|
|
|
|||||||
|
|||||||||
J Biol Chem, Vol. 275, Issue 8, 5512-5520, February 25, 2000
From the a Department of Neurobiology, University of
Heidelberg, Im Neuenheimer Feld 364, D-69120 Heidelberg, and the
Max-Planck-Institute of Molecular Cell Biology and Genetics,
Pfotenhauerstrasse 110, D-01307 Dresden, Germany,
e d'Embryologie, CNRS Unité Propre de Recherche
9064, Nogent-sur-Marne, France, and INSERM U506, Villejuif, France,
f AmCell Corp, Sunnyvale, California 94089, g Medical Research Council Molecular Haematology Unit,
Institute of Molecular Medicine, John Radcliffe Hospital,
Oxford OX3 9DS, United Kingdom, and the i Hanson Center
for Cancer Research, Adelaide, Australia 5000
The human AC133 antigen and mouse prominin are
structurally related plasma membrane proteins. However, their tissue
distribution is distinct, with the AC133 antigen being found on
hematopoietic stem and progenitor cells and prominin on various
epithelial cells. To determine whether the human AC133 antigen and
mouse prominin are orthologues or distinct members of a protein family,
we examined the human epithelial cell line Caco-2 for the possible
expression of the AC133 antigen. By both immunofluorescence and
immunoprecipitation, the AC133 antigen was found to be expressed on the
surface of Caco-2 cells. Interestingly, immunoreactivity for the AC133
antigen, but not its mRNA level, was down-regulated upon
differentiation of Caco-2 cells. The AC133 antigen was specifically
located at the apical rather than basolateral plasma membrane. An
apical localization of the AC133 antigen was also observed in various human embryonic epithelia including the neural tube, gut, and kidney.
Electron microscopy revealed that, within the apical plasma membrane of
Caco-2 cells, the AC133 antigen was confined to microvilli and absent
from the planar, intermicrovillar regions. This specific subcellular
localization did not depend on an epithelial phenotype, because the
AC133 antigen on hematopoietic stem cells, as well as that ectopically
expressed in fibroblasts, was selectively found in plasma membrane
protrusions. Hence, the human AC133 antigen shows the features
characteristic of mouse prominin in epithelial and transfected
non-epithelial cells, i.e. a selective association with
apical microvilli and plasma membrane protrusions, respectively. Conversely, flow cytometry of murine CD34+ bone marrow
progenitors revealed the cell surface expression of prominin. Taken
together, the data strongly suggest that the AC133 antigen is the human
orthologue of prominin.
A characteristic feature of epithelial cells, which is a
prerequisite for their function, is the selective association of certain proteins with specific subdomains of the plasma membrane (1-3). An example is the recently identified protein prominin, a
115-kDa five transmembrane domain protein found to be expressed on the
apical surface of neuroepithelial cells and several other embryonic
epithelia and on brush border membranes of adult kidney proximal
tubules (4). Within the apical plasma membrane domain, prominin is
selectively associated with microvilli and other related plasma
membrane protrusions rather than the planar subdomain of the membrane.
Studies with prominin-transfected Madin-Darby canine kidney cells have
shown that this selective association is due to a combination of apical
sorting and retention in microvilli (5). Remarkably, a plasma membrane
protrusion-specific localization of prominin was also observed in
prominin-transfected non-epithelial cells (i.e. CHO
cells),1 showing that the
mechanism underlying the selective association of membrane proteins
with plasmalemmal protrusions is conserved between epithelial and
non-epithelial cells (4).
These studies were conducted with prominin derived from one species,
the mouse. In independent studies, a novel marker of human
hematopoietic stem and progenitor cells was identified, characterized,
and referred to as AC133 antigen (6, 7). The human AC133 antigen
appears to be related to mouse prominin in size (120 kDa), membrane
topology (five transmembrane domains, two large extracellular loops),
and sequence (8, 9).
However, the tissue distribution reported for the human AC133 antigen
(6, 7) is remarkably distinct from that reported for mouse prominin
(4). In contrast to the epithelial expression of prominin (4), the
AC133 antigen was found to be selectively expressed on human
CD34bright progenitors derived from bone marrow, fetal
liver, and peripheral blood (6) and on subsets of CD34+
leukemia cells, i.e. non-epithelial cells, but not in
epithelial tissues such as colon and kidney (7). Moreover, the sequence identity between mouse prominin and the human AC133 antigen is relatively low (average 60%), in particular in the three extracellular domains (54%), which comprise more than 70% of the polypeptide.
This raises a major question. Is the AC133 antigen the human orthologue
of prominin? If so, an explanation would be required as to why the
AC133 antigen is apparently not expressed in epithelial tissues such as
adult kidney (7), which in the case of mouse does express prominin (4),
although mRNA for the AC133 antigen is well detectable (7). In
addition, one would then expect the AC133 antigen to be selectively
associated with plasma membrane protrusion upon expression in
fibroblasts, as previously observed in the case of prominin (4).
Conversely, prominin should then be expressed also in murine
hematopoietic progenitor cells.
Or, alternatively, are prominin and the AC133 antigen distinct members
of a protein family? In the support of this possibility is the
occurrence of at least three open reading frames in the Caenorhabditis elegans genome related to mouse prominin and
the human AC133 antigen (8). In that case, one would not necessarily expect the AC133 antigen to be selectively associated with plasma membrane protrusions.
Answering this question is particularly important in light of a set of
recent findings (10) that are as follows. First, a frameshift mutation
in the gene encoding the AC133 antigen, which results in a truncated
protein, causes retinal degeneration in humans. Second, a similar
truncated version of mouse prominin does not reach the cell surface.
Third, consistent with the detection of AC133 immunoreactivity in human
retinoblastoma cells (6), in mouse, prominin is found in photoreceptor
cells where it is selectively associated with the plasma membrane
evaginations at the base of the rod outer segment that are
intermediates in disc formation. If the AC133 antigen was the human
orthologue of prominin rather than a distinct member of the prominin
family, this would suggest that the lack of appearance of this protein
in the plasma membrane evaginations of the rod outer segment may lead
to impaired disc formation and, eventually, to retinal degeneration.
Here we report several lines of evidence strongly suggesting that the
AC133 antigen is the human orthologue of prominin. The evidence
includes the expression of the AC133 antigen in certain human
epithelial cells. This apparent contradiction of previous observations
(6, 7) is very likely explained by our finding that the epitope
recognized by monoclonal antibody AC133, which is thought to be a
glycosylated structure (7), is down-regulated upon differentiation of
epithelial cells.
Cell Culture and Transfection--
Caco-2 cells (kindly donated
by Drs. S. Robine and D. Louvard, CNRS-Institut Curie, Paris, France)
were maintained in Dulbecco's minimal essential medium supplemented
with 10% fetal calf serum, 1% non-essential amino acids (Life
Technologies, Inc.), 2 mM L-glutamine, 100 units/ml penicillin, and 100 µg/ml streptomycin and were cultured in
a humidified incubator at 37 °C under 10% CO2
atmosphere. Cultures were used between passages 8 and 18. CHO cells
were cultured in Ham's F-12 medium supplemented with 10% fetal calf
serum and 50 µg/ml gentamycin at 37 °C under 5% CO2.
For experiments studying polarized cell surface delivery, Caco-2 cells
were plated at a density of 4.5 × 105
cells/cm2 on permeable membranes (24-mm
TranswellTM-COL chambers, 0.4-µm pore size). Media were
changed every day, and the experiments were performed 11 days after
seeding to allow the development of a tight monolayer.
CHO cells were transfected with the pCR3.1-Uni plasmid containing the
AC133 antigen-coding cDNA sequence under the control of the
cytomegalovirus promoter (7), using the LipofectAMINE reagent (Life
Technologies, Inc.) according to the supplier's instructions. Cells
expressing the neomycin resistance gene were then selected by
introducing 600 µg/ml of G418 into the incubation medium. Two weeks
later, G418-resistant colonies were pooled and expanded. To enhance the
expression of the transgene, cells were incubated for 17 h with 10 mM sodium butyrate (11). All subsequent steps were
performed in medium lacking butyrate. Under these conditions 10-30%
of neomycin-resistant cells expressed the recombinant AC133 antigen.
