- Email: [email protected]
Lactase persistence, allelic asociation, and possible heterogeneity
and HA
juvenile dermatomyositis). MM has been shown to cause graft-vs.-host-like reactions in several autoimmune disorders. Graft vs. host disease(GVHD) and BA share histologic and immunologic characteristics. Both demonstrate T-helper lymphocyte mediated processes. In mice models of GVHD, both intrahepatic and extraheparic bile duct sclerosis are seen, similar to BA. AIM: To determine the presence and extent of MM in livers of infants with BA. METHODS: Liver biopsies from all male infants with BA from January 1995 - January 2002 and four male infants with neonatal hepatitis(NH) as controls, were examined using fiourescent in situ hybridization(FISH)for X and Y chromosomes. Clinical diagnosis was based upon histologic and/or surgical assessment A total of 15 liver biopsies were evaluated. Paraffin embedded hepatic tissue underwent initial digestion with pepsin and then stained for markers of the X chromosome(Cy5 - red), Y chromosome(FITC - green) and nuclear material(Dubeque - blue). The biopsies were examined for evidence of MM using the following criteria: two red signals without a green signal located within a blue stain, with the distance between the red signals being more then the size of the signal itself which represents a female cell. RESULTS: Ten of 15 liver biopsies were interpretable. The five uninterpretable biopsies(3BA and 2NH) underwent poor digestion and had no discemable signals. Of the ten readable biopsies, 6/8 (75%) of the BA biopsies had evidence of MM. Maternal cells ranged from 1 - 4 cells per biopsy slide examined in positive specimens. None of the NH biopsies(2)had evidence of MM. CONCLUSION: This is the first study documenting MM in BA. Further studies are required to determine the significance of MM in BA. Supported in part by T32 DK07762 and DK59301.
156
Genetic Disruption of apobec-1 Reduces Intestinal Tumorigenesis in Apcmin/+ Mice JeffreyO Henderson, Hanllin Wang, Shrikant Anant, Nicholas O. Davidson Apobee-1is an RNA-specific cytidine dahminase required for C to U editing of apoB mRNA. Wenow find that apobec- 1 binds AU-rich elements in other transcripts, including the YUTR ofcyclooxygenase-2 (Cox-2) mRNA, ~bere binding enhances mRNA stability in vivo. Cox2 mRNA has been shown to be indficed in the polyps of Apcmin/+ mice compared to adlacentuninvolved mucosa. Furthermore, genetic disruption of Cox-2 in Apomin/+ mice greatlyreduces polyp number. Immunoprecipitation of polyp extracts from Apcmin/+ mice ~th anti-apobec-1 IgG demonstrated the presence of Cox-2 mRNA in the immune complex and establish that apobec-1 binds Cox-2 mRNA in-vivo. We therefore hypothesized that genetic ablation of apobec-1 in the Apcmin/+ background would protect against tumor development, possibly through alterations in mRNA stability of AU-rich targets, including Cox-2. Accordingly, we generated co/npound Apcmin/+apobec-l-/- mice in a congenic C57 background and analyzed tumor number and size compared to isogenic Apcmin/+ littermates at 115 days of age. Apcmin/+ apobec-1-/- mice revealed a 35% (p = 0.0014) reductionin small intestinal polyp number compared to Apcmin/+ litterinates. Morphological analysis showed a significant increase (p = 0.0336) in the number of fiat, regressed polypsin Apcmin/+ apobec- 14- mice compared to Apcmin/+ littermates. Analysis of proliferation and apoptosis by BrdU labeling and TUNEL staining, respeeuvely, demonstrated that proliferation was decreased 26% (p = 0.0175) while apoptosis increased 2-fold (p = 0.0062)in polyps of Apcmin/+ apobec-1-/- mice compared to Apcmird + hitermates. These datasuggest that ApcmilV + apobec- 1-/- mice are protected against initiation of tumor formatt0n and that growth of polyps is compromised. Gene chip and real-time PCR analysis revealed significant reductions in the abundance of several AU-rich mRNAs (TNF, VEGF, c-mycand Cox-2) in the polyps of Apcmin/+Apobec-1-/- mice compared to Apcmin/+ littermates. Concomitantly, Cox-2 protein (p = 0.0256) and PGE2 levels (p = 0.0371) werealso significantly reduced in the polyps of the Apcmird+Apobeol-/- mice compared to Aprmird+ littermates. These results suggest that apobec-1 contributes to tumorigenesis bybinding to and altering the stability of AU-nch targets, including Cox-2 mRNA, the first example of an RNA binding protein modulating tumor development.
