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1.
Exp Hematol ; 80: 27-35, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31759073

RESUMO

Spleen colony-forming unit (CFU-s) growth in spleen colonies is a stochastic process in which CFU-s, with each cell division, can either self-renew or differentiate, but not both. The fundamental parameter governing this process is p, or the probability of CFU-s self-renewing. Previously, when CFU-s growth was modeled by Monte Carlo simulations, p was kept constant during the 20 cell cycles required for the modeling. However, it is known that CFU-s self-renewal undergoes decline with proliferation. In the present study, this was taken into consideration, such that p was forced to undergo a small decline with each cell division. These new Monte Carlo calculations give an improved fit to CFU-s cumulative growth curves as compared with those calculations using fixed p. This new model, referred to as the variable p model, offers an explanation as to how large mammals can amplify marrow output from stem cell compartments that are no larger than those found in small mammals. It is a model in which small changes in active stem cell aging generate disproportionally large increases in the size of active stem cell clones.


Assuntos
Simulação por Computador , Células-Tronco Hematopoéticas/citologia , Modelos Biológicos , Animais , Tamanho Corporal , Gatos , Contagem de Células , Divisão Celular , Autorrenovação Celular , Senescência Celular , Ensaio de Unidades Formadoras de Colônias , Humanos , Mamíferos/sangue , Camundongos , Método de Monte Carlo , Papio , Especificidade da Espécie , Processos Estocásticos
2.
Appl Radiat Isot ; 152: 106-108, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31280103

RESUMO

Colony counting by eye is time consuming and subjective. Here comparison between the measurements of proliferative growth inhibition in plates of radiation-treated cells by an imaging station correlated highly significantly with counts determined by eye. This would suggest that an imaging station could be a viable alternative for colony counting for doses over 200KBq.


Assuntos
Contagem de Células/instrumentação , Ensaio de Unidades Formadoras de Colônias/instrumentação , Linhagem Celular Tumoral , Feminino , Humanos , Doses de Radiação , Radiobiologia
3.
Bull Exp Biol Med ; 167(1): 150-153, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-31183651

RESUMO

In diffuse large B-cell lymphoma, bone marrow involvement is rarely diagnosed. We compared the properties of bone marrow stromal progenitor cells and the concentration of fibroblast CFU in patients with diffuse large B-cell lymphoma without bone marrow involvement and in healthy donors. It was found that the properties of multipotent mesenchymal stromal cells in patients in the debut of the disease differed considerably from those in healthy donors. In particular, the total cell production in patients was significantly higher than in donors. In multipotent mesenchymal stromal cells of patients, some cell parameters were changes; the mean fluorescence intensity of the adhesion molecule ICAM1 on the cell surface was increased. The mean fluorescence intensity of mesenchymal stromal cell markers (HLA-ABC, CD73 and CD90) was significantly elevated. The relative expression of BMP4, MMP2, FGFR1, and ICAM1 genes in mesenchymal stromal cell was reduced, while the expression of FGFR2 gene was enhanced. Despite the absence of proven involvement of the bone marrow, the properties of mesenchymal stromal cells, the components in the stromal microenvironment niche regulating hemopoiesis are altered in patients with diffuse large B-cell lymphoma.


Assuntos
Células da Medula Óssea/patologia , Linfoma Difuso de Grandes Células B/patologia , Células-Tronco Mesenquimais/patologia , Adulto , Idoso , Células da Medula Óssea/metabolismo , Proteína Morfogenética Óssea 2/metabolismo , Proteína Morfogenética Óssea 4/metabolismo , Células Cultivadas , Ensaio de Unidades Formadoras de Colônias , Feminino , Antígenos HLA-DR/metabolismo , Humanos , Molécula 1 de Adesão Intercelular/metabolismo , Linfoma Difuso de Grandes Células B/metabolismo , Masculino , Células-Tronco Mesenquimais/metabolismo , Pessoa de Meia-Idade , Receptor Tipo 1 de Fator de Crescimento de Fibroblastos/metabolismo
4.
Toxicon ; 168: 22-31, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31233771

RESUMO

This study was conducted to examine the cytotoxic effects of Nubein6.8 isolated from the venom of the Egyptian Spitting Cobra Naja nubiae on melanoma (A375) and ovarian carcinoma cell lines and to reveal its mode of action. The size of Nubein6.8 (6801.8 Da) and its N-terminal sequence are similar to cytotoxins purified from the venom of other spitting cobras. Nubein6.8 showed a high significant cytotoxic effect on A375 cell line and moderate effect on A2780. A clonogenic assay showed that Nubein6.8 has a significant long-term potency on A375 cell survival when compared to A2780. The molecular intracellular signaling pathways of Nubein6.8 have been investigated using Western blotting analysis, flow cytometry, and microscale protein labeling. This data revealed that Nubein6.8 has DNA damaging effects and the ability to activate apoptosis in both tumor cell lines. Cellular uptake recordings revealed that the labeled-Nubein6.8 was intracellularly present in A375 cells while A2780 displayed resistance against it. SEM examination showed that Nubein6.8 was found to have high accessibility to malignant melanoma cells. The apoptotic effect of Nubein6.8 was confirmed by TEM examination that revealed many evident characteristics for Nubein6.8 apoptotic efficacy on A375 cell sections. Also, TEM reflected many resistant characteristics that faced Nubein6.8 acquisition through ovarian carcinoma cell sections. Accordingly, the snake venom peptide of Nubein6.8 is a promising template for developing potential cytotoxic agents targeting human melanoma and ovarian carcinoma.


