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Maintenance of intestinal mucosal epithelial integrity requires polyamines that are involved in the multiple signaling pathways controlling gene expression and different epithelial cell functions. Integrity of the intestinal epithelial barrier depends on a complex of proteins composing different intercellular junctions, including tight junctions, adherens junctions, and desmosomes. E-cadherin is primarily found at the adherens junctions and plays a critical role in cell-cell adhesions that are fundamental to formation of the intestinal epithelial barrier. The current study determined whether polyamines regulate intestinal epithelial barrier function by altering E-cadherin expression. Depletion of cellular polyamines by α-difluoromethylornithine (DFMO) reduced intracellular free Ca2+concentration ([Ca2+]cyt), decreased E-cadherin expression, and increased paracellular permeability in normal intestinal epithelial cells (IEC-6 line). Polyamine depletion did not alter expression of tight junction proteins such as zona occludens (ZO)-1, ZO-2, and junctional adhesion molecule (JAM)-1. Addition of exogenous polyamine spermidine reversed the effects of DFMO on [Ca2+]cytand E-cadherin expression and restored paracellular permeability to near normal. Elevation of [Ca2+]cytby the Ca2+ionophore ionomycin increased E-cadherin expression in polyamine-deficient cells. In contrast, reduction of [Ca2+]cytby polyamine depletion or removal of extracellular Ca2+not only inhibited expression of E-cadherin mRNA but also decreased the half-life of E-cadherin protein. These results indicate that polyamines regulate intestinal epithelial paracellular barrier function by altering E-cadherin expression and that polyamines are essential for E-cadherin expression at least partially through [Ca2+]cyt.

Mutations within microsatellite sequences, consisting of additions or deletions of repeat units, are known as the replication/repair error positive (RER+) phenotype or micorsatellite instability (MI). Microsatellite instability has been demonstrated in hereditary and sporadic colorectal carcinomas and is usually observed in noncoding regions of genomic DNA. However, relatively few coding region targets of MI have been identified thus far. Using PCR, we amplified regions encompassing (A)8 and (C)8 microsatellite tracts within hMSH3 and hMSH6 from 31 RER+ sporadic colorectal tumors, 8 hereditary colon cancers, 23 RER+ gastric carcinomas, and 32 RER- gastric tumors. Mutations were found in 11 (36%) of 31 sporadic colon carcinomas, 4 (50%) of 8 hereditary colorectal cancers, and 5 (22%) of 23 RER+ gastric carcinomas, but in only 2 (6%) of 32 RER- gastric carcinomas. These frameshift mutations cause premature stop codons downstream that are predicted to abolish normal protein function. Our results and those of others suggest that DNA mismatch repair genes, such as hMSH3 and hMSH6, are targets for the mutagenic activity of upstream mismatch repair gene mutations and that this enhanced genomic instability may accelerate the accumulation of mutations in RER+ tumors.

Frequent loss of heterozygosity (LOH) on human chromosome 7q31 has been reported in numerous malignancies. Suppressor of tumorigenicity 7 (ST7) has been identified as a candidate tumor suppressor gene in this region. To identify whether 7q31 and genetic alterations of ST7 were involved in human esophageal carcinogenesis, we performed LOH mapping of a 5.4 cM region at 7q31-q35 in 43 primary esophageal carcinomas, as well as mutational analyses of the ST7 gene in tumors with LOH in this region. Of 43 tumors, 12 (28%) showed LOH at 7q31–q35. These included four (22%) of 18 squamous cell carcinomas and eight (32%) of 25 adenocarcinomas. The peak LOH locus was D7S480, lying 4.2 Mb telomeric to ST7 and showing LOH in eight of 37 informative tumors, or 22%. No mutations were found in the entire coding or flanking intronic regions of the ST7 gene among 12 tumors with 7q-LOH. In addition, quantitative RT–PCR analyses of ST7 mRNA expression levels in 11/13 normal-tumor pairs failed to show more than a 50% decrease in tumor ST7 mRNA relative to matched normal tissues. These data suggest that LOH at 7q31–q35 is involved in the origin or progression of at least a subset of esophageal carcinomas, but that ST7 is not the target gene of this somatic event.

