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Carcinoma Cell Lines (carcinoma + cell_line)
Kinds of Carcinoma Cell Lines Selected AbstractsEpstein-Barr Virus (EBV) Latent Membrane Protein 1 Induces Interleukin-8 through the Nuclear Factor-,B Signaling Pathway in EBV-Infected Nasopharyngeal Carcinoma Cell LineTHE LARYNGOSCOPE, Issue 5 2004Qingchun Ren MD Abstract Background/Objectives: Nasopharyngeal carcinoma (NPC) is a highly invasive and metastatic malignant tumor and is associated with Epstein-Barr virus (EBV) infection that exhibits type II latency. Angiogenesis is essential for tumor growth, invasion, and metastasis. Our previous studies have indicated that interleukin (IL)-8 was over-expressed in many NPC tissues and was found to be significantly correlated with angiogenesis by immunohistochemistry. Study Design: In vitro design. Methods: The influence of the EBV genome for IL-8 gene expression was studied using the EBV,genome-positive and -negative epithelial/NPC hybrid cell line NPC-KT. The EBV-positive and -negative clones were selected by polymerase chain reaction and in situ hybridization. Results: EBV-positive clones expressed abundant IL-8 mRNA compared with EBV-negative clones. This result indicated that over-expression of IL-8 depended on the presence of EBV genomes in NPC-KT cells. Two encoded genes, latent membrane protein (LMP)1 and EBV-encoded small RNAs (EBERs), expressed in NPC were transfected in EBV-negative NPC-KT cells. LMP1 transactivated the IL-8 promoter, whereas EBERs did not. Moreover, the nuclear factor (NF)- ,B binding site in the IL-8 promoter was essential for the response to LMP1, and the activator protein (AP)-1 binding site played only a partial role. Conclusions: LMP1 induces IL-8 mainly through the activation of NF-,B and partly through AP-1 in NPC model cell lines, NPC-KT, and this suggests that LMP1 plays an important role in the angiogenesis of NPC. [source] Isolation of Differentiated Squamous and Undifferentiated Spindle Carcinoma Cell Lines with Differing Metastatic Potential from a 4-Nitroquinoline N-Oxide-induced Tongue Carcinoma in a F344 RatCANCER SCIENCE, Issue 12 2000Shinichi Takeuchi One differentiated squamous cell carcinoma (SCC) cell line (RSC3-E2) and two undifferentiated tumor cell lines (RSC3-LM and RSC3-E2R) with different metastatic potential were established from a 4-nitroquinoline N-oxide (4NQO)-induced differentiated SCC in F344 rat tongue. The RSC3-E2 subline was isolated from a parental cell line (RSC3-P) by single cell cloning in vitro, whereas the RSC3-LM subline was isolated from a lung metastatic focus after subcutaneous (s.c.) injection of RSC3-P cells. The RSC3-E2R cell line was isolated from a lung metastatic focus following s.c. injection of RSC3-E2 cells after X-irradiation in vitro. The RSC3-E2 cell line is keratinpositive and grows as a keratinizing tumor in nude mice, whereas RSC3-LM and RSC3-E2R cells are keratin-negative, vimentin-positive and form undifferentiated tumors. When s.c. injected into nude mice, the RSC3-E2 cell line proved to be non-metastatic, while the RSC3-LM cell line was metastatic by both hematogenous and lymphogenous routes, and the RSC3-E2R cell line was metastatic only hematogenously. In vitro relative growth rates and in vitro invasion activity of these cell lines were in the order RSC3-LM>RSC3-E2R>RSC3-E2. Chromosome analysis revealed two peaks with modal chromosome numbers of 83 and 78 for RSC3-P cells and single peaks at 83, 78 and 56 for RSC3-LM, RSC3-E2 and RSC3-E2R cell lines, respectively. Common structural abnormalities on chromosome 11 were shared by all cell lines. Mutation analysis of the p53 gene using a yeast functional assay demonstrated RSC3-LM cell line to have a point mutation at codon 269, whereas RSC3-E2 and RSC3-E2R had double mutations at codons 106 and 170 on each allele. These results suggest that the two undifferentiated RSC3-LM and RSC3-E2R tumor cell lines with different metastatic potential were generated from differentiated SCC cells via different genetic pathways as a consequence of tumor progression in vivo and in vitro, respectively. These cell lines should provide a useful model for understanding mechanisms of hematogenous and lymphogenous metastasis, as well as tumor progression of oral SCCs. [source] Schedule-dependent Interactions between Raltitrexed and Cisplatin in Human Carcinoma Cell Lines in vitroCANCER SCIENCE, Issue 4 2000Yasuhiko Kano Raltitrexed (,Tomudex") is a new anticancer agent which inhibits thymidylate synthase. To provide a rational basis for clinical trial design of the combination of raltitrexed and cisplatin, we studied the cytotoxic effects of this combination using various schedules in vitro and four human colon cancer cell lines, Colo201, Colo320, LoVo, and WiDr. Cell growth inhibition after 5 days was determined by using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reduction assay. The effects of drug combinations at the concentration producing 80% cell growth inhibition (IC80) level were analyzed by the isobologram method. Simultaneous exposure to raltitrexed and cisplatin for 24 h, and sequential exposure to raltitrexed followed by cisplatin produced additive effects in the Colo201, Colo320, and LoVo cells, and additive and synergistic effects in WiDr cells. Sequential exposure to cisplatin followed by raltitrexed produced additive effects in the Colo201 cells and antagonistic effects in other three cell lines. Simultaneous and continuous exposure to both agents for 5 days produced additive effects in all four cell lines. These findings suggest that the simultaneous administration of raltitrexed and cisplatin, or the sequential administration of raltitrexed followed by cisplatin, generally produce the expected cytotoxicity at the cellular level and are optimal schedules, while the sequential administration of cisplatin followed by raltitrexed produces antagonistic effects and is inappropriate for this combination. Further in vivo and clinical studies will be necessary to determine the toxicity and antitumor effects of this schedule. [source] Synthesis of 9,9-Dialkyl-4,5-diazafluorene Derivatives and Their Structure,Activity Relationships Toward Human Carcinoma Cell LinesCHEMMEDCHEM, Issue 4 2010Qiwei Wang Abstract A homologous set of 9,9-dialkyl-4,5-diazafluorene compounds were prepared by alkylation of 4,5-diazafluorene with the appropriate alkyl bromide and under basic conditions. The structures of these simple organic compounds were confirmed by spectroscopic techniques (FTIR, NMR, and FABMS). Their biological effects toward a panel of human carcinoma cells, including Hep3B hepatocellular carcinoma, MDAMB-231 breast carcinoma, and SKHep-1 hepatoma cells, were investigated; a structure,activity correlation was established with respect to the length of the alkyl chain and the fluorene ring structure. The relationship between the mean potency [log(1/IC50)] and alkyl chain length was systematically studied. The results