Assuntos
Antineoplásicos/química , Venenos Elapídicos/química , Venenos Elapídicos/toxicidade , Animais , Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Linhagem Celular , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Ensaio de Unidades Formadoras de Colônias , Dano ao DNA/efeitos dos fármacos , Venenos Elapídicos/metabolismo , Humanos , Naja , Neoplasias/tratamento farmacológico , Peptídeos/química , Peptídeos/metabolismo , Peptídeos/toxicidade , Transdução de Sinais
5.
J Shoulder Elbow Surg ; 28(9): 1779-1787, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31036422

RESUMO

BACKGROUND: The rotator cuff (RC) repair failure rate is high. Tendon and bone represent sources of mesenchymal stem cells (MSCs), but the number of MSCs from each has not been compared. Bone channeling may increase bone-derived MSC numbers participating in enthesis re-formation at the "footprint" repair site. The effect of preoperative channeling on increasing bone MSC numbers has never been reported. We asked (1) whether bone contains more MSCs than tendon at the time of arthroscopic repair and (2) whether bone preoperative channeling at the RC repair site increases the number of bone-derived MSCs at the time of surgery. METHODS: In 23 participants undergoing arthroscopic RC repair, bone was sampled from the footprint and tendon was sampled from the distal supraspinatus. We randomized participants to the channeling or no-channeling group 5 to 7 days before surgery. We enumerated MSCs from both tissues using the colony-forming unit-fibroblast (CFU-F) assay (10 per group). We identified MSC identity using flow cytometry and MSC tri-differentiation capacity (n = 3). RESULTS: Tendon CFU-F per gram exceeded bone CFU-F per gram for both groups (479 ± 173 CFU-F/g vs. 162 ± 54 CFU-F/g for channeling [P = .036] and 1334 ± 393 CFU-F/g vs. 284 ± 88 CFU-F/g for no channeling [P = .009]). Ninety-nine percent of cultured cells satisfied the MSC definition criteria. CONCLUSIONS: The distal supraspinatus tendon contained more MSCs per gram than the humeral footprint. Tendon may represent an important and overlooked MSC source for postoperative enthesis re-formation. Further studies are needed to evaluate the repair role of tendon MSCs and to recommend bone channeling in RC repair.


Assuntos
Úmero/patologia , Células-Tronco Mesenquimais , Lesões do Manguito Rotador/patologia , Manguito Rotador/patologia , Idoso , Artroplastia , Artroscopia , Células Cultivadas , Ensaio de Unidades Formadoras de Colônias , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Manguito Rotador/cirurgia , Lesões do Manguito Rotador/cirurgia , Articulação do Ombro/cirurgia
6.
PLoS One ; 14(5): e0216603, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31100067

RESUMO

We examined the impact of statins on Yes-associated Protein (YAP) localization, phosphorylation and transcriptional activity in human and mouse pancreatic ductal adenocarcinoma (PDAC) cells. Exposure of sparse cultures of PANC-1 and MiaPaCa-2 cells to cerivastatin or simvastatin induced a striking re-localization of YAP from the nucleus to the cytoplasm and inhibited the expression of the YAP/TEAD-regulated genes Connective Tissue Growth Factor (CTGF) and Cysteine-rich angiogenic inducer 61 (CYR61). Statins also prevented YAP nuclear import and expression of CTGF and CYR61 stimulated by the mitogenic combination of insulin and neurotensin in dense culture of these PDAC cells. Cerivastatin, simvastatin, atorvastatin and fluvastatin also inhibited colony formation by PANC-1 and MiaPaCa-2 cells in a dose-dependent manner. In contrast, the hydrophilic statin pravastatin did not exert any inhibitory effect even at a high concentration (10 µM). Mechanistically, cerivastatin did not alter the phosphorylation of YAP at Ser127 in either PANC-1 or MiaPaCa-2 cells incubated without or with neurotensin and insulin but blunted the assembly of actin stress fiber in these cells. We extended these findings with human PDAC cells using primary KC and KPC cells, (expressing KrasG12D or both KrasG12D and mutant p53, respectively) isolated from KC or KPC mice. Using cultures of these murine cells, we show that lipophilic statins induced striking YAP translocation from the nucleus to the cytoplasm, inhibited the expression of Ctgf, Cyr61 and Birc5 and profoundly inhibited colony formation of these cells. Administration of simvastatin to KC mice subjected to diet-induced obesity prevented early pancreatic acini depletion and PanIN formation. Collectively, our results show that lipophilic statins restrain YAP activity and proliferation in pancreatic cancer cell models in vitro and attenuates early lesions leading to PDAC in vivo.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/antagonistas & inibidores , Carcinoma Ductal Pancreático/prevenção & controle , Núcleo Celular/metabolismo , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Inibidores de Hidroximetilglutaril-CoA Redutases/farmacologia , Neoplasias Pancreáticas/prevenção & controle , Proteínas Proto-Oncogênicas p21(ras)/genética , Fatores de Transcrição/antagonistas & inibidores , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Carcinoma Ductal Pancreático/genética , Carcinoma Ductal Pancreático/patologia , Proliferação de Células , Ensaio de Unidades Formadoras de Colônias , Humanos , Camundongos , Mutação , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/patologia , Fosforilação , Transporte Proteico , Fatores de Transcrição/metabolismo , Células Tumorais Cultivadas
7.
Biomed Pharmacother ; 114: 108806, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30928804

