[CANCER RESEARCH 48. 6173-6182. November I. 1988|
Modulation of HT-29 Human Colonie Cancer Cell Differentiation with
Calmidazolium and 12-O-Tetradecanoylphorbol-13-acetate1
Cécile
Rochette-Egly,2 MichèleKedinger, and Katy Haffen
INSERM Unite 61, Biologie Cellulaire et Physiopathologie Digestives, 3, avenue Molière,67200 Strasbourg, France
ABSTRACT
The effects of a protein kinase C activator, 12-0-tetradecanoylphorbol13-acetate (TPA), and of a calmodulin antagonist calmidazolium (CZ),
on a human colonie cancer cell line HT-29 were analyzed. HT-29 cells
are undifferentiated in standard culture conditions (HT-29 G+) and
display an enterocytic differentiation when cultured in glucose-deprived
medium (HT-29 G-). Early effects of TPA and CZ on the localization
of cytoskeletal proteins (caldesmon, a-actinin and vinculin) and on cell
proliferation were examined. Differentiation of the cells was assessed
after 4 weeks on the basis of ultrastructural and functional characteristics
of enterocytic polarity, presence of apical brush borders, expression of
brush border membrane antigens (Caco 5/50 and sucrase-isomaltase),
and segregation of calmodulin to the brush border cytoskeleton.
TPA treatment of HT-29 G+ or G- cells induced early morphological
and cytoskeletal alterations: the cells rounded up and lost their stress
fibers with the associated caldesmon, a-actinin, and vinculin. TPA did
not modify the differentiation of G—cells, but induced in G+ cells the
expression, although limited, of enterocytic differentiation characteris
tics. Addition of CZ to HT-29 G—cells enhanced their differentiation
state but did not provoke any early morphological or cytoskeletal alter
ations. No effects of CZ on HT-29 G+ cells were obvious. The results
suggest that protein kinase C, the TPA receptor, is involved in the
triggering of HT-29 G+ cell differentiation whereas calmodulin-dependent functions would be implicated in HT-29 G—cell maturation.
INTRODUCTION
Enterocytes are highly polarized columnar epithelial cells,
characterized by the presence at their luminal surface of thou
sands of microvilli forming the brush border (1,2). Accumulat
ing evidence suggests that in mature enterocytes, various cell
functions are regulated by calcium ions (3). Indeed Ca2+ binds
to two major proteins, the vitamin-D calcium binding protein
named CaBP (4, 5) and calmodulin (5-7), both being concen
trated in the brush border region. Upon binding calcium, cal
modulin regulates the ionized calcium concentration in the
microvilli (3), activates the actomyosin-based contractility sys
tem (8), and interacts with a number of actin-binding proteins
(9). Calcium is also a direct cofactor of the phospholipid and
Ca2+-dependent protein kinase C (10) which has been recently
identified in intestinal epithelial cells (11).
Recently, much attention has been directed towards the as
sembly of the microvillous cytoskeleton during development
(12-14) and in correlation with morphogenetic events (15).
However, nothing is known concerning the mechanisms gov
erning these processes. Among the mechanisms which could be
responsible for microvillous cytoskeleton assembly and more
generally for the biogenesis of enterocytes' polarity, a role for
extracellular matrix components and epithelial-mesenchymal
Received 3/10/88; revised 6/15/88; accepted 7/28/88.
The cosls of publication of this article were defrayed in part by the payment
of page charges. This article must therefore be hereby marked advertisement in
accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
' Financial support was provided by INSERM and a grant from the Ligue
Nationale Françaisecontre le Cancer.
2 Present address. Laboratoire de Génétique
Moléculairedes Eucaryotes du
CNRS, Unité184 de Biologie Moléculaireet de Génie
Génétique
de l'INSERM,
Facultéde Médecine,11 rue Humann, 67085 Strasbourg Cedex, France. To
whom requests for reprints should be addressed.
cell contacts has been suggested (1, 2, 15-17). Concerning the
intracellular events, in many instances, calmodulin levels have
been shown to regulate the differentiation processes (18, 19).
Furthermore, activation of the protein kinase C may stimulate
differentiation (20). Thus, in order to define the possible role
of these two factors in enterocyte differentiation, we examined
the effects of a calmodulin antagonist, CZ ' (21) and of a protein
kinase C activator: the phorbol ester TPA (22). Indeed these
compounds have been shown to mediate the terminal differen
tiation in various cell lines (for review see Reference 23), the
most studied system being the human promyelocytic cell line
HL-60 (24-27).
