Levels Of Nestin Research Articles

In vitro Analysis of Neurospheres Derived from Glioblastoma Primary Culture: A Novel Methodology Paradigm

Glioblastomas are the most lethal primary brain tumor that frequently relapse or progress as focal masses after radiation, suggesting that a fraction of tumor cells are responsible for the tumor regrowth. The identification of a brain tumor cell subpopulation with potent tumorigenic activity supports the cancer stem cell hypothesis in solid tumors. The goal of this study is to determine a methodology for the establishment of primary human glioblastoma cell lines. Our aim is achieved by taking the following approaches: (i) the establishment of primary glioblastoma cell culture; (ii) isolation of neurospheres derived from glioblastoma primary cultures; (iii) selection of CD133 cells from neurospheres, (iv) formation of subspheres in the CD133-positive population, (v) study of the expression level of GFAP, CD133, Nestin, Nanog, CD34, Sox2, CD44, and CD90 markers on tumor subspheres. Hence, we described a successful method for isolation of CD133-positive cell population and establishment of glioblastoma neurospheres from this primary culture, which are more robust than the ones derived straight from the tumor. Pointed out that the neurospheres derived from glioblastoma primary culture showed 29% more cells expressing CD133 then the ones straight tumor-derived, denoting a higher concentration of CD133-positive cells in the neurospheres derived from glioblastoma primary culture. These CD133-positive fractions were able to further generate subspheres. The subspheres derived from glioblastoma primary culture presented a well-defined morphology while the ones derived from the fresh tumor were sparce and less robust. And the negative fraction of CD133 cells was unable to generate subspheres. The tumor subspheres expressed GFAP, CD133, Nestin, Nanog, CD44, and CD90. Also, the present study describes an optimization of neurospheres/subspheres isolation from glioblastoma primary culture by selection of CD133-positive adherent stem cell.

Multipotent neural crest stem cell-like cells from rat vibrissa dermal papilla induce neuronal differentiation of PC12 cells.

Bone marrow mesenchymal stem cells (BMSCs) transplants have been approved for treating central nervous system (CNS) injuries and diseases; however, their clinical applications are limited. Here, we model the therapeutic potential of dermal papilla cells (DPCs) in vitro. DPCs were isolated from rat vibrissae and characterized by immunocytofluorescence, RT-PCR, and multidifferentiation assays. We examined whether these cells could secrete neurotrophic factors (NTFs) by using cocultures of rat pheochromocytoma cells (PC12) with conditioned medium and ELISA assay. DPCs expressed Sox10, P75, Nestin, Sox9, and differentiated into adipocytes, osteoblasts, smooth muscle cells, and neurons under specific inducing conditions. The DPC-conditioned medium (DPC-CM) induced neuronal differentiation of PC12 cells and promoted neurite outgrowth. Results of ELISA assay showed that compared to BMSCs, DPCs secreted more brain-derived neurotrophic factor (BDNF) and glial cell line-derived neurotrophic factor (GDNF). Moreover, we observed that, compared with the total DPC population, sphere-forming DPCs expressed higher levels of Nestin and P75 and secreted greater amounts of GDNF. The DPCs from craniofacial hair follicle papilla may be a new and promising source for treating CNS injuries and diseases.

Nestin expression throughout multistep pathogenesis of multiple myeloma

The stem cell marker nestin (NES) is found in dividing cells of developing and regenerating tissues. Upon terminal differentiation, NES expression is diminished but may be re-expressed following injury or in cancer. Surprisingly, we recently confirmed NES as a tumour-specific marker for mature CD138(+) 38(+) plasma cells (PC) in multiple myeloma (MM). The present study analysed NES expression throughout the spectrum of MM developmental stages, starting with individuals with no haematological malignancy, through monoclonal gammopathy of undetermined significance (MGUS) and MM to plasma cell leukaemia (PCL) and MM cell lines. NES was analysed in bone marrow PC of 163 MM, four PCL and nine MGUS patients, 10 individuals with no haematological malignancy and 6 myeloma cell lines (OPM-2, RPMI-8226, MOLP-8, U-266, EJM, NCI-H929) by flow cytometry and/or real-time polymerase chain reaction or immunochemistry. We observed a tendency of increased NES expression in parallel with disease progression. NES was evaluated as a reliable marker for accurate discrimination between MM patients and the control group. High NES levels were strongly associated with the presence of 1q21 gain. For the first time, NES was demonstrated to predict worse response to conventional therapy/novel agents. These results suggest that NES might become a useful clinical parameter with an important role in MM pathogenesis.

