- Email: [email protected]
p63, a p53 homologue, compensates for functional p53 in the oral-esophageal epithelium in p53 deficient mice
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p161NK4APromoter Hypermethylation of Gastric Cancers and Precursor Lesions Chang-Hun Yang, Dae-ln Kim, Coo Lee, Jung-II Suh, Chang-Woo Lee, Tae-Jung Jang, Kyu-Chun Lee, Univ of Dong-Guk Coil of medicine, Kyung-Ju South Korea; Myung-Gyu Choi, The Catholic Univ Coil of Medicine, Seoul South Korea; Won-Ho Kim, Yonsei Unlv Coil of Medicine, Seoul South Korea BACKGROUND:p161NK4Atumor suppressor gene can be inactivated by promoter region hypermethyiationin many tumor types including gastric cancer. The aim of this study was to elucidatethe timing of this eventand the role of p 161NK4Apromoter region hypermethylation in gastric carcinogenesis.METHODS:we examined46 gastric cancers,correspondingadjacent non-neoplastic tissues and 8 gastric tissues from chronic gastritis as well as 9 adenomas for the methylation status of p161NK4Apromoter by methylation specific PCR and analyzed protein expression using immunohistochemistry and Western blot. RESULTS: plGINK4A promoter hypermethylationwas observed in 43% of gastric cancers, 59% of adjacent nonneoplastic tissues, and 22% of gastric adenomas, however, none of the samples retrieved from chronic gastritis displayedp 161NK4Apromoter hypermethyiation.Gastriccancersshowed an inverse correlation between vascular invasion and p161NK4Apromoter hypermethylation, and adjacentnon-neoplastictissues displayed close associationbetweenthe grade of chronic inflammation, presence of glandular atrophy and pl61NK4A promoter hypermethylation. p161NK4A protein expression was markedly decreasedin samples with p161NK4Apromoter hypermethylationwhen comparedwith sampleswithout p 161NK4Apromoter hypermethylation. CONCLUSION:These results suggest that p161NK4Apromoter hypermethyiation is an early and frequent event in gastric carcinogenesisand may possessesa potential as a new prognostic biomarker.
1519 Genotyping Of Frameshifl Mutations In Early And Metastatic Coloractal Cancer With Microsatellite Instability Luigi Laghi, Dept of Gastroenterology - Inst Clinico Humanitas, RozzanoItaly; Paolo Bianchi, Dept of Pathology - Inst Clinico Humanitas, RozzanoItaly; Ombrefta Orbetegli, Dept of Gastroenterology- Inst Clinico Humanitas, RozzanoItaly; Karl Heinimann, Research Group in Human Genetics - Univ Hosp, Basel Switzerland; Carlo Rossetti, Roberto Doci, Leandro Gennari, Dept of Surg - Inst Clinico Humanitas, RozzanoItaly; Massimo Roncalli, Dept of Pathology - Inst Clinico Humanitas, RozzanoItaly;,AJberto Malesci, Dept of Gastroenterology- Inst Clinico Humanitas, RozzanoItaly
28(40%) cases, respectively.The rates of/~-catenin-positive, cyclin Dl-positive, and COX-2positive caseswere higher in tumors with largersize (>6 mm) (P = 0.048, P = 0.002, P = 0.008, respectively) and were higher in tumors with high grade dyspiasia (P
1521 p63, a p53 Homologue, Compensates for Functional p53 in the Oral-Esophageal Epithelium in 153 Deficient Mice Anjali Avandhani,Yasir Suliman, Oliver Opitz, Noel Williams, Tim Bumes, Wafiq El-Deity, Anil X. Rustgi, Univ of Pennsylvania,Philadelphia,PA Introduction: p63, a p53 homolegue, has been shown to be critical in normal squamous epithelial development, p63 can bind to p53 responsive promoters and thus activate p53 dependent genes in vitro, but its biological properties in vivo have not been explored, p63 might compensatefor the inactivationof p53 in a tissue-specific fashion as in oral-esophegeal cancers. Methods: Immunohistochemistry for full-length p63 and N-terminal truncated isoforms of p63 ware performed in the tongue and esophagusof wild-type and p53 null-mice, the latter of which do not develop oral-esophagealcancers, but do develop sarcomas and lymphomas elsewhere. In addition, the expression of the p63 isoforms ware determined by Westem blot analysis of isolated tongue and esophagealepithelia. The mRNA expression levels of p21, a cell cycle inhibitor, and Bax, a proapoptotic gene, both of which are functional targets of p53 and p63, were determined by a PCR basedTaq Man assay. Results: I)63 was overexpreseedin the oral-esophagealepithelium of p53 deficient mice comparedto wild-type control mice in a statistically significantfashion (p < 0.05) basedupon immunohistochemistry. Importantly, full-length I)63 expressionwas confined to proliferating basal cells whereas the truncated form could be found also in differentiated suprabasalcells. This was independently confirmed by Western blot analysis. Moreover, p21 and Bax mRNA expression were induced in p53 deficient mice that correlated with I)63 expression. Conclusion: The p53 homologue, p63, may play a role in maintaining oral-esophagealepithelial differentiation in p53 null mice, thereby possibly serving as a protective mechanism against tumor development.
