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(PP289) Drastic changes in gene expression caused by Rad51 overexpression affects DNA repair only in S/G2- but not in the G1-phase.
Borgmann, Kerstin*,1, Jend, Camilla1, Streichert, Thomas2, Zschenker, Oliver1, Stürzbecher, Horst3, Dikomey, Ekkehard1, 1 Lab of Radiobiology & Experimental Radiooncology, Clinic of Radiotherapy and Radiooncology, Hamburg, Germany2 Institute of Clinical Chemistry, Hamburg, Germany3 Institute of Human Genetics, Luebeck, Germany
ABSTRACT- Objectives: The repair of DNA double strand breaks in mammalian cells occurs by non- homologous end joining (NHEJ) or homologous recombination (HR). HR mainly acts in the S/G2-phase, whereas NHEJ is active in all cell cycle phases. Deregulation of Rad51, the central protein of HR, can lead to genomic instability as observed for many different tumours as well as to a resistance against chemotherapeutica. This project focuses on the influence of Rad51 overexpression on gene expression and cell cycle dependent genomic instability. Methods: Experiments were carried out with human UiRad5-2 cells transfected with Rad51 under control of a ponasterone-inducible promoter; UiLacZ cells expressing -galactosidase instead of Rad51 were used as the control. The expression of the Rad51 protein was detected quantitatively by Western blot analysis and functionally via foci formation. Cell survival was measured by the colony forming assay, DSB repair by the formation of H2AX-Foci, chromosomal damage both by the metaphase technique and the G2-assay and gene expression profiles were generated by cDNA-microarray technique (Affymetrix). Results: In UiRad5-2 cells, ponasterone treatment caused a 4fold increase in Rad51, which resulted in an immense modification of gene expression with 135 genes being up and 70 being down regulated. These genetic changes caused an increase of spontaneous genomic instability reflected by H2AX foci and G2-Aberrations leading to a pronounced G2-arrest. Rad51 overexpression resulted in a reduced Mitomycin C sensitivity but affected neither the cellular radiosensitivity nor the number of lethal chromosome aberrations. Conclusion: Overexpression of Rad51, which led to a drastic change in gene expression, affects repair of DNA damage of the S/G2-Phase, like chromatid breaks and DNA-cross links but not repair of the G1-Phase like lethal chromosome aberrations and cell survival, which could explain the increased genomic instability observed in many tumours. Supported by grant no. 70-1932 Di I/II from the Deutsche Krebshilfe
Key words: DNA repair, HDR, chromosome aberrations, gene expression
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