RESUMO

Agents that provide protection against irradiation-induced hematopoietic injury are urgently needed for radiotherapy. We examined the effects of the small molecule, 1,2-propanediol (PPD), on total body irradiation (TBI)-induced hematopoietic injury in C57BL/6 mice. PPD administration 1 h before TBI significantly increased hematopoietic parameters such as white blood cell, platelet, red blood cell, and lymphocyte counts in vivo and enhanced the survival of mice exposed to TBI (7.0 and 7.5 Gy). PPD administration 1 h before TBI improved bone marrow (BM) and spleen recovery after TBI, with increases in both BM cellularity and spleen index. The number of colony-forming-units in bone marrow mononuclear cells (BMNCs) in vitro also increased significantly. PPD pretreatment increased the numbers of hematopoietic stem cells and hematopoietic progenitor cells in BM. Importantly, PPD also maintained endogenous antioxidant status by decreasing levels of malondialdehyde and increasing the expression of reduced glutathione, superoxide dismutase and catalase in the serum of irradiated mice. PPD alleviated the levels of apoptosis in HSCs induced by TBI, thus increasing the proportion of dividing BMNCs. These results suggest that PPD protects against TBI-induced hematopoietic injury through the increased activities of antioxidant enzymes and the inhibition of apoptosis in HSCs. PPD increased the serum levels of granulocyte-colony stimulating factor and interleukin-6 irrespective of TBI. In conclusion, these data suggest that PPD acts as a radioprotector against radiation-induced hematopoietic injury.


Assuntos
Células-Tronco Hematopoéticas/efeitos dos fármacos , Propilenoglicol/farmacologia , Lesões Experimentais por Radiação/tratamento farmacológico , Animais , Antioxidantes/metabolismo , Apoptose/efeitos dos fármacos , Medula Óssea/efeitos dos fármacos , Medula Óssea/metabolismo , Células da Medula Óssea/efeitos dos fármacos , Células da Medula Óssea/metabolismo , Ensaio de Unidades Formadoras de Colônias/métodos , Fator Estimulador de Colônias de Granulócitos/metabolismo , Células-Tronco Hematopoéticas/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Lesões Experimentais por Radiação/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Irradiação Corporal Total/métodos
8.
Lasers Med Sci ; 34(9): 1799-1805, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-30929100

RESUMO

Methicillin-resistant Staphylococcus aureus (MRSA) is an important cause of infections in humans. Photodynamic therapy using blue light (450 nm) could possibly be used to reduce MRSA on different human tissue surfaces without killing the human cells. It could be less harmful than 300-400 nm light or common disinfectants. We applied blue light ± riboflavin (RF) to MRSA and keratinocytes, in an in vitro liquid layer model, and compared the effect to elimination using common disinfection fluids. MRSA dilutions (8 × 105/mL) in wells were exposed to blue light (450 nm) ± RF at four separate doses (15, 30, 56, and 84 J/cm2). Treated samples were cultivated on blood agar plates and the colony forming units (CFU) determined. Adherent human cells were cultivated (1 × 104/mL) and treated in the same way. The cell activity was then measured by Cell Titer Blue assay after 24- and 48-h growth. The tested disinfectants were chlorhexidine and hydrogen peroxide. Blue light alone (84 J/cm2) eliminated 70% of MRSA. This dose and riboflavin eradicated 99-100% of MRSA. Keratinocytes were not affected by blue light alone at any dose. A dose of 30 J/cm2 in riboflavin solution inactivated keratinocytes completely. Disinfectants inactivated all cells. Blue light alone at 450 nm can eliminate MRSA without inactivation of human keratinocytes. Hence, a high dose of blue light could perhaps be used to treat bacterial infections without loss of human skin cells. Photodynamic therapy using riboflavin and blue light should be explored further as it may perhaps be possible to exploit in treatment of skin diseases associated with keratinocyte hyperproliferation.


