Christiaan H. Righolt

Dynamic chromosomal rearrangements in Hodgkin’s lymphoma are due to ongoing three-dimensional nuclear remodeling and breakage-bridge-fusion cycles

Background Hodgkin’s lymphoma is characterized by the presence of mono-nucleated Hodgkin cells and bi- to multi-nucleated Reed-Sternberg cells. We have recently shown telomere dysfunction and aberrant synchronous/asynchronous cell divisions during the transition of Hodgkin cells to Reed-Sternberg cells.1 Design and Methods To determine whether overall changes in nuclear architecture affect genomic instability during the transition of Hodgkin cells to Reed-Sternberg cells, we investigated the nuclear organization of chromosomes in these cells. Results Three-dimensional fluorescent in situ hybridization revealed irregular nuclear positioning of individual chromosomes in Hodgkin cells and, more so, in Reed-Sternberg cells. We characterized an increasingly unequal distribution of chromosomes as mono-nucleated cells became multi-nucleated cells, some of which also contained chromosome-poor ‘ghost’ cell nuclei. Measurements of nuclear chromosome positions suggested chromosome overlaps in both types of cells. Spectral karyotyping then revealed both aneuploidy and complex chromosomal rearrangements: multiple breakage-bridge-fusion cycles were at the origin of the multiple rearranged chromosomes. This conclusion was challenged by super resolution three-dimensional structured illumination imaging of Hodgkin and Reed-Sternberg nuclei. Three-dimensional super resolution microscopy data documented inter-nuclear DNA bridges in multi-nucleated cells but not in mono-nucleated cells. These bridges consisted of chromatids and chromosomes shared by two Reed-Sternberg nuclei. The complexity of chromosomal rearrangements increased as Hodgkin cells developed into multi-nucleated cells, thus indicating tumor progression and evolution in Hodgkin’s lymphoma, with Reed-Sternberg cells representing the highest complexity in chromosomal rearrangements in this disease. Conclusions This is the first study to demonstrate nuclear remodeling and associated genomic instability leading to the generation of Reed-Sternberg cells of Hodgkin’s lymphoma. We defined nuclear remodeling as a key feature of Hodgkin’s lymphoma, highlighting the relevance of nuclear architecture in cancer.

Shattered and stitched chromosomes-chromothripsis and chromoanasynthesis-manifestations of a new chromosome crisis?

Chromothripsis (chromosome shattering) has been described as complex rearrangements affecting single chromosome(s) in one catastrophic event. The chromosomes would be “shattered” and “stitched together” during this event. This phenomenon is proposed to constitute the basis for complex chromosomal rearrangements seen in 2‐3% of all cancers and in ∼ 25% of bone cancers. Here we discuss chromothripsis, the use of this term and the evidence presented to support a single catastrophic event that remodels the genome in one step. We discuss why care should be taken in using the term chromothripsis and what evidence is lacking to support its use while describing complex rearrangements.

Nuclear remodeling as a mechanism for genomic instability in cancer.

This chapter focuses on the three-dimensional organization of the nucleus in normal, early genomically unstable, and tumor cells. A cause-consequence relationship is discussed between nuclear alterations and the resulting genomic rearrangements. Examples are presented from studies on conditional Myc deregulation, experimental tumorigenesis in mouse plasmacytoma, nuclear remodeling in Hodgkins lymphoma, and in adult glioblastoma. A model of nuclear remodeling is proposed for cancer progression in multiple myeloma. Current models of nuclear remodeling are described, including our model of altered nuclear architecture and the onset of genomic instability.

Three-Dimensional Quantitative Imaging of Telomeres in Buccal Cells Identifies Mild, Moderate, and Severe Alzheimer's Disease Patients

Using three-dimensional (3D) telomeric analysis of buccal cells of 82 Alzheimers disease (AD) patients and cognitively normal age and gender-matched controls, we have for the first time examined changes in the 3D nuclear telomeric architecture of buccal cells among levels of AD severity based on five 3D parameters: i) telomere length, ii) telomere number, iii) telomere aggregation, iv) nuclear volume, and v) a/c ratio, a measure of spatial telomere distribution. Our data indicate that matched controls have significantly different 3D telomere profiles compared to mild, moderate, and severe AD patients (p < 0.0001). Distinct profiles were also evident for each AD severity group. An increase in telomere number and aggregation concomitant with a decrease in telomere length from normal to severe AD defines the individual stages of the disease (p < 0.0001).

