Interactions of the UVRABC Endonuclease in Vivo and in Vitro with DMA Damage Produced by Antineoplastic Anthracyclines1

[CANCER RESEARCH 44, 3489-3492, August 1984] Vii -.

Barry M. Kacinski2 and W. Dean Rupp3

Departments of Therapeutic Radiology [B. M. K., W. D. R.] and Molecular Biophysics and Biochemistry [W. D. R.], Yale University School of Medicine, New Haven, Connecticut 06510

ABSTRACT clines. However, they do not bind to DNA (31, 34) and do not appear to enter cells to release free drug (31, 32, 34). The anthracycline antineoplastic agents Adriamycin and N- Therefore, Tritton ef al. (30,31 ) and others (22) have proposed trifluoroacetyl-Adriamycin-14-valerate were assayed in vivo and that anthracyclines exert their antitumor effects at the cell surface in vitro for ability to produce DMA lesions recognized by the by generating free radical species which damage membranes. It UVRABC endonuclease, a DNA repair enzyme of Escherichia has also been suggested that they may interfere with nucleic coli which recognizes large, bulky lesions in DNA. We found that, acid metabolism less directly by blocking transcription of RNA while both drugs produce DNA lesions, only the lesions produced from DNA (6, 7, 27). Since data on the biochemical nature of the by Adriamycin were toxic. Hence, anthracycline antineoplastic damage to DNA induced by anthracycline exposure in mammal activity may be related to production of large, bulky lesions in ian cells are scanty (18), it is not yet possible to rule out DNA, while toxicity may correlate with toxicity measured in a anthracycline-DNA interactions in the antineoplastic activity of simple E. coli DNA repair mutant test system. anthracyclines (1). The toxicities of the anthracyclines are even less well-under INTRODUCTION stood than are their antineoplastic activities. Adriamycin and daunorubicin are very toxic (36), but carminomycin (23), AD32 The anthracycline antibiotics are among the most effective (15), aclacinomycin (20), and the daunorubicin macromolecular antineoplastic agents currently in clinical use, with activity both conjugates (32) are reported to have reduced toxicity. It has against solid tumor and lymphomas as single agents and in been recognized that the most toxic anthracyclines have the multidrug combinations (2, 36). Unfortunately, they are extremely most profound effects on DNA structure and metabolism and toxic. The utility of Adriamycin and daunorubicin, in particular, is that this toxicity is additive to that of the DNA-damaging treat often limited by the need to keep the cumulative dose to less ments such as X-irradiation and cyclophosphamide therapy (36). than 500 mg/sq m to avoid irreversible, often fatal, cardiac It would, therefore, seem reasonable to propose that anthracy toxicity (17, 21). The mechanisms of anthracycline antitumor cline toxicity may involve damage to DNA. Since, however, no activity and toxicity are not clearly understood, but they may be specific anthracycline-induced DNA lesions have yet been iden unrelated and dissociable (36). tified in mammalian cells, this proposal will remain hypothetical Certain anthracyclines, such as Adriamycin and daunorubicin, until methods are developed to detect and characterize anthra bind tightly to double-stranded DNA in vitro by intercalating cycline-induced damage in DNA. between base pairs to unwind superhelical DNA molecules (11, In this paper, we present our observations on the toxicity of 29). Anthracyclines also damage DNA in vivo since certain of the anthracyclines Adriamycin and AD32 in a simple bacterial these agents induce prophage X (2) and produce mutations (33) system where the effects of DNA damage and its repair are in bacteria. Many anthracyclines disrupt nucleic acid and protein easily analyzed. We show that such investigations help provide synthesis (6, 7, 9, 16) and fragment DNA molecules (27) in some insight into the mechanisms of anthracycline antineoplastic mammalian cells. and toxic activity as they relate to the interactions of anthracy The antineoplastic activities of anthracyclines were initially clines and DNA. attributed to their effects on the structure and metabolism of DNA in analogy to other antineoplastic drugs which alkylate DNA or otherwise interfere with its metabolic functions (8). This notion MATERIALS AND METHODS was challenged by the discovery of antineoplastic anthracyclines such as AD324 which do not bind to DNA (12,15). Certain other Escherichia coli K-12 strains AB1157, AB1886, and AB2463 have antineoplastic anthracyclines such as marcellomycin have little been described previously (14). AB1886 carried the uvrA6 mutation which effect on DNA synthesis (28), while the clinically effective anthra inactivates the UVRA protein, one of the components of the UVRABC cycline, aclacinomycin, was found not to be a mutagen (33). endonuclease. This enzyme is involved in the repair of pyrimidine dimers Macromolecular conjugates of daunorubicin and Adriamycin and other large, bulky adducts in DNA (24, 26). AB2463 carries the recA13 mutation which leads to a lack of active RECA protein and to appear to be effective antineoplastic agents (31, 32, 34) with deficiencies in recombination, postreplication repair, and induction of enhanced therapeutic ratios compared with the free anthracy- SOS repair (19, 35). CSR603 is a uvrA6 recA1 E. coli strain obtained from Dr. A. Sanear (see Ref. 25). pBR322 is the ampicillin- and tetracycline-resistant E. coli plasmid 1Supported by NIH Grant CA06519. described by Bolivar ef al. (3). 2 Recipient of an award from the American Cancer Society through the Yale Yeast-extract tryptone broth contains 10 g Bacto-Tryptone, 5 g Bacto- Comprehensive Cancer Center. 3 Recipient of American Cancer Society Grant PDT-80. Yeast Extract, and 10 g NaCI/liter. Solutions are autoclaved and stored 4The abbreviation used is: AD32, A/-trifluoroacetyl-Adriamycin-14-valerate. at room temperature. Received October 17,1983; accepted April 17,1984. Phosphate-buffered saline contains 1.36 g anhydrous Na2HPO4,1.42

