Section of Physiology, Cornell University, Ithaca, New York 14853
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Growth arrest and cell differentiation are generally considered temporally and functionally linked phenomena in small intestinal crypt cells and colon tumor cell lines (Caco-2, HT-29). We have derived a Caco-2 subclone (NGI3) that deviates from such a paradigm. In striking contrast with the parental cells, proliferative and subconfluent NGI3 cells were found to express sucrase-isomaltase (SI) mRNA and to synthesize relatively high levels of SI, dipeptidyl peptidase IV, and aminopeptidase N (APN). In postconfluent cells, little difference was seen in SI mRNA levels between Caco-2 and NGI3 cells, but the latter still expressed much higher levels of SI that could be attributed to higher rates of translation. APN expression was also greatly enhanced in NGI3 cells. To determine whether high levels of brush-border enzymes correlated with expression of cell-cycle regulatory proteins, we investigated their relative cellular levels in growing and growth-arrested cells. The results showed that the cyclin-dependent kinase inhibitors (p21 and p27) and D-type cyclins (D1 and D3) were all induced in postconfluent cells, but NGI3 cells expressed much higher levels of p21. This study demonstrated that cell growth and expression of differentiated traits are not mutually exclusive in intestinal epithelial cells and provided evidence indicating that posttranscriptional events play an important role in regulation of SI expression.
cyclins; cyclin-dependent kinases; cyclin-dependent kinase inhibitors; brush-border enzymes
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
COLON TUMOR CELL LINES, in particular Caco-2 (10, 23) and HT-29 (36), have become widely used in in vitro models for studying intestinal enterocyte functions because of their ability to express relatively high levels of digestive brush-border enzymes (12, 23) and to display other structural and functional characteristics of absorptive villus cells (23). These features probably represent reexpression of an oncofetal phenotype brought about by the oncogenic process (35), inasmuch as the human colon, during early and midgestation, resembles the adult small intestine in its structure and cellular organization and expresses a set of digestive enzymes quite similar to that of confluent Caco-2 cells (12). Morphologically, differentiated Caco-2 cells are polarized columnar cells displaying microvilli and tight junctions at their apical membrane (23). These characteristics have been used advantageously to study general features of processing and transporting membrane glycoproteins in polarized epithelial cells (2, 12, 18) and regulation of their expression in intestinal cells by hormones (13, 14) and growth factors (8).
The rationale behind the use of Caco-2 cells as a model for intestinal cell differentiation is that their loss of proliferative activity on reaching confluence and the subsequent appearance of sucrase-isomaltase (SI) and other brush-border enzymes can be considered equivalent to the crypt-to-villus differentiation process occurring in vivo. A closer examination of these phenomena has, however, revealed significant inconsistencies and evidence for complex intracellular regulatory mechanisms. Despite repeated cloning, Caco-2 cells display a marked heterogeneity in morphology and function (3, 34). Their apical microvilli appear quite dense and well organized on some cells but more sparse and irregular on others (34). Even during the period of maximal sucrase activity (typically 2-3 wk after reaching confluence), cells expresssing high levels of SI or none at all can be observed close to each other (3). Although lactase-positive cells are relatively rare and appear scattered at random throughout the monolayer, in striking contrast, essentially all cells can be positive for dipeptidyl peptidase IV (DPPIV) and aminopeptidase N (APN). This heterogeneity is difficult to explain and complicates considerably the interpretation of studies aimed at the investigation of gene-regulatory mechanisms in intestinal cell growth and differentiation.
We herein describe a new subclone of Caco-2 cells, called NGI3, which may present significant advantages over the parental cell line for studying gene regulation during intestinal cell differentiation. The NGI3 cells are much more uniform in morphology and grow as tighter colonies. Among their remarkable characteristics, these cells express relatively high levels of SI, DPPIV, and APN even during their rapid growth phase at subconfluence. On reaching confluence, all cells express much higher levels of SI and APN than the original Caco-2 cells. A detailed study has revealed that the high level of SI expression in NGI3 cells is not simply due to a higher level of mRNA, but rather to increased SI protein synthesis and to more efficient intracellular processing. An analysis of cell cycle regulatory proteins showed that p21, p27, and cyclins D1 and D3 were all induced in both NGI3 and Caco-2 cells after reaching confluence, but the NGI3 cells expressed much higher levels of p21. Our results suggest that posttranslational mechanisms play important roles in the regulation of SI expression and that the NGI3 clone may represent an interesting new model system for future studies centered on regulation of gene expression and differentiation in intestinal cells.
![]() |
MATERIALS AND METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Isolation of NGI3 clone. The Caco-2/15 clone used in this study was originally selected as the one expressing the highest level of SI among 16 obtained by random cloning (3). The NGI3 clone was derived from Caco-2/15 cells transfected with the plasmid pCEP4 (Invitrogen, Carlsbad, CA) by the calcium-phosphate precipitation method (5) and selected in the presence of hygromycin B (380 µg/ml, Sigma) for 3 wk. After selection, NGI3 cells were cultured and passaged up to 25 times in the absence of hygromycin without apparent changes in their characteristics. Both the parental Caco-2/15 and NGI3 cells were routinely cultured in 100-mm-diameter dishes at 37°C in an atmosphere of 95% air-5% CO2 in DMEM containing 10% fetal bovine serum (FBS; vol/vol), 50 U/ml penicillin, 50 µg/ml streptomycin, 20 mM HEPES, and 4 mM glutamine. The cultures were replenished twice weekly with 10 ml of fresh medium and subcultured serially on reaching 70-80% confluence.
