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The Effect of Nicotinamide Dinucleotides on Methotrexate Binding to Proteins in a Methotrexate-Resistant Cell-Line of Petunia hybrida
J.PlantPhysiol. Vol. 136.pp. 611-614(1990)
The Effect of Nicotinamide Dinucleotides on Methotrexate Binding to Proteins in a Methotrexate-Resistant Cell-Line of
Petunia hybrida RIVKA BARGh~, JACQUES 1
2
S. BECKMANN\ and MEIR PERL2
Department of Plant Genetics and Breeding, and Department of Medicinal, Aromatic and Spice Crops, Agricultural Research Organization, The Volcani Center, Bet Dagan 50250, Israel
Received October 31,1989 . Accepted December 7,1989
Summary The present report describes the mode of interaction between MTX and the MTX-binding protein (MTX-BP) of a MTX-resistant cell-line of Petunia. This cell-line was previously shown to produce elevated amounts of a MTX binding protein, which is different from the normal DHFR, the target protein of MTX. Irreversible binding of MTX to this protein depends on the presence of a dialyzable cofactor. In dialyzed protein extracts, the irreversible nature of MTX binding is restored by a supplement of NADPH, and less efficiently by NADH or NADP. The MTX-binding protein shows a greater affinity to MTX than to dihydrofolate, the natural substrate of DHFR. Hence, it is suggested that this MTX-BP contributes to the MTX resistance of the cell-line mainly by acting as a trap for MTX penetrating the cells. The formation of a stable complex of MTX-BP-NADPH-MTX would be essential for this protein to exert its full function as a MTX scavenger.
Key words: Petunia hybrida, cell-culture, DHFR, MTX-binding protein, MTX-resistance. Abbreviations: DHF = dihydrofolate; DHFR = Dihydrofolate Reductase; GSH = Glutathione-reduced form; GSS = Glutathione-oxidized form; MTX = methotrexate; MTX-BP = MTX-binding protein; NAD = J3-Nicotinamide adenine dinucleotide, oxidized form; NADH = J3-Nicotinamide adenine dinucleotide, reduced form; NADP = /3-Nicotinamide adenine dinucleotide 3'-phosphate, oxidized form; NADPH = J3-Nicotinamide adenine dinucleotide 3'-phosphate, reduced form. Introduction Several cell-lines of Petunia hybrida resistant to Methotrexate (MTX) were isolated upon stepwise selection; all of them are characterized by high levels of MTX binding (Barg et al., 1984). One of these, cell-line 1ECB, was compared with the WT cell-line with regard to cell-free MTX-binding capacity and the activity of Dihydrofolate Reductase (DHFR; EC 1.5.1.3), a target protein for MTX (Blakely, 1969; Cossins, 1980; Huennekens et al., 1976). The data obtained indicated that the major MTX-binding protein (MTX-BP) in cell-line
* To whom correspondence should be sent. © 1990 by Gustav Fischer Verlag, Stuttgart
1ECB differs from the normal DHFR. It was suggested that this abundant MTX-BP exerts DHFR activity, and that it also acts as a «trap» for MTX penetrating the cells, thus protecting MTX-sensitive protein(s), the functions of which are essential for cell viability (Barg et al., 1987). In mammals and even in bacteria, the dependency of MTX binding to DHFR on nicotinamide dinucleotide co-factors was studied extensively (e.g. Kamen et al., 1983; Otting and Huennekens, 1972; Perkins and Bertino, 1966; Werkheiser, 1961). In plants, this question was not addressed, though MTX binding to plant proteins was tested in the presence of NADPH (Barg et al., 1984, 1987; Cella et al., 1983, 1984, 1987). It was shown previously that for the MTX-resistant
612
RrvKA BARG, JACQUES S. BECKMANN, and MEIR PERL
petunia cell-line lECB, the presence or absence of NADPH does not affect the KD and BMAX for MTX binding (Barg et al., 1987). However, if this MTX-BP is to serve as a trap, an irreversible binding might be expected. The results described herein support this assumption and demonstrate the specificity of NADPH as a co-factor ensuring a tight binding of MTX to this protein.
