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Sensitivity and resistance in human metastatic melanoma to the new chloroethylnitrosourea anti-tumor drug Fotemustine
277
Biochimica et Biophysica Acta, 1096 (1991) 277-283 '~ 1991 Elsevier Science Publishers B.V. 0925-4439/91/$03.50 A DONIS 092544399100079D
BBADIS 61037
Sensitivity and resistance in human metastatic melanoma to the new chloroethylnitrosourea anti-tumor drug Fotemustine K a r i n U . S c h a l l r e u t e r a n d J o h n M. W o o d Department of Dermatology, Unit,ersity of Hamburg, Hamburg (F. R. G.) (Received 2 August 1990)
Key words: Anti-tumor drug; Drug resistance; (Human melanoma)
Fotemustine is a novel chloroethylnitrosourea derivative currently used in Phase III clinical trials for disseminated metastatic melanoma. This drug has been shown to inhibit enzymes in the ribonucleotide reduction pathway (i.e., thioredoxin reductase, glutathione reductase and ribonucleotide reductase), t4c chloroethyl-labelled Fotemustine covalently labels the thiolate active sites of thioredoxin reductase and glutathione reductase yielding ~4C chloroethylthioether enzyme-inhibitor complexes. Enzyme activities can be restored by a reduced thioredoxin or reduced glutathione mediated fl-elimination of the chloroethyl group. 14C Fotemustine has been used to determine its reactivity and metabolism in drug sensitive and resistant melanoma metastases and in cultures of sensitive and resistant clones of human melanoma cells. Melanoma metastases from four different patients who were treated with Fotemustine could be labelled with radioactive drug only under reducing conditions with N A D P H as electron donor and DTNB as substrate. FPLC analysis of these extracts revealed two radioactive proteins (I) glutathione reductase and (II) an unidentified protein with 95 and 50 kDa subunits. A similar labelling pattern was also found in extracts of Fotemustine sensitive melanoma cells (Cal 1). Fotemustine resistant tumors were melanotic and contained more glutathione reductase than thioredoxin reductase, whereas sensitive tumors were clinically amelanotic with more thioredoxin reductase than glutathione reductase. Fotemustine resistant melanoma cells (Cal 7) showed a slower uptake of 14C-label with 34% less isotope intraceilularly in 1 h compared to sensitive melanoma cells (Cal 1). These results strongly indicate (I) the induction of alternate electron donors thioredoxin reductase or glutathione reductase for ribonucleotide reduction determines tumor and melanoma cell responses to the drug and (II) Fotemustine transport and the intracellular redox status seems to regulate resistance in melanoma cells and tissues.
Introduction The chloroethylnitrosoureas are highly reactive compounds with a general instability in aqueous solution after 1 hour at 37°C [1,2]. This homologous series of drugs are in widespread use for the treatment of gliomas, Hodgkin lymphomas, lung cancer, colorectal cancer and melanoma [1,2]. It has been suggested that the major cytotoxicity of the chloroethylnitrosoureas resides in their formation of crosslinking bridges at DNAguanine-O 6 positions [3-10]. This single reaction has been proposed responsible for their anti-tumor activity [11-13]. A mechanism involving chloroethyl-group transfer from a single DNA-guanine-O 6 position to the
Correspondence: K.U. Schallreuter, Department of Dermatology, University of Hamburg, Martinistrasse 52, D-2000 Hamburg 20, F.R.G.
thiolate active site of the guanine-O6-alkyltransferase enzyme yields a chloroethylthioether enzyme-inhibitor complex [2]. As a consequence, cells with low guanineO6-alkyltransferase activity have been especially sensitive to the chloroethylnitrosoureas [2]. A second mode of action for the chloroethylnitrosoureas has been shown involving its reactivity with other important thioproteins. These drugs alkylate the thiolate active sites of glutathione reductase, thioredoxin reductase and ribonucleotide reductase [14,15]. This inhibition would be expected to prevent the synthesis of deoxyribonucleotides in the S-phase of the growth cycle [16]. However, the covalent deactivation of thioredoxin reductase and glutathione reductase, the alternate electron donors for ribonucleotide reductase, can be reversed by their respective reaction products reduced thioredoxin and reduced glutathione [15]. Reduced thioredoxin and reduced glutathione catalyze a nucleophilic B-elimination of chloride ions
278 from the chloroethylthioether enzyme-inhibitor complexes [15]. A general mechanism for the alkylation of thioproteins by the chloroethylnitrosoureas followed by reactivation as a result of B-elimination is presented in Scheme I. Clearly, the significance of this pathway for the inhibition of DNA-synthesis resides in the inhibitor constants for the reactions of different chloroethylnitrosoureas with the three enzymes involved in ribonucleotide reduction. In enzymes purified from human metastatic melanoma tissue, glutathione reductase was significantly less reactive with Fotemustine than thioredoxin reductase [15]. Based on these results, it was proposed that drug resistance and sensitivity may reside in the dominance of the glutathione reductase/glutaredoxin vs. the thioredoxin reductase/thioredoxin pathways for electron transfer to ribonucleotide reductase in metastatic melanoma tissues or in clones of sensitive or resistant melanoma cells. The aim of this study was to examine drug resistant and drug-sensitive tumors and cells in terms of their thioredoxin reductase and glutathione reductase activities as well as their ability to react with the chloroethylnitrosourea Fotemustine.
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Materials and Methods
Enzymes and cell extracts Thioredoxin reductase (TR) and glutathione reductase (GR) have been purified from human metastatic melanoma tissues by a modified method of Luthman and Holmgren [17,18,24]. Seven metastases were obtained from four patients intravenously treated with Fotemustine (100 m g / m 2 body surface). The treatment cycle included a weekly administration over 3 weeks, followed by a 3 week rest period with a consequent assessment of tumor size. Two patients showed initially a partial remission (i.e., < 50% decrease of tumor size) followed by a progression of the tumor. At this time, metastases were excised. The two other patients showed a progressive disease after the 3 week induction cycle with Fotemustine. At this point, tumors were excised. Cell extracts were prepared for FPLC analyses on a Mono-Q anion-exchange column (HR 10/10) by the following procedure: (1) After surgical excision, tumors were frozen in liquid nitrogen and stored at - 7 2 ° C . (2) Tissues were homogenized and sonicated in 0.1 M Tris-HC1 buffer at pH 7.5 followed by centrifugation at 10000 rpm for 30 min at 0°C. (Upon clinical examination, those tumors initially responding to Fotemustine appeared amelanotic or only slightly pigmented, meanwhile tumors from patients not responding to the drug were significantly darker. Fig. 1 shows a photograph of the membranous pellets from all of the metastases examined.)
