Table of Contents

Volume 11, Issue 6

  March 15, 2012

  Pages 1049 - 1259

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EDITORIALS: CELL CYCLE FEATURES

Open Access Article

The cyclin K/Cdk12 complex: An emerging new player in the maintenance of genome stability

Dalibor Blazek
Pages 1049 - 1050
http://dx.doi.org/10.4161/cc.11.6.19678
Abstract | PDF

Comment on: Blazek D, et al. Genes Dev 2011; 25:2158–72

Adaptation in a eukaryotic pathway: Combining experiments with modeling

Wouter-Jan Rappel and Richard Firtel
Pages 1051 - 1052
http://dx.doi.org/10.4161/cc.11.6.19715
Abstract | PDF

Comment on: Takeda K, et al. Sci Signal 2012; 5:2

Glycogen synthase kinase (GSK)-3 and mammalian target of rapamycin complex 1 (mTORC1) cooperate to regulate protein S6 kinase 1 (S6K1)

Sejeong Shin, Laura Wolgamott and Sang-Oh Yoon
Pages 1053 - 1054
http://dx.doi.org/10.4161/cc.11.6.19784
Abstract | PDF

Comment on: Shin S, et al. Proc Natl Acad Sci USA 2011; 108:1204–13

Finding a needle in the haystack: Recognition of DNA damage by collaboration between DNA repair proteins able to perform DNA charge transport

Robert M. Brosh
Pages 1055 - 1056
http://dx.doi.org/10.4161/cc.11.6.19785
PDF




CELL CYCLE NEWS & VIEWS

Open Access Article

A role of miR-33 for cell cycle progression and cell proliferation

Yasuko Iwakiri
Page 1057
http://dx.doi.org/10.4161/cc.11.6.19744
Abstract | PDF

Comment on: Cirera-Salinas D, et al. Cell Cycle 2012; 11:922–33

Open Access Article

Metformin and reprogramming into iPSCs

Manuel Serrano
Page 1058
http://dx.doi.org/10.4161/cc.11.6.19745
Abstract | PDF

Comment on: Vazquez-Martin A, et al. Cell Cycle 2012; 11:922–33

Open Access Article

Reverse Warburg: Straight to cancer

Lorenzo Galluzzi, Oliver Kepp and Guido Kroemer
Page 1059
http://dx.doi.org/10.4161/cc.11.6.19746
Abstract | PDF

Comment on: Witkiewicz AK, et al. Cell Cycle 2012; 11: In this issue.

Open Access Article

MiR-33 connects cholesterol to the cell cycle

Sachi Inukai and Frank J. Slack
Pages 1060 - 1061
http://dx.doi.org/10.4161/cc.11.6.19786
Abstract | PDF

Comment on: Cirera-Salinas D, et al. Cell Cycle 2012; 11:922–33




EXTRA VIEWS

p97/VCP- and Lys48-linked polyubiquitination form a new signaling pathway in DNA damage response

Kristijan Ramadan
Pages 1062 - 1069
http://dx.doi.org/10.4161/cc.11.6.19446
Abstract | PDF

RNF8/RNF168-dependent Lys63-linked polyubiquitination at sites of DNA double-strand breaks (DSBs) was originally regarded as the sole ubiquitin-signaling pathway involved in the DNA damage response (DDR). However, ubiquitin-dependent p97/VCP segregase activity and RNF8-dependent Lys48-linked polyubiquitin chains at DSB sites have recently been identified as components of an additional and parallel ubiquitin-signaling DDR pathway. This newly identified pathway is essential to spatiotemporal protein turnover and regulates both main branches of DSB repair, homologous recombination and nonhomologous end joining. In this report, the function of the RNF8/Lys48 polyubiquitin chains/p97 pathway is discussed in the context of DSB repair and p97 chromatin-related functions.

Regulation of oxidative DNA damage repair: The adenine:8-oxo-guanine problem

Enni Markkanen, Ulrich Hübscher and Barbara van Loon
Pages 1070 - 1075
http://dx.doi.org/10.4161/cc.11.6.19448
Abstract | PDF

