Table of Contents

Volume 10, Issue 2

  January 15, 2011

  Pages 171 - 357

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EDITORIALS: CELL CYCLE FEATURES

Open Access Article

When Aurora B met p53: Newly revealed regulatory phosphorylation in an old protein

Liming Wu, Chi A. Ma and Ashish Jain
Pages 171 - 172
http://dx.doi.org/10.4161/cc.10.2.14349
Abstract | PDF

Comment on: Wu L, et al. J Biol Chem. 2011; 286:2236-44. PMID: 20959462

Open Access Article

A FAK-PI-3K-mTOR axis is required for Wnt-Myc driven intestinal regeneration and tumorigenesis

Jennifer P. Morton, Kevin B. Myant and Owen J. Sansom
Pages 173 - 175
http://dx.doi.org/10.4161/cc.10.2.14350
Abstract | PDF

Comment on: Ashton GH, et al. Dev Cell 2010; 19:259-69.

Open Access Article

B-cell differentiation stimulated by physiologic DNA double strand breaks

Nicole C. Walsh and Michael Teitell
Pages 176 - 177
http://dx.doi.org/10.4161/cc.10.2.14474
Abstract | PDF

Comment on: Sherman MH, et al. Mol Cell 2010; 39:873-85.

Open Access Article

The emerging role of maternal phosphatidylinositol 3 kinase (PI3K) signaling in manipulating mammalian preimplantation embryogenesis

Wenjing Zheng and Kui Liu
Pages 178 - 179
http://dx.doi.org/10.4161/cc.10.2.14475
Abstract | PDF

Feature on: Zheng W, et al. EMBO Rep 2010; 11:890-5.

Open Access Article

Metabolic cues from the microenvironment act as a major selective factor for cancer progression and metastases formation

Jacinta Serpa and Sergio Dias
Pages 180 - 181
http://dx.doi.org/10.4161/cc.10.2.14476
Abstract | PDF

Feature on: Serpa J, et al. J Biol Chem 2010. [Epub ahead of print]

Open Access Article

Decoding the chromatin modification landscape

Till Bartke and Tony Kouzarides
Page 182
http://dx.doi.org/10.4161/cc.10.2.14477
Abstract | PDF

Feature on: Bartke T, et al. Cell 2010; 143:470-84.

Open Access Article

Mechanistic insights into the action of Bisphenol A on the germline using C. elegans

Patrick Allard and Monica P. Colaiácovo
Pages 183 - 184
http://dx.doi.org/10.4161/cc.10.2.14478
Abstract | PDF

Feature on: Allard P, et al. Proc Natl Acad Sci USA 2010; 107:20405-10.




NEWS & VIEWS

Open Access Article

Release the ink4a/arf growth suppression by “u” and “me”?

Shuo Qie and Nianli Sang
Pages 185 - 190
http://dx.doi.org/10.4161/cc.10.2.14471
Abstract | PDF

Comment on: Roberti et al. Cell Cycle 2011; 10:127–34.
 

Open Access Article

Inhibition of Smad3 activity by cyclin D-CDK4 and cyclin E-CDK2 in breast cancer cells

Fang Liu

http://dx.doi.org/10.4161/cc.10.2.14479
Abstract | PDF

Comment on: Cooley A, et al. Cell Cycle 2010; 9:4900–7.
 

Open Access Article

Breast cancer go sMAD: Cyclin towards aggressive phenotypes

Eric K. Lee and J. Alan Diehl

http://dx.doi.org/10.4161/cc.10.2.14480
Abstract | PDF

Comment on: Cooley A, et al. Cell Cycle 2010; 9:4900–7.
 

Open Access Article

Targeting the metabolic flexibility of cancer cells: Straighten up and die right

Isaac Marin-Valencia and Ralph J. DeBerardinis

http://dx.doi.org/10.4161/cc.10.2.14481
Abstract | PDF

Comment on: Chen V, et al. Cell Cycle 2010; 9:4941–53.
 

Open Access Article

The role of BAFF-R dysregulation in B-lymphoid lineage malignancies

Lan V. Pham and Richard J. Ford

http://dx.doi.org/10.4161/cc.10.2.14570
Abstract | PDF

Comment on: Mihalcik S, et al. Cell Cycle 2010; 9:4884–92.  