Esophagin is a member of the small proline-rich protein family of cell envelope precursor proteins, which are expressed during squamous cell differentiation. Esophagin is expressed at high levels in normal esophageal epithelium, but its expression is absent from esophageal squamous cell carcinomas and adenocarcinomas. Moreover, loss of esophagin expression is present in areas of dysplasia or normal mucosa adjacent to carcinomas, suggesting that absence of esophagin may constitute a harbinger of early esophageal malignant transformation. A greater understanding of transcriptional control of esophagin may provide valuable insights into esophageal malignancy. Therefore, this study was undertaken in order to isolate and carry out initial characterization of a functional promoter for esophagin. A genomic clone containing esophagin was isolated and sequenced, including 2.7 kb of the esophagin promoter region. Esophagin expression was studied in response to various treatments of primary cultured human esophageal epithelial cells and squamous cell carcinoma cell lines. Calcium was the strongest inducer of the endogenous esophagin promoter, with induction occurring at 12–72 hours. In primary cultured esophageal epithelial cells, a region spanning 116 bp upstream of the transcriptional start site to 45 bp downstream was sufficient to direct low, basal, in vitro esophagin expression. However, responsiveness of primary esophageal cells to calcium required inclusion of promoter elements 1688 bp upstream of the transcriptional start site. Site-directed mutagenesis studies suggested a putative role for C/EBP-β, OCT-1, and OCT-3 transcription factor binding sites in the minimal promoter region. In conjunction with published human in vivo studies, these data support the hypothesis that esophagin is a biomarker of esophageal squamous cell differentiation and provide an in vitro model to evaluate regulatory factors involved in this differentiation process.

In order to discover global gene expression patterns characterizing subgroups of colon cancer, microarrays were hybridized to labeled RNAs obtained from seventeen colonic specimens (nine carcinomas and eight normal samples). Using a hierarchical agglomerative method, the samples grouped naturally into two major clusters, in perfect concordance with pathological reports (colon cancer versus normal colon). Using a variant of the unpaired t-test, selected genes were ordered according to an index of importance. In order to confirm microarray data, we performed quantitative, real-time reverse transcriptase-polymerase chain reaction (TaqMan RT-PCR) on RNAs from 13 colorectal tumors and 13 normal tissues (seven of which were matched normal-tumor pairs). RT-PCR was performed on the gro1, B-factor, adlican, and endothelin converting enzyme-1 genes and confirmed microarray findings. Two hundred and fifty genes were identified, some of which were previously reported as being involved in colon cancer. We conclude that cDNA microarraying, combined with bioinformatics tools, can accurately classify colon specimens according to current histopathological taxonomy. Moreover, this technology holds promise of providing invaluable insight into specific gene roles in the development and progression of colon cancer. Our data suggests that a large-scale approach may be undertaken with the purpose of identifying biomarkers relevant to cancer progression.

A significant portion of gastric cancers exhibit defective DNA mismatch repair, manifested as microsatellite instability (MSI). High-frequency MSI (MSI-H) is associated with hypermethylation of the human mut-L homologue 1 (hMLH1) mismatch repair gene promoter and diminished hMLH1 expression in advanced gastric cancers. However, the relationship between MSI and hMLH1 hypermethylation has not been studied in early gastric neoplasms. We therefore investigated hMLH1 hypermethylation, hMLH1 expression and MSI in a group of early gastric cancers and gastric adenomas. Sixty-four early gastric neoplasms were evaluated, comprising 28 adenomas, 18 mucosal carcinomas, and 18 carcinomas with superficial submucosal invasion but clear margins. MSI was evaluated using multiplex fluorescent PCR to amplify loci D2S123, D5S346, D17S250, BAT 25 and BAT 26. Methylation-specific PCR was performed to determine the methylation status of hMLH1. In two hypermethylated MSI-H cancers, hMLH1 protein expression was also evaluated by immunohistochemistry. Six of sixty-four early gastric lesions were MSI-H, comprising 1 adenoma, 4 mucosal carcinomas, and 1 carcinoma with superficial submucosal invasion. Two lesions (one adenoma and one mucosal carcinoma) demonstrated low-frequency MSI (MSI-L). The remaining 56 neoplasms were MSI-stable (MSI-S). Six of six MSI-H, one of two MSI-L, and none of thirty MSI-S lesions showed hMLH1 hypermethylation (P

