Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UMLS:C0025202 (melanoma)
 69,561 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

We applied an enzyme-linked immunospot (ELISPOT) assay for the detection and quantification of blood-derived CD8+ T cells recognizing peptide antigens presented by HLA-A2.1. CD8+ T lymphocytes were isolated from peripheral blood and were stimulated for 40 h with peptide-loaded A2.1-positive 0.174 x CEM.T2 cells. Tumor necrosis factor alpha (TNF-alpha) secreted by single T cells in response to antigen contact was trapped on nitrocellulose membranes precoated with anti-TNF-alpha antibodies and was then immunochemically visualized as spots. With this assay, up to 25% of cloned cytolytic T lymphocytes (CTL) were detected during the test period that recognized defined melanoma antigens in association with HLA-A2.1. CD8+ lymphocytes responsive to a known immunogenic HLA-A2.1-binding peptide from reverse transcriptase of the human immunodeficiency virus (HIV) were only detectable in HIV-infected patients, but not in anti-HIV-negative donors. T cells reacting with a peptide derived from a mutated cyclin-dependent kinase 4 (CDK4-R24C) were exclusively detected among CD8+ lymphocytes isolated from blood of the patient, whose melanoma had previously been found to carry the CDK4-R24C allele. T cells responding to HLA-A2.1-associated peptides of normal melanocyte differentiation antigens tyrosinase and Melan-A/MART-1 were found at low frequencies in almost all donors tested, which might reflect a natural autoimmunity to these antigens. However, in a melanoma patient we found a few days after surgery of melanoma metastases high frequencies of T cells against Melan-A/MART-1 and tyrosinase peptides (up to 38 per 10(5) CD8+ T cells), which gradually decreased during the following months. In an HIV-infected patient with progressive disease we observed a loss of T cells reactive with the HIV reverse transcriptase peptide. These observations provide evidence that peptide-dependent TNF-alpha spot formation in vitro resulted from previous antigen exposure in vivo. Therefore, the TNF-alpha ELISPOT assay might be useful in monitoring antigen-specific T lymphocyte responses during the natural course of diseases as well as during therapeutic interventions aiming at the induction of protective T cell immunity. In addition, it might help to identify immunodominant T cell epitopes.
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PMID:Detection and quantification of blood-derived CD8+ T lymphocytes secreting tumor necrosis factor alpha in response to HLA-A2.1-binding melanoma and viral peptide antigens. 866 32

Choroidal and ciliary body melanomas disseminate exclusively by a hematogenous route because there are no lymphatics inside the eye. Although angiogenesis is an absolute precondition for metastasis in this tumor system, not all morphologic expressions of tumor angiogenesis are associated with metastasis from choroidal and ciliary body melanomas. Specifically, the remodeling of the microcirculation to form vascular networks is very strongly associated with metastasis. Type VI collagen is upregulated in tissue remodeling and the generation of tissue patterns and is either not present in the normal choroid or present at very low levels. This study was designed to investigate the possible expression of type VI collagen in the stroma of choroidal and ciliary body melanomas. Type VI collagen was detected in tissue sections from five primary choroidal melanomas and three melanomas involving the choroid and ciliary body in the subendothelial compartment of the microcirculation and in avascular areas by immunohistochemistry. Melanoma cell lines were established from each of these tumors. Cultured melanoma cells invaded into type I collagen gels and expressed type VI collagen by immunohistochemistry. Using specific primers for human type VI collagen, the expected band size (413 base pairs) was isolated from one of the cell lines by reverse transcriptase PCR. The presence of type VI collagen in the melanoma tumor stroma reflects active remodeling of the uveal extracellular matrix microenvironment by the melanoma cells themselves. Before the formation of the microvasculature, the expression of type VI collagen and of the other matrix components, such as hyaluronan, to which it binds, may erect a scaffold permitting the formation of higher order stromal patterns such as vascular networks. These stromal patterns, which are markers of tumor progression, may be detectable clinically by a specialized form of ultrasonography that detects backscatterers of the same dimension as tissue compartments encircled by vascular loops in networks.
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PMID:Expression of type VI collagen in uveal melanoma: its role in pattern formation and tumor progression. 868 40

