Table of Contents

Volume 6, Issue 18

  September 15, 2007

  Pages 2201 - 2313

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 Issue Catalogue (MARC XML)

EXTRA VIEWS

Open Access Article

Asymmetric Cell Divisions of Human Hematopoietic Stem and Progenitor Cells Meet Endosomes

Bernd Giebel and Julia Beckmann
Pages 2201 - 2204
http://dx.doi.org/10.4161/cc.6.18.4658
Abstract | PDF

Hematopoietic stem cells (HSC) are undifferentiated cells, which self-renew over a long period of time and give rise to committed hematopoietic progenitor cells (HPC) containing the capability to replenish the whole blood system. Since both uncontrolled expansion as well as loss of HSC would be fatal, the decision of self-renewal versus differentiation needs to be tightly controlled. There is good evidence that both HSC niches as well as asymmetric cell divisions are involved in controlling whether HSC self-renew or become committed to differentiate. In this context, we recently identified four proteins which frequently segregate asymmetrically in dividing HSC/HPC. Remarkably, three of these proteins, the tetraspanins CD53 and CD63, and the transferrin receptor are endosome-associated proteins. Here, we highlight these observations in conjunction with recent findings in model organisms which show that components of the endosomal machinery are involved in cell-fate specification processes.

Open Access Article

p53 Reactivation Kills KSHV Lymphomas Efficiently In Vitro and In Vivo: New Hope for Treating Aggressive Viral Lymphomas

Grzegorz Sarek and Päivi M. Ojala
Pages 2205 - 2209
http://dx.doi.org/10.4161/cc.6.18.4730
Abstract | PDF

KSHV infection is the causative agent in three different tumor types; Kaposi’s sarcoma, a plasmablastic variant of multicentric Castelman’s disease and an AIDS-related form of B cell lymphoproliferative disorder called primary effusion lymphoma (PEL). PEL manifests as an effusion malignancy in Kaposi’s sarcoma patients with advanced AIDS, but also occurs in human immunodeficiency virus-negative individuals. PEL is a very aggressive disease, and currently there are no efficient therapies for treating PEL. In our recent paper we report that p53 reactivation by a small molecule inhibitor of p53-MDM2 interaction, Nutlin-3a, induces selective and massive apoptosis in PEL cells, and has striking anti-tumor activity in a mouse xenograft PEL model 1. In the light of current treatment regimens for PEL, we discuss here the benefits of using reactivation of the p53 pathway as a novel principle for the treatment of this virally induced highly aggressive malignancy.

Open Access Article

γH2AX in Bystander Cells: Not Just a Radiation-Triggered Event, a Cellular Response to Stress Mediated by Intercellular Communication

Mykyta V. Sokolov, Jennifer S. Dickey, William M. Bonner and Olga A. Martin
Pages 2210 - 2212
http://dx.doi.org/10.4161/cc.6.18.4682
Abstract | PDF

The recent years have witnessed a rapid accumulation of experimental data showing that ionizing radiation elicits a plethora of biological effects in unirradiated cells receiving bystander signals from hit cells. This so-called radiation-induced bystander effect (RIBE) manifests in various ways including changes in gene expression, genetic and epigenetic alterations, as well as increases in cell transformation and cell death. Our group and others found that DNA double-stranded breaks (DSBs), directly measured by the γ-H2AX focus formation assay, accumulate in bystander cells in a number of experimental systems such as human cultured cells, human 3-dimensional tissue models and in mice. In addition, we recently found that various other sources of cell stress, including media from cancerous cells resulted in a DNA damage response (DDR) in normal human cells that is reminiscent of RIBE. These results suggest that the RIBE may be part of a more general stress response, however, the molecular mechanism underpinning the formation of DNA DSBs in bystander cells is still unclear. This extra view points to some possibilities that might explain why DDR in human cells can be observed under bystander conditions.

