RESUMEN
A series of novel nitroheterocyclic phosphoramidates has been prepared, and the cytotoxicity of these compounds has been evaluated in clonogenic assays against B16, wild-type and cyclophosphamide-resistant MCF-7, and HT-29 cells under aerobic conditions and HT-29 cells under hypoxic conditions. All compounds were comparable in toxicity to wild-type and resistant MCF-7 cells and were also selectively toxic to HT-29 cells under hypoxic conditions (selectivity ratios 1.7 to >20). Analogues lacking the nitro group were not cytotoxic. Electron-withdrawing substituents increased cytotoxicity under aerobic conditions and thereby decreased hypoxic selectivity. In contrast, an electron-donating substituent markedly decreased both aerobic and hypoxic cytotoxicity but enhanced hypoxic selectivity. Chemical reduction of the nitro group resulted in rapid expulsion of the cytotoxic phosphoramide mustard. The most potent of these compounds show significant cytotoxicity under both aerobic and hypoxic conditions.
Asunto(s)
Antineoplásicos Alquilantes/síntesis química , Compuestos Organofosforados/síntesis química , Oxígeno/metabolismo , Animales , Antineoplásicos Alquilantes/química , Antineoplásicos Alquilantes/farmacología , Hipoxia de la Célula , Ensayos de Selección de Medicamentos Antitumorales , Células HT29 , Humanos , Melanoma Experimental , Ratones , Compuestos Organofosforados/química , Compuestos Organofosforados/farmacología , Células Tumorales CultivadasRESUMEN
Glutamate-L-cysteine ligase (GLCL [EC 6.3.2.2], also referred to as gamma-glutamylcysteine synthetase) catalyzes the rate-limiting reaction in the synthesis of the important cellular antioxidant glutathione. GLCL is a heterodimer consisting of a catalytic (GLCLC) and a regulatory (GLCLR) subunit. The structure of the human GLCLC subunit gene, GLCLC, which has been mapped to chromosome 6p12, spans 51.4 kb and consists of 16 exons separated by 15 introns.
Asunto(s)
Exones/genética , Glutamato-Cisteína Ligasa/genética , Intrones/genética , Secuencia de Bases , Clonación Molecular , Fibroblastos , Glutamato-Cisteína Ligasa/química , Humanos , MasculinoRESUMEN
gamma-Glutamylcysteine synthetase (GCS), the rate-limiting enzyme in the de novo synthesis of GSH, is a heterodimer, consisting of a catalytic (GCSh) and a regulatory subunit (GCSl). We previously demonstrated that the constitutive and beta-naphthoflavone (beta-NF)-induced expression of the GCSh gene is mediated by a distal antioxidant response element (ARE), ARE4, located 3.1 kb upstream of the transcriptional start site [Mulcahy, Wartman, Bailey and Gipp (1997) J. Biol. Chem. 272, 7445-7454]. ARE4 consists of a consensus ARE sequence (5'-GTGACTCAGCG-3') containing an embedded PMA-responsive element (TRE, underlined). The relative significance of the two overlapping response elements to constitutive and beta-NF-induced expression of the GCSh gene was determined by mutational analyses. The internal activator protein-1 (AP-1)-binding sequence mediated constitutive expression of promoter/reporter transgenes, but was not required for beta-NF responsiveness. In gel-shift experiments, the TRE was necessary for binding of proteins from nuclear extracts prepared from untreated HepG2 cells. In contrast, induction by beta-NF was dependent on an intact ARE sequence, particularly the terminal GC box of ARE4. The GC box of ARE4 was shown to be essential for both basal and beta-NF-induced expression of reporter constructs. This element also influenced binding of nuclear proteins to ARE4, specifically in extracts isolated from beta-NF-treated HepG2 cells. Because previous studies indicated that ARE4 may co-operate with a separate putative ARE, the role of the neighbouring sequence (ARE3), located 34 bases downstream of ARE4, was also evaluated. Mutation of this element within a GCSh promoter/reporter did not modify the basal or beta-NF-induced expression of the transgene, demonstrating that ARE3 does not influence the constitutive or beta-NF-induced expression of the GCSh gene.
