Markers Of Epithelial Cell Differentiation Research Articles

Polarized Porcine Trophoblastic Cell Lines Spontaneously Secrete Interferon-Gamma

Following the demonstration of high levels of interferon-gamma (IFN-γ) synthesis by the pig trophectoderm around implantation, an adequate experimental system was established so as to study the regulation of endogenous IFN-γ gene. Several stable cell lines have been isolated from Day 14 and 15 pig trophoblast, that could withstand indefinite growth when cultured on collagen-coated supports. Since no feeder cells were used for culture, and cell lines could be successfully cloned, these lines represent the first pure porcine trophoblastic (TB) cell lines isolated so far. These cells were shown to exhibit most differentiation markers of epithelial cells and in addition to express the porcine trophectoderm-specific antigen SN1-38. The TB cell line A (TBA) was characterized in more details upon culture on microporous filters, where a high apico-basal polarity could be obtained. In those conditions, a transient and acute interferon-gamma secretion was detected only in the apical medium. Moreover, the two trophoblast-specific mRNA of 1.3 and 1.4kb that have been described in the blastocyst collected in vivo were shown to be synchronously transcribed. This polarized synthesis and secretion of IFN-γ was correlated with the acquisition of maximal levels of electric resistance of TBA monolayers on filters, and was not observed on the same cells cultured on plastic. This differentiation was maintained over 30 passages, demonstrating that the induction of IFN-γ secretion by pig conceptus is not maternally controlled. This cellular model will be of prime importance for studies on the developmental regulation of the IFN-γ gene, and more generally for studies on relationship between secretion and polarity in transporting epithelia.

Human Immunodeficiency Virus-1 Tat Induces Hyperproliferation and Dysregulation of Renal Glomerular Epithelial Cells

Human immunodeficiency virus-associated nephropathy (HIVAN) is etiologically related to the viral infection, but the mechanisms of virus-induced renal injury remain undetermined. Peculiar histopathological features of HIVAN are the enhanced proliferation and the loss of differentiation markers of glomerular epithelial cells (podocytes). We found that podocytes were not permissive to HIV-1 replication. In this study we investigated the effects of the HIV-1 regulatory protein Tat on primary cultures and on a continuous line of podocytes. Our results demonstrated that Tat induced hyperproliferation of these cells in a dose-dependent manner. This activity was primarily mediated by the basic domain of the viral protein. Proteoglycans were required for this phenomenon because Tat-induced increase of podocyte growth was significantly impaired by inhibition of proteoglycan synthesis with beta-D-xyloside. In podocyte cultures Tat promoted both the transcription and the release of basic fibroblast growth factor, which contributed to the enhanced cell proliferation. Moreover, Tat deregulated the podocyte phenotype causing down-regulation of maturity markers such as WT-1 and synaptopodin, alteration of cytoarchitecture, and impairment of permselectivity. Together, these results demonstrate that the interaction of extracellular Tat with podocytes can induce alterations that mimic the pathological changes of podocytes detected in HIVAN.

Lactase Gene Transcription Is Activated in Response to Hypoxia in Intestinal Epithelial Cells

Lactase-phlorizin hydrolase, a brush-border membrane disaccharidase, is a marker of intestinal epithelial cell differentiation and digestive function. The intestine is susceptible to conditions of hypoxia resulting from vascular perfusion deficits. We hypothesized that lactase gene induction may provide a mechanism to efficiently increase nutrient energy substrates during gut hypoxia. These studies sought to characterize expression of the lactase gene in response to hypoxia and to characterize a role for hypoxia-inducible factor (HIF-1) in mediating the hypoxic response. Microarray analysis and confirmatory RT-PCR identified a 4-fold induction of lactase mRNA abundance in intestinal epithelial Caco-2 cells exposed to hypoxia. Lactase promoter activity was similarly induced by hypoxia in cells stably transfected with a 2.0-kb 5′-flanking region of the rat lactase gene linked to a reporter gene. Transient cotransfection with HIF-1α and β stimulated lactase promoter activity 2.4- and 3.5-fold under conditions of normoxia and hypoxia, respectively. We conclude that HIF-1 can activate the lactase promoter in intestinal epithelial cells exposed to hypoxia. Induction of lactase transcription may represent an adaptive response to gut hypoxia.

