bis(2-chloroethyl)-N-nitrosourea Research Articles

Total Methionine Restriction Treatment of Cancer.

This chapter reviews how total methionine (MET) restriction (MR) of a human brain tumor xenograft, effected by the combination of recombinant L-methionine-α-deamino-γ-lyase (rMETase) and a MET-free diet, greatly potentiates standard chemotherapy for brain tumors in mouse models. The growth of human brain tumor Daoy, SWB77, and D-54 xenografts in nude mice was arrested after the depletion of mouse plasma methionine (MET) with a combination of an MR diet and rMETase and homocysteine to rescue normal cells and tissues. MET was depleted to below 5μm by this treatment. MR for 10-12days inhibited tumor growth, but did not prevent tumor regrowth after treatment cessation. A single dose of N,N'-bis(2-chloroethyl)-N-nitrosourea (BCNU), which was ineffective alone, was administered at the end of the MR regimen, and caused a more than 80-day growth delay for Daoy and D-54 and a 20-day growth delay for SWB77. The total MR treatment regimens also increased the efficacy of temozolomide (TMZ) against the SWB77 xenograft when administered at the end of the MET regimen.

Genomic Phenotyping by Barcode Sequencing Broadly Distinguishes between Alkylating Agents, Oxidizing Agents, and Non-Genotoxic Agents, and Reveals a Role for Aromatic Amino Acids in Cellular Recovery after Quinone Exposure

Toxicity screening of compounds provides a means to identify compounds harmful for human health and the environment. Here, we further develop the technique of genomic phenotyping to improve throughput while maintaining specificity. We exposed cells to eight different compounds that rely on different modes of action: four genotoxic alkylating (methyl methanesulfonate (MMS), N-Methyl-N-nitrosourea (MNU), N,N′-bis(2-chloroethyl)-N-nitroso-urea (BCNU), N-ethylnitrosourea (ENU)), two oxidizing (2-methylnaphthalene-1,4-dione (menadione, MEN), benzene-1,4-diol (hydroquinone, HYQ)), and two non-genotoxic (methyl carbamate (MC) and dimethyl sulfoxide (DMSO)) compounds. A library of S. cerevisiae 4,852 deletion strains, each identifiable by a unique genetic ‘barcode’, were grown in competition; at different time points the ratio between the strains was assessed by quantitative high throughput ‘barcode’ sequencing. The method was validated by comparison to previous genomic phenotyping studies and 90% of the strains identified as MMS-sensitive here were also identified as MMS-sensitive in a much lower throughput solid agar screen. The data provide profiles of proteins and pathways needed for recovery after both genotoxic and non-genotoxic compounds. In addition, a novel role for aromatic amino acids in the recovery after treatment with oxidizing agents was suggested. The role of aromatic acids was further validated; the quinone subgroup of oxidizing agents were extremely toxic in cells where tryptophan biosynthesis was compromised.

Combination of arsenic trioxide and BCNU synergistically triggers redox-mediated autophagic cell death in human solid tumors

Arsenic trioxide (As2O3) is an effective treatment for relapsed or refractory acute promyelocytic leukemia (APL). After the discovery of As2O3 as a promising treatment for APL, several studies investigated the use of As2O3 as a single agent in the treatment of solid tumors; however, its therapeutic efficacy is limited. Thus, the systematic study of the combination of As2O3 with other clinically used chemotherapeutic drugs to improve its therapeutic efficacy in treating human solid tumors is merited. In this study, we demonstrate for the first time, using isobologram analysis, that As2O3 exhibits a synergistic interaction with N,N′-bis(2-chloroethyl)-N-nitrosourea (BCNU). The synergistic augmentation of the cytotoxicity of As2O3 with BCNU is in part through the autophagic cell death machinery in human solid tumor cells. As2O3 and BCNU in combination produce enhanced cytotoxicity via the depletion of reduced glutathione (GSH) and augmentation of reaction oxygen species (ROS) production. Further analysis indicated that the extension of GSH depletion by this combined regimen occurs through the inhibition of the catalytic activity of glutathione reductase. Blocking ROS production with antioxidants or ROS scavengers effectively inhibits cell death and autophagy formation, indicating that redox-mediated autophagic cell death involves the synergism of As2O3 with BCNU. Taken together, this is the first evidence that BCNU could help to extend the therapeutic spectrum of As2O3. These findings will be useful in designing future clinical trials of combination chemotherapy with As2O3 and BCNU, with the potential for broad use against a variety of solid tumors.

