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Clinical Cancer Research Vol. 10, 5111-5122, August 1, 2004
© 2004 American Association for Cancer Research

Molecular Oncology, Markers, Clinical Correlates

Primary Acute Lymphoblastic Leukemia Cells Use a Novel Promoter and 5'Noncoding Exon for the Human Reduced Folate Carrier That Encodes a Modified Carrier Translated from an Upstream Translational Start

Robin M. Flatley1, Scott G. Payton2, Jeffrey W. Taub1,3,4 and Larry H. Matherly1,2

1 Experimental and Clinical Therapeutics Program, Barbara Ann Karmanos Cancer Institute, and the Departments of 2 Pharmacology and 3 Pediatrics, Wayne State University School of Medicine, and the 4 Children’s Hospital of Michigan, Detroit, Michigan


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The human reduced folate carrier (hRFC) is reported to be regulated by up to seven alternatively spliced noncoding exons (A1, A2, A, B, C, D, and E). Noncoding exon and promoter usage was analyzed in RNAs from 27 childhood acute lymphoblastic leukemia (ALL) specimens by real-time PCR and/or 5' rapid amplification of cDNA ends (5' RACE) assay. By real-time PCR, total hRFC transcripts in ALL spanned a 289-fold range. Over 90% of hRFC transcripts were transcribed with A1, A2, and B 5' untranslated regions (UTRs). Analysis of 5' RACE clones showed that the A1 + A2 5'UTRs contained A1 sequence alone or a fusion of A1 and A2, implying the existence of a single, alternatively spliced 1021-bp A1/A2 noncoding region. High frequency sequence polymorphisms (AGG deletion, C/T transition) identified in the A1/A2 region by 5'RACE were confirmed in normal DNAs. By reporter assays in HepG2 hepatoma and Jurkat leukemia cells, A1/A2 promoter activity was localized to a 134-bp minimal region. Translation from an upstream AUG in the A1/A2 noncoding region in-frame with the normal translation start resulted in synthesis of a larger (~7 kDa) hRFC protein with transport properties altered from those for wild-type hRFC. Although there was no effect on transcript or protein stabilities, in vitro translation from A1/A2 transcripts was decreased compared with those with the B 5'UTR. Our results document the importance of the hRFC A1/A2 upstream region in childhood ALL and an intricate transcriptional and posttranscriptional regulation of hRFC-A1/A2 mRNAs. Furthermore, they suggest that use of the A1/A2 5'UTR may confer a transport phenotype distinct from the other 5'UTRs due to altered translation efficiency and transport properties.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
In the current treatment of childhood acute lymphoblastic leukemia (ALL), methotrexate (MTX) is a key component of both intensification and maintenance therapies and is administered intrathecally in the prophylaxis and treatment of central nervous system leukemia (1 , 2) . Major determinants of MTX sensitivity and resistance originally reported for cultured cells have now been described for ALL lymphoblasts (3 , 4) . Reports from multiple labs have described wide variations in the expression of the human reduced folate carrier (hRFC), the major transport process for reduced folates and MTX (5) , in diagnostic and relapsed ALL (6, 7, 8) . Whereas some studies have shown that levels of hRFC transcripts in ALL specimens were generally predictive of MTX uptake capacities (6 , 7) , in other cases hRFC transcripts were disproportionate to levels of MTX transport (7) . Thus, both transcriptional and posttranscriptional controls for hRFC appear to be important determinants of MTX transport in ALL lymphoblasts.

Previous studies from our laboratory suggested a remarkably complex regulation of hRFC gene expression in tissues and tumors (9) . We reported recently that hRFC is ubiquitously but differentially expressed in human tissues and tumors and is regulated by up to seven alternatively spliced noncoding exons (originally designated A1, A2, A, B, C, D, and E), spanning >35 kb upstream of the major translational start site (9) . Including various splice forms, as many as 18 potential hRFC transcripts were identified with unique 5' untranslated regions (UTRs) linked to a common coding sequence. To date, promoter activity has been localized to the 5' regions proximal to exons A, B, and C (9, 10, 11) . Whereas the biological role of each 5'UTR has yet to be established, alternate noncoding exons can influence transcript stabilities, intracellular targeting, and/or translational efficiencies, or even encode modified proteins translated from upstream AUGs (12, 13, 14, 15, 16, 17, 18) . For hRFC, cell- and tissue-specific usage of each promoter and noncoding exon can be envisaged to function in regulating relative levels of hRFC transcripts and protein in tissues and tumors in response to folate cofactor requirements or other cell- or tissue-specific signals.

In this report, we describe the transcriptional and posttranscriptional features of the noncoding region immediately flanking coding exon 1, originally designated as separate A1 and A2 exons (9) but now identified as a single A1/A2 noncoding region. We document the synthesis of heterogeneous hRFC transcripts transcribed from the A1/A2 region as the major forms in primary ALLs under control of a unique promoter. In addition, we report the synthesis and transport characteristics of a novel hRFC protein isoform translated from an upstream AUG in the A1/A2 region.


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Chemicals and Reagents.
[{alpha}-32P]dCTP (3000 Ci/mmol) was obtained from Perkin-Elmer (Boston, MA). [3',5',7-3H]MTX (20 Ci/mmol) was purchased from Moravek Biochemicals (Brea, CA). Unlabeled MTX and (6R,S)5-CHO H4PteGlu (Leucovorin) were provided by the Drug Development Branch, National Cancer Institute (Bethesda, MD). Both labeled and unlabeled MTX were purified by high-performance liquid chromatography before use (19) . Folic acid was purchased from the Sigma Chemical Co. (St. Louis, MO). The sources of the antifolate drugs were as follows: Tomudex [N-(5-[N-(3,4-dihydro-2-methyl-4-oxyquinazolin-6-ylmethyl)-N-methyl-amino]-2-thenoyl)-L-glutamic acid] was obtained from AstraZeneca Pharmaceuticals (Macclesfield, Cheshire, England); Pemetrexed [N-(4-{2-[2-amino-3,4-dihydro-4-oxo-7H-pyrrolo(2,3-D)pyrimidin-5-yl]ethyl}benzoyl)-L-glutamic acid] was from Eli Lilly and Co. (Indianapolis, IN); and GW1843U89 {(S)-2-[5-({[1,2-dihydro-3-methyl-1-oxo-benzo(F)quinazolin-9-yl] methyl} amino)1-oxo-2-isoindolinyl] glutaric acid} was from the GlaxoWellcome-SmithKline Co. (Research Triangle Park, NC). Lipofectin was purchased from Invitrogen (Carlsbad, CA). Restriction and modifying enzymes, reporter gene vectors (pGL3-Basic and pRLSV40), and other molecular biologicals were obtained from Promega (Madison, WI). Synthetic oligonucleotides were obtained from Invitrogen or Sigma-Genosys (The Woodlands, TX). All of the other chemicals were obtained from commercial sources in the highest available purity.

