Integrating cell-based and clinical genome-wide studies to identify genetic variants contributing to treatment failure in neuroblastoma patients.

High-risk neuroblastoma is an aggressive malignancy, with high rates of treatment failure. We evaluated genetic variants associated with in vitro sensitivity to two derivatives of cyclophosphamide for association with clinical response in a separate replication cohort of neuroblastoma patients (n = 2,709). To determine sensitivity, lymphoblastoid cell lines (LCLs) were exposed to increasing concentrations of 4-hydroperoxycyclophosphamide (4HC; n = 422) and phosphoramide mustard (PM; n = 428). Genome-wide association studies were performed to identify single-nucleotide polymorphisms (SNPs) associated with sensitivity to 4HC and PM. SNPs consistently associated with LCL sensitivity were analyzed for associations with event-free survival (EFS) in patients. Two linked SNPs, rs9908694 and rs1453560, were found to be associated with (i) sensitivity to PM in LCLs across populations and (ii) EFS in all patients (P = 0.01) and within the high-risk subset (P = 0.05). Our study highlights the value of cell-based models to identify candidate variants that may predict response to treatment in patients with cancer.

Genome-wide meta-analysis identifies variants associated with platinating agent susceptibility across populations.

Platinating agents are used in the treatment of many cancers, yet they can induce toxicities and resistance that limit their utility. Using previously published and additional world population panels of diverse ancestry totaling 608 lymphoblastoid cell lines (LCLs), we performed meta-analyses of over 3 million single-nucleotide polymorphisms (SNPs) for both carboplatin- and cisplatin-induced cytotoxicity. The most significant SNP in the carboplatin meta-analysis is located in an intron of NBAS (neuroblastoma amplified sequence; P=5.1 × 10(-7)). The most significant SNP in the cisplatin meta-analysis is upstream of KRT16P2 (P=5.8 × 10(-7)). We also show that cisplatin-susceptibility SNPs are enriched for carboplatin-susceptibility SNPs. Most of the variants that associate with platinum-induced cytotoxicity are polymorphic across multiple world populations; therefore, they could be tested in follow-up studies in diverse clinical populations. Seven genes previously implicated in platinating agent response, including BCL2 (B-cell CLL/lymphoma 2), GSTM1 (glutathione S-transferase mu 1), GSTT1, ERCC2 and ERCC6, were also implicated in our meta-analyses.

Clinical translation of cell-based pharmacogenomic discovery.

The use of cell-based models has emerged as a promising means to discover and validate pharmacologic phenotype-genotype relationships. The availability of large-scale genome studies in both human and model systems is now allowing us an unprecedented opportunity to understand how well cell-based models identify clinically relevant genetic variants associated with drug response and toxicity. Here we review these studies and the emerging translational information.

Heritable and non-genetic factors as variables of pharmacologic phenotypes in lymphoblastoid cell lines.

Publicly available genetic and expression data on lymphoblastoid cell lines (LCLs) make them a unique resource for understanding the genetic underpinnings of pharmacological outcomes and disease. LCLs have been used for pharmacogenomic discovery and validation of clinical findings associated with drug response. However, variation in cellular growth rate, baseline Epstein-Barr virus (EBV) copy number and ATP levels can all be confounders in such studies. Our objective is to better define confounding variables that affect pharmacological end points in LCLs. To this end, we evaluated the effect of these three variables on drug-induced cytotoxicity in LCLs. The drugs evaluated included daunorubicin, etoposide, carboplatin, cisplatin, cytarabine, pemetrexed, 5'-deoxyfluorouridine, vorinostat, methotrexate, 6-mercaptopurine, and 5-fluorouracil. Baseline ATP or EBV copy number were not significantly correlated with cellular growth rate or drug-induced cytotoxicity. In contrast, cellular growth rate and drug-induced cytotoxicity were significantly, directly related for all drugs except vorinostat. Importantly, cellular growth rate is under appreciable genetic influence (h²=0.30-0.39) with five suggestive linkage regions across the genome. Not surprisingly, a percentage of SNPs that significantly associate with drug-induced cytotoxicity also associate with cellular growth rate (P ≤ 0.0001). Studies using LCLs for pharmacologic outcomes should therefore consider that a portion of the genetic variation explaining drug-induced cytotoxicity is mediated via heritable effects on growth rate.

