RESUMEN
The paper describes a collection of datasets containing both LEGO brick renders and real photos. The datasets contain around 155,000 photos and nearly 1,500,000 renders. The renders aim to simulate real-life photos of LEGO bricks allowing faster creation of extensive datasets. The datasets are publicly available via the Gdansk University of Technology "Most Wiedzy" institutional repository. The source files of all tools used during the creation of the dataset were made publicly available via GitHub repositories. The images, both photos and the renders were annotated with the unique brick ID and category from the official LEGO catalog. The proposed datasets are stored in easy-to-read formats and are labeled via directory structure allowing easy manipulation and conversion of metadata to other formats.
RESUMEN
Numerous subclinical diseases in sheep occur in the periparturient period and involve inflammatory processes; therefore, determining markers, such as acute-phase proteins (APPs), can allow an early diagnosis. Therefore, the objective of the present study was to assess changes in the plasma concentration of APPs and cortisol in clinically healthy ewes in the periparturient period for use in future studies. At the same time, haematological parameters were monitored. We showed that plasma APPs and cortisol concentrations were significantly higher in pregnant ewes than before insemination. A gradual increase in the SAA concentration was observed from the 14th day before to the day of parturition, while Hp was reduced from 2 weeks before up to 2 weeks after delivery. A significant increase in the Fb concentration was detected from the 14th day before to the 1st week after delivery. The cortisol concentration did not undergo significant changes in the periparturient period. We found an increase in the SAA and Fb concentrations and decrease in Hp in the periparturient period. The direction of the change in APPs of healthy ewes in the current study may be related to their distinct regulatory mechanisms during pregnancy. The APPs are usually altered during infection, inflammation, neoplasia, stress and trauma; therefore, knowing their reference values could help lead to an early diagnosis of subclinical forms of some diseases and pregnancy complications in ewes. The haematological analysis showed that ewes in late pregnancy and postpartum compared to dry period were under metabolic stress related to pregnancy and lactation.
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Proteínas de Fase Aguda/análisis , Hidrocortisona/sangre , Periodo Posparto/fisiología , Oveja Doméstica/fisiología , Animales , Recuento de Células Sanguíneas/veterinaria , Femenino , Embarazo , Oveja Doméstica/sangreRESUMEN
The clinically used antitumor agent mitomycin C (MC) alkylates DNA upon reductive activation, forming six covalent DNA adducts in this process. This paper focuses on differential biological effects of individual adducts in various mammalian cell cultures, observed in the authors' laboratories. Evidence is reviewed that various adducts are capable of inducing different cell death pathways in cancer cells. This evidence is derived from a parallel study of MC and its derivatives 2,7-diaminomitosene (2,7-DAM) which is the main metabolite of MC and forms two monoadducts with DNA, and decarbamoyl mitomycin C (DMC), which alkylates and crosslinks DNA, predominantly with a chirality opposite to that of the DNA adducts of MC. Specifically, 2,7-DAM is not cytotoxic and does not activate the p53 pathway while MC and DMC are cytotoxic and able to activate the p53 pathway. DMC is more cytotoxic than MC and can also kill p53-deficient cells by inducing degradation of Checkpoint 1 protein, which is not seen with MC treatment of the p53-deficient cells. This difference in the cell death pathways activated by the MC and DMC is attributed to differential signaling by the DNA adducts of DMC. We hypothesize that the different chirality of the adduct-to-DNA linkage has a modulating influence on the choice of pathway. Future studies will be directed to elucidate mechanisms of MC- and DMC-DNA adduct signaling in a structure-dependent context.
