time for repair and hence contribute to radioresistance in late-S-phase. The cytoplasmic and intranuclear milieu of the cell appears to have less significant effects on lesions produced by high-LET radiation compared to those made by low-LET radiation. High-LET radiation fails to slow progression through S phase, and there is much less repair of lesions evident at all cell ages; however, high-LET particles cause a more profound block in G2 phase than that observed after low-LET radiation. Hazards posed by the interaction of damage from sequential doses of radiations of different qualities have been evaluated and are shown to lead to a cell-cycle-dependent enhancement of radiobiological effects. A summary comparison of various cell-cycle-dependent endpoints measured with low-or high-LET radiations is given and includes a discussion of the possible additional effects introduced by microgravity.

  19. Are paradoxical cell cycle activities in neurons and glia related to the metabolic theory of Alzheimer's disease? (United States)

    Erol, Adnan


    The progression and outcome of neurological diseases are determined by the balance between neurodegeneration, neuroprotection, and neuroregeneration. In this context, astroglial cells are invariably involved in every kind of neuropathology. Mitotically, active glial cells provide metabolic support to active neurons, contribute to coupling between synaptic activity and local blood flow, and thus protect against oxidative stress. Disturbances of the complex neuron-glia interrelation are increasingly recognized as a potentially important pathophysiological mechanism in a wide variety of neurological disorders including those marked by neurodegeneration. Peripheral insulin resistance-mediated increased oxidative stress in glial cells, and consequent DNA damage, induces senescence in glial cells leads to the development of an inflammatory environment. The immune mediators released by senescent (activated) glial cells are considered to be neurotoxic and ultimately increase the oxidant load of neurons. While the neuron is viewed as the prototypical post-mitotic, fully differentiated cell, certain subsets of neurons reactivate cell-cycle activity in response to triggers of neuronal apoptosis, such as genotoxic stress generated by redox changes due to pathological alterations in supporting astroglial cells. Thus, a paradoxical cell cycle block in glial cells coupled with concomitant cell cycle re-entry in neurons (due to pathological alterations created by peripheral insulin resistance-induced neuroendocrine signaling changes) may cause neurodegeneration, such as seen in Alzheimer's disease.

  20. Evolution of cell cycle control: same molecular machines, different regulation

    DEFF Research Database (Denmark)

    de Lichtenberg, Ulrik; Jensen, Thomas Skøt; Brunak, Søren


    Decades of research has together with the availability of whole genomes made it clear that many of the core components involved in the cell cycle are conserved across eukaryotes, both functionally and structurally. These proteins are organized in complexes and modules that are activated or deacti......Decades of research has together with the availability of whole genomes made it clear that many of the core components involved in the cell cycle are conserved across eukaryotes, both functionally and structurally. These proteins are organized in complexes and modules that are activated...... or deactivated at specific stages during the cell cycle through a wide variety of mechanisms including transcriptional regulation, phosphorylation, subcellular translocation and targeted degradation. In a series of integrative analyses of different genome-scale data sets, we have studied how these different...... layers of regulation together control the activity of cell cycle complexes and how this regulation has evolved. The results show surprisingly poor conservation of both the transcriptional and the post-translation regulation of individual genes and proteins; however, the changes in one layer of regulation...

  1. Refined life-cycle assessment of polymer solar cells

    DEFF Research Database (Denmark)

    Lenzmann, F.; Kroon, J.; Andriessen, R.


    A refined life-cycle assessment of polymer solar cells is presented with a focus on critical components, i.e. the transparent conductive ITO layer and the encapsulation components. This present analysis gives a comprehensive sketch of the full environmental potential of polymer-OPV in comparison...

  2. Maid (GCIP) is involved in cell cycle control of hepatocytes

    DEFF Research Database (Denmark)

    Sonnenberg-Riethmacher, Eva; Wüstefeld, Torsten; Miehe, Michaela;


    . Therefore, we studied the role of Maid during cell cycle progression after partial hepatectomy (PH). Lack of Maid expression after PH was associated with a delay in G1/S-phase progression as evidenced by delayed cyclinA expression and DNA replication in Maid-deficient mice. However, at later time points...

  3. Cycle life status of SAFT VOS nickel-cadmium cells (United States)

    Goualard, Jacques


    The SAFT prismatic VOS Ni-Cd cells have been flown in geosynchronous orbit since 1977 and in low earth orbit since 1983. Parallel cycling tests are performed by several space agencies in order to determine the cycle life for a wide range of temperature and depth of discharge (DOD). In low Earth orbit (LEO), the ELAN program is conducted on 24 Ah cells by CNES and ESA at the European Battery Test Center at temperatures ranging from 0 to 27 C and DOD from 10 to 40 percent. Data are presented up to 37,000 cycles. One pack (X-80) has achieved 49,000 cycles at 10 C and 23 percent DOD. The geosynchronous orbit simulation of a high DOD test is conducted by ESA on 3 batteries at 10 C and 70, 90, and 100 percent DOD. Thirty-one eclipse seasons are completed, and no signs of degradation have been found. The Air Force test at CRANE on 24 Ah and 40 Ah cells at 20 C and 80 percent DOD has achieved 19 shadow periods. Life expectancy is discussed. The VOS cell technology could be used for the following: (1) in geosynchronous conditions--15 yrs at 10-15 C and 80 percent DOD; and (2) in low earth orbit--10 yrs at 5-15 C and 25-30 percent DOD.

  4. Visualizing cell-cycle kinetics after hypoxia/reoxygenation in HeLa cells expressing fluorescent ubiquitination-based cell cycle indicator (Fucci). (United States)

    Goto, Tatsuaki; Kaida, Atsushi; Miura, Masahiko


    Hypoxia induces G1 arrest in many cancer cell types. Tumor cells are often exposed to hypoxia/reoxygenation, especially under acute hypoxic conditions in vivo. In this study, we investigated cell-cycle kinetics and clonogenic survival after hypoxia/reoxygenation in HeLa cells expressing fluorescent ubiquitination-based cell cycle indicator (Fucci). Hypoxic treatment halted cell-cycle progression during mid-S to G2 phase, as determined by the cell cycle-regulated E3 ligase activities of SCF(Skp2) and APC/C(Cdh1), which are regulators of the Fucci probes; however, the DNA content of the arrested cells was equivalent to that in G1 phase. After reoxygenation, time-lapse imaging and DNA content analysis revealed that all cells reached G2 phase, and that Fucci fluorescence was distinctly separated into two fractions 24h after reoxygenation: red cells that released from G2 arrest after repairing DNA double-strand breaks (DSBs) exhibited higher clonogenic survival, whereas most cells that stayed green contained many DSBs and exhibited lower survival. We conclude that hypoxia disrupts coordination of DNA synthesis and E3 ligase activities associated with cell-cycle progression, and that DSB repair could greatly influence cell-cycle kinetics and clonogenic survival after hypoxia/reoxygenation.

  5. Down-regulation of AP-4 inhibits proliferation, induces cell cycle arrest and promotes apoptosis in human gastric cancer cells.

    Directory of Open Access Journals (Sweden)

    Xinghua Liu

    Full Text Available BACKGROUND: AP-4 belongs to the basic helix-loop-helix leucine-zipper subgroup; it controls target gene expression, regulates growth, development and cell apoptosis and has been implicated in tumorigenesis. Our previous studies indicated that AP-4 was frequently overexpressed in gastric cancers and may be associated with the poor prognosis. The purpose of this study is to examine whether silencing of AP-4 can alter biological characteristics of gastric cancer cells. METHODS: Two specific siRNAs targeting AP-4 were designed, synthesized, and transfected into gastric cancer cell lines and human normal mucosa cells. AP-4 expression was measured with real-time quantitative PCR and Western blot. Cell proliferation and chemo-sensitivity were detected by CCK-8 assay. Cell cycle assay and apoptosis assay were performed by flow cytometer, and relative expression of cell cycle regulators were detected by real-time quantitative PCR and Western blot, expression of the factors involved in the apoptosis pathway were examined in mRNA and protein level. RESULTS: The expression of AP-4 was silenced by the siRNAs transfection and the effects of AP-4 knockdown lasted 24 to 96 hrs. The siRNA-mediated silencing of AP-4 suppressed the cellular proliferation, induced apoptosis and sensitized cancer cells to anticancer drugs. In addition, the expression level of p21, p53 and Caspase-9 were increased when AP-4 was knockdown, but the expression of cyclin D1, Bcl-2 and Bcl-x(L was inhibited. It didn't induce cell cycle arrest when AP-4 was knockdown in p53 defect gastric cancer cell line Kato-III. CONCLUSIONS: These results illustrated that gene silencing of AP-4 can efficiently inhibited cell proliferation, triggered apoptosis and sensitized cancer cells to anticancer drugs in vitro, suggesting that AP-4 siRNAs mediated silencing has a potential value in the treatment of human gastric cancer.

  6. Effect of staurosporine on cycle of human gastric cancer cells

    Institute of Scientific and Technical Information of China (English)

    Min-Wen Ha; Ke-Zuo Hou; Yun-Peng Liu; Yuan Yuan


    AIM: To study the effect of staurosporine (ST) on the cell cycle of human gastriccancer cell lines MGC803 and SGC7901.METHODS: Cell proliferation was evaluated by trypan blue dye exclusion method. Apoptotic morphology was observed under a transmission electron microscope. Changes of cell cycle and apoptotic peaks of cells were determined by flow cytometry. Expression of p21WAFI gene was examined using immunohistochemistry and RT-PCR.RESULTS: The growth of MGC803 and SGC7901 cells was inhibited by ST. The inhibitory concentrations against 50% cells (IC50) at 24 h and 48 h were 54 ng/ml and 23 ng/ml for MlGC803, and 61 ng/ml and 37 ng/ml for SGC7901. Typical apoptotic bodies and apoptotic peaks were observed 24 hafter cells were treated wth ST at a concentration of 200ng/ml. The percentage of cells at G0/G1 phase was decreased and that of cells at G2/M was increased significantly in the group treated wth ST at the concentrations of 40ng/ml,60 ng/ml, 100 ng/ml for 24 h, compared with the control group (P<0.01). The expression levels of p21WAFI gene in both MGC803 and SGC7901 cells were markedly up-regulated after treatment with ST.CONCLUSION: ST can cause arrest of gastric cancer cells at G2/M phase, which may be one of the mechanisms that inhibit cell proliferation and cause apoptosis in these cells.Effect of ST on cells at G2/M phase may be attributed to the up-regulattion of p21WAFI gene.

  7. Heat production of mammalian cells at different cell-cycle phases

    NARCIS (Netherlands)

    Loesberg, C.; Miltenburg, J.C. van; Wuk, R. van


    1. 1.|Heat production of Reuber H35 rat hepatoma cells and murine C1300 neuroblastoma cells at different stages of the cell cycle were measured microcalorimetrically. 2. 2.|Reuber H35 monolayer cultures of G1-phase cells and cells in S-phase were trypsinized, reincubated in suspension culture and i

  8. A Coarse Estimation of Cell Size Region from a Mesoscopic Stochastic Cell Cycle Model

    Institute of Scientific and Technical Information of China (English)

    YI Ming; JIA Ya; LIU Quan; ZHU Chun-Lian; YANG Li-Jian


    Based on a deterministic cell cycle model of fission yeast, the effects of the finite cell size on the cell cycle regulation in wee1- cdc25△ double mutant type are numerically studied by using of the chemical Langevin equations. It is found that at a certain region of cell size, our numerical results from the chemical Langevin equations are in good qualitative agreement with the experimental observations. The two resettings to the G2 phase from early stages of mitosis can be induced under the moderate cell size. The quantized cycle times can be observed during such a cell size region. Therefore, a coarse estimation of cell size is obtained from the mesoscopic stochastic cell cycle model.

  9. A Coarse Estimation of Cell Size Region from a Mesoscopic Stochastic Cell Cycle Model (United States)

    Yi, Ming; Jia, Ya; Liu, Quan; Zhu, Chun-Lian; Yang, Li-Jian


    Based on a deterministic cell cycle model of fission yeast, the effects of the finite cell size on the cell cycle regulation in wee1- cdc25Δ double mutant type are numerically studied by using of the chemical Langevin equations. It is found that at a certain region of cell size, our numerical results from the chemical Langevin equations are in good qualitative agreement with the experimental observations. The two resettings to the G2 phase from early stages of mitosis can be induced under the moderate cell size. The quantized cycle times can be observed during such a cell size region. Therefore, a coarse estimation of cell size is obtained from the mesoscopic stochastic cell cycle model.


    Institute of Scientific and Technical Information of China (English)

    李济宇; 全志伟; 张强; 刘建文


    Objective To explore the effect of somatostatin on the cell cycle of human gallbladder cancer cell. Methods Growth curve of gallbladder cancer cell was measured after somatostatin treated on gradient concentration. Simultaneously, the change of gallbladder cancer cell cycle was detected using flow cytometry.Results Concentration-dependent cell growth inhibition caused by somatostatin was detected in gallbladder cancer cell(P<0.05). Cell growth was arrested in S phase since 12h after somatostatin treated, which reached its peak at 24h, then fell down. The changes in apoptosis index of gallbladder cancer cell caused by somatostatin correlated with that's in cell cycle. Conclusion Somatostatin could inhibit the cell growth of human gallbladder cancer cell in vitro on higher concentration. It might result from inducing growth arrest in S phase in early stage and inducing apoptosis in the late stage.

  11. Cell Cycle Regulates Nuclear Stability of AID and Determines the Cellular Response to AID.

    Directory of Open Access Journals (Sweden)

    Quy Le


    Full Text Available AID (Activation Induced Deaminase deaminates cytosines in DNA to initiate immunoglobulin gene diversification and to reprogram CpG methylation in early development. AID is potentially highly mutagenic, and it causes genomic instability evident as translocations in B cell malignancies. Here we show that AID is cell cycle regulated. By high content screening microscopy, we demonstrate that AID undergoes nuclear degradation more slowly in G1 phase than in S or G2-M phase, and that mutations that affect regulatory phosphorylation or catalytic activity can alter AID stability and abundance. We directly test the role of cell cycle regulation by fusing AID to tags that destabilize nuclear protein outside of G1 or S-G2/M phases. We show that enforced nuclear localization of AID in G1 phase accelerates somatic hypermutation and class switch recombination, and is well-tolerated; while nuclear AID compromises viability in S-G2/M phase cells. We identify AID derivatives that accelerate somatic hypermutation with minimal impact on viability, which will be useful tools for engineering genes and proteins by iterative mutagenesis and selection. Our results further suggest that use of cell cycle tags to regulate nuclear stability may be generally applicable to studying DNA repair and to engineering the genome.

  12. Neisseria meningitidis causes cell cycle arrest of human brain microvascular endothelial cells at S phase via p21 and cyclin G2. (United States)

    Oosthuysen, Wilhelm F; Mueller, Tobias; Dittrich, Marcus T; Schubert-Unkmeir, Alexandra


    Microbial pathogens have developed several mechanisms to modulate and interfere with host cell cycle progression. In this study, we analysed the effect of the human pathogen Neisseria meningitidis on cell cycle in a brain endothelial cell line as well as in primary brain endothelial cells. We found that N.  Meningitidis causes an accumulation of cells in the S phase early at 3 and at 24 h post-infection that was paralleled by a decrease of cells in G2/M phase. Importantly, the outer membrane proteins of the colony opacity-associated (Opa) protein family as well as the Opc protein proved to trigger the accumulation of cells in the S phase. A focused cell cycle reverse transcription quantitative polymerase chain reaction-based array and integrated network analysis revealed changes in the abundance of several cell cycle regulatory mRNAs, including the cell cycle inhibitors p21(WAF1/CIP1) and cyclin G2. These alterations were reflected in changes in protein expression levels and/or relocalization in N. meningitidis-infected cells. Moreover, an increase in p21(WAF1/CIP1) expression was found to be p53 independent. Genetic ablation of p21(WAF1/CIP1) and cyclin G2 abrogated N. meningitidis-induced S phase accumulation. Finally, by measuring the levels of the biomarker 8-hydroxydeoxyguanosine and phosphorylation of the histone variant H2AX, we provide evidence that N. meningitidis induces oxidative DNA damage in infected cells.

  13. Amygdala activation by corticosterone alters visceral and somatic pain in cycling female rats. (United States)

    Gustafsson, Jenny K; Greenwood-Van Meerveld, Beverley


    Irritable bowel syndrome (IBS) is often seen in women, and symptom severity is known to vary over the menstrual cycle. In addition, activation of the hypothalamic-pituitary-adrenal (HPA) axis enhances symptomology and patients with IBS have increased activation of the amygdala, a brain region known to facilitate HPA output. However, little is known about the effects of amygdala activation during different stages of the menstrual cycle. We therefore investigated the effects of amygdala activation on somatic and visceral pain perception over the rat estrous cycle. Female Wistar rats were implanted with either corticosterone (Cort) or cholesterol as a control onto the dorsal margin of the central amygdala. Visceral sensitivity was quantified by recording the visceromotor response (VMR) to colorectal distension (CRD) and somatic sensitivity was assessed via the Von Frey test. In cholesterol controls, both visceral and somatic sensitivity varied over the estrous cycle. Rats in proestrus/estrus responded to CRD with an increased VMR compared with rats in metestrus/diestrus. Somatic sensitivity followed a similar pattern with enhanced sensitivity during proestrus/estrus compared with metestrus/diestrus. Elevated amygdala Cort induced visceral hypersensitivity during metestrus/diestrus but had no effect during proestrus/estrus. In contrast, elevated amygdala Cort increased somatic sensitivity during both metestrus/diestrus and proestrus/estrous. These results suggests that amygdala activation by Cort eliminates spontaneously occurring differences in visceral and somatic pain perception, which could explain the lowered pain thresholds and higher incidence of somatic pain observed in women with IBS.

  14. Akt1 intramitochondrial cycling is a crucial step in the redox modulation of cell cycle progression.

    Directory of Open Access Journals (Sweden)

    Valeria Gabriela Antico Arciuch

    Full Text Available Akt is a serine/threonine kinase involved in cell proliferation, apoptosis, and glucose metabolism. Akt is differentially activated by growth factors and oxidative stress by sequential phosphorylation of Ser(473 by mTORC2 and Thr(308 by PDK1. On these bases, we investigated the mechanistic connection of H(2O(2 yield, mitochondrial activation of Akt1 and cell cycle progression in NIH/3T3 cell line with confocal microscopy, in vivo imaging, and directed mutagenesis. We demonstrate that modulation by H(2O(2 entails the entrance of cytosolic P-Akt1 Ser(473 to mitochondria, where it is further phosphorylated at Thr(308 by constitutive PDK1. Phosphorylation of Thr(308 in mitochondria determines Akt1 passage to nuclei and triggers genomic post-translational mechanisms for cell proliferation. At high H(2O(2, Akt1-PDK1 association is disrupted and P-Akt1 Ser(473 accumulates in mitochondria in detriment to nuclear translocation; accordingly, Akt1 T308A is retained in mitochondria. Low Akt1 activity increases cytochrome c release to cytosol leading to apoptosis. As assessed by mass spectra, differential H(2O(2 effects on Akt1-PDK interaction depend on the selective oxidation of Cys(310 to sulfenic or cysteic acids. These results indicate that Akt1 intramitochondrial-cycling is central for redox modulation of cell fate.

  15. Alterations in integrin expression modulates invasion of pancreatic cancer cells.

    LENUS (Irish Health Repository)

    Walsh, Naomi


    BACKGROUND: Factors mediating the invasion of pancreatic cancer cells through the extracellular matrix (ECM) are not fully understood. METHODS: In this study, sub-populations of the human pancreatic cancer cell line, MiaPaCa-2 were established which displayed differences in invasion, adhesion, anoikis, anchorage-independent growth and integrin expression. RESULTS: Clone #3 displayed higher invasion with less adhesion, while Clone #8 was less invasive with increased adhesion to ECM proteins compared to MiaPaCa-2. Clone #8 was more sensitive to anoikis than Clone #3 and MiaPaCa-2, and displayed low colony-forming efficiency in an anchorage-independent growth assay. Integrins beta 1, alpha 5 and alpha 6 were over-expressed in Clone #8. Using small interfering RNA (siRNA), integrin beta1 knockdown in Clone #8 cells increased invasion through matrigel and fibronectin, increased motility, decreased adhesion and anoikis. Integrin alpha 5 and alpha 6 knockdown also resulted in increased motility, invasion through matrigel and decreased adhesion. CONCLUSION: Our results suggest that altered expression of integrins interacting with different extracellular matrixes may play a significant role in suppressing the aggressive invasive phenotype. Analysis of these clonal populations of MiaPaCa-2 provides a model for investigations into the invasive properties of pancreatic carcinoma.

  16. Clinical aspects of urea cycle dysfunction and altered brain energy metabolism on modulation of glutamate receptors and transporters in acute and chronic hyperammonemia. (United States)

    Natesan, Vijayakumar; Mani, Renuka; Arumugam, Ramakrishnan


    In living organisms, nitrogen arise primarily as ammonia (NH3) and ammonium (NH4(+)), which is a main component of the nucleic acid pool and proteins. Although nitrogen is essential for growth and maintenance in animals, but when the nitrogenous compounds exceeds the normal range which can quickly lead to toxicity and death. Urea cycle is the common pathway for the disposal of excess nitrogen through urea biosynthesis. Hyperammonemia is a consistent finding in many neurological disorders including congenital urea cycle disorders, reye's syndrome and acute liver failure leads to deleterious effects. Hyperammonemia and liver failure results in glutamatergic neurotransmission which contributes to the alteration in the function of the glutamate-nitric oxide-cGMP pathway, modulates the important cerebral process. Even though ammonia is essential for normal functioning of the central nervous system (CNS), in particular high concentrations of ammonia exposure to the brain leads to the alterations of glutamate transport by the transporters. Several glutamate transporters have been recognized in the central nervous system and each has a unique physiological property and distribution. The loss of glutamate transporter activity in brain during acute liver failure and hyperammonemia is allied with increased extracellular brain glutamate concentrations which may be conscientious for the cerebral edema and ultimately cell death.

  17. Withaferin a alters intermediate filament organization, cell shape and behavior.

    Directory of Open Access Journals (Sweden)

    Boris Grin

    Full Text Available Withaferin A (WFA is a steroidal lactone present in Withania somnifera which has been shown in vitro to bind to the intermediate filament protein, vimentin. Based upon its affinity for vimentin, it has been proposed that WFA can be used as an anti-tumor agent to target metastatic cells which up-regulate vimentin expression. We show that WFA treatment of human fibroblasts rapidly reorganizes vimentin intermediate filaments (VIF into a perinuclear aggregate. This reorganization is dose dependent and is accompanied by a change in cell shape, decreased motility and an increase in vimentin phosphorylation at serine-38. Furthermore, vimentin lacking cysteine-328, the proposed WFA binding site, remains sensitive to WFA demonstrating that this site is not required for its cellular effects. Using analytical ultracentrifugation, viscometry, electron microscopy and sedimentation assays we show that WFA has no effect on VIF assembly in vitro. Furthermore, WFA is not specific for vimentin as it disrupts the cellular organization and induces perinuclear aggregates of several other IF networks comprised of peripherin, neurofilament-triplet protein, and keratin. In cells co-expressing keratin IF and VIF, the former are significantly less sensitive to WFA with respect to inducing perinuclear aggregates. The organization of microtubules and actin/microfilaments is also affected by WFA. Microtubules become wavier and sparser and the number of stress fibers appears to increase. Following 24 hrs of exposure to doses of WFA that alter VIF organization and motility, cells undergo apoptosis. Lower doses of the drug do not kill cells but cause them to senesce. In light of our findings that WFA affects multiple IF systems, which are expressed in many tissues of the body, caution is warranted in its use as an anti-cancer agent, since it may have debilitating organism-wide effects.

  18. Phase resetting reveals network dynamics underlying a bacterial cell cycle.

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    Yihan Lin

    Full Text Available Genomic and proteomic methods yield networks of biological regulatory interactions but do not provide direct insight into how those interactions are organized into functional modules, or how information flows from one module to another. In this work we introduce an approach that provides this complementary information and apply it to the bacterium Caulobacter crescentus, a paradigm for cell-cycle control. Operationally, we use an inducible promoter to express the essential transcriptional regulatory gene ctrA in a periodic, pulsed fashion. This chemical perturbation causes the population of cells to divide synchronously, and we use the resulting advance or delay of the division times of single cells to construct a phase resetting curve. We find that delay is strongly favored over advance. This finding is surprising since it does not follow from the temporal expression profile of CtrA and, in turn, simulations of existing network models. We propose a phenomenological model that suggests that the cell-cycle network comprises two distinct functional modules that oscillate autonomously and couple in a highly asymmetric fashion. These features collectively provide a new mechanism for tight temporal control of the cell cycle in C. crescentus. We discuss how the procedure can serve as the basis for a general approach for probing network dynamics, which we term chemical perturbation spectroscopy (CPS.

  19. Autophagy and the Cell Cycle: A Complex Landscape (United States)

    Mathiassen, Søs Grønbæk; De Zio, Daniela; Cecconi, Francesco


    Autophagy is a self-degradation pathway, in which cytoplasmic material is sequestered in double-membrane vesicles and delivered to the lysosome for degradation. Under basal conditions, autophagy plays a homeostatic function. However, in response to various stresses, the pathway can be further induced to mediate cytoprotection. Defective autophagy has been linked to a number of human pathologies, including neoplastic transformation, even though autophagy can also sustain the growth of tumor cells in certain contexts. In recent years, a considerable correlation has emerged between autophagy induction and stress-related cell-cycle responses, as well as unexpected roles for autophagy factors and selective autophagic degradation in the process of cell division. These advances have obvious implications for our understanding of the intricate relationship between autophagy and cancer. In this review, we will discuss our current knowledge of the reciprocal regulation connecting the autophagy pathway and cell-cycle progression. Furthermore, key findings involving nonautophagic functions for autophagy-related factors in cell-cycle regulation will be addressed.

  20. Regulation of DNA synthesis and the cell cycle in human prostate cancer cells and lymphocytes by ovine uterine serpin

    Directory of Open Access Journals (Sweden)

    Hansen Peter J


    Full Text Available Abstract Background Uterine serpins are members of the serine proteinase inhibitor superfamily. Like some other serpins, these proteins do not appear to be functional proteinase inhibitors. The most studied member of the group, ovine uterine serpin (OvUS, inhibits proliferation of several cell types including activated lymphocytes, bovine preimplantation embryos, and cell lines for lymphoma, canine primary osteosarcoma and human prostate cancer (PC-3 cells. The goal for the present study was to evaluate the mechanism by which OvUS inhibits cell proliferation. In particular, it was tested whether inhibition of DNA synthesis in PC-3 cells involves cytotoxic actions of OvUS or the induction of apoptosis. The effect of OvUS in the production of the autocrine and angiogenic cytokine interleukin (IL-8 by PC-3 cells was also determined. Finally, it was tested whether OvUS blocks specific steps in the cell cycle using both PC-3 cells and lymphocytes. Results Recombinant OvUS blocked proliferation of PC-3 cells at concentrations as low as 8 μg/ml as determined by measurements of [3H]thymidine incorporation or ATP content per well. Treatment of PC-3 cells with OvUS did not cause cytotoxicity or apoptosis or alter interleukin-8 secretion into medium. Results from flow cytometry experiments showed that OvUS blocked the entry of PC-3 cells into S phase and the exit from G2/M phase. In addition, OvUS blocked entry of lymphocytes into S phase following activation of proliferation with phytohemagglutinin. Conclusion Results indicate that OvUS acts to block cell proliferation through disruption of the cell cycle dynamics rather than induction of cytotoxicity or apoptosis. The finding that OvUS can regulate cell proliferation makes this one of only a few serpins that function to inhibit cell growth.

