Table of Contents

Volume 8, Issue 1

  January 1, 2009

  Pages 3 - 177

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 Issue Catalogue (MARC XML)

NEWS & VIEWS

Open Access Article

Prospective identification and characterization of ovarian cancer stem cells: Implications for the treatment of chemotherapy resistant/recurrent ovarian disease

Alessandro D. Santin
Pages 3 - 5
http://dx.doi.org/10.4161/cc.8.1.7670
PDF

Open Access Article

Apoptosis in space: Facts, not fancies

Mauro Maccarrone and Allessandro Finazzi-Agrò
Pages 3 - 5
http://dx.doi.org/10.4161/cc.8.1.7671
PDF

Open Access Article

Replication factors controlling G₁: A checkpoint for pre-replication complex assembly

Erik S. Knudsen
Pages 3 - 5
http://dx.doi.org/10.4161/cc.8.1.7672
PDF

Open Access Article

More intriguing roles for mammalian homologs of worm heterochronic genes

Eric Moss
Pages 3 - 5
http://dx.doi.org/10.4161/cc.8.1.7673
PDF




EXTRA VIEWS

Open Access Article

Dyskerin, telomerase and the DNA damage response

BaiWei Gu, Monica Bessler and Philip J. Mason
Pages 6 - 10
http://dx.doi.org/10.4161/cc.8.1.7265
Abstract | PDF

The bone marrow failure syndrome Dyskeratosis congenita (DC), though rare, has attracted a great deal of attention in the last few years because it is caused by mutations in genes whose products are involved in telomere maintenance. The disease presents with a variety of features that can all be due to failure of tissues that require constant renewal via stem cell activity. It is thought this is caused by defects in telomere maintenance leading eventually to cell cycle arrest or cell death caused by critically short telomeres. The most common form of DC is the X-linked form caused by mutations in DKC1 encoding the nucleolar protein, dyskerin. We recently reported a mouse model of the X-linked form of the disease in which females heterozygous for a mutation that copies a human pathogenic mutation showed a growth disadvantage in cells expressing the mutant dyskerin. This growth disadvantage, which was associated with an enhanced DNA damage response, was dependent on telomerase but appeared to be independent of telomere shortening. Here we discuss these results in terms of the role of dyskerin in telomere maintenance and the possible role that the DNA damage response plays in the pathogenesis of DC.

Open Access Article

Transforming growth factor β (TGFβ)-induced apoptosis: The rise and fall of Bim

Sneha Ramesh, Gary M. Wildey and Philip H. Howe
Pages 11 - 17
http://dx.doi.org/10.4161/cc.8.1.7291
Abstract | PDF

Transforming growth factor β (TGFβ) regulates essential cellular functions such as cellular proliferation, differentiation and apoptosis. Multiple apoptotic mediators and signaling pathways have been implicated in TGFβ-induced apoptosis. Bim, a BH3-only protein, is critical for apoptosis in a variety of cell types. In resting cells, BimEL expression levels, the major and most abundant isoform, are controlled by Erk1/2-mediated phosphorylation, which targets BimEL for ubiquitination and degradation. We previously reported that TGFβ induces the expression of the pro-apoptotic protein Bim through a Smad3-dependent mechanism to induce cell death in B-lymphocytes. A number of studies have shown TGFβ to cause transcriptional induction of Bim in many cell types. Recently, we demonstrated that, in addition to its transcriptional effects on Bim, TGFβ induces a MAPK phosphatase (MKP), MKP2/DUSP4, to rapidly increase BimEL levels by inactivation of Erk1/2, resulting in dephosphorylation and escape of BimEL from ubiquitin-mediated degradation. Our findings are of importance not only in the context that we implicate TGFβ to increase BimEL levels through both an immediate post-translational regulatory mechanism and a long-term effect through transcriptional induction, but also in the context of implicating MKPs as regulatory players in apoptosis. Here we summarize these recent findings and their significance to our understanding of how TGFβ mediates apoptosis, and we explore the possible regulatory mechanisms controlling Bim expression levels.

