©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Isolation of Human cDNAs That Restore Methotrexate Sensitivity and Reduced Folate Carrier Activity in Methotrexate Transport-defective Chinese Hamster Ovary Cells (*)

(Received for publication, January 18, 1995; and in revised form, April 7, 1995)

So C. Wong (1), Susan A. Proefke (1), Alok Bhushan (3), Larry H. Matherly (1) (2)(§)

From the  (1)Developmental Therapeutics Program, Michigan Cancer Foundation and the (2)Department of Pharmacology, Wayne State University, School of Medicine, Detroit, Michigan 48201 and the (3)Department of Pharmacology and Vermont Cancer Center, University of Vermont, Burlington, Vermont 05405

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

This report describes the isolation, nucleotide sequencing, and functional expression of human cDNAs that restore reduced folate carrier activity in transport-defective cells. Based on homology to a partial murine cDNA probe, two functional cDNAs were isolated from a gt11 library prepared from methotrexate transport up-regulated K562 cells (K562.4CF). A 2.8-kilobase (kb) clone, KS43, contained a 1776-base pair open reading frame. The 2.5-kb clone, KS32, contained an internal deletion (626 base pairs) resulting a shortened open reading frame and 3`-untranslated region. KS43 and KS32 encoded proteins with multiple hydrophobic domains, one consensus N-glycosylation site, and predicted molecular masses of 65 and 58 kDa, respectively. The deduced amino acid sequence of KS43 is 79% and 80% homologous to the mouse and hamster sequences, respectively (Dixon, K. H., Lanpher, B. C., Chiu, J., Kelley, K., and Cowan, K. H.(1994) J. Biol. Chem. 269, 17-20; Williams, F. M. R., Murray, R. C., Underhill, T. M., and Flintoff, W. F.(1994) J. Biol. Chem. 269, 5810-5816). Northern blots identified one primary transcript at 3.1 kb in parental K562, K562.4CF, and transport-impaired K500E cells; transcript levels varied by 7-fold.

The expression of both KS43 and KS32 in methotrexate transport-defective Chinese hamster ovary cells restored methotrexate sensitivity and transport. Certain transport characteristics of the transfected cells resembled both the wild type human (K562) and hamster ``classical'' reduced folate carriers, suggesting the expression of a hybrid system. For instance, based on K values, up to a 4-fold increased affinity for 1843U89 over wild type hamster cells (typical of human cells), and a 19-fold increased affinity for methotrexate over K562 cells (typical of hamster cells) was observed. Further, a photoaffinity probe with high specificity for the reduced folate carrier labeled 94-kDa proteins in K562 cells and the transfectant containing the full-length KS43, and a 85-kDa protein in the transfectant containing the 3`-truncated KS32. No specifically labeled proteins were detected in wild type or mock-transfected hamster cells. Collectively, our results suggest that the KS43/KS32 cDNAs encode the human reduced folate carrier; however, additional modulatory/regulatory factors may be required to manifest the full spectrum of transport substrate activities typical of this system.


INTRODUCTION

Methotrexate (Mtx)()as well as some newer and more potent antifolates are actively transported intracellularly via a carrier-mediated uptake system(1, 2, 3, 4) . In experimental and clinical neoplastic cells, impaired membrane transport has been recognized as one of the major mechanisms of Mtx resistance (4, 5, 6) . The carrier system is characterized by its saturability and marked temperature dependence, as well as its high affinity for Mtx and reduced folates(7, 8) . By contrast, this system exhibits a low affinity for folic acid and, hence, transports this compound poorly(7, 8) . In addition to this ``classical'' Mtx/reduced folate carrier (RFC), a low affinity/high capacity transport system for folic acid has been reported(9) . Other membrane folate ``binding proteins'' have also been described in various cells and tissues(10, 11) . From their cDNAs, the amino acid sequences for these proteins have been deduced (12, 13) . These binding proteins show extremely high affinities (nanomolar) for folic acid, and somewhat lower affinities for reduced folates and Mtx.

In order to better understand the underlying cellular and molecular mechanisms associated with defective membrane transport, a number of studies have focused on the identification and molecular cloning of putative Mtx/reduced folate transport proteins. Matherly et al.(14) used radioaffinity labeling with N-hydroxysuccinimide-[H]Mtx (NHS-[H]Mtx) to identify a 92-kDa glycoprotein carrier (on a 7.5% gel) from Mtx transport up-regulated K562 cells (designated K562.4CF). More recently, a highly glycosylated protein (GP-Mtx), presumably representing the carrier, was isolated from NHS[H]Mtx-labeled membrane proteins from K562.4CF cells on columns of Ricinus communis agglutinin I-agarose(15) . A similar 80-85-kDa putative carrier was identified in transport up-regulated CCRF-CEM cells (CEM-7A) by affinity labeling with N-(4amino-4-deoxy-10-methylpteroyl)-N-(4-azido-5-[I]iodosalicylyl)-L-lysine (APA-[I]ASA-Lys)(16) . Identical labeling methods were used in murine L1210 cells to identify presumptive transport proteins with molecular masses of 42-48 kDa or 38 kDa (17, 18, 19) . Most recently, homologous cDNAs from L1210 cells (20) and Chinese hamster ovary (CHO) cells (21) were isolated and characterized. Both cDNAs encoded 58-kDa proteins and, when expressed in transport-impaired cells, restored Mtx sensitivities and Mtx transport activities. The apparent discrepancies in the molecular masses of the cDNA-encoded products and the membrane proteins identified in intact cells by radioaffinity labeling have raised considerable interest in the potential relationships among these proteins and their respective roles in the overall scheme of carrier-mediated folate transport.

