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  • 1
    Keywords: brain ; CELLS ; EXPRESSION ; SURVIVAL ; tumor ; TUMOR-CELLS ; BLOOD ; CELL ; Germany ; THERAPY ; DISEASE ; LONG-TERM ; TUMORS ; TIME ; PATIENT ; INFECTION ; prognosis ; SKIN ; T cell ; T cells ; T-CELL ; T-CELLS ; cell culture ; culture ; MEMORY ; virus ; NERVOUS-SYSTEM ; NO ; immunohistochemistry ; ASSAY ; NUMBER ; VACCINE ; SAFETY ; CD8(+) ; immune response ; IMMUNE-RESPONSE ; IMMUNITY ; T-LYMPHOCYTES ; vaccination ; T lymphocyte ; side effects ; NEWCASTLE-DISEASE VIRUS ; ESTABLISHMENT ; TUMOR CELLS ; LONG-TERM SURVIVORS ; GLIOMAS ; T lymphocytes ; IMMUNIZATION ; ONCOLOGY ; AUTOLOGOUS TUMOR ; overall survival ; NEWCASTLE-DISEASE-VIRUS ; SURVIVORS
    Abstract: Purpose Prognosis of patients with glioblastoma is poor. Therefore, in glioblastoma patients, we analyzed whether antitumor vaccination with a virus-modified autologous tumor cell vaccine is feasible and safe. Also, we determined the influence on progression-free survival and overall survival and on vaccination-induced antitumor reactivity. Patients and Methods In a nonrandomized study, 23 patients were vaccinated and compared with nonvaccinated controls (n = 87). Vaccine was prepared from patient's tumor cell cultures by infection of the cells with Newcastle Disease Virus, followed by gamma-irradiation, and applied up to eight times. Antitumor immune reactivity was determined in skin, blood, and relapsed tumor by delayed-type hypersensitivity skin reaction, ELISPOT assay, and immunohistochemistry, respectively. Results Establishment of tumor cell cultures was successful in approximately 90% of patients. After vaccination, we observed no severe side effects. The median progression-free survival of vaccinated patients was 40 weeks (v 26 weeks in controls; log-rank test, P =.024), and the median overall survival of vaccinated patients was 100 weeks (v 49 weeks in controls; log-rank test, P 〈.001). Forty-five percent of the controls survived 1 year, 11% survived 2 years, and there were no long-term survivors (greater than or equal to3 years). Ninety-one percent of vaccinated 39% survived 2 years, and 4% were long-term survivors. In the patients survived I year, vaccinated group, immune monitoring revealed significant increases of delayed-type hypersensitivity reactivity, numbers of tumor-reactive memory T cells, and numbers of CD8(+) tumor-infiltrating T-lymphocytes in secondary tumors. Conclusion Postoperative vaccination with virus-modified autologous tumor cells seems to be feasible and safe and to improve the prognosis of patients with gliobiastomas. This could be substantiated by the observed antitumor immune response. (C) 2004 by American Society of Clinical Oncology
    Type of Publication: Journal article published
    PubMed ID: 15452186
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  • 2
    Keywords: CANCER ; CELLS ; EXPRESSION ; tumor ; TUMOR-CELLS ; carcinoma ; CELL ; Germany ; neoplasms ; DIAGNOSIS ; NEW-YORK ; microarray ; transcription ; TISSUE ; DNA ; MICROARRAY DATA ; MUTATIONS ; Jun ; PHENOTYPE ; vimentin ; HEAD ; adenocarcinoma ; pathology ; BEHAVIOR ; MICROARRAY ANALYSIS ; expression profiling ; TUMOR CELLS ; DIFFERENTIAL-DIAGNOSIS ; CELL CARCINOMA ; OF-THE-LITERATURE ; pancreas ; review ; DUCTAL ADENOCARCINOMA ; AUTOPSY ; analysis ; pancreatic ; TUMOR-CELL ; GENOTYPE ; DNA-MICROARRAY ; USA ; pancreatic ductal adenocarcinoma ; MYOEPITHELIAL CARCINOMA ; pancreatic neoplasm ; PSEUDOPAPILLARY TUMORS
