925e Altered intestinal epithelial homeostasis in mice with a target mutation in the gene encoding...
1
AGA Abstracts carcinoma, using whole-genome sequencing and linkage analysis, followed by association analysis in several thousand cases and controls. In some of our largest families we found causal germline mutations in two related genes, POLE and POLD1, that encode the main polymerases involved in DNA replication. The most common mutations, POLE p.Leu424Val and POLD1 p.Ser478Asn, lie at analogous sites in the proofreading domains of the proteins and are predicted to hinder the correction of base mispairs that occur during DNA replication. In keeping with this prediction, the two human mutations cause a mutator phenotype in yeast and in our patients' tumours. This is manifest as a tendency to all types of base substitution, especially G:C.T:A changes. We present data on screening for further germline POLE and POLD1 mutations in a panel of ;sl1000 familial CRC patients as part of an effort to fully characterise the spectrum of pathogenic mutations. In addition to very rare, high- penetrance mutations, we have invesigated an uncommon (freq. ;sl0.1%) germline POLE variant, p.Asp287Glu that appears to act as a moderate-penetrance CRC risk allele. Carriers of germline POLE and POLD1 mutations have a variable phenotype of multiple, early-onset adenomas and CRCs, and POLD1 carriers are also at high risk of endometrial cancer (EC). We propose that this condition be called PPAP, for Polymerase Proofreading-Associated Polyposis. At present, we recommend genetic testing for POLE and POLD1 in any family with a Lynch syndrome or attenuated FAP phenotype. We suggest management of PPAP families in a similar way to Lynch syndrome, but noting a higher risk of multiple adenomas and no currently-proven risk of cancer outside the large bowel and endometrium in PPAP. Our findings on POLE and POLD1 variants in the germ line are supported by data from the TCGA and ourselves showing somatic POLE proofreading mutations in` ultramutant'' CRCs. We present the results of screening for somatic POLE and POLD1 mutations in panels of CRC and EC cases from the UK. These data (i) identify POLE mutations in 5–10% ECs, (ii) show mutation hotspots in POLE shared by CRC and EC, (iii) identify clinicopathological- molecular associations, and (iv) show differential effects of POLE mutations at specific amino acids on mutation rates and spectra. In contrast, somatic POLD1 proofreading mutations are very rare in CRC and EC. Testing for POLE mutations in the soma and for POLE and POLD1 mutations in the germline is likely to have clinical benefits for CRC patients. Germline and somatic POLE and POLD1 mutaions mapped onto a composite crystal structure of yeast and T4 polymerase show their locations close to the ssDNA binding domain. 925d Caveolin–1 Containing Caveolae and Caveolin–1 containing Cytosolic Endocytic Vesicles Are Important for Dietary Fatty Acid Absorption Shahzad Siddiqi, Charles M. Mansbach The mechanism of uptake of dietary fatty acids (FA) by apical brush border membranes (BB) is controversial. A variety of transport proteins and diffusion mechanisms have all been proposed but none have been confirmed. We tested the hypothesis that caveolin–1 containing lipid rafts, caveolae, is the mechanism by which FA are absorbed and are transported in enterocyte cytosol as caveolin–1 containing endocytic vesicles (CEV). Caveolae are resistant to solubilization in Triton X–100. BB were obtained from wild type (WT) and caveolin–1 knock out (KO) mice (enrichment of alkaline phosphatase 17 fold), incubated with 3H- oleate, and solubilized in 1% Triton X–100. The proteins were separated on an OptiPrep gradient, known to be able to separate detergent resistant membranes (DRM) from detergent soluble membranes (DSM). WT mice had 74% of the oleate in DRM compared to only 15% in the KO mice, similar to when oleate alone was put on the gradient. 