Open Access Article

Overcoming resistance to tyrosine kinase inhibitors: Lessons learned from cancer cells treated with EGFR antagonists

Brent N. Rexer, Jeffrey A. Engelman and Carlos L. Arteaga
Pages 18 - 22
http://dx.doi.org/10.4161/cc.8.1.7324
Abstract | PDF

Tyrosine kinase inhibitors (TKIs) are effective anti-cancer therapies but resistance to these agents eventually develops. Several models of resistance to TKIs have been studied including resistance to EGFR inhibitors. Recent studies in EGFR-dependent A431 cells found upregulation of the IGF1R pathway as a mechanism to overcome blockade of EGFR. This was associated with amplification of IGF1R signaling and recovery of downstream PI3K-AKT activation. This work adds to a growing body of cell lines in culture that have provided insights into mechanisms of resistance that can be interrogated in primary tumors in patients. In this review, these model systems and their applicability to human cancers, as well as strategies to identify and overcome resistance to TKIs, are discussed.




PERSPECTIVES

Open Access Article

Epigenetics and cancer without genomic instability

Elizabeth S. McKenna and Charles W. M. Roberts
Pages 23 - 26
http://dx.doi.org/10.4161/cc.8.1.7290
Abstract | PDF

Genomic instability is often considered a hallmark of cancer.  However, it remains unclear whether chromosomal disorganization is most frequently a cause or a consequence of tumorigenesis.  The fact that subsets of many cancers lack chromosomal or microsatellite instability argues against the hypothesis that genomic instability plays an essential role in the initiation and maintenance of oncogenesis.  Cancer-specific, epigenetically-based changes in gene expression caused by abnormalities in DNA methylation, in histone modifications, and in nucleosome positioning are gaining recognition as driving events in tumorigenesis.  But are these changes merely a small piece of the oncogenesis puzzle or could they be the major component, or even sufficient for cancer?    Here we discuss evidence that the progression of aggressive cancers can be driven by epigenetic events without genomic instability.

Open Access Article

NALP3 inflammasome functional polymorphisms and gout susceptibility

Zhi-Min Miao, Shi-Hua Zhao, Sheng-Li Yan, Chang-Gui Li, Yan-Gang Wang, Dong-Mei Meng, Li Zhou and Qing-Sheng Mi
Pages 27 - 30
http://dx.doi.org/10.4161/cc.8.1.7325
Abstract | PDF

Gout is the most common autoinflammatory arthritis characterized by elevated serum urate and recurrent attacks of intra-articular crystal deposition of monosodium urate (MSU). Although the pathogenesis of gout is still unclear, accumulated studies indicate that genetic factors trigger gout development, including some susceptibility genes that control the production and clearance of urate and lead to hyperuricemia. However, the epidemiological evidence suggests that only less than 10% of hyperuricemia patients develop gout, indicating that other genes unrelated to the urate metabolism may also contribute to the diseases susceptibility. Accumulated evidences have implied that MSU crystal-induced inflammation is a paradigm of innate immunity and that NALP3 inflammasome, an innate immune complex containing NALP3, ASC, and CARD-8, is involved in gout development. Recent studies suggest that NALP3 and CARD-8 functional mutations contribute to the development of autoinflammatory diseases including hereditary periodic fever syndrome, arthristis as well as hypertension susceptibility. Taking into account these genetic findings, here we would like to propose a novel hypothesis that functional mutations in NALP3 inflammasome may make NALP3 inflammasome as attractive susceptibility candidates and genetic markers for gout. Further clinical genetic studies need to be performed to confirm the role of NALP3 inflammasome in the etiology of gout.

Open Access Article

The p53 mRNA-Mdm2 interaction

Nadia Naski, Madhavsai Gajjar, Karima Bourougaa, Laurence Malbert-Colas, Robin Fåhraeus and Marco M. Candeias
Pages 31 - 34
http://dx.doi.org/10.4161/cc.8.1.7326
Abstract | PDF

