- Email: [email protected]
Identification and isolation of candidate human colonic stem cells based on cell surface integrin expression
especiallyaround 2 hot spots within exon 2, as colon cancer cells de-differentiate.Inactivation of aberrantly expressed GRPR possibly represents a central mechanism regutating colon cancer de-differentiation.
wild type littermates. Pretreatment of wild-type mice with lipopolysaccharide (LPS) 14 h prior to irradiation protected crypt epithelial cells from apoptosis, by rnarkedly increasing prostaglandinE2 (PGE2)synthesis.Apobec-1 null mice, on the other hand,did not demonstrate an increase in expressionof PGE2yet maintainedan increasedapoptotic index. LPS induces COX-2 mRNA expression resulting in increased PGE2synthesis. These data suggest that in apobec-1 null mice, COX-2 expression is perturbed. To determine whether this is due to changes in COX-2mRNAstability, we performed RNA stability experimentsusing actinomycin D in 293 cells. The half life of COX-2mRNAchangedfrom 60 min to -150 min in the presence of apobec-l. Taken together, these data demonstrate a role for apobec-1 in intestinal stem cell proliferation in response to injury via COX-2 mediated PGE2 overexpression.These are the first findings suggesting a biological target for apobec-1 beyond apoB mRNA editing in the small intestine.
318 Expression of Tight Junction Proteins in Low and High Resistance Epithelial Cell Lines MDCK-C7 and -Cll Salah Amasheh, Noga Meid, Alfred H. Gitter, Joachim Mankertz, Joerg D. Schulzke, Michael Fromm, Freie Univ Berlin, Berlin Germany Epithelialtight junctions consist of the transmembranalproteins occludin and severalclaudins. It is an unresolved puzzle so far, which of these proteins are responsible for forming the paracellular barrier. Expression of the tight junction proteins occludin and claudins 1, 2, 3, 4, and 5 was studied in tight and leaky clones of Madin-Oarbycanine kidney (MDCK) cells. As cell adhesion and the paracellularbarrier function of epithelia depend on the extracellular calcium concentration, we studied effects of extracellular Ca2+ on transepithelial electrical resistance (17) and on the expression of occludin and claudins 1, 2, 3, 4, and 5. Methods: Epithelial cell monolayersof Madin-Darbycanine kidney (MDCK) cell clones C7 and Cll were grown on porous polycarbonatefilters (n = 17-18 / cell strain) at 37° C and the resistance was measured on day 7. Ca~÷ switch experimentswere performed employing a 2 or 4 h low extracellularCa2+ (2.5/~M) incubation step. Subsequently,tissues were used for membrane protein preparation.The expressionof tight junction proteins was detectedby immunoblotting. Results: Monolayers of MDCK-Cll revealedfeatures of a low resistanceepithelium (52 + 1 g.c--.......~,whereas MDCK-C7 cells exhibited a relatively high resistance (1056 _+ 74 O.cm2). Low extracellular Ca2+ decreased ? to less than 50 % of initial/7 in monolayers of both clones, without effect of the inhibitor of protein kinases, H7. Occludin and claudin 1, 2, 3, 4, and 5 were detected in all cell lines under investigation. Expressionof occludin and claudin 3 did not differ in C7 and Cll. However, claudins 1 and 2 were stronger expressed in Cll cells, whereas the expression of claudin 4 was pronounced in C7. Low Ca2+ incubation revealeda decreaseof claudin 2 in Cll cells whereas other tight junction proteins remained constant. Conclusion:Theseobservationsindicate that tight junction contribution by claudins is correlated to barrier properties of mammalian epithelial cell monolayers. In MDCK cells claudins 1 and 2 are strongly expressed in permeable tight junctions, while claudin 4 is predominantlypresent in tight junctions of low permeability.Theseobservationssuggest that barrier properties of mammalian epithelial cells depend on different combinations of claudin proteins. Furthermore, a decrease of claudin 2 during low Ca2+ incubation was detected, indicating a differential effect of extracellularCa2+ on tight junction protein expression.