Efficient and stable MGMT-mediated selection of long-term repopulating stem cells in nonhuman primates.

HSC transplantation using genetically modified autologous cells is a promising therapeutic strategy for various genetic diseases, cancer, and HIV. However, for many of these conditions, the current efficiency of gene transfer to HSCs is not sufficient for clinical use. The ability to increase the percentage of gene-modified cells following transplantation is critical to overcoming this obstacle. In vivo selection with mutant methylguanine methyltransferase (MGMTP140K) has been proposed to overcome low gene transfer efficiency to HSCs. Previous studies have shown efficient in vivo selection in mice and dogs but only transient selection in primates. Here, we report efficient and stable MGMTP140K-mediated multilineage selection in both macaque and baboon nonhuman primate models. Treatment consisting of both O6-benzylguanine (O6BG) and N,N'-bis(2-chloroethyl)-N-nitroso-urea (BCNU) stably increased the percentage of transgene-expressing cells from a range of initial levels of engrafted genetically modified cells, with the longest follow-up after drug treatment occurring over 2.2 years. Drug treatment was well tolerated, and selection occurred in myeloid, lymphoid, and erythroid cells as well as platelets. Retrovirus integration site analysis before and after drug treatments confirmed the presence of multiple clones. These nonhuman primate studies closely model a clinical setting and should have broad applications for HSC gene therapy targeting human diseases of malignant, genetic, and infectious nature, including HIV.

N,N'-Bis(2-chloroethyl)- N-nitrosourea (BCNU)-induced Apoptosis of Neural Progenitor Cells in the Developing Fetal Rat Brain

N,N’-bis(2-chloroethyl)-N-nitrosourea (BCNU) is one of the major drugs used in chemotherapy against malignant gliomas due to its effects, such as induction of bifunctional alkylation of DNA and formation of interstrand DNA cross-linkages, and induces cortical malformations in the fetal and neonatal rat brain. In this study, pregnant rats were treated with 7.5 mg/kg of BCNU on gestational day 13 (GD 13), and their fetuses were collected from 12 to 72 hours after BCNU treatment in order to examine the timecourses of morphological and immunohistochemical changes in neural progenitor cells in the developing brain. The number of pyknotic cells in the telencephalon peaked at 24 h and then gradually decreased until 72 h. The majority of these pyknotic cells were positive for cleaved caspase-3, a key executioner of apoptosis. The pyknotic cells showed the ultrastructural characteristics of apoptosis. The number of p53-positive cells began to increase prior to the appearance of apoptotic cells and p21-positive cells. The number of phosphorylated-histone H3-positive cells (mitotic cells) decreased from 24 to 36 h. The number of Iba1-positive cells (microglial cells) in the telencephalon increased from 12 to 48 h. These results suggest that BCNU induces p53-dependent apoptosis and reduces proliferative activity, resulting in reduction of the weight of the telencephalon and the thickness of the telencephalic wall in the fetal brain. This study will help to clarify the mechanisms of BCNU-induced fetal brain toxicity.

Bone Marrow-Derived Cells Exhibiting Lung Epithelial Cell Characteristics Are Enriched In Vivo Using Methylguanine DNA Methyltransferase-Mediated Drug Resistance

