Cell-enriched Cultures Research Articles

Abstract 210: Id4 inhibition of twist1-mediated autocrine PDGF signaling inhibits growth and promotes apoptosis of glioblastoma-derived stem cell cultures

Abstract Glioblastoma Multiforme (GBM) is the most common, aggressive, and lethal primary brain tumor. A hallmark of GBM is the resistance of tumor stem cells to chemotherapy resulting in tumor recurrence and progression decreasing the quality of life and causing patient death. The prevention of GBM recurrence after chemotherapy by eliminating cancer stem cells enhances survival in genetically engineered mouse models; however, the signal transduction networks that control cancer stem cell survival and proliferation remain poorly understood. We have discovered that decreased expression of Id4, a dominant negative helix-loop-helix transcription factor, is associated with the proliferation and survival of sphere cultures from GBM-derived cell lines, which are enriched for cancer stem cells. In striking contrast, Id4 expression did not affect proliferation or apoptosis in differentiated, adherent cultures of these same cell lines. Moreover, the cell lines we examined required autocrine PDGF signaling for growth. We found that Id4 inhibited autocrine PDGF signaling in cancer stem cell enriched cultures by suppressing the expression of platelet-derived growth factor receptor α (PDGFRα). In these studies we found that Id4 mediated inhibition of PDGFRα expression was mediated through the antagonistic interaction between Id4 and Twist1, a basic helix-loop-helix transcription factor. This observation is consistent with our previously reported finding that Id4, uniquely among the Id family of proteins, binds to and antagonizes Twist1. Further, using the Rembrandt and TCGA databases, we found that Id4 expression levels directly correlate with glioma patient survival and can predict the level of PDGFRα expression. These data provide strong evidence that Id4 may regulate GBM stem cell survival and thereby contribute to GBM tumor progression. Along with our recent report demonstrating that Id4 can block invasion of GBM by silencing Twist1-mediated MMP2 expression, these findings suggest that pharmacological targeting of the Id4/Twist1 complex might provide opportunities to reduce the lethality of GBM by regulating both GBM invasion and tumor stem cell maintenance. Citation Format: Gilbert J. Rahme, Mark A. Israel. Id4 inhibition of twist1-mediated autocrine PDGF signaling inhibits growth and promotes apoptosis of glioblastoma-derived stem cell cultures. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 210. doi:10.1158/1538-7445.AM2014-210

Lipid metabolism enzyme ACSVL3 supports glioblastoma stem cell maintenance and tumorigenicity.

BackgroundTargeting cell metabolism offers promising opportunities for the development of drugs to treat cancer. We previously found that the fatty acyl-CoA synthetase VL3 (ACSVL3) is elevated in malignant brain tumor tissues and involved in tumorigenesis. This study investigates the role of ACSVL3 in the maintenance of glioblastoma multiforme (GBM) stem cell self-renewal and the capacity of GBM stem cells to initiate tumor xenograft formation.MethodsWe examined ACSVL3 expression during differentiation of several GBM stem cell enriched neurosphere cultures. To study the function of ACSVL3, we performed loss-of-function by using small interfering RNAs to target ACSVL3 and examined stem cell marker expression, neurosphere formation and tumor initiation properties.ResultsACSVL3 expression levels were substantially increased in GBM stem cell enriched neurosphere cultures and decreased after differentiation of the neurospheres. Down-regulating ACSVL3 with small inhibiting RNAs decreased the expression of markers and regulators associated with stem cell self-renewal, including CD133, ALDH, Musashi-1 and Sox-2. ACSVL3 knockdown in neurosphere cells led to increased expression of differentiation markers GFAP and Tuj1. Furthermore, ACSVL3 knockdown reduced anchorage-independent neurosphere cell growth, neurosphere-forming capacity as well as self-renewal of these GBM stem cell enriched neurosphere cultures. In vivo studies revealed that ACSVL3 loss-of-function substantially inhibited the ability of neurosphere cells to propagate orthotopic tumor xenografts. A link between ACSVL3 and cancer stem cell phenotype was further established by the findings that ACSVL3 expression was regulated by receptor tyrosine kinase pathways that support GBM stem cell self-renewal and tumor initiation, including EGFR and HGF/c-Met pathways.ConclusionsOur findings indicate that the lipid metabolism enzyme ACSVL3 is involved in GBM stem cell maintenance and the tumor-initiating capacity of GBM stem cell enriched-neurospheres in animals.

