Pancreatic trypsinogen I expression during cell growth and
differentiation of two human colon carcinoma cells
Françoise
Bernard-Perrone1,
Jacqueline
Carrere2,
Wanda
Renaud3,
Christine
Moriscot3,
Karine
Thoreux1,
Patrice
Bernard1,
Alain
Servin4,
Daniel
Balas1, and
Françoise
Senegas-Balas1
1 Groupe de Recherche sur la
Trophicité et le Vieillissement, Faculté de Médecine,
06107 Nice Cedex 2; 3 Groupe
de Recherche sur les Glandes Exocrines, 13385 Marseille Cedex;
2 Hopital Renée Sabran,
Giens, 83406 Hyères Cedex; and
4 Contrat Jeune Formation,
Institut National de la Santé et de la Recherche Médicale
94-07, Unité de Formation et de Recherche-Sciences-Pharmacie,
Paris XI, 92296 Chatenay-Malabry Cedex, France
 |
ABSTRACT |
Pancreatic trypsin has been found to induce
tight junction or dome formation in some colon cancer cell lines
(HT-29, Caco-2), and a tumor-associated trypsinogen, trypsinogen type
II, has been isolated from another colon cancer cell line (COLO
205). We have tried to determine if trypsinogen is present
and how its expression varies during cell culture in HT-29 Glc+/
and Caco-2 cells, which exhibit enterocytic differentiation, and in
HT-29 Glc+ cells, which never differentiate. Trypsinogen
mRNA presence and expression were demonstrated in these cells by mRNA
hybridization, RT-PCR, cytoimmunofluorescence, Western immunoblot
analysis, and gel filtration. Trypsinogen was found to be trypsinogen
type I and was mainly in zymogen form in culture media. Differentiating
cells exhibited variations in trypsinogen I expression, but cells that
remained undifferentiated did not. In the differentiated cells, a high and transient peak in trypsinogen I expression was observed during the
first steps of differentiation.
HT-29; Caco-2
 |
INTRODUCTION |
TIGHT JUNCTIONS CAN BE assembled in a variety of
tissues and cultured cells by treatment of proteases (26, 27, 34, 44). In some colon cancer cell lines (parental HT-29 and Caco-2 cells), pancreatic trypsin, added to the culture cell medium, has been shown to
be a potent inducer of tight junction (1, 9, 16, 33, 40, 46) and dome
formation (HT-29 D4 cells) (11), which are characteristic of cell
differentiation. Moreover, a trypsinogen type II isoenzyme, the
tumor-associated trypsinogen II, has been isolated from the culture
medium of another colon cancer cell line, COLO 205 (20).
We have tried to determine if trypsinogen is present in other colon
cancer cell lines and if its expression is observed during a particular
stage of cell differentiation.
Caco-2 and HT-29 Glc+/
cells exhibit a pattern of enterocytic
differentiation (2, 3, 18, 24, 38). This differentiation has been
demonstrated by the appearance of tight junctions (Caco-2), well-organized brush borders, and intestinal hydrolases (Caco-2 and
HT-29 Glc+/
cells). Thus we considered tight junctions and dipeptidyl-peptidase IV (DPPIV)- and sucrase-specific activities to be
markers of differentiation. We examined Caco-2 and HT-29 Glc+/
cell lines for the presence of trypsinogen by cytoimmunofluorescence, Western blot analysis, and RT-PCR. Trypsinogen expression
variations were assessed in both cell homogenate and culture media by
mRNA hybridization and immunoenzymatic assays. We have also examined whether trypsinogen is present in zymogen form in the medium. All
results have been compared with those obtained in HT-29 Glc+ cells,
which remain undifferentiated.
 |
MATERIALS AND METHODS |
Cell lines and culture conditions.
Caco-2 and HT-29 cells derive from human colorectal adenocarcinoma (Dr.
J. Fogh, Sloan Kettering Memorial Center, Rye, NY). As is well known
(38), Caco-2 cells spontaneously differentiate after confluence. The
following two populations of HT-29 cells were used in this study.
1) Parental HT-29 cells (referred to as HT-29 Glc+ cells) are grown in a medium containing glucose and
remain essentially undifferentiated throughout the cell culture, with
only 2% of postconfluent cells undergoing enterocytic differentiation (23, 38). 2) Permanently
differentiated HT-29 cells (referred to as HT-29 Glc+/
cells)
were obtained from A. Zweibaum (Institut National de la Santé et
de la Recherche Médicale, Villejuif, France). HT-29 Glc+/
cells are a 100% enterocytic subpopulation (18) obtained by selection
through glucose deprivation that maintains its differentiation
characteristics when switched back to standard glucose-containing
medium (24). HT-29 Glc+/
cells also differentiate after
confluence. Phase-contrast microscopy showed a dense and well-organized
brush border, which carpets the apical surface, and with scanning
electron microscopy the presence of uniformly distributed tall and
regular microvilli was observed (3, 24). These cells expressed a low
amount of aminopeptidase N and no sucrase (24). Another
brush-border hydrolase, DPPIV, was found by cytoimmunologic technique
in these cells after 20 days of culture (2, 3, 24).
Cells were grown in DMEM (Sigma Chemical, St. Louis, MO) supplemented
with 10% (HT-29) or 20% (Caco-2) inactivated (30 min at 56°C) FCS
(GIBCO, Grand Island, NY) and 1% nonessential amino acids (Caco-2).
