Alkyltransferase Research Articles

The Role of General Acid Catalysis in the Mechanism of an Alkyl Transferase Ribozyme.

MTR1 is an in vitro-selected alkyl transferase ribozyme that transfers an alkyl group from O 6-alkylguanine to N1 of the target adenine in the RNA substrate (A63). The structure of the ribozyme suggested a mechanism in which a cytosine (C10) acts as a general acid to protonate O 6-alkylguanine N1. Here, we have analyzed the role of the C10 general acid and the A63 nucleophile by atomic mutagenesis and computation. C10 was substituted by n1c and n1c, c5n variants. The n1c variant has an elevated pK a (11.4 as the free nucleotide) and leads to a 104-fold lower activity that is pH-independent. Addition of the second c5n substitution with a lower pK a restored both the rate and pH dependence of alkyl transfer. Quantum mechanical calculations indicate that protonation of O 6-alkylguanine lowers the barrier to alkyl transfer and that there is a significantly elevated barrier to proton transfer for the n1c single substitution. The calculated pK a values are in good agreement with the apparent values from measured rates. Increasing the pK a of the nucleophile by A63 n7c substitution led to a 6-fold higher rate. The increased reactivity of the nucleophile corresponds to a βnuc of ∼0.5, indicating significant C-N bond formation in the transition state. Taken together, these results are consistent with a two-step mechanism comprising protonation of the O 6-alkylguanine followed by alkyl transfer.

Abstract 5070: Landscape of clonal hematopoiesis prior to PARP inhibitor treatment in patients with ovarian cancer

Abstract Background: Poly (ADP-ribose) polymerase inhibitors (PARPi) are an important tool for treatment of ovarian and other cancers, particularly for those with mutations in BRCA1 or 2 genes or homologous recombination deficiency. The risk of developing secondary hematologic malignancy, particularly myelodysplastic syndrome or acute myeloid leukemia (MDS/AML), is substantially higher (4-12%) after PARPi maintenance or treatment in the second line and beyond in patients with ovarian cancer. This elevated risk necessitates additional investigation into the pathogenesis of PARPi-related secondary malignancy. Clonal hematopoiesis of indeterminate potential (CHIP) has been associated with the development of therapy-related myeloid neoplasms. We aim to understand the patterns of CHIP in patients with high grade serous ovarian carcinoma who receive PARPi therapy. Methods: We performed ultra-high-depth whole exome sequencing of plasma derived cfDNA or white blood cells (WBCs) barcoded with unique molecular identifiers from patients with high grade serous ovarian carcinoma who were treated with multiple lines of chemotherapy (n=6) or with PARPi in addition to chemotherapy (n = 10). Using spike-in mutated DNAs as positive controls, we detected variant alleles as low as 1% variant allele frequency. Gene set enrichment analysis was performed on identified groups. Results: We identified 7162 recurrent variants among our patients. We began by comparing quantities of CHIP between the samples from patients who eventually receive PARPi and those who do not. Samples contained a median of 2030 recurrent CHIP variants and 4300 total variants. No differences in total CHIP quantity or mutation type was discernable among samples from patients who will receive PARPi (BRCA1/2 enriched) vs those who will not. Among our 16 patients, 14 ultimately developed secondary hematologic malignancy (t-MN+ Comparison of t-MN+ (n=14) and t-MN- (n=2) defined a group of 14 variants enriched in the t-MN+ population and a group of 85 variants depleted in the t-MN+ population. The t-MN+ enriched variants we identified were not previously associated with CHIP and had significant overlaps with alkyl transferase activity (FDR q val < 0.01) and nucleotide binding gene sets (FDR q val < 0.01). Conclusions: We used a novel methodology to identify unbiased low variant mutational changes in the hematopoietic system. These results suggest that baseline clonal hematopoiesis variants are similar among patients newly diagnosed with ovarian carcinoma. We have also identified a signature potentially related to future risk of leukemia requiring further validation. Citation Format: Amma Asare, Sara Corvigno, Jun Yao, Li Zhao, Nicole D. Fleming, Joseph Celestino, Richard A. Hajek, Mark S. Kim, Alejandra Flores Legarreta, Karen H. Lu, Koichi Takahashi, P Andrew Futreal, Amir A. Jazaeri, Shannon N. Westin, Anil K. Sood, Sanghoon Lee. Landscape of clonal hematopoiesis prior to PARP inhibitor treatment in patients with ovarian cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5070.

