Hypoxia is a common feature of solid tumours and is associated with poor prognosis, resistance to... more Hypoxia is a common feature of solid tumours and is associated with poor prognosis, resistance to radio- and chemotherapy, and tumour aggressiveness. For predictive purposes as well as for improved therapeutic intervention, it is increasingly needed to have direct and specific diagnostic tools in order to measure the extent of, and changes in, tumour hypoxia. In this article, we have investigated the potential of Fourier Transform Infrared (FTIR) microspectroscopy, at cellular and subcellular resolution, for detecting hypoxia-induced metabolic changes in brain tumour (glioblastoma) cell lines and in short term primary cultures derived from patient samples. The most prominent and common changes observed were the increase in glycogen (specifically in the U87MG cell line) and lipids (all cell lines studied). Additionally, each cell line presented specific individual metabolic fingerprints. The metabolic changes did not evolve markedly with time (from 1 to 5 days hypoxic incubation), an...
... Robert Wilson*, David G. Spiller, Alison Beckett§, Ian A. Prior§, and Violaine S e ... less... more ... Robert Wilson*, David G. Spiller, Alison Beckett§, Ian A. Prior§, and Violaine S e ... less well-documented: Li-Shishisdo and colleagues found that 10 mM glutathione more than doubled the luminescence intensity of commercial polymer-coated QDs,(57) and Yu and colleagues ...
Tumors of the Central Nervous System, Volume 8, 2012
ABSTRACT The design of medulloblastoma therapy has become more and more sophisticated over the pa... more ABSTRACT The design of medulloblastoma therapy has become more and more sophisticated over the past 30 years. Despite this, a significant proportion of patients continue to die from this disease, with drug resistance and relapse still observed. Resistance to treatment with anticancer drugs results from a variety of factors including individual genetic variations in patients and acquired resistance. We here review the most common reasons for drug resistance in medulloblastoma cells. We will cover the acquisition of drug resistance towards the classical chemotherapeutic drugs used in medulloblastoma due to modification of enzymatic activities such as alkyl tranferase, gluthatione tranferase, phosphatidyl-inositol-3-kinase or to expression of energy-dependent transporters that eject anticancer drugs from cells. We will also review other mechanisms of resistance including insensitivity to drug-induced cell death due to genetic mutations in crucial signaling pathways such as NF-kappaB and p53.
Neuroblastoma is a paediatric cancer that arises from the sympathetic ganglia (SG) or adrenal gla... more Neuroblastoma is a paediatric cancer that arises from the sympathetic ganglia (SG) or adrenal gland. Tumours that occur in patients under 18 months of age have a particularly good prognosis and frequently undergo spontaneous regression. This led to the hypothesis that developmental cues in the youngest patients may prompt belated differentiation and/or apoptosis of the tumour cells. To test our hypothesis, we have injected MYCN-amplified neuroblastoma cells into the extra embryonic veins of chick embryos at embryonic day 3 (E3) and E6 and analysed the response of these Kelly cells at E10 and E14. Amplification of the MYCN gene occurs in up to 30% of tumours and is normally associated with a very poor prognosis. Kelly cells injected at E3 follow neural crest pathways and integrate into neural locations such as SG and the enteric nervous system although never into the adrenal gland. Additionally they migrate to non-neural locations such as the heart, meninges, jaw regions and tail. Th...
We show that low-intensity laser irradiation of cancer cells containing endosomal gold nanopartic... more We show that low-intensity laser irradiation of cancer cells containing endosomal gold nanoparticles leads to endosome rupture and escape of the nanoparticles into the cytosol without affecting the cells' viability. The low light intensity of our experiments allows us to rule out photothermal effects as the underlying mechanism, and we present results that suggest photoinduced radicals as the photogenerated active species. This nonthermal mechanism may also be important in the context of cell death at higher laser intensities, which had been reported previously. FIGURE 4. Confocal microscopy: laser exposure causes oxidative stress. Reactive oxygen species (ROS) monitored by the appearance of green fluorescence; (a) HeLa cells incubated with gold nanoparticles after laser irradiation; (b) same as (a) but in the absence of gold nanoparticles; (c) same as (a) but without laser irradiation; and (d) control experiment without gold nanoparticles and without laser irradiation. Laser power density in (a) and (b) was 20 Wcm -2 for 1 min exposure time. Scale bars are 20 µm. (e) Quantitative comparison of ROS generation in panels a-d.