In order to examine terminal differentiation in human enter
ocytes an appropriate in vitro model was provided by the human
HT-29 colon carcinoma cell line established by Fogh and
Trempe (28). As shown by Zweibaum's group (29-31), the state
of differentiation of these cells is dependent on the culture
conditions. Indeed HT-29 cells remain undifferentiated (HT29 G+) when grown under standard conditions, in the presence
of glucose. In contrast, when cultured in the total absence of
sugar (HT-29 G-) they express a typical enterocytic differen
tiation starting after confluence (10-15 days) and being maxi
mum after 30 days in culture (32). The morphological and
enzymatic enterocytic features expressed by HT-29 G—cells
remain however irregular and limited compared to small intes
tinal absorptive cells or to another colonie adenocarcinoma cell
line Caco-2 (32).
The present study examines the differentiation response of
both HT-29 G+ and G- cells to the calmodulin antagonist CZ
and to the protein kinase C activator, TPA. The early effects of
TPA and CZ were studied on the basis of morphological and
cytoskeletal alterations. The differentiation of the cells was
assessed by ultrastructural criteria and by the expression and
distribution of microvillous membrane and cytoskeletal anti
gens. The data reported herein emphasize that CZ increases
the maturation of HT-29 G—cells whereas TPA triggers a
"limited" differentiation of HT-29 G+ cells.
MATERIALS
AND METHODS
Dulbecco's modified Eagle's medium was purchased from Eurobio
(Paris, France). Fetal calf serum and trypsin were from GIBCO Biocult
(Glasgow, Scotland). Disposable plastic material used for cell culture
experiments was from Falcon (Los Angeles, CA).
Benzamidine, antipain, pepstatin A, poly(ethylene oxide) 20 sorbitan
monolaurate (Tween 20), inosine, phosphatidylserine, and TPA were
obtained from Sigma Chemical Co. (St. Louis, MO). Leupeptin, aprotinin, and calmidazolium were from Boehringer (Mannheim, W. Ger
many). Sodium dodecyl sulfate, acrylamide, AW'-methylene-bis-acrylamide were obtained from Bio-Rad Laboratories (Richmond, CA). NC
membranes (0.45 /¿m)were purchased from Schleicher and Schuell
(Dassel, W. Germany). Prestained molecular weight standards were
from Bethesda Research Laboratories.
[A/efAy/-'H]thymidine (specific activity, 45 Ci/mmol) was obtained
3The abbreviations used are: CZ. calmidazolium; TPA. 12-0-tetradecanoylphorbol-13-acetate; NC, nitrocellulose; PBS, phosphate buffered saline; TCA,
trichloracetic acid.
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TRA. CZ. AND COLONIC CANCER CELL DIFFERENTIATION
from the Centre d'Energie Atomique (Saclay, France). Normal goat
serum, fluorescein-conjugated goat antirabbit IgG, and fluoresceinconjugated rabbit antimouse IgG were obtained from Nordic Immunological Laboratories (Tilburg, The Netherlands). Polyclonal calmodulin antibodies raised in rabbits were a generous gift of Dr. J. De Mey
(Jansen Pharmaceutica. Beerse, Belgium); caldesmon antibodies were
kindly provided by Dr. M. P. Walsh (Calgary, Canada). The mouse
monoclonal Caco 5/50 antibodies (33) were kindly provided by Dr. A
Quaroni (Cornell University, Ithaca, NY). Monoclonal antibodies
against sucrase-isomaltase (HBB 2/614/88) (34) were kindly provided
by Dr. H. P. I lauri (Biocenter, Basel, Switzerland). Monoclonals anti«-actininand antivinculin were from Bioyeda and Biomekor (Kiryat
Weizman, Rehovot, Israel), respectively.
( aim.nlnlin was prepared as previously described (35) and iodinated
using 125I-labeledBolton and Hunter Reagent (2000 Ci/mmol, Amersham, les Ulis, France); the specific radioactivity of [I25l]calmodulin
ranged from 40 to 80 ßC\/ng.
Cell Cultures and Cell Treatments. The human colon adenocarcinoma
cell line HT-29 originally established by Fogh and Trempe (28) was
obtained from Dr. A. Zweibaum (INSERM, U.I78, Villejuif, France).
The HT-29 G+ cells (passages 148 to 151) were grown in Dulbecco's
modified Eagle's medium supplemented with 10% fetal calf serum and
containing a standard concentration of glucose (25 ITIM).Stock cultures
were subcultured by trypsinization (0.25% trypsin in Ca2+,Mg2+-free
PBS containing 0.54 mM EDTA) and seeded at 1.8 x IO4cells/cm2 in
plastic dishes. The HT-29 G- cells (passages 6 to 12 following the
switch of HT-29 G+ cells to sugar-free medium) were cultured in
Dulbecco's modified Eagle's medium specially prepared without glu
cose by Eurobio and supplemented with 10% dialyzed fetal calf serum
and 2.5 m\i inosine. The cells were dissociated with 0.25% trypsin in
Ca2+,Mg2+-free PBS containing 2.67 mM EDTA and seeded at 3.6 x
IO4cells/cm2.