Base flipping mechanism and binding strength of methyl-damaged DNA during the interaction with AGT.

Methyl damage to DNA bases is common in the cell nucleus. O6-alkylguanine-DNA alkyl transferase (AGT) may be a promising candidate for direct damage reversal in methylated DNA (mDNA) at the O6 point of the guanine. Indeed, atomic-level investigations in the contact region of AGT-DNA complex can provide an in-depth understanding of their binding mechanism, allowing to evaluate the silico-drug nature of AGT and its utility in removing methyl damage in DNA. In this study, molecular dynamics (MD) simulation was utilized to examine the flipping of methylated nucleotide, the binding mechanism between mDNA and AGT, and the comparison of binding strength prior and post methyl transfer to AGT. The study reveals that methylation at the O6 atom of guanine weakens the hydrogen bond (H-bond) between guanine and cytosine, permitting for the flipping of such nucleotide. The formation of a H-bond between the base pair of methylated nucleotide (i.e., cytosine) and the intercalated arginine of AGT also forces the nucleotide to rotate. Following that, electrostatics and van der Waals contacts as well as hydrogen bonding contribute to form the complex of DNA and protein. The stronger binding of AGT with DNA before methyl transfer creates the suitable condition to transfer methyl adduct from DNA to AGT.

Current promising treatment strategy for glioblastoma multiform: A review.

Glioblastoma multiform (GBM) is a heterogeneous group of primary neoplasm resistant to conventional therapies. Due to their infiltrative nature it not fully isolated by aggressive surgery, radiation and chemotherapy showing poor prognosis in glioma patients. Unfortunately, diagnosed patients die within 1.5-2 year treatment schedule. Currently temozolomide (TMZ) is the first choice for the prognosis of GBM patients. TMZ metabolites methyl triazen imidazol carboxamide form complex with alkyl guanine alkyl transferase (O6 MGMT- DNA repair protein) induced DNA damage following resistance properties of TMZ and inhibit the overall survival of the patients. Last few decades different TMZ conjugated strategy is developed to overcome the resistance and enhance the chemotherapy efficacy. The main aim of this review is to introduce the new promising pharmaceutical candidates that significantly influence the therapeutic response of the TMZ in context of targeted therapy of glioblastoma patients. It is hoped that this proposed strategy are highly effective to overcome the current resistance limitations of TMZ in GBM patients and enhance the survival rate of the patients.

A Simple Colorimetric Assay for Specific Detection of Glutathione-S Transferase Activity Associated with DDT Resistance in Mosquitoes

BackgroundInsecticide-based methods represent the most effective means of blocking the transmission of vector borne diseases. However, insecticide resistance poses a serious threat and there is a need for tools, such as diagnostic tests for resistance detection, that will improve the sustainability of control interventions. The development of such tools for metabolism-based resistance in mosquito vectors lags behind those for target site resistance mutations.Methodology/Principal FindingsWe have developed and validated a simple colorimetric assay for the detection of Epsilon class Glutathione transferases (GST)-based DDT resistance in mosquito species, such as Aedes aegypti, the major vector of dengue and yellow fever worldwide. The colorimetric assay is based on the specific alkyl transferase activity of Epsilon GSTs for the haloalkene substrate iodoethane, which produces a dark blue colour highly correlated with AaGSTE2-2-overexpression in individual mosquitoes. The colour can be measured visually and spectrophotometrically.Conclusions/SignificanceThe novel assay is substantially more sensitive compared to the gold standard CDNB assay and allows the discrimination of moderate resistance phenotypes. We anticipate that it will have direct application in routine vector monitoring as a resistance indicator and possibly an important impact on disease vector control.

