RESUMO
Several in vitro models have been developed to mimic chronic lymphocytic leukemia (CLL) proliferation in immune niches; however, they typically do not induce robust proliferation. We prepared a novel model based on mimicking T-cell signals in vitro and in patient-derived xenografts (PDXs). Six supportive cell lines were prepared by engineering HS5 stromal cells with stable expression of human CD40L, IL4, IL21, and their combinations. Co-culture with HS5 expressing CD40L and IL4 in combination led to mild CLL cell proliferation (median 7% at day 7), while the HS5 expressing CD40L, IL4, and IL21 led to unprecedented proliferation rate (median 44%). The co-cultures mimicked the gene expression fingerprint of lymph node CLL cells (MYC, NFκB, and E2F signatures) and revealed novel vulnerabilities in CLL-T-cell-induced proliferation. Drug testing in co-cultures revealed for the first time that pan-RAF inhibitors fully block CLL proliferation. The co-culture model can be downscaled to five microliter volume for large drug screening purposes or upscaled to CLL PDXs by HS5-CD40L-IL4 ± IL21 co-transplantation. Co-transplanting NSG mice with purified CLL cells and HS5-CD40L-IL4 or HS5-CD40L-IL4-IL21 cells on collagen-based scaffold led to 47% or 82% engraftment efficacy, respectively, with ~20% of PDXs being clonally related to CLL, potentially overcoming the need to co-transplant autologous T-cells in PDXs.
Assuntos
Ligante de CD40 , Proliferação de Células , Técnicas de Cocultura , Leucemia Linfocítica Crônica de Células B , Células Estromais , Humanos , Leucemia Linfocítica Crônica de Células B/patologia , Leucemia Linfocítica Crônica de Células B/genética , Leucemia Linfocítica Crônica de Células B/tratamento farmacológico , Animais , Camundongos , Células Estromais/metabolismo , Células Estromais/patologia , Ligante de CD40/metabolismo , Ligante de CD40/genética , Linfócitos T/imunologia , Linfócitos T/metabolismo , Ensaios Antitumorais Modelo de Xenoenxerto , Interleucinas/genética , Interleucinas/metabolismo , Inibidores de Proteínas Quinases/farmacologiaRESUMO
Duchenne muscular dystrophy (DMD) is a devastating condition shortening the lifespan of young men. DMD patients suffer from age-related dilated cardiomyopathy (DCM) that leads to heart failure. Several molecular mechanisms leading to cardiomyocyte death in DMD have been described. However, the pathological progression of DMD-associated DCM remains unclear. In skeletal muscle, a dramatic decrease in stem cells, so-called satellite cells, has been shown in DMD patients. Whether similar dysfunction occurs with cardiac muscle cardiovascular progenitor cells (CVPCs) in DMD remains to be explored. We hypothesized that the number of CVPCs decreases in the dystrophin-deficient heart with age and disease state, contributing to DCM progression. We used the dystrophin-deficient mouse model (mdx) to investigate age-dependent CVPC properties. Using quantitative PCR, flow cytometry, speckle tracking echocardiography, and immunofluorescence, we revealed that young mdx mice exhibit elevated CVPCs. We observed a rapid age-related CVPC depletion, coinciding with the progressive onset of cardiac dysfunction. Moreover, mdx CVPCs displayed increased DNA damage, suggesting impaired cardiac muscle homeostasis. Overall, our results identify the early recruitment of CVPCs in dystrophic hearts and their fast depletion with ageing. This latter depletion may participate in the fibrosis development and the acceleration onset of the cardiomyopathy.
