©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Isolation of a Human cDNA That Complements a Mutant Hamster Cell Defective in Methotrexate Uptake (*)

(Received for publication, October 13, 1994; and in revised form, November 29, 1994)

Frederick M. R. Williams Wayne F. Flintoff (§)

From the Department of Microbiology and Immunology, University of Western Ontario, London, Ontario N6A 5C1, Canada

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

A clone has been isolated from a human lymphoblastic cDNA expression library that complements a mutant Chinese hamster cell defective in the uptake of the folate analogue methotrexate. When transfected with this clone the mutant cells regain the ability to transport the drug and, as a consequence, become sensitive to its cytotoxic action.

The clone is 2863 base pairs long and has an open reading frame of 1770 base pairs that codes for a putative protein of 64 kDa. The putative protein has 51 and 50% identity at the amino acid level with the mouse and hamster functions, respectively, involved in the transport of reduced folates. Together these three proteins share 47% identity and have similar predicted structural features.

The data are consistent with this human clone encoding either the reduced folate transporter or an auxiliary function that interacts with this transporter.


INTRODUCTION

Folates are essential components required for several metabolic pathways involving the biosynthesis of amino acids, purines, and pyrimidines. These compounds cannot be synthesized by eukaryotic cells and thus must be obtained from extracellular sources.

Studies in various systems have identified two transport systems whereby folic acid and its reduced forms including the chemotherapeutic agent methotrexate (Mtx) (^1)enter cells. One system consists of a membrane-bound folate receptor, termed the folate-binding protein, which is a 40-kDa glycoprotein. It has a high affinity for folates, including folic acid and a low affinity for Mtx (1, 2, 3, 4, 5, 6, 7, 8) . It is present in both human and mouse cells and its cDNA has been cloned from several sources(9, 10, 11, 12, 13, 14) .

The second transport system, called the reduced folate carrier, has been identified in mouse(15, 16, 17) , human(18, 19, 20) , and hamster (21) cells. It has an apparent mass ranging from 36 to 78 kDa(16, 20, 22, 23) , is heavily glycosylated in human cells(23, 24) , and shows a preference for binding and transporting reduced folates and Mtx over folic acid.

In some cell systems, both transport systems are present and may interact in tandem to allow the transfer of folates across the membrane (25) . Other studies indicate that the two systems act independently (26, 27) .

In order to develop a better understanding of the folate transport process, we have previously isolated a series of Chinese hamster ovary (CHO) cell lines that are resistant to the cytotoxic action of Mtx because of an inability to take-up the drug(21, 28) . Such mutant cells require higher levels of reduced folates for growth as compared with wild-type cells(29) . This provided a selection scheme whereby we were able to obtain both a genomic DNA fragment (30) and a cDNA clone (pMtxT9) (31) which when introduced into the mutant cells could complement the phenotype. When the cDNA was transfected into mutant cells which did not express an mRNA for this gene, it conferred the ability to grow on low levels of folinic acid as a source of folates, to bind and take up Mtx, and as a consequence become sensitive to the drug's cytotoxic action(31) . The properties of the putative protein encoded by this cDNA are consistent with it being the reduced folate carrier. A function with a high degree of similarity to the hamster gene has also been cloned from mouse cells(32) .

In this report, we describe the isolation of a human cDNA homologous to the hamster gene using the latter as a molecular probe. The nucleotide sequence and predicted amino acid sequence of the putative encoded protein are similar to both the respective sequences from hamster and mouse, although the human protein is larger in its predicted size. When transfected into the mutant CHO cells, the human cDNA is able to complement the Mtx-resistant phenotype and restore wild-type phenotypic properties to the cells. This indicates that the hamster and human proteins are functionally similar.