a b s t r a c t HIF (hypoxia inducible factor) is an oxygen-regulated transcription factor that m... more a b s t r a c t HIF (hypoxia inducible factor) is an oxygen-regulated transcription factor that mediates the intracellular response to hypoxia in human cells. There is increasing evidence that cell signaling pathways encode temporal information, and thus cell fate may be determined by the dynamics of protein levels. We have developed a mathematical model to describe the transient dynamics of the HIF-1α protein measured in single cells subjected to hypoxic shock. The essential characteristics of these data are modeled with a system of differential equations describing the feedback inhibition between HIF-1α and prolyl hydroxylases (PHD) oxygen sensors. Heterogeneity in the single-cell data is accounted through parameter variation in the model. We previously identified the PHD2 isoform as the main PHD sensor responsible for controlling the HIF-1α transient response, and make here testable predictions regarding HIF-1α dynamics subject to repetitive hypoxic pulses. The model is further developed to describe the dynamics of HIF-1α in cells cultured as 3D spheroids, with oxygen dynamics parameterized using experimental measurements of oxygen within spheroids. We show that the dynamics of HIF-1α and transcriptional targets of HIF-1α display a non-monotone response to the oxygen dynamics. Specifically we demonstrate that the dynamic transient behavior of HIF-1α results in differential dynamics in transcriptional targets.
Medulloblastoma (MB) is an embryonic brain tumour that arises in the cerebellum. Using several MB... more Medulloblastoma (MB) is an embryonic brain tumour that arises in the cerebellum. Using several MB cell lines, we have demonstrated that the chemotherapeutic drug etoposide induces a p53- and caspase-dependent cell death. We have observed an additional caspase-independent cell death mechanism involving delayed nuclear factor κB (NF-κB) activity. The delayed induction was controlled by a p53-dependent transcription step and the production of death receptors (especially CD95/Fas). We further demonstrated that in both MB and glioblastoma (GM) cell lines, in which the p53 pathway was not functional, no p65 activation could be detected upon etoposide treatment. MB cell lines that have mutations in p53 or NF-κB are either less sensitive (NF-κB mutant) or even completely resistant (p53 mutant) to chemotherapeutic intervention. The optimal cell death was only achieved when both p53 and NF-κB were switched on. Taken together, our results shed light on the mechanism of NF-κB activation by etoposide in brain tumours and show that the genetic background of MB and GM cells determines their sensitivity to chemotherapy and has to be taken into account for efficient therapeutic intervention.
A critical step of neuronal terminal differentiation is the permanent withdrawal from the cell cy... more A critical step of neuronal terminal differentiation is the permanent withdrawal from the cell cycle that requires the silencing of genes that drive mitosis. Here, we describe that the alpha isoform of the heterochromatin protein 1 (HP1) protein family exerts such silencing on several E2F-targeted genes. Among the different isoforms, HP1alpha levels progressively increase throughout differentiation and take over HP1gamma binding on E2F sites in mature neurons. When overexpressed, only HP1alpha is able to ensure a timed repression of E2F genes. Specific inhibition of HP1alpha expression drives neuronal progenitors either towards death or cell cycle progression, yet preventing the expression of the neuronal marker microtubule-associated protein 2. Furthermore, we provide evidence that this mechanism occurs in cerebellar granule neurons in vivo, during the postnatal development of the cerebellum. Finally, our results suggest that E2F-targeted genes are packaged into higher-order chroma...
SOCIÉTÉ FRANÇAISE DE NEUROPATHOLOGIE toxine cholérique qui se fixe avec une haute affinité au GM1... more SOCIÉTÉ FRANÇAISE DE NEUROPATHOLOGIE toxine cholérique qui se fixe avec une haute affinité au GM1. Ce marqueur a mis en évidence du GM1 à la périphérie du dépôt amyloïde de la plaque sénile, alors que les dépôts diffus en étaient dépourvus. Nous testons actuellement l'hypothèse selon laquelle GM1 serait abondant dans les prolongements microgliaux qui entourent le coeur amyloïde.
Chemotherapeutic drug resistance and relapse remains a major challenge for paediatric (medullobla... more Chemotherapeutic drug resistance and relapse remains a major challenge for paediatric (medulloblastoma) and adult (glioblastoma) brain tumour treatment. Medulloblastoma tumours and cell lines with mutations in the p53 signalling pathway have been shown to be specifically insensitive to DNA damaging agents. The aim of this study was to investigate the potential of triggering cell death in p53 mutated medulloblastoma cells by a direct activation of pro-death signalling downstream of p53 activation. Since non-coding microRNAs (miRNAs) have the ability to fine tune the expression of a variety of target genes, orchestrating multiple downstream effects, we hypothesised that triggering the expression of a p53 target miRNA could induce cell death in chemo-resistant cells. Treatment with etoposide, increased miR-34a levels in a p53dependent fashion and the level of miR-34a transcription was correlated with the cell sensitivity to etoposide. miR-34a activity was validated by measuring the expression levels of one of its well described target: the NADH dependent sirtuin1 (SIRT1). Whilst drugs directly targeting SIRT1, were potent to trigger cell death at high concentrations only, introduction of synthetic miR-34a mimics was able to induce cell death in p53 mutated medulloblastoma and glioblastoma cell lines. Our results show that the need of a functional p53 signaling pathway can be bypassed by direct activation of miR-34a in brain tumour cells.
