Brn-2在人脉络膜黑色素瘤细胞系中的表达及其对MART-1基因启动子活性的影响

目的　探讨黑色素抗原转录子Brn-2在人脉络膜黑色素瘤细胞系中的表达及其对T细胞可识别抗原-1（melanoma antigen recognized by T cells 1,MART-1）启动子活性的影响。方法　采用RT-PCR和Western blot检测人脉络膜黑色素瘤细胞系92-1、92-2、Me1285和Ocm3 细胞中Brn-2的mRNA和蛋白表达水平。将含不同长度MART-1基因启动子序列和荧光素酶报告基因的表达质粒pGL3-Luc构建成融合表达载体pGL3-p286-Luc及pGL3-p2956-Luc,与pEV-Brn-2融合表达质粒共转染脉络膜黑色素瘤细胞系细胞。分别测量四种细胞系中p286组、p2956组、p286+Brn-2组、p2956+Brn-2组细胞荧光素酶表达变化,并观察Brn-2对上述细胞MART-1基因启动子活性的影响。结果　人脉络膜黑色素瘤细胞系92-1、92-2、Me1285、Ocm3细胞系均可检测到Brn-2 mRNA和蛋白的表达,Brn-2蛋白电泳带大致在相对分子质量47 000处。pGL3-p2956-Luc及pGL3-p286-Luc重组质粒经ApaI和NheI双酶切可切出2956 bp和286 bp 2个条带,pEV-Brn-2重组质粒经EcoRI和BamHI双酶切可切出1329 bp条带。pEV-Brn-2重组质粒分别对人脉络膜黑色素瘤细胞系Ocm3、Mel285、92-2、92-1进行磷酸钙法转染,Western blot检测可见四种细胞系均表达Brn-2蛋白。缺乏Brn-2时,所有细胞的MART-1的p286均显示出活性,MART-1阳性细胞系（92-1、92-2、Ocm3）p286活性高于阴性细胞系（Mel285）。p2956活性不同细胞系表现不同,92-1、92-2中较高,Ocm3中其活性与细胞密度相关,MART-1阴性细胞系中p2956近乎无活性。共转染Brn-2后明显下调92-1、92-2、Ocm3中启动子p286及p2956活性（P<0.05）;阴性细胞系Mel285中,Brn-2对启动子活性无影响（P>0.05）。结论　Brn-2表达于人脉络膜黑色素瘤细胞,并下调阳性细胞系MART-1启动子活性。     

Uveal and cutaneous melanoma: shared expression characteristics of melanoma-associated antigens

PURPOSE: Downregulation of melanoma-associated antigens (MAAs), against which natural cytolytic T lymphocytes (CTLs) exist in humans, is one of the mechanisms that aids in evasion of immune surveillance. In view of putative re-expression strategies for MAAs during immunotherapy, this study was conducted to investigate MAA silencing in malignant melanoma. METHODS: The expression of the MAA Melan-A/MART-1 was analyzed in 10 uveal and 10 cutaneous patient-derived melanoma cell lines by Western blot analysis and RT-PCR. Expression characteristics of four other MAAs-Tyr, Tyrp1, Dct, and gp100/Pmel17-were analyzed by RT-PCR. DNA methylation patterns at the Melan-A/MART-1 promoter region were investigated by methylation-sensitive restriction enzyme digestion and subsequent Southern blot analysis. Exogenous promoter activity was assessed in all 20 melanoma cell lines to correlate the DNA methylation patterns with Melan-A/MART-1 expression. RESULTS: MAA expression was observed in 15 of the 20 melanoma cell lines. Furthermore, there is a direct correlation between DNA methylation patterns at the Melan-A/MART-1 promoter region, exogenous Melan-A/MART-1 promoter activity, and Melan-A/MART-1 protein expression. These data reveal the division of patient-derived melanoma cell lines into two distinct subsets, which are identical for both uveal and cutaneous tumor types. CONCLUSIONS: The authors propose a categorization of melanoma cell lines into two different panels based on shared MAA-expression characteristics: panel I, MAA-expressing cell lines, and panel II, MAA-deficient cell lines. This categorization can be used to obtain knowledge about the regulation of MAA-expression and for further research concerning MAA-based immunotherapy.     

