bluuebunny/crossref_metadata_2025 huggingface dataset:
N- and 6-O-Sulfated Heparan Sulfates Mediate Internalization of Coxsackievirus B3 Variant PD into CHO-K1 Cells
Authors: Zautner Andreas E. , Jahn Birgit , Hammerschmidt Elke , Wutzler Peter , Schmidtke Michaela
Published: June 2006

Recently, it was demonstrated that the coxsackievirus B3 variant PD (CVB3 PD) is able to infect coxsackievirus-adenovirus receptor (CAR)-lacking cells by using heparan sulfates (HS) as additional receptors (A. E. Zautner, U. Korner, A. Henke, C. Badorff, and M. Schmidtke, J. Virol.
77:
10071-10077, 2003). For this study, competition experiments with growth factors binding to known HS sequences as well as with specifically desulfated heparins were performed with Chinese hamster ovary cells (CHO-K1) to determine the structural requirements of HS for interaction with CVB3. Hepatocyte growth factor interacting with HS sequences containing [IdUA-GlcNSO
3
(6OSO
3
)]

n

, but not basic fibroblast growth factor binding to [HexUA-GlcNSO
3
-HexUA-GlcNSO
3
-IdUA(2OSO
3
)]

n

, was shown to compete effectively with CVB3 PD for cell surface HS. Whereas unmodified heparin and 2-O-desulfated heparin strongly inhibited the CVB3 PD-induced cytopathic effect, the antiviral activity was markedly reduced after N-, O- and 6-O-desulfation of heparin. Taken together, these results indicate that 6-O- and N-sulfation of GlcNAc of HS is crucial for HS interaction with CVB3 PD and that the disaccharide [IdUA-GlcNSO
3
(6OSO
3
)]

n

is involved in viral binding. Results from experiments with various inhibitors of endocytic pathways suggest that HS-mediated virus internalization is pH dependent. Despite the fact that CVB3 PD initiates infection about four times slower by making use of HS as a receptor than by using CAR, the time required for a complete viral life cycle in Chinese hamster ovary cells was independent of the utilized receptor.
Genotoxic Stress and Cellular Stress Alter the Subcellular Distribution of Human T-Cell Leukemia Virus Type 1 Tax through a CRM1-Dependent Mechanism
Authors: Gatza Michael L. , Marriott Susan J.
Published: June 2006

Human T-cell leukemia virus type 1 Tax is a predominantly nuclear viral oncoprotein that colocalizes with cellular proteins in nuclear foci known as Tax speckled structures (TSS). Tax is also diffusely distributed throughout the cytoplasm, where it interacts with and affects the functions of cytoplasmic cellular proteins. Mechanisms that regulate the distribution of Tax between the cytoplasm and nucleus remain to be identified. Since Tax has been shown to promote genome instability by perturbing cell cycle progression and DNA repair mechanisms following DNA damage, we examined the effect of genotoxic stress on the subcellular distribution and interacting partners of Tax. Tax localization was altered in response to various forms of cellular stress, resulting in an increase in cytoplasmic Tax and a decrease in Tax speckled structures. Concomitantly, colocalization of Tax with sc35 (a TSS protein) decreased following stress. Tax translocation required the CRM1 nuclear export pathway, and a transient interaction between Tax and CRM1 was observed following stress. These results suggest that the subcellular distribution of Tax and the interactions between Tax and cellular proteins respond dynamically to cellular stress. Changes in Tax distribution and interacting partners are likely to affect cellular processes that regulate cellular transformation.
Demonstration of Nicking/Joining Activity at the Origin of DNA Replication Associated with the Rep and Rep′ Proteins of Porcine Circovirus Type 1
Authors: Steinfeldt Tobias , Finsterbusch Tim , Mankertz Annette
Published: June 2006

The replication of porcine circovirus type 1 (PCV1) is thought to occur by rolling-circle replication (RCR), whereby the introduction of a single-strand break generates a free 3′-hydroxyl group serving as a primer for subsequent DNA synthesis. The covalently closed, single-stranded genome of PCV1 replicates via a double-stranded replicative intermediate, and the two virus-encoded replication-associated proteins Rep and Rep′ have been demonstrated to be necessary for virus replication. However, although postulated to be involved in RCR-based virus replication, the mechanism of action of Rep and Rep′ is as yet unknown. In this study, the ability of PCV1 Rep and Rep′ to “nick” and “join” strand discontinuities within synthetic oligonucleotides corresponding to the origin of replication of PCV1 was investigated in vitro. Both proteins were demonstrated to be able to cleave the viral strand between nucleotides 7 and 8 within the conserved nonanucleotide motif (5′-TAGTATTAC-3′) located at the apex of a putative stem-loop structure. In addition, the Rep and Rep′ proteins of PCV1 were demonstrated to be capable of joining viral single-stranded DNA fragments, suggesting that these proteins also play roles in the termination of virus DNA replication. This joining activity was demonstrated to be strictly dependent on preceding substrate cleavage and the close proximity of origin fragments accomplished by base pairing in the stem-loop structure. The dual “nicking/joining” activities associated with PCV1 Rep and Rep′ are pivotal events underlying the RCR-based replication of porcine circoviruses in mammalian cells.
Recombinant Extracellular Domains of Tetraspanin Proteins Are Potent Inhibitors of the Infection of Macrophages by Human Immunodeficiency Virus Type 1
Authors: Ho Siu-Hong , Martin Francine , Higginbottom Adrian , Partridge Lynda J. , Parthasarathy Varadarajan , Moseley Gregory W. , Lopez Peter , Cheng-Mayer Cecilia , Monk Peter N.
Published: June 2006

