Figure 2.

Genomic DNA was extracted from murine mammary tumor cell lines, was bisulfite modified and was PCR amplified using primer pairs specific for the murine methylated (M) (118 bp) product or the murine unmethylated (U) (126 bp) product. (a) Ten microliters of PCR product were loaded per lane of nondenaturing polyacrylamide gel and were stained with ethidium bromide. Lines 66.1, 410.4 and 67 express only the unmethylated band, whereas line 410 contains both methylated and unmethylated cyclooxygenase-2. (b) Genomic DNA was extracted from tumors derived from the transplantation of the indicated cell lines to syngeneic mice and was analyzed as in (a). The methylation pattern observed in cell lines is maintained when cells are transplanted to mice. (c) RNA was extracted from mammary tumor cell lines, was reverse transcribed and was amplified using primers specific for either murine COX-2 or β-actin. The fold increase was determined by densitometry. (d) Clonal populations were derived from line 410, and the DNA extracted, bisulfite modified and PCR amplified using primers as in (a). Each pair of methylated and unmethylated products represents an individual clone.