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BACKGROUND: New therapies are required for castrate-resistant prostate cancer (CRPC), and growth-arrest specific 5 (GAS5) lncRNA, which riborepresses androgen receptor action, may offer novel opportunities in this regard. This lncRNA promotes the apoptosis of prostate cancer cells and its levels decline as prostate cancer cells acquire castrate-resistance, so that enhancing GAS5 expression may improve the effectiveness of chemotherapies. Since GAS5 is a member of the 5' terminal oligopyrimidine gene family, we have examined mTOR inhibition as a strategy to increase GAS5 expression. Furthermore, we have determined if GAS5 itself mediates the action of mTOR inhibitors, as demonstrated for other chemotherapeutic agents in prostate cancer cells. METHODS: The effects of mTOR inhibitors on GAS5 lncRNA levels and cell growth were determined in a range of prostate cancer cell lines. Transfection of cells with GAS5 siRNAs and plasmid constructs was performed to determine the involvement of GAS5 lncRNA in mTOR inhibitor action. RESULTS: First generation mTORC1, combined mTORC1/mTORC2 and dual PI3K/mTOR inhibitors all increased cellular GAS5 levels and inhibited culture growth in androgen-dependent (LNCaP) and androgen-sensitive (22Rv1) cell lines, but not in androgen-independent (PC-3 and DU 145) cell lines. The latter exhibited low endogenous GAS5 expression, and GAS5 silencing in LNCaP and 22Rv1 cells decreased the sensitivity to mTOR inhibitors, whereas transfection of GAS5 lncRNA sensitized PC-3 and DU 145 cells to these agents. CONCLUSION: mTOR inhibition enhances GAS5 transcript levels in certain prostate cancer cell lines. This selectivity is likely to be related to endogenous GAS5 expression levels, since GAS5 lncRNA is itself required for mTOR inhibitor action in prostate cancer cells.
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Regulación Neoplásica de la Expresión Génica/fisiología , Neoplasias Hormono-Dependientes/metabolismo , Neoplasias de la Próstata/metabolismo , ARN Largo no Codificante/metabolismo , Serina-Treonina Quinasas TOR/metabolismo , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Humanos , Masculino , ARN Largo no Codificante/genética , ARN Neoplásico/química , ARN Neoplásico/genética , ARN Interferente Pequeño/farmacología , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Sirolimus/farmacología , Serina-Treonina Quinasas TOR/antagonistas & inhibidoresRESUMEN
The putative tumour suppressor and apoptosis-promoting gene, growth arrest-specific 5 (GAS5), encodes long ncRNA (lncRNA) and snoRNAs. Its expression is down-regulated in breast cancer, which adversely impacts patient prognosis. In this preclinical study, the consequences of decreased GAS5 expression for breast cancer cell survival following treatment with chemotherapeutic agents are addressed. In addition, functional responses of triple-negative breast cancer cells to GAS5 lncRNA are examined, and mTOR inhibition as a strategy to enhance cellular GAS5 levels is investigated. Breast cancer cell lines were transfected with either siRNA to GAS5 or with a plasmid encoding GAS5 lncRNA and the effects on breast cancer cell survival were determined. Cellular responses to mTOR inhibitors were evaluated by assaying culture growth and GAS5 transcript levels. GAS5 silencing attenuated cell responses to apoptotic stimuli, including classical chemotherapeutic agents; the extent of cell death was directly proportional to cellular GAS5 levels. Imatinib action in contrast, was independent of GAS5. GAS5 lncRNA promoted the apoptosis of triple-negative and oestrogen receptor-positive cells but only dual PI3K/mTOR inhibition was able to enhance GAS5 levels in all cell types. Reduced GAS5 expression attenuates apoptosis induction by classical chemotherapeutic agents in breast cancer cells, providing an explanation for the relationship between GAS5 expression and breast cancer patient prognosis. Clinically, this relationship may be circumvented by the use of GAS5-independent drugs such as imatinib, or by restoration of GAS5 expression. The latter may be achieved by the use of a dual PI3K/mTOR inhibitor, to improve apoptotic responses to conventional chemotherapies.