Indo-1 Hybrid Biosensors For Calcium Monitoring In Cellular Organelles

The central role of calcium in signal transduction depends on the precise spatial and temporal control of its concentration. The existing possibilities to detect fluctuations in Ca2+ concentration with adequate temporal and spatial resolution and in specific cellular organelles, are limited. We have developed a method to measure Ca2+ concentrations in defined subcellular locations that uses derivatives of the dye Indo-1 covalently bound to fusions of “SNAP-tag” (a multiply mutated version of human alkylguanyl DNA alkyl transferase) expressed inside cells. SNAP-Indo-1 conjugates retained the Ca2+−sensing ability of Indo-1 in vitro. One of the derivatives of Indo-1 displayed a four-fold higher fluorescence after coupling to SNAP-tag, which improves specificity of Ca2+ sensing in living cells. In a proof-of-principle experiment, local Ca2+ sensing was demonstrated in muscle cells of mice expressing a SNAP fusion localized to nuclei. [Ca2+] inside nuclei ([Ca2+]n) was evaluated by SEER (shifted excitation and emission ratioing) of confocal microscopic images of fluorescence of the sensor. After permeabilizing the plasma membrane, changes to bathing solutions containing different [Ca2+] induced corresponding changes in [Ca2+]n that were readily detected and used for a preliminary calibration of the technique. Similar hybrid sensors using Indo-1 but targeted to the mitochondrial matrix and the SR were also constructed. In principle, these hybrid sensors should combine the spatial specificity of biosensors with the superior kinetics and dynamic range of small synthetic fluorescent monitors. Factors that tended to limit their performance in initial experiments include targeting specificity of SNAP fusions and unspecific staining by the Indo-1 not reacted with SNAP-tag. Overall, the hybrid biosensor approach is a promising tool for organellar Ca2+ imaging. Support: NIAMS/NIH grants to E.R., MDA to J.Z. and a Marie-Curie Fellowship (EC) to M.B.

Phase I Trial of VNP40101M (Cloretazine) in Children with Recurrent Brain Tumors: A Pediatric Brain Tumor Consortium Study

VNP40101M (Cloretazine), a novel DNA alkylating agent, was evaluated in a phase I study in children with recurrent brain tumors. VNP40101M was given i.v. daily for 5 consecutive days every 6 weeks for up to eight cycles. Dose escalation was done independently in patients stratified based on intensity of prior therapy (moderately pretreated, stratum I; heavily pretreated, stratum II). Correlative studies included pharmacokinetics and measurement of O(6)-alkylguanine-DNA alkyl transferase levels in peripheral blood mononuclear cells before and after treatment. Forty-one eligible patients (stratum I, 19; stratum II, 22) were enrolled on this study. The dose-limiting toxicity in 35 evaluable patients was myelosuppression, which occurred in 4 of 16 patients in stratum I and 3 of 19 patients in stratum II. Pharmacokinetic studies showed a median terminal half-life of 30 min (range, 14-39.5). The maximum tolerated dose in stratum I and II were 45 and 30 mg/m(2)/d daily for 5 days every 6 weeks, respectively. Peripheral blood mononuclear cells alkylguanine alkyl transferase levels did not decrease significantly after VNP40101M treatment. Central imaging review confirmed that three patients had stable disease for a median of 45 weeks (range, 37-61+) after therapy. The recommended dose of VNP40101M for phase II studies in children with brain tumors is 45 mg/m(2)/d in moderately pretreated and 30 mg/m(2)/d in heavily pretreated patients when administered for 5 consecutive days every 6 weeks.

