A cDNA microarray study of multiple-gene expression in mouse Ku80 knockout and wild-type cells after single-dose-irradiation

The study aims to compare differences in global gene expression between mouse Ku80-/- (KO) and Ku+/+ (WT) embryonic fibroblasts under irradiated and non-irradiated conditions. Both dual-fluorescence and colorimetric-based cDNA microarrays were employed, containing human 4,600 and mouse 9,600 gene sequences, respectively. Colony formation assay showed that KO cells were much more radiosensitive than WT cells, with nearly shoulderless survival curve. At 12 hrs postirradiation of 10 Gy, KO exhibited extensive G2/M accumulation, compensated by a reduction of G1 cells, while WT demonstrated about two-fold increase in G2/M and 4-fold S reduction. RT-PCR confirmed the knockout of Ku80 gene in the Ku-/-, and its presence in the WT cells. Without irradiation, microarray containing human gene targets showed that in log-growth KO cells, one gene (HXB) had nearly 3-fold expression level compared to WT. However, 18 genes were significantly down regulated in KO. A similar situation was revealed when the mouse-targets microarray was used for hybridization, with differences only in some genes. Similarly down-regulated genes correspond to cytokinesis, transcription, and apoptosis. At 12 hrs postirradiation, very few genes showed differential expression in either KO or WT. Almost none of the genes involved in DNA repair including base excision repair, nucleotide excision repair, mismatch repair, homologous recombination, non-homologous end rejoining, DNA polymerase, and editing and processing nucleases showed altered expression by irradiation at this time. Under the same irradiation protocol, small changes (up) were detected for Cyclin's, CDK7, and Wee1 and for some apoptosis factors and inhibitors in WT, but not in KO. Investigations at earlier postirradiation times may help interpretation of the molecular mechanisms underlying the differences in survival and cell-cycle arrests between KO and WT