Assuntos
Queratinócitos/efeitos dos fármacos , Queratinócitos/efeitos da radiação , Luz , Staphylococcus aureus Resistente à Meticilina/efeitos dos fármacos , Staphylococcus aureus Resistente à Meticilina/efeitos da radiação , Fotoquimioterapia , Riboflavina/farmacologia , Linhagem Celular , Ensaio de Unidades Formadoras de Colônias , Humanos
9.
Mol Med Rep ; 19(6): 4819-4831, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30957187

RESUMO

BLM RecQ like helicase (BLM) has a pivotal role in genetic recombination, transcription, DNA replication and DNA repair, which presents the possibility of using BLM as an anti­cancer target for treatment. However, the post­transcriptional control regulation of BLM gene expression is not fully understood and limits the application of drugs targeting BLM for carcinoma therapy in the future. MicroRNAs (miRNAs) inhibit gene expression through interaction with the 3' untranslated region (3'­UTR) of mRNA at the post­transcriptional stage. Therefore, the current study screened for miRNAs that regulate BLM gene expression, with software predicting that miRNA (miR)­27b­3p, miR­607, miR­361­3p, miR­628­5p and miR­338­3p. BLM gene expression levels in the PC3 prostate cancer cell line and RWPE­2 normal prostate epithelium cell line were detected by reverse transcription­quantitative PCR. Additionally, BLM mRNA levels were following miRNA overexpression for 24 and 48 h. For further miRNA filtration and validation, a dual­luciferase reporter system and western blot analysis were performed, which demonstrated that miR­27b­3p and miR­607 reduce BLM gene expression by directly targeting the BLM mRNA 3'­UTR. A Box­Behnken design experiment suggested that miR­27b­3p and miR­607 have synergetic mutual effects on BLM gene expression. Finally, the suppressive effect of miR­27b­3p and miR­607 on PC3 cell proliferation, colony formation, migration and invasion indicated the benefit of studying BLM as a drug target in cancer. In conclusion, the findings of the current provide evidence that miR­27b­3p and miR­607 have an oncosuppressive function in PC3 cells and cooperatively downregulate BLM expression at the post­transcriptional level.


Assuntos
Regiões 3' não Traduzidas/genética , Regulação da Expressão Gênica , MicroRNAs/genética , RecQ Helicases/genética , Movimento Celular , Proliferação de Células , Ensaio de Unidades Formadoras de Colônias , Humanos , MicroRNAs/metabolismo , Mutação , Células PC-3 , RNA Mensageiro/metabolismo , Deleção de Sequência
10.
PLoS One ; 14(4): e0213452, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30943212

RESUMO

Bone marrow stromal cells (BMSCs) include a subset of stem cells that are considered promising for developmental studies and therapeutic applications. While it is appreciated generally that BMSC populations can exhibit morphological and functional heterogeneity upon in vitro culture expansion, the potential for heterogeneity within a single colony forming unit-generated ostensibly from a single mother cell-is less explored but is critical to design of both fundamental studies and cell therapy production. Here we observed BMSC colony formation in real time via time lapsed optical imaging and analysis, to quantify whether and how heterogeneity emerged over multiple cell divisions spanning the duration of a typical colony formation unit assay. These analyses demonstrate that such colonies are neither homogeneous subpopulations of stem cells nor necessarily derived from single originating cells. While the mechanisms for and causes of this intracolony heterogeneity are not understood fully, we further demonstrate that extensive cell-cell contacts do not correlate with senescence, but that media exchange was concurrent with diversification in even the most uniform single-cell-derived colonies. These direct quantitative observations and visualizations of colony formation provide new insights that are motivated by significant implications for both basic research and stem cell-based therapies.


Assuntos
Células da Medula Óssea/fisiologia , Divisão Celular/fisiologia , Microscopia Intravital , Células-Tronco Mesenquimais/fisiologia , Células Cultivadas , Ensaio de Unidades Formadoras de Colônias , Humanos , Análise de Célula Única , Imagem com Lapso de Tempo
11.
EBioMedicine ; 43: 150-158, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-30975542

