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Many chemoradiation therapies cause DNA damage through oxidative stress. An important cellular mechanism that protects cells against oxidative stress involves DNA repair. One of the primary DNA repair mechanisms for oxidative DNA damage is base excision repair (BER). BER involves the tightly coordinated function of four enzymes (glycosylase, apurinic/apyrimidinic endonuclease, polymerase and ligase), in which 8-oxoguanine DNA glycosylase 1 initiates the cycle. An imbalance in the production of any one of these enzymes may result in the generation of more DNA damage and increased cell killing. In this study, we targeted mitochondrial DNA to enhance cancer chemotherapy by over-expressing a human 8-oxoguanine DNA glycosylase 1 (hOGG1) gene in the mitochondria of human hepatoma cells. Increased hOGG1 transgene expression was achieved at RNA, protein and enzyme activity levels. In parallel, we observed enhanced mitochondrial DNA damage, increased mitochondrial respiration rate, increased membrane potential and elevated free radical production. A greater proportion of the hOGG1-over-expressing hepatoma cells experienced apoptosis. Following exposure to a commonly used chemotherapeutic agent, cisplatin, cancer cells over-expressing hOGG1 displayed much shortened long-term survival when compared with control cells. Our results suggest that over-expression of hOGG1 in mitochondria may promote mitochondrial DNA damage by creating an imbalance in the BER pathway and sensitize cancer cells to cisplatin. These findings support further evaluation of hOGG1 over-expression strategies for cancer therapy. [ABSTRACT FROM PUBLISHER]

Clinically relevant cancer chemotherapeutic alkylating agents such as temozolomide and dacarbazine induce apoptosis and are mutagenic via the formation of O6-alkylguanine adducts in DNA. The DNA repair protein O6-alkylguanine-DNA alkyltransferase (AGT) functions by dealkylating such adducts and can thus prevent apoptosis and mutagenesis. In attempts to maximize the clinical effectiveness of these alkylating agents, inhibitors of AGT such as O6-benzylguanine (BeG) have been developed. We show here that within murine small intestinal crypt cells, BeG administration does not alter the apoptotic response to the direct-acting methylating agents N-methyl-N-nitrosurea (MNU), temozolomide and N-methyl-N′-nitro-N-nitrosoguanidine. Furthermore, we show that BeG pretreatment fails to elevate the mutation frequency at the murine Dlb-1 locus following exposure to MNU. Consistent with these results, we show that intestinal AGT activity is effectively abolished by administration of 100 mg/kg temozolomide, even in the absence of BeG. In contrast, pretreatment with BeG transiently abolished the apoptotic response to the methylating prodrug dacarbazine. Activation of dacarbazine to its reactive intermediate has previously been shown to be cytochrome P450 dependent and we show here that pretreatment of mice with the cytochrome P450 inhibitor metyrapone also inhibits dacarbazine-induced apoptosis. Thus BeG increases neither the prevalence of apoptosis nor mutation frequency in the murine small intestine, but is capable of inhibiting P450-dependent prodrug activation. The positive implication from this study is that BeG treatment may not exacerbate the toxic and mutagenic effects of methylating agents within normal cells, although it may engender other adverse reactions through the suppression of cytochrome P450-dependent processes. [ABSTRACT FROM PUBLISHER]

Chinese hamster V79 lung fibroblasts express low levels (specific activity 2–4 fmol/mg protein) of -alkylguanine (-AG) alkyltransferase (ATase). In cells surviving selection with low doses (10 μg/ml) of the chloroethylating agent, mitozolomide (Mz), ATase activity was increased 5-to 8-fold. Repeated selection of such cells produced a maximal specific activity of 36–40 fmol/mg protein, whilst selection at 20 or 40 μg/ml result in specific activities of ˜50 and 70 fmol/mg respectively. Only slight decreases in ATase activity were seen by 51 days after an initial selection with 10 μg/ml Mz. A similar effect was observed using chlorozotocin. Selected cells had a higher D37 for Mz (2.5–6.0 μg/ml) in comparison with control cell (D37, 0.8 μg/ml) but the D37s for nitrogen mustard and vincristine were closely similar in selected and control cells. Possible explanations for the increase in ATase activity are discussed. [ABSTRACT FROM PUBLISHER]

The lesion -alkylguanine (-AG) is produced in cellular DNA following exposure to monofunctional alkylating agents and its miscoding and mutagenic properties have been demonstrated in specific systems. In order to examine whether this lesion could be responsible for any of the biological effects of alkylating agents in mammalian cells, we have constructed a plasmid containing the -AG alkyltransferase (ATase) region of the gene from , the product of which normally repairs both -AG and alkyl-phosphotriesters in DNA. We have transfected the construction into Chinese hamster fibroblasts which are deficient in endogenous ATase activity and selected a clone that expresses the truncated repair gene. We demonstrate that this protein is functional, acts on damage in host cell DNA and protects the cells from the toxic effects of those alkylating agents that react extensively at oxygen atom positions. [ABSTRACT FROM PUBLISHER]

Many prokaryotic and eukaryotic cells contain enzymes that repair damage introduced into their DNA following exposure to chemical, physical and biological agents. One such lesion that has received considerable attention is the potentially miscoding and mutagenic base O-alkylguanine which is produced in varying amounts in DNA following reaction with monofunctional alkylating agents. As part of a study to assess the role of this lesion and its repair in the processes of cytotoxicity, mutagenicity and transformation, we have recently cloned the gene which codes for the protein responsible for the repair of such damage in DNA. In the present study we describe the construction of a plasmid which allows the efficient expression of the bacterial gene in mammalian cells. [ABSTRACT FROM PUBLISHER]

There is increasing evidence to suggest that O(6)-alkyl guanine DNA-alkyltransferase (MGMT) activity provides protection against alkylating agent induced formation of GC-->AT transition mutations in the K-ras oncogene of colorectal tumours. As this mutagenic event occurs during the growth of adenomas, both biomarkers of exposure (N7-methylguanine levels in DNA) and susceptibility (MGMT activity) were measured in biopsy samples obtained from normal and adenomatous tissue from 34 patients with large adenomas (>10 mm in size). There was no correlation between MGMT activity in the adenoma and in matched normal tissue. However, MGMT activity was significantly lower in adenoma tissue than in adjacent normal mucosa (5.18 versus 7.05 fmol/microg DNA, P = 0.01), particularly in men and those whose age was greater than the median. Upon stratification by K-ras mutational status, MGMT activity was lower in adenomas bearing a K-ras GC-->AT transition mutation (mean 4.21 fmol/microg DNA) than in adjacent normal tissue (mean 7.7 fmol/microg DNA; P < 0.004). In contrast, there was no significant difference in MGMT activity in adenomas lacking a K-ras GC-->AT transition mutation and adjacent normal mucosa. N7-methylguanine levels however did not vary with age, gender, K-ras mutational status or MGMT activity. These results are consistent with the acquisition of K-ras GC-->AT transition mutations in adenomas with low MGMT activity as a result of unavoidable exposure to methylating agents.