Fig. 1, Photograph of melanin deposits in membranes of Fotemustine sensitive (A) and resistant (B) metastases.
(3) Soluble extracts were dialyzed against 5 1 of 0.05 M Tris-HC1 buffer (pH 7.5) for 48 h with three buffer changes. (4) Proteins were determined by the method of Kalb and Bernlohr [16]. (5) 100 mg of protein from each cell extract was applied to a preparative Mono-Q column (HR 10/10) equilibrated with 0.05 M Tris-HC1 buffer (pH 7.5). (6) Thioredoxin reductase and glutathione reductase standards were subjected to FPLC analysis in a 0-0.5 M NaC1 gradient prior to the chromatography of each melanoma cell extract. Enzyme assays Thioredoxin reductase and glutathione reductase were assayed by the reduction of DTNB at 412 nm, using N A D P H as the electron donor, by the method of Luthman and Holmgren [171.
279 Membrane-associated thioredoxin reductase was determined by the method of Schallreuter and Wood [18]. 3 m m punch biopsies were taken from each tumor sample and the reduction of nitroxide radicals on a spin-labelled quaternary a m m o n i u m substrate was followed in quartz tissue cells in a Bruker D-200 EPR spectrometer at 25°C. The amplitude of the nitroxide radical signal was followed with time under conditions of saturating free radical substrate (3.5 • 10-3 M). This assay has been shown to be specific for epidermal thioredoxin reductase [19]. (*U.S. Patent N u m b e r 4,849,346 (1989).)
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lsotope experiments ~4C chloroethyl-labelled Fotemustine was provided by Servier Company (France) (53 m C i / m m o l ) [15]. T u m o r cell extracts (100 mg) were preincubated with N A D P H ( 5 . 1 0 -3 M) and D T N B (2.5.10 -3 M) over 15 min before the addition of 10 ~tl of 14C Fotemustine. These radiolabelled extracts were dialyzed against 0.05 M Tris-HC1 buffer (pH 7.5) for 5 h prior to F P L C analysis. Melanoma cells (2-3 mg of protein) were preincubated for 1 h with 10 /zl of 14C Fotemustine, centrifuged, resuspended in 1.0 ml of 0.05 M Tris-HC1 buffer (pH 7.5), washed with buffer and cell extracts prepared, using a microprobe of a Heat Systems sonicator at the m a x i m u m setting at 0°C. Cell membranes were removed by centrifugation at 5000 rpm for 10 rain at 0°C. Results The interaction of Fotemustine with plasma membrane-associated thioredoxin reductase was examined in 3 m m tissue biopsies taken from human melanoma metastases. The spin-label reduction assay for membrane-associated thioredoxin reductase revealed a time dependent inhibition of this enzyme by Fotemustine (Fig. 2). After 20 min preincubation time with the drug, full enzyme activity returned suggesting rapid degradation of this chloroethylnitrosourea in these experiments. This inhibition/reactivation phenomenon has been repeated in biopsies taken from three other metastatic melanomas from different patients. However, enzyme recovery did not occur in every tumor tested nor in skin biopsies from normal healthy donors. Therefore, these preliminary results suggested that the recovery of membrane-associated thioredoxin reductase from Fotemustine inhibition may be tumor specific. Experiments reported previously [15] on thioredoxin reductase and glutathione reductase showed that Fotemustine is a much more potent inhibitor of thioredoxin reductase ( K I = 3.0 • 10 -5 M) compared to glutathione reductase ( K I = 1.5.10 -3 M). Based on this 500-fold difference in the sensitivity of thioredoxin reductase over glutathione reductase to Fotemustine, it
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Fig. "~. The inhibition of membrane-associated thioredoxin reductase on 3 mm punch biopsies taken from a metastatic melanoma followed by reactivation. Reactions were performed in the presence of 3.5.10-3 M nitroxide radical substrate using 2.5.10 -3 M Fotemustine as inhibitor for incubation times of 0, 3, 5, 10, 15 and 20 min. Specific activities for membrane-associated thioredoxin reductase were determined as the decrease in amplitude of the nitroxide radical signal/10 mg of tissue per 10 min.
was anticipated that those tumors with the thioredoxin reductase/thioredoxin pathway for ribonucleotide reduction should be more prone to a drug response compared to tumors with the alternate glutathione reductase/glutaredoxin system. To test this hypothesis, four patients were selected without previous chemotherapy other than the induction cycle with Fotemustine. Seven tumors were obtained; ( 4 / 2 patients who initially responded to Fotemustine, and 3 / 2 patients who did not respond). F P L C analysis of crude extracts from these tumors revealed that both thioredoxin reductase and glutathione reductase were present at significant levels in all tumors as identified by: (1) pure enzyme standards and (2) the D T N B reduction assay. Fig. 3a shows the analysis of one Fotemustine sensitive tumor where thioredoxin reductase activity was 3.5-fold higher than glutathione reductase activity in the standard D T N B reduction assay. Fig. 3b shows one example of a drug-resistant tumor where glutathione reductase activity was 4.4-fold that of thioredoxin reductase. Similar results were obtained for four responding and three non-responding tumor extracts. These results unambiguously indicate that a switch from the thioredoxin reductase/thioredoxin pathway to the glutathione reductase/glutaredoxin system in metastatic melanoma coincides with resistance to Fotemustine. Since 14C chloroethyl-labelled Fotemustine alkylates the active sites of thioredoxin reductase and glutathione reductase yielding covalently labelled enzyme-inhibitor complexes, this reaction was examined in crude extracts from melanoma tissues [15]. The 14C chloroethyl-etherglutathione reductase complex has been more stable
280 than the corresponding thioredoxin reductase complex [15]. This instability appears to be promoted by the oxidizing conditions on the Mono-Q anion-exchange column during the FPLC procedure (i.e., 14C-labelled pure thioredoxin reductase loses its radioactivity during the FPLC procedure whereas labelled glutathione reductase is stable). Fig. 4a and b present FPLC analyses of crude extracts labelled with ~4C Fotemustine from drug resistant and drug sensitive tumors. It should be noted that the covalent labelling of proteins in these extracts occurs after preincubation with N A D P H as electron donor and D T N B as substrate. Without the addition of cofactor and substrate for optimum reducing conditions, isotope incorporation into these cell extracts has been unsuccessful. Two proteins are labelled with ~4C Fotemustine: (1) glutathione reductase and (2) an unidentified protein eluting at 0.17 M NaC1 under the chosen FPLC conditions. Although glutathione reductase was labelled with ~4C Fotemustine, it was not completely inhibited being still active in the D T N B
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Fig. 3. (a) FPLC separation (Mono-Q HR 10/10) of TR and GR in 0.05 M Tris-HC1 buffer (pH 7.5) using a 0-0.5 M NaC1 gradient. TR eluted at 0.23 M NaC1 and GR at 0.3 I M NaC1. Peak fractions were assayed by DTNB reduction as AA4]z/mg protein per 10 min yielding TR =0.51 and GR =0.15 (colon metastasis, Fotemustine responder). (b) FPLC separation (Mono-Q 10/10) of TR and GR in 0.05 M Tris-HC1 buffer (pH 7.5) using a 0-0.5 M NaC1 gradient. TR eluted at 0.23 M NaCI and GR at 0.3 M NaC1. Peak fractions assayed by DTNB reduction gave specific activities for TR -0.074 and GR - 0.326 (lymph node metastasis, Fotemustine non-responder).
