Cancer and the Cell Cycle

Injection of poly(b- L -malate) into the plasmodium of Physarum polycephalum shortens the cell cycle and increases the growth rate Michael Karl 1 , Roger Anderson 2 and Eggehard Holler 1 1 Institut fu ¨ r Biophysik und Physikalische Biochemie der Universita ¨ t Regensburg, Germany; 2 Molecular Biology and Biotechnology, University of Sheffield, UK Poly(b- L -malate) (PMLA) has been reported as an uncon- ventional, physiologically important biopolymer in plasmo- dia of myxomycetes, and has been proposed to function in the s torage and transport of nuclear proteins by mimicking the phospho(deoxy)ribose backbone of nucleic acids. It is distributed in the cytoplasm a nd especially in the nuclei of these giant, multinucleate cells. We report here f or the first time an increase in growth rate and a shortening of the cell cycle after the injection of purified PMLA. By comparing two strains of Physarum polycephalum that differed in their production levels of PMLA, it was found that growth activation and cell cycle shortening correlated with the relative increases of PMLA levels in the cytoplasm or the nuclei. Growth rates of a low PMLA producer strain (LU897 · LU898) were increased by 40–50% while those of a high producer strain (M 3 CVIII) were increased by only 0–17% in comparison with controls. In both strains, shortening of the cell cycle occurred to a similar extent (7.2– 9.5%), and t his w as associat ed with similar increases in nuclear PMLA levels. The effects showed saturation de- pendences with regard to the amount of injected PMLA. A steep rise of i ntracellular PMLA shortly a fter injection was followed by the appearance of histone H1 in the cytoplasm. The increase i n growth rate, the shortening of the cell cycle duration and the appearance of H1 in the cytoplasm suggest that PMLA competes with nucleic acids in binding to pro- teins that control translation and/or transcription. Thus, PMLA could play an important role in the coordination of molecular p athways t hat a re responsible f or the synchronous functioning of the multinucleate plasmodium. Keywords 1 : cell cycle; g rowth rate; P hysarum polycephalum; plasmodium; polymalic acid. In the absence of cytokinesis, repeated nuclear divisions give rise to giant multinucleate cells (plasmodia) in Physarum polycephalum [1], a well studied representative of the myxomycete family. One of the notable features of plasmodia is the high synchrony of events during the cell cycle. The maintenance of this synchrony over large cellular distances must require an activity that accounts for the rapid and ubiquitous distribution and coordination of protein activities in the periodical cell cycle eve nts. W e h ave previously identified the unusual polyanion poly(b- L -ma- late) (PMLA) a s a specific component of the plasmodium that fulfils the requirements for such a Ôdistributing activityÕ [2,3]. Its level in the nuclei i s kept constant by constitutive synthesis and secretion of excess polymer from the cyto- plasm to the culture medium, and the levels in the nuclei for different strains are of the same magnitude [4]. PMLA binds reversibly to histones, DNA polymerases, a nd other DNA- interacting proteins, thus favouring the formation of large complexes consisting o f a variety of Cancer and the Cell Cycle Cancer and the Cell Cycle Bởi: OpenStaxCollege Cancer comprises many different diseases caused by a common mechanism: uncontrolled cell growth Despite the redundancy and overlapping levels of cell cycle control, errors occur One of the critical processes monitored by the cell cycle checkpoint surveillance mechanism is the proper replication of DNA during the S phase Even when all of the cell cycle controls are fully functional, a small percentage of replication errors (mutations) will be passed on to the daughter cells If changes to the DNA nucleotide sequence occur within a coding portion of a gene and are not corrected, a gene mutation results All cancers start when a gene mutation gives rise to a faulty protein that plays a key role in cell reproduction The change in the cell that results from the malformed protein may be minor: perhaps a slight delay in the binding of Cdk to cyclin or an Rb protein that detaches from its target DNA while still phosphorylated Even minor mistakes, however, may allow subsequent mistakes to occur more readily Over and over, small uncorrected errors are passed from the parent cell to the daughter cells and amplified as each generation produces more non-functional proteins from uncorrected DNA damage Eventually, the pace of the cell cycle speeds up as