Evaluation of hydroxyurea genotoxicity in patients with sickle cell disease / Avaliação da indução de genotoxicidade pela hidroxiureia em pacientes com doença falciforme

ABSTRACT Objective To evaluate the induction of DNA damage in peripheral blood mononuclear cells of patients with sickle cell disease, SS and SC genotypes, treated with hydroxyurea. Methods The study subjects were divided into two groups: one group of 22 patients with sickle cell disease, SS and SC genotypes, treated with hydroxyurea, and a Control Group composed of 24 patients with sickle cell disease who were not treated with hydroxyurea. Peripheral blood samples were submitted to peripheral blood mononuclear cell isolation to assess genotoxicity by the cytokinesis-block micronucleus cytome assay, in which DNA damage biomarkers - micronuclei, nucleoplasmic bridges and nuclear buds - were counted. Results Patients with sickle cell disease treated with hydroxyurea had a mean age of 25.4 years, whereas patients with sickle cell disease not treated with hydroxyurea had a mean age of 17.6 years. The mean dose of hydroxyurea used by the patients was 12.8mg/kg/day, for a mean period of 44 months. The mean micronucleus frequency per 1,000 cells of 8.591±1.568 was observed in the Hydroxyurea Group and 10.040±1.003 in the Control Group. The mean frequency of nucleoplasmic bridges per 1,000 cells and nuclear buds per 1,000 cells for the hydroxyurea and Control Groups were 0.4545±0.1707 versus 0.5833±0.2078, and 0.8182±0.2430 versus 0.9583±0.1853, respectively. There was no statistically significant difference between groups. Conclusion In the study population, patients with sickle cell disease treated with the standard dose of hydroxyurea treatment did not show evidence of DNA damage induction.

RESUMO Objetivo Avaliar o efeito da indução de danos ao DNA em células monocelulares do sangue periférico de pacientes com doença falciforme, genótipos SS e SC, tratados com hidroxiureia. Métodos Os sujeitos da pesquisa foram divididos em dois grupos: um de 22 pacientes com doença falciforme genótipos SS e SC tratados com hidroxiureia, e o outro controle, composto por 24 pacientes com doença falciforme que não eram tratados com o fármaco. As amostras de sangue periférico foram submetidas ao isolamento de células mononucleares do sangue periférico para avaliação da genotoxicidade pelo ensaio de micronúcleo citoma com bloqueio da citocinese, tendo sido quantificados os biomarcadores de danos ao DNA - micronúcleos, pontes nucleoplasmáticas e brotamento nuclear. Resultados Os pacientes com doença falciforme tratados com hidroxiureia apresentaram média de idade de 25,4 anos, enquanto aqueles com doença falciforme não tratados com hidroxiureia tiveram média de idade de 17,6 anos. A dose média de hidroxiureia utilizada pelos pacientes foi de 12,8mg/kg/dia, por período médio de 44 meses. A frequência média de micronúcleos por 1.000 células de 8,591±1,568 foi observada no Grupo Hidroxiureia e de 10,040±1,003 no Grupo Controle. Adicionalmente, a frequência média de pontes nucleoplasmáticas por 1.000 células e brotamento nuclear por 1.000 células para o Grupo Hidroxiureia e Controle foi de 0,4545±0,1707 versus 0,5833±0,2078, e de 0,8182±0,2430 versus 0,9583±0,1853, respectivamente. Não houve diferença estatisticamente significativa entre os grupos. Conclusão Na população estudada de pacientes com doença falciforme com tratamento em dose padrão de hidroxiureia, não houve evidência de indução de danos ao DNA.

