From the McGill Cancer Centre and Departments of
§ Biochemistry and ¶ Pharmacology and Therapeutics,
McGill University, Montréal, Québec H3G 1Y6, Canada
Received for publication, December 9, 2002, and in revised form, January 22, 2003
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ABSTRACT |
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The external domains of
Ig superfamily members are involved in multiple binding interactions,
both homophilic and heterophilic, that initiate molecular events
leading to the execution of diverse cell functions. Human
carcinoembryonic antigen (CEA), an Ig superfamily cell surface
glycoprotein used widely as a clinical tumor marker, undergoes
homophilic interactions that mediate intercellular adhesion. Recent
evidence supports the view that deregulated overexpression of CEA has
an instrumental role in tumorigenesis through the inhibition of cell
differentiation and the disruption of tissue architecture. The
CEA-mediated block of the myogenic differentiation of rat L6 myoblasts
depends on homophilic binding of its external domains. We show here
that L6 transfectant cells expressing CEA can "trans-block" the
myogenesis of juxtaposed differentiation-competent L6 transfectant cells expressing a deletion mutant of CEA ( Tissue architecture is established and maintained to a large
extent by specific affinities of cell surface glycoproteins for molecules in the extracellular matrix or on the surface of adjacent cells. The latter are known collectively as cell adhesion molecules (CAMs)1 (1-3). CAMs function
not only to fix cells in specific locations within tissues and regulate
their movement but also to translate biochemical information from the
extracellular environment through the activation of intracellular
signaling pathways leading to specific functional cell responses (4,
5). CAMs are grouped into several different molecular families; the
majority identified to date belong to the Ig superfamily (1, 6-8).
Although the members of this family are functionally diverse, most are
cell surface molecules involved in the recognition of other soluble or
cell-associated molecules. All members of the Ig superfamily share
conserved amino acid residues. These residues are limited to positions
within the core of the Ig fold that are important for its
structure. In contrast, the functional regions of the various
members are often highly diverse.
Ig superfamily members function in many cases as a result of homophilic
binding between their external domains or heterophilic binding
interactions with other molecules (1, 9, 10). Homophilic interactions
can be either antiparallel or parallel. Antiparallel interactions
between molecules on apposed cell surfaces are required for
intercellular binding (9, 11, 12). Parallel interactions between
adjacent molecules on the same cell surface can facilitate this process
by concentrating the binding molecules into synergistic arrays as
described by the "Velcro" (13) or "zipper" models (14), in
which the concerted action of multiple relatively weak interactions
between individual pairs of molecules can lead to a strong overall
bonding. Both types of interactions can also initiate signaling events
(5, 14, 15). The clustering resulting from their combination might be
expected to amplify these signals and lead to the triggering of
threshold-activated signaling pathways (5, 14, 15).
The human carcinoembryonic antigen (CEA) family is of particular
interest in terms of intermolecular binding because of the multiplicity and diversity of interactions between multiple closely related family members (13). We have investigated previously the
structural requirements for CEA-mediated intercellular adhesion (9,
16). Intercellular adhesion seems to depend mainly on antiparallel
CEA-CEA interactions as indicated by studies with hybrid constructs
between CEA and neural cell adhesion molecule (9). CEA consists of a
V-like Ig amino-terminal domain and three pairs of I-like Ig domains
(denoted AXBX) that are terminated by a
hydrophobic domain, which is processed to allow the addition of
a glycophosphatidylinositol membrane anchor (17-19).
Intercellular adhesion was shown to be mediated by double reciprocal
bonds between the N and A3B3 domains of antiparallel CEA molecules on
apposed cell surfaces (9). Subdomains consisting of 5-6 amino acids that are required for intercellular adhesion, presumably as points of
initial binding, were identified by mutational analysis of the
amino-terminal domain (16).
