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Sa1824 Dietary Pectin Increases Intestinal Crypt Stem Cell Survival Following Radiation Injury
Sa1823
AGA Abstracts
Impact of TNFα and IFNγ on Intestinal Epithelial Stem and Progenitor Cell (Isc/PC) Responses Hyunji Ryu, Elizabeth Managlia, Rebecca B. Katzman, Terrence A. Barrett INTRODUCTION: Inflammatory cytokines are required for mucosal host defense yet during acute colitis contribute to mucosal injury and ulceration. Here we focused on understanding the impact of TNFα and IFNγ on intestinal epithelial stem and progenitor cell (ISC/PC) responses In Vivo during colitis and In Vitro in isolated crypt cultures. METHODS/RESULTS: We examined the regulation of ISC/PC genes using immunohistochemistry (IHC) and qPCR. During the first week of colitis [8 days (d) after 7d 2-5% DSS], tissue histology revealed extensive ulcerations surrounded by regenerative foci of hyperproliferating crypts characterized by increased BrdU-incorporation and Ser552 phosporylated, β-catenin (p-β-catenin552). Outside of these regenerative zones, epithelial proliferation and p-β-catenin552 levels were reduced. mRNA from colonic intestinal epithelial cells (IEC) sorted for the ISC/PC marker CD44 revealed reduced expression of Lgr5, Axin2, and Ascl2 ISC genes. In contrast, IECs from mice after ulcer healing (d20) revealed elevated Lgr5, Axin2, Ki67, CD44, L-rig, and cMyc. We interpreted these data to indicate that IEC responses during acute DSS colitis were heterogeneous with areas of stimulated and repressed stem cell responses. To focus on mechanisms for cytokine-induced ISC/PC responses, we isolated IEC crypts for 3D culture in matrigel as described (Sato et al). Cultures were examined after stimulation with TNFα (1-20 ng/ml) or IFNγ (200, 500 Units/ml) for 1 or 7d. TNFα increased Ascl2, Lgr5, Ki67, Lrig1, and Axin2 2-3 fold whereas IFNγ suppressed Axin2, Lgr5, and Ascl2 expression after 1d. Strikingly, Ascl2 mRNA levels increased 11-fold in crypts cultured with TNFα for 7d. DISCUSSION: In summary, we find that IEC responses in DSS-colitis indicate ISC/PC stimulation or repression depending on their distance from ulcer margins. Data from primary cell culture suggests that whereas TNFα aids in ISC/PC responses, IFNγ attenuates stem cell gene expression. We propose that high levels of IFNγ, seen early in acute colitis, impair ulcer healing, whereas TNFα production assists in repair over time.
Values for TEER = Ω.cm2; USSING Isc of 20 mM glucose solution = μA/cm2; mRNA expression = fold change from control. *p<0.05 v.s. control Sa1821 Abnormal Expression of Epigenetic Regulators in Mesenchymal Stem Cells Explains Helicobacter Induced Gastric Carcinogenesis Ayman M. Metwally, Hanchen Li, Jian Hua Liu, Jean Marie Houghton Background - Epigenetic changes are heritable changes in gene expression without alteration of the coding sequence of the gene. Epigenetic regulators are key players in stem cell biology and carcinogenesis. We have shown that bone marrow derived MSC home to the gastric mucosa during Helicobacter infection and differentiate abnormally as dysplastic cells, can initiate, contribute to and become gastric adenocarcinoma. The time can be dramatically decreased by introducing MSC which have been repeatedly passaged in culture and allowed to spontaneously transform. The aim of the present study was to investigating the abnormal expression of the regulatory elements of polycomb (PcG) and trithorax (TrX) complexes over time in MSC and correlate these changes with tumor forming potential. Materials and Methods- MSC isolated from C57BL/6 mice bone marrow were placed in culture and grown for up to 1 year. Cells at each passage were tested for their ability to contribute to tumor formation in C57BL/6 mice, and frozen for later use and analysis. 