Table of Contents

Volume 8, Issue 19

  October 1, 2009

  Pages 3057 - 3240

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 Issue Catalogue (MARC XML)

EDITORIAL

Open Access Article

Real-time case fatality analysis points to emerging evidence of increasing severity of pandemic (H1N1) 2009

Gennadi V. Glinsky
Pages 3057 - 3062
http://dx.doi.org/10.4161/cc.8.19.9817
Abstract | PDF

Comment on: Fraser C, Donnelly CA, Cauchemez S, Hanage WP, Van Kerkhove MD, Hollingsworth TD, et al. WHO Rapid Pandemic Assessment Collaboration. Pandemic potential of a strain of influenza A (H1N1): early findings. Science 2009; 324:1557-61.




EDITORIALS: CELL CYCLE FEATURES

Open Access Article

The protein tyrosine phosphatase receptor D, a broadly inactivated tumor suppressor regulating STAT function

Timothy A. Chan and Adriana Heguy
Pages 3063 - 3064
http://dx.doi.org/10.4161/cc.8.19.9455
Abstract | PDF

Comment on: Veeriah S, Brennan C, Meng S, Singh B, Fagin JA, Solit DB, et al. The tyrosine phosphatase PTPRD is a tumor suppressor that is frequently inactivated and mutated in glioblastoma and other human cancers. Proc Natl Acad Sci USA 2009; 106:9435-40.

Open Access Article

On the origins of ultra-fine anaphase bridges

Kok Lung Chan and Ian D. Hickson
Pages 3065 - 3066
http://dx.doi.org/10.4161/cc.8.19.9513
Abstract | PDF

Comment on: Chan KL, Palmai-Pallag T, Ying S, Hickson ID. Replication stress induces sister-chromatid bridging at fragile site loci in mitosis. Nat Cell Biol 2009; 11:753-60.

Open Access Article

Co-ordinated regulation of translation following DNA damage

Alexander V. Kondrashov, Keith A. Spriggs, Martin Bushell and Anne E. Willis
Pages 3067 - 3068
http://dx.doi.org/10.4161/cc.8.19.9467
Abstract | PDF

Comment on: Powley IR, Kondrashov A, Young LA, Dobbyn HC, Hill K, Cannell IG, et al. Translational reprogramming following UVB irradiation is mediated by DNA-PKcs and allows selective recruitment to the polysomes of mRNAs encoding DNA repair enzymes. Genes Dev 2009; 15:1207-20.

Open Access Article

The type III TGF-β receptor regulates directional migration: New tricks for an old dog

Karthikeyan Mythreye and Gerard C. Blobe
Pages 3069 - 3070
http://dx.doi.org/10.4161/cc.8.19.9419
Abstract | PDF

Comment on: Mythreye K, Blobe GC. The type III TGF-{beta} receptor regulates epithelial and cancer cell migration through {beta}-arrestin2-mediated activation of Cdc42. Proc Natl Acad Sci USA 2009; 19;106:8221-6.

Open Access Article

BRIT1/MCPH1 is a multifunctional DNA damage responsive protein mediating DNA repair-associated chromatin remodeling

Guang Peng and Shiaw-Yih Lin
Pages 3071 - 3072
http://dx.doi.org/10.4161/cc.8.19.9411
Abstract | PDF

Comment on: Peng, G, et al. BRIT1/MCPH1 links chromatin remodelling to DNA damage response. Nat Cell Biol 2009; 11: 865-872.




CELL CYCLE NEWS & VIEWS

Open Access Article

Replication timing: Findings from fibers

Apolinar Maya-Mendoza and Dean A. Jackson
Pages 3073 - 3077
http://dx.doi.org/10.4161/cc.8.19.9959
Abstract | PDF

Comment on: Temporal differences in DNA replication during the S phase using single fiber analysis of normal human fibroblasts and glioblastoma T98G cells.

Rebecca A. Frum, Zakaria S. Khondker and David G. Kaufman. Cell Cycle 2009; 8; In press.

Open Access Article

Selectively killing transformed cells through proteasome inhibition

Steven Grant
Pages 3073 - 3077
http://dx.doi.org/10.4161/cc.8.19.9815
Abstract | PDF

Comment on: Discovery of a novel proteasome inhibitor selective for cancer cells over non-transformed cells.

