RESUMO
The combination of loss of habitat, human population encroachment, and increased demand of select nonhuman primates for biomedical research has significantly affected populations. There remains a need for knowledge and expertise in understanding background findings as related to the age, source, strain, and disease status of nonhuman primates. In particular, for safety/biomedical studies, a broader understanding and documentation of lesions would help clarify background from drug-related findings. A workshop and a minisymposium on spontaneous lesions and diseases in nonhuman primates were sponsored by the concurrent Annual Meetings of the American College of Veterinary Pathologists and the American Society for Veterinary Clinical Pathology held December 3-4, 2011, in Nashville, Tennessee. The first session had presentations from Drs Lowenstine and Montali, pathologists with extensive experience in wild and zoo populations of nonhuman primates, which was followed by presentations of 20 unique case reports of rare or newly observed spontaneous lesions in nonhuman primates (see online files for access to digital whole-slide images corresponding to each case report at http://www.scanscope.com/ACVP%20Slide%20Seminars/2011/Primate%20Pathology/view.apml). The minisymposium was composed of 5 nonhuman-primate researchers (Drs Bradley, Cline, Sasseville, Miller, Hutto) who concentrated on background and spontaneous lesions in nonhuman primates used in drug safety studies. Cynomolgus and rhesus macaques were emphasized, with some material presented on common marmosets. Congenital, acquired, inflammatory, and neoplastic changes were highlighed with a focus on clinical, macroscopic, and histopathologic findings that could confound the interpretation of drug safety studies.
Assuntos
Animais Selvagens , Animais de Zoológico , Doenças dos Primatas/patologia , Primatas , Experimentação Animal , Animais , Pesquisa Biomédica , Avaliação Pré-Clínica de Medicamentos , Feminino , Macaca fascicularis , Macaca mulatta , Masculino , Modelos AnimaisRESUMO
Unr (upstream of N-ras) is a cytoplasmic RNA-binding protein involved in the regulation of messenger RNA stability and internal initiation of translation. We have used Unr-deficient murine embryonic stem (ES) cells to analyse Unr role in cell proliferation and response to stress. Disruption of both unr gene copies had no effect on ES cell proliferation. However, after ionizing radiation (IR), clonogenic survival of unr(-/-) ES cells was approximately 3-fold enhanced as compared to unr(+/+) cells. We further determined that IR-induced apoptosis was decreased in unr(-/-) ES cells, and that reintroduction of the unr gene in unr(-/-) cells restored normal IR-induced apoptosis. Three pro-apoptotic genes, p53, caspase-3 and Gadd45gamma, were downregulated in unr(-/-) ES cells, indicating that Unr, as other cytoplasmic RNA-binding proteins, regulates a complex genetic program, promoting cell death after IR. In contrast, in the human hepatoma cell line HuH7, Unr knockdown using unr-specific small interfering RNAs induced apoptosis, both in untreated and gamma-irradiated cells. Thus, our results establish that Unr acts as a positive or negative regulator of cell death, depending on the cell type. Manipulating the level of Unr may constitute a specific approach to sensitize cancer cells to anticancer treatments.
