Table of Contents

Volume 8, Issue 10

  May 15, 2009

  Pages 1461 - 1636

View covers archive

 Issue Catalogue (MARC XML)

EDITORIALS: CELL CYCLE FEATURES

Open Access Article

On the origin of cancer-associated fibroblasts

Charlotte Anderberg and Kristian Pietras
Pages 1461 - 1465
http://dx.doi.org/10.4161/cc.8.10.8557
PDF

Open Access Article

Convergent pathways link the endothelin A receptor to the β-catenin: The β-arrestin connection

Laura Rosanò and Anna Bagnato
Pages 1461 - 1465
http://dx.doi.org/10.4161/cc.8.10.8559
PDF

Open Access Article

Arginine methylation in interferon signaling: New light on an old story

Susanne Weber and Uta-Maria Bauer
Pages 1461 - 1465
http://dx.doi.org/10.4161/cc.8.10.8560
PDF




NEWS & VIEWS

Open Access Article

p63 regulation by microRNAs

Giovanni Blandino and Ute M. Moll
Pages 1466 - 1470
http://dx.doi.org/10.4161/cc.8.10.8766
PDF

Open Access Article

A novel player in the p53-mediated autophagy: Sestrin2

Marcello D’Amelio and Francesco Cecconi
Pages 1466 - 1470
http://dx.doi.org/10.4161/cc.8.10.8767
PDF

Open Access Article

End of the Daxx diatribe?

Paolo Salomoni and David Michod
Pages 1466 - 1470
http://dx.doi.org/10.4161/cc.8.10.8774
PDF

Open Access Article

Living with reduced replicative helicase protein

William K. Kaufmann
Pages 1466 - 1470
http://dx.doi.org/10.4161/cc.8.10.8781
PDF

Open Access Article

Linking DNA replication with cell cycle control

Sheng Wang
Pages 1466 - 1470
http://dx.doi.org/10.4161/cc.8.10.8782
PDF

Open Access Article

Dr. Wafik S. El-Deiry hosts the 199th Meeting of the Interurban Clinical Club


Pages 1466 - 1470
http://dx.doi.org/10.4161/cc.8.10.8784
Abstract | PDF

Wafik S. El-Deiry, M.D., Ph.D., American Cancer Society Research Professor at the University of Pennsylvania, organized and hosted in Philadelphia the 199th Meeting of the Interurban Clinical Club (interurbanclinicalclub.org) started by Sir William Osler in 1905. The Scientific Meeting was held at the College of Physicians of Philadelphia and featured outstanding presentations of the most exciting biomedical research in Philadelphia. The evening black-tie dinner event was held at the historic Union League of Philadelphia where Margaret Foti, Ph.D., M.D. (h.c.), Chief Executive Officer of the American Association for Cancer Research addressed the attendees.




EDITOR'S CORNER: IN THEIR OWN WORDS

Open Access Article

An interview with Dr. Zbigniew Darzynkiewicz on his highly cited paper published in Cell Cycle


Pages 1471 - 1472
http://dx.doi.org/10.4161/cc.8.10.8623
Abstract | PDF

Zbigniew Darzynkiewicz is the Director of the Brander Cancer Research Institute and Professor of Pathology and Medicine at the New York Medical College. He received his M.D. (with the highest honors) and Ph.D. degrees from the Medical University of Warsaw in Warsaw, Poland and completed post-graduate studies at the State University of New York at Buffalo and at the Medical Nobel Institute of Karolinska Institute, Stockholm, Sweden. His research is focused on the regulatory mechanisms associated with cell proliferation, apoptosis and sensitivity to anti-tumor drugs. He published over 550 original articles in peer-reviewed journals, over 100 chapters and reviews, authored/edited 12 books and holds 7 US patents. His publications were cited over 23,000 times and 79 of them have over 79 citations each, which ranks him at number 79 in Hirsch’s impact “h-index.”




EXTRA VIEWS

Open Access Article

Activation of hepatitis c virus translation by a liver-specific microRNA

Michael Niepmann
Pages 1473 - 1477
http://dx.doi.org/10.4161/cc.8.10.8349
Abstract | PDF

microRNAs (miRNAs) are involved in the regulation of translation of many mRNAs and affect cell cycle progression, differentiation and metabolism. The liver-specific microRNA-122 (miR-122) contributes to the liver tropism of Hepatitis C Virus (HCV) by accelerating the binding of ribosomes to the viral RNA and thereby stimulating HCV translation. Here, the most recent results on the molecular mechanisms of microRNA action on HCV and the implications for the understanding of translation regulation by microRNAs are discussed.