84 genes representing mouse polycomb and trithorax complexes were studied using quantitative PCR analysis. RNA was isolated quantified and reverse transcribed into cDNA followed by quantitative PCR using Mouse Polycomb & Trithorax Complexes PCR Array (SAbioscience). Sequence analysis for the DNA binding domain of TP53 gene was performed at the core facility of the UMass. Results - Differences in gene expression were detected beginning prior to the third passage. The fold change for each gene was calculated using 2(-ΔΔCT) method. The most significantly regulated genes were kdm5d, Rbp2, RnaseL, Zbtb16, cbx5, cbx7, Dnmt3I, phf19 and smarca1. P53 mutations were detected as early as passage 3. Early in culture, substitution mutations were common while at later passages insertions and deletion were detected. Despite dramatic genetic dysregulation, only cells grown for a year or greater in culture formed tumors in the C57BL/6 mouse. Conclusion and Discussion: Abnormal expression of these epigenetic regulatory elements may be involved in induction of p53 mutation. Tautomeres (C T substitution) are types of mutation that appear after DNA replication. This type of mutation is repaired through DNA proofreading mechanism occur directly after DNA synthesis. The presence of this type of mutation in passages 6, 9 and 12 indicate a defect in the DNA proofreading mechanism which may be linked to abnormal expression of these epigenetic regulatory elements. Also the presence of these tautomeres show that the mutations likely occur randomly along the DNA, not only in the P53 gene. The persistent accumulation of these mutations along the DNA over time, in parallel with the abnormal expression of the epigenetic regulatory elements results into malignant transformation of MSC.
Sa1824 Dietary Pectin Increases Intestinal Crypt Stem Cell Survival Following Radiation Injury Sripathi M. Sureban, Randal May, Dongfeng Qu, Nathaniel Weygant, Parthasarathy Chandrakesan, Shahid Umar, Rama Ramanujam, Courtney W. Houchen Background: The microcolony assay following ionizing irradiation (IR) is a functional assay of intestinal stem cell (ISC) fate. Pectin is a highly complex branched polysaccharide fiber rich in galactoside residues and is present in all plant cell walls. It has been observed that natural polysaccharides including pectin are radioprotectors and detoxicants. We have demonstrated that doublecortin and CAM-kinase-like-1 (DCAMKL-1) marks putative ISCs. We have previously observed that the Notch pathway plays an important role in ISC fate determination. Aim: To determine the effects of dietary pectin on radiation-induced ISC deletion and crypt survival following lethal dose (14 Gy) IR. Methods: Adult C57BL/6 mice were placed on a 6% pectin diet with additional 0.5% pectin in their drinking water for one week prior to IR. Animals were exposed to whole-body γ-IR (14 Gy) utilizing a Gammacell 40 137Cs γ-irradiator with air being pumped into the chamber during exposure. All irradiation treatments were begun in the A.M. Two hours before being killed, each mouse was given an i.p. injection of 5-bromo-2'-deoxyuridine and 5-fluoro-2′-deoxyuridine. Animals were killed 84 h post IR to assess ISC survival by scoring regenerative crypts using the microcolony assay. Surviving crypts were counted from ten cross sections of small intestine per mouse and three mice per experimental group. Mouse small intestine was subjected to real-time RT PCR and immunohistochemical analysis for DCAMKL-1 and Notch-1. Results: We observed a 3-fold increase in ISC/crypt survival following 14 Gy IR in mice treated with pectin compared to irradiated mice on a normal chow diet with regular drinking water. Additionally, we observed a 2-fold increase in surviving DCAMKL-1 positive ISCs at 24 h post IR in mice pretreated with pectin compared to controls. Furthermore and surprisingly, mice pre-treated with pectin survived longer (10 days) compared to mice treated with IR alone (7 days). We also found significantly increased expression of DCAMKL-1 (1.5-fold) and Notch-1 (2-fold) mRNA in the small intestine of mice treated with pectin compared to control animals at 24h post IR, indicating that both DCAMKL-1 and Notch-1 may play an important role in protecting ISCs against radiation injury. Conclusions: These data taken together suggest that dietary pectin acts as a radioprotective agent when administered in the short-term (1week) prior to lethal dose whole body IR. Furthermore pectin prevents IR-induced deletion of ISCs at 24hrs, facilitates crypt regeneration, and ultimately promotes survival. These data support further investigation into the mechanisms by which pectin promotes overall survival following lethal dose whole body IR.