Apoptose em pólipos nasais eosinofílicos submetidos in vitro ao tratamento com mitomicina C / Apoptosis in eosinophilic nasal polyps treated in vitro with mitomycin C

A etiopatogênese da polipose nasal eosinofílica ainda não foi esclarecida. Os eosinófilos constituem as principais células do infiltrado inflamatório e estão relacionados com a perpetuação do processo inflamatório na rinossinusite crônica com pólipos nasais. OBJETIVO: Avaliar a ação in vitro da mitomicina no índice apoptótico de pólipos nasais eosinofílicos. MATERIAL E MÉTODO: Estudo prospectivo experimental autopareado com amostra de biópsia de 15 pacientes com polipose nasal eosinofílica. Cada fragmento foi dividido em dois grupos. No grupo experimental aplicou-se mitomicina por cinco minutos, na dosagem de 400 µg/ml. O grupo controle foi submetido às mesmas manipulações, mas utilizando-se somente meio de cultura. Os fragmentos contidos nos dois primeiros compartimentos, controle e experimento, foram imediatamente submetidas ao preparo para histopatologia. O outro par de amostra, contendo controle e experimento, foi incubado por 12 horas. Após 12 horas, os fragmentos foram retirados para exame histopatológico. O índice apoptótico foi determinado pela morfometria na coloração hematoxilina-eosina e pela análise da fragmentação do DNA. RESULTADO: A comparação do dois grupos demonstrou diferença significativa (p < 0,001) no índice apoptótico das culturas incubadas por 12 horas. CONCLUSÃO: A mitomicina induz in vitro o aumento do índice apoptótico dos eosinófilos dos pólipos nasais eosinofílicos.

The etiopathogenesis of eosinophilic nasal polyps is yet to be explained. Eosinophils are key components in the inflammatory infiltrate and are related to the perpetuation of the inflammatory process in chronic rhinosinusitis with nasal polyps. OBJECTIVE: This paper aims to evaluate the in vitro action of mitomycin upon the apoptotic index of nasal polyps. MATERIALS AND METHODS: This is a self-paired prospective experimental study using biopsy fragments from 15 patients with eosinophilic nasal polyps. Biopsy fragments were divided into two groups. In the case group, the fragments were treated with 400 µg/ml of mitomycin for five minutes. The control group fragments were treated with culture medium. The pair of fragments contained in the two first compartments - control and case - were immediately sent to the histopathologist. The other pair of samples containing control and case fragments was incubated for 12 hours. The fragments were then taken to the histopathologist for testing. The apoptotic index was determined by the morphometry in hematoxylin and eosin staining and DNA fragmentation analysis (TUNEL reaction). RESULTS: The comparison between the two groups showed a statistically significant difference (p < 0,001) in the apoptotic index of the 12-hour incubated cultures. CONCLUSION: Mitomycin acts in vitro upon the eosinophilic nasal polyps inducing the rise of the eosinophilic apoptotic index.

Staurosporine and cytochalasin D induce chondrogenesis by regulation of actin dynamics in different way

Actin cytoskeleton has been known to control and/or be associated with chondrogenesis. Staurosporine and cytochalasin D modulate actin cytoskeleton and affect chondrogenesis. However, the underlying mechanisms for actin dynamics regulation by these agents are not known well. In the present study, we investigate the effect of staurosporine and cytochalasin D on the actin dynamics as well as possible regulatory mechanisms of actin cytoskeleton modulation. Staurosporine and cytochalasin D have different effects on actin stress fibers in that staurosporine dissolved actin stress fibers while cytochalasin D disrupted them in both stress forming cells and stress fiber-formed cells. Increase in the G-/F-actin ratio either by dissolution or disruption of actin stress fiber is critical for the chondrogenic differentiation. Cytochalasin D reduced the phosphorylation of cofilin, whereas staurosporine showed little effect on cofilin phosphorylation. Either staurosporine or cytochalasin D had little effect on the phosphorylation of myosin light chain. These results suggest that staurosporine and cytochalasin D employ different mechanisms for the regulation of actin dynamics and provide evidence that removal of actin stress fibers is crucial for the chondrogenic differentiation.

Effects of mitomycin C on infiltration of polymorphonuclear leukocytes after epithelial scrape injury in the mouse cornea / Efeito da mitomicina C na infiltração de leucócitos polimorfonucleares após lesão epitelial em córnea de camundongo

