bluuebunny/crossref_metadata_2025 huggingface dataset:
Increased spinal reflex excitability is not associated with neural plasticity underlying the cross-education effect
Authors: Lagerquist Olle , Zehr E. Paul , Docherty David
Published: December 2005

The purpose of this study was to examine the effects of a 5-wk unilateral, isometric strength-training program on plasticity in the spinal Hoffmann (H-) reflex in both the trained and untrained legs. Sixteen participants, 22–42 yr old, were assigned to either a control ( n = 6) or an exercise group ( n = 10). Both groups were tested for plantar flexion maximal voluntary isometric contractions (MVIC) and soleus H-reflex amplitude in both limbs, at the beginning and at the end of a 5-wk interval. Participants in the exercise group showed significantly increased MVIC in both legs after training ( P < 0.05), whereas strength was unchanged in the control group for either leg. Subjects in the exercise group displayed increased ( P < 0.05) H-reflex amplitudes on the ascending limb of the recruitment curve (at an equivalent M wave of 5% of the maximal M wave, HA) only in the trained leg. Maximal H-reflex and M-wave remained unchanged with training. Increased amplitude of HA in the trained limb concurrent with increased strength suggests that spinal mechanisms may underlie the changes in strength, possibly because of increased α-motoneuronal excitability or reduced presynaptic inhibition. Despite a similar increase in strength in the contralateral limb of the exercise group, HA amplitude was unchanged. We conclude that the cross-education effect of strength training may be due to supraspinal to a greater extent than spinal mechanisms.
Time course of COX-1 and COX-2 expression during ischemia-reperfusion in rat skeletal muscle
Authors: Dupouy V. M. , Ferre P. J. , Uro-Coste E. , Lefebvre H. P.
Published: December 2005

The aim of this study was to assess cyclooxygenase (COX)-1 and COX-2 expression in skeletal muscle after an ischemia-reperfusion (I/R). Male Sprague-Dawley rats were subjected to unilateral hindlimb ischemia for 2 h and then euthanized after 0, 1, 2, 4, 6, 10, 24, and 72 h of reperfusion. The COX protein and mRNA were assessed in control and injured gastrocnemius muscle. Muscle damage was indirectly determined by plasma creatine kinase activity and edema by weighing wet muscle. Creatine kinase activity in plasma increased as early as 1 h after reperfusion and returned to control levels by 72 h of reperfusion. Edema was observed at 6 and 10 h of reperfusion, but histological investigations showed an absence of tissular inflammatory cell infiltration. COX-1 mRNA was expressed in control muscle and was increased at 72 h of reperfusion, but the levels of associated COX-1 protein detected in control and injured gastrocnemius muscle were similar. COX-2 mRNA was not, or only slightly, detectable in control muscle and after I/R. In contrast, I/R induced major overexpression of COX-2 immunoreactivity at 6 and 10 h of reperfusion with a maximum at 10 h, whereas COX-2 protein was undetectable in control muscle. In conclusion, hindlimb I/R induced a large overexpression of COX-2 but not COX-1 protein between 6 and 10 h after injury. These results suggest a role for COX-2 enzyme in such pathophysiological conditions of the skeletal muscle.
Effect of chronic hypoxia on purinergic synaptic transmission in rat carotid body
Authors: He L. , Chen J. , Dinger B. , Stensaas L. , Fidone S.
Published: December 2005

Recent studies indicate that chemoafferent nerve fiber excitation in the rat carotid body is mediated by acetylcholine and ATP, acting at nicotinic cholinergic receptors and P2X2purinoceptors, respectively. We previously demonstrated that, after a 10- to 14-day exposure to chronic hypoxia (CH), the nicotinic cholinergic receptor blocker mecamylamine no longer inhibits rat carotid sinus nerve (CSN) activity evoked by an acute hypoxic challenge. The present experiments examined the effects of CH (9–16 days at 380 Torr) on the expression of P2X2purinoceptors in carotid body and chemoafferent neurons, as well as the effectiveness of P2X2receptor blocking drugs on CSN activity evoked by hypoxia. In the normal carotid body, immunocytochemical studies demonstrated a dense plexus of P2X2-positive nerve fibers penetrating lobules of type I cells. In addition, type I cells were lightly stained, indicating P2X2receptor expression. After CH, the intensity of P2X2receptor immunostaining was maintained in chemosensory type I cells and in the soma of chemoafferent neurons. P2 receptor expression on type I cells was confirmed by demonstrations of ATP-evoked increased intracellular Ca2+; this response was modulated by simultaneous exposure to hypoxia. In normal preparations, CSN activity evoked by hypoxia in vitro was 65% inhibited in the presence of specific P2X2receptor antagonists. However, unlike the absence of mecamylamine action after CH, P2X2antagonists remained effective against hypoxia-evoked activity after CH. Our findings indicate that ATP acting at P2X2receptors contributes to adjusted chemoreceptor activity after CH, indicating a possible role for purinergic mechanisms in the adaptation of the carotid body in a chronic low-O2environment.
Endothelial influences on cerebrovascular tone
Authors: Andresen Jon , Shafi Nadeem I. , Bryan Robert M.
Published: December 2005

The cerebrovascular endothelium exerts a profound influence on cerebral vessels and cerebral blood flow. This review summarizes current knowledge of various dilator and constrictor mechanisms intrinsic to the cerebrovascular endothelium. The endothelium contributes to the resting tone of cerebral arteries and arterioles by tonically releasing nitric oxide (NO•). Dilations can occur by stimulated release of NO•, endothelium-derived hyperpolarization factor, or prostanoids. During pathological conditions, the dilator influence of the endothelium can turn to that of constriction by a variety of mechanisms, including decreased NO• bioavailability and release of endothelin-1. The endothelium may participate in neurovascular coupling by conducting local dilations to upstream arteries. Further study of the cerebrovascular endothelium is critical for understanding the pathogenesis of a number of pathological conditions, including stroke, traumatic brain injury, and subarachnoid hemorrhage.
Heat intolerance: does gene transcription contribute?
Authors: Moran Daniel S. , Eli-Berchoer Luba , Heled Yuval , Mendel Liran , Schocina Mara , Horowitz Michal
Published: December 2005

