Senescence Associated Metabolic Change And Isolation Of Live Premature Senescent Cells Using FUCCI Technology

Part Ⅰ:Mitochondrial hyper-function accompanied with apoptosis in senescent cellsDefined as stable cell-cycle arrest, cellular senescence plays an important role in diverse biological processes including tumorigenesis, organismal aging, and embryonic development. Although increasing evidence has documented the metabolic changes in senescent cells, mitochondrial function and its potential contribution to the fate of senescent cells remain largely unknown. Here, using two in vitro models of cellular senescence induced by doxorubicin treatment and prolonged passaging of neonatal human foreskin fibroblasts, we report that senescent cells have high ROS level, which accompanied by mitochondrial change and elevated glucose metabolic rate. Also, in the late stage of senescence, mitochondrial membrane potential in senescent cells show obvious depolarization which eventually, led to cellular apoptosis. Our study reveals that mitochondrial hyper-function contributes to the implementation of cellular senescence and we propose a model in which the mitochondrion acts as the key player in promoting fate-determination in senescent cells.Part II:Pyruvate Kinase downregulation induces cellular senescence that is irreversible by the restoration of glucose metabolism Cellular senescence defined as irreversible cell growth arrest is a biological process that results from various stresses. Cellular senescence has been implicated in the onset of a variety of age-related diseases such as cancer. While mounting studies have reported alterations of glucose metabolism associated with senescence and the role of metabolic intermediates in establishing cellular senescence, the causative relationship between glucose metabolism and cellular senescence remains largely unknown. To adress this, here we focused on knockdown of pyruvate kinase (PK), the enzyme that regulates the final rate-limiting step of glycolysis, in human foreskin fibroblasts. Our results showed that knockdown of PK isoenzymes induced premature cellular senescence accompanied by augmented glucose metabolic rate. Restoration of PKM expression reversed metabolic changes but failed to bypass cellular senescence induced by the downregulation of PKM. Our study thus suggests that altered glucose metabolism associated with cellular senescence is not the deterministic mechanism that induces cellular senescence.Part Ⅲ:Isolation of live premature senescent cells using FUCCI technologyCellular senescence plays an important role in diverse biological processes such as tumorigenesis and organismal aging. However, lack of methods to specifically identify and isolate live senescent cells hampers the precise understanding of the molecular mechanisms regulating cellular senescence. Here, we report that utilization of fluorescent ubiquitination-based cell cycle indicator (FUCCI) technology allows isolation of live premature senescent cells induced by doxorubicin treatment. Exposure of human foreskin fibroblasts (HFFs) to a low dose of doxorubicin led to cellular senescent phenotypes including formation of y-H2AX and 53BP1 foci indicative of DNA damage, decreased cell proliferation and increased senescence-associated β-galactosidase (SA-β-gal) activity. Importantly, doxorubicin-induced senescent cells were arrested at S/G2/M phases of cell cycle which can be reported by a construct encoding a fragment of hGeminin fused with monomeric Azami-Green (mAG-hGeminin). Flow cytometric sorting of GFP+ cells from doxorubicin-treated HFFs carrying mAG-hGeminin reporter enabled isolation and enrichment of live senescent cells in the culture. Our study develops a novel method to identify and isolate live premature senescent cells, thereby providing a new tool to study cellular senescence.