Assuntos
Apoptose/efeitos da radiação , Carcinoma Hepatocelular/metabolismo , Proteínas de Ligação a DNA/metabolismo , Embrião de Mamíferos/metabolismo , Proteínas de Ligação a Poli(A)/metabolismo , Proteínas de Ligação a RNA/metabolismo , Animais , Carcinoma Hepatocelular/patologia , Proteínas de Transporte/metabolismo , Caspase 3/metabolismo , Divisão Celular , Proliferação de Células , Reparo do DNA , Embrião de Mamíferos/citologia , Fibroblastos/metabolismo , Fase G2 , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patologia , Camundongos , Radiação Ionizante , Células-Tronco/metabolismo , Proteína Supressora de Tumor p53/metabolismoRESUMO
The DNA intercalator, 4'-(9-acridinylamino)methanesulfon-m-anisidide (m-AMSA) and the nonintercalator, etoposide (VP-16) produce topoisomerase II-mediated protein-linked DNA strand breaks. This function of topoisomerase II was investigated in relation to cell proliferation and cell cycle. Mouse fibroblasts NIH 3T3 and mouse leukemia L1210 cells stop proliferation when they reach a certain density. Nuclei were isolated from proliferative or quiescent cells and then treated with drug for 30 min. DNA modifications were assayed by alkaline elution. We found that the frequencies of m-AMSA- or VP-16-induced DNA-protein links were higher in nuclei from exponentially growing than in those from quiescent cells in both the 3T3 and the L1210 lines. Drug-induced protein-associated DNA breaks were also studied as a function of the cell cycle in 3T3 cells that had been arrested by contact inhibition in medium containing 1% calf serum and then stimulated to proliferate by raplating at a lower cell density in medium containing 10% serum. In these synchronized cells, a large peak of [3H]thymidine incorporation occurred 18-30 h after replating. The yield of DNA-protein cross-links produced by 30-min drug treatments of nuclei isolated at various times after growth initiation increased concomitantly with the peak of the DNA synthesis. The topoisomerase II activity of nuclear extracts, as measured by kinetoplast DNA decatenation followed a similar pattern. Using colony-forming assays, we also observed that m-AMSA and VP-16 were most cytotoxic in proliferative cells and during DNA synthesis. These results suggest that alkaline elution measurement of m-AMSA- or VP-16-induced protein-linked DNA breaks reflects the association of topoisomerase II with DNA. This association is increased during DNA replication, making the cells more vulnerable to m-AMSA and VP-16 at this time.
Assuntos
Sobrevivência Celular/efeitos dos fármacos , Dano ao DNA , DNA Topoisomerases Tipo I/fisiologia , Leucemia L1210/patologia , Amsacrina/farmacologia , Animais , Ciclo Celular/efeitos dos fármacos , Divisão Celular/efeitos dos fármacos , Linhagem Celular , DNA Topoisomerases Tipo I/análise , Etoposídeo/farmacologia , Fibroblastos/efeitos dos fármacos , Camundongos , Proteínas/metabolismoRESUMO
Ditercalinium, a 7H-pyridocarbazole dimer (bisintercalator) belongs to a new class of antineoplastic intercalating agents. To investigate its mechanism of cytotoxicity, the effects of ditercalinium on DNA were assessed using normal (L1210) and drug-resistant (L1210/PyDi1) mouse leukemia cells. Alkaline elution assays demonstrated that ditercalinium produced no DNA strand breaks, DNA-protein cross-links, or DNA-DNA cross-links, eliminating these effects as cytotoxic lesions. This result sets ditercalinium apart from other intercalating agents with respect to its interaction with DNA. Nucleoids (histone-depleted chromatin) from ditercalinium-treated L1210 cells were considerably more compact than those from untreated cells, as determined by sedimentation in neutral sucrose gradients. In contrast, nucleoids from ditercalinium-treated L1210/PyDi1 (resistant) cells were similar in compactness to those from control cells. Thus, ditercalinium altered chromatin structure in vivo. The effect of the bisintercalator on purified DNA topoisomerase II, an intracellular target of monointercalators, was measured in vitro. Ditercalinium (5 X 10(-7) M) completely inhibited both the formation of covalent complexes between this enzyme and simian virus 40 DNA and the enzyme-induced DNA cleavage. In addition, ditercalinium induced DNA catenation in the presence of topoisomerase II and adenosine triphosphate. Thus, the cytotoxicity of ditercalinium may derive from a mechanism that, although involving topoisomerase II, is manifested by condensation of DNA rather than by the induction of protein-associated DNA strand breaks.
