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[Cancer Research 61, 1941-1947, March 1, 2001]
© 2001 American Association for Cancer Research

Experimental Therapeutics

Localization of a Human Reduced Folate Carrier Protein in the Mitochondrial as Well as the Cell Membrane of Leukemia Cells1

Tanya M. Trippett2, Shawna Garcia, Katia Manova, Roshni Mody, Leona Cohen-Gould, Wayne Flintoff and Joseph R. Bertino

Departments of Pediatrics [T. M. T., S. G., R. M.], Molecular Cytology [K. M.], and Molecular Pharmacology and Therapeutics [J. R. B.], Memorial Sloan-Kettering Cancer Center, New York, New York 10021; Department of Anatomy and Physiology, Weill-Cornell University Medical College, New York, New York 10021 [L. C-G.]; and Department of Microbiology and Immunology, University of Western Ontario, London, Ontario, N6A5CI Canada [W. F.]


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
IgG polyclonal antiserum was generated in New Zealand White rabbits immunized with a 16-mer peptide consisting of a specific amino acid sequence at residues corresponding to the sixth to seventh predicted transmembrane domain of the human reduced folate carrier (RFC). Using Western immunoblotting to examine the cytosolic and membrane fractions of the human CCRF-CEM T-cell lymphoblastic leukemia cell line, polyclonal antihuman RFC antiserum recognized two bands in the cytosolic fraction (approximately 60 kDa and approximately 70 kDa) on 10% polyacrylamide gels. In the membrane fraction, an approximately 60-kDa protein was identified. Comparative studies of a panel of human tumor cell lines including the HT1080 fibrosarcoma, 8805 malignant fibrous histiocytoma, and the MCF breast cancer cell lines revealed similar findings. Likewise, a recombinant approximately 60-kDa membrane protein was identified after expression of baculovirus-infected Sf9 insect cells containing cDNA of the human RFC. In the CEM-7A cell line, a variant of the CCRF-CEM cell line that overexpresses the RFC, 21-fold overexpression of the approximately 60-kDa membrane protein (RFC) was shown by Western analysis. To characterize further the cellular distribution of the human RFC, immunohistochemical analyses were performed in CCRF-CEM T-cell lymphoblastic leukemia cells. Predominantly membrane localization of the antibody reacting sites was detected; however, a cytoplasmic component was noted as well. By confocal microscopy and by immunogold electron microscopy, the cytoplasmic expression was found to be largely of mitochondrial origin. These findings were corroborated by Western immunoblotting of mitochondrial membrane isolates from the CCRF-CEM cell line, which demonstrate an approximately 60-kDa protein. The localization of the human RFC to the mitochondrial membrane is a novel finding, and it suggests a role for the mitochondrial membrane in the transport of folates.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Transport of MTX3 into tumor cells occurs through two active processes. The predominant route of influx of MTX and reduced folates is via the RFC. The RFC, described in murine L1210 (1, 2, 3) and human CCRF-CEM lymphoblastic leukemia cells (4) , is a low affinity, Kt = 0.3–4.0 µM, and high capacity membrane transport protein for reduced folates and MTX (5, 6, 7, 8) . The RFC has a lower affinity for folic acid (>=200 µM) and transports folic acid poorly (5, 6, 7, 8) . The second transport system, the folate receptor or FBP, which has been described in T47D human mammary carcinoma, human nasopharyngeal epidermoid carcinoma (KB), MA104 monkey kidney, L1210, and CCRF-CEM cells, transports folic acid and 5-methyltetrahydrofolic acid more efficiently (Kt = 1–5 nM) than MTX (Kt = 300 nM). It plays a role in the uptake of MTX and 5-methyltetrahydrofolate in normal epithelial cells, placenta, and certain neoplasms, such as nonmucinous ovarian carcinoma and some brain tumors (9, 10, 11, 12, 13, 14) . In several established MTX-resistant cell lines, impairment in the function of the RFC (decrease in Vmax or increased Kt) has been shown to account for resistance to MTX (15, 16, 17, 18, 19) . Defective transport of MTX accounts for a major mechanism of acquired resistance in tumor cell lines and also in leukemia cells obtained from patients previously treated with MTX (19, 20, 21) .