PURPOSE: To determine whether mitomycin C (MMC) alters appearance and disappearance of polymorphonuclear leucocytes (PMN) in the cornea stroma, using an epithelial scrape injury in eye mouse model. METHODS: Twenty-mice underwent mechanical epithelium debridement in the central cornea using 20 percent ethanol. After the scrape, the right eye received 0.02 percent MMC for one minute, while the left eye received physiological saline. The animals were sacrificed on days 1, 2, 5, and 14 after surgery, and corneal whole mounts were prepared for histology. PMN distribution was analyzed in digitized microscope images. Cell division in the cornea was determined by immunohistochemical detection of bromodeoxyuridine (BrdU), which was injected intraperitoneally before the mice were sacrificed. RESULTS: Epithelial scrape injury triggered infiltration of PMNs into the corneal stroma. An analysis of PMN distribution revealed that there was no difference between eyes treated with and without MMC at all time points. BrdU labeling showed that 0.02 percent MMC for one minute blocked keratocyte proliferation completely. CONCLUSION: MMC treatment regimen, which is common in clinical practice, inhibits keratocyte proliferation during wound healing, but when used at 0.02 percent for one minute, it does not affect PMN infiltration into the corneal stroma, and subsequent movement toward the injury site, or the disappearance of PMNs from the stroma, in the mouse epithelial injury model.

OBJETIVO: O objetivo do estudo foi determinar se a mitomicina C (MMC) altera o aparecimento dos leucócitos polimorfonucleares (PMN) no estroma corneano após abrasão epitelial central, utilizando olhos de camundongo como modelo. MÉTODOS: Vinte camundongos foram submetidos à abrasão epitelial em córnea central utilizando etanol a 20 por cento. Após a lesão, o olho direito recebeu MMC a 0,02 por cento por 1 minuto, enquanto o olho esquerdo recebeu solução salina. Os animais foram sacrificados em 1, 2, 5 e 14 dias após a cirurgia e a córnea foi preparada para histologia. A distribuição dos PMN foi analisada e digitalizada em imagens microscópicas. A divisão celular na córnea foi detectada pela imuno-histoquímica da bromodeoxirudina (BrdU), injetada intraperitonialmente duas horas antes dos animais serem secrificados. RESULTADOS: A lesão epitelial gerou infiltração de PMN no estroma da córnea. A análise da distribuição dos PMNs revelou que não houve diferença estatisticamente significante entre os olhos tratados e não tratados com MMC, em todos os tempos estudados. O estudo com BrdU mostrou que a MMC quando utilizada a 0,02 por cento por um minuto bloqueou completamente a proliferação de ceratócitos. CONCLUSÃO: O tratamento com MMC, que é utilizada comumente na prática clínica, inibe a proliferação dos ceratócitos durante a cicatrização corneana, porém quando utilizada a 0,02 por cento por um minuto, não altera a infiltração dos PMNs dentro do estroma corneano após lesão epitelial em córneas de camundongos.

Correlation of thyroid and growth hormones to chromosomal instability in Egyptian Fanconi anemia patients.

OBJECTIVE: Fanconi anemia (FA) is a rare inherited genomic instability syndrome and usually associated with endocrine dysfunctions. We aimed to assess the diagnostic standards of chromosomal instability in FA and to correlate the breakage frequency with the severity of endocrinal dysfunctions. METHODS: Twenty seven FA patients were randomly selected from Hematology Unit of Mansoura University Children's Hospital; their mean age 8.8 yr. Sixteen normal children matched for age and sex were used as controls. Cytogenetic studies included peripheral blood lymphocyte cultures using phytohemagglutinin to obtain chromosomal spreads. Chromosomal breakage was induced by (i) Diepoxybutane 0.1 mug/ml. (ii) Mitomycin C 0.1 microg/ml. (iii) Irradiation of cultures to four radiation doses; 75, 150, 300 and 400 rads (rad1, rad2, rad3 and rad4 respectively). Chromosomal aberrations were scored from the previous 6 cultures besides a culture for spontaneous chromosomal breakage; then mean chromosomal breakage was calculated for the seven cultures. Endocrinal evaluation included quantitative determination of thyroid stimulating hormone (TSH) and tetraiodothyronine (T4), serum growth hormone (GH), insulin like growth factor-1 (IGF-1) and insulin levels. RESULTS: Chromosomal breakage was found to be significantly higher in patients than control when induced by Diepoxybutane (p = 0.003), Mitomycin (p = 0.001), rad3 (p = 0.043) and rad4 (p = 0.001). Mean chromosomal breakage was significantly negative correlated to head circumference (r = -0.57) and GH level (r = -0.50), with no significant correlation to other hormonal parameters. Mitomycin and rad4 were found more accurate than DEB test for diagnosis of FA in suspected cases. CONCLUSION: Correction of the frequently associated hormonal dysfunction (reduced GH and T4) should be considered in the treatment discipline of FA patients to improve their final height.