Cell Culture.
Jurkat T-ALL cells were obtained from Dr. Beverly Mitchell (University of North Carolina, Chapel Hill, NC). The wild-type K562 human erythroleukemia and HepG2 human hepatoma cell lines were obtained from the American Type Culture Collection (Manassas, VA) and cultured as described previously (10 , 20) . Jurkat cells were cultured in RPMI 1640 containing 10% (v/v) heat-inactivated iron-supplemented calf serum (Hyclone Laboratories, Inc., Logan, UT), 2 mM L-glutamine, 100 units/ml penicillin, and 100 µg/ml streptomycin in a 37°C humidified atmosphere in the presence of 5% CO2/95% air. The origin and the characteristics of the MTX-resistant (K500E) K562 subline have been described previously (20) .

Patient Specimens.
The childhood ALL specimens used for this study were obtained from the Children’s Hospital of Michigan and are those used in our previous studies of MTX response and resistance in pediatric ALL (7 , 21) . Samples were obtained at diagnosis after informed consent, and in accordance with protocols approved by the Committee on Investigation Involving Human Subjects at Wayne State University. Leukemia blasts were purified by standard Ficoll-Hypaque density centrifugation.

5'Rapid Amplification of cDNA Ends Assay.
Total RNAs were extracted from primary ALL lymphoblasts using the RNEasy Midiprep kit (Qiagen). cDNAs were prepared from 1 µg total RNA using PowerScript reverse transcriptase and 5'-CDS polyadenylic acid and SMART II A primers (Clontech). Dilutions of each cDNA were used in primary and nested secondary PCR amplification reactions with SMART RACE kit sense primers (Universal Primer Mix A and Nested Universal Primer A, respectively) and gene-specific antisense primers [RFC Nest-10 (5'-GACCTGCTCCCGCGTGAAGTTCTTGTCG-3') and RFC Nest-1 (5'-AGCTCCGGAGGGGACGAAGGTGACACTGTG-3'), respectively]. Primary PCR conditions were 94°C for 7 min (1 cycle), 94°C for 2 s, and 70°C for 3 min (7 cycles), and 94°C for 2 s and 67°C for 3 min (35 cycles), followed by 72°C for 7 min (1 cycle). For secondary PCRs, the conditions were 94°C for 7 min (1 cycle), 94°C for 2 s, and 70°C for 3 min (5 cycles), and 94°C for 2 s and 67°C for 3 min (20 cycles), followed by 72°C for 7 min (1 cycle). Secondary PCR products were ligated into pGEM T-Easy (Promega), and ligations were transformed into competent JM109 cells. Plasmid DNAs were prepared from individual clones using the Qiagen Miniprep Spin kit and 5'Rapid Amplification of cDNA Ends Assay (5'-RACE) inserts were sequenced using gene-specific primers.

Real-time PCR Quantitation of hRFC Transcripts.
cDNAs were prepared from 1-µg RNAs using random hexamers and a reverse transcription-PCR (RT-PCR) kit (Perkin-Elmer) and purified with the QIAquick PCR Purification kit (Qiagen). Total hRFC transcripts, hRFC A1/A2, A, B, C, and D 5'UTRs, and 18S RNA levels were quantitated using a LightCycler real-time PCR machine (Roche). Reactions contained 2 µl of purified cDNA or standard plasmid, 4 mM MgCl2, 0.5 µM each of sense and antisense primers, and 2 µl of FastStart DNA Master SYBR Green I enzyme-SYBR reaction mix (Roche). Primers were as follows: hRFC coding sequence sense (5'-CAGATTGCATCTTCTCTGT-3') and antisense (5'-CACGTCCGAGACAATGA-3'); A1/A2 sense (5'-GGCCTGCAGACCATCTT-3') and A1/A2 and C antisense (5'-ATGAAG-CCGTAGAAGCAAAG-3'); exon A sense (5'-CCAGGCACGTGTTGCTTCG-3') and A, B, and D antisense (5'-ACGAGGTGCCGCCAGGACCG-3'); exon B sense (5'-CGAGTCGCAGGCACAGTGTCAG-3'); exon C sense (5'-CTCGGCTA-CACCTGACC-3'); exon D sense (5'-AGGAGGATCTTCTGCGGATT-3'); and 18S RNA sense (GATGCGGGGCGTTATT-3') and antisense (5'-TGAGGTTTCCCGTGTTGTCA-3'). PCR conditions were 95°C for 600 s, 35–55 cycles of 95°C, 59°C (18S and coding region), 63°C (exons A and B), 69°C (A1/A2 and exon C), or 67°C (exon D) for 10 s, and 72°C for 5 s, followed by melting curve analysis from 40°C to 95°C and a final cooling step to 40°C. An external standard curve was constructed using serial dilutions of KpnI-linearized pC43 construct, consisting of the full-length hRFC cDNA including the B 5'UTR cloned into pcDNA3 (22) . An 18S RNA external curve was generated from an 18S RNA amplicon, cloned into pGEM T-Easy vector (Promega), and linearized with ApaI. A1/A2, A, B, C, and D standards were prepared by amplifying exon fragments from cell line cDNAs with the above primers, cloning into pGEM T-Easy vector, and linearizing the vectors with ApaI (A1/A2, A, C, and D) or SalI (B). Serial dilutions were used to create standard curves for each 5'UTR. K562 and patient results were calculated from hRFC and 18S RNA standard curves, and patient total hRFC levels were expressed relative to those in K562 cells. The mean variation between replicate measurements (n = 18) on the same K562 cDNA sample (for hRFC) was 50.4% (± 11.9%; SD). 5'UTR levels by real-time PCR were expressed in the same relative units as the total hRFC and were normalized to 18S RNA. All of the results were calculated as mean values from two to three experiments.

