Cytogenetic characterization and gene expression profiling in the rat reflux-induced esophageal tumor model.

OBJECTIVES: The reasons for the increasing incidence of esophageal adenocarcinoma are not clear. A causal relation between gastroesophageal reflux disease and esophageal adenocarcinoma has been suggested. Support for this comes from the development of esophageal adenocarcinoma in the rat reflux model. However, to date, no systematic characterization of the tumors derived from this model has been reported. METHODS: We induced biliary reflux by creating esophagojejunal anastomoses in 12 Sprague-Dawley rats. The experiment was terminated at 9 months, and rat esophagi were harvested for histopathologic documentation of reflux-associated changes and evidence of tumor formation. Three cell lines were established from 2 of the reflux-associated tumors. We tested the ability of these cells to grow in vitro in tissue culture and in vivo as xenografts in an orthotopic location at the gastroesophageal junction. Furthermore, we performed a cytogenetic analysis and determined the array-based gene expression profiles of these 3 rodent carcinoma lines compared with normal esophageal mucosa. RESULTS: At 9 months, 12 of 12 rodents had histologic features of metaplastic columnar epithelium in the esophagus, with 7 having invasive carcinomas with glandular differentiation (either adenocarcinomas or adenosquamous carcinomas). The 3 cell lines established from 2 reflux-associated tumors were capable of sustained in vitro propagation and grew successfully as xenografts in both subcutaneous and orthotopic locations, confirming the tumorigenic nature of these lines. Despite their establishment from primary tumors with glandular features, the histology of the xenografts was that of well-differentiated squamous carcinomas. Karyotype analyses demonstrated cytogenetic heterogeneity and aneuploidy; furthermore, translocation (7:11) was present in all 3 lines. Array-based gene expression profiling confirmed upregulation of several cancer-related genes important in human esophageal cancer. Quantitative reverse transcription-polymerase chain reaction was used to confirm the differential expression of selected transcripts (vascular endothelial growth factor [VEGF], polo-like kinases [PLK], cyclin dependent kinase 4 [CDK4], hypoxia-inducible factor 1alpha [HIF1alpha], and insulin-like growth factor 1 [IGF-1]) in comparison with nonneoplastic esophageal mucosal scrapings. CONCLUSIONS: The rodent reflux model is capable of inducing metaplastic epithelial changes simulating Barrett esophagus, as well as subsequent neoplastic transformation, at a high frequency. Cell lines have been established from these tumors that are capable of in vitro and in vivo passaging. The rodent reflux model should be a valuable model for studying therapy and chemoprevention efforts for Barrett esophagus, whereas the established cell lines provide a useful resource for drug discovery and other high-throughput studies.

Mitochondrial DNA mutations in preneoplastic lesions of the gastrointestinal tract: a biomarker for the early detection of cancer.

BACKGROUND: Somatic mutations of mitochondrial DNA (mtDNA) are common in many human cancers. We have described an oligonucleotide microarray ("MitoChip") for rapid sequencing of the entire mitochondrial genome (Zhou et al, J Mol Diagn 2006), facilitating the analysis of mtDNA mutations in preneoplastic lesions. We examined 14 precancerous lesions, including seven Barrett esophagus biopsies, with or without associated dysplasia; four colorectal adenomas; and three inflammatory colitis-associated dysplasia specimens. In all cases, matched normal tissues from the corresponding site were obtained as germline control. MitoChip analysis was performed on DNA obtained from cryostat-embedded specimens. RESULTS: A total of 513,639 bases of mtDNA were sequenced in the 14 samples, with 490,224 bases (95.4%) bases assigned by the automated genotyping software. All preneoplastic lesions examined demonstrated at least one somatic mtDNA sequence alteration. Of the 100 somatic mtDNA alterations observed in the 14 cases, 27 were non-synonymous coding region mutations (i.e., resulting in an amino acid change), 36 were synonymous, and 37 involved non-coding mtDNA. Overall, somatic alterations most commonly involved the COI, ND4 and ND5 genes. Notably, somatic mtDNA alterations were observed in preneoplastic lesions of the gastrointestinal tract even in the absence of histopathologic evidence of dysplasia, suggesting that the mitochondrial genome is susceptible at the earliest stages of multistep cancer progression. CONCLUSION: Our findings further substantiate the rationale for exploring the mitochondrial genome as a biomarker for the early diagnosis of cancer, and confirm the utility of a high-throughput array-based platform for this purpose from a clinical applicability standpoint.

