Table of Contents

Volume 8, Issue 3

  February 1, 2009

  Pages 335 - 509

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 Issue Catalogue (MARC XML)

NEWS & VIEWS

Open Access Article

Inhibiting PI-3-K for glioma therapy

Garth Powis, Nathan T. Ihle and W.K. Alfred Yung
Pages 335 - 337
http://dx.doi.org/10.4161/cc.8.3.7884
PDF

Open Access Article

Do checkpoint kinases contribute to HIV-1–mediated apoptosis?

Helin Vakifahmetoglu and Boris Zhivotovsky
Pages 335 - 337
http://dx.doi.org/10.4161/cc.8.3.7885
PDF

Open Access Article

Plk1, a negative regulator of p21?

Xiaoqi Liu
Pages 335 - 337
http://dx.doi.org/10.4161/cc.8.3.7886
PDF

Open Access Article

Checkpoint “Madness”

Michael Brandeis
Pages 335 - 337
http://dx.doi.org/10.4161/cc.8.3.7892
PDF




EXTRA VIEWS

Open Access Article

The spindle assembly checkpoint in Caenorhabditis elegans: One who lacks Mad1 becomes mad one

Risa Kitagawa
Pages 338 - 344
http://dx.doi.org/10.4161/cc.8.3.7448
Abstract | PDF

The spindle assembly checkpoint (SAC) monitors the microtubule attachment status of the kinetochore and arrests cells before anaphase until all pairs of sister kinetochores achieve bipolar attachment of microtubules, thereby ensuring faithful chromosome transmission. The evolutionarily conserved coiled-coil protein MAD1 has been implicated in the SAC signaling pathway. MAD1 forms a complex with another SAC component MAD2 and specifically localizes to unattached kinetochores to facilitate efficient binding of MAD2 to its target, CDC20, the mitotic substrate-specific activator of the anaphase promoting complex or cyclosome (APC/C). Thus, MAD1 connects 2 sequential events in the SAC signaling pathway – recognition of unattached kinetochores and inhibition of APC/C activity. However, the molecular mechanisms by which it specifically localizes to unattached kinetochores are largely unknown. Studies in multicellular organisms have revealed the role of MAD1 in development and tumor suppression, but the precise time at which MAD1 activity is required is unknown. Investigation of cellular and organismic functions of MAD1 in the simple multicellular organism C. elegans identified functional interactors of MAD1 in both kinetochore-oriented SAC signaling and kinetochore-independent cell cycle regulation. Studying the function of SAC components in C. elegans provides a new molecular insight into the SAC-regulated cell cycle progression in a context of a multicellular organism.

Open Access Article

Subnuclear compartmentalization of transiently expressed polyadenylated pri-microRNAs: Processing at transcription sites or accumulation in SC35 foci

Jan M. Pawlicki and Joan A. Steitz
Pages 345 - 356
http://dx.doi.org/10.4161/cc.8.3.7494
Abstract | PDF

MicroRNAs (miRNAs) are small, noncoding RNAs that posttranscriptionally regulate expression of their target messenger RNAs. We recently demonstrated that primary miRNA transcripts (pri-miRNAs) retained at transcription sites are processed with enhanced efficiency, suggesting that pri-miRNA processing is coupled to transcription in mammalian cells. We also observed that transiently expressed pri-miRNAs accumulate in nuclear foci with splicing factor SC35 and Microprocessor components, Drosha and DGCR8. Here, we show that pri-miRNAs containing a self-cleaving hepatitis delta ribozyme accumulate in the nucleoplasm after release from their transcription sites, but are not efficiently processed. Pri-miRNAs with ribozyme-generated 3′ ends do not localize to SC35-containing foci, whereas cleaved and polyadenylated pri-miRNA transcripts with or without the pre-miRNA hairpin do. Pri-miRNA/SC35 foci contain a number of proteins normally associated with SC35 domains, including ASF/SF2, PABII, and the prolyl isomerase, Pin1. In contrast, RNA polymerase II and PM/Scl-100 do not strongly colocalize with pri-miRNAs in SC35-containing foci. These data argue that pri-miRNA/SC35-containing foci are not major sites of pri-miRNA processing and that pri-miRNA processing is coupled to transcription. We discuss the implications of our findings relative to recent insights into miRNA biogenesis, mRNA metabolism, and the nuclear organization of gene expression.