3H-MTX BINDING 125
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Biological material The study was performed using the MTX-resistant cell-line lECB, which was described previously (Barg et aI., 1984, 1987). Proteins were extracted from cells propagated for four growth cycles in the absence of MTX. Crude cell extraction was performed as described previously (Barg et aI., 1984); briefly, vacuum-dried cells were ground to powder under liquid nitrogen, dissolved in extraction buffer (50mM K-phosphate, pH = 7.8/0.15M KCl), mixed thoroughly, and spun at 11,000 g for 15 min at 4°C. Dialyzed extract refers to the water-soluble protein fraction that was precipitated from the crude extract with 100% ammonium sulfate and dialyzed overnight against 2 L of the extraction buffer (Barg et aI., 1987).
JH-MTX binding assay The binding assays were performed essentially as described previously (Barg et aI., 1984). In the 3H-MTX binding and release experiments, the standard pre-incubation was with 0.23 JLM radio-labelled MTX (10Ci/mmole) for 20 min at room temperature. This concentration is approximately 20-fold higher than the apparent KD value for MTX in this line (Barg et aI., 1987); thus, the experiments were performed under saturated conditions for MTX. The actual concentration of unlabelled MTX added and the duration of incubation time with the unlabelled MTX are specified for each set of experiments described.
Protein quantitation
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Fig. 1: The effect of NADPH on the binding of 3H-MTX to and on its release from crude and dialyzed protein extracts. Binding reaction mixtures containing 0.23/LM 3H-MTX were incubated for 20 min in the presence or absence (W/O) of 5JLM NADPH (A). To half of each sample was added 5/LM of unlabelled MTX for an additional 30 min (B), and the bound 3H-MTX was monitored. The percent of 3H-MTX release was calculated as described in Table 1. The data presented are the average of two independent experiments, each performed in duplicate. (Deviation among replicates was less than 10%).
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.
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Proteins were precipitated with 10% trichloroacetic acid and measured according to the Lowry method (Lowry et aI., 1951).
Chemicals Folic acid, DHF, NADPH (enzymatic preparation), NADP, NAD, NADH, nicotinic acid, GSH and GSS were purchased from Sigma (St. Louis, MO). MTX was a generous gift from Lederle Laboratories (Pearl River, NY), and 3H-MTX (10Ci/mmole) was purchased from Amersham (UK).
Results
Effect 0/ NADPH on binding and release 0/MTX The NADPH dependency of MTX binding and release was examined in crude and in dialyzed protein extracts. As demonstrated in Fig. 1 A, the binding capacity of MTX is not affected by the presence or absence of NADPH (5 J.tM). The total amount of MTX bound to dialyzed extract is slightly lower (approx. 10- 15 % less) than that bound to the crude extract. The crude and the dialyzed extracts differ sig-
IfJM) NADPH
Fig. 2: The effect of NADPH concentration on the percent release of 3H-MTX (0.23 JLM) pre-bound to dialyzed protein extracts, following the addition of 5 JLM unlabelled MTX for 45 min (.). The concentration of NADPH added had no significant effect on the level of 3H-MTX binding, as monitored in control samples at the end of the incubation period with the unlabelled MTX (A). The data presented are the average of two independent experiments, each performed in duplicate. (Deviation among replicates was less than 10%).
nificantly, however, with regard to the effect of NADPH on the release of pre-bound 3H-MTX following the addition of 5 J.tM unlabelled MTX (Fig. 1 B): while in the dialyzed extract, MTX is displaced unless NADPH is added; in the crude extract most of the 3H-MTX is not released even if NADPH is not added.
Irreversible binding of MTX to plant proteins
The level of MTX binding to dialyzed protein extracts is not affected by NADPH (up to 10 fLM). Yet, the degree of displacement of the pre-bound 3H-MTX (0.23 fLM) following the addition of 5 fLM unlabelled MTX for 45 min depends on the concentration of NADPH (Fig. 2): adding 0.5 fLM NADPH results in retention of 50 % of the pre-bound MTX, and in the presence of 5 I'M NADPH the binding becomes practically irreversible.
