Table of Contents

Volume 3, Issue 12

  December 2004

  Pages 1477 - 1650

View covers archive

EXTRA VIEWS

Open Access Article

Epithelial-Mesenchymal Transition: NF-κB Takes Center Stage

Margit A. Huber, Hartmut Beug and Thomas Wirth
Pages 1477 - 1480
http://dx.doi.org/10.4161/cc.3.12.1280
Abstract | PDF

Activation of the transcription factor NF-?B occurs in many human tumors, and studies have shown that NF-κB can promote cell proliferation and oncogenesis, possibly by protecting cells from apoptosis. Little is known, however, about whether NF-κB is involved in tumor progression including epithelial-mesenchymal transition (EMT), a central process governing both morphogenesis and carcinoma progression in multicellular organisms. In a combined in vitro/in vivo model of mammary carcinogenesis, NF-κB was essential both for the induction and maintenance of EMT and for in vivo metastasis. NF-κB and the signaling pathways that are involved in its activation should therefore receive attention as potential targets for the development of novel anti-metastatic cancer treatments.

Open Access Article

Regulation of Cell Cycle in Hematopoietic Stem Cells by the Niche

Atsushi Hirao, Fumio Arai and Toshio Suda
Pages 1481 - 1483
http://dx.doi.org/10.4161/cc.3.12.1281
Abstract | PDF

The quiescent state is thought to be an indispensable property for the maintenance of hematopoietic stem cells (HSCs). Interaction of HSCs with their particular microenvironments, known as the stem cell niches, is critical for cell cycle regulation of HSCs. Monitoring of the quiescence of HSCs using by a new stem cell marker, Side Population (SP), revealed that the cell cycle status of HSCs is dynamically controlled by the microenvironments. We have recently revealed a molecular mechanism in which cell cycle of HSCs is regulated by the niche. HSCs expressing the receptor tyrosine kinase Tie2 are adhere to osteoblasts (OBs) in the BM niche. The interaction of Tie2 and its ligand Angiopoietin-1 (Ang-1) leads to tight adhesion of HSCs to stromal cells, resulting in maintainance of long-term repopulating activity of HSCs. Thus, Tie2/Ang-1 signaling pathway plays a critical role in the maintenance of HSCs in a quiescent state in the BM niche. The understanding of cell cycle control in stem cells leads to development of new strategy for progress in regenerative medicine.

Open Access Article

Heat Shock Protein 70 Promotes Cancer Cell Viability by Safeguarding Lysosomal Integrity

Mads Gyrd-Hansen, Jesper Nylandsted and Marja Jäättelä
Pages 1484 - 1485
http://dx.doi.org/10.4161/cc.3.12.1287
Abstract | PDF

The major heat-inducible Hsp70 is a potent survival protein that confers cytoprotection against numerous death-inducing stimuli and increases the tumorigenicity of rodent cells. The depletion of Hsp70 by adenovirus-mediated transfer of antisense cDNA induces caspase-independent death of tumorigenic cells while non-tumorigenic cells are unaffected, suggesting that Hsp70 has cancer-specific function(s). We have recently demonstrated that the depletion of Hsp70 in cancer cells results in a cysteine cathepsin-dependent death, which is preceded by lysosomal destabilization and release of lysosomal constituents to the cytosol. In line with this, Hsp70 localizes to the membranes of lysosomes in human colon carcinoma cells and immortalized murine embryonic fibroblasts (MEFs) and prevents lysosomal membrane permeabilization and cell death induced by tumor necrosis factor (TNF), etoposide and H2O2. These findings identify Hsp70 as the first survival protein that functions by stabilizing the lysosomal membrane.

Open Access Article

Abl Oncogene Bypasses Normal Regulation of JAK/STAT Activation

André Limnander and Paul B. Rothman
Pages 1486 - 1488
http://dx.doi.org/10.4161/cc.3.12.1297
Abstract | PDF

In normal cells, the strength and duration of proliferative signaling pathways are tightly regulated. In oncogenic settings, negative regulation is often bypassed to allow constitutive activation of these pathways. In our recent manuscript, we identify a mechanism that allows the v-Abl oncogene to bypass negative regulation by SOCS-1 to constitutively activate Jak-Stat signaling. The mechanism involves post-translational modifications of SOCS-1 that disrupt its interaction with the proteasome, thereby preventing it from targeting activated Jak kinases for degradation. In this review, we discuss the implications of these findings for our understanding of v-Abl oncogenesis and the regulation of SOCS protein function.

