Table of Contents

Volume 4, Issue 1

  January 2005

  Pages 3 - 195

View covers archive

EXTRA VIEWS

Open Access Article

Altered Epigenetic Patterning Leading to Replicative Senescence and Reduced Longevity: A Role of a Novel SNF2 Factor, PASG

Lin-Quan Sun and Robert J. Arceci
Pages 3 - 5
http://dx.doi.org/10.4161/cc.4.1.1341
Abstract | PDF

Understanding the biological mechanisms underlying aging and cancer predisposition remains a fundamentally important goal in biomedicine. The generation of a PASG hypomorphic mutant mouse model shows that PASG, an SNF2 family member, is essential for properly maintaining normal DNA methylation and gene expression patterns. Disruption of PASG leads to decreased incorporation of BrdU, accumulation of senescence-associated tumor suppressor genes, and increased senescence-associated ?- galactosidase as well as age-related phenotypes. These observations demonstrate that PASG plays a critical role in maintenance of tissue homeostasis, normal growth and longevity.

Open Access Article

Control of Cell Cycle Progression by the Stress-Activated Hog1 MAPK

Meritxell Zapater, Josep Clotet, Xavier Escoté and Francesc Posas
Pages 6 - 7
http://dx.doi.org/10.4161/cc.4.1.1344
Abstract | PDF

Activation of the stress-activated Hog1 kinase is essential for cell survival in response to high osmolarity. The Hog1 SAPK elicits the program for cell adaptation that includes modulation of several aspects of cell biology, such as, gene expression, translation and cell-cycle progression. In response to stress, the Hog1 SAPK mediates a transient cell cycle arrest required for proper cell adaptation. A recent report has described the mechanism underlying the effect of the SAPK at G1. Here, we propose that the mechanisms that involve cell cycle control in response to osmostress are of great complexity and that they could be conserved among eukaryotic cells.

Open Access Article

TrkAIII: A Novel Hypoxia-regulated Alternative TrkA Splice Variant of Potential Physiological and Pathological Importance

Antonella Tacconelli, Antonietta R. Farina, Lucia Cappabianca, Alberto Gulino and Andrew R. Mackay
Pages 8 - 9
http://dx.doi.org/10.4161/cc.4.1.1349
Abstract | PDF

Nerve growth factor receptor TrkA is critical for development and maturation of central and peripheral nervous systems, regulating proliferation, differentiation and apoptosis. In cancer, TrkA frequently exhibits suppressor activity in non-mutated form and oncogenic activity upon mutation. Our identification of a novel hypoxia-regulated alternative TrkAIII splice variant, expressed by neural crest-derived neuroblastic tumors, that exhibits neuroblastoma tumor promoting activity, adds significantly to our understanding of potential TrkA involvement in cancer. Our observation that hypoxia, which characterises the tumor micro-environment, stimulates alternative TrkAIII splicing, provides a way by which TrkA tumor suppressing signals may convert to tumor promoting signals during progression and is consistent with conservation and pathological subversion by neural crest-derived neuroblastic tumors of a mechanism of potential physiological importance to normal neural stem/neural crest progenitors.

Open Access Article

Inactivation of Tumor Suppressor Genes: Choice Between Genetic and Epigenetic Routes

Wen Yong Chen and Stephen B. Baylin
Pages 10 - 12
http://dx.doi.org/10.4161/cc.4.1.1361
Abstract | PDF

Inactivation of tumor suppressor genes can occur via epigenetic or genetic mechanisms. The reasons underlying this choice of gene inactivation routes during tumorigenesis have not been clarified, nor have the precise roles in cancer evolution for genes which are solely affected by epigenetically mediated loss of function. Here we discuss a mouse model in which the disruption of Hic1, a gene solely involved with epigenetic silencing in human cancer, can markedly influence the disruption of the powerful tumor suppressor gene, p53, in determining malignant tumor incidence, spectrum and virulence. Furthermore, the mechanism for inactivation of Hic1 in tumors produced can be switched from an epigenetic to a genetic mode depending on how the Hic1 and p53 knockouts are localized on mouse chromosome 11. The value of such a model and the implications of the findings for choice of epigenetically versus genetically determined loss of gene function in cancer are discussed.

Open Access Article

Heat Shock, Histone H3 Phosphorylation and the Cell Cycle

Mark H. Dyson, Stuart Thomson and Louis C. Mahadevan
Pages 13 - 17
http://dx.doi.org/10.4161/cc.4.1.1362
Abstract | PDF

Genome-wide and gene-specific changes in histone H3 phosphorylation during heat shock have recently been described using two well-established experimental models, the “puffing” of heat shock loci in Drosophila polytene chromosomes and the induction of Hsp70 mRNA transcripts in cultured mouse cells. Despite conservation of the molecular participants and overall stress response in these two organisms, some striking differences have emerged. Here, we summarise accounts of heat shock-modulated histone phosphorylation in Drosophila and mouse cells highlighting these differences. In addition, we describe a further complexity of this response in cultured mouse cells that becomes apparent when the nucleosomal response, referring to histone H3 and HMGN1 phosphorylation, is monitored through the cell cycle. This suggests that some heat shock-induced effects in mouse cells may be indirect and arise as a secondary consequence of the effect of heat shock on the cell cycle, complicating comparisons between the fly and mouse systems.

Open Access Article

Life and Death in Cancer GADD45 α and γ are Critical Regulators of NF- κB Mediated Escape from Programmed Cell Death

Luiz F. Zerbini and Towia A. Libermann
Pages 18 - 20
http://dx.doi.org/10.4161/cc.4.1.1363
Abstract | PDF

The NF-kB/IkB signaling pathway is a critical regulator of cell survival, and constitutive activation of NF-kB is a crucial step for many types of cancers to escape programmed cell death. Furthermore, chemotherapeutic agents activate NF-kB in cancer cells, and this may partially explain the resistance of cancer cells to chemotherapy. The precise mechanism of the anti-apoptotic action of NF-kB is not known, but involves the regulation of several cell cycle regulatory and anti-apoptotic genes. We recently demonstrated that NF-kB mediated cell survival is absolutely dependent on two GADD45 family members, GADD45a and g. In line with this, inhibition of NF-kB in cancer cells results in GADD45a and g dependent induction of apoptosis, JNK activation and inhibition of tumor growth. These findings establish an unambiguous role for the GADD45 family as an essential mediator of cell survival in cancer cells with implications for cancer chemotherapy and novel drug discovery.

