Paneth Cell Markers Research Articles

Notch signaling modulates proliferation and differentiation of intestinal crypt base columnar stem cells

Notch signaling is known to regulate the proliferation and differentiation of intestinal stem and progenitor cells; however, direct cellular targets and specific functions of Notch signals had not been identified. We show here in mice that Notch directly targets the crypt base columnar (CBC) cell to maintain stem cell activity. Notch inhibition induced rapid CBC cell loss, with reduced proliferation, apoptotic cell death and reduced efficiency of organoid initiation. Furthermore, expression of the CBC stem cell-specific marker Olfm4 was directly dependent on Notch signaling, with transcription activated through RBP-Jκ binding sites in the promoter. Notch inhibition also led to precocious differentiation of epithelial progenitors into secretory cell types, including large numbers of cells that expressed both Paneth and goblet cell markers. Analysis of Notch function in Atoh1-deficient intestine demonstrated that the cellular changes were dependent on Atoh1, whereas Notch regulation of Olfm4 gene expression was Atoh1 independent. Our findings suggest that Notch targets distinct progenitor cell populations to maintain adult intestinal stem cells and to regulate cell fate choice to control epithelial cell homeostasis.

Paneth cell marker expression in intestinal villi and colon crypts characterizes dietary induced risk for mouse sporadic intestinal cancer

Nutritional and genetic risk factors for intestinal tumors are additive on mouse tumor phenotype, establishing that diet and genetic factors impact risk by distinct combinatorial mechanisms. In a mouse model of dietary-induced sporadic small and large intestinal cancer in WT mice in which tumor etiology, lag, incidence, and frequency reflect >90% of intestinal cancer in Western societies, dietary-induced risk altered gene expression profiles predominantly in villus cells of the histologically normal mucosa, in contrast to targeting of crypt cells by inheritance of an Apc(1638N) allele or homozygous inactivation of p21(Waf1/cip1), and profiles induced by each risk factor were distinct at the gene or functional group level. The dietary-induced changes in villus cells encompassed ectopic expression of Paneth cell markers (a lineage normally confined to the bottom of small intestinal crypts), elevated expression of the Wnt receptor Fzd5 and of EphB2 (genes necessary for Paneth cell differentiation and localization to the crypt bottom), and increased Wnt signaling in villus cells. Ectopic elevation of these markers was also present in the colon crypts, which are also sites of sporadic tumors in the nutritional model. Elevating dietary vitamin D(3) and calcium, which prevents tumor development, abrogated these changes in the villus and colon cells. Thus, common intestinal cancer driven by diet involves mechanisms of tumor development distinct from those mechanisms that cause tumors induced by the rare inheritance of a mutant adenomatous polyposis coli (Apc) allele. This is fundamental for understanding how common sporadic tumors arise and in evaluating relative risk in the population.

Fibroblast Growth Factor Receptor-3 (FGFR-3) Regulates Expression of Paneth Cell Lineage-specific Genes in Intestinal Epithelial Cells through both TCF4/β-Catenin-dependent and -independent Signaling Pathways

Fibroblast growth factor receptor-3 (FGFR-3) expression in the developing intestine is restricted to the undifferentiated epithelial cells within the lower portion of the crypt. We previously showed that mice lacking functional FGFR-3 have a significant decrease in the number of Paneth cells in the small intestine. Here, we used Caco2 cells to investigate whether FGFR-3 signaling can directly modulate expression of Paneth cell differentiation markers through its effects on TCF4/β-catenin or through other signaling pathways downstream of this receptor. Caco2 cells treated with FGFR-3 ligands or expressing FGFR-3(K650E), a constitutively active mutant, resulted in a significantly increased expression of genes characteristic of mature Paneth cells, including human α-defensins 5 and 6 (HD5 and HD6) and Paneth cell lysozyme, whereas enterocytic differentiation markers were reduced. Activation of FGFR-3 signaling sustained high levels of β-catenin mRNA expression, leading to increased TCF4/β-catenin-regulated transcriptional activity in Caco2 cells. Sustained activity of the TCF4/β-catenin pathway was required for the induction of Paneth cell markers. Activation of the MAPK pathway by FGFR-3 is also required for the induction of Paneth cell markers in addition to and independent of the effect of FGFR-3 on TCF4/β-catenin activity. These studies suggest that coordinate activation of multiple independent signaling pathways downstream of FGFR-3 is involved in regulation of Paneth cell differentiation.