hRFC-Luciferase Reporter Constructs.
An upstream fragment from positions –1935 to –987 [relative to the major translational start (AUG1) in coding exon 1] was amplified by PCR from an hRFC genomic clone (RFCg1–31) described by Zhang et al. (23) . A series of deletion constructs from positions –1935 to –1177 were prepared using the same antisense primer [(5'-CTAGCTAAGCTTGGGCGTGTCTCTTTCAGTCCTGA-TTCTGCCCAGGA-3'), including a HindIII site (italicized) for cloning] and the following sense primers [each with a XhoI site (italicized)]: hRFC-A1/A2(–1935), 5'-ACTGCCTCGAGCTGCCGTGGAGCTCCGGAAACCTCGAGGTT-3'; hRFC-A1/A2(–1590), 5'-ACTGCCTCGAGGGGTCCCTGGACCCCCAAAGCTGGCCCCACA-3'; hRFC-A1/A2(–1463), 5'-ACTGCCTCGAGTGGCGAGGCACAATTGTCCAACTGTCAG-3'; hRFC-A1/A2(–1311), 5'-ACTGCCTCGAGTCCGGGAGCAGAGGTCAGCTGA TACAGA-3'; and hRFC-A1/A2(–1177), 5'-ACTGCCTCGAGTGAAGGAGAACTTTACACCGAGGC-CTTAATG-3'. The amplicons were digested with XhoI and HindIII and subcloned into the multiple cloning site of XhoI/HindIII-digested pGL3-basic vector. Deletion clones spanning the –801 to –242 region flanking the hRFC A1 noncoding region (positions –457 to –50; Ref. 9 ) were prepared by PCR using a single antisense primer (5'-CTAGCTAAGCTTGGAGTCCCCCAGGCCAGGCTGTCACCACCT-3', where the HindIII site is italicized) and the following sense primers (XhoI site is italicized): hRFC-A1(–801), 5'-ACTGCCTCGAGTAACCTGCCGCCGGGGATGCTGGGGT-3'; and hRFC-A1(–532), 5'-ACTGCCTCGAGTGCCTGTGCCCTCCCTGGGCCACCTGGGTGCCGCTA-3'. Amplicons were digested with XhoI and HindIII and cloned into XhoI/HindIII-digested pGL3-basic vector.

Transient Transfections and Reporter Gene Assays.
Transient transfections of hRFC-A1/A2 or hRFC-A1 promoter reporter constructs in pGL3-basic or empty pGL3-basic (3 µg) into HepG2 cells were performed, as described previously (10) . For the Jurkat cell transfections, hRFC-A1/A2 or hRFC-A1 reporter constructs (see above) or empty pGL3-basic (5 µg) were cotransfected with 200 ng of pRLSV40 plasmid using Lipofectamine 2000 (Invitrogen) according to the manufacturer’s protocol. After an additional 48 h, lysates were prepared and luciferase activities assayed using a Dual Luciferase kit (Promega) in a Turner 20/20 luminometer. Firefly luciferase activity was normalized with Renilla luciferase. For all of the transfections, three or more experiments were performed.

Preparation and Expression of A1/A2 hRFC cDNA Constructs.
Splice-overlap-extension PCR (24) was used to generate the hRFCAUG2HA, del-hRFCAUG2HA, and hRFCAUG3HA constructs with GC-rich polymerase (Roche) and either standard PCR buffer (Perkin-Elmer) or GC-rich kit buffer. Initially, fragments containing the A1/A2 region including AUGs 2 and 3 were amplified from 5'RACE clones containing the A1/A2 exon. For AUG2, templates with or without the polymorphic AGG deletion at positions –133 to –131 were used (see below). Primers included upstream exon-specific primers with a KpnI site (italicized) at the 5' end (hRFC-AUG3: 5'-CAAGCTTGGTACCGATTAATTAACTTGGTTTTGATTGGCACTT-3'; hRFC-AUG2: 5'-CAAGCTTGGTACCGATTTGGTGCTACGGGGTGAGGATGGGTCT-3') and antisense hRFC coding sequence primer to Nest-1 (see below). Additionally, a hRFC coding sequence fragment was amplified from the pC43 hRFC construct (22) using a sense Nest-1 primer (5'-CACAGTGTCACCTTCGTCCCCTCCG-3') and hRFC antisense primer rHA8 (5'-GCCCAGCGGATCTTCACGAAG-3'). PCR conditions were 95° for 30 s, 63° for 45 s, and 72° for 60 s (35 cycles). Secondary amplifications were performed under identical conditions with the AUG3/rHA8 or AUG2/rHA8 primer sets and a mixture of primary products as template. After digestion with KpnI and NotI, the fragments were ligated into KpnI/NotI-digested pC43HA construct (hRFCAUG1HA in the present study), which contains a hemagglutinin (HA) tag at the 3' end of the coding sequence (25) .

The khRFCAUG2HA and del-khRFCAUG2HA constructs containing a mutated Kozak consensus sequence for the normal AUG translation start in coding exon 1 (AUG1) were generated from the hRFCAUG2HA and del-hRFCAUG2HA constructs, respectively, using internal mutant primers, kAUG1sense (5'-CACGTGGCCTGAGCCGGATGGTGCCCTCCAGC-3') and kAUG1anti (5'-GCTGGAGGGCACCATCCGGCTCAGGCCACGTG-3'; mutations are italicized). Primary reactions used AUG2/kAUG1anti and kAUG1sense/rHA8 primer sets. Secondary PCRs used a mixture of primary products and the AUG2/rHA8 primers. The amplicons were ligated into the KpnI/NotI-digested hRFCAUG1HA construct. All of the constructs were confirmed by automated DNA sequencing.

Constructs were in vitro translated (see below) and stably transfected into transport-impaired K500E cells with Lipofectin (20) . G418-resistant clones were isolated, expanded, and characterized.

Northern Blot Analysis and Assays of hRFC Transcript Stabilities.
hRFC transcript levels in RNA from transfected cells were determined by Northern blot. hRFC transcript stabilities were measured over 10 h by Northern analysis in cells treated with 10 µg/ml actinomycin D (26) . Equal loading was established with ethidium bromide. Densitometry of the X-ray films was performed on a Kodak Digital Science 440CF Image Station.