Open Access Article

Compacting DNA During the Interphase: Condensin Maintains rDNA Integrity

Chi Kwan Tsang, Yuehua Wei and X.F. Steven Zheng
Pages 2213 - 2218
http://dx.doi.org/10.4161/cc.6.18.4733
Abstract | PDF

During mitosis, condensin is responsible for folding chromatin fibers into highly compact chromosomes, ensuring the faithful segregation of replicated chromosomes into daughter cells after each cell division. Our laboratory has unexpectedly found that condensin is capable of compacting DNA during the interphase: upon nutrient starvation, condensin is loaded to the rDNA array, leading to DNA condensation in this region. This subchromosomal DNA condensation appears to protect the integrity of the rDNA array. These observations provide the first microscopic evidence of DNA compaction by condensin outside mitosis. In addition, they show that condensin is also highly regulated during the interphase.

Open Access Article

Substrate Specificity of Cyclins Determined by Electrostatics

Hui Jun Lee, Gek Huey Chua, Arun Krishnan, David P. Lane and Chandra S. Verma
Pages 2219 - 2226
http://dx.doi.org/10.4161/cc.6.18.4706
Abstract | PDF

Cyclin dependent kinases (CDK) associate with Cyclins to regulate cell cycle progression and gene transcription by phosphorylating key proteins. The different Cyclin-CDK complexes display differences in substrate specificities with substrates binding across a shallow, hydrophobic, substrate binding pocket known as the Cyclin groove. However the mechanism underlying this differential substrate recognition remains largely unknown and cannot be explained merely on the basis of sequence variability. A subset of Cyclins, Cyclins A2, E1 and B1 despite being structurally and functionally similar, show marked differences in their interactions with recruitment peptides derived from their substrate or inhibitor proteins p27, p21, p57, E2F1, p53, pRb, and p107. While these peptides (characterized by a Cyclin Binding motif of four residues ZRXL where Z and X are cationic residues) inhibit the activity of Cyclins A2 and E1, no such inhibition is observed for Cyclin B1. Electrostatic potentials of Cyclins A2, E1 and B1 show that anionic regions of Cyclins A2 and E1 enable them to bind peptides while cationic regions at homologous locations in Cyclin B1 abrogate binding. These arise from charged residues that are conserved. Mutations that switch these characters are suggested. Computed energetics of binding confirms this. Deregulation of the enzymatic activity of this class of enzymes is a ubiquitous feature of human neoplasia, but attempts to exploit this therapeutically have been confounded by a lack of understanding of the precise specificity of the different Cyclin complexes. Here we begin to clarify this issue by explaining the mechanism by which Cyclin B1 escapes regulation by the p21 family of CDKIs.

Open Access Article

Heat Shock Protein 90: A Potential Therapeutic Target in Leukemic Progenitor and Stem Cells Harboring Mutant BCR-ABL Resistant to Kinase Inhibitors

Cong Peng, Dongguang Li and Shaoguang Li
Pages 2227 - 2231
http://dx.doi.org/10.4161/cc.6.18.4722
Abstract | PDF

Development of drug resistance has become a major obstacle for tyrosine kinase inhibitors (TKIs) in the treatment of Philadelphia chromosome-positive (Ph+) chronic myelogenous leukemia (CML) and other cancers. The BCR-ABL-T315I mutant does not respond to clinically available TKIs, although some newly developed anti-BCR-ABL-T315I TKIs are now being tested in patients. TKIs transiently inhibit kinase activity of BCR-ABL, but do not reduce the level of the BCR-ABL protein. Elimination of mutant BCR-ABL protein would provide a new therapeutic strategy for treating Ph+ leukemia. We recently showed that inhibition of heat shock protein 90 (Hsp90) by a novel Hsp90 inhibitor, IPI-504, causes BCR-ABL protein degradation, decreased numbers of leukemia stem cells, and prolonged survival of mice with CML induced by BCR-ABL-T315I. Here we discuss further the mechanisms and effectiveness of Hsp90 inhibition in suppression of survival and proliferation of leukemic progenitor and stem cells in CML mice, and the potential of this anti-Hsp90 strategy in treating CML patients, including those who have developed resistance to TKIs.




