Table of Contents

Volume 9, Issue 22

  November 15, 2010

  Pages 4417 - 4601

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 Issue Catalogue (MARC XML)

EDITORIALS: CELL CYCLE FEATURES

Open Access Article

Follow the leader: LIM kinases pave the way for collective tumor cell invasion

Michael F. Olson
Pages 4417 - 4418
http://dx.doi.org/10.4161/cc.9.22.14049
Abstract | PDF

Comment on: Scott RW, et al. J Cell Biol 2010; 191:169-85.

Open Access Article

Genome-wide RNAi screen discovers functional coupling of alternative splicing and cell cycle control to apoptosis regulation

Qingqing Wang and Pamela A. Silver
Pages 4419 - 4421
http://dx.doi.org/10.4161/cc.9.22.14051
Abstract | PDF

Comment on: Moore MJ, et al. Cell 2010; 142:625-36.

Open Access Article

The importance of being DNA

Alan B. Clark and Thomas A. Kunkel
Pages 4422 - 4424
http://dx.doi.org/10.4161/cc.9.22.14052
Full Text | PDF

Open Access Article

The mitochondrial apoptosis pathway and p66Shc: A regulatory redox enzyme or an adapter protein snuggling around?

Melanie Gertz and Clemens Steegborn
Pages 4425 - 4426
http://dx.doi.org/10.4161/cc.9.22.14053
Abstract | PDF

Comment on: Gertz M, et al. Aging 2009; 1:254-265.




CELL CYCLE NEWS & VIEWS

Open Access Article

The RP-p53-Mdm2 pathway: A new link to genetic integrity?

Rebecca A. Frum and Yanping Zhang
Pages 4427 - 4432
http://dx.doi.org/10.4161/cc.9.22.13606
Abstract | PDF

Comment on: Llanos S, et al. Cell Cycle 2010; 9:4005–12.

Open Access Article

Hedgehog signaling in T-cell development: A non-redundant role for Gli1

Ursula Bommhardt

http://dx.doi.org/10.4161/cc.9.22.13748
Abstract | PDF

Comment on: Drakopoulou E, et al. Cell Cycle 2010; 9:4144–52.

Open Access Article

Dissecting the functions of Gli transcription factors in T-cell development

Alberto Varas and Rosa Sacedón

http://dx.doi.org/10.4161/cc.9.22.13749
Abstract | PDF

Comment on: Drakopoulou E, et al. Cell Cycle 2010; 9:4144–52.

Open Access Article

Small RFC subunits make a big difference

Danielle Daee and Kyungjae Myung

http://dx.doi.org/10.4161/cc.9.22.13821
Abstract | PDF

Comment on: Maradeo ME, et al. Cell Cycle 2010; 9:4370–8.

Open Access Article

MicroRNAs are invading the tumor microenvironment: Fibroblast microRNAs regulate tumor cell motility and invasiveness

Michael R. Shurin

http://dx.doi.org/10.4161/cc.9.22.13822
Abstract | PDF

Comment on: Aprelikova O, et al. Cell Cycle 2010; 9:4387–98.

Open Access Article

Finding markers for cancer stem cells in renal cell carcinoma: Looking beyond CD133

Alessandro Ottaiano

http://dx.doi.org/10.4161/cc.9.22.13823
Abstract | PDF

Comment on: D’Alterio C, et al. Cell Cycle 2010; 9:4492–4500.

Open Access Article

p53, transcriptional repression and drug sensitivity: Fresh perspectives on an old activity

Maureen E. Murphy

http://dx.doi.org/10.4161/cc.9.22.13927
Abstract | PDF

Comment on: McKenzie L, et al. Cell Cycle 2010; 9:4200–12.




EXTRA VIEWS

Open Access Article

Merlin's tumor suppression linked to inhibition of the E3 ubiquitin ligase CRL4^DCAF1

Wei Li and Filippo G. Giancotti
Pages 4433 - 4436
http://dx.doi.org/10.4161/cc.9.22.13838
Abstract | PDF

The mechanism by which the FERM domain protein Merlin, encoded by the tumor suppressor NF2, restrains cell proliferation is poorly understood. Prior studies have suggested that Merlin exerts its antimitogenic effect by interacting with multiple signaling proteins located at or close to the plasma membrane. We have recently observed that Merlin translocates into the nucleus and binds to and inhibits the E3 ubiquitin ligase CRL4^DCAF1. Genetic evidence indicates that inactivation of Merlin induces oncogenic gene expression, hyperproliferation, and tumorigenicity by unleashing the activity of CRL4^DCAF1. In addition to providing a potential explanation for the diverse effects that loss of Merlin exerts in multiple cell types, these findings suggest that compounds inhibiting CRL4^DCAF1 may display therapeutic efficacy in Neurofibromatosis type 2 and other cancers driven by Merlin inactivation.

