Krieger Eye Research Laboratory, Felsenstein Medical Research Center, Petah Tiqwa. Israel.
Invest Ophthalmol Vis Sci. 2012 Oct 3;53(6):2611-9. doi: 10.1167/iovs.11-7730.
RASSF1A inactivation in uveal melanoma (UM) is common and methylation-induced. We investigated the effect of RASSF1A re-expression on the UM phenotype in vivo and in vitro.
The phenotypic effect of methylation-induced inactivation of RASSF1A in UM was explored using a stable RASSF1A-expressing UM-15 clone. RASSF1A expression was assessed using QRT-PCR. Proliferation was evaluated in vitro using MTT assays. Additionally, athymic NOD/SCID mice were injected subcutaneously or intraocularly with RASSF1A-expressing and -non-expressing UM-15 clones, and euthanized when tumors reached a volume of 1500 mm(3), or at 56 or 46 days, respectively. Tumor tissues, eyes, and livers were analyzed histologically.
In vitro analysis confirmed the lack of RASSF1A expression and full methylation of the RASSF1A promoter region in the UM-15 cell line, which was reversible following treatment with 5-Aza-2-deoxycytidine. Cells expressing exogenous RASSF1A showed slower proliferation than controls and regained sensitivity to cisplatin. Compared to mice injected with control cells, mice treated with UM-15 cells expressing exogenous RASSF1A did not acquire intraocular tumors, and their subcutaneous tumors were relatively delayed and small. Neither group had liver metastases.
UM cells reduced tumorigenicity in the presence of activated RASSF1A. RASSF1A apparently has an important role in the development of UM, and its reactivation might be applied in the development of new treatments.
RASSF1A 在葡萄膜黑色素瘤（UM）中失活常见且与甲基化有关。我们研究了 RASSF1A 重新表达对 UM 表型的体内和体外影响。
采用稳定表达 RASSF1A 的 UM-15 克隆研究了 RASSF1A 甲基化诱导失活对 UM 的表型影响。使用 QRT-PCR 评估 RASSF1A 表达。通过 MTT 分析评估体外增殖。此外，将表达和不表达 RASSF1A 的 UM-15 克隆皮下或眼内注射到无胸腺 NOD/SCID 小鼠中，当肿瘤体积达到 1500mm3 或分别在 56 或 46 天时安乐死。分析肿瘤组织、眼睛和肝脏的组织学。
体外分析证实 UM-15 细胞系中缺乏 RASSF1A 表达和 RASSF1A 启动子区域的完全甲基化，经 5-Aza-2-脱氧胞苷处理后可逆转。表达外源性 RASSF1A 的细胞比对照细胞增殖更慢，并恢复对顺铂的敏感性。与注射对照细胞的小鼠相比，注射表达外源性 RASSF1A 的 UM-15 细胞的小鼠未获得眼内肿瘤，其皮下肿瘤相对延迟且较小。两组均无肝转移。
在激活的 RASSF1A 存在的情况下，UM 细胞降低了致瘤性。RASSF1A 在 UM 的发生发展中显然具有重要作用，其重新激活可能应用于新治疗方法的开发。