321 Identification and Isolation of Candidate Human Colonic Stem Cells Based on Cell Surface Integrin Expression. Koji Fujimoto, R Daniel Beauchamp,Robert H. Whitehead, Vanderbilt Univ Medical Ctr, Nashville, TN Introduction:The surface epithelium of the colonic mucosa is constantly being replaced by cells derived from the stem cells located near the base of the crypts. Although the location of the stem cells is known, there are no known markers for these cells. A potential method for identifying stem cells is an in vitro culture method that we have recently described (Gastroenterology,117, 858, 1999) in which single cells obtained from normal colonic crypts will grow and form colonies in soft agar. This study was designedto test the hypothesisthat colonic stem cells might be enriched and cultured on the basis of differential integrin expression. Msthods:immunofluorescence. Cryostatsections of normal human colonic mucosawere immunostained using antibodies to alpha (a1,,~2,(z3,a4,~5,~6 and ~V)and beta (,81,~2,~3,/34,~5) integdns, isolation of crypt cells and FACSanalysis. Basedon the specific integrin expression patterns identified when the tissue sections were stained, a single cell suspension was preparedfrom isolated crypts and stained with anti-integdn antibodies. The cells were analyzedand sorted into strongly staining and weakly staining subpopulations by FACS.Colony assays in soft agar were performed to evaluatethe clonogenic growth potential of the sorted cells. Results: The immunofluorescence study showed that ,81 integdn was expressedmost strongly on the cells of the bottom third of the crypt. A single cell suspension stained with this antibody was sorted and two peaksof fluorescent intensity were seen. Since the cells with the higher fluorescence were from the bottom part of the crypt, the region that contains the stem cells, this cell population was collected and the clonogenic growth of these cells was comparedto that of the unsorted cells by colony assay.As shown below, selection of cells with ,81 integfin antibody produceda cell population with significantly enhancedability to form colonies in soft agar. The results for assays on 5 human samples were:- Unsorted cells 2.4+_0.6 colonies per plate; non-sorted + LIM1863CM 5 +0.8 (p-
319 Up-Regulation Of The o,6,84 Integrin In Human Colon Cancer Cells Hehong Hi, David Gagne, Nuria Besora, Jean-Francois Beaulieu, Univ de Sherbrooke, Sherbrooke, PQ Canada The integrin ~ is one of the main laminin receptors in epithelia. Although unique in its ability to associatewith the intermediatefilament network, c~,6,84shares with other integrins the ability of transducing signals, which ultimately regulate cell growth and differentiation. The express!onof o:6~ had beenpreviouslyanalyzedin colon cancersby indirect immunofluorescence using various antibodies,but raised conflicting data. In the presentstudy,/34 subunit expression was analyzed at both protein and transcript levels in 22 primary tumors and corresponding resectionmargins as well as in 7 adenocarcinomacell lines. For immunodetection, a panel of 4 antibodies recognizing both intestinal forms of ,84 (JBC, 274:29819-25, 1999) was used. In colon cancers,/~4 staining was found to be increasedin 19/22 tumors (all well or moderately differentiated) as compared with their normal counterparts. RT-PCR was used to further investigate ,84 expression in 12 of these paired samples. The integfin /31 subunit and the oncogene c-myc were also investigated. Amounts of transcript were estimated relative to both $14 and/Y-actin. The results showed that the relative amounts of the ,84 and c-myctranscripts were significantly increasedin the primary tumors in comparison to the resection margins (p
322 The/31 lategrin-EGFR later'action May Regulate Cell Fate Decisions in Esophageal Eplthatiat Keratinocyte Stem Cogs. Hiroshi Nakagawa,Takaaki Mizushima, Hideki Harada,Yasir Suliman, Univ of Pennsylvania, Philadelphia, PA; Oliver G. Opitz, Univ of Freiburg, Freiburg Germany; Anti K. Rustgi, Univ of Pennsylvania,Philadelphia, PA BACKGROUND:Integrins constitute cell surface receptors mediating cell-matrix and cell-cell adhesion. Integrin-dependentsignaling pathways include that for adhesion-dependentcell motility, cell growth, and cell survival. The ~ integrin subunit (CD29), a key constituent of the heterodimeric receptors for matrix proteins, activates epidermal growth factor receptor (EGFR) by physical interaction in the absence of EGFRligands. In the epidermis of the skin, the basal layer contains cells with elevated CD29 expression level, and such cells may be slowly cycling stem cells basedon their enhancedability to adhereto matrix and form colonies. To study the role of CD29 and EGFR, we have isolated CD29 bright cells from normal human esophageal keratinocytes, and overexpressed EGFR. METHODS: Primary epithelial keratinocytes (EPC2) were isolated from normal human esophagus and grown in a serum free medium. EPC2cells were stained with fluorescein isothiocyanateconjugatedanti-human CD29 and sorted according to fluorescent intensity. EPC2cells were infected with EGFRand green fluorescent protein expression retroviral vectors and selectedwith G418. Immunocytochemistry, flowcytomatry, and western blotting were employed to characterizethe cells. Cell proliferation was assessed by 3H-thymidine incorporation assay. RESULTS:EPC2cells were fractionated into two groups: the 20% of cells with the highest level of surface CD29 (CD29 bright) and the 20% with the leastCD29 (CD29dull). CD29bright cells enteredthe exponentially growth phase later than CD29 dull cells, suggesting the former cell population may contain slow cycling cells. Western blot analysis showed that CD29 bright cells expressed a higher level of EGFRthan CG29dull cells and the unsorted whole cell population. Interestingly, both CD29 and EGFRwere most intensely expressed at cell-cell contact sites in EPC2 cells. In EGFR transduced EPC2 (EPC2-EGFR)cells, cell-cell contact inhibition appearedearlier than control cells in postconfluent states. Furthermore, DNA synthesis was suppressed by 80% in EPC2-EGFRcells comparing to the control cells in the absence of EGF. CONCLUSIONS: CD29 bright basal keratinocytes in the esophagealsquamous epithelium may contain slowly cycling cells (i.e. stem cells). EGFRmay contribute to cell fate decision by negativelyregulating cell proliferation in conjunction with integrin function.