Open Access Article

To polyadenylate or to deadenylate: That is the question

Xiaokan Zhang, Anders Virtanen and Frida E. Kleiman
Pages 4437 - 4449
http://dx.doi.org/10.4161/cc.9.22.13887
Abstract | PDF

mRNA polyadenylation and deadenylation are important processes that allow rapid regulation of gene expression in response to different cellular conditions. Almost all eukaryotic mRNA precursors undergo a co-transcriptional cleavage followed by polyadenylation at the 3' end. After the signals are selected, polyadenylation occurs to full extent, suggesting that this first round of polyadenylation is a default modification for most mRNAs. However, the length of these poly(A) tails changes by the activation of deadenylation, which might regulate gene expression by affecting mRNA stability, mRNA transport, or translation initiation. The mechanisms behind deadenylation activation are highly regulated and associated with cellular conditions such as development, mRNA surveillance, DNA damage response, cell differentiation and cancer. After deadenylation, depending on the cellular response, some mRNAs might undergo an extension of the poly(A) tail or degradation. The polyadenylation/deadenylation machinery itself, miRNAs, or RNA binding factors are involved in the regulation of polyadenylation/deadenylation. Here, we review the mechanistic connections between polyadenylation and deadenylation and how the two processes are regulated in different cellular conditions. It is our conviction that further studies of the interplay between polyadenylation and deadenylation will provide critical information required for a mechanistic understanding of several diseases, including cancer development.

Open Access Article

Both lipid- and protein-phosphatase activities of PTEN contribute to the p53-PTEN anti-invasion pathway

Jacquelyne S. Poon, Robert Eves and Alan S. Mak
Pages 4450 - 4454
http://dx.doi.org/10.4161/cc.9.22.13936
Abstract | PDF

We have recently identified mutually antagonizing signaling pathways that regulate podosome formation and invasive phenotypes in Src-transformed vascular smooth muscle cells and fibroblasts. Cross-talks between the anti-invasion p53-PTEN, and the pro-invasion Src-Stat3 and Src-PI3K-Akt pathways serve as a check and balance that dictates the outcome of either an invasive or non-invasive phenotype. Using a retrovirus vector encoding PTEN phosphatase mutants that retain either protein- or lipid-phosphatase activity on a Src(Y527F)background, we report here that both lipid- and protein-phosphatase activities of PTEN contribute to the suppression of Src-induced podosome formation and associated invasive phenotypes in rat aortic smooth muscle cells. This data suggests that p53 up-regulation of PTEN inhibits cell invasion via a two-prong mechanism: inactivating podosome agonists by its protein-phosphatase activity on the one hand, and antagonising the PI3K-Akt pathway by its lipid-phosphatase activity on the other.

Open Access Article

Dicer-independent, Ago2-mediated microRNA biogenesis in vertebrates

Jr-Shiuan Yang and Eric C. Lai
Pages 4455 - 4460
http://dx.doi.org/10.4161/cc.9.22.13958
Abstract | PDF

A canonical biogenesis pathway involving sequential cleavage by the Drosha and Dicer RNAse III enzymes governs the maturation of most animal microRNAs. However, there exist a variety of alternative miRNA biogenesis pathways, most of which bypass Drosha processing. Recently, three groups described for the first time a vertebrate microRNA pathway that bypasses Dicer cleavage. This mechanism was characterized with respect to the highly conserved vertebrate gene mir-451, for which Drosha processing yields a short (42 nucleotide) hairpin that is directly loaded into Ago2, the sole vertebrate "Slicer" Argonaute. Ago2-mediated cleavage of this hairpin yields a 30 nucleotide intermediate, whose 3' end is resected to generate the dominantly cloned ~23 nucleotide mature miR-451. Knowledge of this pathway provides an unprecedented tool with which to express microRNAs and small interfering RNAs in Dicer mutant cells. More generally, the mir-451 backbone constitutes a new platform for gene silencing that complements existing shRNA technology.

