Department of Anatomy and Cell Biology, Rush University Medical Center, 1750 W Harrison Ave., Jelke Building, Room 1306, Chicago, Illinois, USA.
J Cell Biochem. 2010 Dec 1;111(5):1280-90. doi: 10.1002/jcb.22852.
Promyelocytic leukemia nuclear bodies (PML-NBs) are multiprotein complexes that include PML protein and localize in nuclear foci. PML-NBs are implicated in multiple stress responses, including apoptosis, DNA repair, and p53-dependent growth inhibition. ALT-associated PML bodies (APBs) are specialized PML-NBs that include telomere-repeat binding-factor TRF1 and are exclusively in telomerase-negative tumors where telomere length is maintained through alternative (ALT) recombination mechanisms. We compared cell-cycle and p53 responses in ALT-positive cancer cells (U2OS) exposed to ionizing radiation (IR) or the p53 stabilizer Nutlin-3a. Both IR and Nutlin-3a caused growth arrest and comparable induction of p53. However, p21, whose gene p53 activates, displayed biphasic induction following IR and monophasic induction following Nutlin-3a. p53 was recruited to PML-NBs 3-4 days after IR, approximately coincident with the secondary p21 increase. These p53/PML-NBs marked sites of apparently unrepaired DNA double-strand breaks (DSBs), identified by colocalization with phosphorylated histone H2AX. Both Nutlin-3a and IR caused a large increase in APBs that was dependent on p53 and p21 expression. Moreover, p21, and to a lesser extent p53, was recruited to APBs in a fraction of Nutlin-3a-treated cells. These data indicate (1) p53 is recruited to PML-NBs after IR that likely mark unrepaired DSBs, suggesting p53 may either be further activated at these sites and/or function in their repair; (2) p53-p21 pathway activation increases the percentage of APB-positive cells, (3) p21 and p53 are recruited to ALT-associated PML-NBs after Nutlin-3a treatment, suggesting that they may play a previously unrecognized role in telomere maintenance.
早幼粒细胞白血病核小体（PML-NBs）是一种包含 PML 蛋白并定位于核斑点的多蛋白复合物。PML-NBs 参与多种应激反应，包括细胞凋亡、DNA 修复和 p53 依赖性生长抑制。与 ALT 相关的 PML 体（APBs）是专门的 PML-NBs，包括端粒重复结合因子 TRF1，仅存在于端粒酶阴性肿瘤中，这些肿瘤通过替代（ALT）重组机制维持端粒长度。我们比较了暴露于电离辐射（IR）或 p53 稳定剂 Nutlin-3a 的 ALT 阳性癌细胞（U2OS）中的细胞周期和 p53 反应。IR 和 Nutlin-3a 均导致生长停滞和 p53 的可比诱导。然而，p53 激活的基因 p21 的表达呈双相诱导，IR 后呈单相诱导，Nutlin-3a 后呈单相诱导。p53 在 IR 后 3-4 天被募集到 PML-NBs，大约与 p21 的二次增加同时发生。这些 p53/PML-NBs 标记了明显未修复的 DNA 双链断裂（DSBs）的部位，通过与磷酸化组蛋白 H2AX 的共定位来识别。Nutlin-3a 和 IR 均导致大量的 APBs 增加，这依赖于 p53 和 p21 的表达。此外，p21，并且在较小程度上依赖 p53，在一部分用 Nutlin-3a 处理的细胞中被募集到 APBs。这些数据表明：（1）IR 后 p53 被募集到 PML-NBs，这些 PML-NBs 可能标记未修复的 DSBs，这表明 p53 可能在这些部位进一步激活和/或在其修复中发挥作用；（2）p53-p21 通路的激活增加了 APB 阳性细胞的百分比；（3）Nutlin-3a 处理后 p21 和 p53 被募集到与 ALT 相关的 PML-NBs，表明它们可能在端粒维持中发挥以前未被认识到的作用。