During exertion in the heat, heat-intolerant (HI) subjects have a physiological disability in metabolic heat dissipation. The HI state is either permanent or temporary, depending on whether it stems from transient predisposing factors or inherent thermoregulatory dysfunction. In this investigation, we studied protein levels of heat shock protein (HSP) 70 and HSP72, HSP90, bcl-2xL, glutathione S-transferase-p, heat shock factor-1, TATA-binding protein-associated factor, and NF-κB transcripts using Western blot and quantitative RT-PCR, respectively, in lymphocytes of HI and tolerant (T) male volunteers of similar anthropometric features. Measurements were made from blood drawn before, during the heat tolerance test (3.5 mph, 40°C, 40% relative humidity, 2 h), and 1 h after recovery at 24°C. Rectal and skin temperatures, as well as heart rate, were continuously recorded. Of 58 subjects, 7 were identified as HI, with a significantly higher physiological strain index than in the T group (6.3 ± 0.9 vs. 3.8 ± 0.6, P < 0.001). The responsiveness of the vasculature to thermal stimuli was decreased in the HI group, as indicated by rectal temperature minus skin temperature. The HSP72 level in the HI group dropped during the recovery session ( P < 0.01), whereas that of the T group continued to rise. A significantly increased expression of the transcription factors in the T subjects and significantly decreased expression in the HI group ( P < 0.009, 0.013, and 0.005 for heat shock factor-1, NF-κB, and TATA-binding protein-associated factor, respectively) points to impaired transcriptional processes in the HI group. Our data suggest that transcriptional malfunction and sluggishness of the vasculature to thermal stimuli are predisposing factors in the HI group.
Spike-Timing Precision Underlies the Coding Efficiency of Auditory Receptor Neurons
Authors: Rokem Ariel , Watzl Sebastian , Gollisch Tim , Stemmler Martin , Herz Andreas V. M. , Samengo Inés
Published: December 2005

Sensory systems must translate incoming signals quickly and reliably so that an animal can act successfully in its environment. Even at the level of receptor neurons, however, functional aspects of the sensory encoding process are not yet fully understood. Specifically, this concerns the question how stimulus features and neural response characteristics lead to an efficient transmission of sensory information. To address this issue, we have recorded and analyzed spike trains from grasshopper auditory receptors, while systematically varying the stimulus statistics. The stimulus variations profoundly influenced the efficiency of neural encoding. This influence was largely attributable to the presence of specific stimulus features that triggered remarkably precise spikes whose trial-to-trial timing variability was as low as 0.15 ms—one order of magnitude shorter than typical stimulus time scales. Precise spikes decreased the noise entropy of the spike trains, thereby increasing the rate of information transmission. In contrast, the total spike train entropy, which quantifies the variety of different spike train patterns, hardly changed when stimulus conditions were altered, as long as the neural firing rate remained the same. This finding shows that stimulus distributions that were transmitted with high information rates did not invoke additional response patterns, but instead displayed exceptional temporal precision in their neural representation. The acoustic stimuli that led to the highest information rates and smallest spike-time jitter feature pronounced sound-pressure deflections lasting for 2–3 ms. These upstrokes are reminiscent of salient structures found in natural grasshopper communication signals, suggesting that precise spikes selectively encode particularly important aspects of the natural stimulus environment.
Motor Coding in Floccular Climbing Fibers
Authors: Winkelman Beerend , Frens Maarten
Published: December 2005

The climbing fibers (CFs) that project from the dorsal cap of the inferior olive (IO) to the flocculus of the cerebellar cortex have been reported to be purely sensory, encoding “retinal slip.” However, a clear oculomotor projection from the nucleus prepositus hypoglossi (NPH) to the IO has been shown. We therefore studied the sensorimotor information that is present in the CF signal. We presented rabbits with visual motion noise stimuli to break up the tight relation between instantaneous retinal slip and eye movement. Strikingly, the information about the motor behavior in the CF signal more than doubled that of the sensory component and was time-locked more tightly. The contribution of oculomotor signals was independently confirmed by analysis of spontaneous eye movements in the absence of visual input. The motor component of the CF code is essential to distinguish unexpected slip from self-generated slip, which is a prerequisite for proper oculomotor learning.
Hypoxia-Regulated Expression of Attenuated Diphtheria Toxin A Fused with Hypoxia-Inducible Factor-1α Oxygen-Dependent Degradation Domain Preferentially Induces Apoptosis of Hypoxic Cells in Solid Tumor
Authors: Koshikawa Nobuko , Takenaga Keizo
Published: December 2005

