- Email: [email protected]
K-ras transgenic mouse model recapitulates early pancreatic ductal carcinogenesis
of/3-catenin in relationship with cyclin D1 overexpression,tumor grade, clinicopathological parameters and patients survival in 43 adenocarcinomasof the pancreas and in 5 normal pancreatic tissues by immunohistochemistry. RESULTS:We could show that, both, reduced membranous fl-catenin expression (25 of 43, 58.1%) and accumulation of ~catenin in the cytoplasm (28 of 43, 65.1%) correlated significantly with cyclin D1 overexpression(both p < 0.0005). Furthermore, we could show a clear correlation between reduced membranous expressionand ectopic cytoplasmic expressionof ,8-catenin (p < 0.0005). More importantly, analysis of postoperative survival revealed a poorer prognosis for patients whose tumors showed irregular expression of/?,-catenin (1-year-survival: no cytoplasmic expression 86.6% vs. cytoplasmic expression 35.7%, p < 0.01). Coprecipitationexperiments revealedreduced /t,-catenin bound to the E-cadherin-catenincomplex in pancreatictumor tissues comparedto normal pancreatictissues. CONCLUSION:Out data may support the supposition that ,8-catenin can be involved in the tumorigensis of pancreatic cancer and exhibits its effects mainly by the transactivaton of cyclin D1.
and/3-catenin form a complex and have DNA binding activity. ESCCcell lines did not show constitutive activation of/3-catenin TCF dependenttranscription. Co-transfection, co-culture or culture with condition media experiments demonstrated that Wnt 1, but not Wnt 2B, Wet 5A, and Wnt 7A, activatedthe Tcf reporter geneas well as the cyclin D1 promoter. Additionally, when cultured with Wnt 1 conditioned media, ESCCcell lines showed an accumulation of catenin in the cytoplasm. RPA demonstratedthat both ESCCcell lines and normal esophageal cells expressedseveral FZDs. Among them, expression of FZD 9 was restricted to ESCCcell lines but absent in normal cells. Conclusions. A comparison of extracelluiar stimuli reveals the novel finding that Wnt 1, but not other Writ family members, stabilizes ~catenin with resulting activation of TCt mediatedtranscription in ESCC.Wnt 1 might contribute to proliferation of ESCC,possibly mediated though FZD 9 expression.
238 ~/-catenin Stabilizes ,6-cetenin and Induces Tcf/Lef Reporter Activity Through a Mechanism that Requires Armadillo Repeats Bat Not the Antlvation Domain of catenin. Shaun G. Weller, RebeccaChinery, William E. Kames Jr., Mayo Clio, Rochester, MN
241 Ckang4is in Wof/~Cofenin Signaling During Regulated Growth in Liver Regeneration George K. Michalopoulos, Satdarshan P. S. Mungo, Peter Pediaditakis,Donna 6. Stolz, Karen Mule, Univ of Pittsburgh, Pittsburgh, PA Wnt/p-catenin pathway is important during embryogenesis and carcinogenesis. ~Catenin interactions with E-cadherin have been shown to be crucial in cell-cell adhesion. We report novel findings in writ pathway during rat liver regeneration after 70% partial hepatectomy utilizing western blot analyses, immunoprecipitation studies and immunofluorescence. Wnt1 and ~-catenin proteins are predominantly localizedin hepatocytes.Immediatelyafter partial hepatectornythere is an initial increase in fl-catenin protein during the first 5 minutes with translocation to the nucleus. This increase is due to decreased degradation of b-catenin (decreasein serine phosphoryletedb-catenin) as seen by immunoprecipitationstudies.Activation of/P,,atenin degradationcomplexcomprising of adenomatouspolyposis coil geneproduct (APC) and serine phosphoryiatedaxin protein begins at 5 minutes after hepatectomy,leading to its decreasedlevelsafter this time. Quantitativechanges in E-cadherinare in generalreverse to those seenin/t~catenin protein during liver regeneration.Also, utilizing immunoprecipitation, we observe elevated levels of tyrosine phosphorylated ~catenin at 6 hours onwards. Thus quantitativechangesas well as stoichiometric changes in serine and tyrosine phosphorylation of the componentsduring regulatedgrowth, are aimed at tightly regulating cytosolic/3-catenin levels and may be contributing to induce and monitor cell proliferation and target gene expression.Thesechanges might atso be intendedto modulatecell-cell adhesionfor structural reorganization that is mandatory during liver mass restoration process after hepatectomy.