The E3 ligase Mdm2 is a key regulator of p53 activity via a complex regulatory feedback system that involves all levels of expression control including transcription, mRNA translation and protein degradation. Best known is the effect of p53 on Mdm2 transcription and the capacity of Mdm2 to target p53 for degradation, but more recently the role of Mdm2 as a positive regulator of p53 activity has also started to emerge.  Mdm2 stimulates p53 mRNA translation by binding the p53 mRNA and, interestingly, this interaction also suppresses Mdm2’s capacity to promote p53 polyubiquitination and degradation. Another interesting aspect of the p53 mRNA-Mdm2 interaction is that the p53 mRNA sequence encoding the amino acids which bind the N-terminus of Mdm2 is the same that interacts with the Mdm2 RING domain. Indeed, the regulatory elements for controlling Mdm2-dependent expression of p53 are derived from the same p53 genomic sequence. In addition, the RNA binding and the E3 ligase domain of Mdm2 overlap, indicating that the two functions of Mdm2 to control p53 synthesis and degradation have co-evolved in parallel in both p53 and Mdm2. Here we illustrate how the p53-Mdm2 protein-protein and p53 mRNA-Mdm2 interactions affect Mdm2-mediated control of p53 expression using the Phe19Ala p53 mutant. We discuss how the new insights into the regulation of p53 expression levels can help to shed light on the origin of this elegant feedback system and on the function of Mdm2 isoforms.

Open Access Article

The many faces of shugoshin, the “guardian spirit,” in chromosome segregation

Brett Macy, Mian Wang and Hong-Guo Yu
Pages 35 - 37
http://dx.doi.org/10.4161/cc.8.1.7327
Abstract | PDF

Since it was first identified in Drosophila as a centromeric protein, MEI-S332/Shugoshin (Sgo1) has been an enigma.  It is neither a kinase nor a phosphatase, but Sgo1’s many functions are often attributable to protein-protein interactions that require Sgo1’s presence to localize and/or function properly in a timely manner.  Though much has been reported about Sgo1's role in ensuring proper genome segregation through its regulatory role in maintaining cohesion through protection of centromeric cohesin, several additional functions have been identified that are less well characterized.  In higher eukaryotes Sgo2 and a splice variant of Sgo1 make up a small family of shugoshin proteins, whereas in budding yeast a single Sgo1 exists, and available data suggest that the many functions carried out by Sgo2 and the splice variant are handled exclusively by the Sgo1 homolog in yeast.  In fact, the function of the mammalian splice variant is the factor that has revealed a new possible role for shugoshin in centrosome regulation, which may be related mechanistically to its task in protection of centromeric cohesin.  New evidence in budding yeast supports this line of reasoning, which may represent an exciting new advance in exposing the myriad faces of the guardian spirit.

Open Access Article

Proteomic identification of carboxypeptidase E connects lipid-induced β-cell apoptosis and dysfunction in type 2 diabetes

James D. Johnson
Pages 38 - 42
http://dx.doi.org/10.4161/cc.8.1.7343
Abstract | PDF

Type 2 diabetes occurs when the endocrine pancreas can no longer secrete enough insulin to maintain glucose and lipid homeostasis. This is likely due to cumulative defects in β-cell fate and function, as well as insulin resistance. A number of recent studies, including ones from our group, have used unbiased proteomic, genomic, and genetic approaches to unravel the mechanisms by which hyperlipidemia causes beta-cell apoptosis and dysfunction. It is clear from these studies and others, that there are multiple pathways by which fatty acids such as palmitate can lead to β-cell dysfunction and death. In the present article, we highlight the role for dysfunction in the ER and secretory pathway in the toxic effects of free fatty acids. Recent work has shown that the rapid degradation of carboxypeptidase E plays a significant role in beta-cell death in response to the free fatty acid palmitate. These newly identified targets of beta-cell lipotoxicity present novel avenues for research and therapeutic intervention.




REVIEWS

Open Access Article

Stem cells do play with dice: A statistical physics view of transcription

Sol Efroni, Shai Melcer, Malka Nissim-Rafinia and Eran Meshorer
Pages 43 - 48
http://dx.doi.org/10.4161/cc.8.1.7216
Abstract | PDF

Embryonic stem cells display wide-spread pervasive transcriptional output. Here, we propose that multiple simultaneous transcriptional states underlay pluripotency.

Open Access Article

YAP: At the crossroad between transformation and tumor suppression

Efrem Bertini, Tsutomu Oka, Marius Sudol, Sabrina Strano and Giovanni Blandino
Pages 49 - 57
http://dx.doi.org/10.4161/cc.8.1.7259
Abstract | PDF

Yap is a small protein that binds to many transcription factors and modulates their activity. Yap was described to increase the ability of p73 in inducing apoptosis as a consequence of damage to the DNA, and therefore its activity was thought to favor tumor-suppression. However, other studies have recently shown a role for Yap in cell differentiation, cell transformation and in the regulation of organ size. It has been demonstrated that the Drosophila m. Hippo pathway has a mammalian equivalent, and that Yap is part of this pathway, where it might stimulate proliferation. In light of these new findings we ought to re-consider the role of Yap, which seems to be in service of several masters, and whose regulation - likely at the level of PTM - and cellular context might have a pivotal role in the choice of its partners and consequently on the final outcome.