Open Access Article

Role of p53 in the anti-proliferative effects of Sirt1 inhibition in prostate cancer cells

Brittney Jung-Hynes and Nihal Ahmad
Pages 1478 - 1483
http://dx.doi.org/10.4161/cc.8.10.8408
Abstract | PDF

Prostate Cancer (PCa), next only to skin cancer, is the most commonly occurring malignancy in men in the USA. Aging is recognized as a major risk factor for this neoplasm as a man’s chance for developing this disease significantly increases with increasing age. Because aging is inevitable, Americans are living longer, and the existing treatments have not been able to manage this neoplasm, novel mechanism-based approaches are needed. We have recently shown that Sirt1, a sirtuin class III histone deacetylases (HDACs) originally linked to aging and longevity in yeast, was overexpressed in human PCa cells and PCa tissues obtained from patients. We also found that chemical inhibition and/or genetic knockdown of Sirt1 caused a FoxO1-mediated inhibition in the growth and viability of human PCa cells. Since p53 is a target for deacetylation by Sirt1, we wanted to determine the involvement of p53 in Sirt1 inhibition mediated responses in PCa. To achieve our objective, we utilized a pair of isogenic PCa cell lines viz. PC3 and PC3-p53, which differ only in p53 status. Our data demonstrated that Sirt1 inhibition caused a decrease in cell growth, cell viability and the colony formation ability of both cell lines. Further, Sirt1 inhibition resulted in an increase in FoxO1 acetylation and subsequent transcriptional activation in both cell types regardless of p53 status. However, an interesting observation of our study was that Sirt1 inhibition resulted in an increase in senescence in PC3-p53 cells whereas its resulted in an increase in apoptosis in PC3 cells. The results of this study compliment our previous study and suggest that Sirt1 inhibition may have different downstream targets in cells with active p53 versus cells where p53 is inactive.

Open Access Article

Rho-GTPase signaling drives melanoma cell plasticity

Victoria Sanz-Moreno and Christopher J. Marshall
Pages 1484 - 1487
http://dx.doi.org/10.4161/cc.8.10.8490
Abstract | PDF

To investigate mechanisms that underlie different modes of tumor cell movement we have studied how regulation of the activity of the Rho family GTPases determines the mode of tumor cell movement. Guanine nucleotide exchange factors (GEFs) and GTPase accelerating proteins (GAPs) are key regulators of the activity of small GTPases with GEFs promoting activation to the GTP bound state and GAPs promoting inactivation by stimulating GTP hydrolysis. We identified two important signaling pathways regulating amoeboid and mesenchymal types of motility in melanoma. Here, we discuss our findings in the context of how specificity of Rho signaling is achieved by GEFs and GAPs.

Open Access Article

Lamin complexes in the nuclear interior control progenitor cell proliferation and tissue homeostasis

Nana Naetar and Roland Foisner
Pages 1488 - 1493
http://dx.doi.org/10.4161/cc.8.10.8499
Abstract | PDF

Lamins are components of the peripheral nuclear lamina that has important mechanical roles in nuclear architecture. Lamins have increasingly been implicated in nuclear processes, including DNA replication, chromatin organization and transcription. Recent data highlight the specific functions of a small pool of lamina-independent A-type lamins, located throughout the nucleoplasm, in the regulation of cell cycle progression in early tissue progenitor cells. This novel role of nucleoplasmic lamins is likely mediated by their regulatory activity towards the tumor suppressor Retinoblastoma protein. We describe how an interaction partner of A-type lamins, lamina-associated polypeptide 2α (LAP2α) affects targeting of lamins A and C to the nuclear interior and how this complex may affect cell cycle progression and tissue homeostasis. Finally, we discuss implications of these novel findings for understanding the pathogenesis of laminopathies, a group of human diseases caused by mutations in lamins.

Open Access Article

Paving the way for H2AX phosphorylation: Chromatin changes in the DNA damage response

Nabieh Ayoub, Anand D. Jeyasekharan, Juan A. Bernal and Ashok R. Venkitaraman
Pages 1494 - 1500
http://dx.doi.org/10.4161/cc.8.10.8501
Abstract | PDF

The dynamics of chromatin-associated proteins control the accessibility of DNA to essential biological transactions like transcription, replication, recombination and repair.  Here, we briefly outline what is known about the chromatin changes that occur during the cellular response to DNA breakage, focusing on our recent findings revealing that the chromatin factor HP1β is mobilized within seconds after DNA damage by an unrecognized signalling cascade mediated by casein kinase 2 (CK2) phosphorylation, paving the way for histone H2AX phosphorylation.  We also show here that HP1β mobilization is neither associated with histone H3 modification on Ser10, an alteration proposed to assist in HP1 ejection from chromatin, nor with evidence of a physical interaction between HP1β and the CK2 regulatory subunit. Interestingly, following its rapid mobilization, we find that HP1β gradually re-accumulates on damaged chromatin over a longer time period, suggesting that temporal changes in HP1β dynamics and interaction with chromatin may assist in different stages of the cellular response to DNA breakage.




