1989 American Association for Cancer cancerres.aacrjournals.org ..... Gibson, N. W., Hartley, J. A., LaFrance, R. J., and Vaughan. K. Differential cytotoxicity and ...
[CANCER RESEARCH 49, 154-157, January 1, 1989]
Characterization of DNA Damage and Cytotoxicity Induced in Two Human Colon Carcinoma Cell Lines by Cyclodisone1 Neil W. Gibson Laboratory of Pharmacology, AMC Cancer Research Center, Denver, Colorado 80214, and School of Pharmacy, university of Colorado, Boulder, Colorado 80309
ABSTRACT Cyclodisone is an active alkylating antitumor agent that is being considered for Phase 1 clinical trials in humans and is currently undergo ing toxicological evaluation. Cyclodisone was found to be more toxic to human colon carcinoma cells of the Mer—phenotype (BE) than cells of the Mer+ phenotype (HT-29). DNA interstrand cross-links were ob served in the sensitive cell line but only at concentrations which were extremely toxic. No DNA interstrand cross-links were observed in the resistant cell line. Total DNA cross-links, which reflect both DNA interstrand and DNA-protein cross-linking, were observed in either cell type but were greater in quantity and persisted longer in the sensitive BE cell line, when compared to those produced in the resistant HT-29 cell line. DNA strand breaks were also observed in both cell types and were found to be protein associated. The mechanism of action of Cyclodisone would appear to be related to the presence of total DNA cross-links and might involve an, as yet, unidentified DNA-protein interaction.
INTRODUCTION 1,5,2,4-Dioxadithiepane-2,2,4,4-tetraoxide (cyclodisone, NSC 348948) is a member of a novel class of compounds which have been shown to inhibit the growth of a variety of tumors in the screening panel of the National Cancer Institute. As a result, this compound is being considered for Phase 1 clinical trials in humans and is currently undergoing toxicological evaluation. Although cyclodisone resembles the structure of two other classes of alkylating antitumor agents, the haloethylsulfonates and the dimethanesulfonic acid esters, its chemistry is quite distinct. Cyclodisone, upon nucleophilic attack, would be ex pected to behave as shown in Fig. 1. The intermediate, formed after the initial nucleophilic attack, carries a negative charge. This is an unusual feature for a bifunctional alkylating agent, and the influence that this may have upon its bifunctional capabilities is not clear at this time. In contrast, the haloethyl sulfonates (e.g., clomesone) and the dimethanesulfonic acid esters (e.g., busulfan) form a neutral intermediate after the nucleophilic attack. The DNA reactivity and in vitro cytotoxicity of cyclodisone in two human embryo cells have recently been published (1). The simian virus 40-transformed human embryonic lung cell line (VA-13) was found to be 3-fold more sensitive to cyclodi sone than the normal human embryonic cell strain (IMR-90) (1). Previous studies, with the VA-13 and IMR-90 cell lines, have shown that the VA-13 cell line is unable to repair alkylations at the guanine Opposition and was designated Mer- (2). In contrast, the IMR-90 cell line is proficient in the repair of these lesions and is designated Mer+ (2). Cyclodisone was different from other bifunctional alkylating agents in that it failed to induce DNA interstrand cross-links in either cell line, even at concentrations which produced a greater than 3 log cell kill in the sensitive VA-13 cell line (1). In this study, two human colon carcinoma cell lines, one Received 5/12/88; revised 8/29/88; accepted 10/4/88. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1Supported by USPHS Grant CA-47844 and by a gift to AMC from Robert L. Cohen.
