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Originally published In Press as doi:10.1074/jbc.M308700200 on September 7, 2003

J. Biol. Chem., Vol. 278, Issue 47, 47156-47165, November 21, 2003
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Regulation of Progenitor Cell Fusion by ABCB5 P-glycoprotein, a Novel Human ATP-binding Cassette Transporter*

Natasha Y. Frank{ddagger}§, Shona S. Pendse§, Peter H. Lapchak¶, Armen Margaryan¶, Debbie Shlain¶, Carsten Doeing¶, Mohamed H. Sayegh§||, and Markus H. Frank§||**

From the {ddagger}Partners Center for Human Genetics, Harvard Medical School, Boston, Massachusetts 02115, the §Laboratory of Immunogenetics and Transplantation, Renal Division, Department of Medicine, Brigham and Women's Hospital, Boston, Massachusetts 02115, and the Nephrology Division, Children's Hospital, Harvard Medical School, Boston, Massachusetts 02115

Received for publication, August 6, 2003


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Cell fusion involving progenitor cells is a newly recognized phenomenon thought to contribute to tissue differentiation. The molecular mechanisms governing cell fusion are unknown. P-glycoprotein and related ATP-binding cassette transporters are expressed by progenitor cells, but their physiological role in these cell types has not been defined. Here, we have cloned ABCB5, a rhodamine efflux transporter and novel member of the human P-glycoprotein family, which marks CD133-expressing progenitor cells among human epidermal melanocytes and determines as a regulator of membrane potential the propensity of this subpopulation to undergo cell fusion. Our findings show that polyploid ABCB5+ cells are generated by cell fusion and that this process is specifically enhanced by ABCB5 P-glycoprotein blockade. Remarkably, multinucleated cell hybrids gave rise to mononucleated progeny, demonstrating that fusion contributes to culture growth and differentiation. Thus, our findings define a molecular mechanism for cell fusion involving progenitor cells and show that fusion and resultant growth and differentiation are not merely spontaneous events, but phenomena regulated by ABCB5 P-glycoprotein.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Several recent reports have demonstrated that co-culture of pluripotent embryonic or mesenchymal stem cells with lineage-committed cell types can give rise to cell hybrids as a result of cell fusion, and it has been shown that such cell hybrids can generate differentiated progeny in vitro and in vivo (1–5). These findings raise the possibility that cell fusion may represent a physiological mechanism by which endogenous progenitor cells participate in tissue plasticity and renewal. A cellular molecular marker identifying progenitor cells participating in cell fusion or associated with the regulation of cell fusion has as of yet not been identified, however. P-glycoproteins (P-gp)1 and related members of the ABC superfamily of active transporters mediate multidrug resistance in mammalian cancers (6–13) and serve physiologic transport (14–21), differentiation (22, 23), and survival (24, 25) functions in nonmalignant cell types. Two known members of the ABC superfamily of transporters, ABCB1 (MDR1) P-gp and the ABCG2 (Bcrp1) transporter, are also expressed at high levels on stem and progenitor cell populations (26, 27), and the efflux capacity for the fluorescent dyes rhodamine-123 (28–30) and Hoechst 33342 (31–34) mediated by these or related ABC transporters has been utilized for the isolation of such cell subsets. A physiologic role of ABC transporters in such progenitor cells has, however, not been defined. A recent study investigated a possible role of ABCB1 P-gp as a determinant of membrane fluidity and membrane potential, but ABCB1 P-gp was found not responsible for the plasma membrane hyperpolarization observed in multidrug resistant cells (35). Here we have cloned and characterized a novel, third member of the human P-gp family encoded on chromosome 7p21-15.3, designated ABCB5 (ATP-binding cassette, subfamily B (MDR/TAP), member 5) P-gp, which functions as a rhodamine-123 efflux transporter and marks CD133-expressing progenitor cells among HEM. ABCB5 P-gp regulates membrane potential in this progenitor cell subset and determines its propensity to undergo cell fusion. Our results define a novel molecular marker and mechanism for cell fusion involving progenitor cells and show that cell fusion and resultant growth and differentiation are not merely spontaneous events but phenomena specifically regulated by ABCB5 P-gp function.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Cell Culture and Isolation—HEM isolated from the foreskins of healthy donors were purchased from Gentaur (Brussels, Belgium) and cultured in Ham's F-10 medium (Clonetics, Walkersville, MD) supplemented with 10% fetal bovine serum, 6 mM HEPES, and 25 µg/ml bovine pituitary extract (Invitrogen), 10 µg/ml insulin (Sigma), 2.8 µg/ml hydrocortisone, 2 mmol/liter L-glutamine, 100 IU/ml penicillin/streptomycin (Sigma), 0.6 ng/ml basic fibroblast growth factor (Sigma), and 10 nmol/liter phorbol 12-myristate 13-acetate (Sigma) as previously described (36). The G3361 human melanoma cell line, its cisplatin-resistant variant G3361/CDDP, and the MCF-7 breast cancer and SCC25 squamous cell carcinoma cell lines were provided by Dr. Emil Frei III (Dana Farber Cancer Institute, Boston, MA) and were cultured as described (37). Peripheral blood mononuclear cells were donated by healthy volunteers and isolated as described (20). CD133+ cell populations were isolated from HEM cultures harvested by EDTA-4Na 0.2 g/liter PBS treatment (Versene 1:5000; Invitrogen), by positive selection using anti-CD133 Ab-coated magnetic microbeads (Miltenyi Biotec, Auburn, CA) and purification in MiniMACS separation columns (Miltenyi Biotec) according to the manufacturer's recommendations. ABCB5 P-gp+ cells were isolated from HEM cultures by incubation with anti-ABCB5 mAb (20 µg/ml) for 30 min at 4 °C, washing for excess antibody removal, incubation with goat anti-mouse Ig-coated magnetic microbeads, and subsequent purification in MiniMACS separation columns (Miltenyi Biotec).