Abstract 2292: Absent E2 liganded AhR inhibits tumor inhibition by ERα in Min mice

Abstract Previously, we showed that E2 and ERα prevent intestinal tumor formation in the Min mouse. Others showed that Aryl hydrocarbon Receptor (AhR) associates with ubiquitin ligase to down-regulate β-catenin and Wnt signaling. AhR activity also may affect ERα. AhR has endogenous, dietary, and xenobiotic ligands. We hypothesized that the tumor inhibiting function of ERα promotes Wnt signaling in vivo and is antagonized by liganded-AhR in the absence of E2. In groups of 10, ovariectomized Min (OvxMin) mice were treated w/wo AhR ligand 5, 6-benzoflavone (BF; 2,000 ppm) by diet for 17 weeks. Treated mice developed more tumors in the small intestine (54.7±11.1) than controls (38.3±4.5 P<0.0001). Protein expression was evaluated in non-tumor ileum of study mice by immunohistochemistry, immunoprecipitation (IP), and immunoblotting (IB). ERα and β-catenin nuclear expression in crypt base cells, total β-catenin and ERα protein amounts, and Wnt signaling-dependent Paneth cell markers (Sox9 and Lysozyme) were significantly reduced in BF-treated OvxMin compared to control mice. Reciprocal IP/IBs using total cell lysates from study mice and antibodies for β-catenin and ubiquitin (Ub) showed increased expression of ubiquitylated β-catenin in BF-treated OvxMin mice relative to controls. Wnt signaling-dependent cell proliferation (Ki67+ nuclei/crypt) was reduced in BF-treated OvxMin (11.4±2.5) relative to controls (16.2±3.0) and untreated intact Min (20.7±3.2) mice (P<0.01). AhR was expressed throughout the crypt-villus unit of OvxMin ileum; its activity was assessed by induction of Cyp1A1. A minimal amount of Cyp1A1, but elevated levels were found in untreated mucosa of intact Min vs. OvxMin mice w/wo BF, respectively. Lower expression of the AhR negative regulator, AhR Repressor, was present in ileum of OvxMin mice regardless of treatment relative to untreated intact Min mice. In study mice mucosa, other xenobiotic responsive regulators (Pxr, Nrf2, Car1/2) were minimally expressed, not affected by BF, hence not implicated in Cyp1A1 induction. In conclusion, these results indicate that E2 loss reduces AhR feedback inhibition and Wnt signaling in Min intestine. Since liganded-AhR promoted β-catenin turnover and reduced ERα expression absent E2, our data suggest that deficient Wnt signaling in the non-tumor mucosa of Min mice provides a selective pressure for Apc functional loss in intestinal stem cells and tumor initiation. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2292. doi:10.1158/1538-7445.AM2011-2292

Cdx2 Levels Modulate Intestinal Epithelium Maturity and Paneth Cell Development

Caudal-related homeobox protein 2 (Cdx2) is an intestine-specific transcription factor that is important for intestinal development and intestine-specific gene expression. Cdx2 regulates intestinal cell-cell adhesion, proliferation, and the transcriptional activities of Wnt and β-catenin in cell culture systems. We generated transgenic mice that overexpress Cdx2 in the small intestinal and colonic epithelium to investigate the role of Cdx2 in differentiation and function of the intestinal epithelium. We established 4 different lines of villin-Cdx2 transgenic mice. Intestines were collected from infant, 3-month old, and wild-type mice. Genes of interest and cell lineage markers were examined by polymerase chain reaction and immunohistochemistry. Villin-Cdx2 transgenic mice had complex phenotypes that were associated with transgene expression levels. The 2 lines that had the greatest levels of transgene expression had significant, preweaning failure to grow and death; these were the result of early epithelial maturation and alterations in nutrient digestion and absorption. Fat malabsorption was a prominent feature. Other effects associated with the transgene expression included loss of Paneth cell markers, increases in goblet cells, and migration of proliferating, EphB2-expressing cells to the crypt base. Loss of Paneth cell markers was associated with reduced nuclear localization of β-catenin but not homeotic posteriorization of the epithelium by Cdx2. Overexpression of Cdx2 in the small intestine is associated with reduced post-natal growth, early epithelial maturation, alterations in crypt base organization, and changes in Paneth and goblet cell lineages. Cdx2 is a critical regulator not only of intestine-specific genes, but also processes that determine epithelial maturity and function.

