A computational platform for robotized fluorescence microscopy (II): DNA damage, replication, checkpoint activation, and cell cycle progression by high‐content high‐resolution multiparameter image‐cytometry

Abstract

Dissection of complex molecular-networks in rare cell populations is limited by current technologies that do not allow simultaneous quantification, high-resolution localization, and statistically robust analysis of multiple parameters. We have developed a novel computational platform (Automated Microscopy for Image CytOmetry, A.M.I.CO) for quantitative image-analysis of data from confocal or widefield robotized microscopes. We have applied this image-cytometry technology to the study of checkpoint activation in response to spontaneous DNA damage in nontransformed mammary cells. Cell-cycle profile and active DNA-replication were correlated to (i) Ki67, to monitor proliferation; (ii) phosphorylated histone H2AX (cH2AX) and 53BP1, as markers of DNA-damage response (DDR); and (iii) p53 and p21, as checkpoint-activation markers. Our data suggest the existence of cell-cycle modulated mechanisms involving different functions of cH2AX and 53BP1 in DDR, and of p53 and p21 in checkpoint activation and quiescence regulation during the cell-cycle. Quantitative analysis, event selection, and physical relocalization have been then employed to correlate protein expression at the population level with interactions between molecules, measured with Proximity Ligation Analysis, with unprecedented statistical relevance. ' 2013 International Society for Advancement of Cytometry

New insights into cell cycle and DNA damage response machineries through high‐resolution AMICO quantitative imaging cytometry

“…Continuation of this progress was recently reported by Furia et al (20, 21), who describe a novel approach in high resolution imaging cytometry (schematic work flow in Figure 1). The authors defined it as Automated Microscopy for Image CytOmetry (AMICO) and utilized to elucidate signaling pathways and molecular mechanisms associated with controls of the cell cycle progression.…”

“…Concurrently analyzed were also the primary markers of DNA damage response by measurement histone H2AX-Ser139 phosphorylation and recruitment of p53BP1. The Proximity Ligation Analysis (22) of the images made it possible to measure proximity of the detected DNA replication sites vis-à-vis DNA damage and repair foci revealing likely the cause-effect association of the molecular processes ongoing in these sites (20, 21). …”

“…The AMICO approach brought several interesting findings (20, 21). The constitutive (spontaneous) DNA damage sites, identified by the presence of γH2AX and 53 binding protein 1 (53BP1), which was reported before to be caused primarily by endogenous oxidants, the by-products of oxidative phosphorylation (23, 24), was found to be strongly correlated spatially with the DNA replication sites.…”

RNAi screens identify CHD4 as an essential gene in breast cancer growth

“…(automated microscopy for image cytometry) technology, which allowed us to perform a multi-parameter analysis, targeting specific cell subpopulations [60, 61]. To analyze effects of CHD4 downregulation on proliferation ability, cell cycle progression and regulation we simultaneously assessed the level of i) CHD4 , ii) Ethinyl-deoxyUridine (EdU), for active DNA synthesis detection, iii) Ki67, as a proliferation marker, and iv) TP53 and CDKN1A , to monitor checkpoint activation, in relation to DNA content.…”

“…An oil immersion 60X 1.3 NA objective was employed for acquisition. Cell cycle statistical analysis was performed as described by Furia and colleagues [61]. …”