RESUMEN
AIMS: Uveal melanomas with or without monosomy 3 have different metastatic potentials. Therefore, it would be of great experimental importance to obtain cell lines of both groups - established and characterized under the same conditions. METHODS: Long-term culture of biopsies derived from untreated primary uveal melanomas was performed, and the status of chromosome 3 in the tumour was determined. Characterization by immunocytochemistry and in vitro assays for cell migration, vasculogenic cord formation and proliferation were performed. RESULTS: Establishment of cell lines succeeded in 6 out of 128 uveal melanomas. No histopathological feature was predictive for cultivation success. Two out of 4 cell lines derived from tumours with monosomy 3 were invasive in vitro. Both cell lines with disomy 3 showed higher proliferation levels (p = 0.002). CONCLUSIONS: The in vitro tendency for invasion did not correlate with the proliferative behaviour of uveal melanoma cell lines, but was partly associated with monosomy of chromosome 3.
Asunto(s)
Melanoma/secundario , Úvea/patología , Neoplasias de la Úvea/patología , Biopsia , Técnicas de Cultivo de Célula/métodos , División Celular , Línea Celular Tumoral , Movimiento Celular , Supervivencia Celular , Cromosomas Humanos Par 3 , Humanos , Melanoma/genética , Monosomía , Metástasis de la Neoplasia , Neoplasias de la Úvea/genéticaRESUMEN
OBJECTIVE: Two conjunctival cell lines (CRMM-1 and CRMM-2) have been established from recurrent conjunctival melanoma. The authors examined the chemosensitivity of these cell lines to cytotoxic substances and combinations to identify substances that inhibit cell growth efficiently in vitro. MATERIAL AND METHODS: CRMM-1 and CRMM-2 were exposed to cisplatin, mitomycin C (MMC), all-trans-retinoic-acid (ATRA), fotemustine or imatinib for 24 h. Sulforhodamine-B assays were used to assess the IC(50). Isobolograms were performed to test possible synergism and antagonism with ATRA or imatinib. RESULTS: Cisplatin and MMC were efficient to inhibit the growth of CRMM-1 and CRMM-2. Combination of imatinib with MMC showed additive antitumoral effect on both cell lines. Combined treatment of imatinib with fotemustine or cisplatin resulted in antagonism. Strong antagonisms were also obtained with ATRA and fotemustine or cisplatin in both cell lines. A synergism was found for ATRA and mitomycin or imatinib in CRMM-2, in contrast to CRMM-1, where antagonism was obtained. CONCLUSIONS: Cisplatin and MMC inhibit cell growth in conjunctival melanoma cell lines. The potential of ATRA was evident only in combination with MMC or imatinib in CRMM-2 cells. Imatinib and mitomycin increased their efficiency under combination therapy.
Asunto(s)
Antineoplásicos/uso terapéutico , Neoplasias de la Conjuntiva/patología , Melanoma/patología , Línea Celular Tumoral , Proliferación Celular , Neoplasias de la Conjuntiva/tratamiento farmacológico , Sinergismo Farmacológico , Quimioterapia Combinada , Humanos , Melanoma/tratamiento farmacológicoRESUMEN
Uveal melanoma (UM) is the most common intraocular malignancy. Approximately 50% of UM patients die of metastases, which mainly arise from primary tumors with loss of an entire chromosome 3 (monosomy 3). To identify cell lines with monosomy 3 that may serve as a model system for UM with high metastatic potential, we determined the chromosome 3 status of previously established and frequently used UM cell lines by microsatellite analysis (Mel202, Mel285, Mel290, 92-1, OMM-1, OCM-1, OCM-3, OCM-8) and cytogenetic analysis (Mel202, Mel285, OCM-8). We found that none of these cell lines has monosomy 3. Therefore we established and characterized two novel cell lines, UPMM-1 and UPMM-2 that are both developed from primary uveal melanoma tissue samples with monosomy 3. The cell line UPMM-1 has retained the chromosome 3 status of the primary tumor. In UPMM-2 chromosome 3 has undergone duplication (isodisomy) and is present on the background of a hypotetraploid karyotype. Our data suggest that, UPMM-1 may serve as a model system to study the mechanisms underlying the metastatic potential of uveal melanomas with monosomy 3.