RNA Extraction and Reverse Transcriptase-PCR—The cells were lysed in TRIzol reagent (Invitrogen) followed by isopropanol precipitation and ethanol washing. Each sample was treated with 10 units of RNase-free DNase I (Roche Applied Science) and incubated at 37 °C for 30 min followed by phenol/chloroform extraction and ethanol precipitation. Standard cDNA synthesis reactions were performed using 5 µg RNA and the SuperScript First-Strand Synthesis System for reverse transcriptase-PCR (Invitrogen) as per the manufacturer's instructions. For PCR analysis, 5 µl of diluted first strand product (~100 ng of cDNA) was added to 45 µl of a 1x PCR mix (50 pmol of each primer, 1x PCR PreMix C (MasterAmp PCR optimization kits; Epicenter, Madison, WI), 2.5 units MasterAmp TAQurate DNA polymerase mix (Epicenter) and denatured at 95 °C for 3 min, then cycled 35 times at 94 °C for 1 min, 58 °C for 30 s and 72 °C for 3 min, and subsequently extended at 72 °C for 10 min. The reaction products were resolved on a 1% LE agarose gel (Ambion, Austin, TX) and photographed. {beta}-Actin primers were used as controls to ensure RNA integrity. The gene-specific oligonucleotide primer pair 5'-CCTAATTCCTCTAATATCTCTCTGTGAGCC-3' (forward primer) and 5'-TCACTGCACTGACTGTGCATTCAC-3' (reverse primer) were used for PCR amplification of reverse transcribed total RNA. The 2906-base cDNA PCR product was sequenced using the amplification primer pair and additional gene-specific sequencing primers (5'-AGTGGGAAGAGTACGGTAGTCCAGCTTCTG-3', 5'-CCTTTAGGGTCACAATCAAATCTGCACTTCG-3', 5'-GAGCAAAGGTCGGACTACAATCGTG-3', 5'-CCCCTGCTCTGCCGTAAAATAATCC-3', 5'-ATTTTGGGTGTTATTTGCTTTGTCAG-3', 5'-CTGCGGCTGTCTATATTTGGTTTC-3', 5'-CCAGAGGGCATGTTCATAGTTTTTAC-3', and 5'-ACGGCTGTTGTCACCATAGGC-3') and the dideoxy chain termination method analyzed by an ABI PRISM 377 DNA Sequencer (PerkinElmer Life Sciences).

ABCB5 Gene Cloning and Transfection—For ABCB5 gene transfection experiments, a 2506-base cDNA containing the full-length ABCB5 open reading frame was amplified by PCR from the product of total HEM RNA using a four base (CACC) 5'-modified forward primer (5'-CACC AGT GGG AAG AGT ACG GTA GTC CAG CTT CTG-3'), the reverse primer (5'-TCA CTG CAC TGA CTG TGC ATT CAC-3'), and the Advantage PCR polymerase mix (Clontech, Palo Alto, CA). The resultant blunt end PCR product was directionally cloned into the pcDNA3.1D/V5-His-TOPO plasmid vector (Invitrogen). Recombinant plasmids were transformed into and amplified in competent TOP10 Escherichia coli cells (Invitrogen) and subsequently purified and sequenced. Recombinant ABCB5 open reading frame-containing plasmids or pcDNA3.1D/V5-His/lacZ expression control plasmids containing the E. coli lacZ gene encoding {beta}-galactosidase (Invitrogen) were transfected into cultured ABCB5 nonexpressing MCF7 breast cancer cells using relative quantities of plasmid and LipofectAMINE 2000 reagent (Invitrogen) as recommended by the manufacturer. ABCB5 gene expression was assessed at 48 h by Western analysis and flow cytometry. As a control, pcDNA3.1D/V5-His/lacZ control plasmid-transfected MCF-7 cancer cells were assayed at 48 h for {beta}-galactosidase expression using a {beta}-galactosidase staining kit (Invitrogen) and bright field microscopic examination.

