bluuebunny/crossref_metadata_2025 huggingface dataset:
Structure of the θ Subunit ofEscherichia coliDNA Polymerase III in Complex with the ε Subunit
Authors: Keniry Max A. , Park Ah Young , Owen Elisabeth A. , Hamdan Samir M. , Pintacuda Guido , Otting Gottfried , Dixon Nicholas E.
Published: June 2006

The catalytic core ofEscherichia coliDNA polymerase III contains three tightly associated subunits, the α, ε, and θ subunits. The θ subunit is the smallest and least understood subunit. The three-dimensional structure of θ in a complex with the unlabeled N-terminal domain of the ε subunit, ε186, was determined by multidimensional nuclear magnetic resonance spectroscopy. The structure was refined using pseudocontact shifts that resulted from inserting a lanthanide ion (Dy3+, Er3+, or Ho3+) at the active site of ε186. The structure determination revealed a three-helix bundle fold that is similar to the solution structures of θ in a methanol-water buffer and of the bacteriophage P1 homolog, HOT, in aqueous buffer. Conserved nuclear Overhauser enhancement (NOE) patterns obtained for free and complexed θ show that most of the structure changes little upon complex formation. Discrepancies with respect to a previously published structure of free θ (Keniry et al., Protein Sci.9:721-733, 2000) were attributed to errors in the latter structure. The present structure satisfies the pseudocontact shifts better than either the structure of θ in methanol-water buffer or the structure of HOT. satisfies these shifts. The epitope of ε186 on θ was mapped by NOE difference spectroscopy and was found to involve helix 1 and the C-terminal part of helix 3. The pseudocontact shifts indicated that the helices of θ are located about 15 Å or farther from the lanthanide ion in the active site of ε186, in agreement with the extensive biochemical data for the θ-ε system.
Spindle cell carcinoma of head and neck: an immunohistochemical and molecular approach to its pathogenesis
Authors: Gupta Ruchika , Singh Sompal , Hedau Suresh , Nigam Sonu , Das Bhudev C , Singh Ishwar , Mandal Ashish Kumar
Published: May 2006

Background:Spindle cell carcinoma (SpCC) is a rare microscopic type of cancer of the mouth and oropharynx. Although SpCC is thought to arise from squamous cell carcinoma (SCC), it carries a worse prognosis.

Aim:To find out the difference in immunohistochemical expression of cytokeratin, vimentin and smooth-muscle actin, and mutational alterations in the K-ras oncogene between the two tumours, in an attempt to characterise SpCC.

Methods:Immunohistochemical analysis was performed by standard avidin–biotin complex method in 35 cases each of SpCCs and SCCs. DNA extracted from paraffin wax-embedded tumours was used for PCR followed by single-strand conformation polymorphism for mutational analysis of K-ras exon 1 and exon 2.

Results:In the SpCC group, cytokeratin positivity was significantly higher in epithelial areas (52.2%) than in spindle cell areas (16.1%), whereas vimentin was more positive in spindle cell areas (18.7%) than epithelial areas (2.7%). Cells intermediate between epithelial and spindle cell areas were consistently positive for both cytokeratin and vimentin. Cytokeratin was found to be significantly more positive in SCC (72.6%) than the squamous component and spindle cell component of SpCC. In this study, no mutation was detected in the K-ras gene of either the SpCC or SCC group.

Conclusions:The spindle cell component of SpCC is intermixed with cells that are morphologically mesenchymal but express dual antigen-positivity characteristic of epithelial (cytokeratin) and mesenchymal (vimentin) cells. These, possibly, are cells in transition suggesting that SpCC may be a sarcomatous metaplasia of SCC.
NEUTRINO MASS AS A CONSEQUENCE OF THE EXACT SOLUTION OF 3-3-1 GAUGE MODELS WITHOUT EXOTIC ELECTRIC CHARGES
Authors: PALCU ADRIAN
Published: June 2006

The unjustly neglected method of exactly solving generalized electroweak models — with an original spontaneous symmetry breaking mechanism based on the gauge group SU (n)L⊗ U (1)Y — is applied here to a particular class of chiral 3-3-1 models. This procedure enables us, without resorting to any approximation, to express the boson mass spectrum and charges of the particles involved therein as a straightforward consequence of both a proper parametrization of the Higgs sector and a new generalized Weinberg transformation. We prove that the resulting values can accommodate the experimental ones just by tuning a sole parameter. Furthermore, if we take into consideration both the left-handed and right-handed components of the neutrino (included in a lepton triplet along with their corresponding left-handed charged partner), then we are in the position to propose an original method for the neutrino to acquire a very small but nonzero mass without spoiling the previously achieved results in the exact solution of the model. In order to be compatible with the existing phenomenological data, the range of that sole parameter imposes a large order of magnitude for the vev〈ϕ〉~104 TeV in our method. Consequently, the new bosons of the model have to be very massive.
Inhibition of in-stent restenosis by oral copper chelation in porcine coronary arteries
Authors: Mandinov L. , Moodie K. L. , Mandinova A. , Zhuang Z. , Redican F. , Baklanov D. , Lindner V. , Maciag T. , Simons M. , de Muinck E. D.
Published: May 2006

