Specific Preferences in Lineage Choice and Phenotypic Plasticity of Glioma Stem Cells Under BMP4 and Noggin Influence

Although BMP4-induced differentiation of glioma stem cells (GSCs) is well recognized, details of the cellular responses triggered by this morphogen are still poorly defined. In this study, we established several GSC-enriched cell lines (GSC-ECLs) from high-grade gliomas. The expansion of these cells as adherent monolayers, and not as floating neurospheres, enabled a thorough study of the phenotypic changes that occurred during their differentiation. Herein, we evaluated GSC-ECLs' behavior toward differentiating conditions by depriving them of growth factors and/or by adding BMP4 at different concentrations. After analyzing cellular morphology, proliferation and lineage marker expression, we determined that GSC-ECLs have distinct preferences in lineage choice, where some of them showed an astrocyte fate commitment and others a neuronal one. We found that this election seems to be dictated by the expression pattern of BMP signaling components present in each GSC-ECL. Additionally, treatment of GSC-ECLs with the BMP antagonist, Noggin, also led to evident phenotypic changes. Interestingly, under certain conditions, some GSC-ECLs adopted an unexpected smooth muscle-like phenotype. As a whole, our findings illustrate the wide differentiation potential of GSCs, highlighting their molecular complexity and paving a way to facilitate personalized differentiating therapies.Fil: Videla Richardson, Guillermo Agustín. Fundación para la Lucha contra las Enfermedades Neurológicas de la Infancia; ArgentinaFil: Garcia, Carolina Paola. Fundación para la Lucha contra las Enfermedades Neurológicas de la Infancia; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; ArgentinaFil: Roisman, Alejandro. Fundación para la Lucha contra las Enfermedades Neurológicas de la Infancia; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Medicina Experimental. Academia Nacional de Medicina de Buenos Aires. Instituto de Medicina Experimental; ArgentinaFil: Slavutsky, Irma Rosa. Fundación para la Lucha contra las Enfermedades Neurológicas de la Infancia; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Medicina Experimental. Academia Nacional de Medicina de Buenos Aires. Instituto de Medicina Experimental; ArgentinaFil: Fernandez Espinosa, Damian Dario. Fundación para la Lucha contra las Enfermedades Neurológicas de la Infancia; ArgentinaFil: Romorini, Leonardo. Fundación para la Lucha contra las Enfermedades Neurológicas de la Infancia; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Medicina Experimental. Academia Nacional de Medicina de Buenos Aires. Instituto de Medicina Experimental; ArgentinaFil: Miriuka, Santiago Gabriel. Fundación para la Lucha contra las Enfermedades Neurológicas de la Infancia; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Medicina Experimental. Academia Nacional de Medicina de Buenos Aires. Instituto de Medicina Experimental; ArgentinaFil: Arakaki, Naomi. Fundación para la Lucha contra las Enfermedades Neurológicas de la Infancia; ArgentinaFil: Martinetto, Horacio Enrique. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; ArgentinaFil: Scassa, Maria Elida. Fundación para la Lucha contra las Enfermedades Neurológicas de la Infancia; ArgentinaFil: Sevlever, Gustavo Emilio. Fundación para la Lucha contra las Enfermedades Neurológicas de la Infancia; Argentin

Regulation of cyclin E1 expression in human pluripotent stem cells and derived neural progeny