Human immunodeficiency virus type 1 (HIV-1) infection of human macrophages can be inhibited by antibodies which bind to the tetraspanin protein CD63, but not by antibodies that bind to other members of the tetraspanin family. This inhibitory response was limited to CCR5 (R5)-tropic virus and was only observed using macrophages, but not T cells. Here, we show that recombinant soluble forms of the large extracellular domain (EC2) of human tetraspanins CD9, CD63, CD81, and CD151 produced as fusion proteins with glutathioneS-transferase (GST) can all potently and completely inhibit R5 HIV-1 infection of macrophages with 50% inhibitory concentration values of 0.11 to 1.2 nM. Infection of peripheral blood mononuclear cells could also be partly inhibited, although higher concentrations of EC2 proteins were required. Inhibition was largely coreceptor independent, as macrophage infections by virions pseudotyped with CXCR4 (X4)-tropic HIV-1 or vesicular stomatitis virus (VSV)-G glycoproteins were also inhibited, but was time dependent, since addition prior to or during, but not after, virus inoculation resulted in potent inhibition. Incubation with tetraspanins did not decrease CD4 or HIV-1 coreceptor expression but did block virion uptake. Colocalization of fluorescently labeled tetraspanin EC2 proteins and HIV-1 virions within, and with CD4 and CXCR4 at the cell surfaces of, macrophages could be detected, and internalized tetraspanin EC2 proteins were directed to vesicular compartments that contained internalized dextran and transferrin. Collectively, the data suggest that the mechanism of inhibition of HIV-1 infection by tetraspanins is at the step of virus entry, perhaps via interference with binding and/or the formation of CD4-coreceptor complexes within microdomains that are required for membrane fusion events.
Herpes Simplex Virus Capsid Structure: DNA Packaging Protein UL25 Is Located on the External Surface of the Capsid near the Vertices
Authors: Newcomb William W. , Homa Fred L. , Brown Jay C.
Published: June 2006

UL25 is one of seven herpes simplex virus-encoded proteins involved specifically in DNA encapsidation. Its role appears to be to stabilize the capsid so that DNA is prevented from escaping once it has entered. To clarify the function of UL25, we have examined capsids with the goal of defining where it is located. Analysis of trypsin-treated capsids showed that UL25 is sensitive to cleavage like other proteins such as the major capsid and portal proteins that are exposed on the capsid surface. Internal proteins such as the scaffolding protein and protease were not affected under the same experimental conditions. Capsids were also examined by electron microscopy after staining with gold-labeled antibody specific for UL25. Images of stained capsids demonstrated that most labeled sites (71% in C capsids) were at capsid vertices, and most stained C capsids had label at more than one vertex. A quantitative immunoblotting method showed that the capsid contents of UL25 were 56, 20, and 75 copies per capsid in A, B, and C capsids, respectively. Finally, soluble UL25 protein was found to bind in vitro to purified capsids lacking it. The amount of bound UL25 corresponded to the amount present in B capsids, and bound UL25 was found by immunoelectron microscopy to be located predominantly at the capsid vertices. The results are interpreted to suggest that five UL25 molecules are found at or near each of the capsid vertices, where they are exposed on the capsid surface. Exposure on the surface is consistent with the view that UL25 is added to the capsid as DNA is packaged or during late stages of the packaging process.
The Capsid-Associated UL25 Protein of the Alphaherpesvirus Pseudorabies Virus Is Nonessential for Cleavage and Encapsidation of Genomic DNA but Is Required for Nuclear Egress of Capsids
Authors: Klupp Barbara G. , Granzow Harald , Keil Günther M. , Mettenleiter Thomas C.
Published: June 2006

Homologs of the UL25 gene product of herpes simplex virus (HSV) have been identified in all three subfamilies of the
Herpesviridae
. However, their exact function during viral replication is not yet known. Whereas earlier studies indicated that the UL25 protein of HSV-1 is not required for cleavage of newly replicated viral DNA but is necessary for stable encapsidation (A. R. McNab, P. Desai, S. Person, L. Roof, D. R. Thompson, W. W. Newcomb, J. C. Brown, and F. L. Homa, J. Virol. 72:1060-1070, 1998), viral DNA packaging has recently been demonstrated to occur in the absence of UL25, although at significantly decreased levels compared to wild-type HSV-1 (N. Stow, J. Virol. 75:10755-10765 2001). To clarify the functional role of UL25 we analyzed the homologous protein of the alphaherpesvirus pseudorabies virus (PrV). PrV UL25 was found to be essential for viral replication, as a mutant virus lacking the UL25 protein required UL25-expressing cells for productive propagation. In the absence of the UL25 protein, newly replicated PrV DNA was cleaved and DNA-containing C-type capsids were detected in infected cell nuclei. However, although capsids were frequently found in close association with the inner nuclear membrane, nuclear egress was not observed. Consequently, no capsids were found in the cytoplasm, resulting in an inhibition of virion morphogenesis. In contrast, the formation of capsidless enveloped tegument structures (L particles) in the cytoplasm was readily observed. Thus, our data demonstrate that the PrV UL25 protein is not essential for cleavage and encapsidation of viral genomes, although both processes occur more efficiently in the presence of the protein. However, the presence of the PrV UL25 protein is a prerequisite for nuclear egress. By immunoelectron microscopy, we detected UL25-specific label on DNA-containing C capsids but not on other intranuclear immature or defective capsid forms. Thus, the PrV UL25 protein may represent the hitherto missing trigger that allows primary envelopment preferably of DNA-filled C capsids.
Hepatic expression of galectin-3 and receptor for advanced glycation end products in patients with liver disease
Authors: Butscheid M , Hauptvogel P , Fritz P , Klotz U , Alscher D M
Published: June 2006

Background:Advanced glycation end products (AGEs) are a heterogeneous group of glycosylated proteins (of which carboxymethyl-lysine (CML) is the most common) which accumulate during ageing processes and play an important role in the pathogenesis of a variety of chronic diseases. Impaired hepatic function might result in elevated levels of AGEs, as the liver represents the major site of AGE metabolism. The actions of AGEs are mediated by various receptors, among which the AGE-receptor complex (including galectin-3 as an essential part) is thought to have a cytoprotective effect, and receptor for advanced glycation end product (RAGE) a cytotoxic effect.