Assuntos
Cardiomiopatia Dilatada/genética , Distrofina/genética , Distrofia Muscular de Duchenne/genética , Miocárdio/metabolismo , Proteínas Proto-Oncogênicas c-kit/genética , Envelhecimento/genética , Envelhecimento/patologia , Animais , Cardiomiopatia Dilatada/metabolismo , Cardiomiopatia Dilatada/patologia , Sistema Cardiovascular/metabolismo , Sistema Cardiovascular/patologia , Dano ao DNA/genética , Modelos Animais de Doenças , Distrofina/deficiência , Regulação da Expressão Gênica/genética , Humanos , Camundongos , Camundongos Endogâmicos mdx/genética , Distrofia Muscular de Duchenne/metabolismo , Distrofia Muscular de Duchenne/patologia , Miocárdio/patologia , Miócitos Cardíacos/metabolismo , Miócitos Cardíacos/patologia , Células-Tronco/metabolismo , Células-Tronco/patologiaRESUMO
In the fast-developing field of tissue engineering there is a constant demand for new materials as scaffolds for cell seeding, which can better mimic a natural extracellular matrix as well as control cell behavior. Among other materials, polysaccharides are widely used for this purpose. One of the main candidates for scaffold fabrication is alginate. However, it lacks sites for cell adhesion. That is why one of the steps toward the development of suitable scaffolds for cells is the introduction of the biofunctionality to the alginate structure. In this work we focused on bone-sialoprotein derived peptide (TYRAY) conjugation to the molecule of alginate. Here the comparison study on four different approaches of peptide conjugation was performed including traditional and novel modification methods, based on 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide/N-hydroxy succinimide (EDC/NHS), 4-(4,6-dimethoxy-1,3,5-triazine-2-yl)-4-methylmorpholinium chloride (DMTMM), thiol-Michael addition and Cu-catalyzed azide-alkyne cycloaddition reactions. It was shown that the combination of the alginate amidation with the use of and subsequent Cu-catalyzed azide-alkyne cycloaddition led to efficient peptide conjugation, which was proven with both NMR and XPS methods. Moreover, the cell culture experiment proved the positive effect of peptide presence on the adhesion of human embryonic stem cells.
Assuntos
Alginatos/química , Biomimética , Peptídeos/química , Engenharia Tecidual , Alicerces Teciduais , Aminas/química , Biomimética/métodos , Adesão Celular , Técnicas de Cultura de Células , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Química Click , Espectroscopia de Ressonância Magnética , Estrutura Molecular , Peptídeos/farmacologia , Engenharia Tecidual/métodosRESUMO
Organ function depends on tissues adopting the correct architecture. However, insights into organ architecture are currently hampered by an absence of standardized quantitative 3D analysis. We aimed to develop a robust technology to visualize, digitalize, and segment the architecture of two tubular systems in 3D: double resin casting micro computed tomography (DUCT). As proof of principle, we applied DUCT to a mouse model for Alagille syndrome (Jag1Ndr/Ndr mice), characterized by intrahepatic bile duct paucity, that can spontaneously generate a biliary system in adulthood. DUCT identified increased central biliary branching and peripheral bile duct tortuosity as two compensatory processes occurring in distinct regions of Jag1Ndr/Ndr liver, leading to full reconstitution of wild-type biliary volume and phenotypic recovery. DUCT is thus a powerful new technology for 3D analysis, which can reveal novel phenotypes and provide a standardized method of defining liver architecture in mouse models.
Many essential parts of the body contain tubes: the liver for example, contains bile ducts and blood vessels. These tubes develop right next to each other, like entwined trees. To do their jobs, these ducts must communicate and collaborate, but they do not always grow properly. For example, babies with Alagille syndrome are born with few or no bile ducts, resulting in serious liver disease. Understanding the architecture of the tubes in their livers could explain why some children with this syndrome improve with time, but many others need a liver transplant. Visualising biological tubes in three dimensions is challenging. One major roadblock is the difficulty in seeing several tubular structures at once. Traditional microscopic imaging of anatomy is in two dimensions, using slices of tissue. This approach shows the cross-sections of tubes, but not how the ducts connect and interact. An alternative is to use micro computed tomography scans, which use X-rays to examine structures in three dimensions. The challenge with this approach is that soft tissues, which tubes in the body are made of, do not show up well on X-ray. One way to solve this is to fill the ducts with X-ray absorbing resins, making a cast of the entire tree structure. The question is, can two closely connected tree structures be distinguished if they are cast at the same time? To address this question, Hankeova, Salplachta et al. developed a technique called double resin casting micro computed tomography, or DUCT for short. The approach involved making casts of tube systems using two types of resin that show up differently under X-rays. The new technique was tested on a mouse model of Alagille syndrome. One resin was injected into the bile ducts, and another into the blood vessels. This allowed Hankeova, Salplachta et al. to reconstruction both trees digitally, revealing their length, volume, branching, and interactions. In healthy mice, the bile ducts were straight with uniform branches, but in mice with Alagille syndrome ducts were wiggly, and had extra branches in the centre of the liver. This new imaging technique could improve the understanding of tube systems in animal models of diseases, both in the liver and in other organs with tubes, such as the lungs or the kidneys. Hankeova, Salplachta et al. also lay a foundation for a deeper understanding of bile duct recovery in Alagille syndrome. In the future, DUCT could help researchers to see how mouse bile ducts change in response to experimental therapies.
