scholarly journals Curcumin Inhibits Growth of Saccharomyces cerevisiae through Iron Chelation

2011 ◽  
Vol 10 (11) ◽  
pp. 1574-1581 ◽  
Author(s):  
Steven Minear ◽  
Allyson F. O'Donnell ◽  
Anna Ballew ◽  
Guri Giaever ◽  
Corey Nislow ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Mammalian Cells ◽  
Cell Cycle Progression ◽  
Iron Chelation ◽  
Yeast Cells ◽  
Iron Starvation ◽  
Intracellular Iron ◽  
Iron Pool ◽  
Yeast Mutants

ABSTRACTCurcumin, a polyphenol derived from turmeric, is an ancient therapeutic used in India for centuries to treat a wide array of ailments. Interest in curcumin has increased recently, with ongoing clinical trials exploring curcumin as an anticancer therapy and as a protectant against neurodegenerative diseases.In vitro, curcumin chelates metal ions. However, although diverse physiological effects have been documented for this compound, curcumin's mechanism of action on mammalian cells remains unclear. This study uses yeast as a model eukaryotic system to dissect the biological activity of curcumin. We found that yeast mutants lacking genes required for iron and copper homeostasis are hypersensitive to curcumin and that iron supplementation rescues this sensitivity. Curcumin penetrates yeast cells, concentrates in the endoplasmic reticulum (ER) membranes, and reduces the intracellular iron pool. Curcumin-treated, iron-starved cultures are enriched in unbudded cells, suggesting that the G1phase of the cell cycle is lengthened. A delay in cell cycle progression could, in part, explain the antitumorigenic properties associated with curcumin. We also demonstrate that curcumin causes a growth lag in cultured human cells that is remediated by the addition of exogenous iron. These findings suggest that curcumin-induced iron starvation is conserved from yeast to humans and underlies curcumin's medicinal properties.

scholarly journals Cpc2, a Fission Yeast Homologue of Mammalian RACK1 Protein, Interacts with Ran1 (Pat1) Kinase To Regulate Cell Cycle Progression and Meiotic Development

2000 ◽  
Vol 20 (11) ◽  
pp. 4016-4027 ◽  
Author(s):  
Maureen McLeod ◽  
Boris Shor ◽  
Anthony Caporaso ◽  
Wei Wang ◽  
Hua Chen ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Fission Yeast ◽  
Mammalian Cells ◽  
Cell Cycle Progression ◽  
Fluorescent Protein ◽  
Yeast Cells ◽  
Anchoring Protein ◽  
Signal Transduction Proteins ◽  
Green Fluorescent

ABSTRACT The Schizosaccharomyces pombe ran1/pat1 gene regulates the transition between mitosis and meiosis. Inactivation of Ran1 (Pat1) kinase is necessary and sufficient for cells to exit the cell cycle and undergo meiosis. The yeast two-hybrid interaction trap was used to identify protein partners for Ran1/Pat1. Here we report the identification of one of these, Cpc2. Cpc2 encodes a homologue of RACK1, a WD protein with homology to the β subunit of heterotrimeric G proteins. RACK1 is a highly conserved protein, although its function remains undefined. In mammalian cells, RACK1 physically associates with some signal transduction proteins, including Src and protein kinase C. Fission yeast cells containing a cpc2 null allele are viable but cell cycle delayed. cpc2Δ cells fail to accumulate in G1 when starved of nitrogen. This leads to defects in conjugation and meiosis. Copurification studies show that although Cpc2 and Ran1 (Pat1) physically associate, Cpc2 does not alter Ran1 (Pat1) kinase activity in vitro. Using a Ran1 (Pat1) fusion to green fluorescent protein, we show that localization of the kinase is impaired in cpc2Δ cells. Thus, in parallel with the proposed role of RACK1 in mammalian cells, fission yeast cpc2 may function as an anchoring protein for Ran1 (Pat1) kinase. All defects associated with loss of cpc2 are reversed in cells expressing mammalian RACK1, demonstrating that the fission yeast and mammalian gene products are indeed functional homologues.

scholarly journals Spy1 Interacts with p27Kip1 to Allow G1/S Progression

2003 ◽  
Vol 14 (9) ◽  
pp. 3664-3674 ◽  
Author(s):  
Lisa A. Porter ◽  
Monica Kong-Beltran ◽  
Daniel J. Donoghue
Keyword(s):  
Cell Cycle ◽  
Mammalian Cells ◽  
Cell Cycle Progression ◽  
Regulatory Protein ◽  
Cell Cycle Regulatory Protein ◽  
Proliferative Effect ◽  
Cycle Arrest ◽  
Mammalian Cell Cycle

