A novel fluorometric oligonucleotide assay to measure O( 6)-methylguanine DNA methyltransferase, methylpurine DNA glycosylase, 8-oxoguanine DNA glycosylase and abasic endonuclease activities: DNA repair status in human breast carcinoma cells overexpressing methylpurine DNA glycosylase.

DNA repair status plays a major role in mutagenesis, carcinogenesis and resistance to genotoxic agents. Because DNA repair processes involve multiple enzymatic steps, understanding cellular DNA repair status has required several assay procedures. We have developed a novel in vitro assay that allows quantitative measurement of alkylation repair via O(6)-methylguanine DNA methyltransferase (MGMT) and base excision repair (BER) involving methylpurine DNA glycosylase (MPG), human 8-oxoguanine DNA glycosylase (hOGG1) and yeast and human abasic endonuclease (APN1 and APE/ref-1, respectively) from a single cell extract. This approach involves preparation of cell extracts in a common buffer in which all of the DNA repair proteins are active and the use of fluorometrically labeled oligonucleotide substrates containing DNA lesions specific to each repair protein. This method enables methylation and BER capacities to be determined rapidly from a small amount of starting sample. In addition, the stability of the fluorometric oligonucleotides precludes the substrate variability caused by continual radiolabeling. In this report this technique was applied to human breast carcinoma MDA-MB231 cells overexpressing human MPG in order to assess whether up-regulation of the initial step in BER alters the activity of selected other BER (hOGG1 and APE/ref-1) or direct reversal (MGMT) repair activities.

DNA repair and gene therapy: implications for translational uses.

Gene therapy has been proposed to have implications in the treatment of cancer. By genetically manipulating the hematopoietic stem cell compartment with genes that confer resistance to chemotherapeutic agents, the dose escalation that is necessary to effectively treat the cancers could potentially be achieved. DNA repair genes are some of the potential candidates to confer increased resistance to chemotherapeutic agents. Although initial focus in this area has been on the direct reversal protein (MGMT), its protective ability is limited to those agents that produce O(6)-methylGuanine cross-links-agents that are not extensively used clinically (e.g., nitrosoureas). Furthermore, most alkylating agents attack more sites in DNA other than O(6)-methylGuanine, such that the protections afforded by MGMT may prevent the initial cytotoxicity, but at a price of increased mutational burden and potential secondary leukemias. Therefore, some of the genes that are being tested as candidates for gene transfer are base excision repair (BER) genes. We and others have found that overexpression of selective BER genes confers resistance to chemotherapeutic agents such as thiotepa, ionizing radiation, bleomycin, and other agents. As these "proof of concept" analyses mature, many more clinically relevant chemotherapeutic agents can be tested for BER protective ability.

Going APE over ref-1.

The DNA base excision repair (BER) pathway is responsible for the repair of cellular alkylation and oxidative DNA damage. A crucial and the second step in the BER pathway involves the cleavage of baseless sites in DNA by an AP endonuclease. The major AP endonuclease in mammalian cells is Ape1/ref-1. Ape1/ref-1 is a multifunctional protein that is not only responsible for repair of AP sites, but also functions as a reduction-oxidation (redox) factor maintaining transcription factors in an active reduced state. Ape1/ref-1 has been shown to stimulate the DNA binding activity of numerous transcription factors that are involved in cancer promotion and progression such as Fos, Jun, NF(B, PAX, HIF-1(, HLF and p53. Ape1/ref-1 has also been implicated in the activation of bioreductive drugs which require reduction in order to be active and has been shown to interact with a subunit of the Ku antigen to act as a negative regulator of the parathyroid hormone promoter, as well as part of the HREBP transcription factor complex. Ape1/ref-1 levels have been found to be elevated in a number of cancers such as ovarian, cervical, prostate, rhabdomyosarcomas and germ cell tumors and correlated with the radiosensitivity of cervical cancers. In this review, we have attempted to try and assimilated as much data concerning Ape1/ref-1 and incorporate the rapidly growing information on Ape1/ref-1 in a wide variety of functions and systems.

Protection of mammalian cells against chemotherapeutic agents thiotepa, 1,3-N,N'-bis(2-chloroethyl)-N-nitrosourea, and mafosfamide using the DNA base excision repair genes Fpg and alpha-hOgg1: implications for protective gene therapy applications.

Chemotherapeutic agents used in the treatment of cancer often lead to dose-limiting bone marrow suppression and may initiate secondary leukemia. N,N',N"-triethylenethiophosphoramide (thiotepa), a polyfunctional alkylating agent, is used in the treatment of breast, ovarian, and bladder carcinomas and is also being tested for efficacy in the treatment of central nervous system tumors. Thiotepa produces ring-opened bases such as formamidopyrimidine and 7-methyl-formamidopyrimidine, which can be recognized and repaired by the formamidopyrimidine glycosylase/AP lyase (Fpg) enzyme of Escherichia coli. Using this background information, we have created constructs using the E. coli fpg gene along with the functional equivalent human ortholog alpha-hOgg1. Although protection with the Fpg protein has been previously observed in Chinese hamster ovary cells, we demonstrate significant (100-fold) protection against thiotepa using the E. coli Fpg or the human alpha-hOgg1 cDNA in NIH3T3 cells. We have also observed a 10-fold protection by both the Fpg and alpha-hOgg1 transgenes against 1,3-N,N'-bis(2-chloroethyl)-N-nitrosourea (BCNU) and, to a lesser extent, mafosfamide (2-fold), an active form of the clinical agent cyclophosphamide. These latter two findings are novel and are particularly significant since the added protection was in an O(6)-methylguanine-DNA methyltransferase-positive background. These results support our general approach of using DNA base excision repair genes in gene therapy for cellular protection of normal cells during chemotherapy, particularly against the severe myelosuppressive effect of agents such as thiotepa, BCNU, and cyclophosphamide.