Proc. Nati. Acad. Sci. USA Vol. 88, pp. 8572-8576, October 1991 Medical Sciences Stimulation of dihydrofolate reductase promoter activity by antimetabolic drugs HELEN B. EASTMAN, ANDREW G. SWICK*, M. CHRISTINE SCHMITTt, AND JANE CLIFFORD AZIZKHAN Lineberger Cancer Research Center and the Departments of Pharmacology and Pediatrics, University of North Carolina, Chapel Hill, NC 27599-7295 Communicated by Gertrude B. Elion, June 17, 1991 (receivedfor review September 19, 1990)
ABSTRACT Dihydrofolate reductase (DHFR; EC 1.5.1.3) Transcriptional activation ofthe DHFR gene by MTX is an is required in folate metabolism for the synthesis of purines, equally plausible hypothesis to explain the increased mRNA thymidine, and glycine. Although there have been several and enzyme levels, and this possibility has not been carefully reports ofinduction ofDHFR enzyme by methotrexate (MTX), addressed. We have previously characterized the protein/ a drug that competitively inhibits DHFR, there are no studies DNA interactions in the DHFR promoter that are required reported that examine the effect of MTX on DHFR gene for efficient and accurate transcription (11-13). There are transcription. We have examined the effect of MTX and other several GC elements (shown in Fig. 1), consensus binding inhibitors of DNA synthesis on DHFR transcription using a sites for the transcription factor Spl, present in the hamster transient expression assay. MTX stimulates transient expres- DHFR promoter that are essential for DHFR transcription sion in a concentration-dependent manner from a hamster (11) and that control transcription start site utilization (12). DHFR promoter construct containing 150 base pairs 5' to the This (G+C)-rich promoter motif is common to many so- start of transcription. Addition of either tetrahydrofolate or called housekeeping genes as well as several growth factor hypoxanthine plus thymidine prevents the promoter induction and growth factor receptor genes and oncogenes (ref. 11 and in response to MTX, suggestiug that stimulation by MTX references therein). There are also two overlapping recogni- results from inhibition of these metabolites. Furthermore, two tion sites for the transcription factor E2F that lie immediately other antimetabolic drugs-fluorodeoxyuridine and hydroxy- 3' to the major start of transcription; mutation of these sites urea-als stimulate the DHFR promoter in a concentration- results in a significant reduction of transcription (13). The dependent manner. In contrast, aphidicolin, which blocks cell functional significance of a nucleotide sequence is often growth through inhibition of DNA polymerase a, has no effect suggested by evolutionary conservation; the 5' flanking re- on theDHFR promoter. The potential relevance ofthese results gions of the human (14), murine (15), and hamster (16, 17) to cross-resistance to chemotherapeutic agents and to the DHFR genes exhibit a high degree of sequence conservation, process of gene amplification is discussed. particularly within the binding sites for the transcription factors Spl and E2F. Because the DHFR promoter is similar Dihydrofolate reductase (DHFR; EC 1.5.1.3) catalyzes the to those ofmany housekeeping genes, it is possible that many reduction of folate to dihydrofolate and then to tetrahydro- other genes in this class may also be transcriptionally acti- folate. Reduced folates are required cofactors for de novo vated by antimetabolic drugs, such as those discussed herein. synthesis of thymidylate, purines, and glycine. DHFR is We have examined the effects of treatment with MTX and required for maintenance ofcell growth and is thus expressed other inhibitors of DNA synthesis on DHFR promoter ac- in virtually all growing cells. The chemotherapeutic agent tivity to address the question of whether these agents tran- methotrexate (MTX) is a dihydrofolate analog that binds scriptionally activate the DHFR gene. In this paper we report DHFR, thereby inhibiting its enzymatic activity and prevent- that treatment of Chinese hamster ovary (CHO) cells with ing cell growth. Chronic MTX