Tumor cells in hypoxic areas of solid tumors are resistant to conventional chemotherapy and radiotherapy and thus are obstacles of cancer therapy. We report here the feasibility of applying hypoxia-regulated expression of diphtheria toxin A (DT-A) for killing hypoxic tumor cells. The expression vector was constructed to express DT-A fused with hypoxia-inducible factor-1α (HIF-1α) oxygen-dependent degradation (ODD) domain under the control of vascular endothelial growth factor gene promoter and contain erythropoietin mRNA-binding protein (ERBP)–binding sequence downstream of the DT-A/ODD sequence. In vitro ubiquitination assay showed that DT-A/ODD, but not DT-A, was ubiquitinated as efficient as HIF-1α under normoxic conditions in a von Hippel-Lindau– and oxygen-dependent manner. DT-A/ODD exhibited a comparable translation inhibitory activity to DT-A. ERBP-binding sequence was effective in stabilizing mRNA under hypoxic conditions in various cell types. Transfection of the vector expressing DT-A/ODD into high-metastatic Lewis lung carcinoma (3LL) A11 cells resulted in induction of apoptosis independently of hypoxia, probably due to its extreme toxicity. However, transfection of the vector expressing attenuated DT-AW153F/ODD or DT-AH21A/ODD resulted in a hypoxia-dependent induction of apoptosis. Liposomal gene transfer of the vector encoding DT-AW153F/ODD induced apoptosis in hypoxic, but not in normoxic, areas of solid tumors established by A11 variant cells with higher resistance to hypoxia-induced apoptosis and inhibited the growth of hypoxic tumors established by 3LL-P29 cells. These results suggest that hypoxia-regulated expression of attenuated DT-AW153F/ODD fusion protein is potentially of use for killing hypoxic tumor cells with minimizing the damage to normoxic normal tissues. (Cancer Res 2005; 65(24): 11622-30)
Epidermal Growth Factor Receptor–Targeted Immunoliposomes Significantly Enhance the Efficacy of Multiple Anticancer DrugsIn vivo
Authors: Mamot Christoph , Drummond Daryl C. , Noble Charles O. , Kallab Verena , Guo Zexiong , Hong Keelung , Kirpotin Dmitri B. , Park John W.
Published: December 2005

We previously reported the development of epidermal growth factor receptor (EGFR)–targeted immunoliposomes that bind and internalize in tumor cells which overexpress EGFR and/or mutant EGFR variant III (EGFRvIII), enabling intracellular delivery of potent anticancer agents in vitro. We now describe in vivo proof-of-concept for this approach for the delivery of multiple anticancer drugs in EGFR-overexpressing tumor models. Anti-EGFR immunoliposomes were constructed modularly with Fab′ fragments of cetuximab (IMC-C225), covalently linked to liposomes containing probes and/or anticancer drugs. Pharmacokinetic and biodistribution studies confirmed long circulation times (t1/2 = 21 hours) and efficient accumulation in tumors (up to 15% ID/g) irrespective of the presence of the targeting ligand. Although total accumulations of anti-EGFR immunoliposomes and nontargeted liposomes in EGFR-overexpressing tumors were comparable, only immunoliposomes internalized extensively within tumor cells (92% of analyzed cells versus <5% for nontargeted liposomes), indicating different mechanisms of delivery at the cellular level. In vivo therapy studies in a series of xenograft models featuring overexpression of EGFR and/or EGFRvIII showed the superiority of immunoliposomal delivery of encapsulated drugs, which included doxorubicin, epirubicin, and vinorelbine. For each of these drugs, anti-EGFR immunoliposome delivery showed significant antitumor effects and was significantly superior to all other treatments, including the corresponding free or liposomal drug (P < 0.001-0.003). We conclude that anti-EGFR immunoliposomes provide efficient and targeted drug delivery of anticancer compounds and may represent a useful new treatment approach for tumors that overexpress the EGFR. (Cancer Res 2005; 65(24): 11631-8)
Novel Role for PDEF in Epithelial Cell Migration and Invasion
Authors: Gunawardane Ruwanthi N. , Sgroi Dennis C. , Wrobel Carolyn N. , Koh Eugene , Daley George Q. , Brugge Joan S.
Published: December 2005

Cell migration and invasion are two critical cellular processes that are often deregulated during tumorigenesis. To identify factors that contribute to oncogenic progression by stimulating cell migration, we conducted a powerful retroviral based migration screen using an MCF7 cDNA library and the immortalized human breast epithelial cell line MCF-10A. We identified prostate derived Ets factor (PDEF), an Ets transcription factor that is overexpressed in both prostate and breast carcinoma, as a candidate promigratory gene from this screen. Whereas PDEF induced limited motility of MCF-10A cells, coexpression of PDEF with the receptor tyrosine kinases (RTK) ErbB2 and colony-stimulating factor receptor (CSF-1R)/CSF-1 significantly enhanced MCF-10A motility. Furthermore, cells coexpressing PDEF with either ErbB2 or CSF-1R/CSF-1 induced a dramatic invasive phenotype in three-dimensional cultures. Constitutive activation of the extracellular signal–regulated kinase (ERK) pathway also enhanced PDEF-induced motility and invasion, suggesting that activation of the ERK/mitogen-activated protein kinase by ErbB2 and CSF-1R/CSF-1 can cooperate with PDEF to promote motility and invasion. Furthermore, PDEF promoted anchorage-independent growth of ErbB2 and CSF-1R/CSF-1–expressing cells. Using laser capture microdissection, we also found that PDEF mRNA is overexpressed in breast tumor epithelia throughout tumor progression. Taken together, these findings suggest that the transcription factor PDEF may play an important role in breast tumorigenesis and that PDEF overexpression may be particularly significant in tumors that exhibit activation of oncogenic RTKs such as ErbB2 and CSF-1R. (Cancer Res 2005; 65(24): 11572-80)
Multiple Repair Pathways Mediate Tolerance to Chemotherapeutic Cross-linking Agents in Vertebrate Cells
Authors: Nojima Kuniharu , Hochegger Helfrid , Saberi Alihossein , Fukushima Toru , Kikuchi Koji , Yoshimura Michio , Orelli Brian J. , Bishop Douglas K. , Hirano Seiki , Ohzeki Mioko , Ishiai Masamichi , Yamamoto Kazuhiko , Takata Minoru , Arakawa Hiroshi , Buerstedde Jean-Marie , Yamazoe Mitsuyoshi , Kawamoto Takuo , Araki Kasumi , Takahashi Jun A. , Hashimoto Nobuo , Takeda Shunichi , Sonoda Eiichiro
Published: December 2005