Cell proliferation assays. For growth curves, cells were seeded in 60-mm-diameter dishes at 1.2 × 105 cells/dish; cells in triplicate dishes were used for determination of cellular DNA (8) every 2 days. The number of cells per dish was calculated using the conversion factor of 19.33 µg DNA/106 cells (8). This indirect cell counting method was necessary with Caco-2 and NGI3 cells because of the difficulty in preparing single cell suspensions from high-density or confluent cultures, due to extensive cell-cell adhesion and clumping.
DNA synthesis was assessed using the 5-bromo-2'-deoxyuridine (BrdU) labeling and detection kit from Boehringer Mannheim (Indianapolis, IN) following the manufacturer's protocol. Briefly, cells were incubated with culture medium containing 10 µM BrdU for 30 min (for triple labeling for SI or ZO-1; see Fig. 2, E-J) or 24 h (for determination of labeling index; see Fig. 3) and then were washed with PBS, fixed with acidic ethanol, and incubated with anti-BrdU antibody in the presence of nucleases for DNA denaturation. BrdU incorporated into cellular DNA was visualized by fluorescence microscopy (see Indirect immunofluorescence staining).Scanning electron microscopy. NGI3 cells grown on coverslips at subconfluence were fixed with 2.5% glutaraldehyde in 0.1 M sodium cacodylate (pH 7.4) at room temperature for 30 min and 4°C for 60 min. The coverslips were then washed with 0.1 M sodium cacodylate (pH 7.4), incubated with 2% osmium tetroxide at room temperature for 1 h, and followed by washing with 0.1 M sodium cacodylate. The coverslips were gradually dehydrated with ethanol: 10% for 10 min, 30% for 10 min, 50% for 10 min, 70% overnight, 70% containing 2% uranyl acetate for 20 min, 90% for 10 min, and 100% for 20 min. Finally, the coverslips were critical point dried and then sputter coated and mounted on stubs. Cells were observed with a Hitachi S4500 field-emission scanning electron microscope.
Indirect immunofluorescence staining. Cells, grown in 35-mm dishes, either subconfluent (60-70% confluent) or 4 days after reaching confluence, were fixed with 3% paraformaldehyde and stained by the indirect immunofluorescence method as previously described (24). The primary antibody specific for human SI used in most experiments was the mouse monoclonal Caco-3/73 antibody produced in our laboratory (24); it was used in the form of ascites fluids diluted 1:50 with PBS (2, 24). In triple-labeling experiments with cells that had incorporated BrdU into their DNA (see Fig. 2, E and G), an affinity-purified rabbit-anti-SI antibody also prepared by us (27) was used at a dilution of 1:25. The rabbit-anti-ZO-1 antibody was obtained from Zymed Laboratories (South San Francisco, CA) and was used at a 1:100 dilution. The secondary antibodies were FITC- or rhodamine-conjugated goat anti-mouse or donkey-anti-rabbit IgG, obtained from Boehringer Mannheim and diluted 1:25 in PBS. DNA was stained with 4',6-diamidino-2-phenylindole (DAPI) at 2 µg/ml for 4 min. Negative controls included nonimmune mouse serum (diluted 1:50) in place of the primary antibody or PBS in place of the secondary antibody. The cells were mounted with glycerol-PBS (9:1) plus 2.5% 1,4-diazabicyclo[2.2.2]octane. Stained cells were examined with a Zeiss Axiovert 10 microscope equipped with epifluorescence optics.
Enzyme assays. Total cell membrane fractions were prepared as previously described (2) from cells grown in 100-mm-diameter dishes. Protein concentrations were determined by the method of Lowry. Sucrase and lactase activities were measured by the method of Messier and Dalqvist (19) with appropriate substrates (100 mM in all cases): sucrose for sucrase and lactose for lactase. APN and DPPIV activities were measured according to Roncari and Zuber (28) with L-leucine p-nitroanilide as APN substrate and glycyl-L-proline p-nitroanilide-p-tosylate as DPPIV substrate. Enzyme activities (units defined as the amount of enzyme transforming one micromole of substrate per minute at 37°C) were usually expressed as units per gram total protein.
Protein immunoprecipitation. Total cell membrane fractions were obtained, solubilized, and immunoprecipitated with antibodies bound to Affigel-10 beads (Bio-Rad) as previously described (24). All buffers used for cell homogenization, membrane purification, solubilization, and immunoprecipitation contained the same cocktail of protease inhibitors (8). Two monoclonal antibodies (MAb) used for SI immunoprecipitation were either Caco-3/73 (recognizing all forms of SI) or HSI-5 [specific for the high-mannose precursor (hmP2) and complex-glycosylated SI precursor (cP) forms (2, 24)]. The MAb DAO7/219 was used for DPPIV and the MAb HBB2/45 was used for APN immunoprecipitation. Antigens were eluted from the Affigel-10 beads and separated by 7.5% SDS-PAGE under reducing conditions (50 mM dithiothreitol) (8).
