Effects of nitrosoureas on human DNA polymerase activities from acute and chronic granulocytic leukemia cells.

In vitro DNA synthesis by isolated cytoplasmic DNA polymerases of human leukemic cells was found to be inhibited by 1,3-bis(2-chloroethyl)-1-nitrosourea, 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea and 1-(2-chloroethyl)-3-(trans-4-methyl-cyclohexyl)-1-nitrosourea. 2-Chloroethyl isocyanate and cyclohexyl isocyanate, the decomposition products of 1,3-bis(2-chloroethyl)-1-nitrosourea and 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea, respectively, are as effective as their parent nitrosoureas in inhibiting the enzyme activity. Preincubation studies indicated that these compounds inhibit DNA synthesis primarily by altering the enzyme DNA polymerases without significantly affecting the DNA template activities.     

Denitrosation of carcinostatic nitrosoureas by purified NADPH cytochrome P-450 reductase and rat liver microsomes to yield nitric oxide under anaerobic conditions

The nitrosoureas, CCNU (1-(2-chloroethyl)-3-(cyclohexyl)-1-nitrosourea) and BCNU (1,3-bis(2-chloroethyl)-1-nitrosourea) are representatives of a class of N-nitroso compounds which undergo denitrosation in the presence of NAD(P)H and deoxygenated hepatic microsomes from rats to yield nitric oxide (NO) and the denitrosated parent compound. Formation of NO during microsomal denitrosation of CCNU and BCNU was determined by three methods. With one procedure, NO was measured and concentration shown to increase over time in the head gas above microsomal incubations with BCNU. Two additional methods utilized NO binding to either ferrous cytochrome P-450 or hemoglobin to form distinct Soret maxima at 444 and 415 nm, respectively. Incubation of either BCNU or CCNU in the presence of NAD(P)H and deoxygenated microsomes resulted in the formation of identical cytochrome P-450 ferrous · NO optical difference spectra. Determination of the P-450 ferrous · NO extinction coefficient by the change in absorbance at 444 minus 500 nm allowed measurement of rates of denitrosation by monitoring the increase in absorbance at 444 nm. The rates of BCNU and CCNU denitrosation were determined to be 4.8 and 2.0 nmol NO/min/mg protein, respectively, for phenobarbital (PB) induced microsomes. For the purpose of comparison, the rate of [¹⁴C]CCNU (1-(2-[¹⁴C]chloroethyl)-3-(cyclohexyl)-1-nitrosourea turnover was examined by the isolation of [¹⁴C]CCU (1-(2-[¹⁴C] chloroethyl)-3-(cyclohexyl)-1-urea) from incubations that contained NADPH and deoxygenated PB-induced microsomes. These analyses showed stoichiometric amounts of NO and [¹⁴C]CCU being formed at a rate of 2.0 nmol/min/mg protein. Denitrosation catalysis by microsomes was enhanced by phenobarbital pretreatment and partially decreased by cytochrome P-450 inhibitors, SKF-525A, α-naphthoflavone (ANF), metyrapone, and CO, suggesting a cytochrome P-450-dependent denitrosation. However, in the presence of NADPH and purified NADPH cytochrome P-450 reductase reconstituted in dilauroylphosphatidylcholine, [¹⁴C]CCNU was shown to undergo denitrosation to [¹⁴C]CCU. Thus, NADPH cytochrome P-450 reductase could support denitrosation in the absence of cytochrome P-450.     

Anti-sickling activity of nitrosoureas

Incubation at physiological conditions of erythrocytes from a person bearing the sickle-cell trait with 1-methyl-1-nitrosourea, 1,3-bis-(2-chloroethyl)-1-nitrosourea, or 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea prevented sickling of the cells upon subsequent treatment of the cells with sodium metabisulfite. In these experiments these compounds were more effective than potassium cyanate as inhibitors of sickling. It is assumed that the inhibition results from the carbamoylation of the hemoglobin S by the isocyanic acid or the isocyanates derived from the nitrosoureas.     