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Neoplasias de la Mama/genética , Neoplasias de la Mama/patología , ARN Largo no Codificante , Apoptosis/genética , Benzamidas/farmacología , Neoplasias de la Mama/tratamiento farmacológico , Línea Celular Tumoral , Supervivencia Celular/genética , Femenino , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Mesilato de Imatinib , Células MCF-7/patología , Morfolinas/farmacología , Piperazinas/farmacología , Pirimidinas/farmacología , Serina-Treonina Quinasas TOR/antagonistas & inhibidores , Serina-Treonina Quinasas TOR/metabolismo , Neoplasias de la Mama Triple Negativas/tratamiento farmacológico , Neoplasias de la Mama Triple Negativas/genética , Neoplasias de la Mama Triple Negativas/patologíaRESUMEN
Safe genetic modification of neural stem cell (NSC) transplant populations is a key goal for regenerative neurology. We describe a technically simple and safe method to increase transfection in NSCs propagated in the neurosphere (suspension culture) model, using magnetic nanoparticles deployed with applied oscillating magnetic fields ('magnetofection technology'). We show that transfection efficiency was enhanced over two-fold by oscillating magnetic fields (frequency=4 Hz). The protocols had no effect on cell viability, cell number, stem cell marker expression and differentiation profiles of 'magnetofected' cultures, highlighting the safety of the technique. As far as we are aware, this is the first successful application of magnetofection technology to suspension cultures of neural cells. The procedures described offer a means to augment the therapeutic potential of NSCs propagated as neurospheres - a culture model of high clinical translational relevance - by safe genetic manipulation, with further potential for incorporation into 'magneto-multifection' (repeat transfection) protocols. FROM THE CLINICAL EDITOR: This team of investigators describe a simple and safe method to increase transfection in neural stem cells using magnetic nanoparticles deployed with oscillating magnetic fields, demonstrating a greater than two-fold transfection efficiency increase by applying low frequency magnetic oscillation.
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Técnicas de Cultivo de Célula/métodos , Nanopartículas de Magnetita/química , Células-Madre Neurales/citología , Transfección , Animales , Diferenciación Celular/genética , Proliferación Celular , Células Cultivadas , Humanos , Campos Magnéticos , Nanopartículas de Magnetita/administración & dosificación , Células-Madre Neurales/químicaRESUMEN
FAU, which encodes a ubiquitin-like protein (termed FUBI) with ribosomal protein S30 as a carboxy-terminal extension, has recently been identified as a pro-apoptotic regulatory gene. This activity may be mediated by Bcl-G (a pro-apoptotic member of the Bcl-2 family) which can be covalently modified by FUBI. FAU gene expression has been shown to be down-regulated in human breast, prostate and ovarian tumours, and this down-regulation is strongly associated with poor prognosis in breast cancer. We demonstrate here that ectopic FAU expression increases basal apoptosis in human T-cell lines and 293T/17 cells, whereas it has only a transient stimulatory effect on ultraviolet-C (UVC)-induced apoptosis. Conversely, siRNA-mediated silencing of FAU gene expression has no effect on basal apoptosis, but attenuates UV-induced apoptosis. Importantly, prior knockdown of Bcl-G expression ablates the stimulation of basal apoptosis by FAU, consistent with an essential downstream role for Bcl-G, itself a candidate tumour suppressor, in mediating the apoptosis regulatory role of FAU. In 293T/17 cells, Bcl-G knockdown also attenuates UV-induced apoptosis, so that Bcl-G may constitute a common factor in the pathways by which both FAU and UV-irradiation induce apoptosis. UV irradiation increases Bcl-G mRNA levels, providing an explanation for the transient nature of the effect of ectopic FAU expression on UV-induced apoptosis. Since failure of apoptosis is fundamental to the development of many cancers, the pro-apoptotic activity of the Fau/Bcl-G pathway offers an attractive explanation for the putative tumour suppressor role of FAU.