Two cellular functions, intercellular adhesion (for review, see
Stanners and Fuks (13)) and the inhibition of cell differentiation (20)
have been shown to be dependent on homophilic CEA family member
interactions. In particular, CEA was shown to block the myogenic
differentiation of rat L6 myoblasts (20) and the neurogenic differentiation of mouse P19 embryonal carcinoma
cells.2 The effect of CEA on
differentiation would be expected to promote tumorigenic behavior and,
in fact, was found to markedly increase the tumorigenicity of L6
myoblasts (21) and Caco-2 cells (22). Deregulated overexpression of CEA
and the closely related CEA family member, CEACAM6 (formerly known as
NCA), at levels closely approximating those found in many
colorectal carcinomas, has been shown recently to block cellular
polarization, disrupt tissue architecture, and block the
differentiation of human colonocyte cell lines (22). Consistent with
these results, the cell surface level of CEA, which was determined by
FACS analysis of highly purified epithelial colonocytes from colorectal
carcinomas and normal colonic tissue, was found to be elevated in the
tumor cells at levels that were inversely correlated with the degree of
differentiation (23). In this study, we have focused on the effects of
CEA on the differentiation of L6 myoblasts because of the relative ease of experimentation. Although the expression of CEA in this system is
ectopic, our experience to date indicates that the results obtained
with this model system are closely mimicked by more biologically relevant systems such as human colonocytes (22, 24).
Because CEA and/or CEACAM6 are overexpressed in more than 50% of human
cancers (25), it would seem experimentally and medically important to
devise a means of releasing the CEA-mediated differentiation block and
to determine whether interference with this function can be made
selective without affecting the intercellular adhesion function. We
show here that although the structural requirements for the
intercellular adhesion and the differentiation block functions overlap,
they can be separated effectively in the case of rat L6 myogenic
differentiation. Using this information, small cyclized peptides and
monovalent Fab fragments of monoclonal antibodies have been designed
that are capable of releasing the myogenic differentiation block.
Materials--
Cyclized and linearly blocked oligopeptides were
obtained (>95% purity) from Multiple Peptide Systems (San Diego, CA).
Linearly blocked peptides were rendered more stable by acetylation of
the amino terminus and amination of the carboxyl terminus. Cyclic peptides contained two cysteine residues joined by sulfide bonds at
their termini. The peptides used were blocked linear
NAc-LFGYSWYKGE-NH2, NAc-VDGNRQIIGY-NH2,
NAc-RIIQNDTGFY-NH2, and NAc-FNVAEGKEV-NH2; and
cyclized H-CGYSWYKC-OH,
H-CGNRQIIC-OH,
H-CQNDTGC-OH, and H-YCTDEKQCY-OH, representing
subdomains 1, 2, and 3 and control peptides, respectively. Sequences
actually present in the amino-terminal domain of CEA for the cyclized
peptides are underlined.
Construction of CEA cDNA Mutants--
Wild type cDNA
coding for CEA (17) was used as a template for all PCR-generated
constructs. The recombinant PCR technique (26) was used to generate
site-directed mutants as described previously (16).
Cell Culture--
Rat L6 myoblasts were grown as monolayer
cultures at 37 °C in a humidified atmosphere with 5%
CO2 in Dulbecco's modified Eagle's medium containing 10%
fetal bovine serum (Invitrogen), 100 units/ml penicillin and 100 µg/ml streptomycin (growth medium (GM); Invitrogen). Cell cultures
were subcultured while subconfluent to avoid the selection of
non-fusing variants. LR-73 cells (27) derived from the Chinese hamster
ovary line were grown in monolayer culture in Transfection--
L6 myoblast cells were seeded at 2 × 105 cells/l00-mm plastic tissue culture Petri dish and
cotransfected 24 h later by the calcium phosphate-mediated
co-precipitation method as described previously (29), with 5 µg of
p91023B expression vector containing CEA (wild type or mutant)
cDNA, 10 µg of LR-73 carrier genomic DNA, and 0.5 µg pSV2neo
plasmid/dish. Stable pooled transfectant colonies were isolated by
selection with 400 µg/ml Geneticin (G418, Invitrogen).