Alteration of tyrosine aminotransferase multiple form patterns by antisickling agents and a decomposition product of antitumor agents

Antineoplastic agents of the nitrosourea class also have antisickling properties. It has been shown that compounds such as BCNU (1,3-bis-(2-chloroethyl)-1-nitrosourea) decompose under physiological conditions and give rise to substituted isocyanates. The latter are carbamylating agents and, presumably, through such activity have antisickling and antineoplastic activity. In fresh liver homogenates (at 0–4°C) cyanate and 2-chloroethyl isocyanate caused conversion of tyrosine aminotransferase form I to form III. This did not occur with the parent compound BCNU. However, liver homogenates prepared from rats pretreated with BCNU contained significantly more tyrosine aminotransferase form III than controls. Considering the effects that cyanate and isocyanate have on hemoglobin and tyrosine aminotransferase, it is possible that the antineoplastic activity of the nitrosoureas is due to carbamylation of specific regulators of cell division.     

Mechanism of action of 2-haloethylnitrosoureas on DNA and its relation to their antileukemic properties

1,3-Bis(2-chloroethyl)-1-nitrosourea (BCNU) and related 2-haloethylnitrosoureas covalently cross-link DNA under physiological conditions. The rate of the cross-linking increases with increasing pH in the range 4–10 and with the (G + C) content of natural DNAs. The reaction leads to stable interstrand cross-links by a two-step process and is strongly dependent on the 2-halogen in the nitrosourea where Cl ? Br > F ? I. Only one 2-haloethyl group is necessary for cross-linking, which is not observed when the halogen is replaced by -OH or -OCH₃. Promoting the acidity of the N³H group by appropriate aryl substitution increases the rate of cross-linking. The position of the halogen is critical since, while 1-(2-chloroethyl)-1-nitrosourea cross-links DNA efficiently, 1-(3-chloropropyl)-1-nitrosourea shows no reactivity. N⁴-(2-Chloroethyl)-1-methylcytosine hydrochloride, very similar to a suggested intermediate in the cross-linking process, alkylates PM2-CCC-DNA readily. The modes of aqueous decomposition of nitrosoureas as they apply to alkylation and cross-linking are discussed. The results are in accord with formation of a haloethonium ion which forms a nitrogen half mustard intermediate with a DNA base then completes the cross-link. A correlation exists between the extent of DNA cross-linking and activity of the nitrosoureas against L1210 leukemia. Based on the results of this work, a new nitrosourea is designed and synthesized which shows more efficient cross-linking.     

Activation of the renal cortical and hepatic guanylate cyclase-guanosine 3′,5′-monophosphate systems by nitrosoureas divalent cation requirements and relationship to thiol reactivity

