DNA strand scission and cross-linking by diaziridinylbenzoquinone (diaziquone) in human cells and relation to cell killing.

The effects of 3,6-diaziridinyl-2,5-bis(carboethoxyamino)-1,4-benzoquinone (diaziquone; AZQ) on various cell types were studied in relation to two chemical reactivities that this drug would be expected to have intracellularly. AZQ can undergo a reduction-oxidation cycle of the quinone function; this could generate free radicals which could produce DNA damage, especially DNA strand scission. The second reactivity, based on the two aziridine groups, could produce alkylation reactions that could produce DNA cross-links. DNA strand breakage and cross-linking were measured by alkaline elution and were compared with cell killing assayed by colony survival. Among the cell strains studied (human IMR-90, VA-13, and HT-29 and mouse L1210), marked differences were found in the magnitudes of DNA strand breakage and interstrand cross-linking produced by AZQ. Most striking, IMR-90 cells exhibited substantial strand breakage and little or no interstrand cross-linking, whereas the reverse was true for HT-29 cells. Cell killing correlated well with interstrand cross-linking but did not correlate with strand scission in these cell lines. It is concluded that AZQ produces DNA strand breaks and interstrand cross-links by different mechanisms which vary independently among different cell lines.

Interstrand DNA crosslinking by 4,5'8-trimethylpsoralen plus monochromatic ultraviolet light. Studies by alkaline elution in mouse L1210 leukemia cells.

DNA crosslinking by 4,5',8-trimethylpsoralen plus monochromatic ultraviolet light of wavelength 365 nm was studied in mouse L1210 leukemia cells. DNA breaks and crosslinking were evaluated by alkaline elution of DNA from poly(vinyl chloride) filters. Trimethylpsoralen plus 365 nm light produced DNA crosslinks but not breaks. The kinetics of crosslinking were linear with respect to concentration and second-order with respect to light exposure time. The latter finding supports the proposed two photon mechanism for the formation of diadducts. In contrast to DNA crosslinking agents such as nitrogen mustard, nitrosoureas and platinums, trimethylpsoralen crosslinks were resistant to proteolytic digestion. Thus, trimethylpsoralen plus 365 nm light produced interstrand crosslinks, as proposed for a bifunctional agent binding to bases on opposite DNA strands.

DNA crosslinking and cytotoxicity in normal and transformed human cells treated with antitumor nitrosoureas.

Normal (IMR-90) and simian virus 40-transformed (VA-13) human embryo cells were treated with antitumor nitrosoureas, and the effects on cell viability and cell DNA were compared. All six nitrosoureas tested were more toxic to VA-13 cells than to IMR-90 cells as measured by decrease in cell proliferation or in colony formation. The nitrosoureas capable of generating alkylisocyanates produced a smaller difference between the cell types than did derivatives lacking this capacity. DNA damage was measured by alkaline elution in cells treated with four chloroethylnitrosoureas. Whereas VA-13 cells exhibited dose-dependent interstrand crosslinking, little or none was detected in IMR-90 cells. The IMR-90 cells, however, exhibited at least as much DNA-protein crosslinking as did VA-13 cells. The results can be interpreted in terms of a possible difference in DNA repair between the cell lines.

DNA-protein crosslinking by trans-platinum(II)diamminedichloride in mammalian cells, a new method of analysis.

DNA-protien crosslinks produced in mouse leukemia L1210 cells by trans-Pt(II)diamminedichloride were quantitated using the technique of DNA alkaline elution. DNA single-strand segments that were or were not linked to protein were separable into distinct components by alkaline elution after exposure of the cells to 2--15 kR of X-ray. Protein-linked DNA strands were separated on the basis of their retention of filters at pH 12 while free DNA strands of the size generated by 2--15 kR of X-ray passed rapidly through the filters. The retention of protein-linked DNA strands was attributable to adsorption of protein to the filter under the conditions of alkaline elution. The results obeyed a simple quantitative model according to which the frequency of DNA-protein crosslinks could be calculated.

DNA-protein cross-linking and DNA interstrand cross-linking by haloethylnitrosoureas in L1210 cells.

Bifunctional alkylating agents are known to produce cross-links between DNA and protein and between paired DNA strands. The possibility of discriminating these two classes of cross-links in L1210 cells treated with haloethylnitrosoureas or nitrogen mustard was explored with the alkaline elution technique. Two classes of cross-links were demonstrated, based on sensitivity to proteinase K; the proteinase-sensitive cross-links appear to be DNA-protein cross-links, and the proteinase-resistant class may include interstrand cross-links. Proteinase-sensitive cross-links form more rapidly than do proteinase-resistant cross-links in cells treated with chloroethylnitrosoureas, perhaps because these agents can chloroethylate protein sulfhydryl or amino groups followed by rapid reaction of these chloroethylated groups with DNA. Although both types of cross-links produced by nitrogen mustard disappeared or were repaired after 24 hr, the removal of cross-links produced by chloroethylnitrosoureas either did not occur or was incomplete in 24 hr. In addition to cross-links, cells treated with haloethylnitrosoureas exhibited DNA strand breaks; a method is suggested for estimating the apparent frequencies of strand breaks and cross-links in the DNA.

Fluorescent light-induced DNA crosslinkage and chromatid breaks in mouse cells in culture.

