ABSTRACT
To identify the component(s) involved in cell cycle control in the protozoan Giardia lamblia, cells arrested at the G1/S- or G2-phase by treatment with nocodazole and aphidicolin were prepared from the synchronized cell cultures. RNA-sequencing analysis of the 2 stages of Giardia cell cycle identified several cell cycle genes that were up-regulated at the G2-phase. Transcriptome analysis of cells in 2 distinct cell cycle stages of G. lamblia confirmed previously reported components of cell cycle (PcnA, cyclin B, and CDK) and identified additional cell cycle components (NEKs, Mad2, spindle pole protein, and CDC14A). This result indicates that the cell cycle machinery operates in this protozoan, one of the earliest diverging eukaryotic lineages.
Subject(s)
Aphidicolin , Cell Culture Techniques , Cell Cycle , Cell Cycle Checkpoints , Cyclin B , Gene Expression Profiling , Genes, cdc , Giardia lamblia , Giardia , Nocodazole , Spindle PolesABSTRACT
<p><b>OBJECTIVE</b>Alkaline comet assay was used to evaluate DNA repair (nucleotide excision repair, NER) capacity of human fresh lymphocytes from 12 young healthy non-smokers (6 males and 6 females).</p><p><b>METHODS</b>Lymphocytes were exposed to UV-C (254 nm) at the dose rate of 1.5 J/m2/sec. Novobiocin (NOV) and aphidicolin (APC), DNA repair inhibitors, were utilized to imitate the deficiency of DNA repair capacity at the incision and ligation steps of NER. Lymphocytes from each donor were divided into three grougs: UVC group, UVC plus NOV group, and UVC plus APC group. DNA single strand breaks were detected in UVC irradiated cells incubated for 0, 30, 60, 90, 120, 180, and 240 min after UVC irradiation. DNA repair rate (DRR) served as an indicator of DNA repair capacity.</p><p><b>RESULTS</b>The results indicated that the maximum DNA damage (i.e. maximum tail length) in the UVC group mainly appeared at 90 min. The ranges of DRRs in the UVC group were 62.84%-98.71%. Average DRR value was 81.84%. The DRR difference between males and females was not significant (P < 0.05). However, the average DRR value in the UVC plus NOV group and the UVC plus APC group was 52.98% and 39.57% respectively, which were significantly lower than that in the UVC group (P < 0.01).</p><p><b>CONCLUSION</b>The comet assay is a rapid, simple and sensitive screening test to assess individual DNA repair (NER) capacity. It is suggested that the time to detect DNA single strand breaks in comet assay should include 0 (before UV irradiation), 90 and 240 min after exposure to 1.5 J x m(-2) UVC at least. The DRR, as an indicator, can represent the individual DNA repair capacity in comet assay.</p>
Subject(s)
Adult , Female , Humans , Male , Aphidicolin , Pharmacology , Comet Assay , Methods , DNA Damage , Radiation Effects , DNA Repair , Genetics , Radiation Effects , Enzyme Inhibitors , Pharmacology , Lymphocytes , Metabolism , Radiation Effects , Novobiocin , Pharmacology , Risk Assessment , Time Factors , Ultraviolet RaysABSTRACT
<p><b>OBJECTIVE</b>To assess DNA repair capacity of human lymphocytes with comet assay.</p><p><b>METHODS</b>Fresh lymphocytes form twelve 26-year old donors (6 males, 6 females) were exposed to ultraviolet C (UVC, 254 nm) at the dose rate of 1.5 J/m(2). The lymphocytes of each donor were divided into three parts: UVC group, UVC + aphidicolin (APC) group, UVC + novobiocin (NOV) group. DNA single strand breaks were detected with comet assay in UVC-irradiated cells and unirradiated cells incubated for 30, 60, 90, 120, 180 and 240 min. DNA repair rate (DRR) was calculated and served as an indicator of DNA repair capacity.</p><p><b>RESULTS</b>The maximum average comet tail length (MTL) in three groups appeared 90 min after UVC exposure. The DRR range of UVC group was 81.84% (62.84% - 98.71%); There was no significant difference in DRR between males and females (P > 0.05). However, the average DRRs of UVC + NOV group and UVC + APC group (52.98% and 39.57% respectively) were significantly lower than that of UVC group (P < 0.01).</p><p><b>CONCLUSION</b>Comet assay is a rapid and simple screening test to assess DNA repair capacity. DRR, as an indicator, may express the individual DNA repair capacity.</p>
Subject(s)
Female , Humans , Male , Aphidicolin , Pharmacology , Comet Assay , Methods , DNA , Genetics , Radiation Effects , DNA Repair , Enzyme Inhibitors , Pharmacology , Lymphocytes , Metabolism , Radiation Effects , Novobiocin , Pharmacology , Ultraviolet RaysABSTRACT