Effects of Paramyxoviral Infection on Airway Epithelial Cell Foxj1 Expression, Ciliogenesis, and Mucociliary Function

To elucidate molecular mechanisms underlying the association between respiratory viral infection and predisposition to subsequent bacterial infection, we used in vivo and in vitro models and human samples to characterize respiratory virus-induced changes in airway epithelial cell morphology, gene expression, and mucociliary function. Mouse paramyxoviral bronchitis resulted in airway epithelial cell infection and a distinct pattern of epithelial cell morphology changes and altered expression of the differentiation markers beta-tubulin-IV, Clara cell secretory protein, and Foxj1. Furthermore, changes in gene expression were recapitulated using an in vitro epithelial cell culture system and progressed independent of the host inflammatory response. Restoration of mature airway epithelium occurred in a pattern similar to epithelial cell differentiation and ciliogenesis in embryonic lung development characterized by sequential proliferation of undifferentiated cells, basal body production, Foxj1 expression, and beta-tubulin-IV expression. The effects of virus-induced alterations in morphology and gene expression on epithelial cell function were illustrated by decreased airway mucociliary velocity and impaired bacterial clearance. Similar changes in epithelial cell Foxj1 expression were also observed in human paramyxoviral respiratory infection. Taken together, these model systems of paramyxoviral respiratory infection mimic human pathology and identify epithelial cell Foxj1 expression as an early marker of epithelial cell differentiation, recovery, and function.

Ectopic expression of guanylyl cyclase C in CD34+ progenitor cells in peripheral blood.

To examine the utility of guanylyl cyclase C (GC-C)-specific nested reverse transcriptase polymerase chain reaction (RT-PCR) to detect circulating tumor cells in patients with colorectal cancer. Peripheral-blood mononuclear cells from 24 patients with Dukes' stage D colorectal cancer were analyzed by GC-C-specific nested RT-PCR using 1 microg of total RNA. Peripheral-blood mononuclear cells from 20 healthy volunteers served as controls. Additionally, peripheral-blood CD34+ progenitor cells were assayed for the expression of both GC-C and other epithelial cell-specific markers. GC-C mRNA was detected in blood mononuclear cells from all 24 patients with colorectal cancer and all healthy volunteers. These unexpected positive results reflected low-level ectopic transcription of GC-C in CD34+ progenitor cells. Moreover, CD34+ progenitor cells expressed other epithelial cell-specific markers, including prostate-specific antigen, prostate-specific membrane antigen, carcinoembryonic antigen, CK-19, CK-20, mucin 1, and GA733.2. Limiting the quantity of mononuclear cell total RNA analyzed to < or = 0.8 microg eliminated detection of GC-C and other tissue-specific transcripts in blood of healthy volunteers. However, under the same conditions, GC-C mRNA was detected in mononuclear cells from all 24 patients with metastatic colorectal cancer. Using 0.5 microg of total RNA and GC-C-specific primers, nested RT-PCR detected a single human colon carcinoma cell (approximately 20 to 200 GC-C transcripts/cell) in 10(6) to 10(7) mononuclear blood cells. These data suggest that GC-C may be useful for detecting circulating colorectal cancer cells. They also demonstrate that CD34+ cells are a source of ectopically expressed epithelial cell-specific markers and that CD34+ cells may contribute to the high false-positive rate generally observed when those markers are used to detect rare circulating metastatic cancer cells by RT-PCR.