PERSPECTIVES

Open Access Article

Hsp90 and the Fanconi Anemia Pathway: A Molecular Link Between Protein Quality Control and the DNA Damage Response

Takayuki Yamashita, Tsukasa Oda and Takayuki Sekimoto
Pages 2232 - 2235
http://dx.doi.org/10.4161/cc.6.18.4653
Abstract | PDF

Heat shock protein 90 (Hsp90) is a molecular chaperone that plays an essential role in cell growth and survival. The chaperone exerts these functions by regulating key signaling proteins involved in cell growth/survival and protecting cells from proteotoxic stress. Importantly, Hsp90 inhibitors including geldanamycin analogues show anti-tumor effects. We recently found that Hsp90 promotes stabilization and nuclear localization of the Fanconi anemia (FA) protein FANCA, which is required for activation of the FA pathway. The FA pathway is a multiprotein biochemical pathway involved in genotoxic signaling, defects in which cause genomic instability, hematopoietic stem cell failure and tumor development. Inhibition of Hsp90 impairs the intracellular homeostasis of FANCA, resulting in disruption of the FA pathway. These findings have important implications for rational cancer chemotherapy using Hsp90 inhibitors. We also discuss the possible functions of Hsp90 in FA pathophysiology and stem cell/cancer biology. Based on our findings and other data, we propose that Hsp90 functions as “a guardian of the genome” through the control of DNA repair proteins.

Open Access Article

Bim and the Pro-Survival Bcl-2 Proteins: Opposites Attract, ERK Repels

Katherine E. Ewings, Ceri M. Wiggins and Simon J. Cook
Pages 2236 - 2240
http://dx.doi.org/10.4161/cc.6.18.4728
Abstract | PDF

Bim (Bcl-2-interacting mediator of cell death) is a BH3-only protein (BOP), a pro-apoptotic member of the Bcl-2 protein family. The Bim mRNA undergoes alternate splicing to give rise to the short, long and extra long protein variants (BimS, BimL and BimEL). These proteins have distinct potency in promoting death and distinct modes of regulation conferred by their interaction with other proteins. Quite how Bim and other BOPs promote apoptosis has been the subject of some debate. Bim was isolated by it’s interaction with pro-survival proteins such as Bcl-2 and it has been suggested that this is key to the ability of Bim to induce apoptosis. However, an alternative model argues that some forms of Bim can bind directly to the pro-apoptotic Bax and Bak proteins to initiate apoptosis. A new study may finally put this debate to rest as it provides strong evidence to suggest that Bim and other BOPs act primarily by binding to pro-survival Bcl-2 proteins, thereby releasing Bax or Bak proteins to promote apoptosis. The importance of the interaction between Bim and the pro-survival Bcl-2 proteins is underlined by our demonstration that it is regulated by ERK1/2-dependent phosphorylation of BimEL. ERK1/2-dependent dissociation of BimEL from pro-survival proteins is the first step in a process by which the pro-survival ERK1/2 pathway promotes the destruction of this most abundant Bim splice variant. In this review we outline the significance of these new studies to our understanding of how BOPs such as Bim initiate apoptosis and how this process is regulated by growth factor-dependent signalling pathways.

Open Access Article

HOX Gene Regulation in Acute Myeloid Leukemia: CDX Marks the Spot?

Stefan Frohling, Claudia Scholl, Dimple Bansal and Brian J.P. Huntly
Pages 2241 - 2245
http://dx.doi.org/10.4161/cc.6.18.4656
Abstract | PDF

Gene targeting and overexpression studies have demonstrated the importance of the clustered homeobox (HOX) genes in hematopoesis. In addition, global HOX gene dysregulation is found in the majority of cases of acute myeloid leukemia (AML) and many cases of acute lymphoblastic leukemia (ALL), and substantial evidence exists to suggest that aberrant expression of HOX genes contributes to the pathogenesis of leukemia. However, although individual HOX genes are rearranged in rare cases of AML and HOX genes are known transcriptional targets of certain leukemia-associated fusion proteins, such as those involving the mixed lineage leukemia (MLL) gene, in the majority of cases, the upstream regulators of HOX genes are unknown. The CDX family of non-clustered homeobox genes are known developmental regulators of HOX gene expression. We have recently demonstrated that Cdx4 is expressed in adult murine bone marrow where its expression pattern follows that of Hox genes. We also demonstrated that CDX2 is expressed in the majority, and that CDX4 is expressed in almost a quarter, of AML patient samples. For CDX2, this expression was predominantly monoallelic but was not associated with coding sequence or promoter mutations, gene amplification, or aberrant promoter methylation. In addition, stable knockdown of CDX2 resulted in a loss of proliferation and clonogenicity in AML cell lines, and bone marrow retrovirally engineered to express either Cdx2 or Cdx4 generated AML in transplant recipients. Cdx4 was shown to cooperate with the known Hox cofactor Meis1a, and structure-function experiments confirmed that the transcription factor function of Cdx4 was required for transformation. Finally, expression of either Cdx2 or Cdx4 generated a dysregulated Hox gene program in normal hematopoietic progenitors and in leukemic tissue. Taken together, these studies implicate CDX proteins as master regulators of HOX gene regulation in AML.

