RESUMO
Analyses of structural genome variation by array-CGH have dramatically enhanced our ability to detect copy number variations (CNVs). De novo CNVs and those co-segregating with disease in a family are generally interpreted as pathogenic. Yet, often CNVs, such as recurrent microdeletions in region 15q13.3, are not so clearly pathogenic. Here we discuss potential confounding mechanisms that may lead to the phenotypic pleiotropy of CNVs, such as unmasking of recessive alleles by hemizygous deletions, interaction of CNVs with other loci and genes, genetic epistasis, allelic exclusion, and somatic mosaicism. We illustrate some of these mechanisms with a detailed analysis of recent studies of CNVs involving MCPH1, AUTS2, CNTNAP2, and mutations in GRIN2B. Next we discuss the clinical ramifications of these findings and urge workers to avoid 'diagnostic fatalism' (i.e., halting all genetic investigation after the detection of a single CNV) and address some of the future challenges likely to result from implementations of next generation sequencing techniques.
Assuntos
Transtorno Autístico/genética , Epilepsia/genética , Esquizofrenia/genética , Transtorno Autístico/diagnóstico , Aberrações Cromossômicas , Cromossomos Humanos Par 15 , Hibridização Genômica Comparativa , Variações do Número de Cópias de DNA , Epilepsia/diagnóstico , Epistasia Genética , Genes Recessivos , Pleiotropia Genética , Estudo de Associação Genômica Ampla , Genômica , Humanos , Esquizofrenia/diagnósticoRESUMO
A theoretical and experimental investigation is presented on the intermodal coupling between the flexural vibration modes of a single clamped-clamped beam. Nonlinear coupling allows an arbitrary flexural mode to be used as a self-detector for the amplitude of another mode, presenting a method to measure the energy stored in a specific resonance mode. The observed complex nonlinear dynamics are quantitatively captured by a model based on coupling of the modes via the beam extension; the same mechanism is responsible for the well-known Duffing nonlinearity in clamped-clamped beams.
RESUMO
We have measured the backaction of a dc superconducting quantum interference device (SQUID) position detector on an integrated 1 MHz flexural resonator. The frequency and quality factor of the micromechanical resonator can be tuned with bias current and applied magnetic flux. The backaction is caused by the Lorentz force due to the change in circulating current when the resonator displaces. The experimental features are reproduced by numerical calculations using the resistively and capacitively shunted junction model.
RESUMO
Autism spectrum disorder (ASD) represents a set of neurodevelopmental disorders with a strong genetic aetiology. Chromosomal rearrangements have been detected in 5-10% of the patients with ASD, and recent applications of array comparative genomic hybridisation (aCGH) are identifying further candidate regions and genes. In this study, we present four patients who implicate microcephalin 1 (MCPH1) in band 8p23.1 as an ASD susceptibility gene. Patient 1 was a girl with a syndromic form of autistic disorder satisfying the Autism Diagnostic Interview-Revised (ADI-R), Autism Diagnostic Observation Schedule (ADOS) and Diagnostic and Statistical Manual of Mental Disorders (DSM-IV) criteria. Oligonucleotide aCGH (oaCGH) showed that she had a classic inv dup del(8)(qter-> p23.1::p23.1-> p21.2) containing at least three candidate genes; MCPH1 and DLGAP2 within the 6.9-Mb terminal deletion and NEF3 within the concomitant 14.1-Mb duplication. Three further patients with MCPH1 copy number changes were found using single-nucleotide polymorphism (SNP) array analysis in a cohort of 54 families with ASD patients. Our results show that ASD can be a component of the classical inv dup del(8) phenotype and identify changes in copy number of MCPH1 as a susceptibility factor for ASD in the distal short arm of chromosome 8.
Assuntos
Transtornos Globais do Desenvolvimento Infantil/genética , Cromossomos Humanos Par 8/genética , Variações do Número de Cópias de DNA/genética , Predisposição Genética para Doença/genética , Proteínas do Tecido Nervoso/genética , Fenótipo , Proteínas de Ciclo Celular , Criança , Transtornos Globais do Desenvolvimento Infantil/patologia , Pré-Escolar , Análise Citogenética , Proteínas do Citoesqueleto , Feminino , Humanos , Masculino , Polimorfismo de Nucleotídeo ÚnicoRESUMO
A female infant with dysmorphic facial features, psychomotor retardation, and clitoris hypertrophy is described. Molecular cytogenetic analyses revealed a de novo unbalanced translocation, causing partial monosomy 1p36 and partial trisomy 18q22. Monosomy 1p was confirmed by FISH, and trisomy of the distal part of chromosome 18q was demonstrated by microFISH. Gene copy number changes in these chromosomal regions were determined by array-CGH. The absence of a number of facial dysmorphic signs, and the presence of clitoris hypertrophy indicate that the combination of a del(1p36->pter) with a dup(18q22->qter) may lead to a unique phenotypic constellation. The findings at birth and at age 12 years in our patient are compared with genotype-phenotype correlations discussed in the literature.
