Table of Contents

Volume 9, Issue 2

  January 15, 2010

  Pages 207 - 418

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EDITORIALS: CELL CYCLE FEATURES

Open Access Article

Hes1 oscillation: Making variable choices for stem cell differentiation

Taeko Kobayashi and Ryoichiro Kageyama
Pages 207 - 208
http://dx.doi.org/10.4161/cc.9.2.10478
Abstract | PDF

Comment on: Kobayashi T, et al. Genes Dev 2009; 23:1870-5.

Open Access Article

FANCD2: A DNA binding protein regulated by MRE11-RAD50-NBS1

Céline Roques and Jean-Yves Masson
Pages 209 - 210
http://dx.doi.org/10.4161/cc.9.2.10492
Abstract | PDF

Comment on: Roques C, et al. EMBO J 2009; 28:2400-13.

Open Access Article

AMPK: A metabolic checkpoint that regulates the growth of EGFR activated glioblastomas

Deliang Guo, Timothy F. Cloughesy, Caius G. Radu and Paul S. Mischel
Pages 211 - 212
http://dx.doi.org/10.4161/cc.9.2.10540
Abstract | PDF

Comment on: Guo et al. PNAS 2009;106(31):12932-7.

Open Access Article

miR-449 regulates CDK-Rb-E2F1 through an auto-regulatory feedback circuit

Min Feng and Qiang Yu
Pages 213 - 214
http://dx.doi.org/10.4161/cc.9.2.10502
Abstract | PDF

Comment on: Yang X, et al. Genes Dev 2009; 23:2388-93.

Open Access Article

Uncovering a tumor-suppressor function for Drosophila Polycomb group genes

Anne-Marie Martinez and Giacomo Cavalli
Pages 215 - 216
http://dx.doi.org/10.4161/cc.9.2.10518
Abstract | PDF

Comment on: Martinez AM, et al. Nat Genet 2009; 41:1076-82.

Open Access Article

Modulating c-Abl nuclear activity as a strategy to preserve female fertility

Stefania Gonfloni
Pages 217 - 218
http://dx.doi.org/10.4161/cc.9.2.10563
Abstract | PDF

Comment on: Gonfloni S, et al. Nat Med 2009; 15:1179-85.

Open Access Article

Foxo1 in hepatic lipid metabolism

Zhiyong Cheng and Morris F. White
Pages 219 - 220
http://dx.doi.org/10.4161/cc.9.2.10567
Abstract | PDF

Comment on: Cheng Z, et al. Nat Med 2009; 15:1307–11.

Open Access Article

An EGOcentric view of TORC1 signaling

Matteo Binda, Grégory Bonfils, Nicolas Panchaud, Marie-Pierre Péli-Gulli and Claudio De Virgilio
Pages 221 - 222
http://dx.doi.org/10.4161/cc.9.2.10585
Abstract | PDF

Comment on: Binda M, et al. Mol Cell 2009; 35:563-73.

Open Access Article

Pivotal role of calcium/calmodulin-dependent protein kinase II in ER stress-induced apoptosis

Lale Ozcan and Ira Tabas
Pages 223 - 224
http://dx.doi.org/10.4161/cc.9.2.10596
PDF

Open Access Article

Linking rDNA transcription to the cellular energy supply

Ingrid Grummt and Renate Voit
Pages 225 - 226
http://dx.doi.org/10.4161/cc.9.2.10614
Abstract | PDF

Comment on: Hoppe S, et al. Proc Natl Acad Sci USA 2009; 106:17781-6.




NEWS & VIEWS

Open Access Article

DDA3: A new dancer at the growing end?

Liangyu Zhang and Xuebiao Yao
Pages 227 - 232
http://dx.doi.org/10.4161/cc.9.2.10980
Abstract | PDF

Comment on: Jang CY, et al. Cell Cycle 2009; 8:3165-71.

Open Access Article

Quinacrine: New anti-tumor application for an old anti-malaria drug

Mehdi Mollapour and Len Neckers
Pages 227 - 232
http://dx.doi.org/10.4161/cc.9.2.10649
Abstract | PDF

Comment on: Anti-malaria drug blocks proteotoxic stress response: Anti-cancer implications. Neznanov N, et al. Cell Cycle 2009; 8:3960-70.


Open Access Article

An experimental model of ovarian carcinoma: More than just genetics

Alice S.T. Wong
Pages 227 - 232
http://dx.doi.org/10.4161/cc.9.2.10607
Abstract | PDF

Comment on: Induction of high grade serous carcinoma in human ovarian surface epithelial cells using combined genetic elements and peritoneal microenvironment. Zheng J, et al. Cell Cycle 2010; 9:In press.