the effectiveness of the control and repair mechanisms decreases Uncontrolled growth of the mutated cells outpaces the growth of normal cells in the area, and a tumor (“-oma”) can result Proto-oncogenes The genes that code for the positive cell cycle regulators are called proto-oncogenes Proto-oncogenes are normal genes that, when mutated in certain ways, become oncogenes, genes that cause a cell to become cancerous Consider what might happen to the cell cycle in a cell with a recently acquired oncogene In most instances, the alteration of the DNA sequence will result in a less functional (or non-functional) protein The result is detrimental to the cell and will likely prevent the cell from completing the cell cycle; however, the organism is not harmed because the mutation will not be carried forward If a cell cannot reproduce, the mutation is not propagated and the damage is minimal Occasionally, however, a gene mutation causes a change that increases the activity of a positive regulator For example, a mutation that allows Cdk to be activated without being partnered with cyclin could push the cell cycle past a checkpoint before all of the required conditions are met If the resulting daughter cells 1/6 Cancer and the Cell Cycle are too damaged to undergo further cell divisions, the mutation would not be propagated and no harm would come to the organism However, if the atypical daughter cells are able to undergo further cell divisions, subsequent generations of cells will probably accumulate even more mutations, some possibly in additional genes that regulate the cell cycle The Cdk gene in the above example is only one of many genes that are considered protooncogenes In addition to the cell cycle regulatory proteins, any protein that influences the cycle can be altered in such a way as to override cell cycle checkpoints An oncogene is any gene that, when altered, leads to an increase in the rate of cell cycle progression Tumor Suppressor Genes Like proto-oncogenes, many of the negative cell cycle regulatory proteins were discovered in cells that had become cancerous Tumor suppressor genes are segments of DNA that code for negative regulator proteins, the type of regulators that, when activated, can prevent the cell from undergoing uncontrolled division The collective function of the best-understood tumor suppressor gene proteins, Rb, p53, and p21, is to put up a roadblock to cell cycle progression until certain events are completed A cell that carries a mutated form of a negative regulator might not be able to halt the cell cycle if there is a problem Tumor suppressors are similar to brakes in a vehicle: Malfunctioning brakes can contribute to a car crash Mutated p53 genes have been identified in more than one-half of all human tumor cells This discovery is not surprising in light of the multiple roles that the p53 protein plays at the G1 checkpoint A cell with a faulty p53 may fail to detect errors present in the genomic DNA ([link]) Even if a partially functional p53 does identify the mutations, it may no longer be able to signal the necessary DNA repair enzymes Either way, damaged DNA will remain uncorrected At this point, a functional p53 will deem the cell unsalvageable and trigger programmed cell death (apoptosis) The damaged version of p53 found in cancer cells, however, cannot trigger apoptosis Art Connection 2/6 Cancer and the Cell Cycle The role of normal p53 is to monitor DNA and the supply of oxygen (hypoxia is a condition of reduced oxygen supply) If damage is detected, p53 triggers repair mechanisms If repairs are unsuccessful, p53 signals apoptosis A cell with an abnormal p53 protein ...Dynamic association of MLL1, H3K4 trimethylation with chromatin and Hox gene expression during the cell cycle Bibhu P. Mishra, Khairul I. Ansari and Subhrangsu S. Mandal Department of Chemistry and Biochemistry, The University of Texas at Arlington, Arlington, TX, USA Histone methyltransferases (HMTs) are key enzymes that post-translationally methylate histones and play critical roles in gene expression, epigenetics and cancer [1–11]. Mixed lineage leukemias (MLLs) are human HMTs that specifically methylate histone H3 at lysine 4 (H3K4) and are linked with gene activation [12–20]. Notably, Set1 is the sole H3K4-specific HMT present in yeast [21–23]. Humans encode six Set1 homologs: MLL1, MLL2, MLL3, MLL4, Set1A and Set1B [12,13,16,19,24–27]. Each of these proteins exists as multiprotein complexes sharing several common subunits, including Ash2, Wdr5, Rbbp5, human CpG-binding protein (CGBP) and Dpy30 [12–14,16,19, 24–31]. MLLs are well known as the master