Antibodies—For anti-ABCB5 mAb production, a 16-mer peptide (RFGAYLIQAGRMTPEG) derived from the extracellular loop-associated amino acid residues 493–508 was synthesized and conjugated to the carrier protein bovine serum albumin via a cysteine residue added for coupling purposes. The immunogen (100 µg) was administered subcutaneously to Balb/c mice during primary immunization, and mice received booster immunizations using 100 µg of immunogen at 4 and 8 weeks. Mouse sera were screened for specific-antibody production against the immunogen by enzyme-linked immunosorbent assay, mice were sacrificed, and the splenocytes were collected for fusion with FO myeloma cells when test bleed serum titers were >1:80000. Specific antibody-secreting fusion products were subcloned to monoclonality by limiting dilution, screened for reactivity with native ABCB5 P-gp by Western analysis and flow cytometry of ABCB5 P-gp-expressing HEM and ABCB5 P-gp gene-transfected, at base-line nonexpressing MCF-7 breast cancer cells. The Ig isotypes of mAbs secreted by selected clones was determined, and spinner flask-produced mAbs were purified by protein A affinity chromatography and tested for lipopolysaccharide content. The IgG1{kappa} anti-ABCB5 P-gp mAb UG3C2–2D12 was used in the studies reported here, and the MOPC-31C mouse isotype control mAb (PharMingen, San Diego, CA) was used as a control.

Western Analysis—For Western analysis, whole cell lysates were mixed with 2x sample buffer (125 mM Tris-HCl, pH 6.8, 20% glycerol, 10% {beta}-mercaptoethanol, 4% SDS, and 0.0025% bromphenol blue), heated at 100 °C for 5 min, and run on 4–15% continuous gradient polyacrylamide gels (Bio-Rad) with Tris-glycine-SDS running buffer (Bio-Rad). The separated proteins were transferred to a polyvinylidene difluoride membrane (NEN Life Science Product, Boston, MA). The membranes were washed and blocked with PBS-Tween 20 containing 5% dry milk and then coated with either anti-ABCB5 mAb or mouse isotype control antibody. After washings, the membranes were incubated with peroxidase-linked secondary goat anti-mouse antibody, and the reactive bands were detected by the addition of chemoluminescent substrate (Pierce).

Flow Cytometry—HEM and purified CD133+ HEM, G3361, and G3361/CDDP human melanoma cells, and untreated, ABCB5 P-gp gene-transfected or control lacZ gene-transfected MCF-7 breast cancer cells were analyzed for surface ABCB5 P-gp expression by incubation of 5 x 105 cells for 60 min at 4 °C with anti-ABCB5 mAb or isotype control mAb (10 µg/ml), followed by counterstaining with fluorescein isothiocyanate-conjugated goat anti-mouse Ig Ab (PharMingen, San Diego, CA) as described (20). Analysis of cell surface marker expression was performed by single color flow cytometry as described (20). Phycoerythrin-conjugated anti-CD133 mAb (clone AC133/2) and anti-CD34 mAb were obtained from Miltenyi.

Rhodamine-123 Transport Studies—ABCB5 P-gp gene- or control lacZ gene-transfected MCF-7 cells (2 x 106) were incubated with rhodamine-123 (1 µg/ml) (Molecular Probes, Eugene, OR) for 60 min at 37 °C and 5% CO2. Subsequently the cells were washed, and serial fluorescence measurements were acquired by flow cytometry at the FL1 emission spectrum on a Becton Dickinson FACScan.

DNA Content Analysis—HEM or purified CD133+ HEM subpopulations were fixed in ice-cold 65% (v/v) ethanol in PBS, washed in cold PBS, and then resuspended in a PI stain mixture containing 50 µg/ml PI, 0.1% Triton X-100, 0.1 mM EDTA(Na)2, and 100 units/ml RNase (all purchased from Sigma) and incubated at 37 °C for 30 min in the dark and an additional 30 min on ice, followed by cellular DNA content determination by flow cytometry as previously described (20). In those experiments where ABCB5 P-gp surface expression and DNA content were examined concurrently, indirect ABCB5 P-gp surface staining was performed immediately prior to PI staining.

Fluorescence Microscopy—To visualize ABCB5 P-gp expression on HEM, CD133+ HEM cells were purified as above and cultured for 24 h in 24-well tissue culture plates (Becton Dickinson, Franklin Lakes, NJ) at 37 °C in a humidified 5% CO2 atmosphere. The cells were then fixed in methanol for 3 min, washed twice in PBS, and incubated with anti-ABCB5 mAb or isotype control Ab (10 µg/ml) at 4 °C for 30 min. After washing, the cells were incubated with fluorescein isothiocyanateconjugated goat anti-mouse secondary antibody for 30 min at 4 °C, washed, followed by counterstaining of nuclei with 4'6-diamidino-2-phenyindole (DAPI) (Molecular Probes). Fluorescence staining was analyzed by fluorescent microscopy using a Mercury-100 Watts fluorescent light source (Microvideo Instruments, Avon, MA) attached to a Nikon Eclipse TE 300 microscope (Nikon Instruments, Melville, NY) with the use of separate filters for each fluorochrome. The images were obtained using a Spot digital camera (Diagnostic Instruments Inc., Sterling Heights, MI), and the Spot 3.3.2. software package was imported into Adobe Photoshop (Adobe Systems, Mountain View, CA).