Reactive oxygen species (ROS) constantly attack DNA. One of the best-characterized oxidative DNA lesions is 7,8-dihydro-8-oxoguanine (8-oxo-G). Many human diseases, such as cancer and neurodegenerative disorders, have been correlated with oxidative DNA damage. In the last few years, DNA polymerase (Pol) λ, one of the 15 cellular Pols, has been identified to play an important role in performing accurate translesion synthesis over 8-oxo-G. This is eminently important, since normally faithful replicative Pols α, δ and ε, with their tight active center, often wrongly incorporate adenine (A) opposite the 8-oxo-G lesion. A:8- oxo-G mispairs are accurately repaired by the pathway identified in our laboratory involving MutY DNA glycosylase homolog (MutYH) and Pol λ. Until now, very little was known about the spatial and temporal regulation of Pol λ and MutYH in active repair complexes. We now showed in our latest publication that the E3 ligase Mule can ubiquitinate and degrade Pol λ, and that the control of Pol λ levels by Mule has functional consequences for the ability of mammalian cells to deal with 8-oxo-G lesions. In contrast, phosphorylation of Pol λ by Cdk2/cyclinA counteracts this degradation by recruiting it to MutYH on chromatin to form active 8-oxo-G repair complexes.

Mining the cancer genome uncovers therapeutic activity of EphA7 against lymphoma

Elisa Oricchio and Hans-Guido Wendel
Pages 1076 - 1080
http://dx.doi.org/10.4161/cc.11.6.19451
Abstract | PDF

The functional annotation of the cancer genome can reveal new opportunities for cancer therapies. The wealth of genomic data on various cancers has not yet been mined for clinically and therapeutically useful information. We use cross-comparisons of genomic data with the results of unbiased genetic screens to prioritize genomic changes for further study. In this manner, we have identified a soluble variant of the ephrin receptor A7 (EPHA7^TR) as a tumor suppressor that is lost in lymphoma. We also developed antibody-based delivery to restore this tumor suppressor to the cancer cells in situ. We will discuss our strategy of screening genomic data, specific findings concerning EPHA7 and the potential for future discoveries.

Vitamin D has wide regulatory effects on histone demethylase genes

Fábio Pereira, Antonio Barbáchano, Prashant K. Singh, Moray J. Campbell, Alberto Muñoz and María Jesús Larriba
Pages 1081 - 1089
http://dx.doi.org/10.4161/cc.11.6.19508
Abstract | PDF

Vitamin D from the diet or synthesized in the skin upon UV-B irradiation is converted in the organism into the active metabolite 1α,25- dihydroxyvitamin D₃ [1,25(OH)₂D₃, calcitriol], a pleiotropic hormone with wide regulatory actions. The classical model of 1,25(OH)2D3 action implies the activation of the vitamin D receptor, which binds specific DNA sequences in its target genes and modulates their transcription rate. We have recently shown that 1,25(OH)₂D₃ induces the expression of the JMJD3 gene coding for a histone demethylase that is involved in epigenetic regulation. JMJD3 mediates the effects of 1,25(OH)₂D₃ on a subset of target genes and affects the expression of ZEB1, ZEB2 and SNAI1, inducers of epithelial-mesenchymal transition. Novel data indicate that 1,25(OH)₂D₃ has an unanticipated wide regulatory action on the expression of genes coding for histone demethylases of the Jumonji C (JmjC) domain and lysine-specific demethylase (LSD) families. Moreover, JMJD3 knockdown decreases the expression of miR‑200b and miR‑200c, two microRNAs targeting ZEB1 RNA. This may explain the upregulation of this transcription factor found in JMJD3-depleted cells. Thus, 1,25(OH)₂D₃ exerts an ample regulatory effect on the expression of histone-modifying enzymes involved in epigenetic regulation that may mediate its actions on gene transcription and cell phenotype.




REVIEWS

YAP oncogene overexpression supercharges colon cancer proliferation

Joseph Avruch, Dawang Zhou and Nabeel Bardeesy
Pages 1090 - 1096
http://dx.doi.org/10.4161/cc.11.6.19453
Abstract | PDF

The transcriptional co-activator YAP is an evolutionarily conserved regulator of organ size and progenitor cell proliferation. YAP is overexpressed at high frequency in many common human cancers and can directly drive cancer development in mouse models. YAP abundance and nuclear localization are negatively regulated by the Hippo kinase cascade, which, in epithelia, is activated by physiological cell-cell contact. Recent work in intestinal epithelium has established that YAP is constitutively inhibited by the Hippo pathway and entirely dispensable for normal development and homeostasis. YAP serves only in a standby capacity; should cell-cell contact be abrogated, as after intestinal damage, the loss of Hippo input permits increased YAP abundance and nuclear residence. In turn, YAP cooperates with β-catenin to transactivate genes that promote stem cell expansion for epithelial repair. This interplay between overexpressed YAP and β-catenin also drives proliferation of colon cancer cells. The dispensability of YAP in normal intestine makes YAP’s expression or outputs attractive targets for cancer therapy.