Open Access Article

Mitochondrial Dynamics Of Survivin And “Four Dimensional” Control Of Tumor Cell Apoptosis

Takehiko Dohi and Dario C. Altieri
Pages 21 - 23
http://dx.doi.org/10.4161/cc.4.1.1367
Abstract | PDF

The intense investigation of cell death pathways over the past few years has uncovered elevated complexity with panoply of molecular interactions, multiple intersecting gene networks and bidirectional crosstalk among disparate cellular regulators. What has remained more elusive is how these interactions are spatially arranged in the “four dimensions” of the cell as a whole, and their true relevance to human diseases. Fresh evidence obtained with survivin has unraveled a process of dynamic subcellular shuttling that is required to inhibit apoptosis during tumor growth, with the mitochondrion playing a central, orchestrating role.

Open Access Article

Borate Transport and Cell Growth and Proliferation: Not Only in Plants

Meeyoung Park, Qin Li, Nikolay Shcheynikov, Shmuel Muallem and Weizong Zeng
Pages 24 - 26
http://dx.doi.org/10.4161/cc.4.1.1394
Abstract | PDF

Boron is an abundant mineral essential for the life cycle of plants and may play a role in animal development and growth. Very little is known about boron homeostasis in plant and animal cells and the physiological roles of boron in animals. The recent identification of boron transporters, BOR1 in plants and NaBC1 in mammals, and that NaBC1 functions as an electrogenic Na+-coupled borate transporter essential for cell growth and proliferation open the way to probe the roles of boron in cellular function and physiology.

Open Access Article

Recruiting Substrates to Cullin 4-Dependent Ubiquitin Ligases by DDB1

Chad M. McCall, Jian Hu and Yue Xiong
Pages 27 - 29
http://dx.doi.org/10.4161/cc.4.1.1396
Abstract | PDF

The ubiqutin-proteasome system is the major pathway by which cells target proteins for degradation in a specific manner. The E3 ubiquitin ligase, which brings targeted proteins (substrates) and activated ubiquitin in close proximity, enabling covalent conjugation of ubiquitin to the substrate, is an essential component of this system. Of the E3 ligases, the cullin (CUL) ligases are of high interest because of their capacity to form multiple distinct E3 complexes to ubiquitinate a potentially large number of substrates. Of the six closely related cullins, very little is known about how specific substrates are recruited to CUL4-dependent ligases. A recent paper in Nature Cell Biology may shed some light on this issue as well as on the function of DDB1, a damaged-DNA binding protein that has long been associated with DNA repair.

Open Access Article

Signalling DNA Damage by Regulating p53 Co-factor Activity

Linda Smith and Nicholas B. La Thangue
Pages 30 - 32
http://dx.doi.org/10.4161/cc.4.1.1397
Abstract | PDF

Abstract not yet available.

Open Access Article

Dominant Negative Effects of the AML1/ETO Fusion Oncoprotein

Timothy S. Fenske, Gina Pengue and Timothy A. Graubert
Pages 33 - 36
http://dx.doi.org/10.4161/cc.4.1.1399
Abstract | PDF

The t(8;21)(q22;q22) translocation, present in 10-15% of acute myeloid leukemia (AML) cases results in the production of the AML1/ETO fusion protein. Expression of AML1/ETO in patients or mouse models is not sufficient to induce AML. Despite convincing evidence that AML1/ETO is directly involved in the pathogenesis of AML, the underlying mechanism is not well understood. Genetic and biochemical experiments suggest that AML1/ETO is a dominant inhibitor of the core binding factor (CBF) transcription complex that includes AML1 (RUNX1), the N-terminal fusion partner in the t(8;21). We generated and recently characterized a novel strain of transgenic mice in which the AML1/ETO cDNA was inserted into the Ly-6A gene that encodes Sca1, a well-characterized marker of murine hematopoietic stem cells. Unexpectedly, transgene expression assessed by flow cytometry was significantly lower than predicted in lymphocytes from these mice. We have confirmed this finding at the mRNA level and suggest that this phenotype is a consequence of dominant inhibition of transgene expression by AML1/ETO. The dominant negative characteristics of AML1/ETO may be important for AML pathogenesis and may provide a molecular target for therapeutic intervention.

Open Access Article

Crosstalk between p53 and FOXO Transcription Factors

Han You and Tak W. Mak
Pages 37 - 38
http://dx.doi.org/10.4161/cc.4.1.1401
Abstract | PDF

Recent studies find striking similarities between p53 and the FOXO family of forkhead transcription factors. Both transcription factors can be posttranslationally modified, for example, by acetylation and phosphorylation. They regulate a number of genes in common. Upregulation of either p53 or FKHRL1 leads to cell cycle arrest or apoptosis, depending on cellular context and type of stress. While these findings suggest that p53 and FOXO may parallel in their functions, our recent report demonstrates cross talk between these two transcription factors in vivo. Since both p53 and FOXO have been described as potential drug targets in cancer therapy, we believe understanding their functional interactions as well as the signaling pathways involved will be important to direct their clinical application.




PERSPECTIVES

Open Access Article

A Transcriptional Pathway for Cell Separation in Fission Yeast

Jürg Bähler
Pages 39 - 41
http://dx.doi.org/10.4161/cc.4.1.1336
Abstract | PDF

Numerous genes are transcriptionally activated and repressed in a cell cycle-dependent manner. We have recently reported the global gene expression program during the cell cycle in fission yeast (S. pombe). Among the periodically expressed fission yeast genes, a large proportion shows peak transcript levels during mitosis. Many of these genes are regulated by a transcriptional cascade involving two transcription factors: the forkhead protein Sep1p which activates the zinc finger protein Ace2p. A main function of the Sep1p-Ace2p transcriptional pathway is to trigger the separation of daughter cells after cytokinesis. Absence of Sep1p, Ace2p, or some of their target genes leads to a hyphal-like growth pattern with chains of connected cells. Yeast cells probably evolved from filamentous fungi. It is possible that the Sep1p-Ace2p pathway contributed to the emergence of proliferation through single cells, and that this regulatory pathway can still be modulated to adjust growth modes depending on environmental conditions. Here, various properties of the Sep1p-Ace2p transcriptional pathway and mechanisms for cell separation are discussed in the context of recent findings.