PD-L1: PD-1 interaction contributes to the functional suppression of T-cell responses to human uveal melanoma cells in vitro

PURPOSE: To assess the expression of PD-L1 on human uveal melanomas and its potential to suppress T-cell function. METHODS: A panel of primary and metastatic uveal melanoma cell lines was evaluated for PD-L1 expression by RT-PCR and flow cytometric analysis. Uveal melanoma-containing eyes were examined for PD-L1 expression by immunohistochemistry. PD-L1 function was tested by coculturing IFN-gamma-pretreated uveal melanoma cells with activated Jurkat T cells for 48 hours and assessing T-cell production of IL-2 by ELISA. RESULTS: Five of the nine primary and one of the five metastatic uveal melanoma cell lines tested constitutively expressed PD-L1 protein at various levels. However, all primary and metastatic uveal melanoma cell lines upregulated PD-L1 expression after stimulation with IFN-gamma. Immunohistochemistry demonstrated that PD-L1 was not expressed by primary uveal melanomas in situ. IL-2 production by activated Jurkat T cells was decreased significantly when the cells were cocultured with IFN-gamma-pretreated uveal melanoma cells. More than 70% of IL-2 production was restored by addition of either anti-PD-L1 or anti-PD-1 antibody to the coculture assays (P < 0.01). CONCLUSIONS: Expression of PD-L1 by uveal melanoma cells regulates T-cell function by suppressing IL-2 production. The results imply that the presence of IFN-gamma in the tumor local microenvironment promotes upregulation of PD-L1 expression by uveal melanoma, which may, in part, promote immune escape by impairing T-cell function. The selective blockade of PD-L1 is a potential strategy in T-cell-based immunotherapy for uveal melanoma.     

Costimulatory molecule expression on human uveal melanoma cells: functional analysis of CD40 and B7-H1

Costimulatory molecules play important roles in regulating T cell function in tumor immunity. In this study, we investigated costimulatory molecule expression on human uveal melanoma cells (a primary culture, and OCM-1, OMM-1 and 92-1 cell lines) and assessed the functional roles of selected costimulatory molecules. Uveal melanoma cells were incubated in the presence or absence of IFN-γ and expression of costimulatory molecules on the cells was measured by flow cytometry. The costimulatory effect of B7-H1-expressing uveal melanoma cells on cytokine production by purified T cells was studied in uveal melanoma/T cell co-culture experiments using a blocking anti-B7-H1 monoclonal antibody (mAb). The functional role of CD40-mediated interactions in modifying immune responses to uveal melanoma cells was assessed in?vitro using recombinant human CD40 ligand (rhCD40L). MHC class I and B7-H1 were consistently detected and further upregulated by IFN-γ stimulation in all human uveal melanoma cell cultures. CD40 was consistently detected and further upregulated by IFN-γ stimulation in primary culture, OCM-1, and OMM-1 but not 92-1. IL-2 production from purified CD3(+) T cells co-stimulated with IFN-γ-treated uveal melanoma cells was significantly enhanced by the addition of anti-B7-H1 mAb. Treatment of primary culture, OCM-1, or OMM-1 with rhCD40L induced or enhanced secretion of chemokines IL-8, MCP-1, IP-10 and RANTES. These results suggest that the expression of B7-H1 on IFN-γ-treated uveal melanoma cells contributes to suppression of T cells by decreasing IL-2 production. In contrast, CD40 expressed on uveal melanoma cells plays an important role in augmenting anti-tumor immunity by stimulating chemokine production. The dual effects of CD40 and B7-H1 may contribute to positive or negative regulation of anti-tumor immune responses to human uveal melanoma.     