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Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 1984 American Association for Cancer Research. B. M. Kacinski and W. D. Rupp g anhydrous KH2PO4, and 0.5 g NaCI/liter. Solutions are autoclaved and stored at room temperature. Agarose gel electrophoresis is carried out at room temperature (125 V and 75 mA) as described previously (26) with 0.6% agarose at pH 7.8 in a buffer containing 4.6 g Trizma base (Sigma Chemical Co.), 4.1 g NaH2PO4, and 0.37 g disodium EDTA/liter. Adriamycin was a gift from Adria Laboratories (10). AD32 (15) was obtained from the National Cancer Institute, courtesy of Dr. Suffness. IODO - ce Ampicillin and ethidium bromide were purchased from Sigma. o V) < RESULTS ce AB1886, the UVRABC endonuclease-deficient mutant of Â£. coli is much more sensitive to Adriamycin than are either AB2463 or AB 1157. Within the concentration range studied, no toxic effects for AB1157 were observed, and AB2463 was only slightly sensitive to the drug (Chart 1A). None of the strains was sensitive to AD32 after 1- (Chart 16) or 36-hr (data not shown) exposure I 0 to this agent. Plasmid DMA was prepared from a strain of E. coli lacking Chart 2. Transformation efficiency of plasmid DNA treated with Adriamycin or AD32. CSR603 (wrA6 recA13) carrying the plasmid pBR322 is grown to a density both excision and postreplication repair pathways after exposure of approximately 1.5 x 108 cells/ml of yeast-extract tryptone broth after which to Adriamycin or AD32 and analyzed for biological activity by a chloramphenicol is added to a concentration of 200 fig/ml to block chromosomal replication but allow replication of the plasmid DNA (3, 4, 5). After 12 hr at 37Â°, cells are washed in phosphate-buffered saline and resuspended in yeast-extract tryptone broth with Adriamycin or AD32 at 20 Â¿ig/ml.Incubation is carried out at 'i 8 37Â°for another hour. Cells are lysed by the method of Godson and Vapnek (13), and plasmid DNA is isolated by CsCI-ethidium bromide isopyknic centrifugation. DNA is dialyzed against Tris (50 mM, pH 7.5) and disodium EDTA (1 HIM)and used to transform competent AB1157 and AB1886 (36). Transformants are assayed by growth at 37Â°after 24 hr on yeast-extract tryptone broth agar plates containing ampicillin at 200 ng/ml. Colonies per ^g DNA treated with Adriamycin (A) and AD32 (B) are plotted above and compared with transformation efficiency of untreated pBR322 DNA. O, AB1157 uvrA* with untreated DNA; â€¢,AB1157 uvrA* with Adriamycin- or AD32-treated DNA; D, AB1886 uvrA' with untreated DNA; â€¢ AB1886 uvrA~ with Adriamycin- or AD32-treated DNA. o cell transformation assay (Chart 2). Adriamycin-treated DNA z Lu efficiently transformed AB1157 to ampicillin resistance when O compared with untreated DNA. For the UVRABC endonuclease- deficient strain AB 1886, however, the efficiency of transforma

