ARTICLE |
Correspondence to: Françoise O. SenegasBalas, Laboratoire d’Histologie, Faculté de Médecine, av. de Valombrose, 06107 Nice Cedex 2, France.
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Summary |
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We localized REG protein in Paneth cells and nonmature columnar cells of the human small intestinal crypts and speculated that this protein was associated with growth and/or differentiation. The aim of this study was to determine whether REG protein is present in two human colon cancer cell lines that exhibit enterocytic differentiation after confluence and to investigate changes in the level of its expression during growth and differentiation. Results were compared to those obtained on cells that remain undifferentiated. Western immunoblotting and immunofluorescence demonstrated the presence of REG protein in the three cell lines. With the antisera against human REG protein, the staining was diffusely spread throughout the cytoplasm at Day 2, and after Days 34 it appeared to have migrated to cell boundaries. After confluence, we observed only a punctate staining array along cell boundaries, which disappeared at Day 15. REG mRNA expression was demonstrated by RTPCR and REG mRNA hybridization until Day 13, but not after, in the three cell types. REG protein may be involved in cellular junctions. Its presence appears to be associated with the cell growth period and the protein must be downregulated when growth is achieved and differentiation is induced. (J Histochem Cytochem 47:863870, 1999)
Key Words: REG protein, Caco-2, HT-29, growth, differentiation, human
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Introduction |
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In 90% DEPANCREATIZED, NICOTINAMIDE-TREATED RATS, regeneration of pancreatic islets was associated with expression of the REG gene (
In the small intestine, we demonstrated the presence of REG protein (
In an attempt to verify this hypothesis, we use some human colon carcinoma cell lines (Caco-2 and HT-29 Glc-/+ cell lines) in which the occurrence of enterocytic differentiation has been reported (
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Materials and Methods |
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Cell Lines and Culture Conditions
Caco-2 cells and HT-29 cells derive from human colorectal adenocarcinoma (Dr. J. Fogh; SloanKettering Memorial Center, Rye, NY) (
Cells were grown in Dulbecco's modified Eagle's minimal essential medium (DMEM) (Sigma Chemical; St Louis, MO) supplemented with 10% (HT-29) or 20% (Caco-2) inactivated (56C, 30 min) fetal calf serum (FCS) (Gibco, Grand Island, NY) and 1% nonessential amino acids (Caco-2). Cells were seeded at 8 x 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 the maintenance of cells were carried out at 37C in 10% CO290% air. The culture medium was changed daily. In our culture conditions (
Preparation of Cell Lysates
After washing the cells in PBS, cell lysates (Days 8 and 15) were recovered in PBS containing 2 mM EDTA. The cells were pelleted by centrifugation at 1000 rpm for 15 min and then resuspended in a lysis buffer [50 mM Tris-HCl (pH 7.5), 1% Triton X-100, 2 mM ethylene glycol bis (ß-aminoethyl ether)-N, N, N', N'-tetraacetic acid, 10 mM EDTA, 100 mM NaF, 1 mM Na4P2O7, 2 mM Na3VO4, 1 mM PMSF, 1 µg/ml aprotinin, 1µg/ml pepstatin A, and 1 µg/ml leupeptin]. The supernatant fractions obtained by centrifugation of the lysates at 14,000 x g for 10 min were harvested and stored at -80C for Western immunoblot analysis.
Preparation of Antibodies
Antisera against REG protein and its proteolysis product were prepared by injecting into rabbits the two protein bands (19 kD and 14 kD) obtained by SDS-gel electrophoresis of the fractions containing these proteins, co-purified by DEAETrisacryl chromatography. The antibodies tested by immunoelectrophoresis against human pancreatic juice gave one single precipitating line (
To avoid false-positive reactions resulting from the rabbit blood groups, we checked that antibodies against human erythrocytes from group A and O were absent from all rabbit sera (
Immunofluorescence Staining
Indirect immunofluorescence was performed on cell monolayers grown on glass coverslips. Cells were fixed in paraformaldehyde (3.5%) (Days 220) for 2 min for HT-29 cells and for 10 min for Caco-2 cells, and then were quenched in 50 mM ammonium chloride. These cells were permeabilized in various concentrations of saponin (0.1%, 0.05%, 0.075%) or Triton (0.05%, 0.1%) for 515 min. We obtained good staining and the best cell preservation for saponin (0.1%, 10 min). For Caco-2 at Day 15, we also used pepsin solution (0.1% in HCl 0.001 N, pH 2, 10 min) (Sigma) for retrieval of antigen masked by formalin fixation. Cells were then incubated sequentially with 3% nonimmune goat serum, rabbit IgG against human REG protein, or against its nonglycosylated proteolysis product (IgG against 14-kD protein) (1:201:100 in PBS/3% goat serum/0.2% gelatin; 12 hr, 4C) and an FITC-conjugated goat anti-rabbit IgG (1:100, 1 hr) (Dako; Carpinteria, CA).