Assuntos
Transtornos Cromossômicos/genética , Cromossomos Humanos Par 18 , Cromossomos Humanos Par 1 , Clitóris/anormalidades , Deficiência Intelectual/genética , Translocação Genética , Virilismo , Anormalidades Múltiplas/genética , Transtornos Cromossômicos/patologia , Feminino , Humanos , Hibridização in Situ Fluorescente , Lactente , Recém-NascidoRESUMO
Autocrine-secreted melanoma tumor growth-inhibiting activity (MIA, approximately Mr 8000) was isolated from supernatants of a malignant melanoma cell line HTZ-19 dM, established from a central nervous system-melanoma metastasis. Cell cycle kinetic analysis performed with bromodeoxyuridine/Hoechst flow cytometry revealed a MIA-sensitive period at the G0/G1 to S traverse; MIA mediated prolongation of the S-phase and increased arrest of cells in the G2 compartment. Growth inhibition by MIA is cell-density dependent; maximal effect is seen at low densities, and the effect may be partially antagonized by whole serum. MIA may cause growth stimulation at high cell densities and low MIA concentrations. The effect of MIA on different histological neuroectodermal cell types was compared by the same methodology: proliferation of a second malignant melanoma was inhibited, and no effect was observed with an ependymoma; 2 glioblastomas were slightly stimulated. Effects on human fibroblast-like cell strains were inconsistent. The mechanism of MIA is discussed in relation to other endogenous autocrine growth inhibitors.
Assuntos
Divisão Celular/efeitos dos fármacos , Inibidores do Crescimento/farmacologia , Melanoma/patologia , Fase S/efeitos dos fármacos , Contagem de Células/efeitos dos fármacos , Ciclo Celular/efeitos dos fármacos , Humanos , Cinética , Células Tumorais CultivadasRESUMO
Quantum information processing holds great promise for communicating and computing data efficiently. However, scaling current photonic implementation approaches to larger system size remains an outstanding challenge for realizing disruptive quantum technology. Two main ingredients of quantum information processors are quantum interference and single-photon detectors. Here we develop a hybrid superconducting-photonic circuit system to show how these elements can be combined in a scalable fashion on a silicon chip. We demonstrate the suitability of this approach for integrated quantum optics by interfering and detecting photon pairs directly on the chip with waveguide-coupled single-photon detectors. Using a directional coupler implemented with silicon nitride nanophotonic waveguides, we observe 97% interference visibility when measuring photon statistics with two monolithically integrated superconducting single-photon detectors. The photonic circuit and detector fabrication processes are compatible with standard semiconductor thin-film technology, making it possible to implement more complex and larger scale quantum photonic circuits on silicon chips.
RESUMO
DNA replication has been studied in in vitro cultured bovine liver cells permeabilized in 0.02% Triton X-100. The Km for TTP was 20 microM. The initial incorporation rate at 10 microM TTP concentration was about 12% of the in vivo synthesis and declined very strongly within 1 h. A similar decline of the incorporation rate was found at 0.12 microM TTP concentration. DNAase I digestion of DNA-matrix complexes obtained from isolated nuclei in 2 M NaCl revealed that newly replicated DNA was preferentially bound to the nuclear matrix. A similar digestion with S1 nuclease caused a selective release of short duplexes of Okazaki fragments with the complementary parental strand. The results show that in vivo replication continues in permeabilized cells in an almost unchanged way, except for a gradual decline of its rate which is mainly due to inactivation of one or more essential components.
