RESUMO
The neuronal calcium- and voltage-activated BK potassium channel is modulated by ethanol, and plays a role in behavioral tolerance in vertebrates and invertebrates. We examine the influence of temporal parameters of alcohol exposure on the characteristics of BK molecular tolerance in the ventral striatum, an important component of brain reward circuitry. BK channels in striatal neurons of C57BL/6J mice exhibited molecular tolerance whose duration was a function of exposure time. After 6 h exposure to 20 mm (0.09 mg%) ethanol, alcohol sensitivity was suppressed beyond 24 h after withdrawal, while after a 1 or 3 h exposure, sensitivity had significantly recovered after 4 h. This temporally controlled transition to persistent molecular tolerance parallels changes in BK channel isoform profile. After withdrawal from 6 h, but not 3 h alcohol exposure, mRNA levels of the alcohol-insensitive STREX (stress axis-regulated exon) splice variant were increased. Moreover, the biophysical properties of BK channels during withdrawal from 6 h exposure were altered, and match the properties of STREX channels exogenously expressed in HEK 293 cells. Our results suggest a temporally triggered shift in BK isoform identity. Once activated, the transition does not require the continued presence of alcohol. We next determined whether the results obtained using cultured striatal neurons could be observed in acutely dissociated striatal neurons, after alcohol administration in the living mouse. The results were in remarkable agreement with the striatal culture data, showing persistent molecular tolerance after injections producing 6 h of intoxication, but not after injections producing only 3 h of intoxication.
Assuntos
Depressores do Sistema Nervoso Central/farmacologia , Etanol/farmacologia , Ativação do Canal Iônico/efeitos dos fármacos , Canais de Potássio Ativados por Cálcio de Condutância Alta/metabolismo , Dinâmica não Linear , Regulação para Cima/efeitos dos fármacos , 8-Bromo Monofosfato de Adenosina Cíclica/farmacologia , Animais , Animais Recém-Nascidos , Cálcio/metabolismo , Sobrevivência Celular , Células Cultivadas , Corpo Estriado/citologia , DNA Recombinante/efeitos dos fármacos , DNA Recombinante/genética , Éxons/efeitos dos fármacos , Éxons/genética , Humanos , Ativação do Canal Iônico/genética , Canais de Potássio Ativados por Cálcio de Condutância Alta/genética , Masculino , Potenciais da Membrana/efeitos dos fármacos , Potenciais da Membrana/genética , Camundongos , Camundongos Endogâmicos C57BL , Neurônios/efeitos dos fármacos , Splicing de RNA , Ratos , Ratos Sprague-Dawley , Síndrome de Abstinência a Substâncias/genética , Síndrome de Abstinência a Substâncias/fisiopatologia , Fatores de TempoRESUMO
Many commonly used drugs, such as psoralen and cisplatin, can generate a very unique type of DNA damage, namely ICL (interstrand cross-link). An ICL can severely block DNA replication and transcription and cause programmed cell death. The molecular mechanism of repairing the ICL damage has not been well established. We have studied the role of XPF (xeroderma pigmentosum group F) protein in psoralen-induced ICL-mediated DNA repair and mutagenesis. The results obtained from our mutagenesis studies revealed a very similar mutation frequency in both human normal fibroblast cells and XPF cells. The mutation spectra generated in both cells, however, were very different: most of the mutations generated in the normal fibroblast cells were T167-->A transversions, whereas most of the mutations generated in the XPF cells were T167-->G transversions. When a wild-type XPF gene cDNA was stably transfected into the XPF cells, the T167-->A mutations were increased and the T167-->G mutations were decreased. We also determined the DNA repair capability of the XPF cells using both the host-cell reactivation and the in vitro DNA repair assays. The results obtained from the host-cell reactivation experiments revealed an effective reactivation of a luciferase reporter gene from the psoralen-damaged plasmid in the XPF cells. The results obtained from the in vitro DNA repair experiments demonstrated that the XPF nuclear extract is normal in introducing dual incisions during the nucleotide excision repair process. These results suggest that the XPF protein has important roles in the psoralen ICL-mediated DNA repair and mutagenesis.
Assuntos
Reagentes de Ligações Cruzadas/farmacologia , Dano ao DNA , Reparo do DNA/fisiologia , DNA Recombinante/efeitos dos fármacos , Proteínas de Ligação a DNA/fisiologia , Mutagênese , Oligonucleotídeos/farmacologia , Trioxsaleno/farmacologia , Xeroderma Pigmentoso/genética , Sequência de Bases , Células Cultivadas/efeitos dos fármacos , Células Cultivadas/metabolismo , Células Cultivadas/efeitos da radiação , Adutos de DNA/metabolismo , Reparo do DNA/genética , DNA Complementar/genética , DNA Recombinante/genética , DNA Recombinante/efeitos da radiação , Proteínas de Ligação a DNA/deficiência , Proteínas de Ligação a DNA/genética , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Fibroblastos/efeitos da radiação , Genes Reporter , Genes Supressores , Genes Sintéticos/efeitos dos fármacos , Genes Sintéticos/efeitos da radiação , Humanos , Luciferases/biossíntese , Luciferases/genética , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Fotoquímica , Plasmídeos/efeitos dos fármacos , Plasmídeos/efeitos da radiação , RNA de Transferência/genética , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/genética , Transfecção , Trioxsaleno/análogos & derivados , Raios Ultravioleta , Xeroderma Pigmentoso/metabolismo , Xeroderma Pigmentoso/patologiaRESUMO
In erythroid tissues the chromatin structure of the beta-globin gene locus is extensively remodeled. Changes include the formation of DNase I hypersensitive sites (HSs) over the promoters of actively expressed genes. To test the hypothesis that such "opening" of promoter chromatin structure is important for beta-globin gene expression, we placed a 101-bp erythroid-specific hypersensitive-site forming element (HSFE) from the core of LCR HS4 immediately upstream of a minimal beta-globin gene promoter. We then studied the effects of this element alone and in combination with other cis-acting elements on globin gene chromatin structure and gene expression in MEL cells and transgenic mice. Single or tandem HSFEs increased the size of the portion of the promoter accessible to DNase digestion, increased the proportion of promoters in an accessible conformation, and increased gene expression approximately 5-fold. These were equivalent to expression levels attained using a 2.8-kb microLCR construct. Inclusion of the LCR HS2 enhancer did not increase expression further. In transgenic mouse fetal liver cells the HSFE increased average expression 2.5-fold compared to the minimal promoter alone. These results indicate that a small cis-acting element is capable of remodeling local beta-globin promoter chromatin structure and producing expression similar to that seen with a microLCR construct.
