Calretinin and calretinin-22k increase resistance toward sodium butyrate-induced differentiation in CaCo-2 colon adenocarcinoma cells.

Calretinin (CR) and the alternatively spliced form calretinin-22k (CR-22k) are members of the EF-hand family of Ca(2+)-binding proteins (CaBPs). CR is expressed in more than 60% of poorly differentiated human colon tumors and both isoforms are present in several colon carcinoma cell lines (e.g., WiDr). They are absent in normal enterocytes and in well-differentiated adenocarcinoma cell lines such as CaCo-2. Calretinins are thought to act as Ca(2+) buffers and to be involved in the regulation of Ca(2+)-dependent processes. Down-regulation of calretinins in WiDr cells by antisense oligonucleotides leads to growth inhibition and treatment with sodium butyrate (NaBt, an inducer of differentiation) leads to a blockage of the cell cycle and, in parallel, to down-regulation of CR. It has been proposed that CR and/or CR-22k may be involved in maintaining the undifferentiated phenotype of WiDr cells and contributing to the transformation of enterocytes. Expression levels and distribution of CR-22k were investigated in WiDr cells. CR-22k was down-regulated in NaBt-treated cells and the normally cytoplasmic protein was preferentially localized in the nucleus either as a result of translocation or selective nuclear maintenance, a process more pronounced than in the case of CR. To compare functional differences of calretinins, CR-negative Caco-2 cells were stably transfected with cDNAs encoding CR or CR-22k. Cell growth of CR-transfected cells was increased, an effect that was not observed in CR-22k-transfected ones. The CR-expressing clones were almost completely resistant to treatment with 0.5 mM NaBt, a concentration significantly reducing cell growth in control cells. The same effect was obtained in the CR-22k-expressing clones, although to a lesser extent. This implicates that expression of CR and/or CR-22k in colon tumor cells may contribute to tumorigenesis by blocking differentiation-promoting signals.