Immunofluorescent labeling with anti-CEA monoclonal antibody J22 (30)
and FACS sorting were carried out to select for populations of
transfectants stably expressing desired levels of mutant or wild type
CEA on the cell surface. At least two independent pooled populations of
transfectant clones were isolated for each transfected cDNA. All
transfectant populations were maintained in GM containing 400 µg/ml
G418. G418 was removed from the medium 24 h before each functional
assay was performed.
FACS Analysis--
Cells were removed from culture vessels by
light trypsinization (a treatment that does not affect cell surface
levels of CEA) and resuspended in ice-cold phosphate-buffered saline
plus 2% fetal bovine serum (PBSF). 2.5 × 105 cells
were incubated with polyclonal rabbit or monoclonal anti-CEA antibodies
(J22) at a dilution of 1:100 in PBSF for 35 min on ice. Cells were
washed with 2.5 ml of PBSF, centrifuged, and resuspended in 0.5 ml of
PBSF containing fluorescein isothiocyanate-conjugated goat anti-rabbit
or anti-mouse antibody at a dilution of 1:100. After 30 min of
incubation on ice, cells were washed, centrifuged, resuspended in 0.75 ml of PBSF, and analyzed using a BD Biosciences FACScan® instrument.
Adhesion Assays--
LR-73 cells were seeded at 1 × 106/80-cm2 culture flask (Nalge Nunc Inc.,
Naperville, IL) in LR-73 growth medium. After 2 days in monolayer
culture, the cultures were rendered as single cell suspensions
by 3 min of incubation at 37 °C with 0.12% Bacto trypsin in
phosphate-buffered saline lacking Mg2+ and Ca2+
and containing 15 mM sodium citrate. The cells were
incubated at 106 cells/ml in Differentiation Assays--
To initiate fusion and
differentiation, L6 cultures were seeded at 104
cells/cm2 at day 0 in 60- or 35-mm tissue culture Petri
dishes or 7 × 103 cells/cm2 in multiwell
plastic chamber slides (Nalge Nunc Inc.) and grown in GM. The medium
was replaced after 3 days with Dulbecco's modified Eagle's medium plus 2% horse serum (differentiation medium (DM)), and
the cells were cultured for an additional 5-7 days. To co-culture L6
(CEA) and L6 (
As a biochemical measure of myogenic differentiation, cells were fixed
in methanol:acetone (3:7) at
To study the effect of cyclized or linearly blocked peptides on the
CEA-mediated L6 differentiation block, cells were seeded at 7 × 103 cells/cm2 in 8-well chamber slides (Nalge
Nunc Inc.) in GM on day 0. After 3 days of incubation, the medium was
replaced with DM-containing peptide at the indicated concentrations.
Purification of Antibodies--
Rabbit polyclonal anti-CEA
antibody and mouse monoclonal anti-CEA antibodies (A20, B18, and D14)
(30) were purified with the Bio-Rad Affi-Gel protein A MAPS II kit.
Antibodies were added to differentiation medium to a final
concentration of 1 mg/ml. Fab fragments of monoclonal anti-CEA
antibodies were prepared as described previously (30). Fab fragments
were added to differentiation medium to a final concentration of 100 µg/ml. The values for fusion indices shown in Fig. 10 represent the
averages of three independent determinations.
Nature of CEA Homophilic Intermolecular Interactions Required for
Differentiation Block--
To explore the nature of the intermolecular
interactions involved in the CEA-mediated block of the myogenic
differentiation of L6 myoblasts (20), experiments were carried out to
distinguish the requirements for parallel versus
antiparallel intermolecular binding. For antiparallel interactions,
non-differentiating stable L6 (CEA) transfectants were co-cultured with
differentiation competent L6 (
To test for the role of parallel CEA-CEA interactions on the same cell
surface, differentiation-competent L6 (
These experiments support the hypothesis that both antiparallel and
parallel binding between CEA molecules are involved in the CEA-mediated
myogenic differentiation block.
Structural Requirements for Myogenic Differentiation Blocking
Function of CEA--
Deletions and substitutions in three subdomains
of the amino-terminal domain of CEA (Fig.