Previous studies have suggested that donor bone marrow-derived cells can differentiate into lung epithelial cells at low frequency. We investigated whether we could enrich the number of donor-derived hematopoietic cells that have type II pneumocyte characteristics by overexpression of the drug resistance gene methylguanine DNA methyltransferase (MGMT). MGMT encodes O(6)-alkylguanine DNA alkyltransferase (AGT), a drug resistance protein for DNA damage induced by N,N'-bis(2-chloroethyl)-N-nitrosourea (BCNU), and the mutant P140K MGMT confers resistance to BCNU and the AGT inactivator O(6)-benzylguanine (BG). For this study, we used two MGMT selection models: one in which donor cells had a strong selection advantage because the recipient lung lacked MGMT expression, and another in which drug resistance was conferred by gene transfer of P140K MGMT. In both models, we saw an increase in the total number of donor-derived cells in the lung after BCNU treatment. Analysis of single-cell suspensions from 28 mice showed donor-derived cells with characteristics of type II pneumocytes, determined by surfactant protein C (SP-C) expression. Furthermore, an increase in the percentage of donor-derived SP-C cells was noted after BCNU or BG and BCNU treatment. This study demonstrates that bone marrow cells expressing MGMT can engraft in the lung and convert into cells expressing the type II pneumocyte protein SP-C. Furthermore, these cells can be enriched in response to alkylating agent-mediated lung injury. These results suggest that expression of MGMT could enhance the capacity of bone marrow-derived cells to repopulate lung epithelium, and when used in combination with a gene of interest, MGMT could have therapeutic applications.

Combined effect of 2-5A-linked antisense against telomerase RNA and conventional therapies on human malignant glioma cells in vitro and in vivo

We recently showed that therapy with 2'-5'-oligoadenylate (2-5A)-linked antisense against human telomerase RNA component (2-5A-anti-hTR) is a novel telomerase-targeting strategy against malignant gliomas. In this study, we investigated conventional chemotherapeutic agents and gamma-irradiation (IR) to determine whether they could augment the efficacy of 2-5A-anti-hTR against these tumors in vitro and in vivo. Treatment with 2-5A-anti-hTR inhibited the viability of U373-MG and U87-MG malignant glioma cells in a dose-dependent manner; the antitumor effect resulted from induction of apoptosis. Also, telomerase-positive astrocytes with oncogenic Ras were more sensitive to 2-5A-anti-hTR than were those without oncogenic Ras. In addition, we sought to determine the combined effect of 2-5A-anti-hTR with N, N'-bis (2-chloroethyl)-N-nitrosourea (BCNU), cisplatin (CDDP), paclitaxel (PTX), temozolomide (TMZ), or IR. When we administered the combination treatments on the same day, PTX and IR showed a greater combined effect with 2-5A-anti-hTR on both tumor cell lines than did BCNU, CDDP and TMZ. However, all of the combination regimens were synergistic when we first treated tumor cells with 2-5A-anti-hTR for 24 h and then exposed them to the conventional treatments. Apoptosis-inducing agents (CDDP and PTX) but not autophagy-inducing therapies (TMZ and IR) enhanced the incidence of apoptosis caused by 2-5A-anti-hTR. Lastly, we observed a combinatorial effect of 2-5A-anti-hTR and TMZ in vivo in subcutaneous U87-MG tumors in nude mice. Interestingly, treatment with TMZ increased the incidence of apoptosis in subcutaneous tumor cells treated with 2-5A-anti-hTR. These results suggest that 2-5A-anti-hTR is preferable in combination with established cancer therapies.

IN VIVO IMAGING IN A MURINE MODEL OF GLIOBLASTOMA

To use in vivo imaging methods in mice to quantify intracranial glioma growth, to correlate images and histopathological findings, to explore tumor marker specificity, to assess effects on cortical function, and to monitor effects of chemotherapy. Mice with DBT glioma cell tumors implanted intracranially were imaged serially with a 4.7-T small-animal magnetic resonance imaging (MRI) scanner. MRI tumor volumes were measured and correlated with postmortem histological findings. Different nonspecific and specific positron emission tomography radiopharmaceuticals, [18F]2-fluoro-2-deoxy-d-glucose, [18F]3'-deoxy-3'-fluorothymidine, or [11C]RHM-I, a sigma2-receptor ligand, were visualized with microPET (CTI-Concorde MicroSystems LLC, Knoxville, TN). Intrinsic optical signals were imaged serially during contralateral whisker stimulation to study the impact of tumor growth on cortical function. Other groups of mice were imaged serially with MRI after one or two doses of the antimitotic N,N'-bis(2-chloroethyl)-N-nitrosourea (BCNU). MRI and histological tumor volumes were highly correlated (r2 = 0.85). Significant binding of [11C]RHM-I was observed in growing tumors. Over time, tumors reduced and displaced (P # 0.001) whisker-activated intrinsic optical signals but did not change intrinsic optical signals in the contralateral hemisphere. Tumor growth was delayed 7 days after a single dose of BCNU and 18 days after two doses of BCNU. Mean tumor volume 15 days after DBT implantation was significantly smaller for treated mice (1- and 2-dose BCNU) compared with controls (P = 0.0026). Mouse MRI, positron emission tomography, and optical imaging provide quantitative and qualitative in vivo assessments of intracranial tumors that correlate directly with tumor histological findings. The combined imaging approach provides powerful multimodality assessments of tumor progression, effects on brain function, and responses to therapy.