159 Plasminogen Deficiency Results in a Pancreatic Switch during Liver Repair in Adult Mice Kumar Shanmukhappa, Gregg Sabla, Jay L. Degen, Jorge A. 8ezerra The development of the liver and pancreas from endodermal cells obeys hierarchical rules governed by transcripuon and growth factors during embryogenesis. Although some of these rules also direct repair posmatally, the cellular plasticity in fully differentiated tissue remains poorly defined. We demonstrated that plasminogen regulates postnatal liver repair in vivo, with defective removal of necrotic cells after a toxic injury. Here, we aimed to identify the molecular consequences of plasminogen deficiency m the injured microenvironment. To this end, we performed large-scale gene expression analyses in livers of adult plasminogendeficient mice and control littermates 2-14 days after one dose of carbon tetrachloride. Hepatic biotinylated cRNA pools were generated for each time point and hybridized in duplicate against Affymetrix U94Av2 gene chips (12,500 genes). Levels of gene expression were analyzed using GeneSpring software. Among the genes uniquely overexpressed in plasminogen-deficient livers, the expression levels of 5 genes were increased 3.4-50 fold exclusively at 14 days after injury, a time of prominent defect in repair. Notably, these genes encoded the pancreatic-specific hydrolasesdproteases trypsinogen (increased 50 fold), alphaamylase-2 (50 fold), elastases 1 (4 fold) and 2 (13 fold), and bile salt-activated lipase precursor (3.4 fold). The findings were validated by PCR, showing a temporal production of gene transcripts restricted to the peak of defective repair (14 days). To further define the molecular mechanism of reprogramming, we searched for genetic regulatory elements that are evolutionarily conserved and common to all five hydrolasesdproteases. This strategy identified GATA3 and p48 elements within 350 bp from the initiating ATG. p48 is a critical regulator of pancreatic acinar differentiation during embryogenesis. Therefore, we determined the activation of p48, and found a temporal-rest~'icted pattern of expression in plasminogendeficient livers 14 days after injury. In conclusion, plasminogen deficiency results in a genetic reprogramming to a pancreatic phenotype through the activation of p48 to drive a compensatory increase in bydrolases/proteases during defective liver repair. Together, these data reveal a unique regulatory role of plasminogen in the maintenance of cellular differentiation, and underscore the postnatal inter-tissue plasticity of the digestive system.