The present study describes the isolation and expression of human cDNAs which encode a function involved in the membrane transport of Mtx and reduced folates. The cDNAs were isolated from a cDNA library prepared from Mtx transport up-regulated K562.4CF cells, based on homology to the mouse cDNA. Mtx transport-defective CHO cells transfected with the human cDNAs regain their Mtx sensitivities and transport activities. The transport characteristics of the transfected cells resemble both the wild type human and hamster classical RFCs, suggesting the expression of a hybrid transport system. In addition, the cDNA encoded proteins can be radiolabeled with APA-[I]ASA-Lys, a known photoaffinity inhibitor of the RFC. Taken together, the results strongly suggest that these cDNAs encode the human RFC (hRFC).


EXPERIMENTAL PROCEDURES

Materials

[3`,5`,7-H]Mtx (20 Ci/mmol) was purchased from Moravek Biochemicals (Brea, CA). Unlabeled MTX and (6R,S)-5-formyltetrahydrofolate (leucovorin) were provided from the Drug Development Branch, National Cancer Institute, Bethesda, MD. Both labeled and unlabeled MTX were purified prior to use as described previously(22) . [-P]dCTP (3000 Ci/mmol) and [-S-thio]dATP (1400 Ci/mmol) were obtained from DuPont NEN. Fetal bovine serum and iron-supplemented calf serum were from Life Technologies, Inc./BRL and Hyclone Laboratories, Inc. (Logan, UT), respectively. The antifolate drug, 1843U89, ((S)-2-(5-(((1,2-dihydro-3-methyl-1-ocobenzo(F)quinazolin-9-yl)amino)1-oxo-2-isoindolinyl)glutaric acid)) was provided by Burroughs Wellcome Company (Research Triangle Park, NC). Sequencing reagents and enzymes were from United States Biochemicals (Cleveland, OH). Restriction and modifying enzymes were purchased from Life Technologies, Inc./BRL or Promega (Madison, WI). Sequencing and PCR primers were obtained from Genosys Biotechnologies Inc. (The Woodlands, TX).

Cell Culture

The K562 erythroleukemia line was obtained from American Type Culture Collection (Rockville, MD). Wild type K562, transport up-regulated K562.4CF, and transport down-regulated K500E cells were cultured as described previously(14) . Culture conditions for murine L1210 leukemia cells were those of Schuetz et al.(18) .

CHO wild type Pro3 and transport-defective Mtx-resistant (Mtx RII Oua 2-4) cells(23) , gifts of Dr. Wayne F. Flintoff (University of Western Ontario), were grown in -minimal essential medium with 10% iron-supplemented bovine calf serum, penicillin (100 units/ml), and streptomycin (100 µg/ml). CHO cells were grown as monolayers for transfection experiments and cytotoxicity assays; for transport experiments, cells were grown in suspension in spinner flasks. Transfected CHO clones were selected and maintained in the presence of 1.5 mg/ml G418 (see below).

For cytotoxicity determinations, cells were cultured in RPMI 1640 supplemented with dialyzed fetal bovine serum and antibiotics, as described above, in 24-well dishes at 20,000 (K562) or 10,000 (CHO) cells/well. After 96 h of continuous exposure to various concentrations of Mtx and 1843U89, cell viabilities were determined by direct microscopic counting with a hemacytometer, either directly (K562) or following trypsinization (CHO). IC values were calculated as the concentrations of inhibitors required to inhibit growth by 50% compared to control cells grown under identical conditions, except that growth inhibitors were omitted.

Isolation of Human cDNA Clones

A 5` fragment of the murine putative RFC cDNA(20) , flanking positions 173 and 823 of the coding sequence, was prepared by reverse transcription-polymerase chain reaction (RT-PCR) from mouse L1210 cell total RNA. The oligonucleotides used were 5`-tccaggaattcgCCAAGGAGCAGGTGACTAACGAA-3` and 5`-cggtatcgatCCCACCAGAGGCACCAGAGAGACGC-3`. The EcoRI and ClaI restriction sites (lower case bases) were added to the up and down-stream primers, respectively, for subcloning the PCR product into the pBluescript SK(-) vector (Stratagene). RT-PCR was performed using a Perkin-Elmer RT-PCR kit and an Ericomp thermal cycler. The partial cDNA fragment, designated M-D6, was partially sequenced to confirm its identity.