Kazi A, et al. Cell Cycle 2009; 8:1940-1951.

Open Access Article

c-Myc and telomerase activation

Goberdhan P. Dimri
Pages 3073 - 3077
http://dx.doi.org/10.4161/cc.8.19.9966
Abstract | PDF

Comment on: Telomerase activation by c-Myc in human mammary epithelial cells requires additional genomic changes.

Alexey V. Bazarov, William C. Hines, Rituparna Mukhopadhyay, Alain Beliveau, Sonya Melodyev, Yuri Zaslavsky, Paul Yaswen. Cell Cycle 2009; 8:In press.

Open Access Article

Aurora-C: The youngest of “The Three (Aurora kinase) Tenors" of mitotic symphony

Subrata Sen
Pages 3073 - 3077
http://dx.doi.org/10.4161/cc.8.19.10150
Abstract | PDF

Comment on: Slattery SD, et al. Cell Cycle 2009; 8:2984-94.




EXTRA VIEWS

Open Access Article

Induced pluripotent stem cells from swine (Sus scrofa): Why they may prove to be important

R. Michael Roberts, Bhanu Prakash V.L. Telugu and Toshihiko Ezashi
Pages 3078 - 3081
http://dx.doi.org/10.4161/cc.8.19.9589
Abstract | PDF

Three recent papers, published almost simultaneously by different groups, have described the generation of induced pluripotent stem (iPS) cells from the pig, a species whose size, anatomy, and physiology render them attractive as clinical models for the human. The approach used in each case was to infect somatic cells with integrating retroviral vectors designed to express four reprogramming genes (POU5F1, SOX2, cMYC and KLF4). The cell lines generated met the standard criteria for pluripotency, including the ability to differentiate along multiple tissue lineages. In most respects, the porcine iPS cells more resembled human embryonic stem cells and human iPS cells than their murine equivalents. Provided such porcine iPS cells can be “personalized” to specific pigs and then coaxed to differentiate along specific lineages, it should be possible to use such animals to test transplantation therapies with iPS cells for safety and efficacy before the procedures are applied to human patients.

Open Access Article

The My(c)stery of iNKT cell ontogeny

Marei Dose and Fotini Gounari
Pages 3082 - 3085
http://dx.doi.org/10.4161/cc.8.19.9615
Abstract | PDF

Invariant Vα14 bearing natural killer T cells (iNKT) cells constitute a subset of lymphocytes that recognize lipid-based ligands presented by the non-classical MHC class I-like molecule CD1d and responds with rapid cytokine production. Despite their multiple implications in regulating immune responses, pertaining to cancer and auto-immunity, the molecular requirements for their development in the thymus are poorly understood. Here we discuss recent evidence that c-Myc mediates an intrathymic proliferation wave immediately following agonist selection of iNKT cells that is vital for the generation of mature iNKT cells in vivo. We review aspects of early iNKT ontogeny in light of our findings and speculate about possible mechanistic links controlling c-Myc activity in newly selected cells.

Open Access Article

The apoptosis modulating role of SAP (SLAM associated protein) contributes to the symptomatology of the X linked lymphoproliferative disease

Noemi Nagy, Liudmila Matskova, Ulf Hellman, George Klein and Eva Klein
Pages 3086 - 3090
http://dx.doi.org/10.4161/cc.8.19.9636
Abstract | PDF

Deletion or mutation of the SH2D1A gene located at Xq25 is responsible for the development of the X-linked lymphoproliferative disease, XLP. Primary infection of the affected individuals with EBV leads to fulminant and often fatal infectious mononucleosis, FIM. Moreover, they run a 200 fold elevated risk for lymphoma development. Due to the critical role of the immune response for the outcome of EBV infection and the detection of EBV genomes in several malignancies, XLP studies have been mainly focused on the immunological aspects.
The involvement of SAP in the apoptotic machinery provides a further aspect in the complex syndrome of XLP. Functional impairment of SAP leads to defective apoptotic responses. Activation induced apoptosis plays a pivotal role in the termination of the lymphocyte proliferation in IM. This mechanism is inefficient in XLP patients. In addition, in the absence of SAP, lymphoma development may be promoted by the illegitimate survival of lymphocytes with damaged DNA that would be normally eliminated by apoptosis.


