Table of Contents

Volume 9, Issue 24

  December 15, 2010

  Pages 4771 - 4953

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EDITORIALS: CELL CYCLE FEATURES

Open Access Article

TIS11/TTP gene family: It's never too late for tumor suppressors 

Tommaso Zanocco-Marani
Page 4771
http://dx.doi.org/10.4161/cc.9.24.14217
Abstract | PDF

Comment on: Vignudelli T, et al. Mol Biol Cell 2010; 21:3340-51.

Open Access Article

Dicing up chromosomes: The unexpected role of Dicer in apoptosis 

Brian L. Harry, Akihisa Nakagawa and Ding Xue
Pages 4772 - 4773
http://dx.doi.org/10.4161/cc.9.24.14240
Abstract | PDF

Comment on: Nakagawa A, et al. Science 2010; 328:327-34.

Open Access Article

Phosphorylation of the p52 NF-κB subunit 

Benjamin Barré and Neil D. Perkins
Pages 4774 - 4775
http://dx.doi.org/10.4161/cc.9.24.14246
Abstract | PDF

Comment on: Barré B, et al. Mol Cell 2010; 38:524-38.

Open Access Article

Lessons learned from testicular cancer: Identification of cytoplasmic p21 as an Achilles' heel of cisplatin resistance 

Roelof Koster and Steven de Jong
Pages 4776 - 4777
http://dx.doi.org/10.4161/cc.9.24.14247
Abstract | PDF

Comment on: Koster R, et al. J Clin Invest 2010; 120:3594-605; DOI:10.1172/JCI41939.

Open Access Article

Oncogene-induced senescence pathways in melanocytes 

Sebastian Haferkamp and Helen Rizos
Pages 4778 - 4779
http://dx.doi.org/10.4161/cc.9.24.14248
Abstract | PDF

Comment on: Haferkamp S, et al. Aging 2009; 1:542-56.

Open Access Article

Metabolism meets autophagy 

J. Julie Wu, Celia Quijano, Jiamei Wang and Toren Finkel
Pages 4780 - 4781
http://dx.doi.org/10.4161/cc.9.24.14273
Abstract | PDF

Comment on: Wu, et al. Aging 2009; 1:425-37.




CELL CYCLE NEWS & VIEWS

Open Access Article

Cyclin E goes nuts: A cell cycle regulator affects male fertility 

Juan Méndez
Pages 4782 - 4787
http://dx.doi.org/10.4161/cc.9.24.14060
Abstract | PDF

Comment on: Liberal V, et al. Cell Cycle 2010; 9:4222-7.

Open Access Article

When enough is enough: Detrimental effects of excess histones 

Valérie Villeneuve and Alain Verreault

http://dx.doi.org/10.4161/cc.9.24.14062
Abstract | PDF

Comment on: Singh RK, et al. Cell Cycle 2010; 9:4236-44.

Open Access Article

Skirmish between the mighty p53 protein and the complex retrovirus HIV-1 

Alagarsamy Srinivasan

http://dx.doi.org/10.4161/cc.9.24.14095
Abstract | PDF

Comment on: Mukerjee R, et al. Cell Cycle 2010; 9(22):in press.

Open Access Article

CXCR4 and renal cancer: From lab to bedside 

Luigi Schips and Alessandro Volpe

http://dx.doi.org/10.4161/cc.9.24.14165
Abstract | PDF

Comment on: D'Alterio C, et al. Cell Cycle 2010; 9: In press.

Open Access Article

Research highlights on a notable retrovirus and a popular guardian gene 

Paraskevi Vogiatzi and Philip J. Mason

http://dx.doi.org/10.4161/cc.9.24.14166
Abstract | PDF

Comment on: Mukerjee R, et al. Cell Cycle 2010; 9:4569-78.

Open Access Article

Linking Cdc7 with the replication checkpoint 

Cyrus Vaziri

http://dx.doi.org/10.4161/cc.9.24.14167
Abstract | PDF

Comment on: Matsumoto S, et al. Cell Cycle 2010; 9: In press.