Epigenetic regulation identifies RASEF as a tumor-suppressor gene in uveal melanoma

PURPOSE: Recently, a segregation study in families with uveal and cutaneous melanoma identified 9q21 as a potential locus harboring a tumor-suppressor gene (TSG). One of the genes in this area, RASEF, was then analyzed as a candidate TSG, but lack of point mutations and copy number changes could not confirm this. In this study, the RASEF gene was investigated for potential mutations and gene silencing by promoter methylation in uveal melanoma. METHODS: Eleven uveal melanoma cell lines and 35 primary uveal melanoma samples were screened for mutations in the RASEF gene by high-resolution melting-curve and digestion analysis. Expression of RASEF was determined by real-time RT-PCR in all cell lines and 16 primary uveal melanoma samples, and the methylation status of the promoter of the RASEF gene was analyzed and confirmed by direct sequencing. RESULTS: Mutation screening revealed a known polymorphism (R262C; C-->T) in exon 5 of the RASEF gene that displayed a normal frequency (54%). Of the primary uveal melanomas, 46% presented a heterozygous genotype, and 10 (91%) of 11 cell lines showed a homozygous genotype. Melting-curve analysis indicated loss of heterozygosity in at least two primary tumors. Low RASEF expression in the cell lines and primary tumors correlated with methylation of the RASEF promoter region. Homozygosity and methylation of the RASEF gene in primary tumors were associated with decreased survival (P = 0.019). CONCLUSIONS: Homozygosity, in combination with methylation, appears to be the mechanism targeting RASEF in uveal melanoma, and allelic imbalance at this locus supports a TSG role for RASEF.     

Expression and possible function of IL-2 and IL-15 receptors on human uveal melanoma cells

PURPOSE: Interleukin-2 (IL-2) and IL-15 receptors have been detected on some murine neoplasms. Accordingly, the expression of these receptors on human uveal melanoma cell lines was examined, and the effect of exogenous IL-2 and -15 on melanoma cell proliferation, susceptibility to natural killer (NK) cell-mediated cytolysis, and sensitivity to apoptosis were assessed. METHODS: Nine human uveal melanoma cell lines and three cell lines from uveal melanoma metastases were tested by flow cytometry for the expression of human IL-2R and -15Ralpha. Melanoma cells were cultured, with or without recombinant human IL-2 or -15, cell proliferation was determined by tritiated thymidine incorporation, and IL-2 and -15 receptor expression was assessed by flow cytometry. The effect of these cytokines on NK activity was evaluated with a standard (51)Cr-release assay. RESULTS: All the melanoma cell lines expressed IL-2R and -15R. IL-2 induced a three- to eightfold upregulation of IL-2R expression in all the melanoma cell lines. Although IL-2 did not affect the proliferation of six of the seven uveal melanoma cell lines, it induced a 32% and 57% increase in the proliferation of both metastatic cell lines. IL-15 induced proliferation on all tested cell lines (4%-68%). Both IL-2 and -15 reduced melanoma cell sensitivity to NK-cell-mediated cytolysis and cisplatin-induced apoptosis. CONCLUSIONS: The results suggest that IL-2 and -15 elaborated by tumor-infiltrating lymphocytes and macrophages may affect the malignant behavior of human uveal melanoma by stimulating proliferation and reducing uveal melanoma cell susceptibility to NK-cell-mediated cytolysis and cisplatin-induced apoptosis.     