Isolation of Human and Mouse Hematopoietic Cells From Bone
Marrow--
Human bone marrow was aspirated into preservative-free
heparin from the posterior iliac crest of normal adult volunteers
according to procedures approved by the ethics committee of the Royal
Adelaide Hospital. Bone marrow mononuclear cells (BMNC) were obtained
following separation over Ficoll (Lymphoprep, Nygaard) and washed twice at 4 °C in HHF buffer (comprising calcium and magnesium-free Hanks' balanced salt solution (HBSS) supplemented with 5% human AB serum and
5% fetal calf serum). CD34+ cells were isolated from BMNC
suspensions by means of immunomagnetic beads (Dynal Pty. Ltd). Briefly,
BMNC were pelleted by centrifugation, and the pellet was suspended in
100 µl of CD34 Dynabeads to give a final cell:bead ratio of 4:1.
After gentle vortexing for 2 min, the cells were resuspended in 4 ml of
HHF and placed on a rotator for a further 30 min at 4 °C to ensure
complete capture of CD34+ cells. Bead-rosetted
CD34+ cells were isolated and purified by five sequential
rounds of washing with HHF followed by capture on a magnet. The CD34
antibody-coated beads were released from the CD34+ cells by
the addition of 200 µl of the CD34 Detachabead reagent (supplied with
the kit) followed by incubation in a shaking waterbath for 45 min at
37 °C. After removal of the detached beads using the magnet, the
cells were washed three times in HHF, and the aliquots were analyzed
for purity using HPCA-2-PE (Becton Dickinson) and for expression of
AC133 antigen using AC133-PE conjugates (Amcell). In all experiments,
the purity of the CD34+ cell preparations obtained in this
manner was >98%.
Mouse bone marrow cells were obtained from 8-10-week-old BALB/c mice
by flushing dissected femurs, using a syringe with 27G needle, with
Hanks' balanced salt solution supplemented with 0.5% bovine serum
albumin (Sigma). Red blood cells were lysed by a 5-min incubation with
red cell lysing buffer (Sigma), and BMNC were isolated on a Histopaque
1077 gradient (Sigma). Interface cells were harvested and washed twice
with HBSS/bovine serum albumin at 4 °C and adjusted to a
concentration of 2 × 107/ml prior to staining for
flow cytometric analysis.
Flow Cytometric Analysis--
Antibodies were purified from
murine ascites or hollow fiber culture harvests (Cellmax QUAD
artificial capillary system, Cellco Inc. Germantown, MD) by protein A
affinity chromatography and phycoerythrin (PE) conjugates prepared as
described previously (6).
106 murine BMNC were stained for 30 min at 4 °C with
biotinylated anti-mouse CD34 (RAM 34) and, after washing with
HBSS/bovine serum albumin, stained with streptavidin-Cy-Chrome
(Pharmingen). Cells were washed twice with HBSS/bovine serum albumin
before incubation with 150 ng/test of PE-conjugated 13A4 antibody
(anti-mouse prominin). Stained cells were analyzed on a FACScan flow
cytometer (BDIS, San Jose, CA) with the scatter gates set on the
lymphoid population. 50,000 events were acquired and analyzed using
Cell Quest software (BDIS).
Northern Blot Analysis--
Total RNA from Caco-2 cells was
prepared using the acidic phenol/chloroform phase separation method
(12). Northern blot analyses were performed using
CLONTECH multiple tissue Northern blot membrane and
by resolving 20 µg of total RNA from Caco-2 cells on
formaldehyde-agarose gels followed by RNA transfer to nylon membranes
and hybridization with the EcoRI-EcoRI fragment (nucleotides 756-1195) derived from the human AC133 hematopoietic stem
cell antigen cDNA (GenBankTM accession number
AF027208). The probe was labeled with [ Cell Surface Biotinylation and Immunoprecipitation--
All
steps of cell surface biotinylation and immunoprecipitation were
carried out at 4 °C. Just prior to use, the membrane-impermeable sulfo-NHS-LC-biotin agent (Pierce) was dissolved in Ca/Mg-PBS (PBS
containing 1 mM CaCl2 and 0.5 mM
MgCl2) to a final concentration of 0.2 mM.
After repeated washing (three times) with Ca/Mg-PBS, Caco-2 cells (on
100-mm dishes) were incubated for 1 h in 3 ml of the biotin
solution. The cells were washed three times with Ca/Mg-PBS and then
incubated for 10 min with Ca/Mg-PBS containing 20 mM
glycine to quench the residual biotin. Cells were then lysed in
ice-cold solubilization buffer (1% Triton X-100, 0.1% SDS, 150 mM NaCl, 5 mM EGTA, 50 mM Tris/HCl,
pH 7.5, 1 mM phenylmethylsulfonyl fluoride, 2 µg/ml
leupeptin, 10 µg/ml aprotinin). Cell lysates obtained after
centrifugation (10 min, 10,000 × g) were subjected to
immunoprecipitation. For domain-selective cell surface biotinylation (14), 11-day-old monolayers on duplicate Transwell filters were washed
three times with Ca/Mg-PBS and then biotinylated from either the apical
(1.5 ml) or basolateral (2.6 ml) chamber compartment. The compartment
not receiving the sulfo-NHS-LC-biotin was filled with Ca/Mg-PBS. After
quenching, cells were scraped into ice-cold solubilization buffer, and
cell lysates were prepared as described above.
Cell lysates corresponding to one confluent 100-mm dish or half of one
24-mm filter were diluted 4-fold in immunoprecipitation buffer (1%
Triton X-100, 0.1% SDS, 150 mM NaCl, 10 mM
EDTA, 50 mM Tris/HCl, pH 7.8, 10 µg/ml aprotinin, 2 µg/ml leupeptin, and 1 mM phenylmethylsulfonyl fluoride),
and monoclonal antibody (mAb) AC133 (10 µg/ml) was added. Samples
were incubated overnight on an end-over-end shaker. Immune complexes
were collected with protein A-Sepharose CL-4B beads (Amersham Pharmacia
Biotech), pre-adsorbed with rabbit anti-mouse IgG, and used for
deglycosylation, SDS-polyacrylamide gel electrophoresis, and
NeutrAvidin blotting (see below).
Endoglycosidase Digestions and NeutrAvidin Blotting--
Immune
complexes containing the cell surface-biotinylated proteins were eluted
from protein A-Sepharose beads using glycosidase buffer (1% Triton
X-100, 0.1% SDS, 25 mM EDTA, 1% 2-mercaptoethanol, 50 mM sodium phosphate, pH 7.2) at 95 °C. Eluates were
incubated for 6 h at room temperature in the absence or presence
of 1 unit PNGase F (Roche Molecular Biochemicals). Proteins were then
analyzed by SDS-polyacrylamide gel electrophoresis and transferred to
nitrocellulose membranes (Schleicher and Schuell, pore size 0.45 µm)
using standard procedures. After transfer, nitrocellulose membranes
were incubated for 2 h in PBS containing 5% low fat milk powder
and 0.3% Tween 20 and then washed extensively with PBS. Biotinylated
proteins were identified with horseradish peroxidase-conjugated
NeutrAvidin (Pierce) (diluted in PBS containing 0.1% Tween 20)
followed by incubation with the SuperSignal ULTRA chemiluminescent
substrate (Pierce).
Immunofluorescence and Confocal Microscopy--
Cell surface
immunofluorescence was carried out as described previously (5). Cells
grown on glass coverslips were washed with Ca/Mg-PBS, first at room
temperature and then on ice, and surface-labeled for 30 min at 4 °C
by the addition of the mAb AC133 (10 µg/ml) or mAb AC141 (10 µg/ml)
or double surface-labeled by an additional incubation for 10 min with
fluorescein isothiocyanate-conjugated wheat germ agglutinin (Sigma).
