Browsing by Author "Wilson, Glenn L."
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Item Altering DNA Base Excision Repair: Use of Nuclear and Mitochondrial-Targeted N-Methylpurine DNA Glycosylase to Sensitize Astroglia to Chemotherapeutic Agents(2007-11) Harrison, Jason F.; Rinne, Mikael L.; Kelley, Mark R.; Druzhyna, Nadiya M.; Wilson, Glenn L.; Ledoux, Susan P.Primary astrocyte cultures were used to investigate the modulation of DNA repair as a tool for sensitizing astrocytes to genotoxic agents. Base excision repair (BER) is the principal mechanism by which mammalian cells repair alkylation damage to DNA and involves the processing of relatively nontoxic DNA adducts through a series of cytotoxic intermediates during the course of restoring normal DNA integrity. An adenoviral expression system was employed to target high levels of the BER pathway initiator, N-methylpurine glycosylase (MPG), to either the mitochondria or nucleus of primary astrocytes to test the hypothesis that an alteration in BER results in increased alkylation sensitivity. Increasing MPG activity significantly increased BER kinetics in both the mitochondria and nuclei. Although modulating MPG activity in mitochondria appeared to have little effect on alkylation sensitivity, increased nuclear MPG activity resulted in cell death in astrocyte cultures treated with methylnitrosourea (MNU). Caspase-3 cleavage was not detected, thus indicating that these alkylation sensitive astrocytes do not undergo a typical programmed cell death in response to MNU. Astrocytes were found to express relatively high levels of antiapoptotic Bcl-2 and Bcl-XL and very low levels of proapoptotic Bad and Bid suggesting that the mitochondrial pathway of apoptosis may be blocked making astrocytes less vulnerable to proapoptotic stimuli compared with other cell types. Consequently, this unique characteristic of astrocytes may be responsible, in part, for resistance of astrocytomas to chemotherapeutic agents.Item Conditional Targeting of the DNA Repair Enzyme hOGG1 into Mitochondria(2002-11) Rachek, Lyudmila I.; Grishko, Valentina I.; Musiyenko, Sergiy I.; Kelley, Mark R.; LeDoux, Susan P.; Wilson, Glenn L.Oxidative damage to mitochondrial DNA (mtDNA) has been suggested to be a key factor in the etiologies of many diseases and in the normal process of aging. Although the presence of a repair system to remove this damage has been demonstrated, the mechanisms involved in this repair have not been well defined. In an effort to better understand the physiological role of recombinant 8-oxoguanine DNA glycosylase/apurinic lyase (OGG1) in mtDNA repair, we constructed an expression vector containing the gene for OGG1 downstream of the mitochondrial localization sequence from manganese-superoxide dismutase. This gene construct was placed under the control of a tetracycline-regulated promoter. Transfected cells that conditionally expressed OGG1 in the absence of the tetracycline analogue doxycycline and targeted this recombinant protein to mitochondria were generated. Western blots of mitochondrial extracts from vector- and OGG1-transfected clones with and without doxycycline revealed that removal of doxycycline for 4 days caused an approximate 8-fold increase in the amount of OGG1 protein in mitochondria. Enzyme activity assays and DNA repair studies showed that the doxycycline-dependent recombinant OGG1 is functional. Functional studies revealed that cells containing recombinant OGG1 were more proficient at repairing oxidative damage in their mtDNA, and this increased repair led to increased cellular survival following oxidative stress.Item Enhanced mtDNA repair and cellular survival following oxidative stress by targeting the hOGG repair enzyme to mitochondria.(2000-12) Dobson, Allison W.; Xu, Yi; Kelley, Mark R.; LeDoux, Susan P.; Wilson, Glenn L.Oxidative damage to mtDNA has been implicated as a causative factor in many disease processes and in aging. We have recently discovered that different cell types vary in their capacity to repair this damage, and this variability correlates with their ability to withstand oxidative stress. To explore strategies to enhance repair of oxidative lesions in mtDNA, we have constructed a vector containing a mitochondrial transport sequence upstream of the sequence for human 8-oxoguanine glycosylase. This enzyme is the glycosylase/AP lyase that participates in repair of purine lesions, such as 8-oxoguanine. Western blot analysis confirmed this recombinant protein was targeted to mitochondria. Enzyme activity assays showed that mitochondrial extracts from cells transfected with the construct had increased enzyme activity compared to cells transfected with vector only, while nuclear enzyme activity was not changed. Repair assays showed that there was enhanced repair of oxidative lesions in mtDNA. Additional studies revealed that this augmented repair led to enhanced cellular viability as determined by reduction of tetrazolium compound to formazan, Trypan blue dye exclusion, and clonogenic assays. Therefore, targeting of DNA repair enzymes to mitochondria may be a viable approach for the protection of cells against some of the deleterious effects of oxidative stress.Item Yeast apurinic/apyrimidinic endonuclease Apn1 protects mammalian neuronal cell line from oxidative stress(2007-07) Ho, Renee; Rachek, Lyudmila I.; Xu, Yi; Kelley, Mark R.; LeDoux, Susan P.; Wilson, Glenn L.Reactive oxygen species (ROS) have been implicated as one of the agents responsible for many neurodegenerative diseases. A critical target for ROS is DNA. Most oxidative stress-induced DNA damage in the nucleus and mitochondria is removed by the base excision repair pathway. Apn1 is a yeast enzyme in this pathway which possesses a wider substrate specificity and greater enzyme activity than its mammalian counterpart for removing DNA damage, making it a good therapeutic candidate. For this study we targeted Apn1 to mitochondria in a neuronal cell line derived from the substantia nigra by using a mitochondrial targeting signal (MTS) in an effort to hasten the removal of DNA damage and thereby protect these cells. We found that following oxidative stress, mitochondrial DNA (mtDNA) was repaired more efficiently in cells containing Apn1 with the MTS than controls. There was no difference in nuclear repair. However, cells that expressed Apn1 without the MTS showed enhanced repair of both nuclear and mtDNA. Both Apn1-expressing cells were more resistant to cell death following oxidative stress compared with controls. Therefore, these results reveal that the expression of Apn1 in neurons may be of potential therapeutic benefit for treating patients with specific neurodegenerative diseases.