Open Access Article

Metformin against TGFβ-induced epithelial-to-mesenchymal transition (EMT): From cancer stem cells to aging-associated fibrosis

Sílvia Cufí, Alejandro Vazquez-Martin, Cristina Oliveras-Ferraros, Begoña Martin-Castillo, Jorge Joven and Javier A. Menendez
Pages 4461 - 4468
http://dx.doi.org/10.4161/cc.9.22.14048
Abstract | PDF

Transforming Growth Factor-b (TGFb) is a major driving force of the Epithelial-to-Mesenchymal (EMT) genetic program, which becomes overactive in the pathophysiology of many age-related human diseases.  TGFb-driven EMT is sufficient to generate migrating cancer stem cells by directly linking the acquisition of cellular motility with the maintenance of tumor-initiating (stemness) capacity.  Chronic diseases exhibiting excessive fibrosis can be caused by repeated and sustained infliction of TGFb-driven EMT, which increases collagen and extracellular matrix synthesis.  Pharmacological prevention and/or reversal of TGFb-induced EMT may therefore have important clinical applications in the management of cancer metastasis as well as in the prevention and/or treatment of end-state organ failures.  Earlier studies from our group have revealed that clinically-relevant concentrations of the biguanide derivative metformin, the most widely used oral agent to lower blood glucose concentration in patients with type 2 diabetes and metabolic syndrome, notably decreased both the self-renewal and the proliferation of trastuzumab-refractory breast cancer stem cell populations.  Given that: a.) tumor-initiating cancer stem cells display a significant enrichment in the expression of basal/mesenchymal or myoepithelial markers, including an increased secretion of TGFb; b.) metformin treatment impedes the ontogeny of generating the stem cell phenotype by transcriptionally repressing key drivers of the EMT genetic program (e.g. ZEB1, TWIST1, SNAIL2 [Slug], TGFbs), we recently hypothesized that prevention of TGFb-induced EMT might represent a common molecular mechanism underlying the anti-cancer stem cells and anti-fibrotic actions of metformin.  Remarkably, metformin exposure not only impedes TGFb-promoted loss of the epithelial marker E-cadherin in MCF-7 breast cancer cells but it prevents further TGF-induced cell scattering and accumulation of the mesenchymal marker vimentin in Madin-Darby canine kidney (MDCK) cells.  We now propose that metformin, by weakening the ability of TGFb signaling to fully induce mesenchymal cell states in a variety of pathological processes including fibrosis (e.g. chronic renal disease, non-alcoholic steatohepatitis, heart failure or sclerosis) and malignant progression (and likely by reducing TGFb-regulated inflammation and immune responses -inflamm-aging-), molecularly behaves as a bona fide anti-aging modality.




PERSPECTIVES

Open Access Article

The centrosome and mitotic spindle apparatus in cancer and senescence

Stephan Schmidt, Frank Essmann, Ion C. Cirstea, Fabian Kuck, Harish C. Thakur, Madhurendra Singh, Anja Kletke, Reiner U. Jänicke, Constanze Wiek, Helmut Hanenberg, M. Reza Ahmadian, Klaus Schulze-Osthoff, Bernd Nürnberg and Roland P. Piekorz
Pages 4469 - 4473
http://dx.doi.org/10.4161/cc.9.22.13684
Abstract | PDF

Altered cell division is associated with overproliferation and tumorigenesis, however, mitotic aberrations can also trigger antiproliferative responses leading to postmitotic cell cycle exit. Here, we focus on the role of the centrosome and in particular of centrosomal TACC (transforming acidic coiled coil) proteins in tumorigenesis and cellular senescence. We have compiled recent evidence that inhibition or depletion of various mitotic proteins which take over key roles in centrosome and kinetochore integrity and mitotic checkpoint function is sufficient to activate a p53-p21^WAF driven premature senescence phenotype. These findings have direct implications for proliferative tissue homeostasis as well as for cellular and organismal aging.

Open Access Article

Fasting and differential chemotherapy protection in patients

Lizzia Raffaghello, Fernando Safdie, Giovanna Bianchi, Tanya Dorff, Luigi Fontana and Valter D. Longo
Pages 4474 - 4476
http://dx.doi.org/10.4161/cc.9.22.13954
Abstract | PDF

Chronic calorie restriction has been known for decades to prevent or retard cancer growth, but its weight-loss effect and the potential problems associated with combining it with chemotherapy have prevented its clinical application. Based on the discovery in model organisms that short term starvation (STS or fasting) causes a rapid switch of cells to a protected mode, we described a fasting-based intervention that causes remarkable changes in the levels of glucose, IGF-I and many other proteins and molecules and is capable of protecting mammalian cells and mice from various toxins, including chemotherapy. Because oncogenes prevent the cellular switch to this stress resistance mode, starvation for 48 hours or longer protects normal yeast and mammalian cells and mice but not cancer cells from chemotherapy, an effect we termed Differential Stress Resistance (DSR). In a recent article, 10 patients who fasted in combination with chemotherapy, reported that fasting was not only feasible and safe but caused a reduction in a wide range of side effects accompanied by an apparently normal and possibly augmented chemotherapy efficacy. Together with the remarkable results observed in animals, these data provide preliminary evidence in support of the human application of this fundamental biogerontology finding, particularly for terminal patients receiving chemotherapy. Here we briefly discuss the basic, pre-clinical, and clinical studies on fasting and cancer therapy.