Progression through the G1/S transition commits cells to synthesize DNA. Cyclin dependent kinase 2 (CDK2) is the major kinase that allows progression through G1/S phase and subsequent replication events. p27 is a CDK inhibitor (CKI) that binds to CDK2 to prevent premature activation of this kinase. Speedy (Spy1), a novel cell cycle regulatory protein, has been found to prematurely activate CDK2 when microinjected into Xenopus oocytes and when expressed in mammalian cells. To determine the mechanism underlying Spy1-induced proliferation in mammalian cell cycle regulation, we used human Spy1 as bait in a yeast two-hybrid screen to identify interacting proteins. One of the proteins isolated was p27; this novel interaction was confirmed both in vitro, using bacterially expressed and in vitro translated proteins, and in vivo, through the examination of endogenous and transfected proteins in mammalian cells. We demonstrate that Spy1 expression can overcome a p27-induced cell cycle arrest to allow for DNA synthesis and CDK2 histone H1 kinase activity. In addition, we utilized p27-null cells to demonstrate that the proliferative effect of Spy1 depends on the presence of endogenous p27. Our data suggest that Spy1 associates with p27 to promote cell cycle progression through the G1/S transition.

scholarly journals Intracellular Dynamics of the Ubiquitin-Proteasome-System

F1000Research ◽  
2015 ◽  
Vol 4 ◽  
pp. 367 ◽  
Author(s):  
Maisha Chowdhury ◽  
Cordula Enenkel
Keyword(s):  
Cell Cycle ◽  
Protein Degradation ◽  
Mammalian Cells ◽  
Cell Cycle Progression ◽  
Current Knowledge ◽  
Ubiquitin Proteasome System ◽  
Yeast Cells ◽  
Ubiquitin Chain ◽  
Dividing Cells ◽  
Ubiquitin Proteasome

The ubiquitin-proteasome system is the major degradation pathway for short-lived proteins in eukaryotic cells. Targets of the ubiquitin-proteasome-system are proteins regulating a broad range of cellular processes including cell cycle progression, gene expression, the quality control of proteostasis and the response to geno- and proteotoxic stress. Prior to degradation, the proteasomal substrate is marked with a poly-ubiquitin chain. The key protease of the ubiquitin system is the proteasome. In dividing cells, proteasomes exist as holo-enzymes composed of regulatory and core particles. The regulatory complex confers ubiquitin-recognition and ATP dependence on proteasomal protein degradation. The catalytic sites are located in the proteasome core particle. Proteasome holo-enzymes are predominantly nuclear suggesting a major requirement for proteasomal proteolysis in the nucleus. In cell cycle arrested mammalian or quiescent yeast cells, proteasomes deplete from the nucleus and accumulate in granules at the nuclear envelope (NE) / endoplasmic reticulum ( ER) membranes. In prolonged quiescence, proteasome granules drop off the nuclear envelopeNE / ER membranes and migrate as droplet-like entitiesstable organelles  throughout the cytoplasm, as thoroughly investigated in yeast. When quiescence yeast cells are allowed to resume growth, proteasome granules clear and proteasomes are rapidly imported into the nucleus.Here, we summarize our knowledge about the enigmatic structure of proteasome storage granules and the trafficking of proteasomes and their substrates between the cyto- and nucleoplasm.Most of our current knowledge is based on studies in yeast. Their translation to mammalian cells promises to provide keen insight into protein degradation in non-dividing cells, which comprise the majority of our body’s cells.

scholarly journals Identification of MoKA, a Novel F-Box Protein That Modulates Krüppel-Like Transcription Factor 7 Activity

2004 ◽  
Vol 24 (3) ◽  
pp. 1058-1069 ◽  
Author(s):  
Silvia Smaldone ◽  
Friedrich Laub ◽  
Cindy Else ◽  
Cecilia Dragomir ◽  
Francesco Ramirez
Keyword(s):  
Cell Cycle ◽  
Nervous System ◽  
Transcription Factor ◽  
Mammalian Cells ◽  
Cell Cycle Progression ◽  
Direct Interaction ◽  
Proximal Promoter ◽  
F Box Protein ◽  
Developing Nervous System