Background.In colorectal cancers with microsatellite instability (MSI CRC), frameshift mutations at exonic mononucleotidetracts of cancer related genes (target genes) are thought to play a role in the molecular pathogenesisof the disease. However, it is not clear whether early and advancedMSI CRC differ as to the patternand/or the number of framnshiff mutations in target genes. Aim. To investigateframeshift mutations in known target genes in MSI CRC of different pathological stages. Materials and methods. TGF~RII,CASP-5,BAX,hMSH3,and hMSHGframeshiftsmutations were studied in MSI CRC, 21 being non-metastatic (all Dukes 8), and 14 being metastatic(9 DukesC and 5 Dukes D cancers); 20 MSI CRCarose in patients either fullfilling Amsterdam criteria for hereditary non-polyposis colorectal cancer (HNPCC) or with proven hMLHI(5 patients)or hMSH2(7 patients) germline mutations. Results.Frameshifts of TGF~RIIoccurredin 86% of tumors, of CASP-5in 63%, of B/IX in 57%, of hMSH3 and of hMSH6in 40% and 34%, respectively.29 out of 35 (83%) MSI CRC harbored more than one mutation, and CASP-blrameshiftswere always associatedto TGF~RIImutations.No mutational pattern was otherwise recognizablein early or in advancedcases,and the number of frameshift mutations per tumor was not significantly different betweenearly and metastatic MSI CRC.However,the number of frameshifts/tumor was higher in CRCarising in patientswith hMSH2germlinemutations (>3 hits/tumor) than in CRC from patients with hMLHlgermlina mutation (<2 hits/tumor;Gill square, p
APC and p-salenin Protein Expression in Adenomas of Patients with Familial Pu~osis. Michiko Iwamoto, National Inst of Health, Bethesda,MD; Francis M. Giardiello, Johns Hopldns Univ, Baltimore, MD; C Richard Boland, Univ of CA, San Diego, La Jolla, CA; Kathy Romans, Johns Hopkins Univ, Baltimore, MD; Dharam P. Chauhan, Univ of CA, San Diego, La Jolia, CA; Dennis J. Aboen, Univ of Colorado, Denver, CO Background:Patientswith familial adenomatouspolyposis(FAP) haveinherited an inactivating garmline mutation in one allele of the APC geneand develophundredsto thousands of colonic adenomasand eventual carcinoma of the large bowel. Somatic mutational inactivation of the second APC allele is frequently detected in adenomasand biallelic loss of APC is thought to be requiredfor adenomaformation in patientswith FAP.All of the common somatic alterations known to occur in the APC gene during colonic carcinogenesis (truncating mutations, loss of heterozygosity,promoter methylation)would be expectedto result in the loss of expression of full length APC protein and the accumulation of nuclear and cytosolic/3-catenin. Goal- To determinethe relationshipbetweenfull length APC and/3-catenin protein expressionin normal and adenomatous colonic mucosa of patients with FAP due to known truncating mutations of one APC allele. Methods-lmmunohistochemietrywas performed using a rabbit polyclonal antibody to the C-terminal 20 amino acids of the full length APC protein and a mouse monoclonal antibody to/3-catenin. Immunoreactivityfor both proteins was evaluatedin serial sections of colonic adenomasand the adjacent normal mucoea from 39 FAP patients known to have truncating garmline mutations of the APC gene. Results-In all 39 samples of normal colonic mucoea both APC and ~catenin immunoreactivity were present in the colonocytes. APC expression was cytoplasmic, with maximal immunoreactivity in goblet cells, p-catenin was predominantlylocatedon the plasmamembrane.No nuclearimmunoreactivitywas present in the normal mucoea.As expected,almost all (97%)of the 39 adenomashad nuclear~-catenin immunoreactlvity. Surprisingly, however, 64% of the adenomas had abundant expression of full length APC protein in all areasof the adenoma,23% had focalareasof APC immunoreactlvity and only 13% of the adenomaswere completely negativefor full length APC protein. There was no difference in ff-catenin expression as a function of the presence or absence of APC immonoreactivity within an adenoma.Conclusions:Thepresenceof full length APC protein in most of the adenomas examined suggests that biallelic inactivation of APC does not occur universallyin small adenomasof patientswith FAP.The dissociationbetweenloss of expression of APC protein and the nuclear accumulation of/~-catenin suggests that biallelic APC loss is not required for the nuclear accumulation/3-catenin in FAP.