Characterisation of a P140K mutant O6‐methylguanine‐DNA‐methyltransferase (MGMT)‐expressing transgenic mouse line with drug‐selectable bone marrow

Gene transfer of the P140K mutant of O6-methylguanine-DNA-methyltransferase (MGMT(P140K)) into hematopoietic stem cells (HSC) provides a mechanism for drug resistance and the selective expansion of gene-modified cells in vivo. Possible clinical applications for this strategy include chemoprotection to allow dose escalation of alkylating chemotherapy, or combining MGMT(P140K) expression with a therapeutic gene in the treatment of genetic diseases. Our aim is to use MGMT(P140K)-driven in vivo selection to develop allogeneic micro-transplantation protocols that rely on post-engraftment selection to overcome the requirement for highly toxic pre-transplant conditioning, and to establish and maintain predictable levels of donor/recipient chimerism. Using stably transfected murine embryonic stem (ES) cells, we have generated a C57BL/6 transgenic mouse line with expression of MGMT(P140K) within the hematopoietic compartment for use as a standard source of donor HSC in such models. Functional characterisation of transgene expression was carried out in chemotherapy-treated transgenic mice and in allogeneic recipients of transgenic HSC. Expression of the transgene provided chemoprotection and allowed in vivo selection of MGMT(P140K)-expressing cells in transgenic mice after exposure to O6-benzylguanine (BG) and N,N'-bis(2-chloroethyl)-N-nitrosourea (BCNU). In an allogeneic transplant experiment in which transgenic HSC were engrafted into 129 strain recipients following low intensity conditioning (Busulfan, anti-CD8, anti-CD40Ligand), MGMT(P140K)-expressing cells could be selected using chemotherapy. This MGMT(P140K) transgenic mouse line provides a useful source of drug-selectable donor cells for the development of non-myeloablative allogeneic transplant models in which variation in transplant conditioning elements can be investigated independently of gene transfer efficiency.

Tissue transglutaminase 2 inhibition promotes cell death and chemosensitivity in glioblastomas

Tissue transglutaminase 2 belongs to a family of transglutaminase proteins that confers mechanical resistance from proteolysis and stabilizes proteins. Transglutaminase 2 promotes transamidation between glutamine and lysine residues with the formation of covalent linkages between proteins. Transglutaminase 2 also interacts and forms complexes with proteins important in extracellular matrix organization and cellular adhesion. We have identified the novel finding that treatment of glioblastoma cells with transglutaminase 2 inhibitors promotes cell death and enhances sensitivity to chemotherapy. Treatment with either the competitive transglutaminase 2 inhibitor, monodansylcadaverine, or with highly specific small-molecule transglutaminase 2 inhibitors, KCA075 or KCC009, results in induction of apoptosis in glioblastoma cells. Treatment with these transglutaminase 2 inhibitors resulted in markedly decreased levels of the prosurvival protein, phosphorylated Akt, and its downstream targets. These changes promote a proapoptotic profile with altered levels of multiple intracellular proteins that determine cell survival. These changes include decreased levels of the antiapoptotic proteins, survivin, phosphorylated Bad, and phosphorylated glycogen synthetase kinase 3beta (GSK-3beta), and increased levels of the proapoptotic BH3-only protein, Bim. In vivo studies with s.c. murine DBT glioblastoma tumors treated with transglutaminase 2 inhibitors combined with the chemotherapeutic agent, N-N'-bis (2-chloroethyl)-N-nitrosourea (BCNU), decreased tumor size based on weight by 50% compared with those treated with BCNU alone. Groups treated with transglutaminase 2 inhibitors showed an increased incidence of apoptosis determined with deoxynucleotidyl transferase-mediated biotin nick-end labeling staining. These studies identify inhibition of transglutaminase 2 as a potential target to enhance cell death and chemosensitivity in glioblastomas.