show that compounds with butyl, hexyl, and octyl chains exhibit good growth inhibitory effects toward these three human carcinoma cell lines, and the 9,9-dihexyl-4,5-diazafluorene further exhibits antitumor activity in athymic nude mice Hep3B xenograft models. For the structurally related dialkylfluorenes that lack the diaza functionality, in,vitro cytotoxicity was not observed at clinically relevant concentrations. [source] Measurement of barbed ends, actin polymerization, and motility in live carcinoma cells after growth factor stimulation,CYTOSKELETON, Issue 4 2004Mike Lorenz Abstract Motility is associated with the ability to extend F-actin-rich protrusions and depends on free barbed ends as new actin polymerization sites. To understand the function and regulation of different proteins involved in the process of generating barbed ends, e.g., cofilin and Arp2/3, fixed cell approaches have been used to determine the relative barbed end concentration in cells. The major disadvantages of these approaches are permeabilization and fixation of cells. In this work, we describe a new live-cell time-lapse microscopy assay to determine the increase of barbed ends after cell stimulation that does not use permeabilization and provides a better time resolution. We established a metastatic carcinoma cell line (MTLn3) stably expressing GFP-,-actin at physiological levels. Stimulation of MTLn3 cells with epidermal growth factor (EGF) causes rapid and transient lamellipod protrusion along with an increase in actin polymerization at the leading edge, which can be followed in live cell experiments. By measuring the increase of F-actin at the leading edge vs. time, we were able to determine the relative increase of barbed ends after stimulation with a high temporal resolution. The F-actin as well as the barbed end concentration agrees well with published data for this cell line. Using this newly developed assay, a decrease in lamellipod extension and a large reduction of barbed ends was documented after microinjecting an anti-cofilin function blocking antibody. This assay has a high potential for applications where rapid changes in the dynamic filament population are to be measured. Cell Motil. Cytoskeleton 57:207,217, 2004. © 2004 Wiley-Liss, Inc. [source] Enantioselective estrogenicity of o,p'-dichlorodiphenyltrichloroethane in the MCF-7 human breast carcinoma cell line,ENVIRONMENTAL TOXICOLOGY & CHEMISTRY, Issue 1 2009Lumei Wang Abstract Research increasingly suggests that selectivity between enantiomers may exist in acute and chronic toxicological effects of chiral contaminants. In this study, we used the human breast carcinoma MCF-7 cell line to evaluate enantioselectivity of o,p'-dichlorodiphenyltrichloroethane (o,p'-DDT). Baseline separation of o,p'-DDT enantiomers was achieved on the Chiralcel® OJ chiral column by high-performance liquid chromatography, and the absolute configuration and optical rotation of the resolved enantiomers were further identified. Significant differences in estrogenic potential were observed between the two enantiomers of o,p'-DDT in the MCF-7 cell proliferation assay (i.e., the E-Screen assay) and the real-time quantitative polymerase chain reaction (PCR). In the E-Screen assay, the relative proliferative effect ratios of R -(,)- o,p'-DDT and S -(+)- o,p'-DDT were 89.4 and 27.9%, respectively, and the relative proliferative potency ratios were 0.1 and 0.001%, respectively. Compared to the solvent control, R -(,)- o,p'-DDT induced the maximal increase of 2.31-fold at a concentration of 10,6 mol/L, while S -(+)- o,p'-DDT at 10,5 mol/L induced the maximal increase of 1.65-fold in estrogenic biomarker pS2 mRNA level. The maximal down-regulation of the transcription levels of estrogen receptor a (ER,) and ER, by R -(,)- o,p'-DDT were 49 and 40% at the concentration of 10,6 mol/L, while those by S -(+)- o,p'-DDT were 24 and 26% at the concentration of 10,5 mol/L. The cell proliferation, the up-regulation of pS2, and the down-regulation of ER, and ER, gene expressions induced by the racemate and enantiomers of o,p'-DDT were all reversed by cotreatment with 10,6 mol/L ICI 182,780. Therefore, the enantioselective estrogenicity of o,p'-DDT was likely through the ER, and ER, signaling pathways. Results from this study suggest the need for considering enantioselectivity of chiral contaminants in chronic ecological toxicities. [source] Estrogenic activity of lambda-cyhalothrin in the MCF-7 human breast carcinoma cell line,ENVIRONMENTAL TOXICOLOGY & CHEMISTRY, Issue 5 2008Meirong Zhao Abstract Synthetic pyrethroids are widely used in both agricultural and urban environments for insect control. Lambda-cyhalothrin (LCT) is one of the most common pyrethroids and is used mainly for controlling mosquitoes, fleas, cockroaches, flies, and ants around households. Previous studies have addressed the environmental behaviors and acute toxicities of LCT, but little is known about its chronic toxicity, such as estrogen-like activity. In the present study, the estrogenic potential of LCT was evaluated using the MCF-7 human breast carcinoma cell line. The in vitro E-screen assay showed that 10,7 M LCT could significantly promote MCF-7 cell proliferation, with a relative proliferative effect ratio of 45%. The cell proliferation induced by LCT could be blocked completely, however, by the addition of 10,9 M of the estrogen receptor (ER)-antagonist ICI 182,780. The semiquantitative reverse transcription-polymerase chain reaction (RT-PCR) results showed that the Trefoil factor 1 (pS2) and progesterone receptor gene expression were up-regulated by 10,7 M LCT for 2- and 1.5-fold, respectively. On the other hand, RT-PCR, Western blot analysis, and immunofluorescent assay demonstrated that LCT significantly repressed the mRNA and protein expression levels of ER, and ER,. These observations indicate that LCT possesses estrogenic properties and may function as a xenoestrogen, likely via a mechanism similar to that of 17,-estradiol. The endocrine-disruption potential of LCT should be considered when assessing the safety of this compound in sensitive environmental compartments. [source] Effects of brominated flame retardants and brominated dioxins on steroidogenesis in H295R human adrenocortical carcinoma cell lineENVIRONMENTAL TOXICOLOGY & CHEMISTRY, Issue 4 2007Ling Ding Abstract Brominated flame retardants (BFRs) and brominated dioxins are emerging persistent organic pollutants that are ubiquitous in the environment and can be accumulated by wildlife and humans. These chemicals can disturb endocrine function. Recent studies have demonstrated that one of the mechanisms of endocrine disruption by chemicals is modulation of steroidogenic gene expression or enzyme activities. In this study, an in vitro assay based on the H295R human adrenocortical carcinoma cell line, which possesses most key genes or enzymes involved in steroidogenesis, was used to examine the effects of five bromophenols, two polybrominated