  1. Cell-cycle analyses using thymidine analogues in fission yeast.

    Directory of Open Access Journals (Sweden)

    Silje Anda

    Full Text Available Thymidine analogues are powerful tools when studying DNA synthesis including DNA replication, repair and recombination. However, these analogues have been reported to have severe effects on cell-cycle progression and growth, the very processes being investigated in most of these studies. Here, we have analyzed the effects of 5-ethynyl-2'-deoxyuridine (EdU and 5-Chloro-2'-deoxyuridine (CldU using fission yeast cells and optimized the labelling procedure. We find that both analogues affect the cell cycle, but that the effects can be mitigated by using the appropriate analogue, short pulses of labelling and low concentrations. In addition, we report sequential labelling of two consecutive S phases using EdU and 5-bromo-2'-deoxyuridine (BrdU. Furthermore, we show that detection of replicative DNA synthesis is much more sensitive than DNA-measurements by flow cytometry.

  2. Cell-cycle analyses using thymidine analogues in fission yeast. (United States)

    Anda, Silje; Boye, Erik; Grallert, Beata


    Thymidine analogues are powerful tools when studying DNA synthesis including DNA replication, repair and recombination. However, these analogues have been reported to have severe effects on cell-cycle progression and growth, the very processes being investigated in most of these studies. Here, we have analyzed the effects of 5-ethynyl-2'-deoxyuridine (EdU) and 5-Chloro-2'-deoxyuridine (CldU) using fission yeast cells and optimized the labelling procedure. We find that both analogues affect the cell cycle, but that the effects can be mitigated by using the appropriate analogue, short pulses of labelling and low concentrations. In addition, we report sequential labelling of two consecutive S phases using EdU and 5-bromo-2'-deoxyuridine (BrdU). Furthermore, we show that detection of replicative DNA synthesis is much more sensitive than DNA-measurements by flow cytometry.

  3. Reduced expression of DNA repair and redox signaling protein APE1/Ref-1 impairs human pancreatic cancer cell survival, proliferation, and cell cycle progression. (United States)

    Jiang, Yanlin; Zhou, Shaoyu; Sandusky, George E; Kelley, Mark R; Fishel, Melissa L


    Pancreatic cancer is a deadly disease that is virtually never cured. Understanding the chemoresistance intrinsic to this cancer will aid in developing new regimens. High expression of APE1/Ref-1, a DNA repair and redox signaling protein, is associated with resistance, poor outcome, and angiogenesis; little is known in pancreatic cancer. Immunostaining of adenocarcinoma shows greater APE1/Ref-1 expression than in normal pancreas tissue. A decrease in APE1/Ref-1 protein levels results in pancreatic cancer cell growth inhibition, increased apoptosis, and altered cell cycle progression. Endogenous cell cycle inhibitors increase when APE1/ Ref-1 is reduced, demonstrating its importance to proliferation and growth of pancreatic cancer.

  4. NSA2, a novel nucleolus protein regulates cell proliferation and cell cycle

    Energy Technology Data Exchange (ETDEWEB)

    Zhang, Heyu [Department of Immunology, School of Basic Medical Sciences, Peking University, No. 38 Xueyuan Road, Beijing 100191 (China); Human Disease Genomics Center, Peking University, No. 38 Xueyuan Road, Beijing 100191 (China); Ma, Xi [Department of Immunology, School of Basic Medical Sciences, Peking University, No. 38 Xueyuan Road, Beijing 100191 (China); Human Disease Genomics Center, Peking University, No. 38 Xueyuan Road, Beijing 100191 (China); State Key Lab of Animal Nutrition, China Agricultural University, No. 2 Yuanmingyuan West Road, Beijing 100193 (China); Shi, Taiping [Chinese National Human Genome Center, Beijing. 3-707 North YongChang Road BDA, Beijing 100176 (China); Song, Quansheng [Department of Immunology, School of Basic Medical Sciences, Peking University, No. 38 Xueyuan Road, Beijing 100191 (China); Human Disease Genomics Center, Peking University, No. 38 Xueyuan Road, Beijing 100191 (China); Zhao, Hongshan, E-mail: [Department of Immunology, School of Basic Medical Sciences, Peking University, No. 38 Xueyuan Road, Beijing 100191 (China); Human Disease Genomics Center, Peking University, No. 38 Xueyuan Road, Beijing 100191 (China); Ma, Dalong [Department of Immunology, School of Basic Medical Sciences, Peking University, No. 38 Xueyuan Road, Beijing 100191 (China); Human Disease Genomics Center, Peking University, No. 38 Xueyuan Road, Beijing 100191 (China)


    NSA2 (Nop seven-associated 2) was previously identified in a high throughput screen of novel human genes associated with cell proliferation, and the NSA2 protein is evolutionarily conserved across different species. In this study, we revealed that NSA2 is broadly expressed in human tissues and cultured cell lines, and located in the nucleolus of the cell. Both of the putative nuclear localization signals (NLSs) of NSA2, also overlapped with nucleolar localization signals (NoLSs), are capable of directing nucleolar accumulation. Moreover, over-expression of the NSA2 protein promoted cell growth in different cell lines and regulated the G1/S transition in the cell cycle. SiRNA silencing of the NSA2 transcript attenuated the cell growth and dramatically blocked the cell cycle in G1/S transition. Our results demonstrated that NSA2 is a nucleolar protein involved in cell proliferation and cell cycle regulation.

  5. Andrographolide inhibits prostate cancer by targeting cell cycle regulators, CXCR3 and CXCR7 chemokine receptors. (United States)

    Mir, Hina; Kapur, Neeraj; Singh, Rajesh; Sonpavde, Guru; Lillard, James W; Singh, Shailesh


    Despite state of the art cancer diagnostics and therapies offered in clinic, prostate cancer (PCa) remains the second leading cause of cancer-related deaths. Hence, more robust therapeutic/preventive regimes are required to combat this lethal disease. In the current study, we have tested the efficacy of Andrographolide (AG), a bioactive diterpenoid isolated from Andrographis paniculata, against PCa. This natural agent selectively affects PCa cell viability in a dose and time-dependent manner, without affecting primary prostate epithelial cells. Furthermore, AG showed differential effect on cell cycle phases in LNCaP, C4-2b and PC3 cells compared to retinoblastoma protein (RB(-/-)) and CDKN2A lacking DU-145 cells. G2/M transition was blocked in LNCaP, C4-2b and PC3 after AG treatment whereas DU-145 cells failed to transit G1/S phase. This difference was primarily due to differential activation of cell cycle regulators in these cell lines. Levels of cyclin A2 after AG treatment increased in all PCa cells line. Cyclin B1 levels increased in LNCaP and PC3, decreased in C4-2b and showed no difference in DU-145 cells after AG treatment. AG decreased cyclin E2 levels only in PC3 and DU-145 cells. It also altered Rb, H3, Wee1 and CDC2 phosphorylation in PCa cells. Intriguingly, AG reduced cell viability and the ability of PCa cells to migrate via modulating CXCL11 and CXCR3 and CXCR7 expression. The significant impact of AG on cellular and molecular processes involved in PCa progression suggests its potential use as a therapeutic and/or preventive agent for PCa.

  6. Relation Between the Cell Volume and the Cell Cycle Dynamics in Mammalian cell (United States)

    Magno, A. C. G.; Oliveira, I. L.; Hauck, J. V. S.


    The main goal of this work is to add and analyze an equation that represents the volume in a dynamical model of the mammalian cell cycle proposed by Gérard and Goldbeter (2011) [1]. The cell division occurs when the cyclinB/Cdkl complex is totally degraded (Tyson and Novak, 2011)[2] and it reaches a minimum value. At this point, the cell is divided into two newborn daughter cells and each one will contain the half of the cytoplasmic content of the mother cell. The equations of our base model are only valid if the cell volume, where the reactions occur, is constant. Whether the cell volume is not constant, that is, the rate of change of its volume with respect to time is explicitly taken into account in the mathematical model, then the equations of the original model are no longer valid. Therefore, every equations were modified from the mass conservation principle for considering a volume that changes with time. Through this approach, the cell volume affects all model variables. Two different dynamic simulation methods were accomplished: deterministic and stochastic. In the stochastic simulation, the volume affects every model's parameters which have molar unit, whereas in the deterministic one, it is incorporated into the differential equations. In deterministic simulation, the biochemical species may be in concentration units, while in stochastic simulation such species must be converted to number of molecules which are directly proportional to the cell volume. In an effort to understand the influence of the new equation a stability analysis was performed. This elucidates how the growth factor impacts the stability of the model's limit cycles. In conclusion, a more precise model, in comparison to the base model, was created for the cell cycle as it now takes into consideration the cell volume variation

  7. The Hog1 MAP Kinase Promotes the Recovery from Cell Cycle Arrest Induced by Hydrogen Peroxide in Candida albicans. (United States)

    Correia, Inês; Alonso-Monge, Rebeca; Pla, Jesús


    Eukaryotic cell cycle progression in response to environmental conditions is controlled via specific checkpoints. Signal transduction pathways mediated by MAPKs play a crucial role in sensing stress. For example, the canonical MAPKs Mkc1 (of the cell wall integrity pathway), and Hog1 (of the HOG pathway), are activated upon oxidative stress. In this work, we have analyzed the effect of oxidative stress induced by hydrogen peroxide on cell cycle progression in Candida albicans. Hydrogen peroxide was shown to induce a transient arrest at the G1 phase of the cell cycle. Specifically, a G1 arrest was observed, although phosphorylation of Mkc1 and Hog1 MAPKs can take place at all stages of the cell cycle. Interestingly, hog1 (but not mkc1) mutants required a longer time compared to wild type cells to resume growth after hydrogen peroxide challenge. Using GFP-labeled cells and mixed cultures of wild type and hog1 cells we were able to show that hog1 mutants progress faster through the cell cycle under standard growth conditions in the absence of stress (YPD at 37°C). Consequently, hog1 mutants exhibited a smaller cell size. The altered cell cycle progression correlates with altered expression of the G1 cyclins Cln3 and Pcl2 in hog1 cells compared to the wild type strain. In addition, Hgc1 (a hypha-specific G1 cyclin) as well as Cln3 displayed a different kinetics of expression in the presence of hydrogen peroxide in hog1 mutants. Collectively, these results indicate that Hog1 regulates the expression of G1 cyclins not only in response to oxidative stress, but also under standard growth conditions. Hydrogen peroxide treated cells did not show fluctuations in the mRNA levels for SOL1, which are observed in untreated cells during cell cycle progression. In addition, treatment with hydrogen peroxide prevented degradation of Sol1, an effect which was enhanced in hog1 mutants. Therefore, in C. albicans, the MAPK Hog1 mediates cell cycle progression in response to oxidative

  8. Cell cycle-arrested tumor cells exhibit increased sensitivity towards TRAIL-induced apoptosis


    Ehrhardt, H.; Wachter, F; Grunert, M.; Jeremias, I


    Resting tumor cells represent a huge challenge during anticancer therapy due to their increased treatment resistance. TNF-related apoptosis-inducing ligand (TRAIL) is a putative future anticancer drug, currently in phases I and II clinical studies. We recently showed that TRAIL is able to target leukemia stem cell surrogates. Here, we tested the ability of TRAIL to target cell cycle-arrested tumor cells. Cell cycle arrest was induced in tumor cell lines and xenografted tumor cells in G0, G1 o...

  9. Shielding of the Geomagnetic Field Alters Actin Assembly and Inhibits Cell Motility in Human Neuroblastoma Cells


    Wei-Chuan Mo; Zi-Jian Zhang; Dong-Liang Wang; Ying Liu; Bartlett, Perry F.; Rong-Qiao He


    Accumulating evidence has shown that absence of the geomagnetic field (GMF), the so-called hypomagnetic field (HMF) environment, alters the biological functions in seemingly non-magnetosensitive cells and organisms, which indicates that the GMF could be sensed by non-iron-rich and non-photo-sensing cells. The underlying mechanisms of the HMF effects on those cells are closely related to their GMF sensation but remain poorly understood so far. Previously, we found that the HMF represses expres...

  10. Weight cycling promotes fat gain and altered clock gene expression in adipose tissue in C57BL/6J mice. (United States)

    Dankel, S N; Degerud, E M; Borkowski, K; Fjære, E; Midtbø, L K; Haugen, C; Solsvik, M H; Lavigne, A M; Liaset, B; Sagen, J V; Kristiansen, K; Mellgren, G; Madsen, L


    Repeated attempts to lose weight by temporary dieting may result in weight cycling, eventually further gain of body fat, and possible metabolic adaptation. We tested this with a controlled experiment in C57BL/6J mice subjected to four weight cycles (WC), continuous hypercaloric feeding (HF), or low-fat feeding (LF). To search for genes involved in an adaptive mechanism to former weight cycling and avoid acute effects of the last cycle, the last hypercaloric feeding period was prolonged by an additional 2 wk before euthanization. Total energy intake was identical in WC and HF. However, compared with HF, the WC mice gained significantly more total body mass and fat mass and showed increased levels of circulating leptin and lipids in liver. Both the HF and WC groups showed increased adipocyte size and insulin resistance. Despite these effects, we also observed an interesting maintenance of circulating adiponectin and free fatty acid levels after WC, whereas changes in these parameters were observed in HF mice. Global gene expression was analyzed by microarrays. Weight-cycled mice were characterized by a downregulation of several clock genes (Dbp, Tef, Per1, Per2, Per3, and Nr1d2) in adipose tissues, which was confirmed by quantitative PCR. In 3T3-L1 cells, we found reduced expression of Dbp and Tef early in adipogenic differentiation, which was mediated via cAMP-dependent signaling. Our data suggest that clock genes in adipose tissue may play a role in metabolic adaptation to weight cycling.

  11. Modeling circadian clock-cell cycle interaction effects on cell population growth rates. (United States)

    El Cheikh, R; Bernard, S; El Khatib, N


    The circadian clock and the cell cycle are two tightly coupled oscillators. Recent analytical studies have shown counter-intuitive effects of circadian gating of the cell cycle on growth rates of proliferating cells which cannot be explained by a molecular model or a population model alone. In this work, we present a combined molecular-population model that studies how coupling the circadian clock to the cell cycle, through the protein WEE1, affects a proliferating cell population. We show that the cell cycle can entrain to the circadian clock with different rational period ratios and characterize multiple domains of entrainment. We show that coupling increases the growth rate for autonomous periods of the cell cycle around 24 h and above 48 h. We study the effect of mutation of circadian genes on the growth rate of cells and show that disruption of the circadian clock can lead to abnormal proliferation. Particularly, we show that Cry 1, Cry 2 mutations decrease the growth rate of cells, Per 2 mutation enhances it and Bmal 1 knockout increases it for autonomous periods of the cell cycle less than 21 h and decreases it elsewhere. Combining a molecular model to a population model offers new insight on the influence of the circadian clock on the growth of a cell population. This can help chronotherapy which takes benefits of physiological rhythms to improve anti-cancer efficacy and tolerance to drugs by administering treatments at a specific time of the day.

  12. Propionate Increases Hepatic Pyruvate Cycling and Anaplerosis and Alters Mitochondrial Metabolism

    DEFF Research Database (Denmark)

    Perry, Rachel J; Borders, Candace B; Cline, Gary W;


    In mammals, pyruvate kinase (PK) plays a key role in regulating the balance between glycolysis and gluconeogenesis; however, in vivo regulation of PK flux by gluconeogenic hormones and substrates is poorly understood. To this end, we developed a novel NMR-liquid chromatography....../tandem-mass spectrometry (LC-MS/MS) method to directly assess pyruvate cycling relative to mitochondrial pyruvate metabolism (VPyr-Cyc/VMito) in vivo using [3-(13)C]lactate as a tracer. Using this approach, VPyr-Cyc/VMito was only 6% in overnight fasted rats. In contrast, when propionate was infused simultaneously...... glucagon suppressed VPyr-Cyc/VMito These data show that under fasting conditions, when hepatic gluconeogenesis is stimulated, pyruvate recycling is relatively low in liver compared with VMito flux and that liver metabolism, in particular pyruvate cycling, is sensitive to propionate making it an unsuitable...

  13. Alterations in dopamine system function across the estrous cycle of the MAM rodent model of schizophrenia. (United States)

    Perez, Stephanie M; Chen, Li; Lodge, Daniel J


    Clinical studies have reported differences in the incidence and severity of schizophrenia symptoms between male and female schizophrenia patients. Unfortunately, the cause of these differences is not currently known due, in part, to the fact that preclinical studies largely focus on male subjects. Dopamine neuron activity has been previously demonstrated to change across the estrous cycle, and may therefore be of relevance, as aberrant dopamine signaling is thought to underlie the positive symptoms of schizophrenia. Here we examine dopamine neuron activity across the estrous cycle in the MAM rodent model of schizophrenia. We demonstrate that the elevation in dopamine neuron activity, consistently observed in male MAM-treated rats, is most prominent during estrus and attenuated in met-estrus. Furthermore, this appears to be mediated, in part, by progesterone in the ventral hippocampus, as increases in dopamine neuron population activity (observed in estrus) were normalized by the intra-hippocampal administration of the progesterone receptor antagonist, mifepristone (but not the estrogen receptor antagonists, fulvestrant). Taken together, these data suggest that changes in dopamine system function occur across the estrous cycle in MAM-treated rats and may contribute to the differences in symptomatology between male and female schizophrenia patients.

  14. Molecular ties between the cell cycle and differentiation in embryonic stem cells. (United States)

    Li, Victor C; Kirschner, Marc W


    Attainment of the differentiated state during the final stages of somatic cell differentiation is closely tied to cell cycle progression. Much less is known about the role of the cell cycle at very early stages of embryonic development. Here, we show that molecular pathways involving the cell cycle can be engineered to strongly affect embryonic stem cell differentiation at early stages in vitro. Strategies based on perturbing these pathways can shorten the rate and simplify the lineage path of ES differentiation. These results make it likely that pathways involving cell proliferation intersect at various points with pathways that regulate cell lineages in embryos and demonstrate that this knowledge can be used profitably to guide the path and effectiveness of cell differentiation of pluripotent cells.

  15. Boolean network model predicts cell cycle sequence of fission yeast.

    Directory of Open Access Journals (Sweden)

    Maria I Davidich

    Full Text Available A Boolean network model of the cell-cycle regulatory network of fission yeast (Schizosaccharomyces Pombe is constructed solely on the basis of the known biochemical interaction topology. Simulating the model in the computer faithfully reproduces the known activity sequence of regulatory proteins along the cell cycle of the living cell. Contrary to existing differential equation models, no parameters enter the model except the structure of the regulatory circuitry. The dynamical properties of the model indicate that the biological dynamical sequence is robustly implemented in the regulatory network, with the biological stationary state G1 corresponding to the dominant attractor in state space, and with the biological regulatory sequence being a strongly attractive trajectory. Comparing the fission yeast cell-cycle model to a similar model of the corresponding network in S. cerevisiae, a remarkable difference in circuitry, as well as dynamics is observed. While the latter operates in a strongly damped mode, driven by external excitation, the S. pombe network represents an auto-excited system with external damping.

  16. Immunological control of cell cycle aberrations for the avoidance of oncogenesis: the case of tetraploidy. (United States)

    Senovilla, Laura; Galluzzi, Lorenzo; Castedo, Maria; Kroemer, Guido


    Tetraploid cells--cells that contain twice the normal amount of DNA--are more prone to neoplastic transformation than their normal, diploid counterparts since they are genomically unstable and frequently undergo asymmetric, multipolar cell divisions. Similar to many other genomic aberrations, tetraploidization is normally avoided by multiple, nonredundant cell-intrinsic mechanisms that are tied to cell cycle checkpoints. Unexpectedly, tetraploidization is also under the control of a cell-extrinsic mechanism determined by the immune system. Indeed, oncogene- or carcinogen-induced cancers developing in immunodeficient mice contain cells with a higher DNA content than similar tumors growing in immunocompetent hosts. Moreover, cancer cell lines that have been rendered tetraploid in vitro grow normally in immunodeficient mice, yet almost fail to generate tumors in immunocompetent animals. One of the mechanisms whereby the immune system recognizes tetraploid cells originates from tetraploidy causing an endoplasmic reticulum (ER) stress response that culminates in the exposure of the ER protein calreticulin on the cell surface. Hence, tetraploidy exemplifies a potentially oncogenic alteration that is repressed by a combination of cell-autonomous mechanisms and immunosurveillance. Oncogenesis and tumor progression require the simultaneous failure of both such control systems.

  17. STK31 is a cell-cycle regulated protein that contributes to the tumorigenicity of epithelial cancer cells.

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    Pao-Lin Kuo

    Full Text Available Serine/threonine kinase 31 (STK31 is one of the novel cancer/testis antigens for which its biological functions remain largely unclear. Here, we demonstrate that STK31 is overexpressed in many human colorectal cancer cell lines and tissues. STK31 co-localizes with pericentrin in the centrosomal region throughout all phases of the cell cycle. Interestingly, when cells undergo mitosis, STK31 also localizes to the centromeres, central spindle, and midbody. This localization behavior is similar to that of chromosomal passenger proteins, which are known to be the important players of the spindle assembly checkpoint. The expression of STK31 is cell cycle-dependent through the regulation of a putative D-box near its C-terminal region. Ectopically-expressed STK31-GFP increases cell migration and invasive ability without altering the proliferation rate of cancer cells, whereas the knockdown expression of endogenous STK31 by lentivirus-derived shRNA results in microtubule assembly defects that prolong the duration of mitosis and lead to apoptosis. Taken together, our results suggest that the aberrant expression of STK31 contributes to tumorigenicity in somatic cancer cells. STK31 might therefore act as a potential therapeutic target in human somatic cancers.

  18. Tangeretin induces cell cycle arrest and apoptosis through upregulation of PTEN expression in glioma cells. (United States)

    Ma, Li-Li; Wang, Da-Wei; Yu, Xu-Dong; Zhou, Yan-Ling


    Tangeretin (TANG), present in peel of citrus fruits, has been shown to various medicinal properties such as chemopreventive and neuroprotective. However, the chemopreventive effect of TANG on glioblastoma cells has not been examined. The present study was designed to explore the anticancer potential of TANG in glioblastoma cells and to investigate the related mechanism. Human glioblastoma U-87MG and LN-18 cells were treated with 45μM concentration of TANG and cell growth was measured by MTT assay. The cell cycle distribution and cell death were measured by flow cytometry. The expression of cell cycle and apoptosis related genes were analyzed by quantitative RT-PCR and western blot. The cells treated with TANG were significantly increased cell growth suppression and cell death effects than vehicle treated cells. Further, TANG treatment increases G2/M arrest and apoptosis by modulating PTEN and cell-cycle regulated genes such as cyclin-D and cdc-2 mRNA and protein expressions. Moreover, the ability of TANG to decrease cell growth and to induce cell death was compromised when PTEN was knockdown by siRNA. Taken together, the chemopreventive effect of TANG is associated with regulation of cell-cycle and apoptosis in glioblastoma, thereby attenuating glioblastoma cell growth. Hence, the present findings suggest that TANG may be a therapeutic agent for glioblastoma treatment.

  19. Thermodynamic Analysis of an Integrated Solid Oxide Fuel Cell Cycle with a Rankine Cycle

    DEFF Research Database (Denmark)

    Rokni, Masoud


    Hybrid systems consisting of Solid Oxide Fuel Cells (SOFC) on the top of a Steam Turbine (ST) are investigated. The plants are fired by natural gas (NG). A desulfurization reactor removes the sulfur content in the fuel while a pre-reformer breaks down the heavier hydrocarbons. The pre-treated fuel...... enters then into the anode side of the SOFC. The remaining fuels after the SOFC stacks enter a burner for further burning. The off-gases are then used to produce steam for a Rankine cycle in a Heat Recovery Steam Generator (HRSG). Different system setups are suggested. Cyclic efficiencies up to 67......% are achieved which is considerably higher than the conventional Combined Cycles (CC). Both ASR (Adiabatic Steam Reformer) and CPO (Catalytic Partial Oxidation) fuel pre-reformer reactors are considered in this investigation....

  20. Differential expression and alternative splicing of cell cycle genes in imatinib-treated K562 cells. (United States)

    Liu, Jing; Lin, Jin; Huang, Lin-Feng; Huang, Bo; Xu, Yan-Mei; Li, Jing; Wang, Yan; Zhang, Jing; Yang, Wei-Ming; Min, Qing-Hua; Wang, Xiao-Zhong


    Cancer progression often involves the disorder of the cell cycle, and a number of effective chemotherapeutic drugs have been shown to induce cell cycle arrest. The purpose of this study was to comprehensively investigate the effects of imatinib on the expression profile of cell cycle genes in the chronic myeloid leukemia (CML) K562 cell line. In addition, we also investigated alternative splicing of the cell cycle genes affected by imatinib, since an important relationship has been shown to exist between RNA splicing and cell cycle progression. Exon array analysis was performed using total RNA purified from normal and imatinib-treated K562 cells. We identified 185 differentially expressed genes and 277 alternative splicing events between the two cell groups. A detailed analysis by reverse transcription-PCR (RT-PCR) of key genes confirmed the experimental results of the exon array. These results suggested that treatment of K562 cells with imatinib shifts the expression and alternative splicing profiles of several cell cycle-related genes. Importantly, these findings may help improve imatinib treatment strategies in patients with CML and may be useful for imatinib resistance research and CML drug development.

  1. Lineage-specific interface proteins match up the cell cycle and differentiation in embryo stem cells

    DEFF Research Database (Denmark)

    Re, Angela; Workman, Christopher; Waldron, Levi;


    The shortage of molecular information on cell cycle changes along embryonic stem cell (ESC) differentiation prompts an in silico approach, which may provide a novel way to identify candidate genes or mechanisms acting in coordinating the two programs. We analyzed germ layer specific gene expression...... changes during the cell cycle and ESC differentiation by combining four human cell cycle transcriptome profiles with thirteen in vitro human ESC differentiation studies. To detect cross-talk mechanisms we then integrated the transcriptome data that displayed differential regulation with protein...... interaction data. A new class of non-transcriptionally regulated genes was identified, encoding proteins which interact systematically with proteins corresponding to genes regulated during the cell cycle or cell differentiation, and which therefore can be seen as interface proteins coordinating the two...

  2. Host cell poly(ADP-ribose glycohydrolase is crucial for Trypanosoma cruzi infection cycle.

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    Salomé C Vilchez Larrea

    Full Text Available Trypanosoma cruzi, etiological agent of Chagas' disease, has a complex life cycle which involves the invasion of mammalian host cells, differentiation and intracellular replication. Here we report the first insights into the biological role of a poly(ADP-ribose glycohydrolase in a trypanosomatid (TcPARG. In silico analysis of the TcPARG gene pointed out the conservation of key residues involved in the catalytic process and, by Western blot, we demonstrated that it is expressed in a life stage-dependant manner. Indirect immunofluorescence assays and electron microscopy using an anti-TcPARG antibody showed that this enzyme is localized in the nucleus independently of the presence of DNA damage or cell cycle stage. The addition of poly(ADP-ribose glycohydrolase inhibitors ADP-HPD (adenosine diphosphate (hydroxymethyl pyrrolidinediol or DEA (6,9-diamino-2-ethoxyacridine lactate monohydrate to the culture media, both at a 1 µM concentration, reduced in vitro epimastigote growth by 35% and 37% respectively, when compared to control cultures. We also showed that ADP-HPD 1 µM can lead to an alteration in the progression of the cell cycle in hydroxyurea synchronized cultures of T. cruzi epimastigotes. Outstandingly, here we demonstrate that the lack of poly(ADP-ribose glycohydrolase activity in Vero and A549 host cells, achieved by chemical inhibition or iRNA, produces the reduction of the percentage of infected cells as well as the number of amastigotes per cell and trypomastigotes released, leading to a nearly complete abrogation of the infection process. We conclude that both, T. cruzi and the host, poly(ADP-ribose glycohydrolase activities are important players in the life cycle of Trypanosoma cruzi, emerging as a promising therapeutic target for the treatment of Chagas' disease.

