Normal Uveal Melanocytes Research Articles

Multi-omics comparison of malignant and normal uveal melanocytes reveals molecular features of uveal melanoma

Uveal melanoma (UM) is a rare cancer resulting from the transformation of melanocytes in the uveal tract. Integrative analysis has identified four molecular and clinical subsets of UM. To improve our molecular understanding of UM, we performed extensive multi-omics characterization comparing two aggressive UM patient-derived xenograft models with normal choroidal melanocytes, including DNA optical mapping, specific histone modifications, and DNA topology analysis using Hi-C. Our gene expression and cytogenetic analyses suggest that genomic instability is a hallmark of UM. We also identified a recurrent deletion in the BAP1 promoter resulting in loss of expression and associated with high risk of metastases in UM patients. Hi-C revealed chromatin topology changes associated with the upregulation of PRAME, an independent prognostic biomarker in UM, and a potential therapeutic target. Our findings illustrate how multi-omics approaches can improve our understanding of tumorigenesis and reveal two distinct mechanisms of gene expression dysregulation in UM.

Focal Aggregates of Normal or Near Normal Uveal Melanocytes (FANNUMs) in the Choroid. A Practical Clinical Category of Small Ophthalmoscopically Evident Discrete Melanocytic Choroidal Lesions

Focal aggregate of normal or near normal uveal melanocytes (FANNUM) of the choroid is a term the author has proposed to categorize small melanocytic choroidal lesions that are not detectably thicker than surrounding normal choroid by B-scan ocular ultrasonography. In this article, the author describes the clinical features of small melanotic choroidal lesions he categorizes clinically as FANNUMs and discusses the presumed compositional spectrum of such lesions. He explains why such lesions should not be categorized uniformly as small choroidal nevi. Lesions of this type are detectable in at least one eye of approximately 25% of lightskinned blue-eyed persons over the age of 50 years. They are frequently multifocal in the affected eye, bilateral, or both.

Focal aggregates of normal or near normal uveal melanocytes (FANNUMs) in the choroid: a distinct clinical and histopathological entity?

PurposeTo define, describe, and illustrate a previously unreported category of discrete melanotic choroidal melanocytic lesion.MethodsProspective ophthalmoscopic study of the ocular fundi of 79 light-skinned persons 50 years of age or older not referred for any evident fundus lesion, with detection of all evident discrete melanotic choroidal lesions > 0.3 mm in largest basal diameter.ResultsOne or more discrete dark-brown to gray choroidal lesions > 0.3 mm in largest basal diameter were detected in 27 of the 79 evaluated subjects (34.2%). All but four of the detected lesions were “flat” by both ophthalmoscopy and ultrasonography. A single flat lesion was present in one eye of 14 subjects whose fellow eye was normal, 2 or more flat lesions were evident in one eye of 5 subjects whose other eye was normal, and one or more lesions were evident in both eyes of 6 subjects.ConclusionWhile some of the discrete small, flat melanocytic choroidal lesions detected in this study might have been choroidal nevi, the author hypothesizes that an indeterminate proportion of them may have been focal aggregates of normal or near normal uveal melanocytes (FANNUMs).

Differential modulation and prognostic values of immune-escape genes in uveal melanoma.

Uveal melanoma (UM) is the most common primary intraocular cancer in adults. In the present study, we aimed to characterize the immunological features of primary UM cancer and to provide an association with prognostic markers and outcome. Also, we assessed the influence of the microenvironment on the expression of inhibitory immune checkpoints in UM. Genes of interest included MHC Class I and Class II molecules, as well as inhibitory immune-checkpoints, i.e. PDL1, PDL2, B7-H3, B7-H4, TBFRSF6B, CD47, CD155, GAL9, HVEM and CD200. We observed significant lower levels of MHC genes in UM cells as compared to normal uveal melanocytes. Unexpectedly however, the expression levels of most of the analyzed inhibitory immune-checkpoint genes were not different in cancer cells as compared to normal melanocytes, with the exception of CD200 and HVEM, that resulted significantly reduced. On the other hand, PDL1 inversely correlated with OS, PFS and thickness of the tumor. Also, PDL1, along with PDL2, expression significantly increased under inflammatory conditions. Finally, for the first time, we propose a possible role for CD47 in the immune evasive properties of UM. We show here that CD47 is significantly upregulated by UM cells following inflammatory stimuli and that it represents a good independent predictor of disease progression. The results from this study may propel advances in the development of immune-based therapies for UM patients.

