Formation Of O6-methylguanine Research Articles

Exploring the Potential of Ellagic Acid in Gastrointestinal Cancer Prevention: Recent Advances and Future Directions.

Gastrointestinal (GI) cancers are a significant global health concern with diverse etiologies and limited treatment options. Ellagic acid (EA), a natural polyphenolic compound, exhibits promising anticancer properties against various GI malignancies. In this article, we have reviewed recent research on the anticancer potential of EA across esophageal, gastric, colorectal, pancreatic, and liver cancers. In esophageal cancer, EA inhibits the formation of O6-methylguanine (O6-meGua) adducts induced by carcinogens like N-nitrosomethylbenzylamine (NMBA), thereby suppressing tumor growth. Additionally, EA inhibits STAT3 signaling and stabilizes tumor suppressor proteins, showing potential as an anti-esophageal cancer agent. In gastric cancer, EA regulates multiple pathways involved in cell proliferation, invasion, and apoptosis, including the p53 and PI3K-Akt signaling pathways. It also demonstrates anti-inflammatory and antioxidant effects, making it a promising therapeutic candidate against gastric cancer. In colorectal cancer (CRC), EA inhibits cell proliferation, induces apoptosis, and modulates the Wnt/β-catenin and PI3K/Akt pathways, suggesting its efficacy in preventing CRC progression. Furthermore, EA has shown promise in pancreatic cancer by inhibiting nuclear factor-kappa B, inducing apoptosis, and suppressing epithelial-mesenchymal transition. In liver cancer, EA exhibits radio-sensitizing effects, inhibits inflammatory pathways, and modulates the tumor microenvironment, offering potential therapeutic benefits against hepatocellular carcinoma. Studies on EA potential in combination therapies and the development of targeted delivery systems are required for enhanced efficacy against gastrointestinal cancers.

Chemopreventive effects and anti-tumorigenic mechanisms of 2,6-dimethoxy-1,4-benzoquinone, a constituent of Vitis coignetiae Pulliat (crimson glory vine, known as yamabudo in Japan), toward 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK)-induced lung tumorigenesis in A/J mice

Previously, we isolated and identified anti-mutagenic and anti-inflammatory components from Vitis coignetiae (crimson glory vine, known as yamabudo in Japan) as 2,6-dimethoxy-1,4-benzoquinone (DBQ), fertaric acid and caftaric acid. We also reported that the oral intake of a partially purified fraction from yamabudo juice (yamabudo-fr) or DBQ affords significant protection against two-stage skin carcinogenesis in mice. In this study, we found that oral intake of yamabudo-fr or DBQ affords significant protection against a tobacco-specific nitrosamine, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK)-induced mouse model of lung tumorigenesis. Furthermore, we investigated the anti-tumorigenic mechanisms of yamabudo juice and DBQ. NNK is known to be a DNA-methylating and alkylating agent; thus, we investigated the anti-tumorigenic mechanisms of yamabudo juice and DBQ in relation to DNA methylation. Pretreatment with yamabudo-fr or DBQ dose-dependently decreased formation of O6-methylguanine and N7-methylguanine in DNA of the A549 human lung epithelial-like cell line treated with a methylating agent, 1-methyl-3-nitro-1-nitrosoguanidine. Yamabudo juice and DBQ inhibited the mutagenicity of NNK in the Ames test using Salmonella typhimurium TA1535 but not S. typhimurium YG7108, an alkylguanine DNA alkyltransferase-deficient strain (same as TA1535 but Δadast::Kmr, Δogtst::Cmr). Yamabudo juice and DBQ might accelerate the repair of DNA damage caused by NNK and reduce DNA damage to cells. We also investigated the effects of yamabudo juice and DBQ on signaling pathways in A549 cells. With or without epidermal growth factor stimulation, phosphorylation of Erk1/2, Akt and Stat3 in A549 cells was significantly decreased in the presence of yamabudo juice or DBQ, indicating that yamabudo juice and DBQ suppressed PI3K/AKT, MAPK/ERK and JAK/STAT3 signaling pathways. These results suggest that both initiation and growth/progression steps in carcinogenesis, especially anti-oxidant effects, stimulation of repair of alkyl DNA adducts and suppressed growth signaling pathways are potential anti-tumorigenic targets of yamabudo juice and DBQ in NNK-induced lung tumorigenesis.

Immunological and mass spectrometry-based approaches to determine thresholds of the mutagenic DNA adduct O6-methylguanine in vivo.