  3. DNA repair by nonhomologous end joining and homologous recombination during cell cycle in human cells (United States)

    Mao, Zhiyong; Bozzella, Michael; Seluanov, Andrei; Gorbunova, Vera


    DNA double-strand breaks (DSBs) are dangerous lesions that can lead to potentially oncogenic genomic rearrangements or cell death. The two major pathways for repair of DSBs are nonhomologous end joining (NHEJ) and homologous recombination (HR). NHEJ is an intrinsically error-prone pathway while HR results in accurate repair. To understand the origin of genomic instability in human cells it is important to know the contribution of each DSB repair pathway. Studies of rodent cells and human cancer cell lines have shown that the choice between NHEJ or HR pathways depends on cell cycle stage. Surprisingly, cell cycle regulation of DSB repair has not been examined in normal human cells with intact cell cycle checkpoints. Here we measured the efficiency of NHEJ and HR at different cell cycle stages in hTERT-immortalized diploid human fibroblasts. We utilized cells with chromosomally-integrated fluorescent reporter cassettes, in which a unique DSB is introduced by a rare-cutting endonuclease. We show that NHEJ is active throughout the cell cycle, and its activity increases as cells progress from G1 to G2/M (G1cell cycle stages. We conclude that human somatic cells utilize error-prone NHEJ as the major DSB repair pathway at all cell cycle stages, while HR is used, primarily, in the S phase. PMID:18769152

  4. Systematic identification of yeast cell cycle transcription factors using multiple data sources

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    Li Wen-Hsiung


    Full Text Available Abstract Background Eukaryotic cell cycle is a complex process and is precisely regulated at many levels. Many genes specific to the cell cycle are regulated transcriptionally and are expressed just before they are needed. To understand the cell cycle process, it is important to identify the cell cycle transcription factors (TFs that regulate the expression of cell cycle-regulated genes. Results We developed a method to identify cell cycle TFs in yeast by integrating current ChIP-chip, mutant, transcription factor binding site (TFBS, and cell cycle gene expression data. We identified 17 cell cycle TFs, 12 of which are known cell cycle TFs, while the remaining five (Ash1, Rlm1, Ste12, Stp1, Tec1 are putative novel cell cycle TFs. For each cell cycle TF, we assigned specific cell cycle phases in which the TF functions and identified the time lag for the TF to exert regulatory effects on its target genes. We also identified 178 novel cell cycle-regulated genes, among which 59 have unknown functions, but they may now be annotated as cell cycle-regulated genes. Most of our predictions are supported by previous experimental or computational studies. Furthermore, a high confidence TF-gene regulatory matrix is derived as a byproduct of our method. Each TF-gene regulatory relationship in this matrix is supported by at least three data sources: gene expression, TFBS, and ChIP-chip or/and mutant data. We show that our method performs better than four existing methods for identifying yeast cell cycle TFs. Finally, an application of our method to different cell cycle gene expression datasets suggests that our method is robust. Conclusion Our method is effective for identifying yeast cell cycle TFs and cell cycle-regulated genes. Many of our predictions are validated by the literature. Our study shows that integrating multiple data sources is a powerful approach to studying complex biological systems.

  5. Biochar affects soil organic matter cycling and microbial functions but does not alter microbial community structure in a paddy soil. (United States)

    Tian, Jing; Wang, Jingyuan; Dippold, Michaela; Gao, Yang; Blagodatskaya, Evgenia; Kuzyakov, Yakov


    The application of biochar (BC) in conjunction with mineral fertilizers is one of the most promising management practices recommended to improve soil quality. However, the interactive mechanisms of BC and mineral fertilizer addition affecting microbial communities and functions associated with soil organic matter (SOM) cycling are poorly understood. We investigated the SOM in physical and chemical fractions, microbial community structure (using phospholipid fatty acid analysis, PLFA) and functions (by analyzing enzymes involved in C and N cycling and Biolog) in a 6-year field experiment with BC and NPK amendment. BC application increased total soil C and particulate organic C for 47.4-50.4% and 63.7-74.6%, respectively. The effects of BC on the microbial community and C-cycling enzymes were dependent on fertilization. Addition of BC alone did not change the microbial community compared with the control, but altered the microbial community structure in conjunction with NPK fertilization. SOM fractions accounted for 55% of the variance in the PLFA-related microbial community structure. The particulate organic N explained the largest variation in the microbial community structure. Microbial metabolic activity strongly increased after BC addition, particularly the utilization of amino acids and amines due to an increase in the activity of proteolytic (l-leucine aminopeptidase) enzymes. These results indicate that microorganisms start to mine N from the SOM to compensate for high C:N ratios after BC application, which consequently accelerate cycling of stable N. Concluding, BC in combination with NPK fertilizer application strongly affected microbial community composition and functions, which consequently influenced SOM cycling.

  6. Plasma Ghrelin Concentrations Were Altered with Oestrous Cycle Stage and Increasing Age in Reproductively Competent Wistar Females (United States)

    Saffrey, M. Jill; Taylor, Victoria J.


    Changes in appetite occur during the ovarian cycle in female mammals. Research on appetite-regulatory gastrointestinal peptides in females is limited, because reproductive changes in steroid hormones present additional experimental factors to control for. This study aimed to explore possible changes in the orexigenic (appetite-stimulating) gastrointestinal peptide hormone ghrelin during the rodent oestrous cycle. Fed and fasted plasma and stomach tissue samples were taken from female Wistar rats (32–44 weeks of age) at each stage of the oestrous cycle for total ghrelin quantification using radioimmunoassay. Sampling occurred during the dark phase when most eating takes place in rats. Statistical analysis was by paired-samples t-test, one-way ANOVA on normally distributed data, with Tukey post-hoc tests, or Kruskal-Wallis if not. GLM univariate analysis was used to assess main effects and interactions in ghrelin concentrations in the fed or fasted state and during different stages of the ovarian cycle, with age as a covariate. No consistent fed to fasted ghrelin increases were measured in matched plasma samples from the same animals, contrary to expectations. Total ghrelin concentrations did not significantly change between cycle stages with ANOVA, in either fed or fasted plasma or in stomach tissue. This was despite significantly decreased fasted stomach contents at oestrus (P = 0.028), suggesting decreased food intake. There was however a significant interaction in ghrelin plasma concentrations between fed and fasted proestrus rats and a direct effect of age with rats over 37 weeks old having lower circulating concentrations of ghrelin in both fed and fasted states. The biological implications of altered ghrelin plasma concentrations from 37 weeks of age are as yet unknown, but warrant further investigation. Exploring peripheral ghrelin regulatory factor changes with increasing age in reproductively competent females may bring to light potential effects on

  7. Computational analysis of mammalian cell division gated by a circadian clock: quantized cell cycles and cell size control. (United States)

    Zámborszky, Judit; Hong, Christian I; Csikász Nagy, Attila


    Cell cycle and circadian rhythms are conserved from cyanobacteria to humans with robust cyclic features. Recently, molecular links between these two cyclic processes have been discovered. Core clock transcription factors, Bmal1 and Clock (Clk), directly regulate Wee1 kinase, which inhibits entry into the mitosis. We investigate the effect of this connection on the timing of mammalian cell cycle processes with computational modeling tools. We connect a minimal model of circadian rhythms, which consists of transcription-translation feedback loops, with a modified mammalian cell cycle model from Novak and Tyson (2004). As we vary the mass doubling time (MDT) of the cell cycle, stochastic simulations reveal quantized cell cycles when the activity of Wee1 is influenced by clock components. The quantized cell cycles disappear in the absence of coupling or when the strength of this link is reduced. More intriguingly, our simulations indicate that the circadian clock triggers critical size control in the mammalian cell cycle. A periodic brake on the cell cycle progress via Wee1 enforces size control when the MDT is quite different from the circadian period. No size control is observed in the absence of coupling. The issue of size control in the mammalian system is debatable, whereas it is well established in yeast. It is possible that the size control is more readily observed in cell lines that contain circadian rhythms, since not all cell types have a circadian clock. This would be analogous to an ultradian clock intertwined with quantized cell cycles (and possibly cell size control) in yeast. We present the first coupled model between the mammalian cell cycle and circadian rhythms that reveals quantized cell cycles and cell size control influenced by the clock.

  8. Genetic deletion of Mst1 alters T cell function and protects against autoimmunity.

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    Konstantin V Salojin

    Full Text Available Mammalian sterile 20-like kinase 1 (Mst1 is a MAPK kinase kinase kinase which is involved in a wide range of cellular responses, including apoptosis, lymphocyte adhesion and trafficking. The contribution of Mst1 to Ag-specific immune responses and autoimmunity has not been well defined. In this study, we provide evidence for the essential role of Mst1 in T cell differentiation and autoimmunity, using both genetic and pharmacologic approaches. Absence of Mst1 in mice reduced T cell proliferation and IL-2 production in vitro, blocked cell cycle progression, and elevated activation-induced cell death in Th1 cells. Mst1 deficiency led to a CD4+ T cell development path that was biased toward Th2 and immunoregulatory cytokine production with suppressed Th1 responses. In addition, Mst1-/- B cells showed decreased stimulation to B cell mitogens in vitro and deficient Ag-specific Ig production in vivo. Consistent with altered lymphocyte function, deletion of Mst1 reduced the severity of experimental autoimmune encephalomyelitis (EAE and protected against collagen-induced arthritis development. Mst1-/- CD4+ T cells displayed an intrinsic defect in their ability to respond to encephalitogenic antigens and deletion of Mst1 in the CD4+ T cell compartment was sufficient to alleviate CNS inflammation during EAE. These findings have prompted the discovery of novel compounds that are potent inhibitors of Mst1 and exhibit desirable pharmacokinetic properties. In conclusion, this report implicates Mst1 as a critical regulator of adaptive immune responses, Th1/Th2-dependent cytokine production, and as a potential therapeutic target for immune disorders.

  9. Natural compounds' activity against cancer stem-like or fast-cycling melanoma cells.

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    Malgorzata Sztiller-Sikorska

    Full Text Available BACKGROUND: Accumulating evidence supports the concept that melanoma is highly heterogeneous and sustained by a small subpopulation of melanoma stem-like cells. Those cells are considered as responsible for tumor resistance to therapies. Moreover, melanoma cells are characterized by their high phenotypic plasticity. Consequently, both melanoma stem-like cells and their more differentiated progeny must be eradicated to achieve durable cure. By reevaluating compounds in heterogeneous melanoma populations, it might be possible to select compounds with activity not only against fast-cycling cells but also against cancer stem-like cells. Natural compounds were the focus of the present study. METHODS: We analyzed 120 compounds from The Natural Products Set II to identify compounds active against melanoma populations grown in an anchorage-independent manner and enriched with cells exerting self-renewing capacity. Cell viability, cell cycle arrest, apoptosis, gene expression, clonogenic survival and label-retention were analyzed. FINDINGS: Several compounds efficiently eradicated cells with clonogenic capacity and nanaomycin A, streptonigrin and toyocamycin were effective at 0.1 µM. Other anti-clonogenic but not highly cytotoxic compounds such as bryostatin 1, siomycin A, illudin M, michellamine B and pentoxifylline markedly reduced the frequency of ABCB5 (ATP-binding cassette, sub-family B, member 5-positive cells. On the contrary, treatment with maytansine and colchicine selected for cells expressing this transporter. Maytansine, streptonigrin, toyocamycin and colchicine, even if highly cytotoxic, left a small subpopulation of slow-dividing cells unaffected. Compounds selected in the present study differentially altered the expression of melanocyte/melanoma specific microphthalmia-associated transcription factor (MITF and proto-oncogene c-MYC. CONCLUSION: Selected anti-clonogenic compounds might be further investigated as potential adjuvants

  10. Human Cpr (Cell Cycle Progression Restoration) Genes Impart a Far(-) Phenotype on Yeast Cells


    Edwards, M. C.; Liegeois, N.; Horecka, J.; DePinho, R A; Sprague-Jr., G. F.; Tyers, M; Elledge, S J


    Regulated cell cycle progression depends on the proper integration of growth control pathways with the basic cell cycle machinery. While many of the central molecules such as cyclins, CDKs, and CKIs are known, and many of the kinases and phosphatases that modify the CDKs have been identified, little is known about the additional layers of regulation that impinge upon these molecules. To identify new regulators of cell proliferation, we have selected for human and yeast cDNAs that when overexp...

  11. DNA-damage response gene GADD45A induces differentiation in hematopoietic stem cells without inhibiting cell cycle or survival. (United States)

    Wingert, Susanne; Thalheimer, Frederic B; Haetscher, Nadine; Rehage, Maike; Schroeder, Timm; Rieger, Michael A


    Hematopoietic stem cells (HSCs) maintain blood cell production life-long by their unique abilities of self-renewal and differentiation into all blood cell lineages. Growth arrest and DNA-damage-inducible 45 alpha (GADD45A) is induced by genotoxic stress in HSCs. GADD45A has been implicated in cell cycle control, cell death and senescence, as well as in DNA-damage repair. In general, GADD45A provides cellular stability by either arresting the cell cycle progression until DNA damage is repaired or, in cases of fatal damage, by inducing apoptosis. However, the function of GADD45A in hematopoiesis remains controversial. We revealed the changes in murine HSC fate control orchestrated by the expression of GADD45A at single cell resolution. In contrast to other cellular systems, GADD45A expression did not cause a cell cycle arrest or an alteration in the decision between cell survival and apoptosis in HSCs. Strikingly, GADD45A strongly induced and accelerated the differentiation program in HSCs. Continuous tracking of individual HSCs and their progeny via time-lapse microscopy elucidated that once GADD45A was expressed, HSCs differentiate into committed progenitors within 29 hours. GADD45A-expressing HSCs failed to long-term reconstitute the blood of recipients by inducing multilineage differentiation in vivo. Importantly, γ-irradiation of HSCs induced their differentiation by upregulating endogenous GADD45A. The differentiation induction by GADD45A was transmitted by activating p38 Mitogen-activated protein kinase (MAPK) signaling and allowed the generation of megakaryocytic-erythroid, myeloid, and lymphoid lineages. These data indicate that genotoxic stress-induced GADD45A expression in HSCs prevents their fatal transformation by directing them into differentiation and thereby clearing them from the system.

  12. Mechanistic insights into aging, cell cycle progression, and stress response

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    Troy Anthony Alan Harkness


    Full Text Available The longevity of an organism depends on the health of its cells. Throughout life cells are exposed to numerous intrinsic and extrinsic stresses, such as free radicals, generated through mitochondrial electron transport, and ultraviolet irradiation. The cell has evolved numerous mechanisms to scavenge free radicals and repair damage induced by these insults. One mechanism employed by the yeast Saccharomyces cerevisiae to combat stress utilizes the Anaphase Promoting Complex (APC, an essential multi-subunit ubiquitin-protein ligase structurally and functionally conserved from yeast to humans that controls progression through mitosis and G1. We have observed that yeast cells expressing compromised APC subunits are sensitive to multiple stresses and have shorter replicative and chronological lifespans. In a pathway that runs parallel to that regulated by the APC, members of the Forkhead box (Fox transcription factor family also regulate stress responses. The yeast Fox orthologues Fkh1 and Fkh2 appear to drive the transcription of stress response factors and slow early G1 progression, while the APC seems to regulate chromatin structure, chromosome segregation, and resetting of the transcriptome in early G1. In contrast, under non-stress conditions, the Fkhs play a complex role in cell cycle progression, partially through activation of the APC. Direct and indirect interactions between the APC and the yeast Fkhs appear to be pivotal for lifespan determination. Here we explore the potential for these interactions to be evolutionarily conserved as a mechanism to balance cell cycle regulation with stress responses.

  13. IDH1 mutations alter citric acid cycle metabolism and increase dependence on oxidative mitochondrial metabolism. (United States)

    Grassian, Alexandra R; Parker, Seth J; Davidson, Shawn M; Divakaruni, Ajit S; Green, Courtney R; Zhang, Xiamei; Slocum, Kelly L; Pu, Minying; Lin, Fallon; Vickers, Chad; Joud-Caldwell, Carol; Chung, Franklin; Yin, Hong; Handly, Erika D; Straub, Christopher; Growney, Joseph D; Vander Heiden, Matthew G; Murphy, Anne N; Pagliarini, Raymond; Metallo, Christian M


    Oncogenic mutations in isocitrate dehydrogenase 1 and 2 (IDH1/2) occur in several types of cancer, but the metabolic consequences of these genetic changes are not fully understood. In this study, we performed (13)C metabolic flux analysis on a panel of isogenic cell lines containing heterozygous IDH1/2 mutations. We observed that under hypoxic conditions, IDH1-mutant cells exhibited increased oxidative tricarboxylic acid metabolism along with decreased reductive glutamine metabolism, but not IDH2-mutant cells. However, selective inhibition of mutant IDH1 enzyme function could not reverse the defect in reductive carboxylation activity. Furthermore, this metabolic reprogramming increased the sensitivity of IDH1-mutant cells to hypoxia or electron transport chain inhibition in vitro. Lastly, IDH1-mutant cells also grew poorly as subcutaneous xenografts within a hypoxic in vivo microenvironment. Together, our results suggest therapeutic opportunities to exploit the metabolic vulnerabilities specific to IDH1 mutation.

  14. Biogenesis and dynamics of mitochondria during the cell cycle: significance of 3'UTRs.

    Directory of Open Access Journals (Sweden)

    Marta Martínez-Diez

    Full Text Available Nowadays, we are facing a renaissance of mitochondria in cancer biology. However, our knowledge of the basic cell biology and on the timing and mechanisms that control the biosynthesis of mitochondrial constituents during progression through the cell cycle of mammalian cells remain largely unknown. Herein, we document the in vivo changes on mitochondrial morphology and dynamics that accompany cellular mitosis, and illustrate the following key points of the biogenesis of mitochondria during progression of liver cells through the cycle: (i the replication of nuclear and mitochondrial genomes is synchronized during cellular proliferation, (ii the accretion of OXPHOS proteins is asynchronously regulated during proliferation being the synthesis of beta-F1-ATPase and Hsp60 carried out also at G2/M and, (iii the biosynthesis of cardiolipin is achieved during the S phase, although full development of the mitochondrial membrane potential (DeltaPsim is attained at G2/M. Furthermore, we demonstrate using reporter constructs that the mechanism regulating the accretion of beta-F1-ATPase during cellular proliferation is controlled at the level of mRNA translation by the 3'UTR of the transcript. The 3'UTR-driven synthesis of the protein at G2/M is essential for conferring to the daughter cells the original phenotype of the parental cell. Our findings suggest that alterations on this process may promote deregulated beta-F1-ATPase expression in human cancer.

  15. Local homogeneity of cell cycle length in developing mouse cortex (United States)

    Cai, L.; Hayes, N. L.; Nowakowski, R. S.


    We have measured the amount of variation in the length of the cell cycle for cells in the pseudostratified ventricular epithelium (PVE) of the developing cortex of mice on embryonic day 14. Our measurements were made in three cortical regions (i.e., the neocortex, archicortex, and periarchicortex) using three different methods: the cumulative labeling method (CLM), the percent labeled mitoses (PLM) method, and a comparison of the time needed for the PLM to ascend from 0 to 100% with the time needed for the PLM to descend from 100 to 0%. These 3 different techniques provide different perspectives on the cytokinetic parameters. Theoretically, CLM gives an estimate for a maximum value of the total length of the cell cycle (TC), whereas PLM gives an estimate of a minimum value of TC. The difference between these two estimates indicates that the range for TC is +/-1% of the mean TC for periarchicortex, +/-7% for neocortex, and +/-8% for archicortex. This was confirmed by a lengthening of the PLM descent time in comparison with its ascent time. The sharpness of the transitions and the flatness of the plateau of the PLM curves indicate that 99% of the proliferating cells are within this narrow estimated range for TC; hence, only approximately 1% deviate outside of a relatively restricted range from the average TC of the population. In the context of the possible existence within the cortical PVE of two populations with markedly dissimilar cell cycle kinetics from the mean, one such population must comprise approximately 99% of the total population, and the other, if it exists, is only approximately 1% of the total. This seems to be true for all three cortical regions. The narrow range of TC indicates a homogeneity in the cell cycle length for proliferating cells in three different cortical regions, despite the fact that progenitor cells of different lineages may be present. It further predicts the existence of almost synchronous interkinetic nuclear movements of the

  16. Matrine alters microRNA expression profiles in SGC-7901 human gastric cancer cells. (United States)

    Li, Hailong; Xie, Shoupin; Liu, Xiaojun; Wu, Hongyan; Lin, Xingyao; Gu, Jing; Wang, Huping; Duan, Yongqiang


    Matrine, a major alkaloid extracted from Sophora flavescens, has been reported to possess antitumor properties in several types of cancers, including gastric cancer. However, its mechanisms of action on gastric cancer remain poorly understood. Dysregulation of microRNAs, a class of small, non-coding, regulatory RNA molecules involved in gene expression, is strongly correlated with cancer. The aim of the present study was to demonstrate that matrine treatment altered miRNA expression in SGC7901 cells. Using miRCURY™ microarray analysis, we identified 128 miRNAs substantially exhibiting >2-fold expression changes in matrine-treated cells relative to their expression levels in untreated cells. RT-qPCR was used to show that the levels of 8 miRNAs whose target genes were clustered in the cell cycle pathway increased, while levels of 14 miRNAs whose target genes were clustered in the MAPK signaling pathway decreased. These results were consistent with those from the miRNA microarray experiment. Bioinformatical analysis revealed that the majority of 57 identified enrichment pathways were highly involved in tumorigenesis. In conclusion, the results demonstrated that matrine induces considerable changes in the miRNA expression profiles of SGC7901 cells, suggesting miRNA microarray combined with RT-qPCR validation and bioinformatical analysis provide a novel and promising approach to identify anticancer targets and the mechanisms of matrine involved.

  17. The Cell Cycle Timing of Human Papillomavirus DNA Replication. (United States)

    Reinson, Tormi; Henno, Liisi; Toots, Mart; Ustav, Mart; Ustav, Mart


    Viruses manipulate the cell cycle of the host cell to optimize conditions for more efficient viral genome replication. One strategy utilized by DNA viruses is to replicate their genomes non-concurrently with the host genome; in this case, the viral genome is amplified outside S phase. This phenomenon has also been described for human papillomavirus (HPV) vegetative genome replication, which occurs in G2-arrested cells; however, the precise timing of viral DNA replication during initial and stable replication phases has not been studied. We developed a new method to quantitate newly synthesized DNA levels and used this method in combination with cell cycle synchronization to show that viral DNA replication is initiated during S phase and is extended to G2 during initial amplification but follows the replication pattern of cellular DNA during S phase in the stable maintenance phase. E1 and E2 protein overexpression changes the replication time from S only to both the S and G2 phases in cells that stably maintain viral episomes. These data demonstrate that the active synthesis and replication of the HPV genome are extended into the G2 phase to amplify its copy number and the duration of HPV genome replication is controlled by the level of the viral replication proteins E1 and E2. Using the G2 phase for genome amplification may be an important adaptation that allows exploitation of changing cellular conditions during cell cycle progression. We also describe a new method to quantify newly synthesized viral DNA levels and discuss its benefits for HPV research.

  18. Heterogeneity in mitochondrial morphology and membrane potential is independent of the nuclear division cycle in multinucleate fungal cells. (United States)

    Gerstenberger, John P; Occhipinti, Patricia; Gladfelter, Amy S


    In the multinucleate filamentous fungus Ashbya gossypii, nuclei divide asynchronously in a common cytoplasm. We hypothesize that the division cycle machinery has a limited zone of influence in the cytoplasm to promote nuclear autonomy. Mitochondria in cultured mammalian cells undergo cell cycle-specific changes in morphology and membrane potential and therefore can serve as a reporter of the cell cycle state of the cytoplasm. To evaluate if the cell cycle state of nuclei in A. gossypii can influence the adjacent cytoplasm, we tested whether local mitochondrial morphology and membrane potential in A. gossypii are associated with the division state of a nearby nucleus. We found that mitochondria exhibit substantial heterogeneity in both morphology and membrane potential within a single multinucleated cell. Notably, differences in mitochondrial morphology or potential are not associated with a specific nuclear division state. Heterokaryon mutants with a mixture of nuclei with deletions of and wild type for the mitochondrial fusion/fission genes DNM1 and FZO1 exhibit altered mitochondrial morphology and severe growth and sporulation defects. This dominant effect suggests that the gene products may be required locally near their expression site rather than diffusing widely in the cell. Our results demonstrate that mitochondrial dynamics are essential in these large syncytial cells, yet morphology and membrane potential are independent of nuclear cycle state.

  19. Cell cycle arrest induced by MPPa-PDT in MDA-MB-231 cells (United States)

    Liang, Liming; Bi, Wenxiang; Tian, Yuanyuan


    Photodynamic therapy (PDT) is a medical treatment using a photosensitizing agent and light source to treat cancers. Pyropheophorbidea methyl ester (MPPa), a derivative of chlorophyll, is a novel potent photosensitizer. To learn more about this photosensitizer, we examined the cell cycle arrest in MDA-MB-231. Cell cycle and apoptosis were measured by flow cytometer. Checkpoints of the cell cycle were measured by western blot. In this study, we found that the expression of Cyclin D1 was obviously decreased, while the expression of Chk2 and P21 was increased after PDT treatment. This study showed that MPPa-PDT affected the checkpoints of the cell cycle and led the cells to apoptosis.

  20. Synchronization of Green Algae by Light and Dark Regimes for Cell Cycle and Cell Division Studies. (United States)

    Hlavová, Monika; Vítová, Milada; Bišová, Kateřina


    A synchronous population of cells is one of the prerequisites for studying cell cycle processes such as DNA replication, nuclear and cellular division. Green algae dividing by multiple fission represent a unique single cell system enabling the preparation of highly synchronous cultures by application of a light-dark regime similar to what they experience in nature. This chapter provides detailed protocols for synchronization of different algal species by alternating light-dark cycles; all critical points are discussed extensively. Moreover, detailed information on basic analysis of cell cycle progression in such cultures is presented, including analyses of nuclear, cellular, and chloroplast divisions. Modifications of basic protocols that enable changes in cell cycle progression are also suggested so that nuclear or chloroplast divisions can be followed separately.

  1. Cell-cycle research with synchronous cultures: an evaluation (United States)

    Helmstetter, C. E.; Thornton, M.; Grover, N. B.


    The baby-machine system, which produces new-born Escherichia coli cells from cultures immobilized on a membrane, was developed many years ago in an attempt to attain optimal synchrony with minimal disturbance of steady-state growth. In the present article, we put forward a model to describe the behaviour of cells produced by this method, and provide quantitative evaluation of the parameters involved, at each of four different growth rates. Considering the high level of selection achievable with this technique and the natural dispersion in interdivision times, we believe that the output of the baby machine is probably close to optimal in terms of both quality and persistence of synchrony. We show that considerable information on events in the cell cycle can be obtained from populations with age distributions very much broader than those achieved with the baby machine and differing only modestly from steady state. The data presented here, together with the long and fruitful history of findings employing the baby-machine technique, suggest that minimisation of stress on cells is the single most important factor for successful cell-cycle analysis.

  2. A flow cytometry-based screen of nuclear envelope transmembrane proteins identifies NET4/Tmem53 as involved in stress-dependent cell cycle withdrawal.