Abstract 4348: Methylation analysis of uveal melanoma reveals definitive patterns in tumors harboring BAP1 mutations

Abstract Introduction: Uveal melanoma (UM) is the most common primary intraocular malignancy and can be classified by gene expression profiling into two distinct molecular classes that correspond to metastatic risk: Class 1 (low risk) and Class 2 (high risk). The less aggressive Class 1 UMs express a more differentiated phenotype and exhibit numerous characteristics of normal uveal melanocytes, while the metastasizing Class 2 UMs display loss of melanocytic differentiation and have acquired a primitive, stem-like phenotype. The majority of Class 2 UMs harbor loss of chromosome 3 in addition to inactivating mutations of the tumor suppressor BAP1, which also catalyzes ubiquitin removal from histone H2A and modulates gene expression. Histone modifications have been shown to be associated with DNA methylation and this study investigates the global methylation and gene expression changes in BAP1 mutant tumors. Methods: RNA-Sequencing data and Illumina Human Methyl 450K BeadChip Array data obtained from 92 primary uveal melanoma tumors was analyzed using optimized pipelines. Results: This analysis revealed that the more aggressive Class 2 tumors exhibit a distinct pattern of hypermethylation and silencing of developmental genes involved in neural crest migration and differentiation. Chromosomal regions that were significantly enriched for hypermethylated genes with decreased gene expression in Class 2 tumors included chromosome 3p14-26, 3q12-29 and 8p12-22, whereas the only significantly enriched region for genes that were hypomethylated with increased gene expression in Class 2 tumors was chromosome 8q22-24. Additionally, BAP1 itself was differentially hypermethylated in Class 2 UMs, suggesting that it may regulate its own transcription. Conclusions: Class 2 UMs harboring BAP1 mutations displayed distinct regions of differential hypermethylation in comparison to Class 1 UMs. In these tumors, the most significantly hypermethylated regions occurred on chromosome 3 and in regions that were enriched for genes encoding neural crest guidance cue proteins that regulate homing, migration and invasion. These findings suggest that BAP1 mutations are associated with marked reorganization of the DNA methylome, providing new insights into the pathophysiology of UM and suggesting that methylation may serve as a diagnostic to further stratify metastatic risk. Furthermore, these findings may open new opportunities for targeted therapy of Class 2 UMs. Citation Format: Michael Durante, Matthew Field, Stefan Kurtenbach, Parker Bussies, Christina Decatur, J. William Harbour. Methylation analysis of uveal melanoma reveals definitive patterns in tumors harboring BAP1 mutations [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4348. doi:10.1158/1538-7445.AM2017-4348

Subtoxic Levels of Apigenin Inhibit Expression and Secretion of VEGF by Uveal Melanoma Cells via Suppression of ERK1/2 and PI3K/Akt Pathways

The effects of apigenin on the expression of VEGF in uveal melanoma cells have not been reported. We studied this effect and relevant signaling pathways in two human uveal melanoma cell lines (SP6.5 and C918). ELISA assay revealed that the constitutive secretion of VEGF by uveal melanoma cells was 21-fold higher than that in normal uveal melanocytes. Apigenin at subtoxic levels (1–5 μM) significantly suppressed the secretion of VEGF in a dose- and time-dependent manner in melanoma cells. VEGF levels in the conditioned culture media from SP6.5 and C918 cell lines treated with 5 μM apigenin for 24 h reduced to 29% and 21% of those in cells not treated with apigenin, respectively. RT-PCR analysis found that apigenin also decreased the expression of VEGF mRNA in melanoma cells. ELISA study of various signal pathways showed that apigenin significantly decreased phosphorylated Akt and ERK1/2 but increased phosphorylated JNK1/2 and p38 MAPK levels in melanoma cells. PI3K/Akt or ERK1/2 inhibitors significantly decreased, but JNK1/2 and p38 MAPK inhibitors did not influence the secretion of VEGF by melanoma cells, suggesting that apigenin suppresses the secretion of VEGF mainly through the inhibition of PI3K/Akt and ERK1/2 pathways.