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Apoptosis/efectos de los fármacos , Proteínas Proto-Oncogénicas c-bcl-2/fisiología , Proteínas Ribosómicas/fisiología , Proteínas Supresoras de Tumor/fisiología , Técnicas de Silenciamiento del Gen , Silenciador del Gen , Células HEK293 , Humanos , Células Jurkat , Proteínas Proto-Oncogénicas c-bcl-2/genética , Proteínas Proto-Oncogénicas c-bcl-2/efectos de la radiación , Proteínas Ribosómicas/genética , Proteínas Ribosómicas/efectos de la radiación , Rayos UltravioletaRESUMEN
PURPOSE: To develop Fe(3)O(4)-PEI-RITC magnetic nanoparticles with multimodal MRI-fluorescence imaging and transfection capability, for use in neural cell replacement therapies. METHODS: The Fe(3)O(4)-PEI-RITC MNPs were synthesised through a multi-step chemical grafting procedure: (i) Silanisation of MNPs with 3-iodopropyltrimethoxysilane; (ii) PEI coupling with iodopropyl groups on the MNP surface; and (iii) RITC binding onto the PEI coating. The cell labelling and transfection capabilities of these particles were evaluated in astrocytes derived from primary cultures. RESULTS: Fe(3)O(4)-PEI-RITC MNPs did not exert acute toxic effects in astrocytes (at ≤ 6 days). Cells showed rapid and extensive particle uptake with up to 100% cellular labelling observed by 24 h. MRI and microscopy studies demonstrate that the particles have potential for use in bimodal MR-fluorescence imaging. Additionally, the particles were capable of delivering plasmids encoding reporter protein (approximately 4 kb) to astrocytes, albeit with low efficiencies. CONCLUSIONS: Multifunctional Fe(3)O(4)-PEI-RITC MNPs were successfully prepared using a multi-step synthetic pathway, with the PEI and RITC chemically bound onto the MNP surface. Their combined MR-fluorescence imaging capabilities with additional potential for transfection applications can provide a powerful tool, after further development, for non-invasive cell tracking and gene transfer to neural transplant populations.
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Trasplante de Células , Compuestos Férricos/química , Técnicas de Transferencia de Gen , Iminas/química , Magnetismo , Nanopartículas , Polietilenos/química , Animales , Astrocitos/metabolismo , Astrocitos/trasplante , Células Cultivadas , Inmunohistoquímica , Microscopía Fluorescente , Ratas , Ratas Sprague-Dawley , Espectroscopía Infrarroja por Transformada de Fourier , Propiedades de Superficie , Difracción de Rayos XRESUMEN
The development of chemotherapy resistance by cancer cells is complex, using different mechanisms and pathways. The gene FAU (Finkel-Biskis-Reilly murine sarcoma virus (FBR-MuSV)-associated ubiquitously expressed gene) was identified through functional expression cloning and previous data have shown that overexpression enhances apoptosis in several cell types. We demonstrate that the expression of FAU was reduced in the A2780cis (cisplatin resistant subclone of A2780) cell line compared with the A2780 ovarian cancer cell line, and was directly related to the cell line's sensitivity to carboplatin. Downregulation of FAU in the A2780 cell line by transfection with two predesigned short-interfering RNAs (siRNAs) to FAU resulted in a significant increase in resistance to carboplatin-induced cell death. Downregulation resulted in increased cell viability and reduced apoptosis after 72 hr of drug treatment compared with the negative controls (Kruskal-Wallis P = 0.0002). Transfection of the A2780cis cell line with the pcDNA3 plasmid containing FAU was associated with increased sensitivity to carboplatin-induced apoptosis, with decreased cell viability and increased apoptosis (Mann Whitney P < 0.0001). The expression of FAU was examined by quantitative real-time reverse transcriptase polymerase chain reaction in normal and malignant ovarian tissue. A significant reduction in the expression of FAU was seen in the malignant compared with normal ovarian samples (Kruskal-Wallis P = 0.0261). These data support a role for FAU in the regulation of platinum-resistance in ovarian cancer. Further research is needed into the apoptotic pathway containing FAU to investigate the potential for targeted therapies to increase or restore the platinum sensitivity of ovarian cancer.
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Carboplatino/farmacocinética , Resistencia a Antineoplásicos , Neoplasias Ováricas/tratamiento farmacológico , Proteínas Ribosómicas/fisiología , Apoptosis , Línea Celular Tumoral , Supervivencia Celular , Femenino , Silenciador del Gen/efectos de los fármacos , Humanos , Neoplasias Ováricas/patología , ARN Interferente Pequeño/farmacología , Proteínas Ribosómicas/genéticaRESUMEN
Animal models have been used in preclinical research to examine potential new treatments for spinal cord injury (SCI), including mesenchymal stem cell (MSC) transplantation. MSC transplants have been studied in early human trials. Whether the animal models represent the human studies is unclear. This systematic review and meta-analysis has examined the effects of MSC transplants in human and animal studies. Following searches of PubMed, Clinical Trials and the Cochrane Library, published papers were screened, and data were extracted and analysed. MSC transplantation was associated with significantly improved motor and sensory function in humans, and significantly increased locomotor function in animals. However, there are discrepancies between the studies of human participants and animal models, including timing of MSC transplant post-injury and source of MSCs. Additionally, difficulty in the comparison of functional outcome measures across species limits the predictive nature of the animal research. These findings have been summarised, and recommendations for further research are discussed to better enable the translation of animal models to MSC-based human clinical therapy.
