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Sample records for adrenocortical cell lines

  1. Human Adrenocortical Carcinoma Cell Lines

    PubMed Central

    Wang, Tao; Rainey, William E.

    2011-01-01

    Summary The human adrenal cortex secretes mineralocorticoids, glucocorticoids and adrenal androgens. These steroids are produced from unique cell types located within the three distinct zones of the adrenal cortex. Disruption of adrenal steroid production results in a variety of diseases that can lead to hypertension, metabolic syndrome, infertility and androgen excess. The adrenal cortex is also a common site for the development of adenomas, and rarely the site for the development of carcinomas. The adenomas can lead to diseases associated with adrenal steroid excess, while the carcinomas are particularly aggressive and have a poor prognosis. In vitro cell culture models provide an important tool to examine molecular and cellular mechanisms controlling both the normal and pathologic function of the adrenal cortex. Herein we discuss the human adrenocortical cell lines and their use as model systems for adrenal studies. PMID:21924324

  2. Effects of Type 1 Insulin-Like Growth Factor Receptor Silencing in a Human Adrenocortical Cell Line.

    PubMed

    Ribeiro, T C; Jorge, A A; Montenegro, L R; Almeida, M Q; Ferraz-de-Souza, B; Nishi, M Y; Mendonca, B B; Latronico, A C

    2016-07-01

    Type 1 insulin-like growth factor receptor (IGF-1R) is overexpressed in a variety of human cancers, including adrenocortical tumors. The aim of the work was to investigate the effects of IGF-1R downregulation in a human adrenocortical cell line by small interfering RNA (siRNA). The human adrenocortical tumor cell line NCI H295R was transfected with 2 specific IGF1R siRNAs (# 1 and # 2) and compared with untreated cells and a negative control siRNA. IGF1R expression was determined by quantitative reverse-transcription PCR (qRTPCR) and Western blot. The effects of IGF-1R downregulation on cell proliferation and apoptosis were assessed. IGF-1R levels were significantly decreased in cells treated with IGF-1R siRNA # 1 or # 2. Relative expression of IGF1R mRNA decreased approximately 50% and Western blot analysis revealed a 30% of reduction in IGF-1R protein. Downregulation of this gene resulted in 40% reduction in cell growth in vitro and 45% increase in apoptosis using siRNA # 2. These findings demonstrate that decreasing IGF-1R mRNA and protein expression in NCI H295R cells can partially inhibit adrenal tumor cell growth in vitro. Targeting IGF1R is a promising therapy for pediatric malignant adrenocortical tumor and can still be an option for adult adrenocortical cancer based on personalized genomic tumor profiling. PMID:27246621

  3. Expression of adiponectin receptors in mouse adrenal glands and the adrenocortical Y-1 cell line: adiponectin regulates steroidogenesis.

    PubMed

    Li, Ping; Sun, Fei; Cao, Huang-Ming; Ma, Qin-Yun; Pan, Chun-Ming; Ma, Jun-Hua; Zhang, Xiao-Na; Jiang, He; Song, Huai-Dong; Chen, Ming-Dao

    2009-12-25

    Obesity is frequently associated with malfunctions of the hypothalamus-pituitary-adrenal (HPA) axis and hyperaldosteronism, but the mechanism underlying this association remains unclear. Since the adrenal glands are embedded in adipose tissue, direct cross-talk between adipose tissue and the adrenal gland has been proposed. A previous study found that adiponectin receptor mRNA was expressed in human adrenal glands and aldosterone-producing adenoma (APA). However, the expression of adiponectin receptors in adrenal glands has not been confirmed at the protein level or in other species. Furthermore, it is unclear whether adiponectin receptors expressed in adrenal cells are functional. We found, for the first time, that adiponectin receptor (AdipoR1 and AdipoR2) mRNA and protein were expressed in mouse adrenal and adrenocortical Y-1 cells. However, adiponectin itself was not expressed in mouse adrenal or Y-1 cells. Furthermore, adiponectin acutely reduced basal levels of corticosterone and aldosterone secretion. ACTH-induced steroid secretion was also inhibited by adiponectin, and this was accompanied by a parallel change in the expression of the key genes involved in steroidogenesis. These findings indicate that adiponectin may take part in the modulation of steroidogenesis. Thus, adiponectin is likely to have physiological and/or pathophysiological significance as an endocrine regulator of adrenocortical function.

  4. mTOR pathway is activated by PKA in adrenocortical cells and participates in vivo to apoptosis resistance in primary pigmented nodular adrenocortical disease (PPNAD).

    PubMed

    de Joussineau, Cyrille; Sahut-Barnola, Isabelle; Tissier, Frédérique; Dumontet, Typhanie; Drelon, Coralie; Batisse-Lignier, Marie; Tauveron, Igor; Pointud, Jean-Christophe; Lefrançois-Martinez, Anne-Marie; Stratakis, Constantine A; Bertherat, Jérôme; Val, Pierre; Martinez, Antoine

    2014-10-15

    Primary pigmented nodular adrenocortical disease (PPNAD) is associated with inactivating mutations of the PRKAR1A tumor suppressor gene that encodes the regulatory subunit R1α of the cAMP-dependent protein kinase (PKA). In human and mouse adrenocortical cells, these mutations lead to increased PKA activity, which results in increased resistance to apoptosis that contributes to the tumorigenic process. We used in vitro and in vivo models to investigate the possibility of a crosstalk between PKA and mammalian target of rapamycin (mTOR) pathways in adrenocortical cells and its possible involvement in apoptosis resistance. Impact of PKA signaling on activation of the mTOR pathway and apoptosis was measured in a mouse model of PPNAD (AdKO mice), in human and mouse adrenocortical cell lines in response to pharmacological inhibitors and in PPNAD tissues by immunohistochemistry. AdKO mice showed increased mTOR complex 1 (mTORC1) pathway activity. Inhibition of mTORC1 by rapamycin restored sensitivity of adrenocortical cells to apoptosis in AdKO but not in wild-type mice. In both cell lines and mouse adrenals, rapid phosphorylation of mTORC1 targets including BAD proapoptotic protein was observed in response to PKA activation. Accordingly, BAD hyperphosphorylation, which inhibits its proapoptotic activity, was increased in both AdKO mouse adrenals and human PPNAD tissues. In conclusion, mTORC1 pathway is activated by PKA signaling in human and mouse adrenocortical cells, leading to increased cell survival, which is correlated with BAD hyperphosphorylation. These alterations could be causative of tumor formation. PMID:24865460

  5. In search of adrenocortical stem and progenitor cells.

    PubMed

    Kim, Alex C; Barlaskar, Ferdous M; Heaton, Joanne H; Else, Tobias; Kelly, Victoria R; Krill, Kenneth T; Scheys, Joshua O; Simon, Derek P; Trovato, Alessia; Yang, Wei-Hsiung; Hammer, Gary D

    2009-05-01

    Scientists have long hypothesized the existence of tissue-specific (somatic) stem cells and have searched for their location in different organs. The theory that adrenocortical organ homeostasis is maintained by undifferentiated stem or progenitor cells can be traced back nearly a century. Similar to other organ systems, it is widely believed that these rare cells of the adrenal cortex remain relatively undifferentiated and quiescent until needed to replenish the organ, at which time they undergo proliferation and terminal differentiation. Historical studies examining cell cycle activation by label retention assays and regenerative potential by organ transplantation experiments suggested that the adrenocortical progenitors reside in the outer periphery of the adrenal gland. Over the past decade, the Hammer laboratory, building on this hypothesis and these observations, has endeavored to understand the mechanisms of adrenocortical development and organ maintenance. In this review, we summarize the current knowledge of adrenal organogenesis. We present evidence for the existence and location of adrenocortical stem/progenitor cells and their potential contribution to adrenocortical carcinomas. Data described herein come primarily from studies conducted in the Hammer laboratory with incorporation of important related studies from other investigators. Together, the work provides a framework for the emerging somatic stem cell field as it relates to the adrenal gland.

  6. Adrenocortical endocrine disruption.

    PubMed

    Harvey, Philip W

    2016-01-01

    in vivo ACTH challenge test to prove adrenocortical competency, and the H295R cell line to examine molecular mechanisms of steroidogenic pathway toxicity, are discussed. Finally, because of the central role of the adrenal in the physiologically adaptive stress response, the distinguishing features of stress, compared with adrenocortical toxicity, are discussed with reference to the evidence required to claim that adrenal hypertrophy results from stress rather than adrenocortical enzyme inhibition which is a serious adverse toxicological finding. This article is part of a special issue entitled 'Endocrine disruptors and steroids'.

  7. Acute self-suppression of corticosteroidogenesis in isolated adrenocortical cells.

    PubMed

    Carsia, R V; Malamed, S

    1979-10-01

    The relation between steroidogenesis induced by ACTH and that induced by exogenous concentrations of glucocorticoids was studied in isolated adrenocortical cells. Exogenous corticosterone and cortisol, in concentrations within the production capacity of the adrenal gland, suppressed steroidogenesis induced by ACTH in rat and beef cells, respectively. The precursors pregnenolone and progesterone enhanced steroidogenesis in both rat and beef cells. Aldosterone in rat cells and 17 beta-estradiol in rat and beef cells had little if any effect on steroidogenesis. Either suppression or stimulation by exogenous steroids was acute, that is, after 2-h incubation for rat cells and 1-h incubation for beef cells. A direct suppressive action of end product glucocorticoids is indicated. This observed self-suppression of adrenocortical cells suggests the existence of a mechanism for the find adjustment of steroidogenesis that operates in addition to the classical control exerted by the anterior pituitary.

  8. Effects of PCBs and MeSO2-PCBs on adrenocortical steroidogenesis in H295R human adrenocortical carcinoma cells.

    PubMed

    Xu, Yan; Yu, Richard M K; Zhang, Xiaowei; Murphy, Margaret B; Giesy, John P; Lam, Michael H W; Lam, Paul K S; Wu, Rudolf S S; Yu, Hongxia

    2006-05-01

    Some endocrine disrupting chemicals (EDCs) in the environment have been shown to exert their biological effects through interference with steroidogenesis. In this study, the potential effects of four selected polychlorinated biphenyl (PCB) congeners (PCB101, PCB110, PCB126 and PCB149) as well as several of their environmentally-relevant methylsulfonyl-(MeSO(2)-) PCB metabolites (3'-MeSO(2)-CB101, 4'-MeSO(2)-CB101, 4'-MeSO(2)-CB110, 3'-MeSO(2)-CB149 and 4'-MeSO(2)-CB149) on adrenocortical steroidogenesis were evaluated by in vitro bioassay based on the human adrenocortical carcinoma H295R cell line. The PCBs included in the study represented different structures and potential mechanisms of action. Cells were exposed for 48 h to 10 microM of each PCB congener in the presence or absence of 20% (w/w) of their corresponding MeSO(2)-PCB metabolite(s). After the chemical treatments, changes in mRNA expression of 11 steroidogenic genes (CYP11A, CYP11B1, CYP11B2, CYP17, CYP19, CYP21, 3beta-HSD1, 3beta-HSD2, 17beta-HSD1, StAR and HMGR) were quantified using molecular beacon-based real-time RT-PCR. Genes coding for enzymes involved in the later or final steps of steroid production (CYP11B1, CYP11B2, CYP19, 3beta-HSD1, 3beta-HSD2 and 17beta-HSD1) were up-regulated to various extents by most PCBs. The greatest transcriptional activations (2.8-29.9-fold) were elicited by PCB110 on CYP11B1, CYP11B2, 3beta-HSD2 and CYP19, and PCB149 on CYP11B1, 3beta-HSD1 and 17beta-HSD1. Increased expression of these steroidogenic genes might ultimately lead to a change in hormonal balance through excessive production of steroid hormones including aldosterone, cortisol and estradiol. In addition, co-treatment with 3'- and 4'-MeSO(2)-PCB149 resulted in a significant decrease in PCB149-induced 3beta-HSD1 and 17beta-HSD1 expression. This result indicates that some PCB congeners and their MeSO(2)-metabolites may affect steroidogenesis via different mechanisms. Overall, these findings suggest that

  9. Exposure to the three structurally different PCB congeners (PCB 118, 153, and 126) results in decreased protein expression and altered steroidogenesis in the human adrenocortical carcinoma cell line H295R.

    PubMed

    Tremoen, Nina Hårdnes; Fowler, Paul A; Ropstad, Erik; Verhaegen, Steven; Krogenæs, Anette

    2014-01-01

    Polychlorinated biphenyls (PCB), synthetic, persistent organic pollutants (POP), are detected ubiquitously, in water, soil, air, and sediments, as well as in animals and humans. PCB are associated with range of adverse health effects, such as interference with the immune system and nervous system, reproductive abnormalities, fetotoxicity, carcinogenicity, and endocrine disruption. Our objective was to determine the effects of three structurally different PCB congeners, PCB118, PCB 126, and PCB 153, each at two concentrations, on the steroidogenic capacity and proteome of human adrenocortical carcinoma cell line cultures (H295R) . After 48 h of exposure, cell viability was monitored and estradiol, testosterone, cortisol and progesterone secretion measured to quantify steroidogenic capacity of the cells. Two-dimensional (2D) gel-based proteomics was used to screen for proteome alterations in H295R cells in response to the PCB. Exposure to PCB 118 increased estradiol and cortisol secretion, while exposure to PCB 153 elevated estradiol secretion. PCB 126 was the most potent congener, increasing estradiol, cortisol, and progesterone secretion in exposed H295R cells. Seventy-three of the 711 spots analyzed showed a significant difference in normalized spot volumes between controls (vehicle only) and at least one exposure group. Fourteen of these protein spots were identified by liquid chromatography with mass spectroscopy (LC-MS/MS). Exposure to three PCB congeners with different chemical structure perturbed steroidogenesis and protein expression in the H295R in vitro model. This study represents an initial analysis of the effects on proteins and hormones in the H295R cell model, and additional studies are required in order to obtain a more complete understanding of the pathways disturbed by PCB congeners in H295R cells. Overall, alterations in protein regulation and steroid hormone synthesis suggest that exposure to PCB disturbs several cellular processes, including

  10. Global gene expression response to telomerase in bovine adrenocortical cells

    SciTech Connect

    Perrault, Steven D.; Hornsby, Peter J.; Betts, Dean H. . E-mail: bettsd@uoguelph.ca

    2005-09-30

    The infinite proliferative capability of most immortalized cells is dependent upon the presence of the enzyme telomerase and its ability to maintain telomere length and structure. However, telomerase may be involved in a greater system than telomere length regulation, as recent evidence has shown it capable of increasing wound healing in vivo, and improving cellular proliferation rate and survival from apoptosis in vitro. Here, we describe the global gene expression response to ectopic telomerase expression in an in vitro bovine adrenocortical cell model. Telomerase-immortalized cells showed an increased ability for proliferation and survival in minimal essential medium above cells transgenic for GFP. cDNA microarray analyses revealed an altered cell state indicative of increased adrenocortical cell proliferation regulated by the IGF2 pathway and alterations in members of the TGF-B family. As well, we identified alterations in genes associated with development and wound healing that support a model that high telomerase expression induces a highly adaptable, progenitor-like state.

  11. Cell cycle dependent RRM2 may serve as proliferation marker and pharmaceutical target in adrenocortical cancer

    PubMed Central

    Grolmusz, Vince Kornél; Karászi, Katalin; Micsik, Tamás; Tóth, Eszter Angéla; Mészáros, Katalin; Karvaly, Gellért; Barna, Gábor; Szabó, Péter Márton; Baghy, Kornélia; Matkó, János; Kovalszky, Ilona; Tóth, Miklós; Rácz, Károly; Igaz, Péter; Patócs, Attila

    2016-01-01

    Adrenocortical cancer (ACC) is a rare, but agressive malignancy with poor prognosis. Histopathological diagnosis is challenging and pharmacological options for treatment are limited. By the comparative reanalysis of the transcriptional malignancy signature with the cell cycle dependent transcriptional program of ACC, we aimed to identify novel biomarkers which may be used in the histopathological diagnosis and for the prediction of therapeutical response of ACC. Comparative reanalysis of publicly available microarray datasets included three earlier studies comparing transcriptional differences between ACC and benign adrenocortical adenoma (ACA) and one study presenting the cell cycle dependent gene expressional program of human ACC cell line NCI-H295R. Immunohistochemical analysis was performed on ACC samples. In vitro effects of antineoplastic drugs including gemcitabine, mitotane and 9-cis-retinoic acid alone and in combination were tested in the NCI-H295R adrenocortical cell line. Upon the comparative reanalysis, ribonucleotide reductase subunit 2 (RRM2), responsible for the ribonucleotide dezoxyribonucleotide conversion during the S phase of the cell cycle has been validated as cell cycle dependently expressed. Moreover, its expression was associated with the malignancy signature, as well. Immunohistochemical analysis of RRM2 revealed a strong correlation with Ki67 index in ACC. Among the antiproliferative effects of the investigated compounds, gemcitabine showed a strong inhibition of proliferation and an increase of apoptotic events. Additionally, RRM2 has been upregulated upon gemcitabine treatment. Upon our results, RRM2 might be used as a proliferation marker in ACC. RRM2 upregulation upon gemcitabine treatment might contribute to an emerging chemoresistance against gemcitabine, which is in line with its limited therapeutical efficacy in ACC, and which should be overcome for successful clinical applications. PMID:27725909

  12. Rosiglitazone induces autophagy in H295R and cell cycle deregulation in SW13 adrenocortical cancer cells

    SciTech Connect

    Cerquetti, Lidia; Sampaoli, Camilla; Amendola, Donatella; Bucci, Barbara; Masuelli, Laura; Marchese, Rodolfo; Misiti, Silvia; De Venanzi, Agostino; Poggi, Maurizio; Toscano, Vincenzo; Stigliano, Antonio

    2011-06-10

    Thiazolidinediones, specific peroxisome proliferator-activated receptor-{gamma} (PPAR-{gamma}) ligands, used in type-2 diabetes therapy, show favourable effects in several cancer cells. In this study we demonstrate that the growth of H295R and SW13 adrenocortical cancer cells is inhibited by rosiglitazone, a thiazolidinediones member, even though the mechanisms underlying this effect appeared to be cell-specific. Treatment with GW9662, a selective PPAR-{gamma}-inhibitor, showed that rosiglitazone acts through both PPAR-{gamma}-dependent and -independent mechanisms in H295R, while in SW13 cells the effect seems to be independent of PPAR-{gamma}. H295R cells treated with rosiglitazone undergo an autophagic process, leading to morphological changes detectable by electron microscopy and an increased expression of specific proteins such as AMPK{alpha} and beclin-1. The autophagy seems to be independent of PPAR-{gamma} activation and could be related to an increase in oxidative stress mediated by reactive oxygen species production with the disruption of the mitochondrial membrane potential, triggered by rosiglitazone. In SW13 cells, flow cytometry analysis showed an arrest in the G0/G1 phase of the cell cycle with a decrease of cyclin E and cdk2 activity, following the administration of rosiglitazone. Our data show the potential role of rosiglitazone in the therapeutic approach to adrenocortical carcinoma and indicate the molecular mechanisms at the base of its antiproliferative effects, which appear to be manifold and cell-specific in adrenocortical cancer lines.

  13. Transplantation of bovine adrenocortical cells encapsulated in alginate

    PubMed Central

    Balyura, Mariya; Gelfgat, Evgeny; Ehrhart-Bornstein, Monika; Ludwig, Barbara; Gendler, Zohar; Barkai, Uriel; Zimerman, Baruch; Rotem, Avi; Block, Norman L.; Schally, Andrew V.; Bornstein, Stefan R.

    2015-01-01

    Current treatment options for adrenal insufficiency are limited to corticosteroid replacement therapies. However, hormone therapy does not replicate circadian rhythms and has unpleasant side effects especially due to the failure to restore normal function of the hypothalamic–pituitary–adrenal (HPA) axis. Adrenal cell transplantation and the restoration of HPA axis function would be a feasible and useful therapeutic strategy for patients with adrenal insufficiency. We created a bioartificial adrenal with 3D cell culture conditions by encapsulation of bovine adrenocortical cells (BACs) in alginate (enBACs). We found that, compared with BACs in monolayer culture, encapsulation in alginate significantly increased the life span of BACs. Encapsulation also improved significantly both the capacity of adrenal cells for stable, long-term basal hormone release as well as the response to pituitary adrenocorticotropic hormone (ACTH) and hypothalamic luteinizing hormone-releasing hormone (LHRH) agonist, [D-Trp6]LHRH. The enBACs were transplanted into adrenalectomized, immunodeficient, and immunocompetent rats. Animals received enBACs intraperitoneally, under the kidney capsule (free cells or cells encapsulated in alginate slabs) or s.c. enclosed in oxygenating and immunoisolating βAir devices. Graft function was confirmed by the presence of cortisol in the plasma of rats. Both types of grafted encapsulated cells, explanted after 21–25 d, preserved their morphology and functional response to ACTH stimulation. In conclusion, transplantation of a bioartificial adrenal with xenogeneic cells may be a treatment option for patients with adrenocortical insufficiency and other stress-related disorders. Furthermore, this model provides a microenvironment that ensures 3D cell–cell interactions as a unique tool to investigate new insights into cell biology, differentiation, tissue organization, and homeostasis. PMID:25675525

  14. Role of intramitochondrial arachidonic acid and acyl-CoA synthetase 4 in angiotensin II-regulated aldosterone synthesis in NCI-H295R adrenocortical cell line.

    PubMed

    Mele, Pablo G; Duarte, Alejandra; Paz, Cristina; Capponi, Alessandro; Podestá, Ernesto J

    2012-07-01

    Although the role of arachidonic acid (AA) in angiotensin II (ANG II)- and potassium-stimulated steroid production in zona glomerulosa cells is well documented, the mechanism responsible for AA release is not fully described. In this study we evaluated the mechanism involved in the release of intramitochondrial AA and its role in the regulation of aldosterone synthesis by ANG II in glomerulosa cells. We show that ANG II and potassium induce the expression of acyl-coenzyme A (CoA) thioesterase 2 and acyl-CoA synthetase 4, two enzymes involved in intramitochondrial AA generation/export system well characterized in other steroidogenic systems. We demonstrate that mitochondrial ATP is required for AA generation/export system, steroid production, and steroidogenic acute regulatory protein induction. We also demonstrate the role of protein tyrosine phosphatases regulating acyl-CoA synthetase 4 and steroidogenic acute regulatory protein induction, and hence ANG II-stimulated aldosterone synthesis.

  15. Nitrophenols isolated from diesel exhaust particles regulate steroidogenic gene expression and steroid synthesis in the human H295R adrenocortical cell line

    SciTech Connect

    Furuta, Chie; Noda, Shiho; Li Chunmei; Suzuki, Akira K; Taneda, Shinji; Watanabe, Gen; Taya, Kazuyoshi

    2008-05-15

    Studies of nitrophenols isolated from diesel exhaust particles (DEPs), 3-methyl-4-nitrophenol (PNMC) and 4-nitro-3-phenylphenol (PNMPP) have revealed that these chemicals possess estrogenic and anti-androgenic activity in vitro and in vivo and that PNMC accumulate in adrenal glands in vivo. However, the impacts of exposure to these compounds on adrenal endocrine disruption and steroidogenesis have not been investigated. To elucidate the non-receptor mediated effects of PNMC and PNMPP, we investigated the production of the steroid hormones progesterone, cortisol, testosterone, and estradiol-17{beta} and modulation of nine major enzyme genes involved in the synthesis of steroid hormones (CYP11A, CYP11B1, CYP17, CYP19, 17{beta}HSD1, 17{beta}HSD4, CYP21, 3{beta}HSD2, StAR) in human adrenal H295R cells supplied with cAMP. Exposure to 10{sup -7} to 10{sup -5} M PNMC and 1 mM 8-Br-cAMP for 48 h decreased testosterone, cortisol, and estradiol-17{beta} levels and increased progesterone secretion. At 10{sup -5} M, PNMC with 1 mM 8-Br-cAMP significantly stimulated expression of the 17{beta}HSD4 and significantly suppressed expression of 3{beta}HSD2. In comparison, 10{sup -7} to 2 x 10{sup -5} M PNMPP with 1 mM 8-Br-cAMP for 48 h decreased concentrations of estradiol-17{beta}, increased progesterone levels, but did not affect testosterone and cortisol secretion due to the significant suppression of CYP17 and the non-significant but obvious suppression of CYP19. Our results clarified steroidogenic enzymes as candidates responsible for the inhibition or stimulation for the production of steroid hormones in the steroidogenic pathway, thus providing the first experimental evidence for multiple mechanisms of disruption of endocrine pathways by these nitrophenols.

  16. Different expression of protein kinase A (PKA) regulatory subunits in cortisol-secreting adrenocortical tumors: Relationship with cell proliferation

    SciTech Connect

    Mantovani, G.; Lania, A.G.; Bondioni, S.; Peverelli, E.; Pedroni, C.; Ferrero, S.; Pellegrini, C.; Vicentini, L.; Arnaldi, G.; Bosari, S.; Beck-Peccoz, P.; Spada, A.

    2008-01-01

    The four regulatory subunits (R1A, R1B, R2A, R2B) of protein kinase A (PKA) are differentially expressed in several cancer cell lines and exert distinct roles in growth control. Mutations of the R1A gene have been found in patients with Carney complex and in a minority of sporadic primary pigmented nodular adrenocortical disease (PPNAD). The aim of this study was to evaluate the expression of PKA regulatory subunits in non-PPNAD adrenocortical tumors causing ACTH-independent Cushing's syndrome and to test the impact of differential expression of these subunits on cell growth. Immunohistochemistry demonstrated a defective expression of R2B in all cortisol-secreting adenomas (n = 16) compared with the normal counterpart, while both R1A and R2A were expressed at high levels in the same tissues. Conversely, carcinomas (n = 5) showed high levels of all subunits. Sequencing of R1A and R2B genes revealed a wild type sequence in all tissues. The effect of R1/R2 ratio on proliferation was assessed in mouse adrenocortical Y-1 cells. The R2-selective cAMP analogue 8-Cl-cAMP dose-dependently inhibited Y-1 cell proliferation and induced apoptosis, while the R1-selective cAMP analogue 8-HA-cAMP stimulated cell proliferation. Finally, R2B gene silencing induced up-regulation of R1A protein, associated with an increase in cell proliferation. In conclusion, we propose that a high R1/R2 ratio favors the proliferation of well differentiated and hormone producing adrenocortical cells, while unbalanced expression of these subunits is not required for malignant transformation.

  17. IGF2 Promotes Growth of Adrenocortical Carcinoma Cells, but Its Overexpression Does Not Modify Phenotypic and Molecular Features of Adrenocortical Carcinoma

    PubMed Central

    Guillaud-Bataille, Marine; Ragazzon, Bruno; de Reyniès, Aurélien; Chevalier, Claire; Francillard, Isabelle; Barreau, Olivia; Steunou, Virginie; Guillemot, Johann; Tissier, Frédérique; Rizk-Rabin, Marthe; René-Corail, Fernande; Ghuzlan, Abir Al; Assié, Guillaume; Bertagna, Xavier; Baudin, Eric; Le Bouc, Yves; Bertherat, Jérôme; Clauser, Eric

    2014-01-01

    Insulin-like growth factor 2 (IGF2) overexpression is an important molecular marker of adrenocortical carcinoma (ACC), which is a rare but devastating endocrine cancer. It is not clear whether IGF2 overexpression modifies the biology and growth of this cancer, thus more studies are required before IGF2 can be considered as a major therapeutic target. We compared the phenotypical, clinical, biological, and molecular characteristics of ACC with or without the overexpression of IGF2, to address these issues. We also carried out a similar analysis in an ACC cell line (H295R) in which IGF2 expression was knocked down with si- or shRNA. We found no significant differences in the clinical, biological and molecular (transcriptomic) traits between IGF2-high and IGF2-low ACC. The absence of IGF2 overexpression had little influence on the activation of tyrosine kinase pathways both in tumors and in H295 cells that express low levels of IGF2. In IGF2-low tumors, other growth factors (FGF9, PDGFA) are more expressed than in IGF2-high tumors, suggesting that they play a compensatory role in tumor progression. In addition, IGF2 knock-down in H295R cells substantially impaired growth (>50% inhibition), blocked cells in G1 phase, and promoted apoptosis (>2-fold). Finally, analysis of the 11p15 locus showed a paternal uniparental disomy in both IGF2-high and IGF2-low tumors, but low IGF2 expression could be explained in most IGF2-low ACC by an additional epigenetic modification at the 11p15 locus. Altogether, these observations confirm the active role of IGF2 in adrenocortical tumor growth, but also suggest that other growth promoting pathways may be involved in a subset of ACC with low IGF2 expression, which creates opportunities for the use of other targeted therapies. PMID:25089899

  18. Comparison of the effects of PRKAR1A and PRKAR2B depletion on signaling pathways, cell growth, and cell cycle control of adrenocortical cells.

