Down-Regulation of Reduced Folate Carrier Gene (RFC1) Expression after Exposure to Methotrexate in ZR-75-1 Breast Cancer Cells

Biochemical and Biophysical Research Communications 279, 891– 897 (2000) doi:10.1006/bbrc.2000.4019, available online at http://www.idealibrary.com on

Down-Regulation of Reduced Folate Carrier Gene (RFC1) Expression after Exposure to Methotrexate in ZR-75-1 Breast Cancer Cells Deqin Ma, Hui Huang, and Jeffrey A. Moscow 1 Department of Pediatrics, University of Kentucky, Lexington, Kentucky 40502

Received November 20, 2000

Methotrexate (MTX) is administered in intervals of one week or longer in the treatment of cancer and autoimmune disease. Early studies suggested that daily MTX administration was associated with decreased effectiveness and increased toxicity, leading to schedules of administration that include periodic intervals of rest during chronic MTX therapy. We hypothesized that these observations may be the result of the down-regulation of the reduced folate carrier, the major route of cellular uptake of both MTX and the endogenous folates, after MTX exposure. We exposed folate-depleted ZR-75-1 breast cancer cells to low-dose MTX in the presence of hypoxanthine, adenosine and thymidine. After 72 h, the initial rate of MTX uptake had decreased to 22% of the Day 0 value. Western blot analysis showed down-regulation of RFC1 protein expression, and Northern blot analysis showed a corresponding decrease in RFC1 RNA levels. Using an RTPCR assay, we found that levels of RNA transcripts containing each of the three RFC1 5ⴕ noncoding exons were decreased after exposure to MTX, suggesting that MTX exposure causes transcriptional downregulation of RFC1. Promoter–reporter construct assays demonstrated decreased activity of RFC1 promoter elements upstream of these exons after MTX exposure. Preexposure of the ZR-75-1 cells to 5-azacytine, a DNA methylation inhibitor, further decreased MTX uptake rather than reverse the inhibition of RFC1 activity, indicating that RFC1 downregulation after MTX exposure is not the result of methylation of the RFC1 promoter. In summary, these studies demonstrate that MTX exposure can downregulate RFC1 expression and activity. These acute, inducible, epigenetic changes in RFC1 expression may Abbreviations used: MTX, methotrexate; LCV, leucovorin; ALL, acute lymphoblastic leukemia; HD, high-dose; HAT-supplemented medium, hypoxanthine, adenosine and thymidine supplemented medium; RFC, reduced folate carrier; UTR, untranslated region. 1 To whom correspondence should be addressed at 740 S. Limestone, Room J457, Lexington, KY 40502. Fax: 859-257-6048. E-mail: [email protected].

ultimately be molded into the more permanent genetic changes that result in the transport-mediated MTX resistance that have been observed in MTX-resistant cell lines. © 2000 Academic Press Key Words: antifolates; drug resistance; gene expression.

Methotrexate (MTX) has an unusually large range of dosage and schedule. For maintenance therapy in childhood acute lymphoblastic leukemia (ALL), it may be given at dosages as low as 20 mg/m 2 per dose once every week. On the other hand, it may also be administered in dosages up to 33 grams/m 2 over 24 h with leucovorin (LCV) rescue (1). Current schedules of administration for MTX in cancer therapy vary from weekly intervals to cycles of every three to four weeks or even longer (1). The regulation of MTX uptake may play a role in the effectiveness of both low-dose and high-dose MTX therapy. Early studies which attempted to determine the optimal schedule of administration for MTX in the treatment of ALL demonstrated that MTX administration every four days was superior to daily administration schedules (2, 3). These studies form the basis for the modern practice of administering low-dose MTX at weekly intervals, even though the other antimetabolites used in the treatment of ALL, such as 6-mercaptopurine and 6-thioguanine, are administered on daily schedules. In addition, there is also evidence that daily administration of MTX can be associated with an increased incidence of hepatic cell injury in comparison to intermittent dosage schedules (4). In the administration of high-dose (HD) MTX with LCV rescue, clinical practice has been based on the assumption that, apart from competing for uptake or interacting in anion exchange, these drugs do not autoregulate their own uptake. However, variation in the ability of cancer cells to transport LCV after exposure