A bicistronic SIN-lentiviral vector containing G156A MGMT allows selection and metabolic correction of hematopoietic protoporphyric cell lines.

Erythropoietic protoporphyria (EPP) is an inherited disease characterised by a ferrochelatase (FECH) deficiency, the latest enzyme of the heme biosynthetic pathway, leading to the accumulation of toxic protoporphyrin in the liver, bone marrow and spleen. We have previously shown that a successful gene therapy of a murine model of the disease was possible with lentiviral vectors even in the absence of preselection of corrected cells, but lethal irradiation of the recipient was necessary to obtain an efficient bone marrow engraftment. To overcome a preconditioning regimen, a selective growth advantage has to be conferred to the corrected cells. We have developed a novel bicistronic lentiviral vector that contains the human alkylating drug resistance mutant O(6)-methylguanine DNA methyltransferase (MGMT G156A) and FECH cDNAs. We tested their capacity to protect hematopoietic cell lines efficiently from alkylating drug toxicity and correct enzymatic deficiency. EPP lymphoblastoid (LB) cell lines, K562 and cord-blood-derived CD34(+) cells were transduced at a low multiplicity of infection (MOI) with the bicistronic constructs. Resistance to O(6)-benzylguanine (BG)/N,N'-bis(2-chloroethyl)-N-nitrosourea (BCNU) was clearly shown in transduced cells, leading to the survival and expansion of provirus-containing cells. Corrected EPP LB cells were selectively amplified, leading to complete restoration of enzymatic activity and the absence of protoporphyrin accumulation. This study demonstrates that a lentiviral vector including therapeutic and G156A MGMT genes followed by BG/BCNU exposure can lead to a full metabolic correction of deficient cells. This vector might form the basis of new EPP mouse gene therapy protocols without a preconditioning regimen followed by in vivo selection of corrected hematopoietic stem cells.

Escherichia coli FPG and human OGG1 reduce DNA damage and cytotoxicity by BCNU in human lung cells.

The pulmonary complications of 1,3-N,N'-bis(2-chloroethyl)-N-nitrosourea (BCNU) are among the most important dose-limiting factors of BCNU-containing cancer chemotherapeutic regimens. BCNU damages DNA of both cancer cells and normal cells. To increase the resistance of lung cells to BCNU, we employed gene transfer of Escherichia coli formamidopyrimidine-DNA glycosylase (FPG) and human 8-oxoguanine-DNA glycosylase (hOGG1) to A549 cells, a lung epithelial cell line, using a bicistronic retroviral vector, pSF91-RE, that encoded both FPG/hOGG1 and an enhanced green fluorescent protein. The transduced epithelial cells were sorted by flow cytometry, and expression of FPG/hOGG1 protein was determined by the level of FPG/hOGG1 RNA and enzyme activity. The single-cell gel electrophoresis (comet assay) measured DNA damage induced by BCNU. FPG/hOGG1-expressing A549 cells incubated with 40-500 microg/ml BCNU exhibited significantly less DNA damage than vector-transduced cells. In addition, FPG- and/or hOGG1-expressing cells incubated with 10-40 microg/ml BCNU showed at least a 25% increase in cell survival. Gene transfer of FPG/hOGG1 reduced BCNU-induced DNA damage and cytotoxicity of cultured lung cells and may suggest a new mechanism to reduce BCNU pulmonary toxicity.

Protection of committed murine haemopoietic progenitors against BCNU toxicity does not predict protection of primitive, multipotent spleen colony-forming cells - implications for chemoprotective gene therapy.