Open Access Article

Cancer stem cells: Just sign here!

Emmanuelle Charafe-Jauffret, Christophe Ginestier and Daniel Birnbaum
Pages 227 - 232
http://dx.doi.org/10.4161/cc.9.2.10669
Abstract | PDF

Comment on: Hussenet, et al. Cell Cycle 2010; 9(2); In press.

Open Access Article

A genome wide view of hunchback-like-1 targets

Zoya Kai and Amy E. Pasquinelli
Pages 227 - 232
http://dx.doi.org/10.4161/cc.9.2.10608
Abstract | PDF

Comment on: C. elegans sym-1 is a downstream target of the Hunchback-like-1 developmental timing transcription factor. Niwa R, et al. Cell Cycle 2009; 8:In press.


Open Access Article

Circadian rhythms: Phosphorylating the CLOCK

Christopher L. Baker and Jay C. Dunlap
Pages 227 - 232
http://dx.doi.org/10.4161/cc.9.2.10609
Abstract | PDF

Comment on: A serine cluster mediates BMAL-dependent CLOCK phosphorylation and degradation. Spengler ML, et al. Cell Cycle 2009; 8:In press.





EDITOR'S CORNER: IN THEIR OWN WORDS

Open Access Article

An interview with Dr. George C. Prendergast


Pages 233 - 234
http://dx.doi.org/10.4161/cc.9.2.11150
Abstract | PDF

Cell Cycle congratulates Dr. George C. Prendergast for his recent appointment as the Editor-in-Chief of Cancer Research (AACR). Dr. Prendergast, president and CEO of the Lankenau Institute for Medical Research in Wynnewood, PA, assumed the new post on January 1, 2010.




EXTRA VIEWS

Open Access Article

Crosstalk between planar cell polarity signaling and miR-8 control of NHERF1-mediated actin reorganization

Alex S. Flynt and James G. Patton
Pages 235 - 237
http://dx.doi.org/10.4161/cc.9.2.10434
Abstract | PDF

The response to osmotic stress in developing zebrafish embryos requires proper apical patterning and trafficking of transmembrane ion transporters in ionocytes, specialized cells of the epidermis. The miR-8 family of miRNAs plays a key role in this process by precisely regulating the activity of NHERF1, a regulator of sodium hydrogen exchange that also serves as an adaptor protein linked to the actin cytoskeleton. We have discovered that NHERF1 activity is also coupled to Planar Cell Polarity (PCP) signaling in the zebrafish epidermis. Loss of NHERF1 in wild type fish disrupts actin organization but the observed defects can be largely restored when combined with mutants in the PCP pathway. We propose that proper apical patterning depends on input and coordination between PCP signaling and the response to stress.

Open Access Article

The cephalic neural crest of amniote vertebrates is composed of a large majority of precursors endowed with neural, melanocytic, chondrogenic and osteogenic potentialities

Elisabeth Dupin, Giordano W. Calloni and Nicole M. Le Douarin
Pages 238 - 249
http://dx.doi.org/10.4161/cc.9.2.10491
Abstract | PDF

In the amniote embryo, the neural crest (NC) has the unique capacity to give rise to neuronal and glial cells in the peripheral nervous system (PNS), melanocytes and mesenchymal cells including those forming the head skeleton and connective tissues. In the trunk, mesenchymal cells are derived from the mesoderm. The question was raised whether the NC-derived head mesenchyme arises from a lineage separate from the neural-melanocytic one, or if both skeletogenic and neural-melanocytic derivatives originate from a common putative stem cell in the early cephalic NC. We discuss here these issues and present experimental data that provide evidence for the multipotency of NC cells (NCC), focusing on those at the origin of the craniofacial skeleton. Recent work of in vitro clonal culture revealed that the vast majority (92% of clonogenic cells) of the cephalic quail NCC are capable to yield osteoblasts together with neurones, glial cells and melanocytes. A common pluripotent progenitor for chondrocytes, osteocytes, neurones, glial cells, melanocytes and myofibroblasts has been identified and is present in the early cephalic NC at the frequency of 7 to 13 % of clonogenic cells depending on the environmental conditions. Together with recent reports that multipotent NC-related progenitors persist in adult tissues in rodents and humans, these results reinforce a stem cell model for the generation and maintenance of NC-derived lineages during embryogenesis and in adult tissue homeostasis.