regulators Keywords cell cycle; H3K4 methylation; histone methyltransferase; Hox genes; mixed lineage leukemia Correspondence S. S. Mandal, Department of Chemistry and Biochemistry, The University of Texas at Arlington, Arlington, TX 76019, USA Fax: +1 817 272 3808 Tel: +1 817 272 3804 E-mail: smandal@uta.edu (Received 6 November 2008, revised 3 January 2009, accepted 9 January 2009) doi:10.1111/j.1742-4658.2009.06895.x Mixed lineage leukemias (MLLs) are histone H3 at lysine 4 (H3K4)-spe- cific methylases that play a critical role in regulating gene expression in humans. As chromatin condensation, relaxation and differential gene expression are keys to correct cell cycle progression, we analyzed the dynamic association of MLL and H3K4 trimethylation at different stages of the cell cycle. Interestingly, MLL1, which is normally associated with transcriptionally active chromatins (G1 phase), dissociates from condensed mitotic chromatin and returns at the end of telophase when the nucleus starts to relax. In contrast, H3K4 trimethylation mark, which is also nor- mally associated with euchromatins (in G1), remains associated, even with condensed chromatin, throughout the cell cycle. The global levels of MLL1 and H3K4 trimethylation are not affected during the cell cycle, and H3Ser28 phosphorylation is only observed during mitosis. Interest- ingly, MLL target homeobox-containing (Hox) genes (HoxA5, HoxA7 and HoxA10) are differentially expressed during the cell cycle, and the recruitment of MLL1 and H3K4 trimethylation levels are modulated in the promoter of these Hox genes as a function of their expression. In addition, down-regulation of MLL1 results in cell cycle arrest at the G2 ⁄ M phase. The fluctuation of H3K4 trimethylation marks at specific promoters, but not at the global level, indicates that H3K4 trimethylation marks that are present in the G1 phase may not be the same as the marks in other phases of the cell cycle; rather, old marks are removed and new marks are introduced. In conclusion, our studies demonstrate that MLL1 and H3K4 methylation have distinct dynamics during the cell cycle and play critical roles in the differential expression of Hox genes associated with cell cycle regulation. Abbreviations CGBP, human CpG-binding protein; ChIP, chromatin The inhibition of Ras farnesylation leads to an increase in p27 Kip1 and G1 cell cycle arrest Hadas Reuveni*, Shoshana Klein and Alexander Levitzki Department of Biological Chemistry, Institute of Life Sciences, The Hebrew University of Jerusalem, Jerusalem, Israel HR12 is a novel farnesyltransferase inhibitor (FTI). We have shown previously that HR12 induces phenotypic reversion of H-ras V12 -transformed Rat1 (Rat1/ras) fibroblasts. This reversion was characterized by formation of cell–cell con- tacts, focal adhesions and stress fibers. Here we show that HR12 inhibits anchorage independent and dependent growth of Rat1/ras cells. HR12 also suppresses motility and proliferation of Rat1/ras cells, in a wound healing assay. Rat1 fibroblasts transformed with myristoylated H-ras V12 (Rat1/myr-ras) were resistant to HR12. Thus, the effects of HR12 are due to the inhibition of farnesylation of Ras. Cell growth of Rat1/ras cells was arrested at the G1 phase of the cell cycle. Analysis of cell cycle components showed that HR12 treatment of Rat1/ras cells led to elevated cellular levels of the cyclin-dependent kinase inhibitor p27 Kip1 and inhibition of the kinase activity of the cyclin E/Cdk2 complex. This is the first time an FTI has been shown to lead to a rise in p27 Kip1 levels in ras-transformed cells. The data suggest a new mechanism for FTI action, whereby in ras- transformed cells, the FTI causes an increase in p27 Kip1 levels, which in turn inhibit cyclin E/Cdk2 activity, leading to G1 arrest. Keywords: farnesyl transferase inhibitor (FTI); p27 Kip1 ;Ras; cell cycle. Localization of Ras proteins in the plasma membrane follows a series of post-translational modifications [1] and is crucial to the functioning of these proteins [2,3]. The first and essential step in this process is farnesylation, whereby a farnesyl group (C 15 -isoprenoid) is covalently attached to the cysteine residue of the C-terminal CAAX sequence of Ras [4]. Farnesylation is mediated by the enzyme farnesyltrans- ferase (FT). The three C-terminal residues, AAX, are then proteolytically cleaved and the new carboxy-terminus is methylated. H-Ras, N-Ras and K-Ras4A are also palmi- toylated on one or more upstream cysteine residues. Mutationally activated ras genes are found in  30% of all human cancers. As farnesylation is required for the