At Day 2 (exponential phase of growth) CZ or TPA was added at
concentrations of 0.5 /JMand 10 nM, respectively. These concentrations
were shown to be optimal by a dose-response (morphological behavior
of the cells and [3H]thymidine incorporation) curve ranging from 0.05
to 1 MMfor CZ and 1 to 100 nM for TPA (data not shown). Then the
media supplemented with either CZ or TPA were changed daily. In
control experiments, the cells were treated with the solvent (ethanol,
0.05%).
Morphological Analysis. For transmission electron microscopy, cell
sheets scraped from the dishes were fixed in 0.2 M cacodylate-buffered
2% glutaraldehyde (pH 7.4) for 2 h at +4°C,postfixed in cacodylatebuffered 1% osmium tetroxide (pH 7.4) for 30 min at 4°Cand embedded
in araldite. Ultrathin sections were double stained with uranyl acetate
and lead citrate before their observation with a Philips 300 electron
microscope.
For scanning electron microscopy, cells were grown on plastic culture
coverslips (Thermanox Lux Scientific Corporation, Newbury Park,
PA). The cells were fixed as above, dehydrated, dried in a critical point
drier, coated with gold, and examined with a Philips 50IB scanning
electron microscope.
['I II'l'liymiiliiicIncorporation. ['H]Thymidine was added to the culture
medium (1 /jCi/ml) for a 24-h incubation at 37°C.Then, the radioactive
medium was aspirated and the culture rinsed twice with PBS and
trypsinized. After centrifugation, aliquots of the cell suspension were
spotted onto discs of Whatman paper which were successively treated
with TCA 20% (twice for 5 min), TCA 10% (twice for 5 min), ethanol
(5 min), ethanol/ether (3 min), and ether (twice for 2 min). The discs
were then dried and the adsorbed radioactivity measured in an Inter-
Fig. 1. Phase contrast micrographs of HT29 G+ (a. c, e) and G- (b, d,f) cells cultured
for 3 days. a. b, control cells; c, d. CZ. (0.5 jiM)treated cells; e, f, TPA (10 n,M)-treated cells.
CZ and TPA were added 2 days after plating
and cell morphology was examined 24 h later.
Bar, 50 firn.
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TPA. CZ. AND COLONIC CANCER CELL DIFFERENTIATION
Fig. 2. Effect of TPA on caldesmon. a-actinin, and \ineuiin distribution in HT-29 G+ cells. Cells were grown on glass coverslips and stained as described in
"Materials and Methods" after a 24-h treatment with 0.05^ ethanol (a, d, g). 10 nM TPA (h, e, h) or 0.5 JIMCZ (c, f, /'). Bar. 50 J/M. a, h, c, caldesmon: d, e,f. aactinin: g, h, i. vinculin.
technique Scintillation Counter. The rate of ['H]thymidine incorpora
tion was expressed as dpm/24 h/mg of protein.
Immunofluorescence Microscopy on Cryosections. This technique was
used to demonstrate the expression and localization of specific brush
border antigens (Caco 5/50, sucrase-isomaltase, calmodulin). After
washing the cells twice in Hanks' buffer, a "sheet" of cells was collected,
fixed for 1 h at 4°Cwith 2% paraformaldehyde in 0.1 M piperazinel,4-bis-2-ethanesulfonate at pH 7.0 containing 5% sucrose and em
bedded in Cryoform. Cryosections (4-5 jitn thick) were cut with a
cryostat and incubated with the specific mouse CaCo 5/50 (33) or
sucrase (34) monoclonals or with the polyclonal rabbit calmodulin
antibodies (36). The second antibodies were fluorescein-conjugated
rabbit anti-mouse and goat anti-rabbit sera, respectively. Sections were
mounted in phosphate-buffered glycerol supplemented with paraphenylene-diamine (37) and observed using a Leitz epifluorescence micro
scope. Controls included incubations without monoclonals or after
neutralization of calmodulin antibodies with native calmodulin (35.
36).
Immunofluorescence on Cultured Cell Monolayers. This immunofluorescence technique was used to localize caldesmon, n-actinin. and
vinculin. Cells routinely cultured on glass coverslips were rinsed thrice
in Tris buffered saline, fixed for 10 min in methanol at —20°C,
and
essed for indirect immunofluorescence as described for Cryosections.
The second antibodies were goat anti-rabbit IgG conjugated to fluorescein for caldesmon, and fluorescein-conjugated rabbit anti-mouse IgG
for «-actininand vinculin.