Assuntos
Cromatina/genética , Regulação da Expressão Gênica , Rearranjo Gênico , Globinas/genética , Região de Controle de Locus Gênico , Regiões Promotoras Genéticas , Animais , Cromatina/ultraestrutura , DNA Recombinante/efeitos dos fármacos , DNA Recombinante/genética , Desoxirribonuclease I/farmacologia , Elementos Facilitadores Genéticos , Globinas/biossíntese , Humanos , Leucemia Eritroblástica Aguda/patologia , Região de Controle de Locus Gênico/genética , Camundongos , Camundongos Transgênicos , RNA Mensageiro/biossíntese , Transfecção , Células Tumorais CultivadasRESUMO
Folate receptor (FR) type beta is expressed in the myelomonocytic lineage, predominantly during neutrophil maturation and in myeloid leukemias. FR-beta expression was elevated up to 20-fold by all-trans retinoic acid (ATRA) in KG-1 myeloid leukemia cells in a dose-dependent and reversible manner in the absence of terminal differentiation or cell growth inhibition. ATRA also increased FR-beta expression in vitro in myeloid leukemia cells from patient marrow. FR-beta was not up-regulated in KG-1 cells treated with phorbol ester, dexamethasone, 1,25-dihydroxy vitamin D(3), or transforming growth factor beta. ATRA did not induce FR-beta expression in receptor negative cells of diverse origin. The ATRA-induced increase in FR-beta expression in KG-1 cells occurred at the level of messenger RNA synthesis, and in 293 cells containing a stably integrated FR-beta promoter-luciferase reporter construct, ATRA induced expression of the reporter. From experiments using retinoid agonists and antagonists and from cotransfection studies using the FR-beta promoter and expression plasmids for the nuclear receptors retinoic acid receptor (RAR)alpha, RARbeta, or RARgamma, it appears that the retinoid effect on FR-beta expression could be mediated by ligand binding to RARs alpha, beta, or gamma, but not to retinoid X receptors. Furthermore, there was apparent cross-talk between RARalpha and RARgamma selective agonists or antagonists, suggesting a common downstream target for RAR isoforms in inducing FR-beta expression. Thus, blocks in the RARalpha-specific pathway of retinoid-induced differentiation may be bypassed during retinoid induction of FR-beta expression. The results suggest that to facilitate FR-targeted therapies, retinoids may be used to modulate FR-beta expression in myeloid leukemia cells refractory to retinoid differentiation therapy.
Assuntos
Proteínas de Transporte/efeitos dos fármacos , Leucemia Mieloide/metabolismo , Receptores de Superfície Celular , Tretinoína/farmacologia , Antineoplásicos/farmacologia , Células da Medula Óssea/metabolismo , Células da Medula Óssea/patologia , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Diferenciação Celular/efeitos dos fármacos , Divisão Celular/efeitos dos fármacos , DNA Recombinante/efeitos dos fármacos , Receptores de Folato com Âncoras de GPI , Regulação da Expressão Gênica/efeitos dos fármacos , Glicosilfosfatidilinositóis/metabolismo , Humanos , Leucemia Mieloide/patologia , Proteínas de Neoplasias/efeitos dos fármacos , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Fosfatidilinositol Diacilglicerol-Liase , Regiões Promotoras Genéticas/efeitos dos fármacos , Isoformas de Proteínas/efeitos dos fármacos , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Receptores do Ácido Retinoico/fisiologia , Transcrição Gênica/efeitos dos fármacos , Transfecção , Tretinoína/análogos & derivados , Células Tumorais Cultivadas , Fosfolipases Tipo C/metabolismo , Regulação para Cima/efeitos dos fármacosRESUMO
Agonist binding to G protein-coupled receptors induces the formation of a receptor-G protein complex and subsequent guanosine 5'-diphosphate/guanosine 5'-triphosphate (GDP/GTP) exchange. Some receptors, however, form receptor-G protein complexes and promote GDP/GTP exchange even when not occupied by agonists. Such receptors preferentially activate pertussis toxin-sensitive G proteins (i.e., G(i)/G(o)), and the interactions of receptors and G proteins are affected by monovalent cations (most notably Na(+)), both in the occupied and unoccupied state. We investigated the effects of Na(+) on the intrinsic activity of 5-hydroxytryptamine(1A) (5-HT(1A)) receptor ligands, measured as maximal effect (E(MAX)), using guanosine 5'-0-(3-[35S]thio)-triphosphate ([35S]GTPgammaS) binding to membranes prepared from human epithelioid carcinoma (HeLa) cells, expressing 500 fmol/mg protein of cloned human 5-HT(1A) receptor (HA7 cells). A decrease of the NaCl concentration