RESUMO

BACKGROUND: Systemic mastocytosis (SM) is a haematological disease characterised by organ infiltration by neoplastic mast cells. Almost all SM patients have a mutation in the gene encoding the tyrosine kinase receptor KIT causing a D816V substitution and autoactivation of the receptor. Mast cells and CD34+ haematopoietic progenitors can carry the mutation; however, in which progenitor cell subset the mutation arises is unknown. We aimed to investigate the distribution of the D816V mutation in single mast cells and single haematopoietic stem and progenitor cells. METHODS: Fluorescence-activated single-cell index sorting and KIT D816V mutation assessment were applied to analyse mast cells and >10,000 CD34+ bone marrow progenitors across 10 haematopoietic progenitor subsets. In vitro assays verified cell-forming potential. FINDINGS: We found that in SM 60-99% of the mast cells harboured the KIT D816V mutation. Despite increased frequencies of mast cells in SM patients compared with control subjects, the haematopoietic progenitor subset frequencies were comparable. Nevertheless, the mutation could be detected throughout the haematopoietic landscape of SM patients, from haematopoietic stem cells to more lineage-primed progenitors. In addition, we demonstrate that FcεRI+ bone marrow progenitors exhibit mast cell-forming potential, and we describe aberrant CD45RA expression on SM mast cells for the first time. INTERPRETATION: The KIT D816V mutation arises in early haematopoietic stem and progenitor cells and the mutation frequency is approaching 100% in mature mast cells, which express the aberrant marker CD45RA.


Assuntos
Substituição de Aminoácidos , Predisposição Genética para Doença , Células-Tronco Hematopoéticas/metabolismo , Mastocitose Sistêmica/etiologia , Mutação , Proteínas Proto-Oncogênicas c-kit/genética , Antígenos CD34/metabolismo , Biomarcadores , Células da Medula Óssea/metabolismo , Células Cultivadas , Ensaio de Unidades Formadoras de Colônias , Citometria de Fluxo , Estudos de Associação Genética , Humanos , Imunofenotipagem , Antígenos Comuns de Leucócito/metabolismo , Mastócitos/imunologia , Mastócitos/metabolismo , Mastocitose Sistêmica/diagnóstico , Mastocitose Sistêmica/metabolismo , Análise de Célula Única
12.
Int J Mol Sci ; 20(7)2019 Apr 03.
Artigo em Inglês | MEDLINE | ID: mdl-30987116

RESUMO

Stage-specific embryonic antigen 1 (SSEA-1) is an antigenic epitope (also called CD15 antigen) defined as a Lewis X carbohydrate structure and known to be expressed in murine embryonal carcinoma cells, mouse embryonic stem cells (ESCs), and murine and human germ cells, but not human ESCs/induced pluripotent stem cells (iPSCs). It is produced by α1,3-fucosyltransferase IX gene (FUT9), and F9 ECCs having a disrupted FUT9 locus by gene targeting are reported to exhibit loss of SSEA-1 expression on their cell surface. Mouse ESCs are pluripotent cells and therefore known as "naïve stem cells (NSCs)." In contrast, human ESCs/iPSCs are thought to be epiblast stem cells (EpiSCs) that are slightly more differentiated than NSCs. Recently, it has been demonstrated that treatment of EpiSCs with several reprograming-related drugs can convert EpiSCs to cells similar to NSCs, which led us to speculate that SSEA-1 may have been expressed in these NSC-like EpiSCs. Immunocytochemical staining of these cells with anti-SSEA-1 revealed increased expression of this epitope. RT-PCR analysis also confirmed increased expression of FUT9 transcripts as well as other stemness-related transcripts such as REX-1 (ZFP42). These results suggest that SSEA-1 can be an excellent marker for human NSCs.


Assuntos
Membrana Celular/metabolismo , Polpa Dentária/citologia , Células-Tronco Pluripotentes Induzidas/citologia , Antígenos CD15/metabolismo , Dente Decíduo/citologia , Animais , Ensaio de Unidades Formadoras de Colônias , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Camundongos Endogâmicos BALB C , Camundongos Nus
13.
Acta Haematol ; 141(4): 261-267, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30965317

RESUMO

BCR-ABL1-negative myeloproliferative disorders and chronic myeloid leukaemia are haematologic malignancies characterised by single and mutually exclusive genetic alterations. Nevertheless, several patients co-expressing the JAK2V617F mutation and the BCR-ABL1 transcript have been described in the literature. We report the case of a 61-year-old male who presented with an essential thrombocythaemia phenotype and had a subsequent diagnosis of chronic phase chronic myeloid leukaemia. Colony-forming assays demonstrated the coexistence of 2 different haematopoietic clones: one was positive for the JAK2V617F mutation and the other co-expressed both JAK2V617F and the BCR-ABL1 fusion gene. No colonies displayed the BCR-ABL1 transcript alone. These findings indicate that the JAK2V617F mutation was the founding genetic alteration of the disease, followed by the acquisition of the BCR-ABL1 chimeric oncogene. Our data support the hypothesis that a heterozygous JAK2V617F clone may have favoured the bi-clonal nature of this myeloproliferative disorder, generating clones harbouring a second transforming genetic event.