ÃœJ tion to ampicillin resistance by Adriamycin-treated DNA was o B much lower than that with untreated DNA (Chart 2A). AD32 z treatment of DNA had no effect on transformation efficiency for either strain (Chart 26). Incubation of both AD32- or Adriamycin-treated DNA with purified UVRABC endonuclease produced specific endonucleo- lytic cleavage of both DMAs (Fig. 1, Lanes 7 to 12).

DISCUSSION

Our results show a marked toxic effect of Adriamycin on a strain of E. coli (AB1886) which lacks UVRABC endonuclease but which is proficient in inducible error-prone repair and post- IO' replication repair. A strain which lacks the latter repair pathways (AB263) is only slightly sensitive to the toxic effects of Adriamy 100 200 25 50 cin, whereas the wild-type cells (AB1157) are quite resistant to ADRIAMYCIN (/ig/ml) AD32 this agent. Chart 1. Survivalof AB1157(uv^*rec/\*)(O), AB1886(wM-)(O),and AB2463 In contrast, AD32, an effective anthracycline antitumor agent (recA~) (A) after exposure to Adriamycin (A) and AD32 (B). E. coli was grown to a density of approximately 1.5 x 10" cells/ml from 1 x 10" cells/ml at 37Â° with with little affinity for DNA (28) and with reduced toxicity in animal agitation in yeast-extract tryptone broth (10 g Bactc-Tryptone, 5 g Bacto-Yeast systems when compared to Adriamycin (15) is essentially non- Extract, and 10 g NaCI/liter). Adriamycin (10 mg/ml in distilled water) was added toxic for all 3 Â£.coli strains in the range of concentrations tested. to the cultures at the concentrations indicated, and the incubation was carried out for an additional hr at the same temperature. Cells were then pelleted by centrifu- (Higher concentrations of AD32 could not be tested due to the gation, washed in phosphate-buffered saline (1.36 g anhydrous Na2HPO4, 1.42 g limited solubility of this compound.) This result strongly suggests anhydrous KH2PO4, and 0.5 g NaCI/liter), and plated on agar plates consisting of that Adriamycin produces a lethal lesion in the DNA of Â£.coll yeast-extract tryptone broth solidified with 1% agar. Incubation was carried out overnight at 37Â°,and colonies were counted. The logarithm of surviving fraction is which is repaired by the UVRABC endonuclease and that such plotted versus the concentration of either Adriamycin or AD32. lethal lesions are not produced by AD32.

