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.
Irinotecan (CPT-11), a recently introduced component of a standard chemotherapy for colorectal cancer, induces in colon cancer cell lines in vitro cell cycle arrest and apoptosis. Since sporadic colon carcinomas exhibit in 50 -60% mutations in the p53 gene and in 10 -15% an MSI phenotype due in the great majority of the cases to hMLH1 inactivation, we investigated how these lesions influence the cellular effects of CPT-11 by using colorectal carcinoma cell line HCT116 (which has the genotype p53 ؉/؉ ,hMLH1 -) and 2 derivative cell lines with the genotypes p53 ؉/؉ ,hMLH1 ؉ and p53 -/-,hMLH1 -. CPT-11 treatment induced G2/M arrest in all 3 cell lines within 48 hr. In the p53 ؉/؉ ,hMLH1 ؉ cell line, G2/M arrest was maintained for at least 12 days. There was little concomitant apoptosis, but this was enhanced when the hMLH1 protein was absent. This enhanced apoptosis was accompanied by a shorter duration of the G2/M arrest than in the hMLH1 ؉ cell line. Partial abrogation of G2/M arrest by caffeine enhanced apoptosis in both hMLH1 ؉ and hMLH1 -cells. By contrast, in the p53 -/-cell line, the G2/M arrest was terminated within 4 days. Termination of the G2/M arrest was accompanied by a high level of apoptosis detectable through poly(ADP-ribose)polymerase (PARP) cleavage, DNA fragmentation and by the appearance of cells with a DNA content <2N. The triggering of G2/M arrest was accompanied in the 3 cell lines by a transient phosphorylation of cdc-2, while the maintenance of the arrest in the p53 ؉/؉ cell lines was accompanied by the overexpression of p53 and p21 proteins and, consequently, by the inhibition of cdc-2 kinase activity. These data indicate that: (i) CPT-11 induces long-term arrest in p53 ؉/؉ cells and a short-term arrest followed by apoptosis in p53 -/-cells; (ii) triggering of the arrest is p53 independent and is associated with a brief increase of phosphorylation of cdc-2, while the p53-dependent maintenance of G2/M arrest is associated with the inhibition of cdc-2 kinase activity by p21; and (iii) lack of hMLH1 protein enhances CPT-11-induced apoptosis. These results may be useful for designing rational therapies dependent on the p53 and mismatch-repair status in the tumor.
Summary Colorectal carcinoma cells have recently been shown to express Fas ligand (FasL). This ligand could allow the tumour cells to evade activated tumour-infiltrating lymphocytes (TILs) by inducing their apoptosis and would thus promote tumour survival and possibly metastasis formation. To test this hypothesis in vivo we analysed the expression of FasL mRNA and protein in paired tissue samples of normal colonic mucosa (N), primary colorectal carcinomas (T) and their metastases (M) from a total of 21 patients by four different methods. Additionally, the presence and activation status of infiltrating lymphocytes, which might contribute to the total amount of FasL in the tissue, was determined by semiquantitative reverse transcription-polymerase chain reaction (RT-PCR) in the same samples. The frequency of FasL detection was 30-40% in T and was 60-100% in M, depending on the sensitivity of the method. Simultaneously, the amount of CD25 mRNA, used as a measure of the number of activated TILs, was in 90% of patients lower in M than in T. The increased frequency of FasL detection in liver metastases was therefore not due to the presence of activated TILs. We conclude that metastasizing subpopulations of colorectal tumour cells express FasL more frequently than the primary carcinomas and may be able to eliminate activated TILs in vivo via Fas/FasLinduced apoptosis or other hitherto unknown mechanisms.
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