Human pluripotent stem cells (hPSCs), including embryonic and induced pluripotent stem cells (hESCs and hiPSCs) show unique cell cycle characteristics, such as a short doubling time due to an abbreviated G1 phase. Whether or not the core cell cycle machinery directly regulates the stemness and/or the differentiation potential of hPSCs remains to be determined. To date, several scenarios describing the atypical cell cycle of hPSCs have been suggested, and therefore there is still controversy over how cyclins, master regulators of the cell cycle, are expressed and regulated. Furthermore, the cell cycle profile and the expression pattern of major cyclins in hESCs-derived neuroprogenitors (NP) have not been studied yet. Therefore, herein we characterized the expression pattern of major cyclins in hPSCs and NP. We determined that all studied cyclins mRNA expression levels fluctuate along cell cycle. Particularly, after a thorough analysis of synchronized cell populations, we observed that cyclin E1 mRNA levels increased sharply in G1/S concomitantly with cyclin E1 protein accumulation in hPSCs and NP. Additionally, we demonstrated that cyclin E1 mRNA expression levels involves the activation of MEK/ERK pathway and the transcription factors c-Myc and E2Fs in hPSCs. Lastly, our results reveal that proteasome mediates the marked down-regulation (degradation) of cyclin E1 protein observed in G2/M by a mechanism that requires a functional CDK2 but not GSK3β activity. Abbreviations: hPSCs: human pluripotent stem cells; hESCs: human embryonic stem cells; hiPSCs: human induced pluripotent stem cells; NP: neuroprogenitors; HF: human foreskin fibroblasts; MEFs: mouse embryonic fibroblasts; iMEFs: irradiated mouse embryonic fibroblasts; CDKs: cyclindependent kinases; CKIs: CDK inhibitors; CNS: central nervous system; Oct-4: Octamer-4; EB: embryoid body; AFP: Alpha-fetoprotein; cTnT: Cardiac Troponin T; MAP-2: microtubule-associated protein; TUJ-1: neuron-specific class III β-tubulin; bFGF: basic fibroblastic growth factor; PI3K: Phosphoinositide 3-kinase; KSR: knock out serum replacement; CM: iMEF conditioned medium; E8: Essential E8 medium.Fil: Rodríguez Varela, Maria Soledad. Fundación para la Lucha contra las Enfermedades Neurológicas de la Infancia; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Mucci, Sofia. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Fundación para la Lucha contra las Enfermedades Neurológicas de la Infancia; ArgentinaFil: Videla Richardson, Guillermo Agustín. Fundación para la Lucha contra las Enfermedades Neurológicas de la Infancia; ArgentinaFil: Morris Hanon, Olivia. Fundación para la Lucha contra las Enfermedades Neurológicas de la Infancia; ArgentinaFil: Furmento, Verónica Alejandra. Fundación para la Lucha contra las Enfermedades Neurológicas de la Infancia; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Miriuka, Santiago Gabriel. Fundación para la Lucha contra las Enfermedades Neurológicas de la Infancia; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Sevlever, Gustavo Emilio. Fundación para la Lucha contra las Enfermedades Neurológicas de la Infancia; ArgentinaFil: Scassa, Maria Elida. Fundación para la Lucha contra las Enfermedades Neurológicas de la Infancia; ArgentinaFil: Romorini, Leonardo. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Fundación para la Lucha contra las Enfermedades Neurológicas de la Infancia; Argentin

Chemical hypoxia induces apoptosis of human pluripotent stem cells by a NOXA-mediated HIF-1α and HIF-2α independent mechanism