Jurg Bahler

Open Access Article

The Rb Tumor Suppressor in Stress Responses and Hematopoietic Homeostasis

Benjamin T. Spike and Kay F. Macleod
Pages 42 - 45
http://dx.doi.org/10.4161/cc.4.1.1337
Abstract | PDF

The Rb tumor suppressor pathway is functionally inactivated in most human cancers 1. In human blood cancers, loss of Rb expression has been widely reported in both acute myeloblastic and acute lymphoblastic leukemias, cyclin D1 amplification is common in mantle cell lymphoma, and silencing of the p16/INK4a tumor suppressor gene occurs frequently in AML and various types of lymphoma 2. Silencing of p16/INK4A expression is frequently caused by hyper-methylation of CpG islands in its promoter but may also be achieved through activation of Bmi-1, for example in lymphomas associated with the E2A-Pbx1 translocation 3. Hematopoietic homeostasis ensures a balance between proliferation and differentiation at different levels within the hematopoietic hierarchy such that hematopoietic stem cell self-renewal, progenitor proliferation and terminal differentiation to functional blood cells are maintained throughout the life of the animal 4. Leukemic transformation disrupts this balance favoring proliferation over differentiation, but the mechanisms underlying cell cycle regulation and checkpoint control in the hematopoietic system are not understood 5. Recent data from our laboratory has identified a unique role for the Rb tumor suppressor gene in the response to anemic and oxidative stress 6 that has implications not just for red cell production but for the homeostatic balance in the entire hematopoietic system and for the development of hematopoietic malignancies.

Open Access Article

Tuberous Sclerosis and Insulin Resistance: Unlikely Bedfellows Reveal A TORrid Affair

O. Jameel Shah and Tony Hunter
Pages 46 - 51
http://dx.doi.org/10.4161/cc.4.1.1343
Abstract | PDF

The TSC1-TSC2 tumor suppressor complex serves as an interface between insulin and nutrient signaling pathways and the cell growth machinery. Recent work has indicated that the TSC1-TSC2 complex plays a role in the pathobiology of a number of tumor predisposition syndromes, including tuberous sclerosis (TSC1/2), Peutz-Jeghers syndrome (LKB1), and Cowden’s syndrome (PTEN), in which the TSC/Rheb/mTOR axis is inappropriately active secondary to loss of tumor suppressor function. Recent work has demonstrated that TSC deficiency imposes a negative autoregulatory loop that suppresses insulin signaling at the post-receptor level, effectively resulting in cell autonomous insulin resistance. Exploitation of this insulin signaling deficiency may hold promise among tailored clinical therapies designed to manage tuberous sclerosis.

Open Access Article

Protein Kinase Specificity: A Strategic Collaboration between Kinase Peptide Specificity and Substrate Recruitment

Guozhi Zhu, Yin Liu and Stephen Shaw
Pages 52 - 56
http://dx.doi.org/10.4161/cc.4.1.1353
Abstract | PDF

Specificity of phosphorylation by protein kinases is essential to the integrity of biological signal transduction. Specificity is determined by two critical elements: 1) peptide specificity of the kinase, i.e. preferential phosphorylation of S/T/Y residues surrounded by particular patterns of amino acids; and 2) recruitment, i.e. increasing the frequency of encounter between kinase and substrate. Historically, the importance of peptide specificity was studied first, but it has been somewhat overshadowed by emerging emphasis on the importance of recruitment. Recent studies confirm and extend understanding of the relative contribution of these two elements. Peptide specificity always constrains the range of sites that can be phosphorylated by a kinase. Only when recruitment is very strong, as in the case with autophosphorylation, can markedly suboptimal substrates be phosphorylated.

Open Access Article

The p38-Mediated Stress-Activated Checkpoint: A Rapid Response System for Delaying Progression through Antephase and Entry into Mitosis

Alexei Mikhailov, Mio Shinohara and Conly L. Rieder
Pages 57 - 62
http://dx.doi.org/10.4161/cc.4.1.1357
Abstract | PDF

Cells have evolved a number of control pathways that delay or prevent them from entering mitosis under conditions that can compromise genome integrity. One recently appreciated and versatile control pathway involves the p38 stress activated protein kinase. During late G2 p38 is rapidly activated by diverse stresses (topoisomerase II (topo II)) and histone deacetylase inhibitors, osmotic shock, microtubule disassembly, UV light, etc) via a number of different pathways. Once activated p38 appears to delay entry into mitosis by inhibiting cdc25B phosphatase that, in turn, downregulates cyclin A/CDK2 activity. Depending on the agent and degree of stress, this delay may be transient, or it may last until transcription mediated checkpoint pathways can take over.

Open Access Article

Inhibition of Smad Antiproliferative Function by CDK Phosphorylation

Fang Liu and Isao Matsuura
Pages 63 - 66
http://dx.doi.org/10.4161/cc.4.1.1366
Abstract | PDF

Rb family members were the only demonstrated substrates of CDK4 until it was shown recently that Smad3, which plays a key role in mediating TGF-β antiproliferative responses, is phosphorylated by both CDK4 and CDK2 in vivo and in vitro. CDK phosphorylation of Smad3 inhibits its transcriptional activity and antiproliferative function. The Rb pathway is disrupted essentially in all types of human solid tumors. Most cancers contain high levels of CDK activity due to frequent inactivation of the p16 tumor suppressor or overexpression of cyclin D1. Therefore, disruption of the Rb pathway not only inactivates Rb, but also likely diminishes Smad activity, which may contribute to tumorigenesis and resistance to the TGF-β growth-inhibitory effects in cancers. Although genetic mutation of Smad3 has not been reported, CDK phosphorylation of Smad3 may provide an epigenetic mechanism for inhibition of the tumor suppressive function of Smad3 during the early stages of tumorigenesis.

