In vitro targeting of NG2 antigen by 213Bi-9.2.27 alpha-immunoconjugate induces cytotoxicity in human uveal melanoma cells

PURPOSE: To examine uveal melanoma cell lines for the expression of human melanoma proteoglycan (NG2) using monoclonal antibody (mAb) 9.2.27 and subsequently to assess the in vitro specificity and cytotoxicity of mAb 9.2.27 conjugated to the alpha-particle-emitting radioisotope 213bismuth (213Bi-9.2.27) for uveal melanoma cells. METHODS: Immunocytochemistry and flow cytometry were used to examine OCM-1, OCM-3, OCM-8, OMM-1, Mel202 and 92-1 melanoma cell lines for NG2 expression. Melanoma cells were treated with test (213Bi-9.2.27) or control (213Bi-A2) alpha-immunoconjugates (AICs). The specific cytotoxicity of 213Bi-9.2.27 AIC was evaluated using an MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfenyl)-2H-tetrazolium, inner salt) assay. Cell death was also assessed using TUNEL. RESULTS: OCM-1, OCM-8, OMM-1, and Mel202 cells strongly expressed NG2. OCM-3 cells showed moderate expression and 92-1 cells were NG2-negative. 213Bi-9.2.27 specifically killed NG2-positive OCM-1, OCM-8, and OMM-1 cells in a concentration-dependent manner. D0 values for 37% cell survival of NG2-positive OCM-1, OCM-8, and OMM-1 cells were 5.8, 5.0, and 5.6 microCi, respectively, and the value was 43.4 muCi for NG2-negative 92-1 cells. CONCLUSIONS: The specific cytotoxicity of 213Bi-9.2.27 AIC for NG2-positive, but not NG2-negative, cells suggests NG2 is a suitable target for alpha-immunotherapy in uveal melanoma. 213Bi-9.2.27 AIC used directly or as adjunct therapy may be a promising new agent for treating NG2-positive uveal melanomas or metastases.     

Cripto-1 expression in uveal melanoma: an immunohistochemical study

Human Cripto, the founder member of the epidermal growth factor-Cripto-FRL1-Cryptic (EGF-CFC) family, plays an important role during early embryonic development and in particular in carcinogenesis and the development of cancer metastases. Cripto-1 is over-expressed in most cancers, but is absent or only weakly expressed in normal cells. For this reason, Cripto-1 could be of potential value in the targeted treatment. There is no information on the expression of Cripto-1 in human uveal melanoma. Cripto-1 reactivity was evaluated by immunohistochemistry on 36 archival uveal melanomas using the polyclonal antibody to Cripto-1. The tumors were divided in to 2 groups. There were 18 uveal melanomas with no intrascleral or extrascleral extension and 18 uveal melanomas with intrascleral/extrascleral extension/liver metastasis. Cripto-1 reactivity was correlated with tumor aggressiveness and cell type. Furthermore, we studied the immunolocalization of Cripto-1 in 4 uveal melanoma cell lines OCM-1, OCM-8, and 92-1, and OMM-1 and in 2 primary uveal melanocyte cultures. Cripto-1 was expressed in both the non-invasive and aggressive uveal melanomas. Cripto-1 was positive in the 4 uveal melanoma cell lines and absent in the primary uveal melanocyte cultures. Retinal tissue did not express Cripto-1. The results suggest that Cripto-1 is expressed in uveal melanoma, negative in the non-neoplastic ocular tissue and point to its use as a target for therapy.     

Establishment and characterization of two uveal melanoma cell lines derived from tumors with loss of one chromosome 3

Uveal melanoma (UM) is the most common intraocular malignancy. Approximately 50% of UM patients die of metastases, which mainly arise from primary tumors with loss of an entire chromosome 3 (monosomy 3). To identify cell lines with monosomy 3 that may serve as a model system for UM with high metastatic potential, we determined the chromosome 3 status of previously established and frequently used UM cell lines by microsatellite analysis (Mel202, Mel285, Mel290, 92-1, OMM-1, OCM-1, OCM-3, OCM-8) and cytogenetic analysis (Mel202, Mel285, OCM-8). We found that none of these cell lines has monosomy 3. Therefore we established and characterized two novel cell lines, UPMM-1 and UPMM-2 that are both developed from primary uveal melanoma tissue samples with monosomy 3. The cell line UPMM-1 has retained the chromosome 3 status of the primary tumor. In UPMM-2 chromosome 3 has undergone duplication (isodisomy) and is present on the background of a hypotetraploid karyotype. Our data suggest that, UPMM-1 may serve as a model system to study the mechanisms underlying the metastatic potential of uveal melanomas with monosomy 3.     