Efficacy of various nicotinamide compounds as co/actors conferring irreversible MTX binding under reduced and oxidized conditions Since 5 I'M NADPH was found to restore completely the irreversible nature of MTX binding in dialyzed protein extracts, the efficacy of several other derivatives of nicotinamide in mediating irreversibility of MTX binding was examined. The nicotinamides NADP, NADH, NAD, and nicotinic acid were each added at a concentration of 5 fLM to dialyzed extracts together with 0.23 fLM 3H-MTX; after 20 min, 10 I'M of unlabelled MTX was added and the incubation continued for 45 min. The percent of release of prebound MTX was monitored (Table 1). The various compounds examined can be graded according to their efficiency as co-factors mediating irreversible binding: NADPH is the most potent one, allowing for 90 - 95 % of the MTX to remain irreversibly bound; NADH and NADP, conferred approximately 70 and 50 % irreversible binding respectively. NAD and nicotinic acid did not restore irreversible binding. The fact that NADH and not NADP was the «second best» raised the possibility that NADPH exerts its effect via its influence on the level of reduction of the environment, which affects the configuration of the MTX-BP, assuming that under reduced conditions binding is irreversible. To ex-
Table 1: The effect of various nicotinamide derivatives, oxidizing (GSS) and reducing (GSH) agents on the release (%) of pre-bound 3H-MTX. Dialyzed protein extracts were pre-incubated with the specified compounds for 5 min, at the end of which 0.23 I'M 3H_ MTX was added and the incubation continued for 20 min, and then 10 I'M of unlabelled MTX was added to half of each sample for 45 min. The percent of release was calculated from the ratio between the cpm determined in the two halves of each sample. The results presented are the range found in three independent experiments, each carried out in duplicates. Compound added Nicotinamide GSH derivative (5 I'M) (100 I'M) None None + None NADPH NADPH + NADPH NADP NADP + NADP NADH NAD Nicotinic Acid
GSS (100 I'M)
+ + +
3H-MTX released (%) 90-95 90-95 90-95 5-15 25-30 40-50 35-50 35-50 55-75 30-35 90-95 90-95
613
MTX
100 X
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~ 25
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1000
Fig.3: Release (%) of 3H-MTX (0.23I'M), pre-bound to dialyzed protein extract in the absence of NADPH, following the addition of increasing concentrations of either unlabelled MTX (e) or DHF (0) for 45 min. The data presented are from one of two similar experiments, each performed in duplicate. (Deviation among replicates was less than 10 %).
amine this hypothesis, the effect of reduced (GSH) and of oxidized glutathione (GSS) was tested. It was found (Table 1) that neither GSH nor GSS per se had any effect on the release of pre-bound MTX. None of the compounds added had any effect on the level of 3H-MTX binding (data not shown). The addition of 100 I'M GSH together with 5 I'M NADP had no significant effect on the percent of MTX release; addition of GSH together with NADPH increased the release of prebound MTX. That is, the presence of GSH affected adversely the ability of NADPH to tighten MTX binding.
Displacement ofpre-bound J H-MTX by DHF and MTX The ability of non-radioactive MTX and DHF to displace pre-bound 3H-MTX was examined in dialyzed extract in the absence of NADPH. Increasing concentrations of either MTX or DHF, were added and incubation was continued for 45 min. While 5 I'M MTX was sufficient to release 80 % of the pre-bound MTX, 1000 I'M DHF was required to achieve a similar level of displacement (Fig. 3). This higher displacement ability of MTX was also found when the time course of displacement of pre-bound 3H-MTX by either MTX or DHF (50 I'M) was followed: at each period of incubation (up to 45 min), the unlabelled MTX released comparatively more of the pre-bound 3H-MTX than did DHF (data not shown). Discussion The mode of interaction between the antimetabolite MTX and the major MTX-BP in cell-line lECB, which is highly tolerant to MTX (Barg et al., 1984, 1987), was the subject of the present study. A comparative study with Wild-Type protein extract was practically impossible, because of its low MTX-binding capacity. The binding of MTX to a crude cell
614
RIVKA BARG, ]ACQUES S. BECKMANN, and MEIR PERL
extract of cell-line 1ECB was shown to be extremely tight, and the compound(s) responsible for the irreversible binding was found to be lost upon 100 % ammonium sulfate precipitation and dialysis (Figs. 1 A, B). NADPH-dependent irreversible MTX-binding was demonstrated (Figs. 1 Band 2): a similar observation was made for the binding of MTX to DHFR of mammals (Perkins and Bertino, 1966) and bacteria (Otting and Huennekens, 1972). Displacement of 12 - 20 % of the pre-bound 3H-MTX was always observed, probably representing a non-specific MTX binding. The concentration of NADPH required to insure a complete irreversible binding in the dialyzed ammonium sulfate-precipitated protein extract was approximately five-fold higher than the concentration of the radio-labelled MTX added (Fig. 2). It should be noted that binding of MTX and NADPH to partially purified mammalian DHFR is accomplished on an equimolar basis (Kamen et aI., 1983; Perkins and Bertino, 1966; Werkheiser, 1961). The potential of other nicotinamide derivatives to serve as co-factors for irreversible MTX binding was examined. All the tested derivatives were less efficient than NADPH, and NAD had no influence on the tightness of MTX binding (Table 1). The adverse effect of reduced glutathione on the irreversible MTX binding in the presence of NADPH, and its lack of effect in the presence of NADP (Table 1), indicate that a reducing environment per se is not required for the binding reaction. The positive effect of NADP on irreversible binding might be a result of its reduction to NADPH, a process which is impaired in the presence of
GSS.