Open Access Article

Inflammatory breast cancer cells are constitutively adapted to hypoxia

Deborah Silvera and Robert J. Schneider
Pages 3091 - 3096
http://dx.doi.org/10.4161/cc.8.19.9637
Abstract | PDF

We recently showed that overexpression of translation initiation factor eIF4G partially drives the unusual pathological features of Inflammatory Breast Cancer (IBC), the most lethal form of primary breast cancer. IBC has the peculiar feature that, rather than develop as a solid tumor, it typically generates rapidly metastasizing tight clusters of cancer cells referred to as tumor cell emboli, consisting of cancer cells held together by increased membrane expression of E-cadherin.  Overexpression of eIF4GI in IBC leads to a specific increase in the translation of internal ribosomal entry site (IRES) containing mRNAs, of which two encode key proteins involved in the pathological features of IBC. One of these mRNAs encodes p120 catenin, which mediates E-cadherin retention at the cell surface, and the other encodes VEGF, which accounts for high levels of IBC angiogenesis and resistance to hypoxia. Here we show that IBC cells have adapted to the persistent hypoxia they experience as tumor emboli by reprogramming the protein synthesis machinery to constitutively translate mRNAs required for IBC cell survival during hypoxia even under normal oxygen (normoxic) conditions. Thus, IBC cells have been able to behave as if they are continuously hypoxic even when they are not.

Open Access Article

Somatic hypermutation: Processivity of the cytosine deaminase AID and error-free repair of the resulting uracils

Ursula Storb, Hong Ming Shen and Dan Nicolae
Pages 3097 - 3101
http://dx.doi.org/10.4161/cc.8.19.9658
Abstract | PDF

The somatic hypermutation (SHM) process is initiated in activated B lymphocytes by the cytosine DNA deaminase AID creating uracils that are further treated by DNA replication and/or error-prone base excision repair (BER) and/or error-prone mismatch repair (MMR), resulting in mutations at the targeted cytosines, as well as at all four nucleotides neighboring the AID-induced uracil. In this analysis we investigate two issues that are specific to SHM, the processivity of AID in vivo in vertebrate cells versus in cell free assays, and the error-prone versus error-free repair of the AID-induced uracils. Compilation of published data shows that AID is highly processive in vitro, but shows little, although apparently real, processivity in vivo. We postulate that the combined effects of chromatin and associated transcription factors prevent AID from migrating along extensive tracks in vivo. The comparison of mutation loads in Ig genes at cytosines in wild type mice with those in mice with a combined Ung/MMR-deficiency suggests that Ig genes in wild type mice undergo error-free repair of over 47% of the uracils originally created by AID. The uracil glycosylase Ung which is involved in the error-prone repair during SHM is also involved in the error-free repair.




PERSPECTIVE

Open Access Article

tRNA genes in eukaryotic genome organization and reorganization

Ramsay J. McFarlane and Simon K. Whitehall
Pages 3102 - 3106
http://dx.doi.org/10.4161/cc.8.19.9625
Abstract | PDF

The primary function of tRNA genes is to provide the templates for the transcription of essential tRNA molecules. However, there is now evidence that these dispersed repetitive elements have the potential to mediate the spatial and functional organization of the genome and to drive genome change and evolution. Indeed, tRNA genes and related Pol III promoter elements can occupy distinct subnuclear positions and also provide barriers which functionally separate domains of chromatin. Furthermore, tRNA genes can also represent barriers to DNA replication fork progression and accordingly, tRNA genes can contribute to the formation of genomic fragile sites and have been implicated in genome evolution. Here we give insight into our current understanding of these "extra transcriptional" functions of tRNA genes and discuss how these functions may impact upon genome regulation and evolution.




REVIEWS

Open Access Article

Inflammation: A hidden path to breaking the spell of ovarian cancer

Weiwei Shan and Jinsong Liu
Pages 3107 - 3111
http://dx.doi.org/10.4161/cc.8.19.9590
Abstract | PDF

Epithelial ovarian cancer is a highly lethal gynecological cancer for which overall prognosis has remained poor over the past few decades. A number of theories have been postulated in an effort to explain the etiology of epithelial ovarian cancer, each of which has been both applauded and doubted. Of note, these theories likely are not mutually exclusive, as they all converge more or less on the role of inflammation in promoting ovarian tumorigenesis. In this review, we describe the latest studies on the role of inflammation in the initiation and progression of EOC from three major aspects: physiological functions of a normal ovary, potential involvement of the fallopian tube in the initiation of EOC, and the strong impact of the cellular microenvironment on the development of the disease.