N-nitroso compounds are alkylating agents, which are widespread in our diet and the environment. They induce DNA alkylation adducts such as O6-methylguanine (O6-MeG), which is repaired by O6-methylguanine-DNA methyltransferase (MGMT). Persistent O6-MeG lesions have detrimental biological consequences like mutagenicity and cytotoxicity. Due to its pivotal role in the etiology of cancer and in cytotoxic cancer therapy, it is important to detect and quantify O6-MeG in biological specimens in a sensitive and accurate manner. Here, we used immunological approaches and established an ultra performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) to monitor O6-MeG adducts. First, colorectal cancer (CRC) cells were treated with the methylating anticancer drug temozolomide (TMZ). Immunofluorescence microscopy and an immuno-slot blot assay, both based on an adduct-specific antibody, allowed for the semi-quantitative, dose-dependent assessment of O6-MeG in CRC cells. Using the highly sensitive and specific UPLC-MS/MS, TMZ-induced O6-MeG adducts were quantified in CRC cells and even in peripheral blood mononuclear cells exposed to clinically relevant TMZ doses. Furthermore, all methodologies were used to detect O6-MeG in wildtype (WT) and MGMT-deficient mice challenged with the carcinogen azoxymethane. UPLC-MS/MS measurements and dose-response modeling revealed a non-linear formation of hepatic and colonic O6-MeG adducts in WT, whereas linear O6-MeG formation without a threshold was observed in MGMT-deficient mice. Collectively, the UPLC-MS/MS analysis is highly sensitive and specific for O6-MeG, thereby allowing for the first time for the determination of a genotoxic threshold upon exposure to O6-methylating agents. We envision that this method will be instrumental to monitor the efficacy of methylating chemotherapy and to assess dietary exposures.

Effect of temozolomide on the viability of musculoskeletal sarcoma cells

Musculoskeletal sarcomas (MSS) are a heterogeneous group of malignancies with relatively high mortality rates. The prognosis for patients with MSS is poor, with few drugs inducing measurable activity. Alkylating agents, namely ifosfamide and dacarbazine, which act nonspecifically on proliferating cells, are the typical therapy prescribed for advanced MSS. A novel alkylating agent, temozolomide (TMZ), has several advantages over existing alkylating agents. TMZ induces the formation of O6-methylguanine in DNA, thereby inducing mismatches during DNA replication and the subsequent activation of apoptotic pathways. However, due to conflicting data in the literature, the mechanism of TMZ action has remained elusive. Therefore, the present study aimed to evaluate apoptosis in MSS cells treated with TMZ, and to evaluate the correlation between TMZ action and survival pathways, including the phosphoinositide 3-kinase (PI3K)/Akt and extracellular signal-regulated kinase (ERK)1/2 mitogen activated protein kinase (MAPK) pathways. Cell proliferation was evaluated by performing an XTT (sodium 3'-[1-(phenylaminocarbonyl)-3,4-tetrazolium]-bis (4-methoxy-6-nitro) benzene sulfonic acid hydrate) assay. Apoptotic morphological changes, for example chromatin condensation, were evaluated by fluorescence confocal microscopy. The expression of the apoptosis-associated proteins caspase-3, poly adenosine diphosphate ribose polymerase (PARP), Akt and ERK1/2, was determined by western blotting. The results of the present study indicated that, in certain MSS cells, the IC50 value was lower than that in TMZ-sensitive U-87 MG cells. Furthermore, TMZ treatment was associated with apoptotic morphological changes and the expression levels of pro-apoptotic cleaved caspase-3 and PARP were also increased in TMZ-treated MSS cells. In addition, the results indicated that PI3K/Akt and ERK1/2 MAPK were constitutively phosphorylated in MSS cells, and phosphorylation of PI3K/Akt was suppressed in certain cells, and maintained in other cells, by TMZ. These observations emphasized the plasticity of MSS cells, and suggested that this plasticity may contribute to the variance in cell sensitivity to TMZ and TMZ-resistance in MSS.

Development of a liquid chromatography/tandem mass spectrometry method to investigate the presence of biomarkers of DNA damage in urine related to red meat consumption and risk of colorectal cancer

The consumption of red meat is known to enhance the endogenous formation of N-nitroso compounds (NOCs), which are potent carcinogens. DNA damage related to NOCs, and hence red meat, has been detected in colorectal cells and in blood. We proposed to extend previous studies to a non-invasive approach for the detection of O(6)-carboxymethylguanine (O(6)CMG) and O(6)-carboxymethyl-2'-deoxyguanosine (O(6)CMdG) in urine in relation to red meat intake using liquid chromatography/tandem mass spectrometry (LC/MS/MS). The presence of the adduct in urine samples either as the free base or as 2'-deoxynucleoside could help in determining the repair mechanism involved when such lesions are produced. A non-invasive assessment of DNA adducts could also allow for large-scale analyses in the population and cancer prevention dietary strategies. An LC/MS/MS method for the quantitation of O(6)CMG and O(6)CMdG was developed. Urine samples collected from healthy volunteers on red meat and vegetarian diets were analysed either by direct injection or after purification by solid-phase extraction (SPE). A separate LC/MS/MS method for O(6)-methylguanine (O(6)MeG) and O(6)-methyl-2'-deoxyguanosine (O(6)MedG), which are possible hydrolysis products forming during the sample pre-treatment, was also developed. The developed LC/MS/MS method allowed the simultaneous measurement of O(6)CMG and O(6)CMdG. The limits of detection (LODs) were 0.38 ng/mL for O(6)CMG and 0.18 ng/mL for O(6)CMdG. The direct injection analysis of the clinical samples showed low sensitivity due to high background signal that was improved by SPE purification. However, the concentrations of the adducts in clinical samples were still found to be below the LOD. Novel, reproducible, and accurate LC/MS/MS methods were developed for the determination of the urinary content of O(6)CMG and O(6)CMdG, and of the possible formation of O(6)MeG and O(6)MedG by decarboxylation. Clinical samples from volunteers on different diets were analysed. Further studies are required to discover a link between the presence of these biomarkers in urine and red meat consumption.

