Effectors of senescent states in human fibroblasts

Brown University, Providence RI

Investigators

Linked publications & trials

Abstract

[unreadable] DESCRIPTION (provided by applicant): Replicative cellular senescence is a phenomenon of irreversible growth arrest triggered by the accumulation of a discrete number of cell divisions. The vast majority of normal cell types from all vertebrate species display this response. It is becoming increasingly evident that what has classically been described as cellular senescence is a collection of interrelated states that can be triggered by distinct intrinsic and extrinsic stimuli. We have found that when normal human diploid fibroblasts are subcultured into replicative exhaustion, three [unreadable] essentially independent processes can take place, each of which is sufficient to establish a senescent growth arrest state. The first pathway is initiated by telomere shortening. The second pathway is initiated by an unknown, spontaneous and stochastic process that leads to the upregulation of the cyclin-dependent kinase inhibitor p16 n K4A.The third pathway is initiated by mitochondrial damage leading to the production of sufficient oxidative stress to activate the p53 tumor suppressor protein. We have developed an in vitro cell based model system that allows us to selectively track and study senescent cells in unperturbed cultures, and to prepare homogeneous populations of cells that have activated a single senescence pathway. Aim 1 will examine the role of p53 and upstream effectors in telomere-initiated senescence. Posttranslational modifications of p53 and the activation status of signaling pathways upstream of p53 will be studied. Aim 2 will seek to discover what causes the spontaneous, age-dependent upregulation of pl6. Effectors implicated in regulating the p 16 gene will be examined, pharmacological methods will be used to probe cytoplasmic kinase cascades, and the physiological state elicited by the p16 pathway will be explored by microarray expression profiling. Aim 3 will investigate the causes and consequences of spontaneous upregulation of reactive oxygen species (ROS). The functional relationships between the ROS, p 16 and telomere pathways will be examined, and the effect of the IGF signaling pathway implicated in organismal aging on the pathways of cellular senescence will be studied. In all cases, reverse-genetic interventions utilizing dominant and constitutively active proteins as well as siRNA-mediated ablation will be used to probe the transmission of the senescence signals. Interventions will be sought to elicit senescence responses in naive cells, to prevent a natural senescence response caused by replicative exhaustion, and to reverse an established senescent state. Studies described in this proposal will give us a better understanding of cellular senescence processes, and will begin to shed light on the roles of cellular senescence in organismal aging. [unreadable] [unreadable]