Cross-Linking of DNA by Alkylating Agents and Effects on DNA Function in the Chick Embryo

Home , Crosslinking of DNA, Mitomycin C, Triethylenemelamine

(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 Â¡acceptedJune 15, 1971. phoramide; HN2, methyl-di-(2-chloroethyl)amine.

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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 37Â°, 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 45Â°for 15 min, cooled to DNA, RNA, and protein content. The results show that difunctional alkylating agents can cross-link the DNA of the 0Â°,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 4Â°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. Generally 1 nCi (sometimes 2 fid) of 3H-labeled thymidine and 0.2 nCi of uridine-'"C or L-leucine-I4C were used at dilutions such that RESULTS 0.1 ml was injected. After the appropriate incubation, the embryos were removed, stripped of their extraembryonic Cross-linked DNA. The purity of the dialyzed samples, prior membranes, and washed in 0.9% NaCl solution. Only living to phase separation, was estimated with A2s9:A23o, and embryos were used, generally 5 to 10 per time point. For since this ratio was generally 2.20 or greater it was assumed determination of radioactive incorporation of precursors and that the DNA was quite pure. The alkali treatment was total content of DNA, RNA, and protein, the embryos were expected both to denature the DNA and to hydrolyze the homogenized for 1 min at top speed in the Sorvall Omni-Mixer RNA. These samples were subsequently assayed for DNA, with 5 ml of 0.9% NaCl solution per embryo. Aliquotsof 0.1 RNA, and protein with the Burton assay (6), the orcinol assay ml were added to 0.5 ml of cold 1.0 M trichloroacetic acid, (19), and the assay of Lowry et al. (16), respectively, and were and the precipitates were collected on 2.4-cm Whatman GFA found to contain on the average a content of glass fiber paper. The discs were washed with 95% ethanol, RNA:DNA:protein of 2.8:1.0:0.27. It therefore became dried with a heat lamp, and added to vials containing 5 ml of imperative to know whether these high ratios of RNA and 0.4% Omni-Fluor (New England Nuclear, Boston, Mass.) in protein to DNA would interfere with the partition of DNA in toluene. Radioactivity was determined on triplicate samples the 2-phase system. To test this, a solution of 1 mg/ml of with a Beckman Model 1650 liquid scintillation counter. DNA crude yeast RNA and a chick embryo extract, made by content was estimated with the diphenylamine assay (19), blending 6-day embryos 1 min at top speed in the Sorvall

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.il RNA //g ONA incorporation of each of these precursors was tested with 2.0 4.0 6.0 ap 10 .. 20 40 T T Ã¯ Ã¯"^^^""T^r*^^^â„¢" appropriate enzymes (Table 2). Approximately 25% of the uridine is incorporated into DNA as judged by resistance to hydrolysis by RNase and base. The incorporation of uridine

0.00 Native ONA itomycin per egg -0.50 11.0

-1.00 D Protein â€¢ RNA 10.0

-1.50

6.0

-2.00 i Denatured ONA 100,,,, HN2 per egg I 5-Â° -2.50 a u K -3.00 ! 4.0 0.2 0.4 0-6 0-8 1-0" 2.0 4.0 o tig Protein/"!i/ 'i DNA K u 3.0 50i/g ThioTEPA Chart 1. Effect of RNA and protein on partition of DNA in per egg polyethylene glycol-dextran 2-phase system. Native chick embryo DNA (107 Mg)or heat-denatured chick embryo DNA (358 jug)plus increasing amounts of yeast RNA (1 mg/ml) or embryo extract (2.28 mg 2.0 20/