Mer- (BE) and one Mer+ (HT-29), have been used in order to determine whether the relationship between the Mer phenotype and cyclodisone toxicity exists in another pair of Mer+/Mercell lines. In addition, cyclodisone-induced DNA damage has been characterized in the BE and HT-29 human colon carci noma cells to determine if the failure to detect DNA interstrand cross-linking in the VA-13 cell line is also true in other Mer— cell lines. MATERIALS
AND METHODS
Cell Culture. BE and HT-29 human colon carcinoma cells are main tained by growing cells at 37°Cas monolayers in 75-cm2 tissue culture flasks in Eagle's minimal essential medium. Both cell lines have been maintained in this laboratory for several years. The medium was sup plemented with the following components: 10% fetal bovine serum; kanamycin (0.1 mg/ml); glutamine (0.03 mg/ml); 0.1 mivinonessential amino acids; 1 IÕIM sodium pyruvate; and 0.02 M 4-(2-hydroxyethyl)-lpiperazineethane sulfonic acid. Cells were subcultured once per week. L1210 leukemia cells were grown as a suspension in RPMI 1640 medium, supplemented with 10% fetal bovine serum and 0.1 mg/ml of kanamycin. Drug Treatment. Cyclodisone, obtained from the Drug Synthesis and Chemistry Branch, Division of Cancer Treatment, National Cancer Institute, was dissolved in sterile dimethyl sulfoxide immediately before treatment of cell cultures. The concentration of dimethyl sulfoxide, in either treated or control cells, was never greater than 2% (v/v). In all assays, cells were exposed to the drug for 2 h at 37 °C,and then treatments were terminated by aspiration of the drug-containing me dium and replacement with fresh minimal essential medium. In Vitro Cytotoxicity Assays. Inhibition of the colony-forming ability of BE and HT-29 human colon carcinoma cells was assessed by seeding 3 x IO3cells in T 25-cm2 tissue culture flasks. Cells were preincubated for 12 to 18 h to allow attachment to the surface of the flasks. Cells were then exposed to various concentrations of cyclodisone for 2 h at 37°C.After 10 days of incubation in fresh medium, the flasks were rinsed with Hanks' balanced salt solution, fixed with methanol, and then stained with a solution containing 1 ml of méthylène blue, 1 ml of 0.15 M Na2HPO4, and 1 ml of 0.15 M KH2PO4 diluted to 50 ml with distilled water. Colonies were then counted, and the observed control plating efficiencies were 21% for BE cells and 54% for HT-29 cells. Alkaline Elution Experiments. Cells (1.5 to 2.5 x 10s) were seeded into 25-cm2 flasks in 10 ml of medium which contained 0.02 ¿iCiof [14C]thymidine/ml and grown for 48 h. Alkaline elution experiments were carried out as previously described ( 1, 3). For analysis of inter strand cross-links and DNA strand breaks, cells were lysed on 0.8-Mm pore size polycarbonate filters with 2% sodium dodecyl sulfate:0.025 M EDTA:0.1 M glycine (pH 10.0), which was allowed to flow through the filter by gravity. Following lysis, 2 ml of 2% sodium dodecyl sulfate:0.02 M EDTA:0.1 M glycine (pH 10.0) containing 0.5 mg of proteinase K/ml were added to a reservoir over the filter and pumped through the filter for approximately l h at 2 ml/h. DNA was eluted from filters by pumping 0.02 M EDTA solution adjusted to pH 12.1 with tetrapropylammonium hydroxide, which contained 0.1% sodium dodecyl sulfate. For assays of total DNA cross-links and DNA strand breaks in the absence of proteinase K, cells were lysed on 2-^m pore size Polyvinyl chloride filters with 2% sodium dodecyl sulfate:0.02 M EDTA:0.1 M glycine (pH 10.0). Filters were then washed with 5 ml of 0.02 M EDTA (pH 10.0) prior to elution with tetrapropylammonium hydroxide:EDTA (pH 12.1). In all experiments, internal standards were [3H]154
CYCLODISONE-INDUCED
DNA DAMAGE
1.0 0.8
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S
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Fig. 1. The proposed mechanism of nucleophilic attack of cyclodisone.
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CONCENTRATION (pM) Fig. 2. Inhibition of the colony-forming ability of BE (O) and HT-29 (•) human colon carcinoma cells by 2-h treatments with various concentrations of cyclodisone. Points, mean; bars, S.D.
thymidine-labeled LI210 cells irradiated with 3 Gy of 137Cs7-rays in the cold. For all cross-link assays, control and drug-treated cells were also irradiated with 3 Gy. Results were quantified as previously de scribed (3, 4).