Open Access Article

Defective autophagy control by the p53 rheostat in cancer

Lorenzo Galluzzi, Eugenia Morselli, Oliver Kepp, Maria Chiara Maiuri and Guido Kroemer
Pages 250 - 255
http://dx.doi.org/10.4161/cc.9.2.10493
Abstract | PDF

Autophagy is a finely regulated, lysosomal catabolic pathway that contributes to the turnover of long-lived proteins and to the elimination of old/damaged organelles. Autophagy exerts bona fide oncosuppressive functions by: (1) limiting chromosomal instability; (2) reducing potentially mutagenic oxidative stress; and (3) restraining intratumoral necrosis and local inflammation. Defective autophagy constitutes a hallmark of cancer cells together with: (1) provision of autonomous growth signals, (2) insensitivity to antiproliferative stimuli, (3) disabled apoptosis, (4) limitless replication, (5) production of angiogenic factors, (6) tissue invasion with metastasis, (7) avoidance of the immune response, and (8) enhanced anabolism. p53 is the best-known human oncosuppressor protein, and its genetic/epigenetic inactivation has been observed in more than 50% of all human cancers. p53 mostly mediates tumor suppression by transactivating pro-apoptotic and cell cycle arresting genes, but also by favoring mitochondrial apoptosis in a transcription-independent fashion, by modulating metabolic circuitries and by regulating autophagy. p53 mutations (or epigenetic changes) that simultaneously abolish its pro-apoptotic and autophagy-inhibitory functions behave as "multi-hit" events, as opposed to "single-hit" mutations that only affect the classical (pro-apoptotic and/or cell cycle-arresting) functions of the p53 system. We speculate that, in this latter case, additional genetic/epigenetic events resulting in disabled autophagy are likely to contribute to accelerated oncogenesis.




PERSPECTIVES

Open Access Article

A systematic approach to understand the functional consequences of non-protein coding risk regions

Gerhard A. Coetzee, Li Jia, Baruch Frenkel, Brian E. Henderson, Amos Tanay, Christopher A. Haiman and Matthew L. Freedman
Pages 256 - 259
http://dx.doi.org/10.4161/cc.9.2.10419
Abstract | PDF

A primary goal of genetic association studies is to elucidate genes and novel biological mechanisms involved in disease. Recently, genome-wide association studies have identified many common genetic variants that are significantly associated with complex diseases such as cancer. In contrast to mutation-causing Mendelian disorders, a sizable fraction of the variants lies outside known protein-coding regions; therefore, understanding their biological consequences presents a major challenge in human genetics. Here we describe an integrated framework to allow non-protein coding loci to be annotated with respect to regulatory functions. This will facilitate identification of target genes as well as prioritize variants for functional testing.

Open Access Article

Blocking ovarian cancer progression by targeting tumor microenvironmental leukocytes

Juan R. Cubillos-Ruiz, Melanie Rutkowski and José R. Conejo-Garcia
Pages 260 - 268
http://dx.doi.org/10.4161/cc.9.2.10430
Abstract | PDF

Current therapies for metastatic ovarian carcinoma are based on surgical debulking followed by chemotherapy. After more than three decades implementing treatments that selectively target the tumor cell, the 5-year survival rate for metastatic ovarian cancer patients is still lower than 30%. Novel strategies are therefore urgently needed to complement classical treatments for this malignancy. Recently, leukocytes in the ovarian cancer microenvironment such as regulatory T cells and immature pro-angiogenic/tolerogenic myeloid cells have been demonstrated to play a fundamental role in tumor progression. This review focuses on our recent understanding of the potential of eliminating and/or modulating the phenotype of these leukocytes in vivo and in situ as a novel intervention to complement standard ovarian cancer treatments. The significant effects of targeting these crucial microenvironmental players on cancer vascularization, local tumor growth, distal metastatic spreading and spontaneous anti-tumor immune responses are discussed.

Open Access Article

Apolipoprotein D: An overview of its role in aging and age-related diseases

Julien Muffat and David W. Walker
Pages 269 - 273
http://dx.doi.org/10.4161/cc.9.2.10433
Abstract | PDF

“It’s got to be doing something important!”-Seymour Benzer, reflecting on the observation that ApoD mRNA levels increase 500-fold following neuronal crush injury.

Seymour Benzer’s curiosity was legendary and seemingly limitless. Towards the end of his life, one of the (many) questions that kept him awake at night concerned the emerging role of Apolipoprotein D (ApoD) in aging and neurological disease. In this perspective, we will discuss the clinical and biochemical data on ApoD, and the input from the recent genetic studies in model systems, including those from the Benzer lab.