Assuntos
Permeabilidade da Membrana Celular/efeitos dos fármacos , Núcleo Celular/metabolismo , Replicação do DNA/efeitos dos fármacos , Detergentes/farmacologia , Fígado/metabolismo , Polietilenoglicóis/farmacologia , Tensoativos/farmacologia , Animais , Bovinos , Núcleo Celular/efeitos dos fármacos , Células Cultivadas , Desoxirribonuclease I , Cinética , Fígado/efeitos dos fármacos , OctoxinolRESUMO
A flow cytometric method to determine cellular GSH contents has been developed. This method is fast and simple and enables the determination of GSH contents in intact cells. Results obtained with the new method correlate well with the results obtained by a specific biochemical assay for GSH (r = 0.9984; n = 7). The method has been used to determine GSH recovery rates in cultured fibroblasts from healthy subjects and from patients with Werner's syndrome, Spielmeyer-Vogt syndrome and Fanconi's anemia. No obvious differences in GSH recovery rates were observed. GSH recovery rates were also not affected after in vitro ageing. Experiments with cells deficient in GSH synthetase revealed that the observed GSH recovery is exclusively due to de novo synthesis.
Assuntos
Envelhecimento/metabolismo , Glutationa/biossíntese , Doenças Metabólicas/metabolismo , Adolescente , Adulto , Pré-Escolar , Feminino , Fibroblastos/metabolismo , Citometria de Fluxo , Humanos , Técnicas In Vitro , MasculinoRESUMO
Oxysterols are cytotoxic agents. The gallbladder epithelium is exposed to high concentrations of oxysterols, and so elucidating the mechanisms of cytotoxicity in this organ may enhance our understanding of the pathogenesis of biliary tract disorders. We investigated the cytotoxic effects of the oxysterol cholestan-3beta,5alpha,6beta-triol (TriolC) on dog gallbladder epithelial cells. Apoptosis was the major form of cytotoxicity, as determined by analysis of nuclear morphologic changes and by multiparameter flow cytometry. Hydrophobic bile salts are known to have cytotoxic effects, whereas hydrophilic bile salts have cytoprotective effects. We therefore examined whether the hydrophobic bile acid taurodeoxycholic acid (TDC) and the hydrophilic bile acid tauroursodeoxycholic acid (TUDC) had modifying effects on oxysterol-induced cytotoxicity. TriolC caused an increase in the number of apoptotic cells from 14+/-11% (control) to 48+/-12% of total cells (P<0.01). After combining TriolC with TDC, cell apoptosis increased to 63+/-16% (P<0.05), whereas after addition of TUDC, the number of apoptotic cells decreased to 31+/-12% (P<0.05) of total cells. In summary, oxysterols such as TriolC induce apoptosis. Hydrophobic bile salts enhance TriolC-induced apoptosis, whereas hydrophilic bile salts diminish TriolC-induced apoptosis. These results suggest that interactions between oxysterols and bile salts play a role in the pathophysiology of biliary tract disorders.
Assuntos
Apoptose/efeitos dos fármacos , Ácidos e Sais Biliares/farmacologia , Colestanóis/farmacologia , Vesícula Biliar/efeitos dos fármacos , Hipolipemiantes/farmacologia , Animais , Ciclo Celular/efeitos dos fármacos , Células Cultivadas , Cães , Interações Medicamentosas , Células Epiteliais/efeitos dos fármacos , Citometria de Fluxo , Vesícula Biliar/fisiologiaRESUMO
In combination with other factors, hyperglycemia may cause the accelerated progression of atherosclerosis in people with diabetes. Arterial smooth muscle cell (SMC) proliferation and accumulation contribute to formation of advanced atherosclerotic lesions. Therefore, we investigated the effects of hyperglycemia on SMC proliferation and accumulation in vivo and in isolated arteries and SMCs by taking advantage of a new porcine model of diabetes-accelerated atherosclerosis, in which diabetic animals are hyperglycemic without receiving exogenous insulin. We show that diabetic animals fed a cholesterol-rich diet, like humans, develop severe lesions of atherosclerosis characterized by SMC accumulation and proliferation, whereas lesions in nondiabetic animals contain fewer SMCs after 20 weeks. However, high glucose (25 mmol/l) does not directly stimulate the proliferation of SMCs in isolated arterial tissue from diabetic or nondiabetic animals, or of cultured SMCs from these animals or from humans. Furthermore, the mitogenic actions of platelet-derived growth factor, IGF-I, or serum are not enhanced by high glucose. High glucose increases SMC glucose metabolism through the citric acid cycle and the pentose phosphate pathway by 240 and 90%, respectively, but <10% of consumed glucose is metabolized through these pathways. Instead, most of the consumed glucose is converted into lactate and secreted by the SMCs. Thus, diabetes markedly accelerates SMC proliferation and accumulation in atherosclerotic lesions. The stimulatory effect of diabetes on SMCs is likely to be mediated by effects secondary to the hyperglycemic state.