Nucleolar translocalization of GRA10 of Toxoplasma gondii transfectionally expressed in HeLa cells

Toxoplasma gondii GRA10 expressed as a GFP-GRA10 fusion protein in HeLa cells moved to the nucleoli within the nucleus rapidly and entirely. GRA10 was concentrated specifically in the dense fibrillar component of the nucleolus morphologically by the overlap of GFP-GRA10 transfection image with IFA images by monoclonal antibodies against GRA10 (Tg378), B23 (nucleophosmin) and C23 (nucleolin). The nucleolar translocalization of GRA10 was caused by a putative nucleolar localizing sequence (NoLS) of GRA10. Interaction of GRA10 with TATA-binding protein associated factor 1B (TAF1B) in the yeast two-hybrid technique was confirmed by GST pull-down assay and immunoprecipitation assay. GRA10 and TAF1B were also co-localized in the nucleolus after co-transfection. The nucleolar condensation of GRA10 was affected by actinomycin D. Expressed GFP-GRA10 was evenly distributed over the nucleoplasm and the nucleolar locations remained as hollows in the nucleoplasm under a low dose of actinomycin D. Nucleolar localizing and interacting of GRA10 with TAF1B suggested the participation of GRA10 in rRNA synthesis of host cells to favor the parasitism of T. gondii.

Catechins inhibit angiotensin II-induced vascular smooth muscle cell proliferation via mitogen-activated protein kinase pathway

Catechins, components of green tea, reduce the incidence of cardiovascular diseases such as atherosclerosis. Angiotensin II (Ang II) is highly implicated in the proliferation of vascular smooth muscle cells (VSMC), resulting in atherosclerosis. The acting mechanisms of the catechins remain to be defined in the proliferation of VSMC induced by Ang II. Here we report that catechin, epicatechin (EC), epicatechingallate (ECG) or epigallocatechingallate (EGCG) significantly inhibits the Ang II-induced [3H]thymidine incorporation into the primary cultured rat aortic VSMC. Ang II increases the phosphorylation of the extracellular signal-regulated protein kinase 1/2 (ERK 1/2), c-jun-N-terminal kinase 1/2 (JNK 1/2), or p38 mitogen-activated protein kinases (MAPKs) and mRNA expression of c-jun and c-fos. The EGCG pretreatment inhibits the Ang II-induced phosphorylation of ERK 1/2, JNK 1/2, or p38 MAPK, and the expression of c-jun or c-fos mRNA. U0126, a MEK inhibitor, SP600125, a JNK inhibitor, or SB203580, a p38 inhibitor, attenuates the Ang II-induced [3H]thymidine incorporation into the VSMC. In conclusion, catechins inhibit the Ang II-stimulated VSMC proliferation via the inhibition of the Ang II-stimulated activation of MAPK and activator protein-1 signaling pathways. The antiproliferative effect of catechins may be associated with the reduced risk of cardiovascular diseases by the intake of green tea. Catechins may be useful in the development of prevention and therapeutics of vascular diseases.

Mitomicina C e "Excimer laser" / Mitomycin C and Excimer Laser

A mitomicina C é um antimetabólito que atua em nível celular bloqueando a replicação de DNA e RNA e inibindo a síntese protéica. Utilizada em diversas áreas da oftalmologia, recentemente vem sendo empregada como moduladora da resposta cicatricial corneana em cirurgias ópticas/refrativas por "excimer laser". A aplicação única de mitomicina C associada à cirurgia fotoablativa de superfície corneana tem se mostrado opção segura e eficiente para fins terapêuticos em olhos com opacidade corneana pré-existente e/ou profiláticos em olhos com alto risco de desenvolvimento de opacificação corneana pós-operatória. O uso da droga em cirurgia fotoablativa deve ser cauteloso até que seguimento de longo prazo avalie sua inocuidade tardia. O presente texto faz revisão dos principais estudos sobre modulação da resposta cicatricial corneana com uso de mitomicina C em cirurgias ópticas/refrativas de superfície.

