Model for the Formation of Double Minutes from Prematurely Condensed Chromosomes of Replicating Micronuclei in Drug-treated Chinese Hamster Ovary Cells Undergoing DNA Amplification

Abstract

Double minutes (DM) have been associated with gene amplification in drug-resistant cells and tumor cells. However, the mechanisms by which DM are formed have not been elucidated. We present here a model to describe a possible mechanism of DM formation based on the observations made in two independent early drug-selected multidrug-resistant cell lines and from in vitro somatic cell fusion experiments between synchronized S- and M-phase cells. The multidrug-resistant cell lines contain both DM and amplified mdr (P-glycoprotein) gene. Cytogenetic analyses of cells at early stages of selection revealed the presence of a number of micronuclei in a subpopulation of these cells. These micronuclei were often asynchronous in their progression through the cell cycle. As a result, premature condensation of micronuclear chromatin was often observed in metaphase plates. The pulverized chromatin pattern seen in certain instances of S-phase prematurely condensed chromosomes displays a striking resemblance to DM structures. These DM-like structures are linked by replicating DNA as revealed by DNA labeling experiments. Somatic cell hybrids between S- and M-phase cells when grown in vitro demonstrated that S-phase prematurely condensed chromatin indeed gives rise to extra chromosomal structures in the successive cell generations. It is hypothesized that distinct DM-like structures may arise from the partially replicated and prematurely condensed S-phase chromosomes following their liberation as extra chromosomal entities after replication and/or recombination in the succeeding division cycle(s). The enrichment for DM containing specific genes in drug-resistant cells may result from the subsequent drug selections.