Open Access Article

Delineating the Hepatocyte's Hematopoietic Fusion Partner

Holger Willenbring and Markus Grompe
Pages 1489 - 1491
http://dx.doi.org/10.4161/cc.3.12.1304
Abstract | PDF

Cell fusion has evolved as an explanation of how transplanted bone marrow cells adopt the phenotype of hepatocytes, Purkinje neurons, skeletal and cardiac muscle cells. In vivo nuclear transfer associated with cell fusion has direct implications for regenerative medicine, but the spontaneous frequency of cell fusion is well below the threshold of therapeutic significance. Increased efficiency could be achieved by utilizing cellular factors known to govern fusion but for this the identity of the hematopoietic cell that fuses with the host cell must be known. Using increasingly lineage-restricted donor bone marrow cell populations we have shown in mouse liver that fusion occurs between host hepatocytes and transplanted myelomonocytic cells such as macrophages. Now it should be feasible to increase the efficiency and assess the potential of cell fusion for the correction of a broad range of somatic cell types that can be targeted by fusion.

Open Access Article

Stress-Induced p53 Runs a Direct Mitochondrial Death Program: Its Role in Physiologic and Pathophysiologic Stress Responses In Vivo

Susan Erster and Ute M. Moll
Pages 1492 - 1495
http://dx.doi.org/10.4161/cc.3.12.1318
Abstract | PDF

It is now well established that a fraction of stress-induced wtp53 protein rapidly translocates to mitochondria in immortalized and transformed cells in culture. Mitochondrial p53 interacts with anti-apoptotic proteins of the Bcl 2 family at the outer mitochondrial membrane, resulting in membrane permeabilization, release of death effectors such as cytochrome C and subsequent rapid apoptosis. The significance and relevance of this direct mitochondrial p53 program to the overall p53-mediated stress response in vivo is underlined by a number of recent studies in animals and primary cells. They all support a role for this direct pathway in the physiologic and pathophysiologic response to genotoxic and hypoxic insults and occur precisely in those tissues where p53 plays a critical role in mediating apotpotis rather than cell cycle arrest.

Open Access Article

Inhibition of Mutant EGF Receptors by Gefitinib: Targeting an Achilles’ Heel of Lung Cancer

Jeffrey Settleman
Pages 1496 - 1497
http://dx.doi.org/10.4161/cc.3.12.1325
Abstract | PDF

Recent reports revealed a strong correlation between the presence of somatic activating mutations in the kinase domain of the EGF receptor in some human lung cancers and a striking clinical response to the selective EGF receptor inhibitor, Gefitinib. These oncogenic receptors exhibit altered signaling properties such that they selectively activate downstream survival pathways on which tumor cells have become dependent. Thus, tumor cells are effectively killed by Gefitinib treatment. In addition, the catalytic function of the mutant receptors exhibits increased sensitivity to Gefitinib, raising the possibility that two distinct consequences of Gefitinib action account for its clinical efficacy.

Open Access Article

Protein Phosphorylation as a Key Mechanism for the Regulation of BCL-3 Activity

Patrick Viatour, Marie-Paule Merville, Vincent Bours and Alain Chariot
Pages 1498 - 1501
http://dx.doi.org/10.4161/cc.3.12.1328
Abstract | PDF

Constitutive NF-?B activation, a hallmark of many human cancers, upregulates anti-apoptotic gene expression and therefore disrupts the balance between apoptosis and proliferation. In some lymphomas, this constitutive NF-?B activity is the result of point mutations or translocations of the genes coding for NF-?B inhibitors, namely I?B? or p100. The BCL-3 protein is another member of the I?B family and is overexpressed in a subset of human B-cell chronic lymphocytic leukemias because of a chromosomal translocation. This oncoprotein is phosphorylated by multiple kinases including GSK3 and this phosphorylation regulates BCL-3 function by modulating its oncogenic potential and by regulating the expression of a subset of its target genes. Therefore, deciphering the NF-?B/I?B protein phosphorylations is critical in order to better understand the molecular mechanisms of NF-?B-mediated oncogenesis.

Open Access Article

Pharmacokinetic Resistance to Imatinib Mesylate: Role of the ABC Drug Pumps ABCG2 (BCRP) and ABCB1 (MDR1) in the Oral Bioavailability of Imatinib

Kk Burger and Kees Nooter
Pages 1502 - 1505
http://dx.doi.org/10.4161/cc.3.12.1331
Abstract | PDF

Imatinib mesylate is a selective tyrosine kinase inhibitor that is successfully used in the treatment of chronic myeloid leukaemias and gastrointestinal stromal tumours. The drug is taken orally on a daily basis in order to suppress tumour growth. Unfortunately, the vast majority of patients will eventually progress while on therapy. It is generally thought that this acquired unresponsiveness is due to gene amplification or somatic mutations in the drug’s target genes. However, we have now evidence, based on several in vitro and in vivo observations suggesting that pharmacokinetic resistance may also play a definitive role in the ultimate resistance of patients on chronic imatinib. Our findings may have serious implications for the chronic imatinib treatment of cancer patients.




