[905][906][907][908][909][910]

No abstract

Background-Ulcerative colitis associated colorectal cancer (UCACRC) has several distinctive clinicopathological and genetic features which diVer from sporadic colorectal cancer (SCRC). Hypermethylation of the E-cadherin gene (CDH1) has not been described previously in colorectal cancer. Aims-A panel of SCRC and UCACRC were investigated for mutations in CDH1, and for hypermethylation of the promoter region of CDH1. Subjects and methods-DNA was available from 14 patients with UCACRC and from 14 with SCRC. All exons of CDH1 were amplified with the polymerase chain reaction (PCR) and screened using single strand conformational polymorphism and direct sequencing. Hypermethylation of the CDH1 promoter region was determined by methylation specific PCR following bisulphite modification, and compared with E-cadherin protein expression from a previous immunohistochemistry study. Results-Thirteen of 28 cancers (46%)were hypermethylated in the CDH1 promoter region-eight cancers (57%) in the UCACRC group and five cancers (36%) in the SCRC group (NS)-and this correlated with reduced E-cadherin expression (p<0.05). There was a trend for methylation to be associated with a more advanced stage of cancer although this did not reach statistical significance. There were no mutations in CDH1 in either group although there were several polymorphisms. Conclusion-We have demonstrated hypermethylation of the promoter region in CDH1 in 46% of colorectal cancers studied. There was no diVerence between the UCACRC and SCRC groups. Just as there are specific diVerences in the genetic changes between UCACRC and SCRC, there is also likely to be a large degree of overlap among the genetic pathways of these cancers.

Epithelial (E)-cadherin and its associated cytoplasmic proteins (␣-, ␤-, and ␥-catenins) are important mediators of epithelial cell-cell adhesion and intracellular signaling. Much evidence exists suggesting a tumor͞invasion suppressor role for E-cadherin, and loss of expression, as well as mutations, has been described in a number of epithelial cancers. To investigate whether E-cadherin gene (CDH1) mutations occur in colorectal cancer, we screened 49 human colon carcinoma cell lines from 43 patients by single-strand conformation polymorphism (SSCP) analysis and direct sequencing. In addition to silent changes, polymorphisms, and intronic variants in a number of the cell lines, we detected frameshift single-base deletions in repeat regions of exon 3 (codons 120 and 126) causing premature truncations at codon 216 in four replication-errorpositive (RER؉) cell lines (LS174T, HCT116, GP2d, and GP5d) derived from 3 patients. In LS174T such a mutation inevitably contributes to its lack of E-cadherin protein expression and function. Transfection of full-length E-cadherin cDNA into LS174T cells enhanced intercellular adhesion, induced differentiation, retarded proliferation, inhibited tumorigenicity, and restored responsiveness to the migratory effects induced by the motogenic trefoil factor 2 (human spasmolytic polypeptide). These results indicate that, although inactivating E-cadherin mutations occur relatively infrequently in colorectal cancer cell lines overall (3͞43 ‫؍‬ 7%), they are more common in cells with an RER؉ phenotype (3͞10 ‫؍‬ 30%) and may contribute to the dysfunction of the E-cadherin-catenin-mediated adhesion͞sig-naling system commonly seen in these tumors. These results also indicate that normal E-cadherin-mediated cell adhesion can restore the ability of colonic tumor cells to respond to trefoil factor 2.

Fifteen to twenty-five percent of sporadic colorectal carcinomas are replication error (RER) positive. Because the frequency of mutations in the mismatch repair genes (hMLH1 and hMSH2) is low in these tumors, we have investigated the role of mutational inactivation, methylation of the promoter region, and loss of heterozygosity (LOH) as a possible explanation for the mutator phenotype of RER؉ colorectal cancer cell lines. Genomic DNA was extracted from a panel of 49 human colorectal cancer cell lines. The RER status was determined by amplification of BAT-26. All exons of hMLH1 and hMSH2 were amplified with the PCR and screened by using single-strand conformational polymorphism and direct sequencing. The methylation status was ascertained by methylation-specific PCR after bisulfite modification of DNA. Western blotting for hMLH1 was performed on methylated cell lines before and after the addition of the demethylating agent 5-azacytidine. LOH was sought by GE-NESCAN analysis of amplified CA repeat markers and indirectly by determining the number of homozygotes in the cell lines and human random controls. Twelve cell lines from ten tumors (24%) were RER؉. Hypermethylation of the hMLH1 promoter occurred in five of ten (50%) RER؉ tumors, whereas three of thirty-two (6%) RER tumors showed partial methylation. None of the fully methylated cell lines expressed hMLH1, although all reexpressed hMLH1 after treatment with 5-azacytidine. There was no LOH in the RER؉ tumors in either hMLH1 or hMSH2. Our results suggest that mutations of hMLH1 together with hypermethylation of the promoter region, but not LOH, are the cause of the mutator phenotype in the majority (70%) of RER؉ tumors.

