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, Keli Agama, Amit Roy, David Hersi Smith, Thomas D Pfister, Maria Unni Rømer, Hong-Liang Zhang, James H Doroshow, Birgitta R. Knudsen, Jan Stenvang, Nils Brünner, Yves Pommier

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.
OriginalsprogEngelsk
Artikelnummer56
TidsskriftJournal of Experimental and Clinical Cancer Research (Online)
Vol/bind35
Antal sider14
ISSN1756-9966
DOI
StatusUdgivet - 2016

ID: 160448868