Aim:To assess the relationship between CML and expression of galectin-3 and RAGE in different histological structures in biopsy specimens from patients with varying degrees of liver impairment.

Method:Immunohistochemical staining of 164 biopsies from patients with varying degrees of liver impairment was performed to determine the levels of CML, galectin-3 and RAGE in hepatocytes, Kupffer cells and bile ducts by a semiquantative score.

Results:Independent of diagnosis, CML and RAGEs were detected in hepatocytes, whereas galectin-3 was present only in hepatocytes of cirrhotics. By contrast, CML and galectin-3 were highly expressed in Kupffer cells (well correlating levels, highest scores in cholestasis) whereas expression of RAGEs was not significant. All three assessed biochemical markers showed their highest levels of expression/detection in bile ducts.

Conclusion:These findings indicate an increased susceptibility of hepatocytes to the detrimental effects of AGEs and underline the protective function of Kupffer cells. Furthermore, the biliary system seems to play an important role in the disposition of AGEs.
Nasal natural killer/T-cell lymphoma and its association with type “i”/XhoI loss strain Epstein–Barr virus in Chile
Authors: Cabrera M E , Eizuru Y , Itoh T , Koriyama C , Tashiro Y , Ding S , Rey S , Akiba S , Corvalan A
Published: June 2006

Background:Nasal T/natural killer (NK)-cell lymphoma is an aggressive type of non-Hodking’s lymphoma associated with Epstein–Barr virus (EBV) and striking geographical variations worldwide.

Aim:To characterise nasal NK/T-cell lymphoma associated with genotypes of EBV in Chile, a Latin American country, where multiple strains of EBV, including two new recombinant strains, in healthy individuals were recently found.

Methods:Cases with diagnosis of primary nasal lymphoma were selected for histological and immunohistochemical analysis (CD3, CD3e, CD4, CD8, CD79a, CD56, CD57 and TIA-1) and in-situ hybridisation, serology and genotyping analysis for EBV.

Results:Out of 22 cases, 9 (41%) cases fulfilled the World Health Organization criteria for nasal NK/T-cell lymphoma; of these 7 (78%) cases were positive for EBV. Genotyping analysis revealed 6 cases of type 1 EBV and wildtype F at theBamHI-F region, 4 cases type “i” EBV at theBamHI-W1/I1 region;XhoI wild type was found in 2 andXhoI loss in 4 cases, respectively. Cosegregation analysis of theBamHI-W1/I1 region andXhoI restriction site showed the new recombinant strain type “i”/XhoI loss in 3 cases and type “i”/XhoI wild-type strain in 1 case. Most patients were treated with combined anthracycline-containing regimens. Half of the cases attained complete remission.

Conclusion:Although nasal NK/T-cell lymphomas from Chile share similar clinicopathological features, high association with EBV and unfavourable prognosis with those described elsewhere, genotype analysis shows that the new recombinant type “i”/XhoI loss strain might contribute to explain the intermediate incidence of nasal NK/T-cell lymphomas in Latin America.
Assessment of erythrocyte shape by flow cytometry techniques
Authors: Piagnerelli M , Zouaoui Boudjeltia K , Brohee D , Vereerstraeten A , Piro P , Vincent J-L , Vanhaeverbeek M
Published: June 2006

Background:Red blood cell (RBC) rheology is altered in different diseases, including acute conditions such as patients in intensive care units (ICU) with sepsis or with an inflammatory reaction due to postoperative states or intracerebral haemorrhage, or chronic conditions such as diabetes mellitus or terminal renal failure. Several techniques are available to assess alterations in RBC rheology, especially deformability, but they are too cumbersome to be used on a large number of cells.

Objective:To develop a new, rapid flow cytometry technique for easy assessment of RBC shape in patients.

Methods:In flow cytometry, healthy human RBC shape shows a bimodal distribution related to the biconcave form. On this histogram, the second Pearson coefficient of dissymmetry (PCD) representing the asymmetry of this histogram and the spherical index (M2:M1) were calculated, both representing the spherical shape. This technique was used in healthy volunteers (n = 17) and in diseases characterised by abnormalities in RBC rheology, including terminal renal failure requiring haemodialysis (n = 28), diabetes mellitus (n = 18), sepsis (n = 19) and acute inflammatory states (postoperative, intracerebral haemorrhage, chronic obstructive pulmonary disease, epilepsy or severe drug intoxication; n = 21). Multivariate analysis was performed to determine the factors influencing RBC shape.

Results:Measurement of RBC shape was highly reproducible. A good correlation was observed between the PCD and the spherical index, except in the critically ill patients without sepsis. RBCs were more spherical in patients with terminal renal failure (PCD −0.56 (0.14), p<0.05), diabetes mellitus (PCD −0.59 (0.23), p<0.05), sepsis (PCD −0.58 (0.22), p<0.05) or an acute inflammatory state (PCD −0.65 (0.29), p<0.05) than in healthy volunteers (PCD −0.89 (0.12)). The spherical index was also increased in all populations compared with healthy volunteers (terminal renal failure 2.30 (0.20); diabetes mellitus 2.27 (0.38); sepsis 2.28 (0.37); acute inflammatory state 2.35 (0.42) vs healthy volunteers 2.72 (0.47); all p<0.05). Multivariate analysis demonstrated that the underlying pathology (sepsis, acute inflammatory state, diabetes mellitus, terminal renal failure) was the principal cause of these RBC shape abnormalities.