Etoposide sensitivity does not predict MLL rearrangements or risk of therapy-related acute myeloid leukemia.

Therapy-related acute myeloid leukemia (t-AML) caused by MLL rearrangements (rMLL) can arise from topoisomerase II agents. However, whether rMLL-related leukemogenesis is inextricably linked to drug cytotoxicity remains controversial. We therefore compared (i) rMLL in children with acute lymphoblastic leukemia (ALL) who developed t-AML and those who did not, (ii) epipodophyllotoxin toxicity in patients with t-AML and in controls, and (iii) rMLL in cells sensitive to etoposide and in those resistant to etoposide. In children with ALL, rMLL appeared to be more frequent in children who developed t-AML than in those who did not (seven pairs, P = 0.04), although independent of the cumulative etoposide dose (P = 0.5). Similarly, the frequency of epipodophyllotoxin-related toxicities did not differ between patients with t-AML and controls (26 pairs, P > 0.17). Moreover, in 25 cell lines, etoposide-induced MLL fusions did not differ in sensitive vs. resistant lines at equitoxic concentrations (P = 0.65). Together, these results indicate that epipodophyllotoxin-mediated leukemogenesis is not directly linked to drug cytotoxicity.

Genome scan implicates adhesion biological pathways in secondary leukemia.

The genetic risk factors for etoposide-induced leukemia with MLL translocations remain largely unknown. To identify genetic risk factors for and novel characteristics of secondary leukemia, we profiled 116,204 single nucleotide polymorphisms (SNPs) in germline and paired leukemic cell DNA from 13 secondary leukemia/myelodysplasia cases and germline DNA from 13 matched and 156 unmatched controls, all with acute lymphoblastic leukemia treated with etoposide. We analyzed global gene expression from a partially overlapping cohort. No single locus was altered in most cases. We discovered 81 regions of loss of heterozygosity (LOH) in leukemic blasts and 309 SNPs whose allele frequencies differed in cases vs controls. Candidate genes were prioritized on the basis of genes whose SNPs or expression differentiated cases from controls or showed LOH or copy number change in germline vs paired blast DNA from the 13 cases. Three biological pathways were altered: adhesion, Wnt signaling and regulation of actin. Validation experiments using a genome scan for etoposide-induced leukemogenic MLL chimeric fusions in 15 HapMap cell lines also implicated genes involved in adhesion, a process linked to de novo leukemogenesis. Independent clinical epidemiologic and in vitro genome-wide approaches converged to identify novel pathways that may contribute to therapy-induced leukemia.

The pharmacogenetics research network: from SNP discovery to clinical drug response.

The NIH Pharmacogenetics Research Network (PGRN) is a collaborative group of investigators with a wide range of research interests, but all attempting to correlate drug response with genetic variation. Several research groups concentrate on drugs used to treat specific medical disorders (asthma, depression, cardiovascular disease, addiction of nicotine, and cancer), whereas others are focused on specific groups of proteins that interact with drugs (membrane transporters and phase II drug-metabolizing enzymes). The diverse scientific information is stored and annotated in a publicly accessible knowledge base, the Pharmacogenetics and Pharmacogenomics Knowledge base (PharmGKB). This report highlights selected achievements and scientific approaches as well as hypotheses about future directions of each of the groups within the PGRN. Seven major topics are included: informatics (PharmGKB), cardiovascular, pulmonary, addiction, cancer, transport, and metabolism.

A phase II trial of oral temozolomide in patients with metastatic renal cell cancer.