decreased the maximal effect of serotonin, increased basal [35S]GTPgammaS binding, and increased the negative intrinsic activity of spiperone and N-[2-[4-(2-methoxyphenyl)-1-piperazinyl]-N-(2-pyridinyl)cyclohexaneca rboxamide (WAY 100635). This ability of WAY 100635 to decrease basal [35S]GTPgammaS binding was antagonized by (s)-N-tert-butyl-3-(4-(2-methoxyphenyl)piperazine-1-yl)-2-phenylpropa namide ((s)-WAY 100135) (pA(2)=7.77). Further, WAY 100635 was able to antagonize carboxamidotryptamine (5-CT)-stimulated [35S]GTPgammaS binding with a pA(2) of 9.9, in standard NaCl conditions, and of 9.7, in the absence of NaCl. Changes in membrane concentration did not affect the ability of WAY 100635 to decrease [35S]GTPgammaS binding. WAY 100635 did not affect basal [35S]GTPgammaS binding to membranes from untransfected HeLa cells. Pertussis toxin (200 ng/ml) prevented WAY 100635 and spiperone to decrease [35S]GTPgammaS binding, showing that their effects were mediated by G proteins of the G(i)/G(o) family. In conclusion, the constitutive and stimulated activity of human 5-HT(1A) receptors expressed in HA7 cells is sodium-dependent, which allowed to confirm the 5-HT(1A) inverse agonist properties of spiperone, and to show that WAY 100635 is an inverse agonist at 5-HT(1A) receptors that inhibits basal [35S]GTPgammaS binding to a lesser extent than spiperone. [35S]GTPgammaS binding to membranes from HA7 cells under low NaCl conditions appears to be especially suitable to evidence and pharmacologically analyze the inverse agonist properties of 5-HT(1A) receptor ligands.
Assuntos
Piperazinas/farmacologia , Piridinas/farmacologia , Receptores de Serotonina/efeitos dos fármacos , Antagonistas da Serotonina/farmacologia , Agonistas do Receptor de Serotonina/farmacologia , Serotonina/análogos & derivados , Ligação Competitiva/efeitos dos fármacos , Membrana Celular/metabolismo , DNA Recombinante/efeitos dos fármacos , DNA Recombinante/genética , Relação Dose-Resposta a Droga , Guanosina 5'-O-(3-Tiotrifosfato)/metabolismo , Células HeLa , Humanos , Receptores de Serotonina/genética , Receptores 5-HT1 de Serotonina , Serotonina/farmacologia , Cloreto de Sódio/farmacologia , Espiperona/farmacologia , Radioisótopos de EnxofreRESUMO
The distribution of DNA adducts formed by the two main components, aristolochic acid I (AAI) and aristolochic acid II (AAII), of the carcinogenic plant extract aristolochic acid (AA) was examined in a plasmid containing exon 2 of the mouse c-H-ras gene by a polymerase arrest assay. AAI and AAII were reacted with plasmid DNA by reductive activation and the resulting DNA adducts were identified as the previously characterized adenine adducts (dA-AAI and dA-AAII) and guanine adducts (dG-AAI and dG-AAII) by the (32)P-post-labeling method. In addition, a structurally unknown adduct was detected in AAII-modified DNA and shown to be derived from reaction with cytosine (dC-AAII). Sites at which DNA polymerase progress along the template was blocked were assumed to be at the nucleotide 3' to the adduct. Polymerase arrest spectra showed a preference for reaction with purine bases in the mouse H-ras gene for both activated compounds, consistent with previous results that purine adducts are the principal reaction products of AAI and AAII with DNA. Despite the structural similarities among AAI-DNA and AAII-DNA adducts, however, the polymerase arrest spectra produced by the AAs were different. According to the (32)P-post-labeling analyses reductively activated AAI showed a strong preference for reacting with guanine residues in plasmid DNA, however, the polymerase arrest assay revealed arrest sites preferentially at adenine residues. In contrast, activated AAII reacted preferentially with adenine rather than guanine residues and to a lesser extent with cytosine but DNA polymerase was arrested at guanine as well as adenine and cytosine residues with nearly the same average relative intensity. Thus, the polymerase arrest spectra obtained with the AA-adducted ras sequence do not reflect the DNA adduct distribution in plasmid DNA as determined by (32)P-post-labeling. Arrest sites of DNA polymerase associated with cytosine residues confirmed the presence of a cytosine adduct in DNA modified by AAII. For both compounds adduct distribution was not random; instead, regions with adduct hot spots and cold spots were observed. Results from nearest neighbor binding analysis indicated that flanking pyrimidines displayed the greatest effect on polymerase arrest and therefore on DNA binding by AA.