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to HD MTX could result in differences in survival after HD MTX. Therefore, if MTX exposure acutely decreased its own uptake, then the role of acute changes in folate transport in determining the toxicity and effectiveness of HD MTX would have to be considered. The major route of uptake of folates and antifolates at these pharmacologic concentrations is the reduced folate carrier (RFC). Decreased RFC activity has been observed in several in vitro models of transportmediated MTX resistance (5–13). In different cell lines, MTX transport deficiency has been attributed either to mutations in the RFC gene (RFC1) or to decreased expression of the RFC gene product (14 –18). RFC1 RNA levels also have correlated with MTX sensitivity in a panel of nonselected cell lines (19). The possibility that MTX may regulate its own uptake by down-regulating theRFC1 gene is therefore of importance in both low-dose MTX regimens and for HD MTX with LCV rescue. We used a well-characterized human breast cancer cell line, ZR-75-1, to study the effects of MTX exposure on MTX uptake. MATERIALS AND METHODS Cell culture. Media, serum and antibiotics were purchased from Gibco BRL (Gaithersburg, MD). WT ZR-75-1 cells were maintained in IMEM supplemented with 5% FBS. Prior to all studies, ZR-75-1 cells were plated in folate-free RPMI 1640 supplemented with 10% dialyzed FBS, 100 ␮M hypoxathine, 100 ␮M adenosine, and 16 ␮M thymidine (HAT). After 72 h, cells were exposed to 30 nM MTX in this medium. MTX uptake studies. MTX uptake study was performed according to a previous described method (14, 20) with minor modifications. Wild-type ZR-75-1 (WT ZR-75-1) cells were plated in 6-well plate at a density of 1 ⫻ 10 5/well in HAT-supplemented folate-free medium containing 10% dialyzed FBS. After 72 h incubation, the cells were exposed to 30 nM MTX in the above medium. At different time points, cells were washed three times with folate-free RPMI and then incubated in folate-free RPMI containing 1 ␮M [ 3H]MTX (Moravek Biochemicals, Brea, CA) and 20 mM Hepes (pH 7.2) for 8 min at 37°C. The uptake was terminated by aspiration of the transport medium and the plates were washed immediately with ice-cold PBS for three times. Cells were solubilized in 0.2 N NaOH overnight and neutralized by 0.2 N HCl. Radioactivity was measured by scintillation counting. Protein concentration was determined by the Bradford method using a kit from Bio-Rad (Hercules, CA). Radioactivity measured at the 0 time point was subtracted from 8 min time point to determine the rate of initial MTX uptake. RT-PCR analysis of RFC1 RNA levels. WT ZR-75-1 cells were plated at a density of 2 ⫻ 10 6 per 150 mm dish for 72 h prior to the addition of MTX as described above. At the indicated time points, total cellular RNA was extracted from cells using a RNA isolation column supplied by Qiagen (Santa Clarita, CA). Reverse transcription of whole cellular RNA (4 ␮g) was performed by using the Superscript preamplification system (Gibco BRL) according to the manufacturer’s instruction with minor modifications. The reaction was carried out at 45°C for 1 h, 70°C for 15 min and then digested by RNase H for 30 min at 37°C. Amplification of different 5⬘ non-coding exons was performed by using the following primers flanking part of the RFC1 5⬘ cDNA: the downstream common primer in the RFC1 coding region was 5⬘-GTA GGA GGA ATA GGC GAT GCG CG-3⬘. UTR specific primers were: UTR1b, 5⬘-AGC CCC AGG GCA GCC GCC-3⬘; UTR1c, 5⬘-GGC CCT GGG GTG AGT GCG GGG-3⬘; and