3490 CANCER RESEARCH VOL. 44

To test this hypothesis, we exposed plasmici DNA to Adria- cells In vitro. Cancer Res., 32: 1307-1314, 1972. 7. Daskal, Y., Woodward, C., Crooke, S. T., and Busch, H. Comparative ultra- mycin and AD32 in vivo in a strain of E. coli deficient in excision structural studies on nucleoli of tumor cells treated with Adriamycin and the and postreplication repair of DNA damage. We purified the new anthracyclines carminomycin and marcellomycin. Cancer Res., 38: 467- 473,1978. plasmid DNA and were able to show that the transformation 8. DiMarco, A. Adriamycin (NSC-123127) mode and mechanism of action. Cancer efficiency of DNA treated with Adriamycin was markedly de Chemother. Rep., 6: 91-105. 1975. creased (relative to untreated DNA) in a strain of E. coli which 9. DiMarco, A., Casazza, A. M., Gambetta, R., Supino, R., and Zunino, F. Relationship between activity and amino sugar stereochemistry of daunorubi- lacks the UVRABC endonuclease (12). Treatment of DNA with cin and Adriamycin derivatives. Cancer Res., 36:1962-1966,1976. AD32 produced no such reduction in transformation efficiencies 10. DiMarco, A., Soldati, M., Fioretti, A., and Dasdia, T. Richerche sull'attivitÃ della daunomicina su cellule normale et neoplastia coltivate. Tumori, 49: 235-243, (Chart 28). 1963. We also incubated these DNAs (Fig. 1) in vitro with purified 11. DuVemay, V. H., Pachter, J. A., and Crooke, S. T. Deoxyribonucleic acid UVRABC endonuclease under conditions in which the enzyme binding studies on several new anthracycline antitumor antibiotics. Sequence preference and structure-activity relationships of marcellomycin and its ana readily cleaved DNA molecules containing UV-induced pyrimidine logues compared to Adriamycin. J. Bid. Chem., 18: 4024-4030,1979. dimers. In this experiment, we found that covalently closed 12. DuVemay, V. H., Prestayko, A. W., Galvan, L., Busch, Hâ€žand Crooke, S. circular DNA molecules treated with Adriamycin or AD32 were Studies on the structure-activity relationships of several new anthracyclines. readily cleaved to the nicked-circular form by the UVRABC Proc. Am. Assoc. Cancer Res., 78:182, 1977. 13. Godson, G. N., and Vapnek, D. A simple method of preparing large amounts endonuclease. of Ox174 RFI supercoiled DNA. Biochem. Biophys. Acta, 299. 516-530,1973. To us, these results strongly suggest that (a) anthracyclines 14. Howard-Flander, P., and Boyce, R. P. DNA repair and genetic recombination: studies on mutants of Escherichia coli defective in these processes. RadiÃ¢t. can produce lesions in DNA, even anthracyclines such as AD32 Res. (Suppl. 6): 156-184,1966. which have little physical affinity for DNA; and (b) that these 15. Israel, M., Modest, E. J., and Frei, E., III. A/-triftuoroacetyladriamycin-14- valerate, an analog with greater experimental antitumor activity and less lesions produce distortions in the double helical structure of DNA toxicity than Adriamycin. Cancer Res., 35: 1365-1368, 1975. which are recognized and cleaved by the UVRABC endonucle 16. Kanter, P. M., and Schwartz, H. S. Effects of N-trifluoroacetyladriamycin-14- ase, an enzyme which has been implicated in the repair of large valerate and related agents on DNA strand damage and thymidine incorpora tion in CCRF-CEM cells. Cancer Res., 39: 448-451,1979. bulky lesions in DNA such as pyrimidine dimers, psoralen ad- 17. Lefrak, E. A., Pitha, J., Rosenheim, S., and Gottlieb, J. A. Clinicopathologic ducts, and adducts produced by diaminodichloro-c/s-platinum analysis of Adriamycin cardiotoxicity. Cancer (Phila.), 32: 302-314, 1973. 18. Levin, M., Silber, R., Israel, M., Goldfeder, A., Khetarpal, V. K., and Potmesil, (24, 26). M. Protein-associated DNA breaks and DNA-protein cross-links caused by Comparison of the in vitro and in vivo experimental results DNA nonbinding derivatives of Adriamycin in L1210 cells. Cancer Res., 47: suggests that not all anthracycline-induced lesions recognized 1006-1010,1981. 