A gt11 K562.4CF cDNA library was prepared by Clontech (Palo Alto, CA). This library was plated at a density of 10,000 plaque-forming units/100-mm Petri dish and phage DNA was lifted onto nylon filters (Magna Lift Nylon, MSI). The filters were prehybridized in 50% formamide, 6 SSPE (1 SSPE contained 0.15 M NaCl, 10 mM NaHPO, 1 mM EDTA, pH 7.4), 0.5% SDS, 50 µg/ml denatured sheared salmon sperm DNA, and 5 Denhardt's solution for 2 h at 42 °C. Hybridization was carried out at 42 °C overnight with the M-D6 probe radiolabeled with [-P]dCTP by random priming. Filters were washed (in 2 SSC (1 SSC contained 0.15 M NaCl, 15 mM Nacitrate, pH 7.0), 0.1% SDS for 20 min; twice at room temperature and twice at 42 °C), and positive phage were identified by overnight autoradiography. From confirmed positives, phage DNA was prepared using LambdaSorb phage adsorbent (Promega) with the plate lysate method as described by the supplier. Restriction mapping of phage DNA was performed, and cDNA inserts (KS6, KS32, and KS43) were subcloned into the pBluescript SK(-) vector for sequencing.

DNA Sequencing

Unidirectional nested deletion sets were prepared from both sense and antisense KS32 cDNA/pBluescript plasmid constructs using the Erase-a-Base system from Promega. Single-stranded DNA templates were prepared from deletion set subclones by infecting plasmid-containing XL-1 Blue cells with the helper phage VCSM13 (Stratagene). Utilizing the universal T3 primer, both the sense and antisense strands were sequenced completely using the dideoxynucleotide chain termination method (24) with a Sequenase Version 2 kit. Routinely, dGTP and dITP reactions were run in parallel to avoid problems associated with compression caused by G/C-rich sequences. Nucleotide sequence of the KS43 insert was obtained primarily with primers designed from the KS32 sequence and single-stranded template from the SalI-digested KS43 insert in pBluescript. In addition, EcoRI fragments of KS43 and KS6 in pBluescript were sequenced at both the 5`- and 3`-ends using T3 and T7 universal primers. Sequence data was analyzed using the University of Wisconsin Genetic Computer Group (GCG) package. Homology searches in nucleotide and protein sequence data bases were performed using the BLAST network service at the National Center for Biotechnology Information. Expression of KS32 and KS43 cDNA Inserts in CHO Mtx RII Oua 2-4 Cells-The KS32 and KS43 cDNA inserts in pBluescript were directionally ligated into the expression vector pcDNA3 (Invitrogen) utilizing the XbaI and KpnI multi-cloning sites. The resulting plasmid constructs, designated pC32 and pC43, respectively, were used to transfect Mtx-resistant Mtx RII Oua 2-4 cells; a mock transfection using the pcDNA3 vector was also performed. Transfection was carried out by the polybrene procedure (25) using 2-4 µg of plasmid/3 10 cells in 60-mm Petri dishes. Seventy-two h post transfection, cells were trypsinized, washed with Dulbecco's phosphate-buffered saline (DPBS; 26), and replated at densities ranging from 0.1 to 1 10 cells in 4 ml of fresh -media containing 1.5 mg/ml G418. G418-resistant CHO clones that exhibited increased Mtx sensitivities over Mtx RII Oua 2-4 cells were selected for further analysis.

Membrane Transport Methodology

Logarithmically growing cells were washed with DPBS and resuspended into Hank's balanced salts solution. Transport experiments with [H]Mtx were performed exactly as described previously (14) using cell densities of 1-2 10 cells/ml. The levels of intracellular radioactivity were expressed as picomoles/milligram protein or pmol/10 cells, calculated from direct measurements of radioactivity and protein contents of the cell homogenates. Protein assays were by the method of Lowry et al.(27) . Intracellular accumulations were corrected for surface adsorption as described by Sirotnak(2) . Kinetic constants (i.e.K, V, and K values) for assorted folate and antifolate substrates were calculated from Lineweaver-Burk and Dixon plots, respectively.

Northern Analysis

Total RNA was isolated from log phase cells using the single step method of Chomczynski and Nicoletta(28) . Total RNA from each cell line (20 µg) was electrophoresed on 0.9% agarose gels containing 2.2 M formaldehyde and 1 MOPS buffer. The gel was equilibrated in 10 SSC and capillary transferred to GeneScreen Plus membrane (DuPont) in 10 SSC; the membrane was baked at 80 °C under vacuum for 1.5 h. Membranes were prehybridized in QuickHyb solution (Stratagene) for 15 min then hybridized for 1 h with the addition of [P]dCTP-labeled KS43 cDNA insert (labeled by random priming). Nonspecific hybridization was removed by washing membranes in 2 SSC, 0.1% SDS at 42 °C and, finally, in 0.1 SSC, O.1% SDS at 60 °C. After autoradiography, the filters were stripped and rehybridized with a [P]dCTP-labeled -actin cDNA probe (Clontech). Densitometry of the autoradiograms was performed on a Molecular Dynamics Computing Densitometer and analyzed using the ImageQuant software (Molecular Dynamics, Sunnyvale, CA).