Assuntos
Carbazóis/farmacologia , Dano ao DNA , DNA de Neoplasias/efeitos dos fármacos , Substâncias Intercalantes/farmacologia , Inibidores da Topoisomerase II , Amsacrina/farmacologia , Animais , Reagentes de Ligações Cruzadas/farmacologia , Etoposídeo/farmacologia , Leucemia L1210/enzimologia , Camundongos , Conformação de Ácido Nucleico/efeitos dos fármacos , Relação Estrutura-AtividadeRESUMO
Tyrosine phosphorylation plays a crucial role in cell proliferation and cell transformation which suggests that tyrosine kinase-specific inhibitors might be used as anticancer agents. When the cytotoxic effect of the potent tyrosine kinase inhibitor genistein on various cell lines was studied, we observed that 9-hydroxyellipticine-resistant Chinese hamster lung cells (DC-3F/9-OH-E) were markedly more resistant to genistein than the parental cell line (DC-3F). The DC-3F/9-OH-E cells have been shown to have an altered DNA topoisomerase II activity. We therefore examined the effects of genistein on DNA topoisomerase II-related activities of nuclear extracts from DC-3F cells as well as on purified DNA topoisomerase II from calf thymus. Our results show that genistein (a) inhibits the decatenation activity of DNA topoisomerase II and (b) stimulates DNA topoisomerase II-mediated double strand breaks in pBR322 DNA on sites different from those of 4'-(9-acridinylamino)methanesulfon-m-anisidide, etoposide, and 2-methyl-9-hydroxyellipticinium. Structure-activity studies with six chemically related compounds show that only genistein has an effect on the cleavage activity of DNA topoisomerase II in the concentration range studied. Finally, genistein treatment of DC-3F cells results in the occurrence of protein-linked DNA strand breaks as shown by DNA filter elution. Viscometric (lengthening) studies demonstrate that genistein is not a DNA intercalator. Genistein is therefore an interesting compound because it induces cleavable complexes without intercalation. Taken together, our results show that genistein is an inhibitor of both protein tyrosine kinases and mammalian DNA topoisomerase II. This could be accounted for by the sharing of a common structure sequence between the two proteins at the ATP binding site.
Assuntos
Dano ao DNA , DNA Topoisomerases Tipo II/metabolismo , Flavonoides/farmacologia , Isoflavonas/farmacologia , Proteínas Tirosina Quinases/antagonistas & inibidores , Sequência de Aminoácidos , Animais , Divisão Celular/efeitos dos fármacos , Linhagem Celular , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/metabolismo , Sobrevivência Celular/efeitos dos fármacos , DNA Super-Helicoidal , Receptores ErbB/efeitos dos fármacos , Receptores ErbB/metabolismo , Genisteína , Humanos , Cinética , Dados de Sequência Molecular , Fosforilação , Plasmídeos , Inibidores da Topoisomerase IIRESUMO
In the Chinese hamster lung cell line DC-3F/9-OH-E, selected for resistance to 9-OH-ellipticine and cross-resistant to other topoisomerase II inhibitors, the amount of topoisomerase IIalpha is 4-5-fold lower than in the parental DC-3F cells, whereas topoisomerase IIbeta is undetectable. Cloning and sequencing of topoisomerase IIalpha cDNAs from DC-3F and DC-3F/9-OH-E cells revealed an allele polymorphism, one allele differing from the other by the presence of seven silent mutations and three mutations in the noncoding region. In addition, the mutated allele contains three missense mutations located close to the ATP binding site (Thr371Ser) or to the catalytic site (Ala751Gly; Ile863Thr). To analyze the contribution of these topoisomerase IIalpha alterations to their resistance phenotype, DC-3F/9-OH-E cells were transfected with an eukaryotic expression vector containing the human topoisomerase IIalpha cDNA. In one transfected clone, the amount of topoisomerase IIalpha isoform and the catalytic activity were similar to that in the parental DC-3F cells. These cells, which contain only topoisomerase IIalpha, are then a unique mammalian cell line to analyze the physiological and pharmacological properties of this enzyme. However, the restoration of a nearly normal topoisomerase IIalpha activity in the DC-3F/9-OH-E cells did not have the same effect on their sensitivity to different enzyme inhibitors; a 75% reversion of the resistance, associated with a 2-3-fold increased stabilization of the cleavable complex, was observed with both etoposide and m-AMSA, two drugs that inhibit the DNA religation step in the enzyme catalytic cycle; in contrast, the transfected cells remained fully resistant to ellipticine derivatives that did not induce the stabilization of the cleavable complex. We hypothesized that a trans-acting factor, inhibiting the induction of cleavable complex formation by drugs that are not religation inhibitors, might be present in the resistant cells. However, such a factor was not detected in in vitro experiments, and other hypotheses are discussed.