Clinicopathological significance of expression of nestin, a neural stem/progenitor cell marker, in human glioma tissue

The purpose of the study was to investigate the pathological and clinical significance of the expression of nestin, a type-VI intermediate filament transiently expressed during brain development, in glioma tissue. This study was conducted in 70 patients with newly diagnosed adult supratentorial gliomas who underwent multimodality treatment in our department, including surgery. The pathological diagnosis was grade II in 6 patients, grade III in 21 patients, and grade IV in 43 patients. Two specimen sections, one from the bulk of the removed tumor and one from the border between the tumor and normal brain tissue, were subjected to immunostaining with a mouse anti-human nestin monoclonal antibody. Analyses were performed to investigate possible correlation with pathological features, the relationship between nestin expression and the continuity of tumor with the subventricular zone (SVZ), correlation with the therapeutic prognosis, etc. Nestin was expressed specifically in astrocytoma lineage cells. In oligodendroglial tumors, nestin was expressed only in less-differentiated cells and cells suggestive of the presence of astrocytoma. In astrocytic tumors, the rate and level of nestin expression increased as the degree of malignancy increased. There was no significant correlation between the expression level of nestin and the continuity of tumor with the SVZ in the contrast-enhanced imaging before surgery. In addition, no correlation with the therapeutic prognosis was observed. Nestin, a neural stem cell marker, was specifically expressed in astrocytoma lineage cells in glioma tissue. A positive correlation was observed between the degree of malignancy and the level of nestin expression. However, the level of nestin expression was not related to the tumor localization in the SVZ and was not correlated with the therapeutic prognosis.

Glioma Spheroids Obtained via Ultrasonic Aspiration Are Viable and Express Stem Cell Markers

Ultrasonic aspirators allow safe, rapid, and accurate removal of brain tumors. However, the tissue fragments removed are used surprisingly little in research. To investigate whether such tissue fragments could be cultured as organotypic multicellular spheroids because access to biopsy tissue is often limited. Tissue fragments obtained by ultrasonic aspiration from 10 glioblastomas and tumor biopsy tissue from 7 of these tumors were cultured in serum-containing and serum-free medium. On culturing, the fragments formed spheroids, which were prepared for histology. Two glioblastoma cell lines from ultrasonic fragments and biopsy tissue were established as well. Hematoxylin and eosin staining showed viable glioma spheroids obtained from both ultrasonic and biopsy tissue in both types of medium. Endothelial growth factor receptor and PTEN/chromosome 10 status was found to be preserved in most spheroids (7-8 of 10 tumors), together with the level of glial fibrillary acidic protein, von Willebrand factor, and Ki-67. The levels of stem cell markers CD133, Bmi-1, nestin, and Sox2 also were preserved. The ultrasonic spheroids had higher levels of glial fibrillary acidic protein and von Willebrand factor and lower levels of Bmi-1, nestin, Sox2, and Olig2 compared with conventional biopsy spheroids. For both types of spheroids, the stem cell medium seemed to favor expression of stem cell markers. The established cell lines were capable of both spheroid formation at clonal density and tumor formation in vivo. Viable organotypic and proliferating spheroids were easily obtained from ultrasonic tissue fragments. The preservation of markers and the establishment of cell lines with tumor-initiating cell properties suggest ultrasonic spheroids as a new tissue resource for glioma research.