Stress-induced release of IL-1α and fibroblast growth factor-1 is dependent on intracellular copper and is a major driver of neointimal hyperplasia. Therefore, we assessed the effect of tetrathiomolybdate (TTM), a clinically proven copper chelator, on in-stent restenosis. Nine pigs were treated with TTM (5 mg/kg po) twice daily for 2 wk before stent implantation and for 4 wk thereafter, and nine pigs served as controls. In-stent restenosis was assessed by quantitative coronary angiography (QCA), intravascular ultrasound (IVUS), and histomorphometry. Serum ceruloplasmin activity was used as a surrogate marker of copper bioavailability. In TTM-treated animals, ceruloplasmin dropped 70 ± 10% below baseline levels. Baseline characteristics were comparable in TTM-treated and control animals. At 4-wk follow-up, all parameters relevant to in-stent restenosis were significantly reduced in TTM-treated animals: minimal lumen diameter by QCA was 2.03 ± 0.57 and 1.47 ± 0.45 mm in TTM-treated and control animals, respectively ( P < 0.05), percent stenosis diameter was 39% less in TTM-treated animals (27.1 ± 16.6% vs. 44.5 ± 16.1%, P < 0.05), minimal lumen area by IVUS was 60% larger in TTM-treated animals (4.27 ± 1.56 vs. 2.67 ± 1.19 mm2, P < 0.05), and neointimal volume by histomorphometry was 37% less in TTM-treated animals (34.9 ± 11.5 vs. 55.2 ± 19.6 mm3, P < 0.05). We conclude that systemic copper chelation with a clinically approved chelator significantly inhibits in-stent restenosis.
Plasma viscosity regulates systemic and microvascular perfusion during acute extreme anemic conditions
Authors: Cabrales Pedro , Tsai Amy G.
Published: May 2006

The hamster window chamber model was used to study systemic and microvascular hemodynamic responses to extreme hemodilution with low- and high-viscosity plasma expanders (LVPE and HVPE, respectively) to determine whether plasma viscosity is a factor in homeostasis during extreme anemic conditions. Moderated hemodilution was induced by two isovolemic steps performed with 6% 70-kDa dextran until systemic hematocrit (Hct) was reduced to 18% ( level 2). In a third isovolemic step, hemodilution with LVPE (6% 70-kDa dextran, 2.8 cP) or HVPE (6% 500-kDa dextran, 5.9 cP) reduced Hct to 11%. Systemic parameters, cardiac output (CO), organ flow distribution, microhemodynamics, and functional capillary density, were measured after each exchange dilution. Fluorescent-labeled microspheres were used to measure organ (brain, heart, kidney, liver, lung, and spleen) and window chamber blood flow. Final blood and plasma viscosities after the entire protocol were 2.1 and 1.4 cP, respectively, for LVPE and 2.8 and 2.2 cP, respectively, for HVPE (baseline = 4.2 and 1.2 cP, respectively). HVPE significantly elevated mean arterial pressure and CO compared with LVPE but did not increase vascular resistance. Functional capillary density was significantly higher for HVPE [87% (SD 7) of baseline] than for LVPE [42% (SD 11) of baseline]. Increases in mean arterial blood pressure, CO, and shear stress-mediated factors could be responsible for maintaining organ and microvascular perfusion after exchange with HVPE compared with LVPE. Microhemodynamic data corresponded to microsphere-measured perfusion data in vital organs.
Internalizing Agency of Self-Action: Perception of One's Own Hand Movements Depends on an Adaptable Prediction About the Sensory Action Outcome
Authors: Synofzik Matthis , Thier Peter , Lindner Axel
Published: June 2006

Extensive work on learning in reaching and pointing tasks has demonstrated high degrees of plasticity in our ability to optimize goal-directed motor behavior. However, studies focusing on the perceptual awareness of our own actions during motor adaptation are still rare. Here we present the first simultaneous investigation of sensorimotor adaptation on both levels, i.e., action and action perception. We hypothesized that self-action perception relies on internal predictions about the sensory action outcome that are updated in a way similar to that of motor control. Twenty human subjects performed out-and-back pointing movements that were fed back visually. Feedback was initially presented in spatiotemporal correspondence with respect to the actual finger position, but later rotated by a constant angle. When distorted feedback was applied repetitively, subjects' perceived pointing direction shifted in the direction of the trajectory rotation. A comparable perceptual reinterpretation was observed in control trials without visual feedback, indicating that subjects learned to predict the new visual outcome of their actions based on nonvisual, internal information. The perception of the world, however, remained unchanged. The changes in perception of one's own movements were accompanied by adaptive changes in motor performance of the same amount, i.e., a secondary motor compensation opposite to the direction of the imposed visual rotation. Our results show that the perception of one's own actions depends on adaptable internal predictions about the sensory action outcome, allowing us to attribute new sensory consequences of our actions to our own agency. Furthermore, they indicate that the updated sensory prediction can be used to optimize motor control.
Early Visuomotor Representations Revealed From Evoked Local Field Potentials in Motor and Premotor Cortical Areas
Authors: O'Leary John G. , Hatsopoulos Nicholas G.
Published: June 2006

Local field potentials (LFPs) recorded from primary motor cortex (MI) have been shown to be tuned to the direction of visually guided reaching movements, but MI LFPs have not been shown to be tuned to the direction of an upcoming movement during the delay period that precedes movement in an instructed-delay reaching task. Also, LFPs in dorsal premotor cortex (PMd) have not been investigated in this context. We therefore recorded LFPs from MI and PMd of monkeys ( Macaca mulatta) and investigated whether these LFPs were tuned to the direction of the upcoming movement during the delay period. In three frequency bands we identified LFP activity that was phase-locked to the onset of the instruction stimulus that specified the direction of the upcoming reach. The amplitude of this activity was often tuned to target direction with tuning widths that varied across different electrodes and frequency bands. Single-trial decoding of LFPs demonstrated that prediction of target direction from this activity was possible well before the actual movement is initiated. Decoding performance was significantly better in the slowest-frequency band compared with that in the other two higher-frequency bands. Although these results demonstrate that task-related information is available in the local field potentials, correlations among these signals recorded from a densely packed array of electrodes suggests that adequate decoding performance for neural prosthesis applications may be limited as the number of simultaneous electrode recordings is increased.
Cannabinoids Modulate the P-Type High-Voltage-Activated Calcium Currents in Purkinje Neurons
Authors: Fisyunov Alexander , Tsintsadze Vera , Min Rogier , Burnashev Nail , Lozovaya Natalia
Published: June 2006