Open Access Article

Exploiting synthetic lethal interactions for targeted cancer therapy

H. Christian Reinhardt, Hai Jiang, Michael T. Hemann and Michael B. Yaffe
Pages 3112 - 3119
http://dx.doi.org/10.4161/cc.8.19.9626
Abstract | PDF

Emerging data suggests that synthetic lethal interactions between mutated oncogenes/tumor suppressor genes and molecules involved in DNA damage signaling and repair can be therapeutically exploited to preferentially kill tumor cells.   In this review, we discuss the concept of synthetic lethality, and describe several recent examples in which this concept was successfully implemented to target tumor cells in culture, in mouse models, and in human cancer patients.

Open Access Article

The p53 tumor suppressor protein is a critical regulator of hematopoietic stem cell behavior

Yan Liu, Shannon E. Elf, Takashi Asai, Yasuhiko Miyata, Yuhui Liu, Goro Sashida, Gang Huang, Silvana Di Giandomenico, Andrew Koff and Stephen D. Nimer
Pages 3120 - 3124
http://dx.doi.org/10.4161/cc.8.19.9627
Abstract | PDF

In response to diverse stresses, the tumor suppressor p53 differentially regulates its target genes, variably inducing cell-cycle arrest, apoptosis or senescence. Emerging evidence indicates that p53 plays an important role in regulating hematopoietic stem cell (HSC) quiescence, self-renewal, apoptosis and aging. The p53 pathway is activated by DNA damage, defects in ribosome biogenesis, oxidative stress and oncogene induced p19ARF upregulation. We present an overview of the current state of knowledge about p53 (and its target genes) in regulating HSC behavior, with the hope that understanding the molecular mechanisms that control p53 activity in HSCs and how p53 mutations affect its role in these events may facilitate the development of therapeutic strategies for eliminating leukemia (and cancer) propagating cells.

Open Access Article

NFκB inhibitors: Strategies from poxviruses

Mohamed Ragaa Mohamed and Grant McFadden
Pages 3125 - 3132
http://dx.doi.org/10.4161/cc.8.19.9683
Abstract | PDF

The orchestration of the inflammatory responses to both infection and tissue damage is arguably the key physiological function of NF-κB, and thus interference with the activation of NF-κB represents an exceptional strategy for a successful pathogen to exploit to counter multiple host innate defense processes through the targeting of a single host regulatory pathway. Because of their large genomes, which typically encode ~200 proteins, and their unusual independence from the host nuclear transcriptional machinery, poxviruses are especially well suited to manipulate the cytoplasmic activation of NF-κB. Indeed, poxviruses are known to encode multiple proteins that regulate the activation of NF-κB in a variety of different ways and these can be considered potential paradigms for the development of novel anti-inflammatory therapies and more effective vaccines. Given the renewed interest in the pathogenesis of orthopoxviruses like smallpox and monkeypox, we review the current understanding of how the various classes of poxviral immunomodulatory proteins target and manipulate the NF-κB pathway.




REPORTS

Open Access Article

Temporal differences in DNA replication during the S phase using single fiber analysis of normal human fibroblasts and glioblastoma T98G cells

Rebecca A. Frum, Zakaria S. Khondker and David G. Kaufman
Pages 3133 - 3148
http://dx.doi.org/10.4161/cc.8.19.9682
Abstract | PDF