RESULTS
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FRACTION OF (°H] DNA RETAINED ON FILTER Fig. 3. Alkaline elution profiles to show the dependence of DNA strand break formation on the use of proteinase K (Pro-K) in the alkaline elution assay. BE (left) and HT-29 (right) cells were exposed to cyclodisone for 2 h and then incubated in drug-free medium for 12 h. Cyclodisone exposures were: O control; •,25 ¿IM; A, 50 IIM; A, 100 JIM; Ü,200 >iM. Top, presence of proteinase K, bottom, absence of proteinase K in the elution experiments. These profiles are representative of at least two independent experiments.
Cytotoxicity assays, based upon inhibition of colony forma tion, showed the BE (Mer—)cells to be 6-fold more sensitive to cyclodisone than HT-29 (Mer+) cells (Fig. 2). Twenty-five ¿JM cyclodisone produced a 1-log cell kill in the BE cell line, whereas 125 to 150 n\t cyclodisone was required to produce the same degree of cell kill in the HT-29 cell line. Cyclodisone induced DNA strand breaks to a similar degree in both cell types when compared at equimolar concentrations (Fig. 3). Fig. 3 shows the alkaline elution profiles obtained 12 h after a 2-h drug exposure. To determine whether the appear ance of these DNA strand breaks was due to the destruction of a drug-induced DNA-protein interaction, we compared the elution profiles obtained with or without the use of a proteinase K digestion step in the elution assay. No strand breaks were .0 0.5 0.2 0.1 observed in either the BE or HT-29 cell lines when proteinase FRACTION OF [3H] DNA RETAINED ON FILTER K was not used in the elution experiments. Similar levels of strand breaks were observed at 0, 6, and 18 h after cyclodisone Fig. 4. Alkaline elution profiles to detect the presence of DNA interstrand cross-links in BE (left) and HT-29 (right) cells after a 2-h treatment with exposure (data not shown). In all cases, these strand breaks cyclodisone; O, control; •,25 UM;A, 50 *iM;A, 100 >IM;D, 200 nM. In each case, cells were irradiated with 3 Gy of "7Cs -y-rays. These profiles are representative appeared to be protein associated. of at least two independent experiments. The ability of cyclodisone to induce DNA interstrand cross links, in either BE or HT-29 colon carcinoma cells, was ana lyzed by the technique of alkaline elution. Fig. 4 shows that 12 be emphasized that 100 JIM cyclodisone is extremely toxic to h after a 2-h drug treatment, a small quantity of DNA interthe BE cells. In comparison, a variety of other alkylating agents, strand cross-links was formed in the BE cell line with 100 MM at concentrations which give a 2-log cell kill in the BE cell line, cyclodisone. In contrast, no evidence of DNA interstrand cross- produce peak levels of DNA interstrand cross-linking greater linking was observed in the HT-29 cell line, even at concentra than the value shown by the dotted line in Fig. 5, left (1,5, 6). tions of 200 /¿Mcyclodisone (Fig. 4). The increased rate of The negative DNA interstrand cross-link indices, obtained in elution of the HT-29 drug-treated cells, relative to the 3-Gy the HT-29 cell line, are again a reflection of the large number control cells, is due to the large number of DNA strand breaks of DNA strand breaks produced in this cell line. Alkaline elution assays, which measure both DNA inter produced at the same time, after drug treatment (see Fig. 3). The kinetics of formation of DNA interstrand cross-links strand cross-linking and DNA-protein cross-linking in both cell showed that these cyclodisone-induced lesions peaked 12 to 18 lines 12 h after a 2-h drug treatment, are shown in Fig. 6. Fig. 6 shows that there is an increase in total DNA cross-linking h after a 2-h drug treatment (Fig. 5). DNA interstrand cross link indices were calculated as described earlier (3, 4). It must with increasing concentrations of cyclodisone in both the sen155
CYCLODISONE-INDUCED DNA DAMAGE
phenotype than cells of the Mer+ phenotype (5, 7). The mech anism behind this differential toxicity is thought to be due to the formation of DNA interstrand cross-links. Mer—cells have 0.08-$ been shown to be deficient in a DNA repair process (guanine °-06"---0--repair. In contrast, the DNA-protein cross-links formed in the • —•C^
