(CANCER RESEARCH 43, 5718-5724, December 1983] Effects of Dimethyl Sulfoxide and Thiourea upon Intercalator-induced DMA Single-Strand Breaks in Mouse Leukemia (L1210) Cells Yves Pommier,1 Leonard A. Zwelling, Michael R. Mattern, Leonard C. Erickson, Donna Kerrigan, Ronald Schwartz, and Kurt W. Kohn Laboratory of Molecular Pharmacology. Developmental Therapeutics Program, Division of Cancer Treatment, National Cancer Institute, NIH, Bethesda. Maryland 20205 ABSTRACT bleomycin, generate free radical species which produce DNA breaks (5, 6), and it has been proposed that some intercalating The free radical scavengers, dimethyl sulfoxide (MezSO) and agents also break DNA by a free radical mechanism. In particular, thiourea, were used to assess the role of free radicals in the ADM2 can generate free radicals during NADPH and O2 reaction production of intercalator-induced DNA breaks and cytotoxicity with reducíaseenzymes (2, 3, 14), and DNA strand breaks can in mouse leukemia L1210 cells. Both agents decreased X-ray be generated by this mechanism (4). break production, and this decrease was comparable in magni Free radical formation depends on reductive reactions which tude to the degree of inhibition of X-ray-induced cell killing. By are most likely to occur in those intercalating agents, incuding contrast, Me2SO increased the DNA breaks produced by the ADM, which contain quinone groups. However, several interca intercalators, Adriamycin, 5-iminodaunorubicin, and 4'-(9-acridi- lating agents, such as ellipticine, 2-Me-9-OH-E+, 5-ID, or m- nylamino)methanesulforHn-anisidide. This was not due to an AMSA, do not contain quinone groups and exhibit relatively little enhancement of Adriamycin or 4'-(9-acridinylamino)methane- free radical production (19, 23) yet still stimulate the formation sulfon-m-anisidide uptake by Me2SO. Strand break production of protein-associated DNA strand breaks in cells (27,33,36,37). by intercalators was decreased by thiourea. This was not due to Thus, intercalating agents could produce DNA strand breaks by an inactivation of the intercalators or to a decrease of Adriamycin 2 distinct mechanisms, but it is not dear whether the free radical or 4'-{9-acridinylamino)methanesulfon-/n-anisidide uptake by thi mechanism may make an appreciable contribution in living cells. ourea. Experiments using nudeokJ sedimentation to assess the Therefore, we investigated the effects of the free radical DNA linking number and domain size from cells treated with scavengers, Me2SO and thiourea, on intercalator-induced DNA Me2SO and thiourea indicated that these chemicals alter chro- breakage and cytotoxicity. These compounds reduce the extent matin structure in a fashion which may account for effects on of DNA breakage in cells exposed to ionizing radiation or hydro intercalator-induced DNA scission. The alterations in intercalator- gen peroxide (5, 22). In the course of these experiments, we induced DNA scission were not accompanied by corresponding found unexpectedly that Me2SO actually increased the interca alterations in cytotoxicity, thus dissociating intercalator-induced lator-induced DNA breakage. We propose that this phenomenon strand break production from lethality and the mechanism of X- may involve an effect on chromatin structure. ray break production. MATERIALS AND METHODS INTRODUCTION Materials. [2-'4C]Thymidine (58 mCi/mmol) and [mef/jy/-3H]thymidine A variety of DNA intercalating agents cause single- and double- (20 Ci/mmol) were purchased from New England Nuclear, Boston, Mass. strand DNA scission in mammalian cells (25-27, 33-37). This MejSO and thiourea were purchased from Fisher Scientific Co., Fairtawn, N. J. and from Eastman Organic Chemicals, Rochester, N. Y., respec DNA scission differs from the DNA breakage produced by other tively. ADM (NSC 123127) and m-AMSA (NSC 249992) were obtained DNA damaging agents in several respects, (a) The intercalator- from the Drug Synthesis and Chemistry Branch, Division of Cancer induced DNA breaks are associated with an approximately stoi- Treatment, National Cancer Institute; m-AMSA was dissolved in 100% chiometric amount of covalently bound protein which dissociates M62SO at 10 mM, and ADM was dissolved in glass distilled water at 1 at the same time the breaks are reseated (27, 36). (b) The mg/ml. 5-ID (NSC 254681) was a gift from Dr. Robert I. Glazer, Applied formation of the DNA breaks is saturable and is inhibited at low Pharmacology Section, Laboratory of Medical Chemistry and Biology, temperature but wilt occur subsequently in the presence of drug National Cancer Institute, who obtained the compound from Dr. E. Acton, Stanford Research Institute. 