Apoptosis of unstimulated human lymphocytes and DNA strand breaks induced by the topoisomerase II inhibitor etoposide (VP-16).
Etoposide (VP-16)-induced DNA strand breaks and repair and apoptosis of unstimulated human lymphocytes have been studied using DNA comet assay, electrophoresis of low-molecular-weight DNA extracts, and fluorescence microscopy. Incubation of unstimulated human lymphocytes with VP-16 (50-200 microg/ml) for 3 or 24 h induced apoptosis. This conclusion is supported by results of morphological studies, evaluation of the proportion of hypodiploidy and internucleosomal degradation of DNA in lymphocytes. Etoposide-induced formation of DNA strand breaks preceded the appearance of these conventional apoptotic manifestations. The number of single-strand breaks depended on VP-16 concentration, and 2-3 h after its removal from the incubation medium they were repaired. The hydroxyl group at the C-4; position of the etoposide dimethoxyphenol ring may be responsible for the formation of single-strand breaks. Double-strand breaks were unrepaired 20 h after the change of the incubation medium. The number of double-strand breaks and a proportion of apoptotic cells did not exhibit any dependence on VP-16 concentration and/or duration of cell exposure to this agent. We suggest that the cytotoxic effect of VP-16 on unstimulated lymphocytes is mediated by a topoisomerase II isoform, topoisomerase II-beta, which is localized in the nucleolus and is not related to the cell cycle.
Tronov
V A
VA
Institute of Chemical Physics, Russian Academy of Sciences, Moscow, 117977, Russia. tronov@chph.ras.ru
Konoplyannikov
M A
MA
Nikolskaya
T A
TA
Konstantinov
E M
EM
D017209:Apoptosis / Q000187:drug effects* / Q000502:physiology; D004249:DNA Damage; D053938:DNA Fragmentation / Q000187:drug effects; D004260:DNA Repair / Q000187:drug effects; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005047:Etoposide / Q000494:pharmacology*; D006801:Humans; D066298:In Vitro Techniques; D008214:Lymphocytes / Q000166:cytology* / Q000187:drug effects* / Q000378:metabolism; D059005:Topoisomerase II Inhibitors
D004791:Enzyme Inhibitors; D059005:Topoisomerase II Inhibitors; D005047:Etoposide