RESUMEN
Mitomycin C induces both MC-mono-dG and cross-linked dG-adducts in vivo. Interstrand cross-linked (ICL) dG-MC-dG-DNA adducts can prevent strand separation. In Escherichia coli cells, UvrABC repairs ICL lesions that cause DNA bending. The mechanisms and consequences of NER of ICL dG-MC-dG lesions that do not induce DNA bending remain unclear. Using DNA fragments containing a MC-mono-dG or an ICL dG-MC-dG adduct, we found (i) UvrABC incises only at the strand containing MC-mono-dG adducts; (ii) UvrABC makes three types of incisions on an ICL dG-MC-dG adduct: type 1, a single 5' incision on 1 strand and a 3' incision on the other; type 2, dual incisions on 1 strand and a single incision on the other; and type 3, dual incisions on both strands; and (iii) the cutting kinetics of type 3 is significantly faster than type 1 and type 2, and all of 3 types of cutting result in producing DSB. We found that UvrA, UvrA+UvrB and UvrA+UvrB+UvrC bind to MC-modified DNA specifically, and we did not detect any UvrB- and UvrB+UvrC-DNA complexes. Our findings challenge the current UvrABC incision model. We propose that DSBs resulted from NER of ICL dG-MC-dG adducts contribute to MC antitumor activity and mutations.
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Aductos de ADN/metabolismo , Reparación del ADN , Endodesoxirribonucleasas/metabolismo , Proteínas de Escherichia coli/metabolismo , Mitomicina/metabolismo , Modelos Genéticos , Aductos de ADN/química , Roturas del ADN de Doble Cadena , Mitomicina/químicaRESUMEN
The mitomycin derivative 10-decarbamoyl mitomycin C (DMC) more rapidly activates a p53-independent cell death pathway than mitomycin C (MC). We recently documented that an increased proportion of mitosene1-beta-adduct formation occurs in human cells treated with DMC in comparison to those treated with MC. Here, we compare the cellular and molecular response of human cancer cells treated with MC and DMC. We find the increase in mitosene 1-beta-adduct formation correlates with a condensed nuclear morphology and increased cytotoxicity in human cancer cells with or without p53. DMC caused more DNA damage than MC in the nuclear and mitochondrial genomes. Checkpoint 1 protein (Chk1) was depleted following DMC, and the depletion of Chk1 by DMC was achieved through the ubiquitin proteasome pathway since chemical inhibition of the proteasome protected against Chk1 depletion. Gene silencing of Chk1 by siRNA increased the cytotoxicity of MC. DMC treatment caused a decrease in the level of total ubiquitinated proteins without increasing proteasome activity, suggesting that DMC mediated DNA adducts facilitate signal transduction to a pathway targeting cellular proteins for proteolysis. Thus, the mitosene-1-beta stereoisomeric DNA adducts produced by the DMC signal for a p53-independent mode of cell death correlated with reduced nuclear size, persistent DNA damage, increased ubiquitin proteolysis and reduced Chk1 protein.
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Aductos de ADN/química , Mitomicinas/química , Complejo de la Endopetidasa Proteasomal/metabolismo , Proteínas Quinasas/metabolismo , Proteína p53 Supresora de Tumor/metabolismo , Apoptosis , Línea Celular Tumoral , Quinasa 1 Reguladora del Ciclo Celular (Checkpoint 1) , Daño del ADN , Silenciador del Gen , Humanos , Mitomicina/toxicidad , Mitomicinas/toxicidad , Interferencia de ARN , ARN Interferente Pequeño/metabolismo , Proteína p53 Supresora de Tumor/genéticaRESUMEN
Fanconi anemia (FA) is characterized by cellular hypersensitivity to DNA crosslinking agents, but how the Fanconi pathway protects cells from DNA crosslinks and whether FA proteins act directly on crosslinks remain unclear. We developed a chromatin-IP-based strategy termed eChIP and detected association of multiple FA proteins with DNA crosslinks in vivo. Interdependence analyses revealed that crosslink-specific enrichment of various FA proteins is controlled by distinct mechanisms. BRCA-related FA proteins (BRCA2, FANCJ/BACH1, and FANCN/PALB2), but not FA core and I/D2 complexes, require replication for their crosslink association. FANCD2, but not FANCJ and FANCN, requires the FA core complex for its recruitment. FA core complex requires nucleotide excision repair proteins XPA and XPC for its association. Consistent with the distinct recruitment mechanism, recombination-independent crosslink repair was inversely affected in cells deficient of FANC-core versus BRCA-related FA proteins. Thus, FA proteins participate in distinct DNA damage response mechanisms governed by DNA replication status.
