Cancer Chemother Pharmacol. (1990) 26: 340-342 ~ ancer hemotherapyand harmacology © Springer-Verlag 1990 Cytotoxicity and DNA damage caused by 4-demethoxydaunorubicin and its metabolite 4-demethoxy-13-hydroxydaunorubicin in human acute myeloid leukemia cells Monica Limontal, 2, Andrea Biondi2, Giovanni Giudici2*, Giorgina Specchia 3, Carlo Catapan01, Giuseppe Masera 2, Tiziano Barbni4, and Maurizio D'Incaicil t Istituto di Ricerche Farmacologiche "Mario Negri", Via Eritrea, 62, 1-20157 Milan, Italy 2 Ctinica Pediatrica, Universiflt di Milano, Ospedale S. Gerardo, Monza, Milan, Italy 3 Ematologia, Universi~ di Bari, Italy '*Divisione di Ematologia, Ospedale di Bergamo, Italy Received 18 April 1989/Accepted 2 March 1990 Summary. 4-Demethoxydaunorubicin (4-DMDR.) and its major metabolite 4-demethoxy-13-hydroxydaunorubicin (4-DMDRol) were investigated for their cytotoxicity and mode of action against human leukemic cells. The drug and its metabolite appeared to be equally potent as both inhibitors of cell proliferation and inducers of DNA dou- ble-strand breaks in the OCI AML-3 cell line and cells derived directly from patients with acute myeloid leukemia (AML). This suggests that 4-DMDRol plays an important role in the antileukemic activity of 4-DMDR. Introduction 4-Demethoxydaunorubicin (4-DMDR) is an anthracycline [1] with proven clinical antileukemic activity [5, 7, 8]. Clinical pharmacokinetic studies have shown that 4-DMDR is extensively biotransformed to 4-demethoxy- 13-hydroxydaunorubicin (4-DMDRol), a metabolite that is eliminated much more slowly than the parent drug [11]. To elucidate the possible role of 4-DMDRol in the anti- leukemic activity of 4-DMDR, we carried out a series of experiments comparing the cytotoxicity of these drugs and the DNA damage they induce. Materials and methods Cells. Peripheral blood was obtained from five adult patients with AML after they had given their informed consent to participate in the study. Mononuclear cells were isolated by Ficoll-Hypaque centrifugation. At this point, the blast-cell content of the samples was >90%. The OCI AML-3 cell line was derived from a patient with acute myelomonocytic leukemia (FAB M4) (Biondi, manuscript in preparation) and was main- tained at 37"C in a humidified atmosphere containing 5% CO2 and cultured in lscove's medium with 10% fetal calf serum. Four patients had * Supported by Fondazione Tettamanti Offprhtt requests to: M. D'lncalci previously been treated with chemotherapeutic agents including ara-C, vincristine, doxorubicin, daunorubicin, mitoxanthrone, VPI6 and 4-DMDR; the other subject (patient 5) had not received prior treatment. Drug treatment. 4-DMDR and 4-DMDRol were synthesized and gener- ously provided by Dr. Suarato of Farmitalia Carlo Erba (Milan, Italy). They were pure as assessed by HPLC. Before their use, the drugs were dissolved in sterile water at a concentration of 1 mg/ml and then diluted in medium at the indicated concentrations. Leukemic blast cells were resuspended in lscove's medium with glutamine, 25 mM HEPES, sodium bicarbonate, 1% sodium piruvate, 20% fetal calf serum and were stimulated with 10% GCT conditioned medium (Gibco) for 24 h before drug treatment. In all experiments, cells were exposed to the drugs for4 h. Determination of DNA double-strand breaks. DNA double-strand breaks (DNA-DSB) were determined according to the method described and recently reviewed by Kohn et al. [6]. OCI AML-3 cells were labeled for 48 h using medium supplemented with 0.04 I.tCi/ml [14C]-thymidine (specific activity, 61 m Ci/mmol), then were washed, resuspended in medium and exposed for 4 h to several concentrations (0.005, 0.01,0.05, 0.1 and 1 [.tg/ml) of DMDR or DMDRol. Cell were washed, resuspended in cold PBS and layered on polycarbonate filters (pore size, 0.8 p.m; diameter, 47 ram) (Nucleopore Corp., Pleasanton, Calif). Cells (2-3 × 105 cells/sample) were lysed with 2 ml lysis solution containing proteinase K and the elution buffer [20 mM ethylenediaminetetraacetate (EDTA) solution (adjusted to pH 9.6) containing 0. !% sodium dodecyl sulfate (SDS)] and were pumped for 15 h through the filter at approxi- mately 2 ml/h. Fractions were collected every 3 h and processed as previously described [8]. Colony assays. Colony assays were carried out according to previously described methods [4, 9]. In brief, 103 OCI AML-3 cells were plated in 0.5-ml cultures in Iscove's modified Dulbecco's medium (IMDM; Gibco Laboratories, Grand Island, N. Y.) containing 20% heat-inactivated fetal calf serum (FCS; Flow Laboratories, UK) and 0.3% agar (Agar Noble; Difco Laboratories, Detroit, Mich.) over an underlayer containing 0.5% agar in the same medium. After 6-7 days, the agar overlayers were removed from the undertayers, dried on glass slides and fixed in methanol and the colonies were stained with hematoxylin and counted. Each experimental point was determined in quadruplicate. [.~H]-Thymidine incorporation. After treatment with DMDR and DMDRol (0. I- I p.g/ml), cells were washed with PBS and incubated in drug-free medium for 72 h. Between 48 and 72 h after the end of drug treatment, cells were exposed to 0. I ktCi/ml [3H]-thymidine (specific activity. 83 Ci/mmol). At 72 h, ceils were harvested, washed with PBS