unknown protein (peak 1) and GR (peak 2). Fotemustine incorporation into GR caused delayed elution at 0.34 M NaCI instead of the 0.3 1 M NaCI observed in controls. GR was still active in the DTNB reduction assay (specific activity -0.085). (b) FPLC separation (Mono-Q 10/10) of cell extracts from a Fotemustine-resistant tumor showing 14C Fotemustine uptake into unknown protein (peak 1) and GR (peak 2). GR had a residual specific activity of 0.215 in the DTNB reduction assay.
reduction assay. The unidentified protein was purified further by FPLC and yielded subunits of 95 and 50 Kda upon S D S - P A G E analysis (Fig. 5). Since this protein was not labelled in the absence of N A D P H and DTNB, it is assumed to be another thioprotein capable of nucleophilic displacement of the chloroethyl-group from Fotemustine. H u m a n melanoma cell lines which are sensitive (Cal 1) and resistant (Cal 7) to Fotemustine were used in the following experiments. Suspensions of Cal 1 and Cal 7 melanoma cells were incubated with ~4C Fotemustine and the intracellular radioactivity was determined after 1 h. Cal 1 incorporated 8773 c p m / m g protein per hr and Cal 7 5811 c p m / m g protein per hr. FPLC analysis of Cal 1 extracts yielded incorporation of the ~4C label into: (1) glutathione reductase; (2) unknown protein (95 k D a / 5 0 kDa); and (3) a small radioactive peak eluting at 0.39 M NaC1, whereas cell extracts from Cal 7 revealed no clearly defined radioactive incorporation
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drugs with individual thioenzymes, (2) the intracellular levels of enzymes which can alternate for each other in important metabolic pathways (e.g., thioredoxin reductase and glutathione reductase in ribonucleotide reduction), (3) the cellular reducing potential v i s a vis N A D P H synthesis via the pentose phosphate shunt for glucose metabolism ( N A D P H controls the reactivity of the chloroethylnitrosoureas) and (4) the more rapid uptake of Fotemustine in drug-sensitive cells c o m p a r e d to resistant cell lines. Since glutathione reductase is 500-fold less sensitive to inhibition by Fotemustine than thioredoxin reductase [15,22], then both a kinetic effect based on drug concentration, and the replacement of the thioredoxin reductase system for the glutathione reductase p a t h w a y for ribonucleotide reduction m a y be expected to confer resistance in tumors and m e l a n o m a cells. The reactiva-
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Discussion
It is well established that the instability of the chloroethylnitrosourea anti-tumor drugs resides in their susceptibility to nucleophilic attack causing a c o m m o n mechanism of alkylation by the displacement of a chloroethylcarbonium ion [20]. It is now recognized that several important thioenzymes are covalently deactivated by these drugs by the formation of chloroethylthioether-enzyme-inhibitor complexes. Thioenzymes involved in ribonucleotide reduction, and as a consequence D N A synthesis, are primary targets for these drugs (i.e., TR, G R and RR). Also, the thioprotein guanine-O6-alkyl transferase, an important enzyme in preventing ethyl-bridged D N A crosslinks, is alkylated by both the chloroethylnitrosoureas and by chloroethylD N A adducts [2]. The Class M u glutathione transferases appear to be important in the metabolism of chloroethylnitrosoureas assuming importance over the other glutathione transferase isozymes in drug resistant cells [21]. However, all the above mentioned reactions point to a c o m m o n mechanism for inhibition a n d / o r resistance to this homologous series of drugs: (1) alkylation of active site thiolate groups by chloroethyl-group transfer and (2) reductive fl-elimination of the chloroethyl-group to reactivate these enzymes (Scheme I). As a consequence of these two reactions, four independent factors could be critically important in chloroethylnitrosourea sensitivity/resistance in t u m o r cells and tissues: (1) the kinetics for the reaction of these
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Fig. 6. (a) FPLC separation of 14C Fotemustine labelled proteins from cell extracts of a human melanoma cell clone Cal 1 (Fotemustine-sensitive) on a Mono-Q HR 5/5 column in 0.05 M Tris-HCl buffer (pH 7.5) using a 0-0.5 M NaCI gradient. Radioactive peaks were corrected for background and eluted at 0.08, 0.18 and 0.39 M NaC1. The peak at 0.18 M NaCI was identified as GR and the small peak at 0.39 M NaCI revealed similar chromatographic properties to ribonucleotide reductase. The peak at 0.08 was the unidentified protein (95 kDa/50 kDa). (b) FPLC profile of 14C Fotemustine labelled proteins from cell extracts of human melanoma cell clone Cal 7 (Foiemustine resistant). There was no discernible radioactive incorporation into glutathione reductase and the total intracellular lac incorporation was 34% less in Cal 7 compared to Cal 1.