骨桥蛋白在不同侵袭转移潜能葡萄膜黑色素瘤中的表达及意义

背景 骨桥蛋白(OPN)在多种转移性肿瘤组织中表达增高,但在葡萄膜黑色素瘤(UM)中的表达与临床病理特征及侵袭转移是否相关尚不清楚. 目的 研究UM组织及不同转移潜能人UM细胞系MUM-2B、C918和OCM-1A中OPN的表达情况,分析OPN的组织及细胞系表达水平与UM患者临床病理特征与转移预后之间的关系.方法 收集2004年1月至2007年12月在北京同仁医院行眼球摘除手术并已经病理证实为脉络膜黑色素瘤组织的标本共50例,应用免疫组织化学法检测石蜡标本中OPN的表达,分析其与临床资料的相关性.应用定量聚合酶链反应(Q-PCR)技术检测不同侵袭性转移潜能的人UM细胞系中OPN mRNA的表达情况.结果 50例UM组织中发生肝转移者13例,其中10例OPN表达阳性,未发生肝转移的37例中14例OPN表达阳性;上皮型与非上皮型脉络膜黑色素瘤OPN表达阳性者分别为11/15和13/35;肿瘤累及睫状体和未累及者OPN表达阳性者分别为20/30和4/20;上述指标的比较差异均有统计学意义(X2=5.888、5.510、10.470,P＜0.05).患者不同性别、年龄、眼别、肿瘤最大基底径以及是否侵犯巩膜导管间OPN表达阳性例数的比较差异均无统计学意义(P=0.536、0.256、0.802、0.848、0.555).转移潜能细胞系由高到低依次为MUM-2B、C918、OCM-1A,其OPN mRNA相对表达量分别为1.00±0.04、0.91±0.03、0.08±0.01,差异有统计学意义(F=33.135,P＜0.05),MUM-2B、C918中OPN mRNA的表达水平较OCM-1A中明显增高,差异均有统计学意义(P=0.00),而MUM-2B、C918中OPN mRNA的表达水平差异无统计学意义(P=0.804).结论 OPN与UM侵袭能力及转移潜能密切相关,发生转移的UM组织及高侵袭转移性细胞系中OPN表达水平明显升高,提示OPN可能作为预测UM侵袭能力、转移潜能以及患者预后的指标.     

Uveal melanoma expression of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) receptors and susceptibility to TRAIL-induced apoptosis

PURPOSE: The study had two purposes: to examine the expression of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) receptors on uveal melanoma cells and metastases arising from uveal melanoma and to determine the susceptibility of uveal melanoma cells to TRAIL-induced apoptosis. METHODS: Nine human uveal melanoma cell lines and three cell lines derived from uveal melanoma metastases were examined for TRAIL receptor expression by flow cytometry. In vitro apoptosis assays were performed to determine the relative susceptibility of uveal melanoma cells to TRAIL-induced apoptosis. Annexin V staining was also used to determine the capacity of either cycloheximide or interferon-beta to enhance TRAIL-induced apoptosis. RESULTS: Five of the nine uveal melanoma cell lines expressed TRAIL-R2 on more than 60% of the cells. All three of the cell lines derived from uveal melanoma metastases expressed TRAIL-R2 on more than 50% of the cells. Cycloheximide exerted a profound effect in enhancing TRAIL-induced apoptosis in all but two of the uveal melanoma cell lines and in all three of the metastases cell lines. Interferon-beta produced a similar enhancement of TRAIL-induced apoptosis, even in cell lines that were previously shown to be resistant. CONCLUSIONS: TRAIL is a potentially useful therapeutic modality for the management of uveal melanomas and their metastases. Moreover, pharmacological agents and biological response modifiers that independently display antineoplastic properties can enhance TRAIL-induced apoptosis in resistant uveal melanoma cells.     