A critical step of neuronal terminal differentiation is the permanent withdrawal from the cell cy... more A critical step of neuronal terminal differentiation is the permanent withdrawal from the cell cycle that requires the silencing of genes that drive mitosis. Here, we describe that the alpha isoform of the heterochromatin protein 1 (HP1) protein family exerts such silencing on several E2F-targeted genes. Among the different isoforms, HP1alpha levels progressively increase throughout differentiation and take over HP1gamma binding on E2F sites in mature neurons. When overexpressed, only HP1alpha is able to ensure a timed repression of E2F genes. Specific inhibition of HP1alpha expression drives neuronal progenitors either towards death or cell cycle progression, yet preventing the expression of the neuronal marker microtubule-associated protein 2. Furthermore, we provide evidence that this mechanism occurs in cerebellar granule neurons in vivo, during the postnatal development of the cerebellum. Finally, our results suggest that E2F-targeted genes are packaged into higher-order chromatin structures in mature neurons relative to neuroblasts, likely reflecting a transition from a 'repressed' versus 'silenced' status of these genes. Together, these data present new epigenetic regulations orchestrated by HP1 isoforms, critical for permanent cell cycle exit during neuronal differentiation.
Iron-oxide based contrast agents play an important role in magnetic resonance imaging (MRI) of la... more Iron-oxide based contrast agents play an important role in magnetic resonance imaging (MRI) of labelled cells in vivo. Currently, a wide range of such contrast agents is available with sizes varying from several nanometers up to a few micrometers and consisting of single or multiple magnetic cores. Here, we evaluate the effectiveness of these different particles for labelling and imaging stem cells, using a mouse mesenchymal stem cell line to investigate intracellular uptake, retention and processing of nano-and microsized contrast agents. The effect of intracellular confinement on transverse relaxivity was measured by MRI at 7 T and in compliance with the principles of the '3Rs', the suitability of the contrast agents for MR-based cell tracking in vivo was tested using a chick embryo model. We show that for all particles tested, relaxivity was markedly reduced following cellular internalisation, indicating that contrast agent relaxivity in colloidal suspension does not accurately predict performance in MR-based cell tracking studies. Using a bimodal imaging approach comprising fluorescence and MRI, we demonstrate that labelled MSC remain viable following in vivo transplantation and can be tracked effectively using MRI. Importantly, our data suggest that larger particles might confer advantages for longer-term imaging.
Functional nanomaterials have recently attracted strong interest from the biology community, not ... more Functional nanomaterials have recently attracted strong interest from the biology community, not only as potential drug delivery vehicles or diagnostic tools, but also as optical nanomaterials. This is illustrated by the explosion of publications in the field with more than 2,000 publications in the last 2 years (4,000 papers since 2000; from ISI Web of Knowledge, 'nanoparticle and cell' hit). Such a publication boom in this novel interdisciplinary field has resulted in papers of unequal standard, partly because it is challenging to assemble the required expertise in chemistry, physics, and biology in a single team. As an extreme example, several papers published in physical chemistry journals claim intracellular delivery of nanoparticles, but show pictures of cells that are, to the expert biologist, evidently dead (and therefore permeable). To attain proper cellular applications using nanomaterials, it is critical not only to achieve efficient delivery in healthy cells, but also to control the intracellular availability and the fate of the nanomaterial. This is still an open challenge that will only be met by innovative delivery methods combined with rigorous and quantitative characterization of the uptake and the fate of the nanoparticles. This review mainly focuses on gold nanoparticles and discusses the various approaches to nanoparticle delivery, including surface chemical modifications and several methods used to facilitate cellular uptake and endosomal escape. We will also review the main detection methods and how their optimum use can inform about intracellular localization, efficiency of delivery, and integrity of the surface capping.
uclear factor kappa B (NF-B) has been implicated in the regulation of cell proliferation and tran... more uclear factor kappa B (NF-B) has been implicated in the regulation of cell proliferation and transformation. We investigated the role of the serum-induced intracellular calcium increase in the NF-B-dependent cell cycle progression in Swiss 3T3 fibroblasts. Noninvasive photoactivation of a calcium chelator (Diazo-2) was used to specifically disrupt the transient rise in calcium induced by serum stimulation of starved Swiss 3T3 cells. The seruminduced intracellular calcium peak was essential for subsequent NF-B activation (measured by real-time imaging of the dynamic p65 and I B ␣ fluorescent fusion proteins), N cyclin D1 (CD1) promoter-directed transcription (measured by real-time luminescence imaging of CD1 promoter-directed firefly luciferase activity), and progression to cell division. We further showed that the serum-induced mitogen-activated protein kinase (MAPK) phosphorylation is calcium dependent. Inhibition of the MAPK-but not the PtdIns3K-dependent pathway inhibited NF-B signaling, and further, CD1 transcription and cell cycle progression. These data suggest that a serum-dependent calcium signal regulates the cell cycle via a MAPK-NF-B pathway in Swiss 3T3 cells.