Epidermal growth factor receptor and hedgehog signaling pathways are active in esophageal cancer cells from rat reflux model.

BACKGROUND: Advancements in experimental therapeutics for esophageal cancers have been hampered by the lack of a reliable preclinical model that recapitulates the biology of human cancer, including in vivo growth in an animal model. METHODS: Bilious reflux was induced by esophago-jejunostomy in Sprague-Dawley rats. Nine of 12 (75%) Sprague-Dawley rats developed squamous or adenosquamous cancers, and three cell lines were created by in vitro propagation of freshly resected tumors, JA and JB lines from one cancer, and the AMY cell line from another. We subsequently tested the ability of these cell lines to propagate long-term in vitro and form xenografts in vivo, both hallmarks of transformed cells. In addition, we determined the effects of small molecule inhibitors of two important oncogenic pathways-the epidermal growth factor receptor (EGFR) and Hedgehog (Hh) signaling pathways, in vitro, as a "proof of principle" of using these unique cell lines for developing targeted therapies for esophageal cancer. Mechanism-based growth inhibition was assessed by down-regulation of activated downstream targets of EGFR in the case of Iressa, and by Hh luciferase reporter activity with cyclopamine. RESULTS: JA, JB, and AMY cell lines were able to grow continuously in vitro and consistently form xenografts in vivo in athymic mice, both subcutaneously, as well as in the "orthotopic" location at the gastroesophageal serosal junction (n = 2 mice per line, six of six engrafted). By histology, the tumors grow in vivo as well-differentiated keratinizing squamous cell carcinomas. JB cells had the highest expression of EGFR protein and also the most profound response to Iressa (gefitinib), an EGFR inhibitor (IC50 < 1 microm). Growth inhibition by Iressa was mirrored functionally by down-regulation of activated targets of the EGFR pathway, phospho-ERK1/2 and phospho-MEK levels. AMY cells expressed approximately 900-fold elevation of the Hh ligand, Indian Hh (Ihh), compared with normal esophageal epithelium, whereas expression of another Hh ligand, Sonic Hh (Shh), was not detected. On treatment with the specific Hh small molecule inhibitor cyclopamine, AMY cells demonstrated growth inhibition, which was accompanied by significant down-regulation of endogenous Hh luciferase reporter activity at 24 h and increased apoptosis in treated cells. CONCLUSIONS: We have established a model of esophageal carcinogenesis, capable of long-term in vitro and in vivo passage, and demonstrated therapeutic potential of targeting the EGFR and Hh pathways in the cell lines created from the rodent cancers. These unique cell lines should provide a platform for rapid preclinical validation of novel therapeutics for esophageal cancers.

Genomic alterations in cultured human embryonic stem cells.

Cultured human embryonic stem cell (hESC) lines are an invaluable resource because they provide a uniform and stable genetic system for functional analyses and therapeutic applications. Nevertheless, these dividing cells, like other cells, probably undergo spontaneous mutation at a rate of 10(-9) per nucleotide. Because each mutant has only a few progeny, the overall biological properties of the cell culture are not altered unless a mutation provides a survival or growth advantage. Clonal evolution that leads to emergence of a dominant mutant genotype may potentially affect cellular phenotype as well. We assessed the genomic fidelity of paired early- and late-passage hESC lines in the course of tissue culture. Relative to early-passage lines, eight of nine late-passage hESC lines had one or more genomic alterations commonly observed in human cancers, including aberrations in copy number (45%), mitochondrial DNA sequence (22%) and gene promoter methylation (90%), although the latter was essentially restricted to 2 of 14 promoters examined. The observation that hESC lines maintained in vitro develop genetic and epigenetic alterations implies that periodic monitoring of these lines will be required before they are used in in vivo applications and that some late-passage hESC lines may be unusable for therapeutic purposes.