EXTRA VIEWS

Open Access Article

Selective control of Pax7 expression by TNF-activated p38α/polycomb repressive complex 2 (PRC2) signaling during muscle satellite cell differentiation

Chiara Mozzetta, Silvia Consalvi, Valentina Saccone, Sonia V. Forcales, Pier Lorenzo Puri and Daniela Palacios
Pages 191 - 198
http://dx.doi.org/10.4161/cc.10.2.14441
Abstract | PDF

Muscle regeneration relies on adult muscle stem (satellite) cells. Inflammatory cues released within the regenerative microenvironment, such as TNFα, instruct different components of the satellite cell niche toward specialized tasks by regulating specific subsets of genes in each individual cell type. However, how regeneration cues are deciphered and interpreted by the multitude of cell types within the regenerative environment is unknown. We have recently identified an inflammation-activated signaling, consisting of p38α-mediated recruitment of polycomb repressive complex 2 (PRC2) to the Pax7 promoter, in satellite cells. Here we show that p38α-PRC2 regulation of Pax7 expression is restricted to a discrete stage of satellite cell-mediated regeneration. In activated satellite cells, Pax7 locus shows a “bivalent” chromatin signature, with co-existence of H3-K273me and H3-K43me, that appears to confer responsiveness to p38α-PRC2 signaling. p38α activation resolves bivalence to H3-K273me which results in Pax7 repression, while p38α blockade promotes Pax7 expression by preventing PRC2-mediated H3-K273me and leading to relative increase in H3-K43me. Interestingly, in satellite cell-derived myotubes Pax7 expression cannot be re-induced by p38α blockade, revealing a post-mitotic resistance of Pax7 gene to inflammatory cues. Likewise, in other cell types, such as muscle-derived fibroblasts, Pax7 locus is constitutively repressed by PRC2 and is unresponsive to p38α signaling. Finally, we show that Pax7 repression in embryonic stem cells is not directed by p38α signaling, although it is mediated by PRC2. This evidence indicates a cell type- and differentiation-stage specific control of Pax7 transcription by the p38α-PRC2.

Open Access Article

Centrosomal localization of cyclins E and A: Structural similarities and functional differences

Gaetan Pascreau, Mair E.A. Churchill and James L. Maller
Pages 199 - 205
http://dx.doi.org/10.4161/cc.10.2.14444
Abstract | PDF

Recent identification of the modular CLS motifs responsible for cyclins A and E localization on centrosomes has revealed a tight linkage between the nuclear and centrosomal cycles. These G1/S cyclins must localize on the centrosome in order for DNA replication to occur in the nucleus, whereas essential DNA replication factors also function on the centrosome to prevent centrosome overduplication. Both events are dependent on the presence of an intact CLS within each cyclin. Here we compare the cyclins A and E CLSs at the structural and functional levels and identify a new cyclin A CLS mutant that disrupts all CLS functions and reduces the affinity of cyclin A for Cdk2. Analysis of interactions of the CLS motif within the cyclin molecules highlights the importance of the cyclin CBOX1 region for Cdk2 binding.

Open Access Article

Hypoxia: Are stem cells in it for the long run?

Philip Davy and Richard Allsopp
Pages 206 - 211
http://dx.doi.org/10.4161/cc.10.2.14535
Abstract | PDF

Adult stem cells must persist throughout life to ensure continuous replenishment of dead or damaged cells in various tissues of the body. While numerous studies have already begun to identify some of the factors and mechanisms that regulate long term function and survival of stem cells, there is still much to learn in this regard. A growing body of evidence suggests that various types of stem cells exist in a hypoxic microenvironment, which may be conducive to stem cell longevity. We have recently shown that the oxygen dependent transcription factor hypoxia inducible factor 1alpha (Hif1α) is essential for maintenance of functional levels of telomerase in murine embryonic stem cells (mES). Importantly, long-term proliferation of mES cells with reduced Hif1α levels led to telomere shortening and ultimately cell senescence. Studies by others over the past 10 years has also indicated that hypoxia and Hif expression are essential for self-renewal and are involved in the regulation of proliferation for some types of stem cells. We now report our view regarding the effects and mechanisms by which hypoxia may regulate the long-term maintenance of stem cell populations.