Open Access Article

Mitotic crisis: The unmasking of a novel role for RPA

Rachel William Anantha and James A. Borowiec
Pages 357 - 361
http://dx.doi.org/10.4161/cc.8.3.7496
Abstract | PDF

Mitotic DNA damage is a constant threat to genomic integrity, yet understanding of the cellular responses to this stress remain incomplete. Recent work by Anantha et al. (PNAS 2008; 105:12903–8) has found surprising evidence that RPA, the primary eukaryotic single-stranded DNA-binding protein, can stimulate the ability of cells to exit mitosis into a 2N G₁ phase. Along with providing additional discussion of this study, we review evidence suggesting that DNA replication and repair factors can modulate mitotic transit by acting through Polo-like kinase-1 (Plk1) and the centrosome. “A crisis unmasks everyone.”—Mason Cooley, US aphorist.

Open Access Article

Targeting CDC37: An alternative, kinase-directed strategy for disruption of oncogenic chaperoning

Jennifer R. Smith and Paul Workman
Pages 362 - 372
http://dx.doi.org/10.4161/cc.8.3.7531
Abstract | PDF

Interest in HSP90 inhibitors has grown rapidly in the last decade.  The heightened dependence of malignant cells on molecular chaperones to maintain multiple oncogenic signalling pathways gives HSP90 broad anti-cancer appeal.  New HSP90-directed agents are continually emerging, several of which are under clinical evaluation. In parallel, dissection of the functional mechanism of the chaperone system has emphasised the importance of cochaperones that regulate HSP90.  As we begin to fully elucidate the roles of these HSP90 accessory proteins, it is becoming increasingly clear that they too have potential as additional routes to disrupt chaperone activity.  CDC37, a predominantly kinase client-associated cochaperone that promotes malignant transformation, has particular promise.  Recently, we demonstrated that, similar to HSP90 inhibitors, siRNA-mediated CDC37 silencing caused the proteasomal degradation of kinase client proteins and inhibited the proliferation of cancer cells.  Importantly, depleting CDC37 does not induce the unwanted, antiapoptotic heat shock response that is characteristic of pharmacologic HSP90 inhibition.  Furthermore, CDC37 silencing sensitises cancer cells to HSP90 inhibitors by potentiating kinase client depletion and the induction of apoptosis, suggesting that simultaneously modulating HSP90 and CDC37 could be beneficial.  Here we discuss the therapeutic possibilities of targeting CDC37 for cancer treatment in light of recent significant findings.




PERSPECTIVES

Open Access Article

In vivo measurement of signaling cascade dynamics

Megan N. McClean, Pascal Hersen and Sharad Ramanathan
Pages 373 - 376
http://dx.doi.org/10.4161/cc.8.3.7450
Abstract | PDF

Genetic and biochemical studies yield information about the component proteins and interactions involved in a cellular signaling pathway.  However this parts inventory often does not immediately reveal the in vivo signal processing capabilities and function of the pathway.  Signaling pathways are complex systems with dynamic behavior and a systems level approach is needed to understand the physiological roles they play within in the cell.  We recently used such an approach to measure the signal processing behavior of the budding yeast HOG MAP kinase pathway in response to precisely varied temporal stimuli controlled with a microfluidic device.  Despite being a well-studied pathway with well-known components the signaling dynamics and biochemical parameters of this pathway were not known.   Our approach allowed us to characterize the pathway’s in vivo signal processing and put bounds on all of the in vivo reaction rates.  The experimental and theoretical techniques used in our study are general and can be applied to understanding other signaling pathways in a range of biological systems.

Open Access Article

How epigenetics can explain human metastasis: A new role for microRNAs

Amaia Lujambio and Manel Esteller
Pages 377 - 382
http://dx.doi.org/10.4161/cc.8.3.7526
Abstract | PDF

The metastatic process is characterized by the dissemination of tumoral cells throughout the bloodstream to distal sites, where these transformed cells proliferate and give rise to secondary tumors, which are the principal cause of mortality in cancer patients. In recent years, a significant number of metastasis-related genes have been described, such as cadherins, laminins, heparan sulfates, and protease and angiogenesis inhibitors, among others. However, the mechanisms by which these genes are altered in metastasis remain unclear, since genetic alterations occur rarely, despite their widespread downregulation. Epigenetic alterations, and specifically CpG island hypermethylation-associated silencing, can potentially explain the aberrant expression of many of these genes. The disruption of histone modifiers and chromatin-remodeling factors also contributes to the alteration of metastasis genes. Members of a new class of regulatory RNAs, microRNAs (miRNAs), have an important role in cancer and metastasis and are also regulated by epigenetic mechanisms in both malignancies. As we gain insight into the epigenetic mechanisms orchestrating all the metastatic steps, we broaden the therapeutic possibilities of epigenetic drugs, such as DNA demethylating drugs and histone deacetylase inhibitors, which can act upon metastasis-related genes and miRNAs, restoring their expression. In this review, the latest studies relating cancer epigenetics and metastasis are analyzed, and we emphasize the importance of miRNAs and their epigenetic regulation in tumoral progression.