Genomic Instability: The Driving Force behind Refractory/Relapsing Hodgkin's Lymphoma

In classical Hodgkin’s lymphoma (HL) the malignant mononuclear Hodgkin (H) and multinuclear, diagnostic Reed-Sternberg (RS) cells are rare and generally make up <3% of the total cellular mass of the affected lymph nodes. During recent years, the introduction of laser micro-dissection techniques at the single cell level has substantially improved our understanding of the molecular pathogenesis of HL. Gene expression profiling, comparative genomic hybridization analysis, micro-RNA expression profiling and viral oncogene sequencing have deepened our knowledge of numerous facets of H- and RS-cell gene expression deregulation. The question remains whether disturbed signaling pathways and deregulated transcription factors are at the origin of refractory/relapsing Hodgkin’s lymphoma or whether these hallmarks are at least partially related to another major factor. We recently showed that the 3D nuclear organization of telomeres and chromosomes marked the transition from H- to RS-cells in HL cell lines. This transition is associated with progression of telomere dysfunction, shelterin disruption and progression of complex chromosomal rearrangements. We reported analogous findings in refractory/relapsing HL and identified the shelterin proteins TRF1, TRF2 and POT1 as targets of the LMP1 oncogene in post-germinal center B-cells. Here we summarize our findings, including data not previously published, and propose a model in which progressive disruption of nuclear integrity, a form of genomic instability, is the key-player in refractory/relapsing HL. Therapeutic approaches should take these findings into account.

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

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.

Image filtering in structured illumination microscopy using the Lukosz bound

Various aspects of image filtering affect the final image quality in Structured Illumination Microscopy, in particular the regularization parameter and type of regularization function, the relative height of the side bands, and the shape of the apodization function. We propose an apodization filter without adjustable parameters based on the application of the Lukosz bound in order to guarantee a non-negative point spread function. Simulations of digital resolution charts and experimental data of chromatin structures and of actin filaments show artefact free reconstructions for a wide range of filter parameters. In general, a trade-off is observed between sharpness and noise suppression.

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

Recent developments in microscopy have led to superresolution microscopy images of cells. Structured illumination microscopy was used before to reveal new details in the DNA structure and the structure of the DNA‐free space in the DAPI‐stained cell nuclei of the Hodgkins lymphoma HDLM‐2 cell line. This study extends this technology to primary pre‐treatment classical Hodgkins lymphoma samples of ten patients. Significant differences in both the DNA structure and the structure of the DNA‐free space were detected between lymphocytes and malignant cells. Both types of structures were similar for lymphocytes of different patients. When the patients were un‐blinded and grouped based on their clinical outcome, either non‐relapsed or relapsed, a significant difference in the DNA structure of their Reed–Sternberg (RS) cells was found. Since, RS cells develop from mono–nucleated Hodgkin (H) cells, these data suggest distinct architectural restructuring of nuclei during RS cell formation in patients going to long‐lasting remission versus relapse. J. Cell. Biochem. 117: 1633–1637, 2016.

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

The mammalian nucleus has a distinct substructure that cannot be visualized directly by conventional microscopy. In this study, the organization of the DNA within the nucleus of multiple myeloma (MM) cells, their precursor cells (monoclonal gammopathy of undetermined significance; MGUS) and control lymphocytes of the representative patients is visualized and quantified by superresolution microscopy. Three‐dimensional structured illumination microscopy (3D‐SIM) increases the spatial resolution beyond the limits of conventional widefield fluorescence microscopy. 3D‐SIM reveals new insights into the nuclear architecture of cancer as we show for the first time that it resolves organizational differences in intranuclear DNA organization of myeloma cells in MGUS and in MM patients. In addition, we report a significant increase in nuclear submicron DNA structure and structure of the DNA‐free space in myeloma nuclei compared to normal lymphocyte nuclei. Our study provides previously unknown details of the nanoscopic DNA architecture of interphase nuclei of the normal lymphocytes, MGUS and MM cells. This study opens new avenues to understanding the disease progression from MGUS to MM. J. Cell. Biochem. 116: 704–710, 2015.

Molecular Image Analysis: Quantitative Description and Classification of the Nuclear Lamina in Human Mesenchymal Stem Cells

The nuclear lamina is an intermediate filament network that provides a structural framework for the cell nucleus. Changes in lamina structure are found during changes in cell fate such as cell division or cell death and are associated with human diseases. An unbiased method that quantifies changes in lamina shape can provide information on cells undergoing changes in cellular functions. We have developed an image processing methodology that finds and quantifies the 3D structure of the nuclear lamina. We show that measurements on such images can be used for cell classification and provide information concerning protein spatial localization in this structure. To demonstrate the efficacy of this method, we compared the lamina of unmanipulated human mesenchymal stem cells (hMSCs) at passage 4 to cells activated for apoptosis. A statistically significant classification was found between the two populations.