Assuntos
Proteínas de Fusão bcr-abl , Regulação Enzimológica da Expressão Gênica , Regulação Leucêmica da Expressão Gênica , Janus Quinase 2 , Leucemia Mielogênica Crônica BCR-ABL Positiva , Mutação de Sentido Incorreto , Trombocitemia Essencial , Substituição de Aminoácidos , Ensaio de Unidades Formadoras de Colônias , Proteínas de Fusão bcr-abl/biossíntese , Proteínas de Fusão bcr-abl/genética , Humanos , Janus Quinase 2/biossíntese , Janus Quinase 2/genética , Leucemia Mielogênica Crônica BCR-ABL Positiva/enzimologia , Leucemia Mielogênica Crônica BCR-ABL Positiva/genética , Leucemia Mielogênica Crônica BCR-ABL Positiva/patologia , Masculino , Pessoa de Meia-Idade , Trombocitemia Essencial/enzimologia , Trombocitemia Essencial/genética , Trombocitemia Essencial/patologia
14.
J Microbiol Biotechnol ; 29(4): 571-576, 2019 Apr 28.
Artigo em Inglês | MEDLINE | ID: mdl-30955254

RESUMO

Microenvironmental stress, which is naturally observed in solid tumors, has been implicated in anticancer drug resistance. This tumor-specific stress causes the degradation of topoisomerase IIα, rendering cells resistant to topoisomerase IIα-targeted anticancer agents. In addition, microenvironmental stress can induce the overexpression of 78kDa glucose regulated protein (GRP78), which can subsequently block the activation of apoptosis induced by treatment with anticancer agents. Therefore, inhibition of topoisomerase IIα degradation and reduction in GRP78 expression may be effective strategies for inhibiting anticancer drug resistance. In this study, we investigated the active compound arctigenin, which inhibited microenvironmental stress-induced etoposide resistance in HT-29 cells. Arctigenin was also highly toxic to etoposide-resistant HT-29 cells, with an IC50 value of 10 µM for colony formation. We further showed that arctigenin inhibited the degradation of topoisomerase IIα and reduced the expression of GRP78. Thus, these results suggest that arctigenin is a novel therapeutic agent that inhibits resistance to etoposide associated with microenvironmental stress conditions.


Assuntos
Antineoplásicos/farmacologia , Neoplasias do Colo/tratamento farmacológico , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Etoposídeo/farmacologia , Furanos/antagonistas & inibidores , Células HT29/efeitos dos fármacos , Lignanas/antagonistas & inibidores , Estresse Fisiológico , Microambiente Tumoral/efeitos dos fármacos , Apoptose/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Ensaio de Unidades Formadoras de Colônias , DNA Topoisomerases Tipo II , Furanos/química , Células HT29/citologia , Proteínas de Choque Térmico/metabolismo , Humanos , Lignanas/química
15.
PLoS One ; 14(3): e0213782, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30870474

RESUMO

Myelopoiesis was evaluated in 66 pediatric patients with chronic neutropenia who were positive for anti-neutrophil antibodies (median age at diagnosis: 11 months, median neutrophil count at diagnosis: 419/µl). Other causes of neutropenia were excluded. Bone marrow morphology, clonogenic tests and/or the peripheral blood CD 34+ cell count, and apoptotic rate were evaluated in 61 patients with neutropenia lasting > 12 months or severe infections. The peripheral blood CD 34+ cell count and apoptotic rate were evaluated in five patients with shorter neutropenia. The median follow-up time was 29 months (range 7-180 months). Forty-seven patients (71.2%) had a spontaneous recovery after 7-180 months (median 29 months). The group of patients younger than 24 months at diagnosis (n = 50) had a higher probability of recovery (40/50 vs. 7/16 χ2 p<0.01) with a shorter period of neutropenia (median 26 versus 47 months, Kaplan-Meier analysis p = 0.001). The colony-forming units-granulocyte-macrophage (CFU-GM) were significantly decreased in 26/35 patients (74%) evaluated for clonogenic tests. All patients with normal CFU-GM recovered (9/9 patients); whereas, neutropenia persisted in 12/26 patients with reduced CFU-GM (46%, Pearson χ2 p = 0.02). In 36/55 (65%) patients evaluated by flow cytometry we observed reduced circulating CD34+ cells compared with controls of the same age. An increase in the circulating CD34+ cell apoptotic rate was observed in 28/55 patients (51%). Infections requiring hospitalization were observed in 9/18 (50%; Pearson χ2, p = 0.03) patients with both decreased circulating CD34+ cells and increased CD34+ apoptotic rates. In the group aged < 24 months, we observed a significant correlation between the persistence of neutropenia and decreased circulating CD34+ cells (Pearson χ2 p = 0.008). In conclusion, reduced CFU-GM and circulating hematopoietic progenitors were observed in a subgroup of children with chronic neutropenia who were positive for anti-neutrophil antibodies and had a higher incidence of severe infections and delayed spontaneous remission.