Differential gene expression profiling of enriched human spermatogonia after short- and long-term culture.

This study aimed to provide a molecular signature for enriched adult human stem/progenitor spermatogonia during short-term (<2 weeks) and long-term culture (up to more than 14 months) in comparison to human testicular fibroblasts and human embryonic stem cells. Human spermatogonia were isolated by CD49f magnetic activated cell sorting and collagen−/laminin+ matrix binding from primary testis cultures obtained from ten adult men. For transcriptomic analysis, single spermatogonia-like cells were collected based on their morphology and dimensions using a micromanipulation system from the enriched germ cell cultures. Immunocytochemical, RT-PCR and microarray analyses revealed that the analyzed populations of cells were distinct at the molecular level. The germ- and pluripotency-associated genes and genes of differentiation/spermatogenesis pathway were highly expressed in enriched short-term cultured spermatogonia. After long-term culture, a proportion of cells retained and aggravated the “spermatogonial” gene expression profile with the expression of germ and pluripotency-associated genes, while in the majority of long-term cultured cells this molecular profile, typical for the differentiation pathway, was reduced and more genes related to the extracellular matrix production and attachment were expressed. The approach we provide here to study the molecular status of in vitro cultured spermatogonia may be important to optimize the culture conditions and to evaluate the germ cell plasticity in the future.

Tff3 is Expressed in Neurons and Microglial Cells

Background/Aims: The trefoil factor family (TFF) peptide TFF3 is typically secreted by mucous epithelia, but is also expressed in the immune system and the brain. It was the aim of this study to determine the cerebral cell types which express Tff3. Methods: Primary cultures from rat embryonic or neonatal cerebral cortex and hippocampus, respectively, were studied by means of RT-PCR and immunofluorescence. Moreover, Tff3 expression was localized by immunocytochemistry in sections of adult rat cerebellum. Results: Tff3 transcripts were detectable in neural cultures of both the cortex and the hippocampus as well as in glial cell-enriched cultures. Tff3 peptide co-localized with Map2 indicating an expression in neurons in vitro. The neuronal expression was confirmed by immunofluorescence studies of adult rat cerebellum. Furthermore, Tff3 peptide showed also a clear co-localization with Iba-1 in vitro typical of activated microglial cells. Conclusion: The neuronal expression of Tff3 is in line with a function of a typical neuropeptide influencing, e.g., fear, memory, depression and motoric skills. The expression in activated microglial cells, which is demonstrated here for the first time, points towards a possible function for Tff3 in immune reactions in the CNS. This opens a plethora of additional possible functions for Tff3 including synaptic plasticity and cognition as well as during neuroinflammatory diseases and psychiatric disorders.

Comparison of innate immune activation after prolonged feeding of milk fermented with three species of Lactobacilli

The present investigation aimed at identifying the abilities of three different species of probiotic lactobacilli to modulate cellular immune responses in mouse neutrophils and macrophages in vivo over a study period of 60 days. Neutrophil respiratory burst enzymes (cytochrome c reductase and MPO) showed remarkable increased activity (P ≤ 0.01) after consumption of milks fermented by different species of probiotics over 30 and 60 days of feeding trials. Enzyme activities (β-galactosidase and β-glucuronidase) and nitric oxide production also increased considerably (P ≤ 0.01) in macrophages, both in peritoneal fluid and in enriched cell cultures. The effects of enhanced enzyme activities were corroborated by simultaneous increases in the phagocytic activities of neutrophils and macrophages. The increases in cellular functions were invariably maximal during the first 30 days of study and were maintained, but did not increase, over the next 30 days. Further, Lactobacillus helveticus-fed groups were most effective at modulating neutrophil functions whereas Lactobacillus paracasei-fed groups were more potent at enhancing macrophage functions. Together, our results indicate that probiotics have strain specific effects on stimulating cellular functions while not causing excessive stimulation of the immune system over longer feeding periods, thereby resulting in maximum and stable health benefits.