The nuclear factor kappaB (NF-kappaB) transcription factor regulates cellular stress responses an... more The nuclear factor kappaB (NF-kappaB) transcription factor regulates cellular stress responses and the immune response to infection. NF-kappaB activation results in oscillations in nuclear NF-kappaB abundance. To define the function of these oscillations, we treated cells with repeated short pulses of tumor necrosis factor-alpha at various intervals to mimic pulsatile inflammatory signals. At all pulse intervals that were analyzed, we observed synchronous cycles of NF-kappaB nuclear translocation. Lower frequency stimulations gave repeated full-amplitude translocations, whereas higher frequency pulses gave reduced translocation, indicating a failure to reset. Deterministic and stochastic mathematical models predicted how negative feedback loops regulate both the resetting of the system and cellular heterogeneity. Altering the stimulation intervals gave different patterns of NF-kappaB-dependent gene expression, which supports the idea that oscillation frequency has a functional role.
phenotype in tissues other than the stem and leaf and accumulation of residual surface wax on the... more phenotype in tissues other than the stem and leaf and accumulation of residual surface wax on the stem of cer5-2 knockout line suggest that additional wax export mechanisms must exist in plants. Chemical analysis of the mutant wax demonstrated that CER5, like many ABC transporters, has broad substrate specificity and is capable of transporting a variety of wax substrates. We conclude that in plants, as in other eukaryotes, proteins of the WBC/ ABCG subfamily are key components of lipid transport systems.
Background: Vascular endothelial growth factor (VEGF) has previously been shown to upregulate the... more Background: Vascular endothelial growth factor (VEGF) has previously been shown to upregulate the expression of the endogenous calcineurin inhibitor, regulator of calcineurin 1, variant 4 (RCAN1.4). The aim of this study was to determine the role and regulation of VEGF-mediated RCAN1.4 expression, using human dermal microvascular endothelial cells (HDMECs) as a model system.
In this study, we examined the effects of oxidative stress on a nitric oxide (NO)-regulated neuro... more In this study, we examined the effects of oxidative stress on a nitric oxide (NO)-regulated neuroendocrine function, the release of arginine vasopressin (AVP) by the hypothalamo-neurohypophyseal axis. Treatment of mouse-isolated hypothalami and neurointermediate lobes (NIL) with H2O2 increased AVP release. This effect was inhibited by copper-zinc superoxide dismutase-1 (SOD1) analogs. By measuring cGMP accumulation as an indicator of biologically active NO, we found that H2O2 treatment decreased cGMP formation in both hypothalami and NIL. We have previously shown that NO inhibits AVP release by a cGMP-independent mechanism. Given that H2O2 stimulated AVP release, while it reduced cGMP production, our findings strongly suggest that oxidative damage affects neurosecretion by reducing NO availability. To test whether such a mechanism may operate under pathological conditions with pronounced oxidative stress, we compared neurosecretion in wild-type and transgenic mice carrying a mutated form of SOD1 associated with human familial amyotrophic lateral sclerosis. Reminiscent of the data obtained from H2O2-treated tissues, hypothalami and NIL from SOD1 mutants displayed decreased cGMP accumulation and increased AVP release, compared with tissues from wild-type littermates. Since neuronal NO synthase expression was not modified, we conclude that the perturbed free radical metabolism associated with the SOD1 mutation is likely to trap NO, and thereby alter neurosecretion, a mechanism that can be exacerbated in specific physiopathological conditions.
Alzheimer's disease (AD) is an age-related neurodegenerative disorder characterized by the accumu... more Alzheimer's disease (AD) is an age-related neurodegenerative disorder characterized by the accumulation of extracellular depositions of fibrillar B-amyloid (AB), which is derived from the alternative processing of B-amyloid precursor protein (APP). Although APP is thought to function as a cell surface receptor, its mode of action still remains elusive. In this study, we found that the culture medium derived from cortical neurons treated with an anti-APP antibody triggers the death of naive neurons. Biochemical and immunocytochemical analyses revealed the presence, both in the conditioned medium and in neurons, of increased levels of tumor necrosis factor-a and monocyte chemoattractant protein-1. Furthermore, the expression of these proinflammatory mediators occurred through a c-Jun Nterminal protein kinase/c-Jun-dependent mechanism. Taken together, our findings provide evidence for a novel mechanism whereby neuronal APP in its full-length configuration induces neuronal death. Such a mechanism might be relevant to neuroinflammatory processes as those observed in AD.