REVIEW OF CONCEPT

Open Access Article

Increasing healthy lifespan by suppressing aging in our lifetime: Preliminary proposal 

Mikhail V. Blagosklonny
Pages 4788 - 4794
http://dx.doi.org/10.4161/cc.9.24.14360
Abstract | PDF

Although we do not know everything about aging, we now know enough to start its pharmacologic suppression using clinically approved drugs. Aging turns out to be driven by sensing-signaling pathways (such as the mTOR pathway). Given that some inhibitors of the mTOR pathway are already in clinical use, there is a unique opportunity to suppress aging, while treating and preventing diseases. By itself this will answer some burning questions in gerontology. Here I discuss a proposal, starting from retrospective clinical studies to animal and cellular models to drug screens in order to develop non-toxic and effective schedules and drug combinations for extending healthy life span in our lifetime.




EXTRA VIEWS

Open Access Article

Regulation of activity and function of the p52 NF-κB subunit following DNA damage 

Benjamin Barré, Olivier Coqueret and Neil D. Perkins
Pages 4795 - 4804
http://dx.doi.org/10.4161/cc.9.24.14245
Abstract | PDF

We have previously shown that after DNA-damage, the p52 NF-kB subunit can function cooperatively with the p53 tumor suppressor to both repress and induce Skp2 expression.  However, the wider role and activation of p52 after DNA-damage has not been determined.  Activation of NF-kB in response to DNA break inducers can be mediated by ATM (ataxia telangiectasia mutated)-dependent phosphorylation of NEMO (NF-kB essential modulator), resulting in IKKβ mediated induction of the classical NF-kB pathway, leading to the induction of RelA(p65)/p50 dimers. Here, we show that DNA damage also induces p100 (NF-kB2) processing to generate active p52. We further demonstrate that p52 generation is dependent not only on IKKα but also on atypical activation by NEMO/ATM.  Moreover, we identify a post-DNA damage, positive feedback loop of p52 activation through induction of NF-kB2 gene expression, involving both the classical and alternative NF-kB pathways. Gene expression and chromatin immunoprecipitation analyses indicated DNA damage induced p52 dimer recruitment on multiple, p53 dependent and independent, target genes associated with promoting cell cycle arrest and cell death. These results demonstrate an important role for the alternative, p52 NF-kB pathway after DNA-damage distinct from its functions as a regulator of adaptive immunity.

Open Access Article

ATM activation in the presence of oxidative stress 

Zhi Guo, Rajashree Deshpande and Tanya T. Paull
Pages 4805 - 4811
http://dx.doi.org/10.4161/cc.9.24.14323
Abstract | PDF

The Ataxia-Telangiectasia mutated (ATM) kinase is regarded as the major regulator of the cellular response to DNA double strand breaks (DSBs). In response to DSBs, ATM dimers dissociate into active monomers in a process promoted by the Mre11-Rad50-Nbs1 (MRN) complex. ATM can also be activated by oxidative stress directly in the form of exposure to H₂O₂. The active ATM in this case is a disulfide-crosslinked dimer containing 2 or more disulfide bonds. Mutation of a critical cysteine residue in the FATC domain involved in disulfide bond formation specifically blocks ATM activation by oxidative stress. Here we show that ATM activation by DSBs is inhibited in the presence of H₂O₂ because oxidation blocks the ability of MRN to bind to DNA. However, ATM activation via direct oxidation by H₂O₂ complements the loss of MRN/DSB-dependent activation and contributes significantly to the overall level of ATM activity in the presence of both DSBs and oxidative stress.

Open Access Article

Characterization of genome-nucleoporin interactions in Drosophila links chromatin insulators to the nuclear pore complex 

Bernike Kalverda and Maarten Fornerod
Pages 4812 - 4817
http://dx.doi.org/10.4161/cc.9.24.14328
Abstract | PDF

Nuclear pore complexes (NPCs) are multiprotein complexes consisting of nucleoporins and function in transport between the nucleus and the cytoplasm. In yeast, nucleoporins have also been linked to gene expression as well as to chromatin insulating activity. Recently, we identified genomic regions that interact with nucleoporins in Drosophila using DamID technology. We found that nucleoporins in the nucleoplasm interact with active genes and stimulate gene expression. However, genes interacting with nucleoporins at the NPC itself show average gene expression and it remains unclear why they interact with the NPC. Here, we further investigated the function of the genome-NPC interactions. First, to investigate whether a different technique would lead to similar results, we compared our nucleoporin DamID data to recently published nucleoporin chromatin immunoprecipitation (ChIP) data. Then, to further understand the function of interactions between the genome and NPCs, we analyzed the relationship between NPC-interacting genomic regions and chromatin insulators. We found that the insulator protein Su(Hw) was enriched within and near NPC-interacting genomic regions, suggesting a role of this protein in chromatin architecture close to the NPC. This suggests that the NPC may have a function in the structural organization of the genome.