Efeito da mitomicina C na secreção de fator estimulador de granulócitos macrófagos e interleucina-5 em cultura de estroma de pólipos nasais eosinofílicos / Effect of mitomycin C on the secretion of granulocyte macrophages colonies stimulating factor and interleukin-5 in eosinophilic nasal polyps stromal culture

O estudo de fatores teciduais, como a concentração de fator estimulador de colônias de macrófagos (GM-CSF) e interleucina 5 (IL-5), aponta para os mecanismos envolvidos na manutenção da eosinofilia em pólipos nasossinusais eosinofílicos. A mitomicina C (MMC) tem sido utilizada com bons resultados em otorrinolaringologia. OBJETIVO: Este estudo teve como objetivo avaliar a ação da Mitomicina C sobre a secreção de GM-CSF e IL-5 em pólipos eosinofílicos. FORMA DE ESTUDO: caso-controle. MATERIAL E MÉTODO: O estudo foi comparativo experimental autopareado, com amostras de pólipos biopsiados de pacientes portadores de polipose nasossinusal eosinofílica. Os fragmentos semeados como grupo experimental receberam mitomicina C por 5 minutos na dosagem de 400microg/ml e então lavadas em meio RPMI. Nos tempos zero, 12 e 24 horas, o sobrenadante foi retirado para determinação dos níveis de GM-CSF em 22 pacientes e IL-5, em 19 pacientes, utilizando o método de ELISA. RESULTO: Diminuição de secreção de GM-CSF nos grupos tratados com mitomicina C no tempo 24h (p<= 0,05); no grupo tratado houve expressão significativa de GM-CSF entre zero e 12 horas (p=0,013) demonstrando a viabilidade da cultura igualmente ao grupo não tratado; tendência à queda dos níveis de IL-5 no grupo tratado em 24h. CONCLUSÃO: O estudo demonstrou que a mitomicina C foi capaz de inibir a síntese de GM-CSF em culturas de pólipos nasais eosinofílicos e com provável ação sobre a secreção de IL-5, necessitando de estudos complementares.

Distinct Patterns of Cleavage and Translocation of Cell Cycle Control Proteins in CD95-induced and p53-induced apoptosis

Apoptotic cell death induced by p53 occurs at a late G1 cell cycle checkpoint termed the restriction(R)point, and it has been proposed that p53-induced apoptosis causes upregulation of CD95. However, as cells with defective in CD95 signaling pathway are still sensitive to p53-induced apoptosis, CD95 cannot be the sole factor resulting in apoptosis. In addition, unlike p53-induced apoptosis, the relationship between CD95-mediated apoptosis and the cell cycle is not clearly understood. It would there-fore be worth investigating whether CD95-mediated cell death is pertinent with p53-induced apoptosis in view of cell cycle related molecules. In this report, biochemical analysis showed that etoposide-induced apoptosis caused the induction and the nuclear translocation of effector molecules involved in G1 cell cycle checkpoint. However, there was no such translocation in the case of CD95-mediated death. Thus, although both types of apoptosis involved caspase activation, the cell cycle related proteins responded differently. This argues against the idea that p53-induced apoptosis occurs through the induction of CD95/CD95L expression.

Resultado a longo prazo do uso de drogas antiproliferativas na trabeculectomia primária / Long-term outcomes of antiproliferative agents in primary trabeculectomy

Objetivo: Avaliar comparativamente a longo prazo os resultados cirúrgicos de trabeculectomias nas quais foram utilizadas injeções subconjuntivais de 5-fluorouracil (5-FU) no pós-operatório ou aplicações de mitomicina C (MMC) peroperatória. Métodos: Estudo retrospectivo de 133 olhos submetidos a trabeculectomia primária. Resultados: A pressão ocular não apresentou valores estatisticamente significantes em nenhuma das visitas de pós-operatório quando comparados os grupos em que se utilizou a mitomicina C ou 5-fluorouracil. Em cada um dos grupos analisados separadamente, os níveis da pressão ocular não apresentaram diferenças estatisticamente significantes nos períodos pré e pós-operatório. A incidência de complicações pós-operatórias também não apresentou diferença estatisticamente significante à exceção de alterações epiteliais na córnea, mais freqüentes no grupo tratado com 5-fluorouracil, Nos dois grupos, destacou-se a elevada incidência de desenvolvimento/progressão de catarata. Conclusão: O uso de mitomicina C ou 5-fluorouracil promoveu redução importante e estável da pressão ocular, com índices de complicações semelhantes.