19. MacEntee, K. Protein X is the product of the recA gene of Escherichia coli. by the UVRABC endonuclease have the same consequences for Proc. Nati. Acad. Sci. USA, 74: 5275-5279, 1977. the cells as lesions produced by Adriamycin appear to be more 20. Mathe, G., Bayssas. M., and Goveia, J. Preliminary results of a phase II trial toxic for Â£.coli than do those produced by AD32. of aclacinomycin in acute leukemia and lymphosarcoma: an oncostatic anthra cycline that is rarely cardiotoxic and induces no alopecia. Cancer Chemother. To us, these results also imply a possible correlation between Pharmacol., 7: 259-262,1978. DNA repair in E. coli and anthracycline antineoplastic activity and 21. Minow, R. A., Benjamin, R. S., and Gottlieb, J. A. Adriamycin cardiomyopa- thyâ€”an overview with determination of risk factors. Cancer Chemotherap. toxicity in animal and human tumor systems, since both agents Rep. Part III, 6: 195-201, 1975. are good antitumor agents, and both produce lesions in DNA 22. Myers, C. E., McGuire, W. P., Ã¼ss,R. H., Ifrim, I., Grotzinger, K., and Young, recognized by the UVRABC endonuclease. AD32 appears much R. C. Adriamycin: the role of lipid peroxidation in cardiac toxicity and tumor response. Science (Wash. DC), 797: 165-169,1977. less toxic in animal studies and is also essentially nontoxic to E. 23. Perevodchikova, N. I., Uchnitser, M. R., and Gorbunova, V. A. Phase I clinical coli. study of carminomycin: its activity against soft tissue sarcomas. Cancer Treat. More anthracyclines need to be analyzed in our system, but Rep., 67: 1705-1707,1977. 24. Rupp, W. D., Sanear, A., and Sanear, G. B. Properties and regulation of the our results suggest that production by anthracyclines of DNA UVRABC endonuclease. Biochimie (Paris), 64:595-598,1982. lesions which are substrates in vitro for the UVRABC endonucle 25. Sanear, A., and Rupert, C. S. Determination of plasmid molecular weights from ase positively correlates with their antitumor activity, while an ultraviolet sensitivities. Nature (Lond.), 272: 471-472, 1978. 26. Sanear, A., and Rupp, W. D. A novel repair enzyme: UVRABC excision thracycline toxicity for E. coli strains deficient in UVRABC endo nuclease of Escherichia coli cuts a DNA strand on both sides of the damaged nuclease correlates with clinical toxicity. region. Cell, 33: 249-260,1983. Further investigations into the nature of anthracycline-induced 27. Schwartz, H. S. DNA breaks in P288 tumor cells in mice after treatment with daunorubicin and Adriamycin. Res. Commun. Chem. Pathol. Pharmacol., 70: lesions of DNA will be necessary before this correlation can be 51-64, 1975. explained. 28. Sengupta, S. K., Seshadri, R., Modest, E. J., and Israel, M. Comparative DNA- binding studies with Adriamycin (ADR), W-trifluoroacetyladriamycin-14-valerate (AD32) and related compounds. Proc. Am. Assoc. Cancer Res., 77:109,1976. REFERENCES 29. Strong, E., and Crooke, S. T. Interaction of anthracyclines with covalently- closed circular DNA. Mol. Pharmacol., 77: 100-104, 1980. 1. Anderson, W. A., Moreau, P. L., and Devoret, R. Induction of prophage lambda 30. Tritton, T. R., Murphee, S. A., and Sartorelli, A. C. Adriamycin: a proposal on and derivatives: correlation with antineoplastic activity. MutÃ¢t.Res., 77: 197- the specificity of drug action. Biochem. Biophys. Res. Commun., 84: 802-808, 208, 1980. 1978. 2. Blum, R. H.. and Carter, S. K. Adriamycin: a new anticancer drug with 31. Tritton, T. R., and Yee, G. The anticancer agent Adriamycin can be actively significant clinical activity. Ann. Intern. Med., 80: 249-259,1980. cytotoxic without entering cells. Science (Wash. DC), 277: 248-250,1982. 3. Bolivar. F., Rodriguez, P. J., Greene, M. C., Betlach, H. L., Heynecker, H. W., 32. Trouet, A., and Sokal, G. Clinical studies with daunorubicin DNA and Adria Boyer, H. W., Crosa, J. H., and Falkow, S. Construction and characterization mycin DNA complexes. A review. Cancer Treat. Rep., 63: 895-898,1974. of new cloning vehicles. II. A multipurpose cloning system. Gene (Amst.), 2: 33. Umezawa, K., Sawamura, M., Matsushima, T., and Sugimura, T. Mutagenicity 95-113,1977. of aclacinomycin A and daunomycin derivatives. Cancer Res., 38:1782-1785, 4. Clewed, D. M. Nature of colE1 plasmid replication in Escherichia coli in the 1978. presence of chloramphenicol. J. Bacterio!., 770: 667-676,1972. 34. Wiesehahn, G., Varga, J. M., and Hearst, E. J. Interactions of daunomycin and 5. Cohen, S. N., and Chang, A. C. Y. Recircularization and autonomous replication melanotropin-daunomycin with DNA. Nature (Lond.), 292: 467-469, 1981. of a sheared R-factor DNA segment in Escherichia coli transformants. Proc. 35. Witkin, E. Ultraviolet mutagenesis and inducible DNA repair in Escherichia coli. Nati. Acad. Sci. USA, 70: 1293-1297,1973. Bacterio!. Rev., 40: 869-907, 1976. 6. DaÃ±o,K., Freidriksen, M. S., and Hellung-Larsen, P. Inhibition of DNA and 36. Young, R. C., Ozols, R. F., and Myers, C. E. The anthracycline antineoplastic RNA synthesis by daunomycin Â¡nsensitive and resistant Ehrlich ascites tumor drugs. N. Engl. J. Med., 305: 139-153,1981.