Assuntos
Ácidos Aristolóquicos , Carcinógenos/metabolismo , Adutos de DNA/análise , Replicação do DNA/efeitos dos fármacos , DNA Polimerase Dirigida por DNA/metabolismo , Genes ras/efeitos dos fármacos , Fenantrenos/metabolismo , Adenina/química , Animais , Composição de Bases , Carcinógenos/farmacologia , Códon/efeitos dos fármacos , Códon/genética , Dano ao DNA , DNA Recombinante/química , DNA Recombinante/efeitos dos fármacos , Desoxiadenosinas/análise , Desoxiguanosina/análogos & derivados , Desoxiguanosina/análise , Guanina/química , Camundongos , Oligodesoxirribonucleotídeos/metabolismo , Fenantrenos/análise , Fenantrenos/farmacologia , Plasmídeos/química , Plasmídeos/efeitos dos fármacos , Moldes GenéticosRESUMO
Mammalian cell extracts have been shown to carry out damage-specific DNA repair synthesis induced by a variety of lesions, including those created by UV and cisplatin. Here, we show that a single psoralen interstrand cross-link induces DNA synthesis in both the damaged plasmid and a second homologous unmodified plasmid coincubated in the extract. The presence of the second plasmid strongly stimulates repair synthesis in the cross-linked plasmid. Heterologous DNAs also stimulate repair synthesis to variable extents. Psoralen monoadducts and double-strand breaks do not induce repair synthesis in the unmodified plasmid, indicating that such incorporation is specific to interstrand cross-links. This induced repair synthesis is consistent with previous evidence indicating a recombinational mode of repair for interstrand cross-links. DNA synthesis is compromised in extracts from mutants (deficient in ERCC1, XPF, XRCC2, and XRCC3) which are all sensitive to DNA cross-linking agents but is normal in extracts from mutants (XP-A, XP-C, and XP-G) which are much less sensitive. Extracts from Fanconi anemia cells exhibit an intermediate to wild-type level of activity dependent upon the complementation group. The DNA synthesis deficit in ERCC1- and XPF-deficient extracts is restored by addition of purified ERCC1-XPF heterodimer. This system provides a biochemical assay for investigating mechanisms of interstrand cross-link repair and should also facilitate the identification and functional characterization of cellular proteins involved in repair of these lesions.
Assuntos
Reagentes de Ligações Cruzadas/farmacologia , Dano ao DNA , Reparo do DNA , DNA Recombinante/biossíntese , Proteínas de Ligação a DNA/fisiologia , Endonucleases , Ficusina/farmacologia , Mamíferos/genética , Plasmídeos/genética , Proteínas/fisiologia , Animais , Linhagem Celular , Sistema Livre de Células , DNA Recombinante/efeitos dos fármacos , Proteínas de Ligação a DNA/genética , Anemia de Fanconi/genética , Anemia de Fanconi/patologia , Células HeLa , Humanos , Mamíferos/metabolismo , Camundongos , Plasmídeos/efeitos dos fármacos , Proteínas/genética , Rad51 Recombinase , Recombinação Genética , Extratos de Tecidos , Xeroderma Pigmentoso/genética , Xeroderma Pigmentoso/patologiaRESUMO
BACKGROUND: An approach to improve current chemotherapy is the selective transduction of tumor cells with suicide genes to sensitize these cells to prodrugs of cytostatic agents. METHODS: In this study, gene transfer was accomplished with the cationic polymer poly(2-(dimethylamino)ethyl methacrylate) (PDMAEMA), able to condense plasmid-DNA by electrostatic interaction. OVCAR-3 cells were transfected with plasmids encoding E. coli-derived or human beta-glucuronidase and the transfection efficiency and inhibition by serum was determined. Next, we measured the sensitivity of OVCAR-3 cells transiently expressing beta-glucuronidase to the glucuronide prodrug of doxorubicin (DOX-GA3) or to doxorubicin. RESULTS: OVCAR-3 cells were efficiently transfected with a plasmid encoding E. coli-derived beta-glucuronidase. The degree of transfection (30% of cells) was higher than that achieved with commercially available cationic lipids (DOTAP, Lipofectamine) without inhibition by serum. OVCAR-3 cells transiently expressing beta-glucuronidase were equally sensitive to the glucuronide prodrug of doxorubicin (DOX-GA3) or to doxorubicin itself, indicating complete conversion of prodrug to drug. Similar studies were performed with the plasmid encoding for human beta-glucuronidase, which is likely to be less immunogenic. Also in this case, OVCAR-3 cells showed an increased sensitivity to the prodrug DOX-GA3, although less pronounced than when the bacterial enzyme was used. A strong bystander effect was observed when OVCAR-3 cells transfected with beta-glucuronidase were mixed with non-transfected cells at different ratios. Complete tumor cell growth inhibition was already observed when only 15% of the cells expressed the activating enzyme. CONCLUSION: These studies suggest that beta-glucuronidase gene therapy using PDMAEMA as a carrier system and DOX-GA3 as the prodrug has a potential application in cancer gene therapy.
