O6 Position Of Guanine Research Articles

Synthesis of 131I-Labeled Glucose-Conjugated Inhibitors of O6-Methylguanine-DNA Methyltransferase (MGMT) and Comparison with Nonconjugated Inhibitors as Potential Tools for in Vivo MGMT Imaging

O(6)-Substituted guanine derivatives are powerful agents used for tumor cell sensitization by inhibition of the DNA repair enzyme O(6)-methylguanine-DNA methyltransferase (MGMT). To provide targeted accumulation of MGMT inhibitors in tumor tissue as well as tools for in vivo imaging, we synthesized iodinated C(8)-alkyl-linked glucose conjugates of 2-amino-6-(5-iodothenyl)-9H-purine (O(6)-(5-iodothenyl) guanine, ITG) and 2-amino-6-(3-iodobenzyloxy)-9H-purine (O(6)-(5-iodobenzyl) guanine, IBG). These compounds have MGMT inhibitor constants (IC(50) values) of 0.8 and 0.45 microM for ITGG and IBGG, respectively, as determined in HeLa S3 cells after 2-h incubation with inhibitor. To substantiate that the (131)I-(hetero)arylmethylene group at the O(6)-position of guanine is transferred to MGMT, both the glucose conjugated inhibitors ITGG and IBGG and the corresponding nonglucose conjugated compounds ITG and IBG were labeled with iodine-131. The radioiodinations of all compounds with [(131)I]I(-) were performed with radiochemical yields of >70% for the destannylation of the corresponding tri-n-butylstannylated precursors. The binding ability of [(131)I]ITGG, [(131)]IBGG, [(131)I]ITG, and [(131)I]IBG to purified MGMT was tested. All radioactive compounds were substrates for MGMT, as demonstrated using a competitive repair assay. The newly synthesized radioactive inhibitors were utilized to study ex vivo biodistribution in mice, and the tumor-to-blood ratio of tissue uptake of [(131)I]IBG and [(131)I]IBGG was determined to be 0.24 and 0.76 after 0.5 h, respectively.

Phase II Trial of the O6-Alkylguanine DNA Alkyltransferase Inhibitor O6-Benzylguanine and 1,3-Bis(2-Chloroethyl)-1-Nitrosourea in Advanced Melanoma

1,3-Bis(2-chloroethyl)-1-nitrosourea (BCNU) induces DNA damage via a chloroethyl adduct at the O(6) position of guanine, which can be repaired by O(6)-alkylguanine DNA alkyltransferase (AGT) expressed in melanoma. We postulated that the addition of O(6) benzylguanine (O(6)BG), a potent inactivator of AGT, would improve the clinical response to BCNU in melanoma. Patients had measurable disease, adequate organ function, and a corrected Diffusing capacity of the lung for carbon monoxide (DLCO) of > or =70% predicted. They were accrued into two cohorts based on prior chemotherapy. O(6)BG (120 mg/m(2)) was administered i.v. followed by BCNU (40 mg/m(2)) on an outpatient basis. Peripheral blood mononuclear cells (PBMC) were collected pre- and 18 hours post-O(6)BG to analyze AGT depletion. Treatment was every 6 weeks, and clinical response was assessed after every two cycles. Forty-two patients were enrolled, 22 of these patients were chemotherapy-naïve. In the chemotherapy-naïve cohort, there was a patient with a complete response (CR), 4 with stable disease (SD), 13 with progressive disease (PD), and 4 nonevaluable patients; the median time to progression was 80 days and the median survival was 211 days. In the prior-chemotherapy cohort, there were no responses, 3 SD, 15 PD, and 2 nonevaluable patients; median time to progression was 54 days and median survival was 120 days. AGT was depleted from PBMC in the 15 patients tested. Grades 3 to 4 myelosuppression was seen in 57% of patients; toxicities were similar between the two cohorts. O(6)BG/BCNU was successfully administered on an outpatient basis and depleted AGT from PBMC. However, significant myelosuppression was observed and the clinical outcome was not improved. Alternative mechanisms of resistance to melanoma cell death need to be investigated.