Endocannabinoids released by postsynaptic cells inhibit neurotransmitter release in many central synapses by activating presynaptic cannabinoid CB1 receptors. In particular, in the cerebellum, endocannabinoids inhibit synaptic transmission at granule cell to Purkinje cell synapses by modulating presynaptic calcium influx via N-, P/Q-, and R-type calcium channels. Using whole cell patch-clamp techniques, we show that in addition to this presynaptic action, both synthetic and endogenous cannabinoids inhibit P-type calcium currents in isolated rat Purkinje neurons independent of CB1 receptor activation. The IC50for the anandamide (AEA)-induced inhibition of P-current peak amplitude was 1.04 ± 0.04 μM. In addition, we demonstrate that all the tested cannabinoids in a physiologically relevant range of concentrations strongly accelerate inactivation of P currents. The effects of AEA cannot be attributed to the metabolism of AEA because a nonhydrolyzing analogue of AEA, methanandamide inhibited P-type currents with a similar efficacy. All effects of cannabinoids on P-type Ca2+currents were insensitive to antagonists of CB1 cannabinoid or vanilloid TRPV1 receptors. In cerebellar slices, WIN 55,212–2 significantly affected spontaneous firing of Purkinje neurons in the presence of CB1 receptor antagonist, in a manner similar to that of a specific P-type channel antagonist, indicating a possible functional implication of the direct effects of cannabinoids on P current. Taken together these findings demonstrate a functionally important direct action of cannabinoids on P-type calcium currents.
Epidermal Growth Factor Receptor Signaling Is Up-regulated in Human Colonic Aberrant Crypt Foci
Authors: Cohen Greg , Mustafi Reba , Chumsangsri Anusara , Little Nathaniel , Nathanson Jeff , Cerda Sonia , Jagadeeswaran Sujatha , Dougherty Urszula , Joseph Loren , Hart John , Yerian Lisa , Tretiakova Maria , Yuan Weihua , Obara Piotr , Khare Sharad , Sinicrope Frank A. , Fichera Alessandro , Boss Gerry R. , Carroll Robert , Bissonnette Marc
Published: June 2006

Aberrant crypt foci (ACF) are collections of abnormal colonic crypts with heterogeneous molecular and pathologic characteristics. Large and dysplastic ACF are putative precursors of colon cancer with neoplastic risk related to increased proliferation. In this study, we examined the role of epidermal growth factor receptor (EGFR) signaling in regulating ACF proliferation. Using magnification chromoendoscopy, we collected large ACF with endoscopic features of dysplasia and separately biopsied adjacent mucosa. Transcript levels were measured by real-time PCR, proteins were assessed by Western blotting, and levels were expressed as fold changes of adjacent mucosa. K-ras and B-Raf mutations were assessed by PCR and Ras activation by the ratio Ras-GTP / (Ras-GTP + Ras-GDP). At the RNA level, 38% of ACF were hyperproliferative, with proliferating cell nuclear antigen (PCNA) mRNA ≥2-fold of adjacent mucosa. Hyperproliferative ACF had significantly increased mRNA levels of EGFR (6.0 ± 1.7–fold), transforming growth factor-α (14.4 ± 5.0–fold), heparin-binding EGF-like growth factor (4.5 ± 1.4–fold), cyclin D1 (4.6 ± 0.7–fold), and cyclooxygenase-2 (COX-2; 9.3 ± 4.2–fold; P < 0.05). At the protein level, 46% of ACF were hyperproliferative (PCNA, 3.2 ± 1.2–fold). In hyperproliferative ACF, 44% possessed significant increases in four EGFR signaling components: EGFR (9.5 ± 1.3–fold), phosphoactive ErbB2 (2.6 ± 0.4–fold), phosphoactive extracellular signal-regulated kinase (3.7 ± 1.1–fold), and cyclin D1 (3.4 ± 0.8–fold; P < 0.05). Ras was activated in 46% of ACF (3.2 ± 0.4–fold; P < 0.05), but K-ras mutations were present in only 7% of ACF. In contrast to COX-2 mRNA, the protein was not increased in hyperproliferative ACF. In summary, we have shown that ACF with up-regulated PCNA possess increased EGFR signaling components that likely contribute to the enhanced proliferative state of dysplastic-appearing ACF. (Cancer Res 2006; 66(11): 5656-64)
SV40 T/t-Common Polypeptide Specifically Induces Apoptosis in Human Cancer Cells that Overexpress HER2/neu
Authors: Wen Chun-Chiang , Cheng Shih-An , Hsuen Shu-Ping , Huang Ya-Ling , Kuo Zong-Keng , Lee Hsin-Fang , Kuo Chou-Hua , Du Jia-Ling , Wang Won-Bo
Published: June 2006

Previously, we reported that SV40 T/t-common polypeptide, which contains the NH2-terminal common domain of SV40 large T and small t antigens, can repress HER2/neu (also known as erbB-2) expression and consequently suppress the tumorigenic potential of the HER2/neu-overexpressing ovarian carcinoma cells. Here we report that T/t-common could specifically induce apoptosis in HER2/neu-overexpressing human cancer cell lines but not in nontransformed cell lines and HER2/neu low-expressing human cancer cell lines. The ability of T/t-common to induce apoptosis in HER2/neu-overexpressing cancer cells was derived from its ability to inhibit HER2/neu because reexpression of a large amount of HER2/neu could block apoptosis induced by T/t-common. T/t-common expression in HER2/neu-overexpressing SK-OV-3 cancer cells led to down-regulation of Bcl-2 and Bcl-XL, and overexpression of Bcl-2 could inhibit the ability of T/t-common to induce apoptosis in these cells. Therefore, the apoptosis-inducing activity of T/t-common is related to its ability to inhibit Bcl-2 expression in HER2/neu-overexpressing cancer cells. Consistent with the apoptosis-inducing activity of T/t-common, we found that T/t-common could specifically inhibit the soft-agarose colony-forming ability of the HER2/neu-overexpressing human cancer cell lines but not that of the HER2/neu low-expressing human cancer cell lines. Finally, we showed that T/t-common could specifically sensitize HER2/neu-overexpressing human cancer cell lines, but not HER2/neu low-expressing human cancer cell lines, to chemotherapeutic agent etoposide. Together, these data suggest that T/t-common alone or in combination with chemotherapy may provide a new approach for treatment of cancers that overexpress HER2/neu. (Cancer Res 2006; 66(11): 5847-57)
BCR-ABL Is Not an Immunodominant Antigen in Chronic Myelogenous Leukemia
Authors: Grünebach Frank , Mirakaj Valbona , Mirakaj Valdete , Müller Martin R. , Brümmendorf Tim , Brossart Peter
Published: June 2006