Preparation of Cultured Cell Lysates. Cultured cells were washed
twice in ice-cold Ca:*,Mg;+-free Hanks' balanced salt solution (20
mM 4-(2-hydroxyethyl)-l-piperazineethanesulfonic
acid, 0.3 mM
Na2H PCX, 0.4 mM KH:PO4, 140 mM NaCI, 5.4 mM KC1, pH 7.3)
containing 1 m\i ethyleneglycol bis(ff-aminoethyl ether)-jV,jV,/V',/V'tetraacetic acid and a cocktail of protease inhibitors [pepstatin (1 jjg/
ml), antipain (l Mg/ml), benzamidine (15 ¿ig/ml),leupeptin (10 jjg/ml),
and aprotinin (10 jjg/ml)]. Cells were then scraped in Hanks' buffer,
sonicated, and stored at -80°Cbefore analysis.
Determination of Calmodulin Content. The cell lysates were treated
at 80°Cfor 5 min, rapidly cooled on ice, and centrifuged at 8000 x g
for 5 min. Then the supernatants were assayed for calmodulin content
using a commercially available [l;l;I]calmodulin radioimmunoassay kit
from New England Nuclear (Frankfurt. Germany). Results were ex
pressed as the mean of six replicates in ng/mg protein. Proteins were
estimated according to Lowry et al. (38).
Detection of Calmodulin-binding Proteins. Proteins from cell lysates
were resolved by electrophoresis in 5-15% linear gradient polyacrylpermeabilized in cold acetone (10 min). Then the cultures were proc
amide gels (0.75 mm thickness) in the presence of 0.1% sodium dodecyl
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TPA. CZ, AND COLONIC CANCER CELL DIFFERENTIATION
250
200-
Q
controls
H
TPA 100 nM
0 TPA 10
nMEl
15O
uMIi1•II1C
CZ 0,5
^^¿t?.K1¡
100
¡|\^¡C
50
24hG*|^ÃŒi72hIj^
24h 72hG-
Fig. 3. Effect of CZ and TPA on ['HJthymidine incorporation of HT-29 G+
and G- cells. CZ (0.5 »IM)
and TPA (10 and 100 nM) were added at Day 2 after
plating. ['HjThymidine was added for a 24-h incubation at the time of treatment
and 2 days later as described in "Materials and Methods." Results represent the
rates of thymidinc incorporation 24 and 72 h after TPA or CZ addition and are
expressed as a percentage of the controls without TPA or CZ. The values
presented herein are the mean ±SD of two experiments performed in duplicate.
sulfate (39) and electrophoretically transferred to NC membranes as
already described (40). The calmodulin-binding proteins bound to NC
membranes were visualized by ['"Ijcalmodulin overlay as already re
ported (13).
Determination of Sucrase Activity. Sucrase activity was measured
according to Messer and Dahlqvist (41) in a fraction enriched in brush
border membranes (42). The results are expressed as milliunits per mg
of proteins. One unit is defined as the activity that hydrolyzes 1
of sucrase per min under the experimental conditions.
RESULTS
Cell Morphology and Cystoskeleton Organization after CZ
and TPA Treatment. HT29 G+ and G—cells grow normally as
tight colonies of polygonal cells (Fig. 1, a and b). Their treat
ment in exponential phase of growth (Day 2 after seeding) with
CZ (0.05-0.5 MM)for several days did not result in any change
in morphology when examined under the phase contrast micro
scope (Fig. 1, c and d). However, they showed a clear response
to TPA (1-100 nM) as soon as 24 h (Fig. 1, e and /): the
colonies extended and the cells rounded up showing large
extracellular spaces. These effects were no more obvious when
cells reached confluency. When cells that had been treated with
TPA for 24 h were washed and subsequently cultured in the
absence of TPA, they recovered a morphology similar to that
of controls, thus demonstrating that the rapid effect of TPA on
HT-29 cell morphology was reversible.
Concomitantly caldesmon, o-actinin, and vinculin organiza
tion were studied in HT-29 cells. Caldesmon is a calmodulinand actin-binding protein whose distribution is associated with
that of actin microfilaments; a-actinin and vinculin are cytoskeletal proteins present at focal contacts of the cellular ventral
surface. Control HT-29 G+ cells contain a prominent system
of stress fibers labeled with caldesmon antibodies throughout
the cells (Fig. la). Very shortly (24 h) after TPA was added to
the culture medium, changes in caldesmon organization were
perceived (Fig. 2b). The stress fiber system disappeared and
staining accumulated within the cytoplasm. Furthermore, aactinin fluorescence localized in control cells at the plasma
membrane level mostly in the intercellular contact regions, and
in the cytoplasm in association with microfilaments (Fig. 2d),
disappeared after addition of TPA. However, some punctuate
and patchy arrays were retained throughout most of the cyto
plasm (Fig. 2e). Concerning vinculin distribution in control
HT-29 G+ cells (Fig. 2#), the corresponding antibodies re
vealed small focal patches of fluorescence at the end of stress
fibers, corresponding to adhesion-plaque structures situated
both on the ventral cell surface and at the periphery of the
cellular colonies. In response to TPA, the characteristic spear
head shape of the fluorescent vinculin patches disappeared (Fig.