Kinetic Analysis of Steps in the Repair of Damaged DNA by Human O6-Alkylguanine-DNA Alkyltransferase

Rates of individual steps in the removal of alkyl groups from O6-methyl (Me) and -benzyl (Bz) guanine in oligonucleotides by human O6-alkylguanine DNA alkyltransferase (AGT) were estimated using rapid reaction kinetic methods. The overall reaction yields hyperbolic plots of rate versus AGT concentration for O6-MeG but linear plots for the O6-BzG reaction, which is approximately 100-fold faster. The binding of AGT and DNA (double-stranded 30-mer/36-mer complex) appears to be diffusion-limited. The rate of dissociation of the complex is approximately 25-fold slower (approximately 1 s(-1)) for DNA containing O6-MeG or O6-BzG than unmodified DNA. The fluorescent dC-analog 6-methylpyrrolo[2,3-d]pyrimidine-2(3H) one deoxyribonucleoside (pyrrolo dC), which pairs with G, was positioned opposite G, O6-MeG, or O6-BzG and used as a probe of the rate of base flipping. A rapid increase of fluorescence (k approximately 200 s(-1)) was observed with O6-MeG and O6-BzG and AGT but not with a Gly mutation at Arg128, which has been implicated in base flipping with crystal structures. Only weak and slower fluorescence changes were observed with G:pyrrolo dC or T:2-aminopurine pairs. These rate estimates were used in a kinetic model in which AGT binds and scans DNA rapidly, flips O6-alkylG residues, transfers the alkyl group in a chemical step that is rate-limiting in the case of O6-MeG but not O6-BzG, and releases the dealkylated DNA. The results explain the overall patterns of rates of alkyl group removal versus AGT concentration and the effects of the mutations, as well as the greater affinity of AGT for DNA with O6-alkylG lesions.

Gene expression profile induced by BCNU in human glioma cell lines with differential MGMT expression

Chemotherapy with the alkylating agent BCNU (1,3-bis (2-chloroethyl)-1-nitrosourea) is the most commonly used chemotherapeutic agent for gliomas. However, the usefulness of this agent is limited because tumor cell resistance to BCNU is frequently found in clinical brain tumor therapy. The O6-methylguanine-DNA methyltransferase protein (MGMT) reverses alkylation at the O6 position of guanine and we have reported the role of MGMT in the response of brain tumors to alkylating agents. However, the different mechanisms underlying the patterns related to MGMT remain unclear. To better understand the molecular mechanism by which BCNU exerts its effect in glioma cell lines according MGMT expression, we used microarray technology to interrogate 3800 known genes and determine the gene expression profiles altered by BCNU treatment. Our results showed that treatment with BCNU alters the expression of a diverse group of genes in a time-dependent manner. A subset of gene changes was found common in both glioma cell lines and other subset is specific of each cell line. After 24 h of BCNU treatment, up-regulation of transcription factors involved in the nucleation of both RNA polymerase II and III transcription initiation complexes was reported. Interestingly, BCNU promoted the expression of actin-dependent regulators of chromatin. Similar effects were found with higher BCNU doses in MGMT+ cell line showing a similar mechanism that in MGMT-deficient cell with standard doses. Our data suggest that human glioma cell lines treated with BCNU, independently of MGMT expression, show changes in the expression of cell cycle and survival-related genes interfering the transcription mechanisms and the chromatin regulation.