In the present study, we analyzed the involvement of the BCR-ABL protein in the induction of antigen-specific CTL in order to develop an immunotherapeutic approach in patients with chronic myelogenous leukemia (CML). To accomplish this, we generated dendritic cells (DC) in vitro and electroporated them with various sources of RNA harboring the chimeric bcr-abl transcript. These genetically engineered DCs were used as antigen-presenting cells for the induction of CTLs. By applying this approach, we found that the CTLs induced by DCs transfected with RNA extracted from bcr-abl–positive K-562 cells or CML blasts lysed DCs transfected with the corresponding RNA, but failed to recognize epitopes derived from the chimeric BCR-ABL fusion protein in 51Cr-release assays. In contrast, they were able to lyse autologous DCs electroporated with RNA isolated from patients with acute myeloid leukemia, indicating that antigens shared among these malignant cells are involved and recognized by these CTLs. In patients with CML in complete cytogenetic remission during IFN-α treatment, we detected some reactivity of CD8+ T cells against BCR-ABL in IFN-γ ELISPOT assays, which was weaker as compared with proteinase 3 (PR3)- or prame-directed responses, suggesting that the BCR-ABL protein is less immunogenic as compared with other CML-derived antigens. (Cancer Res 2006; 66(11): 5892-900)
Elevated Expression of Mitogen-Activated Protein Kinase Phosphatase 3 in Breast Tumors: A Mechanism of Tamoxifen Resistance
Authors: Cui Yukun , Parra Irma , Zhang Mao , Hilsenbeck Susan G. , Tsimelzon Anna , Furukawa Toru , Horii Akira , Zhang Zhong-Yin , Nicholson Robert I. , Fuqua Suzanne A.W.
Published: June 2006

Antiestrogen resistance is a major clinical problem in the treatment of breast cancer. Altered growth factor signaling with estrogen receptor (ER)-α is associated with the development of resistance. Gene expression profiling was used to identify mitogen-activated protein kinase (MAPK) phosphatase 3 (MKP3) whose expression was correlated with response to the antiestrogen tamoxifen in both patients and in vitro–derived cell line models. Overexpression of MKP3 rendered ER-α-positive breast cancer cells resistant to the growth-inhibitory effects of tamoxifen and enhanced tamoxifen agonist activity in endometrial cells. MKP3 overexpression was associated with lower levels of activated extracellular signal-regulated kinase 1/2 (ERK1/2) phosphorylation in the presence of estrogen but that estrogen deprivation and tamoxifen treatment decreased MKP3 phosphatase activity, leading to an up-regulation of pERK1/2 MAPK, phosphorylated Ser118-ER-α, and cyclin D1. The MAPK/ERK kinase inhibitor PD98059 blocked tamoxifen-resistant growth. Accumulation of reactive oxygen species was observed with tamoxifen treatment of MKP3-overexpressing cells, and antioxidant treatment increased MKP3 phosphatase activity, thereby blocking resistance. Furthermore, PD98059 increased the levels of phosphorylated c-Jun NH2-terminal kinase (JNK) in tamoxifen-treated MKP3-overexpressing cells, suggesting an interaction between MKP3 levels, activation of ERK1/2 MAPK, and JNK signaling in human breast cancer cells. MKP3 represents a novel mechanism of resistance, which may be a potential biomarker for the use of ERK1/2 and/or JNK inhibitors in combination with tamoxifen treatment. (Cancer Res 2006; 66(11): 5950-9)
Oncoprotein EWS-FLI1 Activity Is Enhanced by RNA Helicase A
Authors: Toretsky Jeffrey A. , Erkizan Verda , Levenson Amy , Abaan Ogan D. , Parvin Jeffrey D. , Cripe Timothy P. , Rice Anna M. , Lee Sean Bong , Üren Aykut
Published: June 2006

RNA helicase A (RHA), a member of the DEXH box helicase family of proteins, is an integral component of protein complexes that regulate transcription and splicing. The EWS-FLI1 oncoprotein is expressed as a result of the chromosomal translocation t(11;22) that occurs in patients with the Ewing's sarcoma family of tumors (ESFT). Using phage display library screening, we identified an EWS-FLI1 binding peptide containing homology to RHA. ESFT cell lines and patient tumors highly expressed RHA. GST pull-down and ELISA assays showed that EWS-FLI1 specifically bound RHA fragment amino acids 630 to 1020, which contains the peptide region discovered by phage display. Endogenous RHA was identified in a protein complex with EWS-FLI1 in ESFT cell lines. Chromatin immunoprecipitation experiments showed both EWS-FLI1 and RHA bound to EWS-FLI1 target gene promoters. RHA stimulated the transcriptional activity of EWS-FLI1 regulated promoters, including Id2, in ESFT cells. In addition, RHA expression in mouse embryonic fibroblast cells stably transfected with EWS-FLI1 enhanced the anchorage-independent phenotype above that with EWS-FLI1 alone. These results suggest that RHA interacts with EWS-FLI1 as a transcriptional cofactor to enhance its function. (Cancer Res 2006; 66(11): 5574-81)
Cardiac Glycosides Initiate Apo2L/TRAIL-Induced Apoptosis in Non–Small Cell Lung Cancer Cells by Up-regulation of Death Receptors 4 and 5
Authors: Frese Steffen , Frese-Schaper Manuela , Andres Anne-Catherine , Miescher Daniela , Zumkehr Beatrice , Schmid Ralph A.
Published: June 2006