REVIEWS

Open Access Article

Integrating DNA replication with trans-lesion synthesis via Cdc7 

Cyrus Vaziri and Hisao Masai
Pages 4818 - 4823
http://dx.doi.org/10.4161/cc.9.24.14241
Abstract | PDF

It has long been appreciated that Cdc7 is an essential protein kinase that phosphorylates Mcm2-7 helicase subunits to promote initiation of DNA replication. In addition to its well-elucidated role in DNA replication, recent studies suggest that DDK is active in genotoxin-treated cells and may mediate aspects of the DNA damage response. However, specific role(s) of DDK and its effector targets in DNA damage signaling have not been defined. A recent study from our laboratories has identified the E3 ubiquitin ligase Rad18 as novel substrate of DDK in vitro and in human cells. Rad18 plays a central role in a post-replication DNA repair pathway termed ‘Trans-Lesion Synthesis’ (TLS) by promoting recruitment of DNA Polymerase eta (Polη) and other TLS polymerases to stalled replication forks. DDK-mediated Rad18 phosphorylation promotes Rad18-Polη complex formation and facilitates Rad18-dependent recruitment of Polη to stalled replication forks. The mechanisms that regulate Rad18-dependent TLS are incompletely understood. Our study provides the first demonstration of Rad18 regulation by direct phosphorylation and defines a novel mechanism for Rad18-dependent recruitment of TLS polymerases to stalled forks. This study also demonstrates a molecular basis for integration of TLS with S-phase progression via the essential Cdc7 kinase. These findings reveal unexpected mechanistic insights to the regulation of the TLS pathway and Polη recruitment.

Open Access Article

Alternative activation of tumor-associated macrophages by IL-4: Priming for protumoral functions 

Hao-Wei Wang and Johanna A. Joyce
Pages 4824 - 4835
http://dx.doi.org/10.4161/cc.9.24.14322
Abstract | PDF

Although macrophages were originally recognized as major immune effector cells, it is now appreciated that they also play many important roles in the maintenance of tissue homeostasis, and are involved in a variety of pathological conditions including cancer. Several studies have demonstrated the contributions of tumor-associated macrophages (TAMs) to tumor initiation, progression, and metastasis. However, the detailed mechanisms underlying how TAMs differ molecularly from their normal counterparts and how the conversion to TAMs occurs have only just begun to be understood. TAMs have been proposed to exhibit phenotypes of ‘alternatively activated’ macrophages, though there has been limited evidence directly linking the phenotypes of TAMs to the alternative activation of macrophages. This review will focus on IL-4, the prototypic cytokine that induces the alternative activation of macrophages, and review current knowledge regarding the contributions of IL-4 to the phenotypes of TAMs and its effects on tumorigenesis.

Open Access Article

Do predictive signatures really predict response to cancer chemotherapy? 

Piet Borst and Lodewyk Wessels
Pages 4836 - 4840
http://dx.doi.org/10.4161/cc.9.24.14326
Abstract | PDF

Predictive signatures are gene expression profiles that should predict the response of tumors to chemotherapy in patients. Such signatures have been derived from the response of tumor cell lines to chemotherapy, but their usefulness in patients remains controversial, as the most spectacular published signatures are based on unreliable data. We discuss why it is difficult to derive meaningful predictive signatures from cell line panels and we argue that it is implausible that fully predictive signatures can be obtained for classical chemotherapy from oligo-based gene expression arrays. One reason is that resistance to chemotherapy can be caused by alterations in (the expression of) a single gene. We do not expect that such subtle alterations will be reliably picked up by standard gene expression profiling. We delineate alternative approaches that should be able to yield predictive markers that can be used for optimizing patient treatment.




