RESUMEN
Realizamos el analisis de 75 melanomas primarios(25 por ciento ) que dieron metastasis,de una serie continua de 296 con el objeto de catalogarlos segun su estadio. Analizamos edad,sexo color de piel y de ojos,lesiones pigm,entadas previas,sindrome del nevo displasico,melanomas multiples,melanomas familiares,localizacion del primitivo,tipo,nivel,espesor,ulceracion,tipo celular predominante,indice miotico,infiltrado linfositario,regresion,estadio,tiempo que transcurre hasta el diagnostico del primario y de este al final del seguimiento.El estadio se determino segun la clasificacion utilizada por la American Joint Committee on Cancer-Union Internationale contre le Cancer(A.J.C.C.-U.I.C.C.) corregida en 1992. En nuestra serie obtuvimos los siguientes en 65 casos:estadop 1,7,(IA:pt1 No Mo y IB 5 pt2 No Mo);II,19,(IIA:7 pt3 No Mo y IIB 12 pt4 No Mo);III,30,(cualquier pt N1 Mo 11,cualquier pt N2 Mo 8 y en 11 casos no se determ,ino el tamanio de las metastasis ganglionares);IV,9,(cualquier pt,cualquier N,M1): Fueron esxcluidos 10 melanomas porque a)dicha clasificacion no contempla ciertas situaciones:ocular(2),recaida(2),secundario a nevo intradermico (1) y a nevo azul(1),polipoide(1),y escrotal(1),[por invacion del musculo dartoico,b)por falta de detreminacion del espesor (2). La clasificacion de la A.J.C.C.-U.I.C.C.tiene como objeto uniformar estadios para permitir obtener resultados equiparables,con el fin de podercomparar la eficacia terapeutica. Es sucinta y concreta. Seria deseable agregar datos sobre el estudio clinico de la piel,factores desencadenantes ,lesiones precursoras,genetica e inmunologia. Es solo aparentemente multidisciplinaria,aunque creemos sigue siendo la mas util
Asunto(s)
Niño , Adolescente , Adulto , Persona de Mediana Edad , Humanos , Masculino , Femenino , Melanoma/análisis , Melanoma/clasificación , Melanoma/diagnóstico , Metástasis de la Neoplasia , Estadificación de Neoplasias , NevoRESUMEN
Realizamos el analisis de 75 melanomas primarios(25 por ciento ) que dieron metastasis,de una serie continua de 296 con el objeto de catalogarlos segun su estadio. Analizamos edad,sexo color de piel y de ojos,lesiones pigm,entadas previas,sindrome del nevo displasico,melanomas multiples,melanomas familiares,localizacion del primitivo,tipo,nivel,espesor,ulceracion,tipo celular predominante,indice miotico,infiltrado linfositario,regresion,estadio,tiempo que transcurre hasta el diagnostico del primario y de este al final del seguimiento.El estadio se determino segun la clasificacion utilizada por la American Joint Committee on Cancer-Union Internationale contre le Cancer(A.J.C.C.-U.I.C.C.) corregida en 1992. En nuestra serie obtuvimos los siguientes en 65 casos:estadop 1,7,(IA:pt1 No Mo y IB 5 pt2 No Mo);II,19,(IIA:7 pt3 No Mo y IIB 12 pt4 No Mo);III,30,(cualquier pt N1 Mo 11,cualquier pt N2 Mo 8 y en 11 casos no se determ,ino el tamanio de las metastasis ganglionares);IV,9,(cualquier pt,cualquier N,M1): Fueron esxcluidos 10 melanomas porque a)dicha clasificacion no contempla ciertas situaciones:ocular(2),recaida(2),secundario a nevo intradermico (1) y a nevo azul(1),polipoide(1),y escrotal(1),[por invacion del musculo dartoico,b)por falta de detreminacion del espesor (2). La clasificacion de la A.J.C.C.-U.I.C.C.tiene como objeto uniformar estadios para permitir obtener resultados equiparables,con el fin de podercomparar la eficacia terapeutica. Es sucinta y concreta. Seria deseable agregar datos sobre el estudio clinico de la piel,factores desencadenantes ,lesiones precursoras,genetica e inmunologia. Es solo aparentemente multidisciplinaria,aunque creemos sigue siendo la mas util
Asunto(s)
Niño , Adolescente , Adulto , Persona de Mediana Edad , Anciano , Humanos , Masculino , Femenino , Estudio Comparativo , Melanoma/diagnóstico , Melanoma/clasificación , Melanoma/análisis , Estadificación de Neoplasias , Metástasis de la Neoplasia , NevoRESUMEN