Open Access Article

DNA double-strand breaks and ATM activation by transcription-blocking DNA lesions

Olivier Sordet, Asako J. Nakamura, Christophe E. Redon and Yves Pommier
Pages 274 - 278
http://dx.doi.org/10.4161/cc.9.2.10506
Abstract | PDF

Ataxia telangiectasia mutated (ATM), the deficiency of which causes a severe neurodegenerative disease, is a crucial mediator for the DNA double-strand break (DSB) response. We recently showed that transcription-blocking topoisomerase I cleavage complexes (TOP1cc) produce DSBs related to R-loop formation and activate ATM in post-mitotic neurons and lymphocytes. Here we discuss how TOP1cc can produce transcription arrest with R-loop formation and generate DSBs that activate ATM, as well as data suggesting that those transcription-dependent DSBs tend to form at the IgH locus and at specific genomic sites. We also address the potential roles of ATM in response to transcription-blocking TOP1cc.




REVIEWS

Open Access Article

Targeting the checkpoint kinase Chk1 in cancer therapy

Callie Merry, Kang Fu, Jingna Wang, I-Ju Yeh and Youwei Zhang
Pages 279 - 283
http://dx.doi.org/10.4161/cc.9.2.10445
Abstract | PDF

A paramount objective of the eukaryotic cell division cycle is to overcome numerous internal and external insults to faithfully duplicate the genetic information once per every cycle. This is carried out by elaborate networks of genome surveillance signaling pathways, termed replication checkpoints. Central to replication checkpoints are two protein kinases, the upstream kinase ATR, and its downstream target kinase, Chk1. When the DNA replication process is interrupted, the ATR-Chk1 pathway transmits signals to delay cell cycle progression, and to maintain fork viability so that DNA duplication can resume after the initial damage is corrected. Previous studies showed that replicative stress not only activated Chk1, but also triggered the ubiquitin-dependent destruction of Chk1 in cultured human cells. In a recent study, we identified the F-box protein, Fbx6, as the mediator that regulates Chk1 ubiquitination and degradation in both normally cycling cells and during replication stress. We further showed that expression levels of Chk1 and Fbx6 exhibited an overall inverse correlation in both cultured cancer cell lines and in breast tumor tissues, and that defects in Chk1 degradation, for instance, due to reduced expression of Fbx6, rendered tumor cells resistant to anticancer treatment. Here we highlight those findings and their implications in the replication checkpoint and cellular sensitivity to cancer therapies.

Open Access Article

Cdk5, the multifunctional surveyor

Vassiliki Lalioti, Diego Pulido and Ignacio V. Sandoval
Pages 284 - 311
http://dx.doi.org/10.4161/cc.9.2.10466
Abstract | PDF

Cyclin-dependent kinases (Cdks) are a family of proline-directed Ser/Thr kinases known for their role in the control of cell cycle progression. In 1992, this family was joined by Cdk5, which is an atypical member in that it uses its own activators and is multifunctional, playing important regulatory roles in multiple cellular functions. Here, we review the structure of Cdk5 and its various mechanisms of activation within the context of the Cdk family. We also address the functional significance of the numerous and varied targets of Cdk5 within pathways that control the organization of the cytoskeleton and focal adhesions, signaling cascades, membrane dynamics and function, cell metabolism, cycle arrest in postmitotic cells, gene transcription and cell survival, all within the context of the adaptation of cells and life systems to changing internal and external environments.




REPORTS

Open Access Article

A role for Chk2 in DNA damage induced mitotic delays in human colorectal cancer cells

Hanne Varmark, Seongae Kwak and William E. Theurkauf
Pages 312 - 320
http://dx.doi.org/10.4161/cc.9.2.10418
Abstract | PDF

Progression into mitosis in the presence of DNA damage leads to spindle checkpoint (SAC) dependent mitotic delays and cytokinesis failure. In Drosophila embryos, DNA damage does not delay mitotic entry but triggers Chk2 kinase dependent delays in mitotic exit. It is unclear if damage associated mitotic delays in human cells result from kinase signaling or breaks in centromere DNA that disrupt kinetochore function and activate the SAC. We show that transgenic expression of Human Chk2 in a Drosophila chk2 mutant background restores damage induced mitotic delays during early embryogenesis. Parental HCT116 colorectal cancer cells that progress into mitosis following DNA damage, due to either G2 checkpoint adaptation or G2 checkpoint abrogation by caffeine or the Chk1 inhibitor UCN-01, delay in mitosis and show high rates of cytokinesis failure. Significantly, these mitotic responses are suppressed in HCT116 chk2 knockout cells, and the response is restored by transgenic expression of wild type Chk2. However, both parental and chk2-/-HCT116 cells arrested in G2 for prolonged periods by DNA damage prior to release from the G2 block do show significant mitotic delays. Chk2 thus appears to have a conserved function in control of mitotic progression following G2/M transition with DNA damage. However, prolonged G2 arrest with DNA damage can trigger Chk2-independent mitotic delays that may be secondary to kinetochore disruption.