Tumor necrosis factor (TNF)–related apoptosis-inducing ligand (Apo2L/TRAIL) belongs to the TNF family known to transduce their death signals via cell membrane receptors. Because it has been shown that Apo2L/TRAIL induces apoptosis in tumor cells without or little toxicity to normal cells, this cytokine became of special interest for cancer research. Unfortunately, cancer cells are often resistant to Apo2L/TRAIL-induced apoptosis; however, this can be at least partially negotiated by parallel treatment with other substances, such as chemotherapeutic agents. Here, we report that cardiac glycosides, which have been used for the treatment of cardiac failure for many years, sensitize lung cancer cells but not normal human peripheral blood mononuclear cells to Apo2L/TRAIL-induced apoptosis. Sensitization to Apo2L/TRAIL mediated by cardiac glycosides was accompanied by up-regulation of death receptors 4 (DR4) and 5 (DR5) on both RNA and protein levels. The use of small interfering RNA revealed that up-regulation of death receptors is essential for the demonstrated augmentation of apoptosis. Blocking of up-regulation of DR4 and DR5 alone significantly reduced cell death after combined treatment with cardiac glycosides and Apo2L/TRAIL. Combined silencing of DR4 and DR5 abrogated the ability of cardiac glycosides and Apo2L/TRAIL to induce apoptosis in an additive manner. To our knowledge, this is the first demonstration that glycosides up-regulate DR4 and DR5, thereby reverting the resistance of lung cancer cells to Apo2/TRAIL-induced apoptosis. Our data suggest that the combination of Apo2L/TRAIL and cardiac glycosides may be a new interesting anticancer treatment strategy. (Cancer Res 2006; 66(11): 5867-74)
Deletion of the COOH-Terminal Domain of CXC Chemokine Receptor 4 Leads to the Down-regulation of Cell-to-Cell Contact, Enhanced Motility and Proliferation in Breast Carcinoma Cells
Authors: Ueda Yukiko , Neel Nicole F. , Schutyser Evemie , Raman Dayanidhi , Richmond Ann
Published: June 2006

The CXC chemokine receptor 4 (CXCR4) contributes to the metastasis of human breast cancer cells. The CXCR4 COOH-terminal domain (CTD) seems to play a major role in regulating receptor desensitization and down-regulation. We expressed either wild-type CXCR4 (CXCR4-WT) or CTD-truncated CXCR4 (CXCR4-ΔCTD) in MCF-7 human mammary carcinoma cells to determine whether the CTD is involved in CXCR4-modulated proliferation of mammary carcinoma cells. CXCR4-WT-transduced MCF-7 cells (MCF-7/CXCR4-WT cells) do not differ from vector-transduced MCF-7 control cells in morphology or growth rate. However, CXCR4-ΔCTD-transduced MCF-7 cells (MCF-7/CXCR4-ΔCTD cells) exhibit a higher growth rate and altered morphology, potentially indicating an epithelial-to-mesenchymal transition. Furthermore, extracellular signal-regulated kinase (ERK) activation and cell motility are increased in these cells. Ligand induces receptor association with β-arrestin for both CXCR4-WT and CXCR4-ΔCTD in these MCF-7 cells. Overexpressed CXCR4-WT localizes predominantly to the cell surface in unstimulated cells, whereas a significant portion of overexpressed CXCR4-ΔCTD resides intracellularly in recycling endosomes. Analysis with human oligomicroarray, Western blot, and immunohistochemistry showed that E-cadherin and Zonula occludens are down-regulated in MCF-7/CXCR4-ΔCTD cells. The array analysis also indicates that mesenchymal marker proteins and certain growth factor receptors are up-regulated in MCF-7/CXCR4-ΔCTD cells. These observations suggest that (a) the overexpression of CXCR4-ΔCTD leads to a gain-of-function of CXCR4-mediated signaling and (b) the CTD of CXCR4-WT may perform a feedback repressor function in this signaling pathway. These data will contribute to our understanding of how CXCR4-ΔCTD may promote progression of breast tumors to metastatic lesions. (Cancer Res 2006; 66(11): 5665-75)
A Critical Role for the Inflammatory Response in a Mouse Model of Preneoplastic Progression
Authors: Schwertfeger Kathryn L. , Xian Wa , Kaplan Alan M. , Burnett Sandra H. , Cohen Donald A. , Rosen Jeffrey M.
Published: June 2006

The tumor microenvironment, which includes inflammatory cells, vasculature, extracellular matrix, and fibroblasts, is a critical mediator of neoplastic progression and metastasis. Using an inducible transgenic mouse model of preneoplastic progression in the mammary gland, we discovered that activation of inducible fibroblast growth factor receptor-1 (iFGFR1) in the mammary epithelium rapidly increased the expression of several genes involved in the inflammatory response. Further analysis revealed that iFGFR1 activation induced recruitment of macrophages to the epithelium and continued association with the alveolar hyperplasias that developed following long-term activation. Studies using HC-11 mammary epithelial cells showed that iFGFR1-induced expression of the macrophage chemoattractant osteopontin was required for macrophage recruitment in vitro. Finally, conditional depletion of macrophages inhibited iFGFR1-mediated epithelial cell proliferation and lateral budding. These findings show that inflammatory cells, specifically macrophages, are critical for mediating early events in an inducible transgenic mouse model of preneoplastic progression. (Cancer Res 2006; 66(11): 5676-85)
A New Anticancer Glycolipid Monoclonal Antibody, SC104, which Directly Induces Tumor Cell Apoptosis
Authors: Durrant Lindy G. , Harding Stephen J. , Green Nicola H. , Buckberry Lorraine D. , Parsons Tina
Published: June 2006