Phase I trial of VNP40101M in children with recurrent brain tumors—A Pediatric Brain Tumor Consortium (PBTC) study

2059 Background: VNP40101M, a novel DNA alkylating agent and a potent inhibitor of alkylguanine alkyl transferase (AGT) in pre- clinical studies, was evaluated in a phase I study in children with recurrent brain tumors. Methods: VNP40101M was given intravenously (i.v) daily for 5 days q 6 weeks up to 8 cycles. Using the continual reassessment method, dose escalation was performed independently in pts stratified based on intensity of prior therapy (stratum I-less-heavily pre-treated; stratum II- heavily pre-treated). Dose limiting toxicities (DLTs) and responses were assessed at the end of the first and 2nd cycles of treatment, respectively. Correlative studies included pharmacokinetics and measurement of AGT activity in peripheral blood mononuclear cells (PBMC) before and after treatment. Results: 41 eligible pts (stratum I- 19, stratum II- 22; median age 9.3 yrs, range 0.9 to 21.5) were enrolled on this study. Dose levels (in mg/m2/day) evaluated in Stratum I were 45, 60, and 78 mg; In Stratum II 20, 30, 45, and 60 mg. DLT in evaluable pts was myelosuppression (grade IV neutropenia for > 7 days or any grade IV thrombocytopenia) and occurred in 4/16 pts in stratum I [45 mg (n=1/12), 60 mg (n=1/2), 78 mg (n= 2/2)] and 3/19 pts in stratum II [45 mg (n= 3/4), 60 mg (n=0/1), 30 mg (n= 0/12), and 20 mg (n= 0/2)] respectively. Other significant toxicities post first course included renal failure (n=2), pulmonary (n=2), and fatal infection (n=1). PK studies showed median (range) terminal half-life of 62 mins (7.3 to 522 min). The MTDs in stratum I and II were 45 mg/m2/day and 30 mg/m2/day daily for 5 days q 6 weeks, respectively. Objective responses were observed in one pt each with brain stem glioma and medulloblastoma respectively. PBMC AGT levels did not decrease following VNP40101M treatment. Conclusions: The recommended pediatric phase II dose of VNP40101M given i.v daily for 5 days q 6 weeks is 45 mg/m2/day in less-heavily treated and 30 mg/m2/day in heavily treated pts. [Table: see text]

Imaging drug resistance with radiolabeled molecules.

A major obstacle to successful cancer chemotherapy is drug resistance. Multidrug resistance (MDR) is often seen with chemotherapeutic agents such as anthracycline derivatives, vinca alkaloids and taxanes. Multiple aspects of cellular biochemistry have been implicated in the MDR process. Cellular mechanisms of resistance are due to the presence of efflux pumps, P-glycoprotein (P-gp) and multiple resistance-associated protein (MRP), which belong to the ATP-binding cassette (ABC) family of transporters. Another form of drug resistance is involved in the chemotherapy of cancers with alkylating agents such as nitrosourea derivatives and nitrogen mustards. The cytotoxicity of these agents is primarily due to alkylation of the DNA guanine residues at their O6-position, which leads, via a cascade of events, to DNA strand breaks. The DNA repair protein, alkylguanine-DNA alkyl transferase (AGT) removes the alkyl groups from the lesions stoichiometrically to a cysteine in its active site. This process is irreversible and results in the degradation of the protein and its recovery is entirely from de novo synthesis. Noninvasive methodologies for monitoring the transport activity of these efflux pumps and determining tumor content of AGT could serve as critical tools for optimizing chemotherapeutic protocols on a patient-specific basis and gaining an understanding of the dynamics of resistance in living patients. In this review, we will describe the efforts made to date to synthesize radioactive probes of chemotherapy resistance and their use to quantitate these transporters and DNA repair protein by radionuclide imaging.

Toxicity of intracranial and intraperitoneal O6-benzyl guanine in combination with BCNU delivered locally in a mouse model.

The DNA-repair protein, O6-alkylguanine-DNA alkyl transferase, may account for resistance of CNS tumors to DNA-alkylating drugs, such as bis-(2-chloroethyl)-1-nitrosourea (BCNU). The therapeutic effects of BCNU can be potentiated by inhibiting the repair protein with an alkylated guanine analog, O6-benzyl guanine (O6BG). To investigate potential toxicity of this inhibition, we examined the effects of O6BG in mice treated with intracranial (i.c.) BCNU given via a biodegradable polymer. Mice were treated with escalating doses of BCNU chronically delivered i.c., and with chronically delivered O6BG. The O6BG was delivered via a 7-day intraperitoneal (i.p.) or i.c. osmotic minipump. Toxicity of the combination therapies was measured from survival data. Bone marrow response was estimated from white blood cell counts. Combining systemic (i.p.) O6BG with locally (i.c.) delivered BCNU resulted in a decrease in the maximum tolerated dose (MTD) of local BCNU. With local delivery of O6BG, the MTD of BCNU in combination with O6BG was increased. Based on the results of this study, a dose escalation study will be necessary when combining systemic O6BG with the higher doses of i.c. BCNU.