15% of 3H-oleate incubated with WT-BB were taken up by the BB which was reduced to 1% by prior cholesterol depletion of the BB (methyl- β-cyclodextrin treatment). KO-BB took up only 1% of the oleate. Caveolin–1 was present in BB of WT but not KO mice. By immunoblot, caveolin–1, CD36, and intestinal alkaline phosphatase (IAP) were in the DRM fraction, clathrin was absent as expected for caveolae. To determine if the oleate-caveolae were endocytosed from BB as CEV, we incubated 3H-oleate with intestinal sacs, isolated enterocyte cytosol, solubilized it in 1% Triton X–100, and fractionated it on the OptiPrep gradient. In WT mice, 47% of the 3H-oleate dpm were in the DRM fraction compared to 17% in the KO mice. When 3H- oleate was added to cytosol, 7% was in the DRM. By immunoblot, caveolin–1, 2, 3, IAP, CD36 were present in the DRM, but intestinal fatty acid binding protein (FABP2), liver S-162 AGA Abstracts FABP (FABP1), and FATP4 were absent in the DRM. After 3 rounds of caveolin–1 immuno- depletion, 91% of the 3H-oleate was removed from the cytosol. By electron microscopy (EM), CEV were 60 nm in diameter, and contained caveolin–1 by immuno-EM. CEV did not increase in size on oleate feeding but their numbers increased 5 fold. No vesicles were seen in the KO mice. WT and KO mice gained weight equally on a chow (4% fat) diet but on a 23% fat diet, female WT mice gained 16gm compared to 7.5gm for the KO mice over 7 weeks. On the high fat diet, WT mice had 12mg fat in their stool but KO mice had 28mg/ 3 days. We conclude that most dietary oleate is absorbed by associating with caveolae in apical BB. The caveolae are endocytosed and appear in cytosol as CEV. We propose CEVs deliver the dietary oleate to the ER for esterification, independent of FABP. We speculate that most dietary FA are sequestered in cytosol in CEV resulting in prevention of enterocyte membrane disruption. 925e Altered intestinal epithelial homeostasis in mice with a target mutation in the gene encoding vasoactive intestinal peptide (VIP) Xiujuan Wu, Kevan Jacobson AIMS: VIP is a neuropeptide known to modulate intestinal epithelial barrier integrity and mucosal immune function during colitis. However, the mechanisms responsible for the biological activities of VIP on intestinal homeostasis and epithelial cell crypt development under physiology condition remain unclear. The aim of the study was to examine the role of VIP on intestinal homeostasis, including epithelial cell proliferation, migration, apoptosis and differentiation under physiology condition.METHODS: Eight-week- old C57BL/6 wild type and VIP-/- mice were studied. BrdU (5;pr-bromo–2;prdeoxyruidine) was injected i.p. into mice at 2 h and 72 h, respectively. Colonic cross sections and swiss roll sections were used to evaluate colonocyte proliferation and migration. Ki–67 immunostaining and the terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay were used to evaluate epithelial cell proliferation and apoptosis respectively. Goblet cells were examined by staining for acidic, neutral and Muc2 mucins using Alcin blue (AB), Periodic-acid Schiff (PAS), and anti-Muc2 stains, respectively. TFF3 expression was assessed by immunoreactivity and real-time PCR, respectively. VIP treatment was performed by i.p. injection to wild type and VIP-/- mice for 10 days.RESULTS: VIP-/- mice have significantly reduced colonic crypt height compared to wild type mice (147.2 ;pm 2.8 ;gmm vs. 168.5 ;pm 2.6 ;gmm, n=6, p,0.001). In addition, VIP-/- mice had reduced rates of proliferation (BrdU positive cells: 3.7 ;pm 0.2/crypt vs. 5.4 ;pm 2.2/crypt, n=4, p ,0.01) and migration compared to wild type mice. The percentage of PAS-positive goblet cells was also significantly decreased in the colon of VIP-/- mice (18.22 ;pm 1.48 vs. 26.58 ;pm 2.23, respectively, n=6, p ,0.05). Moreover, Muc2 and TFF3 expression in goblet cells in VIP-/- mice was significantly decreased at both transcript level (42% decrease, p ,0.05) and translational level compared to wild type mice (3.6 ;pm 0.4/crypt vs. 7.2 ;pm 0.3/crypt, p ,0.001). Treatment with exogenous VIP to VIP-/- mice increased the expression of Muc2 and TFF3 (1.6 fold increase, p ,0.01) and the number of Ki–67 positive cells (6.4 ;pm 0.6/crypt vs. 3.0 ;pm 0.7/crypt, n=5, p ,0.05) compared to untreated mice, resulting in the improvement of histological morphology and function of intestine.CONCLUSIONS: These data provide new sights into the function of VIP establishing its role in regulating colonic crypt development and epithelial cell function and in maintaining intestinal epithelial homeostasis under physiological conditions in