Cell Surface Labeling of the Transport Protein

A photoaffinity analogue of Mtx, APA-[I]ASA-Lys, was synthesized from the unlabeled compound under subdued light using the procedure of Price and Freisheim(17) . The radiospecific activity of the APA-[I]ASA-Lys was 2.9 mCi/µmol. Cells (K562, wild type CHO, L1210, and the transfected CHO lines) were incubated at a density of 1 10 cells/ml in ice-cold 20 mM HEPES, and 225 mM sucrose (pH adjusted to 7.4 with MgO). Cells were irradiated with a long wave-length UV lamp for 3 min, washed with DPBS (two times), and the radiolabeled proteins extracted with 500 µl of 10 mM Tris-HCl, pH 7.0, 1% Triton X-100 in the presence of a proteolytic inhibitor mixture(14) . Aliquots were electrophoresed on a 7.5% denaturing gel(14) . The gel was dried and exposed to film. Specificity of labeling was established by performing identical incubations in the presence of 100 µM aminopterin (AMT).

Western Analysis with Anti-GP-Mtx Antiserum

Plasma membranes from K562 wild type, CHO cells, and transfected clones were prepared as described previously(15) . Membrane proteins (50-100 µg) were electrophoresed on a 4-10% linear gradient gel in the presence of SDS and electroblotted onto Immobilon P (Millipore). Immunoblot analysis was performed as described previously using rabbit antiserum to GP-Mtx, a putative glycoprotein component of the hRFC(15) .


RESULTS

Isolation and Characterization of cDNA Clones

A partial cDNA to the putative RFC from murine cells (M-D6) was synthesized by RT-PCR and hybridized to a single 2.5-kilobase (kb) transcript in L1210 total RNA(20) . Based on the increased expression of a cross-hybridizing 3.1-kb transcript in K562.4CF over wild type K562 cells in low stringency washes (2 SSC, 0.1% SDS at 42 °C; not shown), a human K562.4CF cDNA library constructed in gtll was screened with M-D6 under similar conditions. Approximately 500,000 phage were screened, and eight clones were selected from 40 positives. Phage DNAs were prepared from these eight clones and restriction digest analysis revealed that all inserts contained two identical SacI restriction fragments (300 and 800 base pairs (bp), gel not shown). The cDNA inserts for two clones, KS6 and KS32 (1.4 and 2.5 kb, respectively), were subcloned into the EcoRI multicloning site of the pBluescript SK(-) vector. Four other phage inserts contained an internal EcoRI restriction site. The longest, KS43 (2.8 kb), was subcloned into pBluescript as two separate EcoRI fragments (1.1 and 1.7 kb) and also in its entirety as a SalI restriction fragment (a SalI restriction site was present in the adaptors used in cDNA library construction). The resulting plasmids were sequenced. The physical map and sequencing strategies for clones KS6, KS32, and KS43 are shown in Fig. 1A.


Figure 1: Sequencing strategy, restriction map, nucleotide sequence, and deduced amino acid sequence of the human RFC cDNAs. A, a restriction map for the composite sequence of KS32 and KS43 is shown. The solid horizontal bars represent the open reading frame. The shaded horizontal bars represent the heterogeneous 5`-untranslated sequences. The break in the KS32 line indicates deletion in the sequence with respect to KS43. The arrows indicate the direction and extent of nucleotide sequence obtained from each primer as described. B, the complete nucleotide sequence (upper line) and the deduced amino acid sequence (lower line) for the KS43 cDNA clone are shown. The numbering system is relative to the putative start codon (29) at position 1. The termination codon is indicated by a plus (+) sign. The putative polyadenylation signal is indicated by double underlined bases. Single underlined bases represent the deleted sequence in clone KS32. The 14 bold faced and italicized bases, starting at position 2194, are included in the KS32 putative coding region.



The nucleotide sequence and the deduced amino acid sequence for the KS43 cDNA insert is shown in Fig. 1B. The sequence contains a 98-bp 5`-untranslated region, an open reading frame (ORF) of 1776 bp preceded by a Kozak consensus sequence(29) , and a 3`-untranslated region of 864 bp which is followed by a poly(A) sequence. The predicted molecular mass of the translated protein is 65 kDa. One consensus N-glycosylation site was identified (at asparagine 58). For all three clones (KS6, KS32, and KS43), the upstream 5`-noncoding regions were highly heterogeneous starting at position -53 (Fig. 2). The nucleotide sequence of KS32 is identical to KS43 commencing with position -52, with the exception of a 626 base pair deletion starting at position 1568 (underlined sequence in Fig. 1B) and an additional four bases (TGTG) in the 3`-untranslated region immediately upstream from the poly(A) tail at position 2718 (not shown in Fig. 1B). As a result of the deletion in the 3`-ORF of KS32, the in-frame translational stop codon (position 1774) is lost. The KS32 coding sequence continues into the 3`-non-coding region with the utilization of a new stop codon at position 2205 (Fig. 1B). The result is that the KS32 ORF contains 14 bp (including the stop codon; bold face in Fig. 1B) not used in KS43, encoding 4 new carboxyl-terminal amino acids (LRCS). Clone KS6 appeared to be a partial cDNA. Only the 5`- and 3`-ends of the KS6 insert were sequenced (Fig. 1A).