Status of RASSF1A in Uveal Melanocytes and Melanoma Cells

RASSF1A gene, found at the 3p21.3 locus, is a tumor suppressor gene frequently hypermethylated in human cancers. In this study, we report that compared with melanocytes in normal choroid, RASSF1A is downregulated in uveal melanoma samples and in uveal melanoma cell lines. LOH at 3p21.3 was detected in 50% of uveal melanoma. Moreover, methylation of the RASSF1A promoter was detected in 35 of 42 tumors (83%) and RASSF1A was also weakly expressed at the mRNA level. These data indicate that LOH at the RASSF1A locus or RASSF1A promoter methylation may partly account for the suppression of RASSF1A expression observed in uveal melanoma. Furthermore, following ectopic expression in three RASSF1A-deficient melanoma cell lines (OCM-1, Mel270, and 92.1), RASSF1A weakly reduces cell proliferation and anchorage-independent growth of uveal melanoma cells without effect on ERK1/2 activation, cyclin D1 and p27(Kip1) expression. This study explored biological functions and underlying mechanisms of RASSF1A in the ERK1/2 pathway in normal uveal melanocytes. We showed that siRNA-mediated depletion of RASSF1A increased ERK1/2 activation, cyclin D1 expression, and also decreased p27(Kip1) expression in normal uveal melanocytes. Moreover, that the depletion of RASSF1A induced senescence-associated β-galactosidase activity and increased p21(Cip1) expression suggests that RASSF1A plays a role in the escape of cellular senescence in normal uveal melanocytes. Interestingly, we found that RASSF1A was epigenetically inactivated in long-term culture of uveal melanocytes. Taken together, these data show that depletion of RASSF1A could be an early event observed during senescence of normal uveal melanocytes and that additional alterations are acquired during malignant transformation to uveal melanoma.

A Comparative Transcriptomic Analysis of Uveal Melanoma and Normal Uveal Melanocyte

BackgroundUveal melanoma is the most common primary intraocular tumor in adults in western countries. It is associated with very severe visual morbidity and may lead to distant metastases even after successful treatment of the primary tumor. In order to gain better insight into molecular mechanisms related to tumorigenesis and metastasis of uveal melanoma, we used next-generation sequencing technology (SOLiD, Life Technologies) to acquire global transcriptome alteration between posterior uveal melanoma cells and normal uveal melanocyte.ResultsFrom mRNAs of the cultured uveal melanoma cells and normal uveal melanocytes, we annotated more than 3.7×107 and 2.7×107 sequencing tags based on human Ensembl databases, respectively. For detailed analysis, we chose 5155 well-annotated genes mainly involved in the MAPK signaling pathway, cell cycle, cell adhesion junction, apoptosis, and P53 signaling pathways as well as melanogenesis. In an effort to confirm the authenticity of our sequencing results, we validated twenty-one identically differentially expressed genes by using quantitative real time PCR from cultured cell lines of other posterior uveal melanoma cells and normal uveal melanocytes.ConclusionWe have identified a large number of potentially interesting genes for biological investigation of uveal melanoma. The expression profiling also provides useful resources for other functional genomic and transcriptome studies. These 21 potential genes could discriminate between uveal melanoma cells and normal uveal melanocyte, which may be indicative of tumorigenesis process. Our results further suggest that high-throughput sequencing technology provides a powerful tool to study mechanisms of tumogenesis in the molecular level.

Comparison of eumelanin and pheomelanin content between cultured uveal melanoma cells and normal uveal melanocytes

Levels of eumelanin (EM) and pheomelanin (PM) of uveal melanoma cells have not been measured and compared with those of normal uveal melanocytes. EM and PM amounts in four immortal human uveal melanoma cell lines were measured by chemical degradation and microanalytical high-performance liquid chromatography and compared with those from 39 normal human uveal melanocyte cell lines reported earlier by us. Uveal melanoma cells had a very low EM/PM ratio (0.41), which was very significantly lower than that from normal melanocytes isolated both from eyes with light-colored irides (1.31) or dark-colored irides (7.32). The low EM/PM ratio was caused by a low level of EM in melanoma cells, which was only 1/8 and 1/31 of that in melanocytes from eyes with light-colored irides and dark-colored irides, respectively. The PM level in uveal melanoma cells was not statistically different from normal melanocytes from eyes with light-colored irides or dark-colored irides. The total quantity of EM and PM in uveal melanoma cells was significantly less than that in normal melanocytes. This difference was because of the low level of EM in uveal melanoma cells. The results of these studies indicate that the changes of melanin content in uveal melanoma cells mainly relate to the decrease of EM content. Low melanin and EM content may make melanoma cells more susceptible to mutagenic effects of ultraviolet radiation and oxidative stress, which may enhance the proliferation of melanoma cells and accelerate progression of melanoma.

