Die Rolle von LIN-9 und B-MYB in der Zellzyklusregulation humaner Zellen

The human LIN-9 Protein was first identified as a novel pRB-interacting Protein which acts as a tumorsuppressor in context of the pRB-pathway. The homologs of LIN-9 in D. melanogaster and C. elegans are required for the transcriptional regulation of different genes. B-MYB is a direct target-gene of E2F in D. melanogaster and acts as a transcriptionfactor and participates in the activation of differentiation specific and cellcycle regulating genes. Also, in vertebrates (zebra fish) B-MYB was described as a haploinsufficient Tumorsuppressor and depletion of B-MYB leads to increased numbers of an- and polyploid cells. This and the fact, that both LIN-9 and B-MYB cooperate with pRB in the activation of differentiation specific genes let to the hypothesis, that these genes could play an important role in the transcriptional regulation of genes. Thus, the primary goal of this thesis was to identify the function of LIN-9 and B-MYB, specifically during mitosis. For that purpose, the progression through the cell cycle of LIN-9 or B-MYB depleted SHEP-cells in comparison to control cells should be analyzed (FACS). Therefore an RNAi-based system was established, that efficiently represses the posttranscriptional expression of LIN-9 and B-MYB in SHEP cells. It was possible to reproduce results which showed that the loss of LIN-9 or B-MYB lead to a reduced expression of a cluster of G2/M-specific genes, whose products are required for the mitotic checkpoint. The reduced expression of these genes is accompanied by phenotypically changes, such as a defect of the mitotic spindle checkpoint, which leads to an early termination of mitosis after nocodazole treatment. With an additional trigger LIN-9 and B-MYB depleted cells show a high rate of apoptotic cells, while the G2/M-Block is less characteristic but still existent. There was no significant increase of an- or polyploidy and no dependency on p53 or p21. Cell cycle kinetics generated by flowcytometry revealed that the progression of LIN-9 or B-MYB depleted cells from S-phase to G2/M-phase and into the next G1-Phase is significantly delayed. In summary, depletion of LIN-9 or B-MYB results neither in an arrest in mitosis nor in a significantly changed S-phase length of these cells. This indicates that the slowed progression through the cell cycle is most likely due to a defect in the late G2-phase, which results in a delayed entry into mitosis. The homologes of LIN-9 and B-MYB in D. melanogaster and C. elegans act together as subunits of highly conserved RB/E2F-complexes in the regulation of genes, which suggests, that LIN-9 and B-MYB are also components of a similar complex in humans and thereby mediate the regulation of the cell cycle. Keywords: LIN-9; B-MYB; cell cycle; G2/M-transition; mitosis; transcription