Table of Contents

Volume 11, Issue 16

  August 15, 2012

  Pages 2959 - 3142

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 Issue Catalogue (MARC XML)

SPECIAL FEATURE

Niche science: The aging stem cell

Heinrich Jasper and Brian K. Kennedy
Pages 2959 - 2960
http://dx.doi.org/10.4161/cc.21558
Abstract | Full Text | PDF

Studies on stem cell aging are uncovering molecular mechanisms of regenerative decline, providing new insight into potential rejuvenating therapies.




EDITORIALS: CELL CYCLE FEATURES

Open Access Article

MALT lymphoma meets stem cells

Carolina Vicente-Dueñas, César Cobaleda, José Ángel Martínez-Climent and Isidro Sánchez-García
Pages 2961 - 2962
http://dx.doi.org/10.4161/cc.21264
Abstract | Full Text | PDF

Comment on: Vicente-Dueñas C, et al. Proc Natl Acad Sci USA 2012; 109:10534-9.

Open Access Article

IF1 reprograms energy metabolism and signals the oncogenic phenotype in cancer

María Sánchez-Aragó, Laura Formentini, Javier García-Bermúdez and José M. Cuezva
Pages 2963 - 2964
http://dx.doi.org/10.4161/cc.21387
Abstract | Full Text | PDF

Comment on: Formentini L, et al. Mol Cell 2012; 45:731-42.

Open Access Article

A long way to stemness

Igor M. Samokhvalov
Pages 2965 - 2966
http://dx.doi.org/10.4161/cc.21388
Abstract | Full Text | PDF

Comment on: Tanaka Y, et al. Proc Natl Acad Sci USA 2012; 109:4515-20.

Open Access Article

Manipulation of single polymerase-DNA complexes: A mechanical view of DNA unwinding during replication

Jose A. Morin, Francisco J. Cao, José M. Valpuesta, José L. Carrascosa, Margarita Salas and Borja Ibarra
Pages 2967 - 2968
http://dx.doi.org/10.4161/cc.21389
Abstract | Full Text | PDF

Comment on: Morin JA, et al. Proc Natl Acad Sci USA 2012; 109:8115-20.

Open Access Article

Through the looking-glass: Observing HSCs through the lens of infection reveals unexpected insights into HSC function

Christopher Hall, Kathryn Crosier and Philip Crosier
Pages 2969 - 2970
http://dx.doi.org/10.4161/cc.21390
Abstract | Full Text | PDF

Comment on: Hall CJ, et al. Cell Stem Cell 2012; 10:198-209.

Open Access Article

Drosophila growth and development: Keeping things in proportion

Ditte S. Andersen, Julien Colombani and Pierre Léopold
Pages 2971 - 2972
http://dx.doi.org/10.4161/cc.21466
Abstract | Full Text | PDF

Comment on: Colombani J, et al. Science 2012; 336:582-5.

Open Access Article

T-ARG-eting E2F-1 growth control

Jutta Moehlenbrink and Nicholas B. La Thangue
Pages 2973 - 2974
http://dx.doi.org/10.4161/cc.21467
Abstract | Full Text | PDF

Comment on: Cho EC, et al. EMBO J 2012; 31:1785-97.




CELL CYCLE NEWS & VIEWS

Open Access Article

The p53-HAT connection: PCAF rules?

Oleg Laptenko and Carol Prives
Page 2975
http://dx.doi.org/10.4161/cc.21528
Abstract | Full Text | PDF

Comment on: Love IM, et al. Cell Cycle 2012; 11:2458-66.

Open Access Article

A critical role for TORC1 in cellular senescence

Eros Lazzerini Denchi
Page 2976
http://dx.doi.org/10.4161/cc.21529
Abstract | Full Text | PDF

Comment on: Kolesnichenko M, et al. Cell Cycle 2012; 11:2391-401 and Pospelova TV, et al. Cell Cycle 2012; 11:2402-407.

