Restoration of epithelial cell polarity in a colorectal cancer cell line by suppression of beta-catenin/T-cell factor 4-mediated gene transactivation.

Beta-catenin acts as a transcriptional coactivator by forming a complex with T-cell factor/lymphoid enhancer factor (TCF/LEF) DNA-binding proteins. Aberrant transactivation of a certain set of target genes by beta-catenin and TCF4 complexes has been implicated in familial and sporadic colorectal tumorigenesis. A colorectal cancer cell line, DLD-1, becomes irregularly multilayered, when maintained confluent for 2-3 weeks, and forms numerous dome-like polypoid foci piled-up over the surface of cell sheets. By the use of a strict tetracycline-regulation system, we found that the continuous suppression of beta-catenin/TCF4-mediated gene transactivation by dominant-negative TCF4B (deltaN30) reduced these piled-up foci and restored a simple monolayer of polarized columnar cells resembling normal intestinal epithelium. The restoration of epithelial cell polarity was evident in two ways: (a) the formation of microvilli over the apical surface; and (b) the distribution of a tight junction protein, ZO-1, to the lateral plasma membrane. Retroviral expression of stabilized beta-catenin (deltaN89) induced the formation of similar piled-up foci in untransformed IEC6 intestinal epithelial cells. Sulindac, a nonsteroidal antiinflammatory drug effective against colorectal tumorigenesis in familial adenomatous polyposis syndrome, suppressed the formation of foci. The loss of epithelial cell polarity may be a critical cellular event driving beta-catenin/TCF4-mediated intestinal tumorigenesis.

Transactivation of the multidrug resistance 1 gene by T-cell factor 4/beta-catenin complex in early colorectal carcinogenesis.

The mutational inactivation of a tumor suppressor gene, adenomatous polyposis coli (APC), results in the accumulation of cytoplasmic beta-catenin protein and the activation of T-cell factor (TCF)/lymphoid enhancer factor transcriptional factors. A colorectal carcinoma cell line, DLD-1, was engineered to suppress transactivation by the TCF4/beta-catenin complex in a dominant-negative manner under the strict control of the tetracycline regulatory system. A large-scale comparison of the expression profiles, using two-color fluorescence hybridization of cDNA microarray, led to the identification of MDR1 as a target gene of the TCF4/beta-catenin complex. Luciferase reporter and gel retardation assays revealed the TCF4/beta-catenin responsive elements in the promoter of the human MDR1 gene. Corresponding to the accumulation of beta-catenin, expression of the MDR1 gene product was steadily up-regulated in adenomas and adenocarcinomas of 10 patients with familial adenomatous polyposis. In combination with cell proliferative activities of c-myc and cyclin D1, MDR1 may initiate colorectal tumorigenesis by suppressing cell death pathways programmed in intestinal epithelial cells.

Cloning and characterization of the human beta4-integrin gene promoter and enhancers.

The cell-surface adhesion molecule alpha6beta4-integrin is a receptor for laminins and a component of hemidesmosomes. beta4-Integrin expression is restricted to proliferating basal keratinocytes in the epidermis and is suppressed when differentiation commences. Altered beta4-integrin expression levels correlate significantly with the aggressive behavior of cancers. In order to clarify the mechanisms that regulate transcription of the beta4-integrin gene, we cloned its 5'-flanking region. This 5'-flanking region was found to have a high G + C content and not to contain either TATA or CAAT boxes. Nested delimitation and reporter analyses mapped a basal promoter to nucleotides -106 to +105, surrounding the most proximal transcription initiation site. Gel retardation and mutational analyses revealed that cooperation between AP1 and Ets, interacting with other factors, mediated the promoter activity. In addition to the promoter element, enhancer activity was found in the first intron (+1905/+3933) and in a sequence upstream of the promoter region (-414/-107). These findings should facilitate our understanding of the regulation of beta4-integrin gene expression in processes such as cell growth and differentiation, apoptosis, and cancer development and metastasis.

Infrequent alterations of the p16 (MTS-1) gene in human gastric cancer.

p16 (MTS-1, multiple tumor suppressor gene 1), a putative tumor suppressor gene, is one of the cyclin-dependent kinase inhibitors (CDI) and it regulates the G1/S transition of the cell cycle. To clarify the role of p16 in primary gastric cancer, we have investigated somatic mutations of this gene by using the polymerase chain reaction/single strand conformation polymorphism (PCR-SSCP) method. In 23 surgical specimens of primary gastric cancer, none were detected in exon1 and exon 2. Among the 6 human gastric cancer cell lines examined, PCR products were not found in 2, MKN28 and MKN45, suggesting the presence of homozygous deletions. No mutation was found in the other 4 cell lines. Furthermore, decreased expression levels were not observed in 13 gastric cancer tissues by reverse transcription PCR (RT-PCR). Considering the above results of PCR-SSCP and RT-PCR, genetic alterations of the p16 gene are rarely implicated in human gastric cancer tumorigenesis.