Abstract 262: O6-methylguanine:Deoxythymidine mismatches sensitize cells to DNA strand break-induced cytotoxicity

Abstract Background: O6-methylguanine (O6-MeG) is the primary cytotoxic lesion formed by SN1-methylating agents such as temozolomide. Cytotoxicity is induced following the mispairing of O6-MeG with deoxythymidine (T) residues and the subsequent activation of a mismatch repair (MMR)-mediated process. While it is generally recognized that MMR-induced DNA double strand breaks (DSBs) are central to O6-MeG-induced cell death, our previous work demonstrates that signaling via the p50 subunit of nuclear factor-κB (NF-κB) is also necessary for efficient SN1-methylation-induced cytotoxicity. In the current study, we isolated O6-MeG:T mismatches using a series of oligonucleotide duplexes and examined the hypothesis that O6-MeG:T mismatches induce a p50-dependent signaling pathway that sensitizes cells to DNA strand break-mediated cell death. Methods: A series of 40bp oligonucleotide duplex substrates bearing a unique central O6-MeG:T, G:T, O6-MeG:C, or G:C base pair were used. Cell lines included both human glioma cells and mouse embryonic fibroblasts. Loss of function was performed with shRNA-mediated p50 knockdown and targeted p50 knockout. Techniques used include EMSA, NF-κB-dependent luciferase reporter assays, in-vitro kinase assay, indirect immunofluorescence, annexin V binding and colony formation assays. Results: When administered to nuclear extracts, O6-MeG:T substrate inhibits NF-κB DNA binding substantially more than control duplex, and, when used in live cells, inhibition of NF-kB-dependent activity is seen only in response to O6-MeG:T substrate. The inhibition of NF-kB by O6-MeG:T substrate is noted only in p50-proficient cells. O6-MeG:T, but not control substrate, induces Chk1-mediated phosphorylation of p50 at a novel serine residue, a pathway that is shown to be necessary for inhibition of NF-kB DNA binding. O6-MeG:T substrate, used at a concentration sufficient to induce MMR- and p50-mediated signaling, does not increase apoptosis or decrease clonogenic survival relative to control duplexes. However, O6-MeG:T, but not control duplex, sensitizes p50-proficient, but not p50-deficent, cells to non-cytotoxic doses of ionizing radiation. O6-MeG:T-induced sensitization to cytotoxicity occurs without changing the level of IR-induced DSB and is only seen when duplex substrate is given prior to, but not after, IR. Conclusion: While an O6-MeG-induced signaling pathway has been previously described, the role of such a pathway to the damage response is disputed. The above findings suggest that O6-MeG:T-induced, p50-dependent signaling sensitizes cells to cytotoxicity that is ultimately induced by DNA DSBs. Such a model is temporally consistent with the response to O6-MeG in that MMR-induced signaling is induced after the first S-phase following O6-MeG formation while DSBs are formed after the second S-phase. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 262. doi:1538-7445.AM2012-262

Pre-Screening for Low Methyl Guanine Methyltransferase (MGMT) Expression Results in High Response Rates to Temozolomide (TMZ) Therapy in AML and MDS Patients.

Abstract 422 Introduction:Many patients with acute myeloid leukemia (AML) and high-risk myelodysplasia (MDS) are not suitable candidates for standard induction therapy, and newer treatment approaches are needed. TMZ anti-tumor activity is attributed to methylation of DNA, leading to the formation of O6-methylguanine that eventually leads to apoptosis. Expression of MethylGuanine DNA MethylTransferase (MGMT) restores normal guanine, decreasing DNA alkylation and thus anti-tumor activity of TMZ. Brandwein et al. (Leukemia 2007;21:821–4) previously demonstrated that response to TMZ in AML patients could be correlated with low MGMT expression. Thus MGMT expression prescreening could be used to identify patients more likely to respond to TMZ and provide an alternative for patients who are not candidates for standard induction therapy. Methods:We conducted a prospective multicenter study of patients with previously untreated AML and high risk MDS who were not candidates for standard induction therapy. Patient selection was based on MGMT expression as determined by Western blot in bone marrow or peripheral blood. Screening was performed by comparing the expression ratio between MGMT and b-actin protein levels. Patients with a ratio <0.25 were eligible to receive TMZ therapy 200 mg/m2/day × 7 days. Patients achieving a complete response (CR) could receive up to 12 monthly cycles of temozolomide 200 mg/m2/day × 5 days. Partial responders received a modified schedule of temozolomide 100 mg/m2/day × 21 days. Results:MGMT expression was tested in 188 patients; median age was 75 years old (range 46–94 years);62% were male. Primary diagnoses were AML (86%) and MDS (14%). Low or weak MGMT expression was detected in 46% of patient that were therefore potential candidates for TMZ therapy. Responses were evaluated in 42 patients, and were divided into 4 categories: Complete (CR/CRp), Morphologic Leukemia-Free State (MLFS), Partial (PR) Minimal (MR). Response was observed in 74% of cases including 29% CR, 10% MLFS, 19% PR and 17% MR. Prognostic factors associated with a higher response rate to temozolomide included MDS presentation, ECOG PS 0 and favorable/standard cytogenetic risk group. Induction and post-remission cycles were well-tolerated and most patients could be successfully treated on an outpatient basis.Patient who reached CR/CRp had a significantly longer median survival (>18 months) compared to those who reached MLFS, PR and MR (5.6 months). Median duration of therapy for patients reaching CR/CRp was 255 days and a majority (80%) became transfusion independent. Grade 3 and 4 toxicities were rare with the most common being fatigue (17%) and diarrhea (6%). Discussion:Pre-selection of patients based on MGMT expression level was associated with a higher response rate to TMZ as compared to previously reported unselected patients. Complete response was associated with increased survival and treatment had a favorable toxicity profile even in frail elderly patients. Targeted therapy with TMZ is feasible in patients with low MGMT expression and represents a new option for AML and MDS patients who are not candidate for standard induction therapy. Disclosures:Brandwein:Merck: Research Funding. Off Label Use: Temozolomide therapy for AML and MDS. Kassis:Merck: Research Funding. Leber:Merck: Research Funding. Howson-Jan:Merck: Research Funding. Minden:Merck: Research Funding. Galarneau:Merck: Employment. Pouliot:Merck: Employment.