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â€¢"â€¢Â»-ne DNA â€¢â€”-cccDNA

Fig. 1. Cutting of anthracycline-treated plasmid DNA by the UVRABC endonuclease. 100 ng of untreated pBR322 [covalently closed circular (ccc)] plasmid DNA. UV- irradiated (60 J/sq m, germicidal UV) pBR322 plasmid DNA, or anthracycline-treated pBR322 plasmid DNA (ci. Chart 2) were incubated (25 ^l at 37Â°for 60 min) with purified UVRABC endonuclease, Mg2*, and ATP as described previously (27). Reaction products were analyzed by agarose gel electrophoresis (0.6%, pH 7.8) and visualized for photography with ethidium bromide fluorescent staining as described previously (27). The lower bands in each lane contain uncut superhelical covalently closed circular plasmid DNA while the upper bands contain endonucleolytically cleaved, nicked circular (ne) DNA. Lane 1, untreated pBR322 DNA. no enzyme; Lane 2, untreated pBR322 DNA. â€¢incomplete"UVRABC endonuclease (UVRB and UVRC proteins only); Lane 3, untreated pBR322 DNA and "complete" UVRABC endonuclease (UVRA, UVRB, and UVRC proteins); Lane 4, UV-irradiated pBR322 DNA, no enzyme; Lane 5. UV-irradiated pBR322 DNA and incomplete UVRABC endonuclease; Lane 6, UV-irradiated pBR322 DNA and complete UVRABC endonuclease; Lane 7. Adriamycin-treated pBR322 DNA, no enzyme; Lane 8, Adriamycin-treated pBR322 DNA and incomplete UVRABC endonuclease; Lane 9, Adriamycin-treated pBR322 DNA and complete UVRABC endonuclease; Lane 70, AD32-treated pBR322 DNA, no enzyme; Lane 77, AD32-treated pBR322 DNA, incomplete UVRABC endonuclease; Lane 72, AD32-treated pBR322 DNA, complete UVRABC endonuclease.

3492 CANCER RESEARCH VOL. 44

Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 1984 American Association for Cancer Research. Interactions of the UVRABC Endonuclease in Vivo and in Vitro with DNA Damage Produced by Antineoplastic Anthracyclines

Barry M. Kacinski and W. Dean Rupp

Cancer Res 1984;44:3489-3492.

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