Cross-linking agents that induce DNA interstrand cross-links (ICL) are widely used in anticancer chemotherapy. Yeast genetic studies show that nucleotide excision repair (NER), Rad6/Rad18-dependent postreplication repair, homologous recombination, and cell cycle checkpoint pathway are involved in ICL repair. To study the contribution of DNA damage response pathways in tolerance to cross-linking agents in vertebrates, we made a panel of gene-disrupted clones from chicken DT40 cells, each defective in a particular DNA repair or checkpoint pathway, and measured the sensitivities to cross-linking agents, including cis-diamminedichloroplatinum (II) (cisplatin), mitomycin C, and melphalan. We found that cells harboring defects in translesion DNA synthesis (TLS), Fanconi anemia complementation groups (FANC), or homologous recombination displayed marked hypersensitivity to all the cross-linking agents, whereas NER seemed to play only a minor role. This effect of replication-dependent repair pathways is distinctively different from the situation in yeast, where NER seems to play a major role in dealing with ICL. Cells deficient in Rev3, the catalytic subunit of TLS polymerase Polζ, showed the highest sensitivity to cisplatin followed by fanc-c. Furthermore, epistasis analysis revealed that these two mutants work in the same pathway. Our genetic comprehensive study reveals a critical role for DNA repair pathways that release DNA replication block at ICLs in cellular tolerance to cross-linking agents and could be directly exploited in designing an effective chemotherapy. (Cancer Res 2005; 65(24): 11704-11)
ABCC Drug Efflux Pumps and Organic Anion Uptake Transporters in Human Gliomas and the Blood-Tumor Barrier
Authors: Bronger Holger , König Jörg , Kopplow Kathrin , Steiner Hans-Herbert , Ahmadi Rezvan , Herold-Mende Christel , Keppler Dietrich , Nies Anne T.
Published: December 2005

Delivery of therapeutic agents to the brain and its neoplasms depends on the presence of membrane transport proteins in the blood-brain barrier and in the target cells. The cellular and subcellular localization of these membrane transporters determines the drug accessibility to the brain and its tumors. We therefore analyzed the expression and localization of six members of the multidrug resistance protein family of ATP-dependent efflux pumps (ABCC1-ABCC6, formerly MRP1-MRP6) and of six organic anion uptake transporters (OATP1A2, OATP1B1, OATP1B3, OATP1C1, OATP2B1, and OATP4A1) in 61 human glioma specimens of different histologic subtypes. Real-time PCRs indicated expressions of ABCC1, ABCC3, ABCC4, and ABCC5. In addition, we detected expressions of the OATP uptake transporter genes SLCO1A2, SLCO1C1, SLCO2B1, and SLCO4A1. At the protein level, however, only OATP1A2 and OATP2B1 were detectable by immunofluorescence microscopy in the luminal membrane of endothelial cells forming the blood-brain barrier and the blood-tumor barrier, but not in the glioma cells. ABCC4 and ABCC5 proteins were the major ABCC subfamily members in gliomas, localized both at the luminal side of the endothelial cells and in the glioma cells of astrocytic tumors and in the astrocytic portions of oligoastrocytomas. These results indicate that expression of ABCC4 and ABCC5 is associated with an astrocytic phenotype, in accordance with their expression in astrocytes and with the higher chemoresistance of astrocytic tumors as compared with oligodendrogliomas. Our data provide a basis for the assessment of the role of uptake transporters and efflux pumps in the accessibility of human gliomas for chemotherapeutic agents. (Cancer Res 2005; 65(24): 11419-28)
Lysyl Oxidase Regulates Breast Cancer Cell Migration and Adhesion through a Hydrogen Peroxide–Mediated Mechanism
Authors: Payne Stacey L. , Fogelgren Ben , Hess Angela R. , Seftor Elisabeth A. , Wiley Elizabeth L. , Fong Sheri F.T. , Csiszar Katalin , Hendrix Mary J.C. , Kirschmann Dawn A.
Published: December 2005

We have previously shown that lysyl oxidase (LOX) mRNA is up-regulated in invasive breast cancer cells and that catalytically active LOX facilitates in vitro cell invasion. Here we validate our in vitro studies by showing that LOX expression is up-regulated in distant metastatic breast cancer tissues compared with primary cancer tissues. To elucidate the mechanism by which LOX facilitates cell invasion, we show that catalytically active LOX regulates in vitro motility/migration and cell-matrix adhesion formation. Treatment of the invasive breast cancer cell lines, Hs578T and MDA-MB-231, with β-aminopropionitrile (βAPN), an irreversible inhibitor of LOX catalytic activity, leads to a significant decrease in cell motility/migration and adhesion formation. Conversely, poorly invasive MCF-7 cells expressing LOX (MCF-7/LOX32-His) showed an increase in migration and adhesion that was reversible with the addition of βAPN. Moreover, a decrease in activated focal adhesion kinase (FAK) and Src kinase, key proteins involved in adhesion complex turnover, was observed when invasive breast cancer cells were treated with βAPN. Additionally, FAK and Src activation was increased in MCF-7/LOX32-His cells, which was reversible on βAPN treatment. Hydrogen peroxide was produced as a by-product of LOX activity and the removal of hydrogen peroxide by catalase treatment in invasive breast cancer cells led to a dose-dependent loss in Src activation. These results suggest that LOX facilitates migration and cell-matrix adhesion formation in invasive breast cancer cells through a hydrogen peroxide–mediated mechanism involving the FAK/Src signaling pathway. These data show the need to target LOX for treatment of aggressive breast cancer. (Cancer Res 2005; 65(24): 11429-36)
ANLN Plays a Critical Role in Human Lung Carcinogenesis through the Activation of RHOA and by Involvement in the Phosphoinositide 3-Kinase/AKT Pathway
Authors: Suzuki Chie , Daigo Yataro , Ishikawa Nobuhisa , Kato Tatsuya , Hayama Satoshi , Ito Tomoo , Tsuchiya Eiju , Nakamura Yusuke
Published: December 2005