Open Access Article

A new link between epigenetic progenitor lesions in cancer and the dynamics of signal transduction

Winston Timp, Andre Levchenko and Andrew P. Feinberg
Pages 383 - 390
http://dx.doi.org/10.4161/cc.8.3.7542
Abstract | PDF

Our recent study of the mechanism by which an epigenetic alteration, loss of imprinting (LOI) of Igf2, increases tumor risk, revealed a strong relationship between IGF2 dosage, the dynamics of signaling along the IGF2 axis, cell proliferation and tumor risk.¹ Colon epithelia in a mouse model with LOI of Igf2 showed increased sensitivity to IGF1R blockade and abrogation of premalignant lesion development in LOI(+) mice. These results are consistent with the epigenetic progenitor model of cancer,² in which epigenetic changes precede and heighten risk of cancer in response to oncogenic mutations. Thus, one can envision a highly targeted and focused chemoprevention strategy targeted to signaling pathways in nonmalignant cells that have undergone an epigenetic lesion, rather than a broad approach toward reversing epigenetic lesions that may have unintended consequences affecting the whole epigenome.

Open Access Article

Autophagy in mineralizing tissues: Microenvironmental perspectives

Vickram Srinivas, Jolene Bohensky, Adam M. Zahm and Irving M. Shapiro
Pages 391 - 393
http://dx.doi.org/10.4161/cc.8.3.7545
Abstract | PDF

Chondrocytes in the growth plate and articular cartilage and osteocytes subsumed in Haversian bone exist in environmental niches that are characterized by a limited oxygen supply. In these tissues, cells display a hitherto unrecognized state in which there is evidence of autophagy. The autophagic condition serves to promote cell survival. When the response is triggered, the cell cannibalizes itself to generate energy; if extended, then it can activate Type II apoptosis. We opine that survival is dependent on niche conditions and regulated by crosstalk between mTOR, AMPK and HIF-1 and HIF-2.  Recent studies suggest that HIF-2 is a potent regulator of chondrocyte autophagy and that this protein acts as a brake to the stimulatory function of HIF-1. Accordingly, the oxemic state of the tissue, its nutrient supply as well as the energetic state of the cells regulates autophagic flux. From a clinical viewpoint, it may be possible to enhance skeletal cell survival through drugs that modulate the autophagic state and prevent the induction of apoptosis.


Open Access Article

The miR-302-367 cluster as a potential stemness regulator in ESCs

Alicia Barroso-del Jesus, Gema Lucena-Aguilar and Pablo Menendez
Pages 394 - 398
http://dx.doi.org/10.4161/cc.8.3.7554
Abstract | PDF

Increasing experimental evidence suggests an important role of miRNAs in embryonic stem cell (ESC) biology. The miR-302-367 cluster is exclusively expressed at high levels in ESCs but not in either somatic stem cells or adult/embryonic differentiated cells. The human miR-302-367 gene structure has been recently described and its promoter has been identified, characterized and functionally validated in human stem cells. The miR-302-367 promoter activity depends on the ontogeny and hierarchical cellular stage. The miR-302-367 promoter is transcriptionally regulated by the ESC-specific transcription factors Oct3/4, Sox2 and Nanog and, its activity restricted to the ESC compartment. Functionally, this cluster regulates cell cycle in ESCs promoting self-renewal and pluripotency, therefore representing a master regulator in the maintenance of hESC stemness. We envision this data may open up new avenues to investigate the transcriptional regulators upstream miR-302-367 cluster and to dissect the complex interplay by which this miR-302-367 cluster integrates in the molecular network conferring pluripotency to ESCs. In this perspective, we summarize recent progress in the genomic and functional characterization of the miR-302-367 cluster and discuss its potential as a stemness determinant.