Neuronal cell differentiation of mesenchymal stem cells originating from canine amniotic fluid

The amniotic fluid contains mesenchymal stem cells (MSCs) and can be readily available for tissue engineering. Regenerative treatments such as tissue engineering, cell therapy, and transplantation show potential in clinical trials of degenerative diseases. Disease presentation and clinical responses in the Canis familiaris not only are physiologically similar to human compared with other traditional mammalian models but is also a suitable model for human diseases. The aim of this study was to investigate whether canine amniotic-fluid-derived mesenchymal stem cells (cAF-MSCs) can differentiate into neural precursor cells in vitro when exposed to neural induction reagent. During neural differentiation, cAF-MSCs progressively acquire neuron-like morphology. Messenger RNA (mRNA) expression levels of neural-specific genes, such as NEFL, NSE, and TUBB3 (βIII-tubulin) dramatically increased in the differentiated cAF-MSCs after induction. In addition, protein expression levels of nestin, βIII-tubulin, and tyrosine hydroxylase remarkably increased in differentiated cAF-MSCs. This study demonstrates that cAF-MSCs have great potential for neural precursor differentiation in vitro. Therefore, amniotic fluid may be a suitable alternative source of stem cells and can be applied to cell therapy in neurodegenerative diseases.

Two outward potassium current types are expressed during the neural differentiation of neural stem cells.

The electrophysiological properties of potassium ion channels are regarded as a basic index for determining the functional differentiation of neural stem cells. In this study, neural stem cells from the hippocampus of newborn rats were induced to differentiate with neurotrophic growth factor, and the electrophysiological properties of the voltage-gated potassium ion channels were observed. Immunofluorescence staining showed that the rapidly proliferating neural stem cells formed spheres in vitro that expressed high levels of nestin. The differentiated neurons were shown to express neuron-specific enolase. Flow cytometric analysis revealed that the neural stem cells were actively dividing and the percentage of cells in the S + G2/M phase was high. However, the ratio of cells in the S + G2/M phase decreased obviously as differentiation proceeded. Whole-cell patch-clamp recordings revealed apparent changes in potassium ion currents as the neurons differentiated. The potassium ion currents consisted of one transient outward potassium ion current and one delayed rectifier potassium ion current, which were blocked by 4-aminopyridine and tetraethylammonium, respectively. The experimental findings indicate that neural stem cells from newborn rat campus could be cultured and induced to differentiate into functional neurons under defined conditions in vitro. The differentiated neurons expressed two types of outward potassium ion currents similar to those of mature neurons in vivo.

Evaluation of mRNA expression levels and electrophysiological function of neuron-like cells derived from canine bone marrow stromal cells

To investigate the in vitro differentiation of canine bone marrow stromal cells (BMSCs) into functional, mature neurons. Bone marrow from 6 adult dogs. BMSCs were isolated from bone marrow and chemically induced to develop into neurons. The morphology of the BMSCs during neuronal induction was monitored, and immunocytochemical analyses for neuron markers were performed after the induction. Real-time PCR methods were used to evaluate the mRNA expression levels of markers for neural stem or progenitor cells, neurons, and ion channels, and western blotting was used to assess the expression of neuronal proteins before and after neuronal induction. The electrophysiological properties of the neuron-like cells induced from canine BMSCs were evaluated with fluorescent dye to monitor Ca(2)+ influx. Canine BMSCs developed a neuron-like morphology after neuronal induction. Immunocytochemical analysis revealed that these neuron-like cells were positive for neuron markers. After induction, the cells' mRNA expression levels of almost all neuron and ion channel markers increased, and the protein expression levels of nestin and neurofilament-L increased significantly. However, the neuron-like cells derived from canine BMSCs did not have the Ca(2)+ influx characteristic of spiking neurons. Although canine BMSCs had neuron-like morphological and biochemical properties after induction, they did not develop the electrophysiological characteristics of neurons. Thus, these results have suggested that canine BMSCs could have the capacity to differentiate into a neuronal lineage, but the differentiation protocol used may have been insufficient to induce development into functional neurons.