Open Access Article

Timeless tunes: Replicating happy endings

Fuyuki Ishikawa
Page 2977
http://dx.doi.org/10.4161/cc.21530
Abstract | Full Text | PDF

Comment on: Leman AR, et al. Cell Cycle 2012; 11:2337-47.

Open Access Article

A new mode of cell cycle stimulation: Cyclin E and CDK2-mediated cytoplasmic retention of repressive E2F complexes

Alexandre Blais
Page 2978
http://dx.doi.org/10.4161/cc.21531
Abstract | Full Text | PDF

Comment on: Kolupaeva V, et al. Cell Cycle 2012; 11:2557-66.

Open Access Article

“Double hit” makes the difference

Frédéric Bost
Page 2979
http://dx.doi.org/10.4161/cc.21532
Abstract | Full Text | PDF

Comment on: Menendez JA, et al. Cell Cycle 2012; 11:2782–92.




EXTRA VIEWS

R-loops and genomic instability in Bre1 (RNF20/40)-deficient cells

Sophia B. Chernikova and J. Martin Brown
Pages 2980 - 2984
http://dx.doi.org/10.4161/cc.21090
Abstract | Full Text | PDF

We have proposed that maintenance of genomic stability may constitute the basis for the tumor-suppressing activity of the Bre1 (RNF20/RNF40) complex. Revisiting the evidence we presented in our recent publication, we discuss the mechanism by which maintenance of genomic stability by the Bre1 complex is achieved through coordination of events during transcription. Among many functions of Bre1, we focus on the two that, when defective, could lead to the formation of R-loops, the RNA:DNA hybrid structures regarded as a major source of genomic instability. Specifically, we discuss the role of Bre1-mediated H2B ubiquitination in the 3′-end processing of replication-associated histone mRNA and in heterochromatic gene silencing and show how disturbance of these two functions may result in the specific pattern of chromosomal abnormalities we observe in the Bre1-depleted cells.

Induced pluripotent stem cells as a tool for gaining new insights into Fanconi anemia

Lars U.W. Müller, Thorsten M. Schlaeger, Alexander L. DeVine and David E. Williams
Pages 2985 - 2990
http://dx.doi.org/10.4161/cc.21109
Abstract | Full Text | PDF

Induced pluripotent stem cells (iPSC) hold significant promise for advancing biomedical research. In the case of monogenic diseases, patient-iPSC and their derivatives contain the disease-causing mutation, suggesting the possibility of recapitulating salient disease features in vitro. Fanconi anemia (FA) is the most common inherited bone marrow failure syndrome. The etiology of bone marrow failure in FA remains largely unclear, but limited studies on patient bone marrow cells indicate cell intrinsic defects as causative. We examined the feasibility of modeling FA in a system based on hematopoietic differentiation of patient-specific iPSC. An informative iPSC-based model is predicated on the ability to derive disease-specific (uncorrected) patient iPSC that contain the disease-causing mutation, are pluripotent, maintain a normal karyotype and are capable of hematopoietic differentiation. Careful analysis of hematopoietic differentiation of such iPSC holds the promise of uncovering new insights into bone marrow failure and may enable high-throughput screening with the goal of identifying compounds that ameliorate hematopoietic failure. Ultimately, genetic correction, molecular characterization and successful engraftment of iPSC-derived cells may provide an attractive alternative to current hematopoietic stem cell-targeted gene therapy in some monogenic diseases, including FA.