Asunto(s)
Antioxidantes/farmacología , Regulación de la Expresión Génica/efectos de los fármacos , Glutamato-Cisteína Ligasa/genética , beta-naftoflavona/farmacología , Sitios de Unión/fisiología , Análisis Mutacional de ADN , Elementos de Facilitación Genéticos/genética , Genes Sobrepuestos , Humanos , Hígado/enzimología , Proteínas Nucleares/análisis , Oligodesoxirribonucleótidos/genética , Regiones Promotoras Genéticas/genética , ARN Mensajero/metabolismo , Acetato de Tetradecanoilforbol/farmacología , Factor de Transcripción AP-1/farmacología , Células Tumorales CultivadasRESUMEN
Glutathione (GSH) is an abundant cellular non-protein sulfhydryl that functions as an important protectant against reactive oxygen species and electrophiles, is involved in the detoxification of xenobiotics, and contributes to the maintenance of cellular redox balance. The rate-limiting enzyme in the de novo synthesis of glutathione is gamma-glutamylcysteine synthetase (GCS), a heterodimer consisting of heavy and light subunits expressing catalytic and regulatory functions, respectively. Exposure of HepG2 cells to beta-naphthoflavone (beta-NF) resulted in a time- and dose-dependent increase in the steady-state mRNA levels for both subunits. In order to identify sequences mediating the constitutive and induced expression of the heavy subunit gene, a series of deletion mutants created from the 5'-flanking region (-3802 to +465) were cloned into a luciferase reporter vector (pGL3-Basic) and transfected into HepG2 cells. Constitutive expression was maximally directed by sequences between -202 and +22 as well as by elements between -3802 to -2752. The former sequence contains a consensus TATA box. Increased luciferase expression following exposure to 10 microM beta-NF was only detected in cells transfected with a reporter vector containing the full-length -3802:+465 fragment. Hence, elements directing constitutive and induced expression of the GCS heavy subunit are present in the distal portion of the 5'-flanking region, between positions -3802 and -2752. Sequence analysis revealed the presence of several putative consensus response elements in this region, including two potential antioxidant response elements (ARE3 and ARE4), separated by 34 base pairs. When cloned into the thymidine kinase-luciferase vector, pT81-luciferase, and transfected into HepG2 cells, both ARE3 and ARE4 increased basal luciferase expression approximately 20-fold. When cloned in tandem in their native arrangement the increase in luciferase activity was in excess of 100-fold, suggesting a strong interaction between the two sequences. Luciferase expression was elevated in beta-NF-treated cells transfected with the ARE4-tk-luciferase vector and all DNA fragments containing ARE4. In contrast, ARE3 did not direct increased luciferase expression in response to beta-NF nor did it significantly modify the magnitude of induction directed by ARE4. The influence of the ARE4 oligonucleotide on constitutive and induced expression was eliminated by introduction of a single base mutation, converting the core ARE sequence in ARE4 from 5'-GTGACTCAGCG-3' to 5'-GGGACTCAGCG-3'. When introduced into the full-length -3802:+465 segment, the same single base mutation also eliminated both functions. Collectively the data indicate that the constitutive and beta-NF-induced expression of the human GCS heavy subunit gene is mediated by a distal ARE sequence containing an embedded tetradecanoylphorbol-13-acetate-responsive element.
Asunto(s)
Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Glutamato-Cisteína Ligasa/genética , beta-naftoflavona/farmacología , Antioxidantes , Secuencia de Bases , Análisis Mutacional de ADN , Humanos , Datos de Secuencia Molecular , Alineación de SecuenciaRESUMEN
Although glutathione (GSH) has long been implicated in resistance to certain common chemotherapeutic agents, including alkylating agents, platinum analogues, and doxorubicin, evidence establishing a direct role in the resistant phenotype has been lacking. We cotransfected COS cells with the cDNAs for the two subunits of gamma-glutamylcysteine synthetase (GCS), which catalyzes the rate-limiting step in the de novo synthesis of GSH and is itself up-regulated in some drug-resistant tumor cells. Transfection resulted in increased GCS activity and elevated GSH levels (up to 2.6-fold). Cotransfection with the two subunits greatly enhanced the synthetic efficiency of the heavy subunit. A direct correlation (P < 0.01) between intracellular GSH levels and the LD99 dose of melphalan was observed, signifying that elevation of the thiol secondary to GCS expression is sufficient to confer the resistance phenotype.