Although many reports on chromosome changes in human malignant melanoma (HMM) have been published, it is still impossible to define the significance of the different markers reported. In fact, we think that the difficulties in interpreting the chromosomal abnormalities could be due to poorly defined clinical conditions and a lack of correlation with cytological and histological analyses. To verify this hypothesis, we studied 10 cell lines obtained from 8 patients affected by cutaneous malignant melanoma that were well defined for their clinical, histologic, and cytogenetic aspects. No significant correlation was found among these parameters, and, hence, the cytogenetics findings cannot be used to determine a more detailed diagnosis or a more definite prognosis.
Asunto(s)
Aberraciones Cromosómicas , Melanoma/genética , Humanos , Queratinas/análisis , Melanoma/análisis , Melanoma/patología , Células Tumorales Cultivadas , Vimentina/análisisRESUMEN
Immunohistochemical localization of S-100 protein alpha and beta subunits in the cells of melanocytic nevi and malignant melanomas was studied by using monoclonal antibodies directed against each subunit. Although polyclonal anti-S-100 reactivities have been demonstrated uniformly in all nevus cells and melanoma cells, monoclonal anti-S-100 alpha and anti-S-100 beta reactivities were either absent or rarely found in ordinary junctional nevi or junctional nests of ordinary compound nevi. However, in the junctional nests of dysplastic junctional nevi and junctional components of dysplastic compound nevi, monoclonal anti-S-100 alpha reactivity become more frequent, whereas monoclonal anti-S-100 beta reactivity remains negative. In the superficial variety of melanomas such as superficial spreading melanoma and lentigo maligna melanoma, monoclonal anti-S-100 beta is nonreactive until vertical growth or invasiveness begins. Most nodular melanomas are positively stained with both monoclonal anti-S-100 alpha and anti-S-100 beta. It is suggested that monoclonal anti-S-100 alpha can be an indicator of active junctional nevus of melanocytic nevi and the reactivity with monoclonal anti-S-100 beta may be related to vertical progression of superficial spreading melanomas and lentigo maligna melanomas.
Asunto(s)
Anticuerpos Monoclonales , Biomarcadores , Proteínas de Unión al Calcio/análisis , Melanoma/análisis , Nevo/análisis , Proteínas S100/análisis , Neoplasias Cutáneas/análisis , Glándulas Ecrinas/análisis , Humanos , Técnicas para Inmunoenzimas , Factores de Crecimiento Nervioso , Subunidad beta de la Proteína de Unión al Calcio S100RESUMEN
The existence of beta-adrenoceptors on intact cells of the human malignant melanoma cell line A-375 was investigated using the binding properties of the tritiated radioligand (-)-[3H]CGP-12177, a hydrophilic non-selective beta-adrenoceptor antagonist. Displacement experiments of the radioligand from its binding site were performed with antagonists and agonists to determine the beta-adrenoceptor subtype selectivity. The binding of (-)-[3H]CGP-12177 was saturable, of high affinity (KD = 0.025 nmol/l, n = 12) and was rapid and readily reversible. The maximal number of binding sites (Bmax) was 33.5 +/- 1.9 fmol/10(7) cells or 2018 +/- 114 receptors per cell. beta-adrenoceptor antagonists inhibited binding of the radioligand with monophasic displacement curves. IC50 values were (nmol/l): propranolol (non-selective) 2.82, alprenolol (non-selective) 2.0, ICI 118,551 (beta 2-selective) 3.5 and bisoprolol (beta 1-selective) 2200. Agonists inhibited binding in the order of potency of isoprenaline greater than adrenaline greater than noradrenaline. It is concluded that cells of the melanoma cell line A-375 contain a homogeneous population of beta 2-adrenoceptors.