Identification of AML and MDS Patients Who Could Potentially Benefit From Temozolomide Therapy: Proportion of Patients with Low Methyl Guanine Methyltransferase (MGMT) Expression Level

AbstractAbstract 3985 Introduction:Temozolomide is an imidazotetrazine derivative of dacarbazine that functions as an alkylating agent. Temozolomide anti-tumor activity is attributed to methylation of DNA, leading to the formation of O6-methylguanine that eventually leads to apoptosis. Resistance to temozolomide may largely be conferred by O6-methylguanine DNA methyltransferase (MGMT) over-expression. The level of MGMT expression will determine the ability of a cell to withstand damage from an alkylating chemotherapeutic agent. Brandwein et al. (Leukemia 2007;21:821-4) previously showed that the complete response rate to temozolomide therapy in patients with acute myeloid leukemia (AML) was higher when MGMT expression was low. Methods:In this prospective multicenter study we evaluated MGMT expression by Western blot in bone marrow or peripheral blood from older patients with previously untreated AML and high risk myelodysplastic syndrome (MDS). Expression was characterized by comparison with b-actin and results were expressed as an MGMT:b-actin ratio: Negative: < 0.2; Weak: 0.2 – 0.25; and Strong:> 0.25 MGMT promoter methylation was also evaluated using methylation-specific PCR. Both protein expression and promoter methylation were tested in a central lab. Results:Data were obtained for 152 patients; median age was 76 years old (range 46–94 years). A majority (63%) were male. Primary diagnosis was AML (84%) and MDS (16%). MGMT expression level could be determined in 128 of the 152 patients tested (84%). Expression ratios were: Negative (41%), Weak (11%), and Strong (48%). MGMT promoter methylation could be detected in 16% of cases in patients with either Negative or Weak expression ratio and in 2% of patients with Strong expression ratio. A majority of patients with promoter methylation (82%) had low MGMT expression. Discussion:One-half of patients with AML/MDS had either a Negative or Weak MGMT expression level, and would therefore be potential candidates for temozolomide therapy. These low levels could be explained by promoter methylation in a minority of cases. Thus, low level of MGMT expression in AML/MDS may be due to direct alteration of the MGMT gene or modulation of expression at the post-transcriptional level. Disclosures:Brandwein:Merck: Honoraria, Research Funding. Off Label Use: temozolomide for acute myeloid leukemia. Kassis:Merck: Research Funding. Leber:Merck: Research Funding. Howson-Jan:Merck: Research Funding. Minden:Merck: Research Funding. Greaves:Schering-Plough/Merck: Employment. Pouliot:Schering-Plough/Merck: Employment.

An O6-methylguanine-DNA Methyltransferase-like Protein from Thermus thermophilus Interacts with a Nucleotide Excision Repair Protein

The major damage to DNA caused by alkylating agents involves the formation of O6-methylguanine (O6-meG). Almost all species possess O6-methylguanine-DNA-methyltransferase (Ogt) to repair such damage. Ogt repairs O6-meG lesions in DNA by stoichiometric transfer of the methyl group to a cysteine residue in its active site (PCHR). Thermus thermophilus HB8 has an Ogt homologue, TTHA1564, but in this case an alanine residue replaces cysteine in the putative active site. To reveal the possible function of TTHA1564 in processing O6-meG-containing DNA, we characterized the biochemical properties of TTHA1564. No methyltransferase activity for synthetic O6-meG-containing DNA could be detected, indicating TTHA1564 is an alkyltransferase-like protein. Nevertheless, gel shift assays showed that TTHA1564 can bind to DNA containing O6-meG with higher affinity (9-fold) than normal (unmethylated) DNA. Experiments using a fluorescent oligonucleotide suggested that TTHA1564 recognizes O6-meG in DNA using the same mechanism as other Ogts. We then investigated whether TTHA1564 functions as a damage sensor. Pull-down assays identified 20 proteins, including a nucleotide excision repair protein UvrA, which interacts with TTHA1564. Interaction of TTHA1564 with UvrA was confirmed using a surface plasmon resonance assay. These results suggest the possible involvement of TTHA1564 in DNA repair pathways.