Asunto(s)
Antineoplásicos Alquilantes/farmacología , ADN Complementario/genética , Glutamato-Cisteína Ligasa/genética , Glutatión/biosíntesis , Melfalán/farmacología , Animales , Catálisis , Línea Celular , Resistencia a Antineoplásicos/fisiología , Humanos , Líquido Intracelular/metabolismo , Riñón/citología , Sustancias Macromoleculares , TransfecciónRESUMEN
We have cloned the human gamma-glutamylcysteine synthetase heavy subunit gene (GCSh) from a P1 library and isolated a 5.5kb fragment (P1-GCS5') from the 5'-end of the P1 clone. P1-GCS5' has been sequenced from -1460 to +547. Multiple transcription start sites were identified by primer extension and S1 nuclease protection. Two start sites were identified by primer extension analysis within 23 bp (+1 and +10) of a consensus TATAAAA box; all sequences were numbered relative to the 5'-most of these two sites. Two additional major start sites were identified at -106 and +398. This latter site was the most prominent of all the initiation sites. In addition to a TATA box, the promoter contains a CCAAT box at -125 and GC boxes up- and down-stream of the TATAAAA. In addition, the first few hundred base pairs of the sequence are highly GC-rich (approximately 75%). This sequence also contains several Sp-1 binding sites, a consensus AP-1 site and several AP-1-like binding sites, as well as putative AP-2 sites. A consensus metal responsive element (MRE) was identified at position +198. Sequence analysis also identified a putative core (5'-TGACnnnGCA-3') antioxidant response element (ARE) at -862 to -853. As is typical of other AREs, a second AP-1-like sequence is located adjacent to the core sequence. These results suggest that GCSh gene expression in response to oxidative challenge may be regulated through an antioxidant response element similar to those recently detected in the promoter region of several Phase II enzymes.
Asunto(s)
Antioxidantes/farmacología , Glutamato-Cisteína Ligasa/genética , Secuencias Reguladoras de Ácidos Nucleicos , Secuencia de Bases , Clonación Molecular , ADN Complementario , Humanos , Datos de Secuencia Molecular , Regiones Promotoras Genéticas , Transcripción GenéticaRESUMEN
We have cloned and sequenced a full length cDNA for the light subunit of human liver gamma-glutamylcysteine synthetase (GCS1). The GCS1 cDNA consists of 1628 bp, containing an open reading frame of 822 bp which encodes a protein of 274 amino acids having a calculated M(r) of 30,729 Daltons. Human liver GCS1 shares 91% and 96% sequence homology at the nucleotide and amino acid levels, respectively, with its rat counterpart (Huang, Anderson and Meister, J. Biol. Chem. 268:20578-20583, 1993). Transcripts of 1.4 and 4.1 kb in length were detected by Northern blot analysis of mRNA isolated from each of sixteen different human tissues. Relative expression of the two GCS1 RNA transcripts was highly tissue-dependent. High steady-state levels of the smaller transcript were detected in colon, while very high levels of both transcripts were found in skeletal muscle. Expression of mRNA transcripts for the GCS heavy subunit were likewise tissue-dependent, and did not necessarily correlate with the level of GCS1 transcripts in any tissue.
Asunto(s)
Glutamato-Cisteína Ligasa/biosíntesis , Hígado/enzimología , ARN Mensajero/metabolismo , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Clonación Molecular , ADN Complementario/biosíntesis , ADN Complementario/química , Femenino , Expresión Génica , Biblioteca de Genes , Humanos , Riñón/enzimología , Sustancias Macromoleculares , Masculino , Datos de Secuencia Molecular , Especificidad de Órganos , Reacción en Cadena de la Polimerasa , ARN Mensajero/análisis , Ratas , Valores de Referencia , Homología de Secuencia de Ácido NucleicoRESUMEN
To test the hypothesis that elevated expression of the glutathione (GSH) salvage enzyme, gamma-glutamyl transpeptidase (gamma-GT) can confer resistance to chemotherapeutic agents, the cDNA for human gamma-GT was introduced into human prostate carcinoma cells by calcium phosphate precipitation. The sensitivity of a stable clone expressing an 18-fold increase in gamma-GT activity to melphalan (L-PAM), cisplatinum and doxorubicin was compared to that of the parent cell line and a clone transfected with gamma-GT cDNA in the antisense orientation. Despite increased gamma-GT expression and the ability of intact cells to metabolize exogenous GSH, transfection did not result in increased intracellular GSH levels even when an exogenous source of GSH was provided. Furthermore, no change in sensitivity attributable to the transfection and increased expression of gamma-GT was detected with any of the three drugs. Our data indicate that an increase in gamma-GT expression, exceeding that typically associated with resistance phenotypes, is not sufficient to confer resistance to L-PMA, cisplatinum or doxorubicin in the absence of other alterations in GSH homeostasis.
