Oh Jin Ho, Gertych Arkadiusz, Tajbakhsh Jian
Translational Cytomics Group, Department of Surgery, Cedars-Sinai Medical Center, Los Angeles, CA, USA.
Oncotarget. 2013 Mar;4(3):474-93. doi: 10.18632/oncotarget.942.
This study reports on probing the utility of in situ chromatin texture features such as nuclear DNA methylation and chromatin condensation patterns - visualized by fluorescent staining and evaluated by dedicated three-dimensional (3D) quantitative and high-throughput cell-by-cell image analysis - in assessing the proliferative capacity, i.e. growth behavior of cells: to provide a more dynamic picture of a cell population with potential implications in basic science, cancer diagnostics/prognostics and therapeutic drug development. Two types of primary cells and four different cancer cell lines were propagated and subjected to cell-counting, flow cytometry, confocal imaging, and 3D image analysis at various points in culture. Additionally a subset of primary and cancer cells was accelerated into senescence by oxidative stress. DNA methylation and chromatin condensation levels decreased with declining doubling times when primary cells aged in culture with the lowest levels reached at the stage of proliferative senescence. In comparison, immortal cancer cells with constant but higher doubling times mostly displayed lower and constant levels of the two in situ-derived features. However, stress-induced senescent primary and cancer cells showed similar levels of these features compared with primary cells that had reached natural growth arrest. With regards to global DNA methylation and chromatin condensation levels, aggressively growing cancer cells seem to take an intermediate level between normally proliferating and senescent cells. Thus, normal cells apparently reach cancer-cell equivalent stages of the two parameters at some point in aging, which might challenge phenotypic distinction between these two types of cells. Companion high-resolution molecular profiling could provide information on possible underlying differences that would explain benign versus malign cell growth behaviors.
本研究报告了通过荧光染色可视化并经专门的三维（3D）定量和高通量逐细胞图像分析评估的原位染色质纹理特征，如核DNA甲基化和染色质凝聚模式，在评估细胞增殖能力即细胞生长行为方面的效用：以提供细胞群体更动态的图景，这在基础科学、癌症诊断/预后及治疗药物开发中可能具有潜在意义。两种原代细胞和四种不同的癌细胞系在培养的不同时间点进行传代培养，并进行细胞计数、流式细胞术、共聚焦成像和3D图像分析。此外，一部分原代细胞和癌细胞通过氧化应激加速进入衰老状态。当原代细胞在培养中老化时，DNA甲基化和染色质凝聚水平随着倍增时间的下降而降低，在增殖性衰老阶段达到最低水平。相比之下，具有恒定但较高倍增时间的永生癌细胞大多表现出较低且恒定的这两种原位衍生特征水平。然而，与已达到自然生长停滞的原代细胞相比，应激诱导衰老的原代细胞和癌细胞表现出相似的这些特征水平。关于整体DNA甲基化和染色质凝聚水平，快速生长的癌细胞似乎处于正常增殖细胞和衰老细胞之间的中间水平。因此，正常细胞在衰老的某个阶段显然会达到与癌细胞相当的这两个参数水平，这可能会挑战这两种细胞类型之间的表型区分。配套的高分辨率分子谱分析可以提供有关可能的潜在差异的信息，这些差异可以解释良性与恶性细胞的生长行为。