The effect of expression of an O6-benzylguanine (O6-beG)-resistant mutant (hATPA/GA) of human O6-alkylguanine-DNA alkyltransferase (ATase) on the in vivo toxicity and clastogenicity of the anti-tumour agent N,N'-bis(2-chloroethyl)-N-nitrosourea (BCNU) to murine bone marrow has been investigated. When compared with control animals, the bipotent granulocyte-macrophage colony-forming (GM-CFC) progenitor population of the hATPA/GA transduced mice were somewhat more resistant to BCNU (1.4-fold, P = 0.047) and this effect was more significant in the presence of the ATase inactivator O6-beG (3. 5-fold, P = 0.001). The polychromatic erythrocytes were also significantly protected against BCNU-induced clastogenicity both in the presence (P < 0.001) and absence of O6-beG (P < 0.05). The primitive, multipotent spleen colony-forming cells (CFU-S) in these animals also showed moderate (1.6-fold, P = 0.034) protection in the absence of O6-beG but in the presence of the inactivator they remained as sensitive to BCNU toxicity as those in the control animals (P = 0.133). This result contrasts with previous findings demonstrating significant hATPA/GA-mediated, O6-beG-resistant protection against the toxicity and clastogenicity of a number of O6-alkylating agents, including temozolomide, fotemustine and chlorozotocin. The possibility that our strategy for protective gene therapy may be highly agent and cell-type specific is unexpected and has possible implications for clinical trials of this approach using BCNU or related agents.

Cytotoxicity of Fluoroethylating Agents Is Potentiated by O6-Benzylguanine.

O6-Benzylguanine (BG) is a potent depleter of a repair enzyme O6-alkylguanine-DNA alkyltransferase. Pretreatment of cells with BG potentiates the cytotoxicity of chloroethylating anti-cancer agents. In this study we used HeLa S3 cells to examine the cytotoxic potentiation of 39 compounds after BG pretreatment. Compounds tested included anti-cancer agents and carcinogens, and among them only the cytotoxicity of methylating, chloroethylating and fluoroethylating agents was potentiated. This is the first description of the cytotoxic potentiation of fluoroethylating agents. Potentiation ratios were found to vary even among compounds possessing the same alkylating group. By pretreatment with 10 microM of BG, the cytotoxicity of methylating agents such as N-methyl-N-nitrosourea and streptozotocine was potentiated 3.7 and 9.4 fold, respectively. For chloroethylating agents, the potentiation ratios were 3.5 for N-chloroethyl-N-nitrosourea, 8.6 for N-[(4-amino-2-methyl-5-pyrimidinyl)methyl]-N'-(2-chloroethyl)-N'-nitroso urea (ACNU), 2.2 for N,N'-bis(2-chloroethyl)-N-nitrosourea (BCNU), 3.0 for N-(2-chloroethyl)-N'-cyclohexyl-N-nitrosourea (CCNU) and 5.2 for chloroethyl methanesulfonate. With respect to fluoroethylating agents, the potentiation ratios were 7.2 for N-fluoroethyl-N-nitrosourea, 2.0 for N-cyclohexyl-N'-fluoroethyl-N'-nitrosourea and 5.5 for fluoroethyl methanesulfonate. No effect was observed with the bromoethylating agent, N-bromoethyl-N-nitrosourea. There was no potentiation of the cytotoxicity of anti-cancer agents such as mitomycin C (MMC), cisplatin (CDDP), 5-fluorouracil (5FU), bleomycin (BLM), prednisolone, camptothecin, etoposide, methotrexate or vinblastine. A possible mechanism for the cytotoxic potentiation of the test compounds by BG pretreatment is discussed.