Assuntos
Antimetabólitos Antineoplásicos/toxicidade , DNA Topoisomerases Tipo II/genética , DNA Topoisomerases Tipo II/metabolismo , Elipticinas/toxicidade , Isoenzimas/genética , Isoenzimas/metabolismo , Polimorfismo Genético , Amsacrina/toxicidade , Animais , Antígenos de Neoplasias , Linhagem Celular , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Cricetinae , Cricetulus , Sondas de DNA , DNA Complementar , DNA de Cinetoplasto/metabolismo , Proteínas de Ligação a DNA , Resistência a Medicamentos , Etoposídeo/toxicidade , Fibroblastos , Humanos , Fenótipo , Mutação Puntual , Reação em Cadeia da Polimerase , Proteínas Recombinantes/metabolismo , Transcrição Gênica , TransfecçãoRESUMO
We have used a mouse bone marrow transplantation (BMT) model to study the safety of retrovirus-mediated transfer of anti-HIV genes (RevM10 and HIV-1 pol antisense) into hematopoietic stem/progenitor cells (HSPCs). In particular, we have monitored the hematologic recovery post-BMT and transgene expression in myeloid and lymphoid lineages, and analyzed tissue sections for evidence of any transgene-related pathological condition. Expression of anti-HIV genes had no effect on kinetics of hematologic recovery post-BMT. The average time to reach 20% of normal cell counts was 15-17 days for white blood cells and 12-14 days for platelets, and the average time to reach complete recovery was 42-56 days for leukocytes and 104-161 days for platelets. Hematocrit levels were not significantly affected by irradiation and transplantation procedures. Donor chimerism was uniformly > or =90% in all transplanted animals. At 4-5 weeks post-BMT transgene expression was detected in peripheral blood leukocytes in 100% of the animals and ranged from 4.5 to 44.7%. In a majority of the animals the percentage of transgene-expressing cells in circulation decreased over time but remained detectable for the length of the study (>6 months). Expression was detected in all analyzed cell lineages (RBCs, platelets, monocytes, granulocytes, and T and B cells). Relative counts of various leukocytes (Mac1+ monocytes, Gr1+ granulocytes, CD3+ T cells, and B220+ B cells) were normal. There were no treatment-related histopathological changes in a wide range of tissues examined. In addition, there were no treatment effects on differential leukocyte counts, and morphology of peripheral blood and bone marrow brush smears. In summary, transfer and expression of the RevM10 and the HIV-1 antisense genes into hematopoietic stem/progenitor cells in vivo appears safe. We propose that the mouse bone marrow transplantation model could be used to evaluate some safety aspects of HSPC-based gene therapies.
Assuntos
Transplante de Medula Óssea , DNA Antissenso/genética , Produtos do Gene pol/genética , Terapia Genética , Vetores Genéticos , HIV/genética , Células-Tronco Hematopoéticas/fisiologia , Animais , Linhagem da Célula , DNA Viral/genética , Feminino , Produtos do Gene pol/biossíntese , Terapia Genética/métodos , Vetores Genéticos/genética , Vetores Genéticos/fisiologia , HIV/fisiologia , Células-Tronco Hematopoéticas/patologia , Masculino , Camundongos , Modelos Biológicos , Retroviridae/genética , Distribuição Tecidual , Transfecção , Produtos do Gene pol do Vírus da Imunodeficiência HumanaRESUMO
The DNA binding characteristics of a mono-, di- and trimeric derivative of 9-aminoacridine were studied. The length of the linking carboxamidoalkyl chains was selected to allow bis- or tris-intercalation according to the excluded-site model. Measurements of DNA unwinding angle using closed circular DNA showed that the trimeric derivative behaves as a tris-intercalating agent. Nevertheless the increase of DNA binding affinity on going from dimer to trimer was found to be relatively small. This is probably related to the large structural constraint for DNA binding of the trimeric derivative. The nature of the linking chain for the design of high-affinity DNA poly-intercalating agents appears therefore critical.
Assuntos
Aminacrina/metabolismo , Aminoacridinas/metabolismo , DNA/metabolismo , Animais , Bovinos , Fluorometria , Substâncias Macromoleculares , Poli dA-dT/metabolismo , ViscosidadeRESUMO
The presence of apurinic/apyrimidinic (AP) sites in cell genomes is known to be toxic and mutagenic. These lesions are therefore repaired in cells by efficient enzymatic systems. However, a report (Nakamura and Swenberg, Cancer Res. 59 (1999) 2522-2526) indicates an unexpected high rate of endogenous apurinic/apyrimidinic (AP) sites in genomic DNA in mammalian tissues. The technology used does not allow the authors to distinguish between intact AP sites and 3'cleaved AP sites. The corresponding values range between 2 and 4 sites per million of nucleotides in various human and rat tissues. Using a modified alkaline elution method we show here that the stationary level of intact AP sites is about 0.16 per million of nucleotides in leukemic mouse L1210 cells.