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Proteína BRCA2/metabolismo , Factores de Transcripción con Cremalleras de Leucina de Carácter Básico/metabolismo , Neoplasias de la Mama/genética , Daño del ADN , Replicación del ADN , Proteína del Grupo de Complementación D2 de la Anemia de Fanconi/metabolismo , Proteínas del Grupo de Complementación de la Anemia de Fanconi/metabolismo , Proteínas Nucleares/metabolismo , Proteínas Supresoras de Tumor/metabolismo , Proteínas Reguladoras de la Apoptosis , Proteína BRCA2/genética , Factores de Transcripción con Cremalleras de Leucina de Carácter Básico/genética , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Línea Celular Tumoral , Inmunoprecipitación de Cromatina/métodos , Reactivos de Enlaces Cruzados/farmacología , Proteínas de Unión al ADN/metabolismo , Proteína del Grupo de Complementación D2 de la Anemia de Fanconi/genética , Proteína del Grupo de Complementación N de la Anemia de Fanconi , Proteínas del Grupo de Complementación de la Anemia de Fanconi/genética , Femenino , Ficusina/farmacología , Regulación Neoplásica de la Expresión Génica , Humanos , Mutación , Proteínas Nucleares/genética , Plásmidos/metabolismo , Recombinación Genética , Factores de Tiempo , Proteínas Supresoras de Tumor/genética , Proteína de la Xerodermia Pigmentosa del Grupo A/metabolismoRESUMEN
The antitumor antibiotic and cancer chemotherapeutic agent mitomycin C (MC) alkylates and crosslinks DNA, forming six major MC-deoxyguanosine adducts of known structures in vitro and in vivo. Two of these adducts are derived from 2,7-diaminomitosene (2,7-DAM), a nontoxic reductive metabolite of MC formed in cells in situ. Several methods have been used for the analysis of MC-DNA adducts in the past; however, a need exists for a safer, more comprehensive and direct assay of the six-adduct complex. Development of an assay, based on mass spectrometry, is described. DNA from EMT6 mouse mammary tumor cells, Fanconi Anemia-A fibroblasts, normal human fibroblasts, and MCF-7 human breast cancer cells was isolated after MC or 10-decarbamoyl mitomycin C (DMC) treatment of the cells, digested to nucleosides, and submitted to liquid chromatography electrospray-tandem mass spectrometry. Two fragments of each parent ion were monitored ("multiple reaction monitoring"). Identification and quantitative analysis were based on a standard mixture of six adducts, the preparation of which is described here in detail. The lower limit of detection of adducts is estimated as 0.25 pmol. Three initial applications of this method are reported as follows: (i) differential kinetics of adduct repair in EMT6 cells, (ii) analysis of adducts in MC- or DMC-treated Fanconi Anemia cells, and (iii) comparison of the adducts generated by treatment of MCF-7 breast cancer cells with MC and DMC. Notable results are the following: Repair removal of the DNA interstrand cross-link and of the two adducts of 2,7-DAM is relatively slow; both MC and DMC generate DNA interstrand cross-links in human fibroblasts, Fanconi Anemia-A fibroblasts, and MCF-7 cells as well as EMT6 cells; and DMC shows a stereochemical preference of linkage to the guanine-2-amino group opposite from that of MC.