Increased killing of prostate, breast, colon, and lung tumor cells by the combination of inactivators of O6-alkylguanine-DNA alkyltransferase and N, N′- bis(2-chloroethyl)- N-nitrosourea

The ability of a number of compounds that act as inactivators of O 6-alkylguanine-DNA alkyltransferase (AGT) to sensitize human tumor cell lines to the effects of N, N′- bis(2-chloroethyl)- N-nitrosourea (BCNU) were examined. The AGT inactivators tested included O 6-benzylguanine (BG) and its 8-aza-, 8-bromo-, 8-methyl-, 8-oxo, and 8-amino-derivatives and O 6-[ p-(hydroxymethyl)benzyl]guanine. All of these compounds except the 8-amino-derivative were active in greatly increasing the killing of HT29 colon, Du 145 prostate, MCF-7 breast and A549 lung tumor cells by BCNU. Their activities were comparable to those of BG. Two pyrimidines, 2,4-diamino-6-benzyloxy-5-nitrosopyrimidine and 2,4-diamino-6-benzyloxy-5-nitropyrimidine, were found to be considerably more potent than BG in enhancing BCNU-induced cell killing. The addition of a steroid group to the 9-position of BG forming either O 6-benzyl-9-[3-oxo-4-androsten-17 β-yloxycarbonyl)methyl]guanine or O 6-benzyl-9-[3-oxo-5 α-androstan-17 β-yloxycarbonyl)methyl]guanine also produced compounds effective in enhancing the cytotoxicity of BCNU when added at 10 μM. These results indicate that a range of potent compounds with potentially different pharmacokinetics is available to test the hypothesis that inactivation of AGT overcomes the resistance of many tumor cells to nitrosoureas.

Studies on the formation of reactive intermediates from the antineoplastic agent N,N′Bis(2‐chloroethyl)‐N‐nitrosourea (BCNU) in vitro and in Vivo. Characterization of novel glutathione adducts by ionspray tandem mass spectrometry

AbstractIn a study designed to examine the nature of short‐lived, electrophilic intermediates liberated during decomposition of N,N′‐bis(2‐chloroethyl)‐N‐nitrosourea (BCNU) in vitro and also on administration of BCNU (140 μmol i.p.) to rats in vivo, both on‐line and off‐line LC/MS/MS techniques were employed to detect and characterize the corresponding glutathione (GSH) adducts present in incubation media and excreted into bile, respectively. In vitro, four GSH conjugates were formed and these were identified, on the basis of their product ion spectra, as products of S‐and N‐carbamoylation and alkylation reactions. Although the relative proportions of these in vitro adducts were found to depend on the molar ratios of GSH and BCNU, the major adduct under all conditions studied proved to be S‐(2‐chloroethylcarbamoyl)glutathione (SCG). Analysis of untreated bile samples by means of on‐line LC/MS/MS with constant neutral loss (129 u) and precursor ion (m/z 179) scanning techniques again led to the detection of four GSH conjugates, although only one of these (SCG) was common to the group of adducts identified in vitro. All of the GSH conjugates detected in bile represented products of S‐carbamoylation, indicating that the alkylating moiety released from BCNU undergoes reactions in vivo with nucleophiles other than GSH.

The advantageous use of hypoxic tumour cells in cancer therapy: identical chemosensitization by metronidazole and misonidazole at moderately elevated temperatures.

Chemosensitization by two nitroimidazoles (NIs), metronidazole (METRO) and misonidazole (MISO), of the anti-tumour effect of alkylating agents was studied at three different temperatures: room temperature (RT), 37 and 41.5 degrees C. Three alkylating agents, cyclophosphamide (CY), 1,3 bis(2-chloroethyl)-N-nitrosourea (BCNU) and melphalan (L-PAM) were tested when the tumours reached an average diameter of 4 mm. Tumours were 4th generation isotransplants of a spontaneous fibrosarcoma, FSa-II. Treatment at 37 or 41.5 degrees C was given by immersing the tumour-bearing foot for 60 min in a water bath set at these temperatures. The test agents were injected ip immediately before immersing the foot in the water bath, whereas METRO or MISO (2.5 mmol/kg) was given ip 30 min before the injection of a test agent. Following treatment the tumour growth (TG) time, i.e. the time required for one-half of treated tumours to reach 1000 mm3 after the treatment day, was studied. For CY, MISO was a better sensitizer than METRO at RT and 37 degrees C, but the magnitude of the chemosensitization by MISO and METRO became identical at 41.5 degrees C. Notably, the chemosensitization was substantially enhanced at 41.5 degrees C, whereas neither 41.5 degrees C-heat, NIs or combined NI and heat prolonged the TG time. Although no chemosensitization was observed for BCNU at RT, both METRO and MISO equally enhanced the effect of BCNU at 41.5 degrees C.(ABSTRACT TRUNCATED AT 250 WORDS)

