Prominin-1/CD133, a neural and hematopoietic stem cell marker, is expressed in adult human differentiated cells and certain types of kidney cancer
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- Florek, M., Haase, M., Marzesco, A. et al. Cell Tissue Res (2005) 319: 15. doi:10.1007/s00441-004-1018-z
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Human prominin-1/CD133 has been reported to be expressed in neural and hematopoietic stem/progenitor cells and in embryonic, but not adult, epithelia. This lack of detection of human prominin-1, as defined by its glycosylation-dependent AC133 epitope, is surprising given the expression of the murine ortholog in adult epithelia. Here, we demonstrate, by using a novel prominin-1 antiserum (αhE2), that the decrease of AC133 immunoreactivity observed during differentiation of the colonic adenocarcinoma-derived Caco-2 cells is not paralleled by a down-regulation of prominin-1. We have also shown that αhE2 immunoreactivity, but not AC133 immunoreactivity, is present in several adult human tissues, such as kidney proximal tubules and the parietal layer of Bowman’s capsule of juxtamedullary nephrons, and in lactiferous ducts of the mammary gland. These observations suggest that only the AC133 epitope is down-regulated upon cell differentiation. Furthermore, αhE2 immunoreactivity has been detected in several kidney carcinomas derived from proximal tubules, independent of their grading. Interestingly, in one particular case, the AC133 epitope, which is restricted to stem cells in normal adult tissue, was up-regulated in the vicinity of the tumor. Our data thus show that (1) in adults, the expression of human prominin-1 is not limited to stem and progenitor cells, and (2) the epitopes of prominin-1 might be useful for investigating solid cancers.
KeywordsProminin-1 (CD133)Epithelial cellsDifferentiationCancerHuman
Human prominin-1 (CD133; Fargeas et al. 2003a) was originally identified as a novel cell surface marker (AC133 antigen) expressed on a subpopulation of the CD34+ hematopoietic stem and progenitor cells (Miraglia et al. 1997; Yin et al. 1997) that have the capacity to repopulate xenogeneic transplantation models (Yin et al. 1997; de Wynter et al. 1998). This antigen has also been detected in several malignant hematopoietic diseases, including acute and chronic myeloid lymphoblastic leukemias and myelodysplastic syndrome (Waller et al. 1999; Baersch et al. 1999; Buhring et al. 1999; Green et al. 2000; Wuchter et al. 2001). Since its first characterization, several reports have shown that prominin-1 can be used to identify and isolate human stem cells from various sources, including the hematopoietic system and central nervous system (Yin et al. 1997; Uchida et al. 2000; for a review, see Bhatia 2001).
Human prominin-1 displays, on average, 60% amino acid identity to mouse prominin-1 (Corbeil et al. 1998; Miraglia et al. 1998), a 115-kDa glycoprotein originally described as being expressed on the apical surface of neuroepithelial stem cells and in several other epithelia, including kidney proximal tubules (Weigmann et al. 1997). Prominin-1 belongs to a new family of pentaspan membrane glycoproteins expressed throughout the animal kingdom (Corbeil et al. 2001b; Fargeas et al. 2003a, 2003b). Irrespective of the cell type, prominin-1 is specifically associated with microvilli and other plasma membrane protrusions (Weigmann et al. 1997; Corbeil et al. 2000; Fargeas et al. 2004; Giebel et al. 2004). Prominin-1 is a cholesterol-binding protein, and recent studies involving morphological, biochemical, and genetic approaches suggest a role of this molecule in the morphogenesis and/or physiology of plasma membrane protrusions (Maw et al. 2000; Röper et al. 2000; Corbeil et al. 2001b).