Cells were seeded at 8 × 104 cells/ml in 35-mm petri dishes
or on glass coverslips, which were placed in six-well tissue culture
plates (Corning Glass Works, Corning, NY). All the experiments and cell
maintenance were carried out at 37°C in 10%
CO2-90% air. The culture medium
was changed daily.
Growth curves were determined to define the exponential and stationary
phases of growth and confluence. Four wells were carefully washed with
PBS, and the attached cells were then removed with trypsin-EDTA
(0.15%/0.1%) and counted in the presence of trypan blue using a
Malassez counting cell.
Preparation of proteins and antibodies.
Human trypsin type I was prepared by autoactivation of partially
purified human pancreatic trypsinogen I. DP-trypsin I was obtained by
incubating purified trypsin I with diisopropyl fluorophosphate at pH
7.8 (24 h). Antiserum against DP-trypsin I was raised in rabbits
(kindly provided by O. Guy-Crotte, Groupe de Recherche sur les Glandes
Exocrines, Marseille, France).
An antiserum against human trypsinogen I was also prepared by injecting
the 23-kDa band isolated by SDS-gel electrophoresis of the partially
purified zymogen (kindly provided by O. Guy-Crotte). The antibodies
tested by Western blot analysis against human pancreatic juice revealed
both trypsinogens I and II (13).
To avoid false-positive reactions resulting from the rabbit blood
groups, we ascertained that antibodies against human erythrocytes were
absent from the two rabbit sera used (35).
Antiserum against human ZO-1, which is associated with the cytoplasmic
face of the tight junction, was supplied by Zymed Laboratories (San
Francisco, CA). Antiserum was raised in rabbits against a 69-kDa fusion
protein, corresponding to amino acids 463-1109 of human ZO-1 cDNA
(48). This antibody will react with ZO-1+ and ZO-1. No reactivity with
PSD-95 and p55 was observed, and the antibody reacted with human,
mouse, rat, guinea pig, and canine ZO-1 on Western blot. ZO-1 was
immunoprecipitated from Caco-2 cells.
Immunofluorescence staining.
Indirect immunofluorescence was performed on cell monolayers (Caco-2,
HT-29 Glc+, and HT-29 Glc+/
cells) grown on glass coverslips. Cells were fixed in paraformaldehyde (3.5%) for 2 (HT-29 cells) or 10 min (Caco-2 cells). They were permeabilized in saponin (0.1%) for 10 min and rinsed in 0.05 M phosphate buffer with 8.5 g/l of NaCl (pH 7.2)
(PBS).
Cells were incubated sequentially with 3% nonimmune goat serum,
primary rabbit antibodies against human trypsinogen I or ZO-1 (1:20 to
1:100 in PBS, 3% goat serum, 0.2% gelatin, for 12 h at 4°C) and
an FITC-conjugated goat anti-rabbit IgG (1:100, 1 h) (Dako).
HT-29 Glc+/
cells (day
3) were also observed with the use
of a confocal laser microscope (LSM 410; Carl Zeiss, Iena, Germany) after using primary rabbit antibodies against human trypsinogen I. An
argon ion laser adjusted at 488 nm was used for the analysis of
fluorescence. Optical sectioning was used to collect four horizontal views (5 µm).
The following controls were performed.
1) The primary antibodies were
replaced with PBS or preimmune rabbit serum.
2) FITC-conjugated goat anti-rabbit
IgG was replaced with PBS. 3)
Immunoadsorption tests were also performed by incubating 0.2 mg/ml of
human DP-trypsin I (at 4°C for 24 h) with the antiserum against
human trypsinogen I.
Preparation of cell homogenates and cell culture media.
The cell culture media (Caco-2, HT-29 Glc+, and HT-29 Glc+/
cells) were harvested with phenylmethylsulfonyl fluoride (PMSF; 1 mM),
lyophilized, and stored at
80°C. After the cells were washed
in PBS, cell homogenates were obtained by freezing the cells at
80°C for 20 min and then adding 2 ml of a solution composed of 10 mM Tris, pH 7.5, 1 mM EDTA, pH 7, 100 mM NaCl, and 1 mM PMSF at
37°C. After scraping, the cell homogenates were sonicated, lyophilized, and stored at
80°C. These cell homogenates were used for enzyme immunoassays of trypsinogen I as culture media on which
we also performed gel filtrations. Some supernatant fractions obtained
by centrifugation of these homogenates at 14,000 g for 10 min were harvested and stored
at
80°C for Western blot analysis.
For brush-border hydrolase assays, cell homogenates were obtained as
described above, but without PMSF.
Brush-border hydrolase assays.
DPPIV (EC 3.4.14.5) was assayed, according to the method of Nagatsu et
al. (30), in Caco-2, HT-29 Glc+, and HT-29 Glc+/
cells, using
Gly-Pro p-nitroanilide tosylate as
substrate (4 dishes/cell line/experimental time). The results of each
cell homogenate were expressed as nanomoles of substrate hydrolyzed per
minute at 37°C (milli-international units; mIU) and assessed with
respect to the protein levels in this cell homogenate (microassay
procedure; Bio-Rad Laboratories, Münschen, Germany) (3 assays/homogenate).
Sucrase (EC 3.2.1.48) activity was measured according to the method of
Dahlquist (10) (3 dishes/cell line/experimental time). The results of
each cell homogenate were expressed as nanomoles of substrate
hydrolyzed per minute at 37°C (mIU) per milligram of
protein.