Western Blot Analysis.
Plasma membranes were prepared from transfected K562 cells by differential centrifugation (27) . Membrane proteins were electrophoresed on 7.5% polyacrylamide gels in the presence of SDS (28) , and electroblotted onto polyvinylidene difluoride membranes (Pierce; Ref. 29 ). The blots were developed with hRFC-specific antibody (30) or anti-HA antibody (BabCo, Berkeley, CA) and enhanced chemiluminescence (Roche) and exposed to X-ray film with multiple exposures. Densitometry was performed on a Kodak Digital Science Image Station 440CF. For N-glycosidase F (New England Biolabs) digestions, samples were incubated at 37°C overnight after denaturation (10 min, 0.5% SDS and 1% ß-mercaptoethanol) and the addition of 50 mM sodium phosphate (pH 7.5), 1% (v/v) NP40, and N-glycosidase F (0.5 unit).

To measure stabilities of the hRFCAUG1HA and hRFCAUG2HA proteins, cells were treated with cycloheximide (0.2 mg/ml), and at intervals up to 36 h, plasma membrane fractions (25 µg) were analyzed on Western blots with anti-HA antibody.

In Vitro Translation of hRFCAUG1HA and hRFCAUG2HA Proteins.
Translation efficiencies were determined using the TNT Quick T7 Coupled Transcription/Translation System (Promega). Equal amounts (1 µg) of empty pCDNA3, hRFCAUG1HA, hRFCAUG2HA, khRFCAUG2HA, and del khRFCAUG2HA were incubated with the TNT Quick master mix and methionine for 90 min at 30°C. The reactions were analyzed on Western blots with anti-HA antibody.

Transport Methodology.
Initial rates of [3H]MTX (0.5 µM) uptake in the K562 transfectants were measured over 180 s, as described previously (20 , 22) . Uptake was expressed as pmol/mg protein, calculated from direct measurements of radioactivity and protein contents of the cell homogenates. Protein assays were by the method of Lowry et al. (31) . Kinetic constants (Kt and maximum velocity, Vmax) were calculated from Lineweaver-Burk plots for [3H]MTX, and inhibition constant Ki values for assorted transport substrates were determined from Dixon plots, with [3H]MTX (1 µM).

Confocal Microscopy.
For confocal microscopy, cells were fixed with 3.3% paraformaldehyde/Dulbecco’s PBS, permeabilized with 0.1% Triton X-100, and stained with anti-HA clone 12CA5 monoclonal antibody (Roche), followed by a secondary goat antimouse IgG conjugated with Alexa Fluor 488 (Molecular Probes) as described previously (32) . Detection was performed with Zeiss laser scanning microscope 310 using a x63 water-immersion lens.

Genotype Analysis.
Genomic DNAs from 27 normal (nondisease) individuals were provided by Murray Norris and Michelle Haber (Children’s Cancer Institute Australia, Randwick, Australia). Genomic DNAs were purified from ALL blasts using the PureGene DNA Isolation kit (Gentra). The A1/A2 region containing the putative exon A1/A2 polymorphisms (i.e., AGG at positions –133 to –131, T/C transition at position –109) was amplified with Easy A polymerase (Stratagene). Primary PCRs used gene-specific primers, hRFC-AUG3 (see above) and KS43UTR323A (5'-CCAGCACGGCCAGGTAGGAGTA-3'), and the secondary nested PCRs used the A1-RT (5'-CAGCCTCGGAGACCCTGGAGGTGGTGAC-3') and RFCpoly1 (5'-GGGTGATGAAGCTCTCCCCTGG-3') primers. PCR conditions were 95°C for 30 s, 60°C for 45 s, and 72°C for 60 s (35 cycles). PCR reactions were separated on agarose gels, and amplicons were excised and eluted from the gels using the Rapid Gel Extraction System (Marlingen Biosciences). Purified PCR products were sequenced and the chromatographs were analyzed to determine frequencies for each polymorphism.


   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Analysis of hRFC 5'UTRs from Pediatric ALLs by 5'RACE and Real-Time RT-PCR.
We previously used 5'RACE to identify up to 7 noncoding regions (A1, A2, A, B, C, D, and E)5 for hRFC in RNAs from a selection of normal human tissues (9) . We then extended these experiments to include RNAs from 21 pediatric ALL specimens (15 B-precursor ALL and 6 T-ALL) and sequences from 292 independent clones. For 220 clones from 15 B-precursor ALL specimens, 136 (62%) contained exon B sequence, with 67 (30%) derived from the A1 + A2 region. A few clones originated from exons A (4%) and C (4%). Exons D and E were not detected. Virtually identical results were obtained for 6 T-ALLs (72 total 5'RACE clones; 57% B, 33% A1 + A2, 8% C, and 1% A).

The A1 and A2 sequences were of interest given their high frequencies in ALL specimens compared with most nonmalignant tissues (9) . Moreover, the 5'RACE sequence data were inconsistent with our earlier interpretation of separate A1 and A2 noncoding exons (9) , because all of our 5'RACE clones contained only A1 sequence (positions –458 to –50; 67% of total) or a fusion of the A1 and A2 regions (positions –1070 to –50; 33% of total; Fig. 1Citation ). By this interpretation, the A1 and A2 regions would provide the 3' and 5' boundaries, respectively, for a single ~1021-bp noncoding sequence (designated hereafter as A1/A2) included in an expanded ~1258-bp first coding exon (Fig. 1)Citation . Thus, the exclusive use of distal A2 sequence (positions –1070 to –803 in Fig. 1Citation ) in 5'UTRs identified in liver hRFC transcripts (9) must arise from alternative splicing of this larger A1/A2 sequence.



