The ratio of single- to double-strand DNA breaks and their absolute values determine cell death pathway

Abstract

Summary Bleomycin is a cytotoxic antibiotic that generates DNA double-strand breaks (DSB) and DNA single-strand breaks (SSB). It is possible to introduce known quantities of bleomycin molecules into cells. Low amounts kill the cells by a slow process termed mitotic cell death, while high amounts produce a fast process that has been termed pseudoapoptosis. We previously showed that these types of cell death are a direct consequence of the DSB generated by bleomycin. Here, we use deglyco-bleomycin, a bleomycin derivative lacking the carbohydrate moiety. Although this molecule performs the same nucleophilic attacks on DNA as bleomycin, we show that deglyco-bleomycin is at least 100 times less toxic to Chinese hamster fibroblasts than bleomycin. In fact, deglyco-bleomycin treatment results in apoptosis induction. In contrast, however, deglyco-bleomycin was found to generate almost exclusively SSB. Our results suggest that more than 150 000 SSB per cell are required to trigger apoptosis in Chinese hamster fibroblasts and that SSB are 300 times less toxic than DSB. Taken together with previous studies on bleomycin, our data demonstrates that cells can die by apoptosis, mitotic cell death, or pseudoapoptosis, depending on the number of DNA breaks and on the ratio of SSB to DSB. MATERIALS AND METHODS Cells and chemicalsChinese hamster DC-3F fibroblasts were maintained in the previously described culture conditions (Orlowski et al, 1988). Cell culture materials were obtained from Gibco Laboratories (CergyPontoise, France). Lyophilized BLM (Lab. Roger Bellon, Neuilly, France) was dissolved in NaCl 0.9% at pH 7 and stored at -20˚C. BLM-A 2 was kindly provided by Nippon Kayaku (Tokyo, Japan). Deglyco-BLM-A 2 was prepared by BLM-A 2 solvolysis using fluorhydric acid (Kenani et al, 1988b). Deglyco-BLM purity (i.e. the complete deglycosylation of the batch of BLM) was checked by high-pressure liquid chromatography and by ion spray/mass spectrometry, according to Kenani et al (1988). DNase-free RNase and proteinase K were purchased from Boehringer Mannheim (Meylan, France) and all other products from Sigma Chemical Co (La Verpillière, France) except when otherwise stated. Cell electropermeabilization proceduresCell electropermeabilization was performed using a square wave pulse generator (PS-15 electropulsator, Jouan, Saint-Herblain, France). After harvest by trypsinization of exponentially growing cells and inactivation of trypsin by complete medium, cells were washed 3 times in 0.5 mM Ca 2+ -supplemented serum-free S-MEM (Gibco Lab). Cells were then resuspended in the same ice-cooled medium at 2.2 × 10 7 cells ml -1 . Aliquots of 67.5 µl of the cell suspension were mixed with 7.5 µl of 10-fold concentrated drug solutions. 50 µl of the mixture were immediately deposited between two electrodes (2 mm apart) and subjected to the electric treatment (8 pulses of 100 µs and 1250 V cm -1 delivered at a frequency of 1 Hz). Then, cells were kept for 5 min at 24˚C, diluted in complete culture medium and seeded or treated as described b...

In vivo evolution of tumour cells after the generation of double-strand DNA breaks

“…Single-strand DNA breaks (SSB) in very large numbers also result in cell death (Yoshida et al, 1993;CohenJonathan et al, 1999). We also reported that not only the absolute numbers of DSB or SSB but also their ratio determine cell death pathway: true apoptosis, pseudoapoptosis or mitotic cell death (Tounekti et al, 2001).…”

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SU11752 inhibits the DNA-dependent protein kinase and DNA double-strand break repair resulting in ionizing radiation sensitization

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SUPPRESSOR OF GAMMA RESPONSE1 Links DNA Damage Response to Organ Regeneration

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