biphenyls (PBBs 77 and 169), 2,3,7,8-tetrabromodibenzo- p -dioxin, and 2,3,7,8-tetrabromodibenzofuran on the expression of 10 key steroidogenic genes. The H295R cells were exposed to various BFR concentrations for 48 h, and the expression of specific genes,cytochrome P450 (CYP11A, CYP11B2, CYP17, CYP19, and CYP21), 3,-hydroxysteroid dehydrogenase (3,HSD2), 17,-hydroxysteroid dehydrogenase (17,HSD1 and 17,HSD4), steroidogenic acute regulatory protein (StAR), and 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR),was quantitatively measured using real-time polymerase chain reaction. Cell viability was not affected at the doses tested. Most of the genes were either up- or down-regulated, to some extent, by BFR exposure. Among the genes tested, 3,HSD2 was the most markedly up-regulated, with a range of magnitude from 1.6- to 20-fold. The results demonstrate that bromophenol, bromobiphenyls, and bromodibenzo- p -dioxin/furan are able to modulate steroidogenic gene expression, which may lead to endocrine disruption. [source] Tumour cell,dendritic cell fusion for cancer immunotherapy: comparison of therapeutic efficiency of polyethylen-glycol versus electro-fusion protocolsEUROPEAN JOURNAL OF CLINICAL INVESTIGATION, Issue 3 2002M. Lindner Abstract Background ,Fusion of tumour cells with dendritic cells (DC) is a powerful new technology to increase tumour vaccine immunogenicity. The aim of this study was to compare fusion protocols with syngenic DCs with respect to the efficiency of polyethylen-glycol-(PEG) and electric pulse-mediated fusions for induction of protective anti-tumour immune responses. As a model we chose a low immunogenic and metastatic murine mammary carcinoma cell line, which mimics clinically relevant tumour features. Methods FACS-staining, chromium release assay, therapeutic immunization, adoptive transfer. Results ,We show that the parental line with low cell surface expression of MHC molecules as well as a lacZ transfectant becomes highly immunogenic upon fusion with DCs. This was true for PEG- as well as for electro-fused cells. Immunization with products of DCs and tumour cells cocultivated for 16 h without the fusing agent PEG also caused induction of profound anti-tumour immunity, while this was not the case when using parental tumour cells or their lacZ transfectants as vaccines. Immune protection against the parental tumour cells after vaccination with fused cells was long-lasting and could be transferred via immune spleen cells into immuno-incompetent nude (nu/nu) mice. Conclusion ,Fusion products of DA3hi mammary carcinoma cells and DCs produced by an electric pulse were similar to those produced by PEG fusion with regard to vaccine potency in prophylactic antitumour immunization assays in vivo. Therefore, both techniques seem to be promising for clinical application. [source] Synthesis of Polyamines from Ethylenediamine and Their Platinum(II) Complexes, Potential Antitumor AgentsEUROPEAN JOURNAL OF INORGANIC CHEMISTRY, Issue 9 2006Mara Rubia Costa Couri Abstract This work describes the synthesis and characterization of five new amine ligands and also the preparation and characterization of their respective platinum(II) complexes by reaction with K2PtCl4 in water. These ligands were obtained by treatment of different halides or epoxides with ethylenediamine. Cytotoxic activity and cellular accumulation of three complexes were investigated in a human small-cell lung carcinoma cell line and its cisplatin resistant subline. The introduction of a spacer (cycle) between the two platinum atoms leads to a significant decrease in cytotoxic activity. At equitoxic doses, the intracellular platinum concentrations found for compounds 12 and 15 were significantly higher than those found for the reference compounds, cisplatin, carboplatin, or compound 9. This fact suggests that the formation of adducts between compounds 12 and 15 and the putative pharmacological target, DNA, is less favored. If these compounds bind more slowly to DNA, interaction with other intracellular ligands such as sulfur-containing molecules will become relevant and it may be the reason for the elevated intracellular platinum concentrations. (© Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2006) [source] Efficient killing of SW480 colon carcinoma cells by a signal transducer and activator of transcription (STAT) 3 hairpin decoy oligodeoxynucleotide , interference with interferon-,-STAT1-mediated killingFEBS JOURNAL, Issue 9 2009Ali Tadlaoui Hbibi The signal transducers and activators of transcription (STATs) convey signals from the membrane to the nucleus in response to cytokines or growth factors. STAT3 is activated in response to cytokines involved mostly in cell proliferation; STAT1 is activated by cytokines, including interferon-,, involved in defence against pathogens and the inhibition of cell proliferation. STAT3, which is frequently activated in tumour cells, is a valuable target with respect to achieving inhibition of tumour cell proliferation. Indeed, its inhibition results in cell death. We previously observed that inhibition of the transcription factor nuclear factor-,B, a key regulator of cell proliferation, with decoy oligodeoxynucleotides results in cell death. We used a similar approach for STAT3. A hairpin STAT3 oligodeoxynucleotide was added to a colon carcinoma cell line in which it induced cell death as efficiently as the STAT3 inhibitor stattic. The hairpin STAT3 oligodeoxynucleotide co-localized with STAT3 within the cytoplasm, prevented STAT3 localization to the nucleus, blocked a cyclin D1 reporter promoter and associated with STAT3 in pull-down assays. However, the same cells were efficiently killed by interferon-,. This effect was counteracted by the STAT3 oligodeoxynucleotide, which was found to efficiently inhibit STAT1. Thus, although it can inhibit STAT3, the hairpin STAT3 oligodeoxynucleotide appears also to inhibit STAT1-mediated interferon-, cell killing, highlighting the need to optimize STAT3-targeting oligodeoxynucleotides. [source] Novel genes in cell cycle control and lipid metabolism with dynamically regulated binding sites for sterol regulatory element-binding protein 1 and RNA polymerase II in HepG2 cells detected by chromatin immunoprecipitation with microarray detectionFEBS JOURNAL, Issue 7 2009Mehdi Motallebipour Sterol regulatory element-binding proteins 1 and 2 (SREBP-1 and SREBP-2) are important regulators of genes involved in cholesterol and fatty acid metabolism, but have also been implicated in the regulation of the cell cycle and have been associated with the pathogenesis of type 2 diabetes, atherosclerosis and obesity, among others. In this study, we aimed to characterize the binding sites of SREBP-1 and RNA polymerase II through chromatin immunoprecipitation and microarray analysis in 1% of the human genome, as defined by the Encyclopaedia of DNA Elements consortium, in a hepatocellular carcinoma cell line (HepG2). Our data identified novel binding