3) were produced by site-directed mutagenesis as described previously (16). The rationale for choosing
these particular subdomains can be summarized as follows. The
requirement for amino-terminal domain amino acids 32-106, deleted in
mutant
Concerning subdomain 1, G30YSWYK,
substitutions at the carboxyl terminus, Y34A, the more conservative
Y34F, and K35A had a profound effect on the myogenic differentiation
blocking activity of CEA, whereas mutation Y31A at the amino terminus
had no effect (Fig. 5). As expected,
deletion of the entire subdomain 1 (
Similarly, the deletion of subdomain 2, N42RQII, had less
effect on CEA function than some of the substitutions within this domain, notably the double mutation Q44R,I46V, for which the degree of
differentiation of 100%, a reproducible effect, actually exceeded that
of parental L6 cells (Fig. 6). A single
mutation at the amino terminus of the subdomain, N42D, partially
removed the differentiation blocking activity of CEA.
The mouse analogs of subdomains 1 and 2, in a crystallized soluble
mouse construct of CEACAM1 consisting of the amino-terminal domain
linked to one internal domain, have been shown by Tan et al.
(32) to contribute to an unusual structure involving residues 35-44
that projects from the surface of the molecule, with
Ile41 (Gly41 in human CEA) at its tip.
This protrusion represents the binding site of murine hepatitis virus.
Tyr34 in murine CEACAM1 is postulated to contribute
critically to this structure. It is of interest that the above results
show critical effects of Tyr34 on the differentiation
blocking function of CEA, although the adjacent Lys35
residue seems to have even greater effects. It remains to be seen, of
course, whether CEA has a similar structure at this site.
The third subdomain, Q80NDTG, was found to play a critical
role in the CEA-mediated differentiation block, because mutation Q80A
resulted in a complete loss of this function and mutation D82N, like
Q44R,I46V in subdomain 2, gave 100% differentiation, thus exceeding
that of the parental cells (Fig. 7).
Mutations Q80R, giving 81% differentiation, and D82N, giving 100%
differentiation, are of particular interest, because Q80R and D82N
actually enhanced the intercellular adhesion function of CEA expressed
in LR-73 cells (Fig. 8). These mutations
therefore separate the intercellular adhesion and differentiation
blocking functions of CEA.
Two other mutations at residue Arg64, a site shown by
Sippel et al. (33) to have a marked effect on the
intercellular adhesion function of CEA expressed in insect cells, had a
relatively small effect on the differentiation blocking function of CEA
in rat L6 myoblasts (Fig. 7).
Effects of Monovalent Monoclonal Anti-CEA Antibody Fragments on
CEA-mediated Differentiation Block--
The epitope of the
adhesion-inhibitory anti-CEA mAb A20 (30) was shown previously to
bridge the carboxyl-terminal Lys35 residue of the first
subdomain and the amino-terminal Asn42 residue of the
second subdomain (16). The binding epitope of anti-CEA mAb B18, which
also inhibits intercellular adhesion (although less effectively than
A20; data not shown), was found to be shifted slightly upstream from
that of A20 in that the K35A mutation completely abrogated B18 binding,
but unlike A20 (16) the N42D mutation was without effect (Fig.
9). Binding to CEACAM8, which differs from all other CEA family members by the presence of an Ala
versus a Gly residue at position 41, was completely absent
in both B18 and A20 (data not shown) and binding to Y34F was reduced
somewhat for both (Fig. 9 and Ref. 16). All other point mutations in the first two subdomains (Fig. 9 and Ref. 16) and the third subdomain
(data not shown) were without effect.
The effects of monovalent Fab fragments of A20 and B18 on
the CEA-mediated myogenic differentiation block are shown in Fig. 10. Both partially released the block
and, as in their effects on intercellular adhesion, A20 was more
effective than B18, giving an average value of 41% fusion at a
concentration of 100 µg/ml in three independent experiments. Fab
fragments of mAb J22, which binds to the A1B1 and A2B2 domains of CEA
(30), had no significant effect (Fig 10). Whole divalent mAbs were
without effect on the differentiation block (data not shown).