    Directory of Open Access Journals (Sweden)

    Nadia Korfali

    Full Text Available Disruption of cell cycle regulation is one mechanism proposed for how nuclear envelope protein mutation can cause disease. Thus far only a few nuclear envelope proteins have been tested/found to affect cell cycle progression: to identify others, 39 novel nuclear envelope transmembrane proteins were screened for their ability to alter flow cytometry cell cycle/DNA content profiles when exogenously expressed. Eight had notable effects with seven increasing and one decreasing the 4N:2N ratio. We subsequently focused on NET4/Tmem53 that lost its effects in p53(-/- cells and retinoblastoma protein-deficient cells. NET4/TMEM53 knockdown by siRNA altered flow cytometry cell cycle/DNA content profiles in a similar way as overexpression. NET4/TMEM53 knockdown did not affect total retinoblastoma protein levels, unlike nuclear envelope-associated proteins Lamin A and LAP2α. However, a decrease in phosphorylated retinoblastoma protein was observed along with a doubling of p53 levels and a 7-fold increase in p21. Consequently cells withdrew from the cell cycle, which was confirmed in MRC5 cells by a drop in the percentage of cells expressing Ki-67 antigen and an increase in the number of cells stained for ß-galactosidase. The ß-galactosidase upregulation suggests that cells become prematurely senescent. Finally, the changes in retinoblastoma protein, p53, and p21 resulting from loss of NET4/Tmem53 were dependent upon active p38 MAP kinase. The finding that roughly a fifth of nuclear envelope transmembrane proteins screened yielded alterations in flow cytometry cell cycle/DNA content profiles suggests a much greater influence of the nuclear envelope on the cell cycle than is widely held.

  3. Analysis of cell surface alterations in Legionella pneumophila cells treated with human apolipoprotein E. (United States)

    Palusinska-Szysz, Marta; Zdybicka-Barabas, Agnieszka; Cytryńska, Małgorzata; Wdowiak-Wróbel, Sylwia; Chmiel, Elżbieta; Gruszecki, Wiesław I


    Binding of human apolipoprotein E (apoE) to Legionella pneumophila lipopolysaccharide was analysed at the molecular level by Fourier-transform infrared spectroscopy, thereby providing biophysical evidence for apoE-L. pneumophila lipopolysaccharide interaction. Atomic force microscopy imaging of apoE-exposed L. pneumophila cells revealed alterations in the bacterial cell surface topography and nanomechanical properties in comparison with control bacteria. The changes induced by apoE binding to lipopolysaccharide on the surface of L. pneumophila cells may participate in: (1) impeding the penetration of host cells by the bacteria; (2) suppression of pathogen intracellular growth and eventually; and (3) inhibition of the development of infection.

  4. Genome wide transcriptome analysis of dendritic cells identifies genes with altered expression in psoriasis.

    Directory of Open Access Journals (Sweden)

    Kata Filkor

    Full Text Available Activation of dendritic cells by different pathogens induces the secretion of proinflammatory mediators resulting in local inflammation. Importantly, innate immunity must be properly controlled, as its continuous activation leads to the development of chronic inflammatory diseases such as psoriasis. Lipopolysaccharide (LPS or peptidoglycan (PGN induced tolerance, a phenomenon of transient unresponsiveness of cells to repeated or prolonged stimulation, proved valuable model for the study of chronic inflammation. Thus, the aim of this study was the identification of the transcriptional diversity of primary human immature dendritic cells (iDCs upon PGN induced tolerance. Using SAGE-Seq approach, a tag-based transcriptome sequencing method, we investigated gene expression changes of primary human iDCs upon stimulation or restimulation with Staphylococcus aureus derived PGN, a widely used TLR2 ligand. Based on the expression pattern of the altered genes, we identified non-tolerizeable and tolerizeable genes. Gene Ontology (GO and Kyoto Encyclopedia of Genes and Genomes (Kegg analysis showed marked enrichment of immune-, cell cycle- and apoptosis related genes. In parallel to the marked induction of proinflammatory mediators, negative feedback regulators of innate immunity, such as TNFAIP3, TNFAIP8, Tyro3 and Mer are markedly downregulated in tolerant cells. We also demonstrate, that the expression pattern of TNFAIP3 and TNFAIP8 is altered in both lesional, and non-lesional skin of psoriatic patients. Finally, we show that pretreatment of immature dendritic cells with anti-TNF-α inhibits the expression of IL-6 and CCL1 in tolerant iDCs and partially releases the suppression of TNFAIP8. Our findings suggest that after PGN stimulation/restimulation the host cell utilizes different mechanisms in order to maintain critical balance between inflammation and tolerance. Importantly, the transcriptome sequencing of stimulated/restimulated iDCs identified

  5. From quiescence to proliferation : Cdk oscillations drive the mammalian cell cycle

    Directory of Open Access Journals (Sweden)

    Claude eGérard


    Full Text Available We recently proposed a detailed model describing the dynamics of the network of cyclin-dependent kinases (Cdks driving the mammalian cell cycle [Gérard, C. and Goldbeter, A. (2009. Temporal self-organization of the cyclin/Cdk network driving the mammalian cell cycle. Proc. Natl. Acad. Sci. USA 106, 21643-21648]. The model contains four modules, each centered around one cyclin/Cdk complex. Cyclin D/Cdk4-6 and cyclin E/Cdk2 promote progression in G1 and elicit the G1/S transition, respectively; cyclin A/Cdk2 ensures progression in S and the transition S/G2, while the activity of cyclin B/Cdk1 brings about the G2/M transition. This model shows that in the presence of sufficient amounts of growth factor the Cdk network is capable of temporal self-organization in the form of sustained oscillations, which correspond to the ordered, sequential activation of the various cyclin/Cdk complexes that control the successive phases of the cell cycle. The results suggest that the switch from cellular quiescence to cell proliferation corresponds to the transition from a stable steady state to sustained oscillations in the Cdk network. The transition depends on a finely tuned balance between factors that promote or hinder progression in the cell cycle. We show that the transition from quiescence to proliferation can occur in multiple ways that alter this balance. By resorting to bifurcation diagrams, we analyze the mechanism of oscillations in the Cdk network. Finally, we show that the complexity of the detailed model can be greatly reduced, without losing its key dynamical properties, by considering a skeleton model for the Cdk network. Using such a skeleton model for the mammalian cell cycle we show that positive feedback loops enhance the amplitude and the robustness of Cdk oscillations with respect to molecular noise. We compare the relative merits of the detailed and skeleton versions of the model for the Cdk network driving the mammalian cell cycle.

  6. Effects of mimosine on Wolbachia in mosquito cells: cell cycle suppression reduces bacterial abundance. (United States)

    Fallon, Ann M


    The plant allelochemical L-mimosine (β-[N-(3-hydroxy-4-pyridone)]-α-aminopropionic acid; leucenol) resembles the nonessential amino acid, tyrosine. Because the obligate intracellular alphaproteobacterium, Wolbachia pipientis, metabolizes amino acids derived from host cells, the effects of mimosine on infected and uninfected mosquito cells were investigated. The EC50 for mimosine was 6-7 μM with Aedes albopictus C7-10 and C/wStr cell lines, and was not influenced by infection status. Mosquito cells responded to concentrations of mimosine substantially lower than those used to synchronize the mammalian cell cycle; at concentrations of 30-35 μM, mimosine reversibly arrested the mosquito cell cycle at the G1/S boundary and inhibited growth of Wolbachia strain wStr. Although lower concentrations of mimosine slightly increased wStr abundance, concentrations that suppressed the cell cycle reduced Wolbachia levels.


    Institute of Scientific and Technical Information of China (English)

    XU Zhou-min; WANG Yi-qun; MEI Qi; CHEN Jian; DU Jia; WEI Yan; XU Ying-chun


    Objective: The histone deacetylase inhibitors (HDACIS) have been shown to inhibit cancer cell proliferation, stimulate apoptosis, an induce cell cycle arrest. Our purpose was to investigate the antiproliferative effects of a HDACI, trichostatin A (TSA), against human cervical cancer cells (HeLa). Methods: HeLa cells were treated in vitro with various concentrations of TSA. The inhibitory effect of TSA on the growth of HeLa cells was measured by MTT assay. To detect the characteristic of apoptosis chromatin condensation, HeLa cells were stained with Hoechst 33258 in the presence of TSA. Induction of cell cycle arrest was studied by flow cytometry. Changes in gene expression of p53, p21Waf1 and p27Kip1 were studied by semiquantitative RT-PCR. Results: TSA inhibited cell growth in a time- and dose-dependent manner. Hoechst 33258 staining assay showed that TSA induced apoptosis. Cell cycle analysis indicated that treatment with TSA decreased the proportion of cells in S phase and increased the proportion of cells in G0/G1 and/or G2/M phases of the cell cycle. This was concomitant with overexpression of genes related to malignant phenotype, including an increase in p53, p21Waf1 and p27Kip1. Conclusion: These results suggest that TSA is effective in inhibiting growth of HeLa cells in vitro. The findings raise the possibility that TSA may prove particularly effective in treatment of cervical cancers.

  8. Effects of Trichostatin A on HDAC8 Expression, Proliferation and Cell Cycle of Molt-4 Cells

    Institute of Scientific and Technical Information of China (English)

    HE Jing; LIU Hongli; CHEN Yan


    The effects of Trichostatin A (TSA) on histone deacetylase 8 (HDAC8) expression, proliferation and cell cycle arrest in T-lymphoblastic leukemia cell line Molt-4 cells in vitro were investigated. The effect of TSA on the growth of Molt-4 cells was studied by MTT assay. Flow cytometry was used to examine the cell cycle. The expression of HDAC8 was detected by using immunocytochemistry and Western blot. The results showed that proliferation of Molt-4 cells was inhibited in TSA-treated group in a time- and dose-dependent manner. The IC50 of TSA exposures for 24 h and 36 h were 254.3236 and 199.257 μg/L respectively. The cell cycle analysis revealed that Molt-4 was mostly in G0/G1 phase, and after treatment with TSA from 50 to 400 μg/L for 24 h, the percents of G0/G1 cells were decreased and cells were arrested in G2/M phase. Treatment of TSA for 24 h could significantly inhibit the expression of HDAC8 protein in Molt-4 cells (P<0.01). It was concluded that TSA could decrease the expression of HDAC8 in Molt-4 cells, which contributed to the inhibition of proliferation and induction of cell cycle arrest in Molt-4 cells.

  9. Linalool Induces Cell Cycle Arrest and Apoptosis in Leukemia Cells and Cervical Cancer Cells through CDKIs. (United States)

    Chang, Mei-Yin; Shieh, Den-En; Chen, Chung-Chi; Yeh, Ching-Sheng; Dong, Huei-Ping


    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.

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

    Directory of Open Access Journals (Sweden)

    Mei-Yin Chang


    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.

  11. cGMP-independent nitric oxide signaling and regulation of the cell cycle

    Directory of Open Access Journals (Sweden)

    Cintron Ana


    Full Text Available Abstract Background Regulatory functions of nitric oxide (NO• that bypass the second messenger cGMP are incompletely understood. Here, cGMP-independent effects of NO• on gene expression were globally examined in U937 cells, a human monoblastoid line that constitutively lacks soluble guanylate cyclase. Differentiated U937 cells (>80% in G0/G1 were exposed to S-nitrosoglutathione, a NO• donor, or glutathione alone (control for 6 h without or with dibutyryl-cAMP (Bt2cAMP, and then harvested to extract total RNA for microarray analysis. Bt2cAMP was used to block signaling attributable to NO•-induced decreases in cAMP. Results NO• regulated 110 transcripts that annotated disproportionately to the cell cycle and cell proliferation (47/110, 43% and more frequently than expected contained AU-rich, post-transcriptional regulatory elements (ARE. Bt2cAMP regulated 106 genes; cell cycle gene enrichment did not reach significance. Like NO•, Bt2cAMP was associated with ARE-containing transcripts. A comparison of NO• and Bt2cAMP effects showed that NO• regulation of cell cycle genes was independent of its ability to interfere with cAMP signaling. Cell cycle genes induced by NO• annotated to G1/S (7/8 and included E2F1 and p21/Waf1/Cip1; 6 of these 7 were E2F target genes involved in G1/S transition. Repressed genes were G2/M associated (24/27; 8 of 27 were known targets of p21. E2F1 mRNA and protein were increased by NO•, as was E2F1 binding to E2F promoter elements. NO• activated p38 MAPK, stabilizing p21 mRNA (an ARE-containing transcript and increasing p21 protein; this increased protein binding to CDE/CHR promoter sites of p21 target genes, repressing key G2/M phase genes, and increasing the proportion of cells in G2/M. Conclusion NO• coordinates a highly integrated program of cell cycle arrest that regulates a large number of genes, but does not require signaling through cGMP. In humans, antiproliferative effects of NO• may rely

  12. Stretching Fibroblasts Remodels Fibronectin and Alters Cancer Cell Migration (United States)

    Ao, Mingfang; Brewer, Bryson M.; Yang, Lijie; Franco Coronel, Omar E.; Hayward, Simon W.; Webb, Donna J.; Li, Deyu


    Most investigations of cancer-stroma interactions have focused on biochemical signaling effects, with much less attention being paid to biophysical factors. In this study, we investigated the role of mechanical stimuli on human prostatic fibroblasts using a microfluidic platform that was adapted for our experiments and further developed for both repeatable performance among multiple assays and for compatibility with high-resolution confocal microscopy. Results show that mechanical stretching of normal tissue-associated fibroblasts (NAFs) alters the structure of secreted fibronectin. Specifically, unstretched NAFs deposit and assemble fibronectin in a random, mesh-like arrangement, while stretched NAFs produce matrix with a more organized, linearly aligned structure. Moreover, the stretched NAFs exhibited an enhanced capability for directing co-cultured cancer cell migration in a persistent manner. Furthermore, we show that stretching NAFs triggers complex biochemical signaling events through the observation of increased expression of platelet derived growth factor receptor α (PDGFRα). A comparison of these behaviors with those of cancer-associated fibroblasts (CAFs) indicates that the observed phenotypes of stretched NAFs are similar to those associated with CAFs, suggesting that mechanical stress is a critical factor in NAF activation and CAF genesis.

  13. Membrane alterations in irreversibly sickled cells: hemoglobin--membrane interaction. (United States)

    Lessin, L S; Kurantsin-Mills, J; Wallas, C; Weems, H


    Irreversibly sickled cells (ISCs) are sickle erythrocytes which retain bipolar elongated shapes despite reoxygenation and owe their biophysical abnormalities to acquired membrane alterations. Freeze-etched membranes both of ISCs produced in vitro and ISCs isolated in vivo reveal microbodies fixed to the internal (PS) surface which obscure spectrin filaments. Intramembranous particles (IMPs) on the intramembrane (PF) surface aggregate over regions of subsurface microbodies. Electron microscopy of diaminobenzidine-treated of ISC ghosts show the microbodies to contain hemoglobin and/or hemoglobin derivatives. Scanning electron microscopy and freeze-etching demonstrate that membrane--hemoglobin S interaction in ISCs enhances the membrane loss by microspherulation. Membrane-bound hemoglobin is five times greater in in vivo ISCs than non-ISCs, and increases during ISC production, parallelling depletion of adenosine triphosphate. Polyacrylamide gel electrophoresis of ISC membranes shows the presence of high-molecular-weight heteropolymers in the pre--band 1 region, a decrease in band 4.1 and an increase in bands 7, 8, and globin. The role of cross-linked membrane protein polymers in the generation of ISCs is discussed and is synthesized in terms of a unified concept for the determinants of the genesis of ISCs.

  14. Three Gorges Dam alters the Changjiang (Yangtze) river water cycle in the dry seasons: Evidence from H-O isotopes. (United States)

    Deng, Kai; Yang, Shouye; Lian, Ergang; Li, Chao; Yang, Chengfan; Wei, Hailun


    As the largest hydropower project in the world, the Three Gorges Dam (TGD) has attracted great concerns in terms of its impact on the Changjiang (Yangtze) River and coastal marine environments. In this study, we measured or collected the H-O isotopic data of river water, groundwater and precipitation in the mid-lower Changjiang catchment during the dry seasons of recent years. The aim was to investigate the changes of river water cycle in response to the impoundment of the TGD. Isotopic evidences suggested that the mid-lower Changjiang river water was ultimately derived from precipitation, but dominated by the mixing of different water masses with variable sources and isotopic signals as well. The isotopic parameter "deuterium excess" (d-excess) yielded large fluctuations along the mid-lower mainstream during the initial stage of the TGD impoundment, which was inherited from the upstream water with inhomogeneous isotopic signals. However, as the reservoir water level rising to the present stage, small variability of d-excess was observed along the mid-lower mainstream. This discrepancy could be explained that the TGD impoundment had significantly altered the water cycle downstream the dam, with the rising water level increasing the residence time and enhancing the mixing of reservoir water derived from upstream. This eventually resulted in the homogenization of reservoir water, and thus small fluctuations of d-excess downstream the dam after the quasi-normal stage (2008 to present). We infer that the retention effect of large reservoirs has greatly buffered the d-excess natural variability of water cycle in large river systems. Nevertheless, more research attention has to be paid to the damming effect on the water cycle in the river, estuarine and coastal areas, especially during the dry seasons.

  15. Endothelial adhesion of synchronized gastric tumor cells changes during cell cycle transit and correlates with the expression level of CD44 splice variants

    Institute of Scientific and Technical Information of China (English)

    Anton Oertl; Jens Castein; Tobias Engl; Wolf-Dietrich Beecken; Dietger Jonas; Richard Melamed; Roman A. Blaheta


    AIM: To study adhesion capacity and CD44 expression of human gastric adenocarcinoma MKN45 cells at different stages of a first cell cycle.METHODS: MKN45 cells were synchronized by aphidicolin and assayed for adhesion to an endothelial cell (HUVEC)monolayer. Surface expression of CD44 and CD44 splice variants on MKN45 cells was evaluated by flow cytometry.Functional relevance of CD44 adhesion receptors was investigated by blocking studies using anti CD44 monoclonal antibodies or by hyaluronan digestion.RESULTS: Adhesion of MKN45 to HUVEC was increased during G2/M transit, after which adhesion returned to baseline levels with cell cycle completion. In parallel, CD44splice variants CD44v4, CD44v5, and CD44v7 were all upregulated on MKN45 during cell cycle progression with a maximum effect in G2/M. The function of CD44 surface receptors was assessed with specific receptor blocking monodonal antibodies or removal of hyaluronan by digestion with hyaluronidase. Both strategies inhibited tumor cell adhesion to HUVEC by nearly 50%, which indicates that MKN45-HUVEC-interaction is CD44 dependent.CONCLUSION: CD44 expression level is linked to the cell cycle in gastrointestinal tumor cells, which in turn leads to cell cyde dependent alterations of their adhesion behaviour to endothelium.

  16. A conserved DNA damage response pathway responsible for coupling the cell division cycle to the circadian and metabolic cycles. (United States)

    Chen, Zheng; McKnight, Steven L


    The circadian clock drives endogenous oscillations of cellular and physiological processes with a periodicity of approximately 24 h. Progression of the cell division cycle (CDC) has been found to be coupled to the circadian clock, and it has been postulated that gating of the CDC by the circadian cycle may have evolved to protect DNA from the mutagenic effects of ultraviolet light. When grown under nutrient-limiting conditions in a chemostat, prototrophic strains of budding yeast, Saccharomyces cerevisiae, adopt a robust metabolic cycle of ultradian dimensions that temporally compartmentalizes essential cellular events. The CDC is gated by this yeast metabolic cycle (YMC), with DNA replication strictly segregated away from the oxidative phase when cells are actively respiring. Mutants impaired in such gating allow DNA replication to take place during the respiratory phase of the YMC and have been found to suffer significantly elevated rates of spontaneous mutation. Analogous to the circadian cycle, the YMC also employs the conserved DNA checkpoint kinase Rad53/Chk2 to facilitate coupling with the CDC. These studies highlight an evolutionarily conserved mechanism that seems to confine cell division to particular temporal windows to prevent DNA damage. We hypothesize that DNA damage itself might constitute a "zeitgeber", or time giver, for both the circadian cycle and the metabolic cycle. We discuss these findings in the context of a unifying theme underlying the circadian and metabolic cycles, and explore the relevance of cell cycle gating to human diseases including cancer.

  17. Hubble Space Telescope solar cell module thermal cycle test (United States)

    Douglas, Alexander; Edge, Ted; Willowby, Douglas; Gerlach, Lothar


    The Hubble Space Telescope (HST) solar array consists of two identical double roll-out wings designed after the Hughes flexible roll-up solar array (FRUSA) and was developed by the European Space Agency (ESA) to meet specified HST power output requirements at the end of 2 years, with a functional lifetime of 5 years. The requirement that the HST solar array remain functional both mechanically and electrically during its 5-year lifetime meant that the array must withstand 30,000 low Earth orbit (LEO) thermal cycles between approximately +100 and -100 C. In order to evaluate the ability of the array to meet this requirement, an accelerated thermal cycle test in vacuum was conducted at NASA's Marshall Space Flight Center (MSFC), using two 128-cell solar array modules which duplicated the flight HST solar array. Several other tests were performed on the modules. The thermal cycle test was interrupted after 2,577 cycles, and a 'cold-roll' test was performed on one of the modules in order to evaluate the ability of the flight array to survive an emergency deployment during the dark (cold) portion of an orbit. A posttest static shadow test was performed on one of the modules in order to analyze temperature gradients across the module. Finally, current in-flight electrical performance data from the actual HST flight solar array will be tested.

  18. Pitx2 expression promotes p21 expression and cell cycle exit in neural stem cells. (United States)

    Heldring, Nina; Joseph, Bertrand; Hermanson, Ola; Kioussi, Chrissa


    Cortical development is a complex process that involves many events including proliferation, cell cycle exit and differentiation that need to be appropriately synchronized. Neural stem cells (NSCs) isolated from embryonic cortex are characterized by their ability of self-renewal under continued maintenance of multipotency. Cell cycle progression and arrest during development is regulated by numerous factors, including cyclins, cyclin dependent kinases and their inhibitors. In this study, we exogenously expressed the homeodomain transcription factor Pitx2, usually expressed in postmitotic progenitors and neurons of the embryonic cortex, in NSCs with low expression of endogenous Pitx2. We found that Pitx2 expression induced a rapid decrease in proliferation associated with an accumulation of NSCs in G1 phase. A search for potential cell cycle inhibitors responsible for such cell cycle exit of NSCs revealed that Pitx2 expression caused a rapid and dramatic (≉20-fold) increase in expression of the cell cycle inhibitor p21 (WAF1/Cip1). In addition, Pitx2 bound directly to the p21 promoter as assessed by chromatin immunoprecipitation (ChIP) in NSCs. Surprisingly, Pitx2 expression was not associated with an increase in differentiation markers, but instead the expression of nestin, associated with undifferentiated NSCs, was maintained. Our results suggest that Pitx2 promotes p21 expression and induces cell cycle exit in neural progenitors.

  19. Effect of Lithium on Cell Cycle Progression of Pig Airway Epithelial Cells

    Institute of Scientific and Technical Information of China (English)

    陈文书; 吴人亮; 王曦; 李媛; 郝天玲


    To investigate the effect of lithium on cell cycle progression of airway epithelial cells,primary pig tracheobronchial epithelial cells were incubated with lithium chloride (LiCl) at different concentrations (0, 5 mmol/L, and 10 mmol/L) and time (12 h, 16 h and 24 h). After the treatment, cells were counted, cell cycle profile was measured by BrdU labeling and flow cytometry, and expression of cyclin D1 and cyclin B1 were detected by Western blotting. The results showed that after 24h of 10mmol/L but not 5mmol/L LiCl treatment, proliferation of cells was slowed down as manifested by delayed confluence and cell number accumulation (P<0.05). Lithium did not change the percentage of cells in S phase (P>0.05), but 24 h incubation with 10 mmol/L LiCl induced a G2/M cell cycle arrest. Furthermore, 10mmol/L LiCl elevated cyclin D1 expression after 12h treatment, while expression of cyclin B1 increased more significantly after 24h incubation. These data demonstrate that lithium inhibits proliferation of pig airway epithelial cells by inhibiting cell cycle progression, and suggest that lithium-sensitive molecule(s) such as glycogen synthase kinase 3 may have a role in the regulation of growth of airway epithelial cells.

  20. One-carbon cycle alterations induced by Dyrk1a dosage

    Directory of Open Access Journals (Sweden)

    Jean-Maurice Delabar


    Full Text Available Hyperhomocysteinemia due to cystathionine beta synthase deficiency confers diverse clinical manifestations. It is characterized by elevated plasma homocysteine levels, a common amino acid metabolized by remethylation to methionine or transsulfuration to cysteine. We recently found a relationship between hepatic Dyrk1A protein expression, a serine/threonine kinase involved in signal transduction in biological processes, hepatic S-adenosylhomocysteine activity, and plasma homocysteine levels. We aimed to study whether there is also a relationship between Dyrk1a and cystathionine beta synthase activity. We used different murine models carrying altered gene coy numbers for Dyrk1a, and found a decreased cystathionine beta synthase activity in the liver of mice under-expressing Dyrk1a, and an increased in liver of mice over-expressing Dyrk1a. For each model, a positive correlation was found between cystathionine beta synthase activity and Dyrk1a protein expression in the liver of mice, which was confirmed in a non-modified genetic context. The positive correlation found between liver Dyrk1a protein expression and CBS activity in modified and non-modified genetic context strengthens the role of this kinase in one carbon metabolism.

  1. (p)ppGpp and the bacterial cell cycle

    Indian Academy of Sciences (India)

    Aanisa Nazir; Rajendran Harinarayanan


    Genes of the Rel/Spo homolog (RSH) superfamily synthesize and/or hydrolyse the modified nucleotides pppGpp/ppGpp (collectively referred to as (p)ppGpp) and are prevalent across diverse bacteria and in plant chloroplasts. Bacteria accumulate (p)ppGpp in response to nutrient deprivation (generically called the stringent response) and elicit appropriate adaptive responses mainly through the regulation of transcription. Although at different concentrations (p)ppGpp affect the expression of distinct set of genes, the two well-characterized responses are reduction in expression of the protein synthesis machinery and increase in the expression of genes coding for amino acid biosynthesis. In Escherichia coli, the cellular (p)ppGpp level inversely correlates with the growth rate and increasing its concentration decreases the steady state growth rate in a defined growth medium. Since change in growth rate must be accompanied by changes in cell cycle parameters set through the activities of the DNA replication and cell division apparatus, (p)ppGpp could coordinate protein synthesis (cell mass increase) with these processes. Here we review the role of (p)ppGpp in bacterial cell cycle regulation.

  2. (p)ppGpp and the bacterial cell cycle. (United States)

    Nazir, Aanisa; Harinarayanan, Rajendran


    Genes of the Rel/Spo homolog (RSH) superfamily synthesize and/or hydrolyse the modified nucleotides pppGpp/ ppGpp (collectively referred to as (p)ppGpp) and are prevalent across diverse bacteria and in plant chloroplasts. Bacteria accumulate (p)ppGpp in response to nutrient deprivation (generically called the stringent response) and elicit appropriate adaptive responses mainly through the regulation of transcription. Although at different concentrations (p)ppGpp affect the expression of distinct set of genes, the two well-characterized responses are reduction in expression of the protein synthesis machinery and increase in the expression of genes coding for amino acid biosynthesis. In Escherichia coli, the cellular (p)ppGpp level inversely correlates with the growth rate and increasing its concentration decreases the steady state growth rate in a defined growth medium. Since change in growth rate must be accompanied by changes in cell cycle parameters set through the activities of the DNA replication and cell division apparatus, (p)ppGpp could coordinate protein synthesis (cell mass increase) with these processes. Here we review the role of (p)ppGpp in bacterial cell cycle regulation.

  3. Altered functional brain asymmetry for mental rotation: effect of estradiol changes across the menstrual cycle. (United States)

    Zhu, Xun; Kelly, Thomas H; Curry, Thomas E; Lal, Chitra; Joseph, Jane E


    Mental rotation is a visuospatial task associated with pronounced sex differences. Performance is also affected by gonadal hormones such as testosterone and estradiol. To better understand hormonal modulation of the neural substrates of mental rotation, the present study examined the influence of estradiol using functional MRI. Ten premenopausal women were tested on a 3D mental rotation task during the early follicular and late follicular phases of the menstrual cycle. Change in estradiol between the two phases was confirmed by hormone assays. Brain activation patterns were similar across the two phases, but the change in estradiol had different associations with the two hemispheres. Better performance in the late follicular than the early follicular phase was associated with a pattern of reduced recruitment of the right hemisphere and increased recruitment of the left hemisphere. The increased recruitment of the left hemisphere was directly associated with greater changes in estradiol. Given that the right hemisphere is the dominant hemisphere in visuospatial processing, our results suggest that estradiol is associated with reduced functional asymmetry, consistent with recent accounts of hormonal modulation of neurocognitive function.

