Mutations in the adenomatous polyposis coli or ␤-catenin gene lead to cytosolic accumulation of ␤-catenin and, subsequently, to increased transcriptional activity of the ␤-catenin-T cell-factor/lymphoid-enhancer-factor complex. This process seems to play an essential role in the development of most colorectal carcinomas. To identify genes activated by ␤-catenin overexpression, we used colorectal cell lines for transfection with the ␤-catenin gene and searched for genes differentially expressed in the transfectants. There are four genes affected by ␤-catenin overexpression; three overexpressed genes code for two components of the AP-1 transcription complex, c-jun and fra-1, and for the urokinase-type plasminogen activator receptor (uPAR), whose transcription is activated by AP-1. The direct interaction of the ␤-catenin-T cell-factor͞lymphoid-enhancerfactor complex with the promoter region of c-jun and fra-1 was shown in a gel shift assay. The concomitant increase in ␤-catenin expression and the amount of uPAR was confirmed in primary colon carcinomas and their liver metastases at both the mRNA and the protein levels. High expression of ␤-catenin in transfectants, as well as in additionally analyzed colorectal cell lines, was associated with decreased expression of ZO-1, which is involved in epithelial polarization. Thus, accumulation of ␤-catenin indirectly affects the expression of uPAR in vitro and in vivo. Together with the other alterations, ␤-catenin accumulation may contribute to the development and progression of colon carcinoma both by dedifferentiation and through proteolytic activity.

The contribution of SLNB to conventional nodal staging of colon cancer patients is still unspecified. Technical problems have to be resolved before a definite conclusion can be drawn in this regard. However, SLNB identifies about one fourth of stage II patients to reveal MM/ITC in lymph nodes. Further studies must clarify the clinical impact of these findings in terms of prognosis and the indication of adjuvant therapy.

The detection of disseminated tumor cells in peripheral blood from colorectal cancer patients by RT-PCR could be an attractive method for selecting patients for adjuvant therapy. We here report on real-time RT-PCR assays (LightCycler) to quantitate potential mRNA markers. We investigated specimens from colon carcinoma and normal colon mucosa tissues, cell lines, blood samples from 129 patients with colorectal cancer (all stages) and 58 reference blood samples (healthy donors, persons suffering from inflammatory bowel or infectious diseases). The expression profile in tissues showed high values for CEA and CK20, whereas in cell lines ProtM was predominant. All markers were detected in reference and patient blood samples (ProtM, 22, 17%; CEA, 84, 86%; CK20, 85, 88%). After quantitative analysis, the definition of cutoff values for each marker and the combination of markers, 13% of patients were judged to have elevated marker concentrations in their blood, from which only 6 had values significantly differing from cutoff value. There were no differences between stages of disease. In the case of 19 patients, investigated prior to and 1 week after surgery, 2 samples revealed a significant postoperative increase in CEA or CK20 mRNA concentration. In spite of high expression levels in tissues and cell lines, we were not able to differentiate satisfyingly mRNA markers originating from tumor cells and those from illegitimate transcription in hematopoetic cells in blood. We conclude that either copy numbers of analyzed markers in circulating tumor cells are not sufficient for detection or, more probably, peripheral blood is not a suitable compartment for detection of tumor cells in colorectal cancer. © 2003 Wiley-Liss, Inc. Key words: colorectal cancer; real-time RT-PCR; minimal residual disease; carcinoembryonic antigene; cytokeratin 20; protease MThe molecular monitoring of circulating tumor cells by RT-PCR, routinely applied in patients with certain leukemias and lymphomas, 1-4 is still under debate for patients with solid malignancies. In colorectal cancer, where indication for adjuvant therapy without metastasis is yet performed by histologic investigation of lymph nodes, 5 the immunocytochemical identification of epithelial cells in bone marrow 6 encouraged the detection of epithelial mRNA markers in blood and bone marrow by RT-PCR. 7 However, a series of subsequent investigations by a number of groups with conventional nested PCR led to conflicting reports on both the frequency of gene transcripts in blood of patients and the specificity of the method. 8 -20 The recent availability of real-time PCR equipment has obviously changed the situation. 21-23 The quantification of low-level background transcription allows the definition of cutoff values for marker expression in blood and thus improves specificity. 16,22,24,25 Furthermore, the reliable quantification of housekeeping gene expression allows excellent quality control on a per-sample basis and relates absolute marker concentration to sample quality.We now devel...

Differential expression of genes encoding claudins in CRC suggests that these tight junction proteins may be associated to and involved in tumorigenesis. CLDN1 is frequently up-regulated in large proportion of CRC and may represent potential target molecule for blocking studies in CRC.