Assuntos
Antibióticos Antineoplásicos/administração & dosagem , Carcinoma/patologia , Doxorrubicina/análogos & derivados , Doxorrubicina/administração & dosagem , Vetores Genéticos/efeitos dos fármacos , Glucuronatos/administração & dosagem , Glucuronidase/metabolismo , Metacrilatos/farmacologia , Nylons/farmacologia , Neoplasias Ovarianas/patologia , Plasmídeos/administração & dosagem , Pró-Fármacos/administração & dosagem , Antibióticos Antineoplásicos/metabolismo , Antibióticos Antineoplásicos/farmacocinética , Antimetabólitos Antineoplásicos/administração & dosagem , Antimetabólitos Antineoplásicos/metabolismo , Antimetabólitos Antineoplásicos/farmacocinética , Biotransformação , Resinas de Troca de Cátion , Morte Celular , Divisão Celular/efeitos dos fármacos , Meios de Cultura Livres de Soro , Citosina Desaminase , DNA Recombinante/química , DNA Recombinante/efeitos dos fármacos , Doxorrubicina/metabolismo , Doxorrubicina/farmacocinética , Portadores de Fármacos/administração & dosagem , Portadores de Fármacos/farmacologia , Ensaios de Seleção de Medicamentos Antitumorais , Feminino , Flucitosina/administração & dosagem , Flucitosina/metabolismo , Flucitosina/farmacocinética , Genes Reporter , Vetores Genéticos/química , Vetores Genéticos/genética , Glucuronatos/metabolismo , Glucuronatos/farmacocinética , Glucuronidase/genética , Humanos , Lipídeos , Nucleosídeo Desaminases/genética , Tamanho da Partícula , Pró-Fármacos/metabolismo , Pró-Fármacos/farmacocinética , Eletricidade Estática , Transfecção , Células Tumorais Cultivadas/enzimologiaRESUMO
Big Blue Rat2 embryonic fibroblasts carry the lambda-Liz shuttle vector which is also present in the Big Blue mouse and rat. Mutations in the Big Blue systems have most often been measured at the lacI locus. However, a method for positive selection of mutations at the lambda cII locus was recently described. This assay appears to have many advantages over the use of lacI as a mutational target, but it has yet to be well characterized in mammalian mutagenesis studies. The objective of these studies was to determine the spontaneous and ethylnitrosourea (ENU)-induced mutant frequencies (MFs) and mutational spectra at cII using Big Blue Rat2 embryonic fibroblasts. The average spontaneous MF was 13 +/- 1.4 x 10(-5). The average induced MF was 60 +/- 10 x 10(-5) 10 days following a 30 min treatment with 0.1 mg/ml ENU. Eighty four independent spontaneous mutants were sequenced: 23 (27.4%) were frameshift mutations and 61 (72.6%) were base substitutions. Two spontaneous frameshift hotspots were detected, both in mononucleotide runs. G:C-->A:T transitions were the most common type of base substitution in cII; of these 71% occurred at CpG sites. The ENU-induced mutational spectrum at cII (44 mutants) consisted of 42 base substitutions (95.5%) and two -1 frameshift mutations (4.5%). Compared with the spontaneous spectrum, the ENU-induced spectrum had significantly fewer frameshift mutations (4.5 versus 27%) and base substitutions occurred predominantly at A:T base pairs (71 versus 34%). Overall, the spontaneous cII mutational spectrum reported here differs slightly from spontaneous spectra reported at the Big Blue lacI locus, but the mutational spectra and base substitution MFs following treatment with ENU were comparable at both loci. These data support the continued use of cII as a selectable marker in mutagenesis studies involving cells or tissues that carry a lambda transgene.
Assuntos
Animais Geneticamente Modificados/genética , Bacteriófago lambda/genética , DNA Recombinante/efeitos dos fármacos , Proteínas de Escherichia coli , Etilnitrosoureia/toxicidade , Genes Virais/efeitos dos fármacos , Vetores Genéticos/genética , Mutação , Fatores de Transcrição/genética , Transgenes/efeitos dos fármacos , Proteínas Estruturais Virais/genética , Sequência de Aminoácidos , Animais , Proteínas de Bactérias/genética , Bacteriófago lambda/efeitos dos fármacos , Sequência de Bases , Ilhas de CpG , Adutos de DNA , Dano ao DNA , Análise Mutacional de DNA , DNA Recombinante/genética , Fibroblastos , Mutação da Fase de Leitura , Vetores Genéticos/efeitos dos fármacos , Óperon Lac/efeitos dos fármacos , Repressores Lac , Lisogenia/genética , Camundongos , Camundongos Endogâmicos C57BL , Dados de Sequência Molecular , Mutação Puntual , Reação em Cadeia da Polimerase , Ratos , Proteínas Repressoras/genética , Deleção de Sequência , Proteínas ViraisRESUMO
Leukaemia inhibitory factor (LIF) is a pleiotropic cytokine which has been found to be expressed in the human endometrium and to play an important role in human reproduction. In the present study we investigated expression and regulation of the human LIF promoter in HEC-1B endometrial adenocarcinoma cells using a luciferase reporter plasmid bearing a 666 bp promoter fragment (h666LIF-Luc) in transient transfection assays. HEC-1B cells were first shown by reverse transcription-polymerase chain reaction (RT-PCR) to be able to produce endogenous LIF mRNA. The LIF promoter was efficiently transcribed in HEC-1B cells, showing much higher levels of basal activity than in the previously studied Jurkat T-lymphoma cells and SKUT-1B uterine mesodermal tumour cells. The activity of the LIF promoter was stimulated in HEC-1B cells by a combination of phorbol ester (TPA) and ionomycin, which we had previously found to strongly induce its activity in Jurkat T-lymphoma cells. We next studied the effect of progestin (medroxyprogesterone acetate; MPA) on the LIF promoter activity in HEC-1B cells. The LIF promoter was not stimulated by MPA treatment in the presence of transfected progesterone receptor B (PR-B) expression vector in HEC-1B cells, while we had previously described its induction by MPA in SKUT-1B cells. This indicates that progestin-dependent regulation of the LIF promoter in uterine tumour cells is different in cells of epithelial and mesodermal origin.