PERSPECTIVES

Open Access Article

Developmentally programmed endoreduplication in animals

Zakir Ullah, Chrissie Y. Lee, Mary A. Lilly and Melvin L. DePamphilis
Pages 1501 - 1509
http://dx.doi.org/10.4161/cc.8.10.8325
Abstract | PDF

Development of a fertilized egg into an adult human requires trillions of cell divisions, the vast majority of which duplicate their genome once and only once. Nevertheless, trophoblast giant cells and megakaryocytes in mammals circumvent this rule by duplicating their genome multiple times without undergoing cell division, a process generally referred to as 'endoreduplication.' In contrast, arthropods such as Drosophila endoreduplicate their genome in most larval tissues, as well as in many adult tissues. Endoreduplication requires that cells prevent entrance into or completion of mitosis and cytokinesis under conditions that permit assembly of prereplication complexes. In addition, cells must prevent induction of apoptosis in response to incomplete DNA replication or DNA damage that may occur during the ensuing sequence of ‘endocycles’. Thus, developmentally regulated endoreduplication results in terminal cell differentiation. Recent progress has revealed both differences and similarities in the mechanisms employed by flies and mammals to change from mitotic cell cycles to 'endocycles.' The critical step, however, appears to be switching from a CDK-dependent form of the anaphase promoting complex (APC) to one that functions only in the absence of CDK activity.

Open Access Article

Division of labor: DNA repair and the cell cycle specific functions of the Mre11 complex

Carrie A. Adelman and John H.J. Petrini
Pages 1510 - 1514
http://dx.doi.org/10.4161/cc.8.10.8380
Abstract | PDF

Genomic integrity is maintained via the concerted action of proteins that coordinate and control DNA replication and those that respond to DNA damage.  The Mre11 complex is a mediator of the DNA damage response through its functions in DNA double strand break (DSB) sensing, checkpoint activation and recombinational DNA repair.  The complex responds to mitotic and meiotic DSBs, and is also activated in cells experiencing DNA replication stress.  The Mre11 complex’s role in recombinational repair primarily concerns the promotion of homologous recombination (HR), but it is also implicated in non-homologous end joining (NHEJ)- a DSB repair mechanism prevalent in non-dividing cells.  We recently characterized deletion of the Mre11 complex member, Rad50, in a number of postmitotic and proliferative tissues of the mouse.  These studies indicated that the complex is dispensable in postmitotic tissues, but loss of Rad50 in proliferating cells resulted in accumulation of unrepaired, DNA replication-dependent lesions.  The data suggest that the Mre11 complex is not a major contributor to NHEJ and support the interpretation that its role in recombinational DNA repair is largely restricted to dividing cells, in which repair involving sister chromatids predominates.  An exception to this concept is manifest in previous work from our laboratory revealing that the mammalian Mre11 complex promotes meiotic DSB repair, an event involving recombination between sister chromatids of homologous chromosomes and taking place in cells not undergoing replication.  Together these studies highlight the importance of cell cycle and cell type specific modulation of the Mre11 complex’s repair activities in vivo.

Open Access Article

Defining pathways that enforce cell lineage specification in early development and stem cells

Stephen Roper and Myriam Hemberger
Pages 1515 - 1525
http://dx.doi.org/10.4161/cc.8.10.8381
Abstract | PDF

The molecular processes that govern the first cell lineage decisions after fertilization also dictate the developmental potency of stem cells derived from the early mouse embryo. Our understanding of these mechanisms is therefore instrumental for stem cell biology and regenerative medicine. A number of transcription factors are known that determine a cell’s fate towards either the embryonic or extraembryonic trophoblast lineages. Recent insights have shown that the definitive fixation of cell lineage fate is achieved by an epigenetic restriction through DNA methylation of the transcription factor Elf5. Lineage crossover can be induced, however, by manipulation of determinants and gatekeepers, or their epigenetic regulation. Here we summarize the accumulating number of experimental conditions where such ‘transdifferentiation’ is observed that shed light onto the genetic and epigenetic pathways involved in lineage separation and the developmental potential of stem cells.