Glutamate-induced epigenetic and morphological changes allow rat Müller cell dedifferentiation but not further acquisition of a photoreceptor phenotype

Müller cells are not only the main glial cell type in the retina but also latent progenitor/stem cells, which in pathological conditions can transdifferentiate to a neuronal phenotype and regenerate the neurons lost in a mature retina. Several signal transduction pathways can induce the dedifferentiation of mature Müller cells to a progenitor-like state, including that stimulated by glutamate. However, the precise molecular mechanisms by which terminally differentiated cells are initially primed to acquire multipotency remain unclear. In the present study, we have characterized early genetic and epigenetic events that occur immediately after glutamate-induced dedifferentiation of fully differentiated Müller cells is initiated. Using Müller cell-enriched cultures from postnatal rats, we demonstrate that glutamate triggers a rapid dedifferentiation response characterized by changes in cell morphology coupled to the induction of progenitor cell marker gene expression (e.g., nestin, lin28 and sox2) within 1h. Dedifferentiation involved the activation of N-methyl-d-aspartate and type II metabotropic glutamate receptors, as well as global DNA demethylation (evident through the decrease in methyl-CpG-binding protein 2 immunoreactivity) and an increase in gadd45-β gene expression; although, early progenitor gene expression was only partially inhibited by pharmacological impairment of DNA methylation. Importantly, the expression of Müller glia identity genes (i.e., glutamine synthetase; cellular retinaldehyde binding protein, CRALBP) is retained through the process. Dedifferentiated Müller cells held an early neuronal differentiation potential similar to that observed in retinal progenitor-enriched cultures but, contrary to the latter, dedifferentiated Müller cells failed to further differentiate into mature photoreceptor lineages. We speculate that, in spite of the initial triggering of the dedifferentiation pathways, these cells may exhibit a certain degree of epigenetic memory that precludes them from further differentiation.

Targeting the Cytosolic Innate Immune Receptors RIG-I and MDA5 Effectively Counteracts Cancer Cell Heterogeneity in Glioblastoma

Cellular heterogeneity, for example, the intratumoral coexistence of cancer cells with and without stem cell characteristics, represents a potential root of therapeutic resistance and a significant challenge for modern drug development in glioblastoma (GBM). We propose here that activation of the innate immune system by stimulation of innate immune receptors involved in antiviral and antitumor responses can similarly target different malignant populations of glioma cells. We used short-term expanded patient-specific primary human GBM cells to study the stimulation of the cytosolic nucleic acid receptors melanoma differentiation-associated gene 5 (MDA5) and retinoic acid-inducible gene I (RIG-I). Specifically, we analyzed cells from the tumor core versus "residual GBM cells" derived from the tumor resection margin as well as stem cell-enriched primary cultures versus specimens without stem cell properties. A portfolio of human, nontumor neural cells was used as a control for these studies. The expression of RIG-I and MDA5 could be induced in all of these cells. Receptor stimulation with their respective ligands, p(I:C) and 3pRNA, led to in vitro evidence for an effective activation of the innate immune system. Most intriguingly, all investigated cancer cell populations additionally responded with a pronounced induction of apoptotic signaling cascades revealing a second, direct mechanism of antitumor activity. By contrast, p(I:C) and 3pRNA induced only little toxicity in human nonmalignant neural cells. Granted that the challenge of effective central nervous system (CNS) delivery can be overcome, targeting of RIG-I and MDA5 could thus become a quintessential strategy to encounter heterogeneous cancers in the sophisticated environments of the brain.

Sonic hedgehog promotes generation and maintenance of human forebrain Olig2 progenitors

Function of oligodendrocytes (OLs), myelin forming cells in the CNS, is disrupted in demyelinating diseases such as periventricular leukomalacia or multiple sclerosis. It is, thus, important to better understand factors that can affect generation or differentiation of human OLs. In rodents, Sonic hedgehog (Shh) is influencing expression of Olig2, a helix-loop-helix transcription factor required for development of OLs. In humans, Olig2 is present in cortical progenitors at midgestation, however the role of Shh in the specification of human OLs, including Olig2 positive (Olig2+) progenitors, is not fully understood. Here we studied in vitro effects of Shh signaling on proliferation and specification of human cortical Olig2+ progenitors at midgestation. First, we established that the spatial pattern of Olig2 expression in the human developing CNS, described on cryosections, was preserved in mixed and enriched radial glia cell (RGC) cultures. Next, we demonstrated that in vitro treatment with Shh induced an increase in the number of Olig2+ progenitors. Shh treatment increased the density of early oligodendrocyte progenitors (OPCs) at the expense of RGC, while the number of late OPCs, did not change. However, inhibition of endogenous Shh with cyclopamine did not reduce the density of Olig2+ cells, implying the presence of an additional Shh-independent mechanism for OLs generation in vitro. These results suggest that the primary role of Shh signaling in the human dorsal oligodendrogenesis is the expansion and specification of multipotent radial glia progenitors into Olig2+ early OPCs. These results obtained in vitro are relevant to understand primary myelination during CNS development, as well as remyelination in demyelinating diseases.