Assuntos
Neoplasias do Endométrio/genética , Inibidores do Crescimento/genética , Interleucina-6 , Linfocinas/genética , Regiões Promotoras Genéticas/genética , DNA Recombinante/efeitos dos fármacos , DNA Recombinante/genética , Neoplasias do Endométrio/patologia , Feminino , Regulação da Expressão Gênica , Humanos , Ionomicina/farmacologia , Fator Inibidor de Leucemia , Ésteres de Forbol/farmacologia , Progestinas/farmacologia , Regiões Promotoras Genéticas/efeitos dos fármacos , RNA Mensageiro/análise , RNA Mensageiro/genética , Células Tumorais CultivadasRESUMO
Two series of analogues of the clinical antileukemic drug and DNA-intercalating ligand amsacrine have been prepared, containing aniline mustard sidechains of varying reactivity, linked either at the 4-position of the intercalating acridine chromophore (type A) or at the 1'-position of the 9-anilino group (type B). DNase I footprinting assays showed that compounds of type B had stronger reversible binding to DNA than did compounds of type A. Compounds of each type showed similar patterns of alkylation-induced cleavage of DNA, and alkylate at the N7 of guanines in runs of guanines (similar to the pattern for untargeted mustards) as well as some adenines. Both classes of compounds crosslinked DNA, although those bearing relatively inactive mustards did so only at high drug/base pair ratios. However, while the patterns of DNA alkylation were broadly similar, the compounds were considerably more cytotoxic than analogous untargeted mustards. Comparison of their cytotoxicities in wild-type and DNA repair-deficient lines indicated this toxicity was due to DNA crosslinks (except for the least reactive SO2-linked mustards). The 4-linked analogues showed slightly higher in vivo antileukemic activity than the corresponding 1'-linked analogues.
Assuntos
Amsacrina/farmacologia , Mostarda de Anilina/análogos & derivados , DNA Recombinante/efeitos dos fármacos , Substâncias Intercalantes/farmacologia , Amsacrina/química , Mostarda de Anilina/química , Mostarda de Anilina/farmacologia , Animais , Células CHO , Cricetinae , DNA Recombinante/química , Substâncias Intercalantes/química , Leucemia Experimental/patologia , Espectroscopia de Ressonância Magnética , Espectrometria de Massas , Camundongos , Células Tumorais CultivadasRESUMO
The thyroid hormone (T3) was shown to down regulate the level of alpha-foetoprotein (AFP) mRNA in hepatoma cells HepG2. Recombinant plasmids containing segments from the mouse AFP gene promoter were transfected in HepG2 cells and transient expression assays showed that the T3 inhibitory effect depends on the sequence limited by positions -80 and -38, upstream from the TATA box. This sequence is able to confer T3 sensitivity to a heterologous promoter and contains a putative T3-responsive element, as well as likely CEBP- and HNF1-responsive elements. These observations suggest that T3 is a good candidate for hormonal control of the AFP gene expression and especially for the neonatal shut off of the gene.
Assuntos
Hormônios Tireóideos/farmacologia , alfa-Fetoproteínas/genética , Animais , Sequência de Bases , Sítios de Ligação , Divisão Celular/efeitos dos fármacos , DNA Recombinante/efeitos dos fármacos , DNA Recombinante/genética , Elementos Facilitadores Genéticos/genética , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/genética , Humanos , Camundongos , Oligonucleotídeos/genética , Reação em Cadeia da Polimerase , Regiões Promotoras Genéticas/efeitos dos fármacos , Regiões Promotoras Genéticas/genética , RNA Mensageiro/efeitos dos fármacos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Sequências Reguladoras de Ácido Nucleico/genética , Transcrição Gênica/efeitos dos fármacos , Transcrição Gênica/genética , Células Tumorais Cultivadas , alfa-Fetoproteínas/efeitos dos fármacosRESUMO
Genetic studies revealed that DNA gyrase seems to catalyze immediate and transient DNA relaxation after Escherichia coli cells are exposed to heat shock (Ogata, Y., Mizushima, T., Kataoka, K., Miki, T., and Sekimizu, K. (1994) Mol. Gen. Genet. 244, 451-455). We have now obtained biochemical evidence to support this hypothesis. DNA gyrase catalyzed an increase in the linking number of DNA and relaxation of negatively supercoiled DNA, under physiological concentrations of ATP. Analyses by gel filtration chromatography of each subunit revealed that DNA relaxation activity co-migrated with each subunit. The linking number of DNA increased as the temperature increased. Further, the reaction was inhibited by nalidixic acid or by oxolinic acid. Based on these results, we propose that DNA gyrase participates in a concerted reaction with DNA topoisomerases in the immediate relaxation of DNA in cells exposed to heat shock.
Assuntos
Trifosfato de Adenosina/farmacologia , DNA Topoisomerases Tipo II/metabolismo , DNA Recombinante/metabolismo , Escherichia coli/enzimologia , Temperatura Alta , Conformação de Ácido Nucleico , Catálise , DNA Recombinante/efeitos dos fármacos , DNA Recombinante/genética , Mutação , Deleção de SequênciaRESUMO
A tumor-inhibiting antibiotic, 6-diazo-5-oxo-L-norleucine (DON), caused DNA single-strand breaks. Thus, supercoiled plasmid DNA was transformed into an open circular relaxed form DNA by incubation with DON at pH 7.4. DNA strand cleavage by DON was not inhibited by superoxide dismutase, but inhibited by catalase. The inhibition by catalase may not be due to the destruction of hydrogen peroxide, but to the masking DON by the interaction with the heme moiety of the enzyme. DNA strand cleavage by DON was inhibited by azide, mannitol, ethanol, cysteine and 2-mercaptoethanol, suggesting the involvement of radical species in the cleavage. The cleavage, however, was not suppressed by removal of dissolved oxygen from the reaction mixture, indicating that no oxygen-derived radicals participated in the cleavage. Electron spin resonance spin-trapping technique using 5,5-dimethyl-1-pyrroline N-oxide (DMPO) and N-tert-butyl-alpha-phenylnitrone (PBN) elucidated the generation of a carbon-centered radical from DON. Hence, the carbon-centered radical may participate in DNA strand cleavage by DON.