320 A Novel Function for Protaoncegene apobec-l: Increased Apoptosis of Stem Cells Resulting from Decreased Prostaglandin E2 Expression in apobec-1 Null Mice. Shrikant Anant, Courtney Houchen, Terry Riehl, ThiruvengadamArumugam, Debnath Mukhopadhyay, William F. Stenson, Nicholas O. Davidson,Washington Univ, St. Louis, MO Apobeo-1,an RNA-specificcytidine deaminase,is expressedexclusivelyin the human intestine where it binds a variety of AU-rich target RNAs. Apobec-1 binds with high affinity (-50 nM) to a consensus binding sequence UUUN(A/U)U in c-myc, TNF-~, VEGFand cyclooxygenase2 (COX-2).Apobec-1 binding to its consensussite in 3'UTR of c-myc increasesc-myc mRNA stability suggesting that apobec-1 is an RNA stabilizing protein. Overexpressionof apobec1 in livers of transgenic mice causeshepatocellularcarcinomasuggestingthat apobec-1 plays a critical role in modulating cellular proliferation. Given its restricted expressionto the human intestine, we examined whether apobec-1 may regulate genes involved in proliferation of intestinal stem cells. We have generated apobec-1 null mice and back-crossedto over 10 generations into the C57BU6 strain, initially the mice appeared normal with no obvious morphological changes in intestinal epithelium. However, upon -/-radiation injury (12 Gy), stem cells in the apobec-1 null mice underwent increased apoptosis (3-fold) compared to
A-61
wild type littermates. Pretreatment of wild-type mice with lipopolysaccharide (LPS) 14 h prior to irradiation protected crypt epithelial cells from apoptosis, by rnarkedly increasing prostaglandinE2 (PGE2)synthesis.Apobec-1 null mice, on the other hand,did not demonstrate an increase in expressionof PGE2yet maintainedan increasedapoptotic index. LPS induces COX-2 mRNA expression resulting in increased PGE2synthesis. These data suggest that in apobec-1 null mice, COX-2 expression is perturbed. To determine whether this is due to changes in COX-2mRNAstability, we performed RNA stability experimentsusing actinomycin D in 293 cells. The half life of COX-2mRNAchangedfrom 60 min to -150 min in the presence of apobec-l. Taken together, these data demonstrate a role for apobec-1 in intestinal stem cell proliferation in response to injury via COX-2 mediated PGE2 overexpression.These are the first findings suggesting a biological target for apobec-1 beyond apoB mRNA editing in the small intestine.