Comparison of Calcium Supplementation or Low-Fat Dairy Foods on Epithelial Cell Proliferation and Differentiation

Epidemiological evidence suggests that dietary calcium and vitamin D intake are inversely related to incidence of colon cancer. Previous studies have demonstrated that supplementation of the diet with calcium in the form of calcium tablets or low-fat dairy foods alters colonic epithelial cell proliferation from a higher- to a lower-risk pattern. The present study compared relative effects of administration of calcium carbonate at ~900 mg/day (calcium) with those of a low-fat dairy food diet providing about the same amount of calcium (dairy) in a cross-over "head-to-head" study of 40 subjects at risk for colonic neoplasia. Dietary intake of macronutrients was similar in the two study periods, except for a slight increase in protein intake during dairy calcium supplementation. Rectal epithelial cell proliferation was studied in flat endoscopically normal-appearing mucosa at baseline and at the end of each of the two study periods and showed a significant reduction in epithelial crypt cell labeling index from 12.5% to 9.1% (calcium) or 9.3% (dairy) as well as in proliferating cells in the upper 40% of the crypt from 0.09 to 0.03 in the calcium- and low-fat dairy-supplemented intervention groups. No significant changes in two epithelial cell differentiation markers, cytokeratin AE1 and acidic mucins, were found. Furthermore, there were no differences in epithelial cell apoptosis or expression of the proapoptotic gene product BAK. These data indicate that increased dietary calcium given as supplements or in the diet in low-fat dairy foods lowers epithelial cell proliferation indexes from a higher- to a lower-risk pattern. Because supplemental calcium has been shown to reduce the recurrence of colonic adenomatous polyps in patients at increased risk for colonic neoplasia, our data suggest that supplemental low-fat dairy foods may also be effective.

Expression and function of CCAAT/enhancer binding proteinbeta (C/EBPbeta) LAP and LIP isoforms in mouse mammary gland, tumors and cultured mammary epithelial cells.

CCAAT/Enhancer binding proteins (C/EBPs) play important roles in the regulation of cell growth and differentiation. This study investigated the expression and function of C/EBPbeta isoforms in the mouse mammary gland, mammary tumors, and a nontransformed mouse mammary epithelial cell line (HC11). C/EBPbeta mRNA levels are 2-5-fold higher in mouse mammary tumors derived from MMTV/c-neu transgenic mice compared with lactating and involuting mouse mammary gland. The "full-length" 38 kd C/EBPbeta LAP ("Liver-enriched Activator Protein") isoform is the predominant C/EBPbeta protein isoform in mammary tumor whole cell lysates, however, the truncated 20 kd C/EBPbeta LIP ("Liver-enriched Inhibitory Protein") isoform is also present at detectable levels (mean LAP:LIP ratio 5.3:1). The mammary tumor C/EBPbeta LAP:LIP ratio decreases 70% (from 5.3:1 to 1.6:1) when lysate preparation is switched from a rapid whole cell lysis protocol to a multistep nuclear/cytoplasmic fractionation protocol. In contrast to mammary tumors, only the C/EBPbeta LAP isoform is detectable in the mammary gland whole cell and nuclear lysates; the truncated "LIP" isoform is undetectable regardless of isolation protocol. Ectopic over expression of C/EBPbeta LIP or C/EBPbeta LAP did not alter HC11 growth rates. However, C/EBPbeta LIP over expressing HC11 cells (LAP:LIP ratio of approximately 1:1) exhibited a consistent 2-4 h delay in G(0)/S phase transition. C/EBPbeta LIP overexpressing HC11 cells did not express beta-casein mRNA (mammary epithelial cell differentiation marker) in response to lactogenic hormones. This defect in beta-casein expression was not corrected by carrying out the differentiation protocol in the presence of an artificial extracellular matrix. These results demonstrate that the "full-length" C/EBPbeta LAP isoform is the predominant C/EBPbeta protein isoform expressed in mouse mammary gland in vivo and mouse mammary epithelial cell cultures in vitro. C/EBPbeta LIP detected in mammary tumor lysates may result from in vivo production or ex vivo isolation-induced proteolysis of C/EBPbeta LAP. Ectopic overexpression of C/EBPbeta LIP (LAP:LIP ratio of approximately 1:1) inhibits mammary epithelial cell differentiation (beta-casein expression).

Phenotypic analyses of limbal epithelial cell cultures derived from donor corneoscleral rims.