We have recently shown that replication forks pause near origins in normal human fibroblasts (NHF1-hTERT) but not glioblastoma T98G cells.  This observation led us to question whether other differences in the replication program may exist between these cell types that may relate to their genetic integrity. To identify differences, we detected immunoflourescently the sequential incorporation of the nucleotide analogs IdU and CldU into replicating DNA at the start of every hour of a synchronized S phase. We then characterized the patterns of labeled replicating DNA tracks and quantified the percentages and lengths of the tracks found at these hourly intervals.  From the directionality of labeling in single extended replicating DNA fibers, tracks were categorized as single bidirectional origins, unidirectional elongations, clusters of origins firing in tandem, or merging forks (terminations).  Our analysis showed that the start of S phase is enriched in single bidirectional origins in NHF1-hTERT cells, followed by an increase in clustering during mid S phase and an increase in merging forks during late S phase.  Early S phase in T98G cells also largely consisted of single bidirectional origin initiations; however, an increase in clustering was delayed until an hour later, and clusters were shorter in mid/late S phase than in NHF1-hTERT cells. The spike in merging forks also did not occur until an hour later in T98G cells.  Our observations suggest models to explain the temporal replication of single and clustered origins, and suggest differences in the replication program in a normal and cancer cell line.

Open Access Article

GHRH antagonist causes DNA damage leading to p21 mediated cell cycle arrest and apoptosis in human colon cancer cells

Florian Hohla, Stefan Buchholz, Andrew V. Schally, Stefan Seitz, Ferenc G. Rick, Luca Szalontay, Jozsef L. Varga, Marta Zarandi, Gabor Halmos, Irving Vidaurre, Awtar Krishan, Metin Kurtoglu, Sudhir Chandna, Elmar Aigner and Christian Datz
Pages 3149 - 3156
http://dx.doi.org/10.4161/cc.8.19.9698
Abstract | PDF

We investigated the mechanisms of inhibitory effect of growth hormone-releasing hormone (GHRH) antagonist JMR-132 on the growth of HT29, HCT-116 and HCT-15 human colon cancer cells in vitro and in vivo.  High-affinity binding sites for GHRH and mRNA for GHRH and splice variant-1 (SV1) of the GHRH receptor were found in all three cell lines tested. Proliferation of HT-29, HCT-116 and HCT-15 cells was significantly inhibited in vitro by JMR-132. Time course studies revealed that the treatment of human HCT-116 colon cancer cells with 10μM GHRH antagonist JMR-132 causes a significant DNA damage as shown by an increase in olive tail moment (OTM) and loss of inner mitochondrial membrane potential (∆Ψm). Western blotting demonstrated a time-dependent increase in protein levels of phospho-p53 (Ser46), Bax, cleaved caspase-9, -3, cleavage of poly(ADP-ribose)polymerase (PARP) and a decrease in Bcl-2 levels. An augmentation in cell cycle checkpoint protein p21Waf1/Cip1 was accompanied by a cell cycle arrest in S-phase. DNA fragmentation visualized by the comet assay and the number of apoptotic cells increased time dependently as determined by flow cytometric annexinV and PI staining assays. In vivo, JMR-132 decreased the volume of HT-29, HCT-116 and HCT-15 tumors xenografted into athymic mice up to 75% (p<0.05) and extended tumor doubling time (p<0.001).
Our observations suggest that GHRH antagonist JMR-132 exerts its antiproliferative effect on experimental colon cancer cells through p21Waf1/Cip1 mediated S-phase arrest along with apoptosis involving the intrinsic pathway.


Open Access Article

Hypoxia-mediated regulation of Cdc25A phosphatase by p21 and miR-21

Pierre E. Queiroz de Oliveira, Lin Zhang, Zhenghe Wang and Celeste Reese
Pages 3157 - 3164
http://dx.doi.org/10.4161/cc.8.19.9704
Abstract | PDF

Hypoxia is a common feature of solid tumors and represents a critical factor in their progression and responsiveness to chemotherapy and radiotherapy.   We now report that hypoxic exposure of colon cancer cells decreased the protein levels of the cell cycle-controlling phosphatase Cdc25A. Hypoxia decreased the mitotic population and caused S-phase arrest in these cells.  Suppression of Cdc25A was phosphatase family member-specific, as a similar decrease was not observed with closely related Cdc25B or Cdc25C phosphatases.  Pharmacological and genetic blockade of Chk1 and Chk2 failed to inhibit the hypoxia-mediated loss of Cdc25A, indicating this process was not regulated by a traditional ATM/ATR checkpoint response. In addition, hypoxia did not affect ectopically expressed Cdc25A levels suggesting independence from an increase in proteasomal degradation.  Cdc25A mRNA levels also decreased in human colon cancer cells 24 hr after hypoxia supporting a mechanistic role for decreased Cdc25A expression or mRNA stability. The reduction in Cdc25A mRNA and protein was dependent on the cyclin-dependent kinase inhibitor p21 and miR-21, which were upregulated in HCT116 colon cancer cells during hypoxia. These results reveal previously unknown mechanisms for the transient suppression of Cdc25A, providing a coordinated and fundamental adaptive change that may be exploited by cancer cells conferring proliferative and survival advantages.