Asunto(s)
Resistencia a Medicamentos , Neoplasias de la Próstata/enzimología , gamma-Glutamiltransferasa/metabolismo , Cisplatino/farmacología , Relación Dosis-Respuesta a Droga , Doxorrubicina/farmacología , Humanos , Masculino , Melfalán/farmacología , Neoplasias de la Próstata/genética , Transfección , Células Tumorales Cultivadas , gamma-Glutamiltransferasa/genéticaRESUMEN
Tumor cell resistance to many chemotherapeutic agents, including alkylating agents, cisplatin, and doxorubicin, is frequently associated with increased intracellular levels of the nonprotein sulfhydryl glutathione (GSH). Recent evidence has demonstrated that increased GSH levels can be accompanied by an increase in the activity of gamma-glutamylcysteine synthetase (GCS), which catalyzes the rate-limiting step in de novo synthesis of GSH, and by an increase in the steady state level of mRNA for the catalytic subunit of GCS. Using melphalan-resistant DU 145/M4.5 human prostate carcinoma cells, which express elevated GSH levels, GCS enzyme activity, and GCS mRNA levels, we sought to determine the mechanism(s) responsible for the increased GCS mRNA expression. As determined by Northern analyses and RNase protection assays, the steady state level of GCS message in the resistant cells was increased 10-20-fold, in comparison with the drug-sensitive parent DU 145 cells. No significant difference in gene copy number or evidence of rearrangement was detected in the resistant cell line by Southern analyses. The GCS-specific mRNA isolated from the resistant cells was less stable than that isolated from the drug-sensitive cells (half-lives of 6 hr and 9 hr, respectively), indicating that this difference does not contribute to the increased steady state levels in the resistant cells. Nuclear run-on experiments revealed that the GCS transcription rate in the DU 145/M4.5 cells was increased approximately 12-fold, in comparison with that detected in the DU 145 cells. This difference in transcription rate was comparable in magnitude to the difference in steady state mRNA levels detectable in the two cell populations. Similar correlations between steady state GCS mRNA levels and transcription rates were also observed in other DU 145 lines expressing intermediate degrees of resistance to melphalan and correspondingly intermediate GCS mRNA elevations. These data suggest that GCS expression is transcriptionally regulated in these melphalan-resistant tumor cells.
Asunto(s)
Regulación Enzimológica de la Expresión Génica/fisiología , Regulación Neoplásica de la Expresión Génica/fisiología , Glutamato-Cisteína Ligasa/biosíntesis , Melfalán/farmacología , Neoplasias de la Próstata/enzimología , Resistencia a Medicamentos/genética , Humanos , Masculino , Transcripción Genética/fisiología , Células Tumorales Cultivadas , Regulación hacia Arriba/fisiologíaRESUMEN
A series of novel nitrobenzyltetrakis(chloroethyl)phosphorodiamidates has been prepared, and its cytotoxicity has been evaluated against HT-29 cells under aerobic and hypoxic conditions and against murine bone marrow progenitor cells under aerobic conditions. All compounds were selectively toxic to HT-29 cells under hypoxic conditions, and the selectivity ratios varied from 1.6 to > 90. Analogs lacking either the nitro group or the tetrakis(chloroethyl) moiety were not cytotoxic, confirming that the presence of both nitro and incipient alkylating groups are essential for activity. Surprisingly, some analogs were far more toxic to bone marrow progenitors than to HT-29 cells under aerobic conditions, suggesting that other activation mechanisms must exist in these hematopoietic cells. Cytotoxicity increased with increasing depth in the HT-29 spheroid model, consistent with the preferential hypoxic toxicity of these compounds. Alkaline elution experiments showed a greater number of DNA interstrand cross-links under hypoxic compared to aerobic conditions. The extent of cross-linking in hypoxic cells was essentially identical to that produced by an equitoxic dose of melphalan, suggesting that the cytotoxicity of these compounds results from phosphorodiamidate release and alkylation of DNA.