Phase II study of VNP40101M for adults with recurrent malignant glioma

3128 Introduction: VNP40101M is newly synthesized alkylating agent belonging to a novel class of alkylating agents called (1,2-bis(sulfonyl)hydrazines (BCHs). Agents that belong to this class do not produce vinylation and chloroethylation species and hence this class of alkylating agents is thought to have minimal systemic toxicty. VNP40101M does produces two short-lived active species, 1,2-bis(methylsulfonyl)-1-(2-chloroethyl) hydrazine (t1/2=30 seconds at physiologic state) and a thiophilic carbamoylating methylisocyanate species. This agent preferentially produces lesions at the O6-position of guanine. The methyl-isocyanate species may inhibit an important mechanism of resistance against alkylating agents, alkyl-guanine alkyltransferase (AGT). The purpose of this study was to determine the efficacy and tolerability of VNP40101M in patients with recurrent malignant glioma. Methods: Patients were divided into two stratum: Stratum 1-Recurrent glioblastoma multiforme, Stratum 2-Recurrent anaplastic astocytoma or anaplastic oligodendrogliomas. Patients received an intravenous admistration of VNP40101M (300mg/m2) every 6 weeks. Radiographic response, survival data and toxicity were assessed. Results: A total of 38 patients have been enrolled to date of which 36 are evaluable. The median age is 55 years. Seventy-one percent were males. There have been no objective radiographic responses and 15 patients have had a best response of stable disease. Ten patients experienced grade 4 thrombocytopenia and three patients experienced grade 3 neutropenia. Conclusion: VNP40101M administered every 6 weeks seems relatively well tolerated though this schedule may have limited activity for patients with recurrent glioblastoma multiforme. Response and survival data for anaplastic astrocytomas and anaplastic oligodendrogliomas is pending. No significant financial relationships to disclose.

Phase II trial of O6-benzylguanine (O6BG) and BCNU in patients with advanced melanoma

7524 Background: While alkylating agents such as BCNU are active in melanoma, they give response rates of only 10–15%. BCNU induces DNA damage by formation of a chloroethyl adduct at the O6 position of guanine. This modification can be repaired by alkylguanine DNA alkyltransferase (AGT), and melanoma tumor cells can express high levels of AGT. O6BG is a potent inhibitor of AGT. We hypothesized that addition of O6BG would improve the clinical response to BCNU in patients with advanced melanoma. Methods: Patients had measurable disease, adequate organ function, PS≤ 2, and a corrected DLCO of ≥ 70% predicted. Patients were accrued into 2 cohorts based on whether or not they received prior chemotherapy. O6BG (120 mg/m2) was administered i.v. over 1 hour, followed by BCNU (40 mg/m2) i.v. over 1 hour, as an outpatient. PBMC were collected pre- O6BG and 18 hours later to assess for AGT depletion. Treatment cycles were repeated every 6 weeks and response using RECIST criteria was assessed every 2 cycles. Results: 42 patients were enrolled, 22 chemo-naive and 20 with prior chemo. In the chemo-naive cohort there were 1 CR, 4 SD, 13 PD, and 4 non-evaluable (NE) patients; the median time to progression (TTP) was 80d and the median survival (MS) was 211d. In the prior chemo cohort there were no objective responses, 3 SD, 15 PD, and 2 NE patients; the median TTP was 40d and the MS was 120d. The NE patients had clinical disease progression. AGT was effectively depleted from PBMC in the 15 patients tested (p<0.001). Grade 3/4 toxicities included thrombocytopenia (n=24), neutropenia (19), anemia (10), lymphopenia (7), fever (2), fatigue (2), hyponatremia (2), neuropathy (1), vomiting (1), decreased DLCO (1), and death (1). Toxicities were similar in the two cohorts. Conclusions: O6BG/BCNU was successfully administered on an outpatient basis and depleted AGT from PBMC. However, significant myelosuppression was seen and the clinical response rate was not greater than what is expected with BCNU alone. Although this regimen should not be explored further, administration of O6BG with other alkylating agents with greater single agent activity should be considered. No significant financial relationships to disclose.

Phase II trial of O6-benzylguanine (O6BG) and BCNU in patients with advanced melanoma

7524 Background: While alkylating agents such as BCNU are active in melanoma, they give response rates of only 10–15%. BCNU induces DNA damage by formation of a chloroethyl adduct at the O6 position of guanine. This modification can be repaired by alkylguanine DNA alkyltransferase (AGT), and melanoma tumor cells can express high levels of AGT. O6BG is a potent inhibitor of AGT. We hypothesized that addition of O6BG would improve the clinical response to BCNU in patients with advanced melanoma. Methods: Patients had measurable disease, adequate organ function, PS≤ 2, and a corrected DLCO of ≥ 70% predicted. Patients were accrued into 2 cohorts based on whether or not they received prior chemotherapy. O6BG (120 mg/m2) was administered i.v. over 1 hour, followed by BCNU (40 mg/m2) i.v. over 1 hour, as an outpatient. PBMC were collected pre- O6BG and 18 hours later to assess for AGT depletion. Treatment cycles were repeated every 6 weeks and response using RECIST criteria was assessed every 2 cycles. Results:...