Extracellular Signal-regulated Kinase-dependent Proliferation Is Mediated through the Protein Kinase A/B-Raf Pathway in Human Uveal Melanoma Cells

Mutated B-Raf-mediated constitutive activation of ERK1/2 is involved in about 66% of cutaneous melanoma. By contrast, activating mutations in B-RAF are rare in ocular melanoma. This study aimed to determine the role of wild-type B-Raf ((WT)B-Raf) in uveal melanoma cell growth. We used cell lines derived from primary tumors of uveal melanoma to assess the role of (WT)B-Raf in cell proliferation and to characterize its upstream regulators and downstream effectors. Melanoma cell lines expressing (WT)B-Raf and (WT)Ras grew with similar proliferation rates, showed constitutive activation of ERK1/2, and had similar levels of B-Raf expression and B-Raf kinase activity as melanoma cell lines expressing the activating V600E mutation ((V600E)B-Raf). They were equally as sensitive to pharmacological inhibition of MEK1/2 for cell proliferation and transformation as (V600E)B-Raf cells. siRNA-mediated depletion of Raf-1 did not affect either ERK1/2 activation, whereas siRNA-mediated depletion of B-Raf reduced cell proliferation by up to 65% through the inhibition of ERK1/2 activation, irrespective of the mutational status of B-Raf. Pharmacological inhibition of cAMP-dependent protein kinase (PKA) and siRNA-mediated depletion of PKA greatly reduced B-Raf activity, ERK1/2 activation, and cell proliferation in (WT)B-Raf cells, whereas it did not affect (V600E)B-Raf cells, demonstrating a key role of PKA in mediating (WT)B-Raf/ERK signaling for uveal melanoma cell growth. Moreover, inactivation or depletion of PKA did not affect Rap-1 activity, and Rap-1 depletion did not affect either B-Raf activity or ERK1/2 activation. This ruled out a role for Rap1 in the PKA-mediated B-Raf/ERK activation in (WT)B-Raf cells. Finally, we demonstrated the importance of cyclin D1 in mediating PKA/(WT)B-Raf signaling for cell proliferation. Altogether, our results suggest that the PKA/B-Raf pathway is a potential target for therapeutic strategies against (WT)B-Raf-expressing uveal melanoma.

Functional Analysis of the p53 Pathway in Response to Ionizing Radiation in Uveal Melanoma

Uveal melanomas are notoriously radioresistant and thus necessitate treatment with extremely high radiation doses that often cause ocular complications. The p53 tumor suppressor pathway is a major mediator of the cellular response to radiation-induced DNA damage, suggesting that this pathway may be defective in uveal melanoma. The current study was conducted to analyze the functional integrity of the p53 pathway in primary uveal melanoma cells. The p53 gene was sequenced in three primary uveal melanoma cells lines. Cultured primary uveal melanoma cells (MM28, MM50, Mel202, Mel270, and Mel290), MCF7 breast carcinoma cells, normal uveal melanocytes (UM47), and normal human diploid fibroblasts (NHDFs) were irradiated at 250 kVp and 12 mA at a dose rate of 1.08 Gy/min for a total dose of up to 20 Gy. Cell viability was analyzed with trypan blue exclusion. Western blot analysis was used to analyze the expression of p53, p53-phospho-Ser15, p21, Bax, PUMA, and Bcl-x(L). No p53 gene mutations were found in MM28, MM50, or Mel270 cells. Upstream signaling to p53 was intact, with normal induction of p53 and phosphorylation of p53-Ser15, in all five cell lines. Radiation-induced downstream activation of p21 was defective in MM28 and MM50 cells, and activation of Bax was defective in MM50 and Mel290 cells. MM28, MM50, and Mel202 cells failed to deamidate Bcl-x(L) in response to radiation-induced DNA damage. Overall, four of the five uveal melanoma cell lines exhibited at least one downstream defect in the p53 pathway. Expression of p53 and upstream signaling to p53 in response to radiation-induced DNA damage appear to be intact in most uveal melanomas. In contrast, functional defects in the p53 pathway downstream of p53 activation appear to be common. Further elucidation of p53 pathway abnormalities in uveal melanoma may allow therapeutic interventions to increase the radiosensitivity of the tumors.

