The long-term objective of this application is to understand the mechanism by which tumors transcriptionally silence CpG island-containing genes. CpG islands are regions of DNA associated with promoters of approximately 50 percent of human genes. In normal cells, CpG islands are devoid of cytosine methylation and have a unique chromatin structure, which supports expression of associated genes. In tumors and SV-40-transformed cells, CpG island-containing genes can be aberrantly silenced by a mechanism involving methylation and alteration of chromatin structure. While methylation-associated gene silencing plays a role in many processes important in cancer biology, the events that trigger CpG island methylation are not understood. The presence of methylated CpG islands in SV40-infected, T antigen-expressing cells suggests that inactivation of pRb and/or p53 may be a prerequisite for aberrant methylation. Changes in expression/activity of other proteins may also be involved, however, as CpG island methylation does not seem to occur immediately after pRb/p53 inactivation, but rather following growth of pRb and/or p53 deficient cells to near the end of their lifespan. Among the proteins upregulated as cells near the end of their lifespans are the cyclin-dependent kinase inhibitors p16 and p21. p16 and p21 block the phosphorylation of proteins such as pRb, block entry of cells into S-phase, and may block the action of proteins that function in the reassembly of appropriately methylated chromatin that occurs following DNA replication. Cells lacking functional pRb/p53 cannot respond to p16/p21-mediated growth arrest signals and replicate their DNA under conditions in which chromatin restructuring, and the closely linked process of methylation, may be disregulated. The applicant hypothesizes that p53 and/or pRb inactivation, in combination with high levels of p16 or p21, triggers CpG island methylation. This hypothesis will be tested by the following specific aims: 1) To determine if functional inactivation of p53/pRb is alone sufficient to trigger CpG island methylation. 2) To determine if over-expression of p16 and/or p21 are alone sufficient to trigger CpG island methylation. 3) To determine if functional inactivation of p53/pRb in combination with over-expression of p16 and/or p21 predisposes cells to inappropriate methylation of CpG islands. These studies represent a first step in understanding the gene silencing process and will ultimately contribute to the development of therapies addressed at controlling gene expression.
| Hirose, Yuichi; Kreklau, Emiko L; Erickson, Leonard C et al. (2003) Delayed repletion of O6-methylguanine-DNA methyltransferase resulting in failure to protect the human glioblastoma cell line SF767 from temozolomide-induced cytotoxicity. J Neurosurg 98:591-8 |
| Hirose, Y; Berger, M S; Pieper, R O (2001) p53 effects both the duration of G2/M arrest and the fate of temozolomide-treated human glioblastoma cells. Cancer Res 61:1957-63 |
| Hirose, Y; Berger, M S; Pieper, R O (2001) Abrogation of the Chk1-mediated G(2) checkpoint pathway potentiates temozolomide-induced toxicity in a p53-independent manner in human glioblastoma cells. Cancer Res 61:5843-9 |
