Anthony E. Pegg

Repair of O6-alkylguanine by alkyltransferases

Abstract The predominant pathway for the repair of O 6 -methylguanine in DNA is via the activity of an alkyltransferase protein that transfers the methyl group to a cysteine acceptor site on the protein itself. This review article describes recent studies on this alkyltransferase. The protein repairs not only methyl groups but also 2-chloroethyl-, benzyl- and pyridyloxobutyl-adducts. It acts on double-stranded DNA by flipping the O 6 -guanine adduct out of the DNA helix and into a binding pocket. The free base, O 6 -benzylguanine, is able to bind in this pocket and react with the cysteine, rendering it an effective inactivator of mammalian alkyltransferases. The alkylated form of the protein is rapidly degraded by the ubiquitin/proteasomal system. Some tumor cells do not express alkyltransferase despite having an intact gene. Methylation of key sites in CpG-rich islands in the promoter region are involved in this silencing and a change in the nuclear localization of an enhancer binding protein may also contribute. The alkyltransferase promoter contains Sp1, GRE and AP-1 sites and is slightly inducible by glucocorticoids and protein kinase C activators. There is a complex relationship between p53 and alkyltransferase expression with p53 mediating a rise in alkyltransferase in response to ionizing radiation but having no clear effect on basal levels. DNA adducts at the O 6 -position of guanine are a major factor in the carcinogenic, mutagenic, apoptopic and clastogenic actions of methylating agents and chloroethylating agents. Studies with transgenic mice in which alkyltransferase levels are increased or decreased confirm the importance of this repair pathway in protecting against carcinogenesis. Alkyltransferase activity in tumors protects them from therapeutic agents such as temozolomide and BCNU. This resistance is abolished by O 6 -benzylguanine and this drug is currently in clinical trials to enhance cancer chemotherapy by these agents. Studies are in progress to reduce the toxicity of such therapy towards the bone marrow by gene therapy to express alkyltransferases with mutations imparting resistance to O 6 -benzylguanine at high levels in marrow stem cells. Several polymorphisms in the human alkyltransferase gene have been identified but the significance of these in terms of alkyltransferase action is currently unknown.

Structure, function, and inhibition of O6-alkylguanine-DNA alkyltransferase.

Publisher Summary This chapter focuses on the structure and function of alkyltransferase protein. O 6 -alkylguanine-DNA alkyltransferase is a remarkable protein that alone can, in a single step, remove adducts from DNA that are formed at the O 6 -position of guanine and the O 4 -position of thymine and can, thus, restore the original DNA. Production of such adducts is a major contributor to the toxic, mutagenic, and carcinogenic effects of alkylating agents. Alkyltransferase activity has been detected in many species, including microorganisms, insects, fish, and mammals. The unique activity of alkyltransferase suggests that it is an ideal target for biochemical modulation. The efficient repair of toxic lesions formed at the O 6 -position of guanine without additional enzymes or cofactors provides a less complex target to modulate than other DNA repair proteins. Furthermore, the high degree of correlation that exists between alkyltransferase activity and sensitivity to nitrosoureas indicates that elimination of this protein may reverse resistance in many cases. Two methods have been used to overcome alkylnitrosourea resistance by inactivation of alkyltransferase. One uses methylating agents that indirectly decrease alkyltransferase levels by introducing O 6 -methylguanine residues in DNA that are then repaired by the alkyltransferase. The second method uses direct alkyltransferase inactivators such as O 6 -methylguanine.

Spermidine/spermine N1-acetyltransferase--the turning point in polyamine metabolism.