treatment can lead to MTX MTX increases expression from a DHFR promoter chloram- resistance; one mechanism whereby cells become resistant is phenicol acetyltransferase (CAT) construct. We show that amplification of the DHFR gene, which results in increased 5-meth- DHFR mRNA and protein levels (1). Very little is known stimulation by MTX can be inhibited by addition of about the early events that occur between MTX treatment yltetrahydrofolate, which obviates the requirement for and gene amplification. DHFR to reduce folates. This increase can also be prevented Several laboratories have reported that DHFR enzyme by addition of hypoxanthine plus thymidine, which supplies levels are elevated by treatment of cells with MTX (2-4) or the two end-products ofthe biosynthetic pathway involved in hydroxyurea (4) and that the stimulation by hydroxyurea is DNA replication that is inhibited by MTX. We also examined due to elevated mRNA levels (4). The drug-induced increase the effect of three other DNA synthesis inhibitors in DHFR mRNA levels may result from increased transcrip- aphidicolin, fluorodeoxyuridine, and hydroxyurea-on the tion, decreased mRNA degradation, or accumulation of DHFR promoter. We present data to demonstrate that treat- mRNA due to blockage of cells at the G1/S boundary of the ment of cells with aphidicolin does not affect DHFR pro- cell cycle, when DHFR gene transcription is maximal (5, 6). moter activity, whereas hydroxyurea or fluorodeoxyuridine The stimulation ofDHFR by MTX and hydroxyurea may also treatments stimulate the DHFR promoter in a concentration- be the result of inhibition of DNA synthesis, resulting in dependent manner. Thus, the DHFR promoter is stimulated rereplication of the gene (i.e., gene amplification) and sub- by at least three different antimetabolites, and the stimulation sequent elevated expression from the increased number of is not simply the result of inhibition of cell growth. transcription templates (7). Consistent with this idea, hy- droxyurea, which blocks DNA replication, and hypoxia and Abbreviations: DHFR, dihydrofolate reductase; MTX, methotrex- UV irradiation, which damage DNA, all have been found to ate; CAT, chloramphenicol acetyltransferase; CHO, Chinese ham- increase the frequency of DHFR gene amplification (8-10). ster ovary; AdMLP, adenovirus major late promoter; DMSO, di- methyl sulfoxide; ANOVA, analysis of variance. *Present address: Department of Biological Chemistry, Johns Hop- The publication costs of this article were defrayed in part by page charge kins University, Baltimore, MD 21205. payment. This article must therefore be hereby marked "advertisement" tPresent address: Vanderbilt University School of Medicine, Nash- in accordance with 18 U.S.C. §1734 solely to indicate this fact. ville, TN 37215. 8572 Downloaded by guest on September 29, 2021 Medical Sciences: Eastman et al. Proc. Natl. Acad. Sci. USA 88 (1991) 8573
1 11 1 LE2F-e IIn I I II I ------ATG 0o r. 0 FIG. 1. DHFR promoter transient expression vector. pDHF/CAT was constructed by cloning the hamster DHFR promoter fragment from nucleotide position -210 to -23 (relative to ATG = position 1) 5' to the bacterial CAT gene and simian virus 40 poly(A) signal in a pUC18 vector. The major start site of DHFR transcription (bold arrow) is at nucleotide position -63, and nucleotide position -107 is the minor start site. GC boxes are indicated by stippled boxes and are numbered in the text I-IV, proximal to distal; open boxes indicate additional conserved sequence elements. Binding sites for the transcription factor E2F are indicated by the hatched box. EXPERIMENTAL PROCEDURES CAT) reported in this manuscript contained DNA sequence from the positions -210 to -23 bp relative to ATG. The Construction of Clones. The DHFR promoter constructs were derived from a genomic CHO clone previously de- sequence elements in this construct that are highly conserved scribed (16). The construct that is used in the present studies, in the mouse and human DHFR promoters (shown in Fig. 1) designated pDHF/CAT, contains hamster DHFR promoter consist