Open Access Article

Leukemia-Associated Fusion Proteins: Multiple Mechanisms of Action to Drive Cell Transformation

Alessandra Insinga, Pier Giuseppe Pelicci and Saverio Minucci
Pages 67 - 69
http://dx.doi.org/10.4161/cc.4.1.1400
Abstract | PDF

Leukemic cells are defined by two main biological features: arrest of differentiation at a specific stage compatible with continued proliferation, and enhanced resistance to stress. Recent work shows that the leukemia-associated fusion protein PML-RAR can mediate both biological effects targeting independent pathways, through a unifying mechanism. Differentiation block is achieved through transcriptional silencing of genes physiologically regulated by RAR, which are involved in hematopoietic differentiation. In contrast, enhanced resistance to stress is due to the capacity of the fusion protein to cause degradation of the tumor suppressor p53, thus explaining the puzzling observation that mutations of p53 are remarkably rare in acute myeloid leukemias (AMLs). Interestingly, this latter phenomenon depends on expression of wild-type PML, acting as a molecular bridge between p53 and the fusion protein. Strikingly, both effects require a unifying molecular mechanism: aberrant recruitment of histone deacetylases (HDACs). Therefore, the study of this form of leukemia appears also of interest for a better understanding of the action of HDAC inhibitors, potential antitumor drugs that are at the early stages of clinical studies.




REVIEWS

Open Access Article

Cell Cycle Dependent Regulation of the Origin Recognition Complex

Melvin L. DePamphilis
Pages 70 - 79
http://dx.doi.org/10.4161/cc.4.1.1333
Abstract | PDF

The eukaryotic origin recognition complex (ORC) not only selects the sites where pre-replication complexes are assembled and DNA replication begins, it is the first in a series of multiple coherent pathways that determines when pre-replication complexes are assembled. Data from yeast, frogs, flies and mammals present a compelling case that one or more of the six ORC subunits undergoes cell cycle dependent modifications involving phosphorylation and ubiquitination that repress ORC activity during S, G2 and M-phases. ORC activity is not restored until mitosis is complete and a nuclear membrane is present. In yeast, frogs and mammals, the same cyclin-dependent protein kinase [Cdk1(Cdc2)] that initiates mitosis also inhibits assembly of functional ORC/chromatin sites. In yeast, ORC remains bound to chromatin throughout cell division, but in the metazoa either ORC or the Orc1 subunit appears to cycle on and off the chromatin. Thus, this “ORC cycle” is the premier step in preventing re-replication of DNA during a single cell division cycle.

Open Access Article

New Advances in the DNA Damage Response Network of Fanconi Anemia and BRCA proteins: FAAP95 Replaces BRCA2 as the True FANCB Protein

Peiwen Fei, Jinhu Yin and Weidong Wang
Pages 80 - 86
http://dx.doi.org/10.4161/cc.4.1.1358
Abstract | PDF

Fanconi anemia (FA) proteins function in a DNA damage response pathway that appears to be part of the network including breast cancer susceptibility gene products, BRCA1 and BRCA2. In response to DNA damage or replication signals, a nuclear FA core complex of at least 6 FA proteins (FANCA, FANCC, FANCE, FANCF, FANCG, and FANCL) is activated and leads to monoubiquitination of the downstream FA protein, FANCD2. One puzzling question for this pathway is the role of BRCA2. A previous study has proposed that BRCA2 could be identical to two FA proteins: FANCD1, which functions either downstream or in a parallel pathway; and FANCB, which functions upstream of the FANCD2 monoubiquitination. Now, a new study shows that the real FANCB protein is not BRCA2, but a previously uncharacterized component of the FA core complex, FAAP95, suggesting that BRCA2 does not act upstream of the FA pathway. Interestingly, the newly discovered FANCB gene is X-linked and subject to X-inactivation. The presence of a single active copy of FANCB and its essentiality for a functional FA-BRCA pathway make it a potentially vulnerable component of the cellular machinery that maintains genomic integrity.

Open Access Article

HER2-targeting Antibodies Modulate the Cyclin-dependent Kinase Inhibitor p27Kip1 via Multiple Signaling Pathways

Xiao-Feng Le, Franz Pruefer and Robert C. Bast
Pages 87 - 95
http://dx.doi.org/10.4161/cc.4.1.1360
Abstract | PDF

Anti-HER2 antibody trastuzumab is emerging as a frontline therapy for patients with metastatic breast cancers that overexpress HER2. Understanding the molecular mechanisms by which the antibody inhibits tumor growth should permit the design of even more effective trastuzumab-based protocols. Several groups including our own have demonstrated that induction of cyclin-dependent kinase (CDK) inhibitor p27Kip1 protein is one of the key mechanisms of action of HER2-targeting antibodies. In this review, we discuss currently available data regarding the multiple signaling targets and pathways by which HER2-targeting antibodies upregulate p27Kip1 protein in breast cancer cells that overexpress HER2. Anti-HER2 antibodies inhibits HER2-mediated signaling in cancer cells, ultimately upregulating the levels and activity of p27Kip1 protein. At least six signaling targets and pathways are modulated by trastuzumab. By inhibiting CDK2 and decreasing Thr187 phosphorylation of p27Kip1, trastuzumab abrogates targeting of SCFubiquitin E3 ligase and minimizes proteasome degradation of p27Kip1. By inhibiting AKT and human kinase interacting stathmin (hKIS), trastuzumab blocks Thr157-, Thr198-, and Ser10-induced p27Kip1 translocation from the nucleus to the cytosol, which increases the inhibitory effect of p27Kip1. By inhibiting Jun activation domain-binding protein 1 (Jab1) trastuzumab increases nuclear retention of p27Kip1. By inhibiting cyclin D and c-Myc, trastuzumab releases the sequestrated p27Kip1 protein from cyclin D-CDK4/6 complexes and increase the effect of p27Kip1 on CDK2-cyclin E complexes. By stimulating minibrain related kinase (MIRK), trastuzumab stabilizes p27Kip1 in the nucleus, which increases inhibitory action of p27Kip1 on CDK2. The targets and pathways affected by trastuzumab work in concert to maximize the expression and inhibitory effect of p27Kip1, which leads to cell cycle G1 arrest and growth inhibition.

