Normal and malignant epithelial cells with stem-like properties have an extended G2 cell cycle phase that is associated with apoptotic resistance.
Normal and malignant epithelial cells with stem-like properties have an extended G2 cell cycle phase that is associated with apoptotic resistance.
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DOI：
10.1186/1471-2407-10-166
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发表时间：
2010-04-28
期刊：
影响因子：
3.8
通讯作者：
Mackenzie IC
中科院分区：
文献类型：
--
作者：
Harper LJ;Costea DE;Gammon L;Fazil B;Biddle A;Mackenzie IC
具有干细胞样特性的细胞亚群先前已经从人类上皮癌中分离出来，它们对凋亡诱导刺激的抵抗与癌症复发和治疗失败有关。本研究的目的是探讨正常和恶性人上皮中具有干细胞样特性的细胞对凋亡诱导剂的抗性机制。从新鲜人头颈癌（n = 11）、头颈癌、前列腺癌和乳腺癌（n = 7）和正常人口腔粘膜（n = 5）中分离的细胞暴露于各种诱导凋亡的刺激（紫外线、肿瘤坏死因子、顺铂、依托泊苷和新ocarzinostatin）中。流式细胞术检测CD44和上皮特异性抗原（ESA）表达、集落形态、肿瘤球形成和快速粘附试验用于鉴定具有干细胞样特性的细胞亚群。检测细胞凋亡、细胞周期和各种细胞周期检查点蛋白的表达（Western Blot, qPCR）。利用去溴膜醇醚（DBH）和siRNA研究了g2检查点调节因子Chk1和Chk2的作用。在癌症活检和癌细胞系中，cd44高的细胞亚群显示出克隆原性增加，细胞凋亡率显著降低，细胞周期g2期的细胞比例显著提高。g2期细胞百分比与细胞凋亡率呈负相关。碘脱氧尿苷（IdU）脉冲追踪显示，即使在未治疗的对照组中，cd44高水平的癌细胞在G2中停留的时间也更长。这些细胞表达了更高水平的G2检查点蛋白，并且它们与BDH或Chk1 siRNA一起从G2中释放增加了它们的凋亡率。正常角质形成细胞的低传代培养也发现含有cd44高细胞亚群，显示出增加的克隆原性，以及与凋亡抵抗相关的类似g2阻滞模式。这些数据表明，具有干细胞样特性的正常和恶性人上皮细胞都表现出更强的抗凋亡能力，这与G2细胞周期期延长有关，并且这种特性不是肿瘤转化的结果。靶向G2检查点蛋白将这些细胞从G2阻滞中释放出来，使它们更容易凋亡，这意味着改进治疗方法的机会。
Subsets of cells with stem-like properties have been previously isolated from human epithelial cancers and their resistance to apoptosis-inducing stimuli has been related to carcinoma recurrence and treatment failure. The aim of this study was to investigate the mechanisms of resistance to apoptosis-inducing agents of cells with stem-like properties in both normal and malignant human epithelia. Cells isolated from fresh human head and neck carcinomas (n = 11), cell lines derived from head and neck, prostate and breast human carcinomas (n = 7), and from normal human oral mucosa (n = 5), were exposed to various apoptosis-inducing stimuli (UV, Tumour Necrosis Factor, Cisplatin, Etoposide, and Neocarzinostatin). Flow cytometry for CD44 and epithelial-specific antigen (ESA) expression, colony morphology, tumour sphere formation and rapid adherence assays were used to identify the subset of cells with stem-like properties. Apoptosis, cell cycle and expression of various cell cycle checkpoint proteins were assessed (Western Blot, qPCR). The role of G2-checkpoint regulators Chk1 and Chk2 was investigated by use of debromohymenialdisine (DBH) and siRNA. In both cancer biopsies and carcinoma cell lines a subset of CD44high cells showed increased clonogenicity, a significantly lower rate of apoptosis, and a significantly higher proportion of cells in the G2-phase of the cell cycle. An inverse correlation between the percentage of cells in G2-phase and the rate of apoptosis was found. Pulse-chase with iododeoxyuridine (IdU) demonstrated that CD44high carcinoma cells spent longer time in G2, even in un-treated controls. These cells expressed higher levels of G2 checkpoint proteins, and their release from G2 with BDH or Chk1 siRNA increased their rate of apoptosis. Low passage cultures of normal keratinocytes were also found to contain a subset of CD44high cells showing increased clonogenicity, and a similar pattern of G2-block associated with apoptotic resistance. These data indicate that both normal and malignant human epithelial cells with stem-like properties show greater resistance to apoptosis associated with extended G2 cell cycle phase, and that this property is not a consequence of neoplastic transformation. Targeting G2 checkpoint proteins releases these cells from the G2-block and makes them more prone to apoptosis, implying an opportunity for improved therapeutic approaches.
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