Assuntos
Síndrome de Alagille/fisiopatologia , Ductos Biliares/fisiopatologia , Microtomografia por Raio-X/métodos , Animais , Ductos Biliares/crescimento & desenvolvimento , Modelos Animais de Doenças , Camundongos , Camundongos Transgênicos , Microtomografia por Raio-X/classificaçãoRESUMO
High-mobility group box (HMGB)1 and HMGB2 proteins are the subject of intensive research because of their involvement in DNA replication, repair, transcription, differentiation, proliferation, cell signaling, inflammation, and tumor migration. Using inducible, stably transfected human embryonic stem cells (hESCs) capable of the short hairpin RNA-mediated knockdown (KD) of HMGB1 and HMGB2, we provide evidence that deregulation of HMGB1 or HMGB2 expression in hESCs and their differentiated derivatives (neuroectodermal cells) results in distinct modulation of telomere homeostasis. Whereas HMGB1 enhances telomerase activity, HMGB2 acts as a negative regulator of telomerase activity in the cell. Stimulation of telomerase activity in the HMGB2-deficient cells may be related to activation of the PI3K/protein kinase B/ glycogen synthase kinase-3ß/ß-catenin signaling pathways by HMGB1, augmented TERT/telomerase RNA subunit transcription, and possibly also because of changes in telomeric repeat-containing RNA (TERRA) and TERRA-polyA+ transcription. The impact of HMGB1/2 KD on telomerase transcriptional regulation observed in neuroectodermal cells is partially masked in hESCs by their pluripotent state. Our findings on differential roles of HMGB1 and HMGB2 proteins in regulation of telomerase activity may suggest another possible outcome of HMGB1 targeting in cells, which is currently a promising approach aiming at increasing the anticancer activity of cytotoxic agents.-Kucírek, M., Bagherpoor, A. J., Jaros, J., Hampl, A., Stros, M. HMGB2 is a negative regulator of telomerase activity in human embryonic stem and progenitor cells.
Assuntos
Proteína HMGB2/fisiologia , Células-Tronco Embrionárias Humanas/enzimologia , Células-Tronco/enzimologia , Telomerase/metabolismo , Diferenciação Celular , Proteína HMGB1/genética , Proteína HMGB2/genética , Células-Tronco Embrionárias Humanas/citologia , Humanos , Células-Tronco/citologia , Transcrição Gênica , TransfecçãoRESUMO
Introduction of small-molecule inhibitors of B-cell receptor signaling and BCL2 protein significantly improves therapeutic options in chronic lymphocytic leukemia. However, some patients suffer from adverse effects mandating treatment discontinuation, and cases with TP53 defects more frequently experience early progression of the disease. Development of alternative therapeutic approaches is, therefore, of critical importance. Here we report details of the anti-chronic lymphocytic leukemia single-agent activity of MU380, our recently identified potent, selective, and metabolically robust inhibitor of checkpoint kinase 1. We also describe a newly developed enantioselective synthesis of MU380, which allows preparation of gram quantities of the substance. Checkpoint kinase 1 is a master regulator of replication operating primarily in intra-S and G2/M cell cycle checkpoints. Initially tested in leukemia and lymphoma cell lines, MU380 significantly potentiated efficacy of gemcitabine, a clinically used inducer of replication stress. Moreover, MU380 manifested substantial single-agent activity in both TP53-wild type and TP53-mutated leukemia and lymphoma cell lines. In chronic lymphocytic leukemia-derived cell lines MEC-1, MEC-2 (both TP53-mut), and OSU-CLL (TP53-wt) the inhibitor impaired cell cycle progression and induced apoptosis. In primary clinical samples, MU380 used as a single-agent noticeably reduced the viability of unstimulated chronic lymphocytic leukemia cells as well as those induced to proliferate by anti-CD40/IL-4 stimuli. In both cases, effects were comparable in samples harboring p53 pathway dysfunction (TP53 mutations or ATM mutations) and TP53-wt/ATM-wt cells. Lastly, MU380 also exhibited significant in vivo activity in a xenotransplant mouse model (immunodeficient strain NOD-scid IL2Rγnull ) where it efficiently suppressed growth of subcutaneous tumors generated from MEC-1 cells.
