Late onset of CAD gene amplification in unamplified PALA resistant Chinesehamster mutants

In rodent cells, resistance to PALA (N-phosphonacetyl-L-aspartate) has alwa ys been found associated with amplification of the CAD gene (carbamyl-P syn thetase, 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 i n the presence of the same drug concentration used for selection, cells wit h increased CAD gene copy number and higher levels of resistance overrode t he original population. In these cell populations, a heterogeneous organiza tion 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-bri dge cycles. The clastogenic effect of PALA, evidenced as chromosomal aberra tions in the cells grown in the presence of the drug, could have favored th e late onset of the amplified mutants. It is tempting to speculate that, du ring the expansion of tumor populations, different drug resistance mechanis ms, including gene amplification, could occur in succession and lead to the generation of cells highly resistant to chemotherapeutic agents. (C) 2000 Elsevier Science Ireland Ltd. All rights reserved.