title: How a replication origin and matrix attachment region accelerate gene amplification under replication stress in mammalian cells.
abstract: The gene amplification plays a critical role in the malignant transformation of mammalian cells. The most widespread method for amplifying a target gene in cell culture is the use of methotrexate (Mtx) treatment to amplify dihydrofolate reductase (Dhfr). Whereas, we found that a plasmid bearing both a mammalian origin of replication (initiation region; IR) and a matrix attachment region (MAR) was spontaneously amplified in mammalian cells. In this study, we attempted to uncover the underlying mechanism by which the IR/MAR sequence might accelerate Mtx induced Dhfr amplification. The plasmid containing the IR/MAR was extrachromosomally amplified, and then integrated at multiple chromosomal locations within individual cells, increasing the likelihood that the plasmid might be inserted into a chromosomal environment that permits high expression and further amplification. Efficient amplification of this plasmid alleviated the genotoxicity of Mtx. Clone-based cytogenetic and sequence analysis revealed that the plasmid was amplified in a chromosomal context by breakage-fusion-bridge cycles operating either at the plasmid repeat or at the flanking fragile site activated by Mtx. This mechanism explains how a circular molecule bearing IR/MAR sequences of chromosomal origin might be amplified under replication stress, and also provides insight into gene amplification in human cancer.
authors: Graduate School of Biosphere Science, Hiroshima University, Higashi-hiroshima, Hiroshima, Japan.
authors: Shun-suke
authors: SS
authors: Tanaka
authors: Graduate School of Biosphere Science, Hiroshima University, Higashi-hiroshima, Hiroshima, Japan.
authors: Sho-hei
authors: SH
authors: Mitsuda
authors: Graduate School of Biosphere Science, Hiroshima University, Higashi-hiroshima, Hiroshima, Japan.
authors: Noriaki
authors: N
authors: Shimizu
mesh_terms: D000818:Animals; D016466:CHO Cells; D006224:Cricetinae; D003412:Cricetulus; D004261:DNA Replication; D005784:Gene Amplification; D045170:Matrix Attachment Regions; D018741:Replication Origin; D013312:Stress, Physiological; D013762:Tetrahydrofolate Dehydrogenase / Q000235:genetics
chemical_list: D013762:Tetrahydrofolate Dehydrogenase