Open Access Article

The Ribosome Filter Redux

Vincent P. Mauro and Gerald M. Edelman
Pages 2246 - 2251
http://dx.doi.org/10.4161/cc.6.18.4739
Abstract | PDF

The ribosome filter hypothesis postulates that ribosomes are not simply translation machines but also function as regulatory elements that differentially affect or filter the translation of particular mRNAs. On the basis of new information, we take the opportunity here to review the ribosome filter hypothesis, suggest specific mechanisms of action, and discuss recent examples from the literature that support it.

Open Access Article

The Transcription Stress Response

Mats Ljungman
Pages 2252 - 2257
http://dx.doi.org/10.4161/cc.6.18.4751
Abstract | PDF

The RNA polymerase II transcription machinery acts as a molecular motor that traverses large parts of the genome on a regular basis. It has been suggested that the transcription machinery may play an important role in sensing DNA damage and activating DNA repair and stress response pathways when stalled at blocking lesions. We have collectively termed the activation of these different pathways as the transcription stress response. Recently, it was shown that the ATR kinase and the single-strand DNA-binding protein RPA mediate the phosphorylation of p53 following blockage of transcription elongation. This ATR-mediated phosphorylation occurs even when transcription elongation is blocked in the absence of DNA damage suggesting that ATR and RPA senses the consequences of blocked transcription elongation rather than sensing DNA lesions directly. It is proposed that the transcription stress response activated by blockage of transcription may play an important role in safeguarding the genome from DNA damage and thus act to suppress tumorigenesis.




BRIEF REPORTS

Open Access Article

Development of Fetal Testicular Cells in Androgen Receptor Deficient Mice

Jorge Merlet, Evelyne Moreau, René Habert and Chrystèle Racine
Pages 2258 - 2262
http://dx.doi.org/10.4161/cc.6.18.4654
Abstract | PDF

During mammalian development, androgens produced by the fetal testis are the most important hormones controlling the masculinization of the reproductive tract and the genitalia. New findings show that the male germ line is the most sensitive to anti-androgenic endocrine disruptors during the embryonic period. In a recent study, we reported that endogenous androgens physiologically control germ cell growth in the male mouse fetus during early fetal life. In the present study, we extended this result by showing the presence of a functional androgen receptor in the gonocytes in the latter part of the fetal life. We also studied the effect of androgens on the development of the somatic testicular cells using the Tfm mice which carry a naturally inactivating mutation of the androgen receptor. Fetal Leydig cell are largely independent of endogenous androgens during fetal development whereas fetal Sertoli cell number is decreased following a default of peritubular myoid cells differenciation. They also point to the gonocyte as a special target for androgens during the embryonic period and indicate a novel mechanism of androgen action on gonocytes. Elucidation of this new pathway in the fetal testis will clarify not only fetal testis physiology but also the effects of environmental anti-androgens that act during fetal life and open new perspectives for future investigations into the sensitivity of fetal germ cell to androgens.

Open Access Article

Cell Cycle-Dependent Induction of Autophagy, Mitophagy and Reticulophagy

Ezgi Tasdemir, M. Chiara Maiuri, Nicolas Tajeddine, Ilio Vitale, Alfredo Criollo, José Miguel Vicencio, John A. Hickman, Olivier Geneste and Guido Kroemer
Pages 2263 - 2267
http://dx.doi.org/10.4161/cc.6.18.4681
Abstract | PDF

When added to cells, a variety of autophagy inducers that operate through distinct mechanisms and target different organelles for autophagic destruction (mitochondria in mitophagy, endoplasmic reticulum in reticulophagy) rarely induce autophagic vacuolization in more than 50% or the cells. Here we show that this heterogeneity may be explained by cell cycle-specific effects. The BH3 mimetic ABT737, lithium, rapamycin, tunicamycin or nutrient depletion stereotypically induce autophagy preferentially in the G1 and S phases of the cell cycle, as determined by simultaneous monitoring of cell cycle markers and the cytoplasmic aggregation of GFP-LC3 in autophagic vacuoles. These results point to a hitherto neglected crosstalk between autophagic vacuolization and cell cycle regulation.