Recently, the cDNA for the putative RFC (RFC1) gene from the mouse, hamster, and human was cloned and mapped by fluorescence in situ hybridization in human cells to the long arm of chromosome 21 (q22.2 and q22.3; Refs. 22, 23, 24, 25 ). Transfection of the RFC cDNA in MTX transport-defective hamster and human cell lines reconstitutes the ability of these cells to transport MTX (24, 25, 26, 27) .

Sequence homology exists between species (65% homology between the human, murine, and hamster homologues) for the RFC gene, which encodes a glycoprotein belonging to a superfamily of transmembrane-spanning transport proteins with 12 predicted transmembrane spanning domains. The predicted molecular core mass of the human RFC (65 kDa) has been shown to differ from that of the mouse (58 kDa) and hamster (59 kDa). Little information is currently available regarding the expression of the RFC protein in human tumors. To study the characteristics of RFC expression in human leukemia cell lines, we generated an IgG polyclonal antibody in New Zealand rabbits immunized separately with a 16-mer MAP peptide. This peptide corresponded to a specific region in transmembrane domain 6–7 of the human RFC.


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Cell Culture.
The CCRF-CEM human T-cell lymphoblastic leukemia and fibrosarcoma HT1080 cell lines obtained from American Type Culture Collection and the malignant fibrous histiocytoma cell line (8805; established in this laboratory) were all maintained in RPMI 1640 with 10% fetal bovine serum, 1% L-glutamine, 100 units/ml penicillin, and 100 µg/ml streptomycin. The wild-type MCF breast cancer cell line was maintained in improved MEM with 10% fetal bovine serum, 1% L-glutamine, 100 units/ml penicillin, and 100 µg/ml streptomycin. The CEM-7A cell line, a variant of the CCRF-CEM lymphoblastic leukemia cell line with 30-fold overexpression of the RFC as shown by affinity labeling with NHS-[3H]MTX, was kindly provided by Dr. Gerrit Jansen, (University Hospital of Utrecht, Utrecht, the Netherlands; Ref. 28 ). The cell line was maintained in suspension culture in RPMI 1640, 10% horse serum, 1% L-glutamine, and 0.15 nM 5-methyltetrahydrofolate.

Generation of Polyclonal RFC Antiserum.
Computer analysis for determination of the Hopp and Wood hydrophilicity plot of the human RFC protein sequence was performed using the Hopwood Algorithm (Oxford Molecular Group, Campbell, CA). A highly antigenic region was identified at amino acid residues 205–220 located between the predicted transmembrane domains 6 and 7 of the human RFC located adjacent to the long internal cytoplasmic loop of the protein (Fig. 1)Citation . The resultant 16-mer was synthesized by Fmoc solid phase methods, using MAP resin technology (29 , 30) . MAP (0.5 mg) was emulsified with an equal volume of Freund’s adjuvant, and 1 ml was injected into three s.c. dorsal sites of two 3–9-month-old New Zealand White rabbits for primary immunization. Boosters with 0.5 mg of MAP emulsified in Freund’s incomplete adjuvant were performed at 3, 7, and 9 weeks after the primary immunization. The animals were bled before primary immunization (preimmune) and at 5, 9, and 11 weeks after immunization. Anti-MAP antibody titers were determined on all bleeds by ELISA with MAP on solid phase (1 µg/ml). The antibody titer is expressed as the reciprocal of the serum dilution resulting in an A492 nm of 0.2. (Detection with goat antirabbit IgG-horseradish peroxidase conjugate and peroxidase dye.)



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  		Fig. 1. Hopp and Wood hydrophilicity plot of the RFC protein sequence. Analysis of the human RFC protein sequence using the Hopp and Wood Algorithm. Highly antigenic region putatively expressed at the membrane surface and corresponding to amino acid residues 205–220 in transmembrane domain 6 and 7 adjacent to the internal loop of the human RFC was used to generate the polyclonal antisera (the region is designated by the hatch mark).