Gene expression profile analysis of non–small cell lung cancers (NSCLC) and subsequent functional analyses revealed that human ANLN, a homologue of anillin, an actin-binding protein in Drosophila, was transactivated in lung cancer cells and seemed to play a significant role in pulmonary carcinogenesis. Induction of small interfering RNAs against ANLN in NSCLC cells suppressed its expression and resulted in growth suppression; moreover, treatment with small interfering RNA yielded cells with larger morphology and multiple nuclei, which subsequently died. On the other hand, induction of exogenous expression of ANLN enhanced the migrating ability of mammalian cells by interacting with RHOA, a small guanosine triphosphatase, and inducing actin stress fibers. Interestingly, inhibition of phosphoinositide 3-kinase/AKT activity in NSCLC cells decreased the stability of ANLN and caused a reduction of the nuclear ANLN level. Immunohistochemical staining of nuclear ANLN on lung cancer tissue microarrays was associated with the poor survival of NSCLC patients, indicating that this molecule might serve as a prognostic indicator. Our data imply that up-regulation of ANLN is a common feature of the carcinogenetic process in lung tissue, and suggests that selective suppression of ANLN could be a promising approach for developing a new strategy to treat lung cancers. (Cancer Res 2005; 65(24): 11314-25)
Immunomodulatory Drug Lenalidomide (CC-5013, IMiD3) Augments Anti-CD40 SGN-40–Induced Cytotoxicity in Human Multiple Myeloma: Clinical Implications
Authors: Tai Yu-Tzu , Li Xian-Feng , Catley Laurence , Coffey Rory , Breitkreutz Iris , Bae Jooeun , Song Weihua , Podar Klaus , Hideshima Teru , Chauhan Dharminder , Schlossman Robert , Richardson Paul , Treon Steven P. , Grewal Iqbal S. , Munshi Nikhil C. , Anderson Kenneth C.
Published: December 2005

SGN-40, a humanized immoglobulin G1 (IgG1) anti-CD40 monoclonal antibody, mediates cytotoxicity against human multiple myeloma (MM) cells via suppression of interleukin (IL)-6–induced proliferative and antiapoptotic effects as well as antibody-dependent cell-mediated cytotoxicity (ADCC). Here, we studied the clinical significance of an immunomodulatory drug lenalidomide on SGN-40–induced cytotoxicity against CD138+CD40+ MM lines and patient MM cells. Pretreatment with lenalidomide sensitized MM cells to SGN-40–induced cell death. Combined lenalidomide and SGN-40 significantly induced MM apoptosis, evidenced by enhanced cleavage of caspase-3/8/poly(ADP-ribose)polymerase and increased sub-G0 cells, compared with either single agent at the same doses. Pretreatment of effector cells with lenalidomide augmented SGN-40–induced MM cell lysis, associated with an increased number of CD56+CD3− natural killer (NK) cells expressing CD16 and LFA-1. Importantly, pretreatment with lenalidomide or lenalidomide and SGN-40 markedly enhanced NK-cell–mediated lysis of autologous patient MM cells triggered by SGN-40. Lenalidomide also up-regulated CD40L on CD56+CD3− NK cells, facilitating IL-2–mediated activation of NK cells. In addition, lenalidomide induced the CD56dim NK subset, which are more potent mediators of ADCC against target MM cells than the CD56bright NK subset. Finally, pretreatment of both effector and target MM cells with lenalidomide markedly enhanced SGN-40–mediated ADCC against CD40-expressing MM cells. These studies, therefore, show that the addition of lenalidomide to SGN-40 enhances cytotoxicity against MM cells, providing the framework for combined lenalidomide and SGN-40 in a new treatment paradigm to both target MM cells directly and induce immune effectors against MM. (Cancer Res 2005; 65(24): 11712-20)
Inhibition of DNA Methylation Sensitizes Glioblastoma for Tumor Necrosis Factor–Related Apoptosis-Inducing Ligand–Mediated Destruction
Authors: Eramo Adriana , Pallini Roberto , Lotti Fiorenza , Sette Giovanni , Patti Mariella , Bartucci Monica , Ricci-Vitiani Lucia , Signore Michele , Stassi Giorgio , Larocca Luigi M. , Crinò Lucio , Peschle Cesare , De Maria Ruggero
Published: December 2005

Life expectancy of patients affected by glioblastoma multiforme is extremely low. The therapeutic use of tumor necrosis factor–related apoptosis-inducing ligand (TRAIL) has been proposed to treat this disease based on its ability to kill glioma cell lines in vitro and in vivo. Here, we show that, differently from glioma cell lines, glioblastoma multiforme tumors were resistant to TRAIL stimulation because they expressed low levels of caspase-8 and high levels of the death receptor inhibitor PED/PEA-15. Inhibition of methyltransferases by decitabine resulted in considerable up-regulation of TRAIL receptor-1 and caspase-8, down-regulation of PED/PEA-15, inhibition of cell growth, and sensitization of primary glioblastoma cells to TRAIL-induced apoptosis. Exogenous caspase-8 expression was the main event able to restore TRAIL sensitivity in primary glioblastoma cells. The antitumor activity of decitabine and TRAIL was confirmed in vivo in a mouse model of glioblastoma multiforme. Evaluation of tumor size, apoptosis, and caspase activation in nude mouse glioblastoma multiforme xenografts showed dramatic synergy of decitabine and TRAIL in the treatment of glioblastoma, whereas the single agents were scarcely effective in terms of reduction of tumor mass, apoptosis induction, and caspase activation. Thus, the combination of TRAIL and demethylating agents may provide a key tool to overcome glioblastoma resistance to therapeutic treatments. (Cancer Res 2005; 65(24): 11469-77)
Differentiation of Androgen-Independent Prostate Cancer PC-3 Cells Is Associated with Increased Nuclear Factor-κB Activity
Authors: Floryk Daniel , Huberman Eliezer
Published: December 2005