The expression of podocyte-specific proteins in parietal epithelial cells is regulated by protein degradation

The role of parietal epithelial cells (PECs) in glomerular disease is unclear because they also express podocyte proteins under pathophysiological conditions. To help resolve this, we established a novel PEC isolation technique in rats and mice to investigate which regulatory mechanisms lead to podocyte protein expression in PECs. This pure pool of naive PECs was then compared with PECs in primary culture and immortalized PECs in permanent culture. The naive PECs expressed low levels of podocyte-specific mRNA. Accordingly, in crescentic glomerulonephritis, single PECs activated the podocin promoter in vivo. In primary culture, PECs expressed a distinct morphology from podocytes but with high transcript and protein levels of PEC markers. In contrast to naive PECs, cultured PECs also expressed podocyte proteins, and this correlated with reduced proteolytic activity but not with increased transcript levels. Activation of autophagy or proteasomal degradation decreased the levels of podocyte proteins in PECs, whereas inhibition of proteasomal degradation led to the stabilization of podocyte proteins in PECs. Thus, naive PECs express podocyte transcripts physiologically and these podocyte proteins are stable under pathological conditions through decreased proteolysis.

Nrf2 is required to maintain the self-renewal of glioma stem cells

BackgroundGlioblastomas are deadly cancers that display a functional cellular hierarchy maintained by self-renewing glioma stem cells (GSCs). Self-renewal is a complex biological process necessary for maintaining the glioma stem cells. Nuclear factor rythroid 2-related factor 2(Nrf2) plays a significant role in protecting cells from endogenous and exogenous stresses. Nrf2 is a key nuclear transcription factor that regulates antioxidant response element (ARE)-containing genes. Previous studies have demonstrated the significant role of Nrf2 in the proliferation of glioblastoma, and in their resistance to radioactive therapies. We examined the effect of knocking down Nrf2 in GSCs.MethodsNrf2 expression was down-regulated by shRNA transinfected with lentivirus. Expression levels of Nestin, Nrf2, BMI-1, Sox2 and Cyclin E were assessed by western blotting, quantitative polymerase chain reaction (qPCR) and immunohistochemistry analysis. The capacity for self-renewal in vitro was assessed by genesis of colonies. The capacity for self-renewal in vivo was analyzed by tumor genesis of xenografts in nude mice.ResultsKnockdown of Nrf2 inhibited the proliferation of GSCs, and significantly reduced the expression of BMI-1, Sox2 and CyclinE. Knocking down of Nrf2 changed the cell cycle distribution of GSCs by causing an uncharacteristic increase in the proportion of cells in the G2 phase and a decrease in the proportion of cells in the S phase of the cell cycle.ConclusionsNrf2 is required to maintain the self-renewal of GSCs, and its down-regulation can attenuate the self-renewal of GSCs significantly.

Neural differentiation of human embryonic stem cells as an in vitro tool for the study of the expression patterns of the neuronal cytoskeleton during neurogenesis

The neural differentiation of human embryonic stem cells (ESCs) is a potential tool for elucidating the key mechanisms involved in human neurogenesis. Nestin and β-III-tubulin, which are cytoskeleton proteins, are marker proteins of neural stem cells (NSCs) and neurons, respectively. However, the expression patterns of nestin and β-III-tubulin in neural derivatives from human ESCs remain unclear. In this study, we found that neural progenitor cells (NPCs) derived from H9 cells express high levels of nestin and musashi-1. In contrast, β-III-tubulin was weakly expressed in a few NPCs. Moreover, in these cells, nestin formed filament networks, whereas β-III-tubulin was distributed randomly as small particles. As the differentiation proceeded, the nestin filament networks and the β-III-tubulin particles were found in both the cell soma and the cellular processes. Moreover, the colocalization of nestin and β-III-tubulin was found mainly in the cell processes and neurite-like structures and not in the cell soma. These results may aid our understanding of the expression patterns of nestin and β-III-tubulin during the neural differentiation of H9 cells.