Aphidicolin (APC)-induced chromosomal breakage was analyzed for women representing three generations of a single family and carrying a Robertsonian translocation rob(14q21q). Fluorescence in situ hybridization (FISH) analysis confirmed the dicentric constitution of the derived chromosome and indicated the absence of beta-satellite signal at the translocation region. Per-individual analysis of metaphases from APC-treated peripheral blood lymphocyte cultures identified significantly nonrandom chromosomal breakage at the translocation region in all three individuals examined. The APC-inducible fragility at the 14q21q translocation region suggests that this rearrangement was the result of chromosomal mutation at fragile site(s) in the progenitor chromosomes, or that this fragility was the result of the fusion of nonfragile progenitor chromosomes
Subject(s)
Humans , Female , Adult , Middle Aged , Aphidicolin , Chromosomes, Human, Pair 14 , Chromosomes, Human, Pair 21 , Chromosome Fragility/genetics , In Situ Hybridization, Fluorescence , Translocation, GeneticABSTRACT
BACKGROUND: Mutations in the human Cu, Zn-superoxide dismutase (SOD1) gene have been identified in some cases of familial amyotrophic lateral sclerosis (ALS). Neuronal cells with mutant SOD1 gene promoted cell death during differentiation by dibutyryl cAMP and aphidicolin. The aim of this study is to delineate if there is an impairment of the neural differentiation process in mutant SOD1 cells. METHODS: We studied the motoneuron-neuroblastoma hybrid cells (VSC 4.1) expressing wild-type or mutant SOD1 (G93A) during the differentiation by dibutyryl cAMP and aphidicolin. RESULTS: Mutant SOD1 cell (G93A) showed an impairment in the neurite formation. Western blot analysis revealed that the amount of neurofilament decreased before differentiation. A decrease in the amount of MAP-2 is observed during differentiation. CONCLUSIONS: Our results suggest that the impairment in the neurite formation of mutant SOD1 cell (G93A) is a differentiation failure and is associated with neuronal cell death.
Subject(s)
Humans , Amyotrophic Lateral Sclerosis , Aphidicolin , Blotting, Western , Cell Death , Hybrid Cells , Neurites , NeuronsABSTRACT
BACKGROUND: Mutations in the human Cu, Zn-superoxide dismutase(SOD1) gene have been identified in some cases of familial amyotrophic lateral sclerosis(ALS). The aim of this study is to delineate the effect of the SOD1 mutation on neural differentiation, and to investigate the mechanism of neuronal death. METHODS: We studied motorneuron-neurob-lastoma hybrid cells(VSC 4.1) expressing wild type or mutant SOD1(G93A, A4V) during differentiation by dibutyryl cAMP and aphidicolin. RESULTS: Mutant cells(G93A) revealed a decreased viability compared with the control cells, mainly in the early stage ofdifferentiation. The release of cytochrome c and increased nuclear fragmentation were observed. However, cell death was not protected by nonselective caspase inhibitor(z-VAD-fmk), but by the antioxi-dant( Trolox). CONCLUSIONS: The results suggest that oxidative stress may be the main mechanism of neuronal death, particularly in the early stage of differentiation.
Subject(s)
Humans , Aphidicolin , Cell Death , Cytochromes c , Motor Neurons , Neurons , Oxidative StressABSTRACT
An aphidicolin is a chemical agent which selectively inhibits DNA polymerase alpha in S phase of cell cycle. The purpose of this study is toinvestigate of chromosomal abnormalities including fragile sites induced by 0.2 microgram/ml and 0.4 ng/ml aphidicolin in lymphocyte cultures of six healthy individuals. The results were follows. 1. A significant decreasing in mitotic indexes in respect to control culture was observed with both aphidicolin concentrations used. 2. The cells showing chromosome aberrations and the total number of cytogeneticic alterations were significantly increased both aphidicolin treated cultures than control cultures. 3. The total numbers of chromosomal aberrations were increased in the concentration of 0.4 microgram/ml aphidicolin compared to 0.2 microgram/ml treated groups. 4. The most frequent type of chromosomal aberration is a gap. 5. A site showing a gap or break was defined as common fragile sites (c-fra) if it appeared more than 1% of cells analyzed and in at least three of six individuals studied with the same culture treatment. Using these criteria, 3p14, 4q12, 5p13, 6q16, 9p13, and 16q23 were induced in different proportions by different concentration of aphidicolin and four of these c-fras, 4q12, 5p13, 6q16, 9p13 have not been reported so far. This results support that aphidicolin induced fragile sites differently according to cultured cell or cultured conditions, and also suggest the mechanism that common fragile sites caused be closely related with the defect of DNA synthesis in the S phase of cell cycle.
