Double-strand breakage in the extrachromosomal double minutes triggers their aggregation in the nucleus, micronucleation, and morphological transformation.

Abstract

Gene amplification plays a pivotal role in malignant transformation. Amplified genes often reside on extrachromosomal double minutes (DMs). Low-dose hydroxyurea induces DM aggregation in the nucleus which, in turn, generates micronuclei composed of DMs. Low-dose hydroxyurea also induces random double-strand breakage throughout the nucleus. In the present study, we found that double-strand breakage in DMs is sufficient for induction of DM aggregation. Here, we used CRISPR/Cas9 to introduce specific breakages in both natural and artificially tagged DMs of human colorectal carcinoma COLO 320DM cells. Aggregation occurred in the S phase but not in the G1 phase within 4 hours after breakage, which suggested the possible involvement of homologous recombination in the aggregation of numerous DMs. Simultaneous detection of DMs and the phosphorylated histone H2AX revealed that the aggregation persisted after breakage repair. Thus, the aggregate generated cytoplasmic micronuclei at the next interphase. Our data also suggested that micronuclear entrapment eliminated the DMs or morphologically transformed them into giant DMs or homogeneously staining regions (HSRs). In this study, we obtained a model explaining the consequences of DMs after double-strand breakage in cancer cells. Because double-strand breakage is frequently involved in cancer therapy, the model suggests how it affects gene amplification.