of four GC boxes (stippled boxes), two 20-bp regions sequence from positions -210 to -23 base pairs (bp) relative 3' to boxes I and II (open boxes), and two overlapping to the start of translation (i.e., -147 to +40 relative to the recognition sites for the transcription factor E2F (hatched major transcription start) cloned 5' to the coding sequence for box). Each of these elements has been shown to be func- the bacterial CAT gene fused to a poly(A) sequence from the tionally important for constitutive activity of the hamster simian virus 40 large tumor antigen (11). The clone used as a DHFR promoter (11-13). Maximal promoter activity is ob- control consists of the adenovirus major late promoter fused tained with this construct; constructs with additional 5' to CAT (AdMLP/CAT) (18) in the same expression vector sequence are equally active in transfection experiments (11), construct. whereas deletion or mutation of GC boxes I, II, or III Cell Culture and Transfections. CHO cells were grown in significantly reduces transcriptional efficiency (12). monolayer in Eagle's minimal essential medium (MEM) To determine the effect of MTX on the DHFR promoter, supplemented with 5% fetal bovine serum, nonessential CHO cells were transfected with pDHF/CAT and treated amino acids, glutamine, and penicillin/streptomycin. The with MTX at 0.05, 0.1, 0.15, 0.2, and 0.25 /ug/ml. A linear doubling time of CHO cells is -20 hr under these growth increase in CAT activity was observed in response to MTX conditions. Twenty-four hours prior to transfection, cells treatment from 0.05 to 0.2 pug/ml (Fig. 2). In cells treated with were plated at -5 x 105 per 10-cm plate in Eagle's MEM 0.2 ,gg of MTX per ml of medium, there was an -5-fold supplemented with 5% dialyzed fetal bovine serum (dFBS). stimulation of CAT expression in this experiment. The max- Promoter-CAT constructs were transfected by the calcium in the 20 times that MTX has been tested phosphate coprecipitation method essentially as described by imal stimulation Graham and van der Eb (19). Within each experiment, a ranged from 4- to 20-fold. single DNA precipitate at a concentration of 10 Ag/ml was To determine whether general transcription is stimulated formed for each DNA construct. The individual plates were by MTX treatment, the AdMLP (18), which bears no se- rinsed and the monolayer was drained; 0.5 ml of DNA quence homology to the DHFR promoter, was tested. As precipitate (5 ,ug) was added dropwise to the individual shown in Fig. 2, CAT expression under the control of the plates. Complete medium containing 5% dFBS was added AdMLP was not stimulated by MTX treatment ofCHO cells. after 30 min at room temperature. Four hours later, the Thus, stimulation of the DHFR promoter by MTX is not the monolayer was washed and complete medium containing result of a general transcriptional response. dFBS was added to the cells. Test agents were added directly Metabolic Signals. In an effort to understand the signal(s) to the medium as indicated in the figure legends. MTX was involved in the elevation ofDHFR promoter activity, various obtained from the Drug Synthesis and Chemistry Branch of substances that obviate the requirement for certain metabolic the National Cancer Institute; MTX was reconstituted in 0.1 cofactors were added in addition to MTX. Addition of 2 ,uM M NaOH in a 50 mg/ml stock solution that was diluted in water for use and stored in the dark at -20°C. Aphidicolin, 6.0 hydroxyurea, and fluorodeoxyuridine were obtained from Sigma. Aphidicolin was reconstituted in dimethyl sulfoxide 5.0 DHFR (DMSO) at 10 mg/ml; DMSO was added to cells in the > volumes used for aphidicolin treatment to control for vehicle :t 4.0 - effects. Hydroxyurea and fluorodeoxyuridine were reconsti- tuted in water at 1 M and 100 mM, respectively. 