Asunto(s)
Cromosomas Humanos Par 3/genética , Melanoma/genética , Monosomía , Neoplasias de la Úvea/genética , Línea Celular Tumoral , Proliferación Celular , Humanos , Cariotipificación , Melanoma/patología , Melanoma/secundario , Repeticiones de Microsatélite , Neoplasias de la Úvea/patologíaRESUMEN
Based on gene profiling, two entities of uveal melanomas exist. So far, these two entities can be distinguished by the chromosome 3 status which strongly associates with the metastatic potential of the tumours. Reorganization of the extracellular matrix is one of the steps towards dissemination of tumour cells. In the present study, we examined the tissue inhibitor of matrix metalloproteinases (TIMP) 3 expression in 19 uveal melanomas and compared the results with histopathological and genetic features. The expression level of TIMP-3 mRNA as determined by microarray analysis was associated with the chromosome 3 status of the tumour (p = 0.003). All tumours with disomy 3 showed moderate to high expression of TIMP-3 mRNA, whereas TIMP-3 was highly expressed in one tumour, less expressed in 3 tumours and absent in the remaining 6 tumours with monosomy 3. Immunohistochemistry for TIMP-3 was positive in 9/19 tumours, but only in 3 tumours were more than 5% of the tumour cells stained positive. There was no association between immunohistochemical detection of TIMP-3 and chromosome 3 status. In tumours with disomy 3, we found none or very few TIMP-3-positive cells though the mRNA level was high which indirectly postulates posttranscriptional problems in protein biosynthesis in this entity of uveal melanomas. There was a trend between TIMP-3 protein expression and both cell type (p = 0.11) and presence of loops and/or networks (p = 0.06) in tumour which may indicate a role of TIMP-3 in the biology of uveal melanoma.
Asunto(s)
Melanoma/metabolismo , Inhibidor Tisular de Metaloproteinasa-3/metabolismo , Neoplasias de la Úvea/metabolismo , Cromosomas Humanos Par 3/genética , Perfilación de la Expresión Génica , Regulación Neoplásica de la Expresión Génica/fisiología , Humanos , Técnicas para Inmunoenzimas , Melanoma/patología , Monosomía/genética , Análisis de Secuencia por Matrices de Oligonucleótidos , ARN Mensajero/metabolismo , Inhibidor Tisular de Metaloproteinasa-3/genética , Neoplasias de la Úvea/patologíaRESUMEN
The small leucine-rich proteoglycan decorin can bind via its core protein to different types of collagens such as type I and type VI. To test whether decorin can act as a bridging molecule between these collagens, the binding properties of wild-type decorin, two full-length decorin species with single amino acid substitutions (DCN E180K, DCN E180Q), which previously showed reduced binding to collagen type I fibrils, and a truncated form of decorin (DCN Q153) to the these collagens were investigated. In a solid phase assay dissociation constants for wild-type decorin bound to methylated, therefore monomeric, triple helical type I collagen were in the order of 10(-10) m, while dissociation constants for fibrillar type I collagen were approximately 10(-9) m. The dissociation constant for type VI was approximately 10(-7) m. Using real-time analysis for a more detailed investigation DCN E180Q and DCN E180K exhibited lower association and higher dissociation constants to type I collagen, compared to wild-type decorin, deviating by at least one order of magnitude. In contrast, the affinities of these mutants to type VI collagen were 10 times higher than the affinity of wild-type decorin (K(D) approximately 10(-8) m). Further investigations verified that complexes of type VI collagen and decorin bound type I collagen and that the affinity of collagen type VI to type I was increased by the presence of decorin. These data show that decorin not only can regulate collagen fibril formation but that it also can act as an intermediary between type I and type VI collagen and that these two types of collagen interact via different binding sites.