    Abstract: Pancreatic neoplasms have been reliably classified on the basis of their histopathology and immunophenotype. In this study, we report on a pancreatic tumor whose phenotype and genotype could not be assigned to any known tumor entity. The tumor was observed in the pancreatic head of a 54-year-old woman. It was found to be a solid infiltrating carcinoma with abundant clear cells. Apart from cytokeratin, the tumor cells expressed vimentin, S100, and MUC-1. DNA microarray analysis revealed a transcription profile clearly differing from that of normal pancreatic tissue and pancreatic ductal adenocarcinoma. Despite metastatic behavior, the tumor displayed a more favorable course than conventional pancreatic ductal adenocarcinoma. We suggest that this tumor be called solid type clear cell carcinoma of the pancreas
    Type of Publication: Journal article published
    PubMed ID: 17453235
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  • 3
    Keywords: CANCER ; tumor ; CELL ; Germany ; human ; SYSTEM ; SYSTEMS ; DISEASE ; liver ; PROTEIN ; PROTEINS ; SAMPLE ; SAMPLES ; MOLECULES ; TISSUE ; TISSUES ; BIOLOGY ; MOLECULE ; IDENTIFICATION ; MEMBRANE ; INSTABILITY ; ELECTROPHORESIS ; pancreatic cancer ; SECTIONS ; molecular biology ; pancreas ; PANCREATIC-CANCER ; EXTRACTION ; SEPARATION ; USA ; protein fractionation ; CELL-CELL ; EFFECTOR MOLECULE ; ISLETS ; protein extraction
    Abstract: Proteins are the major class of effector molecules in cellular systems. For the identification of functional differences between normal and diseased tissues, a reliable analysis of their protein content is essential. Reproducible isolation and fractionation of intact proteins are important in this respect, but their complexity in structure and concentration, their close interaction, and their instability represent major challenges. For protein isolation in tissues, the breakdown of cell-cell and cell-matrix connections within a tissue without affecting protein quality is a critical factor. We compared different processes for a compartmental protein preparation from pancreatic tissue, one of the most challenging tissues for protein isolation because of its high protease content. Success of the different procedures varied greatly. Based on a scheme of tissue-slicing and subsequent cell isolation, we established a reliable workflow for the fractional extraction of cytosolic proteins, membrane and organelle proteins, nuclear proteins, and cytoskeletal filaments. The tissue slices also allow for a representative confirmation of individual samples' cellular status by histochemical processes, and a proper separation or mixing of cellular material from across a tumor if required
    Type of Publication: Journal article published
    PubMed ID: 19450236
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  • 4
    Keywords: RECEPTOR ; EXPRESSION ; GROWTH ; IN-VITRO ; INHIBITOR ; CELL ; Germany ; INHIBITION ; PATHWAY ; PATHWAYS ; DISEASE ; GENE ; GENE-EXPRESSION ; GENES ; microarray ; PROTEIN ; METABOLISM ; LINES ; NF-KAPPA-B ; NITRIC-OXIDE ; MACROPHAGES ; TARGET ; MOUSE ; ASSAY ; microarrays ; NECROSIS-FACTOR-ALPHA ; CELL-LINE ; LINE ; PROGNOSTIC VALUE ; TARGETS ; inflammation ; CYTOTOXICITY ; INCREASED EXPRESSION ; INHIBITORS ; CELL-GROWTH ; signaling ; INTERFERENCE ; SCIENCE ; pharmacology ; nitric oxide ; NITROSATIVE STRESS ; INTERLEUKIN-10 ; TUMOR BIOLOGY ; NATURAL-PRODUCTS ; POOR SURVIVAL ; natural product ; Glucocorticoid receptor signaling pathway ; Griess assay ; Interleukin-10 signaling pathway ; RAW-264.7 CELLS ; THIOREDOXIN SYSTEM