  4. Alterations in the carnitine cycle in a mouse model of Rett syndrome (United States)

    Mucerino, Sabrina; Di Salle, Anna; Alessio, Nicola; Margarucci, Sabrina; Nicolai, Raffaella; Melone, Mariarosa A. B.; Galderisi, Umberto; Peluso, Gianfranco


    Rett syndrome (RTT) is a neurodevelopmental disease that leads to intellectual deficit, motor disability, epilepsy and increased risk of sudden death. Although in up to 95% of cases this disease is caused by de novo loss-of-function mutations in the X-linked methyl-CpG binding protein 2 gene, it is a multisystem disease associated also with mitochondrial metabolic imbalance. In addition, the presence of long QT intervals (LQT) on the patients’ electrocardiograms has been associated with the development of ventricular tachyarrhythmias and sudden death. In the attempt to shed light on the mechanism underlying heart failure in RTT, we investigated the contribution of the carnitine cycle to the onset of mitochondrial dysfunction in the cardiac tissues of two subgroups of RTT mice, namely Mecp2+/− NQTc and Mecp2+/− LQTc mice, that have a normal and an LQT interval, respectively. We found that carnitine palmitoyltransferase 1 A/B and carnitine acylcarnitine translocase were significantly upregulated at mRNA and protein level in the heart of Mecp2+/− mice. Moreover, the carnitine system was imbalanced in Mecp2+/− LQTc mice due to decreased carnitine acylcarnitine transferase expression. By causing accumulation of intramitochondrial acylcarnitines, this imbalance exacerbated incomplete fatty acid oxidation, which, in turn, could contribute to mitochondrial overload and sudden death. PMID:28150739

  5. MicroRNA-497 impairs the growth of chemoresistant neuroblastoma cells by targeting cell cycle, survival and vascular permeability genes (United States)

    Soriano, Aroa; París-Coderch, Laia; Jubierre, Luz; Martínez, Alba; Zhou, Xiangyu; Piskareva, Olga; Bray, Isabella; Vidal, Isaac; Almazán-Moga, Ana; Molist, Carla; Roma, Josep; Bayascas, José R.; Casanovas, Oriol; Stallings, Raymond L.; de Toledo, José Sánchez; Gallego, Soledad; Segura, Miguel F.


    Despite multimodal therapies, a high percentage of high-risk neuroblastoma (NB) become refractory to current treatments, most of which interfere with cell cycle and DNA synthesis or function, activating the DNA damage response (DDR). In cancer, this process is frequently altered by deregulated expression or function of several genes which contribute to multidrug resistance (MDR). MicroRNAs are outstanding candidates for therapy since a single microRNA can modulate the expression of multiple genes of the same or different pathways, thus hindering the development of resistance mechanisms by the tumor. We found several genes implicated in the MDR to be overexpressed in high-risk NB which could be targeted by microRNAs simultaneously. Our functional screening identified several of those microRNAs that reduced proliferation of chemoresistant NB cell lines, the best of which was miR-497. Low expression of miR-497 correlated with poor patient outcome. The overexpression of miR-497 reduced the proliferation of multiple chemoresistant NB cell lines and induced apoptosis in MYCN-amplified cell lines. Moreover, the conditional expression of miR-497 in NB xenografts reduced tumor growth and inhibited vascular permeabilization. MiR-497 targets multiple genes related to the DDR, cell cycle, survival and angiogenesis, which renders this molecule a promising candidate for NB therapy. PMID:26824183

  6. Systematic Characterization of Cell Cycle Phase-dependent Protein Dynamics and Pathway Activities by High-content Microscopy-assisted Cell Cycle Phenotyping

    Institute of Scientific and Technical Information of China (English)

    Christopher Bruhn; Torsten Kroll; Zhao-Qi Wang


    Cell cycle progression is coordinated with metabolism, signaling and other complex cel-lular functions. The investigation of cellular processes in a cell cycle stage-dependent manner is often the subject of modern molecular and cell biological research. Cell cycle synchronization and immunostaining of cell cycle markers facilitate such analysis, but are limited in use due to unphysiological experimental stress, cell type dependence and often low flexibility. Here, we describe high-content microscopy-assisted cell cycle phenotyping (hiMAC), which integrates high-resolution cell cycle profiling of asynchronous cell populations with immunofluorescence microscopy. hiMAC is compatible with cell types from any species and allows for statistically pow-erful, unbiased, simultaneous analysis of protein interactions, modifications and subcellular locali-zation at all cell cycle stages within a single sample. For illustration, we provide a hiMAC analysis pipeline tailored to study DNA damage response and genomic instability using a 3–4-day protocol, which can be adjusted to any other cell cycle stage-dependent analysis.

  7. The retinoblastoma protein: a master tumor suppressor acts as a link between cell cycle and cell adhesion

    Directory of Open Access Journals (Sweden)

    Engel BE


    Full Text Available Brienne E Engel,1 W Douglas Cress,1 Pedro G Santiago-Cardona2 1Molecular Oncology Program, H Lee Moffitt Cancer Center and Research Institute, Tampa, FL, USA; 2Department of Biochemistry, Ponce School of Medicine, Ponce, Puerto Rico, USA Abstract: RB1 was the first tumor suppressor gene discovered. Over 4 decades of work have revealed that the Rb protein (Rb is a master regulator of biological pathways influencing virtually every aspect of intrinsic cell fate including cell growth, cell-cycle checkpoints, differentiation, senescence, self-renewal, replication, genomic stability, and apoptosis. While these many processes may account for a significant portion of RB1's potency as a tumor suppressor, a small but growing stream of evidence suggests that RB1 also significantly influences how a cell interacts with its environment, including cell-to-cell and cell-to-extracellular matrix interactions. This review will highlight Rb’s role in the control of cell adhesion and how alterations in the adhesive properties of tumor cells may drive the deadly process of metastasis. Keywords: cadherin, integrin, Rb, cancer, aggressiveness, metastasis

  8. The cell cycle, cell death, and cell morphology during retinoic acid-induced differentiation of embryonal carcinoma cells

    NARCIS (Netherlands)

    Mummery, C.L.; Brink, C.E. van den; Saag, P.T. van der; Laat, S.W. de


    Abstract Time-lapse films were made of PC13 embryonal carcinoma cells, synchronized by mitotic shake off, in the absence and presence of retinoic acid. Using a method based on the transition probability model, cell cycle parameters were determined during the first five generations following synchron

  9. Particle acceleration in sub-cycle optical cells

    CERN Document Server

    Terranova, F


    A single laser pulse with spot size smaller than half its wavelength ($w_0 < \\lambda/2$) can provide a net energy gain to ultra-relativistic particles. In this paper, we discuss the properties of an optical cell consisting of $N$ sub-cycle pulses that propagate in the direction perpendicular to the electron motion. We show that the energy gain produced by the cell is proportional to $N$ and it is sizable even for $\\mathcal{O}(1\\mathrm{ TW})$ pulses. The optical cell acts as a defocusing lens with chromatic aberration and can be treated as a linear component in conventional accelerators if the transverse size of the beam is of the order of $\\lambda$.

  10. Cell-cycle regulatory proteins in human wound healing

    DEFF Research Database (Denmark)

    Bartkova, Jirina; Grøn, Birgitte; Dabelsteen, Erik


    Proper healing of mucosal wounds requires careful orchestration of epithelial cell migration and proliferation. To elucidate the molecular basis of the lack of cellular proliferation in the migrating 'epithelial tongue' during the re-epithelialization of oral mucosal wounds, the expression of cell......-cycle regulators critical for G(1)-phase progression and S-phase entry was here analysed immunohistochemically. Compared to normal human mucosa, epithelia migrating to cover 2- or 3-day-old wounds made either in vivo or in an organotypic cell culture all showed loss of the proliferation marker Ki67 and cyclins D(1...... the abundance of most of the CKIs, including p27Kip1, p57Kip2, p15ink4b and p18ink4c, was relatively maintained in the migrating epithelial tongue. These data indicate that downmodulation of several G(1)/S-phase cyclins and a relative excess of CKIs may cooperate to ensure the quiescent state of migrating...

  11. An Invasive Grass Species Alters Carbon Cycling in Hawaiian Dry Forest (United States)

    Litton, C. M.; Sandquist, D. R.; Cordell, S.


    At lower elevations on the leeward side of the island of Hawaii, remnant native forests are heavily invaded by an introduced African bunchgrass, Pennisetum setaceum (fountain grass). Our research is designed to determine the consequences of this invasion for carbon (C) cycling in Hawaiian dry forests. We examined above- and belowground C pools and fluxes in 400 m2 replicated forest plots (n = 4) with fountain grass (grass plots) and in areas where fountain grass had been removed for ˜3 years (removal plots). C pools were estimated with direct sampling and allometric equations developed in situ for the dominant tree species. Aboveground net primary productivity (ANPP) was estimated as aboveground biomass increment plus litterfall minus loss from mortality (trees) and with clip plots (grass and herbaceous species); total belowground carbon allocation (TBCA) was estimated using a conservation of mass, C balance approach. Our results indicate that the invasion of a non-native grass in this ecosystem has considerable impacts on both C pools and fluxes. Aboveground, tree biomass did not differ between treatments (P = 0.57) but the presence of fountain grass led to a 7.5-fold increase in understory biomass in grass plots compared to removal plots (P < 0.01). Tree ANPP was significantly higher in removal plots for both foliage (0.10 and 0.06 kg C m-2 yr-1 for removal and grass plots, respectively; P = 0.02) and wood (0.13 and 0.05 kg C m-2 yr-1 for removal and grass plots, respectively; P < 0.01). However, grass ANPP was ˜35% greater than tree foliage productivity in grass plots. Despite this added foliar productivity, total ANPP (Tree + Grass ANPP) was significantly higher in removal plots (P = 0.04). Belowground, grass plots exhibited higher rates of soil-surface CO2 efflux (1.09 and 1.38 kg C m-2 yr-1 for removal and grass plots, respectively; P = 0.03 ). Likewise, TBCA was significantly higher in grass plots (1.21 kg C m-2 yr-1) than in removal plots (0.97 kg C m-2

  12. Modifications in cell cycle kinetics and in expression of G1 phase-regulating proteins in human amniotic cells after exposure to electromagnetic fields and ionizing radiation. (United States)

    Lange, S; Viergutz, T; Simkó, M


    Low-frequency electromagnetic fields are suspected of being involved in carcinogenesis, particularly in processes that could be related to cancer promotion. Because development of cancer is associated with deregulated cell growth and we previously observed a magnetic field-induced decrease in DNA synthesis [Lange et al. (2002) Alterations in the cell cycle and in the protein level of cyclin D1p, 21CIP1, and p16INK4a after exposure to 50 HZ. MF in human cells. Radiat. Environ. Biophys.41, 131], this study aims to document the influence of 50 Hz, 1 mT magnetic fields (MF), with or without initial gamma-ionizing radiation (IR), on the following cell proliferation-relevant parameters in human amniotic fluid cells (AFC): cell cycle distribution, expression of the G1 phase-regulating proteins Cdk4, cyclin D1, p21CIP1 and p16INK4a, and Cdk4 activity. While IR induced a G1 delay and a dose-dependent G2 arrest, no discernible changes in cell cycle kinetics were observed due to MF exposure. However, a significant decrease in the protein expression of cyclin D1 and an increase in p21CIP1- and p16INK4a-expression could be detected after exposure to MF alone. IR-exposure caused an augmentation of p21CIP1- and p16INK4a- levels as well, but did not alter cyclin D1 expression. A slight diminution of Cdk4 activity was noticed after MF exposure only, indicating that Cdk4 appears not to act as a mediator of MF- or IR-induced changes in the cell cycle of AFC cells. Co-exposure to MF/IR affected neither cell cycle distribution nor protein expression or kinase activity additionally or synergistically, and therefore MF seems not to modify the mutagenic potency of IR.

  13. Fe3 O4 nanoparticle redox system modulation via cell-cycle progression and gene expression in human mesenchymal stem cells. (United States)

    Periasamy, Vaiyapuri S; Athinarayanan, Jegan; Alhazmi, Mohammad; Alatiah, Khalid A; Alshatwi, Ali A


    The use of engineered nanoparticles (NPs) across multiple fields and applications has rapidly increased over the last decade owing to their unusual properties. However, there is an increased need in understanding their toxicological effect on human health. Particularly, iron oxide (Fe3 O4 ) have been used in various sectors, including biomedical, food, and agriculture, but the current understanding of their impact on human health is inadequate. In this investigation, we assessed the toxic effect of Fe3 O4 NPs on human mesenchymal stem cells (hMSCs) adopting cell viability, cellular morphological changes, mitochondrial transmembrane potential, and cell-cycle progression assessment methodologies. Furthermore, the expression of oxidative stress, cell death, and cell-cycle regulatory genes was assessed using quantitative polymerase chain reaction. The Fe3 O4 NPs induced cytotoxicity and nuclear morphological changes in hMSCs by dose and time exposure. Cell-cycle analysis indicated that Fe3 O4 NPs altered the cell-cycle progression through a decrease in the proportion of cells in the G0 -G1 phase. The hMSC mitochondrial membrane potential loss increased with an increase in the concentration of Fe3 O4 NPs exposure. The observed expression levels of the CYP1A, TNF3, TNFSF10, E2F1, and CCNC genes were significantly upregulated in hMSCs in response to Fe3 O4 NPs exposure. Our findings suggest that Fe3 O4 NPs caused metabolic stress through altered cell cycle, oxidative stress, and cell death regulatory gene expression in hMSCs. The results of this investigation revealed that Fe3 O4 NPs exhibited moderate toxicity on hMSCs and that Fe3 O4 NPs may have biomedical applications at low concentrations. © 2014 Wiley Periodicals, Inc. Environ Toxicol 31: 901-912, 2016.

  14. Coupling between the circadian clock and cell cycle oscillators: implication for healthy cells and malignant growth

    Directory of Open Access Journals (Sweden)

    Celine eFeillet


    Full Text Available Uncontrolled cell proliferation is one of the key features leading to cancer. Seminal works in chronobiology have revealed that disruption of the circadian timing system in mice, either by surgical, genetic or environmental manipulation, increased tumor development. In humans, shift work is a risk factor for cancer. Based on these observations, the link between the circadian clock and cell cycle has become intuitive. But despite identification of molecular connections between the two processes, the influence of the clock on the dynamics of the cell cycle has never been formally observed. Recently, two studies combining single live cell imaging with computational methods have shed light on robust coupling between clock and cell cycle oscillators. We recapitulate here these novel findings and integrate them with earlier results in both healthy and cancerous cells. Moreover, we propose that the cell cycle may be synchronized or slowed down through coupling with the circadian clock, which results in reduced tumour growth. More than ever, systems biology has become instrumental to understand the dynamic interaction between the circadian clock and cell cycle, which is critical in cellular coordination and for diseases such as cancer.

  15. Coupling between the Circadian Clock and Cell Cycle Oscillators: Implication for Healthy Cells and Malignant Growth. (United States)

    Feillet, Celine; van der Horst, Gijsbertus T J; Levi, Francis; Rand, David A; Delaunay, Franck


    Uncontrolled cell proliferation is one of the key features leading to cancer. Seminal works in chronobiology have revealed that disruption of the circadian timing system in mice, either by surgical, genetic, or environmental manipulation, increased tumor development. In humans, shift work is a risk factor for cancer. Based on these observations, the link between the circadian clock and cell cycle has become intuitive. But despite identification of molecular connections between the two processes, the influence of the clock on the dynamics of the cell cycle has never been formally observed. Recently, two studies combining single live cell imaging with computational methods have shed light on robust coupling between clock and cell cycle oscillators. We recapitulate here these novel findings and integrate them with earlier results in both healthy and cancerous cells. Moreover, we propose that the cell cycle may be synchronized or slowed down through coupling with the circadian clock, which results in reduced tumor growth. More than ever, systems biology has become instrumental to understand the dynamic interaction between the circadian clock and cell cycle, which is critical in cellular coordination and for diseases such as cancer.

  16. Impairment of cell cycle progression by sterigmatocystin in human pulmonary cells in vitro. (United States)

    Huang, Shujuan; Wang, Juan; Xing, Lingxiao; Shen, Haitao; Yan, Xia; Wang, Junling; Zhang, Xianghong


    Sterigmatocystin (ST) is a carcinogenic mycotoxin that is commonly found in human food, animal feed and in the indoor environment. Although the correlation between ST exposure and lung cancer has been widely reported in many studies, the cytotoxicity of ST on human pulmonary cells is not yet fully understood. In the current study, we found that ST could induce DNA double-strand breaks in a human immortalized bronchial epithelial cell line (BEAS-2B cells) and a human lung cancer cell line (A549 cells). In addition, the effects of ST on cell cycle arrest were complex and dependent on the tested ST concentration and cell type. Low concentrations of ST arrested cells in the G2/M phase in BEAS-2B cells and in the S phase in A549 cells, while at high concentration both cells lines were arrested in S and G2/M phases. Furthermore, we observed that the modulation of cyclins and CDK expression showed concomitant changes with cell cycle arrest upon ST exposure in BEAS-2B and A549 cells. In conclusion, ST induced DNA damage and affected key proteins involved in cell cycle regulation to trigger genomic instability, which may be a potential mechanism underlying the developmental basis of lung carcinogenesis.

  17. Effects of cell cycle on the uptake of water soluble quantum dots by cells (United States)

    Zheng, Shen; Chen, Ji-Yao; Wang, Jun-Yong; Zhou, Lu-Wei; Peng, Qian


    Quantum dots (QDs) with excellent optical properties have become powerful candidates for cell imaging. Although numerous reports have studied the uptake of QDs by cells, little information exists on the effects of cell cycle on the cellular QD uptake. In this report, the effects of cell cycle on the uptake of water soluble thiol-capped CdTe QDs by the human cervical carcinoma Hela cell line, human hepatocellular carcinoma QGY7701 cell line, and human embryonic kidney 293T cell line were studied by means of laser scanning confocal microscopy and flow cytometry. All three cell lines show to take up CdTe QDs via endocytosis. After arresting cells at specific phases with pharmacological agents, the cells in G2/M phase take up the most CdTe QDs, probably due to an increased membrane expansion during mitosis; whereas the cells in G1 phase do the least. A mathematical physics model was built to calculate the relative uptake rates of CdTe QDs by cells in different phases of the cell cycle, with the result as the uptake rate in G2/M phase is 2-4 times higher than that in G1 phase for these three cell lines. The results obtained from this study may provide the information useful for intracellular delivery of QDs.

  18. Cytotoxic effects of incense particles in relation to oxidative stress, the cell cycle and F-actin assembly. (United States)

    Chuang, Hsiao-Chi; Jones, Tim; Chen, Tzu-Tao; BéruBé, Kelly


    Epidemiological studies have suggested that combustion-derived smoke, such as that produced during incense burning, is a deleterious air pollutant. It is capable of initiating oxidative stress and mutation; however, the related apoptotic processes remain unclear. In order to elucidate the biological mechanisms of reactive oxygen species (ROS)-induced respiratory toxicology, alveolar epithelial A549 cells were exposed to incense particulate matter (PM), with and without antioxidant N-acetyl-l-cysteine (NAC). The cross-linking associations between oxidative capacity, cell cycle events, actin cytoskeletal dynamics and intracellular calcium signals were investigated. An incense PM suspension caused significant oxidative stress in A549 cells, as shown by inhibition of the cell cycle at G1 and G2/M check-points, and the induction of apoptosis at Sub-G1. At the same time, alterations in the F-actin filamentous assemblies were observed. The levels of intracellular Ca(2+) were increased after incense PM exposure. Antioxidant NAC treatment revealed that oxidative stress and F-actin remodelling was significantly mitigated. This suggests that ROS accumulation could alter cell cycle regulation and anomalous remodelling of the cortical cytoskeleton that allowed impaired cells to enter into apoptosis. This study has elucidated the integral patho-physiological interactions of incense PM and the potential mechanisms for the development of ROS-driven respiratory impairment.

  19. Effect of silencing HOXA5 gene expression using RNA interference on cell cycle and apoptosis in Jurkat cells. (United States)

    Huang, Hui-Ping; Liu, Wen-Jun; Guo, Qu-Lian; Bai, Yong-Qi


    Acute lymphocytic leukemia (ALL) is a common malignant tumor with a high morbidity rate among children, accounting for approximately 80% of leukemia cases. Although there have been improvements in the treatment of patients frequent relapse lead to a poor prognosis. The aim of the present study was to determine whether HOXA5 may be used as a target for gene therapy in leukemia in order to provide a new treatment. Mononuclear cells were extracted from the bone marrow according to the clinical research aims. After testing for ALL in the acute stage, the relative mRNA and protein expression of HOXA5 was detected in the ALL remission groups (n=25 cases per group) and the control group [n=20 cases, immune thrombocytopenia (ITP)]. Gene silencing by RNA interference (RNAi) was used to investigate the effect of silencing HOXA5 after small interfering RNA (siRNA) transfection to Jurkat cells. The HOXA5-specific siRNA was transfected to Jurkat cells using lipofectamine. The experiment was divided into the experimental group (liposomal transfection of HOXA5 targeting siRNA), the negative control group (liposomal transfection of cells with negative control siRNA) and the control group (plus an equal amount of cells and culture media only). Western blotting and quantitative fluorescent polymerase chain reaction (QF‑PCR) were used to detect the relative HOXA5 mRNA expression and protein distribution in each cell group. Cell distribution in the cell cycle and the rate of cells undergoing apoptosis were determined using flow cytometry. The expression of HOXA5 at the mRNA and protein levels in the acute phase of ALL was significantly higher than that in ALL in the remission and control groups. In cells transfected with HOXA5-specific siRNA, the expression of HOXA5 at the mRNA and protein levels decreased significantly (Pcells in the cell cycle was also altered. Specifically, more cells were present in the G0/G1 phase compared to the S phase (Pcells transfected with HOXA5

  20. Gene expression alteration during redox-dependent enhancement of arsenic cytotoxicity by emodin in HeLa cells

    Institute of Scientific and Technical Information of China (English)

    Xiao Jing WANG; Jie YANG; Hui CANG; Yan Qiong ZOU; Jing YI


    Emodin (1,3,8-trihydroxy-6-methylanthraquinone) could enhance the sensitivity of tumor cells to arsenic trioxide (As2O3)-induced apoptosis via generation of ROS,but the molecular mechanism has not been elucidated.Here,we carried out cDNA microarray-based global transcription profiling of HeLa cells in response to As2O3/emodin cotreatment,comparing with As2O3-only treatment.The results showed that the expression of a number of genes was substantially altered at two time points.These genes are involved in different aspects of cell function.In addition to redox regulation and apoptosis,ROS affect genes encoding proteins associated with cell signaling,organelle functions,cell cycle,cytoskeleton,etc.These data suggest that based on the cytotoxicity of As2O3,emodin mobilize every genomic resource through which the As2O3-induced apoptosis is facilitated.

  1. Deforestation for oil palm alters the fundamental balance of the soil N cycle (United States)

    Hamilton, Liz; Trimmer, Mark; Bradley, Chris; Pinay, Gilles


    soils were first disturbed. These results are an important precursor to studies providing improved estimates of regional N turnover and loss in Southeast Asia which will have global implications for N biogeochemical cycling.

  2. [The role of cortical microtubules in moss protonemal cells during dehydration/rehydration cycle]. (United States)

    Chen, Zhi-Ling; Ouyang, Hao-Miao; Liu, Xiang-Lin; Xia, Gui-Xian


    Plant cells response to water deficit through a variety of physiological processes. In this work, we studied the function of microtubule cytoskeleton during dehydration/rehydration cycle in moss (Atrichum undulatum) protonemal cells as a model system. The morphological and cytological change of protonemal cells during dehydration and rehydration cycle were first investigated. Under normal conditions, protonemal cells showed bright green colour and appeared wet and fresh. Numerous chloroplasts distributed regularly throughout the cytoplasm in each cell. After dehydration treatment, protonemal cells lost most of their chlorophylls and turned to look yellow and dry. In addition, dehydration caused plasmolysis in these cells. Upon rehydration, the cells could recover completely from the dehydrated state. These results indicated that moss had a remarkable intrinsic ability to survive from the extreme drought stress. Microtubule, an important component of cytoskeleton, is considered to play crucial roles in the responses to some environmental stresses such as cold and light. To see if it is also involved in the drought tolerance, dynamic organization of microtubules in protonemal cells of Atrichum undulatum subjected to drought and rehydration were examined by indirect immunofluorescence combined with confocal lasersharp scanning microscopy. The cortical microtubules were arranged into a fine structure with a predominant orientation parallel to the long axis of the cells in the control cells. After dehydration, the microtubule organization was remarkablly altered and the fine microtubule structure disappeared whereas some thicker cables formed. When the cells were grown under rehydration conditions, the fine microtubule arrays reappeared. These results provided a piece of evidence that microtubules play a role in the cellular responses to drought stress in moss. Furthermore, we analyzed the effects of the microtubule-disrupting agent colchicine on the morphology recovery

  3. Genome Wide Expression Profiling of Cancer Cell Lines Cultured in Microgravity Reveals Significant Dysregulation of Cell Cycle and MicroRNA Gene Networks.

    Directory of Open Access Journals (Sweden)

    Prasanna Vidyasekar

    Full Text Available Zero gravity causes several changes in metabolic and functional aspects of the human body and experiments in space flight have demonstrated alterations in cancer growth and progression. This study reports the genome wide expression profiling of a colorectal cancer cell line-DLD-1, and a lymphoblast leukemic cell line-MOLT-4, under simulated microgravity in an effort to understand central processes and cellular functions that are dysregulated among both cell lines. Altered cell morphology, reduced cell viability and an aberrant cell cycle profile in comparison to their static controls were observed in both cell lines under microgravity. The process of cell cycle in DLD-1 cells was markedly affected with reduced viability, reduced colony forming ability, an apoptotic population and dysregulation of cell cycle genes, oncogenes, and cancer progression and prognostic markers. DNA microarray analysis revealed 1801 (upregulated and 2542 (downregulated genes (>2 fold in DLD-1 cultures under microgravity while MOLT-4 cultures differentially expressed 349 (upregulated and 444 (downregulated genes (>2 fold under microgravity. The loss in cell proliferative capacity was corroborated with the downregulation of the cell cycle process as demonstrated by functional clustering of DNA microarray data using gene ontology terms. The genome wide expression profile also showed significant dysregulation of post transcriptional gene silencing machinery and multiple microRNA host genes that are potential tumor suppressors and proto-oncogenes including MIR22HG, MIR17HG and MIR21HG. The MIR22HG, a tumor-suppressor gene was one of the highest upregulated genes in the microarray data showing a 4.4 log fold upregulation under microgravity. Real time PCR validated the dysregulation in the host gene by demonstrating a 4.18 log fold upregulation of the miR-22 microRNA. Microarray data also showed dysregulation of direct targets of miR-22, SP1, CDK6 and CCNA2.

  4. The same, only different - DNA damage checkpoints and their reversal throughout the cell cycle

    NARCIS (Netherlands)

    Shaltiel, Indra A.; Krenning, Lenno; Bruinsma, Wytse; Medema, René H.