Abstract 4157: Genetic and dietary complementation in risk for mouse intestinal tumorigenesis

Abstract Dietary factors are significant in determining probability for intestinal tumor development even in the presence of strong genetic initiation. For example, a rodent Western-style diet (WD1), modeled to reproduce intake of nutrient risk factors in the human diet associated with sporadic colon cancer, potentiates the formation of intestinal tumors in Apc1638N/+ mice, even when the genetic initiation by Apc is augmented by targeted inactivation of the p21Waf1/cip1 locus. To investigate mechanisms of this synergistic effect, we profiled gene expression along the mouse crypt-villus axis (CVA) using cells isolated as a function of their position along this axis from WT, Apc1638N/+ and p21−/− mice fed control AIN76A diet, and WT mice fed the WD1. Modulation of sequence expression by the WD1 was enriched in the villus compartment, while changes induced by targeting either genetic locus (Apc1638N/+ or p21−/−) were largely in the crypt, where the overlap in altered gene sets was greatest for the genetic models. There was greater overlap among expression changes between Apc1638N/+ and p21−/− mice, than between these genetic mice models and mice fed the WD1, in both the villus and crypt. Paneth cell markers, whose deregulation has been reported in intestinal tumors, were up-regulated in the p21−/− mouse crypt and in WD1 mouse villus. Moreover, there was attenuated expression of secretory cell lineage markers (ie goblet and enteroendocrine cells), but enhanced expression of enterocyte markers, for both the genetic and dietary models. Genes that encode tricarboxylic acid (TCA) cycle enzymes were uniformly decreased in the crypt of both genetic models, with fewer changes in this pathway altered in the WD1 mouse. Gene ontology and biological pathway analysis also showed that Wnt pathway and c-myc target genes were overrepresented in the villus of the WD1 mouse. Supplementation of WD1 with calcium and vitamin D (WD2) reversed aberrantly up-regulated cyclin D1, cyclin D2, defensins and lysozymes in the WD1 villus back to control levels. In summary, complementary changes in cell reprogramming contributed by a western style diet and targeted inactivation of the p21 gene are important in the synergy of these factors in elevating probability of intestinal tumorigenesis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4157.

IL-9 Promotes IL-13-Dependent Paneth Cell Hyperplasia and Up-Regulation of Innate Immunity Mediators in Intestinal Mucosa

IL-9 contributes to lung inflammatory processes such as asthma, by promoting mast cell differentiation, B cell activation, eosinophilia, and mucus production by lung epithelial cells. The observation that IL-9 overexpressing mice show increased mast cell numbers in the intestinal mucosa suggests that this cytokine might also play a role in intestinal inflammation. In colons from IL-9 transgenic mice, the expression of Muc2, a major intestinal mucin gene, was up-regulated, together with that of CLCA3 chloride channel and resistin like alpha, which are goblet cell-associated genes. Additional IL-9 up-regulated genes were identified and included innate immunity genes such as angiogenin 4 and the PLA2g2a phospholipase A(2), which are typical Paneth cell markers. Histochemical staining of Paneth cells by phloxine/tartrazine showed that IL-9 induces Paneth cell hyperplasia in Lieberkühn glands of the small intestine, and in the colonic mucosa, where this cell type is normally absent. Expression of Paneth cell markers, including angiogenin 4, PLA2g2a, and cryptdins, was induced in the colon of wild-type mice after two to four daily administrations of IL-9. By crossing IL-9 transgenic mice with IL-13(-/-) mice, or by injecting IL-9 into IL-4R(-/-) mice, we showed that IL-13 was required for the up-regulation of these Paneth cell-specific genes by IL-9. Taken together, our data indicate that Paneth cell hyperplasia and expression of their various antimicrobial products contribute to the immune response driven by TH2 cytokines, such as IL-9 and IL-13 in the intestinal mucosa.

