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

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 wildtype 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. © 2016 jensen et al.