Studies on the irreversibility of cellular senescence and its role in vivo

细胞衰老是一种永久性的细胞周期停滞状态，可由各种潜在的致癌应激诱导。因此，它被认为是一种故障保护机制，可以保护正常细胞不会失去对细胞分裂的控制，从而形成肿瘤。虽然在组织培养细胞中诱导细胞衰老的分子机制已经得到了很好的研究，但对于如何在体内稳定地维持衰老细胞周期停滞还知之甚少。为了进一步了解体内细胞衰老的作用和机制，我们试图阐明衰老细胞周期停滞不可逆转的分子机制。为此，我们利用了一种表达温度敏感的SV4-Large T抗原的条件永生化细胞系。使用该细胞系，我们发现肿瘤抑制因子不仅通过激活衰老的人类细胞中的活性氧簇(ROS)-PKCδ信号来阻止dna复制，而且还阻止细胞质分裂。重要的是，一旦被ROS激活，PKCδ就会促进ROS的进一步产生，从而建立一个正反馈环来维持ROS-PKCδ信令。ROS-PKCδ信号的持续激活不可逆转地阻止胞质分裂，至少部分是通过阻断LATS1/WARTS的功能，LATS1/WART是胞质分裂所需的有丝分裂退出网络激酶。我们还建立了在衰老相关基因启动子控制下表达萤火虫荧光素酶的转基因小鼠系。结合非侵入性生物发光成像技术，这些转基因小鼠品系使我们能够监测体内衰老相关基因的表达。使用这个系统，我们能够揭示体内整个生命周期中衰老信号的动态变化。我们希望这些研究将为癌症的发展提供新的见解，并为控制癌症开辟新的可能性。

Cellular senescence is a state of permanent cell cycle arrest that can be induced by a variety of potentially oncogenic stresses. It is therefore proposed as a fail-safe mechanism that protects normal cells from losing control of cell division and forming tumors. Although molecular mechanism inducing cellular senescence has been well studied in tissue culture cells, very little is known about how senescent cell cycle arrest is stably maintained in vivo. To gain further insight into the roles and mechanisms of cellular senescence in vivo, we attempted to elucidate molecular mechanisms underlying irreversibility of senescent cell cycle arrest. Towards this end, we utilized a conditionally immortalized cell line that express temperature-sensitive SV4- large T antigen. Using this cell line, we uncovered that p 16^<INK4a> tumor suppressor blocks not only DNA replication but also cytokinesis through activating reactive oxygen species (ROS) -PKCδsignalling in senescent human cells. Importantly, once activated by ROS, PKCδ promotes further generation of ROS, thus establishing a positive feedback loop to sustain ROS-PKCδsignalling. Sustained activation of ROS-PKCδ signalling irreversibly blocks cytokinesis, at least partly through blocking the function of LATS1/WARTS, a mitotic exit network kinase required for cytokinesis. We also established the transgenic mice lines expressing the firefly luciferase under the control of the senescence-associated gene promoter. In conjunction with a noninvasive bioluminescence imaging technique, these transgenic mice lines enabled us to monitor the senescence-associated gene expression in vivo. Using this system, we were able to unveil the dynamics of senescence signaling throughout entire lifespan in vivo. We hope that these studies will provide new insights into the development of cancer and open up new possibilities for its control.