Translational control of cell fate: From integration of environmental signals to breaching anticancer defense

Peter B. Bitterman and Vitaly A. Polunovsky
Pages 1097 - 1107
http://dx.doi.org/10.4161/cc.11.6.19610
Abstract | PDF

Despite their genetic diversity, different cancers manifest common features at the protein pathway level. They share a core group of perturbed pathways that converge upon a few regulatory hubs linking the cellular signaling network with the basic metabolic machinery. Available evidence indicates that one such hub is the eIF4F-mediated cap-dependent mRNA translation initiation apparatus, whose integrity is required for physiological control of growth, proliferation and viability. However, when hyperactivated by upstream oncogenic signaling, eIF4F selectively stimulates the translation of a group of mRNAs required for cancer genesis and progression. Here, we describe a model that links the pro-neoplastic function of eIF4F to its ability to disable oncogene-activated tumor surveillance programs and propose a novel therapeutic strategy for cancer based upon targeting aberrant eIF4F with small-molecule antagonists.




REPORTS

Open Access Article

Using the “reverse Warburg effect” to identify high-risk breast cancer patients: Stromal MCT4 predicts poor clinical outcome in triple-negative breast cancers

Agnieszka K. Witkiewicz, Diana Whitaker-Menezes, Abhijit Dasgupta, Nancy J. Philp, Zhao Lin, Ricardo Gandara, Sharon Sneddon, Ubaldo E. Martinez-Outschoorn, Federica Sotgia and Michael P. Lisanti
Pages 1108 - 1117
http://dx.doi.org/10.4161/cc.11.6.19530
Abstract | PDF

We have recently proposed a new model of cancer metabolism to explain the role of aerobic glycolysis and L-lactate production in fueling tumor growth and metastasis. In this model, cancer cells secrete hydrogen peroxide (H2O2), initiating oxidative stress and aerobic glycolysis in the tumor stroma. This, in turn, drives L-lactate secretion from cancer-associated fibroblasts. Secreted L-lactate then fuels oxidative mitochondrial metabolism (OXPHOS) in epithelial cancer cells, by acting as a paracrine onco-metabolite. We have previously termed this type of two-compartment tumor metabolism the “Reverse Warburg Effect,” as aerobic glycolysis takes place in stromal fibroblasts, rather than epithelial cancer cells. Here, we used MCT4 immuno-staining of human breast cancer tissue microarrays (TMAs; > 180 triple-negative patients) to directly assess the prognostic value of the “Reverse Warburg Effect.” MCT4 expression is a functional marker of hypoxia, oxidative stress, aerobic glycolysis, and L-lactate efflux. Remarkably, high stromal MCT4 levels (score = 2) were specifically associated with decreased overall survival (< 18% survival at 10 y post-diagnosis). In contrast, patients with absent stromal MCT4 expression (score = 0), had 10-y survival rates of ~97% (p-value < 10⁻³²). High stromal levels of MCT4 were strictly correlated with a loss of stromal Cav-1 (p-value < 10⁻¹⁴), a known marker of early tumor recurrence and metastasis. In fact, the combined use of stromal Cav-1 and stromal MCT4 allowed us to more precisely identify high-risk triple-negative breast cancer patients, consistent with the goal of individualized risk-assessment and personalized cancer treatment. However, epithelial MCT4 staining had no prognostic value, indicating that the “conventional” Warburg effect does not predict clinical outcome. Thus, the “Reverse Warburg Effect” or “parasitic” energy-transfer is a key determinant of poor overall patient survival. As MCT4 is a druggable-target, MCT4 inhibitors should be developed for the treatment of aggressive breast cancers, and possibly other types of human cancers. Similarly, we discuss how stromal MCT4 could be used as a biomarker for identifying high-risk cancer patients that could likely benefit from treatment with FDA-approved drugs or existing MCT-inhibitors (such as, AR-C155858, AR-C117977, and AZD-3965).