    PubMed

    Basso, F; Rocchetti, F; Rodriguez, S; Nesterova, M; Cormier, F; Stratakis, C A; Ragazzon, B; Bertherat, J; Rizk-Rabin, M

    2014-11-01

    The cyclic AMP/protein kinase A signaling cascade is one of the main pathways involved in the pathogenesis of adrenocortical tumors. The PKA R1A and R2B proteins are the most abundant regulatory subunits in endocrine tissues. Inactivating mutations of PRKAR1A are associated with Carney complex and a subset of sporadic tumors and the abundance of R2B protein is low in a subset of secreting adrenocortical adenomas. We previously showed that PRKAR1A and PRKAR2B inactivation have anti-apoptotic effects on the adrenocortical carcinoma cell line H295R. The aim of this study was to compare the effects of PRKAR1A and PRKAR2B depletion on cell proliferation, apoptosis, cell signaling pathways, and cell cycle regulation. We found that PRKAR2B depletion is compensated by an upregulation of R1A protein, whereas PRKAR1A depletion has no effect on the production of R2B. The depletion of either PRKAR1A or PRKAR2B promotes the expression of Bcl-xL and resistance to apoptosis; and is associated with a high percentage of cells in S and G2 phase, activates PKA and MEK/ERK pathways, and impairs the expression of IkB leading to activate the NF-κB pathway. However, we observed differences in the regulation of cyclins. The depletion of PRKAR1A leads to the accumulation of cyclin D1 and p27kip, whereas the depletion of PRKAR2B promotes the accumulation of cyclin A, B, cdk1, cdc2, and p21Cip. In conclusion, although the depletion of PRKAR1A and PRKAR2B in adrenocortical cells has similar effects on cell proliferation and apoptosis; loss of these PKA subunits differentially affects cyclin expression. PMID:25268545

  19. Comparison of the Effects of PRKAR1A and PRKAR2B Depletion on Signaling Pathways, Cell Growth, and Cell Cycle Control of Adrenocortical Cells

    PubMed Central

    Basso, F.; Rocchetti, F.; Rodriguez, S.; Nesterova, M.; Cormier, F.; Stratakis, C.; Ragazzon, B.; Bertherat, J.; Rizk-Rabin, M.

    2016-01-01

    The cyclic AMP/protein kinase A signaling cascade is one of the main pathways involved in the pathogenesis of adrenocortical tumors. The PKA R1A and R2B proteins are the most abundant regulatory subunits in endocrine tissues. Inactivating mutations of PRKAR1A are associated with Carney complex and a subset of sporadic tumors and the abundance of R2B protein is low in a subset of secreting adrenocortical adenomas. We previously showed that PRKAR1A and PRKAR2B inactivation have anti-apoptotic effects on the adrenocortical carcinoma cell line H295R. The aim of this study was to compare the effects of PRKAR1A and PRKAR2B depletion on cell proliferation, apoptosis, cell signaling pathways, and cell cycle regulation. We found that PRKAR2B depletion is compensated by an upregulation in the abundance of R1A protein, whereas PRKAR1A depletion has no effect on the production of R2B. The depletion of either PRKAR1A or PRKAR2B promotes the expression of Bcl-xL and resistance to apoptosis; and is associated with a high percentage of cells in S and G2 phase, activates PKA and MEK/ERK pathways, and impairs the expression of IkB leading to activate the NF-κB pathway. Nonetheless, we observed differences in the regulation of cyclins. The depletion of PRKAR1A leads to the accumulation of cyclin D1 and p27kip, whereas the depletion of PRKAR2B promotes the accumulation of cyclin A, B, cdk1, cdc2, and p21Cip. In conclusion, although the depletion of PRKAR1A and PRKAR2B in adrenocortical cells has similar effects on cell proliferation and apoptosis; loss of these PKA subunits differentially affects cyclin expression. PMID:25268545

  20. Loss of sensitivity to ACTH of adrenocortical cells isolated from maturing domestic fowl.

    PubMed

    Carsia, R V; Scanes, C G; Malamed, S

    1985-07-01

    Maturation of domestic fowl corticosteroidogenesis was evaluated using purified adrenocortical cells. Basal corticosterone production decreased steadily from 2 days to 26 weeks after hatching. However, maximally stimulated corticosterone production was not changed. In contrast, the half-maximal steroidogenic concentrations (ED50 values or effective doses for 50% maximal effect) of ACTH analogs increased approximately 40 times by 26 weeks, but the ED50 values of 8-bromo-cyclic AMP and pregnenolone were not changed. This suggests that adrenocortical cell sensitivity to ACTH decreases with maturation of the domestic fowl.

  1. Properties of calcium and potassium currents of clonal adrenocortical cells

    PubMed Central

    1989-01-01

    The ionic currents of clonal Y-1 adrenocortical cells were studied using the whole-cell variant of the patch-clamp technique. These cells had two major current components: a large outward current carried by K ions, and a small inward Ca current. The Ca current depended on the activity of two populations of Ca channels, slow (SD) and fast (FD) deactivating, that could be separated by their different closing time constants (at -80 mV, SD, 3.8 ms, and FD, 0.13 ms). These two kinds of channels also differed in (a) activation threshold (SD, approximately - 50 mV; FD, approximately -20 mV), (b) half-maximal activation (SD, between -15 and -10 mV; FD between +10 and +15 mV), and (c) inactivation time course (SD, fast; FD, slow). The total amplitude of the Ca current and the proportion of SD and FD channels varied from cell to cell. The amplitude of the K current was strongly dependent on the internal [Ca2+] and was almost abolished when internal [Ca2+] was less than 0.001 microM. The K current appeared to be independent, or only slightly dependent, of Ca influx. With an internal [Ca2+] of 0.1 microM, the activation threshold was -20 mV, and at +40 mV the half- time of activation was 9 ms. With 73 mM external K the closing time constant at -70 mV was approximately 3 ms. The outward current was also modulated by internal pH and Mg. At a constant pCa gamma a decrease of pH reduced the current amplitude, whereas the activation kinetics were not much altered. Removal of internal Mg produced a drastic decrease in the amplitude of the Ca-activated K current. It was also found that with internal [Ca2+] over 0.1 microM the K current underwent a time- dependent transformation characterized by a large increase in amplitude and in activation kinetics. PMID:2539432

  2. Role of ALADIN in Human Adrenocortical Cells for Oxidative Stress Response and Steroidogenesis

    PubMed Central

    Jühlen, Ramona; Idkowiak, Jan; Taylor, Angela E.; Kind, Barbara; Arlt, Wiebke; Huebner, Angela; Koehler, Katrin

    2015-01-01

    Triple A syndrome is caused by mutations in AAAS encoding the protein ALADIN. We investigated the role of ALADIN in the human adrenocortical cell line NCI-H295R1 by either over-expression or down-regulation of ALADIN. Our findings indicate that AAAS knock-down induces a down-regulation of genes coding for type II microsomal cytochrome P450 hydroxylases CYP17A1 and CYP21A2 and their electron donor enzyme cytochrome P450 oxidoreductase, thereby decreasing biosynthesis of precursor metabolites required for glucocorticoid and androgen production. Furthermore we demonstrate that ALADIN deficiency leads to increased susceptibility to oxidative stress and alteration in redox homeostasis after paraquat treatment. Finally, we show significantly impaired nuclear import of DNA ligase 1, aprataxin and ferritin heavy chain 1 in ALADIN knock-down cells. We conclude that down-regulating ALADIN results in decreased oxidative stress response leading to alteration in steroidogenesis, highlighting our knock-down cell model as an important in-vitro tool for studying the adrenal phenotype in triple A syndrome. PMID:25867024

  3. Role of ALADIN in human adrenocortical cells for oxidative stress response and steroidogenesis.

    PubMed

    Jühlen, Ramona; Idkowiak, Jan; Taylor, Angela E; Kind, Barbara; Arlt, Wiebke; Huebner, Angela; Koehler, Katrin

    2015-01-01

    Triple A syndrome is caused by mutations in AAAS encoding the protein ALADIN. We investigated the role of ALADIN in the human adrenocortical cell line NCI-H295R1 by either over-expression or down-regulation of ALADIN. Our findings indicate that AAAS knock-down induces a down-regulation of genes coding for type II microsomal cytochrome P450 hydroxylases CYP17A1 and CYP21A2 and their electron donor enzyme cytochrome P450 oxidoreductase, thereby decreasing biosynthesis of precursor metabolites required for glucocorticoid and androgen production. Furthermore we demonstrate that ALADIN deficiency leads to increased susceptibility to oxidative stress and alteration in redox homeostasis after paraquat treatment. Finally, we show significantly impaired nuclear import of DNA ligase 1, aprataxin and ferritin heavy chain 1 in ALADIN knock-down cells. We conclude that down-regulating ALADIN results in decreased oxidative stress response leading to alteration in steroidogenesis, highlighting our knock-down cell model as an important in-vitro tool for studying the adrenal phenotype in triple A syndrome.

  4. Silencing diacylglycerol kinase-theta expression reduces steroid hormone biosynthesis and cholesterol metabolism in human adrenocortical cells.

    PubMed

    Cai, Kai; Lucki, Natasha C; Sewer, Marion B

    2014-04-01

    Diacylglycerol kinase theta (DGKθ) plays a pivotal role in regulating adrenocortical steroidogenesis by synthesizing the ligand for the nuclear receptor steroidogenic factor 1 (SF1). In response to activation of the cAMP signaling cascade nuclear DGK activity is rapidly increased, facilitating PA-mediated, SF1-dependent transcription of genes required for cortisol and dehydroepiandrosterone (DHEA) biosynthesis. Based on our previous work identifying DGKθ as the enzyme that produces the agonist for SF1, we generated a tetracycline-inducible H295R stable cell line to express a short hairpin RNA (shRNA) against DGKθ and characterized the effect of silencing DGKθ on adrenocortical gene expression. Genome-wide DNA microarray analysis revealed that silencing DGKθ expression alters the expression of multiple genes, including steroidogenic genes, nuclear receptors and genes involved in sphingolipid, phospholipid and cholesterol metabolism. Interestingly, the expression of sterol regulatory element binding proteins (SREBPs) was also suppressed. Consistent with the suppression of SREBPs, we observed a down-regulation of multiple SREBP target genes, including 3-hydroxy-3-methylglutary coenzyme A reductase (HMG-CoA red) and CYP51, concomitant with a decrease in cellular cholesterol. DGKθ knockdown cells exhibited a reduced capacity to metabolize PA, with a down-regulation of lipin and phospholipase D (PLD) isoforms. In contrast, suppression of DGKθ increased the expression of several genes in the sphingolipid metabolic pathway, including acid ceramidase (ASAH1) and sphingosine kinases (SPHK). In summary, these data demonstrate that DGKθ plays an important role in steroid hormone production in human adrenocortical cells.

  5. Effects of ToxCast Phase I Chemicals on Steroidogenesis in H295R Human Adrenocortical Carcinoma cells (SOT)

    EPA Science Inventory

    Steroid hormones are essential for proper development and reproduction. Disruption of steroidogenesis by environmental toxicants results in altered hormone levels causing adverse reproductive and developmental effects. H295R human adrenocortical carcinoma cells were used to evalu...

  6. Toying with fate: Redirecting the differentiation of adrenocortical progenitor cells into gonadal-like tissue

    PubMed Central

    Röhrig, Theresa; Pihlajoki, Marjut; Ziegler, Ricarda; Cochran, Rebecca S.; Schrade, Anja; Schillebeeckx, Maximiliaan; Mitra, Robi D.; Heikinheimo, Markku; Wilson, David B.

    2014-01-01

    Cell fate decisions are integral to zonation and remodeling of the adrenal cortex. Animal models exhibiting ectopic differentiation of gonadal-like cells in the adrenal cortex can shed light on the molecular mechanisms regulating steroidogenic cell fate. In one such model, prepubertal gonadectomy (GDX) of mice triggers the formation of adrenocortical neoplasms that resemble luteinized ovarian stroma. Transcriptomic analysis and genome-wide DNA methylation mapping have identified genetic and epi-genetic markers of GDX-induced adrenocortical neoplasia. Members of the GATA transcription factor family have emerged as key regulators of cell fate in this model. Expression of Gata4 is pivotal for the accumulation of gonadal-like cells in the adrenal glands of gonadectomized mice, whereas expression of Gata6 limits the spontaneous and GDX-induced differentiation of gonadal-like cells in the adrenal cortex. Additionally, Gata6 is essential for proper development of the adrenal X-zone, a layer analogous to the fetal zone of the human adrenal cortex. The relevance of these observations to developmental signaling pathways in the adrenal cortex, to other animal models of altered adrenocortical cell fate, and to human diseases is discussed. PMID:25498963

  7. Ultrastructural Localization of Endogenous Exchange Factor for ARF6 in Adrenocortical Cells In Situ of Mice.

    PubMed

    Chomphoo, Surang; Mothong, Wilaiwan; Sawatpanich, Tarinee; Kanla, Pipatphong; Sakagami, Hiroyuki; Kondo, Hisatake; Hipkaeo, Wiphawi

    2016-06-28

    EFA6 (exchange factor for ARF6) activates Arf6 (ADP ribosylation factor 6) by exchanging ADP to ATP, and the resulting activated form of Arf6 is involved in the membrane dynamics and actin re-organization of cells. The present study was attempted to localize EFA6 type D (EFA6D) in mouse adrenocortical cells in situ whose steroid hormone secretion is generally considered not to depend on the vesicle-involved regulatory mechanism. In immunoblotting, an immunoreactive band with the same size as brain EFA6D was detected in homogenates of adrenal cortical tissues almost free of adrenal capsules and medulla. In immuno-light microscopy, EFA6D-immunoreactivity was positive in adrenocortical cells and it was often distinct along the plasmalemma, especially along portions of the cell columns facing the interstitium. In immuno-electron microscopy, the gold-labeling was more dense in the peripheral intracellular domains than the central domain of the immunopositive cells. The labeling was deposited on the plasma membranes in a discontinuous pattern and in cytoplasmic domains rich in filaments. It was also associated with some, but not all, of pleiomorphic vesicles and coated pits/vesicles. No labeling was seen in association with lipid droplets or smooth endoplasmic reticulum. The present finding is in support of the importance of EFA6D for activation of Arf6 in adrenocortical cells. PMID:27462133

  8. Aging of the rat adrenocortical cell: response to ACTH and cyclic AMP in vitro.

    PubMed

    Malamed, S; Carsia, R V

    1983-03-01

    To study intrinsic age-related changes in adrenocortical steroid production, cells isolated from rats of different ages (3 to 24 months) were used. Acute (2 hour) corticosterone production in response to stimulation by adrenocorticotrophic hormone (ACTH) and adenosine 3':5'-cyclic monophosphate (cAMP) was measured by radioimmunoassay. With age, adrenocortical cells lose much of their ability to produce corticosterone in the absence or presence of ACTH or cAMP. The loss is progressive from 6 to 24 months of age. Analysis of the data suggests that from 6 to 12 months, an intracellular steroidogenic lesion develops; in addition there may be a loss in ACTH receptors on the plasma membrane. After 12 months these defects increase and are accompanied by a decrease in receptor sensitivity to ACTH.

  9. Isolated adrenocortical cells of the domestic fowl (Gallus domesticus): steroidogenic and ultrastructural properties.

    PubMed

    Carsia, R V; Scanes, C G; Malamed, S

    1985-02-01

    Isolated adrenocortical cells from White Leghorn chickens (Gallus domesticus) were compared to those from rats (Rattus norvegicus). Cells were prepared from collagenase-dispersed adrenal glands of sexually mature male animals. Corticosterone was measured by radioimmunoassay after incubation for 2 h with steroidogenic agents. Of the four ACTH analogues used, three were 6-17 times more potent with rat cells than with fowl cells (potencies were indicated by half-maximal steroidogenic concentrations). However, 9-tryptophan (O-nitrophenylsulfenyl) ACTH was 8 times more potent with fowl cells than with rat cells, thus suggesting that ACTH receptor differences exist between the two cell types. In addition, cAMP analogues were 10 times more potent with rat cells than with fowl cells suggesting that fowl corticosteroidogenesis is less dependent on cAMP than is rat corticosteroidogenesis. At equal cell concentrations, rat cells secreted 20-40 times more corticosterone than did chicken cells when they were maximally stimulated. Although rat cells converted 8 times more pregnenolone to corticosterone than did fowl cells, the half-maximal steroidogenic concentration for pregnenolone-supported corticosterone synthesis was the same for both cell types (about 5 microM). This suggests that fowl cells have lower steroidogenic enzyme content rather than lower steroidogenic enzyme activity. An unusual feature seen in the isolated fowl adrenocortical cells was an abundance of intracellular filaments.

  10. Cloning of DNA from double minutes of Y1 mouse adrenocortical tumor cells: evidence for gene amplification.

    PubMed

    George, D L; Powers, V E

    1981-04-01

    We have isolated a metaphase chromosome fraction highly enriched in double minutes (dm) from a mouse adrenocortical tumor cell line (Y1-DM). We have cloned DNA from this dm-enriched fraction in the lambda vector Charon 4A, and have characterized two randomly chosen recombinant bacteriophage clones from this dm DNA library. When 32 P-labeled DNA from each recombinant was hybridized to Southern blots of restriction endonuclease-digested DNA from different mouse cell lines, large differences were seen in the intensity of the resulting autoradiographic images, depending on the source of the genomic DNA. A very strong signal was obtained with DNA from the Y1-DM cells and with DNA from a related Y1 subline that lacks dm but contains a marker chromosome bearing a large homogeneously staining region (HSR). Hybridization to DNA from parental inbred mice and from two unrelated mouse cell lines produced a significantly weaker signal than that obtained with DNA from the Y1 cells, but the DNA fragments from the sources were of similar size. Based on results from filter hybridization analysis, we estimate that sequences homologous to the cloned fragments are approximately 100- to 200-fold more abundant in the genome of the Y1-DM cells than in the parental mouse cells. The data are consistent with the hypothesis that dm and HSRs in these cells contain amplified genes.

  11. Steroid control of steroidogenesis in isolated adrenocortical cells: molecular and species specificity.

    PubMed

    Carsia, R V; Macdonald, G J; Malamed, S

    1983-06-01

    The molecular and species specificity of glucocorticoid suppression of corticosteroidogenesis was investigated in isolated adrenocortical cells. Trypsin-isolated cells from male rat, domestic fowl and bovine adrenal glands were incubated with or without steroidogenic agents and with or without steroids. Glucocorticoids were measured by radioimmunoassay or fluorometric assay after 1-2 h incubation. Glucocorticoids suppressed ACTH-induced steroidogenesis of isolated rat cells with the following relative potencies: corticosterone greater than cortisol = cortisone greater than dexamethasone. The mineralocorticoid, aldosterone did not affect steroidogenesis. Suppression by glucocorticoids was acute (within 1-2 h), and varied directly with the glucocorticoid concentration. Testosterone also suppressed ACTH-induced steroidogenesis. Glucocorticoid-type steroids have equivalent suppressive potencies, thus suggesting that these steroids may induce suppression at least partly by a common mechanism. Although corticosterone caused the greatest suppression, testosterone was more potent. The steroid specificity of suppression of cyclic AMP (cAMP)-induced and ACTH-induced steroidogenesis were similar, suggesting that suppression is not solely the result of interference with ACTH receptor function or the induction of adenylate cyclase activity. Exogenous glucocorticoids also suppressed ACTH-induced steroidogenesis of cells isolated from domestic fowl and beef adrenal glands, thus suggesting that this observed suppression may be a general mechanism of adrenocortical cell autoregulation.

  12. Acute effects of ACTH on dissociated adrenocortical cells: quantitative changes in mitochondria and lipid droplets.

    PubMed

    Zoller, L C; Malamed, S

    1975-08-01

    To study the role of certain organelles in steroidogenesis, dissociated rat adrenocortical cells were incubated for two hours with ACTH at a concentration that induces a high level of steroid production. Sections of ACTH treated and untreated cells were photographed in the electron microscope, and morphometric analysis was undertaken to assess possible ACTH-induced changes in total cell volume, volume density and numerical denisty of lipid droplets and mitochondria. There was no change in total cell volume. Lipid droplet volume density and numerical density decreased. Mitochondrial volume density did not change, but numerical density increased. The decrease in lipid droplet volume density indicates a rapid depletion of cholesterol for steroid production. This depletion is almost entirely due to the disappearance of lipid droplets, rather than to an overall diminution in their size, as shown by the decrease in lipid droplet numerical density. The mitochondrial data suggest that the adrenocortical cell has an adedquate mitochondrial apparatus to respond to acute ACTH stimulation with increased steroid output without an increase inmitochondrial volume.

  13. Direct inhibitory effect of etomidate on corticosteroid secretion in human pathologic adrenocortical cells.

    PubMed

    Varga, I; Rácz, K; Kiss, R; Fütö, L; Tóth, M; Sergev, O; Gláz, E

    1993-02-01

    Etomidate has been shown to inhibit corticosteroid secretion in the normal adrenal gland, but its direct effect in human pathologic adrenals has not been clearly established. In the present study the effect of varying doses of etomidate (10(-11)-10(-5) M) was investigated on basal and adrenocorticotrophic hormone (ACTH)-stimulated corticosteroid secretions in isolated adrenocortical cells obtained from two patients with primary aldosteronism (adenoma and micronodular hyperplasia) and in those from a patient with Cushing's syndrome (adenoma). In cells from primary aldosteronism, increasing concentrations of etomidate (10(-11)-10(-5) M) produced a dose-dependent decrease of basal and ACTH-stimulated cortisol, aldosterone, 18-hydroxycorticosterone, and corticosterone secretions (ED50: 10(-9)-10(-8) M for each of these corticosteroids). In the same cells, the secretions of 11-deoxycortisol and deoxycorticosterone were increased in the presence of low (10(-9)-10(-7) M) but not high doses of etomidate (10(-6)-10(-5) M). In cells from Cushing's syndrome the changes in corticosteroid secretion were similar to those found in primary aldosteronism except that aldosterone and 18-hydroxycorticosterone could not be determined due to their low levels. Thus the potent inhibition of corticosteroids in human pathologic adrenocortical cells in the presence of low concentrations of etomidate may be predominantly due to inhibition of the 11 beta-hydroxylase enzyme, whereas higher doses of the drug may inhibit earlier steps of the corticosteroid biosynthetic pathway. PMID:8387232

  14. A Case of Cushing's Syndrome with Multiple Adrenocortical Adenomas Composed of Compact Cells and Clear Cells.

    PubMed

    Asakawa, Masahiro; Yoshimoto, Takanobu; Ota, Mitsutane; Numasawa, Mitsuyuki; Sasahara, Yuriko; Takeuchi, Takato; Nakano, Yujiro; Oohara, Norihiko; Murakami, Masanori; Bouchi, Ryotaro; Minami, Isao; Tsuchiya, Kyoichiro; Hashimoto, Koshi; Izumiyama, Hajime; Kawamura, Naoko; Kihara, Kazunori; Negi, Mariko; Akashi, Takumi; Eishi, Yoshinobu; Sasano, Hironobu; Ogawa, Yoshihiro

    2016-06-01

    A 58-year-old woman was referred to our hospital for Cushingoid features and diagnosed as adrenal Cushing's syndrome due to a right adrenocortical mass (60 × 55 mm). The mass was composed of three different tumors; the first one was homogeneously lipid-poor neoplasm measuring 20 × 13 mm located at the most dorsal region, the second one was heterogeneous and lipid-rich tumor containing multiple foci of calcification measuring 50 × 32 mm located at the central region, and the last one was heterogeneous harboring dilated and tortuous vessels and lipid-poor one measuring 35 × 18 mm at the most ventral region of the adrenal gland. A right adrenalectomy was subsequently performed by open surgery. Macroscopic and microscopic analyses revealed that all three tumors were adrenocortical adenomas; the first one represents a pigmented adrenocortical adenoma, the second one adrenocortical adenoma associated with degeneration, and the third one adrenocortical adenoma harboring extensive degeneration. Immunohistochemical analysis of the steroidogenic enzymes also revealed that all of the tumors had the capacity of synthesizing cortisol. This is a very rare case of Cushing's syndrome caused by multiple adrenocortical adenomas including a pigmented adenoma. Immunohistochemical analysis of steroidogenic enzymes contributed to understanding of steroidogenesis in each of these three different adrenocortical adenomas in this case.

  15. Inhibition of the Tcf/beta-catenin complex increases apoptosis and impairs adrenocortical tumor cell proliferation and adrenal steroidogenesis

    PubMed Central

    Leal, Letícia F.; Bueno, Ana Carolina; Gomes, Débora C.; Abduch, Rafael; de Castro, Margaret; Antonini, Sonir R.

    2015-01-01

    Background To date, there is no effective therapy for patients with advanced/metastatic adrenocortical cancer (ACC). The activation of the Wnt/beta-catenin signaling is frequent in ACC and this pathway is a promising therapeutic target. Aim To investigate the effects of the inhibition of the Wnt/beta-catenin in ACC cells. Methods Adrenal (NCI-H295 and Y1) and non-adrenal (HeLa) cell lines were treated with PNU-74654 (5–200 μM) for 24–96 h to assess cell viability (MTS-based assay), apoptosis (Annexin V), expression/localization of beta-catenin (qPCR, immunofluorescence, immunocytochemistry and western blot), expression of beta-catenin target genes (qPCR and western blot), and adrenal steroidogenesis (radioimmunoassay, qPCR and western blot). Results In NCI-H295 cells, PNU-74654 significantly decreased cell proliferation 96 h after treatment, increased early and late apoptosis, decreased nuclear beta-catenin accumulation, impaired CTNNB1/beta-catenin expression and increased beta-catenin target genes 48 h after treatment. No effects were observed on HeLa cells. In NCI-H295 cells, PNU-74654 decreased cortisol, testosterone and androstenedione secretion 24 and 48 h after treatment. Additionally, in NCI-H295 cells, PNU-74654 decreased SF1 and CYP21A2 mRNA expression as well as the protein levels of STAR and aldosterone synthase 48 h after treatment. In Y1 cells, PNU-74654 impaired corticosterone secretion 24 h after treatment but did not decrease cell viability. Conclusions Blocking the Tcf/beta-catenin complex inhibits the Wnt/beta-catenin signaling in adrenocortical tumor cells triggering increased apoptosis, decreased cell viability and impairment of adrenal steroidogenesis. These promising findings pave the way for further experiments inhibiting the Wnt/beta-catenin pathway in pre-clinical models of ACC. The inhibition of this pathway may become a promising adjuvant therapy for patients with ACC. PMID:26515592

  16. Adrenocortical carcinoma

    MedlinePlus

    ... this tumor. Adrenocortical carcinoma can produce the hormones cortisol, aldosterone, estrogen, or testosterone, as well as other ... Symptoms of increased cortisol or other adrenal gland hormones: ... high on the back just below the neck ( buffalo hump ) Flushed, ...

  17. High-throughput screening of chemical effects on steroidogenesis using H295R human adrenocortical carcinoma cells

    EPA Science Inventory

    Disruption of steroidogenesis by environmental chemicals can result in altered hormone levels causing adverse reproductive and developmental effects. A high-throughput assay using H295R human adrenocortical carcinoma cells was used to evaluate the effect of 2,060 chemical samples...

  18. Adrenocortical hemorrhagic necrosis: the role of catecholamines and retrograde medullary-cell embolism

    SciTech Connect

    Szabo, S.; McComb, D.J.; Kovacs, K.; Huettner, I.

    1981-10-01

    We investigated the pathogenesis of adrenal necrosis using animal models of the disease (induced by administration of acrylonitrile, cysteamine, or pyrazole) and human cases. Results of electron-microscopic and histochemical time-response studies with rat models revealed an early, retrograde embolization of medullary cells and cell fragments in the cortical capillaries that showed prominent endothelial injury. The experimental adrenal lesions were prevented by surgical removal of the medulla one month before administration of adrenocorticolytic chemicals, or by the administration of the alpha-adrenergic antagonist phenoxybenzamine hydrochloride. Histochemical staining for medullary (argyrophil) granules in human cases of adrenal necrosis demonstrated tissue fragments that stained positively for silver in vascular cortical spaces in nine of ten autopsy specimens and in all four surgical cases we reviewed. Thus, catecholamines released from the adrenal medulla and from the retrograde medullary emboli in the cortex may have a role in the pathogenesis of adrenocortical necrosis.