Immunoblotting analysis. For SI analysis, proteins separated on SDS-PAGE gels were electrophoretically transferred to nitrocellulose membranes. The membranes were blocked with 3% BSA in PBS at room temperature overnight and then incubated for 2 h at room temperature with Caco-3/73 (ascites fluids, diluted 500-fold in PBS containing 0.2% BSA). After being washed, the membranes were incubated with alkaline phosphatase-conjugated goat anti-mouse IgG (H + L), diluted 1:7,500 in PBS containing 0.2% BSA, further washed, and incubated with freshly prepared nitro blue tetrazolium-5-bromo-4-chloro-3-indolylphosphate p-toluidine salt substrate for alkaline phosphatase detection (Promega, Madison, WI). Finally, the blots were rinsed with water, photographed, and air dried.
For Western blot analysis of cell cycle regulatory proteins, total cell lysates solubilized in protein sample buffer were subjected to SDS-PAGE and immunoblotting. After electrophoresis, proteins were electrically transferred onto nitrocellulose membranes (high-bond nitrocellulose, Amersham Life Science) using a transblot system (Bio-Rad) at 100 V, 5°C for 90 min. The membranes were blocked at 4°C overnight in blocking buffer containing PBS, 0.1% Tween 20, and 3% BSA, incubated with primary antibody [p21, p27, pRb, p53, cyclin-dependent kinase (Cdk) 2, Cdk4, Cdk6, cyclin D1, cyclin D3, all antibodies obtained from Santa Cruz Biotechnology, Santa Cruz, CA], diluted in blocking buffer at room temperature for 2 h, and washed three times in washing buffer containing PBS and 0.1% Tween 20. Appropriate secondary antibodies (1:3,000 dilution of either horseradish peroxidase-linked sheep anti-mouse immunoglobulin or donkey anti-rabbit immunoglobulin, Amersham) were incubated with membranes for 1 h at room temperature. Specific proteins were detected using an enhanced chemiluminescence system (ECL protocol, Amersham). When necessary, the band intensities were analyzed using an LKB Ultroscan densitometer (LKB, Bromma, Sweden). As required, the membranes were stripped, blocked, and reprobed with different antibodies (ECL protocol, Amersham).Pulse-chase and metabolic labeling. Methionine labeling experiments were performed with 1-wk postconfluent cells. Cells grown on Transwell filters (3.0 µm pore size, Costar, Cambridge, MA) were preincubated twice with methionine-free DMEM (GIBCO, Grand Island, NY) containing 4% dialyzed FBS, antibiotics, and glutamine for 20 min each time to deplete intracellular methionine pools. Cells were then incubated with 4 ml of fresh methionine-free medium containing 280 µCi L-[35S]methionine per Transwell filter as previously described (8). The incubation time was 6 h for continuous labeling and 30 min for pulse-chase labeling. In pulse-chase experiments, the cells, after incubation with the radioactive methionine, were rinsed twice with standard complete medium and further incubated with the complete medium supplemented with 5 mM unlabeled methionine for the desired chase time points. In both continuous and pulse-chase labeling experiments, the cells were finally rinsed with cold PBS three times and processed for the preparation of total cell membrane fractions. Membrane fractions were solubilized and immunoprecipitated with antibodies bound to Affigel-10 beads. Antigens were eluted from the antibody-conjugated beads and analyzed by SDS-PAGE under reducing conditions (50 mM dithiothreitol). SDS-PAGE and detection of labeled proteins by fluorography were performed as previously described (24) using 7.5% acrylamide gels. Gels impregnated with EN3HANCE (DuPont NEN) were dried and exposed to Kodak XAR-5 films for 1 to 30 days. Multiple exposures of the same gel for different lengths of time were obtained in all cases. The intensity of the bands on the fluorographs was quantified within the limits of linearity by using a laser densitometer (Ultroscan XL, Pharmacia LKB, Piscataway, NJ).
Analysis of SI mRNA by Northern blotting.
Total cellular RNA was extracted from the cells as described by
Chomczynski and Sacchi (7). The integrity of the RNA was verified by
ethidium bromide staining, and the RNA concentration was determined
spectrophotometrically. RNA was denatured with formaldehyde and
resolved on a 1.2% agarose gel, transferred to a nylon membrane
(Genescreen Plus from DuPont) by electroblotting, and cross-linked to
the membrane by ultraviolet irradiation. Prehybridization was performed
in a solution consisting of 5× Denhardt's solution [5× saline-sodium citrate (SSC; 750 mM NaCl, 75 mM sodium
citrate, pH 7.0), 0.1% sodium pyrophosphate, 8 mM EDTA, 0.5% SDS, and
100 µg/ml heat-denatured salmon sperm DNA at 65°C
overnight]. Hybridization was carried out in the above solution
with the addition of
[32P]dCTP-labeled and
heat-denatured full-length SI cDNA (see below) at 65°C for 24 h.
Membranes were washed finally at 60°C in 0.1× SSC and 1% SDS
and subjected to fluorography at 70°C.
SI cDNA probe.