Assuntos
Arteriosclerose/patologia , Diabetes Mellitus Experimental/patologia , Angiopatias Diabéticas/patologia , Músculo Liso Vascular/patologia , Animais , Aorta Torácica/patologia , Arteriosclerose/induzido quimicamente , Glicemia/análise , Divisão Celular/efeitos dos fármacos , Divisão Celular/fisiologia , Células Cultivadas , Colesterol na Dieta/administração & dosagem , Glucose/metabolismo , Glucose/farmacologia , Humanos , Recém-Nascido , Masculino , Músculo Liso Vascular/metabolismo , SuínosRESUMO
Werner Syndrome is a rare autosomal recessive disorder characterized by an increased cancer risk and by symptoms suggestive of premature aging. Cells from these patients demonstrate a typical pattern of chromosomal instability and a spontaneous hypermutability with a high rate of unusually large deletions. We have studied the in vivo DNA ligation in three lymphoblast cell lines from Werner syndrome patients and three from normal donors. In our host cell ligation assay we transfected linearized plasmid pZ189 and measured the amount of plasmid DNA ends rejoined by these host cells as the ability of the recovered plasmid to transform bacteria. A mutagenesis marker gene close to the ligation site allowed screening for mutations. Subsequent mutation analysis provided information about the accuracy of the ligation process. The cells from Werner syndrome patients were as effective as normal cells in ligating DNA ends. However, mutation analysis revealed that the three Werner syndrome cell lines introduced 2.4-4.6 times more mutations (p < 0.001) than the normal cell lines during ligation of the DNA ends: the mutation rates were 69.4, 97.2, and 58.7%, as compared to 23.6, 21.7, and 24.4% in the normal cell lines. These increased mutation frequencies in plasmids ligated during passage through Werner syndrome cells were mainly due to a significant (p < 0.001) increase in deletions. This error-prone DNA ligation might be responsible for the spontaneous hypermutability and the genomic instability in Werner syndrome cells and related to the apparently accelerated aging and high cancer risk in affected patients.
Assuntos
DNA Bacteriano/genética , Mutação/genética , Plasmídeos , Síndrome de Werner/genética , Linhagem Celular , DNA/análise , DNA/genética , Deleção de Genes , Humanos , Linfócitos/química , Linfócitos/patologia , Transfecção , Síndrome de Werner/patologiaRESUMO
To analyse the putative role of methylation of cytosine residues in the nuclear DNA as a regulatory step during cellular ageing, we incubated ageing human amniotic fluid derived fibroblast-like cells and non-ageing NIH-3T3 cells with 5-azacytidine. BrdUrd/Hoechst and acridine orange (AO) flow cytometry was used to compare the effects of the base analogue on cell proliferation and cell differentiation. In NIH-3T3 cultures, 96h exposures to 4 microM 5-azacytidine caused diminished cell proliferation due to cell arrest in the G1 compartments of the second and third cell cycles of serum stimulated cells. The exit from the G0/G1 compartment was not affected. The 5-azacytidine induced cell kinetic disturbances were unstable in NIH-3T3 cultures, such that pre-treated cells reverted to normal cell cycle transit within 2-3 days after termination of treatment. In contrast, 5-azacytidine pre-treated amniotic fluid derived fibroblast-like cell cultures showed persistently elevated G2 phase arrests and delayed G0/G1 phase exit kinetics, which explain the premature cessation of proliferation observed in these primary cultures. In both cell systems, 5-azacytidine exposed cultures showed elevated numbers of G1 phase cells with increased RNA content as revealed by AO flow cytometry. Again, this effect was reversible in NIH-3T3 cells but not in amniotic fluid derived fibroblast-like cells. These contrasting responses to 5-azacytidine are likely to reflect intrinsic differences in methylation patterns or de novo methylase activity between ageing cell strains and non-ageing cell lines.