oncogenic activity of activated Ras, there has been much interest in the development of FT inhibitors (FTIs) for anticancer treatment. We have developed an FTI, cysteine-N-methyl-valine- N-cyclohexyl-glycine-methionine-methyl ester, called HR12 [5]. We have demonstrated recently [6] the compound’s ability to completely reverse the transformed phenotype of oncogenic H-Ras-transformed Rat1 (Rat1/ras) fibroblasts. This reversion entailed the assembly of adheren junctions, concomitant with induction of cadherin and b-catenin. Focal adhesions and actin stress fibers were formed, and the overall cell morphology was indistinguishable from that of nontransformed Rat1 cells. Cell adhesion affects cell growth and invasion. Cadherin, the primary cell–cell adhesion molecule, acts as a suppressor of cancer cell invasion [7,8], and the loss of cadherin function is required for tumor progression in vivo [9,10]. Moreover, the activation Genome Biology 2009, 10:R123 Open Access 2009Castellanoet al.Volume 10, Issue 11, Article R123 Research Serum-dependent transcriptional networks identify distinct functional roles for H-Ras and N-Ras during initial stages of the cell cycle Esther Castellano *† , Carmen Guerrero * , Alejandro Núñez * , Javier De Las Rivas * and Eugenio Santos * Addresses: * Centro de Investigación del Cáncer, IBMCC (CSIC-USAL), University of Salamanca, Campus Unamuno, 37007 Salamanca, Spain. † Current address: Signal Transduction Laboratory, Cancer Research UK London Research Institute, 44 Lincoln's Inn Fields, London WC2A 3PX, UK. Correspondence: Eugenio Santos. Email: esantos@usal.es © 2009 Castellano et al.; licensee BioMed Central Ltd. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Ras isoforms and the cell cycle<p>Transcriptional and functional analysis reveals that the H-Ras and N-Ras isoforms have different roles in the initial phases of the mouse cell cycle</p> Abstract Background: Using oligonucleotide microarrays, we compared transcriptional profiles corresponding to the initial cell cycle stages of mouse fibroblasts lacking the small GTPases H-Ras and/or N-Ras with those of matching, wild-type controls. Results: Serum-starved wild-type and knockout ras fibroblasts had very similar transcriptional profiles, indicating that H-Ras and N-Ras do not significantly control transcriptional responses to serum deprivation stress. In contrast, genomic disruption of H-ras or N-ras, individually or in combination, determined specific differential gene expression profiles in response to post- starvation stimulation with serum for 1 hour (G0/G1 transition) or 8 hours (mid-G1 progression). The absence of N-Ras caused significantly higher changes than the absence of H-Ras in the wave of transcriptional activation linked to G0/G1 transition. In contrast, the absence of H-Ras affected the profile of the transcriptional wave detected during G1 progression more strongly than did the absence of N-Ras. H-Ras was predominantly functionally associated with growth and proliferation, whereas N-Ras had a closer link to the regulation of development, the cell cycle, immunomodulation and apoptosis. Mechanistic analysis indicated that extracellular signal-regulated kinase (ERK)-dependent activation of signal transducer and activator of transcription 1 (Stat1) mediates the regulatory effect of N-Ras on defense and immunity, whereas the pro-apoptotic effects of N-Ras are mediated through ERK and p38 mitogen-activated protein kinase signaling. Conclusions: Our observations confirm the notion of an absolute requirement for different peaks of Ras activity during the initial stages of the cell cycle and document the functional specificity of H- Ras and N-Ras during those processes. Published: 6 November 2009 Genome Biology 2009, 10:R123 (doi:10.1186/gb-2009-10-11-r123) Received: 2 July 2009 Accepted: 6 November 2009 The electronic version of this article is the complete one and can be found online at http://genomebiology.com/2009/10/11/R123 http://genomebiology.com/2009/10/11/R123 Genome Biology 2009, Volume 10, Issue 11, Article R123 Castellano et al. R123.2 Genome Biology 2009, 10:R123 Background The mammalian H-Ras, N-Ras and K-Ras proteins are highly related small GTPases functioning as critical components of cellular signaling pathways controlling proliferation, differ- entiation or survival. They act as molecular switches cycling between inactive (GDP-bound) and active (GTP-bound) states in a process modulated under physiological conditions by a variety of specific regulatory proteins, including GAPs (GTPase activating proteins) and GEFs (guanine nucleotide exchange