Polyamines are thought to have several vital roles in cell growth and differentiation. The highly regulated polyamine metabolic pathway provides cells with the ability to finely control the intracellular concentration of these ubiquitous polycations. Although earlier studies of regulation of polyamine content were concentrated on the biosynthetic reactions, recently the importance of the catabolic processes, particularly the highly regulated acetylation step in polyamine degradation, has become apparent. This work has led to an understanding of how a cell may, in a tightly controlled manner, facilitate the breakdown, excretion, cycling, and/or intracellular shuttling of the polyamines. This myriad of possibilities appears to be regulated initially at a single rate‐limiting enzymatic step, the N1‐acetylation of spermidine or spermine, by spermidine/spermine N1‐acetyltransferase (SSAT). Recent cloning of the human SSAT gene has facilitated a more detailed study of this enzyme. SSAT appears to have a role in the determination of tumor sensitivity to a new class of antineoplastic agents. The further study of SSAT and the associated polyamine metabolism should provide a better understanding of the regulation and function of these cations.—Casero, R. A., Jr., Pegg, A. E. Spermidine/spermine N1‐acetyltransferase—the turning point in polyamine metabolism. FASEB J. 7: 653‐661; 1993.

Mammalian Polyamine Metabolism and Function

Polyamines are ubiquitous small basic molecules that play multiple essential roles in mammalian physiology. Their cellular content is highly regulated and there is convincing evidence that altered metabolism is involvement in many disease states. Drugs altering polyamine levels may therefore have a variety of important targets. This review will summarize the current state of understanding of polyamine metabolism and function, the regulation of polyamine content, and heritable pathological conditions that may be derived from altered polyamine metabolism.

Regulation of Ornithine Decarboxylase

Ornithine decarboxylase (ODC) initiates the polyamine biosynthetic pathway. The amount of ODC is altered in response to many growth factors, oncogenes, and tumor promoters and to changes in polyamine levels. Susceptibility to tumor development is increased in transgenic mice expressing high levels of ODC and is decreased in mice with reduced ODC due to loss of one ODC allele or elevated expression of antizyme, a protein that stimulates ODC degradation. This review describes key factors that contribute to the regulation of ODC levels, which can occur at the levels of transcription, translation, and protein turnover.

Formation and Metabolism of Alkylated Nucleosides: Possible Role in Carcinogenesis by Nitroso Compounds and Alkylating Agents

Publisher Summary Alkylation of nucleic acids occurs both physiologically within living cells and after the administration of compounds that are either themselves direct chemical alkylating agents or are converted into alkylating agents by metabolic activation. Some of these compounds are highly potent carcinogens. Carcinogenicity of these agents is due to the alkylation of certain cellular components because no other degradation product nor is the compound itself oncogenic. This chapter deals with the formation and metabolism of alkylated purines in nucleic acids. It briefly discusses other alkylation reactions leading to the alkylphosphate triester production and alkylated pyrimidines. It also presents evidences favoring particular critical targets for the action of alkylating carcinogens. The attack on nucleic acids by carcinogenic alkylating agents is not entirely random and generally leads to the formation of alkylated nucleosides at many different sites distributed throughout the cellular nucleic acids. Carcinogenesis is not necessarily mediated through mutagenesis in somatic cells. However, it is observed that carcinogenic action could be mediated through a distinct action of the electrophilic reactant.

Phase I Trial of Temozolomide Plus O6-Benzylguanine for Patients With Recurrent or Progressive Malignant Glioma

PURPOSE We conducted a two-phase clinical trial in patients with progressive malignant glioma (MG). The first phase of this trial was designed to determine the dose of O6-BG effective in producing complete depletion of tumor AGT activity for 48 hours. The second phase of the trial was designed to define the maximum tolerated dose (MTD) of a single dose of temozolomide when combined with O6-BG. In addition, plasma concentrations of O6-BG and O6-benzyl-8-oxoguanine were evaluated after O6-BG. PATIENTS AND METHODS For our first phase of the clinical trial, patients were scheduled to undergo craniotomy for AGT determination after receiving a 1-hour O6-BG infusion at 120 mg/m2 followed by a continuous infusion at an initial dose of 30 mg/m2/d for 48 hours. The dose of the continuous infusion of O6-BG escalated until tumor AGT was depleted. Once the O6-BG dose was established a separate group of patients was enrolled in the second phase of clinical trial, in which temozolomide, administered as a single dose at the end of the 1-hour O6-BG infusion, was escalated until the MTD was determined. RESULTS The O6-BG dose found to be effective in depleting tumor AGT activity at 48 hours was an IV bolus of 120 mg/m2 over 1 hour followed by a continuous infusion of 30 mg/m2/d for 48 hours. On enrolling 38 patients in six dose levels of temozolomide, the MTD was established at 472 mg/m2 with dose-limiting toxicities limited to myelosuppression. CONCLUSION This study provides the foundation for a phase II trial of O6-BG plus temozolomide in temozolomide-resistant MG.