Assuntos
Quinase 1 do Ponto de Checagem/antagonistas & inibidores , Sinergismo Farmacológico , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Leucemia Linfocítica Crônica de Células B/tratamento farmacológico , Mutação , Piperidinas/farmacologia , Pirazóis/farmacologia , Pirimidinas/farmacologia , Proteína Supressora de Tumor p53/genética , Animais , Antimetabólitos Antineoplásicos/farmacologia , Apoptose , Biomarcadores Tumorais/genética , Ciclo Celular , Proliferação de Células , Desoxicitidina/análogos & derivados , Desoxicitidina/farmacologia , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Feminino , Humanos , Leucemia Linfocítica Crônica de Células B/genética , Leucemia Linfocítica Crônica de Células B/patologia , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Inibidores de Proteínas Quinases/farmacologia , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de Xenoenxerto , GencitabinaRESUMO
Checkpoint-mediated dependency of tumor cells can be deployed to selectively kill them without substantial toxicity to normal cells. Specifically, loss of CHK1, a serine threonine kinase involved in the surveillance of the G2-M checkpoint in the presence of replication stress inflicted by DNA-damaging drugs, has been reported to dramatically influence the viability of tumor cells. CHK1's pivotal role in maintaining genomic stability offers attractive opportunity for increasing the selectivity, effectivity, and reduced toxicity of chemotherapy. Some recently identified CHK1 inhibitors entered clinical trials in combination with DNA antimetabolites. Herein, we report synthesis and profiling of MU380, a nontrivial analogue of clinically profiled compound SCH900776 possessing the highly unusual N-trifluoromethylpyrazole motif, which was envisioned not to undergo metabolic oxidative dealkylation and thereby provide greater robustness to the compound. MU380 is a selective and potent inhibitor of CHK1 which sensitizes a variety of tumor cell lines to hydroxyurea or gemcitabine up to 10 times. MU380 shows extended inhibitory effects in cells, and unlike SCH900776, does not undergo in vivo N-dealkylation to the significantly less selective metabolite. Compared with SCH900776, MU380 in combination with GEM causes higher accumulation of DNA damage in tumor cells and subsequent enhanced cell death, and is more efficacious in the A2780 xenograft mouse model. Overall, MU380 represents a novel state-of-the-art CHK1 inhibitor with high potency, selectivity, and improved metabolic robustness to oxidative N-dealkylation. Mol Cancer Ther; 16(9); 1831-42. ©2017 AACR.
Assuntos
Antineoplásicos/síntese química , Antineoplásicos/farmacologia , Quinase 1 do Ponto de Checagem/antagonistas & inibidores , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Inibidores de Proteínas Quinases/síntese química , Inibidores de Proteínas Quinases/farmacologia , Animais , Apoptose/efeitos dos fármacos , Biomarcadores , Ciclo Celular/efeitos dos fármacos , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Remoção de Radical Alquila/efeitos dos fármacos , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Humanos , Metilação , Camundongos , Estrutura Molecular , Pirazóis/farmacologia , Pirimidinas/farmacologia , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
BACKGROUND: Lipomodeling is a technique that uses the patient's own fat for tissue regeneration and augmentation. The extent of regenerative effect is reported to be determined by the numbers of adipose-derived stem cells and the viability of cells in processed adipose tissue which, together with other factors, influence the degree of graft retention. This study addresses whether differences exist in properties of fat graft obtained by three commonly used techniques. METHODS: Adipose tissue harvested from the hypogastric regions of 14 patients was processed by decantation, centrifugation, and membrane-based tissue filtration. The morphology of each preparation was assessed by electron microscopy and overall cell viability was assessed by live/dead assay. The number of adipose-derived stem cells was determined and their stem cell character was assessed by the presence of cell surface molecules (i.e., CD105, CD90, CD31, and CD45) and by their capacity to differentiate into adipogenic and osteogenic lineages. RESULTS: First, morphologies of processed fat samples obtained by individual procedures differed, but no preparation caused obvious damage to cellular or acellular components. Second, although the highest numbers of adipose-derived stem cells were contained in the upper fraction of centrifuged lipoaspirates, the difference between preparations was marginal. Third, the maximal concentration of adipose fraction (removal of watery component) of lipoaspirate was achieved by membrane-based tissue filtration. Finally, no significant differences in overall viability were detected. CONCLUSIONS: Properties of processed lipoaspirate were influenced by the preparation procedure. However, the differences were not dramatic; both centrifugation and membrane-based filtration are methods of choice whose selection depends on other criteria (e.g., practicality) for individual surgical settings.