Asunto(s)
Antineoplásicos/síntesis química , Hipoxia de la Célula , Nitrobencenos/síntesis química , Mostazas de Fosforamida/química , Aerobiosis , Alquilación , Animales , Antineoplásicos/farmacología , Células de la Médula Ósea , Supervivencia Celular/efectos de los fármacos , Neoplasias del Colon , Reactivos de Enlaces Cruzados , ADN/química , ADN/efectos de la radiación , Rayos gamma , Granulocitos/efectos de los fármacos , Células Madre Hematopoyéticas/efectos de los fármacos , Células Madre Hematopoyéticas/fisiología , Humanos , Macrófagos/efectos de los fármacos , Ratones , Nitrobencenos/farmacología , Oxígeno/administración & dosificación , Mostazas de Fosforamida/farmacología , Células Tumorales CultivadasRESUMEN
Levels of intracellular glutathione (GSH) and the GSH-related enzymes gamma-glutamylcysteine synthetase (gamma-GCS) and gamma-glutamyltranspeptidase (gamma-GT) were measured in the melphalan-resistant human multiple myeloma cell line 8226/LR-5 and were compared to those measured in the drug-sensitive 8226/S and doxorubicin-resistant 8226/Dox40 cell lines. Both GSH and gamma-GCS activity, the rate-limiting step in the de novo synthesis of GSH, were elevated by a factor of approximately 2 in the melphalan-resistant 8226/LR-5 cells relative to the other two lines. gamma-GT activity was not elevated significantly in the /LR-5 cells. Northern analysis with a probe specific for the large subunit of human liver gamma-GCS identified two bands (3.2 and 4.0 kb), both of which were increased by a factor of 2-3 in the 8226/LR-5 line. Levels of gamma-GCS mRNA expression were comparable in the /S and /Dox40 cell lines. Levels of gamma-GT mRNA were similar in the /S and /LR-5 lines but were reduced in the /Dox40 cells. These data suggest that the increased GSH levels associated with resistance to melphalan in the 8226/LR-5 myeloma cells is attributable to up-regulation of gamma-GCS. This observation is consistent with recent demonstrations of up-regulation of gamma-GCS in melphalan-resistant prostate carcinoma cells and cisplatinum-resistant ovarian carcinoma cells, suggesting that increased expression of gamma-GCS may be an important mediator of GSH-associated resistance mechanisms.
Asunto(s)
Glutamato-Cisteína Ligasa/fisiología , Glutatión/metabolismo , Melfalán/farmacología , Mieloma Múltiple/tratamiento farmacológico , Mieloma Múltiple/metabolismo , Regulación hacia Arriba/fisiología , Northern Blotting , Supervivencia Celular/efectos de los fármacos , Doxorrubicina/farmacología , Resistencia a Medicamentos/fisiología , Humanos , Mieloma Múltiple/enzimología , ARN/análisis , Células Tumorales Cultivadas , gamma-Glutamiltransferasa/fisiologíaRESUMEN
The biochemical and molecular basis for the elevation of glutathione (GSH) levels commonly detected in many drug-resistant cells has not been elucidated. In a series of L-phenylalanine mustard-resistant human prostate carcinoma cell lines (DU-145), resistance was associated with elevated GSH levels, increased activity of gamma-glutamylcysteine synthetase (GCS), the rate-limiting enzyme in GSH biosynthesis, and a marked increase in the steady-state levels of GCS-specific transcripts (4.0 and 3.2 kilobases). Loss of the resistant phenotype was accompanied by a reduction in GSH and a return of GCS activity and transcript levels to values comparable to those detected in the drug-sensitive parent cells. These data strongly implicate up-regulation of GCS activity as an important mechanism in the evolution of drug resistance associated with increased levels of intracellular GSH. The results further suggest that the ability to synthesize GSH may be more indicative of resistance than steady-state GSH levels per se.