Fibroblast Growth Factor Receptor 3 (FGFR3) modulates Paneth cell lineage allocation through a β‐catenin/Tcf‐4 dependent signaling mechanism

Fibroblast growth factor receptors are receptor tyrosine kinases that mediate cellular growth and differentiation during normal gut development and injury‐repair. Robust FGFR3 expression is restricted to the lower portion of the crypt where stem cells reside during normal intestinal development. Activation of the β‐catenin/Tcf‐4 complex is crucial for maintaining the intestinal stem cell compartment and for Paneth cell lineage allocation/differentiation. Previously we showed that FGFR3‐/‐ mice have fewer intestinal crypts, have a reduction in the total number of clonogenic stem cells, and decreased β‐catenin/Tcf‐4 activation. FGFR3‐/‐ mice at post‐natal days 14 and 21 display both a significant reduction in the number of intestinal Paneth cells and a reduction in mRNA levels of Paneth cell products. Since Paneth cell lineage allocation/differentiation is dependent upon β‐catenin/Tcf‐4 activation, we examined whether FGFR3 signaling can directly modulate Paneth cell specification. Caco2 cells treated with FGF9, a FGFR3 ligand, or co‐transfected with a constituitively active mutant (K650E) of FGFR3 had sustained β‐catenin/Tcf‐4 transcriptional activity. FGF9 increased mRNA levels of two Paneth cell markers, lysozyme and human defensin 5. These findings suggest that FGFR3 signaling can directly modulate Paneth cell specification possibly through the β‐catenin/Tcf‐4 signaling complex.

Proteomic changes during intestinal cell maturation in vivo

Intestinal epithelial cells undergo progressive cell maturation as they migrate along the crypt-villus axis. To determine molecular signatures that define this process, proteins differentially expressed between the crypt and villus were identified by 2D-DIGE and MALDI-MS. Forty-six differentially expressed proteins were identified, several of which were validated by immunohistochemistry. Proteins upregulated in the villus were enriched for those involved in brush border assembly and lipid uptake, established features of differentiated intestinal epithelial cells. Multiple proteins involved in glycolysis were also upregulated in the villus, suggesting increased glycolysis is a feature of intestinal cell differentiation. Conversely, proteins involved in nucleotide metabolism, and protein processing and folding were increased in the crypt, consistent with functions associated with cell proliferation. Three novel paneth cell markers, AGR2, HSPA5 and RRBP1 were also identified. Notably, significant correlation was observed between overall proteomic changes and corresponding gene expression changes along the crypt-villus axis, indicating intestinal cell maturation is primarily regulated at the transcriptional level. This proteomic profiling analysis identified several novel proteins and functional processes differentially induced during intestinal cell maturation in vivo. Integration of proteomic, immunohistochemical, and parallel gene expression datasets demonstrate the coordinated manner in which intestinal cell maturation is regulated.

Does Small Intestinal Atresia Affect Epithelial Protein Expression in Human Newborns?

Bowel segments distal to a congenital intestinal obstruction have been suggested to be immature. In other words, luminal components such as amniotic fluid (before birth) and/or enteral nutrition (after birth) may be required to activate intestinal epithelial protein expression, thereby influencing epithelial differentiation. We investigated cell-type-specific protein expression proximal and distal to jejunal and ileal atresias in human newborns. We immunohistochemically studied intestinal tissue specimens of 16 newborns who had undergone surgery for jejunal or ileal atresia. Sections were taken from both the proximal and distal sides of the atresias. For all patients, the enterocyte-specific markers lactase, sucrase-isomaltase, sodium glucose cotransporter 1, glucose transporters 2 and 5, intestinal fatty acid-binding protein and alkaline phosphatase were expressed at a mean 3 +/- 1 days after birth, both proximal and distal to jejunal and ileal atresias. Expression of goblet cell-specific markers mucin 2 and trefoil factor 3 and that of the Paneth cell marker lysozyme was maintained at either side of the atretic segment. With respect to the markers used, the human small intestinal epithelium is already differentiated shortly after birth. The absence of intestinal continuity in case of a jejunal or ileal atresia does not affect epithelial protein expression. This would seem to indicate that the developing small intestinal epithelium matures independently of luminal components.

