Human Choroidal Melanoma Cells Research Articles

An Orthotopic Model for Human Uveal Melanoma in SCID Mice

The microcirculation of primary uveal melanomas, their precursors, and their metastases is distinctive. Medium-sized and even large primary uveal melanomas typically lack significant zones of necrosis, suggesting that either these tumors are relatively well perfused or they are capable of growth in a severely blood-deprived microenvironment. In addition to normal choroidal vessels that are incorporated into nevi and most primary uveal melanomas, aggressive primary and metastatic uveal melanomas tend to contain patterns of extracellular matrix that surround spheroidal or cylindrical packets of tumor cells. Some components of this branching, looping, and interconnected system of matrix may be perfused. It is now known that the generation of this patterning is a characteristic of genetically dysregulated melanoma cells (nonaggressive tumor cells do not form these patterns and melanomas lacking branching, looping, or interconnected matrix patterns tend to follow a relatively indolent course). We developed an orthotopic model of an aggressive human uveal melanoma by injecting suspensions of the primary human choroidal melanoma cell line (OCM1) into the subretinal space of one eye of 20 SCID mice. All mice were examined daily for tumor growth and tumors developed in every eye within 3 weeks of injection. The tumors were characterized by extraocular extension and the development of looping matrix patterns characteristic of those seen in aggressive human uveal melanoma. As in human uveal melanomas, these patterns were perfused by blood in areas. The orthotopic injection of human uveal melanoma cells into the SCID mouse eye generates a model reproducing the matrix-associated microcirculatory patterns of aggressive primary human uveal melanomas. This model can be used to explore the molecular pathogenesis and modulation of this novel circulation in vivo, to facilitate our understanding of the blood flow to these tumors providing insight into perfusion and drug delivery, to enable testing of pharmacologic modulation of pattern formation and intratumoral blood flow, and to refine noninvasive methods such as confocal scanning laser ophthalmoscopy to detect the presence of these patterns by which ophthalmologists might assess the biological behavior of tumors as noninvasive substitute for biopsy.

Alterations of sensory retinal explants exposed to choroidal melanoma cells ex vivo.

Cultures of retinal explants have been established as a useful tool to investigate effects of pathogenic agents in vitro. We used such cultures as a model to study the effects of choroidal melanoma on retinal organisation and function. Rabbit retinal explants were co-cultured with human choroidal melanoma cells, or exposed to supernatants from choroidal melanoma cell cultures, for various periods from 1 day to 10 days. The retinal explants were then studied by histology and immunocytochemistry for glial fibrillary acidic protein (GFAP) and vimentin. The release of the pro-inflammatory interleukins IL-6 and IL-8 into the media was measured by enzyme-linked immunosorbent assay. Both in the co-cultures and after treatment with choroidal melanoma cell supernatants for more than 1 week, the layered structure of the retinae became disorganised. Retinal glial (Müller) cells displayed gliosis as indicated by increased GFAP immunoreactivity and decreased immunoreactivity for vimentin. Additionally, the secretion of cytokines, particularly of IL-8, was significantly modulated. The retinal explants produced much less IL-8 than the melanoma cells in separate cultures but increased their IL-8 release significantly after a few days' exposure to melanoma cell-conditioned medium. The results show that in cases of choroidal melanoma, the well-known morphological and inflammatory alterations of the retina are accompanied by glial cell reactivity and up-regulated retinal cytokine secretion, and may be caused by soluble factors secreted and induced by the melanoma.

P21CIP1 Is Dispensable for the G2 Arrest Caused by Genistein in Human Melanoma Cells

We have investigated the effect of genistein on cell cycle distribution of the human choroidal melanoma cell line OCM-1. We report that this isoflavonoid arrested cells in G2. This effect was correlated with the induction of the CDK inhibitor p21CIP1. However, while CDK1 activity was markedly reduced following genistein treatment, CDK2 activity was not affected. This was in agreement with the absence of G1 arrest that we observed but caused some doubt about the functionality of p21CIP1. Attempts to demonstrate mutation or post-translational modification of p21CIP1 from OCM-1 cells were unsuccessful. In fact, the level of p21CIP1 induced by genistein was shown to be unsufficient to cause CDK2 inhibition. The role of p21CIP1 in the inhibition of CDK1 was questionable, as we demonstrated that genistein impaired Tyr15 dephosphorylation of CDK1 and because CDK1–cyclin B1 complexes from treated cells could be reactivated upon exposure to CDC25 phosphatase. Finally, we report that p21CIP1 was not absolutely required for the genistein-induced G2 arrest, as the isoflavone caused at least partial G2 arrest in p21-deficient Rat-1 fibroblasts as well as in p21−/− mouse embryo fibroblasts.

