Amanda Guffei

Chromosomal rearrangements after ex vivo Epstein-Barr virus (EBV) infection of human B cells

The Epstein–Barr virus (EBV) is carried by more than 90% of the adult world population and has been implicated in several human malignancies. Its ability to induce unlimited in vitro proliferation of B cells is frequently used to generate lymphoblastoid cell lines (LCLs). In this study, we have investigated the evolution of two LCLs up to 25 weeks after EBV infection. LCLs were karyotyped once a month by spectral karyotyping (SKY). LCLs but not mitogen-activated B cells showed evidence of DNA damage and DNA damage response within the first 2 weeks. After 4 weeks, the former, but not the latter, showed a high level of non-clonal structural aberrations, mainly deletions, fragments, dicentric chromosomes and unbalanced translocations. Genomic instability decreased thereafter over time. Nonrandom aneuploidy 12 weeks after infection showed clonal evolution in culture. After 25 weeks post-infection, most cells exhibited karyotypic stability. Chromosomal aberrations were compatible with telomere dysfunction, although in the absence of telomere shortening. The telomere capping protein TRF2 was partially displaced from telomeres in EBV-infected cells, suggesting an EBV-mediated uncapping problem. In conclusion, this study suggests that DNA damage and telomere dysfunction contribute to EBV-related chromosomal instability in early LCLs.

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.

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

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.

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. J. Cell. Biochem. 104: 2040–2058, 2008.

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

Burkitts 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 Δ106-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 Δ106-Myc protein. To examine the tumorigenic potential of WT or Δ106-driven Ba/F3 cells, in vivo tumorigenesis studies were performed in SCID mice. Under the experimental conditions of this study, WT but not Δ106-Myc expressing Ba/F3 cells triggered tumorigenesis in SCID mice. Therefore, the genomic instability phenotype induced by Δ106-Myc can be genetically uncoupled from its tumorigenic potential.

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.

c-Myc Deregulation Promotes a Complex Network of Genomic Instability

c-Myc is a versatile protein that has many different functions. With respect to genomic instability, it affects many endpoints that include gene amplification, translocation, deletions, insertions, long-range rearrangements, DNA breakage, and point mutations. This overall promotion of genomic instability suggests that c-Myc may have a central role in destabilizing the genome. We propose that c-Myc is a structural modifier of the genome and an important initiator and progressor molecule in neoplastic transformation.