* McGill Cancer Centre and Department of Biochemistry, McGill University, Montreal, Quebec, Canada H3G 1Y6; and Department of Medical Biophysics, University of Toronto, Ontario Cancer Institute, Toronto, Ontario, Canada M5G 2M9
Carcinoembryonic antigen (CEA) is a tumor marker that is overexpressed in many human cancers and functions in vitro as a homotypic intercellular adhesion molecule. We have investigated the possibility of synergy between CEA, v-Myc, and Bcl-2 in the transformation of cells with differentiation capacity. We find that v-Myc increases the cell division rate and maximum density of rat L6 myoblasts but also markedly stimulates both apoptosis and surprisingly, differentiation, thus preventing transformation. The superposition of Bcl-2 blocks the apoptotic stimulation of v-Myc and independently promotes further cell division at confluence, but still allows differentiation. The further expression of CEA has a dominant effect in blocking differentiation, regardless of the presence of the other activated oncogenes, generating cells that enter a reversible quiescent G0-like state in medium promoting differentiation. Transfectants expressing CEA with or without v-myc and bcl-2 allow the emergence of cells with the property of heritable, efficient, anchorageindependent growth in soft agar and the ability to markedly reduce the latency for tumor formation in nude mice. We propose that by prolonging cell survival in the presence of differentiation signals, CEA represents a novel class of dominant differentiation-blocking oncogene.
Malignant transformation is believed to require
four critical events: acquisition of increased proliferative activity, abrogation of apoptosis, immortalization, and deletion of access to differentiation programs. Differentiation has generally been assumed to
be attenuated by an increase in proliferative behavior, if
only because these cellular programs are considered mutually inhibiting. A major focus of oncobiology, therefore,
has been to identify and characterize genes that influence
cellular proliferation and recently, genes that govern cell
death. Prototypic members of these two classes of oncogenes are, respectively, c-myc (for reviews see Evan and Littlewood, 1993 c-myc is an early response gene implicated in emergence
from quiescence and normal progression through the cell
cycle, in the establishment and propagation of neoplasia
and, paradoxically, in initiating apoptosis (for review see
Evan and Littlewood, 1993 Our laboratory has used a well characterized in vitro differentiation system, the immortal rat L6 skeletal myoblast
cell line (Yaffe, 1968 We have proposed a model in which overproduction of
CEA plays an active role in carcinogenesis by disrupting
the normal tissue architecture of colonic epithelium, leading to inhibition of differentiation and tissue dysplasia
(Benchimol et al., 1989 To test the proposed differentiation-inhibiting effect of
CEA directly in a well defined differentiation system, rat
L6 myoblasts were used. In low serum, L6 cells withdraw
irreversibly from the cell cycle, align, and form multinucleated myotubes while concomitantly upregulating musclespecific gene products (Nadal-Ginard, 1978 Extensive evidence has led to the prevailing view that
activation of the cellular proliferative machinery results in
the obligatory disruption of the myogenic differentiation
program, which is normally accessed after arrest in the G1
phase of the cell cycle. Specifically, myogenesis can be prevented by maintaining cells in high concentrations of soluble growth factors such as PDGF and IGF-I (Screaton,
R.A., and C.P. Stanners, unpublished) and by the forced
expression of cellular or viral oncogenes (for review see
Alema and Tato, 1994 Cell Culture
Rat L6 (Yaffe, 1968 Infections
L6 cells were incubated with replication-defective recombinant retrovirus
containing either pBabe(avian v-myc)hygro (Facchini et al., 1994 Transfections
L6 or C2 myoblasts were seeded at 2 × 105 cells/100-mm dish and cotransfected 24 h later by calcium phosphate coprecipitation with 5 µg of
p91023B expression vector (courtesy of Kaufman, R., Genetics Institute,
Boston, MA) or PKJ expression vector containing full-length CEA cDNA
(clone 17; Beauchemin et al., 1987 Differentiation Assays
To induce differentiation, L6 and C2 cells were seeded on day 0 at 7 × 103
and 3.5 × 103 cells/cm2, respectively, in 60-mm plastic tissue culture petri
dishes in 4 ml DME plus 10% FBS. On day 3, when cells were nearly confluent, the medium was changed to DME containing 2% horse serum (differentiation medium [DM]). Cells were assessed for differentiation/fusion
on day 7 unless otherwise noted. Fusion index was determined as described previously (Eidelman et al., 1993 Cell Lysis and Immunoblotting
Cells at a concentration of 107/ml were boiled in SDS lysis buffer (100 mM
Tris, pH 8.0, 10% glycerol, 2% SDS) for 5 min and clarified by centrifugation. Protein determinations were made using BCA reagent (Pierce,
Rockford, IL). 50-100 µg total protein was resolved by SDS-PAGE and
transferred electrophoretically to a 0.45 µm PVDF membrane (Millipore
Corp., Bedford, MA). Membranes were incubated with polyclonal panMyc antibody (generously provided by Evan, G.I., Imperial Cancer Research Fund, London, UK) at a dilution of 1:1,000 or polyclonal anti-Bcl-2
antibody (kind gift of Andrews, D., McMaster University, ON, Canada) at
a dilution of 1:5,000 for 1 h at 25°C in TBST/M (25 mM Tris, pH 8.0, 62.5 mM NaCl, 0.4% Tween, 10% milk). After extensive washing, membranes were developed with HRP-conjugated anti-rabbit antibody and visualized by ECL reagent (Amersham Life Science, Pittsburgh, PA), which was
used according to manufacturer's instructions.
Growth Curves
2 × 104 L6 cells were seeded in 60-mm dishes in GM. Triplicate cultures
were rendered single cell suspensions by trypsinization every 24 h and
their cell numbers individually determined using a particle counter
(Coulter Electronics Inc., Hialeah, FL). Plates were not refed fresh medium during the course of the experiment. Cell size distributions were obtained using a channelyzer (Coulter Electronics Inc.), and their mean values were determined. All distributions were essentially symmetrical such
that mean and mode values were equivalent.
Cell Viability Assay
200 cells (or nuclei equivalent, where cell fusion was appreciable) were
seeded in GM in replicate 60-mm dishes and incubated at 37°C for 9-10 d.
The resultant colonies were rinsed with PBS, fixed with 3.7% formaldehyde in PBS, and stained with 0.1% methylene blue. The number of colonies of >25 cells was determined by eye and expressed as a percentage of
the number of cells seeded to give the plating efficiency.
Colony Formation in Soft Agar
103 cells were seeded in 60-mm dishes in 0.3% agar in 5 ml GM on a bottom layer of 0.5% agar in 3 ml GM and incubated at 37°C. Plates were not
refed during the course of the experiment. Uniform, dense colonies of
>100 or >500 cells were counted by microscopic observation 3-4 wk after
seeding, as indicated.