    Cell cycle checkpoints activated by DNA double-strand breaks (DSBs) are essential for the maintenance of the genomic integrity of proliferating cells. Following DNA damage, cells must detect the break and either transiently block cell cycle progression, to allow time for repair, or exit the cell cyc

  5. Berberine induces cell cycle arrest and apoptosis in human gastric carcinoma SNU-5 cell line

    Institute of Scientific and Technical Information of China (English)

    Jing-Pin Lin; Jai-Sing Yang; Jau-Hong Lee; Wen-Tsong Hsieh; Jing-Gung Chung


    AIM: To investigate the relationship between the inhibited growth (cytotoxic activity) of berberine and apoptotic pathway with its molecular mechanism of action.METHODS: The in vitro cytotoxic techniques were complemented by cell cycle analysis and determination of sub-G1 for apoptosis in human gastric carcinoma SNU-5 cells. Percentage of viable cells, cell cycle, and sub-G1 group (apoptosis) were examined and determined by the flow cytometric methods. The associated proteins for cell cycle arrest and apoptosis were examined by Western blotting.RESULTS: For SNU-5 cell line, the IC (50) was found to be 48 μmol/L of berberine. In SNU-5 cells treated with 25-200 μmol/L berberine, G2/M cell cycle arrest was observed which was associated with a marked increment of the expression of p53, Wee1 and CDk1 proteins and decreased cyclin B. A concentration-dependent decrease of cells in G0/G1 phase and an increase in G2/M phase were detected. In addition, apoptosis detected as sub-G0 cell population in cell cycle measurement was proved in 25-200 μmol/L berberine-treated cells by monitoring the apoptotic pathway. Apoptosis was identified by sub-G0 cell population, and upregulation of Bax, downregulation of Bcl-2, release of Ca2+, decreased the mitochondrial membrane potential and then led to the release of mitochondrial cytochrome C into the cytoplasm and caused the activation of caspase-3, and finally led to the occurrence of apoptosis.CONCLUSION: Berberine induces p53 expression and leads to the decrease of the mitochondrial membrane potential, Cytochrome C release and activation of caspase-3 for the induction of apoptosis.

  6. Ethanol Metabolism Activates Cell Cycle Checkpoint Kinase, Chk2 (United States)

    Clemens, Dahn L.; Mahan Schneider, Katrina J.; Nuss, Robert F.


    Chronic ethanol abuse results in hepatocyte injury and impairs hepatocyte replication. We have previously shown that ethanol metabolism results in cell cycle arrest at the G2/M transition, which is partially mediated by inhibitory phosphorylation of the cyclin-dependent kinase, Cdc2. To further delineate the mechanisms by which ethanol metabolism mediates this G2/M arrest, we investigated the involvement of upstream regulators of Cdc2 activity. Cdc2 is activated by the phosphatase Cdc25C. The activity of Cdc25C can, in turn, be regulated by the checkpoint kinase, Chk2, which is regulated by the kinase ataxia telangiectasia mutated (ATM). To investigate the involvement of these regulators of Cdc2 activity, VA-13 cells, which are Hep G2 cells modified to efficiently express alcohol dehydrogenase, were cultured in the presence or absence of 25 mM ethanol. Immunoblots were performed to determine the effects of ethanol metabolism on the activation of Cdc25C, Chk2, and ATM. Ethanol metabolism increased the active forms of ATM, and Chk2, as well as the phosphorylated form of Cdc25C. Additionally, inhibition of ATM resulted in approximately 50% of the cells being rescued from the G2/M cell cycle arrest, and ameliorated the inhibitory phosphorylation of Cdc2. Our findings demonstrate that ethanol metabolism activates ATM. ATM can activate the checkpoint kinase Chk2, resulting in phosphorylation of Cdc25C, and ultimately in the accumulation of inactive Cdc2. This may, in part, explain the ethanol metabolism-mediated impairment in hepatocyte replication, which may be important in the initiation and progression of alcoholic liver injury. PMID:21924579

  7. Cell-cycle-dependent PC-PLC regulation by APC/C(Cdc20)-mediated ubiquitin-proteasome pathway. (United States)

    Fu, Da; Ma, Yushui; Wu, Wei; Zhu, Xuchao; Jia, Chengyou; Zhao, Qianlei; Zhang, Chunyi; Wu, Xing Zhong


    Phosphatidylcholine-specific phospholipase C (PC-PLC) is involved in the cell signal transduction, cell proliferation, and apoptosis. The mechanism of its action, however, has not been fully understood, particularly, the role of PC-PLC in the cell cycle. In the present study, we found that cell division cycle 20 homolog (Cdc20) and PC-PLC were co-immunoprecipitated reciprocally by either antibody in rat hepatoma cells CBRH-7919 as well as in rat liver tissue. Using confocal microscopy, we found that PC-PLC and Cdc20 were co-localized in the perinuclear endoplasmic reticulum region (the "juxtanuclear quality control" compartment, JUNQ). The expression level and activities of PC-PLC changed in a cell-cycle-dependent manner and were inversely correlated with the expression of Cdc20. Intriguingly, Cdc20 overexpression altered the subcellular localization and distribution of PC-PLC, and caused PC-PLC degradation by the ubiquitin proteasome pathway (UPP). Taken together, our data indicate that PC-PLC regulation in cell cycles is controlled by APC/C(Cdc20)-mediated UPP.

  8. The tumor suppressor p53 connects ribosome biogenesis to cell cycle control: a double-edged sword. (United States)

    Hölzel, Michael; Burger, Kaspar; Mühl, Bastian; Orban, Mathias; Kellner, Markus; Eick, Dirk


    Since its first description more than 30 years ago p53 has become a paradigm for a protein with versatile functions. P53 sensitizes a large variety of genetic alterations and has been entitled the guardian of the genome. Stabilization of p53 upon DNA damage is accompanied by a complex pattern of modifications, which ascertain the cellular response either in the direction of a reversible or irreversible cell cycle arrest or programmed cell death. More recently it became evident that p53 also responds to non-genotoxic cell stress, in particular if ribosome biogenesis is affected.

  9. Impaired Cell Cycle Regulation in a Natural Equine Model of Asthma.

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    Alicja Pacholewska

    Full Text Available Recurrent airway obstruction (RAO is a common and potentially debilitating lower airway disease in horses, which shares many similarities with human asthma. In susceptible horses RAO exacerbation is caused by environmental allergens and irritants present in hay dust. The objective of this study was the identification of genes and pathways involved in the pathology of RAO by global transcriptome analyses in stimulated peripheral blood mononuclear cells (PBMCs. We performed RNA-seq on PBMCs derived from 40 RAO affected and 45 control horses belonging to three cohorts of Warmblood horses: two half-sib families and one group of unrelated horses. PBMCs were stimulated with hay dust extract, lipopolysaccharides, a recombinant parasite antigen, or left unstimulated. The total dataset consisted of 561 individual samples. We detected significant differences in the expression profiles between RAO and control horses. Differential expression (DE was most marked upon stimulation with hay dust extract. An important novel finding was a strong upregulation of CXCL13 together with many genes involved in cell cycle regulation in stimulated samples from RAO affected horses, in addition to changes in the expression of several HIF-1 transcription factor target genes. The RAO condition alters systemic changes observed as differential expression profiles of PBMCs. Those changes also depended on the cohort and stimulation of the samples and were dominated by genes involved in immune cell trafficking, development, and cell cycle regulation. Our findings indicate an important role of CXCL13, likely macrophage or Th17 derived, and the cell cycle regulator CDC20 in the immune response in RAO.

  10. Induction of G1 cell cycle arrest and apoptosis by berberine in bladder cancer cells. (United States)

    Yan, Keqiang; Zhang, Cheng; Feng, Jinbo; Hou, Lifang; Yan, Lei; Zhou, Zunlin; Liu, Zhaoxu; Liu, Cheng; Fan, Yidon; Zheng, Baozhong; Xu, Zhonghua


    Bladder cancer is the ninth most common type of cancer, and its surgery is always followed by chemotherapy to prevent recurrence. Berberine is non-toxic to normal cells but has anti-cancer effects in many cancer cell lines. This study was aimed to determine whether berberine inhibits the cell proliferation and induces cell cycle arrest and apoptosis in BIU-87 and T24 bladder cancer cell line. The superficial bladder cancer cell line BIU-87 and invasive T24 bladder cancer cells were treated with different concentrations of berberine. MTT assay was used to determine the effects of berberine on the viability of these cells. The cell cycle arrest was detected through propidium iodide (PI) staining. The induction of apoptosis was determined through Annexin V-conjugated Alexa Fluor 488 (Alexa488) staining. Berberine inhibited the viability of BIU-87 and T24 cells in a dose- and time-dependent manner. It also promoted cell cycle arrest at G0/G1 in a dose-dependent manner and induced apoptosis. We observed that H-Ras and c-fos mRNA and protein expressionswere dose-dependently and time-dependently decreased by berberine treatment. Also, we investigated the cleaved caspase-3 and caspase-9 protein expressions increased in a dose-dependent manner. Berberine inhibits the cell proliferation and induces cell cycle arrest and apoptosis in BIU-87, bladder cancer cell line and T24, invasive bladder cancer cell line. Berberine can inhibit the oncogentic H-Ras and c-fos in T24 cells, and can induce the activation of the caspase-3 and caspase-9 apoptosis. Therefore, berberine has the potential to be a novel chemotherapy drug to treat the bladder cancer by suppressing tumor growth.

  11. Mechanism of T-oligo-induced cell cycle arrest in Mia-Paca pancreatic cancer cells (United States)

    Rankin, Andrew M.; Sarkar, Sibaji; Faller, Douglas V.


    DNA oligonucleotides with sequence homology to human telomeric DNA (T-oligo) induce cell cycle arrest, followed by apoptosis, senescence, or autophagy in a human cancer cell type-specific manner. T-oligo has potential as a new therapeutic strategy in oncology because of its ability to target certain types of tumor cells while sparing normal ones. In the present study, we demonstrate the T-oligo-induced S-phase cell cycle arrest in four pancreatic cancer cell lines. To further contribute to the mechanistic understanding of T-oligo, we also identify cyclin dependent kinase 2 (cdk2) as a functional mediator in the T-oligo-induced cell cycle arrest of pancreatic cancer cells. Ectopic expression of a constitutively-active cdk2 mutant abrogates T-oligo-induced cell cycle arrest in these tumor cells while knockdown of cdk2 expression alone recapitulates the T-oligo effect. Finally, we demonstrate the dispensability of T-oligo-induced ATM/ATR-mediated DNA damage response-signaling pathways, which have long been considered functional in the T-oligo signaling mechanism. PMID:21898405

  12. Cytokine-dependent and–independent gene expression changes and cell cycle block revealed in Trypanosoma cruzi-infected host cells by comparative mRNA profiling

    Directory of Open Access Journals (Sweden)

    Burleigh Barbara A


    Full Text Available Abstract Background The requirements for growth and survival of the intracellular pathogen Trypanosoma cruzi within mammalian host cells are poorly understood. Transcriptional profiling of the host cell response to infection serves as a rapid read-out for perturbation of host physiology that, in part, reflects adaptation to the infective process. Using Affymetrix oligonucleotide array analysis we identified common and disparate host cell responses triggered by T. cruzi infection of phenotypically diverse human cell types. Results We report significant changes in transcript abundance in T. cruzi-infected fibroblasts, endothelial cells and smooth muscle cells (2852, 2155 and 531 genes respectively; fold-change ≥ 2, p-value T. cruzi-infected fibroblasts and endothelial cells transwell plates were used to distinguish cytokine-dependent and -independent gene expression profiles. This approach revealed the induction of metabolic and signaling pathways involved in cell proliferation, amino acid catabolism and response to wounding as common themes in T. cruzi-infected cells. In addition, the downregulation of genes involved in mitotic cell cycle and cell division predicted that T. cruzi infection may impede host cell cycle progression. The observation of impaired cytokinesis in T. cruzi-infected cells, following nuclear replication, confirmed this prediction. Conclusion Metabolic pathways and cellular processes were identified as significantly altered at the transcriptional level in response to T. cruzi infection in a cytokine-independent manner. Several of these alterations are supported by previous studies of T. cruzi metabolic requirements or effects on the host. However, our methods also revealed a T. cruzi-dependent block in the host cell cycle, at the level of cytokinesis, previously unrecognized for this pathogen-host cell interaction.

  13. Effects of 50 Hz pulsed electromagnetic fields on the growth and cell cycle arrest of mesenchymal stem cells: an in vitro study. (United States)

    Li, Xinping; Zhang, Mingsheng; Bai, Liming; Bai, Wenfang; Xu, Weicheng; Zhu, Hongxiang


    Mesenchymal stem cells (MSCs) are capable of self-renew and multipotent differatiation which allows them to be sensitive to microenvironment is altered. Pulsed electromagnetic fields (PEMF) can affect cellular physiology of some types of cells. This study was undertaken to investigate the effects of PEMF on the growth and cell cycle arrest of MSCs expanded in vitro. To achieve this, cultured of normal rat MSCs, the treatment groups were respectively irradiated by 50 Hz PEMF at 10 mT of flux densities for 3 or 6 h. The effects of PEMF on cell proliferation, cell cycle arrest, and cell surface antigen phenotype were investigated. Our results showed that exposed MSCs had a significant proliferative capacity (P cell growth was not different (P>0.05) at an earlier phase after PEMF treatment. Exposure to PEMF had a significant increase the percentage of MSCs in G1 phase compare with the control group, with a higher percentage of cells in G1 phase exposed for 6 h then that for 3 h. At the 16th hour after treatment, PEMF had no significant effect on cell proliferation and cell cycle (P>0.05). These results suggested that PEMF enhanced MSCs proliferation with time-independent and increased the percentage of cells at the G1 phase of the cell cycle in a time-dependent manner, and the effect of PEMF on the cell proliferation and cell cycle arrest of MSCs was temporal after PEMF treatment.

  14. Shielding of the Geomagnetic Field Alters Actin Assembly and Inhibits Cell Motility in Human Neuroblastoma Cells. (United States)

    Mo, Wei-Chuan; Zhang, Zi-Jian; Wang, Dong-Liang; Liu, Ying; Bartlett, Perry F; He, Rong-Qiao


    Accumulating evidence has shown that absence of the geomagnetic field (GMF), the so-called hypomagnetic field (HMF) environment, alters the biological functions in seemingly non-magnetosensitive cells and organisms, which indicates that the GMF could be sensed by non-iron-rich and non-photo-sensing cells. The underlying mechanisms of the HMF effects on those cells are closely related to their GMF sensation but remain poorly understood so far. Previously, we found that the HMF represses expressions of genes associated with cell migration and cytoskeleton assembly in human neuroblastoma cells (SH-SY5Y cell line). Here, we measured the HMF-induced changes on cell morphology, adhesion, motility and actin cytoskeleton in SH-SY5Y cells. The HMF inhibited cell adhesion and migration accompanied with a reduction in cellular F-actin amount. Moreover, following exposure to the HMF, the number of cell processes was reduced and cells were smaller in size and more round in shape. Furthermore, disordered kinetics of actin assembly in vitro were observed during exposure to the HMF, as evidenced by the presence of granule and meshed products. These results indicate that elimination of the GMF affects assembly of the motility-related actin cytoskeleton, and suggest that F-actin is a target of HMF exposure and probably a mediator of GMF sensation.

  15. Mirk/Dyrk1B mediates G0/G1 to S phase cell cycle progression and cell survival involving MAPK/ERK signaling in human cancer cells

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    Gao Jingchun


    Full Text Available Abstract Background Mirk/Dyrk1B contributes to G0 arrest by destabilization of cyclin D1 and stabilization of p27kip1 to maintain the viability of quiescent human cancer cells, and it could be negatively regulated by mitogenic-activated protein kinase (MAPK/extracellular signal-regulated kinase (ERK signaling. This study was performed to investigate the effect of Mirk/Dyrk1B on cell cycle and survival of human cancer cells involving MAPK/ERK signaling. Methods The correlations between Mirk/Dyrk1B expression and active ERK1/2 detected by western blot in both ovarian cancer and non-small cell lung cancer (NSCLC cells were analyzed by simple regression. Mirk/Dyrk1B unique phosphopeptides with sites associated with Mirk/Dyrk1B protein were isolated and quantitated by liquid chromatography coupled to tandem mass/mass spectrometry (LC-MS/MS proteomics analysis. The human cancer cells were treated with small interfering RNAs (siRNAs and/or U0126, an inhibitor of MEK for indicated duration, followed by investigating the alterations of cell cycle and apoptosis as well as related proteins examined by flow cytometry and Western blot, respectively. Results Our study demonstrated the widely expressed Mirk/Dyrk1B proteins in the human cancer cells were positively correlated with the levels of activated ERK1/2. Moreover, Mirk/Dyrk1B protein expressions consistent with the tyrosine autophosphorylated levels in the human cancer cells were increased by U0126 or growth factor-depleted culture. Conversely, knockdown of Mirk/Dyrk1B by siRNA led to up-regulated activation of c-Raf-MEK-ERK1/2 pathway and subsequent changes in cell cycle proteins (cyclin D1, p27kip1, accompanied by increased growth rate and cells from G0/G1 into S of cell cycle which could be blocked by U0126 in a dose-dependent manner, indicating Mirk/Dyrk1B may sequester MAPK/ERK pathway, and vice versa. Whereas, combined Mirk siRNA and U0126 induced cell apoptosis in the human cancer cells

  16. Regulation of the G1 phase of the mammalian cell cycle

    Institute of Scientific and Technical Information of China (English)


    In any multi-cellular organism, the balance between cell division and cell death maintains a constant cell num ber. Both cell division cycle and cell death are highly regulated events. Whether the cell will proceed through the cycle or not, depends upon whether the conditions re quired at the checkpoints during the cycle are filfilled. In higher eucaryotic cells, such as mammalian cells, signals that arrest the cycle usually act at a G1 checkpoint. Cells that pass this restriction point are committed to complete the cycle. Regulation of the G1 phase of the cell cycle is extremely complex and involves many different families of proteins such as retinoblastoma family, cyclin dependent kinases, cyclins, and cyclin kinase inhibitors.

  17. S-phase-dependent cell cycle disturbances caused by Aleutian mink disease parvovirus

    DEFF Research Database (Denmark)

    Oleksiewicz, M.B.; Alexandersen, Søren


    We examined replication of the autonomous parovirus Aleutian mink disease parovirus (ADV) in relation to cell cycle progression of permissive Crandell feline kidney (CRFK) cells. Flow cytometric analysis showed that ADV caused a composite, binary pattern of cell cycle arrest. ADV-induced cell cycle...... with subthreshold levels of ADV products through the late S/G(2) block and, consequently, that the binary pattern of ADV-induced cell cycle arrest may be governed merely by viral replication levels within a single S phase. Flow cytometric analysis of propidium iodide fluorescence and bromodeoxyuridine uptake showed...... that population A cells sustained significantly higher levels of DNA replication than population B cells during the ADV-induced cell cycle arrest. Therefore, the type of ADV-induced cell cycle arrest was not trivial and could have implications for subsequent viral replication in the target cell....


    Energy Technology Data Exchange (ETDEWEB)

    FuelCell Energy


    With about 50% of power generation in the United States derived from coal and projections indicating that coal will continue to be the primary fuel for power generation in the next two decades, the Department of Energy (DOE) Clean Coal Technology Demonstration Program (CCTDP) has been conducted since 1985 to develop innovative, environmentally friendly processes for the world energy market place. The 2 MW Fuel Cell Demonstration was part of the Kentucky Pioneer Energy (KPE) Integrated Gasification Combined Cycle (IGCC) project selected by DOE under Round Five of the Clean Coal Technology Demonstration Program. The participant in the CCTDP V Project was Kentucky Pioneer Energy for the IGCC plant. FuelCell Energy, Inc. (FCE), under subcontract to KPE, was responsible for the design, construction and operation of the 2 MW fuel cell power plant. Duke Fluor Daniel provided engineering design and procurement support for the balance-of-plant skids. Colt Engineering Corporation provided engineering design, fabrication and procurement of the syngas processing skids. Jacobs Applied Technology provided the fabrication of the fuel cell module vessels. Wabash River Energy Ltd (WREL) provided the test site. The 2 MW fuel cell power plant utilizes FuelCell Energy's Direct Fuel Cell (DFC) technology, which is based on the internally reforming carbonate fuel cell. This plant is capable of operating on coal-derived syngas as well as natural gas. Prior testing (1992) of a subscale 20 kW carbonate fuel cell stack at the Louisiana Gasification Technology Inc. (LGTI) site using the Dow/Destec gasification plant indicated that operation on coal derived gas provided normal performance and stable operation. Duke Fluor Daniel and FuelCell Energy developed a commercial plant design for the 2 MW fuel cell. The plant was designed to be modular, factory assembled and truck shippable to the site. Five balance-of-plant skids incorporating fuel processing, anode gas oxidation, heat recovery

  19. Alkylphospholipids deregulate cholesterol metabolism and induce cell-cycle arrest and autophagy in U-87 MG glioblastoma cells. (United States)

    Ríos-Marco, Pablo; Martín-Fernández, Mario; Soria-Bretones, Isabel; Ríos, Antonio; Carrasco, María P; Marco, Carmen


    Glioblastoma is the most common malignant primary brain tumour in adults and one of the most lethal of all cancers. Growing evidence suggests that human tumours undergo abnormal lipid metabolism, characterised by an alteration in the mechanisms that regulate cholesterol homeostasis. We have investigated the effect that different antitumoural alkylphospholipids (APLs) exert upon cholesterol metabolism in the U-87 MG glioblastoma cell line. APLs altered cholesterol homeostasis by interfering with its transport from the plasma membrane to the endoplasmic reticulum (ER), thus hindering its esterification. At the same time they stimulated the synthesis of cholesterol from radiolabelled acetate and its internalisation from low-density lipoproteins (LDLs), inducing both 3-hydroxy-3-methylglutaryl-CoA reductase (HMGCR) and LDL receptor (LDLR) genes. Fluorescent microscopy revealed that these effects promoted the accumulation of intracellular cholesterol. Filipin staining demonstrated that this accumulation was not confined to the late endosome/lysosome (LE/LY) compartment since it did not colocalise with LAMP2 lysosomal marker. Furthermore, APLs inhibited cell growth, producing arrest at the G2/M phase. We also used transmission electron microscopy (TEM) to investigate ultrastructural alterations induced by APLs and found an abundant presence of autophagic vesicles and autolysosomes in treated cells, indicating the induction of autophagy. Thus our findings clearly demonstrate that antitumoural APLs interfere with the proliferation of the glioblastoma cell line via a complex mechanism involving cholesterol metabolism, cell-cycle arrest or autophagy. Knowledge of the interrelationship between these processes is fundamental to our understanding of tumoural response and may facilitate the development of novel therapeutics to improve treatment of glioblastoma and other types of cancer.

  20. Altered epigenetic regulation of homeobox genes in human oral squamous cell carcinoma cells

    Energy Technology Data Exchange (ETDEWEB)

    Marcinkiewicz, Katarzyna M.; Gudas, Lorraine J., E-mail:


    To gain insight into oral squamous cell carcinogenesis, we performed deep sequencing (RNAseq) of non-tumorigenic human OKF6-TERT1R and tumorigenic SCC-9 cells. Numerous homeobox genes are differentially expressed between OKF6-TERT1R and SCC-9 cells. Data from Oncomine, a cancer microarray database, also show that homeobox (HOX) genes are dysregulated in oral SCC patients. The activity of Polycomb repressive complexes (PRC), which causes epigenetic modifications, and retinoic acid (RA) signaling can control HOX gene transcription. HOXB7, HOXC10, HOXC13, and HOXD8 transcripts are higher in SCC-9 than in OKF6-TERT1R cells; using ChIP (chromatin immunoprecipitation) we detected PRC2 protein SUZ12 and the epigenetic H3K27me3 mark on histone H3 at these genes in OKF6-TERT1R, but not in SCC-9 cells. In contrast, IRX1, IRX4, SIX2 and TSHZ3 transcripts are lower in SCC-9 than in OKF6-TERT1R cells. We detected SUZ12 and the H3K27me3 mark at these genes in SCC-9, but not in OKF6-TERT1R cells. SUZ12 depletion increased HOXB7, HOXC10, HOXC13, and HOXD8 transcript levels and decreased the proliferation of OKF6-TERT1R cells. Transcriptional responses to RA are attenuated in SCC-9 versus OKF6-TERT1R cells. SUZ12 and H3K27me3 levels were not altered by RA at these HOX genes in SCC-9 and OKF6-TERT1R cells. We conclude that altered activity of PRC2 is associated with dysregulation of homeobox gene expression in human SCC cells, and that this dysregulation potentially plays a role in the neoplastic transformation of oral keratinocytes. - Highlights: • RNAseq elucidates differences between non-tumorigenic and tumorigenic oral keratinocytes. • Changes in HOX mRNA in SCC-9 vs. OKF6-TERT1R cells are a result of altered epigenetic regulation. • RNAseq shows that retinoic acid (RA) influences gene expression in both OKF6-TERT1R and SCC-9 cells.

  1. ADAM17 deletion in thymic epithelial cells alters aire expression without affecting T cell developmental progression.

    Directory of Open Access Journals (Sweden)

    David M Gravano

    Full Text Available BACKGROUND: Cellular interactions between thymocytes and thymic stromal cells are critical for normal T cell development. Thymic epithelial cells (TECs are important stromal niche cells that provide essential growth factors, cytokines, and present self-antigens to developing thymocytes. The identification of genes that mediate cellular crosstalk in the thymus is ongoing. One candidate gene, Adam17, encodes a metalloprotease that functions by cleaving the ectodomain of several transmembrane proteins and regulates various developmental processes. In conventional Adam17 knockout mice, a non-cell autonomous role for ADAM17 in adult T cell development was reported, which strongly suggested that expression of ADAM17 in TECs was required for normal T cell development. However, knockdown of Adam17 results in multisystem developmental defects and perinatal lethality, which has made study of the role of Adam17 in specific cell types difficult. Here, we examined T cell and thymic epithelial cell development using a conditional knockout approach. METHODOLOGY/PRINCIPAL FINDINGS: We generated an Adam17 conditional knockout mouse in which floxed Adam17 is deleted specifically in TECs by Cre recombinase under the control of the Foxn1 promoter. Normal T cell lineage choice and development through the canonical αβ T cell stages was observed. Interestingly, Adam17 deficiency in TECs resulted in reduced expression of the transcription factor Aire. However, no alterations in the patterns of TEC phenotypic marker expression and thymus morphology were noted. CONCLUSIONS/SIGNIFICANCE: In contrast to expectation, our data clearly shows that absence of Adam17 in TECs is dispensable for normal T cell development. Differentiation of TECs is also unaffected by loss of Adam17 based on phenotypic markers. Surprisingly, we have uncovered a novel genetic link between Adam17and Aire expression in vivo. The cell type in which ADAM17 mediates its non-cell autonomous impact and

  2. DNA Damage, Cell Cycle Arrest, and Apoptosis Induction Caused by Lead in Human Leukemia Cells. (United States)

    Yedjou, Clement G; Tchounwou, Hervey M; Tchounwou, Paul B


    In recent years, the industrial use of lead has been significantly reduced from paints and ceramic products, caulking, and pipe solder. Despite this progress, lead exposure continues to be a significant public health concern. The main goal of this research was to determine the in vitro mechanisms of lead nitrate [Pb(NO₃)₂] to induce DNA damage, apoptosis, and cell cycle arrest in human leukemia (HL-60) cells. To reach our goal, HL-60 cells were treated with different concentrations of Pb(NO₃)₂ for 24 h. Live cells and necrotic death cells were measured by the propidium idiode (PI) assay using the cellometer vision. Cell apoptosis was measured by the flow cytometry and DNA laddering. Cell cycle analysis was evaluated by the flow cytometry. The result of the PI demonstrated a significant (p cell death in Pb(NO₃)₂-treated cells, indicative of membrane rupture by Pb(NO₃)₂ compared to the control. Data generated from the comet assay indicated a concentration-dependent increase in DNA damage, showing a significant increase (p cells (apoptotic cells) compared to the control. The flow cytometry assessment also indicated Pb(NO₃)₂ exposure caused cell cycle arrest at the G₀/G₁ checkpoint. The result of DNA laddering assay showed presence of DNA smear in the agarose gel with little presence of DNA fragments in the treated cells compared to the control. In summary, Pb(NO₃)₂ inhibits HL-60 cells proliferation by not only inducing DNA damage and cell cycle arrest at the G₀/G₁ checkpoint but also triggering the apoptosis through caspase-3 activation and nucleosomal DNA fragmentation accompanied by secondary necrosis. We believe that our study provides a new insight into the mechanisms of Pb(NO₃)₂ exposure and its associated adverse health effects.