Analysis of protein level and RNA expression of the DNA-repair enzyme MGMT and p53 in human glioblastomas

Introduction: Despite the improvement in neurosurgery and radiotherapy the prognosis of malignant glioma remains poor. Migration and high invasiveness as well as the poor sensitivity to radio- and chemotherapy play key roles in the progression of this tumor. Inhibition of chemotherapy resistance, particularly against alkylating agents, might be new therapeutic approaches. The DNA-repair enzyme O6-methylguanine-DNA methyltransferase (MGMT) reverses alkylation at the O6 position of guanine. In brain tumors MGMT status decreases with increasing grading but only a minority of glioblastomas showed sensitivity to alkylants. Our aim was the investigation of MGMT protein by immunohistochemistry and the RNA expression in glioblastomas. Simultaneously expression of MGMT with the tumor suppressor p53 was examined by immunostaining.

Mutation analysis of K-ras and β-catenin genes related toO6-methylguanin-DNA methyltransferase and mismatch repair protein status in human gallbladder carcinoma

O6-methylguanin-DNA methyltransferase (MGMT) is a DNA repair enzyme that transfers methyl groups from O6-methylguanine to itself. Alkylation of DNA at the O6 position of guanine is the first step by alkylating agents in inducing DNA mutations in an organism. When MGMT and the mismatch repair (MMR) system are impaired, O6-methylguanine mispairs with thymine during DNA replication, resulting in a G:C right curved arrow A:T transitional mutation in DNA. We obtained cancer lesions by manual micro-dissection (MMD) from 26 paraffin-embedded formalin-fixed gallbladder carcinoma and Laser Capture Micro-dissection (LCM) method from 10 fresh frozen specimens. Mutation analysis was performed on the micro-dissected samples for K-ras and beta-catenin genes. At codon 12 of the K-ras gene, the MMD and LCM methods detected mutations in 3 (11.5%) and 1 (10%) case, respectively. In exon 3 of beta-catenin gene, only 1 (3.8%) case revealed a mutation in MMD cancer foci. Two cases without MGMT or MMR expression revealed a G right curved arrow A transition mutation in the K-ras gene. The findings suggested that negative MGMT and MMR status contributed to a G:C right curved arrow A:T transitional mutation in the K-ras gene. However, K-ras and beta-catenin mutations were actually rare in GB carcinoma. Other gene mutations frequently occurring in gallbladder carcinoma might be affected by this negative MGMT and MMR status.

Inhibition of O6-alkylguanine-DNA alkyltransferase by O6-benzyl-N2-acetylguanosine increases chloroethylnitrosourea-induced apoptosis in Mer+ human melanoma cells.

The exposure of cells to -benzyl- 2-acetylguanosine (BNAG) and several guanine derivatives is known to reduce -alkylguanine-DNA alkyltransferase (AGAT) activity and to decrease the resistance of methyl enzyme repair positive (Mer ) cells to chloroethylnitrosoureas (CENUs) and. We evaluated the influence of AGAT activity inhibition by BNAG on the ability of two CENUs, 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) and 3-(2-chloroethyl)-1-(2-methylsulphonyl)ethyl-3-nitrosourea (cystemustine), to induce an apoptotic response in two melanoma cell lines, M3 Dau (Mer ) and IPC 227F (Mer ). The apoptotic morphology of cells was assessed by microscopy after Wright-Giemsa or Hoechst 33342 staining of cells, and DNA internucleosomal cleavage was demonstrated by the ladder-like pattern of DNA separated by agarose gel electrophoresis. The concentration-dependent number of apoptotic cells assessed using a terminal deoxynucleotidyl transferase-mediated dUTP-fluorescein nick-end labelling (TUNEL) assay 72 h after BCNU or cystemustine treatment (0-400 microM for 2 h) was increased by prior AGAT depletion with BNAG pretreatment (300 microM for 4 h) in Mer cells. These results suggest that the DNA lesions on the position of guanine are a key event in inducing an apoptotic response in melanoma cells. We also observed that cystemustine was a more potent inducer of apoptosis than BCNU, and that the synergism with BNAG was more potent with cystemustine than with BCNU. These results suggest that the nature of the CENUs associated with an AGAT inhibitor is a determinant factor in forecasting the clinical efficacy of the association, especially in melanoma.