Open Access Article

The N-terminal domain of DDA3 regulates the spindle-association of the microtubule depolymerase Kif2a and controls the mitotic function of DDA3

Chang-Young Jang and Guowei Fang
Pages 3165 - 3171
http://dx.doi.org/10.4161/cc.8.19.9724
Abstract | PDF

DDA3 is a microtubule-associated protein that controls chromosome congression and segregation by regulating the dynamics of the mitotic spindle. Depletion of DDA3 alters spindle structure, generates unaligned chromosomes at metaphase, and delays the mitotic progression. DDA3 interacts with the microtubule depolymerase Kif2a and controls the association of Kif2a to the mitotic spindle and the dynamic turnover of microtubules in the spindle. To understand the function and regulation of DDA3, we analyzed its domain structure and found that the C-terminal domain of DDA3 directly binds to microtubules in vitro and associates with the mitotic spindle in vivo. The N-terminal domain of DDA3 does not interact with microtubules, but acts dominant negatively over the wild-type protein. Ectopic expression of this domain prevents the endogenous DDA3 from association with the spindle and results in a high frequency of unaligned chromosomes in metaphase cells, a phenotype similar to that in metaphase cells depleted of DDA3. Mechanistically, expression of N-terminal DDA3 reduces the amount of spindle-associated Kif2a and increases the spindle microtubule density, pheno-copying those in DDA3-depleted cells. We conclude that DDA3 has a distinct domain structure. The C-terminal domain confers its ability to associate with the mitotic spindle, while the regulatory N-terminal domain controls the microtubule-binding by the C-terminal domain and determines the cellular activity of the DDA3 protein.

Open Access Article

The Aurora B kinase inhibitor AZD1152 sensitizes cancer cells to fractionated irradiation and induces mitotic catastrophe

Yungan Tao, Céline Leteur, Julien Calderaro, Fiona Girdler, Ping Zhang, Valérie Frascogna, Mariana Varna, Paule Opolon, Maria Castedo, Jean Bourhis, Guido Kroemer and Eric Deutsch
Pages 3172 - 3181
http://dx.doi.org/10.4161/cc.8.19.9729
Abstract | PDF

AZD1152, an Aurora kinase inhibitor with selectivity for Aurora B kinase, can enhance the effect of ionizing radiation (IR). The aim of this study was to evaluate and to mechanistically explore scheduling effects of AZD1152 on tumor responses to IR, in three different settings: neoadjuvant (AZD1152 before IR), adjuvant (IR before AZD1152), or concomitant treatments (AZD1152 plus one single IR dose). A more pronounced tumor growth delay was observed in the neoadjuvant and adjuvant schedules as compared to the concomitant schedule. However, AZD1152 enhanced the efficacy of IR when concomitant IR was fractionated over several days. Histopathological examination revealed that AZD1152 + IR induced polyploidy, multinucleation and micronuclei in vivo. Time-lapse videomicroscopy confirmed that cell death induced by AZD1152 + IR was preceded by multinucleation and the formation of micronuclei, which both are hallmarks of mitotic catastrophe. Caspase inhibition or removal of the pro-apoptotic protein Bax did not ameliorate the long-term cell survival of AZD1152-treated cancer cells. In contrast, a chemical inhibitor of CHK1, Chir124, sensitized cancer cells to the lethal effect of AZD1152. Altogether, these data support the contention that AZD1152 mediates radiosensitization in vivo by enhancing mitotic catastrophe, which can be used as a biomarker of treatment efficacy.