Grafted cultures of limbal epithelial cells aid repair of the corneal epithelium, but their phenotype is unclear. In this study, the phenotype of cultures that were similar in age to those used clinically were analysed. Limbal epithelial cells were isolated from donor corneoscleral rims and grown in various media, including those designed for keratinocytes. Successful cultures in each medium developed predominantly small (10 microm) tightly packed cells. Immunocytochemistry and western blotting revealed expression of keratins 3, 14 and 19. Expression of these keratins in situ was confirmed by immunohistochemistry. Basal limbal epithelial cells were positive for keratins 14 and 19, and suprabasal cells were positive for keratin 3. However intense staining for keratin 14 was also observed at the inner cut edge of corneoscleral rims. These findings demonstrate the potential importance of keratins 14 and 19 as markers of epithelial cell differentiation in the human cornea.

Altered enteroendocrine cell expression in T cell receptor alpha chain knock-out mice

Mice lacking T cell receptor alpha chain (TCRalpha(-/-)) develop inflammation of the colon. We have examined the effect of this inflammation on the colonic epithelium by studying markers of epithelial cuff, enteroendocrine, and immune cell differentiation. Using immunohistochemical techniques, colons were compared in normal C57/BL6 and murine TCR alpha(-/-) mice aged 2 and 3 weeks and 3-11 months. TCR alpha(-/-) mice aged 3-11 months had histologic evidence of inflammation with increased expression of CD45, CD4+, CD8+, and B220+ cells and a decrease in expression of IgA+ cells. There was a decrease in the number of cholecystokinin, serotonin, and neurotensin enteroendocrine expressing cells in the colon of TCR alpha(-/-) mice. These changes were not present in 2-3-week-old suckling/weaning mice. In contrast, peptide tyrosine tyrosine (PYY), glucagon-like peptide-1, and gastrin expression did not change and small intestinal enteroendocrine cells remained unaltered. The change in colonic enteroendocrine cell expression appears to be a specific response, since only a subset of these cells was altered, and the epithelium was intact by histologic analysis. The absence of functional T cells in TCR alpha(-/-) colon has a marked effect on differentiation of a specific subpopulation of enteroendocrine cells, prior to loss of integrity of the epithelium.

Role of Bcl-xL protein in differentiation and apoptosis of human middle ear cholesteatoma epithelium.

To compare the mechanisms of proliferation, differentiation, and apoptosis in middle ear cholesteatoma epithelium with those of normal external ear canal epithelium. The localizations of the expression of Bcl-xL protein and involucrin and the presence of apoptotic cells were determined for tissue slices of middle ear cholesteatoma epithelium and compared with the findings for normal external ear canal epithelium. In addition, SCC-25/bcl-xL transfectants showing the overexpression of Bcl-xL were used to investigate the effect of this protein on the expression of involucrin, which is a marker of epithelial cell differentiation. Cholesteatoma tissue specimens were surgically excised from 10 patients. Normal skin specimens collected from the external ear canal of the 10 patients were used as control specimens. The expression of Bcl-xL was detected in the vicinity of the basal cell layer of both the cholesteatoma epithelium and the normal external ear canal epithelium. Conversely, the expression of involucrin (ie, a marker of epithelial cell differentiation) increased in proportion to the shallowness of the epithelial layer. In situ labeling detected apoptotic cells in the spinous and granular cell layers of cholesteatoma tissue sections and similar findings in the normal external skin specimens. Western blot analysis confirmed that the expression of involucrin protein was the same in both wild-type SCC-25 cells and the SCC-25/bcl-xL transfectants. In both the cholesteatoma epithelium and the normal external ear canal epithelium, differentiation and apoptosis begin when the epithelial cells separate from the basal cells. The mechanisms behind these changes, at least in apoptosis, appear to be controlled by the expression of the Bcl-xL protein.

Tissue interaction regulates expression of a spasmolytic polypeptide gene in chicken stomach epithelium.

