LMP1 and Dynamic Progressive Telomere Dysfunction: A Major Culprit in EBV-Associated Hodgkin's Lymphoma

Abstract

Epstein-Barr virus (EBV)-encoded latent membrane protein 1 (LMP1) is expressed in germinal-center-derived, mononuclear Hodgkin (H) and multinuclear, diagnostic Reed-Sternberg (RS) cells in classical EBV-positive Hodgkin's lymphoma (cHL). LMP1 expression in EBV-negative H-cell lines results in a significantly increased number of RS cells. In a conditional, germinal-center-derived B-cell in vitro system, LMP1 reversibly down-regulates the shelterin proteins, telomeric repeat binding factor (TRF)1, TRF2, and protection of telomeres (POT)1. This down-regulation is associated with progressive 3D shelterin disruption, resulting in telomere dysfunction, progression of complex chromosomal rearrangements, and multinuclearity. TRF2 appears to be the key player. Thus, we hypothesize that the 3D interaction of telomeres and TRF2 is disrupted in H cells, and directly associated with the formation of H and RS cells. Using quantitative 3D co-immuno-TRF2-telomere fluorescent in situ hybridization (3D TRF2/Telo-Q-FISH) applied to monolayers of primary H and RS cells, we demonstrate TRF2-telomere dysfunction in EBV-positive cHL. However, in EBV-negative cHL a second molecular mechanism characterized by massive up-regulation of TRF2, but attrition of telomere signals, is also identified. These facts point towards a shelterin-related pathogenesis of cHL, where two molecularly disparate mechanisms converge at the level of 3D Telomere-TRF2 interactions, leading to the formation of RS cells.

Keywords: 3D TRF2/Telo-Q-FISH; EBV; Hodgkin’s lymphoma; LMP1; Reed–Sternberg cell; TRF2; shelterin; telomere.

Figure 1

Latent membrane protein 1 (LMP1) expression in BJAB-tTA-LMP1 Burkitt’s lymphoma cells is associated with multinuclearity. Original magnification 640×, Zeiss AxioImager Z1 microscope (Zeiss, Toronto, ON, Canada). (A) LMP1-suppressed transfectants at day 14 still reveal uniform Burkitt cell morphology with only rare bi-nucleated or large mononuclear cells. Immunostaining with anti-LMP1 MoAb CS1-4 confirms successful LMP1 suppression through tetracycline. (B) LMP1-expressing transfectants at day 14 contain multiple Reed–Sternberg-like giant cells. Strong LMP1 expression is confirmed with anti-LMP1 MoAb CS1-4. Only one small mononuclear cell (arrow) appears not to express LMP1. Note several LMP1-positive vesicles (exosomes) at the surface of the top two polycaria. In vivo, such vesicles may influence the tumour microenvironment [48]. Photomicrograph performed in parallel during the experiments shown in Figure 2 of Lajoie et al. [46].

Figure 2

LMP1-induced telomere dynamics of multinucleated Reed–Sternberg (RS)-like cells. (A) 3D identification of disturbed nuclear telomere organization in a tri-nuclear LMP1-expressing Reed–Sternberg-like BJAB-tTA-LMP1 cell (upper left). Three-dimensional reconstruction of nuclear DNA (DAPI, blue) in surface mode reveals three nuclei (1–3). Three-dimensional telomere (red) reconstruction in surface mode (lower left) reveals abundant, irregularly distributed telomeres and two aggregates (asterix). Three-dimensional telomere identification in surface mode (right) against a white background (increases contrast and enhances visibility of short telomeres) identifies a total of 409 telomeres and confirms two large aggregates (asterix). (B). Telomere distribution according to size. Results are based on 3D analysis of 30 cells for each time point. Frequency (y-axis) and relative fluorescent intensity (i.e., size of telomeres (x-axis)) reveal individual telomere profiles at day 9. Telomeres with a relative fluorescent intensity (x-axis) ranging from 0 to 5000 arbitrary fluorescent units are classified as very short, with an intensity ranging from 5000 to 15,000 units classified as short, an intensity from 15,000 to 30,000 units classified as mid-sized, and an intensity >30,000 units classified as large [47]. LMP1 expression induces multinucleated RS-like cells with abundant very short and short telomeres already at day 9 when compared to LMP1-suppressed cells. Photomicrograph (A) and telomere plot (B) performed in parallel during the experiment shown in Figure 3 and Figure 4 of Lajoie et al. [46].