Figure 2: The heterogenous 5` sequences of hRFC cDNAs. The heterogenous untranslated 5` sequences of clones KS6, KS32 and KS43 are shown. The numbering indicates the nucleotide position relative to the putative translational start site (assigned as position 1). The underlined sequences indicate where the identical 5`-untranslated regions for all three clones commence.



As expected, GenBank data base homology searches identified the mouse (20; GenBank accession no. L23755) and hamster (21; GenBank accession no. U03031) RFC sequences as highly homologous to KS43. No other homologous sequence was identified at the time this article was submitted. Amino acid sequence alignment analysis (Fig. 3), comparing the KS43 amino acid sequence with the homologous mouse (20) and hamster (21) sequences, revealed homologies of 79 and 80%, respectively; although the human RFC contained 73-79 more amino acids. All 9 of the tryptophans contained in the human sequence are conserved in the homologous hamster and mouse RFCs. Likewise, 4 of 6 cysteines are conserved. Interestingly, the single N-glycosylation site in the hRFC (at asparagine 58) was only conserved in the hamster RFC, even though two potential N-glycosylation sites were present in both the mouse and hamster sequences(20, 21) . Computer analysis of the deduced amino acid sequences, according to the criteria described by Kyte and Doolittle (30; not shown), predicted multiple hydrophobic domains similar to the homologous mouse and hamster cDNAs(20, 21) .


Figure 3: Amino acid sequence alignment for the human, mouse, and hamster RFCs. The predicted amino acid sequence of human RFC from KS43 is compared to the predicted amino acid sequences from the mouse (20) and hamster (21) cDNAs. Gaps, indicated by a dash(- - -), are introduced for the alignment. Where the mouse or hamster amino acid is conserved relative to the human sequence, it is indicated by a period.



KS43 was used as a probe for Northern blots of total RNA from parental K562 cells, transport up-regulated K562.4CF cells, and a variant line (K500E) with 70-fold Mtx resistance associated with 82% impaired Mtx uptake(15) . A major 3.1-kb transcript was detected in all three lines with an intensity, in part, paralleling relative transport. Compared to wild type K562 cells, the 3.1-kb transcript was 3.3-fold increased in K562.4CF and 2.2-fold decreased in K500E cells (Fig. 4). A faint 4.2-kb transcript was also detected in the K562.4CF cells.


Figure 4: Northern blot analysis of RNAs prepared from K562 sublines. Upper panel, Total RNA (20 µg) was electrophoresed, blotted onto a nylon membrane, and hybridized with the [P]dCTP-labeled KS43 cDNA insert as described. Lane 1, wild type K562 cells; lane 2, Mtx transport up-regulated K562.4CF cells; lane 3, Mtx transport impaired K500E cells. Lower panel, the membrane was stripped and reprobed with a [P]dCTP-labeled mouse -actin cDNA probe.



Expression of KS32 and KS43 cDNAs in Mtx Uptake-resistant CHO Cells

To characterize the potential role of the human cDNA-encoded RFCs in Mtx membrane transport, the KS43 and KS32 pBluescript plasmid constructs were digested with XbaI and KpnI and the inserts directionally ligated into the XbaI and KpnI multicloning sites of the expression vector, pcDNA3. These pcDNA3 constructs, containing the KS43 and KS32 inserts and designated pC43 and pC32, respectively, were transfected into Mtx-resistant Mtx RII Oua 2-4 CHO cells. Stable transfectants were selected with G418 (1.5 mg/ml) and screened for Mtx sensitivities over a range of 10-200 nM. Based on prior reports of an enhanced membrane transport and increased cytotoxicity of the benzoquinazoline antifolate, 1843U89, for human over murine cells(3) , we assessed the growth inhibitory effects for this compound, as well.

Both Mtx RII Oua 2-4 cells and mock-transfected cells (designated pC29) were approximately 60-fold resistant to Mtx compared to wild type CHO (Pro3) cells (Table 1). Mtx sensitivities for two transfectants, pC43/10 and pC32/2 (Table 1), are nearly identical to that of the Pro3 cells. Mtx RII Oua 2-4 cells and mock transfected pC29 cells were completely insensitive to 1843U89, up to 2000 nM. Sensitivities to 1843U89 for both pC43/10 and pC32/2 transfectants were approximately 16-fold greater than for wild type Pro3 cells (Table 1) and only 2-fold less than for the wild type K562 line (data not shown). These data strongly suggest the presence of a functional human transport system in the transfected CHO lines.