  19. The effect of pioglitazone on aldosterone and cortisol production in HAC15 human adrenocortical carcinoma cells.

    PubMed

    Pan, Zhi-qiang; Xie, Ding; Choudhary, Vivek; Seremwe, Mutsa; Tsai, Ying-Ying; Olala, Lawrence; Chen, Xunsheng; Bollag, Wendy B

    2014-08-25

    Pioglitazone belongs to the class of drugs called thiazolidinediones (TZDs), which are widely used as insulin sensitizers in the treatment of diabetes. A major side effect of TZDs is fluid retention. The steroid hormone aldosterone also promotes sodium and fluid retention; however, the effect of pioglitazone on aldosterone production is controversial. We analyzed the effect of pioglitazone alone and in combination with angiotensin II (AngII) on the late rate-limiting step of adrenocortical steroidogenesis in human adrenocortical carcinoma HAC15 cells. Treatment with pioglitazone for 24 h significantly increased the expression of CYP11B2 and enhanced AngII-induced CYP11B2 expression. Despite the observed changes in mRNA levels, pioglitazone significantly inhibited AngII-induced aldosterone production and CYP11B2 protein levels. On the other hand, pioglitazone stimulated the expression of the unfolded protein response (UPR) marker DDIT3, with this effect occurring at early times and inhibitable by the PPARγ antagonist GW9962. The levels of DDIT3 (CHOP) and phospho-eIF2α (Ser51), a UPR-induced event that inhibits protein translation, were also increased. Thus, pioglitazone promotes CYP11B2 expression but nevertheless inhibits aldosterone production in AngII-treated HAC15 cells, likely by blocking global protein translation initiation through DDIT3 and phospho-eIF2α. In contrast, pioglitazone promoted AngII-induced CYP11B1 expression and cortisol production. Since cortisol enhances lipolysis, this result suggests the possibility that PPARs, activated by products of fatty acid oxidation, stimulate cortisol secretion to promote utilization of fatty acids during fasting. In turn, the ability of pioglitazone to stimulate cortisol production could potentially underlie the effects of this drug on fluid retention. PMID:25038520

  20. The effect of pioglitazone on aldosterone and cortisol production in HAC15 human adrenocortical carcinoma cells.

    PubMed

    Pan, Zhi-qiang; Xie, Ding; Choudhary, Vivek; Seremwe, Mutsa; Tsai, Ying-Ying; Olala, Lawrence; Chen, Xunsheng; Bollag, Wendy B

    2014-08-25

    Pioglitazone belongs to the class of drugs called thiazolidinediones (TZDs), which are widely used as insulin sensitizers in the treatment of diabetes. A major side effect of TZDs is fluid retention. The steroid hormone aldosterone also promotes sodium and fluid retention; however, the effect of pioglitazone on aldosterone production is controversial. We analyzed the effect of pioglitazone alone and in combination with angiotensin II (AngII) on the late rate-limiting step of adrenocortical steroidogenesis in human adrenocortical carcinoma HAC15 cells. Treatment with pioglitazone for 24 h significantly increased the expression of CYP11B2 and enhanced AngII-induced CYP11B2 expression. Despite the observed changes in mRNA levels, pioglitazone significantly inhibited AngII-induced aldosterone production and CYP11B2 protein levels. On the other hand, pioglitazone stimulated the expression of the unfolded protein response (UPR) marker DDIT3, with this effect occurring at early times and inhibitable by the PPARγ antagonist GW9962. The levels of DDIT3 (CHOP) and phospho-eIF2α (Ser51), a UPR-induced event that inhibits protein translation, were also increased. Thus, pioglitazone promotes CYP11B2 expression but nevertheless inhibits aldosterone production in AngII-treated HAC15 cells, likely by blocking global protein translation initiation through DDIT3 and phospho-eIF2α. In contrast, pioglitazone promoted AngII-induced CYP11B1 expression and cortisol production. Since cortisol enhances lipolysis, this result suggests the possibility that PPARs, activated by products of fatty acid oxidation, stimulate cortisol secretion to promote utilization of fatty acids during fasting. In turn, the ability of pioglitazone to stimulate cortisol production could potentially underlie the effects of this drug on fluid retention.

  1. GPER agonist G-1 decreases adrenocortical carcinoma (ACC) cell growth in vitro and in vivo.

    PubMed

    Chimento, Adele; Sirianni, Rosa; Casaburi, Ivan; Zolea, Fabiana; Rizza, Pietro; Avena, Paola; Malivindi, Rocco; De Luca, Arianna; Campana, Carmela; Martire, Emilia; Domanico, Francesco; Fallo, Francesco; Carpinelli, Giulia; Cerquetti, Lidia; Amendola, Donatella; Stigliano, Antonio; Pezzi, Vincenzo

    2015-08-01

    We have previously demonstrated that estrogen receptor (ER) alpha (ESR1) increases proliferation of adrenocortical carcinoma (ACC) through both an estrogen-dependent and -independent (induced by IGF-II/IGF1R pathways) manner. Then, the use of tamoxifen, a selective estrogen receptor modulator (SERM), appears effective in reducing ACC growth in vitro and in vivo. However, tamoxifen not only exerts antiestrogenic activity, but also acts as full agonist on the G protein-coupled estrogen receptor (GPER). Aim of this study was to investigate the effect of a non-steroidal GPER agonist G-1 in modulating ACC cell growth. We found that G-1 is able to exert a growth inhibitory effect on H295R cells both in vitro and, as xenograft model, in vivo. Treatment of H295R cells with G-1 induced cell cycle arrest, DNA damage and cell death by the activation of the intrinsic apoptotic mechanism. These events required sustained extracellular regulated kinase (ERK) 1/2 activation. Silencing of GPER by a specific shRNA partially reversed G-1-mediated cell growth inhibition without affecting ERK activation. These data suggest the existence of G-1 activated but GPER-independent effects that remain to be clarified. In conclusion, this study provides a rational to further study G-1 mechanism of action in order to include this drug as a treatment option to the limited therapy of ACC.

  2. GPER agonist G-1 decreases adrenocortical carcinoma (ACC) cell growth in vitro and in vivo

    PubMed Central

    Zolea, Fabiana; Rizza, Pietro; Avena, Paola; Malivindi, Rocco; De Luca, Arianna; Campana, Carmela; Martire, Emilia; Domanico, Francesco; Fallo, Francesco; Carpinelli, Giulia; Cerquetti, Lidia; Amendola, Donatella; Stigliano, Antonio; Pezzi, Vincenzo

    2015-01-01

    We have previously demonstrated that estrogen receptor (ER) alpha (ESR1) increases proliferation of adrenocortical carcinoma (ACC) through both an estrogen-dependent and -independent (induced by IGF-II/IGF1R pathways) manner. Then, the use of tamoxifen, a selective estrogen receptor modulator (SERM), appears effective in reducing ACC growth in vitro and in vivo. However, tamoxifen not only exerts antiestrogenic activity, but also acts as full agonist on the G protein-coupled estrogen receptor (GPER). Aim of this study was to investigate the effect of a non-steroidal GPER agonist G-1 in modulating ACC cell growth. We found that G-1 is able to exert a growth inhibitory effect on H295R cells both in vitro and, as xenograft model, in vivo. Treatment of H295R cells with G-1 induced cell cycle arrest, DNA damage and cell death by the activation of the intrinsic apoptotic mechanism. These events required sustained extracellular regulated kinase (ERK) 1/2 activation. Silencing of GPER by a specific shRNA partially reversed G-1-mediated cell growth inhibition without affecting ERK activation. These data suggest the existence of G-1 activated but GPER-independent effects that remain to be clarified. In conclusion, this study provides a rational to further study G-1 mechanism of action in order to include this drug as a treatment option to the limited therapy of ACC. PMID:26131713

  3. GPER agonist G-1 decreases adrenocortical carcinoma (ACC) cell growth in vitro and in vivo.

    PubMed

    Chimento, Adele; Sirianni, Rosa; Casaburi, Ivan; Zolea, Fabiana; Rizza, Pietro; Avena, Paola; Malivindi, Rocco; De Luca, Arianna; Campana, Carmela; Martire, Emilia; Domanico, Francesco; Fallo, Francesco; Carpinelli, Giulia; Cerquetti, Lidia; Amendola, Donatella; Stigliano, Antonio; Pezzi, Vincenzo

    2015-08-01

    We have previously demonstrated that estrogen receptor (ER) alpha (ESR1) increases proliferation of adrenocortical carcinoma (ACC) through both an estrogen-dependent and -independent (induced by IGF-II/IGF1R pathways) manner. Then, the use of tamoxifen, a selective estrogen receptor modulator (SERM), appears effective in reducing ACC growth in vitro and in vivo. However, tamoxifen not only exerts antiestrogenic activity, but also acts as full agonist on the G protein-coupled estrogen receptor (GPER). Aim of this study was to investigate the effect of a non-steroidal GPER agonist G-1 in modulating ACC cell growth. We found that G-1 is able to exert a growth inhibitory effect on H295R cells both in vitro and, as xenograft model, in vivo. Treatment of H295R cells with G-1 induced cell cycle arrest, DNA damage and cell death by the activation of the intrinsic apoptotic mechanism. These events required sustained extracellular regulated kinase (ERK) 1/2 activation. Silencing of GPER by a specific shRNA partially reversed G-1-mediated cell growth inhibition without affecting ERK activation. These data suggest the existence of G-1 activated but GPER-independent effects that remain to be clarified. In conclusion, this study provides a rational to further study G-1 mechanism of action in order to include this drug as a treatment option to the limited therapy of ACC. PMID:26131713

  4. H295R Human Adrenocortical Carcinoma Cells as a Screening Platform for Steroidogenesis (NC SOT)

    EPA Science Inventory

    Proper biosynthesis and metabolism of steroid hormones is essential for development and reproduction. Disruption of steroidogenesis by environmental toxicants results in altered hormone levels causing adverse reproductive and developmental effects. H295R human adrenocortical carc...

  5. Mutual effects of melatonin and activin on induction of aldosterone production by human adrenocortical cells.

    PubMed

    Hara, Takayuki; Otsuka, Fumio; Tsukamoto-Yamauchi, Naoko; Inagaki, Kenichi; Hosoya, Takeshi; Nakamura, Eri; Terasaka, Tomohiro; Komatsubara, Motoshi; Makino, Hirofumi

    2015-08-01

    Melatonin has been reported to suppress adrenocorticotropin (ACTH) secretion in the anterior pituitary and cortisol production in the adrenal by different mechanisms. However, the effect of melatonin on aldosterone production has remained unknown. In this study, we investigated the role of melatonin in the regulation of aldosterone production using human adrenocortical H295R cells by focusing on the activin system expressed in the adrenal. Melatonin receptor MT1 mRNA and protein were expressed in H295R cells and the expression levels of MT1 were increased by activin treatment. Activin increased ACTH-induced, but not angiotensin II (Ang II)-induced, aldosterone production. Melatonin alone did not affect basal synthesis of either aldosterone or cortisol. However, melatonin effectively enhanced aldosterone production induced by co-treatment with ACTH and activin, although melatonin had no effect on aldosterone production induced by Ang II in combination with activin. These changes in steroidogenesis became apparent when the steroid production was evaluated by the ratio of aldosterone/cortisol. Melatonin also enhanced dibutyryl-AMP-induced aldosterone/cortisol levels in the presence of activin, suggesting a functional link to the cAMP-PKA pathway for induction of aldosterone production by melatonin and activin. In accordance with the data for steroids, ACTH-induced, but not Ang II-induced, cAMP synthesis was also amplified by co-treatment with melatonin and activin. Furthermore, the ratio of ACTH-induced mRNA level of CYP11B2 compared with that of CYP17 was amplified in the condition of treatment with both melatonin and activin. In addition, melatonin increased expression of the activin type-I receptor ALK-4 but suppressed expression of inhibitory Smads6/7, leading to the enhancement of Smad2 phosphorylation. Collectively, the results showed that melatonin facilitated aldosterone production induced by ACTH and activin via the cAMP-PKA pathway. The results also

  6. StAR Protein Stability in Y1 and Kin-8 Mouse Adrenocortical Cells.

    PubMed

    Clark, Barbara J; Hudson, Elizabeth A

    2015-03-04

    The steroidogenic acute regulatory protein (STAR) protein expression is required for cholesterol transport into mitochondria to initiate steroidogenesis in the adrenal and gonads. STAR is synthesized as a 37 kDa precursor protein which is targeted to the mitochondria and imported and processed to an intra-mitochondrial 30 kDa protein. Tropic hormone stimulation of the cAMP-dependent protein kinase A (PKA) signaling pathway is the major contributor to the transcriptional and post-transcriptional regulation of STAR synthesis. Many studies have focused on the mechanisms of cAMP-PKA mediated control of STAR synthesis while there are few reports on STAR degradation pathways. The objective of this study was to determine the effect of cAMP-PKA-dependent signaling on STAR protein stability. We have used the cAMP-PKA responsive Y1 mouse adrenocortical cells and the PKA-deficient Kin-8 cells to measure STAR phosphorylation and protein half-life. Western blot analysis and standard radiolabeled pulse-chase experiments were used to determine STAR phosphorylation status and protein half-life, respectively. Our data demonstrate that PKA-dependent STAR phosphorylation does not contribute to 30 kDa STAR protein stability in the mitochondria. We further show that inhibition of the 26S proteasome does not block precursor STAR phosphorylation or steroid production in Y1 cells. These data suggest STAR can maintain function and promote steroidogenesis under conditions of proteasome inhibition.

  7. Human Adrenocortical Remodeling Leading to Aldosterone-Producing Cell Cluster Generation

    PubMed Central

    Hayashi, Yuichiro; Al-Eyd, Ghaith; Nakagawa, Ken; Morita, Shinya; Kosaka, Takeo; Oya, Mototsugu; Mitani, Fumiko; Suematsu, Makoto; Kabe, Yasuaki

    2016-01-01

    Background. The immunohistochemical detection of aldosterone synthase (CYP11B2) and steroid 11β-hydroxylase (CYP11B1) has enabled the identification of aldosterone-producing cell clusters (APCCs) in the subcapsular portion of the human adult adrenal cortex. We hypothesized that adrenals have layered zonation in early postnatal stages and are remodeled to possess APCCs over time. Purposes. To investigate changes in human adrenocortical zonation with age. Methods. We retrospectively analyzed adrenal tissues prepared from 33 autopsied patients aged between 0 and 50 years. They were immunostained for CYP11B2 and CYP11B1. The percentage of APCC areas over the whole adrenal area (AA/WAA, %) and the number of APCCs (NOA, APCCs/mm2) were calculated by four examiners. Average values were used in statistical analyses. Results. Adrenals under 11 years old had layered zona glomerulosa (ZG) and zona fasciculata (ZF) without apparent APCCs. Some adrenals had an unstained (CYP11B2/CYP11B1-negative) layer between ZG and ZF, resembling the rat undifferentiated cell zone. Average AA/WAA and NOA correlated with age, suggesting that APCC development is associated with aging. Possible APCC-to-APA transitional lesions were incidentally identified in two adult adrenals. Conclusions. The adrenal cortex with layered zonation remodels to possess APCCs over time. APCC generation may be associated with hypertension in adults. PMID:27721827

  8. Adrenocortical Carcinoma

    PubMed Central

    Kim, Alex C.; Sabolch, Aaron; Raymond, Victoria M.; Kandathil, Asha; Caoili, Elaine M.; Jolly, Shruti; Miller, Barbra S.; Giordano, Thomas J.

    2014-01-01

    Adrenocortical carcinoma (ACC) is a rare endocrine malignancy, often with an unfavorable prognosis. Here we summarize the knowledge about diagnosis, epidemiology, pathophysiology, and therapy of ACC. Over recent years, multidisciplinary clinics have formed and the first international treatment trials have been conducted. This review focuses on evidence gained from recent basic science and clinical research and provides perspectives from the experience of a large multidisciplinary clinic dedicated to the care of patients with ACC. PMID:24423978

  9. Comparative CYP-dependent binding of the adrenocortical toxicants 3-methylsulfonyl-DDE and o,p'-DDD in Y-1 adrenal cells.

    PubMed

    Hermansson, Veronica; Asp, Vendela; Bergman, Ake; Bergström, Ulrika; Brandt, Ingvar

    2007-11-01

    The environmental pollutant 3-MeSO(2)-DDE [2-(3-methylsulfonyl-4-chlorophenyl)-2-(4-chlorophenyl)-1,1-dichloroethene] is an adrenocortical toxicant in mice, specifically in the glucocorticoid-producing zona fasciculata, due to a cytochrome P450 11B1 (CYP11B1)-catalysed bioactivation and formation of covalently bound protein adducts. o,p'-DDD [2-(2-chlorophenyl)-2-(4-chlorophenyl)-1,1-dichloroethane] is toxic and inhibits steroidogenesis in the human adrenal cortex after bioactivation by unidentified CYPs, but does not exert any toxic effects on the mouse adrenal. As a step towards determining in vitro/in vivo relationships for the CYP-catalysed binding and toxicity of 3-MeSO(2)-DDE and o,p'-DDD, we have investigated the irreversible protein binding of these two toxicants in the murine adrenocortical cell line Y-1. The irreversible binding of 3-MeSO(2)-DDE previously demonstrated in vivo was successfully reproduced and could be inhibited by the CYP-inhibitors etomidate, ketoconazole and metyrapone. Surprisingly, o,p'-DDD reached similar levels of binding as 3-MeSO(2)-DDE. The binding of o,p'-DDD was sensitive to etomidate and ketoconazole, but not to metyrapone. Moreover, GSH depletion increased the binding of 3-MeSO(2)-DDE, but not of o,p'-DDD, indicating an important role of GSH conjugation in the detoxification of the 3-MeSO(2)-DDE-derived reactive metabolite. In addition, the specificity of CYP11B1 in activating 3-MeSO(2)-DDE was investigated using structurally analogous compounds. None of the analogues produced histopathological lesions in the mouse adrenal in vivo following a single i.p. injection of 100 mg/kg body weight, but two of the compounds were able to decrease the irreversible binding of 3-MeSO(2)-DDE to Y-1 cells. These results indicate that the bioactivation of 3-MeSO(2)-DDE by CYP11B1 is highly structure-dependent. In conclusion, both 3-MeSO(2)-DDE and o,p'-DDD bind irreversibly to Y-1 cells despite differences in binding and adrenotoxicity in mice

  10. Molecular pathways of human adrenocortical carcinoma - translating cell signalling knowledge into diagnostic and treatment options.

    PubMed

    Szyszka, Paulina; Grossman, Ashley B; Diaz-Cano, Salvador; Sworczak, Krzysztof; Dworakowska, Dorota

    2016-01-01

    Adrenocortical carcinoma is associated with a low cure rate and a high recurrence rate. The prognosis is poor, and at diagnosis 30-40% of cases are already metastatic. The current therapeutic options (surgical resection, followed by adjuvant mitotane treatment +/- chemotherapy) are limited, and the results remain unsatisfactory. Key molecular events that contribute to formation of adrenocortical cancer are IGF2 overexpression, TP53-inactivating mutations, and constitutive activation of the Wnt/b-catenin signalling pathway via activating mutations of the b-catenin gene. The underlying genetic causes of inherited tumour syndromes have provided insights into molecular pathogenesis. The increased occurrence of adrenocortical tumours in Li-Fraumeni and Beckwith-Wiedemann syndromes, and Carney complex, has highlighted the roles of specific susceptibility genes: TP53, IGF2, and PRKAR1A, respectively. Further studies have confirmed that these genes are also involved in sporadic tumour cases. Crucially, transcriptome-wide studies have determined the differences between malignant and benign adrenocortical tumours, providing potential diagnostic tools. In conclusion, enhancing our understanding of the molecular events of adrenocortical tumourigenesis, especially with regard to the signalling pathways that may be disrupted, will greatly contribute to improving a range of available diagnostic, prognostic, and treatment approaches. (Endokrynol Pol 2016; 67 (4): 427-440). PMID:27387247

  11. Loss of expression of a differentiated function gene, steroid 17 alpha-hydroxylase, as adrenocortical cells senescence in culture.

    PubMed Central

    Hornsby, P J; Hancock, J P; Vo, T P; Nason, L M; Ryan, R F; McAllister, J M

    1987-01-01

    Senescence in cultured adrenocortical cells involves changes in expression of differentiated functions as well as changes in responses to mitogenic stimulation. Steroid 17 alpha-hydroxylase (steroid 17 alpha-monooxygenase, EC 1.14.99.9) is an adrenal-specific enzyme, the expression of which is dependent on the presence of stimulators of cyclic AMP production, such as cholera toxin. Dot-blot hybridization of RNA from bovine adrenocortical cells that had been incubated with cholera toxin showed a marked decline in 17 alpha-hydroxylase mRNA levels as a function of population doubling level, closely paralleling the decline in induction of 17 alpha-hydroxylase enzyme activity. The lower levels of 17 alpha-hydroxylase induction did not result from a requirement for a longer time period for induction or from a specific defect in response to cholera toxin and were not caused by a general failure of enzyme induction in response to cyclic AMP. The decreased growth rate in older cells results from a general decline in response to several growth factors. However, the decline in 17 alpha-hydroxylase induction did not result from a loss of response of the cells to mitogens, since quiescent cells at a low population doubling level showed stimulation of 17 alpha-hydroxylase mRNA by cholera toxin to levels similar to those in nonquiescent cultures and added mitogens either had no effect on 17 alpha-hydroxylase mRNA levels or decreased them. There was, however, a specific posttranscriptional effect of insulin on 17 alpha-hydroxylase. The loss of 17 alpha-hydroxylase induction is unlikely to result from overgrowth of a minority cell type lacking the ability to induce 17 alpha-hydroxylase, because adrenocortical cell clones that had high levels of 17 alpha-hydroxylase induction gave rise to cells with lower levels of induction on subcloning. Thus, loss of 17 alpha-hydroxylase activity in adrenocortical cellular senescence results from a primary failure of accumulation of 17 alpha

  12. Adrenocortical carcinoma.

    PubMed

    Baudin, Eric

    2015-06-01

    Recent developments in the treatment of adrenocortical carcinoma (ACC) include diagnostic and prognostic risk stratification algorithms, increasing evidence of the impact of historical therapies on overall survival, and emerging targets from integrated epigenomic and genomic analyses. Advances include proper clinical and molecular characterization of all patients with ACC, standardization of proliferative index analyses, referral of these patients to large cancer referral centers at the time of first surgery, and development of new trials in patients with well-characterized ACC. Networking and progress in the molecular characterization of ACC constitute the basis for significant future therapeutic breakthroughs. PMID:26038209

  13. Combined steroidogenic characters of fetal adrenal and Leydig cells in childhood adrenocortical carcinoma.

    PubMed

    Fujisawa, Yasuko; Sakaguchi, Kimiyoshi; Ono, Hiroyuki; Yamaguchi, Rie; Kato, Fumiko; Kagami, Masayo; Fukami, Maki; Ogata, Tsutomu

    2016-05-01

    Although childhood adrenocortical carcinomas (c-ACCs) with a TP53 mutation are known to produce androgens, detailed steroidogenic characters have not been clarified. Here, we examined steroid metabolite profiles and expression patterns of steroidogenic genes in a c-ACC removed from the left adrenal position of a 2-year-old Brazilian boy with precocious puberty, using an atrophic left adrenal gland removed at the time of tumorectomy as a control. The c-ACC produced not only abundant dehydroepiandrosterone-sulfate but also a large amount of testosterone via the Δ5 pathway with Δ5-androstenediol rather than Δ4-androstenedione as the primary intermediate metabolite. Furthermore, the c-ACC was associated with elevated expressions of CYP11A1, CYP17A1, POR, HSD17B3, and SULT2A1, a low but similar expression of CYB5A, and reduced expressions of AKR1C3 (HSD17B5) and HSD3B2. Notably, a Leydig cell marker INSL3 was expressed at a low but detectable level in the c-ACC. Furthermore, molecular studies revealed a maternally inherited heterozygous germline TP53 mutation, and several post-zygotic genetic aberrations in the c-ACC including loss of paternally derived chromosome 17 with a wildtype TP53 and loss of maternally inherited chromosome 11 and resultant marked hyperexpression of paternally expressed growth promoting gene IGF2 and drastic hypoexpression of maternally expressed growth suppressing gene CDKN1C. These results imply the presence of combined steroidogenic properties of fetal adrenal and Leydig cells in this patient's c-ACC with a germline TP53 mutation and several postzygotic carcinogenic events.

  14. The effect of mitotane on viability, steroidogenesis and gene expression in NCI‑H295R adrenocortical cells.

    PubMed

    Lehmann, Tomasz P; Wrzesiński, Tomasz; Jagodziński, Paweł P

    2013-03-01

    Mitotane, also known as o,p'‑DDD or (RS)‑1‑chl-oro‑2‑[2,2‑dichloro‑1‑(4‑chlorophenyl)‑ethyl]‑benzene, is an adrenal cortex-specific cytotoxic drug used in the therapy of adrenocortical carcinoma (ACC). The drug also inhibits steroidogenesis, however, the mechanisms of its anticancer and antisteroidogenic effects remain unknown. At present, data on the impact of mitotane on cell viability and the regulation of genes encoding proteins associated with steroids synthesis in the adrenal cortex, including cortisol and dehydroepiandrosterone sulfate (DHEAS), are limited and contradictory. In the present study, the effect of 24‑h mitotane treatment on viability of the ACC cell line, NCI‑H295R, was analyzed, identifying a decrease in cell viability and an increase in caspase‑3 and ‑7 activities. Mitotane treatment also led to decreased cortisol and DHEAS concentration in the culture media. Concomitantly, mitotane resulted in decreased mRNA levels of two cytochromes P450 (CYP11A1 and CYP17A1), mRNAs encoding proteins involved in the synthesis of cortisol and DHEAS. Mitotane did not affect mRNA levels of cyclin dependent kinase inhibitor 1A (encoding p21) and MYC (encoding cMyc). cMyc and p21 are key transcription factors associated with cell cycle regulation. However, mitotane inhibited expression of transforming growth factor β1 gene, encoding a potent inhibitor of cell proliferation and steroidogenesis. PRKAR1A, a protein kinase A regulatory subunit, is involved in the activation of steroidogenesis. PRKAR1A mRNA levels were reduced following 24‑h treatment with mitotane. Results indicate that mitotane markedly inhibited expression of genes involved in steroidogenesis, secretion of cortisol and DHEAS. Reduced expression of TGFB1 cannot account fully for the effect of mitotane on CYP11A1 and CYP17A1. We hypothesized that reduced viability of NCI‑H295R cells in the presence of mitotane may be a result of apoptosis triggered by increased

  15. The effect of types I and III interferons on adrenocortical cells and its possible implications for autoimmune Addison's disease.

    PubMed

    Hellesen, A; Edvardsen, K; Breivik, L; Husebye, E S; Bratland, E

    2014-06-01

    Autoimmune Addison's disease (AAD) is caused by selective destruction of the hormone-producing cells of the adrenal cortex. As yet, little is known about the potential role played by environmental factors in this process. Type I and/or type III interferons (IFNs) are signature responses to virus infections, and have also been implicated in the pathogenesis of autoimmune endocrine disorders such as type 1 diabetes and autoimmune thyroiditis. Transient development of AAD and exacerbation of established or subclinical disease, as well as the induction of autoantibodies associated with AAD, have been reported following therapeutic administration of type I IFNs. We therefore hypothesize that exposure to such IFNs could render the adrenal cortex susceptible to autoimmune attack in genetically predisposed individuals. In this study, we investigated possible immunopathological effects of type I and type III IFNs on adrenocortical cells in relation to AAD. Both types I and III IFNs exerted significant cytotoxicity on NCI-H295R adrenocortical carcinoma cells and potentiated IFN-γ- and polyinosine-polycytidylic acid [poly (I : C)]-induced chemokine secretion. Furthermore, we observed increased expression of human leucocyte antigen (HLA) class I molecules and up-regulation of 21-hydroxylase, the primary antigenic target in AAD. We propose that these combined effects could serve to initiate or aggravate an ongoing autoimmune response against the adrenal cortex in AAD.

  16. PROFILING GENE EXPRESSION IN HUMAN H295R ADRENOCORTICAL CARCINOMA CELLS AND RAT TESTES TO IDENTIFY PATHWAYS OF TOXICITY FOR CONAZOLE FUNGICIDES

    EPA Science Inventory

    Profiling Gene Expression in Human H295R Adrenocortical Carcinoma Cells and Rat Testes to Identify Pathways of Toxicity for Conazole Fungicides
    Ren1, H., Schmid1, J., Retief2, J., Turpaz2, Y.,Zhang3, X.,Jones3, P., Newsted3, J.,Giesy3, J., Wolf1, D.,Wood1, C., Bao1, W., Dix1, ...

  17. Expression of the epithelial Na(+) channel and other components of an aldosterone response pathway in human adrenocortical cells.

    PubMed

    Burton, Timothy J; Cope, Georgina; Wang, Jing; Sim, Joalice C; Azizan, Elena A B; O'Shaughnessy, Kevin M; Brown, Morris J

    2009-06-24

    We have unexpectedly found expression of the epithelial Na(+) channel (ENaC) in human adrenocortical cells and tested the hypothesis that these cells contain the components of an aldosterone response pathway. Tissue was obtained from patients undergoing adrenalectomy and mRNA and protein expression of recognised components of an aldosterone-response pathway were determined by RT-PCR and Western blotting. The effects of mineralocorticoid receptor agonists and antagonists, amiloride analogues, and extracellular Na(+) on basal and stimulated aldosterone release from immortalised (H295R) cells were determined by radioimmunoassay. Expression of mRNA for alpha-, beta- and gamma-subunits of ENaC, the mineralocorticoid receptor, Nedd4L, Sgk1 and 11beta hydroxysteroid dehydrogenase type II was confirmed in human adrenal cortex. Using Western blotting alpha-, beta- and gamma-ENaC expression was demonstrated in adrenocortical cells. Measurements of 24 h aldosterone release from H295R cells showed stimulation by K(+) and angiotensin II, suppression by both Na(+) and high-concentration 5-(N-ethyl-N-isopropyl) amiloride (EIPA, blocker of Na(+)-H(+) exchange) and no change with benzamil (ENaC blocker). (22)Na-uptake into H295R cells was inhibited by EIPA, but not by benzamil. Our experiments suggest that the components of an aldosterone response pathway are present in human adrenal cortex. Studies in H295R cells, however, suggest that ENaC is not an important mediator of (22)Na-uptake or aldosterone production. Further studies are required to determine the importance of an adrenal aldosterone response pathway. PMID:19371736

  18. p53 Mutations in human adrenocortical neoplasms: Immunohistochemical and molecular studies

    SciTech Connect

    Reincke, M.; Allolio, B.; Travis, W.H.; Linehan, H.M.; Karl, M.; Mastorakos, G.; Chrousos, G.P.