Open Access Article

An adult tissue-specific stem cell molecular phenotype is activated in epithelial cancer stem cells and correlated to patient outcome

Thomas Hussenet, Doulaye Dembélé, Nadine Martinet, Jean-Michel Vignaud and Stanislas du Manoir
Pages 321 - 327
http://dx.doi.org/10.4161/cc.9.2.10421
Abstract | PDF

Recent studies have shown that embryonic stem cell-like molecular phenotypes are commonly activated in human epithelial primary tumors and are linked to adverse patient prognosis. However it remains unclear whether these correlations to outcome are linked to the differentiation status of the human primary tumours1 or represent molecular reminiscences of epithelial cancer stem cells. In addition, while it has been demonstrated that leukemic cancer stem cells re-acquire an embryonic stem cell-like phenotype, the molecular basis of stem cell function in epithelial cancer stem cells has not been investigated. Here we show that a normal adult tissue-specific stem cell molecular phenotype is commonly activated in epithelial cancer stem cells and for the first time provide evidence that enrichment in cancer stem cells-specific molecular signatures are correlated to highly aggressive tumor phenotypes in human epithelial cancers.

Open Access Article

Phospho-ΔNp63α/NF-Y protein complex transcriptionally regulates DDIT3 expression in squamous cell carcinoma cells upon cisplatin exposure

Yiping Huang, Alice Y. Chuang, Rose A. Romano, Nanette J. Liégeois, Satrajit Sinha, Barry Trink, Edward A. Ratovitski and David Sidransky
Pages 328 - 338
http://dx.doi.org/10.4161/cc.9.2.10432
Abstract | PDF

Cisplatin remains the most important chemotherapeutic agent for patients with human head and neck cancer. However, tumor cells often develop resistance to cisplatin-induced apoptosis. We previously found that head and neck squamous cell carcinoma (HNSCC) cells exposed to cisplatin display a marked ATM-induced phosphorylation of ΔNp63α. However, the mutated ΔNp63α-S385G failed to undergo phosphorylation by ATM kinase. We used HNSCC cell lines expressing the wild type ΔNp63α or mutated ΔNp63α-S385G to determine the effect of S385G mutation on the ΔNp63α transcriptional activity and protein-protein interactions. The S385G mutation in ΔNp63α dramatically abolished the up-regulation/down-regulation of downstream gene targets and the binding of ΔNp63α−S385G to certain promoters. In contrast to the non-phosphorylated ΔNp63α-S385G, the phospho-ΔNp63α forms protein-protein complexes with NF-YA transcription factor and regulates the transcription of DDIT3 gene implicated in the programmed cell death of HNSCC cells upon cisplatin exposure. We suggest that the transcriptional activation of ΔNp63α through its phosphorylation by ATM kinase in HNSCC cells exposed to cisplatin is a critical step in the subsequent sensitivity of certain human head and neck cancers to platinum therapy.

Open Access Article

Epigenetic status determines germ cell meiotic commitment in embryonic and postnatal mammalian gonads

Ning Wang and Jonathan L. Tilly
Pages 339 - 349
http://dx.doi.org/10.4161/cc.9.2.10447
Abstract | PDF

The meiotic cell cycle is required for production of fertilization-competent gametes. Germ cell meiotic commitment requires expression of Stimulated by retinoic acid gene 8 (Stra8), which is transcriptionally activated by retinoic acid (RA). Meiotic suppression in embryonic male germ cells is believed to result from sex-specific differences in CYP26B1-catalyzed RA metabolism in the developing gonads. Here we show in mice that RA-induced Stra8 transcription is epigenetically controlled and requires a co-activator that binds proximal to the RA response elements (RAREs) in the Stra8 promoter. Embryonic male germ cells exposed in utero to the class I/II histone deacetylase (HDAC) inhibitor, trichostatin-A (TSA), show premature Stra8 activation and meiotic entry without altered Cyp26b1 expression. We also show that Stra8 expression is detectable and physiologically regulated in adult mouse ovaries. Further, oogenesis induction in adult females using TSA is associated with Stra8 activation, and these events are absent in mice deficient in the RA precursor vitamin A. Finally, all of the actions of TSA in premeiotic germ cells in vitro and in mouse ovaries in vivo can be reproduced with the small molecule HDAC inhibitor, suberoylanilide hydroxamic acid (SAHA). Thus, the ability of RA to transcriptionally induce expression of the meiosis-commitment gene, Stra8, is epigenetically controlled and this process involves a novel co-activator that functions upstream of the RAREs. These events not only coordinate the sex-specific timing of meiotic entry during embryogenesis, but also contribute to the regulation of oogenesis in adult female mammals.