sites for SREBP-1 in genes directly or indirectly involved in cholesterol metabolism, e.g. apolipoprotein C-III (APOC3). The most interesting biological findings were the binding sites for SREBP-1 in genes for host cell factor C1 (HCFC1), involved in cell cycle regulation, and for filamin A (FLNA). For RNA polymerase II, we found binding sites at classical promoters, but also in intergenic and intragenic regions. Furthermore, we found evidence of sterol-regulated binding of SREBP-1 and RNA polymerase II to HCFC1 and FLNA. From the results of this work, we infer that SREBP-1 may be involved in processes other than lipid metabolism. [source] Mass spectrometric detection of tyrosine sulfation in human pancreatic trypsinogens, but not in tumor-associated trypsinogenFEBS JOURNAL, Issue 2 2008Outi Itkonen Trypsinogen-1 and -2 are well-characterized enzymes that are expressed in the pancreas and also in several other tissues. Many cancers produce trypsinogen isoenzymes that differ from the pancreatic ones with respect to substrate specificity and isoelectric point. These tumor-associated trypsinogens play a pivotal role in cancer progression and metastasis. The differences between these and the pancreatic isoenzymes have been suggested to be caused by post-translational modification, either sulfation or phosphorylation of a tyrosine residue. We aimed to elucidate the cause of these differences. We isolated trypsinogens from pancreatic juice and conditioned medium from a colon carcinoma cell line. Intact proteins, and tryptic and chymotryptic peptides were characterized by electrospray ionization mass spectrometry. We also used immunoblotting with antibody against phosphotyrosine and N-terminal sequencing. The results show that pancreatic trypsinogen-1 and -2 are sulfated at Tyr154, whereas tumor-associated trypsinogen-2 is not. Detachment of a labile sulfogroup could be demonstrated by both in-source dissociation and low-energy collision-induced dissociation in a tandem mass spectrometer. Tyrosine sulfation is an ubiquitous protein modification occurring in the secretory pathway, but its significance is often underestimated due to difficulties in its analysis. Sulfation is an almost irreversible modification that is thought to regulate protein,protein interactions and the activity of proteolytic enzymes. We conclude that the previously known differences in charge, substrate specificity and inhibitor binding between pancreatic and tumor-associated trypsinogens are probably caused by sulfation of Tyr154 in pancreatic trypsinogens. [source] Identification of amplified and expressed genes in breast cancer by comparative hybridization onto microarrays of randomly selected cDNA clonesGENES, CHROMOSOMES AND CANCER, Issue 1 2002Jeremy Clark Microarray analysis using sets of known human genes provides a powerful platform for identifying candidate oncogenes involved in DNA amplification events but suffers from the disadvantage that information can be gained only on genes that have been preselected for inclusion on the array. To address this issue, we have performed comparative genome hybridization (CGH) and expression analyses on microarrays of clones, randomly selected from a cDNA library, prepared from a cancer containing the DNA amplicon under investigation. Application of this approach to the BT474 breast carcinoma cell line, which contains amplicons at 20q13, 17q11,21, and 17q22,23, identified 50 amplified and expressed genes, including genes from these regions previously proposed as candidate oncogenes. When considered together with data from microarray expression profiles and Northern analyses, we were able to propose five genes as new candidate oncogenes where amplification in breast cancer cell lines was consistently associated with higher levels of RNA expression. These included the HB01 histone acetyl transferase gene at 17q22,23 and the TRAP100 gene, which encodes a thyroid hormone receptor-associated protein coactivator, at 17q11,21. The results demonstrate the utility of this microarray-based CGH approach in hunting for candidate oncogenes within DNA amplicons. © 2002 Wiley-Liss, Inc. [source] Integrative molecular characterization of head and neck cancer cell model genomesHEAD & NECK: JOURNAL FOR THE SCIENCES & SPECIALTIES OF THE HEAD AND NECK, Issue 9 2010Ivy F. L. Tsui BSc Abstract Background. Cell lines are invaluable model systems for the investigation of cancer. Knowledge of the molecular alterations that exist within cell models is required to define the mechanisms governing cellular phenotypes. Methods. Five tongue squamous cell carcinomas cell lines and 1 submaxillary salivary gland epidermoid carcinoma cell line were analyzed for copy number and mRNA expression by tiling-path DNA microarrays and Agilent Whole Human Genome Oligoarrays, respectively. Results. Integrative analysis of genetic and expression alterations revealed the molecular landscape of each cell line. Molecular results for individual cell lines and across all samples have been summarized and made available for easy reference. Conclusion. Our integrative genomic analyses have defined the DNA and RNA alterations for each individual line. These data will be useful to anyone modeling oral cancer behavior, providing a molecular context that will be useful for deciphering cell phenotypes. © 2009 Wiley Periodicals, Inc. Head Neck, 2010 [source] Priming of immune responses against transporter associated with antigen processing (TAP)-deficient tumours: tumour direct primingIMMUNOLOGY, Issue 3 2009Xiao-Lin Li Summary We previously showed that introduction of transporter associated with antigen processing (TAP) 1 into TAP-negative CMT.64, a major histocompatibility complex class I (MHC-I) down-regulated mouse lung carcinoma cell line, enhanced T-cell immunity against TAP-deficient tumour cells. Here, we have addressed two questions: (1) whether such immunity can be further augmented by co-expression of TAP1 with B7.1 or H-2Kb genes, and (2) which T-cell priming mechanism (tumour direct priming or dendritic cell cross-priming) plays the major role in inducing an immune response against TAP-deficient tumours. We introduced the B7.1 or H-2Kb gene into TAP1-expressing CMT.64 cells and determined which gene co-expressed with TAP1 was able to provide greater protective immunity against TAP-deficient tumour cells. Our results show that immunization of mice with B7.1 and TAP1 co-expressing but not H-2Kb and TAP1 co-expressing CMT.64 cells dramatically augments T-cell-mediated immunity, as shown by an increase in survival of mice inoculated with live CMT.64 cells. In addition, our results suggest that induction of T-cell-mediated immunity against TAP-deficient tumour cells could be mainly through tumour direct priming rather than dendritic cell cross-priming as they show that T cells generated by tumour cell-lysate-loaded dendritic cells recognized TAP-deficient tumour cells much less than TAP-proficient tumour cells. These data suggest