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  		Fig. 1. Schematic representation of the human reduced folate carrier (hRFC) upstream region showing alternate noncoding exons and partial sequence of hRFC noncoding exon A1/A2 and coding exon 1. A, the upstream region of the hRFC gene is shown including six noncoding regions (A1/A2 and A-E) and the first coding exon (Exon 1) including the ATG translation start site described by Zhang et al. (23) . The approximate distances between each of the noncoding exons were determined from those in chromosome 21 contig HS21C102 (accession no. AL163302). Sequence of the 1021 bp A1/A2 region is shown in its entirety, with partial 5' sequence of coding exon 1 (italicized). Locations of the major transcription start sites identified by 5' rapid amplification of cDNA ends in multiple acute lymphoblastic leukemia specimens are indicated by the arrows. AUGs (ATGs) 1, 2, and 3 and the two sequence variants [AGG(–133/–131), C/T-109] are in bold. B, amino acid sequence encoded from the upstream AUG2, and continuing into the common sequence for hRFC is shown. Translation initiation encoded by AUG1 is indicated by *, and the Gly(G)21 deletion accompanying AGG(–133/–131) is underlined.

 
Other notable features of the A1/A2 noncoding sequence in our 5'RACE clones included multiple transcriptional starts, suggested by multiple reproducible 5' boundaries for clones isolated from >1 patient, 2 ATGs (AUGs) upstream of the normal (exon 1) translation start and in-frame with normal hRFC coding sequence, and high frequency sequence variants (deletion of AGG at positions –133 to –131 and a T/C transition at position –109). These features are all noted in Fig. 1Citation .

5' RACE is not sufficiently reliable for quantitating frequencies of transcript forms, because it is based on PCR and may be affected by template secondary structures (15) . Accordingly, we used real-time RT-PCR, with primers to the individual 5'UTRs identified in ALL specimens by 5'RACE and downstream primers to hRFC coding sequence to quantitate levels of the individual 5'UTRs in 26 ALL specimens (includes 14 of 15 B-precursor ALL and 6 T-ALL samples used for 5'RACE and 6 additional T-ALL samples). Total hRFC transcripts were also measured by real-time RT-PCR assay using coding sequence primers and normalized to 18S RNA.

Total hRFC transcripts in our ALL specimens spanned a wide range (289-fold); there were no significant differences in median levels of hRFC transcripts between 15 B-precursor ALL and T-ALL lymphoblasts (relative median values of 0.18 and 0.35, respectively; Fig. 2ACitation ). Similar to 5'RACE, our real time RT-PCR assays of 5'UTR usage showed A1/A2 and B to be the major 5'UTRs in primary ALLs (>90% of hRFC transcripts for both immunophenotypes; Fig. 2, B and CCitation ). Levels of transcripts with 5'UTRs derived from exons A, C, and D were very low. The exon A primer set amplified only ~45% of the A splice variants identified by 5'RACE. However, when another primer was used to amplify an additional subset of exon A forms, this did not significantly change the overall usage patterns indicated by real time RT-PCR. Between B-precursor ALL - and T-ALL samples, there were notable differences in the median values for certain 5'UTRs (i.e., B, C, and D); however, there was significant overlap. Only for exon C was this difference statistically significant (medians of 0.00024 and 0.00569, respectively; P = 0.0234 by the Mann-Whitney test).



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  		Fig. 2. Real-time reverse transcription-PCR analysis of patient samples. A, human reduced folate carrier (hRFC) values of 14 BP-acute lymphoblastic leukemia (ALL) and 12 T-ALL patient samples, based on real-time reverse transcription-PCR analysis, are shown. B and C, hRFC 5' untranslated region levels in BP-ALL (B) and T-ALL (C) samples are shown. For both total hRFC and 5' untranslated regions, results are expressed in the same relative units and are normalized to 18S RNA. Median values are indicated by horizontal lines.

 
Identification of a Novel Promoter Activity.
If the A1 and A2 regions are actually composed of a single noncoding region, we predicted that the region immediately proximal to the 5' end of A1/A2 region (beginning at position –1071) would exhibit promoter activity in transient reporter gene assays. Conversely, the sequence immediately flanking the 5' end of the A1 region (positions –457 to –50) from our earlier report (9) should be transcriptionally inert. Because HepG2 human hepatoma and Jurkat leukemia cells expressed transcripts with A1/A2 sequence by RT-PCR (9) , we transiently transfected these cell line models with progressively 5' deleted fragments [hRFC-A1/A2(–1935), hRFC-A1/A2(–1590), hRFC-A1/A2(–1463), hRFC-A1/A2(–1311), and hRFC-A1/A2(–1177)] that included the 5' flanking region and the 5' end of the A1/A2 region (from positions –1070 to –987) in pGL3-Basic. Firefly luciferase reporter assays were performed and normalized to Renilla luciferase. Parallel experiments were performed with 5' deleted fragments [hRFC-A1(–801) and hRFC-A1(–532)] flanking the 5' end of putative exon A1 (9) in pGL3-Basic. Whereas the A1 flanking fragments were all transcriptionally inert (data not shown), for the hRFC-A1/A2(–1935) construct, an ~3-fold increased luciferase activity over pGL3-Basic was detected (Fig. 3ACitation shows only results with HepG2; essentially identical results were obtained in Jurkat cells). Maximal promoter activity was detected for the hRFC-A1/A2(–1311) construct (~4.4-fold over pGL3-Basic). Additional deletion from the 5' end of hRFC-A1/A2(–1311), generating hRFC-A1/A2(–1177), completely abolished luciferase activity (Fig. 3A)Citation .



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  		Fig. 3. Deletion analysis of human reduced folate carrier (hRFC)-A1/A2 promoter activity in HepG2 cells and nucleotide sequence for the hRFC-A1/A2 (–1311) bp promoter. A, 5' deletions were introduced into a 948-bp fragment including 83 bp of exon A1/A2, as described in the text. Promoter constructs in pGL3-Basic were transiently transfected into HepG2 hepatoma cells for reporter gene assays. Results are presented as relative firefly luciferase activities, normalized to Renilla luciferase activities and are expressed as mean values from three to four experiments; bars, ±SE. B, the nucleotide sequence for the 324-bp region between positions –1311 and –987 [hRFC-A1/A2(–1311)], corresponding to the maximal luciferase activity in the reporter gene assays, is shown. The numbering is relative to the AUG1 translation start site in coding exon 1. Potential cis- elements are noted. Noncoding exon A1/A2 is given in italics. Luciferase activity was abolished upon deletion of 134 bp of 5' sequence [hRFC-A1/A2(–1177); noted by •].

