View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by PubMed Central Br. J. Cancer 205-211 Macmillan Press Ltd., 1994 Br. J. Cancer (1994), 69, 205-211 (D 1994 Mechanism of cytotoxicity of 5,10-dideazatetrahydrofolic acid in human ovarian carcinoma cells in vitro and modulation of the drug activity by folic or folinic acid E. Erbal, S. Sen', C. Sessa2, F.L. Vikhanskayal* & M. D'Incalcil 'Istituto di Ricerche Farmacologiche 'Mario Negri' Via Eritrea 62, 20157 Milan, Italy; 2Ospedale San Giovanni, CH 6500 Bellinzona, Switzerland. Summary Inhibition of clonogenic potential by the glycinamideribonucleosyl transformylase inhibitor 5,10- dideazatetrahydrofolic acid (DDATHF, Lometrexol) was evaluated in vitro in a human ovarian carcinoma cell line, SW626. Drug-induced inhibition of clonogenic potential is a function of the dose and time of exposure and is independent of the formation of DNA single-strand breaks or de novo synthesis of protein. Simul- taneous treatment with 1OOiLM hypoxanthine completely prevented the inhibition of clonogenic potential caused by 0.5 ILM DDATHF. DDATHF blocked cells in the early-middle S-phases of the cell cycle, and there was a correponding marked reduction in the rate of DNA synthesis after drug withdrawal. The cytotoxic potential of DDATHF was modulated by the folic acid concentration present in the medium. In a medium containing 0.22 gM folic acid, DDATHF cytotoxicity was at least 100 times that in a regular medium containing 2.221LM folic acid, levels which, however, are about 100 times those found in human plasma. DDATHF cytotoxicity differed moderately when folic acid concentrations varied between 0.22 and 011M, suggesting that folic acid does not necessarily antagonise DDATHF anti-tumour activity. Folinic acid at a concentration as low as 0.1IJM can completely rescue cells when given simultaneously with 0.5 gAM DDATHF. When folinic acid was given 24 h after DDATHF, a reversal of cytotoxicity was observed at 0.5 and I gAM, but to a much lesser extent than simultaneous treatment. When folinic acid was added after 48 or 72 h of DDATHF washout, even at a high concentration and for a long time, no reduction in DDATHF cytotoxicity was found. In conclusion, the study highlights the modulation of DDATHF cytotoxicity by folic acid or by folinic acid and provides further rationale for in vivo clinical investigation with these combinations. 5,10-Dideazatetrahydrofolic acid (DDATHF, Lometrexol) is The aim of this study was to investigate whether and how an anti-cancer agent under early clinical investigation in DDATHF affects the normal cell cycle distribution and Europe and in the USA. It is the first clinically investigated DNA integrity of tumour cells exposed to cytotoxic concent- antifolate whose mode of action is related to the inhibition of rations of the drug and to obtain information on the glycinamideribonucleosyl (GAR) transformylase, a key influence of folic and folinic acid on the drug cytotoxicity. enzyme in the de novo synthesis of purines (Moran et al., 1985; Beardsley et al., 1989; Taylor et al., 1989; Baldwin et al., 1991). Many aspects of the cellular pharmacology of DDATHF have already been investigated in detail. Materials and methods DDATHF appears to a be good substrate for membrane Cells and folate-binding proteins (mFBP) (Kane et al., 1988; Jansen et culture conditions al., 1991; Westerhof et al., 1991), which probably act as a The SW626 human ovarian carcinoma cell line was used (Sen relevant carrier for its intracellular transport. The intracel- et al., 1990). For all these experiments cells were grown as lular transport can also be mediated by the reduced folate monolayers in RPMI-1640 medium supplemented with 10% carrier (Pizzorno et al., 1993). Once in the cell, DDATHF is dialysed fetal bovine serum (FBS) (cut-off point 3,500 Da). In efficiently biotransformed to polyglutamated metabolites, order to assess the role of folic acid in the medium, cells were which are much more potent inhibitors of GAR transfor- grown in. progressively lower folate-containing media. From mylase than the monoglutamated parent compound (Piz- normal RPMI-1640 containing 2.2 lM folic acid, the cells zorno et al., 1991a). were conditioned to grow in low-folate medium by reducing What is not yet known is the mechanism of cytotoxicity the folic acid concentration from 2.2 gsm to 0.22 iAM in at consequent to GAR transformylase inhibition. Like other least six passages, then stepwise from 0.22 to 0.11 to 0.05 to antifolates (Lorico et al., 1988) DDATHF could cause DNA 0.025 to 0.0125 liM. From the last passage, cells were condi- damage, which will eventually result in cell death, but this tioned to grow in completely folate-free conditions in at least hypothesis requires experimental verification. six passages. Under low-folate conditions, the cells grew During phase I clinical