Chromatin Staining and Apoptotic
Frequency Determination
Cells were seeded for differentiation in 60-mm dishes in GM. The culture
medium was changed to DM on day 2 and incubated for an additional 24 h, at
which time the cultures were trypsinized and the resultant cell suspension
pooled with nonadherent cells in the medium and centrifuged. Cell pellets
were resuspended in 1 ml DME, and an equal volume of ice-cold fixative
(3:1 ratio of 95% EtOH to glacial acetic acid) was added dropwise with intermittent vortexing. Cells were collected by centrifugation, resuspended in 1 ml of fixative, and incubated on ice for 10 min. Fixed cells were centrifuged, resuspended in 200 µl fixative, mounted on cold slides, and quick
dried over a boiling water bath. Slides were immersed in a saturated solution of orcein in 50% glacial acetic acid for 30-60 min at 25°C. Damaged
cells with condensed chromatin, confirmed by electron microscopy to be
apoptotic, were easily distinguished; their numbers relative to undamaged
cells were determined in six separate fields. A minimum of 300 cells was counted per field.
Transmission Electron Microscopy
Cells were incubated in DM for 24 h, as described above for chromatin
staining, before fixation in 2.5% glutaraldehyde and processed for electron microscopy using standard procedures. Cells were viewed under an
electron microscope (Philips Electronic Instruments, Inc., Mahwah, NJ).
FACS Analysis
Cultures were trypsinized and resuspended in ice cold PBS plus 2% FBS
(PBSF). 2.5 × 105 cells were incubated with polyclonal rabbit or goat antiCEA antibodies diluted 1:100 (rabbit) or 1:600 (goat) in a total volume of
0.5 ml PBSF for 30 min on ice. Cells were centrifuged, rinsed with 2 ml
PBSF, and resuspended in 0.5 ml PBSF containing FITC-conjugated rabbit anti-goat or goat anti-rabbit antibody diluted 1:100. After 30 min incubation on ice, cells were rinsed and resuspended in 0.75 ml PBSF for cytofluorometric analysis (FACScan®; Becton Dickinson, Bedford, MA).
Tumorigenicity Assay
To assay for tumorigenicity, 8-9 wk old nude mice (Swiss nu/nu; Taconic
Farms Inc., Germantown, NY) were injected intramuscularly in both hind
legs with cells resuspended in 100 µl serum-free PBS. The average latent
periods for tumor formation by 105 and 3 × 105 injected cells (or 5 × 105
cells in the case of M alone) were determined as the time of appearance of
a visible mass (>0.5 cm in diameter) at the site of injection or, for cases in
which tumors developed inside the leg, the time of visible swelling of the
leg and/or an alteration in gait. Differences in tumor latency were evaluated using the Student's t test.
Tumor Resection and Dissociation
Freshly excised tumor tissue was rinsed well with cold PBS, finely minced,
and incubated in a sterile-filtered solution containing 50 U/ml purified collagenase (CLSPA) and 0.13% CLS-2 collagenase (Worthington Biochem.
Corp., Freehold, NJ) in PBS supplemented with 10 µg/ml DNAseI for 30-
45 min at 37°C. After washing with GM to stop the dissociation, cells were
filtered through a 70-µm filter and collected by centrifugation. Cells were
seeded in GM containing 50 µg/ml gentamycin. 24-36 h later, cultures
were divided by trypsinization and introduced into selective media containing either G418 (CEA+ lines) or puromycin (Bcl-2+ lines). After 3 d
growth under selection, these tumor lines (denoted t) were expanded and
then reintroduced into soft agar and nude mice.
The block in myogenic differentiation of L6 cells stably expressing CEA in DM (Eidelman et al., 1993
Ectopic Myc Expression Accelerates
Proliferation and Induces Both Apoptosis and
Differentiation in Myoblasts
To investigate the effects of v-Myc alone on the growth
behavior of L6 myoblasts, M and P cells were seeded in
replicate culture dishes in GM and their cell numbers determined daily without change of medium; the results for a
single experiment typical of three separate experiments,
which included transfectants expressing Bcl-2 and CEA
(see below), are shown in Fig. 2. The population doubling
times are revealed by the slopes of the curves and are reported along with initial (day 5) and final (day 15) saturation densities in the inset of Fig. 2. v-Myc expression increased both the proliferation rate and the maximum
culture density (Fig. 2, inset, compare P and M). These relatively small differences were highly significant and reproducible in repeated experiments. Cells expressing v-Myc
were reduced in size during the exponential growth phase
(Fig. 3), which is consistent with the observed increase in
proliferative rate (Murray and Hunt, 1993
On day 5, cultures expressing ectopic Myc alone began
to acidify the medium and display cell death. Coincident
with this, marked cell elongation and alignment and the
formation of multinucleated myotubes were also apparent.
These changes are reflected in the complete loss of viability as measured by colony-forming ability after 15 d in culture (Fig. 2, inset) and in the increase in average size of M
cells nearing saturation (Fig. 3). To investigate the effect
of v-Myc on differentiation, P and M cells were tested in
two different assays in which serum growth factors become limiting. First, we quantified the effect of growth factor depletion in the nonproliferating centers of cell colonies (Fig. 4). 75% of M colonies, obtained by plating viable
M cells from the exponential growth phase, showed myotubes in their centers after 10 d of incubation in GM,
whereas P colonies showed no myotubes under these conditions (Fig. 4), thus indicating a dramatic increase in the
propensity for differentiation. Second, cells were seeded
directly into DM and stained 4 d later. In spite of significant cell loss that reduced the cell density of M cultures, M
cells formed myotubes; in this case P cells, as expected,
readily differentiated (Fig. 5 A, compare M and P).
The increased cell mortality in response to depletion of
growth factors was suggestive of an apoptotic response, as
seen with Rat-1 fibroblasts overexpressing c-myc (Evan et
al., 1992
The transformation potential of M cells was determined
in vitro using a soft agar assay to assess the loss of anchorage dependence of growth. M cells formed colonies in soft
agar with an efficiency of 1%, whereas P cells were incapable of anchorage-independent growth (Table I). No macrocolonies of >500 cells were formed by either P or M
cells. Thus, v-Myc alone does not induce a highly transformed phenotype, consistent with previous results of Falcone et al. (1985) Table I.
Anchorage Independent Growth of L6 Transfectants
and Derived Macrocolony (m) and Tumor (t) Lines
Bcl-2 Expression Blocks Myc-induced Apoptosis and
Promotes Cell Overgrowth
In fibroblast cultures overexpressing c-myc, bcl-2 expression can delay cell death for several days (Bissonette et al.,
1992; Fanidi et al., 1992 Table II.
Medium pH of L6 Transfectants
; Evan et al., 1994
) and bcl-2 (for reviews
see Korsmeyer, 1992
; 1995).
