Overwhelming evidence has demonstrated that the major mechanism of tumor cell resistance to the chloroethyl-nitrosoureas (CENU) results from the DNA repair activity of O-6 methylguanine DNA methyltransferase (MGMT). This DNA repair protein is thought to protect cells from the cytotoxic DNA interstrand crosslink (ISC) produced by the CENU by removing chloroethyl adducts from the O-6 position of guanine before these adducts can rearrange to form a lethal crosslink. Studies conducted over the previous eight years of support have demonstrated that this DNA repair system can be temporarily inhibited by a variety of biochemical strategies including pre-incubation of tumor cells with DNA methylating agents such as streptozotocin (STZ) which produce the natural substrate for MGMT, O-6 methylguanine. Repair of this lesion depletes the tumor cell of MGMT due to MGMT's suicide repair activity. In addition, the free bases O-6 methylguanine (MG) and O-6 benzylguanine (BG) can also deplete cells of MGMT activity and subsequently sensitize tumor cells to treatment with BCNU. During the most recent funding period the applicant has demonstrated that STZ combined with BG and BCNU can produce a prolonged sensitization of resistant tumor cells in vitro and in xenograft tumors in vivo. Soon BG plus BCNU will be tested in Phase I clinical trials at other institutions and BG plus STZ plus BCNU will be tested at this institution. However, biochemical modulation strategies are not selective for tumor cells over normal cells. In this application the applicant proposes to develop molecular strategies for modulating resistance to the CENU which might be applied to tumor-specific therapy in the future.
Aim 1 will determine the optimum ribozyme target sequence(s) by transfecting human tumor cells with pooled MGMT ribozymes constitutively expressed by a high expression promoter. This approach will identify the optimum ribozyme target sequence(s), and examine the loss of resistance phenotype by identification of clones sensitive to BCNU.
Aim 2 will optimize expression of antisense ribozymes designed to degrade long-lived MGMT mRNA using inducible promoters. These studies, continued from the previous period of support, will utilize ribozymes transcribed from inducible promoters such as the heat-shock promoter (HSP) or the glucocorticoid promoter (GCP).
Aim 3 will identify random oligoribonucleotides capable of direct inhibition of MGMT using the SELEX system developed by Cold and co-workers. The SELEX (Systematic Evolution of Ligands by Exponential enrichment) procedure identifies highly specific, high affinity ligands from a heterogeneous pool of random RNA molecules.
Aim 4 will identify Genetic Suppressor Elements (GSEs) generated from random MGMT EDNA sequences which lead to inhibition or down-regulation of MGMT function or expression. GSE technology utilizes partial cDNA sequences of MGMT to express partial peptides in the sense orientation, and anti-sense RNAs in the opositer orientation. The applicant strongly believes that any molecular reagents (ribozyme, antisense RNA, GSE, or SELEX oligo) identified in the above Specific Aims will be powerful candidates for gene therapy in the future as tumor-specific delivery vectors are developed by others. His molecular reagents will be poised for testing in those systems. Collaborations have been established with laboratories with outstanding expertise in liposome and retroviral vector development for future studies of his anti-MGMT reagents.
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