318 Expression of Tight Junction Proteins in Low and High Resistance Epithelial Cell Lines MDCK-C7 and -Cll Salah Amasheh, Noga Meid, Alfred H. Gitter, Joachim Mankertz, Joerg D. Schulzke, Michael Fromm, Freie Univ Berlin, Berlin Germany Epithelialtight junctions consist of the transmembranalproteins occludin and severalclaudins. It is an unresolved puzzle so far, which of these proteins are responsible for forming the paracellular barrier. Expression of the tight junction proteins occludin and claudins 1, 2, 3, 4, and 5 was studied in tight and leaky clones of Madin-Oarbycanine kidney (MDCK) cells. As cell adhesion and the paracellularbarrier function of epithelia depend on the extracellular calcium concentration, we studied effects of extracellular Ca2+ on transepithelial electrical resistance (17) and on the expression of occludin and claudins 1, 2, 3, 4, and 5. Methods: Epithelial cell monolayersof Madin-Darbycanine kidney (MDCK) cell clones C7 and Cll were grown on porous polycarbonatefilters (n = 17-18 / cell strain) at 37° C and the resistance was measured on day 7. Ca~÷ switch experimentswere performed employing a 2 or 4 h low extracellularCa2+ (2.5/~M) incubation step. Subsequently,tissues were used for membrane protein preparation.The expressionof tight junction proteins was detectedby immunoblotting. Results: Monolayers of MDCK-Cll revealedfeatures of a low resistanceepithelium (52 + 1 g.c--.......~,whereas MDCK-C7 cells exhibited a relatively high resistance (1056 _+ 74 O.cm2). Low extracellular Ca2+ decreased ? to less than 50 % of initial/7 in monolayers of both clones, without effect of the inhibitor of protein kinases, H7. Occludin and claudin 1, 2, 3, 4, and 5 were detected in all cell lines under investigation. Expressionof occludin and claudin 3 did not differ in C7 and Cll. However, claudins 1 and 2 were stronger expressed in Cll cells, whereas the expression of claudin 4 was pronounced in C7. Low Ca2+ incubation revealeda decreaseof claudin 2 in Cll cells whereas other tight junction proteins remained constant. Conclusion:Theseobservationsindicate that tight junction contribution by claudins is correlated to barrier properties of mammalian epithelial cell monolayers. In MDCK cells claudins 1 and 2 are strongly expressed in permeable tight junctions, while claudin 4 is predominantlypresent in tight junctions of low permeability.Theseobservationssuggest that barrier properties of mammalian epithelial cells depend on different combinations of claudin proteins. Furthermore, a decrease of claudin 2 during low Ca2+ incubation was detected, indicating a differential effect of extracellularCa2+ on tight junction protein expression.
321 Identification and Isolation of Candidate Human Colonic Stem Cells Based on Cell Surface Integrin Expression. Koji Fujimoto, R Daniel Beauchamp,Robert H. Whitehead, Vanderbilt Univ Medical Ctr, Nashville, TN Introduction:The surface epithelium of the colonic mucosa is constantly being replaced by cells derived from the stem cells located near the base of the crypts. Although the location of the stem cells is known, there are no known markers for these cells. A potential method for identifying stem cells is an in vitro culture method that we have recently described (Gastroenterology,117, 858, 1999) in which single cells obtained from normal colonic crypts will grow and form colonies in soft agar. This study was designedto test the hypothesisthat colonic stem cells might be enriched and cultured on the basis of differential integrin expression. Msthods:immunofluorescence. Cryostatsections of normal human colonic mucosawere immunostained using antibodies to alpha (a1,,~2,(z3,a4,~5,~6 and ~V)and beta (,81,~2,~3,/34,~5) integdns, isolation of crypt cells and FACSanalysis. Basedon the specific integrin expression patterns identified when the tissue sections were stained, a single cell suspension was preparedfrom isolated crypts and stained with anti-integdn antibodies. The cells were analyzedand sorted into strongly staining and weakly staining subpopulations by FACS.Colony assays in soft agar were performed to evaluatethe clonogenic growth potential of the sorted cells. Results: The immunofluorescence study showed that ,81 integdn was expressedmost strongly on the cells of the bottom third of the crypt. A single cell suspension stained with this antibody was sorted and two peaksof fluorescent intensity were seen. Since the cells with the higher fluorescence were from the bottom part of the crypt, the region that contains the stem cells, this cell population was collected and the clonogenic growth of these cells was comparedto that of the unsorted cells by colony assay.As shown below, selection of cells with ,81 integfin antibody produceda cell population with significantly enhancedability to form colonies in soft agar. The results for assays on 5 human samples were:- Unsorted cells 2.4+_0.6 colonies per plate; non-sorted + LIM1863CM 5 +0.8 (p-