A single 20-hr exposure of mouse cells derived from embryonic or lung tissue to cool-white fluorescent light (4.6 W/m2) causes both DNA damage and chromosome aberrations including chromatid breaks, exchanges, and minutes. In Kohn's alkaline elution technique, the DNA from exposed cells elutes more slowly than that from shielded cells. Because larger molecular weight DNA elutes slower than smaller, we interpret these results to mean that the DNA in cells exposed to light is crosslinked. The estimated frequency of crosslinks is sufficient to account for the number of chromatid breaks observed. The types of chromosome aberrations produced by light indicate that the primary lesion results in chromatid rather than chromosome breaks, and the results suggest an influence of cell density in that cells in densely populated cultures showed few or no chromatid breaks after irradiation. The present results, together with observations from the literature, suggest that the DNA crosslinkage and the chromosome aberrations produced by light may be related.

Differences between melphalan and nitrogen mustard in the formation and removal of DNA cross-links.

The formation of DNA cross-links is thought to represent the lethal lesion following exposure of cells to bifunctional alkylating agents. Since differences in rates of formation and repair of cross-links may explain differences in activity of these agents, we have studied these events following exposure of L1210 cells to nitrogen mustard (HN2) and melphalan. With the technique of alkaline elution, it was possible to measure cross-linking at doses that result in relatively little cell kill. Following a 30-min exposure to HN2, DNA cross-links increased for 1 to 2 hr and were then removed by a process that was virtually complete in 24 hr. In contrast, following a 30-min exposure to melphalan, cross-link formation increased for 12 hr and removal was much slower than it was for HN2. Comparison of cell survival with cross-linking kinetics suggests that persistence of the cross-links with time is an important factor in determining lethality.

Kinetics of formation and disappearance of a DNA cross-linking effect in mouse leukemia L1210 cells treated with cis- and trans-diamminedichloroplatinum(II).

cis- and trans-diamminedichloroplatinum(II) (PDD) produced a DNA cross-linking effect in mouse leukemia L1210 cells, which was demonstrable after subtoxic treatments with the DNA alkaline elution technique. Cross-linking effects developed following treatments with concentrations as low as 1 micron for cis-PDD and 5 micron for trans-PDD, which permitted over 80% survival of colony-forming ability. The maximum cross-linking effect by cis-PDD required about 12 hr of posttreatment incubation before it was fully developed, whereas the cross-linking effect of trans-PDD was fully developed at the end of the 1-hr drug exposure. The cross-linking effects of both agents were reversed upon further incubation of the cells.

Differential cytotoxicity between transformed and normal human cells with combinations of aminonucleoside and hydroxyurea.

Normal human WI-38 cells can be protected from killing by hydroxyurea if proliferation is arrested during drug treatment. This protection was demonstrated both in cells arrested by density-dependent inhibition and by 3'-amino-3'deoxy-N6,N6-dimethyladenosine (puromycin aminonucleoside). In contrast, VA-13 cells (a simian virus 40-transformed clone of WI-38) were not arrested under these conditions, and continued to be sensitive to hydroxyurea. These results suggest that a search for agents that selectively and reversibly inhibit normal cycling human cells might lead to an enhancement of differential tumor toxicity.

Single-strand scission and repair of DNA in mammalian cells by bleomycin.

DNA single-strand breakage by bleomycin treatment of cultured mammalian cells was demonstrated by the method of alkaline elution. Elution patterns from treated L1210 cells indicated that part of the DNA was extensively broken while the remainder was affected to a lesser degree. This biphasic effect, which was less prominent in human fibroblasts, may reflect a selective sensitivity either of part of the cell population or of part of the DNA within individual cells. In both cell types, the DNA damage was at least partially repaired upon incubation of the cells after removal of drug. Bleomycin did not inhibit the rejoining of X-ray-induced single-strand breaks. The production and repair of DNA single-strand breaks after bleomycin treatment were the same in normal human and xeroderma pigmentosum fibroblasts, indicating that these events do not require the excision endonuclease that appears to be defective in these ultraviolet light-sensitive xeroderma cells.

Effect of pH on the bleomycin-induced DNA single-strand scission in L1210 cells and the relation to cell survival.

The susceptibility of cultured L1210 cells to bleomycin was investigated as a function of pH of the medium, and was compared with DNA damage measured by alkaline elution. With increasing pH of the medium, both cytotoxicity and DNA damage increased. This was observed in the effects of bleomycin on cell proliferation, colony-forming ability, and DNA elutability. The reduction of colony-forming ability correlated with DNA single-strand scission, and this correlation was independent of pH.

Fractionation of DNA from mammalian cells by alkaline elution.

The method of alkaline elution provides a sensitive measure of DNA single-strand length distribution in mamalian cells and is applicable to a variety of problems concerning DNA damage, repair, and replication. The physical basis of the elution process was studied. The kinetics of elution above the alkaline transition pH were found to occur in two phases: an initial phase in which single-strand length is rate limiting, followed by a phase in which elution is accelerated due to the accumulation of alkali-induced strand breaks. The range of DNA single-strand lengths that can be discriminated by elution above the alkaline transition pH was estimated by calibration relative to the effects of x ray, and was found to be 5 X 10(8)-10(10) daltons. Shorter DNA strands elute within the pH transition zone, which extended from pH 11.3 to 11.7 when tetrapropylammonium hydroxide was used as base. This elution was relatively rapid, but was sharply limited by pH, according to the length of the strands: the length of the strands eluted increased with increasing pH. Alkaline elution was inhibited by treatment of cells with low concentrations of nitrogen mustard, a bifunctional alkylating known to cross-link DNA. On investigation of the possibility that DNA subclasses may differ in their elution behavior, satellite L strands were found to elute more slowly from cells exposed to a low dose of x ray than did the bulk DNA.