3.0- Assay for CAT Activity. Following a 48-hr incubation, cells 0 were harvested and subjected to freeze-thaw lysis. Lysates .;5lY 2.0- were assayed for protein content by the Bradford (20) assay. AdMLP CAT activity was measured by a fluor-diffusion assay using 1.0e [acetyl-3H]acetyl CoA (200 mCi/mmol; 1 Ci = 37 GBq; NEN), essentially as described by Neumann et al. (21), 0.0 - except that 0.4 ,Ci of [3H]acetyl CoA plus 40 AM unlabeled 0.00 0.05 0.10 0.15 0.20 0.25 acetyl CoA were used per assay. Twenty micrograms of protein cell extract was assayed, and CAT activity was Methotrexate (jug/ml) calculated as pmol ofproduct acetylated per ,ug ofprotein. To make comparisons between experiments, data are presented FIG. 2. Effect of MTX on transient expression from the DHFR as relative CAT activity, with wild-type untreated CAT promoter. pDHF/CAT or AdMLP/CAT plasmid constructs (5 ,g each) were transfected by the calcium phosphate procedure into expression normalized to 1. CHO cells. MTX was added to the medium at the concentrations indicated. After 48 hr, cells were harvested and assayed for protein RESULTS concentration and CAT activity was measured in cellular lysates on equal amounts of protein. CAT activity of untreated cells was Effects of MTX on DHFR Promoter Activity. The hamster assigned a relative value of 1. These results are representative of DHFR promoter construct used in the experiments (pDHF/ three repetitions of this experiment. Downloaded by guest on September 29, 2021 8574 Medical Sciences: Eastman et al. Proc. Natl. Acad. Sci. USA 88 (1991) 5-methyltetrahydrofolate, which circumvents the cellular re- A quirement for DHFR, restored cell growth (Fig. 3B) and 10 - --.__ ..-
blocked the stimulation ofDHFR promoter activity (Fig. 3A). r 5-Methyltetrahydrofolate restores tetrahydrofolate pools, MT X (Ig rnI 8 0 0 which are specifically blocked by MTX; however, 5-meth- F: E.025 yltetrahydrofolate also competitively inhibits MTX transport 6 El .05 into cells (22,23). To address the possibility that the inhibition -El -10 20 ofCAT activity was due to decreased MTX uptake rather than C) 4 to activation of the promoter, we added hypoxanthine and H- thymidine, alone and in combination with MTX, to circumvent 2 Er the inhibition of purine and thymidine biosynthesis, respec- LU tively, by MTX. Neither hypoxanthine nor thymidine has been 0 shown to affect MTX transport, but both of these compounds C H 4 F HX Thy HX+ThV have been shown to rescue cells from MTX treatment (24). Both 17.6 ,uM hypoxanthine and 100 gM thymidine alone inhibited stimulation of the DHFR promoter by MTX, hypo- B xanthine to a greater extent than thymidine. When both agents 1.5 - ---- were added together, stimulation by MTX was completely MTX (uLg ml) blocked, suggesting that the stimulated transcription is medi- MO E .025 ated by decreased purine and thymidine metabolism (Fig. 3A). z 1.0 l .05 When added in the absence of MTX, neither 5-methyltetrahy- Li .10 drofolate, thymidine, nor hypoxanthine significantly affected -n 20 CAT expression (data not shown). U' 0.5 Relatioship Between Effects on Cell Growth and Promoter Stimniation. Treatment ofcells with MTX alone stimulated the DHFR promoter in a concentration-dependent manner (Figs. Lu 2 and 3A, groupC). In an effort to determine whether the effect 0. of MTX was due to cell killing and a stress-related response, C H 4F HX Thy HX.Jhy especially at the higher concentrations, cells treated as shown and the protein content ofthe cellular FIG. 3. Effect of 5-methyltetrahydrofolate (H4F), hypoxanthine in Fig. 3A were counted, (HX), and/or thymidine (Thy) on MTX stimulation of the DHFR lysate was compared to the cell number. We have found that promoter and on cell growth. CHO cells were transfected with there is a linear relationship between cell number and protein pDHF/CAT (5 ,ug) by the calcium phosphate transfection method. concentration (data not shown); we therefore routinely deter- Four hours later, the following reagents were added to the medium mined protein concentration as a measure of cell growth. as indicated: 0.025-0.2,ug of MTX per ml; 2 jAM H4F; 17.6,uM HX; These data are graphed normalizing the protein content (or cell 100 AtM Thy. All agents were added at 0 and 24 hr. C, control number) ofthe untreated sample to 1.0 to readily compare one (treatment with MTX alone). (A) Results