282 Inhibition and reactivation of chloroethyl-nitrosourea inhibited thioproteins (1) Inhibition O
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reductase to thioredoxin [24,25]. Reduced thioredoxin is a specific inhibitor of tyrosinase, and therefore calcium-binding to thioredoxin reductase could increase melanin biosynthesis in m e l a n o m a tissues [25]. Thioredoxin reductase purified from melanotic melanoma has been isolated with b o u n d calcium, meanwhile this enzyme from amelanotic m e l a n o m a has been calcium free in its fully active form [24,26]. C a l c i u m - b o u n d thioredoxin reductase has been shown to be protected against inhibition by Fotemustine [15]. Consistent with these observations, we find that Fotemustine sensitive melanomas (n = 4) were primarily amelanotic with the drug resistant metastases (n = 3) being more melanotic (Fig. 1). These observations strongly suggest that the calcium status of malignant melanomas contributes significantly to intracellular redox conditions as observed previously for primary melanomas and keratinocyte cell cultures [24,26]. Since the reactivity of the nitrosoureas depends on the availability of sensitive thiolate enzyme active sites, it can be generally concluded that tumor cells with a higher redox-potential should be more resistant to these drugs than lower redox cells. This is also reflected in the resistance of melanotic melanomas where more oxidizing conditions must exist for the oxygen-dependent synthesis of the melanins via tyrosinase.
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Acknowledgements Active Enzyme Scheme I. A general reaction pathway for the inhibition and reactivation of thioproteins by the chloroethylnitrosourea anti-tumor drugs. The thiolate active site is involved in nucleophilic attack to displace a chloroethylcarbonium ion to deactivate the enzyme by forming a chloroethylthioether-enzyme inhibitor complex. This complex is susceptible to nucleophilic attack by thiols, such as GSH, with B-elimination of C1- with the formation of the disulfide. The disulfide is reduced (by NADPH) to reactivate the enzyme. tion of the weakly inhibited chloroethyl-thioetherglutathione reductase complex by reduced glutathione [15] should increase the intracellular concentration of oxidized glutathione (Scheme I). This p h e n o m e n o n has been reported previously in BCNU-resistant rat glioma cells (9L2) [23]. Clearly an increase in the intracellular redox conditions to a more oxic situation should slow down the reactivity of the chloroethylnitrosoureas with sensitive thiolate enzyme active sites yielding cells more resistant to chemotherapy with these drugs. This has been observed in the low ~4C incorporation in Cal 7 cells compared to Cal 1 cells. A second conclusion from the results of this study, together with our previously published work [18], stems from the sensitivity of the thioredoxin r e d u c t a s e / thioredoxin system to Fotemustine. The thioredoxin r e d u c t a s e / t h i o r e d o x i n system has been shown to regulate pigmentation with calcium functioning as an allosteric inhibitor for the electron transfer from thioredoxin
The human melanoma cell lines Ca1 1 and Cal 7 were established by Dr. DeGioanni and were kindly provided by Servier, Paris, France. Continuation of the cell cultures has been accomplished by Dr. M.R. Pittelkow, Mayo Clinic, Rochester, MN. We would like to thank Dr. Sarah McFarlane for technical assistance with the FPLC HR 10/10 system. We thank Louise Mohn for typing this manuscript and Doris Lewandowski for drawing the figures. This research was supported by a grant to K.U.S. from Servier, Paris, France. References 1 Wasserman, T.H., Slavik, M. and Carter, S.K. (1975) Cancer 36, 1258-1266. 2 Kohn, K.W. (1988) Adv. Neur.-Onc., 19, 491-513. 3 Brent, T.P. (1984) Cancer Res. 44, 1887-1892. 4 Brent, T.P. (1985) Pharmacol. Ther. 31, 121-140. 5 Erickson, L.C., Laurert, G., Sharkey, N.A. and Kohn, K.W. (1980) Nature 288, 727-729. 6 Ewig, R.A. and Kohn, K.W. (1978) Cancer Res. 38, 3197-3203. 7 Kohn, K.W. (1981) Cancer Res. 76, 141-152. 8 Lown, J.W., McLaughlin, L.W. and Chang, Y.M. (1978) Bio-org. Chem. 7, 97-110. 9 Ludlum, D.B. (1986) Cancer Res. 46, 3353-3357. 10 Tong, W.P., Kirk, M.C. and Ludlum, D.B. (1982) Cancer Res. 42, 3102-3105. 11 Aida, T. and Bodell, W.J. (1987) Cancer Res. 47, 5052-5058. 12 Aida, T., Cheitlin, R.A. and Bodell, W.J. (1987) Carcinogenesis 8, 1219-1223.
283 13 Thomas, C.B., Osieka, R. and Kohn, K.W. (1978) Cancer Res. 38, 2448-2454. 14 Babson, J.R. and Reed, D.J. (1978) Biochim. Biophys. Res. Commun. 83, 754-762. 15 Schallreuter, K.U., Gleason, F.K. and Wood, J.M. (1990) Biochim. Biophys. Acta 1054, 14-20. 16 Kalb, V.F., Jr. and Bernlohr, R.W. (1977) Anal. Biochem. 82, 362-371. 17 Luthman, M. and Holmgren, A. (1982) Biochemistry 21, 66286633. 18 Schallreuter, K.U. and Wood, J.M. (1988) Biochim. Biophys. Acta 967, 103-109. 19 Schallreuter, K.U. and Witkop, C.J., Jr. (1988) J. Invest. Dermatol. 90, 372-377. 20 Goodman, L.S. and Gilman S.G. (1985) The Pharmacological Basis of Therapeutics, MacMillan New York, pp. 1260-1262.
21 Smith, M.T., Evans, C.G., Doane-Seker, P., Castro, V.M., Kalin Tahir, M. and Mannervik, B. (1989) Cancer Res. 49, 2621-2625. 22 Boutin, J.A., Norbeck, K., Moldeus, P., Yenton, A., Paraire, M., Bizzari J.P., LaVielle, C.T. and Cudennec, C.A. (1989) Eur. J Cancer Clin. Oncol. 23, 1311-1316. 23 Evans, C.G., Bodell, W.J., Tokuda, K., Doane-Setzer, P. and Smith, M.T. (1987) Cancer Res. 2525-2530. 24 Schallreuter, K.U. and Wood, J.M. (1989) Biochim. Biophys. Acta 997, 242-247. 25 Wood, J.M. and Schallreuter, K.U. (1988) Inorg. Chim. Acta 151, 7. 26 Schallreuter, K.U., Pittelkow, M.R. and Wood, J.M. (1989) Biochim. Biophys. Res. Commun. 162, 113-1316.
