As the cells progressed further towards malignancy and became immortalized, DNA methylation was observed at several additional gene loci including, RASSF1A, SFRP1, p57, and MGMT. In order to determine whether this panel of four genes represented a program of epigenetic alterations important for the immortalization of mammary epithelial cells, we examined whether these genes were methylated in the immortalized, but non-tumorigenic 184-A1 and MCF-10A mammary cells, as well as in the MCF-7 and MDA-231 breast cancer cell lines. The gene discussed is RASSF1; the disease is breast cancer.