    1994-03-01

    p53 is a recessive tumor suppressor gene located on chromosome 17p. Mutations in the p53 gene play an important role in the tumorigenesis of diverse types of human neoplasms including breast and colon cancers. More than 90% of all mutations discovered in such tumors have been detected in 4 hot spot areas that lie between exons 5 and 8. In contrast to wild-type p53, mutant p53 accumulates intracellularly and can be easily detected by immunohistochemistry. The authors therefore investigated the frequency of p53 mutations in human adrenocortical neoplasms using molecular biology and immunohistochemistry techniques. Five patients with adrenocortical adenomas (5 female; ages 39-72 yr), 11 patients with adrenocortical carcinomas (8 female, 3 male; ages 15-50 yr), and two adrenocortical tumor cell lines were studied. After DNA extraction from frozen tumor tissue or paraffin-embedded material, exons 5 through 8 were amplified using the polymerase chain reaction and directly sequenced by the dideoxy termination method. Immunohistochemistry was performed on paraffin-embedded tumor specimens obtained during adrenalectomy using a monoclonal antibody reacting with both wild-type and mutant p53. Prevalence of mutations was adenomas, 0/5, carcinomas, 3/11, and adrenocortical cell lines, 2/2. Single point mutations were detected in 3 cases (exons 5, 6, and 7, respectively), and rearrangements of exon 7/8 and 8 were found in 2 cases. Immunohistochemistry detected strong nuclear and/or cytoplasmic p53 immunoreactivity in all adrenocortical carcinomas with point mutations of the p53 gene but not in adenomas and carcinomas with the wild-type sequence or with deletion/rearrangement of the p53 gene. They conclude that p53 plays a role in the tumorigenesis of adrenocortical carcinomas but is of less importance to benign adenomas. 27 refs., 3 figs., 2 tabs.

  19. Orexin-A regulates cell apoptosis in human H295R adrenocortical cells via orexin receptor type 1 through the AKT signaling pathway.

    PubMed

    Chang, Xiaocen; Zhao, Yuyan; Ju, Shujing; Guo, Lei

    2015-11-01

    Numerous studies have demonstrated the ability of orexin-A to regulate adrenocortical cells through the mitogen-activated protein kinase signaling pathway. In the present study, human H295R adrenocortical cells were exposed to orexin‑A (10‑10-10‑6 M), with orexin receptor type 1 (OX1 receptor) antagonist SB334867 or AKT antagonist PF‑04691502. It was found that orexin‑A stimulated H295R cell proliferation, reduced the pro‑apoptotic activity of caspase‑3 to protect against apoptotic cell death and increased cortisol secretion. Furthermore, phospho‑AKT protein was increased by orexin‑A. SB334867 (10‑6 M) and PF‑04691502 (10‑6 M) abolished the effects of orexin‑A (10‑6 M). These results suggested that the orexin‑A/OX1 receptor axis has a significant pro-survival function in adrenal cells, which is mediated by AKT activation. Further studies investigating the effects of orexin-A-upregulation may further elucidate the diverse biological effects of orexin-A in adrenal cells.

  20. The prolonged stimulatory effect of ACTH on 11 beta-hydroxylation, and its contribution to the steroidogenic potency of adrenocortical cells.

    PubMed

    Lambert, F; Lammerant, J; Kolanowski, J

    1984-04-01

    The mechanism of the prolonged stimulatory influence of corticotropin (ACTH) on the capacity of adrenocortical cells to produce cortisol in response to ACTH and more specifically the role of 11 beta-hydroxylation, was studied on guinea-pig adrenocortical cells dispersed from control and ACTH-treated animals. As a result of the previous in vivo exposure to ACTH, the net maximal production of glucocorticoids in response to ACTH (by 10(5) cells and 2 h incubation) increased from 660 +/- 33.9 ng (control group) to 1105 +/- 117.9 ng for cells from ACTH-treated animals (P less than 0.001), whereas the apparent affinity of the steroidogenic response remained unchanged. In addition there occurred an increased conversion of exogenous pregnenolone into cortisol by cells from ACTH-treated animals, indicating a prolonged stimulatory influence of ACTH on the post-pregnenolone pathway of cortisol biosynthesis. The activity of 11 beta-hydroxylation step was therefore examined by incubating the adrenocortical cells from control and ACTH-treated animals in the presence of increasing amounts of 11-deoxycortisol. The maximal capacity of 11-deoxycortisol conversion into cortisol was increased as a result of the in vivo exposure to ACTH, averaging 3423 +/- 211 ng cortisol formed from 5 micrograms 11-deoxycortisol by 10(5) cells from ACTH-treated animals vs 2074 +/- 185 ng for cells from control guinea-pigs (P less than 0.001). However, the conversion of lower amounts of 11-deoxycortisol into cortisol, reproducing quantitatively the maximal effect of ACTH on cortisol biosynthesis, was only barely increased in cells from ACTH-treated animals (P greater than 0.05). Therefore it was concluded that ACTH increases in a lasting way not only the overall steroidogenic capacity of adrenocortical cells but also the maximal efficiency of 11 beta-hydroxylation. Since the latter effect cannot account quantitatively for the magnitude of the lasting effect of ACTH on the maximal capacity of

  1. Origin and Molecular Pathology of Adrenocortical Neoplasms

    PubMed Central

    Bielinska, M.; Parviainen, H.; Kiiveri, S.; Heikinheimo, M.; Wilson, D.B.

    2008-01-01

    Neoplastic adrenocortical lesions are common in humans and several species of domestic animals. Although there are unanswered questions about the origin and evolution of adrenocortical neoplasms, analysis of human tumor specimens and animal models indicates that adrenocortical tumorigenesis involves both genetic and epigenetic alterations. Chromosomal changes accumulate during tumor progression, and aberrant telomere function is one of the key mechanisms underlying chromosome instability during this process. Epigenetic changes serve to expand the size of the uncommitted adrenal progenitor population, modulate their phenotypic plasticity (i.e., responsiveness to extracellular signals), and increase the likelihood of subsequent genetic alterations. Analyses of heritable and spontaneous types of human adrenocortical tumors have documented alterations in either cell surface receptors or their downstream effectors that impact neoplastic transformation. Many of the mutations associated with benign human adrenocortical tumors result in dysregulated cyclic AMP signaling, whereas key factors/signaling pathways associated with adrenocortical carcinomas include dysregulated expression of the IGF2 gene cluster, activation of the Wnt/β-catenin pathway, and inactivation of the p53 tumor suppressor. A better understanding of the factors and signaling pathways involved in adrenal tumorigenesis is necessary to develop targeted pharmacologic and genetic therapies. PMID:19261630

  2. Adrenocortical reactivity and central serotonin and dopamine turnover in young chicks from a high and low feather-pecking line of laying hens.

    PubMed

    van Hierden, Yvonne M; Korte, S Mechiel; Ruesink, E Wim; van Reenen, Cornelis G; Engel, Bas; Korte-Bouws, Gerdien A H; Koolhaas, Jaap M; Blokhuis, Harry J

    2002-04-15

    Feather pecking in domestic fowl is a behavioral abnormality that consists of mild or injurious pecking at feathers of conspecifics. Previously, it was shown that chicks from a high feather-pecking (HFP) and low feather-pecking (LFP) line of laying hens already differ in their propensity to feather peck at 14 and 28 days of age. As a first step in investigating a possible relationship between the development of feather pecking and physiological and neurobiological characteristics of laying hens, two subsequent experiments were carried out. Firstly, we investigated the development of adrenocortical (re)activity in HFP and LFP chicks during the first 8 weeks of life. Secondly, we studied dopamine (DA) and serotonin (5-HT) turnover in the brain of 28-day-old HFP and LFP chicks. In both experiments, chicks were exposed to manual restraint (placing the chicks on its side for 5 min). Plasma corticosterone levels were lower (baseline on Days 3 and 56; restraint-induced on Days 3, 14 and 28) in HFP chicks. Both brain DA and 5-HT turnover were lower in the HFP chicks, as well. Possible consequences for the observed differences in (stress) physiology and neurobiology between the two lines in relation to the feather pecking are discussed. PMID:12020730

  3. Steroid hormone related effects of marine persistent organic pollutants in human H295R adrenocortical carcinoma cells.

    PubMed

    van den Dungen, Myrthe W; Rijk, Jeroen C W; Kampman, Ellen; Steegenga, Wilma T; Murk, Albertinka J

    2015-06-01

    Persistent organic pollutants (POPs) such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), polychlorobiphenyl (PCB) 126 and 153, perfluorooctanesulfonic acid (PFOS), hexabromocyclododecane (HBCD), 2,2',4,4'-tetrabromodiphenyl ether (BDE-47), tributyltin (TBT), and methylmercury (MeHg) can be accumulated in seafood and then form a main source for human exposure. Some POPs have been associated with changes in steroid hormone levels in both humans and animals. This study describes the in vitro effects of these POPs and mixtures thereof in H295R adrenocortical carcinoma cells. Relative responses for 13 steroid hormones and 7 genes involved in the steroidogenic pathway, and CYP1A1, were analyzed. PFOS induced the most pronounced effects on steroid hormone levels by significantly affecting 9 out of 13 hormone levels measured, with the largest increases found for 17β-estradiol, corticosterone, and cortisol. Furthermore, TCDD, both PCBs, and TBT significantly altered steroidogenesis. Increased steroid hormone levels were accompanied by related increased gene expression levels. The differently expressed genes were MC2R, CYP11B1, CYP11B2, and CYP19A1 and changes in gene expression levels were more sensitive than changes in hormone levels. The POP mixtures tested showed mostly additive effects, especially for DHEA and 17β-estradiol levels. This study shows that some seafood POPs are capable of altering steroidogenesis in H295R cells at concentrations that mixtures might reach in human blood, suggesting that adverse health effects cannot be excluded.

  4. 3βHSD and CYB5A double positive adrenocortical cells during adrenal development/aging

    PubMed Central

    Nakamura, Yasuhiro; Fujishima, Fumiyoshi; Hui, Xiao-Gang; Felizola, Saulo J.A.; Shibahara, Yukiko; Akahira, Jun-ichi; McNamara, Keely M.; Rainey, William E.; Sasano, Hironobu

    2014-01-01

    Androstenedione is a common precursor of sex steroids produced and secreted in the human adrenal gland and produced by 3β-hydroxysteroid dehydrogenase (3βHSD), 17α-hydroxylase/17-20 lyase (CYP17) and cytochrome b5 (CYB5A). 3βHSD is expressed in the zona glomerulosa (ZG) and fasciculata (ZF), CYP17 in the ZF and zona reticularis (ZR) and CYB5A in the ZR, respectively. We previously demonstrated the presence of cortical parenchymal cells co-expressing 3βHSD and CYB5A with hybrid features of both ZF and ZR in human adrenal cortex and hypothesized that these cells may play an important role in androstenedione production in human adrenal gland. Age-related morphologic development of these hybrid cells has, however, not been studied. Therefore, in this study, 48 human adrenal specimens from various age groups were retrieved. Double-immunohistochemical analyses were used in order to study the correlation between this hybrid cell type and age. In both male and female adrenal cortex, the mean of total adrenocortical area, the area of CYB5A positive cells and the mean of its ratio reached highest peak in the 21–40 year-old (y.o.). The greatest overlap between 3βHSD and CYB5A in both total and relative area was present in the 13–20 y.o. group. For all of the markers above, statistically significant differences were detected among the different age groups examined (P<0.05). These findings all indicated that both area and ratio of 3βHSD and CYB5A double positive cells, which could represent the hybrid cells of ZF and ZR, are correlated with human adrenal development and could subsequently influence age-related serum androstenedione levels. PMID:24832628

  5. Species-specific sensitivity to selenium-induced impairment of cortisol secretion in adrenocortical cells of rainbow trout (Oncorhynchus mykiss) and brook trout (Salvelinus fontinalis)

    SciTech Connect

    Miller, L.L. Hontela, A.

    2011-06-01

    Species differences in physiological and biochemical attributes exist even among closely related species and may underlie species-specific sensitivity to toxicants. Rainbow trout (RT) are more sensitive than brook trout (BT) to the teratogenic effects of selenium (Se), but it is not known whether all tissues exhibit this pattern of vulnerability. In this study, primary cultures of RT and BT adrenocortical cells were exposed to selenite (Na{sub 2}SO{sub 3}) and selenomethionine (Se-Met) to compare cell viability and ACTH-stimulated cortisol secretion in the two fish species. Cortisol, the primary stress hormone in fish, facilitates maintenance of homeostasis when fish are exposed to stressors, including toxicants. Cell viability was not affected by Se, but selenite impaired cortisol secretion, while Se-Met did not (RT and BT EC{sub 50} > 2000 mg/L). RT cells were more sensitive (EC{sub 50} = 8.7 mg/L) to selenite than BT cells (EC{sub 50} = 90.4 mg/L). To identify the targets where Se disrupts cortisol synthesis, selenite-impaired RT and BT cells were stimulated with ACTH, dbcAMP, OH-cholesterol, and pregnenolone. Selenite acted at different steps in the cortisol biosynthesis pathway in RT and BT cells, confirming a species-specific toxicity mechanism. To test the hypothesis that oxidative stress mediates Se-induced toxicity, selenite-impaired RT cells were exposed to NAC, BSO and antioxidants (DETCA, ATA, Vit A, and Vit E). Inhibition of SOD by DETCA enhanced selenite-induced cortisol impairment, indicating that oxidative stress plays a role in Se toxicity; however, modifying GSH content of the cells did not have an effect. The results of this study, with two closely related salmonids, provided additional evidence for species-specific differences in sensitivity to Se which should be considered when setting thresholds and water quality guidelines. - Research Highlights: > We investigated species-specific sensitivity to Se in trout adrenocortical cells. > Selenite

  6. Stages of Adrenocortical Carcinoma

    MedlinePlus

    ... of Childhood Treatment for more information.) Having certain genetic conditions increases the risk of adrenocortical carcinoma. Anything ... can be a sign of disease. CT scan (CAT scan) : A procedure that makes a series of ...

  7. Opposite regulation of thrombospondin-1 and corticotropin-induced secreted protein/thrombospondin-2 expression by adrenocorticotropic hormone in adrenocortical cells.

    PubMed

    Lafeuillade, B; Pellerin, S; Keramidas, M; Danik, M; Chambaz, E M; Feige, J J

    1996-04-01

    Corticotropin-induced secreted protein (CISP) is a trimeric glycoprotein secreted by primary cultures of bovine adrenortical cells in response to adrenocorticotropic hormone (ACTH). This protein was recently purified in our laboratory, and its N-terminal amino-acid sequence revealed a significant similarity with thrombospondin-2 (TSP2). We report here the nucleotide sequence of a 386 bp RT-PCR fragment specific for CISP. The deduced protein sequence shares 84% identity with the N-terminal portion of mature human TSP2, suggesting that CISP is its bovine counterpart. Northern analysis of adrenocortical cell RNA using the above cDNA fragment as a probe revealed a 6.0 kb CISP/TSP2 mRNA whose abundance was increased nearly fivefold following a 24 h cell treatment with 10(-7) M ACTH. Under the same conditions, the expression of TSP1 mRNA was reduced by tenfold. The protein levels of TSP1 and CISP/TSP2 varied accordingly with their respective mRNA levels, as shown by immunoprecipitation and immunofluorescence experiments. Taken together, these data show that ACTH induces a dramatic shift in the pattern of adrenocortical cell thrombospondin expression from TSP1 to CISP/TSP2. This observation suggests that these two members of the thrombospondin family exert distinct biological functions in the adrenal cortex. This hypothesis is further supported by the observation that anti-CISP antibodies inhibit the maintenance of the morphological changes of bovine adrenocortical cells induced by ACTH, whereas anti-TSP1 antibodies do not. PMID:8698834

  8. High-Throughput Screening of Chemical Effects on Steroidogenesis Using H295R Human Adrenocortical Carcinoma Cells.

    PubMed

    Karmaus, Agnes L; Toole, Colleen M; Filer, Dayne L; Lewis, Kenneth C; Martin, Matthew T

    2016-04-01

    Disruption of steroidogenesis by environmental chemicals can result in altered hormone levels causing adverse reproductive and developmental effects. A high-throughput assay using H295R human adrenocortical carcinoma cells was used to evaluate the effect of 2060 chemical samples on steroidogenesis via high-performance liquid chromatography followed by tandem mass spectrometry quantification of 10 steroid hormones, including progestagens, glucocorticoids, androgens, and estrogens. The study employed a 3 stage screening strategy. The first stage established the maximum tolerated concentration (MTC; ≥ 70% viability) per sample. The second stage quantified changes in hormone levels at the MTC whereas the third stage performed concentration-response (CR) on a subset of samples. At all stages, cells were prestimulated with 10 µM forskolin for 48 h to induce steroidogenesis followed by chemical treatment for 48 h. Of the 2060 chemical samples evaluated, 524 samples were selected for 6-point CR screening, based in part on significantly altering at least 4 hormones at the MTC. CR screening identified 232 chemical samples with concentration-dependent effects on 17β-estradiol and/or testosterone, with 411 chemical samples showing an effect on at least one hormone across the steroidogenesis pathway. Clustering of the concentration-dependent chemical-mediated steroid hormone effects grouped chemical samples into 5 distinct profiles generally representing putative mechanisms of action, including CYP17A1 and HSD3B inhibition. A distinct pattern was observed between imidazole and triazole fungicides suggesting potentially distinct mechanisms of action. From a chemical testing and prioritization perspective, this assay platform provides a robust model for high-throughput screening of chemicals for effects on steroidogenesis.

  9. High-Throughput Screening of Chemical Effects on Steroidogenesis Using H295R Human Adrenocortical Carcinoma Cells

    PubMed Central

    Toole, Colleen M.; Filer, Dayne L.; Lewis, Kenneth C.; Martin, Matthew T.

    2016-01-01

    Disruption of steroidogenesis by environmental chemicals can result in altered hormone levels causing adverse reproductive and developmental effects. A high-throughput assay using H295R human adrenocortical carcinoma cells was used to evaluate the effect of 2060 chemical samples on steroidogenesis via high-performance liquid chromatography followed by tandem mass spectrometry quantification of 10 steroid hormones, including progestagens, glucocorticoids, androgens, and estrogens. The study employed a 3 stage screening strategy. The first stage established the maximum tolerated concentration (MTC; ≥ 70% viability) per sample. The second stage quantified changes in hormone levels at the MTC whereas the third stage performed concentration-response (CR) on a subset of samples. At all stages, cells were prestimulated with 10 µM forskolin for 48 h to induce steroidogenesis followed by chemical treatment for 48 h. Of the 2060 chemical samples evaluated, 524 samples were selected for 6-point CR screening, based in part on significantly altering at least 4 hormones at the MTC. CR screening identified 232 chemical samples with concentration-dependent effects on 17β-estradiol and/or testosterone, with 411 chemical samples showing an effect on at least one hormone across the steroidogenesis pathway. Clustering of the concentration-dependent chemical-mediated steroid hormone effects grouped chemical samples into 5 distinct profiles generally representing putative mechanisms of action, including CYP17A1 and HSD3B inhibition. A distinct pattern was observed between imidazole and triazole fungicides suggesting potentially distinct mechanisms of action. From a chemical testing and prioritization perspective, this assay platform provides a robust model for high-throughput screening of chemicals for effects on steroidogenesis. PMID:26781511

  10. Adrenocortical Zonation, Renewal, and Remodeling

    PubMed Central

    Pihlajoki, Marjut; Dörner, Julia; Cochran, Rebecca S.; Heikinheimo, Markku; Wilson, David B.

    2015-01-01

    The adrenal cortex is divided into concentric zones. In humans the major cortical zones are the zona glomerulosa, zona fasciculata, and zona reticularis. The adrenal cortex is a dynamic organ in which senescent cells are replaced by newly differentiated ones. This constant renewal facilitates organ remodeling in response to physiological demand for steroids. Cortical zones can reversibly expand, contract, or alter their biochemical profiles to accommodate needs. Pools of stem/progenitor cells in the adrenal capsule, subcapsular region, and juxtamedullary region can differentiate to repopulate or expand zones. Some of these pools appear to be activated only during specific developmental windows or in response to extreme physiological demand. Senescent cells can also be replenished through direct lineage conversion; for example, cells in the zona glomerulosa can transform into cells of the zona fasciculata. Adrenocortical cell differentiation, renewal, and function are regulated by a variety of endocrine/paracrine factors including adrenocorticotropin, angiotensin II, insulin-related growth hormones, luteinizing hormone, activin, and inhibin. Additionally, zonation and regeneration of the adrenal cortex are controlled by developmental signaling pathways, such as the sonic hedgehog, delta-like homolog 1, fibroblast growth factor, and WNT/β-catenin pathways. The mechanisms involved in adrenocortical remodeling are complex and redundant so as to fulfill the offsetting goals of organ homeostasis and stress adaptation. PMID:25798129

  11. Beacon[47-73] inhibits glucocorticoid secretion and growth of cultured rat and human adrenocortical cells.

    PubMed

    Ziolkowska, Agnieszka; Carraro, Gianni; Rebuffat, Piera; Spinazzi, Raffaella; Nussdorfer, Gastone G; Rucinski, Marcin; Malendowicz, Ludwik K

    2004-09-01

    Evidence has been recently provided that beacon, an ubiquitin-like protein overexpressed in the hypothalamus of Israeli sand rat, is also expressed in several endocrine glands of the Wistar rat, including adrenal cortex. Moreover, it has been shown that the in vivo administration of beacon[47-73] (hereinafter, beacon) evokes within 60 min a marked decrease in the plasma concentrations of ACTH and corticosterone. Hence, we have investigated the effect of beacon (4x10(-9) or 4x10(-7) M) on the secretion and growth of cultured rat and human zona fasciculata/reticularis (ZF/R) cells. Reverse transcription-polymerase chain reaction detected beacon mRNA in all human adrenal cortexes examined. A 3-h exposure to beacon was ineffective, but prolonged (24 and 96 h) exposures significantly lowered basal corticosterone and cortisol secretion from cultured rat and human ZF/R cells, respectively. Moreover, beacon (4x10(-7) M) counteracted the secretagogue action of 10(-8) M ACTH on cultured cells. The 96-h exposure to beacon concentration-dependently decreased basal proliferation rate of cultured cells, without inducing significant changes in the number of apoptotic and necrotic cells. Beacon (4x10(-7) M) significantly inhibited the proliferogenic effect of 10(-8) M adrenomedullin. In light of the involvement of ubiquitin-like proteins in the control of cell cycle and protein sorting and degradation, the hypothesis is advanced that the inhibitory effect of beacon on the secretion and growth of cultured rat ZF/R cells may be connected to its stimulating effect on proteolysis of steroidogenic enzymes and proteins involved in cell replication.

  12. Corticotropin (ACTH) regulates alternative RNA splicing in Y1 mouse adrenocortical tumor cells.

    PubMed

    Schimmer, Bernard P; Cordova, Martha

    2015-06-15

    The stimulatory effect of ACTH on gene expression is well documented and is thought to be a major mechanism by which ACTH maintains the functional and structural integrity of the gland. Previously, we showed that ACTH regulates the accumulation of over 1200 transcripts in Y1 adrenal cells, including a cluster with functions in alternative splicing of RNA. On this basis, we postulated that some of the effects of ACTH on the transcription landscape of Y1 cells are mediated by alternative splicing. In this study, we demonstrate that ACTH regulates the alternative splicing of four transcripts - Gnas, Cd151, Dab2 and Tia1. Inasmuch as alternative splicing potentially affects transcripts from more than two-thirds of the mouse genome, we suggest that these findings are representative of a genome-wide effect of ACTH that impacts on the mRNA and protein composition of the adrenal cortex.

  13. CLO: The cell line ontology

    PubMed Central

    2014-01-01

    Background Cell lines have been widely used in biomedical research. The community-based Cell Line Ontology (CLO) is a member of the OBO Foundry library that covers the domain of cell lines. Since its publication two years ago, significant updates have been made, including new groups joining the CLO consortium, new cell line cells, upper level alignment with the Cell Ontology (CL) and the Ontology for Biomedical Investigation, and logical extensions. Construction and content Collaboration among the CLO, CL, and OBI has established consensus definitions of cell line-specific terms such as ‘cell line’, ‘cell line cell’, ‘cell line culturing’, and ‘mortal’ vs. ‘immortal cell line cell’. A cell line is a genetically stable cultured cell population that contains individual cell line cells. The hierarchical structure of the CLO is built based on the hierarchy of the in vivo cell types defined in CL and tissue types (from which cell line cells are derived) defined in the UBERON cross-species anatomy ontology. The new hierarchical structure makes it easier to browse, query, and perform automated classification. We have recently added classes representing more than 2,000 cell line cells from the RIKEN BRC Cell Bank to CLO. Overall, the CLO now contains ~38,000 classes of specific cell line cells derived from over 200 in vivo cell types from various organisms. Utility and discussion The CLO has been applied to different biomedical research studies. Example case studies include annotation and analysis of EBI ArrayExpress data, bioassays, and host-vaccine/pathogen interaction. CLO’s utility goes beyond a catalogue of cell line types. The alignment of the CLO with related ontologies combined with the use of ontological reasoners will support sophisticated inferencing to advance translational informatics development. PMID:25852852

  14. Role of EPAC in cAMP-Mediated Actions in Adrenocortical Cells.

    PubMed

    Lewis, Aurélia E; Aesoy, Reidun; Bakke, Marit

    2016-01-01

    Adrenocorticotropic hormone regulates adrenal steroidogenesis mainly via the intracellular signaling molecule cAMP. The effects of cAMP are principally relayed by activating protein kinase A (PKA) and the more recently discovered exchange proteins directly activated by cAMP 1 and 2 (EPAC1 and EPAC2). While the intracellular roles of PKA have been extensively studied in steroidogenic tissues, those of EPACs are only emerging. EPAC1 and EPAC2 are encoded by the genes RAPGEF3 and RAPGEF4, respectively. Whereas EPAC1 is ubiquitously expressed, the expression of EPAC2 is more restricted, and typically found in endocrine tissues. Alternative promoter usage of RAPGEF4 gives rise to three different isoforms of EPAC2 that vary in their N-termini (EPAC2A, EPAC2B, and EPAC2C) and that exhibit distinct expression patterns. EPAC2A is expressed in the brain and pancreas, EPAC2B in steroidogenic cells of the adrenal gland and testis, and EPAC2C has until now only been found in the liver. In this review, we discuss current knowledge on EPAC expression and function with focus on the known roles of EPAC in adrenal gland physiology. PMID:27379015

  15. Role of EPAC in cAMP-Mediated Actions in Adrenocortical Cells

    PubMed Central

    Lewis, Aurélia E.; Aesoy, Reidun; Bakke, Marit

    2016-01-01

    Adrenocorticotropic hormone regulates adrenal steroidogenesis mainly via the intracellular signaling molecule cAMP. The effects of cAMP are principally relayed by activating protein kinase A (PKA) and the more recently discovered exchange proteins directly activated by cAMP 1 and 2 (EPAC1 and EPAC2). While the intracellular roles of PKA have been extensively studied in steroidogenic tissues, those of EPACs are only emerging. EPAC1 and EPAC2 are encoded by the genes RAPGEF3 and RAPGEF4, respectively. Whereas EPAC1 is ubiquitously expressed, the expression of EPAC2 is more restricted, and typically found in endocrine tissues. Alternative promoter usage of RAPGEF4 gives rise to three different isoforms of EPAC2 that vary in their N-termini (EPAC2A, EPAC2B, and EPAC2C) and that exhibit distinct expression patterns. EPAC2A is expressed in the brain and pancreas, EPAC2B in steroidogenic cells of the adrenal gland and testis, and EPAC2C has until now only been found in the liver. In this review, we discuss current knowledge on EPAC expression and function with focus on the known roles of EPAC in adrenal gland physiology. PMID:27379015

  16. Celecoxib reduces glucocorticoids in vitro and in a mouse model with adrenocortical hyperplasia.

    PubMed

    Liu, Sisi; Saloustros, Emmanouil; Berthon, Annabel; Starost, Matthew F; Sahut-Barnola, Isabelle; Salpea, Paraskevi; Szarek, Eva; Faucz, Fabio R; Martinez, Antoine; Stratakis, Constantine A

    2016-01-01

    Primary pigmented nodular adrenocortical disease (PPNAD), whether in the context of Carney complex (CNC) or isolated, leads to ACTH-independent Cushing's syndrome (CS). CNC and PPNAD are caused typically by inactivating mutations of PRKAR1A, a gene coding for the type 1a regulatory subunit (R1α) of cAMP-dependent protein kinase (PKA). Mice lacking Prkar1a, specifically in the adrenal cortex (AdKO) developed CS caused by bilateral adrenal hyperplasia (BAH), which is formed from the abnormal proliferation of fetal-like adrenocortical cells. Celecoxib is a cyclooxygenase 2 (COX2) inhibitor. In bone, Prkar1a inhibition is associated with COX2 activation and prostaglandin E2 (PGE2) production that, in turn, activates proliferation of bone stromal cells. We hypothesized that COX2 inhibition may have an effect in PPNAD. In vitro treatment of human cell lines, including one from a patient with PPNAD, with celecoxib resulted in decreased cell viability. We then treated AdKO and control mice with 1500 mg/kg celecoxib or vehicle. Celecoxib treatment led to decreased PGE2 and corticosterone levels, reduced proliferation and increased apoptosis of adrenocortical cells, and decreased steroidogenic gene expression. We conclude that, in vitro and in vivo, celecoxib led to decreased steroidogenesis. In a mouse model of PPNAD, celecoxib caused histological changes that, at least in part, reversed BAH and this was associated with a reduction of corticosterone levels. PMID:26438728

  17. Celecoxib reduces glucocorticoids in vitro and in a mouse model with adrenocortical hyperplasia

    PubMed Central

    Liu, Sisi; Saloustros, Emmanouil; Berthon, Annabel; Starost, Matthew F.; Sahut-Barnola, Isabelle; Salpea, Paraskevi; Szarek, Eva; Faucz, Fabio R.; Martinez, Antoine; Stratakis, Constantine A.