REVIEWS

Open Access Article

REST and CoREST are transcriptional and epigenetic regulators of seminal neural fate decisions

Irfan A. Qureshi, Solen Gokhan and Mark F. Mehler
Pages 4477 - 4486
http://dx.doi.org/10.4161/cc.9.22.13973
Abstract | PDF

Complementary transcriptional and epigenetic regulatory factors (e.g., histone and chromatin modifying enzymes and non-coding RNAs) regulate genes responsible for mediating neural stem cell maintenance and lineage restriction, neuronal and glial lineage specification, and progressive stages of lineage maturation. However, an overall understanding of the mechanisms that sense and integrate developmental signals at the genomic level and control cell type-specific gene network deployment has not emerged. REST and CoREST are central players in the transcriptional and epigenetic regulatory circuitry that is responsible for modulating neural genes, and they have been implicated in establishing cell identity and function, both within the nervous system and beyond it. Herein, we discuss the emerging context-specific roles of REST and CoREST and highlight our recent studies aimed at elucidating their neural developmental cell type- and stage-specific actions. These observations support the conclusion that REST and CoREST act as master regulators of key neural cell fate decisions.

Open Access Article

Same partners, different dance: Involvement of DNA replication proteins in centrosome regulation

James Knockleby and Hoyun Lee
Pages 4487 - 4491
http://dx.doi.org/10.4161/cc.9.22.14047
Abstract | PDF

Genome replication is the most fundamental element of the continuity of life. In eukaryotes, DNA replication is regulated by an elegant network of many different protein factors to ensure the timely and accurate copying of their entire genome once per cell cycle. The replication factors include the maintenance (MCM) proteins, Cdt1, Cdc6, Cdc7, Cdc45, and geminin. All of these proteins are involved in the regulation of DNA replication at the initiation step. Interestingly, recent studies have shown that some of these replication proteins also localize to the centrosome, often throughout the entire cell cycle. These centrosomally localized replication proteins appear to play essential roles in the regulation of centrosome biogenesis, suggesting that genome replication and segregation are regulated interdependently. In this review, we summarize and discuss the inter-dependent regulation played by some of the replication proteins.




REPORTS

Open Access Article

Differential role of CD133 and CXCR4 in renal cell carcinoma

Crescenzo D’Alterio and 22 others
Pages 4492 - 4500
http://dx.doi.org/10.4161/cc.9.22.13680
Abstract | PDF

The chemokine receptor CXCR4 and CD133, putative stem cell markers, were previously described in renal cancer (RCC). To evaluate the biological and prognostic role of CD133 and CXCR4 in RCC the expression  was  evaluated through qPCR and immunoblotting in human renal cancer cell lines (786-O, A498, ACHN, CAKI-1, SN12C, TK10, UO31) and patients biopsies.  Renal cancer cells and surgical biopsies expressed functional CXCR4 while CD133 was not detectable. CXCR4 and CD133 expression was then evaluated in 240 renal cancer patients through immunohistochemistry. CXCR4 and CD133 were low in 19.1% and 59.6%; intermediate in 20% and 17.9%; high in 60.8% and 22.5% of the cases, respectively. CXCR4 was overexpressed in tumours (p= 0.02), while CD133 was over expressed in healthy tissues (p= 0.04). Disease free survival Kaplan Meier plots suggest that prognosis is unfavourable for patients whose primary tumours express CXCR4 (p= 0.0199) but nor CD133 (p= 0.151) neither the concomitant CXCR4-CD133 (p=0.848) high expression affected prognosis. Analysis of prognostic factors suggests that age, clinical presentation, AJCC stage and CXCR4 had a significant prognostic value at the univariate analysis. The CXCR4 predictive ability was confirmed at  the multivariate analysis while no prognostic role was identified for CD133. Thus concomitant CD133 and CXCR4 evaluation  is not worth in RCC patient while the CXCR4 prognostic role encourage CXCR4 antagonists as promising therapeutic option.

