Angiostatin inhibits endothelial and melanoma cellular invasion by blocking matrix-enhanced plasminogen activation.
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Angiostatin, a kringle-containing fragment of plasminogen, is a potent inhibitor of angiogenesis. The mechanism(s) responsible for the anti-angiogenic properties of angiostatin are unknown. We now report that human angiostatin blocks plasmin(ogen)-enhanced in vitro invasion of tissue plasminogen activator (t-PA)-producing endothelial and melanoma cells. Kinetic analyses demonstrated that angiostatin functions as a non-competitive inhibitor of extracellular-matrix (ECM)-enhanced, t-PA-catalysed plasminogen activation, with a Ki of 0.9+/-0.03 microM. This mechanism suggests that t-PA has a binding site for the inhibitor angiostatin, as well as for its substrate plasminogen that, when occupied, prevents ternary complex formation between t-PA, plasminogen and matrix protein. Direct binding experiments confirmed that angiostatin bound to t-PA with an apparent Kd [Kd(app)] of 6.7+/-0.7 nM, but did not bind with high affinity to ECM proteins. Together, these data suggest that angiostatin in the cellular micro-environment can inhibit matrix-enhanced plasminogen activation, resulting in reduced invasive activity, and suggest a biochemical mechanism whereby angiostatin-mediated regulation of plasmin formation could influence cellular migration and invasion. (+info)
An antigen-targeted approach to adoptive transfer therapy of cancer.
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Previous attempts to treat human malignancies by adoptive transfer of tumor-specific CTLs have been limited by the difficulty of isolating T cells of defined antigen specificity. The recent development of MHC class I/antigenic peptide tetrameric complexes that allow direct identification of antigen-specific T cells has opened new possibilities for the isolation and in vitro expansion of tumor-specific T cells. In the present study, we have derived polyclonal monospecific cell lines from circulating Melan-A-specific CTL precursors of HLA-A*0201+ melanoma patients by combining stimulation with recently identified peptide analogues of the immunodominant epitope from the melanoma-associated antigen Melan-A with staining with fluorescent HLA-A*0201/Melan-A peptide tetramers. In vitro expansion of antigen-specific CD8+ T cells was monitored by flow cytometry with the fluorescent tetramers and anti-CD8 monoclonal antibody. This analysis revealed that Melan-A 26-35 peptide analogues were much more efficient than the parental peptides in stimulating a rapid in vitro expansion of antigen-specific CD8+ T cells. These cells were then isolated by tetramer-guided cell sorting and subsequently expanded in vitro by mitogen stimulation. The resulting polyclonal but monospecific CTLs fully cross-recognized the parental peptides and were able to efficiently lyse Melan-A-expressing tumor cells. Altogether, these results pave the way to a molecularly defined approach to antigen-specific adoptive transfer therapy of cancer. (+info)
Overexpression of sialomucin complex, a rat homologue of MUC4, inhibits tumor killing by lymphokine-activated killer cells.
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Sialomucin complex (SMC) is a large heterodimeric glycoprotein complex composed of a mucin subunit ascites sialoglycoprotein-1 and a transmembrane subunit ascites sialoglycoprotein-2. It is a rat homologue of human mucin gene MUC4 and is abundantly expressed on the cell surface of highly metastatic ascites 13762 rat mammary adenocarcinoma cells. Because of their extended and rigid structures, mucin-type glycoproteins are suggested to have suppressing effects on cell-cell and cell-matrix interactions. During the metastatic process, these effects presumably cause tumor cell detachment from the primary tumor mass and facilitate escape of the tumor cells from immunosurveillance. Analyses of human breast cancer cells in solid tumors and tumor effusions showed that the more aggressive cells in effusions are stained with polyclonal antibodies against SMC more frequently than cells in solid tumors, suggesting a role for MUC4/SMC in tumor progression and metastasis. Previously, we generated recombinant cDNAs for SMC that vary in the number of mucin repeats to study the putative functions of SMC in tumor metastasis. These cDNAs were transfected into human cancer cell lines and tested for the effect of the expression of this gene. Here, using a tetracycline-responsive inducible expression system, we demonstrate that overexpression of SMC masks the surface antigens on target tumor cells and effectively suppresses tumor cell killing by cytotoxic lymphocytes. This effect results from the ability of SMC to block killer cell binding to the tumor cells and is dependent on both overexpression of the mucin and the number of mucin repeats in the expressed SMC. These results provide an explanation for the proposed role of SMC/MUC4 in tumor progression. (+info)
Specific lysis of melanoma cells by receptor grafted T cells is enhanced by anti-idiotypic monoclonal antibodies directed to the scFv domain of the receptor.