Both antibodies and wheat germ agglutinin were diluted in ice-cold
immunofluorescence buffer (Ca/Mg-PBS containing 0.2% gelatin). Unbound
antibodies or free wheat germ agglutinin were removed by five washes
with ice-cold immunofluorescence buffer. Fixative (3% paraformaldehyde
(Sigma) in PBS) was added to the cells on ice, and the coverslips were placed at room temperature for 30 min. The fixative was removed by
three washes with Ca/Mg-PBS, and the residual formaldehyde was quenched
for 30 min with 0.1 M glycine in PBS. Cells were then
incubated for 30 min at room temperature with lissamine
rhodamine-conjugated goat anti-mouse IgG (H + L) (Dianova). Coverslips
were rinsed sequentially with immunofluorescence buffer, PBS, and
distilled water and mounted in Mowiol 4.88 (Calbiochem).
In experiments with permeabilized cells, cells grown on glass
coverslips were washed with PBS and fixed with 3% paraformaldehyde in
PBS for 30 min at room temperature. Coverslips were then rinsed with
and incubated for 10 min in PBS containing 50 mM ammonium chloride. Cells were permeabilized and blocked with 0.2% saponin/0.2% gelatin in PBS (blocking solution) for 30 min. Cells were then incubated sequentially with mAb AC133 (10 µg/ml), 1 mg/ml RNase A for
10 min at room temperature (to digest cytoplasmic mRNA), and
CyTM2-conjugated goat anti-mouse IgG (H + L) (Dianova), all
in blocking solution. Nuclei were labeled with propidium iodide (0.3 µg/ml) during the incubation of secondary antibody. Coverslips were
rinsed and mounted as described above. Cells were observed with a Leica TCS4D confocal laser scanning microscope. The confocal
microscope settings were such that the photomultipliers were within
their linear range. The images shown were prepared from the confocal
data files using Adobe photoshop software.
Immunocytochemistry of Human Embryonic Tissues--
A human
embryo at 32 days of development was obtained immediately after
voluntary termination of pregnancy induced with the RU 486 antiprogestative compound. Informed consent for the use of the embryo
in research was obtained from the patient, and the embryo was collected
according to the guidelines and with the approval of both national and
institutional ethics committees.
Human embryonic tissue, fixed in 4% paraformaldehyde (v/v) in PBS for
1 h at 4 °C, was rinsed in PBS for several hours and then twice
in 15% sucrose in PBS for at least 24 h. The tissue was then
embedded in 15% sucrose, 7.5% gelatin in PBS, and frozen at
Immunogold Electron Microscopy of Caco-2 Cells and
AC133-transfected CHO Cells--
Caco-2 cells or CHO cells stably
transfected with the human AC133 gene cultured on collagen-coated 60-mm
Petri dishes (Collagen R, Serva) were washed with 200 mM
Hepes-NaOH, pH 7.4, and fixed with 4% paraformaldehyde (Caco-2 cells)
or 8% paraformaldehyde (CHO cells) in Hepes buffer for 4 h at
4 °C. CHO cells were embedded in 10% gelatin in PBS. Ultrathin
cryosections were prepared as described previously (5). Immunogold
labeling of cryosections using either mAb AC133 (0.5-1.5 mg/ml) or mAb
AC141 (0.15-2.3 mg/ml) followed by goat anti-mouse antibody coupled to
15-nm gold particles (Aurion) was performed according to the
manufacturer's instruction (Aurion) with minor modifications.
Immunogold Electron Microscopy of Human CD34+
Hematopoietic Progenitor Cells--
Human CD34+
hematopoietic progenitor cells were incubated for 45 min at 4 °C
with saturating concentrations of AC133 antibody and with an equivalent
concentration of an IgG1 isotype-matched nonbinding control antibody,
1B5 (generously supplied by Dr. Graham Mayerhofer, Hanson Center for
Cancer Research). After two washes with HHF buffer, the cells were
incubated with goat anti-mouse IgG coupled to colloidal gold particles
of average diameter of 10 nm (GAM-Au10, Janssen Pharmaceutica) for
1 h at 4 °C with gentle rocking, washed three times with HHF
buffer, and then fixed in a 3% (v/v) solution of EM-grade
glutaraldehyde (Agar Aids) in 150 mM cacodylate buffer (pH
7.3). Additional aliquots of CD34+ cells were stained in
parallel using an identical protocol with mAb DREG-56 (anti-CD62L) and
with mAb 7E10 (an antibody prepared in P. J. Simmons' laboratory,
which identifies a class 3 epitope on the CD34 molecule (15)).
Immunolabeled cells were then washed three times in cacodylate buffer,
post-fixed for 1 h in 1% osmium tetroxide (Johnson Matthey
Chemical Ltd.) in the same buffer, and then dehydrated by exposure to a
graded series of ethanol. Following dehydration in 100% ethanol, the
cells were transferred to propylene oxide (BDH) prior to infiltration
with epon-araldite resin overnight at room temperature. The resin was
polymerized for 8 h at 80 °C after which ultrathin sections
were cut and examined without counterstaining in a transmission
electron microscope (Jeol) operating at 80 kV. The qualitative and
quantitative statements about the distribution of the various antigens
between protrusions and planar regions of the plasma membrane are based
on the examination of 80-100 cells and the counting of at least 250 gold particles for each antibody.
The AC133 Antigen Is Endogenously Expressed in the Human
Intestine-derived Epithelial Cell Line Caco-2--
Both previously
described (6) mouse mAb against the human AC133 hematopoietic stem cell
antigen, mAb AC133 (Fig. 1A)
and mAb AC141 (Fig. 1B) were found to stain, on cell surface
immunofluorescence, Caco-2 cells, a human intestine-derived epithelial
cell line (16). The staining showed a punctate pattern, which is
characteristic of a microvilli-associated antigen. Caco-2 cells did not
show any staining when primary antibodies were omitted (Fig.
1C).
The antigen recognized by mAbs AC133 and AC141 on the surface of Caco-2
cells was characterized. Both mAb AC133 (7) and mAb
AC1412 recognize a
glycosylated structure. We therefore labeled the surface of Caco-2
cells with the membrane-impermeant sulfo-NHS-LC-biotin and subjected
the antigen immunoprecipitated with mAb AC133 to incubation with and
without PNGase F, followed by SDS-polyacrylamide gel electrophoresis
and blotting with horseradish peroxidase-conjugated NeutrAvidin (Fig.
2A). Without PNGase F
treatment, a band with an apparent molecular mass of
Northern blot analysis of Caco-2 cell RNA, using an EcoRI
fragment derived from the human AC133 antigen cDNA as probe,
revealed the presence of a
We examined the presence of the AC133 antigen mRNA in various human
tissues. AC133 antigen mRNA was detected in brain, heart, kidney,
liver, lung, and placenta, as reported previously (7), as well as in
the colon and small intestine (Fig. 2C). Consistent with the
presence of AC133 antigen mRNA in the human colon, six expressed
sequence tags derived from this source were identified (GenBankTM accession numbers AI469575, AI273443, AI308936,
D25789, AA622198, and AA577606). It should be noted that the presence of the AC133 antigen mRNA in adult colon is in contrast to the lack
of AC133 immunoreactivity in that tissue (see Table II within Ref.
7).
Polarized Distribution of the AC133 Antigen at the Plasma Membrane
of Caco-2 Cells--
Consistent with their epithelial origin, Caco-2
cells exhibit an apical-basal polarity with distinct, apical
versus basolateral, plasma membrane domains (17, 18). To
determine the localization of AC133 antigen with regard to the apical
versus basolateral plasma membrane domain of these cells,
indirect immunofluorescence using mAb AC133 followed by confocal laser
scanning microscopy was performed on Caco-2 cells grown on glass
coverslips for 16 days after reaching confluence (Fig.