Evaluation of the Human Choroidal Melanoma Rabbit Model for Studying Microcirculation Patterns with Confocal ICG and Histology

The aim of this study was to develop consistently focal elevated choroidal masses of human choroidal melanoma in immunosuppressed rabbits and to correlate the visualization of prognostically significant microcirculation patterns from confocal indocyanine green angiography with histologic microcirculation patterns. A human choroidal melanoma cell line (OCM1) was implanted in the choroid of 40 rabbit eyes using three different techniques: transscleral choroidal injection of a cell suspension, injection of a cell suspension in a surgically induced cyclodialysis cleft, and implantation of solid tumor fragments in a surgically induced cyclodialysis cleft. The rabbits were immunosuppressed with daily injections of Cyclosporin A to prevent host versus graft reaction. The eyes were studied weekly with indirect ophthalmoscopy and fundus photography to monitor tumor growth and indocyanine green angiography using a confocal scanning laser ophthalmoscope to identify microcirculation patterns in vivo and correlate these findings with the histologic demonstration of tumor microcirculation patterns. A tumor mass was identified by indirect ophthalmoscopy in 16 of the 40 implanted rabbit eyes (40%). Each of these tumors was confirmed histologically to represent a focal elevated choroidal mass. All 16 elevated choroidal masses grow in eyes in which solid tumor fragments were implanted. In total, a melanoma was identified histologically in 28 of the implanted 40 eyes (70%). In addition to the 16 eyes where the melanoma appeared as a focal elevated choroidal mass, 4 eyes contained a focal elevated mass in the sclera and 8 eyes contained a flat choroidal tumor. Histologically, microcirculation patterns were identified only in the 16 eyes with focal elevated choroidal masses. Confocal indocyanine green angiography imaged microcirculation patterns in 13 of these 16 eyes (81%). The surgical implantation of small solid fragments of human choroidal melanoma in immunosuppressed rabbit eyes provides the best method to consistently obtain focal elevated choroidal masses. These focal elevated choroidal masses resemble booth the localization and the growth pattern of choroidal melanomas in humans. In addition, they also contain microcirculation patterns similar to those seen in humans that are detectable with confocal indocyanine green angiography. The use of indocyanine green angiography with this animal model may be especially useful in designing and evaluating anti-microcirculation treatments directed at uveal melanoma.

G1 phase arrest by the phosphatidylinositol 3-kinase inhibitor LY 294002 is correlated to up-regulation of p27 Kip1 and inhibition of G1 CDKs in choroidal melanoma cells

We have investigated the effect of the flavonoid derivative LY 294002, a potent and selective phosphatidylinositol 3-kinase inhibitor, on cell cycle progression in human choroidal melanoma cells. We demonstrate that LY 294002 induces a specific G1 block in asynchronously growing cells leading to an almost complete inhibition of cell proliferation after three days of treatment. When melanoma cells are released from a nocodazole-induced G2/M block, LY 294002 is shown to delay and greatly restrain the G1/S transition. The inhibitor is able to exert its action as long as it is added during the G1 progression and before the cells enter in S phase. We report that the LY 294002-induced G1 arrest is closely correlated to inhibition of CDK4 and CDK2 activities leading to the impairment of pRb phosphorylation which normally occurs during G1 progression. While the inhibition of CDK4 may be attributed at least in part to the decline in CDK4 protein level, CDK2 activity reduction is rather due to the up-regulation of the CDK inhibitor p27 Kip1 and to its increased association to CDK2.

Ki-67 and bromodeoxyuridine labeling of human choroidal melanoma cells

Understanding tumor growth patterns has implications for prognosis as well as for response and susceptibility to treatment. The antibody Ki-67 was used as a marker of cycling cells and bromodeoxyuridine (BrdUrd) was used as a marker of proliferating cells to characterize the cycling and proliferative rates of cells from human choroidal melanoma. The BrdUrd labeling indices varied from 0-1.1% and the Ki-67 labeling indices ranged from 0-3.0.3%. Linear regression modeling showed good correlation defined by the equation: Ki-67 index = 0.237 + 1.63 x BrdUrd labeling index with r = 0.919. Correlations between these indices and clinical and histologic parameters were not significant.

Differences in Nuclear Pore Density among Human Choroidal Melanoma Cell Types

Two cases of mixed cell type choroidal melanoma were examined by means of conventional and freeze-fracture electron microscopy. Data on average nuclear pore density were readily collected from freeze-fracture replicas of spindle A, spindle B, and epithelioid cells. Highly significant (p less than 0.001) differences in pore densities were found among the three cell types. Pore density was lowest in spindle A cells, intermediate in spindle B cells, and highest in epithelioid cells. The possible relationship between nuclear pore density and nucleolar activity as well as the potential usefulness of this new finding as a prognostic indicator in cases of choroidal melanoma are discussed.

Tissue culture of human choroidal melanoma cells obtained by fine-needle aspiration biopsy.

The optimal treatment of choroidal melanoma is controversial. Many patients are now being treated with nonenucleation therapies. Fine-needle aspiration biopsy of intraocular tumors may well prove to be safe; we used a needle biopsy technique on three freshly enucleated globes and established choroidal melanoma cell lines in culture from these specimens. This report describes our needle biopsy and tissue culture techniques.