vivo.S- upported by CCFC (Canada) 925f Epigenetic remodeling and transmissible murine colonic hyperplasia Badal C. Roy, Ishfaq Ahmed, Shrikant Anant, Shahid Umar Background: Epigenetic mechanisms help establish cellular identities, and failure of proper preservation of epigenetic marks can result in inappropriate activation or inhibition of signaling pathways leading to various pathologies. In an in vivo model of transmissible murine colonic hyperplasia (TMCH) caused by Citrobacter rodentium (CR), we showed previously that CR infection induces Wnt/ β-catenin signaling to regulate crypt hyperplasia and that the increases in β-catenin were not due to mutations in either Apc or CTNNB1 gene. Aim: To evaluate epigenetic mechanism in the regulation of β-catenin induced colonic crypt hyperplasia. Method: TMCH was induced by CR infection in NIH:Swisss mice. Colonic crypts and crypt-denuded lamina propria (CLP) were isolated and cutting edge molecular and biochemical techniques were employed for analysis of epigenetic events. Results: Follow- ing CR infection, analysis of histone deacetylase (HDAC) profile in the crypts revealed significant increases in HDAC1–3 at days 6 and 12 (progression of hyperplasia) and HDAC4 at day–12 (peak hyperplasia) and day–34 (regression of hyperplasia) compared to uninfected control. In the CLP, HDAC3 levels remained elevated at days 6–34 while HDAC4 levels were higher at days 6–12 before declining at day–34. Increases in HDAC1–3 at day–6 and 12 correlated with robust expression of H3K4m3 in the crypt suggesting an active chromatin signature while changes in the CLP were subtle. Expression of H3K4m3 methyltransferase
Transcript of 925e Altered intestinal epithelial homeostasis in mice with a target mutation in the gene encoding...
AG
AA
bst
ract
scarcinoma, using whole-genome sequencing and linkage analysis, followed by associationanalysis in several thousand cases and controls. In some of our largest families we foundcausal germline mutations in two related genes, POLE and POLD1, that encode the mainpolymerases involved in DNA replication. The most common mutations, POLE p.Leu424Valand POLD1 p.Ser478Asn, lie at analogous sites in the proofreading domains of the proteinsand are predicted to hinder the correction of base mispairs that occur during DNA replication.In keeping with this prediction, the two human mutations cause a mutator phenotype inyeast and in our patients' tumours. This is manifest as a tendency to all types of basesubstitution, especially G:C.T:A changes. We present data on screening for further germlinePOLE and POLD1 mutations in a panel of ;sl1000 familial CRC patients as part of an effortto fully characterise the spectrum of pathogenic mutations. In addition to very rare, high-penetrance mutations, we have invesigated an uncommon (freq. ;sl0.1%) germline POLEvariant, p.Asp287Glu that appears to act as a moderate-penetrance CRC risk allele. Carriersof germline POLE and POLD1 mutations have a variable phenotype of multiple, early-onsetadenomas and CRCs, and POLD1 carriers are also at high risk of endometrial cancer (EC).We propose that this condition be called PPAP, for Polymerase Proofreading-AssociatedPolyposis. At present, we recommend genetic testing for POLE and POLD1 in any familywith a Lynch syndrome or attenuated FAP phenotype. We suggest management of PPAPfamilies in a similar way to Lynch syndrome, but noting a higher risk of multiple adenomasand no currently-proven risk of cancer outside the large bowel and endometrium in PPAP.Our findings on POLE and POLD1 variants in the germ line are supported by data fromthe TCGA and ourselves showing somatic POLE proofreading mutations in`̀ ultramutant''CRCs. We present the results of screening for somatic POLE and POLD1 mutations in panelsof CRC and EC cases from the UK. These data (i) identify POLE mutations in 5–10% ECs,(ii) showmutation hotspots in POLE shared by CRC and EC, (iii) identify clinicopathological-molecular associations, and (iv) show differential effects of POLE mutations at specific aminoacids on mutation rates and spectra. In contrast, somatic POLD1 proofreading mutationsare very rare in CRC and EC. Testing for POLE mutations in the soma and for POLE andPOLD1 mutations in the germline is likely to have clinical benefits for CRC patients.