Open Access Article

Cell cycle-dependent phosphorylation of Rad53 kinase by Cdc5 and Cdc28 modulates checkpoint adaptation

Thomas Schleker, Kenji Shimada, Ragna Sack, Brietta L. Pike and Susan M. Gasser
Pages 350 - 363
http://dx.doi.org/10.4161/cc.9.2.10448
Abstract | PDF

In budding yeast the evolutionarily conserved checkpoint response varies in its sensitivity to DNA damaging agents through the cell cycle. Specifically, higher amounts of damage are needed to activate the downstream checkpoint kinase Rad53 in S-phase cells. We examined here whether phosphorylation of Rad53 itself by cell cycle-dedicated kinases regulates Rad53 activation. We found that during unperturbed growth Rad53 exhibits a small phosphorylation-dependent electrophoretic mobility shift in G2, M and G1 phases of the cell cycle that is lost in S phase. We show that Rad53 is phosphorylated in vitro by Cdc5, a mitotic Polo-like kinase, and by the yeast cyclin-dependent kinase, Cdc28. Consistently, the cell cycle-dependent Rad53 mobility shift requires both Cdc5 and Cdc28 activities. We mapped the in vitro targeted phosphorylation sites by mass spectrometry and confirmed with mass spectroscopy that serines 774, 789 and 791 within Rad53 are phosphorylated in vivo in M-phase arrested cells. By creating nonphosphorylatable mutations in the endogenous RAD53 gene, we confirmed that the CDK and Polo kinase target sites are responsible for the observed cell cycle-dependent shift in protein mobility. The loss of phospho-acceptor sites does not interfere with Rad53 activation but accelerates checkpoint adaptation after induction of a single irreparable double-strand break. We thus demonstrate that cell cycle-dependent phosphorylation can fine-tune the response of Rad53 to DNA damage.

Open Access Article

H2A.Z overexpression promotes cellular proliferation of breast cancer cells

Amy Svotelis, Nicolas Gévry, Gilles Grondin and Luc Gaudreau
Pages 364 - 370
http://dx.doi.org/10.4161/cc.9.2.10465
Abstract | PDF

We recently showed that histone H2A.Z, as well as members of the ATP-dependent p400 chromatin remodeling complex (p400.com), are essential components of estrogen receptor a (ERa) signaling.  More specifically, we showed that H2A.Z and p400.com are incorporated into the promoter regions of ERα target genes only upon gene induction, and also in a cyclic fashion.  RNAi-mediated cellular depletion of H2A.Z and p400.com strongly impedes estrogen-dependent growth of breast cancer cells as well as strongly affect ERa-target gene expression.  Two mechanisms emerged from our studies of how H2A.Z incorporation within ERa-target regulatory regions can actually regulate estrogen-mediated signaling: (1) by stabilizing nucleosomes within the translational DNA axis, a process that allows general transcription factors to be efficiently recruited to promoter regions; 2) by allowing estrogen-responsive enhancer function.  In the current study, we now show that in MCF7 cells, ectopic overexpression of H2A.Z increases proliferation, and such in conditions where estrogen levels are low.  Also, immunohistochemical studies of breast cancer biopsies show that the presence of H2A.Z correlates highly with that of ERa, but is associated with high-grade ER-negative cancers.  Finally we show that ERa directly associates to the H2A.Z promoter, and consequently modulates its expression.  Our study provides a possible link between H2A.Z and endocrine resistance by showing that H2A.Z overexpression leads to increased growth, particularly when estrogen levels are very low.

Open Access Article

pRB and E2F4 play distinct cell-intrinsic roles in fetal erythropoiesis

Jing Zhang, Eunice Y. Lee, Yangang Liu, Seth D. Berman, Harvey F. Lodish and Jacqueline A. Lees
Pages 371 - 376
http://dx.doi.org/10.4161/cc.9.2.10467
Abstract | PDF