Assuntos
Azacitidina/farmacologia , Divisão Celular/efeitos dos fármacos , Fase G1/efeitos dos fármacos , Fase G2/efeitos dos fármacos , Células 3T3/efeitos dos fármacos , Laranja de Acridina , Líquido Amniótico/citologia , Animais , Bromodesoxiuridina , Separação Celular , Células Cultivadas/efeitos dos fármacos , Senescência Celular/efeitos dos fármacos , Citometria de Fluxo , Humanos , Metilação , Camundongos , RNA/análiseRESUMO
The glutathione level of cultured human fibroblasts was determined with a micromodification of a spectrophotometric glutathione cycling method. There was a slight increase in reduced glutathione (GSH) content during in vitro ageing of normal human fibroblasts. Fibroblasts from patients with Werner's syndrome or ceroid lipofuscinosis (Spielmeyer-Vogt syndrome) and healthy individuals exhibited similar patterns of GSH levels during in vitro ageing. The GSH content of non-proliferating confluent cultures of normal fibroblasts and of proliferating normal fibroblasts was identical. Moreover, autofluorescent "aged" cells isolated by cell sorting did not differ in GSH content from the non-autofluorescent cells in the same culture. It was concluded that the GSH content does not play a role in in vitro ageing, nor in the accumulation of autofluorescent material in human skin fibroblasts.
Assuntos
Fibroblastos/metabolismo , Glutationa/metabolismo , Sobrevivência Celular , Células Cultivadas , Humanos , Lipofuscinoses Ceroides Neuronais/metabolismo , Pele/metabolismo , Síndrome de Werner/metabolismoRESUMO
A 240 000 molecular weight protein was found to accumulate in sorted autofluorescent (AF) cells, and during growth inhibition and in vitro ageing of cultures of human skin fibroblasts. Vitamin E, a lipophilic free radical scavenger which suppressed completely the formation of cellular autofluorescence, did not affect the accumulation of this protein. So, this accumulation is not related to cellular autofluorescence and lipid peroxidation, the major cause of this autofluorescence. This protein was also found in cells from a patient with the Spielmeyer-Vogt syndrome with a high percentage of maximal lifespan (MLS), while it was completely absent from all cells of a patient with Werner's syndrome. On two-dimensional gel electrophoresis the protein showed a heterogeneous acidic isoelectric point (IEP) of around 5.3. Neuraminidase treatment caused the IEP of this protein to shift towards a less acidic pH value (5.85). Upon differential centrifugation of a cell homogenate the protein was found to be located in the microsomal pellet and the cytosol. Chromatography on gelatin-sepharose revealed that the protein was not fibronectin. It is concluded that in human skin fibroblasts a high molecular weight glycoprotein accumulates as a result of impaired proliferation and that this accumulation is not related to cellular lipid peroxidation.
Assuntos
Fibroblastos/metabolismo , Glicoproteínas/metabolismo , Adulto , Divisão Celular , Linhagem Celular , Sobrevivência Celular , Pré-Escolar , Citosol/metabolismo , Feminino , Fibroblastos/citologia , Fibroblastos/efeitos dos fármacos , Fluorescência , Humanos , Masculino , Microssomos/metabolismo , Peso Molecular , Lipofuscinoses Ceroides Neuronais/metabolismo , Síndrome , Fatores de Tempo , Vitamina E/farmacologia , Síndrome de Werner/metabolismoRESUMO
To test the possible role of lipid peroxidation in the process of in vitro ageing, human diploid skin fibroblasts were cultured with the lipophilic hydroperoxide cumene hydroperoxide (Chp) or the breakdown product of lipid peroxidation 4-hydroxy-2,3-trans-nonenal (HNE). Both compounds inhibited cellular DNA and protein synthesis in a dose-dependent way. Cells exposed to Chp or to HNE during growth inhibition recovered DNA and protein synthesis within 24 h upon removal of Chp or HNE from the culture medium. Continuously proliferating cells showed only a partial recovery of DNA and protein synthesis. Pre-culturing cells with the lipophilic free radical scavenger vitamin E did not abolish the effect of Chp upon DNA synthesis. Cellular levels of reduced glutathione (GSH) rose slightly during 1 week of culture with HNE, but remained unaltered with Chp. Neither ATP levels nor cellular energy charges were affected during culture with Chp or HNE. So, DNA synthesis is not impaired due to a shortage of nucleotides nor does GSH protect DNA synthesis against the effects of Chp or HNE. These results suggest that oxygen free-radical induced lipid peroxidation is not the cause of the irreversible loss of proliferation occurring during in vitro ageing.