A novel monoclonal antibody was raised by immunization of mice with colorectal tumor cell lines. The fusion was screened by immunohistochemistry for binding to primary colorectal tumors. Subsequent analysis on primary disaggregated colorectal tumors show that the antibody recognizes a cell surface antigen expressed by the majority of colorectal tumors. Antigen characterization has shown that the antibody recognizes a sialyltetraosylceramide but does not bind to GM1, GD1a, GT1b, or sialyl LewisX antigens. Binding to a frozen panel of tumor and normal tissue sections revealed that the antigen was also strongly expressed on esophageal, gastric, and endometrial tumors. Its normal tissue distribution was largely restricted to moderate staining of large intestine. Surprisingly, SC104 antibody directly induces tumor cell death without the need for immune effector cells or complement. This may be related in part to its homophilic binding properties that allow cross-linking of antibody and receptors on the cell surface. Caspase activation can be detected following SC104 treatment of colorectal cells, and cotreatment with caspase inhibitors has been shown to inhibit cell death. This suggests that SC104 induces death by a classic apoptotic pathway. Furthermore, SC104 antibody shows additive killing with complement and 5-fluorouracil/leucovorin in vivo, suggesting a new therapeutic approach for this class of antibodies. (Cancer Res 2006; 66(11): 5901-9)
D4-GDI, a Rho GTPase Regulator, Promotes Breast Cancer Cell Invasiveness
Authors: Zhang Yaqin , Zhang Baolin
Published: June 2006

D4-GDI is a Rho GDP dissociation inhibitor that is widely expressed in hematopoietic cells. Its possible expression and function in breast cancer cells has not been described. Here, we found that D4-GDI is expressed in a panel of breast cancer cell lines, but not in benign-derived mammary epithelial cells. Knockdown of D4-GDI expression in MDA-MB-231 cells by RNA interference blocks cell motility and invasion. The cells lacking D4-GDI grown on Matrigel revert to a normal breast epithelial phenotype characterized by the formation of cavitary structures. Silencing D4-GDI expression inhibits β1-integrin expression and cell-matrix adhesion. Reintroduction of D4-GDI fully restored both β1-integrin expression and cellular invasion. Knockdown of D4-GDI in BT549 cells results in a similar effect. These results show that D4-GDI modulates breast cancer cell invasive activities. (Cancer Res 2006; 66(11): 5592-8)
SSeCKS Metastasis-Suppressing Activity in MatLyLu Prostate Cancer Cells Correlates with Vascular Endothelial Growth Factor Inhibition
Authors: Su Bing , Zheng Qiao , Vaughan Mary M. , Bu Yahao , Gelman Irwin H.
Published: June 2006

SSeCKS, a Src-suppressed protein kinase C substrate with metastasis suppressor activity, is the rodent orthologue of human gravin/AKAP12, a scaffolding protein for protein kinase A and protein kinase C. We show here that the tetracycline-regulated reexpression of SSeCKS in MatLyLu (MLL) prostate cancer cells suppressed formation of macroscopic lung metastases in both spontaneous and experimental models of in vivo metastasis while having minimal inhibitory effects on the growth of primary-site s.c. tumors. SSeCKS decreased angiogenesis in vitro and in vivo by suppressing vascular endothelial growth factor (VEGF) expression in MLL tumor cells as well as in stromal cells. The forced reexpression of VEGF165 and VEGF121 isoforms was sufficient to reverse aspects of SSeCKS metastasis-suppressor activity in both the experimental and spontaneous models. SSeCKS reexpression in MLL cells resulted in the down-regulation of proangiogenic genes, such as osteopontin, tenascin C, KGF, angiopoietin, HIF-1α, and PDGFRβ, and the up-regulation of antiangiogenic genes, such as vasostatin and collagen 18a1, a precursor of endostatin. These results suggest that SSeCKS suppresses formation of metastatic lesions by inhibiting VEGF expression and by inducing soluble antiangiogenic factors. (Cancer Res 2006; 66(11): 5599-607)
Tumor-Driven Evolution of Immunosuppressive Networks during Malignant Progression
Authors: Kim Ryungsa , Emi Manabu , Tanabe Kazuaki , Arihiro Koji
Published: June 2006

Tumors evolve mechanisms to escape immune control by a process called immune editing, which provides a selective pressure in the tumor microenvironment that could lead to malignant progression. A variety of tumor-derived factors contribute to the emergence of complex local and regional immunosuppressive networks, including vascular endothelial growth factor, interleukin-10, transforming growth factor-β, prostaglandin E2, and soluble phosphatidylserine, soluble Fas, soluble Fas ligand, and soluble MHC class I–related chain A proteins. Although deposited at the primary tumor site, these secreted factors could extend immunosuppressive effects into the local lymph nodes and the spleen, promoting invasion and metastasis. Vascular endothelial growth factors play a key role in recruiting immature myeloid cells from the bone marrow to enrich the microenvironment as tumor-associated immature dendritic cells and tumor-associated macrophages. The understanding of the immunosuppressive networks that evolve is incomplete, but several features are emerging. Accumulation of tumor-associated immature dendritic cells may cause roving dendritic cells and T cells to become suppressed by the activation of indoleamine 2,3-dioxygenase and arginase I by tumor-derived growth factors. Soluble phosphatidylserines support tumor-associated macrophages by stimulating the release of anti-inflammatory mediators that block antitumor immune responses. Soluble Fas, soluble FasL, and soluble MHC class I–related chain A proteins may help tumor cells escape cytolysis by cytotoxic T cells and natural killer cells, possibly by counterattacking immune cells and causing their death. In summary, tumor-derived factors drive the evolution of an immunosuppressive network which ultimately extends immune evasion from the primary tumor site to peripheral sites in patients with cancer. (Cancer Res 2006; 66(11): 5527-36)
COOH-Terminal Src Kinase–Mediated c-Jun Phosphorylation Promotes c-Jun Degradation and Inhibits Cell Transformation
Authors: Zhu Feng , Choi Bu Young , Ma Wei-Ya , Zhao Zhongliang , Zhang Yiguo , Cho Yong Yeon , Choi Hong Seok , Imamoto Akira , Bode Ann M. , Dong Zigang
Published: June 2006