MATERIALS AND METHODS

Chemicals

Unlabeled Mtx was purchased from Sigma, N^5-formyltetrahydrofolate (folinic acid) from ICN Biomedicals, and polybrene from Aldrich. [3`,5`,7`-^3H]Mtx (20 Ci/mmol) was purchased from Amersham Corp. Immediately prior to use, it was purified by thin-layer chromatography as described previously(21) . [P]dCTP (3000 Ci/mmol) was purchased from ICN Biomedicals. Restriction endonucleases were obtained from Pharmacia Biotech Inc. All other reagents were obtained in the highest purity available from various commercial sources.

Cells and Cell Culture

Wild-type and mutant Mtx-resistant (MtxRII 5-3) CHO cells (21, 28) and human Hep G2 and normal female primary fibroblasts were maintained in monolayer cultures in alpha-medium supplemented with 10% fetal bovine serum as described previously(21, 28) . For transfection studies, the CHO cells were maintained under selective pressure in folic acid-free medium (selective medium) with 10% dialyzed fetal bovine serum and 2 nM folinic acid.

Phenotype Testing

The resistance of various cell lines to Mtx was determined by dose-response curves as described previously (28) .

Mtx Uptake

The time-dependent intracellular accumulation of 0.4 µM [^3H]Mtx into various cell lines was determined on monolayer cultures as described previously(29) .

DNA Transfection

Transfection of plasmid DNA into the recipient MtxRII 5-3 cells was carried out using 10 µg of purified DNA/2 times 10^5 cells as described previously(30, 31) . After transfection with DNA, cells able to grow in low levels of folinic acid (2 nM) were selected by plating in selective medium as described previously(30, 31) .

DNA Isolation

Plasmid clones were propagated in LB medium supplemented with ampicillin. DNA was isolated from overnight cultures by the Qiagen procedure as described by the supplier. In some cases, the insert from plasmid DNAs for probe preparation was purified from agarose gels by the Geneclean procedure (BIO 101, Inc.).

RNA Isolation

Poly(A) RNA was isolated from approximately 10^7 exponentially growing cells using the Quick Prep® Micro mRNA purification kit supplied by Pharmacia.

Northern Blotting

The analysis of 5 µg of poly(A) RNA from the various cell lines was carried out as described previously(31) .

Probe Labeling

DNA for probes was labeled using [P]dCTP by the random priming method described by Feinberg and Vogelstein (34) using either purified DNA fragments or DNA in low melting point agarose. Routinely, labeled DNA was obtained at a specific activity of 5-10 times 10^8 cpm/µg.

cDNA Expression Library and Screening

A human cDNA expression library was obtained from Dr. M. Buchwald, Hospital for Sick Children, Toronto. The cDNA was prepared from RNA isolated from the human lymphoblastic cell line HSC93 and cloned into the pREP4 vector in which the cloned sequences are expressed from the Rous sarcoma virus long terminal repeat promoter(35) .

For screening, approximately 2 times 10^5 colony-forming units were plated, transferred to Biotrans membranes, processed according to the directions supplied by the manufacturer, and hybridized with a radiolabeled 2.5-kb DNA fragment from the plasmid pMtxT9, which contains the hamster gene for Mtx transport(31) , using the hybridization conditions described previously(33) .

DNA Sequencing

For DNA sequencing, the BamHI fragment containing the insert in plasmid pHuMtxT4 was subcloned into pGEM3. Overlapping deletion clones were obtained by exonuclease III digestions using the double-stranded nested deletion kit from Pharmacia. Double-stranded DNA sequencing was performed by the dideoxy chain termination method (36) using the T7 Sequencing(TM) kit supplied by Pharmacia with either T7 or SP6 primers or synthetic 20-mer primers derived from the sequence of various clones.

Sequence information was obtained from clones sequenced in both directions and/or from sequences of multiple clones covering the same regions such that the sequences obtained were unambiguously determined.

Computer Analyses

Computer analyses of the nucleotide and predicted amino acid sequences were performed using the Genetics Computer Group sequence analysis software package(37) .


