Decreased Expression of DNA Topoisomerase I in Camptothecin-resistant Tumor Cell Lines as Determined by a Monoclonal Antibody

DNA topoisomerase I (topo I) has been identified as a principal target of a plant alkaloid camptothecin (CPT) and its derivative (CPT-11). The latter compound is expected to be a clinically useful antitumor agent. Three human tumor cell lines resistant to CPT (A549/CPT, HT-29/CPT, St-4/CPT) were isolated in vitro, and a murine tumor cell line resistant to CPT-11 (P388/CPT) was isolated in vivo by continuous exposure of the drugs. A549/CPT, HT-29/CPT, and St-4/CPT showed 1.8-, 6.9-, and 8.8-fold more resistance to CPT, and P388/CPT showed 45-fold more resistance to CPT than did the parental line. To examine the possible involvement of topo I in drug-resistant mechanisms, a monoclonal antibody was developed by using purified human topo I as antigen. The antibody T14C (immunoglobulin Gl) recognized both human and murine topo I, as shown by Western blot analysis. By using this monoclonal antibody, cellular contents of topo I were examined in CPT-resistant tumor lines. Respective contents of topo I in HT-29/CPT, St-4/CPT, and P388/CPT were approximately 8-, 4-, and 3-fold less than those in their parental cell lines. A549/CPT, a weak CPT-resistant line, possessed amounts of topo I similar to those of the parental line. HT-29/CPT showed lower topo I activity than did the parental HT-29 in the nuclear extracts and in the hydroxylapatite column-eluted fractions. Purified topo I from HT-29 and HT-29/CPT showed similar catalytic activity when the same amounts of protein were used. These results indicate that the quantitative reduction of topo I content seems to be the most frequently occurring event in the development of resistance to camptothecin.