Streptozotocin, 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) and N-methyl nitrosourea, compounds with both oncogenic and cytotoxic properties, increased guanylate cyclase activity in the 100 000 × g soluble fractions of rat renal cortex and liver 35- to 65-fold over basal values. Particulate enzyme activities of these tissues were increased 2- to 4-fold by a maximally effective concentration of the nitrosoureas. In the presence of the cyclic nucleotide phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine, maximally effective concentrations of these nitrosoureas increased cyclic GMP accumulation of hepatic and renal cortical slices to peak levels 7- to 10-fold over control in 30 min. By contrast, with the structurally related carcinogen N-methyl-N′-nitro-N-nitrosoguanidine (MNNG) peak increases occurred in 5–10 min and were 40- to 70-fold over control levels in renal cortex and liver, respectively. Unlike the Ca²⁺-dependent actions of cholinergic stimuli on cyclic GMP, the nitrosoureas and MNNG increased cyclic GMP in either the presence or absence of extracellular Ca²⁺. Moreover, while basal soluble guanylate cyclase of renal cortex was highly Mn²⁺-dependent and decreased 85% when either Mg²⁺ or Ca²⁺ was employed as sole divalent cation in reaction mixtures, the actions of nitrosoureas on enzyme activity were well expressed with either Mn²⁺ or Mg²⁺, but not with Ca²⁺, as sole divalent cation. Improved utilization of Mg²⁺ by guanylate cyclase in the presence of nitrosoureas would favor enhanced enzyme activity under cellular conditions where Mg²⁺ is abundant. In the presence of maximally stimulatory concentrations of streptozotocin or BCNU, high concentrations of Mg²⁺ or Mn²⁺ further increased soluble guanylate cyclase, suggesting important differences in metal and nitrosourea stimulation of enzyme activity.Preincubation of supernatant fractions with nitrosoureas plus dithiothreitol inhibited the action of the N-nitroso compounds to increase renal cortical guanylate cyclase. Glutathione and cysteine were also inhibitory, but less effective than dithiothreitol. Initial incubation of nitrosoureas with dithiothreitol in buffer alone similarly suppressed the subsequent action of the N-nitroso compounds on guanylate cyclase, and implicated direct chemical interactions. Prior incubation of renal cortical supernatant fractions with the SH blockers N-ethylmaleimide or maleimide significantly suppressed guanylate cyclase activation mediated by streptozotocin or BCNU. Direct drug interactions seemed unlikely, since effects of the inhibitors were optimally expressed by initial exposure of the supernatant fraction of tissue to the SH blockers and were not potentiated by a 30 min preincubation of the SH blockers and nitrosoureas in buffer alone.Thus, nitrosoureas activate and alter the metal requirements of soluble guanylate cyclase and increase cellular cyclic GMP in the presence or absence of extracellular Ca²⁺. Activation of soluble guanylate cyclase by nitrosoureas may involve an interaction of these agents with tissue SH groups, and possibly SH to SS transformation. Stimulation of the guanylate cyclase system by nitrosoureas could be related to the oncogenic actions of these agents.     

The effectiveness of the O(6)-alkylguanine-DNA alkyltransferase encoded by the ogt(ST) gene from S. typhimurium in protection against alkylating drugs, resistance to O(6)-benzylguanine and sensitisation to dibromoalkane genotoxicity

Here we demonstrate that the Ogt(ST) from Salmonella typhimurium is a highly efficient O(6)-alkylguanine-DNA alkyltransferase (AGT) in affording protection against antitumour chloroethylating drugs (1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) and 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea (CCNU)). In addition, Ogt(ST) is refractory to O(6)-benzylguanine (BG) inactivation and its expression provides only minor sensitisation to genotoxicity by environmental dibromoalkanes (DBE). No other of the assayed bacterial or human AGTs displayed such advantageous properties for chemoprotective gene therapy strategy. Our observations indicate that the Ogt(ST) AGT might be, under some circumstances, of potential use to improve cancer chemotherapy. At least, its properties may provide further insight into the design of human AGT variants that could be expressed in normal or tumour cells to provide either protection or ablation.     

Characteristics of reovirus-mediated chemoimmunotherapy of murine L1210 leukemia

Summary We have previously demonstrated the ability of reovirus to function synergistically with chemotherapy in the treatment of murine EL-4 lymphoma. This study characterizes this treatment regimen in the therapy of L1210 leukemia. Animals with an estimated tumor burden of 10⁷ cells were treated with 9 mg/kg 1,3-bis(2-chloroethyl)-1-nitrosourea. Reovirus type 3, which had been quantitated either by particles or plaque-forming units (pfu), was administered 48 h after chemotherapy. Complete remission of tumor was observed in 80% of the animals which received either 10¹¹ particles or 10⁹ pfu of reovirus. Cured animals were resistant to challenge with homologous tumor, but were susceptible to challenge with heterologous tumor. Reovirus undergoes limited replication at the tumor site, and virus-specific antibody appears only after disappearance of reovirus-infected cells and virus from the ascites fluid. Reovirus appears to function therapeutically by inducing a tumor-specific cytolytic immune response.     

