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

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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 -. Interactions of the UVRABC Endonuclease in Vivo and in Vitro with DMA Damage Produced by Antineoplastic Anthracyclines1 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 AUGUST 1984 3489 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 £. 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 37°, cells are washed in phosphate-buffered saline and resuspended in yeast-extract tryptone broth with Adriamycin or AD32 at 20 ¿ig/ml.Incubation is carried out at 'i 8 37°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 37°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; •,AB1157 uvrA* with Adriamycin- or AD32-treated DNA; D, AB1886 uvrA' with untreated DNA; • 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
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