 
Potential transcription elements identified within the maximally active promoter region [hRFC-A1/A2(–1311)] by database analysis (Transfac) are indicated in Fig. 3BCitation . Although neither a TATA box nor Inr element was identified, a number of potential transcription elements, including E-box, Ikaros, and GATA, were identified (Fig. 3B)Citation .

An Upstream AUG in the hRFC-A1/A2 Noncoding Exon Encodes a Modified hRFC Protein.
hRFC is typically translated from the first AUG in coding exon 1 (position 1; designated AUG1), where the exon 1 acceptor junction is at position –49 (Ref. 23 ; Fig. 1Citation ). The noncoding A1/A2 region is juxtaposed to coding exon 1 and spans positions –1070 to –50, relative to the AUG1 start site. Analysis of the nucleotide sequence for the A1/A2 region identified 2 additional AUGs in-frame with the hRFC coding sequence (GenBank accession no. U19720), beginning 309 bp (AUG3) and 192 bp (AUG2) upstream from AUG1 (Fig. 1)Citation . If translated, the predicted molecular masses would be ~11 kDa and ~7 kDa larger than the normal hRFC translated from AUG1. AUG3 (TCCATGT) was a poor fit to the consensus Kozak sequence for optimal translation initiation (e.g., GCCA/GCCAUGG, where the purine at position –3 and the G at +4 have the strongest effects; Ref. 33 ). However, AUG2 (AGGATGG) was a reasonable match to the optimal consensus sequence.

To assess the extent to which translation was initiated from these upstream AUGs, hRFC cDNA constructs were prepared, including A1/A2 5'UTR sequence with AUGs 3 and 2 (designated hRFCAUG3HA and hRFCAUG2HA, respectively) fused to full-length coding sequence at position –49. An additional construct was prepared (khRFCAUG2HA) in which the "normal" sequence flanking AUG1 was mutated (to CGGATGG) to additionally reduce translation initiation at this site. This change had no effect on the amino acid sequence of the putative polypeptide translated from AUG2, because both AGG and CGG encode Arg. All of the constructs included a HA epitope at amino acid 587. The HA-tagged KS43 hRFC cDNA (25) , including the B 5'UTR and translated from AUG1 (designated hRFCAUG1HA), was prepared as a positive control. By in vitro translation from transcripts transcribed from constant (1 µg) plasmid DNAs with the T7 promoter, the hRFCAUG1HA construct generated a 65 kDa protein, and a 72 kDa protein was expressed from the hRFCAUG2HA and khRFCAUG2HA constructs (Fig. 4)Citation . However, there was an ~12-fold difference between the levels of hRFCAUG1HA, and the hRFCAUG2HA and khRFCAUG2HA proteins. No translation appeared to initiate from AUG3, because hRFC protein translated from the hRFCAUG3HA construct was indistinguishable from hRFCAUG2HA and hRFCAUG1HA (data not shown). The amino acid sequence encoded from the upstream AUG2 and continuing into the common sequence for hRFC is shown in Fig. 1BCitation .



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  		Fig. 4. In vitro translation of human reduced folate carrier (hRFC)AUG1HA, hRFCAUG2HA, and khRFCAUG2HA proteins. Empty pCDNA3, and hRFCAUG1HA, hRFCAUG2HA, and khRFCAUG2HA inserts cloned in pCDNA3 (1 µg each) were transcribed from the T7 promoter and translated using the TNT Quick T7 Coupled Transcription/Translation system (Promega). Ten microliters of each reaction and 30 µg of deglycosylated hRFCAUG1HA membrane protein (deG hRFCAUG1HA) as a control were separated on a 7.5% SDS-PAGE gel, transferred to polyvinylidene difluoride membrane, and probed with antihemagglutinin primary and antimouse-horseradish peroxidase-conjugated secondary antibodies. The sizes of the immunoreactive proteins are noted.

 
hRFCAUG2HA, khRFCAUG2HA, and hRFCAUG1HA constructs were each transfected into hRFC-null K500E cells. G418-resistant clones were selected in soft agar, and plasma membrane proteins were screened for hRFC expression on Western blots with hRFC- or HA-specific antibodies (Fig. 5)Citation . Despite differences in translation efficiency, we were able to select clones expressing similar levels of hRFC proteins. Predictably, hRFC transcripts were ~2.8-fold lower in hRFCAUG1HA transfectants than khRFCAUG2HA transfectants (Fig. 5A)Citation . hRFCAUG2HA and khRFCAUG2HA transfectants expressed proteins with apparent masses slightly larger (~92 kDa) than that for the wild-type hRFCAUG1HA (~85 kDa; Fig. 5, B and DCitation ). Deglycosylation with N-glycosidase F decreased the molecular masses for both hRFCAUG2HA and khRFCAUG2HA to ~72 kDa, whereas the hRFCAUG1HA protein decreased to ~65 kDa (Fig. 5, C and D)Citation . By densitometry, the percentages of the ~72 kDa form to total hRFC (including both ~65 kDa and ~72 kDa forms) ranged from ~60% for hRFCAUG2HA to ~90% for khRFCAUG2HA. Thus, mutation of the AUG1 Kozak sequence augmented the level of the ~72 kDa form over the ~65 kDa species.



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  		Fig. 5. Northern and Western blots for human reduced folate carrier (hRFC)AUG1HA, hRFCAUG2HA, and khRFCAUG2HA cells. A, total RNAs (20 µg) were fractionated on a 0.8% agarose/formaldehyde gel, capillary transferred to nitrocellulose, and probed with a 32P-labeled full-length hRFC probe (top). Equal loading was established with ethidium bromide staining (bottom). B, proteins (30 µg) were fractionated on a 7.5% gel in the presence of SDS and electroblotted onto a polyvinylidene difluoride membrane. Detection was with primary anti-hRFC and secondary antirabbit-horseradish peroxidase-conjugated antibodies and an enhanced chemiluminescence kit. C, membrane fractions were deglycosylated with N-glycosidase F before separation by SDS-PAGE, blotting, and detection with anti-hRFC antibody. For B and C, Western blotting results are also shown for the hRFC-null K500E cells. D, glycosylated (lanes 1 and 2) and deglycosylated (lanes 3 and 4) membrane fractions were separated by SDS-PAGE and probed with primary antihemagglutinin and secondary antimouse peroxidase-conjugated antibodies. Differential migrations of hemagglutinin-tagged hRFCAUG2 and hRFCAUG1 proteins are apparent in both glycosylated and deglycosylated samples.