 
Reagents.
The MOv19 mouse monoclonal antibody and human placenta folate receptor protein (FBP) were generously provided by Dr. Barton Kamen (Robert Wood Johnson School of Medicine, New Brunswick, NJ). The MOv19 monoclonal antibody recognizes a 38-kDa glycosyl-phosphatidylinositol-anchored glycoprotein corresponding to the membrane-bound form of the folate receptor protein (31) .

Preparation of Plasma Membranes.
Membranes were prepared as described by Henderson and Zevely (8) . Crude membranes were suspended in lysis buffer and applied to a discontinuous gradient consisting of 12 ml each of 60% and 20% sucrose in lysis buffer. After centrifugation at 100,000 x g (1 h, 4°C), the 20–60% interface was collected, diluted 5-fold with lysis buffer, centrifuged at 16,000 x g, and resuspended in HEPES-buffered saline.

Mitochondrial Isolation.
Samples were prepared for mitochondrial isolation as described by Lin et al. (32) . All procedures were carried out at 4°C. CCRF-CEM cells (4 x 108 cells) were suspended in 1 ml of HMS and homogenized in a glass-Teflon homogenizer. Nuclei and unbroken cells were sedimented at 900 x g for 10 min. The supernatant was carefully removed without disturbing the pellet and transferred to another centrifuge tube. The pellet was resuspended in 1 ml of HMS, homogenized for four strokes, and then centrifuged at 900 x g for 10 min. The supernatant was combined with the first supernatant. The postnuclear supernatant was then centrifuged at 10,000 x g for 15 min, and the pellet was stored on ice. The supernatant was recentrifuged (10,000 x g, 15 min), and the second pellet was combined with the first, washed with 2 ml of HMS, and then resuspended in 1 ml of HMS to yield the mitochondrial fraction.

SDS-PAGE and Immunoblotting.
Aliquots of the cell membrane mitochondrial and cytosolic fractions from the human tumor cell lines mentioned above (30 µg) were resolved on a 10% SDS-PAGE reducing gel and transferred electrophoretically to nitrocellulose membranes (33) . Membranes were immersed in milk blocking buffer for 24 h and then washed with 1x TBST [25 mM Tris-HCl, 500 mM NaCl, and 0.1% Tween 20 (pH 7.5)] for 30 min. The blots were probed for 2 h with a 1:500 dilution of rabbit antihuman RFC antiserum as a primary antibody. The immunoblots were then incubated for 1 h with a 1:10,000 dilution of a horseradish peroxidase-conjugated goat antirabbit specific IgG (Sigma, St. Louis, MO) and visualized with enhanced chemiluminescence detection reagents (Amersham, Buckinghamshire, United Kingdom).

Nonreducing PAGE.
An aliquot of the FBP from human placenta (1 µg) was resolved on a nonreducing PAGE gel (12% running and 8% stacking gel) and transferred electrophoretically onto a nitrocellulose membrane. The blot was incubated for 1 h in blocking solution containing TBST-3% fish gel (34) . The blot was probed with a 1:10,000 dilution of MOv19 for 1 h. The blot was then incubated for 1 h with a horseradish peroxidase-conjugated goat antimouse Fab-specific IgG at a 1:10,000 dilution (Sigma). A complementary blot was also prepared and incubated for 2 h with rabbit antihuman RFC antiserum (1:500) and then incubated for 1 h with horseradish peroxidase-conjugated goat antirabbit IgG (1:10,000). Both blots were visualized with enhanced chemiluminescence detection reagents (Amersham).

Generation of Recombinant Baculovirus and Expression of RFC Gene.
The Bac-to Bac Baculovirus Expression system (Life Technologies, Inc., Grand Island, NY) was used for generation of recombinant baculovirus and RFC gene expression. The coding region of the human RFC cDNA was cloned into the multiple cloning site of a pFASTBAC donor plasmid. The recombinant plasmid was transformed into DH10BAC competent cells containing bacmid with a mini-attTn7 target site and a helper plasmid. The recombinant baculovirus was generated by site-specific transposon-mediated insertion of the RFC expression cassette from the pFASTBAC donor plasmid and the recombinant bacmid propagated in Escherichia coli. (35) . Cell lines derived from Spodoptera frugiperda (Sf 9) were then transfected with baculovirus, and expressed membrane protein was analyzed by Western immunoblotting.