Reconciling the different roles of Gsk3β in “naïve” and “primed” pluripotent stem cells

Amar M. Singh, Matthew Bechard, Keriayn Smith and Stephen Dalton
Pages 2991 - 2996
http://dx.doi.org/10.4161/cc.21110
Abstract | Full Text | PDF

Signaling pathways orchestrated by PI3K/Akt, Raf/Mek/Erk and Wnt/β-catenin are known to play key roles in the self-renewal and differentiation of pluripotent stem cells. The serine/threonine protein kinase Gsk3β has roles in all three pathways, making its exact function difficult to decipher. Consequently, conflicting reports have implicated Gsk3β in promoting self-renewal, while others suggest that it performs roles in the activation of differentiation pathways. Different thresholds of Gsk3β activity also have different biological effects on pluripotent cells, making this situation even more complex. Here, we describe a further level of complexity that is most apparent when comparing “naïve” murine and “primed” human pluripotent stem cells. In naïve cells, Gsk3β activity is restrained by PI3K/Akt, but when released from inhibitory signals it antagonizes self-renewal pathways by targeting pluripotency factors such as Myc and Nanog. This situation also applies in primed cells, but, in addition, a separate pool of Gsk3β is required to suppress canonical Wnt signaling. These observations suggest that different Gsk3β-protein complexes shift the balance between naïve and primed pluripotent cells and identify fundamental differences in their cell signaling. Altogether, these findings have important implications for the mechanisms underpinning the establishment of different pluripotent cell states and for the control of self-renewal and differentiation.




PERSPECTIVES

DNA excision repair: Where do all the dimers go?

Michael G. Kemp and Aziz Sancar
Pages 2997 - 3002
http://dx.doi.org/10.4161/cc.21126
Abstract | Full Text | PDF

Exposure of cells to UV light from the sun causes the formation of pyrimidine dimers in DNA that have the potential to lead to mutation and cancer. In humans, pyrimidine dimers are removed from the genome in the form of ~30 nt-long oligomers by concerted dual incisions. Though nearly 50 y of excision repair research has uncovered many details of UV photoproduct damage recognition and removal, the fate of the excised oligonucleotides and, in particular, the ultimate fate of the chemically very stable pyrimidine dimers remain unknown. Physiologically relevant UV doses introduce hundreds of thousands of pyrimidine dimers in diploid human cells, which are excised from the genome within ~24 h. Once removed from the genome, “where do all the dimers go?” In a recent study we addressed this question. Although our study did not determine the fate of the dimer itself, it revealed that the excised ~30-mer is released from the duplex in a tight complex with the transcription/repair factor TFIIH. This finding combined with recent reports that base and oligonucleotide products of the base and double-strand break repair pathways also make stable complexes with the cognate repair enzymes, and that these complexes activate the MAP kinase and checkpoint signaling pathways, respectively, raises the possibility that TFIIH-30-mer excision complexes may play a role in signaling reactions in response to UV damage.




REVIEW

Open Access Article

Cell cycle regulation of Rho signaling pathways

Muriel David, Dominique Petit and Jacques Bertoglio
Pages 3003 - 3010
http://dx.doi.org/10.4161/cc.21088
Abstract | Full Text | PDF

The dynamics of the actin cytoskeleton and its regulation by Rho GTPases are essential to maintain cell shape, to allow cell motility and are also critical during cell cycle progression and mitosis. Rho GTPases and their effectors are involved in cell rounding at mitosis onset, in chromosomes alignment and are required for contraction of the actomyosin ring that separates daughter cells at the end of mitosis. Recent studies have revealed how a number of nucleotide exchange factors and GTPase-activating proteins regulate the activity of Rho GTPases during these processes. This review will focus on how the cell cycle machinery, in turn, regulates expression of proteins in the Rho signaling pathways through transcriptional activation, ubiquitylation and proteasomal degradation and modulates their activity through phosphorylation by mitotic kinases.