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BACKGROUND: The molecular control of cell death through apoptosis is compromised in prostate cancer cells, resulting in inappropriate cell survival and resistance to cytotoxic therapy. Reduced expression of the functionally connected apoptosis-regulators and candidate tumor suppressors Fau and Bcl-G has recently been implicated in oncogenesis in other tissues. The present study examines the hypothesis that reduced expression of these genes may be involved in prostate cancer. METHODS: Fau and Bcl-G mRNA levels were determined by real time RT-PCR in two independent prostate tissue collections. In experiments in vitro, Fau and Bcl-G levels in prostate cancer cell lines were reduced using RNA interference and the effects on sensitivity to UVC irradiation were determined. RESULTS: Fau and Bcl-G mRNA levels were both lower in prostate cancer tissue than in normal prostate and Benign Prostate Hyperplasia. Active down-regulation of Fau and Bcl-G expression in vitro resulted in decreased sensitivity to UVC-induced cytotoxicity. Simultaneous down-regulation of Fau and Bcl-G produced a decrease in sensitivity which was similar to either gene alone. CONCLUSIONS: Fau and Bcl-G mRNA levels are both decreased in prostate cancer. In prostate cancer cell lines in vitro such down-regulation results in reduced sensitivity to UVC-induced cytotoxicity, consistent with the putative roles of these genes as candidate prostate tumor suppressors. The absence of an additive effect when Fau and Bcl-G were down-regulated simultaneously is consistent with the two genes acting in the same apoptosis pathway, for example, with the pro-apoptotic effects of Fau being mediated through modulation of Bcl-G.
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Neoplasias de la Próstata/genética , Proteínas Ribosómicas/genética , Anciano , Apoptosis/efectos de la radiación , Línea Celular Tumoral , Supervivencia Celular/efectos de la radiación , Regulación Neoplásica de la Expresión Génica , Genes bcl-1 , Humanos , Masculino , Persona de Mediana Edad , Estadificación de Neoplasias , Próstata/fisiología , Hiperplasia Prostática/genética , Hiperplasia Prostática/patología , Neoplasias de la Próstata/patología , Proteínas Proto-Oncogénicas c-bcl-2/genética , Interferencia de ARN , ARN Mensajero/genética , ARN Interferente Pequeño/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Rayos UltravioletaRESUMEN
Magnetic nanoparticles (MNPs) are important contrast agents used to monitor a range of neuropathological processes; microglial cells significantly contribute to MNP uptake in sites of pathology. Microglial activation occurs following most CNS pathologies but it is not known if such activation alters MNP uptake, intracellular processing and toxicity. We assessed these parameters in microglial cultures with and without experimental 'activation'. Microglia showed rapid and extensive MNP uptake under basal conditions with no changes found following activation; significant microglial toxicity was observed at higher particle concentrations. Based on our findings, we suggest that avid MNP uptake by endogenous CNS microglia could significantly limit uptake by other cellular subtypes in mixed neural cell populations.
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Nanopartículas de Magnetita , Microglía/metabolismo , Neuronas/metabolismo , Animales , Transporte Biológico , Células Cultivadas , Corteza Cerebral/citología , RatasRESUMEN
Emerging evidence indicates that a strong relationship exists between brain regenerative therapies and nutrition. Early life nutrition plays an important role during embryonic brain development, and there are clear consequences to an imbalance in nutritional factors on both the production and survival of mature neuronal populations and the infant's risk of diseases in later life. Our research and that of others suggest that vitamins play a fundamental role in the formation of neurons and their survival. There is a growing body of evidence that nicotinamide, the water-soluble amide form of vitamin B3, is implicated in the conversion of pluripotent stem cells to clinically relevant cells for regenerative therapies. This study investigated the ability of nicotinamide to promote the development of mature catecholaminergic neuronal populations (associated with Parkinson's disease) from mouse embryonic stem cells, as well as investigating the underlying mechanisms of nicotinamide's action. Nicotinamide selectively enhanced the production of tyrosine hydroxylase-expressing neurons and serotonergic neurons from mouse embryonic stem cell cultures (Sox1GFP knock-in 46C cell line). A 5-Ethynyl-2´-deoxyuridine (EdU) assay ascertained that nicotinamide, when added in the initial phase, reduced cell proliferation. Nicotinamide drove tyrosine hydroxylase-expressing neuron differentiation as effectively as an established cocktail of signalling factors, reducing the proliferation of neural progenitors and accelerating neuronal maturation, neurite outgrowth and neurotransmitter expression. These novel findings show that nicotinamide enhanced and enriched catecholaminergic differentiation and inhibited cell proliferation by directing cell cycle arrest in mouse embryonic stem cell cultures, thus driving a critical neural proliferation-to-differentiation switch from neural progenitors to neurons. Further research into the role of vitamin metabolites in embryogenesis will significantly advance cell-based regenerative medicine, and help realize their role as crucial developmental signalling molecules in brain development.