    Abstract: Nitric oxide (NO) plays a role in various physiological and pathophysiological conditions such as immunoregulatory and inflammatory processes. Hence, NO and its generating enzyme, inducible nitric oxide synthase (iNOS) may not only be of diagnostic and prognostic value, but may also serve as targets for novel therapeutic options. In the present investigation, we have screened a phytochemical library by correlating the IC50 values for 531 natural products of 60 cell lines with the microarray-based mRNA expression of 95 genes known to be involved in NO metabolism and signaling with the aim to identify candidate compounds as inhibitors for NO metabolism and signaling. We identified bis(helenalinyl)glutarate (BHG) as putative candidate compound. Indeed. BHG inhibited NO production (IC50 value: 0.90 +/- 0.04 mu M) and down-regulated iNOS protein expression (IC50 value: 1.12 +/- 0.16 mu M) of RAW264.7 mouse macrophages in the presence of lipopolysaccharide. Performing XTT cytotoxicity assays, we found that BHG inhibited cell growth in a dose-dependent manner with an IC50 value of 5.6 mu M. To gain insight into molecular pathways involved in NO inhibition and cytotoxicity, we performed microarray experiments which were exemplarily validated by real-time RT-PCR. A total of 227 genes (67 up- and 160 down-regulated) were obtained, which exhibited significant differences in mRNA regulation between BHG-treated and untreated RAW264.7 macrophages. Sixteen of 227 genes are known to be involved in NO-signaling. Pathway analyses revealed that further five and four down-regulated genes belong to the glucocorticoid receptor and interleukin-1 and interleukin-10 pathways, respectively. An interference of these two pathways and NO is known for inflammation and auto-immune diseases. The therapeutic potential of this compound has to be explored in the future. (C) 2010 Elsevier Inc. All rights reserved
    Type of Publication: Journal article published
    PubMed ID: 20105431
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  • 5
    Keywords: CANCER ; EXPRESSION ; CELL ; Germany ; GENOME ; microarray ; PROTEIN ; PROTEINS ; PATIENT ; COMPLEX ; COMPLEXES ; QUALITY ; BIOLOGY ; antibodies ; antibody ; ASSAY ; microarrays ; DESIGN ; ARRAYS ; REPRODUCIBILITY ; CANCER-PATIENTS ; CANCER PATIENTS ; pancreatic cancer ; PROTEOMICS ; PROTEOMIC ANALYSIS ; SERUM ; FEATURES ; PANCREATIC-CANCER ; antibody microarray ; HEALTHY-SUBJECTS ; methods ; HUMAN PLASMA ; DEPLETION ; QUANTITATIVE PROTEOMICS ; proteomic ; ABUNDANCE PROTEINS ; BIOMARKER DISCOVERY
    Abstract: Antibody microarrays have the potential to enable comprehensive proteomic analysis of small amounts of sample material. Here, protocols are presented for the production, quality assessment, and reproducible application of antibody microarrays in a two-color mode with an array of 1,800 features, representing 810 antibodies that were directed at 741 cancer-related proteins. In addition to measures of array quality, we implemented indicators for the accuracy and significance of dual-color detection. Dual-color measurements outperform a single-color approach concerning assay reproducibility and discriminative power. In the analysis of serum samples, depletion of high-abundance proteins did not improve technical assay quality. On the contrary, depletion introduced a strong bias in protein representation. In an initial study, we demonstrated the applicability of the protocols to proteins derived from urine samples. We identified differences between urine samples from pancreatic cancer patients and healthy subjects and between sexes. This study demonstrates that biomedically relevant data can be produced. As demonstrated by the thorough quality analysis, the dual-color antibody array approach proved to be competitive with other proteomic techniques and comparable in performance to transcriptional microarray analyses.