Metformin selectively affects human glioblastoma tumor-initiating cell viability

Cancer stem cell theory postulates that a small population of tumor-initiating cells is responsible for the development, progression and recurrence of several malignancies, including glioblastoma. In this perspective, tumor-initiating cells represent the most relevant target to obtain effective cancer treatment. Metformin, a first-line drug for type II diabetes, was reported to possess anticancer properties affecting the survival of cancer stem cells in breast cancer models. We report that metformin treatment reduced the proliferation rate of tumor-initiating cell-enriched cultures isolated from four human glioblastomas. Metformin also impairs tumor-initiating cell spherogenesis, indicating a direct effect on self-renewal mechanisms. Interestingly, analyzing by FACS the antiproliferative effects of metformin on CD133-expressing subpopulation, a component of glioblastoma cancer stem cells, a higher reduction of proliferation was observed as compared with CD133-negative cells, suggesting a certain degree of cancer stem cell selectivity in its effects. In fact, glioblastoma cell differentiation strongly reduced sensitivity to metformin treatment. Metformin effects in tumor-initiating cell-enriched cultures were associated with a powerful inhibition of Akt-dependent cell survival pathway, while this pathway was not affected in differentiated cells. The specificity of metformin antiproliferative effects toward glioblastoma tumor-initiating cells was confirmed by the lack of significant inhibition of normal human stem cells (umbilical cord-derived mesenchymal stem cells) in vitro proliferation after metformin exposure. Altogether, these data clearly suggest that metformin exerts antiproliferative activity on glioblastoma cells, showing a higher specificity toward tumor-initiating cells, and that the inhibition of Akt pathway may represent a possible intracellular target of this effect.

Rat and mouse testicular testin is different from the human tumor suppressor gene TESTIN (Tes)

In response to a “Letter to the Editor” from Dr. Shailendra Kapoor with regard to a paper published on testin in Spermatogenesis,1 we wish to clarify some misinformation on this protein in the literature. In the late 1980s, two papers were published, reporting a new testicular protein purified from rat Sertoli cell-enriched culture media. It was designated testin based on its unique N-terminal amino acid sequence.2,3 Specifically, testin is a testis-specific protein,4 composed of two isoforms of testin I (35 kDa) and testin II (37 kDa) where testin II has three extra N-terminal amino acids of Thr-Ala-Pro.3 The full-length cDNA encoding rat testicular testin was cloned, sequenced and published shortly thereafter (GenBank™ Accession Numbers: {type:entrez-nucleotide,attrs:{text:U16858,term_id:577429}}U16858; {type:entrez-nucleotide,attrs:{text:NM_173132,term_id:402693052}}NM_173132, {type:entrez-protein,attrs:{text:NP_775155,term_id:27465595}}NP_775155).5 Testin was subsequently cloned and sequenced in the mouse (GenBank™ Accession Numbers: {type:entrez-nucleotide,attrs:{text:NM_178098,term_id:31342510}}NM_178098, {type:entrez-protein,attrs:{text:NP_835199,term_id:30017423}}NP_835199) and shown to display 90.1% similarity with rat testin.6 Testin is also a gonad-specific protein,4 with its expression restricted largely to the testis and ovary in adult rats.7,8 Interestingly, its expression is upregulated during disruption of the testis-specific anchoring junction known as the apical ectoplasmic specialization (ES).8-10 The recent report published in Spermatogenesis has shown that testin is also an actin-binding protein at the ES.1 While the rat testin primary amino acid sequence contains an apparent His- and an Asn-active site, a characteristic feature of cysteine proteases, and is ~60% homologous with human cathepsin L ({type:entrez-protein,attrs:{text:NP_666023,term_id:22202619}}NP_666023; NM-145918), testin does not possess protease nor protease inhibitor activity.11 Equally important, Sertoli cell testin originally identified in the rat testis is entirely different from a tumor suppressor gene called TESTIN (gene name Tes) (GenBank™ Accession Numbers: {type:entrez-nucleotide,attrs:{text:NM_015641,term_id:325197160}}NM_015641, {type:entrez-protein,attrs:{text:NP_056456,term_id:7661666}}NP_056456), which was identified and cloned around the same time rat Sertoli cell testin was identified.12-14 The tumor suppressor TESTIN14 shares no significant homology in its amino acid sequence (only ~1–3%) with rat Sertoli cell testin. Moreover, Sertoli cell testin does not contain any characteristic LIM domains or zinc finger motifs, which are found in the tumor suppressor TESTIN, illustrating that these two proteins are distinctly different and evolutionarily unrelated. A mouse genetic model is available for the tumor suppressor TESTIN, and its deletion was found to associate with hematopoietic malignancies and epithelial tumors.15 However, findings from this genetic model are not applicable to Sertoli cell testin since they are two entirely different proteins.

