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.
PMID 23463591 · Cytometry. Part A : the journal of the International Society for Analytical Cytology · 2013 · DOI 10.1002/cyto.a.22265
Authors: Laura Furia; Pier Giuseppe Pelicci; Mario Faretta
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 (γH2AX) 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 γH2AX 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.
Cell Cycle Checkpoints; Cell Line; DNA; DNA Damage; DNA Replication; Epithelial Cells; Female; Gene Expression; Histones; Humans; Image Cytometry; Image Processing, Computer-Assisted; Intracellular Signaling Peptides and Proteins; Ki-67 Antigen; Mammary Glands, Human; Microscopy, Confocal; Microscopy, Fluorescence; Robotics; Software; Tumor Suppressor Protein p53; Tumor Suppressor p53-Binding Protein 1; p21-Activated Kinases
Cite
Laura Furia; Pier Giuseppe Pelicci; Mario Faretta. 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. Cytometry. Part A : the journal of the International Society for Analytical Cytology (2013). PMID 23463591. DOI 10.1002/cyto.a.22265.