Xiao Yin, Zou Ping, Wang Jie, Song Hui, Zou Jing, Liu Lingbo
Department of Hematology, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, 430022, China.
J Huazhong Univ Sci Technolog Med Sci. 2012 Jun;32(3):328-333. doi: 10.1007/s11596-012-0057-z. Epub 2012 Jun 9.
Leukemia seems to depend on a small population of "leukemia stem cells (LSCs)" for its growth and metastasis. However, the precise surviving mechanisms of LSCs remain obscure. Cellular senescence is an important obstacle for production and surviving of tumor cells. In this study we investigated the activated state of a pathway, in which reactive oxygen species (ROS) induces cellular senescence through DNA damage and phophorylation of p38 MAPK (p38), in myeloid leukemic CD34(+)CD38(-) cells. Bone marrow samples were obtained from patients with acute myeloid leukemia (AML, n=11) and chronic myeloid leukemia (CML, n=9). CD34(+)CD38(-) cells were isolated from mononuclear cells from these bone marrow samples, and K562 and KG1a cells (two kinds of myeloid leukemia cell lines) by mini-magnetic activated cell sorting. Hematopoietic stem cells (HSCs) from human cord blood served as controls. Intracellular ROS level was detected by flow cytometry. DNA damage defined as the γH2AX level was measured by immunofluorescence staining. Real-time RT-PCR was used to detect the expression of p21, a senescence-associated gene. Western blotting and immunofluorescence staining were employed to determine the p38 expression and activation. The proliferation and apoptosis of CD34(+)CD38(-) cells were detected by MTT assay and flow cytometry. Our results showed that ROS and DNA damage were substantially accumulated and p38 was less phosphorated in myeloid leukemic CD34(+)CD38(-) cells as compared with HSCs and H(2)O(2)-induced senescent HSCs. Furthermore, over-phosphorylation of p38 by anisomycin, a selective activator of p38, induced both the senescence-like growth arrest and apoptosis of CD34(+)CD38(-) cells from K562 and KG1a cell lines. These findings suggested that, although excessive accumulation of oxidative DNA damage was present in LSCs, the relatively decreased phosphorylation of p38 might help leukemic cells escape senescence and apoptosis.
白血病的生长和转移似乎依赖于一小部分“白血病干细胞（LSCs）”。然而，LSCs确切的存活机制仍不清楚。细胞衰老对于肿瘤细胞的产生和存活是一个重要障碍。在本研究中，我们调查了一条信号通路的激活状态，即活性氧（ROS）通过DNA损伤和p38丝裂原活化蛋白激酶（p38）磷酸化诱导细胞衰老，该通路存在于髓系白血病CD34(+)CD38(-)细胞中。从急性髓系白血病（AML，n = 11）和慢性髓系白血病（CML，n = 9）患者中获取骨髓样本。通过微量磁珠激活细胞分选从这些骨髓样本的单个核细胞以及K562和KG1a细胞（两种髓系白血病细胞系）中分离出CD34(+)CD38(-)细胞。来自人脐带血的造血干细胞（HSCs）作为对照。通过流式细胞术检测细胞内ROS水平。通过免疫荧光染色测量定义为γH2AX水平的DNA损伤。使用实时RT-PCR检测衰老相关基因p21的表达。采用蛋白质免疫印迹法和免疫荧光染色法测定p38的表达和活化情况。通过MTT法和流式细胞术检测CD34(+)CD38(-)细胞的增殖和凋亡。我们的结果表明，与HSCs和H2O2诱导衰老的HSCs相比，髓系白血病CD34(+)CD38(-)细胞中ROS和DNA损伤大量积累，且p38磷酸化程度较低。此外，p38的选择性激活剂茴香霉素使p38过度磷酸化，可诱导来自K562和KG1a细胞系的CD34(+)CD38(-)细胞出现类似衰老的生长停滞和凋亡。这些发现表明，尽管LSCs中存在氧化性DNA损伤的过度积累，但p38相对减少的磷酸化可能有助于白血病细胞逃避衰老和凋亡。