PURPOSE: To determine the activity of temozolomide, an oral imidazotetrazine alkylating agent that has exhibited broad antitumor activity in preclinical studies, in renal cell cancer (RCC) patients. METHODS. Metastatic RCC patients were treated with temozolomide, 200 mg/m(2) per day orally, and traditional radiologic response endpoints were assessed. O(6)-Alkylguanine-DNA alkyltransferase (AGT) activity was measured in four pretreatment biopsies. RESULTS: Among 12 patients, there were no responses. High AGT activity was observed in all four biopsies analyzed. CONCLUSIONS: Temozolomide is not active against RCC and this clinical observation may be due to high levels of AGT in this tumor.

Role of cytochrome P450 isoenzymes in metabolism of O(6)-benzylguanine: implications for dacarbazine activation.

O(6)-Benzylguanine (BG) effectively inactivates the DNA repair protein O(6)-alkylguanine-DNA alkyltransferase and enhances the effectiveness of alkylating agents, such as 1,3-bis(2-chloroethyl)-1-nitrosourea and temozolomide, in vitro and in vivo. BG is presently in clinical trials with 1,3-bis(2-chloroethyl)-1-nitrosourea and temozolomide. Preclinical data demonstrate that BG enhances the sensitivity of cells to 5-[3-methyl-triazen-1-yl]-imidazole-4-carboxamide, the active intermediate of dacarbazine (DTIC), making the combination BG plus DTIC attractive for additional clinical development. DTIC requires metabolism by cytochrome P450 (CYP450) isoforms, 1A1, 1A2, and 2E1 to form two reactive N-demethylated metabolites, 5-[3-hydroxy-methyl-3-methyl-triazen-1-yl]-imidazole-4-carboxamide and 5-[3-methyl-triazen-1-yl]-imidazole-4-carboxamide, ultimately forming a methylating species responsible for its cytotoxicity. The objective of this study was to examine the role of CYP450 1A1 and 1A2 in the metabolism of BG and identify possible drug-drug interactions with DTIC. Our data show that CYP450 isoforms 1A1 and 1A2 are primarily responsible for both BG oxidation to O(6)-benzyl-8-oxoguanine (8-oxoBG) and additional debenzylation to 8-oxoguanine. The catalytic efficiency of BG oxidation is 16 times lower for CYP1A1 than CYP1A2; however, the catalytic efficiency of 8-oxoBG debenzylation is 11 times greater for CYP1A1 than CYP1A2. Furthermore, BG inhibits CYP1A1 and 1A2 catalyzed conversion of DTIC to active methylating species. 8-OxoBG also inhibited conversion of DTIC to active methylating species but to a much lesser extent. The concentrations of BG required to inhibit 50% of DTIC N-demethylation were 2.8, 0.13, and 3.8 microM in human liver microsomes, baculovirus overexpressed CYP1A1, and CYP1A2, respectively. Our results suggest that treating patients with BG as a means to enhance the effectiveness of DTIC is unlikely to result in a therapeutic benefit as a result of inhibition of the enzymes responsible for conversion of DTIC to its active methylating species.

High efficiency electroporation of human umbilical cord blood CD34+ hematopoietic precursor cells.

Human umbilical cord blood provides an alternative source of hematopoietic cells for purposes of transplantation or ex vivo genetic modification. The objective of this study was to evaluate electroporation as a means to introduce foreign genes into human cord blood CD34+ cells and evaluate gene expression in CD34+/CD38(dim) and committed myeloid progenitors (CD33+, CD11b+). CD34+ cells were cultured in X-VIVO 10 supplemented with thrombopoietin, stem cell factor, and Flt-3 ligand. Electroporation efficiency and cell viability measured by flow cytometry using enhanced green fluorescent protein (EGFP) as a reporter indicated 31% +/- 2% EGFP+ /CD34+ efficiency and 77% +/- 3% viability as determined 48 hours post-electroporation. The addition of allogeneic cord blood plasma increased the efficiency to 44% +/- 5% with no effect on viability. Of the total CD34+ cells 48 hours post-electroporation, 20% were CD38(dim)/EGFP+. CD34+ cells exposed to interleukin-3, GM-CSF and G-CSF for an additional 11 days differentiated into CD33+ and CD11b+ cells, and 9% +/- 3% and 8% +/- 7% were expressing the reporter gene, respectively. We show that electroporation can be used to introduce foreign genes into early hematopoietic stem cells (CD34+/CD38(dim)), and that the introduced gene is functionally expressed following expansion into committed myeloid progenitors (CD33+, CD11b+) in response to corresponding cytokines. Further investigation is needed to determine the transgene expression in functional terminal cells derived from the genetically modified CD34+ cells, such as T cells and dendritic cells.