RESULTS

Isolation of cDNA Clones

Since the systems involved in the transport of folates are expressed in a wide variety of cells, this raised the possibility of isolating the human homologue to the hamster function that is involved in the transport of Mtx. In preliminary experiments, Southern blotting analysis using hybridization conditions under reduced stringency indicated that the hamster cDNA could detect human DNA sequences. Thus, if the DNA were expressed into RNA, then it should be possible to isolate the human counterpart from a cDNA library.

To identify cDNA clones, approximately 2 times 10^5 recombinant colony-forming units from the human lymphoblast library were screened. Five clones giving strong hybridization signals were isolated and designated as pHuMtxT1 to T5. The inserts from these clones could be removed intact by digestion of the DNA with BamHI and were shown to have approximate sizes of 4.3, 2.0, 2.5, 2.6, and 2.5 kb for the clones T1 to T5, respectively.

To determine whether any of these clones could complement mutant CHO cells, DNA from each plasmid clone was transfected into the mutant cells (MtxRII 5-3) which are Mtx-resistant because of defective drug uptake and lack the message for the gene involved in the transport of the drug(31) . The resulting transfectants were selected for growth in a low level of folinic acid. As shown in Table 1, DNA from plasmids pHuMtxT3, pHuMtxT4, and pHuMtxT5 were able to complement the mutant CHO phenotype to permit growth in 2 nM folinic acid. The frequency of colonies recovered with these three plasmid DNAs was similar to or higher than that obtained with the DNA from the plasmid containing the hamster gene (pMtxT9). Several transfectants tested were sensitive to Mtx. This indicates that these three plasmids contained full-length copies of a human cDNA capable of correcting the defect in hamster cells and consequently rendering them sensitive to Mtx.



Since plasmids pHuMtxT1 and pHuMtxT2 did not complement the mutant phenotype, no further analysis was carried out on these clones.

Drug Uptake

Since the transfectants described above had regained the wild-type sensitivity to Mtx, it was important to determine that these cells had regained the ability to take up the drug. As shown in Fig. 1, a representative transfectant obtained from each of the plasmid transfections was able to accumulate Mtx. At an extracellular concentration of 0.4 µM, one transfectant accumulated the drug in a similar manner as the wild-type CHO cells, whereas the other transfectants accumulated slightly more. These results indicate that the human cDNA sequences in the plasmid were able to function in the CHO cell and restore the ability of these mutant cells to accumulate Mtx.


Figure 1: Cellular uptake of 0.4 µM [^3H]Mtx by various cell lines. Cells were incubated at 37 °C and [^3H]Mtx uptake was measured as described under ``Materials and Methods.'' circle, wild-type CHO; bullet, mutant CHO MtxRII 5-3; , MtxRII 5-3 transfected with pHuMtxT3 DNA; box, MtxRII 5-3 transfected with pHuMtxT4 DNA; , MtxRII 5-3 transfected with pHuMtxT5 DNA; up triangle, MtxRII 5-3 transfected with pMtxT9 DNA.



Northern Analysis

Northern analysis was carried out with poly(A) RNA isolated from the various cell lines to determine the nature of the transcripts in human and transfected CHO cells detected by the sequences encoded by the plasmids. For these studies the 2.5-kb BamHI fragment from pHuMtxT5 was used as probe. As shown in Fig. 2, a major transcript of 2.8 kb was detected in the RNAs from the human fibroblasts, Hep G2, and the CHO cells transfected with the two plasmid DNAs containing the human cDNA sequences. In the original autoradiogram, a faint band of 3.7 kb was also visible in the RNAs from the two human cell lines. The transfectant generated using DNA from plasmid pHuMtxT4 also produced a message at 5.2 kb. This different size message may represent the effect of a site of integration of the plasmid and its effect on the transcription of the transfected gene.


Figure 2: Autoradiogram of Northern hybridizations. A, approximately 5 µg of poly(A) RNA isolated from human Hep G2 (lane 1), human fibroblasts (lane 2), CHO line MtxRII 5-3 (lane 3), CHO line MtxRII 5-3 transfected with plasmid pHuMtxT3 DNA (lane 4), and CHO line MtxRII 5-3 transfected with plasmid pHuMtxT4 DNA (lane 5) cells were separated on agarose gels, blotted, and hybridized with the 2.5-kb BamHI fragment from pHuMtxT5 as described under ``Materials and Methods.'' RNA molecular mass markers (in kilobases) from Life Technologies, Inc. are shown. B, the blot in A was stripped and rehybridized with radiolabeled hamster dihydrofolate reductase cDNA.