Melatonin treatment increases the transcription of cell proliferation-related genes prior to inducing cell death in C6 glioma cells in vitro

A number of studies have suggested that melatonin possesses anticancer properties. However, conflicting data exists with regard to the role of melatonin in the treatment of cancer. In the present study, the effects of melatonin on the transcriptional regulation of three genes associated with cell proliferation (Nestin, Bmi-1 and Sox2), and on C6 glioma cell survival and viability, were investigated in vitro to evaluate the use of melatonin in cancer therapy. Melatonin was shown to increase the mRNA levels of Nestin, Bmi-1 and Sox2 in a similar pattern, with the highest mRNA levels noted at a concentration of 3 mM. At higher concentrations of melatonin (5 mM), the mRNA levels of Nestin, Bmi-1 and Sox2 were reduced from their peak levels, and were correlated with changes observed in immunofluorescence morphology studies, cell viability and survival assays. Immunofluorescence studies of Nestin-stained cells demonstrated that treatment with a higher concentration of melatonin (3 and 5 mM) led to the Nestin filaments condensing and rearranging around the cell nuclei, and an alteration in the cell morphology. C6 cell viability was also significantly decreased at 3 mM melatonin, and cell death was observed at 5 and 10 mM melatonin. These results suggested that Nestin, Bmi-1 and Sox2 were strongly correlated with the survival of C6 cells following treatment with melatonin, and that high therapeutic concentrations of melatonin (>5 mM) were required to induce cell death. These findings suggested that the implementation of melatonin in the treatment of glioma and other types of cancer may be inhibited by conflicting cell growth signals in cells. Therefore, adjunct therapy is required to improve the efficacy of melatonin in the treatment of cancer.

Changes of the Enteric Nervous System in Amyloid-β Protein Precursor Transgenic Mice Correlate with Disease Progression

In Alzheimer's disease (AD), fatal neuronal cell loss occurs long before relevant evidence can lead to a reliable diagnosis. If characteristic pathological alterations take place in the enteric nervous system (ENS), it could be one of the most promising targets for an early diagnosis, using submucosal biopsies from the gut. We therefore investigated time- and spatial-dependent changes in an amyloid-β protein precursor (AβPP) overexpressing transgenic mouse model to examine early changes within the ENS. Wholemount preparations and paraffin sections were analyzed for the expression of neuronal, glial, and innate immunity markers. Isolated myenteric networks were screened for differences in overall protein expression, and a motility analysis delivered functional data. The level of AβPP in the gut was significantly higher in the AD mouse model than in wild-type mice and also higher in the gut than in the brain at all ages investigated. The transcriptional level of Nestin, GFAP, and TLR4 increased with age with a peak at 3 months. At the protein level, human amyloid-β was located in myenteric neurons. Myenteric networks showed a reduction of the neuronal density in AβPP compared to wild-type mice, which was functionally relevant as revealed by motility analysis. The ENS undergoes significant changes during the early onset of AβPP expression in AD mouse models that appear before those seen in the brain as demonstrated in this study. Thus, there is a chance of determining similar alterations in the human gut of AD patients, which could be used to develop early diagnostic approaches.

The Expression of Intermediate Filament Protein Nestin and Its Association With Cyclin-dependent Kinase 5 in the Glomeruli of Rats With Diabetic Nephropathy

BackgroundPodocyte injury plays a crucial role in the development of diabetic nephropathy (DN), but its underlying mechanism remains poorly understood. Emerging evidences suggest that the cytoskeleton disruption is related to podocyte injury. The aim of this study was to investigate whether nestin, a cytoskeleton-associated intermediate filament protein, is involved in the development of DN. MethodsRat diabetes was induced by intraperitoneal injection of streptozotocin. The renal histological changes were investigated by light microscopy and transmission electron microscopy. The location of nestin and vimentin in renal tissues was observed by immunohistochemistry. The protein or messenger RNA levels of nestin and cyclin-dependent kinase 5 (Cdk5) were detected by Western blot and real-time polymerase chain reaction. The relationship between nestin and vimentin was detected by co-immunoprecipitation. ResultsCompared with controls, diabetic rats showed significant characteristics of renal damage. The expression of nestin and vimentin in the glomeruli was increased at the early stage of diabetes, which then gradually decreased. Co-immunoprecipitation assays demonstrated that nestin disassembled with vimentin in diabetic rats. The expression of Cdk5 was increased in a time-dependent manner in diabetic rats. The degree of albuminuria in diabetic rats was negatively correlated with nestin and positively correlated with Cdk5. Roscovitine, a Cdk5 inhibitor, reduced the degradation of nestin. Moreover, podocyte injuries were significantly ameliorated by treatment with roscovitine. ConclusionsThe intermediate filament protein nestin is associated with development of DN. Blockage of Cdk5 increases the level of nestin and attenuates renal damage, which would provide a useful target for DN therapy.

