Identification of breast cancer resistant protein/mitoxantrone resistance/placenta-specific, ATP-binding cassette transporter as a transporter of NB-506 and J-107088, topoisomerase I inhibitors with an indolocarbazole structure.

The antitumor drugs NB-506 and J-107088 are potent topoisomerase I inhibitors with an indolocarbazole structure. To clarify the factors involved in resistance to these drugs, we established two NB-506-resistant mouse fibroblast cell lines (LY/NR1 and LY/NR2), a human colon carcinoma cell line (HCT116/NR1), and a lung cancer cell line (PC13/NR1). These cell lines were highly resistant to NB-506 and J-107088, and LY/NR2 cells showed markedly reduced accumulation and strong efflux of NB-506, suggesting activation of a drug efflux pump in the resistant cells. To identify the molecules responsible for efflux of NB-506, we compared the gene expressions of the mouse resistant LY/NR1 cells, LY/NR2 cells, and their parental cells by oligonucleotide microarray. Of 34,020 genes analyzed, we found that an ATP-binding cassette transporter BCRP/MXR/ABCP (BCRP) gene showed the highest increase in the expression, 31-fold higher in the LY/NR2-resistant cells than in their parental cells. The selective overexpression of this gene was also detected in the two human resistant cell lines, suggesting the involvement of breast cancer resistant protein (BCRP) in the resistance and efflux of these drugs. Finally, a PC-13 cell line transfected with BCRP expression vector displayed 22- and 17-fold resistance to NB-506 and J-107088 and enhanced efflux activity of J-107088. However, the transfectants were not resistant to mitoxantrone or topotecan, the drugs previously thought to be the substrates of BCRP. Thus, our study presents a novel mechanism of drug resistance mediated by BCRP.

A new mechanism of acquisition of drug resistance by partial duplication of topoisomerase I.

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-(beta-D-glucopyranosyl)- 5H-indolo[2,3-alpha]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.

Complete nucleotide sequence of the genomic RNA of tobacco mosaic virus strain Cg.

Tobacco mosaic virus (TMV)-Cg is a crucifer-infecting tobamovirus that was isolated from field-grown garlic. We determined the complete nucleotide sequence of the genomic RNA of TMV-Cg. The genomic RNA of TMV-Cg consists of 6303 nucleotides and encodes four large open reading frames, organized basically in the same way as that of other tobamoviruses. The nucleotide and deduced amino acid sequences are very similar to those of the other crucifer-infecting tobamoviruses that have been sequenced so far.

Sequence-selective DNA cleavage by a topoisomerase I poison, NB-506.

An indolocarbazole compound, NB-506, inhibits the activity of topoisomerase I by stabilizing the DNA-topoisomerase I complex (cleavable complex). NB-506 inhibited the religation step of topoisomerase I activity more potently than camptothecin or its derivative, topotecan. A cleavage assay using an end-labeled fragment of DNA revealed that the pattern of cleavage induced by NB-506 was different from that induced by camptothecin. The preferred cleavage sites of NB-506 were found to be not only T but also A or C at the 3'-terminus of the cleaved DNA (position -1), while the DNA cleavage sites of camptothecin always had T at position -1. At the 5'-terminus of the cleaved DNA (position +1), NB-506 showed a preference for G, which is a feature shared in common with camptothecin. Therefore, the difference in cleavage patterns was most likely due mainly to the preferred base at position -1. Moreover, the re-ligation rate was significantly slower at NB-506-selective sites, which had C at position-1, than at camptothecin-selective sites or at sites cleaved by both NB-506 and camptothecin. Our data suggest that NB-506 is an unique topoisomerase I poison and that its potent inhibition of topoisomerase I is partly dependent on retardation of re-ligation at sites selectively induced by NB-506.