PERSPECTIVES

Open Access Article

Red Light, Green Light: Signals That Control Endothelial Cell Proliferation during Embryonic Vascular Development

Brenda L. Bohnsack and Karen K. Hirschi
Pages 1506 - 1511
http://dx.doi.org/10.4161/cc.3.12.1334
Abstract | PDF

The proper regulation of endothelial cell proliferation is critical for vascular development in the embryo. VEGF-A and bFGF, which are important in the induction of mesodermal progenitors to form a capillary plexus, are also key mitogenic signals. Disruption in VEGF-A or bFGF decreases endothelial cell proliferation and halts vascular development. While stimulation of endothelial cell proliferation is necessary during vasculogenesis, inhibitory signals such as TGF-?1 and retinoic acid are equally important and required to inhibit endothelial cell proliferation. These signals and activation of numerous downstream pathways must be properly integrated with extracellular matrix proteins and integrin receptor signaling in order to form the embryonic vasculature. This coordination of mitogenic and anti-proliferative signals needed to form a circulatory network in the embryo may be unique relative to neovascularization in adult tissues where mitogenic stimulation promotes proliferation of previously quiescent endothelial cells to repair and expand existing vasculatures.

Open Access Article

Wnt and Cyclooxygenase-2 Cross-talk Accelerates Adenoma Growth

Dingzhi Wang, Jason R. Mann and Raymond N. DuBois
Pages 1512 - 1515
http://dx.doi.org/10.4161/cc.3.12.1288
Abstract | PDF

Both Wnt and cyclooxygenase (COX-2) pathways are activated in most sporadic and familial colorectal cancers, especially in those with chromosomal instability. We have recently shown that a common target of both signaling pathways, the peroxisome proliferator-activated receptor (PPAR)-?, is involved in intestinal adenoma growth. Activation of this receptor by synthetic agonist (GW501516) or COX-2-derived prostaglandin E2 (PGE2) accelerates intestinal adenoma growth in ApcMin mice. Moreover, these effects are lost in ApcMin mice lacking PPAR?. These findings implicate PPAR? as a focal point of cross-talk between the Wnt and prostaglandin signaling pathways. Based on this work it looks as if PPAR? agonists currently in development for treatment of dyslipidemias and obesity may increase the risk of tumor formation in humans. By contrast, antagonists of PPAR? may provide a novel approach for prevention and treatment of colorectal cancer.

Open Access Article

Multiple Roles for Cysteine Cathepsins in Cancer

Johanna A. Joyce and Douglas Hanahan
Pages 1516 - 1519
http://dx.doi.org/10.4161/cc.3.12.1289
Abstract | PDF

Cysteine cathepsins are a family of proteases that have recently emerged as important players in cancer, and have variously been reported to be involved in apoptosis, angiogenesis, cell proliferation, and invasion. In normal cells, cysteine cathepsins are typically localized in lysosomes and other intracellular compartments, and are involved in protein degradation and processing. However, in certain tumors, cathepsins are translocated from their intracellular compartments to the cell surface, and can even be secreted. In addition, the expression and activity levels of some cysteine cathepsins are upregulated in human and mouse cancers. Understanding which cathepsins are critically involved, what their substrates are, and how they may be mediating these complex roles in cancer are important questions to address. We highlight recent results that begin to answer some of these questions, illustrating in particular the lessons from studying a mouse model of multistage carcinogenesis, which suggests distinctive roles for individual cysteine cathepsins in tumor progression.

Open Access Article

JNK2: A Negative Regulator of Cellular Proliferation

Kanaga Sabapathy and Erwin F. Wagner
Pages 1520 - 1523
http://dx.doi.org/10.4161/cc.3.12.1315
Abstract | PDF

The three different c-Jun N-terminal kinases (JNKs) are activated in multiple cell types by both apoptotic and mitogenic signals, and in turn regulate the activity of transcription factors such as c-Jun. Being highly homologous and ubiquitously expressed, the JNK1 and JNK2 proteins have been thought to perform redundant functions in many physiological process. However, our data from Jnk1-/- or Jnk2-/- cells and mice suggest that both JNK isozymes perform distinct functions in regulating cellular proliferation via differential regulation of c-Jun, which is a critical regulator of cell-cycle progression. Absence of JNK1, the positive regulator of c-Jun, leads to decreased fibroblast proliferation. In contrast, JNK2 deficiency leads to reduced c-Jun degradation, thereby augmenting c-Jun levels and cellular proliferation. Various cell types including fibroblasts, erythroblasts and hepatocytes from Jnk2-/- mice exhibit increased proliferation rates compared to their wild-type counterparts. These data therefore suggests that JNK2, in contrast to JNK1, is a negative regulator of cellular proliferation in multiple cell types.