Prediction of the relative in vitro sensitivity of 9L rat brain tumor cells to nitrosoureas by the sister chromatid exchange assay

In an earlier study we showed that there is a good correlation between sister chromatid exchange induction and cell kill in 9L cells treated with certain nitrosoureas. In the study reported here, we treated four 9L cell lines that have different sensitivities to chloroethylnitrosoureas with 1,3-bis (2-chloroethyl)-1-nitrosourea, chlorozotocin, and ethylnitrosourea and determined the number of sister chromatid exchanges induced. Cell lines that were most sensitive to the drugs with respect to cell kill were also most sensitive to induction of sister chromatid exchanges for a given drug, and the assay based on sister chromatid exchange is therefore predictive of the relative sensitivity of these cells to the drugs used.     

Modification of calmodulin on Lys-75 by carbamoylating nitrosoureas

This paper describes characterization of the reaction of calmodulin with a series of nitrosoureas which are capable of releasing amine-reactive isocyanates of varying hydrophobic character. The site of calcium-dependent carbamoylation on calmodulin by the antineoplastic agent 1-(2-chloroethyl)-3-(4-methylcyclohexyl)-1-nitrosourea (methyl CCNU) was determined to be Lys-75 as demonstrated using [ring-14C]methyl CCNU and sequence analysis of the sole labeled peptide obtained from tryptic digestion of reversed-phase high pressure liquid chromatography (HPLC)-purified radiolabeled calmodulin. CCNU, the 4-desmethylcyclohexyl derivative of methyl CCNU, and its reactive hydrolysis product, cyclohexyl isocyanate, were also determined to modify calmodulin in a similar manner and at the same site, as demonstrated by specific blockade of modification by the calmodulin antagonist calmidazolium. Nitrosoureas which release the less hydrophobic 4-hydroxy- and 4-carboxycyclohexyl isocyanates are unable to modify calmodulin at 25-fold higher concentrations than those required for modification with methyl CCNU, CCNU, or cyclohexyl isocyanate. With this monomodified Lys-75 derivative, purified to homogeneity by HPLC, differential effects of modification on the activation of bovine brain 3',5'-cyclic nucleotide phosphodiesterase (phosphodiesterase) and human erythrocyte Ca2+,Mg2+-ATPase were observed. Compared to the amounts of native calmodulin needed, phosphodiesterase required 7-fold higher amounts of this derivative to reach maximal activation, whereas the activation of the ATPase was unaffected. Clearly, different regions of calmodulin are responsible for the activation of phosphodiesterase and the ATPase. We conclude that Lys-75 is not essential for the function of calmodulin but is in a region of the molecule involved in interaction with phosphodiesterase as well as the binding of certain hydrophobic calmodulin antagonists.     

Reaction of 1,3-bis(2-chloroethyl)-1-nitrosourea with synthetic polynucleotides.

The antitumor agent BCNU (1,3-bis(2-chloroethyl)-1-nitrosourea) was incubated with poly(C) and poly(G) in aqueous solution at 37 degrees and pH 7 to produce approximately 0.33 and 0.07% substitution, respectively. Under the same conditions, there was relatively little reaction with poly(A) and poly(U). Poylnucleotides reacted with [14C]BCNU were digested by chemical and enzymatic methods, and the derivative nucleotides were isolated by column chromatography. There were identified by a combination of ultraviolet and mass spectroscopy as 3-(beta-hydroxyethyl)CMP, 3,N4-ethano-CMP, and 7-(beta-hydroxy-ethyl)GMP. This would indicate that BCNU generates active two carbon fragments, probably chloroethyl carbonium ions, which are free to react with nucleotides. The production of these substituted bases may be important to the mechanism of action of the therapeutic nitrosoureas since they would probably alter the function of any nucleic acid which contained them.     