Asunto(s)
Melanoma/análisis , Receptores Adrenérgicos beta/análisis , Neoplasias Cutáneas/análisis , 4-Cloro-7-nitrobenzofurazano/análogos & derivados , Agonistas Adrenérgicos beta/farmacología , Antagonistas Adrenérgicos beta/farmacología , Humanos , Cinética , Propanolaminas , Ensayo de Unión Radioligante , Células Tumorales Cultivadas/análisisRESUMEN
Treatment of four A375 human melanoma sublines (A375, A375P, A375P-5, A375M), exhibiting distinct metastatic potentials in vivo, with beta-all-trans-retinoic acid in vitro caused a dose- and time-dependent inhibition of the ability of these cells to penetrate Matrigel-coated filters using a reconstituted basement membrane invasion assay. The possible mechanisms of action responsible for the antiinvasive effect were further investigated, and the data showed that compared with untreated cells the retinoic acid-treated cells: (a) secreted lower levels of collagenolytic enzymes, as demonstrated by a decreased ability of the cells to degrade [3H]proline-labeled type IV collagen substrate and by a reduction in the activity of a secreted Mr 64,000 collagenolytic enzyme detected in type IV collagen-containing polyacrylamide gels; (b) expressed lower levels of the human type IV collagenase mRNA (except in the A375P cells), as detected by Northern blot analysis; (c) exhibited decreased levels of tissue plasminogen activator activity, as demonstrated by a chromogenic assay; (d) were 10-40% less adhesive to a reconstituted basement membrane matrix, as determined by a 60-min Na2(51)CrO4-labeled cell attachment assay; (e) exhibited an increase in the high affinity metastasis-associated cell surface laminin receptor, as determined by flow cytometry after binding of fluorescently labeled laminin receptor antibody; and (f) expressed decreased amounts of gp78, a cell surface receptor for motility factor, demonstrated by immunoblotting and immunofluorescence. Collectively, these data suggest that retinoic acid inhibits tumor cell invasion through a basement membrane-like matrix by suppressing matrix degradation and by altering cell surface receptors.
Asunto(s)
Melanoma/patología , Colagenasa Microbiana/análisis , Invasividad Neoplásica , Activadores Plasminogénicos/análisis , ARN Mensajero/análisis , Receptores de Superficie Celular/análisis , Receptores Inmunológicos/análisis , Tretinoina/farmacología , Membrana Basal/efectos de los fármacos , Adhesión Celular/efectos de los fármacos , División Celular/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Inducción Enzimática/efectos de los fármacos , Humanos , Melanoma/análisis , Melanoma/enzimología , Colagenasa Microbiana/genética , Metástasis de la Neoplasia , Receptores de Laminina , Células Tumorales CultivadasRESUMEN
The experiments described in this paper were designed to examine the specific binding of tissue plasminogen activator (tPA) to cultured human aortic endothelial (HAE) cells. When 125I-labelled tPA was incubated with the cells at 4 degrees C, binding was found to plateau within 90 min after incubations were begun. Binding was saturable and the bound enzyme dissociated from the sites with a half-time of approx. 48 min. Scatchard analyses were performed using tPA molecules isolated from human melanoma and colon cells as well as from C127 and Chinese hamster ovary cells that had been transfected with the human tPA gene. These enzymes showed very similar binding characteristics in spite of the fact that they differ substantially in the types of sugars which comprise their side chains. Neither the chainedness of the molecules (one-chain or two-chain) nor the sites at which they are glycosylated (type I or type II) appear to affect their ability to interact with binding sites. The tPA molecules were found to have an average equilibrium dissociation constant of (1.15 +/- 0.10) x 10(-9) M and HAE cells appeared to have a single, homogeneous population of independent binding sites present at a concentration of (1.57 +/- 0.13) x 10(6) sites per cell. Lowering the pH of the binding buffer from 7.4 to 6.5 resulted in a reversible increase in specific binding of between 2-fold and 7-fold depending upon the particular preparation of cells. Preincubation of tPA with plasminogen activator inhibitor 1 (PAI-1) was found to have little effect on binding, suggesting that tPA interacts at sites distinct from surface-bound PAI-1. No evidence for either internalization or degradation of tPA was observed in assays run at 37 degrees C. This suggests that, like urokinase, tPA remains on cell surfaces for an extended period of time.
