EPIGENETICS OF CANCER

Peter A. Jones,* Gangning Liang, Carvell Nguyen, Christine Yoo, Jonathan Cheng
Department of Biochemistry and Molecular Biology,
USC/Norris Comprehensive Cancer Center,
The Keck School of Medicine of the University of Southern California,
1441 Eastlake Avenue, Los Angeles, California 90089-9181, USA
*jones_p@ccnt.hsc.usc.edu

INTRODUCTION. It is becoming increasingly clear that the epigenetic silencing of cancer related genes by cytosine methylation represents a common pathway for their inactivation (1,2). Heritably silenced tumor suppressor genes adopt a chromatin configuration which is associated with increased binding of methyl binding proteins and also increased methylation of the lysine-9 residue of histone H3. This epigenetic silencing can be heritably maintained over a long period yet is subject to reversal by drugs which inhibit cytosine methylation in DNA. Our research is focused on the mechanisms for abnormal methylation of CpG islands and in the development of strategies to reverse silencing so that increased growth control can be exerted on cancer cells.

METHOD. Methylation of cytosine residues in human DNA can be measured following bisulfite treatment of DNA which converts cytosine residues to uracil yet leaves 5-methylcytosine residues intact. We have developed quantitative methods to take advantage of this procedure and have used the quantitative Ms-SNuPE (methylation sensitive single nucleotide primer extension) assay to measure methylation of defined sites in uncultured tumors in cell lines. Additionally, we sequence individual DNA molecules to determine the patterns of methylation. To study de novo methylation, which normally takes place over a long period of time, we utilize cell lines exposed transiently to the DNA methylation inhibitor 5-aza-2’-deoxycytidine and measure the kinetics and details of remethylation following removal of the drug.

RESULTS. Quantitative bisulfite analysis of DNA from uncultured tumors shows that CpG islands located in the promoters of growth regulatory genes are frequently subject to de novo methylation and silencing in human cancer (3). Studies with cell lines have shown that these changes are reinforced when cells are cultured and result in an altered chromatin structure which is consistent with the heritable silencing of cancer related genes (4,5). These changes can be rapidly reversed by transient treatment with 5-aza-2’-deoxcytidine and robust gene expression seen at loci which have been silenced over a long period of time in culture. Re-expression is not permanent, since methylation tends to reoccur in the promoter regions following culture and passage of the cells. This can result in a significant problem for the future chemotherapeutic application of these compounds since tumor cells might be expected to revert to their original epigenetic state following drug treatment.

More recently we have focused our attention on the stable cytidine analog, zebularine, which is incorporated into DNA and forms covalent bond with DNA methyltransferase enzymes. Zebularine can be given continuously to cells and keeps genes actively expressed over a prolonged period of time (6,7). The drug also results in the strong knockdown in the level of active DNA methyltransferase protein within treated cells. Importantly, zebularine can be given to animals orally and we have shown that it can result in the re-expression of silenced tumor suppressor genes in human tumor xenografts growing in nude mice. These results suggest that epigenetic therapy in which silenced genes are reactivated may be a viable therapeutic option for the treatment of human cancer.

ACKNOWLEDGMENT. This work was supported by grant CA082422 from the National Cancer Institute.

REFERENCES

1. Jones, P.A., and Laird, P. (1999) Nat. Genet. 21, 163-167.
2. Jones, P.A., and Baylin, S.B. (2002) Nat. Rev. Genet. 3, 415-428.
3. Bender, C.M., Gonzalgo, M.L., Gonzales, F.A., Nguyen, C.T., Robertson, K.D., and Jones, P.A. (1999) Mol. Cell. Biol. 19, 6690-6698.
4. Nguyen, C.T., Gonzales, F.A., and Jones, P.A. (2001) Nucl. Acids Res. 29, 4598-4606.
5. Nguyen, C.T., Weisenberger, D.J., Velicescu, M., Gonzales, F.A., Lin, J.C., Liang, G., and Jones, P.A. (2002) Cancer Res. 62, 6456-6461.
6. Cheng, J.C., Matsen, C.B., Gonzales, F.A., Ye, W., Greer, S., Marquez, V.E., Jones, P.A., and Selker, E.U. (2003) J. Natl. Cancer Inst. 95, 399-409.
7. Cheng, J.C., Weisenberger, D.J., Gonzales, F.A., Liang, G., Xu, G-L., Hu, Y-G., Marquez, V.E., and Jones, P.A. (in press) Mol. Cell. Biol.