O6-methylguanine (O6-MeG) is the primary cytotoxic lesion induced by SN1-methylating agents such as temozolomide (TMZ), a chemotherapeutic agent used in the treatment of malignant glioma and other human tumors. Unrepaired O6-MeG adducts cause mismatch repair (MMR)-directed apoptosis or, if tolerated, can lead to the induction of mutations. In investigating the mechanism of action of TMZ, we recently reported that TMZ inhibits the activity of nuclear factor-:B (NF-:B) through damage specific attenuation of DNA-binding. The overall goal of our work is to examine the role of NF-:B in the response to chemotherapeutic methylating agents as a basis to improving their anti-tumor action and ameliorating their carcinogenic effects. Preliminary studies demonstrate that the p50 (NF-:B1) subunit is required for inhibition of NF-:B by TMZ and that deletion of p50 renders cells highly resistant to TMZ-induced killing. Based on the above data, we hypothesize that the DNA damage response to O6-MeG-inducing agents is mediated by p50. The experiments outlined below will determine whether p50 acts as an effector specific to the O6-MeG-induced DNA damage response pathway and whether p50 expression level is important in regulating the chemotherapeutic and carcinogenic actions of TMZ.
In Aim 1, experiments will be performed to examine the mechanism by which p50 mediates TMZ-induced inhibition of NF-:B DNA-binding. Studies will first look upstream up p50 at the Chk1/p50 interaction and then look downstream of p50 at the :B-site DNA sequence In Aim 2, experiments will examine whether p50-mediated inhibition of NF-:B is a specific response to O6- MeG damage. The O6-MeG lesion will be isolated using oligonucleotide duplexes and studies performed both in vitro and in intact cells. We will first look upstream of p50 to examine if inhibition of NF-:B is a specific response to O6-MeG damage or a general consequence of DNA mismatch recognition. Next, experiments will examine if O6-MeG-induced inhibition of NF-:B, mediated by p50, is sufficient to elicit the downstream damage response involving apoptosis.
In Aim 3, to determine if loss of p50 may be clinically relevant in the treatment of glioma with TMZ, animal experiments will examine if depletion of p50 renders glioma xenografts resistant to the anti-tumor effect of TMZ.
In Aim 4, to examine if loss of p50 predisposes to chemotherapeutic-induced carcinogenesis, experiments will be performed to determine whether deletion of p50 renders cells prone to mutation and malignant transformation following TMZ treatment.
The effectiveness of alkylating chemotherapeutic agents is dependent on the expression of specific molecules within the tumor cells. In this proposal, the ability of a specific intermediate, nuclear factor-:B, to enable killing by alkylating agents will be investigated in an attempt to determine whether this molecule plays a role in the overall response of patients to alkylating chemotherapy.
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