BACKGROUND:/3-cateninand -y-cateninare vertebratehomologuesof the Drosophilaarmadillo protein, and play critical roles in cell adhesion and Wnt-signal transduction. In colorectal cancer, mutations in the APCtumor suppressoror in ~catenin stabilize/~:atenin and enhance its ability to activate transcription of Tcf/Lef target genes. Recent evidencesuggests that -ycatenin can also activate transcription of Tcf/Lef target genes in rodent kidney cells. AIMS: To determinethe effects of ~/-cateninon ~-catenin levels and the activity of a Tct/Lef reporter in RKO human colorectal cancer cells, which have wild-type APC and ~catenin. METHODS: RKO colon cancer cells were transfected with wild-type and deletion mutants of FLAG-tagged ~/-catenin and/or FLAG-tagged/3-cateninand assayedfor endogenousand FLAG-tagged,8catenin levels by immunopracipitation and Western analysis. Tct/Lef transcriptional activity was assayed using TopFlash luciterase reporter constructs. RESULTS: Over-expressionof wild-type ~,-catenin doubled the protein levels of endogenous/8-catenin and increased the protein levels of transiently expressed FLAG-tagged~-catenin several-fold. These effects of ~-catenin on/3-catenin stability were enhancedby deletion of the activation domain (~C687), and were nearly abolished by deletion of the central armadillo repeat domain of x-catenin (Arm-). TCt/Lef reporter activity was also induced several-fold by -y-catenin expression and was highest with the AC687 mutant, which lacks the activation domain. CONCLUSION:-ycatenin stabilizes/3-cateninand inducesTcf-Lef reporter activity. Transcriptionaleffects correlate with ~catenin levelsand occur independentlyof ~t-catenins activation domain, suggesting that ~,-catenininducesTct-Lef reporter activity indirectly by stabilizing ff-catenin. We speculate that the armadillo repeatdomain of -y-cateninmay stabilize/?~cateninby competitivelydisplacing/3-catenin from APC.
242 239
K-ras Trons0enic Mouse Model Recapitulates Early Pancreatic Ductal
Cand~eoa~s
FasL Is a/3-cetenin/TCF-4 Target GeH Repressed by Smads in RK0-1 Cofonictof Cancer Cells. Rebecca Chinery, Shaun G. Weller, Mayo Clio, Rochester, MN
Felix H. Brembeck, Franz S. Schraiber, Linden E. Craig, Anti K. Rustgi, Univ of Pennsylvania, Philadelphia,PA Introduction: Pancreatic ductal adenocarcinomais the fourth leading cause of cancer death in Western countries. The most common genetic alteration is mutation in the K-ras oncogene including the preneoplastic stages of pancreatic cancer. Geneticallybased animal models of pancreaticductal adenocaroinomaare lacking. We haveestablishedthat the keratin 19 promoter is transcriptionally regulated in pancreatic ductal cells and it targets the lacz reporter gene in these calls.Therafore, we sought to generate and characterize a K19 - Ki-ras transgenic mouse model. Methods: A 2.1 kb K19 5 UTR fragment was fused to the mutated human Kras (Gly • Val at codon 12) and this purified DNA fragment was used as transgene. Three lines of transgenic mice were established.Genotypingwas determined by PCR and Southern Blotting. Age-matched wild-type and transgenic mice were characterizedby histology, BrdU immunohisto-chemistry and Western blot analyses,the latter for evidenceof activated K-ras and ERK1/2 as well as expression of cyclin DI. Results: Activated K-ras and Erk 1/2 as well as cyclin D1 expressionwere increasedin transgenic mice comparedto age-matchedwildtype mice. Sixteen transgenic mice at the age of 4, 6, 9 and 12 months were sacrificed. BrdUimmunostaining revealed increased pmliteration within ductal cells starting at 6 months. A subset of mice also demonstrated histologic evidenceof ductal hyperplasiaand an increased stromal reaction.Conclusion:Targetoverexprassionof mutated K-ras in transgenic mice leads to biochemical evidence of K-ras and ERK 1/2 activation and overexpressionof cyclin D1, a downstreamtarget of this pathway.Preliminaryanalysisof transgenic mice indicatesevidence of ductal hyperplasiaand increasedcell proliferation. This unique model will serve as a novel approach to understanding the genetic basis of pancreatic cancer.
