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To the Editor:

Many subcellular phenomena occur in discrete spatial regions of the nucleus or cytoplasm. Nuclear processes like DNA replication, transcription, or splicing take place in such small regions defined as foci or microfoci. Using the methods of modern optical microscopy these processes can be visualized in 3D space and time. Numerous labeling techniques are available for visualization of various cellular components and processes that occur in these structures. As new microscopy methods are being developed, resolution of images improves and currently several approaches make it possible to record images with resolution exceeding the Abbe limit. An ability to image subcellular microfoci in 3D space, and often also in time, using techniques of tagging with fluorescent proteins that report time-related events opens new avenues of investigation of cellular processes. The article by Bernaś et al. [1] provides a novel protocol allowing correct spatiotemporal observation of events in close proximity, as well as the ability to judge their association.

DNA damage response (DDR) is the primary guardian of our genetic stability, good health, and longevity. It is a set of events initiated by insults causing loss of DNA-integrity that ultimately leads (when possible) to full restoration of the damaged DNA fragment. DDR is initiated primarily by the post-translational modification of several proteins, which are directly or indirectly playing important roles in different signaling pathways, not only DNA repair, but also cell cycle progression, cell death, and cell senescence [2]. While the primary response is mostly of protein modification type (mainly phosphorylations and poly-ADP-ribosylations), modulation of transcription and translation also occurs, especially in later phases of the response [3].

When two nuclear processes such as DNA damage and replication are causally related, the microfoci corresponding to these events are expected to overlap. The causal relationship may be strong or weak, i.e. there may be a one-to-one relationship between both types of foci, or—more often—just a weak tendency or a preference, which depends on various modifying conditions. How should one measure the strengths, likelihood, or temporal dependence of such causal relationships? There is a need to put metrics on spatial relationships between foci representing the phenomena of both classes. This means, in practice, measuring the number of foci that are overlapping, closely spaced, and located far from each other, in each cell, and repeating this process in many cells. The report by Bernaś et al. [1] describes a combined cytometric approach to this problem. It employs image analysis of confocal images, and combines these data with information derived from laser scanning cytometry.

In most instances, cancer cells proliferate with much higher frequency as compared to their surrounding tissues. This feature of cancer cells is explored, among others, by a set of drugs called topoisomerase inhibitors (i.e., camptothecin, etoposide). Those drugs freeze the action of topoisomerases (the DNA-unwinding enzymes) causing formation of a “cleavable complex,” thus leading to the generation of double stranded breaks in proliferating cells [2, 4]. Thus, especially when topoisomerase-inhibitors are used, the two nuclear processes, DNA replication and DNA-damage, are spatially, temporary, and casually related to each other, while taking place in small regions (foci or microfoci). Depending on the insult causing DNA-damage, the relationship (the distance) between them could be either strong or weak. This highly depends from the type of conditions that cause—or favor—DNA damage, like the type of physical (UV, X-rays, gamma-irradiation), chemical insults (various carcinogens), or natural propensities of DNA at certain regions [5, 6].

In the article by Bernaś et al. [1], the authors have measured a number of foci that are overlapping, closely spaced, and located far from each other in a cell. Theoretically, counting the distance between foci looks easy. But in reality, it is complicated because of its structural dissimilarity. We could even find from their results that foci do not represent perfect spheres always and do not overlap, ideally. The researchers have come across difficulties like this while counting the foci in self-clustered regions, especially in S phase and during random distribution which provides distance value zero, when both the foci are independent from each other. To avoid all these above errors, it is necessary to set proper scanning conditions on the laser scanning cytometer.

The other issue that should be considered while scanning is the application of “watershedding” function. Upon application of this algorithm, the amount of foci may increase by 20–30% in a cell, under some experimental circumstances. Hence, the chances of increased overlapping between foci, which might be actually not related to each other, are increased.

In the discussed study here [1], the strong correlation between DNA damage and DNA replication are most evident in late S phase after treatment with camptothecin. On the other hand, in cells treated with H2O2, result in low correlation of microfoci stained for γH2AX (DNA-damage) and DNA replication (EdU signal). Thus, the experimental conditions may affect the number of foci and the association score. These results are consistent with the previous study, which also have shown that H2O2 treatment-triggered DNA damage does not correlate with the DNA replicating sites [7].