Open Access Article

Sir2-dependent daughter-to-mother transport of the damaged proteins in yeast is required to prevent high stress sensitivity of the daughters

Dmitry A. Knorre, Irina A. Kulemzina, Maxim I. Sorokin, Sergey A. Kochmak, Natalya A. Bocharova, Sviatoslav S. Sokolov and Fedor F. Severin
Pages 4501 - 4505
http://dx.doi.org/10.4161/cc.9.22.13683
Abstract | PDF

The budding yeast Saccharomyces cerevisiae actively transports adverse factors (e.g. oxidized proteins) from the daughter to mother cells. The transport is believed to ensure that the daughters are born "young", thus preventing clonal senescence. Is this the only reason for the existence of such transport? We subjected yeast cells to various stress conditions and compared survival of mother and daughter cells. It was found that replicative age-dependent mortality under our experimental stress conditions was U-shaped: the resistance of both virgin daughters and old mother cells (more than three bud scars) was lower compared to the young mothers. SIR2 mutants were shown to fail to maintain the mother-daughter asymmetry. We showed that sir2 knockout affects the relative stress resistance in favor of the mothers. Thus, daughter cells are more vulnerable to a variety of stresses than the young mothers, and Sir2-dependent transport of the adverse factors acts to equalize the resistance.

Open Access Article

FBG1 is a promiscuous ubiquitin ligase that sequesters APC2 and causes S-phase arrest

Hsiang Wen, Namhun Kim, Ernesto J. Fuentes, Adam Mallinger, Pedro Gonzalez-Alegre and Kevin A. Glenn
Pages 4506 - 4517
http://dx.doi.org/10.4161/cc.9.22.13743
Abstract | PDF

During cell proliferation, protein degradation is strictly regulated by the cell cycle and involves two complementary ubiquitin ligase complexes, the SCF (Skp, Cullin, F-box) and APC/C (Anaphase Promoting Complex/Cyclosome) ubiquitin ligases. SCF ligases are constitutively active and generally target only proteins after they have been selected for degradation, usually by phosphorylation. In contrast, APC/C complexes are themselves activated by phosphorylation and their substrates contain a targeting signal known as degron, a consensus amino acid sequence such as a D-Box. SCF complexes degrade proteins during the G1 phase. However, as DNA synthesis begins, the SCF complexes are degraded and APC/C complexes are activated. APC-2, a protein crucial to cell division, initiates anaphase by triggering the degradation of multiple proteins. This study explores an unexpected interaction between APC-2 and SCF^FBG1. We found that FBG1 is a promiscuous ubiquitin ligase with many partners. Immunoprecipitation experiments demonstrate that FBG1 and APC2 interact directly. Mutagenesis-based experiments show that this interaction requires a D-Box found within the FBG1 F-box domain. Unexpectedly, we demonstrate that co-expression with FBG1 increases total APC2 levels. However, free APC2 is decreased, inhibiting cell proliferation. Finally, FACS analysis of cell populations expressing different forms of FBG1 demonstrate that this ubiquitin ligase induces S-phase arrest, illustrating the functional consequences of the interaction described. In summary, we have discovered a novel APC2 inhibitory activity of FBG1 independent from its function as ubiquitin ligase, providing the basis for future studies of FBG1 in aging and cancer.

Open Access Article

Vessel destruction by tumor-targeting Salmonella typhimurium A1-R is enhanced by high tumor vascularity

Fang Liu, Lei Zhang, Robert M. Hoffman and Ming Zhao
Pages 4518 - 4524
http://dx.doi.org/10.4161/cc.9.22.13744
Abstract | PDF

Our laboratory has previously developed a tumor-targeting double-auxotrophic mutant of Salmonella typhimurium termed A1-R. The present report demonstrates that S. typhimurium A1-R destroys tumor blood vessels and this is enhanced in tumors with high vascularity. Red fluorescent protein (RFP)-expressing Lewis lung cancer cells (LLC-RFP) were transplanted subcutaneously in the ear, back skin, and footpad of nestin-driven green fluorescent protein (ND-GFP) transgenic nude mice, which selectively express GFP in nascent blood vessels. Color-coded in vivo imaging demonstrated that the LLC-RFP ear tumor had the highest cell density and the footpad tumor had the least with the ear tumor having more abundant blood vessels than that on the back or footpad. The tumor-bearing mice were treated with A1-R bacteria via tail-vein injection. Tumors in the ear were the earliest responders to bacterial therapy and hemorrhaged severely the day after A1-R administration. Tumors growing in the back were the second fastest responders to bacterial treatment and appeared necrotic 3 days after A1-R administration. Tumors growing in the footpad had the least vascularity and were the last responders to A1-R. Therefore, tumor vascularity correlated positively with tumor efficacy of A1-R.  The present study suggests that bacteria efficacy on tumors involved vessel destruction which depends on the extent of vascularity of the tumor.