A full-length human SI cDNA was cloned into pBluescript
SK phagemid (Stratagene)
using the Sma I and
Kpn I sites. Total cellular RNA was
isolated from the confluent Caco-2/15 cells according to Chomczynski
and Sacchi (7), and the mRNA was then purified using an oligo(dT)
cellulose column (GIBCO BRL) and transcribed into complementary DNA
(cDNA) with an avian myeloblastosis virus reverse transcriptase
(Stratagene). A cDNA library was finally constructed in the lambda
ZAPII vector (Stratagene) and screened with a partial human SI cDNA
probe, pSI2 (provided by Dr. Dallas Swallow, MRC Human Biochemical
Genetics Unit, University College, London, UK). Positive plaques were
picked, subcloned, and then in vivo excised to generate inserts into
pBluescript SK
. The size of the clone was determined,
and a full-length clone was selected. Its identity was confirmed by
transcription of mRNA using T3 RNA polymerase and cell-free translation
in a reticulocyte lysate. The radiolabeled probe was obtained by
labeling the insert with
[32P]dCTP using a
random primer labeling kit according to the manufacturer's instructions (GIBCO BRL).
RPA.
SI and DPPIV mRNAs were quantified by ribonuclease protection assay
(RPA) following the manufacturer's protocol (1). The human DPPIV
(CD26) cDNA was obtained from Drs. S. Zhang and B. U. Pauli (Department
of Molecular Medicine, College of Veterinary Medicine, Cornell
University, Ithaca, NY). Anti-sense riboprobes for human SI (285 bp,
EcoR
I-Hind III fragment covering bp
1809-2094 of the cDNA) and DPPIV (a 411 bp,
XbaI-NdeI
fragment covering bp 1964-2375 of the cDNA) and control probes
(actin, 175 bp; 18S, 80 bp; both supplied by Ambion) were prepared
using an in vitro RNA transcription kit (Stratagene) with the addition
of [32P]rUTP in the
reaction. All anti-sense riboprobes were purified by electrophoresis on
a 5% acrylamide-8 M urea gel. All probes had specific activities
greater than 9 × 109
cpm/µg RNA. In each reaction, 30 µg of total RNA were hybridized to
8 × 104 cpm SI probe, 1 × 105 cpm of DPPIV probe, 4 × 104 cpm actin, and 18S
probes at 42°C for 24 h. RNase digestion of the unhybridized,
single-strand RNA was performed at 37°C for 40 min after adding a
mixture of RNase A and T1 at concentrations of 0.5 and 20 units,
respectively. The protected fragments were precipitated with ethanol
and ammonium acetate, resuspended in the sample buffer, and separated
on a 5% acrylamide-8 M urea gel. The gels were directly exposed to
X-ray film at 70°C for periods ranging from a few hours to 3 days. The intensity of bands for each probe was quantified with a laser
densitometer (LKB).
Statistical analysis. ANOVA was used as a statistical test. Results are expressed as means ± SE and were considered significant at P < 0.001.
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Isolation and general characteristics of NGI3 cells.
NGI3 cells grew significantly slower than Caco-2 cells, with a
population doubling time of 2-3 days, and formed much more compact
colonies than the parental cells (compare
Fig. 1, A
and B). Small colonies often
displayed a pseudolumen at their center (see
inset in Fig.
1C). Independent of their location
within such colonies individual cells appeared tall, columnar, and
covered with a thick layer of microvilli (Fig.
1C), lacking the structural diversity observed in parental Caco-2 cells (3, 23, 34). The morphology
and organization of NGI3 cells did not change significantly on their
reaching confluence, with the exception that pseudolumens were usually
no longer observed.
|
|
Growth characteristics of NGI3 cells.
Considering the slower growth rate of NGI3 cells compared with
Caco-2/15, a considerable fraction of nonproliferative cells in the
former clone could have at least in part explained their higher level
of expression of differentiated traits. We have therefore compared the
growth curves of NGI3 and Caco-2/15 cells and determined the
proliferative fraction at different times after cell seeding (4-14
days). Cells subconfluent (4 days), in their most rapid phase of growth
(7 days), approaching confluency (10 days), and confluent (14 days)
were incubated with BrdU for 24 h and then double stained for BrdU and
total nuclei (DAPI). The duration of BrdU labeling was based on the
population doubling time of Caco-2/15 cells (23.6 h during their most
rapid stage of growth). From these data we have derived the labeling
index for the two cell lines at each culture time (Table
1). Parallel cultures were stained for SI
to confirm the presence of differentiated cells in NGI3 cultures at all
times examined (data not shown).
|
|
Expression and biosynthesis of brush-border enzymes.
We have previously obtained evidence indicating that proliferative
crypt cells in vivo, and some clones of Caco-2 cells, express conformationally immature forms of SI endowed with much-reduced enzymatic activity (2, 3). To determine whether this could explain the
presence of immunoreactive SI in proliferative NGI3 cells, we compared
the sucrase, DPPIV, and APN activities in NGI3 and Caco-2/15 cells
under different culture conditions: subconfluent (50-75%
confluent), just-confluent, or postconfluent for 3 to 9 days (when
maximal levels of sucrase activity are usually observed in Caco-2/15
cells). As illustrated in Fig. 4, sucrase
activities paralleled the immunofluorescence staining results. In
subconfluent, just-confluent, or 3-days postconfluent cultures, sucrase
could only be detected in NGI3 cells. With prolonged postconfluence (6 or 9 days), as expected, sucrase activity became measurable in
Caco-2/15 cells, but its specific activity was still ~7-10-fold lower than in NGI3 cells.
|
|
|
Analysis of SI mRNA expression by RPA.