Asunto(s)
Endotelio Vascular/metabolismo , Activador de Tejido Plasminógeno/metabolismo , Aorta , Sitios de Unión , Línea Celular , Colon/análisis , Glicosilación , Humanos , Concentración de Iones de Hidrógeno , Cinética , Melanoma/análisis , Estructura Molecular , Inactivadores Plasminogénicos/farmacología , Activador de Tejido Plasminógeno/genética , TransfecciónRESUMEN
A case of Spitz's nevus with eosinophilic globules was examined using antibodies for several components of the basement membrane. Aggregated tumor cells revealed the same characteristics as normal nevocytic nevi, that is, they were surrounded by laminin and type-IV collagen, whereas type-VII collagen was absent. All of these components of basement membranes, including type-VII collagen, were also found in eosinophilic globules, which were densely stained by these antibodies. It is assumed that these eosinophilic globules are essentially composed of basement membrane components, which are probably synthesized by epidermal and possibly also by melanocytic tumor cells.
Asunto(s)
Membrana Basal/análisis , Cuerpos de Inclusión/análisis , Melanoma/ultraestructura , Nevo Pigmentado/ultraestructura , Neoplasias Cutáneas/ultraestructura , Membrana Basal/ultraestructura , Colágeno/análisis , Eosina Amarillenta-(YS) , Humanos , Laminina/análisis , Masculino , Melanocitos/análisis , Melanocitos/ultraestructura , Melanoma/análisis , Persona de Mediana Edad , Nevo Pigmentado/análisis , Neoplasias Cutáneas/análisis , Coloración y EtiquetadoRESUMEN
Two autologous human melanoma cell lines were studied to determine their capacities to bind wheat germ agglutinin (WGA). Both cell lines were derived from the same patient, the first, IGR 39, originated from the primary tumor, the second, IGR 37, was established from a metastatic lymph node. WGA binding sites on the surface of these cell lines were compared before and after sialidase and/or tunicamycin treatments. IGR 39 cells exhibited two classes of WGA binding sites with high and low affinities, whereas IGR 37 cells had only one class of high affinity binding sites. After tunicamycin treatment, the capacity of IGR 39 cells to bind WGA was markedly altered, since only one class of WGA binding sites with high affinity was observed under these conditions, whereas tunicamycin did not induce significant changes in the lectin binding of IGR 37 cells. The low affinity WGA binding sites, which were only found on IGR 39 cells, corresponded to sialyl residues present in N-linked glycoproteins. The high affinity binding sites present on both cell lines probably involved sialyl and N-acetyl-glucosaminyl residues associated with O-linked glycoproteins and/or glycolipids. No direct correlation could be drawn between the number of WGA binding sites and the overall sialic acid levels exposed to sialidase treatment. The 3-fold increase in the amount of cell surface glycopeptides obtained after pronase digestion and specifically binding to WGA-Sepharose was in good agreement with the overall higher number of WGA binding sites on IGR 39 compared to IGR 37 cells. Thus, subtle carbohydrate changes of cell surface glycoconjugates might account for the differences between the biological properties of human melanoma cell lines of low and high tumorigenicity.