Human embryonic and induced pluripotent stem cells (hESCs and hiPSCs) are self-renewing human pluripotent stem cells (hPSCs) that can differentiate to a wide range of specialized cells. Notably, hPSCs enhance their undifferentiated state and self-renewal properties in hypoxia (5% O2). Although thoroughly analyzed, hypoxia implication in hPSCs death is not fully determined. In order to evaluate the effect of chemically mimicked hypoxia on hPSCs cell survival, we analyzed changes in cell viability and several aspects of apoptosis triggered by CoCl2 and dimethyloxalylglycine (DMOG). Mitochondrial function assays revealed a decrease in cell viability at 24 h post-treatments. Moreover, we detected chromatin condensation, DNA fragmentation and CASPASE-9 and 3 cleavages. In this context, we observed that P53, BNIP-3, and NOXA protein expression levels were significantly up-regulated at different time points upon chemical hypoxia induction. However, only siRNA-mediated downregulation of NOXA but not HIF-1α, HIF-2α, BNIP-3, and P53 did significantly affect the extent of cell death triggered by CoCl2 and DMOG in hPSCs. In conclusion, chemically mimicked hypoxia induces hPSCs cell death by a NOXA-mediated HIF-1α and HIF-2α independent mechanism.Fil: Isaja, Luciana. Fundación para la Lucha contra las Enfermedades Neurológicas de la Infancia. Instituto de Neurociencias - Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Neurociencias; Argentina. Fundación para la Lucha contra las Enfermedades Neurológicas de la Infancia; ArgentinaFil: Mucci, Sofía. Fundación para la Lucha contra las Enfermedades Neurológicas de la Infancia; ArgentinaFil: Vera, Jonathan. Fundación para la Lucha contra las Enfermedades Neurológicas de la Infancia; ArgentinaFil: Rodríguez Varela, Maria Soledad. Fundación para la Lucha contra las Enfermedades Neurológicas de la Infancia. Instituto de Neurociencias - Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Neurociencias; Argentina. Fundación para la Lucha contra las Enfermedades Neurológicas de la Infancia; ArgentinaFil: Marazita, Mariela Claudia. Fundación para la Lucha contra las Enfermedades Neurológicas de la Infancia. Instituto de Neurociencias - Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Neurociencias; Argentina. Fundación para la Lucha contra las Enfermedades Neurológicas de la Infancia; ArgentinaFil: Morris Hanon, Olivia. Fundación para la Lucha contra las Enfermedades Neurológicas de la Infancia. Instituto de Neurociencias - Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Neurociencias; Argentina. Fundación para la Lucha contra las Enfermedades Neurológicas de la Infancia; ArgentinaFil: Videla Richardson, Guillermo Agustín. Fundación para la Lucha contra las Enfermedades Neurológicas de la Infancia; ArgentinaFil: Sevlever, Gustavo Emilio. Fundación para la Lucha contra las Enfermedades Neurológicas de la Infancia; ArgentinaFil: Scassa, Maria Elida. Fundación para la Lucha contra las Enfermedades Neurológicas de la Infancia; ArgentinaFil: Romorini, Leonardo. Fundación para la Lucha contra las Enfermedades Neurológicas de la Infancia. Instituto de Neurociencias - Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Neurociencias; Argentina. Fundación para la Lucha contra las Enfermedades Neurológicas de la Infancia; Argentin

Isolation and in vitro study of cancer stem/progenitor cell lines derived from high grade glioma specimens as a renewable source for therapeutic design