157 The Mismatch Repair (MMR) Complex hMutSat Recognizes 5-Fluorouracil (SFU) Modified DNA: a Mechanism for 5FU Sensitivity AkihiroTajima, Betty L. Cabrera, Heather McMahon, Ryan Doctolero, Sherry Huang, John Carethers Backgrounds&Aims: Patients with advanced stages of colorectal cancer are treated with 5FU-based chemotherapy, which can improve mortality. The cellular mechanism for the elkcdveness of 5FU has traditionally been thought to be a function of inhibition of thymidylate symhetase and interference with RNA metabolism. Previously, we reported that MMRdeficientcells are more resistant to 5FU treatment compared to MMR-proficient cells, and that 5FU is incorporated into DNA. Here, we examined if the MMR system could recognize 5FU incorporated into DNA as a potential mechanism for targeting cancer cell death. Methods:We synthesized oligomers containing one or more molecules of 5FU incorporated into the DNA strand, and annealed its complementary strand to yield double stranded DNA (I)S-DNA). We purified hMutSa (a heterodimer of hMSH2 and hMSH6 MMR proteins) by usinga recombinant Baculovirus system containing the two MMR proteins, followed by fast proteinliquid chromatography using two purification columns. We performed electromobility gel shift assays (EMSA) using radiolabelled DS-DNA molecules and purified hMutSa complex.A single GT mismatch in DS-DNA was used as a positive control, and the perfect complement was used as a negative control. Results:We prepared hMutSQ to >95% purity as detected by Western blotting with hMSH2 and hMSH6 antibodies and Coomassie staining. EMSA demonstrated binding of hMutS~x i0 the GT mismatched DNA and to the 5FU-containing DNA Since hMutSa hydrolyzes AIP to separate from bound DNA, we demonstrated this specificity of binding of purified hMutSn to 5FU-containing DS-DNA by addition of 4mM ATP, with subsequent resolution 0l the protein-DNA complex. Conclusions: The MMR complex hMutSa binds specifically to DNA containing 5FU molecules The selectivity of 5FU for the treatment of advanced CRC may be in part due to this .~MRrecognition ol 5FU incorporated into DNA.
160 Lactase Persistence, AUelic Association, and Possible Heterogeneity Mark PoulteL Edward J. Hollox, Clare B. Harvey, Charlotte Mulcare, Katri Peuhkuri, Kajsa Kajander, Martin Sarner, Riitta Korpela, Dallas M Swallow Expression of lactase in the intestine persists into adult life in some people and not others and this is due to a cis-acting regulatory polymorphism Persistence is associated with a haplntype of the lactase gene (LCT) known as A (Harvey et al. 1998, Arm Hum. Genet.62, 215, Hofiox et al. 2001, Amer. J. Hum. Genet. 68,160). Recent studies by others reported complete sequencing of a 47kb region upstream of LCT, identified by linkage disequilibrium mapping, in individuals of known lactase persistence status (Enattah et al. 2002, Nat. Genet. 30, 233). A 100% correlation was found of lactase persistence with the presence of the T allele of a CT SNP at -14kb from LCT, in individuals of Finnish origin and also a very tight association with a second SNP (GA -22kb). Here we report that the -14kb T allele and the 22kb A allele both occur on the background of a very extended (700kb) A haplotype. In a series of Finnish individuals we found a strong correlation (40/41 people) with lactose digestion and the presence of the T allele at -14kb. The T allele was present in all of a series of 36 lactase persistent individuals from the UK (phenotyped by enzyme assay), 31/36 of whom were of Northern European ancestry, but not in 11 non-persistent individuals, who were mainly of non UK ancestry All the lactase persistence heterozygotes previously phenotyped by determining allefic transcript expression were CT and showed intermediate lactase activity. However the additonal CT heterozygotes of unknown transcript status did not show intermediate lactase enzyme activity. Furthermore one lactase persistent homozygote identified by having equal high expression of A and B haplotype transcripts, was beterozygous CT at the -14kb site. SNP analysis in this person showed no evidence of recombination on either chromosome between the -14kb SNP and LCT. Thus although the -14kb CT SNP is an excellent candidate for the cause of the lactase persistence polymorphism, it does not, on its own, explain all the variation in expression of LCT, suggesting the possibility of genetic heterogeneity, or that the CT SNP is simply a highly associated marker. We have used the combined genetic data to identify other regions in the vicinity of LCT for further investigation.
