(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 The free radical scavengers, dimethyl sulfoxide (MezSO) and thiourea, were used to assess the role of free radicals in the production of intercalator-induced DNA breaks and cytotoxicity in mouse leukemia L1210 cells. Both agents decreased X-ray break production, and this decrease was comparable in magni tude to the degree of inhibition of X-ray-induced cell killing. By contrast, Me2SO increased the DNA breaks produced by the intercalators, Adriamycin, 5-iminodaunorubicin, and 4'-(9-acridi- nylamino)methanesulforHn-anisidide. This was not due to an enhancement of Adriamycin or 4'-(9-acridinylamino)methane- sulfon-m-anisidide uptake by Me2SO. Strand break production by intercalators was decreased by thiourea. This was not due to an inactivation of the intercalators or to a decrease of Adriamycin or 4'-{9-acridinylamino)methanesulfon-/n-anisidide uptake by thi ourea. Experiments using nudeokJ sedimentation to assess the DNA linking number and domain size from cells treated with Me2SO and thiourea indicated that these chemicals alter chro- matin structure in a fashion which may account for effects on intercalator-induced DNA scission. The alterations in intercalator- induced DNA scission were not accompanied by corresponding alterations in cytotoxicity, thus dissociating intercalator-induced strand break production from lethality and the mechanism of X- ray break production. INTRODUCTION A variety of DNA intercalating agents cause single- and double- strand DNA scission in mammalian cells (25-27, 33-37). This DNA scission differs from the DNA breakage produced by other DNA damaging agents in several respects, (a) The intercalator- induced DNA breaks are associated with an approximately stoi- chiometric amount of covalently bound protein which dissociates at the same time the breaks are reseated (27, 36). (b) The formation of the DNA breaks is saturable and is inhibited at low temperature but wilt occur subsequently in the presence of drug when the temperature is raised (36). (c) The formation and resealing of the breaks is not accompanied by stimulation of poly(adenosine diphosphoribose) synthesis (35). (d) The break resealing can occur in isolated nuclei and permeabilized cells in the absence of nucleoside triphosphates (24, 35). These findings taken together suggest a mechanism of DNA scission coordi nated with protein-DNA binding having its origins in an enzyme constituent of the cell nucleus, perhaps a topoisomerase (27, 36). Some DNA damaging agents, such as ionizing radiation and bleomycin, generate free radical species which produce DNA breaks (5, 6), and it has been proposed that some intercalating agents also break DNA by a free radical mechanism. In particular, ADM2 can generate free radicals during NADPH and O2 reaction with reducÃ-aseenzymes (2, 3, 14), and DNA strand breaks can be generated by this mechanism (4). Free radical formation depends on reductive reactions which are most likely to occur in those intercalating agents, incuding ADM, which contain quinone groups. However, several interca lating agents, such as ellipticine, 2-Me-9-OH-E+, 5-ID, or m- AMSA, do not contain quinone groups and exhibit relatively little free radical production (19, 23) yet still stimulate the formation of protein-associated DNA strand breaks in cells (27,33,36,37). Thus, intercalating agents could produce DNA strand breaks by 2 distinct mechanisms, but it is not dear whether the free radical mechanism may make an appreciable contribution in living cells. Therefore, we investigated the effects of the free radical scavengers, Me2SO and thiourea, on intercalator-induced DNA breakage and cytotoxicity. These compounds reduce the extent of DNA breakage in cells exposed to ionizing radiation or hydro gen peroxide (5, 22). In the course of these experiments, we found unexpectedly that Me2SO actually increased the interca lator-induced DNA breakage. We propose that this phenomenon may involve an effect on chromatin structure. MATERIALS AND METHODS Materials. [2-'4C]Thymidine (58 mCi/mmol) and [mef/jy/-3H]thymidine (20 Ci/mmol) were purchased from New England Nuclear, Boston, Mass. MejSO and thiourea were purchased from Fisher Scientific Co., Fairtawn, N. J. and from Eastman Organic Chemicals, Rochester, N. Y., respec tively. ADM (NSC 123127) and m-AMSA (NSC 249992) were obtained from the Drug Synthesis and Chemistry Branch, Division of Cancer Treatment, National Cancer Institute; m-AMSA was dissolved in 100% M62SO at 10 mM, and ADM was dissolved in glass distilled water at 1 mg/ml. 5-ID (NSC 254681) was a gift from Dr. Robert I. Glazer, Applied Pharmacology Section, Laboratory of Medical Chemistry and Biology, National Cancer Institute, who obtained the compound from Dr. E. Acton, Stanford Research Institute. 5-ID was dissolved in glass distilled water at 1 mM. 2-Me-9-OH-E+ was a gift from Dr. J. B. Le Pecq, Laboratoire de Pharmacologie Moléculaireau Centre National de la Recherche Scien tifique, Institut Gustave-Roussy, Villejuif, France, and was dissolved in glass distilled water at 8.25 mw. [14C]m-AMSA (19.6 mCi/mmol) and [MC]ADM (17.2 mCi/mmol) were synthesized by SRI International, Menlo Park, Calif., and were obtained through the Chemical Resources Section, National Cancer Institute. [14C]m-AMSA and [14C]ADM were dissolved in 100% Me2SO and in glass-distilled water, respectively. ADM, 5-ID, m- 1To whom requests for reprints should be addressed, at Building 37, Room 5D17, 9000 Rockvilte Pike, Bethesda, Md. 20205. Received May 26,1983; accepted August 25,1983. 2The abbreviations used are: ADM, Adriamycin; 2-Me-9-OH-E+, 2-methyl-9- 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- chloride-(14-"C]; RPMI 1630, Roswell Park Memorial Institute tissue culture Me dium 1630; DPC, DNA-protein cross-links; SSB, single-strand breaks; SDS, sodium dodecyl sulfate. 5718 CANCER RESEARCH VOL. 43 Research. on November 27, 2021. © 1983 American Association for Cancer cancerres.aacrjournals.org Downloaded from