Caco-2 cells (days 3 and 10) were also observed with a confocal laser microscope LSM 410 (Carl Zeiss; Jena, Germany) after using IgG against 14-kD protein. An argon ion laser adjusted to 488 nm was used for analysis of fluorescence. Optical sectioning was used to collect four en face images (8 µm) at Day 3. At Day 10, nuclei were labeled by propidium iodide and a series of en face views (1 µm) and lateral sections (xy axis) (8 µm) were performed.
Several controls were performed: (a) PBS or whole preimmune rabbit serum replacing primary antisera; (b) PBS replacing FITC-conjugated goat anti-rabbit IgG; and (c) immunoadsorption tests by incubating 0.2 mg/ml of the 14-kD proteolytic product of REG protein (4C, 24 hr) with the two primary antisera.
Immunocytochemical reactions were also carried out on living Caco-2, HT-29 Glc -/+, and HT-29 Glc+ cells at Days 3 and 7. They were incubated for 1 hr with IgG against 14-kD protein (1:100) in culture medium supplemented with 10% FCS (CM). After three rinses in culture medium without FCS, cells were incubated for 30 min with an FITC-conjugated goat anti-rabbit IgG (1:100) in CM. After rinses, cells were treated in methanol/acetic acid (95/5, v/v). Fixed cells were treated in the same fashion.
PeroxidaseAnti-peroxidase Staining
Multiple human duodenum and colon biopsy specimens were taken from five consenting subjects. Fragments of duodenum, jejunum, ileum, and colon from five Wistar rats and five Swiss albino mice were removed. These specimens were fixed in Baker's formalin at 4C, embedded in cytoparaffin, and treated with PAP techniques using IgG against 14-kD protein.
Western Immunoblotting
SDS-polyacrylamide gel (10%) electrophoresis (SDS-PAGE) was performed (
Reverse Transcription Polymerase Chain Reaction (RT-PCR)
After being washed twice in PBS, the Caco-2, HT-29 Glc+, and HT-29 Glc-/+ cells (Day 8) were scraped and dispersed in 4 M guanidium isothiocyanate (3 dishes/cell line). Total RNAs were purified (
Quantitative Analysis of REG mRNA Expression by Dot-Blot Hybridization
We used total RNA of Caco-2, HT-29 Glc+, and HT-29 Glc-/+ cells of human jejunum, pancreas (positive control), and spleen (negative control;
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Results |
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Cytoimmunofluorescence Demonstration of REG Protein in Cell Lines
The presence of REG protein in the three cancer cell lines was demonstrated because, with the two primary antisera, intense labeling was seen until Day 11. The staining was diffusely spread throughout the cytoplasm at Day 2 (Figure 1A, Figure 1G, Figure 1J, and Figure 1M). At Days 3 to 4, the labeling was always diffusely spread in some cells, whereas in other cells it already seemed to migrate to cellular junctions (Figure 1B). After confluence (Days 7 and 11), we observed a punctate staining array along cell boundaries (Figure 1C, Figure 1D, Figure 1H, Figure 1K, Figure 1O, and Figure 1Q). The labeling disappeared at Day 15 (Figure 1E) even after pepsin treatment (not shown). All the control tests were negative (Figure 1F, Figure 1I, Figure 1L, Figure 1N, Figure 1P, and Figure 1R).
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No staining was seen in living cells at Days 3 and 7 (not shown). On the contrary, fixed cells treated for 1 hr with the IgG against REG protein or against its proteolytic product exhibited similar but slight labeling compared to cells treated for 12 hr (not shown). This absence of staining on living cells demonstrated that the labeling seen on fixed cells is clearly localized inside these cells before confluence. After confluence, the staining observed on fixed cells could not correspond to labeling of the external part of the apical membrane because nonpermeabilized living cells were not labeled. In these living cells, we could not exclude that the antibodies might not have access to basolateral intercellular spaces where the REG protein could be present.