Inhibition by Allyl Sulfides and Crushed Garlic of O6-methylguanine Formation in Liver DNA of Dimethylnitrosamine-Treated Rats

: Garlic consumption is linked with lower incidences of certain cancers perhaps because garlic-derived allyl sulfides inhibit nitrosamine activation by cytochrome P450s. To help evaluate this view, effects of allyl sulfides on O6-methylguanine (O6MG) levels were examined in liver of rats injected with 20 mg/kg of liver carcinogen dimethylnitrosamine (DMN) and killed 3 h later. DNA was isolated and hydrolyzed, and O6MG/guanine ratios were determined by HPLC-fluorescence. Mean inhibition of O6MG formation fell from 89% for 200 to 33% for 12 mg diallyl sulfide (DAS) per kilogram gavaged 18 h before DMN injection. Gavage of DAS 3 or 6 h (instead of 18 h) before DMN injection significantly reduced inhibitions. Mean inhibitions for diallyl disulfide, diallyl sulfoxide, and diallyl sulfone (75-100 mg/kg) gavaged 18 h before DMN were 39%, 72%, and 82%. In lung and kidney, DAS produced mean inhibitions of 98% and 74% compared with 89% in liver. When methylnitrosourea was injected instead of DMN, neither DAS nor DADS inhibited O6MG formation in liver DNA. Feeding 2.5% garlic for 7 days inhibited DMN-induced O6MG formation in liver DNA by 46%, similar to that expected from the estimated yield of allyl sulfides from garlic. Hence, dosing with DAS or feeding garlic may be useful chemopreventive strategies against nitrosamine-induced cancers.

Comparison of the Chemopreventive Efficacies of 1,4-phenylenebis(methylene)selenocyanate and Selenium-Enriched Yeast on 4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone Induced Lung Tumorigenesis in A/J Mouse

Epidemiological studies, clinical intervention trials (including the trial with selenium-enriched yeast by Clark et al. JAMA 276, 1957, 1996) and assays in laboratory animals provide evidence for a protective role of selenium against the development of several cancers, including lung cancer. We have demonstrated that selenium in the form of 1,4 phenylenebis (methylene) selenocyanate (p-XSC) is a promising chemopreventive agent in the A/J mouse lung tumor model induced with the carcinogenic tobacco-specific nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK); under identical conditions, selenomethionine (SM), a component of selenium-enriched yeast, had no effect. The lack of an effect of SM suggests that other forms of selenium, or selenium-enriched yeast as a whole, are essential for lung cancer prevention; moreover, various species may respond differently to a given form of selenium. Therefore, in this study, we compared the chemopreventive efficacies of p-XSC with selenium-enriched yeast. Groups of 5-wk-old mice were fed either control diet or experimental diet containing p-XSC (5 or 10 ppm as selenium, equivalent to 20% and 40% maximum tolerated dose [MTD], respectively) or selenium-enriched yeast (5 or 10 ppm). Beginning at Wk 7, each mouse received NNK (3 ?mol) in 0.1 ml cottonseed oil by intragastric intubation, once weekly for 8 wk. Twenty-six weeks after the first NNK administration, mice were killed and tumors in lung and forestomach were counted. p-XSC at 5 and 10 ppm doses significantly reduced lung tumor induction by NNK from 10.4 ±6.0 (multiplicity) to 2.7±1.5 (P<0.001) and 1.8±2.0 (P< 0.0001) respectively, whereas selenium-enriched yeasthadno effect. p-XSC at 10 ppm also significantly reduced the incidence level from 96% to 68% (P < 0.01). The amounts of selenium that reach the target organ (lung) after dietary administration of p-XSC (326 ± 69 ng Se/g lung tissue) were significantly higher than that from selenium-enriched yeast (34±8.5 ng Se/g lung tissue). However, the levels of selenium in plasma from selenium-enriched yeast (620 ± 54 ng Se/g plasma) were twofold higher than those fromp-XSC (355±85 ng Se/g plasma). In biochemical studies, p-XSC was shown to significantly inhibit formation ofO6-methylguanine (O6-MG) and 7-methylguanine (7-MG) in the lungs and livers of mice treated with NNK. The lack of effect of selenium-enriched yeast on these lesions agrees with the results of the bioassay. Collectively, the results of this study clearly indicate that as a chemopreventive agent, p-XSC is superior to selenium-enriched yeast under the conditions of the present protocol. The inhibition ofDNA methylation and the significantly higher retention of selenium from p-XSC as compared with selenium- enriched yeast in the target organ may in part account

Genotoxic methylating agents modulate extracellular signal regulated kinase activity through MEK-dependent, glutathione-, and DNA methylation-independent mechanisms in lung epithelial cells.