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Reactivos de Enlaces Cruzados/farmacología , Aductos de ADN/biosíntesis , Mitomicina/farmacología , Mitomicinas/farmacología , Espectrometría de Masa por Ionización de Electrospray/métodos , Espectrometría de Masas en Tándem/métodos , Animales , Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/genética , Neoplasias de la Mama/metabolismo , Línea Celular Tumoral , Cromatografía Líquida de Alta Presión , Reactivos de Enlaces Cruzados/química , Reactivos de Enlaces Cruzados/metabolismo , Aductos de ADN/química , Daño del ADN , Reparación del ADN , ADN de Neoplasias/química , ADN de Neoplasias/efectos de los fármacos , Fibroblastos/efectos de los fármacos , Fibroblastos/metabolismo , Humanos , Neoplasias Mamarias Animales/tratamiento farmacológico , Neoplasias Mamarias Animales/genética , Neoplasias Mamarias Animales/metabolismo , Ratones , Mitomicina/química , Mitomicina/metabolismo , Mitomicinas/química , Mitomicinas/metabolismoRESUMEN
The cancer chemotherapeutic agent mitomycin C (MC) alkylates and cross-links DNA monofunctionally and bifunctionally in vivo and in vitro, forming six major MC-deoxyguanosine adducts of known structures. The synthesis of one of the monoadducts (8) by the postoligomerization method was accomplished both on the nucleoside and oligonucleotide levels, the latter resulting in the site-specific placement of 8 in a 12-mer oligodeoxyribonucleotide 26. This is the first application of this method to the synthesis of a DNA adduct of a complex natural product. Preparation of the requisite selectively protected triaminomitosenes 14 and 24 commenced with removal of the 10-carbamoyl group from MC, followed by reductive conversion to 10-decarbamoyl-2,7-diaminomitosene 10. This substance was transformed to 14 or 24 in several steps. Both were successfully coupled to the 2-fluoro-O(6)-(2-trimethylsilylethyl)deoxyinosine residue of the 12-mer oligonucleotide. The N(2)-phenylacetyl protecting group of 14 after its coupling to the 12-mer oligonucleotide could not be removed by penicillinamidase as expected. Nevertheless, the Teoc protecting group of 24 after coupling to the 12-mer oligonucleotide was removed by treatment with ZnBr2 to give the adducted oligonucleotide 26. However, phenylacetyl group removal was successful on the nucleoside-level synthesis of adduct 8. Proof of the structure of the synthetic nucleoside adduct included HPLC coelution and identical spectral properties with a natural sample, and (1)H NMR. Structure proof of the adducted oligonucleotide 26 was provided by enzymatic digestion to nucleosides and authentic adduct 8, as well as MS and MS/MS analysis.
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Aductos de ADN/síntesis química , Mitomicina/síntesis química , Oligodesoxirribonucleótidos/síntesis química , Cromatografía Líquida de Alta Presión , Dicroismo Circular , Aductos de ADN/química , Inosina/análogos & derivados , Inosina/química , Mitomicina/química , Mitomicinas/síntesis química , Mitomicinas/química , Nucleósidos/síntesis química , Nucleósidos/química , Oligodesoxirribonucleótidos/química , Espectrofotometría Ultravioleta , Espectrometría de Masas en Tándem , Compuestos de Trimetilsililo/químicaRESUMEN
10-Decarbamoyl-mitomycin C (DMC), a mitomycin C (MC) derivative, generates an array of DNA monoadducts and interstrand cross-links stereoisomeric to those that are generated by MC. DMC was previously shown in our laboratory to exceed the cytotoxicity of MC in a human leukemia cell line that lacks a functional p53 pathway (K562). However, the molecular signal transduction pathway activated by DMCDNA adducts has not been investigated. In this study, we have compared molecular targets associated with signaling pathways activated by DMC and MC in several human cancer cell lines. In cell lines lacking wild-type p53, DMC was reproducibly more cytotoxic than MC, but it generated barely detectable signal transduction markers associated with apoptotic death. Strikingly, DMCs increased cytotoxicity was not associated with an increase in DNA double-strand breaks but was associated with early poly(ADP-ribose) polymerase (PARP) activation and Chk1 kinase depletion. Alkylating agents can induce increased PARP activity associated with programmed necrosis, and the biological activity of DMC in p53-null cell lines fits this paradigm. In cell lines with a functional p53 pathway, both MC and DMC induced apoptosis. In the presence of p53, both MC and DMC activate procaspases; however, the spectrum of procaspases involved differs for the two drugs, as does induction of p73. These studies suggest that in the absence of p53, signaling to molecular targets in cell death can shift in response to different DNA adduct structures to induce non-apoptotic cell death.