Conclusion:RBCs are characterised by an increased spherical shape in many disease states. The measure of the second PCD in flow cytometry is a new, easy method to investigate RBC shape in various diseases. This technique could facilitate the investigation of abnormalities of RBC rheology.
Endocytosis, Endosome Trafficking, and the Regulation ofDrosophilaDevelopment
Authors: Fischer Janice A. , Eun Suk Ho , Doolan Benjamin T.
Published: June 2006

Endocytosis and endosome trafficking regulate cell signaling in unexpected ways. Here we review the contribution that Drosophila research has made to this exciting field. In addition to attenuating signaling, endocytosis shapes morphogen gradients, activates ligands, and regulates spatially receptor activation within a single cell. Moreover, some receptors signal from within endosomes, and the ability of a specific type of endosome to form controls the ability of cells to signal. Experiments in Drosophila reveal that through regulation of a variety of cell signaling pathways, endocytosis controls cell patterning and cell fate.
Intracellular angiotensin II induces cell proliferation independent of AT1 receptor
Authors: Baker Kenneth M. , Kumar Rajesh
Published: June 2006

We recently reported intracrine effects of angiotensin II (ANG II) on cardiac myocyte growth and hypertrophy that were not inhibited by the ANG II type 1 receptor (AT1) antagonist, losartan. To further determine the role of AT1 in intracrine effects, we studied the effect of intracellular ANG II (iANG II) on cell proliferation in native Chinese hamster ovary (CHO) cells and those stably transfected with AT1 receptor (CHO-AT1). CHO-AT1, but not CHO cells, showed enhanced proliferation following exposure to extracellular ANG II (eANG II). However, when transiently transfected with an iANG II expression vector, both cell types showed significantly enhanced proliferation, compared with those transfected with a scrambled peptide. Losartan blocked eANG II-induced cell proliferation, but not that induced by iANG II. To further confirm these findings, CHO and CHO-AT1 cells were stably transfected for iANG II expression (CHO-iA and CHO-AT1-iA, respectively). Cells grown in serum-free medium were counted every 24 h, up to 72 h. CHO-iA and CHO-AT1-iA cells showed a steeper growth curve compared with CHO and CHO-AT1, respectively. These observations were confirmed by Wst-1 assay. The AT1 receptor antagonists losartan, valsartan, telmisartan, and candesartan did not attenuate the faster growth rate of CHO-iA and CHO-AT1-iA cells. eANG II showed an additional growth effect in CHO-AT1-iA cells, which could be selectively blocked by losartan. These data demonstrate that intracrine ANG II can act independent of AT1 receptors and suggest novel intracellular mechanisms of action for ANG II.
HMG-CoA reductase inhibitor simvastatin mitigates VEGF-induced “inside-out” signaling to extracellular matrix by preventing RhoA activation
Authors: Xu Hanshi , Zeng Lixia , Peng Hui , Chen Sheldon , Jones Jonathan , Chew Teng-Leong , Sadeghi Mehran M. , Kanwar Yashpal S. , Danesh Farhad R.
Published: June 2006

The 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors exert modulatory effects on a number of cell signaling cascades by preventing the synthesis of various isoprenoids derived from the mevalonate pathway. In the present study, we describe a novel pleiotropic effect of HMG-CoA reductase inhibitors, also commonly known as statins, on vascular endothelial growth factor (VEGF)-induced type IV collagen accumulation. VEGF is an angiogenic polypeptide that is also known to play a central role in endothelial cell permeability and differentiation. Recently, VEGF has also been implicated in promoting extracellular matrix (ECM) accumulation, although the precise signaling mechanism that mediates VEGF-induced ECM expansion remains poorly characterized. Elucidation of the mechanisms through which VEGF exerts its effect on ECM is clearly a prerequisite for both understanding the complex biology of this molecule as well as targeting VEGF in several pathological processes. To this end, this study explored the underlying molecular mechanisms mediating VEGF-induced ECM expansion in mesangial cells. Our findings show that VEGF stimulation elicits a robust increase in ECM accumulation that involves RhoA activation, an intact actin cytoskeleton, and β1- integrin activation. Our data also indicate that simvastatin, via mevalonate depletion, reverses VEGF-induced ECM accumulation by preventing RhoA activation.
BK channels in the kidney: role in K+secretion and localization of molecular components
Authors: Pluznick Jennifer L. , Sansom Steven C.
Published: June 2006

Although it is generally accepted that ROMK is the K+secretory channel in the mammalian distal nephron, recent in vitro and in vivo studies have provided evidence that large-conductance Ca2+-activated K+channels (BK, or maxi K) also secrete K+in renal tubules. This review assesses the current evidence relating BK channels with K+secretion. We shall consider the component proteins of the BK channel, their localization with respect to segment and cell type, and the electrophysiological forces involved in K+secretion. Although the majority of studies have focused on a role for BK channels in flow-mediated K+secretion, this review also considers a potential role for BK channels in high-K diet-induced K+secretion. The division of workload between ROMK and BK is discussed as a mechanism for ensuring a constant plasma K+concentration.
Development of Gap Junctions in Hippocampal Astrocytes: Evidence That Whole Cell Electrophysiological Phenotype Is an Intrinsic Property of the Individual Cell
Authors: Schools Gary P. , Zhou Min , Kimelberg Harold K.
Published: June 2006