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.     

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.     

Pyrophosphorolysis detects B-RAF mutations in primary uveal melanoma

PURPOSE: Mutations in the genes that control cell proliferation in cutaneous melanoma are generally uncommon in uveal melanoma. Despite the absence of known activating mutations, the RAF-MEK-ERK, or mitogen-activated protein kinase (MAPK), pathway is usually activated in uveal melanoma. An assay with increased potential to identify mutations is now available, and this study was therefore conducted to reanalyze uveal melanoma cell lines and primary tumors for this mutation. METHODS: Eleven uveal melanoma cell lines and 45 primary uveal melanomas were analyzed for mutations in exon 15 of the B-RAF gene by using pyrophosphorolysis-activated polymerization (PAP). Mutations were validated by sequencing of the PAP product. RESULTS: B-RAF mutations were detected in cell lines OCM-1 and -3 (V600E) and in six primary uveal melanomas. The V600K mutation was detected in one primary uveal melanoma, for which the V600E assay turned out to be sensitive as well. Direct sequencing of the exon 15 PCR product did not reveal the mutations found with the PAP-assay, indicating a low frequency of the mutant allele in primary samples. CONCLUSIONS: Because of the very sensitive PAP technology, B-RAF mutations were found in cell lines and primary uveal melanomas, which suggests that they may occasionally play a role in the activation of the MAPK pathway in uveal melanoma and indicates a higher prevalence of B-RAF mutations in uveal melanoma than was reported earlier. However, the relative scarcity of the B-RAF mutation excludes an elemental role for this mutation in uveal melanoma.     

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.     

Oral picropodophyllin (PPP) is well tolerated in vivo and inhibits IGF-1R expression and growth of uveal melanoma

PURPOSE: The cyclolignan picropodophyllin (PPP) efficiently blocks the activity of insulinlike growth factor-1 receptor (IGF-1R) and inhibits the growth of uveal melanoma cells in vitro and in vivo. In this study, the authors investigated the efficiency of orally administered PPP on the growth of uveal melanoma xenografts. In addition, they focused on the effect of PPP on vascular endothelial growth factor (VEGF) in vivo and evaluated its effects in combination with other established antitumor agents in vitro. METHODS: Four different uveal melanoma cell lines (OCM-1, OCM-3, OCM-8, 92-1) were treated with PPP alone and in combination with imatinib mesylate, cisplatin, 5-fluorouracil, and doxorubicin. Cell viability was determined by XTT assay. SCID mice that underwent xenografting with uveal melanoma cells were used to determine antitumor efficacy of oral PPP in vivo. Five mice were used per group. Tumor samples obtained from the in vivo experiments were analyzed for VEGF and IGF-1R expression by Western blotting. RESULTS: PPP was found to be superior to the other antitumor agents in killing uveal melanoma cells in all four cell lines (IC50 < 0.05 microM). Oral PPP inhibited uveal melanoma growth in vivo in OCM-3 (P = 0.03) and OCM-8 (P = 0.01) xenografts and was well tolerated by the animals. PPP decreased VEGF expression in the OCM-1 (P = 0.006) and OCM-8 (P = 0.01) tumors. CONCLUSIONS: Oral PPP was well tolerated in vivo, caused total growth inhibition of uveal melanoma xenografts, and decreased VEGF levels in the tumors.     