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Aductos de ADN/fisiología , Mitomicinas/farmacología , Transducción de Señal/fisiología , Proteína p53 Supresora de Tumor/fisiología , Animales , Muerte Celular/efectos de los fármacos , Muerte Celular/fisiología , Línea Celular Tumoral , Aductos de ADN/genética , Humanos , Ratones , Transducción de Señal/efectos de los fármacosRESUMEN
Mitomycin C (MC) is a cytotoxic and mutagenic antitumor agent that alkylates and cross-links DNA. These effects are dependent on reductive bioactivation of MC. 2,7-Diaminomitosene (2,7-DAM) is the major metabolite of MC in tumor cells, generated by the reduction of MC. 2,7-DAM alkylates DNA in the cell in situ, forming an adduct at the N7 position of 2'-deoxyguanosine (2,7-DAM-dG-N7). To determine the biological effects of this adduct, we have synthesized an oligonucleotide containing a single 2,7-DAM-dG-N7 adduct and inserted it into an M13 bacteriophage genome. Replication of this construct in repair-competent Escherichia coli showed that the adduct was only weakly toxic and generated approximately 50% progeny as compared to control. No mutant was isolated after analysis of more than 4000 progeny phages from SOS-induced or uninduced host cells; therefore, we estimate that the mutation frequency of 2,7-DAM-dG-N7 was less than 2 x 10(-4) in E. coli. Subsequently, to determine if this adduct might be mutagenic in mammalian cells, it was incorporated into a single-stranded shuttle phagemid vector, pMS2, and replicated in simian kidney (COS-7) cells. Analysis of the progeny showed that mutational frequency of a site specific 2,7-DAM-dG-N7 was not higher than the spontaneous mutation frequency in simian kidney cells. In parallel experiments in cell free systems, template oligonucleotides containing a single 2,7-DAM-dG-N7 adduct directed selective incorporation of cytosine in the 5'-32P-labeled primer strands opposite the adducted guanine, catalyzed by Klenow (exo-) DNA polymerase. The adducted templates also supported full extension of primer strands by Klenow (exo-) and T7 (exo-) DNA polymerases and partial extension by DNA polymerase eta. The innocuous behavior of the 2,7-DAM-dG-N7 monoadduct in vivo and in vitro is in sharp contrast to that of the toxic MC-dG-N2 monoadduct reported earlier.