Biochimica et Biophysica Acta, 1096 (1991) 277-283 '~ 1991 Elsevier Science Publishers B.V. 0925-4439/91/$03.50 A DONIS 092544399100079D
BBADIS 61037
Sensitivity and resistance in human metastatic melanoma to the new chloroethylnitrosourea anti-tumor drug Fotemustine K a r i n U . S c h a l l r e u t e r a n d J o h n M. W o o d Department of Dermatology, Unit,ersity of Hamburg, Hamburg (F. R. G.) (Received 2 August 1990)
Key words: Anti-tumor drug; Drug resistance; (Human melanoma)
Fotemustine is a novel chloroethylnitrosourea derivative currently used in Phase III clinical trials for disseminated metastatic melanoma. This drug has been shown to inhibit enzymes in the ribonucleotide reduction pathway (i.e., thioredoxin reductase, glutathione reductase and ribonucleotide reductase), t4c chloroethyl-labelled Fotemustine covalently labels the thiolate active sites of thioredoxin reductase and glutathione reductase yielding ~4C chloroethylthioether enzyme-inhibitor complexes. Enzyme activities can be restored by a reduced thioredoxin or reduced glutathione mediated fl-elimination of the chloroethyl group. 14C Fotemustine has been used to determine its reactivity and metabolism in drug sensitive and resistant melanoma metastases and in cultures of sensitive and resistant clones of human melanoma cells. Melanoma metastases from four different patients who were treated with Fotemustine could be labelled with radioactive drug only under reducing conditions with N A D P H as electron donor and DTNB as substrate. FPLC analysis of these extracts revealed two radioactive proteins (I) glutathione reductase and (II) an unidentified protein with 95 and 50 kDa subunits. A similar labelling pattern was also found in extracts of Fotemustine sensitive melanoma cells (Cal 1). Fotemustine resistant tumors were melanotic and contained more glutathione reductase than thioredoxin reductase, whereas sensitive tumors were clinically amelanotic with more thioredoxin reductase than glutathione reductase. Fotemustine resistant melanoma cells (Cal 7) showed a slower uptake of 14C-label with 34% less isotope intraceilularly in 1 h compared to sensitive melanoma cells (Cal 1). These results strongly indicate (I) the induction of alternate electron donors thioredoxin reductase or glutathione reductase for ribonucleotide reduction determines tumor and melanoma cell responses to the drug and (II) Fotemustine transport and the intracellular redox status seems to regulate resistance in melanoma cells and tissues.
Introduction The chloroethylnitrosoureas are highly reactive compounds with a general instability in aqueous solution after 1 hour at 37°C [1,2]. This homologous series of drugs are in widespread use for the treatment of gliomas, Hodgkin lymphomas, lung cancer, colorectal cancer and melanoma [1,2]. It has been suggested that the major cytotoxicity of the chloroethylnitrosoureas resides in their formation of crosslinking bridges at DNAguanine-O 6 positions [3-10]. This single reaction has been proposed responsible for their anti-tumor activity [11-13]. A mechanism involving chloroethyl-group transfer from a single DNA-guanine-O 6 position to the
Correspondence: K.U. Schallreuter, Department of Dermatology, University of Hamburg, Martinistrasse 52, D-2000 Hamburg 20, F.R.G.
thiolate active site of the guanine-O6-alkyltransferase enzyme yields a chloroethylthioether enzyme-inhibitor complex [2]. As a consequence, cells with low guanineO6-alkyltransferase activity have been especially sensitive to the chloroethylnitrosoureas [2]. A second mode of action for the chloroethylnitrosoureas has been shown involving its reactivity with other important thioproteins. These drugs alkylate the thiolate active sites of glutathione reductase, thioredoxin reductase and ribonucleotide reductase [14,15]. This inhibition would be expected to prevent the synthesis of deoxyribonucleotides in the S-phase of the growth cycle [16]. However, the covalent deactivation of thioredoxin reductase and glutathione reductase, the alternate electron donors for ribonucleotide reductase, can be reversed by their respective reaction products reduced thioredoxin and reduced glutathione [15]. Reduced thioredoxin and reduced glutathione catalyze a nucleophilic B-elimination of chloride ions
278 from the chloroethylthioether enzyme-inhibitor complexes [15]. A general mechanism for the alkylation of thioproteins by the chloroethylnitrosoureas followed by reactivation as a result of B-elimination is presented in Scheme I. Clearly, the significance of this pathway for the inhibition of DNA-synthesis resides in the inhibitor constants for the reactions of different chloroethylnitrosoureas with the three enzymes involved in ribonucleotide reduction. In enzymes purified from human metastatic melanoma tissue, glutathione reductase was significantly less reactive with Fotemustine than thioredoxin reductase [15]. Based on these results, it was proposed that drug resistance and sensitivity may reside in the dominance of the glutathione reductase/glutaredoxin vs. the thioredoxin reductase/thioredoxin pathways for electron transfer to ribonucleotide reductase in metastatic melanoma tissues or in clones of sensitive or resistant melanoma cells. The aim of this study was to examine drug resistant and drug-sensitive tumors and cells in terms of their thioredoxin reductase and glutathione reductase activities as well as their ability to react with the chloroethylnitrosourea Fotemustine.