that direct priming by TAP1 and B7.1 co-expressing tumour cells is potentially a major mechanism to facilitate immune responses against TAP-deficient tumour cells. [source] Apoptosis induction by interleukin-2-activated cytotoxic lymphocytes in a squamous cell carcinoma cell line and Daudi cells , involvement of reactive oxygen species-dependent cytochrome c and reactive oxygen species-independent apoptosis-inducing factorsIMMUNOLOGY, Issue 2 2003Tetsuya Yamamoto Summary Investigation of the induction of apoptosis by cytotoxic lymphocytes has mainly focused on the signalling associated with Fas and its adaptor proteins. The signal pathway via mitochondria, however, has not been sufficiently elucidated in cytotoxic lymphocyte-induced apoptosis. We examined the release of mitochondrial proapoptotic factors by lymphokine-activated killer (LAK) cells in two cell lines. LAK cell-induced DNA fragmentation of the target cells was suppressed to approximately 50% of control levels by the addition of neutralizing monoclonal antibody to Fas and a granzyme B inhibitor. When intracellular reactive oxygen species (ROS) were scavenged, the LAK cell-induced DNA fragmentation was decreased to approximately 60% of the non-treated cell level. Co-cultivation of Daudi cells with LAK cells increased cytosolic and mitochondrial ROS levels. Activation of procaspase-3 and apoptosis by treatment of oral squamous cell carcinoma cells (OSC) with LAK cells was partially inhibited by pretreatment of OSC cells with ROS scavengers and mitochondrial complex inhibitors. Furthermore, cytochrome c and apoptosis-inducing factor (AIF) were released from mitochondria by OSC cell treatment with supernatants of LAK cells. The supernatant-induced cytochrome c release was suppressed by mitochondrial complex inhibitors, but the inhibitors did not inhibit the release of AIF. These results indicate that LAK cells induce target cell apoptosis via not only the Fas/Fas ligand system and granzyme B, but also ROS-dependent cytochrome c and ROS-independent AIF release. [source] Establishment, characterization and drug sensitivity testing in primary cultures of human thymoma and thymic carcinomaINTERNATIONAL JOURNAL OF CANCER, Issue 12 2008Volker Ehemann Abstract Thymomas and thymic carcinomas are peculiar epithelial tumors of the anterior mediastinum. They may show aggressive clinical behavior and are a paradigm for the interaction between the tumor and the immune system. So far, adequate functional studies enabling a better understanding of this malignancy have not been performed, since human thymoma/thymic carcinoma cell lines have not been available. Here, the authors describe the establishment, characterization and functional analyses of epithelial cell lines from a Type B1-thymoma and a poorly differentiated thymic carcinoma. By Fluorescence-activated cell sorting (FACS) analyses, both cell lines were aneuploid. The aneuploid cell fraction of the thymic carcinoma cell line was characterized by a high proliferation index of 55.9%, in contrast to a lower proliferation rate of the aneuploid cell fraction of the thymoma (19.7%). Array-based comparative genomic hybridization (aCGH) and conventional cytogenetic analysis of the thymoma revealed only minor imbalances whereas the thymic carcinoma was characterized by a complex karyotype in the hyperdiploid range that was readily defined with multicolor FISH (mFISH). Application of a selective COX-2 inhibitor reduced cell viability in both cell lines in a dose-dependent manner. In conclusion, these first cell lines of a thymoma and a CD5-positive thymic carcinoma are useful tools for further in vitro studies of cellular, molecular and genetic aspects of the disease and for functional tests to evaluate new therapeutic targets. © 2008 Wiley-Liss, Inc. [source] 2,-Deoxyadenosine causes apoptotic cell death in a human colon carcinoma cell lineJOURNAL OF BIOCHEMICAL AND MOLECULAR TOXICOLOGY, Issue 6 2003Michela Giannecchini Abstract The combination of 2,-deoxyadenosine and 2,-deoxycoformycin is toxic for the human colon carcinoma cell line LoVo. In this study we investigated the mode of action of the two compounds and have found that they promote apoptosis. The examination by fluorescence microscopy of the cells treated with the combination revealed the characteristic morphology associated with apoptosis, such as chromatin condensation and nuclear fragmentation. The occurrence of apoptosis was also confirmed by the release of cytochrome c and the proteolytic processing of procaspase-3 in cells subjected to the treatment. To exert its triggering action on the apoptotic process, 2,-deoxyadenosine enters the cells through an equilibrative nitrobenzyl-thioinosine-insensitive carrier, and must be phosphorylated by intracellular kinases. Indeed, in the present work we demonstrate by analysis of the intracellular metabolic derivatives of 2,-deoxyadenosine that, as suggested by our previous findings, in the incubation performed with 2,-deoxyadenosine and 2,-deoxycoformycin, an appreciable amount of dATP was formed. Conversely, when also an inhibitor of adenosine kinase was added to the incubation mixture, dATP was not formed, and the toxic and apoptotic effect of the combination was completely reverted. © 2003 Wiley Periodicals, Inc. J Biochem Mol Toxicol 17:329,337, 2003; Published online in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/jbt.10095 [source] Establishment of OC3 oral carcinoma cell line and identification of NF-,B activation responses to areca nut extractJOURNAL OF ORAL PATHOLOGY & MEDICINE, Issue 2 2004Shu-Chun Lin Background:, Cell lines derived from oral squamous cell carcinoma (OSCC) exposed to variable etiological factors can bestow advantages in understanding the molecular and cellular alterations pertaining to environmental impacts. Most OSCC cell lines have been established from smoker patients or areca chewing/smoker patients, carrying the genomic alterations in p53. Methods:, A new cell line, oral carcinoma 3 (OC3), was established from an OSCC in a long-term areca (betel) chewer who does not smoke. Cellular and molecular features of OC3 were determined by variable assays. Results:, The cultured monolayer cells were mainly polygonal and had the expression of cytokeratin 14. The chromosomal analysis using comparative genomic hybridization has revealed the gain in chromosomes 1q, 5q, and 8q, the loss in 4q, 6p, and 8p as well as the gain of entire chromosome 20. Loss of heterozygosity and instability in multiple microsatellite markers in chromosome 4q were also noted. OC3 cells bear wild-type p53 coding sequence and have a high level of p53 expression. Its p21 expression was similar to that in normal human oral keratinocyte (NHOK). Interestingly, activation of nuclear factor ,B (NF-,B) in OC3 cells following the treatment of areca nut extract was observed. Conclusion:, OC3 cell line could be valuable in understanding the genetic impairments and phenotypic changes associated with