REPORTS

Open Access Article

The Epstein-Barr virus replication and transcription activator, Rta/BRLF1, induces cellular senescence in epithelial cells

Yu-Lian Chen, Yen-Ju Chen, Wan-Hua Tsai, Ying-Chieh Ko, Jen-Yang Chen and Su-Fang Lin
Pages 58 - 65
http://dx.doi.org/10.4161/cc.8.1.7411
Abstract | PDF

Epstein-Barr Virus (EBV) replication and transcription activator (Rta/BRLF1) is an immediate-early transcription factor that controls the conversion of the latent viral genome into one undergoing lytic replication. By using a doxycycline-inducible expression system, the present study demonstrates that EBV Rta efficiently elicits growth arrest in the human epithelial cell line HEK293. In cells arrested by EBV Rta, the expression of p21 (CDKN1A), p27 (CDKN1B) and cyclin E were increased. In contrast, the levels of cyclin D1, CDK4 and CDK6 were sharply decreased. Activation of the host cell DNA damage response (DDR), indicated by the increasing phosphorylation of H2AX and p53 Ser15, was observed on day 3 and day 5 after EBV Rta expression, respectively. Finally, EBV Rta arrested cells exhibited strong senescence-associated β-galactosidase staining on day 10 after doxycycline induction. Together, these results indicate that, in addition to triggering viral lytic replication in epithelial cells, EBV Rta concurrently initiates a cellular senescence program that was previously undocumented. This finding, showing Rta may be centrally involved in inducing a host cell state amenable to efficient viral reproduction, in addition to its previously characterized regulation of viral transcription, provides new perspectives in understanding EBV pathogenesis.

Open Access Article

The atypical CDK activator Spy1 regulates the intrinsic DNA damage response and is dependent upon p53 to inhibit apoptosis

Christopher W. McAndrew, Randy F. Gastwirt and Daniel J. Donoghue
Pages 66 - 75
http://dx.doi.org/10.4161/cc.8.1.7451
Abstract | PDF

The intrinsic damage response is activated by DNA damage that arises during the cell division process. The ability of the cell to repair this damage during proliferation is important for normal cell growth and, when disrupted, may lead to increased mutatagenesis and tumorigenesis. The atypical CDK activator, Spy1, was previously shown to promote cell survival, prevent apoptosis, and inhibit checkpoint activation in response to DNA damage. Prior studies have shown that Spy1 is up-regulated in breast carcinomas and accelerates mammary tumorigenesis in vivo. In this report, first, we demonstrate that the ability of Spy1 to inhibit apoptosis and bypass UV-induced checkpoint activation is dependent on the presence of the gene regulatory protein p53 and the CKI p21. Second, we demonstrate that Spy1 expression has the following effects: prevents repair of cyclobutane pyrimidine dimers through bypass of nucleotide excision repair; increases the cellular mutation frequency; and reduces the formation of cyclin E induced γH2A.X foci. Lastly, we show that knockdown of endogenous Spy1 leads to γH2A.X foci formation, Chk1 phosphorylation, and proliferation defects, demonstrating a functional role for Spy1 in the intrinsic DNA damage response. These results also demonstrate that Spy1 fulfills a novel regulatory role in the intrinsic DNA damage response and maintains the balance between checkpoint activation, apoptosis, repair, and cell cycle progression in response to exogenous or intrinsic damage. Furthermore, the overexpression of Spy1 as a contributing factor in cancer progression will most likely be confined to p53-positive cells.