The oncoprotein c-Jun is a component of the activator protein-1 transcription factor complex, which is involved in cellular proliferation, transformation, and death. The stabilization of c-Jun is critically important for its function. The phosphorylation of c-Jun by c-Jun NH2-terminal kinase 1 and extracellular signal-regulated protein kinases reduces c-Jun ubiquitination resulting in increased stabilization of c-Jun. In this report, we showed that COOH-terminal Src kinase (CSK) binds with and phosphorylates c-Jun at Y26 and Y170. Phosphorylation of c-Jun by CSK, in opposition to c-Jun NH2-terminal kinase 1 and extracellular signal-regulated protein kinases, promoted c-Jun degradation and reduced stability. By promoting c-Jun degradation, CSK helps to maintain a low steady-state level of c-Jun, thereby inhibiting activator protein-1 activity and cell transformation caused by c-Jun. These results indicated that this function of CSK controls cell proliferation under normal growth conditions and may have implications for CSK loss of function in carcinogenesis. (Cancer Res 2006; 66(11): 5729-36)
Dasatinib (BMS-354825) Selectively Induces Apoptosis in Lung Cancer Cells Dependent on Epidermal Growth Factor Receptor Signaling for Survival
Authors: Song Lanxi , Morris Mark , Bagui Tapan , Lee Francis Y. , Jove Richard , Haura Eric B.
Published: June 2006

Mutations of the epidermal growth factor receptor (EGFR) selectively activate Akt and signal transducer and activator of transcription (STAT) pathways that are important in lung cancer cell survival. Src family kinases can cooperate with receptor tyrosine kinases to signal through downstream molecules, such as phosphatidylinositol 3-kinase/PTEN/Akt and STATs. Based on the importance of EGFR signaling in lung cancer, the known cooperation between EGFR and Src proteins, and evidence of elevated Src activity in human lung cancers, we evaluated the effectiveness of a novel orally bioavailable Src inhibitor dasatinib (BMS-324825) in lung cancer cell lines with defined EGFR status. Here, we show that cell fate (death versus growth arrest) in lung cancer cells exposed to dasatinib is dependent on EGFR status. In cells with EGFR mutation that are dependent on EGFR for survival, dasatinib reduces cell viability through the induction of apoptosis while having minimal apoptotic effect on cell lines with wild-type (WT) EGFR. The induction of apoptosis in these EGFR-mutant cell lines corresponds to down-regulation of activated Akt and STAT3 survival proteins. In cell lines with WT or resistant EGFR mutation that are not sensitive to EGFR inhibition, dasatinib induces a G1 cell cycle arrest with associated changes in cyclin D and p27 proteins, inhibits activated FAK, and prevents tumor cell invasion. Our results show that dasatinib could be effective therapy for patients with lung cancers through disruption of cell growth, survival, and tumor invasion. Our results suggest EGFR status is important in deciding cell fate in response to dasatinib. (Cancer Res 2006; 66(11): 5542-8)
Targeted Deletion of MKK4 in Cancer Cells: A Detrimental Phenotype Manifests as Decreased Experimental Metastasis and Suggests a Counterweight to the Evolution of Tumor-Suppressor Loss
Authors: Cunningham Steven C. , Gallmeier Eike , Hucl Tomas , Dezentje David A. , Calhoun Eric S. , Falco Geppino , Abdelmohsen Kotb , Gorospe Myriam , Kern Scott E.
Published: June 2006

Tumor-suppressors have commanded attention due to the selection for their inactivating mutations in human tumors. However, relatively little is understood about the inverse, namely, that tumors do not select for a large proportion of seemingly favorable mutations in tumor-suppressor genes. This could be explained by a detrimental phenotype accruing in a cell type–specific manner to most cells experiencing a biallelic loss. For example, MKK4, a tumor suppressor gene distinguished by a remarkably consistent mutational rate across diverse tumor types and an unusually high rate of loss of heterozygosity, has the surprisingly low rate of genetic inactivation of only ∼5%. To explore this incongruity, we engineered a somatic gene knockout of MKK4 in human cancer cells. Although the null cells resembled the wild-type cells regarding in vitro viability and proliferation in plastic dishes, there was a marked difference in a more relevant in vivo model of experimental metastasis and tumorigenesis. MKK4−/− clones injected i.v. produced fewer lung metastases than syngeneic MKK4-competent cells (P = 0.0034). These findings show how cell type-specific detrimental phenotypes can offer a paradoxical and yet key counterweight to the selective advantage attained by cells as they experiment with genetic null states during tumorigenesis, the resultant balance then determining the observed biallelic mutation rate for a given tumor-suppressor gene. (Cancer Res 2006; 66(11): 5560-4)
Importance of the Stress Kinase p38α in Mediating the Direct Cytotoxic Effects of the Thalidomide Analogue, CPS49, in Cancer Cells and Endothelial Cells
Authors: Warfel Noel A. , Lepper Erin R. , Zhang Chunyu , Figg William D. , Dennis Phillip A.
Published: June 2006

Purpose: Thalidomide has gained renewed interest as a cancer therapeutic due to its potential antiangiogenic effects. The thalidomide analogues CPS11 and CPS49 are active in preclinical angiogenesis assays and xenograft model systems, but the biochemical basis for these observations is unclear.