PPARβ/δ Regulates Paneth Cell Differentiation Via Controlling the Hedgehog Signaling Pathway

All 4 differentiated epithelial cell types found in the intestinal epithelium derive from the intestinal epithelial stem cells present in the crypt unit, in a process whose molecular clues are intensely scrutinized. Peroxisome proliferator-activated receptor beta (PPARbeta) is a nuclear hormone receptor activated by fatty acids and is highly expressed in the digestive tract. However, its function in intestinal epithelium homeostasis is understood poorly. To assess the role of PPARbeta in the small intestinal epithelium, we combined various cellular and molecular approaches in wild-type and PPARbeta-mutant mice. We show that the expression of PPARbeta is particularly remarkable at the bottom of the crypt of the small intestine where Paneth cells reside. These cells, which have an important role in the innate immunity, are strikingly affected in PPARbeta-null mice. We then show that Indian hedgehog (Ihh) is a signal sent by mature Paneth cells to their precursors, negatively regulating their differentiation. Importantly, PPARbeta acts on Paneth cell homeostasis by down-regulating the expression of Ihh, an effect that can be mimicked by cyclopamine, a known inhibitor of the hedgehog signaling pathway. We unraveled the Ihh-dependent regulatory loop that controls mature Paneth cell homeostasis and its modulation by PPARbeta. PPARbeta currently is being assessed as a drug target for metabolic diseases; these results reveal some important clues with respect to the signals controlling epithelial cell fate in the small intestine.

Gene expression profiling of intestinal epithelial cell maturation along the crypt-villus axis

To define the genetic reprogramming that drives intestinal epithelial cell maturation along the crypt-villus axis, enterocytes were sequentially isolated from the villus tip to the crypts of mouse small intestine. Changes in gene expression were assessed using 27,405-element complementary DNA microarrays (14,685 unique genes) and specific changes validated by Western blotting. A total of 1113 genes differentially expressed between the crypt and villus were identified. Among these, established markers of absorptive and goblet cell differentiation were up-regulated in villus cells, whereas Paneth cell markers were maximally expressed in crypt cells. The 1113 differentially expressed genes were significantly enriched for genes involved in cell cycle progression, RNA processing, and translation (all predominantly down-regulated during maturation) and genes involved in cytoskeleton assembly and lipid uptake (predominantly up-regulated during maturation). No enrichment for apoptosis-regulating genes was observed. We confirmed that Wnt signaling was maximal in the proliferative compartment and observed a decrease in MYC and an increase in MAD and MAX expression during the maturation program. Consistent with these changes, the 1113 genes were enriched for MYC targets, establishing the importance of this network in intestinal cell maturation. This database serves as a resource for understanding the molecular mechanisms of intestinal cell maturation and for dissection of how perturbations in the maturation process can lead to changes in gastrointestinal physiology and pathology, particularly intestinal tumorigenesis.

Differential Labeling by Monoclonal Antibodies Adnab-9 and Anti-?-Defensin 5 Based on the Distribution and Adenomatous Tissue Content of Colonic Polyps

We sought a correlation between site and morphology of colonic polyps by labeling with neoplastic and general Paneth cell markers, monoclonal antibodies Adnab-9 and anti-alpha-defensin 5, respectively. Proportions labeled by Adnab-9 and anti-alpha-defensin 5 were, respectively, 42 and 85% for adenomas, 39 and 63% for early tubular adenomas, 41 and 44% for serrated, 34 and 20% for mixed, and 11 versus 2.7% for hyperplastic polyps. Compared with hyperplastic polyps, the proportion of other polyps labeled by Adnab-9 or anti-alpha-defensin 5 was higher but this difference was more significant for distal (P = 0.008 for Adnab-9 and P = 0.0001 for anti-alpha-defensin 5) than proximal (P = 0.645 and P = 0.154, respectively) polyps. While increased labeling of all proximal polyps compared to distal ones mirrored the colonic distribution of Paneth cells, distal adenomas tended to have a higher proportion labeled by Adnab-9, suggesting that Adnab-9 labels Paneth cells associated with increased neoplastic potential.