miR-194在葡萄膜黑色素瘤细胞系中的表达及对细胞增殖、凋亡、迁移和侵袭的影响

目的　探讨miR-194在葡萄膜黑色素瘤细胞系中的表达及对细胞增殖、凋亡、迁移和侵袭的影响。方法　RT-PCR检测miR-194在正常视网膜上皮细胞系ARPE-19及葡萄膜黑色素瘤细胞系SP6.5、M23及OM431中的表达。取SP6.5、M23及OM431细胞分成两组,miR-194过表达组（miR-194组）及阴性对照组（NC组）,经Lipofectamine^TM 2000分别转染miR-194 mimic及阴性对照序列scramble。CCK-8实验及流式细胞仪分别测定细胞增殖和凋亡能力,细胞划痕及Transwell实验分别测定细胞迁移和侵袭能力。Western blot测定叉头盒蛋白A1（forkhead box,FOXOA1)、多聚ADP-核糖聚合酶（poly ADP-ribose polymerase,PARP)蛋白表达。结果　正常视网膜上皮细胞系ARPE-19中miR-194的相对表达量为1.03±0.04,葡萄膜黑色素瘤细胞系SP6.5、M23及OM431相对表达量分别为0.25±0.02、0.37±0.02及0.47±0.03,葡萄膜黑色素瘤细胞系SP6.5、M23及OM431中miR-194相对表达量低于正常视网膜上皮细胞系ARPE-19(均为P<0.001 )。转染0 h、24 h、48 h、72 h、96 h后,miR-194组和 NC组A值分别为:0.23±0.02、0.21±0.03（t=0.960,P=0.391）,0.38±0.02、0.37±0.03（t=0.480,P=0.656）,0.75±0.04、0.78±0.06（t=-0.720,P=0.511）,0.87±0.09、1.59±0.12（t=-8.313,P=0.001）,1.53±0.14、3.05±0.22（t=-10.096,P=0.000）;流式细胞仪检测结果显示:miR-194组细胞凋亡率为23.30%±2.10%,NC组为2.47%±0.30%,miR-194组细胞凋亡率高于NC组（t=17.007,P=0.000）。细胞划痕实验结果显示:miR-194组细胞划痕愈合率为25.3%±3.5%,NC组为72.9%±6.4%,miR-194组细胞划痕愈合率低于NC组（t=-11.302,P=0.000）;Transwell实验结果显示:200倍视野下,miR-194组侵袭细胞数为（188.6±20.7）个,NC组为（352.9±32.8）个,miR-194组侵袭细胞数少于NC组（t=-7.337,P=0.001）。miR-194组FOX A1蛋白相对表达量为0.29±0.03,NC组为1.03±0.06,miR-194组FOX A1蛋白相对表达量低于NC组（t=-19.106,P=0.000）;miR-194组裂解型PARP蛋白相对表达量为2.87±0.24,NC组为1.03±0.06,miR-194组裂解型PARP蛋白相对表达量高于NC组（t=12.882,P=0.000）。结论　miR-194在葡萄膜黑色素瘤细胞系中低表达,上调miR-194表达可抑制葡萄膜黑色素瘤细胞增殖,促进凋亡,抑制迁移和侵袭,其机制可能与下调FOX A1及上调裂解型PARP蛋白表达有关。     

c-Kit-dependent growth of uveal melanoma cells: a potential therapeutic target?

PURPOSE: This study was conducted to investigate the expression and functional impact of the proto-oncogene c-kit in uveal melanoma. METHODS: Based on immunohistochemical (IHC) study of paraffin-embedded specimens from 134 uveal melanomas and Western blot analysis on eight fresh-frozen samples the expression of c-kit in uveal melanoma was studied. Furthermore, the phosphorylation of c-kit and the impact of the tyrosine kinase inhibitor STI571 was examined in the three uveal melanoma cell lines OCM-1, OCM-3, and 92-1. RESULTS: Eighty-four of 134 paraffin-embedded samples and six of eight fresh-frozen samples expressed c-kit. c-Kit was strongly expressed and tyrosine phosphorylated in cultured uveal melanoma cells compared with cutaneous melanoma cells. Moreover, in contrast to cutaneous melanoma cell lines c-kit maintained a high phosphorylation level in serum-depleted uveal melanoma cells. No activation-related mutations in exon 11 of the KIT gene were found. On the contrary, expression of the stem cell growth factor (c-kit ligand) was detected in all three uveal melanoma cell lines, suggesting the presence of autocrine (paracrine) stimulation pathways. Treatment of uveal melanoma cell lines with STI571, which blocks c-kit autophosphorylation, resulted in cell death. The IC(50) of the inhibitory effects on c-kit phosphorylation and cell proliferation was of equal size and less than 2.5 microM. CONCLUSIONS: The results confirm that c-kit is vastly expressed in uveal melanoma, suggest that the c-kit molecular pathway may be important in uveal melanoma growth, and point to its use as a target for therapy with STI571.     