As shown in B in Fig. 2, under these hybridization conditions, the hamster dihydrofolate reductase cDNA does not detect the human dihydrofolate reductase homologue.

Nucleotide Sequence and Predicted Amino Acid Sequence

Since pHuMtxT4 contained the largest insert and was capable of complementing the CHO mutant phenotype, it was chosen for DNA sequence analysis. The complete nucleotide sequence of the human cDNA in this plasmid is shown in Fig. 3. The 2863-base pair pHuMtxT4 clone contains a 1770-base pair open reading frame that is flanked by 94- and 999-nucleotide 5`- and 3`-noncoding regions, respectively. The translation start site is assigned position 1 according to its similarity to the eukaryotic consensus start sequence as described by Kozak(38) . The termination signal TGA is at position 1768 and a putative polyadenylation signal at position 2692.


Figure 3: Nucleotide and predicted amino acid sequence of pHuMtxT4. The nucleotide sequence is numbered on the left, and the amino acid sequence is numbered on the right. The putative Kozak sequence (38) at position 1 and the putative polyadenylation signal at position 2692 are underlined. Consensus site for N-linked glycosylation is marked with a +.



Sequence analysis of clones pHuMtxT3 and pHuMtxT5, which can also complement the mutant CHO phenotype, indicate that they possess identical sequences to pHuMtxT4 in the 5`-noncoding and coding region but are truncated in the 3`-noncoding region (data not shown).

The deduced amino acid composition of the putative protein encoded by pHuMtxT4 is shown in Fig. 3. The open reading frame contains 589 amino acids, the predicted molecular mass is 63,954 Da, and the pI is 9.12. A consensus site for N-linked glycosylation occurs at amino acid residue 58. An amino acid hydropathy plot using the criteria of Hopp and Woods (39) (Fig. 4) indicates that a large part of the molecule is hydrophobic, with hydrophilic regions near both the NH(2) and COOH termini as well as in the middle of the molecule. Secondary structure predictions according to the criteria of Chou and Fasman (40) indicate that a majority of the molecule conforms to beta-sheets. These predicted indices for the putative human protein are similar to those of the hamster protein(31) .


Figure 4: Hydropathy plot and predicted secondary structure of the putative protein encoded by pHuMtxT4. A, Hopp and Woods (39) analysis of the deduced amino acid sequences using a window size of 6 is shown. B, the secondary structure predictions using the criteria of Chou and Fasman (40) is indicated.



Data Base Searches

Searches were made in the current nucleotide and protein data bases at the National Center for Biotechnology Information using the BLAST network service. Significant matches were found with the hamster (31) and mouse (32) functions involved in the transport of reduced folates. At the nucleotide level, the putative coding region of the human cDNA has 57 and 56% identity with the coding regions for the mouse and hamster cDNAs, respectively. At the amino acid level, the putative human protein has 51 and 50% identity with the similar protein from mouse and hamster, respectively. Together these three predicted proteins have 47% identity at the amino acid level (Fig. 5). These regions of identity occur over several stretches scattered throughout the protein. The longer stretches, up to 26 amino acid residues, appear for the most part to be in hydrophobic regions and thus probably buried in the cell membrane.


Figure 5: Comparison of the amino acid sequences for the mouse, hamster, and human cDNAs. The numbers refer to the amino acid sequences in the predicted coding region for the human gene as contained in pHuMtxT4. The dots indicate spaces in order to maximize the homology. The boxed areas indicate regions of identity.




DISCUSSION

Using a hamster cDNA clone as a molecular probe for a function involved in the transport of reduced folates, we have been able to isolate the corresponding human cDNA from a lymphoblastic cDNA expression library. This human cDNA codes for a putative hydrophobic protein with an apparent molecular mass of 64 kDa and with considerable predicted beta-sheet structure.

