Bleomycin-induced aggregation of presolubilized and nuclear chromatin from L1210 cells

doi:10.1016/0167-4781(87)90067-4

Biochimica et Biophysica Acta (BBA) - Gene Structure and Expression

Volume 910, Issue 2, 20 November 1987, Pages 149-156

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Abstract

These studies have identified a new activity of bleomycin (in addition to the well-known DNA cleavage): drug-induced chromatin aggregation. Bleomycin treatment of presolubilized chromatin from L1210 nuclei resulted in two types of effect as shown by sedimentation analysis of intact nucleoprotein. The first effect was a dose-dependent fragmentation of chromatin to mononucleosomes (12 S) and subnucleosomal fragments (under 5 S). The second effect was aggregation manifested by the generation of large chromatin particles (over 120 S) that sedimented faster than the original material (20–40 S). Bleomycin treatment of nuclei from L1210 cells resulted in a similar, almost bimodal, size distribution of drug-released chromatin fragments. Increasing levels of bleomycin produced a gradual enhancement of the amount of small fragments (under 5 S) accompanied by generation of large, aggregated particles. Aggregation caused by high drug concentrations significantly reduced the overall extent of chromatin solubilization and allowed only the release of the most degraded frgments from nuclei. The aggregation required intact nucleoprotein, since it was not detected after high-salt deproteinization of bleomycin-treated presolubilized or nuclear chromatin. The aggregation phenomenon reflects a novel activity of bleomycin which may contribute to the drug's antiproliferative properties.

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Schedule dependent variation in human lymphocyte sensitivity to bleomycin and repair of chromosomal aberrations in G<inf>2</inf>
1992, Mutation Research - Fundamental and Molecular Mechanisms of Mutagenesis
Bleomycin (BLM) induced chromosomal damage in G₂ phase and its repair kinetics in normal human lymphocytes were studied following different treatment schedules. As a first step, a dose-response curve was obtained (concentrations of 5–50 μg/ml). For repair kinetics studies, blood samples were treated with BLM at a concentration of 20 μg/ml. Continuous treatment produced equal numbers of breaks per cell (br/c) when the cells were treated 3, 4 or 5 h before fixation. If the treatment time was extended to 6 h, the level of br/c was increased 2-fold (p < 0.001) as a result of an increased number of cells with more than 3 br/c. The curves obtained after pulse treatment showed maximal chromosome damage at time 3 (45 min BLM treatment, followed by 2 h repair in drug free medium). When the time after treatment was extended to 4 h (treatment time 5), a 50% reduction in chromosome damage was measured. It was found out that at treatment points 3, 4 and 5 the differences in breaks per cell at the different schedules applied were statistically highly significant. If caffeine (CAF) was added, the continuous treatment, BLM + CAF, induced a statistically significant increase in the frequency of br/c at every treatment point, but the shape of the curve illustrating the kinetics of chromosomal damage remained unchanged. Moreover, the addition of CAF at continuous BLM treatment brings the level of br/c close to that measured at the pulse BLM treatment except for treatment time 3. When applied in a combination with BLM, CAF considerably modified the kinetics of chromosome damage for a pulse (BLM alone) treatment. The possible reasons for the changes in the level of br/c as well as a tentative scheme for assessment of chromosome damage repair capacity after BLM treatment are discussed.
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1989, BBA - Gene Structure and Expression
This study determined whether nascent chromatin in nuclei from leukemia L1210 cells constitutes a preferential target for bleomycin. No differences were seen in fragmentation of nascent and bulk DNA as judged by DNA double-stranded cleavage and the release of acid-soluble material or subnucleosomal (under 8 S) fragments. In contrast, bleomycin-induced chromatin aggregation (Woynarowski, J.M., Gawron, L.S. and Beerman, T.A. (1987) Biochim. Biophys. Acta 910, 149–156) occurred preferentially in nascent chromatin as indicated by a retarded solubilization of nascent chromatin and generation of a fast-sedimenting material (above 45 S) in the sedimentation profiles of drug-released nascent chromatin. This preferential aggregation disappeared completely when chromatin became older than 10 min. The drug aggregation activity did not distinguish nascent and mature presolubilized oligonucleosomes. The results suggest that bleomycin recognizes higher-order structures of nascent chromatin.
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Regular paperBleomycin-induced aggregation of presolubilized and nuclear chromatin from L1210 cells

Abstract

Biochem. Pharmacol.

J. Biol. Chem.

J. Free Radiol. Biol. Med.

J. Mol. Biol.

Biochim. Biophys. Acta

Anal. Biochem.

Biochim. Biophys. Acta

Chem. Biol. Interact.

Mutat. Res.

Pharmacol. Ther.

Nucleic Acids Res.

Regular paper
Bleomycin-induced aggregation of presolubilized and nuclear chromatin from L1210 cells