DiI/DiO Membrane Dye Labeling Studies—To examine cell fusion, HEM cells were harvested and labeled in suspension with either the DiI or DiO fluorescent lipophilic carbocyanine dyes (VybrantCell labeling solution kit; Molecular Probes) by incubation for 20 min at 37 °C according to the manufacturer's instructions. The cells were subsequently washed three times followed by remixing of DiI- or DiO-labeled HEM populations and co-culture for 1 h at 37 °C in a humidified 5% CO2 atmosphere, in the presence or absence of anti-ABCB5 mAb (20 µg/ml), anti-ABCB1 mAb (Hyb-241 clone, 20 µg/ml), or isotype control Ab (20 µg/ml). Dual color flow cytometry was subsequently performed at the Fl2 (DiI) and Fl1 (DiO) detection channels on a Becton Dickinson FACScan flow cytometer. For the detection of cell fusion by fluorescent microscopy, ABCB5 P-gp+ HEM were purified from DiI- or DiO-labeled mixtures by incubation with anti-ABCB5 mAb (20 µg/ml) for 30 min at 4 °C, washing for excess antibody removal, incubation with goat anti-mouse Ig-coated magnetic microbeads, and subsequent purification in MiniMACS separation columns (Miltenyi Biotec). ABCB5 P-gp+ cells were cultured in C24 multiwell plates, and nuclear counterstaining with Hoechst 33342 was performed as a time course. Fluorescent microscopy with the use of separate filters for each fluorochrome was performed as described above.

Membrane Potential Measurements—Membrane potential was assessed by flow cytometry using the anionic DiBaC4 (3) oxonol dye (Molecular Probes) as previously described (35, 38). Oxonol fluorescence intensity is known to vary as a function of cellular membrane potential, with membrane depolarization resulting in increased fluorescence intensity. Briefly, cell suspensions of 1 x 106 cells/ml PBS were incubated with anti-ABCB5, anti-ABCB1, or isotype control mAbs (50 µg/ml) for 30 min at 4 °C, followed by the addition of 150 nM oxonol dye. After an equilibration time of 2 min, oxonol fluorescence intensity of 5 x 104 cells/sample was measured by flow cytometry, with acquisition of fluorescence emission at the FL1 spectrum on a Becton Dickinson FACScan.

Statistical Analysis—The unpaired two-sided Student's t test was used for the statistical analysis of the results. Differences with p < 0.05 were considered statistically significant.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
ABCB5 Is a Novel Human P-gp Family Member—We used the NCBI tblastn application to compare conserved amino acid sequences derived from the known ABCB1 (MDR1) P-gp structure against the NCBI nonredundant Homo sapiens nucleotide sequence data base dynamically translated in all reading frames to detect genes encoding P-gp-homologous structures and identified a candidate gene on chromosome 7p21-15.3. Using a gene-specific oligonucleotide primer pair and PCR amplification of reverse transcribed total mRNA isolated from primary HEM or the G3361 human malignant melanoma cell line and its CDDP-resistant variant G3361/CDDP (37), we amplified a 2906-base cDNA (Fig. 1A) encoding a novel human P-gp family member, designated ABCB5 P-gp. ABCB5 P-gp mRNA transcripts were not detected in peripheral blood mononuclear cells or additional nonmelanoma human tumors, including the MCF-7 breast cancer and SCC-25 squamous cell carcinoma cell lines (37) (Fig. 1A), indicating a selective tissue expression pattern of ABCB5 P-gp among human physiologic tissues and malignant cancers. Sequencing of the 2906-base cDNA demonstrated that the cDNA contained the complete coding sequence for ABCB5 P-gp, because the 467-base 5'-untranslated region contained five in-frame stop codons upstream of the translation initiation codon. The ABCB5 P-gp cDNA sequence is available from NCBI GenBankTM under accession number AY234788 [GenBank] . The ABCB5 gene (Fig. 1B) contains 19 exons and spans 108 kb of genomic DNA at the 7p21-15.3 locus on the contiguous human genomic clones CTA-367017 (AC002486 [GenBank] , 79611 bp, exons 1–5) and, sequenced to the right, CTB-86D3 (AC005060 [GenBank] , 120169 bp, exons 6–19). Comparison of the encoded 812 amino acid ABCB5 primary sequence revealed that the molecule is highly homologous (73%) to both of the known human P-gp isoforms ABCB1 (MDR1) and ABCB4 (MDR3) (54 and 56% amino acid identity, respectively) (Fig. 1C), its closest human relatives. Primary structure analysis using the PIR International Protein Family Classification system algorithm confirmed the protein to be a novel MDR P-gp member of the ABC transport protein superfamily.



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  		FIG. 1.
ABCB5 P-gp gene expression and structure. A, reverse transcriptase-PCR amplification of ABCB5 P-gp cDNA in G3361 (lane 2) and G3361/CDDP (lane 3) melanoma cells and human epidermal melanocytes (HEM)(lane 6); MCF-7 (lane 4); SCC-25 (lane 5); peripheral blood mononuclear cells (lane 7); molecular size markers (lane 1). B, ABCB5 gene exon-intron structure. Exons 1–5 are located on clone CTA-367017 (join bases 61205–61431, 61751–61861, 63805–63930, 65203–65406, and 72289–72459), exons 6–19 on CTB-86D3 (join bases 15877–16038, 20068–20208, 32779–32922, 34197–34301, 34430–34507, 39096–39179, 57388–57591, 61412–61510, 62685–62828, 73356–73511, 77249–77446, 79604–79810, 87732–87878, and 89799–89996). C, comparison of the ABCB5 P-gp primary amino acid sequence with the homologous sequence portions of ABCB1 (MDR1) and ABCB4 (MDR3) P-gp. Amino acid identity (red), substitution of highly similar (green), similar (purple), or unrelated (black) amino acid residues are illustrated.