TEM is an appropriate surgical treatment option for benign rectal tumours. For carcinomas, it is oncologically safe provided that resection margins are clear, but strict patient selection is required.

CDC4/FBXW7 is part of a ubiquitin ligase complex which targets molecules such as cyclin E, c-myc, and c-jun for destruction. CDC4 mutations occur in several cancer types and are best described in colorectal tumors. Knockout of CDC4 in vitro in colorectal cancer cells causes changes suggestive of chromosomal instability (CIN). In p53 +/À mice, radiation-induced lymphomas show deletion or mutation of one copy of CDC4 and knockdown of CDC4 leads to increased aneuploidy in mouse fibroblasts. We screened 244 colorectal tumors and 40 cell lines for CDC4 mutations and allelic loss. Six percent (18 of 284) of tumors, including near-diploid (CINÀ) lesions, harbored CDC4 mutations and there was no association between mutation and CIN (polyploidy). The CDC4 mutation spectrum in colorectal tumors was heavily biased towards C:G>T:A changes, either missense mutations at critical arginine residues or nonsense changes in the 5V half of the gene. The reasons for this odd mutation spectrum were unclear but C:G>T:A changes were not found more often than expected at APC, K-ras, or p53 in the same tumors and we found no specific defects in DNA repair to account for the observations. No colorectal tumor was found to carry two CDC4 mutations predicted to abolish protein function; partial loss of CDC4 function may therefore cause tumorigenesis. The in vitro studies, therefore, did not assess the functional effects of mutant alleles which are found in vivo. CDC4 mutations may be selected primarily to drive progression through the cell cycle although CIN might be an important secondary effect in some cancers. (Cancer Res 2005; 65(24): 11361-6)

Introduction-Hypermethylation of the promoter region of the hMLH1 gene is associated with absent expression of MLH1 protein in sporadic colorectal cancers with microsatellite instability (MSI+), and it has been proposed that methylation may be a mechanism of inactivation in Knudson's hypothesis. The incidence of hypermethylation of the hMLH1 promoter in hereditary nonpolyposis colorectal cancer (HNPCC) versus MSI+ sporadic colorectal cancer was investigated and compared. Methods-DNA was available from 10 HNPCC colorectal cancers (median age 58 years, range 39-67) with germline mutations in hMLH1 and 10 MSI+ sporadic colorectal cancers (mean age 79 years, range 41-85). MSI was determined by amplification of BAT26 and TGF-RII. The methylation status of the hMLH1 promoter was studied by the polymerase chain reaction (PCR) based HpaII restriction enzyme assay technique. Evidence of allelic loss at hMLH1 was searched for in the HNPCC colorectal cancers. Results-All cases were confirmed to be MSI+. The promoter region of hMLH1 was hypermethylated in seven of 10 MSI+ sporadic cancers versus 0 of 10 HNPCC cancers (p<0.002). Evidence of loss of heterozygosity at hMLH1 was observed in eight of the 10 HNPCC colorectal cancers. Conclusion-While mutations and allelic loss are responsible for the MSI+ phenotype in HNPCC cancers, the majority of MSI+ sporadic cancers are hypermethylated in the promoter region of hMLH1. These data further support our argument that tumours from HNPCC patients, which almost always acquire a raised mutation rate, mostly follow a diVerent pathway from MSI+ sporadic tumours. (J Med Genet 2000;37:588-592)