Effects of Peptides on CEA-mediated Differentiation
Block--
To determine whether peptides representing the three
subdomains of CEA could release the CEA-imposed myogenic
differentiation block, peptides both terminally blocked (for improved
stability) and cyclized (for both improved stability and conformation)
were tested by adding them to L6 (CEA) cells cultured in the
presence of DM. All linear peptides were virtually ineffective (data
not shown) but were effective when cyclized (Fig. 10), a conformation that is expected to mimic a Using the myogenic differentiation of rat L6 myoblasts as a model
system, we investigated the structural requirements for the
differentiation blocking activity of CEA with the goal of designing
agents that were capable of releasing this activity. The basic approach
was to identify small domains in which integrity was essential for
function and then to test the effect of peptides representing
these domains and of mAb monovalent Fab fragments that bind to them.
Three such subdomains were identified, and both cyclized peptides and
mAb Fab fragments were found to be effective in releasing the CEA
differentiation block in L6 myoblasts.
The rationale used to focus this structure-function study was
based on previous results showing that the amino-terminal and A3B3
domains of CEA were required for the differentiation blocking effect
(20), thus implicating the involvement of double reciprocal bonds
between antiparallel molecules on apposite cell surfaces, as observed
for the intercellular adhesion function of CEA (9). A direct experiment
showing that L6 (CEA) transfectants could "trans-block" the
differentiation of differentiation-competent L6 ( CEA expression inhibits molecular events occurring very early in the
myogenic differentiation process, notably the up-regulation of the
myogenic transcriptional regulator, myogenin (20). Recent evidence
indicates that the molecular basis for the pan-inhibition of cellular
differentiation mediated by CEA involves perturbation of the function
of certain integrins ( With the knowledge that subdomains exist in the CEA molecule that can
be differentially antagonized to affect the differentiation blocking
function while leaving the intercellular adhesive function intact, the
possibility of designing agents with functionally selective blocking
activity can be entertained. Subdomain 3 was identified as such a
region, but although its corresponding peptide was effective in
releasing the CEA-imposed differentiation block, it was also effective
in inhibiting CEA-mediated intercellular adhesion (16). Thus, although
QNDTG appears to represent an experimentally useful agent with
potential for medical application, further application of the methods
outlined here might be expected to yield even more potent and selective agents.
Finally, even though our results to date have shown that the L6
myogenic differentiation model system used here is accurately predictive of the results obtained with more medically relevant systems, the findings bear repeating in other less experimentally convenient systems such as human Caco-2, LS-180, and SW-1222
colonocytes, in which several CEA family members are normally expressed
in a regulated fashion (40). In the latter cells, deregulated
overexpression of both CEA and CEACAM6 at levels observed in freshly
excised human tumor colonocytes has been shown to block cell
polarization and disrupt tissue architecture (22). Present findings
indicate that Fab fragments of mAb A20 are capable of reversing these
tumorigenic effects,5 thus
providing an appealing strategy for the reversal of the malignant
phenotype in medically relevant situations.
NCEA). This result implies the efficacy of antiparallel CEA-CEA interactions between cells
in the differentiation block. In addition,
NCEA can acquire differentiation blocking activity by cross-linking with specific anti-CEA antibodies, thus implying the efficacy of parallel CEA-CEA interactions on the same cell surface. The myogenic differentiation blocking activity of CEA was demonstrated by site-directed mutations to
involve three subdomains of the amino-terminal domain, shown previously
to be critical for its intercellular adhesion function. Monovalent Fab
fragments of monoclonal antibodies binding to the region bridging
subdomains 1 and 2 could both inhibit intercellular adhesion and
release the myogenic differentiation block. Amino acid substitutions
Q80A, Q80R, and D82N in subdomain 3, QNDTG, however, were found to
completely ablate the differentiation blocking activity of CEA but had
no effect on intercellular adhesion activity. A cyclized peptide
representing this subdomain was the most effective at releasing the
differentiation block.