REPORTS

Open Access Article

Regulation of ΔNp63α by NFκΒ 

Tanusree Sen, Xiaofei Chang, David Sidransky and Aditi Chatterjee
Pages 4841 - 4847
http://dx.doi.org/10.4161/cc.9.24.14093
Abstract | PDF

ΔNp63α, the dominant negative isoform of the p63 family is an essential survival factor in head and neck squamous cell carcinoma. This isoform has been shown to be down regulated in response to several DNA damaging agents, thereby enabling an effective cellular response to genotoxic agents. Here, we identify a key molecular mechanism underlying the regulation of ΔNp63α expression in response to extrinsic stimuli, such as chemotherapeutic agents. We show that ΔNp63α interacts with NF-κΒ in presence of cisplatin. We find that NF-κΒ promotes ubiquitin-mediated proteasomal degradation of ΔNp63α. Chemotherapy-induced stimulation of NF-κΒ leads to degradation of ΔNp63α and augments trans-activation of p53 family-induced genes involved in the cellular response to DNA damage. Conversely, inhibition of NF-κΒ with siRNA-mediated silencing NF-κΒ expression attenuates chemotherapy induced degradation of ΔNp63α . These data demonstrate that NF-κΒ plays an essential role in regulating ΔNp63α in response to extrinsic stimuli. Our findings suggest that the activation of NF-κΒ may be a mechanism by which levels of ΔNp63α are reduced, thereby rendering the cells susceptible to cell death in the face of cellular stress or DNA damage.

Open Access Article

The interacting domains of hCdt1 and hMcm6 involved in the chromatin loading of the MCM complex in human cells 

Jingjing Zhang, Lan Yu, Xing Wu, Lan Zou, Kelvin K.L. Sou, Zhun Wei, Xue Cheng, Guang Zhu and Chun Liang
Pages 4848 - 4857
http://dx.doi.org/10.4161/cc.9.24.14136
Abstract | PDF

The stepwise assembly of pre-replicative complexes (pre-RCs) is essential for the initiation of DNA replication. Cdt1, a component of the pre-RC, is required for the loading of the minichromosome maintenance (MCM) complex onto chromatin. Cdt1 physically interacts with the MCM complex, and this interaction mainly occurs between Cdt1 and Mcm6 in human cells. Here we show by yeast two-hybrid analysis, co-immunoprecipitation and GST pull-down assays that the extreme C-terminal region of hMcm6 (a.a. 708-821) interacts with a short C-terminal region in hCdt1 (a.a. 392-471), while the large N-terminal part of hMcm6 (a.a. 1-707) interacts with some other MCM subunits. Furthermore, our functional studies show that ectopic expression of either of the interacting domains of hCdt1 and hMcm6 in human cells reduces chromatin association of the MCM complex and DNA replication, inhibits cell proliferation, and leads to cell apoptosis. These dominant negative effects indicate that the interaction between hCdt1 and hMcm6 through their interacting domains we identified is the key for hCdt1 in facilitating the MCM hetero-hexamer to load onto chromatin for replication licensing. The newly indentified interacting domains of hCdt1 and hMcm6 may become targets for identification of novel anticancer drugs.

Open Access Article

Duplication of cyb-3 (cyclin B3) suppresses sterility in the absence of mdf-1/MAD1 spindle assembly checkpoint component in Caenorhabditis elegans

Maja Tarailo-Graovac, Jun Wang, Domena Tu, David L. Baillie, Ann M. Rose and Nansheng Chen
Pages 4858 - 4865
http://dx.doi.org/10.4161/cc.9.24.14137
Abstract | PDF

mdf-1/MAD1 is a conserved spindle assembly checkpoint component that is essential for the survival of Caenorhabditis elegans. Previously, using a dog-1(gk10)/FANCJ mutator strain, we have isolated a suppressor of mdf-1(gk2) sterility. This suppressor, named such-4, was demonstrated to be a tandem duplication that contained 62 putative protein coding genes. We apply here the recently developed Mos1-mediated single-copy insertion (MosSCI) method to study this copy number variation (CNV) in C. elegans and show that such-4 is caused by the duplication of a single gene cyb-3, illustrating the power of MosSCI-mediated single-gene duplications for uncovering gene dosage genetic interactions. Importantly, we show here, for the first time, that doubling the CYB-3 (Cyclin B3) dosage suppresses sterility in the absence of the essential spindle assembly checkpoint component MDF-1 without causing a delay in the onset of anaphase.

