[Loss and stabilization of aminopterin resistance in murine cell lines]

Mouse L-cell lines (B-82, tk-) were obtained using the stepwise selection method, their aminopterin (AP) resistance being 10(3)-5 X 10(4) times higher than that of parental cells. This resistance increase results from dihydrofolate reductase (DHFR) gene amplification which was determined from the 15-120-fold rise of the enzyme activity and with the cytogenetical techniques. The development and loss of AP resistance have been studied and karyological analysis of the lines obtained carried out. Two types of karyological changes were found in stable DM and HSR cells which correspond to extrachromosomal and intrachromosomal forms of the amplified material organization. Localization of the DHFR gene in HSR was proved using the in situ hybridization technique. Extrachromosomal localization of the amplified genes in DM providing unstable AP resistance is dominant at the early stages of the development of resistance and for a long time. It was demonstrated that DM and HSR can exist in one cell during the prolonged period. DHFR gene copy number in such cells is regulated by a change in the DM number, whereas the HSR size and localization are highly stable. HSR covers 1.7-1.9% of the genome length and 38-40% of the marker chromosome length. The genes localized in HSR provide stable AP resistance. Evidence on some intermediate, relative stabilization of the resistance has been obtained. This stabilization is mediated by temporary integration of DHFR copies into other chromosomal sites, in addition to HSR.