  3. Is the time dimension of the cell cycle re-entry in AD regulated by centromere cohesion dynamics? (United States)

    Bajić, Vladan P; Spremo-Potparević, Biljana; Zivković, Lada; Djelić, Ninoslav; Smith, Mark A


    Chromosomal involvement is a legitimate, yet not well understood, feature of Alzheimer disease (AD). Firstly, AD affects more women than men. Secondly, the amyloid-β protein precursor genetic mutations, responsible for a cohort of familial AD cases, reside on chromosome 21, the same chromosome responsible for the developmental disorder Down's syndrome. Thirdly, lymphocytes from AD patients display a novel chromosomal phenotype, namely premature centromere separation (PCS). Other documented morphological phenomena associated with AD include the occurrence of micronuclei, aneuploidy, binucleation, telomere instability, and cell cycle re-entry protein expression. Based on these events, here we present a novel hypothesis that the time dimension of cell cycle re-entry in AD is highly regulated by centromere cohesion dynamics. In view of the fact that neurons can re-enter the cell division cycle, our hypothesis predicts that alterations in the signaling pathway leading to premature cell death in neurons is a consequence of altered regulation of the separation of centromeres as a function of time. It is well known that centromeres in the metaphase-anaphase transition separate in a non-random, sequential order. This sequence has been shown to be deregulated in aging cells, various tumors, syndromes of chromosome instability, following certain chemical inductions, as well as in AD. Over time, premature chromosome separation is both a result of, and a driving force behind, further cohesion impairment, activation of cyclin dependent kinases, and mitotic catastrophe, a vicious circle resulting in cellular degeneration and death.

  4. Life-cycle analysis of product integrated polymer solar cells

    DEFF Research Database (Denmark)

    Espinosa Martinez, Nieves; García-Valverde, Rafael; Krebs, Frederik C


    economics through design to functionality. An LCA study was performed to quantify the energy use and greenhouse gas (GHG) emissions from electricity use in the manufacture of a light-weight lamp based on a plastic foil, a lithium-polymer battery, a polymer solar cell, printed circuitry, blocking diode......A life cycle analysis (LCA) on a product integrated polymer solar module is carried out in this study. These assessments are well-known to be useful in developmental stages of a product in order to identify the bottlenecks for the up-scaling in its production phase for several aspects spanning from...... on the complete product integrated polymer solar cell. We have compared this portable lighting system with other lighting solutions, namely: a kerosene lamp in a remote rural area in Africa (Ethiopia), as a replacement of a silicon PV based lamp, in place of a torch with non-rechargeable lead-acid battery...

  5. Robust synchronization of coupled circadian and cell cycle oscillators in single mammalian cells. (United States)

    Bieler, Jonathan; Cannavo, Rosamaria; Gustafson, Kyle; Gobet, Cedric; Gatfield, David; Naef, Felix


    Circadian cycles and cell cycles are two fundamental periodic processes with a period in the range of 1 day. Consequently, coupling between such cycles can lead to synchronization. Here, we estimated the mutual interactions between the two oscillators by time-lapse imaging of single mammalian NIH3T3 fibroblasts during several days. The analysis of thousands of circadian cycles in dividing cells clearly indicated that both oscillators tick in a 1:1 mode-locked state, with cell divisions occurring tightly 5 h before the peak in circadian Rev-Erbα-YFP reporter expression. In principle, such synchrony may be caused by either unidirectional or bidirectional coupling. While gating of cell division by the circadian cycle has been most studied, our data combined with stochastic modeling unambiguously show that the reverse coupling is predominant in NIH3T3 cells. Moreover, temperature, genetic, and pharmacological perturbations showed that the two interacting cellular oscillators adopt a synchronized state that is highly robust over a wide range of parameters. These findings have implications for circadian function in proliferative tissues, including epidermis, immune cells, and cancer.

  6. Cell mass and cell cycle dynamics of an asynchronous budding yeast population

    DEFF Research Database (Denmark)

    Lencastre Fernandes, Rita; Carlquist, Magnus; Lundin, Luisa


    consumption observed during batch cultivation. The good agreement between the proposed multi-scale model (a population balance model [PBM] coupled to an unstructured model) and experimental data (both the overall physiology and cell size and cell cycle distributions) indicates that a mechanistic model...... of model predictions for cell property distributions against experimental data is scarce. This study focuses on the experimental and mathematical description of the dynamics of cell size and cell cycle position distributions, of a population of Saccharomyces cerevisiae, in response to the substrate......Despite traditionally regarded as identical, cells in a microbial cultivation present a distribution of phenotypic traits, forming a heterogeneous cell population. Moreover, the degree of heterogeneity is notably enhanced by changes in micro-environmental conditions. A major development...

  7. Timing robustness in the budding and fission yeast cell cycles.

    KAUST Repository

    Mangla, Karan


    Robustness of biological models has emerged as an important principle in systems biology. Many past analyses of Boolean models update all pending changes in signals simultaneously (i.e., synchronously), making it impossible to consider robustness to variations in timing that result from noise and different environmental conditions. We checked previously published mathematical models of the cell cycles of budding and fission yeast for robustness to timing variations by constructing Boolean models and analyzing them using model-checking software for the property of speed independence. Surprisingly, the models are nearly, but not totally, speed-independent. In some cases, examination of timing problems discovered in the analysis exposes apparent inaccuracies in the model. Biologically justified revisions to the model eliminate the timing problems. Furthermore, in silico random mutations in the regulatory interactions of a speed-independent Boolean model are shown to be unlikely to preserve speed independence, even in models that are otherwise functional, providing evidence for selection pressure to maintain timing robustness. Multiple cell cycle models exhibit strong robustness to timing variation, apparently due to evolutionary pressure. Thus, timing robustness can be a basis for generating testable hypotheses and can focus attention on aspects of a model that may need refinement.

  8. Host cell kinases and the hepatitis C virus life cycle. (United States)

    Colpitts, Che C; Lupberger, Joachim; Doerig, Christian; Baumert, Thomas F


    Hepatitis C virus (HCV) infection relies on virus-host interactions with human hepatocytes, a context in which host cell kinases play critical roles in every step of the HCV life cycle. During viral entry, cellular kinases, including EGFR, EphA2 and PKA, regulate the localization of host HCV entry factors and induce receptor complex assembly. Following virion internalization, viral genomes replicate on endoplasmic reticulum-derived membranous webs. The formation of membranous webs depends on interactions between the HCV NS5a protein and PI4KIIIα. The phosphorylation status of NS5a, regulated by PI4KIIIα, CKI and other kinases, also acts as a molecular switch to virion assembly, which takes place on lipid droplets. The formation of lipid droplets is enhanced by HCV activation of IKKα. In view of the multiple crucial steps in the viral life cycle that are mediated by host cell kinases, these enzymes also represent complementary targets for antiviral therapy. This article is part of a Special Issue entitled: Inhibitors of Protein Kinases.

  9. Trypsin-induced proteome alteration during cell subculture in mammalian cells

    Directory of Open Access Journals (Sweden)

    Lin Cheng-Wen


    Full Text Available Abstract Background It is essential to subculture the cells once cultured cells reach confluence. For this, trypsin is frequently applied to dissociate adhesive cells from the substratum. However, due to the proteolytic activity of trypsin, cell surface proteins are often cleaved, which leads to dysregulation of the cell functions. Methods In this study, a triplicate 2D-DIGE strategy has been performed to monitor trypsin-induced proteome alterations. The differentially expressed spots were identified by MALDI-TOF MS and validated by immunoblotting. Results 36 proteins are found to be differentially expressed in cells treated with trypsin, and proteins that are known to regulate cell metabolism, growth regulation, mitochondrial electron transportation and cell adhesion are down-regulated and proteins that regulate cell apoptosis are up-regulated after trypsin treatment. Further study shows that bcl-2 is down-regulated, p53 and p21 are both up-regulated after trypsinization. Conclusions In summary, this is the first report that uses the proteomic approach to thoroughly study trypsin-induced cell physiological changes and provides researchers in carrying out their experimental design.

  10. Genistein sensitizes ovarian carcinoma cells to chemotherapy by switching the cell cycle progression in vitro

    Institute of Scientific and Technical Information of China (English)

    Huang Yanhong; Yuan Peng; Zhang Qinghong; Xin Xiaoyan


    Objective: To address how genistein sensitizes the chemotherapy-resistant ovarian carcinoma cells and promotes apoptosis in the respect of cell cycle and the regulation of survivin expression in the process. Methods: Ovarian SKOV-3 carcinoma cell line was treated with genistein or cisplatin either alone or in combination. Cell viability was showed by MTT method. Cell cycle and apoptosis were detected by flow cytometry. Survivin mRNA and protein were revealed by RT-PCR and immunocytochemistry, respectively. Results: Genistein could reduce the cell viability in a dose-dependent manner, while cisplatin did so at a much higher level. In contrast, if the two agents were treated in combination, half growth inhibition (IC50) value for cisplatin was reduced remarkably and the effect was synergistic as analyzed by isobologram. In particular, the reduced cell viability was exhibited by a switch in cell cycle progression, as the cells were arrested in G2/M phase and the G0/G1 phase-fraction was significantly decreased. The reduced cell viability appeared to involve apoptosis, based on our results from flow cytometry and Hoechst 33258 staining. In the meanwhile, genistein performed the inhibitory effect on cisplatin-induced survivin expression. Conclusion: Genistein can sensitize ovarian carcinoma cells to cisplatin therapy with the inhibition of survivin expression as the potential mechanism.

  11. Andrographolide inhibits hepatoma cells growth and affects the expression of cell cycle related proteins. (United States)

    Shen, Kai-Kai; Liu, Tian-Yu; Xu, Chong; Ji, Li-Li; Wang, Zheng-Tao


    The present study is aimed to investigate the toxic effects of andrographolide (Andro) on hepatoma cells and elucidate its preliminary mechanisms. After cells were treated with different concentrations of Andro (0-50 micromol x L(-1)) for 24 h, cell viability was evaluated with 3-(4,5-dimethylthiazol-2-yl) 2,5-diphenyltetrazolium bromide (MTT) assay. Furthermore, after hepatoma cells (Hep3B and HepG2) were treated with different concentrations of Andro (0-30 micromol x L(-1)) for 14 d, the number of colony formation was accounted under microscope. Cell cycle related proteins such as Cdc-2, phosphorylated-Cdc-2, Cyclin B and Cyclin D1 were detected with Western blotting assay and the cell cycle was analyzed by flow cytometry using propidium iodide staining. MTT results showed that Andro induced growth inhibition of hepatoma cells in a concentration-dependent manner but had no significant effects on human normal liver L-02 cells. Andro dramatically decreased the colony formation of hepatoma cells in the concentration-dependent manner. Moreover, Andro induced a decrease of Hep3B cells at the G0-G1 phase and a concomitant accumulation of cells at G2-M phase. At the molecular level, Western blotting results showed that Andro decreased the expression of Cdc-2, phosphorylated-Cdc-2, Cyclin D1 and Cyclin B proteins in a time-dependent manner, which are all cell cycle related proteins. Taken together, the results demonstrated that Andro specifically inhibited the growth of hepatoma cells and cellular cell cycle related proteins were possibly involved in this process.

  12. DUBbing cancer: Deubiquitylating enzymes involved in epigenetics, DNA damage and the cell cycle as therapeutic targets

    Directory of Open Access Journals (Sweden)

    Benedikt M Kessler


    Full Text Available Controlling cell proliferation is one of the hallmarks of cancer. A number of critical checkpoints ascertain progression through the different stages of the cell cycle, which can be aborted when perturbed, for instance by errors in DNA replication and repair. These molecular checkpoints are regulated by a number of proteins that need to be present at the right time and quantity. The ubiquitin system has emerged as a central player controlling the fate and function of such molecules such as cyclins, oncogenes and components of the DNA repair machinery. In particular, proteases that cleave ubiquitin chains, referred to as deubiquitylating enzymes (DUBs, have attracted recent attention due to their accessibility to modulation by small molecules. In this review, we describe recent evidence of the critical role of DUBs in aspects of cell cycle checkpoint control, associated DNA repair mechanisms and regulation of transcription, representing pathways altered in cancer. Therefore, DUBs involved in these processes emerge as potentially critical targets for the treatment of not only hematological, but potentially also solid tumors.

  13. DUBbing Cancer: Deubiquitylating Enzymes Involved in Epigenetics, DNA Damage and the Cell Cycle As Therapeutic Targets. (United States)

    Pinto-Fernandez, Adan; Kessler, Benedikt M


    Controlling cell proliferation is one of the hallmarks of cancer. A number of critical checkpoints ascertain progression through the different stages of the cell cycle, which can be aborted when perturbed, for instance by errors in DNA replication and repair. These molecular checkpoints are regulated by a number of proteins that need to be present at the right time and quantity. The ubiquitin system has emerged as a central player controlling the fate and function of such molecules such as cyclins, oncogenes and components of the DNA repair machinery. In particular, proteases that cleave ubiquitin chains, referred to as deubiquitylating enzymes (DUBs), have attracted recent attention due to their accessibility to modulation by small molecules. In this review, we describe recent evidence of the critical role of DUBs in aspects of cell cycle checkpoint control, associated DNA repair mechanisms and regulation of transcription, representing pathways altered in cancer. Therefore, DUBs involved in these processes emerge as potentially critical targets for the treatment of not only hematological, but potentially also solid tumors.

  14. Albumin Suppresses Human Hepatocellular Carcinoma Proliferation and the Cell Cycle

    Directory of Open Access Journals (Sweden)

    Shunsuke Nojiri


    Full Text Available Many investigations have revealed that a low recurrence rate of hepatocellular carcinoma (HCC is associated with high serum albumin levels in patients; therefore, high levels of serum albumin are a major indicator of a favorable prognosis. However, the mechanism inhibiting the proliferation of HCC has not yet been elucidated, so we investigated the effect of serum albumin on HCC cell proliferation. Hep3B was cultured in MEM with no serum or containing 5 g/dL human albumin. As control samples, Prionex was added to generate the same osmotic pressure as albumin. After 24-h incubation, the expressions of α-fetoprotein (AFP, p53, p21, and p57 were evaluated with real-time PCR using total RNA extracted from the liver. Protein expressions and the phosphorylation of Rb (retinoblastoma were determined by Western blot analysis using total protein extracted from the liver. For flow cytometric analysis of the cell cycle, FACS analysis was performed. The percentages of cell cycle distribution were evaluated by PI staining, and all samples were analyzed employing FACScalibur (BD with appropriate software (ModFit LT; BD. The cell proliferation assay was performed by counting cells with using a Scepter handy automated cell counter (Millipore. The mRNA levels of AFP relative to Alb(−: Alb(−, Alb(+, and Prionex, were 1, 0.7 ± 0.2 (p < 0.001 for Alb(−, and 1 ± 0.3, respectively. The mRNA levels of p21 were 1, 1.58 ± 0.4 (p = 0.007 for Alb(− and p = 0.004 for Prionex, and 0.8 ± 0.2, respectively. The mRNA levels of p57 were 1, 4.4 ± 1.4 (p = 0.002 for Alb(− and Prionex, and 1.0 ± 0.1, respectively. The protein expression levels of Rb were similar in all culture media. The phosphorylation of P807/811 and P780 of Rb protein was reduced in Alb(+. More cells in the G0/G1 phase and fewer cells in S and G2/M phases were obtained in Alb(+ than in Alb(− (G0/G1: 60.9%, 67.7%, 61.5%; G2/M: 16.5%, 13.1%, 15.6%; S: 22.6%, 19.2%, 23.0%, Alb(−, Alb

  15. Effects of cell-cycle-dependent expression on random fluctuations in protein levels. (United States)

    Soltani, Mohammad; Singh, Abhyudai


    Expression of many genes varies as a cell transitions through different cell-cycle stages. How coupling between stochastic expression and cell cycle impacts cell-to-cell variability (noise) in the level of protein is not well understood. We analyse a model where a stable protein is synthesized in random bursts, and the frequency with which bursts occur varies within the cell cycle. Formulae quantifying the extent of fluctuations in the protein copy number are derived and decomposed into components arising from the cell cycle and stochastic processes. The latter stochastic component represents contributions from bursty expression and errors incurred during partitioning of molecules between daughter cells. These formulae reveal an interesting trade-off: cell-cycle dependencies that amplify the noise contribution from bursty expression also attenuate the contribution from partitioning errors. We investigate the existence of optimum strategies for coupling expression to the cell cycle that minimize the stochastic component. Intriguingly, results show that a zero production rate throughout the cell cycle, with expression only occurring just before cell division, minimizes noise from bursty expression for a fixed mean protein level. By contrast, the optimal strategy in the case of partitioning errors is to make the protein just after cell division. We provide examples of regulatory proteins that are expressed only towards the end of the cell cycle, and argue that such strategies enhance robustness of cell-cycle decisions to the intrinsic stochasticity of gene expression.

  16. Sinorhizobium meliloti CpdR1 is critical for co-ordinating cell cycle progression and the symbiotic chronic infection. (United States)

    Kobayashi, Hajime; De Nisco, Nicole J; Chien, Peter; Simmons, Lyle A; Walker, Graham C


    ATP-driven proteolysis plays a major role in regulating the bacterial cell cycle, development and stress responses. In the nitro -fixing symbiosis with host plants, Sinorhizobium meliloti undergoes a profound cellular differentiation, including endoreduplication of the ome. The regulatory mechanisms governing the alterations of the S. meliloti cell cycle in planta are largely unknown. Here, we report the characterization of two cpdR homologues, cpdR1 and cpdR2, of S. meliloti that encode single-domain response regulators. In Caulobacter crescentus, CpdR controls the polar localization of the ClpXP protease, thereby mediating the regulated proteolysis of key protein(s), such as CtrA, involved in cell cycle progression. The S. meliloti cpdR1-null mutant can invade the host cytoplasm, however, the intracellular bacteria are unable to differentiate into bacteroids. We show that S. meliloti CpdR1 has a polar localization pattern and a role in ClpX positioning similar to C. crescentus CpdR, suggesting a conserved function of CpdR proteins among alpha-proteobacteria. However, in S. meliloti, free-living cells of the cpdR1-null mutant show a striking morphology of irregular coccoids and aberrant DNA replication. Thus, we demonstrate that CpdR1 mediates the co-ordination of cell cycle events, which are critical for both the free-living cell division and the differentiation required for the chronic intracellular infection.

  17. A cell cycle timer for asymmetric spindle positioning.

    Directory of Open Access Journals (Sweden)

    Erin K McCarthy Campbell


    Full Text Available The displacement of the mitotic spindle to one side of a cell is important for many cells to divide unequally. While recent progress has begun to unveil some of the molecular mechanisms of mitotic spindle displacement, far less is known about how spindle displacement is precisely timed. A conserved mitotic progression mechanism is known to time events in dividing cells, although this has never been linked to spindle displacement. This mechanism involves the anaphase-promoting complex (APC, its activator Cdc20/Fizzy, its degradation target cyclin, and cyclin-dependent kinase (CDK. Here we show that these components comprise a previously unrecognized timer for spindle displacement. In the Caenorhabditis elegans zygote, mitotic spindle displacement begins at a precise time, soon after chromosomes congress to the metaphase plate. We found that reducing the function of the proteasome, the APC, or Cdc20/Fizzy delayed spindle displacement. Conversely, inactivating CDK in prometaphase caused the spindle to displace early. The consequence of experimentally unlinking spindle displacement from this timing mechanism was the premature displacement of incompletely assembled components of the mitotic spindle. We conclude that in this system, asymmetric positioning of the mitotic spindle is normally delayed for a short time until the APC inactivates CDK, and that this delay ensures that the spindle does not begin to move until it is fully assembled. To our knowledge, this is the first demonstration that mitotic progression times spindle displacement in the asymmetric division of an animal cell. We speculate that this link between the cell cycle and asymmetric cell division might be evolutionarily conserved, because the mitotic spindle is displaced at a similar stage of mitosis during asymmetric cell divisions in diverse systems.

  18. Live-cell monitoring of periodic gene expression in synchronous human cells identifies Forkhead genes involved in cell cycle control. (United States)

    Grant, Gavin D; Gamsby, Joshua; Martyanov, Viktor; Brooks, Lionel; George, Lacy K; Mahoney, J Matthew; Loros, Jennifer J; Dunlap, Jay C; Whitfield, Michael L


    We developed a system to monitor periodic luciferase activity from cell cycle-regulated promoters in synchronous cells. Reporters were driven by a minimal human E2F1 promoter with peak expression in G1/S or a basal promoter with six Forkhead DNA-binding sites with peak expression at G2/M. After cell cycle synchronization, luciferase activity was measured in live cells at 10-min intervals across three to four synchronous cell cycles, allowing unprecedented resolution of cell cycle-regulated gene expression. We used this assay to screen Forkhead transcription factors for control of periodic gene expression. We confirmed a role for FOXM1 and identified two novel cell cycle regulators, FOXJ3 and FOXK1. Knockdown of FOXJ3 and FOXK1 eliminated cell cycle-dependent oscillations and resulted in decreased cell proliferation rates. Analysis of genes regulated by FOXJ3 and FOXK1 showed that FOXJ3 may regulate a network of zinc finger proteins and that FOXK1 binds to the promoter and regulates DHFR, TYMS, GSDMD, and the E2F binding partner TFDP1. Chromatin immunoprecipitation followed by high-throughput sequencing analysis identified 4329 genomic loci bound by FOXK1, 83% of which contained a FOXK1-binding motif. We verified that a subset of these loci are activated by wild-type FOXK1 but not by a FOXK1 (H355A) DNA-binding mutant.

  19. Effect of Docosahexaenoic Acid on Cell Cycle Pathways in Breast Cell Lines With Different Transformation Degree. (United States)

    Rescigno, Tania; Capasso, Anna; Tecce, Mario Felice


    n-3 polyunsaturated fatty acids (PUFAs), such as eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), abundant in fish, have been shown to affect development and progression of some types of cancer, including breast cancer. The aim of our study was to further analyze and clarify the effects of these nutrients on the molecular mechanisms underlying breast cancer. Following treatments with DHA we examined cell viability, death, cell cycle, and some molecular effects in breast cell lines with different transformation, phenotypic, and biochemical characteristics (MCF-10A, MCF-7, SK-BR-3, ZR-75-1). These investigations showed that DHA is able to affect cell viability, proliferation, and cell cycle progression in a different way in each assayed breast cell line. The activation of ERK1/2 and STAT3 pathways and the expression and/or activation of molecules involved in cell cycle regulation such as p21(Waf1/Cip1) and p53, are very differently regulated by DHA treatments in each cell model. DHA selectively: (i) arrests non tumoral MCF-10A breast cells in G0 /G1 cycle phase, activating p21(Waf1/Cip1) , and p53, (ii) induces to death highly transformed breast cells SK-BR-3, reducing ERK1/2 and STAT3 phosphorylation and (iii) only slightly affects each analyzed process in MCF-7 breast cell line with transformation degree lower than SK-BR-3 cells. These findings suggest a more relevant inhibitory role of DHA within early development and late progression of breast cancer cell transformation and a variable effect in the other phases, depending on individual molecular properties and degree of malignancy of each clinical case.

  20. Glucose capped silver nanoparticles induce cell cycle arrest in HeLa cells. (United States)

    Panzarini, Elisa; Mariano, Stefania; Vergallo, Cristian; Carata, Elisabetta; Fimia, Gian Maria; Mura, Francesco; Rossi, Marco; Vergaro, Viviana; Ciccarella, Giuseppe; Corazzari, Marco; Dini, Luciana


    This study aims to determine the interaction (uptake and biological effects on cell viability and cell cycle progression) of glucose capped silver nanoparticles (AgNPs-G) on human epithelioid cervix carcinoma (HeLa) cells, in relation to amount, 2×10(3) or 2×10(4) NPs/cell, and exposure time, up to 48h. The spherical and well dispersed AgNPs (30±5nm) were obtained by using glucose as reducing agent in a green synthesis method that ensures to stabilize AgNPs avoiding cytotoxic soluble silver ions Ag(+) release. HeLa cells take up abundantly and rapidly AgNPs-G resulting toxic to cells in amount and incubation time dependent manner. HeLa cells were arrested at S and G2/M phases of the cell cycle and subG1 population increased when incubated with 2×10(4) AgNPs-G/cell. Mitotic index decreased accordingly. The dissolution experiments demonstrated that the observed effects were due only to AgNPs-G since glucose capping prevents Ag(+) release. The AgNPs-G influence on HeLa cells viability and cell cycle progression suggest that AgNPs-G, alone or in combination with chemotherapeutics, may be exploited for the development of novel antiproliferative treatment in cancer therapy. However, the possible influence of the cell cycle on cellular uptake of AgNPs-G and the mechanism of AgNPs entry in cells need further investigation.

  1. Genetic alteration in notch pathway is associated with better prognosis in renal cell carcinoma. (United States)

    Feng, Chenchen; Xiong, Zuquan; Jiang, Haowen; Ding, Qiang; Fang, Zujun; Hui, Wen


    Notch signaling was associated with a variety of cancers but was not comprehensively studied in clear-cell renal cell carcinoma (ccRCC). We have in this study studied the genetic alteration (mutation and copy number variance) of Notch gene set in the Cancer Genome Atlas (TCGA) Kidney Renal Clear Cell Carcinoma (KIRC) database. We found that Notch pathway was frequently altered in ccRCC. The Notch gene set was genetically altered in 182 (44%) of the 415 ccRCC patients. CNV was the predominant type of alteration in most genes. Alterations in KAT2B and MAML1 occurred in 13% and 19% of patients, respectively, both of which were functionally active in ccRCC. Deletion of VHL was exclusively found in cases with Notch alteration. Overall survival was longer in ccRCC patients with altered-Notch pathway. The median survival was 90.41 months in Notch-altered cases and 69.15 in Notch-unaltered cases (P = 0.0404). The median disease free time was 89.82 months in Notch-altered cases and 77.27 months in in Notch-unaltered cases (P = 0.935). Conclusively, Notch signaling was altered in almost half of the ccRCC patients and copy number variances in MAML1 and KAT2B were predominant changes. These findings broadened our understanding of the role of Notch in ccRCC.

  2. Cell cycle synchronization of canine ear fibroblasts for somatic cell nuclear transfer. (United States)

    Koo, Ok Jae; Hossein, Mohammad Shamim; Hong, So Gun; Martinez-Conejero, Jose A; Lee, Byeong Chun


    Cycle synchronization of donor cells in the G0/G1 stage is a crucial step for successful somatic cell nuclear transfer. In the present report, we evaluated the effects of contact inhibition, serum starvation and the reagents - dimethyl sulphoxide (DMSO), roscovitine and cycloheximide (CHX) - on synchronization of canine fibroblasts at the G0/G1 stage. Ear fibroblast cells were collected from a beagle dog, placed into culture and used for analysis at passages three to eight. The population doubling time was 36.5 h. The proportion of G0/G1 cells was significantly increased by contact inhibition (77.1%) as compared with cycling cells (70.1%); however, extending the duration of culture did not induce further synchronization. After 24 h of serum starvation, cells were effectively synchronized at G0/G1 (77.1%). Although synchronization was further increased gradually after 24 h and even showed significant difference after 72 h (82.8%) of starvation, the proportion of dead cells also significantly increased after 24 h. The percentage of cells at the G0/G1 phase was increased (as compared with controls) after 72 h treatment with DMSO (76.1%) and after 48 h treatment with CHX (73.0%) or roscovitine (72.5%). However, the rate of cell death was increased after 24 and 72 h of treatment with DMSO and CHX, respectively. Thus, we recommend the use of roscovitine for cell cycle synchronization of canine ear fibroblasts as a preparatory step for SCNT.