To form distant metastases, tumour cells must stabilize adhesive interactions that prevent detachment at secondary sites. Primary receptor-ligand interactions alone may not maintain prolonged adhesive contacts without secondary events that lead to adhesion stabilization. Computerized imaging methods enable us to examine various substrates for: (i) the wall shear adhesion threshold (WSAT), a measure of the dynamic adhesive potential of tumour cells; (ii) the number of tumour cells that adhered; and (iii) the adhesion stabilization lag time (ASLT) or length of time required for tumour cells to stabilize adhesive contacts capable of withstanding high wall shear force (up to 100 dynes/cm2). The relative WSAT ratios found were: wheat germ agglutinin (WGA) > laminin > fibronectin > vitronectin > collagen I > collagen IV > von Willebrand factor (vWF) (the greater the shear rate the higher the adhesive potential). The relative stabilization ratios found were as follows: laminin < fibronectin < vitronectin < collagen IV < collagen I < vWF < WGA (shorter times correlate with greater stabilization potential). Stabilization data using fibronectin as a substrate correlated the best with metastatic potential. Using three melanoma lines of different metastatic potential semiquantitative reverse transcriptase-polymerase chain reaction (PCR) showed a two- to four-fold increase in alpha1, alpha3, alpha4, alpha5, alpha6, and ICAM-1 in the highly metastatic 70W cells compared to the MeWo and non-metastatic 3S5 melanoma cells. There were no differences in alphav, beta1 and beta3 levels among the three melanoma lines, and PCR products for alphaIIb, alpha2, CD36, or ICAM-2 were not detected. The 70W cells also had higher levels of alphax and beta2 (CD11/CD18 and p150 leukocyte antigen) than either the MeWo or 3S5 cells. The data indicate that melanoma cells exhibit differences in the adhesion properties under fluid shear and differences in the expression of adhesion components that correlate with their metastatic potential.
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PMID:Human melanoma integrins contribute to arrest and stabilization potential while flowing over extracellular matrix. 871 81

Sequence analysis of two clones found repressed in melanoma cell lines in earlier studies showed 9F2 to be identical with the TRP-1 gene and 6F5 with TRP-2 containing a long untranslated 3' end. For further investigation of the expression of the tyrosinase gene family in normal and malignant melanocytic cells, a series of melanoma cell lines and of cultured melanocytes were analyzed by Northern blotting and by reverse transcriptase-polymerase chain reaction (RT-PCR). The Northern blots were probed with cDNA fragments specific for TRP-1, TRP-2, and tyrosinase, for nested tyrosinase-PCR the outer primers specified a 284 bp and the nested primers a 207 bp fragment. Investigations on 14 established melanoma cell lines grown in different media compared with seven normal human melanocyte (NHM) cultures revealed that all three pigment genes were expressed in NHM, whereas pigment gene expression was found repressed in nearly all melanoma cell lines and was completely absent in 4 of 14 specimen. In particular, tyrosinase and TRP-2 genes were found always to be expressed together, and TRP-1 mRNA alone was absent in four melanoma cell lines. Negativity of cultured melanoma cells for tyrosinase mRNA was confirmed by nested RT-PCR, and gene deletion was ruled out by genomic Southern blots. The gene expression seemed independent from the type of medium used for cultivation. These findings indicate repressed or lacking expression of pigment genes in melanoma cell lines, most likely due to regulatory mechanisms, and that differences may exist between tyrosinase and TRP-2 on one hand and TRP-1 on the other. Overall, it seemed that RT-PCR for tyrosinase has limited value for identifying melanoma cells in the peripheral blood of melanoma patients; TRP-1, TRP-2, and other, additional markers may be required.
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PMID:Incomplete expression of the tyrosinase gene family (tyrosinase, TRP-1, and TRP-2) in human malignant melanoma cells in vitro. 878 39