ABSTRACT KLF7, a member of the Krüppel-like transcription factor family, is believed to regulate neurogenesis and cell cycle progression. Here, a yeast two-hybrid screen for KLF7 cofactors in the developing nervous system identified a novel 140-kDa protein named MoKA, for modulator of KLF7 activity. Interaction between MoKA and KLF7 was confirmed by the in vitro glutathione S-transferase pull-down assay and by coimmunoprecipitation of the proteins overexpressed in mammalian cells. Functional assays documented that MoKA is a KLF7 coactivator, and in situ hybridizations identified the developing nervous system and the adult testes as two sites of MoKA and Klf7 coexpression. Chromatin immunoprecipitation experiments demonstrated KLF7 binding to the p21WAF1/Cip1 gene while transient transfection assays documented KLF7 stimulation of the p21WAF1/Cip1 proximal promoter. Additional tests revealed that distinct structural motifs of MoKA direct interaction with KLF7 and shuttling between the nucleus and cytoplasm of asynchronously cycling cells. Altogether, our results strongly suggest that MoKA and KLF7 interact functionally to regulate gene expression during cell differentiation and identify the cell cycle regulator p21WAF1/Cip1 as one of the targeted genes.

scholarly journals Only Akt1 Is Required for Proliferation, while Akt2 Promotes Cell Cycle Exit through p21 Binding

2006 ◽  
Vol 26 (22) ◽  
pp. 8267-8280 ◽  
Author(s):  
Lisa Héron-Milhavet ◽  
Celine Franckhauser ◽  
Vanessa Rana ◽  
Cyril Berthenet ◽  
Daniel Fisher ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cell Proliferation ◽  
Mammalian Cells ◽  
Cell Cycle Progression ◽  
Specific Interaction ◽  
Cyclin A ◽  
Cell Cycle Exit ◽  
Cycle Progression ◽  
Negative Effect

ABSTRACT Protein kinase B (PKB/Akt) is an important modulator of insulin signaling, cell proliferation, and survival. Using small interfering RNA duplexes in nontransformed mammalian cells, we show that only Akt1 is essential for cell proliferation, while Akt2 promotes cell cycle exit. Silencing Akt1 resulted in decreased cyclin A levels and inhibition of S-phase entry, effects not seen with Akt2 knockdown and specifically rescued by microinjection of Akt1, not Akt2. In differentiating myoblasts, Akt2 knockout prevented myoblasts from exiting the cell cycle and showed sustained cyclin A expression. In contrast, overexpression of Akt2 reduced cyclin A and hindered cell cycle progression in M-G1 with increased nuclear p21. p21 is a major target in the differential effects of Akt isoforms, with endogenous Akt2 and not Akt1 binding p21 in the nucleus and increasing its level. Accordingly, Akt2 knockdown cells, and not Akt1 knockdown cells, showed reduced levels of p21. A specific Akt2/p21 interaction can be reproduced in vitro, and the Akt2 binding site on p21 is similar to that in cyclin A spanning T145 to T155, since (i) prior incubation with cyclin A prevents Akt2 binding, (ii) T145 phosphorylation on p21 by Akt1 prevents Akt2 binding, and (iii) binding Akt2 prevents phosphorylation of p21 by Akt1. These data show that specific interaction of the Akt2 isoform with p21 is key to its negative effect on normal cell cycle progression.

Applications of Human Peripheral Blood Lymphocytes in Genotoxicity and Cytotoxicity

1990 ◽  
Vol 18 (1_part_1) ◽  
pp. 231-241
Author(s):  
Lucia Celotti ◽  
Vera Bianchi
Keyword(s):  
Cell Cycle ◽  
Peripheral Blood ◽  
Mammalian Cells ◽  
Cell Cycle Progression ◽  
Human Peripheral Blood ◽  
Quantitative Difference ◽  
Peripheral Blood Lymphocytes ◽  
Blood Lymphocytes