The retinoblastoma tumor suppressor protein pRB functions, at least in part, by directly binding to and modulating the activity of the E2F transcription factors. Previous studies have shown that both E2F4 and pRB play important roles in fetal erythropoiesis. Given that these two proteins interact directly we investigated the overlap of E2F4 and pRB function in this process by analyzing E2f4^-/-, conditional Rb knockout (Rb^1lox/1lox), and compound E2f4^-/-;Rb^1lox/1lox embryos. At E15.5 E2f4^-/- and Rb^1lox/1lox fetal erythroid cells display distinct abnormalities in their differentiation profiles. When cultured in vitro, both E2f4^-/- and Rb^1lox/1lox erythroid cells show defects in cell cycle progression. Surprisingly, analysis of cell cycle profiling suggests that E2F4 and pRB control cell cycle exit through different mechanisms. Moreover, only pRB, but not E2F4, promotes cell survival in erythroid cells.  We observed an additive rather than a synergistic impact upon the erythroid defects in the compound E2f4^-/-;Rb^1lox/1lox embryos. We further found that fetal liver macrophage development is largely normal regardless of genotype. Taken together, our results show that E2F4 and pRB play independent cell-intrinsic roles in fetal erythropoiesis.

Open Access Article

Defective autophagy associated with LC3 puncta in epothilone-resistant cancer cells

Shensi Shen, Oliver Kepp, Isabelle Martins, Ilio Vitale, Sylvie Souquère, Maria Castedo, Gérard Pierron and Guido Kroemer
Pages 377 - 383
http://dx.doi.org/10.4161/cc.9.2.10468
Abstract | PDF

Autophagy is commonly characterized by the redistribution of the microtubule-associated light chain 3 (LC3) protein into cytoplasmic puncta, coinciding with its lipidation, as well as by a decrease in the abundance of autophagic substrates including p62 and ubiquitinylated proteins. Here, we describe a cell line, A549-B480, which, in contrast to its parental A549 line, exhibits massive accumulation of LC3 (or a GFP-LC3 fusion protein) in cytoplasmic puncta. These puncta co-localize with accumulated p62 and ubiquitinylated proteins, yet are not enwrapped by membranes. Indeed, LC3 is not lipidated in A549-B480, even when these cells are cultured in conditions in which A549 cells would develop autophagy. A549-B480 cells have been selected for their resistance against the microtubule-stabilizing agent epothilone B and actually require the continuous presence of epothilone B for their survival. Parental A549 cells treated with epothilone B manifested all signs of bona fide autophagy. In contrast, the autophagic program of A549-B480 was defective, irrespective of the absence or presence of epothilone B, and correlated with the complete absence of Atg7, a protein that is reputed to be essential for autophagy. These results establish novel functional links between microtubules and autophagy, identify a new chemotherapy resistance-associated autophagic defect, and describe the existence of LC3 puncta outside from autophagosomes.

Open Access Article

Identification of a functional nuclear export sequence in diacyl glycerol kinase-ζ

Camilla Evangelisti, Gian Carlo Gaboardi, Anna Maria Billi, Andrea Ognibene, Kaoru Goto, Pier Luigi Tazzari, James A. McCubrey and Alberto M. Martelli
Pages 384 - 388
http://dx.doi.org/10.4161/cc.9.2.10469
Abstract | PDF

Diacylglycerol kinases (DGKs) are key regulators of diacylglycerol-dependent signaling pathways. Among the 10 DGK isoforms, DGK-ζ is the only nuclear form that contains a nuclear localization signal. Here, by site-directed mutagenesis, we showed that DGK-ζ also displays a functional independent nuclear export signal (NES) sequence between the amino acid residues 363-370. Indeed, the NES mutant forms of DGK-ζ accumulated in the nucleus to a much greater extent than wild type DGK-ζ. Moreover, treatment with leptomycin B, an inhibitor of leucine-rich type NES, resulted in accumulation of both endogenous and ectopically expressed DGK-ζ in the nucleus, demonstrating that nuclear export of DGK-ζ is chromosome regional maintenance protein 1 (CRM1) -dependent. Previously, we reported that nuclear DGK-ζ is a negative regulator of cell cycle progression in C2C12 mouse myoblasts. In this paper, we documented that enhancement of DGK-ζ nuclear localization by NES sequence mutation, increases G0/G1 block in C2C12 cells. Overall, our data demonstrate that DGK-ζ export from nucleus to cytoplasm is regulated by a leucine-rich NES through the exportin CRM1 and suggest that the nuclear localization of DGK-ζ could finely tune its function as a regulator of G1/S cell cycle transition.