Open Access Article

The novel BTB/POZ and zinc finger factor Zbtb45 is essential for proper glial differentiation of neural and oligodendrocyte progenitor cells 

Erik Södersten, Tobias Lilja and Ola Hermanson
Pages 4866 - 4875
http://dx.doi.org/10.4161/cc.9.24.14154
Abstract | PDF

Understanding the regulatory mechanisms controlling the fate decisions of neural stem cells (NSCs) is a crucial issue to shed new light on mammalian central nervous system (CNS) development in health and disease. We have investigated a possible role for the previously uncharacterized BTB/POZ-domain containing zinc finger factor Zbtb45 in the differentiation of NSCs and postnatal oligodendrocyte precursors. In situ hybridization histochemistry and RT-qPCR analysis revealed that Zbtb45 mRNA was ubiquitously expressed in the developing CNS in mouse embryos at embryonic day (E) 12.5 and 14.5. Zbtb45 mRNA knockdown in embryonic forebrain NSCs by siRNA resulted in a rapid decrease in the expression of oligodendrocyte-characteristic genes after mitogen (FGF2) withdrawal, whereas the expression of astrocyte-associated genes such as CD44 and GFAP increased compared to control. Accordingly, the number of astrocytes was significantly increased seven days after Zbtb45 siRNA delivery to NSCs, in contrast to the numbers of neuronal and oligodendrocyte-like cells. Surprisingly, mRNA knockdown of the Zbtb45-associated factor Med31, a subunit of the Mediator complex, did not result in any detectable effect on NSC differentiation. Similar to NSCs, Zbtb45 mRNA knockdown in oligodendrocyte precursors (CG-4) reduced oligodendrocyte maturation upon mitogen withdrawal associated with down-regulation of the mRNA expression and protein levels of markers for oligodendrocytic differentiation. Zbtb45 mRNA knockdown did not significantly affect proliferation or cell death in any of the cell types. Based on these observations, we propose that Zbtb45 is a novel regulator of glial differentiation.

Open Access Article

Phenotypic enhancement of thymidylate synthetase pathway inhibitors following ablation of Neil1 DNA glycosylase/lyase 

Lorena Taricani, Frances Shanahan, Rob H. Pierce, Timothy J. Guzi and David Parry
Pages 4876 - 4883
http://dx.doi.org/10.4161/cc.9.24.14155
Abstract | PDF

Inhibition of thymidine biosynthesis is a clinically-validated therapeutic approach for multiple cancers. Inhibition of thymidylate synthetase (TS) leads to a decrease in cellular TTP levels, replication stress and increased genomic incorporation of uridine (dUMP). Thus, inhibitors of this pathway (such as methotrexate) can drive a multitude of downstream cell cycle checkpoint and DNA repair processes. Genomic dUMP is recognized by the base excision repair (BER) pathway. Using a synthetic lethal siRNA-screening approach, we systematically screened for components of BER that, when ablated, enhanced methotrexate response in a high content γ-H2A.X bioassay. We observed specific ablation of the mixed function DNA glycosylase/lyase Neil1 phenotypically enhanced several inhibitors of thymidine biosynthesis, as well as cellular phenotypes downstream of gemcitabine, cytarabine and clofarabine exposure. These synthetic lethal interactions were associated with significantly enhanced accumulation of γ-H2A.X and improved growth inhibition. Significantly, following TS pathway inhibition, addition of exogenous dTTP complemented the primary Neil1 γ-H2A.X phenotypes. Similarly, co-depletion of Neil1 with Cdc45, Cdc6, Cdc7 or DNA polymerase β (PolB) suppressed Neil1 phenotypes. Conversely, co-depletion of Neil1 with the Rad17, Rad9 ATR, ATM and DNA-PK checkpoint/sensor proteins appears primarily epistatic to Neil1. These data suggest Neil1 may be a critical mediator of BER of incorporated dUMP following TS pathway inhibition