Involvement of intermediate filament nestin in cell growth of small-cell lung cancer

BackgroundNestin is a class VI intermediate filament protein expressed in stem/progenitor cells during the development of the central nervous system. Nestin is detected in various types of tumors and is involved in malignant processes. This study investigated the expression and function of nestin in small-cell lung cancer (SCLC). MethodsExpression of nestin and achaete-scute homolog 1 (ASH1) was studied in 21 lung cancer cell lines. To assess the function of nestin, a short hairpin RNA (shRNA) targeting nestin was transfected into two SCLC cell lines (DMS53 and SBC3), and cloned cells that showed apparent down-regulation of nestin were obtained. Nestin expression was also studied immunohistochemically in surgically resected SCLC primary tumors and metastatic SCLC tumors obtained from autopsy cases. ResultNestin was expressed in nine of 10 SCLC cell lines. The nestin expression level was significantly higher in SCLC cell lines than in NSCLC cell lines (P<0.01). There was a statistically significant positive correlation between the expression levels of nestin and ASH1 in SCLC cell lines. Nestin knock-down cells created by transfection with shRNA exhibited decreased invasion and cell proliferation capabilities. Furthermore, nestin was detected in SCLC tumor cells and tumor vessels in all clinical tumor specimens. ConclusionNestin is expressed in SCLC in association with neuroendocrine features and participates in malignant phenotypes, including cell growth. Therefore, nestin may be a novel therapeutic target for SCLC.

Abstract 3750: Inhibition of phosphorylation of nestin decrease pancreatic cancer cell growth.

Abstract Background: Nestin is a class VI intermediate filament, first described as a central nervous system (CNS) progenitor/stem cell marker and recently reported as a tumor stem cell marker in several malignant tumors, including glioblastoma and melanoma. In the normal pancreas, nestin is expressed in exocrine progenitor cells. Previously, we have reported that 30% of pancreatic cancer cases expressed nestin in cancer cells, and its expression correlated with nerve invasion and the presence of cancer cells in the tumor resection margins (Kawamoto, et al. Hum Pathol 2009). Knockdown of nestin exhibited a sheet-like appearance with tight cell-cell adhesion, increased expression of filamentous-actin and E-cadherin, and attenuated migration and invasion, both of which recovered following nestin re-expression (Matsuda, et al. Cancer Biol Ther 2011). Furthermore, knockdown of nestin suppressed sphere-forming ability, and altered the expression levels of stemness-related genes such as NANOG and transcription factor 4 (Matsuda, et al. AACR 2012). Therefore, nestin is considered to be a novel therapeutic target for pancreatic cancer via suppression of cell migration, invasion, metastasis and stemness. To develop nestin-targeting therapy, we focused on the phosphorylation of nestin protein. Phosphorylation of nestin at two different sites of progenitor cells of the CNS and myocytes has been reported to regulate the cell cycle via activation of cyclin-dependent kinase 5 and cdc2 kinase (Sahlgren, et al. Mol Cell Biol 2003). In this study, we analyzed the expression and roles of nestin phosphorylation in pancreatic cancer cells. Methods: Expression levels of phosphorylated nestin in pancreatic cancer cells were determined by flow cytometer, fluorescent staining and Western blot analyses. To assess the functions of phosphorylated nestin, we prepared three mutated nestin expression vectors which possess mutations at two different phosphorylated sites, both in nestin. A human pancreatic cancer cell line, MIA PaCa-2, which express very low levels of nestin was transfected with the mutated or wild-type nestin-expression vector. Results: Expression level of phosphorylated nestin was higher in M-phase cells than S- or G0/G1-phase cells. Phosphorylated nestin was strongly and diffusely localized in M-phase cells, while it was weakly detected in the nuclei of cells in other phases. Mutated nestin in phosphorylated site-transfected cells showed lower levels of cell growth, migration and invasion than wild-type nestin-transfected cells. Furthermore, mutated nestin-transfected cells had a tendency towards increasesed multinuclear cells. Conclusion: Phosphorylation of nestin affects the cell growth, migration and invasion of pancreatic cancer cells, and phosphorylated nestin may play important roles in the regulation of the cell cycle. Inhibition of phosphorylated nestin may be a novel target for pancreatic cancer treatment. Citation Format: Yoko Matsuda, Hisashi Yoshimura, Taeko Suzuki, Zenya Naito, Kiyoko Kawahara, Yuji Yanagisawa, Yoko Kawamoto, Toshiyuki Ishiwata. Inhibition of phosphorylation of nestin decrease pancreatic cancer cell growth. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3750. doi:10.1158/1538-7445.AM2013-3750

