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J Exp Clin Cancer Res. 2016 Mar 31;35:56. doi: 10.1186/s13046-016-0335-x.

Characterization of DNA topoisomerase I in three SN-38 resistant human colon cancer cell lines reveals a new pair of resistance-associated mutations.

Author information

1
Department of Veterinary Disease Biology, Faculty of Health and Medical Sciences, Section for Molecular Disease Biology, University of Copenhagen, Strandboulevarden 49, DK-2100, Copenhagen, Denmark.
2
National Institutes of Health, National Cancer Institute, Center for Cancer Research, Laboratory of Molecular Pharmacology, 37 Convent Drive, Building 37, Room 5068, Bethesda, MD, 20892-4255, USA.
3
Department of Molecular Biology and Genetics, Aarhus University, C.F. Møllers Allé 3, Building 1130, DK-8000, Aarhus C, Denmark.
4
Department of Biotechnology, National Institute of Pharmaceutical Education and Research (NIPER), Hajipur, Vaishali, 844102, India.
5
Dako Denmark A/S, R&D, Produktionsvej 42, DK-2600, Glostrup, Denmark.
6
Laboratory of Human Toxicology and Pharmacology, Applied/Developmental Directorate, Leidos Biomedical Research, Inc., Frederick National Laboratory for Cancer Research, Frederick, MD, 21702, USA.
7
Department for Clinical Physiology and Nuclear Medicine, Frederiksberg Hospital, Nordre Fasanvej 57, DK-2000, Frederiksberg C, Denmark.
8
Department of Veterinary Disease Biology, Faculty of Health and Medical Sciences, Section for Molecular Disease Biology, University of Copenhagen, Strandboulevarden 49, DK-2100, Copenhagen, Denmark. stenvang@sund.ku.dk.

Abstract

BACKGROUND:

DNA topoisomerase I (Top1) is a DNA unwinding protein and the specific target of the camptothecin class of chemotherapeutic drugs. One of these, irinotecan, acting through its active metabolite SN-38, is used in the treatment of metastatic colorectal cancer. However, resistance to irinotecan represents a major clinical problem. Since molecular alterations in Top1 may result in resistance to irinotecan, we characterized Top1 in three human colon cancer cell lines with acquired resistance to SN-38.

METHODS:

Three SN-38 resistant (20-67 fold increased resistance) cell lines were generated and compared to wild-type parental cells with regards to: TOP1 gene copy number and gene sequence, Top1 expression (mRNA and protein), Top1 enzymatic activity in the absence and presence of drug, and Top1-DNA cleavage complexes in drug treated cells. TOP1 mutations were validated by PCR using mutant specific primers. Furthermore, cross-resistance to two indenoisoquinoline Top1-targeting drugs (NSC 725776 and NSC 743400) and two Top2-targeting drugs (epirubicin and etoposide) was investigated.

RESULTS:

Two of three SN-38 resistant cell lines carried TOP1 gene copy number aberrations: A TOP1 gene copy gain and a loss of chromosome 20, respectively. One resistant cell line harbored a pair of yet unreported TOP1 mutations (R364K and G717R) in close proximity to the drug binding site. Mutant TOP1 was expressed at a markedly higher level than wild-type TOP1. None or very small reductions were observed in Top1 expression or Top1 activity in the absence of drug. In all three SN-38 resistant cell lines Top1 activity was maintained in the presence of high concentrations of SN-38. None or only partial cross-resistance were observed for etoposide and epirubicin, respectively. SN-38 resistant cells with wild-type TOP1 remained sensitive to NSC 743400, while cells with mutant TOP1 was fully cross-resistant to both indenoisoquinolines. Top1-DNA cleavage complex formation following drug treatment supported the other findings.

CONCLUSIONS:

This study adds to the growing knowledge about resistance mechanisms for Top1-targeting chemotherapeutic drugs. Importantly, two yet unreported TOP1 mutations were identified, and it was underlined that cross-resistance to the new indenoisoquinoline drugs depends on the specific underlying molecular mechanism of resistance to SN-38.

KEYWORDS:

Colon cancer; DNA topoisomerase I; Irinotecan; Mutation; Resistance; SN-38; TOP1

PMID:
27029323
PMCID:
PMC4815242
DOI:
10.1186/s13046-016-0335-x
[Indexed for MEDLINE]
Free PMC Article
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