5-ID was dissolved in glass distilled water when the temperature is raised (36). (c) The formation and at 1 mM. 2-Me-9-OH-E+ was a gift from Dr. J. B. Le Pecq, Laboratoire resealing of the breaks is not accompanied by stimulation of de Pharmacologie Moléculaireau Centre National de la Recherche Scien poly(adenosine diphosphoribose) synthesis (35). (d) The break tifique, Institut Gustave-Roussy, Villejuif, France, and was dissolved in resealing can occur in isolated nuclei and permeabilized cells in glass distilled water at 8.25 mw. [14C]m-AMSA (19.6 mCi/mmol) and the absence of nucleoside triphosphates (24, 35). These findings [MC]ADM (17.2 mCi/mmol) were synthesized by SRI International, Menlo taken together suggest a mechanism of DNA scission coordi Park, Calif., and were obtained through the Chemical Resources Section, nated with protein-DNA binding having its origins in an enzyme National Cancer Institute. [14C]m-AMSA and [14C]ADM were dissolved constituent of the cell nucleus, perhaps a topoisomerase (27, in 100% Me2SO and in glass-distilled water, respectively. ADM, 5-ID, m- 36). 2The abbreviations used are: ADM, Adriamycin; 2-Me-9-OH-E+, 2-methyl-9- Some DNA damaging agents, such as ionizing radiation and hydroxyellipticinium; 5-ID, 5-iminodaunorubicin; m-AMSA, 4'-(9-acridinylam- ino)methanesulfon-m-aniskJ"tde; Me^O, dimethyl sulfoxide; [14C]m-AMSA, 4'-(9- acridinyl-[9-'4C]amino)methanesulfon-m-anisidide; [14C]ADM, Adriamycin hydro- 1To whom requests for reprints should be addressed, at Building 37, Room chloride-(14-"C]; RPMI 1630, Roswell Park Memorial Institute tissue culture Me 5D17, 9000 Rockvilte Pike, Bethesda, Md. 20205. dium 1630; DPC, DNA-protein cross-links; SSB, single-strand breaks; SDS, sodium Received May 26,1983; accepted August 25,1983. dodecyl sulfate. 5718 CANCER RESEARCH VOL. 43 Downloaded from cancerres.aacrjournals.org on September 25, 2021. © 1983 American Association for Cancer Research. Effects of Me^SO and Thiourea on Intercalator-induced DMA Breaks AMSA, and 2-Me-9-OH-E+ were stored frozen in stock solutions. [log (f,/ro)] [tog (flo/ro)] ' Cell Labeling, Irradiation, and Drug Treatment. L1210 mouse leu kemia cells were grown in suspension culture in RPMI 1630 supple mented with 15% fetal calf serum. Stock cultures were maintained in were PB is the DNA break frequency produced by the X-ray (300 or 1000 static bottles without antibiotics and were used to initiate suspension rad-equivalents), and r,, ro, and ñoare the retention (16) of DNA from drug-treated, untreated, and 300- or 1000-rad-treated [14C]thymidine- cultures. Cultures used to assess drug effects were in exponential growth phase with a doubling time of 13 to 15 hr. labeled cells. Retention was evaluated at the time corresponding to retention of 0.35 of the [3H]DNA in the high-sensitivity assay or 0.60 of Cellular DNA was radioactively labeled in exponentially growing cells by incubation with [2-uC]- or [mef/7y/-3H]thymidine for 20 hr at 37°.For the [3H]DNA in the low-sensitivity assay. The exact choice of this end experiments in which DNA SSB and DPC were measured by alkaline point was not critical, since the elution kinetics were nearly first-order elution, [14C]thymidine labeling of DNA was at 0.01 ¿tCi/ml,and the DNA following all drug treatments. Results are expressed as "rad-equivalents," of cells used as internal standard (16) was labeled with [mef/?y/-3H]- indicating that the elution rate of DNA from drug-treated cells is equal to thymidine(0.1 //Ci/ml; 10"6 M unlabeled thymidine added). In experiments that produced by that X-ray dose. in which DNA SSB were measured by alkaline sucrose sedimentation, DNA-Protein Cross-Linking. Drug-treated (or untreated control) [14C]- and [3H]thymidine labeling concentrations were increased 20- and [14C]thymidine-labeled cells were X-irradiated at ice temperature (4°)with 10-fold, respectively, as compared to alkaline elution experiments. In the 3000 rads. These cells were combined with an equal number (approxi mately 5 x 105) of [3H]thymidine-labeled cells which had received no nucleoid sedimentation experiments, cellular DNA was labeled with [mef/)y/-3H]thymidine (0.4 ¿tCi/ml;10"6 M unlabeled thymidine added). In drug treatment and had been concurrently irradiated with 3000 rads in all cases, radioactive label was removed by centrifugation príorto drug ice. Cells were deposited on a 2-^m polyvinyl chloride filter (Type BS; treatment or irradiation of the cells. Millipore) and lysed with a solution consisting of 2 M NaCI, 0.2% sarkosyl, L1210 cells at a concentration between 1 and 1.5 x I06/ml in iced and 0.04 M disodium EDTA, pH 10.0 (5 ml). This lysis solution was RPM11630 plus 15% fetal calf serum were irradiated with either a 137Cs removed by washing the filter with 0.04 M EDTA, pH 10.0 (3 ml). DNA or a 200-kV X-ray source as described previously (16, 36). Cells were elution was performed with tetrapropylammonium hydroxide-EDTA, pH maintained at ice temperature until they were assayed either by alkaline 12.1, at a pump speed of 0.03 to 0.04 ml/min. Fractions (6 ml) were elution or alkaline sucrose sedimentation.