Abnormal cell cycle regulation in primary human uveal melanoma cultures

Uveal malignant melanoma is the most frequent primary intraocular tumor in adult humans. The cellular events leading to neoplasic transformation of normal uveal melanocytes are not well known when compared to other cancers. In this study, we investigated the role of G1 and G1/S regulatory proteins of the cell cycle in human uveal melanoma (UM) primary cell cultures, since these proteins are common targets in tumor development. Further, freshly established and characterized tumor cells are a better model for in vitro studies when compared to cell lines established long ago. Human primary cell cultures from eight different UM were established, as well as one primary culture from rhesus uveal normal melanocytes (UNM). Primary human UM cultures were characterized by a low establishment and growing rate. From four successful cultures, three showed a high expression of cyclin D1, cyclin E, p16NK4A, and p27KIP1 with no variations in cyclin A, cyclin-dependent kinase 2 (CDK2), and CDK4. Interestingly, in one of the cultured tumors, tumor suppressor protein retinoblastoma (Rb) did not bind E2F despite the fact that Rb was found in its hypophosphorylated form. No mutations in either RB1 or the Rb-binding pocket of E2F-1 were detected. Furthermore, we identified seven proteins co-immunoprecipitating with Rb in this tumor, including Lamin A/C and six proteins not previously reported to bind Rb: Hsc70, high mobility group protein 1 (HMG-1), hnRPN, glyceraldehyde 3 phosphate dehydrogenase (G3PDH), EF-1, and EF-2. Our results indicate that the overexpression of cyclins D1/E and CDKIs p16 and p27, together with a deregulation of the Rb/E2F pathway, may be implicated in the development of human UM.

Roles of Stem Cell Factor/c-Kit and Effects of Glivec®/STI571 in Human Uveal Melanoma Cell Tumorigenesis

The B-Raf(V599E)-mediated constitutive activation of ERK1/2 is involved in establishing the transformed phenotype of some uveal melanoma cells (Calipel, A., Lefevre, G., Pouponnot, C., Mouriaux, F., Eychene, A., and Mascarelli, F. (2003) J. Biol. Chem. 278, 42409-42418). We have shown that stem cell factor (SCF) is involved in the proliferation of normal uveal melanocytes and that c-Kit is expressed in 75% of primary uveal melanomas. This suggests that the acquisition of autonomous growth during melanoma progression may involve the SCF/c-Kit axis. We used six human uveal melanoma tumor-derived cell lines and normal uveal melanocytes to characterize the SCF/c-Kit system and to assess its specific role in transformation. We investigated the possible roles of activating mutations in c-KIT, the overexpression of this gene, and ligand-dependent c-Kit overactivation in uveal melanoma cell tumorigenesis. Four cell lines (92.1, SP6.5, Mel270, and TP31) expressed both SCF and c-Kit, and none harbored the c-KIT mutations in exons 9, 11, 13, and 17 that have been shown to induce SCF-independent c-Kit activation. Melanoma cell proliferation was strongly inhibited by small interfering RNA-mediated depletion of c-Kit in these cells, despite the presence of (V599E)B-Raf in SP6.5 and TP31 cells. We characterized the signaling pathways involved in SCF/c-Kit-mediated cell growth and survival in normal and tumoral melanocytes and found that constitutive ERK1/2 activation played a key role in both the SCF/c-Kit autocrine loop and the gain of function of (V599E)B-Raf for melanoma cell proliferation and transformation. We also provide the first evidence that Glivec/STI571, a c-Kit tyrosine kinase inhibitor, could be used to treat uveal melanomas.

Gene expression profiling identifies tumour markers potentially playing a role in uveal melanoma development.

