Caspase3&7非致死性激活促进细胞基因组不稳定和诱导肿瘤发生

Apoptosis is normally considered a barrier for genetic instability and carcinogenesis because of its widely perceived role in getting rid of unwanted or damaged cells. However, this paradigm is premised on the assumption that once the apoptotic program is initiated, it is irreversible leads to cell death. However, evidence for this assumption is lacking. In fact, recent discoveries in our laboratory suggest that in many cells initiation of the apoptotic program does not always lead to cell death. For example, caspase 3&8 activation is involved in a cell-autonomous manner to clear epigenetic barriers during the reprogramming of differentiated human fibroblasts into pluripotent stem cells (iPSCs). Moreover, our preliminary data indicate that caspase 3 activation is responsible for a significant amount of DNA damage and genetic instability in surviving cells after exposure to radiation. Therefore, we need to re-examine the roles of apoptosis, especially those of apoptotic caspases, in carcinogenesis. In this project, we will examine the hypothesis that non-lethal caspase 3 or 7 activation induces genomic instability to facilitate radiation and other stress-induced carcinogenesis. Our hypothesis is a bold one that goes against the current paradigm. It will have wide-ranging implications since many endogenous and external stimuli could activate apoptotic caspases. Examples of such stimuli include exposure to ionizing radiation, UV, chemicals, and oncogene expression (e.g., myc). A facilitative role for caspases in carcinogenesis would provide exciting new insights into how those diverse environmental insults cause cancer. We will use a variety of in vitro and in vivo models, in combination with state-of-the-art molecular technologies, to examine the roles of caspases 3&7 in radiation- and other stress-induced genetic instability and carcinogenesis. We expect our studies to provide a comprehensive evaluation of the roles of caspases 3&7 in promoting carcinogenesis through inducing genetic instability. Upon completion of the project, we hope we can gain significant insights into the roles of apoptotic caspases in carcinogenesis. Such insights may provide novel targets for future cancer prevention strategies.

细胞凋亡能主动地清除机体内的受损细胞，通常认为细胞凋亡抑制肿瘤发生。然而，我们最近的研究发现，在诱导iPS细胞形成过程中需要细胞自身caspase3&8激活，而这种激活程度是非致死性的。进一步研究表明，非致死地激活caspase3能诱导细胞内DNA损伤和基因组不稳定。因此，caspase激活并不一定会导致细胞死亡。这促使我们重新审视caspases与细胞凋亡、肿瘤发生的关系及其在其中的作用。因此，我们提出一个全新的假说：caspase3&7非致死性激活能诱导基因组不稳定，促进射线、化学药物和癌基因等诱导的肿瘤发生。我们将利用多种体外和体内模型检测caspases 3&7在应激条件下激活,诱导基因组不稳定和肿瘤发生过程中的作用。由于多种外界和内部因素都能激活caspase，所以这一研究具有广泛意义。完成这一研究，不仅为阐明caspase的新功能提供实验证据，也为解释环境因素致癌机制提供新视角

细胞凋亡能主动地清除机体内的受损细胞，通常认为细胞凋亡抑制肿瘤发生。然而，课题组前期研究发现， iPS细胞形成过程中非致死性激活了细胞自身Caspase3&8，非致死地激活Caspase3能诱导细胞内DNA损伤和基因组不稳定。因此，提出与传统观点Caspase激活诱导细胞凋亡，抑制肿瘤细胞生长相反的假说：Caspase3&7非致死性激活能诱导基因组不稳定，促进放射线、化学药物和癌基因等诱导的肿瘤发生。本研究（1）从分子水平、细胞水平验证外界应激条件（放射线、化学药物）刺激或内源性原癌基因激活下，Caspase3&7非致死性激活对细胞基因组稳定性的调控以及肿瘤的发生。本研究通过Casp3/7-GFP-Luc报告基因检测、western blot等实验方法明确了放射线（X射线、铁离子射线）照射、化学药物（细胞凋亡诱导剂）等外源性刺激以及内源性原癌基因（Myc等）激活都能造成持续性、非致死性的caspase3/7激活，通过γH2AX Foci染色、comet assay检测及染色体Giemsa 染色分析检测等方法证实了caspase3/7非致死性激活能导致细胞DNA双链断裂、基因组不稳定，并在Caspase3/7基因敲除细胞系中证实了caspase3/7是造成持续性DNA损失、基因组不稳定的来源，明确了外源性刺激造成的细胞线粒体外膜通透（MOMP）能导致细胞凋亡通路caspase持续性的低水平激活以及EndoG从线粒体转位到细胞核造成DNA损伤和基因组不稳定。（2）通过soft agar克隆形成体外实验证实了Caspase3/7促进放射线、化学药物及原癌基因导致的正常细胞恶性转化;通过Casp3转基因小鼠模型证实Caspase3促进化学药物(DMBA/TPA)及原癌基因（MMTV-Pymt）导致的肿瘤发生。综上，本课题通过体外、体内研究揭示Caspase3/7参与基因组稳定性和肿瘤发生的肿瘤生物学新功能，阐明直接过间接损失DNA的药物或刺激能诱导细胞基因组不稳定，从而促进正常细胞的恶性转化和肿瘤发生。由于多种外界和内部因素都能激活caspase，这一研究成果具有广泛的科学意义，也为解释环境因素致癌机制提供了新视角。