Open Access Article

Dual association by TFAP2A during activation of the p21cip/CDKN1A promoter

Angelo G. Scibetta, Ping-Pui Wong, KaYi V. Chan, Monica Canosa and Helen C. Hurst
Pages 4525 - 4532
http://dx.doi.org/10.4161/cc.9.22.13746
Abstract | PDF

The cyclin-dependent kinase inhibitor p21cip/CDKN1A is induced  to promote growth arrest in response to a variety of stimuli in normal cells and loss of correct regulation of this gene is frequently observed in cancer. In particular, the upregulation of CDKN1A by p53 is considered to be a central mechanism of tumour suppression. Other transcription factors with tumour suppressor activity can also regulate CDKN1A, including the developmentally regulated factor, TFAP2A. Here we identify a novel AP-2 binding site within the proximal promoter of the CDKN1A gene and show this is required for optimal, p53-independent expression of p21cip/CDKN1A. We further describe a non-tumourgenic breast epithelial cell line model to study the role of endogenous TFAP2A and p53 in the control of drug-induced p21cip expression using ChIP. Maximal expression of CDKN1A requires TFAP2A which binds to two regions of the promoter: the proximal region where the AP-2 site lies and upstream near the major p53 binding site. The pattern of binding alters with time post-induction, with the proximal, p53-independent site becoming more important at later stages of p21cip induction. This pattern of promoter interaction by TFAP2A is distinct from that seen for the TFAP2C family member which represses CDKN1A expression.

Open Access Article

Activation of Cdk2/Cyclin E complexes is dependent on the origin of replication licensing factor Cdc6 in mammalian cells

Cara L. Lunn, John C. Chrivia and Joseph J. Baldassare
Pages 4533 - 4541
http://dx.doi.org/10.4161/cc.9.22.13789
Abstract | PDF

Cyclin E-associated CDK2 activity is required for the initiation of DNA synthesis in human cells. CDK2 activity is tightly regulated; CDK2 must be in the nucleus, bound to a cyclin, phosphorylated on T160, and dephosphorylated on T14/Y15 for complete kinase activation. Nuclear localization exposes CDK2 to activating enzymes (CAK, Cdc25A) in stimulated cells. Previous studies from our lab indicate CDK2 nuclear localization and cyclin E co-expression are insufficient to cause CDK2 activation or T160 phosphorylation in stimulated IIC9 cells; these activities still require serum stimulation and ERK kinase activity. Recent studies have implicated a role for origin of replication (ORC) licensing proteins in the activation of G1/S Cdks. In this study, we show that CDK2 associates with chromatin and Cdc6 in an ERK-dependent manner following stimulation of IIC9 CHEF cells. We show that nuclear-localized CDK2 (CDK2-NLS) ectopically expressed with cyclin E requires mitogenic stimulation and ERK activation for chromatin association, in addition to previously shown kinase activation and T160 phosphorylation in IIC9 cells. Additionally, we show that expression of Cdc6 in stimulated IIC9 cells treated with ERK inhibitor rescues CDK2-NLS chromatin association, kinase activation, and T160 phosphorylation. From the above data, we deduce ERK-dependent CDK2 activation is due in part to ERK-dependent Cdc6 expression. To examine the role of Cdc6 directly in stimulated primary human fibroblasts, we used RNA interference to attenuate the expression of Cdc6. We show that Cdc6 expression is required for CDK2 chromatin association and kinase activation in stimulated primary human fibroblasts. Additionally, we show that Cdc6 expression is required for the initiation of DNA synthesis and S phase entry in stimulated primary human fibroblasts. Ultimately, this data implicates Cdc6 expression as an important mitogen-induced mechanism in the activation of CDK2/cyclin E, the initiation of DNA synthesis, and the regulation of G1-S phase progression.

Open Access Article

Inhibition of Cot1/Tlp2 oncogene in AML cells reduces ERK5 activation and upregulates p27^Kip1 concomitant with enhancement of differentiation and cell cycle arrest induced by silibinin and 1,25-dihydroxyvitamin D₃

Xuening Wang, Elzbieta Gocek, Victoria Novik, Jonathan S. Harrison, Michael Danilenko and George P. Studzinski
Pages 4542 - 4551
http://dx.doi.org/10.4161/cc.9.22.13790
Abstract | PDF

