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A novel bioassay was developed to permit the identification of cytotoxic natural principles that bind to DNA. A hexane extract of Schoepfia californica cytotoxic to cultured KB cells displayed much less cytotoxic potential when the culture medium contained exogenously added calf thymus DNA. Fractionation of the extract afforded a purified principle shown to be 9-octadecynoic acid, an 18-carbon, unbranched acetylenic fatty acid. 9-Octadecynoic acid had an apparent DNA dissociation constant of 1.8 mM; it inhibited topoisomerase I mediated DNA filter binding but did not inhibit the DNA topoisomerase I mediated relaxation of a supercoiled plasmid DNA. The fatty acid was weakly inhibitory to DNA polymerase alpha. 9-Octadecynoic acid possesses none of the structural characteristics of known DNA binding molecules and may bind to DNA by some novel mechanism.

Growth inhibition and cell killing by bleomycin are believed to be related to the ability of this antibiotic to cleave chromosomal DNA. Because bleomycin has an intracellular site of action, its ability to cross biological membranes must be critical to its overall effectiveness as an antitumor agent. The local anesthetic dibucaine acts to enhance membrane fluidity; therefore, the reported ability of this local anesthetic to modulate bleomycin effects on KB cells was investigated. Cells were treated with various bleomycin congeners in the presence or absence of dibucaine for 24 h. Dibucaine enhanced the inhibition of cell growth mediated by bleomycin A2, demethylbleomycin A2, bleomycin B2, and isobleomycin A2. N-Acetylbleomycin A2 did not inhibit cell growth in the absence of dibucaine, but it was inhibitory in the presence of dibucaine. Cells treated simultaneously for analysis of DNA breakage on alkaline sucrose gradients revealed that breakage was also enhanced in the presence of dibucaine. The degree of enhancement varied with dose and bleomycin congener. N-Acetylbleomycin A2 did not induce DNA breakage in either the absence or the presence of dibucaine. While growth inhibition and net DNA breakage correlated reasonably well in the absence of dibucaine for each bleomycin analogue tested, proportionality was lost in the presence of dibucaine, and very little DNA breakage was present when growth inhibition was complete. These observations imply that, at least in the presence of dibucaine, bleomycin may mediate growth inhibition at some locus in addition to chromosomal DNA and, also, that a given net amount of bleomycin analogue induced DNA damage per se does not produce a specific degree of growth inhibition.

Bleomycin is hypothesized to cause cell growth inhibition and cell death via DNA cleavage. We have attempted to determine if net DNA cleavage is directly related to growth inhibition by measuring whether both parameters vary in parallel. Of primary importance to these studies was use of several bleomycin congeners. We have shown that these congeners vary in their abilities both to inhibit cell growth and to cause DNA damage. Bleomycin B2, tallysomycin, and phleomycin were the most potent growth inhibitors, and bleomycin B2 caused the most DNA damage. N-Acetylbleomycin A2 was inactive in both assays. The net amount of DNA damage measured at two levels of growth inhibition was compared for each congener and was found to vary widely among the congeners. Similarly, the degree of growth inhibition at a given level of submaximal DNA damage was found to vary widely when individual congeners were compared to each other. Hence, growth inhibition and net DNA damage due to bleomycin are not directly correlated with each other when individual congeners are compared to each other.

We have investigated the structure of parental DNA as a function of the cell cycle phase of HeLa cells. DNA was isolated from synchronized HeLa cells 0, 5, 8, and 12 h after release from a second exposure to 2 mM thymidine. These DNA preparations were characterized by CS2SO4/AgClO4 buoyant density, sensitivity to a single-strand specific nuclease, sedimentation in neutral and alkaline sucrose gradients, and sedimentation in neutral sucrose gradients after digestion with S1 nuclease. The cultures were staged according to cell cycle phase by measurements of DNA content per cell by flow microfluorometry. The cell cycle phases were G1/S (0-h culture), S (5-h culture), G2 (8-h culture), and G1 (12-h culture). There are no nuclease-sensitive sites in G2. As the cells enter G1, the number increases, with a maximum being reached in the S phase. The number of breaks in DNA with respect to cell cycle phase follows the same pattern. The amount of single strandedness, measured by buoyant density and nuclease sensitivity, is also minimal in G2, increases in G1, with a maximum achieved in the S phase. It appears that there is a chromosomal cycle, reflected as continuous structural changes in the DNA molecule, as cells traverse the cell cycle.