Initial rates of [H]Mtx influx (at 0.5 µM) were assayed over 180 s in wild type CHO, Mtx RII Oua 2-4, and the pC29, pC43/10 and pC32/2 transfectants (Fig. 5). By this analysis, the initial uptake rate for pC32/2 was not significantly different than wild type CHO cells, whereas a 37% increase was observed for pC43/10. Neither Mtx RII Oua 2-4 nor mock-transfected pC29 cells accumulated significant [H]Mtx over this interval. Hence, both the full-length and 3`-truncated cDNAs completely restored Mtx membrane transport.


Figure 5: Initial uptake rates of [H]Mtx by various CHO cell lines. Cells were incubated at 37 °C, and 0.5 µM [H]Mtx uptake was measured as described under ``Experimental Procedures.''



Kinetic analyses of the influx rates over a range of [H]Mtx concentrations were performed; the kinetic constants were calculated from Lineweaver-Burk plots and are summarized in Table 2. Whereas wild type CHO cells exhibited a similar V to K562 cells, the K for [H]Mtx binding was decreased greater than 6-fold (Table 2). Notably, the CHO lines transfected with either the full-length (pC43/10) or 3`-truncated (pC32/2) human cDNAs had K values more similar to the wild type CHO than to K562 cells. The V values for the transfectants were similar to (pC32/2) or slightly higher (pC43/10) than for the wild type lines. Due to their extremely poor Mtx uptake (<3% of wild type), kinetic parameters for the Mtx RII Oua 2-4 CHO cells were impossible to measure accurately and, therefore, are not included in Table 2.



To further characterize the structural specificities of the restored transport system in the CHO transfectants, experiments were performed to assess the inhibitions of [H]Mtx influx by assorted transport substrates (unlabeled Mtx, leucovorin, 1843U89, folic acid). K values were calculated from Dixon plots (not shown) and the K and V values in Table 2. K values for these inhibitions are summarized in Table 3. A characteristic feature of the classical RFC from both murine and human cells relates to increased affinities for Mtx and reduced folates compared to folic acid (Table 3). Relative to wild type CHO cells, [H]Mtx transport in K562 cells exhibited a somewhat increased (3-fold) sensitivity to inhibition by 1843U89 and a markedly decreased (up to 19-fold) inhibition by unlabeled Mtx. For Mtx, the calculated K values approximated the Kvalues in Table 2.



The pC43/10 and pC32/2 transfectants exhibited transport characteristics of both the human and hamster cells. Hence, K values (Table 3) for Mtx were nearly identical to wild type hamster cells. For 1843U89, the Kvalues are virtually identical with that for human K562 cells, consistent with the results of the growth inhibition experiments (Table 1). For folic acid, the K for pC43/10 transfectant was nearly identical to that for wild type CHO cells; however, the K for the 3`-truncated clone, pC32/2, most resembled that for K562 cells. An increased K for leucovorin binding to the pC32/2 transporter was measured, as well. The relative affinities of the restored transport in the CHO transfectants for folic acid versus other transport substrates strongly suggest that the uptake involves the classical RFC system. The differences in relative substrate binding between the pC43/10 and pC32/2 transfectants demonstrate that while the deletion of a terminal portion of the KS43 ORF results in some differences in the binding of particular transport substrates, its effects on the overall transport properties of the restored transport are minor. This is further considered below.

The restored drug sensitivities and [H]Mtx transport in the pC43/10 and pC32/2 transfectants were accompanied by the expression of hRFC transcripts (3.5 and 3.2 kb, respectively) on Northern blots probed with KS43 insert (Fig. 6, lanes 4 and 5). When normalized to -actin mRNA levels, the hRFC mRNA level in pC43/10 was 35% higher than that of the pC32/2 line. The KS43 cDNA probe did not hybridize to the homologous 2.5-kb hamster transcript (21) in wild type CHO cells, nor was any signal detected in either Mtx RII Oua 2-4 or mock-transfected pC29 cells (Fig. 6, lanes 1-3, respectively). There was a 13-18-fold disparity in the relative expression of the hRFC transcript between the transfected CHO lines and K562 cells (Fig. 6, lanes 4-6). Cell Surface Labeling of hRFC with APA-[I]ASA-Lys-Apparently identical 94-kDa surface proteins (Fig. 7) were specifically labeled in wild type K562 cells and pC43/10 transfectants with APA-[I]ASA-Lys, a documented photoaffinity inhibitor for the RFC(16, 17) . For the pC32/2 transfectant, an 85 kDa band was detected. The disparity between the sizes of these I-labeled bands in the CHO transfectants and those predicted from the KS43/KS32 cDNA sequences (65 and 58 kDa) were mostly, if not entirely, due to their N-glycosylation (data not shown). Interestingly, the L1210 labeling control (Fig. 7) exhibited a band centered around 46 kDa, differing from the predicted 58 kDa value from the published murine cDNA sequence(20) ; this value is similar to that reported from other laboratories by affinity labeling(17, 18, 19) . No specific labeling was detected in wild type CHO and the mock-transfected pC29 cells, even after prolonged autoradiography.