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Materials and Methods
Enzymes and cell extracts Thioredoxin reductase (TR) and glutathione reductase (GR) have been purified from human metastatic melanoma tissues by a modified method of Luthman and Holmgren [17,18,24]. Seven metastases were obtained from four patients intravenously treated with Fotemustine (100 m g / m 2 body surface). The treatment cycle included a weekly administration over 3 weeks, followed by a 3 week rest period with a consequent assessment of tumor size. Two patients showed initially a partial remission (i.e., < 50% decrease of tumor size) followed by a progression of the tumor. At this time, metastases were excised. The two other patients showed a progressive disease after the 3 week induction cycle with Fotemustine. At this point, tumors were excised. Cell extracts were prepared for FPLC analyses on a Mono-Q anion-exchange column (HR 10/10) by the following procedure: (1) After surgical excision, tumors were frozen in liquid nitrogen and stored at - 7 2 ° C . (2) Tissues were homogenized and sonicated in 0.1 M Tris-HC1 buffer at pH 7.5 followed by centrifugation at 10000 rpm for 30 min at 0°C. (Upon clinical examination, those tumors initially responding to Fotemustine appeared amelanotic or only slightly pigmented, meanwhile tumors from patients not responding to the drug were significantly darker. Fig. 1 shows a photograph of the membranous pellets from all of the metastases examined.)
Fig. 1, Photograph of melanin deposits in membranes of Fotemustine sensitive (A) and resistant (B) metastases.
(3) Soluble extracts were dialyzed against 5 1 of 0.05 M Tris-HC1 buffer (pH 7.5) for 48 h with three buffer changes. (4) Proteins were determined by the method of Kalb and Bernlohr [16]. (5) 100 mg of protein from each cell extract was applied to a preparative Mono-Q column (HR 10/10) equilibrated with 0.05 M Tris-HC1 buffer (pH 7.5). (6) Thioredoxin reductase and glutathione reductase standards were subjected to FPLC analysis in a 0-0.5 M NaC1 gradient prior to the chromatography of each melanoma cell extract. Enzyme assays Thioredoxin reductase and glutathione reductase were assayed by the reduction of DTNB at 412 nm, using N A D P H as the electron donor, by the method of Luthman and Holmgren [171.
279 Membrane-associated thioredoxin reductase was determined by the method of Schallreuter and Wood [18]. 3 m m punch biopsies were taken from each tumor sample and the reduction of nitroxide radicals on a spin-labelled quaternary a m m o n i u m substrate was followed in quartz tissue cells in a Bruker D-200 EPR spectrometer at 25°C. The amplitude of the nitroxide radical signal was followed with time under conditions of saturating free radical substrate (3.5 • 10-3 M). This assay has been shown to be specific for epidermal thioredoxin reductase [19]. (*U.S. Patent N u m b e r 4,849,346 (1989).)
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lsotope experiments ~4C chloroethyl-labelled Fotemustine was provided by Servier Company (France) (53 m C i / m m o l ) [15]. T u m o r cell extracts (100 mg) were preincubated with N A D P H ( 5 . 1 0 -3 M) and D T N B (2.5.10 -3 M) over 15 min before the addition of 10 ~tl of 14C Fotemustine. These radiolabelled extracts were dialyzed against 0.05 M Tris-HC1 buffer (pH 7.5) for 5 h prior to F P L C analysis. Melanoma cells (2-3 mg of protein) were preincubated for 1 h with 10 /zl of 14C Fotemustine, centrifuged, resuspended in 1.0 ml of 0.05 M Tris-HC1 buffer (pH 7.5), washed with buffer and cell extracts prepared, using a microprobe of a Heat Systems sonicator at the m a x i m u m setting at 0°C. Cell membranes were removed by centrifugation at 5000 rpm for 10 rain at 0°C. Results The interaction of Fotemustine with plasma membrane-associated thioredoxin reductase was examined in 3 m m tissue biopsies taken from human melanoma metastases. The spin-label reduction assay for membrane-associated thioredoxin reductase revealed a time dependent inhibition of this enzyme by Fotemustine (Fig. 2). After 20 min preincubation time with the drug, full enzyme activity returned suggesting rapid degradation of this chloroethylnitrosourea in these experiments. This inhibition/reactivation phenomenon has been repeated in biopsies taken from three other metastatic melanomas from different patients. However, enzyme recovery did not occur in every tumor tested nor in skin biopsies from normal healthy donors. Therefore, these preliminary results suggested that the recovery of membrane-associated thioredoxin reductase from Fotemustine inhibition may be tumor specific. Experiments reported previously [15] on thioredoxin reductase and glutathione reductase showed that Fotemustine is a much more potent inhibitor of thioredoxin reductase ( K I = 3.0 • 10 -5 M) compared to glutathione reductase ( K I = 1.5.10 -3 M). Based on this 500-fold difference in the sensitivity of thioredoxin reductase over glutathione reductase to Fotemustine, it
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Fig. "~. The inhibition of membrane-associated thioredoxin reductase on 3 mm punch biopsies taken from a metastatic melanoma followed by reactivation. Reactions were performed in the presence of 3.5.10-3 M nitroxide radical substrate using 2.5.10 -3 M Fotemustine as inhibitor for incubation times of 0, 3, 5, 10, 15 and 20 min. Specific activities for membrane-associated thioredoxin reductase were determined as the decrease in amplitude of the nitroxide radical signal/10 mg of tissue per 10 min.