Insulin and phorbol ester regulation of gene 33 expression in CHO cells

Rat gene 33 (g33) mRNA has a widespread tissue distribution. Insulin and various agents such as glucocorticoids, phorbol esters and plant lectins regulate G33 expression in rat hepatoma cells. The regulation of g33 by insulin and a phorbol ester was examined in two Chinese Hamster ovary (CHO) cell lines, CHO-T cells (which overexpress human insulin receptors (hIR)) and wild type CHOwt cells. These cell lines were used to determine how expression of the hIR influences the capacity of g33 to respond to insulin and phorbol myristate acetate (PMA). Treatment of CHOwt and CHO-T cells with insulin increased mRNAg33 levels three to four-fold, with a maximum effect reached after three hours of treatment. PMA treatment of CHOwt and CHO-T cells caused a similar elevation of mRNAg33 levels after three hours. Insulin had no effect on mRNAg33 stability in both CHO cell lines. Additionally, the effects of insulin and PMA on mRNAg33 levels were additive only in CHO-T cells. Insulin or PMA-pretreated CHO-T cells were able to respond to both agents, but elevation ofmRNAg33 levels was maximal. In contrast, when insulin and/or PMA-pretreated CHOwt cells were exposed to insulin or PMA, g33 was able to respond maximally. These results suggest that insulin and phorbol esters act through different signaling mechanisms in CHOwt cells. Additionally, insulin's ability to stimulate g33 expression in CHOwt cells suggests that this insulin effect may be independent of the insulin eceptor. There are differences in the regulation pattern of g33 by insulin and PMA in rat hepatoma and among the two CHO cell lines used in this study

Effects of interleukin-10 on chemokine KC gene expression by mouse peritoneal macrophages in response to Candida albicans

Chemokine KC has been considered to be a murine homologue of human GRO/MGSA and was identified as chemoattractant for monocytes and neutrophils. This study examined the expression of KC mRNA in thioglycollate-elicited mouse peritoneal macrophages that were stimulated in vitro with Candida albicans (CA). Also examined were the inhibitory effects of IL-10 on the CA-induced expression of KC gene by Northern blot analysis. CA was found to induce chemokine gene expression in a gene-specific manner, CXC chemokine IP-10 mRNA expression was not detected in CA-stimulated macrophages. Maximum KC mRNA expression was observed approximately 2 hr after adding CA. The inhibitory action of IL-10 to CA-induced KC mRNA expression on mouse peritoneal macrophages was independent on concentration and stimulation time of IL-10 and was observed approximately one hour after adding IL-10 and CA simultaneously. IL-10 produced a decrease in the stability of KC mRNA, and CA-stimulated macrophages with cycloheximide blocked the suppressive effect of IL-10. These results suggest that CA also induces chemokine KC from macrophages, and IL-10 acts to destabilize CA-induced KC mRNA and de novo synthesis of an intermediate protein is a part of the IL-10 suppressive mechanism.

Characterization of primed lymphocytes on the basic of sensitivity of chromatin to DNase I.

Certain qualitative criteria for primed lymphocytes in the expression of cytotoxic function have been studied. Unlike normal lymphocytes, primed lymphocytes expressed cytotoxicity even when DNA synthesis and new gene expression were inhibited by hydroxyurea (HU) and bromodeoxyuridine (BU) respectively. Such differential cytotoxic expression in presence of HU and BU by primed lymphocytes might have their basis in conformational change within the chromatin. Chromatin from primed lymphocytes was more susceptible to DNase I digestion than virgin lymphocytes indicating exposition of more DNase I sensitive sites in primed state. The result suggest the presence of more ready to act sites for the polymerases in the genomic material of primed lymphocytes even at quiescent state.