1520 Expression of Nuclear p-catenin Correlated with Cyclin Ol and COX-2 in Colorectal Adenomas Atsushi Tatsuguchi, Choitsu Sakamoto,Teruyuki Kishida, Shunji Fujimori, Jun Sato, Yuh Fukuda, Masafumi Kobayashi,Nippon Medical Sch, Tokyo Japan Background:/3-catenirVlcf-medietedtranscriptional activation has been implicated in human carcinogenesis. Although in vitro studies utilizing cultured cell lines have demonstratedthat c-myc, cyclin D1, and PPAR#are direct target genes of the ,8-catenin/Tctsignaling pathway, these data may not reflect an actual relationship between/3-cateninand target gene products in colorectal tumor tissues. In addition, COX-2 seems critical for tumor formation resulting from APC mutation. Aim: To examine whether the expression of cyclin D1 and COX-2 is altered by/3-catenin deregulation in the early stage of human colorectal tumorigenesis. Methods: Formalin-fixed,paraffin-embeddedtissues from 70 human colorectal adenomas were used in this study. The size of adenomasvaried from 2 mm to 2.5 cm (avr. 6.6 mm). The grade of dysplasia was divided into two groups as follows; low grade dysplasia (mild and moderate) and high grade dysplasia (severe and carcinoma in adenoma). Sections were stained using single-label (peroxidase/ABCcomplex) and dual-label (immunolluorescencei confocal laser scanning microscopy) methods with antibodiesto detect ~eatenin, cyclin Dr, and COX-2. Results:The reactionfor ~catenin was positive in the cell membranes,cytoplasm, and nuclei of tumor cells. The reaction for cyclin D1 was also positive in the nuclei of tumor cells. Staining for COX-2 labeledboth tumor cells and stromal cells. The reactionfor,8-catenin (in the nuclei), cyclin D1, and COX-2 (in tumor cells) was positive in 29 (41%), 26 (37%),
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p161NK4APromoter Hypermethylation of Gastric Cancers and Precursor Lesions Chang-Hun Yang, Dae-ln Kim, Coo Lee, Jung-II Suh, Chang-Woo Lee, Tae-Jung Jang, Kyu-Chun Lee, Univ of Dong-Guk Coil of medicine, Kyung-Ju South Korea; Myung-Gyu Choi, The Catholic Univ Coil of Medicine, Seoul South Korea; Won-Ho Kim, Yonsei Unlv Coil of Medicine, Seoul South Korea BACKGROUND:p161NK4Atumor suppressor gene can be inactivated by promoter region hypermethyiationin many tumor types including gastric cancer. The aim of this study was to elucidatethe timing of this eventand the role of p 161NK4Apromoter region hypermethylation in gastric carcinogenesis.METHODS:we examined46 gastric cancers,correspondingadjacent non-neoplastic tissues and 8 gastric tissues from chronic gastritis as well as 9 adenomas for the methylation status of p161NK4Apromoter by methylation specific PCR and analyzed protein expression using immunohistochemistry and Western blot. RESULTS: plGINK4A promoter hypermethylationwas observed in 43% of gastric cancers, 59% of adjacent nonneoplastic tissues, and 22% of gastric adenomas, however, none of the samples retrieved from chronic gastritis displayedp 161NK4Apromoter hypermethyiation.Gastriccancersshowed an inverse correlation between vascular invasion and p161NK4Apromoter hypermethylation, and adjacentnon-neoplastictissues displayed close associationbetweenthe grade of chronic inflammation, presence of glandular atrophy and pl61NK4A promoter hypermethylation. p161NK4A protein expression was markedly decreasedin samples with p161NK4Apromoter hypermethylationwhen comparedwith sampleswithout p 161NK4Apromoter hypermethylation. CONCLUSION:These results suggest that p161NK4Apromoter hypermethyiation is an early and frequent event in gastric carcinogenesisand may possessesa potential as a new prognostic biomarker.