The nucleotide sequence of this cDNA and the predicted amino acid sequence of the putative encoded protein show a high degree of similarity and identity to both the hamster and the mouse counterparts that have been shown to be involved in the uptake of reduced folates (31, 32) . The putative human protein, however, is larger than either the mouse or hamster proteins which both have predicted sizes of 52 kDa (31, 32) . The human protein appears to have a majority of its additional amino acid residues at the COOH-terminal end. Changes may occur post-translationally and it will be of interest to determine the sizes of the proteins present in the cells.

These proteins are also functionally similar. Transfection of mutant CHO cells, lacking the message for this function, with the human cDNA clones, restores wild-type properties to these cells. They take up the drug and thus become sensitive to its cytotoxic action. This is the same result obtained when these cells were transfected with the homologous hamster cDNA clone(31) . This implies that this function is conserved between hamster and human.

At present the exact role that similar proteins from hamster, mouse, and human cells play in folate transport is not clear. The data are consistent that these putative proteins are the reduced folate transporters from the three species. CHO cells do not appear to express the folate-binding protein component of folate transport (41, 42) and obtain folates via the reduced folate transporter. Mutant CHO cells, which are defective in this transport, when transfected with either the hamster cDNA (31) or the human cDNAs, regain the ability to transport Mtx. Similarly, the mouse cDNA can restore reduced folate carrier activity to human cells that are defective in this mode of folate transport(32) . It is also possible that these cloned functions are not the reduced folate carrier but rather an additional component that interacts with it to facilitate the transport of Mtx. Clearly, further biochemical studies are required to distinguish between these alternatives.

The reduced folate transporter from human cells is extensively glycosylated containing both N- and O-linked oligosaccharides(23, 24) . In its N-deglycosylated form in human K562 cells it has an apparent molecular mass of 68 kDa(23) . The similar protein from human CCRF-CEM cells when grown in the presence of tunicamycin and subsequently treated with N-glycanase had a molecular mass of 50 kDa(24) . The predicted size of the protein encoded by pHuMtxT4 is 64 kDa. These differences may be due in part to processing and additional post-translational modifications. It is of interest to note that the putative protein has a single consensus site for N-linked glycosylation at residue 58 which appears to be in an exposed region of the protein.

The hydropathy and secondary structure prediction plots for both the hamster and human putative proteins are similar. Both proteins contain stretches of hydrophobic amino acids, suggesting that these regions are buried in the membrane. At present it is too early to estimate the number and extent of the transmembrane regions until more is know about the protein in its mature, native form in the cell. A structural comparison has been made between the presumed mouse reduced folate carrier and the human GLUT1 glucose transporter(32) . These two proteins showed remarkable similarity and 12 potential transmembrane regions were identified in the mouse reduced folate carrier. The structure description of the GLUT1 transporter was based on the idea that the transmembrane regions formed alpha-helices (43) and recent work supports this(44) . Other work, however, suggests that these regions may form beta-barrels(45) . This latter observation would be consistent with the predicted structure of the hamster and human putative reduced folate carrier proteins described here in which a majority of the hydrophobic regions have a predicted beta-sheet structure.

The availability of a human cDNA for a function involved in the transport of Mtx will allow an investigation of the role that this gene product may have in the development of clinical resistance to this important chemotherapeutic agent.


FOOTNOTES

*
This work was supported by grants from the Cancer Research Society and the Medical Research Council of Canada (to W. F. F.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) U17566[GenBank].

§
To whom correspondence and reprint requests should be addressed. Tel.: 519-661-3438; Fax: 519-661-3499.

(^1)
The abbreviations used are: Mtx, methotrexate; CHO, Chinese hamster ovary; kb, kilobases.


ACKNOWLEDGEMENTS

We thank R. C. Murray for helpful discussions and Dr. M. Buchwald for the cDNA library.


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