158 Evidence of Maternal Microchimerism in Livers of Infants with Bfliary Atresia Dalad L Suskind, Philip Rosenthal, Melvin B. Heyman, Greg Magrane, Marcus O. ~uench While many hypotheses, including infectious and developmental, have been proposed, the etiologyof biliary atresia(BA) remains enigmatic. Maternal microchimerism(MM), caused by the translocation of maternal hematopoietic cells in utero to the fetus, has been found in the tissues of patients with autoimmune disorders(e.g, systemic lupus erythromatosis and
A-2 1
AGA
Abstracts
juvenile dermatomyositis). MM has been shown to cause graft-vs.-host-like reactions in several autoimmune disorders. Graft vs. host disease(GVHD) and BA share histologic and immunologic characteristics. Both demonstrate T-helper lymphocyte mediated processes. In mice models of GVHD, both intrahepatic and extraheparic bile duct sclerosis are seen, similar to BA. AIM: To determine the presence and extent of MM in livers of infants with BA. METHODS: Liver biopsies from all male infants with BA from January 1995 - January 2002 and four male infants with neonatal hepatitis(NH) as controls, were examined using fiourescent in situ hybridization(FISH)for X and Y chromosomes. Clinical diagnosis was based upon histologic and/or surgical assessment A total of 15 liver biopsies were evaluated. Paraffin embedded hepatic tissue underwent initial digestion with pepsin and then stained for markers of the X chromosome(Cy5 - red), Y chromosome(FITC - green) and nuclear material(Dubeque - blue). The biopsies were examined for evidence of MM using the following criteria: two red signals without a green signal located within a blue stain, with the distance between the red signals being more then the size of the signal itself which represents a female cell. RESULTS: Ten of 15 liver biopsies were interpretable. The five uninterpretable biopsies(3BA and 2NH) underwent poor digestion and had no discemable signals. Of the ten readable biopsies, 6/8 (75%) of the BA biopsies had evidence of MM. Maternal cells ranged from 1 - 4 cells per biopsy slide examined in positive specimens. None of the NH biopsies(2)had evidence of MM. CONCLUSION: This is the first study documenting MM in BA. Further studies are required to determine the significance of MM in BA. Supported in part by T32 DK07762 and DK59301.
156
Genetic Disruption of apobec-1 Reduces Intestinal Tumorigenesis in Apcmin/+ Mice JeffreyO Henderson, Hanllin Wang, Shrikant Anant, Nicholas O. Davidson Apobee-1is an RNA-specific cytidine dahminase required for C to U editing of apoB mRNA. Wenow find that apobec- 1 binds AU-rich elements in other transcripts, including the YUTR ofcyclooxygenase-2 (Cox-2) mRNA, ~bere binding enhances mRNA stability in vivo. Cox2 mRNA has been shown to be indficed in the polyps of Apcmin/+ mice compared to adlacentuninvolved mucosa. Furthermore, genetic disruption of Cox-2 in Apomin/+ mice greatlyreduces polyp number. Immunoprecipitation of polyp extracts from Apcmin/+ mice ~th anti-apobec-1 IgG demonstrated the presence of Cox-2 mRNA in the immune complex and establish that apobec-1 binds Cox-2 mRNA in-vivo. We therefore hypothesized that genetic ablation of apobec-1 in the Apcmin/+ background would protect against tumor development, possibly through alterations in mRNA stability of AU-rich targets, including Cox-2. Accordingly, we generated co/npound Apcmin/+apobec-l-/- mice in a congenic C57 background and analyzed tumor number and size compared to isogenic Apcmin/+ littermates at 115 days of age. Apcmin/+ apobec-1-/- mice revealed a 35% (p = 0.0014) reductionin small intestinal polyp number compared to Apcmin/+ litterinates. Morphological analysis showed a significant increase (p = 0.0336) in the number of fiat, regressed polypsin Apcmin/+ apobec- 14- mice compared to Apcmin/+ littermates. Analysis of proliferation and apoptosis by BrdU labeling and TUNEL staining, respeeuvely, demonstrated that proliferation was decreased 26% (p = 0.0175) while apoptosis increased 2-fold (p = 0.0062)in polyps of Apcmin/+ apobec-1-/- mice compared to Apcmird + hitermates. These datasuggest that ApcmilV + apobec- 1-/- mice are protected against initiation of tumor formatt0n and that growth of polyps is compromised. Gene chip and real-time PCR analysis revealed significant reductions in the abundance of several AU-rich mRNAs (TNF, VEGF, c-mycand Cox-2) in the polyps of Apcmin/+Apobec-1-/- mice compared to Apcmin/+ littermates. Concomitantly, Cox-2 protein (p = 0.0256) and PGE2 levels (p = 0.0371) werealso significantly reduced in the polyps of the Apcmird+Apobeol-/- mice compared to Aprmird+ littermates. These results suggest that apobec-1 contributes to tumorigenesis bybinding to and altering the stability of AU-nch targets, including Cox-2 mRNA, the first example of an RNA binding protein modulating tumor development.
