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Topoisomerase I amino acid substitutions, Gly185Arg and Asp325Glu, confer camptothecin resistance in Leishmania donovani.

ABSTRACT: The antitumor compound camptothecin (CPT) is also recognized for its specific activity against Leishmania donovani topoisomerase I (Topo-I). In consequence, defining CPT resistance mechanisms represents an important strategic tool in the acquisition of a better understanding of its mode of action. In the present study, we selected a single highly resistant L. donovani strain termed LdRCPT.160 by stepwise exposure to CPT. Gene sequencing revealed two single nucleotide mutations in the LdRCPT.160 LdTOP1A gene, resulting in two amino acid substitutions (Gly185Arg and Asp325Glu) in the protein. Moreover, these two substitutions observed in the LdTOP1A protein were correlated with a decreased Topo-I DNA relaxation activity in these resistant parasites. Nevertheless, there was no change in the LdTOP1A gene expression level. Interestingly, transfection studies of the LdRCPT.160 LdTOP1A gene in its wild-type counterpart showed that it induced CPT resistance. Site-directed mutagenesis studies demonstrated that, despite a substantial level of resistance conferred by the Gly185Arg and Asp325Glu substitutions separately, both were essential to reach a high-resistance phenotype. Of interest, the amino acid substitutions observed in LdRCPT.160 LdTOP1A protein occurred near the amino acids previously predicted to interact with CPT, providing new insight into the mechanism of CPT molecular action.

SUBMITTER: Marquis JF

PROVIDER: S-EPMC1068627 | biostudies-literature | 2005 Apr

REPOSITORIES: biostudies-literature

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Topoisomerase I amino acid substitutions, Gly185Arg and Asp325Glu, confer camptothecin resistance in Leishmania donovani.

Marquis Jean-François JF Hardy Isabelle I Olivier Martin M

Antimicrobial agents and chemotherapy 20050401 4

The antitumor compound camptothecin (CPT) is also recognized for its specific activity against Leishmania donovani topoisomerase I (Topo-I). In consequence, defining CPT resistance mechanisms represents an important strategic tool in the acquisition of a better understanding of its mode of action. In the present study, we selected a single highly resistant L. donovani strain termed LdRCPT.160 by stepwise exposure to CPT. Gene sequencing revealed two single nucleotide mutations in the LdRCPT.160 ...[more]

PMID: 15793124

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Project description:BACKGROUND: Topoisomerase I (TOP1) is a nuclear enzyme that catalyzes the relaxation of supercoiled DNA during DNA replication and transcription. TOP1 is the molecular target of camptothecin and related drugs such as irinotecan and SN38 (irinotecan's active metabolite). Irinotecan is widely used as an anti-cancer agent in the treatment of metastatic colon cancer. However, its efficacy is often limited by the development of resistance. METHODS: We previously established several SN38 resistant HCT116-derived clones to study the mechanisms underlying resistance to SN38. Here, we investigated whether resistance to SN38 in these cell lines could be linked to the presence of TOP1 mutations and changes in its expression and activity. Functional analyses were performed on these cell lines challenged with SN38 and we specifically monitored the double strands breaks with ?H2AX staining and replication activity with molecular combing. RESULTS: In SN38 resistant HCT116 clones we identified three new TOP1 mutations, which are located in the core subdomain III (p.R621H and p.L617I) and in the linker domain (p.E710G) and are packed together at the interface between these two domains. The presence of these TOP1 mutations in SN38 resistant HCT116 cells did not modify TOP1 expression or intrinsic activity. Conversely, following challenge with SN38, we observed a decrease of TOP1-DNA cleavage complexes and a reduction in double-stranded break formation). In addition, we showed that SN38 resistant HCT116 cells present a strong decrease in the SN38-dependent asymmetry of replication forks that is characteristic of SN38 sensitive HCT116 cells. CONCLUSIONS: These results indicate that the TOP1 mutations are involved in the development of SN38 resistance. We hypothesize that p.L617, p.R621 and p.E710 TOP1 residues are important for the functionality of the linker and that mutation of one of these residues is sufficient to alter or modulate its flexibility. Consequently, linker fluctuations could have an impact on SN38 binding by reducing the enzyme affinity for the drug.

Dataset Information

Topoisomerase I amino acid substitutions, Gly185Arg and Asp325Glu, confer camptothecin resistance in Leishmania donovani.

Publications

Topoisomerase I amino acid substitutions, Gly185Arg and Asp325Glu, confer camptothecin resistance in Leishmania donovani.

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