Starvation for arginine and glutamine sensitizes human diploid cells to the transforming effects of N-acetoxy-2-acetylaminofluorene.

We have examined the influence of the method of synchronization on the transformation of normal human diploid fibroblasts treated in mid-S phase with N-acetylaminofluorene (N-Ac-AAF), and on the subsequent kinetics of the removal of carcinogen damage. At early times post-treatment, the transformation frequency in cultures synchronized by a 24 h starvation for arginine and glutamine was enhanced over that of cultures synchronized by release from confluence. In cells synchronized by either method, transformation was enhanced at all times when the cells were incubated after treatment in medium containing high concentrations of nonessential amino acids, vitamins and serum. Synchronized cells treated with 3 microM [3H]N-Ac-AAF in mid-S removed significantly less DNA damage than cultures treated and held under confluent, noncycling conditions. Cells synchronized by amino acid starvation removed less damage than cells synchronized by release from confluence. S-phase treatment of synchronized cells resulted in a dose-dependent delay of DNA replication. The early, rapid phase of repair was temporally correlated with the delay in S-phase progression. We conclude from these data that synchrony achieved by amino acid starvation renders human cells more sensitive to N-Ac-AAF-induced transformation in vitro than does that achieved by release from density-inhibition of growth. The S-phase treated, cycling cells lost only about 10% of their initial carcinogen damage before reinitiation of treatment-delayed bulk DNA synthesis. These results support the hypothesis that the persistence of carcinogen damage in replicating DNA may be an important factor in cell transformation.