SENP1 deficiency promotes ER stress-induced apoptosis by increasing XBP1 SUMOylation

Zhou Jiang, Qiuju Fan, Zhening Zhang, Yanqiong Zou, Rong Cai, Qi Wang, Yong Zuo and Jinke Cheng
Pages 1118 - 1122
http://dx.doi.org/10.4161/cc.11.6.19529
Abstract | PDF

The transcription factor X box-binding protein 1 (XBP1) is a key component of the endoplasmic reticulum (ER) stress response. Recently, it has been reported that the spliced XBP1 (XBP1s), an activated XBP1 during ER stress, can be SUMOylated. Here, we identify Sentrin/SUMO-specific protease 1 (SENP1) as a specific de-SUMOylation protease for XBP1. SENP1 can increase the transcriptional activity of XBP1. In Senp1^-/- cells, the SUMOylated XBP1 is accumulated, and the expression of XBP1 target genes is downregulated in response to ER stress. Moreover, SENP1 deficiency significantly increases ER stress-induced apoptosis through accumulating XBP1 SUMOylation. These results reveal an essential function of SENP1 in ER stress response through regulating XBP1 SUMOylation.

The ubiquitin-specific protease USP2a enhances tumor progression by targeting cyclin A1 in bladder cancer

Jayoung Kim, Wun-Jae Kim, Zhiqian Liu, Massimo Loda and Michael R. Freeman
Pages 1123 - 1130
http://dx.doi.org/10.4161/cc.11.6.19550
Abstract | PDF

The deubiquitinating enzyme USP2a has shown oncogenic properties in many cancer types by impairing ubiquitination of FASN, MDM2, MDMX or Aurora A. Aberrant expression of USP2a has been linked to progression of human tumors, particularly prostate cancer. However, little is known about the role of USP2a or its mechanism of action in bladder cancer. Here, we provide evidence that USP2a is an oncoprotein in bladder cancer cells. Enforced expression of USP2a caused enhanced proliferation, invasion, migration and resistance to several chemotherapeutic reagents, while USP2a loss resulted in slower proliferation, greater chemosensitivity and reduced migratory/invasive capability compared with control cells. USP2a, but not a catalytically inactive mutant, enhanced proliferation in immortalized TRT-HU1 normal human bladder epithelial cells. USP2a bound to cyclin A1 and prevented cyclin A1 ubiquitination, leading to accumulation of cyclin A1 by a block in degradation. Enforced expression of wild type USP2a, but not an inactive USP2a mutant, resulted in cyclin A1 accumulation and increased cell proliferation. We conclude that USP2a impairs ubiquitination and stabilizes an important cell cycle regulator, cyclin A1, raising the possibility of USP2a targeting as a therapeutic strategy against bladder tumors in combination with chemotherapy.

Rad18 is a transcriptional target of E2F3

Lakshman Varanasi, Phi Do, Elzbieta Goluszko and Luis A. Martinez
Pages 1131 - 1141
http://dx.doi.org/10.4161/cc.11.6.19558
Abstract | PDF

The E2F family of transcription factors responds to a variety of intracellular and extracellular signals and, as such, are key regulators of cell growth, differentiation and cell death. The cellular response to DNA damage is a multistep process generally involving the initial detection of DNA damage, propagation of signals via posttranslational modifications (e.g., phosphorylation and ubiquitination) and, finally, the implementation of a response. We have previously reported that E2F3 can be induced by DNA damage, and that it plays an important role in DNA damage-induced apoptosis. Here, we demonstrate that E2F3 knockdown compromises two canonical DNA damage modification events, the ubiquitination of H2AX and PCNA. We find that the defect in these posttranscriptional modifications after E2F3 knockdown is due to reduced expression of important DNA damage responsive ubiquitin ligases. We characterized the regulation of one of these ligases, Rad18, and we demonstrated that E2F3 associates with the Rad18 promoter and directly controls its activity. Furthermore, we find that ectopic expression of Rad18 is sufficient to rescue the PCNA ubiquitination defect resulting from E2F3 knockdown. Our study reveals a novel facet of E2F3’s control of the DNA damage response.

The ubiquitin E1 enzyme Ube1 mediates NEDD8 activation under diverse stress conditions

Orsolya Leidecker, Ivan Matic, Bidesh Mahata, Emmanuelle Pion and Dimitris P. Xirodimas
Pages 1142 - 1150
http://dx.doi.org/10.4161/cc.11.6.19559
Abstract | PDF

