PMID: 9533521May 14, 1998Paper

Pharmacodynamics of topoisomerase I inhibition: Western blot determination of topoisomerase I and cleavable complex in patients with upper gastrointestinal malignancies treated with topotecan

Clinical Cancer Research : an Official Journal of the American Association for Cancer Research
L LiebesH Hochster

Abstract

Analogues of camptothecins are specific inhibitors of eukaryotic DNA topoisomerase I (topo I) that lead to DNA damage and, eventually, cellular cytotoxicity. Camptothecin analogues bind to this target enzyme in the course of its normal function and stabilize the DNA-enzyme adduct to form a "cleavable complex." Preclinical experiments using Western blot analyses have shown cleavable complex formation to be the key intermediate step in topo I inhibition. In this series of experiments, it was our goal to convert this laboratory technique into a useful clinical assay, allowing measurement of the target enzyme and detection of the key intermediate in clinical specimens taken from patients being treated with the topo I inhibitor topotecan. Because available antibodies were not sufficiently sensitive at the start of this project, we identified a highly specific human SCL-70 antibody from a patient with scleroderma, which allowed quantitative determination of topo I copy number in HeLa and HT-29 cell lines. Additional refinements of the Western blot technique were accomplished to improve signal:noise ratio. In surgical tumor specimens, we found the median topo I level to be 30.1 x 10(5) copies/cell for gastric adenocarcinomas, compared...Continue Reading

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