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
minimum essential
medium (28) containing 10% fetal bovine serum, 100 units/ml
penicillin, and 100 µg/ml streptomycin at 37 °C in a humidified
atmosphere with 5% CO2.
minimum essential medium
containing 0.8% fetal bovine serum and 10 µg/ml DNase I at 37 °C
with stirring at 100 rpm (29). The percentage of cells remaining as
single cells, which declines over time because of formation of
aggregates, was determined as a function of time by visual counting
using a hemocytometer.
NCEA) cell transfectants, cells were seeded at 3 × 105 cells of each type per 35-mm plastic tissue culture
Petri dish in GM. 24 h later the medium was replaced with DM. For
fusion index determinations, cells were fixed with 2.5% glutaraldehyde and stained with hematoxylin. The fusion index was calculated as the
percentage of total nuclei contained in fused myotubes having more than
three nuclei/myotube, as described previously (20). Fusion
determinations were repeated three times (independent experiments) for
each of two independently obtained transfectant populations for
each mutant. The values reported in Figs. 5-7 represent the
averages of these determinations.
20 °C and processed for immunofluorescent staining with anti-myosin mAb (31).
RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
NCEA) transfectants.
NCEA is a CEA
cDNA deletion mutant lacking two-thirds of the amino-terminal
domain (from amino acid 32 to 106) and is completely defective in
mediating both intercellular adhesion (9) and the differentiation block
of L6 myoblasts (20). CEA-expressing cells can bind to
NCEA-expressing cells, consistent with the model that the
amino-terminal domain (intact in CEA) on a CEA-expressing cell can bind
to the A3B3 domain (intact in
NCEA) on a juxtaposed
NCEA-expressing cell by an antiparallel mechanism (9). If such
antiparallel interactions are sufficient for the differentiation
blocking function of CEA, the CEA-expressing myoblasts should be able
to "trans-inhibit" the differentiation of the
NCEA-expressing
myoblasts. Co-culturing CEA-expressing L6 cells with an equal number of
NCEA-expressing L6 cells inhibited the overall differentiation
(assessed by fusion into multinucleated myotubes) to a significantly
lower level than co-culturing the same CEA-expressing cells with an
equal number of non-CEA-expressing parental L6 cells for which
trans-binding mediated by CEA would be impossible (Fig.
1). The latter co-culture controls for
the dilution of differentiating cells with non-differentiating L6 (CEA)
cells. This experiment therefore supports the contention that
antiparallel CEA-CEA interactions between cells are sufficient for the
CEA-imposed myogenic differentiation block.
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Fig. 1.
Trans-cellular blocking of the myogenesis of
differentiation-competent L6 transfectants expressing a deletion mutant
of CEA, NCEA, by L6 transfectants expressing
CEA. L6 (CEA) cells were co-cultured with an equal number of L6
(
NCEA) cells in GM, and 24 h later the medium was replaced with
DM. After 5-7 days of incubation, fusion indices were measured as
described under "Experimental Procedures." Values shown represent
the mean and standard error of measurements from three independent
experiments.
NCEA) transfectants were
treated with cross-linking polyclonal and monoclonal anti-CEA antibodies. Antibodies for which the binding epitopes are still intact
in the
NCEA molecule, rabbit polyclonal and D14 (binding epitope at
the B2-A3 junction (30)), converted
NCEA to a differentiation blocking molecule, whereas control antibodies directed to binding epitopes that are missing in
NCEA, A20 and B18, two N
domain-specific mAbs (binding epitopes at residues 35-42 in the
amino-terminal domain (16)) were without effect (Fig.
2). To further control for nonspecific
effects, one of the effective antibodies, D14, was shown to have no
effect on the differentiation of the parental L6 cells (Fig.
2).
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Fig. 2.
Cross-linking of
NCEA molecules in differentiation-competent L6
(
NCEA) transfectants with polyclonal and
monoclonal anti-CEA antibodies. Purified rabbit polyclonal and mAb
D14 with intact epitopes in
NCEA converted
NCEA to a
differentiation blocking molecule, whereas control antibodies A20 and
B18 with missing epitopes in
NCEA had no effect. Values shown
represent the mean and standard error of measurements from two
independent experiments.