areca in oral keratinocyte. [source] Alteration of argyrophilic nucleolar organizer region associated (Ag-NOR) proteins in apoptosis-induced human salivary gland cells and human oral squamous carcinoma cellsJOURNAL OF ORAL PATHOLOGY & MEDICINE, Issue 4 2001Yasuhiro Morimoto Abstract: The level of argyrophilic nucleolar organizer regions (AgNORs) and AgNOR-associated proteins (Ag-NOR proteins) varies with cell activity, including ribosomal biogenesis occurring in proliferating cells. Proteins associated with some AgNORs are detected by a specific silver staining. To investigate a possible relationship between apoptosis and the AgNORs or Ag-NOR proteins, we examined the changes of AgNORs and Ag-NOR proteins during apoptosis in a human salivary gland cell line, HSG cells, and a human oral squamous carcinoma cell line, SCC-25 cells. Apoptosis was induced by treatment of HSG and SCC-25 cells with okadaic acid. Proteins prepared from HSG and SCC-25 cells treated with varying concentrations of okadaic acid (OA) were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) followed by transferring to transfer membranes and staining for Ag-NOR proteins by modified Western blot analysis. Four major bands (110 kDa, 43 kDa, 39kDa, and 37 kDa) were detected in the proteins obtained from the control cells. The level of the 110-kDa protein decreased in the proteins prepared from OA-induced apoptotic cells; however, the reaction intensity of the other three bands was changed in apoptotic cells. An additional band of an 80-kDa Ag-NOR protein appeared and increased in the apoptotic cells. Cellular fractionation of HSG cells and SCC-25 cells was done with or without apoptotic induction. An 80-kDa Ag-NOR protein was detected in the nuclear fraction prepared from the apoptotic cells, while the 110-kDa protein decreased in the nuclear fraction of these cells. The 110-kDa Ag-NOR protein may be nucleolin (C23) as deduced from its AgNOR staining features, including molecular weight. The 80-kDa protein may be the cleavage product of the 110-kDa protein. In the cell-free apoptotic system, in which intact nuclei of HSG cells were incubated with the cytosol fraction of apoptotic HSG and SCC-25 cells, the 80-kDa Ag-NOR protein was detected in nuclei incubated with the cytosol fraction of apoptotic cells, while the level of the 110-kDa protein decreased. The changes of Ag-NOR proteins in nuclei prepared from SCC-25 cells incubated with cytosol fractions prepared from HSG and SCC-25 cells were identical to those of the HSG cells. The alternation of AgNORs in apoptosis-induced HSG cells was also examined using double staining with Hoechst 33342 and silver nitrate. Hoechst staining revealed typical apoptotic nuclei, which exhibited highly fluorescent condensed chromatin in OA-treated HSG cells. Silver grains representing AgNORs were not detected in the cells undergoing apoptosis. The dual-imposition view confirmed that AgNORs, which are visible as dots in nucleoli in the control cells, disappeared from the apoptotic nuclei of HSG cells. Our results indicate that the 110-kDa nucleolar Ag-NOR protein is associated with apoptosis and is cleaved during apoptosis. [source] Reversal of cancer multidrug resistance by green tea polyphenolsJOURNAL OF PHARMACY AND PHARMACOLOGY: AN INTERNATI ONAL JOURNAL OF PHARMACEUTICAL SCIENCE, Issue 10 2004Yuying Mei The aim of this study was to examine the effect and mechanism of green tea polyphenols (TP) on reversal of multidrug resistance (MDR) in a carcinoma cell line. Using the MTT assay, TP was examined for its modulating effects on the drug-resistant KB-A-1 cells and drug-sensitive KB-3,1 cells. When 10 ,g mL,1 (-)-epigallocatechin gallate (EGCG) or 40 ,g mL,1 TP were present simultaneously with doxorubicin (DOX), the IC50 of DOX on KB-A-1 cells decreased from 10.3 ± 0.9 ,g mL,1 to 4.2 ± 0.2 and 2.0 ± 0.1 ,g mL,1, respectively. TP and EGCG enhanced the DOX cytotoxicity on KB-A-1 cells by 5.2-and 2.5-times, respectively, but did not show a modulating effect on KB-3,1 cells. This indicated that both TP and EGCG had reversal effects on the MDR phenotype in-vitro. A KB-A-1 cell xenograft model was established, and the effect of TP on reversing MDR in-vivo was determined. Mechanistic experiments were conducted to examine the uptake, efflux and accumulation of DOX. Cloning and expression of the nucleotide binding domain of the human MDR1 gene in Escherichia coli was established, and by using colorimetry to examine the activity of ATPase to hydrolyse ATP, the ATPase activity of target nucleotide binding domain protein was determined. TP exerted its reversal effects through the inhibition of ATPase activity, influencing the function of P-glycoprotein, and causing a decreased extrusion of anticancer drug and an increased accumulation of anticancer drug in drug resistant cells. Using reverse transcription-polymerase chain reaction, the inhibitory effect of TP on MDR1 gene expression was investigated. Down-regulation of MDR1 gene expression was the main effect, which resulted in the reversal effect of TP on the MDR phenotype. TP is a potent MDR modulator with potential in the treatment of P-glycoprotein mediated MDR cancers. [source] Long-term ethanol exposure causes human liver cancer cells to become resistant to mitomycin C treatment through the inactivation of bad-mediated apoptosis,MOLECULAR CARCINOGENESIS, Issue 8 2010Ching-Shui Huang Abstract The aim of this study was to test whether long-term ethanol consumption confers therapeutic resistance to human liver cancer patients infected with hepatitis B virus (HBV). Chronic ethanol-treated cells were established by consecutively culturing a human hepatocellular carcinoma cell line, Hep 3B, which contains integrated HBV sequences, for 20,40 passages with or without 10,mM ethanol (designated as E20,E40 and C20,C40, respectively). Flow cytometry analysis demonstrated that a growth promoting effect of long-term ethanol treatment was induced in the E40 cells through preferential acceleration of S-phase in these cells. Lower protein expression levels of p16, p21/Cip1, and p27/Kip1 were detected in the ethanol-treated E40 cells. We further demonstrated that long-term ethanol-treated E40 cells develop drug resistance in response to mitomycin C (MMC) treatment (>8,µM). Immunoblot analysis revealed that caspase-8-mediated mitochondrial apoptotic signals (such as Bad) were inactivated in the MMC-resistant E40 cells. Immunoprecipitation experiments demonstrated that the sequestration of phosphorylated Bad (Ser-112) through its binding with 14-3-3 was detected more profoundly in the MMC-resistant E40 cells. Next, we examined the therapeutic efficacy of MMC (10,mg MMC/kg body weight, three times per week) in severe combined immunodeficient (SCID) mice bearing E40- and C40-xenografted tumors. Significant reductions (>3-fold) in tumor growth were detected in MMC-treated C40-xenografted mice. In vivo and in vitro studies demonstrated that AKT- and extracellular