    2015-01-01

    Primary pigmented nodular adrenocortical disease (PPNAD), whether in the context of Carney complex (CNC) or isolated, leads to adrenocorticotropin hormone (ACTH) - independent Cushing’s syndrome (CS). CNC and PPNAD are caused typically by inactivating mutations of PRKAR1A, a gene coding for the type 1a regulatory subunit (R1α) of cAMP–dependent protein kinase (PKA). Mice lacking Prkar1a, specifically in the adrenal cortex (AdKO) developed CS caused by bilateral adrenal hyperplasia (BAH), which is formed from the abnormal proliferation of fetal-like adrenocortical cells. Celecoxib is a cyclooxygenase-2 (COX2) inhibitor. In bone, Prkar1a inhibition is associated with COX2 activation and prostaglandin E2 (PGE2) production that, in turn, activates proliferation of bone stromal cells. We hypothesized that COX2 inhibition may have an effect in PPNAD. In vitro treatment of human cell lines, including one from a patient with PPNAD, with Celecoxib resulted in decreased cell viability. We then treated AdKO and control mice with 1,500 mg/kg Celecoxib or vehicle. Celecoxib treatment led to decreased PGE2 and corticosterone levels, reduced proliferation and increased apoptosis of adrenocortical cells, and decreased steroidogenic gene expression. We conclude that, in vitro and in vivo, Celecoxib led to decreased steroidogenesis. In a mouse model of PPNAD, Celecoxib caused histological changes that reversed, at least in part, BAH and this was associated with a reduction of corticosterone levels. PMID:26438728

  18. Orexin-A stimulates 3β-hydroxysteroid dehydrogenase expression and cortisol production in H295R human adrenocortical cells through the AKT pathway.

    PubMed

    Chang, Xiaocen; Zhao, Yuyan; Ju, Shujing; Guo, Lei

    2014-12-01

    Orexin-A is a regulatory peptide involved in the regulation of food intake, sleep-wakefulness, and it has various endocrine and metabolic functions. It orchestrates diverse central and peripheral processes through the stimulation of two G-protein coupled receptors, orexin receptor type 1 (OX1 receptor) and orexin receptor type 2 (OX2 receptor). In this study, human adrenocortical cells (NCI-H295R cells) were incubated with various concentrations of orexin-A (10-10 to 10-6 M) in vitro, and the mRNA and protein expression of OX1 receptor was determined in the cells. In addition, NCI-H295R cells treated with 10-6 M orexin-A were then treated with or without OX1 receptor specific antagonist (SB334867), AKT antagonist (PF-04691502), or a combination of both. Subsequently, cell proliferation, the cortisol content in the medium and the mRNA and protein expression expression of 3β-hydroxysteroid dehydrogenase (3β-HSD) were analyzed. The activity of the AKT signaling pathway was also determined in the NCI-H295R cells. We observed that the increase in the mRNA and protein expression of OX1 receptor was orexin-A concentration-dependent, with 10-6 M orexin-A exerting the most potent effect. Orexin-A enhanced cell proliferation and cortisol production, and increased the mRNA and protein expression of 3β-HSD in the NCI-H295R cells; however, these effects were partly blocked by the OX1 receptor antagonist, the AKT antagonist and the combination of both. Furthermore, orexin-A significantly increased the phosphorylation of AKT, with the levels of total AKT protein remaining unaltered. This effect was blocked in the presence of PF-04691502 (10-6 M), SB334867 (10-6 M) and the combination of both. On the whole, our data demonstrate that the effects of orexin-A on the survival and function of human adrenocortical cells are mediated through the AKT signaling pathway.

  19. Biology of SNU Cell Lines

    PubMed Central

    Ku, Ja-Lok

    2005-01-01

    SNU (Seoul National University) cell lines have been established from Korean cancer patients since 1982. Of these 109 cell lines have been characterized and reported, i.e., 17 colorectal carcinoma, 12 hepatocellular carcinoma, 11 gastric carcinoma, 12 uterine cervical carcinoma, 17 B-lymphoblastoid cell lines derived from cancer patients, 5 ovarian carcinoma, 3 malignant mixed Mllerian tumor, 6 laryngeal squamous cell carcinoma, 7 renal cell carcinoma, 9 brain tumor, 6 biliary tract, and 4 pancreatic carcinoma cell lines. These SNU cell lines have been distributed to biomedical researchers domestic and worldwide through the KCLB (Korean Cell Line Bank), and have proven to be of value in various scientific research fields. The characteristics of these cell lines have been reported in over 180 international journals by our laboratory and by many other researchers from 1987. In this paper, the cellular and molecular characteristics of SNU human cancer cell lines are summarized according to their genetic and epigenetic alterations and functional analysis. PMID:19956504

  20. Low DICER1 expression is associated with poor clinical outcome in adrenocortical carcinoma.

    PubMed

    de Sousa, Gabriela Resende Vieira; Ribeiro, Tamaya C; Faria, Andre M; Mariani, Beatriz M P; Lerario, Antonio M; Zerbini, Maria Claudia N; Soares, Iberê C; Wakamatsu, Alda; Alves, Venancio A F; Mendonca, Berenice B; Fragoso, Maria Candida B V; Latronico, Ana Claudia; Almeida, Madson Q

    2015-09-01

    Low DICER1 expression was associated with poor outcome in several cancers. Recently, hot-spot DICER1 mutations were found in ovarian tumors, and TARBP2 truncating mutations in tumor cell lines with microsatellite instability. In this study, we assessed DICER1 e TRBP protein expression in 154 adult adrenocortical tumors (75 adenomas and 79 carcinomas). Expression of DICER1 and TARBP2 gene was assessed in a subgroup of 61 tumors. Additionally, we investigated mutations in metal biding sites located at the RNase IIIb domain of DICER1 and in the exon 5 of TARBP2 in 61 tumors. A strong DICER1 expression was demonstrated in 32% of adenomas and in 51% of carcinomas (p = 0.028). Similarly, DICER1 gene overexpression was more frequent in carcinomas (60%) than in adenomas (23%, p = 0.006). But, among adrenocortical carcinomas, a weak DICER1 expression was significantly more frequent in metastatic than in non-metastatic adrenocortical carcinomas (66% vs. 31%; p = 0.002). Additionally, a weak DICER1 expression was significantly correlated with a reduced overall (p = 0.004) and disease-free (p = 0.005) survival. In the multivariate analysis, a weak DICER1 expression (p = 0.048) remained as independent predictor of recurrence. Regarding TARBP2 gene, its protein and gene expression did not correlate with histopathological and clinical parameters. No variant was identified in hot spot areas of DICER1 and TARBP2. In conclusion, a weak DICER1 protein expression was associated with reduced disease-free and overall survival and was a predictor of recurrence in adrenocortical carcinomas.

  1. Alterations of Phosphodiesterases in Adrenocortical Tumors

    PubMed Central

    Hannah-Shmouni, Fady; Faucz, Fabio R.; Stratakis, Constantine A.

    2016-01-01

    Alterations in the cyclic (c)AMP-dependent signaling pathway have been implicated in the majority of benign adrenocortical tumors (ACTs) causing Cushing syndrome (CS). Phosphodiesterases (PDEs) are enzymes that regulate cyclic nucleotide levels, including cyclic adenosine monophosphate (cAMP). Inactivating mutations and other functional variants in PDE11A and PDE8B, two cAMP-binding PDEs, predispose to ACTs. The involvement of these two genes in ACTs was initially revealed by a genome-wide association study in patients with micronodular bilateral adrenocortical hyperplasia. Thereafter, PDE11A or PDE8B genetic variants have been found in other ACTs, including macronodular adrenocortical hyperplasias and cortisol-producing adenomas. In addition, downregulation of PDE11A expression and inactivating variants of the gene have been found in hereditary and sporadic testicular germ cell tumors, as well as in prostatic cancer. PDEs confer an increased risk of ACT formation probably through, primarily, their action on cAMP levels, but other actions might be possible. In this report, we review what is known to date about PDE11A and PDE8B and their involvement in the predisposition to ACTs. PMID:27625633

  2. Alterations of Phosphodiesterases in Adrenocortical Tumors

    PubMed Central

    Hannah-Shmouni, Fady; Faucz, Fabio R.; Stratakis, Constantine A.

    2016-01-01

    Alterations in the cyclic (c)AMP-dependent signaling pathway have been implicated in the majority of benign adrenocortical tumors (ACTs) causing Cushing syndrome (CS). Phosphodiesterases (PDEs) are enzymes that regulate cyclic nucleotide levels, including cyclic adenosine monophosphate (cAMP). Inactivating mutations and other functional variants in PDE11A and PDE8B, two cAMP-binding PDEs, predispose to ACTs. The involvement of these two genes in ACTs was initially revealed by a genome-wide association study in patients with micronodular bilateral adrenocortical hyperplasia. Thereafter, PDE11A or PDE8B genetic variants have been found in other ACTs, including macronodular adrenocortical hyperplasias and cortisol-producing adenomas. In addition, downregulation of PDE11A expression and inactivating variants of the gene have been found in hereditary and sporadic testicular germ cell tumors, as well as in prostatic cancer. PDEs confer an increased risk of ACT formation probably through, primarily, their action on cAMP levels, but other actions might be possible. In this report, we review what is known to date about PDE11A and PDE8B and their involvement in the predisposition to ACTs.

  3. Alterations of Phosphodiesterases in Adrenocortical Tumors.

    PubMed

    Hannah-Shmouni, Fady; Faucz, Fabio R; Stratakis, Constantine A

    2016-01-01

    Alterations in the cyclic (c)AMP-dependent signaling pathway have been implicated in the majority of benign adrenocortical tumors (ACTs) causing Cushing syndrome (CS). Phosphodiesterases (PDEs) are enzymes that regulate cyclic nucleotide levels, including cyclic adenosine monophosphate (cAMP). Inactivating mutations and other functional variants in PDE11A and PDE8B, two cAMP-binding PDEs, predispose to ACTs. The involvement of these two genes in ACTs was initially revealed by a genome-wide association study in patients with micronodular bilateral adrenocortical hyperplasia. Thereafter, PDE11A or PDE8B genetic variants have been found in other ACTs, including macronodular adrenocortical hyperplasias and cortisol-producing adenomas. In addition, downregulation of PDE11A expression and inactivating variants of the gene have been found in hereditary and sporadic testicular germ cell tumors, as well as in prostatic cancer. PDEs confer an increased risk of ACT formation probably through, primarily, their action on cAMP levels, but other actions might be possible. In this report, we review what is known to date about PDE11A and PDE8B and their involvement in the predisposition to ACTs. PMID:27625633

  4. Treatment Option Overview (Adrenocortical Carcinoma)

    MedlinePlus

    ... of Childhood Treatment for more information.) Having certain genetic conditions increases the risk of adrenocortical carcinoma. Anything ... can be a sign of disease. CT scan (CAT scan) : A procedure that makes a series of ...

  5. Thyroid cell lines in research on goitrogenesis.

    PubMed

    Gerber, H; Peter, H J; Asmis, L; Studer, H

    1991-12-01

    Thyroid cell lines have contributed a lot to the understanding of goitrogenesis. The cell lines mostly used in thyroid research are briefly discussed, namely the rat thyroid cell lines FRTL and FRTL-5, the porcine thyroid cell lines PORTHOS and ARTHOS, The sheep thyroid cell lines OVNIS 5H and 6H, the cat thyroid cell lines PETCAT 1 to 4 and ROMCAT, and the human thyroid cell lines FTC-133 and HTh 74. Chinese hamster ovary (CHO) cells and COS-7 cells, stably transfected with TSH receptor cDNA and expressing a functional TSH receptor, are discussed as examples for non-thyroidal cells, transfected with thyroid genes. PMID:1726925

  6. Adrenocortical function in cane toads from different environments.

    PubMed

    Hernández, Sandra E; Sernia, Conrad; Bradley, Adrian J

    2016-05-01

    The adrenocortical function of cane toads (Rhinella marina) exposed to different experimental procedures, as well as captured from different environments, was assessed by challenging the hypothalamic-pituitary-adrenal (HPA) axis. It was found that restriction stress as well as cannulation increased plasma corticosterone (B) levels for up to 12h. A single dose of dexamethasone (DEX 2mg/kg) significantly reduced B levels demonstrating its potential for use in the evaluation of the HPA axis in amphibia. We also demonstrate that 0.05 IU/g BW (im) of synthetic adrenocorticotropic hormone (ACTH) significantly increased plasma B levels in cane toads. Changes in size area of the cortical cells were positively associated with total levels of B after ACTH administration. We also found differences in adrenal activity between populations. This was assessed by a DEX-ACTH test. The animals captured from the field and maintained in captivity for one year at the animal house (AH) present the highest levels of total and free B after ACTH administration. We also found that animals from the front line of dispersion in Western Australia (WA) present the weakest adrenal response to a DEX-ACTH test. The animals categorized as long established in Queensland Australia (QL), and native in Mexico (MX), do not shown a marked difference in the HPA activity. Finally we found that in response to ACTH administration, females reach significantly higher levels of plasma B than males. For the first time the adrenocortical response in cane toads exposed to different experimental procedures, as well as from different populations was assessed systematically. PMID:26877241

  7. Estrogen related receptor α (ERRα) a promising target for the therapy of adrenocortical carcinoma (ACC).

    PubMed

    Casaburi, Ivan; Avena, Paola; De Luca, Arianna; Chimento, Adele; Sirianni, Rosa; Malivindi, Rocco; Rago, Vittoria; Fiorillo, Marco; Domanico, Francesco; Campana, Carmela; Cappello, Anna Rita; Sotgia, Federica; Lisanti, Michael P; Pezzi, Vincenzo

    2015-09-22

    The pathogenesis of the adrenocortical cancer (ACC) involves integration of molecular signals and the interplay of different downstream pathways (i.e. IGFII/IGF1R, β-catenin, Wnt, ESR1). This tumor is characterized by limited therapeutic options and unsuccessful treatments. A useful strategy to develop an effective therapy for ACC is to identify a common downstream target of these multiple pathways. A good candidate could be the transcription factor estrogen-related receptor alpha (ERRα) because of its ability to regulate energy metabolism, mitochondrial biogenesis and signalings related to cancer progression. In this study we tested the effect of ERRα inverse agonist, XCT790, on the proliferation of H295R adrenocortical cancer cell line. Results from in vitro and in vivo experiments showed that XCT790 reduced H295R cell growth. The inhibitory effect was associated with impaired cell cycle progression which was not followed by any apoptotic event. Instead, incomplete autophagy and cell death by a necrotic processes, as a consequence of the cell energy failure, induced by pharmacological reduction of ERRα was evidenced. Our results indicate that therapeutic strategies targeting key factors such as ERRα that control the activity and signaling of bioenergetics processes in high-energy demanding tumors could represent an innovative/alternative therapy for the treatment of ACC. PMID:26312764

  8. Estrogen related receptor α (ERRα) a promising target for the therapy of adrenocortical carcinoma (ACC)

    PubMed Central

    Chimento, Adele; Sirianni, Rosa; Malivindi, Rocco; Rago, Vittoria; Fiorillo, Marco; Domanico, Francesco; Campana, Carmela; Cappello, Anna Rita; Sotgia, Federica; Lisanti, Michael P.; Pezzi, Vincenzo

    2015-01-01

    The pathogenesis of the adrenocortical cancer (ACC) involves integration of molecular signals and the interplay of different downstream pathways (i.e. IGFII/IGF1R, β-catenin, Wnt, ESR1). This tumor is characterized by limited therapeutic options and unsuccessful treatments. A useful strategy to develop an effective therapy for ACC is to identify a common downstream target of these multiple pathways. A good candidate could be the transcription factor estrogen-related receptor alpha (ERRα) because of its ability to regulate energy metabolism, mitochondrial biogenesis and signalings related to cancer progression. In this study we tested the effect of ERRα inverse agonist, XCT790, on the proliferation of H295R adrenocortical cancer cell line. Results from in vitro and in vivo experiments showed that XCT790 reduced H295R cell growth. The inhibitory effect was associated with impaired cell cycle progression which was not followed by any apoptotic event. Instead, incomplete autophagy and cell death by a necrotic processes, as a consequence of the cell energy failure, induced by pharmacological reduction of ERRα was evidenced. Our results indicate that therapeutic strategies targeting key factors such as ERRα that control the activity and signaling of bioenergetics processes in high-energy demanding tumors could represent an innovative/alternative therapy for the treatment of ACC. PMID:26312764

  9. Adrenocortical Gap Junctions and Their Functions

    PubMed Central

    Bell, Cheryl L.; Murray, Sandra A.

    2016-01-01

    Adrenal cortical steroidogenesis and proliferation are thought to be modulated by gap junction-mediated direct cell–cell communication of regulatory molecules between cells. Such communication is regulated by the number of gap junction channels between contacting cells, the rate at which information flows between these channels, and the rate of channel turnover. Knowledge of the factors regulating gap junction-mediated communication and the turnover process are critical to an understanding of adrenal cortical cell functions, including development, hormonal response to adrenocorticotropin, and neoplastic dedifferentiation. Here, we review what is known about gap junctions in the adrenal gland, with particular attention to their role in adrenocortical cell steroidogenesis and proliferation. Information and insight gained from electrophysiological, molecular biological, and imaging (immunocytochemical, freeze fracture, transmission electron microscopic, and live cell) techniques will be provided. PMID:27445985

  10. Adrenocortical Activity and Emotion Regulation.

    ERIC Educational Resources Information Center

    Stansbury, Kathy; Gunnar, Megan R.

    1994-01-01

    This essay argues that the activity of the hypothalamic-pituitary-adrenocortical (HPA) system does not appear to be related to emotion regulation processes in children, although individual differences in emotion processes related to negative emotion temperaments appear to be associated with individual differences in HPA reactivity among normally…

  11. Integrated genome-wide analysis of genomic changes and gene regulation in human adrenocortical tissue samples

    PubMed Central

    Gara, Sudheer Kumar; Wang, Yonghong; Patel, Dhaval; Liu-Chittenden, Yi; Jain, Meenu; Boufraqech, Myriem; Zhang, Lisa; Meltzer, Paul S.; Kebebew, Electron

    2015-01-01

    To gain insight into the pathogenesis of adrenocortical carcinoma (ACC) and whether there is progression from normal-to-adenoma-to-carcinoma, we performed genome-wide gene expression, gene methylation, microRNA expression and comparative genomic hybridization (CGH) analysis in human adrenocortical tissue (normal, adrenocortical adenomas and ACC) samples. A pairwise comparison of normal, adrenocortical adenomas and ACC gene expression profiles with more than four-fold expression differences and an adjusted P-value < 0.05 revealed no major differences in normal versus adrenocortical adenoma whereas there are 808 and 1085, respectively, dysregulated genes between ACC versus adrenocortical adenoma and ACC versus normal. The majority of the dysregulated genes in ACC were downregulated. By integrating the CGH, gene methylation and expression profiles of potential miRNAs with the gene expression of dysregulated genes, we found that there are higher alterations in ACC versus normal compared to ACC versus adrenocortical adenoma. Importantly, we identified several novel molecular pathways that are associated with dysregulated genes and further experimentally validated that oncostatin m signaling induces caspase 3 dependent apoptosis and suppresses cell proliferation. Finally, we propose that there is higher number of genomic changes from normal-to-adenoma-to-carcinoma and identified oncostatin m signaling as a plausible druggable pathway for therapeutics. PMID:26446994

  12. Integrated genome-wide analysis of genomic changes and gene regulation in human adrenocortical tissue samples.

    PubMed

    Gara, Sudheer Kumar; Wang, Yonghong; Patel, Dhaval; Liu-Chittenden, Yi; Jain, Meenu; Boufraqech, Myriem; Zhang, Lisa; Meltzer, Paul S; Kebebew, Electron

    2015-10-30

    To gain insight into the pathogenesis of adrenocortical carcinoma (ACC) and whether there is progression from normal-to-adenoma-to-carcinoma, we performed genome-wide gene expression, gene methylation, microRNA expression and comparative genomic hybridization (CGH) analysis in human adrenocortical tissue (normal, adrenocortical adenomas and ACC) samples. A pairwise comparison of normal, adrenocortical adenomas and ACC gene expression profiles with more than four-fold expression differences and an adjusted P-value < 0.05 revealed no major differences in normal versus adrenocortical adenoma whereas there are 808 and 1085, respectively, dysregulated genes between ACC versus adrenocortical adenoma and ACC versus normal. The majority of the dysregulated genes in ACC were downregulated. By integrating the CGH, gene methylation and expression profiles of potential miRNAs with the gene expression of dysregulated genes, we found that there are higher alterations in ACC versus normal compared to ACC versus adrenocortical adenoma. Importantly, we identified several novel molecular pathways that are associated with dysregulated genes and further experimentally validated that oncostatin m signaling induces caspase 3 dependent apoptosis and suppresses cell proliferation. Finally, we propose that there is higher number of genomic changes from normal-to-adenoma-to-carcinoma and identified oncostatin m signaling as a plausible druggable pathway for therapeutics.

  13. The combination of insulin-like growth factor receptor 1 (IGF1R) antibody cixutumumab and mitotane as a first-line therapy for patients with recurrent/metastatic adrenocortical carcinoma: a multi-institutional NCI-sponsored trial.

    PubMed

    Lerario, Antonio M; Worden, Francis P; Ramm, Carole A; Hesseltine, Elizabeth A; Hasseltine, Elizabeth A; Stadler, Walter M; Else, Tobias; Shah, Manisha H; Agamah, Edem; Rao, Krishna; Hammer, Gary D

    2014-08-01

    Adrenocortical carcinoma (ACC) is an aggressive malignancy, which lacks an effective systemic treatment. Abnormal activation of insulin-like growth factor receptor 1 (IGF1R) has been frequently observed. Preclinical studies demonstrated that pharmacological inhibition of IGF1R signaling in ACC has antiproliferative effects. A previous phase I trial with an IGF1R inhibitor has demonstrated biological activity against ACC. The objective of this study is to assess the efficacy of the combination of the IGF1R inhibitor cixutumumab (IMC-A12) in association with mitotane as a first-line treatment for advanced/metastatic ACC. We conducted a multicenter, randomized double-arm phase II trial in patients with irresectable recurrent/metastatic ACC. The original protocol included two treatment groups: IMC-A12 + mitotane and mitotane as a single agent, after an initial single-arm phase for safety evaluation with IMC-A12 + mitotane. IMC-A12 was dosed at 10 mg/kg intravenously every 2 weeks. The starting dose for mitotane was 2 g daily, subsequently adjusted according to serum levels/symptoms. The primary endpoint was progression-free survival (PFS) according to RECIST (Response Evaluation Criteria in Solid Tumors). This study was terminated before the randomization phase due to slow accrual and limited efficacy. Twenty patients (13 males, 7 females) with a median age of 50.2 years (range 21.9-79.6) were enrolled for the single-arm phase. Therapeutic effects were observed in 8/20 patients, including one partial response and seven stable diseases. The median PFS was 6 weeks (range 2.66-48). Toxic events included two grade 4 (hyperglycemia and hyponatremia) and one grade 5 (multiorgan failure). Although the regimen demonstrated activity in some patients, the relatively low therapeutic efficacy precluded further studies with this combination of drugs. PMID:24849545

  14. Effects of bisphenol A-related diphenylalkanes on vitellogenin production in male carp (Cyprinus carpio) hepatocytes and aromatase (CYP19) activity in human H295r adrenocortical carcinoma cells

    SciTech Connect

    Letcher, Robert J. . E-mail: robert.letcher@ec.gc.ca; Sanderson, J. Thomas; Bokkers, Abraham; Giesy, John P.; Berg, Martin van den

    2005-12-01

    The present study investigated the effects of the known xenoestrogen bisphenol A (BPA) relative to eight BPA-related diphenylalkanes on estrogen receptor (ER)-mediated vitellogenin (vtg) production in hepatocytes from male carp (Cyprinus carpio), and on aromatase (CYP19) activity in the human adrenocortical H295R carcinoma cell line. Of the eight diphenylalkanes, only 4,4'-(hexafluoropropylidene)diphenol (BHF) and 2,2'-bis(4-hydroxy-3-methylphenyl)propane (BPRO) induced vtg, i.e., to a maximum of 3% to 4% (at 100 {mu}M) compared with 8% for BPA relative to the maximum induction by 17{beta}-estradiol (E2, 1 {mu}M). Bisphenol A diglycidyl ether (BADGE) was a potent antagonist of vtg production with an IC50 of 5.5 {mu}M, virtually 100% inhibition of vtg at 20 {mu}M, and an inhibitive (IC50) potency about one-tenth that of the known ER antagonist tamoxifen (IC50, 0.6 {mu}M). 2,2'-Diallyl bisphenol A, 4,4'-(1,4-phenylene-diisopropylidene)bisphenol, BPRO, and BHF were much less inhibitory with IC50 concentrations of 20-70 {mu}M, and relative potencies of 0.03 and 0.009 with tamoxifen. Bisphenol ethoxylate showed no anti-estrogenicity (up to 100 {mu}M), and 4,4'-isopropylidene-diphenol diacetate was only antagonistic at 100 {mu}M. When comparing the (anti)estrogenic potencies of these bisphenol A analogues/diphenylalkanes, anti-estrogenicity occurred at lower concentrations than estrogenicity. 4,4'-Isopropylidenebis(2,6-dimethylphenol) (IC50, 2.0 {mu}M) reduced E2-induced (EC50, 100 nM) vtg production due to concentration-dependent cytotoxicity as indicated by a parallel decrease in MTT activity and vtg, whereas the remaining diphenylalkanes did not cause any cytotoxicity relative to controls. None of the diphenylalkanes (up to 100 {mu}M) induced EROD activity indicating that concentration-dependent, CYP1A enzyme-mediated metabolism of E2, or any Ah-receptor-mediated interaction with the ER, was not a likely explanation for the observed anti-estrogenic effects. At

  15. Effects of bisphenol A-related diphenylalkanes on vitellogenin production in male carp (Cyprinus carpio) hepatocytes and aromatase (CYP19) activity in human H295R adrenocortical carcinoma cells.

    PubMed

    Letcher, Robert J; Sanderson, J Thomas; Bokkers, Abraham; Giesy, John P; van den Berg, Martin

    2005-12-01

    The present study investigated the effects of the known xenoestrogen bisphenol A (BPA) relative to eight BPA-related diphenylalkanes on estrogen receptor (ER)-mediated vitellogenin (vtg) production in hepatocytes from male carp (Cyprinus carpio), and on aromatase (CYP19) activity in the human adrenocortical H295R carcinoma cell line. Of the eight diphenylalkanes, only 4,4'-(hexafluoropropylidene)diphenol (BHF) and 2,2'-bis(4-hydroxy-3-methylphenyl)propane (BPRO) induced vtg, i.e., to a maximum of 3% to 4% (at 100 microM) compared with 8% for BPA relative to the maximum induction by 17beta-estradiol (E2, 1 microM). Bisphenol A diglycidyl ether (BADGE) was a potent antagonist of vtg production with an IC50 of 5.5 microM, virtually 100% inhibition of vtg at 20 microM, and an inhibitive (IC50) potency about one-tenth that of the known ER antagonist tamoxifen (IC50, 0.6 microM). 2,2'-Diallyl bisphenol A, 4,4'-(1,4-phenylene-diisopropylidene)bisphenol, BPRO, and BHF were much less inhibitory with IC50 concentrations of 20-70 microM, and relative potencies of 0.03 and 0.009 with tamoxifen. Bisphenol ethoxylate showed no anti-estrogenicity (up to 100 microM), and 4,4'-isopropylidene-diphenol diacetate was only antagonistic at 100 microM. When comparing the (anti)estrogenic potencies of these bisphenol A analogues/diphenylalkanes, anti-estrogenicity occurred at lower concentrations than estrogenicity. 4,4'-Isopropylidenebis(2,6-dimethylphenol) (IC50, 2.0 microM) reduced E2-induced (EC50, 100 nM) vtg production due to concentration-dependent cytotoxicity as indicated by a parallel decrease in MTT activity and vtg, whereas the remaining diphenylalkanes did not cause any cytotoxicity relative to controls. None of the diphenylalkanes (up to 100 microM) induced EROD activity indicating that concentration-dependent, CYP1A enzyme-mediated metabolism of E2, or any Ah-receptor-mediated interaction with the ER, was not a likely explanation for the observed anti-estrogenic effects. At

  16. Isolation of rat adrenocortical mitochondria

    SciTech Connect

    Solinas, Paola; Fujioka, Hisashi; Tandler, Bernard; Hoppel, Charles L.