Open Access Article

Identification of two independent SUMO-interacting motifs in Daxx: Evolutionary conservation from Drosophila to humans and their biochemical functions

Aleixo Santiago, Adam C. Godsey, Jamil Hossain, Lisa Y. Zhao and Daiqing Liao
Pages 76 - 87
http://dx.doi.org/10.4161/cc.8.1.7493
Abstract | PDF

Daxx is essential for embryonic development and implicated in apoptosis and transcriptional regulation. It is found only in the animal kingdom and appears to arise first in insects. In the Drosophila genus, the Daxx orthologs are much larger than those in other species. Here we show that in addition to a conserved core of ~200 residues, Daxx possesses several conserved domains and two essentially invariable short SUMO-interacting motifs (SIMs), each located at one or the other terminus of the protein. Both can independently interact with SUMO. The Daxx I7/733K double mutant with one mutation in each of the two SIMs no longer interacts with SUMO. Daxx interacts with Ubc9 and this interaction strictly requires at least one SIM. Interestingly, the Ubc9 H20D mutation that abolishes non-covalent Ubc9-SUMO interaction also interrupts Daxx-Ubc9 interaction. Thus, SUMO serves as the intermediate for Daxx-Ubc9 interaction. Surprisingly, Daxx I7/733K double mutant could still colocalize with PML. Furthermore, wt Daxx also strongly colocalizes with PMLΔS mutant, in which all three sumoylation sites are mutated, whereas PMLΔS only weakly colocalizes with Daxx I7/733K mutant, suggesting that SIM-SUMO interaction is not essential for but enhances PML-Daxx interaction. Remarkably, Daxx strongly stimulates c-Jun-mediated transcription and both SIMs are required for this stimulation. PML also activates c-Jun, which requires all three sumoylation sites. Coexpression of Daxx and PML revealed that they independently regulate c-Jun, with Daxx exerting a dominant role. These results suggest that the conserved SIMs are involved in mediating protein-protein interactions that underlie Daxx’s diverse cellular functions.

Open Access Article

The antidiabetic drug metformin suppresses HER2 (erbB-2) oncoprotein overexpression via inhibition of the mTOR effector p70S6K1 in human breast carcinoma cells

Alejandro Vazquez-Martin, Cristina Oliveras-Ferraros and Javier A. Menendez
Pages 88 - 96
http://dx.doi.org/10.4161/cc.8.1.7499
Abstract | PDF

Population studies have revealed that treatment with the anti-diabetic drug metformin significantly associates with reduced breast cancer risk.  Animal studies have shown that metformin suppresses the development of mammary carcinomas in transgenic female mice carrying a HER2 oncogene, but not that of spontaneous tumors.  We herein demonstrate that HER2 oncoprotein itself may represent a key cellular target involved in the anti-breast cancer actions of metformin.  First, ectopical overexpression of HER2 oncogene significantly enhances metformin-induced breast cancer cell growth inhibition.  Second, metformin treatment drastically down-regulates HER2 protein levels (up to 85% reduction) in a dose- and time-dependent manner. Metformin-induced inhibition of HER2 take places regardless the molecular mechanism contributing to HER2 overexpression (i.e., human HER2 cDNA exogenously driven by a viral promoter and naturally occurring endogenous HER2 gene amplification).  Mechanistically, metformin-induced suppression of HER2 overexpression appears to occur via direct (AMPK-independent) inhibition of p70S6K1 activity.  Compound C- and small interference RNA (siRNA)-induced blockade of AMPK activity/expression fail to prevent the anti-HER2 effect of metformin while AMPK hyperactivation following exposure to the AMP analog AICAR is not sufficient to down-regulate HER2 expression. HER2-positive breast cancer cells transfected with p70S6K1 siRNA become completely refractory to metformin-induced HER2 suppression. Of note, co-incubation with agents that block reactive oxygen species (ROS) production (e.g. N-acetylcisteine) dramatically enhanced the ability of metformin to decrease HER2 expression. From the perspective of chemoprevention, these findings altogether suggest that metformin might exert a protective mostly confined to the HER2-positive breast cancer subtype. From the perspective of intervention, the presence/absence of molecular hallmarks such as HER2 overexpression and/or p70S6K1 hyperactivation might dictate alternative responses in metformin-based treatment of early breast cancer. The importance of mTOR/p70S6K1-sensed ROS status at mediating the anti-oncogenic effects of metformin might represent a previously unrecognized linkage molecularly connecting its anti-aging and anti-cancer actions.