For Caco-2 cells at Day 3, we could see diffuse labeling from the basal part of the cells to the apical one (Figure 1S and Figure 1T) with confocal microscopy. We observed intense staining in the first section of some cells, which could correspond to endoplasmic reticulum (Figure 1S1 and 1S2) as we have already seen in human intestine (
At Day 10, lateral views clearly show that the staining is restricted to a small zone near or on the lateral plasma membrane (Figure 1U), located on a level with the upper part of the nucleus (Figure 1W); this labeling could be seen in seven successive en face sections of 1 µm (not shown).
PeroxidaseAnti-peroxidase Staining
Paneth and crypt cells of duodenal sections, but not human colon or rat and mouse small intestine and colon, were labeled (not shown). Therefore, the two antisera reacted specifically with the human small intestine, as with human pancreas (
Western Immunoblotting
A 19-kD band was observed with pancreatic juice and with the three cell line cell lysates at Day 8, which is consistent with the presence of REG protein (Figure 2). On the contrary, no band could be observed at Day 15, confirming immunofluorescence studies.
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REG mRNA Expression in Cell Lines at Day 8
PCR analysis of cDNA samples generated from the three cell lines indicated the presence of a specific signal with the expected length (321 BP) for REG mRNA (Figure 3, Lane c, Caco-2 cells; Lane e, HT-29 Glc-/+ cells; Lane g, HT-29 Glc+ cells) as in human pancreas (Figure 3, Lane i). Cross-tissue contamination could be excluded because no PCR product was detected when primers were used alone in liquid controls (Figure 3, Lanes b, d, f, and h). REG mRNA expression was demonstrated in the three colon cancer cell lines.
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REG mRNA Levels
Dot-blot assays enabled us to show that REG mRNA was present in human jejunum and in the three cell lines from Day 3 to Day 13 and was absent at Day 21 (Figure 4). As expected, it was present in human pancreatic tissue, and no hybridization for human spleen was detected (Figure 4). Similar amounts of mRNA were detected at Day 3 and Day 13 of culture in the three colon cancer cell lines (Figure 5). Similar amounts of mRNA coding for REG protein were found in HT-29 Glc-/+ and HT-29 Glc+. The levels of Caco-2 REG mRNA were significantly higher than those of HT-29 cell lines (+25% and +29% at Days 3 and 13 compared to HT-29 Glc-/+ levels; + 35% and + 29% at Days 3 and 13 compared to HT-29 Glc+ levels; p<0.05).
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Discussion |
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We confirmed our previous cytoimmunological results in human small intestine (
The presence of REG protein in colon cancer cell lines was in accordance with the hybridization of REG cDNA with RNA from some human colon and rectal tumors (
The staining localization varied during the culture, with similar patterns in the three colon cancer cell lines, differentiated or not. Labeling was observed until Day 11 and had disappeared at Day 15. REG protein was detected at Day 8 but not at Day 15 by Western blot. The REG mRNA expression was observed until Day 13. Days 1113 correspond to the first days of the stationary phase of growth. Therefore, expression of REG protein and REG mRNA was observed during the growth period of colon cancer cell lines and also during the first days of the stationary phase. This REG protein expression during the growth period agrees with the results observed in regenerating pancreatic islets, in pancreatic exocrine tissue, in stomach, and in cornea, where REG protein appears to be increased during the growth period (
In colon cancer cell lines that differentiate (Caco-2 and HT-29 Glc-/+), the beginning of differentiating processes was observed after Day 5 (
The localization of the staining varied during the culture of the three colon cancer cell lines. Labeling was observed diffusely in the cytoplasm during the first days of culture. After confluence, it was restricted to cell junction zones and presented a punctate staining array along cell boundaries. By confocal microscopy, this staining was confirmed to be near the cell boundaries and in a very specific zone on a level with the upper part of nucleus. These results suggest that REG protein is implicated in cell junctions. In this respect, it is interesting to note that REG protein was integrated into Group VII of the calcium-dependent lectin (C-type animal lectins) classification sharing a single carbohydrate recognition domain linked to an amino acid sequence corresponding to a signal peptide (
We clearly demonstrated the presence of REG protein in the three colon cancer cell lines used, where this protein seemed to be involved in cell junctions. REG expression was concomitant with the cell growth period. We can also hypothesize that REG protein will be downregulated after the induction of differentiation.
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Acknowledgments |
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We are indebted to Dr A. Servin (Laboratoire de Microbiologie; ChatenayMalabry, France) for the kind gift of the cell lines Caco-2 and HT-29, and to Dr Hirsh (Centre d'Immunologie de Luminy; Marseille, France) for GAPDH cDNA.
Received for publication December 28, 1998; accepted January 19, 1999.
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