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Diferenciación Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Células Madre Embrionarias de Ratones/citología , Células-Madre Neurales/citología , Neurogénesis/efectos de los fármacos , Niacinamida/farmacología , Animales , Células Cultivadas , Ratones , Neuronas/citologíaRESUMEN
INTRODUCTION: Programmed cell death through apoptosis plays an essential role in the hormone-regulated physiological turnover of mammary tissue. Failure of this active gene-dependent process is central both to the development of breast cancer and to the appearance of the therapy-resistant cancer cells that produce clinical relapse. Functional expression cloning in two independent laboratories has identified Finkel-Biskis-Reilly murine sarcoma virus-associated ubiquitously expressed gene (Fau) as a novel apoptosis regulator and candidate tumour suppressor. Fau modifies apoptosis-controller Bcl-G, which is also a key target for candidate oncoprotein maternal embryonic leucine zipper kinase (MELK). METHODS: We have used RNA interference to downregulate Fau and Bcl-G expression, both simultaneously and independently, in breast cancer cells in vitro to determine the importance of their roles in apoptosis. Expression of Fau, Bcl-G and MELK was measured by quantitative RT-PCR in breast cancer tissue and in matched breast epithelial tissue from the same patients. Expression data of these genes obtained using microarrays from a separate group of patients were related to patient survival in Kaplan-Meier analyses. RESULTS: siRNA-mediated downregulation of either Fau or Bcl-G expression inhibited apoptosis, and the inhibition produced by combining the two siRNAs was consistent with control of Bcl-G by Fau. Fau expression is significantly reduced in breast cancer tissue and this reduction is associated with poor patient survival, as predicted for a candidate breast cancer tumour suppressor. In addition, MELK expression is increased in breast cancer tissue and this increase is also associated with poor patient survival, as predicted for a candidate oncogene. Bcl-G expression is reduced in breast cancer tissue but decreased Bcl-G expression showed no correlation with survival, indicating that the most important factors controlling Bcl-G activity are post-translational modification (by Fau and MELK) rather than the rate of transcription of Bcl-G itself. CONCLUSIONS: The combination of in vitro functional studies with the analysis of gene expression in clinical breast cancer samples indicates that three functionally interconnected genes, Fau, Bcl-G and MELK, are crucially important in breast cancer and identifies them as attractive targets for improvements in breast cancer risk prediction, prognosis and therapy.
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Adenocarcinoma/genética , Neoplasias de la Mama/genética , Regulación Neoplásica de la Expresión Génica , Proteínas de Neoplasias/fisiología , Proteínas Serina-Treonina Quinasas/fisiología , Proteínas Proto-Oncogénicas c-bcl-2/fisiología , Proteínas Ribosómicas/fisiología , Adenocarcinoma/mortalidad , Apoptosis/genética , Apoptosis/efectos de la radiación , Neoplasias de la Mama/mortalidad , Línea Celular Tumoral/citología , Línea Celular Tumoral/efectos de la radiación , Células Epiteliales/citología , Células Epiteliales/metabolismo , Femenino , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Técnicas de Silenciamiento del Gen , Estudios de Asociación Genética , Humanos , Estimación de Kaplan-Meier , Proteínas de Neoplasias/antagonistas & inhibidores , Proteínas de Neoplasias/genética , Pronóstico , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Proteínas Serina-Treonina Quinasas/genética , Proteínas Proto-Oncogénicas c-bcl-2/biosíntesis , Proteínas Proto-Oncogénicas c-bcl-2/genética , Interferencia de ARN , ARN Interferente Pequeño/farmacología , Proteínas Ribosómicas/antagonistas & inhibidores , Proteínas Ribosómicas/genética , Ensayo de Tumor de Célula Madre , Rayos UltravioletaRESUMEN
Neural stem cells (NSCs) have high translational potential in transplantation therapies for neural repair. Enhancement of their therapeutic capacity by genetic engineering is an important goal for regenerative neurology. Magnetic nanoparticles (MNPs) are major non-viral vectors for safe bioengineering of NSCs, offering critical translational benefits over viral vectors, including safety, scalability, and ease of use. This unit describes protocols for the production of suspension (neurosphere) and adherent (monolayer) murine NSC cultures. Genetic engineering of NSCs with MNPs and the application of 'magnetofection' (magnetic fields) or 'multifection' (repeat transfection) approaches to enhance gene delivery are described. Magnetofection of monolayer cultures achieves optimal transfection, but neurospheres offer key advantages for neural graft survival post-transplantation. A protocol is presented which allows the advantageous features of each approach to be combined into a single procedure for transplantation. The adaptation of these protocols for other MNP preparations is considered, with emphasis on the evaluation of procedural safety. © 2017 by John Wiley & Sons, Inc.