BACKGROUND:Fas ligand (FasL) is a well-characterizeddeath ligeod in lymphoid tissues bat is also expressed in coloractat neoplasms, where it may play a role in immune evasion. Coexpressionof FasL, nuclear ~-catenin and the matrix metalloproteinasematrilysin is common among colorectal neoplasms, and strongly correlates with decreased CD8-bearing tumorinfiltrating lymphocytes, increasedtumor vascularity and poor prognosis. The concordance betweenFasLand matrilysin expressionsuggeststhat these genesmay sharesimilar transcriptional regulation. AIMS: To determinewhether FasL, like matrilysin, is a transcriptional target of the APC//3-catenin pathway. METHODS:Transfection studies were performed in RKO-1 (wild-type ~catenin and APC) human coloractal cancer cells. Effects of ~-catenin and Smads (2 and 4) on FasL expression were determined by Western analysis. Human FasL promoter activity was assessed using a luciterase reporter gene under the control of a 1.28kbp FasL promoter or deletion mutants. Specificity of ~catenin/rCF effects was assessed by cotransfection with dominant negative (dn) TCF-4 or the E-cadherin cytoplasmic tail (ECCT). Binding of TCF//3-cateninto putativeTCFresponseelements (TCFREs)was verified by electrophoratic mobility shift assay (EMSA). RESULTS:Transient expression of/3-catenin and Tcf4 in RKO-1 cells resulted in 2-5 fold increased mRNA and protein levels of FasL that were blocked by coexprassion with ECCTor dnTCF-4. ,8-catenin/i'CF transactivation of the FasL promoterwas localizedto a TCFRE(-210bp). Site-mutationof this TCFREand EMSAsconfirmed that TCF-4 and ~-catenin competitively bound to this element as a complex. Using deletion mutants, a repressor element was located between -926 and -817bp containing a putative Smad responseelement.Co-transfectionof Smad-2/4abolishedTCF/,8-cateninresponsiveness of the FasLpromoter and this effect was also mappedbetween-926 and -817. CONCLUSIONS: FasL is a/~-catenin/TCF-4 target gene upregulated in colorectal cancer. A putative Smad response element negatesactivation by ,8-catenin/TCF-4.Thesestudies provide new insights into how loss of TGF-fl signaling (mutant Smads or TGF/3RII) and activation of the APC/#catenin pathway may combine to promote colorectal carcinogenesisthrough transcriptional regulation of target genes such as FasL.
243
Differentiating Role Of B7-CD28/CTLA-4 Interaction in The Induction Of Mucosal Inflammation And Regulatory T Cell Functions In Chronic Experimental Colitis Zhanju Liu, Univ Hosp Gasthuisberg, Univ of Leuven, Lauven Belgium; Philippe Maerten, Stefann Colpaert, Jan L. Ceuppens, Lab of ExperimentalImmunology, Lauven Belgium; Karel Geboes,Dept of Pathology, Leuven Belgium; Paul Rutgeerts, Dept of Gastroenterology, Leuven Belgium 67-CD28/CTLA-4interaction plays a role in the regulation of immune responses.Our previous results have demonstratedthat in vivo treatment with anti-B7.1 but not anti-B72 effectively prevents intestinal mucosal inflammation in colitic SCID mice, and that anti-CTLA-4 leads to the deterioration of colitis. Aims: To further evaluatethe in vivo relevanceof B7-CD28/CTLA4 interaction in the induction of mucosal inflammation and of regulatory T cell activity in chronic colitis. Methods: CD45RBh~h,CD45RB"~ and CD25+CD4÷ T cells were isolated from the splenic CD4+ cells of wild-type (WT) and CD28-~ mice. SCID mice were reconstituted with either WT or CD28 ~ CD45RB"~" cells alone, or cotransferredwith regulatoryCD25÷CD4+ T cells from WT or CD28 / mice. Clinical,histological and cytological analysiswere performed.