Open Access Article

Handcuff for sisters: A new model for sister chromatid cohesion

Nenggang Zhang and Debananda Pati
Pages 399 - 402
http://dx.doi.org/10.4161/cc.8.3.7586
Abstract | PDF

High fidelity chromosome transmission requires the pairing of sister chromatids by a group of conserved proteins called cohesins during S-phase. Sister chromatid cohesion is maintained until anaphase onset.  Presently, there are two sets of models of cohesin complex popular in the cohesin biology field: one model predicts that single cohesin rings entrap both sister chromatids, and the other model proposes that cohesin complexes associate with each sister chromatid and become paired during DNA replication.  It is the first model that currently predominate the field -- in part because prior efforts failed to detect higher order cohesin-cohesin interactions.  However, the static configuration and size limitation of the one ring embrace model are the major limitations of the embrace model, and cannot explain various functions of cohesin in DNA replication, DNA repair, and gene expression.  In a recent study published by Zhang et al in the Journal of Cell Biology describes a two-ring handcuff model for the cohesin complex, and provides new information regarding how sister chromatid cohesion and separation are achieved in vertebrate cell systems.




REVIEWS

Open Access Article

PI3K/mTORC1 activation in hamartoma syndromes: Therapeutic prospects

Vera P. Krymskaya and Elena A. Goncharova
Pages 403 - 413
http://dx.doi.org/10.4161/cc.8.3.7555
Abstract | PDF

Dysregulated activity of phosphatidylinositol 3-kinase (PI3K) and mammalian target of rapamycin complex 1 (mTORC1) is characteristic feature of hamartoma syndromes. Hamartoma syndromes, dominantly inherited cancer predisposition disorders, affect multiple organs and are manifested by benign tumors consisting of various cell types native to the tissues in which they arise. In the past few years, three inherited hamartoma syndromes, Cowden syndrome (CS), tuberous sclerosis complex (TSC) syndrome, and Peutz-Jeghens syndrome (PJS), have all been linked to a common biochemical pathway: the hyperactivation of PI3K/mTORC1 intracellular signaling. Three tumor suppressors, PTEN (phosphatases and tensin homolog), tuberous sclerosis complex TSC1/TSC2, and LKB1, are negative regulators of PI3K/mTORC1 signaling; disease-related inactivation of these tumor suppressors results in the development of PTEN-associated hamartoma syndromes, TSC, and PJS, respectively. The goal of this review is to provide a roadmap for navigating the inherently complex regulation of PI3K/mTORC1 signaling while highlighting the progress that has been made in elucidating the cellular and molecular mechanisms of hamartoma syndromes and identificating potential therapeutic targets for their treatment. Importantly, because the PI3K/mTORC1 pathway is activated in the majority of common human cancers, the identification of novel molecular target(s) for the treatment of hamartoma syndromes may have a broader translational potential, and is critically important not only for therapeutic intervention in hamartoma disorders, but also for the treatment of cancers.

Open Access Article

Multiple roles for Pur-α in cellular and viral regulation

Martyn K. White, Edward M. Johnson and Kamel Khalili
Pages 414 - 420
http://dx.doi.org/10.4161/cc.8.3.7585
Abstract | PDF

Pur-alpha is a ubiquitous multifunctional protein that is strongly conserved throughout evolution, binds to both DNA and RNA and functions in the initiation of DNA replication, control of transcription and mRNA translation.  In addition, it binds to several cellular regulatory proteins including the retinoblastoma protein, E2F-1, Sp1, YB-1, cyclin T1/Cdk9 and cyclin A/Cdk2.  These observations and functional studies provide evidence that Purα is a major player in the regulation of the cell cycle and oncogenic transformation.  Purα also binds to viral proteins such as the large T-antigen of JC virus (JCV) and the Tat protein of human immunodeficiency virus-1 (HIV-1) and plays a role in the cross-communication of these viruses in the opportunistic polyomavirus JC (JCV) brain infection, progressive multifocal leukoencephalopathy (PML).  The creation of transgenic mice with inactivation of the PURA gene that encodes Purα has revealed that Purα is critical for postnatal brain development and has unraveled an essential role of Purα in the transport of specific mRNAs to the dendrites and the establishment of the postsynaptic compartment in the developing neurons.  Finally, the availability of cell cultures from the PURA knockout mice has allowed studies that have unraveled a role for Purα in DNA repair.