319 Up-Regulation Of The o,6,84 Integrin In Human Colon Cancer Cells Hehong Hi, David Gagne, Nuria Besora, Jean-Francois Beaulieu, Univ de Sherbrooke, Sherbrooke, PQ Canada The integrin ~ is one of the main laminin receptors in epithelia. Although unique in its ability to associatewith the intermediatefilament network, c~,6,84shares with other integrins the ability of transducing signals, which ultimately regulate cell growth and differentiation. The express!onof o:6~ had beenpreviouslyanalyzedin colon cancersby indirect immunofluorescence using various antibodies,but raised conflicting data. In the presentstudy,/34 subunit expression was analyzed at both protein and transcript levels in 22 primary tumors and corresponding resectionmargins as well as in 7 adenocarcinomacell lines. For immunodetection, a panel of 4 antibodies recognizing both intestinal forms of ,84 (JBC, 274:29819-25, 1999) was used. In colon cancers,/~4 staining was found to be increasedin 19/22 tumors (all well or moderately differentiated) as compared with their normal counterparts. RT-PCR was used to further investigate ,84 expression in 12 of these paired samples. The integfin /31 subunit and the oncogene c-myc were also investigated. Amounts of transcript were estimated relative to both $14 and/Y-actin. The results showed that the relative amounts of the ,84 and c-myctranscripts were significantly increasedin the primary tumors in comparison to the resection margins (p
322 The/31 lategrin-EGFR later'action May Regulate Cell Fate Decisions in Esophageal Eplthatiat Keratinocyte Stem Cogs. Hiroshi Nakagawa,Takaaki Mizushima, Hideki Harada,Yasir Suliman, Univ of Pennsylvania, Philadelphia, PA; Oliver G. Opitz, Univ of Freiburg, Freiburg Germany; Anti K. Rustgi, Univ of Pennsylvania,Philadelphia, PA BACKGROUND:Integrins constitute cell surface receptors mediating cell-matrix and cell-cell adhesion. Integrin-dependentsignaling pathways include that for adhesion-dependentcell motility, cell growth, and cell survival. The ~ integrin subunit (CD29), a key constituent of the heterodimeric receptors for matrix proteins, activates epidermal growth factor receptor (EGFR) by physical interaction in the absence of EGFRligands. In the epidermis of the skin, the basal layer contains cells with elevated CD29 expression level, and such cells may be slowly cycling stem cells basedon their enhancedability to adhereto matrix and form colonies. To study the role of CD29 and EGFR, we have isolated CD29 bright cells from normal human esophageal keratinocytes, and overexpressed EGFR. METHODS: Primary epithelial keratinocytes (EPC2) were isolated from normal human esophagus and grown in a serum free medium. EPC2cells were stained with fluorescein isothiocyanateconjugatedanti-human CD29 and sorted according to fluorescent intensity. EPC2cells were infected with EGFRand green fluorescent protein expression retroviral vectors and selectedwith G418. Immunocytochemistry, flowcytomatry, and western blotting were employed to characterizethe cells. Cell proliferation was assessed by 3H-thymidine incorporation assay. RESULTS:EPC2cells were fractionated into two groups: the 20% of cells with the highest level of surface CD29 (CD29 bright) and the 20% with the leastCD29 (CD29dull). CD29bright cells enteredthe exponentially growth phase later than CD29 dull cells, suggesting the former cell population may contain slow cycling cells. Western blot analysis showed that CD29 bright cells expressed a higher level of EGFRthan CG29dull cells and the unsorted whole cell population. Interestingly, both CD29 and EGFRwere most intensely expressed at cell-cell contact sites in EPC2 cells. In EGFR transduced EPC2 (EPC2-EGFR)cells, cell-cell contact inhibition appearedearlier than control cells in postconfluent states. Furthermore, DNA synthesis was suppressed by 80% in EPC2-EGFRcells comparing to the control cells in the absence of EGF. CONCLUSIONS: CD29 bright basal keratinocytes in the esophagealsquamous epithelium may contain slowly cycling cells (i.e. stem cells). EGFRmay contribute to cell fate decision by negativelyregulating cell proliferation in conjunction with integrin function.
320 A Novel Function for Protaoncegene apobec-l: Increased Apoptosis of Stem Cells Resulting from Decreased Prostaglandin E2 Expression in apobec-1 Null Mice. Shrikant Anant, Courtney Houchen, Terry Riehl, ThiruvengadamArumugam, Debnath Mukhopadhyay, William F. Stenson, Nicholas O. Davidson,Washington Univ, St. Louis, MO Apobeo-1,an RNA-specificcytidine deaminase,is expressedexclusivelyin the human intestine where it binds a variety of AU-rich target RNAs. Apobec-1 binds with high affinity (-50 nM) to a consensus binding sequence UUUN(A/U)U in c-myc, TNF-~, VEGFand cyclooxygenase2 (COX-2).Apobec-1 binding to its consensussite in 3'UTR of c-myc increasesc-myc mRNA stability suggesting that apobec-1 is an RNA stabilizing protein. Overexpressionof apobec1 in livers of transgenic mice causeshepatocellularcarcinomasuggestingthat apobec-1 plays a critical role in modulating cellular proliferation. Given its restricted expressionto the human intestine, we examined whether apobec-1 may regulate genes involved in proliferation of intestinal stem cells. We have generated apobec-1 null mice and back-crossedto over 10 generations into the C57BU6 strain, initially the mice appeared normal with no obvious morphological changes in intestinal epithelium. However, upon -/-radiation injury (12 Gy), stem cells in the apobec-1 null mice underwent increased apoptosis (3-fold) compared to
A-61