(CANCER RESEARCH 31, 1573-1579, November 1971] Cross-linking of DNA by Alkylating Agents and Effects on DNA Function in the Chick Embryo Jerome J. McCann, Timothy M. Lo, and D. A. Webster Department of Biology, Illinois Institute of Technology, Chicago, Illinois 60616 SUMMARY single-stranded DNA partitions into the dextran-rich lower phase (3). This assay has been used to demonstrate that many Drug-induced cross-links are found in the DNA of chick preparations of DNA normally contain some cross-linked embryos within 6 hr after injection of mitomycin C and DNA, which was postulated to arise when DNA is sheared methyl-di-(2-chloroethyl)amine into the egg and 24 hr after during purification (2, 3). In Escherichia coli, an enzyme injection of triethylene thiophosphoramide. Effects on the system, consisting of an exonuclease and a ligase, has been rates of synthesis of DNA, RNA, and protein were studied shown to cross-link DNA terminally (25). with chemical assays for total content of these When difunctional alkylating agents were injected onto the macromolecules as well as radioactive precursor incorporation. area vasculosa of 4-day chick embryos in ovo, within 24 hr a All three drugs inhibited DNA synthesis before RNA and specific type of macrophage was formed over the entire protein synthesis were affected, but there were discrepancies embryo (24). These macrophages were indistinguishable by between the two methods of measuring macromolecular light and electron microscopy from macrophages which are synthesis; thus, radioactive precursor incorporation is not formed during the process of "programmed cell death," a always a reliable measurement of macromolecular synthesis in phenomenon believed important for sculpturing the normal the chick embryo. When the effects of contours of the limbs (18). The macrophages arise from methyl-di-(2-chloroethyl)amine were compared to those of mesodermal cells adjacent to the dead or dying cells, dimethyl-2-chloroethylamine, a monofunctional analog, the presumably in response to their moribund state, and engulf former was a more effective inhibitor of DNA and RNA and digest the cells and cell debris associated with cell death synthesis at equivalent alkylating doses; however, the fact that (18). Besides the difunctional alkylating agents, only the monofunctional analog had some inhibitory activity hydroxyurea and daunomycin could induce the formation of suggests that not all the effects of difunctional alkylating macrophages in 4- and 5-day chick embryos; all other tested agents are due to their DNA cross-linking activity. metabolic poisons did not have this effect. The latter included monofunctional alkylating agents, actinomycin D, cycloheximide, colchicine, cyanide, and many others (24). On INTRODUCTION the basis of these results, it was postulated that programmed cell death may occur by a mechanism similar to that produced Alkylating agents are inhibitors of cell division, and for this by the difunctional alkylating agents, hydroxyurea, and reason they have been valuable tools for both basic research in daunomycin. With the polyethylene glycol-dextran 2-phase cell biology and cancer chemotherapy. It had been speculated system, DNA from chick embryos treated with mitomycin C, that the difunctional (and polyfunctional) alkylating agents thioTEPA,1 or Carzinophilin was demonstrated to contain a exerted their effects because of their ability to cross-link DNA. higher percentage of cross-linked DNA than untreated Although there was evidence for the ability of these agents to embryos, but DNA from chick embryo tissues containing cross-link DNA in vitro (4, 8, 12, 15), no such evidence fora prospective necrotic cells and necrotic cells contained no similar activity in vivo was available until Szybalski and Iyer higher percentage of cross-linked DNA than control tissues (21) using cesium salt density gradient centrifugation showed (24). Although these direct experiments failed to demonstrate that mitomycin C in the growth medium cross-linked the DNA a role for cross-linked DNA in programmed cell death, they are of bacterial cells and mammalian tissue culture cells. They also inconclusive because of the relatively few cells involved, the demonstrated that the amount of cross-linking increased with fact that they comprised only about 1% of the extirpated increasing amounts of mitomycin C in the medium. tissues, and limitations of the assay, which would have Cross-linked DNA, when denatured, will spontaneously required about 700 more cross-links per nucleus to have "zipper up" to reform double-stranded helical DNA; this detected an increase in cross-linked DNA (24). The positive process is concentration independent, and one needs only to effects of hydroxyurea, a specific inhibitor of DNA synthesis be able to detect a small fraction of double-stranded DNA in (13), and daunomycin, which also affects nucleic acid the presence of a large amount of single-stranded