UTR1d, 5⬘-TCC AAC ACC GCT ACA GCA GGA AAG-3⬘. The RFC1 coding region upstream primer was 5⬘-AGG CAC AGT GTC ACC TTC GTC CCC-3⬘. The actin primers were 5⬘-CGC ACC TGC ATT GTG ATT GC-3⬘ and 5⬘-TCA TTC CCC CCT TTT TCT GG-3⬘. All RFC1 5⬘ non-coding exons were amplified for 30 cycles using a denaturing temperature of 95°C for 30 s, and an elongation temperature 72°C for 70 s. The annealing time was 30 s and the annealing temperatures were 70°C for UTR1b, 68°C for UTR1c, and 67°C for UTR1d. The RFC1 coding region fragment was amplified for 26 cycles (95°C for 30 s, 68°C for 30 s and 72°C for 60 s). The actin control was amplified for 25 cycles at: 95°C for 30 s, 58°C for 30 s and 72°C for 60 s. All PCR were performed in a Perkin–Elmer 9700 thermocycler (Perkin–Elmer, Foster, CA). Aliquots of the PCRs were size-fractionated on a 1.5% agarose gel in Tris-Borate-EDTA buffer. The gels were stained with SYBRgreen I (FMC Bioproducts, Rockland, ME), visualized under ultraviolet light, and the images were captured with an electronic gel documentation system. Northern blot analysis. Whole cellular RNA (20 ␮g) was size fractionated on a 1% agarose gel in the Mops gel running buffer (Ambion, Austin, TX), transferred to a nylon membrane for 2 h according to the instructions from Ambion. DIG-labeled RFC1 cDNA probe was synthesized using a kit provided by Boehringer-Mannheim as described by the supplier. After an overnight hybridization, the membrane was washed with the washing buffers supplied in NorthernMax kit (Ambion), and detected using a chemiluminescence detection kit supplied by Boehringer-Mannheim (Indianapolis, IN). Western blot. Cell pellets were resuspended in M-PER Mammalian Protein Extraction Reagent (Pierce, Rockford, IL). Protein (15 ␮g) from each cellular lysate was resolved by gel electrophoresis on a 10% ready-made polyacrylamide gel (Bio-Rad) according to the method of Laemmli (21). Proteins were electroblotted onto a nitrocellulose membrane (Bio-Rad) in transfer buffer containing 48 mM Tris, 39 mM glycine, 0.037% SDS and 20% methanol for 1 h. The nitrocellulose membrane was then blocked with a blocking solution (10 mM Tris-buffered saline, 5% nonfat milk, 0.01% Tween 20) for 2 h then incubated with a rabbit polyclonal antibody which had been raised against a hydrophilic region of RFC1 using a peptide sequence leu-phe-phe-asn-arg-asp-asp-arg-gly-arg-cys-glu-thr-ser-ala-ser-gluleu as previously described (14) at a dilution of 1:10,000 for 2 h at room temperature. The membrane was washed with Tris buffered saline containing 0.1% Tween 20 and then incubated with an antirabbit antibody-horseradish peroxidase conjugate (Pierce), washed with Tris buffered saline containing 0.1% Tween 20 again and developed using a chemiluminescence detection kit according to the manufacturer’s instructions (ECL-plus kit, Amersham-Pharmacia, Arlington Heights, IL). Cell cycle analysis. ZR-75-1 cells were treated as described in MTX uptake. At different time points, cells were collected and flow cytometry analysis was performed by the core facility lab for flow cytometry at the University of Kentucky. Promoter–reporter constructs. pGL3-Basic, a promoter-less vector and pGL3-Control, an SV40-driven vector, were obtained from Promega. The constructs pRFC1b and 1c were named according to a numbering system previously described in which nt 1 is located 1005 nt upstream of exon 1a (22). The plasmid pRFC-1d-877 is a 877 bp fragment that covers the entire sequence of exon 1d and the intron between exon 1c and 1d. pRFC-1b-651 and pRFC-1b-518 were generated by restriction digestions. The genomic fragment RFC-1b-651 was inserted between KpnI and SmalI sites of pGL3-Basic vector and the RFC-1b-518 fragment was inserted into the KpnI site. The plasmids pRFC-1c-192 and pRFC-1d-877 were generated by PCR. The fragment RFC-1c-192 was inserted between SacI and NheI sites, and RFC-1d-877 was inserted between XhoI and BglII sites. All constructs were confirmed by restriction digestions and sequencing. ZR-75-1 cells were plated in folate-free RPMI 1640 supplemented with 10% dialyzed FBS and HAT medium for 3 days to deplete folate supply. The cells were then plated at a density of 3 ⫻ 10 5/well in 6