Expression of somatostatin receptors in uveal melanomas

PURPOSE: To investigate the distribution of somatostatin receptor (SSR) subtypes 2, 3, and 5 in uveal melanomas and their diagnostic and possible therapeutic value. METHODS: SSRs were investigated in 25 paraffin-embedded eyes with uveal melanomas and in 6 normal eyes without any disease, by using polyclonal antiserum directed to SSR2A, -2B, -3, and -5. Antigen expression was evaluated by a semiquantitative method. The expression pattern of SSR was correlated with the patients' ad vitam prognosis by use of the Kaplan-Meier survival curve. Six different human melanoma cell lines were incubated with octreotide and vapreotide, and a proliferation assay was performed by determining [(3)H]-TdR uptake. [111-Indium-DTPA-D-Phe1]-octreotide scintigraphy was performed in the eyes of four patients with known uveal melanomas. RESULTS: All uveal melanomas were positive for SSR2. SSR2A was expressed in 15 of 25, SSR2B in 23 of 25, SSR3 in 7 of 25, and SSR5 in 13 of 25 uveal melanomas. A Kaplan-Meier survival curve showed a significantly better ad vitam prognosis for patients with tumors expressing high levels of SSR2. Cell proliferation was inhibited up to 36% +/- 6% in three of six melanoma cell lines at a concentration of 10(-4) M octreotide or vapreotide. Eyes of two patients with uveal melanomas showed positive uptake of [111-Indium-DTPA-D-Phe1]-octreotide. CONCLUSIONS: SSR2, -3, and -5 are expressed in human uveal melanomas and patients with a high amount of SSR2 in the melanoma tissue have a better ad vitam prognosis. Because a melanoma cell proliferation assay showed an inhibitory effect of up to 36% +/- 6% using octreotide or vapreotide, somatostatin analogues may be beneficial in the treatment of patients with ocular melanomas.     

Tumorigenicity of the mixed spindle-epithelioid SP6.5 and epithelioid TP17 uveal melanoma cell lines is differentially related to alpha5beta1 integrin expression.

PURPOSE: It has been suggested that the epithelioid morphology and high aggressiveness that is typical of the uveal melanoma cell line TP17 is dependent on the loss of alpha5beta1 integrin expression at the cell surface. The purpose of the current study was to test this hypothesis in the TP17 cell line and investigate the role this integrin may play in the tumorigenicity of the SP6.5 cells, a mixed spindle-epithelioid culture-type human uveal melanoma that shows tumorigenic properties clearly distinct from that of TP17 cells. METHODS: Expression of the alpha5 integrin subunit was restored in the alpha5-TP17 cell line by stably transfecting the cells with a recombinant plasmid encoding the integrin subunit. Flow cytometry and adhesion assays on fibronectin (FN)-coated culture plates were used to monitor alpha5 expression in the cells. The effect of alpha5 expression on both tumorigenicity and cell proliferation was evaluated in vivo in nude mice. In vitro growth properties of the alpha5(+) TP17 cells was evaluated by cell counting and compared with that of the alpha5 parental TP17 cell line. The influence exerted by the alpha5 integrin subunit on the tumorigenic and proliferative properties of the SP6.5 cells was evaluated in vivo in nude mice by exposing the cells to increasing doses of a blocking antibody directed against the alpha5-subunit before subcutaneous injection, and compared with the results obtained with untreated SP6.5 cells. RESULTS: Expression of the alpha5 integrin subunit in the alpha5-TP17 cells was successfully achieved, as evidenced by both flow cytometry and adhesion assays on FN-coated culture plates. Restoring expression of alpha5 in TP17 cells enhanced epithelioid cell morphology and increased the growth properties of this cell line in vivo. The ability of the SP6.5 cells to yield subcutaneous tumors was found to be concentration dependent and was reduced in a dose-dependent manner when the cells were exposed to the anti-alpha5 blocking antibody. CONCLUSIONS: Restoring expression of alpha5 in the alpha5-negative TP17 uveal melanoma cell line influenced the proliferative properties of these cells but did not alter its tumorigenic potential. In contrast, the ability of the SP6.5 cells to yield tumors in vivo in nude mice appeared to be related to expression of this integrin.     

