MGMT is a DNA-repair protein that contributes to resistance to certain mono-and bifunctional alkylating agents such as CENU. The 'long term objective' of this study is to enhance the efficacy of CENUs by silencing the MGMT gene from being expressed. Normally, when expressed, the functional gene is associated with open chromatin structure and lack of cytosine methylation in the 5'CpG island/promoter region of the gene. In addition, transcription factor binding is also present. When MGMT expression is lost, which occurs in as many as 20 percent of tumor cell lines and primary tumors, transcription factor binding is excluded due to diffuse methylation and closed chromatin structure of the promoter region CpG island. It is hypothesized in this application that the promoter region chromatin structure and methylation status is controlled at distal sites where certain DNA-protein interactions occur. These are nuclease-hypersensitive regions in or near the MGMT gene. Changes in these distal site interactions result in tumor-controlled silencing of the MGMT gene. This hypothesis will be tested by identifying and isolating the nuclease hypersensitive sites, and determining if these regions control the chromatin structure/methylation of the MGMT promoter. It will also be determined if the DNA-protein interactions in these nuclease sensitive sites differ between MGMT- and + cells. These studies could lead to the development of therapies which sensitize the tumor to CENUs. These findings could also contribute to the development of strategies directed at tumor-selective silencing or reactivation of other therapeutically-relevant, CpG island containing genes, e.g., mdr1, GST, metallothionein, p16/INK4A, p53, estrogen receptor, whose expression is likely to be similarly controlled by chromatin structure/methylation.