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Table 1 into the DNA of E. coli occurs primarily after its conversion to Assay for cross-linked DNA in 4-day chick embryos treated cytidine (9), and a similar pathway may exist in chick with hydroxyurea or dimethyl-2-chloroethylamine embryos. This should not affect the results reported here as long as the relative rates of incorporation into RNA and DNA Drug treatment % DNA in top phase were the same in control and drug-treated embryos. None 0.86 The effect of drugs on incorporation of these precursors was Hydroxyurea, 500 /ig/egg, 24 hr 0.71 investigated in double-labeling experiments with 3H-labeled Hydroxyurea, 500 fig/egg, 48 hr 0.75 thymidine and uridine-14 C or leucine-14C. Since an inhibition Dimethyl-2-chloroethylamine, 200 Â¿ig/egg,24hi 1.03 of DNA synthesis might cause a secondary inhibition of RNA Dimethyl-2-chloroethylamine, 200 /ig/egg, 48 hr 0.70 synthesis, because there will be less DNA template on which RNA can be synthesized, we tested hydroxyurea, a known Table 2 specific inhibitor of DNA synthesis (13), to ascertain whether Specificity of incorporation of thymidine, uridine, and a primary inhibition of DNA synthesis could be distinguished leucine into DNA, RNA, and protein, respectively from a secondary inhibition of RNA synthesis. Hydroxyurea Four-day incubated eggs were given injections of does inhibit DNA synthesis, as measured by both 3H-labeled thymidine-methyl-3 H (5 Â¿iCi/egg),uridine-5-3H (0.5 juCi/egg), or L-leucine-4,5-3 H (0.5 /iCi/egg), and after 24-hr incubation homogenates thymidine incorporation and total DNA content per embryo, were prepared from the embryos as described in the text. After when injected into 4-day chick embryos (Chart 4). The effects treatment with the following enzymes and reagents, the trichloroacetic of this drug on 3H-labeled thymidine and uridine-14 C acid-precipitable cpm were determined on the treated homogenates and incorporation in double-labeling experiments were primarily untreated controls with the procedures described in the text. All enzyme incubations were at 37Â°. an inhibition of 3H-labeled thymidine incorporation, beginning between 10 and 20 hr, and secondarily an inhibition of uridine-14C incorporation, beginning between 20 and 30 hr Radioactive of cpm precursorThymidineUridineTreatmentDNase solubilized67Â° (Chart 5). In this and subsequent charts, the data are expressed as percentage of inhibition relative to controls so that several \, 50 Mg/ml, l hr experiments could be averaged because of variations in RNase, 42 Â¿ig/ml,l hr hrDNase0.96MNaOH, 50Â°,1 6 026 incorporation of precursors from experiment to experiment. At this stage of development, the embryos are approximately I, 50 fig/ml, 1 hr doubling in mass every 24 hr. An inhibition of synthesis of a RNase, 42 Mg/ml, l hr 7576 macromolecule of 50% between any 24-hr time point, 0.96 M NaOH, 50Â°,1hi% therefore, represents a complete inhibition of accumulation of that macromolecule. For time points differing by 12 and 48 Leucine Pronase, 500 /ig/ml, plus 1% 95 sodium dodecyl sulfate. 4 hr hr, the corresponding values are approximately 30 and 75%, respectively. Thus, for hydroxyurea there is little 0 After 14 hr, 82% of the cpm were solubilized. accumulation of DNA between 8 and 33 hr, and the inhibition is approximately 50% at 33 hr (Charts 4 and 5). 60.0001- 1 200 Mitomycin C also inhibited DNA synthesis before RNA

60 50.000 â€¢

50 40.000 â€¢ 800 * o 40

30.000 / HU 600 o " 30h ik o 20.000 - 400

10.000 200

10 20 30 40 50 60 10 20 30 40 50 60 HOURS AFTER INJECTIONS HOURS AFTER INJECTIONS Chart 4. Inhibition of DNA synthesis by hydroxyurea (500 jig/egg) in 4-day chick embryos. Average of 2 experiments, o, control; â€¢, Chart 5. Inhibition of incorporation of precursors into nucleic acids hydroxyurea (HU); , thymidine-3H incorporation per embryo; , in 4-day chick embryos by hydroxyurea (500 Mg/egg).â€¢,thymidine-3H; MgDNA per embryo, o, uridine-1 4C.

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for each cross-linking drug. Further, DNA from embryos treated with mitomycin C and HN2 was cross-linked within 6 sor hr after treatment (Chart 2), and 3H-labeled thymidine incorporation was inhibited by 10 to 12 hr (Chart 6), while 40 â€¢ DNA from embryos treated with thioTEPA was not cross-linked until 24 hr after treatment (Chart 2), and 3H-labeled thymidine incorporation was not inhibited until 30 hr. E 30 An important question is whether drugs with 2 or more eg alkylating sites per molecule (i.e., cross-linking ability) are i more efficacious in inhibiting the synthesis of macromolecules. Â¿v 20 . thymidine To answer this question, we compared HN2 and a monofunctional analog, dimethyl-2-chloroethylamine,

10 â€¢ DNA 50r

10 20 30 40 50 60 70

HOURS AFTER INJECTIONS Chart 6. Effects of mitomycin C (20 jug/egg) on incorporation of macromolecular precursors in 4-day chick embryos.