Asunto(s)
Glutamato-Cisteína Ligasa/metabolismo , Glutatión/metabolismo , Melfalán/farmacología , Neoplasias de la Próstata/metabolismo , Resistencia a Medicamentos , Expresión Génica , Glutamato-Cisteína Ligasa/genética , Humanos , Técnicas In Vitro , Masculino , Neoplasias de la Próstata/genética , ARN Mensajero/genética , ARN Neoplásico/genética , Células Tumorales CultivadasRESUMEN
We have cloned and sequenced a full-length cDNA for human liver gamma-glutamylcysteine synthetase (GCS), the rate-limiting enzyme in glutathione biosynthesis. The cDNA consists of 2634 bp containing an open reading frame encoding a protein of 367 amino acids and having a calculated M(r) = 72,773. The nucleotide sequence of the cDNA for human liver GCS shares an 84% overall similarity with the composite rat GCS sequence deduced from three overlapping partial cDNAs (Yan and Meister, JBC 265: 1588-1593, 1990). The deduced amino acid sequences are 94% similar. Comparison of Northern blots of total RNA isolated from rat kidney or liver with that from human kidney revealed the GCS mRNA to be larger in the human tissue (approximately 4.0 kb vs. approximately 3.7 kb). (The sequence for the human liver GCS cDNA has been assigned accession number M90656 in GenBank/EMBL databases.
Asunto(s)
Glutamato-Cisteína Ligasa/genética , Hígado/enzimología , Secuencia de Aminoácidos , Secuencia de Bases , Clonación Molecular , ADN de Cadena Simple/genética , Biblioteca de Genes , Humanos , Datos de Secuencia Molecular , ARN Mensajero/análisis , Mapeo Restrictivo , Especificidad de la EspecieRESUMEN
Exposure of HT-29 colon carcinoma cells to 1-methyl-2-nitrosoimidazole (INO), a reductive metabolite of a model 2-nitroimidazole, induced concentration-dependent DNA damage detectable by conventional alkaline (single-strand breaks) and neutral (double-strand breaks) filter elution techniques. Elution of DNA from the filters under alkaline conditions was distinctly biphasic. No evidence of DNA damage was detected when cellular DNA was incubated directly with INO prior to filter elution. DNA damage was enhanced markedly in HT-29 cells incubated with buthionine sulfoximine to deplete intracellular glutathione levels prior to INO treatment. The biphasic shape of the elution profiles was not attributable to loss of labeled thymidine mononucleotides or to the formation of DNA-protein crosslinks. Rather, the data suggest the existence of two subpopulations of cells differing in sensitivity to the DNA-damaging effects of INO exposure. Based upon differential adherence, two populations of cells, differing with respect to the rate and extent of elution from the filters during alkaline elution assays, were detected, although they could not be purified sufficiently by this technique to permit biochemical characterization. The results suggest that the nitroso intermediate is either an active metabolite, or a proximate form of the ultimate DNA-reactive species, responsible for DNA damage in cells exposed to 2-nitroimidazoles under reducing conditions.
Asunto(s)
Daño del ADN , Imidazoles/farmacología , Nitroimidazoles/metabolismo , Compuestos Nitrosos/farmacología , Butionina Sulfoximina , Neoplasias del Colon , ADN/efectos de los fármacos , ADN de Neoplasias/aislamiento & purificación , Endopeptidasa K , Humanos , Concentración de Iones de Hidrógeno , Imidazoles/toxicidad , Metionina Sulfoximina/análogos & derivados , Nitroimidazoles/toxicidad , Compuestos Nitrosos/toxicidad , Oxidación-Reducción , Serina Endopeptidasas , Células Tumorales Cultivadas/efectos de los fármacos , Células Tumorales Cultivadas/efectos de la radiaciónRESUMEN
Chemosensitization of bifunctional alkylators by misonidazole (MISO) and related nitroimidazoles in vitro has been shown to require hypoxic exposures. Presumably, reductive metabolism of the nitroimidazole under hypoxic conditions results in generation of a chemosensitizing intermediate(s) in a manner analogous to that described for the hypoxic toxicity of these compounds. In an attempt to identify these intermediates, we examined the ability of reductive metabolites of a model 2-nitroimidazole compound, 1-methyl-2-nitroimidazole (INO2), to enhance the toxicity of melphalan (t-PAM) in HT-29 human colon cancer cells. INO2 was a modest chemosensitizing agent, enhancing L-PAM only under hypoxic conditions. The 2-electron reduction product, 1-methyl-2-nitrosoimidazole (INO), was a potent chemosensitizer, enhancing L-PAM toxicity at micromolar concentrations under either aerobic or hypoxic conditions. In contrast, the 4- and 6-electron reduction products, 1-methyl-2-[hydroxylamino]imidazole and 1-methyl-2-aminoimidazole, respectively, failed to modify cell kill by L-PAM even at millimolar concentration. These results suggest that nitrosoimidazoles may be the active chemosensitizing species generated upon the reductive metabolism of nitroimidazoles.