Mitogen-activated protein kinases (MAPKs) play a central role in transmitting stress-induced signals stimulated by genotoxic agents. The present study is the first to investigate the mechanisms by which genotoxic alkylating agents modulate MAPKs by directly measuring the effects of methylating agents on MAPK activity, DNA methylation, and intracellular glutathione levels. The effects of acetoxymethylmethylnitrosamine (AMMN), N-nitroso-N-methylurethane (NMUR), and N-methyl-N-nitrosourea (MNU) on these parameters were compared in a fetal rat lung cell line model (MP48). These compounds were chosen because they methylate DNA via a methanediazonium intermediate and, therefore, should induce similar cellular methylation patterns, although they produce different side products upon decomposition. All three compounds stimulated the activation of the stress-activated MAPKs, c-Jun N-terminal kinase, and p38. In contrast to what has been reported for other methylating agents, these compounds also stimulated the activation of extracellular signal regulated kinase (ERK), a MAPK typically activated by mitogenic agents. O6-methylguanine (O6-mG) is widely considered to be the critical toxic lesion induced by methylating agents, including AMMN, NMUR, and MNU, which form DNA adducts through SN1 reactions. O6-mG does not appear to be a key regulator of MAPK activity by these compounds, however. There is no direct relationship between the levels of O6-mG and the levels of MAPK activation, and formation of O6-mG does not appear to be sufficient to stimulate MAPK activation. The present studies also indicate that depletion of glutathione is not required or sufficient to stimulate MAPK activation by the methylating agents investigated here. The use of a pharmacological inhibitor indicates that these methylating agents activate ERK through a signaling pathway that requires the ERK kinase MEK. Altogether, these data indicate that genotoxic methylating agents activate MAPKs through mechanisms that are likely to involve the alkylation of cellular targets other than DNA.

Inhibition of N-nitrosomethylbenzylamine-induced tumorigenesis in the rat esophagus by dietary freeze-dried strawberries.

In the present study, we examined the ability of dietary freeze-dried strawberries to inhibit N-nitrosomethylbenzylamine (NMBA)-induced tumorigenesis in the rat esophagus. Initially, we conducted a bioassay to determine the effects of dietary freeze-dried strawberries on esophageal tumor development. Two weeks prior to NMBA treatment, animals were placed on a control diet or diets containing 5 and 10% freeze-dried strawberries. NMBA treatment was once per week for 15 weeks. At 30 weeks, 5 and 10% freeze-dried strawberries in the diet caused significant reductions in esophageal tumor multiplicity of 24 and 56%, respectively. Based on these results, we conducted studies to determine potential mechanisms by which freeze-dried strawberries inhibit tumorigenesis. In a short-term bioassay, we evaluated the effects of dietary freeze-dried strawberries on the formation of O6-methylguanine in the rat esophagus. Animals were placed on control diet or diets containing 5 and 10% freeze-dried strawberries for two weeks. At the end of this period, animals received a single subcutaneous dose of NMBA and were killed 24 h later. A significant decrease in O6-methylguanine levels was observed in the esophageal DNA of animals fed strawberries, suggesting that one or more components in strawberries influence the metabolism of NMBA to DNA-damaging species. Finally, in order to evaluate post-initiation effects, we conducted a study where freeze-dried strawberries were administered in the diet only following NMBA treatment. Animals were placed on control diet and dosed with NMBA three times per week for 5 weeks. Immediately following NMBA treatment, animals were placed on control diet or diets containing 5 and 10% freeze-dried strawberries. At 25 weeks, 5 and 10% freeze-dried strawberries in the diet significantly reduced tumor multiplicity by 38 and 31%, respectively. Our data suggest that dietary freeze-dried strawberries effectively inhibit NMBA-induced tumorigenesis in the rat esophagus.

O6-methylguanine formation, repair protein depletion and clinical outcome with a 4 hr schedule of temozolomide in the treatment of advanced melanoma: Results of a phase II study

O6-Methylguanine-DNA methyltransferase (MGMT) is a major determinant of resistance to temozolomide. Its levels are depleted in lymphocytes after drug administration, but there is partial recovery by 24 hr, the usual time of subsequent dosing. Administering subsequent doses of temozolomide at the MGMT nadir could enhance its effectiveness, by increasing the amount of O6-methylguanine (O6-meG) in DNA. We evaluated the efficacy of such a schedule of temozolomide and determined the kinetics of MGMT depletion and O6-meG formation in DNA following treatment. Thirty patients with advanced malignant melanoma were treated with temozolomide 1,000 mg/m2 equally split into 5 doses over a 16 hr period every 28 days. O6-meG formation was determined in peripheral blood mononuclear cell (PBMC) DNA and, in a subset of patients, in tumor tissue during the first treatment cycle. MGMT levels fell rapidly with dosing, reaching a nadir in PBMCs of 18.0 +/- 2.26% of initial levels. O6-meG levels increased during the treatment period, peaking at 11.1 +/- 1.25 micromol/mol dG in PBMCs and at 4.25 +/- 0.79 micromol/mol dG in tumor biopsies. The main toxicities were grade IV thrombocytopenia in 12 patients (42.8%) and grade IV neutropenia in 11 patients (39.2%), associated with fever in 8 cases. There were 7 responses (1 complete), for an overall response rate of 23.3%; median overall survival was 6.1 months. The compressed schedule has activity against melanoma, with greater MGMT depletion and O6-meG formation than previously reported for O6-alkylating agent regimens. Myelosuppression precludes its wider application, but MGMT in PBMCs predicted the dose intensity of temozolomide that patients could sustain, suggesting a means by which individuals suitable for this approach might be identified.