Sa1822 Hyperproliferation is an Intrinsic Property of the Intestinal Epithelium in the CFTR-Null Mouse Intestine Jinghua Liu, Nancy M. Walker, Lane L. Clarke Cystic fibrosis (CF) patients have a 6-fold increased risk for gastrointestinal cancer, a significant statistic in a relatively young population (N Engl J Med 1995;332:494). The risk is multifactorial, resulting from both disease and therapeutic interventions. Importantly, studies of Cftr-null mice have found increased epithelial cell proliferation in the intestine In Vivo (Am J Physiol 2001, 281: G681). Although many co-morbidities and therapeutic factors are eliminated in Cftr-null mice, they demonstrate significant intestinal disease including dysmotility, altered bacterial flora, bacterial overgrowth, abnormal Toll-like receptor signaling and low-grade inflammation. We questioned whether epithelial hyperproliferation in the Cftr-null intestine is dependent on the abnormal intestinal environment or is an intrinsic property of Cftr-null epithelium. To test this hypothesis, we employed a method for highlydifferentiated primary culture of the mouse CF intestinal epithelium (termed ‘enteroids') to determine whether increased proliferation persisted in the absence of bacteria or other endogenous cells. Since proliferation in the stem cell population of the intestine is maintained by Wnt/β-catenin signaling, we investigated whether there was evidence of altered signaling in the Cftr-null intestine In Vivo and in the enteroid cultures. Western blots demonstrated significantly increased β-catenin protein (~50%) in both freshly isolated Cftr-null epithelium and in cultured Cftr-null enteroids as compared to WT. Immunofluorescence studies of freshly isolated crypts revealed increased nuclear β-catenin and marginalization of disheveled 2 (Dvl2) to the cell borders in the Cftr-null as compared to WT crypts. Proliferation studies using EdU incorporation revealed increased proliferation in the Cftr-null enteroids relative to WT in growth medium (+10%). However, when growth factors (Rspondin1, Noggin, EGF) were removed from the medium, the Cftr-null enteroids showed +40% greater proliferation than WT enteroids, albeit both WT and Cftr-null were at reduced proliferation rates. We had previously reported that intracellular pH is increased in the Cftr-null crypts, which may facilitate cell proliferation by increasing cell cycle transitions (J Biol Chem 2003, 278: 44645). Therefore Cftr-null enteroids were treated with 1 μM EIPA (amiloride analog) to block Nhe1 Na+/H+ exchange for cell acidification, which reduced cell proliferation to a level commiserate with WT enteroids. We conclude that hyperproliferation in the Cftr-null intestine exists in the absence of the intestinal microenvironment and therefore is an intrinsic property of Cftr-null intestinal epithelium. Hyperproliferation in the Cftr-null epithelium is associated with increased Wnt/β-catenin signaling and an alkaline intracellular pH. Supported by NIH R01DK48816 and the CF Foundation.
AGA Abstracts
Sa1825 The Epithelial Cell Response After Ileocecal Resection (ICR) Impacts p53 and Survivin Expression Valeria C. Cohran, Zheng J. Zhang, Elizabeth Managlia, Tatiana Goretsky, Rebecca B. Katzman, Terrence A. Barrett Introduction : Short gut syndrome (SGS) or intestinal failure results from intestinal resection or inflammation from conditions such as Necrotizing enterocolitis. The epithelial layer responds to small bowel (SB) loss with expansion of absorptive surface area through crypt fissioning. It has been postulated that increased absorptive surface results from a balance between intestinal epithelial cell (IEC) proliferation and apoptosis in the small bowel remnant. Given that the PI3K signaling pathway mediates downstream events for a number of growth factors, we examined PI3K signaling in ICR mice. Western blot (WB) data from IECs revealed no increase in pAKTser473 levels; however, there was a significant reduction of the p85 alpha subunit of PI3K. The p85α subunit was recently shown to mediate p53 transcriptional activation. We therefore examined the potential role of p53 in the epithelial cell response to ICR. Methods; 8-12 week old C57BL/6J (WT) mice underwent sham surgery or ICR.