The insulin-like growth factor II receptor (IGFIIR) has been implicated as a tumor suppressor gene in human malignancy. Frequent mutation, loss of heterozygosity, and microsatellite instability (MSI) directly affecting the IGFIIR gene have been reported in several primary human tumor types. However, to our knowledge, dynamic functional evidence of a growth-suppressive role for IGFIIR has not yet been provided. We identified one MSI-positive colorectal carcinoma cell line, SW48, with monoallelic mutation in IGFIIR identical to that seen in primary colorectal carcinomas. A zinc-inducible construct containing the wild-type IGFIIR cDNA was stably transfected into SW48 cells. Growth rate and apoptosis were compared between zinc-treated, untreated, and untransfected cells. A twofold increase in IGFIIR protein expression was detected after zinc treatment in discrete clonal isolates of transfected SW48 cells. Moreover, zinc induction of exogenous wild-type IGFIIR expression reproducibly decreased growth rate and increased apoptosis. These data prove that wild-type IGFIIR functions as a growth suppressor gene in colorectal cancer cells and provide dynamic in vitro functional support for the hypothesis that IGFIIR is a human growth suppressor gene.

FHIT (fragile histidine triad gene), a candidate tumor suppressor gene, was recently identified and cloned at chromosome 3p14.2. Alterations of this gene have been reported in a number of primary human tumors, including colorectal, esophageal, gastric and lung carcinomas. However, some reports have found no abnormalities in this gene. We investigated a total of 63 primary esophageal tumors, nine esophageal cancer cell lines and 17 ulcerative colitis-associated neoplasms (UCANs) for alterations of FHIT. In 13 esophageal tumors, we employed overlapping reverse transcriptase-PCRs (RT-PCRs) to amplify and sequence the complete open reading frame of FHIT. One of 13 primary esophageal tumors analysed by RT-PCR expressed no detectable FHIT transcript; the remaining 12 expressed normal-sized transcripts with wild-type open reading frame sequences. In an additional 50 esophageal tumors, the polymorphic microsatellite loci D3S1300 and D3S1313 were used to evaluate loss of heterozygosity (LOH) at 3p14.2. Eleven of these 50 tumors showed LOH at one or both loci. In all these 11 tumors, genomic PCR and direct sequencing of FHIT exons 5-9 was performed. This analysis revealed that none of these 11 primary esophageal tumors contained any alterations in the FHIT open reading frame or adjacent intron sequences. Finally, among 17 UCANs, the in vitro synthesized protein (IVSP) assay detected no truncated protein products, nor were there any abnormalities in size or DNA sequence of FHIT RT-PCR products. However, in six of nine esophageal carcinoma cell lines, no FHIT RT-PCR product was detectable using either of the overlapping primer sets. Genomic PCR and direct sequencing of exons 5-9, also performed in these nine cell lines, revealed wild-type sequence in eight cell lines; however, one cell line contained no exon 5 PCR product. This cell line also lacked detectable FHIT transcript. These data suggest that the open reading frame of FHIT is not important in the development or progression of most primary esophageal carcinomas or UCANs, although lack of expression of the FHIT transcript may be common in esophageal cancer-derived cell lines. The possibility of an additional tumor suppressor gene at chromosome 3p14.2 remains to be evaluated.

Genomic instability at simple repeated sequences has been observed in various types of human cancers and is considered an important mechanism in tumorigenesis. Alterations at microsatellite loci have been reported scattered throughout the genome. Recently, the transforming growth factor-beta receptor type II (TGF-beta RII) and the insulin-like growth factor II receptor (IGF-IIR) genes were shown to have inactivating mutations within coding microsatellite sequences. The demonstration of mutations in two growth regulatory genes supports the idea that other regulatory genes with repeat sequences may also be targets in tumours with defective mismatch repair. We examined genes involved in tumour suppression, cell adhesion and cell cycle regulation for mutations at small repeat sequences in replication error positive gastrointestinal cancers. Several polymorphisms were found which exhibited instability, but no other instability was present in the regions examined.