The expression profile of human prominin-1 is generally similar to that of the murine molecule, being expressed in several embryonic epithelia, including neuroepithelial progenitor cells, and adult hematopoietic stem cells (Weigmann et al. 1997; Corbeil et al. 2000). However, a notable exception has been observed (Miraglia et al. 1997; Weigmann et al. 1997; Corbeil et al. 2000). Whereas rodent prominin-1 is highly expressed in the adult kidney (Weigmann et al. 1997; Corbeil et al. 2001a), the same tissue in humans appears to lack prominin-1 (Miraglia et al. 1997). The apparent absence of prominin-1 in human adult kidney is particularly puzzling given the high expression of its mRNA in this tissue (Miraglia et al. 1997; Corbeil et al. 2000) and the isolation of several expressed sequence tag (EST) clones derived therefrom (GenBank accession numbers AI766048, AW614306, BE501788).
The widespread expression of prominin-1 in human embryonic (Corbeil et al. 2000), but apparently not adult, tissues (Miraglia et al. 1997) may be explained, at least in part, because the monoclonal antibody (mAb) AC133 (Miltenyi Biotech) used to detect prominin-1 recognizes a glycosylated epitope (Miraglia et al. 1997), and the glycosylation of this protein may change depending on the state of cellular differentiation (Corbeil et al. 2000). The glycosylation of prominin-1 could also be altered in cells that have undergone malignant transformation. Such alteration of glycosylation of prominin-1 may explain the apparently discordant expression of prominin-1 in patients with acute myelogenous leukemia and myelodysplastic syndrome that have been observed with two mAbs recognizing distinct glycosylation-dependent epitopes (mAbs AC133 and AC141; Green et al. 2000) and could reflect the different stages of differentiation of these malignant cells. The aim of the present study has been to resolve the apparent discrepancy of prominin-1 expression in adult humans by using a novel antibody that recognizes prominin-1 independently of glycosylation.
Materials and methods
Antiserum against recombinant human prominin-1/CD133
The bacterial expression plasmid pGEX-hE2-prominin-1, containing a cDNA fragment encoding part of the second extracellular domain of human prominin-1 fused in-frame to glutathione S-transferase (GST), was constructed by subcloning the EcoRI–EcoRI fragment (nucleotides 756–1195) derived from the human prominin-1 cDNA (GenBank accession number AF027208; Miraglia et al. 1997) into the corresponding site of pGEX-2T (Amersham Biosciences, Uppsala, Sweden). The GST-hE2 fusion protein encoded by the pGEX-hE2-prominin-1 plasmid was expressed in HB 101 E. coli, purified on glutathione Sepharose 4B beads according to the batch method (Corbeil et al. 1999), and eluted from the column at room temperature by addition of 10 mM glutathione in 50 mM Tris–HCl, pH 8.0. Purified GST-hE2 fusion protein was then used to generate the αhE2 antiserum by immunizing a New Zealand white rabbit.
Cell culture and sample preparation
Chinese hamster ovary (CHO) cells transiently transfected with human prominin-1 cDNA and Caco-2 cells endogenously expressing prominin-1 were cultured as described (Corbeil et al. 2000). CD34+ cells were isolated from G-CSF-mobilized peripheral blood of human healthy donors by using the Direct CD34 Progenitor Cell Isolation Kit (Miltenyi Biotec, Gladbach, Germany). CD3+, CD19+, CD14+, and CD15+ cells were enriched from peripheral blood of human healthy donors by using FACSVantage SE (BD Biosciences; San Jose, Calif.) with appropriate fluorophore-conjugated antibodies (CD3+, CD19+, and CD14+, all from BD Biosciences; CD15+ from Beckman Coulter). Informed consent in all cases was obtained and approved by the institutional review boards. Detergent cell lysates were prepared as described previously (Corbeil et al. 1999). Samples of normal tissue taken some distance (>1 cm) from a tumor region (conventional carcinoma) of kidney were obtained from anonymous archival tissues (Department of Pathology, University of Dresden), and membranes were prepared according to procedures reported previously (Corbeil et al. 2001a). Protein concentrations were determined by using BCA Protein Assay Reagent (Pierce, Rockford, Ill.).