Open Access Article

A CAAX motif can compensate for the PH domain of Num1 for cortical dynein attachment

Xianying Tang, Jesse J. Punch and Wei-Lih Lee
Pages 3182 - 3190
http://dx.doi.org/10.4161/cc.8.19.9731
Abstract | PDF

During mitosis in budding yeast, cortically anchored dynein exerts pulling forces on cytoplasmic microtubules, moving the mitotic spindle into the mother-bud neck. Anchoring of dynein requires the cortical patch protein Num1, which is hypothesized to interact with PI(4,5)P₂ via its C-terminal pleckstrin homology (PH) domain. Here we show that the PH domain and PI(4,5)P₂ are required for the cortical localization of Num1, but are not sufficient to mediate the cortical assembly of Num1 patches. A GFP fusion to the PH domain localizes to the cortex in foci containing ~2 molecules, whereas patches of full-length Num1-GFP contain ~14 molecules. A membrane targeting sequence containing the CAAX motif from the yeast Ras2 protein can compensate for the PH domain to target Num1 to the plasma membrane as discrete patches. The CAAX-targeted Num1 exhibits overlapping but largely distinct localization from wild-type Num1. However, it is fully functional in the dynein pathway. More importantly, cortical PI(4,5)P₂ is dispensable for the localization and function of the CAAX-targeted Num1. Together, these results demonstrate that cortical assembly of Num1 into functional dynein-anchoring patches is independent of its interaction with PI(4,5)P₂.

Open Access Article

Involvement of MKP-1 and Bcl-2 in acquired cisplatin resistance in ovarian cancer cells

Juan Wang, Jun-Ying Zhou, Lianfeng Zhang and Gen Sheng Wu
Pages 3191 - 3198
http://dx.doi.org/10.4161/cc.8.19.9751
Abstract | PDF

Although cisplatin is a very effective anticancer agent against several types of cancer including ovarian cancer, the mechanisms of acquired resistance are not fully understood. By chronically exposing cisplatin to ovarian cancer cell lines, we established two cisplatin-resistant cell lines OV433 and TOV112D. Our results indicate that the mechanisms underlying their cisplatin resistance are distinct. In OV433 cells, cisplatin resistance is associated with increased expression of mitogen-activated protein kinase (MAPK) phosphatase-1 (MKP-1). By knocking down MKP-1 expression by siRNA or inhibiting MKP-1 expression by its pharmacological inhibitor triptolide, cisplatin-resistant OV433 cells became cisplatin-sensitive and subsequently increased cisplatin-induced apoptosis. In TOV112D cells, on the other hand, acquired cisplatin resistance is associated with increased levels of Bcl-2 protein. By inhibiting the activity of Bcl-2 protein with its pharmacological inhibitor gossypol or knocking down Bcl-2 expression by siRNA, cisplatin-resistant TOV112D cells became cisplatin-sensitive and subsequently increased cisplatin-induced apoptosis. Therefore, our data suggest that the mechanisms of acquired cisplatin resistance vary among ovarian cancer cells, which involve up-regulation of molecules associated with the cell survival pathways.

Open Access Article

DNA damage responses by human ELG1 in S phase are important to maintain genomic integrity

Nilabja Sikdar, Soma Banerjee, Kyoo-young Lee, Stephen Wincovitch, Evgenia Pak, Koji Nakanishi, Maria Jasin, Amalia Dutra and Kyungjae Myung
Pages 3199 - 3207
http://dx.doi.org/10.4161/cc.8.19.9752
Abstract | PDF

Genomic integrity depends on DNA replication, recombination, and repair, particularly in S phase. We demonstrate that a human homologue of yeast Elg1 plays an important role in S phase to preserve genomic stability. The level of ELG1 is induced during recovery from a variety of DNA damage. In response to DNA damage, ELG1 forms distinct foci at stalled DNA replication forks that are different from DNA double strand break foci. Targeted gene knockdown of ELG1 resulted in spontaneous foci formation of γ-H2AX, 53BP1, and phosphorylated-ATM that mark chromosomal breaks.  Abnormal chromosomes including fusions, inversions and hypersensitivity to DNA damaging agents were also observed in cells expressing low level of ELG1 by targeted gene knockdown. Knockdown of ELG1 by siRNA reduced homologous recombination frequency in the I-SceI induced double strand break-dependent assay. In contrast, spontaneous homologous recombination frequency and sister chromatin exchange rate were up-regulated when ELG1 was silenced by shRNA. Taken together, we propose that ELG1 would be a new member of proteins involved in maintenance of genomic integrity.

