Gene amplification in human cells knocked down for RAD54

We constructed human cell lines in which the expression of RAD54 and/or DNA-PKcs was constitutively knocked-down by RNA interference. We analyzed their radiosensitivity and their capacity to generate amplified DNA. Our results showed that both RAD54 and DNA-PKcs deficient cells are hypersensitive to γ-irradiation and generate methotrexate resistant colonies at a higher frequency compared to the proficient cell lines. In addition, the analysis of the cytogenetic organization of the amplicons revealed that isochromosome formation is a prevalent mechanism responsible for copy number increase in RAD54 defective cells.Defects in the DSBs repair mechanisms can influence the organization of amplified DNA. The high frequency of isochromosome formation in cells deficient for RAD54 suggests that homologous recombination proteins might play a role in preventing rearrangements at the centromeres.Different pathways, mainly controlling either the cell cycle in response to DNA damage or the repair of the damage itself, maintain genome stability in mammalian cells. Mutations in genes implicated in these pathways cause genetic lesions that can give rise to cellular transformation. Gene amplification, the increase in the copy number of a portion of the genome, is a common manifestation of genome instability in tumour cells and an important mechanism of oncogene activation as well as drug resistance, since it leads to over-expression of relevant genes. Amplification of DNA sequences containing cancer genes has been described in several types of solid tumours and lymphomas [1,2]. The fact that gene amplification has never been detected in cells of normal origin [3,4] suggests that either control mechanisms that prevent the occurrence of gene amplification are active (such as the p53-mediated damage-sensing pathway), or cells carrying gene amplifications do not survive.Cytogenetic manifestations of amplified DNA include self-replicating extrachromosomal elements called double minutes (D