Deligezer Ugur, Akisik Ebru E, Erten Nilgün, Dalay Nejat
Department of Basic Oncology, Oncology Institute, Istanbul University, Istanbul, Turkey.
Clin Chem. 2008 Jul;54(7):1125-31. doi: 10.1373/clinchem.2007.101766. Epub 2008 May 16.
alterations in DNA methylation and histone modifications have been implicated in carcinogenesis. Although tumor-specific alterations in DNA methylation can be detected in the serum and plasma of cancer patients, no data are available on the presence of histone modifications in circulating blood. We investigated whether histone methylation, as a model of histone modifications, is detectable in plasma. Because methylation at histone 3 lysine 9 (H3K9) has been demonstrated to be enriched at sites of repetitive ALU elements, we addressed the specificity of histone-methylation detection and hypothesized that if monomethylated H3K9 (H3K9me1) is detectable in plasma, the concentrations in mononucleosomes and oligonucleosomes would be different. We also analyzed a single-copy gene, CDKN2A.
we enrolled 21 multiple myeloma patients in the study. We used ELISA and real-time PCR analysis to evaluate nucleosomes and cell-free DNA, respectively, as evidence of the presence of histones and associated DNA in circulating blood. H3K9me1 was analyzed by chromatin immunoprecipitation.
ELISA and real-time PCR assays indicated the presence of free nucleosomes and DNA in plasma, and the results were quantitatively correlated (P < 0.001). The detection of histone methylation on free nucleosomes was sequence dependent. Fragments representing mono- and oligonucleosomes differed with respect to H3K9me1 concentrations (P = 0.004), in accordance with our hypothesis. In addition, the detection rate and concentrations of H3K9me1 were significantly higher on the fragment covering both mononucleosomes and oligonucleosomes than on the CDKN2A promoter (P < 0.001).
if validated in further studies, our findings may be a basis for investigations of cancer-specific alterations in histone modifications in the circulation.
DNA甲基化和组蛋白修饰的改变与癌症发生有关。尽管在癌症患者的血清和血浆中可检测到肿瘤特异性的DNA甲基化改变，但关于循环血液中组蛋白修饰的存在情况尚无数据。我们研究了作为组蛋白修饰模型的组蛋白甲基化是否可在血浆中检测到。由于已证明组蛋白3赖氨酸9（H3K9）的甲基化在重复性ALU元件位点富集，我们探讨了组蛋白甲基化检测的特异性，并假设如果血浆中可检测到单甲基化H3K9（H3K9me1），那么单核小体和寡核小体中的浓度会有所不同。我们还分析了一个单拷贝基因CDKN2A。
我们招募了21名多发性骨髓瘤患者参与研究。我们分别使用酶联免疫吸附测定（ELISA）和实时聚合酶链反应（PCR）分析来评估核小体和游离DNA，作为循环血液中组蛋白及相关DNA存在的证据。通过染色质免疫沉淀分析H3K9me1。
ELISA和实时PCR检测表明血浆中存在游离核小体和DNA，且结果在数量上具有相关性（P < 0.001）。游离核小体上组蛋白甲基化的检测具有序列依赖性。代表单核小体和寡核小体的片段在H3K9me1浓度方面存在差异（P = 0.004），与我们的假设一致。此外，覆盖单核小体和寡核小体的片段上H3K9me1的检测率和浓度显著高于CDKN2A启动子上的检测率和浓度（P < 0.001）。
如果在进一步研究中得到验证，我们的发现可能为研究循环中组蛋白修饰的癌症特异性改变奠定基础。
