Differential induction of secondary DNA fragmentation by topoisomerase II inhibitors in human tumor cell lines with amplified c-myc expression.
In order to understand the cellular events associated with cell death after the formation of topoisomerase II-DNA cleavable complexes, we compared the induction of endonucleolytic DNA fragmentation by etoposide and its more potent analog, teniposide (VM-26) in the human cell lines HT-29 and HL-60. A new filter-binding assay is described, which allows rapid quantification of nonprotein-linked DNA fragmentation involved in apoptosis. Both cell lines showed similar loss of colony formation ability following 30 min of treatment with various VM-26 concentrations even though the initial topoisomerase II-mediated DNA single-strand break frequency was higher in HL-60 cells. DNA repair studies following drug removal indicated that VM-26-induced DNA breaks reversed rapidly and completely in HT-29 cells, while in HL-60 cells, the initial lesions persisted at and above 5 microM VM-26. In both cell lines, topoisomerase II cleavage complexes, as measured by DNA-protein cross-links by alkaline elution, reversed rapidly and completely within 2-3 h. Secondary DNA fragmentation resembling chromatin endonucleolytic cleavage by apoptosis could be detected in HL-60 cells 3 h after VM-26 or etoposide treatment but not in HT-29 cells. Secondary DNA fragmentation was also induced in the human colon cancer cell lines COLO 320, which have c-myc amplification. Since HL-60 cells also have c-myc amplification and HT-29 do not, it is possible that c-myc overexpression may be involved in secondary DNA fragmentation. Finally, our results indicate heterogeneity of cell death mechanisms after exposure to topoisomerase II inhibitors among human cancer cell lines.
Laboratory of Molecular Pharmacology, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20892.
R
R
Bertrand
M
M
Sarang
J
J
Jenkin
D
D
Kerrigan
Y
Y
Pommier
D000230:Adenocarcinoma / Q000235:genetics; D003110:Colonic Neoplasms / Q000235:genetics; D004249:DNA Damage; D004260:DNA Repair; D004273:DNA, Neoplasm / Q000187:drug effects*; D005047:Etoposide / Q000494:pharmacology*; D005784:Gene Amplification; D016259:Genes, myc; D006801:Humans; D015473:Leukemia, Promyelocytic, Acute / Q000235:genetics; D013713:Teniposide / Q000494:pharmacology*; D014407:Tumor Cells, Cultured / Q000187:drug effects; D014410:Tumor Stem Cell Assay
D004273:DNA, Neoplasm; D005047:Etoposide; D013713:Teniposide