Recently, we have reported that inosine 5′-monophosphate dehydrogenase inhibitors, such as mycophenolic acid (MPA), induce the differentiation of PC-3 cells, which are derived from a human androgen-independent prostate cancer, into cells with a phenotype resembling maturing prostate secretory cells. Here, we describe such differentiation induced by the histone deacetylase inhibitor tributyrin. The maturation was defined by cytoplasmic vacuole production and induction of CD10, CD46, CD55, GRP78, keratin 17, and zinc-α-2-glycoprotein. To identify additional genes associated with tributyrin-induced PC-3 cell differentiation and to gain some insight into the mechanism that underlies this differentiation, we have, by means of microarray analyses, compared tributyrin-induced gene expression patterns with those of MPA, which initiates PC-3 cell differentiation by a dissimilar mode of action. We suggested that genes induced by both tributyrin and MPA would be most likely associated with differentiation rather than with the unique action of each particular inducer. Our results indicated that tributyrin or MPA induced the expression of a large number of common genes, including genes known or assumed to be NF-κB dependent. The NF-κB dependency of a group of these genes, which included the PC-3 cell differentiation marker keratin 17, was confirmed by using two common NF-κB activation inhibitors, Bay11-082 and TMB-8, and p65 subunit of NF-κB complex specific small interfering RNA. Taken together, our results implicate both NF-κB–dependent and NF-κB–independent genes in the processes leading to PC-3 cell differentiation induced by tributyrin and MPA. (Cancer Res 2005; 65(24): 11588-96)
BGC 945, a Novel Tumor-Selective Thymidylate Synthase Inhibitor Targeted to α-Folate Receptor–Overexpressing Tumors
Authors: Gibbs David D. , Theti Davinder S. , Wood Nadya , Green Matthew , Raynaud Florence , Valenti Melanie , Forster Martin D. , Mitchell Fraser , Bavetsias Vassilios , Henderson Elisa , Jackman Ann L.
Published: December 2005

BGC 945 is a cyclopenta[g]quinazoline–based, thymidylate synthase inhibitor specifically transported into α-folate receptor (α-FR)–overexpressing tumors. Affinity of BGC 945 for the α-FR is 70% of the high-affinity ligand folic acid. In contrast to conventional antifolates, BGC 945 has low affinity for the widely expressed reduced-folate carrier (RFC). The Ki for isolated thymidylate synthase is 1.2 nmol/L and the IC50 for inhibition of the growth of α-FR-negative mouse L1210 or human A431 cells is ∼7 μmol/L. In contrast, BGC 945 is highly potent in a range of α-FR-overexpressing human tumor cell lines (IC50 ∼1-300 nmol/L). Pharmacokinetic variables measured following i.v. injection of 100 mg/kg BGC 945 to KB tumor–bearing mice showed rapid plasma clearance (0.021 L/h) and tissue distribution. The terminal half-lives in plasma, liver, kidney, spleen, and tumor were 2, 0.6, 5, 21, and 28 hours, respectively. Tumor BGC 945 concentration at 24 hours was ∼1 nmol/g tissue, at least 10-fold higher than that in plasma or normal tissues. Inhibition of thymidylate synthase in tissues leads to increased incorporation of 5-[125I]-iodo-2′-deoxyuridine ([125I]dUrd) into DNA. Forty-eight hours after injection of 100 mg/kg 6RS-BGC 945 ([125I]dUrd injected at 24 hours), tumor was the only tissue with incorporation above control level (6-fold). The RFC-mediated thymidylate synthase inhibitor plevitrexed also increased uptake of [125I]dUrd in tumor (10-fold) but, in contrast, also caused increased incorporation in other normal tissues such as spleen and small bowel (4.5- and 4.6-fold, respectively). These data suggest that BGC 945 selectively inhibits thymidylate synthase in α-FR-overexpressing tumors and should cause minimal toxicity to humans at therapeutic doses. (Cancer Res 2005; 65(24): 11721-8)
Sequence Variants of Toll-Like Receptor 4 and Susceptibility to Prostate Cancer
Authors: Chen Yen-Ching , Giovannucci Edward , Lazarus Ross , Kraft Peter , Ketkar Shamika , Hunter David J.
Published: December 2005

Chronic inflammation has been hypothesized to be a risk factor for prostate cancer. The Toll-like receptor 4 (TLR4) presents the bacterial lipopolysaccharide (LPS), which interacts with ligand-binding protein and CD14 (LPS receptor) and activates expression of inflammatory genes through nuclear factor-κB and mitogen-activated protein kinase signaling. A previous case-control study found a modest association of a polymorphism in the TLR4 gene [11381G/C, GG versus GC/CC: odds ratio (OR), 1.26] with risk of prostate cancer. We assessed if sequence variants of TLR4 were associated with the risk of prostate cancer. In a nested case-control design within the Health Professionals Follow-up Study, we identified 700 participants with prostate cancer diagnosed after they had provided a blood specimen in 1993 and before January 2000. Controls were 700 age-matched men without prostate cancer who had had a prostate-specific antigen test after providing a blood specimen. We genotyped 16 common (>5%) single nucleotide polymorphisms (SNP) discovered in a resequencing study spanning TLR4 to test for association between sequence variation in TLR4 and prostate cancer. Homozygosity for the variant alleles of eight SNPs was associated with a statistically significantly lower risk of prostate cancer (TLR4_1893, TLR4_2032, TLR4_2437, TLR4_7764, TLR4_11912, TLR4_16649, TLR4_17050, and TLR4_17923), but the TLR4_15844 polymorphism corresponding to 11381G/C was not associated with prostate cancer (GG versus CG/CC: OR, 1.01; 95% confidence interval, 0.79-1.29). Six common haplotypes (cumulative frequency, 81%) were observed; the global test for association between haplotypes and prostate cancer was statistically significant (χ2 = 14.8 on 6 degrees of freedom; P = 0.02). Two common haplotypes were statistically significantly associated with altered risk of prostate cancer. Inherited polymorphisms of the innate immune gene TLR4 are associated with risk of prostate cancer. (Cancer Res 2005; 65(24): 11771-8)
Endothelin-1 Promotes Epithelial-to-Mesenchymal Transition in Human Ovarian Cancer Cells
Authors: Rosanò Laura , Spinella Francesca , Di Castro Valeriana , Nicotra Maria Rita , Dedhar Shoukat , de Herreros Antonio Garcia , Natali Pier Giorgio , Bagnato Anna
Published: December 2005

