Antioxidant pattern in uveal melanocytes and melanoma cell cultures.

PURPOSE: To investigate the antioxidant status of cultured uveal melanocytes from patients with uveal melanoma and uveal melanoma cells to characterize some of the biochemical properties of these cells in respect to the normal cutaneous melanocytes. METHODS: The fatty acid pattern of membrane phospholipids, intracellular vitamin E level, and superoxide dismutase (SOD) and catalase activities were studied in uveal melanocytes (n = 10) and uveal melanoma cell (n = 10) cultures, by gas chromatography mass spectrometry or by spectrophotometer. RESULTS: Among the uveal melanocyte cultures, two groups were differentiated, according to catalase activity: group A with catalase values comparable to those of cutaneous ones and higher SOD activity and group B with catalase values 2 SD lower (P<0.001) and lower SOD activity. Vitamin E concentration was not significantly different between melanoma cells and melanocytes, whereas a significantly higher percentage of polyunsaturated fatty acids was found in melanoma cells and the B group of melanocytes (P = 0.022). In uveal melanoma cells SOD activity was significantly lower than that detected in uveal melanocytes (P< 0.005). CONCLUSIONS: These results show a different pattern of antioxidants in uveal melanocytes with respect to cutaneous ones, possibly related to the anatomic distribution. However, as in cutaneous melanocytes, two subgroups were identified on the basis of the antioxidant pattern that could be the expression of a constitutional increased susceptibility to oxidative stress in some subjects. Moreover, an imbalance of the antioxidants was observed in melanoma cells, possibly related to the disease status and progression.

Glycosphingolipid domains on cell plasma membrane.

In this study we analyzed by immunofluorescence, laser confocal microscopy, immunoelectron microscopy and label fracture technique the ganglioside distribution on the plasma membrane of several different cell types: human peripheral blood lymphocytes (PBL), Molt-4 lymphoid cells, and NIH 3T3 fibroblasts, which mainly express monosialoganglioside GM3, and murine NS20Y neuroblastoma cells, which have been shown to express a high amount of monosialoganglioside GM2. Our observations showed an uneven distribution of both GM3 and GM2 on the plasma membrane of all cells, confirming the existence of ganglioside-enriched microdomains on the cell surface. Interestingly, in lymphoid cells the clustered immunolabeling appeared localized over both the microvillous and the nonvillous portions of the membrane. Similarly, in cells growing in monolayer, the clusters were distributed on both central and peripheral regions of the cell surface. Therefore, glycosphingolipid clusters do not appear confined to specific areas of the plasma membrane, implying general functions of these domains, which, as structural components of a cell membrane multimolecular signaling complex, may be involved in cell activation and adhesion, signal transduction and, when associated to caveolae, in endocytosis of specific molecules.

Receptor-mediated endocytosis of keratinocyte growth factor.

Keratinocyte growth factor (KGF) is a fibroblast growth factor which acts specifically on epithelial cells, regulating their proliferation and differentiation. KGF elicits its activity through binding to and activation of KGF receptor, a splicing transcript variant of fibroblast growth factor receptor 2 (FGFR2). Here we analyzed the pathway of internalization of KGF and its receptor using several approaches, including the utilization in immunofluorescence and in immunoelectron microscopy of a functional KGF-HFc chimeric protein as a specific tool to follow the endocytosis of the growth factor and of its receptor. Western blot analysis with anti-FGFR2 and anti-phosphotyrosine antibodies, as well as parallel double immunofluorescence and confocal analysis of NIH3T3 KGFR transfectants treated with KGF at 4 degrees C, followed by incubations at 37 degrees C for different time points, showed that KGF induced endocytosis of tyrosine activated KGFRs. The use of KGF-HFc in immunofluorescence and in immunogold electron microscopy on KGFR transfectants, A253 epithelial tumor cells and human cultured keratinocytes allowed us to follow the early steps of KGF internalization and revealed that this process occurred through clathrin-coated pits. A quantitative ELISA assay confirmed that KGF-HFc binding on the cell surface rapidly decreased because of internalization. Our results demonstrate that KGF is internalized by receptor-mediated endocytosis and illustrate the involvement of clathrin-coated pits in this process.

Cerebral autosomal dominant arteriopathy with subcortical infarcts and leucoencephalopathy (CADASIL). Neuropathological and in vitro studies of abnormal elastogenesis.

This study was performed on a family of CADASIL (cerebral autosomal dominant arteriopathy with subcortical infarcts and leucoencephalopathy) subjects. Neuropathological alterations of small arteries consisting in thickening, reduplication and fragmentation of the internal elastic lamella, and granular periodic acid-Schiff-positive material deposited in the arterial media were demonstrated in 1 autopsy case by histochemistry and electron microscopy. This material reacted with a monoclonal antibody anti-elastin (aE), as demonstrated by immunohistochemistry and immunoelectron microscopy. Significant increases of aE-immunoreactivity and elastin mRNA expression were found in cultured skin fibroblasts from 5 family members genetically affected by CADASIL, but not genetically and clinically healthy members. These results suggest that alterations of the elastic apparatus are associated with CADASIL genotype and related to the clinical expression of the disease.

Mechanisms of the platelet proaggregating activity of human carcinoma a431 cells.

An unusual mechanism has been demonstrated for the in vitro proaggregating interaction between human platelets and human epidermoid carcinoma A431 cells. A431 cells induce platelet aggregation in a dodependent manner, depending on the rate of ADP release from tumour cells, which occurs in the presence not only of platelet rich plasma (PRP) but also of platelet poor plasma (PPP) or serum. This ADP release appears to be correlated to C(3) cleavage and binding of C(3c) to the A431 cell membrane. The interaction between A431 cells and PRP is characterized by typical morphological changes of A431 cells, leading to formation of mixed aggregates showing long projections of tumour cells deeply penetrating into the aggregate. These features, lacking in the presence of gel-filtered platelets (GFP), and reduced in the presence of thrombin degranulated platelets (TDP), are inhibited by cytochalasin and RGDS. The same activation of A431 cell cytoskeleton is induced by PDGF, but not by ADP or thromboxane receptor agonist U46619 or TGFP. These findings suggest a cooperative mechanism of tumour cell platelet interaction, in which a complementdependent ADP release from A431 cells induces platelet degranulation, PDGF release and aggregation. PDGF may induce in A431 cells Ca(2+) influx, cytoskeleton activation and changes in exposition of surface adhesion molecules, while fibrinogen binding causes mixed tumour cell-platelet aggregates to form.