Figure 6: Northern blot analysis of CHO and K562 RNAs. Upper panel, total RNA (20 µg) was electrophoresed, blotted onto a nylon membrane, and hybridized with the [P]dCTP-labeled KS43 cDNA insert as described. Lane 1, CHO wild type (Pro3) cells; lane 2, Mtx RII Oua 2-4 cells; lane 3, clone pC29; lane 4, clone pC43/10; lane 5, clone pC32/2; and lane 6, wild type K562. Lower panel, membrane was stripped and reprobed with a [P]dCTP-labeled mouse -actin cDNA probe.




Figure 7: Photoaffinity labeling of hRFC with APA-[I]ASA-Lys. For each cell line, 1 10 cells were treated with 1 µM of APA-[I]ASA-Lys in the presence (+) or absence(-) of 100 µM of AMT. Cells were irradiated, washed with DPBS, and the radiolabeled proteins extracted with 500 µl of 10 µM Tris-HCl, pH 7.0, 1% Triton X-100. Equal aliquots (based on starting cell numbers) were electrophoresed in the presence of SDS. The figure shows an autoradiograph of the dried gel. The molecular masses (in kDa) of standard proteins are indicated.



In sharp contrast to the 13-18 fold differences in hRFC transcript levels between the pC43/10 and pC32/2 CHO transfectants and wild type K562 cells (Fig. 6), affinity labeled hRFC protein levels were increased only 1.7- and 3.4-fold higher than K562 cells, respectively (based on densitometric readings normalized to actual micrograms of protein loaded). This indicates that hRFC translation is relatively inefficient in the transfected lines. The increased labeling of the pC32/2 cells suggests that the truncated hRFC may be less efficient in mediating transport (3-fold based on their respective V values) than the form expressed in pC43/10 cells.

Do KS43 and KS32 cDNAs Encode for GP-Mtx?

Previous studies from this laboratory described the characteristics and isolation of a highly glycosylated membrane protein (i.e. GP-Mtx), following its radiolabeling with NHS-[H]Mtx. Antiserum prepared to GP-Mtx in rabbits detected a single membrane glycoprotein whose expression for a series of K562 sublines paralleled Mtx transport over a 12-fold range(15) . The possibility that hRFCs expressed in the pC43/10 and pC32/2 transfectants are identical to GP-MTX was directly considered by Western blotting, using specific antiserum to GP-Mtx (15) . In this experiment, GP-Mtx was readily detected in plasma membranes from wild type K562 cells; however, no immunoreactive proteins were detected in either wild type CHO cells or the pC43/10 and pC32/2 CHO transfectants (data not shown).


DISCUSSION

The present study describes the isolation, nucleotide sequence analysis, and functional expression of human cDNAs which restore Mtx sensitivity and membrane transport in Mtx-resistant Mtx RII Oua 2-4 CHO cells. Mtx RII Oua 2-4 cells are characterized by severely defective Mtx transport and low levels of surface [H]Mtx binding(23) , associated with the expression of a non-functional hamster RFC mRNA(21) . Moreover, CHO cells express no detectable membrane folate-binding proteins(31, 32) . Restored transport in the CHO transfectants appeared to involve the classical reduced folate/Mtx transport system which has been extensively documented in a variety of mammalian cells(4, 7) .

The nucleotide sequence and predicted protein structure of the encoded hRFC showed close homology to the recently described mouse (20) and hamster (21) cDNAs. The larger size of the human transcript (3.1 versus 2.5 kb) appears to reflect not only a larger ORF but also differences in both 5`- and 3`-untranslated regions. The prediction of multiple hydrophobic domains with membrane spanning potential and the presence of a putative N-glycosylation site with high surface probability substantiates our earlier studies of a glycosylated 92-kDa integral membrane carrier protein in transport up-regulated K562 cells which can be affinity labeled with NHS-[H]Mtx(14) . In the present report, similar sized proteins were detected in the pC43/10 and pC32/2 CHO transfectants, by photoaffinity labeling with APA-[I]ASA-Lys.

The restoration of Mtx sensitivities and capacities for Mtx transport by both the full-length and the 3`-truncated human cDNAs suggests that the deleted 3`-ORF sequence in KS32 has little direct role on membrane insertion. The hydrophilic peptide encoded by the deleted ORF in KS32, nonetheless, appears to influence the binding of certain transport substrates (i.e. folic acid, leucovorin) and overall transport efficiency.

The extensive heterogeneity in the 5`-non-coding sequences of the hRFC cDNAs may reflect the alternative splicing of transcripts transcribed from multiple promoters or transcription initiation sites(33) . The 3`-truncated KS32 clone may have a similar origin or represent a cloning artifact. Of course, the detection of a major 3.1-kb mRNA in K562 cells and sublines with widely disparate capacities for Mtx transport implies that only the hRFC encoded by the longer KS43 transcript is significantly expressed in these lines. Furthermore, only a single labeled protein was detected in K562 cells treated with APA-[I]ASA-Lys.