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FIG. 1. The acute effects of incubation of WT ZR-75-1 cells on RFC1 activity. WT ZR-75-1 cells were exposed to 30 nM MTX in HAT-supplemented folate-free medium as described under Materials and Methods. At the indicated time points cells were washed 3 times with folate-free RPMI and then incubated in folate-free RPMI containing 1 ␮M [ 3H] MTX and supplemented with 20 mM Hepes (pH 7.2) for 8 min. The uptake was terminated by aspiration of the transport medium and the plates were washed immediately with ice-cold PBS for three times. Cells were solubilized in 0.2 N NaOH overnight and neutralized by 0.2 N HCl. Protein concentration was determined by the Bradford method. The radioactivity that was bound nonspecifically to the cells was measured at 0 time incubation of the cells in the transport medium. The results shown are the average of 3 experiments.

well plates and transfected using Lipofectamine (Gibco-BRL) the next day. The cells were incubated in medium at the presence or absence of 30 nM MTX 24 h after transfection. Cells were harvested 24 h after MTX exposure, and cell lysate was prepared and luciferase assays were performed according to the manufacturer’s instructions (Promega). Protein concentration was measured by the Bradford method using a kit from Bio-Rad (Hercules, CA). The results were normalized to the protein concentration of the sample and the activity was expressed relative to the activity of pGL3-Control.

are shown in Fig. 1, which shows the rate of initial uptake of 1 ␮M MTX during a time course after exposure to 30 nM MTX. In these studies, RFC function decreased gradually over the first 48 h. The rate of initial MTX uptake after the exposure to MTX decreased to 56% of the pretreatment value after 1 day (from 0.104 to 0.058 pmol/mg/min, and further decreased to 25% of the pretreatment value after 2 days (0.026 pmol/mg/min). By the third day, the rate of initial MTX uptake was reduced to 22% of the pretreatment value (0.023 pmol/mg/min). To confirm that the decrease in RFC activity was associated with a change in RFC1 protein expression, we performed Western blot analysis on cells exposed to the same time course of MTX exposure in HATsupplemented folate-free medium. As can be seen in Fig. 2, a polyclonal antibody raised against an RFC1 peptide showed a detectable decrease in RFC1 protein by 72 h, indicating that the decrease in MTX uptake observed is a result of decreased expression of the RFC1 gene product. We then looked at whether the decrease in RFC1 protein expression was correlated with a change in RFC1 RNA levels. The effect of MTX preexposure was determined both in standard medium, which contains 1 ␮M folic acid (Figs. 3A and 3B) and in HATsupplemented folate-free medium as in the previous studies (Fig. 3C). RT-PCR of RFC1 RNA (Fig. 3A) and Northern blot analysis (Figs. 3B and 3C) showed a decrease in RFC1 RNA levels in ZR-75-1 cells after MTX preexposure, regardless of whether the medium contains folic acid. It appears that RFC1 RNA levels decreased faster than protein and function. The lag of functional activity behind the RNA levels may be the consequence of a relatively long half-life of the RFC1 protein. Densitometry analysis of the RT-PCR in Fig. 3A demonstrates that RFC1 RNA levels decreased to approximately 30% of the pretreatment value (when normalized to actin) after only one day of exposure to a