The irreversible binding of MTX to the crude extract (Fig. 1 A) might be caused by the existence of a binary complex of NADPH and MTX-BP; similar complexes were described before (Cohen et al., 1978; Huennekens et aI., 1973; Kamen et al., 1976, 1983; Otting and Huennekens, 1972). NADH might also contribute to the irreversible MTX binding found in the crude cell extract (Table 1). Since the native compound conferring irreversible binding in the crude extract was not identified, the existence of yet another unknown compound can not be ruled out. The higher efficiency of MTX as compared with DHF in displacing pre-bound radio-labelled MTX points to a greater affinity of the MTX-BP to the non-metabolizable analogue (Fig. 3). This is in agreement with previously reported results obtained from DHF and MTX competitive binding experiments (Barg et al., 1987). In summary, the data obtained from the MTX-displacement experiments, together with the previous characterization studies, strongly suggest that the MTX-binding protein, present in elevated amounts in cell-line 1ECB, contributes to the MTX resistance of this line mainly by acting as a trap for MTX penetrating the cells. The formation of a stable complex ofMTX-BP-NADPH-MTX seems essential forthis protein to exert its full function as a MTX scavenger. Whether NADPH is the co-factor acting in vivo remains to be demonstrated. Acknowledgements This is contribution No. 2646-E, 1989 series, from the Agricultural Research Organization, The Volcani Center, Bet Dagan
50250, Israel. Methotrexate was a generous gift from Lederle Laboratories (Pearl River, NY). This work was supported in part by a grant to J.S.B. from the US-Israel Binational Agricultural Research and Development Fund (BARD) and in part by a grant to R.B. from the US·Israel Binational Science Foundation (84-149).
References BARG, R., N. PELEG, M. PERL, and ]. S. BECKMANN: Isolation of methotrexate-resistant cell-lines in Petunia hybrida upon stepwise selection procedure. Plant Mol. BioI. 3,303-311 (1984). BARG, R., M. PERL, and J. S. BECKMANN: Elevated amounts of methotrexate binding protein, different from normal dihydrofolate reductase, in a petunia MTXR-celliine. Plant Mol. BioI. 8, 87 -94 (1987). BLAKELY, R. L.: The biological reduction of pteridines. In: NEUBERGER, A. and E. L. TATUM (Eds.). The biochemistry of folic acid and related pteridines. Frontiers of Biology, vol. 13, pp. 139-187. North Holland, Amsterdam (1969). CELLA, R., D. ALBANI, M. G. BIASINI, D. CARBONERA, and B. PARISI: Isolation and characterization of carrot cell line resistant to methotrexate. J. Exp. Bot. 35, 1390-1397 (1984). CELLA, R., D. ALBANI, D. CARBONERA, L. ETTERI, E. MAESTRI, and B. PARISI: Selection of methotrexate resistant cell lines in Daucus carota: Biochemical analysis and genetic characterization by protoplast fusion. J. Plant Physiol. 