50p

40 â€¢ z O t: 30 m z z 10 20 30 40 60

20 HOURS AFTER INJECTION

Chart 8. Effects of HN2 (100 Mg/egg) on net synthesis of DNA, 10 RNA, and protein in 4-day chick embryos.

10 20 30 40 50 60 70

HOURS AFTER INJECTIONS 40 â€¢ Chart 7. Effect of mitomycin C (20 jug/egg) on net synthesis of DNA, RNA, and protein in 4-day chick embryos. o 30 â€¢ i- synthesis, but both were inhibited to the same extent after about 30 hr (Chart 6). Incorporation of leucine-14C was not inhibited by mitomycin C, although total protein content per 20 embryo relative to controls decreased, as did total DNA and RNA content (Chart 7). The effects of HN2 and thioTEPA on 200 ug 10 â€¢ â€¢Dimethyl-2-chloroethylamine these same parameters were also investigated. There was no Per egg significant difference in the inhibition of incorporation of 3H-labeled thymidine and uridine-14C by either of these drugs; the incorporation of leucine-14C into acid-precipitable 10 20 30 40 50 material was inhibited, but the development of this inhibition HOURS AFTER INJECTIONS took longer than the inhibition of incorporation of the nucleic Chart 9. Comparison of effects of HN2 and acid precursors. Embryos treated with both drugs showed dime thy I-2-chloroe thy lamine on DNA synthesis in 4-day chick primarily a decrease relative to controls of total DNA content, embryos. Â»,inhibition of incorporation of thymidine-3H by HN2; â€¢, followed by a decrease in RNA and protein content. These inhibition of incorporation of thymidine-3 H by dimethyl-2-chloro- data are presented for HN2 (Chart 8). ethylamine; o, inhibition of net DNA synthesis (total DNA per embryo) There is a good correlation between the amount of DNA by HN2; â€¢,inhibition of net DNA synthesis by dimethyl-2-chloro- cross-linked and the percentage of inhibition of DNA synthesis ethylamine.