REPORTS

Open Access Article

Dual Inhibition of PI3K/Akt Signaling and the DNA Damage Checkpoint in p53-Deficient Cells with Strong Survival Signaling: Implications for Cancer Therapy

Andrzej Skladanowski, Przemyslaw Bozko, Michal Sabisz and Annette K. Larsen
Pages 2268 - 2275
http://dx.doi.org/10.4161/cc.6.18.4705
Abstract | PDF

Natural (intrinsic) resistance of many tumor types to DNA damaging agents is closely associated with their capacity to undergo robust cell cycle arrest in G2/M. G2 arrest is regulated by the DNA damage checkpoint and by survival signaling, with a potential role of PI3K/Akt in checkpoint function. In this work, we wanted to clarify if inhibition of multiple checkpoint/survival pathways may confer better efficacy in the potentiation of genotoxic agents compared to inhibition of either pathway alone. We compared the influence of UCN-01, which affects both the DNA damage checkpoint and PI3K/Akt-mediated survival signaling, with the PI3K inhibitors wortmannin and LY294002 in p53-deficient M1 acute myeloid leukemia cells treated with the DNA damaging agent cisplatin. Our results show that direct inhibition of PI3K/Akt in G2-arrested cells by wortmannin or LY294002 strongly enhanced the cytotoxicity of cisplatin without influencing the G2 checkpoint. Unexpectedly, dual inhibition of both survival and checkpoint signaling by UCN-01, also increased the cytotoxicity of cisplatin, but to a lesser degree than wortmannin or LY294002. The differences in cytotoxicity were accompanied by differences in cell death pathways: direct inhibition of PI3K/Akt was accompanied by rapid apoptotic cell death during G2, whereas cells underwent mitotic transit and cell division followed by cell death during G1 when both checkpoint and survival signaling were inhibited. Our results elucidate a novel function for PI3K/Akt as a survival factor during DNA damage-induced G2 arrest and could have important pharmacological consequences for the application of response modulators in p53-deficient tumors with strong survival signaling.

Open Access Article

A Transgenic Mouse Model for High Content, Cell Cycle Phenotype Screening in Live Primary Cells

Richard O. Burney, Alan I. Lee, Denise E. Leong, Joshua T. Jones, Angela T. Hahn, Tobias Meyer and Mylene W.M. Yao
Pages 2276 - 2283
http://dx.doi.org/10.4161/cc.6.18.4718
Abstract | PDF

High content cell-based genetic and small molecule library screens are powerful strategies in drug discovery and investigations of disease mechanisms. We report that primary cells derived from a transgenic mouse model expressing a fluorescence mitosis biosensor provide unambiguous phenotype readouts without the need for transfection or immunocytochemistry. Phenotype profiles of cell cycle disruption and of apoptosis are easily detectable at a single time point selected from time-lapse live fluorescence microscopy. Most importantly, this transgenic mouse model may be crossed with cancer mouse models to derive biosensor-expressing primary cancer cells for use in high content screening strategies targeting discovery of tumor-specific chemotherapeutic compounds.

Open Access Article

Inhibition of the Canonical IKK/NFκB Pathway Sensitizes Human Cancer Cells to Doxorubicin

Maria A. Tapia, Irene González-Navarrete, Alba Dalmases, Marta Bosch, Vanesa Rodriguez-Fanjul, Mark Rolfe, Jeffrey S. Ross, Jovita Mezquita, Cristobal Mezquita, Oriol Bachs, Pere Gascón, Federico Rojo, Rosario Perona, Ana Rovira and Joan Albanell
Pages 2284 - 2292
http://dx.doi.org/10.4161/cc.6.18.4721
Abstract | PDF