The primitive epithelium of embryonic chicken proventriculus (glandular stomach) differentiates, after day 6 of incubation, into luminal epithelium, which faces the lumen and abundantly secretes mucus, and glandular epithelium, which invaginates into mesenchyme and later expresses embryonic chicken pepsinogen (ECPg). So far it is not well understood how undifferentiated epithelial cells differentiate into these two distinct cell populations. Spasmolytic polypeptide (SP) is known to be expressed in surface mucous cells of mammalian stomach. In order to obtain the differentiation marker for proventricular luminal epithelial cells, we cloned a cDNA encoding chicken SP (cSP). Sequence analysis indicated that cSP has the duplicated cysteine-rich domain characteristic of SP. Examination of the spatial and temporal expression pattern of cSP gene revealed that, during embryogenesis, cSP was expressed in lumina epithelial cells of the proventriculus, gizzard, small intestine, and lung, but not the esophagus. In the proventriculus, cSP mRNA was first detected on day 8 of incubation and was localized to differentiated luminal epithelial cells. By using cSP as a molecular marker, the effects of mesenchyme on the differentiation of epithelium were analyzed in vitro. On the basis of these data, a model is presented concerning the differentiation of proventricular epithelium.

Levels of expression of hRPB11, a core subassembly subunit of human RNA polymerase II, affect doxorubicin sensitivity and cellular differentiation

We have previously shown that the human RNA polymerase II subunit 11 (hRPB11) is among the proteins specifically downregulated upon Doxorubicin (Dox) treatment of human cancer cell lines, and that Dox resistant clones derived upon drug selection express about 20% of the protein present in the original parental cell line. Given the prominent role that this subunit appears to have in eukaryotic cells, and the fact that its deletion causes lethality in yeast, we wanted to test the effect of the reintroduction of parental cell line levels of this subunit in Dox resistant colon cancer cells (LoVoDX). Stable transfectants of LoVoDX expressing parental (LoVoH) levels of hRPB11 showed a reduced sensitivity to the drug without changing the response of these cells to other chemotherapeutic agents, confirming a specific inverse correlation between cellular Dox sensitivity anti-hRPB11 levels of expression. In addition we show here that the levels of expression of this same RNA polymerase II subunit directly affect cellular differentiation, reducing the rate of cell proliferation, clonogenicity and increasing the expression of E-cadherin, a marker of epithelial cell differentiation. As expected from cells with these characteristics, upon in vivo administration of these clones in nude mice, we detected a significant reduction in the size and time of appearance of the primary tumors and overall metastatic capability. Finally, the role played by hRPB11 in regulating the transcription of specific genes is underlined by transient transfection experiments that show transactivation of the E-cadherin promoter by this protein.

Autocrine Insulin-like Growth Factor Binding Protein-2 (IGFBP-2) Modulates the Effects of Insulin-like Growth Factor-II (IGF-II) on Differentiating Human Fetal Glomerular Epithelial Cells † 1823

We have previously implicated the IGF system in the process of normal mammalian nephrogenesis. IGF-II is an important fetal mitogen and is abundantly expressed in the undifferentiated metanephric blastema, while the IGF binding proteins, which modulate IGF biological effects, display specific spatial and temporal expression in the developing human fetal kidney. To study the role of IGF-II and IGFBP-2 in renal epithelial cell differentiation, we developed and characterized a primary fetal glomerular epithelial cell line and a kidney explant model from human fetal kidneys obtained at 12-18 weeks gestation. Stimulation of the cell monolayers with IGF-II resulted in a dose-dependent increase in 3H-thymidine incorporation to 126 +/- 4.6% control at a concentration of 200 ng/ml for 24 hours. This stimulation was abrogated by addition of a Type I IGF receptor (IGF-IR) antibody. Stimulation with IGFBP-2, the major endogenous IGFBP of this cell type, did not affect mitogenesis, however co-incubation of the monolayers with IGFBP-2 inhibited IGF-II-stimulated mitogenesis in a dose-dependent fashion at all concentrations of IGF-II tested (1-200 ng/ml). Furthermore, incubation of the unstimulated monolayers with a neutralizing IGFBP-2 antibody (1:4000) resulted in an increase in mitogenesis to 121 +/- 2.5% of control. By Northern blot analysis, IGF-II upregulated IGFBP-2 and IGFBP-4 mRNAs, markers of glomerular epithelial cell differentiation (119% and 137% respectively vs control), while IGFBP-2 also upregulated IGFBP-4 mRNA (141% vs control) in the cell monolayers. Finally, incubation of the kidney explants with a neutralizing antibody to IGFBP-2 resulted in an inhibition of explant differentiation at 24 hours, with an expanded nephrogenic zone and abundant metanephric blastema. These data suggest that IGF-II stimulated IGFBP-2 production in the developing fetal glomerular epithelial cell plays a role in further differentiation of this cell type.