Open Access Article

Double null cells reveal that CBP and p300 are dispensable for p53 targets p21 and Mdm2 but variably required for target genes of other signaling pathways

Lawryn H. Kasper, Mary C. Thomas, Gerard P. Zambetti and Paul K. Brindle
Pages 212 - 221
http://dx.doi.org/10.4161/cc.10.2.14542
Abstract | PDF

The histone acetyltransferase coactivators CBP (CREBBP) and p300 (EP300) have more than 400 described protein interaction partners and are implicated in numerous transcriptional pathways. We have shown previously that CBP and p300 double knockout mutations in mouse embryonic fibroblasts (dKO MEFs) result in mixed effects on cAMP-inducible gene expression, with many CREB target genes requiring CBP/p300 for full expression, while others are unaffected or expressed better in their absence. Here we used CBP and p300 dKO MEFs to examine gene expression in response to four other signals: DNA damage (via p53), double-stranded RNA, serum, and retinoic acid. We found that while retinoic acid-inducible gene expression tends to be uniformly dependent on CBP/p300, dsRNA- and serum-inducible genes displayed non-uniform requirements for CBP/p300, with the dsRNA-inducible expression of Ifnb1 (interferon-β) being particularly dependent on CBP/p300. Surprisingly, the p53-dependent genes Cdkn1a (p21/CIP/WAF) and Mdm2 did not require CBP/p300 for their expression. As with cAMP-responsive CREB targets, we propose that the signal-responsive recruitment of CBP and p300 does not necessarily indicate a requirement for these coactivators at a locus. Rather, target gene context (e.g. DNA sequence) influences the extent to which transcription requires CBP/p300 versus other coactivators, which may not be HATs.

Open Access Article

Sister chromatid exchanges occur in G₂-irradiated cells

Sandro Conrad, Julia Künzel and Markus Löbrich
Pages 222 - 228
http://dx.doi.org/10.4161/cc.10.2.14639
Abstract | PDF

DNA double-strand breaks (DSBs) are arguably the most important lesions induced by ionizing radiation (IR) since unrepaired or mis-repaired DSBs can lead to chromosomal aberrations and cell death. The two major pathways to repair IR-induced DSBs are non-homologous end-joining (NHEJ) and homologous recombination (HR). Perhaps surprisingly, NHEJ represents the predominant pathway in the G1 and G2 phases of the cell cycle, but HR also contributes and repairs a subset of IR-induced DSBs in G2. Following S-phase-dependent genotoxins, HR events give rise to sister chromatid exchanges (SCEs), which can be detected cytogenetically in mitosis. Here, we describe that HR occurring in G2-irradiated cells also generates SCEs in ~50% of HR events. Since HR of IR-induced DSBs in G2 is a slow process, SCE formation in G2-irradiated cells requires several hours. During this time, irradiated S-phase cells can also reach mitosis, which has contributed to the widely held belief that SCEs form only during S phase. We describe procedures to measure SCEs exclusively in G2-irradiated cells and provide evidence that following IR cells do not need to progress through S phase in order to form SCEs.




REVIEWS

Open Access Article

Cellular IRES-mediated translation: The war of ITAFs in pathophysiological states

Anton A. Komar and Maria Hatzoglou
Pages 229 - 240
http://dx.doi.org/10.4161/cc.10.2.14472
Abstract | PDF

Translation of cellular mRNAs via initiation at Internal Ribosome Entry Sites (IRESs) has received increased attention during recent years due to its emerging significance for many physiological and pathological stress conditions in eukaryotic cells. Expression of genes bearing IRES elements in their mRNAs is controlled by multiple molecular mechanisms, with IRES-mediated translation favored under conditions when cap-dependent translation is compromised. In this review, we discuss recent advances in the field and future directions that may bring us closer to understanding the complex mechanisms that guide cellular IRES-mediated expression. We present examples in which the competitive action of IRES-transacting factors (ITAFs) plays a pivotal role in IRES-mediated translation and thereby controls cell-fate decisions leading to either pro-survival stress adaptation or cell death.

Open Access Article

CRL4^Cdt2: Master coordinator of cell cycle progression and genome stability

Tarek Abbas and Anindya Dutta
Pages 241 - 249
http://dx.doi.org/10.4161/cc.10.2.14530
Abstract | PDF

Polyubiquitin-mediated degradation of proteins plays an essential role in various physiological processes including cell cycle progression, transcription and DNA replication and repair. Increasing evidence supports a vital role for the E3 ubiquitin ligase cullin-4, in conjunction with the substrate recognition factor Cdt2 (CRL4Cdt2), for the degradation of multiple cell cycle-regulated proteins to prevent genomic instability. In addition, it is critical for normal cell cycle progression by ensuring the timely destruction of various cell cycle proteins whose deregulated expression impairs cell cycle progression. Here, we summarize our current knowledge about the various roles of the CRL4Cdt2 E3 ubiquitin ligase, and how its activity contributes both to the preservation of genome integrity and to normal cell cycle progression, and how its deregulation may contribute to human cancer.