159 Plasminogen Deficiency Results in a Pancreatic Switch during Liver Repair in Adult Mice Kumar Shanmukhappa, Gregg Sabla, Jay L. Degen, Jorge A. 8ezerra The development of the liver and pancreas from endodermal cells obeys hierarchical rules governed by transcripuon and growth factors during embryogenesis. Although some of these rules also direct repair posmatally, the cellular plasticity in fully differentiated tissue remains poorly defined. We demonstrated that plasminogen regulates postnatal liver repair in vivo, with defective removal of necrotic cells after a toxic injury. Here, we aimed to identify the molecular consequences of plasminogen deficiency m the injured microenvironment. To this end, we performed large-scale gene expression analyses in livers of adult plasminogendeficient mice and control littermates 2-14 days after one dose of carbon tetrachloride. Hepatic biotinylated cRNA pools were generated for each time point and hybridized in duplicate against Affymetrix U94Av2 gene chips (12,500 genes). Levels of gene expression were analyzed using GeneSpring software. Among the genes uniquely overexpressed in plasminogen-deficient livers, the expression levels of 5 genes were increased 3.4-50 fold exclusively at 14 days after injury, a time of prominent defect in repair. Notably, these genes encoded the pancreatic-specific hydrolasesdproteases trypsinogen (increased 50 fold), alphaamylase-2 (50 fold), elastases 1 (4 fold) and 2 (13 fold), and bile salt-activated lipase precursor (3.4 fold). The findings were validated by PCR, showing a temporal production of gene transcripts restricted to the peak of defective repair (14 days). To further define the molecular mechanism of reprogramming, we searched for genetic regulatory elements that are evolutionarily conserved and common to all five hydrolasesdproteases. This strategy identified GATA3 and p48 elements within 350 bp from the initiating ATG. p48 is a critical regulator of pancreatic acinar differentiation during embryogenesis. Therefore, we determined the activation of p48, and found a temporal-rest~'icted pattern of expression in plasminogendeficient livers 14 days after injury. In conclusion, plasminogen deficiency results in a genetic reprogramming to a pancreatic phenotype through the activation of p48 to drive a compensatory increase in bydrolases/proteases during defective liver repair. Together, these data reveal a unique regulatory role of plasminogen in the maintenance of cellular differentiation, and underscore the postnatal inter-tissue plasticity of the digestive system.