    2012-10-12

    Highlights: Black-Right-Pointing-Pointer A method for isolation of adrenocortical mitochondria from the adrenal gland of rats is described. Black-Right-Pointing-Pointer The purified isolated mitochondria show excellent morphological integrity. Black-Right-Pointing-Pointer The properties of oxidative phosphorylation are excellent. Black-Right-Pointing-Pointer The method increases the opportunity of direct analysis of adrenal mitochondria from small animals. -- Abstract: This report describes a relatively simple and reliable method for isolating adrenocortical mitochondria from rats in good, reasonably pure yield. These organelles, which heretofore have been unobtainable in isolated form from small laboratory animals, are now readily accessible. A high degree of mitochondrial purity is shown by the electron micrographs, as well as the structural integrity of each mitochondrion. That these organelles have retained their functional integrity is shown by their high respiratory control ratios. In general, the biochemical performance of these adrenal cortical mitochondria closely mirrors that of typical hepatic or cardiac mitochondria.

  17. Cholesterol and steroid synthesizing smooth endoplasmic reticulum of adrenocortical cells contains high levels of proteins associated with the translocation channel.

    PubMed

    Black, Virginia H; Sanjay, Archana; van Leyen, Klaus; Lauring, Brett; Kreibich, Gert

    2005-10-01

    Steroid-secreting cells are characterized by abundant smooth endoplasmic reticulum whose membranes contain many enzymes involved in sterol and steroid synthesis. Yet they have relatively little morphologically identifiable rough endoplasmic reticulum, presumably required for synthesis and maintenance of the smooth membranes. In this study, we demonstrate that adrenal smooth microsomal subfractions enriched in smooth endoplasmic reticulum membranes contain high levels of translocation apparatus and oligosaccharyltransferase complex proteins, previously thought confined to rough endoplasmic reticulum. We further demonstrate that these smooth microsomal subfractions are capable of effecting cotranslational translocation, signal peptide cleavage, and N-glycosylation of newly synthesized polypeptides. This shifts the paradigm for distinction between smooth and rough endoplasmic reticulum. Confocal microscopy revealed the proteins to be distributed throughout the abundant tubular endoplasmic reticulum in these cells, which is predominantly smooth surfaced. We hypothesize that the broadly distributed translocon and oligosaccharyltransferase proteins participate in local synthesis and/or quality control of membrane proteins involved in cholesterol and steroid metabolism in a sterol-dependent and hormonally regulated manner.

  18. Molluscan cells in culture: primary cell cultures and cell lines

    PubMed Central

    Yoshino, T. P.; Bickham, U.; Bayne, C. J.

    2013-01-01

    In vitro cell culture systems from molluscs have significantly contributed to our basic understanding of complex physiological processes occurring within or between tissue-specific cells, yielding information unattainable using intact animal models. In vitro cultures of neuronal cells from gastropods show how simplified cell models can inform our understanding of complex networks in intact organisms. Primary cell cultures from marine and freshwater bivalve and gastropod species are used as biomonitors for environmental contaminants, as models for gene transfer technologies, and for studies of innate immunity and neoplastic disease. Despite efforts to isolate proliferative cell lines from molluscs, the snail Biomphalaria glabrata Say, 1818 embryonic (Bge) cell line is the only existing cell line originating from any molluscan species. Taking an organ systems approach, this review summarizes efforts to establish molluscan cell cultures and describes the varied applications of primary cell cultures in research. Because of the unique status of the Bge cell line, an account is presented of the establishment of this cell line, and of how these cells have contributed to our understanding of snail host-parasite interactions. Finally, we detail the difficulties commonly encountered in efforts to establish cell lines from molluscs and discuss how these difficulties might be overcome. PMID:24198436

  19. Combined transcriptome studies identify AFF3 as a mediator of the oncogenic effects of β-catenin in adrenocortical carcinoma

    PubMed Central

    Lefèvre, L; Omeiri, H; Drougat, L; Hantel, C; Giraud, M; Val, P; Rodriguez, S; Perlemoine, K; Blugeon, C; Beuschlein, F; de Reyniès, A; Rizk-Rabin, M; Bertherat, J; Ragazzon, B

    2015-01-01

    Adrenocortical cancer (ACC) is a very aggressive tumor, and genomics studies demonstrate that the most frequent alterations of driver genes in these cancers activate the Wnt/β-catenin signaling pathway. However, the adrenal-specific targets of oncogenic β-catenin-mediating tumorigenesis have not being established. A combined transcriptomic analysis from two series of human tumors and the human ACC cell line H295R harboring a spontaneous β-catenin activating mutation was done to identify the Wnt/β-catenin targets. Seven genes were consistently identified in the three studies. Among these genes, we found that AFF3 mediates the oncogenic effects of β-catenin in ACC. The Wnt response element site located at nucleotide position −1408 of the AFF3 transcriptional start sites (TSS) mediates the regulation by the Wnt/β-catenin signaling pathway. AFF3 silencing decreases cell proliferation and increases apoptosis in the ACC cell line H295R. AFF3 is located in nuclear speckles, which play an important role in RNA splicing. AFF3 overexpression in adrenocortical cells interferes with the organization and/or biogenesis of these nuclear speckles and alters the distribution of CDK9 and cyclin T1 such that they accumulate at the sites of AFF3/speckles. We demonstrate that AFF3 is a new target of Wnt/β-catenin pathway involved in ACC, acting on transcription and RNA splicing. PMID:26214578

  20. Cell line: 2004-2014.

    PubMed

    2014-11-20

    2014 marks Cell's 40th anniversary, and over the year we have looked back at how discoveries of the last four decades have molded our understanding of biology. The final decade of the Cell Line features a selection of the exceptional scientific work-both landmark papers and essential reviews. Select entries can be read as an "Annotated Classic," which includes the original paper and accompanying reflections of a leading scientist, considering the work from our current vantage point. Our last installment includes a harbinger of the interplay between microbiota and mammalian hosts in 2004, revolutionary papers in 2006 and 2007 unlocking cellular reprogramming, the discovery of beige adipocytes in 2012, and the first example of CRISPR-based genome editing in a nonhuman primate in 2014. In addition to landmark publications, there were innovative developments at the journal in this decade, with the complete redesign of the print journal and the creation of Leading Edge in late 2005 and the restructuring of the online display of the article in 2010. Keeping pace with the changing nature of biological research, over the decade Cell added new article types, introduced guidelines for the organization of supplementary material, and expanded the journal's web-based content to bring editors' and authors' excitement and perspective on individual papers to the readership. An interactive version of the timeline, with links to the papers, full author lists, and Annotated Classics, is available at http://dx.doi.org/10.1016/j.cell.2014.11.004. PMID:25416957

  1. Pathogenesis of benign adrenocortical tumors.

    PubMed

    Vezzosi, Delphine; Bertherat, Jérôme; Groussin, Lionel

    2010-12-01

    Most adrenocortical tumors (ACT) are benign unilateral adrenocortical adenomas, often discovered incidentally. Exceptionally, ACT are bilateral. However bilateral ACT have been very helpful to progress in the pathophysiology of ACT. Although most ACT are of sporadic origin, they may also be part of syndromic and/or hereditary disorders. The identification of the genetics of familial diseases associated with benign ACT has been helpful to define somatic alterations in sporadic ACT: for example, identification of PRKAR1A mutations in Carney complex or alterations of the Wnt/β-catenin pathway in Familial Adenomatous Polyposis Coli. Components of the cAMP signaling pathway-for example, adrenocorticotropic-hormone receptors and other membrane receptors, Gs protein, phosphodiesterases and protein kinase A-can be altered to various degrees in benign cortisol-secreting ACT. These progress have been important for the understanding of the pathogenesis of benign ACT, but already have profound implications for clinical management, for example in unraveling the genetic origin of disease in some patients with ACT. They also have therapeutic consequences, and should help to develop new therapeutic options. PMID:21115158

  2. HLA expression in hepatocellular carcinoma cell lines.

    PubMed

    Wadee, A A; Paterson, A; Coplan, K A; Reddy, S G

    1994-08-01

    The present study undertook to investigate the biological significance of human leucocyte antigen expression in hepatocellular carcinoma and to elucidate the role of potential modulating agents on human leucocyte antigen expression. These studies used several hepatic tumour-derived cell lines as in vitro model systems. The cell lines included PLC/PRF/5 (Alexander cell line), Hep3B, HepG2, TONG PHC, HA22T/VGH, HA59T/VGH and Mahlavu. The cell lines K562 and Raji were used as negative and positive controls, respectively. K562, a B lymphoid-derived cell line, was shown to express negligible amounts of human leucocyte antigens, while Raji, an erythromyeloid-derived cell line, expressed both class I and class II human leucocyte antigens as well as their respective invariant chains, beta 2-microglobulin and Ii. Using an ELISA, experiments performed on these cell lines confirmed the natural expression of class I and class II antigens by the HA22T/VGH and HA59T/VGH cell lines, whereas PLC/PRF/5 displayed class II surface antigens only. The effects of modulating agents such as interferon-gamma sodium butyrate and clofazimine on human leucocyte antigen expression were investigated using the HA22T/VGH, HA59T/VGH and TONG PHC cell lines. These agents increased class II and class II human leucocyte antigen expression on HA22T/VGH and TONG PHC cells, but had no effect on the HA59T/VGH cell line. The results suggest a potential use for these agents as modulators of human leucocyte antigen expression by human heptocellular cell lines.

  3. Treatment Options by Stage (Adrenocortical Carcinoma)

    MedlinePlus

    ... of Childhood Treatment for more information.) Having certain genetic conditions increases the risk of adrenocortical carcinoma. Anything ... can be a sign of disease. CT scan (CAT scan) : A procedure that makes a series of ...

  4. TCGA analysis of adrenocortical carcinoma - TCGA

    Cancer.gov

    In the most comprehensive molecular characterization to date of adrenocortical carcinoma, a rare cancer of the adrenal cortex, researchers extensively analyzed 91 cases for alterations in the tumor genomes.

  5. GATA4 is a critical regulator of gonadectomy-induced adrenocortical tumorigenesis in mice.

    PubMed

    Krachulec, Justyna; Vetter, Melanie; Schrade, Anja; Löbs, Ann-Kathrin; Bielinska, Malgorzata; Cochran, Rebecca; Kyrönlahti, Antti; Pihlajoki, Marjut; Parviainen, Helka; Jay, Patrick Y; Heikinheimo, Markku; Wilson, David B

    2012-06-01

    In response to gonadectomy certain inbred mouse strains develop sex steroidogenic adrenocortical neoplasms. One of the hallmarks of neoplastic transformation is expression of GATA4, a transcription factor normally present in gonadal but not adrenal steroidogenic cells of the adult mouse. To show that GATA4 directly modulates adrenocortical tumorigenesis and is not merely a marker of gonadal-like differentiation in the neoplasms, we studied mice with germline or conditional loss-of-function mutations in the Gata4 gene. Germline Gata4 haploinsufficiency was associated with attenuated tumor growth and reduced expression of sex steroidogenic genes in the adrenal glands of ovariectomized B6D2F1 and B6AF1 mice. At 12 months after ovariectomy, wild-type B6D2F1 mice had biochemical and histological evidence of adrenocortical estrogen production, whereas Gata4(+/-) B6D2F1 mice did not. Germline Gata4 haploinsufficiency exacerbated the secondary phenotype of postovariectomy obesity in B6D2F1 mice, presumably by limiting ectopic estrogen production in the adrenal glands. Amhr2-cre-mediated deletion of floxed Gata4 (Gata4(F)) in nascent adrenocortical neoplasms of ovariectomized B6.129 mice reduced tumor growth and the expression of gonadal-like markers in a Gata4(F) dose-dependent manner. We conclude that GATA4 is a key modifier of gonadectomy-induced adrenocortical neoplasia, postovariectomy obesity, and sex steroidogenic cell differentiation.

  6. GATA4 Is a Critical Regulator of Gonadectomy-Induced Adrenocortical Tumorigenesis in Mice

    PubMed Central

    Krachulec, Justyna; Vetter, Melanie; Schrade, Anja; Löbs, Ann-Kathrin; Bielinska, Malgorzata; Cochran, Rebecca; Kyrönlahti, Antti; Pihlajoki, Marjut; Parviainen, Helka; Jay, Patrick Y.; Heikinheimo, Markku

    2012-01-01

    In response to gonadectomy certain inbred mouse strains develop sex steroidogenic adrenocortical neoplasms. One of the hallmarks of neoplastic transformation is expression of GATA4, a transcription factor normally present in gonadal but not adrenal steroidogenic cells of the adult mouse. To show that GATA4 directly modulates adrenocortical tumorigenesis and is not merely a marker of gonadal-like differentiation in the neoplasms, we studied mice with germline or conditional loss-of-function mutations in the Gata4 gene. Germline Gata4 haploinsufficiency was associated with attenuated tumor growth and reduced expression of sex steroidogenic genes in the adrenal glands of ovariectomized B6D2F1 and B6AF1 mice. At 12 months after ovariectomy, wild-type B6D2F1 mice had biochemical and histological evidence of adrenocortical estrogen production, whereas Gata4+/− B6D2F1 mice did not. Germline Gata4 haploinsufficiency exacerbated the secondary phenotype of postovariectomy obesity in B6D2F1 mice, presumably by limiting ectopic estrogen production in the adrenal glands. Amhr2-cre-mediated deletion of floxed Gata4 (Gata4F) in nascent adrenocortical neoplasms of ovariectomized B6.129 mice reduced tumor growth and the expression of gonadal-like markers in a Gata4F dose-dependent manner. We conclude that GATA4 is a key modifier of gonadectomy-induced adrenocortical neoplasia, postovariectomy obesity, and sex steroidogenic cell differentiation. PMID:22461617

  7. Genetics of adrenocortical tumors: Carney complex.

    PubMed

    Stratakis, C A

    2001-04-01

    Adrenal cancer is a rare neoplasm; however, up to 1 in 1 500 adrenal incidentalomas may hide a carcinoma, which, if diagnosed late or left untreated, is associated with significant morbidity and mortality. Despite extensive investigation of the molecular mechanisms involved in adrenal carcinogenesis and significant improvements in diagnostic imaging, efforts to cure advanced adrenal cancer remain largely unsuccessful. Thus, the investigation of the genetics of adrenocortical cancer by the candidate or positional cloning gene approach is essential in the development of new therapies for this disease. We propose that adrenocortical tumorigenesis follows a pattern similar to that in other organs: As the pathology of the adrenocortical tumor increases towards malignancy, the genetic changes that are observed also increase. Known genetic associations, like TP53 gene changes, occur during the latest stages of adrenocortical tumorigenesis. Thus, it is essential to study the relatively few genes that are affected at the beginning of this process, at the stages of benign tumorigenesis in the cortex. We have studied primary pigmented adrenocortical disease (PPNAD), a benign, bilateral, adrenocortical hyperplasia, which either in its isolated form or as part of Carney complex (CNC), is inherited in an autosomal dominant manner and, therefore, the gene(s) responsible for this disorder could be identified by positional cloning approaches. Indeed, we have identified two genetic loci harboring genes for PPNAD and/or CNC on chromosomal loci 2p16 and 17q22-24. The chromosome 17 gene, PRKAR1A, was recently cloned and the identification of other responsible genes is currently under way in our, and collaborating laboratories. The present report reviews the genetics of adrenocortical cancer first, followed by what is known today about the genetics of PPNAD and/or CNC. PMID:11353891

  8. Adenosine 5'-(gamma-thio) triphosphate (ATPgammaS) stimulates both P2Y receptors linked to inositol phosphates production and cAMP accumulation in bovine adrenocortical fasciculata cells.

    PubMed

    Nishi, Haruhisa; Hori, Seiji; Niitsu, Akiyoshi; Kawamura, Masahiro

    2004-01-16

    The study was aimed to investigate the existence of at least two kinds of P2Y receptors linked to steroidogenesis in bovine adrenocortical fasciculata cells (BAFCs). Extracellular nucleotides facilitated steroidogenesis in BAFCs. The potency order was UTP > adenosine 5'-(gamma-thio) triphosphate (ATPgammaS) > ATP > 2-methylthio ATP (2MeSATP) > adenosine 5'-(beta-thio) diphosphate (ADPbetaS) > alpha,beta-methylene ATP (alpha,beta-me-ATP), beta,gamma-methylene ATP (beta,gamma -me-ATP). ATPgammaS (10-100 microM) remarkably stimulated both total inositol phosphates (IPs) production and cyclic AMP (cAMP) accumulation. Competitive displacement experiments by using [35S]ATPgammaS as a radioactive ligand in BAFCs showed that the potency under these unlabelled ligands was ATPgammaS > ATP > ADPbetaS > 2MeSATP > UTP > alpha,beta-me-ATP, beta,gamma-me-ATP. These suggest that two different binding sites of [35S]ATPgammaS, namely P2Y receptors, exist in BAFCs, and that these receptors are linked to steroidogenesis via distinct second messenger systems in the cells.

  9. Familial predisposition to adrenocortical tumors: clinical and biological features and management strategies.

    PubMed

    Ribeiro, Raul C; Pinto, Emilia M; Zambetti, Gerard P

    2010-06-01

    The incidence of adrenocortical tumors (ACTs) is increased in several familial cancer syndromes resulting from abnormalities in genes that encode transcription factors implicated in cell proliferation, differentiation, senescence, apoptosis, and genomic instability. These include P53, MEN1, APC, and PRKAR1A. Adenomas are the most common ACTs, but adrenocortical carcinomas occur rarely as well. The clinical manifestations of ACTs, which result from increased secretion of adrenocortical hormones, are similar in the familial and sporadic forms of the disease. However, their management may differ because of unique aspects of the constitutional syndromes. The analysis of gene expression profiles of ACTs in these constitutional syndromes have contributed to our understanding of adrenal tumorigenesis and revealed new molecular diagnostic and prognostic markers and candidate genes for targeted therapies. This chapter summarizes the clinical and biological features, pathogenesis, and management strategies for ACTs that develop in patients with familial cancer syndrome. PMID:20833338

  10. Protein kinase A alterations in adrenocortical tumors.

    PubMed

    Espiard, S; Ragazzon, B; Bertherat, J

    2014-11-01

    Stimulation of the cAMP pathway by adrenocorticotropin (ACTH) is essential for adrenal cortex maintenance, glucocorticoid and adrenal androgens synthesis, and secretion. Various molecular and cellular alterations of the cAMP pathway have been observed in endocrine tumors. Protein kinase A (PKA) is a central key component of the cAMP pathway. Molecular alterations of PKA subunits have been observed in adrenocortical tumors. PKA molecular defects can be germline in hereditary disorders or somatic in sporadic tumors. Heterozygous germline inactivating mutations of the PKA regulatory subunit RIα gene (PRKAR1A) can be observed in patients with ACTH-independent Cushing's syndrome (CS) due to primary pigmented nodular adrenocortical disease (PPNAD). PRKAR1A is considered as a tumor suppressor gene. Interestingly, these mutations can also be observed as somatic alterations in sporadic cortisol-secreting adrenocortical adenomas. Germline gene duplication of the catalytic subunits Cα (PRKACA) has been observed in patients with PPNAD. Furthermore, exome sequencing revealed recently activating somatic mutations of PRKACA in about 40% of cortisol-secreting adrenocortical adenomas. In vitro and in vivo functional studies help in the progress to understand the mechanisms of adrenocortical tumors development due to PKA regulatory subunits alterations. All these alterations are observed in benign oversecreting tumors and are mimicking in some way cAMP pathway constitutive activation. On the long term, unraveling these alterations will open new strategies of pharmacological treatment targeting the cAMP pathway in adrenal tumors and cortisol-secretion disorders. PMID:25105543

  11. Neuroblastoma cell lines showing smooth muscle cell phenotypes.

    PubMed

    Sugimoto, T; Mine, H; Horii, Y; Takahashi, K; Nagai, R; Morishita, R; Komada, M; Asada, Y; Sawada, T

    2000-12-01

    Neuroblastoma is a tumor that is derived from the neural crest. Recent studies demonstrated that several human neuroblastoma cell lines exhibit at least three morphologic types: neuroblastic (N)-type, substrate-adhesive (S)-type and intermediate (I)-type cells. However, the origin of the S-type cells has not been clearly identified. In this study, the expressions of smooth muscle-specific proteins (desmin, alpha-smooth muscle actin, basic calponin and the smooth muscle myosin heavy-chain isoforms of SM1 and SM2) in three parent and four cloned neuroblastoma cell lines, composed of S-type cells, were examined by indirect immunofluorescence, Western blot and/or by reverse transcription-polymerase chain reaction (RT-PCR). Desmin was found in two of the seven cell lines, and alpha-smooth muscle actin and basic calponin were detected in all of seven of the cell lines. In three parent cell lines and one cloned cell line composed of N-type cells, none of three smooth muscle-specific proteins were detected. In smooth muscle myosin heavy-chain isoforms, SM1 was detected in two parent cell lines composed of S-type cells (MP-N-MS and KP-N-YS) by immunofluorescence, Western blot and/or by RT-PCR, whereas the SM2 isoform was detected in one parent cell line (MP-N-MS) by RT-PCR. These findings indicate that S-type cells have either the immature or mature smooth muscle cell phenotype, and neural crest cells very likely have the ability of to differentiate into smooth muscle cells in the human system.

  12. Virus Discovery Using Tick Cell Lines

    PubMed Central

    Bell-Sakyi, Lesley; Attoui, Houssam

    2016-01-01

    While ticks have been known to harbor and transmit pathogenic arboviruses for over 80 years, the application of high-throughput sequencing technologies has revealed that ticks also appear to harbor a diverse range of endogenous tick-only viruses belonging to many different families. Almost nothing is known about these viruses; indeed, it is unclear in most cases whether the identified viral sequences are derived from actual replication-competent viruses or from endogenous virus elements incorporated into the ticks’ genomes. Tick cell lines play an important role in virus discovery and isolation through the identification of novel viruses chronically infecting such cell lines and by acting as host cells to aid in determining whether or not an entire replication-competent, infective virus is present in a sample. Here, we review recent progress in tick-borne virus discovery and comment on the actual and potential applications for tick cell lines in this emerging research area. PMID:27679414

  13. Virus Discovery Using Tick Cell Lines

    PubMed Central

    Bell-Sakyi, Lesley; Attoui, Houssam

    2016-01-01

    While ticks have been known to harbor and transmit pathogenic arboviruses for over 80 years, the application of high-throughput sequencing technologies has revealed that ticks also appear to harbor a diverse range of endogenous tick-only viruses belonging to many different families. Almost nothing is known about these viruses; indeed, it is unclear in most cases whether the identified viral sequences are derived from actual replication-competent viruses or from endogenous virus elements incorporated into the ticks’ genomes. Tick cell lines play an important role in virus discovery and isolation through the identification of novel viruses chronically infecting such cell lines and by acting as host cells to aid in determining whether or not an entire replication-competent, infective virus is present in a sample. Here, we review recent progress in tick-borne virus discovery and comment on the actual and potential applications for tick cell lines in this emerging research area.

  14. Virus Discovery Using Tick Cell Lines.

    PubMed

    Bell-Sakyi, Lesley; Attoui, Houssam

    2016-01-01

    While ticks have been known to harbor and transmit pathogenic arboviruses for over 80 years, the application of high-throughput sequencing technologies has revealed that ticks also appear to harbor a diverse range of endogenous tick-only viruses belonging to many different families. Almost nothing is known about these viruses; indeed, it is unclear in most cases whether the identified viral sequences are derived from actual replication-competent viruses or from endogenous virus elements incorporated into the ticks' genomes. Tick cell lines play an important role in virus discovery and isolation through the identification of novel viruses chronically infecting such cell lines and by acting as host cells to aid in determining whether or not an entire replication-competent, infective virus is present in a sample. Here, we review recent progress in tick-borne virus discovery and comment on the actual and potential applications for tick cell lines in this emerging research area. PMID:27679414

  15. Killer cell lines against Shope carcinoma cells in rabbits.

    PubMed

    Takahashi, M; Yamade, I; Seto, A

    1991-09-01

    Killer cell activity against Shope carcinoma cells was not detected in PBL nor in spleen cells from tumor-bearing B/J rabbits, but was induced by in vitro culture of these cells in the presence of IL-2 and X-irradiated carcinoma cells. HTLV-I-transformed killer cell lines were successfully obtained by the culturing of PBL from an HTLV-I-infected and tumor-bearing Chbb:HM rabbit. These killer cells included large cells with azurophilic granules in the cytoplasm and with a reniform nucleus, thus resembling large granular lymphocytes. The killer activity was similar against the Vx2K cell line from a random-bred rabbit and SCB cell lines from an B/J rabbit, suggesting the absence of MHC restriction. PMID:1655241

  16. The effects of the standardized extracts of Ginkgo biloba on steroidogenesis pathways and aromatase activity in H295R human adrenocortical carcinoma cells

    PubMed Central

    2016-01-01

    Objectives Aromatase inhibitors that block estrogen synthesis are a proven first-line hormonal therapy for postmenopausal breast cancer. Although it is known that standardized extract of Ginkgo biloba (EGb761) induces anti-carcinogenic effects like the aromatase inhibitors, the effects of EGb761 on steroidogenesis have not been studied yet. Therefore, the effects of EGb761 on steroidogenesis and aromatase activity was studied using a H295R cell model, which was a good in vitro model to predict effects on human adrenal steroidogenesis. Methods Cortisol, aldosterone, testosterone, and 17β-estradiol were evaluated in the H295R cells by competitive enzyme-linked immunospecific assay after exposure to EGb761. Real-time polymerase chain reaction were performed to evaluate effects on critical genes in steroid hormone production, specifically cytochrome P450 (CYP11/ 17/19/21) and the hydroxysteroid dehydrogenases (3β-HSD2 and 17β-HSD1/4). Finally, aromatase activities were measured with a tritiated water-release assay and by western blotting analysis. Results H295R cells exposed to EGb761 (10 and 100 μg/mL) showed a significant decrease in 17β-estradiol and testosterone, but no change in aldosterone or cortisol. Genes (CYP19 and 17β-HSD1) related to the estrogen steroidogenesis were significantly decreased by EGb761. EGb761 treatment of H295R cells resulted in a significant decrease of aromatase activity as measured by the direct and indirect assays. The coding sequence/ Exon PII of CYP19 gene transcript and protein level of CYP19 were significantly decreased by EGb761. Conclusions These results suggest that EGb761 could regulate steroidogenesis-related genes such as CYP19 and 17β-HSD1, and lead to a decrease in 17β-estradiol and testosterone. The present study provides good information on potential therapeutic effects of EGb761 on estrogen dependent breast cancer. PMID:27188280

  17. Primary aldosteronism and malignant adrenocortical neoplasia.

    PubMed Central

    Salassa, T. M.; Weeks, R. E.; Northcutt, R. C.; Carney, J. A.

    1975-01-01

    Our experience indicates that although adrenal carcinoma is not a common cause of primary aldosteronism, 4 to 5% of patients in a single large series may have a malignant adrenocortical tumor. The magnitude of the hypokalemia and the hyperaldosteronuria tends to be greater in patients with malignant tumors, but these patients cannot be clearly separated from those with benign tumors or hyperplasia on this basis. Patients who have malignant tumors may have no chemical evidence of adrenocortical dysfunction other than excessive aldosterone secretion. Finally, a good response to spironolactone for months does not exclude adrenal carcinoma as the cause of primary aldosteronism. Images Fig. 1 PMID:1179589

  18. Adrenocortical carcinoma in pregnancy: A diagnostic dilemma

    PubMed Central

    Jairath, Ankush; Aulakh, Baldev S.

    2014-01-01

    Adrenocortical carcinoma is a rare disease. Additionally, in the case of coexisting pregnancy, there are diagnostic difficulties due to associated physiological hormonal changes as well as imaging limitations. Cushing's syndrome and virilization during pregnancy is a rare entity with few cases reported in the literature. Misdiagnosis is common as the syndrome may be easily confused with preeclampsia or gestational diabetes. We present the case of a 31-year-old pregnant woman with rapidly developing symptoms related to hormonally active adrenocortical cancer. PMID:25097324

  19. Refractory lining for electrochemical cell

    DOEpatents

    Blander, Milton; Cook, Glenn M.