Open Access Article

Transcriptional and post-transcriptional mechanisms of BAFF-receptor dysregulation in human B lineage malignancies 

Stephen A. Mihalcik, Renee C. Tschumper and Diane F. Jelinek
Pages 4884 - 4892
http://dx.doi.org/10.4161/cc.9.24.14156
Abstract | PDF

Together, circulating BAFF and dominant receptor BAFF-R homeostatically regulate the humoral immune system. Consistently aberrant BAFF-R expression in leukemic cells reveals an intimate connection of these cells’ malignant physiology to the BAFF/BAFF-R axis and also provides an additional survival mechanism to the expressing cells. In this study, we used primary cells and cell lines to interrogate the mechanisms underlying aberrant BAFF-R expression in precursor B acute lymphoblastic leukemia (precursor B-ALL) and mature B chronic lymphocytic leukemia (CLL). Here we demonstrate the aberrant expression of BAFF-R in precursor B-ALL cell lines and reveal that these cells acquire BAFF-R expression through premature transcriptional activation of the BAFF-R promoter in coordination with regulatory transcription factor c-Rel. Investigations using primary CLL cells provide a crucial counterpoint through their paucity of BAFF-R relative to their benign mature B cell counterparts, which we establish as functionally significant in its depletion of the CLL cells’ BAFF-binding capacity. Furthermore, BAFF-R downregulation in CLL patients is revealed here to be restricted to the malignant compartment and mediated post-transcriptionally in order to compensate for the consistently unchanged levels of transcription factor c-Rel and BAFF-R mRNA. Finally, we present evidence that CLL cells retain endogenous mechanisms of BAFF-R regulatory control despite active receptor dysregulation.

Open Access Article

MCPH1 patient cells exhibit delayed release from DNA damage-induced G₂/M checkpoint arrest 

Ioannis Gavvovidis, Charlotte Pöhlmann, Juan Alberto Marchal, Markus Stumm, Daisuke Yamashita, Tatsuya Hirano, Detlev Schindler, Heidemarie Neitzel and Marc Trimborn
Pages 4893 - 4899
http://dx.doi.org/10.4161/cc.9.24.14157
Abstract | PDF

Mutations in the MCPH1 gene cause primary microcephaly associated with a unique cellular phenotype of misregulated chromosome condensation. The encoded protein contains three BRCT domains, and accumulating data show that MCPH1 is involved in the DNA damage response. However, most of this evidence has been generated by experiments using RNA interference (RNAi) and cells from non-human model organisms. Here, we demonstrate that patient-derived cell lines display a proficient G2/M checkpoint following ionizing irradiation (IR) despite homozygous truncating mutations in MCPH1. Moreover, chromosomal breakage rates and the relocation to DNA repair foci of several proteins functioning putatively in an MCPH1-dependent manner are normal in these cells. However, the MCPH1-deficient cells exhibit a slight delay in re-entering mitosis and delayed resolution of γH2AX foci following IR. Analysis of chromosome condensation behavior following IR suggests that these latter observations may be related to hypercondensation of the chromatin in cells with MCPH1 mutations. Our results indicate that the DNA damage response in human cells with truncating MCPH1 mutations differs significantly from the damage responses in cells of certain model organisms and in cells depleted of MCPH1 by RNAi. These subtle effects of human MCPH1 deficiency on the cellular DNA damage response may explain the absence of cancer predisposition in patients with biallelic MCPH1 mutations.

Open Access Article

Impact of cyclin E overexpression on Smad3 activity in breast cancer cell lines 

Anne Cooley, Stanislav Zelivianski and Jacqueline S. Jeruss
Pages 4900 - 4907
http://dx.doi.org/10.4161/cc.9.24.14158
Abstract | PDF