; Packham and Cleveland,
1995
). Cellular transformation induced by c-myc deregulation is, however, often incomplete and depends on the activation of another oncogene, such as ras (Land et al., 1983
; Adams et al., 1985
). In contrast, forced bcl-2 expression promotes cell survival both in vitro (Bissonnette et al.,
1992
; Fanidi et al., 1992
) and in vivo (McDonnell et al.,
1989
; McDonnell and Korsmeyer, 1991
) by preventing apoptosis. Cultured fibroblasts coexpressing myc and bcl-2 continue to proliferate at low mitogen concentrations and
experience reduced cell death but are not morphologically transformed (Fanidi et al., 1992
). Mice expressing both
Eµ-myc and Eµ-bcl-2 transgenes (Strasser et al., 1990
) accumulate cycling pre-B and differentiated B cells, display
an increased frequency of monoclonal tumors, and die earlier than Eµ-myc mice (Adams et al., 1985
), observations
that are attributed to a tumorigenic cooperativity between
Myc and Bcl-2. The latent period and monoclonal nature
of the tumors suggest, however, that additional mutations are required. Thus, Myc and Bcl-2 together result in the
expansion of a population of benign cells that retain, at
least in part, the capacity for differentiation.
), to investigate oncogene action in
the context of aberrant expression of human carcinoembryonic antigen (CEA).1 First discovered to be overexpressed in human colon tumors (Gold and Freedman,
1965
), CEA has since been widely used as a marker in the
clinical management of many cancers, including carcinomas of the colon, breast, and lung (Averbach and Sugarbaker, 1995
). CEA is a glycophosphatidylinositol-linked
(Hefta et al., 1988
; Takami et al., 1988
) cell surface glycoprotein of the immunoglobulin gene superfamily that has
been shown to mediate homotypic intercellular adhesion
(Benchimol et al., 1989
; Oikawa et al., 1989
).
). We contend that overproduction
and redistribution of CEA from apical to the lateral and
basal surfaces of colonic crypt epithelial cells perturbs normal intercellular interactions leading to the adoption of a multilayered ("stratified") epithelial configuration, perhaps mimicking the multilayered configuration seen in
embryonic colonic epithelium. The predicted effect of this
distortion in tissue architecture is the inhibition of normal
colonocyte terminal differentiation, thus potentiating increased cell division. In support of this model we have recently shown that the cell surface levels of CEA determined by cytofluorometric analysis of highly purified
epithelial colonocytes from freshly excised colon carcinomas and adjacent normal tissue are elevated in the tumor
cells and at levels that are inversely correlated with the degree of differentiation of the tumors (Ilantzis et al., 1997
).
). Myogenesis
of L6 cells requires a complex series of cell-cell recognition and membrane fusion events that are mediated by a number of adhesion systems (Knudsen, 1990
) and the upregulation of functional Myf5 and myogenin, members of
the MyoD family of skeletal muscle transcriptional regulators (for review see Olson and Klein, 1994
). Ectopic expression of cell surface CEA blocks biochemical and morphological myogenic differentiation in L6 myoblasts (Eidelman
et al., 1993
); since the CEA-imposed differentiation block
can be released by peptides representing the two adhesion domains of CEA, the effect depends on CEA-CEA interactions (Eidelman et al., 1993
; Zhou et al., 1993
). We hypothesized that L6 cells stably expressing CEA, which retain their viability and proliferative potential while remaining
in a quiescent nondifferentiated state for weeks in differentiation medium (Eidelman et al., 1993
; Screaton, R.A.,
and C.P. Stanners, unpublished observations), should
show increased susceptibilty to further malignant progression induced by oncogenic activation. To directly test this
idea we have examined cellular phenotypes in single and
multiple transfectants of the rat L6 and mouse C2 (Yaffe
and Saxel, 1977
) myoblast cell lines to examine possible
synergism between Myc, Bcl-2, and CEA in transformation.
), including c-myc (Denis et al.,
1987
; La Rocca et al., 1994) and v-myc (La Rocca et al.,
1989). The mechanism of disruption has been suggested to
involve either the stimulation of proliferative behavior,
which precludes cell cycle withdrawal, or the direct inactivation of Myf5 (Braun et al., 1992
). We present data showing that v-myc expression in L6 cells increases the division
rate and saturation density, promotes cell death by apoptosis in limiting serum, and contrary to previous reports,
accelerates myogenic differentiation. Superimposed expression of bcl-2 relieves cell loss due to apoptosis and independently promotes further cell division at confluence
but does not prevent differentiation. In low serum, CEA
induces a reversible quiescent G0-like state that is incompatible with progression to terminal differentiation. We
demonstrate that cells expressing CEA in addition to v-myc and bcl-2 proliferate faster, reach higher saturation densities, neither undergoes apoptosis nor differentiates in low
serum, and furthermore, are capable of giving rise to large
anchorage-independent colonies that form tumors in nude
mice. CEA is the critical factor in allowing the emergence
of these colonies and in reducing the latent period of tumor formation. These results suggest that CEA may contribute to malignancy in myoblasts by interfering exclusively with access to the differentiation program, inducing
reversible quiescence in the presence of differentiation
stimuli and increased susceptibility to proliferative oncogene activation.
Materials and Methods
) and mouse C2 (Yaffe and Saxel, 1977
) myoblasts
were grown as monolayer cultures in DME containing 10 (L6) or 20%
(C2) FBS growth medium (GM; GIBCO BRL, Gaithersburg, MD) supplemented with 100 U/ml penicillin and 100 µg/ml streptomycin (GIBCO
BRL) and were incubated at 37°C in a humidified atmosphere with 5%
CO2. v-myc and CEA coexpressing transfectant cells were maintained in
medium containing 20% FBS to minimize cell death by apoptosis in medium depleted of growth factors. All cells were passaged while subconfluent to avoid selection of nonfusing variants.
),
pBabe(human bcl-2)puro (Zhu et al., 1996
), or the vector alone controls
(Morgenstern and Land, 1990
) in 2.5 ml
-MEM plus 10% FBS supplemented with 8 µg/ml polybrene for 2 h at 37°C. Cells were then washed
and incubated with GM overnight. The cultures were subcultured 1:5 to 1:20
and selection started 24 h later with GM containing hygromycin B (v-myc)
or puromycin (bcl-2) at 100 µg/ml and 2 µg/ml, respectively. After 7 d of
selection, selective drugs (hygromycin or puromycin) were removed, and
total populations of resistant clones were pooled and cultured thereafter
without selection. v-Myc and Bcl-2 expression was verified by immunoblotting. Total populations were used to avoid the possibility of clonal variation in cellular properties unrelated to the phenotypic effects of the
gene being tested. Proof of the polyclonality of these populations was provided by the presence of multiple integration sites in genomic DNA from
the appropriate cell populations as revealed by Southern analysis using
cDNA-specific probes for v-myc and bcl-2 (not shown). Cultures used for
experiments were obtained from frozen stocks incubated for relatively
few doubling times. The observed effects on cellular phenotype were reproduced with distinct total populations generated by repeating the infections for v-myc and bcl-2. No loss of v-Myc and Bcl-2 was noted during the
course of the experiments (not shown). A triple selection with G418 (for
CEA, see below), hygromycin, and puromycin done in separate steps allowed for the isolation of a population of cells expressing CEA, v-myc,
and bcl-2.