RESULTS To determine whether MTX-pre-exposure could influence RFC activity, we incubated WT ZR-75-1 cells in non-toxic concentrations (30 nM) of MTX in HATsupplemented folate-free medium. In these studies, it was important to use concentrations of MTX that would not affect cell growth in order to prevent changes in RFC1 RNA levels that would be secondary to the effects of toxic drug concentrations. Jansen et al. (22) have shown that alteration in exogenous folate level could affect RFC1 mediated [ 3H]MTX transport. Therefore, we depleted the cells of folate before exposing them to MTX. We first determined whether RFC1 activity changed after exposure to MTX. These results

FIG. 2. Western blot for RFC1 protein during incubation of WT ZR-75-1 cells in MTX (30 nM) using a polyclonal anti-RFC1 antibody with an actin control. MTX R ZR-75-1 cells transfected with an expression vector for RFC1 were used for a positive control, and nontransfected MTX R ZR-75-1 cells which do not express detectable levels of RFC1 were used as a negative control for RFC1 expression.

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FIG. 3. The acute effects of incubation of WT ZR-75-1 cells on RFC1 RNA levels. WT ZR-75-1 cells were plated in HAT-supplemented folate-free RPMI 1640 containing 10% dialyzed FBS for 72 h. Cells were then exposed to 30 nM MTX in either standard medium (A and B) or in HAT-supplemented folate-free medium (C). Cells were then collected for examination at the indicated time points. A and B represent independent experiments. (A) RNA was isolated from cells harvested from cells at the indicated time points and analyzed with the RT-PCR assay using primers for the RFC1 ORF and actin. The PCR assay shown is one of two independent experiments with nearly identical results. (B) WT ZR-75-1 breast cancer cells exposed to 30 nM MTX in standard medium as described above were analyzed for RFC1 RNA levels by Northern blot. (C) WT ZR-75-1 breast cancer cells exposed to 30 nM MTX in HAT-supplemented folate-free medium as described under Materials and Methods. The results of densitometric analysis of these figures are discussed in the text.

sublethal (30 nM) concentration of MTX in the presence of 1 ␮M folic acid. These low RNA levels remain constant over the next 48 h. In Fig. 3B, densitometric analysis of this Northern demonstrates that the RFC1 RNA levels 24 h after MTX exposure decreased to 9% of hour 0 level when controlled for the expression of actin RNA. In Fig. 3C, densitometry analysis demonstrates that, 24 h after exposure to MTX in HATsupplemented folate-free medium, the level of RFC1 RNA had decreased to only 21% of the hour 0 level, when normalized to the expression of actin RNA. We have previously shown that RFC1 RNA levels are cell cycle regulated in immortalized breast epithelial cells, with peak levels occurring near the G1 to S transition (22). Therefore, we wanted to determine whether the decrease in RFC1 RNA was an indirect consequence of alteration of the cell cycle distribution after MTX exposure. By flow cytometry analysis, however, there was no significant difference in the cell cycle distribution 24 h after the addition of low-dose MTX (Table 1). Therefore, it appears that the apparent decrease in RFC1 RNA levels and protein expression after exposure to low-dose MTX does not result from a change in cell cycle distribution.

The RFC1 RNA transcript may contain one of four 5⬘ noncoding exons (22–24). These exons span 2.9 kb on the genome and are spliced to the same acceptor site upstream from the translation start site (22). RFC1 transcripts containing three of these four exons (1b, 1c, and 1d) are found in ZR-75-1 cells. Exons 1b and 1c are in very close proximity to each other, separated by only 19 nucleotides (nt), suggesting that transcription of these two exons may be under control of the same promoter. Exon 1d is located 445 nt downstream from the splice donor site of exon 1c. To determine if there was selectivity in the decrease in the level of transcripts containing each of the three 5⬘ exons expressed in these cells, we used an RT-PCR assay to look at transcripts containing each of the upstream exons (22). As shown in Fig. 4, there was a dramatic decrease in the level of RNA transcripts containing each of the 5⬘ exons after 3 days of exposure to low-dose MTX. These results suggest that there may be a common element controlling transcription of each of these exons. We therefore examined the activity of RFC1 promoter-reporter constructs using 5⬘ flanking sequences of exons 1b, 1c, and 1d. We performed the experiment using a luciferase reporter, normalized the results to the pGLControl vector treated under identi-