RANK (TNFRSF11A) Is Epigenetically Inactivated and Induces Apoptosis in Gliomas

Alterations of DNA methylation play an important role in gliomas. In a genome-wide screen, we identified a CpG-rich fragment within the 5′ region of the tumor necrosis factor receptor superfamily, member 11A gene (TNFRSF11A) that showed de novo methylation in gliomas. TNFRSF11A, also known as receptor activator of NF-κB (RANK), activates several signaling pathways, such as NF-κB, JNK, ERK, p38α, and Akt/PKB. Using pyrosequencing, we detected RANK/TNFRSF11A promoter methylation in 8 (57.1%) of 14 diffuse astrocytomas, 17 (77.3%) of 22 anaplastic astrocytomas, 101 (84.2%) of 120 glioblastomas, 6 (100%) of 6 glioma cell lines, and 7 (100%) of 7 glioma stem cell-enriched glioblastoma primary cultures but not in four normal white matter tissue samples. Treatment of glioma cell lines with the demethylating agent 5-aza-2′-deoxycytidine significantly reduced the methylation level and resulted in increased RANK/TNFRSF11A mRNA expression. Overexpression of RANK/TNFRSF11A in glioblastoma cell lines leads to a significant reduction in focus formation and elevated apoptotic activity after flow cytometric analysis. Reporter assay studies of transfected glioma cells supported these results by showing the activation of signaling pathways associated with regulation of apoptosis. We conclude that RANK/TNFRSF11A is a novel and frequent target for de novo methylation in gliomas, which affects apoptotic activity and focus formation thereby contributing to the molecular pathogenesis of gliomas.

Ascitic derived primary pancreatic cancer cell cultures from different patients as a platform for personalized medicine.

e14646 Background: In Pancreatic Cancer (PC) challenges in drug development include: obtaining metastatic cancer tissue for research, developing and validating biomarkers predicative for personalized therapeutic decisions. We have recently developed a novel therapeutic model for PC to address these challenges based on the isolation of viable PC cells derived from ascitic fluid. Methods: Ascitic fluid was obtained from PC patients undergoing palliative paracentesis. In several patients ascites was obtained several times during the course of their disease. Ascitic-derived primary PC cells were isolated, cultured and identified for each patient in vitro. Informed consent was obtained for all patients. Results: We successfully established ascitic-derived primary cell cultures in 87% (53/61) of the ascites obtained. Homogeneous epithelial PC enriched cell cultures were identified; CK 7/8 (epithelial marker) staining by FACS in 90-95% of the cells, IHC strong staining of CK 7 and negative staining for CK 5/6 in the majority of cells in the cultures. We observed a wide range in doubling time and migration properties among the patients' cell cultures. The diverse nature of each individual patient's cell cultures were further demonstrated by varying chemo-sensitivity to gemcitabine and additional therapeutic agents. Morphological changes were observed at different stages of disease, most cells initially displayed a characteristic epithelial morphology. With more advanced disease a mixed morphological appearance "epithelial-mesenchymal" was seen and up regulation of various epithelial-mesenchymal-transition markers were observed by real-time PCR. Conclusions: We have developed a unique primary ascitic-derived PC cell culture model. These cell lines have the potential to serve as a relevant model to study signaling pathways in PC progression, and to assess the sensitivity to therapeutic agents in a short time frame, therefore supporting personalized treatment decisions.

Abstract B124: Isolation and characterization of ascitic derived primary pancreatic cancer cell cultures from different patients as a platform for personalized medicine.