Asunto(s)
Acetilglucosamina/análisis , Glucosamina/análogos & derivados , Glicoconjugados/análisis , Melanoma/metabolismo , Ácidos Siálicos/análisis , Aglutininas del Germen de Trigo/metabolismo , Sitios de Unión , Cromatografía de Afinidad , Glucolípidos/análisis , Humanos , Melanoma/análisis , Glicoproteínas de Membrana/análisis , Neuraminidasa/farmacología , Células Tumorales Cultivadas , Tunicamicina/farmacologíaRESUMEN
Because its expression appears to be largely restricted to human melanomas, 9-O-acetyl-GD3 is a candidate antigen for vaccine construction. Searching for potential sources, we compared chemically O-acetylated calf brain GD3 and 9-O-acetyl-GD3 extracted from bovine buttermilk with 9-O-acetyl-GD3 from human melanoma. Three fractions (F1-F3) of chemically O-acetylated GD3 differed in the number and position of O-acetyl groups. O-Acetylation sites were the lactose portion in F1 and lactose as well as sialic acid in F2 and F3. Natural (melanoma- or buttermilk-derived) 9-O-acetyl-GD3 was O-acetylated solely on the sialic acid moiety. While F1 was not reactive with monoclonal antibodies against 9-O-acetyl-GD3, F2 and F3 were as reactive as the natural products. Immunization with the natural products induced high-titer antibodies against natural 9-O-acetyl-GD3 as well as F2 and F3. In contrast, mice immunized with the synthetic fractions produced antibodies only against the immunogen but not against natural 9-O-acetyl-GD3. Only immunization with the natural products induced production of antibodies reactive with surface antigens of melanoma cells expressing 9-O-acetyl-GD3. The findings suggest (a) that C-9 of the subterminal sialic acid is the site of chemical O-acetylation in F2 and F3, as opposed to C-9 of the terminal sialic acid in the natural products; (b) that O-acetylation of both the terminal and subterminal sialic acid moieties of GD3 results in recognition by three murine monoclonal antibodies (D1.1, ME 311, and Jones) reactive with human melanoma cells; (c) that O-acetylation of the terminal sialic acid is critical, on the other hand, for inducing an immune response against melanoma 9-O-acetyl-GD3; and (d) that O-acetyl GD3 from bovine buttermilk can substitute as immunogen for inducing an immune response against human melanoma cell surface antigens in the mouse.
Asunto(s)
Gangliósidos/análisis , Inmunoglobulina G/biosíntesis , Melanoma/análisis , Acetilación , Animales , Mantequilla/análisis , Cromatografía Líquida de Alta Presión , Cromatografía en Capa Delgada , Femenino , Gangliósidos/síntesis química , Gangliósidos/inmunología , Gangliósidos/metabolismo , Humanos , Inmunización , Melanoma/inmunología , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BLRESUMEN
To determine the potential utility of antigen-specific immune complex analysis, we developed a competitive enzyme-linked immunosorbent assay utilizing polyclonal human antibody to detect tumor-associated antigen-specific immune complexes. Sera from 10 normal volunteers and 19 patients with recurrent melanoma were studied. Patients with recurrent melanoma had a mean +/- SD percent inhibition of 27.6% +/- 29.8% in contrast to normal individuals with a mean value of 8.4% +/- 17.8%. A monoclonal antibody (MAb JSI) was developed following immunization with a partially purified antigen. Utilizing MAb JSI, we developed a "sandwich" enzyme-linked immunosorbent assay and studied sera from 45 normal volunteers and 44 patients with cancer with recurrent melanoma. Results were expressed as a percent maximum binding of a positive control. The mean +/- SD percent maximum binding for normal subjects was 4.9% +/- 7.7% in contrast to sera from patients with melanoma who had a mean of 38.3% +/- 33.3%. Serial analysis of four patients with melanoma with tumor-associated antigen-specific immune complexes demonstrated the presence of tumor-associated antigen-specific immune complexes up to 12 years prior to clinical recurrence.