The analysis of spatial relationship between microfoci of two or more classes within individual cells in a quantitative manner using 3D optical microscopy as described in the companion paper [7] has also been adopted in the current study. The high-resolution 3D images of cell nuclei provide a lot of information about the spatial relations among foci. Ideally, the data should be verified by other methods that could quickly provide information about large numbers of cells (i.e., automated microscopy, or image-capturing flow cytometry of a sufficient resolution) [8].

In conclusion, this wide-reaching approach opens the way to investigation of two or more classes of cellular processes that are represented by microfoci. The article by Bernaś et al. provides an example of an investigation for two cellular processes of which detection signatures (microfoci) may be located in close proximity, yet not necessarily associated with each other. To visualize the technology, the authors simultaneously analyzed DNA damage signaling triggered by known double-strand breaks inducers, camptothecin, or oxidative stress, as well as DNA-synthesis (campthotectin may cause damage close to DNA-synthesis foci, whereas oxidative stress would act more randomly). The proposed approach is, however, potentially useful in analyzing any subcellular processes or structures that appear in images as microfoci representing endosomes, FISH signals, individual receptors on plasma membrane and many others. It is anticipated that this method of analysis may also become useful in analyzing images recorded using modern high-resolution techniques like PALM, STORM, SIM, or STED. Hence, the study by Bernaś et al. discussed here offers a new, brilliant approach of developing a quantitative assessment method for amount of correlation between these two patterns that could serve as an important tool to analyze any subcellular process that upon staining, could be visualized as microfoci.

  • Mayur V. Jain

  • Division of Cell Biology

  • Department Clinical and Experimental Medicine (IKE)

  • Linköping University, Sweden

  • Integrative Regenerative Medicine Center (IGEN)

  • Linköping University, Sweden

  • Marek J. Łos*

  • Division of Cell Biology

  • Department Clinical and Experimental Medicine (IKE)

  • Linköping University, Sweden

  • Integrative Regenerative Medicine Center (IGEN)

  • Linköping University, Sweden

  • BioApplications Ent.

  • Winnipeg, MB, Canada

  • Department of Pathomorphology

  • Pomeranian Medical University

  • Szczecin, Poland

Literature Cited

  1. Top of page
  2. Literature Cited
  • 1
    Bernas T, Berniak K, Rybak P, Zarebski M, Zhao H, Darzynkiewicz Z, Dobrucki JW. Analysis of spatial correlations between patterns of DNA damage response and DNA replication in nuclei of cells subjected to replication stress or oxidative damage. Cytometry A 2013;83A:925932.
  • 2
    Kastan MB. DNA damage responses: Mechanisms and roles in human disease: 2007 G.H.A. Clowes Memorial Award Lecture. Mol Cancer Res 2008;6:517524.
  • 3
    Los M, Mozoluk M, Ferrari D, Stepczynska A, Stroh C, Renz A, Herceg Z, Wang ZQ, Schulze-Osthoff K. Activation and caspase-mediated inhibition of PARP: A molecular switch between fibroblast necrosis and apoptosis in death receptor signaling. Mol Biol Cell 2002;13:978988.
  • 4
    Zhao H, Rybak P, Dobrucki J, Traganos F, Darzynkiewicz Z. Relationship of DNA damage signaling to DNA replication following treatment with DNA topoisomerase inhibitors camptothecin/topotecan, mitoxantrone, or etoposide. Cytometry A 2012;81A:4551.
  • 5
    Los M. New, exciting developments in experimental therapies in the early 21st century. Eur J Pharmacol 2009;625:15.
  • 6
    Wiechec E. Implications of genomic instability in the diagnosis and treatment of breast cancer. Expert Rev Mol Diagn 2011;11:445453.
  • 7
    Berniak K, Rybak P, Bernas T, Zarebski M, Biela E, Zhao H, Darzynkiewicz Z, Dobrucki JW. Relationship between DNA damage response, initiated by camptothecin or oxidative stress, and DNA replication, analyzed by quantitative 3D image analysis. Cytometry A 2013;83A:913924.
  • 8
    Furia L, Pelicci PG, Faretta M. A computational platform for robotized fluorescence microscopy (I): High-content image-based cell-cycle analysis. Cytometry A 2013;83A:333343.