Department of Cellular and Environmental Biology, University of Perugia via Pascoli, 06123, Perugia, Italy.
Clinic for Diagnostics and Intervention, Oslo University Hospital-Rikshospitalet, Oslo, Norway.
Cell Div. 2013 Aug 6;8:11. doi: 10.1186/1747-1028-8-11. eCollection 2013.
We previously reported that a pool of low molecular weight peptides can be extracted by alkali treatment of DNA preparations obtained from prokaryotic and eukaryotic cells after intensive deproteinization. This class of peptides, isolated from wheat bud chromatin, induces growth inhibition, DNA damage, G2 checkpoint activation and apoptosis in HeLa cells. In this work we studied their mechanism of action by investigating their ability to interfere with DNA synthesis.
BrdUrd comet assays were used to detect DNA replication defects during S phase. DNA synthesis, cell proliferation, cell cycle progression and DNA damage response pathway activation were assessed using 3H-thymidine incorporation, DNA flow cytometry and Western blotting, respectively.
BrdUrd labelling close to DNA strand discontinuities (comet tails) detects the number of active replicons. This number was significantly higher in treated cells (compared to controls) from entry until mid S phase, but markedly lower in late S phase, indicating the occurrence of defective DNA synthesis. In mid S phase the treated cells showed less 3H-thymidine incorporation with respect to the controls, which supports an early arrest of DNA synthesis. DNA damage response activation was also shown in both p53-defective HeLa cells and p53-proficient U2OS cells by the detection of the phosphorylated form of H2AX after peptide treatment. These events were accompanied in both cell lines by an increase in p21 levels and, in U2OS cells, of phospho-p53 (Ser15) levels. At 24 h of recovery after peptide treatment the cell cycle phase distribution was similar to that seen in controls and CDK1 kinase accumulation was not detected.
The data reported here show that the antiproliferative effect exhibited by these chromatin peptides results from their ability to induce genomic stress during DNA synthesis. This effect seems to be S-phase specific since surviving cells are able to progress through their normal cell cycle when the peptide fraction is removed from the culture medium. It is likely that the subsequent apoptosis is a consequence of the failed attempt of the tumour cells to repair the DNA damage induced by the peptides.
我们之前报道过，通过对来自原核和真核细胞的 DNA 制剂进行强烈的去蛋白化处理，用碱提取可以得到一组低分子量肽。从小麦芽染色质中分离出的这类肽可诱导 HeLa 细胞生长抑制、DNA 损伤、G2 检验点激活和细胞凋亡。在这项工作中，我们通过研究其干扰 DNA 合成的能力来研究其作用机制。
使用 BrdUrd 彗星试验检测 S 期 DNA 复制缺陷。通过 3H-胸苷掺入、DNA 流式细胞术和 Western blot 分别评估 DNA 合成、细胞增殖、细胞周期进程和 DNA 损伤反应途径的激活。
BrdUrd 标记靠近 DNA 链中断处（彗星尾部）检测活跃复制子的数量。与对照组相比，处理组细胞在进入 S 期直到中期 S 期时，该数量显著增加，但在晚期 S 期时明显减少，表明 DNA 合成存在缺陷。在中期 S 期，与对照组相比，处理组细胞的 3H-胸苷掺入量较少，这支持 DNA 合成的早期停滞。在用肽处理后，p53 缺陷型 HeLa 细胞和 p53 功能型 U2OS 细胞中均检测到 H2AX 磷酸化形式，表明 DNA 损伤反应被激活。在这两种细胞系中，p21 水平增加，在 U2OS 细胞中，磷酸化 p53（Ser15）水平增加。在用肽处理后 24 小时的恢复期间，细胞周期相分布与对照组相似，未检测到 CDK1 激酶的积累。
本文报道的数据表明，这些染色质肽表现出的抗增殖作用是由于它们在 DNA 合成过程中诱导基因组应激的能力。这种效应似乎是 S 期特异性的，因为当肽从培养基中去除时，存活的细胞能够正常通过其细胞周期。随后的细胞凋亡很可能是肿瘤细胞试图修复肽诱导的 DNA 损伤失败的结果。