Open Access Article

DNA excision repair proteins and Gadd45 as molecular players for active DNA demethylation

Dengke K. Ma, Junjie U. Guo, Guo-li Ming and Hongjun Song
Pages 1526 - 1531
http://dx.doi.org/10.4161/cc.8.10.8500
Abstract | PDF

DNA cytosine methylation represents an intrinsic modification signal of the genome that plays important roles in heritable gene silencing, heterochromatin formation and certain transgenerational epigenetic inheritance. In contrast to the process of DNA methylation that is catalyzed by specific classes of methyltransferases, molecular players underlying active DNA demethylation have long been elusive. Emerging biochemical and functional evidence suggests that active DNA demethylation in vertebrates can be mediated through DNA excision repair enzymes, similar to the well-known repair-based DNA demethylation mechanism in Arabidopsis. As key regulators, non-enzymatic Gadd45 proteins function to recruit enzymatic machineries and promote coupling of deamination, base and nucleotide-excision repair in the process of DNA demethylation. In this article, we review recent findings and discuss functional and evolutionary implications of such mechanisms underlying active DNA demethylation.




REVIEWS

Open Access Article

Solving the RIDDLE of 53BP1 recruitment to sites of damage

Grant S. Stewart
Pages 1532 - 1538
http://dx.doi.org/10.4161/cc.8.10.8351
Abstract | PDF

The cellular response to DNA double strand breaks is a complex, integrated network of pathways, coordinated by the PI-3-kinase-like family of kinases, which includes ATM, ATR and DNA-PK, that function to preserve the integrity of the genome. Mutations in genes that control these pathways are associated with increased genomic instability, neurodegeneration, immunodeficiency, premature aging and tumour predisposition. Indeed a significant proportion of our understanding regarding the mechanisms controlling DNA double strand break (DSB) repair has come from the study of cells derived from patients with inherited mutations in these genes. The discovery of the E3 ubiquitin ligase, RNF8, as a regulator of DNA DSB repair has brought to light a critical role for the ubiquitin system in regulating the cellular DSBs. Recently, identification of mutations in a second E3 ubiquitin ligase, RNF168, as the underlying genetic cause of the DNA repair deficiency disorder, RIDDLE syndrome, has provided the first link between ubiquitin-dependent DSB repair and immune system development in man. The finding that RNF168 functions downstream of RNF8 to orchestrate the recruitment of repair proteins, such as BRCA1 and 53BP1, to sites of DNA damage suggests that these two E3 ligases define a ubiquitylation cascade that regulates the spatial relocalisation of DSB repair proteins.

Open Access Article

Pathogenesis of rheumatoid arthritis and c-Fos/AP-1

Shunichi Shiozawa and Ken Tsumiyama
Pages 1539 - 1543
http://dx.doi.org/10.4161/cc.8.10.8411
Abstract | PDF

c-Fos/AP-1 controls the expression of inflammatory cytokines and matrix-degrading matrix metalloproteinases (MMPs) important in arthritis via promoter AP-1 binding motif. Among inflammatory cytokines, IL-1β is the most important inducer of a variety of MMPs, and mainly responsible for cartilage breakdown and osteoclastogenesis. IL-1β and c-Fos/AP-1 influence each other’s gene expression and activity, resulting in an orchestrated cross-talk that is crucial to arthritic joint destruction, where TNFα can act synergistically with them. While how to stop the degradation of bone and cartilage, i.e., to control MMP, has long been the central issue in the research of rheumatoid arthritis (RA), selective inhibition of c-Fos/AP-1 does resolve arthritic joint destruction. Thus, the blockade of IL-1β and/or c-Fos/AP-1 can be promising as an effective therapy for rheumatoid joint destruction in addition to the currently available TNFα blocking agents that act mainly on arthritis.




REPORTS

Open Access Article

Daxx is a predominately nuclear protein that does not translocate to the cytoplasm in response to cell stress

Cory R. Lindsay, Serena Giovinazzi and Alexander M. Ishov
Pages 1544 - 1551
http://dx.doi.org/10.4161/cc.8.10.8379
Abstract | PDF