REPORTS

Open Access Article

Lack of response to unaligned chromosomes in mammalian female gametes

Jaroslava Sebestova, Anna Danylevska, Lucia Novakova, Michal Kubelka and Martin Anger
Pages 3011 - 3018
http://dx.doi.org/10.4161/cc.21398
Abstract | Full Text | PDF

Chromosome segregation errors are highly frequent in mammalian female meiosis, and their incidence gradually increases with maternal age. The fate of aneuploid eggs is obviously dependent on the stringency of mechanisms for detecting unattached or repairing incorrectly attached kinetochores. In case of their failure, the newly formed embryo will inherit the impaired set of chromosomes, which will have severe consequences for its further development. Whether spindle assembly checkpoint (SAC) in oocytes is capable of arresting cell cycle progression in response to unaligned kinetochores was discussed for a long time. It is known that abolishing SAC increases frequency of chromosome segregation errors and causes precocious entry into anaphase; SAC, therefore, seems to be essential for normal chromosome segregation in meiosis I. However, it was also reported that for anaphase-promoting complex (APC) activation, which is a prerequisite for entering anaphase; alignment of only a critical mass of kinetochores on equatorial plane is sufficient. This indicates that the function of SAC and of cooperating chromosome attachment correction mechanisms in oocytes is different from somatic cells. To analyze this phenomenon, we used live cell confocal microscopy to monitor chromosome movements, spindle formation, APC activation and polar body extrusion (PBE) simultaneously in individual oocytes at various time points during first meiotic division. Our results, using oocytes from aged animals and interspecific crosses, demonstrate that multiple unaligned kinetochores and severe congression defects are tolerated at the metaphase to anaphase transition, although such cells retain sensitivity to nocodazole. This indicates that checkpoint mechanisms, operating in oocytes at this point, are essential for accurate timing of APC activation in meiosis I, but they are insufficient in detection or correction of unaligned chromosomes, preparing thus conditions for propagation of the aneuploidy to the embryo.

Metabolic reprogramming of cancer-associated fibroblasts by TGF-β drives tumor growth: Connecting TGF-β signaling with “Warburg-like” cancer metabolism and L-lactate production

Carmela Guido, Diana Whitaker-Menezes, Claudia Capparelli, Renee Balliet, Zhao Lin, Richard G. Pestell, Anthony Howell, Saveria Aquila, Sebastiano Andò, Ubaldo Martinez-Outschoorn, Federica Sotgia and Michael P. Lisanti
Pages 3019 - 3035
http://dx.doi.org/10.4161/cc.21384
Abstract | Full Text | PDF

We have previously shown that a loss of stromal Cav-1 is a biomarker of poor prognosis in breast cancers. Mechanistically, a loss of Cav-1 induces the metabolic reprogramming of stromal cells, with increased autophagy/mitophagy, mitochondrial dysfunction and aerobic glycolysis. As a consequence, Cav-1-low CAFs generate nutrients (such as L-lactate) and chemical building blocks that fuel mitochondrial metabolism and the anabolic growth of adjacent breast cancer cells. It is also known that a loss of Cav-1 is associated with hyperactive TGF-β signaling. However, it remains unknown whether hyperactivation of the TGF-β signaling pathway contributes to the metabolic reprogramming of Cav-1-low CAFs. To address these issues, we overexpressed TGF-β ligands and the TGF-β receptor I (TGFβ-RI) in stromal fibroblasts and breast cancer cells. Here, we show that the role of TGF-β in tumorigenesis is compartment-specific, and that TGF-β promotes tumorigenesis by shifting cancer-associated fibroblasts toward catabolic metabolism. Importantly, the tumor-promoting effects of TGF-β are independent of the cell type generating TGF-β. Thus, stromal-derived TGF-β activates signaling in stromal cells in an autocrine fashion, leading to fibroblast activation, as judged by increased expression of myofibroblast markers, and metabolic reprogramming, with a shift toward catabolic metabolism and oxidative stress. We also show that TGF-β-activated fibroblasts promote the mitochondrial activity of adjacent cancer cells, and in a xenograft model, enhancing the growth of breast cancer cells, independently of angiogenesis. Conversely, activation of the TGF-β pathway in cancer cells does not influence tumor growth, but cancer cell-derived-TGF-β ligands affect stromal cells in a paracrine fashion, leading to fibroblast activation and enhanced tumor growth. In conclusion, ligand-dependent or cell-autonomous activation of the TGF-β pathway in stromal cells induces their metabolic reprogramming, with increased oxidative stress, autophagy/mitophagy and glycolysis, and downregulation of Cav-1. These metabolic alterations can spread among neighboring fibroblasts and greatly sustain the growth of breast cancer cells. Our data provide novel insights into the role of the TGF-β pathway in breast tumorigenesis, and establish a clear causative link between the tumor-promoting effects of TGF-β signaling and the metabolic reprogramming of the tumor microenvironment.