Enzyme immunoassay of trypsinogen I.
Human trypsinogen I immunoreactivity (IRT) was measured by a
noncompetitive "sandwich" enzyme immunoassay using the antibody against human DP-trypsin I in cell culture media and in cell
homogenates (4 dishes/cell line/experimental time) (5).
It was verified that this immunoassay does not recognize purified human
pancreatic trypsinogen I or trypsins from other species (28).
The results in each cell homogenate (3 assays/homogenate) were
expressed per microgram of protein measured in this cell line homogenate. The IRT level in cell lysate was also
assessed with respect to the cell number for each petri dish. After the
IRT levels in each cell medium were measured, the quantity of IRT found
was divided by the amount of cell homogenate proteins assayed in a dish
(3 assays/medium). The amount of trypsinogen I released in the culture
medium by 1 µg of cell homogenate protein could thus be compared with
the quantity synthesized by 1 µg of cell homogenate protein.
Gel filtration experiments.
The molecular size distribution of IRT found in the three cell line
culture media (day
10) was determined by Sephadex G100 SF filtration in a 50 mM Tris · HCl buffer containing
200 mM NaCl (pH 7.6, 4°C). Column calibration was made with the
following reference proteins: RNase A [relative molecular weight
(Mr)
13,700],
-chymotrypsinogen A
(Mr 25,000),
ovalbumin (Mr
43,000), and BSA (Mr 67,000)
(Pharmacia Fine Chemicals). Loaded samples were 500 µl, and collected
fractions were 2 ml with an elution rate of 3 ml/h. Aliquots of 50 µl
of each fraction were assayed twice by immunoassay.
Activation by enterokinase.
The fraction corresponding to the highest IRT for HT-29 at
day
10 for Glc+/
cells was
filtrated on 0.22-µm filter units (Millex-GS; Millipore, Molssheim,
France). We exposed 200 µl of this IRT solution (6 ng of IRT) to
enterokinase (in excess amount) in Tris · HCl buffer
at pH 7.6 containing 10 mM CaCl2
at 4°C (2 h). This solution was loaded on Kohn gelatin (API system
20E; BioMerieux, Marcy l'Etoile, France) in which india ink was
incorporated (for 24 h at 37°C). The result was compared with those
obtained without enterokinase in the same conditions.
Western immunoblot analysis (Caco-2, HT-29 Glc+/
, and HT-29
Glc+ cell homogenate, day
5).
SDS-PAGE was performed according to the method of Laemmli (22) in the
presence of urea (8 M), on a 10% acrylamide concentration slab gel. As
positive control, immunoblots were also performed with nonactivated
human pancreatic juice (kindly provided by O. Guy-Crotte). After the
proteins were transferred to polyvinylidene difluoride membranes (250 mA; Millipore, Bradford, MA), the membranes were incubated overnight in
PBS containing 10% (wt/vol) dry skim milk. Immobilized proteins were
characterized by using the antibody against human trypsinogen I (1:250)
in PBS containing 5% (wt/vol) dry skim milk or whole preimmune rabbit
serum in the same conditions. The blots were incubated (1 h) with
horseradish peroxidase-labeled anti-rabbit antibody (1:5,000; Bio-Rad,
Hercules, CA). Then the membranes were washed three times
with PBS, 0.1% (vol/vol) Tween 20. The immunoreactive bands were
detected by enhanced chemiluminescence (Amersham, Buckinghamshire, UK).
The standard prestained proteins (Bio-Rad prestained SDS-PAGE
standards; Hercules, CA) used were as follows: phosphorylase b (105 kDa), BSA (82 kDa), ovalbumin (49 kDa), carbonic anhydrase (33.3 kDa),
soybean trypsin inhibitor (28.6 kDa), and lysozyme (19.4 kDa).
RT-PCR.
Purification of mRNA from total RNA (Caco-2, HT-29 Glc+, and HT-29
Glc+/
cell homogenates at day
8) was performed with an mRNA
separator kit (Clontech Laboratories, Palo Alto, CA). mRNA were reverse
transcribed into cDNA using
oligo(dT)15 (Boehringer Mannheim)
as primer, with or without SuperScript II RT (Life Technologies, Gaithersburg, MD) according to the manufacturer's recommendation, and
the preparation qualities were monitored by agarose gel
electrophoresis. PCR amplifications were accomplished
(Taq DNA polymerase; Life Technologies) with an MJ Research, PT-100 DNA thermal cycler as suggested by the manufacturer. The RNA were used for
future trypsinogen mRNA PCR analysis only if human actin amplification
was observed and none were used without the RT indicating the absence
of DNA contamination. The sense primer was
5'-GCCAGTGGCTGGAGGGGCGGTGGGC-3' (nt 390-414), and the
antisense primer was 5'-GGCAGCTGCTCTTGCTGCCCCC-3' (nt
36-57). Samples from 0.5 µg of cell mRNA and 1 µg of human total pancreatic RNA were initially denatured at 95°C for 1 min. Cycling parameters (35 cycles) were as follows: denaturation at 95°C for 1 min, annealing at 69°C for 1 min, and extension at 72°C for 1 min. A final extension at 72°C for 5 min was
performed. Portions of the amplified products were analyzed by agarose
gel electrophoresis (1.5%) in the presence of ethidium bromide (for 1 h at 100 V).
Quantitative analysis of trypsinogen mRNAs by dot-blot
hybridization.