Open Access Article

Cdk5 acts as a mediator of neuronal cell cycle re-entry triggered by amyloid-β and prion peptides

João P. Lopes, Catarina R. Oliveira and Paula Agostinho
Pages 97 - 104
http://dx.doi.org/10.4161/cc.8.1.7506
Abstract | PDF

Cyclin-dependent kinase 5 (Cdk5) is a serine-threonine kinase important for different cellular processes. Involved in tau protein hyperphosphorylation and apoptotic neuronal death, two main neuropathological markers of Alzheimer’s disease (AD) and Prion-related encephalopathies (PRE), Cdk5 also participates in cell cycle regulation. However, the precise relationship between cell cycle reactivation and Cdk5 dysregulation in AD and PRE remains unclear. To determine Cdk5 involvement in the triggering of an abortive cell cycle by amyloid-beta (Aβ) and prion (PrP) peptides, associated with AD and PRE pathogenesis, we examined the levels/activation of several cell cycle-associated proteins in cultured cortical neurons treated with Aβ1-40 and PrP106-126 peptides. Peptide treatments significantly increased Cdk4, phospho-retinoblastoma and proliferating cell nuclear antigen (PCNA) levels, whereas phospho-histone H3 remained invariable, suggesting cell cycle arrest before the M phase. Moreover, Aβ1-40 and PrP106-126 largely augmented the number of PCNA-immunoreactive cells with fragmented nuclei. The Cdk5 inhibitor roscovitine and the calpain inhibitor MDL28170 prevented the alterations in cell cycle markers induced by both peptides. The data obtained suggest that Aβ and PrP peptides induced neuronal cell cycle re-entry through a mechanism involving Cdk5 dysregulation. Therefore, cell cycle reactivation mediated by Cdk5 can underlie the neurodegenerative processes that occur in AD and PRE.

Open Access Article

Loss of p21^CDKN1A impairs entry to quiescence and activates a DNA damage response in normal fibroblasts induced to quiescence

Paola Perucca, Ornella Cazzalini, Mark Madine, Monica Savio, Ronald A. Laskey, Vanio Vannini, Ennio Prosperi and Lucia Anna Stivala
Pages 105 - 114
http://dx.doi.org/10.4161/cc.8.1.7507
Abstract | PDF

The cell cycle inhibitor p21^CDKN1A induces cell cycle arrest under different conditions, including senescence and terminal differentiation. Still debated is its involvement in the reversible transition from proliferation to a non-dividing quiescent state (G0), in which a significant role has been attributed to cell cycle inhibitor p27^CDKN1B. Here we provide evidence showing that high p21 protein levels are necessary to enter and maintain the quiescence state following contact inhibition and growth factor withdrawal. In fact, entry into quiescence was impaired, both in human fibroblasts in which p21 gene has been deleted, or protein expression knocked-down by RNA interference. Importantly, in the absence of p21, human fibroblasts activate a DNA damage-like signalling pathway, as shown by phosphorylation of histone H2AX and Chk1 proteins. In addition, we show that in the absence of p21, checkpoint is activated by an unscheduled entry into S phase, with a reduced efficiency in DNA maturation, in the presence of high c-myc protein levels. These results highlight the role of p21 in counteracting inappropriate proliferation stimuli for genome stability maintenance.

Open Access Article

Inhibition of p27^Kip1 gene transcription by mitogens

Tapan K. Bagui, Dongming Cui, Sangita Roy, Subhra Mohapatra, Audrey C. Shor, Le Ma and W. Jackson Pledger
Pages 115 - 124
http://dx.doi.org/10.4161/cc.8.1.7527
Abstract | PDF

How mitogens reduce the abundance of the cell cycle inhibitor p27^Kip1 is an important question, and regulation of p27^Kip1 translation and turnover has been described.  Here we show that platelet-derived growth factor (PDGF) reduces the activity of the p27^Kip1 promoter and the abundance of the p27^Kip1 transcript in density-arrested mouse fibroblasts.  Inhibition of p27^Kip1 gene expression by PDGF required protein synthesis and histone deacetylase activity but not Akt or ERK activity.  PDGF increased the expression of c-Myc in the absence but not presence of a histone deacetylase inhibitor, and c-Myc inhibited p27^Kip1 promoter activity when ectopically expressed in fibroblasts.  c-Myc targeted the same region of the p27^Kip1 promoter as did PDGF (deletion analysis) and interacted with this region in vivo (chromatin immunoprecipitation assay).  Collectively, these findings suggest that c-Myc mediates the inhibitory effects of PDGF on the p27^Kip1 promoter.  We also demonstrate reductions in p27^Kip1 mRNA abundance in primary splenocytes exposed to concanavalin A and in T cells exposed to interleukin-2 (IL-2).  In contrast to PDGF in fibroblasts, IL-2 required Akt activity for maximal reductions in p27^Kip1 promoter activity and mRNA abundance in T cells.  Thus, mitogens repress p27^Kip1 gene transcription in multiple systems and by multiple mechanisms.