Open Access Article

Impact of BCR-ABL mutations on patients with chronic myeloid leukemia

Andreas Hochhaus, Paul La Rosée, Martin C. Müller, Thomas Ernst and Nicholas C.P. Cross
Pages 250 - 260
http://dx.doi.org/10.4161/cc.10.2.14537
Abstract | PDF

Therapies that target BCR-ABL in chronic myeloid leukemia, including imatinib, dasatinib and nilotinib, have dramatically improved patient outcome. BCR-ABL mutations, however, contribute to treatment resistance by disrupting drug contact sites or causing conformational changes thus making contact sites inaccessible. Clinical data indicate that developing BCR-ABL mutations during imatinib treatment is predictive for shorter progression-free survival, and that outcomes may depend on mutation type or location. In vitro, dasatinib and nilotinib inhibit most imatinib-resistant BCR-ABL mutations, except for T315I. In clinical studies, other mutations associated with treatment resistance include V299L, T315A, and F317I/L for dasatinib and Y253F/H, E255K/V, and F359C/V for nilotinib. Evaluating patients with clinical signs of resistance for BCR-ABL mutations is an important component of disease monitoring, potentially facilitating selection of subsequent therapy. First-line treatment with dasatinib or nilotinib instead of imatinib may reduce emergence of resistance but novel agents are needed to overcome the problematic T315I mutation.

Open Access Article

Chromatin dynamics and the repair of DNA double strand breaks

Ye Xu and Brendan D. Price
Pages 261 - 267
http://dx.doi.org/10.4161/cc.10.2.14543
Abstract | PDF

DNA double-strand breaks (DSBs) arise through both replication errors and from exogenous events such as exposure to ionizing radiation. DSBs are potentially lethal, and cells have evolved a highly conserved mechanism to detect and repair these lesions. This mechanism involves phosphorylation of histone H2AX (γH2AX) and the loading of DNA repair proteins onto the chromatin adjacent to the DSB. It is now clear that the chromatin architecture in the region surrounding the DSB has a critical impact on the ability of cells to mount an effective DNA damage response. DSBs promote the direct the formation of open, relaxed chromatin domains which are spatially confined to the area surrounding the break. These relaxed chromatin structures are created through the coupled action of the p400 SWI/SNF ATPase and histone acetylation by the Tip60 acetyltransferase. The resulting destabilization of nucleosomes at the DSB by Tip60 and p400 is required for ubiquitination of the chromatin by the RNF8 ubiquitin ligase, and for the subsequent recruitment of the brca1 complex. Chromatin dynamics at DSBs can therefore exert a powerful influence on the process of DSB repair. Further, there is emerging evidence that the different chromatin structures in the cell, such as heterochromatin and euchromatin, utilize distinct remodeling complexes and pathways to facilitate DSB. The processing and repair of DSB is therefore critically influenced by the nuclear architecture in which the lesion arises.




REPORTS

Open Access Article

Transgenic overexpression of PKCε in the mouse prostate induces preneoplastic lesions

Fernando Benavides, Jorge Blando, Carlos J. Perez, Rachana Garg, Claudio J. Conti, John DiGiovanni and Marcelo G. Kazanietz
Pages 268 - 277
http://dx.doi.org/10.4161/cc.10.2.14469
Abstract | PDF

It is well established that protein kinase C (PKC) isozymes play distinctive roles in mitogenic and survival signaling as well as in cancer progression. PKCe, the product of the PRKCE gene, is up-regulated in various types of cancers including prostate, lung and breast cancer. To address a potential role for PKCs in prostate cancer progression we generated three mouse transgenic lines expressing PKCa, PKCd, or PKCe in the prostate epithelium under the control of the rat probasin (PB) promoter. Whereas PB-PKCa and PB-PKCd mice did not show any evident phenotype, PB-PKCe mice developed prostate hyperplasia as well as prostate intraepithelial neoplasia (PIN) that displayed enhanced phospho-Akt, phospho-S6, and phospho-Stat3 levels, as well as enhanced resistance to apoptotic stimuli. PKCe overexpression was insufficient to drive neoplastic changes in the mouse prostate. Notably, overexpression of PKCe by adenoviral means in normal immortalized RWPE-1 prostate cells confers a growth advantage and hyperactivation of Erk and Akt. Our results argue for a causal link between PKCe overexpression and prostate cancer development.