Hypoxia is a common feature of solid tumours and is associated with poor prognosis, resistance to... more Hypoxia is a common feature of solid tumours and is associated with poor prognosis, resistance to radio- and chemotherapy, and tumour aggressiveness. For predictive purposes as well as for improved therapeutic intervention, it is increasingly needed to have direct and specific diagnostic tools in order to measure the extent of, and changes in, tumour hypoxia. In this article, we have investigated the potential of Fourier Transform Infrared (FTIR) microspectroscopy, at cellular and subcellular resolution, for detecting hypoxia-induced metabolic changes in brain tumour (glioblastoma) cell lines and in short term primary cultures derived from patient samples. The most prominent and common changes observed were the increase in glycogen (specifically in the U87MG cell line) and lipids (all cell lines studied). Additionally, each cell line presented specific individual metabolic fingerprints. The metabolic changes did not evolve markedly with time (from 1 to 5 days hypoxic incubation), an...
... Robert Wilson*, David G. Spiller, Alison Beckett§, Ian A. Prior§, and Violaine S e ... less... more ... Robert Wilson*, David G. Spiller, Alison Beckett§, Ian A. Prior§, and Violaine S e ... less well-documented: Li-Shishisdo and colleagues found that 10 mM glutathione more than doubled the luminescence intensity of commercial polymer-coated QDs,(57) and Yu and colleagues ...
Tumors of the Central Nervous System, Volume 8, 2012
ABSTRACT The design of medulloblastoma therapy has become more and more sophisticated over the pa... more ABSTRACT The design of medulloblastoma therapy has become more and more sophisticated over the past 30 years. Despite this, a significant proportion of patients continue to die from this disease, with drug resistance and relapse still observed. Resistance to treatment with anticancer drugs results from a variety of factors including individual genetic variations in patients and acquired resistance. We here review the most common reasons for drug resistance in medulloblastoma cells. We will cover the acquisition of drug resistance towards the classical chemotherapeutic drugs used in medulloblastoma due to modification of enzymatic activities such as alkyl tranferase, gluthatione tranferase, phosphatidyl-inositol-3-kinase or to expression of energy-dependent transporters that eject anticancer drugs from cells. We will also review other mechanisms of resistance including insensitivity to drug-induced cell death due to genetic mutations in crucial signaling pathways such as NF-kappaB and p53.
Neuroblastoma is a paediatric cancer that arises from the sympathetic ganglia (SG) or adrenal gla... more Neuroblastoma is a paediatric cancer that arises from the sympathetic ganglia (SG) or adrenal gland. Tumours that occur in patients under 18 months of age have a particularly good prognosis and frequently undergo spontaneous regression. This led to the hypothesis that developmental cues in the youngest patients may prompt belated differentiation and/or apoptosis of the tumour cells. To test our hypothesis, we have injected MYCN-amplified neuroblastoma cells into the extra embryonic veins of chick embryos at embryonic day 3 (E3) and E6 and analysed the response of these Kelly cells at E10 and E14. Amplification of the MYCN gene occurs in up to 30% of tumours and is normally associated with a very poor prognosis. Kelly cells injected at E3 follow neural crest pathways and integrate into neural locations such as SG and the enteric nervous system although never into the adrenal gland. Additionally they migrate to non-neural locations such as the heart, meninges, jaw regions and tail. Th...
We show that low-intensity laser irradiation of cancer cells containing endosomal gold nanopartic... more We show that low-intensity laser irradiation of cancer cells containing endosomal gold nanoparticles leads to endosome rupture and escape of the nanoparticles into the cytosol without affecting the cells' viability. The low light intensity of our experiments allows us to rule out photothermal effects as the underlying mechanism, and we present results that suggest photoinduced radicals as the photogenerated active species. This nonthermal mechanism may also be important in the context of cell death at higher laser intensities, which had been reported previously. FIGURE 4. Confocal microscopy: laser exposure causes oxidative stress. Reactive oxygen species (ROS) monitored by the appearance of green fluorescence; (a) HeLa cells incubated with gold nanoparticles after laser irradiation; (b) same as (a) but in the absence of gold nanoparticles; (c) same as (a) but without laser irradiation; and (d) control experiment without gold nanoparticles and without laser irradiation. Laser power density in (a) and (b) was 20 Wcm -2 for 1 min exposure time. Scale bars are 20 µm. (e) Quantitative comparison of ROS generation in panels a-d.