Cytokeratins and cell differentiation in the pancreas.

Keratins, or cytokeratins, represent a family of more than 20 different polypeptides which are important markers of epithelial cell differentiation. This review deals with the use of keratin immunohistochemistry in the study of pancreatic cell differentiation. Exocrine acinar cells and endocrine islet cells are well-differentiated cells which express the keratin combination 8 and 18, whereas the less-differentiated cells of the ductal tree are characterized by the additional expression of keratin 7, keratin 19, and, in the rat, keratin 20. Keratin expression is stable and can be used for cell identification after isolation and culture, and in clinical or experimental injury. The intercalated ductal cells and centroacinar cells are inconspicuous unless specific immunohistochemical markers, such as keratins, are used. In conditions where there is morphogenetic differentiation such as in fetal life, or where transdifferentiation is occurring, keratins have been used to trace the origin and fate of pancreatic cells.

Bleomycin Induces Strain-Dependent Alterations in the Pattern of Epithelial Cell-Specific Marker Expression in Mouse Lung

Clinical use of the antineoplastic agent bleomycin is restricted due to pulmonary toxicity. Murine models of bleomycin-induced pulmonary fibrosis have been developed in an attempt to understand the mechanisms involved in the fibrotic process. Studies have shown that the alveolar epithelium is damaged early after bleomycin treatment. The purpose of this study was to evaluate the pattern of gene expression in airway and alveolar epithelial cells after bleomycin exposure in mice that vary in susceptibility to bleomycin-induced fibrosis. Surfactant protein C (SPC) and Clara cell-specific protein (CC10) mRNA were used as cell-specific markers of alveolar type II cells and airway Clara cells, respectively. Mice were treated with a single intratracheal dose of bleomycin and the pattern of SPC and CC10 transcripts was examined byin situhybridization. The pattern of SPC mRNA 28 days after treatment was uniform in controls and resistant mice but exhibited a patchy appearance in sensitive mice. Bleomycin treatment also resulted in a strain-dependent loss of CC10 mRNA-expressing cells. In sensitive mice 28 days after treatment, SPC mRNA was ectopically expressed in the distal bronchiolar epithelium in a morphologically distinct cell type. Serial sections revealed that these cells either coexpressed CC10 mRNA or were located adjacent to CC10 mRNA-containing cells. This unique cell population may represent a progenitor cell type important in epithelial repair. The strain-dependent changes in CC10 and SPC gene expression after bleomycin treatment are suggestive of a role for the epithelium in pulmonary fibrosis versus repair.

The effect of tibolone on proliferation, differentiation and apoptosis in normal breast cells

Tissue homeostasis is the result of a balance between proliferation, differentiation and apoptosis, with apoptosis, or spontaneous programmed cell death, being thought to play a major role in the growth and regulation of both normal and tumor tissue. The expression of bcl-2protein plays an important role in controlling apoptosis, and this protein has been shown to be influenced by fluctuations in hormone levels during the menstrual cycle. Although considerable work has been carried out investigating the effect of estrogens on normal breast cells and breast cancer cell lines, less is known about the effect of progestins, and very little has been published on the tissue-specific hormone tibolone, which exhibits estrogenic, progestogenic and androgenic characteristics. Studies with tibolone, using normal human breast epithelium cells, demonstrate that this hormone displays a progestin-likeprofile and has favorable effects with respect to breast tumorigenesis. Tibolone decreases cell proliferation rates, promotes 17ß-hydroxysteroid dehydrogenase activity (a marker of epithelial cell differentiation) and increases apoptosis in normal as well as breast cancer cells. These findings indicate a possible future role for tibolone in the treatment of breast cancer and the need for further detailed investigations.