Sequential administration of temozolomide and fotemustine: Depletion of O6-alkyl guanine-DNA transferase in blood lymphocytes and in tumours

Background: The DNA repair protein O6-alkylguanine-DNA alkyl transferase (AT) mediates resistance to chloroethylnitro-soureas. Agents depleting AT such as DTIC and its new analogue temozolomide (TMZ) can reverse resistance to chloro-ethylnitrosoureas. We report the results of a dose finding study of TMZ in association with fotemustine.Patients and methods: Twenty-four patients with metastatic melanoma or recurrent glioma were treated with escalating dose of oral or intravenous TMZ ranging from 300 to 700 mg/m2, divided over two days. Fotemustine 100 mg/m2 was given intravenously on day 2, 4 hours after TMZ. AT depletion was measured in peripheral blood mononuclear cells (PBMCs) and in selected cases in melanoma metastases and was compared to TMZ pharmacokinetics.Results: The maximum tolerated dose (MTD) of TMZ was 400 mg/m2 (200 mg/m2/d) when associated with fotemustine the 2nd day with myelosuppression as dose limiting toxicity. The decrease of AT level in PBMCs was progressive and reached 34% of pretreatment values on day 2. There was however wide interindividual variability. AT reduction was neither dose nor route dependent and did not appear to be related to TMZ systemic exposure (AUC). In the same patients, AT depletion in tumour did not correlate with the decrease of AT observed in PBMCs.Conclusions: PBMCs may not be used as a surrogate of tumour for AT depletion. Further study should concentrate on the pharmacokinetic pharmacodynamic relationship in tumour to provide the basis for individually tailored therapy.

Pharmacokinetics of temozolomide in association with fotemustine in malignant melanoma and malignant glioma patients: comparison of oral, intravenous, and hepatic intra-arterial administration

Depletion of the DNA repair enzyme O6-alkylguanine-DNA alkyltransferase (AT) has been shown to increase tumor sensitivity to chloroethylnitrosoureas. Temozolomide (TMZ), an analogue of dacarbazine, can deplete AT, suggesting that it may be used to sensitize tumors to chloroethylnitrosoureas. However, the influence of nitrosoureas on the pharmacokinetics of TMZ is unknown, and a pilot study was performed to assess the pharmacokinetics of TMZ given via, various routes to 29 patients (27 malignant melanomas, 2 gliomas) with or without sequential administration of i.v. fotemustine. On day 1, TMZ was given intravenously (i.v.), orally (p.o.), or by intra-hepatic arterial infusion (h.i.a.) at four ascending dose levels (150 to 350 mg/m2 per day). On day 2 the same dose of TMZ was given by the same route (or by another route in six patients for determination of its bioavailability), followed 4 h later by fotemustine infusion at 100 mg/m2. Plasma and urinary levels of TMZ were determined on days 1 and 2 by high-performance liquid chromatography after solid-phase extraction. The pharmacokinetics of i.v. TMZ appeared linear, with the area under the curve (AUC) increasing in proportion to the dose expressed in milligrams per square meter (r = 0.86 and 0.91 for days 1 and 2, respectively). The clearance after i.v. administration was 220 +/- 48 and 241 +/- 39 ml/min on days 1 and 2, respectively. The apparent clearance after p.o. and h.i.a. administration was 290 +/- 86 and 344 +/- 77 ml/min, respectively. The volume of distribution of TMZ after i.v., p.o., and h.i.a. administration was 0.4, 0.6, and 0.6 l/kg on day 1 and 0.5, 0.5, and 0.6 l/kg on day 2, respectively. The absolute bioavailability of TMZ was 0.96 +/- 0.1, regardless of the sequence of the i.v.-p.o. or p.o.-i.v. administration, confirming that TMZ is not subject to a marked first-pass effect. A comparison of TMZ pharmacokinetics after i.v. and h.i.a. treatment at the same infusion rate revealed little evidence of hepatic extraction of TMZ. However, the systemic exposure to TMZ (AUC) appeared to decrease at a lower infusion rate. TMZ excreted unchanged in the urine accounted for 5.9 +/- 3.4% of the dose, with low within-patient and high interpatient variability. TMZ crosses the blood-brain barrier and the concentration detected in CSF amounted to 9%, 28%, and 29% of the corresponding plasma levels (three patients). The equilibrium between plasma and ascitic fluid was reached after 2 h (assessed in one patient). The sequential administration of fotemustine at 4 h after TMZ treatment had no clinically relevant influence on the pharmacokinetics of TMZ. The potential clinical effect of TMZ given by h.i.a. or by locoregional administration has yet to be established, as has the impact of the infusion duration on patients' tolerance and response rate.