Open Access Article

14-3-3σ (sigma) regulates proliferation and differentiation of multipotent p63-positive cells isolated from human breastmilk

Elizabeth Thomas, Nikolajs Zeps, Mark Cregan, Peter Hartmann and Thomas Martin
Pages 278 - 284
http://dx.doi.org/10.4161/cc.10.2.14470
Abstract | PDF

The mammary gland is a dynamic organ that only undergoes complete differentiation during pregnancy. Differentiation is fuelled by asymmetric division of stem cells that reside in normally quiescent niches in the resting gland in response to pregnancy-associated hormones. Loss of regulation of stem cells is believed to underlie some breast cancers. This process is poorly understood in humans since it is difficult to extract stem cells from the lactating gland. We have identified a p63-positive population in breastmilk that proliferates and differentiates into at least two separate mammary lineages in culture. Nuclear translocation of p63 coincides with expression of the cell-cycle arrest protein 14-3-3σ (Sigma) and precedes differentiation. Transient down-regulation of Sigma promotes maintenance of the p63-positive population without affecting normal differentiation. We propose that p63-postive cells from breastmilk represent a novel source of cells to model regulation of mammary gland development and tumorigenesis.

Open Access Article

Nuclear translocation of Skp2 facilitates its destruction in response to TGFβ signaling

Dong Hu, Weijun Liu, George Wu and Yong Wan
Pages 285 - 292
http://dx.doi.org/10.4161/cc.10.2.14517
Abstract | PDF

Skp2, a F-box protein that determines the substrate specificity for SCF ubiquitin ligase, has recently been demonstrated to be degraded by Cdh1/APC in response to TGFβ signaling. The TGFβ-induced Skp2 proteolysis results in the stabilization of p27 that is necessary to facilitate TGFβ cytostatic effect. Previous observation from immunocytochemistry indicates that Cdh1 principally localizes in the nucleus while Skp2 mainly localizes in the cytosol, which leaves us a puzzle on how Skp2 is recognized and then ubiquitylated by Cdh1/APC in response to TGFβ stimulation. Here, we report that Skp2 is rapidly translocated from the cytosol to the nucleus upon the cellular stimulation with TGFβ. Using a combinatorial approach of immunocytochemistry, biochemical-fraction-coupled immunoprecipitation, mutagenesis as well as protein degradation assay, we have demonstrated that the TGFβ-induced Skp2 nucleus translocation is critical for TGFβ cytostatic effect that allows physical interaction between Cdh1 and Skp2 and in turn facilitates the Skp2 ubquitylation by Cdh1/APC. Disruption of nuclear localization motifs on Skp2 stabilizes Skp2 in the presence of TGF-β signaling, which attenuates TGFβ-induced p27 accumulation and antagonizes TGFβ-induced growth inhibition. Our finding reveals a cellular mechanism that facilitates Skp2 ubiquitylation by Cdh1/APC in response to TGFβ.

Open Access Article

γH2A is a component of yeast heterochromatin required for telomere elongation

Tasuku Kitada, Thomas Schleker, Adam S. Sperling, Wei Xie, Susan M. Gasser and Michael Grunstein
Pages 293 - 300
http://dx.doi.org/10.4161/cc.10.2.14536
Abstract | PDF

Histones of heterochromatin are deacetylated in yeast and methylated in more complex eukaryotes to regulate heterochromatin structure and gene silencing. Here, we report that histone H2A phosphorylated at serine 129 (γH2A) in Saccharomyces cerevisiae is a conceptually new type of heterochromatin modification that functions downstream of silent chromatin assembly. We show that γH2A is enriched throughout yeast telomeric and silent mating locus (HM) heterochromatin where γH2A results from the action of kinases Tel1 and Mec1. Interestingly, mutation of γH2A has no apparent effect on the binding of Sir (silent information regulator) complex or on gene silencing. In contrast, deletion of SIR3 abolishes the formation of γH2A at heterochromatin. To address the function of γH2A, we used a Δrif1 mutant strain in which telomeres are excessively elongated to show that γH2A is required for the optimal recruitment of Cdc13, a regulator of telomere elongation, and for telomere elongation itself. Thus, a histone modification that parallels Sir3 protein binding is shown here to be dispensable for the formation of a silent structure but is important for a crucial heterochromatin-specific downstream function in telomere homeostasis.

