Sander, Petra (2010)
Frequency distribution of telomere maintenance mechanisms in soft tissue sarcoma.
Technische Universität Darmstadt
Dissertation, Erstveröffentlichung

Kurzbeschreibung (Abstract)

Molecular mechanisms, which cause changes of the ends of chromosomes, the telomeres, play an important role in the generation of genomic instability. In normal somatic cells, the length of the telomeres are shortened during cell division due to the inability of the cells to replicate their chromosome ends completely. Telomeres below a critical length are dysfunctional and undergo apoptosis or permanent growth arrest referred to as replicative senescence. One possibility to bypass telomere dysfunction and maintenance of stable telomere length is the activation of a telomere maintenance mechanism. The currently known telomere maintenance mechanisms in humans are telomerase activity (TA-activity) and the alternative lengthening of telomeres (ALT-Mechanism). The preliminary goal of the thesis was to assess the frequency distribution of the activation of telomerase and of the ALT-mechanism in the major soft tissue sarcomas subtypes, which had been genetically well characterized in preceding studies. A first step of the thesis was the establishment of a method to assess the ALT-mechanism. One hallmark of ALT positive tumors/cells is the presence of ALT-associated promyelocytic leukemia (PML) bodies (APBs). APBs are characterized by promyelocytic leukemia (PML) bodies, which colocalize with telomeric DNA or telomere-specic binding proteins. A further marker of ALT is their highly heterogeneous telomere length with some exceptionally long telomeres. In order to assess ALT in human soft tissue sarcoma subtypes by combined telomere uorescence in situ hybridization and PML immunouorescence, confocal laser scanning microscope was used. 3D images were acquired, which visualize telomere spots in the rst channel, PML bodies in the second channel and DAPI stained nuclei in the third channel. The central task of image analysis was to automatically detect and classify APBs within the cell nucleus as well as to detect and quantify very large telomeres. Telomerase activity was evaluated by the TRAP assay (telomeric repeat amplication protocol). Afterwards, these data were correlated with the number of chromosomal imbalances detected by comparative genomic hybridization (CGH). This study contributed to the understanding of the frequency distribution of telomere maintenance mechanisms in soft tissue sarcoma subgroups and whether these mechanisms are associated with specic chromosomal imbalances. The most important results based on this thesis are: 1. The study demonstrates that the occurrence of the telomere maintenance mechanism is characteristic for the subtype of soft tissue sarcoma. The presence of telomerase activity ranges from 100% in synovial sarcoma to 46% in pleomorphic liposarcomas. The frequency of the marker for the ALT mechanism depends on the subtype of soft tissue sarcoma. While the telomeres detected in synovial sarcomas were equal in length, 92% of all cases of pleomorphic liposarcomas showed exceptionally long telomere signals. Furthermore, the correlation of both telomere maintenance mechanisms has shown that in tumors with high telomerase activity, the markers for the ALT-mechanism are significantly reduced. 2. In order to investigated an abundant number of healthy tissue with regard to markers for the ALT mechanism automatic quantication were performed. Healthy tissue, as well as our investigated tumor samples, show the appearance of ALT-associated PML bodies, whereas no telomeres with heterogeneous size and exceptionally long telomeres could be detected in healthy tissue. Therefore an important questions remains: how tightly are APBs linked to the ALT mechanism? 3. Comparative genomic hybridization (CGH) allows for a genome-wide screen for chromosomal imbalances in tumor samples. The correlation of the CGH data and the telomere maintenance mechanisms revealed that tumors with telomerase activity show a dierent cytogenetic prole compared to those tumors with markers for ALT. In respect to the frequency of chromosomal imbalances, such as gains and losses, there were no dierences between both telomere maintenance mechanisms. 4. A new approach for an automated quantication of telomere and PML spots as well as colocalization in multi-channel 3D microscopy images to assess the ALT mechanism were developed. To discriminate between spots inside and outside the cell nucleus, DAPI staining was used for the nuclear segmentation. With our program more than 500 confocal images were successfully analyzed. The characterization of tumors is highly relevant for tumor diagnostics and treatment planning. Due to the appearances of APBs in healthy tissue, one important question remains. How tightly are these structures linked to the ALT-mechanism? Further investigation is needed to clarify if in healthy tissue APBs are induced by the ALT mechanism or other nuclear processes besides ALT. Therefore, the characterization and understanding of telomere maintenance mechanism in tumorigenesis could have important implications in the development new treatments for these malignancies.

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