A number of features make peripheral blood lymphocytes an excellent system for studying both genotoxicity and cytotoxicity in humans. They are an abundant and readily accessible source of somatic cells, mostly in a non-proliferative state, but able to be stimulated by mitogens to enter the cell cycle. The blastocyte transformation of lymphocytes is a useful model for investigating the mechanisms which regulate cell-cycle progression in mammalian cells. By stimulating lymphocytes in vitro, it is possible to detect the genetic damages they have sustained in vivo, which become manifest as chromosomal aberrations, sister-chromatid exchanges or gene mutations. The metabolic properties of lymphocytes have been extensively studied, especially with reference to their characteristic collection of enzymes involved in nucleotide turnover, which makes them exquisitely sensitive to changes in intracellular levels of DNA precursors. The data collected on the ability of lymphocytes to metabolise xenobiotics show a marked quantitative difference between resting and proliferating lymphocytes, and minor qualitative differences between lymphocytes and other cell types, e.g. hepatocytes. An indirect approach to detect the metabolism of genotoxic xenobiotics by lymphocytes is the analysis of DNA adducts in their chromatin after in vivo or in vitro exposure. Lymphocytes can be employed to identify the (cyto)genetic consequences of in vivo genotoxic exposure and inter-individual variation in sensitivity to genotoxic agents. The analysis of mutations at the hgprt locus in lymphocytes is a promising approach for the study of somatic-cell mutations in humans and of the possible mechanisms of in vivo selection against mutants. In the field of cytotoxicity, the applications of lymphocytes are, as yet, still few: the main effect measured is the impairment of the proliferative response to mitogens. But lymphocytes can be employed as primary human cells to be treated in vitro with mutagenic or toxic chemicals in standard genotoxicity and cytotoxicity assays, and offer the advantage of avoiding the problems of inter-species extrapolation of results by testing in a human system. Moreover, the (geno)toxic effects detected in lymphocytes after treatments in vitro may give information on the spontaneous or environmentally-determined susceptibility of the individual donors to xenobiotics.

scholarly journals G1/S control of anchorage-independent growth in the fibroblast cell cycle.

1991 ◽  
Vol 115 (5) ◽  
pp. 1419-1425 ◽  
Author(s):  
T M Guadagno ◽  
R K Assoian
Keyword(s):  
Cell Cycle ◽  
Mammalian Cells ◽  
Cell Cycle Progression ◽  
Fibroblast Cell ◽  
Close Correlation ◽  
Anchorage Independent Growth ◽  
Mammalian Cell Cycle ◽  
Nih 3T3

We have developed methodology to identify the block to anchorage-independent growth and position it within the fibroblast cell cycle. Results with NRK fibroblasts show that mitogen stimulation of the G0/G1 transition and G1-associated increases in cell size are minimally affected by loss of cell anchorage. In contrast, the induction of G1/S cell cycle genes and DNA synthesis is markedly inhibited when anchorage is blocked. Moreover, we demonstrate that the anchorage-dependent transition maps to late G1 and shortly before activation of the G1/S p34cdc2-like kinase. The G1/S block was also detectable in NIH-3T3 cells. Our results: (a) distinguish control of cell cycle progression by growth factors and anchorage; (b) indicate that anchorage mediates G1/S control in fibroblasts; and (c) identify a physiologic circumstance in which the phenotype of mammalian cell cycle arrest would closely resemble Saccharomyces cerevisiae START. The close correlation between anchorage independence in vitro and tumorigenicity in vivo emphasizes the key regulatory role for G1/S control in mammalian cells.

scholarly journals Cyclin E Associates with Components of the Pre-mRNA Splicing Machinery in Mammalian Cells

1998 ◽  
Vol 18 (8) ◽  
pp. 4526-4536 ◽  
Author(s):  
Wolfgang Seghezzi ◽  
Katrin Chua ◽  
Frances Shanahan ◽  
Or Gozani ◽  
Robin Reed ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Mammalian Cells ◽  
Cell Cycle Progression ◽  
Cyclin E ◽  
Specific Inhibitor ◽  
Mrna Splicing ◽  
Core Proteins ◽  
Associated Proteins ◽  
Cdk Regulation

ABSTRACT Cyclin E-cdk2 is a critical regulator of cell cycle progression from G1 into S phase in mammalian cells. Despite this important function little is known about the downstream targets of this cyclin-kinase complex. Here we have identified components of the pre-mRNA processing machinery as potential targets of cyclin E-cdk2. Cyclin E-specific antibodies coprecipitated a number of cyclin E-associated proteins from cell lysates, among which are the spliceosome-associated proteins, SAP 114, SAP 145, and SAP 155, as well as the snRNP core proteins B′ and B. The three SAPs are all subunits of the essential splicing factor SF3, a component of U2 snRNP. Cyclin E antibodies also specifically immunoprecipitated U2 snRNA and the spliceosome from splicing extracts. We demonstrate that SAP 155 serves as a substrate for cyclin E-cdk2 in vitro and that its phosphorylation in the cyclin E complex can be inhibited by the cdk-specific inhibitor p21. SAP 155 contains numerous cdk consensus phosphorylation sites in its N terminus and is phosphorylated prior to catalytic step II of the splicing pathway, suggesting a potential role for cdk regulation. These findings provide evidence that pre-mRNA splicing may be linked to the cell cycle machinery in mammalian cells.