Abstract 4360: Involvement of intermediate filament nestin in cell growth of small-cell lung cancer.

Abstract Small-cell lung cancer (SCLC) represents 13% of all newly diagnosed cases of lung cancer. Clinical progression of SCLC is rapid and aggressive. Unfortunately, 70% of SCLC cases have already metastasized at the time of diagnosis, and the recent median survival time and two-year survival rate in SCLC patients with extensive-stage disease is only 8-13 months and 5%, respectively. Clinical trials of SCLC treatments have been conducted since the mid-1980s, but have not achieved prolonged survival; this lack of success is likely due to a lack of appropriate target molecules. To improve outcomes for SCLC patients, new therapeutic strategies including novel molecular targets for SCLC are desired. Nestin is a class VI intermediate filament protein expressed in stem/progenitor cells during the development of the central nervous system. Nestin is detected in various types of tumors and reported to be involved in malignant phenotypes. Here, we investigated the expression and function of nestin in SCLC. At the beginning, protein expression of nestin and achaete-scute homolog 1 (ASH1) was studied in 21 lung cancer cell lines. Nestin was expressed in 9 of 10 SCLC cell lines. The nestin expression level in SCLC cell lines was significantly higher than that in non-small-cell lung cancer (NSCLC) cell lines (P&amp;lt;0.01). There was a statistically significant positive correlation between the expression levels of nestin and ASH1 in SCLC cell lines, but not in NSCLC cell lines. To assess the function of nestin, we transfected a short hairpin RNA (shRNA) targeting nestin into two SCLC cell lines, DMS53 and SBC3, and then obtained cloned cells that showed apparent down-regulation of nestin. Nestin knock-down cells created by transfection with shRNA exhibited decreased invasion and cell proliferation capabilities. In conclusion, Nestin is expressed in SCLC in association with neuro-endocrine feature and participates in malignant phenotypes including cell growth. Therefore, nestin may be a novel therapeutic target of SCLC. Additional in vivo study will be needed. Citation Format: Eiji Kunii, Osamu Takakuwa, Ken Maeno, Tetsuya Oguri, Midori Yokoyama, Hisatoshi Hijikata, Takehiro Uemura, Daishi Kasai, Hirotsugu Ohkubo, Mikinori Miyazaki, Akio Niimi. Involvement of intermediate filament nestin in cell growth of small-cell lung cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4360. doi:10.1158/1538-7445.AM2013-4360

Abstract 257: In vitro tumorigenicity and stemness characterization of the U87MG glioblastoma cell line: Monolayer cultures, neurospheres, and CD133+ and CD133- sorted fractions.