Optimization of culture conditions to enhance transfection of human CD34+ cells by electroporation.

The ability to culture CD34+ stem cells, while maintaining their pluripotency, is essential for manipulations such as gene transfection for therapeutic trials. Human peripheral blood (PB) CD34+ cells (> or = 90% purity) were cultured for up to 4 days in serum-free culture medium supplemented with thrombopoietin (TPO), stem cell factor (SCF), Flt-3 ligand (Flt-3L), with or without PIXY321 (IL-3/GM-CSF fusion protein) and human serum. The CD34 mean fluorescence intensity (MFI) and cell cycle status were evaluated daily using flow cytometry and hypotonic propidium iodide. Prior to culture (day 0), 97.0 +/- 0.9%, 1.9 +/- 0.3% and 1.0 +/- 0.6% of the selected CD34+ cells were in G0-G1, S-phase, or G2-M, respectively. After 2-4 days in culture with TPO/SCF/Flt-3L, there was an increase in the percent of cells in S-phase to 26.4 +/- 0.1% without significant loss of CD34 MFI. The addition of PIXY321 increased.the percentage of CD34+ cells in S-phase to 36.3 +/- 4.0%, but the CD34 MFI and numbers of CFU (colony-forming units) were significantly decreased at day 3 when cultured with PIXY321 or various recombinant cytokine combinations that included IL-3 and IL-6. There is an increase from day 0 to day 4 in the percentages of CD34+ with CD38-, HLA-DR-, and c-kit(low), but not Thy-1+ cells. Electroporation with EGFP reporter gene showed that 1-2 days of pre-stimulation in X-VIVO 10 supplemented with TPO/SCF/Flt-3L was necessary and sufficient for efficient transfection. Flow cytometry analysis demonstrated that 22% of the viable cells are CD34+/EGFP+ 48 h post electroporation. The introduced reporter gene appears to be stable as determined by EGFP+/LTC-IC (long-term colony-initiating cells), at 30-40 positive colonies (16 +/- 7%) per 1 x 10(5) electroporated CD34+ cells.

Efficient expression of foreign genes in human CD34(+) hematopoietic precursor cells using electroporation.

Introduction of foreign genes into human CD34(+) hematopoietic precursor cells offers a means to correct inborn errors or to protect human stem cells from chemotherapeutic damage. Electroporation is a non-chemical, nonviral, highly reproducible means to introduce foreign genes into mammalian cells that has been used primarily for rapidly dividing cells. CD34(+) cells isolated from mobilized peripheral blood of patients were cultured for 48 h in serum-free culture medium supplemented with Flt-3 ligand, stem cell factor and thrombopoietin. Cell cycle analysis showed an increase in % S-phase from 2% on day 0 to 28% on day 2 without significant loss of mean fluorescence intensity (MFI). Optimal electroporation conditions for CD34(+) cells were 550 V/cm, 38 ms, 30 microg DNA/500 microl at cell densities between 0.2 x 10(6) and 10 x 10(6) cells/ml resulting in transient EGFP gene expression in 21% (+/- 1%) of CD34(+) precursor cells, as determined by flow cytometry 48 h after electroporation. The more primitive cells were also found to be EGFP(+) as determined by subset analysis using Thy1, CD38, AC133 and c-kit conjugated monoclonal antibodies. Methylcellulose assays on electroporated CD34(+) cells yielded 20% (+/- 7%) EGFP(+) colonies (CFU-GM, BFU-E and CFU-mix) and 22% (+/- 5%) EGFP(+) long-term colony-initiating cells (LTC-IC). The reporter gene was found to be integrated into the LTC-IC genomic DNA as determined by inverse PCR and DNA sequencing. These results suggest that electroporation has the potential to effectively and stably deliver exogenous genes into human hematopoietic precursor cells.