    1987-01-01

    Apparatus for processing a metallic fluid containing iron oxide, container for a molten metal including an electrically conductive refractory disposed for contact with the molten metal which contains iron oxide, an electrolyte in the form of a basic slag on top of the molten metal, an electrode in the container in contcat with the slag electrically separated from the refractory, and means for establishing a voltage across the refractory and the electrode to reduce iron oxide to iron at the surface of the refractory in contact with the iron oxide containing fluid. A process is disclosed for refining an iron product containing not more than about 10% by weight oxygen and not more than about 10% by weight sulfur, comprising providing an electrolyte of a slag containing one or more of calcium oxide, magnesium oxide, silica or alumina, providing a cathode of the iron product in contact with the electrolyte, providing an anode in contact with the electrolyte electrically separated from the cathode, and operating an electrochemical cell formed by the anode, the cathode and the electrolyte to separate oxygen or sulfur present in the iron product therefrom.

  20. Umbelliprenin Induces Apoptosis in CLL Cell Lines.

    PubMed

    Ziai, Seyed Ali; Gholami, Omid; Iranshahi, Mehrdad; Zamani, Amir Hassan; Jeddi-Tehrani, Mahmood

    2012-01-01

    Chronic lymphocytic leukemia (CLL) remains an incurable disease that requires innovative new approaches to improve therapeutic outcome. Many Ferula species, including F. asa-foetida, synthesize terpenyloxy coumarins. One of these coumarins is umbelliprenin, which has been implicated with induction of apoptosis in some cancer cell lines. In this study induction of apoptosis by umbelliprenin on Jurkat T-CLL and Raji B-CLL cell lines was studied. In this regard, cells were incubated with various concentrations of umbelliprenin in-vitro for different times and assayed for apoptosis with annexin V-FITC/PI double staining flowcytometry method. Results showed that umbelliprenin induced apoptosis in leukemic cells in a dose- and time-dependent manner and that CLL cells were more susceptible to umbelliprenin induced cell death than normal peripheral blood mononuclear cell (PBMCs). Moreover, we study the induction of apoptosis in Jurkat cells by umbelliprenin in the presence of interleukin 4 (IL-4) as an agent that causes resistance to apoptosis in CLL cells, was also student. We showed that IL-4 can not reduce apoptotic effect of umbelliprenin. The preferential toxicity of umbelliprenin for CLL cells, supports the hypothesis that oral administration of umbelliprenin in the form of foods or folk medicines containing this coumarin, might enhance protection against the development of CLL in man with little side effects. In conclusion, umbelliprenin may be an effective therapeutic agent in the treatment of CLL, and thus clinical studies with umbelliprenin may be appropriate.

  1. Umbelliprenin Induces Apoptosis in CLL Cell Lines

    PubMed Central

    Ziai, Seyed Ali; Gholami, Omid; Iranshahi, Mehrdad; Zamani, Amir Hassan; Jeddi-Tehrani, Mahmood

    2012-01-01

    Chronic lymphocytic leukemia (CLL) remains an incurable disease that requires innovative new approaches to improve therapeutic outcome. Many Ferula species, including F. asa-foetida, synthesize terpenyloxy coumarins. One of these coumarins is umbelliprenin, which has been implicated with induction of apoptosis in some cancer cell lines. In this study induction of apoptosis by umbelliprenin on Jurkat T-CLL and Raji B-CLL cell lines was studied. In this regard, cells were incubated with various concentrations of umbelliprenin in-vitro for different times and assayed for apoptosis with annexin V–FITC/PI double staining flowcytometry method. Results showed that umbelliprenin induced apoptosis in leukemic cells in a dose- and time-dependent manner and that CLL cells were more susceptible to umbelliprenin induced cell death than normal peripheral blood mononuclear cell (PBMCs). Moreover, we study the induction of apoptosis in Jurkat cells by umbelliprenin in the presence of interleukin 4 (IL-4) as an agent that causes resistance to apoptosis in CLL cells, was also student. We showed that IL-4 can not reduce apoptotic effect of umbelliprenin. The preferential toxicity of umbelliprenin for CLL cells, supports the hypothesis that oral administration of umbelliprenin in the form of foods or folk medicines containing this coumarin, might enhance protection against the development of CLL in man with little side effects. In conclusion, umbelliprenin may be an effective therapeutic agent in the treatment of CLL, and thus clinical studies with umbelliprenin may be appropriate. PMID:24250490

  2. [Characterization of a liver metastatic cell line derived from a human gastric cancer cell line].

    PubMed

    Wakasugi, J

    1990-08-01

    This study was carried out to investigate whether there is any difference of biological characteristics between a gastric cancer cell line (KATOIII) and another cell line derived from liver metastasis of the same cell line (KATOIII-H2). The liver metastasis was produced by intrasplenic injection of the fluid containing of KATOIII in nude mouse and new cell line was established using the cells of metastatic site. The results are as follows. 1) Inoculation of KATOIII-H2 into the spleen produced liver metastases in all of the experimental animals, whereas the same procedure with KATOIII produced metastasis only in 30% of the animals. 2) KATOIII-H2 exhibited more prominent platelet-aggregating activity than KATOIII. 3) There is no difference between two cell lines on doubling time, histological findings of the xenografts and chromosomal number. 4) DNA index of KATOIII-H2 is lower than KATOIII and the trisomy in NO. 20 chromosome of KATOIII-H2 was noted. The results indicate that metastatic potential is different between two cell lines and this fact is probably in a part because of the different platelet-aggregating activity of each cell line. PMID:2233668

  3. microRNA-7 as a tumor suppressor and novel therapeutic for adrenocortical carcinoma

    PubMed Central

    Gill, Anthony J.; Weiss, Jocelyn; Mugridge, Nancy; Kim, Edward; Feeney, Alex L.; Ip, Julian C.; Reid, Glen; Clarke, Stephen; Soon, Patsy S.H.; Robinson, Bruce G.; Brahmbhatt, Himanshu; MacDiarmid, Jennifer A.; Sidhu, Stan B.

    2015-01-01

    Adrenocortical carcinoma (ACC) has a poor prognosis with significant unmet clinical need due to late diagnosis, high rates of recurrence/metastasis and poor response to conventional treatment. Replacing tumor suppressor microRNAs (miRNAs) offer a novel therapy, however systemic delivery remains challenging. A number of miRNAs have been described to be under-expressed in ACC however it is not known if they form a part of ACC pathogenesis. Here we report that microRNA-7–5p (miR-7) reduces cell proliferation in vitro and induces G1 cell cycle arrest. Systemic miR-7 administration in a targeted, clinically safe delivery vesicle (EGFREDVTM nanocells) reduces ACC xenograft growth originating from both ACC cell lines and primary ACC cells. Mechanistically, miR-7 targets Raf-1 proto-oncogene serine/threonine kinase (RAF1) and mechanistic target of rapamycin (MTOR). Additionally, miR-7 therapy in vivo leads to inhibition of cyclin dependent kinase 1 (CDK1). In patient ACC samples, CDK1 is overexpressed and miR-7 expression inversely related. In summary, miR-7 inhibits multiple oncogenic pathways and reduces ACC growth when systemically delivered using EDVTM nanoparticles. This data is the first study in ACC investigating the possibility of miRNAs replacement as a novel therapy. PMID:26452132

  4. A morphometric analysis of adrenocortical actin localized by immunoelectron microscopy: the effect of adrenocorticotropin.

    PubMed

    Loesser, K E; Malamed, S

    1987-10-01

    The localization of actin and the effect of ACTH on its concentration was examined in freshly isolated rat adrenocortical cells. Lowicryl K4M-embedded cells were used for the immunoelectron localization of actin; gold was used as a label for immunoreactive sites. Actin was at least 4 times as concentrated at the cortical cytoplasm as in the lipid droplets and at least 5 times as concentrated in the microvilli as in the lipid droplets. ACTH stimulation approximately doubled the concentration of actin in the cortical cytoplasm and increased by 50% the concentration of actin in the microvilli. The microvillar contribution to the cell surface area was 40% higher in ACTH-stimulated cells than it was in unstimulated cells. These results provide quantitative evidence suggesting that actin and the microvilli participate in steroid secretion by the adrenocortical cell.

  5. Cancer stem cell-like cells from a single cell of oral squamous carcinoma cell lines

    SciTech Connect

    Felthaus, O.; Ettl, T.; Gosau, M.; Driemel, O.; Brockhoff, G.; Reck, A.; Zeitler, K.; Hautmann, M.; Reichert, T.E.; Schmalz, G.; Morsczeck, C.

    2011-04-01

    Research highlights: {yields} Four oral squamous cancer cell lines (OSCCL) were analyzed for cancer stem cells (CSCs). {yields} Single cell derived colonies of OSCCL express CSC-marker CD133 differentially. {yields} Monoclonal cell lines showed reduced sensitivity for Paclitaxel. {yields} In situ CD133{sup +} cells are slow cycling (Ki67-) indicating a reduced drug sensitivity. {yields} CD133{sup +} and CSC-like cells can be obtained from single colony forming cells of OSCCL. -- Abstract: Resistance of oral squamous cell carcinomas (OSCC) to conventional chemotherapy or radiation therapy might be due to cancer stem cells (CSCs). The development of novel anticancer drugs requires a simple method for the enrichment of CSCs. CSCs can be enriched from OSCC cell lines, for example, after cultivation in serum-free cell culture medium (SFM). In our study, we analyzed four OSCC cell lines for the presence of CSCs. CSC-like cells could not be enriched with SFM. However, cell lines obtained from holoclone colonies showed CSC-like properties such as a reduced rate of cell proliferation and a reduced sensitivity to Paclitaxel in comparison to cells from the parental lineage. Moreover, these cell lines differentially expressed the CSC-marker CD133, which is also upregulated in OSCC tissues. Interestingly, CD133{sup +} cells in OSCC tissues expressed little to no Ki67, the cell proliferation marker that also indicates reduced drug sensitivity. Our study shows a method for the isolation of CSC-like cell lines from OSCC cell lines. These CSC-like cell lines could be new targets for the development of anticancer drugs under in vitro conditions.

  6. Pathological and molecular features of adrenocortical carcinoma: an update.

    PubMed

    Volante, M; Buttigliero, C; Greco, E; Berruti, A; Papotti, M

    2008-07-01

    The pathological diagnosis of adrenocortical carcinoma (ACC), which is based on gross and microscopic criteria, is subjective. None of the features are absolutely indicative of malignancy, although their combination in a scoring system may correctly identify ACC. The Weiss system, which is currently the most popular, combines nine morphological parameters, of which three are structural ("dark" cytoplasm, diffuse architecture, necrosis), three are cytological (atypia, mitotic count, atypical mitotic figures) and three are related to invasion (of sinusoids, veins and tumour capsule). Although there are strictly defined criteria for each feature, some are straightforward and objective, while others are potentially more problematic (diffuse architecture, necrosis, sinusoidal, venous and capsular invasions). The classification of oncocytic and paediatric adrenocortical tumours is even more challenging, as not all of the above morphological parameters are predictors of malignancy in these tumour types. As an alternative to the morphological approach, a wide array of chromosomal, genetic, molecular and immunohistochemical markers have been tested in ACC to identify reliable diagnostic and prognostic factors. Genetic and epigenetic alterations of p53, IGF-2 and molecules involved in cancer cell invasive properties seem the most promising. These molecular markers may not only play a role in the biology of these tumours and have prognostic implications, but may also be used as potential targets for treatment. However, these markers are not sufficiently sensitive and specific to replace conventional morphological criteria. PMID:18430754

  7. Spontaneous Cell Competition in Immortalized Mammalian Cell Lines

    PubMed Central

    Penzo-Méndez, Alfredo I.; Chen, Yi-Ju; Li, Jinyang; Witze, Eric S.; Stanger, Ben Z.

    2015-01-01

    Cell competition is a form of cell-cell interaction by which cells compare relative levels of fitness, resulting in the active elimination of less-fit cells, “losers,” by more-fit cells, “winners.” Here, we show that in three routinely-used mammalian cell lines – U2OS, 3T3, and MDCK cells – sub-clones arise stochastically that exhibit context-dependent competitive behavior. Specifically, cell death is elicited when winner and loser sub-clones are cultured together but not alone. Cell competition and elimination in these cell lines is caspase-dependent and requires cell-cell contact but does not require de novo RNA synthesis. Moreover, we show that the phenomenon involves differences in cellular metabolism. Hence, our study demonstrates that cell competition is a common feature of immortalized mammalian cells in vitro and implicates cellular metabolism as a mechanism by which cells sense relative levels of “fitness.” PMID:26200654

  8. Experience with the Vero cell line.

    PubMed

    Montagnon, B J; Vincent-Falquet, J C

    1998-01-01

    The Vero cell line has been managed with the Cell Bank system to produce at the 142nd passage IPV, OPV and rabies vaccines since 1982 by Pasteur Mérieux Serums & Vaccins (PMsv). The safety of the cell line was regularly validated at the Working Cell Bank (WCB) level according to the WHO and European Pharmacopoeia requirements for absence of bacteria, fungi, mycoplasma and viruses. A special emphasis was devoted to research on the absence of simian viruses (SV40, SIV, Retro-D virus and simian CMV). All these specific researches were negative. At a low level of passage, the Vero cells are not tumorigenic. Vaccines have been prepared in low passage level Vero cells and together with the excellent downstream purification have resulted in excellent safety as attested by pharmacovigilance of more than 100 million doses of IPV during 12 years, more than 20 million doses of rabies vaccine during 10 years and more than 1 billion of OPV during eight years.

  9. Giant adrenal pseudocyst harbouring adrenocortical cancer

    PubMed Central

    Wilkinson, Michael; Fanning, Deirdre Mary; Moloney, James; Flood, Hugh

    2011-01-01

    The authors report a very rare case of adreno-cortical carcinoma arising in a giant adrenal pseudocyst. A 64-year-old woman presented to the emergency department with a 6 week history of progressively worsening severe left abdominal pain, anorexia, anergia and constipation. On examination, she was cachectic with tenderness over the left abdomen and flank. Medical history was significant for gastritis and anaemia. During her investigation, a well-defined para-renal 12×6 centimetre multi-loculated cyst, of uncertain origin was identified on CT. Ultrasound-guided biopsy was not diagnostic. MRI showed the cyst to be likely adrenal in origin. Serum and urinary catecholamines were unremarkable. At laparotomy an unresectable large, tense, fixed, cystic mass was seen to occupy the left side of the abdomen. The cyst was de-roofed. Pathology showed a high-grade poorly differentiated adreno-cortical carcinoma with a pseudo-capsule. She died 2 months postoperatively. PMID:22679267

  10. Quantitative RT-PCR methods for evaluating toxicant-induced effects on steroidogenesis using the H295R cell line.

    PubMed

    Zhang, Xiaowei; Yu, Richard M K; Jones, Paul D; Lam, Gabriel K W; Newsted, John L; Gracia, Tannia; Hecker, Markus; Hilscherova, Klara; Sanderson, Thomas; Wu, Rudolf S S; Giesy, John P

    2005-04-15

    Gene expression profiles show considerable promise for the evaluation of the toxic potential of environmental contaminants. For example, any alterations in the pathways of steroid synthesis or breakdown have the potential to Cause endocrine disruption. Therefore monitoring these pathways can provide information relative to a chemical's ability to impact endocrine function. One approach to monitoring these pathways has been to use a human adrenocortical carcinoma cell line (H295R) that expresses all the key enzymes necessary for steroidogenesis. In this study we have further developed these methods using accurate and specific quantification methods utilizing molecular beacon-based quantitative RT-PCR (Q-RT-PCR). The assay system was used to analyze the expression patterns of 11 steroidogenic genes in H295R cells. The expression of gene transcripts was measured using a real-time PCR system and quantified based on both a standard curve method using a dilution series of RNA standards and a comparative Ct method. To validate the optimized method, cells were exposed to specific and nonspecific model compounds (inducers and inhibitors of various steroidogenic enzymes) for gene expression profiling. Similar gene expression profiles were exhibited by cells treated with chemicals acting through common mechanisms of action. Overall, our findings demonstrated that the present assay can facilitate the development of compound-specific response profiles, and will provide a sensitive and integrative screen for the effects of chemicals on steroidogenesis.

  11. Mechanistic Computational Model of Steroidgenesis in H295R Cells: Role of (Oxysterols and Cell Proliferation to Improve Predictability of Biochemical Response to Endocrine Active Chemical-Metyrapone

    EPA Science Inventory

    The human adrenocortical carcinoma cell line H295R is being used as an in vitro steroidogenesis screening assay to assess the impact of endocrine active chemicals (EACs) capable of altering steroid biosynthesis. To enhance the interpretation and quantitative application of measur...

  12. Genomic landscape of paediatric adrenocortical tumours.

    PubMed

    Pinto, Emilia M; Chen, Xiang; Easton, John; Finkelstein, David; Liu, Zhifa; Pounds, Stanley; Rodriguez-Galindo, Carlos; Lund, Troy C; Mardis, Elaine R; Wilson, Richard K; Boggs, Kristy; Yergeau, Donald; Cheng, Jinjun; Mulder, Heather L; Manne, Jayanthi; Jenkins, Jesse; Mastellaro, Maria J; Figueiredo, Bonald C; Dyer, Michael A; Pappo, Alberto; Zhang, Jinghui; Downing, James R; Ribeiro, Raul C; Zambetti, Gerard P

    2015-01-01

    Paediatric adrenocortical carcinoma is a rare malignancy with poor prognosis. Here we analyse 37 adrenocortical tumours (ACTs) by whole-genome, whole-exome and/or transcriptome sequencing. Most cases (91%) show loss of heterozygosity (LOH) of chromosome 11p, with uniform selection against the maternal chromosome. IGF2 on chromosome 11p is overexpressed in 100% of the tumours. TP53 mutations and chromosome 17 LOH with selection against wild-type TP53 are observed in 28 ACTs (76%). Chromosomes 11p and 17 undergo copy-neutral LOH early during tumorigenesis, suggesting tumour-driver events. Additional genetic alterations include recurrent somatic mutations in ATRX and CTNNB1 and integration of human herpesvirus-6 in chromosome 11p. A dismal outcome is predicted by concomitant TP53 and ATRX mutations and associated genomic abnormalities, including massive structural variations and frequent background mutations. Collectively, these findings demonstrate the nature, timing and potential prognostic significance of key genetic alterations in paediatric ACT and outline a hypothetical model of paediatric adrenocortical tumorigenesis. PMID:25743702

  13. Adjuvant and Definitive Radiotherapy for Adrenocortical Carcinoma

    SciTech Connect

    Sabolch, Aaron; Feng, Mary; Griffith, Kent; Hammer, Gary; Doherty, Gerard; Ben-Josef, Edgar

    2011-08-01

    Purpose: To evaluate the impact of both adjuvant and definitive radiotherapy on local control of adrenocortical carcinoma. Methods and Materials: Outcomes were analyzed from 58 patients with 64 instances of treatment for adrenocortical carcinoma at the University of Michigan's Multidisciplinary Adrenal Cancer Clinic. Thirty-seven of these instances were for primary disease, whereas the remaining 27 were for recurrent disease. Thirty-eight of the treatment regimens involved surgery alone, 10 surgery plus adjuvant radiotherapy, and 16 definitive radiotherapy for unresectable disease. The effects of patient, tumor, and treatment factors were modeled simultaneously using multiple variable Cox proportional hazards regression for associations with local recurrence, distant recurrence, and overall survival. Results: Local failure occurred in 16 of the 38 instances that involved surgery alone, in 2 of the 10 that consisted of surgery plus adjuvant radiotherapy, and in 1 instance of definitive radiotherapy. Lack of radiotherapy use was associated with 4.7 times the risk of local failure compared with treatment regimens that involved radiotherapy (95% confidence interval, 1.2-19.0; p = 0.030). Conclusions: Radiotherapy seems to significantly lower the risk of local recurrence/progression in patients with adrenocortical carcinoma. Adjuvant radiotherapy should be strongly considered after surgical resection.

  14. Cytosine methylation profiling of cancer cell lines

    PubMed Central

    Ehrich, Mathias; Turner, Julia; Gibbs, Peter; Lipton, Lara; Giovanneti, Mara; Cantor, Charles; van den Boom, Dirk

    2008-01-01

    DNA-methylation changes in human cancer are complex and vary between the different types of cancer. Capturing this epigenetic variability in an atlas of DNA-methylation changes will be beneficial for basic research as well as translational medicine. Hypothesis-free approaches that interrogate methylation patterns genome-wide have already generated promising results. However, these methods are still limited by their quantitative accuracy and the number of CpG sites that can be assessed individually. Here, we use a unique approach to measure quantitative methylation patterns in a set of >400 candidate genes. In this high-resolution study, we employed a cell-line model consisting of 59 cancer cell lines provided by the National Cancer Institute and six healthy control tissues for discovery of methylation differences in cancer-related genes. To assess the effect of cell culturing, we validated the results from colon cancer cell lines by using clinical colon cancer specimens. Our results show that a large proportion of genes (78 of 400 genes) are epigenetically altered in cancer. Although most genes show methylation changes in only one tumor type (35 genes), we also found a set of genes that changed in many different forms of cancer (seven genes). This dataset can easily be expanded to develop a more comprehensive and ultimately complete map of quantitative methylation changes. Our methylation data also provide an ideal starting point for further translational research where the results can be combined with existing large-scale datasets to develop an approach that integrates epigenetic, transcriptional, and mutational findings. PMID:18353987

  15. CellLineNavigator: a workbench for cancer cell line analysis.

    PubMed

    Krupp, Markus; Itzel, Timo; Maass, Thorsten; Hildebrandt, Andreas; Galle, Peter R; Teufel, Andreas

    2013-01-01

    The CellLineNavigator database, freely available at http://www.medicalgenomics.org/celllinenavigator, is a web-based workbench for large scale comparisons of a large collection of diverse cell lines. It aims to support experimental design in the fields of genomics, systems biology and translational biomedical research. Currently, this compendium holds genome wide expression profiles of 317 different cancer cell lines, categorized into 57 different pathological states and 28 individual tissues. To enlarge the scope of CellLineNavigator, the database was furthermore closely linked to commonly used bioinformatics databases and knowledge repositories. To ensure easy data access and search ability, a simple data and an intuitive querying interface were implemented. It allows the user to explore and filter gene expression, focusing on pathological or physiological conditions. For a more complex search, the advanced query interface may be used to query for (i) differentially expressed genes; (ii) pathological or physiological conditions; or (iii) gene names or functional attributes, such as Kyoto Encyclopaedia of Genes and Genomes pathway maps. These queries may also be combined. Finally, CellLineNavigator allows additional advanced analysis of differentially regulated genes by a direct link to the Database for Annotation, Visualization and Integrated Discovery (DAVID) Bioinformatics Resources.

  16. High prevalence of side population in human cancer cell lines

    PubMed Central

    Boesch, Maximilian; Zeimet, Alain G.; Fiegl, Heidi; Wolf, Barbara; Huber, Julia; Klocker, Helmut; Gastl, Guenther

    2016-01-01

    Cancer cell lines are essential platforms for performing cancer research on human cells. We here demonstrate that, across tumor entities, human cancer cell lines harbor minority populations of putative stem-like cells, molecularly defined by dye extrusion resulting in the side population phenotype. These findings establish a heterogeneous nature of human cancer cell lines and argue for their stem cell origin. This should be considered when interpreting research involving these model systems. PMID:27226981

  17. Growth Patterns of Placental and Paraovarian Adrenocortical Heterotopias Are Different

    PubMed Central

    Xu, Bo; Popiolek, Dorota A.

    2013-01-01

    Two cases of adrenocortical heterotopia are reported. One is in a full-term placenta. The other is adjacent to the ovarian hilum of an adult. Both are incidental findings. Despite sharing similar histological and immunological features, they show different growth patterns. The literature is reviewed and adrenocortical heterotopias of different locations are compared. New hypotheses of its histogenesis are discussed. PMID:24383032

  18. Comprehensive Pan-Genomic Characterization of Adrenocortical Carcinoma.

    PubMed

    Zheng, Siyuan; Cherniack, Andrew D; Dewal, Ninad; Moffitt, Richard A; Danilova, Ludmila; Murray, Bradley A; Lerario, Antonio M; Else, Tobias; Knijnenburg, Theo A; Ciriello, Giovanni; Kim, Seungchan; Assie, Guillaume; Morozova, Olena; Akbani, Rehan; Shih, Juliann; Hoadley, Katherine A; Choueiri, Toni K; Waldmann, Jens; Mete, Ozgur; Robertson, A Gordon; Wu, Hsin-Ta; Raphael, Benjamin J; Shao, Lina; Meyerson, Matthew; Demeure, Michael J; Beuschlein, Felix; Gill, Anthony J; Sidhu, Stan B; Almeida, Madson Q; Fragoso, Maria C B V; Cope, Leslie M; Kebebew, Electron; Habra, Mouhammed A; Whitsett, Timothy G; Bussey, Kimberly J; Rainey, William E; Asa, Sylvia L; Bertherat, Jérôme; Fassnacht, Martin; Wheeler, David A; Hammer, Gary D; Giordano, Thomas J; Verhaak, Roel G W

    2016-05-01

    We describe a comprehensive genomic characterization of adrenocortical carcinoma (ACC). Using this dataset, we expand the catalogue of known ACC driver genes to include PRKAR1A, RPL22, TERF2, CCNE1, and NF1. Genome wide DNA copy-number analysis revealed frequent occurrence of massive DNA loss followed by whole-genome doubling (WGD), which was associated with aggressive clinical course, suggesting WGD is a hallmark of disease progression. Corroborating this hypothesis were increased TERT expression, decreased telomere length, and activation of cell-cycle programs. Integrated subtype analysis identified three ACC subtypes with distinct clinical outcome and molecular alterations which could be captured by a 68-CpG probe DNA-methylation signature, proposing a strategy for clinical stratification of patients based on molecular markers. PMID:27165744

  19. On the Ontology Based Representation of Cell Lines

    PubMed Central

    Ganzinger, Matthias; He, Shan; Breuhahn, Kai; Knaup, Petra

    2012-01-01

    Cell lines are frequently used as highly standardized and reproducible in vitro models for biomedical analyses and assays. Cell lines are distributed by cell banks that operate databases describing their products. However, the description of the cell lines' properties are not standardized across different cell banks. Existing cell line-related ontologies mostly focus on the description of the cell lines' names, but do not cover aspects like the origin or optimal growth conditions. The objective of this work is to develop an ontology that allows for a more comprehensive description of cell lines and their metadata, which should cover the data elements provided by cell banks. This will provide the basis for the standardized annotation of cell lines and corresponding assays in biomedical research. In addition, the ontology will be the foundation for automated evaluation of such assays and their respective protocols in the future. To accomplish this, a broad range of cell bank databases as well as existing ontologies were analyzed in a comprehensive manner. We identified existing ontologies capable of covering different aspects of the cell line domain. However, not all data fields derived from the cell banks' databases could be mapped to existing ontologies. As a result, we created a new ontology called cell culture ontology (CCONT) integrating existing ontologies where possible. CCONT provides classes from the areas of cell line identification, origin, cell line properties, propagation and tests performed. PMID:23144907

  20. 77 FR 5489 - Identification of Human Cell Lines Project

    Federal Register 2010, 2011, 2012, 2013, 2014

    2012-02-03

    ... National Institute of Standards and Technology Identification of Human Cell Lines Project AGENCY: National... tandem repeat (STR) profiling up to 1500 human cell line samples as part of the Identification of Human Cell Lines Project. All data and corresponding information will be posted in a publically held...

  1. EXAFS studies of prostate cancer cell lines

    NASA Astrophysics Data System (ADS)

    Czapla, J.; Kwiatek, W. M.; Lekki, J.; Kisiel, A.; Steininger, R.; Goettlicher, J.

    2013-04-01

    Sulphur plays a vital role in every human organism. It is known, that sulphur-bearing compounds, such as for example cysteine and glutathione, play critical roles in development and progression of many diseases. Any alteration in sulphur's biochemistry could become a precursor of serious pathological conditions. One of such condition is prostate cancer, the most frequently diagnosed malignancy in the western world and the second leading cause of cancer related death in men. The purpose of presented studies was to examine what changes occur in the nearest chemical environment of sulphur in prostate cancer cell lines in comparison to healthy cells. The Extended X-ray Absorption Fine Structure (EXAFS) spectroscopy was used, followed by theoretical calculations. The results of preliminary analysis is presented.