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Aductos de ADN/química , Daño del ADN , Mitomicina/química , Mitomicinas/química , Animales , Células COS , ADN/efectos de los fármacos , Escherichia coli/química , Escherichia coli/efectos de los fármacos , Mitomicinas/síntesis química , Mitomicinas/farmacología , Estructura Molecular , Pruebas de Mutagenicidad , Oligonucleótidos/síntesis química , Oligonucleótidos/química , Oligonucleótidos/farmacologíaRESUMEN
Overexpression of endoplasmic reticulum-localized NADPH: cytochrome c (P450) reductase (NPR) in Chinese hamster ovary cells increases the hypoxic/aerobic differential toxicity of the mitomycins. Because considerable evidence indicates that DNA cross-links are the major cytotoxic lesions generated by the mitomycins, we proposed that bioactivation of the mitomycins in the nucleus close to the DNA target would influence the cytotoxicity of these drugs. The simian virus 40 large T antigen nuclear localization signal was fused to the amino-terminal end of a human NPR protein that lacked its membrane anchor sequence. Immunofluorescent imaging of transfected cell lines expressing the fusion protein confirmed the nuclear location of the enzyme. Regardless of the oxygenation state of the cell, mitomycin C (MC) cytotoxicity was enhanced in cells with overexpressed NPR localized to the nuclear compartment compared with cells overexpressing an endoplasmic reticulum localized enzyme. Enhanced cytotoxicity in cells treated under hypoxic conditions correlated with increases in genomic DNA alkylations, with more MC-DNA adducts being formed when the enzyme was expressed closer to its DNA target. No change was observed in the hypoxic/aerobic differential toxicity as a function of enzyme localization. These findings indicate that drug efficacy is increased when the subcellular site of drug activation corresponds to its site of action.
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Núcleo Celular/efectos de los fármacos , Núcleo Celular/enzimología , Mitomicina/toxicidad , NADPH-Ferrihemoproteína Reductasa/metabolismo , Transporte Activo de Núcleo Celular/efectos de los fármacos , Transporte Activo de Núcleo Celular/fisiología , Animales , Células CHO , Cricetinae , Sinergismo Farmacológico , NADPH-Ferrihemoproteína Reductasa/toxicidadRESUMEN
The three dimers 3, 4, and 5 of mitomycin C (MC), a natural antibiotic and cancer chemotherapeutic agent, were synthesized in which two MC molecules were linked with -(CH(2))(4)-, -(CH(2))(12)-, and -(CH(2))(3)N(CH(3))(CH(2))(3)- tethers, respectively. The dimeric mitomycins were designed to react as polyfunctional DNA alkylators, generating novel types of DNA damage. To test this design, their in vitro DNA alkylating and interstrand cross-linking (ICL) activities were studied in direct comparison with MC, which is itself an ICL agent. Evidence is presented that 3-5 multifunctionally alkylate and cross-link extracellular DNA and form DNA ICLs more efficiently than MC. Reductive activation, required for these activities, is catalyzed by the same reductases and chemical reductants that activate MC. Dimer 5, but not MC, cross-linked DNA under activation by low pH also. Sequence specificities of cross-linking of a 162-bp DNA fragment (tyrT DNA) by MC, 3, and 5 were determined using DPAGE. The dimers and MC cross-linked DNA with the same apparent CpG sequence specificity, but 5 exhibited much greater cross-linking efficacy than MC. Greatly enhanced regioselectivity of cross-linking to G.C rich regions by 5 relative to MC was observed, for which a mechanism unique to dimeric MCs is proposed. Covalent dG adducts of 5 with DNA were isolated and characterized by their UV and mass spectra. Tri- and tetrafunctional DNA adducts of 5 were detected. Although the dimers were generally less cytotoxic than MC, dimer 5 was highly and uniformly cytotoxic to all 60 human tumor cell cultures of the NCI screen. Its cytotoxicity to EMT6 tumor cells was enhanced under hypoxic conditions. These findings together verify the expected features of the MC dimers and warrant further study of the biological effects of dimer 5.