Abstract The cancer stem cells (CSC) hypothesis is nowadays the most widely accepted theory regarding tumor formation and self-renewal capacity. It states that one of the different tumorigenic phenotypes inside a tumor mass is capable of generating new tumors if transplanted onto a host and it is able to self-generate and regenerate the rest of the tumor cells. The necessity to find pharmacological compounds or biological agents capable of eliminating CSC makes the search for methods of recognition and isolation of these cells a matter of great urgency. Our working hypothesis is that the selection of cancer cells with the CD133 marker and their subsequent isolation and characterization will allow us to assess the capacity of the different CD133 subpopulations as CSC. The aim of the study was to separate CD133+ and CD133- cell subpopulations from the U87MG glioblastoma cell line by magnetic activated cell sorting (MACS), and to analyze the tumorigenic and stem cell differences among four sets of cells: the cell line grown in serum (monolayer group), the neurospheres of the same cell line, and the CD133+ and CD133- sorted cell fractions from the neurospheres. Our results showed that the neurospheres and the CD133+ cells overexpressed stem cell marker genes like SOX2, Nestin, Musashi1 and CD133, and formed more colonies in soft agar than the cell groups with less CD133 expression (monolayer and CD133- group). On the other hand the CD133- and the monolayer groups had similar expression levels of CD133, while the CD133- cells expressed higher levels of Nestin, SOX2 and Musashi1 than the monolayer cells. Besides, cell migration assessed by the scratching test showed that the CD133- and the monolayer cells migrated more than the neurospheres and the CD133+ cells. Moreover, the CD133- cells had a higher proliferation rate than the CD133+ cells and the neurospheres, being all these three groups cultured in the medium for neurospheres. In conclusion, we might point to the presence of CSC in the CD133- group, as previously suggested by others. Then, CD133+ cells are not necessarily equivalent to CSC. Maybe CD133+ cells present a higher probability of including CSC than CD133- do. Therefore, further functional and genetic analyses must be performed to reach an optimal isolation of CSC. Citation Format: A Vacas-Oleas, J de la Rosa, R Garcia-Lopez, B Vera-Cano, G Gallo-Oller, M Alfaro, M H. Shahi, X Fan, I J. Encio, J A. Rey, Javier S. Castresana. In vitro tumorigenicity and stemness characterization of the U87MG glioblastoma cell line: Monolayer cultures, neurospheres, and CD133+ and CD133- sorted fractions. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 257. doi:10.1158/1538-7445.AM2013-257

Alteration of Podocyte Protein Expression and Localization in the Early Stage of Various Hemodynamic Conditions

Given that podocalyxin (PCX) and nestin play important roles in podocyte morphogenesis and the maintenance of structural integrity, we examined whether the expression and localization of these two podocyte proteins were influenced in the early stage of various hemodynamic conditions. Mice kidney tissues were prepared by in vivo cryotechnique (IVCT). The distribution of glomeruli and podocyte proteins was visualized with DAB staining, confocal laser scanning microscopy and immunoelectron microscopy. The mRNA levels were examined by real-time quantitative PCR. The results showed the following: Under the normal condition, PCX stained intensely along glomerular epithelial cells, whereas nestin was clearly staining in the endothelial cells and appeared only weakly in the podocytes. Under the acute hypertensive and cardiac arrest conditions, PCX and nestin staining was not clear, with a disarranged distribution, but the colocalization of PCX and nestin was apparent under this condition. In addition, under the acute hypertensive and cardiac arrest conditions, the mRNA levels of PCX and nestin were significantly decreased. Collectively, the abnormal redistribution and decreased mRNA expressions of PCX and nestin are important molecular events at the early stage of podocyte injury during hemodynamic disorders. IVCT may have more advantages for morphological analysis when researching renal diseases.

Nestin is highly expressed in advanced-stage melanomas and neurotized nevi

Nestin, a class VI intermediate filament protein, was originally described as a neural stem cell/progenitor cell marker. Expression of nestin has been reported to be associated with the migration and metastasis of various types of tumor. In the present study, we examined the expression of nestin in malignant melanomas and nevi. Immunohistochemically, nestin was detected in all compound nevi, but not in the majority of junctional nevi. Nestin was expressed at particularly high levels in nevi with neurotization. In melanoma, nestin was expressed in all T3 and T4 cases, but only in half of the cases of T2 or less severe disease. These results indicate that the expression levels of nestin in melanoma are associated with advanced disease. In conclusion, nestin was expressed in advanced melanoma tissues and neurotized nevi. Nestin may be an important marker of melanocytic neoplasms. Further studies are required to elucidate the regulatory mechanisms of nestin expression and to examine the possibility of nestin-targeted therapy for malignant melanomas.