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AGA Abstracts
Impact of TNFα and IFNγ on Intestinal Epithelial Stem and Progenitor Cell (Isc/PC) Responses Hyunji Ryu, Elizabeth Managlia, Rebecca B. Katzman, Terrence A. Barrett INTRODUCTION: Inflammatory cytokines are required for mucosal host defense yet during acute colitis contribute to mucosal injury and ulceration. Here we focused on understanding the impact of TNFα and IFNγ on intestinal epithelial stem and progenitor cell (ISC/PC) responses In Vivo during colitis and In Vitro in isolated crypt cultures. METHODS/RESULTS: We examined the regulation of ISC/PC genes using immunohistochemistry (IHC) and qPCR. During the first week of colitis [8 days (d) after 7d 2-5% DSS], tissue histology revealed extensive ulcerations surrounded by regenerative foci of hyperproliferating crypts characterized by increased BrdU-incorporation and Ser552 phosporylated, β-catenin (p-β-catenin552). Outside of these regenerative zones, epithelial proliferation and p-β-catenin552 levels were reduced. mRNA from colonic intestinal epithelial cells (IEC) sorted for the ISC/PC marker CD44 revealed reduced expression of Lgr5, Axin2, and Ascl2 ISC genes. In contrast, IECs from mice after ulcer healing (d20) revealed elevated Lgr5, Axin2, Ki67, CD44, L-rig, and cMyc. We interpreted these data to indicate that IEC responses during acute DSS colitis were heterogeneous with areas of stimulated and repressed stem cell responses. To focus on mechanisms for cytokine-induced ISC/PC responses, we isolated IEC crypts for 3D culture in matrigel as described (Sato et al). Cultures were examined after stimulation with TNFα (1-20 ng/ml) or IFNγ (200, 500 Units/ml) for 1 or 7d. TNFα increased Ascl2, Lgr5, Ki67, Lrig1, and Axin2 2-3 fold whereas IFNγ suppressed Axin2, Lgr5, and Ascl2 expression after 1d. Strikingly, Ascl2 mRNA levels increased 11-fold in crypts cultured with TNFα for 7d. DISCUSSION: In summary, we find that IEC responses in DSS-colitis indicate ISC/PC stimulation or repression depending on their distance from ulcer margins. Data from primary cell culture suggests that whereas TNFα aids in ISC/PC responses, IFNγ attenuates stem cell gene expression. We propose that high levels of IFNγ, seen early in acute colitis, impair ulcer healing, whereas TNFα production assists in repair over time.
Values for TEER = Ω.cm2; USSING Isc of 20 mM glucose solution = μA/cm2; mRNA expression = fold change from control. *p<0.05 v.s. control Sa1821 Abnormal Expression of Epigenetic Regulators in Mesenchymal Stem Cells Explains Helicobacter Induced Gastric Carcinogenesis Ayman M. Metwally, Hanchen Li, Jian Hua Liu, Jean Marie Houghton Background - Epigenetic changes are heritable changes in gene expression without alteration of the coding sequence of the gene. Epigenetic regulators are key players in stem cell biology and carcinogenesis. We have shown that bone marrow derived MSC home to the gastric mucosa during Helicobacter infection and differentiate abnormally as dysplastic cells, can initiate, contribute to and become gastric adenocarcinoma. The time can be dramatically decreased by introducing MSC which have been repeatedly passaged in culture and allowed to spontaneously transform. The aim of the present study was to investigating the abnormal expression of the regulatory elements of polycomb (PcG) and trithorax (TrX) complexes over time in MSC and correlate these changes with tumor forming potential. Materials and Methods- MSC isolated from C57BL/6 mice bone marrow were placed in culture and grown for up to 1 year. Cells at each passage were tested for their ability to contribute to tumor formation in C57BL/6 mice, and frozen for later use and analysis. 