Open Access Article

NF-κB and JNK: An Intricate Affair

Concetta Bubici, Salvatore Papa, Can G. Pham, Francesca Zazzeroni and Guido Franzoso
Pages 1524 - 1529
http://dx.doi.org/10.4161/cc.3.12.1321
Abstract | PDF

NF-κB/Rel transcription factors block apoptosis or programmed cell death (PCD) induced by tumor necrosis factor (TNF)α. The antiapoptotic activity of NF-κB is also crucial for immunity, lymphocyte development, tumorigenesis, and cancer chemoresistance. With respect to TNF?, the NF-κB-mediated suppression of apoptosis involves inhibition of the c-Jun-N-terminal kinase (JNK) cascade. This inhibitory activity of NF-κB depends upon transcriptional upregulation of blockers of the JNK cascade such as the caspase inhibitor XIAP, the zinc-finger protein A20, and the inhibitor of the MKK7/JNKK2 kinase Gadd45β/Myd118. Moreover, NF-κB blunts accumulation of reactive oxygen species (ROS) induced by TNF?, and this antioxidant effect of NF-κB is also critical for inhibition of TNFα-induced JNK activation. Suppression of ROS by NF- κB is mediated by Ferritin heavy chain (FHC)—the primary iron-storage mechanism in cells—and possibly, by the mitochondrial enzyme Mn++ superoxide dismutase (Mn- SOD). Thus, induction of FHC and Mn-SOD represents an additional, indirect means by which NF-κB controls proapoptotic JNK signaling. These findings identify potential new targets for anti-inflammatory and anti-cancer therapy.




REVIEWS

Open Access Article

Hsp90 Activation and Cell Cycle Regulation

Francis Burrows, Hong Zhang and Adeela Kamal
Pages 1530 - 1536
http://dx.doi.org/10.4161/cc.3.12.1277
Abstract | PDF

The widely-expressed molecular chaperone heat shock protein 90 (Hsp90) regulates several important cellular processes via its’ repertoire of ‘client’ proteins. Signal transduction pathways controlled by Hsp90 contribute to all major components of the malignant phenotype, so Hsp90 inhibitors are under investigation as anticancer agents. Since Hsp90 is also expressed at high levels in many normal tissues, it was unclear why Hsp90 inhibitors such as 17-allylamino-geldanamycin (17-AAG) have selective antitumor activity in animals and are well tolerated clinically. Recent findings indicate that Hsp90 is largely latent in unstressed normal cells, but tumor Hsp90 becomes completely utilized during malignant progression, resulting in an activation-dependent conformational shift that radically increases 17-AAG binding affinity in cancer cells. In this article, the implications of this discovery are discussed, with particular reference to cell cycle regulation in normal and malignant cells, and the consequences of inducing cell cycle arrest with Hsp90 inhibitors.

Open Access Article

Flavopiridol, An Inhibitor of Transcription: Implications, Problems and Solutions

Mikhail V. Blagosklonny
Pages 1537 - 1542
http://dx.doi.org/10.4161/cc.3.12.1278
Abstract | PDF

After a decade of exciting promises, the CDK inhibitor flavopiridol has quietly failed in most clinical trials. This review discusses that flavopiridol is a potent inhibitor of global transcription. This explains not only downregulation of numerous proteins, cell cycle arrest and apoptosis but also all pleiotropic and mysterious effects of flavopiridol. Yet, flavopiridol is not just a second actinomycin D. As an inhibitor of transcription with a unique mechanism of action, flavopiridol may have tremendous clinical potentials. This article reviews the molecular and cellular effects of flavopiridol as well as mechanisms of therapeutic and side effects, suggesting its novel clinical applications as a single agent and in drug combinations.