factors) [1-3]. Hyperactivating point mutations Genome Biology 2006, 7:219 comment reviews reports deposited research interactions information refereed research Minireview RNA interference pinpoints regulators of cell size and the cell cycle Megan J Cully* and Sally J Leevers † Addresses: *Signal Transduction Laboratory and † Growth Regulation Laboratory, Cancer Research UK London Research Institute, 44 Lincoln’s Inn Fields, London WC2A 3PX, UK. Correspondence: Sally J Leevers. Email: sally.leevers@cancer.org.uk Abstract Cell-based genome-wide RNA interference screens are being used to address an increasingly broad spectrum of biological questions. In one recent screen, Drosophila cell cultures treated with double-stranded RNA were analyzed by flow cytometry, providing a wealth of new information and identifying 488 regulators of the cell cycle, cell size, and cell death. Published: 30 May 2006 Genome Biology 2006, 7:219 (doi:10.1186/gb-2006-7-5-219) The electronic version of this article is the complete one and can be found online at http://genomebiology.com/2006/7/5/219 © 2006 BioMed Central Ltd The growth of an organism is the net result of a variety of processes, including changes in cell size, cell division and apoptosis. These processes are regulated by intricate, inter- related molecular networks, and their disruption can have major biological consequences. In particular, the relation- ship between changes in cell size and the cell cycle has long fascinated researchers. It is complex, poorly understood, and varies according to the organism, tissue type and develop- mental context. In yeast, large-scale genetic screens have uncovered many genes involved in cell growth and the initia- tion of DNA synthesis (S phase) [1,2]. It is now clear that yeast cells must grow to a certain minimal size before start- ing DNA synthesis, providing a ‘cell size checkpoint’ at the transition from the preceding G1 phase to S phase (the G1/S transition). Yeast is a unicellular organism, however, and there is increasing evidence that the relationship between cell growth and cell division may be different in metazoans. Excitingly, recent technical advances in high-throughput RNA interference (RNAi) mean that large-scale screening approaches, somewhat analogous to the genetic screens in yeast, can now be applied to cultured metazoan cells. Drosophila hemocyte cell lines have emerged as popular cell systems for this experimental approach for a number of reasons. First, they are very amenable to RNAi mediated by double-stranded RNA (dsRNA): dsRNA molecules of more than 500 bp can be easily introduced into these cells and are rapidly processed into short interfering RNAs (siRNAs). Second, there are significantly fewer genes in Drosophila than in mammals, making the mammoth undertaking of a genome- wide screen a little less daunting. Finally, there is less genetic redundancy in Drosophila than in mammals, so depletion of just one gene is more likely to reveal a phenotype. Genomic screens for the total complement of protein kinases (the kinome) and general genome-wide screens have been performed in Drosophila cell cultures using diverse readouts such as cell shape, resistance to bacterial infection and tran- scriptional activity [3-8]. Bjorklund et al. [9] have recently published one of the most comprehensive screens to date, in which they searched on a genome-wide scale for dsRNAs that alter cell size, cell-cycle distribution and cell death. The dataset they generated provides an excellent starting point for many new avenues of research. At the same time, this massive undertaking highlights some of the bioinformatic challenges associated with screens on this scale. For example, the data generated can be analyzed and presented in various ways to highlight the different phenotypic effects (see the supplementary data accompanying [9]). The Taipale lab [9] used dsRNAs corresponding to 11,971 individual cDNAs to target the silencing of approximately 70% of known Drosophila genes. After 4 days .. .Cancer and the Cell Cycle are too damaged to undergo further cell divisions, the mutation would not be propagated and no harm would come to the organism However, if the atypical daughter cells... of how cancer results from errors in the cell cycle Section Summary Cancer is the result of unchecked cell division caused by a breakdown of the mechanisms that regulate the cell cycle The loss... addition to the faulty tumor 3/6 Cancer and the Cell Cycle suppressor gene Cells such as these daughter cells quickly accumulate both oncogenes and non-functional tumor suppressor genes Again, the result