Differences in Nuclear DNA Organization Between Lymphocytes, Hodgkin and Reed–Sternberg Cells Revealed by Structured Illumination Microscopy

Abstract

Advances in light microscopy have enabled the visualization of DNA in the interphase nucleus with more detail than is visible with conventional light microscopy. The nuclear architecture is assumed to be different in cancer cells compared to normal cells. In this paper we have studied, for the first time, the organization of nuclear DNA and that of DNA-free space in control lymphocytes, Hodgkin cells and Reed–Sternberg cells using 3D structured illumination microscopy (SIM). We have observed detail in these SIM images that was not observed in conventional widefield images. We have measured the size distribution of the DNA structure using granulometry and noted a significant, progressive increase in the amount of sub-micron structures from control lymphocytes to Hodgkin cells to Reed–Sternberg cells. The DNA-free space changes as well; “holes” in the DNA distribution start to appear in the malignant cells. We have studied whether these “holes” are nucleoli by staining for upstream binding factor (UBF), a protein associated with the nucleolus. We have found that the relative UBF content progressively and significantly decreases—or is absent—in the DNA-free space when measured as either the Pearson correlation coefficient with the DNA-free space or as the number of “holes” that contain UBF. Similar differences exist within the population of Reed–Sternberg cells between binucleated and multinucleated cells with four or more subnuclei. To our knowledge, this is the first study that investigates the changes of the nuclear DNA structure in any disease with superresolution light microscopy. J. Cell. Biochem. 115: 1441–1448, 2014. © 2014 The Authors. Journal of Cellular Biochemistry published by Wiley Periodicals, Inc. This is an open access article under the terms of the Creative Commons Attribution-NonCommercial-NoDerivs License, which permits use and distribution in any medium, provided the original work is properly cited, the use is non-commercial and no modifications or adaptations are made.

Distinct and shared three‐dimensional chromosome organization patterns in lymphocytes, monoclonal gammopathy of undetermined significance and multiple myeloma

“…Our findings of a larger size of CTs and nuclear volume in myeloma are consistent with our previous work that revealed a significant increase in the intranuclear submicron structure in myeloma interphase nuclei by using 3D‐SIM as well as increased DNA‐free/poor nuclear space compared to control lymphocyte . DNA‐free space refers to areas within the nucleus that have a low DNA density or no DNA at all …”

Quantitative Superresolution Microscopy Reveals Differences in Nuclear DNA Organization of Multiple Myeloma and Monoclonal Gammopathy of Undetermined Significance

“…). “Holes” refer to DNA poor or DNA‐free nuclear space [Righolt et al, 2014]. While most of the myeloma nuclei have the large scale “holes” within their nuclei, we have hardly detected these structures in lymphocyte nuclei.…”

“…To quantify whether there are differences between normal lymphocytes and myeloma nuclei, we have used granulometry to evaluate the size distribution of the DNA structure and the DNA‐free space [Righolt et al, 2014]. The granulometry shows the differences in nuclear size in the microns, whereas the differences in the DNA‐structure occur at submicron size.…”

“…The application of this technique is compatible with both fixed and live cells [Hirvonen et al, 2009]. Using 3D‐SIM, Righolt et al (2014) had measured the DNA organization in the interphase nuclei of Hodgkin's lymphoma and revealed a significant increase in submicron DNA structures of Hodgkin cells and Reed‐Sternberg cells compared to normal lymphocytes that clearly distinguish the three cell types from each other.…”

DNA Superresolution Structure of Reed–Sternberg Cells Differs Between Long‐Lasting Remission Versus Relapsing Hodgkin's Lymphoma Patients

“…The progressive change in internal structure from lymphocyte to H cell to RS cell that was previously described for a HL cell line [Righolt et al, ] was, however, not detected in these primary pre‐treatment Hodgkin's lymphoma samples. This might be explained by the fact that this cell line was kept in culture and the cells might no longer be fully representative of cells found in primary HL samples as the HDLM‐2 cell line was cultured from a pleural effusion of a stage IV HL patient.…”