Open Access Article

The complexity of phosphorylated H2AX foci formation and DNA repair assembly at DNA double-strand breaks

Asako J. Nakamura, V. Ashutosh Rao, Yves Pommier and William M. Bonner
Pages 389 - 397
http://dx.doi.org/10.4161/cc.9.2.10475
Abstract | PDF

The maintenance of genome stability requires efficient DNA double-stranded break (DSB) repair mediated by the phosphorylation of multiple histone H2AX molecules near the break sites. The phosphorylated H2AX (γ-H2AX) molecules form foci covering many megabases of chromatin. The formation of g-H2AX foci is critical for efficient DNA damage response (DDR) and for the maintenance of genome stability, however, the mechanisms of protein organization in foci is largely unknown. To investigate the nature of γ-H2AX foci formation, we analyzed the distribution of γ-H2AX and other DDR proteins at DSB sites using a variety of techniques to visualize, expand and partially disrupt chromatin. We report here that γ-H2AX foci change composition during the cell cycle, with proteins 53BP1, NBS1 and MRE11 dissociating from foci in G2 and mitosis to return at the beginning of the following G1. In contrast, MDC1 remained colocalized with g-H2AX during mitosis. In addition, while γ-H2AX was found to span large domains flanking DSB sites, 53BP1 and NBS1 were more localized and MDC1 colocalized in doublets in foci. H2AX and MDC1 were found to be involved in chromatin relaxation after DSB formation. Our data demonstrates that the DSB repair focus is a heterogeneous and dynamic structure containing internal complexity.

Open Access Article

The EphB2 tumor suppressor induces autophagic cell death via concomitant activation of the ERK1/2 and PI3K pathways

Mustapha Kandouz, Khadidja Haidara, Jing Zhao, Marie-Laure Brisson and Gerald Batist
Pages 398 - 407
http://dx.doi.org/10.4161/cc.9.2.10505
Abstract | PDF

EphB2 is a tyrosine kinase receptor that has been shown to be a tumor suppressor gene in various cancers. However the mechanisms of this function are unknown. We report that EphB2 induces a form of cell death that does not involve the formation of apoptotic bodies or nuclear fragmentation and is instead accompanied by extensive vacuolization. Transmission electron microscopy demonstrates cytoplasmic vacuoles in EphB2-overexpressing cells that resembled autophagosomes. Using an EYFP-LC3 fusion protein and immunoblotting, we detected LC3 aggregation and conversion from form I to form II, both hallmarks of autophagy, in EphB2-transfected cells. Silencing of the autophagy regulating genes ATG5 or ATG7 using shRNAs, strongly prevented EphB2-induced cell death, further confirming its autophagic nature. EphB2 expression results in mitochondrial depolarization and translocation of cytochrome c from the mitochondria to the cytosol. Mapping of signaling pathways revealed novel information about the mechanisms of action of EphB2. We demonstrated that the MAPK pathway is important in the pro-death action of EphB2, through ERK1/2 phosphorylation and inhibition of this pathway using PD98059 counters EphB2-driven cell death. In addition, we found that inhibition of class III PI3K pathway, using the autophagy inhibitor 3MA, but not class I PI3K inhibition using LY294002, also effectively blocks EphB2-induced cell death. Finally, EphB2 expression inactivates Akt, which is a known inhibitor of autophagy. In conclusion, the EphB2 receptor induces an autophagic cell death that is mediated through the ERK1/2 and PI3K/Akt pathways.

Open Access Article

Regulation of the female mouse germ cell cycle during entry into meiosis

Denise C. Miles, Jocelyn A. van den Bergen, Andrew H. Sinclair and Patrick Western
Pages 408 - 418
http://dx.doi.org/10.4161/cc.9.2.10691
Abstract | PDF

During mouse embryonic development germ cells proliferate extensively until they commit to the male or female pathway and arrest in mitosis or meiosis respectively. Whilst the transition of female germ cells exiting the mitotic cell cycle and entering meiosis is well defined histologically, the essential cell cycle proteins involved in this process have remained unresolved. Using flow cytometry we have examined the entry of female germ cells into meiosis, their termination of DNA synthesis and entry into prophase I. Analysis of key G₂/M cell cycle proteins revealed that entry into meiosis and cell cycle exit at G₂/M involves repression of G₂/M promoting Cyclin B1, coincident upregulation of G₂/M repressing Cyclin B3 and robust establishment of the ATM/CHK2 pathway. By contrast we show that the ATR/CHK1 pathway is activated in male and female germ cells. This data indicates that an important G₂/M surveillance mechanism operates during germ cell proliferation and that passage into meiotic G₂/M involves the combined repression of G₂/M through Cyclin B3 and activation of the key G₂/M checkpoint regulatory network modulated through ATM and CHK2. This work shows that the core regulatory machinery that controls G₂/M progression in mitotic cells is activated in female mouse germ cells as they enter meiosis.