REPORTS

Open Access Article

TAp73α binds the kinetochore proteins Bub1 and Bub3 resulting in polyploidy

Patrizia Vernole, Michael H. Neale, Daniela Barcaroli, Eliana Munarriz, Richard Knight, Richard Tomasini, Tak W. Mak, Gerry Melino and Vincenzo De Laurenzi
Pages 421 - 429
http://dx.doi.org/10.4161/cc.8.3.7623
Abstract | PDF

Aneuploidy is a characteristic of most solid tumors, often associated with negative prognosis. It can arise from two principal mechanisms: from a tetraploid intermediate state, or directly from errors at cell division. The control of cell division, crucial to maintain genomic stability, is still poorly understood in its relationship to aneuploidy. Here we show that the TAp73α  isoform induces polyploidy when over-expressed. This is possibly due to the interaction of TAp73α with kinetochore-related proteins leading to the alteration of mitotic checkpoint abilities. TAp73α but not p53 or any of the other p73 isoforms binds Bub1 and Bub3. Since TAp73α  is frequently over-expressed in cancer, this interaction may contribute to the aneuploidy observed in cancer progression. Our results suggest a novel molecular mechanism leading to aneuploidy involving interference of TAp73α with Bub1 and Bub3 resulting in an altered mitotic checkpoint.

Open Access Article

Mdm2 and PCAF increase Chk2 ubiquitination and degradation independently of their intrinsic E3 ligase activities

Elizabeth M. Kass, Masha V. Poyurovsky, Yan Zhu and Carol Prives
Pages 430 - 437
http://dx.doi.org/10.4161/cc.8.3.7624
Abstract | PDF

We have previously shown that Chk2 stability, which is increased after DNA damage in some cells, is regulated in part through phosphorylation at S456 and that a mutant  Chk2 (S456A)  is hyperubiquitinated and turned over more rapidly than wild-type Chk2. (Kass J. Biol Chem 2007)  Here we show that the S456A mutant preferentially binds to Mdm2 compared to wild-type Chk2 and down-regulation of Mdm2 with siRNA can rescue the observed destabilization of S456A Chk2 following DNA damage.  Ubiquitination of wild-type Chk2 is increased by co-expression of Mdm2, and increasing amounts of Mdm2 enhance degradation of both wild-type and mutant S456A Chk2. Surprisingly, however, an E3 ligase defective version of Mdm2 (ΔC7) also increases ubiquitination and degradation of Chk2 when transfected into cells, suggesting that the ability of Mdm2 to affect Chk2 turnover is independent of its intrinsic E3 ligase function.  Recently the acetyltransferase PCAF was shown to have intrinsic E3 ligase activity against itself and Mdm2.  We found that PCAF interacts with both Mdm2 and Chk2 and that PCAF increases Chk2 ubiquitination and degradation.  Interestingly as well, a PCAF deletion mutant that lacks E3 ligase activity towards Mdm2 also enhances Chk2 degradation. We hypothesize that Mdm2 and PCAF may function as part of a multi-subunit E3 complex in their regulation of Chk2 turnover.

Open Access Article

Pro-apoptotic function of checkpoint kinase-2 in syncytia elicited by the HIV-1 envelope

Claire Séror, Syed Qasim Raza, Fanélie Brottes, Frédéric Subra, Mauro Piacentini, Jean-Luc Perfettini and Guido Kroemer
Pages 438 - 442
http://dx.doi.org/10.4161/cc.8.3.7642
Abstract | PDF

Fusogenic HIV-1 isolates induce the fusion of infected and bystander cells. Such syncytia can be found as "multinucleated giant cells" in the brain from HIV-1-infected individuals, as well as in lymphoid tissues. Syncytia elicited by the HIV-1 envelope glycoprotein (Env) manifest the aggregation of PML in discrete nuclear bodies and the recruitment of TopBP1, NBS1 and ATM to DNA damage foci containing phosphorylated ATM and histone H2AX (γ-H2AX). This DNA damage response then culminates in p53-dependent activation of the mitochondrial pathway of apoptosis. Here, we show that Env-elicited syncytia also manifest activating phosphorylations of the checkpoint kinases 1 and 2 (Chk1 and Chk2), and both Chk1 and Chk2 colocalize with γ-H2AX foci. However, only the siRNA-mediated knockdown of Chk2, not the depletion of Chk1, inhibits mitochondrial outer membrane permeabilization and subsequent syncytial apoptosis. Depletion of PML, TopBP1, NBS1 or ATM inhibit the activating phosphorylation of Chk2. Altogether, these results indicate that Chk2 (but not Chk1) participates in the DNA damage-elicited pro-apoptotic cascade that leads to the demise of Env-elicited syncytia.