3). To facilitate the analysis, nuclei
were labeled with propidium iodide (red). A series of single optical xy-plane sections from the apical (Fig. 3A) to the
basolateral (Fig. 3E) side revealed that AC133
immunostaining (green) was predominantly located at the
apical domain. Two single optical xz-plane sections through the AC133
antigen-positive cells shown in panels A-E confirmed the
apical localization of the AC133 antigen (Fig. 3, e' and
e").
The staining for the AC133 antigen was not uniform across the entire
postconfluent cell monolayer (Fig. 3), in contrast to the observations
on subconfluent cultures where almost all cells were stained (Fig.
1A). Approximately 20% of the postconfluent Caco-2 cells
showed immunofluorescence staining at the apical membrane (Fig. 3,
A-E and Fig. 4,
A-C), whereas
To quantitate the cell surface distribution of the AC133 antigen in
Caco-2 cells, domain-selective cell surface biotinylation was
performed. 11-day-old postconfluence Caco-2 cell monolayers grown on
Transwell filters were biotinylated with sulfo-NHS-LC-biotin agent on
either the apical or basolateral membrane. The AC133 antigen was
immunoprecipitated from cell extracts, and cell surface-labeled AC133
antigen was then quantified by probing the immunoprecipitates with
horseradish peroxidase-conjugated avidin (Fig.
5A). Biotinylated AC133
antigen was recovered from the apical (Fig. 5A, lane
1) but not basolateral (Fig. 5A, lane 2),
plasma membrane. The absence of biotinylated AC133 antigen at the
basolateral cell surface suggested that either the lateral staining for
the AC133 antigen detected by light microscopy in some cells (Fig.
4E) occurred in an intracellular membrane compartment
closely associated with the basolateral membrane, or the amount of
AC133 antigen in the lateral plasma membrane was below the level of
biochemical detection.
To rule out the possibility that the biotinylation agent did not have
access to the basolateral membrane due to steric hindrance, we analyzed
the protein composition of the apical and basolateral membranes after
domain-selective cell surface biotinylation. Visualization of
biotinylated proteins with horseradish peroxidase-conjugated NeutraAvidin revealed that both membrane domains contain a distinct set
of cell surface proteins (Fig. 5B, lanes 1 and
2), which indicated that the biotinylation agent reached
both the apical and basolateral surface of Caco-2 cells grown on
permeable supports. These data also document the integrity of the cell
monolayer used in our assays.
Down-regulation of the AC133 Epitope upon Differentiation of Caco-2
Cells--
Enterocytic differentiation of Caco-2 cells is a
growth-related process, which starts about 7 days after the cells reach
confluence (17, 18) and is complete within 20-30 days (20). To further analyze the mosaic expression of the AC133 antigen in Caco-2 cells, we
investigated in greater detail its expression pattern in relation to
the time cells spent in a subconfluent/confluent state in culture. AC133 antigen expression was monitored by indirect cell surface immunofluorescence using either mAb AC133 (Fig.
6A) or mAb AC141 (which gave
the same results as mAb AC133 (data not shown)). As mentioned above,
virtually all cells in subconfluent culture were labeled (Fig.
6A). After confluence, AC133 immunoreactivity persisted for
about a week in 70-80% of the cells (Fig. 6A).
Interestingly, upon differentiation of Caco-2 cells, the AC133
immunoreactivity decreased rapidly, and at 15 days post-confluence and
thereafter only 10-20% of the cells were stained by the mAb AC133
(Fig. 6A).
To determine whether down-regulation of AC133 immunoreactivity is
associated with a change in mRNA expression, we examined transcripts by Northern blot analysis on days 3, 17, and 33 post-confluence (Fig. 6B). Remarkably, a small increase,
rather than a decrease, in the relative level of the AC133 antigen
mRNA over time was detected (Fig. 6B).
Expression of the AC133 Antigen in Human Embryonic
Tissues--
Prominin immunoreactivity has previously been detected in
various murine embryonic epithelia (4). Given that the AC133 antigen is
expressed in a human epithelial cell line, Caco-2, we examined the
expression of the AC133 antigen in human embryonic epithelia.
32-day-old human embryonic tissues were stained with mAb AC133 (Fig.
7). Interestingly, AC133 immunoreactivity
was detected on the apical, but not basolateral, side of the neural tube (Fig. 7A). AC133 immunoreactivity was also observed on
the apical side of the mesonephros (Fig. 7B) and gut (Fig.
7C). Hence, the tissue expression of the AC133 antigen in
the human embryo is similar to that of prominin in the mouse embryo
(4).
The AC133 Antigen Is Associated with Apical Microvilli in
Epithelial Cells and with Plasma Membrane Protrusions in Non-epithelial
Cells--
The remarkable feature of mouse prominin, i.e.
its specific localization in microvillar membranes of epithelial cells
(4, 5) and related plasma membrane protrusions of non-epithelial cells
(4), prompted us to investigate the ultrastructural localization of the
AC133 antigen in Caco-2 cells and, upon transfection, in CHO cells
(Fig. 8). Immunogold EM revealed that
AC133 immunoreactivity was specifically associated with microvilli at
the apical surface of Caco-2 cells and lacking from the small
intermicrovillar regions of the plasma membrane (Fig. 8, A
and B). The preferential localization of the AC133 antigen
to plasma membrane protrusions was also observed in AC133
antigen-transfected fibroblasts (Fig. 8, C and
D). In this case, gold particles were conspicuously
concentrated on microvilli-like protrusions, whereas the large
neighboring planar areas of the plasma membrane were labeled to a much
lesser extent, if at all.
The AC133 Antigen Is Enriched in Plasma Membrane Protrusions of
Human CD34+ Hematopoietic Progenitor Cells--
To
investigate the ultrastructural localization of the AC133 antigen in
hematopoietic progenitor cells, CD34+ cells were isolated
from human bone marrow, subjected to cell surface immunogold labeling,
and analyzed by EM (Fig. 9). Remarkably, cell surface AC133 immunoreactivity was preferentially associated with
plasma membrane protrusions (Fig. 9A, arrows),
where it appeared to be concentrated at the tip (Fig. 9B).
Specifically, about half of the gold particles were associated with
protrusions, which however constituted only a minor proportion of the
plasma membrane. Control experiments performed with mAb 7E10
(anti-CD34) revealed that the CD34 antigen was randomly distributed
over the surface of CD34+ cells (Fig. 9C), with
a minor fraction ( Prominin Is Found on Murine Hematopoietic Progenitor
Cells--
The data presented above show that the human AC133 antigen
exhibits the same key features as previously reported for mouse prominin. This in turn raises the possibility that prominin is expressed by murine hematopoietic progenitor cells. To examine this
possibility, we analyzed by flow cytometry the expression of prominin
in mouse bone marrow CD34+ cells (22-24) (Fig.
10). Double cell surface staining of
the lymphoid-gated cell population with mAb anti-CD34 and mAb 13A4
(anti-mouse prominin) (4) revealed that 36% (n = 3) of
the CD34+ cells (which represented 2.85% of nucleated
cells in bone marrow) express prominin. This expression level was
comparable to that reported for the AC133 antigen in human bone marrow
cells (6, 25).
The present data indicate that the human AC133 antigen shows a
very similar, if not identical, cellular distribution and subcellular localization as previously reported for mouse prominin (4, 5). The
human AC133 antigen, previously found in flow cytometry to be
restricted to the CD34bright population of progenitors
cells from adult and fetal bone marrow, fetal liver, cord, or
peripheral blood (6, 25), and to subsets of CD34+ leukemias
(7), is like prominin (4) expressed in epithelial cells. Conversely,
mouse prominin is also expressed on a subset of CD34+
progenitors isolated from murine bone marrow, consistent with the
expression of the AC133 antigen on human hematopoietic stem cells (6,
25). This finding suggests that prominin could be of use as a marker of
murine hematopoietic progenitors, and therefore its value in studies of
murine hematopoiesis may be considerable. Further studies will be
needed to characterize the specific subpopulation of mouse bone marrow
cells expressing prominin.
