3D Nuclear Organization of Telomeres in Hodgkin and Reed-Sternberg Cells.

Abstract

Telomerase activity is high in Hodgkin (H) and Reed-Sternberg (RS) cells and abundant telomerase RNA template (hTR) is identified in both by in situ hybridization. However, nothing is known about the three-dimensional (3D) structure of the telomeres in H and RS and the molecular events associated with the transition from the mononuclear H to the multinuclear diagnostic RS are still unclear. We analyzed the 3D structure of telomeres in interphase nuclei in the Hodgkin cell lines HDLM-2, L-428 and L-1236, where about 90 to 99% are mononuclear H and only 1 to 10% are multinuclear RS. We also analyzed the 3D structure of telomeres within 5 mm thin sections of three lymph node biopsies diagnostic for Hodgkin's disease (HD). The stereometric (3D) organization of telomeres was investigated in 30 H and 30 RS of each cell line and in 30 H and 30 RS of each lymph node biopsy specimen as previously described (Proc Natl Acad Sci USA , 102: 9613). Cellular localization of key-proteins of the telomere localized shelterin-complex (TRF1 and TRF2), of the mitotic spindle (centrin and gamma-tubulin), and of double-stranded DNA breaks (gamma-H2AX), was also analyzed. In all three cell lines the multinuclear RS showed overall significantly shorter and also significantly less telomeres in relation to the total nuclear mass when compared to their mononuclear H precursors. Visualization of their 3D telomeric structure revealed that this difference was due to partial or nearly total loss of telomeres within single nuclei of multinuclear RS; in particular, one or two nearly telomere free nuclei were often adjacent to one or two nuclei displaying several huge telomeric aggregates. TRF1 and TRF2 were mainly cytoplasmic in H and RS whereas gamma-H2AX accumulated in the nuclei of RS but not H. Multiple pairs of centrosomes not correlating with the number of nuclei, as well as high numbers of multi-polar spindles and incomplete spindles were identified in RS. Importantly, results analogous to those described in the HD cell lines were found in the HD lymph node biopsy specimen. In conclusion, our results suggest that multinuclear RS represent end stage tumour cells, where further nuclear division gets impossible due to sustained 3D telomere aggregation, shortening or loss. In particular, the number of nuclei within RS correlates closely with the 3D organization of telomeres, thus allowing for the first time a mechanistic understanding of the transition from H to RS cells. This process is initiated in H and advances to end stage telomere free “ghost” nuclei as observed in many RS. The shelterin complex appears to be disrupted and the mitotic cycle is profoundly disturbed.