was anticipated that those tumors with the thioredoxin reductase/thioredoxin pathway for ribonucleotide reduction should be more prone to a drug response compared to tumors with the alternate glutathione reductase/glutaredoxin system. To test this hypothesis, four patients were selected without previous chemotherapy other than the induction cycle with Fotemustine. Seven tumors were obtained; ( 4 / 2 patients who initially responded to Fotemustine, and 3 / 2 patients who did not respond). F P L C analysis of crude extracts from these tumors revealed that both thioredoxin reductase and glutathione reductase were present at significant levels in all tumors as identified by: (1) pure enzyme standards and (2) the D T N B reduction assay. Fig. 3a shows the analysis of one Fotemustine sensitive tumor where thioredoxin reductase activity was 3.5-fold higher than glutathione reductase activity in the standard D T N B reduction assay. Fig. 3b shows one example of a drug-resistant tumor where glutathione reductase activity was 4.4-fold that of thioredoxin reductase. Similar results were obtained for four responding and three non-responding tumor extracts. These results unambiguously indicate that a switch from the thioredoxin reductase/thioredoxin pathway to the glutathione reductase/glutaredoxin system in metastatic melanoma coincides with resistance to Fotemustine. Since 14C chloroethyl-labelled Fotemustine alkylates the active sites of thioredoxin reductase and glutathione reductase yielding covalently labelled enzyme-inhibitor complexes, this reaction was examined in crude extracts from melanoma tissues [15]. The 14C chloroethyl-etherglutathione reductase complex has been more stable
280 than the corresponding thioredoxin reductase complex [15]. This instability appears to be promoted by the oxidizing conditions on the Mono-Q anion-exchange column during the FPLC procedure (i.e., 14C-labelled pure thioredoxin reductase loses its radioactivity during the FPLC procedure whereas labelled glutathione reductase is stable). Fig. 4a and b present FPLC analyses of crude extracts labelled with ~4C Fotemustine from drug resistant and drug sensitive tumors. It should be noted that the covalent labelling of proteins in these extracts occurs after preincubation with N A D P H as electron donor and D T N B as substrate. Without the addition of cofactor and substrate for optimum reducing conditions, isotope incorporation into these cell extracts has been unsuccessful. Two proteins are labelled with ~4C Fotemustine: (1) glutathione reductase and (2) an unidentified protein eluting at 0.17 M NaC1 under the chosen FPLC conditions. Although glutathione reductase was labelled with ~4C Fotemustine, it was not completely inhibited being still active in the D T N B
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unknown protein (peak 1) and GR (peak 2). Fotemustine incorporation into GR caused delayed elution at 0.34 M NaCI instead of the 0.3 1 M NaCI observed in controls. GR was still active in the DTNB reduction assay (specific activity -0.085). (b) FPLC separation (Mono-Q 10/10) of cell extracts from a Fotemustine-resistant tumor showing 14C Fotemustine uptake into unknown protein (peak 1) and GR (peak 2). GR had a residual specific activity of 0.215 in the DTNB reduction assay.
reduction assay. The unidentified protein was purified further by FPLC and yielded subunits of 95 and 50 Kda upon S D S - P A G E analysis (Fig. 5). Since this protein was not labelled in the absence of N A D P H and DTNB, it is assumed to be another thioprotein capable of nucleophilic displacement of the chloroethyl-group from Fotemustine. H u m a n melanoma cell lines which are sensitive (Cal 1) and resistant (Cal 7) to Fotemustine were used in the following experiments. Suspensions of Cal 1 and Cal 7 melanoma cells were incubated with ~4C Fotemustine and the intracellular radioactivity was determined after 1 h. Cal 1 incorporated 8773 c p m / m g protein per hr and Cal 7 5811 c p m / m g protein per hr. FPLC analysis of Cal 1 extracts yielded incorporation of the ~4C label into: (1) glutathione reductase; (2) unknown protein (95 k D a / 5 0 kDa); and (3) a small radioactive peak eluting at 0.39 M NaC1, whereas cell extracts from Cal 7 revealed no clearly defined radioactive incorporation
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drugs with individual thioenzymes, (2) the intracellular levels of enzymes which can alternate for each other in important metabolic pathways (e.g., thioredoxin reductase and glutathione reductase in ribonucleotide reduction), (3) the cellular reducing potential v i s a vis N A D P H synthesis via the pentose phosphate shunt for glucose metabolism ( N A D P H controls the reactivity of the chloroethylnitrosoureas) and (4) the more rapid uptake of Fotemustine in drug-sensitive cells c o m p a r e d to resistant cell lines. Since glutathione reductase is 500-fold less sensitive to inhibition by Fotemustine than thioredoxin reductase [15,22], then both a kinetic effect based on drug concentration, and the replacement of the thioredoxin reductase system for the glutathione reductase p a t h w a y for ribonucleotide reduction m a y be expected to confer resistance in tumors and m e l a n o m a cells. The reactiva-
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Discussion
It is well established that the instability of the chloroethylnitrosourea anti-tumor drugs resides in their susceptibility to nucleophilic attack causing a c o m m o n mechanism of alkylation by the displacement of a chloroethylcarbonium ion [20]. It is now recognized that several important thioenzymes are covalently deactivated by these drugs by the formation of chloroethylthioether-enzyme-inhibitor complexes. Thioenzymes involved in ribonucleotide reduction, and as a consequence D N A synthesis, are primary targets for these drugs (i.e., TR, G R and RR). Also, the thioprotein guanine-O6-alkyl transferase, an important enzyme in preventing ethyl-bridged D N A crosslinks, is alkylated by both the chloroethylnitrosoureas and by chloroethylD N A adducts [2]. The Class M u glutathione transferases appear to be important in the metabolism of chloroethylnitrosoureas assuming importance over the other glutathione transferase isozymes in drug resistant cells [21]. However, all the above mentioned reactions point to a c o m m o n mechanism for inhibition a n d / o r resistance to this homologous series of drugs: (1) alkylation of active site thiolate groups by chloroethyl-group transfer and (2) reductive fl-elimination of the chloroethyl-group to reactivate these enzymes (Scheme I). As a consequence of these two reactions, four independent factors could be critically important in chloroethylnitrosourea sensitivity/resistance in t u m o r cells and tissues: (1) the kinetics for the reaction of these
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Fig. 6. (a) FPLC separation of 14C Fotemustine labelled proteins from cell extracts of a human melanoma cell clone Cal 1 (Fotemustine-sensitive) on a Mono-Q HR 5/5 column in 0.05 M Tris-HCl buffer (pH 7.5) using a 0-0.5 M NaCI gradient. Radioactive peaks were corrected for background and eluted at 0.08, 0.18 and 0.39 M NaC1. The peak at 0.18 M NaCI was identified as GR and the small peak at 0.39 M NaCI revealed similar chromatographic properties to ribonucleotide reductase. The peak at 0.08 was the unidentified protein (95 kDa/50 kDa). (b) FPLC profile of 14C Fotemustine labelled proteins from cell extracts of human melanoma cell clone Cal 7 (Foiemustine resistant). There was no discernible radioactive incorporation into glutathione reductase and the total intracellular lac incorporation was 34% less in Cal 7 compared to Cal 1.