As previously reported for mouse prominin in epithelial cells (4, 5),
the human AC133 antigen is mainly confined to the apical plasma
membrane, where it appears to be concentrated in microvilli. This
microvillar localization is independent of the epithelial phenotype of
the cell, as shown by the ectopic expression of the AC133 antigen in
fibroblasts and its physiological expression in hematopoietic
progenitor cells. Considering also the sequence homology between the
human AC133 antigen and mouse prominin (8, 9), the present results
strongly suggest that the AC133 antigen is the human orthologue of
prominin rather than a distinct member of the prominin family.
Following a suggestion of the HUGO Nomenclature Committee, we propose
to refer to the AC133 antigen as "prominin (mouse)-like 1" (PROML1)
until completion of the sequencing of the human genome; should the
latter fail to reveal the existence of a human open reading frame more
closely related to mouse prominin than the human AC133 antigen, the
latter should be called prominin like its mouse orthologue.
The presence of mRNA for the AC133 antigen/PROML1 in adult colon is
in contrast to the lack of AC133 immunoreactivity in this tissue
reported previously (7). This discrepancy may be explained by the fact
that mAb AC133 binds to a glycosylation-dependent epitope
(7) and that processing of asparagine-linked oligosaccharides has been
reported to change upon differentiation of HT-29 cells (26), a model
cell line to study enterocytic differentiation (18). Our results with
Caco-2 cells, showing a decrease of AC133 immunoreactivity, but not of
the mRNA level for the AC133 antigen/PROML1, upon differentiation
are consistent with differential glycosylation depending on the state
of enterocytic differentiation. Perhaps the more widespread presence of
AC133 immunoreactivity in embryonic as compared with adult human
epithelia reflects, at least in part, differential glycosylation rather
than a variation in protein expression of the AC133 antigen/PROML1.
Further investigation of this issue will require the generation of
antibodies directed against the AC133 antigen/PROML1 polypeptide.
The likely orthologue relationship between the human AC133
antigen/PROML1 and mouse prominin has important implications for studies addressing the as yet unknown function of this protein. In
particular, insight into the molecular basis of prominin's preference
for plasma membrane protrusions, derived from murine models, can now be
used to understand human disease caused by mutation of the AC133
antigen/PROML1 gene, as exemplified by that causing retinal
degeneration (10).
S. M. W. thanks Professor Sir David J. Weatherall for his support.
*
The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
b
Joint first authors.
c
Recipient of a fellowship from the
Max-Planck-Society.
d
Supported by the Studienstiftung des
Deutschen Volkes.
h
Supported by the Medical Research Council,
UK, the Leukemia Research Fund, UK, and EU Biotech and INTAS/RFBR grants.
j
Supported by the National Health and Medical
Research Council (NHMRC) of Australia.
k
Supported by Grants SFB 317, D2 and SFB 352, C1 from the Deutsche Forschungsgemeinschaft, the German-Israeli
Foundation for Scientific Research and Development, and the Fonds der
Chemischen Industrie. To whom reprint requests and correspondence
should be addressed: Dept. of Neurobiology, University of Heidelberg, Im Neuenheimer Feld 364, D-69120 Heidelberg, Germany. Tel.:
49-6221-548218; Fax: 49-6221-546700; E-mail:
whuttner@sun0.urz.uni-heidelberg.de.
2
D. W. Buck, unpublished data.
The abbreviations used are:
CHO, Chinese hamster
ovary;
BMNC, bone marrow mononuclear cells;
HBSS, Hanks' balanced salt
solution;
PE, phycoerythrin;
sulfo-NHS-LC-biotin, sulfosuccinimidyl-6(biotinamido)-hexanoate biotinylating agent;
PBS, phosphate-buffered saline;
mAb, monoclonal antibody.
The Human AC133 Hematopoietic Stem Cell Antigen Is also
Expressed in Epithelial Cells and Targeted to Plasma Membrane
Protrusions*
ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-32P]dCTP using
the Rediprime kit (Amersham Pharmacia Biotech). Hybridization and
washing were performed either at 65 °C using Church buffer as
described elsewhere (13) or at 68 °C using the Expresshyb hybridization solution (CLONTECH) according to the
manufacturer's instructions. Blots were analyzed using a Fuji phosphoimager.
70 °C. Five-micron-thick frozen sections were thawed, hydrated in
PBS, and endogenous peroxidases were inhibited for 20 min in PBS
containing 0.2% hydrogen peroxide. Sections were then washed with
0.25% Triton X-100 in PBS and the mAb AC133 (20 µg/ml) was added
overnight at 4 °C. After washing with PBS-Triton X-100, incubation
was performed for 1 h at room temperature with biotinylated rabbit
anti-mouse antibody (DAKO) and subsequently with peroxidase-coupled streptavidin (DAKO). Peroxidase activity was revealed with 0.025% (v/v) 3.3-diaminobenzidine (Sigma) in PBS containing 0.015% hydrogen peroxide. Slides were counterstained with Harris' hematoxylin, and
mounted in XAM neutral medium (BDH).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (175K):
[in a new window]
Fig. 1.
Cell surface expression of human AC133
antigen in Caco-2 cells. Intact subconfluent Caco-2 cells grown on
glass coverslips were incubated at 4 °C with the mAb AC133
(A), mAb AC141 (B), or, as a control, without
primary antibody (C), paraformaldehyde-fixed, and then
incubated with lissamine rhodamine-conjugated anti-mouse secondary
antibody. The samples were viewed with a confocal microscope, and
composite pictures of all optical sections are shown. D,
Nomarski image of the cells examined in C. Bar in
D = 10 µm. In A and B, note the
punctate cell surface staining characteristic of microvilli-associated
antigens.
120 kDa was
detected (Fig. 2A, lane 1). Deglycosylation
yielded a 95-kDa band (Fig. 2A, lane 2). These
apparent molecular masses are those expected for the AC133 antigen (7).
The same results were obtained using mAb AC141 instead of mAb AC133 for
the immunoprecipitation (data not shown). Furthermore, native and
deglycosylated protein immunoprecipitated from Caco-2 cells with mAb
AC133 exhibited the same molecular mass as authentic human AC133
antigen obtained from CHO cells transfected with the AC133 antigen
cDNA (data not shown).
View larger version (34K):
[in a new window]
Fig. 2.
Characterization of the AC133 antigen in
Caco-2 cells and tissue distribution of its mRNA.
A, AC133 antigen in Caco-2 cells is a 120-kDa glycoprotein.
Confluent Caco-2 cells were incubated at 4 °C with
sulfo-NHS-LC-biotin and solubilized, and AC133 antigen was
immunoprecipitated using mAb AC133 (AC133), or, as a control, no
antibody (no mAb). Solubilized immunoprecipitates were incubated in the
absence ( ) or presence (F) of PNGase F followed by
NeutrAvidin blotting. Asterisk, 120-kDa AC133 antigen;
arrowhead, product after N-deglycosylation. B and
C, Northern blots of total RNA isolated from Caco-2 cells
(B) or poly(A)+ RNA (2 µg each) from
various human tissues (C) and probed with human AC133
antigen cDNA.
5.0-kilobase mRNA (Fig.
2B), as expected for the AC133 antigen (7). Taken together,
the data of Fig. 1 and Fig. 2, A and B, show that
the AC133 antigen is endogenously expressed in the epithelial cell line
Caco-2.
View larger version (39K):
[in a new window]
Fig. 3.