Assuntos
Antibióticos Antineoplásicos/farmacologia , DNA Recombinante/efeitos dos fármacos , DNA de Cadeia Simples/efeitos dos fármacos , Diazo-Oxo-Norleucina/farmacologia , Antibióticos Antineoplásicos/antagonistas & inibidores , Catalase/metabolismo , DNA Recombinante/metabolismo , DNA de Cadeia Simples/metabolismo , Diazo-Oxo-Norleucina/antagonistas & inibidores , Espectroscopia de Ressonância de Spin Eletrônica , Sequestradores de Radicais Livres , Hidrólise , Espécies Reativas de Oxigênio , Superóxido Dismutase/metabolismoRESUMO
The ability of a number of respirable industrial fibres, amosite and crocidolite asbestos, refractory ceramic fibres (RCFs) and man-made vitreous fibres (MMVFs) to cause free radical injury to plasmid phi X174 RFI DNA was assessed. The oxidative DNA damage was observed as depletion of supercoiled DNA after fibre treatment was quantified by scanning laser densitometry. The mechanism of fibre-mediated damage was determined by the use of the specific hydroxyl radical scavenger mannitol and the iron chelator desferrioxamine-B. The amosite and crocidolite asbestos caused substantial damage to DNA that was dose-related. The free radicals responsible for the asbestos-mediated DNA damage were hydroxyl radicals, as determined by inhibition with mannitol. Asbestos fibre-mediated damage to DNA was completely ameliorated by the chelation of fibre-associated iron with desferrioxamine-B. The amount of Fe(II) and Fe(III) released by equal numbers of the different fibre types at equal fibre number was determined. The fibres released very small amounts of Fe(II) and there were no significant differences between the fibre types. The fibres released substantial amounts of Fe(III); MMVF 21 released significantly more Fe(III) than any of the other fibres and short fibre amosite also released more Fe(III) than three of the MMVFs and two of the RCFs. When ability to release Fe(II) and Fe(III) was compared with ability to cause DNA damage there was not a good correlation, because only the long amosite and crocidolite caused substantial free radical injury to DNA; this contrasts with MMVF 21 and short amosite being the two fibres that released the greatest amounts of iron. The loss of ability to damage DNA in DSF-B-treated asbestos fibres shows that iron at the surface of asbestos fibres definitely has a role in generating hydroxyl radicals. However, it is clear that some fibres, such as short amosite and MMVF 21, release large quantities of iron without causing free radical damage, whilst neither long amosite nor crocidolite released more iron than the other fibres. The exact role of iron in fibre reactivity therefore remains unresolved, but fibre-bound iron not released from the surface of asbestos could be important. Further research is under way to investigate this possibility.
Assuntos
Poluentes Ocupacionais do Ar/toxicidade , Dano ao DNA , DNA Super-Helicoidal/efeitos dos fármacos , Radical Hidroxila , Plasmídeos , Amianto/toxicidade , Bacteriófago phi X 174/genética , DNA Recombinante/efeitos dos fármacos , Sequestradores de Radicais Livres , Radicais Livres , Quelantes de Ferro/farmacologiaRESUMO
Nucleotide excision repair (NER) functions to remove DNA damage caused by ultraviolet light and by other agents that distort the DNA helix. The NER machinery has been conserved in structure and function from yeast to humans, and in humans, defective NER is the underlying cause of the cancer-prone disease xeroderma pigmentosum. Here, we reconstitute the incision reaction of NER in Saccharomyces cerevisiae using purified protein factors. The Rad14 protein, the Rad4-Rad23 complex, the Rad2 nuclease, the Rad1-Rad10 nuclease, replication protein A, and the RNA polymerase II transcription factor TFIIH were purified to near homogeneity from yeast. We show that these protein factors are both necessary and sufficient for dual incision of DNA damaged by either ultraviolet light or N-acetoxy-2-aminoacetylfluorene. Incision in the reconstituted system requires ATP, which cannot be substituted by adenosine 5'-O-(3-thiotriphosphate), suggesting that the hydrolysis of ATP is indispensable for the incision reaction. The excision DNA fragments formed as a result of dual incision are in the 24-27-nucleotide range.
Assuntos
Reparo do DNA , Proteínas de Ligação a DNA/metabolismo , Proteínas Fúngicas/metabolismo , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/genética , Fatores Associados à Proteína de Ligação a TATA , Fator de Transcrição TFIID , Fatores de Transcrição TFII , Fatores de Transcrição/metabolismo , 2-Acetilaminofluoreno/toxicidade , Dano ao DNA , Enzimas Reparadoras do DNA , Replicação do DNA , DNA Recombinante/efeitos dos fármacos , DNA Recombinante/isolamento & purificação , DNA Recombinante/efeitos da radiação , Proteína de Replicação A , Fator de Transcrição TFIIH , Raios UltravioletaRESUMO
Mutations in tumor suppressor genes are intricately associated with the etiology of neoplasia. Often, such mutations are followed by the loss of the second, functional alleles of tumor suppressor genes, a phenomenon known as loss of heterozygosity. Loss of heterozygosity may occur by different molecular mechanisms, including mitotic recombination, and it is conceivable that these molecular events are influenced by endogenous as well as exogenous factors. To test whether mitotic recombination is induced by certain carcinogens, we genetically engineered a Saccharomyces cerevisiae tester strain so that it metabolizes two important classes of carcinogens, polycyclic aromatic hydrocarbons and heterocyclic arylamines. This was accomplished by expressing human cDNA's coding for the cytochrome P450 (CYP) enzymes CYP1A1 or CYP1A2 in combination with NADPH-CYP oxidoreductase in a strain heterozygous for two mutations in the trp5 gene. Microsomes isolated from the transformed yeast strains activated various xenobiotics to powerful mutagens that were detected in the Ames test. Of these, the mycotoxin aflatoxin B1, when activated intracellularly in the strains containing either human CYP enzyme, significantly induced mitotic recombination. These results are discussed in light of possible mechanisms that are involved in aflatoxin B1-mediated hepatocarcinogenesis. Similarly, benzo[a]pyrene-trans-7,8-dihydrodiol and 3-amino-1-methyl-5H-pyrido[4,3-b]indole were activated to recombinagenic products, whereas benzo[a]pyrene and 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline were negative in this assay. Our results argue that the constructed yeast strains may be a valuable tool for the investigation of drug-induced mitotic recombination.