To evaluate the relative importance of transcriptional efficiency in SI
expression by NGI3 and Caco-2/15 cells, their relative SI mRNA levels
were quantified by RPA. Actin and DPPIV mRNAs were also evaluated for
comparison. To normalize RNA sample loads, 18S ribosomal RNA was used
as a reference. Cells used in these studies were
subconfluent, just-confluent, or 3-9 days postconfluent. Preliminary Northern blot analysis showed that SI mRNA of the same size
was produced in these two cell lines (data not shown). The results of
these studies are shown in Fig. 7. In
subconfluent and just-confluent cultures, SI mRNA could only be
detected in NGI3 cells, where its level increased at most 60% with
confluence. In contrast, DPPIV mRNA was detected in both Caco-2/15 and
NGI3 cells under all culture conditions; its level increased with
prolonged confluence in both cell lines, and in confluent cells it was
~2.5-fold higher in NGI3 than in Caco-2/15. These data indicate that
transcription of the SI gene is constantly active in NGI3 cells and is
only modestly affected by cell growth or confluence state. Importantly, in postconfluent cultures, SI mRNA levels were not significantly different between NGI3 and Caco-2/15 cells.
|
Expression of cell-cycle regulatory proteins in NGI3 and Caco-2/15
cells.
Recent studies (9, 11) have suggested that cell-cycle regulatory
proteins, in particular the cyclin-dependent kinase inhibitor p21
(WAF1/Cip1), may play an important role in the differentiation of
Caco-2 as well as intestinal cells. To determine whether p21, its
related p27, and their associated cell-cycle proteins might be
responsible for the different behavior of the NGI3 and Caco-2/15 cells,
we evaluated the relative cellular levels of these proteins in
subconfluent, just-confluent, and 3-9 days postconfluent cultures. The results of these studies are summarized in Fig.
8. As previously reported, both cell lines
were found to lack significant levels of p53, whose gene is known to be
mutated in the parental cell line. Caco-2/15 cells expressed moderately
higher (1.5- to 3-fold) amounts of pRb, cyclin-dependent kinases Cdk2
and Cdk6, but neither of these proteins nor Cdk4 changed in cellular
concentration with growth arrest or later stages of confluence (Fig.
8). Both p21 and p27 were increased in concentration with confluence in
both cell lines. In the case of p21, this observation has already been reported for uncloned Caco-2 cells (9, 11). Interestingly, postconfluent NGI3 cells were found to express relatively higher levels
of p21 (Fig. 8). The two D-type cyclins, D1 and D3, were found to
increase markedly in expression in 9-days postconfluent cultures of
both NGI3 and Caco-2/15 cells.
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The NGI3 cells we have characterized in this study exhibit distinctive features that challenge the current model of intestinal cell differentiation based on Caco-2 and other colon tumor cell lines. Central to such models is the concept that cell proliferation and differentiation are mutually exclusive processes, and formation of a confluent monolayer in vitro represents a comparable physiological change to that which triggers terminal cell differentiation in the upper region of the crypts. However, this analogy overlooks the fact that under normal physiological conditions the epithelial cells lining crypts and villi alike form a tight monolayer, where occasional cell losses are rapidly repaired by migration of neighboring cells (restitution, Refs. 6, 17). Even when the epithelial lining is disrupted in a major way, for example by X-ray irradiation killing most crypt cells, or by temporary ischemia leading to loss of most villus cells, the fundamental features of the cell replication and differentiation process are not altered (14). In contrast to subconfluent Caco-2 cells, there is also evidence that undifferentiated proliferative crypt cells can express SI, although in an immature form, in addition to low levels of other brush-border enzymes such as APN (2). Whereas neither the parental Caco-2 cells nor NGI3 cells can be considered similar to stem cells, nor do they replicate in vitro key aspects of crypt cell dynamics, the greater homogeneity in NGI3 morphology, both at subconfluence (Fig. 1) and postconfluence (not shown), and in SI expression (Fig. 2) should greatly facilitate their use in a variety of applications. These include study of the molecular mechanisms regulating cell differentiation and gene expression in enterocytes and drug transport. The phenotypic heterogeneity of postconfluent Caco-2 cells has been stressed in many studies (3, 12) and has been interpreted as the in vitro equivalent of the "mosaic" or "patchy" pattern of differentiation observed in some regions of the intestinal tract in animals (31), in a subgroup of lactase-deficient human adults (30), and in malnourished children (22). The NGI3 cells may therefore recapitulate a distinct physiological state of human enterocytes. In this respect, it is noteworthy that Vachon and Beaulieu (34) have classified Caco-2 cells into three stages with respect to cell differentiation: 1) homogeneously undifferentiated, 2) heterogeneously polarized and differentiated, and 3) homogeneously polarized and differentiated. Based on such a classification, the clone NGI3 would belong to the third stage, displaying the highest levels of SI expression.