Immunohistochemistry.
Cytospin preparations of the CCRF-CEM lymphoblastic leukemia cell line were fixed in precooled methanol:acetone (3:1) for 10 min at -20°C, dried vertically in a hood for 1–1.5 h, and washed three times in PBS with 0.1% BSA for 3 min each. To quench endogenous peroxidases, the slides were incubated for 15 min at room temperature in 0.1% H2O2 in PBS. Washes were repeated three times in PBS with 0.1% BSA (3 min). The slides were incubated for 30 min at room temperature with a 1:50 dilution of primary antibody. Immunostaining was performed using the Vectastain Elite ABC kit (Vector Laboratories, Burlingame, CA) following the kit instructions. Incubations with secondary and tertiary antibodies were performed for 1 h at room temperature. After the diaminobenzidine tetrahydrochloride (DAB) reaction, the slides were counterstained with Gill’s hematoxylin, dehydrated in ethanol, and mounted in Permount.

Electron Microscopy.
Ultrastructural analysis was performed in Spurrs resin according to the procedure described by Berryman et al. (36) .

Immunogold Electron Microscopy.
Cells were prepared by cryoultramicrotomy as described by Tokuyasu (37) . Cells were fixed in 4% paraformaldehyde and 0.1% glutaraldehyde in PBS at room temperature for 15 min, washed in cold PBS, and resuspended in 10% gelatin (porcine skin 75-100; Sigma). Cells were then centrifuged at 2000 rpm for 10 min, chilled to solidify the gelatin, and then infused with 2.3 M sucrose at 4°C overnight. Ultrathin sections were prepared using a RMC MT7000 microtome equipped with a CRX cryostage (Ventana-RMC, Tucson, AZ), collected on nickel grids, and immunolabeled with a 1:50 dilution of antihuman RFC antiserum and a 1:50 dilution of IgG goat antirabbit gold particles (10 nm).

Immunofluorescence.
A cell suspension was prepared at a concentration of 200,000 cells/ml, attached to slides by cytospin, and fixed in 4% paraformaldehyde. The slides were blocked in 5% normal goat serum in PBS for 20 min and then incubated in the presence of a 1:50 dilution of rabbit antihuman RFC antiserum with 2% normal goat serum at 4°C overnight. The slides were washed three times in PBS with 0.5% BSA for 5 min and then incubated for 1 h with a 1:10,000 dilution of a FITC-conjugated goat antirabbit IgG (Dako Corp., Carpenteria, CA) at 4°C. Washes were repeated three times in PBS containing 0.5% BSA for 5 min, followed by a single wash in PBS. Nuclear staining was then performed by incubation with 0.1 µg/ml Hoechst No. 33342 trihydrochloride (Sigma) for 3 min. For the triple immunofluorescence staining, live cells were labeled in suspension with 25 nM MitoTracker Red CMXRos (Molecular Probes, Eugene, OR). The cells were then attached to slides by cytospin, fixed, and processed for immunofluorescence with each of the two RFC polyclonal antibodies and an antibody for chromatin (38 , 39) . A Zeiss (Thornwood, NY) Axiophot 2 microscope equipped for epifluorescence and with a DCS5C Kodak digital camera and a Zeiss LSM510 confocal microscope were used for image acquisition.

Measurement of Succinate Dehydrogenase Activity in Mitochondria.
The mitochondrial preparation was diluted with 0.5 ml of sucrose-Tris-EDTA isolation buffer. The sample was then assayed at room temperature for succinate dehydrogenase activity (40) . The following reagents were added to a cuvette: 1.4 ml of 0.3 M sodium azide; 0.1 ml of 0.5 mg/ml DCPIP; 0.3 ml of water; and 0.1 ml of cell fraction. To measure the endogenous rate of DCPIP reduction, the rate of change in absorbance at 600 nm was recorded. After determining the endogenous rate, 0.1 ml of 1 M sodium succinate was added to the same cuvette, the sample was mixed gently, and the initial change in absorbance at 600 nm with time was recorded. The endogenous rate was then subtracted from the initial rate with succinate to quantitate the activity of succinate dehydrogenase.


