Project description:We observe preferential accumulation of gH2AX at chromosome ends upon irradiation comparison of quiescent vs senescent human fibroblasts
Project description:Cellular stress responses are crucial for survival in sub-optimal conditions, and almost invariably involve transcriptome- and proteome-wide alterations. However, most of our understanding of human cell stress responses stems from experiments in rapidly-dividing cell culture models. Here, we used primary human diploid fetal lung fibroblasts (IMR-90) in proliferating, contact-inhibited quiescent, or doxorubicin-induced senescent states, to explore their responses to a severe heat-shock (44 degrees Celsius for 2 hours) at the transcriptome, proteome, poly-ubiquitylated proteome, and insoluble proteome levels. We identify remarkable conservation in stress responses at the transcriptome, proteome, and poly-ubiquitylated proteome levels, albeit with slight dampening in the magnitude of changes in both quiescent and senescent state. By contrast, there was a considerable impairment in stress-induced sedimentation of RNA-binding proteins in the senescent state, several of which are linked to neurodegenerative disease pathology. We propose a senescence-specific divergence in proteostasis strategies at the level of stress-induced protein and RNA granule formation, which could contribute to the frailty associated with senescent-cell accumulation in ageing and disease.
Project description:H2AX phosphorylation at Ser139 (gH2AX) is an early key event of the DNA damage response (DDR) following DNA double strand breaks (DSB) induction. Although gH2AX distribution has been extensively used as a damage marker, genome-wide investigation of the subsequent DNA repair has been poorly addressed. Here we present ChIP-Seq-based distributions of Histone H3, H2AX and gH2AX under physiological conditions in HeLa cells. Additionally the same histones were studied after a single exposure to 10 Gy X-ray at 0.5, 3 and 24 hours post irradiation
Project description:Keloid radiotherapy is clinically effective but has an incomplete underlying molecular mechanism and limited accessibility; we used X-ray exposure to identify effector pathways replicating its antifibrotic benefits without further radiation. After collecting patient keloid samples, primary human keloid fibroblasts and normal skin fibroblasts, non-targeted metabolomics and RNA-seq were performed, identifying phytosphingosine (PHS) and KIF20A as key mediators of fibrosis and cell death in keloid radiotherapy. X-ray irradiation increased PHS release in keloid fibroblasts; exogenous PHS and X-ray irradiation independently induced cell death and reduced fibrosis, their combination exerted radiosensitization by boosting G2/M arrest and apoptosis while lowering fibrosis progression, and both PHS and irradiation reduced KIF20A whose loss induced cell cycle arrest and apoptosis. Our findings reveal that X-ray irradiation enhances keloid fibroblast PHS secretion that downregulates KIF20A to exert keloid therapeutic effects, and topical PHS or KIF20A-targeting agents are immediately translatable alternatives for radiotherapy-ineligible patients.
Project description:The action of RB as a tumor suppressor has been difficult to define, in part, due to the redundancy of the related proteins p107 and p130. By coupling advanced RNAi technology to suppress RB, p107 or p130 with a genome wide analysis of gene expression in growing, quiescent or ras-senescent cells, we identified a unique and specific activity of RB in repressing DNA replication as cells exit the cell cycle into senescence, a tumor suppressive program. Experiment Overall Design: Expression profiles of IMR90 cells before and after RNAi-mediated supppression of RB, p107 or p130 in growing, quiescent or ras-induced senescent conditions. RNA was extracted from growing, low serum (0.1% FBS), confluent, or ras-senescent cells.
