Differential reactivity of the rat S100A4(p9Ka) gene to sodium bisulfite is associated with differential levels of the S100A4 (p9Ka) mRNA in rat mammary epithelial cells

Elevated intracellular levels of S100A4, an S100-related calcium-binding protein, induce metastatic capability in benign mammary tumor-derived epithelial cells and in transgenic mice bearing oncogene-induced benign mammary tumors. The S100A4(p9Ka) gene in rat mammary epithelial cells expressing low levels of S100A4 yields a reduced number of fragments upon digestion with the methylation-sensitive restriction enzyme, HpaII, compared with the gene from high S100A4-expressing cells. Genomic sequencing of two potential regulatory elements in the S100A4 gene, an intronic enhancer and TATA box region, revealed that in low S100A4-expressing cells, most cytosine bases exhibited high levels of resistance to conversion to thymine by sodium bisulfite. In derivative cell lines, which express high levels of S100A4, only a small number of cytosine bases were resistant to treatment with sodium bisulfite. In contrast, cytosine bases in the DNA surrounding an upstream regulatory region, which binds inhibitory GC factor in the low-expressing cell lines, are sensitive to conversion to thymine by sodium bisulfite in both low- and high-expressing cell lines. The results suggest that the rat S100A4 gene is maintained in a different state in the low-expressing cell lines and that this state might be a consequence of the pattern of methylation in this regulated gene that does not contain a CpG island.