Heterogeneity of O6-alkylguanine DNA-alkyltransferase expression in human breast tumours.

An important determinant of cellular resistance to chemotherapeutic O6-alkylating agents, which comprise methylating and chloroethylating agents, is the ability of cells to repair alkylation damage at the O6-position of guanine in DNA. This is achieved by a specific DNA repair enzyme O6-alkylguanine DNA-alkyltransferase. In this study O6-alkylguanine DNA-alkyltransferase expression was measured in human breast tumours using both biochemical and immunohistochemical techniques. O6-alkylguanine DNA-alkyltransferase activity was then compared with known clinical prognostic indices to assess the potential role of O6-alkylguanine DNA-alkyltransferase in predicting the behaviour of this common malignancy. The application of both biochemical and immunohistochemical techniques was feasible and practical. Most breast tumours expressed high levels of O6-alkylguanine DNA-alkyltransferase. Immunohistochemical analysis showed marked variation in expression not only between individuals but also within individual tumours, and in the same patient, between metastases and between primary tumour and metastatic site. O6-alkylguanine DNA-alkyltransferase activity in tissue extracts significantly correlated not only with immunohistochemical staining intensity determined by subjective quantitation, but also with measures of protein levels using a computerised image analysis system including mean grey (P<0.001), percentage of cells positive for O6-alkylguanine DNA-alkyltransferase (P<0.001), and integrated optical density (P<0.001). O6-alkylguanine DNA-alkyltransferase expression did not correlate with any of the established clinical prognostic indicators for current treatment regimens. However, immunohistochemical offers a rapid and convenient method for assessing potential utility of O6-alkylating agents or O6-alkylguanine DNA-alkyltransferase inactivating agents in future studies of breast cancer treatment.British Journal of Cancer (2002) 86, 1797–1802. doi:10.1038/sj.bjc.6600324 www.bjcancer.com© 2002 Cancer Research UK

Repair of O6-methylguanine is not affected by thymine base pairing and the presence of MMR proteins

Methylation at the O6-position of guanine (O6-MeG) by alkylating agents is efficiently removed by O6-methylguanine–DNA methyltransferase (MGMT), preventing from cytotoxic, mutagenic, clastogenic and carcinogenic effects of O6-MeG-inducing agents. If O6-MeG is not removed from DNA prior to replication, thymine will be incorporated instead of cytosine opposite the O6-MeG lesion. This mismatch is recognized and processed by mismatch repair (MMR) proteins which are known to be involved in triggering the cytotoxic and genotoxic response of cells upon methylation. In this work we addressed three open questions. (1) Is MGMT able to repair O6-MeG mispaired with thymine (O6-MeG/T)? (2) Do MMR proteins interfere with the repair of O6-MeG/T by MGMT? (3) Does MGMT show a protective effect if it is expressed after replication of DNA containing O6-MeG? Using an in vitro assay we show that oligonucleotides containing O6-MeG/T mismatches are as efficient as oligonucleotides containing O6-MeG/C in competing for MGMT repair activity, indicating that O6-MeG mispaired with thymine is still subject to repair by MGMT. The addition of MMR proteins from nuclear extracts, or of recombinant MutSα, to the in vitro repair assay did not affect the repair of O6-MeG/T lesions by MGMT. This indicates that the presence of MutSα still allows access of MGMT to O6-MeG/T lesions. To elucidate the protective effect of MGMT in the first and second replication cycle after N-methyl-N′-nitro-N-nitrosoguanidine (MNNG) treatment, MGMT transfected CHO cells were synchronized and MGMT was inactivated by pulse-treatment with O6-benzylguanine (O6-BG). Thereafter, the recovered cells were treated with MNNG and subjected to clonogenic survival assays. Cells which expressed MGMT in the first and second cell cycle were more resistant than cells which expressed MGMT only in the second (post-treatment) cell cycle. Cells which did not express MGMT in both cell cycles were most sensitive. This indicates that repair of O6-MeG can occur both in the first and second cell cycle after alkylation protecting cells from the killing effect of the lesion.