Protection of the Peyer's patch-associated crypt and villus epithelium against methotrexate-induced damage is based on its distinct regulation of proliferation.

The crypt and villus epithelium associated with Peyer's patches (PPs) is largely spared from methotrexate (MTX)-induced damage, compared with the non-patch (NP) epithelium. To assess the mechanism(s) preventing damage to the PP epithelium after MTX treatment, epithelial proliferation, apoptosis, and cell functions were studied in a rat-MTX model. Small intestinal segments containing PPs were excised after MTX treatment. Epithelial proliferation and apoptosis were assessed by detection of incorporated BrdU and cleaved caspase-3, respectively. Epithelial functions were determined by the expression of cell type-specific gene products at mRNA and protein level. Before and after MTX treatment, the number of BrdU-positive cells was higher in PP crypts than in NP crypts. BrdU incorporation was diminished in NP crypts, while in PP crypts incorporation was hardly affected. In PP and NP crypts, similar and increased levels of cleaved caspase-3-positive cells were observed after MTX. The enterocyte markers, sucrase-isomaltase, sodium-glucose co-transporter 1, glucose transporters 2 and 5, and intestinal and liver fatty acid binding protein, were down-regulated after MTX in NP epithelium but not in PP epithelium. In contrast, expression of the goblet cell markers, Muc2 and trefoil factor 3, and the Paneth cell marker, lysozyme, was maintained after MTX in both PP and NP epithelium. In conclusion, as MTX-induced apoptosis was similar in PP and NP crypts, the protection of the PP epithelium seems to be based on differences in the regulation of epithelial proliferation. Enterocyte function in the PP epithelium was unaffected by MTX treatment. Goblet and Paneth cell function was maintained in both NP and PP epithelium.

Identification of Paneth cells in pyloric glands associated with gastric and intestinal mixed-type intestinal metaplasia of the human stomach.

We have proposed that intestinal metaplasia (IM) of the human stomach be divided into two types on the basis of cell differentiation status: a gastric and intestinal (GI) mixed type and a solely intestinal (I) type. In the GI mixed type, gastric (foveolar epithelial and pyloric gland cells) and intestinal (goblet, intestinal absorptive, and Paneth cells) phenotype cells coexist in the same intestinalized gastric glands in various combinations and degrees. Consequently, intestinalized gastric glands are hybrids. Although we have described the rare appearance of Paneth-like cells in pyloric glands of GI mixed-type IM, the absence of an appropriate Paneth cell marker leaves room for doubt as to their true character. The purpose of this study was to clearly identify Paneth cells in pyloric glands in IM lesions using a new Paneth cell marker, a polyclonal antibody human defensin (HD)-5, raised against HD-5, which is included in granules of Paneth cells. A total of 105 gastric samples (4 biopsy and 101 surgical resected specimens) were examined. In only nine cases (8.6%), the antibody allowed demonstration of Paneth cells in pyloric glands in GI mixed-type IM, confirming our previous finding. Analysis of the proliferative cell (P) zone indicated that a common stem cell might generate both GI phenotype cells by upward and downward migration. No Paneth cells were found above the P zone. The results suggest that the stem cells show abnormal cell differentiation in IM lesions but preserve their normal direction of migration.

Absence of group II phospholipase A2, a Paneth cell marker, from the epididymis.

Paneth cell-like metaplasia has been reported in the epithelium of the epididymis and prostatic adenocarcinomas. We studied the expression of group II phospholipase A2 (PLA2), a marker of Paneth cell differentiation, in six orchiectomy specimens with Paneth cell-like metaplasia. Both immunohistochemistry for group II PLA2 protein and in situ hybridization for the mRNA of group II PLA2 gave negative results in all six cases but positive reaction for lysozyme. The results show that the cells of the Paneth cell-like metaplasia are not true Paneth cells.

