Figure 3.

4-nitroquinoline 1-oxide (4-NQO)-induced methylation of Rarβ₂ gene promoter in murine tumors. Mice were treated with 45 μg/ml of 4-NQO in the drinking water for eight weeks and then received regular drinking water for another 16 weeks. The mice were then sacrificed, and five cases of normal and cancerous tissues were used for this analysis. A: Methylation-specific PCR (MSP). Murine tissue specimens were subjected to DNA extraction, and genomic DNA was then subjected to bisulfate treatment using a methylation kit from Zymed and PCR analysis of Rarβ₂ gene promoter methylation. Note: M, Methylated DNA primers; U, Un-methylated DNA primers. B: DNA sequencing. The MSP products from A were cloned, amplified, and then sequenced in our Institutional DNA sequencing facility. Compared with normal tissues (not treated with 4-NQO), tissues from the 4-NQO-treated mice showed methylation (arrows). Note: Bisulfite converted all C to T, but methylated C cannot be converted, which is the principle of the MSP assay. The graphs show the antisense sequences due to the DNA cloning process.