Endoglycosidase digestions and immunoblotting
CHO cell detergent extracts corresponding to one-fifth of an 80%-confluent 60-mm dish, detergent extracts (150 μg protein) derived from Caco-2 cells harvested at various stages of confluency, or membranes from adult human kidney (100 μg protein) were incubated overnight at 37°C in the absence or presence of 10 mU endo-β-N-acetylglucosaminidase H (endo H) or 1 U PNGase F according to the manufacturer’s instructions (Roche Molecular Biochemicals, Mannheim, Germany). Proteins were analyzed by SDS-polyacrylamide gel electrophoresis (SDS-PAGE; 7.5%) and transferred to polyvinylidene difluoride membranes (Millipore, Belford, Mass.; pore size: 0.45 µm) by standard procedures (Corbeil et al. 2001a). Immunoblotting was performed essentially as described by Corbeil et al. (2001a) with, as primary antibody, either αhE2 antiserum (1:3,000) or mAb AC133 (1 μg/ml; Miltenyi Biotec, Gladbach, Germany).
Pulse-chase studies and immunoprecipitation
Fourteen-day-old Caco-2 cell monolayers grown on permeable Transwell filters (24-mm Transwell-COL chambers, 0.4-μm pore size) were rinsed once with met/cys-free medium, viz., methionine-free, cysteine-free DMEM supplemented with 1% FCS dialysed against phosphate-buffered saline (PBS), and incubated for 1 h at 37°C in this medium. The cells were then pulse-labeled for 30 min at 37°C with fresh met/cys-free medium containing 350 μCi/ml [35S] Easytag express protein labeling mix (PerkinElmer Life Sciences, Boston, Mass.; 1175.0 Ci/mmol). After the pulse, cells were chased for the indicated times in fresh DMEM supplemented with 5% FCS. At the end of the chase period, the filters were cut into four pieces and incubated in 2 ml ice-cold lysis buffer, viz., 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS, 50 mM TRIS–HCl, pH 8.0, and a protease inhibitor cocktail (Sigma, St. Louis, Mo.), for at least 1 h at 4°C on an end-over-end shaker. Labeled prominin-1 was then immunoprecipitated from the detergent extract obtained after centrifugation (10 min, 10,000g, 4°C) by using αhE2 antiserum pre-absorbed to Protein A-Sepharose 4 Fast Flow (Amersham Biosciences, Uppsala, Sweden), followed by SDS-PAGE and phosphoimaging.
In the experiments with unlabeled prominin-1, 10-day-old Caco-2 cell monolayers grown on 100-mm Petri dishes were incubated in 1 ml ice-cold lysis buffer for 1 h at 4°C. Prominin-1 was immunoprecipitated from detergent-cell extracts obtained after centrifugation (10 min, 10,000g, 4°C) by using either mAb AC133 (10 μg/ml) or mAb AC141 (10 μg/ml; Miltenyi Biotec) pre-absorbed to Protein G-Sepharose 4 Fast Flow (Amersham Biosciences) and analyzed by immunoblotting with αhE2 antiserum (see above).
Immunofluorescence of Caco-2 cells
Confluent Caco-2 cells grown on glass coverslips were washed with PBS and fixed with 4% paraformaldehyde in PBS for 30 min at room temperature. Coverslips were then rinsed and incubated for 10 min in PBS containing 50 mM ammonium chloride. Fixed cells were incubated in SDS buffer (0.005% SDS and 0.2% gelatin in PBS) for 1 h at room temperature and then washed with PBS containing 0.2% gelatin for 10 min to remove the residual SDS. Cells were then double-labeled for 30 min at room temperature with αhE2 antiserum (1:500) and mouse mAb AC133 (1 μg/ml) followed by Cy3-conjugated goat anti-rabbit IgG (H + L) and Cy2-conjugated goat anti-mouse IgG (H + L; Jackson ImmunoResearch Labs, West Grove, Pa.), all diluted in PBS containing 0.2% gelatin. Coverslips were rinsed and mounted in Moviol 4.88 (Calbiochem). The samples were observed with an Olympus BX61 microscope.