    Type of Publication: Journal article published
    PubMed ID: 20164060
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  • 6
    Keywords: CELLS ; INHIBITOR ; CELL ; Germany ; human ; INHIBITION ; SYSTEM ; ENZYMES ; GENE ; GENOME ; GENOME SEQUENCE ; PROTEIN ; CONTRAST ; SEQUENCE ; SEQUENCES ; chromosome ; VARIANTS ; SUBUNIT ; ACTIVE-SITE ; DESIGN ; DIFFERENCE ; Drosophila ; DROSOPHILA-MELANOGASTER ; ERYTHROCYTES ; ESCHERICHIA-COLI ; genome analysis ; GLUTATHIONE ; GLUTATHIONE-REDUCTASE ; INSECTICIDE RESISTANCE ; MELANOGASTER ; PHAGOCYTOSIS ; resistance ; SELENOCYSTEINE ; SINGLE-COPY ; VECTOR ; Plasmodium falciparum ; Anopheles gambiae ; Drosophila melanogaster ; Diptera ; insect redox metabolism
    Abstract: The mosquito, Anopheles gambiae, is an important vector of Plasmodium falciparum malaria. Full genome analysis revealed that, as in Drosophila melanogaster, the enzyme glutathione reductase is absent in A. gambiae and functionally substituted by the thioredoxin system. The key enzyme of this system is thioredoxin reductase-1, a homodimeric FAD-containing protein of 55.3 kDa per subunit, which catalyses the reaction NADPH + H+ + thioredoxin disulfide. NADP+ + thioredoxin dithiol. The A. gambiae trxr gene is located on chromosome X as a single copy; it represents three splice variants coding for two cytosolic and one mitochondrial variant. The predominant isoform, A. gambiae thioredoxin reductase-1, was recombinantly expressed in Escherichia coli and functionally compared with the wild-type enzyme isolated in a final yield of 1.4 U.ml(-1) of packed insect cells. In redox titrations, the substrate A. gambiae thioredoxin-1 (K-m = 8.5 muM, k(cat) = 15.4 s(-1) at pH 7.4 and 25degreesC) was unable to oxidize NADPH-reduced A. gambiae thioredoxin reductase-1 to the fully oxidized state. This indicates that, in contrast to other disulfide reductases, A. gambiae thioredoxin reductase-1 oscillates during catalysis between the four-electron reduced state and a two-electron reduced state. The thioredoxin reductases of the malaria system were compared. A. gambiae thioredoxin reductase-1 shares 52% and 45% sequence identity with its orthologues from humans and P. falciparum, respectively. A major difference among the three enzymes is the structure of the C-terminal redox centre, reflected in the varying resistance of catalytic intermediates to autoxidation. The relevant sequences of this centre are Thr-Cys-Cys-SerOH in A. gambiae thioredoxin reductase, Gly-Cys-selenocysteine-GlyOH in human thioredoxin reductase, and Cys-X-X-X-X-Cys-GlyOH in the P. falciparum enzyme. These differences offer an interesting approach to the design of species-specific inhibitors. Notably, A. gambiae thioredoxin reductase-1 is not a selenoenzyme but instead contains a highly unusual redoxactive Cys-Cys sequence
    Type of Publication: Journal article published
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  • 7
    Keywords: APOPTOSIS ; EXPRESSION ; GROWTH ; GROWTH-FACTOR ; IN-VITRO ; CELL ; CELL LUNG-CANCER ; Germany ; INHIBITION ; KINASE ; PATHWAY ; PATHWAYS ; VITRO ; GENERATION ; SYSTEM ; SYSTEMS ; GENE ; DIFFERENTIATION ; TRANSDUCTION ; NF-KAPPA-B ; ACTIVATION ; COMPLEX ; LIGAND ; RESPONSES ; COMPLEXES ; MECHANISM ; INDUCTION ; hepatocytes ; INTERVENTION ; mechanisms ; BIOLOGY ; PHOSPHORYLATION ; signal transduction ; SIGNAL ; culture ; TARGET ; MAP KINASE ; MOUSE ; STRESS ; SIGNAL-TRANSDUCTION ; BETA ; NF-kappa B ; beta-catenin ; RAT HEPATOCYTES ; TARGETS ; ESTABLISHMENT ; FAS-LIGAND ; glutathione-S-transferase ; CYTOKINE ; RE ; SYNTHETASE ; INCREASE ; secretion ; FAS ; REPORTER GENE ; FUNCTIONAL-CHARACTERIZATION ; function ; MAP ; EXTENT ; modelling ; regeneration ; regulatory mechanism ; PREDICT ; CRYOPRESERVED HEPATOCYTES ; DRUG-METABOLISM ; ENZYME-INDUCTION ; GP130 ; LIVER-REGENERATION ; MEDIATE APOPTOSIS ; Stat3