a b s t r a c t HIF (hypoxia inducible factor) is an oxygen-regulated transcription factor that m... more a b s t r a c t HIF (hypoxia inducible factor) is an oxygen-regulated transcription factor that mediates the intracellular response to hypoxia in human cells. There is increasing evidence that cell signaling pathways encode temporal information, and thus cell fate may be determined by the dynamics of protein levels. We have developed a mathematical model to describe the transient dynamics of the HIF-1α protein measured in single cells subjected to hypoxic shock. The essential characteristics of these data are modeled with a system of differential equations describing the feedback inhibition between HIF-1α and prolyl hydroxylases (PHD) oxygen sensors. Heterogeneity in the single-cell data is accounted through parameter variation in the model. We previously identified the PHD2 isoform as the main PHD sensor responsible for controlling the HIF-1α transient response, and make here testable predictions regarding HIF-1α dynamics subject to repetitive hypoxic pulses. The model is further developed to describe the dynamics of HIF-1α in cells cultured as 3D spheroids, with oxygen dynamics parameterized using experimental measurements of oxygen within spheroids. We show that the dynamics of HIF-1α and transcriptional targets of HIF-1α display a non-monotone response to the oxygen dynamics. Specifically we demonstrate that the dynamic transient behavior of HIF-1α results in differential dynamics in transcriptional targets.
Medulloblastoma (MB) is an embryonic brain tumour that arises in the cerebellum. Using several MB... more Medulloblastoma (MB) is an embryonic brain tumour that arises in the cerebellum. Using several MB cell lines, we have demonstrated that the chemotherapeutic drug etoposide induces a p53- and caspase-dependent cell death. We have observed an additional caspase-independent cell death mechanism involving delayed nuclear factor κB (NF-κB) activity. The delayed induction was controlled by a p53-dependent transcription step and the production of death receptors (especially CD95/Fas). We further demonstrated that in both MB and glioblastoma (GM) cell lines, in which the p53 pathway was not functional, no p65 activation could be detected upon etoposide treatment. MB cell lines that have mutations in p53 or NF-κB are either less sensitive (NF-κB mutant) or even completely resistant (p53 mutant) to chemotherapeutic intervention. The optimal cell death was only achieved when both p53 and NF-κB were switched on. Taken together, our results shed light on the mechanism of NF-κB activation by etoposide in brain tumours and show that the genetic background of MB and GM cells determines their sensitivity to chemotherapy and has to be taken into account for efficient therapeutic intervention.
A critical step of neuronal terminal differentiation is the permanent withdrawal from the cell cy... more A critical step of neuronal terminal differentiation is the permanent withdrawal from the cell cycle that requires the silencing of genes that drive mitosis. Here, we describe that the alpha isoform of the heterochromatin protein 1 (HP1) protein family exerts such silencing on several E2F-targeted genes. Among the different isoforms, HP1alpha levels progressively increase throughout differentiation and take over HP1gamma binding on E2F sites in mature neurons. When overexpressed, only HP1alpha is able to ensure a timed repression of E2F genes. Specific inhibition of HP1alpha expression drives neuronal progenitors either towards death or cell cycle progression, yet preventing the expression of the neuronal marker microtubule-associated protein 2. Furthermore, we provide evidence that this mechanism occurs in cerebellar granule neurons in vivo, during the postnatal development of the cerebellum. Finally, our results suggest that E2F-targeted genes are packaged into higher-order chroma...
SOCIÉTÉ FRANÇAISE DE NEUROPATHOLOGIE toxine cholérique qui se fixe avec une haute affinité au GM1... more SOCIÉTÉ FRANÇAISE DE NEUROPATHOLOGIE toxine cholérique qui se fixe avec une haute affinité au GM1. Ce marqueur a mis en évidence du GM1 à la périphérie du dépôt amyloïde de la plaque sénile, alors que les dépôts diffus en étaient dépourvus. Nous testons actuellement l'hypothèse selon laquelle GM1 serait abondant dans les prolongements microgliaux qui entourent le coeur amyloïde.
Chemotherapeutic drug resistance and relapse remains a major challenge for paediatric (medullobla... more Chemotherapeutic drug resistance and relapse remains a major challenge for paediatric (medulloblastoma) and adult (glioblastoma) brain tumour treatment. Medulloblastoma tumours and cell lines with mutations in the p53 signalling pathway have been shown to be specifically insensitive to DNA damaging agents. The aim of this study was to investigate the potential of triggering cell death in p53 mutated medulloblastoma cells by a direct activation of pro-death signalling downstream of p53 activation. Since non-coding microRNAs (miRNAs) have the ability to fine tune the expression of a variety of target genes, orchestrating multiple downstream effects, we hypothesised that triggering the expression of a p53 target miRNA could induce cell death in chemo-resistant cells. Treatment with etoposide, increased miR-34a levels in a p53dependent fashion and the level of miR-34a transcription was correlated with the cell sensitivity to etoposide. miR-34a activity was validated by measuring the expression levels of one of its well described target: the NADH dependent sirtuin1 (SIRT1). Whilst drugs directly targeting SIRT1, were potent to trigger cell death at high concentrations only, introduction of synthetic miR-34a mimics was able to induce cell death in p53 mutated medulloblastoma and glioblastoma cell lines. Our results show that the need of a functional p53 signaling pathway can be bypassed by direct activation of miR-34a in brain tumour cells.