Open Access Article

Poly(ADP-ribosyl)ation and Epigenetics: Is CTCF PARt of the Plot?

Elena Klenova and Rolf Ohlsson
Pages 96 - 101
http://dx.doi.org/10.4161/cc.4.1.1398
Abstract | PDF

Despite the fact that the poly(ADP-ribose) (PAR) modification of proteins has been known for more than four decades, there is no unifying picture of the pathways governed by this process. While the function of poly(ADP-ribosyl)ation (PARlation) has shown to be traditionally associated with DNA repair and genotoxic stress, there is an emerging view that PARlation is also important in epigenetic regulation of chromatin structure and gene expression in the normal context. This view has been exemplified by the recent demonstration that PARlation is essential for the manifestation of the imprinted state of the Igf2 gene. In particular, the PARlation mark was shown to associate preferentially with the maternally inherited H19 ICR allele, this association depended on functional target sites of the chromatin insulator protein CTCF and, importantly, CTCF itself was found to be PARlated. Given that CTCF is currently the only known factor common for all known vertebrate chromatin insulators, it is not surprising that the derepression of the maternal Igf2 allele by 3-aminobenzamide (an inhibitor of PAR polymerases) could be linked to a perturbed chromatin insulator function at the H19 ICR. This feature appears to extend to more than 150 chromatin insulators that were isolated due to their in vivo interaction with CTCF. In this review, we discuss in more depth these results and point out that the turnover of the PARlation mark at DNA-bound CTCF is indicative of a novel mode of rheostat control of expression domains possibly by regulating the stability of higher order chromatin conformations.




INTRO: SPOTLIGHT

Open Access Article

The Nucleolus: Playing by Different Rules?

peter shaw and John Doonan
Pages 102 - 105
http://dx.doi.org/10.4161/cc.4.1.1467
Abstract | PDF

The nucleolus, although an integral part of all eukaryotic nuclei, plays by its own rules in many respects. Cytologically, it is the most prominent subnuclear domain; functionally, it is the site of transcription of 3 of the ribosomal RNAs from the tandemly repeated rDNA, and subsequently of ribosome biogenesis; biochemically, it possesses transcriptional and post-transcriptional machinery not shared with the rest of the nucleus. Making the huge number of ribosomes required by the cell represents an enormous investment of metabolic activity, and so nucleolar function can easily become a de facto limit to cell growth: nucleolar activity must also be actively regulated, but the detailed regulatory networks linking the nucleolus with cellular metabolism are still unclear. Several recent reports have now shown that segregation of the rDNA in yeast, along with telomeric DNA, is also controlled differently from the rest of the genome. This short review describes some of the features of the nucleolus and highlights recent progress in understanding this important, but enigmatic nuclear structure.




SPOTLIGHT

Open Access Article

Nucleolus in the Spotlight

D. Hernandez-Verdun
Pages 106 - 108
http://dx.doi.org/10.4161/cc.4.1.1355
Abstract | PDF

The recent contributions on chromatid segregation at the metaphase/anaphase transition demonstrate two distinct pathways in budding yeast. While segregation of most of the genome is a direct consequence of cohesin cleavage by separase, rDNA segregation requires a novel pathway involving Cdc14 phosphatase activation. This activation induces targeting of condensin to rDNA which in association with Aurora B kinase modulates rDNA compaction during anaphase. The resolution of rDNA sequences occurs after this step.

Open Access Article

CDC14 and the Temporal Coordination between Mitotic Exit and Chromosome Segregation

Jordi Torres-Rosell, Félix Machín and Luis Aragón
Pages 109 - 112
http://dx.doi.org/10.4161/cc.4.1.1356
Abstract | PDF

Cell division involves the inheritance of a complete set of the genome in the form of chromosomes. One of the strategies employed by eukaryotic cells is to maintain replicated sister chromatids together until the anaphase onset. A protein complex named cohesin holds sisters together following replication until anaphase when cleavage of cohesin by the protease separase initiates segregation. Recent studies in budding yeast have shown that cohesin cleavage alone is not sufficient for the segregation of the entire genome. Instead, repetitive regions, such as the ribosomal DNA (rDNA) array and telomeres, require additional mechanisms during mitotic disjunction. The segregation of such chromosome regions is delayed and needs specific cell cycle regulators such as the FEAR network and the conserved phosphatase Cdc14, all of which orchestrate the timely completion of chromosome segregation to mitotic exit. Future studies will be targeted towards unravelling the nature of the additional segregation requirements for repetitive regions and the specifics of its cell cycle control.

Open Access Article

A Case of Selfish Nucleolar Segregation

Alexander V. Strunnikov
Pages 113 - 117
http://dx.doi.org/10.4161/cc.4.1.1488
Abstract | PDF

Mitotic segregation of nucleolus in fission and budding yeast proceeds without disassembling its complex structure, creating challenging problems for transmission of nucleolus-organizing regions during nuclear division. The SMC complex called condensin, which plays a leading role in organizing mitotic structure of chromosomes in all eukaryotes, is essential for nucleolar segregation in budding yeast, where rDNA chromatin is the main target of mitotic condensin activity. Mitosis-specific condensin targeting to the nucleolus presents an attractive model to study mechanisms controlling condensin binding to specific chromatin domains. Recent reports suggest that the early-anaphase release of Cdc14 from the nucleolus (FEAR pathway) controls the proficiency of nucleolar segregation by promoting the mitotic condensin function in rDNA. This finding uncovers an essential function for the FEAR pathway and postulates the unique nucleolar self-regulatory mechanism, which evolved to recruit two essential enzymatic activities, Cdc14 phosphatase and condensin ATP-dependent supercoiling, for the specific task of segregating nucleoli without their disassembly.