NCEA, for the myogenic differentiation block was demonstrated
previously (20). Within this deletion, subdomains 1 and 2 were
implicated by the fact that mAb A20 can release the CEA-imposed
myogenic differentiation block (see "Effects of Monovalent Monoclonal
Anti-CEA Antibody Fragments on CEA-mediated Differentiation Block") and has a binding epitope that bridges them. This
epitope includes the carboxyl-terminal amino acid of subdomain 1 and
the amino-terminal amino acid of subdomain 2 (16). Also, these
subdomains (1 and 2) and subdomain 3 were all shown to be important in
CEA-mediated intercellular adhesion in LR-73 cells; all were
demonstrated to be adjacent and exposed in a three-dimensional
structural model based on the known structures of CD2 (16) and
CD4.3 Pooled stable
transfectant clones of L6 cells expressing comparable cell surface
levels, as assessed by FACS analysis (Fig.
4), were isolated for each of the
deletion and substitution mutants.
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Fig. 3.
Schematic diagram of the structure of CEA
showing the major domains and subdomains in the amino-terminal domain
selected for intensive study.
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Fig. 4.
FACS distributions relating the number of
cells with the level of cell surface expression of wild type and mutant
CEA in L6 myoblast transfectant and control (Neo)
populations. All cells were labeled with the monoclonal anti-CEA
antibody, J22. Immunoreactivity was detected with goat anti-mouse
fluorescein isothiocyanate-conjugated antibody.
GK) also affected this function
of CEA but, curiously, was not as effective as substitutions Y34A and
K35A.
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Fig. 5.
Photomicrographs and fusion indices of L6
(Neo), L6 (CEA), and L6 subdomain 1 mutant CEA transfectants subjected
to the myogenic differentiation assay.
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Fig. 6.
Photomicrographs and fusion indices of L6
subdomain 2 mutant CEA transfectants subjected to myogenic
differentiation assay.
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Fig. 7.
Photomicrographs and fusion indices of L6
subdomain 3 mutant CEA transfectants subjected to myogenic
differentiation assay.
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Fig. 8.
Effect of amino acid substitutions at
positions Gln80 and Asp82
on the kinetics of CEA-mediated aggregation of corresponding
LR-73 cell transfectants. LR Neo, negative control;
LR CEA, positive control. FACS profiles indicating
expression levels of CEA and mutant CEA in these transfectants using
mAb J22 are given in Ref. 16. Mean levels of expression in arbitrary
units were: LR CEA, 420; LR Q80R, 388; and
LR D82N, 249.
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Fig. 9.
Immunoblot results for SDS-PAGE of extracts
of LR-73 cells stably transfected with the indicated CEA mutants in
subdomains 1 and 2. CEA mutant protein was detected with mAbs A20,
B18, and J22 with qualitative results indicated in A and gel
patterns for B18 shown in B. Primary gel data for A20 are
given in Taheri et al. (16).
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Fig. 10.
Photomicrographs of L6 (Neo), L6 (CEA), and
L6 (CEA) cultures treated with cyclized peptides at a concentration of
100 µM representing subdomains 1, 2, and 3 and with Fab fragments of mAbs A20 and B18 and control J22 at
a concentration of 100 µg/ml after growth in
DM. Differentiation is indicated by immunofluorescence with
anti-myosin antibody. The background fluorescence seen in the L6
(CEA) culture in the mAb series was caused by the loss of
CEA expression by occasional cells in the cultures used for this series
of experiments. Values for the fusion index represent averages of three
independent experiments.
-turn configuration in the native molecule, the configuration predicted for the three subdomains. Maximum
activity in releasing the differentiation block was found at a
concentration of approximately 100 µM; higher
concentrations of the peptides were less effective because of
nonspecific toxicity. At 100 µM, cyclized peptide QNDTG
released the myogenic differentiation block to the greatest extent,
with an average of 36% fusion in three experiments versus
28 and 18% for cyclized peptides NRQII and GYSWYK, respectively,
relative to 94% for control L6 (Neo) myoblasts. Experiments done with
combinations of the three different peptides showed that there was
synergy between NRQII and QNDTG; lower concentrations of these peptides
(40 µM each) had the same effect as 100 µM
QNDTG (data not shown).
