Amplification of the human dihydrofolate reductase gene via double minutesis initiated by chromosome breaks

DNA sequence amplification is one of the most frequent manifestations of ge nomic instability in human tumors. We have shown previously that amplificat ion of the dihydrofolate reductase (DHFR) gene in Chinese hamster cells is initiated by chromosome breaks, followed by bridge-breakage-fusion cycles t hat generate large intrachromosomal repeats; these are ultimately trimmed b y an unknown process to smaller, more homogenous units manifested as homoge nously staining chromosome regions (HSRs), However, in most human tumor cel ls, amplified DNA sequences are borne on unstable, extrachromosomal double minutes (DMs), which suggests the operation of a different amplification me chanism. In this study, we have isolated a large number of independent meth otrexate-resistant human cell lines, all of which contained DHFR-bearing DM s, Surprisingly, all but one of these also had suffered partial or complete loss of one of the parental DHFR-bearing chromosomes. Cells in a few popul ations displayed what could be transient intermediates in the amplification process, including an initial HSR, its subsequent breakage, the appearance of DHFR-containing fragments, and, finally, DMs, Our studies suggest that HSRs and DMs both are initiated by chromosome breaks, but that cell types d iffer in how the extra sequences ultimately are processed and/or maintained .