Gap junction communication between astrocytes is prevalent and has been proposed to be involved in several astrocyte functions. One such proposal involves gap junctions in potassium spatial buffering. However, little is known about the developmental time course of gap junction coupling and how much the syncytium affects whole cell measurements of ion currents. Our previous work described three types of hippocampal astrocyte, each with a distinct electrophysiological profile when recorded in whole cell voltage-clamp mode. In the current study we correlated post–whole cell recording immunohistochemistry for GLAST and the spread of injected dye from the recorded cell with the measured electrophysiological phenotype to quantify cell coupling of astrocytes and the type of astrocyte coupled, in the rat hippocampus. We found that passive astrocytes, which predominate after 3 wk postnatally, have much lower membrane resistances ( Rm) and are more frequently dye coupled and to more cells, than outwardly and variably rectifying astrocytes that predominate in early postnatal development. Dye coupling in GLAST(+) cells was first detected in the first postnatal week and the degree of coupling peaked before the complete transition to the low Rm, passive electrophysiological type. Also, the degree of dye coupling did not correlate with the passive electrophysiological phenotype. Passive cells were also detected after pretreatment with a gap junction inhibitor. Further evidence that cell coupling does not contribute to the mature astrocyte electrophysiological phenotype came from recording of excised membrane patches, which predominantly corresponded to the ion channel expression profiles of their cells of origin. These findings imply that in the hippocampus, interastrocyte cell coupling likely contributes little to the overall whole cell current profile of diverse glia, and the electrophysiological passivity reflects the intrinsic ion channel expression of the mature astrocyte.
Ankyrin-G Regulates Inactivation Gating of the Neuronal Sodium Channel, Nav1.6
Authors: Shirahata Emi , Iwasaki Hirohide , Takagi Masahiro , Lin Changqing , Bennett Vann , Okamura Yasushi , Hayasaka Kiyoshi
Published: June 2006

Ankyrin-G, a modular protein, plays a critical role in clustering voltage-gated sodium channels (Nav channels) in nodes of Ranvier and initial segments of mammalian neurons. However, direct effects of ankyrin-G on electrophysiological properties of Nav channels remain elusive. In this study, we explored whether ankyrin-G has a role in modifying gating properties of the neuronal Nav1.6 channel that is predominantly localized at nodes of Ranvier and initial segments. TsA201 cells transfected with the human Nav1.6 cDNA alone exhibited significant persistent sodium current (Ina-p). On the other hand, Ina-p was barely detected on co-expression with ankyrin-G. Ankyrin-B, another ankyrin, did not show such an effect. Expression of chimeras between the two isoforms of ankyrin suggests that the membrane-binding domain of ankyrin-G is critical for reducing the Ina-p of Nav1.6. These results suggest that ankyrin-G regulates neuronal excitability not only through clustering Nav channels but also by directly modifying their channel gating.
Transduction of Tumor Necrosis Factor–Related Apoptosis-Inducing Ligand into Hematopoietic Cells Leads to Inhibition of Syngeneic Tumor GrowthIn vivo
Authors: Song Keli , Benhaga Nordine , Anderson Robin L. , Khosravi-Far Roya
Published: June 2006

Tumor necrosis factor (TNF)–related apoptosis-inducing ligand (TRAIL) is a member of the TNF family of cytokines and has been shown to induce cell death in many types of tumor and transformed cells but not in normal cells. This tumor-selective property has made TRAIL a promising candidate for the development of cancer therapy. However, safety issues are a concern because certain preparations of recombinant TRAIL protein were reported to induce toxicity in normal human hepatocytes in culture. In addition, previous studies on tumor selectivity of exogenous TRAIL protein were carried out in xenograft models, which do not directly address the tumor selectivity issue. It was not known whether exogenous or overexpression of TRAIL in a syngeneic system could induce tumor cell death while leaving normal tissue cells unharmed. Thus, the tumor selectivity of TRAIL-induced apoptosis remains to be further characterized. In our study, we established mice that overexpress TRAIL by retroviral-mediated gene transfer in bone marrow cells followed by bone marrow transplantation. Our results show that TRAIL overexpression is not toxic to normal tissues, as analyzed by hematologic and histologic analyses of tissue samples from TRAIL-transduced mice. We show for the first time that TRAIL overexpression in hematopoietic cells leads to significant inhibition of syngeneic tumor growth in certain tumor lines. This approach may be used further to identify important molecules that regulate the sensitivity of tumor cells to TRAIL-induced cell death in vivo. (Cancer Res 2006; 66(12): 6304-11)
Different Progression of Tumor Xenografts between Mucin-Producing and Mucin–Non-Producing Mammary Adenocarcinoma-Bearing Mice
Authors: Sugihara Ippei , Yoshida Masanobu , Shigenobu Tatsuro , Takagi Hiroaki , Maruyama Koji , Takeuchi Noriko , Toda Munetoyo , Inoue Mizue , Nakada Hiroshi
Published: June 2006

Previously, we found that MUC2 mucins could activate monocytes/macrophages through a scavenger receptor leading to cyclooxygenase (COX) 2 induction and overproduction of prostaglandin E2 (PGE2). To investigate the role of mucins in the tumor-bearing state, we compared s.c. tumor formation by using mucin-producing (TA3-Ha) and mucin–non-producing (TA3-St) cloned variants of mouse mammary adenocarcinomas. Expression of COX2 mRNA and protein and production of PGE2 were elevated in peritoneal macrophages stimulated with epiglycanin, which is a mucin-like glycoprotein produced by TA3-Ha cells. S.c. tumor tissues comprising TA3-Ha cells grew much faster than tissues comprising TA3-St cells. COX2 protein and vascular endothelial growth factor in TA3-Ha tumor tissues were elevated compared with the TA3-St tumor tissues. Although similar numbers of macrophages were observed immunochemically in the two types of tumor tissues, COX2 was induced prominently in the infiltrating macrophages in TA3-Ha tumor tissues but only faintly in TA3-St tumor tissues. Furthermore, angiogenesis progressed remarkably in TA3-Ha tumor tissues but only slightly in TA3-St tumor tissues. Epiglycanin-induced overproduction of PGE2 down-regulated interleukin-12 production by macrophages. IFN-γ-producing CD4 T cells in spleens obtained from TA3-Ha tumor-bearing mice were significantly reduced compared with TA3-St tumor-bearing mice, suggesting that mucins cause PGE2-mediated immune suppression. Actually, the tumor growth of a TA3-Ha cell xenograft was suppressed effectively by oral administration of a COX2 inhibitor but that of a TA3-St cell one was not. These results suggest that mucins play an important role in tumor progression through overproduction of PGE2. (Cancer Res 2006; 66(12): 6175-82)
The Small α5β1 Integrin Antagonist, SJ749, Reduces Proliferation and Clonogenicity of Human Astrocytoma Cells
Authors: Maglott Anne , Bartik Petr , Cosgun Sedat , Klotz Philippe , Rondé Philippe , Fuhrmann Guy , Takeda Kenneth , Martin Sophie , Dontenwill Monique
Published: June 2006