After being washed twice in PBS, the Caco-2, HT-29 Glc+, and HT-29
Glc+/
cells were scraped and dispersed in 4 M guanidinium isothiocyanate (3 dishes/cell line/experimental time). Human pancreas (obtained from organ donor) and the HL-60 cell line were used as
positive and negative controls, respectively (29). Total RNAs were
purified according to the method of Chirgwin et al. (8). The
preparation qualities were monitored by agarose gel electrophoresis in
the presence of formaldehyde.
Human pancreatic trypsinogen cDNA was produced by RT-PCR from 1 µg of
human pancreatic RNA by using the sense and antisense primers
previously used for PCR (29). The trypsinogen insert length was 379 bp.
This cDNA can hybridize with trypsinogen I and II mRNAs. Its
specificity was checked by Northern blot on total RNA, purified from a
pancreas obtained from an organ donor. A single band of the expected
size (0.9 kb) was observed. This cDNA was labeled by
"nick-translation" with
[32P]dCTP.
Dot-blot assays were performed according to the techniques of White and
Bancroft (47) and Favarolo et al. (12). Sequential dilutions
(5-0.156 µg for the cells and 0.5-0.0156 µg for
pancreatic tissue) were loaded onto nitrocellulose membrane (3 assays).
mRNA transcript levels were quantified by dot-blot analysis and
densitometry after hybridization with the trypsinogen probe. The
slopes, calculated by least-square regression analysis, gave an
estimate of the relative amount of trypsinogen mRNA in total RNA. mRNA
concentrations were expressed in arbitrary units per microgram of total
RNA. The regression coefficient was always >0.98.
Statistical methods.
IRT and mRNA levels and hydrolase specific activities were expressed as
means ± SE. Statistical analysis was made with ANOVA followed by
Scheffe's multiple comparison test using Stat View software (Brain
Power, Calabasas, CA).
 |
RESULTS |
Cell growth.
Cell confluence was reached between
days
5 and
6 for HT-29 Glc+ and HT-29
Glc+/
cells and between days
6 and
7 for Caco-2 cells. A stationary phase
was noted at days
11,
12, and
13 for HT-29 Glc+, HT-29 Glc+/
,
and Caco-2 cells, respectively. Figure 1
depicts the growth profile of the cell cultures.

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Fig. 1.
Growth curve of HT-29 Glc+/ , HT-29 Glc+, and Caco-2 cells.
Values are means ± SE of determinations on 4 wells. Cell confluence
was reached at days 5-6
for HT-29 Glc+ and HT-29 Glc+/ cells and at
days 6-7
for Caco-2 cells. A stationary phase appeared at
days 11,
12, and
13 for HT-29 Glc+, HT-29 Glc+/ ,
and Caco-2 cells, respectively. , HT-29 Glc+. , HT-29
Glc+/ . , Caco-2.
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DPPIV and sucrase activities.
Sucrase and DPPIV activities were demonstrated after
day 5 in Caco-2 cells and then increased as expected (15, 38)
(Fig. 2). In HT-29 Glc+ cells, very weak
DPPIV specific activity was detected, as previously described (23). In
HT-29 Glc+/
cells, DPPIV activity was shown at
day
6, confirming that enterocytic differentiation occurs after confluence. This activity increased significantly in HT-29 Glc+/
cells from
day 6 to days
15-16; at this time, the confluent cells showed a well-organized brush border
(2, 24). Afterward, these activities moderately increased. Our assay
confirmed previous cytoimmunologic results with an anti-DPPIV monoclonal antibody applied to HT-29 Glc+/
cells after
day
20 (2, 24). DPPIV activity was lower
in HT-29 Glc+/
cells than in Caco-2 cells.

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Fig. 2.
Variation of specific activities of intestinal brush-border hydrolases
[dipeptidyl-peptidase IV (DPPIV) and sucrase] in HT-29
Glc+/ , HT-29 Glc+, and Caco-2 cell homogenates over the 21 days
of culture (4 dishes/cell line/experimental time). For each homogenate,
results were expressed as nmol of substrate hydrolyzed/min (mIU) and
assessed with respect to the protein level in this homogenate. Values
are means ± SE of 4 determinations. For HT-29 Glc+/ and
Caco-2 cells, these hydrolase activities increased after
day 5, demonstrating that these cells
differentiated. * P < 0.05, ** P < 0.01, significantly
different from day 3.
Top: , HT-29 Glc+; , HT-29
Glc+/ ; , Caco-2. Bottom:
sucrase activities were only measured in Caco-2 cells ( ).
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Cytoimmunofluorescence demonstration of ZO-1 in cell cultures
(day 10).
Gasketlike labeling, typical of tight junctions, is clearly evident on
HT-29 Glc+/
and Caco-2 cell monolayers with anti-ZO-1 antiserum,
demonstrating that these cells differentiated (Fig. 3).

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Fig. 3.
Demonstration of tight junctions in Caco-2 and HT-29 Glc+/ cell
lines at day 10 using immunocytofluorescence.
Paraformaldehyde-fixed and saponin-treated cell monolayers were exposed
to rabbit anti-human ZO-1 antiserum and FITC-conjugated goat
anti-rabbit IgG. Gasketlike staining of tight junctions is clearly
evident in Caco-2 cells (A) and
HT-29 Glc+/ cells (B).
A: bar, 7 µm.
B: bar, 13 µm.
