As described in our study, the karyotypic evolution of the osteosarcoma cell line U2-OS under chronic doxorubicin exposure or CDT1 overexpression suggests that despite ongoing double-strand breaks (DSBs) and illegitimate recombination, the extent of novel chromosomal changes that can be mitotically tolerated in immortalized cells in culture is not unlimited. The gene discussed is CDT1; the disease is osteosarcoma.