    Abstract: Complex cellular networks regulate regeneration, detoxification and differentiation of hepatocytes. By combining experimental data with mathematical modelling, systems biology holds great promises to elucidate the key regulatory mechanisms involved and predict targets for efficient intervention. For the generation of high-quality quantitative data suitable for mathematical modelling a standardised in vitro system is essential. Therefore the authors developed standard operating procedures for the preparation and cultivation of primary mouse hepatocytes. To reliably monitor the dynamic induction of signalling pathways, the authors established starvation conditions and evaluated the extent of starvation-associated stress by quantifying several metabolic functions of cultured primary hepatocytes, namely activities of glutathione-S-transferase, glutamine synthetase, CYP3A as well as secretion of lactate and urea into the culture medium. Establishment of constant metabolic activities after an initial decrease compared with freshly isolated hepatocytes showed that the cultured hepatocytes achieve a new equilibrium state that was not affected by our starving conditions. To verify the highly reproducible dynamic activation of signalling pathways in the in vitro system, the authors examined the JAK-STAT, SMAD, P13 kinase, MAP kinase, NF-kappa B and Wnt/beta-catenin signalling pathways. For the induction of gp130, JAK1 and STAT3 phosphorylation IL6 was used, whereas TGF beta was applied to activate the phosphorylation of SMAD1, SMAD2 and SMAD3. Both Akt/PKB and ERK1/2 phosphorylation were stimulated by the addition of hepatocyte growth factor. The time-dependent induction of a pool of signalling competent B-catenin was monitored in response to the inhibition of GSK3 beta. To analyse whether phosphorylation is actually leading to transcriptional responses, luciferase reporter gene constructs driven by multiple copies of TGF beta-responsive motives were applied, demonstrating a dose-dependent increase in luciferase activity. Moreover, the induction of apoptosis by the TNF-like cytokine Fas ligand was studied in the in vitro system. Thus, the mouse hepatocyte in vitro system provides an important basis for the generation of high-quality quantitative data under standardised cell culture conditions that is essential to elucidate critical hepatocellular functions by the systems biology approach
    Type of Publication: Journal article published
    PubMed ID: 17186705
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  • 8
    Keywords: CANCER ; CELLS ; tumor ; CELL ; Germany ; HYBRIDIZATION ; chromosome ; FREQUENCY ; polymorphism ; single nucleotide polymorphism ; FREQUENCIES ; DELETION ; IN-SITU ; COMPARATIVE GENOMIC HYBRIDIZATION ; EXPERIENCE ; NUMBER ; CHROMOSOMAL-ABERRATIONS ; genetics ; SNP ; ABERRATIONS ; DELETIONS ; EPITHELIAL-CELLS ; FLUORESCENCE ; IMBALANCES ; heredity ; CHRONIC LYMPHOCYTIC-LEUKEMIA ; CHROMOSOMES ; in situ hybridization ; SINGLE ; ONCOLOGY ; ARRAY ; GRADE ; CYTOGENETIC ANALYSIS ; analysis ; methods ; single-nucleotide ; paranasal sinuses ; CHROMOSOMAL-ABNORMALITIES ; EWING SARCOMA ; aberration ; OLFACTORY NEUROBLASTOMA ; SOFT-TISSUE TUMORS ; TRISOMY-8
    Abstract: Esthesioneuroblastoma is a malignant neuroectodermal tumor originating from olfactory epithelial cells in the nasal vault. Due to the rarity of this tumor entity, cytogenetic data are very limited. Therefore, we performed comprehensive cytogenetic analyses of an esthesioneuroblastoma, Hyam's grade III-IV, using trypsin-Giemsa staining (GTG banding), multicolor fluorescence in situ hybridization (M-FISH), and locus-specific FISH complemented by molecular karyotyping using high-density single nucleotide polymorphism arrays. GTG banding of 25 metaphases revealed 54 structural intrachromosomal aberrations, predominantly located on 2q, 6q, 21q, and 22q, which were confirmed by FISH analysis. Interestingly, we found two novel, so far not described deletions, del(2)(q37) and del(21)(q22). Using GTG banding, locus-specific FISH, and