A molecular explanation or interpretation of such "stages" or patterns of enterocyte differentiation both in vivo and in vitro has implicated multiple levels of regulation of brush-border enzyme expression in intestinal cells, including transcriptional, translational, and posttranslational (16, 31, 32). Regulation of SI expression has been particularly well studied in both experimental animals and humans during pre- and postnatal development and in disease states. In different studies, multiple genetic, environmental, or physiological factors have been highlighted (14, 29). In most cases, a good correlation has been observed among cellular or tissue SI mRNA levels, sucrase-specific activities, and SI synthesis (32, 33), but important exceptions have also been described, such as in several cases of human SI deficiency (20). The presence of conformationally distinct forms of SI in small intestinal crypt cells (2) and in human colon (4) has been attributed to incomplete conformational maturation probably centered on the conversion between the hmP1 and hmP2 forms of this enzyme. The conversion normally takes place within 30 to 60 min from synthesis in the endoplasmic reticulum (2). Importantly, such a conversion was found to be inhibited by heat shock (26) and sensitive to exogenous levels of growth factors or hormones, such as epididymal growth factor (8). These findings indicate the importance of physiological modulation in SI expression.
A comparison of the SI biosynthetic ability between NGI3 and Caco-2/15 cells has provided further evidence for such a complex set of regulatory pathways and their importance in determining the differentiated phenotype of intestinal epithelial cells. Clearly, the ability of subconfluent NGI3 but not Caco-2/15 cells to express relatively high amounts of SI could be attributed primarily to transcriptional control, inasmuch as SI mRNA could be detected only in the former (Fig. 7). This control appeared to be specific for SI, inasmuch as DPPIV was expressed in both cell lines, subconfluent or confluent, although its level was higher in NGI3 and increased with differentiation (Figs. 4, 6, and 7). In postconfluent cultures, however, SI mRNA levels were not significantly different between the two lines (Fig. 7). Moreover, a three- to fivefold difference in steady-state protein levels (Fig. 5B) and biosynthetic rates (Figs. 5A and 6B) and a greater than 10-fold difference in sucrase-specific activities (Fig. 4) were observed between the two lines. These differences in SI protein level and its enzyme activity could not be attributed to a more efficient intracellular processing or conversion among different high-mannose precursors (Fig. 6, A-D) as observed in other Caco-2 clones (3), but they still imply the presence of enzymatically and probably conformationally different forms of SI in NGI3 and Caco-2/15 cells. Furthermore, these considerations do not exclude the possible contribution of differences in protein turnover rates after cell-surface expression of the enzymes, a likely explanation for the observation that the vastly different APN biosynthetic activity (Fig. 6, G and H) between postconfluent NGI3 and Caco-2/15 cells was not reflected in the corresponding steady-state enzyme levels (Fig. 4).
Although the considerations discussed point to different and complex levels of biochemical regulation specific for SI expression, it is important to note that all three brush-border enzymes (SI, DPPIV, and APN) were significantly elevated in NGI3 cells. These findings suggest that the process(es) that generated these changes impinged on regulatory factor(s) or mechanisms that are important in intestinal cell differentiation in general. In an attempt to identify potential candidates for such a function, we have been attracted to recent studies conducted both in vivo and in Caco-2 cells that have implicated an important role of a variety of cell-cycle regulatory proteins in intestinal cell differentiation. For example, the expression of the cyclin-dependent kinase inhibitor p21 was induced in newly nondividing cells in diverse epithelia, including the intestine, and also during differentiation of Caco-2 cells (11). Furthermore, it has also been demonstrated that protein levels of the E-type cyclin and of the cyclin-dependent kinases Cdk2 and Cdk4 declined in postconfluent Caco-2 cells (9). Therefore, we have compared expression of these proteins in subconfluent and postconfluent NGI3 and Caco-2/15 cells. Interestingly, p21 expression was very low in the parental Caco-2/15 cell line, albeit induced two- to threefold in postconfluent cells (Fig. 8). The observation that p21 levels were low but detectable in subconfluent NGI3 cells, and much more strongly induced starting around 6 days after confluence, would be consistent with a role for this protein in intestinal cell differentiation. The concomitant increase in differentiated cells of cyclins D1 and D3, normally associated with growth stimulation and progress through the G1 phase of the cell cycle, is surprising but has also been previously noted in parental Caco-2 cells (11). Interestingly, we have also found these cyclins (D1 and D3) to increase together with the cyclin-dependent kinase inhibitors p21 and p27 in a conditionally immortalized human intestinal cell line (tsFHI cells) (25) induced to differentiate at the nonpermissive temperature (unpublished observation). Thus an increase in some D-type cyclins appears to represent a widespread feature in differentiated intestinal epithelial cells. It is noteworthy that new roles have been recently attributed to cyclin D1, independent of its ability to regulate pRb phosphorylation. For example, cyclin D1 was found to potentiate transcription of estrogen-receptor regulated genes and to mediate this activation independent of complex formation with a Cdk partner (21). Similarly, cyclin D1, like B-Myb, was found to strongly activate the HSP70 promoter via the heat shock element, again independently of pRb phosphorylation (15). The observed increase in D1 cyclin in postconfluent Caco-2/15 and NGI3 cells may therefore underscore an important, although still unknown, role for this cyclin in intestinal cell differentiation. Although slight differences in relative levels of the other proteins investigated were observed between NGI3 and Caco-2/15 cells, no firm conclusions could be reached. Caco-2/15 cells consistently expressed higher levels of cyclin-dependent kinases (Cdk2 and Cdk6 in particular), but these proteins are unlikely to have contributed to the distinctive behavior of NGI3 cells and their greater degree of differentiation.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank Dr. Eileen C. A. Paul for cloning the full-length SI cDNA into pBS plasmid. We are very grateful to Daniel Quaroni for excellent computer graphic and photographic work.