Germline and somatic POLE and POLD1mutaions mapped onto a composite crystal structureof yeast and T4 polymerase show their locations close to the ssDNA binding domain.
925d
Caveolin–1 Containing Caveolae and Caveolin–1 containing CytosolicEndocytic Vesicles Are Important for Dietary Fatty Acid AbsorptionShahzad Siddiqi, Charles M. Mansbach
The mechanism of uptake of dietary fatty acids (FA) by apical brush border membranes(BB) is controversial. A variety of transport proteins and diffusion mechanisms have all beenproposed but none have been confirmed. We tested the hypothesis that caveolin–1 containinglipid rafts, caveolae, is the mechanism by which FA are absorbed and are transported inenterocyte cytosol as caveolin–1 containing endocytic vesicles (CEV). Caveolae are resistantto solubilization in Triton X–100. BB were obtained from wild type (WT) and caveolin–1knock out (KO) mice (enrichment of alkaline phosphatase 17 fold), incubated with 3H-oleate, and solubilized in 1% Triton X–100. The proteins were separated on an OptiPrepgradient, known to be able to separate detergent resistant membranes (DRM) from detergentsoluble membranes (DSM). WT mice had 74% of the oleate in DRM compared to only 15%in the KO mice, similar to when oleate alone was put on the gradient. 15% of 3H-oleateincubated withWT-BB were taken up by the BB which was reduced to 1% by prior cholesteroldepletion of the BB (methyl-β-cyclodextrin treatment). KO-BB took up only 1% of the oleate.Caveolin–1 was present in BB of WT but not KO mice. By immunoblot, caveolin–1, CD36,and intestinal alkaline phosphatase (IAP) were in the DRM fraction, clathrin was absent asexpected for caveolae. To determine if the oleate-caveolae were endocytosed from BB asCEV, we incubated 3H-oleate with intestinal sacs, isolated enterocyte cytosol, solubilized itin 1% Triton X–100, and fractionated it on the OptiPrep gradient. In WT mice, 47% of the3H-oleate dpm were in the DRM fraction compared to 17% in the KO mice. When 3H-oleate was added to cytosol, 7% was in the DRM. By immunoblot, caveolin–1, 2, 3, IAP,CD36 were present in the DRM, but intestinal fatty acid binding protein (FABP2), liver
S-162AGA Abstracts
FABP (FABP1), and FATP4 were absent in the DRM. After 3 rounds of caveolin–1 immuno-depletion, 91% of the 3H-oleate was removed from the cytosol. By electron microscopy(EM), CEV were 60 nm in diameter, and contained caveolin–1 by immuno-EM. CEV didnot increase in size on oleate feeding but their numbers increased 5 fold. No vesicles wereseen in the KO mice. WT and KO mice gained weight equally on a chow (4% fat) diet buton a 23% fat diet, female WT mice gained 16gm compared to 7.5gm for the KO mice over7 weeks. On the high fat diet, WT mice had 12mg fat in their stool but KO mice had 28mg/3 days. We conclude that most dietary oleate is absorbed by associating with caveolae inapical BB. The caveolae are endocytosed and appear in cytosol as CEV. We propose CEVsdeliver the dietary oleate to the ER for esterification, independent of FABP. We speculatethat most dietary FA are sequestered in cytosol in CEV resulting in prevention of enterocytemembrane disruption.