Despite considerable efforts to improve early detection and advances in chemotherapy, metastatic relapses remain a major challenge in the management of ovarian cancer. The endothelin A receptor (ETAR)/endothelin-1 (ET-1) axis has been shown to have a significant role in ovarian carcinoma by promoting tumorigenesis. Here we show that the ET-1/ETAR autocrine pathway drives epithelial-to-mesenchymal transition (EMT) in ovarian tumor cells by inducing a fibroblastoid and invasive phenotype, down-regulation of E-cadherin, increased levels of β-catenin, Snail, and other mesenchymal markers, and suppression of E-cadherin promoter activity. Activation of ETAR by ET-1 triggers an integrin-linked kinase (ILK)–mediated signaling pathway leading to glycogen synthase kinase-3β (GSK-3β) inhibition, Snail and β-catenin stabilization, and regulation of transcriptional programs that control EMT. Transfection of dominant negative ILK or exposure to an ILK inhibitor suppresses the ET-1-induced phosphorylation of GSK-3β as well as Snail and β-catenin protein stability, activity, and invasiveness, indicating that ET-1/ETAR–induced EMT-promoting effects depend on ILK. ETAR blockade by specific antagonists or reduction by ETAR RNA interference reverses EMT and cell invasion by inhibiting autocrine signaling pathways. In ovarian carcinoma xenografts, ABT-627, a specific ETAR antagonist, suppresses EMT determinants and tumor growth. In human ovarian cancers, ETAR expression is associated with E-cadherin down-regulation, N-cadherin expression, and tumor grade. Collectively, these findings provide evidence of a critical role for the ET-1/ETAR axis during distinct steps of ovarian carcinoma progression and identify novel targets of therapeutic intervention. (Cancer Res 2005; 65(24): 11649-57)
Mutually Exclusive Cyclin-Dependent Kinase 4/Cyclin D1 and Cyclin-Dependent Kinase 6/Cyclin D2 Pairing Inactivates Retinoblastoma Protein and Promotes Cell Cycle Dysregulation in Multiple Myeloma
Authors: Ely Scott , Di Liberto Maurizio , Niesvizky Ruben , Baughn Linda B. , Cho Hearn J. , Hatada Eunice N. , Knowles Daniel M. , Lane Joseph , Chen-Kiang Selina
Published: December 2005

Multiple myeloma, the second most common hematopoietic cancer, ultimately becomes refractory to treatment when self-renewing multiple myeloma cells begin unrestrained proliferation by unknown mechanisms. Here, we show that one, but not more than one, of the three early G1 D cyclins is elevated in each case of multiple myeloma. Cyclin D1 or D3 expression does not vary in the clinical course, but that alone is insufficient to promote cell cycle progression unless cyclin-dependent kinase 4 (cdk4) is also elevated, in the absence of cdk6, to phosphorylate the retinoblastoma protein (Rb). By contrast, cyclin D2 and cdk6 are coordinately increased, thereby overriding the inhibition by cdk inhibitors p18INK4c and p27Kip1 and phosphorylating Rb in conjunction with the existing cdk4. Thus, cyclin D1 pairs exclusively with cdk4 and cdk6 pairs only with cyclin D2, although cyclin D2 can also pair with cdk4 in multiple myeloma cells. The basis for this novel and specific cdk/D cyclin pairing lies in differential transcriptional activation. In addition, cyclin D1– or cyclin D3–expressing multiple myeloma cells are uniformly distributed in the bone marrow, whereas cdk6-specific phosphorylation of Rb occurs in discrete foci of bone marrow multiple myeloma cells before proliferation early in the clinical course and is then heightened with proliferation and disease progression. Mutually exclusive cdk4/cyclin D1 and cdk6/cyclin D2 pairing, therefore, is likely to be a critical determinant for cell cycle reentry and progression and may play a pivotal role in the expansion of self-renewing multiple myeloma cells. (Cancer Res 2005; 65(24): 11345-53)
The Critical Role of 15-Lipoxygenase-1 in Colorectal Epithelial Cell Terminal Differentiation and Tumorigenesis
Authors: Shureiqi Imad , Wu Yuanqing , Chen Dongning , Yang Xiu L. , Guan Baoxiang , Morris Jeffrey S. , Yang Peiying , Newman Robert A. , Broaddus Russell , Hamilton Stanley R. , Lynch Patrick , Levin Bernard , Fischer Susan M. , Lippman Scott M.
Published: December 2005

Terminal differentiation is an important event for maintaining normal homeostasis in the colorectal epithelium, and the loss of apoptosis is an important mechanism underlying colorectal tumorigenesis. The very limited current data on the role of lipoxygenase (LOX) metabolism in tumorigenesis suggests that the oxidative metabolism of linoleic and arachidonic acid possibly shifts from producing antitumorigenic 15-LOX-1 and 15-LOX-2 products to producing protumorigenic 5-LOX and 12-LOX products. We examined whether this shift occurs in vitro in the human colon cancer cell line Caco-2 in association with the loss of terminal differentiation and apoptosis, or in vivo during the formation of colorectal adenomas in patients with familial adenomatous polyposis (FAP). Restoring terminal differentiation and apoptosis of Caco-2 cells increased the mRNA levels of 5-LOX, 15-LOX-2, and 15-LOX-1, but the only significant increases in protein expression and enzymatic activity were of 15-LOX-1. In FAP patients, 15-LOX-1 expression and activity were significantly down-regulated in adenomas (compared with paired nonneoplastic epithelial mucosa), whereas 5-LOX and 15-LOX-2 protein expressions and enzymatic activities were not. We conducted a validation study with immunohistochemical testing in a second group of FAP patients; 15-LOX-1 expression was down-regulated in colorectal adenomas (compared with nonneoplastic epithelial mucosa) in 87% (13 of 15) of this group. We confirmed the mechanistic relevance of these findings by demonstrating that ectopically restoring 15-LOX-1 expression reestablished apoptosis in Caco-2 cells. Therefore, 15-LOX-1 down-regulation rather than a shift in the balance of LOXs is likely the dominant alteration in LOX metabolism which contributes to colorectal tumorigenesis by repressing apoptosis. (Cancer Res 2005; 65(24): 11486-92)
Expression of Endogenous OncogenicV600EB-rafInduces Proliferation and Developmental Defects in Mice and Transformation of Primary Fibroblasts
Authors: Mercer Kathryn , Giblett Susan , Green Stuart , Lloyd David , DaRocha Dias Silvy , Plumb Mark , Marais Richard , Pritchard Catrin
Published: December 2005