Werner syndrome protein suppresses the formation of large deletions during the replication of human telomeric sequences

Rama Rao Damerla, Kelly E. Knickelbein, Steven Strutt, Fu-Jun Liu, Hong Wang and Patricia L. Opresko
Pages 3036 - 3044
http://dx.doi.org/10.4161/cc.21399
Abstract | Full Text | PDF

Werner syndrome (WS) is a disorder characterized by features of premature aging and increased cancer that is caused by loss of the RecQ helicase WRN. Telomeres consisting of duplex TTAGGG repeats in humans protect chromosome ends and sustain cellular proliferation. WRN prevents the loss of telomeres replicated from the G-rich strand, which can form secondary G-quadruplex (G4) structures. Here, we dissected WRN roles in the replication of telomeric sequences by examining factors inherent to telomeric repeats, such as G4 DNA, independently from other factors at chromosome ends that can also impede replication. For this we used the supF shuttle vector (SV) mutagenesis assay. We demonstrate that SVs with [TTAGGG]₆ sequences are stably replicated in human cells, and that the repeats suppress the frequency of large deletions despite G4 folding potential. WRN depletion increased the supF mutant frequency for both the telomeric and non-telomeric SVs, compared with the control cells, but this increase was much greater (27-fold) for telomeric SVs. The higher SV mutant frequencies in WRN-deficient cells were primarily due to an increase in large sequence deletions and rearrangements. However, WRN depletion caused a more dramatic increase in deletions and rearrangements arising within the telomeric SV (70-fold), compared with non-telomeric SV (8-fold). Our results indicate that WRN prevents large deletions and rearrangements during replication, and that this role is particularly important in templates with telomeric sequence. This provides a possible explanation for increased telomere loss in WS cells.

Low-dose laulimalide represents a novel molecular probe for investigating microtubule organization

Melissa J. Bennett, Gordon K. Chan, J.B. Rattner and David C. Schriemer
Pages 3045 - 3054
http://dx.doi.org/10.4161/cc.21411
Abstract | Full Text | PDF

Laulimalide is a natural product that has strong taxoid-like properties but binds to a distinct site on β-tubulin in the microtubule (MT) lattice. At elevated concentrations, it generates MTs that are resistant to depolymerization, and it induces a conformational state indistinguishable from taxoid-treated MTs. In this study, we describe the effect of low-dose laulimalide on various stages of the cell cycle and compare these effects to docetaxel as a representative of taxoid stabilizers. No evidence of MT bundling in interphase was observed with laulimalide, in spite of the fact that MTs are stabilized at low dose. Cells treated with laulimalide enter mitosis but arrest at prometaphase by generating multiple asters that coalesce into supernumerary poles and interfere with the integrity of the metaphase plate. Cells with a preformed bipolar spindle exist under heightened tension under laulimalide treatment, and chromosomes rapidly shear from the plate, even though the bipolar spindle is well-preserved. Docetaxel generates a similar phenotype for HeLa cells entering mitosis, but when treated at metaphase, cells undergo chromosomal fragmentation and demonstrate reduced centromere dynamics, as expected for a taxoid. Our results suggest that laulimalide represents a new class of molecular probe for investigating MT-mediated events, such as kinetochore-MT interactions, which may reflect the location of the ligand binding site within the interprotofilament groove.