Los gliomas de alto grado son los tumores cerebrales primarios más frecuentes y son altamente agresivos e invasivos. Éstos parecen exhibir una organización jerárquica y se ha postulado que su generación ocurre a partir de células madre tumorales (CMT), las cuales corresponderían a una subpoblación minoritaria capaz de generar y propagar tumores al ser inyectadas en ratones inmunodeficientes. De esta forma, el aislamiento y la caracterización de células tumorales derivadas de pacientes con fenotipo de células madre o progenitoras permite contar con un valioso modelo de estudio para comprender en mayor profundidad la biología de los gliomas. En el presente trabajo logramos establecer con éxito nueve líneas de células madre/progenitoras tumorales (CMPT) a partir de biopsias de gliomas de alto grado. Éstas fueron capaces de generar tumores luego de ser inyectadas en el sistema nervioso central en ratones inmunodeficientes y presentaron las mismas alteraciones genéticas que se encontraron en los tumores parentales. Además, todas ellas fueron positivas para los marcadores de CMT: Nestina, Vimentina, CD44 y Sox2. Sin embargo, el grado de expresión de CD133 resultó ser variable en todas las líneas analizadas. A pesar de las similitudes entre las CMPT y las células madre neurales, es concebible pensar que pueden existir diferencias en sus respectivos potenciales de diferenciación. Para responder este interrogante hemos sometido a estas líneas celulares a diversos protocolos de diferenciación basados principalmente en el agregado de BMP4 recombinante humano y en la deprivación de factores de crecimiento. Luego de analizar la expresión de distintos marcadores de diferenciación y de evaluar cambios en la morfología celular, determinamos que estas líneas de CMPT poseen un grado variable de multipotencia, en donde cada una de ellas presenta un grado de compromiso particular hacia los linajes astroglial y neuronal. Además, pudimos observar que la adición de BMP4 recombinante redujo marcadamente la tasa de proliferación de todas las líneas celulares estudiadas. Por último, hemos determinado los niveles de expresión del ARNm de distintos ligandos y receptores involucrados en la señalización dependiente de BMPs en seis líneas de CMPT, encontrando en ellas ciertos patrones de expresión característicos. Dado que las CMT probablemente dependan de determinadas señales del microambiente tumoral para mantener su identidad, un tratamiento efectivo podría basarse en el bloqueo de mediadores generados en el nicho perivascular. Por este motivo y dado el gran potencial de este modelo experimental para el testeo de drogas, decidimos estudiar los niveles de expresión de los ARNm de los componentes del eje endotelinérgico en diferentes líneas de CMPT. Pudimos observar que todas las líneas celulares analizadas mostraron una expresión elevada de los transcriptos del receptor ETRB, mientras que el grado de expresión de los componentes restantes del sistema fue dependiente de cada línea celular. Más aún, en dos de cuatro líneas analizadas, el agregado de endotelinas recombinantes produjo una disminución significativa de los niveles de apoptosis generados en condiciones de deprivación de factores de crecimiento y nutrientes. Dada la variabilidad observada en las respuestas celulares a diferentes tratamientos en las distintas líneas de CMPT, este trabajo pone de menifiesto la relevancia de contar con un modelo in vitro basado en cultivos derivados de gliomas que permita contribuir al desarrollo de terapias personalizadas.High grade gliomas are the most frequent and malignant primary brain tumors in adults and appear to have a hierarchical organization suggestive of a stem cell foundation. Glioma stem cells (GSC) have been recently isolated from tumors of patients and were characterized as a small subset of stem-like tumor cells capable of initiating and sustaining tumor growth when grafted into mice. Thus, isolation and expansion of these putative cancer stem/progenitor cells, harboring tumor-specific phenotypes, constitutes a powerful model to study glioma biology. Significantly, GSC lines from different tumors exhibit divergent gene expression signatures and distinct differentiation behavior. In the present study, we successfully established nine glioma stem/progenitor cell (GSPC) lines obtained from malignant gliomas which display stem cell properties and initiate high-grade gliomas following xenotransplantation. Importantly, all the established cell lines presented the same genomic alterations as parental tumors. Additionally, they were all positive for the stem cell markers: Nestin, Vimentin, CD44 and Sox2. In contrast, CD133 expression levels widely varied among GSPC lines. Despite similarities between GSPC and normal neural stem cells, we hypothesized that there may be differences in their differentiation potentials. To address this issue, first, we exposed these cell lines to several differentiation conditions that principally include BMP4 treatment and growth factors withdrawal. After analyzing the expression of various markers of differentiation and assessing changes in cell morphology, we determined that these GSPC lines possess a varying degree of multipotency. Moreover, we observed that the addition of recombinant BMP4 markedly reduced the rate of proliferation of all the studied cell lines. Finally, we determined the expression levels of different ligands and receptors involved in BMP-signaling, and found that the GSPC lines show characteristic patterns of expression. As many cancer stem cells might depend on a niche to maintain their identity, targeting molecules triggered by the perivascular niche could be a strategy to deplete or differentiate them. For this reason and given the potential of the present experimental model for drug screening, we studied the expression profile of the endothelin (EDN) axis members in different GSPC lines grown in vitro. Our findings revealed that all tested cell lines robustly express ETRB receptor. Nevertheless, the expression levels of the other components of this axis resulted cell line specific. Furthermore, in two from four GSPC lines, the addition of recombinant EDNs (1, 2 and 3) reduced apoptotic rates upon qrowth factors and nutrients withdrawal. Given the diversity of the responses displayed by patient-derived CSC lines, the present study highlights the relevance of the in vitro model presented herein for the development of tailor-made therapies.Fil:Videla Richardson, Guillermo Agustín. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina

Topoisomerase I inhibitor, camptothecin, induces apoptogenic signaling in human embryonic stem cells

Embryonic stem cells (ESCs) need to maintain their genomic integrity in response to DNA damage to safeguard the integrity of the organism. DNA double strand breaks (DSBs) are one of the most lethal forms of DNA damage and, if not repaired correctly, they can lead to cell death, genomic instability and cancer. How human ESCs (hESCs) maintain genomic integrity in response to agents that cause DSBs is relatively unclear. In the present study we aim to determine the hESC response to the DSB inducing agent camptothecin (CPT). We find that hESCs are hypersensitive to CPT, as evidenced by high levels of apoptosis. CPT treatment leads to DNA-damage sensor kinase (ATM and DNA-PKcs) phosphorylation on serine 1981 and serine 2056, respectively. Activation of ATM and DNA-PKcs was followed by histone H2AX phosphorylation on Ser 139, a sensitive reporter of DNA damage. Nuclear accumulation and ATM-dependent phosphorylation of p53 on serine 15 were also observed. Remarkably, hESC viability was further decreased when ATM or DNA-PKcs kinase activity was impaired by the use of specific inhibitors. The hypersensitivity to CPT treatment was markedly reduced by blocking p53 translocation to mitochondria with pifithrin-μ. Importantly, programmed cell death was achieved in the absence of the cyclin dependent kinase inhibitor, p21Waf1, a bona fide p53 target gene. Conversely, differentiated hESCs were no longer highly sensitive to CPT. This attenuated apoptotic response was accompanied by changes in cell cycle profile and by the presence of p21Waf1. The results presented here suggest that p53 has a key involvement in preventing the propagation of damaged hESCs when genome is threatened. As a whole, our findings support the concept that the phenomenon of apoptosis is a prominent player in normal embryonic development

miR-302 family, miR-145 and miR-296 temporal expression profile along the cell cycle of human pluripotent stem cells

Human pluripotent stem cells (hPSCs), like embryonic (hESCs) and induced pluripotent stem cells (hiPSCs), exhibit an unusual cell cycle structure characterized by a short G1 phase and cells being most of time in S phase. hPSCs are receptive to differentiation cues during their transition through G1 phase when lineage determination is decided. Although several MicroRNAs (miRNAs) have been shown to target transcripts that directly or indirectly coordinate the cell cycle of pluripotent cells, its temporal expression profile along hPSCs cell cycle remains poorly characterized. miR-145 and miR-296 are induced during differentiation and silence the self-renewal and pluripotency program. miR-302 family is essential for hPSCs stemness and its expression decreases during differentiation. We aimed to study how the aforementioned miRNAs are regulated along the cell cycle of hPSCs. We demonstrated by pharmacological synchronization and block and release experiments that miR-145, miR-296 and miR-302 family are periodically expressed in hPSCs. Importantly, miR-302 family expression is induced at G1/S boundary and remained high at S phase, presumably to impede differentiation onset. Besides, we confirmed by a gene ontology analysis that many validated miR-302 family target genes are involved in cell cycle regulation.Fil: Rodríguez Varela, Maria Soledad. Fundación para la Lucha contra las Enfermedades Neurológicas de la Infancia; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Mucci, Sofia. Fundación para la Lucha contra las Enfermedades Neurológicas de la Infancia; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Videla Richardson, Guillermo Agustín. Fundación para la Lucha contra las Enfermedades Neurológicas de la Infancia; ArgentinaFil: Isaja, Luciana. Fundación para la Lucha contra las Enfermedades Neurológicas de la Infancia; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Sevlever, Gustavo Emilio. Fundación para la Lucha contra las Enfermedades Neurológicas de la Infancia; ArgentinaFil: Scassa, Maria Elida. Fundación para la Lucha contra las Enfermedades Neurológicas de la Infancia; ArgentinaFil: Romorini, Leonardo. Fundación para la Lucha contra las Enfermedades Neurológicas de la Infancia; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentin

Palbociclib Effectively Halts Proliferation but Fails to Induce Senescence in Patient-Derived Glioma Stem Cells

Glioblastoma multiforme is the most aggressive primary brain tumor. Current knowledge suggests that the growth and recurrence of these tumors are due in part to the therapy-resistant glioma stem cell subpopulation, which possesses the ability for self-renewal and proliferation, driving tumor progression. In many cancers, the p16INK4a-CDK4/6-pRb pathway is disrupted in favor of cell cycle progression. In particular, the frequent deregulation of CDK4/6 in cancer positions these kinases as promising targets. Palbociclib, a potent and selective CDK4/6 inhibitor, has been approved by the FDA as a first-line treatment of advanced breast cancer and there is currently interest in evaluating its effect on other cancer types. Palbociclib has been reported to be efficient, not only at halting proliferation, but also at inducing senescence in different tumor types. In this study, we evaluated the effect of this inhibitor on four patient-derived glioma stem cell-enriched cell lines. We found that Palbociclib rapidly and effectively inhibits proliferation without affecting cell viability. We also established that in these cell lines CDK6 is the key interphase CDK for controlling cell cycle progression. Prolonged exposure to Palbociclib induced a senescent-like phenotype characterized by flattened morphology, cell cycle arrest, increased β-galactosidase activity and induction of other senescent-associated markers. However, we found that after Palbociclib removal cell lines resumed normal proliferation, which implies they conserved their replicative potential. As a whole, our results indicate that in patient-derived glioma stem cell-enriched cell lines, Palbociclib induces a senescent-like quiescence rather than true senescence.Fil: Morris Hanon, Olivia. Fundación para la Lucha contra las Enfermedades Neurológicas de la Infancia; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Marazita, Mariela Claudia. Fundación para la Lucha contra las Enfermedades Neurológicas de la Infancia; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Romorini, Leonardo. Fundación para la Lucha contra las Enfermedades Neurológicas de la Infancia; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Isaja, Luciana. Fundación para la Lucha contra las Enfermedades Neurológicas de la Infancia; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Fernandez Espinosa, Damian Dario. Fundación para la Lucha contra las Enfermedades Neurológicas de la Infancia; ArgentinaFil: Sevlever, Gustavo. Fundación para la Lucha contra las Enfermedades Neurológicas de la Infancia; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Scassa, Maria Elida. Fundación para la Lucha contra las Enfermedades Neurológicas de la Infancia; ArgentinaFil: Videla Richardson, Guillermo Agustín. Fundación para la Lucha contra las Enfermedades Neurológicas de la Infancia; Argentin

Human Pluripotent Stem Cells and Derived Neuroprogenitors Display Differential Degrees of Susceptibility to BH3 Mimetics ABT-263, WEHI-539 and ABT-199.