We have previously reported that expression of the melanoma-associated antigen (MAA) recognized by MM2-9B6 monoclonal antibody in B16 melanoma was closely associated with C-type ecotropic retroviral particle production. Our present data show that this MAA is encoded by the env gene of an ecotropic retrovirus produced by B16 melanoma cells. Transfection of H-2Kb or H-2Kd genes into two individual clones isolated from B16BL6 melanoma, BL6-8 (H-2Kb-, H-2Db+) and BL6-2 (H-2Kb-, H-2Db-), resulted in a loss of MAA expression. Electron immunohistochemical analysis of melanoma cells and reverse transcriptase assay revealed that the loss of MAA expression in the H-2K gene-transfected cells paralleled the elimination of retroviral particles. In contrast, expression of the endogenous H-2Db gene or transfection with the H-2Dd or H-2Ld gene had no effect on MAA expression or retrovirus production. Northern blot analysis showed equivalent retroviral messages in retrovirus-producing and -nonproducing BL6 melanoma clones. Southern blot analysis revealed that H-2Kb-negative BL6 melanoma cells contain at least four different ecotropic retroviruses with different insertion sites that somatically emerged during malignant transformation or progression. Restriction enzyme analysis showed various changes in proviral DNAs from the H-2Kb- and H2Kd-transfected cells that failed to produce retroviral particles. The observed alterations in the patterns of PstI- and HindIII-digested proviral DNA were found to be due to the appearance of PstI and loss of HindIII restriction sites in the pol region as a result of several nucleotide substitutions. Thus, BL6 melanoma cells produce melanoma-specific ecotropic murine leukemia viruses that encode serologically detectable cell surface MAA. The transfection of BL6 melanoma cells with H-2Kb or H2Kd genes but not H-2Dd or H-2Ld genes resulted in a loss of MAA expression that was attributed to the changes in proviral DNA and loss of melanoma-specific ecotropic retrovirus particle production.
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PMID:Inhibition of melanoma-associated antigen expression and ecotropic retrovirus production in B16BL6 melanoma cells transfected with major histocompatibility complex class I genes. 881 42

Mechanisms of phagocytosis are complex and incompletely understood. The retinal pigment epithelium provides an ideal system to study the specific aspects of phagocytosis since an important function of this cell is the ingestion of packets of membranous discs that are normally discarded at the apical ends of rod and cone cells during outer segment renewal. Here we provide evidence that rod outer segment phagocytosis by retinal pigment epithelium is mediated by CD36, a transmembrane glycoprotein which has been previously characterized on hematopoietic cells as a receptor for apoptotic neutrophils and oxidized low density lipoprotein. Immunocytochemical staining with monoclonal and polyclonal antibodies demonstrated CD36 expression by both human and rat retinal pigment epithelium in transverse cryostat sections of normal retina and in primary cultured cells. By western blot analysis of retinal pigment epithelial cell lysates, polyclonal and monoclonal antibodies to CD36 recognized an 88 kDa protein which comigrated with platelet CD36. Furthermore, the synthesis of CD36 mRNA by retinal pigment epithelium was confirmed by reverse transcriptase-PCR using specific CD36 oligonucleotides. The addition of CD36 antibodies to cultured retinal pigment epithelial cells reduced the binding and internalization of 125I-labeled rod outer segments by 60%. Immunofluorescence confocal microscopy confirmed that outer segment uptake was significantly diminished by an antibody to CD36. Moreover, we found that transfection of a human melanoma cell line with CD36 cDNA enabled these cells to bind and internalize isolated photoreceptor outer segments as seen by double immunofluorescent staining for surface bound and total cell-associated rod outer segments, and by measurement of cell-associated 125I-labeled rod outer segments. We conclude that the multifunctional scavenger receptor CD36 participates in the clearance of photoreceptor outer segments by retinal pigment epithelium and thus, participates in the visual process.
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PMID:CD36 participates in the phagocytosis of rod outer segments by retinal pigment epithelium. 883 62

Interleukin-8 (IL-8) is a cytokine that is thought to promote melanoma tumour progression. We evaluated and adapted a non-radioactive, reverse transcriptase-polymerase chain reaction method for semiquantitative analysis of IL-8 mRNA expression. Using this technique we studied the regulation of IL-8 levels in the melanoma cell line Colo 38. Seeding of melanoma cells into culture dishes resulted in a significant increase of IL-8 expression, which could be attributed to adherence. A pronounced increase of IL-8 mRNA expression and protein production was induced by tumour necrosis factor-alpha (TNF alpha). Interferon-gamma (IFN gamma) partially inhibited TNF alpha-induced IL-8 secretion, whereas no influence on IL-8 mRNA levels was detected. The inhibitory affect of IFN gamma on melanoma cells is in contrast to its stimulatory effect on melanocytes.
Melanoma Res 1996 Aug
PMID:Regulation of interleukin-8 mRNA expression and protein secretion in a melanoma cell line by tumour necrosis factor-alpha and interferon-gamma. 887 50

