PHOSPHORYLATION OF DNA TOPOISOMERASE-I IS INCREASED DURING THE RESPONSE OF MAMMALIAN-CELLS TO MITOGENIC STIMULI

DNA topoisomerase I is phosphorylated after mitogenic stimulation of 3 T3-L1 mouse fibroblasts by 12-O-tetradecanoylphorbol 13-acetate (TPA), a phorbol ester tumor promoter. In vivo labeling with [P-32]orthophos phate and immunoprecipitation with an anti-DNA topoisomerase I antibod y has demonstrated an increase in the phosphorylation of DNA topoisome rase I in Swiss/3T3 mouse fibroblasts treated with epidermal growth fa ctor (EGF) and H35 rat hepatoma cells treated with insulin. The only p hosphorylated form of DNA topoisomerase I observed was the 100-kDa pro tein. Digestion of DNA topoisomerase I with trypsin revealed two phosp hopeptides. In addition, VT-1, a non-responsive genetic variant of 3T3 -L1, and the DNA topoisomerase I inhibitor camptothecin were used to f urther study TPA-induced DNA topoisomerase I phosphorylation. Our resu lts indicate that the phosphorylation of DNA topoisomerase I may be an ubiquitous response of cultured mammalian cells to mitogenic agents, even in the absence of DNA replication.