Assuntos
Tecido Adiposo/transplante , Coleta de Tecidos e Órgãos/métodos , Adipócitos , Adolescente , Adulto , Células Cultivadas , Técnicas Citológicas , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Células-Tronco , Adulto JovemRESUMO
Novel hydrolytically stable gelatin nanofibers modified with sodium or calcium salt of oxycellulose were prepared by electrospinning method. The unique inhibitory effect of these nanofibers against Escherichia coli bacteria was examined by luminometric method. Biocompatibility of these gelatin/oxycellulose nanofibers with eukaryotic cells was tested using human lung adenocarcinoma cell line NCI-H441. Cells firmly adhered to nanofiber surface, as determined by scanning electron microscopy, and no signs of cell dying were detected by fluorescent live/dead assay. We propose that the newly developed gelatin/oxycellulose nanofibers could be used as promising scaffold for lung disease modeling and anti-cancer drug testing.
Assuntos
Adenocarcinoma/tratamento farmacológico , Celulose Oxidada , Gelatina , Neoplasias Pulmonares/tratamento farmacológico , Nanofibras/química , Adenocarcinoma/metabolismo , Adenocarcinoma/patologia , Linhagem Celular Tumoral , Celulose Oxidada/química , Celulose Oxidada/farmacologia , Escherichia coli/crescimento & desenvolvimento , Gelatina/química , Gelatina/farmacologia , Humanos , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologiaRESUMO
Reactive oxygen species (ROS) are important regulators of cellular functions. In embryonic stem cells, ROS are suggested to influence differentiation status. Regulated ROS formation is catalyzed primarily by NADPH-dependent oxidases (NOXs). Apocynin and diphenyleneiodonium are frequently used inhibitors of NOXs; however, both exhibit uncharacterized effects not related to NOXs inhibition. Interestingly, in our model of mouse embryonic stem cells we demonstrate low expression of NOXs. Therefore we aimed to clarify potential side effects of these drugs. Both apocynin and diphenyleneiodonium impaired proliferation of cells. Surprisingly, we observed prooxidant activity of these drugs determined by hydroethidine. Further, we revealed that apocynin inhibits PI3K/Akt pathway with its downstream transcriptional factor Nanog. Opposite to this, apocynin augmented activity of canonical Wnt signaling. On the contrary, diphenyleneiodonium activated both PI3K/Akt and Erk signaling pathways without affecting Wnt. Our data indicates limits and possible unexpected interactions of NOXs inhibitors with intracellular signaling pathways.
Assuntos
Acetofenonas/farmacologia , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Células-Tronco Embrionárias Murinas/metabolismo , Oniocompostos/farmacologia , Estresse Oxidativo/efeitos dos fármacos , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Animais , Proliferação de Células/efeitos dos fármacos , Sinergismo Farmacológico , Camundongos , Células-Tronco Embrionárias Murinas/efeitos dos fármacos , NADPH Oxidases/genética , NADPH Oxidases/metabolismo , Fosforilação/efeitos dos fármacos , Espécies Reativas de Oxigênio/metabolismo , Fator de Transcrição STAT3/metabolismo , Proteínas Wnt/metabolismoRESUMO
CD133 (also known as prominin-1) is a cell surface glycoprotein that is widely used for the identification of stem cells. Furthermore, its glycosylated epitope, AC133, has recently been discussed as a marker of cancer stem cells in various human malignancies. During our recent experiments on rhabdomyosarcomas (RMS), we unexpectedly identified an atypical nuclear localization of CD133 in a relatively stable subset of cells in five RMS cell lines established in our laboratory. To the best of our knowledge, this atypical localization of CD133 has not yet been proven or analyzed in detail in cancer cells. In the present study, we verified the nuclear localization of CD133 in RMS cells using three independent anti-CD133 antibodies, including both rabbit polyclonal and mouse monoclonal antibodies. Indirect immunofluorescence and confocal microscopy followed by software cross-section analysis, transmission electron microscopy and cell fractionation with immunoblotting were also employed, and all the results undeniably confirmed the presence of CD133 in the nuclei of stable minor subpopulations of all five RMS cell lines. The proportion of cells showing an exclusive nuclear localization of CD133 ranged from 3.4 to 7.5%, with only minor differences observed among the individual anti-CD133 antibodies. Although the role of CD133 in the cell nucleus remains unclear, these results clearly indicate that this atypical nuclear localization of CD133 in a minor subpopulation of cancer cells is a common phenomenon in RMS cell lines.