The direct introduction of foreign genes into tumors shows promise as a therapeutic modality to enhance tumor immunogenicity. Hence, melanoma nodules were directly injected with a vector encoding an allogeneic MHC class I molecule, HLA-B7. Tumor-infiltrating lymphocytes (TIL) were isolated from cutaneous melanoma biopsies before and after HLA-B7 gene transfer. TIL were expanded in interleukin-2 (IL-2) by standard techniques for approximately 4 weeks, then analyzed for T cell receptor V beta usage by quantitative reverse transcriptase polymerase chain reaction (RT-PCR). Prior to gene transfer. TIL V beta usage was found to be highly restricted, the only one to four V beta families being expressed and one or two of these families representing more than 90% of the repertoire. As anticipated, TIL V beta usage varied among patients expressing different HLA types. However, V beta 13 was over-represented in that six of eight patients utilized V beta 13 as a dominant family, regardless of HLA type. Following HLA-B7 gene transfer, TIL V beta usage was markedly altered: (1) V beta families that dominated following gene transfer differed from the V beta families utilized by TIL prior to treatment, and (2) introduction of the HLA-B7 gene resulted in a more diverse repertoire with an increase in the number of V beta families represented. In two patients, TIL were evaluated before treatment and from multiple, distinct melanoma nodules following gene transfer. In these two patients, a comparison was made between TIL V beta profiles obtained after treatment from nodules that had been injected with the HLA-B7 gene or left untreated. Interestingly, the V beta repertoires of TIL from uninjected nodules following gene transfer were similar to that of TIL from injected nodules, rather than pretreatment TIL. These data demonstrate a direct immunological effect of foreign MHC gene transfer into human melanoma, and suggest that local expression of an allogeneic MHC molecule generates systemic alterations in the antitumor immune response.
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PMID:Direct transfer of a foreign MHC gene into human melanoma alters T cell receptor V beta usage by tumor-infiltrating lymphocytes. 891 36

To evaluate the specificity and applicability to the study of human tumor cells of the reverse transcription (RT) in situ PCR and RT polymerase chain reaction (PCR) in situ hybridization techniques, we examined five melanoma cell lines and five nonmelanoma lines for tyrosinase mRNA using primers specific for tyrosinase. Each procedural step was optimized and minutely controlled, and results from the in situ techniques and solution-phase RT-PCR were compared. All melanoma lines showed a specific pattern of perinuclear cytoplasmic reaction not seen in nonmelanoma lines. There was exact agreement between the results from the RT in situ PCR and RT-PCR in situ hybridization techniques and those from solution-phase RT-PCR. Ribonuclease digestion abolished cytoplasmic staining, as did omission of the reverse transcriptase step. Nuclear staining was seen in melanoma and nonmelanoma lines, apparently as a result of DNA synthesis from repair-replication and mispriming or nonspecific amplification. Neither high concentrations of deoxyribonuclease nor long incubation periods abolished this effect completely. Demonstration of cytoplasmic mRNA by RT in situ PCR and RT-PCR in situ hybridization specifically identifies cells of melanocytic lineage.
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PMID:Demonstration of cytoplasmic tyrosinase mRNA in tissue-cultured cells by reverse transcription (RT) in situ polymerase chain reaction (PCR) and RT PCR in situ hybridization. 902 34

While not all circulating tumor cells survive, one could postulate that patients with circulating tumor cells might be candidates for adjuvant chemotherapy because of the risk of relapse. Recently, reverse transcriptase-polymerase chain reaction (RT-PCR) for the detection of circulating tumor cells has been suggested as a potential technique for staging of cancer. In malignant melanoma, the detection of circulating melanoma cells by tyrosinase RT-PCR correlated with the clinical stage of patients and was an independent prognostic factor for recurrence. A strong association was found between the detection of neuroblastoma cells in circulation by tyrosine hydroxylase RT-PCR with bone marrow micrometastasis. This method may be of use to identify early signs of relapse in disease-free patients. In prostatic cancer, the frequency of positivity for detection of circulating tumor cells in peripheral blood by PSA RT-PCR increased with tumor stages but a significant proportion of patients with metastatic disease were negative. The prognostic significance of the detection of tumor cell in blood in other epithelial tumors is not established and will require further long-term follow-up study.
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PMID:[Molecular diagnostic detection of circulating tumor cells and their prognostic implications]. 905 Nov 25


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