Chemical Pathology of 5-aza-2'-deoxycytidine

点击翻译按钮获取中文摘要

DESCRIPTION (provided by applicant): The nucleoside analogue, 5-aza-2'-deoxycytidine (DAC) is an investigational agent for the treatment of sickle cell anemia as well as advanced human malignancies. When administered, DAC is incorporated into the DNA of replicating cells. In DNA, DAC can act as an inhibitor of cytosine methyltransferase, resulting in the reactivation of the fetal hemoglobin gene in sickle cell anemia and tumor suppressor genes in human cancer. In addition to its effects on DNA methylation, DAC also induces cellular toxicity and triggers DNA repair responses. We are interested in studying the underlying chemical properties of DAC that may account for both the therapeutic and toxic activities. This amended proposal is divided into five aims. In the first aim, we will examine quantitatively the degradation of DAC in aqueous solution as a function of both temperature and pH, using a battery of UV, NMR, GC/MS and HPLC/MS methods. In the second aim, we will build DAC into synthetic oligonucleotides using established phosphoramidite methods. We will then study the stability of DAC in duplex DNA using a novel isotope-edited NMR technique. We suspect that DAC in DNA exists as an equilibrium between ring-closed and ring-opened forms, given that this equilibrium is known to occur for DAC in solution. A solid understanding of this equilibrium is essential to determining the potential mutagenicity, cytotoxicity and mechanism of demethylation induced by DAC. In aim three, we will examine the coding properties of DAC in oligonucleotides prepared by either chemical synthesis or DAC-triphosphate incorporation using purified DNA polymerases in an in vitro assay. We expect that the ring-closed form will base pair normally. The ring-opened forms are expected to direct rniscoding or act as a non-informational lesion. In aim four, we will examine the repair of DAC using an in vitro assay. DAC-containing oligonucleotides will be incubated with purified DNA glycosylases and whole cell extracts. We anticipate that the ring-opened forms of DAC will be rapidly removed by glycosylases. In aim five, we will measure DAC incorporation and 5-methylcytosine levels in cells grown in culture. This aim should establish an inverse correlation between incorporated DAC and methylated cytosine levels. We further propose testing a novel stable-isotope mass spectrometry (GC/MS) method that might reveal additional mechanisms by which DAC could induce genome demethylation. The results of the studies proposed here should shed significant light on the chemical properties of DAC that could be responsible for the array of biochemical effects induced by this compound in human cells.