Mechanistic studies on bleomycin-mediated DNA damage: multiple binding modes can result in double-stranded DNA cleavage

Abstract

The bleomycins (BLMs) are a family of natural glycopeptides used clinically as antitumor agents. In the presence of required cofactors (Fe(2+) and O(2)), BLM causes both single-stranded (ss) and double-stranded (ds) DNA damage with the latter thought to be the major source of cytotoxicity. Previous biochemical and structural studies have demonstrated that BLM can mediate ss cleavage through multiple binding modes. However, our studies have suggested that ds cleavage occurs by partial intercalation of BLM's bithiazole tail 3' to the first cleavage site that facilitates its re-activation and re-organization to the second strand without dissociation from the DNA where the second cleavage event occurs. To test this model, a BLM A5 analog (CD-BLM) with beta-cyclodextrin attached to its terminal amine was synthesized. This attachment presumably precludes binding via intercalation. Cleavage studies measuring ss:ds ratios by two independent methods were carried out. Studies using [(32)P]-hairpin technology harboring a single ds cleavage site reveal a ss:ds ratio of 6.7 +/- 1.2:1 for CD-BLM and 3.4:1 and 3.1 +/- 0.3:1 for BLM A2 and A5, respectively. In contrast with BLM A5 and A2, however, CD-BLM mediates ds-DNA cleavage through cooperative binding of a second CD-BLM molecule to effect cleavage on the second strand. Studies using the supercoiled plasmid relaxation assay revealed a ss:ds ratio of 2.8:1 for CD-BLM in comparison with 7.3:1 and 5.8:1, for BLM A2 and A5, respectively. This result in conjunction with the hairpin results suggest that multiple binding modes of a single BLM can lead to ds-DNA cleavage and that ds cleavage can occur using one or two BLM molecules. The significance of the current study to understanding BLM's action in vivo is discussed.

Figure 1.

Structures of BLM A2, A5, Phleomycin D1 (PLM), chlorinated BLMs and CD-BLM.

Figure 2.

Pathways of BLM-mediated DNA damage.

Figure 3.

Sequence of GT-2. The predicted ss-cleavage sites of GT-2 are numbered. The G in bold denotes the internal [³²P] labeling site.

Figure 4.

Synthesis of CD-BLM (1).

Figure 5.

Cleavage of internal-[³²P] GT-2 by CD-BLM analyzed by PAGE. Lane 1: Maxim-Gilbert G+A reaction. Lane 2–5: 0, 12.5, 25, 37.5, 50 μM CD-BLM.

Figure 6.

Quantitation of ss- and ds-cleavage products of internal-[³²P] GT-2. (A), by 7.5 μM BLM A5; (B), by 50 μM CD-BLM.

Figure 7.

The concentration dependence of %ss-cleavage versus %ds-cleavage at T13 and T38 by CD-BLM.

Figure 8.

Competitive inhibition of CD-BLM-mediated DNA cleavage by Co-BLM. Lane 1: Control. Lane 2–4: 5, 10, 20 μM CD-BLM. Lane 5–7: 5, 10, 20 μM CD-BLM with 0.25 equivalent Co-BLM. Lane 8–10: 5, 10, 20 μM CD-BLM with 0.5 equivalent Co-BLM. Lane 11: 5 μM BLM A5. Lane 12: 5 μM BLM A5 with five equivalents Co-BLM.

Figure 9.

Quantitation of CD-BLM mediated cleavage and its attenuation by BLM-Co(III)-OOH. (A), 20 μM CD-BLM; (B), 20 μM CD-BLM + 5 μM Co-BLM; (C), 20 μM CD-BLM + 10 μM Co-BLM; (D), 5 μM BLM A5; (E), 5 μM BLM A5 + 25 μM Co-BLM.