Late onset of CAD gene amplification in unamplified PALA resistant Chinese hamster mutants

In rodent cells, resistance to PALA (N-phosphonacetyl-L-aspartate) has always been found associated with amplification of the CAD gene (carbamyl-P synthetase, aspartate transcarbamylase, dihydro-orotase). We describe two PALA resistant Chinese hamster mutant cell lines in which amplification of the CAD gene was not present. The PALA resistant phenotype was stable when the cells were grown in non-selective medium. However, after prolonged growth in the presence of the same drug concentration used for selection, cells with increased CAD gene copy number and higher levels of resistance overrode the original population. In these cell populations, a heterogeneous organization of the CAD genes was revealed by fluorescence in situ hybridization on mitotic chromosomes indicating that the additional copies of the gene were generated in several ways, such as non-disjunction and breakage-fusion-bridge cycles. The clastogenic effect of PALA, evidenced as chromosomal aberrations in the cells grown in the presence of the drug, could have favored the late onset of the amplified mutants. It is tempting to speculate that, during the expansion of tumor populations, different drug resistance mechanisms, including gene amplification, could occur in succession and lead to the generation of cells highly resistant to chemotherapeutic agents.