Abstract Discovery and development of effective cancer treatments in pancreatic cancer (PC) has been hampered by the lack of reliable predictive preclinical models to assess the therapeutic efficacy of candidate agents, mostly due to the genomic heterogeneity in these patients. Therefore alternative models that recapitulate this complex disease need to be developed. Approximately a third of patients with PC have a tendency to produce ascites in advanced disease. The ascitic fluid contains viable tumor cells, and therefore we decided to develop an investigational model based on primary tumor cells obtained from patients' ascites. Ascitic fluid was obtained from 20 patients with PC undergoing palliative paracentesis, in 4 patients ascites was obtained several times during the course of their disease (totaling 44 ascitic fluids obtained). Ascitic-derived primary pancreatic cancer cells were isolated and cultures were grown for each individual patient. We identified the PC cells and examined the cells' morphological characteristics, phenotypes, invasive properties and the effect of various therapeutic agents. We successfully established ascitic-derived primary cell cultures in 84% (37/44). Positive staining by IHC of epithelial marker (CK-7) and negative staining for CK-5/6 was demonstrated in the majority of cells in the culture. Cytokeratin staining by FACS was observed in 90–95% of the isolated cells supporting the presence of homogeneous epithelial PC enriched cell culture. We observed a wide range in doubling time and migration properties among the patients' ascites derived cell cultures and even among cultures from the same patient along the course of his disease. A correlation between high migratory potential and long doubling time was observed. The cells were treated with different chemotherapies and biological agents. The diverse nature of each individual patient's primary pancreatic cancer cell cultures were further demonstrated by varying responses to the therapeutic agents examined. Morphological changes were also observed at different stages of disease, most cells initially displayed a characteristic epithelial morphology, but with more advanced disease a mixed morphological appearance ‘epithelial-mesenchymal’ was seen. The expression of epithelial-mesenchymal-transition (EMT) markers was examined by real-time PCR. Up regulation of Zeb-1, Zeb-2, Twist, Slug and Dab2 were observed in the cells displaying ‘epithelial-mesenchymal’ like appearance. We have developed a unique primary ascitic-derived pancreatic cancer cell culture model that enables a diverse range of patients-related cell cultures. These cell lines has the potential to serve as a relevant model to study signaling pathways in PC progression. Additionally, this model could be used to assess the sensitivity to therapeutic agents in a short time frame, therefore supporting personalized treatment decisions. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr B124.

Metabolic modulation induced by oestradiol and DHT in immature rat Sertoli cells culturedin vitro

Sertoli cells actively metabolize glucose that is converted into lactate, which is used by developing germ cells for their energy metabolism. Androgens and oestrogens have general metabolic roles that reach far beyond reproductive processes. Hence, the main purpose of this study was to examine the effect of sex hormones on metabolite secretion/consumption in primary cultures of rat Sertoli cells. Sertoli cell-enriched cultures were maintained in a defined medium for 50h. Glucose and pyruvate consumption, and lactate and alanine secretion were determined, by 1H-NMR (proton NMR) spectra analysis, in the presence or absence of 100nM E2 (17β-oestradiol) or 100nM 5α-DHT (dihydrotestosterone). Cells cultured in the absence (control) or presence of E2 consumed the same amount of glucose (29±2pmol/cell) at similar rates during the 50h. After 25h of treatment with DHT, glucose consumption and glucose consumption rate significantly increased. Control and E2-treated cells secreted similar amounts of lactate during the 50h, while the amount of lactate secreted by DHT-treated cells was significantly lower. Such a decrease was concomitant with a significant decrease in LDH A [LDH (lactate dehydrogenase) chain A] and MCT4 [MCT (monocarboxylate transporter) isoform 4] mRNA levels after 50h treatment in hormonally treated groups, being more pronounced in DHT-treated groups. Finally, alanine production was significantly increased in E2-treated cells after 25h treatment, which indicated a lower redox/higher oxidative state for the cells in those conditions. Together, these results support the existence of a relation between sex hormones action and energy metabolism, providing an important assessment of androgens and oestrogens as metabolic modulators in rat Sertoli cells.