DISCUSSION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
NCEA) transfectants
supports the notion that antiparallel CEA-CEA interactions between
cells can contribute to the myogenic differentiation block. Parallel
CEA-CEA interactions, probably on the same cell surface, were also
implicated by the demonstration that the deletion mutant
NCEA,
normally incapable of mediating the differentiation block, could be
rendered capable of blocking L6 differentiation by cross-linking with
specific antibodies. Although parallel interactions can be envisioned
to improve intercellular adhesive forces by clustering
antiparallel-interacting intercellular molecular pairs (the Velcro
effect) (13), their relative contribution could be greater for the
differentiation blocking function (34). These considerations may
underlie the observation that the intercellular adhesion and
differentiation blocking functions of CEA could be separated by certain
amino acid substitutions in subdomain 3 that removed the
differentiation blocking activity while leaving the intercellular
adhesive activity intact. This result could therefore be interpreted by
the suggestion that subdomain 3 is primarily necessary for parallel
binding between CEA molecules. Consistent with this, peptide QNDTG,
representing the third subdomain, was the most effective in releasing
the CEA-imposed differentiation block.
5
1 in L6 myoblasts
and human colonocytes)4 known
to affect the earliest steps in differentiation (35-37). The results
reported here showing the involvement of subdomains of the CEA
molecule required for CEA-CEA binding favor a clustering model in which CEA and specific integrins inhabit the same membrane rafts defined by the glycophosphatidylinositol anchor of CEA (38). Clustering of CEA thereby causes clustering of integrin molecules, which in turn results in a change in integrin function (39). Clustering
of CEA would be expected to be effected mainly by parallel interactions
on the same cell surface, but antiparallel interaction may be required
to initiate the process of clustering. We favor a clustering rather
than dimerization model, because the effects of divalent monoclonal
antibodies on integrin perturbation can be further enhanced by the
addition of secondary anti-mouse antibodies. In addition, co-clustering
of CEA and
5
1 has been observed directly by confocal microscopy.4 This model explains why monovalent
Fab fragments of specific mAbs were found to be required to release the
CEA-mediated differentiation block. Whole divalent mAbs have the effect
of enhancing clustering and would thus be expected to increase the
tumorigenic effects of CEA.
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ACKNOWLEDGEMENTS |
---|
We thank Dr. Lynne LeSauteur and Dr. Barbara Gour for critical advice in the design of the peptides and Dr. A. Fuks for antibodies. We thank Luisa De Marte for help in the preparation of the manuscript.
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FOOTNOTES |
---|
* This work was supported by in part by grants from the Canadian Institutes of Health Research and the National Cancer Institute of Canada with funds from the Canadian Cancer Society.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.
Laboratory was supported in part by the Jeanne and J. Louis
Lévesque Chair for Cancer Research at McGill University. To whom correspondence should be addressed: Dept. of Biochemistry, McGill University, 3655 Promenade Sir William Osler, Montréal,
Québec H3G 1Y6, Canada. Tel.: 514-398-7279; Fax: 514-398-6769;
E-mail: cliff.stanners@mcgill.ca.
Published, JBC Papers in Press, February 5, 2003, DOI 10.1074/jbc.M212500200
2 B. Malette and C. P. Stanners, submitted for publication.
3 H. U. Saragovi, unpublished data.
4 C. Ordoñez, A. B. Zhai, M. Fan, R. A. Screaton, C. Ilantzis, and C. P. Stanners, submitted.
5 C. Ilantzis and C. P. Stanners, unpublished data.
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ABBREVIATIONS |
---|
The abbreviations used are: CAM, cell adhesion molecule; CEA, carcinoembryonic antigen; DM, differentiation medium; GM, growth medium; mAb, monoclonal antibody; FACS, fluorescence-activated cell sorter; PBSF, phosphate-buffered saline plus 2% fetal bovine serum.
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