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Keywords:

  • flow cytometry;
  • epigenetics;
  • histones;
  • biomarkers

Commentary

  1. Top of page
  2. Commentary
  3. Literature Cited

Recent advances in the field of cancer epigenetics (heritable changes in gene expression that occur without alteration in DNA sequence) with the increased understanding of the mechanisms of epigenetic silencing of tumor suppressor genes and activation of oncogenes—island methylation patterns, histone modifications, and dysregulation of DNA binding proteins―present great promise for future identification of novel biomarkers of application to cancer detection, prognosis, and therapy response [1, 2]. In particular, histones are essential for chromatin stability. They undergo many modifications, which seem to play important roles in transcriptional regulation and therefore potentially oncogenic if, for example, deregulation leads to the loss of expression of a tumor suppressor gene. Indeed, anomalous patterns of histone modifications are recognized as a hallmark of cancer [3]. Histone H3 phosphorylation at serine 10 (and serine 28) occurs at mitosis and the Aurora Kinases that perform this H3 phosphorylation are frequently implicated in cancer [4]. Similarly, signatures of histone modifications patterns, such as trimethyl-k9H3, are associated with patient prognosis in acute myeloid leukemia (AML) [5].

Epigenetic biomarkers can be detected in tissue samples as well as in biofluids. Chromatin immunoprecipitation (ChIP) using antibodies raised against individual histone marks represents a useful tool for identification of epigenetic biomarkers in basic research and highly specific ChIP grade antibodies are readily available [6]. However, this technology does not allow the characterization of epigenetic patterns in heterogeneous clinical samples. Multiparameter flow cytometry, in contrast, allows one to evaluate the expression of markers of interest on a per cell bases. As early as the late 1990s, we developed flow cytometric techniques to identify modifications in cells undergoing mitosis (H3S10 phosphorylation) that have been proven useful not only to identify mitotic cells, but also to study molecular mechanisms associated with the G2 to M transition and as a tool to screen in vivo Aurora Kinase inhibitors [7, 8]. However, and as noted in the work by Watson et al. (page xxx) in the present issue of Cytometry A, condensed chromatin may negatively affect accessibility of antibodies to histone modifications involved in epigenetic regulation. Initial attempts to detect histone marks by flow cytometry were done in cell lines by Obier et al. [9]. The authors in the present report extended this work by utilizing antibodies available for ChIP and adapting a protocol that allowed simultaneous measurement of surface immune phenotype, light scatter, and intracellular antigens for normal and leukemic blood evaluation [10]. Increased trimethylation of histone H3 Lysine 27 (H3K27me3) has also been associated with tumorigenesis since can silence tumor suppressor genes despite the drop in methylation of the gene's CpG island (an event that normally activates genes) [11]. Interestingly, this is one of the clinically relevant histone marks tested in the Watson's paper. As presented, the overall MFIs of the leukemic blast samples compared to any of the normal blood cell population seems to indicate increased trimethylation in at least some of the AML samples despite the clear heterogeneity observed.

Although, further refinements in the fixation/staining protocol may be needed, this study represents a genuinely novel approach to cancer epigenetics using flow cytometry as the analytical tool. One can only expect an increased number of additional antibodies and applications in the future.

The innovative work by Watson et al. in this issue of Cytometry A describes an approach to epigenetics that allows for the study of heterogeneous cell populations on a cell-by-cell bases. The authors took advantage of antibodies developed for ChIP to detect a number of histone marks by flow cytometry and several fixation and staining protocols for both cell lines and blood samples to finally apply them to basic research and clinical applications. The study very nicely outlines the different steps required to develop such assays, describing an original flow cytometry assay that was finally applied to leukemia patient samples (AML). Although, the authors acknowledge the lack of correlation between the histone marks and the leukemic subtype, due to the small dataset, the clear variability in MFIs for at least one of the markers (H3K27me3) in the leukemia samples may become an important stratification marker for therapeutic intervention. In short, the study represents a novel application of flow cytometry, emphasizing the advantages of the platform with great potential in biomedical research.

Literature Cited

  1. Top of page
  2. Commentary
  3. Literature Cited
  • 1
    Sandoval J, Esteller M. Cancer epigenomics: Beyond genomics. Curr Opin Genet Dev 2012;22:5055.
  • 2
    Verma M. Epigenetic biomarkers in cancer epidemiology. Methods Mol Biol 2012;863:467480.
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    Fraga MF, Ballestar E, Villar-Garea A, Boix-Chornet M, Espada J, Schotta G, Bonaldi T, Haydon C, Ropero S, Petrie K, et al. Loss of acetylation at Lys16 and trimethylation at Lys20 of histone H4 is a common hallmark of human cancer. Nat Genet 2005;37:391340.
  • 4
    Zhang K, Dent SY. Histone modifying enzymes and cancer: going beyond histones. J Cell Biochem 2005;96:11371144.
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    Müller-Tidow C, Klein H-U, Hascher A, Isken F, Tickenbrock L, Thoennissen N, Agrawal-Singh S, Tschanter P, Disselhoff C, Wang Y, et al. Profiling of histone H3 lysine 9 trimethylation levels predicts transcription factor activity and survival in acute myeloid leukemia. Blood 2010;116:35643567.
  • 6
    Nady N, Min J, Kareta MS, Chedin F, Arrowsmith CH. A SPOT on the chromatin landscape? Histone peptide arrays as a tool for epigenetic research. Trends Biochem Sci 2008;33:73057313.
  • 7
    Juan G, Traganos F, James WM, Ray JM, Roberge M, Sauve DM, Anderson H, Darzynkiewicz Z. Histone H3 phosphorylation and expression of cyclins A and B1 measured in individual cells during their progression through G2 and mitosis. Cytometry 1998;32:18.
  • 8
    Payton M, Bush TL, Chung G, Ziegler B, Eden P, McElroy P, Ross S, Cee VJ, Deak HL, Hodous BL, et al. Preclinical Evaluation of AMG 900, a Novel Potent and Highly Selective Pan-Aurora Kinase Inhibitor with Activity in Taxane-Resistant Tumor Cell Lines. Cancer Res 2010;70:98469854.
  • 9
    Obier N, Muller AM. Chromatin flow cytometry identifies changes in epigenetic cell states. Cells Tissues Organs 2010;191:167174.
  • 10
    Chow S, Hedley D, Grom P, Magari R, Jacobberger JW, Shankey TV. Whole blood fixation and permeabilization protocol with red blood cell lysis for flow cytometry of intracellular phosphorylated epitopes in leukocyte subpopulations. Cytometry A 2005;67A:417.
  • 11
    Viré E, Brenner C, Deplus R, Blanchon L, Fraga M, Didelot C, Morey L, Van Eynde A, Bernard D, Vanderwinden J-M, et al. The polycomb group protein EZH2 directly controls DNA methylation. Nature 2006;439:871874.