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Antineoplásicos Alquilantes/síntesis química , Reactivos de Enlaces Cruzados/síntesis química , ADN/química , Mitomicinas/síntesis química , Animales , Antineoplásicos Alquilantes/química , Antineoplásicos Alquilantes/farmacología , Catálisis , Reactivos de Enlaces Cruzados/química , Reactivos de Enlaces Cruzados/farmacología , Dimerización , Ensayos de Selección de Medicamentos Antitumorales , Electroforesis en Gel de Poliacrilamida , Humanos , Espectrometría de Masas , Ratones , Micrococcus luteus , Mitomicina/síntesis química , Mitomicina/química , Mitomicina/farmacología , Mitomicinas/química , Mitomicinas/farmacología , Oligonucleótidos/química , Oxidación-Reducción , Espectrofotometría Ultravioleta , EstereoisomerismoRESUMEN
The effects of the subcellular localization of overexpressed bioreductive enzyme NAD(P)H:quinone oxidoreductase 1 (NQO1) on the activity of the antineoplastic agent mitomycin C (MC) under aerobic and hypoxic conditions were examined. Chinese hamster ovary (CHO-K1/dhfr(-)) cells were transfected with NQO1 cDNA to produce cells that overexpressed NQO1 activity in the nucleus (148-fold) or the cytosol (163-fold) over the constitutive level of the enzyme in parental cells. Subcellular localization of the enzyme was confirmed using antibody-assisted immunofluorescence. Nuclear localization of transfected NQO1 activity increased the cytotoxicity of MC over that produced by overexpression in the cytosol under both aerobic and hypoxic conditions, with greater cytotoxicity being produced under hypoxia. The greater cytotoxicity of nuclear localized NQO1 was not attributable to greater metabolic activation of MC but instead was the result of activation of the drug in close proximity to its target, nuclear DNA. A positive relationship existed between the degree of MC-induced cytotoxicity and the number of MC-DNA adducts produced. The findings indicate that activation of MC proximal to nuclear DNA by the nuclear localization of transfected NQO1 increases the cytotoxic effects of MC regardless of the degree of oxygenation and support the concept that the mechanism of action of MC involves alkylation of DNA.
Asunto(s)
Mitomicina/toxicidad , NAD(P)H Deshidrogenasa (Quinona)/metabolismo , Aerobiosis , Animales , Secuencia de Bases , Biotransformación , Células CHO , Hipoxia de la Célula , Núcleo Celular/enzimología , Supervivencia Celular/efectos de los fármacos , Cricetinae , Citosol/enzimología , Aductos de ADN/metabolismo , ADN Complementario/genética , Expresión Génica , Cinética , Mitomicina/farmacocinética , NAD(P)H Deshidrogenasa (Quinona)/genética , Ratas , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , TransfecciónRESUMEN
NADH:cytochrome b(5) reductase (FpD) is an enzyme capable of converting the prodrug mitomycin C (MC) into a DNA alkylating agent via reduction of its quininone moiety. In this study, Chinese hamster ovary (CHO) cells were transfected with a cDNA encoding rat FpD. Despite the demonstrated ability of this enzyme to reduce MC in vitro, a modest 5-fold level of overexpression of FpD activity in CHO cells did not increase the cytotoxicity of the drug over that seen with the parental cell line under either aerobic or hypoxic conditions. When the enzyme, which is predominantly localized in the mitochondria, was instead directed to the nucleus of cells by the fusion of the SV40 large T antigen nuclear localization signal sequence to the amino terminus of an FpD gene that lacked the membrane anchor domain, drug sensitivity was significantly enhanced at all concentrations of MC examined (2-10 microm) under both aerobic and hypoxic conditions, with greater cell kill occurring under hypoxia. The marked increase in drug sensitivity under hypoxia at 10 microm MC corresponded to a measurable increase in total MC-DNA adducts at the same concentration. The results indicate that the cytotoxicity of MC is modulated by the subcellular location of FpD, with greater cell kill occurring when bioactivation occurs in the proximity of its target, nuclear DNA.