Of particular interest was the finding that the restored [H]Mtx uptake in the CHO transfectants exhibited both hamster and human characteristics. Based on K values, up to a 4-fold increased affinity for 1843U89 over wild type CHO cells (typical of human cells), and a 19-fold increased affinity for Mtx over K562 cells (typical of hamster cells) was observed. This anomaly may reflect differences in protein folding, post-translational processing, and/or membrane insertion of the hRFCs between hamster and human cells. Alternatively, additional regulatory/modulatory factors may be required for the expression of a human carrier system with a full spectrum of transport substrate specificities. Similar concepts were previously advanced by Underhill et al.(34) based on studies of reversion analyses and somatic cell hybrids between transport-defective CHO lines including the Mtx RII Oua 2-4 subline.

Our earlier studies suggested a causal role for a GP-Mtx, a highly glycosylated membrane component, in Mtx transport(15) . GP-Mtx was originally believed to be a Mtx carrier since it could be radioaffinity labeled with NHS-[H]Mtx, and its expression on Western blots approximated Mtx transport for a series of K562 sublines over a 12-fold range(15) . GP-Mtx exhibited an apparent molecular mass (92 kDa) on Western blots nearly identical to those of the APA-[I]ASA-Lys-labeled proteins in the CHO transfectants. No immunoreactive proteins were detected with anti-GP-Mtx in either pC43/10 or pC32/2 lines which expressed hRFC. This strongly suggests that GP-Mtx and hRFC are two separate glycoproteins with identical migration characteristics in SDS gels. Alternatively, the same differences in post-translational processing which may account for the hamster/human hybrid transport phenotype may also render the hRFC isoforms expressed in hamster cells non-immunoreactive with GP-Mtx antibodies.

Whereas the identity of GP-Mtx is presently uncertain, it is interesting that most of the immunoreactive GP-Mtx protein from K562 plasma membranes partitions into the detergent-poor phase in Triton X-114 phase separation experiments,()suggesting its peripheral membrane localization(35) . A separate 38 kDa cytosolic or peripheral membrane protein has also been implicated in RFC function in CCRF-CEM and L1210 cells affinity labeled with APA-[I]ASA-Lys(16, 17) .

In conclusion, our results show that the hRFC cDNAs are capable of restoring Mtx sensitivity and carrier-mediated Mtx membrane transport in Mtx transport defective CHO cells, apparently by expressing the ``classical'' RFC. Surface hRFC proteins were specifically labeled with APA-[I]ASA-Lys, a documented radioaffinity inhibitor of the RFC(16, 17) , confirming their capability to bind (and apparently transport) antifolate substrates. Although the unusual kinetic profiles of the CHO transfectants imply that additional factors may be required to manifest the full spectrum of transport substrate specificities, these may also be attributable to the host cell employed for our studies. We are currently transfecting transport-impaired human cells with the hRFC cDNAs to clarify this point. In any case, the availability of cDNAs involved in the transport of Mtx and reduced folates in human cells will undoubtedly facilitate future studies into the structure and function of this critical membrane system and the molecular basis of transport-mediated Mtx resistance.


FOOTNOTES

*
This work was supported by National Institutes of Health Grant CA 53535. 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®/EMBL Data Bank with accession number(s) U19720[GenBank® Link].

§
Recipient of a Scholar Award from the Leukemia Society of America, Inc. To whom correspondence and reprint request should be addressed: Developmental Therapeutics Program, Michigan Cancer Foundation, 110 E. Warren Ave., Detroit, MI 48201. Tel.: 313-833-0715 (ext. 407); Fax: 313-831-7518.

The abbreviations used are: Mtx, methotrexate; AMT, aminopterin; APA-[I]ASA-Lys, N-(4-amino-4-deoxy-10-methylpteroyl)-N-(4-azido-5-[I]iodosalicylyl)-L-lysine; bp, base pair(s); CHO, Chinese hamster ovary; DPBS, Dulbecco's phosphate-buffered-saline; hRFC, human reduced folate carrier; kb, kilobase; NHS-[H]Mtx, N-hydroxysuccinimide ester of [H]methotrexate; ORF, open reading frame; RFC, reduced folate carrier; RT-PCR, reverse transcription-polymerase chain reaction; MOPS, 4-morpholinepropanesulfonic acid.

S. C. Wong, S. A. Proefke, and L. H. Matherly, unpublished data.


ACKNOWLEDGEMENTS

We thank Daryel Taliaferro for secretarial assistance in preparing this manuscript. We also thank Dr. Wayne Flintoff for providing the wild type (Pro3) and Mtx transport defective Mtx RII Oua 2-4 CHO cells.

Note Added in Proof-Since the submission of this article, two other homologous human cDNAs have been reported (Refs. 36, 37). In both of these instances, numerous differences at the nucleotide level within the open reading frames were noted compared to the full-length hRFC cDNA sequence. These differences were represented by rearrangements, substitutions, or deletions. Due to these differences, the amino acid sequences encoded by these cDNAs are 90-95% homologous to that being reported here.


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