REPORTS

Open Access Article

p53 Prevents the Accumulation of Double-Strand DNA Breaks at Stalled-Replication Forks Induced by UV in Human Cells

Shoshana Squires, Julia A. Coates, Michal Goldberg, Lorraine H. Toji, Stephen P. Jackson, Duncan J. Clarke and Robert T. Johnson
Pages 1543 - 1557
http://dx.doi.org/10.4161/cc.3.12.1272
Abstract | PDF

To investigate the mechanism by which UV irradiation causes S-phase-dependent chromosome aberrations and thereby genomic instability, we have developed an assay to study the DNA structure of replication forks (RFs) in UV-irradiated mammalian cells, using pulse-field gel electrophoresis for the DNA analysis. We demonstrate that replication stalling at UV-induced pyrimidine dimers results in the formation of single-strand DNA (ssDNA) regions and incomplete RF structures. In normal and in excision-repair-defective xeroderma pimentosum (XP) cells, stalling at dimers is rapid and prolonged and recovery depends on dimer repair or bypass. By contrast, XP variant (XPV) cells, defective in replication of a UV-damaged template due to mutation of bypass-polymerase ?, fail to arrest at dimers, resulting in a much higher frequency of ssDNA regions in the stalled RFs. We show that the stability of UV-arrested RFs depends directly on functional p53, and indirectly on NER and pol ?. In p53-deficient cells, the stalled sites give rise to double-strand DNA breaks (DSBs), at a frequency inversely correlated with repair capacity of the cell. In normal cells only a fraction of the stalled sites give rise to DSBs, while in XPASV, XPDSV and also XPVSV all the sites do. XPVSV cells, although repair proficient, accumulate almost double the number of DSBs, suggesting that a high frequency of ssDNA regions in UV-arrested forks cause RF instability. These replication-associated DSBs do not accumulate in p53-proficient human cells. We propose that a major mechanism by which p53 maintains genome stability is the prevention of DSB accumulation at long-lived ssDNA regions in stalled-replication forks.

Supplemental material for this paper can be found at the following link:

http://www.landesbioscience.com/journals/cc/squiresCC3-12-sup.pdf

Open Access Article

Initiation of Colorectal Cancer: Where do the Two Hits Hit?

Natalia L. Komarova and Liming Wang
Pages 1558 - 1565
http://dx.doi.org/10.4161/cc.3.12.1186
Abstract | PDF

It is widely believed that stem cells are of special importance for colorectal cancer initiation. The earliest event being the inactivation of both alleles of the Adenomatous Polyposis Coli (APC) gene, it is thought that the stem cells are the most likely target for these two ?rst hits. Indeed, at the ?rst glance, short-lived differentiated cells cannot sustain a mutation longenough for the second hit to occur, because of the constantapoptosis/renewal process in epithelial tissues. Using a straightforward calculation, we show that this intuitive argument is incorrect.Our model basedon the conventional view of colon cryptarchitecture, suggests thatat least one ofthe two hits mayoccur in the migrating compartment. We suggest that a possible role of differentiating cells in cancer initiation cannot be discarded simply based on the fact that theyare short–lived. More evidence is neededto understandthe cellular origins of cancer and to identify whether or not a double hit in a daughter cell can be “immortalizing”. In this study we discuss several scenarios and propose some experiments which can shed light on these questions.

Open Access Article

Pro- and Anti-Apoptotic Effects of an Inhibitor of Chymotrypsin-Like Serine Proteases

Malcolm A. King, Dorota Halicka and Zbigniew Darzynkiewicz
Pages 1566 - 1571
http://dx.doi.org/10.4161/cc.3.12.1276
Abstract | PDF

The irreversible inhibitor of chymotrypsin-like serine proteases, N-tosyl –L-phenylalanine chloromethylketone (TPCK), was shown to prevent internucleosomal DNA cleavage caused by inducers of apoptosis. The pro-apoptotic properties of TPCK have been studied less thoroughly. The aim of the present study was to investigate the pro- and anti-apoptotic activities of TPCK on HL-60 cells and compare them with the actions of the mitochondrial electron transport inhibitor antimycin A (AMA). The results showed that TPCK alone caused activation of cell cycle checkpoints, mitochondrial cytochrome c release, caspase-3 activation, and chromatin condensation. Caspase-8 was not required for cytochrome c release but was crucial to caspase-3 activation. TPCK synergistically enhanced AMA-induced cytochrome c release and caspase-3 activation while completely blocking AMA-induced internucleosomal DNA fragmentation for at least 8 hours. Rather than blocking AMA-induced DNA fragmentation, the general serine protease inhibitor 4-(2-aminoethyl)-benzenesulphonyl fluoride (AEBSF) actually enhanced it. The pro-apoptotic effect of TPCK may be due to activation of cell cycle checkpoints via inhibition of the proteasome. The apoptotic pathways activated by TPCK and AMA probably converge at the level of the mitochondria. The mode by which TPCK prevents internucleosomal DNA fragmentation is probably not through serine protease inhibition.

