title: 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 (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.
authors: Department of Experimental Oncology, European Institute of Oncology, IFOM-IEO Campus for Oncogenomics, Milan 20139, Italy.
authors: Laura
authors: L
authors: Furia
authors: Pier Giuseppe
authors: PG
authors: Pelicci
authors: Mario
authors: M
authors: Faretta
mesh_terms: D059447:Cell Cycle Checkpoints / Q000235:genetics*; D002460:Cell Line; D004247:DNA / Q000032:analysis*; D004249:DNA Damage; D004261:DNA Replication / Q000235:genetics*; D004847:Epithelial Cells / Q000166:cytology / Q000378:metabolism*; D005260:Female; D015870:Gene Expression; D006657:Histones / Q000235:genetics / Q000378:metabolism; D006801:Humans; D019044:Image Cytometry / Q000295:instrumentation / Q000592:standards; D007091:Image Processing, Computer-Assisted / Q000706:statistics & numerical data; D047908:Intracellular Signaling Peptides and Proteins / Q000235:genetics / Q000378:metabolism; D019394:Ki-67 Antigen / Q000235:genetics / Q000378:metabolism; D042361:Mammary Glands, Human / Q000166:cytology / Q000378:metabolism*; D018613:Microscopy, Confocal / Q000295:instrumentation / Q000592:standards; D008856:Microscopy, Fluorescence / Q000295:instrumentation / Q000592:standards; D012371:Robotics / Q000295:instrumentation / Q000592:standards; D012984:Software; D016159:Tumor Suppressor Protein p53 / Q000235:genetics / Q000378:metabolism; D000071857:Tumor Suppressor p53-Binding Protein 1; D054462:p21-Activated Kinases / Q000235:genetics / Q000378:metabolism
chemical_list: C468783:H2AX protein, human; D006657:Histones; D047908:Intracellular Signaling Peptides and Proteins; D019394:Ki-67 Antigen; C498301:TP53BP1 protein, human; D016159:Tumor Suppressor Protein p53; D000071857:Tumor Suppressor p53-Binding Protein 1; D004247:DNA; D054462:p21-Activated Kinases