Therefore, the number of metabolic H2AX foci in proliferating cells reflects the proliferative activity of cells

Therefore, the number of metabolic H2AX foci in proliferating cells reflects the proliferative activity of cells. same cells of up to 30 years old donors. The number of H2AX foci demonstrated a statistically significant positive correlation with the fraction of proliferating cells in fibroblast cultures. On average, proliferating cells have twice as many the H2AX foci in comparison with the quiescent cells. Within a population of proliferating (Ki67+) cells, the degree of senescence correlated with a relative declining of constitutive H2AX foci number, whereas in the population of quiescent (Ki67-) cells, it was proportional to augmenting the number of the H2AX foci. Our data on a statistically significant (p=0.001) correlation between the age of the donor and the number of constitutive H2AX foci in quiescent cells, could point out the ongoing DNA-damage response due EC1454 in the maintenance of the senescent state of cells. Keywords: dermal fibroblasts, aging, H2AX foci, proliferation, cellular senescence, \galactosidase, lonogenic assay Introduction Skin aging is a multi-factorial process that affects nearly every aspect of its biology and function [1]. However fundamental molecular mechanisms of skin aging are associated with fibroblasts, the primary dermal cell EC1454 population C the ATP1A1 main function of which is to produce and organize an extracellular matrix (ECM) that provides the skin with structural integrity and elasticity [2C4]. The young skin fibroblasts produce and adhere to ECM, consisting of mainly of type I collagen fibrils it [5]. Aging decrease the number of functionally active fibroblasts in the skin, reduce their biosynthetic activity and content of collagen, the main structural dermal component [6]. The structural alterations and degradation of ECM, thought to result in dermal thinning, increasing wrinkling and loss of elasticity [6,7]. This imbalance of ECM homeostasis further stimulates fragmentation of collagen fibrils in a self-sustaining vicious circle. The accumulation of genetic alterations arising from the ineffective or incorrect repair of spontaneous DNA damage (attacks of metabolic free radicals produced, replication and recombination errors, spontaneous chemical changes) is one of the most significant causes of aging and oncotransformation of cells [8]. Some authors consider the accumulation of DNA damage in cells as a universal cause of age-dependent changes at cellular level [9C11]. DNA double-strand breaks (DSBs) are the most intensively investigated form of the damage among the variety of spontaneous DNA lesions. Indeed, DSBs are the most critical DNA alterations that can define the fate of cells and, if repaired incorrectly or inefficiently, can lead to serious cytogenetic abnormalities, cell death, inactivation of tumor suppressor genes and/or activation EC1454 of oncogenes [12C15]. Moreover, the formation of a particular phenotype inherent to senescence linked to the functional state of repair of DSBs, as well as to the accumulation of DSBs [16]. Recently, an indirect method based on the immunocytochemical (ICC) analysis of proteins involved in the repair of DSBs became increasingly popular for studying changes in a small amount of DSBs in living cells. During DSBs DNA repair, complex dynamic microstructures, consisting of thousands of copies of proteins can be visualized by ICC staining in the form of bright dots, so-called DNA repair protein foci [17,18]. It is believed that one focus corresponds to a single or multiple DSBs repair site(s) [19]. The DNA damage response kinases of the PI3-family (Ataxia telangiectasia mutated, ATR, DNA-PKcs and especially ATM) phosphorylate core histone H2AX(2) on serine EC1454 139 in response to the formation of DSBs. EC1454 Therefore, the ICC-assisted measurement of 2 foci indicates the presence of DNA double-strand breaks and DNA repair site(s) [20,21]. The constitutive level of H2AX expression in live cells, untreated by exogenous agents, likely represents DNA damage by endogenous oxidants generated during cellular respiration [22]. We studied spontaneous (constitutive) H2AX foci formation in human dermal fibroblasts in relation to proliferation activity and aging of the donors of the fibroblasts. To.