Distribution and functional consequences of nucleotide polymorphisms in the 3'-untranslated region of the human Sep15 gene.

Selenium has been shown to prevent cancer in a variety of animal model systems. Both epidemiological studies and supplementation trials have supported its efficacy in humans. However, the mechanism by which selenium suppresses tumor development remains unknown. Selenium is present in known human selenoproteins as the amino acid selenocysteine (Sec). Sec is inserted cotranslationally in response to UGA codons within selenoprotein mRNAs in a process requiring a sequence within the 3'-untranslated region (UTR), referred to as a Sec insertion sequence (SECIS) element. Recently, a human Mr 15,000 selenoprotein (Sep15) was identified that contains an in-frame UGA codon and a SECIS element in the 3'-UTR. Examination of the available cDNA sequences for this protein revealed two polymorphisms located at position 811 (C/T) and at position 1125 (G/A) located within the 3'-UTR. Here, we demonstrate significant differences in Sep15 allele frequencies by ethnicity and that the identity of the nucleotides at the polymorphic sites influences SECIS function in a selenium-dependent manner. This, together with genetic data indicating loss of heterozygosity at the Sep15 locus in certain human tumor types, suggests that Sep15 may be involved in cancer development, risk, or both.

Debenzylation of O(6)-benzyl-8-oxoguanine in human liver: implications for O(6)-benzylguanine metabolism.

O(6)-Benzylguanine (BG) effectively inactivates the DNA repair protein O(6)-alkylguanine-DNA alkyltransferase, and enhances the effectiveness of 1,3-bis(2-chloroethyl)-1-nitrosourea in cells in culture and tumor-bearing animals. BG is presently in phase II clinical trials. In humans, BG is converted to O(6)-benzyl-8-oxoguanine (8-oxoBG), a longer-lived, yet equally potent inactivator. We have isolated and identified the debenzylated product, 8-oxoguanine, in plasma and urine of patients following administration of BG. The purpose of this work was to determine the human liver enzymes responsible for the debenzylation of 8-oxoBG. Therefore, 8-oxoBG was incubated with human liver microsomes and cytosol, and the concentration of 8-oxoguanine was determined. No appreciable product was formed in the cytosol; however, increasing amounts of 8-oxoguanine were formed with increasing concentrations of pooled human liver microsomes. The amount of 8-oxoguanine formed increased with time and substrate concentration. Co-incubation of human liver microsomes with 8-oxoBG and various cytochrome P450 isoform-selective inhibitors suggested the possible involvement of CYP1A2, 2E1, and/or 2A6 in this reaction. Incubation of 8-oxoBG with baculovirus cDNA-overexpressed CYP1A2, 2E1, 2A6, and 3A4 demonstrated that formation of 8-oxoguanine was due mainly to CYP1A2. Debenzylation of 8-oxoBG complied with Michaelis-Menten kinetics with K(m) and V(max) values of 35.9 microM and 0.59 pmol/min/pmol of CYP1A2, respectively. CYP1A2 appears to be mainly responsible for the debenzylation of 8-oxoBG in human liver.

Inactivation of O6-alkylguanine-DNA alkyltransferase by 8-substituted O6-benzylguanine analogs in mice.