A Molecular Screening Approach to Identify and Characterize Inhibitors of Glioblastoma Stem Cells

Glioblastoma (GBM) is among the most lethal of all cancers. GBM consist of a heterogeneous population of tumor cells among which a tumor-initiating and treatment-resistant subpopulation, here termed GBM stem cells, have been identified as primary therapeutic targets. Here, we describe a high-throughput small molecule screening approach that enables the identification and characterization of chemical compounds that are effective against GBM stem cells. The paradigm uses a tissue culture model to enrich for GBM stem cells derived from human GBM resections and combines a phenotype-based screen with gene target-specific screens for compound identification. We used 31,624 small molecules from 7 chemical libraries that we characterized and ranked based on their effect on a panel of GBM stem cell-enriched cultures and their effect on the expression of a module of genes whose expression negatively correlates with clinical outcome: MELK, ASPM, TOP2A, and FOXM1b. Of the 11 compounds meeting criteria for exerting differential effects across cell types used, 4 compounds showed selectivity by inhibiting multiple GBM stem cells-enriched cultures compared with nonenriched cultures: emetine, n-arachidonoyl dopamine, n-oleoyldopamine (OLDA), and n-palmitoyl dopamine. ChemBridge compounds #5560509 and #5256360 inhibited the expression of the 4 mitotic module genes. OLDA, emetine, and compounds #5560509 and #5256360 were chosen for more detailed study and inhibited GBM stem cells in self-renewal assays in vitro and in a xenograft model in vivo. These studies show that our screening strategy provides potential candidates and a blueprint for lead compound identification in larger scale screens or screens involving other cancer types.

Pharmacological targeting of the constitutively activated MEK/MAPK-dependent signaling pathway in glioma cells inhibits cell proliferation and migration

Activated mitogen-activated protein kinase MAPK cascade leading to ERK1/2 phosphorylation is expressed in the majority of glial neoplasms and negatively correlates with survival time of patients. Here we show that ERK1/2 kinases are constitutively activated in glioma cell lines and stem cell-enriched primary cultures of glioblastoma. Pharmacological targeting of the activated MEK/ERK1/2 module with the MEK inhibitor U0126 attenuates cell cycle progression (11 out of 11 cell lines), impairs single (7 out of 10) and collective cell migration (9 out of 11) and abolishes single cell emigration from monolayers (4 out of 9). Attacking the activated MEK/ERK1/2 module thus partially blocks the tumorigenic potential of glial cancer cells on different levels and strongly suggests the application of combination molecularly targeted therapies to interfere more efficiently with glial tumor development and progression.

Influence of 5α-dihydrotestosterone and 17β-estradiol on human Sertoli cells metabolism

Sertoli cells metabolize glucose, converting it to lactate that is used by developing germ cells for their energy metabolism. Androgens and oestrogens have metabolic roles that reach far beyond reproductive processes. So, the main purpose of this study was to examine the effect of sex steroid hormones on metabolite secretion/consumption in human Sertoli cells. Human Sertoli cell-enriched primary cultures were maintained in a defined medium for 50 h and glucose, pyruvate, lactate and alanine variations were determined using (1) H-NMR spectra analysis, in the absence or presence of 100 nm 17β-estradiol (E(2) ) or 100 nm 5α-dihydrotestosterone (DHT). The mRNA expression levels of glucose transporters, lactate dehydrogenase and monocarboxylate transporters were also determined using semi-quantitative RT-PCR. Cells cultured in the absence (control) or presence of E(2) consumed the same amounts of glucose at similar rates during the 50 h. During the first 15 h of treatment with DHT, glucose consumption and glucose consumption rate were significantly higher. Nevertheless, DHT-treated cells secreted a significantly lower amount of lactate than control and E(2) -treated cells. Such a decrease was concomitant with a significant decrease in lactate dehydrogenase A mRNA levels after 50 h treatment in DHT-treated groups. Finally, alanine production was significantly increased in E(2) -treated cells after 25 h treatment, which indicated a lower redox/higher oxidative state for the cells on those conditions. These results support the existence of a relationship between sex steroid hormones action and energy metabolism, providing the first assessment of androgens and oestrogens as metabolic modulators of human Sertoli cells.

Dopamine‐dependent ectodomain shedding and release of epidermal growth factor in developing striatum: target‐derived neurotrophic signaling (Part 2)