A critical step of neuronal terminal differentiation is the permanent withdrawal from the cell cy... more A critical step of neuronal terminal differentiation is the permanent withdrawal from the cell cycle that requires the silencing of genes that drive mitosis. Here, we describe that the alpha isoform of the heterochromatin protein 1 (HP1) protein family exerts such silencing on several E2F-targeted genes. Among the different isoforms, HP1alpha levels progressively increase throughout differentiation and take over HP1gamma binding on E2F sites in mature neurons. When overexpressed, only HP1alpha is able to ensure a timed repression of E2F genes. Specific inhibition of HP1alpha expression drives neuronal progenitors either towards death or cell cycle progression, yet preventing the expression of the neuronal marker microtubule-associated protein 2. Furthermore, we provide evidence that this mechanism occurs in cerebellar granule neurons in vivo, during the postnatal development of the cerebellum. Finally, our results suggest that E2F-targeted genes are packaged into higher-order chromatin structures in mature neurons relative to neuroblasts, likely reflecting a transition from a 'repressed' versus 'silenced' status of these genes. Together, these data present new epigenetic regulations orchestrated by HP1 isoforms, critical for permanent cell cycle exit during neuronal differentiation.
Iron-oxide based contrast agents play an important role in magnetic resonance imaging (MRI) of la... more Iron-oxide based contrast agents play an important role in magnetic resonance imaging (MRI) of labelled cells in vivo. Currently, a wide range of such contrast agents is available with sizes varying from several nanometers up to a few micrometers and consisting of single or multiple magnetic cores. Here, we evaluate the effectiveness of these different particles for labelling and imaging stem cells, using a mouse mesenchymal stem cell line to investigate intracellular uptake, retention and processing of nano-and microsized contrast agents. The effect of intracellular confinement on transverse relaxivity was measured by MRI at 7 T and in compliance with the principles of the '3Rs', the suitability of the contrast agents for MR-based cell tracking in vivo was tested using a chick embryo model. We show that for all particles tested, relaxivity was markedly reduced following cellular internalisation, indicating that contrast agent relaxivity in colloidal suspension does not accurately predict performance in MR-based cell tracking studies. Using a bimodal imaging approach comprising fluorescence and MRI, we demonstrate that labelled MSC remain viable following in vivo transplantation and can be tracked effectively using MRI. Importantly, our data suggest that larger particles might confer advantages for longer-term imaging.
Functional nanomaterials have recently attracted strong interest from the biology community, not ... more Functional nanomaterials have recently attracted strong interest from the biology community, not only as potential drug delivery vehicles or diagnostic tools, but also as optical nanomaterials. This is illustrated by the explosion of publications in the field with more than 2,000 publications in the last 2 years (4,000 papers since 2000; from ISI Web of Knowledge, 'nanoparticle and cell' hit). Such a publication boom in this novel interdisciplinary field has resulted in papers of unequal standard, partly because it is challenging to assemble the required expertise in chemistry, physics, and biology in a single team. As an extreme example, several papers published in physical chemistry journals claim intracellular delivery of nanoparticles, but show pictures of cells that are, to the expert biologist, evidently dead (and therefore permeable). To attain proper cellular applications using nanomaterials, it is critical not only to achieve efficient delivery in healthy cells, but also to control the intracellular availability and the fate of the nanomaterial. This is still an open challenge that will only be met by innovative delivery methods combined with rigorous and quantitative characterization of the uptake and the fate of the nanoparticles. This review mainly focuses on gold nanoparticles and discusses the various approaches to nanoparticle delivery, including surface chemical modifications and several methods used to facilitate cellular uptake and endosomal escape. We will also review the main detection methods and how their optimum use can inform about intracellular localization, efficiency of delivery, and integrity of the surface capping.
uclear factor kappa B (NF-B) has been implicated in the regulation of cell proliferation and tran... more uclear factor kappa B (NF-B) has been implicated in the regulation of cell proliferation and transformation. We investigated the role of the serum-induced intracellular calcium increase in the NF-B-dependent cell cycle progression in Swiss 3T3 fibroblasts. Noninvasive photoactivation of a calcium chelator (Diazo-2) was used to specifically disrupt the transient rise in calcium induced by serum stimulation of starved Swiss 3T3 cells. The seruminduced intracellular calcium peak was essential for subsequent NF-B activation (measured by real-time imaging of the dynamic p65 and I B ␣ fluorescent fusion proteins), N cyclin D1 (CD1) promoter-directed transcription (measured by real-time luminescence imaging of CD1 promoter-directed firefly luciferase activity), and progression to cell division. We further showed that the serum-induced mitogen-activated protein kinase (MAPK) phosphorylation is calcium dependent. Inhibition of the MAPK-but not the PtdIns3K-dependent pathway inhibited NF-B signaling, and further, CD1 transcription and cell cycle progression. These data suggest that a serum-dependent calcium signal regulates the cell cycle via a MAPK-NF-B pathway in Swiss 3T3 cells.