 
ABCB5 P-gp Functions as a Rhodamine-123 Efflux Transporter—Western analysis, using a mouse anti-ABCB5 mAb (clone 3C2–2D12) generated in the laboratory against a synthetic immunogen derived from a predicted extracellular loop-associated ABCB5 epitope contained in amino acid residues 493–508 of the molecule, detected a 89-kDa protein band of the predicted size (ExPASy server Compute pI/Mw Tool) in HEM, G3361 melanoma cells, and ABCB5 gene-transfected MCF-7 breast cancer cells, but not lacZ gene-transfected MCF-7 controls (Fig. 2A), demonstrating binding of this antibody to ABCB5 P-gp. The LipofectAMINE-mediated gene transfection efficiency varied from 5 to 20% in MCF-7 cells in repeated experiments (Fig. 2B). To characterize cellular localization and membrane topology, we performed indirect surface immunostaining and flow cytometry of nonpermeabilized ABCB5 gene-transfected MCF7 cells or lacZ gene-transfected MCF-7 controls. Labeling with anti-ABCB5 P-gp 3C2–2D12 mAb revealed 14% immunopositivity versus ABCB5- controls (Fig. 2C; M1 gate), consistent with the determined transfection efficiency. These findings demonstrate a surface plasma membrane localization of ABCB5 P-gp and support a predicted (TMHMM1.O software algorithm) membrane topology of ABCB5 P-gp characterized by five transmembrane helices flanked by both extracellular and intracellular ATP-binding domains (Fig. 2D), in contrast to six transmembrane helices and one intracellular ATP-binding domain per homologous half found in other known mammalian P-gp isoforms (reviewed in Ref. 39). When assessing the role of ABCB5 P-gp in fluorescent rhodamine transport, a hallmark function of the closely related ABCB1 (MDR1) P-gp, ABCB5 gene transfection induced a de novo rhodamine-123 efflux capacity in 15% of MCF-7 breast carcinoma compared with lacZ gene-transfected MCF-7 controls (Fig. 2E; M1-gated rhodaminelow phenotype), consistent with the transfection efficiency and demonstrating that plasma membrane-expressed ABCB5 P-gp functions as a rhodamine efflux transporter.



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  		FIG. 2.
ABCB5 P-gp expression and rhodamine-123 efflux function. A, Western analysis of ABCB5 P-gp expression in HEM, G3361 melanoma cells, MCF-7 breast carcinoma control cells, and ABCB5 gene-transfected MCF-7 cells, with molecular size markers (kDa) shown on the left. B, transfection efficiency of lacZ gene-transfected MCF-7 cultures versus controls, with {beta}-galactosidase-positive cells identified by blue staining. C, flow cytometry analysis of surface anti-ABCB5 mAb staining (solid line) versus isotype control Ab staining (shading) on control lacZ gene- or ABCB5 gene-transfected MCF-7 cells. D, transmembrane helix formation probability of ABCB5 P-gp, as determined using the TMHMM1.O software algorithm for the prediction of transmembrane helix formation in mammalian proteins. E, flow cytometry analysis of ABCB5 P-gp-mediated rhodamine-123 transport function. Rhodamine-123 fluorescence of ABCB5 gene-transfected MCF-7 cells (solid line) versus lacZ gene-transfected MCF-7 control cells (shading) is shown as a time course (minutes after loading).

 
ABCB5 P-glycoprotein Expression Marks CD133+ Progenitor Cells—We next examined ABCB5 P-gp surface expression in native HEM, the G3361 melanoma cell line, and its cisplatin-resistant variant G3361/CDDP, which transcribe ABCB5 mRNA as determined by PCR analysis. Indirect immunostaining using 3C2–2D12 mAb revealed ABCB5 P-gp to be expressed on the plasma membrane of 11% of HEM, 3% of G3361 melanoma cells, and, augmented compared with the parent cell line, on 13% of CDDP-resistant G3361/CDDP melanoma cells (Fig. 3A; M1 gate). Based on our finding that ABCB5 P-gp marked distinct cellular subsets among HEM and human melanoma cells, and on the previous evidence that homologous ABC transporters identify physiologic progenitor cells (26, 27) and tumor stem cells (40), we next examined whether ABCB5 P-gp marked a progenitor phenotype-expressing subpopulation. Among purified CD133+ cells (41, 42), which constitute a stem cell subset in human skin (43) and comprised from 0.2 to 0.5% of cells among HEM cultures in our studies as determined by flow cytometry (p < 0.05), ABCB5 P-gp high expressing cells were markedly enriched, with the majority of cells (56%) expressing the molecule versus unseparated controls (Fig. 3B). CD133+ABCB5 P-gp+ cells did not express the hematopoietic progenitor cell antigen CD34 (44), as determined by flow cytometry (data not shown). Immunofluorescence examination of purified, cultured CD133+ HEM revealed a focal expression pattern of ABCB5 P-gp, with immunostaining localizing predominantly to the cellular poles and additional focal membrane domains (Fig. 3C). Unexpectedly, and in contrast to unseparated HEM cultures, CD133+ABCB5 P-gp+ HEM also comprised abundant numbers of multinucleated cells, including cells with four, three, and two nuclei (Fig. 3C). These findings demonstrated that ABCB5 P-gp marks a distinct cell subset among cultured HEM characterized by mono- and multinucleated cells of CD133+ progenitor phenotype.



