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.
Bertrand
R
R
Laboratory of Molecular Pharmacology, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20892.
Sarang
M
M
Jenkin
J
J
Kerrigan
D
D
Pommier
Y
Y
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