BRIEF REPORTS

Open Access Article

A Subtle Change in p38 MAPK Activity is Sufficient to Suppress In Vivo Tumorigenesis

Oleg Timofeev, Ting Yi Lee and Dmitry V. Bulavin
Pages 118 - 120
http://dx.doi.org/10.4161/cc.4.1.1342
Abstract | PDF

Emerging evidence supports a role for p38 MAPK in negative regulation of tumorigenesis. Here we show that a subtle activation of p38 MAPK is sufficient to suppress tumorigenesis as measured by the ability to form tumors when MKK6-inducible cells were explanted into nude mice. On the other hand, this activation of p38 MAPK did not necessarily cause an immediate inhibition of cell growth in vitro as measured by standard MTS assay. This data uncovers a new methodology for anti-cancer drugs screening and suggests that a substantial number of potential anti-tumor compounds, such as activators of MKK6/p38 signaling, was missed out in previous high throughput screens based on conventional growth inhibition assays.

Open Access Article

The Telomere Repeat Binding Protein Trf1 Interacts with the Spindle Checkpoint Protein Mad1 and Nek2 Mitotic Kinase

Graham Prime and David Markie
Pages 121 - 124
http://dx.doi.org/10.4161/cc.4.1.1351
Abstract | PDF

The mitotic spindle checkpoint arrests cells in metaphase until all kinetochores are correctly attached to spindle fibres. In a search for new components of the spindle checkpoint, based on protein interaction, we have identified mouse Telomeric Repeat Binding Factor 1 (Trf1) as a protein that interacts directly with the spindle checkpoint protein Mad1 and the mitotic kinase Nek2. Trf1 has a role in the regulation of telomere length, but has also been implicated in cell cycle regulation. These interactions now provide a direct link between telomere length regulation and the control of mitotic progression.

Open Access Article

Two-Faced Cyclins with Eyes on the Targets

Vincent Archambault, Nicolas E. Buchler, Gwendolyn M. Wilmes, Matthew D. Jacobson and Frederick R. Cross
Pages 125 - 130
http://dx.doi.org/10.4161/cc.4.1.1402
Abstract | PDF

We recently reported that the ‘hydrophobic patch’ (HP) of the Saccharomyces cerevisiae S-phase cyclin Clb5 facilitates its interaction with Orc6 (via its Cy or RXL motif), providing a mechanism that helps prevent re-replication from individual origins.1 This is the first finding of a biological function for an interaction between a cyclin and a cyclin-binding motif (Cy or RXL motif) in a target protein in Saccharomyces cerevisiae. It is also the first such example involving a B-type cyclin in any organism. Yet, some of our observations as well as work from other groups suggest that HP-RXL interactions are functionally important for cyclin-Cdk signaling to other targets. The evolutionary conservation of the HP motif suggests that it allows cyclins to carry out important and specialized functions.




REPORTS

Open Access Article

Chk1 is Essential for Tumor Cell Viability Following Activation of the Replication Checkpoint

Song H. Cho, Christian D. Toouli, Gregory H. Fujii, Chad Crain and David Parry
Pages 131 - 139
http://dx.doi.org/10.4161/cc.4.1.1299
Abstract | PDF

Chk1 (checkpoint kinase 1) is an evolutionarily conserved serine/threonine kinase involved in DNA damage responses. Originally identified as a kinase regulating the G2/ M transition checkpoint, its role has broadened to include the S-phase checkpoint response and essential functions in early embryonic development. In this manuscript we investigated the potential of chemo-sensitization via ablation of Chk1 in cells treated with anti-metabolite cancer drugs, hydroxyurea (HU) and cytosine arabinoside (ara-C). Exposure to these replication interfering drugs in cells carrying Chk1 targeted siRNA provoked markedly increased rates of apoptosis. Although cell death was accompanied by an increase in p53 and activation of Chk2, the increased susceptibility to apoptosis was not dependent on p53 or Chk2. Additionally, we found that cells with reduced Chk1 expression displayed increased ?-H2A.X expression, a marker for damaged DNA, and phosphorylated 32kDa subunit of replication protein A (RPA). Thus, Chk1 may play an essential role in maintaining DNA integrity during the replication block. Significantly, normal cells such as WS1 did not exhibit increased DNA damage or subsequent increases in apoptosis following replication stress, in the absence of Chk1. Thus, the essential role Chk1 plays in maintaining viability during the replication block in cancer cell lines can be exploited to sensitize cancer cells when abrogation of Chk1 is combined with DNA anti-metabolite chemotherapeutic drugs. Taken together, these data suggest that inhibition of Chk1 in combination with DNA anti-metabolite chemotherapy is a viable therapeutic strategy.

Open Access Article

Novel Assay Utilizing Fluorochrome-Tagged Physostigmine (Ph-F) to In Situ Detect Active Acetylcholinesterase (AChE) Induced during Apoptosis

Xuan Huang, Brian Lee, Gary Johnson, John Naleway, Anthony Guzikowski, Wei Dai and Zbigniew Darzynkiewicz
Pages 140 - 147
http://dx.doi.org/10.4161/cc.4.1.1322
Abstract | PDF

