RESUMEN
The murine melanoma cell line BL-6-beta m, which is a stable cell line transfected with a gene coding a unique actin subspecies called beta m to the BL-6 cell line, has low metastatic potentials as compared with those of the parent cell line. BL-6-beta m melanomas were found to be sensitive to in vivo local injection of IL-2, while BL-6 melanomas showed almost no response. Ganglioside analysis of BL-6 and BL-6-beta melanomas revealed that the main ganglioside of both melanomas was GM3, which suggested that different sensitivities between BL-6 and BL-6-beta m melanomas to the injection of IL-2 did not relate to the different compositions of main gangliosides. However, minor components of the gangliosides such as GM2 and GM1 emerged only in BL-6-beta m melanomas after treatment with IL-2. Local injection of IL-2 caused considerable infiltration of anti-asialo GM1-positive cells into the nests as well as the interstitials of BL-6-beta m melanomas. In contrast, in the BL-6 melanomas treated with IL-2, infiltration of the anti-asialo GM1-positive cells was hardly seen, although anti-Thy1,2 and anti-macrophage-positive cells were found to more or less the same extent as observed in BL-6-beta m melanomas. These results suggest that the murine metastatic variant melanoma cell lines BL-6 and BL-6-beta m have different properties in terms of sensitivity to in vivo IL-2 treatment, and a slight enhancement of the ganglioside components GM2 and GM1 expression only in BL-6-beta m after IL-2 treatment may play a role in the IL-2-mediated attraction of immune cells or may explain the different sensitivities of the two lines to treatment with IL-2.
Asunto(s)
Actinas/fisiología , Gangliósidos/análisis , Factores Inmunológicos/uso terapéutico , Interleucina-2/uso terapéutico , Melanoma Experimental/terapia , Transfección , Actinas/genética , Animales , Antígenos de Neoplasias/análisis , Movimiento Celular , Femenino , Gangliósido G(M1)/análisis , Factores Inmunológicos/farmacología , Vigilancia Inmunológica , Inyecciones Intralesiones , Interleucina-2/farmacología , Células Asesinas Activadas por Linfocinas/inmunología , Melanoma Experimental/química , Melanoma Experimental/inmunología , Melanoma Experimental/patología , Ratones , Ratones Endogámicos C57BL , Metástasis de la Neoplasia , Trasplante de Neoplasias , Proteínas Recombinantes de Fusión , Antígenos Thy-1/análisisRESUMEN
We analyzed the biochemical nature of beta m-actin protein found in mouse B16 melanoma. When we carried out immunostaining with the antibody specific to beta m-actin, filamentous immunofluorescence was observed in B16-F1, a low-metastatic cell line expressing beta m-actin, but not in highly metastatic B16-F10 that did not express beta m-actin. When a purified actin fraction containing beta m-actin was polymerized and immunoprecipitated with anti-beta m-actin antibody, the immunoprecipitate contained beta m-, beta- and gamma-actin. This indicated that the beta m-actin was incorporated into an actin filament together with beta- and gamma-actin in vitro, and this phenomenon was consistently suggested by cellular double immunostaining with anti-beta m-actin and common anti-actin antibody. When the actin fraction containing beta m-actin under a regular depolymerizing condition was subjected to immuno-adsorption assay using anti-beta m antibody and protein-A Sepharose, the immunoadsorbed aggregates contained beta m-, beta- and gamma-actin. This indicates that the actin fraction was not completely depolymerized and contained beta m-actin-containing oligomers, which were too small to be precipitated with anti-beta m-actin antibody alone. The incomplete depolymerization of the beta m-actin-containing fraction was also suggested by the much lower DNase 1 inhibition activity of the beta m-actin-containing fraction than that of beta- and gamma-actin fraction. Furthermore, a DNase 1 binding assay showed that cytoplasmic supernatant prepared from B16-F1 under a low-ionic condition contained less monomeric actin than the cytoplasmic preparation from B16-F10. These results suggested that beta m-actin protein in B16 melanoma probably inhibits the dynamic conversion between the monomeric and polymerized forms of actin, leading to a decrease in cell motility and consequently the suppression of invasiveness and metastasis.
Asunto(s)
Actinas/análisis , Melanoma Experimental/patología , Metástasis de la Neoplasia , Actinas/inmunología , Actinas/fisiología , Secuencia de Aminoácidos , Animales , Anticuerpos/inmunología , Especificidad de Anticuerpos , Desoxirribonucleasa I/metabolismo , Fluoresceína-5-Isotiocianato , Técnica del Anticuerpo Fluorescente , Colorantes Fluorescentes , Técnicas de Inmunoadsorción , Ratones , Ratones Endogámicos C57BL , Datos de Secuencia Molecular , Células Tumorales CultivadasRESUMEN
We recently reported an acidic actin co-expressed with beta and gamma actin in mouse B16 melanoma, whose expression was inversely correlated with the metastatic potential. The cDNA for this actin is slightly different from the hitherto recognized mouse beta actin cDNA, and we designated it beta m actin. In order to directly investigate the effects of beta m actin on metastasis, we transfected the beta m actin cDNA into a re-cloned B16-BL6 cell line which is more invasive than the highly metastatic cell line, B16-F10; we have already reported the suppressive effect of beta m actin on the invasiveness of B16-F10. Here we report on the decline in the metastatic ability of beta m-transfected cells. In the beta m-transfected B16-BL6 cell line, we observed an increase in the organization of actin stress fibers, accompanied by a decrease in metastasis to the lung, in the invasion of collagen gels, in in vivo invasiveness, and in cell migration on a glass plate covered with colloidal gold particles. We observed no correlation of beta m actin expression either with cell attachment to Matrigel, or with type-IV collagenase expression. These results suggest that beta m actin can play a role in reducing the invasiveness of mouse B16 melanoma, most probably through decreasing cell motility, which may thus result in suppression of the metastatic ability of cells.