The potential role of α5β1 integrins in cancer has recently attracted much interest. However, few α5β1-selective antagonists have been developed compared with other integrins. The most specific nonpeptidic α5β1 antagonist described thus far, SJ749, inhibits angiogenesis by affecting adhesion and migration of endothelial cells. We investigated the effects of SJ749 in two human astrocytoma cell lines, A172 and U87, which express different levels of α5β1. SJ749 dose-dependently inhibited adhesion of both cell types on fibronectin. Application of SJ749 to spread cells led to formation of nonadherent spheroids for A172 cells but had no effect on U87 cell morphology. SJ749 also reduced proliferation of A172 cells due to a long lasting G0-G1 arrest, whereas U87 cells were only slightly affected. However, under nonadherent culture conditions (soft agar), SJ749 significantly reduced the number of colonies formed only by U87 cells. As U87 cells express more α5β1 than A172 cells, we specifically examined the effect of SJ749 on A172 cells overexpressing α5. Treatment of α5-A172 cells with SJ749 decreased colony formation similarly to that observed in U87 cells. Therefore, in nonadherent conditions, the effect of SJ749 on tumor cell growth characteristics depends on the level of α5β1 expression. Our study highlights the importance of α5β1 as an anticancer target and shows for the first time that a small nonpeptidic α5β1-specific antagonist affects proliferation of tumor cells. (Cancer Res 2006; 66(12): 6002-7)
Nitric Oxide Regulates Cell Sensitivity to Cisplatin-Induced Apoptosis through S-Nitrosylation and Inhibition of Bcl-2 Ubiquitination
Authors: Chanvorachote Pithi , Nimmannit Ubonthip , Stehlik Christian , Wang Liying , Jiang Bing-Hua , Ongpipatanakul Boonsri , Rojanasakul Yon
Published: June 2006

Cisplatin is a potent cytotoxic agent commonly used for the treatment of solid tumors. However, tumor cell–acquired resistance to cisplatin-induced apoptosis is a major limitation for efficient therapy, as frequently observed in human lung cancer. Nitric oxide (NO) is a key regulator of apoptosis, but its role in cisplatin-induced cell death and the underlying mechanism are largely unknown. Previous studies indicate increased NO synthase activity and elevated NO production in lung carcinomas, which correlate with the incidence of chemotherapeutic resistance. Here, we show that NO impairs the apoptotic function of cells and increases their resistance to cisplatin-induced cell death in human lung carcinoma H-460 cells. The NO donors sodium nitroprusside and dipropylenetriamine NONOate were able to inhibit cisplatin-induced cell death, whereas the NO inhibitors aminoguanidine and 2-(4-carboxyphenyl)-4,4,5,5-tetra-methylimidazoline-1-oxyl-3-oxide had opposite effect. Cisplatin resistance in H-460 cells is mediated by Bcl-2, and NO up-regulates its expression by preventing the degradation of Bcl-2 via ubiquitin-proteasome pathway. Cisplatin-induced generation of reactive oxygen species causes dephosphorylation and degradation of Bcl-2. In contrast, generation of NO has no effect on Bcl-2 phosphorylation but induces S-nitrosylation of the protein, which inhibits its ubiquitination and subsequent proteasomal degradation. These findings indicate a novel pathway for NO regulation of Bcl-2, which provides a key mechanism for cisplatin resistance and its potential modulation for improved cancer chemotherapy. (Cancer Res 2006; 66(12): 6365-60)
Apoptosis Induction by Retinoids in Eosinophilic Leukemia Cells: Implication of Retinoic Acid Receptor-α Signaling in All-Trans-Retinoic Acid Hypersensitivity
Authors: Robert Carine , Delva Laurent , Balitrand Nicole , Nahajevszky Sarolta , Masszi Tamàs , Chomienne Christine , Papp Béla
Published: June 2006

Hypereosinophilic syndrome (HES) has recently been recognized as a clonal leukemic lesion, which is due to a specific oncogenic event that generates hyperactive platelet-derived growth factor receptor-α–derived tyrosine kinase fusion proteins. In the present work, the effect of retinoids on the leukemic hypereosinophilia-derived EoL-1 cell line and on primary HES-derived cells has been investigated. We show that all-trans-retinoic acid (ATRA) inhibits eosinophil colony formation of HES-derived bone marrow cells and is a powerful inducer of apoptosis of the EoL-1 cell line. Apoptosis was shown in the nanomolar concentration range by phosphatidylserine externalization, proapoptotic shift of the Bcl-2/Bak ratio, drop in mitochondrial membrane potential, activation of caspases, and cellular morphology. Unlike in other ATRA-sensitive myeloid leukemia models, apoptosis was rapid and was not preceded by terminal cell differentiation. Use of isoform-selective synthetic retinoids indicated that retinoic acid receptor-α–dependent signaling is sufficient to induce apoptosis of EoL-1 cells. Our work shows that the scope of ATRA-induced apoptosis of malignancies may be wider within the myeloid lineage than thought previously, that the EoL-1 cell line constitutes a new and unique model for the study of ATRA-induced cell death, and that ATRA may have potential for the management of clonal HES. (Cancer Res 2006; 66(12): 6336-44)
Combined DNA Methyltransferase and Histone Deacetylase Inhibition in the Treatment of Myeloid Neoplasms
Authors: Gore Steven D. , Baylin Stephen , Sugar Elizabeth , Carraway Hetty , Miller Carole B. , Carducci Michael , Grever Michael , Galm Oliver , Dauses Tianna , Karp Judith E. , Rudek Michelle A. , Zhao Ming , Smith B. Douglas , Manning Jasper , Jiemjit Anchalee , Dover George , Mays Abbie , Zwiebel James , Murgo Anthony , Weng Li-Jun , Herman James G.
Published: June 2006