Downregulation of survivin expression enhances sensitivity of cultured uveal melanoma cells to cisplatin treatment

The purpose of this study is to evaluate the effect of a novel anti-apoptotic gene, survivin, on the resistance and susceptibility of human uveal melanoma cells to apoptosis induced by cisplatin. The sensitivity of human uveal melanoma cell lines to apoptosis induced by cisplatin was analyzed by caspase-3 assays. The expression of the anti-apoptotic protein, survivin, was examined by flow cytometry. Melanoma cells were transfected with either survivin cDNA or survivin anti-sense cDNA and examined for susceptibility to cisplatin-induced apoptosis. Six human uveal melanoma cell lines were incubated with or without cisplatin and cellular proliferation was determined by MTT assays. Significant growth inhibition was observed in 3 melanoma cell lines (OMM1, OCM3, and MEL 270). By contrast, 3 cell lines (OMM2.5, OMM2.3, and 92-1) were resistant to cisplatin-induced apoptosis. However, a positive association was observed between resistance to cisplatin-induced apoptosis and high expression of the anti-apoptotic protein, survivin. Up-regulation of survivin by gene transfer enhanced resistance to cisplatin-induced apoptosis, while transfection with survivin anti-sense rendered resistant melanoma cells susceptible to cisplatin. The combination of cisplatin and actinomycin D significantly decreased survivin expression and enhanced the cisplatin-induced apoptosis of uveal melanoma cells in vitro. These data indicate that resistance of some uveal melanoma cells to cisplatin-induced apoptosis is controlled by anti-apoptotic proteins, such as survivin, that are sensitive to actinomycin D treatment.     

Differential expression of chemokine receptors on uveal melanoma cells and their metastases

PURPOSE: To determine the expression of the chemokine receptors CXCR4 and CCR7 on human uveal melanoma cells and their metastases and the effect of liver-borne factors on the chemotactic responses of uveal melanoma cells. METHODS: Four human uveal melanoma cell lines and three cell lines of uveal melanoma metastases were examined by RT-PCR and flow cytometry for their constitutive expression of CXCR4 and CCR7. The effect of the liver and liver-borne factors on the expression of CXCR4 and CCR7 was determined after intracameral, intrasplenic, and subcutaneous transplantation of uveal melanoma cells in nude mice. Chemotactic responses of melanoma cells to liver-borne factors were determined by in vitro chemotaxis assays using protein extracts of hepatocytes and striated muscle tissue. RESULTS: All the primary uveal melanoma cell lines expressed CXCR4 and CCR7 message and protein, whereas the metastases cell lines expressed little or no chemokine receptor. Extracts of human liver cells stimulated chemotaxis of uveal melanoma cells, which could be inhibited by anti-CXCR4 antibody. Liver-borne factors also induced the downregulation of CXCR4 and CCR7 on uveal melanoma cells. Uveal melanoma cells maintained their high expression of CXCR4 and CCR7 after intracameral transplantation. However, CXCR4 and CCR7 expression was sharply reduced in liver metastases arising from intraocular melanomas. CONCLUSIONS: CXCR4 and CCR7 provide directional migration of uveal melanoma cells toward the liver, the most common site for the formation of uveal melanoma metastases. However, soluble factors elaborated by hepatocytes induce the downregulation of CXCR4 and CCR7 on metastatic uveal melanoma cells.     

