Sustained activation of DNA damage response in irradiated apoptosis-resistant cells induces reversible senescence associated with mTOR downregulation and expression of stem cell markers.

Cells respond to genotoxic stress by activating the DNA damage response (DDR). When injury is severe or irreparable, cells induce apoptosis or cellular senescence to prevent transmission of the lesions to the daughter cells upon cell division. Resistance to apoptosis is a hallmark of cancer that challenges the efficacy of cancer therapy. In this work, the effects of ionizing radiation on apoptosis-resistant E1A + E1B transformed cells were investigated to ascertain whether the activation of cellular senescence could provide an alternative tumor suppressor mechanism. We show that irradiated cells arrest cell cycle at G 2/M phase and resume DNA replication in the absence of cell division followed by formation of giant polyploid cells. Permanent activation of DDR signaling due to impaired DNA repair results in the induction of cellular senescence in E1A + E1B cells. However, irradiated cells bypass senescence and restore the population by dividing cells, which have near normal size and ploidy and do not express senescence markers. Reversion of senescence and appearance of proliferating cells were associated with downregulation of mTOR, activation of autophagy, mitigation of DDR signaling, and expression of stem cell markers.

An integrated approach for monitoring cell senescence.

Cellular senescence is considered as a crucial mechanism of tumor suppression that helps to prevent the growth of cells at risk for neoplastic transformation. In normal cells, cellular senescence induces an irreversible cell cycle arrest in response to telomere dysfunction, oncogene activation, genotoxic stress and a persistent DNA damage response (DDR). This process is accompanied by dramatic changes in cell morphology as well as in the activity of several signaling pathways. The senescent phenotype is multifaceted. In addition to an obligatory proliferation arrest, senescent cells manifest various senescence markers: mTOR-mediated hypertrophic growth (cell size increase), cell flattening, senescence-associated ß galactosidase (SA-ß gal) staining, expression of negative cell cycle regulators p53, p21(Waf1) and p16(Ink4a), specific chromatin reorganization including DNA segments with chromatin alterations reinforcing senescence (DNA-SCARS), senescence-associated secretory phenotype (SASP) and other features. Here, we describe the protocols that are used to study histone deacetylase inhibitor (HDACI)-induced cellular senescence in transformed cells with a special emphasis on the morphological features of senescence.