Open Access Article

PRIMA-1^Met/APR-246 induces wild-type p53-dependent suppression of malignant melanoma tumor growth in 3D culture and in vivo

Wenjie Bao, Ming Chen, Xu Zhao, Ravjiv Kumar, Clemens Spinnler, Minna Thullberg, Natalia Issaeva, Galina Selivanova and Staffan Stromblad
Pages 301 - 307
http://dx.doi.org/10.4161/cc.10.2.14538
Abstract | PDF

Disseminating malignant melanoma is a lethal disease highly resistant to radio- and chemotherapy. Therefore, the development of new treatment strategies is strongly needed. Tumor suppressor p53-mediated apoptosis is essential for the response to radio- and chemotherapy. Although p53 is not frequently mutated in melanoma, it is inactivated by integrin αv-mediated signaling, as we previously demonstrated 1, which may account, at least partially, for increased apoptosis resistance of malignant melanoma. In this study we addressed the question whether functional restoration of p53 by APR-246 (PRIMA-1Met), which can reactivate mutant p53 and induce massive apoptosis in cancer cells, is able to restore the function of inactive p53 in melanoma. Using a three-dimensional collagen gel (3D-collagen) to culture melanoma cells carrying wild-type p53, we found that APR-246 treatment resulted in activation of p53, leading to increased expression of p53 pro-apoptotic targets Apaf1 and PUMA and activation of caspase- 9 and -3. Moreover, APR-246 triggered melanoma cell apoptosis that was mediated by p53 and caspase 9. Importantly, APR-246 treatment also suppressed human melanoma xenograft tumors in vivo in a p53-dependent manner. Thus, wild-type p53 reactivation may provide a novel approach for malignant melanoma treatment, with APR-246 as a candidate drug for such a development.

Open Access Article

KAP1 depletion increases PML nuclear body number in concert with ultrastructural changes in chromatin

Rosemarie Kepkay, Kathleen M. Attwood, Yael Ziv, Yosef Shiloh and Graham Dellaire
Pages 308 - 322
http://dx.doi.org/10.4161/cc.10.2.14551
Abstract | PDF

The promyelocytic leukemia (PML) protein is the main structural component of subnuclear domains termed PML nuclear bodies (PML NBs), which are implicated in tumor suppression by regulating apoptosis, cell senescence, and DNA repair. Previously, we demonstrated that ATM kinase can regulate changes in PML NB number in response to DNA double-strand breaks (DSBs). PML NBs make extensive contacts with chromatin and ATM mediates DNA damage-dependent changes in chromatin structure in part by the phosphorylation of the KRAB-associated protein 1 (KAP1) at S824. We now demonstrate that in the absence of DNA damage, reduced KAP1 expression results in a constitutive increase in PML NB number in both human U2-OS cells and normal human diploid fibroblasts. This increase in PML NB number correlated with decreased nuclear lamina-associated heterochromatin and a 30% reduction in chromatin density as observed by electron microscopy, which is reminiscent of DNA damaged chromatin. These changes in chromatin ultrastructure also correlated with increased histone H4 acetylation, and treatment with the HDAC inhibitor TSA failed to further increase PML NB number. Although PML NB number could be restored by complementation with wild-type KAP1, both the loss of KAP1 or complementation with phospho-mutants of KAP1 inhibited the early increase in PML NB number and reduced the fold induction of PML NBs by 25-30% in response to etoposide-induced DNA DSBs. Together these data implicate KAP1-dependent changes in chromatin structure as one possible mechanism by which ATM may regulate PML NB number in response to DNA damage.

Open Access Article

S. pombe replication protein Cdc18 (Cdc6) interacts with Swi6 (HP1) heterochromatin protein: Region specific effects and replication timing in the centromere

Pao-Chen Li, Louise Chretien, Jocelyn Côté, Thomas J. Kelly and Susan L. Forsburg
Pages 323 - 336
http://dx.doi.org/10.4161/cc.10.2.14552
Abstract | PDF

Heterochromatin in S. pombe is associated with gene silencing at telomeres, the mating locus and centromeres. The compact heterochromatin structure raises the question how it unpacks and reforms during DNA replication. We show that the essential DNA replication factor Cdc18 (CDC6) associates with heterochromatin protein 1 (Swi6) in vivo and in vitro. Biochemical mapping and mutational analysis of the association domains show that the N-terminus of Cdc18 interacts with the chromoshadow domain of Swi6. Mutations in Swi6 that disrupt this interaction disrupt silencing and delay replication in the centromere. A mutation cdc18-I43A that reduces Cdc18 association with Swi6 has no silencing defect at the centromere, but changes Swi6 distribution and accelerates the timing of centromere replication. We suggest that fine tuning of Swi6 association at replication origins is important for negative as well as positive control of replication initiation.