) and 0.5 µg pSV2neo plasmid per dish.
Neomycin (G418)-resistant total transfectant populations were selected
with 400 µg/ml G418 for 10-14 d. G418-resistant clones were pooled and
sorted for high surface CEA levels by FACS using polyclonal rabbit anti-
human CEA antibody. To enrich for stable CEA-producing cells, sorted
populations were cultured without G418 selection for ~20 doubling times,
resorted for residual high CEA producers, and subsequently maintained
in GM containing G418. Proof of polyclonality was provided by Southern
analysis of genomic DNA using a CEA cDNA probe: pooled, sorted, and
stabilized populations showed numerous integration sites and distinct patterns when using both CEA and neo specific probes in three out of the five
independent clones isolated from the final sorted L6C population (not
shown). Transfectant clone L6(CEA-1) was generated previously and designated L6-17 (Eidelman et al., 1993
). G418 was removed from growth
media 24 h before the application of the various functional assays. No loss
of surface expression of CEA was detected over the culture period required for the assays (not shown).
). To investigate proliferation and
differentiation capabilities of cells in medium containing low serum, cells
were seeded on day 0 directly in DM at concentrations listed above and
after 4 d incubation, fixed with 2.5% glutaraldehyde in PBS, and stained
with hematoxylin. As a further assay of differentiation, cells were seeded
in GM at 200 cells/60-mm petri dish, incubated for 10 d without a change
of medium, as described for the viability assay, and then stained as above.
The percentage of colonies with fused cells in their nonproliferating centers was then determined. For anti-myosin immunofluorescent staining,
cultures were induced to differentiate in multiwell chamber slides (Nunclon®; Nunc, Inc., Naperville, IL) and prepared as described (De Giovanni et al., 1993).
Results
) led to the hypothesis that ectopic CEA expression could contribute to
transformation of myoblasts in conjunction with a proliferative stimulus provided by an activated oncogene. Avian
v-Myc and its cellular homolog, c-Myc, are cell cycle activators that promote G0-G1 and G1-S transitions (Evan
and Littlewood, 1993
, and references therein). Pooled
populations of L6 cells stably expressing v-Myc in a retroviral vector conferring hygromycin B resistance (M) or of
L6 cells expressing the drug resistance gene of the vector
alone (P, parental) were therefore generated; expression
of the p110 gag-v-Myc viral protein was verified by immunoblot (Fig. 1 A).
Fig. 1.
Immunoblot analysis of parental and total transfectant
cell populations. Total protein lysates were separated by SDSPAGE and transferred to a membrane for immunodetection using (A) pan-myc and (B) Bcl-2 polyclonal antisera. Lane assignments are as follows: P, parental L6, M, v-Myc+ cells, B, Bcl-2+
cells. Migration of molecular weight (kD) markers is shown.
[View Larger Version of this Image (29K GIF file)]
).
Fig. 2.
Growth parameters of L6 transfectants.
Growth curves are shown
giving the proliferative rate,
saturation density, and viability at saturation of L6 parental (P), v-Myc (M), Bcl-2
(B), and CEA (C) transfectants and double or triple
transfectants. Cells were seeded in replicate 60-mm
dishes as described in Materials and Methods. The data
shown were obtained in one
experiment and are representative of three separate
experiments in which the
rates, saturation densities,
and viabilities were all reproducible. The standard error
of the means of three independent determinations at
each point was 4% for all
data points, i.e., smaller than the diameters of the symbols
themselves. (Inset) Doubling
times, initial and 15-d saturation densities, and viabilities
of L6 cell transfectants were
obtained from the data
shown in Fig. 2. Culture viability (see Materials and
Methods) is presented as an
average of the viabilities on
days 10 and 15 and is expressed as a percentage of
the viability of exponentially
growing cultures. The latter values varied from 76 to
93%. To normalize for the
effects of differentiation and/
or apoptosis, the number of
cells/plate on days 10 and 15 was taken to be equivalent to
the maximum reached during
the course of the growth
curve.
[View Larger Version of this Image (25K GIF file)]
Fig. 3.
Cell sizes of L6 transfectants corresponding to the
growth curve data of Fig. 2. Cell diameters, as determined by
pulse height analysis of particle count data, are shown.
[View Larger Version of this Image (22K GIF file)]
Fig. 4.
Differentiation in the nonproliferating centers of cell colonies. (A) Photomicrographs of colonies arising from cells
seeded at clonal density and grown for 10 d
in GM. Representative parental (P) and
v-Myc+ (M) colonies, showing the presence of myotubes in the centers of only
v-Myc+ colonies. (B) Lower magnification of plates from (A). (C) The percentage of colonies showing myotubes. Colonies arising after 10-d incubations in GM
were scored for the presence of myotubes.
Average results for two, and for some lines three, experiments are shown. The
plating efficiencies for colony formation
varied from 50 to 92% between individual
experiments and varied no more than 10%
between cell lines in a given experiment. Bar: (A) 0.4 mm; (B) 1.7 mm.
[View Larger Versions of these Images (13 + 148K GIF file)]
Fig. 5.
Effects of v-Myc, Bcl-2, and CEA on culture density and differentiation. (A) Photomicrographs of cultures seeded directly in
DM and incubated for 4 d. (B) Photomicrographs of P, C, MB, and MC cultures incubated for 4 d in DM showing immunofluorescence with anti-myosin antibody and of corresponding fields by phase contrast. Bar: (A) 180 µm; (B) 36 µm.
[View Larger Versions of these Images (112 + 98K GIF file)]
). To maximize cell death and minimize myotube
formation, cells were grown in GM until one day before
confluence, when the medium was changed to DM. After
24 h of incubation in DM, 40% of subconfluent M cells
compared to 6% of P cells displayed condensed chromatin and damaged nuclei, hallmarks of apoptotic cell death as
confirmed by electron microscopy (Fig. 6) by the degradation of DNA into discrete bands visible as a "ladder" on
electrophoretic gels (data not shown) and by the fact that
coexpression of Bcl-2 abrogated the myc-induced increase.
The remaining M cells were sparse but multinucleated, indicative of myogenic fusion (Fig. 5 A).
Fig. 6.
Frequency of apoptosis of L6 transfectants in
DM. (A) Electron micrograph showing chromatin
condensation and compaction at the periphery of the
nuclear membrane characteristic of apoptosis in L6
v-Myc+ cells incubated under low serum conditions.
(B) The percentage of nuclei
in L6 transfectant cultures
demonstrating chromatin
condensation when stained
with orcein. Subconfluent cultures in GM were washed
and incubated in DM for 24 h
before fixation and staining
with orcein. Results are representative of several independent experiments. Two
cell suspensions and three fields per cell suspension
were scored, and the results
were averaged. Photomicrographs of subconfluent (C)
parental L6 and (D) v-Myc+
cells stained with orcein showing condensation of chromatin and damaged nuclei (arrows) in v-Myc+ cells. Bar:
(A) 4 µm; (C and D) 40 µm.