Smad3, a component of the TGFβ signaling pathway, contributes to G1 arrest in breast cancer cells. Overexpression of the cell cycle mitogen, cyclin E, is associated with poor prognosis in breast cancer, and cyclin E/CDK2 mediated phosphorylation of Smad3 has been linked with inhibition of Smad3 activity. We hypothesized that the biological aggressiveness of cyclin E overexpressing breast cancer cells would be associated with CDK2 phosphorylation and inhibition of the tumor suppressant action of Smad3. Expression constructs containing empty vector, wild type (WT) Smad3, or Smad3 with CDK phosphorylation site mutations were co-transfected with a Smad3-responsive reporter construct into parental, vector control (A1), or cyclin E overexpressing (EL1) MCF7 cells. Smad3 function was evaluated by luciferase reporter assay and mRNA analysis. The impact of a Cdk2 inhibitor and cdk2 siRNA on Smad3 activity was also assessed. Cells expressing Smad3 containing mutations of the CDK phosphorylation sites had higher p15 and p21 and lower c-myc mRNA levels, as well as higher Smad3-responsive reporter activity, compared with controls or cells expressing WT Smad3. Transfection of cdk2 siRNA resulted in a significant increase in Smad3-responsive reporter activity compared with control siRNA; reporter activity was also increased after the treatment with a Cdk2 inhibitor. Thus, cyclin E-mediated inhibition of Smad3 is regulated by CDK2 phosphorylation of the Smad3 protein in MCF7 cells. Inhibition of CDK2 may lead to restoration of Smad3 tumor suppressor activity in breast cancer cells, and may represent a potential treatment approach for cyclin E overexpressing breast cancers.

Open Access Article

Proteomic profiling of Myc-associated proteins 

Pooja Agrawal, Kebing Yu, Arthur R. Salomon and John M. Sedivy
Pages 4908 - 4921
http://dx.doi.org/10.4161/cc.9.24.14199
Abstract | PDF

Mammalian c-Myc is a member of a small family of three closely related transcription factors. The Myc family of proto-oncogenes are among the most potent activators of tumorigenesis, and are frequently overexpressed in diverse cancers. c-Myc has an unusually broad array of regulatory functions, which include, in addition to roles in the cell cycle and apoptosis, effects on a variety of metabolic functions, cell differentiation, senescence, and stem cell maintenance. A significant number of c-Myc interacting proteins have already been defined, but it is widely believed that the c-Myc interactome is vastly larger than currently documented. In addition to interactions with components of the transcription machinery, transcription independent nuclear interactions with the DNA replication and RNA processing pathways have been reported. Cytoplasmic roles of c-Myc have also been recently substantiated. Recent advances in proteomics have opened new possibilities for the isolation of protein complexes under native conditions and confidently identifying the components using ultrasensitive, high mass accuracy and high resolution mass spectrometry techniques. In this communication we report a new tandem affinity purification (TAP) c-Myc interaction screen that employed new cell lines with near-physiological levels of c-Myc expression with multi-dimensional protein identification techniques (MudPIT) for the detection and quantification of proteins. Both label-free and the recently developed stable isotope labeling with amino acids in cell culture (SILAC) methodologies were used. Combined data from multiple biological replicates provided a dataset of 418 non-redundant proteins, 389 of which are putative novel interactors. This new information should significantly advance our understanding of this interesting and important master regulator.

Open Access Article

Role of Cdk4 in lymphocyte function and allergen response

Yu Hua Chow, Xiao Dong Zhu, Li Liu, Barbara R. Schwartz, Xiao Zhu Huang, John M. Harlan and Lynn M. Schnapp
Pages 4922 - 4930
http://dx.doi.org/10.4161/cc.9.24.14209
Abstract | PDF

We recently described a new adhesion pathway in lymphocytes that is dependent on Cyclin-dependent kinase (Cdk) 4 activity and mediates lymphocyte interactions with endothelial matrix. We showed that Cdk4^-/- mice had impaired recruitment of lymphocytes following bleomycin model of acute lung injury. In this study, we characterized the development and function of hematopoietic cells in Cdk4^-/- mice and assessed the response of Cdk4^-/- mice to allergen challenge. Cdk4^-/- mice had hypoplastic thymuses with decreased total thymocyte cell numbers and increased CD4/CD8 double negative cells. Cdk4^-/- bone marrow (BM) chimeric mice showed similar findings. Thymocytes from either Cdk4^-/- or Cdk4^-/- BM chimeric mice proliferated equally well as wild type controls in response to IL-2 activation. However Cdk4^-/- thymocytes had decreased adhesion to both endothelial cell matrix and fibronectin compared to wildtype (WT) controls, whereas Cdk4^-/- and WT splenocytes had similar adhesion. When Cdk4^-/- BM chimeric mice and wild type BM chimeric mice were sensitized and challenged by intranasal administration of ovalbumin, we found no differences in allergic responses in the lung and airways between the two groups, as measured by inflammatory cell infiltrate, airway hyperreactivity, IgE levels and cytokine levels. In summary, we show that Cdk4 plays a previously unrecognized role in thymocyte maturation and adhesion, but is not required for thymocyte proliferation. In addition, Cdk4 is not required for lymphocyte trafficking to the lung following allergen sensitization and challenge.