Open Access Article

Molecular pharmacology of phosphatidylinositol 3-kinase inhibition in human glioma

Sandrine Guillard, Paul A. Clarke, Robert te Poele, Zahra Mohri, Lynn Bjerke, Melanie Valenti, Florence Raynaud, Suzanne A. Eccles and Paul Workman
Pages 443 - 453
http://dx.doi.org/10.4161/cc.8.3.7643
Abstract | PDF

Gliomas are primary brain tumors with poor prognosis that exhibit frequent abnormalities in phosphatidylinositol 3-kinase (PI3 kinase) signaling. We investigated the molecular mechanism of action of the isoform-selective Class I PI3 kinase and mTOR inhibitor PI-103 in human glioma cells. The potent inhibitory effects of PI-103 on the PI3 kinase pathway were quantified. PI-103 and the mTOR inhibitor rapamycin both inhibited RPS6 phosphorylation but there were clear differences in the response of upstream components of the PI3 kinase pathway, such as phosphorylation of Thr308-AKT, that were inhibited by PI-103 but not rapamycin. Gene expression profiling identified altered expression of genes encoding regulators of the cell cycle and cholesterol metabolism, and genes modulated by insulin or IGF1 signaling, rapamycin treatment or nutrient starvation. PI-103 decreased expression of positive regulators of G1/S phase progression and increased expression of the negative cell cycle regulator p27kip1. A reversible PI-103-mediated G1 cell cycle arrest occurred without significant apoptosis, consistent with the altered gene expression detected. PI-103 induced vacuolation and processing of LC-3i to LC-3ii, which are features of an autophagic response. In contrast to PI-103, LY294002 and PI-387 induced apoptosis, indicative of likely off-target effects. PI-103 interacted synergistically or additively with cytotoxic agents used in the treatment of glioma, namely vincristine, BCNU and temozolomide. Compared to individual treatments, the combination of PI-103 with temozolomide significantly improved the response of U87MG human glioma xenografts. Our results support the therapeutic potential for PI3 kinase inhibitors with PI-103-like profile as therapeutic agents for the treatment of glioma.

Open Access Article

The homeotic protein HOXC13 is a member of human DNA replication complexes

Laura Comelli, Laura Marchetti, Daniele Arosio, Silvano Riva, Gulnara Abdurashidova, Fabio Beltram and Arturo Falaschi
Pages 454 - 459
http://dx.doi.org/10.4161/cc.8.3.7649
Abstract | PDF

The homeotic (and oncogenic) HOXC13 protein was shown to have an affinity for a DNA fragment corresponding to the sequence covered by the pre-replicative complex of the human lamin B2 replication origin.  We show here that HOXC13 is a member of human replicative complexes.  Our fluorescent fusion-protein data demonstrate that it co-localizes with replication foci of early-S cells and that this peculiar behaviour is driven by the homeodomain.  By ChIP analysis we also show that HOXC13 binds the lamin B2 replication origin and the origins located near the TOP1 and MCM4 genes in asynchronously growing cells, whereas it does not bind these origins in G0 resting cells, consistently with its involvement in origin function.

Open Access Article

Long-term downregulation of Polo-like kinase 1 increases the cyclin-dependent kinase inhibitor p21^WAF1/CIP1

Nina-Naomi Kreis, Katharina Sommer, Mourad Sanhaji, Andrea Krämer, Yves Matthess, Manfred Kaufmann, Klaus Strebhardt and Juping Yuan
Pages 460 - 472
http://dx.doi.org/10.4161/cc.8.3.7651
Abstract | PDF

