Abstract

Purpose

The aim of the present study was to evaluate the effect of the histone lysine-methyltransferase (HKMT) inhibitor chaetocin on chromatin structure and its effect on ionising radiation (IR) induced DNA damage response.

Methods

Concentration and time dependent effects of chaetocin on chromatin clustering, its reversibility and association with cell cycle and chromatin markers were analysed by immunofluorescent assays in the non-small cell lung carcinoma (NSCLC) cell lines H460 and H1299 and in human skin fibroblasts. In addition, IR induced damage response (γH2AX, 53BP1, and pATM foci formation) was studied by immunofluorescent assays. The effect on survival was determined by performing single cell clonogenic assays.

Results

Chaetocin significantly increased the radiation sensitivity of H460 (F test on nonlinear regression, p < 0.0011) and of H1299 (p = 0.0201). In addition, treatment with 15 nM chaetocin also decreased the total radiation doses that control 50% of the plaque monolayers (TCD50) from 17.2 ± 0.3 Gy to 7.3 ± 0.4 Gy (p < 0.0001) in H1299 cells and from 11.6 ± 0.1 Gy to 6.5 ± 0.3 Gy (p < 0.0001). Phenotypically, chaetocin led to a time and concentration dependent clustering of the chromatin in H1299 as well as in fibroblasts, but not in H460 cells. This phenotype of chaetocin induced chromatin clustering (CICC) was reversible and depended on the expression of the HKMTs SUV39H1 and G9a. Treatment with siRNA for SUV39h1 and G9a significantly reduced the CICC phenotype. Immunofluorescent assay results showed that the CICC phenotype was enriched for the heterochromatic marker proteins H3K9me3 and HP1α. γH2AX foci formation was not affected, neither in cells with normal nor with CICC phenotype. In contrast, repair signalling with 53BP1 and pATM foci formation was significantly reduced in the CICC phenotype.

Conclusions

Treatment with chaetocin increased the radiation sensitivity of cells in vitro and DNA damage response, especially of 53BP1 and ATM dependent repair by affecting chromatin structure. The obtained results support the potential use of natural HKMT inhibitors such as chaetocin or other bioactive compounds in improving radiosensitivity of cancer cells.

中文翻译：

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Chaetocin诱导染色质浓缩：对DNA修复信号和生存的影响

摘要

目的

本研究的目的是评估组蛋白赖氨酸甲基转移酶（HKMT）抑制剂chaetocin对染色质结构的影响及其对电离辐射（IR）诱导的DNA损伤反应的影响。

方法

在非小细胞肺癌（NSCLC）细胞系H460和H1299以及人皮肤成纤维细胞中，通过免疫荧光分析法分析了壳聚糖对染色质簇的浓度和时间依赖性效应，其可逆性以及与细胞周期和染色质标记的关联。此外，还通过免疫荧光试验研究了IR诱导的损伤反应（γH2AX，53BP1和pATM灶形成）。通过进行单细胞克隆形成测定来确定对存活的影响。

结果

Chaetocin显着提高了H460（非线性回归的F检验，p <0.0011）和H1299（p = 0.0201）的辐射敏感性。此外，用15 nM壳聚糖进行处理还可以将控制50％斑块单层（TCD50）的总辐射剂量从H1299细胞中的17.2±0.3 Gy降低到7.3±0.4 Gy（p <0.0001），从11.6±0.1 Gy降低至6.5±0.3 Gy（p <0.0001）。从表型上看，chaetocin导致H1299和成纤维细胞中染色质的时间和浓度依赖性聚集，但在H460细胞中却没有。chaetocin诱导染色质聚类（CICC）的这种表型是可逆的，并且取决于HKMT SUV39H1和G9a的表达。SUV39h1和G9a的siRNA处理显着降低了CICC表型。免疫荧光分析结果表明，CICC表型富含异色标记蛋白H3K9me3和HP1α。γH2AX灶的形成不受影响，无论是正常细胞还是CICC表型的细胞。相反，在CICC表型中，具有53BP1和pATM灶形成的修复信号显着降低。

结论

脂蛋白的处理通过影响染色质结构提高了体外细胞的放射敏感性和DNA损伤反应，尤其是53BP1和ATM依赖性修复。所获得的结果支持了天然HKMT抑制剂（如chaetocin或其他生物活性化合物）在改善癌细胞的放射敏感性方面的潜在用途。