The NFκB family is composed by five subunits (p65/RelA, c-Rel, RelB, p105-p50/NFκB1, p100-p52/NF-κB2) and controls the expression of many genes that participate in cell cycle, apoptosis, and other key cellular processes. In a canonical pathway, NF-κB activation depends on the IKK complex activity, which is formed by three subunits (IKKα and IKKβ and IKKγ/NEMO). There is an alternative NFκB activation pathway that does not require IKKβ or IKKγ/NEMO, in which RelB is a major player. We report in a panel of human breast cancer cells that the IKK/NFκB system is generally overexpressed in breast cancer cells and there is heterogeneity in expression levels of individual members between different cell lines. Doxorubicin, an anticancer agent used in patients with breast cancer, activated NFκB and appeared to be less effective in cells expressing predominantly members of the canonical IKK/NFκB. Two NFκB inhibitors, bortezomib and NEMO-Binding Domain Inhibitory Peptide, prevented doxorubicin-induced NFκB activation and increased doxorubicin antitumor effects in BT-474 cells. Transient downregulation of members of the canonical pathway (p65, p52, c-Rel and IKKγ/NEMO) by siRNA in HeLa cells increased doxorubicin cytotoxicity. In contrast, silencing of RelB, a key subunit of the alternative pathway, had no evident effects on doxorubicin cytotoxicity. To conclude, NFκB inhibition sensitized cells to doxorubicin, implying directly p65, p52, c-Rel and IKKγ/NEMO subunits in chemoresistance, but not RelB. These findings suggest that selective inhibition of the canonical NFκB pathway is sufficient to improve doxorubicin antitumor effects.

Open Access Article

Natural Human Gene Correction by Small Extracellular Genomic DNA Fragments

Leonid A. Yakubov, Vladimir A. Rogachev, Anastasia C. Likhacheva, Sergei S. Bogachev, Tamara E. Sebeleva, Alexander G. Shilov, Sergei I. Baiborodin, Natalia A. Petrova, Ludmila V. Mechetina, Mikhail A. Shurdov and Eric Wickstrom
Pages 2293 - 2301
http://dx.doi.org/10.4161/cc.6.18.4729
Abstract | PDF

Classical gene targeting employs natural homologous recombination for a gene correction using a specially designed and artificially Delivered DNA construct but the method is very inefficient. On the other hand, small DNA fragments in the form of tiny chromatin-like articles naturally present in blood plasma can spontaneously penetrate into human cells and cell nuclei. We hypothesized that these natural DNA nanoparticles with recombinagenic free ends might be effective agents for gene replacement therapy. We demonstrate that a mixture of small fragments of total human chromatin from non-mutant cells added to a culture medium without transfection agents efficiently repaired a 47 base pair deletion in the CASP3 gene in 30% of treated human MCF7 breast cancer cells, as shown by restoration of caspase-3 apoptotic function and CASP3 DNA and mRNA structure. Such an innate gene replacement mechanism might function naturally in an organism using its own apoptotic DNA fragments. This mechanism might enable human cancer cell phenotype normalization in the presence of excess normal Cells.




LETTERS TO THE EDITOR

Open Access Article

Gcn2p Regulates a G1/S Cell Cycle Checkpoint in Response to DNA Damage

Mauricio Menacho-Márquez, Jorge Perez-Valle, Joaquin Arino, Jose Gadea and José R Murguía
Pages 2302 - 2305
http://dx.doi.org/10.4161/cc.6.18.4668
Abstract | PDF

Not yet available

Open Access Article

Bone Marrow Derived Cells and Alternative Pathways of Oogenesis in Adult Rodents

Antonin Bukovsky, Maria E. Ayala, Roberto Dominguez, Marta Svetlikova and Rachel Selleck-White
Pages 2306 - 2309
http://dx.doi.org/10.4161/cc.6.18.4707
Abstract | PDF

Not yet available.

Open Access Article

Comparisons Between DT40 Wild Type and DT40-Cre1 Cells as Suitable Model Systems for Studying the DNA Damage Response

Ronan T. Bree, Xian-Yang Lai, Lynn E. Canavan and Noel F. Lowndes
Pages 2310 - 2313
http://dx.doi.org/10.4161/cc.6.18.4723
Abstract | PDF

Not yet available.