Assuntos
Células Progenitoras de Granulócitos e Macrófagos/patologia , Células-Tronco Hematopoéticas/patologia , Neutropenia/patologia , Recuperação de Função Fisiológica , Adolescente , Antígenos CD34/análise , Células Cultivadas , Criança , Pré-Escolar , Ensaio de Unidades Formadoras de Colônias , Feminino , Humanos , Lactente , Masculino , Neutropenia/etiologia
16.
Lasers Med Sci ; 34(9): 1755-1761, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-30891656

RESUMO

Infections caused by Acinetobacter baumannii have become a challenge for healthcare professionals because of the rapid increase in Gram-negative bacteria resistant to carbapenem antibiotics. The objective of this study was to evaluate the effect of antimicrobial photodynamic therapy (aPDT) against different strains of A. baumannii isolated from patients with infectious process and hospitalized at the intensive care unit of the hospitals of São Jose dos Campos, São Paulo. These isolates were obtained from the Valeclin Clinical Analysis Laboratory (SP, Brazil) and were tested for susceptibility to the carbapenems imipenem and meropenem by determination of the minimal inhibitory concentration (MIC) using the broth microdilution method. The strains susceptible and resistant to these antibiotics were submitted to aPDT using methylene blue and a low-level laser with a wavelength of 660 nm and fluence of 39.5 J/cm2 (energy of 15 J and time of 428 s). The number of colony-forming units (CFU/mL) was analyzed by ANOVA and the Tukey test. The laboratory of origin of the clinical isolates identified 1.54% of 13,715 strains tested over a period of 8 months as A. baumannii. Among the A. baumannii isolates, 58% were resistant to carbapenems by the disk diffusion test. Susceptible isolates exhibited MIC of 0.5 to 1 µg/mL and resistant isolates of 64 to > 128 µg/mL. PDT reduced the number of A. baumannii cells for all isolates tested, with this reduction ranging from 63 to 88% for susceptible isolates and from 26 to 97% for resistant isolates. The percentage of viability was dependent on the strain analyzed. In conclusion, these data indicate that PDT could be an alternative strategy for the control of infections caused by carbapenem-resistant A. baumannii.


Assuntos
Acinetobacter baumannii/isolamento & purificação , Carbapenêmicos/farmacologia , Resistência Microbiana a Medicamentos , Fotoquimioterapia , Infecções por Acinetobacter , Acinetobacter baumannii/efeitos dos fármacos , Antibacterianos/farmacologia , Ensaio de Unidades Formadoras de Colônias , Humanos , Azul de Metileno/farmacologia , Testes de Sensibilidade Microbiana , Viabilidade Microbiana/efeitos dos fármacos , Fármacos Fotossensibilizantes/farmacologia
17.
Cancer Sci ; 110(4): 1317-1330, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-30767320

RESUMO

Adult long-term hematopoiesis depends on sustaining hematopoietic stem/progenitor cells (HSPC) in bone marrow (BM) niches, where their balance of quiescence, self-renewal, and hematopoietic differentiation is tightly regulated. Although various BM stroma cells that produce niche factors have been identified, regulation of the intrinsic responsiveness of HSPC to the niche factors remains elusive. We previously reported that mice deficient for Sipa1, a Rap1 GTPase-activating protein, develop diverse hematopoietic disorders of late onset. Here we showed that transplantation of BM cells expressing membrane-targeted C3G (C3G-F), a Rap1 GTP/GDP exchanger, resulted in the progressive decline of the numbers of HSPC repopulated in BM with time and impaired long-term hematopoiesis of all cell lineages. C3G-F/HSPC were sustained for months in spleen retaining hematopoietic potential, but these cells inefficiently contributed to overall hematopoietic reconstitution. C3G-F/HSPC showed enhanced proliferation and differentiation with accelerated progenitor cell exhaustion in response to stem cell factor (SCF). Using a Ba/F3 cell line, we confirmed that the increased basal Rap1GTP levels with C3G-F expression caused a markedly prolonged activation of c-Kit receptor and downstream signaling through SCF ligation. A minor population of C3G-F/HSPC also showed enhanced proliferation in the presence of thrombopoietin (TPO) compared to Vect/HSPC. Current results suggest an important role of basal Rap1 activation status of HSPC in their maintenance in BM for sustaining long-term adult hematopoiesis.


