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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. © 2012 International Society for Advancement of Cytometry
Quantification of cellular DNA damage (DD) is a main target in cancer research (1, 2) as genomic instability is a hallmark of most human cancers. Endogenous and exogenous DNA damaging agents continuously threaten the integrity of our genome, but cells have evolved a tightly regulated network of molecular mechanisms to protect it, known as DNA damage response (DDR). Multiple pathways start from the recognition of a single double-strand break (DSB) by the MRN complex (Mre11-Rad50-Nbs1). The activated signaling cascade then leads to differential recruitment of repair effectors according to the type of lesion (3–7).
Localization of damaged sites in the genome is usually based on the detection of the phosphorylation of the histone isoform H2AX (γH2AX) that extends over a broad genomic area around the lesion (8–12). However, accumulating evidence is challenging the notion of a univocal link between serine 139 phosphorylation of H2AX and DNA-breaks (13–17).
Thanks to the increasing availability of antibodies directed towards the DDR molecular machinery, new surrogate markers have been proposed. For example, monitoring of 53BP1 spatial distribution provides a first alternative to γH2AX detection. 53BP1 rapidly relocalizes to DSB sites, activating the ATM-driven signaling cascade (18, 19). It plays a fundamental function in nonhomologous end joining (NHEJ) repair, which is mainly active in resting cells and repressed during the S-phase (20). The 53BP1 fundamental role in inducing genomic instability through error-prone repair activation has been recently shown in a mutated BRCA background (21, 22). A specific function in the processing of damaged DNA fragile sites has been also attributed to 53BP1 (23, 24). Both γH2AX and 53BP1 are essential to activate the cell-cycle checkpoint and to arrest cell cycle progression (25), and their colocalization might be an effective marker of DSBs (26, 27). However, due to their participation to multiple DDR and repair pathways, a direct correlation between their expression, localization, and interaction in the different phases of the cell cycle is very much required.
Detection of physiological DD is challenging because of the high sensitivity and spatial resolution required for its quantification. Flow cytometry, laser-scanning cytometry and image-streaming cytometry can all provide statistically significant measurements and have been employed to quantify γH2AX and/or other DDR effectors (8, 26, 28–34). The recognition and quantification of DD foci by H2AX phosphorylation and/or 53BP1 relocalization demand an image-based approach, but they still represent a challenging task for automatic image processing. In particular, detection and quantification of endogenous DD foci call for the use of high Numerical Aperture (NA) objectives to increase the amount of collected photons and to detect small foci with size close to and below the diffraction limit. Moreover, the molecular crowding typical of the detected signals requires maximal resolution to resolve clusters of foci and to evaluate the colocalization of the involved DDR components. Many efforts have been made to provide efficient computational tools for foci quantification and description (9, 27, 35–40). However, a high-content image-analysis on a high number of cells is required for results to be statistically significant to: (i) target specific subpopulations, (ii) quantitatively describe phenotypes related not only to DDR but also to checkpoint activation, and (iii) monitor cell-cycle position and progression.
Here, we present a seven-parameter quantitative image-based analysis of the cell cycle with unprecedented spatial resolution and statistical sampling. Combining the flexibility offered by the automation of multiple fluorescence microscopy platforms with our novel analysis software (A.M.I.CO., see the accompanying technical note in this issue), we examined the connections between DDR and cell-cycle checkpoints during exponential cell growth, focusing on the effect of proliferation on genome integrity.
For the first time, we provide a detailed kinetics of both protein content and protein interactions during unperturbed cell-cycle progression. Our results show correlations between DDR and cell proliferation, revealing both accumulation of γH2AX during DNA replication, and colocalization between γH2AX-foci and replication factories. The spatial proximity between γH2AX-foci and the newly replicated DNA, shown by in-situ proximity ligation assay (PLA), indicates the immediate activation of DDR in response to replication errors. We detected distinct expression profiles for γH2AX and 53BP1, together with variable colocalization during the cell-cycle. DDR and checkpoint-activation markers showed either correlated or independent expression profiles. Finally, we show that 53BP1 and γH2AX interact with p53 independently from each other, and that these interactions are quantitatively modulated across the cell cycle.
The data collected contribute to delineate a hierarchy in DDR, providing a most exhaustive view of the complex molecular network regulating tumor-suppressing mechanisms.
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Oncogene expression induces a hyperproliferation burst that causes an enhanced DNA-replication stress. The resulting DD can induce gene mutations and/or chromosomal aberrations, which may eventually lead to malignant transformation (46, 47). To contrast this event the cells have developed physiological barriers by inducing a replicative senescence process, which is triggered by checkpoint activation via DDR. The goal of this study was to carry out a multiparameter analysis of replication-induced damage, cell-cycle regulation, proliferation, and DDR. Until now, this kind of high-content analysis was hindered by the lack of a comprehensive experimental approach, which could, at the same time, maintain single-cell resolution, high spatial-resolution, high-content, and statistical significance with respect to poorly represented phenotypes. In addition, cell-sorting manipulation and/or enhancement of specific cell features, usually employed to study DDR, carry the risk of potentially altering the physiological level of DNA-damage (29).
Herein, for the first time, we employed an image-cytometry computational platform to produce a multi-correlated quantitative cell-cycle profiling of both content and spatial distribution of DNA-replication (EdU), proliferation (KI67), DDR (γH2AX, 53BP1) and checkpoint activation (p53, p21) markers. Multiple cycles of data acquisition-analysis enabled us to assess the expression levels of these markers in the entire cell population, and their molecular interactions in single cells at the tens of nanometers range.