Apical localization of AC133 antigen in
polarized Caco-2 cells. Confluent Caco-2 cells grown on glass
coverslips were paraformaldehyde-fixed, saponin-permeabilized, and
incubated with mAb AC133 followed by CyTM2-conjugated
anti-mouse secondary antibody (green). Nuclei were labeled
with propidium iodide (red). A-E, a series of
single optical xy-plane sections from the apical (A) to the
basal (E) side of the cells. e' and
e", two single optical xz-plane sections of the AC133
antigen-positive cells shown in A-E. The white
triangular indentations at the margins of E indicate the
approximate positions of the xz sections shown in e' and
e". Bar in e" = 10 µm. Note that in
the AC133 antigen-expressing cells, immunoreactivity is confined to the
apical surface.
80% of the cells did not show any
prominent immunofluorescence staining (Fig. 4, J-L). Mosaic
expression of the AC133 antigen in postconfluent Caco-2 cells is
reminiscent of that reported for several brush-border hydrolases (19,
20) and transporters (21). The intensity of immunostaining for the
AC133 antigen at the apical membrane was variable (compare Fig. 4,
A, D, and G). In addition, some Caco-2
cells showed staining for the AC133 antigen also in intracellular compartments (Fig. 4H) and on the lateral domain of the
cells (see ring-like staining in Fig. 4E). The latter
observation suggested that the AC133 antigen in these cells was located
either at the lateral plasma membrane and/or in vesicles closely
associated with this membrane.
View larger version (76K):
[in a new window]
Fig. 4.
Variable cellular expression and subcellular
localization of the AC133 antigen in Caco-2 cells. 16-day-old
post-confluent Caco-2 cells grown on glass coverslips were treated and
stained as in Fig. 3. Four sets of three panels each, A-C,
D-F, G-I, and J-L, show distinct
areas of the same cell monolayer. Single optical xy-plane sections at
the apical surface of the cells (top panels), at the level
of the nuclei (middle panels), and single optical xz-plane
sections (bottom panels) are shown. AC133 antigen are
pseudocolored in green and nuclei are in red.
Bar in K = 10 µm.
View larger version (47K):
[in a new window]
Fig. 5.
Steady-state distribution of the AC133
antigen between the apical and basolateral plasma membrane of Caco-2
cells. Transwell filter-grown Caco-2 cells were
surface-biotinylated at 4 °C from either the apical (a)
or basolateral (bl) side using sulfo-NHS-LC-biotin.
A, cell lysates were subjected to immunoprecipitation using
either mAb AC133 (AC133) or, as a control, no antibody ( ), and the
immunoprecipitates were analyzed by NeutrAvidin blotting.
Asterisk, 120-kDa AC133 antigen. B, cell lysates
were directly analyzed by NeutrAvidin blotting. Open
circles, apical plasma membrane-specific proteins; filled
circles, basolateral plasma membrane-specific proteins.
View larger version (17K):
[in a new window]
Fig. 6.
Expression of the AC133 antigen and its
mRNA during differentiation of Caco-2 cells. A,
down-regulation of AC133 immunoreactivity. Intact Caco-2 cells, grown
on glass coverslips for the indicated periods of time, were surface
double-labeled at 4 °C with the mAb AC133 and fluorescein
isothiocyanate-conjugated wheat germ agglutinin,
paraformaldehyde-fixed, and incubated with lissamine
rhodamine-conjugated anti-mouse secondary antibody. AC133
antigen-positive cells are expressed as the percentage of wheat germ
agglutinin-labeled cells. Values represent the mean of two to four
independent coverslips; bars indicate S.D. or the variation
of the individual values from the mean. More than 400 cells were
counted for each time point. S, quantification of cells in
subconfluent state. B, Northern blot of total RNA ( 20
µg) isolated from Caco-2 cells, grown for the indicated periods of
time, and probed with human AC133 antigen cDNA followed by
re-probing with a glyceraldehyde-3-phosphate dehydrogenase
(GAPDH) cDNA. The bottom panel shows the ratio of the
two RNA signals quantified by densitometric scanning.
View larger version (99K):
[in a new window]
Fig. 7.
Immunoperoxidase localization of the AC133
antigen in human embryonic tissue. Transverse sections of
32-day-old embryonic tissue stained with mAb AC133. A,
neural tube; B, mesonephros; C, gut. In all
panels, AC133 immunoreactivity is confined to the apical, luminal side
of the epithelia. Panels are the same magnification.
View larger version (98K):
[in a new window]
Fig. 8.
Immunogold electron microscopy of the AC133
antigen on the apical plasma membrane of Caco-2 cells and on the plasma
membrane of AC133 antigen-transfected CHO cells. Ultrathin
cryosections of paraformaldehyde-fixed Caco-2 cells (A and
B) or AC133 antigen-transfected CHO cells (C and
D) were stained with mAb AC133 (A, C,
D) or mAb AC141 (B) followed by goat anti-mouse
IgG coupled to 15-nm gold particles. Arrowheads,
intermicrovillar regions of the plasma membrane. Magnifications:
A, × 15,800; B, × 24,200; C, × 34,200; D, × 25,300.
5%) of the gold particles being associated with
protrusions. In contrast, cell surface staining with mAb DREG 56 (anti-CD62L) showed, as anticipated, the preferential association of
L-selectin (CD62L) with plasma membrane protrusions (Fig.
9D, arrows), with about half of the gold
particles being associated with these structures, as was the case for
the AC133 antigen.
View larger version (141K):
[in a new window]
Fig. 9.
Immunogold electron microscopy of the AC133
antigen on the cell surface of human CD34+ hematopoietic
cells. CD34+ hematopoietic progenitor cells, isolated
from human adult bone marrow, were incubated at 4 °C with mAb AC133
(A and B), mAb anti-CD34 (C), or mAb
anti-CD62L (D) followed by goat anti-mouse IgG coupled to
10-nm gold particles. Immunolabeled cells were fixed with
glutaraldehyde and post-fixed with osmium tetroxide, and ultrathin
plastic sections were examined without counterstaining.
Arrows indicate AC133 antigen (A) and
L-selectin (D) immunoreactivity at the tip of
plasma membrane protrusions. Magnifications: A,
C, and D, × 2500; B, × 5000. Note
that the AC133 antibody selectively binds to plasma membrane
protrusions of the cell surface (A and B).
View larger version (38K):
[in a new window]
Fig. 10.
Cell surface expression of prominin in mouse
bone marrow CD34+ cells. Mouse bone marrow cells were
double-labeled with biotinylated mAb anti-CD34 followed by
streptavidin-Cy-Chrome (CD34 CY) and mAb 13A4 conjugated to
phycoerythrin (13A4 PE). Labeled cells were analyzed by flow
cytometry. The upper right quadrant shows cells stained for
both CD34 and prominin.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
ACKNOWLEDGEMENTS
FOOTNOTES
ABBREVIATIONS
REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1.
Rodriguez-Boulan, E.,
and Nelson, J.
(1989)
Science
245,
718-725 2.
Nelson, W. J.
(1992)
Science
258,
948-955 3.
Simons, K.,
Dupree, P.,
Fiedler, K.,
Huber, L. A.,
Kobayashi, T.,
Kurzchalia, T.,
Olkkonen, V.,
Pimplikar, S.,
Parton, R.,
and Dotti, C.
(1992)
Cold Spring Harbor Symp. Quant. Biol.
57,
611-619 4.
Weigmann, A.,
Corbeil, D.,
Hellwig, A.,
and Huttner, W. B.
(1997)
Proc. Natl. Acad. Sci. U. S. A.
94,
12425-12430 5.
Corbeil, D.,
Röper, K.,
Hannah, M. J.,
Hellwig, A.,
and Huttner, W. B.
(1999)
J. Cell Sci.
112,
1023-1033[Abstract]
6.
Yin, A. H.,
Miraglia, S.,
Zanjani, E. D.,
Almeida-Porada, G.,
Ogawa, M.,
Leary, A. G.,
Olweus, J.,
Kearney, J.,
and Buck, D. W.
(1997)
Blood
90,
5002-5012 7.
Miraglia, S.,
Godfrey, W.,
Yin, A. H.,
Atkins, K.,
Warnke, R.,
Holden, J. T.,
Bray, R. A.,
Waller, E. K.,
and Buck, D. W.