Polo-like kinase 1 (Plk1) is overexpressed in tumor tissues and its expression level is tightly associated with the malignancy of tumors and prognosis of tumor patients. Thus, Plk1 is considered as one of the most attractive molecular targets for anticancer therapy. Recently, several small molecule inhibitors of Plk1 have been identified and characterized, and the first generation of Plk1 inhibitors has been investigated in clinical trials. However, the long-term effect of the downregulation of Plk1 on tumor cells has not yet been studied. In this work we have investigated the phenotype of HeLa cells, in which Plk1 is continuously downregulated by constitutive expression of shRNA. The data demonstrate that the long-term suppression of Plk1 increases the levels of cyclin-dependent kinase inhibitor p21^WAF1/CIP, which is partially induced by the elevated tumor suppressor p73 in p53-inactivated HeLa cells. The increased kinase inhibitor p21^WAF1/CIP1 localizes in both cyctoplasm as well as in nucleus and interacts directly with Cdk1/cyclin B1. Moreover, the knockdown of Plk1 leads to a decreased oncoprotein MDM2 and an elevated pro-apoptotic protein Bax in HeLa cells. Importantly, HeLa cells with reduced level of Plk1, which induces an increase of p21, p73 and Bax, are more sensitive to some chemotherapeutic agents, such as cisplatin.

Open Access Article

Interleukin-6 affects cell death escaping mechanisms acting on Bax-Ku70-Clusterin interactions in human colon cancer progression

Sabina Pucci, Paola Mazzarelli, Fabiola Sesti, David A. Boothman and Luigi G. Spagnoli
Pages 473 - 481
http://dx.doi.org/10.4161/cc.8.3.7652
Abstract | PDF

Activation of pro-survival pathways and apoptotic cell death escape are considered hallmarks of oncogenic cell transformation. Tissue microenvironment strongly influences tumorigenesis, redirecting some pathways versus a persisting pro-survival state. Here, we report evidence on the role of interleukin 6 (IL-6)  in affecting  pro-survival pathways in  colon cancer progression, modulating the expression and the molecular interactions among the pro-apoptotic factor Bax, the  DNA repair proteins Ku70/86 and Clusterin isoforms.

Open Access Article

Replication stress activates DNA polymerase alpha-associated Chk1

Lorena Taricani, Frances Shanahan and David Parry
Pages 482 - 489
http://dx.doi.org/10.4161/cc.8.3.7661
Abstract | PDF

Chk1 contributes to both intra-S and DNA damage checkpoint responses.  Here, we show that depletion of DNA Polα and not Polε or Polδ by siRNA induces phosphorylation of Chk1 on Ser345, thus phenocopying antimetabolite exposure.  Combinatorial ablation of DNA Polα and Chk1 causes an accumulation of γ-H2A.X, a marker of double-strand DNA breaks, suggesting that activation of Chk1 in this context is essential for suppression of DNA damage.  Co-depletion of DNA Polα with ATR yields similar phenotypes, suggesting that ATR and Chk1 are epistatic and required for maintenance of genomic integrity following replication stress.  Significantly, Chk1 and DNA Polα can be co-immunoprecipated from native cell extracts.  Moreover, following replication stress, Polα-associated Chk1 becomes rapidly phosphorylated on Ser345 in a TopBP1 and ATR-dependent manner.  Hence, the ability to efficiently phosphorylate Chk1 in the context of DNA Polα complexes is correlated with suppression of DNA damage following replication stress.  These findings identify DNA Polα as an important component of the signal transduction cascade that activates the intra-S checkpoint.

Open Access Article

RNA-binding motif protein 35A is a novel tumor suppressor for colorectal cancer

Olga V. Leontieva and Yurij Ionov
Pages 490 - 497
http://dx.doi.org/10.4161/cc.8.3.7679
Abstract | PDF

The frequent occurrence of inactivating gene mutations in tumors suggests a tumor suppressor function of the mutated gene.  The RNA binding motif protein 35A (RBM35A) is mutated in ~50% of analyzed primary colon tumors with microsatellite instability.  The Tet-off regulated ectopic expression of RBM35A gene in RBM35A-null LS180 colon carcinoma cells inhibited anchorage-independent growth in vitro, suppressed tumorigenic potential in vivo and enhanced adhesiveness of these cancer cells.  Using microarray hybridization we found that in response to RBM35A expression a small fraction of genes showed a decrease in polysome-associated mRNA.  Studies using cell-free in vitro translation system demonstrated that RBM35A differentially affects translation of luciferase reporter mediated by various 5’untranslated regions (UTR).  We proposed that Gibbs energy value (ΔG) of secondary structure formed by 5’UTRs of mRNAs can account for differential effect of RBM35A on reporter translation efficiency.  Targeted mutation in the FOS 5’UTR sequence, which increased the ΔG value of hairpin stem formation, resulted in a stronger inhibitory effect of RBM35A on reporter translation efficiency mediated by this UTR.  Immunoblotting revealed that ectopic expression of RBM35A in LS180 cells caused alterations in protein levels for several cancer related genes.  Our results demonstrate for the first time that RBM35A functions as a tumor suppressor in colon cancer cells.  We propose that RBM35A is involved in posttranscriptional regulation of a number of genes by exerting a differential effect on protein translation via 5’UTRs of mRNAs.