O6-Benzylguanine potentiates BCNU but not busulfan toxicity in hematopoietic stem cells.

Busulfan (BU) is often used in conditioning regimens prior to bone marrow transplantation, but its mechanism of action remains to be resolved. We have examined the possibility that BU may exert part of its toxic effects via DNA alkylation at the O6 position of guanine as this might provide an approach to improving the conditioning regimen. Survival of LAMA-84 and RJKO cells was assessed by colony-forming assay and cell counting, respectively. O6-alkylguanine-DNA alkyltransferase (ATase) activity was assayed by transfer of radioactivity from [3H]-methylated DNA. Colony-forming potential of normal human bone marrow cells (BMC) was measured in the presence of appropriate growth factors as the formation of both granulocyte-macrophage colony-forming units (CFU-GM) or burst-forming unit erythroids (BFU-E) within the same assay. Murine hematopoietic precursors were grown under a bone marrow stromal cell line to allow measurement of the frequency of cobblestone area-forming cells (CAFC) that correspond to CFU-GM, spleen colony-forming units (CFU-S), and the primitive stem cells with long-term repopulating ability. Inactivation of ATase by O6-benzylguanine (O6-BeG) sensitized a human erythromegakaryocytic cell line (LAMA-84) and normal human bone marrow progenitors to 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) but not to BU toxicity. BCNU, but not BU, inactivated ATase in LAMA-84 cells. Overexpression of human ATase in cDNA transfected Chinese hamster cells attenuated the toxicity of BCNU but not BU. Finally, the in vivo treatment of mice showed that the depletion of primitive stem cells by BU as measured in the CAFC assay was not affected by addition of O6-BeG. O6-BeG did, however, dramatically potentiate BCNU toxicity in all CAFC subsets, leading to depletion of more than 99% stem cells. These data suggest that BU does not elicit toxicity via alkylation at the O6 position of guanine in DNA in a way that can be influenced by ATase modulation.

Reduction of BCNU toxicity to lung cells by high-level expression of O(6)-methylguanine-DNA methyltransferase.

1,3-Bis(2-chloroethyl)-1-nitrosourea (BCNU) is an important cause of pulmonary toxicity. BCNU alkylates DNA at the O(6) position of guanine. O(6)-methylguanine-DNA methyltransferase (MGMT) is a DNA repair protein that removes alkyl groups from the O(6) position of guanine. To determine whether overexpression of MGMT in a lung cell reduces BCNU toxicity, the MGMT gene was transfected into A549 cells, a lung epithelial cell line. Transfected A549 cell populations demonstrated high levels of MGMT RNA, MGMT protein, and DNA repair activity. The overexpression of MGMT in lung epithelial cells provided protection from the cytotoxic effects of BCNU. Control A549 cells incubated with 100 microM BCNU had a cell survival rate of 12.5 +/- 1.2%; however, A549 cells overexpressing MGMT had a survival rate of 71.8 +/- 2.7% (P < 0.001). We also demonstrated successful transfection of MGMT into human pulmonary artery endothelial cells and a primary culture of rat type II alveolar epithelial cells with overexpression of MGMT, resulting in significant protection from BCNU toxicity. These data suggest that overexpression of DNA repair proteins such as MGMT in lung cells may protect the lung cells from cytotoxic effects of cancer chemotherapy drugs such as BCNU.

Novel DNA repair alkyltransferase from Caenorhabditis elegans

O6-alkylguanine DNA-alkyltransferase (AGT) is a widely distributed DNA repair protein that protects living organisms from endogenous and exogenous alkylation damage to DNA at the O6-position of guanine. The search of the C. elegans genome database for an AGT protein revealed the presence of a protein (cAGT-2) with some similarity to known AGTs in addition to the easily recognized cAGT-1 protein. The predicted protein sequence of cAGT-2 contains the amino acid sequence -ProCysHisPro- at the presumed active site of the protein, whereas all other known AGTs have -ProCysHisArg-. A truncated version of the cAGT-2 protein was expressed in E. coli. This purified recombinant protein was able to repair O6-methylguanine and O4-methylthymine adducts in DNA in vitro and also reacted with the bulky benzyl adduct in O6-benzylguanine. This fragment of cAGT-2 (104 amino acids) is the smallest protein possessing AGT activity yet described. The full-length cAGT-2 protein (274 amino acids) totally lacks the N-terminal domain present in all other known AGTs but has a long C-terminal extension that has significant homology to histone 1C. Expression of cAGT-2 in an E. coli strain lacking endogenous AGT activity provided modest but statistically significant resistance to the toxicity of N-methyl-N'-nitro-N-nitrosoguanidine, confirming that cAGT-2 is an alkyltransferase.

