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.

Three-dimensional Nuclear Telomere Organization in Multiple Myeloma.

Multiple myeloma (MM) is preceded by monoclonal gammopathy of undetermined significance (MGUS). Up to date, it is difficult to predict an individual's time to disease progression and the treatment response. To examine whether the nuclear telomeric architecture will unravel some of these questions, we carried out. Three-dimensional (3D) telomere analysis on samples from patients diagnosed with MGUS and MM, as well as from patients who went into relapse. Telomere signal intensity, number of telomere aggregates, nuclear volume, and the overall nuclear telomere distribution (a/c ratio) were analyzed. The telomeric profiles allowed for the differentiation of the disease stages. The telomeric profiles of myeloma cells obtained from blood and bone marrow aspirates were identical. Based on this study, we discuss the use of 3D telomere profiling as a potential future tool for risk stratification and personalized treatment decisions.

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.

Telomere-centromere-driven genomic instability contributes to karyotype evolution in a mouse model of melanoma.

Aneuploidy and chromosomal instability (CIN) are hallmarks of most solid tumors. These alterations may result from inaccurate chromosomal segregation during mitosis, which can occur through several mechanisms including defective telomere metabolism, centrosome amplification, dysfunctional centromeres, and/or defective spindle checkpoint control. In this work, we used an in vitro murine melanoma model that uses a cellular adhesion blockade as a transforming factor to characterize telomeric and centromeric alterations that accompany melanocyte transformation. To study the timing of the occurrence of telomere shortening in this transformation model, we analyzed the profile of telomere length by quantitative fluorescent in situ hybridization and found that telomere length significantly decreased as additional rounds of cell adhesion blockages were performed. Together with it, an increase in telomere-free ends and complex karyotypic aberrations were also found, which include Robertsonian fusions in 100% of metaphases of the metastatic melanoma cells. These findings are in agreement with the idea that telomere length abnormalities seem to be one of the earliest genetic alterations acquired in the multistep process of malignant transformation and that telomere abnormalities result in telomere aggregation, breakage-bridge-fusion cycles, and CIN. Another remarkable feature of this model is the abundance of centromeric instability manifested as centromere fragments and centromeric fusions. Taken together, our results illustrate for this melanoma model CIN with a structural signature of centromere breakage and telomeric loss.

Centromeres in cell division, evolution, nuclear organization and disease.

As the spindle fiber attachment region of the chromosome, the centromere has been investigated in a variety of contexts. Here, we will review current knowledge about this unique chromosomal region and its relevance for proper cell division, speciation, and disease. Understanding the three-dimensional organization of centromeres in normal and tumor cells is just beginning to emerge. Multidisciplinary research will allow for new insights into its normal and aberrant nuclear organization and may allow for new therapeutic interventions that target events linked to centromere function and cell division.

c-Myc-dependent formation of Robertsonian translocation chromosomes in mouse cells.

Robertsonian (Rb) translocation chromosomes occur in human and murine cancers and involve the aberrant joining of two acrocentric chromosomes in humans and two telocentric chromosomes in mice. Mechanisms leading to their generation remain elusive, but models for their formation have been proposed. They include breakage of centromeric sequences and their subsequent fusions, centric misdivision, misparing between highly repetitive sequences of p-tel or p-arm repeats, and recombinational joining of centromeres and/or centromeric fusions. Here, we have investigated the role of the oncoprotein c-Myc in the formation of Rb chromosomes in mouse cells harboring exclusively telocentric chromosomes. In mouse plasmacytoma cells with constitutive c-Myc deregulation and in immortalized mouse lymphocytes with conditional c-Myc expression, we show that positional remodeling of centromeres in interphase nuclei coincides with the formation of Rb chromosomes. Furthermore, we demonstrate that c-Myc deregulation in a myc box II-dependent manner is sufficient to induce Rb translocation chromosomes. Because telomeric signals are present at all joined centromeres of Rb chromosomes, we conclude that c-Myc mediates Rb chromosome formation in mouse cells by telomere fusions at centromeric termini of telocentric chromosomes. Our findings are relevant to the understanding of nuclear chromosome remodeling during the initiation of genomic instability and tumorigenesis.