studies DDATHF showed severe normally; the doubling time and cell morphology remained and unexpected haematological and gastrointestinal toxicity unaffected. The doubling time of SW626 cells after appropri- in some patients (Muggia et al., 1990; Sessa et al., 1990; Ray ate adaptation in different medium was 23 h in medium et al., 1992). Two approaches are currently under clinical containing normal serum and 2.27 tLM folic acid, 23 h in investigation to reduce the risk of toxicity: (i) the con- medium containing dialysed serum and 2.27 tLM folic acid, comitant administration of folic acid and (ii) the use of 23 h in medium containing dialysed serum and 0.22 ftM folic folinic acid as an antidote. Both approaches are based on acid in 25 h in medium containing dialysed serum and 0 gLM findings in mice, but the mechanism by which folic and folic acid. During conditioning in complete folate-free folinic acid counteract DDATHF-induced toxicity is not yet medium, the size of the cells increased initially and their clear (Alati et al., 1992; Grindey et al., 1992). doubling time also increased, but after a few passages in this folate-free medium, they regained their original morphology and started growing, as in the presence of folate. Correspondence: M. D'Incalci *Visiting scientist from the Institute of Cytology, Acad. Sci. Russia, Reagents and culture ware 194064 St. Peterburg, Russia, and now fellow of Consorzio Mario Negri Sud, Santa Maria Imbaro, Italy. 5,10-Dideazatetrahydrofolic acid (DDATHF) (batch Received 26 March 1993; and in revised form 13 September 1993. 235MH8) was obtained from Eli Lilly (Indianapolis, IN, 206 E. ERBA et al. USA). Folinic acid (batch LFP956) was provided by PBS then another 1 h incubation with 100 gld of fluorescin- Cyanamid Italia. Methotrexate was obtained from the Drug conjugated goat anti-mouse IgG diluted 1:50 in 5% Tween Synthesis and Chemistry Branch, Division of Cancer Treat- 20 in PBS. After washing with PBS, the cells were resus- ment, National Cancer Insititute, Bethesda, MD, USA. pended in a solution of 5Lgml-' propidium iodide in PBS RPMI-1640 medium and custom prepared folic acid-free and 10,000 U of ribonuclease for at least 2 h in the dark. RPMI-1640 medium (Cat. No. 041-90735 M) were purchased from Gibco Europe, Paisley, UK. Fetal bovine serum (batch 669141) was from Biological Industries, Israel. Cyclohex- Flow cytometric immunofluorescence analysis on MO V18 imide, propidium iodide and ribonuclease were purchased MOV18 expression was detected in SW626 cells growing in from Calbiochem Corporation. Bromodeoxyuridine (BrdU) RPMI with 2.2 lJM or without folic acid after 1 h incubation and goat anti-mouse IgG conjugated with fluorescein with 100 gl of MOV18 antibody diluted 10 tg ml-' in PBS isothiocyanate (FITC) were purchased from Sigma (St Louis, with 0.3% bovine serum albumin (BSA). After washing with MO, USA). Anti-BrdU was from Becton Dickinson (Moun- PBS, the cells were incubated for 1 h with 100 gIl of fluo- tain View, CA, USA) and normal goat serum was a product rescein-conjugated anti-mouse IgG developed in goats diluted of Dakopatts, Denmark. '4C-labelled thymidine (specific 1:50 in 5% Tween 20 in PBS. activity 2.11 GBq mmol ') was obtained from Amersham. Spectra/Por 3 (molecular weight cut-off 3,500) membrane from Spectrum Medical Industries (Los Angeles, CA, USA) Alkaline elution was used as dialysis bag. Plastic flasks and plastic Petri dishes Exponentially growing cells were incubated with '4C-labelled used for tissue culture were from Nunclon (Nunc, Denmark) thymidine for 24 h. The radioactive label was removed and and Falcon (Becton Dickinson, USA) respectively. the cells were chased for a further 24 h. DNA single-strand breaks were assessed by alkaline elution methods slightly DDA THF treatment modified previously (Kohn et al., 1981). DNA breaks were assessed in parallel in samples X-irradiated with 300 rad as DDATHF was dissolved in medium containing dialysed positive controls. serum immediately prior to use. The concentrations of DDATHF tested in different experiments ranged between 1 nM and 1 gLM. Results Several phase I clinical trials with DDATHF are in pro- gress and we still do not know the maximal tolerated dose of The clonogenic inhibitory effect of DDATHF is shown in this drug. At a dose of 45 mg m-2 the plasma concentrations Figure 1. Dose-dependent inhibition was seen 14 days after of DDATHF ranged approximately from 20 tLM to 0.2 tLM 24h drug exposure. The IC50 was approximately 0.25 AM. during the 24 h following drug infusion (D.R. Newell, per- Continuous exposure to 1-50nM DDATHF for 48, 72, 96 sonal communication). Therefore the concentrations used by or 192 h caused concentration- and time-dependent inhibition us were in a pharmacologically reasonable range. of clonogenicity in sets receiving 5, 10, 25 or 50 nM DDATHF (Figure 2).