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  		FIG. 3.
Native ABCB5 P-gp expression. A, flow cytometry analysis of anti-ABCB5 P-gp mAb staining (solid line) or isotype control Ab staining (shading) on HEM or G3361 and G3361/CDDP human melanoma cells. B, ABCB5 P-gp expression on unseparated or purified CD133+ HEM (solid line, anti-ABCB5 P-gp mAb; shading, isotype control Ab). C, ABCB5 P-gp expression on CD133+ HEM. Indirect immunofluorescence labeling (green, primary anti-ABCB5 mAb followed by fluorescein isothiocyanate-labeled goat anti-mouse secondary Ab) and counterstaining of nuclei with DAPI (blue) revealed cells with four nuclei (row 1), three nuclei (row 2), two nuclei (rows 3 and 4), or single nuclei (row 5). The left column shows DAPI staining, the middle column shows anti-ABCB5 mAb staining, and the right column shows the merged image (200x magnification). The scale bars represent 50 µm.

 
ABCB5 P-glycoprotein Expression Identifies Polyploid Cells—To determine the frequency of multinucleated ABCB5 P-gp+ cells among HEM and to examine their cellular DNA content, we next analyzed PI- and ABCB5 P-gp-co-stained cultures by dual color flow cytometry. Among R1-gated cells of the FL2-A versus FL2-W scatter plot (Fig. 4A), which comprise 90% of all cells but do not contain cell aggregates, ABCB5 P-gp+ (R2-gated) cells (upper panels) exhibited markedly increased relative frequencies of cells with 4n DNA content (25% versus 15%, M1 gate) and 6n or 8n DNA (10% versus 1%, M2 gate) compared with all HEM (lower panel). Significantly, ABCB5 P-gp expression marked predominantly a low FSC/low SSC subpopulation (Fig. 4B; R3 gate) of 4n DNA content (Fig. 4b, R4 gate), with 71% of cells of this phenotype, comprising 0.4% of all cells, expressing ABCB5 P-gp (Fig. 4B; M1 gate). The less frequently occurring 6n or 8n DNA-containing ABCB5 P-gp+ cells exhibited a larger, more granular phenotype. Analysis of purified ABCB5 P-gp+ cells revealed a similar DNA content pattern (results not shown). Furthermore, DNA content analysis of purified CD133+ cells, which also exhibited a predominant low FSC/low SSC phenotype compared with unseparated HEM (Fig. 4C), revealed similarly increased frequencies of 4n DNA-containing cells (17% versus 13%; M1 gate) and 6n or 8n DNA-containing cells (4% versus 1%; M2 gate) compared with controls, consistent with the observed co-expression of ABCB5 P-gp and CD133.



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  		FIG. 4.
DNA content/cell cycle analysis of ABCB5 P-gp+ and CD133+ HEM. A, dual color flow cytometry analysis for DNA content (PI staining, Fl2) of anti-ABCB5 P-gp mAb-stained (Fl1) HEM (upper panel) versus isotype control Ab-stained, whole HEM populations (lower panel). B, anti-ABCB5 P-gp mAb staining (solid line) versus isotype control Ab staining HEM gated for (R3*R4 gate) the low FSC/low SSC 4n DNA-containing phenotype. C, DNA content analysis of CD133+-purified HEM (upper panel) versus unseparated HEM populations (lower panel).

 
ABCB5 P-glycoprotein Regulates Cell Fusion—To examine whether ABCB5+/CD133+ cells with >4n DNA content arose by cell fusion and to examine a functional role of ABCB5 P-gp in this process, we harvested and labeled HEM in suspension with either the fluorescent lipophilic carbocyanine dye DiI or DiO, followed by mixing and co-culture of DiI- and DiO-labeled populations and subsequent DiI/DiO co-expression analysis, an established method to study cell fusion based on the resistance of these fluorescent dyes to intercellular transfer (45). Dual color flow cytometric analysis of DiI- or DiO-labeled HEM populations, mixed and co-cultured in the presence of anti-ABCB5 P-gp mAb or isotype control Ab, revealed 7 ± 0.8% (mean ± S.D.) double-labeled, fused cells among low FSC/low SSC-gated HEM (1.6% of all cells) in anti-ABCB5 P-gp mAb-treated cultures (Fig. 5A, row 3) versus 2.7 ± 0.3% and 2.5 ± 0.3% (0.6% and 0.6% of all cells) in untreated (Fig. 5A, panel 1) and control Ig-treated cultures (Fig. 5A, panel 2), respectively (p < 0.05), demonstrating that specific ABCB5 P-gp blockade promotes the frequency of cell fusion among primary HEM. An anti-ABCB1 (MDR1) mAb (Hyb-241) (20, 46) exerted no significant effects beyond control on the frequency of cell fusion (2.7 ± 1.2%, 0.6% of all cells) (Fig. 5A, panel 4), indicating a specific role of ABCB5 P-gp in this process.