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Cytoimmunofluorescence demonstration of trypsinogen in cell
cultures.
Caco-2, HT-29 Glc+, and HT-29 Glc+/
cell cytoplasms exhibited
diffuse staining when exposed to anti-human trypsinogen I antibody at
days
3, 7,
10, and
21 (Fig.
4). However, striking differences in
apparent trypsinogen expression were revealed when we observed the same
coverslip, because the labeling intensity varied from one group of
cells to another (Fig. 4). This is consistent with the fact
that Caco-2, HT-29 Glc+, and HT-29 Glc+/
cells are not clonal.
However, these variations could also reflect different levels of
trypsinogen synthesis or secretion.

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Fig. 4.
Demonstration of trypsinogen presence in Caco-2, HT-29 Glc+, and HT-29
Glc+/ cell lines using immunocytofluorescence.
Paraformaldehyde-fixed and saponin-treated cell monolayers were exposed
to rabbit anti-human trypsinogen I antiserum and FITC-conjugated goat
anti-rabbit IgG. Strong labeling could be seen in all cells on all
days. However, it should be noted that labeling intensity varied from 1 group of cells to another. A: HT-29
Glc+/ cells at day 3. B:
HT-29 Glc+/ cells at day 7. C:
HT-29 Glc+/ cells at day 21.
D: Caco-2 cells at
day 3. E:
HT-29 Glc+ cells at day 10.
F: HT-29 Glc+/ cells at
day 7; cell monolayers were treated with
antiserum previously adsorbed with DP-trypsin I and showed negative
results.
A-C
and E: bar, 6 µm.
D: bar, 13 µm.
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Front views of fixed HT-29 Glc+/
cell monolayers showed that
labeling could be observed in all the cell cytoplasms (Fig. 5).

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Fig. 5.
Front-facing views of immunolocalization of trypsinogen by
laser-confocal microscopy of HT-29 Glc+/ cell line
(day 3). Images shown are from the apical
part of the cells (A) to the basal
end of the cells (D); they are
separated by 5 µm. Labeling is uniformly distributed throughout the
cell cytoplasm area.
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All the control tests were negative. Cell monolayers treated with the
antitrypsinogen previously adsorbed with human DP-trypsin I showed
negative results, demonstrating that the labeling observed with the
anti-human trypsinogen I antibody was specific (Fig. 4F).
Demonstration of IRT.
IRT levels expressed in homogenate protein contents did not
statistically vary in HT-29 Glc+ cells during the cell culture (Fig.
6A) and
decreased with respect to the cell number (Fig. 6C). No variation
of IRT levels in cell homogenate could be seen between
days
3 and
5 for HT-29 Glc+/
cells (Table
1). In the HT-29 Glc+/
and Caco-2
cell homogenates, IRT levels were much higher at
day
10 than at
days
3 and
5 (HT-29 Glc+/
cells, +325%; Caco-2 cells, +147%) (Fig. 6,
A-D).
They strongly decreased from days
10 to
13 (HT-29 Glc+/
cells,
34%; Caco-2 cells,
94%) (Fig. 6,
A-D).
This decrease was particularly strong in Caco-2 cells in which the
levels fell to much lower than those observed at day
3. In HT-29 Glc+/
cells, IRT
levels at day
13 remained higher than those at
days
3 and
5; between
days
13 and
21, IRT levels did not vary (Fig. 6,
A-D).
The highest levels were observed for HT-29 Glc+/
cells.

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Fig. 6.
Kinetics of immunoreactive trypsinogen (IRT) in cell homogenates of
HT-29 Glc+ and HT-29 Glc+/ (A,
C) and Caco-2 cell lines
(B,
D) and in cell culture media of
HT-29 Glc+ and HT-29 Glc+/
(E) and Caco-2 cells
(F) (4 dishes/cell line/experimental
time). Results in each cell homogenate (3 assays/homogenate) were
expressed per µg of proteins measured in this cell line homogenate
(A,
B). IRT levels in cell homogenate
were also assessed with respect to the cell number for each petri dish
(C,
D). After measuring the IRT level in
each cell medium, the quantity of IRT found was divided by the amount
of cell homogenate proteins assayed in a dish (3 assays/medium)
(E,
F). Results were expressed per µg
of cell line homogenate proteins. Values are means ± SE of 4 determinations/dish. * P < 0.05, ** P < 0.01, *** P < 0.001 significantly
different from day 3:
P < 0.001, significantly
different from day 10.
P < 0.01, significantly
different from day 10. Solid bars,
day 3; open bars,
day 10; hatched bars,
day 13; crosshatched bars, day 21.
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IRT levels were also characterized in all cell culture media (Fig. 6,
E and
F). They varied according to the IRT
variation patterns in cell homogenates. The amount of released IRT was
much smaller than in cell homogenates. Moreover, the variations in cell
culture media were smaller than those observed in cell homogenates, especially in HT-29 Glc+/
cells
(day
10, +60%;
day
13, +25%; day
21, no increase in release).
Molecular size distribution of IRT in cell culture media.
Identical profiles of IRT molecular size distribution were observed for
the three cell line culture media. Typical profiles of HT-29
Glc+/
and Caco-2 cells are presented in Fig.
7. Fractions containing IRT were eluted at
the end of the second void volume with the 25-kDa proteins. This
elution profile is consistent with the presence of trypsinogen (24 kDa)
in the cell culture media. If active trypsin had been present, it would
have been mainly eluted in the first chromatographic fractions as
complexes associated with serum trypsin-
1-proteinase inhibitor.