The effect of ultraviolet radiation on choroidal melanocytes and melanoma cell lines: cell survival and matrix metalloproteinase production

Background Ultraviolet radiation (UVR) can induce DNA damage and regulate the expression of factors important for tumour growth and metastasis, including matrix metalloproteinases (MMPs). Epidemiological studies suggest that chronic UVR exposure, especially during early adulthood, may be a risk factor in patients with choroidal melanoma. However, the effects of UV(R)-B on human choroidal melanocyte survival and growth are unknown. In this study, we investigated if UV(R)-B affected the in vitro survival, growth and MMP production of choroidal melanocytes and melanoma cells. Methods Cultures of primary choroidal melanocytes and melanoma cell lines (OCM-1 and OCM-8) were exposed to UV(R)-B (0–30 mJ/cm²). The cell morphology and growth were examined, and cell viability was assessed using an MTT assay. Gelatin zymography was used to assess the enzymatic activity for MMP-2 and -9 in conditioned media following UV(R)-B treatment. Results UV(R)-B ≥20 mJ/cm² was cytotoxic for choroidal melanocytes. Cytotoxic doses of 5 to 10 mJ/cm² were found for OCM-8 and OCM-1 melanoma cell lines. Low levels of UV(R)-B (2.5 and 3.5 mJ/cm²) significantly reduced melanoma cell viability after 48 h, although melanocyte viability was not affected by doses of UV(R)-B <10 mJ/cm². Conditioned media from melanoma cells and melanocytes displayed pro-MMP-2 activity independent of UV(R)-B. Control and UV(R)-B-treated OCM-1 cells secreted active MMP-2 up to 72 h. Pro-MMP-9 activity was seen from 36 h for control and UV(R)-B-treated OCM-1 and OCM-8 cells. Conclusions Melanocytes appeared more resistant to physiological doses of UV(R)-B than melanoma cells; the potential of melanocytes to initially survive DNA damage following UV(R)-B exposure may be relevant to the subsequent transformation of melanocytes to melanomas. Although UV(R)-B did not induce the production and/or activation of MMP-2 and -9 in melanocytes or melanoma cells, we are currently investigating whether DNA damage-response genes such as p53 and p21 can be regulated following UVR exposure, and whether they are important for choroidal melanoma development. This study was supported in part by grants from the Sydney Foundation for Medical Research (MCM) and the National Health and Medical Research Council, Australia (RMC).     

Characterization of uveal melanoma cell lines that grow as xenografts in rabbit eyes

Two cell lines, OCM-1 and OCM-2, were established from biopsied specimens of choroidal melanomas of spindle B and mixed cell type morphologies. Both cell lines were phenotypically malignant. Karyotypic analyses revealed human chromosomes with a modal number of 95 and 85, respectively. These cell lines have been passaged for over 2 years and are essentially immortal. The cells grew without contact inhibition as monolayers in liquid culture or as clones in soft agar. Electron microscopy revealed melanoma cells containing premelanosomes and cultures free from contamination by fibroblasts. To categorize the morphologies of these cultured cells better by the Callender classification, they were grown as xenografts in the anterior chambers of rabbits immunosuppressed with daily i.m. doses of Cyclosporin A (10 mg/kg). Tumor plaques were detected after 10 days. The eyes were enucleated and fixed in formalin and stained with hematoxylin and eosin for histopathological evaluation. The xenograft from OCM-1 was found to consist predominantly of spindle B-type melanoma cells. In contrast, the xenograft from OCM-2 contained epithelioid, spindle B and clear cell ("balloon") melanoma cells. The ability of these cell lines to grow as xenografts confirmed their neoplastic origin. In fact, the types of the uveal melanoma cells in the xenografts resembled those in the original biopsied tumors. This suggests that the morphology of human uveal melanoma cells is an inherited trait and may be genetically fairly stable.     

Altered expression of Rb and p53 in uveal melanomas following plaque radiotherapy.

PURPOSE: To examine the expression of proteins in the Rb and p53 tumor suppressor pathways in uveal melanomas following plaque radiotherapy. METHODS: Immunohistochemistry and cell culture studies. Immunohistochemistry for Rb, p16, cyclin D1, p53, HDM2, and Bcl-2 was performed on twelve eyes containing posterior uveal melanomas that were enucleated following plaque radiotherapy. Cell culture studies were performed in three cases. RESULTS: The irradiated eyes were enucleated for radiation complications (five cases), local tumor recurrence (three cases), and other reasons (four cases). On histopathologic examination, all cases showed evidence of tumor cell loss. However, residual tumor cells were present in all cases, including those that were clinically regressed. Residual cells from three of the clinically regressed cases were cultured and demonstrated minimal cell division, marked cell death, and extensive chromosomal damage. Strong p53 staining was observed in six cases (50%) and was significantly associated with recent radiotherapy (P = .04). Abnormal cytoplasmic staining for Rb was observed in four cases (33%). CONCLUSIONS: Plaque radiotherapy of uveal melanomas induces DNA damage, inhibits cell division, and promotes cell death. These changes may be due, at least in part, to induction of p53, which activates genes involved in both cell cycle arrest and apoptosis. Plaque radiotherapy can also cause alterations in the expression of Rb, but the significance of this finding will require further study.     

