Optimization of hammerhead ribozymes for the cleavage of S100A4 (CAPL) mRNA.

Previously, suppression of the S100A4 mRNA by an endogenously expressed ribozyme in osteosarcoma cells was shown to inhibit their metastasis in rats. As a prelude to performing similar studies with exogenous, synthetic ribozymes, we compared a series of hammerhead ribozymes targeted against different sites in the mRNA. The ribozymes differed only in the 7-base flanking sequences complementary to the substrate and were protected against nucleases by chemical modification. Cleavage efficiency varied widely and was not obviously related to the predicted secondary structure of the target RNA. The most active ribozyme of the series was chosen for further optimization. Lengthening its flanking sequences was counterproductive and reduced cleavage even when using excess ribozyme. Using excess substrate (multiple-turnover kinetics), cleavage was fastest with the (6+8) ribozyme having 6 nucleotides (nt) in stem III and 8 nt in stem I. Although these stems strongly influence ribozyme performance, their optimization is still empirical. Faster cleavage was obtained by adding facilitator oligonucleotides to ribozymes with shorter stems of (6+6) and (5+5) nt. Stimulation was particularly strong in the case of the (5+5) ribozyme, which was poorly active by itself. The enhancement caused by different facilitator oligonucleotides paralleled their expected ability to hybridize to RNA as a function of length and chemical modification.

Differential expression patterns of S100A2, S100A4 and S100A6 during progression of human malignant melanoma.

Three members of the S100 gene family, S100A2, S100A4 and S100A6, have been suggested to be associated with cancer development and metastasis. To study their involvement in the tumorigenesis of human melanoma, we examined the mRNA expression levels of the 3 genes in 45 melanoma metastases and in 20 benign nevi. Interestingly, whereas none of the metastases expressed S100A2 mRNA, and the expression level was low in 6 cell lines established from primary melanomas, all nevi showed moderate to high expression levels. Our results suggest that loss of S100A2 gene expression may be an early event in melanoma development. A significant correlation was found between the expression of S100A6 in melanoma metastases and both the survival time of the patients and the thickness of the corresponding primary tumors. For the S100A4 gene, however, no relationship was found between gene expression and clinical parameters of melanoma malignancy. The observed differences in expression patterns of the 3 S100 genes suggest distinct roles of their products in melanoma tumorigenesis and/or metastasis, and the results encourage studies to evaluate the potential value of using S100A2 and S100A6 expression levels as markers in the clinical management of melanoma.