Assuntos
Aflatoxina B1/farmacocinética , Aflatoxina B1/toxicidade , Sistema Enzimático do Citocromo P-450/metabolismo , DNA Fúngico/efeitos dos fármacos , DNA Fúngico/genética , DNA Recombinante/efeitos dos fármacos , DNA Recombinante/genética , Oxirredutases/metabolismo , Recombinação Genética/efeitos dos fármacos , Saccharomyces cerevisiae/enzimologia , Biotransformação , Citocromo P-450 CYP1A1 , Sistema Enzimático do Citocromo P-450/biossíntese , Líquido Intracelular/enzimologia , NADPH-Ferri-Hemoproteína Redutase/biossíntese , Oxirredutases/biossíntese , Saccharomyces cerevisiae/efeitos dos fármacosRESUMO
Butyric acid has many strong effects on gene expression in mammalian and viral systems, as well as in increasing the expression of recombinant DNAs artificially introduced into cultured cells. We screened 14 analogues of butyric acid for their ability to upregulate expression from 3 different recombinant chloramphenicol acetyltransferase expression vectors stably integrated into NIH 3T3 cells that had been transformed by calcium phosphate transfection or electroporation. Butyric acid, 2-bromobutyric acid, 3-bromopropionic acid, 3-mercaptopropionic acid, vinylacetic acid, and butyraldehyde were found to upregulate human immunodeficiency viral long terminal repeat-, SV40 early gene promoter-, and glucocerebrosidase promoter-directed expression of heterologous genes in cultured cells. Three other analogues had lesser effects; and 6 additional analogues had very little, if any, effect.
Assuntos
Butiratos/farmacologia , DNA Recombinante/genética , Expressão Gênica/efeitos dos fármacos , Transfecção , Células 3T3 , Animais , Divisão Celular/efeitos dos fármacos , Cloranfenicol O-Acetiltransferase/genética , DNA Recombinante/efeitos dos fármacos , Vetores Genéticos , Repetição Terminal Longa de HIV , Humanos , Camundongos , Regiões Promotoras Genéticas , Células Tumorais CultivadasRESUMO
A plasmid pSV2neo (5.6 kb) which is a recombinant SV40-based vector was subjected to hydrogen peroxide treatment in presence of iron in vitro (Fenton reaction). Transmission electron microscopy revealed that H2O2 through the Fenton reaction introduced different types of damages in DNA. Besides the expected nicked circular and linear molecules, single-stranded loops and plasmid multimers could be characterized. These observations on DNA damages might be helpful for better understanding of the effect of hydrogen peroxide on living cells in vivo.
Assuntos
Dano ao DNA , DNA Recombinante/efeitos dos fármacos , Peróxido de Hidrogênio/farmacologia , DNA Recombinante/ultraestrutura , Ferro/farmacologia , Microscopia Eletrônica , Plasmídeos , Vírus 40 dos Símios/genéticaRESUMO
A method for measuring nucleotide excision repair in response to UV irradiation and chemical-induced DNA damage has been developed, validated, and field tested in cultured human lymphocytes. The methodology is amenable to population-based screening and should facilitate future epidemiological studies seeking to investigate associations between DNA repair proficiency and cancer susceptibility. The impetus for such endeavors derives from the suggestion that the high incidence of skin cancer in the genetic disorder xeroderma pigmentosum is manifested as a result of the reduced capacity of patients' cells to repair DNA damaged by UV-mimetic agents. For the assay, damaged, nonreplicating, recombinant plasmid DNA harboring a chloramphenicol acetyltransferase (cat) reporter gene is introduced into lymphocytes by using a DEAE-dextran/DNA complex short-term transfection conditions. Excision repair of the damaged bacterial cat gene is monitored proportionately as a function of reactivated CAT enzyme activity following a 40-h repair/expression incubation period. The validity of the approach was indicated by the ability of the assay to discriminate xeroderma pigmentosum virus-transformed lymphocyte cell lines of both severe (complementation groups A and D) and moderate (complementation group C) excision repair deficiencies from repair-proficient cell lines. Similar results were observed when a mitogen-stimulated peripheral blood lymphocyte culture from an xeroderma pigmentosum A patient was assayed concurrently with mitogen-stimulated peripheral blood lymphocytes obtained from healthy individuals. Adaptation of this DNA repair assay as a field test in a pilot-tested select group of basal cell carcinoma patients and cancer-free controls led to the preliminary identification of a specific subset at risk for this disease as a consequence of significant reduction to the repair of photochemically (UV)-damaged plasmid DNA.