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Malignant transformation of melanocytes is frequently associated with abnormalities in antigen processing and in human leukocyte antigen class I antigen expression. Here, we evaluated a human leukocyte antigen class I antigen-independent approach to target cytotoxic T lymphocytes to melanoma cells by grafting cytotoxic T lymphocytes with a chimeric receptor that consists of both a domain binding to high molecular weight-melanoma associated antigen and a cellular activation domain. The binding domain is a single-chain antibody fragment (scFv) derived from the monoclonal anti-high molecular weight-melanoma associated antigen antibody 763.74 by phage display techniques. The cellular activation domain is the signaling unit of the FcepsilonRI receptor gamma chain. Both domains constitute the chimeric receptor scFv763.74-gammaR. Cytotoxic MD45 T cells grafted with the scFv763.74-gammaR receptor bind specifically to high molecular weight-melanoma associated antigen-positive melanoma cells and lyse melanoma cells in a human leukocyte antigen class I independent fashion. Pre-incubation of receptor grafted T cells with immobilized anti-idiotypic (id) monoclonal antibody MK2-23 binding to the scFv domain of the receptor enhanced the lysis of melanoma cells indicating that the specific cytolytic activity of receptor grafted T cells can be increased by costimulation with cross-linked anti-idiotypic monoclonal antibodies that recognize the antigen binding domain of the chimeric receptor. (+info)
Down-regulation of tumor necrosis factor alpha expression by activating transcription factor 2 increases UVC-induced apoptosis of late-stage melanoma cells.
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To identify mechanisms whereby activating transcription factor 2 (ATF2) alters the radiation resistance of human melanoma cells, we examined the possible role of ATF2 in UVC-induced apoptosis. Forced expression of full-length or truncated (Delta1-195 amino acids) forms of ATF2 in LU1205, a late-stage human melanoma cell line, elevated the levels of UVC-induced apoptosis. At the same time, either truncated or full-length forms of ATF2 reduced UVC-induced activation of the tumor necrosis factor-alpha (TNFalpha) promoter and decreased expression of TNFalpha. Forced expression of c-Jun in ATF2-expressing melanoma cells restored TNFalpha expression, suggesting that both forms of ATF2 sequestered transcription factors that positively regulate TNFalpha expression in response to UV irradiation. Antagonistic antibodies to Fas, but not to TNFR1, efficiently suppressed UVC-induced apoptosis, suggesting that the Fas pathway mediates the primary apoptotic signal in melanoma cells whereas the TNFR1 pathway elicits a survival signal. Indeed, treatment of melanoma cells with TNFalpha before UVC irradiation partially suppressed UVC-induced apoptosis, further supporting the protective role of TNFalpha in UVC-treated melanoma cells. Taken together, our findings suggest that ATF2 contributes to UVC-induced apoptosis through transcriptional silencing of TNFalpha, which balances Fas-mediated cell death in melanoma. (+info)
Pharmacological reactivity of neoplastic and non-neoplastic associated neovasculature to vasoconstrictors.
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Angiogenesis and the pharmacological responses of the tumour and non-tumour associated neovasculature have been investigated. Cannulated sponge discs in mice were used to host the angiogenic stimulators, while 133Xe washout was employed to assess local blood flow. Enhancement of blood flow was detected in implants bearing B16 cells, 3T3 cells and angiotensin II (AII)-treated at day 7. The responses of non-neoplastic associated neovasculature at day 14 post sponge implantation to the vasoconstrictors used endothelin-1 (Et-1), AII, platelet activating factor (PAF) and 5-hydroxytryptamine (5-HT) were dose-dependent. By contrast, the newly formed blood vessels induced by tumour cells were markedly insensitive to the vasoconstrictors agonists Et-1 and AII, while fully responsive to PAF and 5-HT. The vessels resulting from neoplastic stimulus exhibited altered pharmacological reactivity, suggesting that the characteristics of the neovasculature are dependent on the nature of the angiogenic stimuli. (+info)
Imaging features of intraventricular melanoma.
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We present the MR imaging findings in a patient with symptoms of increased intracranial pressure and a mass in the left lateral ventricle. The mass showed increased signal intensity on T1-weighted images and low signal intensity on T2-weighted images. The histologic diagnosis was that of melanoma, and detailed physical and funduscopic examinations disclosed no evidence of a primary lesion. We believe that the mass was a primary intraventricular melanoma, possibly arising from the choroid plexus, and we discuss the mechanisms that may be responsible for its occurrence in this location. (+info)
Identification of a novel major histocompatibility complex class II-restricted tumor antigen resulting from a chromosomal rearrangement recognized by CD4(+) T cells.
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CD4(+) T cells play an important role in antitumor immune responses and autoimmune and infectious diseases. Although many major histocompatibility complex (MHC) class I-restricted tumor antigens have been identified in the last few years, little is known about MHC class II- restricted human tumor antigens recognized by CD4(+) T cells. Here, we describe the identification of a novel melanoma antigen recognized by an human histocompatibility leukocyte antigen (HLA)-DR1-restricted CD4(+) tumor-infiltrating lymphocyte (TIL)1363 using a genetic cloning approach. DNA sequencing analysis indicated that this was a fusion gene generated by a low density lipid receptor (LDLR) gene in the 5' end fused to a GDP-L-fucose:beta-D-galactoside 2-alpha-L-fucosyltransferase (FUT) in an antisense orientation in the 3' end. The fusion gene encoded the first five ligand binding repeats of LDLR in the NH2 terminus followed by a new polypeptide translated in frame with LDLR from the FUT gene in an antisense direction. Southern blot analysis showed that chromosomal DNA rearrangements occurred in the 1363mel cell line. Northern blot analysis detected two fusion RNA transcripts present only in the autologous 1363mel, but not in other cell lines or normal tissues tested. Two minimal peptides were identified from the COOH terminus of the fusion protein. This represents the first demonstration that a fusion protein resulting from a chromosomal rearrangement in tumor cells serves as an immune target recognized by CD4(+) T cells. (+info)