Optimal reexpression of most genes silenced through promoter methylation requires the sequential application of DNA methyltransferase inhibitors followed by histone deacetylase inhibitors in tumor cell cultures. Patients with myelodysplastic syndrome or acute myeloid leukemia (AML) were treated with the methyltransferase inhibitor 5-azacitidine (aza-CR) followed by the histone deacetylase inhibitor sodium phenylbutyrate. Major responses associated with cytogenetic complete response developed in patients receiving prolonged dosing schedules of aza-CR. Bisulfite sequencing of the p15 promoter in marrow DNA during the first cycle of treatment showed heterogeneous allelic demethylation in three responding patients, suggesting ongoing demethylation within the tumor clone, but no demethylation in two nonresponders. Six of six responding patients with pretreatment methylation of p15 or CDH-1 promoters reversed methylation during the first cycle of therapy (methylation-specific PCR), whereas none of six nonresponders showed any demethylation. Gene demethylation correlated with the area under the aza-CR plasma concentration-time curve. Administration of both drugs was associated with induction of acetylation of histones H3 and H4. This study provides the first demonstration that molecular mechanisms responsible for responses to DNA methyltransferase/histone deacetylase inhibitor combinations may include reversal of aberrant epigenetic gene silencing. The promising percentage of major hematologic responses justifies the testing of such combinations in prospective randomized trials. (Cancer Res 2006; 66(12): 6361-9)
Silencing of Peroxiredoxin 2 and Aberrant Methylation of 33 CpG Islands in Putative Promoter Regions in Human Malignant Melanomas
Authors: Furuta Junichi , Nobeyama Yoshimasa , Umebayashi Yoshihiro , Otsuka Fujio , Kikuchi Kanako , Ushijima Toshikazu
Published: June 2006

Aberrant methylation of promoter CpG islands (CGI) is involved in silencing of tumor suppressor genes and is also a potential cancer biomarker. Here, to identify CGIs aberrantly methylated in human melanomas, we did a genome-wide search using methylation-sensitive representational difference analysis. CGIs in putative promoter regions of 34 genes (ABHD9, BARHL1, CLIC5, CNNM1, COL2A1, CPT1C, DDIT4L, DERL3, DHRS3, DPYS, EFEMP2, FAM62C, FAM78A, FLJ33790, GBX2, GPR10, GPRASP1, HOXA9, HOXD11, HOXD12, HOXD13, p14ARF, PAX6, PRDX2, PTPRG, RASD1, RAX, REC8L1, SLC27A3, TGFB2, TLX2, TMEM22, TMEM30B, and UNC5C) were found to be methylated in at least 1 of 13 melanoma cell lines but not in two cultured normal melanocytes. Among these genes, Peroxiredoxin 2 (PRDX2) was expressed in normal melanocytes, and its expression was lost in melanomas with methylation. The loss of expression was restored by treatment of melanomas with a demethylating agent 5-aza-2′-deoxycytidine. In surgical melanoma specimens, methylation of PRDX2 was detected in 3 of 36 (8%). Furthermore, immunohistochemical analysis of PRDX2 showed that disappearance of immunoreactivity tends to associate with its methylation. PRDX2 was recently reported to be a negative regulator of platelet-derived growth factor signaling, and its silencing was suggested to be involved in melanomas. On the other hand, 12 CGIs were methylated in ≥9 of the 13 melanoma cell lines and are considered as candidate melanoma biomarkers. (Cancer Res 2006; 66(12): 6080-6)
Cytokinesis-Blocked Micronucleus Assay as a Novel Biomarker for Lung Cancer Risk
Authors: El-Zein Randa A. , Schabath Matthew B. , Etzel Carol J. , Lopez Mirtha S. , Franklin Jamey D. , Spitz Margaret R.
Published: June 2006

