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A New Mechanism of Acquisition of Drug Resistance by Partial Duplication of Topoisomerase I

Banyu Tsukuba Research Institute in collaboration with Merck Research Laboratories, Tsukuba-shi, Ibaraki, 300-2611, Japan

Topoisomerase (topo)-I targeting antitumor agents are very effective in vivo against various human cancers. The indolocarbazole compound 6-N-formylamino-12,13-dihydro-1,11-dihydroxy-13-(ß-D-glucopyranosyl)-5H-indolo[2,3-a]pyrrolo-[3,4-c]carbazole-5,7(6H)-dione (NB-506) is a potent inhibitor of the religation step of topo I reaction, like camptothecin (CPT). We established a NB-506-resistant cell line from murine leukemia cell line P388. This resistant cell line, P388/F11, exhibited 73-fold higher resistance to NB-506 and 3.5-fold higher cross-resistance to CPT than the parental cell line. No induction of cleavable complex formations induced by NB-506 and CPT were detected by K-SDS precipitation assays in P388/F11 cells. Analysis of nuclear extracts from P388/F11 cells revealed that the relaxation activity of topo I was one-quarter of that of the parental cells, and that the activity was resistant to induction of DNA cleavage by these drugs. Furthermore, Western blot and Northern blot analyses showed the expression of an abnormal-sized 170-kDa topo I protein and its 6.0-kb transcript and the absence of the normal topo I protein and transcript in P388/F11 cells. Analyses of the structure of the abnormal topo I transcript by reverse transcription-PCR and direct sequencing methods revealed that a large portion of the gene from codon 21 to codon 609 was duplicated in its coding region. This internal duplication resulted in in-frame fusion and, thus, production of a partially duplicated protein of 1357 amino acids. Finally, we expressed and purified the recombinant P388/F11 topo I in a baculovirus system. P388/F11 topo I showed similar catalytic activity to wild-type topo I, but reduced sensitivities to NB-506 and CPT. These results show that the altered sensitivity of duplicated topo I is involved in the NB-506 resistance of P388/F11 cells and indicate a novel resistant mechanism which involves duplication of the topo I gene.

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