Modification of proteins with ubiquitin and ubiquitin-like molecules is involved in the regulation of almost every biological process. Historically, each conjugation pathway has its unique set of E1, E2 and E3 enzymes that lead to activation and conjugation of their cognate molecules. Here, we present the unexpected finding that under stress conditions, the ubiquitin E1 enzyme Ube1 mediates conjugation of the ubiquitin-like molecule NEDD8. Inhibition of the 26S proteasome, heat shock and oxidative stress cause a global increase in NEDDylation. Surprisingly, this does not depend on the NEDD8 E1-activating enzyme, but rather on Ube1. A common event in the tested stress conditions is the depletion of “free” ubiquitin. A decrease in “free” ubiquitin levels in the absence of additional stress is sufficient to stimulate NEDDylation through Ube1. Further analysis on the NEDD8 proteome shows that the modified NEDDylated proteins are simultaneously ubiquitinated. Mass spectrometry on the complex proteome under stress reveals the existence of mixed chains between NEDD8 and ubiquitin. We further show that NEDDylation of the p53 tumor suppressor upon stress is mediated mainly through Ube1. Our studies reveal an unprecedented interplay between NEDD8 and ubiquitin pathways operating in diverse cellular stress conditions.

Origin of metastases: Subspecies of cancers generated by intrinsic karyotypic variations

Peter Duesberg, Christine Iacobuzio-Donahue, Jacqueline A. Brosnan, Amanda McCormack, Daniele Mandrioli and Lewis Chen
Pages 1151 - 1166
http://dx.doi.org/10.4161/cc.11.6.19580
Abstract | PDF

Conventional mutation theories do not explain (1) why the karyotypes of metastases are related to those of parental cancers but not to those of metastases of other cancers and (2) why cancers metastasize at rates that often far exceed those of conventional mutations. To answer these questions, we advance here the theory that metastases are autonomous subspecies of cancers, rather than mutations. Since cancers are species with intrinsically flexible karyotypes, they can generate new subspecies by spontaneous karyotypic rearrangements. This phylogenetic theory predicts that metastases are karyotypically related to parental cancers but not to others. Testing these predictions on metastases from two pancreatic cancers, we found: (1) Metastases had individual karyotypes and phenotypes. The karyotypes of metastases were related to, but different from, those of parental cancers in 11 out of 37 and 26 out of 49 parental chromosomal units. Chromosomal units are defined as intact chromosomes with cancer-specific copy numbers and marker chromosomes that are > 50% clonal. (2) Metastases from the two different cancers did not share chromosomal units. Testing the view that multi-chromosomal rearrangements occur simultaneously in cancers, as opposed to sequentially, we found spontaneous non-clonal rearrangements with as many new chromosomal units as in authentic metastases. We conclude that metastases are individual autonomous species differing from each other and parental cancers in species-specific karyotypes and phenotypes. They are generated from parental cancers by multiple simultaneous karyotypic rearrangements, much like new species. The species-specific individualities of metastases explain why so many searches for commonalities have been unsuccessful.

Dynamic inhibition of ATM kinase provides a strategy for glioblastoma multiforme radiosensitization and growth control

Sarah E. Golding, Elizabeth Rosenberg, Bret R. Adams, Shayalini Wignarajah, Jason M. Beckta, Mark J. O’Connor and Kristoffer Valerie
Pages 1167 - 1173
http://dx.doi.org/10.4161/cc.11.6.19576
Abstract | PDF

Glioblastoma multiforme (GBM) is notoriously resistant to treatment. Therefore, new treatment strategies are urgently needed. ATM elicits the DNA damage response (DDR), which confers cellular radioresistance; thus, targeting the DDR with an ATM inhibitior (ATMi) is very attractive. Herein, we show that dynamic ATM kinase inhibition in the nanomolar range results in potent radiosensitization of human glioma cells, inhibits growth and does not conflict with temozolomide (TMZ) treatment. The second generation ATMi analog KU-60019 provided quick, reversible and complete inhibition of the DDR at sub-micromolar concentrations in human glioblastoma cells. KU-60019 inhibited the phosphorylation of the major DNA damage effectors p53, H2AX and KAP1 as well as AKT. Colony-forming radiosurvival showed that continuous exposure to nanomolar concentrations of KU-60019 effectively radiosensitized glioblastoma cell lines. When cells were co-treated with KU-60019 and TMZ, a slight increase in radiation-induced cell killing was noted, although TMZ alone was unable to radiosensitize these cells. In addition, without radiation, KU-60019 with or without TMZ reduced glioma cell growth but had no significant effect on the survival of human embryonic stem cell (hESC)-derived astrocytes. Altogether, transient inhibition of the ATM kinase provides a promising strategy for radiosensitizing GBM in combination with standard treatment. In addition, without radiation, KU-60019 limits growth of glioma cells in co-culture with human astrocytes that seem unaffected by the same treatment. Thus, inter-fraction growth inhibition could perhaps be achieved in vivo with minor adverse effects to the brain.

