J. Biosci., Vol. 10, Number 1, March 1986, pp. 37–47. © Printed in India. Large scale production and characterisations of dihydrofolate reductase from a methotrexate-resistant human lymphoid cell line

AFTAB ALAM* Astbury Department of Biophysics, University of Leeds, Leeds LS2 9JT, England * Present address: BioKalfs-research and development, Suite 8, 2nd Floor, Royal House, 28 Sovereign Street, Leeds LS1 4BJ, England

MS received 10 March 1983; revised 28 October 1985

Abstract. Dihydrofolate reductase has been purified from a methotrexate-resistant human lymphoid cell line (CCRF/CEM-R3) and up to 1 mg of enzyme has been obtained from 5 litres of culture. The enzyme has a molecular weight of 22000 ± 500 as determined by gel filtration.The pH activity profile shows a single optimum at pH 7·7, where marked activation is observed by addition of 0·2 Μ NaCl. The Km for NADPH is 3 µΜ and dihydrofolate 0·7 µΜ. The binding constant for the inhibitor, methotrexate, is 29 pM.

Keywords. Human dihydrofolate reductase; purification; characterization.

Introduction Dihydrofolate reductase (5,6,7,8-tetrahydrofolate NADPH oxidoreductase, EC 1.5.1.1.3.) acts in concert with thymidylate synthetase, and both enzymes are required for de nove synthesis of thymidylate, a DNA precursor. Inhibitors of these enzymes are among the most useful anti-cancer agents employed clinically. In addition, differences between dihydrofolate reductase from human tissue and micro-organisms have been exploited in the design of bacteriocides and anti-malarials. For the rational design of a new generation of inhibitors, precise details on the mode of inhibition at the atomic structural level are required. The 3-dimensional structures of dihydrofolate reductase from Lactobacillus casei, Escherichia coli and chicken lever in a variety of binary and ternary complexes have been reported recently (Matthews et al., 1977, 1978; Volz et al., 1982), but the design of new, clinically useful inhibitors would benefit greatly from a detailed knowledge of the human enzyme. Unfortunately, only very small amounts of dihydrofolate reductase are present in normal human cells and tissues (Jarabak and Bachur, 1971; Lindquist and Bertino, 1976), and none of these tissues would seem a viable source of enzyme for sequence and crystallographic studies.

It is known that cells gradually develop a resistance to the drug Methotrexate (Bertino et al., 1970; Harrap et al., 1971) and one of the mechanisms of the resistance is increased production of dihydrofolate reductase. Alam et al. (1983) have produced a methotrexate-resistant human cell line (CCRF/CEM-R3) with over 200-fold enhanced production of dihydrofolate reductase so that sufficient material can be obtained for Abbreviations used: SDS, Sodium dodecyl sulphate; Mr, molecular weight.

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38 Alam structural studies. The present work describes the characterisation of dihydrofolate reductase from these cells and its production in milligram quantities.

The CCRF/CEM cell line is a line of lymphoid cells isolated from the peripheral blood of a young girl with acute leukemia (Foley et al., 1965). Since it is conceivable that the dihydrofolate reductase from these cells, or from the methotrexate-resistant overproducing cells, might be different from that obtained from normal cells, it is important that dihydrofolate reductase from other human cell lines should also be characterised and sequenced. Materials and methods Materials Methotrexate, folate, NADPH and other chemicals were obtained from Sigma Chemical Co, St. Louis, Missouri, USA; RPMI 1640 medium, donor calf serum and other tissue culture materials from Flow Laboratories Ltd. Aminohexyl-Sepharose 4B from Pharmacia Fine Chemicals and Ultragel AcA44 from LKB-Produkter.