The intracellular translocation of Daxx to the cytoplasm is a phenomenon often attributed to cells undergoing stress, opposite to predominant nuclear localization of this protein under normal homeostatic conditions. Moreover, a number of reports have suggested that export to the cytosol upon several stress conditions, including oxidative stress, glucose deprivation and beta-amyloid peptide treatment, is indispensable for the proper execution of Daxx-induced apoptosis.  On the contrary, other studies have described translocation of Daxx from cytoplasm to nucleus upon stress application.   Here, we examined cellular distribution of Daxx by sub-cellular fractionation and immunofluorescent localization of endogenous protein, using SH-SY5Y neuroblastoma cell line previously reported to exhibit cytoplasmic translocation of Daxx after oxidative stress and beta-amyloid exposure. In control conditions, Daxx is an exclusively nuclear protein in SH-SY5Y cells. Short treatment by either H2O2 or beta-amyloid did not show any significant change in nuclear localization of Daxx. Prolonged exposure of cells to stress compounds did not alter the intracellular deposition of Daxx that remains exclusively in the nucleus.  A cohort of other cell lines, including human prostate cancer cell line DU-145, previously reported to exhibit stress-induced cytosol translocation was examined for Daxx distribution and none were confirmed to show re-localization of Daxx to the cytoplasm after either short or long stress.  Time-lapse visualization of Daxx-GFP upon H2O2 treatment or glucose deprivation did not show cytoplasmic translocation either. Thus, while several Daxx-dependent apoptotic mechanisms have been described, the cytosolic association and function of this protein is questionable in light of these findings.

Open Access Article

Annexin A5 is involved in migration and invasion of oral carcinoma

Liane Wehder, Stephanie Arndt, Ulrike Murzik, Anja K. Bosserhoff, Robert Kob, Ferdinand von Eggeling and Christian Melle
Pages 1552 - 1558
http://dx.doi.org/10.4161/cc.8.10.8404
Abstract | PDF

Annexin A5 is a Ca2+-binding protein which is involved in membrane organization and dynamics. As recent data suggest a role of annexin A5 in cancer we aimed to gain more insight into the biological function of endogenous annexin A5 and assessed its possible influence on proliferation and invasion capacity. We down-regulated annexin A5 by RNA interference in HaCaT keratinocytes, squamous carcinoma cell line A431 as well as in a primary cell culture of a human oral carcinoma. Hereby, we detected reduced migration and invasion capacity of HaCaT cells which was even stronger in the oral carcinoma. To determine target genes of annexin A5 we used a metastasis specific microarray. Thereby, genes implicated in cell motility including S100A4, TIMP-3, and RHOC were observed to be regulated. These deregulations were confirmed by RT-PCR or western blots, respectively. These observations suggest that the invasion capacity, a main characteristic of tumors, is at least partially regulated by annexin A5 in oral carcinoma.

Open Access Article

Targeting transcription factor NFκB: comparative analysis of proteasome and IKK inhibitors

Alexander V. Gasparian, Olga A. Guryanova, Dmitry V. Chebotaev, Alexander A. Shishkin, Alexander Y. Yemelyanov and Irina V. Budunova
Pages 1559 - 1566
http://dx.doi.org/10.4161/cc.8.10.8415
Abstract | PDF

Nuclear factor-κB (NF-κB) plays a critical role in cancer development and progression. Thus, the NF-κB signaling pathway provides important targets for cancer chemoprevention and anti-cancer chemotherapy. The central steps in NF-κB activation are phosphorylation and proteasome-dependent degradation of its inhibitory proteins termed IκBs. Consequently, the major pharmacological approaches to target NF-κB include (i) repression of IκB kinases (IKKs) and (ii) blocking the degradation of IκBs by proteasome inhibitors. We quantitatively compared the efficacy of various proteasome inhibitors (MG132, lactacystin and epoxomicin) and IKK inhibitors (BAY 11-7082 and PS1145) to block NF-κB activity induced by TNFα or TPA and to sensitize LNCaP prostate carcinoma cells to apoptosis. Our studies revealed significant differences between these two classes of NF-κB inhibitors. We found that proteasome inhibitors epoxomicin and MG132 attenuated NF-κB induction much more effectively than the IKK inhibitors. Furthermore, in contrast to IKK inhibitors, all studied proteasome inhibitors specifically blocked TPA-induced generation de novo of NF-κB p50 homodimers – (p50/p50). These results suggest that the proteasome plays a dominant role in TPA-induced formation of functional p50 homodimers, while IKK activity is less important for this process. Interestingly, profound attenuation of p50/p50 DNA-binding does not reduce the high potency of proteasome inhibitors to suppress NF-κB-dependent transcription. Finally, proteasome inhibitors were much more effective in sensitizing LNCaP cells to TNFα-induced apoptosis compared to IKK inhibitors at the concentrations when both types of agents similarly attenuated NF-κB activity. We conclude that this remarkable pro-apoptotic potential of proteasome inhibitors is partially mediated through NF-κB-independent mechanism.