2A); the staining became more granular, amorphous and asso
ciated to the juxtanuclear region of the cell. CZ completely
failed to mimic the series of changes induced by TPA in HT29 G+ cells: caldesmon, «-actinin,and vinculin organization in
CZ-treated cells (Fig. 2, c,/, and /) was not different from that
found in control cells (Fig. 2, a, d, and g).
In a parallel set of experiments performed with the HT-29
G—cells (data not shown), TPA caused very similar changes in
cytoskeletal protein organization whereas CZ was also uneffective.
Effects of CZ and TPA on HT-29 Cell Proliferation. When
undifferentiated HT-29 G+ cells were treated at Day 2 after
seeding with 0.5 UMCZ, their growth rate was 50% reduced as
assessed by the decrease of ['H]thymidine incorporation during
the 24 h following (Fig. 3). Treatment of G+ cells with 10-100
nM TPA caused a similar inhibition of cell growth. In contrast,
when added to HT-29 G- cells, both TPA and CZ acted as
stimulators of ['H]thymidine incorporation (50-150%). These
responses, detectable as soon as 24 h of treatment, were still
visible after 72 h. Thus, in the HT-29 cell line, TPA and CZ
acted either as growth inhibitors or as mitogens, depending on
the differentiation potentiality of the cells linked to their culture
conditions.
Effects of CZ and TPA on HT-29 Cell Differentiation. The
ultrastructural features of both HT-29 G+ and G—cells viewed
at the transmission or scanning electron microscope have al
ready been described (32). Briefly, HT-29 G+ cells display after
3-4 weeks of culture, multilayers of polymorphous cells with
sparse and irregular cytoplasmic processes (Figs. 4a and 5a).
In contrast, grown in the absence of glucose, HT-29 G—cells
form a monolayer of polarized polygonal cells linked by apical
junctions. They exhibit at their apex numerous true microvilli
projecting towards the medium (Figs. 4b and 5b). In addition,
intercellular cysts bordered with microvilli are found within the
cell layer of either G+ or G—cells (Fig. 4, a and b).
When CZ (0.5 MM)was present for 4-5 weeks in the culture
medium of the HT-29 G+ cells, no change in the ultrastructural
characteristics of these cells could be observed (Figs. 4c and
5c). However, CZ caused important modifications in G—cells.
Fig. 4d shows that CZ-treated HT-29 G—cells depicted more
dense and more regular microvilli uniformly distributed at their
apical surface and exhibiting distinct cytoskeletal rootlets.
Treatment of HT-29 cells with TPA (10 nM) led to opposite
results. Indeed, TPA did not modify the ultrastructural pattern
of the differentiating G—cells (Fig. 4/). However after 4 weeks,
TPA-treated HT-29 G+ cells, although still growing in multi
layers exhibited numerous and dense organized microvilli at
the upper surface of approximately 50% of the culture (Fig.
Se/), the remaining cells facing the medium being not signifi
cantly different from the control ones (Fig. 4e).
Thus, from these results it appears that the effects of CZ and
TPA on the ultrastructural characteristics of HT-29 cells differ
depending on the differentiation potential of these cells. It must
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TPA. CZ. AND COLON1C CANCER CELL DIFFERENTIATION
Fig. 4. Ultrastructural features of HT-29
G+ (a, c, e) and G- (b, </,/) cells, cultured for
4 weeks in control medium (a, ft), in the pres
ence of 0.5 MMCZ (c, a1)or of 10 nM TPA (e,
/). In CZ-treated HT-29 G- cells (d) the
apical micro\ ¡Hi
are more dense and organized,
and the cytoskeletal rootlets more visible as
compared to the controls (b). Insert in b, an
intercellular cyst exhibiting numerous microvilli. Bars, 5 /jm (a, c, e and insert); 2 MM(b, d.
,
V*
•¿è
P •
^
& •
e „ ,;* vf.
be stressed that these CZ- and TPA-induced changes in the
differentiation state of G—and G+ cells, respectively, occurred
only when the compounds were present in the medium during
the whole culture period (4-5 weeks). Indeed a single 24-h
exposure of growing cells to CZ or TPA was uneffective on
these late effects. Moreover the effects of CZ and TPA on the
differentiation of HT-29 cells were maintained during several
passages when the cells were continuously cultured in their
presence but were reversible if the cells were subcultured in
control medium.