The analysis of γH2AX expression in relation to the cell-cycle phase and checkpoint activation showed, in an unperturbed context, an increased phosphorylation of this histone isoform during DNA replication. The analysis of different cell lines confirmed that this increase was not restricted to the MCF10A mammary cells but also present in other tissues and in a malignant background. The pioneering work by P. Olive's group on the flow-cytometry analysis of γH2AX (26, 34) had suggested cell-cycle modulation of H2AX phosphorylation. Our multiparameter approach has provided, through direct EdU labeling of S-phase cells, the detailed kinetics underlying this process: G1-early S and late S-G2 transition showed respectively the lowest and highest average γH2AX content within the cell-cycle. By scaling down from a cell-population to a molecular proximity level, we demonstrated that the observed γH2AX increase was partially responsible for activation of the DDR cascade, since only a fraction of foci exhibited simultaneous recruitment of the DDR-effector 53BP1. This provides further evidence of the limitations in the applicability of γH2AX as readout of the occurrence of DNA breaks (13–16).
However, if one considers the simultaneous presence of γH2AX and 53BP1 as an index of DDR activation following a DNA break, our results show a number of foci associated to replication that is very close to the number of endogenous DSBs expected for a single cell-cycle (about 50), and which has never been quantified before in-vivo at a single-cell resolution (48, 49). PLA analysis also revealed the proximity of replication factories and γH2AX foci, thus suggesting that i) γH2AX S-phase signaling maybe associated to impaired progression of the replication-fork and that ii) the induced damage is immediately recognized by the DDR machinery. This interpretation is consistent with recent works, which, by laser-scanning cytometry and confocal microscopy, showed an S-phase specific γH2AX-induction at the replication sites in response to exogenous-oxidants treatment (32).
We demonstrated that the cell-cycle modulation of DDR, in the absence of exogenous DNA-damaging agents, cannot be inferred from the analysis of a single factor, e.g. γH2AX foci. DDR foci showed a higher level of heterogeneity in comparison to ionizing radiation induced foci, which show marked colocalization of 53BP1 and γH2AX. The comparison of γH2AX- and 53BP1-foci cell-cycle kinetics showed distinct profiles. In contrast with γH2AX-foci, no S-phase-induced increase in 53BP1-foci was detected, but we observed a strong accumulation of 53BP1-foci with specific properties (i.e., limited number, large average size, and high mean intensity) in G1. A detailed statistical analysis of the γH2AX- and 53BP1-foci as independent statistical entities showed the presence of distinct subpopulations as γH2AX accumulation was not necessarily accompanied by 53BP1 recruitment. This may be due to a temporally distinct recruitment of the two factors or to selective activation of a specific pathway restricted to γH2AX. Indeed, mitotic regulation of DDR signaling is reported limited to γH2AX accumulation with no 53BP1 binding to condensed chromosomes (50).
The activation of multiple repair modalities and the interplay with cell-cycle regulation mechanisms may justify the heterogeneity we observed by PLA in the content and protein interaction profile. 53BP1 protein is a key element in the NHEJ repair pathway, which is suppressed during DNA replication (51). However, the cell-cycle profile of γH2AX-53BP1 proximity showed a consistent number of PLA-foci suggesting the active participation of a fraction of 53BP1 molecules in DDR signaling during the S-phase. PLA of p53 provided a further confirmation of the complexity of the DDR network, which generates heterogeneous phenotypes in the γH2AX and 53BP1 response. Our data support p53 involvement in the replication-stress response in physiological conditions, up to now only measured under drug-induced replication-fork stalling (52). In fact, by PLA, we detected both p53 spatial proximity to γH2AX and 53BP1 foci and maintenance of high levels of p53 in a fraction of replicating cells. However, we observed a consistently higher number of p53-53BP1-foci than p53-γH2AX foci. This difference suggests that the interaction between p53 and 53BP1 may involve other pathway than the DDR (e.g., transcriptional modulation) and can extend to the whole cell-cycle. Moreover, measurement of the p21 content depicted a similar heterogeneous scenario evidencing both a p53-dependent role in the activation of the checkpoint and a damage independent function. Only a fraction of the cells with high p21 expression showed a simultaneous increase in γH2AX- and/or 53BP1-foci. p21 high-expressing cells not enriched in DDR foci could be the result of either p21 DNA repair activity or a p21, DDR-independent, function in cell cycle control. Strikingly, high levels of p21 were detected in a rare diploid cell-population not expressing the proliferation marker KI67, in the absence of detectable DDR activation. In this G0 subfraction, we also measured high levels of the p63 stem-cell marker, suggesting that p21 is required in these cells for the maintenance of quiescence and self-renewing capacity. Unexpectedly, as it has never been reported before, they also showed an increase in the average level of 53BP1. Since we did not detect any significant foci-accumulation, the higher concentration of 53BP1 in this quiescent population might be representative of a function of this protein in the maintenance of a stem-cell compartment and deserves further investigation.
In conclusion, the application of our high-content, high-resolution, automated Image Cytometry protocol allows the identification of rare cell subpopulations, such as stem cells, and can provide an invaluable quantitative tool for cell biology studies. Importantly, biological heterogeneity can be analyzed without the introduction of exogenous agents, thus avoiding any potential interference with the selected molecular targets. The intrinsic high-content potential of this technique might also be used to create an unprecedented data-reservoir for in silico system biology.