Methotrexate-aminohexyl-Sepharose 4B was prepared by adding 80 mg of solid methotrexate powder to a suspension of AH-Sepharose 4B in distilled water which had been prepared previously by swelling 4 gm of AH-Sepharose 4B in 50 mM NaCl overnight and then washing with distilled water. With the pH adjusted to between 4·5 and 6, solid carbodiimide powder was added to a final concentration of 100 mM. The pH was maintained between 4·5 and 6 for another hour and then the reaction was allowed to proceed for 24 h at room temperature with gentle stirring. The slurry was poured on to a column (2 × 10 cm) and washed with 100 mM NaHCO3 solution overnight at a flow rate of 100–150 ml/h. The column was equilibrated with 50 mM sodium phosphate, pH 6·5. Cell culture Cells were grown in suspension in RPMI 1640 medium supplemented with 10 % donor calf serum, penicillin (100 units/ml) and streptomycin (100 µg/ml), to a density of 1 × 106 cells/ml. Cells were grown in suspension of 0·5–1 litre. Occasionally larger suspensions were also grown. Cells were harvested by centrifugation at mid-log phase, and the cell pellet was washed with saline buffer pH 7·2, spun down and stored at –20°C. A methotrexate resistant human cell line (CCRF/CEM-R3) was produced by treating sensitive, non-resistant cells (CCRF/CEM) with gradually increasing doses of methotrexate (Alam et al., 1983). Enzyme assay Dihydrofolate reductase was assayed by spectrophotometric monitoring at 340 nm of the conversion of NADPH to NADP+. One unit of enzyme is defined as that amount of enzyme which oxidizes 1 µmol of NADPH/min in a cuvette of 1·0 cm light path at 30°C, calculated from a molar absorbance change in the reaction of 12350/mol/cm (see, for example, Jarabak and Bachur, 1971). Assays were performed at 30°C in 50 mM Tris/HCl, pH 7·0, containing 200 mM NaCl, 100 µΜ NADPH and 100 µΜ dihydro-

Dihydrofolate reductase from human lymphoid cell 39 folate. For Ki estimation, methotrexate was added to a mixture of enzyme, buffer and NADPH, and the mixture incubated at 30°C for 5 min. The reaction was started by the addition of 50 µΜ dihydrofolate. Protein was estimated by the method of Lowry et al. (1951). Enzyme purification

Cell pellets were suspended in 20 mM Tris/HCl, pH 7·0, containing 20 mM NaCl and subjected to freezing and thawing 3 times. The lysate was spun at 160,000 g for 1 h and the supernatant collected. The cell free extract was brought to pH 5·2 by dropwise addition of 10 Μ acetic acid with gentle stirring. After 20 min incubation at 5°C the extract was spun at 160,000 g for 20 min and the supernatant collected and titrated back to pH 7·0 by dropwise addition of 5 Μ NaOH. A 50–90% ammonium sulphate precipitate was obtained (Green and Hughes, 1955). These acid precipitation and ammonium sulphate fractionation steps each removed about 40% of additional protein.

The 50–90% ammonium sulphate precipitate was dissolved in 1–2 ml of 50 mM sodium phosphate buffer, pH 6·5, and applied to a column of Ultragel AcA44, (figure 1). The active enzyme fractions from gel filtration were applied to a methotrexate-Sepharose column; the enzyme activity was located in a single peak of protein eluted with folate (figure 2). The fraction containing dihydrofolate reductase activity were bulked and concentrated using an Amicon ultrafiltration cell with a

Figure 1. Gel filtration of CCRF/CEM-R3 dihydrofolate reductase on Ultragel AcA44.

The column (2·5 × 90 cm) was equilibrated with 50 mM sodium phosphate, pH 6·5. The 50–90 % ammonium sulphate precipitate was dissolved in 1–2 ml of buffer and applied to the column. Fractions (11 ml) were collected at a flow rate of 20 ml per h. Fractions were monitored for A280 (Δ) and dihydrofolate reductase activity ( ).

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Figure 2. Afinity chromatography of CCRF/CEM-R3 dihydrofolate reductase.