DNA. A new metabolism although the mechanism of its action is not known assay, which can do this, uses polyethylene glycol and dextran (11), suggest that an inhibition of DNA synthesis is the in a 2-phase system in which double-stranded DNA partitions into the polyethylene glycol-rich upper phase and 'The abbreviations used are: thioTEPA, triethylene thiophos Received April 8, 1971 ¡acceptedJune 15, 1971. phoramide; HN2, methyl-di-(2-chloroethyl)amine. NOVEMBER 1971 1573 Downloaded from cancerres.aacrjournals.org on September 29, 2021. © 1971 American Association for Cancer Research. Jerome J. McCann, Timothy M. Lo, and D. A. Webster primary mechanism by which all these drugs act and may also RNA with the orcinol assay (19), and protein with thebiuret be a primary mechanism of cell death in programmed cell assay. death during normal chick embryo development. Thus, it For the estimation of cross-linked DNA, the embryos were became imperative to study the effect of these drugs on homogenized in 0.15 M NaCl : 0.1 M EDTA, pH 8.1, 2 macromolecular synthesis, especially DNA synthesis, during ml/embryo, with a glass homogenizer fitted with a Teflon chick embryo development. First, we undertook a detailed pestle. Pronase (Calbiochem, Los Angeles, Calif.), and a 20% study of the cross-linking activity of various drugs with the solution of sodium dodecyl sulfate were added to give final polyethylene glycol-dextran 2-phase system but with an concentrations of 500 ng/ml and 1%, respectively. After incubation for 20 to 24 hr at 37°, the solutions were abridged procedure relative to that used formerly (24). The drugs tested were mitomycin C, HN2, thioTEPA, deproteinized with an equal volume of chloroformibutanol hydroxyurea, and dimethyl-2-chloroethylamine. The effects of (4:1, v/v) and centrifuged to separate the phases, and the these drugs on the incorporation of specific radioactive nucleic acids were precipitated from the aqueous phase with precursors into DNA, RNA, and protein as a function of time 95% ethanol. The nucleic acids were collected on a stirring rod after injection was determined as well as their effects on total and dissolved in cold 0.2 M NaOH:0.02 M EDTA (trisodium salt). The solutions were warmed to 45°for 15 min, cooled to DNA, RNA, and protein content. The results show that difunctional alkylating agents can cross-link the DNA of the 0°,and neutralized with 0.3 volume of 1.0 M KH2P04. After chick embryos within 6 hr after their injection and that DNA another deproteinization step with chlorofornrbutanol, the is the first macromolecule for which synthesis is inhibited by solutions were dialyzed against 1 liter of 0.01 M sodium these drugs. phosphate, pH 7.0, for ca. 24 hr at 4°with 1 change of buffer. Chloroform was added to the dialysis buffer, and the dialysis MATERIALS AND METHODS tubing was boiled for 30 min in 0.1 M acetic acid to remove UV-absorbing materials. Cross-linked DNA content of the The drugs were obtained from the same sources that were dialyzed samples was estimated with the polyethylene used previously (24). The radioactive compounds were glycol:dextran 2-phase system (1) with a stock made up of purchased from International Chemical and Nuclear Corp., 23% (w/w) polyethylene glycol and 3 volumes 34% (w/w) Irvine, Calif., and had the following specific activities: dextran. One volume of sample was added to 0.8 volume of thymidine-methyl-3H (10.5 Ci/mmole), uridine-5-3H (21.7 stock solution and mixed intermittently with a Vortex mixer Ci/mmole), L-leucine-4,5-3H (29.8 Ci/mmole), uridine-2-14C for 1 to 2 hr. Low-speed centrifugation separated the phases, (30 mCi/mmole), L-leucine-14C (uniformly labeled, 210 and the upper phase containing the double-stranded DNA was mCi/mmole). The fertile eggs were obtained from C. L. Sharp carefully removed and mixed with an approximately equal (Glen Ellyn, 111.). DNase 1 (bovine pancreas) and RNase volume of chloroform to precipitate the phase polymers. After (bovine pancreas) were purchased from Worthington Bio centrifugation, the upper aqueous phase was removed and chemical Corp. (Freehold, N.J.). We used Pharmacia T500 assayed directly for DNA content with the Burton dextran, Lot 4024, primarily, and General Biochemicals, Inc. modification (6) of the diphenylamine assay except that the (Chagrin Falls, Ohio) polyethylene glycol (M.W.5700 to precipitation step was omitted. The DNA content of the 6700), Lot 88452. dialyzed samples before phase separation was simultaneously Eggs were incubated for 4 days and candled to determine determined; all determinations were in duplicate. Appropriate the position of the embryo, and the drugs were injected on the controls were run simultaneously in the 2-phase system, area vasculosa as previously described (24). The radioactive including native DNA, heat-denatured DNA, and phosphate precursors were injected into the yolk sac through a hole buffer alone. drilled in the narrow end of the egg.