(1997)
Blood
90,
5013-5021 8.
Corbeil, D.,
Röper, K.,
Weigmann, A.,
and Huttner, W. B.
(1998)
Blood
91,
2625-2626 9.
Miraglia, S.,
Godfrey, W.,
and Buck, D.
(1998)
Blood
91,
4390-4391 10.
Maw, M. A.,
Corbeil, D.,
Koch, J.,
Hellwig, A.,
Wilson-Wheeler, J. C.,
Bridges, R. J.,
Kumaramanickavel, G.,
John, S.,
Nancarrow, D.,
Röper, K.,
Weigmann, A.,
Huttner, W. B.,
and Denton, M. J.
(2000)
Hum. Mol. Genet.
9,
27-34 11.
Gorman, C.,
Howard, B.,
and Reeves, R.
(1983)
Nucleic Acids Res.
11,
7631-7648 12.
Chomczynski, P.,
and Sacchi, N.
(1987)
Anal. Biochem.
162,
156-159[Medline]
[Order article via Infotrieve]
13.
Church, G. M.,
and Gilbert, W.
(1984)
Proc. Natl. Acad. Sci. U. S. A.
81,
1991-1995 14.
Le Bivic, A.,
Real, F. X.,
and Rodriguez-Boulan, E.
(1989)
Proc. Natl. Acad. Sci. U. S. A.
86,
9313-9317 15.
Nishio, H.,
Tada, J.,
Hashiyama, M.,
Hirn, J.,
Ingles-Esteve, J.,
and Suda, T.
(1997)
in
Sixth International Workshop and Conference, Kobe, Japan, November 10-14, 1996
(Kishimoto, T.
, Kikutani, H.
, von dem Borne, A. E. G. K.
, Goyert, S. M.
, Miyasaka, M.
, Moretta, L.
, Okumura, K.
, Shaw, S.
, Springer, T. A.
, Sugamura, K.
, and Zola, H., eds)
, pp. 974-976, Garland Publishing, Inc., New York
16.
Fogh, J.,
Fogh, J. M.,
and Orfeo, T.
(1977)
J. Natl. Cancer Inst.
59,
221-226
17.
Pinto, M.,
Robine-Leon, S.,
Appay, M. D., M., K.,
Triadou, N.,
Dussaulx, E.,
Lacroix, B.,
Simon-Assmann, P.,
Haffen, K.,
Fogh, J.,
and Zweibaum, A.
(1983)
Biol. Cell
47,
323-330
18.
Rousset, M.
(1986)
Biochimie (Paris)
68,
1035-1040[Medline]
[Order article via Infotrieve]
19.
Howell, S.,
Kenny, A. J.,
and Turner, A. J.
(1992)
Biochem. J.
284,
595-601
20.
Vachon, P. H.,
and Beaulieu, J. F.
(1992)
Gastroenterology
103,
414-423[Medline]
[Order article via Infotrieve]
21.
Harris, D. S.,
Slot, J. W.,
Geuze, H. J.,
and James, D. E.
(1992)
Proc. Natl. Acad. Sci. U. S. A.
89,
7556-7560 22.
Lin, G.,
Finger, E.,
and Gutierrez-Ramos, J. C.
(1995)
Eur. J. Immunol.
25,
1508-1516[Medline]
[Order article via Infotrieve]
23.
Krause, D. S.,
Ito, T.,
Fackler, M. J.,
Smith, O. M.,
Collector, M. I.,
Sharkis, S. J.,
and May, W. S.
(1994)
Blood
84,
691-701 24.
Krause, D. S.,
Fackler, M. J.,
Civin, C. I.,
and May, W. S.
(1996)
Blood
87,
1-13 25.
de Wynter, E. A.,
Buck, D.,
Hart, C.,
Heywood, R.,
Coutinho, L. H.,
Clayton, A.,
Rafferty, J. A.,
Burt, D.,
Guenechea, G.,
Bueren, J. A.,
Gagen, D.,
Fairbairn, L. J.,
Lord, B. I.,
and Testa, N. G.
(1998)
Stem Cells
16,
387-396 26.
Ogier-Denis, E.,
Codogno, P.,
Chantret, I.,
and Trugnan, G.
(1988)
J. Biol. Chem.
263,
6031-6037
Copyright © 2000 by The American Society for Biochemistry and Molecular Biology, Inc.
CiteULike 
Complore 
Connotea 
Del.icio.us 
Digg 
Reddit 
Technorati What's this?
This article has been cited by other articles:
|
|
 |
|
  D. Germano, P. Blyszczuk, A. Valaperti, G. Kania, S. Dirnhofer, U. Landmesser, T. F. Luscher, L. Hunziker, H. Zulewski, and U. Eriksson Prominin-1/CD133+ Lung Epithelial Progenitors Protect from Bleomycin-induced Pulmonary Fibrosis Am. J. Respir. Crit. Care Med., May 15, 2009; 179(10): 939 - 949. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. P. Kusumbe, A. M. Mali, and S. A. Bapat CD133-Expressing Stem Cells Associated with Ovarian Metastases Establish an Endothelial Hierarchy and Contribute to Tumor Vasculature Stem Cells, March 1, 2009; 27(3): 498 - 508. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. Karbanova, E. Missol-Kolka, A.-V. Fonseca, C. Lorra, P. Janich, H. Hollerova, J. Jaszai, J. Ehrmann, Z. Kolar, C. Liebers, et al. The Stem Cell Marker CD133 (Prominin-1) Is Expressed in Various Human Glandular Epithelia J. Histochem. Cytochem., November 1, 2008; 56(11): 977 - 993. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
Y. Hori, M. Fukumoto, and Y. Kuroda Enrichment of Putative Pancreatic Progenitor Cells from Mice by Sorting for Prominin1 (CD133) and Platelet-Derived Growth Factor Receptor {beta} Stem Cells, November 1, 2008; 26(11): 2912 - 2920. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. Jaksch, J. Munera, R. Bajpai, A. Terskikh, and R. G. Oshima Cell Cycle-Dependent Variation of a CD133 Epitope in Human Embryonic Stem Cell, Colon Cancer, and Melanoma Cell Lines Cancer Res., October 1, 2008; 68(19): 7882 - 7886. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. M. Yi, H.-C. Tsai, S. C. Glockner, S. Lin, J. E. Ohm, H. Easwaran, C. D. James, J. F. Costello, G. Riggins, C. G. Eberhart, et al. Abnormal DNA Methylation of CD133 in Colorectal and Glioblastoma Tumors Cancer Res., October 1, 2008; 68(19): 8094 - 8103. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 K.E. Schwab, P. Hutchinson, and C.E. Gargett Identification of surface markers for prospective isolation of human endometrial stromal colony-forming cells Hum. Reprod., April 1, 2008; 23(4): 934 - 943. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
H. B. Huttner, P. Janich, M. Kohrmann, J. Jaszai, F. Siebzehnrubl, I. Blumcke, M. Suttorp, M. Gahr, D. Kuhnt, C. Nimsky, et al. The Stem Cell Marker Prominin-1/CD133 on Membrane Particles in Human Cerebrospinal Fluid Offers Novel Approaches for Studying Central Nervous System Disease Stem Cells, March 1, 2008; 26(3): 698 - 705. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 K. Ieta, F. Tanaka, N. Haraguchi, Y. Kita, H. Sakashita, K. Mimori, T. Matsumoto, H. Inoue, H. Kuwano, and M. Mori Biological and Genetic Characteristics of Tumor-Initiating Cells in Colon Cancer Ann. Surg. Oncol., February 1, 2008; 15(2): 638 - 648. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 P. B Dirks Brain tumour stem cells: the undercurrents of human brain cancer and their relationship to neural stem cells Phil Trans R Soc B, January 12, 2008; 363(1489): 139 - 152. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. B. Rountree, L. Barsky, S. Ge, J. Zhu, S. Senadheera, and G. M. Crooks A CD133-Expressing Murine Liver Oval Cell Population with Bilineage Potential Stem Cells, October 1, 2007; 25(10): 2419 - 2429. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Brenner, M. F. Ryser, N. L. Whiting-Theobald, M. Gentsch, G. F. Linton, and H. L. Malech The Late Dividing Population of {gamma}-Retroviral Vector Transduced Human Mobilized Peripheral Blood Progenitor Cells Contributes Most to Gene-Marked Cell Engraftment in Nonobese Diabetic/Severe Combined Immunodeficient Mice Stem Cells, July 1, 2007; 25(7): 1807 - 1813. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. Markovic-Lipkovski, C. A. Muller, G. Klein, T. Flad, T. Klatt, S. Blaschke, J. T. Wessels, and G. A. Muller Neural cell adhesion molecule expression on renal interstitial cells Nephrol. Dial. Transplant., June 1, 2007; 22(6): 1558 - 1566. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