127, 135-146 (1987). CELLA, R., P. CROSTI, E. NIELSEN, and B. PARISI: Biochemical basis of different sensitivity to methotrexate in Daucus carota and Oryza sativa cell cultures. J. Exp. Bot. 34, 1189-1195 (1983). COHEN, M., R. A. BENDER, R. DONEHOWER, C. E. MYERS, and B. A. CHABNER: Reversibility of high-affinity binding of methotrexate in L1210 murine leukemia cells. Cancer Res. 38, 2866-2870 (1978). COSSINS, E. A.: One carbon metabolism. In: DAVIES, D. D. (Ed.). The Biochemistry of Plants. pp. 365-418. Academic Press, New York (1980). HUENNEKENS, F. M., K. S. VITOLS,]. M. WHITELELY, and V. G. NEEF: Dihydrofolate reductase. In: BUSCH, H. (Ed.). Methods in Cancer Research. Vol. 13, pp. 199-225, Academic Press, New York (1976). KAMEN, B. A., P. L. TAKACH, R. VATEV, and]. D. CASTON: A rapid radio-chemical-ligand binding assay for methotrexate. Analyt. Biochem. 70, 54-63 (1976). KAMEN, B. A., W. WHYTE-BAURE, and ]. R. BERTINO: A mechanism of resistance to Methotrexate, NADPH but not NADH stimulation of methotrexate binding to dihydrofolate reductase. Biochern. Pharm. 32,1837-1841 (1983). LOWRY, O. H., N.]. ROSEBROUGH, A. L. FARR, and R.]. RANDALL: Protein measurements with the folin phenol reagent. J. BioI. Chern. 193,265-275 (1951). OTTING, F. and F. M. HUENNEKENS: TPNH-Dependent binding of amethopterin by dihydrofolate reductase from Lactobacillus casei. Arch. Biochem. Biophys. 152, 429-431 (1972). PERKINS,]. P. and J. R. BERTINO: Dihydrofolate reductase from the L1210R Murine lymphoma. Fluorometric measurements of the interaction of the enzyme, substrates and inhibitors. Biochemistry 5, 1005-1012 (1966). WERRHEISER, W. c.: Specific binding of 4-amino folic acid analogues to folic acid reductase, studies on the binding of substrates and inhibitors to the enzyme. J. BioI. Chern. 242, 4777 -4781 (1961).
The Effect of Nicotinamide Dinucleotides on Methotrexate Binding to Proteins in a Methotrexate-Resistant Cell-Line of
Petunia hybrida RIVKA BARGh~, JACQUES 1
2
S. BECKMANN\ and MEIR PERL2
Department of Plant Genetics and Breeding, and Department of Medicinal, Aromatic and Spice Crops, Agricultural Research Organization, The Volcani Center, Bet Dagan 50250, Israel
Received October 31,1989 . Accepted December 7,1989
Summary The present report describes the mode of interaction between MTX and the MTX-binding protein (MTX-BP) of a MTX-resistant cell-line of Petunia. This cell-line was previously shown to produce elevated amounts of a MTX binding protein, which is different from the normal DHFR, the target protein of MTX. Irreversible binding of MTX to this protein depends on the presence of a dialyzable cofactor. In dialyzed protein extracts, the irreversible nature of MTX binding is restored by a supplement of NADPH, and less efficiently by NADH or NADP. The MTX-binding protein shows a greater affinity to MTX than to dihydrofolate, the natural substrate of DHFR. Hence, it is suggested that this MTX-BP contributes to the MTX resistance of the cell-line mainly by acting as a trap for MTX penetrating the cells. The formation of a stable complex of MTX-BP-NADPH-MTX would be essential for this protein to exert its full function as a MTX scavenger.