Microarray is a powerful tool to compare the gene expression of different tumour specimens and cell lines simultaneously and quantitatively. To get a better insight into genes that are involved in uveal melanoma tumorigenesis, we compared the gene expression profiles of 12 different uveal melanoma cell lines with three melanocyte cell cultures obtained from healthy donor eyes. Gene expression profiles were obtained by nylon filter arrays, containing 1176 gene spots related to cancer development. The expression levels of selected genes were validated on cell lines and primary uveal melanomas by real time RT–PCR, and were subsequently included in cluster analysis. Four candidate tumour markers, Laminin Receptor 1, Endothelin 2, Von Hippel Lindau Binding protein 1 and Cullin 2, have been selected from genes that were differentially expressed in the uveal melanoma cell lines compared to the normal uveal melanocytes. In primary uveal melanomas, these four markers could discriminate between two classes of uveal melanoma, which may be indicative of a differential disease process.

Status of the NF1 tumor suppressor locus in uveal melanoma.

A clinical association has been observed between uveal melanoma and neurofibromatosis type 1 (NF1). This study aims to determine whether the NF1 tumor suppressor gene is mutated in uveal melanoma. Thirty-eight uveal melanomas, as well as normal uveal melanocytes, were examined for NF1 deletions by dual-color fluorescence in situ hybridization, and for expression of the NF1 protein (neurofibromin) by immunohistochemistry and Western blot analysis. Normal uveal melanocytes strongly express neurofibromin. Eighteen (47%) of uveal melanomas demonstrated weak expression of neurofibromin. One large tumor contained a deletion of the NF1 locus and lacked neurofibromin expression. Two other tumors contained additional copies of the NF1 chromosomal region. Mutations of the NF1 gene may occasionally play a role in the pathogenesis of uveal melanoma. Clinical Relevance A search for biallelic NF1 mutations in uveal melanomas from patients with neurofibromatosis will be of interest to determine whether germline NF1 mutations may predispose to uveal melanoma.

Expression of angiogenic factors Cyr61 and tissue factor in uveal melanoma.

To study the expression of angiogenic factors Cyr61 and tissue factor (TF) in uveal melanoma and its correlation with blood vessel density. Suppression subtractive hybridization was used to identify genes that are differentially expressed between cell lines of uveal melanoma and normal uveal melanocytes. Expression of these genes was subsequently verified in primary uveal melanomas and correlated with the number of blood vessels in archival specimens by immunohistochemical analysis. Cyr61 and TF are expressed at elevated levels in cell lines of uveal melanoma compared with normal uveal melanocytes. Duplication of a region of chromosome arm 1p, encompassing the genes encoding Cyr61 and TF, was observed in the melanoma cell line used in the initial subtractive hybridization. Both genes are also expressed in primary uveal melanomas, and a correlation was found between expression of TF and the number of blood vessels in archival specimens. Cyr61 and TF may contribute to the angiogenic phenotype associated with uveal melanoma. A region of chromosome arm 1p also may contain oncogenes or tumor suppressor genes pertinent to the origins of this type of ocular tumor. New immunotherapies have been devised for the treatment of cancer based on the expression of TF. Similar approaches may be effective in treating uveal melanoma.

Tissue plasminogen activator is released into cultured medium by cultured human uveal melanocytes.

Melanoma cells produce tissue plasminogen activator (t-PA) that plays an important role in tumor invasion and metastasis. The production of t-PA by normal human uveal melanocytes has not been reported previously. In order to explore this possibility, we studied the production of t-PA by cultured human uveal melanocytes and compared that with the production by cultured human uveal melanoma cells and epidermal melanocytes. Human adult uveal melanocytes were isolated and cultured from donor eyes. The cells were cultured in serum-free medium for 48 h and the conditioned medium then collected for the plasminogen activator (PA) activity assay. Free PA activity was tested in an amidolytic assay using a t-PA standard curve. PA type was identified by fibrinography and antihuman t-PA and urokinase plasminogen activator (u-PA) blocking antibodies. Free PA activity was found in the conditioned medium of normal melanocytes and melanoma cells. The predominant PA activity was t-PA. Normal uveal melanocytes produced more t-PA (3.23 +/- 0.73 IU/105 cells/24 h) than that of epidermal melanocytes (1.25 IU/105 cells/24 h) but much less than uveal melanoma cells (11.0 +/- 3.39 IU/105 cells/24 h). Western blot analysis revealed that most t-PA in conditioned media were one-chain t-PA with molecular weight of 69 kDa. Our study indicates that uveal melanocytes may contribute to the free t-PA activity previously found in aqueous humor and choroidal eye cup superfusions. Therefore, this function of uveal melanocytes may play a role in intraocular matrix remodeling, fibrinolysis and aqueous humor outflow.