Expression of the metastasis suppressor gene KISS1 in uveal melanoma

PURPOSE: Uveal melanoma (UM) is the most common primary malignant intraocular tumour in adults. Forty-five percent of UM patients develop metastasis within 15 years of initial diagnosis. KISS1, a human metastasis suppressor gene, has been reported to play a role in various human malignancies. The purpose of this study was to investigate the expression of KISS1 in UM and its potential value as a prognostic marker. METHODS: Thirty-seven cases of paraffin-embedded human UM specimens were immunostained with a KISS1 antibody. Clinical-pathological data were obtained. The relationship between the clinical-pathological data and the expression of KISS1 was evaluated. Moreover, the survival rates of the patients were also assessed. Five UM cell lines (92.1, OCM-1, MKTBR, UW1 and SP6.5) were assayed for KISS1 expression. In addition, real-time PCR was used to determine mRNA levels of KISS1and its receptor GPR54in these cell lines. RESULTS: The immunohistochemical results of KISS1 expression displayed cytoplasmic staining in 84% of UM specimens. Low KISS1 expression was associated with a higher risk of metastatic disease (P<0.05). Furthermore, we found that KISS1 was expressed in all five UM cells lines. Real-time PCR analysis confirmed the presence of both KISS1and its receptor GPR54in all five human UM cell lines. CONCLUSIONS: To the best of our knowledge, this is the first time that KISS1has been characterized in UM. The correlation between KISS1 expression and UM survival rate suggests an important role for KISS1as a prognostic marker in this particular tumour.     

Amfenac increases the radiosensitivity of uveal melanoma cell lines

PURPOSE: To evaluate the proliferation rates of five human uveal melanoma (UM) cell lines after treatment with amfenac, a cyclooxygenase (COX)-2 inhibitor, and subsequent radiation exposure. METHODS: Five human UM cell lines (92.1, SP6.5, MKT-BR, OCM-1, and UW-1) and one human fibroblast cell line (BJ) were incubated with amfenac. Treated and non-treated cell lines were then exposed to various doses of gammaradiation: 0, 2, 4, 6, and 8 Gy. Sulphorhodamine-B assay was used to assess proliferation rates 48 h post-radiation. RESULTS: Treatment of UM cell lines with amfenac prior to radiation led to a marked reduction in proliferation rates. This difference was statistically significant in all cell lines at every radiation dose (P<0.005), with the exception of 92.1 at 2 Gy (P=0.157). Fibroblasts treated with amfenac showed significantly higher proliferation rates after 2 and 8 Gy, with no significant differences at 0, 4, and 6 Gy. CONCLUSIONS: The radiosensitivity of UM cell lines was increased by the administration of amfenac, the active metabolite of nepafenac. There appears to be a radioprotective effect of amfenac on human fibroblasts. The topical administration of nepafenac may decrease tumour recurrence and radiation-induced complications while broadening the indications for radiotherapy by treating larger tumours.     

Multidrug resistance in ocular melanoma.

AIMS/BACKGROUND: Metastatic disease in patients with ocular melanoma is resistant to chemotherapy. One of the main mechanisms of modulating multidrug resistance is the expression of the multidrug resistance gene 1 (MDR1) product (p-glycoprotein) by tumour cells. The purpose of this study was to evaluate the frequency of expression of the MDR1 gene in ocular melanoma whose primary treatment was surgical excision or enucleation. METHODS: Twelve recent ocular melanomas were received fresh, snap frozen and cryostat sections of tumour were analysed for expression of MDR1 by immunohistochemistry using a well characterised monoclonal antibody to MDR1. Tumour explants were established in short term tissue culture from four tumours and cell blocks were examined by immunohistochemistry. RESULTS: MDR1 expression was present in five of 12 ocular melanomas. Upregulation of protein expression was found in four cell lines established in short term culture from tumour explants. A recurrent tumour, initially treated by local excision and radioactive plaque, showed overexpression of MDR1 mRNA. CONCLUSIONS: These results suggest that significant level of MDR1 may be intrinsically present in ocular melanomas before exposure to drugs involved in multidrug resistance, and indicate the possible importance of MDR1 in modulating chemoresistance in ocular melanoma. Chemosensitisation may be of potential value in planning adjuvant chemotherapy for patients with metastatic disease.     