Modulating effect of thiol-disulfide status on [14C]aminopyrine accumulation in the isolated parietal cell

Thiol-oxidizing agents were found to stimulate [14C] aminopyrine accumulation, a reliable index of acid secretory function of isolated canine parietal cells. Glutathione is the predominant intracellular free thiol; thus, its oxidation status largely determines the thiol-disulfide status of the cell by thiol-disulfide interchange reactions. Three agents which alter glutathione oxidation status by different mechanisms were applied to parietal cells in vitro to investigate whether enhanced formation of GSSG alters acid secretory function. The agents studied were diamide (which nonenzymatically oxidizes GSH to GSSG), tert-butyl hydroperoxide (an organic peroxide specifically reduced by glutathione peroxidase, thereby generating GSSG for GSH), and 1,3-bis(2-chloroethyl)-1-nitrosourea (an inhibitor of NADPH:GSSG reductase, which presumably allows the accumulation of GSSG). Each of these agents stimulated aminopyrine accumulation in a dose-dependent fashion. Simple depletion of GSH by diethyl maleate or 2-cyclohexene-1-one did not stimulate aminopyrine accumulation. Likewise, enhanced aminopyrine accumulation occurred at diamide concentrations which did not cause significant depletion of total cellular glutathione. The thiol-reducing agent, dithiothreitol, prevented enhanced aminopyrine accumulation by 1,3-bis(2-chloroethyl)-1-nitrosourea and tert-butyl hydroperoxide. These observations support the hypothesis that thiol-disulfide interchange reactions involving GSSG modulate the acid secretory function of the isolated parietal cell.     

Effect of treatment with dimethyl triazeno imidazole carboxamide (DTIC,NSC-45388) or 1,3-bis (2-chloroethyl)-1-nitrosourea (BCNU,NSC-409962) plus vincristine (NSC-67574) on lymphocyte reactivity of melanoma patients

Summary Fourteen patients with surgically incurable malignant melanoma treated with dimethyl triazeno imidazole carboxamide (DTIC) (2 mg/kg/day i.v. × 10) and 18 patients treated with the combination of 1,3-bis (2-chloroethyl)-1-nitrosourea (BCNU) (150 mg/m² i.v.) plus vincristine (2 mg/m² i.v.) on day 1 only, were tested for lymphocyte reactivity against melanoma cells before and after treatment. Neither chemotherapeutic regimen regularly affected lymphocyte reactivity, either in the presence of fetal calf serum or in the presence of the patients' autologous serum when tested 1 month after treatment. Three patients receiving repeated DTIC therapy and six patients receiving repeated BCNU plus vincristine therapy exhibited little variation in lymphocyte reactivity throughout the course of treatment.     

Alkylation and carbamoylation intermediates from the carcinostatic 1-(2-chloroethyl)-3-cyclohexyl -1-nitrosourea (CCNU)

Evidence is presented which shows that 1-(2-chloroethyl) -3-cyclohexyl-1-nitrosourea (CCNU) upon degradation provides a 2-chloroethyl alkylating intermediate, possibly 2-chloroethyl carbonium ion, and 2-chloroethanol. Thiol alkylation occurs $\text{in vivo}$ and a major urinary metabolite of CCNU is thiodiacetic acid. A rapid microsomal hydroxylation of the cyclohexyl ring occurs which yields varying ratios of at least five metabolites: cis or trans 2-hydroxy, trans- 3-hydroxy, cis-3-hydroxy, cis-4-hydroxy and trans-4- hydroxy-CCNU. $\text{In vivo}$ carbamoylation appears to not be due to cyclohexylisocyanate but to the various hydroxy-cyclohexylisocyanates which are formed from hydroxy CCNU metabolites.     

