Apoptosis of Unstimulated Human Lymphocytes and DNA Strand Breaks Induced by the Topoisomerase II Inhibitor Etoposide (VP-16)

V. A. Tronov^1*, M. A. Konoplyannikov¹, T. A. Nikolskaya², and E. M. Konstantinov¹

¹Institute of Chemical Physics, Russian Academy of Sciences, ul. Kosygina 4, Moscow, 117977 Russia; fax: (095) 938-2156; E-mail: tronov@chph.ras.ru

²Institute of Biochemical Physics, Russian Academy of Sciences, ul. Kosygina 4, Moscow, 117977 Russia; fax: (095) 137-4101

^* To whom correspondence should be addressed.

Received April 16, 1998; Revision received June 25, 1998

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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 µg/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.

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KEY WORDS: lymphocytes, etoposide, topoisomerase II, DNA breaks, DNA comets

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