![]() |
FOOTNOTES |
---|
This research was supported by the National Institute of Diabetes and Digestive and Kidney Diseases Grant DK-48331.
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Address for reprint requests and other correspondence: A. Quaroni, Section of Physiology, T8-008A VRT, Cornell Univ., Ithaca, NY 14853 (E-mail: aq10{at}cornell.edu).
Received 21 September 1998; accepted in final form 21 January 1999.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1.
Ambion.
RPAII: Ribonuclease Protection Assay Kit. Austin, TX: Ambion, 1994.
2.
Beaulieu, J. F.,
B. Nichols,
and
A. Quaroni.
Posttranslational regulation of sucrase-isomaltase expression in intestinal crypt and villus cells.
J. Biol. Chem.
264:
20000-20011,
1989
3.
Beaulieu, J. F.,
and
A. Quaroni.
Clonal analysis of sucrase-isomaltase expression in the human colon adenocarcinoma Caco-2 cells.
Biochem. J.
280:
599-608,
1991[Medline].
4.
Beaulieu, J. F.,
M. M. Weiser,
L. Herrera,
and
A. Quaroni.
Detection and characterization of sucrase-isomaltase in adult human colon and in colonic polyps.
Gastroenterology
98:
1467-1477,
1990[Medline].
5.
Chen, C.,
and
H. Okayama.
High-efficiency transformation of mammalian cells by plasmid DNA.
Mol. Cell. Biol.
7:
2745-2752,
1987[Medline].
6.
Cheng, C.,
and
C. P. LeBlond.
Origin, differentiation and renewal of the four main epithelial cell types in the mouse small intestine. I. Columnar cells.
Am. J. Anat.
141:
461-480,
1974[Medline].
7.
Chomczynski, P.,
and
N. Sacchi.
Single-step method of RNA isolation by acid quanidinium thiocyanate phenol-chloroform extraction.
Anal. Biochem.
162:
156-159,
1987[Medline].
8.
Cross, H. S.,
and
A. Quaroni.
Inhibition of sucrase-isomaltase expression by EGF in the human colon adenocarcinoma cells Caco-2.
Am. J. Physiol.
261 (Cell Physiol. 30):
C1173-C1183,
1991
9.
Evers, B. M.,
T. C. Ko,
J. Li,
and
E. A. Thompson.
Cell cycle proteins suppression and p21 induction in differentiating Caco-2 cells.
Am. J. Physiol.
271 (Gastrointest. Liver Physiol. 34):
G722-G727,
1996
10.
Fogh, J.,
J. M. Fogh,
and
T. Orfeo.
One hundred and twenty seven cultured human tumor cell lines producing tumors in nude mice.
J. Natl. Cancer Inst.
59:
221-226,
1977[Medline].
11.
Gartel, A. L.,
M. S. Serfas,
M. Gartel,
E. Goufman,
G. S. Wu,
W. S. El-Deiry,
and
A. L. Tyner.
P21 (WAF1/SCIP1) expression is induced in newly nondividing cells in diverse epithelia and during differentiation of the Caco-2 intestinal cell line.
Exp. Cell Res.
227:
171-181,
1996[Medline].
12.
Hauri, H. P.,
E. E. Sterchi,
D. Benise,
J. A. M. Fransen,
and
A. Marxer.
Expression and intracellular transport of microvillus membrane hydrolases in human intestinal epithelial cells.
J. Cell Biol.
101:
838-851,
1985[Abstract].
13.
Henning, S. J.
Ontogeny of enzymes in the small intestine.
Annu. Rev. Physiol.
47:
231-245,
1985[Medline].
14.
Henning, S. J.
Functional development of the gastrointestinal tract.
In: Physiology of the Gastrointestinal Tract (3rd ed.), edited by L. R. Johnson. New York: Raven, 1994, p. 571-610.
15.
Kamano, H.,
and
K. H. Klempauer.
B-Myb and cyclin D1 mediate heat shock element dependent activation of the human HSP70 promoter.
Oncogene
14:
1223-1229,
1997[Medline].
16.
Keller, P. K.,
E. Zwicker,
N. Mantei,
and
G. Semenza.
The levels of lactase and of sucrase-isomaltase along the rabbit small intestine are regulated both at the mRNA level and post-translationally.
FEBS Lett.
313:
265-269,
1992[Medline].
17.
Madara, L. L.,
and
J. S. Trier.
Functional morphology of the mucosa of the small intestine.
In: Physiology of the Gastrointestinal Tract (2nd ed.), edited by L. R. Johnson. New York: Raven, 1987, p. 1209-1249.
18.
Matter, K.,
and
H. P. Hauri.
Intracellular transport and conformational maturation of intestinal brush border hydrolases.
Biochemistry
30:
1916-1923,
1991[Medline].
19.
Messier, M.,
and
A. Dalqvist.
A one-step ultramicro method for the assay of intestinal diassacharidases.
Anal. Biochem.
14:
376-392,
1966[Medline].