[View Larger Versions of these Images (80 + 16K GIF file)]
with v-Myc transformed quail myoblasts.
Moreover, v-myc expression had no effect on the latency
of tumor formation in nude mice (see Fig. 9). We hypothesized that the failure of v-Myc to enhance transformation
and tumorigenicity was due to the differentiation and
death responses observed in v-myc-expressing myoblast
monolayer cultures in depleted GM or in DM, which
would compound the effect of decreasing local growth factor concentrations during colony growth. From these data
we conclude that v-Myc stimulates the cell cycle "engine"
of L6 cells, yet the concomitant increase in the potential to
differentiate or die aborts full transformation.
Fig. 9.
Tumorigenicity of L6 transfectants. The average latent
period in weeks of the different transfectant cell lines for tumor formation is displayed as solid bars. Results represent the average of two separate experiments. MBC-m is a cell population
generated by pooling four different MBC macrocolonies isolated
from soft agar and is termed MBC-mpool#1 in Table I.
[View Larger Version of this Image (15K GIF file)]
; Wagner et al., 1993
; Zhu et al.,
1996
). Therefore, to circumvent cell death and to consolidate the stimulation of proliferation induced by v-Myc in
GM we introduced human bcl-2 into parental and v-myc-
expressing lines and selected for total populations of cells
stably expressing bcl-2 (B) and both v-myc and bcl-2
(MB). Bcl-2 expression was verified by immunoblot analysis (Fig. 1 B). In the L6 system, Bcl-2 did not alter the division rates of P or M cells (Fig. 2, inset, compare P and B, M
and MB). However, a striking increase in cell saturation
density and long term retention of colony forming ability
was observed in all cell populations expressing bcl-2 (Fig.
2, inset, compare M and MB). This was presumably affected by blocking v-Myc-induced apoptosis and by a reduction in acidification of the medium (Table II). These
more benign culture conditions promoted further cell proliferation beyond normal serum growth factor-limited saturation density resulting in a smaller cell size (Fig. 3). The
percentage of subconfluent cells displaying chromatin condensation after 24 h in DM is shown in Fig. 6 B. MB cells
demonstrated only 5.5% damaged nuclei (compared with
M at 40%), indicating complete suppression of M cell
death by coexpression of bcl-2. Since P and B cells show
the same low level of apoptosis (Fig. 6 B, and data not
shown), the effect of Bcl-2 in stimulating division at confluence (Fig. 2, compare P and B) is presumably not a consequence of attenuated cell loss. We conclude that Bcl-2
blocks apoptosis and independently allows further cell division of L6 cells in GM.
To investigate the effects of Bcl-2 on differentiation, P, B, and MB cells were tested in the low serum assays. Bcl-2 alone did not disrupt myotube formation in monolayer culture (Fig. 5), but bcl-2 coexpression with v-myc reduced the increased fusion index of M in the clonal assay from 75 to 19% (Fig. 4). MB myotubes were smaller and more densely packed than parental myotubes, making them more difficult to identify (Fig. 5 A). The majority of MB cells expressed myosin, however (Fig. 5 B). bcl-2 expression can therefore abrogate apoptosis induced by v-Myc, but differentiation is allowed to proceed. In fact, the superposition of Bcl-2 did not increase the efficiency of colony formation of P or M cells in soft agar (Table I), and MB cells showed a reduction in latency for tumor formation of only a few weeks (see Fig. 9). We attribute this to the persistence of differentiation in B and MB cell populations. In fact, MB tumors contained many myosin-positive cells, indicating extensive differentiation (Ordonez, C., R.A. Screaton, and C.P. Stanners, unpublished observations).
CEA Blocks Differentiation and Maintains Proliferative Potential of L6 and C2 Myoblasts
To affect complete transformation of L6 myoblasts by
Myc and Bcl-2, a gene product that could block myogenic
differentiation was required. We previously reported that
CEA could provide a complete block of both morphological and biochemical myogenic differentiation of L6 cells
while leaving the cells with proliferative potential (Eidelman et al., 1993). We decided first to characterize the cell
biology of CEA-expressing L6 cells, to check for the generality of the phenomenon in another myogenic cell line,
and finally to superimpose CEA on MB cells to determine
whether this combination of genes would result in full malignant transformation. To this end, L6 rat and C2 mouse
myoblasts were transfected with expression vectors containing CEA cDNA (Beauchemin et al., 1987
) by calcium
phosphate precipitation. Stable transfectant L6 and C2 total cell populations were enriched for cell surface CEA by cytofluorometric sorting after labeling with anti-human
CEA antibody. These total transfectant populations (L6C
or C, C2C) and individual clones (L6C-1) were analyzed
for CEA expression by FACS analysis (Fig. 7, A and C).
The levels of cell surface expression of CEA were within
the range seen by FACS analysis of purified colonocytes from freshly excised human colon carcinomas (Ilantzis et
al., 1997
).
The ability of L6C-1 and C2C myoblasts to differentiate
terminally was evaluated by determining the percentage of
nuclei present in myotubes with increasing time in DM
(Fig. 7, B and D). On day 7 in DM, L6 cells demonstrated
a maximum fusion index of 85%. C2 cells acidified DM
more rapidly than L6 cells and by day 7 in DM were detaching from the plate. On day 5 in DM, however, C2 cells
showed a fusion index of 57%. In contrast, cultures of both
L6 and C2 cells stably expressing cell surface CEA showed dramatically reduced fusion indices; in fact, L6C and L6C-1
cells showed no myotube formation whatsoever (Figs. 5
and 7 B, and data not shown). L6C-1 (Fig. 7 B) and C2C
(Fig. 7 D) myoblasts retained the ability to form colonies
at high efficiency when seeded in GM, even after culture
for several weeks in DM (data not shown). In DM, L6C-1
cells fail to express myogenin mRNA (Eidelman et al.,
1993), and both L6C-1 and C2C cells did not express myosin (Rojas et al., 1996
; Fig. 5 B, and data not shown). Thus
myoblasts expressing CEA cannot access even the earliest
stages of the parental differentiation program.