   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
RFC Protein Expression in the Cytosolic and Membrane Fractions of the CCRF-CEM T-Cell Lymphoblastic Leukemia Cell Line.
Protein extracts from the CCRF-CEM cell line were examined by Western blot analysis. Resolution on 10% SDS-PAGE demonstrated the presence of two bands of approximately 60 and 70 kDa, respectively (Fig. 2A)Citation . In membrane preparations prepared by sucrose density gradient centrifugation, an approximately 60-kDa protein was also demonstrated on Western immunoblotting (Fig. 2B)Citation . Comparative analyses of protein extracts from a variety of human tumor cell lines including the HT1080 fibrosarcoma cell line, a 8805 malignant fibrous histiocytoma cell line, and the MCF human breast cancer cell line demonstrated similar results. The recombinant membrane protein generated after expression of the RFC cDNA in baculovirus-infected insect cells migrated at the same location (60 kDa) on 10% SDS-PAGE (Fig. 2B)Citation , confirming the specificity of the antibody for the RFC.



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  		Fig. 2. Western analysis of cytosolic and membrane preparations from a panel of human tumor cell lines. A, cytosolic proteins (30 µg) from a panel of human tumor cell lines were resolved by 10% SDS-PAGE and probed with rabbit polyclonal antihuman RFC antiserum (peptide 205–220; see "Materials and Methods"). Lane 1, CCRF-CEM; Lane 2, MCF; Lane 3, malignant fibrous histiocytoma cell line 8805; Lane 4, fibrosarcoma cell line HT1080. The standard molecular mass markers are indicated. No immunoreactive bands were noted with identical samples incubated with preimmune serum data (data not shown). B, membrane preparations (30 µg) were prepared by sucrose density gradient centrifugation as described in "Materials and Methods" and electrophoresed on a 10% SDS-polyacrylamide gel for immunoblot analysis using rabbit polyclonal antihuman RFC antiserum (peptide 205–220). Lane 1, CCRF-CEM; Lane 2, fibrosarcoma cell line HT1080; Lane 3, MCF; Lane 4, malignant fibrous histiocytoma cell line 8805; Lane 5, untransfected Sf9 insect cells (control); Lane 6, recombinant protein from Sf9 insect cells (see "Materials and Methods"). No immunoreactive bands were noted with identical samples incubated with preimmune serum (data not shown). Standard molecular weight mass are indicated. C, membrane preparations (30 µg) prepared by sucrose density gradient centrifugation from the human T-cell lymphoblastic leukemia cell lines CCRF-CEM and CEM-7A were electrophoresed on a 10% SDS-polyacrylamide gel for immunoblot analysis using rabbit polyclonal anti-human RFC antiserum (peptide 205–220). Lanes 1 and 2 represent membrane proteins from the CCRF-CEM and CEM-7A cell lines, respectively. Standard molecular mass markers are indicated.

 
RFC Protein Overexpression in the CCRF-CEM T-Cell Lymphoblastic Leukemia Cell and CEM-7A Cell Lines.
In the CEM-7A cell line (Fig. 2C)Citation , 21-fold overexpression of an approximately 60-kDa protein was detected in the membrane fraction by densitometry after immunoblotting in comparison to the parental CCRF-CEM cell line. There was no evidence of overexpression in the cytosolic fraction, although a similar doublet was seen in the lysates of both cell lines.

To verify that the antiserum only recognized RFC protein, a Blast/Beauty search was performed using a basic local alignment search tool. Sequence homology at the protein level for the respective peptides at amino acid residues 205–220 was determined to be 100% between the RFC and the FBP, raising the possibility that the RFC antibody might cross-react with the FBP. To clarify the specificity of the antiserum raised against the peptide localized in the sixth to seventh transmembrane domain, nonreducing PAGE was used to test for cross-reactivity of the polyclonal antihuman RFC antiserum to the FBP. One µg of purified human placenta folate receptor was resolved on a nonreducing PAGE gel (12% running and 8% stacking gel) and probed with the anti-folate receptor mouse monoclonal antibody, MOv19 (1:10,000). A broad band was observed at 38 kDa corresponding to the anticipated mass of the folate receptor. A complementary blot was prepared and probed with the RFC polyclonal antibody (1:500). Immunoreactive bands were not observed on probing with the human RFC antiserum, thus confirming the lack of cross-reactivity of the antibody with the FBP (data not shown).