157 The Mismatch Repair (MMR) Complex hMutSat Recognizes 5-Fluorouracil (SFU) Modified DNA: a Mechanism for 5FU Sensitivity AkihiroTajima, Betty L. Cabrera, Heather McMahon, Ryan Doctolero, Sherry Huang, John Carethers Backgrounds&Aims: Patients with advanced stages of colorectal cancer are treated with 5FU-based chemotherapy, which can improve mortality. The cellular mechanism for the elkcdveness of 5FU has traditionally been thought to be a function of inhibition of thymidylate symhetase and interference with RNA metabolism. Previously, we reported that MMRdeficientcells are more resistant to 5FU treatment compared to MMR-proficient cells, and that 5FU is incorporated into DNA. Here, we examined if the MMR system could recognize 5FU incorporated into DNA as a potential mechanism for targeting cancer cell death. Methods:We synthesized oligomers containing one or more molecules of 5FU incorporated into the DNA strand, and annealed its complementary strand to yield double stranded DNA (I)S-DNA). We purified hMutSa (a heterodimer of hMSH2 and hMSH6 MMR proteins) by usinga recombinant Baculovirus system containing the two MMR proteins, followed by fast proteinliquid chromatography using two purification columns. We performed electromobility gel shift assays (EMSA) using radiolabelled DS-DNA molecules and purified hMutSa complex.A single GT mismatch in DS-DNA was used as a positive control, and the perfect complement was used as a negative control. Results:We prepared hMutSQ to >95% purity as detected by Western blotting with hMSH2 and hMSH6 antibodies and Coomassie staining. EMSA demonstrated binding of hMutS~x i0 the GT mismatched DNA and to the 5FU-containing DNA Since hMutSa hydrolyzes AIP to separate from bound DNA, we demonstrated this specificity of binding of purified hMutSn to 5FU-containing DS-DNA by addition of 4mM ATP, with subsequent resolution 0l the protein-DNA complex. Conclusions: The MMR complex hMutSa binds specifically to DNA containing 5FU molecules The selectivity of 5FU for the treatment of advanced CRC may be in part due to this .~MRrecognition ol 5FU incorporated into DNA.
160 Lactase Persistence, AUelic Association, and Possible Heterogeneity Mark PoulteL Edward J. Hollox, Clare B. Harvey, Charlotte Mulcare, Katri Peuhkuri, Kajsa Kajander, Martin Sarner, Riitta Korpela, Dallas M Swallow Expression of lactase in the intestine persists into adult life in some people and not others and this is due to a cis-acting regulatory polymorphism Persistence is associated with a haplntype of the lactase gene (LCT) known as A (Harvey et al. 1998, Arm Hum. Genet.62, 215, Hofiox et al. 2001, Amer. J. Hum. Genet. 68,160). Recent studies by others reported complete sequencing of a 47kb region upstream of LCT, identified by linkage disequilibrium mapping, in individuals of known lactase persistence status (Enattah et al. 2002, Nat. Genet. 30, 233). A 100% correlation was found of lactase persistence with the presence of the T allele of a CT SNP at -14kb from LCT, in individuals of Finnish origin and also a very tight association with a second SNP (GA -22kb). Here we report that the -14kb T allele and the 22kb A allele both occur on the background of a very extended (700kb) A haplotype. In a series of Finnish individuals we found a strong correlation (40/41 people) with lactose digestion and the presence of the T allele at -14kb. The T allele was present in all of a series of 36 lactase persistent individuals from the UK (phenotyped by enzyme assay), 31/36 of whom were of Northern European ancestry, but not in 11 non-persistent individuals, who were mainly of non UK ancestry All the lactase persistence heterozygotes previously phenotyped by determining allefic transcript expression were CT and showed intermediate lactase activity. However the additonal CT heterozygotes of unknown transcript status did not show intermediate lactase enzyme activity. Furthermore one lactase persistent homozygote identified by having equal high expression of A and B haplotype transcripts, was beterozygous CT at the -14kb site. SNP analysis in this person showed no evidence of recombination on either chromosome between the -14kb SNP and LCT. Thus although the -14kb CT SNP is an excellent candidate for the cause of the lactase persistence polymorphism, it does not, on its own, explain all the variation in expression of LCT, suggesting the possibility of genetic heterogeneity, or that the CT SNP is simply a highly associated marker. We have used the combined genetic data to identify other regions in the vicinity of LCT for further investigation.
158 Evidence of Maternal Microchimerism in Livers of Infants with Bfliary Atresia Dalad L Suskind, Philip Rosenthal, Melvin B. Heyman, Greg Magrane, Marcus O. ~uench While many hypotheses, including infectious and developmental, have been proposed, the etiologyof biliary atresia(BA) remains enigmatic. Maternal microchimerism(MM), caused by the translocation of maternal hematopoietic cells in utero to the fetus, has been found in the tissues of patients with autoimmune disorders(e.g, systemic lupus erythromatosis and
A-2 1
AGA
Abstracts