Epidermal growth factor (EGF) and structurally related peptides promote neuronal survival and the development of midbrain dopaminergic neurons; however, the regulation of their production has not been fully elucidated. In this study, we found that the treatment of striatal cells with dopamine agonists enhances EGF release both in vivo and in vitro. We prepared neuron-enriched and non-neuronal cell-enriched cultures from the striatum of rat embryos and challenged those with various neurotransmitters or dopamine receptor agonists. Dopamine and a dopamine D(1) -like receptor agonist (SKF38393) triggered EGF release from neuron-enriched cultures in a dose-dependent manner. A D(2) -like agonist (quinpirole) increased EGF release only from non-neuronal cell-enriched cultures. The EGF release from striatal neurons and non-neuronal cells was concomitant with ErbB1 phosphorylation and/or with the activation of a disintegrin and metalloproteinase and matrix metalloproteinase. The EGF release from neurons was attenuated by an a disintegrin and metalloproteinase/matrix metalloproteinase inhibitor, GM6001, and a calcium ion chelator, BAPTA/AM. Transfection of cultured striatal neurons with alkaline phosphatase-tagged EGF precursor cDNA confirmed that dopamine D(1) -like receptor stimulation promoted both ectodomain shedding of the precursor and EGF release. Therefore, the activation of striatal dopamine receptors induces shedding and release of EGF to provide a retrograde neurotrophic signal to midbrain dopaminergic neurons.

Expression of genes related to the hypothalamic-pituitary-adrenal axis in murine fetal lungs in late gestation

BackgroundLung maturation is modulated by several factors, including glucocorticoids. Expression of hypothalamic-pituitary-adrenal (HPA) axis-related components, with proposed or described local regulatory systems analogous to the HPA axis, was reported in peripheral tissues. Here, HPA axis-related genes were studied in the mouse developing lung during a period overlapping the surge of surfactant production.MethodsExpression of genes encoding for corticotropin-releasing hormone (CRH), CRH receptors (CRHR) 1 and 2beta, CRH-binding protein, proopiomelanocortin (POMC), melanocortin receptor 2 (MC2R), and glucocorticoid receptor was quantified by real-time PCR and localized by in situ hydridization in fetal lungs at gestational days (GD) 15.5, 16.5, and 17.5, and was also quantified in primary mesenchymal- and epithelial cell-enriched cultures. In addition, the capability of CRH and adrenocorticotropic hormone (ACTH) to stimulate pulmonary expression of enzymes involved in the adrenal pathway of glucocorticoid synthesis was addressed, as well as the glucocorticoid production by fetal lung explants.ResultsWe report that all the studied genes are expressed in fetal lungs according to different patterns. On GD 15.5, Mc2r showed peaks in expression in samples that have previously presented high mRNA levels for glucocorticoid synthesizing enzymes, including 11beta-hydroxylase (Cyp11b1). Crhr1 mRNA co-localized with Pomc mRNA in cells surrounding the proximal epithelium on GD 15.5 and 16.5. A transition in expression sites toward distal epithelial cells was observed between GD 15.5 and 17.5 for all the studied genes. CRH or ACTH stimulation of genes involved in the adrenal pathway of glucocorticoid synthesis was not observed in lung explants on GD 15.5, whereas CRH significantly increased expression of 21-hydroxylase (Cyp21a1) on GD 17.5. A deoxycorticosterone production by fetal lung explants was observed.ConclusionsTemporal and spatial modulations of expression of HPA axis-related genes in late gestation are consistent with roles for these genes in lung development. Our data are likely to lead to valuable insights in relation to lung diseases originating from lung immaturity.

Plasma membrane calcium ATPase isoform 3 expression in single cells isolated from rat liver

The plasma membrane Ca(2+)-ATPase (PMCA) located in the hepatocyte is a controversial molecule in itself since it displays different features to those regarded as canonical for P-type Ca(2+)-ATPases, and from which transcript expression as well as catalytic activity continues to be under active investigation. Our aim in this study was to explore at a first glance, pmca isoform distribution using isolated parenchymal and non-parenchymal cells from rat liver tissue. Expression of pmca transcripts was analyzed in fresh or cell-enriched culture preparations, confirming pmca1 and pmca4 as the housekeeping isoforms in all cell types studied (hepatocytes, Kupffer cells, and stellate cells). However, for the first time we show expression of pmca3 transcripts edited at two different sites in both hepatocytes and non-parenchymal cells. Interestingly, employing non-parenchymal cells we demonstrate the specific expression of pmca3e transcripts previously considered nearly exclusive of excitable tissues. Real-time PCR quantification shows a significant decrease of pmca3 transcripts in cultured Kupffer and hepatic stellate cells in comparison with fresh cells. The presence of pmca2 along with pmca3 in all liver cell types studied suggests that high affinity isoforms are relevant to the adequate management of calcium in liver tissue, particularly when hepatic cells become activated by diverse stimuli.