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Técnicas de Cultivo de Célula/métodos , Nanopartículas de Magnetita/química , Células-Madre Neurales/citología , Transfección/métodos , Animales , Células Cultivadas , Humanos , Laminina/farmacología , Ratones , Péptidos/farmacología , Esferoides Celulares/citologíaRESUMEN
INTRODUCTION: Vitamin B3 has been shown to play an important role during embryogenesis. Specifically, there is growing evidence that nicotinamide, the biologically active form of vitamin B3, plays a critical role as a morphogen in the differentiation of stem cells to mature cell phenotypes, including those of the central nervous system (CNS). Detailed knowledge of the action of small molecules during neuronal differentiation is not only critical for uncovering mechanisms underlying lineage-specification, but also to establish more effective differentiation protocols to obtain clinically relevant cells for regenerative therapies for neurodegenerative conditions such as Huntington's disease (HD). Thus, this study aimed to investigate the potential of nicotinamide to promote the conversion of stem cells to mature CNS neurons. METHODS: Nicotinamide was applied to differentiating mouse embryonic stem cells (mESC; Sox1GFP knock-in 46C cell line) during their conversion towards a neural fate. Cells were assessed for changes in their proliferation, differentiation and maturation; using immunocytochemistry and morphometric analysis methods. RESULTS: Results presented indicate that 10 mM nicotinamide, when added at the initial stages of differentiation, promoted accelerated progression of ESCs to a neural lineage in adherent monolayer cultures. By 14 days in vitro (DIV), early exposure to nicotinamide was shown to increase the numbers of differentiated ßIII-tubulin-positive neurons. Nicotinamide decreased the proportion of pluripotent stem cells, concomitantly increasing numbers of neural progenitors at 4 DIV. These progenitors then underwent rapid conversion to neurons, observed by a reduction in Sox 1 expression and decreased numbers of neural progenitors in the cultures at 14 DIV. Furthermore, GABAergic neurons generated in the presence of nicotinamide showed increased maturity and complexity of neurites at 14 DIV. Therefore, addition of nicotinamide alone caused an accelerated passage of pluripotent cells through lineage specification and further to non-dividing mature neurons. CONCLUSIONS: Our results show that, within an optimal dose range, nicotinamide is able to singly and selectively direct the conversion of embryonic stem cells to mature neurons, and therefore may be a critical factor for normal brain development, thus supporting previous evidence of the fundamental role of vitamins and their metabolites during early CNS development. In addition, nicotinamide may offer a simple effective supplement to enhance the conversion of stem cells to clinically relevant neurons.
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Diferenciación Celular/efectos de los fármacos , Células Madre Embrionarias/efectos de los fármacos , Niacinamida/farmacología , Animales , Linaje de la Célula , Proliferación Celular/efectos de los fármacos , Células Madre Embrionarias/citología , Proteínas Fluorescentes Verdes/genética , RatonesRESUMEN
The E3 ubiquitin ligase RNF168 is a ring finger protein that has previously been identified to play an important regulatory role in the repair of double-strand DNA breaks. In the present study, an unbiased forward genetics functional screen in mouse granulocyte/ macrophage progenitor cell line FDCP1 has identified E3 ubiquitin ligase RNF168 as a key regulator of cell survival and proliferation. Our data indicate that RNF168 is an important component of the mechanisms controlling cell fate, not only in human and mouse haematopoietic growth factor-dependent cells, but also in the human breast epithelial cell line MCF-7. These observations therefore suggest that RNF168 provides a connection to key pathways controlling cell fate, potentially through interaction with PML nuclear bodies and/or epigenetic control of gene expression. Our study is the first to demonstrate a critical role for RNF168 in the in the mechanisms regulating cell proliferation and survival, in addition to its well-established role in DNA repair.