TABLE 1

Cell Cycle Distribution after Exposure to Low-Dose MTX % Total cells WT ZR-75-1 cells after exposure to 30 nM MTX In medium containing folic acid (Figs. 3A and 3B) 0h 24 h In HAT-supplemented folate-free medium (Fig. 3C) 0h 24 h

G0–G1

G2–M

S

50 50

11 14

39 37

49 45

10 14

41 41

FIG. 4. WT ZR-75-1 human breast cancer cells incubated in HAT-supplemented folate-free medium which either contained MTX (25 nM) or did not contain MTX. Cells were harvested after 3 days and the RFC1 5⬘ UTR-specific RT-PCR assay was performed as described under Materials and Methods.

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FIG. 5. Activity of RFC1 promoter–reporter constructs in WT ZR-75-1 cells after exposure to 30 nM MTX in HAT-supplemented folate-free medium as described under Materials and Methods. The cells were preincubated in HAT-supplemented folate-free medium for 3 days and then transiently transfected with pGL3-based vectors containing RFC1 genomic fragments upstream of exons 1b, 1c, and 1d. After exposing the cells to 30 nM MTX for 24 h, the cells were harvested and luciferase assay was performed. The numbering system for the RFC1 genomic fragments starts 1005 nt upstream of exon 1a as described under Materials and Methods. pRFC-1d-877 is a 877 bp fragment that includes exon 1d and its 5⬘ flanking region sequence up to the 3⬘ end of exon 1c. The results were normalized by protein added and the activity of pGL3-Control.

cal conditions, and expressed the results as the percent of promoter activity relative to the control condition. As can be seen in Fig. 5, we found a modest and consistent decrease of promoter-reporter activity of approximately 60% of the control values in vectors incorporating the 5⬘ flanking sequences of exons 1b, 1c, and 1d in three of four constructs, and a decrease to 80% of control values in the remaining construct. The changes in RFC1 promoter activity after MTX exposure, together with the decrease in RFC1 RNA levels, suggest that the decrease in RFC1 activity after MTX exposure partially results from transcriptional down-regulation of RFC1. Further studies are needed to define the relevant regulatory regions of the RFC1 promoter. We also investigated the possibility that methylation of the RFC1 promoter might be a mechanism of RFC1 down-regulation after MTX exposure. ZR-75-1 cells exposed to MTX were co-incubated with 5-azacytidine, a methylation inhibitor. If methylation was a mechanism of down-regulation of RFC1, then 5-azacytidine would have been expected to prevent the decrease in MTX uptake. However, incubation of ZR-75-1 cells in 1 ␮M 5-azacytidine alone decreased the rate of initial MTX uptake to 13% of the control value by 72 h, and incubation of the cells in 30 nM MTX in combination with 1 ␮M 5-azacytidine completely eliminated specific MTX uptake at 72 h (data not shown). Therefore, there was no evidence that methylation plays a role in the immediate down-regulation of RFC1 after MTX exposure. Analysis of cell cycle data from

this experiment indicated that 5-azacytdine exposure resulted in a block at the end of G2-M, suggesting that the observed decreased MTX uptake after 5-azacytidine exposure was the result of altered cell cycle distribution. DISCUSSION These studies of ZR-75-1 breast cancer cells exposed to low-dose MTX demonstrate that MTX pre-exposure can result in down-regulation of RFC1 RNA levels as well as RFC1 protein expression and activity. This phenomenon was observed in an experimental model of RFC1 gene regulation utilizing a cell line exposed to a non-cytotoxic dose of MTX in the presence of hypoxanthine, adenosine and thymidine, so that the effect of MTX could be determined independently of potential non-specific toxic effects of the drug. There are important practical implications of the autoregulation of uptake of folates and antifolates. Both the efficacy and toxicity of MTX therapy could be affected by the relative propensity of different types of cells to down-regulate RFC1 expression after exposure to MTX. With chronic low-dose schedules of MTX administration, cancer cells with decreased RFC1 expression after MTX exposure may be less sensitive to the regimen, while normal cells that do not down-regulate RFC1 activity may have increased toxicity to the regimen. In high-dose schedules of MTX administration with LCV rescue, cells that down-regulate RFC1 after