Cellular Localization of the Human RFC Immunohistochemistry.
The immunohistochemical staining of CCRF-CEM leukemia cells using the peptide 205–220 RFC antiserum also demonstrated staining of the cell membrane and some cytoplasmic staining (Fig. 3)Citation , confirming the previously described results of the Western immunoblotting.



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  		Fig. 3. Immunohistochemistry of CCRF-CEM T-cell lymphoblastic leukemia cell line. A, immunohistochemical staining on incubation with preimmune serum is shown as a control. B, immunohistochemical staining of blasts from the CCRF-CEM cell line incubated with a 1:50 dilution of human RFC antiserum demonstrated localization of the staining to the membrane. The nucleus was counterstained with Gill’s hematoxylin.

 
Intracellular Localization of the RFC to Mitochondria by Immunogold Electron Microscopy.
To further clarify the cytoplasmic expression of the RFC protein observed and to determine the intracellular localization of the antigen binding of the RFC antiserum, experiments were conducted using immunogold electron microscopy. Both the parental CCRF-CEM cell line and the RFC overexpressing CEM-7A subline were used. A similar pattern of localization of immunogold labeling was noted in the CCRF-CEM and CEM-7A cell lines, with labeling observed not only at the cell membrane but also in the cytoplasm. However, the antigenic distribution in the CCRF-CEM cell line was sparse compared with that in the CEM-7A cells (data not shown). Localization of the immunogold labeling in CEM-7A cells is shown in Fig. 4Citation . Thin sections of LR-White-embedded CEM-7A cells were incubated with undiluted antihuman peptide 205–220 RFC antiserum followed by a 1:50 dilution of IgG goat antirabbit gold particles (10 nm). The ultrastructural analysis of the intracellular compartment revealed prominent localization of the immunogold complexes to the mitochondria membrane as well as to the plasma membrane (Fig. 4, C and D)Citation .



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  		Fig. 4. Immunogold electron microscopy in CEM-7A T-cell lymphoblastic leukemia cell lines. Ultrathin sections of CEM-7A T-cell lymphoblastic leukemia cells prepared by cryoultramicrotomy and immunolabeled with a undiluted of anti-human RFC antiserum and a 1:50 dilution of IgG antirabbit gold particles (10 nm) are shown in B–D. A, ultrastructure of CEM-7A leukemic blasts in Spurr’s resin. Magnification, x27,000. B, membrane localization of the RFC antiserum (arrows) in the CEM-7A blasts. C, ultrastructure of CEM-7A by cryoultramicrotomy. D, prominent localization of 10 nm of gold particles to the mitochondria (arrows) is shown. Magnification, x 49,000. M, mitochondria; N, nucleus.

 
Immunofluorescence.
To confirm the expression pattern detected by the immunogold staining, colocalization analyses were performed using confocal microscopy. For this purpose, vital staining of CCRF-CEM T-cell lymphoblastic leukemia cells with a mitochondrion-selective fluorescent probe (25 nM MitoTracker Red CMXRos), followed by immunofluorescence detection with the antisera and Hoechst No. 33342 trihydrochloride counterstaining, was applied. The results shown in (Fig. 5)Citation indicate distinctive membrane and cytoplasmic localization in all cells, with prominent colocalization to the mitochondrial membrane (yellow or yellow-orange color). In Fig. 5DCitation , a single cell is shown at higher magnification (x100) that clearly demonstrates the degree of colocalization. Of interest is the perinuclear localization of the mitochondria also seen on electron microscopy. This pattern was absent when cells were exposed to a similar dilution of preimmune serum. Thus, confocal microscopy substantiated the findings on immunogold electron microscopy and confirmed the presence of RFC protein in the mitochondrial as well as the plasma membrane.