Saquinavir-NO-targeted S6 protein mediates sensitivity of androgen-dependent prostate cancer cells to TRAIL

Marija Mojic, Sanja Mijatovic, Danijela Maksimovic-Ivanic, Svetlana Dinic, Nevena Grdovic, Djordje Miljkovic, Stanislava Stosic-Grujicic, Salvatore Tumino, Paolo Fagone, Katia Mangano, Mai-Britt Zocca, Yousef Al-Abed, James A. McCubrey and Ferdinando Nicoletti
Pages 1174 - 1182
http://dx.doi.org/10.4161/cc.11.6.19611
Abstract | PDF

We previously reported that the NO-modified form of HIV protease inhibitor Saquinavir (Saq) is a potent antitumoral agent efficient against numerous tumor cell lines in vitro and in vivo. In acute toxicity studies, doses of Saq-NO equivalent to DL100 of the parental drug were completely nontoxic. Beside direct effect on malignant cell growth, Saq-NO sensitizes certain type of cells to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-mediated cell death. In this study, we evaluated the effects of Saq-NO on androgen-dependent prostate cancer LNCaP. Saq-NO inhibited both the growth of LNCaP cells in vitro and in xenograft models. Suppression of tumor growth was accompanied with cell cycle arrest in G₀/G₁ phase and established a persistent inhibition of proliferation. Furthermore, Saq-NO reverted sensitivity of LNCaP cells to TRAIL but not to TNF. Treatment of cells with Saq-NO induced transient upregulation of Akt and ERK1/2. This, however, did not represent the primary mode of action of Saq-NO, as elimination with specific inhibitors did not compromise the chemotherapic efficacy of the drug. However, permanent abrogation of phosphorylation of the S6 protein, which is the downstream target of both signaling pathways, was observed. Diminished S6 phosphorylation was associated with re-established sensitivity to TRAIL and reduction of X-linked inhibitor of apoptosis protein (XIAP). In summary, NO modification of Saq led to a new chemical entity with stronger and more pleiotropic antitumor activity than the parental drug.

Role for Rif1 in the checkpoint response to damaged DNA in Xenopus egg extracts

Sanjay Kumar, Hae Yong Yoo, Akiko Kumagai, Anna Shevchenko, Andrej Shevchenko and William G. Dunphy
Pages 1183 - 1194
http://dx.doi.org/10.4161/cc.11.6.19636
Abstract | PDF

TopBP1 is critical for both DNA replication and checkpoint regulation in vertebrate cells. In this study, we have identified Rif1 as a binding partner of TopBP1 in Xenopus egg extracts. In addition, Rif1 also interacts with both ATM and the Mre11-Rad50-Nbs1 (MRN) complex, which are key regulators of checkpoint responses to double-stranded DNA breaks (DSBs). Depletion of Rif1 from egg extracts compromises the activation of Chk1 in response to DSBs but not stalled replication forks. Removal of Rif1 also has a significant impact on the chromatin-binding behavior of key checkpoint proteins. In particular, binding of TopBP1, ATR and the MRN complex to chromatin containing DSBs is reduced in the absence of Rif1. Rif1 interacts with chromatin in a highly regulated and dynamic manner. In unperturbed egg extracts, the association of Rif1 with chromatin depends upon formation of replication forks. In the presence of DSBs, there is elevated accumulation of Rif1 on chromatin under conditions where the activation of ATM is suppressed. Taken together, these results suggest that Rif1 plays a dynamic role in the early steps of a checkpoint response to DSBs in the egg-extract system by promoting the correct accumulation of key regulators on the DNA.

p38α MAPK regulates myocardial regeneration in zebrafish

Chris Jopling, Guillermo Suñe, Cristina Morera and Juan Carlos Izpisua Belmote
Pages 1195 - 1201
http://dx.doi.org/10.4161/cc.11.6.19637
Abstract | PDF