The nuclear factor kappaB (NF-kappaB) transcription factor regulates cellular stress responses an... more The nuclear factor kappaB (NF-kappaB) transcription factor regulates cellular stress responses and the immune response to infection. NF-kappaB activation results in oscillations in nuclear NF-kappaB abundance. To define the function of these oscillations, we treated cells with repeated short pulses of tumor necrosis factor-alpha at various intervals to mimic pulsatile inflammatory signals. At all pulse intervals that were analyzed, we observed synchronous cycles of NF-kappaB nuclear translocation. Lower frequency stimulations gave repeated full-amplitude translocations, whereas higher frequency pulses gave reduced translocation, indicating a failure to reset. Deterministic and stochastic mathematical models predicted how negative feedback loops regulate both the resetting of the system and cellular heterogeneity. Altering the stimulation intervals gave different patterns of NF-kappaB-dependent gene expression, which supports the idea that oscillation frequency has a functional role.
phenotype in tissues other than the stem and leaf and accumulation of residual surface wax on the... more phenotype in tissues other than the stem and leaf and accumulation of residual surface wax on the stem of cer5-2 knockout line suggest that additional wax export mechanisms must exist in plants. Chemical analysis of the mutant wax demonstrated that CER5, like many ABC transporters, has broad substrate specificity and is capable of transporting a variety of wax substrates. We conclude that in plants, as in other eukaryotes, proteins of the WBC/ ABCG subfamily are key components of lipid transport systems.
Background: Vascular endothelial growth factor (VEGF) has previously been shown to upregulate the... more Background: Vascular endothelial growth factor (VEGF) has previously been shown to upregulate the expression of the endogenous calcineurin inhibitor, regulator of calcineurin 1, variant 4 (RCAN1.4). The aim of this study was to determine the role and regulation of VEGF-mediated RCAN1.4 expression, using human dermal microvascular endothelial cells (HDMECs) as a model system.
In this study, we examined the effects of oxidative stress on a nitric oxide (NO)-regulated neuro... more In this study, we examined the effects of oxidative stress on a nitric oxide (NO)-regulated neuroendocrine function, the release of arginine vasopressin (AVP) by the hypothalamo-neurohypophyseal axis. Treatment of mouse-isolated hypothalami and neurointermediate lobes (NIL) with H2O2 increased AVP release. This effect was inhibited by copper-zinc superoxide dismutase-1 (SOD1) analogs. By measuring cGMP accumulation as an indicator of biologically active NO, we found that H2O2 treatment decreased cGMP formation in both hypothalami and NIL. We have previously shown that NO inhibits AVP release by a cGMP-independent mechanism. Given that H2O2 stimulated AVP release, while it reduced cGMP production, our findings strongly suggest that oxidative damage affects neurosecretion by reducing NO availability. To test whether such a mechanism may operate under pathological conditions with pronounced oxidative stress, we compared neurosecretion in wild-type and transgenic mice carrying a mutated form of SOD1 associated with human familial amyotrophic lateral sclerosis. Reminiscent of the data obtained from H2O2-treated tissues, hypothalami and NIL from SOD1 mutants displayed decreased cGMP accumulation and increased AVP release, compared with tissues from wild-type littermates. Since neuronal NO synthase expression was not modified, we conclude that the perturbed free radical metabolism associated with the SOD1 mutation is likely to trap NO, and thereby alter neurosecretion, a mechanism that can be exacerbated in specific physiopathological conditions.
Alzheimer's disease (AD) is an age-related neurodegenerative disorder characterized by the accumu... more Alzheimer's disease (AD) is an age-related neurodegenerative disorder characterized by the accumulation of extracellular depositions of fibrillar B-amyloid (AB), which is derived from the alternative processing of B-amyloid precursor protein (APP). Although APP is thought to function as a cell surface receptor, its mode of action still remains elusive. In this study, we found that the culture medium derived from cortical neurons treated with an anti-APP antibody triggers the death of naive neurons. Biochemical and immunocytochemical analyses revealed the presence, both in the conditioned medium and in neurons, of increased levels of tumor necrosis factor-a and monocyte chemoattractant protein-1. Furthermore, the expression of these proinflammatory mediators occurred through a c-Jun Nterminal protein kinase/c-Jun-dependent mechanism. Taken together, our findings provide evidence for a novel mechanism whereby neuronal APP in its full-length configuration induces neuronal death. Such a mechanism might be relevant to neuroinflammatory processes as those observed in AD.
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Papers by Violaine See