Key words: Petunia hybrida, cell-culture, DHFR, MTX-binding protein, MTX-resistance. Abbreviations: DHF = dihydrofolate; DHFR = Dihydrofolate Reductase; GSH = Glutathione-reduced form; GSS = Glutathione-oxidized form; MTX = methotrexate; MTX-BP = MTX-binding protein; NAD = J3-Nicotinamide adenine dinucleotide, oxidized form; NADH = J3-Nicotinamide adenine dinucleotide, reduced form; NADP = /3-Nicotinamide adenine dinucleotide 3'-phosphate, oxidized form; NADPH = J3-Nicotinamide adenine dinucleotide 3'-phosphate, reduced form. Introduction Several cell-lines of Petunia hybrida resistant to Methotrexate (MTX) were isolated upon stepwise selection; all of them are characterized by high levels of MTX binding (Barg et al., 1984). One of these, cell-line 1ECB, was compared with the WT cell-line with regard to cell-free MTX-binding capacity and the activity of Dihydrofolate Reductase (DHFR; EC 1.5.1.3), a target protein for MTX (Blakely, 1969; Cossins, 1980; Huennekens et al., 1976). The data obtained indicated that the major MTX-binding protein (MTX-BP) in cell-line
* To whom correspondence should be sent. © 1990 by Gustav Fischer Verlag, Stuttgart
1ECB differs from the normal DHFR. It was suggested that this abundant MTX-BP exerts DHFR activity, and that it also acts as a «trap» for MTX penetrating the cells, thus protecting MTX-sensitive protein(s), the functions of which are essential for cell viability (Barg et al., 1987). In mammals and even in bacteria, the dependency of MTX binding to DHFR on nicotinamide dinucleotide co-factors was studied extensively (e.g. Kamen et al., 1983; Otting and Huennekens, 1972; Perkins and Bertino, 1966; Werkheiser, 1961). In plants, this question was not addressed, though MTX binding to plant proteins was tested in the presence of NADPH (Barg et al., 1984, 1987; Cella et al., 1983, 1984, 1987). It was shown previously that for the MTX-resistant
612
RrvKA BARG, JACQUES S. BECKMANN, and MEIR PERL
petunia cell-line lECB, the presence or absence of NADPH does not affect the KD and BMAX for MTX binding (Barg et al., 1987). However, if this MTX-BP is to serve as a trap, an irreversible binding might be expected. The results described herein support this assumption and demonstrate the specificity of NADPH as a co-factor ensuring a tight binding of MTX to this protein.
3H-MTX BINDING 125
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Biological material The study was performed using the MTX-resistant cell-line lECB, which was described previously (Barg et aI., 1984, 1987). Proteins were extracted from cells propagated for four growth cycles in the absence of MTX. Crude cell extraction was performed as described previously (Barg et aI., 1984); briefly, vacuum-dried cells were ground to powder under liquid nitrogen, dissolved in extraction buffer (50mM K-phosphate, pH = 7.8/0.15M KCl), mixed thoroughly, and spun at 11,000 g for 15 min at 4°C. Dialyzed extract refers to the water-soluble protein fraction that was precipitated from the crude extract with 100% ammonium sulfate and dialyzed overnight against 2 L of the extraction buffer (Barg et aI., 1987).
JH-MTX binding assay The binding assays were performed essentially as described previously (Barg et aI., 1984). In the 3H-MTX binding and release experiments, the standard pre-incubation was with 0.23 JLM radio-labelled MTX (10Ci/mmole) for 20 min at room temperature. This concentration is approximately 20-fold higher than the apparent KD value for MTX in this line (Barg et aI., 1987); thus, the experiments were performed under saturated conditions for MTX. The actual concentration of unlabelled MTX added and the duration of incubation time with the unlabelled MTX are specified for each set of experiments described.
Protein quantitation
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Fig. 1: The effect of NADPH on the binding of 3H-MTX to and on its release from crude and dialyzed protein extracts. Binding reaction mixtures containing 0.23/LM 3H-MTX were incubated for 20 min in the presence or absence (W/O) of 5JLM NADPH (A). To half of each sample was added 5/LM of unlabelled MTX for an additional 30 min (B), and the bound 3H-MTX was monitored. The percent of 3H-MTX release was calculated as described in Table 1. The data presented are the average of two independent experiments, each performed in duplicate. (Deviation among replicates was less than 10%).
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.
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Proteins were precipitated with 10% trichloroacetic acid and measured according to the Lowry method (Lowry et aI., 1951).
Chemicals Folic acid, DHF, NADPH (enzymatic preparation), NADP, NAD, NADH, nicotinic acid, GSH and GSS were purchased from Sigma (St. Louis, MO). MTX was a generous gift from Lederle Laboratories (Pearl River, NY), and 3H-MTX (10Ci/mmole) was purchased from Amersham (UK).