Asunto(s)
Melfalán/toxicidad , Nitroimidazoles/administración & dosificación , Butionina Sulfoximina , Supervivencia Celular/efectos de los fármacos , Sinergismo Farmacológico , Glutatión/metabolismo , Humanos , Técnicas In Vitro , Metionina Sulfoximina/análogos & derivados , Metionina Sulfoximina/farmacología , Nitroimidazoles/metabolismo , Oxidación-Reducción , Relación Estructura-Actividad , Células Tumorales CultivadasRESUMEN
A mixed-function compound (I-278) combining 2-nitroimidazole and chloroethylnitrosourea has been shown to be greater than 2-fold more toxic to hypoxic HeLa-MR cells than to cells similarly exposed under aerobic conditions, consistent with chemosensitization of nitrosourea toxicity by the 2-nitroimidazole Misonidazole (MISO). However, in the case of I-278, the enhancement resulted from micromolar concentrations of 2-nitroimidazole as opposed to the millimolar quantities required for a similar enhancement by MISO. These experiments provide evidence (1) that the enhanced hypoxic toxicity of I-278 is not attributable to additional, independent hypoxic cell killing by the nitroimidazole group and (2) that the interaction between the two functions under hypoxic conditions results in increased crosslink formation typical of chemosensitization. The data strongly suggest that the chemosensitizing efficiency of nitroimidazoles can be dramatically improved by covalent linkage to a chloroethylating species.
Asunto(s)
Misonidazol/farmacología , Nitroimidazoles/farmacología , Compuestos de Nitrosourea/farmacología , Supervivencia Celular/efectos de los fármacos , Reactivos de Enlaces Cruzados , Células HeLa , Humanos , Misonidazol/administración & dosificaciónRESUMEN
The biological effects of 1-methyl-2-nitrosoimidazole (INO), the 2 electron reduction product of biologically active 1-methyl-2-nitroimidazole, were examined in HT-29 human colon cancer cells by clonogenic assay and glutathione (GSH) determination. INO was very toxic towards HT-29 cells and was equally toxic under aerobic and hypoxic conditions. Cytotoxicity was highly dependent on cell concentration, decreasing as cell concentration increased. INO also resulted in an initial dose-dependent depletion of intracellular GSH which plateaued when the GSH content of INO-treated cells reached approximately 8% of control levels. As was the case for cytotoxicity, the magnitude of GSH depletion by any given INO dose was highly dependent on cell concentration, being greatest at low cell densities. Both toxicity and GSH depletion were more pronounced when cells were exposed in culture medium without the reducing agent, ascorbate. HT-29 cells preincubated with the GSH synthesis inhibitor, buthionine sulfoximine (BSO), to reduce GSH levels to approximately 10% of control levels were more sensitive to subsequent INO exposure. These data suggest that the nitroso- reduction product of 2-nitroimidazoles may be responsible for cytotoxicity and glutathione depletion associated with hypoxic exposure to 2-nitroimidazoles.
Asunto(s)
Glutatión/análisis , Nitroimidazoles/farmacología , Butionina Sulfoximina , Supervivencia Celular/efectos de los fármacos , Neoplasias del Colon/análisis , Neoplasias del Colon/patología , Humanos , Metionina Sulfoximina/análogos & derivados , Metionina Sulfoximina/farmacología , Células Tumorales Cultivadas/efectos de los fármacosRESUMEN
Thermal radiosensitization was studied in two human T-cell acute lymphoblastic leukemia cell lines (JM and MOLT3) with regard to heat-irradiation sequence and heating duration. In MOLT3 thermal radiosensitization was maximal when 43.5 degrees C hyperthermia immediately preceded or followed irradiation; at 41.5 degrees C, radiosensitization was maximal with hyperthermia immediately before or up to 3 h after irradiation. In JM, enhancement of radiation killing was unexpectedly maximal when 41.5 or 43.5 degrees C hyperthermia preceded irradiation by 2 to 4 h. Thermal radiosensitization increased exponentially with increasing duration of heating at 41.5 degrees C for at least 3 h in MOLT3. In contrast, in JM, radiosensitization increased exponentially for 1.6 h but additional heating (up to 3 h net heating) had no appreciable further effect on radiation killing. For JM, repair of single and double stranded DNA breaks was investigated using alkaline and neutral elution techniques to determine whether the unusual results regarding heat-irradiation sequencing were related to effects of heat on repair of DNA damage. These studies were unable to detect significant differences in repair of single or double stranded DNA breaks between unheated control cells and cells heated at 41.5 degrees C for 1 h ending 4 h before irradiation. The direct cytotoxicity of hyperthermia was also studied in both cell lines.