Mutations of the human B-RAF gene are detected in ∼8% of cancer samples, primarily in cutaneous melanomas (70%). The most common mutation (90%) is a valine-to-glutamic acid mutation at residue 600 (V600E; formerly V599E according to previous nomenclature). Using a Cre/Lox approach, we have generated a conditional knock-in allele of V600EB-raf in mice. We show that widespread expression of V600EB-Raf cannot be tolerated in embryonic development, with embryos dying ∼7.5 dpc. Directed expression of mutant V600EB-Raf to somatic tissues using the IFN-inducible Mx1-Cre mouse strain induces a proliferative disorder and bone marrow failure with evidence of nonlymphoid neoplasia of the histiocytic type leading to death within 4 weeks of age. However, expression of mutant B-Raf does not alter the proliferation profile of all somatic tissues. In primary mouse embryonic fibroblasts, expression of endogenous V600EB-Raf induces morphologic transformation, increased cell proliferation, and loss of contact inhibition. Thus, V600EB-Raf is able to induce several hallmarks of transformation in some primary mouse cells without evidence for the involvement of a cooperating oncogene or tumor suppressor gene. (Cancer Res 2005; 65(24): 11493-500)
Negative Regulation of TSC1-TSC2 by Mammalian D-Type Cyclins
Authors: Zacharek Sima J. , Xiong Yue , Shumway Stuart D.
Published: December 2005

The metazoan cell cycle is driven by the timely and composite activities of cyclin-dependent kinases (CDKs). Among these, cyclin D- and cyclin E-dependent kinases phosphorylate the pRb family proteins during G1 phase of the cell cycle and thereby advance cells beyond the restriction point. Increasing evidence suggests that cyclin D-dependent kinases might affect events other than Rb pathway-mediated entry into S phase, such as accumulation of cell mass. However, little is known about cyclin D activity toward Rb-independent pathway(s) or non-pRb substrates. In this article, we show that the tumor suppressor TSC2 is a cyclin D binding protein. Coexpression of cyclin D1-CDK4/6 in cultured cells leads to increased phosphorylation and decreased detection of both TSC2 and TSC1, and promotes the phosphorylation of the mTOR substrates, 4E-BP1 and S6K1, two key effectors of cell growth that are negatively regulated by the TSC1-TSC2 complex. At the cellular level, ectopic expression of cyclin D1 restores the cell size decrease caused by TSC1-TSC2 expression. Intriguingly, down-regulation of TSC proteins was also observed by the expression of a mutant cyclin D1 that is unable to bind to CDK4/6, or by the coexpression of cyclin D1 with either an INK4 inhibitor or with catalytically inactive CDK6, indicating that cyclin D may regulate TSC1-TSC2 independently of CDK4/6. Together, these observations suggest that mammalian D-type cyclins participate in cell growth control through negative regulation of TSC1-TSC2 function. (Cancer Res 2005; 65(24): 11354-60)
Headpin: A Serpin with Endogenous and Exogenous Suppression of Angiogenesis
Authors: Shellenberger Thomas D. , Mazumdar Abhijit , Henderson Ying , Briggs Katrina , Wang Mary , Chattopadhyay Chandrani , Jayakumar Arumugam , Frederick Mitchell , Clayman Gary L.
Published: December 2005

Headpin is a novel serine proteinase inhibitor (serpin) with constitutive mRNA expression in histologically normal oral mucosa but with lost or down-regulated expression in head and neck squamous cell carcinoma. Several serpin family members are similarly lost in multiple cancer types and hold tumor suppressor functions including the inhibition of angiogenesis. However, the functional significance for the loss of headpin expression in cancer is not known. Using immunohistochemical analysis of invasive squamous cell carcinoma and matched normal squamous mucosa of patient specimens, headpin expression was lost or down-regulated in the vast majority of tumor specimens. We investigated the functions of exogenous recombinant headpin and endogenously expressed headpin related to angiogenesis. In a rat corneal assay of neovascularization, recombinant headpin protein blocked in vivo angiogenesis mediated by interleukin 8 (IL-8) and vascular endothelial growth factor (VEGF). In assays of cellular events in angiogenesis, headpin blocked the invasion, migration, and tube formation of endothelial cells. In light of our findings of nuclear subcellular localization of headpin, we investigated the expression and secretion of angiogenic factors and found reduced mRNA, protein, and promoter activities of IL-8 and VEGF. Finally, using a murine flank tumor model, headpin expression reduced growth and microvessel density in tumors derived from headpin-expressing UMSCC1 cells relative to those from vector control cells. These findings of nuclear regulatory functions of a serpin in the inhibition of angiogenesis bring new understanding to the cellular and molecular mechanisms of serpins. Therefore, this novel serpin targets diverse mechanisms against tumor angiogenesis on which to base therapeutic strategies. (Cancer Res 2005; 65(24): 11501-9)