Retinoic-acid-induced differentiation prevents gene amplification in teratocarcinoma stem cells

In an attempt to study the regulatory properties of cells required for gene amplification, the capacity for amplification of the dehydrofolate reductase gene (dhfr) was determined in F9 teratocarcinoma stem cells during differentiation. By stepwise selection of surviving cells exposed to progressively increasing concentrations of methotrexate (MTX) up to 1,000 microM within 4 months, non-differentiated F9 cells reached a more than 40-fold amplification of their dhfr gene, elevated levels of dhfr mRNA and a 5-log increase in MTX resistance. Exposure to 5 Gy of 60Co-gamma-irradiation accelerated the process of amplification. In contrast, F9 cells that had been differentiated to endodermal cells by treatment with retinoic acid (RA) did not grow in MTX concentrations above 0.1 microM, either with or without radiation pretreatment and did not amplify the dhfr gene to any measurable extent over the same period of time. Upon treatment of methotrexate-resistant (F9-MTXr) cells with retinoic acid, loss of the amplified DNA in the absence of selection was significantly retarded. The ability to amplify the dhfr gene was correlated with the occurrence of origin binding activity in vitro (early domain of SV40 minimal origin of replication). These data indicate an increase in genomic stability and rigorous control of origin activity by differentiation.