84 genes representing mouse polycomb and trithorax complexes were studied using quantitative PCR analysis. RNA was isolated quantified and reverse transcribed into cDNA followed by quantitative PCR using Mouse Polycomb & Trithorax Complexes PCR Array (SAbioscience). Sequence analysis for the DNA binding domain of TP53 gene was performed at the core facility of the UMass. Results - Differences in gene expression were detected beginning prior to the third passage. The fold change for each gene was calculated using 2(-ΔΔCT) method. The most significantly regulated genes were kdm5d, Rbp2, RnaseL, Zbtb16, cbx5, cbx7, Dnmt3I, phf19 and smarca1. P53 mutations were detected as early as passage 3. Early in culture, substitution mutations were common while at later passages insertions and deletion were detected. Despite dramatic genetic dysregulation, only cells grown for a year or greater in culture formed tumors in the C57BL/6 mouse. Conclusion and Discussion: Abnormal expression of these epigenetic regulatory elements may be involved in induction of p53 mutation. Tautomeres (C T substitution) are types of mutation that appear after DNA replication. This type of mutation is repaired through DNA proofreading mechanism occur directly after DNA synthesis. The presence of this type of mutation in passages 6, 9 and 12 indicate a defect in the DNA proofreading mechanism which may be linked to abnormal expression of these epigenetic regulatory elements. Also the presence of these tautomeres show that the mutations likely occur randomly along the DNA, not only in the P53 gene. The persistent accumulation of these mutations along the DNA over time, in parallel with the abnormal expression of the epigenetic regulatory elements results into malignant transformation of MSC.
Sa1824 Dietary Pectin Increases Intestinal Crypt Stem Cell Survival Following Radiation Injury Sripathi M. Sureban, Randal May, Dongfeng Qu, Nathaniel Weygant, Parthasarathy Chandrakesan, Shahid Umar, Rama Ramanujam, Courtney W. Houchen Background: The microcolony assay following ionizing irradiation (IR) is a functional assay of intestinal stem cell (ISC) fate. Pectin is a highly complex branched polysaccharide fiber rich in galactoside residues and is present in all plant cell walls. It has been observed that natural polysaccharides including pectin are radioprotectors and detoxicants. We have demonstrated that doublecortin and CAM-kinase-like-1 (DCAMKL-1) marks putative ISCs. We have previously observed that the Notch pathway plays an important role in ISC fate determination. Aim: To determine the effects of dietary pectin on radiation-induced ISC deletion and crypt survival following lethal dose (14 Gy) IR. Methods: Adult C57BL/6 mice were placed on a 6% pectin diet with additional 0.5% pectin in their drinking water for one week prior to IR. Animals were exposed to whole-body γ-IR (14 Gy) utilizing a Gammacell 40 137Cs γ-irradiator with air being pumped into the chamber during exposure. All irradiation treatments were begun in the A.M. Two hours before being killed, each mouse was given an i.p. injection of 5-bromo-2'-deoxyuridine and 5-fluoro-2′-deoxyuridine. Animals were killed 84 h post IR to assess ISC survival by scoring regenerative crypts using the microcolony assay. Surviving crypts were counted from ten cross sections of small intestine per mouse and three mice per experimental group. Mouse small intestine was subjected to real-time RT PCR and immunohistochemical analysis for DCAMKL-1 and Notch-1. Results: We observed a 3-fold increase in ISC/crypt survival following 14 Gy IR in mice treated with pectin compared to irradiated mice on a normal chow diet with regular drinking water. Additionally, we observed a 2-fold increase in surviving DCAMKL-1 positive ISCs at 24 h post IR in mice pretreated with pectin compared to controls. Furthermore and surprisingly, mice pre-treated with pectin survived longer (10 days) compared to mice treated with IR alone (7 days). We also found significantly increased expression of DCAMKL-1 (1.5-fold) and Notch-1 (2-fold) mRNA in the small intestine of mice treated with pectin compared to control animals at 24h post IR, indicating that both DCAMKL-1 and Notch-1 may play an important role in protecting ISCs against radiation injury. Conclusions: These data taken together suggest that dietary pectin acts as a radioprotective agent when administered in the short-term (1week) prior to lethal dose whole body IR. Furthermore pectin prevents IR-induced deletion of ISCs at 24hrs, facilitates crypt regeneration, and ultimately promotes survival. These data support further investigation into the mechanisms by which pectin promotes overall survival following lethal dose whole body IR.