Assuntos
Aldeídos/farmacologia , Derivados de Benzeno/farmacologia , DNA/biossíntese , Biossíntese de Proteínas , Envelhecimento/metabolismo , Envelhecimento/patologia , Divisão Celular/efeitos dos fármacos , Células Cultivadas , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Humanos , Peróxidos Lipídicos/metabolismoRESUMO
One method for examining cell cycle kinetics by flow cytometry uses continuous DNA labeling with bromodeoxyuridine (BrdU), a thymidine analogue. Upon incorporation into DNA, BrdU causes stoichiometric quenching of the DNA fluorochrome Hoechst 33258. After counterstaining with a secondary DNA fluorochrome (e.g., ethidium bromide), the analyst can distinguish cells in different phases of the cell cycle over a number of mitotic cycles with flow cytometry. In this report, we describe a modification of the flow cytometric BrdU-Hoechst assay that allows combined analysis of cell proliferation and immunophenotyping at the single cell level. To demonstrate an application of this method, human peripheral blood mononuclear cells were stimulated with tetanus toxoid or interleukin-2 for up to 6 days in the presence of BrdU, harvested, and immunostained for the cell surface markers CD3, CD4, CD8, CD14, CD19, and the cytokine receptor, CCR5. We used four-color flow cytometry analyses to simultaneously measure cell proliferation and surface marker expression, for the purpose of immunophenotyping and identifying specific cell subsets responding to antigen stimulation. Our successful application of this method suggests that it may be used to study immune responses at the molecular and cellular level and to identify mechanisms of immune system modulation.
Assuntos
Antígenos de Diferenciação de Linfócitos B/análise , Antígenos de Diferenciação de Linfócitos T/análise , Imunofenotipagem/métodos , Subpopulações de Linfócitos/classificação , Bisbenzimidazol/química , Bromodesoxiuridina/química , Ciclo Celular , Permeabilidade da Membrana Celular , Células Cultivadas , DNA/análise , Dactinomicina/análogos & derivados , Dactinomicina/química , Etídio/química , Fixadores/química , Citometria de Fluxo , Corantes Fluorescentes/química , Formaldeído/química , Humanos , Ativação Linfocitária , Subpopulações de Linfócitos/química , Subpopulações de Linfócitos/citologia , Polímeros/químicaRESUMO
Investigation of mitochondrial morphology and function has been hampered because photostable, mitochondrion-specific stains that are retained in fixed, permeabilized cells have not been available. We found that in live cell preparations, the CMXRos and H2-CMXRos dyes were more photostable than rhodamine 123. In addition, fluorescence and morphology of mitochondria stained with the CMXRos and CMXRos-H2 dyes were preserved even after formaldehyde fixation and acetone permeabilization. Using epifluorescence microscopy, we showed that CMXRos and H2-CMXRos dye fluorescence fully co-localized with antibodies to subunit I of cytochrome c oxidase, indicating that the dyes specifically stain mitochondria. Confocal microscopy of these mitochondria yielded colored banding patterns, suggesting that these dyes and the mitochondrial enzyme localize to different suborganellar regions. Therefore, these stains provide powerful tools for detailed analysis of mitochondrial fine structure. We also used poisons that decrease mitochondrial membrane potential and an inhibitor of respiration complex II to show by flow cytometry that the fluorescence intensity of CMXRos and H2-CMXRos dye staining responds to changes in mitochondrial membrane potential and function. Hence, CMXRos has the potential to monitor changes in mitochondrial function. In addition, CMXRos staining was used in conjunction with spectrally distinct fluorescent probes for the cell nucleus and the microtubule network to concomitantly evaluate multiple features of cell morphology.
Assuntos
Corantes Fluorescentes , Mitocôndrias , Rodaminas , Células 3T3 , Animais , Bovinos , Membranas Intracelulares/metabolismo , Camundongos , Microscopia Confocal , Mitocôndrias/metabolismo , Permeabilidade , Rodamina 123RESUMO
Bromodeoxyuridine/Hoechst flow cytometry was used to analyse the interference of common cytostatic agents with cell activation and cell cycle progression of human B-cell lines. Bleomycin impaired both cell activation and G2 transit, the latter effect being oxygen dependent. The DNA alkylating agents cyclophosphamide, chlorambucil and mitomycin C caused G2 arrest, whereas cisplatin arrested cells in both the S and G2 phase of the cell cycle. Vinblastin interfered with mitosis, but in addition arrested cells in all phases of the cell cycle. The growth inhibitory action of bleomycin, cisplatin and chlorambucil was dependent upon the bromodeoxyuridine (BrdU) concentration in the culture medium. No interaction was found between BrdU and cyclophosphamide, mitomycin C and vinblastin. The cell cycle kinetic mechanism of the interaction between BrdU and bleomycin, cisplatin and chlorambucil was a potentiation of the G2 arrest. In conclusion, BrdU may be useful in clinical chemotherapy as a chemosensitizer for selected cytostatic agents.