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MTX exposure may be less capable of being rescued by leucovorin, while cells that resist RFC1 downregulation may be more efficiently rescued. Thus, differences in the acute regulation of RFC1 may be important in determining the sensitivity of tumors to both low-dose and high-dose MTX therapy. It is important to note that studies which measure constitutive RFC1 RNA levels or activity would not detect differences in the ability of cells to acutely respond to MTX exposure. Nevertheless, clinical studies have suggested a relation between constitutive RFC1 activity and prognosis. Whitehead et al. reported children whose blasts took up ⬎100 pmol MTX and formed ⬎500 pmol MTX polyglutamates/10 9 cells had a 5-year event-free survival (EFS) of 65% vs and EFS of 22% for those children whose ALL blasts took up and polyglutamylated lesser amounts of MTX (25). Bertino and colleagues have shown evidence that defective uptake is an important mechanism of clinical drug resistance in acute lymphoblastic leukemia (26). In this study, 13% of patients (4/30) had evidence of impaired MTX uptake at the time of diagnosis, but 70% of patients (25/35) showed impaired MTX uptake at the time of relapse. Furthermore, the decrease in MTX uptake was correlated with a decrease in RFC1 RNA levels, as 6 of 9 specimens with decreased uptake showed a decrease in RFC1 RNA levels, but none of 15 specimens with no change in MTX uptake showed any change in RFC1 RNA levels (26). Zhang et al. have recently shown a relationship between MTX uptake and RFC1 RNA levels in B-precursor ALL blasts as measured by a quantitative RT-PCR assay, suggesting that RFC1 RNA levels might predict clinical response to MTX (27). We have also examined three pairs of RNA specimens from leukemic blasts of children with pre-B cell acute lymphoblastic leukemia by using an RT-PCR assay for detecting RFC1 RNA levels. In two of three patients there was a marked decrease in RFC1 expression from the time of diagnosis compared to the relapse specimens. In the third patient there was no detectable RFC1 RNA at the time of diagnosis or at the time of relapse (28). In addition, a series of osteosarcoma tumors examined for RFC1 RNA levels revealed that decreased constitutive RFC1 RNA levels also correlated with poor response in osteosarcoma (29). Among 42 patients treated with a multi-agent chemotherapy regimen that included highdose MTX, the likelihood of decreased RFC1 RNA levels among poor responders was 65%, compared to only 36% among those patients with a good response to chemotherapy (29). Our studies suggest that constitutive levels of RFC1 expression may be only part of the story of transportmediated MTX resistance. Another group has also found evidence for acute regulation of RFC activity by folate compounds. Jansen, Assaraf and colleagues have previously described the acute effects of folate and