Assuntos
Ácido Apurínico/química , Dano ao DNA , DNA de Neoplasias/química , DNA/química , Animais , Ácido Apurínico/isolamento & purificação , DNA/isolamento & purificação , DNA de Neoplasias/isolamento & purificação , Humanos , Concentração de Íons de Hidrogênio , Cinética , Leucemia L1210/metabolismo , Leucemia L1210/patologia , Mamíferos , Camundongos , Oligopeptídeos , Ratos , Células Tumorais CultivadasRESUMO
The 8-methoxy- and 8-hydroxy-11H-pyrido[2,3-a]-, -[3,4-a]-, -[4,3-a]-, and [3,2-a]carbazoles were synthesized as potential DNA intercalating antitumor drugs. The structure of these compounds was confirmed by 1H NMR study including NOE experiments. The DNA binding properties of substituted and unsubstituted (8-H) heterocycles were determined by using their hydrochlorides or methiodides. These derivatives are able to bind to DNA with an affinity varying from 2.0 X 10(4) to 1.0 X 10(6) M-1, but most of them are unable to intercalate in contrast with the behavior of 6H- and 7H-pyridocarbazole analogues. The cytotoxicity of 11H-pyridocarbazoles, measured on L1210 cells in vitro, is much lower than those of 6H- and 7H-pyridocarbazole analogues.
Assuntos
Alcaloides/uso terapêutico , Antineoplásicos/uso terapêutico , DNA/metabolismo , Elipticinas/uso terapêutico , Animais , Bovinos , Fenômenos Químicos , Química , Elipticinas/síntese química , Elipticinas/metabolismo , Substâncias Intercalantes , Leucemia L1210/tratamento farmacológico , Espectroscopia de Ressonância MagnéticaRESUMO
An attempt was made to analyze the mechanism by which 4'-(9-acridinylamino)methanesulfon-m-anisidide (m-AMSA) inhibits mammalian DNA topoisomerase II. The effects of various 9-aminoacridine derivatives differing by their DNA affinities and DNA sequence selectivity of binding were compared in the presence of purified mouse leukemia L1210 DNA topoisomerase II. No correlation was found between DNA unwinding and topoisomerase II inhibition. 9-Aminoacridine was inactive as a topoisomerase II inhibitor and o-AMSA was only weakly active. The location of L1210 topoisomerase II mediated DNA breaks produced in the absence or presence of 9-aminoacridines were studied in [32P]-end-labeled pBR 322 DNA. All 9-aminoacridines, even those differing by their DNA sequence selectivity of binding, produced similar DNA cleavage patterns. Most drug-induced topoisomerase II mediated DNA breaks appeared at sites that were already cleaved by the enzyme in the absence of drug. The present results suggest that 9-aminoacridines inhibit L1210 DNA topoisomerase II by interacting at or near enzyme-DNA complexes by some unknown DNA effect or by direct protein interaction.
Assuntos
Aminoacridinas/farmacologia , Amsacrina/farmacologia , Substâncias Intercalantes/farmacologia , Leucemia L1210/enzimologia , Inibidores da Topoisomerase II , Animais , Sequência de Bases , DNA/análise , Dano ao DNA , CamundongosRESUMO
Inhibitors of protein tyrosine kinases (PTK) and DNA topoisomerases are potential antitumour agents. Drugs which bind to the ATP site of PTK, such as genistein, are common inhibitors to both types of enzymes. Eleven erbstatin and tyrphostin derivatives, which inhibit epidermal growth factor receptor PTK activity by competing with both the peptide substrate and ATP were tested for their capacity to inhibit DNA topoisomerases I and II. Erbstatin, two synthetic derivatives with a modified side chain and the tyrphostin AG 786 inhibited both topoisomerases in the same range of concentrations (20-50 microM). The tyrphostin AG 213 inhibited only topoisomerase II. In this series, absence of PTK inhibitory effect was correlated with the absence of DNA topoisomerase inhibition, while the detection of PTK inhibition may or may not be associated with DNA topoisomerase inhibition. In contrast to genistein, none of these molecules induced the stabilization of the topoisomerase-DNA cleavable complex, either in vitro or in vivo. Alcaline elution analysis revealed that erbstatin did not induce the formation of protein associated DNA strand breaks. However, an extensive degradation of the cellular DNA was observed which was shown to result from an internucleosomal fragmentation. Furthermore, typical morphological modifications associated with apoptosis were observed in the erbstatin treated cells by electron microscopy. These data indicate that erbstatin induces an apoptotic cell death.