925e
Altered intestinal epithelial homeostasis in mice with a target mutation in thegene encoding vasoactive intestinal peptide (VIP)Xiujuan Wu, Kevan Jacobson
AIMS: VIP is a neuropeptide known to modulate intestinal epithelial barrier integrity andmucosal immune function during colitis. However, the mechanisms responsible for thebiological activities of VIP on intestinal homeostasis and epithelial cell crypt developmentunder physiology condition remain unclear. The aim of the study was to examine the roleof VIP on intestinal homeostasis, including epithelial cell proliferation, migration, apoptosisand differentiation under physiology condition.METHODS: Eight-week- old C57BL/6 wildtype and VIP-/- mice were studied. BrdU (5;pr-bromo–2;prdeoxyruidine) was injected i.p.into mice at 2 h and 72 h, respectively. Colonic cross sections and swiss roll sections wereused to evaluate colonocyte proliferation and migration. Ki–67 immunostaining and theterminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay were used toevaluate epithelial cell proliferation and apoptosis respectively. Goblet cells were examinedby staining for acidic, neutral and Muc2 mucins using Alcin blue (AB), Periodic-acid Schiff(PAS), and anti-Muc2 stains, respectively. TFF3 expression was assessed by immunoreactivityand real-time PCR, respectively. VIP treatment was performed by i.p. injection to wild typeand VIP-/- mice for 10 days.RESULTS: VIP-/- mice have significantly reduced colonic cryptheight compared to wild type mice (147.2 ;pm 2.8 ;gmm vs. 168.5 ;pm 2.6 ;gmm, n=6,p,0.001). In addition, VIP-/- mice had reduced rates of proliferation (BrdU positive cells:3.7 ;pm 0.2/crypt vs. 5.4 ;pm 2.2/crypt, n=4, p ,0.01) and migration compared to wildtype mice. The percentage of PAS-positive goblet cells was also significantly decreased inthe colon of VIP-/- mice (18.22 ;pm 1.48 vs. 26.58 ;pm 2.23, respectively, n=6, p ,0.05).Moreover,Muc2 and TFF3 expression in goblet cells in VIP-/-mice was significantly decreasedat both transcript level (42% decrease, p,0.05) and translational level compared to wildtype mice (3.6 ;pm 0.4/crypt vs. 7.2 ;pm 0.3/crypt, p ,0.001). Treatment with exogenousVIP to VIP-/- mice increased the expression of Muc2 and TFF3 (1.6 fold increase, p ,0.01)and the number of Ki–67 positive cells (6.4 ;pm 0.6/crypt vs. 3.0 ;pm 0.7/crypt, n=5, p ,0.05)compared to untreated mice, resulting in the improvement of histological morphology andfunction of intestine.CONCLUSIONS: These data provide new sights into the function ofVIP establishing its role in regulating colonic crypt development and epithelial cell functionand in maintaining intestinal epithelial homeostasis under physiological conditions in vivo.S-upported by CCFC (Canada)
925f
Epigenetic remodeling and transmissible murine colonic hyperplasiaBadal C. Roy, Ishfaq Ahmed, Shrikant Anant, Shahid Umar
Background: Epigenetic mechanisms help establish cellular identities, and failure of properpreservation of epigenetic marks can result in inappropriate activation or inhibition ofsignaling pathways leading to various pathologies. In an in vivo model of transmissiblemurine colonic hyperplasia (TMCH) caused by Citrobacter rodentium (CR), we showedpreviously that CR infection induces Wnt/β-catenin signaling to regulate crypt hyperplasiaand that the increases in β-catenin were not due to mutations in either Apc or CTNNB1gene. Aim: To evaluate epigenetic mechanism in the regulation of β-catenin induced coloniccrypt hyperplasia. Method: TMCH was induced by CR infection in NIH:Swisss mice. Coloniccrypts and crypt-denuded lamina propria (CLP) were isolated and cutting edge molecularand biochemical techniques were employed for analysis of epigenetic events. Results: Follow-ing CR infection, analysis of histone deacetylase (HDAC) profile in the crypts revealedsignificant increases in HDAC1–3 at days 6 and 12 (progression of hyperplasia) and HDAC4at day–12 (peak hyperplasia) and day–34 (regression of hyperplasia) compared to uninfectedcontrol. In the CLP, HDAC3 levels remained elevated at days 6–34 while HDAC4 levelswere higher at days 6–12 before declining at day–34. Increases in HDAC1–3 at day–6 and12 correlated with robust expression of H3K4m3 in the crypt suggesting an active chromatinsignature while changes in the CLP were subtle. Expression of H3K4m3 methyltransferase