Asunto(s)
Complejo Antígeno-Anticuerpo/análisis , Antígenos de Neoplasias/análisis , Biomarcadores de Tumor/análisis , Melanoma/inmunología , Adulto , Anticuerpos Monoclonales , Unión Competitiva , Ensayo de Inmunoadsorción Enzimática/métodos , Femenino , Proteínas Fetales/análisis , Humanos , Masculino , Melanoma/análisis , Recurrencia Local de NeoplasiaRESUMEN
The microtubulus system as a part of the cellular cytoskeleton contributes to cell movement. Microtubulus assembly and disassembly is considered to be essential for tumor invasion and serves as a target for tumor chemotherapy. Using immunohistochemical methods, we investigated the distribution of tubulin in normal skin and 34 melanocytic skin tumors. In normal skin, tubulin was strongly expressed in dermal nerves, melanocytes, fibroblasts within the papillary dermis and in myoepithelial cells. In melanocytic skin tumors, nevus cells and melanoma cells stained positive, particularly at the periphery of the lesions, where there were single cells and small nests. The main difference between benign and malignant melanocytic tumors was found in the stromal cells: In melanocytic nevi, the stromal fibroblasts were entirely tubulin negative; whereas, adjacent to the invasive edge in primary and metastatic malignant melanoma, the stroma fibroblasts were strongly positive. Our results show that tubulin is regularly expressed in melanocytic skin tumors and may serve as a prerequisite for cell movement. The pronounced expression of tubulin in fibroblasts surrounding malignant melanocytic skin lesions reflects a stromal alteration that might contribute to tumor invasion.
Asunto(s)
Melanocitos/análisis , Neoplasias Cutáneas/análisis , Tubulina (Proteína)/análisis , Fibroblastos/análisis , Fibroblastos/patología , Humanos , Inmunohistoquímica , Melanoma/análisisRESUMEN
Immunochemical studies have shown that the monoclonal antibody (MoAb) CL203.4, elicited with immune interferon treated cultured human melanoma cells Colo 38, recognizes intercellular adhesion molecule 1 (ICAM-1). The determinant defined by MoAb CL203.4 is distinct and spatially distant from that defined by anti-ICAM-1 MoAb RR1/1, which had been elicited with Epstein-Barr virus-transformed B-lymphocytes from a lymphocyte function associated antigen 1 deficient patient. Immunohistochemical testing with MoAb CL203.4 of surgically removed lesions of melanocyte origin has shown a markedly lower reactivity with benign than with malignant lesions. Among the latter, a higher percentage of metastatic than of primary lesions was stained by MoAb CL203.4. The higher expression of ICAM-1 in metastases than in primary lesions is unique to melanoma, since no difference was found in its distribution in primary and metastatic lesions of a variety of malignancies of different embryological origin. Reactivity with MoAb CL203.4 of primary lesions removed from patients with stage I melanoma showed a highly significant correlation with the lesion thickness and with the clinical course of the disease. The disease free interval in patients without detectable reactivity of their primary lesion with MoAb CL203.4 was significantly (P = 0.004) longer than that of patients whose primary lesion was stained with MoAb CL203.4. These results suggest that ICAM-1 may be a useful marker in the analysis of the molecular mechanism underlying the association between lesion thickness and clinical course of the disease.