Open Access Article

Wnt/β-catenin signaling pathway may regulate cell cycle and expression of cyclin A and cyclin E protein in hepatocellular carcinoma cells

Xinhong Wang, Xiangwei Meng, Xun Sun, Mingna Liu, Shanling Gao, Jing Zhao, Fenghua Pei and Hongying Yu
Pages 1567 - 1570
http://dx.doi.org/10.4161/cc.8.10.8489
Abstract | PDF

Wnt/β-catenin signaling pathway and cell cycle play the key roles during the genesis and development of hepatocellular carcinoma (HCC). The cytoplasmic protein β-catenin is a multifunctional protein and a central molecule in the Wnt signaling pathway. Cell cycle is regulated by a a series of regulatory factors. Current researches indicated that expression of cyclin D1 and c-myc decreased after silencing β-catenin gene in HCC, but it is unclear if other cyclins are affected. To determine the relation, small interference RNA(siRNA) against β-catenin was transfected into HCC cell line HepG2, and cell cycle and cyclin A and cyclin E protein expression were detected. We demonstrated that cell cycle was arrested in G0/G1 at 72 h after the transfection and with the time passing, the cell cycle began to transfer from G0/G1 to G2/M through S and had a trend to revert at 96 h. In addition, β-catenin protein expression was decreased at both 72 and 96 h, although the level was slightly higher at 96 h than that at 72 h. However, cyclin A and cyclin E protein expression increased at 72 h and decreased at 96 h. These findings suggest that silencing β-catenin gene may induce the changes of cell cycle and cyclin A and cyclin E expression. Wnt/β-catenin signaling pathway probably takes part in the genesis and development of HCC through regulating cell cycle and the expression of cyclin A and cyclin E.

Open Access Article

Stimulation of autophagy by the p53 target gene Sestrin2

Maria Chiara Maiuri, Shoaib A. Malik, Eugenia Morselli, Oliver Kepp, Alfredo Criollo, Pierre-Luc Mouchel, Rosa Carnuccio and Guido Kroemer
Pages 1571 - 1576
http://dx.doi.org/10.4161/cc.8.10.8498
Abstract | PDF

The oncosuppressor protein p53 regulates autophagy in a dual fashion. The pool of cytoplasmic  p53 protein represses autophagy in a transcription-independent fashion, while the pool of nuclear p53 stimulates autophagy through the transactivation of specific genes. Here we report the discovery that Sestrin2, a novel p53 target gene, is involved in the induction of autophagy. Depletion of Sestrin2 by RNA interference reduced the level of autophagy in a panel of p53-sufficient human cancer cell lines responding to distinct autophagy inducers. In quantitative terms, Sestrin2 depletion was as efficient in preventing autophagy induction as was the depletion of Dram, another p53 target gene. Knockout of either Sestrin2 or Dram reduced autophagy elicited by nutrient depletion, rapamycin, lithium or thapsigargin. Moreover, autophagy induction by nutrient depletion or pharmacological stimuli led to an increase in Sestrin2 expression levels in p53-proficient cells. In strict contrast, the depletion of Sestrin2 or Dram failed to affect autophagy in p53-deficient cells and did not modulate the inhibition of baseline autophagy by a cytoplasmic p53 mutant that was reintroduced into p53-deficient cells. We conclude that Sestrin2 acts as a positive regulator of autophagy in p53-proficient cells.

Open Access Article

Reduced expression of GINS complex members induces hallmarks of pre-malignancy in primary untransformed human cells

Laura R. Barkley, Ihn Young Song, Ying Zou and Cyrus Vaziri
Pages 1577 - 1588
http://dx.doi.org/10.4161/cc.8.10.8535
Abstract | PDF