Although adult mammals are unable to significantly regenerate their heart, this is not the case for a number of other vertebrate species. In particular, zebrafish are able to fully regenerate their heart following amputation of up to 20% of the ventricle. Soon after amputation, cardiomyocytes dedifferentiate and proliferate to regenerate the missing tissue. More recently, identical results have also been obtained in neonatal mice. Ventricular amputation of neonates leads to a robust regenerative response driven by the proliferation of existing cardiomyocytes in a similar manner to zebrafish. However, this ability is progressively lost during the first week of birth. The fact that adult zebrafish retain the capacity to regenerate their heart suggests that they either possess a unique regenerative mechanism, or that adult mammals lose/ inhibit this process. p38α ΜAPK has previously been shown to negatively regulate the proliferation of adult mammalian cardiomyocytes. We sought to determine whether a similar mechanism exists in adult zebrafish, and whether this needs to be overcome to allow regeneration to proceed. To determine whether p38α ΜAPK also regulates zebrafish cardiomyocytes in a similar manner, we generated conditional transgenic zebrafish in which either dominant-negative or active p38α ΜAPK are specifically expressed in cardiomyocytes. We found that active p38α ΜAPK but not dominantnegative p38α ΜAPK blocks proliferation of adult zebrafish cardiomyocytes and, consequently, heart regeneration as well. It appears that adult zebrafish cardiomyocytes share many characteristics with adult mammalian cardiomyocytes, including p38α MAPK-mediated cell cycle inhibition. These findings raise the possibility that zebrafish-like heart regeneration could be achieved in adult mammals.

Flavopiridol induces phosphorylation of AKT in a human glioblastoma cell line, in contrast to siRNA-mediated silencing of Cdk9: Implications for drug design and development

Valentina Caracciolo, Giulio Laurenti, Gaetano Romano, Vincenzo Carnevale, Anna Maria Cimini, Catena Crozier-Fitzgerald, Emilio Gentile Warschauer, Giuseppe Russo and Antonio Giordano
Pages 1202 - 1216
http://dx.doi.org/10.4161/cc.11.6.19663
Abstract | PDF

Cdk9 and Cdk7 are cdc2-like serine/threonine kinases that stabilize RNA transcript elongation through RNA polII carboxyl terminal domain (CTD) phosphorylation and are considered suitable targets for cancer therapy.

The effects of flavopiridol and of siRNA-mediated inhibition of Cdk9 and/or Cdk7 were analyzed in human glioblastoma and human prostate cancer cell lines.

One finding revealed that Cdk9 and Cdk7 could substitute each other in RNA polII CTD phosphorylation in contrast to the in vitro system. Thus, a simultaneous inhibition of Cdk9 and Cdk7 might be required both for targeting malignant cells and developing a platform for microarray analysis. However, these two pathways are not redundant, as indicated by differential effects observed in cell cycle regulation following siRNA-mediated inhibition of Cdk9 and/or Cdk7 in human PC3 prostate cancer cell line. Specifically, siRNA-mediated inhibition of Cdk9 caused a shift from G₀/G₁ to G₂/M phase in human PC3 prostate cancer cell line.

Another finding showed that flavopiridol treatment induced a substantial AKT-Ser473 phosphorylation in human glioblastoma T98G cell line in contrast to siRNA-mediated inhibition of Cdk9 and Cdk9 combined with Cdk7, whereas siRNA-mediated silencing of Cdk7 caused a minor increase in AKT-Ser473 phosphorylation. AKT-Ser473 is a hallmark of AKT pathway activation and may protect cells from apoptosis. This finding also shows that Cdk9 and Cdk7 pathways are not redundant and may have important implications in drug development and for studying the mechanism of chemoresistance in malignant cells.

HIV-1 Tat impairs cell cycle control by targeting the Tip60, Plk1 and cyclin B1 ternary complex

Shi-Meng Zhang, Maoyong Song, Tian-Yi Yang, Rong Fan, Xiao-Dan Liu and Ping-Kun Zhou
Pages 1217 - 1234
http://dx.doi.org/10.4161/cc.11.6.19664
Abstract | PDF

HIV-1 Tat triggers intrinsic and extrinsic apoptosis pathways in both infected and uninfected cells and plays an important role in the pathogenesis of AIDS. Knocking down Tip60, an interactive protein of Tat, leads to the impairment of cell cycle progression, indicating a key role of Tip60 in cell cycle control. We found that Tip60 interacts with Plk1 through its ZnFMYST domain, and that this interaction is enhanced in the G₂/M phase. In addition, cyclin B1 was confirmed to interact with the ZnF domain of Tip60. Immunofluorescence imaging showed that Tip60 co-localizes with both Plk1 and cyclin B1 at the centrosome during the mitotic phase and to the mid-body during cytokinesis. Further experiments revealed that Tip60 forms a ternary complex with Plk1 and cyclin B1 and acetylates Plk1 but not cyclin B1. HIV-1 Tat likely forms a quaternary complex with Tip60, cyclin B1 and Plk1. Fluorescent microscopy showed that Tat causes an unscheduled nuclear translocation of both cyclin B1 and Plk1, causing their co-localization with Tip60 in the nucleus. Tat, Tip60, cyclin B1 and Plk1 interactions provide new a mechanistic explanation for Tat-mediated cell cycle dysregulation and apoptosis.