Differential amplification of gene expression in lens cell lines conditioned to survive peroxide stress

PURPOSE: The response of lens systems to oxidative stress is confusing. Antioxidative defense systems are not mobilized as expected, and unanticipated defenses appear important. Therefore, mouse lens cell lines conditioned to survive different peroxide stresses have been analyzed to determine their global changes in gene expression. METHODS: The immortal mouse lens epithelial cell line alphaTN4-1 was conditioned to survive 125 microM H2O2 (H cells) or a combination of both 100 microM tertiary butyl hydroperoxide (TBHP) and 125 microM H2O2 (HT cells), by a methodology previously described. The total RNA was isolated from the different cell lines and analyzed with oligonucleotide mouse expression microarrays. Four microarrays were used for each cell line. Microarray results were confirmed by real-time RT-PCR. RESULTS: A new cell line resistant to both 125 microM H2O2 and 100 micro M TBHP was developed, because cells resistant to H2O2 were killed by TBHP. Analysis of classic antioxidative enzyme activities showed little change between cells that survive H2O2 (H) and those that survive H2O2 and TBHP (HT). Therefore, the global change in gene expression in these cell lines was determined with gene expression microarrays. The fluorescent signal changes of the genes within the three cell lines, H, HT, and control (C), were analyzed by statistical methods including Tukey analysis. It was found that from the 12,422 gene fragments and expressed sequence tags (ESTs) analyzed--based on a one-way ANOVA with a stringent cutoff of one false positive per 1000 genes and correcting for microarray background and noise--approximately 950 (7.6%) genes had a significant change in expression in comparing the C, H, and HT groups. A small group of antioxidative defense genes were found in this population, including catalase, members of the glutathione (GSH)-S-transferase family, NAD(P)H menadione oxidoreductase 1, and the ferritin light chain. The remaining genes are involved in a broad spectrum of other biological systems. In the HT versus H comparison, only a few genes were found that had increased expression in the HT line compared with expression in the H line, including GSH-S-transferase alpha 3 and hephaestin. Many genes that are frequently considered antioxidative defense genes, including most of the GSH peroxidases, unexpectedly showed little change. CONCLUSIONS: An unusual and generally unexpected small group of antioxidative defense genes appear to have increased expression in response to H2O2 stress. Cell lines resistant to H2O2 do not appear to survive challenge with another type of peroxide, TBHP, a lipid peroxide prototype. However, acquisition of TBHP resistance by H cells was found to be accompanied by significantly amplified expression of only a few additional antioxidative defense genes. Many of the amplified genes do not appear to be involved with antioxidative systems, reflecting the complexity of the cells' response to oxidative stress.     

经绿色荧光蛋白基因修饰的人脉络膜黑色素瘤细胞株OCM-1-gfp的生物学行为与基因表达

目的 研究经绿色荧光蛋白（GFP）基因修饰的人脉络膜黑色素瘤细胞株OCM-1-gfp在小鼠体内的生长过程、转移规律，以及可能影响肿瘤生长和转移的因素。 方法 用脂质体将GFP基因导入人脉络膜黑色素瘤细胞OCM-1，建立稳定、高水平表达GFP的克隆;分别接种到Balb/c裸鼠视网膜下和后大腿皮下，建立原位和异位肿瘤模型。眼内肿瘤生长情况用荧光显微镜直接观察，皮下肿瘤大小用游标卡尺测量;采用免疫组织化学方法对肿瘤内13种基因的表达进行检测。 结果 稳定表达GFP的脉络膜黑色素瘤细胞OCM-1-gfp基本保持了亲代细胞的特征;能在裸鼠体内形成肿瘤并继续生长和转移;在基因表达方面，肿瘤抑制基因p16染色呈阴性，p53染色呈强阳性。其他的肿瘤抑制基因:视网膜母细胞瘤易感基因（Rb）、p21，转录调控因子（E-2F）、核因子κB（NFκB），细胞增生相关基因细胞周期素D1（cyclin D1）、增生细胞核抗原（PCNA），细胞凋亡相关基因bcl-2、bcl-XL/S和bax，以及表皮生长因子（EGF）及其受体（EGFR）呈现不同程度的阳性表达。 结论 GFP为直接观察脉络膜黑色素瘤在体内的生长和转移提供了标记;脉络膜黑色素瘤原位与异位移植瘤模型在成瘤率和生长情况方面无明显差异;由OCM-1-gfp生长形成的肿瘤p16、p53及NFκB、cyclin D1、PCNA及EGF和EGFR等多种基因表达异常。 (中华眼底病杂志, 2006, 22: 170-173)     