Open Access Article

Regulation of Cyclin E Protein Levels through E2F-Mediated Inhibition of Degradation

Deborah Pajalunga and Marco Crescenzi
Pages 1572 - 1578
http://dx.doi.org/10.4161/cc.3.12.1279
Abstract | PDF

Cyclin E is a key cell cycle regulator, whose accumulation is believed to be positively modulated chiefly at the transcriptional level. We show that forced expression of E2F family members specifically stabilizes cyclin E protein by reducing its conjugation with ubiquitin, leading to increased steady-state levels. The stabilized protein shows enhanced association with cdk2 and higher kinase activity, indicating that cyclin E stabilization bears functional consequences. Although the activity of E2F on the cyclin E protein does not entail increased cyclin E gene transcription, it does require the E2F transactivation capacity, as demonstrated by E2F and DP-1 mutant analysis. However, such activity does not require E2F binding to pRb. Furthermore, E2F stabilizes cyclin E even in non-proliferating cells. Our results bear significance for the understanding of tumor progression, in light of the well-known autoregulatory loop between E2F and cyclin E and the disregulation of E2F that is one consequence of the almost universal impairment of the pRb pathway in cancer. Constitutive pRb inactivation leads to enhanced E2F activity through loss of binding. We propose that such increased E2F activity stabilizes cyclin E and contributes to establish the high and persistent levels of the protein commonly found in human neoplasias.

Open Access Article

ATR and ATM-Dependent Movement of BLM Helicase during Replication Stress Ensures Optimal ATM Activation and 53BP1 Focus Formation

Albert R. Davalos, Patrick Kaminker, Rhonda K. Hansen and Judith Campisi
Pages 1579 - 1586
http://dx.doi.org/10.4161/cc.3.12.1286
Abstract | PDF

The BLM helicase, a deficiency in which markedly increases cancer incidence in humans, is required for optimal repair during DNA replication. We show that BLM rapidly moves from PML nuclear bodies to damaged replication forks, returning to PML bodies several hours later, owing to activities of the DNA damage response kinases ATR and ATM, respectively. Immunofluorescence and cellular fractionation demonstrate that BLM partitions to different subcellular compartments after replication stress. Unexpectedly, fibroblasts lacking BLM were deficient in phospho-ATM (S-1981) and 53-binding protein-1 (53BP1), and these proteins failed to form foci following replication stress. Expression of a dominant p53 mutant or helicasedeficient BLM restored replication stress-induced 53BP1 foci, but only mutant p53 restored optimal ATM activation. Thus, optimal repair of damaged replication fork lesions likely requires both ATR and ATM, BLM recruits 53BP1 to these lesions independent of its helicase activity, and optimal activation of ATM requires both p53 and BLM helicase activities.

Supplemental material for this paper can be found at the following link:

http://www.landesbioscience.com/journals/cc/davalosCC3-12-sup.pdf

Open Access Article

Altered Sumoylation of p63α Contributes to the Split-Hand/Foot Malformation Phenotype

Yi-Ping Huang, Guojun Wu, Zhongmin Guo, Motonobu Osada, Tanya Fomenkov, Hannah Lui Park, Barry Trink, David Sidransky, Alexey Fomenkov and Edward A. Ratovitski
Pages 1587 - 1596
http://dx.doi.org/10.4161/cc.3.12.1290
Abstract | PDF

p63 mutations have been identified in several developmental abnormalities, including splithand/ foot malformation (SHFM). In this study, we demonstrate that the C-terminal domain of p63α associates with the E2 ubiquitin conjugating enzyme, Ubc9. A p63α mutation, Q634X, which naturally occurs in SHFM modulated the interaction of p63α with Ubc9 in yeast genetic assay. Furthermore, Ubc9 catalyzed the conjugation of p63α with small ubiquitin modifier-1 (SUMO-1), which covalently modified p63α in vitro and in vivo at two positions (K549E and K637E), each situated in a SUMO-1 modification consensus site (?KXD/E). In addition, p63? mutations (K549E and K637E) abolished sumoylation of p63α, dramatically activated transactivation properties of TAp63α, and inhibited the dominant-negative effect of ΔNp63α. These p63? mutations also affected the transcriptional regulation of gene targets involved in bone and tooth development (e.g., RUNX2 and MINT) and therefore might contribute to the molecular mechanisms underlying the SHFM phenotype.