Immunohistochemistry of human adult tissues
Samples from various types of kidney cancer and normal tissues of kidney and mammary glands taken some distance (>1 cm) from tumor regions came from the Department of Pathology (University of Dresden) and were archival materials that had not been used for genetic analysis. They were fixed in 4% buffered formaldehyde (pH 8.0) for 24 h at room temperature, dehydrated with increasing concentrations of ethanol (70%, 80%, 2×96%, 2×100%) for 1 h each at 37°C, and then treated twice with xylene (Carl Roth GmbH+Co, Germany) for 45 min at room temperature. The dehydrated samples were incubated in paraffin for 1.5 h at 60°C and then for an additional 2.5 h with fresh paraffin. Finally, they were embedded in tissue blocks and stored at room temperature. Thin sections (4 μm) were cut, mounted on silanized slides, and dried for 3 h at 37°C or overnight at room temperature. They were deparaffinized overnight by xylene treatment, hydrated with decreasing concentrations of ethanol (2×100%, 95%, 80%, 70%, 45%) for 1 min (each at room temperature), and then rinsed with distilled water (Millipore) for 5 min. The samples were washed with PBS containing 0.3% Tween 20 for 10 min, placed in blocking solution (10% FCS, 0.2% saponin in PBS) for 15 min at room temperature, and then incubated for 1 h at 37°C with αhE2 antiserum (1:500) and mAb AC133 (1 μg/ml) followed by Cy3-conjugated goat anti-rabbit IgG (H + L) and Cy2-conjugated goat anti-mouse IgG (H + L), all diluted in blocking solution. Nuclei were stained with 4,6-diamidino-2-phenylindole (DAPI; 1 μg/ml; Molecular Probes) for 10 min at room temperature. The sections were rinsed, mounted in Moviol 4.88, and examined with a fluorescence microscope (Olympus BX61). The images shown were prepared from IPLAB data files (version 3.5) by using Adobe photoshop software.
In experiments with immunoperoxidase, sections were deparaffinized by two successive xylene treatments (10 min each) and then hydrated with decreasing concentrations of ethanol. Cells were permeabilized, and sections were blocked in blocking solution for 20 min. Endogenous peroxidase was neutralized with 1% H2O2 for 15 min. After being washed with PBS containing 0.3% Tween 20, the sections were incubated sequentially with either αhE2 antiserum (1:500) or mAb AC133 (1 μg/ml) followed by peroxidase-coupled goat anti-rabbit or goat anti-mouse antibody IgG (1:300; Jackson) for 1 h at 37°C. Color reactions were performed with the peroxidase substrate DAB (3,3′-diaminobenzidine tablet sets, 0.7 mg/ml; Sigma) according to the manufacturer’s protocol. All sections were counterstained with Mayer’s hematoxylin (Merck). Stained sections were observed with an Olympus BX61 microscope.
mRNA expression analysis
Northern dot blot analysis was performed by using the Human Multiple Tissue Expression (MTE) Array membrane (Clontech, catalog number 7775-1). The amount of poly(A)+ RNA in each dot of the array was normalized by the manufacturer to yield similar hybridization signals for eight housekeeping genes. The amounts of mRNA in each dot varied from 50 ng to 956 ng, and the array allowed the comparative analysis of gene expression in various tissues. The blot was hybridized with a human prominin-1 [α-32P] dCTP-labeled probe (Corbeil et al. 2000) for 6 h at 65°C and washed according to the protocol of Clontech Laboratories (manual PT3307-1). The blot was exposed for 2 days at −80°C.
Prominin-1/CD133 mRNA is widely distributed in adult human tissues
Generation and characterization of an antiserum directed against the human prominin-1/CD133 polypeptide
Direct evidence showing that the αhE2 antiserum recognized the same protein that carried the AC133 and AC141 epitopes (Miraglia et al. 1997) was obtained by immunoprecipitation of prominin-1 with either mAb AC133 or mAb AC141 from Caco-2 cell lysates followed by immunoblotting with αhE2 antiserum (Fig. 2b, lanes 2 and 3, respectively).