Y. Yamada, S.-i. Yokoyama, X.-D. Wang, N. Fukuda, and N. Takakura Cardiac Stem Cells in Brown Adipose Tissue Express CD133 and Induce Bone Marrow Nonhematopoietic Cells to Differentiate into Cardiomyocytes Stem Cells, May 1, 2007; 25(5): 1326 - 1333. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 G Perrella, P Brusini, R Spelat, P Hossain, A Hopkinson, and H S Dua Expression of haematopoietic stem cell markers, CD133 and CD34 on human corneal keratocytes Br. J. Ophthalmol., January 1, 2007; 91(1): 94 - 99. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Yoshida, S. Shimmura, N. Nagoshi, K. Fukuda, Y. Matsuzaki, H. Okano, and K. Tsubota Isolation of Multipotent Neural Crest-Derived Stem Cells from the Adult Mouse Cornea Stem Cells, December 1, 2006; 24(12): 2714 - 2722. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 D. A. Hess, L. Wirthlin, T. P. Craft, P. E. Herrbrich, S. A. Hohm, R. Lahey, W. C. Eades, M. H. Creer, and J. A. Nolta Selection based on CD133 and high aldehyde dehydrogenase activity isolates long-term reconstituting human hematopoietic stem cells Blood, March 1, 2006; 107(5): 2162 - 2169. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. Gotz and L. Sommer Cortical development: the art of generating cell diversity Development, August 1, 2005; 132(15): 3327 - 3332. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A.-M. Marzesco, P. Janich, M. Wilsch-Brauninger, V. Dubreuil, K. Langenfeld, D. Corbeil, and W. B. Huttner Release of extracellular membrane particles carrying the stem cell marker prominin-1 (CD133) from neural progenitors and other epithelial cells J. Cell Sci., July 1, 2005; 118(13): 2849 - 2858. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
G. Kania, D. Corbeil, J. Fuchs, K. V. Tarasov, P. Blyszczuk, W. B. Huttner, K. R. Boheler, and A. M. Wobus Somatic Stem Cell Marker Prominin-1/CD133 Is Expressed in Embryonic Stem Cell-Derived Progenitors Stem Cells, June 1, 2005; 23(6): 791 - 804. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 B Dome, J Timar, G Ostoros, and S Paku Endothelial progenitor cells in non-small cell lung cancer J. Clin. Pathol., April 1, 2005; 58(4): 447 - 447. [Full Text] [PDF]
|
|
|
|
 |
|
 B. Bussolati, S. Bruno, C. Grange, S. Buttiglieri, M. C. Deregibus, D. Cantino, and G. Camussi Isolation of Renal Progenitor Cells from Adult Human Kidney Am. J. Pathol., February 1, 2005; 166(2): 545 - 555. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
B. Giebel, D. Corbeil, J. Beckmann, J. Hohn, D. Freund, K. Giesen, J. Fischer, G. Kogler, and P. Wernet Segregation of lipid raft markers including CD133 in polarized human hematopoietic stem and progenitor cells Blood, October 15, 2004; 104(8): 2332 - 2338. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. A. Fargeas, A. Joester, E. Missol-Kolka, A. Hellwig, W. B. Huttner, and D. Corbeil Identification of novel Prominin-1/CD133 splice variants with alternative C-termini and their expression in epididymis and testis J. Cell Sci., August 15, 2004; 117(18): 4301 - 4311. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
W. Wagner, A. Ansorge, U. Wirkner, V. Eckstein, C. Schwager, J. Blake, K. Miesala, J. Selig, R. Saffrich, W. Ansorge, et al. Molecular evidence for stem cell function of the slow-dividing fraction among human hematopoietic progenitor cells by genome-wide analysis Blood, August 1, 2004; 104(3): 675 - 686. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
G. D. Richardson, C. N. Robson, S. H. Lang, D. E. Neal, N. J. Maitland, and A. T. Collins CD133, a novel marker for human prostatic epithelial stem cells J. Cell Sci., July 15, 2004; 117(16): 3539 - 3545. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. V. Shmelkov, L. Jun, R. St Clair, D. McGarrigle, C. A. Derderian, J. K. Usenko, C. Costa, F. Zhang, X. Guo, and S. Rafii Alternative promoters regulate transcription of the gene that encodes stem cell surface protein AC133 Blood, March 15, 2004; 103(6): 2055 - 2061. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. A. Fargeas, D. Corbeil, and W. B. Huttner AC133 Antigen, CD133, Prominin-1, Prominin-2, Etc.: Prominin Family Gene Products in Need of a Rational Nomenclature Stem Cells, July 1, 2003; 21(4): 506 - 508. [Full Text] [PDF]
|
|
|
|
 |
|
 C. A. Fargeas, M. Florek, W. B. Huttner, and D. Corbeil Characterization of Prominin-2, a New Member of the Prominin Family of Pentaspan Membrane Glycoproteins J. Biol. Chem., February 28, 2003; 278(10): 8586 - 8596. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 Q. Zhang, R. Haleem, X. Cai, and Z. Wang Identification and Characterization of a Novel Testosterone-Regulated Prominin-Like Gene in the Rat Ventral Prostate Endocrinology, December 1, 2002; 143(12): 4788 - 4796. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. J.G. Potgens, U. Schmitz, P. Kaufmann, and H.-G. Frank Monoclonal Antibody CD133-2 (AC141) Against Hematopoietic Stem Cell Antigen CD133 Shows Crossreactivity with Cytokeratin 18 J. Histochem. Cytochem., August 1, 2002; 50(8): 1131 - 1134. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
Y. Yu, A. Flint, E. L. Dvorin, and J. Bischoff AC133-2, a Novel Isoform of Human AC133 Stem Cell Antigen J. Biol. Chem., May 31, 2002; 277(23): 20711 - 20716. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. J. Shamblott, J. Axelman, J. W. Littlefield, P. D. Blumenthal, G. R. Huggins, Y. Cui, L. Cheng, and J. D. Gearhart Human embryonic germ cell derivatives express a broad range of developmentally distinct markers and proliferate extensively in vitro PNAS, December 22, 2000; (2000) 21537998. [Abstract] [Full Text]
|
|
|
|
|
|
N. Uchida, D. W. Buck, D. He, M. J. Reitsma, M. Masek, T. V. Phan, A. S. Tsukamoto, F. H. Gage, and I. L. Weissman Direct isolation of human central nervous system stem cells PNAS, December 19, 2000; 97(26): 14720 - 14725. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. J. Shamblott, J. Axelman, J. W. Littlefield, P. D. Blumenthal, G. R. Huggins, Y. Cui, L. Cheng, and J. D. Gearhart Human embryonic germ cell derivatives express a broad range of developmentally distinct markers and proliferate extensively in vitro PNAS, January 2, 2001; 98(1): 113 - 118. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| All ASBMB Journals | Molecular and Cellular Proteomics |
| Journal of Lipid Research | ASBMB Today |