A new actinoleukin-like antitumor antibiotic, BBM-928A, has been shown to interact with isolated DNA molecules. BBM-928A contains two substituted quinolines linked by a cyclic decapeptide. Quenching effects of the covalently closed superhelical PM2 DNA on the BBM-928A fluorescence revealed a strong interaction with an apparent association constant of 1.93 x 10(7) M-1 and with 11 deoxyribonucleic acid (DNA) nucleotides per BBM-928A binding site. Viscometric studies indicated the BBM-928A induced an unwinding-rewinding process of the closed superhelical PM2 DNA typically observed for DNA intercalators. The unwinding angle (43 degrees) induced by BBM-928A was almost twice that of the ethidium bromide (26 degrees), a monofunctional intercalator. The BBM-928A-induced increase of the helix length of sonicated rodlike calf thymus DNA was approximately 1.5-fold that induced by the ethidium bromide. On the basis of these observations, we concluded that BBM-928A bifunctionally intercalated with DNA in a manner similar to the bifunctional intercalation of echinomycin.

Production of single-strand breaks (ssb) and double-strand breaks (dsb) of PM2 phage DNA by several structurally related bleomycin (BLM) analogues was studied by gel electrophoresis. BLM A2 and BLM B2 produced a comparable extent of dsb. In various experiments, BLM A2 and BLM B2, at 22-41 ng/mL, degraded 50% of the form I DNA into 33-38% form II and 12-17% form III DNA. BLM B1' produced ssb and dsb at a ratio similar to that of BLM A2, but both at a rate less than half that of BLM A2. Phleomycin (PLM) D1 induced an equivalent amount of ssb but only one-eighth of dsb induced by BLM B2. The relatively lower extent dsb production for PLM D1 was observed either in borate buffer (pH 9.5) or in Tris-HCl buffer (pH 7.5) and in the presence or absence of exogenous Fe(II). Deamido-BLM A2 produced ssb to an extent approximately half that of BLM A2 and dsb to less than one-eighth that of BLM A2. The following conclusions were drawn. (1) BLM analogues produced ssb and dsb to different extents and ratios. (2) The ratio of dsb to ssb varied depending on the analogue, indicating a lack of a direct correlation between ssb and dsb. (3) The extent of ssb and dsb was affected by modifications on both the C- and N-terminal half-molecules of BLM: modification of either the N-terminal amide or the bithiazole greatly reduced dsb, whereas changes in structure or charge in the C-terminal amine affected ssb and dsb to a similar extent.

We developed an assay in which single-strand breakage (ssb) and double-strand breakage (dsb) of intracellular DNA by chemical agents can be accurately quantitated and differentiated. Escherichia coli cells containing plasmid pBR322 DNA were incubated with the antitumor antibiotics bleomycin A2 (BLM A2) or talisomycin A (TLM A). The plasmid DNA was isolated and then analyzed by electrophoresis on 1% agarose gels to separate the following conformational forms of plasmid DNA: (i) native, covalently closed, super helical, form I; (ii) nicked, relaxed circular, form II; and (iii) double-strand broken, linear, form III. Quantitation by densitometric analysis of the gels showed that BLM A2 and TLM A were equally active in terms of the concentrations of drug necessary to reduce equivalent amounts of form I DNA in the cells, whereas in vitro (using isolated pBR322 DNA as a drug substrate) twofold more TLM A than BLM A2 was required to produce an equivalent amount of reduction in form I DNA. TLM A produced more intracellular dsb than did BLM A2. The intracellular dsb activities (dsb/ssb ratio) measured from BLM A2 and TLM A were equivalent to those measured for the respective agents when isolated pBR322 DNA was used as the substrate. In E. coli both ssb and dsb were repaired, but TLM A damage was repaired more slowly and to a lesser extent, which may reflect the relative frequency of dsb.The bleomycins (BLM) are a group of glycopeptide antibiotics which were first isolated by Umezawa et al. (36) as copper complexes from culture media of Streptomyces verticillus. BLM was found to be a potent antibiotic against a variety of microorganisms (10) and active against several neoplasms in humans, both as a single agent and in combination chemotherapy (7). The primary mechanism of action of BLM cytotoxicity appears to be interaction with and subsequent breakage of cellular DNA (3, 9; for reviews see references 35 and 39).Talisomycin (TLM) is an antitumor antibiotic related structurally to BLM, which contains two new amino acids and a unique sugar, 4-amino-4,6-dideoxy-L-talose that have not been found previously in the BLM complex (24). A TLM congener, TLM S10 is the first bleomycinic acid-substituted BLM analog to enter clinical trials (R. Comis, R. Gaver, S. Ginsberg, B. Poiesz, A. Louie, S. D. DiFino, A. Scalzo, Abstr. Proc. Am. Assoc. Cancer Res. 25:116). Although the mechanism by which TLM produces DNA breaks appears to be similar to BLM, i.e., complex formation with Fe(II) and oxygen and subsequent free radical formation (2, 15, 33), a number of differences between these two groups of antitumor antibiotics have been reported, including differences in their respective antibacterial and antifungal activities (11), in vivo animal antitumor activities (1, 24, 29), pharmacokinetics (37), and cell-cycle phase specificities (18).Studies which distinguish it structurally from BLM, result in a reduction of the total DNA breakage activity when using isolated PM2 DNA as a substrate (19,33), enhanced production of double-strand breaks (...

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