  3. Stromal interaction molecule 1 regulates growth, cell cycle, and apoptosis of human tongue squamous carcinoma cells. (United States)

    Cui, Xiaobo; Song, Laixiao; Bai, Yunfei; Wang, Yaping; Wang, Boqian; Wang, Wei


    Oral tongue squamous cell carcinoma (OTSCC) is the most common type of oral carcinomas. However, the molecular mechanism by which OTSCC developed is not fully identified. Stromal interaction molecule 1 (STIM1) is a transmembrane protein, mainly located in the endoplasmic reticulum (ER). STIM1 is involved in several types of cancers. Here, we report that STIM1 contributes to the development of human OTSCC. We knocked down STIM1 in OTSCC cell line Tca-8113 with lentivirus-mediated shRNA and found that STIM1 knockdown repressed the proliferation of Tca-8113 cells. In addition, we also showed that STIM1 deficiency reduced colony number of Tca-8113 cells. Knockdown of STIM1 repressed cells to enter M phase of cell cycle and induced cellular apoptosis. Furthermore, we performed microarray and bioinformatics analysis and found that STIM1 was associated with p53 and MAPK pathways, which may contribute to the effects of STIM1 on cell growth, cell cycle, and apoptosis. Finally, we confirmed that STIM1 controlled the expression of MDM2, cyclin-dependent kinase 4 (CDK4), and growth arrest and DNA damage inducible α (GADD45A) in OTSCC cells. In conclusion, we provide evidence that STIM1 contributes to the development of OTSCC partially through regulating p53 and MAPK pathways to promote cell cycle and survival.

  4. Cycle life test and failure model of nickel-hydrogen cells (United States)

    Smithrick, J. J.


    Six ampere hour individual pressure vessel nickel hydrogen cells were charge/discharge cycled to failure. Failure as used here is defined to occur when the end of discharge voltage degraded to 0.9 volts. They were cycled under a low earth orbit cycle regime to a deep depth of discharge (80 percent of rated ampere hour capacity). Both cell designs were fabricated by the same manufacturer and represent current state of the art. A failure model was advanced which suggests both cell designs have inadequate volume tolerance characteristics. The limited existing data base at a deep depth of discharge (DOD) was expanded. Two cells of each design were cycled. One COMSAT cell failed at cycle 1712 and the other failed at cycle 1875. For the Air Force/Hughes cells, one cell failed at cycle 2250 and the other failed at cycle 2638. All cells, of both designs, failed due to low end of discharge voltage (0.9 volts). No cell failed due to electrical shorts. After cell failure, three different reconditioning tests (deep discharge, physical reorientation, and open circuit voltage stand) were conducted on all cells of each design. A fourth reconditioning test (electrolyte addition) was conducted on one cell of each design. In addition post cycle cell teardown and failure analysis were performed on the one cell of each design which did not have electrolyte added after failure. Previously announced in STAR as N83-25038

  5. Mechanisms involved in ceramide-induced cell cycle arrest in human hepatocarcinoma cells

    Institute of Scientific and Technical Information of China (English)

    Jing Wang; Xiao-Wen Lv; Jie-Ping Shi; Xiao-Song Hu


    AIM:To investigate the effect of ceramide on the cell cycle in human hepatocarcinoma Bel7402 cells.Possible molecular mechanisms were explored.METHODS:[3-(4,5)-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide(MTT)assay,plasmid transfection,reporter assay,FACS and Western blotting analyses were employed to investigate the effect and the related molecular mechanisms of C2-ceramide on the cell cycle of Bel7402 cells.RESULTS:C2-ceramide was found to inhibit the growth of Bel7402 cells by inducing cell cycle arrest.During the process,the expression of p21 protein increased,while that of cyclinD1,phospho-ERK1/2 and c-myc decreased.Furthermore,the level of CDK7 was downregulated,while the transcriptional activity of PPARγ was upregulated.Addition of GW9662,which is a PPARγ specific antagonist,could reserve the modulation action on CDK7.CONCLUSION:Our results support the hypothesis that cell cycle arrest induced by C2-ceramide may be mediated via accumulation of p21 and reduction of cyclinD1 and CDK7,at least partly,through PPARγ activation.The ERK signaling pathway was involved in this process.

  6. Discovery of a Splicing Regulator Required for Cell Cycle Progression

    Energy Technology Data Exchange (ETDEWEB)

    Suvorova, Elena S.; Croken, Matthew; Kratzer, Stella; Ting, Li-Min; Conde de Felipe, Magnolia; Balu, Bharath; Markillie, Lye Meng; Weiss, Louis M.; Kim, Kami; White, Michael W.


    In the G1 phase of the cell division cycle, eukaryotic cells prepare many of the resources necessary for a new round of growth including renewal of the transcriptional and protein synthetic capacities and building the machinery for chromosome replication. The function of G1 has an early evolutionary origin and is preserved in single and multicellular organisms, although the regulatory mechanisms conducting G1 specific functions are only understood in a few model eukaryotes. Here we describe a new G1 mutant from an ancient family of apicomplexan protozoans. Toxoplasma gondii temperature-sensitive mutant 12-109C6 conditionally arrests in the G1 phase due to a single point mutation in a novel protein containing a single RNA-recognition-motif (TgRRM1). The resulting tyrosine to asparagine amino acid change in TgRRM1 causes severe temperature instability that generates an effective null phenotype for this protein when the mutant is shifted to the restrictive temperature. Orthologs of TgRRM1 are widely conserved in diverse eukaryote lineages, and the human counterpart (RBM42) can functionally replace the missing Toxoplasma factor. Transcriptome studies demonstrate that gene expression is downregulated in the mutant at the restrictive temperature due to a severe defect in splicing that affects both cell cycle and constitutively expressed mRNAs. The interaction of TgRRM1 with factors of the tri-SNP complex (U4/U6 & U5 snRNPs) indicate this factor may be required to assemble an active spliceosome. Thus, the TgRRM1 family of proteins is an unrecognized and evolutionarily conserved class of splicing regulators. This study demonstrates investigations into diverse unicellular eukaryotes, like the Apicomplexa, have the potential to yield new insights into important mechanisms conserved across modern eukaryotic kingdoms.

  7. Transcription of ribosomal RNA genes is initiated in the third cell cycle of bovine embryos

    DEFF Research Database (Denmark)

    Jakobsen, Anne Sørig; Avery, Birthe; Dieleman, Steph J.


    polymerase I. In conclusion, rRNA transcription is initiated during the third cell cycle at a low level in both in vivo developed and in vitro produced bovine embryos. Transcription seems to be interrupted during the G1 phase of the fourth cell cycle, but reinitiates in the late half of the cycle...

  8. Mechanisms involved in alternariol-induced cell cycle arrest

    Energy Technology Data Exchange (ETDEWEB)

    Solhaug, A., E-mail: [Norwegian Veterinary Institute, Oslo (Norway); Vines, L.L. [Michigan State University, Department of Food Science and Human Nutrition, East Lansing, MI (United States); Ivanova, L.; Spilsberg, B. [Norwegian Veterinary Institute, Oslo (Norway); Holme, J.A. [Norwegian Institute of Public Health, Division of Environmental Medicine, Oslo (Norway); Pestka, J. [Michigan State University, Department of Food Science and Human Nutrition, East Lansing, MI (United States); Collins, A. [University of Oslo, Department of Nutrition, Faculty of Medicine, Oslo (Norway); Eriksen, G.S. [Norwegian Veterinary Institute, Oslo (Norway)


    Alternariol (AOH), a mycotoxin produced by Alternaria sp, is often found as a contaminant in fruit and cereal products. Here we employed the murine macrophage cell line RAW 264.7 to test the hypothesis that AOH causes toxicity as a response to DNA damage. AOH at concentrations of 15-30 {mu}M almost completely blocked cell proliferation. Within 30 min treatment, AOH (30 {mu}M) significantly increased the level of reactive oxygen species (ROS). Furthermore, DNA base oxidations as well as DNA strand breaks and/or alkaline labile sites were detected by the comet assay after 2 h exposure of AOH. Cell death (mostly necrosis) was observed after prolonged exposure to the highest concentration of AOH (60 {mu}M for 24 and 48 h) in our study. The DNA damage response involved phosphorylation (activation) of histone H2AX and check point kinase-1- and 2 (Chk-1/2). Moreover, AOH activated p53 and increased the expression of p21, Cyclin B, MDM2, and Sestrin 2; likewise the level of several miRNA was affected. AOH-induced Sestrin 2 expression was regulated by p53 and could at least partly be inhibited by antioxidants, suggesting a role of ROS in the response. Interestingly, the addition of antioxidants did not inhibit cell cycle arrest. Although the formation of ROS by itself was not directly linked cell proliferation, AOH-induced DNA damage and resulting transcriptional changes in p21, MDM2, and Cyclin B likely contribute to the reduced cell proliferation; while Sestrin 2 would contribute to the oxidant defense.

  9. Meiotic and Mitotic Cell Cycle Mutants Involved in Gametophyte Development in Arabidopsis

    Institute of Scientific and Technical Information of China (English)

    Jingjing Liu; Li-Jia Qu


    The alternation between diploid and haploid generations is fundamentalin the life cycles of both animals and plants.The meiotic cell cycle is common to both animals and plants gamete formation, but in animals the products of meiosis are gametes,whereas for most plants,subsequent mitotic cell cycles are needed for their formation. Clarifying the regulatory mechanisms of mitotic cell cycle progression during gametophyte development will help understanding of sexual reproduction in plants.Many mutants defective in gametophyte development and,in particular,many meiotic and mitotic cell cycle mutants in Arabidopsis male and female gametophyte development were identified through both forward and reverse genetics approaches.

  10. Effects of anti-IgM suppression on polyclonally activated murine B cells: analysis of immunoglobulin mRNA, gene specific nuclear factors and cell cycle distribution. (United States)

    Marcuzzi, A; Van Ness, B; Rouse, T; Lafrenz, D


    Polyclonal activation of murine B cells with bacterial lipopolysaccharide (LPS) and dextran sulfate (DxS) results in cell proliferation as well as increased immunoglobulin gene transcription and antibody secretion. When added to B cell cultures during mitogen activation, anti-mu antibody suppresses the rate of proliferation and immunoglobulin gene expression. Using this model system we show that co-cultures of B cells with LPS/DxS and anti-mu resulted in a decrease of both mu and kappa chain mRNA. Suppression did not prevent B cell entry into cycle nor a significant alteration in the distribution of cells in phases of cell cycle, although it did prolong the cycle transit time in a dose dependent fashion as determined by bromodeoxyuridine pulse labelling. Analysis of B cell specific nuclear binding factors, which previously have been shown to be important in regulating immunoglobulin gene transcription were examined. Results show that the kappa-specific enhancer binding activity of NF-kappa B was induced in activated as well as suppressed cultures. The lymphoid specific factor NF-A2, which recognizes the octamer sequence motif in the promoters of immunoglobulin genes, was induced by the polyclonal activation but was selectively lost in extracts from suppressed cells. Thus, specific regulation of the nuclear factor which plays a critical role in both heavy and light chain immunoglobulin gene expression may contribute to the transcriptional suppression observed in anti-mu treated B cells. Images PMID:2481271

  11. Altered status of glutathione and its metabolites in cystinotic cells.

    NARCIS (Netherlands)

    Levtchenko, E.N.; Graaf-Hess, A.C. de; Wilmer, M.J.G.; Heuvel, L.P.W.J. van den; Monnens, L.A.H.; Blom, H.J.


    BACKGROUND: Cystinosis is an autosomal recessive disorder, caused by mutations of the lysosomal cystine carrier cystinosin, encoded by the CTNS gene (17p13). The concomitant intralysosomal cystine accumulation leads to multi-organ damage, with kidneys being the first affected. Altered mitochondrial

  12. Virus Innexins induce alterations in insect cell and tissue function (United States)

    Polydnaviruses are dsDNA viruses that induce immune and developmental alterations in their caterpillar hosts. Characterization of polydnavirus gene families and family members is necessary to understand mechanisms of pathology and evolution of these viruses, and may aid to elucidate the role of host...

  13. Alterations in the growth and adhesion pattern of Vero cells induced by nutritional stress conditions. (United States)

    Genari, S C; Gomes, L; Wada, M L


    The pattern of growth, adhesion and protein synthesis in Vero cells submitted to nutritional stress conditions was investigated. The control cells presented a characteristic pattern, with monolayer growth, while the stressed cells presented multilayered growth, with aggregate or spheroid formation which detached on the flask surface and continued their growth in another region. In the soft agar assay, with reduced amount of nutrients, only the stressed cells presented growth, indicating physical and nutritional independence. A 44-kDa protein was observed in stressed cells and was absent in non-stressed cells. The adhesion index and fibronectin synthesis and distribution were altered in stressed cells. After confluence, control cells presented fibronectin accumulation in lateral cell-cell contact regions, while this fibronectin accumulation pattern was not observed in stressed cells. These alterations may be responsible for the multilayered growth and decreased adhesion index observed in stressed cells which were transformed by nutritional stress conditions.

  14. Quantitative imaging with Fucci and mathematics to uncover temporal dynamics of cell cycle progression. (United States)

    Saitou, Takashi; Imamura, Takeshi


    Cell cycle progression is strictly coordinated to ensure proper tissue growth, development, and regeneration of multicellular organisms. Spatiotemporal visualization of cell cycle phases directly helps us to obtain a deeper understanding of controlled, multicellular, cell cycle progression. The fluorescent ubiquitination-based cell cycle indicator (Fucci) system allows us to monitor, in living cells, the G1 and the S/G2/M phases of the cell cycle in red and green fluorescent colors, respectively. Since the discovery of Fucci technology, it has found numerous applications in the characterization of the timing of cell cycle phase transitions under diverse conditions and various biological processes. However, due to the complexity of cell cycle dynamics, understanding of specific patterns of cell cycle progression is still far from complete. In order to tackle this issue, quantitative approaches combined with mathematical modeling seem to be essential. Here, we review several studies that attempted to integrate Fucci technology and mathematical models to obtain quantitative information regarding cell cycle regulatory patterns. Focusing on the technological development of utilizing mathematics to retrieve meaningful information from the Fucci producing data, we discuss how the combined methods advance a quantitative understanding of cell cycle regulation.

  15. Altering β-cell number through stable alteration of miR-21 and miR-34a expression

    DEFF Research Database (Denmark)

    Backe, Marie Balslev; Novotny, Guy Wayne; Christensen, Dan Ploug;


    RNAs, miR-21 and miR-34a, may be involved in mediating cytokine-induced β-cell dysfunction. Therefore, manipulation of miR-21 and miR-34a levels may potentially be beneficial to β cells. To study the effect of long-term alterations of miR-21 or miR-34a levels upon net β-cell number, we stably overexpressed...... miR-21 and knocked down miR-34a, and investigated essential cellular processes. Materials and Methods: miRNA expression was manipulated using Lentiviral transduction of the β-cell line INS-1. Stable cell lines were generated, and cell death, NO synthesis, proliferation, and total cell number were...... monitored in the absence or presence of cytokines. Results: Overexpression of miR-21 decreased net β-cell number in the absence of cytokines, and increased apoptosis and NO synthesis in the absence and presence of cytokines. Proliferation was increased upon miR-21 overexpression. Knockdown of miR-34a...

  16. Taxol induces concentration-dependent phosphatidylserine (PS) externalization and cell cycle arrest in ASTC-a-1 cells (United States)

    Guo, Wen-jing; Chen, Tong-sheng


    Taxol (Paclitaxel) is an important natural product for the treatment of solid tumors. Different concentrations of taxol can trigger distinct effects on both the cellular microtubule network and biochemical pathways. Apoptosis induced by low concentrations (5-30 nM) of taxol was associated with mitotic arrest, alteration of microtubule dynamics and/or G2/M cell cycle arrest, whereas high concentrations of this drug (0.2-30 μM) caused significant microtubule damage, and was found recently to induce cytoplasm vacuolization in human lung adenocarcinoma (ASTC-a-1) cells. In present study, cell counting kit (CCK-8) assay, confocal microscope, and flow cytometry analysis were used to analyze the cell death form induced by 35 nM and 70 μM of taxol respectively in human lung adenocarcinoma (ASTC-a-1) cells. After treatment of 35 nM taxol for 48 h, the OD450 value was 0.80, and 35 nM taxol was found to induce dominantly cell death in apoptotic pathway such as phosphatidylserine (PS) externalization, G2/M phase arrest after treatment for 24 h, and nuclear fragmentation after treatment for 48 h. After 70 μM taxol treated the cell for 24 h, the OD450 value was 1.01, and 70 μM taxol induced cytoplasm vacuolization programmed cell death (PCD) and G2/M phase as well as the polyploidy phase arrest in paraptotic-like cell death. These findings imply that the regulated signaling pathway of cell death induced by taxol is dependent on taxol concentration in ASTC-a-1 cells.

  17. Change of the cell cycle after flutamide treatment in prostate cancer cells and its molecular mechanism

    Institute of Scientific and Technical Information of China (English)

    Yong Wang; Wei-Jun Qin; He Wang; Guo-Xing Shao; Chen Shao; Chang-Hong Shi; Lei Zhang; Hong-Hong Yue; Peng-Fei Wang; Bo Yang; Yun-Tao Zhang; Fan Liu


    Aim: To explore the effect of androgen receptor (AR) on the expression of the cell cycle-related genes, such as CDKN1A and BTG1, in prostate cancer cell line LNCaP. Methods: After AR antagonist flutamide treatment and confirmation of its effect by phase contrast microscope and flow cytometry, the differential expression of the cell cycle-related genes was analyzed by a cDNA microarray. The flutamide treated cells were set as the experimental group and the LNCaP cells as the control. We labeled cDNA probes of the experimental group and control group with Cy5 and Cy3 dyes, respectively, through reverse transcription. Then we hybridized the cDNA probes with cDNA microarrays, which contained 8 126 unique human cDNA sequences and the chip was scanned to get the fluorescent values of Cy5 and Cy3 on each spot. After primary analysis, reverse transcription polymerase chain reaction (RTPCR) tests were carried out to confirm the results of the chips. Results:After AR antagonist flutamide treatment,three hundred and twenty-six genes (3.93 %) expressed differentially, 97 down-regulated and 219 up-regulated.Among them, eight up-regulated genes might be cell cycle-related, namely CDC10, NRAS, BTG1, Weel, CLK3,DKFZP564A122, CDKN1A and BTG2. The CDKN1A and BTG1 gene mRNA expression was confirmed to be higher in the experimental group by RT-PCR, whilep53 mRNA expression had no significant changes. Conclusion: Flutamide treatment might up-regulate CDKN1A and BTG1 expression in prostate cancer cells. The protein expressions of CDKN1A and BTG1 play an important role in inhibiting the proliferation of cancer cells. CDKN1A has a great impact on the cell cycle of prostate cancer cells and may play a role in the cancer cells in a p53-independent pathway. The prostate cancer cells might affect the cell cycle-related genes by activating AR and thus break the cell cycle control.

  18. Alterations in Cell Signaling Pathways in Breast Cancer Cells after Environmental Exposure

    Energy Technology Data Exchange (ETDEWEB)

    Kulp, K; McCutcheon-Maloney, S M; Bennett, L M


    Recent human epidemiological studies suggest that up to 75% of human cancers can be attributed to environmental exposures. Understanding the biologic impact of being exposed to a lifetime of complex environmental mixtures that may not be fully characterized is currently a major challenge. Functional endpoints may be used to assess the gross health consequences of complex mixture exposures from groundwater contamination, superfund sites, biologic releases, or nutritional sources. Such endpoints include the stimulation of cell growth or the induction of a response in an animal model. An environmental exposure that upsets normal cell growth regulation may have important ramifications for cancer development. Stimulating cell growth may alter an individual's cancer risk by changing the expression of genes and proteins that have a role in growth regulatory pathways within cells. Modulating the regulation of these genes and their products may contribute to the initiation, promotion or progression of disease in response to environmental exposure. We are investigating diet-related compounds that induce cell proliferation in breast cancer cell lines. These compounds, PhIP, Flor-Essence{reg_sign} and Essiac{reg_sign}, may be part of an everyday diet. PhIP is a naturally occurring mutagen that is formed in well-cooked muscle meats. PhIP consistently causes dose-dependent breast tumor formation in rats and consumption of well-done meat has been linked to increased risk of breast cancer in women. Flor-Essence{reg_sign} and Essiac{reg_sign} herbal tonics are complementary and alternative medicines used by women who have been diagnosed with breast cancer as an alternative therapy for disease treatment and prevention. The long-term goal of this work is to identify those cellular pathways that are altered by a chemical or biologic environmental exposure and understand how those changes correlate with and or predict changes in human health risk. This project addressed this goal

  19. Imaging Nuclear Morphology and Organization in Cleared Plant Tissues Treated with Cell Cycle Inhibitors. (United States)

    de Souza Junior, José Dijair Antonino; de Sa, Maria Fatima Grossi; Engler, Gilbert; Engler, Janice de Almeida


    Synchronization of root cells through chemical treatment can generate a large number of cells blocked in specific cell cycle phases. In plants, this approach can be employed for cell suspension cultures and plant seedlings. To identify plant cells in the course of the cell cycle, especially during mitosis in meristematic tissues, chemical inhibitors can be used to block cell cycle progression. Herein, we present a simplified and easy-to-apply protocol to visualize mitotic figures, nuclei morphology, and organization in whole Arabidopsis root apexes. The procedure is based on tissue clearing, and fluorescent staining of nuclear DNA with DAPI. The protocol allows carrying out bulk analysis of nuclei and cell cycle phases in root cells and will be valuable to investigate mutants like overexpressing lines of genes disturbing the plant cell cycle.

  20. Down-regulation of DNA topoisomerase IIalpha leads to prolonged cell cycle transit in G2 and early M phases and increased survival to microtubule-interacting agents. (United States)

    Skladanowski, Andrzej; Côme, Marie-George; Sabisz, Michal; Escargueil, Alexandre E; Larsen, Annette K


    Microtubule binders are cell cycle-specific agents with preferential cytotoxicity toward mitotic cells. We have characterized vincristine-selected human leukemia cells to establish whether development of vincristine resistance was accompanied by changes in cell cycle kinetics and distribution. Our results indicate that vincristine resistance is accompanied by delayed G2 transit and prolonged early mitosis in both the absence and the presence of the microtubule binder nocodazole. The altered G2/M regulation is accompanied by resistance to short-term (12 h) but not continuous nocodazole exposure in agreement with the transient nature of the observed cell cycle alterations. Western blot analysis indicates that vincristine-selection is accompanied by down-regulation of topoisomerase IIalpha without detectable alterations of the other mitotic regulators studied, including Cdk1, p21, 14-3-3sigma, and 14-3-3epsilon. This was associated with at least 7-fold less chromosome-associated topoisomerase IIalpha, decreased catalytic activity, and cross-resistance to topoisomerase II inhibitors. Characterization of isogenic cell lines expressing different levels of topoisomerase II proteins shows that cellular levels of topoisomerase IIalpha, but not the closely related topoisomerase IIbeta, directly influence the cell cycle kinetics in G2 and early mitosis as well as the resistance to nocodazole. These results underline the importance of topoisomerase IIalpha in late G2 and early M phases and provide evidence for an as-yet-unsuspected interaction between topoisomerase II and microtubule-directed agents.

  1. Notch Pathway Is Activated via Genetic and Epigenetic Alterations and Is a Therapeutic Target in Clear Cell Renal Cancer. (United States)

    Bhagat, Tushar D; Zou, Yiyu; Huang, Shizheng; Park, Jihwan; Palmer, Matthew B; Hu, Caroline; Li, Weijuan; Shenoy, Niraj; Giricz, Orsolya; Choudhary, Gaurav; Yu, Yiting; Ko, Yi-An; Izquierdo, María C; Park, Ae Seo Deok; Vallumsetla, Nishanth; Laurence, Remi; Lopez, Robert; Suzuki, Masako; Pullman, James; Kaner, Justin; Gartrell, Benjamin; Hakimi, A Ari; Greally, John M; Patel, Bharvin; Benhadji, Karim; Pradhan, Kith; Verma, Amit; Susztak, Katalin


    Clear cell renal cell carcinoma (CCRCC) is an incurable malignancy in advanced stages and needs newer therapeutic targets. Transcriptomic analysis of CCRCCs and matched microdissected renal tubular controls revealed overexpression of NOTCH ligands and receptors in tumor tissues. Examination of the TCGA RNA-seq data set also revealed widespread activation of NOTCH pathway in a large cohort of CCRCC samples. Samples with NOTCH pathway activation were also clinically distinct and were associated with better overall survival. Parallel DNA methylation and copy number analysis demonstrated that both genetic and epigenetic alterations led to NOTCH pathway activation in CCRCC. NOTCH ligand JAGGED1 was overexpressed and associated with loss of CpG methylation of H3K4me1-associated enhancer regions. JAGGED2 was also overexpressed and associated with gene amplification in distinct CCRCC samples. Transgenic expression of intracellular NOTCH1 in mice with tubule-specific deletion of VHL led to dysplastic hyperproliferation of tubular epithelial cells, confirming the procarcinogenic role of NOTCH in vivo Alteration of cell cycle pathways was seen in murine renal tubular cells with NOTCH overexpression, and molecular similarity to human tumors was observed, demonstrating that human CCRCC recapitulates features and gene expression changes observed in mice with transgenic overexpression of the Notch intracellular domain. Treatment with the γ-secretase inhibitor LY3039478 led to inhibition of CCRCC cells in vitro and in vivo In summary, these data reveal the mechanistic basis of NOTCH pathway activation in CCRCC and demonstrate this pathway to a potential therapeutic target.

  2. Dynamical modeling of the cell cycle and cell fate emergence in Caulobacter crescentus.

    Directory of Open Access Journals (Sweden)

    César Quiñones-Valles

    Full Text Available The division of Caulobacter crescentus, a model organism for studying cell cycle and differentiation in bacteria, generates two cell types: swarmer and stalked. To complete its cycle, C. crescentus must first differentiate from the swarmer to the stalked phenotype. An important regulator involved in this process is CtrA, which operates in a gene regulatory network and coordinates many of the interactions associated to the generation of cellular asymmetry. Gaining insight into how such a differentiation phenomenon arises and how network components interact to bring about cellular behavior and function demands mathematical models and simulations. In this work, we present a dynamical model based on a generalization of the Boolean abstraction of gene expression for a minimal network controlling the cell cycle and asymmetric cell division in C. crescentus. This network was constructed from data obtained from an exhaustive search in the literature. The results of the simulations based on our model show a cyclic attractor whose configurations can be made to correspond with the current knowledge of the activity of the regulators participating in the gene network during the cell cycle. Additionally, we found two point attractors that can be interpreted in terms of the network configurations directing the two cell types. The entire network is shown to be operating close to the critical regime, which means that it is robust enough to perturbations on dynamics of the network, but adaptable to environmental changes.

  3. Dynamical modeling of the cell cycle and cell fate emergence in Caulobacter crescentus. (United States)

    Quiñones-Valles, César; Sánchez-Osorio, Ismael; Martínez-Antonio, Agustino


    The division of Caulobacter crescentus, a model organism for studying cell cycle and differentiation in bacteria, generates two cell types: swarmer and stalked. To complete its cycle, C. crescentus must first differentiate from the swarmer to the stalked phenotype. An important regulator involved in this process is CtrA, which operates in a gene regulatory network and