signal-regulated kinase (ERK)-mediated survival factors inhibited the Bad-induced mitochondrial apoptotic signals that were involved in E40 tumor cells and that conferred resistance to MMC. © 2010 Wiley-Liss, Inc. [source] A double three-step theory of brain metastasis in mice: the role of the pia mater and matrix metalloproteinasesNEUROPATHOLOGY & APPLIED NEUROBIOLOGY, Issue 3 2007N. Saito The brain is frequently affected by the spread of lung cancer, and haematogenous metastasis is a common route to brain metastasis. We therefore developed an isogenic brain metastasis model of lung cancer to use the Lewis lung carcinoma cell line and analysed dynamics of neoplastic cells after extravasation. Histological analysis revealed two characteristic patterns: metastatic foci exhibiting an angiocentric pattern were designated ,perivascular proliferations'; neoplastic cells infiltrating the brain parenchyma were designated ,invasive proliferations'. Electron microscopic observation of perivascular proliferations showed that neoplastic cells were confined to the perivascular space. In invasive proliferations, however, fragments of collagen fibre were observed in the gaps between neoplastic cells, indicating that the neoplastic cells had disintegrated the pia-glial membrane. We analysed the expressions of matrix metalloproteinase-2 (MMP-2) and MMP-9 by using both immunohistochemical analysis and real-time polymerase chain reaction analysis. MMP-2 expression was significantly higher in invasive proliferations. MMP-9 expression was significantly higher in day 7, but there was no significant difference in day 11. The pia-glial membrane and perivascular space are the barriers that neoplastic cells must overcome to infiltrate the brain. In conclusion, our findings suggest that brain metastasis requires two distinct processes. [source] Induction of apoptosis in the LNCaP human prostate carcinoma cell line and prostate adenocarcinomas of SV40T antigen transgenic rats by the Bowman,Birk inhibitorPATHOLOGY INTERNATIONAL, Issue 11 2009MingXi Tang The soybean-derived serine protease inhibitor, Bowman,Birk inhibitor (BBI), has been reported as a potent chemoprevention agent against several types of tumors. The present study was undertaken to evaluate the effects of BBI on androgen-sensitive/dependent prostate cancers using a human prostate cancer cell (LNCaP) and the transgenic rats developing adenocarcinoma of the prostate (TRAP) model. Treatment of LNCaP prostate cancer cells with 500 µg/mL BBI resulted in inhibition of viability measured on WST-1 assays, with induction of connexin 43 (C×43) and cleaved caspase-3 protein expression. Feeding of 3% roughly prepared BBI (BBIC) to TRAP from the age 3 weeks to 13 weeks resulted in significant reduction of the relative epithelial areas within the acinus and multiplicity of the adenocarcinomas in the lateral prostate lobes. C×43- and terminal deoxynucleotidyl transferase mediated dUTP-biotin end labeling of fragmented DNA (TUNEL)-positive apoptotic cancer cells were more frequently observed in the lateral prostates treated with BBIC than in the controls. These in vivo and in vitro results suggest that BBI possesses chemopreventive activity associated with induction of C×43 expression and apoptosis. [source] Polymeric Photosensitizer Prodrugs for Photodynamic TherapyPHOTOCHEMISTRY & PHOTOBIOLOGY, Issue 4 2007Marino A. Campo ABSTRACT A targeting strategy based on the selective enzyme-mediated activation of polymeric photosensitizer prodrugs (PPP) within pathological tissue has led to the development of agents with the dual ability to detect and treat cancer. Herein, a detailed study of a simple model system for these prodrugs is described. We prepared "first-generation" PPP by directly tethering the photosensitizer (PS) pheophorbide a to poly-(l)-lysine via epsilon amide links and observed that by increasing the number of PS on a polymer chain, energy transfer between PS units improved leading to better quenching efficiency. Fragmentation of the PPP backbone by trypsin digestion gave rise to a pronounced fluorescence increase and to more efficient generation of reactive oxygen species upon light irradiation. In vitro tests using the T-24 bladder carcinoma cell line and ex vivo experiments using mouse intestines illustrated the remarkable and selective ability of these PPP to fluoresce and induce phototoxicity upon enzymatic activation. This work elucidated the basic physicochemical parameters, such as water solubility and quenching/activation behavior, required for the future elaboration of more adaptable "second-generation" PPP, in which the PS is tethered to a proteolytically stable polymer backbone via enzyme-specific peptide linkers. This polymer architecture offers great flexibility to tailor make the PPP to target any pathological tissue known to over-express a specific enzyme. [source] p120 catenin is associated with desmogleins when desmosomes are assembled in high-Ca2+ medium but not when disassembled in low-Ca2+ medium in DJM-1 cellsTHE JOURNAL OF DERMATOLOGY, Issue 6 2008Miho KANNO ABSTRACT We recently showed that p120 catenin (p120ctn), which is an armadillo family protein member that binds to E-cadherin (E-cad), is also localized to desmosomes by directly or indirectly binding to desmogleins (Dsg). We examined whether p120ctn is associated with Dsg1 and Dsg3, as compared with E-cad and plakoglobin (PG), in keratinocytes grown in high or low Ca2+, using a human squamous cell carcinoma cell line, DJM-1 cells. The cell lysate of DJM-1 cells grown in high- or low-Ca2+ media was immunoprecipitated with anti-Dsg1/2 and Dsg3 antibodies, and we examined whether p120ctn is associated with Dsg1 and Dsg3. Then, we observed the co-localization between Dsg3 and p120ctn in cells grown in high- or low-Ca2+ medium on double-staining immunofluorescence microscopy using anti-p120ctn and anti-Dsg3 antibodies. Immunoprecipitates with anti-Dsg1/2 and Dsg3 antibodies in cells grown in high-Ca2+ medium contained p120ctn. In contrast, in low-Ca2+ medium, p120ctn was co-immunoprecipitated with neither Dsg1 nor Dsg3, but was co-immunoprecipitated with E-cad in cells grown in both high- and low-Ca2+ media. Dsg3 was associated with PG in cells grown in both low- and high-Ca2+ media. On immunofluorescence microscopy, p120ctn and Dsg3 were independently observed in cells grown in low-Ca2+ medium; p120ctn, but not Dsg3, was observed in a linear pattern at the cell,cell boundary. However, they were co-localized at cell,cell contacts in cells grown in high-Ca2+ medium. Thus, these proteins are not co-localized in low Ca2+ medium. These results suggest that p120ctn plays an important role in Ca2+ -induced desmosome formation. [source] Enhanced transthyretin tetramer stability following expression of an amyloid disease transsuppressor variant in mammalian cellsTHE JOURNAL OF GENE MEDICINE, Issue 2 2009Masakazu Kamata Abstract Background The transthyretin (TTR) amyloidosis is an incurable fatal