CHARACTERIZATION OF A TOPOISOMERASE-II GENE REARRANGEMENT IN A HUMAN SMALL-CELL LUNG-CANCER CELL-LINE

Background: Small-cell lung cancer (SCLC) is a highly chemosensitive tumor, but the recurrent disease that is common after initial response is often unresponsive to further chemotherapy. Although the mechanisms of drug resistance in SCLC have not been established, studies suggest that alterations of the nuclear enzyme DNA topoisomerase II may reduce the sensitivity of the cell to drug action. This enzyme is recognized as a primary target for cytotoxic activity of important antitumor agents. Purpose: In this study, we attempted to determine if altered forms of DNA topoisomerase II are responsible for reduced drug sensitivity. Methods: We characterized a rearrangement of the topoisomerase II p170 gene (also known as TOP2) in a relatively chemoresistant SCLC cell line, NCI-H69, and compared topoisomerase II expression and activity in this line with those in the chemosensitive NCI-H187 cell line. Fragments of complementary DNA from the topoisomerase II gene were generated by polymerase chain reaction. Immunodetection was accomplished by using the monoclonal antibody 7E6 against the human topoisomerase II p170 isoform. Using DNA probes corresponding to different complementary DNA regions, we showed that the rearrangement was localized at the 3'-terminus of one allele of the topoisomerase II gene. Results: In addition to the normal 6.2-kilobase (kb) topoisomerase II messenger RNA (mRNA), the NCI-H69 line expressed a 7.4-kb topoisomerase II transcript, presumably encoded by the rearranged allele. Moreover, this transcript, although longer than the normal mRNA, lacked a substantial portion of the 3'-terminal p170 gene coding sequence. Topoisomerase II activity in nuclear extracts, as determined by the P4 phage DNA-unknotting assay, was more easily detected and measured at lower NaCl concentrations in NCI-H69 than in NCI-H187 cells. Conclusion: These results are consistent with the hypothesis that the chemoresistant NCI-H69 cell line may express. in addition to the normal enzyme, an altered topoisomerase II enzyme possibly encoded by the 7.4-kb mRNA, which in turn may be transcribed from the rearranged gene allele. Implication: These observations emphasize the role of topoisomerase II in determining drug sensitivity and suggest that such gene rearrangements may contribute to resistance of SCLC cells to topoisomerase II inhibitors.