Pulse-chase experiments performed with Caco-2 cells showed that the αhE2 antiserum immunoprecipitated the newly synthesized, 105-kDa form of prominin-1 (Fig. 2c, lane 1, asterisk) and the mature, 120-kDa form of prominin-1 (Fig. 2c, lanes 2 and 3, arrowhead).
Expression of the human prominin-1/CD133 protein, but not AC133 immunoreactivity, in differentiated Caco-2 cells
We have previously reported that, in the human colon-derived epithelial cell line Caco-2, the expression of prominin-1, as revealed by AC133 immunoreactivity, is down-regulated after differentiation of the cells (Corbeil et al. 2000). This enterocytic differentiation of Caco-2 cells is a growth-related process that starts about 7 days after the cells reach confluence and is complete within 20–30 days (Pinto et al. 1983). Interestingly, in contrast to the decrease of AC133 immunoreactivity, the prominin-1 transcript appears to be up-regulated under the same conditions (Corbeil et al. 2000), suggesting that the loss of AC133 immunoreactivity reflects either an inhibition of prominin-1 translation or a loss of the AC133 epitope, which is thought to be dependent, at least in part, on glycosylation (Miraglia et al. 1997), because of the alternative processing of asparagine-linked oligosaccharides upon differentiation. Such differential glycosylation has been previously reported (Ogier-Denis et al. 1988). To address this issue, we have analyzed the expression of prominin-1 before and after differentiation of Caco-2 cells by using αhE2 antiserum.
To corroborate that the persistence of αhE2 immunoreactivity in differentiated Caco-2 cells, as observed by immunofluorescence microscopy, was attributable to prominin-1, we performed immunoblotting experiments with Caco-2 cells harvested at various days after reaching confluence. As shown in Fig. 4b, the 120-kDa form of prominin-1 detected in undifferentiated Caco-2 cells was still observed after differentiation (Fig. 4b, top panel, arrowhead). Quantification revealed that more than 70% of αhE2 immunoreactivity observed in undifferentiated cells (day 2 or 4) persisted in older differentiated cultures (e.g., day 22 or 23; Fig. 4c). Together, these data showed that the loss of AC133 immunoreactivity upon differentiation of Caco-2 cells was not the result of a down-regulation of the prominin-1 protein.
Expression of prominin-1/CD133 in adult human kidney and mammary gland
Immunohistological analysis of adult human kidney revealed αhE2 immunoreactivity on the apical, but not basolateral, side of epithelial cells lining proximal tubules (Fig. 5b, αhE2) and the parietal layer of Bowman’s capsule (Fig. 5c, αhE2) of nephrons located in the juxtamedullary region of the cortex. The vast majority of nephrons in the outermost region of the cortex were unstained (data not shown). No staining was observed in distal tubules or in collecting duct epithelia. Hence, the expression and localization of prominin-1 in adult human kidney was similar to that in adult mouse kidney (Weigmann et al. 1997). As previously reported (Miraglia et al. 1997), no specific prominin-1 immunoreactivity could be detected in the adult kidney with mAb AC133 (Fig. 5b, c, AC133; data not shown).
The multiple tissue expression array analysis (Fig. 1) suggested that the expression of prominin-1 in adult humans might extend to epithelial tissues other than the kidney. To address this issue, we analyzed the expression of the prominin-1 protein in the mammary gland, a tissue with a high prominin-1 mRNA level (Fig. 1, F9). Immunohistochemistry revealed αhE2, but not AC133, immunoreactivity on the apical side of lactiferous ducts (Fig. 5d, αhE2).
Differential αhE2 versus AC133 immunoreactivity in human kidney cancer
Expression of prominin-1 in various human kidney cancer samples (+ presence or − absence of prominin-1 immunoreactivity in the tumor region)
Cytomorphology of tumor
Conventional carcinoma (clear cell carcinoma)
Collecting duct carcinoma
Two major points can be made in light of the results of the present investigation. First, the expression of prominin-1 in adult human tissues is more widespread than previously assumed from studies with the mAb AC133. Second, in cancer originating from prominin-1-expressing epithelial cells, the presence or absence of prominin-1 and of the AC133 epitope may provide information about the state of de-differentiation of the tumor.