Open Access Article

Genistein Induces G2 Arrest in Malignant B Cells by Decreasing IL-10 Secretion

Amal Mansour, Brian McCarthy, Stephan K Schwander, Victor Chang, Sergei Kotenko, Sreekrishna Donepudi, Janet Lee and Elizabeth Raveche
Pages 1597 - 1605
http://dx.doi.org/10.4161/cc.3.12.1293
Abstract | PDF

Chronic B cell malignancies are often chemoresistant and the development of new therapeutic modalities is a high priority. Many B cell malignancies have autocrine production of IL-10, which regulates B cell growth and differentiation. Here we demonstrated that the soy isoflavone genistein, a tyrosine kinase inhibitor, rapidly decreased IL-10 secretion followed by upregulation of IFN-gamma and inhibition of cell proliferation with predominantly G2 arrest. The antiproliferative effects of genistein could be reversed by the addition of exogenous IL-10. Genistein downregulated cdc25C and cdk1 as well as anti-apoptotic proteins survivin and Ian-5. After genistein withdrawal, the G2M arrested cells re-entered the cell cycle and underwent apoptosis, which was significantly augmented by fludarabine. We conclude that genistein can sensitize malignant B cells to the action of other chemotherapeutic agents by modulating the cytokine profile and controlling cell cycle progression.

Open Access Article

Cell Death Inhibiting RNA (CDIR) Modulates IFN-γ-Stimulated Sensitization to Fas/CD95/Apo-1 and TRAIL/Apo-2L-Induced Apoptosis

Ksenya Shchors, Fruma Yehiely and Louis P. Deiss
Pages 1606 - 1611
http://dx.doi.org/10.4161/cc.3.12.1295
Abstract | PDF

We have previously demonstrated that over-expression of Cell Death Inhibiting RNA (CDIR), a portion of the 3’untranslated region (UTR) of KIAA0425, inhibits Interferon-γ (IFN-γ) induced apoptosis in HeLa cells (Shchors et al., J Biol Chem. 2002 277(49):47061-72). IFN-γ is known to sensitize cells to killing induced by the death receptor ligands such as Fas/APO-1/CD95 and TNF-related apoptosis–inducing ligand (TRAIL/Apo-2L). Here we report that while CDIR does not alter the response of cells to Fas or TRAIL, it significantly modulates IFN-γ-induced sensitization of HeLa cells to these death-inducing ligands. Interestingly, while CDIR abrogates the IFN-γ-modulated sensitization to Fas, it enhances the sensitization to TRAIL. Expression of CDIR did not alter initial steps of IFN-γ signaling including induction of Signal Transducer and Activator-1 (Stat1), caspase-1 or Interferon Regulatory Factor -1 (IRF1) transcription. In contrast, although expression of CDIR does not affect the protein level of caspase-1 or STAT1, it does significantly reduce the level of IRF1 protein. Thus, CDIR mediates IFN-γ-induced apoptosis, at least in part, by reducing the level of the pro-apoptotic tumor suppressor gene IRF1 via a post-transcriptional mechanism. Since tumor cells are often less sensitive to Fas and more sensitive to TRAIL than normal cells, we suggest that CDIR or CDIR-like activity could contribute to such a phenotype of tumor cells.

Open Access Article

Ufd2, a Novel Autoantigen in Scleroderma, Regulates Sister Chromatid Separation

Sarah Spinette, Christoph Lengauer, James A. Mahoney, Prasad V. Jallepalli, Zhenghe Wang, Livia Casciola-Rosen and Antony Rosen
Pages 1612 - 1618
http://dx.doi.org/10.4161/cc.3.12.1345
Abstract | PDF

The characterization of molecules recognized by patients with autoimmune diseases has provided significant insights into important biological pathways. In these studies, we define Ufd2 (a novel E4 polyubiquitylating enzyme) as a new autoantigen in scleroderma, and show that it regulates chromosome condensation and separation during mitosis in human cells. Ufd2 is regulated by phosphorylation in mitosis. Inhibition of Ufd2 expression results in mitotic arrest at the metaphase-anaphase transition, where cells manifest abnormal chromosome morphology, missegregated chromosomes, irregular spindles, and premature separation of sister chromatids. This is accompanied by premature separase activation, and accumulation of securin in a novel modified form. We further demonstrate that Ufd2 directly and efficiently ubiquitylates securin in vitro and is required for securin polyubiquitylation in vivo. This is the first description of a physiologic substrate for Ufd2, establishing this E4 enzyme as an important regulator of chromosome condensation and separation during mitosis in human cells. Its targeting in scleroderma, together with many other components of the mitotic machinery, reinforces the concept that mitotic cells may be an important focus of the autoimmune response in this disease.

