title: Characterization of DNA topoisomerase I in three SN-38 resistant human colon cancer cell lines reveals a new pair of resistance-associated mutations.
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.
authors.affiliation: 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.
authors.forename: Niels Frank
authors.initials: NF
authors.lastname: Jensen
authors.affiliation: 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.
authors.forename: Keli
authors.initials: K
authors.lastname: Agama
authors.affiliation: Department of Molecular Biology and Genetics, Aarhus University, C.F. Møllers Allé 3, Building 1130, DK-8000, Aarhus C, Denmark.|Department of Biotechnology, National Institute of Pharmaceutical Education and Research (NIPER), Hajipur, Vaishali, 844102, India.
authors.forename: Amit
authors.initials: A
authors.lastname: Roy
authors.affiliation: 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.|Dako Denmark A/S, R&D, Produktionsvej 42, DK-2600, Glostrup, Denmark.
authors.forename: David Hersi
authors.initials: DH
authors.lastname: Smith
authors.affiliation: Laboratory of Human Toxicology and Pharmacology, Applied/Developmental Directorate, Leidos Biomedical Research, Inc., Frederick National Laboratory for Cancer Research, Frederick, MD, 21702, USA.
authors.forename: Thomas D
authors.initials: TD
authors.lastname: Pfister
authors.affiliation: 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.|Department for Clinical Physiology and Nuclear Medicine, Frederiksberg Hospital, Nordre Fasanvej 57, DK-2000, Frederiksberg C, Denmark.
authors.forename: Maria Unni
authors.initials: MU
authors.lastname: Rømer
authors.affiliation: 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.
authors.forename: Hong-Liang
authors.initials: HL
authors.lastname: Zhang
authors.affiliation: 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.|Laboratory of Human Toxicology and Pharmacology, Applied/Developmental Directorate, Leidos Biomedical Research, Inc., Frederick National Laboratory for Cancer Research, Frederick, MD, 21702, USA.
authors.forename: James H
authors.initials: JH
authors.lastname: Doroshow
authors.affiliation: Department of Molecular Biology and Genetics, Aarhus University, C.F. Møllers Allé 3, Building 1130, DK-8000, Aarhus C, Denmark.
authors.forename: Birgitta R
authors.initials: BR
authors.lastname: Knudsen
authors.affiliation: 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.
authors.forename: Jan
authors.initials: J
authors.lastname: Stenvang
authors.affiliation: 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.
authors.forename: Nils
authors.initials: N
authors.lastname: Brünner
authors.affiliation: 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.
authors.forename: Yves
authors.initials: Y
authors.lastname: Pommier
mesh_terms: D052117:Benzodioxoles / Q000494:pharmacology; D001665:Binding Sites; D002166:Camptothecin / Q000031:analogs & derivatives* / Q000494:pharmacology; D045744:Cell Line, Tumor; D002872:Chromosome Deletion; D003110:Colonic Neoplasms / Q000235:genetics* / Q000378:metabolism; D004264:DNA Topoisomerases, Type I / Q000235:genetics* / Q000378:metabolism; D019008:Drug Resistance, Neoplasm; D015251:Epirubicin / Q000494:pharmacology; D005047:Etoposide / Q000494:pharmacology; D018628:Gene Dosage; D006146:Guanidines / Q000494:pharmacology; D045325:HCT116 Cells; D019073:HT29 Cells; D006801:Humans; D006835:Hydrazones / Q000494:pharmacology; D000077146:Irinotecan; D007546:Isoquinolines / Q000494:pharmacology; D009154:Mutation
chemical_list: C545735:7-nitro-1H-indole-2-carboxylic acid (4-(1-(guanidinohydrazone)ethyl)phenyl)amide; D052117:Benzodioxoles; D006146:Guanidines; D006835:Hydrazones; D007546:Isoquinolines; C551306:NSC 725776; D015251:Epirubicin; D005047:Etoposide; D000077146:Irinotecan; D004264:DNA Topoisomerases, Type I; C528909:TOP1 protein, human; D002166:Camptothecin
keywords: Colon cancer; DNA topoisomerase I; Irinotecan; Mutation; Resistance; SN-38; TOP1