Assuntos
Catecóis/farmacologia , Hidroquinonas/farmacologia , Nitrilas/farmacologia , Inibidores da Topoisomerase I , Inibidores da Topoisomerase II , Tirfostinas , Sequência de Aminoácidos , Animais , Apoptose , Sítios de Ligação , Linhagem Celular/efeitos dos fármacos , Cricetinae , Genisteína , Isoflavonas/farmacologia , Camundongos , Dados de Sequência Molecular , Proteínas Tirosina Quinases/antagonistas & inibidores , Células Tumorais Cultivadas/efeitos dos fármacosRESUMO
Genistein, an isoflavonoid derivative initially described as an in vitro protein tyrosine kinase inhibitor, also inhibits mammalian DNA topoisomerase II both in vitro and in vivo. From a human leukaemic T cell line (CCRF-CEM), two genistein-resistant cell lines, which grow in the presence of 50 and 150 microM genistein, respectively, were selected and designated CEM/GN50 and CEM/GN150. Flow cytometry and karyotype analyses revealed that more than 95% of the parental cells were tetraploid whereas both resistant sublines were essentially diploid and were likely derived from the diploid fraction in the initial population. The CEM/GN cells were 3- to 4-fold resistant to genistein, and highly cross-resistant to certain metabolic inhibitors such as cytosine-arabinoside (50-fold) and 5-fluoro-2'-deoxyuridine (5000-fold). This resistance was associated with a markedly decreased uptake of thymidine and a 10-fold reduction in thymidine kinase activity. The CEM/GM cells were also 15- to 30-fold cross-resistant to topoisomerase inhibitors (etoposide, m-AMSA, 2-Me-9-OH-ellipticinium). Comparison of topoisomerase II activities in the sensitive and resistant cells showed: (i) an approximately 2-fold reduced decatenation activity in nuclear extracts from the resistant cells; (ii) an approximate 30% reduction in DNA-protein cross-links in etoposide-treated resistant cells; and (iii) a markedly reduced expression of the topoisomerase II beta isoform. These data, consistent with our previous results, indicate that the cytotoxicity of genistein is at least in part related to its capacity to inhibit DNA topoisomerase II.
Assuntos
Isoenzimas/antagonistas & inibidores , Isoflavonas/farmacologia , Inibidores da Topoisomerase II , Animais , DNA Topoisomerases Tipo II/genética , Diploide , Resistência a Medicamentos , Feminino , Citometria de Fluxo , Floxuridina/metabolismo , Floxuridina/farmacologia , Genisteína , Humanos , Cariotipagem , Camundongos , Camundongos Nus , Transplante de Neoplasias , Células Tumorais Cultivadas/efeitos dos fármacos , Células Tumorais Cultivadas/enzimologia , Células Tumorais Cultivadas/ultraestruturaRESUMO
The effects of vitamin K3 (VK3) on DNA synthesis, cell proliferation and mitogen-activated protein kinase pathway were investigated in G0-arrested NIH 3T3 fibroblasts. VK3 (5 microM) alone stimulates DNA synthesis by 40% and moderately increases the mitogenic effects of EGF, which is preceded by a rapid phosphorylation of the extracellular signal-regulated kinases (ERKs). At 20 microM, VK3 had an antiproliferative effect. VK3 alone (5 and 50 microM) or in concert with EGF increases the activity of ERK2 (by 2.5 and 5 fold, respectively). Our studies demonstrate that the activation of ERKs by VK3 alone, or VK3 plus EGF can promote either stimulatory or inhibitory effects on the mitogenic signal.