 
There were no apparent differences between the turnover rates for both hRFCAUG1HA and hRFCAUG2HA transcripts (half-life, ~ 6 h) on Northern blots after treatment of transfected cells with 10 µg/ml actinomycin D or for hRFCAUG1HA and hRFCAUG2HA proteins (half-life, ~ 24 h) after treatment with 0.2 mg/ml cycloheximide (data not shown). By confocal analysis of transfected cells with HA-specific antibody and Alexa488 staining, there was no detectable difference in relative membrane trafficking of khRFCAUG2HA from hRFCAUG1HA (Fig. 6)Citation . Furthermore, no significant differential staining of intracellular structures was detected.



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  		Fig. 6. Confocal microscopy of human reduced folate carrier (hRFC)AUG1HA (A), khRFCAUG2HA (B), and hRFC-null K500E (C) cells, showing localization to the plasma membrane. Cells were fixed with 3.3% paraformaldehyde, permeabilized with 0.1% Triton X-100, incubated with mouse antihemagglutinin primary antibody followed by antimouse IgG-Alexa Fluor 488-conjugated secondary antibody, and spun onto microscope slides. Slides were visualized with a Zeiss laser scanning microscope 310 using a x63 water immersion lens.

 
Transport Characteristics of the hRFCAUG1HA and hRFCAUG2HA Proteins.
Cellular uptake of [3H]MTX (at 0.5 µM) were assayed over 180 s in the hRFCAUG1HA, hRFCAUG2HA, and khRFCAUG2HA transfectants for comparison with K500E cells (Fig. 7)Citation . Whereas total levels of the HA-tagged hRFC proteins were comparable, uptakes for the hRFCAUG2HA and khRFCAUG2HA transfectants were ~82% and ~61%, respectively, of that for wild-type hRFCAUG1HA, thus paralleling the increasing levels of ~72 kDa deglycosylated hRFC in these sublines (Fig. 5)Citation . Kinetic parameters were determined for the hRFCAUG1HA and khRFCAUG2HA transfectants, expressing the maximal levels of proteins translated from AUG1 and AUG2, respectively. Distinct, albeit subtle, changes in both Kt and Vmax parameters were measured for the khRFCAUG2HA cells (Table 1)Citation that resulted in a statistically significant (P = 0.0072) decreased Vmax/Kt (~72% of the value for hRFCAUG1HA). These functional differences between the hRFCs translated from AUG2 additionally manifest as shifts in the inhibition constants for folate and antifolate substrates, reflecting slightly decreased binding affinities. As summarized in Table 1Citation , these inhibition constant effects were most notable for the novel antifolates, GW1843U89 and Pemetrexed.



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  		Fig. 7. Methotrexate uptake in K500E cells, and human reduced folate carrier (hRFC)AUG1HA, hRFCAUG2HA, and khRFCAUG2HA transfectants. Uptake of 0.5 µM [3H]methotrexate was measured over 180 s as described in "Materials and Methods." Transport values are the mean values of five experiments; bars, ±SE. The difference in uptake between the hRFCAUG1HA and khRFCAUG2HA transfectants was statistically significant (P = 0.0067).

 

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  		Table 1 Kinetic constants for hRFCAUG1HA and khRFCAUG2HA*

 
Identification of High Frequency Sequence Polymorphisms in the A1/A2 Noncoding Exon.
The finding of high frequency sequence variants in our 5'RACE clones (Fig. 1)Citation from primary ALLs suggested the occurrence of polymorphisms in the hRFC A1/A2 gene sequence. Accordingly, we amplified genomic sequence from positions –289 to 148 from DNAs from 27 peripheral blood or umbilical cord blood samples and 8 ALL samples, and sequenced the amplicons.

The –131 to –133 AGG deletion and the C/T –109 transition were confirmed at the genomic level. Deletion of the AGG appeared to cosegregate with the C/T –109 transition in the normal DNAs (9 of 27 homozygous +AGG/T, 7 of 27 heterozygous, 11 of 27 homozygous –AGG/C; allelic frequencies of 46% +AGG/T, 54% –AGG/C) and in the ALL DNAs (3 of 8 homozygous +AGG/T, 5 of 8 heterozygous; allelic frequencies of 69% AGG/T, 31% AGG/C).

The –131 to –133 AGG deletion would result in loss of Gly21 from the A1/A2 leader peptide translated from AUG2 (Fig. 1)Citation ; however, C/T –109 is silent (both TGT and TGC encode Cys). To assess the possible consequences from the loss of Gly21, the mutant Kozak construct, khRFCAUG2HA, was additionally mutated to delete the AGG codon from positions –133 to –131, generating del-khRFCAUG2HA. del-khRFCAUG2HA was in vitro translated and transfected into K500E cells and clonal isolates assayed for hRFC expression and transport function for comparison with hRFCAUG1HA and khRFCAUG2HA. del-khRFCAUG2HA was in vitro translated at comparable levels to khRFCAUG2HA and was expressed in K500E cells at an approximately equal level to that of the khRFCAUG2HA transfectant (data not shown). Furthermore, [3H]MTX uptakes (at 0.5 µM) for del-khRFCAUG2HA and khRFCAUG2HA transfectants were essentially identical and were decreased from the uptake for hRFCAUG1HA (data not shown). del-khRFCAUG2HA transfected cells also exhibited similar MTX kinetics [Kt = 2.46 + 0.170 µM (SE), Vmax = 19.3 ± 1.37 pmol/mg/3 min (SE), Vmax/Kt = 7.92 ± 1.25; n = 3] to those for the khRFCAUG2HA transfectant (Table 1)Citation . Thus, the deletion of Gly21 from the AUG2 leader peptide does not additionally alter the transport properties of the hRFCAUG2HA protein.