Asunto(s)
Supervivencia Celular/efectos de la radiación , Hipertermia Inducida , Células Tumorales Cultivadas/efectos de la radiación , Daño del ADN , Reparación del ADN , Relación Dosis-Respuesta en la Radiación , Rayos gamma , Humanos , Leucemia Linfoide , Tolerancia a Radiación , Linfocitos T , Factores de TiempoRESUMEN
Experiments were designed to determine whether heat treatment could sensitize nitrosourea-resistant human tumor cell lines expressing a repair system (O6-alkylguanine DNA alkyltransferase; Mer+) capable of removing monoadducts from the DNA of treated cells prior to the formation of lethal interstrand cross-links. Effects of temperatures compatible with systemic hyperthermia were of particular interest, and, consequently, the effect of 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea (CCNU) exposure in vitro for 4 h at 37 degrees C was compared with that for 1 h at 41 degrees C followed by 3 h at 37 degrees C. CCNU toxicity was significantly enhanced by heat treatment in the Mer+ HT-29 human colon carcinoma, and in HeLa-S3 and HeLa-CCL2 cell lines [thermal enhancement factor (ratio of CCNU doses required to reduce cell survival to 0.001 at 37 degrees C and 41 degrees C) = 1.3-1.4]. Pharmacokinetic studies indicated that the effect of heat treatment on CCNU toxicity was not attributable to exposure to increased concentrations of reactive species, nor was the enhancement due to a direct effect of heat and/or drug on alkyltransferase activity. A similar enhancement of CCNU toxicity was also observed in a Mer- line, HeLa-MR (thermal enhancement factor = 1.3). Heat-sequencing experiments clearly demonstrate that heat and CCNU must be administered concurrently. Alkaline elution experiments were designed to examine DNA-DNA cross-link formation in Mer+ and Mer- cells exposed to CCNU at 37 degrees C and 41 degrees C, but quantitation of cross-link formation was not possible owing to the persistence of single strand breaks in the DNA of drug-treated cells. Nevertheless, collectively the data indicate that thermal enhancement of CCNU toxicity is independent of effects on alkytransferase activity and indicate that hyperthermia could provide an effective strategy for improving the nitrosourea response of resistant Mer+ tumors.
Asunto(s)
Reparación del ADN , Calor , Lomustina/farmacología , Células Tumorales Cultivadas/efectos de los fármacos , ADN/metabolismo , Resistencia a Medicamentos , Humanos , Metiltransferasas/análisis , O(6)-Metilguanina-ADN MetiltransferasaRESUMEN
Experiments were designed to test the hypothesis that mild heat treatment would selectively increase misonidazole (MISO) chemopotentiation of CCNU toxicity in hypoxic versus aerobic cells in vitro and in tumours in vivo via an augmentation of nitroreduction. EMT-6 cells were exposed to CCNU +/- 1.0 mM MISO under aerobic or hypoxic conditions for 4 h either at a constant 37 degrees C or at 41 degrees C for the first hour followed by 37 degrees C for the remaining 3 h. Chemopotentiation was not observed under aerobic conditions and heat treatment did not modify CCNU toxicity. Co-incubation with MISO and CCNU under hypoxic conditions resulted in enhanced toxicity (i.e. chemopotentiation) with either incubation protocol; however, the magnitude of the enhancement was significantly larger (P less than 0.025) when 41 degrees C incubation was included. Systemic heat treatment produced a similar enhancement of chemopotentiation in KHT tumours in C3H/HeN mice treated with MISO (0.5 mg g-1) and whole body hyperthermia (41 degrees C, 1 h) prior to administration of CCNU (15 mg kg-1). Heating had no effect on CCNU response but doubled the median growth delay produced by the CCNU-MISO combination. Heat treatment did not enhance myelosuppression of the combination. Both the in vitro and in vivo data indicate that mild hyperthermia can selectively enhance the magnitude of MISO chemopotentiation.