Because cell culture media were treated in the same conditions for all
the cell lines, these gel filtrations confirmed that the quantity of
IRT released was smaller in Caco-2 and HT-29 Glc+ cells than in HT-29
Glc+/
cells.

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Fig. 7.
Identification of molecular forms of IRT by gel filtration on Sephadex
G 100SF. The representative profiles obtained with HT-29 Glc+/
(A) and Caco-2 cell
(B) culture media at
day 10 show that fractions containing IRT
eluted with 25-kDa proteins. Column, 1.5 × 90 cm; load, 500 µl
of supernatant; fraction volume, 2 ml. V0 is the void of
the column of gel filtration. It represents the volume of liquid
present in the column outside the gel. It is measured by the elution
volume of proteins of high molecular weight that do not enter the gel.
V1 is 2 × V0.
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When HT-29 Glc+/
gel filtration fractions were applied on
gelatin, no release of india ink was observed with the fraction of IRT
devoid of enterokinase. In contrast, india ink was spread out in the
fraction previously incubated with enterokinase (Fig. 8), demonstrating that gelatin was digested
and thus that the IRT present in HT-29 Glc+/
cell culture medium
was in the form of trypsinogen that was activated in trypsin by
enterokinase.

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Fig. 8.
Demonstration that IRT was trypsinogen. The fraction corresponding to
the highest IRT for HT-29 Glc+/ cells
(day 10) (6 ng of immunoreactive
trypsinogen) was exposed to enterokinase (in excess) at 4°C (2 h).
This solution was loaded on Kohn gelatin (24 h, 37°C) into which
india ink was incorporated (A). The
result was compared with results obtained without enterokinase in the
same conditions (B). No release of
india ink was observed without enterokinase. In contrast, india ink
spread into the solution with enterokinase
(A), demonstrating that gelatin was
digested and thus that the IRT present in HT-29 Glc+/ cell
culture medium was trypsinogen that was activated in trypsin by
enterokinase.
|
|
Western immunoblot of cell homogenates.
Western blot analysis demonstrated two bands of 25 and 23 kDa,
corresponding to trypsinogens I and II, respectively, in nonactivated human pancreatic juice. In the cell homogenates of HT-29
Glc+/
, HT-29 Glc+, and Caco-2 cells, we observed only a band
correponding to trypsinogen I that is consistent with the presence of
only trypsinogen I in these cells (Fig. 9).
These blots confirmed that the quantity of trypsinogen is greater in
HT-29 Glc+/
cells than in HT-29 Glc+ cells and greater even than
in Caco-2 cells. No band could be seen with preimmune serum.

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|
Fig. 9.
Representative Western immunoblots of trypsinogen isolated from Caco-2
(lane 1), HT-29 Glc+/
(lane 2), and HT-29 Glc+ cell
(lane 3) lysates (60, 20, and 40 µg of
proteins, respectively) (day 5), using rabbit anti-human
trypsinogen I antibody. As positive control, immunoblots were also
performed with nonactivated human pancreatic juice
(lane 4, 0.1 µg of proteins). As negative
controls, Western immunoblots were also performed simultaneously with
the same lysates of Caco-2 (lane 5), HT-29 Glc+/
(lane 6), and HT-29 Glc+ cells
(lane 7) and pancreas
(lane 8) by using preimmune rabbit serum
as primary antibody. The immunoreactive bands were detected by
chemiluminescence. Trypsinogens I and II were seen in human pancreatic
juice, whereas only trypsinogen I was demonstrated in cancer colon cell
lines.
|
|
RT-PCR.
PCR analysis of cDNA samples generated from the three cell lines
indicated the presence of a specific trypsinogen signal with the
expected length (379 bp) (Fig. 10,
lanes
C, E,
and G). The product observed was
identical to the one demonstrated in human pancreas used as a positive
control (Fig. 10, lane
I). Cross-tissue contamination could
be excluded because no RT-PCR product was detected when primers were
used alone in liquid controls (Fig. 10,
lanes
B, D,
F, H,
and J). Trypsinogen
mRNA presence was thus demonstrated in the three colon cancer cell
lines.

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Fig. 10.
Expression of trypsinogen mRNA detected by PCR in the 3 cell lines and
in the pancreas. mRNA samples (0.5 µg for the cells and 1 µg for
the pancreas) were subjected to RT-PCR as described in
MATERIALS AND METHODS. The expected
size of PCR product is 379 bp (lane C, Caco-2 cells;
lane E, HT-29 Glc+/ cells;
lane G, HT-29 Glc+ cells;
lane I, pancreas). PCR amplifications were
performed without cDNA sample (lanes B, D,
F, and
H), and the molecular size marker is
shown in lanes A
and K. No PCR product was loaded in
lane J.
|
|
Quantitative analysis of trypsinogen mRNAs by dot-blot
hybridization.
Dot-blot assays confirmed that trypsinogen mRNA was expressed by HT-29
Glc+/
, HT-29 Glc+, and Caco-2 cell lines as in human pancreatic
tissue (positive control) (Fig. 11). As
expected, there are ~100 times less trypsinogen mRNA in colon cancer
cell lines than in pancreatic tissue, and no hybridization was observed
for the HL-60 cell line (negative control).

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Fig. 11.