In this case-control study, we modified the cytokinesis-block micronucleus (CBMN) assay, an established biomarker for genomic instability, to evaluate susceptibility to the nicotine-derived nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) by measuring the frequency of NNK-induced chromosomal damage endpoints (micronuclei, nucleoplasmic bridges, and nuclear buds) per 1,000 binucleated lymphocytes. Spontaneous and NNK-induced chromosomal damage were significantly higher in lung cancer patients compared with controls. Forty-seven percent of cases (versus 12% of controls) had ≥4 spontaneous micronuclei, 66% of cases (and no controls) had ≥4 spontaneous nucleoplasmic bridges, and 25% of cases (versus 5% of controls) had ≥1 spontaneous nuclear bud (P < 0.001). Similarly, 40% of cases (versus 6% of the controls) had ≥5 NNK-induced micronuclei, 89% of cases (and no controls) had ≥6 induced nucleoplasmic bridges, and 23% of cases (versus 2% of controls) had ≥2 induced nuclear buds (P < 0.001). When analyzed on a continuous scale, spontaneous micronuclei, nucleoplasmic bridges, and nuclear buds were associated with 2-, 29-, and 6-fold increases in cancer risk, respectively. Similarly, NNK-induced risks were 2.3-, 45.5-, and 10-fold, respectively. We evaluated the use of CBMN assay to predict cancer risk based on the numbers of micronuclei, nucleoplasmic bridges, and nuclear buds defined by percentile cut points in controls. Probabilities of being a cancer patient were 96%, 98%, and 100% when using the 95th percentiles of spontaneous and NNK-induced micronuclei, nucleoplasmic bridges, and nuclear buds, respectively. Our study indicates that the CBMN assay is extremely sensitive to NNK-induced genetic damage and may serve as a strong predictor of lung cancer risk. (Cancer Res 2006; 66(12): 6449-56)
Genome-Wide Profiling of CpG Methylation Identifies Novel Targets of Aberrant Hypermethylation in Myeloid Leukemia
Authors: Gebhard Claudia , Schwarzfischer Lucia , Pham Thu-Hang , Schilling Elmar , Klug Maja , Andreesen Reinhard , Rehli Michael
Published: June 2006

The methylation of CpG islands is associated with transcriptional repression and, in cancer, leads to the abnormal silencing of tumor suppressor genes. Because aberrant hypermethylation may be used as a marker for disease, a sensitive method for the global detection of DNA methylation events is of particular importance. We describe a novel and robust technique, called methyl-CpG immunoprecipitation, which allows the unbiased genome-wide profiling of CpG methylation in limited DNA samples. The approach is based on a recombinant, antibody-like protein that efficiently binds native CpG-methylated DNA. In combination with CpG island microarrays, the technique was used to identify >100 genes with aberrantly methylated CpG islands in three myeloid leukemia cell lines. Interestingly, within all hypermethylation targets, genes involved in transcriptional regulation were significantly overrepresented. More than half of the identified genes were absent in microarray expression studies in either leukemia or normal monocytes, indicating that hypermethylation in cancer may be largely independent of the transcriptional status of the affected gene. Most individually tested genes were also hypermethylated in primary blast cells from acute myeloid leukemia patients, suggesting that our approach can identify novel potential disease markers. The technique may prove useful for genome-wide comparative methylation analysis not only in malignancies. (Cancer Res 2006; 66(12): 6118-28)
Amphiregulin Contributes to the Transformed Phenotype of Human Hepatocellular Carcinoma Cells
Authors: Castillo Josefa , Erroba Elena , Perugorría María J. , Santamaría Mónica , Lee David C. , Prieto Jesús , Avila Matías A. , Berasain Carmen
Published: June 2006

Hepatocellular carcinoma is a major cause of cancer-related deaths. Current treatments are not effective, and the identification of relevant pathways and novel therapeutic targets are much needed. Increasing evidences point to the activation of the epidermal growth factor receptor (EGFR) as an important mechanism in the development of hepatocarcinoma. We previously described that amphiregulin (AR), a ligand of the EGFR, is not expressed in healthy liver but is up-regulated during chronic liver injury, the background on which most liver tumors develop. Now, we have studied the expression and role of AR in human hepatocarcinoma. AR expression and function was studied in human liver tumors and cell lines. AR is expressed in human hepatocellular carcinoma tissues and cell lines and behaves as a mitogenic and antiapoptotic growth factor for hepatocarcinoma cells. We provide several lines of evidence, including AR silencing by small interfering RNAs and inhibition of amphiregulin by neutralizing antibodies, showing the existence of an AR-mediated autocrine loop that contributes to the transformed phenotype. Indeed, interference with endogenous AR production resulted in reduced constitutive EGFR signaling, inhibition of cell proliferation, anchorage-independent growth, and enhanced apoptosis. Moreover, knockdown of AR potentiated transforming growth factor-β and doxorubicin-induced apoptosis. Conversely, overexpression of AR in SK-Hep1 cells enhanced their proliferation rate, anchorage-independent growth, drug resistance, and in vivo tumorigenic potential. These observations suggest that AR is involved in the acquisition of neoplastic traits in the liver and thus constitutes a novel therapeutic target in human hepatocarcinoma. (Cancer Res 2006; 66(12): 6129-38)
Ras Stimulation of E2F Activity and a Consequent E2F Regulation of Integrin α6β4 Promote the Invasion of Breast Carcinoma Cells
Authors: Yoon Sang-Oh , Shin Sejeong , Mercurio Arthur M.
Published: June 2006

Active Ras proteins contribute to breast carcinogenesis and progression. Here, we provide evidence that active H-Ras regulates the expression and activity of the E2F family of transcription factors in SUM-159 breast carcinoma cells. In addition, we show by using a DNA-binding mutant of E2F, as well as expression of specific E2Fs that are transcriptionally active, that the active E2Fs1-3 can mediate the H-Ras-dependent invasion of SUM-159 cells. The inhibitory E2Fs4-5, in contrast, do not influence invasion. One mechanism by which the active E2Fs promote H-Ras-dependent invasion seems to be their ability to increase expression of the β4 integrin subunit, a component of the α6β4 integrin that is known to enhance carcinoma invasion. Specifically, expression of E2Fs1-3 increased β4 mRNA, protein, and cell surface expression. The active E2Fs were unable to stimulate invasion in cells that expressed a β4 short hairpin RNA. This effect of the active E2Fs on β4 expression does not seem to result from E2F-mediated β4 transcription because the β4 promoter lacks known E2F binding motifs. In summary, the data reported here indicate a novel mechanism by which H-Ras can promote the invasion of breast carcinoma cells. This mechanism links active H-Ras, transcriptionally active E2F, and the α6β4 integrin in a common pathway that culminates in enhanced α6β4-dependent invasion. (Cancer Res 2006; 66(12): 6288-95)