Alterations of centromere positions in nuclei of immortalized and malignant mouse lymphocytes.

BACKGROUND: The three-dimensional (3D) positions of centromeres have been studied in several cell systems. However, data on centromere positions during cellular transformation remain elusive. This study has focused on mouse lymphocytes and investigated the centromere positions in primary, immortalized, and tumor cells. METHODS: Eighty-to-ninety z-slices of each mouse lymphocyte nucleus were acquired using a sampling distance of 107 nm in the xy plane and 200 nm along z for each z-stack, using an Axioplan 2 microscope, an AxioCam HR CCD, a 63x/1.4 oil objective, and the Axiovision 3.1 software (Carl Zeiss, Canada). A constrained iterative algorithm (Schaefer et al., J Microsc 2001;204:99-107) was used for deconvolution. Centromere positions in 3D images were analyzed using CentroView, a program we developed to measure nuclear centromere positions. RESULTS: Using CentroView we determined the positions of centromeres in primary lymphocytes, immortalized and malignant mouse B cells. We show that centromeres exhibit altered nuclear positions in immortalized and malignant B cells. These changes are independent of previously described cell cycle-dependent centromere dynamics. CONCLUSIONS: The 3D positions of centromeres are altered during cellular transformation. In lymphocytes, centromeres are found in more central nuclear positions following immortalization and transformation. These nuclear changes reflect structural remodeling of mammalian nuclei during oncogenesis and may impact on the structural organization of chromosomes. How centromeric changes are linked to nuclear remodeling can now be quantitatively examined using the tools of this study.

c-Myc induces chromosomal rearrangements through telomere and chromosome remodeling in the interphase nucleus.

In previous work, we showed that telomeres of normal cells are organized within the 3D space of the interphase nucleus in a nonoverlapping and cell cycle-dependent manner. This order is distorted in tumor cell nuclei where telomeres are found in close association forming aggregates of various numbers and sizes. Here we show that c-Myc overexpression induces telomeric aggregations in the interphase nucleus. Directly proportional to the duration of c-Myc deregulation, we observe three or five cycles of telomeric aggregate formation in interphase nuclei. These cycles reflect the onset and propagation of breakage-bridge-fusion cycles that are initiated by end-to-end telomeric fusions of chromosomes. Subsequent to initial chromosomal breakages, new fusions follow and the breakage-bridge-fusion cycles continue. During this time, nonreciprocal translocations are generated. c-Myc-dependent remodeling of the organization of telomeres thus precedes the onset of genomic instability and subsequently leads to chromosomal rearrangements. Our findings reveal that c-Myc possesses the ability to structurally modify chromosomes through telomeric fusions, thereby reorganizing the genetic information.

Uncoupling of genomic instability and tumorigenesis in a mouse model of Burkitt's lymphoma expressing a conditional box II-deleted Myc protein.

Burkitt's lymphomas (BL) are characterized by the constitutive expression of c-Myc protein. In total, 50-60% of all BL cells carry mutant c-Myc proteins. Using a mouse model of spontaneously immortalized pro-B-lymphocytes (Ba/F3), we have investigated genomic instability mediated by the conditional expression of either wild-type (WT) or deletion box II Delta106-Myc proteins. We found that both proteins mediate common as well as differing types of chromosomal rearrangements as documented by spectral karyotyping (SKY). A higher level of genomic instability is induced by the Delta106-Myc protein. To examine the tumorigenic potential of WT or Delta106-driven Ba/F3 cells, in vivo tumorigenesis studies were performed in SCID mice. Under the experimental conditions of this study, WT but not Delta106-Myc expressing Ba/F3 cells triggered tumorigenesis in SCID mice. Therefore, the genomic instability phenotype induced by Delta106-Myc can be genetically uncoupled from its tumorigenic potential.