Sa1822 Hyperproliferation is an Intrinsic Property of the Intestinal Epithelium in the CFTR-Null Mouse Intestine Jinghua Liu, Nancy M. Walker, Lane L. Clarke Cystic fibrosis (CF) patients have a 6-fold increased risk for gastrointestinal cancer, a significant statistic in a relatively young population (N Engl J Med 1995;332:494). The risk is multifactorial, resulting from both disease and therapeutic interventions. Importantly, studies of Cftr-null mice have found increased epithelial cell proliferation in the intestine In Vivo (Am J Physiol 2001, 281: G681). Although many co-morbidities and therapeutic factors are eliminated in Cftr-null mice, they demonstrate significant intestinal disease including dysmotility, altered bacterial flora, bacterial overgrowth, abnormal Toll-like receptor signaling and low-grade inflammation. We questioned whether epithelial hyperproliferation in the Cftr-null intestine is dependent on the abnormal intestinal environment or is an intrinsic property of Cftr-null epithelium. To test this hypothesis, we employed a method for highlydifferentiated primary culture of the mouse CF intestinal epithelium (termed ‘enteroids') to determine whether increased proliferation persisted in the absence of bacteria or other endogenous cells. Since proliferation in the stem cell population of the intestine is maintained by Wnt/β-catenin signaling, we investigated whether there was evidence of altered signaling in the Cftr-null intestine In Vivo and in the enteroid cultures. Western blots demonstrated significantly increased β-catenin protein (~50%) in both freshly isolated Cftr-null epithelium and in cultured Cftr-null enteroids as compared to WT. Immunofluorescence studies of freshly isolated crypts revealed increased nuclear β-catenin and marginalization of disheveled 2 (Dvl2) to the cell borders in the Cftr-null as compared to WT crypts. Proliferation studies using EdU incorporation revealed increased proliferation in the Cftr-null enteroids relative to WT in growth medium (+10%). However, when growth factors (Rspondin1, Noggin, EGF) were removed from the medium, the Cftr-null enteroids showed +40% greater proliferation than WT enteroids, albeit both WT and Cftr-null were at reduced proliferation rates. We had previously reported that intracellular pH is increased in the Cftr-null crypts, which may facilitate cell proliferation by increasing cell cycle transitions (J Biol Chem 2003, 278: 44645). Therefore Cftr-null enteroids were treated with 1 μM EIPA (amiloride analog) to block Nhe1 Na+/H+ exchange for cell acidification, which reduced cell proliferation to a level commiserate with WT enteroids. We conclude that hyperproliferation in the Cftr-null intestine exists in the absence of the intestinal microenvironment and therefore is an intrinsic property of Cftr-null intestinal epithelium. Hyperproliferation in the Cftr-null epithelium is associated with increased Wnt/β-catenin signaling and an alkaline intracellular pH. Supported by NIH R01DK48816 and the CF Foundation.
AGA Abstracts
Sa1825 The Epithelial Cell Response After Ileocecal Resection (ICR) Impacts p53 and Survivin Expression Valeria C. Cohran, Zheng J. Zhang, Elizabeth Managlia, Tatiana Goretsky, Rebecca B. Katzman, Terrence A. Barrett Introduction : Short gut syndrome (SGS) or intestinal failure results from intestinal resection or inflammation from conditions such as Necrotizing enterocolitis. The epithelial layer responds to small bowel (SB) loss with expansion of absorptive surface area through crypt fissioning. It has been postulated that increased absorptive surface results from a balance between intestinal epithelial cell (IEC) proliferation and apoptosis in the small bowel remnant. Given that the PI3K signaling pathway mediates downstream events for a number of growth factors, we examined PI3K signaling in ICR mice. Western blot (WB) data from IECs revealed no increase in pAKTser473 levels; however, there was a significant reduction of the p85 alpha subunit of PI3K. The p85α subunit was recently shown to mediate p53 transcriptional activation. We therefore examined the potential role of p53 in the epithelial cell response to ICR. Methods; 8-12 week old C57BL/6J (WT) mice underwent sham surgery or ICR.
S-334