CDC6 expression is regulated by lineage-specific transcription factor GATA1

Bárbara Fernández-Morales, Leticia Pavón and Carmela Calés
Pages 3055 - 3066
http://dx.doi.org/10.4161/cc.21471
Abstract | Full Text | PDF

GATA1 is a hematopoietic transcription factor essential for expression of most genes encoding erythro-megakaryocytic proteins, i.e., globins and platelet glycoproteins. A role for GATA1 as a cell proliferation regulator has been proposed, as some of its bona fide targets comprise global regulators, such as c-KIT or c-MYC, or cell cycle factors, i.e., CYCLIN D or p21CIP1. In this study, we describe that GATA1 directly regulates the expression of replication licensing factor CDC6. Using reporter transactivation, electrophoretic mobility shift and chromatin immunoprecipitation assays, we show that GATA1 stimulates CDC6 transcription by binding to a canonical binding site located within a 166bp enhancer region upstream CDC6 promoter. This evolutionary conserved GATA binding site conforms to recently described chromatin occupancy rules, i.e., preferred bases within core WGATAR (TGATAA), 5' and 3' flanking bases (GGTGATAAGG) and distance to the transcription initiation site. We also found adjacent conserved binding sites for ubiquitously expressed transcription factor CP2, needed for GATA activity on CDC6 enhancer. Our results add to the growing evidence for GATA1 acting as a direct transcriptional regulator of the cell cycle machinery, thus linking cell proliferation control and specific gene expression programs during lineage differentiation.

Open Access Article

E2F1 confers anticancer drug resistance by targeting ABC transporter family members and Bcl-2 via the p73/DNp73-miR-205 circuitry

Vijay Alla, Bhavani S. Kowtharapu, David Engelmann, Stephan Emmrich, Ulf Schmitz, Marc Steder and Brigitte M. Pützer
Pages 3067 - 3078
http://dx.doi.org/10.4161/cc.21476
Abstract | Full Text | PDF

Resistance to anti-neoplastic agents is the major cause of therapy failure, leading to disease recurrence and metastasis. E2F1 is a strong inducer of apoptosis in response to DNA damage through its capacity to activate p53/p73 death pathways. Recent evidence, however, showed that E2F1, which is aberrantly expressed in advanced malignant melanomas together with antagonistic p73 family members, drives cancer progression. Investigating mechanisms responsible for dysregulated E2F1 losing its apoptotic function, we searched for genomic signatures in primary and late clinical tumor stages to allow the prediction of downstream effectors associated with apoptosis resistance and survival of aggressive melanoma cells. We identified miR-205 as specific target of p73 and found that upon genotoxic stress, its expression is sufficiently abrogated by endogenous DNp73. Significantly, metastatic cells can be rescued from drug resistance by selective knockdown of DNp73 or overexpression of miR-205 in p73-depleted cells, leading to increased apoptosis and the reduction of tumor growth in vivo. Our data delineate an autoregulatory circuit, involving high levels of E2F1 and DNp73 to downregulate miR-205, which, in turn, controls E2F1 accumulation. Finally, drug resistance associated to this genetic signature is mediated by removing the inhibitory effect of miR-205 on the expression of Bcl-2 and the ATP-binding cassette transporters A2 (ABCA2) and A5 (ABCA5) related to multi-drug resistance and malignant progression. These results define the E2F1-p73/DNp73-miR-205 axis as a crucial mechanism for chemoresistance and, thus, as a target for metastasis prevention.

CDK1 differentially regulates G-overhang generation at leading- and lagging-strand telomeres in telomerase-negative cells in G₂ phase

Xueyu Dai, Chenhui Huang and Weihang Chai
Pages 3079 - 3086
http://dx.doi.org/10.4161/cc.21472
Abstract | Full Text | PDF