Open Access Article

Single-vector inducible lentiviral RNAi system for oncology target validation

Dmitri Wiederschain, Wee Susan, Lin Chen, Alice Loo, Guizhi Yang, Alan Huang, Yan Chen, Giordano Caponigro, Yung-mae Yao, Christoph Lengauer, William R. Sellers and John D. Benson
Pages 498 - 504
http://dx.doi.org/10.4161/cc.8.3.7701
Abstract | PDF

The use of RNA interference (RNAi) has enabled loss-of-function studies in mammalian cancer cells and has hence become critical for  identifying and validating cancer drug targets.  Current transient siRNA and stable shRNA systems, however, have limited utility in accurately assessing the cancer dependency due to their short-lived effects and limited in vivo utility, respectively.  In this study, a single-vector lentiviral, Tet-inducible shRNA system (pLKO-Tet-On) was generated to allow for the rapid generation of multiple stable cell lines with regulatable shRNA expression.  We demonstrate the advantages and versatility of this system by targeting two polycomb group proteins, Bmi-1 and Mel-18, in a number of cancer cell lines.  Our data show that pLKO-Tet-On-mediated knockdown is tightly regulated by the inducer tetracycline and its derivative, doxycycline, in a concentration- and time-dependent manner.  Furthermore, target gene expression is fully restored upon withdrawal of the inducing agent.  An additional, 17 distinct gene products have been targeted by inducible shRNAs with robust regulation in all cases.  Importantly, we functionally validate the ability of the pLKO-Tet-On vector to reversibly silence targeted transcripts in vivo.  The versatile and robust inducible lentiviral RNAi system reported herein can therefore serve as a powerful tool to rapidly reveal tumor cell dependence.




LETTER TO THE EDITOR

Open Access Article

Does the cell number 10⁹ still really fit one gram of tumor tissue?

Ugo Del Monte
Pages 505 - 506
http://dx.doi.org/10.4161/cc.8.3.7608
Abstract | PDF

A tumor reaching the size of 1 cm³ (approximately 1 g wet weight) is commonly assumed to contain 1 x 10⁹ cells. This paper comments on the probable origin of this “magic” number and on some possible reasons why it has remained in use until now. However, mostly in epithelial tumors (85% of all human tumors) a cell number one order of magnitude smaller would be more realistic.




ERRATA

Open Access Article

Erratum to Melissa R. Junttila et al., Cell Cycle Volume 7, Issue 5; pp. 592-596

Melissa R. Junttila and Jukka Westermarck
Page 507
http://dx.doi.org/10.4161/cc.8.3.7856
Abstract | PDF

In the review “Mechanisms of MYC stabilization in human malignancies” by Melissa R. Junttila and Jukka Westermarck Cell Cycle 2008; 7:592–6) there was a minor error in the text.  The original statement on page 593, line 17 reads: A prolyl isomerase, Pin-1, binds phosphorylated S62 and T58 of MYC, and isomerizes MYC at Proline 59. The author’s correction states that in contrast to proline 59 mentioned in the text, based on the available knowledge Pin-1 rather isomerizes proline 63 on c-Myc.

Open Access Article

Erratum to Wolfgang Benetka et al., Cell Cycle Volume 7, Issue 23; pp. 3709-3719

Wolfgang Benetka, Norbert Mehlmer, Sebastian Maurer-Stroh, Michaela Sammer, Manfred Koranda, Ralph Neumüller, Jörg Betschinger, Jürgen A. Knoblich, Markus Teige and Frank Eisenhaber
Pages 508 - 509
http://dx.doi.org/10.4161/cc.8.3.7857
Abstract | PDF

In the paper, "Experimental testing of predicted myristoylation targets involved in asymmetric cell division and calcium-dependent signalling" by Wolfgang Benetka, Norbert Mehlmer, Sebastian Maurer-Stroh, Michaela Sammer, Manfred Koranda, Ralph Neumüller, Jörg Betschinger, Jürgen A. Knoblich, Markus Teige and Frank Eisenhaber (Cell Cycle 2008; 7:3709-19), Figure 5 was published two times but Figure 2 was not included. Figure 2 and 5 appear correct below.