冬凌草甲素协同TRAIL诱导OCM-1A细胞凋亡的作用机制

目的：通过实验研究考察肿瘤坏死因子相关的凋亡诱导配体（TRAIL）与冬凌草甲素联合使用时对脉络膜黑色素瘤细胞株OCM-1A细胞和C918细胞的抑制作用。方法：实验研究。以体外培养OCM-1A细胞和C918 细胞为研究对象,加入不同浓度的冬凌草甲素（终浓度分别为0.625、1.25、2.5、5、10、20 μmol/L）和TRAIL（终浓度分别为6.25、12.5、25、50、100、200 ng/ml）,利用MTT法检测细胞增殖活性,用PI单染结合流式细胞技术检测细胞周期分布,Annexin-V/PI双染检测细胞凋亡,用免疫印迹检测凋亡相关蛋白如死亡受体DR5、A-Caspase-3 蛋白、XIAP蛋白表达量变化。数据采用单因素方差分析和独立样本t检验进行分析。结果：TRAIL单独使用时,对OCM-1A细胞有一定程度的抑制,TRAIL浓度为200 ng/ml时抑制率最高为36.5%;冬凌草甲素单独作用时,对OCM-1A细胞的抑制不明显;当二者联合使用时,对OCM-1A的抑制作用明显增强,TRAIL 200 ng/ml联合冬凌草甲素20μmol/L作用于OCM-1A时抑制作用最强,抑制率为87%,而对C918的抑制增强效果不明显。流式细胞仪检测结果发现处于G2 期细胞显著增多,提示细胞被阻滞在G2/M期,免疫印迹结果显示DR5、A-Caspase-3 表达增强,而凋亡抑制蛋白XIAP表达减弱。TRAIL与冬凌草甲素联合使用后对C918细胞的诱导凋亡作用不明显,差异没有统计学意义。结论：冬凌草甲素和TRAIL共同作用于OCM-1A细胞时,明显增强各自单独作用时候诱导的凋亡作用。     

Orthotopic human choroidal melanoma xenografts in nude rats with aggressive and nonaggressive PAS staining patterns

PURPOSE: Choroidal melanoma is the most common primary ocular cancer among the adult population. Patient survival has been linked to the periodic acid-Schiff base (PAS)-positive vascular patterns in the tumors. The presence of PAS-positive loops or cross-linking parallel channels is a marker of an aggressive tumor. The purpose of this study was to develop new xenograft models of human choroidal melanoma that predictably demonstrate the PAS staining patterns associated with nonaggressive and aggressive tumors in humans. METHODS: Three human choroidal melanoma cell lines (C918, M619, and OCM-1) were used. C918 and M619 are considered aggressive, based on their ability to form PAS-positive channels in vitro. The nonaggressive OCM-1 cells do not form these channels. C918, M619, and OCM-1 spheroids were grown and implanted in the suprachoroidal space of 20, 17, and 16 WAG/RijHs-rnu nude rats, respectively. Tumors were grown for 1 to >4 weeks, and histology was performed to evaluate tumor growth and determine PAS labeling patterns. RESULTS: Growth of C918, M619, and OCM-1 xenografts were histologically verified in 20/20, 15/17, and 16/16 rats, respectively. PAS staining revealed loops and cross-linking parallel channels, typical of aggressive tumors in patients, in 90% of C918 and 100% of M619 xenografts. Only 4 of 16 OCM-1 xenografts showed PAS-positive loops. The rest showed no PAS staining or only perivascular staining, indicative of nonaggressive tumors. CONCLUSIONS: It is possible to grow human choroidal melanoma orthotopic xenografts in nude rats that reproduce the PAS staining patterns associated with aggressive and nonaggressive choroidal melanomas in patients.