Open Access Article

Replication licensing promotes cyclin D1 expression and G₁ progression in untransformed human cells

Peijun Liu, Damien M. Slater, Marc Lenburg, Kathleen Nevis, Jeanette Gowan Cook and Cyrus Vaziri
Pages 125 - 136
http://dx.doi.org/10.4161/cc.8.1.7528
Abstract | PDF

Defects in DNA replication are implicated as early and causal events in malignancy.  However, the immediate effects of impaired DNA replication licensing on cell cycle progression of non-malignant human cells are unknown.  Therefore, we have investigated the acute effects of Mcm7 ablation using synchronized cultures of untransformed Human Dermal Fibroblasts (HDF).  Mcm7 ablation elicited a G1 delay associated with impaired activation of CDK4 and CDK2 and reduced Rb phosphorylation.  The cell cycle delay of Mcm7-ablated cells was not associated with a DNA damage response.  However, levels of cyclin D1 mRNA were specifically reduced and binding of RNA Polymerase II to the CYCD1 promoter was decreased in Mcm7-depleted cells.  Similar to Mcm7-deficiency, Mcm2- or Cdc6-depletion led to impaired cyclin D expression.  Ectopic over-expression of Cdc6 in quiescent cells promoted cyclin D1 expression, CDK4 activation and G1 progression.  Therefore timely and efficient expression of cyclin D1 during G1 phase requires replication licensing.  Reconstitution of cyclin D1 expression was insufficient to correct the G1 delay of Mcm7-depleted cells, indicating that additional cell cycle events during G1are dependent on replication licensing.  However, ectopic expression of the HPV-E7 oncoprotein, and the resulting bypass of the requirement for cyclin D1-Rb signaling enabled Mcm7-depleted cells to enter S-phase.  HPV-E7-induced S-phase entry of Mcm7-depleted cells led to a DNA damage response, a hallmark of pre-malignancy.  Taken together, our results suggest the existence of a 'replication licensing restriction point' that couples pre-RC assembly with G1 progression in normal cells to minimize replication stress, DNA damage and tumorigenesis.

Open Access Article

Dimerization of the core domain of the p53 family: A computational study

Arumugam Madhumalar, Lee Hui Jun, David P. Lane and Chandra S. Verma
Pages 137 - 148
http://dx.doi.org/10.4161/cc.8.1.7530
Abstract | PDF

Computational models reveal the structural origins of cooperativity in the association of the DNA binding domains (DBD) of p53 (and its two homologues p63 and p73) with consensus DNA. In agreement with experiments they show that cooperativity, as defined by sequential binding of monomers to DNA is strong for p53 and weak for homologues p63 and p73. Computations also suggest that cooperativity can arise from the dimerization of the DBD prior to binding the DNA for all 3 family members.   Dimerization between the DBDs is driven by packing interactions originating in residues of helix H1 and loop L3, while DNA binding itself is dominated by local and global electrostatics. Calculations further suggest that low affinity oligomerization of the p53 DBD can precede the oligomerization of the tetramerization domain (TD). During synthesis of multiple chains on the polysome, this may increase fidelity by reducing the possibility of the highly hydrophobic TD from nonspecific aggregation. Mutations have been suggested to test these findings.

Open Access Article

Androgenic regulation of hedgehog signaling pathway components in prostate cancer cells

Mengqian Chen, Matthew Tanner, Alice C. Levine, Elina Levina, Patrice Y. Ohouo and Ralph Buttyan
Pages 149 - 157
http://dx.doi.org/10.4161/cc.8.1.7532
Abstract | PDF