Acute myelogenous leukemia (AML) is a disease characterized by dysregulated cell proliferation associated with impaired cell differentiation, and current treatment regimens rarely save the patient. Thus, new mechanism-based approaches are needed to improve prognosis of this disease. We have investigated in preclinical studies the potential anti-leukemia use of the plant-derived polyphenol Silibinin (SIL) in combination with 1,25-dihydroxyvitamin D3 (1,25D). Although most of the leukemic blasts ex vivo responded by differentiation to treatment with this combination, the reasons for the absence of SIL-1,25D synergy in some cases were unclear. Here we report that failure of SIL to enhance the action of 1,25D is likely due to the SIL-induced increase in the activity of differentiation-antagonizing cell components, such as ERK5. This kinase is under the control of Cot1/Tlp2, and inhibition of Cot1 activity by a specific pharmacological inhibitor 4-(3-chloro-4-fluorophenylamino)-6-(pyridin-3-yl-methylamino-3-cyano-[1–7]-naphthyridine, or by Cot1 siRNA, increases the differentiation by SIL/1,25D combinations. Conversely, over-expression of a Cot1 construct increases the cellular levels of P-ERK5, and SIL/1,25D-induced differentiation and cell cycle arrest are diminished. It appears that reduction in ERK5 activity by inhibition of Cot1 allows SIL to augment the expression of 1,25D-induced differentiation promoting factors and cell cycle regulators such as p27^Kip1, which leads to cell cycle arrest. This study shows that in some cell contexts SIL/1,25D can promote expression of both differentiation-promoting and differentiation-inhibiting genes, and that the latter can be neutralized by a highly specific pharmacological inhibitor, suggesting a potential for supplementing treatment of AML with this combination of agents.

Open Access Article

Perturbation of Spc25 expression affects meiotic spindle organization, chromosome alignment and spindle assembly checkpoint in mouse oocytes.

Shao-Chen Sun, Seung-Eun Lee, Yong-Nan Xu and Nam-Hyung Kim
Pages 4552 - 4559
http://dx.doi.org/10.4161/cc.9.22.13815
Abstract | PDF

Spc25 is a component of the Ndc80 complex which consists of Ndc80, Nuf2, Spc24, and Spc25. Previous work has shown that Spc25 is involved in regulation of kinetochore microtubule attachment and the spindle assembly checkpoint in mitosis. The roles of Spc25 in meiosis remain unknown. Here, we report its expression, localization and functions in mouse oocyte meiosis. The Spc25 mRNA level gradually increased from the GV to MI stage, but decreased by MII during mouse oocyte meiotic maturation. Immunofluorescent staining showed that Spc25 was restricted to the germinal vesicle, and associated with chromosomes during all stages after GVBD. Overexpression of Spc25 by mRNA injection resulted in oocyte meiotic arrest, chromosome misalignment and spindle disruption. Conversely, Spc25 RNAi by siRNA injection resulted in precocious polar body extrusion and caused severe chromosome misalignment and aberrant spindle formation. Our data suggest that Spc25 is required for chromosome alignment, spindle formation, and proper spindle checkpoint signaling during meiosis.

Open Access Article

Stapled peptides in the p53 pathway: Computer simulations reveal novel interactions of the staples with the target protein

Thomas Leonard Joseph, David Lane and Chandra S. Verma
Pages 4560 - 4568
http://dx.doi.org/10.4161/cc.9.22.13816
Abstract | PDF

Atomistic simulations of a set of stapled peptides derived from the transactivation domain of p53 (designed by Verdine & colleagues, JACS 2007 129:2456) and complexed to MDM2 reveal that the good binders are uniquely characterized by higher helicity and by extensive interactions between the hydrocarbon staples and the MDM2 surface; in contrast the poor binders have reduced helicity and their staples are mostly solvent exposed. Our studies also find that the best binders can also potentially inhibit MDMX with similar affinities, suggesting that such stapled peptides can be evolved for dual inhibition with therapeutic potential.

Open Access Article

Transcriptional regulation of HIV-1 gene expression by p53

Ruma Mukerjee, Pier Paolo Claudio, J. Robert Chang, Luis Del Valle and Bassel E. Sawaya
Pages 4569 - 4578
http://dx.doi.org/10.4161/cc.9.22.13836
Abstract | PDF

Several reports have pointed to the negative involvement of p53 in transcriptional regulation of the human immunodeficiency virus type 1 long terminal repeat (HIV-1 LTR). However, the mechanisms of this negative effect remain unclear. In here, we showed that over expression of p53 wild type prevented the phosphorylation of serine 2 in the carboxyl terminal domain (CTD) of RNA polymerase II. As a result of this inhibition, p53 stalled transcriptional elongation on the HIV-1 LTR leading to a significant reduction of HIV-1 replication in primary microglia and astrocytes. However, despite the delay/pause caused by p53, viral transcription and replication decreased and then salvaged. These studies suggest that the negative effect of p53 is alleviated by a third factor. In this regard, our Preliminary Data point to the involvement of the Pirh2 protein in p53 inhibition. Therefore, we suggest that p53 may be a novel therapeutic target for the inhibition of HIV-1 gene expression and replication and the treatment of AIDS.