Open Access Article

Mechanisms of nitric oxide-mediated inhibition of EMT in cancer: Inhibition of the metastasis-inducer Snail and induction of the metastasis-suppressor RKIP

Stavroula Baritaki, Sara Huerta-Yepez, Anna Sahakyan, Iordanis Karagiannides, Kyriaki Bakirtzi, Ali Jazirehi and Benjamin Bonavida
Pages 4931 - 4940
http://dx.doi.org/10.4161/cc.9.24.14229
Abstract | PDF

The role of nitric oxide (NO) in cancer has been controversial and is based on the levels of NO and the responsiveness of the tumor type. It remains unclear whether NO can inhibit the epithelial to mesenchymal transition (EMT) in cancer cells. EMT induction is mediated, in part, by the constitutive activation of the metastasis-inducer transcription factor, Snail and EMT can be inhibited by the metastasis-suppressor Raf-1 kinase inhibitor protein (RKIP) and E-cadherin. Snail is transcriptionally regulated by NF-κB and in turn, Snail represses RKIP transcription. Hence, we hypothesized that high levels of NO, that inhibit NF-κB activity, may also inhibit Snail and induce RKIP and leading to inhibition of EMT. We show that treatment of human prostate metastatic cell lines with the NO donor, DETANONOate, inhibits EMT and reverses both the mesenchymal phenotype and the cell invasive properties. Further, treatment with DETANONOate inhibits Snail expression and DNA-binding activity in parallel with the upregulation of RKIP and E-cadherin protein levels. The pivotal roles of Snail inhibition and RKIP induction in DETANONOate-mediated inhibition of EMT were corroborated by both Snail silencing by siRNA and by ectopic expression of RKIP. The in vitro findings were validated in vivo in mice bearing PC-3 xenografts and treated with DETANONOate. The present findings show, for the first time, the novel role of high subtoxic concentrations of NO in the inhibition of EMT. Thus, NO donors may exert therapeutic activities in the reversal of EMT and metastasis.

Open Access Article

CC3/TIP30 regulates metabolic adaptation of tumor cells to glucose limitation 

Vivian Chen and Emma Shtivelman
Pages 4941 - 4953
http://dx.doi.org/10.4161/cc.9.24.14230
Abstract | PDF

CC3/TIP30 is a metastasis and tumor suppressor, with reduced or absent expression in a variety of aggressive tumors. Overexpression of CC3 in tumor cells predisposes them to apoptosis in response to different death signals. We found that silencing of CC3 expression does not increase apoptotic resistance of cells. However, it strongly improves survival of tumor cells in response to glucose limitation. HeLa cells with silenced CC3 survive long-term in low glucose, and, in comparison to control HeLa cells, show superior metabolic adaptation to glucose limitation. First, unlike the parental HeLa cells, HeLa with silenced CC3 activate and maintain high levels of mitochondrial respiration that is critical for their ability to thrive in low glucose. Second, silencing of CC3 leads to higher expression levels of mitochondrial proteins in respiration complexes when cells are continuously cultured in limiting glucose. Third, HeLa cells with silenced CC3 maintain higher levels of c-MYC and the M2 isoform of pyruvate kinase in low glucose, contributing to more efficient glycolysis. Fourth, HeLa cells with silenced CC3 fail to fully activate AMPK in response to glucose limitation. Inhibition of AMPK, either pharmacologic or via siRNA, protects control HeLa cells from death in low glucose. The metabolic flexibility acquired by cells after silencing of CC3 could be directly relevant to the development of metastatic and aggressive human tumors that frequently have low or absent expression of CC3.