Hedgehog signaling is thought to play a role in several human cancers including prostate cancer.  Although prostate cancer cells express many of the gene products involved in hedgehog signaling, these cells are refractory to the canonical signaling effects of exogenous hedgehog ligands or to activated Smoothened, the hedgehog-regulated mediator of Gli transcriptional activation.  Here, we show that the expression of hedgehog ligands and some hedgehog target genes are regulated by androgen in the human prostate cancer cell line, LNCaP and its more metastatic variants (C4-2 and C4-2B).  Androgen (R1881) strongly suppressed the expression of hedgehog ligands in these cells and their prolonged maintenance in androgen-deficient medium upregulated Sonic and Indian hedgehog mRNA and protein levels by up to 30,000-fold.  Hedgehogs were released into the conditioned medium of androgen-deprived LNCaP cells and this medium was able to increase hedgehog target gene expression in hedgehog-responsive mouse fibroblasts (MC3T3-E1).  Moreover, this activity was accompanied by increased expression of Gli target genes, Patched 1 and Gli2, in LNCaP that could be suppressed by cyclopamine, indicating that chronic androgen-deprivation also re-awakens the autocrine responsiveness of the cancer cells to hedgehog.  In contrast to the suppressive effects of R1881 on hedgehog ligand and Gli2 expression, we found that Gli1 expression in LNCaP cells was induced by R1881.  Given the ability of androgen to modulate the expression and release of hedgehog ligands and the activity of the autocrine hedgehog signaling pathway in these prostate cancer cells, our results imply that chronic androgen deprivation therapy (ADT) for prostate cancer might create a hedgehog signaling environment in the region of the tumor that could ultimately impact on the long term effectiveness of this treatment.  This consideration supports the idea of clinically testing hedgehog-blocking drugs in conjunction with ADT in patients with advanced prostate cancer.

Open Access Article

Molecular phenotyping of human ovarian cancer stem cells unravels the mechanisms for repair and chemoresistance

Ayesha B. Alvero, Rui Chen, Han-Hsuan Fu, Michele Montagna, Peter E. Schwartz, Thomas Rutherford, Dan-Arin Silasi, Karina D. Steffensen, Marianne Waldstrom, Irene Visintin and Gil Mor
Pages 158 - 166
http://dx.doi.org/10.4161/cc.8.1.7533
Abstract | PDF

A major burden in the treatment of ovarian cancer is the high percentage of recurrence and chemoresistance. Cancer stem cells (CSCs) provide a reservoir of cells that can self-renew, can maintain the tumor by generating differentiated cells [non-stem cells (non-CSCs)] which make up the bulk of the tumor and may be the primary source of recurrence. We describe the characterization of human ovarian cancer stem cells (OCSCs). These cells have a distinctive genetic profile that confers them with the capacity to recapitulate the original tumor, proliferate with chemotherapy, and promote recurrence. CSC identified in EOC cells isolated form ascites and solid tumors are characterized by: CD44⁺, MyD88⁺, constitutive NFκB activity and cytokine and chemokine production, high capacity for repair, chemoresistance to conventional chemotherapies, resistance to TNFα-mediated apoptosis, capacity to form spheroids in suspension, and the ability to recapitulate in vivo the original tumor.
Chemotherapy eliminates the bulk of the tumor but it leaves a core of cancer cells with high capacity for repair and renewal. The molecular properties identified in these cells may explain some of the unique characteristics of CSCs that control self-renewal and drive metastasis. The identification and cloning of human OCSCs can aid in the development of better therapeutic approaches for ovarian cancer patients.


Open Access Article

APC/C- and Mad2-mediated degradation of Cdc20 during spindle checkpoint activation

Sheng Ge, Jeffrey R. Skaar and Michele Pagano
Pages 167 - 171
http://dx.doi.org/10.4161/cc.8.1.7606
Abstract | PDF

The spindle assembly checkpoint (SAC) is an important mechanism that prevents the separation of sister chromatids until the microtubules radiating from the spindle poles are correctly attached to the kinetochores.  Cdc20, an activator of the Anaphase Promoting Complex/Cyclosome (APC/C), is known as a major downstream target for inhibition by the SAC through the binding of mitotic checkpoint proteins, such as Mad2 and BubR1.  Here, we report that the SAC also negatively regulates the stability of Cdc20 by targeting it for proteasome-dependent degradation.  Once the checkpoint is activated by spindle poisons, a major population of Cdc20 is degraded via APC/C, an event that requires the binding of Cdc20 to Mad2.  We propose that the degradation of Cdc20 represents a critical control mechanism to ensure inactivation of APC/C^Cdc20 in response to the SAC.




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