Open Access Article

Cancer stem cells and escape from drug-induced premature senescence in human lung tumor cells: Implications for drug resistance and in vitro drug screening models

Michal Sabisz and Andrzej Skladanowski
Pages 3208 - 3217
http://dx.doi.org/10.4161/cc.8.19.9758
Abstract | PDF

In this study, using an in vitro human tumor model, we show that non-small lung adenocarcinoma A549 cells after treatment with DNA damaging antitumor drugs become permanently growth-arrested as a result of so-called drug-induced premature senescence (pseudo-senescence). However, a small fraction of drug-treated cells escapes pseudo-senescence that leads to re-growth of tumor cell population after drug treatment. We show that this re-growth is associated with the presence of cancer stem cells (CSCs) in lung tumor cell population. We also document that re-growth of CSCs can be greatly delayed if lung tumor cells are treated with drug/caffeine combination that leads to the inhibition of the ATM/ATR pathway and decreased phosphorylation of PKB/Akt at Ser473. We show that in non-treated A549 cells caffeine by itself induces a reversible growth arrest that is associated with increased fraction of so-called side population cells, containing CSCs. These results point to the existence of an unknown, caffeine-sensitive mechanism that controls the number of CSCs in lung tumor cell population. Full characterization of this mechanism may lead to the development of innovative cancer therapies which are based on small molecular weight inhibitors of CSC differentiation and self-renewal, that mimic caffeine action. Our results have also important implications for drug screening tumor models in vitro.

Open Access Article

IRS-2, but not IRS-1, can sustain proliferation and rescue UBF stabilization in InR or InR defective signaling of 32D myeloid cells

Shufang Wu, Bo Zhou, Lin Xu and Hongzhi Sun
Pages 3218 - 3226
http://dx.doi.org/10.4161/cc.8.19.9759
Abstract | PDF

Despite both IRS-1 and IRS-2 are two important adaptor molecules essential for intracellular signaling of insulin and IGF-I, the distinct biological pattern of IRS-2 versus IRS-1 remains as a concernful issue to be clarified. We demonstrated here an important evidence that in 32D myeloid cells expressing the insulin receptor (InR) or selected mutants of the InR, IRS-2, but not IRS-1, is required for promoting the proliferation and inhibiting the granulocytic differentiation, thus restore ERKs phosphorylation and UBF1 stabilization. In addition, unlike IRS-1, IRS-2 can effectively compensate the InR defective signaling and lead to the activation of cell cycle progression and proliferation genes in 32D myeloid cells. These results indicate a predominant role of IRS-2 involved in InR signaling of 32 D myeloid cells.

Open Access Article

Mechanism of dominant-negative telomerase function

Binh N. Nguyen, Lynne W. Elmore and Shawn E. Holt
Pages 3227 - 3233
http://dx.doi.org/10.4161/cc.8.19.9788
Abstract | PDF

Human telomerase uses its integral core components, hTR and hTERT, to maintain telomeres in many cell types. Expression of a dominant-negative mutant of the catalytic subunit of telomerase, DN-hTERT, has been shown to cause telomere shortening and ultimately cell death in a number of tumor-derived cell lines. However, the mechanism of dominant-negative hTERT function and its fate inside the cell are still unknown. In order to understand the effect of the dominant-negative on wild-type hTERT, each was fused with GFP and expressed in telomerase-positive cells. GFP-DN-hTERT expression resulted in cytoplasmic exportation and degradation via ubiquitination. Co-expression of wild-type GFP-hTERT with an untagged DN-hTERT resulted in decreased wild-type hTERT levels, export to the cytoplasm, and increased ubiquitination, suggesting that DN-hTERT complexes with wild-type hTERT to induce cytoplasmic localization. Based on the cytoplasmic degradation, we propose two new mechanisms of dominant-negative hTERT, employing the theory of interactive dimerization. First, the heterodimer of DN-hTERT with wild-type hTERT is exported to the cytoplasm for ubiquitin-mediated protein degradation, and second, the heterodimer may be degraded at a faster rate than the wild-type hTERT homodimer. Understanding mechanisms of telomerase degradation will guide future drug design to target sites on telomerase important for catalytic activity and protein stability.




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