A glutathione depletion selectively imposed on μglutathione S-transferase overproducing cells increases nitrogen mustard toxicity

Glutathione (GSH) contributes to the detoxification of anticancer drugs through the operation of specific glutathione S-transferases (GST) and innate, or acquired, overexpression of this enzyme family has been frequently observed in tumor cell lines. In the GMA32 line of Chinese hamster fibroblasts, we showed that GSH starvation produced by exposing cells to buthionine sulfoximine (BSO) increased the toxicity of chlorambucil and melphalan, but not that of N, N′-bis(2-chloroethyl)- N-nitrosourea (BCNU), cisplatine and doxorubicin. This indicates that efficient mechanisms of detoxification using GSH operate for chlorambucil and melphalan, but not for the other drugs in these cells. We then showed that GSH depletion could be selectively and transiently induced in the μGST overexpressing cell line derived from GMA32, HC474, by exposing cells to substrates specific to the overexpressed isozyme. Exposing cells to such a substrate, trans-stilbene oxide, does not alter the sensibility of GMA32 cells to melphalan and chlorambucil, but increases that of HC474 cells to these drugs, to an extent comparable to that obtained with BSO. This observation highlights the possibility of exploiting GST overexpression, a frequent feature of tumor cells, to selectively sensitize these undesirable cells to anticancer drugs.

Selective and irreversible inhibition of glutathione reductase in vitro by carbamate thioester conjugates of methyl isocyanate

Exposure of yeast glutathione reductase (GR) in vitro to S-( N-methylcarbamoyl)glutathione (SMG) and S-( N-methylcarbamoyl)cysteine (SMC), two carbamoylating metabolites of methyl isocyanate (MIC), led to a time-dependent, irreversible loss of enzyme activity (50–90%) over a period of 3 hr. The extent of inhibition was dependent upon the concentration of these carbamate thioester conjugates (0.1 to 1.0 mM) and on the presence of NADPH (100 μM). Omission of NADPH markedly attenuated the inhibitory effects of both SMG and SMC, while oxidized glutathione (GSSG), the natural substrate of the enzyme, protected against the inhibition. Parallel experiments with the antineoplastic drug N, N′-bis-(2-chloroethyl)- N-nitrosourea (BCNU), a carbamoylating agent which is known to inhibit GR selectively, gave results that were similar to those obtained with the above conjugates. When analogs of SMG and SMC labeled with 14C in the carbamoyl group were incubated with GR, radioactivity became bound covalently to the enzyme. These findings, together with the results of kinetic experiments on the release of GSH from SMG and cysteine from SMC, suggested that while both conjugates inhibit GR by carbamoylation of an active-site thiol(s), sMG exhibits a greater affinity for the active site than SMC. In contrast to the studies with GR, SMG and SMC failed to inhibit either glutathione- S-transferase (GST) or glutathione peroxidase (GPO) enzymes in vitro. It is concluded, therefore, that these conjugates most likely inhibit GR by carbamoylating free thiol groups in the active site of this enzyme, which are absent (or inaccessible) at the active-site of GST and GPO.

Induction of intracellular glutathione in alveolar type II pneumocytes following BCNU exposure.

N,N'-bis(2-chloroethyl)-N-nitro-sourea (BCNU) is a potent inhibitor of glutathione reductase (GSSG-Red) activity in both tissues and cells. We examined the effects of treating alveolar type II cells with BCNU and found that a marked decrease in cellular GSSG-Red activity occurred in these cells associated with a time-dependent increase in cellular glutathione (GSH) concentrations. The increase in GSH was not found to be related to changes in cellular gamma-glutamyl transpeptidase activity, gamma-glutamylcysteine synthetase activity, nor increased intracellular transport of cystine. When the BCNU-exposed cells were incubated with hydrogen peroxide to produce oxidant stress, the cells exhibited increased susceptibility to oxidant damage when compared with controls, despite the fact that cellular concentrations of GSH were markedly elevated.