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injecting twice as much of the latter to have comparable rate-limiting step and not affected by mitomycin C treatment. alkylating activities. Dimethyl-2-chloroethylamine should not Kay and Handmaker (10) have reached similar conclusions and does not have cross-linking activity (Table 1), and it is not regarding uridine incorporation and RNA synthesis in as effective as its difunctional relative, HN2, in inhibiting DNA lymphocytes. They found that the incorporation of uridine synthesis at comparable alkylating concentrations (Chart 9). was probably limited by 1 of the steps prior to the entry of The effects of these 2 analogs on total RNA content and uridine into the pool of intracellular nucleotides and uridine-' 4C incorporation were similar to the results for DNA, concluded that the incorporation of 3H-labeled uridine into i.e., HN2 was a more effective inhibitor as in Chart 9, whereas RNA was not a valid measure of the rate of RNA synthesis. they had similar inhibitory effects on protein synthesis. Dimethyl-2-chloroethylamine was not as effective an in hibitor of DNA and RNA synthesis as HN2 at even slightly more than the equivalent alkylating dose, but it still inhibited DISCUSSION the synthesis of both to some extent (Chart 9), and its effects on total protein content were similar to those of HN2. Wheeler Our results strongly implicate the inhibition of DNA et al. (28) have concluded from their studies of the effects of replication as a primary effect of difunctional alkylating drugs monofunctional and difunctional alkylating agents on H.Ep. on cell division. Assays for drug-induced cross-linked DNA No. 2 cells that cross-linking of DNA is not a prerequisite for show its presence within 6 hr after treatment (Chart 2). toxicity by alkylating agents. They did find that higher Although there is evidence for the repair of mitomycin concentrations of the monofunctional agents were required to C-induced cross-links in E. coli (22), there is no known repair exert effects similar to those of difunctional agents. The mechanism for covalent interstrand cross-links in DNA in greater toxicity of the difunctional drugs at low concentra higher cells, and since DNA replication requires strand tions is presumably due to their cross-linking ability. Brookes separation the former will be blocked because the latter is and Lawley (5), studying T2 and T4 bacteriophages, suggested prevented by the covalent cross-links introduced by the drugs. that inactivation by difunctional alkylating agents is due to This conclusion is supported by direct measurement of DNA interstrand cross-linking of DNA, while inactivation by synthesis with 3H-labeled thymidine incorporation and total monofunctional alkylating agents is due to degradation of DNA content per embryo. The results with mitomycin C were DNA consequent upon its alkylation. Perhaps this holds true particularly conclusive; both types of measurements showed for procaryotes and eucaryotes also. Dimethyl-2-chJoroethyl- that DNA synthesis was inhibited before RNA and protein amine, with only 1 alkylating site per molecule, is incapable of synthesis were inhibited (Charts 6 and 7). Reduction in rates cross-linking DNA (Table 1), but it does cause the same strand of RNA and protein synthesis could arise as a consequence of scission and depurination reactions that result predominantly the fact that there is less DNA template on which RNA can be from alkylating of guanine at the N-7 position by the synthetic synthesized and subsequently less RNA to direct the synthesis alkylating agents (4, 14); mitomycin C apparently reacts at of proteins. In E. coli, it was shown early that the primary another site (23). action of mitomycin C was a selective inhibition of DNA Not all of the effects of alkylating drugs can be ascribed to formation (20). Although incorporation studies with the other lesions induced in DNA, however. Mitomycin C, for example, 2 cross-linking agents, HN2 and thioTEPA, were not as is known to react with RNA and ribosomes (26); HN2 is even conclusive, we could demonstrate that net DNA synthesis was more reactive and should be even less specific as it does not inhibited before net synthesis of RNA and protein was require prior reduction as does mitomycin C (21). DeCosse affected (Chart 8), suggesting that cross-links affect DNA and Gelfant (7) concluded that a major biological result of synthesis primarily. nitrogen mustard was independent of DNA replication. It is That precursor incorporation is not always a good likely that a wide range of cell functions are affected, indication of net synthesis is well illustrated in some of the depending not only on the specific drug and tissue but also on results reported here. Thus, mitomycin C had no effect on the dose, duration of treatment, and the stage of the cell when incorporation of leucine-14C into protein for up to 48 hr the drug is first used (7, 27). (Chart 6), but obviously net protein synthesis was being inhibited as judged by total protein per embryo (Chart 7). The simplest explanation is a mitomycin C-induced breakdown of REFERENCES proteins so that incorporation of leucine-14C is not inhibited out net protein content is reduced relative to controls. There is 1. Alberts, B. M. Efficient Separation of Single-stranded and an alternative explanation: the rate of precursor incorporation Double-stranded Deoxyribonucleic Acid in a Dextran-Polyethylene depends on pool size and on the accessibility of the precursor Glycol Two-Phase System. Biochemistry, 6: 2527-2532, 1967. to the cell and permeability into the cell, and these 2. Alberts, B. M. Characterization of a Naturally Occurring, considerations must be important here as the precursors are Crosslinked Fraction of DNA. II. Origin of the Crosslinkage. J. Mol. Biol., 32: 405-421, 1968. injected into the yolk sac and there is a difference in the rates 3. Alberts B. M., and Doty, P. M. Characterization of a Naturally of incorporation of the 3 precursors. After 24 hr, the total incorporation of uridine-14C, 3H-labeled thymidine, and Occurring, Crosslinked Fraction of DNA. I. Nature of the Cross-linkage. J. Mol. Biol., 32: 379-403, 1968. leucine-14C was about 14, 6, and 3%, respectively. Some early 4. Brookes, P., and Lawley, P. D. The Reaction of Mono- and step on leucine incorporation, such as entrance of leucine into Di-functional Aklylating Agents with Nucleic Acids. Biochem. J., the circulating blood from the yolk, could be the slow and 80: 496-503, 1961.