CEA Induces a Reversible G0 Arrest in DM
To determine the characteristics of the cell cycle arrest effected by CEA, cells from L6C-1 cultures incubated for increasing time in DM were harvested, stained with propidium iodide, and their nuclear DNA content analyzed by
cytofluorometric analysis. Fig. 8 shows that the L6C-1 cells
arrested exclusively with a postmitotic, preDNA synthetic
G1 DNA content. Furthermore, upon addition of FBS
(20%) or PDGF-BB (not shown) to these cells on day 9 in
DM, DNA replication was reinitiated with a lag of 18 h
(Fig. 8). The length of the 18 h lag period was unchanged
after an additional 7 d (not shown), suggesting that L6C-1
cells reach a quiescent state after 9 d that does not deepen
with increasing time in DM. The length of a normal G1
phase was calculated from FACS profiles of DNA content
of exponentially growing L6C-1 cells, by determining the
percentage of cells in either G1, S, or G2/M (Stanners and
Till, 1960), giving a value of 9.6-10.0 h. A lag of 18 h therefore represents an 8 h prolongation of the normal G1 period and suggests that L6C-1 cells enter a G0-like quiescent state on or before day 9 in DM. Thus CEA expression
appears to induce cells to enter a viable, reversible G0-like
quiescent state that is incompatible with differentiation
and permanent cell cycle withdrawal. Such a state should
be susceptible to oncogenic activation.
CEA, in Concert with Myc and Bcl-2, Leads to Full Transformation
Effect on Growth Parameters.We next tested the effect of CEA expression alone (C) and in combination with v-Myc and Bcl-2 (MC, BC, and MBC) on the growth behavior, transformation, and tumorigenicity of the transfectant cells. The presence of CEA, unlike v-Myc, increased the doubling time of L6 cells, i.e., slowed the cell cycle, increased the average cell size, and decreased initial saturation density (Figs. 2, inset, and 3). After prolonged culture, however, CEA caused a surprising slow rise in saturation density for C, BC, and MBC but not MC cultures (Fig. 2, inset). This effect was not due to a loss of cell surface CEA expression as CEA levels remained unchanged during the course of the growth cycle (data not shown). CEA did not alter the increase in proliferation rate or the stimulation of apoptosis induced by v-Myc (compare MC with M in Figs. 2 and 6 B) but did, however, override the stimulatory effect of v-Myc in stationary phase by reducing the initial culture saturation density (Fig. 2, compare MC with M).
When CEA expression was superimposed on MB cells (MBC), the doubling time was shorter and typical of M cells (Fig. 2, inset), confirming the overriding effect of v-Myc. The effect of Bcl-2 to annul the v-Myc-induced increase in apoptosis was also preserved (Fig. 6 B, compare MB and MBC), as was the ability of both Bcl-2 and CEA to increase saturation density (Fig. 2, inset). However, CEA profoundly modified the effects of v-Myc in confluent serum-limited conditions by blocking the enhanced differentiation phenotype (Fig. 5). In the growth (Fig. 2), colony (Fig. 4), and low serum (Fig. 5) assays, the presence of CEA in every population of transfectants, regardless of the presence of v-Myc and/or Bcl-2, completely prevented differentiation as measured by the appearance of multinucleated and/or myosin-positive cells. Thus the Myc, Bcl-2, and CEA combination seemed to endow L6 cells with the requisite attributes for full transformation. We tested this supposition directly using soft agar and tumorigenicity assays.
Effect on Transformation.CEA expression neither conferred anchorage independence upon L6 cells when expressed alone nor interfered with the low-level soft agar colony formation induced by v-Myc (Table I). However, MBC cells produced rare macrocolonies (>500 cells) in soft agar which did not form in MB plates. These MBC macrocolonies (MBC-m), when rendered single cells and reintroduced into soft agar, bred true with an efficiency for colony formation ranging from 7 to 78% (Table I, and data not shown). It seemed possible that the macrocolonies could represent a minority of the MBC population with a particular set of heritable expression levels for the three transfected gene products that allowed rapid growth in soft agar and tumorigenicity in nude mice. Whereas this hypothesis is impossible to rule out completely, we consider it unlikely since, for CEA at least, nonheritable variation in expression levels in individual cells exceeds by far any heritable variability we have been able to reveal by cloning or FACS sorting. Furthermore, MBC-m and tumor-derived cell lines did not show an increase in the levels of expression of v-Myc and Bcl-2 by immunoblot analysis (data not shown). Thus we suggest that MBC-m cells represent members of the population with additional inherited changes endowing them with the ability to grow more efficiently when deprived of anchorage. Interestingly, very rare macrocolonies were also observed on plates seeded with cells other than MBC but only with C, BC, or MC cells, i.e., cells expressing CEA, regardless of the other genes expressed. These macrocolonies, when picked and replated, also bred true (Table I), indicating again the presence of further heritable transforming lesions. The expression of CEA thus greatly facilitates the emergence of cells bearing such lesions.
Effect on Tumorigenicity.Tumorigenicity was assessed by injection of cells into the thigh muscles of nude mice (Fig. 9). Interestingly, shorter latent periods were seen for all cell types expressing CEA (Fig. 9, compare P with C, B with BC, and MB with MBC), indicating that the presence of CEA favors a more rapid emergence of malignant cells. In fact, the presence of CEA alone was capable of reducing the tumor latent period by 50%. This effect was highly statistically significant (P < 0.0005 for P vs. C, n = 6; P < 0.01 for B vs. BC, n = 6; P < 0.0005 for MB vs. MBC, n = 8) and agrees with the observation that soft agar macrocolonies were observed only for cell lines expressing CEA, as noted above. The tumors were examined for their gross morphology and microscopic histology. Cell lines capable of differentiation in vitro retained this property in the tumors they formed (Ordonez, C., R.A. Screaton, and C.P. Stanners, unpublished results). Tumors produced by C and BC cells appeared within 8 wk but grew slowly and were contained within the leg muscle (not shown); each consisted of an amorphous mass of nondifferentiated cells. MB, MBC, and MBC-m tumors migrated out of the site of injection to the muscle surface (not shown), indicating a more aggressive phenotype associated with v-Myc.
The question arose whether the tumors, especially those with extended latent periods, were composed of cells that possessed additional genetic lesions selected for during the growth period in vivo. To test for this, isolated tumors were rendered suspensions of single cells by collagenase treatment and evaluated for their resistance to the cytotoxic agent(s) used to isolate the original transfectants. A high percentage of the cells derived from tumors demonstrated resistance with the expected specificity. The drug resistant cells were subsequently tested for anchorage independence of growth. Nearly all, except for those produced by MBC-m cells, showed an increase in plating efficiency for colonies and macrocolonies in soft agar (Table I), implicating selection for preexisting or additional genetic changes during tumor growth. Finally, there is the question whether the MBC-m clones picked from soft agar themselves exhibited maximum tumorigenicity. Both drug resistant cells selected from excised MBC-m tumors and the original MBC-m cells used to produce them were reinjected intramuscularly into nude mice. A small but statistically insignificant decrease in latency was observed for the tumor-derived cells (not shown). When tested for growth characteristics in GM, a progressive increase in saturation density (MBC-m-t > MBC-m > MBC) was noted, indicating a step-wise evolution of the transformed phenotype (not shown).