Asunto(s)
Moléculas de Adhesión Celular/análisis , Melanoma/análisis , Proteínas de Neoplasias/análisis , Adolescente , Adulto , Anciano , Animales , Anticuerpos Monoclonales , Antígenos de Neoplasias , Femenino , Humanos , Molécula 1 de Adhesión Intercelular , Interferón gamma/biosíntesis , Masculino , Melanoma/inmunología , Melanoma/patología , Antígenos Específicos del Melanoma , Ratones , Ratones Endogámicos BALB C , Persona de Mediana Edad , Metástasis de la Neoplasia , PronósticoRESUMEN
Ganglioside composition of human melanoma was correlated with sensitivity of melanoma to antitumor treatment with chemotherapeutic agents and radiation. The cytotoxic effect of each treatment was evaluated on 16 melanoma cell lines using the human tumor colony-forming assay. Ganglioside fractions were extracted and purified from each cell line and analyzed for the four major gangliosides in melanoma (GM3, GM2, GD3, and GD2) by thin-layer chromatography (TLC) and TLC scanner. GD2 content positively correlated with sensitivity to radiation (r = 0.753, p less than 0.001) and vincristine (r = 0.779, p less than 0.001). In contrast, GM3 content inversely correlated with sensitivity to radiation (r = -0.658, p less than 0.01) and vincristine (r = -0.692, less than 0.01). The gangliosides GD3 and GM2 were shown to have no significant correlation with any of these treatments.
Asunto(s)
Antineoplásicos/uso terapéutico , Ensayo de Unidades Formadoras de Colonias , Gangliósidos/análisis , Melanoma/análisis , Tolerancia a Radiación , Ensayo de Tumor de Célula Madre , Antineoplásicos/farmacología , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/efectos de la radiación , Humanos , Melanoma/tratamiento farmacológico , Melanoma/patología , Melanoma/radioterapia , Células Tumorales Cultivadas/análisis , Células Tumorales Cultivadas/efectos de los fármacos , Células Tumorales Cultivadas/efectos de la radiaciónRESUMEN
Three human melanoma cell lines of differing invasive and metastatic potentials were cultured on either a plastic surface or a denuded amniotic basement membrane, and alterations in their cell surface proteins, invasive profiles, and the presence or absence of the 69 Kd high-affinity metastasis-associated laminin receptor were examined. Our data indicate that the labeled, precipitable cell surface proteins are different from the three cell lines when they are cultured on the same substrate, and change when the cells are cultured on a different substrate. Furthermore, the invasive potential (as measured in the in vitro Membrane Invasion Culture System) is decreased for all of the cell lines after culturing the cells on a basement membrane matrix compared to a plastic surface. Finally, we show that the 69 Kd high-affinity metastasis-associated laminin receptor can be isolated from all three cell lines cultured on the two different substrates by labeling the cell surface with trinitrobenzene sulfonic acid and immunoprecipitating these targeted proteins.
Asunto(s)
Melanoma/análisis , Proteínas de la Membrana/análisis , Proteínas de Neoplasias/análisis , Medios de Cultivo , Electroforesis en Gel de Poliacrilamida , Humanos , Immunoblotting , Invasividad Neoplásica , Receptores Inmunológicos/análisis , Receptores de Laminina , Células Tumorales CultivadasRESUMEN
We retrospectively studied the nuclear RNA content in uveal melanomas with known, long-term follow-up. Thirty patients had spindle cell melanomas, 25 had mixed cell tumors, and 9 had epithelioid cell neoplasms. Epithelioid cell types had a higher nuclear RNA content compared to mixed or spindle cell types (p less than 0.05). Using the Cox proportional hazards model, the nuclear RNA content appeared to be an independent prognostic indicator in uveal melanomas, and an increased nuclear RNA content was associated with a worse prognosis (p = 0.001).