Open Access Article

Overlapping and distinct pRb pathways in the mammalian auditory and vestibular organs

Mingqian Huang, Cyrille Sage, Yong Tang, Sang Goo Lee, Marco Petrillo, Philip W. Hinds and Zheng-Yi Chen
Pages 337 - 351
http://dx.doi.org/10.4161/cc.10.2.14640
Abstract | PDF

Retinoblastoma gene (Rb1) is required for proper cell cycle exit in the developing mouse inner ear and its deletion in the embryo leads to proliferation of sensory progenitor cells that differentiate into hair cells and supporting cells. In a conditional hair cell Rb1 knockout mouse, Pou4f3-Cre-pRb^-/-, pRb^-/- utricular hair cells differentiate and survive into adulthood whereas differentiation and survival of pRb^-/- cochlear hair cells are impaired. To comprehensively survey the pRb pathway in the mammalian inner ear, we performed microarray analysis of ^pRb-/- cochlea and utricle. The comparative analysis shows that the core pathway shared between pRb^-/- cochlea and utricle is centered on E2F, the key pathway that mediates pRb function. A majority of differentially expressed genes and enriched pathways are not shared but uniquely associated with pRb^-/- cochlea or utricle. In pRb^-/- cochlea, pathways involved in early inner ear development such as Wnt/β-catenin and Notch were enriched, whereas pathways involving in proliferation and survival are enriched in pRb^-/- utricle. Clustering analysis showed that the pRb^-/- inner ear has characteristics of a younger control inner ear, an indication of delayed differentiation. We created a transgenic mouse model (ER-Cre-pRb^flox/flox) in which Rb1 can be acutely deleted postnatally. Acute Rb1 deletion in the adult mouse fails to induce proliferation or cell death in inner ear, strongly indicating that Rb1 loss in these postmitotic tissues can be effectively compensated for, or that pRb-mediated changes in the postmitotic compartment result in events that are functionally irreversible once enacted. This study thus supports the concept that pRb-regulated pathways relevant to hair cell development, encompassing proliferation, differentiation and survival, act predominantly during early development.

Open Access Article

Radiosensitization and production of DNA double-strand breaks in U87MG brain tumor cells induced by tetraiodothyroacetic acid (tetrac)

Aleck H. Hercbergs, Hung-Yun Lin, Faith B. Davis, Paul J. Davis and John T. Leith
Pages 352 - 357
http://dx.doi.org/10.4161/cc.10.2.14641
Abstract | PDF

We describe the steady-state levels and molecular and cellular repair of DNA double-strand breaks (DSBs) in tetraiodothyroacetic acid (tetrac)-treated human U87MG glioblastoma cells after x-irradiation in vitro. This study was conducted to provide a basis for our previous observation of radiosensitization and inhibition of cellular recovery after irradiation of tetrac-exposed GL261 murine brain tumor cells. We used the neutral comet assay to assess DSBs, and found that the steady-state DSB levels as indicated by the mean tail moment after a 1 h application of 2 nM tetrac at 37oC was increased from a value of 6.1 in control cells to 12.4 in tetrac treated cells at 0 radiation dose. However, at all radiation doses, the induction curves of DSBs were parallel, suggesting that no interaction of tetrac with the initial physical-chemical actions of ionizing radiation occurred. Flow cytometric measurements indicated that this increase was not due to alterations in the relative percentages of U87MG cells throughout the cell cycle. In split-dose DNA repair studies we found that tetrac decreased the repair rate of U87 cells by a factor of 72.5%. This suggests that the radiosensitization from graded single doses of x-rays occurs as a consequence of tetrac inhibition of the post-irradiation repair process. These results link the previously noted changes in cellular endpoints to a molecular endpoint. That is, tetrac produces increased numbers of DSBs in the unirradiated steady-state coupled with a decreased repair rate of DSBs in fractionated radiation experiments.