Topotecan-induced topoisomerase IIalpha expression increases the sensitivity of the CML cell line K562 to subsequent etoposide plus mitoxantrone treatment.

Abstract

A new chemotherapy regimen was designed for leukemia to improve response to therapy and elucidate the possible underlying mechanisms responsible for its efficacy. Cells of the chronic myelogenous leukemia (CML) cell line, K562, were treated singly, in combination, and in sequence with clinically equivalent dosages of topotecan, which targets topoisomerase I (Topo I), and etoposide and mitoxantrone, which target topoisomerase II (Topo II), to determine the best treatment. Apoptosis, early cell deaths, and cell cytotoxicities in drug-treated cells were determined with annexin V and propidium iodide staining and MTT assays, respectively. Confocal microscopy and RT-PCR showed the cellular locations and relative increases in Topo IIalpha in topotecan-treated cells. The double comet assay of individual cells showed simultaneously free Topo proteins, non-Topo-associated DNA, and Topo-DNA complexes in drug-induced DNA fragments. Sequential treatment with topotecan on days 1-3, followed by etoposide+mitoxantrone on days 4, 5, 9 and 10 resulted in 100% cell death whereas treatments involving administration of drugs singly or simultaneously resulted in less cell kill. The cytotoxicity results in cells treated for fewer days with the same sequential chemotherapy regimen showed the same trend, and adequate surviving cells for the experiments on the cellular and molecular mechanisms of drug action were produced. An increase in Topo IIalpha mRNA from RT-PCR 1 h after topotecan treatment was observed. Observations on K562 cells treated sequentially with topotecan followed by etoposide, mitoxantrone or etoposide+mitoxantrone were as follows: (1) Topo IIalpha protein levels increased and relocated from the cytoplasm into the nucleus as detected by confocal microscopy, (2) Topo IIalpha-DNA complexes increased and were associated with fragmented DNA (positive double comets) as detected by protein-DNA double comet assay, and (3) Topo I and Topo IIbeta proteins were not associated with fragmented DNA. Topotecan-induced Topo IIalpha protein levels correlated with increased numbers of positive double comets and reduction of cell viability. Our results showed that Topo IIalpha protein induction after Topo I-directed drug treatment enhanced the sensitivity of cells to subsequent exposure to Topo II-directed drugs. Timed sequential chemotherapy with topotecan followed by etoposide+mitoxantrone is an effective regimen to ablate CML cancer cells.