Human telomeres contain single-stranded 3' G-overhangs that function in telomere end protection and telomerase action. Previously we have demonstrated that multiple steps involving C-strand end resection, telomerase elongation and C-strand fill-in contribute to G-overhang generation in telomerase-positive cancer cells. However, how G-overhangs are generated in telomerase-negative human somatic cells is unknown. Here, we report that C-strand fill-in is present at lagging-strand telomeres in telomerase-negative human cells but not at leading-strand telomeres, suggesting that C-strand fill-in is independent of telomerase extension of G-strand. We further show that while cyclin-dependent kinase 1 (CDK1) positively regulates C-strand fill-in, CDK1 unlikely regulates G-overhang generation at leading-strand telomeres. In addition, DNA polymerase α (Polα) association with telomeres is not altered upon CDK1 inhibition, suggesting that CDK1 does not control the loading of Polα to telomeres during fill-in. In summary, our results reveal that G-overhang generation at leading- and lagging-strand telomeres are regulated by distinct mechanisms in human cells.

pH neutralization protects against reduction in replicative lifespan following chronological aging in yeast

Christopher Murakami and 35 others
Pages 3087 - 3096
http://dx.doi.org/10.4161/cc.21465
Abstract | Full Text | PDF

Chronological and replicative aging have been studied in yeast as alternative paradigms for post-mitotic and mitotic aging, respectively. It has been known for more than a decade that cells of the S288C background aged chronologically in rich medium have reduced replicative lifespan relative to chronologically young cells. Here we report replication of this observation in the diploid BY4743 strain background. We further show that the reduction in replicative lifespan from chronological aging is accelerated when cells are chronologically aged under standard conditions in synthetic complete medium rather than rich medium. The loss of replicative potential with chronological age is attenuated by buffering the pH of the chronological aging medium to 6.0, an intervention that we have previously shown can extend chronological lifespan. These data demonstrate that extracellular acidification of the culture medium can cause intracellular damage in the chronologically aging population that is asymmetrically segregated by the mother cell to limit subsequent replicative lifespan.

Open Access Article

An anti-let-7 sponge decoys and decays endogenous let-7 functions

Xiangling Yang, Zina Jeyapalan Rutnam, Chunwei Jiao, Duo Wei, Yizhen Xie, Jun Du, Ling Zhong and Burton B. Yang
Pages 3097 - 3108
http://dx.doi.org/10.4161/cc.21503
Abstract | Full Text | PDF

The let-7 family contains 12 members, which share identical seed regions, suggesting that they may target the same mRNAs. It is essential to develop a means that can regulate the functions of all members. Using a DNA synthesis technique, we have generated an anti-let-7 sponge aiming to modulate the function of all members. We found that products of the anti-let-7 construct could bind and inactivate all members of the let-7 family, producing decoy and decay effects. To test the role of the anti-let-7 sponge, we stably expressed the anti-let-7 construct in two types of cells, the breast carcinoma cells MT-1 and the oldest and most commonly used human cervical cancer cell line, HeLa cells. We found that expression of anti-let-7 increased cell survival, invasion and adhesion, which corroborate with known functions of let-7 family members. We further identified a novel target site across all species of the let-7 family in hyaluronan synthase 2 (HAS2). HAS2 overexpression produced similar effects as the anti-let-7 sponge. Silencing HAS2 expression by siRNAs produced opposite effects to anti-let-7 on cell survival and invasion. The ability of anti-let-7 to regulate multiple members of the let-7 family allows us to observe their multiple functions using a single reagent. This approach can be applied to other family members with conserved sequences.

The MEN mediates the effects of the spindle assembly checkpoint on Kar9-dependent spindle pole body inheritance in budding yeast

Manuel Hotz, Jette Lengefeld and Yves Barral
Pages 3109 - 3116
http://dx.doi.org/10.4161/cc.21504
Abstract | Full Text | PDF

Many asymmetrically dividing cells segregate the poles of the mitotic spindle non-randomly between their two daughters. In budding yeast, the protein Kar9 localizes almost exclusively to the astral microtubules emanating from the old spindle pole body (SPB) and promotes its movement toward the bud. Thereby, Kar9 orients the spindle relative to the division axis. Here, we show that beyond perturbing Kar9 distribution, activation of the spindle assembly checkpoint (SAC) randomizes SPB inheritance. Inactivation of the B-type cyclin Clb5 led to a SAC-dependent defect in Kar9 orientation and SPB segregation. Furthermore, unlike the Clb4-dependent pathway, the Clb5- and SAC-dependent pathways functioned genetically upstream of the mitotic exit network (MEN) in SPB specification and Kar9-dependent SPB inheritance. Together, our study indicates that Clb5 functions in spindle assembly and that the SAC controls the specification and inheritance of yeast SPBs through inhibition of the MEN.