   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
This study significantly extends our prior reports of a complex transcriptional and posttranscriptional regulation of the hRFC gene (9, 10, 11) . hRFC is ubiquitously yet differentially expressed in human tissues and tumors and is regulated by multiple, alternatively spliced noncoding exons (originally designated A1, A2, A, B, C, D, and E) and promoters (9) . Apparent alternate splice forms were detected in normal human tissues and cell lines at various frequencies for four of these seven forms, including exons A, B, C, and D. Whereas evidence for separate A1 and A2 exons was also presented (9) , in our current analysis of primary ALL specimens, we found that the A1/A2 5'RACE clones contained A1 sequence alone or as a fusion of A1 and A2. This implies that these were likely derived from a single ~1021 bp A1/A2 noncoding region. Accordingly, we have revised our model of hRFC gene structure to include five upstream noncoding exons and an expanded (~1258 bp) coding exon 1 including the A1/A2 sequence (Fig. 1)Citation .

Our 5'RACE data suggested that hRFC transcripts including 5'UTRs derived from exons B and A1/A2 were the predominant forms in primary ALLs. This conclusion was confirmed by real-time PCR assays of 5'UTR usage. Total hRFC transcripts spanned a wide range (289-fold). Similar results were reported previously by Zhang et al. (7) for primary ALLs using a competitive RT-PCR assay.

In this report, we provide the first description of the transcriptional and posttranscriptional controls for the hRFC-A1/A2 noncoding region and promoter. Maximal promoter activity was localized to a 134-bp 5' fragment flanking the A1/A2 noncoding region by transient transfections of deletion constructs into HepG2 hepatoma and Jurkat leukemia cells. Potential transcriptional elements were identified within this region including E-box, Ikaros, and GATA elements. Studies are under way to additionally characterize the functional cis- elements and transcription factors that regulate the hRFC-A1/A2 promoter.

The A1/A2 noncoding region contains two upstream AUGs in-frame with the hRFC coding sequence, one (AUG2) of which includes a near optimal Kozak sequence for translation initiation. In vitro translation from transcripts including the A1/A2 and AUG2 resulted in a modified 72-kDa polypeptide, distinguishable from the 65-kDa form translated from AUG1. The size difference is exactly that predicted from the addition of 64 amino acids to the NH2 terminus of hRFC. Whereas there were no differences in stabilities of transcripts including the B and A1/A2 5'UTRs, differences in translation efficiency were detected. Transfection of hRFC-null K500E cells with a full-length HA-tagged hRFC cDNA construct, including the A1/A2 UTR and AUG2, permitted isolation of clones with similar levels of expression of the hRFCAUG2HA and hRFCAUG1HA proteins despite differences in hRFC transcripts. Deglycosylation of the glycosylated hRFCAUG2HA protein with N-glycosidase F resulted in a predominant ~72-kDa isoform that was again distinguishable from the ~65-kDa deglycosylated hRFCAUG1HA protein. Significantly, there were no apparent differences in protein stability or in targeting of hRFCAUG2HA to the plasma membrane from that for the wild-type hRFCAUG1HA form. The latter is consistent with previous reports that an intact NH2 terminus is not essential for intracellular trafficking and that removal of 16 amino acids (residues 7–22) from the hamster RFC or 27 NH2-terminal amino acids (residues 1–27) from hRFC does not appreciably affect membrane targeting or carrier function (34 , 35) .

Interestingly, the hRFCAUG2HA protein was competent for MTX transport, although transport properties differed somewhat from those of comparably expressed wild-type (AUG1) clones. Most notable was a statistically significant decrease in MTX Vmax/Kt for hRFCAUG2HA compared with hRFCAUG1HA and slight increases in the inhibition constants for certain transport substrates such as Pemetrexed and GW1843U89. Accordingly, utilization of the A1/A2 5'UTR in tissues and tumors, including ALLs, may confer an altered pattern of antifolate sensitivities, reflecting the relative level of the hRFCAUG2 compared with hRFCAUG1. Whereas the magnitude of this effect could conceivably be additionally affected by the presence of high frequency sequence polymorphisms in the A1/A2 noncoding region, such as the 3-bp deletion from positions –133 to –131 that results in loss of Gly21 from the A1/A2 leader peptide, no functional differences were detected between hRFCAUG2 proteins with or without Gly21.

In conclusion, our results further document the use of a novel noncoding region in hRFC, designed A1/A2. The A1/A2 region was originally believed to be divided into two noncoding segments; however, the present study demonstrates a single ~1-kb noncoding sequence juxtaposed to the original hRFC coding exon 1 with a proximal promoter activity localized to within 134 bp. Our previous 5' RACE results suggested that transcripts including the A1/A2 5'UTR could be detected in normal tissues including liver, heart, lung, and bone marrow (9) . Our present real-time PCR data demonstrate that A1/A2 forms are major hRFC transcript forms in primary ALL lymphoblasts from patients. Importantly, use of the A1/A2 5'UTR results in the synthesis of a novel hRFC isoform from an upstream AUG with significantly impaired translation efficiency and with somewhat altered transport properties from those for the wild-type carrier. These characteristics could possibly explain our earlier findings of disparate levels of hRFC transcripts and MTX transport in a significant number of ALL lymphoblast specimens (7) . Future studies will focus on additionally identifying the major transcriptional and posttranscriptional regulatory features of the A1/A2 noncoding exon and promoters and on the pharmacological and physiological ramifications of expressing the novel hRFCAUG2 protein in human tissues and tumors including ALL.


   ACKNOWLEDGMENTS
 
We thank Drs. Murray Norris and Michelle Haber for providing genomic DNAs for our studies.


   FOOTNOTES
 
Grant support: Grants CA76641 and CA53535 from the National Cancer Institute, NIH, and a grant from Leukemia Research Life, Inc.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Requests for reprints: Larry H. Matherly, Experimental and Clinical Therapeutics Program, Karmanos Cancer Institute, 110 East Warren Avenue, Detroit, MI 48201. Phone: (313) 833-0715, extension 2407; Fax: (313) 832-7294; E-mail: matherly{at}karmanos.org

5 The nucleotide sequences for noncoding exons A1, A2, C, D, and E appear in GenBank and European Molecular Biology Laboratory databases with accession numbers AY089985, AY089986, AY089987, and AY089988. Exons A and B appear under accession no. AF046920. AY89985 for noncoding exons A1 and A2 has been amended to denote a single A1/A2 noncoding exon as described in this report. Back

Received 1/20/04; revised 4/ 8/04; accepted 4/16/04.


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 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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