Effects of CZ and TPA on Sucrase-lsomaltase Immunofluorescence. As already demonstrated by Zweibaum's group (30),
the differentiation of HT-29 G—cells was proved by the expres
sion of an intestinal microvillar hydrolase. sucrase-isomaltase:
the enzyme is detectable at the apical surface of 50-60er of the
cells (31) as shown by immunofluorescence with monoclonal
anti-sucrase-isomaltase antibodies (Fig. 6/>). No positive stain
ing could be detected in the pluristratified undifferentiated G+
cells (Fig. 6a).
As expected from the above mentioned results, CZ did not
modify the negative immunofluorescence pattern of G+ cells
(Fig. 6c) but increased the number of G—cells labeled at their
apex (Fig. 6d). On the other hand, TPA did not modify the
sucrase-isomaltase immunofluorescence staining of G—cells
(Fig. 6f). However. Fig. 6e shows that surprisingly, TPA did
not induce the appearance of any labeling in G+ cells.
These immunofluorescence results were confirmed by deter
mination of sucrase activity in brush border membrane frac
tions: CZ increased sucrase activity in G—cells after 4 weeks
of culture (13.3 ml'/mg protein) as compared to controls (7.5
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TPA. C/, AND ( OI.ONIC CANCER CTil.I. DIFFERENTIATION
Fig. 5. Scanning electron microscopic views of HT-29 G+ (a) and G- (A) cells cultured for 4 weeks. HT-29 G+ cells cultured in the presence of 0.5 /*MCZ (c) or
10 n\i TPA (</).Note that TPA-treated G+ cells (</)become polygonal and display more dense and more abundant microvilli at their surface, as compared to controls
(a). Bar, 5 ¿/m.
mU/mg protein), whereas TPA was uneffective; neither CZ nor
TPA was able to induce sucrase activity in G+ cells.
Immunofluorescence of the Caco 5/50 Antigen. As another
approach, the differentiation of HT-29 cells has been studied
by immunofluorescence of the Caco 5/50 antigen, using specific
monoclonal antibodies. The Caco 5/50 antibodies recognize
exclusively an antigen located at the surface membrane in
human jejunum epithelial cells as well as in the spontaneously
differentiated human colonie cancer cell line Caco-2 (33). As
shown in Fig. Id, these antibodies stained, although discontinuously, the apical surface of HT-29 G—cells after 4 weeks of
culture. These cells also exhibited some strongly positive inter
cellular cysts already visible around 10 days of culture (Fig. 70).
In contrast, the pluristratified undifferentiated HT-29 G-t- cells
did not depict any Caco 5/50 staining up to 4-5 weeks of
culture (Fig. 7, a and c).
The presence of CZ during 4 weeks did not modify the
negative immunofluorescence pattern of HT-29 G+ cells (Fig.
le). However CZ increased strongly the number of G—cells
labeled at their apex, the interruptions of the apical staining
corresponding to morphologically less differentiated cells (Fig.
If). TPA treatment, in contrast to CZ, did not modify the Caco
5/50 apical staining of G- cells (Fig. Ih) but induced the
staining of some intercellular cysts in HT-29 G+ cells (Fig.
Ig).
Effects of CZ and TPA on (a ImoduIin Levels and Localization.
To determine whether calmodulin was of regulatory signifi
cance during HT-29 cells' differentiation, calmodulin levels
were measured by radioimmunoassay in growing and postconfluent HT-29 G+ and G—cells submitted or not to CZ or
TPA treatment. As shown in Fig. 8, 2 days after seeding, both
HT-29 G+ and G—cells exhibited low calmodulin concentra
tions. Then calmodulin increased progressively, reaching max
imal values at confluency for HT-29 G+ cells and later (around
3 weeks) for G—cells. 24 h after their addition, both CZ and
TPA decreased calmodulin concentrations in G+ cells (respec
tively, 29.9 ±7.7 and 33.5 ±6.1 ng/mg protein) as compared
to controls (71.7 ±7.5 ng/mg protein). These effects were no
more obvious after 4 weeks. However, neither CZ nor TPA did
modify calmodulin concentrations of HT-29 G—cells up to 4
weeks of culture.
Since it had been previously demonstrated that in rats and
humans (14, 36, 43) the differentiation of intestinal epithelial
cells was accompanied by the segregation of calmodulin to their
apical side, we also studied by immunofluorescence on cryosections the effects of CZ and TPA on calmodulin distribution.
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Tl'A. C/.. AND COI.ONIC CANCER Cl-l.l. Dll-'H-RENTIATION
Fig. 6. Immunofluorcsccnt staining of HT-29 G+ (a, c, e) and G—(b, d, /)
cell sheets with monoclonal antibodies specific for sucrase-isomaltase after 4
weeks of culture in control medium (a, b) or in the presence of 0.5 MMCZ (c, d)
or 10 n.MTPA («•,/).
