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 pr otein, induce metastatic capability in benign mammary tumor-derived epithel ial cells and in transgenic mice bearing oncogene-induced benign mammary tu mors. The S100OA4(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 reg ulatory elements in the S100A4 gene, an intronic enhancer and TATA box regi on, revealed that in low S100A4-expressing cells, most cytosine bases exhib ited high levels of resistance to conversion to thymine by sodium bisulfite . In derivative cell lines, which express high levels of S100A4, only a sma ll number of cytosine bases were resistant to treatment with sodium bisulfi te. In contrast, cytosine bases in the DNA surrounding an upstream regulato ry region, which binds inhibitory GC factor in the low-expressing cell line s, are sensitive to conversion to thymine by sodium bisulfite in both low- and high-expressing cell lines. The results suggest that the rat S100A4 gen e is maintained in a different state in the low-expressing cell lines and t hat this state might be a consequence of the pattern of methylation in this regulated gene that does not contain a CPG island.