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  		Fig. 5. Immunofluorescence of CCRF-CEM T-cell lymphoblastic leukemia cell line. Methanol-fixed CCRF-CEM T cell lymphoblastic leukemia cells were incubated with polyclonal RFC antisera. A, mitochondrial staining pattern with 25 nM MitoTracker Red. B, antigen-antibody complexes detected with a 1:50 dilution of FITC-conjugated IgG secondary antibody. RFC protein was detected at the cellular membrane as well as the cytoplasm in all cells. C, nuclear staining with 0.1 µg/ml Hoescht No. 33342 trihydrochloride. D, triple staining with FITC-conjugated secondary antibody, 0.1 µg/ml Hoescht No. 33342 trihydrochloride, and 25 nM MitoTracker Red shows prominent membrane staining at the cellular membrane and colocalization at the mitochondrial membrane, indicated by simultaneous fluorescence producing a yellow-orange signal (arrows) wherever RFC protein is detected. E, a single leukemic blast is shown at a higher magnification (x100) that shows distinct colocalization of the RFC protein to the mitochondrial membrane. Bar, 10 µM.

 
To validate these findings even further, mitochondria from CCRF-CEM cells were isolated and analyzed for the presence of the RFC protein by immunoblotting. To confirm the presence of mitochondria, the activity of succinate dehydrogenase, a unique enzyme of the Krebs cycle that is tightly bound to the inner mitochondrial membrane, was measured. In the mitochondrial fraction, reduction of DCPIP was noted, and the measured specific activity of succinic dehydrogenase was 0.176 unit/mg protein, confirming the presence of mitochondria in the preparation. Western analysis (Fig. 6)Citation demonstrated evidence of a single, approximately 60-kDa protein that migrates on 10% polyacrylamide gels in the same location as the lower molecular mass band seen in previous Western analyses of the cytosolic fraction.



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  		Fig. 6. Western analysis of the mitochondrial isolates from the CCRF-CEM cell line. Western immunoblotting was performed after electrophoresis of mitochondrial isolates from the CCRF-CEM cell line and was compared to whole cell lysate, plasma membrane, and cytosolic preparations. Lane 1, plasma membrane; Lane 2, mitochondrial membrane; Lane 3, whole cell lysate.

 

   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The RFC is the predominant route of entry of MTX and endogenous reduced folates into most mammalian cells. The open reading frame of the RFC cDNA predicts a protein with a molecular mass of 65 kDa. The RFC is an integral membrane protein that, based on predictions by hydropathy plotting, consists of 12 putative transmembrane-spanning domains flanked by NH2 and COOH termini and a long internal loop between transmembrane domains 6 and 7 localized adjacent to the cytoplasm. In this study, rabbit polyclonal antisera was generated against a 16-mer MAP peptide, corresponding to a sequence localized to a highly antigenic region between transmembrane domains 6 and 7 (adjacent to the internal loop). The antibody was used to characterize the expression of RFC in human tumor cell lines.

Western analysis of membrane preparations isolated from a panel of cell lines by sucrose density gradient centrifugation demonstrated an approximately 60-kDa membrane protein in the CCRF-CEM, MCF, malignant fibrous histiocytoma (8805), and fibrosarcoma (HT1080) cell lines. These findings were similar to and consistent with those reported by Moscow et al. (24) , who also used peptide-specific antiserum and Western blotting. Expression of the full-length RFC cDNA in the baculovirus system also resulted in the generation of an approximately 60-kDa recombinant membrane protein also recognized by this antibody. Interestingly, a second band at approximately 70 kDa was also detected in the cytosolic fractions of the CCRF-CEM, MCF, malignant fibrous histiocytoma (8805), and fibrosarcoma (HT1080) cell lines. Recent studies suggest that transcription of the murine RFC may be regulated by the use of alternative transcriptional start sites that result in the generation of RFC splice variants (41 , 42) . The murine RFC gene comprises eight exons, six primary exons and alternates to exon 1 and 5. In the L1210 and murine erythroleukemia cell lines, the first two exons encode alternative 5' termini. Based on sequence identity analysis of cDNAs encoding the RFC, a cryptic splice donor site within exon 4 and a cryptic splice acceptor site within exon 5 have been identified in the hamster and rat. This may account for the generation of several alternatively spliced transcripts. Additionally, an alternative exon located between exons 5 and 6 has been identified that encodes the largest protein. These splice variants appear to vary based on the cell line or tissue of origin. The presence of two forms of the protein in the cytosolic fraction of the CCRF-CEM T-cell lymphoblastic leukemia cell lines as well as the panel of representative cytosolic fractions from a variety of human tumor cell lines may be explained by the presence of isoforms, one of which is targeted to the membrane. Alternatively, the larger size protein could be the precursor of the membrane protein or a posttranslationally modified form of the RFC. Studies are under way to distinguish between these possibilities. Analysis of the human leukemia cell line CEM-7A, which is known to have 30-fold expression of the RFC compared to the wild-type CCRF-CEM cell line, demonstrated 21-fold overexpression of RFC in membrane preparations, a finding that was consistent with previously published reports (28) .