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Antibióticos Antineoplásicos/farmacología , Reductasas del Citocromo/genética , Aductos de ADN/genética , Mitomicina/farmacología , Animales , Células CHO , Muerte Celular/efectos de los fármacos , Núcleo Celular/metabolismo , Cricetinae , Reductasas del Citocromo/biosíntesis , Citocromo-B(5) Reductasa , Aductos de ADN/efectos de los fármacos , Resistencia a Medicamentos/genética , Regulación Enzimológica de la Expresión Génica , RatasRESUMEN
Mitomycin C (MC), a cytotoxic anticancer drug and bifunctional DNA DNA alkylating agent, induces cross-linking of the complementary strands of DNA. The DNA interstrand cross-links (ICLs) are thought to be the critical cytotoxic lesions produced by MC. Decarbamoyl mitomycin C (DMC) has been regarded as a monofunctional mitomycin, incapable of causing ICLs. Paradoxically, DMC is slightly more toxic than MC to hypoxic EMT6 mouse mammary tumor cells as well as to CHO cells. To resolve this paradox, EMT6 cells were treated with MC or DMC under hypoxia at equimolar concentrations and the resulting DNA adducts were analyzed using HPLC and UV detection. MC treatment generated both intrastrand and interstrand cross-link adducts and four monoadducts, as shown previously. DMC generated two stereoisomeric monoadducts and two stereoisomeric ICL adducts, all of which were structurally characterized; one was identical with that formed with MC, the other was new and unique to DMC. Overall, adduct frequencies were strikingly higher (20-30-fold) with DMC than with MC. Although DMC monoadducts greatly exceeded DMC cross-link adducts ( approximately 10:1 ratio), the latter were equal or higher in number than the cross-link adducts from MC. DMC displayed a much higher monoadduct:cross-link ratio than MC. The similar cytotoxicities of the two drug show a correlation with their similar DNA cross-link adduct frequencies, but not with their total adduct or monoadduct frequencies. This provides specific experimental evidence that the ICLs rather than the monoadducts are critical factors in the cell death induced by MC. In vitro, overall alkylation of calf thymus DNA by DMC was much less efficient than by MC. Nevertheless, ICLs formed with DMC were clearly detectable. The chemical pathway of the cross-linking was shown to be analogous to that occurring with MC. These results also suggest that the differential sensitivity of Fanconi's Anemia cells to MC and DMC is related to factors other than a selective defect in cross-link repair.
Asunto(s)
Reactivos de Enlaces Cruzados/toxicidad , Aductos de ADN/toxicidad , Mitomicina/toxicidad , Mitomicinas/toxicidad , Alquilación/efectos de los fármacos , Animales , Antibióticos Antineoplásicos/toxicidad , Antineoplásicos Alquilantes/toxicidad , Neoplasias de la Mama/metabolismo , Bovinos , Supervivencia Celular/efectos de los fármacos , Aductos de ADN/aislamiento & purificación , Aductos de ADN/ultraestructura , Daño del ADN , Hipoxia , Ratones , Relación Estructura-Actividad , Timo/citología , Células Tumorales CultivadasRESUMEN
Mitomycin C (MC) is a cytotoxic chemotherapeutic agent that causes DNA damage in the form of DNA cross-links as well as a variety of DNA monoadducts and is known to induce p53. The various DNA adducts formed upon treatment of mouse mammary tumor cells with MC as well as 10-decarbamoyl MC (DMC) and 2,7-diaminomitosene (2,7-DAM), the major MC metabolite, have been elucidated. The cytotoxicity of DMC parallels closely that of MC in a number of rodent cell lines tested, whereas 2,7-DAM is relatively noncytotoxic. In this study, we investigate the ability of MC, DMC, and 2,7-DAM to activate p53 at equidose concentrations by treating tissue culture cell lines with the three mitomycins. Whereas MC and DMC induced p53 protein levels and increased the levels of p21 and Gadd45 mRNA, 2,7-DAM did not. Furthermore, MC and DMC, but not 2,7-DAM, were able to induce apoptosis efficiently in ML-1 cells. Therefore the 2,7-DAM monoadducts were unable to activate the p53 pathway. Interestingly, DMC was able to initiate apoptosis via a p53-independent pathway whereas MC was not. This is the first finding that adducts of a multiadduct type DNA-damaging agent are differentially recognized by DNA damage sensor pathways.