Conserved structural motifs governing the stoichiometric repair of alkylated DNA by O6-alkylguanine-DNA alkyltransferase

O6-alkylguanine-DNA alkyltransferase (AGT) directly repairs alkylation damage at the O6-position of guanine in a unique, stoichiometric reaction. Crystal structures of AGT homologs from the three kingdoms of life reveal that despite their extremely low primary sequence homology, the topology and overall structure of AGT has been remarkably conserved. The C-terminal domain of the two-domain, α/β fold bears a helix-turn-helix (HTH) motif that has been implicated in DNA-binding by structural and mutagenic studies. In the second helix of the HTH, the recognition helix, lies a conserved RAV[A/G] motif, whose “arginine finger” promotes flipping of the target nucleotide from the base stack. Recognition of the extrahelical guanine is likely predominantly through interactions with the protein backbone, while hydrophobic sidechains line the alkyl-binding pocket, as defined by product complexes of human AGT. The irreversible dealkylation reaction is accomplished by an active-site cysteine that participates in a hydrogen bond network with invariant histidine and glutamic acid residues, reminiscent of the serine protease catalytic triad. Structural and biochemical results suggest that cysteine alkylation opens the domain-interfacing “Asn-hinge”, which couples the active-site to the recognition helix, providing both a mechanism for release of repaired DNA and a signal for the observed degradation of alkylated AGT.

Real-Time Fluorescence Assay for O6-Alkylguanine-DNA Alkyltransferase

O6-Alkylguanine-DNA alkyltransferase (AGT) is a DNA-repair protein that reverses the effects of alkylating agents by removing DNA adducts from the O6-position of guanine. We developed a real-time AGT assay that utilizes a fluorescent guanosine analog (3-methylisoxantopterin, 3-MI). 3-MI fluorescence is quenched in DNA and fluorescence intensity increases substantially with digestion of the oligonucleotide and release of 3-MI. The substrate is a doubled-stranded oligonucleotide with 3′-overhangs on each end and a PvuII recognition site. PvuII is inhibited by O6-methylguanine, positioned within the restriction site. 3-MI is incorporated in the opposite strand just outside of the PvuII restriction site. AGT repairs O6-methylguanine; PvuII cleaves at its restriction site, yielding a blunt-ended double strand, which is then digested by exonuclease III. This releases 3-MI from the oligonucleotide, resulting in an increase in fluorescence intensity. All reaction components (100-μL volume) are monitored in a single microcuvette. Rate of increase in fluorescence intensity is related to the amount of AGT in the reaction mixture. We measured AGT levels in extracts from a leukemia cell line, from leukemic lymphoblasts from patients, and from peripheral blood mononuclear cells from normal controls. This method may prove useful for mechanistic studies of AGT.

Mechanistic aspects of the cytotoxic activity of glufosfamide, a new tumour therapeutic agent.