PURPOSE: The purpose of this study was to determine the usefulness of various 8-substituted O6-benzylguanine (BG) analogs as modulators of the DNA repair protein. O6-alkylguanine-DNA alkyltransferase (AGT). More specifically, the degree of inactivation of AGT in mouse brain, liver, kidney and tumor by O6-benzyl-8-oxoguanine (8-oxoBG), 8-aza-O6-benzylguanine (8-azaBG), O6-benzyl-8-bromoguanine (8-bromoBG) and O6-benzyl-8-trifluoromethylguanine (8-tfmBG) was compared to inactivation by BG, a modulator in phase II clinical trials. BG is converted rapidly to 8-oxoBG in rodents, monkeys and humans. It was reasoned that 8-substituted analogs of BG would exhibit different pharmacological properties compared to BG which could influence tissue bioavailability and, thus, the extent of AGT inactivation in vivo. We compared the tissue distribution of these agents and AGT activity following administration of the 8-substituted analogs. MATERIALS AND METHODS: At various time points up to 24 h after i.p. administration of the BG analogs, tissues (i.e. brain, liver, kidney), A549 lung tumor xenografts (i.p.) or D456 brain tumor xenografts (i.c.) were harvested from athymic nude mice for AGT analysis. AGT activity was quantified in tissue extracts using a biochemical assay with [3H]methylated DNA as a substrate. In addition, concentrations of BG and 8-oxoBG were determined by HPLC with fluorescence detection in mouse tissues following administration of drug. RESULTS: Each of the 8-substituted analogs of BG demonstrated variable AGT inactivation capabilities that were comparable to or better than those of BG especially in kidney and brain tissues. There was a more pronounced depletion of AGT inactivation in brain and D456 brain tumor xenografts following administration of BG compared to 8-oxoBG that could be explained by a much greater concentration of AGT-inactivating drug (BG plus the metabolite 8-oxoBG for mice treated with BG versus 8-oxoBG for mice treated with 8-oxoBG) present in these tissues. The AUCs for brain, kidney and liver were 3.2, 6.9 and 1 1.8 times greater for BG than for 8-oxoBG. CONCLUSIONS: 8-substituted analogs of BG possess unique AGT-inactivation profiles in vivo that are different from that of BG. The AGT-inhibitory activities of BG and its major metabolite, 8-oxoBG, are related to tissue disposition of both drugs.

Effect of O6-benzylguanine on nitrogen mustard-induced toxicity, apoptosis, and mutagenicity in Chinese hamster ovary cells.

O6-Benzylguanine (BG) inactivates O6-alkylguanine-DNA alkyltransferase (AGT), resulting in an increase in the sensitivity of cells to the toxic effects of O6-alkylating agents. BG significantly enhances the cytotoxicity and decreases the mutagenicity of nitrogen mustards [i.e., phosphoramide mustard (PM), melphalan, and chlorambucil], a group of alkylating agents not known to produce O6-adducts in DNA. The enhancement is observed in cells irrespective of AGT activity. Exposure of Chinese hamster ovary cells to 100 microM BG results in enhancement in the cytotoxicity of PM (300 microM), chlorambucil (40 microM), and melphalan (10 microM) by 9-, 7-, and 18-fold, respectively. In contrast, mutation frequency after treatment with 300 microM PM is decreased from 259 mutants/10(6) cells to 22 mutants/10(6) cells when cells are pretreated with BG. The enhancement of toxicity of these bis-alkylating agents appears to involve cross-link formation, because neither cytotoxicity nor mutagenicity of a monoalkylating PM analogue is significantly altered when combined with BG. Enhanced cytotoxicity and decreased mutagenicity is concomitant with a dramatic increase in the number of cells undergoing apoptosis when BG is combined with PM, melphalan, or chlorambucil at 72-94 h after treatment. Cell cycle analysis demonstrates that BG alone or combined with nitrogen mustards arrests cells in G1 phase of the cell cycle. At 16 h after treatment, 11 and 57% of cells treated with PM alone or with BG plus PM are in G1 phase, respectively. Our data suggest that treatment with BG causes G1 arrest and drives noncycling cells treated with nitrogen mustards into apoptosis, thus protecting against mutagenic DNA damage introduced by nitrogen mustards.

O6-benzylguanine potentiates the antitumor effect of locally delivered carmustine against an intracranial rat glioma.