inherited disease that is characterized by progressive peripheral and autonomic neuropathy. It is caused by missense amyloidogenic mutations in the TTR gene that destabilize the native tetrameric state and lead to the cytotoxic misfolded monomeric state. One interesting variant (T119M) stabilizes heterotetramers with amyloidogenic TTR and, in the reported heterozygous individuals, protects the carriers from disease. In the present study, we characterize in vitro and in vivo the ectopic expression of the human T119M mutant, termed a transsuppressor for TTR amyloid disease. Methods Lentiviral vectors encoding wild or mutant forms of human TTR were constructed and transduced to the human hepatocellular carcinoma cell line, HepG2, or mice. Heterooligomerization between T119M TTR and amyloidogenic variants was analysed by immunoprecipitation following western blotting. Results T119M TTR was stably expressed in transduced HepG2 cells and was secreted as an oligomer that can interact with its native partner, retinol-binding protein. Importantly, the T119M TTR formed secreted heterooligomers with amyloidogenic TTR variants, V30M, L55P and V122I, in HepG2 cells that were more stable than the homooligomers of the same amyloidogenic TTR variants. Human T119M TTR also formed heterooligomers with V30M TTR in transduced mice. Conclusions The results obtained in the present study demonstrate the stabilization of heterotetramers by T119M TTR in human cells and suggest that gene transfer of T119M TTR may have potential as a gene therapy for TTR amyloidosis. Copyright © 2008 John Wiley & Sons, Ltd. [source] Allogeneic retrovirally transduced, IL-2- and IFN-,-secreting cancer cell vaccine in patients with hormone refractory prostate cancer,a phase I clinical trialTHE JOURNAL OF GENE MEDICINE, Issue 7 2007T. H. Brill Background The purpose of this vaccine study was to determine the safety and feasibility of vaccination with an allogeneic prostate carcinoma cell line, LNCaP, expressing recombinant interleukin-2 (IL-2) and interferon-, (IFN-,) and to evaluate the efficacy of inducing tumor-specific immune responses in HLA-A2-matched patients with hormone refractory prostate cancer (HRPC). Methods In a dose-escalating phase I study, HLA-A2-matched HRPC patients received four vaccinations of irradiated allogeneic LNCaP cells retrovirally transduced to secrete IL-2 and IFN-, at study day 1, 15, 29 and 92 and subsequently every 91 days unless tumor progression was evident. Results Three patients receiving the first dose level (7.5 million cells) showed no evidence of dose-limiting toxicity or vaccine-related adverse events including autoimmunity. One of three patients receiving the second dose level (15 million cells) developed a transient self-limiting grade 3 local injection site reaction (ulceration) after the eighth vaccination. Vaccine-induced immune responses against a broad array of prostate tumor associated antigens were detected in all six patients. Two of the three patients receiving the higher dose showed a decline in serum prostate-specific antigen (PSA) values of more than 50%, with one patient remaining on protocol for 3 years. Conclusions Immunisation with the allogeneic LNCaP/IL-2/IFN-, vaccine is safe and feasible without any dose-limiting toxicity or autoimmunity. A 50% PSA decline was achieved in two of the six patients. This encouraging data provides the scientific rationale for further investigation of the vaccine in a phase II trial. Copyright © 2007 John Wiley & Sons, Ltd. [source] Role of Nitric Oxide in the Development of Distant Metastasis From Squamous Cell Carcinoma,THE LARYNGOSCOPE, Issue 2 2007Richard L. Scher MD Abstract Background: Metastasis, the dissemination of malignant cells to distant sites, remains one of the most significant factors responsible for death from cancer. Recent studies have shown some improvement in the rate of distant metastasis (DM) with the addition of chemotherapy to surgery and radiation for treatment of head and neck squamous cell carcinoma (HNSCC). However, diagnosis and treatment at an early stage ultimately leads to a better prognosis. The prediction of which patients will develop metastasis and the selection of treatment most effective at preventing and treating metastasis remains dependent on an incomplete understanding of prognostic factors and the biological and molecular basis for metastatic development. This study was undertaken using an in vivo model to investigate the possible role of nitric oxide (NO) in the development of metastasis from HNSCC. The findings will result in better understanding of the metastatic process for HNSCC, with the potential to develop and implement therapies that could prevent and treat metastasis in patients. Objectives/Hypothesis: 1) To analyze whether in vivo videomicroscopy (IVVM) is useful for the study of DM from squamous cell carcinoma of the head and neck; 2) with use of IVVM, investigate the effect of the biological mediators NO and interleukin (IL)-1 on the adhesion of circulating human HNSCC cells in the hepatic microcirculation. Study Design: Prospective study using an animal model. Methods: Phase 1: athymic nude rats and mice were used for IVVM experiments. The cremaster muscle and liver, used as arterial and venous flow models, were tested to determine whether IVVM was useful for the study of human HNSCC interactions with the microcirculation. A human squamous cell carcinoma cell line (FaDu) labeled with the intracytoplasmic fluorescent marker BCECF-am. was used for all experiments. Videomicroscopic images of FaDu cells in the microcirculation were analyzed for cell adhesion, morphology, deformation, circulation, location of adhesion within the microcirculation, and alteration of microvascular circulation. Phase 2: the effect of IL-1, NO, and NO inhibitors on HNSCC cell adhesion in the hepatic microcirculation of nude mice was analyzed by IVVM. This was followed by histologic determination of the ratio of FaDu cells present for liver area analyzed. Nude mice were treated with 1) IL-1; 2) L-arginine (an NO substrate); or 3) L-N-monomethyl-L-arginine (an NO synthase inhibitor) alone or in combination. These data were analyzed statistically to determine the effect on cell adhesion in the liver. Results: IVVM provided a method for the study of circulating HNSCC with the microcirculation in both the cremaster and liver models. FaDu cells were arrested at the inflow side of the circulation, with maintenance of cell integrity. L-arginine and IL-1 both increased FaDu cell arrest in the liver above baseline (P = .00008 and P = .03), and the combination of these agents potentiated the effect (P = .000009). Conclusions: IVVM allows direct assessment of circulating HNSCC with the microcirculation and is a powerful model for the study of DM. NO and IL-1 play a role in increasing the arrest of HNSCC in the liver and are important in the generation of DM in patients with HNSCC. [source] | |||||||||||||||||||||||||||||||||||||