Melatonin receptors in human uveal melanocytes and melanoma cells.

Previous work has demonstrated that melatonin inhibits growth of cultured human uveal melanoma cells. The goal of this study was to determine the expression of mRNA encoding the melatonin receptor subtypes and the effect of specific melatonin receptor agonists on cell growth of uveal melanoma cells and melanocytes. RNA expression of the human melatonin Mel1a and Mel1b receptor subtypes was determined by reverse transcription-polymerase chain reaction (RT-PCR) amplification of RNA isolated from two melanoma cell lines and from one cell line of normal melanocytes. PCR-amplified cDNA encoding the Mel1b melatonin receptor subtype, but not the Mel1a subtype, was detected in reverse-transcribed RNA obtained from both normal uveal melanocytes and melanoma cell lines. Uveal melanoma cells and melanocytes were cultured for 24 hr, then melatonin or one of its membrane receptor agonists, 6-chloromelatonin (Mel1a-1b) or S-20098 (Mel1b) or its putative nuclear agonist, CGP-52608 (Mel2), was added to the medium. After 5 days, the cells were detached, counted, and compared to untreated controls. Melatonin and its membrane receptor agonists (Mel1a-1b and Mel1b), but not its putative nuclear receptor agonist (Mel2), inhibited the growth of uveal melanoma cells, but not normal melanocytes, at very low concentrations. In uveal melanoma cells, the expression of RNA encoding the Mel1b receptor suggests that the growth inhibiting effect of melatonin on uveal melanoma cells is related to activation of the melatonin Mel1b membrane receptor. Furthermore, the expression of RNA encoding melatonin receptors in normal uveal melanocytes suggests that melatonin may play a role in the function of these cells.

The identification and differential expression of calcium-binding proteins associated with ocular melanoma

Calcium-binding proteins may endow tumor cells with properties related to their malignancy and metastatic phenotype. Chromatographic procedures and amino acid sequence analysis were used in this study to identify seven calcium-binding proteins, annexin VI, cap g, annexin V, calmodulin, S100A11, S100B and S100A6, associated with uveal melanoma, the primary ocular tumor of adults. This series of calcium-binding proteins was identified in both primary tumors and cell lines of uveal melanoma. Several of the proteins were shown by immunochemical methods to be differentially expressed between normal uveal melanocytes and malignant melanomas of the uvea. In addition, the expression of S100A6 may correlate with the malignant properties of the tumor.

Attachment of human uveal melanocytes and melanoma cells to extracellular matrix proteins involves intracellular calcium and calmodulin.

The purpose of this study was to examine which intracellular signalling systems influence the attachment of normal uveal melanocytes and uveal melanoma cells to extracellular matrix (ECM) proteins in vitro. Uveal melanocytes were found to attach strongly to fibronectin in preference to plastic, collagens type I, III or IV, or laminin. In contrast, uveal melanoma cells attached equally well to fibronectin and collagens I, III and IV in preference to plastic or laminin. Manipulation of intracellular cyclic AMP or protein kinase C had little, if any, effect on the attachment of either cell to fibronectin. In contrast, inhibition of calmodulin significantly inhibited the attachment of both normal and transformed cells, as did manipulating intracellular free calcium. We noted that the intracellular free calcium in melanoma cells was less than half that seen in melanocytes. Fibronectin, laminin and Arg-Gly-Asp (RGD) were all capable of acutely increasing the intracellular free calcium in both cells. ECM-induced increases in calcium were more apparent in low density than high density cells and appeared more sustained in melanocytes than in melanoma cells. We conclude that both normal and neoplastic uveal melanocytes require an intracellular signal or signals which involves calcium and calmodulin in the few minutes following cell binding to ECM proteins in order for successful cell attachment to occur. While the transformed cell does not differ significantly from the normal cell in this respect, this dependency on calcium and calmodulin may nevertheless offer an approach for pharmacological intervention in the prevention or arrest of metastatic spread and merits further investigation.