Identification of novel highly expressed genes in pancreatic ductal adenocarcinomas through a bioinformatics analysis of expressed sequence tags.

In most microarray experiments, a significant fraction of the differentially expressed mRNAs identified correspond to expressed sequence tags (ESTs) and are generally discarded from further analyses. We used careful bioinformatics analyses to characterize those ESTs that were found to be highly overexpressed in a series of pancreatic adenocarcinomas. cDNA was prepared from 60 non-neoplastic samples (normal pancreas [n = 20], normal colon [n = 10], or normal duodenal mucosal [n = 30]) and from 64 pancreatic cancers (resected cancers [n = 50] or cancer cell lines [n = 14]) and hybridized to the complete Affymetrix Human Genome U133 GeneChip(R) set (arrays U133A and B) for simultaneous analysis of 45,000 fragments corresponding to 33,000 known genes and 6,000 ESTs. The GeneExpress(R) software system Fold Change Analysis Tool was used and 60 ESTs were identified that were expressed at levels at least 3-fold greater in the pancreatic cancers as compared to normal tissues. Searches against the human genomic sequence and comparative genomic analysis of human and mouse genomes was carried out using basic local alignment search tools (BLAST), BLASTN, and BLASTX, for identifying protein coding genes corresponding to the ESTs. Subsequently, in order to pick the most relevant candidate genes for a more detailed analysis, we looked for domains/motifs in the open reading frames using SMART and Pfam programs. We were able to definitively map 43 of the 60 ESTs to known or novel genes, and 15 of the ESTs could be localized in close proximity to a gene in the human genome although we were unable to establish that the EST was indeed derived from those genes. The differential expression of a subset of genes was confirmed at the protein level by immunohistochemical labeling of tissue microarrays (inhibin beta A [INHBA] and CD29) and/or at the transcript level by RT-PCR (INHBA, AKAP12, ELK3, FOXQ1, EIF5A2, and EFNA5). We conclude that bioinformatics tools can be used to characterize differentially overexpressed ESTs, and that some of these ESTs may represent diagnostically and therapeutically useful targets that might be missed using data solely from currently annotated databases.

[Correlation between polymorphisms of interleukin-1beta and RN genes and risk of gastric carcinoma: a case-control study].

OBJECTIVE: To investigate the correlation between polymorphisms of interleukin-1beta and RN genes and risk of gastric carcinoma. METHODS: PCR and denaturing high-performance liquid chromatography (DHPLC) were used to analyze the IL-1beta-31 and -511 C/T polymorphisms and to genotype the IL-1RN penta-allelic variable number of tandem repeats (VNTR): of the DNA from the peripheral blood of 143 patients of gastric carcinoma, 97 from Linqu County, Shandong Province, and 46 cases from the specimen bank of Beijing Cancer Hospital, and 337 controls without gastric carcinoma from Linqu County, most of which suffered from other gastric diseases. RESULTS: IL-1-511 polymorphisms (carriers of IL-1beta-511T) were correlated with the increased risk of interstitial gastric carcinoma in both sexes (OR = 3.833, 95% CI: 2.282 approximately 6.439) and increased risk of diffuse gastric carcinoma in males (OR = 3.464, 95% CI: 1.394 approximately 8.608). The frequencies of IL-1beta-31 polymorphisms and IL-1IRN VNTR were not different between the gastric carcinoma group and the control group, IL-1beta-511 T carrier could be seen in the controls at different precancerous stages, for chronic atrophic gastritis (OR = 5.164, 95% CI: 2.661 approximately 10022), for interstitial metaplasia (OR = 5.093, 95% CI: 2.708 approximately 9.463), and for dysphasia (OR = 8.438, 95% CI: 3.939 approximately 18.073). CONCLUSION: IL-1beta-31 genotypes and. IL-2RN VNTR do not increase the risk of gastric carcinoma. The IL-1beta-511 T genotype increases the risk of gastric carcinoma in every precancerous stage.