240 Ectopic Cytoplasmic Expression of 18-cetenin Conelates with Cyctin D1 Overexpression in Pancreatic Cancer. Marco Ramadani, Univ of UIm, UIm Germany; Qilu Ciao, Univ of Beijing, 6eijing People'S Rep Of China; Frank Gansauge,SusanneGaneauge,Gerd Leder, Univ of UIm, UIm Germany BACKGROUND:fl-catenin is a component of the E-caitherin-catenincell adhesion complex. It plays also a role in the intracellular signalling and can function as an oncogene when it binds to the T cell factor 4 (Tcf4)-binding site in the promoter region of cyclin D1 and transactivates,genesafter translocationto the nucleus. METHODS:We evaluatedthe expression
A-46
and/3-catenin form a complex and have DNA binding activity. ESCCcell lines did not show constitutive activation of/3-catenin TCF dependenttranscription. Co-transfection, co-culture or culture with condition media experiments demonstrated that Wnt 1, but not Wnt 2B, Wet 5A, and Wnt 7A, activatedthe Tcf reporter geneas well as the cyclin D1 promoter. Additionally, when cultured with Wnt 1 conditioned media, ESCCcell lines showed an accumulation of catenin in the cytoplasm. RPA demonstratedthat both ESCCcell lines and normal esophageal cells expressedseveral FZDs. Among them, expression of FZD 9 was restricted to ESCCcell lines but absent in normal cells. Conclusions. A comparison of extracelluiar stimuli reveals the novel finding that Wnt 1, but not other Writ family members, stabilizes ~catenin with resulting activation of TCt mediatedtranscription in ESCC.Wnt 1 might contribute to proliferation of ESCC,possibly mediated though FZD 9 expression.
238 ~/-catenin Stabilizes ,6-cetenin and Induces Tcf/Lef Reporter Activity Through a Mechanism that Requires Armadillo Repeats Bat Not the Antlvation Domain of catenin. Shaun G. Weller, RebeccaChinery, William E. Kames Jr., Mayo Clio, Rochester, MN
241 Ckang4is in Wof/~Cofenin Signaling During Regulated Growth in Liver Regeneration George K. Michalopoulos, Satdarshan P. S. Mungo, Peter Pediaditakis,Donna 6. Stolz, Karen Mule, Univ of Pittsburgh, Pittsburgh, PA Wnt/p-catenin pathway is important during embryogenesis and carcinogenesis. ~Catenin interactions with E-cadherin have been shown to be crucial in cell-cell adhesion. We report novel findings in writ pathway during rat liver regeneration after 70% partial hepatectomy utilizing western blot analyses, immunoprecipitation studies and immunofluorescence. Wnt1 and ~-catenin proteins are predominantly localizedin hepatocytes.Immediatelyafter partial hepatectornythere is an initial increase in fl-catenin protein during the first 5 minutes with translocation to the nucleus. This increase is due to decreased degradation of b-catenin (decreasein serine phosphoryletedb-catenin) as seen by immunoprecipitationstudies.Activation of/P,,atenin degradationcomplexcomprising of adenomatouspolyposis coil geneproduct (APC) and serine phosphoryiatedaxin protein begins at 5 minutes after hepatectomy,leading to its decreasedlevelsafter this time. Quantitativechanges in E-cadherinare in generalreverse to those seenin/t~catenin protein during liver regeneration.Also, utilizing immunoprecipitation, we observe elevated levels of tyrosine phosphorylated ~catenin at 6 hours onwards. Thus quantitativechangesas well as stoichiometric changes in serine and tyrosine phosphorylation of the componentsduring regulatedgrowth, are aimed at tightly regulating cytosolic/3-catenin levels and may be contributing to induce and monitor cell proliferation and target gene expression.Thesechanges might atso be intendedto modulatecell-cell adhesionfor structural reorganization that is mandatory during liver mass restoration process after hepatectomy.