Together with previous studies, the present data indicate that human and mouse prominin-1 share the same properties, i.e., (1) a pentaspan membrane topology (Miraglia et al. 1997; Weigmann et al. 1997); (2) a widespread distribution in embryonic and adult tissues (Weigmann et al. 1997; Yin et al. 1997; Maw et al. 2000; the present study); (3) their targeting to the apical domain of epithelial cells (Weigmann et al. 1997; Corbeil et al. 1999, 2000), and (4) their concentration in plasma membrane protrusions (Weigmann et al. 1997; Corbeil et al. 2000). Thus, human and mouse proteins clearly represent an orthologous pair, and the open question regarding their relationship (Corbeil et al. 1998; Miraglia et al. 1998) has been answered. Hence, the murine model, including the valuable mAb 13A4 directed against mouse prominin-1 (Weigmann et al. 1997), should constitute a useful and complementary system for studying hematopoiesis.
In both human and mouse, prominin-1 is confined to the brush-border membrane of the kidney proximal tubules and is absent in the distal tube and collecting duct epithelia. The lack of prominin-1 in these epithelial cells, which also contain microvilli, is surprising considering the postulated function of prominin-1, i.e., organizing functional plasma membrane protrusions (Corbeil et al. 2001b). However, we have recently identified prominin-2, a paralog of prominin-1 (Fargeas et al. 2003b). Prominin-2 shows a similar tissue distribution to prominin-1, being highly expressed in the adult kidney and detected all along the digestive tract and in various other epithelia (Fargeas et al. 2003b). Preliminary data from epithelial Madin–Darby canine kidney cells transfected with prominin-2-GFP have revealed that prominin-2 is also found in microvilli (M. Florek and D. Corbeil, unpublished). Therefore, the absence of prominin-1 in a given epithelial cell type does not necessary correspond to the lack of a prominin molecule. Prominin-2 may exert a similar function and hence compensate for the lack of prominin-1 in certain epithelia. Further studies are needed to determine the complete expression pattern of prominin-2 and eventually the role of prominins in kidney physiology.
Given the apparently inverse correlation between the expression of the AC133 epitope and cell differentiation, we have investigated the possibility of exploiting immunoreactivity to αhE2 and AC133 as indicators of de-differentiation of cells endogenously expressing prominin-1. With regard to human kidney cancer, our pilot screen shows that prominin-1, as detected by αhE2 immunoreactivity, is expressed in several cancers thought to be derived from proximal tubules, e.g., conventional carcinoma. This differential expression profile of prominin-1 might provide new information about the biology of tumors arising from one histological family. In one particular case, the up-regulation of the AC133 epitope in cells in the vicinity of a conventional carcinoma is particularly interesting as it is consistent with the notion that tumor progression is associated with cell de-differentiation, such as a change in glycosylation toward a state characteristic of stem/progenitor cells as in the present case of the AC133 epitope (see Fig. 7). The AC133-positive cells may represent a unique population of cancer stem cells that possess the ability to proliferate and maintain their self-renewal capacity extensively, whereas the αhE2-positive cells detected in the tumor region may have lost this ability and are therefore negative for AC133 immunoreactivity. This agrees with the cancer stem cell hypothesis that suggests that not all cells in a tumor have the same capacity for proliferation (Reya et al. 2001). Expression data on the AC133 epitope could therefore be useful tools in the diagnosis and monitoring of malignant disease. Clearly, the clinical significance of prominin-1 and the AC133 epitope is not limited to the hematopoietic field (Handgretinger et al. 2003; Bornhäuser et al. 2004). Large-scale studies are warranted to determine the application of prominin-1 with its various epitopes as a novel prognostic and/or predictive clinical marker.
We thank D. Buck for the human prominin-1 cDNA, G. Baretton for human tissue samples, I. Peterson for her excellent sample preparation, I. Nüβlein for her valuable assistance with FACS, and S. Langer for valuable technical assistance with some immunohistochemistry experiments.