Polyamine catabolism and disease

In addition to polyamine homoeostasis, it has become increasingly clear that polyamine catabolism can play a dominant role in drug response, apoptosis and the response to stressful stimuli, and contribute to the aetiology of several pathological states, including cancer. The highly inducible enzymes SSAT (spermidine/spermine N1-acetyltransferase) and SMO (spermine oxidase) and the generally constitutively expressed APAO (N1-acetylpolyamine oxidase) appear to play critical roles in many normal and disease processes. The dysregulation of polyamine catabolism frequently accompanies several disease states and suggests that such dysregulation may both provide useful insight into disease mechanism and provide unique druggable targets that can be exploited for therapeutic benefit. Each of these enzymes has the potential to alter polyamine homoeostasis in response to multiple cell signals and the two oxidases produce the reactive oxygen species H2O2 and aldehydes, each with the potential to produce pathological states. The activity of SSAT provides substrates for APAO or substrates for the polyamine exporter, thus reducing the intracellular polyamine concentration, the net effect of which depends on the magnitude and rate of any increase in SSAT. SSAT may also influence cellular metabolism via interaction with other proteins and by perturbing the content of acetyl-CoA and ATP. The goal of the present review is to cover those aspects of polyamine catabolism that have an impact on disease aetiology or treatment and to provide a solid background in this ever more exciting aspect of polyamine biology.

Ornithine decarboxylase as an enzyme target for therapy

Interest in ornithine decarboxylase (ODC) and the therapeutic effects of its inhibition with the consequent depletion of polyamine biosynthesis has been widespread since the late 1970s and 1980s. This review covers new information about the properties of ODC, recent findings with ODC inhibitors and a discussion of the mechanism of inactivation of ODC by eflornithine. Recent in vivo therapeutic approaches of ODC inhibition are also discussed including: cancer and cancer chemoprevention; autoimmune diseases; polyamines and the blood-brain barrier, ischemia and hyperplasia; the NMDA receptor and modulation by polyamines; hearing loss; African trypanosomiasis; Pneumocystis carinii pneumonia and Cryptosporidium in AIDS; and other infectious diseases/organisms.

Resuscitation of the monkey brain after one hour complete ischemia. III. Indications of metabolic recovery

Adult rhesus monkeys were subjected to complete cerebral ischemia for one hour and subsequent recirculation for up to 24 h. Animals with signs of functional recovery (e.g. spontaneous EEG activity) exhibited a partial replenishment of cellular energy sources (ATP, phosphocreatine) and a progressive normalization of cerebral lactate levels. Glucose and pyruvate concentrations showed a transient increase over control values during the early stages of postischemic recirculation. Monkeys without functional recovery lacked a significant resynthesis of energy-rich compounds; adenine nucleotides continued to decrease and lactate concentrations were higher than in animals subjected to ischemia without recirculation. Cerebral polysome profiles remained unaltered during the ischemic period but in all animals a marked disaggregation of polyribosomes with a concomitant increase in ribosomal subunits occurred after the onset of recirculation. In monkeys with indications of functional recovery these changes were reversible but a normal polysome profile was only observed after 24 h of recirculation. The results obtained indicate a postischemic depression of protein synthesis due to an inhibition of peptide chain initiation. After recirculation of the brain for 3-6 h there was evidence for an induction of enzymes involved in polyamine synthesis (ornithine decarboxylase and S-adenosylmethionine decarboxylase). No changes in the activity of these enzymes were observed at the end of the ischemic period, indicating that during complete cerebral ischemia not only the synthesis but also the catabolism of proteins is inhibited.