scholarly journals Intracellular Dynamics of the Ubiquitin-Proteasome-System

F1000Research ◽  
2015 ◽  
Vol 4 ◽  
pp. 367 ◽  
Author(s):  
Maisha Chowdhury ◽  
Cordula Enenkel
Keyword(s):  
Cell Cycle ◽  
Protein Degradation ◽  
Mammalian Cells ◽  
Cell Cycle Progression ◽  
Current Knowledge ◽  
Ubiquitin Proteasome System ◽  
Yeast Cells ◽  
Ubiquitin Chain ◽  
Dividing Cells ◽  
Ubiquitin Proteasome

The ubiquitin-proteasome system is the major degradation pathway for short-lived proteins in eukaryotic cells. Targets of the ubiquitin-proteasome-system are proteins regulating a broad range of cellular processes including cell cycle progression, gene expression, the quality control of proteostasis and the response to geno- and proteotoxic stress. Prior to degradation, the proteasomal substrate is marked with a poly-ubiquitin chain. The key protease of the ubiquitin system is the proteasome. In dividing cells, proteasomes exist as holo-enzymes composed of regulatory and core particles. The regulatory complex confers ubiquitin-recognition and ATP dependence on proteasomal protein degradation. The catalytic sites are located in the proteasome core particle. Proteasome holo-enzymes are predominantly nuclear suggesting a major requirement for proteasomal proteolysis in the nucleus. In cell cycle arrested mammalian or quiescent yeast cells, proteasomes deplete from the nucleus and accumulate in granules at the nuclear envelope (NE) / endoplasmic reticulum (ER) membranes. In prolonged quiescence, proteasome granules drop off the NE / ER membranes and migrate as stable organelles throughout the cytoplasm, as thoroughly investigated in yeast. When quiescence yeast cells are allowed to resume growth, proteasome granules clear and proteasomes are rapidly imported into the nucleus.Here, we summarize our knowledge about the enigmatic structure of proteasome storage granules and the trafficking of proteasomes and their substrates between the cyto- and nucleoplasm.Most of our current knowledge is based on studies in yeast. Their translation to mammalian cells promises to provide keen insight into protein degradation in non-dividing cells which comprise the majority of our body’s cells.

Prolonged arrest of mammalian cells at the G1/S boundary results in permanent S phase stasis

2002 ◽  
Vol 115 (14) ◽  
pp. 2829-2838
Author(s):  
Franck Borel ◽  
Françoise B. Lacroix ◽  
Robert L. Margolis
Keyword(s):  
Cell Cycle ◽  
Dna Replication ◽  
Mammalian Cells ◽  
Cell Cycle Progression ◽  
T Antigen ◽  
S Phase ◽  
Inhibitory Circuits ◽  
Cycle Arrest ◽  
Mcm Proteins

Mammalian cells in culture normally enter a state of quiescence during G1 following suppression of cell cycle progression by senescence, contact inhibition or terminal differentiation signals. We find that mammalian fibroblasts enter cell cycle stasis at the onset of S phase upon release from prolonged arrest with the inhibitors of DNA replication, hydroxyurea or aphidicolin. During arrest typical S phase markers remain present, and G0/G1 inhibitory signals such as p21WAF1 and p27 are absent. Cell cycle stasis occurs in T-antigen transformed cells, indicating that p53 and pRB inhibitory circuits are not involved. While no DNA replication is evident in arrested cells, nuclei isolated from these cells retain measurable competence for in vitro replication. MCM proteins are required to license replication origins, and are put in place in nuclei in G1 and excluded from chromatin by the end of replication to prevent rereplication of the genome. Strikingly, MCM proteins are strongly depleted from chromatin during prolonged S phase arrest,and their loss may underlie the observed cell cycle arrest. S phase stasis may thus be a `trap' in which cells otherwise competent for S phase have lost a key component required for replication and thus can neither go forward nor retreat to G1 status.

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