It was recently reported that acetylcholinesterase (AChE) is expressed in cells undergoing apoptosis and that its presence is essential for assembly of the apoptosome and subsequent caspase-9 activation. To obtain a marker of active AChE that could assay this enzyme in live intact cells and be applicable to fluorescence microscopy and cytometry, the fluorescein-tagged physostigmine (Ph-F), high affinity ligand (inhibitor) reactive with the active center of AChE, was constructed and tested for its ability to in situ label AChE and measure its induction during apoptosis. Ph-F inhibited cholinesterase activity in vitro (IC50 = 10-6 and 5x10-6 M for equine butyrycholinesterase and human erythrocyte AChE, respectively) and was a selective marker of cells and structures that were AChE-positive. Thus, exposure of mouse bone marrow cells to Ph-F resulted in the exclusive labeling of megakaryocytes, and of the diaphragm muscle, preferential labeling of the nerve-muscle junctions (end-plates). During apoptosis of carcinoma HeLa cells and leukemic HL-60 or Jurkat cells triggered either by the DNA topoisomerase 1 inhibitor topotecan (TPT) or by oxidative stress (H2O2), the cells become reactive with Ph-F. Their Ph-F derived fluorescence was measured by flow and laser scanning cytometry. The appearance of Ph-F binding sites during apoptosis was preceded by the loss of mitochondrial potential, was concurrent with the presence of activated caspases, and was followed by loss of membrane integrity. At a very early stage of apoptosis, when nucleolar segregation was apparent, the Ph-F binding sites were distinctly localized within the nucleolus and at later stages of apoptosis in the cytoplasm. During apoptosis triggered by TPT, Ph-F binding was preferentially induced in S-phase cells. Our data on megakarocytes and end-plates indicate that Ph-F reacts with active sites of AChE, and can be used to reveal the presence of this enzyme in live cells and possibly to study its expression in disorders of the neurological cholinergic system. The findings are also compatible with the reports that AChE may be induced during apoptosis. In fact, the simple and rapid Ph-F binding assay may serve as a convenient marker of apoptotic cells. However, the proposed role of active AChE as an essential factor for assembly of the apoptosome and caspase activation is arguable because the AChE inhibitors Ph, Ph-F and BW284c51did not protect the cells from apoptosis induced by TPT or H2O2. Further studies are thus needed to ascertain the induction and role of AChE in apoptosis.

Open Access Article

Phosphorylation of Cdc25C by pp90Rsk Contributes to a G2 Cell Cycle Arrest in Xenopus Cycling Egg Extracts

Justin Chun, Andrew S-S Chau, Ferdinand G. Maingat, Stuart D. Edmonds, Hanne L. Ostergaard and Ellen K. Shibuya
Pages 148 - 154
http://dx.doi.org/10.4161/cc.4.1.1323
Abstract | PDF

In unfertilized Xenopus eggs, the p42 mitogen activated protein kinase (p42MAPK) pathway is known to maintain cell cycle arrest at metaphase of meiosis II. However, constitutive activation of p42MAPK in post-meiotic, cycling Xenopus egg extracts can lead to either a G2 or M-phase arrest of the cell cycle, depending on the timing of p42MAPK activation. Here, we examined the molecular mechanism by which activation of the p42MAPK pathway during interphase leads to cell cycle arrest in G2. When either a recombinant wild type Cdc25C(WT) or a mutated form of Cdc25C, in which serine 287 was replaced by an alanine (S287A), was added to cycling egg extracts, S287A accelerated entry into M-phase. Furthermore, the addition of S287A overcame the G2 arrest caused by p42MAPK, driving the extract into M-phase. p90Rsk, a kinase that is the target of p42MAPK, was phosphorylated and activated (pp90Rsk) in the G2-arrested egg extracts, and was able to phosphorylate WT but not S287A in vitro. 14-3-3 proteins were associated with endogenous Cdc25C in G2-arrested extracts. Cdc25C(WT) that had been phosphorylated by pp90Rsk bound 14- 3-3?, whereas S287A could not. These data suggest that the link between the p42MAPK signaling pathway and Cdc25C involves the activation of pp90Rsk and its phosphorylation of Cdc25C at S287, causing the binding of 14-3-3 proteins. We propose that the binding of 14-3-3 proteins to pp90Rsk phosphorylated-Cdc25C results in a G2 arrest in a manner similar to the cell cycle delays induced by differentiation signals that occur later in embryonic development.

Open Access Article

Identification and Comparative Analysis of Multiple Mammalian Speedy/Ringo Proteins

Aiyang Cheng, Wen Xiong, James E. Ferrell, Jr. and Mark J. Solomon
Pages 155 - 165
http://dx.doi.org/10.4161/cc.4.1.1347
Abstract | PDF

In addition to their activation via binding to cyclins, cyclin-dependent kinases (CDKs) can be activated via binding to a novel cell cycle regulator termed Speedy or Ringo, which shows no apparent similarity to cyclins. The first Speedy/Ringo protein was found to be essential for Xenopus oocyte maturation and a human homolog (Spy1, herein called Speedy/Ringo A1) regulates S-phase entry and cell survival after DNA damage in cultured somatic cells. We have identified a Speedy/Ringo-like gene in the most primitive branching clade of chordates (Ciona intestinalis), as well as four mammalian homologs. Of the mammalian proteins, two, Speedy/Ringo A and C, bind to Cdc2 and Cdk2, whereas Speedy/Ringo B binds preferentially to Cdc2. Despite their distinct CDK-binding preferences, both Speedy/Ringo A and B can promote the maturation of Xenopus oocytes and all three Speedy/Ringo proteins can bind to and activate CDKs in vivo. These mammalian Speedy/Ringo proteins exhibit distinct tissue expression patterns, though all three are enriched in testis, consistent with the initial observation that Xenopus Speedy/Ringo functions during meiosis. Speedy/Ringo A is widely expressed in tissues and cell lines. Speedy/Ringo B expression appears to be testis-specific. Speedy/Ringo C is expressed in diverse tissues, particularly those that undergo polyploidization. All Speedy/Ringo proteins share a highly conserved ~140-aa domain we term the Speedy/Ringo box that is essential for CDK binding. Point mutations in this domain abolish CDK binding. Besides the central Speedy/Ringo box, Speedy/Ringo A contains a C-terminal portion, which promotes CDK activation, and an N-terminal portion, which is dispersible for both CDK binding and activation but that influences protein expression. The existence of this growing family of CDK activators suggests that Speedy/Ringo proteins may play as complex a role in cell cycle control as the diverse family of cyclins.