Representative dot-blot autoradiogram of trypsinogen mRNA in Caco-2,
HT-29 Glc+/ , and HT-29 Glc+ cell lines and in human pancreas
(positive control) and HL-60 cell line (negative control). Increasing
amounts of total RNA (0.156-5 µg for cell lines; 0.0156-0.5
µg for pancreatic tissue) were loaded into nitrocellulose filters and
hybridized with the radiolabeled specific trypsinogen cDNA probe.
Lane A: HT-29 Glc+/ cells at
day 3.
Lane B: HT-29 Glc+/ cells at
day 10.
Lane C: HT-29 Glc+/ cells at
day 13.
Lane D: HT-29 Glc+/ cells at
day 21.
Lane E: HT-29 Glc+ cells at
day 3.
Lane F: HT-29 Glc+ cells at
day 10.
Lane G: HT-29 Glc+ cells at
day 13.
Lane H: HT-29 Glc+ cells at
day 21.
Lane I: Caco-2 cells at
day 3.
Lane J: Caco-2 cells at
day 10.
Lane K: Caco-2 cells at
day 13.
Lane L: Caco-2 cells at
day 21.
Lane N: HL-60 cells.
Lane P: human pancreas. No total RNAs were
loaded on lanes M and
O.
|
|
The relative amount of trypsinogen mRNA in total RNA with respect to
the successive days of culture is presented in Fig.
12. The relative quantification of
trypsinogen mRNA shows that similar amounts of trypsinogen mRNA in the
three colon cancer cell lines are present at
day 3 and that the amount in Caco-2 cells was greater than in HT-29
Glc+/
cells at day
10. Trypsinogen mRNA expression did
not vary in HT-29 Glc+ cells. HT-29 Glc+/
and Caco-2 cell lines
exhibited a similar pattern of trypsinogen mRNA expression during cell
culture. A strong increase at day
10 (HT-29 Glc+/
cells, +107%;
Caco-2 cells, +347%) was followed at
day
13 by a strong decrease to the values
observed at day
3. Between
day
13 and day
21 these mRNA expressions did not
vary.

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|
Fig. 12.
Semiquantitative determination of trypsinogen mRNA levels by dot-blot
hybridization in HT-29 Glc+ cells
(A), HT-29 Glc+/ cells
(B), and Caco-2 cells
(C) at
days 3,
10,
13, and
21 (3 dishes/cell line/experimental
time). Increasing concentrations of total RNA were loaded into filters
and hybridized with the labeled specific probes. Spots were then
scanned and analyzed by least-square regression. mRNA concentrations
are expressed in arbitrary units/µg total RNA (AU). Means ± SE
from 3 experiments are shown.
*** P < 0.001, significantly
different from day 3.
P < 0.001, significantly
different from day 10. Solid bars,
day 3; open bars,
day 10; hatched bars,
day 13; crosshatched bars,
day 21.
|
|
 |
DISCUSSION |
The presence of trypsinogen in Caco-2, HT-29 Glc+/
, and HT-29
Glc+ cells was clearly shown over the 21 days of culture. Staining in
colon cancer cells was seen with the antibody against human trypsinogen
I, and trypsinogen mRNA expression was demonstrated until
day
21. The expression of trypsinogen in
these cells was also confirmed by RT-PCR techniques, Western immunoblot
analysis, gel filtration, and digestion of gelatin. Our immunoassay
could only recognize trypsinogen I (28). Moreover, we demonstrated through Western immunoblot analysis that these cells expressed only
trypsinogen I, whereas in another colon cancer cell line (COLO 205),
trypsinogen II is mainly expressed (20). In addition, we showed that
trypsinogen was released in culture medium.
IRT levels in cell homogenates and culture media and mRNA expression
presented similar patterns of variation over the period studied for
each cell line. However, IRT levels were strikingly higher in HT-29
Glc+/
cells than in Caco-2 cells, although the relative amounts
of trypsinogen mRNA were similar in the two cell lines at
day 3 and higher in Caco-2 cells at day
10. These results imply trypsinogen
mRNA posttranscriptional regulation in Caco-2 cells. Moreover, the
decrease in IRT level in cell homogenate after the 10th day was not as
rapid as the decrease in mRNA levels in HT-29 Glc+/
cells. This
slowdown can be accounted for by the fact that the amount of
trypsinogen released was lower than the amount synthesized and thus
trypsinogen is stored in the cells.
Nevertheless, the pattern of IRT variation and trypsinogen mRNA
expression was different in cells undergoing differentiation (HT-29
Glc+/
and Caco-2 cells) and in permanently undifferentiated cells (HT-29 Glc+ cells). No significant variation could be seen in
HT-29 Glc+ cells when a strong increase in IRT levels and trypsin mRNA
expression was observed in HT-29 Glc+/
and Caco-2 cells at
day
10.
The differentiation of these cancer cell lines appeared (after
day
5), when proliferation could still
be observed, and lasted until the beginning of the stationary phase of
growth, as is the case in the small intestine crypt proliferative
compartment, where differentiating cells are also known to divide (7).
In HT-29 Glc+/
and in Caco-2 cells, the increase in IRT levels
and trypsinogen mRNA expression might be associated with cellular
proliferation since growth could still be observed at
day
10. However, no variation in these
parameters could be observed in HT-29 Glc+/
cells between days
3 and
5, corresponding to the exponential
phase of growth (Tables 1 and 2). In
addition, IRT levels were higher during the stationary phase of growth
of HT-29 Glc+/
cells (days
12-20) than at days
3 or
5 (Tables 1 and 2). Moreover, the
greatest increase in cell proliferation was observed in HT-29 Glc+
cells, and no variation of their trypsinogen mRNA and protein
expression was demonstrated (Tables 1 and 2).