The effect of cotinine or cigarette smoke co-administration on the formation of O6-methylguanine adducts in the lung and liver of A/J mice treated with 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK)

4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), a tobacco-specific nitrosamine, induces lung adenomas in A/J mice, following a single intraperitoneal (i.p.) injection. However, inhalation of tobacco smoke has not induced or promoted tumors in these mice. NNK-induced lung tumorigenesis is thought to involve O6-methylguanine (O6MeG) formation, leading to GC-->AT transitional mispairing and an activation of the K-ras proto-oncogene in the A/J mouse. NNK can be metabolized by several different cytochromes P450, resulting in a number of metabolites. Formation of the promutagenic DNA adduct O6MeG is believed to require metabolic activation of NNK by cytochrome P450-mediated alpha-hydroxylation of the methylene group adjacent to the N-nitroso nitrogen to yield the unstable intermediate, methanediazohydroxide. Nicotine, cotinine (the major metabolite of nicotine), and aqueous cigarette tar extract (ACTE) have all been shown to effectively inhibit metabolic activation of NNK to its mutagenic form, most likely due to competitive inhibition of the cytochrome P450 enzymes involved in alpha-hydroxylation of NNK. The objective of the current study was to monitor the effects of cotinine and cigarette smoke (CS) on the formation of O6MeG in target tissues of mice during the acute phase of NNK treatment. To test the effect of cotinine, mature female A/J mice received a single intraperitoneal injection of NNK (0, 2.5, 5, 7.5, or 10 mumole/mouse) with cotinine administered at a total dose of 50 mumole/mouse in 3 separate i.p. injections, administered 30 min before, immediately after, and 30 min after NNK treatment. To test the effect of whole smoke exposure on NNK-related O6MeG formation, mice were exposed to smoke generated from Kentucky 1R4F reference cigarettes at 0, 0.4, 0.6, or 0.8 mg wet total particulate matter/liter (WTPM/L) for 2 h, with a single i.p. injection of NNK (0, 3.75, or 7.5 mumole/mouse) midway through the exposure. Cigarette smoke alone failed to yield detectable levels of O6MeG. The number of O6MeG adducts following i.p. injection of NNK was significantly (p < 0.05) reduced in both lung and liver by cotinine and by cigarette smoke exposure. Our results demonstrate that NNK-induced O6MeG DNA adducts in A/J mice are significantly reduced when NNK is administered together with either cotinine, the major metabolite of nicotine, or the parental complex mixture, cigarette smoke.

The inhibitory effect of vitamin E on 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone-induced DNA injury and the fixation of the DNA injury in mouse lungs.

In order to estimate the effect of vitamin E on DNA injury and K-ras point mutation at an early stage of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone(NNK)-induced lung tumorigenesis in mice, the present study was carried out. Presupplement with vitamin E about 15 times more than control for a week significantly inhibited NNK-induced O6-methylguanine formation in the lungs of mice at 4 and 168 h after the injection. At 30 days after the NNK injection. the activation of K-ras oncogene with a 12th codon GC-->AT transition was detected in 56% of lung samples tested by mutant-allele-specific amplification. Vitamin E supplement reduced the frequency of the mutation to 30%. These results suggest that vitamin E suppresses NNK-induced DNA injury and subsequent fixation of the injury during the initiation and post-initiation phases of the lung tumorigenesis in mice.

Possible rare involvement of O6-methylguanine formation as a significant mutational factor in mouse urinary bladder carcinogenesis models.

O6-methylguanine is known as one of the major premutagenic lesions in the human and rodent carcinogenesis process. O6-methylguanine-DNA methyltransferase (MGMT), which repairs methylated guanine bases, might prevent the G:C to A:T transition, and transgenic mice carrying this MGMT gene have been reported to be less sensitive to the carcinogenicity of certain alkylating agents. Here we utilized MGMT transgenic mice to assess the significance of O6-methylguanine formation during urinary bladder carcinogenesis. In experiment 1, 100 and 60 ppm N-butyl-N(4-hydroxybutyl)nitrosamine was given for 20 weeks to transgenic and non-transgenic mice in their drinking water. The incidences of urinary bladder carcinomas were not different between transgenic mice and non-transgenic mice. The mutational spectrum of the p53 gene was evaluated by polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) analysis and direct sequencing. The pattern of p53 mutations of transgenic and non-transgenic mice did not differ, and the frequencies of mutations were 40% and 42%, respectively. G:C to A:T transition mutations were particularly infrequent (1 of 14 mutations, 7%). In experiment 2, N-methyl-N-nitrosourea, which might induce O6-methylguanine in affected alleles, was given once a week, 3 times (total 5 mg) by direct instillation into the urinary bladder through an abdominal incision. No significant neoplastic lesions were detected, although the experiment was limited by severe toxicity of the treatment. p53 immunostaining was done and there was no difference in transgenic and non-transgenic mice. These results suggest that O6-methylguanine formation might not be a significant mutational factor in these mouse urinary bladder carcinogenesis models.