Molecular basis of the functional distinction between Cln1 and Cln2 cyclins

Inma Quilis and Juan Carlos Igual
Pages 3117 - 3131
http://dx.doi.org/10.4161/cc.21505
Abstract | Full Text | PDF

Cln1 and Cln2 are very similar but not identical cyclins. In this work, we tried to describe the molecular basis of the functional distinction between Cln1 and Cln2. We constructed chimeric cyclins containing different fragments of Cln1 and Cln2 and performed several functional analysis that make it possible to distinguish between Cln1 or Cln2. We identified that region between amino acids 225 and 299 of Cln2 is not only necessary but also sufficient to confer Cln2 specific functionality compared with Cln1. We also studied Cln1 and Cln2 subcellular localization identifying additional differences between them. Both cyclins are distributed between the nucleus and the cytoplasm, but Cln1 shows stronger nuclear accumulation. Nuclear import of both cyclins is mediated by the classical nuclear import pathway and by sequences in the N-terminal end of the proteins. For Cln2, but not for Cln1, a nuclear export mechanism mediated by karyopherin Msn5 has been identified. Strikingly, Cln2 export depends on a Msn5-dependent NES between amino acids 225 and 299. In fact, the introduction of this region confers to Cln1 an export mechanism dependent on Msn5; importantly, this causes the gain of Cln2-specific cytosolic functions and the impairment of nuclear function. In short, a region from Cln2 controlling an Msn5-dependent nuclear export mechanism confers a specific functionality to Cln2 compared with Cln1.

Persistent DNA damage caused by low levels of mitomycin C induces irreversible cell senescence

Elise McKenna, Frank Traganos, Hong Zhao and Zbigniew Darzynkiewicz
Pages 3132 - 3140
http://dx.doi.org/10.4161/cc.21506
Abstract | Full Text | PDF

Mutations of oncogenes and tumor suppressor genes which activate mTOR through several downstream signaling pathways are common to cancer. Activation of mTOR when combined with inhibition of cell cycle progression or DNA replication stress has previously been shown to promote cell senescence. In the present study, we examined the conditions under which human non-small cell lung carcinoma A549 cells can undergo senescence when treated with the DNA alkylating agent mitomycin C (MMC). While exposure of A549 cells to 0.1 or 0.5 µg/ml of MMC led to their arrest in S phase of the cell cycle and subsequent apoptosis, exposure to 0.01 or 0.02 µg/ml for 6 d resulted in induction of cell senescence and near total (0.01 µg/ml) or total (0.02 µg/ml) elimination of their reproductive potential. During exposure to these low concentrations of MMC, the cells demonstrated evidence of DNA replication stress manifested by expression of γH2AX, p21^WAF1 and a very low level of EdU incorporation into DNA. The data are consistent with the notion that enduring DNA replication stress in cells known to have activated oncogenes leads to their senescence. It is reasonable to expect that tumors having constitutive activation of oncogenes triggering mTOR signaling may be particularly predisposed to undergoing senescence following prolonged treatment with low doses of DNA damaging drugs.




LETTER TO THE EDITOR

Alterations of polycomb gene BMI1 in human myeloproliferative neoplasms

Mandy Brecqueville, José Adélaïde, François Bertucci, Pascal Finetti, Max Chaffanet, Daniel Birnbaum and Anne Murati
Pages 3141 - 3142
http://dx.doi.org/10.4161/cc.21114
Full Text | PDF