It is well known that the burst of differentiation in Caco-2 cells is
characterized first by the appearance of tight junctions inducing cell
polarity and afterward, by those of microvilli and brush-border
hydrolase activities (15, 38). The presence of well-formed brush
borders and DPPIV activities was also demonstrated in HT-29
Glc+/
cells after confluence (23). We confirmed these results by
the assay of sucrase and DPPIV and the presence of tight junctions,
demonstrated for the first time in HT-29 Glc+/
cells. The
increase in IRT levels and trypsinogen mRNA expression was observed at
day
10 in Caco-2 and HT-29 Glc+/
cells and might instead be associated with the first steps of cell
differentiation. This hypothesis also corresponds well with the fact
that no increase in IRT level and trypsinogen mRNA expression was seen
in nondifferentiating HT-29 Glc+ cells. These results also agree with
studies demonstrating that trypsin added in differentiating HT-29 D4
cell culture medium induced dome formation (11), which is considered to
be a functional differentiation (25). Moreover, these results were in
line with those demonstrating that trypsin added in nondifferentiating
HT-29 Glc+ cell culture medium induced the formation of tight junction strands (16, 40). In addition, serine protease inhibitors, leupeptin,
and antipain inhibited cesium sulfate-induced tight junction strand
formation in HT-29 Glc+ and Caco-2 cells, and the formation of tight
junction strands after the removal of leupeptin was not affected by
cycloheximide (1). It was suggested that tight junction fibrils are
assembled from protein precursors in the cell membrane and that limited
proteolyse by an endogenous cell membrane protease is required to
transform these proteins into functional elements of the tight
junction. They also suggested that this protease activity might be
nonfunctional in HT-29 Glc+ cells and might be substituted by trypsin
added to the medium. We demonstrated that trypsinogen was released in
HT-29 Glc+/
cell medium at a rate of 500 ng/ml at
day
10, which is a dose similar to the
quantity of trypsin added in all the experiments showing induction of
tight junctions. In addition, the staining with ZO-1 antiserum at
day
10 showed a large amount of tight
junctions. At this time, many differentiating cells were present (Table
1), requiring a large quantity of tight junction inducer, thus
concording with the peak trypsinogen level observed at
day
10.
Nevertheless, this hypothesis could be contested, because in our study
trypsinogen remained mainly in zymogen form in the culture medium. This
is the case with many proteases in cancer cells: trypsinogen I or II or
related protein in pancreatic cancer cell lines (Capan-1 and CFPAC-1
cells), in pancreatic ductal adenocarcinoma, in cyst fluid of mucinous
ovarian tumors, in COLO 205 colon cancer cell line, in gastric
adenocarcinoma, in K-562 erythroleukemia, and in HT-1080 fibrocarcinoma
cell lines (19, 21, 28, 32) and pepsinogen C in breast tumors (41). In
addition, procathepsin B was found in the culture medium of several
human colon carcinoma cells (Caco-2, HT-29, and COLO 205 cell lines)
(17). It should be noted that in tumor invasion, invading cells utilize
integral membrane proteases to cleave and activate secreted
metalloproteinases or serine proteases on the tumor cell surface (6,
42). We hypothesize that trypsinogen I was activated by a protease on the surface of HT-29 Glc+/
cells and that trypsin could induce tight
junction formation and cell differentiation. This hypothesis agrees
with the presence and release of trypsinogen by Paneth cells (43).
These cells are located in the bottom of small intestinal crypts, in
close contact with stem cells, and the presence of trypsinogen could
trigger the differentiation of the immature cells.
Another interesting finding in this study was that trypsinogen
expression was revealed in cancer colon cell lines, as in many benign
or malignant tumor cells, when it could not be demonstrated in the
corresponding healthy tissue (4, 28, 32). Moreover, trypsinogen levels
appeared to correlate with the degree of malignancy in ovarian tumors
(19). In addition, pancreatic-secretory trypsin inhibitor, also
referred to as tumor-associated trypsin inhibitor, was considered to be
a marker of hepatocellular, pancreatic, colorectal, and ovarian cancers
(14, 31, 36, 37, 45). It may be assumed that trypsinogen expression in
the human colon carcinoma cell lines that we studied could be
associated with benign or malignant processes in the colon.
HT-29 Glc+/
, HT-29 Glc+, and Caco-2 cells expressed trypsinogen
mRNA and synthesized trypsinogen I and released it mainly in
nonactivated form in the culture media over the 21 days studied. However, the trypsinogen expression profiles during the culture were
not the same in cells eliciting differentiation as in cells that remain
undifferentiated. In differentiated cells, the peak in trypsinogen I
expression was observed during the first stages of differentiation.
 |
ACKNOWLEDGEMENTS |
We thank O. Guy Crotte, C. Figarella, and M. Geribaldi for fruitful
discussion.
 |
FOOTNOTES |
Address for reprint requests: F. Senegas-Balas, Groupe de Recherche sur
la Trophicite et le Vieillissement, Faculté de Médecine, 16 av. Vallombrose, 06107 Nice Cedex 2, France.
Received 2 December 1996; accepted in final form 2 February 1998.
 |
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