Probing of conformational changes in human O6-alkylguanine-DNA alkyl transferase protein in its alkylated and DNA-bound states by limited proteolysis.

Human O6-alkylguanine-DNA alkyl transferase (hAGT) is a DNA repair protein that protects cells from alkylation damage by transferring an alkyl group from the O6-position of guanine to a cysteine residue in the active site (-PCHR-) of the protein. The structure of the hAGT protein (23 kDa) has been probed by limited proteolysis with trypsin and Glu-C endoproteases and analysis of the polypeptide fragments by SDS/PAGE. The native hAGT protein had limited accessibility to digestion with trypsin and Glu-C in spite of a number of potential cleavage sites. Initial cleavage by trypsin occurred at residue Lys-193 to give a 21 kDa polypeptide fragment, and this polypeptide underwent further cleavage at residues Arg-128 and Lys-165. These trypsin-cleavage sites became more accessible to digestion in the presence of double-stranded DNA (dsDNA), indicating that hAGT undergoes a change in its conformation on binding to DNA. However, the trypsin cutting site at the Arg-128 position was less available for digestion in the presence of single-stranded DNA (ssDNA), suggesting that the hAGT protein has a different conformation when bound to ssDNA compared with dsDNA. When protease digestion was carried out on wild-type protein, preincubated with the low-molecular-mass pseudosubstrate O6-benzylguanine, increased susceptibility to proteases was observed. A mutant C145A hAGT protein, which cannot repair O6-alkylguanine because the Cys-145 acceptor site in the active site of the protein is changed to Ala, showed identical trypsin cleavage to the wild type, but its digestion was not affected by O6-benzylguanine. These results suggest that alkylation of hAGT leads to an altered conformation. The acquisition of increased susceptibility to proteases upon DNA binding and alkylation demonstrates that hAGT undergoes considerable conformational changes in its structure upon binding to DNA and after repair of alkylation damage.

Caffeine-DNA interactions: biochemical investigations comprising DNA-repair enzymes and nucleic acid synthesis.

Chicken embryo cells were treated with caffeine (0.5-8.0 mM) alone or combined with various chemical and physical DNA-and/or chromatin-interactive agents. Analytical procedures comprised scheduled (SDS) and unscheduled (UDS) DNA synthesis, RNA synthesis (RNS), the activities of O6-alkylguanine-DNA alkyltransferase (AT) and poly (ADP-ribose) polymerase (PARP) as well as nucleoid sedimentation. Additional investigations were done in rat thymic and splenic cells. The effect of caffeine on DNase-I activity served as an in vitro-model system. When present in the PARP-, SDS-, UDS- and RNS-assays, caffeine inhibited the corresponding tracer (14C-NAD, dT-3H, 3H-U) incorporation in a dose-dependent manner. The AT activity was slightly stimulated. At concentrations of 0.06-0.3 mM, caffeine inhibited DNase-I activity by excess substrate. No specific effects of caffeine could be shown by nucleoid sedimentation. Besides the reduced permeability of the cells to nucleic acid precursors, the results obtained with the PARP- and DNase-I assays give evidence for the formation of a DNA-caffeine adduct as a prominent mechanism of cellular caffeine effects including DNA repair inhibition.

