Attenuation of replication stress-induced premature cellular senescence to assess anti-aging modalities

Abstract

Described is an in vitro model of premature senescence in pulmonary adenocarcinoma A549 cells induced by persistent DNA replication stress in response to treatment with the DNA damaging drug mitoxantrone (Mxt). The degree of cellular senescence, based on characteristic changes in cell morphology, is measured by laser scanning cytometry. Specifically, the flattening of cells grown on slides (considered the hallmark of cellular senescence) is measured as the decline in local intensity of DNA-associated DAPI fluorescence (represented by maximal pixels). This change is paralleled by an increase in nuclear area. Thus, the ratio of mean intensity of maximal pixels to nuclear area provides a very sensitive morphometric biomarker for the degree of senescence. This analysis is combined with immunocytochemical detection of senescence markers, such as overexpression of cyclin kinase inhibitors (e.g., p21(WAF1) ) and phosphorylation of ribosomal protein S6 (rpS6), a key marker associated with aging/senescence that is detected using a phospho-specific antibody. These biomarker indices are presented in quantitative terms defined as a senescence index (SI), which is the fraction of the marker in test cultures relative to the same marker in exponentially growing control cultures. This system can be used to evaluate the anti-aging potential of test agents by assessing attenuation of maximal senescence. As an example, the inclusion of berberine, a natural alkaloid with reported anti-aging properties and a long history of use in traditional Chinese medicine, is shown to markedly attenuate the Mxt-induced SI and phosphorylation of rpS6. The multivariate analysis of senescence markers by laser scanning cytometry offers a promising tool to explore the potential anti-aging properties of a variety agents.

Keywords: berberine; cell cycle; laser scanning cytometry; mTOR signaling; nuclear size; ribosomal protein S6.

Figure 9.47.1

Induction of premature cellular senescence in A549 cells measured by laser scanning cytometry (LSC; iCys Research Imaging Cytometer; Zhao et al., 2010). A549 cells were untreated (Ctrl) or treated with 2 nM Mxt for 48 or 72 hr. (A) Cell morphometric features revealed by nuclear DNA (DAPI) fluorescence reporting on the bivariate distributions (scatter plots) of nuclear area versus mean intensity of maximal pixels of fluorescence per nucleus, respectively. In Ctrl cells, intensity of the mean value of maximal pixels is highest in mitotic (M) and immediately post-mitotic (pM) G₁ cells; nuclei of these cells also have a small area of DAPI fluorescence, consistent with their high degree of chromatin condensation (Bedner et al., 1999; Darzynkiewicz et al., 1999; Henriksen et al., 2011). In senescing cells, nuclear area increases while maximal pixel intensity decreases (Zhao et al., 2010, 2013; McKenna et al., 2012). These morphometric changes reflect enlargement of the projected nuclear area and decreased intensity of DAPI local staining per unit area due to a flattened cellular appearance, the hallmark of cellular senescence (Dimri et al., 1995; Itahana et al., 2007). The insets show DNA content frequency histograms of cells from the respective cultures. Persistent DNA damage by Mxt (replication stress) leads to a decrease in the frequency of S phase cells and cell arrest in G₂. (B) Bivariate distributions of DNA content versus p21^WAF1, another marker of cellular senescence (Dimri et al., 1995; Itahana et al., 2007), report expression of this CKI with respect to cell cycle phase. In the Mxt-treated cells, note that the increase in p21 expression is concurrent with the reduction in frequency of S-phase cells. (C) Mean values (± SD) of DAPI (nuclear) area, DAPI mean maximal pixels, and the ratio of maximal pixels to nuclear area (mp/area) in control and Mxt-treated cells. Numbers above bars in the ratio graph indicate the senescence index (SI) of Mxt-treated cells (ratio of mp/area in Mxt-treated cells to mp/area ratio in Ctrl cells).

Figure 9.47.2

Attenuation of Mxt-induced senescence in A549 cells by BRB as measured by cell morphometric features and expression of p21^WAF1 and rpS6^P. Exponentially growing cells were untreated (Ctrl) or treated with 2 nm Mxt in the absence or presence of 5 to 60 µM BRB for 5 days. (A) Bivariate distributions of nuclear area (DNA-DAPI) versus mean intensity of maximal pixels of DAPI fluorescence per cell. The ratio of maximal pixels to nuclear area (mp/A) is expressed as SI (i.e., relative to the same ratio in Ctrl cells). Note that SI is progressively reduced (in a dose-dependent manner) in BRB-treated cells relative to cells treated with Mxt alone. Arrows are percent increase in mp/A in BRB-treated cultures (i.e., percent reduction of SI) with respect to Mxt alone. (B) Bivariate distributions of p21^WAF1 and rpS6^P versus cellular DNA content. x is fold increase in mean expression for all cells with respect to Ctrl cells; arrows are percent reduction of expression in BRB-treated cultures compared to Mxt alone. Since aging and induction of premature senescence is considered to be driven by constitutive mTOR signaling, which converges on activation of rpS6 (Blagosklonny, 2008; Zoncu et al., 2011; Magnuson et al., 2012), potential anti-aging agents are expected to reduce the level of rpS6 phosphorylation.