The most intriguing result of this study was the demonstration of colocalization of the RFC to the mitochondrial membrane. One explanation for these findings may be that the protein may not be the RFC itself but a related protein or an unknown protein with a similar motif and molecular mass. Recently, a protein with a molecular mass of 35 kDa that transports folates in mitochondria was identified in CHO cells (43) . In mammalian cells, mitochondrial as well as cytosolic forms of the constituents of folate metabolism have been demonstrated, including serine hydroxymethyltransferase, folylpolyglutamate synthetase, and dihydrofolate reductase (32 , 44) . In CHO cells, studies have demonstrated that the mitochondrial folate pool is not in equilibrium with the cytosolic pool (32) . In CHO cells expressing only human folylpolyglutamyl synthetase, MTX was metabolized to polyglutamates to the same extent as human cells; however, the mitochondrial forms of the folylpolyglutamates differed in length (44) and were longer in chain length than the cytosolic folates. Mitochondrial folate accumulation was found to be significantly impaired by MTX treatment, which suggested the presence of a mitochondrial folate transport system (44) . Previous studies in rat liver mitochondria characterized the uptake of reduced folates as a carrier-mediated transport system (45) , first saturable at low concentrations of 5-formyltetrahydrofolate (Km = 2.8 µmol/liter), and second a linear function of concentration from 10–30 µmol/liter. Additional evidence to support these findings was the inhibition of 5-formyltetrahydrofolate uptake into mitochondria by 5-methyltetrahydrofolate with half-maximal inhibition at ~4 µmol/liter; however, in this study, neither MTX nor folic acid was a significant inhibitor. Our findings show that the RFC or a RFC-related protein is also found in the mitochondrial membrane as well as in the cellular membrane. The recent finding of a folate transport protein in CHO mitochondria with a lower molecular mass may indicate species differences or the presence of more than one carrier protein for folates in mitochondria.


   ACKNOWLEDGMENTS
 
We thank Rob Seitz and the staff of the Antibody Division of Research Genetics, Inc. for assistance in the generation of the RFC polyclonal antibody. We thank Dr. Gerrit Jansen for providing the CEM-7A cell line. We also thank Scott Kerns and Ali McBright (Molecular Cytology Core Facility, Memorial Sloan-Kettering Cancer Center, New York, NY) for assistance with microscopy.


   FOOTNOTES
 
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.

1 Supported by USPHS Grant CA08010 from the National Cancer Institute. Back

2 To whom requests for reprints should be addressed, at Department of Pediatrics, Memorial Sloan-Kettering Cancer Center, 1275 York Avenue, New York, NY 10021. Phone: (212) 639-8267; Fax: (212) 639-2767; E-mail: Trippet1{at}MSKCC.org Back

3 The abbreviations used are: MTX, methotrexate; RFC, reduced folate carrier; MAP, multiple antigenic peptide; FBP, folate-binding protein; HMS, homogenization solution [0.25 M sucrose and 1 mM EDTA (pH 6.9)]; DCPIP, 2,6-dichloroindophenol; CHO, Chinese hamster ovary. Back

Received 6/30/00. Accepted 12/21/00.


   REFERENCES
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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