Sequence-dependent alkylation of guanine residues in duplex DNA Molecular dynamics study

We present data to explain the sequence dependence of alkylation at the O 6 position of guanine. Both methylation and ethylation of O 6 -guanine occur at either the first or the second position of the middle codon, GGA, in the DNA double helix d(5′-GGCGCTGGAGGCGTG-3′). From our molecular dynamics studies, parameters such as hydrogen bonding, surface accessibility and root-mean-square deviation, provide an atomic rationale for the fact that the second guanine of the middle codon of the sequence is the preferred site for alkylation in this sequence. Furthermore, the recognition and repair by O 6 -alkylguanine-DNA alkyl transferase (AGT) is less effective for alkylation at the second position than the first, and is also less effective for O 6 -ethylguanine (O 6 -etG) than for O 6 -methylguanine (O 6 -meG).

Binding and repair of O6-ethylguanine in double-stranded oligodeoxynucleotides by recombinant human O6-alkylguanine-DNA alkyltransferase do not exhibit significant dependence on sequence context.

Double-stranded (ds) oligodeoxynucleotides (29mers) containing an O6-ethylguanine (O6-EtGua) flanked 5' and 3' by different bases (5'..TGT..3'; 5'..CGG..3', 5'..GGT..3'; 5'..GGG..3'; 5'..GGA..3') were synthesized to investigate the binding and repair characteristics of recombinant human O6-alkylguanine-DNA alkyltransferase (AT) in vitro. The apparent association constant (KA(app)) of AT to the oligomers and the repair rate constant for O6-EtGua (k) respectively, were determined by gel retardation and a monoclonal antibody-based filter binding assay. When ds- or single-stranded (ss) oligomers with or without O6-EtGua were used, no major differences in KA(app) values were observed with either substrate: KA(app) values for native AT were 7.1 and 8.4 x 10(5) M(-1) respectively, for unmodified and [O6-EtGua]-containing ds-oligomers. The corresponding values for ss-oligomers were 1.0 and 4.9 x 10(5) M(-1). The N-terminal first 56 amino acids of AT only exert a limited influence on DNA binding; the KA(app) values for an N-terminally truncated AT protein (1.1 x 10(5) M(-1)) and native AT were of the same order. Moreover, KA(app) was hardly affected by Cys(145)-methylated AT (2.0 x 10(5) M(-1)). The k-values (6.5-11.5 x 10(6) M(-1)s(-1)) were not significantly dependent on nucleotide sequence. k-values of 5.3 and 4.0 x 10(6) M(-1)s(-1) respectively, were obtained with the N-terminally truncated AT protein and for repair of the postreplicative mispair [O6-EtGua]: T by native AT. The low KA(app), the negligible influence on O6 of ethylation, and the minor modulation KA(app) and k by varying the bases flanking O6-EtGua, all indicate that the binding of AT to DNA is non-specific and mediated mainly by ionic interactions [reduced KA(app) and k-values at increased ionic strength]. Surplus DNA reduces the rate of O6-EtGua repair in ds-oligomers by competitive binding of AT molecules. The reaction mechanism of AT with DNA in vivo requires further investigation.

Formation and persistence of N7- and O6-methyl-guanine in DNA of chick embryo brain cells in ovo following administration of N-nitroso-N-methylurea.