ofthe CAT assay performed using equal amounts of protein in cell lysates 48 hr after transfection. experiment with another. As can be seen in Fig. 3B, when cells For each treatment group, CAT activity for cells not treated with are treated with MTX alone (group C) cell growth is blocked MTX was assigned a relative value of 1. Absolute values for CAT completely by MTX at 0.025,ug/ml, and very little additional activity in this experiment ranged from 12 pmol of acetylated product effect on cell number is seen at the higher concentrations, formed per /Ag of protein in the untreated control to 99.5 pmol/g at where there is a dose-dependent increase in CAT activity (Fig. 0.20 jtg of MTX per ml. (B) Effect ofthese treatments on cell growth. 3A, group C). From 0.025 to 0.2 gg/ml, the cell number is Protein concentrations in the cellular lysates were determined by the -20% of the untreated sample. This result is consistent with Bradford assay (20) and normalized to a control value of 1.0 as cell growth during the 48-hr treatment, since during described for CAT assays. Protein concentration is a measure of cell blocked number as discussed in Results. These results are representative of this period the cell number of the untreated sample increased three repetitions of this experiment. Two-way analysis of variance 5-fold, corresponding to 2.3 doublings. Thus, within the range (ANOVA) ofthe data inA revealed that the MTX-treated samples are where a linear relationship between drug concentration and significantly different from one another and from the untreated promoter activity is observed, MTX blocked proliferation sample (P c 0.05) at all of the concentrations tested. Furthermore, equally but did not kill cells. Addition of the various metab- the addition of H4F, HX, or HX plus Thy with MTX at all of the olites did not equivalently affect proliferation and DHFR concentrations eliminated this difference (P s 0.05), whereas the promoter activity, suggesting that the effect ofMTX on DHFR addition of Thy had no significant effect as compared with MTX transcription is not due simply to inhibition of cell growth. alone at all of the concentrations tested, with the exception of0.2 ,ug Effect of Other Inhibitors of DNA Synthesis. To determine of MTX per ml (P c 0.05). if the DHFR promoter was stimulated by other inhibitors of DNA synthesis and cell growth, we tested the effect of three Taken together, these data suggest that DHFR promoter other agents on the DHFR promoter. Cells were transfected activity is significantly stimulated in cells treated with MTX, with pDHF/CAT and treated with aphidicolin (10-100 ,ug/ hydroxyurea, and fluorodeoxyuridine. This stimulation is not ml), an inhibitor of DNA polymerase a. Aphidicolin blocked simply the result of inhibition of DNA synthesis and cell cell proliferation (Fig. 4B), but had no effect on CAT expres- proliferation; aphidicolin, which blocks proliferation as ef- sion from the DHFR promoter (Fig. 4A). The effects of two fectively as MTX, does not affect promoter activity. Fur- other antimetabolites on the DHFR promoter were also thermore, MTX stimulation of CAT activity is dose depen- tested. Fluorodeoxyuridine, an inhibitor of thymidylate syn- dent at drug concentrations that have an equal antiprolifer- thetase, stimulated the DHFR promoter in a concentration- ative effect. dependent manner (Fig. SA). Hydroxyurea, an inhibitor of ribonucleotide reductase, in the range of 0.05-1 mM, also DISCUSSION stimulated the DHFR promoter in a concentration-dependent We have explored the response of the DHFR promoter to manner to a maximum of 2.5-fold (Fig. SC). Both of these MTX, and to other inhibitors of DNA synthesis, and found agents inhibited cell growth (Fig. 5 B and D), although not as that MTX as well as the antimetabolites fluorodeoxyuridine completely as MTX or aphidicolin. and hydroxyurea stimulate the DHFR promoter, whereas Downloaded by guest on September 29, 2021 Medical Sciences: Eastman et al. Proc. Natl. Acad. Sci. USA 88 (1991) 8575
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