Metformin lowers the threshold for stress-induced senescence: A role for the microRNA-200 family and miR-205

Sílvia Cufí, Alejandro Vazquez-Martin, Cristina Oliveras-Ferraros, Rosa Quirantes, Antonio Segura-Carretero, Vicente Micol, Jorge Joven, Joaquim Bosch-Barrera, Sonia Del Barco , Begoña Martin-Castillo, Luciano Vellon and Javier A. Menendez
Pages 1235 - 1246
http://dx.doi.org/10.4161/cc.11.6.19665
Abstract | PDF

We have tested the hypothesis that the antidiabetic biguanide metformin can be used to manipulate the threshold for stress-induced senescence (SIS), thus accelerating the onset of cancer-protective cellular senescence in response to oncogenic stimuli. Using senescence-prone murine embryonic fibroblasts (MEFs), we assessed whether metformin treatment modified the senescence phenotype that is activated in response to DNA damaging inducers. Metformin significantly enhanced the number of MEFs entering a senescent stage in response to doxorubicin, an anthracycline that induces cell senescence by activating DNA damage signaling pathways (e.g., ATM/ATR) in a reactive oxygen species (ROS)-dependent manner. Using WI-38 and BJ-1 human diploid fibroblasts (HDFs), we explored whether metformin supplementation throughout their entire replicative lifespan may promote the early appearance of the biomarkers of replicative senescence. Chronic metformin significantly reduced HDFs’ lifespan by accelerating both the loss of replicative potential and the acquisition of replicative senescence-related biomarkers (e.g., enlarged and flattened cell shapes, loss of arrayed arrangement, accumulation of intracellular and extracellular debris and SA-β-gal-positive staining). Metformin functioned as a bona fide stressful agent, inducing monotonic, dose-dependent, SIS-like responses in BJ-1 HDFs, which are highly resistant to ROS-induced premature senescence. Metformin-induced SIS in BJ-1 fibroblasts was accompanied by the striking activation of several microRNAs belonging to the miR-200s family (miR-200a, miR-141 and miR429) and miR-205, thus mimicking a recently described ability of ROS to chemosensitize cancer cells by specifically upregulating anti-EMT (epithelial-to-mesenchymal transition) miR-200s. Because the unlimited proliferative potential of stem cells results from their metabolic refractoriness to SIS, we finally tested if metformin treatment could circumvent the stress (e.g., ROS)-resistant phenotype of induced pluripotent stem cells (iPSCs). Metformin treatment drastically reduced both the number and the size of iPSC colonies and notably diminished the staining of the pluripotency marker alkaline phosphatase. Our current findings, altogether, reveal for the first time that metformin can efficiently lower the threshold for SIS to generate an “stressed” cell phenotype that becomes pre-sensitized to oncogenic-like stimuli, including DNA damaging, proliferative and/or stemness inducers.

Phospho-ΔNp63α-dependent regulation of autophagic signaling through transcription and micro-RNA modulation

Yiping Huang, Rafael Guerrero-Preston and Edward A. Ratovitski
Pages 1247 - 1259
http://dx.doi.org/10.4161/cc.11.6.19670
Abstract | PDF

Cisplatin was shown to induce the ataxia telangiectasia mutated (ATM)-dependent phosphorylation of tumor protein p63 isoform, (ΔNp63α), leading to a transcriptional regulation of specific genes implicated in the control of cell death of squamous cell carcinoma (SCC) cells. We previously observed that the cisplatin-induced phosphorylated (p)-ΔNp63α transcriptionally regulates the expression of specific microRNAs (miRNAs) in SCC cells. We found here that cisplatin exposure of SCC cells led to modulation of the members of the autophagic pathway, such as Atg1/Ulk1, Atg3, Atg4A, Atg5, Atg6/Becn1, Atg7, Atg9A and Atg10, by a direct p-ΔNp63α-dependent transcriptional regulation. We further found that specific miRNAs (miR-181a, miR-519a, miR-374a and miR-630), which are critical downstream targets of the p-ΔNp63α, modulated the protein levels of ATG5, ATG6/BECN1, ATG10, ATG12, ATG16L1 and UVRAG, adding another level of expression control for autophagic pathways in SCC cells upon cisplatin exposure. Our data support the notion that the cisplatin-induced p-ΔNp63α could regulate key pathways implicated in response of cancer cells to chemotherapeutics.