BACKGROUND:/3-cateninand -y-cateninare vertebratehomologuesof the Drosophilaarmadillo protein, and play critical roles in cell adhesion and Wnt-signal transduction. In colorectal cancer, mutations in the APCtumor suppressoror in ~catenin stabilize/~:atenin and enhance its ability to activate transcription of Tcf/Lef target genes. Recent evidencesuggests that -ycatenin can also activate transcription of Tcf/Lef target genes in rodent kidney cells. AIMS: To determinethe effects of ~/-cateninon ~-catenin levels and the activity of a Tct/Lef reporter in RKO human colorectal cancer cells, which have wild-type APC and ~catenin. METHODS: RKO colon cancer cells were transfected with wild-type and deletion mutants of FLAG-tagged ~/-catenin and/or FLAG-tagged/3-cateninand assayedfor endogenousand FLAG-tagged,8catenin levels by immunopracipitation and Western analysis. Tct/Lef transcriptional activity was assayed using TopFlash luciterase reporter constructs. RESULTS: Over-expressionof wild-type ~,-catenin doubled the protein levels of endogenous/8-catenin and increased the protein levels of transiently expressed FLAG-tagged~-catenin several-fold. These effects of ~-catenin on/3-catenin stability were enhancedby deletion of the activation domain (~C687), and were nearly abolished by deletion of the central armadillo repeat domain of x-catenin (Arm-). TCt/Lef reporter activity was also induced several-fold by -y-catenin expression and was highest with the AC687 mutant, which lacks the activation domain. CONCLUSION:-ycatenin stabilizes/3-cateninand inducesTcf-Lef reporter activity. Transcriptionaleffects correlate with ~catenin levelsand occur independentlyof ~t-catenins activation domain, suggesting that ~,-catenininducesTct-Lef reporter activity indirectly by stabilizing ff-catenin. We speculate that the armadillo repeatdomain of -y-cateninmay stabilize/?~cateninby competitivelydisplacing/3-catenin from APC.
242 239
K-ras Trons0enic Mouse Model Recapitulates Early Pancreatic Ductal
Cand~eoa~s
FasL Is a/3-cetenin/TCF-4 Target GeH Repressed by Smads in RK0-1 Cofonictof Cancer Cells. Rebecca Chinery, Shaun G. Weller, Mayo Clio, Rochester, MN
Felix H. Brembeck, Franz S. Schraiber, Linden E. Craig, Anti K. Rustgi, Univ of Pennsylvania, Philadelphia,PA Introduction: Pancreatic ductal adenocarcinomais the fourth leading cause of cancer death in Western countries. The most common genetic alteration is mutation in the K-ras oncogene including the preneoplastic stages of pancreatic cancer. Geneticallybased animal models of pancreaticductal adenocaroinomaare lacking. We haveestablishedthat the keratin 19 promoter is transcriptionally regulated in pancreatic ductal cells and it targets the lacz reporter gene in these calls.Therafore, we sought to generate and characterize a K19 - Ki-ras transgenic mouse model. Methods: A 2.1 kb K19 5 UTR fragment was fused to the mutated human Kras (Gly • Val at codon 12) and this purified DNA fragment was used as transgene. Three lines of transgenic mice were established.Genotypingwas determined by PCR and Southern Blotting. Age-matched wild-type and transgenic mice were characterizedby histology, BrdU immunohisto-chemistry and Western blot analyses,the latter for evidenceof activated K-ras and ERK1/2 as well as expression of cyclin DI. Results: Activated K-ras and Erk 1/2 as well as cyclin D1 expressionwere increasedin transgenic mice comparedto age-matchedwildtype mice. Sixteen transgenic mice at the age of 4, 6, 9 and 12 months were sacrificed. BrdUimmunostaining revealed increased pmliteration within ductal cells starting at 6 months. A subset of mice also demonstrated histologic evidenceof ductal hyperplasiaand an increased stromal reaction.Conclusion:Targetoverexprassionof mutated K-ras in transgenic mice leads to biochemical evidence of K-ras and ERK 1/2 activation and overexpressionof cyclin D1, a downstreamtarget of this pathway.Preliminaryanalysisof transgenic mice indicatesevidence of ductal hyperplasiaand increasedcell proliferation. This unique model will serve as a novel approach to understanding the genetic basis of pancreatic cancer.