Formation and repair ofO6-methylguanine in recombination hot spots of plant chromosomes

Mutagen-induced chromatid aberrations are not randomly distributed along the metaphase chromosomes. In the field bean (Vicia faba), defined late-replicating and transcriptionally inactive heterochromatic regions are preferentially involved. After exposure to the alkylating agent N-methyl-N-nitrosourea (MNU) (10(-3) M, 1 hour), 70% of all aberrations are clustered within 6 segments containing tandemly repeated FokI elements of 59 bp, which comprise approximately 10% of the genome. Using immuno-slot-blot analyses, we have studied the frequency of O6-methylguanine (O6-MeG), a mutagenic lesion important for aberration induction, in total genomic DNA as well as in FokI sequences of the field bean after exposure to MNU. In either case, similar numbers of adducts per nucleotide were found immediately after treatment as well as after 18 hours of recovery, when most adducts were removed and significant amounts of chromatid aberrations were detectable. Peculiarities of long FokI element arrays (e.g., formation of specific tertiary structures), resulting in error-prone recombination repair, rather than preferential formation or delayed repair of O6-MeG are apparently responsible for aberration clustering in these hot spot regions.

Induction of hprt gene mutations in splenic T-lymphocytes from the rat exposed in vivo to DNA methylating agents is correlated with formation of O6-methylguanine in bone marrow and not in the spleen.

The suitability of splenic T-lymphocytes as a substitute tissue for detection of genotoxic effects induced in vivo by chemical agents that are organ-specifically activated was tested in rats exposed to single doses at the potent lung-carcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), acetoxymethylmethylnitrosamine (AMMN) or N-methyl-N-nitrosourea (MNU). NNK, AMMN and MNU methylate DNA most likely via the formation of a methanediazohydroxide ion that decomposes to a methyl diazonium ion. For all three agents, an increase in the levels of 06-methylguanine and 7-methylguanine in DNA of rat liver and lung was detected by reverse phase HPLC and electrochemical detection. Treatment with NNK did not result in the formation of O6-methylguanine and 7-methylguanine in DNA of bone marrow and spleen, corresponding with the absence of metabolic activation pathways for this compound in these tissues. For AMMN formation of both 06-methylguanine and 7-methylguanine was detectable in DNA of the spleen, whereas in DNA of bone marrow only very low frequencies of 7-methylguanine were found at a toxic dose. MNU induced O6-methylguanine and 7-methylguanine in both spleen and bone marrow. Using splenic T-lymphocytes from the rat no increase above control levels of the hprt mutant frequencies was found for NNK and AMMN for all exposure levels tested, 32 days after chemical exposure. For MNU a dose-dependent increase in hprt mutant frequency was found at exposure levels of 0.097 mmol/kg up to 0.582 mmol/kg. DNA sequence analysis was performed on PCR products of hprt cDNA from 39 MNU-induced 6-thioguanine-resistant T-lymphocyte clones. Single base pair substitutions were found in 25 of these mutants (64%), GC-->AT transitions being the predominant type of mutation (19 of 25; 76%). These mutations are probably caused by mispairing of 06-methylguanine with thymine during DNA replication. The results indicate that formation of mutagenic lesions in the spleen is not correlated with an enhanced frequency of 6-thioguanine-resistant splenic T-lymphocyte clones from rats, 32 days after exposure in vivo to DNA damaging agents. This suggests that mutation-fixation in T-lymphocytes does not occur in the spleen but at other sites in the body such as bone marrow, after which these mutated cells migrate to the spleen.

Structure-function relationships of the dietary anticarcinogen ellagic acid.

Ellagic acid is a complex planar molecule which demonstrates a variety of anticarcinogenic activities. Ellagic acid has been shown to inhibit the CYP1A1-dependent activation of benzo[a]pyrene; to bind to and detoxify the diolepoxide of benzo[a]pyrene; to bind to DNA and reduce the formation of O6-methylguanine by methylating carcinogens; and to induce the phase II detoxification enzymes glutathione S-transferase Ya and NAD(P)H:quinone reductase. Chemical analogs of ellagic acid were synthesized to examine the relationship between the hydroxyl and lactone groups of the ellagic acid molecule and its different anticarcinogenic activities. These studies demonstrated that both the 3-hydroxyl and the 4-hydroxyl groups were required for ellagic acid to directly detoxify the diolepoxide of benzo[a]pyrene, while only the 4-hydroxyl groups were necessary for ellagic acid to inhibit CYP1A1-dependent benzo[a]pyrene hydroxylase activity. Induction of glutathione S-transferase Ya and NAD(P):quinone reductase required the lactone groups of ellagic acid, but the hydroxyl groups were not required for the induction of these phase II enzymes. In addition, the lactone groups, but not the hydroxyl groups, were required for the analogs to reduce the carcinogen-induced formation of O6-methylguanine. Thus, different portions of the ellagic acid molecule are responsible for its different putative anticarcinogenic activities.