Studies towards the optimization of the knock-down and the detection of the retrotransposon LINE-1

Abstract

The retrotransposon LINE-1 comprises 17% of the human genome and encodes the proteins necessary for its own distribution within the genome by a "copy-paste" mechanism. This makes it the only autonomously active retrotransposon in human. Its retrotransposition was not only found to have had an important impact on the human genome variability during the evolution, but also to be responsible for numerous diseases. Among them it also favors the development and progression of various cancer types and was found to support the cancers "superpower": the telomere maintenance mechanism. The intense study of LINE-1 and its proteins is essential to gain further information concerning its role as a chemotherapeutic target. This master thesis was part of a project studying the connection between LINE-1 and the stabilization and maintenance of telomeres in telomerase-positive and ALT tumor cell lines. It was aimed to enhance the success rate of the LINE-1 knock-down and its detection on mRNA and protein level to provide a robust base for further functional assays. The transfection protocol and mRNA expression influencing factors were therefore analyzed and optimized and a new anti-LINE-1 ORF2p targeting, monoclonal mouse antibody was tested for its performance in immunofluorescence and immunoblot assays. The apoptosis and cell cycle patterns of LINE-1 silenced cell lines with appropriate controls were compared to further prove the cancer promoting function of LINE-1. Despite the analysis of various factors and an intense optimization effort, a consistent monitoring of the LINE-1 mRNA expression was not achieved. However, a partial improvement of the immunoblot assay was attained enabling the detection of the LINE-1 protein content. Although the monoclonal mouse anti-LINE1 antibody provided an improved fluorescence detection of ORF2p in the immunocytochemistry assay, the low specificity led to contradictory results. This is why the immunoblot assay was preferred and further optimized for protein quantification. While the cell cycle analysis did not show any effects of the LINE-1 knock-down, the apoptosis pattern was altered. A shift towards early and late apoptosis, additionally to the effect of an induced DNA damage, provided further evidence for the cancerogenic potential of LINE-1.