1519 Genotyping Of Frameshifl Mutations In Early And Metastatic Coloractal Cancer With Microsatellite Instability Luigi Laghi, Dept of Gastroenterology - Inst Clinico Humanitas, RozzanoItaly; Paolo Bianchi, Dept of Pathology - Inst Clinico Humanitas, RozzanoItaly; Ombrefta Orbetegli, Dept of Gastroenterology- Inst Clinico Humanitas, RozzanoItaly; Karl Heinimann, Research Group in Human Genetics - Univ Hosp, Basel Switzerland; Carlo Rossetti, Roberto Doci, Leandro Gennari, Dept of Surg - Inst Clinico Humanitas, RozzanoItaly; Massimo Roncalli, Dept of Pathology - Inst Clinico Humanitas, RozzanoItaly;,AJberto Malesci, Dept of Gastroenterology- Inst Clinico Humanitas, RozzanoItaly
28(40%) cases, respectively.The rates of/~-catenin-positive, cyclin Dl-positive, and COX-2positive caseswere higher in tumors with largersize (>6 mm) (P = 0.048, P = 0.002, P = 0.008, respectively) and were higher in tumors with high grade dyspiasia (P
1521 p63, a p53 Homologue, Compensates for Functional p53 in the Oral-Esophageal Epithelium in 153 Deficient Mice Anjali Avandhani,Yasir Suliman, Oliver Opitz, Noel Williams, Tim Bumes, Wafiq El-Deity, Anil X. Rustgi, Univ of Pennsylvania,Philadelphia,PA Introduction: p63, a p53 homolegue, has been shown to be critical in normal squamous epithelial development, p63 can bind to p53 responsive promoters and thus activate p53 dependent genes in vitro, but its biological properties in vivo have not been explored, p63 might compensatefor the inactivationof p53 in a tissue-specific fashion as in oral-esophegeal cancers. Methods: Immunohistochemistry for full-length p63 and N-terminal truncated isoforms of p63 ware performed in the tongue and esophagusof wild-type and p53 null-mice, the latter of which do not develop oral-esophagealcancers, but do develop sarcomas and lymphomas elsewhere. In addition, the expression of the p63 isoforms ware determined by Westem blot analysis of isolated tongue and esophagealepithelia. The mRNA expression levels of p21, a cell cycle inhibitor, and Bax, a proapoptotic gene, both of which are functional targets of p53 and p63, were determined by a PCR basedTaq Man assay. Results: I)63 was overexpreseedin the oral-esophagealepithelium of p53 deficient mice comparedto wild-type control mice in a statistically significantfashion (p < 0.05) basedupon immunohistochemistry. Importantly, full-length I)63 expressionwas confined to proliferating basal cells whereas the truncated form could be found also in differentiated suprabasalcells. This was independently confirmed by Western blot analysis. Moreover, p21 and Bax mRNA expression were induced in p53 deficient mice that correlated with I)63 expression. Conclusion: The p53 homologue, p63, may play a role in maintaining oral-esophagealepithelial differentiation in p53 null mice, thereby possibly serving as a protective mechanism against tumor development.
Background.In colorectal cancers with microsatellite instability (MSI CRC), frameshift mutations at exonic mononucleotidetracts of cancer related genes (target genes) are thought to play a role in the molecular pathogenesisof the disease. However, it is not clear whether early and advancedMSI CRC differ as to the patternand/or the number of framnshiff mutations in target genes. Aim. To investigateframeshift mutations in known target genes in MSI CRC of different pathological stages. Materials and methods. TGF~RII,CASP-5,BAX,hMSH3,and hMSHGframeshiftsmutations were studied in MSI CRC, 21 being non-metastatic (all Dukes 8), and 14 being metastatic(9 DukesC and 5 Dukes D cancers); 20 MSI CRCarose in patients either fullfilling Amsterdam criteria for hereditary non-polyposis colorectal cancer (HNPCC) or with proven hMLHI(5 patients)or hMSH2(7 patients) germline mutations. Results.Frameshifts of TGF~RIIoccurredin 86% of tumors, of CASP-5in 63%, of B/IX in 57%, of hMSH3 and of hMSH6in 40% and 34%, respectively.29 out of 35 (83%) MSI CRC harbored more than one mutation, and CASP-blrameshiftswere always associatedto TGF~RIImutations.No mutational pattern was otherwise recognizablein early or in advancedcases,and the number of frameshift mutations per tumor was not significantly different betweenearly and metastatic MSI CRC.However,the number of frameshifts/tumor was higher in CRCarising in patientswith hMSH2germlinemutations (>3 hits/tumor) than in CRC from patients with hMLHlgermlina mutation (<2 hits/tumor;Gill square, p