Figure 2

LMP1-induced telomere dynamics of multinucleated Reed–Sternberg (RS)-like cells. (A) 3D identification of disturbed nuclear telomere organization in a tri-nuclear LMP1-expressing Reed–Sternberg-like BJAB-tTA-LMP1 cell (upper left). Three-dimensional reconstruction of nuclear DNA (DAPI, blue) in surface mode reveals three nuclei (1–3). Three-dimensional telomere (red) reconstruction in surface mode (lower left) reveals abundant, irregularly distributed telomeres and two aggregates (asterix). Three-dimensional telomere identification in surface mode (right) against a white background (increases contrast and enhances visibility of short telomeres) identifies a total of 409 telomeres and confirms two large aggregates (asterix). (B). Telomere distribution according to size. Results are based on 3D analysis of 30 cells for each time point. Frequency (y-axis) and relative fluorescent intensity (i.e., size of telomeres (x-axis)) reveal individual telomere profiles at day 9. Telomeres with a relative fluorescent intensity (x-axis) ranging from 0 to 5000 arbitrary fluorescent units are classified as very short, with an intensity ranging from 5000 to 15,000 units classified as short, an intensity from 15,000 to 30,000 units classified as mid-sized, and an intensity >30,000 units classified as large [47]. LMP1 expression induces multinucleated RS-like cells with abundant very short and short telomeres already at day 9 when compared to LMP1-suppressed cells. Photomicrograph (A) and telomere plot (B) performed in parallel during the experiment shown in Figure 3 and Figure 4 of Lajoie et al. [46].

Figure 3

Three-dimensional Telomere de-protection in Epstein–Barr virus (EBV)-associated LMP1-expressing classical Hodgkin’s lymphoma (cHL) (disruption pattern B). Progressive attrition of telomeric repeat binding factor 2 (TRF2) spots occurs in LMP1-positive cHL during the transition from mononuclear Hodgkin (H) to RS cells. (A) Complete 3D reconstitution of large mononuclear H cell and lymphocyte corona (left) with nuclear DNA (blue), telomere (red), TRF2 (green), and telomere–TRF2 overlay (orange) signals is shown in transparency mode. The H cell shows several DNA-poor spaces and only few telomere and TRF2 signals, whereas the surrounding reactive lymphocytes (1,2) contain numerous small to midsized orange signals serving as internal control for tight 1:1 association of telomere/TRF2 signals. The TRF2 signal spots of lymphocytes 1 and 2 (upper right) and telomere signals (lower right) in surface mode show identical intensity and congruent localization in benign lymphoid cells. (B) On the left, the same complete 3D nuclear reconstitution (DAPI: white for better contrast) of large mononuclear H cell and lymphocyte corona (1,2) as in A. The two arrows serve as sentinel tags for better localization of the H cell on the right panel. Three-dimensional TRF2 (upper right) and telomere (lower right) identification in surface mode against a white background increases contrast and enhances visibility of short telomeres. The large H cell shows numerous small telomere signals without associated TRF2 signals and a partial dissociation of some TRF2 signals from the telomeres. Photomicrograph performed during case analysis as shown in Figure 4 of Knecht et al. [53].

Figure 4

Substantial increase of unbound (free) TRF2 signals (disruption pattern A). Progressive shortening and loss of telomeres but increase of unbound (free) TRF2 spots occurs during the transition from H to RS cells as shown in this EBV-negative, aggressive cHL case. Complete 3D reconstitution of tri-nuclear RS cell and lymphocyte corona (upper left) with nuclear DNA (blue), telomere (red), TRF2 (green), and telomere–TRF2 overlay (orange) signals is shown in transparency mode. Mainly unbound (free) TRF2 signals are identified in the RS cell, whereas 1:1 telomere/TRF2 complexes are identified in the reactive lymphocytes. Lower left shows the same complete 3D nuclear reconstitution of the RS cell and lymphocyte corona (DAPI: white for better contrast). Arrows identify two satellite nuclei. Three-dimensional TRF2 reconstitution in surface mode (upper right) confirms numerous unbound (free) TRF2 signals in the RS cell when compared 3D telomeres (lower right). Again, the reactive lymphocytes show a tight 1:1 association of telomere/TRF2 signals. Photomicrograph performed during case analysis as shown in Figure 2 of Knecht et al. [53].