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  		FIG. 5.
Detection of cell fusion by flow cytometry and fluorescence microscopy. A, DiI- or DiO-labeled HEM were mixed and co-cultured for 1h either untreated (panel 1) or in the presence of isotype control Ab (20 µg/ml; panel 2), anti-ABCB5 mAb (20 µg/ml; panel 3), or anti-ABCB1 (MDR1) mAb (Hyb-241 clone; 20 µg/ml; panel 4), and dual color flow cytometry was performed at the Fl2 (DiI) and Fl1 (DiO) detection channels. Fused DiI/DiO double-positive cells are detected in the right upper quadrants. B, detection of cell fusion by fluorescent microscopy. ABCB5+ HEM were purified from mixtures of DiI- or DiO-labeled HEM (rows 1–3) and cultured 3 days (rows 4 and 5) or 5 days (row 6) before counterstaining with Hoechst 33342 and fluorescent microscopy. The left column shows nuclear Hoechst 33342 staining (blue), the second column shows membrane DiO staining (green), the third column shows membrane DiI staining (red), and the fourth column shows the merged image. The scale bar represents 25 µm. Row 1, mono- and multinucleated ABCB5+ HEM; rows 2 and 3, unattached, fused DiO/DiI double-positive ABCB5+ HEM with four nuclei; row 4, attached, fused DiO/DiI double-positive ABCB5+ HEM with four nuclei; row 5, attached, fused DiO/DiI double-positive ABCB5+ HEM with two nuclei; row 6, representative cluster of attached, fused DiO/DiI double-positive ABCB5+ HEM with single nuclei. C, unseparated DiI/DiO-labeled HEM cultured for 5 days before counterstaining with Hoechst 33342 and fluorescent microscopy.

 
Fluorescence microscopic examination of ABCB5 P-gp+ cells purified from DiI/DiO-labeled co-cultures revealed the presence of either DiI or DiO single-labeled mononucleated, binucleated, and polynucleated cells (Fig. 5B, row 1) and, in addition, from 10 to 15% polynucleated DiI/DiO double-labeled cells (Fig. 5B, rows 2 and 3) immediately following isolation. The presence of ABCB5 P-gp+ single-labeled polynucleated cells is consistent with the DNA content analysis of unseparated HEM cultures and confirmed the presence of this phenotype among physiologic HEM cultures prior to membrane labeling. The presence of double-labeled ABCB5 P-gp+ polynucleated cells confirmed our flow cytometry findings and directly demonstrates that such cells were generated as a result of cell fusion. Further examination of DiI/DiO-labeled ABCB5 P-gp+ cells in the course of continued culture revealed that double-labeled, fused cells containing multiple nuclei attached after 24 h to the culture surface (Fig. 5B, rows 4 and 5), indicating cellular viability and the capacity for differentiation. After 5 days of culture (Fig. 5B, row 6), clusters of double-labeled, mono- or binucleated attached cells could be discerned, indicating that polynucleated, fused cells had given rise to mono- or binucleated progeny by cell division. Examination of unseparated DiI/DiO-labeled HEM cultures also revealed the presence of double-labeled, fused cells, albeit at lower frequencies (Fig. 5C), demonstrating that cell fusion occurs also spontaneously among cultured HEM.

ABCB5 P-glycoprotein Regulates Membrane Potential—To elicit a possible mechanism by which ABCB5 P-gp regulates cell fusion, we next examined the role of ABCB5 P-gp as a determinant of plasma membrane potential. Plasma membrane potential has been found to critically regulate cell fusion of other human progenitor cells, as evidenced by the recent demonstration that membrane depolarization precedes and triggers cell fusion among human myoblasts (47). Using the membrane potential sensing dye oxonol, an established method to measure membrane potential (35), we found that anti-ABCB5 P-gp mAb (Fig. 6, C and F) but not isotype control (Fig. 6B, F) or anti-ABCB1 mAb (Fig. 6, D and F) significantly depolarized (p < 0.05) a 2.7 ± 0.3% low FSC/low SSC cell subset among HEM compared with untreated controls (Fig. 6, A and F), demonstrating that ABCB5 P-gp functions to maintain membrane potential in the expressing progenitor cell subset.



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  		FIG. 6.
Regulation of membrane potential by ABCB5 P-glycoprotein. Oxononol fluorescence intensity (acquired at the Fl1 emission spectrum) versus SSC is shown in the upper panels for untreated (A), isotype control Ab-treated (B), anti-ABCB5 mAb-treated (C), or anti-ABCB1 mAb-treated (D) HEM. The FSC/SSC characteristics of R1-gated depolarized cells (red) versus all cells (gray) in each sample are shown in the respective lower panels. E, HEM without addition of oxonol (negative control). F, means ± S.E. of R1-gated depolarized cell fractions (%) for each treatment group (n = 3 independent experiments).

