5-Aza-2'-deoxycytidine (DAC) is an investigational agent with demonstrated activity in the treatment of acute myeloid leukemia and likely therapeutic utility in other leukemias and myelodysplastic syndromes. After incorporation into DNA, DAC inhibits the enzyme DNA (cytosine-5)-methyltransferase (MT'ase). This inhibition has generally been assumed to be responsible for its activity. While this action may be necessary, our preliminary results suggest that MT'ase inhibition alone does not fully explain DAC's molecular biological effects. In neoplastic cells in culture, DAC has been shown to decrease the methylation of 5' regulatory regions and to increase the expression of tumor suppressor genes silenced by methylation. As a result, it could inhibit carcinogenesis or tumor progression. DAC has been shown to reduce polyp formation in a murine model for colonic preneoplasia when only one copy of the MT'ase gene is present. 5- Fluoro-2'-deoxycytidine (FdCyd) is another analog which, when present in DNA, binds irreversibly to MT'ase in vitro. Despite the fact that both analogs are irreversible inhibitors of the MT'ase, we have obtained preliminary evidence that FdCyd incorporated into cellular DNA does not decrease the methylation of the tumor suppressor gene p16. DAC and FdCyd do differ significantly in their chemical stability; DAC is far less stable because of its triazine ring structure. Our, Hypothesis is that, in addition in inhibiting MT'ase, unstable DAC residues in DNA contribute to the loss of methylation through DNA damage-induced excision and repair.
Our Specific Aims to test this hypothesis are: (1) to compare the reactivation of p16 by DAC and FdCyd as measured by the methylation of p16 exon and the expression of p16 mRNA, (2) to compare the incorporation of DAC and FdCyd into DNA in cells, and (3) to compare the inhibition of MT'ase by DAC and FdCyd in cells as measured by the MT'ase activity in cellular extracts and the methylation of newly- incorporated deoxycytidine residues in DNA. As part of specific aim 3, we will develop a new assay for MT'ase which does not require measuring the incorporation of radioactivity from [CH3-3H]-S-adenosylmethionine into DNA. This new assay will expedite the analysis of samples in this project and other studies, including future clinical trials. If our hypothesis is correct, DNA-damaging agents combined with FdCyd should produce the same effects as DAC does through its two different actions, MT'ase inhibition and DNA damage. Increasing DNA damage with another agent might also potentiate the effects of DAC. Thus, our final specific aim in this project (4) is to determine the effects of DNA- damaging agents combined with FdCyd and DAC on the methylation and expression of specific genes. The overall goal of this proposal is to understand the actions of MT'ase inhibitors and to use this understanding to develop more effective methods of modulating DNA methylation in human diseases.