20.
Naim, H. Y.,
J. Roth,
E. E. Sterchi,
M. Lentze,
P. Milla,
J. Schmitz,
and
H. P. Hauri.
Sucrase-isomaltase deficiency in humans: different mutations disrupt intracellular transport, processing, and function of an intestinal brush border enzyme.
J. Clin. Invest.
82:
667-679,
1988[Medline].
21.
Neuman, E.,
M. H. Ladha,
N. Lin,
T. M. Upton,
S. J. Miller,
J. Direnzo,
R. G. Pestell,
P. W. Hinds,
S. F. Dowdy,
M. Brown,
and
M. E. Ewen.
Cyclin D1 stimulation of estrogen receptor transcriptional activity independent of cdk4.
Mol. Cell. Biol.
17:
5338-5347,
1997[Abstract].
22.
Nichols, B. L.,
M. A. Dudley,
V. N. Nichols,
M. Putman,
S. E. Avery,
J. K. Fraley,
A. Quaroni,
M. Shiner,
and
F. Carrazza.
Effects of malnutrition on expression and activity of lactase in children.
Gastroenterology
112:
742-751,
1997[Medline].
23.
Pinto, M.,
S. Robine-Leon,
M. D. Appay,
M. Kedinger,
N. Triadou,
E. Dussaulx,
B. V. Lacroix,
P. Simon-Assmann,
K. Haffen,
J. Fogh,
and
A. Zweibaum.
Enterocyte-like differentiation and polarization of the human colon carcinoma cell line Caco-2 in culture.
Biol. Cell
47:
323-330,
1983.
24.
Quaroni, A.
Crypt cell antigen expression in human colon tumor cell lines: analysis with a panel of monoclonal antibodies to Caco-2 luminal membrane components.
J. Natl. Cancer Inst.
76:
571-585,
1986[Medline].
25.
Quaroni, A.,
and
J. F. Beaulieu.
Cell dynamics and differentiation of conditionally immortalized human intestinal epithelial cells.
Gastroenterology
113:
1198-1213,
1997[Medline].
26.
Quaroni, A.,
E. C. A. Paul,
and
B. Nichols.
Intracellular degradation and reduced cell-surface expression of sucrase-isomaltase in heat-shocked Caco-2 cells.
Biochem. J.
292:
725-734,
1993[Medline].
27.
Quaroni, A.,
J. Wands,
R. L. Trelstad,
and
K. J. Isselbacher.
Epithelioid cell cultures from rat small intestine. Characterization by morphologic and immunologic criteria.
J. Cell Biol.
80:
248-265,
1979[Abstract].
28.
Roncari, G.,
and
H. Zuber.
Thermophilic aminopeptidase from Bacillus Stearothermiphilus. I. Isolation, specificity, and general properties of the thermostable aminopeptidase.
Int. J. Protein Res.
1:
45-61,
1969[Medline].
29.
Semenza, G.
Anchoring and biosynthesis of stalked brush border membrane proteins: glycosidases and peptidases of enterocytes and renal tubuli.
Annu. Rev. Cell Biol.
2:
255-313,
1986.
30.
Semenza, G.,
and
S. Auricchio.
Small intestinal disaccharidases.
In: The Metabolic Basis of Inherited Disease (7th ed.), edited by C. R. Scriver,
A. L. Beaudet,
W. S. Sly,
and D. Valle. New York: McGraw-Hill, 1995, p. 4451-4480.
31.
Semenza, G.,
P. Keller,
E. Zwicker,
H. Wacker,
and
N. Mantei.
Lactase-phlorizin hydrolase and sucrase-isomaltase along the small intesine.
In: Common Food Intolerances. 2: Milk in Human Nutrition and Adult-type Hypolactasia, edited by S. Auricchio,
and G. Semenza. Basel: Karger, 1993, p. 66-75.
32.
Traber, P. G.
Regulation of sucrase-isomaltase gene expression along the crypt-villus axis of rat small intestine.
Biochem. Biophys. Res. Commun.
173:
765-773,
1990[Medline].
33.
Traber, P. G.,
G. D. Wu,
and
A. J. Markowitz.
Transcriptional regulation of the sucrase-isomaltase gene.
In: Mammalian Brush-Border Membrane Proteins. II. Symposium Konigswinter, edited by M. J. Lentze,
H. Y. Naim,
and R. J. Grand. New York: Thieme Medical, 1993, p. 56-64.
34.
Vachon, P. H.,
and
J. F. Beaulieu.
Transient mosaic patterns of morphological and functional differentiation in the Caco-2 cell line.
Gastroenterology
103:
414-423,
1992[Medline].
35.
Wiltz, O.,
C. J. O'Hara,
G. D. Steele, Jr.,
and
A. M. Mercurio.
Expression of enzymatically active sucrase-isomaltase is a ubiquitous property of colon adenocarcinomas.
Gastroenterology
100:
1266-1278,
1991[Medline].
36.
Zweibaum, A.,
N. Triadou,
M. Kedinger,
C. Augeron,
S. Robine-Leon,
M. Pinto,
M. Rousset,
and
K. Haffen.
Sucrase-isomaltase: a marker of foetal and malignant epithelial cells of the human colon.
Int. J. Cancer
32:
407-412,
1983[Medline].