Assuntos
Proteínas Quinases Dependentes de Cálcio-Calmodulina/metabolismo , Proteínas de Ciclo Celular , Fator de Crescimento Epidérmico/metabolismo , Fosfoproteínas Fosfatases , Proteínas Tirosina Fosfatases/metabolismo , Vitamina K/farmacologia , Células 3T3 , Animais , Sítios de Ligação , Divisão Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , DNA/biossíntese , DNA/efeitos dos fármacos , Fosfatase 1 de Especificidade Dupla , Fator de Crescimento Epidérmico/fisiologia , Proteínas Imediatamente Precoces/metabolismo , Camundongos , Proteína Quinase 1 Ativada por Mitógeno , Mitógenos/farmacologia , Peptídeos/metabolismo , Proteína Fosfatase 1 , Transdução de Sinais/efeitos dos fármacos , Fatores de TempoRESUMO
DNA topoisomerases regulate the organization of DNA and are important targets for many clinically used antineoplastic agents. In addition, DNA topoisomerases modulate the cellular sensitivity toward a number of DNA damaging agents. Increased topoisomerase II activities were shown to contribute to the resistance of both nitrogen mustard- and cisplatin-resistant cells. Similarly, cells with decreased topoisomerase II levels show increased sensitivity to cisplatin, carmustine, mitomycin C and nitrogen mustard. Recent studies propose that topoisomerases may be involved in damage recognition and DNA repair at several different levels including: 1) the initial recognition of DNA lesions; 2) DNA recombination; and 3) regulation of DNA structure. The stress-activated oncogene suppressor protein p53 can modulate the activity of at least three different human topoisomerases, either directly by molecular associations or by transcriptional regulation. Since DNA topoisomerases have considerable recombinase activities, inappropriately activated topoisomerases in tumor cells lacking functional p53 may contribute to the genetic instability of these cells.
Assuntos
Reparo do DNA , DNA Topoisomerases Tipo I , Proteína Supressora de Tumor p53 , Animais , Cricetinae , DNA/genética , Humanos , Recombinação Genética , Células Tumorais CultivadasRESUMO
Two 1-nitro-9-aminoacridine dimers were prepared: one bearing a spermine flexible linking chain, compound 4, the other a rigid dipiperidine-type linker, compound 7. Both dimers elicited a higher affinity constant for DNA than the parent monomeric drug nitracrine 2. This affinity was several orders lower than what was found for other dimeric compounds having the same linkers and no nitro group on the acridine ring (3, 5, 6 and 8). Bisintercalation was evidenced for compound 4 by viscosimetric measurements. In the absence of dithiothreitol, an inhibitory effect of RNA synthesis in vitro was observed for all the tested compounds except 2 and 7. In the presence of dithiothreitol, 4 and 7 formed irreversible complexes with DNA of decreased template properties. The level of the dimers binding was lower than that of the parent compound 2. Cross-links were detected by means of hydroxylapatite chromatography in a complex of the dimer bearing a flexible linking chain, compound 4 with DNA, while the compound 7-DNA complex eluted in the single-stranded DNA region. The extent of cytotoxicity of the two 1-nitro-9-aminoacridine dimers against L1210 cultured cells was different.
Assuntos
Aminoacridinas/farmacologia , DNA/metabolismo , RNA/biossíntese , Aminoacridinas/síntese química , Aminoacridinas/metabolismo , Animais , Antineoplásicos/síntese química , Ditiotreitol/farmacologia , Células HeLa , Humanos , Substâncias Intercalantes/síntese química , Leucemia L1210/tratamento farmacológico , Camundongos , Células Tumorais CultivadasRESUMO
The effect of dimeric DNA intercalating compounds was assayed on a purified AP endonuclease from Microccoccus luteus using apurinic supercoiled PM2 DNA as a substrate. Binding on apurinic sites was estimated through the competition with the intercalating compound, 9-NH2-ellipticine, which displays great specificity for apurinic sites. An acridine dimer with a spermine linker is at 0.1 microM the best inhibitor of cleavage at the apurinic site induced either by the AP endonuclease or by 9-NH2-ellipticine. Bisintercalating agents are more effective inhibitors of AP endonuclease than monointercalating ones. Most effective inhibitors among dimers have acridine residues.