APC and p-salenin Protein Expression in Adenomas of Patients with Familial Pu~osis. Michiko Iwamoto, National Inst of Health, Bethesda,MD; Francis M. Giardiello, Johns Hopldns Univ, Baltimore, MD; C Richard Boland, Univ of CA, San Diego, La Jolla, CA; Kathy Romans, Johns Hopkins Univ, Baltimore, MD; Dharam P. Chauhan, Univ of CA, San Diego, La Jolia, CA; Dennis J. Aboen, Univ of Colorado, Denver, CO Background:Patientswith familial adenomatouspolyposis(FAP) haveinherited an inactivating garmline mutation in one allele of the APC geneand develophundredsto thousands of colonic adenomasand eventual carcinoma of the large bowel. Somatic mutational inactivation of the second APC allele is frequently detected in adenomasand biallelic loss of APC is thought to be requiredfor adenomaformation in patientswith FAP.All of the common somatic alterations known to occur in the APC gene during colonic carcinogenesis (truncating mutations, loss of heterozygosity,promoter methylation)would be expectedto result in the loss of expression of full length APC protein and the accumulation of nuclear and cytosolic/3-catenin. Goal- To determinethe relationshipbetweenfull length APC and/3-catenin protein expressionin normal and adenomatous colonic mucosa of patients with FAP due to known truncating mutations of one APC allele. Methods-lmmunohistochemietrywas performed using a rabbit polyclonal antibody to the C-terminal 20 amino acids of the full length APC protein and a mouse monoclonal antibody to/3-catenin. Immunoreactivityfor both proteins was evaluatedin serial sections of colonic adenomasand the adjacent normal mucoea from 39 FAP patients known to have truncating garmline mutations of the APC gene. Results-In all 39 samples of normal colonic mucoea both APC and ~catenin immunoreactivity were present in the colonocytes. APC expression was cytoplasmic, with maximal immunoreactivity in goblet cells, p-catenin was predominantlylocatedon the plasmamembrane.No nuclearimmunoreactivitywas present in the normal mucoea.As expected,almost all (97%)of the 39 adenomashad nuclear~-catenin immunoreactlvity. Surprisingly, however, 64% of the adenomas had abundant expression of full length APC protein in all areasof the adenoma,23% had focalareasof APC immunoreactlvity and only 13% of the adenomaswere completely negativefor full length APC protein. There was no difference in ff-catenin expression as a function of the presence or absence of APC immonoreactivity within an adenoma.Conclusions:Thepresenceof full length APC protein in most of the adenomas examined suggests that biallelic inactivation of APC does not occur universallyin small adenomasof patientswith FAP.The dissociationbetweenloss of expression of APC protein and the nuclear accumulation of/~-catenin suggests that biallelic APC loss is not required for the nuclear accumulation/3-catenin in FAP.
1520 Expression of Nuclear p-catenin Correlated with Cyclin Ol and COX-2 in Colorectal Adenomas Atsushi Tatsuguchi, Choitsu Sakamoto,Teruyuki Kishida, Shunji Fujimori, Jun Sato, Yuh Fukuda, Masafumi Kobayashi,Nippon Medical Sch, Tokyo Japan Background:/3-catenirVlcf-medietedtranscriptional activation has been implicated in human carcinogenesis. Although in vitro studies utilizing cultured cell lines have demonstratedthat c-myc, cyclin D1, and PPAR#are direct target genes of the ,8-catenin/Tctsignaling pathway, these data may not reflect an actual relationship between/3-cateninand target gene products in colorectal tumor tissues. In addition, COX-2 seems critical for tumor formation resulting from APC mutation. Aim: To examine whether the expression of cyclin D1 and COX-2 is altered by/3-catenin deregulation in the early stage of human colorectal tumorigenesis. Methods: Formalin-fixed,paraffin-embeddedtissues from 70 human colorectal adenomas were used in this study. The size of adenomasvaried from 2 mm to 2.5 cm (avr. 6.6 mm). The grade of dysplasia was divided into two groups as follows; low grade dysplasia (mild and moderate) and high grade dysplasia (severe and carcinoma in adenoma). Sections were stained using single-label (peroxidase/ABCcomplex) and dual-label (immunolluorescencei confocal laser scanning microscopy) methods with antibodiesto detect ~eatenin, cyclin Dr, and COX-2. Results:The reactionfor ~catenin was positive in the cell membranes,cytoplasm, and nuclei of tumor cells. The reaction for cyclin D1 was also positive in the nuclei of tumor cells. Staining for COX-2 labeledboth tumor cells and stromal cells. The reactionfor,8-catenin (in the nuclei), cyclin D1, and COX-2 (in tumor cells) was positive in 29 (41%), 26 (37%),
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