Results
Effect 0/ NADPH on binding and release 0/MTX The NADPH dependency of MTX binding and release was examined in crude and in dialyzed protein extracts. As demonstrated in Fig. 1 A, the binding capacity of MTX is not affected by the presence or absence of NADPH (5 J.tM). The total amount of MTX bound to dialyzed extract is slightly lower (approx. 10- 15 % less) than that bound to the crude extract. The crude and the dialyzed extracts differ sig-
IfJM) NADPH
Fig. 2: The effect of NADPH concentration on the percent release of 3H-MTX (0.23 JLM) pre-bound to dialyzed protein extracts, following the addition of 5 JLM unlabelled MTX for 45 min (.). The concentration of NADPH added had no significant effect on the level of 3H-MTX binding, as monitored in control samples at the end of the incubation period with the unlabelled MTX (A). The data presented are the average of two independent experiments, each performed in duplicate. (Deviation among replicates was less than 10%).
nificantly, however, with regard to the effect of NADPH on the release of pre-bound 3H-MTX following the addition of 5 J.tM unlabelled MTX (Fig. 1 B): while in the dialyzed extract, MTX is displaced unless NADPH is added; in the crude extract most of the 3H-MTX is not released even if NADPH is not added.
Irreversible binding of MTX to plant proteins
The level of MTX binding to dialyzed protein extracts is not affected by NADPH (up to 10 fLM). Yet, the degree of displacement of the pre-bound 3H-MTX (0.23 fLM) following the addition of 5 fLM unlabelled MTX for 45 min depends on the concentration of NADPH (Fig. 2): adding 0.5 fLM NADPH results in retention of 50 % of the pre-bound MTX, and in the presence of 5 I'M NADPH the binding becomes practically irreversible.
Efficacy of various nicotinamide compounds as co/actors conferring irreversible MTX binding under reduced and oxidized conditions Since 5 I'M NADPH was found to restore completely the irreversible nature of MTX binding in dialyzed protein extracts, the efficacy of several other derivatives of nicotinamide in mediating irreversibility of MTX binding was examined. The nicotinamides NADP, NADH, NAD, and nicotinic acid were each added at a concentration of 5 fLM to dialyzed extracts together with 0.23 fLM 3H-MTX; after 20 min, 10 I'M of unlabelled MTX was added and the incubation continued for 45 min. The percent of release of prebound MTX was monitored (Table 1). The various compounds examined can be graded according to their efficiency as co-factors mediating irreversible binding: NADPH is the most potent one, allowing for 90 - 95 % of the MTX to remain irreversibly bound; NADH and NADP, conferred approximately 70 and 50 % irreversible binding respectively. NAD and nicotinic acid did not restore irreversible binding. The fact that NADH and not NADP was the «second best» raised the possibility that NADPH exerts its effect via its influence on the level of reduction of the environment, which affects the configuration of the MTX-BP, assuming that under reduced conditions binding is irreversible. To ex-
Table 1: The effect of various nicotinamide derivatives, oxidizing (GSS) and reducing (GSH) agents on the release (%) of pre-bound 3H-MTX. Dialyzed protein extracts were pre-incubated with the specified compounds for 5 min, at the end of which 0.23 I'M 3H_ MTX was added and the incubation continued for 20 min, and then 10 I'M of unlabelled MTX was added to half of each sample for 45 min. The percent of release was calculated from the ratio between the cpm determined in the two halves of each sample. The results presented are the range found in three independent experiments, each carried out in duplicates. Compound added Nicotinamide GSH derivative (5 I'M) (100 I'M) None None + None NADPH NADPH + NADPH NADP NADP + NADP NADH NAD Nicotinic Acid
GSS (100 I'M)
+ + +
3H-MTX released (%) 90-95 90-95 90-95 5-15 25-30 40-50 35-50 35-50 55-75 30-35 90-95 90-95
613
MTX
100 X
1-
:I:
§
~ ....Is.o
50
'" III
III
'"
~ 25
10 100 [/JMl DHF
1000
Fig.3: Release (%) of 3H-MTX (0.23I'M), pre-bound to dialyzed protein extract in the absence of NADPH, following the addition of increasing concentrations of either unlabelled MTX (e) or DHF (0) for 45 min. The data presented are from one of two similar experiments, each performed in duplicate. (Deviation among replicates was less than 10 %).
amine this hypothesis, the effect of reduced (GSH) and of oxidized glutathione (GSS) was tested. It was found (Table 1) that neither GSH nor GSS per se had any effect on the release of pre-bound MTX. None of the compounds added had any effect on the level of 3H-MTX binding (data not shown). The addition of 100 I'M GSH together with 5 I'M NADP had no significant effect on the percent of MTX release; addition of GSH together with NADPH increased the release of prebound MTX. That is, the presence of GSH affected adversely the ability of NADPH to tighten MTX binding.