Assuntos
Acridinas/farmacologia , Ácido Apurínico/metabolismo , DNA Super-Helicoidal/metabolismo , Endodesoxirribonucleases/antagonistas & inibidores , Micrococcus/enzimologia , Polinucleotídeos/metabolismo , Sítios de Ligação , Ligação Competitiva , DNA Liase (Sítios Apurínicos ou Apirimidínicos) , Desoxirribonuclease IV (Fago T4-Induzido) , Elipticinas/metabolismo , Temperatura Alta , Substâncias Intercalantes , Estrutura MolecularRESUMO
Dactylaria gallopava was isolated from brain tissue of 1-to-3-week-old quail chicks. Successive batches demonstrated elevated (15-20%) mortality preceded by incoordination and lateral recumbency. Chicks exhibited cerebellar and cerebral encephalitis characterized by brown-red discoloration of affected brain tissue. Decontamination of setters and hatchers resulted in abrupt cessation of mortality in subsequent placements, implicating incubators as the source of infection.
Assuntos
Encefalite/veterinária , Micoses/veterinária , Doenças das Aves Domésticas/microbiologia , Animais , Coturnix , Encefalite/etiologia , Encefalite/patologia , Micoses/complicaçõesRESUMO
Chinese hamster lung cells resistant to 9-OH-ellipticine (DC-3F/9-OH-E) present a complex phenotype. These cells, which are about 150-fold resistant to 9-OH-E, display a cross-resistance to other topo-II inhibitors, such as m-AMSA or VP-16, which stabilize the cleavable complex. In addition, these cells display also a cross-resistance to suramin, which is also a topo-II inhibitor, but does not stabilize the cleavable complex. Finally, DC-3F/9-OH-E present a multidrug-resistant phenotype (MDR) which confers a cross-resistance to natural products such as actinomycin D, taxol or vincristine, due to a decrease of cellular accumulation of these drugs. Analysis of expression of the genes encoding topo-II alpha and beta, and the evaluation of both enzyme forms by immunoblotting, revealed that DC-3F cells contained about 20-fold less of the beta form than of the alpha form. The alpha form was decreased by about 4-5-fold in DC-3F/9-OH-E, whereas the beta form became undetectable. Purification and characterization of topo-II activities in sensitive and resistant cells is presently in progress. Analysis of the expression of pgp1, 2, 3 genes, involved in the MDR phenotype in hamster, by Northern blotting or by immunoblotting, has shown that the MDR phenotype in DC-3F/9-OH-E cells is due to the overexpression of pgp1 gene. In these cells, pgp3 expression is positively regulated by myc oncogene expression. Overexpression of the myc gene is followed by an overexpression of the pgp3 gene and is associated to a reversal of the MDR phenotype.
Assuntos
Resistência a Medicamentos/genética , Pulmão/enzimologia , Inibidores da Topoisomerase II , Animais , Antineoplásicos/antagonistas & inibidores , Antineoplásicos/farmacologia , Divisão Celular , Linhagem Celular , Cricetinae , DNA Topoisomerases Tipo II/genética , Resistência a Múltiplos Medicamentos/genética , Elipticinas/antagonistas & inibidores , Elipticinas/farmacologia , Regulação Enzimológica da Expressão Gênica , Pulmão/citologia , Células Tumorais Cultivadas/efeitos dos fármacosRESUMO
Parasite-free, 4-month-old-calves were inoculated with Ostertagia ostertagi and/or Trichostrongylus axei, followed 6 weeks later by inoculation with increasing doses of O ostertagi for 8 weeks in the 2 groups (n = 4) of calves that had been given O ostertagi. Gastrin immunoreactivity concentration in serum was measured before and after infection and was correlated with changes in mucosal thickness. Gastrin immunoreactivity concentration in preinoculation control sera ranged from 95.2 to 287.1 pg/ml, and increased values were measured in all parasitized calves after 15 weeks. Significantly (P less than 0.05) increased serum gastrin immunoreactivity concentration compared with the preinfection value, was found in calves infected with O ostertagi or T axei, and highly significant (P less than 0.01) values were observed in calves infected with both parasites. Abomasal mucosal hyperplasia was observed in all parasitized calves; increased mucosal thickness and mucosal cross-sectional area were most prominent in calves infected with O ostertagi and T axei.