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

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  • Niels Frank Jensen, Department of Veterinary Disease Biology, Faculty of Health and Medical Sciences, University of Copenhagen, Frederiksberg C
  • ,
  • Keli Agama, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda
  • ,
  • Amit Roy, Department of Biotechnology, National Institute of Pharmaceutical Education and Research (NIPER), India
  • ,
  • David Hersi Smith, University of Copenhagen, Faculty of Health and Medical Sciences, Department of Veterinary Disease Biology, Bülowsvej 17, DK-1870 Frederiksberg, Denmark., R&D, Dako A/S, 2600 Glostrup, Denmark
  • ,
  • Thomas D Pfister, Laboratory of Human Toxicology and Pharmacology, Applied/Developmental Directorate, Leidos Biomedical Research, Inc., Frederick National Laboratory for Cancer Research
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  • Maria Unni Rømer, University of Copenhagen, Faculty of Health and Medical Sciences, Department of Veterinary Disease Biology, Bülowsvej 17, DK-1870 Frederiksberg, Denmark., Department for Clinical Physiology and Nuclear Medicine, Frederiksberg Hospital
  • ,
  • Hong-Liang Zhang, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda
  • ,
  • James H Doroshow, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, Laboratory of Human Toxicology and Pharmacology, Applied/Developmental Directorate, Leidos Biomedical Research, Inc., Frederick National Laboratory for Cancer Research
  • ,
  • Birgitta R Knudsen
  • Jan Stenvang, Department of Veterinary Disease Biology, Faculty of Health and Medical Sciences, University of Copenhagen, Frederiksberg C
  • ,
  • Nils Brünner, Department of Veterinary Disease Biology, Faculty of Health and Medical Sciences, University of Copenhagen, Frederiksberg C
  • ,
  • Yves Pommier, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda

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.

Original languageEnglish
Article number56
JournalJournal of Experimental and Clinical Cancer Research (Online)
Volume35
Issue1
Number of pages14
ISSN1756-9966
DOIs
Publication statusPublished - 31 Mar 2016

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