Assuntos
Antígenos CD/metabolismo , Biomarcadores Tumorais/metabolismo , Núcleo Celular/metabolismo , Glicoproteínas/metabolismo , Peptídeos/metabolismo , Rabdomiossarcoma/metabolismo , Antígeno AC133 , Anticorpos Monoclonais/imunologia , Antígenos CD/imunologia , Linhagem Celular Tumoral , Técnica Indireta de Fluorescência para Anticorpo , Glicoproteínas/imunologia , Humanos , Microscopia Eletrônica de Transmissão , Peptídeos/imunologiaRESUMO
BACKGROUND: Tumor heterogeneity and the plasticity of cancer cells present challenges for effective clinical diagnosis and therapy. Such challenges are epitomized by neuroendocrine transdifferentiation (NED) and the emergence of neuroendocrine-like cancer cells in prostate tumors. This phenomenon frequently arises from androgen-depleted prostate adenocarcinoma and is associated with the development of castration-resistant prostate cancer and poor prognosis. RESULTS: In this study, we showed that NED was evoked in both androgen receptor (AR)-positive and AR-negative prostate epithelial cell lines by growing the cells to a high density. Androgen depletion and high-density cultivation were both associated with cell cycle arrest and deregulated expression of several cell cycle regulators, such as p27Kip1, members of the cyclin D protein family, and Cdk2. Dual inhibition of Cdk1 and Cdk2 using pharmacological inhibitor or RNAi led to modulation of the cell cycle and promotion of NED. We further demonstrated that the cyclic adenosine 3', 5'-monophosphate (cAMP)-mediated pathway is activated in the high-density conditions. Importantly, inhibition of cAMP signaling using a specific inhibitor of adenylate cyclase, MDL-12330A, abolished the promotion of NED by high cell density. CONCLUSIONS: Taken together, our results imply a new relationship between cell cycle attenuation and promotion of NED and suggest high cell density as a trigger for cAMP signaling that can mediate reversible NED in prostate cancer cells.
Assuntos
Transdiferenciação Celular , Células Neuroendócrinas/patologia , Neoplasias da Próstata/patologia , Androgênios/farmacologia , Proteína Quinase CDC2 , Contagem de Células , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Transdiferenciação Celular/efeitos dos fármacos , AMP Cíclico/metabolismo , Quinase 2 Dependente de Ciclina/metabolismo , Quinases Ciclina-Dependentes/metabolismo , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/enzimologia , Células Epiteliais/patologia , Humanos , Imuno-Histoquímica , Masculino , Células Neuroendócrinas/efeitos dos fármacos , Inibidores de Proteínas Quinases/farmacologia , Receptores Androgênicos/metabolismo , Transdução de Sinais/efeitos dos fármacosRESUMO
Death ligands and their tumor necrosis factor receptor (TNFR) family receptors are the best-characterized and most efficient inducers of apoptotic signaling in somatic cells. In this study, we analyzed whether these prototypic activators of apoptosis are also expressed and able to be activated in human pluripotent stem cells. We examined human embryonic stem cells (hESC) and human-induced pluripotent stem cells (hiPSC) and found that both cell types express primarily TNF-related apoptosis-inducing ligand (TRAIL) receptors and TNFR1, but very low levels of Fas/CD95. We also found that although hESC and hiPSC contain all the proteins required for efficient induction and progression of extrinsic apoptotic signaling, they are resistant to TRAIL-induced apoptosis. However, both hESC and hiPSC can be sensitized to TRAIL-induced apoptosis by co-treatment with protein synthesis inhibitors such as the anti-leukemia drug homoharringtonine (HHT). HHT treatment led to suppression of cellular FLICE inhibitory protein (cFLIP) and Mcl-1 expression and, in combination with TRAIL, enhanced processing of caspase-8 and full activation of caspase-3. cFLIP likely represents an important regulatory node, as its shRNA-mediated down-regulation significantly sensitized hESC to TRAIL-induced apoptosis. Thus, we provide the first evidence that, irrespective of their origin, human pluripotent stem cells express canonical components of the extrinsic apoptotic system and on stress can activate death receptor-mediated apoptosis.