M-FISH, we detected numeric changes of chromosomes 5, 17,19, and 22, as well as trisomy 8 at low frequency. Applying SNP array karyotyping, we confirmed the chromosomal aberrations del(2)(q37.3), del(3)(q27.2), del(10)(q26.11), chromosomal imbalance on 17q, del(21)(q22), and revealed a number of so far unknown aberrations (gain of 2q14.3, 13q33.3, and 13q34). While the cytogenetically revealed low frequency mosaic del(6)(q22q24) was not visible using SNP array karyotyping, some of the smaller imbalances (SNP array data) could not have been detected by classic cytogenetic analysis. Therefore, our study supports the usefulness of applying complementary methods for cytogenetic analysis. (c) 2007 Elsevier Inc. All rights reserved
    Type of Publication: Journal article published
    PubMed ID: 17321323
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  • 9
    Keywords: brain ; CANCER ; CELLS ; INHIBITOR ; tumor ; CELL ; Germany ; THERAPY ; DISEASE ; LONG-TERM ; NEW-YORK ; HYBRIDIZATION ; TUMORS ; PATIENT ; primary ; chromosome ; polymorphism ; single nucleotide polymorphism ; IN-SITU ; COMPARATIVE GENOMIC HYBRIDIZATION ; ARRAYS ; CHROMOSOMAL-ABERRATIONS ; genetics ; SNP ; ABERRATIONS ; HUMAN HOMOLOG ; CENTRAL-NERVOUS-SYSTEM ; TRANSLOCATION ; DE-NOVO ; CHILDREN ; FLUORESCENCE ; fluorescence in situ hybridization ; PROTEASOME ; heredity ; TRAIL ; TRAIL-INDUCED APOPTOSIS ; CHROMOSOMES ; in situ hybridization ; SINGLE ; CYTOKINE ; molecular ; ONCOLOGY ; ADULT ; ADULTS ; BRAIN-TUMORS ; HUMAN CANCER ; THERAPIES ; ARRAY ; GRADE ; medulloblastoma ; PRIMITIVE NEUROECTODERMAL TUMORS ; CHEMOTHERAPEUTIC DRUGS ; single-nucleotide ; BASAL-CELL CARCINOMAS ; USA ; RESISTANT ; RARE ; aberration ; chromosomal aberration ; ACUTE MYELOID LEUKEMIAS ; BORTEZOMIB ; CHROMOSOMAL CHANGES ; PARTIAL UNIPARENTAL DISOMY
    Abstract: Medulloblastoma is a malignant invasive embryonal tumor, occurring in children mainly. It is rare in adults (〈 1 % of adult brain tumors), and so comprehensive cytogenetic and molecular biological data on adult medulloblastomas are very limited. Conventional therapies provide disappointing long-term disease control, and new therapeutic options are being tested. We performed comprehensive cytogenetic analyses of an adult medulloblastoma, WHO grade IV, using trypsin-Giemsa staining (GTG-banding), multicolor fluorescence in situ hybridization (M-FISH), and locus-specific FISH, complemented by molecular karyotyping using high-density single nucleotide polymorphism (SNP) arrays. GTG-banding of 25 metaphases revealed 31 structural chromosomal aberrations, predominantly located on chromosomes 4q, 9q, 10q, l l p, and 20q, which were confirmed by M-FISH. Two novel, so far not described translocations were found: t(4;11)(q25;p15) and t(9;20)(p23;p12). GTG-banding, locus-specific FISH, and M-FISH detected numerical changes of chromosomes 8, 14, 18, 19, 20, 21, and 22. Molecular karyotyping by SNP array confirmed chromosomal changes -2p, -10q, -16q, and -Xq and revealed de novo partial uniparental disomy 1q and 9q. Applying an upcoming therapeutic approach, we found that primary medulloblastoma cells were resistant to TRAIL, a novel anticancer cytokine, but could be efficiently sensitized by cotreatment with the proteasome inhibitor bortezomib. Bortezomib-TRAIL cotreatment may serve as a powerful therapeutic option for medulloblastoma patients. (c) 2007 Elsevier Inc. All rights reserved
    Type of Publication: Journal article published
    PubMed ID: 17954265
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  • 10
    Keywords: APOPTOSIS ; CANCER ; CELLS ; EXPRESSION ; CELL ; Germany ; human ; INHIBITION ; ACTIVATION ; primary ; INDUCED APOPTOSIS ; UP-REGULATION ; sensitization ; TRAIL ; ONCOLOGY ; RE ; GLIOMA ; GLIOMA-CELLS ; C-FLIP ; USA ; cancer research ; HEPATOCELLULAR-CARCINOMA CELLS
    Type of Publication: Journal article published
    PubMed ID: 17975170
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