The peak enzyme fractions from the Ultragel AcA44 column were applied to a methotrexate-aminohexyl-Sepharose-48 column at a flow rate of 30 ml/h, followed by 4 bed volumes of equilibration buffer. The column was washed with about 4 bed volume of 50 mM sodium phosphate, pH 6·5, containing 1 Μ NaCl, at a flow rate of 30 ml/h, and then washed with equilibration buffer until the absorption of effluent at 280 nm was negligible. The enzyme was eluted with 50 mM Tris/HCl, pH 8·5, containing 50 mM NaCl and 1 mM folic acid at a flow rate of 40 ml/h. Fractions were monitored for dihydrofolate reductase activity ( ) and protein (Δ).

Diaflo-PM10 membrane. In order to remove folic acid, the concentrated enzyme solution was applied to a Sephadex G-25 column (2·5 × 15 cm) and eluted with 50 mM sodium phosphate buffer, pH 7·2.

Occasionally, it was noticed that some enzyme did not bind to the Afinity column and that the binding was not improved by altering the pH or using a fresh affinity column. In these preparations some activity was also lost at the ultrafiltration step. Poor binding of dihydrofolate reductase to affinity column and subsequent loss of activity at the ultrafiltration step resulted in preparations which came from cell cultures grown in quantities in excess of 1 litre suspensions. It was also found that CCRF/CEM-R3 cell line did not grow well (to a density of 1 × 106 cell/ml) in suspensions larger than 1 litre. Therefore, purification yield of dihydrofolate reductase is influenced by the quality of the cell culture. If there is any intracellular damage of the enzyme, this would probably occur before cell lysis since recovery is occassionally low even when the protease inhibitor phenylmethyl sulphonyl fluoride is used during lysis of the cell pellets and the following steps.

Dihydrofolate reductase from human lymphoid cell 41 Sodium dodecyl sulphate-gel electrophoresis For gel electrophoresis, a 7–17 % polyacrylamide gel containing 0·1 % sodium dodecyl sulphate (SDS) was used and 50 µg of enzyme was loaded (Laemmli, 1970). Protein was boiled for 5 min in a mixture containing 2 % SDS, 10 % mercaptoethanol, 5 % glycerol and a drop of bromophenol blue solution (figure 3). Amino acid analysis The amino acid composition of dihydrofolate reductase was assessed after hydrolysis for 24, 72 and 96 h. Calculations were made using a molecular weight (Mr) of 22,000. Sequence of N-terminal amino acid The Beckman Sequencer 890, Beckman Instrument Inc., USA was used for automatic sequencing. The phenylthiohydantoin derivatives of the amino acid were identified using HPLC, Water Ass. ALC/GPC 202, USA and thin-layer chromatography (Rosmus and Deyl, 1972). Results and discussion Dihydrofolate reductase, purified as described above, showed a single band of protein on SDS-gel electrophoresis (figure 3). Typically, 1 mg of enzyme was obtained from

Figure 3. SDS-gel electrophoresis of purified reductase.

42 Alam 5 litres of cell culture. The enzyme may be stored in 0·2 Μ phosphate buffer, pH 7·0, at – 20°C for several weeks without significant loss of activity.

Estimation of Mr by gel filtration on Ultragel AcA44 using ribonuclease (Mr = 137,000), bovine serum (67,000), ovalbumin (43,000) and chymotrypsin (25,000) as markers, gave a value of 22,000 ± 500. This is similar to the value of 20–22,000 estimated for human placental dihydrofolate reductase (Jarabak and Bachur, 1971) and 21,500 calculated for dihydrofolate reductase from other mammalian species, based on their amino acid sequence (Hitchins and Smith, 1980).

Dihydrofolate reductase from a variety of sources are stimulated by salts (see Hitchins and Smith, 1980). In the present work, sodium chloride gave a maximum (two-fold) stimulation at approx. 200 mM at pH 7·0 (figure 4). The pH optimum for activity was 7·7 (figure 5).