Note that CZ increased the apical labelling of HT-29 Gcells (d). It should be noted that some HT-29 G+ cells (o, c, e) show some
aspecific autofluorescence which is easily distinguished from the greenish specific
immunoreactions by its yellowish color. Bar, 50 urn; arrows point to the apical
staining.
Fig. 9a demonstrates that in undifferentiated HT-29 G+ cells
up to 4 weeks of culture, calmodulin staining was weak and
lined the periphery of individual cells. However in HT-29 G—
cells, in addition to the peripheral staining of the cells, calmod
ulin fluorescence had segregated to the apical side of the differ
entiated cells (Fig. 90). Neither CZ nor TPA treatments led to
modifications in the pericellular calmodulin immunofluorescence pattern of HT-29 G+ cells (Fig. 9, c and e). However the
apical staining of HT-29 G—cells was increased by CZ (Fig.
9d) whereas it was unmodified by TPA (Fig. 9/).
Calmodulin-binding proteins were also investigated after CZ
and TPA treatment. Both HT-29 G+ and G- cells contain
three major calmodulin binding proteins with apparent molec
ular weights of 240,000, 145,000 and 135,000 which have been
previously characterized as fodrin, caldesmon, and an immunoreactive form of the M, 110,000 protein, respectively (13, 14,
44). Neither CZ nor TPA appeared to alter this pattern of
calmodulin-binding proteins in G+ as in G—cells (data not
shown).
DISCUSSION
The present study examined possible roles for calmodulin
and protein kinase C in the differentiation of HT-29 colonie
adenocarcinoma cells by using the calmodulin antagonist calmidazolium and the protein kinase C activator TPA. The HT29 cell line provides a good system for investigating the differ
entiation processes since these cells are able to undergo differ
entiation and to express structural and functional features of
differentiated enterocytes when grown in glucose-free medium
Fig. 7. Immunofluorescent staining of HT-29 Cj-t-(a, r, e, g) and G- (b, d,f,
h) cells with the monoclonal antibodies Caco 5/50. a, ft, control cells after 10
days of culture: r, d. control cells after 30 days of culture; e, f, cells cultured for 4
weeks in the presence of 0.5 n\\ CZ; g, h, cells cultured for 4 weeks in the presence
of 10 n\i TPA. Note in TPA-treated HT-29 G+ cells (#) the appearance of a
positive labelling in intercellular cysts, and in CZ-treated G—cells (/) the
presence of a more regular apical staining as compared to controls (d). It must
be stressed that the apparent staining in c is aspecific. Bar, 50 ,.m.
(30). The message conveyed by the present report is that CZ
and TPA act differentially on HT-29 cells depending on the
differentiation potential of these cells that is associated with
particular culture conditions: CZ favors the enterocytic differ
entiation pathway of HT-29 G- cells cultured in the absence
of sugar whereas TPA triggers a limited differentiation of HT29 G+ cells which in the standard culture conditions (presence
of 25 HIMglucose) remain undifferentiated.
The action of CZ on HT-29 G—cell differentiation was
visualized by increases (a) in the density of microvilli at the cell
surface with well-organized cytoskeletal rootlets, (b) in the
apical Caco 5/50 antigen and sucrase-isomaltase expression,
and (c) in calmodulin apical segregation. It must however be
emphasized that the differentiation degree of CZ-treated HT29 G—cells remains far lower than that described in the
spontaneously differentiating Caco-2 cells (32). The effects of
CZ on HT-29 G—cells corroborate other data which demon
strated that calmodulin antagonists were able to augment dif
ferentiation processes of HL-60 cells (27).
The action of TPA on exponentially growing HT-29 G+ cells
resulted in early morphological and cytoskeletal alterations
6179
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TRA. CZ. AND COLONIC CANCER CELL DIFFERENTIATION
300
I
200
o.
o>
E
•o 100
O
"5
o
10
days
20
30
in culture
Fig. 8. Growth-related calmodulin concentrations (ng/mg protein) in G+
(O - -O) and C- (•--•)HT-29 cells. At the appropriate time points, calmodulin
content was determined by radioimmunoassay as described in "Materials and
Methods." The values presented herein are the mean ±SD of two dishes assayed
in triplicate.
Fig. 9. Immunofluorescent staining of G+ (a, c. e) and G—(b, d, f) HT-29
cells with calmodulin antibodies after 4 weeks of culture in the presence of 0.05'r
ethanol (a. h). 0.5 JIMCZ (c, </)or 10 n\i TPA (e.f). Bar. 50 t¡m.