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5. Brookes, P., and Lawley, P. D. Effects of Alkylating Agents on T2 17. Rosenkranz, H. S. An Anomalous DNA Component in and T4 Bacteriophages. Biochem. J., 89: 138-144, 1963. Hydroxyurea-treated Escherichia coli. Biochim. Biophys. Acta, 6. Burton, K. A Study of the Conditions and Mechanism of the 729:618-621,1966. Diphenylamine Reaction for the Colorimetrie Estimation of 18. Saunders, J. W., Jr., and FallÃ³n,J. F. Cell Death in Morphogenesis. Deoxyribonucleic Acid. Biochem. J., 62: 315-323, 1956. In: M. Locke (ed).. Major Problems in Developmental Biology, pp. 7. DeCosse, J. J., and Gelfant, S. Effects of Nitrogen Mustard during 289-314. New York: Academic Press, Inc., 1966. the Cell Cyle of the Ehrlich Ascites Tumor. Exptl. Cell. Res., 60: 19. Schneider, W. C. Determination of Nucleic Acids in Tissues by 185-190, 1970. Pentose Analysis. Methods Enzymol., 3: 680-684, 1957. 8. Geiduschek, E. P. "Reversible" DNA. Proc. Nati. Acad. Sei. U. S., 20. Shiba, S., Terawaki, A., Taguchi, T., and Kawamata, J. Selective 47: 950-955, 1961. Inhibition of Formation of Deoxyribonucleic Acid in Escherichia 9. KarlstrÃ¶m, O., and Larsson, A. Signficance of Ribonucleotide coli by Mitomycin C. Nature, 183: 1056-1057, 1959. Reduction in the Biosynthesis of Deoxyribonucleotides in 21. Szybalski, W., and Iyer, V. N. Crosslinking of DNA by Escherichia colt. European J. Biochem., 3: 164-170, 1967. Enzymatically or Chemically Activated Mitomycin and 10. Kay, J. E., and Handmaker, S. D. Uridine Incorporation and RNA Porfiromycins, Bifunctionally "Alkylating" Antibiotics. Federation Synthesis during Stimulation of Lymphocytes by PHA. Exptl. Cell Proc., 23: 946-957, 1964. Res., 05:411-421, 1970. 22. Terawaki, A., and Greenberg, J. Post-treatment Breakage of 11. Kim, J. H., Gelbard, A. S., Djordjevic, B., Kim, S. H., and Perez, A. Mitomycin C Induced Cross-links in Deoxyribonucleic Acid of G. Action of Daunomycin on the Nucleic Acid Metabolism and Escherichia coli. Biochim. Biophys. Acta, 779: 540-546, 1966. Viability of HeLa Cells. Cancer Res., 28: 2437-2442, 1968. 23. Tomasz, M. Novel Assay of 7-Alkylation of Guanine Residues in 12. Kohn, K. W., Spears, C. L., and Doty, P. Interstrand Crosslinking DNA. Application to Nitrogen Mustard, Triethylenemelamine and of DNA by Nitrogen Mustard. Appendix: Terminology of Mitomycin C. Biochim. Biophys. Acta, 213: 288-295, 1970. Configurational Changes of DNA in Solution. J. Mol. Biol., 19: 24. Webster, D. A., and Gross, J. Studies on Possible Mechanisms of 266-288, 1966. Programmed Cell Death in the Chick Embryo. Develop. Biol., 22: 157-184,1970. 13. Krakoff, I. H., Brown, N. C., and Reichard, P. Inhibition of 25. Weiss, B. Terminal Cross-linking of DNA Strands by an Enzyme Ribonucleoside Dephosphate Reductose by Hydroxyurea. Cancer Res., 28: 1559-1565,1968. System from Escherichia coli Infected with Bacteriophage T4. Proc. Nati. Acad. Sei. U. S., 65. 652-659, 1970. 14. Laurence, D. J. R. Chain Breakage of Deoxyribonucleic Acid 26. Weissback, A., and Lisio, A. Alkylation of Nucleic Acids by following Treatment with Low Doses of Sulfur Mustard. Proc. Mitomycin C and Porfiromycin. Biochemistry, 4: 196-200, 1965. Roy. Soc. London, Ser. A, 277: 520-530, 1963. 27. Wheeler, G. P. Some Biochemical Effects of Alkylating Agents. 15. Lawley, P. D., and Brookes, P. Interstrand Crosslinking of DNA by Federation Proc., 26: 885-892, 1967. Difunctional Alkylating Agents. J. Mol. Biol., 25: 143-160, 1967. 28. Wheeler, G. P., Bowden, B. J., Adamson, D. J., and Vail, M. H. 16. Lowry, O. H., Rosebrough, N. J., Farr, A. L., and Randall, R. J. Effects of Certain Nitrogen Mustards upon the Progression of Protein Measurement with the Folin Phenol Reagent. J. Biol. Cultured H.Ep. No. 2 Cells through the Cell Cycle. Cancer Res., Chem., 193: 265-275, 1951. 30: 100-111, 1970.

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Downloaded from cancerres.aacrjournals.org on September 29, 2021. © 1971 American Association for Cancer Research. 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

Cancer Res 1971;31:1573-1579.

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