Previously, O6-methyl-guanine-DNA alkyltransferase (AT) of the chicken embryo has been investigated in vitro. In the present studies, the effect of in vivo (in ovo) treatment with methylnitrosourea (MNU) was examined at a developmental stage of 15 days and doses of 1.25-20 mg/egg, yielding about 1-16 mmol MNU/kg embryo weight. At intervals of 1-24 h, DNA of the brain was prepared. N7-methylguanine and O6-methylguanine were quantified by combining a rapid method of DNA isolation, high-pressure-liquid-chromatography (HPLC) and electrochemical detection of the guanine-alkyl adducts. In parallel, the AT activity of brain homogenates was determined. Within the range of the detection limits (N7-methylguanine: 16 nM, O6-methylguanine: 2.5 nM), no repair of the guanine adducts, being about 500 nmol O6-methyl- and 1800 nmol N7-methyl-adducts per mmol guanine 1 h following administration of 10 mg MNU/egg, was evident. The rather low acute toxicity of MNU in the chicken embryo at the 15th day of development DL50/24 h being > 4 mg MNU/embryo, argues, therefore, for an additional repair mechanism, e.g. cell replacement repair.

Modulation of O6-alkylguanine alkyltransferase-directed DNA repair in metastatic colon cancers.

Carmustine (BCNU) resistance has been correlated with tumor expression of the DNA repair enzyme O6-alkylguanine-DNA alkyltransferase (AT). It has been shown that streptozotocin will deplete AT activity of human colon cancer cells in vitro and potentiate BCNU cytotoxicity. This clinical trial was conducted to determine whether streptozotocin can be used as a modulator of AT in metastatic colorectal cancers and thereby overcome clinical resistance to BCNU. Fifteen patients with fluorouracil-resistant metastatic colon or rectal cancers were treated sequentially with 2 g/m2 of streptozotocin followed 5 1/2 hours later by BCNU. Sequential biopsies of metastases before and after streptozotocin were conducted to determine whether streptozotocin depletes tumor AT. Peripheral-blood mononuclear cells (PBMCs) were evaluated as a surrogate tissue for prediction of baseline AT levels and streptozotocin posttreatment modulation of the AT in metastases. Streptozotocin treatment led to a 78% (range, 69% to 89%) decrease in the AT levels in colon cancer metastases; however, myelosuppression and hepatic toxicity limited the BCNU dose to 130 mg/m2. A similar decrease in AT levels of PBMCs was found; however, the absolute levels of AT in PBMCs at baseline and following streptozotocin were not predictive of the levels expressed in metastases from the same patient. Despite the decrease in tumor levels of AT, no clinical responses were observed. Streptozotocin decreases but does not fully deplete AT activity in metastatic colorectal cancers and the residual AT level in metastases is sufficient to maintain clinical resistance to BCNU. We have also demonstrated that sequential computed tomography (CT)-directed biopsies of colorectal cancer metastases can be used to evaluate strategies to investigate modulators of AT-directed repair. AT levels of PBMCs do not predict for the AT level or degree of modulation achieved in the metastatic tumor.

Association of temozolomide (TMZ) and fotemustine (FOTE) in metastatic melanoma. An approach based on 06 alkylguanine-DNA alkyl transferase (AT) depletion

Gander, M.1; Leyvraz, S.1; Perey, L.1; Shen, F.1; Bonfanti, M.3; Collela, G.3; Decosterd, L.; Biollaz, J.2; D'Incalci, M.3; Lejeune, F.1 Author Information

P53 mutations, O6-alkylguanine DNA alkyltransferase activity, and sensitivity to procarbazine in human brain tumors

In human brain tumors, sensitivity to procarbazine as measured by sensitivity in a xenograft tumor model correlated inversely with amounts of the DNA repair enzyme O6-alkylguanine DNA alkyltransferase (AT). To test the hypothesis that mutations of the p53 tumor suppressor gene in human tumors also can correlate with the response to chemotherapy, p53 mutations2 were identified in primary human malignant brain tumors and cell lines in which AT activity and procarbazine sensitivity in a xenograft model was ascertained. Mutations were identified in 7 of 21 (33%) specimens tested. Specimens containing p53 mutations tended to exhibit an increased growth delay in procarbazine-treated xenografts and lower amounts of AT. p53 mutations in brain tumors may contribute to procarbazine sensitivity by failing to induce arrest at the G1/S cell-cycle checkpoint, thereby preventing the repair of procarbazine-induced genetic alterations.