P1798

P1798/ AzaC

Liver

Hepatoma Hepatoma/ AzaC

Fig. 8. PCR sequencing of the amplified MT-1 promoter from bisulfite-converted mouse (A) and rat (B) chromosomal DNA. Arrows indicate methyl cytosines. [From S. Majumder, K. Ghoshal, Z. Li, Y. Bo, and S. T. Jacob, Oncogene 18,6287 (1999); and K. Ghoshal, S. Majumder, Z. Li, X. Dong, and S. T. Jacob, J. Biol. Chem. 275,539 (2000).]

with Taql (T^CGA). This restriction site is not present within the —526 bp to —2 bp region of mouse MT-I DNA, but is generated after CmCGA (internal C methyla-tion) is converted to TCGA on bisulfite conversion. Complete digestion of the amplified product from the lymphosarcoma DNA with Taql and lack of digestion of that from the thymus with Taql (Fig. 7B) demonstrate that MT-1 promoter is methylated in the latter. Similarly, the PCR-amplified DNA from bisulfite-treated liver and hepatoma DNA is completely digested with Apol and Tsp509l.

The amplified products are sequenced by the Femtomol sequencing method (Fig. 8A and B). All cytosines in mouse thymus and rat liver are converted to thymines, whereas only those cytosines that are followed by guanines remain intact in lymphosarcoma cells and hepatoma, indicating methylation in vivo. After treatment with 5-azaC only 20% of the CpGs are demethylated in P1798 cells, whereas in the hepatoma almost all the cytosines are demethylated.

Acknowledgments

The research performed in the authors' laboratory was supported, in part, by Grants ES 10874 and CA 81024 from the National Institute of Environmental Health Sciences and the National Cancer Institute, respectively.

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