Role of O6-alkylguanine-DNA alkyltransferase in the resistance of mouse spermatogenic cells to O6-alkylating agents

The O(6)-alkylguanine-DNA alkyltransferase inactivator O(6)-benzylguanine was administered to BALB/c mice either alone or before exposure to 1,3-bis(2-chloroethyl)-1-nitrosourea to study the role of the DNA repair protein O(6)-alkylguanine-DNA alkyltransferase in the protection of the testis against anti-cancer O(6)-alkylating agents. Exposure of the mice to 1, 3-bis(2-chloroethyl)-1-nitrosourea or O(6)-benzylguanine alone did not produce any marked testicular toxicity at the times studied. Testicular O(6)-alkylguanine-DNA alkyltransferase concentrations were assayed between 0 and 240 min after O(6)-benzylguanine treatment and were shown to be > 95% depleted 15 min after treatment with O(6)-benzylguanine and remained at > 95% at all the times assayed. Histological examination, the reduction in testicular mass and the induction of spermatogenic cell apoptosis showed that this depletion significantly potentiated 1, 3-bis(2-chloroethyl)-1-nitrosourea-induced testicular damage after treatment. Major histological damage was apparent 42 days after treatment, demonstrating that the stem spermatogonia were significantly affected by the combination. These results demonstrate that O(6)-alkylguanine-DNA alkyltransferase plays a significant role in protecting the spermatogenic cells from damage caused by DNA alkylation and indicate that the observed toxicity may result from damage to stem spermatogonia.     

Relation between the changes in the structure and synthesis of DNA in the cells of mouse leukemia L1210 induced by 1-methyl-1-nitrosourea and 1,3-bis(2-chloroethyl)-1-nitrosourea

The damage of DNA structure and synthesis in murine leukemia L1210 cells upon single administration in therapeutic doses of antitumour agents of N-nitrosourea type, such as 1-methyl-1-nitrosourea (MNU) and 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) was studied. MNU and BCNU were characterized by stronger inhibitory effects on de novo DNA synthesis compared to additional pathway of DNA synthesis in leukemia L1210 cells in vivo. Centrifugation in alkaline sucrose density gradients of L1210 cell lysates has revealed persistent single-strand breaks and alkaline-labile sites in newly replicated DNA. Parental DNA structure was more stable to damaging drug effects than that of newly replicated DNA. The results are consistent with our previous data on the differences in the mechanisms of MNU and BCNU action and the absence of complete cross resistance between the drugs.     

Genetic effects of some new bifunctional and water-soluble analogs of the anti-cancer agent 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) in Saccharomyces cerevisiae.

A series of 1,1'-polymethylenebis-3-(2-chloroethyl)-3-nitrosoureas, 1-(omega-hydroxyalkyl)-3-(2-chloroethyl)-3-nitrosoureas, 1,1'(4-methyl-m-phenylenebis)-3-(2-chloroethyl)-3-nitrosourea, 2-[3-(2-chloroethyl)]-3-nitrosoureido-2-deoxy-D-glycopyranose (chlorozotocin and 1-(2-methanesulfonyloxyethyl)-3-(2-chloroethyl)-3-nitrosurea were examined for their genetic activities. BCNU was simultaneously tested as an established, clinically used reference compound. A diploid strain of Saccharomyces cerevisiae, heteroallelic at the gene loci ade2 and trp5, was used as a test system for the induction of mitotic gene conversion (intragenic recombination). All compounds showed strong genetic effects. In the series of aliphatic bifunctional nitrosoureas, 1,1'-ethylenebis-3-(2-chloroethyl)-3-nitrosourea (1) was the most active compound. The water-soluble derivatives, including chlorozotocin, displayed all genetic effects of the same order of magnitude. There was no correlation between genetic activity and chemotherapeutic potential of the test compounds.     

