Wolfson Childhood Cancer Research Centre, Northern Institute for Cancer Research, Newcastle University, Newcastle upon Tyne NE1 7RU, UK.
Newcastle Cancer Centre, Northern Institute for Cancer Research, Newcastle University, Newcastle upon Tyne NE1 7RU, UK.
Int J Oncol. 2018 Mar;52(3):967-977. doi: 10.3892/ijo.2018.4261. Epub 2018 Jan 31.
Cell lines established from the TH-MYCN transgenic murine model of neuroblastoma are a valuable preclinical, immunocompetent, syngeneic model of neuroblastoma, for which knowledge of their p53 pathway status is important. In this study, the Trp53 status and functional response to Nutlin-3 and ionising radiation (IR) were determined in 6 adherent TH-MYCN transgenic cell lines using Sanger sequencing, western blot analysis and flow cytometry. Sensitivity to structurally diverse MDM2 inhibitors (Nutlin-3, MI-63, RG7388 and NDD0005) was determined using XTT proliferation assays. In total, 2/6 cell lines were Trp53 homozygous mutant (NHO2A and 844MYCN+/+) and 1/6 (282MYCN+/-) was Trp53 heterozygous mutant. For 1/6 cell lines (NHO2A), DNA from the corresponding primary tumour was found to be Trp53 wt. In all cases, the presence of a mutation was consistent with aberrant p53 signalling in response to Nutlin-3 and IR. In comparison to TP53 wt human neuroblastoma cells, Trp53 wt murine control and TH-MYCN cell lines were significantly less sensitive to growth inhibition mediated by MI-63 and RG7388. These murine Trp53 wt and mutant TH-MYCN cell lines are useful syngeneic, immunocompetent neuroblastoma models, the former to test p53-dependent therapies in combination with immunotherapies, such as anti-GD2, and the latter as models of chemoresistant relapsed neuroblastoma when aberrations in the p53 pathway are more common. The spontaneous development of Trp53 mutations in 3 cell lines from TH-MYCN mice may have arisen from MYCN oncogenic driven and/or ex vivo selection. The identified species-dependent selectivity of MI-63 and RG7388 should be considered when interpreting in vivo toxicity studies of MDM2 inhibitors.
从 TH-MYCN 转基因神经母细胞瘤小鼠模型中建立的细胞系是一种有价值的临床前、免疫相容、同基因神经母细胞瘤模型，了解其 p53 途径状态非常重要。在这项研究中，使用 Sanger 测序、western blot 分析和流式细胞术确定了 6 种贴壁 TH-MYCN 转基因细胞系的 Trp53 状态和对 Nutlin-3 和电离辐射（IR）的功能反应。使用 XTT 增殖测定法确定对结构不同的 MDM2 抑制剂（Nutlin-3、MI-63、RG7388 和 NDD0005）的敏感性。总共，有 2/6 的细胞系是 Trp53 纯合突变体（NHO2A 和 844MYCN+/+），1/6（282MYCN+/-）是 Trp53 杂合突变体。对于 1/6 的细胞系（NHO2A），发现相应的原代肿瘤 DNA 是 Trp53 wt。在所有情况下，突变的存在与 Nutlin-3 和 IR 反应中的异常 p53 信号一致。与 TP53 wt 人类神经母细胞瘤细胞相比，Trp53 wt 鼠对照和 TH-MYCN 细胞系对 MI-63 和 RG7388 介导的生长抑制明显不敏感。这些鼠 Trp53 wt 和突变的 TH-MYCN 细胞系是有用的同基因、免疫相容的神经母细胞瘤模型，前者可用于测试与免疫疗法（如抗 GD2）联合的 p53 依赖性疗法，后者可作为 p53 途径异常更常见的化学耐药性复发神经母细胞瘤模型。TH-MYCN 小鼠的 3 个细胞系自发发生 Trp53 突变可能是由 MYCN 致癌驱动和/或体外选择引起的。在解释 MDM2 抑制剂的体内毒性研究时，应考虑到 MI-63 和 RG7388 的种属依赖性选择性。