282 Inhibition and reactivation of chloroethyl-nitrosourea inhibited thioproteins (1) Inhibition O
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reductase to thioredoxin [24,25]. Reduced thioredoxin is a specific inhibitor of tyrosinase, and therefore calcium-binding to thioredoxin reductase could increase melanin biosynthesis in m e l a n o m a tissues [25]. Thioredoxin reductase purified from melanotic melanoma has been isolated with b o u n d calcium, meanwhile this enzyme from amelanotic m e l a n o m a has been calcium free in its fully active form [24,26]. C a l c i u m - b o u n d thioredoxin reductase has been shown to be protected against inhibition by Fotemustine [15]. Consistent with these observations, we find that Fotemustine sensitive melanomas (n = 4) were primarily amelanotic with the drug resistant metastases (n = 3) being more melanotic (Fig. 1). These observations strongly suggest that the calcium status of malignant melanomas contributes significantly to intracellular redox conditions as observed previously for primary melanomas and keratinocyte cell cultures [24,26]. Since the reactivity of the nitrosoureas depends on the availability of sensitive thiolate enzyme active sites, it can be generally concluded that tumor cells with a higher redox-potential should be more resistant to these drugs than lower redox cells. This is also reflected in the resistance of melanotic melanomas where more oxidizing conditions must exist for the oxygen-dependent synthesis of the melanins via tyrosinase.
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Acknowledgements Active Enzyme Scheme I. A general reaction pathway for the inhibition and reactivation of thioproteins by the chloroethylnitrosourea anti-tumor drugs. The thiolate active site is involved in nucleophilic attack to displace a chloroethylcarbonium ion to deactivate the enzyme by forming a chloroethylthioether-enzyme inhibitor complex. This complex is susceptible to nucleophilic attack by thiols, such as GSH, with B-elimination of C1- with the formation of the disulfide. The disulfide is reduced (by NADPH) to reactivate the enzyme. tion of the weakly inhibited chloroethyl-thioetherglutathione reductase complex by reduced glutathione [15] should increase the intracellular concentration of oxidized glutathione (Scheme I). This p h e n o m e n o n has been reported previously in BCNU-resistant rat glioma cells (9L2) [23]. Clearly an increase in the intracellular redox conditions to a more oxic situation should slow down the reactivity of the chloroethylnitrosoureas with sensitive thiolate enzyme active sites yielding cells more resistant to chemotherapy with these drugs. This has been observed in the low ~4C incorporation in Cal 7 cells compared to Cal 1 cells. A second conclusion from the results of this study, together with our previously published work [18], stems from the sensitivity of the thioredoxin r e d u c t a s e / thioredoxin system to Fotemustine. The thioredoxin r e d u c t a s e / t h i o r e d o x i n system has been shown to regulate pigmentation with calcium functioning as an allosteric inhibitor for the electron transfer from thioredoxin
The human melanoma cell lines Ca1 1 and Cal 7 were established by Dr. DeGioanni and were kindly provided by Servier, Paris, France. Continuation of the cell cultures has been accomplished by Dr. M.R. Pittelkow, Mayo Clinic, Rochester, MN. We would like to thank Dr. Sarah McFarlane for technical assistance with the FPLC HR 10/10 system. We thank Louise Mohn for typing this manuscript and Doris Lewandowski for drawing the figures. This research was supported by a grant to K.U.S. from Servier, Paris, France. References 1 Wasserman, T.H., Slavik, M. and Carter, S.K. (1975) Cancer 36, 1258-1266. 2 Kohn, K.W. (1988) Adv. Neur.-Onc., 19, 491-513. 3 Brent, T.P. (1984) Cancer Res. 44, 1887-1892. 4 Brent, T.P. (1985) Pharmacol. Ther. 31, 121-140. 5 Erickson, L.C., Laurert, G., Sharkey, N.A. and Kohn, K.W. (1980) Nature 288, 727-729. 6 Ewig, R.A. and Kohn, K.W. (1978) Cancer Res. 38, 3197-3203. 7 Kohn, K.W. (1981) Cancer Res. 76, 141-152. 8 Lown, J.W., McLaughlin, L.W. and Chang, Y.M. (1978) Bio-org. Chem. 7, 97-110. 9 Ludlum, D.B. (1986) Cancer Res. 46, 3353-3357. 10 Tong, W.P., Kirk, M.C. and Ludlum, D.B. (1982) Cancer Res. 42, 3102-3105. 11 Aida, T. and Bodell, W.J. (1987) Cancer Res. 47, 5052-5058. 12 Aida, T., Cheitlin, R.A. and Bodell, W.J. (1987) Carcinogenesis 8, 1219-1223.
283 13 Thomas, C.B., Osieka, R. and Kohn, K.W. (1978) Cancer Res. 38, 2448-2454. 14 Babson, J.R. and Reed, D.J. (1978) Biochim. Biophys. Res. Commun. 83, 754-762. 15 Schallreuter, K.U., Gleason, F.K. and Wood, J.M. (1990) Biochim. Biophys. Acta 1054, 14-20. 16 Kalb, V.F., Jr. and Bernlohr, R.W. (1977) Anal. Biochem. 82, 362-371. 17 Luthman, M. and Holmgren, A. (1982) Biochemistry 21, 66286633. 18 Schallreuter, K.U. and Wood, J.M. (1988) Biochim. Biophys. Acta 967, 103-109. 19 Schallreuter, K.U. and Witkop, C.J., Jr. (1988) J. Invest. Dermatol. 90, 372-377. 20 Goodman, L.S. and Gilman S.G. (1985) The Pharmacological Basis of Therapeutics, MacMillan New York, pp. 1260-1262.
21 Smith, M.T., Evans, C.G., Doane-Seker, P., Castro, V.M., Kalin Tahir, M. and Mannervik, B. (1989) Cancer Res. 49, 2621-2625. 22 Boutin, J.A., Norbeck, K., Moldeus, P., Yenton, A., Paraire, M., Bizzari J.P., LaVielle, C.T. and Cudennec, C.A. (1989) Eur. J Cancer Clin. Oncol. 23, 1311-1316. 23 Evans, C.G., Bodell, W.J., Tokuda, K., Doane-Setzer, P. and Smith, M.T. (1987) Cancer Res. 2525-2530. 24 Schallreuter, K.U. and Wood, J.M. (1989) Biochim. Biophys. Acta 997, 242-247. 25 Wood, J.M. and Schallreuter, K.U. (1988) Inorg. Chim. Acta 151, 7. 26 Schallreuter, K.U., Pittelkow, M.R. and Wood, J.M. (1989) Biochim. Biophys. Res. Commun. 162, 113-1316.