Assuntos
Proteína Reguladora de Apoptosis Semelhante a CASP8 e FADD/genética , Células-Tronco Embrionárias/metabolismo , Células-Tronco Pluripotentes/metabolismo , Receptores do Ligante Indutor de Apoptose Relacionado a TNF/genética , Ligante Indutor de Apoptose Relacionado a TNF/genética , Apoptose/efeitos dos fármacos , Proteína Reguladora de Apoptosis Semelhante a CASP8 e FADD/antagonistas & inibidores , Proteína Reguladora de Apoptosis Semelhante a CASP8 e FADD/metabolismo , Caspase 3/genética , Caspase 3/metabolismo , Caspase 8/genética , Caspase 8/metabolismo , Diferenciação Celular , Proliferação de Células , Sinergismo Farmacológico , Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/efeitos dos fármacos , Regulação da Expressão Gênica , Harringtoninas/farmacologia , Mepesuccinato de Omacetaxina , Humanos , Proteína de Sequência 1 de Leucemia de Células Mieloides/genética , Proteína de Sequência 1 de Leucemia de Células Mieloides/metabolismo , Células-Tronco Pluripotentes/citologia , Células-Tronco Pluripotentes/efeitos dos fármacos , Inibidores da Síntese de Proteínas/farmacologia , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Receptores do Ligante Indutor de Apoptose Relacionado a TNF/metabolismo , Receptores Tipo I de Fatores de Necrose Tumoral/genética , Receptores Tipo I de Fatores de Necrose Tumoral/metabolismo , Transdução de Sinais , Ligante Indutor de Apoptose Relacionado a TNF/metabolismo , Ligante Indutor de Apoptose Relacionado a TNF/farmacologia , Receptor fas/genética , Receptor fas/metabolismoRESUMO
Sonodynamic therapy, an effect of low-power ultrasound field and the anticancer drug cisplatin, was studied in vitro on human melanoma cells A375. The viability of cells has been studied by standard 3-(4,5-dimethylthiazol-2-Yl)-2,5-diphenyltetrazolium bromide viability assay according to different modes of treatment: application of cisplatin alone, exposure of ultrasound field alone, exposure to ultrasound followed by cisplatin and application of cisplatin followed by exposure to ultrasound. Ultrasound was used at a therapeutic intensity of 1 Wâcm(-2) for 10 min. Concentration of cisplatin in the cell suspension was always 2.3 µM. The results show that sonodynamic therapy is one of the possibilities of how to intensify standard cytostatic therapy. This conclusion is supported by reducing the viability of studied cells, especially 72 h after the treatment. The time sequence of application of ultrasonic field and cytostatics appears to be a significant factor affecting the changes in cell viability. Maximum suppression of viability has been found when applying the experimental design involving application of cisplatin followed by exposure to ultrasound; the final value of viability of combined affected cells was more than 10% lower than for cisplatin treatment alone.
Assuntos
Cisplatino/uso terapêutico , Melanoma/diagnóstico por imagem , Melanoma/terapia , Terapia por Ultrassom/métodos , Antineoplásicos/uso terapêutico , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos da radiação , Terapia Combinada , Humanos , Resultado do Tratamento , UltrassonografiaRESUMO
Studies of human embryonic stem cells (hESCs) commonly describe the nonfunctional p53-p21 axis of the G1/S checkpoint pathway with subsequent relevance for cell cycle regulation and the DNA damage response (DDR). Importantly, p21 mRNA is clearly present and upregulated after the DDR in hESCs, but p21 protein is not detectable. In this article, we provide evidence that expression of p21 protein is directly regulated by the microRNA (miRNA) pathway under standard culture conditions and after DNA damage. The DDR in hESCs leads to upregulation of tens of miRNAs, including hESC-specific miRNAs such as those of the miR-302 family, miR-371-372 family, or C19MC miRNA cluster. Most importantly, we show that the hESC-enriched miRNA family miR-302 (miR-302a, miR-302b, miR-302c, and miR-302d) directly contributes to regulation of p21 expression in hESCs and, thus, demonstrate a novel function for miR-302s in hESCS. The described mechanism elucidates the role of miRNAs in regulation of important molecular pathway governing the G1/S transition checkpoint before as well as after DNA damage.