The purified enzyme from the CCRF/CEM-R3 cell line gave a specific activity of 6·5 units/mg, which is about 4 times higher than that reported for human placental enzyme (Jarabak and Bachur, 1971). In the present work only very approximate measurements of specific activity were made prior to affinity chromatography because the inhibitor methotrexate was still present in the preparation. Experience has shown that the enzyme is damaged during the extensive dialysis required to remove methotrexate and this dialysis is not essential since the enzyme exchanges any methotrexate carried over from the cell culture for the methotrexate arms of the affinity column.

The rate of dihydrofolate reduction was measured as a function of concentration of NADPH and dihydrofolate. The Km value for the two substrates, calculated using the

Figure 4. The effect of sodium chloride on dihydrofolate reductase activity.

Dihydrofolate reductase from human lymphoid cell 43

Figure 5. pH-dependence of CCRF/CEM-R3 dihydrofolate reductase.

Buffers used were (O) 200 mM sodium phosphate and ( ) 50 mM Tris/HCl containing 200 mM NaCl to maintain uniform ionic strength.

least square method of Marquardt (1963), were 3·0 µΜ for NADPH and 0·7µΜ for dihydrofolate. Inhibition of dihydrofolate reductase was obtained in the presence of 100 µΜ NADPH and 50 µΜ dihydrofolate (figure 6). The Ki for methotrexate, calculated using the statistical method of Henderson (1973), was 29 pM. In table 1, these values are compared with those of dihydrofolate from other sources and seen to be closely similar to the values reported for pig liver dihydrofolate reductase (Smith et al., 1979).

The amino acid composition of the purified human dihydrofolate reductase is given in table 2. The presence of 4 methionine residues has been confirmed by cleaving the protein with cynogen bromide; this procedure gives 5 peptides. The value for tryptophane and cysteine were not determined. The Mr calculated from amino acid composition is 21,741 and the value is consistent with the value obtained from the gel filtration.

Recently, the nucleotide sequence of the human dihydrofolate reductase has been determined from the analysis of human dihydrofolate reductase cDNA and the amino acid sequence has been derived (figure 7) (Masters and Attardi, 1983). The N-terminal amino acid sequence determined in this work has been compared with those of corresponding nucleotide and amino acid sequences. The sequence determined is consistent with those derived from nucleotide sequencing studies.

There is only one previously reported study on human dihydrofolate reductase and this shows a wide variation in the values of the kinetic parameters (see table 1). No doubt some of these differences can be attributed to differences in experimental conditions and techniques and it is probably too early to conclude that the enzymes are different. Unfortunately, the cell line studied by Jackson and Neithammer (1977) were later found to be a murine cell line and not a human cell line (Alam et al., 1983). There is

44 Alam

Dihydrofolate reductase from human lymphoid cell 45

Figure 6. Inhibition of dihydrofolate reductase with methotrexate.

Activity of dihydrofolate reductase was measured in the presence of increasing amounts of methotrexate. Assays were performed as described in the text with 100 µΜ NADPH and 50 µΜ dihydrofolate.

Table 2. Amino acid composition ofCCRF/CEM-R3 dihydrofolate reductase

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Figure 7. N-Terminal amino acid sequence of human dihydrofolate reductase.

Nucleotide sequence of the human dihydrofolate reductase cDNA coding region (top) and amino acid sequence of the human dihydrofolate reductase as derived from the nucleotide sequence (middle) (Masters and Attardi, 1983). Amino acid sequence of the CCRF/CEM-R3 dihydrofolate reductase as determined in this study (bottom).

a need for further studies to establish whether the enzyme differs in different human tissues and especially in normal and methotrexate-resistant overproducing cell lines. Acknowledgements

This work was supported by funds from the Yorkshire Cancer Research Campaign, Harrogate, West Yorkshire, UK. I am grateful to Professor A. C. T. North and Dr. A. J. Geddes of the Astbury Department of Biophysics, University of Leeds, for their cooperation. References

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