Involvement of FMN and phenobarbital cytochrome P-450 in stimulating a one-electron reductive denitrosation of 1-(2-chloroethyl)-3-(cyclohexyl)-1-nitrosourea catalyzed by NADPH-cytochrome P-450 reductase

Purified hepatic NADPH-cytochrome P-450 reductase, which was reconstituted with dilauroylphosphatidylcholine, catalyzed a one-electron reductive denitrosation of 1-(2-[14C]-chloroethyl)-3-(cyclohexyl)-1-nitrosourea ([14C]CCNU) to give 1-(2-[14C]-chloroethyl)-3-(cyclohexyl)urea at the expense of NADPH. Ambient oxygen or anoxic conditions did not alter the rates of [14C]CCNU denitrosation catalyzed by NADPH-cytochrome P-450 reductase with NADPH. Electron equivalents for reduction could be supplied by NADPH or sodium dithionite. However, the turnover number with NADPH was slightly greater than with sodium dithionite. Enzymatic denitrosation with sodium dithionite or NADPH was observed in anaerobic incubation mixtures which contained NADPH-cytochrome P-450 reductase with or without cytochrome P-450 purified from livers of phenobarbital (PB)-treated rats; PB cytochrome P-450 alone did not support catalysis. PB cytochrome P-450 stimulated reductase activity at molar concentrations approximately equal to or less than NADPH-cytochrome P-450 reductase concentration, but PB cytochrome P-450 concentrations greater than NADPH-cytochrome P-450 reductase inhibited catalytic denitrosation. Cytochrome c, FMN, and riboflavin demonstrated different degrees of stimulation of NADPH-cytochrome P-450 reductase-dependent denitrosation. Of the flavins tested, FMN demonstrated greater stimulation than riboflavin and FAD had no observable effect. A 3-fold stimulation by FMN was not observed in the absence of NADPH-cytochrome P-450 reductase. These studies provided evidence which establish NADPH-cytochrome P-450 reductase rather than PB cytochrome P-450 as the enzyme in the hepatic endoplasmic reticulum responsible for CCNU reductive metabolism.     

Inhibition of Thromboxane Synthase Activity Improves Glioblastoma Response to Alkylation Chemotherapy

Thromboxane synthase (TXSA), an enzyme of the arachidonic acid metabolism, is upregulated in human glial tumors and is involved in glioma progression. Here, we analyzed the in vitro and in vivo effects of pharmacological inhibition of TXSA activity on human glioblastoma cells. Furegrelate, a specific inhibitor of TXSA, significantly inhibited tumor growth in an orthotopic glioblastoma model by inducing proapoptotic, antiproliferative, and antiangiogenic effects. Inhibition of TXSA induced a proapoptotic disposition of glioma cells and increased the sensitivity to the chemotherapeutic agent 1,3-bis(2-chloroethyl)-1-nitrosourea, significantly prolonging the survival time of intracerebral glioma-bearing mice. Our data demonstrate that the targeted inhibition of TXSA activity improves the efficiency of conventional alkylation chemotherapy in vivo. Our study supports the role of TXSA activity for the progression of malignant glioma and the potential utility of its therapeutic modulation for glioma treatment.     

Characterization of DNA reactive and non-DNA reactive anticancer drugs by gene expression profiling

Gene expression profiling technology is expected to advance our understanding of genotoxic mechanisms involving direct or indirect interaction with DNA. We exposed human lymphoblastoid TK6 cells to 14 anticancer drugs (vincristine, paclitaxel, etoposide, daunorubicin, camptothecin, amsacrine, cytosine arabinoside, hydroxyurea, methotrexate, 5-fluorouracil, cisplatin, 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea (CCNU), 1,3-bis (2-chloroethyl)-1-nitrosourea (BCNU), and bleomycin) for 4-h and examined them immediately or after a 20-h recovery period. Cytotoxicity and genotoxicity, respectively, were evaluated by cell counting and by in vitro micronucleus assay at 24h. Effects on the cell cycle were determined by flow cytometry at 4 and 24h. Gene expression was profiled at both sampling times by using human Affymetrix U133A GeneChips (22K). Bioanalysis was done with Resolver/Rosetta software and an in-house annotation program. Cell cycle analysis and gene expression profiling allowed us to classify the drugs according to their mechanisms of action. The molecular signature is composed of 28 marker genes mainly involved in signal transduction and cell cycle pathways. Our results suggest that these marker genes could be used as a predictive model to classify genotoxins according to their direct or indirect interaction with DNA.