[disappearance
of microfilament
stress fibers and re
arrangement of «-actinin and vinculin as already described in
other cell culture systems (45, 46)] followed after 4 weeks by
ultrastructural changes of the cells. The most obvious finding
was the appearance of organized polygonal cells with dense and
regular microvilli at their apical surface. These phenotypic
characteristics were accompanied by the appearance of the Caco
5/50 antigen in the intercellular cysts, the apex of the cells
facing the medium being always devoid of staining. It must be
stressed that other known differentiation criteria of HT-29 cells
were not observed: the cell layer remained pluristratified and
did not display any sucrase-isomaltase either at the apex or in
the intercellular cysts. Furthermore, calmodulin did not segre
gate to the apex of the cells. Thus, these observations suggest
that TPA triggers only a limited expression of differentiation
characteristics in G+.cells. Intercellular cysts within the strati
fied cell layers correspond to the organization of a small number
of cells surrounding a lumen which displays apical membrane
features. It has been proposed that these formations are in
volved in the establishment of cell polarity (47, 48). The obser
vation that Caco 5/50 antigen is found in TPA-treated HT-29
G+ cells exclusively within the cysts, while other brush border
antigens such as sucrase-isomaltase are not expressed, empha
sizes the interest of this Caco 5/50 antigen as an early marker
of intestinal cell differentiation. It must be stressed that induc
tion of HT-29 G+ cell differentiation has already been obtained
with other components such as sodium butyrate (49). However,
while this latter agent triggered the emergence of permanently
differentiated cell clones, TPA effects remained reversible. The
effect of TPA on the differentiation of HT-29 intestinal cancer
cells is interesting since up to now a TPA-induced differentia
tion was described only in leukemia cells (23-26).
In conclusion, both CZ and TPA act as differentiating agents.
However, their mechanism of action may be different. Although
CZ, like all calmodulin antagonists, may have noncalmodulinrelated effects, our results are consistent with the hypothesis
that inhibition of a calmodulin-mediated
function (without
alterations in calmodulin concentrations nor in the nature of
calmodulin-binding proteins) may augment the differentiation
potential of HT-29 G- cells.
The early morphological and cytoskeletal alterations ob
served in HT-29 G+ cells after TPA treatment do not seem to
be related to the differentiating action of TPA since this phorbol
ester provoked the same rapid changes in G—cells, whose
differentiation was unaffected. Furthermore, the observation
that CZ did not induce any cytoskeletal response before increas
ing the maturation of HT-29 G—cells strengthens the hypoth
esis according to which these early alterations are not involved
in the differentiation processes. The "differentiating" action of
TPA on HT-29 G+ cells was preceded by a reduction of their
growth rate. However, the fact that CZ acted similarly without
modifying the ultrastructural characteristics of these cells sug
gests that their differentiation is not linked to a lowering of the
growth rate. The same conclusion could be afforded for the
rapid drop in calmodulin levels which rather appears to be
related to the antiproliferative effect of TPA as well as of CZ.
Recently, phosphorylations have been considered as possible
regulatory events of differentiation (50-52). According to pre
liminary results, HT-29 G+ and G—cells cells exhibited pro
teins whose phosphorylation intensity was increased early after
TPA and CZ treatment, respectively.4 Although the nature of
these phosphorylable substrates remains to be determined, these
observations suggest that phosphorylation processes may be
involved in the mechanisms by which TPA and CZ modulate
HT-29 cell differentiation. The prevailing current hypothesis is
that these agents exert their biological effects by inducing an
altered program of gene expression, a process that involves
protein kinase C, as recently demonstrated for TPA (53). In
" Unpublished
data.
6180
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TRA. C/.. AND COLONIC CANCER CELL DIFFERENTIATION
conclusion, this study of HT-29 G+ and G—cell differentiation
offers the opportunity to investigate, at the molecular level,
which pathway is responsible for transducing the signals gen
erated by TPA and/or CZ from the plasma membrane to the
transcriptional machinery. Thus, the human colon cancer cell
line HT-29 provides a useful system to investigate whether such
differentiating agents could be envisaged in colon cancer chemo
therapy strategies.
ACKNOWLEDGMENTS
23.
24.
25.
We wish to thank C. Lang (preparation of a master degree) for help
with the setting up of some techniques. We also thank Dr. J. De Mey
for the generous gift of calmodulin antibodies. Dr. M. P. Walsh for
caldesmon antibodies, and Dr. H. P. Mauri for sucrase antibodies. We
are very grateful to Dr. A. Quaroni for kindly providing Caco 5/50
antibodies and for his interest in our work. We are indebted to C.
Arnold, D. Daviaud, and C. Leberquier for their skillful technical
assistance and to C. Haffen for photography.
26.
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c .. ..„ 7-Q 71Q „„_
'
Le"' '*y: '•"*-'•"•
1V8/-
6182
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Modulation of HT-29 Human Colonic Cancer Cell Differentiation
with Calmidazolium and 12- O-Tetradecanoylphorbol-13-acetate
Cécile Rochette-Egly, Michèle Kedinger and Katy Haffen
Cancer Res 1988;48:6173-6182.
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