Assuntos
Hematopoese , Células-Tronco Hematopoéticas/metabolismo , Transdução de Sinais , Proteínas de Ligação a Telômeros/metabolismo , Animais , Biomarcadores , Células da Medula Óssea/citologia , Células da Medula Óssea/efeitos dos fármacos , Células da Medula Óssea/metabolismo , Transplante de Medula Óssea , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/genética , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Ensaio de Unidades Formadoras de Colônias , Hematopoese/genética , Células-Tronco Hematopoéticas/citologia , Imunofenotipagem , Camundongos , Proteínas Proto-Oncogênicas c-kit/metabolismo , Fator de Células-Tronco/farmacologia , Proteínas rap de Ligação ao GTP/metabolismo
18.
J Radiat Res ; 60(2): 163-170, 2019 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-30624744

RESUMO

Radiation-induced rescue effect (RIRE) refers to the phenomenon in which detrimental effects in targeted irradiated cells are reduced upon receiving feedback signals from partnered non-irradiated bystander cells, or from the medium previously conditioning these partnered non-irradiated bystander cells. For convenience, in the current review we define two types of RIRE: (i) Type 1 RIRE (reduced detrimental effects in targeted cells upon receiving feedback signals from bystander cells) and (ii) Type 2 RIRE (exacerbated detrimental effects in targeted cells upon receiving feedback signals from bystander cells). The two types of RIRE, as well as the associated mechanisms and chemical messengers, have been separately reviewed. The recent report on the potential effects of RIRE on the traditional colony-formation assays has also been reviewed. Finally, future priorities and directions for research into RIRE are discussed.


Assuntos
Efeito Espectador/efeitos da radiação , Radiação , Animais , Autofagia/efeitos da radiação , Ensaio de Unidades Formadoras de Colônias , Humanos , NF-kappa B/metabolismo , Transdução de Sinais
19.
Radiat Oncol ; 14(1): 11, 2019 Jan 17.
Artigo em Inglês | MEDLINE | ID: mdl-30654822

RESUMO

BACKGROUND: The implementation of magnetic resonance imaging (MRI) guided radiotherapy (RT) continues to increase. Very limited in-vitro data on the interaction of ionizing radiation and magnetic fields (MF) have been published. In these experiments we focused on the radiation response in a MF of the TK6 human lymphoblastoid cells which are known to be highly radiosensitive due to efficient radiation-induced apoptosis. METHODS: Clonogenicity was determined 12-14 days after irradiation with 1-4 Gy 6 MV photons with or without a 1.0 Tesla MF. Furthermore, alterations in cell cycle distribution and rates of radiation induced apoptosis (FACS analysis of cells with sub-G1 DNA content) were analyzed. RESULTS: Clonogenic survival showed an exponential dose-dependence, and the radiation sensitivity parameter (α = 1.57/Gy) was in accordance with earlier reports. Upon comparing the clonogenic survival between the two groups, identical results within error bars were obtained. The survival fractions at 2 Gy were 9% (without MF) and 8.5% (with MF), respectively. CONCLUSION: A 1.0 Tesla MF does not affect the clonogenicity of TK6 cells irradiated with 1-4 Gy 6MV photons. This supports the use of MRI guided RT, however ongoing research on the interaction of MF and radiotherapy is warranted.


Assuntos
Apoptose/efeitos da radiação , Ciclo Celular , Linfócitos/citologia , Linfócitos/efeitos da radiação , Campos Magnéticos , Fótons , Sobrevivência Celular , Células Cultivadas , Ensaio de Unidades Formadoras de Colônias , Humanos
20.
PLoS One ; 14(1): e0205215, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30682016

RESUMO

Ultraviolet A (UVA) radiation is harmful for living organisms but in low doses may stimulate cell proliferation. Our aim was to examine the relationships between exposure to different low UVA doses, cellular proliferation, and changes in cellular reactive oxygen species levels. In human colon cancer (HCT116) and melanoma (Me45) cells exposed to UVA doses comparable to environmental, the highest doses (30-50 kJ/m2) reduced clonogenic potential but some lower doses (1 and 10 kJ/m2) induced proliferation. This effect was cell type and dose specific. In both cell lines the levels of reactive oxygen species and nitric oxide fluctuated with dynamics which were influenced differently by UVA; in Me45 cells decreased proliferation accompanied the changes in the dynamics of H2O2 while in HCT116 cells those of superoxide. Genes coding for proteins engaged in redox systems were expressed differently in each cell line; transcripts for thioredoxin, peroxiredoxin and glutathione peroxidase showed higher expression in HCT116 cells whereas those for glutathione transferases and copper chaperone were more abundant in Me45 cells. We conclude that these two cell types utilize different pathways for regulating their redox status. Many mechanisms engaged in maintaining cellular redox balance have been described. Here we show that the different cellular responses to a stimulus such as a specific dose of UVA may be consequences of the use of different redox control pathways. Assays of superoxide and hydrogen peroxide level changes after exposure to UVA may clarify mechanisms of cellular redox regulation and help in understanding responses to stressing factors.


Assuntos
Proliferação de Células/efeitos da radiação , Oxirredução/efeitos da radiação , Raios Ultravioleta , Linhagem Celular Tumoral , Ensaio de Unidades Formadoras de Colônias/métodos , Regulação da Expressão Gênica/efeitos da radiação , Humanos , Peróxido de Hidrogênio/metabolismo , Redes e Vias Metabólicas/efeitos da radiação , Superóxidos/metabolismo
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