Displacement ofpre-bound J H-MTX by DHF and MTX The ability of non-radioactive MTX and DHF to displace pre-bound 3H-MTX was examined in dialyzed extract in the absence of NADPH. Increasing concentrations of either MTX or DHF, were added and incubation was continued for 45 min. While 5 I'M MTX was sufficient to release 80 % of the pre-bound MTX, 1000 I'M DHF was required to achieve a similar level of displacement (Fig. 3). This higher displacement ability of MTX was also found when the time course of displacement of pre-bound 3H-MTX by either MTX or DHF (50 I'M) was followed: at each period of incubation (up to 45 min), the unlabelled MTX released comparatively more of the pre-bound 3H-MTX than did DHF (data not shown). Discussion The mode of interaction between the antimetabolite MTX and the major MTX-BP in cell-line lECB, which is highly tolerant to MTX (Barg et al., 1984, 1987), was the subject of the present study. A comparative study with Wild-Type protein extract was practically impossible, because of its low MTX-binding capacity. The binding of MTX to a crude cell
614
RIVKA BARG, ]ACQUES S. BECKMANN, and MEIR PERL
extract of cell-line 1ECB was shown to be extremely tight, and the compound(s) responsible for the irreversible binding was found to be lost upon 100 % ammonium sulfate precipitation and dialysis (Figs. 1 A, B). NADPH-dependent irreversible MTX-binding was demonstrated (Figs. 1 Band 2): a similar observation was made for the binding of MTX to DHFR of mammals (Perkins and Bertino, 1966) and bacteria (Otting and Huennekens, 1972). Displacement of 12 - 20 % of the pre-bound 3H-MTX was always observed, probably representing a non-specific MTX binding. The concentration of NADPH required to insure a complete irreversible binding in the dialyzed ammonium sulfate-precipitated protein extract was approximately five-fold higher than the concentration of the radio-labelled MTX added (Fig. 2). It should be noted that binding of MTX and NADPH to partially purified mammalian DHFR is accomplished on an equimolar basis (Kamen et aI., 1983; Perkins and Bertino, 1966; Werkheiser, 1961). The potential of other nicotinamide derivatives to serve as co-factors for irreversible MTX binding was examined. All the tested derivatives were less efficient than NADPH, and NAD had no influence on the tightness of MTX binding (Table 1). The adverse effect of reduced glutathione on the irreversible MTX binding in the presence of NADPH, and its lack of effect in the presence of NADP (Table 1), indicate that a reducing environment per se is not required for the binding reaction. The positive effect of NADP on irreversible binding might be a result of its reduction to NADPH, a process which is impaired in the presence of
GSS.
The irreversible binding of MTX to the crude extract (Fig. 1 A) might be caused by the existence of a binary complex of NADPH and MTX-BP; similar complexes were described before (Cohen et al., 1978; Huennekens et aI., 1973; Kamen et al., 1976, 1983; Otting and Huennekens, 1972). NADH might also contribute to the irreversible MTX binding found in the crude cell extract (Table 1). Since the native compound conferring irreversible binding in the crude extract was not identified, the existence of yet another unknown compound can not be ruled out. The higher efficiency of MTX as compared with DHF in displacing pre-bound radio-labelled MTX points to a greater affinity of the MTX-BP to the non-metabolizable analogue (Fig. 3). This is in agreement with previously reported results obtained from DHF and MTX competitive binding experiments (Barg et al., 1987). In summary, the data obtained from the MTX-displacement experiments, together with the previous characterization studies, strongly suggest that the MTX-binding protein, present in elevated amounts in cell-line 1ECB, contributes to the MTX resistance of this line mainly by acting as a trap for MTX penetrating the cells. The formation of a stable complex ofMTX-BP-NADPH-MTX seems essential forthis protein to exert its full function as a MTX scavenger. Whether NADPH is the co-factor acting in vivo remains to be demonstrated. Acknowledgements This is contribution No. 2646-E, 1989 series, from the Agricultural Research Organization, The Volcani Center, Bet Dagan
50250, Israel. Methotrexate was a generous gift from Lederle Laboratories (Pearl River, NY). This work was supported in part by a grant to J.S.B. from the US-Israel Binational Agricultural Research and Development Fund (BARD) and in part by a grant to R.B. from the US·Israel Binational Science Foundation (84-149).
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