purine exposure on MTX uptake in a CEM leukemia subline which overexpresses RFC1 and which is maintained in a subnanomolar (0.25 nM) concentration of leucovorin (LCV) (30). Preincubation of these cells in 25 nM LCV resulted in significant down-regulation of MTX uptake within 1 h of exposure, an effect inhibited by trimetrexate (TMTX). A similar acute downregulation of RFC1 activity was achieved by preincubation in adenosine or S-adenosylmethionine (SAM), but the effects of these compounds were not reversed by TMTX (30). It is interesting to note that the effects of adenosine and SAM were transient (15–18 h) but the effect of LCV was much longer (⬎3 days), suggesting these observations may have resulted from both an acute effect of intracellular folate pools and a longerterm effect on gene expression. However, unlike the breast cancer cells exposed to MTX observed in our model, RFC1 RNA levels in the CEM cells exposed to LCV reportedly did not change as a result of the preincubations (30). Several groups (22–24) have identified RFC1 mRNA heterogeneity at its 5⬘ end, which suggests that the expression of this gene is controlled by multiple promoters. In our study, after acute exposure to low-dose of MTX, the levels of RFC1 RNA transcripts containing each of the three 5⬘ UTRs were decreased. Furthermore, promoter–reporter assays confirmed that the decrease in promoter activity after MTX exposure was similar among the three different promoter constructs. The difference in magnitude between changes in the levels of RNA transcripts and the degree of change in promoter-reporter construct activity may have been the result of inefficiencies inherent in transient transcription assay systems. However, these studies suggest that there may be common elements regulating the different RFC1 promoters after acute MTX exposure. Methylation has been known to be the mechanism of gene regulation in human multidrug resistant gene (MDR1) (31). The RFC1 promoter region has multiple CpG islands between exons 1 and exon 1b which are the potential sites for methylation (data not shown). In addition, folic acid metabolism is important in the synthesis of S-adenosylmethionine, a substrate for the DNA methylation reaction. Therefore, we explored the possibility that MTX exposure resulted in methylation of the RFC1 promoter. We hypothesized that exposing the ZR-75-1 cells to 5-azacytidine, a drug that inhibits methylation, would increase MTX uptake after exposure to MTX. However, our studies showed that 5⬘azacytidine resulted in a significant, further decrease in MTX uptake, probably due to its effect on the cell cycle of ZR-75-1 cells. Our previous studies have shown that RFC1 RNA levels peak near the G1–S boundary (22), whereas 5-azacytidine appeared to cause these cells to pile-up at the G2–M boundary.

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Reduced transport is one of multiple potential mechanisms of MTX resistance. Our observation that MTX can autoregulate its own uptake, together with previous studies which have demonstrated that RFC1 is a cell-cycle regulated gene (22), provide the rationale for further study of the regulation of RFC1. Acute, epigenetic responses to stresses may precede the more durable genetic changes that are detected in clinical and laboratory studies after drug resistance has been established. Rather than simply examine constitutive levels of RFC1 gene expression and activity, it may ultimately be more informative to examine pathways that control cell cycle regulation and the acute changes to MTX exposure to determine how cancer cells initially learn to adapt to MTX chemotherapy. These studies would give us insight into the initiation of transport-mediated resistance to MTX. REFERENCES 1. Pizzo, P., and Poplack, D. (1989) Principles and Practice of Pediatric Oncology, Lippincott, New York. 2. Vendetti, J., and Goldin, A. (1964) Cancer Res. 24, 1457–1460. 3. Acute, B. G. L. (1965) JAMA 194, 187–193. 4. Dahl, M., Gregor, M., and Scheuer, P. (1972) Br. J. Med. 1, 654 – 656. 5. Sirotnak, F. M., Kurita, F. M., and Hutchison, D. J. (1968) Cancer Res. 28, 75– 80. 6. Fisher, G. A. (1960) Biochem. Pharmacol. 11, 1233–1234. 7. Goldman, I. D., Lichtenstein, N. S., and Oliverio, V. T. (1968) J. Biol. Chem. 243, 5007–5017. 8. Henderson, G. B., and Zevely, E. M. (1984) J. Biol. Chem. 259, 1526 –1531. 9. Flintoff, W. F., and Essani, K. (1980) Biochemistry 18, 4321– 4327. 10. Ohnuma, T., Lo, R. J., Scanlon, K. J., Kamen, B. A., Ohnoshi, T., Wolman, S. R., and Holland, J. F. (1985) Cancer Res. 45, 1815– 1822. 11. Cowan, K. H., and Jolivet, J. (1984) J. Biol. Chem. 259, 10793– 10800. 12. Dixon, K. H., Trepel, J. B., Eng, S. C., and Cowan, K. H. (1991) Cancer Commun. 3, 357–365.

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