Human embryonic stem cells (hESCs) are hypersensitive to genotoxic stress and display lower survival ability relative to their differentiated progeny. Herein, we attempted to investigate the source of this difference by comparing the DNA damage responses triggered by the topoisomerase I inhibitor camptothecin, in hESCs, human induced pluripotent stem cells (hiPSCs) and hESCs-derived neuroprogenitors (NP). We observed that upon camptothecin exposure pluripotent stem cells underwent apoptosis more swiftly and at a higher rate than differentiated cells. However, the cellular response encompassing ataxia-telangiectasia mutated kinase activation and p53 phosphorylation both on serine 15 as well as on serine 46 resulted very similar among the aforementioned cell types. Importantly, we observed that hESCs and hiPSCs express lower levels of the anti-apoptotic protein Bcl-2 than NP. To assess whether Bcl-2 abundance could account for this differential response we treated cells with ABT-263, WEHI-539 and ABT-199, small molecules that preferentially target the BH3-binding pocket of Bcl-xL and/or Bcl-2 and reduce their ability to sequester pro-apoptotic factors. We found that in the absence of stress stimuli, NP exhibited a higher sensitivity to ABT- 263 and WEHI-539 than hESCs and hiPSCs. Conversely, all tested cell types appeared to be highly resistant to the Bcl-2 specific inhibitor, ABT-199. However, in all cases we determined that ABT-263 or WEHI-539 treatment exacerbated camptothecin-induced apoptosis. Importantly, similar responses were observed after siRNA-mediated down-regulation of Bcl-xL or Bcl-2. Taken together, our results suggest that Bcl-xL contrary to Bcl-2 contributes to ensure cell survival and also functions as a primary suppressor of DNA double-strand brake induced apoptosis both in pluripotent and derived NP cells. The emerging knowledge of the relative dependence of pluripotent and progenitor cells on Bcl-2 and Bcl-xL activities may help to predict cellular responses and potentially manipulate these cells for therapeutic purposes in the near future

The Cell Cycle Inhibitors p21 Cip1 and p27 Kip1 Control Proliferation but Enhance DNA Damage Resistance of Glioma Stem Cells

High-grade gliomas are the most prevalent and lethal primary brain tumors. They display a hierarchical arrangement with a population of self-renewing and highly tumorigenic cells called cancer stemcells. These cells are thought to be responsible for tumor recurrence, which make them main candidates for targeted therapies. Unbridled cell cycle progression may explain the selective sensitivity of some cancer cells to treatments. The members of the Cip/Kip family p21Cip1 and p27Kip1 were initially considered as tumor suppressors based on their ability to block proliferation. However, they are currently looked at as proteins with dual roles in cancer: one as tumor suppressor and the other asoncogene. Therefore, the aimof this study was to determine the functions of these cell cycle inhibitors in five patientderived glioma stem cell?enriched cell lines. We found that these proteins are functional in glioma stem cells. They negatively regulate cell cycle progression both in unstressed conditions and in response to genotoxic stress. In addition, p27Kip1 is upregulated in nutrient-restricted and differentiating cells, suggesting that this Cip/Kip is a mediator of antimitogenic signals in glioma cells. Importantly, the lack of these proteins impairs cell cycle halt in response togenotoxic agents, rendering cells more vulnerable to DNA damage. For these reasons, these proteins may operate both as tumor suppressors, limiting cell proliferation, and as oncogenes, conferring cell resistance to DNA damage.Thus, deepening our knowledge on the biological functions of these Cip/Kipsmay shed light on howsomecancer cells develop drug resistance.Fil: Morris Hanon, Olivia. Fundación para la Lucha contra las Enfermedades Neurológicas de la Infancia; ArgentinaFil: Furmento, Verónica Alejandra. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Fundación para la Lucha contra las Enfermedades Neurológicas de la Infancia; ArgentinaFil: Rodríguez Varela, Maria Soledad. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Fundación para la Lucha contra las Enfermedades Neurológicas de la Infancia; ArgentinaFil: Mucci, Sofia. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Fernandez Espinosa, Damian Dario. Fundación para la Lucha contra las Enfermedades Neurológicas de la Infancia; ArgentinaFil: Romorini, Leonardo. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Fundación para la Lucha contra las Enfermedades Neurológicas de la Infancia; ArgentinaFil: Sevlever, Gustavo Emilio. Fundación para la Lucha contra las Enfermedades Neurológicas de la Infancia; ArgentinaFil: Scassa, Maria Elida. Fundación para la Lucha contra las Enfermedades Neurológicas de la Infancia; ArgentinaFil: Videla Richardson, Guillermo Agustín. Fundación para la Lucha contra las Enfermedades Neurológicas de la Infancia; Argentin