Experimental Design: To address this question, we assessed the toxicity of these thalidomide analogues in cancer cells, endothelial cells, and genetically modified cells using assays that measure apoptotic and nonapoptotic cell death. Phosphospecific and native antibodies were used in immunoblotting and immunohistochemical experiments to assess the activation states of kinases that control cellular survival in vitro and in vivo.

Results: CPS49 predominantly induced nonapoptotic cell death in lung cancer cells, prostate cancer cells, and endothelial cells in a dose-dependent manner, whereas CPS11 was not cytotoxic. CPS49 did not inhibit kinases that promote survival, such as Akt or extracellular signal-regulated kinase, but rather rapidly activated the stress kinase p38 pathway in both cancer cells and endothelial cells. CPS49 activated p38 in tumor xenografts. Using p38α−/− cells or an inhibitor of p38, we show that the presence and activation of p38α is important for cytotoxicity in all cell types examined.

Conclusions: Our studies identify a unifying mechanism of action for cytotoxicity of the tetraflourinated thalidomide analogue, CPS49, and suggest that activation of p38 could serve as a biomarker in clinical trials with CPS49.
ABCB1 Modulation Does Not Circumvent Drug Extrusion from Primitive Leukemic Progenitor Cells and May Preferentially Target Residual Normal Cells in Acute Myelogenous Leukemia
Authors: Raaijmakers Marc H.G.P. , de Grouw Elke P.L.M. , van der Reijden Bert A. , de Witte Theo J.M. , Jansen Joop H. , Raymakers Reinier A.P.
Published: June 2006

Purpose: Acute myelogenous leukemia (AML) is a disease originating from normal hematopoietic CD34+CD38− progenitor cells. Modulation of the multidrug ATP-binding cassette transporter ABCB1 has not resulted in improved outcome in AML, raising the question whether leukemic CD34+CD38− cells are targeted by this strategy.

Experimental Design: ABCB1-mediated transport in leukemic CD34+CD38− cells compared with their normal counterparts was assessed by quantitating the effect of specific ABCB1 modulators (verapamil and PSC-833) on mitoxantrone retention [defined as efflux index (EI), intracellular mitoxantrone fluorescence intensity in the presence/absence of inhibitor].

Results: ABCB1 was the major drug transporter in CD34+CD38− cells in normal bone marrow (n = 16), as shown by the abrogation of mitoxantrone extrusion by ABCB1 modulators (EI, 1.99 ± 0.08). Surprisingly, ABCB1-mediated drug extrusion was invariably reduced in CD34+CD38− cells in AML (n = 15; EI, 1.21 ± 0.05; P < 0.001), which resulted in increased intracellular mitoxantrone retention in these cells (mitoxantrone fluorescence intensity, 4.54 ± 0.46 versus 3.08 ± 0.23; P = 0.004). Active drug extrusion from these cells occurred in the presence of ABCB1 modulators in the majority of samples, pointing in the direction of redundant drug extrusion mechanisms. Residual normal CD34+CD38− cells could be identified by their conserved ABCB1-mediated extrusion capacity.

Conclusion: ABCB1-mediated drug extrusion is reduced in leukemic CD34+CD38− progenitor cells compared with their residual normal counterparts. Redundant drug transport mechanisms confer mitoxantrone transport from leukemic progenitors. These data argue that ABCB1 modulation is not an effective strategy to circumvent drug extrusion from primitive leukemic progenitor cells and may preferentially target residual normal progenitors in AML.
Diagnosis of Suspicious Thyroid Nodules Using Four Protein Biomarkers
Authors: Cerutti Janete M. , Latini Flavia R.M. , Nakabashi Claudia , Delcelo Rosana , Andrade Victor P. , Amadei Marcelo João , Maciel Rui M.B. , Hojaij Flavio C. , Hollis Donna , Shoemaker Jennifer , Riggins Gregory J.
Published: June 2006

Purpose: Fine-needle aspiration (FNA) cytology, a standard method for thyroid nodule diagnosis, cannot distinguish between benign follicular thyroid adenoma (FTA) and malignant follicular thyroid carcinoma (FTC). Previously, using expression profiling, we found that a combination of transcript expression levels from DDIT3, ARG2, C1orf24, and ITM1 distinguished between FTA and FTC. The goal of this study was to determine if antibody markers used alone or in combination could accurately distinguish between a wider variety of benign and malignant thyroid lesions in fixed sections and FNA samples.

Experimental Design: Immunohistochemistry was done on 27 FTA, 25 FTC, and 75 other benign and malignant thyroid tissue sections using custom antibodies for chromosome 1 open reading frame 24 (C1orf24) and integral membrane protein 1 (ITM1) and commercial antibodies for DNA damage–inducible transcript 3 (DDIT3) and arginase II (ARG2). FNA samples were also tested using the same antibodies. RNA expression was measured by quantitative PCR in 33 thyroid lesions.

Results: C1orf24 and ITM1 antibodies had an estimated sensitivity of 1.00 for distinguishing FTA from FTC. For the expanded analysis of all lesions studied, ITM1 had an estimated sensitivity of 1.00 for detecting malignancy. Because all four cancer biomarkers did well, producing overlapping confidence intervals, not one best marker was distinguished. Transcript levels also reliably predicted malignancy, but immunohistochemistry had a higher sensitivity. Malignant cells were easily detected in FNA samples using these markers.

Conclusions: We improved this diagnostic test by adding C1orf24 and ITM1 custom antibodies and showing use on a wider variety of thyroid pathology. We recommend that testing of all four cancer biomarkers now be advanced to larger trials. Use of one or more of these antibodies should improve diagnostic accuracy of suspicious thyroid nodules from both tissue sections and FNA samples.