Characterisation of novel uveal melanoma cell lines under serum-free conditions.

BACKGROUND: The establishment of long-term uveal melanoma (UM) cell lines is difficult. However, studying living cells and their behaviour in the presence of other cells and the extracellular matrix is important in terms of understanding tumour biology and malignant behaviour. We have established three UM cell lines and report a first characterisation of these cell lines. METHODS: Three established UM cell lines (UMT2, UMT26 and UMT33) were analysed according to their morphologic characteristics, melanocytic differentiation, adhesion on different extracellular matrices and proliferative activity. Copy number changes of chromosomes 1, 3, 6 and 8 were studied by multiplex ligation-dependent probe amplification (MLPA). Oncogenic mutations in UM involving exons 4 and 5 of GNAQ and GNA11, respectively, were analysed by sequencing. RESULTS: All cell lines grew in suspension. UMT2 cells were homogeneous, UMT26 and UMT33 cells heterogeneous with regard to cell size and pigmentation. All UM cell lines revealed a melanocytic differentiation. UMT2 and 33 adhered on various extracellular matrices, while UMT26 only adhered to basal membrane extract (BME). This difference corresponded to the different expression of various integrins. Ki67 was expressed by 89% of UMT2 and 95% of UMT33 cells, which thus were in a proliferative stage, while only 2% of UMT26 cells revealed immunostaining for this proliferation marker. The doubling time of UMT2 was 3 days, 12 days for UMT33, and circa 3-4 months for UMT26. MLPA revealed disomy 3 in UMT2 and monosomy 3 in UMT33. The same point mutation was found in UMT2, 26 and 33, in exon 5 of GNA11 at codon 209 (p.Q209L). CONCLUSIONS: The establishment of UM cell lines under serum-free conditions is possible. Characterisation of UMT2, 26, and 33 revealed obvious differences in cytomorphology, melanocytic differentiation, adhesion on extracellular matrices, and proliferative activity. UMT2, 26 and 33 showed the same oncogenic mutation in exon 5 of GNA11.

Antiproliferative and cytotoxic properties of bevacizumab on different ocular cells.

AIM: To evaluate the antiproliferative and cytotoxic properties of bevacizumab, a monoclonal antibody against vascular endothelial growth factor (VEGF), on human retinal pigment epithelium (ARPE19) cells, rat retinal ganglion cells (RGC5), and pig choroidal endothelial cells (CEC). METHODS: Monolayer cultures of ARPE19, RGC5, and CEC were used. Bevacizumab (0.008-2.5 mg/ml), diluted in culture medium, was added to cells that were growing on cell culture dishes. Cellular proliferative activity was monitored by 5'-bromo-2'-deoxyuridine (BrdU) incorporation into cellular DNA and the morphology assessed microscopically. For cytotoxicity assays ARPE19, RGC5, and CEC cells were grown to confluence and then cultured in a serum depleted medium to ensure a static milieu. The MTT test was performed after 1 day. The "Live/Dead" viability/cytotoxicity assay was performed and analysed by fluorescence microscopy after 6, 12, 18, 24, 30, 36, and 48 hours of incubation. Expression of VEGF, VEGF receptors (VEGFR1 and VEGFR2) and von Willebrand factor was analysed by immunohistochemistry. RESULTS: No cytotoxicity of bevacizumab on RGC5, CEC, and ARPE19 cells could be observed after 1 day. However, after 2 days at a bevacizumab concentration of 2.5 mg/ml a moderate decrease in ARPE19 cell numbers and cell viability was observed. Bevacizumab caused a dose dependent suppression of DNA synthesis in CEC as a result of a moderate antiproliferative activity (maximum reduction 36.8%). No relevant antiproliferative effect of bevacizumab on RGC5 and ARPE19 cells could be observed when used at a concentration of 0.8 mg/ml or lower. CEC and ARPE 19 cells stained positively for VEGF, VEGFR1, and VEGFR2. More than 95% of the CEC were positive for von Willebrand factor. CONCLUSIONS: These experimental findings support the safety of intravitreal bevacizumab when used at the currently applied concentration of about 0.25 mg/ml. Bevacizumab exerts a moderate growth inhibition on CEC when used in concentrations of at least 0.025 mg/ml. However, at higher doses (2.5 mg/ml) bevacizumab may be harmful to the retinal pigment epithelium.

The polysialic acid moiety of the neural cell adhesion molecule is involved in intraretinal guidance of retinal ganglion cell axons.

We have characterized the antigen recognized by mab10, a monoclonal antibody that has been shown to modify outgrowth of thalamic and cortical axons in vitro, and investigated the influence of this antibody on axonal growth in the chicken retina in vivo. Immunopurification, peptide sequencing, and biochemical characterization proved the epitope recognized by mab10 to be polysialic acid (PSA), associated with the neural cell adhesion molecule (NCAM). Intravitreal injections of antibody-secreting hybridoma cells were combined with whole-mount studies using the fluorescent tracer 1,1'-dioctadecyl-3,3,3', 3'-tetramethylindocarbocyanine perchlorate (DiI). Pathfinding at the optic fissure was affected, resulting in a failure of axons to exit into the nerve. Misprojections also occurred in more peripheral areas of the retina; however, axons eventually oriented toward the center. Similar projection errors were observed after enzymatic removal of PSA by injecting endoneuraminidase N (endo N). Quantitative measurements of the optic nerve diameter as well as the width of the optic fiber layer confirmed that many axons failed to leave the retina and grew back in the optic fiber layer of the retina. Our findings suggest that NCAM-linked PSA is involved in guiding ganglion cell axons in the retina and at the optic fissure.

Progenitor cells from embryonic chick dorsal root ganglia differentiate in vitro to neurons: biochemical and electrophysiological evidence.

We have analyzed the appearance of neurons and glial cells in chick dorsal root ganglia during development. Neurons were identified by the presence of polysialogangliosides recognized by tetanus toxin (GD1b, GT1) or by the monoclonal antibody Q211 directed against polysialogangliosides containing four, five and six sialic acid residues. Glial cells were identified by the presence of 04 antigen. A population of undifferentiated cells, i.e., cells which express neither neuronal nor glial cell surface antigens, present in dorsal root ganglia until embryonic day 7, was separated from the neuronal and glial population. This cell population contains neuronal progenitor cells which differentiate to neurons within 1 day in culture. This differentiation process is characterized by the appearance of neuronal morphology, of neuron-specific gangliosides and by the appearance of voltage-dependent sodium and calcium channels.