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
We have cloned and characterized a novel, third member of the human P-gp family, ABCB5 P-gp, which functions as a rhodamine efflux transporter and regulates membrane potential and cell fusion in a defined progenitor subpopulation of HEM. A P-gp homologous partial cDNA derived from human chromosome 7p21-15.3 has previously been described (48) and the locus has subsequently been designated ABCB5 (NCBI Genome Annotation, LocusLink locus identification 23458); however, ABCB5 P-gp has not been cloned, and its function has not been examined prior to this study. A model reference protein sequence for ABCB5 P-gp providing for a 131-amino acid protein homologous to the N-terminal portion of the 812-amino acid ABCB5 molecule reported here has been predicted by automated computational analysis using gene prediction methods supported by mRNA and expressed sequence tag evidence (GenBankTM GI 29734912), but the function of this truncated putative gene product is unknown. Our findings demonstrate that the 812-amino acid full-length ABCB5 P-gp molecule is expressed in a tissue-restricted manner on the plasma membrane of progenitor cells among HEM and chemoresistant malignant melanoma cells. Specifically, our findings show that ABCB5 P-gp marks an otherwise undistinguishable subset of low FSC, binucleated cells of 4n DNA content and that the transporter is expressed, in addition, on polyploid cells of 6n or 8n DNA content. Our demonstration that ABCB5 P-gp+ 6n or 8n DNA-containing cells are generated by cell fusion indicates that 4n DNA-containing ABCB5 P-gp+ cells fuse with cells of either 2n or 4n DNA content. The relative rarity of low FSC, 4n DNA-containing ABCB5 P-gp+ cells suggests that fusion occurs predominantly not among the ABCB5 P-gp+ progenitor cell subset but rather between ABCB5 P-gp+ cells and ABCB5 P-gp- HEM. Fusion of ABCB5 P-gp+ progenitor cells with differentiated cell types and resultant membrane transfer of ABCB5 P-gp to fusion hybrids and in decreasing amounts to resultant progeny could account for the observation that ABCB5 P-gp is expressed on a significantly larger subpopulation of HEM than is defined by co-expression of the CD133 progenitor phenotype marker.

Cell fusion among pluripotent murine embryonic stem cells and co-cultured murine bone marrow or central nervous system progenitor cells has recently been described, and it has been shown that the resultant cell hybrids give rise to differentiated progeny in vitro (1, 2). Furthermore, cell fusion, including nuclear fusion, occurring among co-cultured human mesenchymal stem cells and human epithelial cells has been shown to contribute to epithelial differentiation and tissue repair in vitro (3). In addition to these findings of cell fusion among co-cultures of heterogeneous cell populations, our results demonstrate that cell fusion involving an endogenous progenitor-type cell subset occurs among cultured human cells of single-tissue origin, in the absence of exogenous stem or progenitor cell populations. Because cell fusion contributed to HEM culture perpetuation in our studies, our findings, in the light of the proposition that cell fusion results in cellular reprogramming and cellular dedifferentiation, raise the possibility that cell fusion involving ABCB5 P-gp+ skin progenitor cells contributes to the inherent turnover and renewal capacity of skin tissue, a known niche of multipotent progenitor cells (49–51).

Regulation of membrane potential and cell fusion by ABCB5 P-gp associates novel functions with the P-gp family of active transporters. The related human P-gp transporters ABCB1 (MDR1) and ABCB4 (MDR3) have previously been implicated in the regulation of plasma membrane currents (52, 53) and phospholipid composition (14, 15), but a recent study addressing the specific role of ABCB1 P-gp in membrane potential regulation did not elicit such a role for this transporter (35). In contrast, our demonstration of a specific role of ABCB5 P-gp in membrane potential regulation and cell fusion indicates that ABCB5 P-gp acts physiologically to counterregulate depolarization-dependent fusogenic membrane processes in specific skin progenitor cells otherwise capable or committed to undergo fusion, such as exist in other tissues (47, 54).

Our findings raise the possibility that ABCB5 P-gp-controlled progenitor membrane potential, fusion propensity, and resultant tissue growth and differentiation are physiologically regulated by factors affecting ABCB5 P-gp function, including availability of ATP for hydrolysis, thus providing for a specific molecular mechanism of preferential growth and differentiation induction at sites of tissue energy depletion or injury, which may contribute to the unique renewal capacity of human skin.


   FOOTNOTES
 
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EBI Data Bank with accession number(s) AY234788 [GenBank] .

* This work was supported by funds provided by NIAID, National Institutes of Health (P01 AI50151 to M. H. S. and K08 AI50783 to M. H. F.). 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. Back

|| Co-senior investigators. Back

** To whom correspondence should be addressed: Laboratory of Immunogenetics and Transplantation, Renal Division, Dept. of Medicine, Brigham and Women's Hospital, 75 Francis St., Boston, MA 02115. Tel.: 617-247-5218; Fax: 617-732-5254; E-mail: mfrank{at}rics.bwh.harvard.edu.

1 The abbreviations used are: P-gp, P-glycoprotein; ABC, ATP-binding cassette; MDR, multidrug resistance; HEM, human epidermal melanocytes; PI, propidium iodide; mAb, monoclonal antibody; Ab, antibody; CDDP, cisplatin; FSC, forward scatter; SSC, side scatter; PBS, phosphate-buffered saline; DAPI, 4'6-diamidino-2-phenyindole. Back


   ACKNOWLEDGMENTS
 
We are grateful to Emil Frei III (Dana-Farber Cancer Institute, Boston, MA) for providing the cancer cell lines used in our studies. We thank our colleague Emanuela Gussoni (Department of Genetics, Children's Hospital, Boston, MA) for critical reading of the manuscript.



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