Open Access Article

A Dynamic Switch in the Replication Timing of Key Regulator Genes in Embryonic Stem Cells upon Neural Induction

Pascale Perry, Stephan Sauer, Nathalie Billon, William D. Richardson, Mikhail Spivakov, Gary Warnes, Frederick J. Livesey, Matthias Merkenschlager, Amanda G. Fisher and Veronique Azuara
Pages 1619 - 1624
http://dx.doi.org/10.4161/cc.3.12.1346
Abstract | PDF

Mammalian embryonic stem (ES) cells can either self-renew or generate progenitor cells that have a more restricted developmental potential. This provides an important model system to ask how pluripotency, cell commitment and differentiation are regulated at the level of chromatin-based changes that distinguish stem cells from their differentiated progeny. Here we show that the differentiation of ES cells to neural progenitors results in dynamic changes in the epigenetic status of multiple genes that encode transcription factors critical for early embryonic development or lineage specification (22 of 43). In particular, we demonstrate that DNA replication at a subset of neural-associated genes including Pax3, Pax6, Irx3, Nkx2.9, and Mash1 is advanced upon neural induction, consistent with increased locus accessibility. Conversely, many ES-associated genes including Oct4, Nanog, Utf1, Foxd3, Cripto and Rex1 that replicate early in ES cells switch their replication timing to later in S-phase in response to differentiation. Detailed analysis of the Rex1 locus reveals that delayed replication extends to a 2.8Mb region surrounding the gene and is associated with substantial reductions in the level of histone H3K9 and H4 acetylation at the promoter. These results show that loss of pluripotency (and lineage choice) is associated with extensive and predictable changes in the replication timing of key regulator genes.

Open Access Article

Comparative Genomics, Evolution and Origins of the Nuclear Envelope and Nuclear Pore Complex

Berend Mans, Vivek Anantharaman, L. Aravind and Eugene V. Koonin
Pages 1625 - 1650
http://dx.doi.org/10.4161/cc.3.12.1316
Abstract | PDF

The presence of a distinct nucleus, the compartment for confining the genome, transcription and RNA maturation, is a central (and eponymous) feature that distinguishes eukaryotes from prokaryotes. Structural integrity of the nucleus is maintained by the nuclear envelope (NE). A crucial element of this structure is the nuclear pore complex (NPC), a macromolecular machine with over 90 protein components, which mediates nucleo-cytoplasmic communication. Given the indispensability of these structures for nuclear function, the natural history of the nucleus can only be understood in terms of the origin and subsequent evolution of NE and NPC components. We investigated the provenance of the conserved domains found in these perinuclear proteins and reconstructed a parsimonious scenario for NE and NPC evolution by means of comparative-genomic analysis of their components from the available sequences of 28 sequenced eukaryotic genomes. We show that the NE and NPC proteins were tinkered together from diverse domains, which evolved from prokaryotic precursors at different points in eukaryotic evolution, divergence from pre-existing eukaryotic paralogs performing other functions, and de novo. It is shown that several central components of the NPC, in particular, the RanGDP import factor NTF2, the HEH domain of Src1p-Man1, and, probably, also the key domains of karyopherins and nucleoporins, the HEAT/ARM and WD40 repeats, have a bacterial, most likely, endosymbiotic origin. The specialized immunoglobulin (Ig) domain in the globular tail of the animal lamins, and the Ig domains in the nuclear membrane protein GP210 are shown to be related to distinct prokaryotic families of Ig domains. This suggests that independent, late horizontal gene transfer events from bacterial sources might have contributed to the evolution of perinuclear proteins in some of the major eukaryotic lineages. Snurportin 1, one of the highly conserved karyopherins, contains a cap-binding domain which is shown to be an inactive paralog of the guanylyl transferase domain of the mRNA-capping enzyme, exemplifying recruitment of paralogs of pre-exsiting proteins for perinuclear functions. We infer an autogenous scenario of nuclear evolution in which the nucleus emerged in the primitive eukaryotic ancestor (the “prekaryote”) as part of cell compartmentalization triggered by archaeo-bacterial symbiosis. A pivotal event in this process was the radiation of Ras-superfamily GTPases yielding Ran, the key regulator of nuclear transport. A primitive NPC with approximately 20 proteins and a Src1p-Man1-like membrane protein with a DNA-tethering HEH domain are inferred to have been integral perinuclear components in the las common ancestor of modern eukaryotes.