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Growth arrest-specific 5 (GAS5) lncRNA promotes apoptosis, and its expression is down-regulated in breast cancer. GAS5 lncRNA is a decoy of glucocorticoid/related receptors; a stem-loop sequence constitutes the GAS5 hormone response element mimic (HREM), which is essential for the regulation of breast cancer cell apoptosis. This preclinical study aimed to determine if the GAS5 HREM sequence alone promotes the apoptosis of breast cancer cells. Nucleofection of hormone-sensitive and -insensitive breast cancer cell lines with a GAS5 HREM DNA oligonucleotide increased both basal and ultraviolet-C-induced apoptosis, and decreased culture viability and clonogenic growth, similar to GAS5 lncRNA. The HREM oligonucleotide demonstrated similar sequence specificity to the native HREM for its functional activity and had no effect on endogenous GAS5 lncRNA levels. Certain chemically modified HREM oligonucleotides, notably DNA and RNA phosphorothioates, retained pro-apoptotic. activity. Crucially the HREM oligonucleotide could overcome apoptosis resistance secondary to deficient endogenous GAS5 lncRNA levels. Thus, the GAS5 lncRNA HREM sequence alone is sufficient to induce apoptosis in breast cancer cells, including triple-negative breast cancer cells. These findings further suggest that emerging knowledge of structure/function relationships in the field of lncRNA biology can be exploited for the development of entirely novel, oligonucleotide mimic-based, cancer therapies.
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Apoptosis/efectos de los fármacos , Hormonas/farmacología , ARN Largo no Codificante/efectos de los fármacos , Elementos de Respuesta/genética , Apoptosis/genética , Apoptosis/efectos de la radiación , Secuencia de Bases , Neoplasias de la Mama/genética , Neoplasias de la Mama/patología , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/genética , Supervivencia Celular/efectos de la radiación , Femenino , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Regulación Neoplásica de la Expresión Génica/efectos de la radiación , Humanos , Células MCF-7 , Oligonucleótidos/genética , ARN Largo no Codificante/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transfección , Rayos UltravioletaRESUMEN
BACKGROUND: The control of breast cell survival is of critical importance for preventing breast cancer initiation and progression. The activity of many proteins which regulate cell survival is controlled by reversible phosphorylation, so that the relevant kinases and phosphatases play crucial roles in determining cell fate. Several protein kinases act as oncoproteins in breast cancer and changes in their activities contribute to the process of transformation. Through counteracting the activity of oncogenic kinases, the protein phosphatases are also likely to be important players in breast cancer development, but this class of molecules is relatively poorly understood. Here we have investigated the role of the serine/threonine protein phosphatase 4 in the control of cell survival of breast cancer cells. METHODS: The breast cancer cell lines, MCF7 and MDA-MB-231, were transfected with expression vectors encoding the catalytic subunit of protein phosphatase 4 (PP4c) or with PP4c siRNAs. Culture viability, apoptosis, cell migration and cell cycle were assessed. The involvement of phosphoprotein enriched in astrocytes 15kDa (PEA15) in PP4c action was investigated by immunoblotting approaches and by siRNA-mediated silencing of PEA15. RESULTS: In this study we showed that PP4c over-expression inhibited cell proliferation, enhanced spontaneous apoptosis and decreased the migratory and colony forming abilities of breast cancer cells. Moreover, PP4c down-regulation produced complementary effects. PP4c is demonstrated to regulate the phosphorylation of PEA15, and PEA15 itself regulates the apoptosis of breast cancer cells. The inhibitory effects of PP4c on breast cancer cell survival and growth were lost in PEA15 knockdown cells, confirming that PP4c action is mediated, at least in part, through the de-phosphorylation of apoptosis regulator PEA15. CONCLUSION: Our work shows that PP4 regulates breast cancer cell survival and identifies a novel PP4c-PEA15 signalling axis in the control of breast cancer cell survival. The dysfunction of this axis may be important in the development and progression of breast cancer.
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Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Fosfoproteínas Fosfatasas/metabolismo , Fosfoproteínas/metabolismo , Transducción de Señal , Apoptosis , Proteínas Reguladoras de la Apoptosis , Línea Celular Tumoral , Movimiento Celular , Proliferación Celular , Supervivencia Celular , Regulación hacia Abajo , Femenino , Técnicas de Silenciamiento del Gen , Silenciador del Gen , Humanos , Fosforilación , Fosfoserina/metabolismo , ARN Interferente Pequeño/metabolismoRESUMEN
AIM: To achieve high and sustained magnetic particle loading in a proliferative and endocytotically active neural transplant population (astrocytes) through tailored magnetite content in polymeric iron oxide particles. MATERIALS & METHODS: MPs of varying magnetite content were applied to primary-derived rat cortical astrocytes ± static/oscillating magnetic fields to assess labeling efficiency and safety. RESULTS: Higher magnetite content particles display high but safe accumulation in astrocytes, with longer-term label retention versus lower/no magnetite content particles. Magnetic fields enhanced loading extent. Dynamic live cell imaging of dividing labeled astrocytes demonstrated that particle distribution into daughter cells is predominantly 'asymmetric'. CONCLUSION: These findings could inform protocols to achieve efficient MP loading into neural transplant cells, with significant implications for post-transplantation tracking/localization.