Open Access Article

MicroRNA-449a levels increase by several orders of magnitude during mucociliary differentiation of airway epithelia

Muriel Lizé, Christian Herr, Alexander Klimke, Robert Bals and Matthias Dobbelstein
Pages 4579 - 4583
http://dx.doi.org/10.4161/cc.9.22.13870
Abstract | PDF

MicroRNAs of the miR-34/449 family mediate cell cycle arrest and tumor suppression.  Here we show that the expression of microRNA miR-449a, unlike its paralogue miR-34a, is highly tissue specific and largely restricted to pulmonary and testicular tissue. MiR-449a levels in the murine lung are particularly high shortly before and after birth, coinciding with terminal differentiation of lung epithelia. Strikingly, miR-449a is upregulated by more than 1000-fold when epithelial cells from human airways are lifted from a liquid environment to air, allowing them to undergo mucociliary differentiation. The induction of miR-449a occurs in parallel to its host gene CDC20B and the transcription factor FoxJ1. Exposure to tobacco smoke induces a moderate further increase in the levels of miR-449a, and also miR-34a, in differentiated airway epithelia. We propose that miR-449a can serve as an exquisitely sensitive and specific biomarker for the differentiation of bronchial epithelia. Moreover, miR-449a may actively promote mucociliary differentiation through its ability to block cell cycle progression, and it may conribute to a first line of defence against genotoxic stress by its proapoptotic functions.

Open Access Article

Interaction of regulators Mdm2 and Mdmx with transcription factors p53, p63 and p73

Michal Zdzalik, Katarzyna Pustelny, Sylwia Kedracka-Krok, Krzysztof Huben, Aleksandra Pecak, Benedykt Wladyka, Stefan Jankowski, Adam Dubin, Jan Potempa and Grzegorz Dubin
Pages 4584 - 4591
http://dx.doi.org/10.4161/cc.9.22.13871
Abstract | PDF

The negative regulation of p53, a major human tumor suppressor, by Mdm2 and Mdmx is crucial for the survival of a cell, whereas its aberrant function is a common feature of cancer.  Both Mdm proteins act through the spatial occlusion of the p53 transactivation (TA) domain and by the ubiquitination of p53, resulting in its degradation.  Two p53 homologues, p63 and p73, have been described in humans.  Unlike p53, these proteins regulate developmental processes rather than genome stability.  Both p63 and p73 contain TA domains homologous to that of p53, but relatively little is known about their regulation by Mdm2 or Mdmx.  Here, we present a detailed characterization of the interaction of Mdm2 and Mdmx with the TA domains of p63 and p73. Earlier reports of Mdm2 and Mdmx interactions with p73 are substantiated by the detailed quantitative characterization reported in this study. Most importantly, earlier contradictions concerning the presumed interaction of the Mdm proteins with p63 are convincingly resolved and for the first time, the affinities of these interactions are determined.  Finally, the contribution of these findings to our understanding of the physiological role of these interactions is discussed.

Open Access Article

Mitotic DNA damage targets the Aurora A/TPX2 complex

Payal Bhatia, Mirco Menigatti, Michele Brocard, Simon J. Morley and Stefano Ferrari
Pages 4592 - 4599
http://dx.doi.org/10.4161/cc.9.22.13665
Abstract | PDF

We have previously shown that the DNA damage-induced G2 arrest is contributed by inhibition of Aurora A (AurA) and that transduction of active AurA into arrested cells allows bypassing the block through reactivation of CDK1. In this study, we investigated the mechanism of DNA damage-induced AurA inhibition. We provide evidence that ionizing radiation (IR) administered in mitosis, a time when AurA protein and enzymatic activity reach peak levels, impairs interaction with the partner TPX2, leading to inactivation of the kinase through dephosphorylation of AurA T-loop residue, T₂₈₈. We find that decreased AurA-TPX2 complex formation in response to irradiation results from reduced cellular levels of TPX2, an effect that is both contributed by increased APC/CDH1-dependent protein degradation and decreased translation of TPX2 mRNA.




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Open Access Article

Histone modifications regulate the norepinephrine transporter gene

Richard Bayles, Emma K. Baker, Nina Eikelis, Assam El-Osta and Gavin Lambert
Pages 4600 - 4601
http://dx.doi.org/10.4161/cc.9.22.13888
PDF