Local delivery of carmustine (BCNU) via biodegradable polymers prolongs survival against experimental brain tumors and in human clinical trials. O6-benzylguanine (O6-BG), a potent inhibitor of the DNA repair protein, O6-alkylguanine-DNA alkyltransferase (AGT), has been shown to reduce nitrosourea resistance and, thus, enhance the efficacy of systemic BCNU therapy in a variety of tumor models. In this report, we demonstrate that O6-BG can potentiate the activity of BCNU delivered intracranially via polymers in rats challenged with a lethal brain tumor. Fischer 344 rats received a lethal intracranial challenge of 100,000 F98 glioma cells (F98 cells have significant AGT activity, 328 fmol/mg protein). Five days later, animals receiving an i.p. injection of O6-BG (50 mg/kg) 2 h prior to BCNU polymer (3.8% BCNU by weight) implantation had significantly improved survival (n = 7; median survival, 34 days) over animals receiving either O6-BG alone (n = 7; median survival, 22 days; P = 0.0002) or BCNU polymer alone (n = 8; median survival, 25 days; P = 0.0001). Median survival for the control group (n = 8) was 23.5 days. Moreover, there was no physical, behavioral, or pathological evidence of treatment-related toxicity. These findings suggest that O6-BG can potentiate the effects of interstitially delivered BCNU and, for tumors expressing significant AGT, may be necessary for the BCNU to provide a meaningful therapeutic benefit. Given the clinical use of BCNU polymers against malignant gliomas, concurrent treatment with O6-BG may provide an important addition to our therapeutic armamentarium.

Phase I trial of carmustine plus O6-benzylguanine for patients with recurrent or progressive malignant glioma.

PURPOSE: The major mechanism of resistance to alkylnitrosourea therapy involves the DNA repair protein O(6)-alkylguanine-DNA alkyltransferase (AGT), which removes chloroethylation or methylation damage from the O(6) position of guanine. O(6)-benzylguanine (O(6)-BG) is an AGT substrate that inhibits AGT by suicide inactivation. We conducted a phase I trial of carmustine (BCNU) plus O(6)-BG to define the toxicity and maximum-tolerated dose (MTD) of BCNU in conjunction with the preadministration of O(6)-BG with recurrent or progressive malignant glioma. PATIENTS AND METHODS: Patients were treated with O(6)-BG at a dose of 100 mg/m(2) followed 1 hour later by BCNU. Cohorts of three to six patients were treated with escalating doses of BCNU, and patients were observed for at least 6 weeks before being considered assessable for toxicity. Plasma samples were collected and analyzed for O(6)-BG, 8-oxo-O(6)-BG, and 8-oxoguanine concentration. RESULTS: Twenty-three patients were treated (22 with glioblastoma multiforme and one with anaplastic astrocytoma). Four dose levels of BCNU (13.5, 27, 40, and 55 mg/m(2)) were evaluated, with the highest dose level being complicated by grade 3 or 4 thrombocytopenia and neutropenia. O(6)-BG rapidly disappeared from plasma (elimination half-life = 0. 54 +/- 0.14 hours) and was converted to a longer-lived metabolite, 8-oxo-O(6)-BG (elimination half-life = 5.6 +/- 2.7 hours) and further to 8-oxoguanine. There was no detectable O(6)-BG 5 hours after the start of the O(6)-BG infusion; however, 8-oxo-O(6)-BG and 8-oxoguanine concentrations were detected 25 hours after O(6)-BG infusion. The mean area under the concentration-time curve (AUC) of 8-oxo-O(6)-BG was 17.5 times greater than the mean AUC for O(6)-BG. CONCLUSION: These results indicate that the MTD of BCNU when given in combination with O(6)-BG at a dose of 100 mg/m(2) is 40 mg/m(2) administered at 6-week intervals. This study provides the foundation for a phase II trial of O(6)-BG plus BCNU in nitrosourea-resistant malignant glioma.

Effect of O6-benzylguanine on alkylating agent-induced toxicity and mutagenicity. In Chinese hamster ovary cells expressing wild-type and mutant O6-alkylguanine-DNA alkyltransferases.