Subject(s)
Humans , Aphidicolin , Cell Cycle , Cells, Cultured , Chromosome Aberrations , Cytogenetics , DNA , DNA Polymerase I , Lymphocytes , Mitotic Index , S PhaseABSTRACT
To investigate fragile sites induced by aphidicolin which is a specific inhibitor of eukaryotic DNA polymerase a which is primarily associated with chromosomal DNA replication in human lymphocytes, HaCat cells (human keratinocytes) and MRC-5 cells (human embryonic lung fibroblast), we cultured each cells in RPMI 1640 with 10% fetal calf serum and 2% PHA. Treatment of the cells with aphidicolin was generally carried out for the last 24 hours of culturing. The drug was dissolved in DMSO and used at final concentrations of 0.05~0.15 mg/ml, corresponding to a maximum DMSO concentration of 0.028%. Karyotypes of each cells were performed by routine method, and 50 metaphases were scored for each culture for analysis of breakage rate. Experimental cells treated with APC showed a dose dependent sensitivity and the amounts of chromosome breakage induced by APC are the highest in concentration of 0.15 mg/ml. The frequency of fragile sites on each cells appeared in MRC-5 cells, lymphocytes and HaCat cells in order. The common fragile sites on all experiments was 16q23, and the common fragile sites on embryonic cells was 1p31. It can be concluded that gene or nucleic acid which is located on 16q23 is the most important factor to induce chromosomal breakage with sensitivity to aphidicolin and 1p31 is important site to induce chromosomal breakage in embryonal cells.
Subject(s)
Humans , Aphidicolin , Chromosome Breakage , Dimethyl Sulfoxide , DNA , DNA Replication , Karyotype , Lung , Lymphocytes , MetaphaseABSTRACT
Amphidicolin (APC) e 3-aminobenzamide (3AB) foram utilizados para elucidar o papel de processos de reparo de DNA no aumento de aberraçöes induzidas por Raio-X em culturas de linfócitos de pacientes com Síndrome de Down, comparadas com controles normais. Estimulados por fitohemaglutinina (PHA) (Fase G1), os controles normais mostraram um aumento significativo na produçäo de dicêntricos, mas näo de acêntricos. Em culturas de Downs as células estimuladas mostraram um aumento significativo na produçäo de dicêntricos e acêntricos. O efeito de APC, em contraste, foi menos pronunciado sem estimulaçäo. Na condiçäo estimulado, como para 3AB, o efeito de APC foi maior em controles normais. O processamento diferencial de dados genéticos induzidos por Raio-X em linfócitos periferais de Downs, é sensível a presença de APL e 3AB. É provável que ambos, DNA polymerase Ó e poly (ADP-ribose) polimerase tem comportamento diferenciado intrínsico nos linfócitos de Downs sensíveis a PHA
Subject(s)
Humans , Animals , Cricetinae , Aphidicolin/pharmacology , Benzamides/pharmacology , Chromosome Aberrations , Lymphocytes , DNA Repair , Down Syndrome/pathology , Cricetulus , DNA Polymerase II , Mammals , PhytohemagglutininsABSTRACT
En la actualidad se acepta que muchas neoplasias humanas están causadas por factores y muchos mutágenos químicos inducen aberraciones cormosómicas (AC), las caules se cree que se origina por roturas en zonas cromosómicas específicas o sitios frágiles (SF). Los SF se estudiaron en cultivos de linfocitos expuestos a diversos inductores químicos, pero todavia no se conoce como influyen los factores ambientales en su expresión. Hasta ahora no hay estudios de SF inducidos con rayos X, ni se conoce la interacción de este agente con los inductores quimicos. Este es el primer trabajo que analiza la expresión de SF inducida por rayos X y por 3 inductores de SF: BUDR, FUDR y anfidicolina. Se identificaron 17 bandas cromosómicas significtivamente afectadas (p<0.oo1), que se definieron como SF. Los SF más frecuentes estaban localizados en las bandas 3p14 y 16q23. Se observó un aumento significativo (p<0.01) de AC y SF en el cultivo expuesto a rayos X y en el tratado con FUDR más radiación, indicando la conveniencia de emplear otro agente radiosensibilizador. Los resultados observados sugerirían que muchos SF pueden ser causados por factores ambientales tales como sustancias químicas o radiación. La alta correlación establecida entre los SF y la ubicación de AC inducidos por radiación, AC en cáncer y oncogenes demostraría la importante interacción entre SF, AC y oncogenes en el proceso neoplásico