Expression of APITD1 is not related to copy number changes of chromosomal region 1p36 or the prognosis of uveal melanoma

PURPOSE: Concurrent loss of chromosome arm 1p, region 36, and chromosome 3 leads to decreased disease-free survival in patients with uveal melanoma. A candidate tumor-suppressor gene APITD1 is located on the critical region on chromosome arm 1p, and it was therefore hypothesized that lower expression levels of this gene could lead to decreased survival in patients, with concurrent loss of a region on chromosome arm 1p and chromosome 3. Using neuroblastoma cells, which, like uveal melanoma, originate from neural crest cells, a former study showed that APITD1 has cell growth and/or cell death properties. In this study, APITD1 expression was analyzed to determine whether it corresponds with the DNA copy number and is related to survival in uveal melanoma. METHODS: To detect whether loss in the copy number of APITD1 results in lowered mRNA expression of the gene, FISH analysis was combined with real-time PCR. In addition, the effect of APITD1 expression on survival was studied by using Kaplan-Meier survival analysis. RESULTS: Expression of APITD1 mRNA was not related to DNA copy number (P = 0.956) or chromosome 3 status (P = 0.958). Kaplan-Meier survival analysis showed very similar survival curves for tumors with high and low APITD1 expression, with a log-rank significance value of P = 0.9682. CONCLUSIONS: These results indicate that APITD1 is not the tumor suppressor gene on 1p36 responsible for the negative prognostic effect in uveal melanoma with concurrent loss of chromosome arm 1p, region 36, and chromosome 3.     

Insulin-like growth factor-1 receptor in uveal melanoma: a predictor for metastatic disease and a potential therapeutic target.

PURPOSE: To investigate the expression of the insulin-like growth factor-1 receptor (IGF-1R) with special focus on its role in cell growth in uveal melanoma. METHODS: Paraffin material from 36 clinicopathologically well characterized cases of primary uveal melanomas (18 of which had metastasized to the liver) with more than 15 years' follow-up was used for immunohistochemical analysis. In the experimental studies, three uveal melanoma cell lines (OCM-1, OCM-3, and 92-1) were used. The expression level of IGF-1R in the cell lines was modulated by glycosylation inhibitors, and the IGF-1R was neutralized with the antibody alphaIR-3. Expression of IGF-1R was assayed by Western blot analysis and immunohistochemistry. Cell growth and survival were analyzed by cell counting, thymidine incorporation, and viability assays. RESULTS: Western blot analysis and immunohistochemistry confirmed that IGF-1R is expressed in uveal melanoma. Although 10 of 18 patients who died of metastasizing disease showed high IGF-1R expression, only 5 of 18 tumors from patients who survived for 15 years or more after enucleation exhibited a high IGF-1R expression. Kaplan-Meier analysis showed a significant association (P = 0.035) between a high IGF-1R expression and death due to metastatic uveal melanoma. Using in vitro experimental models, we found that inhibition of the IGF-1R activity (tyrosine phosphorylation) was associated with a drastic decrease in uveal melanoma cell viability. CONCLUSIONS: These data suggest an important role of IGF-1R in uveal melanoma. The significant association between high IGF-1R expression and death due to metastatic disease may be explained by the fact that IGF-1 is mainly produced in the liver, which is the preferential site for uveal melanoma metastases. These data also point to the possibility of therapeutically interfering with IGF-1R, which appears to be expressed preferentially in uveal melanomas that appear to follow an aggressive clinical course.