The DNA repair protein O6-alkylguanine-DNA alkyltransferase (AGT) has been shown to protect cells from the toxic and mutagenic effect of alkylating agents by removing lesions from the O6 position of guanine. O6-Benzylguanine (BG) is a potent inactivator of AGT, resulting in an increase in the sensitivity of cells to the toxic effects of chemotherapeutic alkylating agents. Chinese hamster ovary (CHO) cells and CHO cells transfected with wild-type AGT (CHOWTAGT) and a mutant AGT [P138 M/V139I/P140K (CHOMIK)] known to be resistant to BG were treated with BG and various alkylating agents. BG treatment alone dramatically decreased AGT activity in CHOWTAGT cells but resulted in no depletion in AGT activity in CHOMIK cells. In the absence of AGT, these cells are highly sensitive to the toxic and mutagenic effects of temozolomide and 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU), and no further sensitization occurs in the presence of BG. In contrast, CHOWTAGT cells are resistant to temozolomide and BCNU, and treatment with BG resulted in a significantly higher cell killing and mutation frequency. CHOMIK cells were completely resistant to temozolomide or BCNU in the presence and absence of BG. Both cell killing and mutation frequency of 4-hydroperoxycyclophosphamide (4-HC) in CHO, CHOWTAGT, and CHOMIK cells were increased in the presence of BG. 4-HC generates two active metabolites, phosphoramide mustard (PM) and acrolein. BG had no effect on 4hydroperoxydidechlorocyclophosphamide (which generates acrolein and a nonalkylating form of PM) in CHO cells and CHOMIK cells, but enhancement of toxicity was observed with PM in both these cell lines. Therefore, we attribute the enhancement to the PM metabolite of 4-HC. Our results demonstrate that wild-type AGT plays an important role in protecting against the toxic and mutagenic effect of O6 alkylating agents and that a mutant AGT resistant to inactivation by BG effectively prevents BG-enhanced toxicity and mutagenicity induced by these agents. Expression of the AGT protein contributes to resistance of 4-HC. BG also enhances the toxicity of 4-HC and PM by a mechanism that may not involve the AGT repair protein.

Chronic daily low dose of 4-methyl-5-(2-pyrazinyl)-1,2-dithiole-3-thione (Oltipraz) in patients with previously resected colon polyps and first degree female relatives of breast cancer patients.

The chemoprevention agent oltipraz, one of the most active chemopreventive compounds in preclinical studies, has been shown to induce glutathione-S-transferase (GST) activity in animals. Oltipraz was evaluated in a Phase I trial at daily oral doses of 20 mg (L1), 50 mg (L2), and 100 mg (L3) and twice weekly doses of 125 mg (L4) taken for 6 months with 6 patients entered at L1 and L2 and 7 patients entered at L3 and L4 (26 subjects: 19 females and 7 males). The subject population included patients with previously resected colon polyps and first-degree female relatives of breast cancer patients. Patients with resected colon polyps underwent rectal biopsy for GST and glutathione (GSH) analyses. Of the 26 subjects, the following completed 6 months of therapy: 4 of 6 patients (L1), 4 of 6 patients (L2), 5 of 7 patients (L3), and 4 of 7 patients (L4). Toxicities were mild to severe and included: gastrointestinal symptoms, photosensitivity/heat intolerance, and neurological symptoms. Monthly plasma samples were obtained 2-3 h after oltipraz ingestion with minimally detectable plasma concentrations at L1. There was a significant difference in mean oltipraz concentration across the four doses, with no significant differences in mean oltipraz concentration over time. Rectal tissue and lymphocyte GSH and GST were variable, with no significant difference in mean levels across doses. At the 100-mg/day dose (L3), 1 patient experienced significant increase in rectal tissue GSH and GST activity, whereas 3 additional patients (L1 and L4) had >50% increase in tissue GSH. Lymphocyte GSH level was significantly related to plasma oltipraz concentration. There were no significant correlations between plasma oltipraz concentration and lymphocyte GST level nor any significant correlation between plasma concentration and percentage of change in tissue GSH or GST. Further investigation of dose/schedule and biological end points is ongoing.