The lysosomal-associated membrane protein LAMP-1 is a novel differentiation marker for HC11 mouse mammary epithelial cells

HC11 cells are a model for mammary epithelial cell differentiation. Following treatment with the lactogenic hormones glucocorticoids, insulin and prolactin, the HC11 cells synthesize milk proteins. Stereological analysis at the ultrastructural level suggested that lysosomal biogenesis was activated following lactogenic hormone treatment of HC11 cells. Differentiation was also accompanied by an increase in the cellular content of tri- and tetra-antennary oligosaccharides, which were reactive with isolectin L4 from Phaseolus vulgaris (L-PHA). The lysosomal-associated membrane glycoproteins LAMP-1 and LAMP-2 are the major carriers of this glycosylation pattern. An analysis of LAMP-1 and LAMP-2 expression levels showed that there was a dramatic increase in LAMP-1 following lactogenic hormone treatment of HC11 cells. The control of LAMP-1 expression is mainly post-transcriptional since the level of LAMP-1 RNA is not affected by lactogenic hormones. Stereological analysis also showed an increase in intermediate filament control of differentiated cells. Analysis of the cytokeratins expressed in differentiated cells suggests that HC11 cells have characteristics of a mammary-specific stem cell. Increase in lysosomal vesicles and their contents might play a role in intra- and extra-cellular remodeling, which is characteristic of cell differentiation.

S-100 protein subunits in bovine oviduct epithelium: in situ distribution and changes during primary cell culture.

Cultures of bovine oviduct epithelial cells are widely used in co-culture systems to improve the results of in vitro fertilization. The aim of the present study was to evaluate the suitability of S-100 protein as a differentiation marker for bovine oviduct epithelial cells in vitro. The distribution of S-100 alpha and S-100 beta was examined immunohistochemically in bovine oviduct epithelium in situ and in primary cell cultures derived from it. Three segments of the Fallopian tube (isthmus, ampulla and fimbriae) were compared and analysed during different stages of the oestrus cycle (luteal phase and follicular phase). Ciliated and non-ciliated cells of the epithelium reacted with anti-S-100 alpha, S-100a (alpha beta) and S-100 beta antibodies, except for isthmic non-ciliated cells, which did not bind anti-S-100 beta or anti-S-100a (alpha beta). In addition, basal cells never showed immunoreactivity for S-100. In confluent monolayers of cultured oviduct epithelial cells, disappearance of reactivity for S-100 paralleled morphological signs of dedifferentiation (loss of cilia, cytoplasmic vacuolization). Free-floating oviduct epithelial cells, in contrast, retained morphological differentiation and still expressed S-100 antigen even after seven days in vitro. The immunohistochemical findings were confirmed by polyacrylamide gel electrophoresis and Western blotting. The results indicate that the presence of S-100 is closely connected to morphological differentiation and to the specific functional condition of bovine oviduct epithelial cells.

5424191 Epithelial cell specific differentiation marker : Prasad Gaddamanugu; Cooper Herbert Rockville, MD, United States Assigned to The United States of America as represented by the Department of Health and Human Services

5424191 Epithelial cell specific differentiation marker : Prasad Gaddamanugu; Cooper Herbert Rockville, MD, United States Assigned to The United States of America as represented by the Department of Health and Human Services

Identification of Vitamin D-Stimulated Alkaline Phosphatase in IEC-6 Cells, a Rat Small Intestine Crypt Cell Line

The IEC-6 cell line was derived from newborn rat small intestinal crypts and maintains characteristics of nontransformed crypt epithelial cells. We have reported that alkaline phosphatase (ALP) activity, a differentiation marker for intestinal epithelial cells, is regulated in IEC-6 cells by 1,25-dihydroxycholecalciferol (1,25(OH) 2D 3). Using reverse transcription polymerase chain reaction, we determined that the ALP in IEC-6 cells is the liver/bone/kidney ALP (LALP) isoenzyme and not the mature enterocyte form. Levamisole, a specific blocker for LALP, blocked the ALP activity of IEC-6 cells. Kinetic studies showed that 1,25(OH) 2D 3 increases V m values of ALP in IEC-6 cells while K m values remain the same. Northern analysis of LALP mRNA levels in IEC-6 cells indicated that 10 -7 M 1,25(OH) 2D 3 acts by increasing alkaline phosphatase activity at the gene expression level.