BACKGROUND:Fas ligand (FasL) is a well-characterizeddeath ligeod in lymphoid tissues bat is also expressed in coloractat neoplasms, where it may play a role in immune evasion. Coexpressionof FasL, nuclear ~-catenin and the matrix metalloproteinasematrilysin is common among colorectal neoplasms, and strongly correlates with decreased CD8-bearing tumorinfiltrating lymphocytes, increasedtumor vascularity and poor prognosis. The concordance betweenFasLand matrilysin expressionsuggeststhat these genesmay sharesimilar transcriptional regulation. AIMS: To determinewhether FasL, like matrilysin, is a transcriptional target of the APC//3-catenin pathway. METHODS:Transfection studies were performed in RKO-1 (wild-type ~catenin and APC) human coloractal cancer cells. Effects of ~-catenin and Smads (2 and 4) on FasL expression were determined by Western analysis. Human FasL promoter activity was assessed using a luciterase reporter gene under the control of a 1.28kbp FasL promoter or deletion mutants. Specificity of ~catenin/rCF effects was assessed by cotransfection with dominant negative (dn) TCF-4 or the E-cadherin cytoplasmic tail (ECCT). Binding of TCF//3-cateninto putativeTCFresponseelements (TCFREs)was verified by electrophoratic mobility shift assay (EMSA). RESULTS:Transient expression of/3-catenin and Tcf4 in RKO-1 cells resulted in 2-5 fold increased mRNA and protein levels of FasL that were blocked by coexprassion with ECCTor dnTCF-4. ,8-catenin/i'CF transactivation of the FasL promoterwas localizedto a TCFRE(-210bp). Site-mutationof this TCFREand EMSAsconfirmed that TCF-4 and ~-catenin competitively bound to this element as a complex. Using deletion mutants, a repressor element was located between -926 and -817bp containing a putative Smad responseelement.Co-transfectionof Smad-2/4abolishedTCF/,8-cateninresponsiveness of the FasLpromoter and this effect was also mappedbetween-926 and -817. CONCLUSIONS: FasL is a/~-catenin/TCF-4 target gene upregulated in colorectal cancer. A putative Smad response element negatesactivation by ,8-catenin/TCF-4.Thesestudies provide new insights into how loss of TGF-fl signaling (mutant Smads or TGF/3RII) and activation of the APC/#catenin pathway may combine to promote colorectal carcinogenesisthrough transcriptional regulation of target genes such as FasL.
243
Differentiating Role Of B7-CD28/CTLA-4 Interaction in The Induction Of Mucosal Inflammation And Regulatory T Cell Functions In Chronic Experimental Colitis Zhanju Liu, Univ Hosp Gasthuisberg, Univ of Leuven, Lauven Belgium; Philippe Maerten, Stefann Colpaert, Jan L. Ceuppens, Lab of ExperimentalImmunology, Lauven Belgium; Karel Geboes,Dept of Pathology, Leuven Belgium; Paul Rutgeerts, Dept of Gastroenterology, Leuven Belgium 67-CD28/CTLA-4interaction plays a role in the regulation of immune responses.Our previous results have demonstratedthat in vivo treatment with anti-B7.1 but not anti-B72 effectively prevents intestinal mucosal inflammation in colitic SCID mice, and that anti-CTLA-4 leads to the deterioration of colitis. Aims: To further evaluatethe in vivo relevanceof B7-CD28/CTLA4 interaction in the induction of mucosal inflammation and of regulatory T cell activity in chronic colitis. Methods: CD45RBh~h,CD45RB"~ and CD25+CD4÷ T cells were isolated from the splenic CD4+ cells of wild-type (WT) and CD28-~ mice. SCID mice were reconstituted with either WT or CD28 ~ CD45RB"~" cells alone, or cotransferredwith regulatoryCD25÷CD4+ T cells from WT or CD28 / mice. Clinical,histological and cytological analysiswere performed.
240 Ectopic Cytoplasmic Expression of 18-cetenin Conelates with Cyctin D1 Overexpression in Pancreatic Cancer. Marco Ramadani, Univ of UIm, UIm Germany; Qilu Ciao, Univ of Beijing, 6eijing People'S Rep Of China; Frank Gansauge,SusanneGaneauge,Gerd Leder, Univ of UIm, UIm Germany BACKGROUND:fl-catenin is a component of the E-caitherin-catenincell adhesion complex. It plays also a role in the intracellular signalling and can function as an oncogene when it binds to the T cell factor 4 (Tcf4)-binding site in the promoter region of cyclin D1 and transactivates,genesafter translocationto the nucleus. METHODS:We evaluatedthe expression
A-46