Doherty Rachel E, Bryant Helen E, Valluru Manoj K, Rennie Ian G, Sisley Karen
Department of Oncology and Metabolism, The Medical School, Beech Hill Road, Sheffield, S10 2RX, UK.
Sheffield Ocular Oncology Service, Sheffield Teaching Hospitals, Royal Hallamshire Hospital, Glossop Road S10 2JF, UK.
Cancers (Basel). 2019 Aug 30;11(9):1278. doi: 10.3390/cancers11091278.
Uveal melanoma (UM) is the most common primary intraocular tumour in adults, with a mean survival of six months following metastasis. The survival rates have not improved in over 30 years. This study has shown that sister chromatid exchange (SCE) is low in UM which is likely due to a reduced expression of . As can function to suppress non-homologous end joining (NHEJ), this study therefore investigated NHEJ in UM. The activation of the catalytic subunit of the NHEJ pathway protein DNA-dependent protein kinase (DNA-PK) was measured by analysing the foci formation and the ligation efficiency by NHEJ determined using a plasmid-based end-joining assay. Using small-interfering RNA (siRNA) knock-down, and chemical inhibitors of DNA-PK, the survival of primary UM cultures and two cell lines were determined. To assess the homologous recombination capacity in response to the inhibition of DNA-PK, a SCE analysis was performed. In addition, to support the findings, the messenger RNA (mRNA) expression of genes associated with NHEJ was analysed using the Cancer Genome Atlas (TCGA)-UM RNAseq data ( = 79). The NHEJ activity and DNA-PKcs activation was upregulated in UM and the inhibition of DNA-PK selectively induced apoptosis and sensitized to ionising radiation and inter-strand cross-linking agents. The inhibition of the NHEJ protein DNA-PK is lethal to UM, indicating a potentially effective therapeutic option, either alone or as a sensitizer for other treatments.
葡萄膜黑色素瘤（UM）是成人中最常见的原发性眼内肿瘤，转移后平均生存期为6个月。30多年来生存率一直没有提高。本研究表明，UM中的姐妹染色单体交换（SCE）较低，这可能是由于……表达降低所致。由于……可抑制非同源末端连接（NHEJ），因此本研究对UM中的NHEJ进行了调查。通过分析灶形成以及使用基于质粒的末端连接测定法测定的NHEJ连接效率，来测量NHEJ途径蛋白DNA依赖性蛋白激酶（DNA-PK）催化亚基的激活情况。使用小干扰RNA（siRNA）敲低和DNA-PK的化学抑制剂，测定原发性UM培养物和两种细胞系的存活率。为了评估对DNA-PK抑制的同源重组能力，进行了SCE分析。此外，为了支持这些发现，使用癌症基因组图谱（TCGA）-UM RNAseq数据（n = 79）分析了与NHEJ相关基因的信使RNA（mRNA）表达。UM中的NHEJ活性和DNA-PKcs激活上调，抑制DNA-PK可选择性诱导细胞凋亡，并使其对电离辐射和链间交联剂敏感。抑制NHEJ蛋白DNA-PK对UM具有致死性，表明这是一种潜在有效的治疗选择，可单独使用或作为其他治疗的增敏剂。