The prevailing view of tumorigenesis holds that conversion from a normal to a completely transformed phenotype involves a series of oncogenic events (Weinberg,
1989). Since overexpression of CEA represents a major
phenotypic change in a wide variety of human cancers, its
potential contribution to malignant progression represents
a significant question. To directly test the contribution of
CEA expression to tumor progression in vivo, L6 myoblasts were used as a model system, as they possess both
proliferation and differentiation potential. The experiments described here provide evidence that CEA can be
considered the prototypic member of a novel class of oncogene. Overexpression results in prolonged cell survival,
in a manner distinct from bcl-2, by the dominant inhibition of terminal differentiation allowing for the outgrowth of
variants bearing genetic alterations, conferring further
growth advantage and subsequent transformation.
The Role of Myc
Deregulated expression of the c-myc gene has been implicated in many types of human cancer (Spencer and Groudine, 1991), and this, in addition to the observed mitogeninduced changes in c-myc expression (Kelly et al., 1983
)
and the requirement for c-myc for the G1-S transition,
strongly supports the model that this transcriptional regulator is central to the control of cellular proliferation.
When v-myc, whose phenotypic and molecular effects have been found to mimic c-myc (Penn et al., 1990
), was
expressed in proliferating L6 cells, a reproducible decrease
in the cellular doubling time and a higher maximum cell
density were noted. Although the observed decrease in
doubling time was relatively small, it would quickly result
in the elimination of P cells in an exponentially increasing
mixed population of P and M cells. These effects could be
due to a more efficient use of serum growth factors by
v-myc-expressing cells than the parental cells, allowing for
more rapid cell growth, further cell division beyond normal saturation, and the production of smaller cells (Figs. 2
and 3).
Two additional cellular responses were also greatly
stimulated by v-myc: apoptosis and myogenic differentiation. These anti-transforming effects were apparent when
growth factors became limiting and were presumably responsible for the failure of M cells to form colonies efficiently in soft agar or to reduce the latency of tumor formation. The commonly observed effect of c-myc triggering apoptosis (Bissonnette et al., 1992; Evan et al., 1992
;
Fanidi et al., 1992
; Hermeking and Eick, 1994
; Wagner et
al., 1994
) is thus duplicated with v-myc in this system. Inhibition of myogenic differentiation at the morphological
(Falcone et al., 1985
; Crescenzi et al., 1994
; La Rocca et al.,
1994) or biochemical (Falcone et al., 1985
; Denis et al.,
1987
; La Rocca et al., 1994) level by Myc, however, has
been reported by most observers under differentiation conditions. Thus, our observation of a dramatic stimulation of differentiation appears to be unique. We feel that
these differences with our findings could be due to systemdependent differences in the balance between Myc-induced
apoptosis and differentiation, which both require reduced
growth factor concentrations for execution. Our results
are not due to a coincidence of myotube formation in a
subpopulation of M cells that escaped apoptosis, since the M population formed colonies with high efficiency in GM,
and 75% of these showed differentiation. This represented
a very significant stimulation of differentiation relative to
the parental cells, which showed 0% differentiated colonies under the same conditions (Fig. 4).
The Role of Bcl-2
The superposition of bcl-2 expression on L6 cells producing v-Myc had no effect on the acceleration of cell division
induced by v-Myc (Fig. 2, inset) but completely suppressed
apoptosis (Fig. 6 C). This is consistent with results from
other systems, as overexpression of c-myc in cultured fibroblasts can induce both accelerated proliferation and
apoptosis (Evan et al., 1992), with the latter being delayed by coexpression of bcl-2 (Bissonnette et al., 1992
; Fanidi et al., 1992
; Wagner et al., 1993
). Our results further indicate a transforming activity of Bcl-2 in addition to its ability to inhibit apoptosis. L6 cells expressing bcl-2 alone reach
higher saturation densities than the parental line (Fig. 2,
inset), independent of any effect on cell death (Fig. 6 B). In
the colony assay, MB colonies on average were twice the
size of M colonies (not shown). In addition, cultures of L6
cells expressing bcl-2, regardless of the other oncogenes
present, remained viable for prolonged culture periods
without medium change (Fig. 2). This evidence, together
with the growth characteristics of these lines (Fig. 2), suggests that Bcl-2 lowers the serum requirement for further cell division at normal saturation and, in v-Myc+ cells,
blocks apoptosis allowing for their overgrowth in increasingly limiting serum. Positive effects of bcl-2 expression on
cell growth parameters have been previously reported for
3T3 fibroblasts (Reed et al., 1991
) and pro-B cells (Miyazaki et al., 1995
). Interestingly, the Bcl-2-mediated block
of apoptosis coincided with a failure to rapidly acidify the
culture medium (Table II). The role of medium acidification in v-Myc-induced apoptosis in L6 cells is currently
under investigation.
B cell directed expression of c-myc and bcl-2 together in
transgenic mice resulted in the formation of monoclonal
tumors (Strasser et al., 1990). By contrast, L6 cells expressing both v-myc and bcl-2 were not fully transformed, as
they were unable to form normal-sized colonies in soft
agar and did not give rise to any larger macrocolonies (Table I). We attribute this failure to the persistence of differentiation, thus abrogating the emergence of variants with
more transformed properties. When tested in vivo, a significant but relatively small reduction in tumor latent period by MB cells was noted relative to M or B cells, though
MB tumors were found to contain many differentiated
cells (Fig. 9, and Ordonez, C., R.A. Screaton, and C.P.
Stanners, unpublished observations).
The Role of CEA
We have shown previously that ectopic cell surface CEA
expression by L6 myoblasts blocks entry to the myogenic
differentiation program at a very early stage, i.e., before
the upregulation of the myogenic transcriptional regulator, myogenin (Eidelman et al., 1993). Cells expressing
CEA alone were slowed in their division rate and reached
lower initial saturation densities (Fig. 2). In cells producing both CEA and v-Myc, v-Myc was dominant in accelerating cell proliferation but succumbed to the effects of
CEA at confluence. We suggest that these effects of CEA
are due to an increase in the probability in DM of cells entering a G0-like state from the G1 period of the cell cycle
(Figs. 7 and 8). Since the parental cells tend to differentiate under these conditions, it is difficult to determine
whether this state is entirely novel or a state transiently traversed by the parental cells during their passage to differentiation. CEA was confirmed to block differentiation
in this study and was dominant over the differentiation
stimulating effect of v-Myc in MC cells, blocking differentiation completely.
Cells expressing v-myc, bcl-2, and CEA together possessed all of the properties expected to yield a fully transformed phenotype: a higher division rate, a higher saturation density, reduced apoptosis, no differentiation, and long term survival (Figs. 2 and 4-6) yet did not produce colonies in soft agar with high efficiency (Table I). MBC cell populations did, however, contain cells at low frequency capable of producing very large anchorage independent macrocolonies which, when picked and dispersed, bred true, forming macrocolonies with high efficiency (Table I). Interestingly, macrocolonies were found in all cell cultures in which CEA was expressed: MBC, MC, BC, and even C cell populations (Table I). These variants also seemed to be responsible for the formation of tumors, since the latent periods for tumor formation were lowest for cell lines containing them (Fig. 9), the cells selected for during tumor growth were highly enriched for them (Table I), and pure populations of variant cells showed the lowest tumor latency.