Asunto(s)
Melanoma/análisis , Neoplasias de Células Germinales y Embrionarias/análisis , ARN Neoplásico/análisis , ARN Nuclear/análisis , Neoplasias de la Úvea/análisis , ADN de Neoplasias/análisis , Citometría de Flujo , Estudios de Seguimiento , Humanos , Melanoma/clasificación , Melanoma/genética , Melanoma/mortalidad , Neoplasias de Células Germinales y Embrionarias/genética , Neoplasias de Células Germinales y Embrionarias/mortalidad , Pronóstico , Modelos de Riesgos Proporcionales , Estudios Retrospectivos , Factores de Riesgo , Neoplasias de la Úvea/genética , Neoplasias de la Úvea/mortalidadRESUMEN
Cloudman S91 mouse melanoma cells express both external (plasma membrane) and internal binding sites for MSH. Using 125I-beta melanotropin (beta-MSH) as a probe, we report here an extensive series of studies on the biological relevance of these internal sites. Cells were swollen in a hypotonic buffer and lysed, and a particulate fraction was prepared by high-speed centrifugation. This fraction was incubated with 125I-beta-MSH with or without excess nonradioactive beta-MSH in the cold for 2 hours. The material was then layered onto a step-wise sucrose gradient (8-80%) and centrifuged (156,000g, 60 min); fractions were collected and counted in a gamma counter or assayed for various enzymatic activities. The following points were established: 1) Specific binding sites for MSH were observed sedimenting at an average density of 50% sucrose in amelanotic cells and at higher densities in melanotic cells. 2) These sites were similar in density to those observed when intact cells were labeled externally with 125I-beta-MSH and then warmed to promote internalization of the hormone. 3) Most of the internal binding sites were not as dense as fully melanized melanosomes. 4) In control experiments, the MSH binding sites were not found in cultured hepatoma cells. 5) Variant melanoma cells, which differed from the wild-type in their responses to MSH, had reduced expression of internal binding sites even though their ability to bind MSH to the outer cell surface appeared normal. (MSH-induced responses included changes in tyrosinase, dopa oxidase, and dopachrome conversion factor activities, melanization, proliferation, and morphology.) 6) Isobutylmethylxanthine, which enhanced cellular responsiveness to MSH, also enhanced expression of internal binding sites. The results indicate that expression of internal binding sites for MSH is an important criterion for cellular responsiveness to the hormone.
Asunto(s)
Hormonas Estimuladoras de los Melanocitos/metabolismo , Melanoma/patología , Animales , Sitios de Unión , Biomarcadores de Tumor/análisis , Centrifugación por Gradiente de Densidad , Variación Genética , Radioisótopos de Yodo , Melanoma/análisis , Melanoma/genética , Ratones , Fenotipo , Receptores de la Hormona Hipofisaria/metabolismo , Células Tumorales Cultivadas/análisis , Células Tumorales Cultivadas/metabolismo , Células Tumorales Cultivadas/patologíaRESUMEN
Calmodulin content and distribution between soluble and particulate fractions were determined by radioimmunoassay in six human melanoma cell lines exhibiting differences in tumor origin (primary or metastatic), degree of tumorigenicity and of pigmentation (amelanotic or melanotic). The results indicate that a) total, soluble and particulate calmodulin levels expressed as ng/10(6) cells or ng/micrograms of proteins remained constant for five out of six cell lines when cells grew from subconfluency to confluency. For IGR 37 line, derived from metastatic melanoma, the calmodulin content decreases from 2.39 to 1.27 ng/micrograms protein for total calmodulin, from 2.17 to 1.52 ng/micrograms protein for soluble calmodulin and from 2.61 to 1.02 ng/micrograms protein for particulate calmodulin, b) total, soluble and particulate calmodulin levels expressed as ng/microgram proteins were twofold (at confluency) to fourfold (at subconfluency) higher in the two cell lines from metastatic origin, IGR 37 and IPC 167. As for example, for total calmodulin, values in IGR 37 and IPC 167 cell lines, were, respectively at subconfluency, 2.39 and 2.31 ng/micrograms protein as compared with the four other cell lines: 0.76 to 0.96 ng/micrograms protein and at confluency: 1.27 and 1.98 ng/micrograms protein as compared with the four other cell lines: 0.76 to 0.90 ng/micrograms protein, c) ratio of calmodulin between soluble and particulate fractions was about 1 for the two autologous cell lines IGR 37 and IGR 39 and varies from 2 to 3 for the four other cell lines.