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Identification of mutations at DNA topoisomerase I responsible for camptothecin resistance

L F Wang1, C Y Ting, C K Lo

  • 1Institute of Molecular Biology, Department of Biochemistry, Taipei Medical College, Taiwan, Republic of China.

Cancer Research
|April 15, 1997
PubMed

Insights

A new camptothecin-resistant ovarian cancer cell line, CPT(R)-2000, shows resistance due to mutations in DNA topoisomerase I. These specific mutations, Gly717 to Val and Thr729 to Ile, confer resistance by altering the enzyme

Area of Science:

  • Molecular Biology
  • Cancer Research
  • Drug Resistance Mechanisms

Background:

  • Established a camptothecin-resistant cell line (CPT(R)-2000) from mutagen-treated A2780 ovarian cancer cells.
  • CPT(R)-2000 exhibits significant resistance to camptothecin and moderate resistance to another DNA topoisomerase I inhibitor, Ho33342.
  • No cross-resistance was observed with unrelated chemotherapeutic agents like Adriamycin and vinblastine.

Purpose of the Study:

  • To investigate the molecular mechanism underlying camptothecin resistance in the CPT(R)-2000 cell line.
  • To identify specific alterations in DNA topoisomerase I responsible for camptothecin resistance.

Main Methods:

  • Comparative analysis of DNA topoisomerase I mRNA, protein levels, and enzyme activity between parental and resistant cells.
  • DNA sequencing to identify mutations in the DNA topoisomerase I gene of CPT(R)-2000 cells.
  • Utilized a yeast system to assess the functional impact of identified mutations on camptothecin resistance.

Main Results:

  • DNA topoisomerase I levels and activity were similar between parental and CPT(R)-2000 cells, but camptothecin could not inhibit the enzyme in resistant cells.
  • CPT(R)-2000 cells showed insensitivity to camptothecin-induced DNA topoisomerase I degradation.
  • DNA sequencing revealed mutations at Gly717 (to Val) and Thr729 (to Ile) in the DNA topoisomerase I of CPT(R)-2000 cells.

Conclusions:

  • The primary mechanism of camptothecin resistance in CPT(R)-2000 cells is attributed to structural mutations in DNA topoisomerase I.
  • Both Gly717Val and Thr729Ile mutations contribute to camptothecin resistance, with a synergistic effect observed in the double mutant.
  • The proximity of these mutation sites to the catalytic active center suggests camptothecin's interaction site may be near this region within the enzyme-DNA complex.

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