Mathew Robin, Karantza-Wadsworth Vassiliki, White Eileen
University of Medicine and Dentistry of New Jersey, Robert Wood Johnson Medical School, Piscataway, New Jersey, USA.
Methods Enzymol. 2009;453:53-81. doi: 10.1016/S0076-6879(08)04004-4.
Autophagy is a survival mechanism activated in response to metabolic stress. In normal tissues autophagy plays a major role in energy homeostasis through catabolic self-digestion of damaged proteins and organelles. Contrary to its survival function, autophagy defects are implicated in tumorigenesis suggesting that autophagy is a tumor suppression mechanism. Although the exact mechanism of this tumor suppressor function is not known, it likely involves mitigation of cellular damage leading to chromosomal instability. The complex role of functional autophagy in tumors calls for model systems that allow the assessment of autophagy status, stress management and the impact on oncogenesis both in vitro as well as in vivo. We developed model systems that involve generation of genetically defined, isogenic and immortal epithelial cells from different tissue types that are applicable to both wild-type and mutant mice. This permits the study of tissue- as well as gene-specific tumor promoting functions. We successfully employed this strategy to generate isogenic, immortal epithelial cell lines from wild-type and mutant mice deficient in essential autophagy genes such as beclin 1 (beclin 1(+/-)) and atg5 (atg 5(-/-)). As these cell lines are amenable to further genetic manipulation, they allowed us to generate cell lines with apoptosis defects and stable expression of the autophagy marker EGFP-LC3 that facilitate in vitro and in vivo assessment of stress-mediated autophagy induction. We applied this model system to directly monitor autophagy in cells and 3D-morphogenesis in vitro as well as in tumor allografts in vivo. Using this model system we demonstrated that autophagy is a survival response in solid tumors that co-localizes with hypoxic regions, allowing tolerance to metabolic stress. Furthermore, our studies have established that autophagy also protects tumor cells from genome damage and limits cell death and inflammation as possible means to tumor suppression. Additionally these cell lines provide an efficient way to perform biochemical analyses, and high throughput screening for modulators of autophagy for potential use in cancer therapy and prevention.
自噬是一种在代谢应激反应中被激活的生存机制。在正常组织中，自噬通过对受损蛋白质和细胞器的分解代谢性自我消化，在能量稳态中发挥主要作用。与其生存功能相反，自噬缺陷与肿瘤发生有关，这表明自噬是一种肿瘤抑制机制。尽管这种肿瘤抑制功能的确切机制尚不清楚，但它可能涉及减轻导致染色体不稳定的细胞损伤。功能性自噬在肿瘤中的复杂作用需要模型系统，以便在体外和体内评估自噬状态、应激管理以及对肿瘤发生的影响。我们开发了模型系统，该系统涉及从不同组织类型生成基因定义的、同基因且永生的上皮细胞，适用于野生型和突变型小鼠。这允许研究组织特异性以及基因特异性的肿瘤促进功能。我们成功地采用这种策略，从缺乏必需自噬基因如贝林1（beclin 1(+/-)）和自噬相关基因5（atg5(-/-)）的野生型和突变型小鼠中生成了同基因、永生的上皮细胞系。由于这些细胞系易于进行进一步的基因操作，它们使我们能够生成具有凋亡缺陷且自噬标记物EGFP-LC3稳定表达的细胞系，这有助于在体外和体内评估应激介导的自噬诱导。我们应用这个模型系统直接监测细胞中的自噬以及体外的三维形态发生和体内肿瘤同种异体移植中的自噬。使用这个模型系统，我们证明自噬是实体瘤中的一种生存反应，与缺氧区域共定位，从而允许对代谢应激的耐受性。此外，我们的研究已经确定，自噬还保护肿瘤细胞免受基因组损伤，并限制细胞死亡和炎症，这可能是肿瘤抑制的手段。此外，这些细胞系提供了一种进行生化分析以及高通量筛选自噬调节剂的有效方法，这些调节剂可能用于癌症治疗和预防。