Open Access Article

Phosphorylation of Plk1 at S137 and T210 is Inhibited in Response to DNA Damage

Lyuben Tsvetkov and David F. Stern
Pages 166 - 171
http://dx.doi.org/10.4161/cc.4.1.1348
Abstract | PDF

Polo-like kinase 1 (Plk1) regulates multiple processes during mitosis. Plk1 is activated by phosphorylation at the G2/M phase boundary. Active Plk1 is involved in promotion of mitotic entry through activation of Cdc25C, and through nuclear import of cyclin B1 that together activate Cdc2/cyclin B kinase. In earlier work, phosphopeptide mapping identified several phosphorylation sites in Plk1. Mutational analysis pinpointed threonine 210, which is located in the activation loop of the kinase domain, as the major activation site of Plk1. In response to DNA damage, ATM/ATR-dependent checkpoint pathways inhibit Plk1 activity. Insensitivity of Plk1T210D, a constitutively active mutant, to DNA damage-induced inhibition of Plk1 indicates that regulation of Plk1 phosphorylation is a potential target of DNA damage checkpoints. In the present paper, we report that in vivo phosphorylation of Plk1 at serine 137 (S137) and threonine 210 (T210) occurs in mitosis. DNA damage prevents phosphorylation of Plk1 at both S137 and T210 in asynchronous cells but not in mitotic cells. Inhibitors of ATM/ATR and Chk1/Chk2 protein kinases avert the inhibition of Plk1 phosphorylation in response to DNA damage. These data suggest a participation of DNA damage checkpoints in regulation of the signaling pathways upstream of Plk1.

Open Access Article

Replication Timing of Human Chromosome 6

Kathryn Woodfine, David M. Beare, Koichi Ichimura, Silvana Debernardi, Andrew J. Mungall, Heike Fiegler, V. Peter Collins, Nigel P. Carter and Ian Dunham
Pages 172 - 176
http://dx.doi.org/10.4161/cc.4.1.1350
Abstract | PDF

Genomic microarrays have been used to assess DNA replication timing in a variety of eukaryotic organisms1-3. A replication timing map of the human genome has already been published at a 1Mb resolution3. Here we describe how the same method can be used to assess the replication timing of chromosome 6 with a greater resolution using an array of overlapping tile path clones. We report the replication timing map of the whole of chromosome 6 in general, and the MHC region in particular. Positive correlations are observed between replication timing and a number of genomic features including GC content, repeat content and transcriptional activity.

Open Access Article

Specific Association of Mouse MDC1/NFBD1 with NBS1 at Sites of DNA-Damage

Alicia C. Lee, Oscar Fernandez-Capetillo, Stephen P. Jackson and André Nussenzweig
Pages 177 - 182
http://dx.doi.org/10.4161/cc.4.1.1354
Abstract | PDF

Human MDC1/NFBD1 has been found to interact with key players of the DNA-damage response machinery. Here, we identify and describe a functional homologue of MDC1/NFBD1 in Mus musculus. The mouse homologue, mMDC1, retains the key motifs identified in the human protein and in response to ionizing radiation forms foci that colocalize with the MRE11-RAD50- NBS1 (MRN) complex and factors such as ?H2AX and 53BP1. In addition, mMDC1 is associated with DNA damage sites generated during meiotic recombination as well as the X and Y chromosomes during the late stages of meiotic prophase I. Finally, whereas MDC1 shows strong colocalization with the MRN complex in response to DNA damage it does not colocalize with the MRN complex on replicating chromatin. These data suggest that mMDC1 is a marker for both exogenously and endogenously generated DNA double-stranded breaks and that its interaction with the MRN complex is initiated exclusively by DNA damage.

Open Access Article

The Protein Stability and Transcriptional Activity of p63α are Regulated by SUMO-1 Conjugation

Pamela Ghioni, Yuri D’Alessandra, Gelsomina Mansueto, Ellis Jaffray, Ronald T. Hay, Girolama La Mantia and Luisa Guerrini
Pages 183 - 190
http://dx.doi.org/10.4161/cc.4.1.1359
Abstract | PDF

Post-translational modification of proteins by the ubiquitin-like molecule SUMO-1 regulates their stability and activity with crucial implications for many cellular processes. Here we show that p63α, but not p63α and α, is sumoylated in vitro and in vivo at a single lysine residue, K637, in the post-SAM domain. SUMO-1 attachment targets ΔNp63α for proteasome mediated degradation while it does not influence p63α intracellular localization, as wild-type protein and a mutant carring the K637 mutated into arginine (K637R), have the same nuclear localization. Four natural p63 mutations, falling within the SAM and post-SAM domain of p63α, were found to be altered in their sumoylation capacity. The transcriptional activities of the natural mutants and of K637R were strongly increased compared to that of wild type p63, suggesting that sumoylation has a negative effect on p63 driven transcription. The findings that ΔNp63α protein levels are regulated by SUMO-1 and that this regulation is altered in natural p63 mutants, suggest that SUMO conjugation to p63 plays a critical role in regulating the biological activity of p63.

Open Access Article

Characterisation and Gene Expression Profiling of a Stable Cell Line Expressing a Cell Cycle GFP Sensor

Nick Thomas, Mike Kenrick, Theresa Giesler, Gretchen Kiser, Hayley Tinkler and Simon Stubbs
Pages 191 - 195
http://dx.doi.org/10.4161/cc.4.1.1405
Abstract | PDF

The use of stable cell lines expressing fusions with green fluorescent protein (GFP) has increased significantly in recent years. In this study we have used a range of complimentary analytical techniques to examine the characteristics of a cell line stably expressing a EGFP cell cycle sensor relative to parental U2OS cells. Analysis of cell cycle duration and cell cycle phase distribution by cell growth assays and flow cytometry revealed that the two cell lines had identical doubling times and cell cycle distributions. Measurement of EGFP fusion protein mRNA by quantitative RT-PCR indicated a EGFP sensor expression level equivalent to endogenous Cyclin B1 (7000 copies/cell in G2). Microarray analysis showed a 0.9% (>2 fold at p<0.001 across 20,000 genes) difference in global gene expression levels between parental and EGFP expressing U2OS cells, with no significant differences in expression of A, B, C, D, E, F, G, H, I, K, L, M or T type Cyclins between the two cell types. These results confirm that engineering a stable cell line for low expression of a EGFP cell cycle sensor is minimally perturbing to the cell cycle and cellular gene expression.