"Fertile Soil" Hypothesis
From these results, we suggest that CEA expression accelerates progression towards transformation and increased
tumorigenicity by blocking terminal differentiation, thus
providing a "fertile soil" for the emergence of cellular
variants with increased proliferative potential. Indeed, after reaching an initial stationary phase, all cell lines expressing CEA tend to increase slowly in cell number (Fig.
2), indicating an increased sensitivity to proliferative stimuli. CEA-expressing cell lines also accumulate tumorigenic macrocolony-producing cells, as noted above. The nature
of the heritable changes giving rise to macrocolonies is
presently unknown. Bcl-2 has also been suggested to provide a cellular state conducive to the emergence of more
autonomous variants, yet accomplishes this by a fundamentally different mechanism, i.e., by the suppression of
apoptosis. In transgenic mice, ectopic bcl-2 expression
alone does not directly result in tumors (McDonnell et al.,
1989) yet generates a population of quiescent cells that
possesses oncogenic potential, perhaps as a consequence
of extended survival (McDonnell and Korsmeyer, 1991
).
In the L6 system, however, Bcl-2 was relatively ineffective
in promoting the emergence of variants or decreasing tumor latency. Thus CEA seems to posess a novel oncogenic
activity, with a distinct mechanism from Bcl-2 for the induction of a long term viable quiescence (Fig. 10).
Relevance In Vivo
CEA is expressed at low levels in the proliferative zone of
the colonic crypt epithelium in normal adult colonocytes
(Ahnen et al., 1987; Jothy et al., 1993
). As colonocytes differentiate, losing division potential, they migrate up the
sides of the crypt towards the lumen of the gut. During this
process, CEA is upregulated on the luminal (apical) surface of these cells (Ahnen et al., 1987
; Jothy et al., 1993
).
Our present model outlining a possible role for CEA in colon tumorigenesis contends that overproduction of CEA
over the entire surface of colonocytes that still possess division potential, i.e., out of the context of a normal cellular differentiation program, could result in such cells retaining inappropriate proliferative capacity. The intercellular adhesion activity of CEA could lead to the disruption of normal cell-cell and cell-substratum interactions that are presumably required both for the maintenance of tissue
architecture and for faithful completion of colonocyte differentiation (Benchimol et al., 1989
). By disrupting cell-
cell interactions expected to be important for normal differentiation, CEA-positive cells would experience extended
survival and remain open to further growth stimuli or oncogenic activation.
Perhaps the most relevant system for examining the effect of CEA is therefore the human colonic epithelium
where CEA family members, CEA, nonspecific cross-reacting antigen, and biliary glycoprotein, undergo dramatic
changes in expression during carcinogenesis (Cournoyer
et al., 1988; Boucher et al., 1989
; Hinoda et al., 1991
; Neumaier et al., 1993
). The technical complexity and poorly defined differentiation process of the colonic system, however, renders it less amenable to experimental investigation in vitro. The results presented in this work refer to a
myoblast model system chosen for ease of experimentation and interpretation and with assumed relevance to systems where CEA is naturally expressed. The results, which
support the above hypothesis, are not species or cell line
specific in that the basic observation of the induction of a
viable quiescent cellular state in which myogenic differentiation is blocked was extended to mouse C2 myoblasts
(Fig. 7). We are currently investigating the effect of CEA
expression on other differentiating systems.
The validity of the use of the myoblast system is supported by our recent observations that CEA and nonspecific cross-reacting antigen, both of which block myogenic
differentiation (Eidelman et al., 1993; Rojas et al., 1996
),
are upregulated on the surface of purified colon carcinoma
cells relative to normal colonocytes from the same patient
and at levels inversely related to the degree of differentiation, while biliary glycoprotein, which has no effect on
myogenic differentiation (Rojas et al., 1996
), shows no increase in expression (Ilantzis et al., 1997
). An upregulation
of CEA was also observed in early dysplastic lesions of the
colon (Ilantzis et al., 1997
). Interestingly, both bcl-2 upregulation (Bedi et al., 1995
; Bosari et al., 1995
; Sinicrope et
al., 1995
) and myc overexpression (Guillem et al., 1995
,
and references therein) have been reported to be involved in
a high proportion of human colon adenomas and carcinomas.
The effect of CEA on myogenesis is similar to that of
oncogenic forms of H-ras and N-ras (Olson et al., 1987), as
the block in differentiation is not dependent on aberrant
proliferation. A major difference in the effects of CEA
and ras, however, is the reduction in saturation density
mediated by CEA (Fig. 2, inset), which precludes placing
CEA and ras in the same category of oncogene. As the
CEA effect is reversible by supplementing DM with peptides corresponding to the CEA adhesion domains (Eidelman et al., 1993
), it is tempting to speculate that the CEA-
CEA interaction may interfere directly with intracellular
signaling events required for activation of the myogenic
differentiation program. In fact, we have shown that CEA
can signal through its GPI anchor to phosphorylate Src
family kinases and other cellular proteins (Draber, P., unpublished observations) and that the GPI anchor is absolutely required for the inhibitory effect on myogenic differentiation (Screaton, R.A., and C.P. Stanners, unpublished
observations).
CEA: A Dominant Differentiation-blocking Oncogene?
Can CEA be considered an oncogene? Often protooncogenes are found to comprise ubiquitous elements of the
cell proliferation or differentiation machinery (Hunter,
1991; 1993). CEA does not have a known counterpart
within the genomes of oncogenic viruses and does not possess overt transforming properties in cultured cells. Does
CEA function as an inducer of quiescence in normal growth control of all cells? The restricted expression pattern of CEA makes this unlikely. However, CEA could be
used as an explicit (as opposed to ligand controlled) differentiation block during embryonic development, in situations where cells are required to remain associated in undifferentiated collectives. We suggest that inappropriate
expression of CEA in adult life in cells with both proliferation and differentiation capacity promotes survival in the
presence of differentiation stimuli, thus facilitating the acquisition of progressive mutations. We therefore propose that CEA defines a new class of gene with dominant, differentiation-blocking oncogenic activity.
Received for publication 19 July 1996 and in revised form 13 December 1996.
1. Abbreviations used in this paper: CEA, carcinoembryonic antigen; DM, differentiation medium; GM, growth medium.We thank Faizan Alawi for the isolation of C2 (CEA) transfectants, Kathy Hewitt for electron microscopy, Luisa DeMarte for technical assistance, and Anthony Sharpe for the preparation of tissue culture medium. We would also like to thank Dr. Jerry Pelletier and Dr. John Silvius for critical reading of the manuscript.
This work was supported by grants from the National Cancer Institute of Canada and the Medical Research Council of Canada to C.P. Stanners and from the Medical Research Council of Canada to L.Z. Penn. R.A. Screaton was supported by a Studentship from the Research Institute of the Royal Victoria Hospital, Montreal, Canada.