In cancer cells ablation of the GINS complex member Psf2 elicits chromosome mis-segregation yet the precise role of Psf2 in mitosis is unknown.  We investigated the putative mitotic role of the GINS complex using synchronized cultures of untransformed Human Dermal Fibroblasts (HDF).  Metaphase spreads from Psf1/Psf2-depleted HDF were normal and mitotic exit of Psf1/Psf2-depleted cells was only slightly delayed, suggesting no direct role for the GINS complex in mitosis of untransformed cells.  Because the GINS complex is required for initiation and elongation events during DNA replication we hypothesized that the mitotic delay of Psf1/Psf2-deficient cells resulted indirectly from defective DNA synthesis during a prior S-phase.  Therefore, we investigated the effects of Psf1/Psf2-depletion on DNA replication.  Recruitment of Mcm7 to chromatin during G1 was unaffected by Psf1/Psf2-ablation, indicating that replication licensing does not require GINS.  However, chromatin-binding of Cdc45 and PCNA, onset of DNA synthesis and accumulation of G2/M markers were delayed in Psf1/Psf2-ablated cells.  The cell cycle delay of Psf1/Psf2-depleted HDF was associated with several hallmarks of pre-malignancy including γH2AX, Thr 68-phosphorylated Chk2, and increased numbers of aberrant fragmented nuclei.  Ectopic expression of catalytically-inactive Chk2 promoted S-phase and G2/M progression in Psf1/Psf2-depleted cells, as evidenced by modestly-increased rates of DNA synthesis and increased dephosphorylation of Cdc2.  Therefore, S-phase progression of untransformed cells containing sub-optimal levels of Psf1/2 is associated with replication stress and acquisition of DNA damage.  The ensuing Chk2-mediated DNA damage signalling likely contributes to maintenance of chromosomal integrity.

Open Access Article

AKT1 mediates bypass of the G1/S checkpoint after genotoxic stress in normal human cells

Madhu A. Lal, Dongsoon Bae, Tura. C. Camilli, Steven R. Patierno and Susan Ceryak
Pages 1589 - 1602
http://dx.doi.org/10.4161/cc.8.10.8547
Abstract | PDF

Certain forms of hexavalent chromium [Cr(VI)] are human carcinogens. Our recent work has shown that a broad range protein tyrosine phosphatase (PTP) inhibitor, sodium orthovanadate (SOV), abrogated both Cr(VI)-induced growth arrest and clonogenic lethality. Notably, SOV enhanced Cr(VI) mutation frequency, ostensibly through forced survival of genetically damaged cells. In the present study, co-treatment with this PTP inhibitor bypassed the Cr(VI)-induced G1/S checkpoint arrest in diploid human lung fibroblasts (HLF). Moreover, the PTP inhibitor abrogated the Cr(VI)-induced decrease in the expression of key effectors of the G1/S checkpoint [Cyclin D1, phospho Ser 807/811 Rb (pRB), p27].  Cr(VI)-induced G1 arrest was associated with the cytoplasmic appearance of pRb and the nuclear localization of p27, both of which were reversed by the PTP inhibitor. The PTP inhibitor’s reversal of G1/S checkpoint effector localization after Cr exposure was found to be Akt1-dependent, as this was abrogated by transfection with either akt1 siRNA or an Akt1-kinase dead plasmid.  Furthermore, Akt1 activation alone was sufficient to induce G1/S checkpoint bypass and to prevent Cr(VI)-induced changes in pRb and p27 localization. In conclusion, this work establishes Akt1 activation to be both sufficient to bypass the Cr(VI)-induced G1/S checkpoint, as well as necessary for the observed PTP inhibitor effects on key mediators of the G1/S transition.  The potential for Akt to bypass G1/S checkpoint arrest in the face of genotoxic damage could increase genomic instability, which is a hallmark of neoplastic progression.

Open Access Article

Dissection of the sequence-specific DNA binding and exonuclease activities reveals a superactive yet apoptotically impaired mutant p53 protein

Jinwoo Ahn, Masha V. Poyurovsky, Nicole Baptiste, Rachel Beckerman, Christine Cain, Melissa Mattia, Kristine McKinney, Jianmin Zhou, Andrew Zupnick, Vanesa Gottifredi and Carol Prives
Pages 1603 - 1615
http://dx.doi.org/10.4161/cc.8.10.8548
Abstract | PDF

Both sequence-specific DNA binding and exonuclease activities have been mapped to the central conserved core domain of p53.  To gain more information about these two activities a series of mutants were generated that changed core domain histidine residues.  Of these mutants, only one, H115N p53, showed markedly reduced exonuclease activity (ca. 15% of wild-type). Surprisingly, purified H115N p53 protein was found to be significantly more potent than wild-type p53 in binding to DNA by several criteria including gel mobility shift assay, filter binding and DNase I footprinting.  Interestingly as well, non-specific DNA binding by the core domain of H115N p53 is superior to that of wild-type p53.  To study H115N p53 in vivo, clones of H1299 cells expressing tetracycline regulated wild-type or H115N p53 were generated.  H115N was both more potent than wild-type p53 in inducing p53 target genes such as p21 and PIG3 and was also more effective in arresting cells in G1.  Unexpectedly, in contrast to wild-type p53, H115N p53 was markedly impaired in causing apoptosis when cells were subjected to DNA damage.  Our results indicate that the exonuclease activity and transcriptional activation functions of p53 can be separated.  They also extend previous findings showing that cell cycle arrest and apoptosis are separable functions of p53. Finally, these experiments confirm that DNA binding and exonuclease activities are distinct features of the p53 core domain.