β- D -Glucosyl-ifosfamide mustard (D 19575, glc-IPM, INN = glufosfamide) is a new agent for cancer chemotherapy. Its mode of action, which is only partly understood, was investigated at the DNA level. In the breast carcinoma cell line MCF7 glufosfamide inhibited both the synthesis of DNA and protein in a dose-dependent manner, as shown by the decreased incorporation of [3H-methyl]-thymidine into DNA and [14C]-methionine into protein of these cells. Treatment of MCF7 cells with 50 μM glufosfamide was sufficient to trigger poly(ADP-ribose) polymerase (PARP) activation, as revealed by immunofluorescence analysis. Both CHO-9 cells, which are O6-methylguanine-DNA methyltransferase (MGMT)-deficient, and an isogenic derivative, which has a high level of MGMT, showed the same cytotoxic response to β- D -glc-IPM, indicating that the O6position of guanine is not the critical target for cytotoxicity. By contrast, a sharp decrease in survival of cross-link repair deficient CL-V5 B cells was observed already at concentrations of 0.1 m Mβ- D -glc-IPM, whereas the wild-type V79 cells showed a 90% reduction in survival only after treatment with 0.5 m M of this compound. The therapeutically inactive β- L -enantiomer of glufosfamide also showed genotoxic effects in the same assays but at much higher doses. This was probably due to small amounts of ifosfamide mustard formed under the conditions of incubation. The results indicate that the DNA crosslinks are the most critical cytotoxic lesions induced by β- D -glc-IPM. © 2000 Cancer Research Campaign

Mutation of the mismatch repair gene hMSH2 and hMSH6 in a human T‐cell leukemia line tolerant to methylating agents

Cell killing by monofunctional methylating agents is due mainly to the formation of adducts at the O6 position of guanine. These methyl adducts are removed from DNA by the O6-alkylguanine DNA alkyltransferase (OGAT). The mechanism by which O6-methylguanine (O6meG) induces cell death in OGAT-deficient cells requires a functional mismatch repair system (MRS). We have previously reported that depletion of OGAT activity in the human T-cell leukemic Jurkat line does not sensitize these cells to the cytotoxic and apoptotic effects of the methylating triazene temozolomide (Tentori et al., 1995). We therefore decided to establish whether the tolerance of Jurkat cells to O6meG could be associated with a defect in MRS. The results of mismatch repair complementation studies indicated that Jurkat cells are defective in hMutSα, a heterodimer of the hMSH2 and hMSH6 proteins. Cytogenetic analysis of two Jurkat clones revealed a deletion in the short arm of chromosome region 2p15–21, indicating an allelic loss of both hMSH2 and hMSH6 genes. DNA sequencing revealed that exon 13 of the second hMSH2 allele contains a base substitution at codon 711, which changes an arginine to a termination codon (CGA→TGA). In addition, a (C)8→(C)7 frameshift mutation in codon 1085–1087 of the hMSH6 gene was also found. Although both hMSH2 and hMSH6 transcripts could be detected in Jurkat clones, the respective polypeptides were absent. Taken together, these data indicate that tolerance of Jurkat cells to methylation damage is linked to a loss of functional hMutSα. Genes Chromosomes Cancer 23:159–166, 1998. © 1998 Wiley-Liss, Inc.

Mutation of the mismatch repair genehMSH2 andhMSH6 in a human T-cell leukemia line tolerant to methylating agents

Cell killing by monofunctional methylating agents is due mainly to the formation of adducts at the O6 position of guanine. These methyl adducts are removed from DNA by the O6-alkylguanine DNA alkyltransferase (OGAT). The mechanism by which O6-methylguanine (O6meG) induces cell death in OGAT-deficient cells requires a functional mismatch repair system (MRS). We have previously reported that depletion of OGAT activity in the human T-cell leukemic urkat line does not sensitize these cells to the cytotoxic and apoptotic effects of the methylating triazene temozolomide (Tentori et al., 1995). We therefore decided to establish whether the tolerance of Jurkat cells to O6meG could be associated with a defect in MRS. The results of mismatch repair complementation studies indicated that Jurkat cells are defective in hMutSalpha, a heterodimer of the hMSH2 and hMSH6 proteins. Cytogenetic analysis of two Jurkat clones revealed a deletion in the short arm of chromosome region 2p15-21, indicating an allelic loss of both hMSH2 and hMSH6 genes. DNA sequencing revealed that exon 13 of the second hMSH2 allele contains a base substitution at codon 711, which changes an arginine to a termination codon (CGA-->TGA). In addition, a (C)8-->(C)7 frameshift mutation in codon 1085-1087 of the hMSH6 gene was also found. Although both hMSH2 and hMSH6 transcripts could be detected in Jurkat clones, the respective polypeptides were absent. Taken together, these data indicate that tolerance of Jurkat cells to methylation damage is linked to a loss of functional hMutSalpha.