Abstract
BackgroundIn mammalian cells gene amplification is a common manifestation of genome instability promoted by DNA double-strand breaks (DSBs). The repair of DSBs mainly occurs through two mechanisms: non-homologous end-joining (NHEJ) and homologous recombination (HR). We previously showed that defects in the repair of DSBs via NHEJ could increase the frequency of gene amplification. In this paper we explored whether a single or a combined defect in DSBs repair pathways can affect gene amplification.ResultsWe 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.ConclusionsDefects 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.
Highlights
In mammalian cells gene amplification is a common manifestation of genome instability promoted by DNA double-strand breaks (DSBs)
Construction of HeLa cell lines with stable inhibition of RAD54 and/or DNA-PKcs expression To inhibit the expression of RAD54, HeLa cells were transfected with the plasmids shRAD54p-1, shRAD54p-2, shRAD54p-3 or shRAD54p-4, each containing the neomycin resistance gene and different oligonucleotides for the production of shRNA against human RAD54, or with the plasmid p-scrambled, containing an oligonucleotide that is not homologous to any human gene
The plasmid containing the scrambled sequence did not induce any reduction in RAD54 expression; plasmids shRNAp-1 and shRNAp-2 did not produce a detectable effect on RAD54 expression while plasmids shRNAp-3 and shRNAp-4 caused a reduction in the levels of the protein
Summary
In mammalian cells gene amplification is a common manifestation of genome instability promoted by DNA double-strand breaks (DSBs). 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. The existence of specific regions of the genome that are hotspots for amplification in cancers with similar cell of origin suggests that they contain genes relevant for tumour formation and progression [6,7]. A large body of evidence indicates that DNA double-strand breaks (DSBs) can promote gene amplification through different processes such as successive breakage-fusion-bridge (BFB) cycles, unequal sister chromatid exchange, rolling circle replication or fold-back priming (for a review see [10])
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