Open Access Article

Robust autophagy/mitophagy persists during mitosis

Leyuan Liu, Rui Xie, Susan Nguyen, Min Ye and Wallace L. McKeehan
Pages 1616 - 1620
http://dx.doi.org/10.4161/cc.8.10.8577
Abstract | PDF

From microscopic observations of autophagosome content it has been argued that autophagy is shut down during mitosis to protect the relative short-lived organelles spindle and chromosomes from the process while they are contiguous with cytosol.  However, without autophagy, buildup of dysfunctional mitochondria arising from the intense energy demands of mitosis potentially poses a hazard to accurate partition of chromosomes.  Here we show using biochemical markers of autophagosomes and mitophagosomes and a blockade at the lysosomal clearance step that autophagy/mitophagy persists during mitosis at robust levels equal to interphase.  This suggests a mechanism that insulates normal spindle and chromosomes from autophagy and potentially recognition of defects in spindle and chromosomes by the autophagic process.

Open Access Article

Prohibitin physically interacts with MCM proteins and inhibits mammalian DNA replication

Wasia Rizwani, Mark G. Alexandrow and Srikumar P. Chellappan
Pages 1621 - 1629
http://dx.doi.org/10.4161/cc.8.10.8578
Abstract | PDF

Prohibitin, a tumor suppressor protein, has been shown to repress E2F-mediated transcription and arrest cell cycle progression.  While prohibitin has been proposed to regulate cell cycle progression by repressing transcriptional targets of E2F1, it is not clear whether other mechanisms are also involved in mediating the growth arrest. Here we demonstrate that prohibitin can function as a potent inhibitor of DNA replication by interacting with members of Minichromosome maintenance complex of proteins (MCM2-7). The data presented here indicates that prohibitin can physically interact with MCM2, MCM5 and MCM7 in in vitro GST binding assays as well as in MCF-7 cells as seen by immunoprecipitation-Western blot experiments.  The association was cell cycle dependent, and more pronounced 4-8 hours after serum stimulation of quiescent cells. Prohibitin associated more robustly with MCM2 and MCM5 compared to MCM7, suggesting that prohibitin mainly interacts with the regulatory subunits of the MCM complex. Confirming these results, prohibitin was found to co-localize with MCM2, MCM5 and MCM7 in MCF-7 cells, as seen by double immunofluorescence experiments. Further, Prohibitin strongly inhibited DNA replication in an in vitro replication assay. These results strongly suggest that prohibitin effectively represses replication by interacting with the components of mammalian replication machinery and this might contribute to the growth regulatory properties of prohibitin.




LETTERS TO THE EDITOR

Open Access Article

Activation of a unique p53-dependent DNA damage response

Jun Yang, Afshan Ahmed and Margaret Ashcroft
Pages 1630 - 1632
http://dx.doi.org/10.4161/cc.8.10.8326
PDF

Open Access Article

Genome-wide inhibitory impact of the AMPK activator metformin on [kinesins, tubulins, histones, auroras and polo-like kinases] M-phase cell cycle genes in human breast cancer cells

Cristina Oliveras-Ferraros, Alejandro Vazquez-Martin and Javier A. Menendez
Pages 1633 - 1636
http://dx.doi.org/10.4161/cc.8.10.8406
Abstract | PDF

Prompted by the ever-growing scientific rationale for examining the antidiabetic drug metformin as a potential antitumor agent in breast cancer disease, we recently tested the hypothesis that the assessment of metformin-induced global changes in gene expression –as identified using 44K (double density) Agilent’s whole human genome arrays- could reveal gene-expression signatures that would allow proper selection of breast cancer patients who should be considered for metformin-based clinical trials.  Using Database for Annotation, Visualization and Integrated Discovery bioinformatics (DAVID) resources we herein reveal that, at doses that lead to activation of the AMP-activated protein kinase (AMPK), metformin not only down-regulates genes coding for ribosomal proteins (i.e. protein and macromolecule biosynthesis) but unexpectedly suppresses numerous mitosis-related gene families including kinesins, tubulins, histones, auroras and polo-like kinases. This is, to our knowledge, the first genome-scale evidence of a mitotic core component in the transcriptional response of human breast cancer cells to metformin. These findings further support a tight relationship between the activation status of AMPK and the chromosomal and cytoskeletal checkpoints of cell mitosis at the transcriptional level.