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Astrocitos/citología , División Celular , Endocitosis , Nanopartículas de Magnetita/administración & dosificación , Animales , Células Cultivadas , Microscopía Fluorescente , Ratas , Ratas Sprague-DawleyRESUMEN
It is increasingly recognised that lncRNAs play essential regulatory roles in fundamental biological processes and, consequently, that their dysregulation may contribute to major human diseases, including cancer. Better understanding of lncRNA biology may therefore offer new insights into pathogenetic mechanisms and thereby offer novel opportunities for diagnosis and therapy. Of particular interest in this regard is GAS5 lncRNA, which is down-regulated in multiple cancers, with expression levels related to both clinico-pathological characteristics and patient prognosis. Functional studies have further shown that GAS5 lncRNA both inhibits the proliferation and promotes the apoptosis of multiple cell types, and that together these cellular mechanisms of action are likely to form the basis of its tumour suppressor action. At the same time, advances have been made in our understanding of the molecular mechanisms of GAS5 lncRNA action in recent years, including riborepression of certain steroid hormone receptors and sequestration of miR-21, impacting key regulatory pathways of cell survival. Overall this accumulating knowledge has the potential to improve both the diagnosis and treatment of cancer, and ultimately patient outcome.
RESUMEN
Genetically engineered neural stem cell (NSC) transplants offer a key strategy to augment neural repair by releasing therapeutic biomolecules into injury sites. Genetic modification of NSCs is heavily reliant on viral vectors but cytotoxic effects have prompted development of non-viral alternatives, such as magnetic nanoparticle (MNPs). NSCs are propagated in laboratories as either 3-D suspension "neurospheres" or 2-D adherent "monolayers". MNPs deployed with oscillating magnetic fields ("magnetofection technology") mediate effective gene transfer to neurospheres but the efficacy of this approach for monolayers is unknown. It is important to address this issue as oscillating magnetic fields dramatically enhance MNP-based transfection in transplant cells (e.g., astrocytes and oligodendrocyte precursors) propagated as monolayers. We report for the first time that oscillating magnetic fields enhanced MNP-based transfection with reporter and functional (basic fibroblast growth factor; FGF2) genes in monolayer cultures yielding high transfection versus neurospheres. Transfected NSCs showed high viability and could re-form neurospheres, which is important as neurospheres yield higher post-transplantation viability versus monolayer cells. Our results demonstrate that the combination of oscillating magnetic fields and a monolayer format yields the highest efficacy for MNP-mediated gene transfer to NSCs, offering a viable non-viral alternative for genetic modification of this important neural cell transplant population.
RESUMEN
Implantable 'structural bridges' based on nanofabricated polymer scaffolds have great promise to aid spinal cord regeneration. Their development (optimal formulations, surface functionalizations, safety, topographical influences and degradation profiles) is heavily reliant on live animal injury models. These have several disadvantages including invasive surgical procedures, ethical issues, high animal usage, technical complexity and expense. In vitro 3-D organotypic slice arrays could offer a solution to overcome these challenges, but their utility for nanomaterials testing is undetermined. We have developed an in vitro model of spinal cord injury that replicates stereotypical cellular responses to neurological injury in vivo, viz. reactive gliosis, microglial infiltration and limited nerve fibre outgrowth. We describe a facile method to safely incorporate aligned, poly-lactic acid nanofibre meshes (±poly-lysine + laminin coating) within injury sites using a lightweight construct. Patterns of nanotopography induced outgrowth/alignment of astrocytes and neurons in the in vitro model were strikingly similar to that induced by comparable materials in related studies in vivo. This highlights the value of our model in providing biologically-relevant readouts of the regeneration-promoting capacity of synthetic bridges within the complex environment of spinal cord lesions. Our approach can serve as a prototype to develop versatile bio-screening systems to identify materials/combinatorial strategies for regenerative medicine, whilst reducing live animal experimentation.