RESPONSE OF THE INTESTINAL EPITHELIUM TO METHOTREXATE INDUCED DAMAGE

One of the major side effects of chemotherapy is severe damage to the intestinal epithelium, leading to impairment of the intestinal function. Especially in pediatric patients, clinical symptoms like malabsorption, malnutrition and anorexia are of great concern. Aim: To gain insight in the regulation of epithelial proliferation, differentiation and cell death during damage and regeneration in order to develop prophylactic/therapeutic regimes to avoid or repair intestinal damage. Methods: Since morphological alterations after chemotherapy are similar in rat and human small intestine, we focussed on rats treated with methotrexate (MTX). Duodenal and jejunal segments were excised on various days after a single i.v. dose of MTX. Next to standard histological evaluation, differentiation of the epithelial cells was assessed by the expression of cell-type specific differentiation markers at mRNA (in situ hybridization) and protein (immunohistochemistry) levels. Sucrase-isomaltase (SI) and intestinal fatty acid binding protein (iFABP) were used as differentiation markers for enterocytes, mucin (MUC2) for goblet cells, and lysozyme for Paneth cells. In addition, proliferating and apoptotic epithelial cells were assessed by immunodetection of proliferating cell nuclear antigen (PCNA) and the terminal uridine deoxynucleotide nick-end labeling (TUNEL) method, respectively. Results: Morphologically, intestinal damage at 2 days after MTX injection is characterized by disturbance of the crypt architecture and shortening of the villi. On day 4 the villus atrophy is even more pronounced, while the crypts are elongated. Both, the crypt- and villus architecture has been restored completely on day 6. Expression of the enterocyte specific differentiation markers showed a decrease of SI while iFABP remained stable at both mRNA- and protein levels. Moreover, expression of the Paneth cell marker lysozyme (on day 2-4) and the goblet cell marker MUC2 (on day 4) are increased at both levels investigated. There is an increase in TUNEL positive cells in the crypts on day 2, which is followed by an increase in PCNA-positive cells on day 4. Conclusions: During MTX-induced damage, lysozyme and MUC2 expression are increased, which implies an increased production of protective molecules. The MTX-induced decrease in SI expression and the unaltered iFABP expression suggest a differential effect on expression of these genes in enterocytes. This might indicate a switch in enterocyte metabolism from sugar absorption to fatty acid absorption. Since alterations in mRNA and corresponding protein levels correlate well for each differentiation marker, the effects of MTX occur mainly at the mRNA level. Furthermore, single dose MTX treatment leads to increased apoptosis in crypts followed by an increase of proliferative activity in crypts as demonstrated by the histological changes, TUNEL method and PCNA expression patterns.

Cell lineage markers in premalignant and malignant colonic mucosa

Normal colonic epithelial cells consist of several cell types or lineages that are thought to arise from a common stem cell precursor. Neoplastic transformation may occur at different stages in the differentiation of a colonic stem cell to produce tumors that may retain characteristic cell lineage phenotypes. In this study, immunohistochemical techniques were used to identify cell lineage-related markers in fetal, normal, hyperplastic, adenomatous, and cancerous colonic tissue. These markers consisted of secretory component (columnar cells), a purified mucin antigen (mucous or goblet cells), chromogranin A (enteroendocrine cells), lysozyme (Paneth cells), and carcinoembryonic antigen (panepithelial cell marker). Colonic neoplasms, like normal mucosa, predominantly expressed the markers of columnar and goblet cell lineages. Chromogranin A was expressed in a small population of cells in most normal and fetal colonic crypts. Chromogranin A reactive cells were found in 55% of hyperplastic polyps, 31% of adenomatous polyps, and 33% of carcinomas. Lysozyme reactivity was rare in fetal, normal, and hyperplastic specimens, but was present in 86% of adenomas and 40% of carcinomas. Of 42 primary carcinomas, 9% were “pluripotent” and expressed markers of all four cell lineages. In addition to columnar and goblet cell markers, 7% expressed both enteroendocrine and Paneth cell markers, 17% expressed enteroendocrine cell markers, and 24% expressed Paneth cell markers. Two cases (5%) lacked expression of any of the cell lineage markers. The remainder expressed only columnar and goblet cell markers. The markers used in this study appear to identify the major cell lineages of fetal and normal colonic epithelium and can be used to delineate the altered cell lineage phenotypes in premalignant and malignant colonic mueosa.