  2. Interaction between Angiotensin II and Insulin/IGF-1 Exerted a Synergistic Stimulatory Effect on ERK1/2 Activation in Adrenocortical Carcinoma H295R Cells

    PubMed Central

    Tong, An-li; Wang, Fen; Cui, Yun-ying; Li, Chun-yan; Li, Yu-xiu

    2016-01-01

    The cross talk between angiotensin II (Ang II) and insulin has been described mainly in cardiovascular cells, hepatocytes, adipocytes, and so forth, and to date no such cross talk was reported in adrenal. In this study, we examined the interaction between Ang II and insulin/IGF-1 in ERK and AKT signaling pathways and expression of steroidogenic enzymes in H295R cells. Compared to the control, 100 nM Ang II increased phospho-ERK1/2 approximately 3-fold. Insulin (100 nM) or IGF-1 (10 nM) alone raised phospho-ERK1/2 1.8- and 1.5-fold, respectively, while, after pretreatment with 100 nM Ang II for 30 min, insulin (100 nM) or IGF-1 (10 nM) elevated phospho-ERK1/2 level 8- and 7-fold, respectively. The synergistic effect of Ang II and insulin/IGF-1 on ERK1/2 activation was inhibited by selective AT1 receptor blocker, PKC inhibitor, and MEK1/2 inhibitor. Ang II marginally suppressed AKT activation under the basal condition, while it had no effect on phospho-AKT induced by insulin/IGF-1. Ang II significantly stimulated mRNA expression of CYP11B1 and CYP11B2, and such stimulatory effects were enhanced when cells were cotreated with insulin/IGF-1. We are led to conclude that Ang II in combination with insulin/IGF-1 had an evident synergistic stimulatory effect on ERK1/2 activation in H295R cells and the effect may be responsible for the enhanced steroid hormone production induced by Ang II plus insulin/IGF-1. PMID:27293433

  3. Generating Rho-0 Cells Using Mesenchymal Stem Cell Lines

    PubMed Central

    Fernández-Moreno, Mercedes; Hermida-Gómez, Tamara; Gallardo, M. Esther; Dalmao-Fernández, Andrea; Rego-Pérez, Ignacio; Garesse, Rafael

    2016-01-01

    Introduction The generation of Rho-0 cells requires the use of an immortalization process, or tumor cell selection, followed by culture in the presence of ethidium bromide (EtBr), incurring the drawbacks its use entails. The purpose of this work was to generate Rho-0 cells using human mesenchymal stem cells (hMSCs) with reagents having the ability to remove mitochondrial DNA (mtDNA) more safely than by using EtBr. Methodology Two immortalized hMSC lines (3a6 and KP) were used; 143B.TK-Rho-0 cells were used as reference control. For generation of Rho-0 hMSCs, cells were cultured in medium supplemented with each tested reagent. Total DNA was isolated and mtDNA content was measured by real-time polymerase chain reaction (PCR). Phenotypic characterization and gene expression assays were performed to determine whether 3a6 Rho-0 hMSCs maintain the same stem properties as untreated 3a6 hMSCs. To evaluate whether 3a6 Rho-0 hMSCs had a phenotype similar to that of 143B.TK-Rho-0 cells, in terms of reactive oxygen species (ROS) production, apoptotic levels and mitochondrial membrane potential (Δψm) were measured by flow cytometry and mitochondrial respiration was evaluated using a SeaHorse XFp Extracellular Flux Analyzer. The differentiation capacity of 3a6 and 3a6 Rho-0 hMSCs was evaluated using real-time PCR, comparing the relative expression of genes involved in osteogenesis, adipogenesis and chondrogenesis. Results The results showed the capacity of the 3a6 cell line to deplete its mtDNA and to survive in culture with uridine. Of all tested drugs, Stavudine (dt4) was the most effective in producing 3a6-Rho cells. The data indicate that hMSC Rho-0 cells continue to express the characteristic MSC cell surface receptor pattern. Phenotypic characterization showed that 3a6 Rho-0 cells resembled 143B.TK-Rho-0 cells, indicating that hMSC Rho-0 cells are Rho-0 cells. While the adipogenic capability was higher in 3a6 Rho-0 cells than in 3a6 cells, the osteogenic and chondrogenic

  4. Adrenocortical LDL receptor function negatively influences glucocorticoid output.

    PubMed

    van der Sluis, Ronald J; Van Eck, Miranda; Hoekstra, Menno

    2015-09-01

    Over 50% of the cholesterol needed by adrenocortical cells for the production of glucocorticoids is derived from lipoproteins. However, the overall contribution of the different lipoproteins and associated uptake pathways to steroidogenesis remains to be determined. Here we aimed to show the importance of LDL receptor (LDLR)-mediated cholesterol acquisition for adrenal steroidogenesis in vivo. Female total body LDLR knockout mice with a human-like lipoprotein profile were bilaterally adrenalectomized and subsequently provided with one adrenal either expressing or genetically lacking the LDLR under their renal capsule to solely modulate adrenocortical LDLR function. Plasma total cholesterol levels and basal plasma corticosterone levels were identical in the two types of adrenal transplanted mice. Strikingly, restoration of adrenal LDLR function significantly reduced the ACTH-mediated stimulation of adrenal steroidogenesis (P<0.001), with plasma corticosterone levels that were respectively 44-59% lower (P<0.01) as compared to adrenal LDLR negative controls. In addition, LDLR positive adrenal transplanted mice exhibited a significant decrease (-39%; P<0.001) in their plasma corticosterone level under fasting stress conditions. Biochemical analysis did not show changes in the expression of genes involved in cholesterol mobilization. However, LDLR expressing adrenal transplants displayed a marked 62% reduction (P<0.05) in the transcript level of the key steroidogenic enzyme HSD3B2. In conclusion, our studies in a mouse model with a human-like lipoprotein profile provide the first in vivo evidence for a novel inhibitory role of the LDLR in the control of adrenal glucocorticoid production. PMID:26136384

  5. Wnt Signaling Inhibits Adrenal Steroidogenesis by Cell-Autonomous and Non–Cell-Autonomous Mechanisms

    PubMed Central

    Walczak, Elisabeth M.; Kuick, Rork; Finco, Isabella; Bohin, Natacha; Hrycaj, Steven M.; Wellik, Deneen M.

    2014-01-01

    Wnt/β-catenin (βcat) signaling is critical for adrenal homeostasis. To elucidate how Wnt/βcat signaling elicits homeostatic maintenance of the adrenal cortex, we characterized the identity of the adrenocortical Wnt-responsive population. We find that Wnt-responsive cells consist of sonic hedgehog (Shh)-producing adrenocortical progenitors and differentiated, steroidogenic cells of the zona glomerulosa, but not the zona fasciculata and rarely cells that are actively proliferating. To determine potential direct inhibitory effects of βcat signaling on zona fasciculata-associated steroidogenesis, we used the mouse ATCL7 adrenocortical cell line that serves as a model system of glucocorticoid-producing fasciculata cells. Stimulation of βcat signaling caused decreased corticosterone release consistent with the observed reduced transcription of steroidogenic genes Cyp11a1, Cyp11b1, Star, and Mc2r. Decreased steroidogenic gene expression was correlated with diminished steroidogenic factor 1 (Sf1; Nr5a1) expression and occupancy on steroidogenic promoters. Additionally, βcat signaling suppressed the ability of Sf1 to transactivate steroidogenic promoters independent of changes in Sf1 expression level. To investigate Sf1-independent effects of βcat on steroidogenesis, we used Affymetrix gene expression profiling of Wnt-responsive cells in vivo and in vitro. One candidate gene identified, Ccdc80, encodes a secreted protein with unknown signaling mechanisms. We report that Ccdc80 is a novel βcat-regulated gene in adrenocortical cells. Treatment of adrenocortical cells with media containing secreted Ccdc80 partially phenocopies βcat-induced suppression of steroidogenesis, albeit through an Sf1-independent mechanism. This study reveals multiple mechanisms of βcat-mediated suppression of steroidogenesis and suggests that Wnt/βcat signaling may regulate adrenal homeostasis by inhibiting fasciculata differentiation and promoting the undifferentiated state of progenitor

  6. Derivation of three new human embryonic stem cell lines.

    PubMed

    Bradley, Cara K; Chami, Omar; Peura, Teija T; Bosman, Alexis; Dumevska, Biljana; Schmidt, Uli; Stojanov, Tomas

    2010-04-01

    Human embryonic stem cells are pluripotent cells capable of extensive self-renewal and differentiation to all cells of the embryo proper. Here, we describe the derivation and characterization of three Sydney IVF human embryonic stem cell lines not already reported elsewhere, designated SIVF001, SIVF002, and SIVF014. The cell lines display typical compact colony morphology of embryonic stem cells, have stable growth rates over more than 40 passages and are cytogenetically normal. Furthermore, the cell lines express pluripotency markers including Nanog, Oct4, SSEA3 and Tra-1-81, and are capable of generating teratoma cells derived from each of the three germ layers in immunodeficient mice. These experiments show that the cell lines constitute pluripotent stem cell lines. PMID:20198447

  7. Neuronal cell lines as model dorsal root ganglion neurons

    PubMed Central

    Yin, Kathleen; Baillie, Gregory J

    2016-01-01

    Background Dorsal root ganglion neuron-derived immortal cell lines including ND7/23 and F-11 cells have been used extensively as in vitro model systems of native peripheral sensory neurons. However, while it is clear that some sensory neuron-specific receptors and ion channels are present in these cell lines, a systematic comparison of the molecular targets expressed by these cell lines with those expressed in intact peripheral neurons is lacking. Results In this study, we examined the expression of RNA transcripts in the human neuroblastoma-derived cell line, SH-SY5Y, and two dorsal root ganglion hybridoma cell lines, F-11 and ND7/23, using Illumina next-generation sequencing, and compared the results with native whole murine dorsal root ganglions. The gene expression profiles of these three cell lines did not resemble any specific defined dorsal root ganglion subclass. The cell lines lacked many markers for nociceptive sensory neurons, such as the Transient receptor potential V1 gene, but expressed markers for both myelinated and unmyelinated neurons. Global gene ontology analysis on whole dorsal root ganglions and cell lines showed similar enrichment of biological process terms across all samples. Conclusions This paper provides insights into the receptor repertoire expressed in common dorsal root ganglion neuron-derived cell lines compared with whole murine dorsal root ganglions, and illustrates the limits and potentials of these cell lines as tools for neuropharmacological exploration. PMID:27130590

  8. Development and characterization of a new human hepatic cell line.

    PubMed

    Ramboer, Eva; De Craene, Bram; De Kock, Joey; Berx, Geert; Rogiers, Vera; Vanhaecke, Tamara; Vinken, Mathieu

    2015-01-01

    The increasing demand and hampered use of primary human hepatocytes for research purposes have urged scientists to search for alternative cell sources, such as immortalized hepatic cell lines. The aim of this study was to develop a human hepatic cell line using the combined overexpression of TERT and the cell cycle regulators cyclin D1 and mutant isoform CDK4R24C. Following transduction of adult human primary hepatocytes with the selected immortalization genes, cell growth was triggered and a cell line was established. When cultured under appropriate conditions, the cell line expressed several hepatocytic markers and liver-enriched transcription factors at the transcriptional and/or translational level, secreted liver-specific proteins and showed glycogen deposition. These results suggest that the immortalization strategy applied to primary human hepatocytes could generate a novel hepatic cell line that seems to retain some key hepatic characteristics. PMID:26869867

  9. Development and characterization of a new human hepatic cell line

    PubMed Central

    Ramboer, Eva; De Craene, Bram; De Kock, Joey; Berx, Geert; Rogiers, Vera; Vanhaecke, Tamara; Vinken, Mathieu

    2015-01-01

    The increasing demand and hampered use of primary human hepatocytes for research purposes have urged scientists to search for alternative cell sources, such as immortalized hepatic cell lines. The aim of this study was to develop a human hepatic cell line using the combined overexpression of TERT and the cell cycle regulators cyclin D1 and mutant isoform CDK4R24C. Following transduction of adult human primary hepatocytes with the selected immortalization genes, cell growth was triggered and a cell line was established. When cultured under appropriate conditions, the cell line expressed several hepatocytic markers and liver-enriched transcription factors at the transcriptional and/or translational level, secreted liver-specific proteins and showed glycogen deposition. These results suggest that the immortalization strategy applied to primary human hepatocytes could generate a novel hepatic cell line that seems to retain some key hepatic characteristics. PMID:26869867

  10. Distinct differentiation characteristics of individual human embryonic stem cell lines

    PubMed Central

    Mikkola, Milla; Olsson, Cia; Palgi, Jaan; Ustinov, Jarkko; Palomaki, Tiina; Horelli-Kuitunen, Nina; Knuutila, Sakari; Lundin, Karolina; Otonkoski, Timo; Tuuri, Timo

    2006-01-01

    Background Individual differences between human embryonic stem cell (hESC) lines are poorly understood. Here, we describe the derivation of five hESC lines (called FES 21, 22, 29, 30 and 61) from frozen-thawed human embryos and compare their individual differentiation characteristic. Results The cell lines were cultured either on human or mouse feeder cells. The cells grew significantly faster and could be passaged enzymatically only on mouse feeders. However, this was found to lead to chromosomal instability after prolonged culture. All hESC lines expressed the established markers of pluripotent cells as well as several primordial germ cell (PGC) marker genes in a uniform manner. However, the cell lines showed distinct features in their spontaneous differentiation patterns. The embryoid body (EB) formation frequency of FES 30 cell line was significantly lower than that of other lines and cells within the EBs differentiated less readily. Likewise, teratomas derived from FES 30 cells were constantly cystic and showed only minor solid tissue formation with a monotonous differentiation pattern as compared with the other lines. Conclusion hESC lines may differ substantially in their differentiation properties although they appear similar in the undifferentiated state. PMID:16895598

  11. Morphological changes in the pituitary-adrenocortical axis in natives of La Paz

    NASA Astrophysics Data System (ADS)

    Gosney, John; Heath, Donald; Williams, David; Rios-Dalenz, Jaime

    1991-03-01

    Increased activity of the hypothalamic-pituitary-adrenocortical axis is part of the response to the stress of initial exposure to hypoxia, but there is evidence to suggest that it persists after homeostatic stability has been regained and acclimatization achieved. The adrenal glands of five lifelong residents of La Paz, Bolivia, who had lived at altitudes in the range 3600 3800 m, were significantly larger than those in age-matched controls from sea level (15.3g vs 10.4g; P<0.001) and appeared hyperplastic. The pituitary glands of the highlanders were not significantly different in size from those of the controls (0.67 g vs 0.51 g), but contained larger populations of corticotrophs expressed in terms of the total cell population of their anterior lobes (25.6% vs 19.4%; P<0.001). In conjunction with other studies of this endocrine axis in man and animals exposed to a hypoxic environment, these data suggest that greater amounts of adrenocorticotrophic hormone (ACTH) are required to maintain normal adrenocortical function under such circumstances, probably as a result of hypoxic inhibition of adrenocortical sensitivity to stimulation. Physiological hyperplasia of the adrenal cortex may be common in people living at high altitude.

  12. The transcendental meditation technique, adrenocortical activity, and implications for stress.

    PubMed

    Jevning, R; Wilson, A F; Smith, W R

    1978-05-15

    The practice of the transcendental meditation technique in subjects eliciting this state regularly for 3--5 years is correlated with acute decline of adrenocortical activity not associated with sleep during the practice.

  13. Continuous human cell lines and method of making same

    DOEpatents

    Stampfer, M.R.

    1985-07-01

    Substantially genetically stable continuous human cell lines derived from normal human mammary epithelial cells (HMEC) and processes for making and using the same. In a preferred embodiment, the cell lines are derived by treating normal human mammary epithelial tissue with a chemical carcinogen such as benzo(a)pyrene. The novel cell lines serve as useful substrates for elucidating the potential effects of a number of toxins, carcinogens and mutagens as well as of the addition of exogenous genetic material. The autogenic parent cells from which the cell lines are derived serve as convenient control samples for testing. The cell lines are not neoplastically transformed, although they have acquired several properties which distinguish them from their normal progenitors. 2 tabs.

  14. Continuous human cell lines and method of making same

    DOEpatents

    Stampfer, Martha R.

    1989-01-01

    Substantially genetically stable continuous human cell lines derived from normal human mammary epithelial cells (HMEC) and processes for making and using the same. In a preferred embodiment, the cell lines are derived by treating normal human mammary epithelial tissue with a chemical carcinogen such as benzo[a]pyrene. The novel cell lines serve as useful substrates for elucidating the potential effects of a number of toxins, carcinogens and mutagens as well as of the addition of exogenous genetic material. The autogenic parent cells from which the cell lines are derived serve as convenient control samples for testing. The cell lines are not neoplastically transformed, although they have acquired several properties which distinguish them from their normal progenitors.

  15. VOLIN and KJON-Two novel hyperdiploid myeloma cell lines.

    PubMed

    Våtsveen, Thea Kristin; Børset, Magne; Dikic, Aida; Tian, Erming; Micci, Francesca; Lid, Ana H B; Meza-Zepeda, Leonardo A; Coward, Eivind; Waage, Anders; Sundan, Anders; Kuehl, W Michael; Holien, Toril

    2016-11-01

    Multiple myeloma can be divided into two distinct genetic subgroups: hyperdiploid (HRD) or nonhyperdiploid (NHRD) myeloma. Myeloma cell lines are important tools to study myeloma cell biology and are commonly used for preclinical screening and testing of new drugs. With few exceptions human myeloma cell lines are derived from NHRD patients, even though about half of the patients have HRD myeloma. Thus, there is a need for cell lines of HRD origin to enable more representative preclinical studies. Here, we present two novel myeloma cell lines, VOLIN and KJON. Both of them were derived from patients with HRD disease and shared the same genotype as their corresponding primary tumors. The cell lines' chromosomal content, genetic aberrations, gene expression, immunophenotype as well as some of their growth characteristics are described. Neither of the cell lines was found to harbor immunoglobulin heavy chain translocations. The VOLIN cell line was established from a bone marrow aspirate and KJON from peripheral blood. We propose that these unique cell lines may be used as tools to increase our understanding of myeloma cell biology. © 2016 Wiley Periodicals, Inc. PMID:27311012

  16. Cell lines used for the selection of recombinant baculovirus.

    PubMed

    Maruniak, J E; Garcia-Canedo, A; Rodrigues, J J

    1994-04-01

    Four insect cell lines were used to isolate two recombinant baculoviruses which had the beta-galactosidase (beta-gal) gene for colorimetric assay purposes. Plaque assays were performed using two Trichoplusia ni cell lines: BTI-TN-5B1-4 and TN-368, and two Spodptera frugiperda cell lines: IPLB-SF-21AE and SF9. The number of plaques (occlusion positive and blue beta-gal+ recombinants) formed in the Trichoplusia cells was higher than in the Spodoptera cells. The appearance of Autographa californica NPV polyhedra was also faster in the T. ni cell lines. The effect of cell passage on the plaque formation proved to be critical when two different passages of the SF9 cells were tested. The higher passage produced a lower viral titration. The size and time of appearance of the plaques was also different.

  17. Authentication of the R06E Fruit Bat Cell Line

    PubMed Central

    Jordan, Ingo; Munster, Vincent J.; Sandig, Volker

    2012-01-01

    Fruit bats and insectivorous bats are believed to provide a natural reservoir for a wide variety of infectious diseases. Several lines of evidence, including the successful isolation of infectious viruses, indicate that Marburg virus and Ravn virus have found a major reservoir in colonies of the Egyptian rousette (Rousettus aegyptiacus). To facilitate molecular studies on virus-reservoir host interactions and isolation of viruses from environmental samples, we established cell lines from primary cells of this animal. The cell lines were given to several laboratories until we realized that a contamination with Vero cells in one of the cultures had occurred. Here we describe a general diagnostic procedure for identification of cross-species contamination with the focus on Vero and Rousettus cell lines, and summarize newly discovered properties of the cell lines that may pertain to pathogen discovery. PMID:22754654

  18. Brain Metastasis in Patients With Adrenocortical Carcinoma: A Clinical Series

    PubMed Central

    Tageja, Nishant; Rosenberg, Avi; Mahalingam, Sowmya; Quezado, Martha; Velarde, Margarita; Edgerly, Maureen; Fojo, Tito

    2015-01-01

    Introduction: Adrenocortical carcinoma (ACC) is a heterogeneous and rare disease. At presentation or at the time of a recurrence, the disease commonly spreads to the liver, lungs, lymph nodes, and bones. The brain has only rarely been reported as a site of metastases. Objective: The aims of this report were to describe the clinical characteristics of patients with ACC who developed brain metastasis and were evaluated at the National Cancer Institute. Methods: We describe the history and clinical presentation of six patients with ACC and metastatic disease in the brain. Images of the six patients and pathology slides were reviewed when available. Results: The median age at the time of the diagnosis of ACC was 42 years. The median time from the initial diagnosis until the presentation of brain metastasis was 43 months. As a group the patients had previously received multiples lines of chemotherapy (median of three), and they presented with one to three metastatic brain lesions. Four patients underwent metastasectomy, one had radiosurgery, and one had both modalities. Two patients are still alive, three died, between 2 and 14 months after the diagnosis of brain metastases, and one was lost to follow-up. Conclusion: Patients with advanced ACC can rarely present with metastasis to the brain, most often long after the initial diagnosis. Timely diagnosis of brain metastasis with appropriate intervention after discussion in a multidisciplinary meeting can improve the prognosis in this particular scenario. PMID:25412413

  19. Establishment and characterization of the Bombyx mandarina cell line.

    PubMed

    Iwanaga, Masashi; Arai, Rika; Shibano, Yuka; Kawasaki, Hideki; Imanishi, Shigeo

    2009-06-01

    A new cell line, designated as NIAS-Boma-529b, was established from the larval fat bodies of Bombyx mandarina (B. mandarina), which is believed to be an ancestor of Bombyx mori (B. mori). This cell line has been cultured for approximately 150 passages during 2years in an IPL-41 medium supplemented with 10% fetal bovine serum at a constant temperature of 26 degrees C. The morphology of this line includes adhesive round and spindle-shaped cells. Random-amplified polymorphic DNA analysis (RAPD) using 7 primers and a statistical analysis based on Nei's genetic distance revealed that this cell line was closely related to B. mori-derived cell lines. An infection study also revealed that this cell line was susceptible to B. mori nucleopolyhedrovirus (BmNPV); however, it had no apparent susceptibility to Autographa californica NPV (AcNPV), which is closely related to BmNPV. Nevertheless, cells infected with AcNPV showed an extensive cytopathic effect (CPE), including a rough cell surface, rounding, nuclear expansion, and cell blebbing. These results suggest that this cell line can be useful to clarify the mechanism of host range determination of BmNPV and AcNPV.

  20. Pharmacogenomic agreement between two cancer cell line data sets.

    PubMed

    2015-12-01

    Large cancer cell line collections broadly capture the genomic diversity of human cancers and provide valuable insight into anti-cancer drug response. Here we show substantial agreement and biological consilience between drug sensitivity measurements and their associated genomic predictors from two publicly available large-scale pharmacogenomics resources: The Cancer Cell Line Encyclopedia and the Genomics of Drug Sensitivity in Cancer databases.

  1. Trichloroethylene toxicity in a human hepatoma cell line

    SciTech Connect

    Thevenin, E.; McMillian, J.

    1994-12-31

    The experiments conducted in this study were designed to determine the usefullness of hepatocyte cultures and a human hepatoma cell line as model systems for assessing human susceptibility to hepatocellular carcinoma due to exposure to trichloroethylene. The results from these studies will then be analyzed to determine if human cell lines can be used to conduct future experiments of this nature.

  2. Hemin toxicity in a human epithelioid sarcoma cell line.

    PubMed

    Braverman, S; Helson, C; Helson, L

    1995-01-01

    The major cytotoxic component of hemin was identified as metal free protoporphyrin IX in an epithelioid sarcoma cell line (VA-ES-BJ) and a glioblastoma cell line (U-373 MG) by exposing the cell lines to the iron chelator deferoxamine, tin-protoporphyrin IX, and protoporphyrin IX. The contribution of lipid peroxidation and free radical generation to toxicity was examined using DL-buthionine-[S,R]-sulfoximine (BSO), and 21-aminosteroid (lazaroid, U74500A). Hemin caused significantly greater toxicity in VA-ES-BJ than in U-373 MG. While exogenous PpIX was more toxic than hemin in both cell lines, this toxicity was not due to iron depletion following intracellular heme formation since ferric citrate did not reverse PpIX toxicity. Pre-treatment with BSO enhanced hemin toxicity in the VA-ES-BJ cell line but not in U-373 MG, suggesting different modes of toxicity in the two cell lines. Exposure to lazaroid protected only VA-ES-BJ from protoporphyrin-induced toxicity implicating a specific sensitivity to lipid peroxidation and/or free radical generation by this cell line. These characteristics of the VA-ES-BJ cell line distinguish it from the glioblastoma and emphasize its utility for exploring cytotoxic effects of hemin and its precursors.

  3. Derivation of the human embryonic stem cell line RCM1.

    PubMed

    De Sousa, P A; Tye, B J; Sneddon, S; Bruce, K; Dand, P; Russell, G; Collins, D M; Greenshields, A; McDonald, K; Bradburn, H; Gardner, J; Downie, J M; Courtney, A; Brison, D R

    2016-03-01

    The human embryonic stem cell line RCM-1 was derived from a failed to fertilise egg undergoing parthenogenetic stimulation. The cell line shows normal pluripotency marker expression and differentiation to three germ layers in vitro and in vivo. It has a normal 46XX female karyotype and microsatellite PCR identity, HLA and blood group typing data is available. PMID:27346018

  4. Regulated expression of erythropoietin by two human hepatoma cell lines

    SciTech Connect

    Goldberg, M.A.; Glass, G.A.; Cunningham, J.M.; Bunn, H.F.

    1987-11-01

    The development of a cell culture system that produces erythropoietin (Epo) in a regulated manner has been the focus of much effort. The authors have screened multiple renal and hepatic cell lines for either constitutive or regulated expression of Epo. Only the human hepatoma cell lines, Hep3B and HepG2, made significant amounts of Epo as measured both by radioimmunoassay and in vitro bioassay (as much as 330 milliunits per 10/sup 6/ cells in 24 hr). The constitutive production of Epo increased dramatically as a function of cell density in both cell lines. At cell densities < 3.3 x 10/sup 5/ cells per cm/sup 2/, there was little constitutive release of Epo in the medium. With Hep3B cells grown at low cell densities, a mean 18-fold increase in Epo expression was seen in response to hypoxia and a 6-fold increase was observed in response to incubation in medium containing 50 ..mu..M cobalt(II) chloride. At similar low cell densities, Epo production in HepG2 cells could be enhanced an average of about 3-fold by stimulation with either hypoxia or cobalt(II) chloride. Upon such stimulation, both cell lines demonstrated markedly elevated levels of Epo mRNA. Hence, both Hep3B and HepG2 cell lines provide an excellent in vitro system in which to study the physiological regulation of Epo expression.

  5. GREG cells, a dysferlin-deficient myogenic mouse cell line

    SciTech Connect

    Humphrey, Glen W.; Mekhedov, Elena; Blank, Paul S.; Morree, Antoine de; Pekkurnaz, Gulcin; Nagaraju, Kanneboyina; Zimmerberg, Joshua

    2012-01-15

    The dysferlinopathies (e.g. LGMD2b, Myoshi myopathy) are progressive, adult-onset muscle wasting syndromes caused by mutations in the gene coding for dysferlin. Dysferlin is a large ({approx} 200 kDa) membrane-anchored protein, required for maintenance of plasmalemmal integrity in muscle fibers. To facilitate analysis of dysferlin function in muscle cells, we have established a dysferlin-deficient myogenic cell line (GREG cells) from the A/J mouse, a genetic model for dysferlinopathy. GREG cells have no detectable dysferlin expression, but proliferate normally in growth medium and fuse into functional myotubes in differentiation medium. GREG myotubes exhibit deficiencies in plasma membrane repair, as measured by laser wounding in the presence of FM1-43 dye. Under the wounding conditions used, the majority ({approx} 66%) of GREG myotubes lack membrane repair capacity, while no membrane repair deficiency was observed in dysferlin-normal C2C12 myotubes, assayed under the same conditions. We discuss the possibility that the observed heterogeneity in membrane resealing represents genetic compensation for dysferlin deficiency.

  6. CACO-2 CELL LINES IN DRUG DISCOVERY- AN UPDATED PERSPECTIVE

    PubMed Central

    Kumar, Kalyan K.V; Karnati, Swathi; Reddy, Mamatha B; Chandramouli, R

    2010-01-01

    Cell lines are the invitro models used for the drug permeability studies in the preclinical and clinical phases of the drug discovery. Cell line models are simple and quick to use and avoids the usage of animal models for pharmacological and toxicological studies and hence cost effective, produce reliable and reproducible results for understanding and evaluating the permeability characteristics of the potential lead drug candidates. Different cell line models used in the drug permeability studies, their characteristics has been summarized emphasizing on CACO-2. By virtue of its merits, CACO-2 cell line development, transport experiments, automated assays, optimization of experimental conditions and mechanistic uses of CACO-2 cell lines dealt comprehensively in the following context. PMID:24825967

  7. Derivation of Genea057 human embryonic stem cell line.

    PubMed

    Dumevska, Biljana; Chami, Omar; McKernan, Robert; Goel, Divya; Schmidt, Uli

    2016-01-01

    The Genea057 human embryonic stem cell line was derived from a donated, fully commercially consented ART blastocyst, through ICM outgrowth on inactivated human feeders. The line showed pluripotent cell morphology and genomic analysis verified a 46, XX karyotype and female allele pattern through traditional karyotyping, CGH and STR analysis. Pluripotency of Genea057 was demonstrated with 97% of cells expressing Nanog, 81% Oct4, 75% Tra1-60 and 97% SSEA4, a PluriTest Pluripotency score of 27.59 and Novelty score of 1.32. The cell line was negative for Mycoplasma and any visible contamination. PMID:27345782

  8. Derivation of Genea042 human embryonic stem cell line.

    PubMed

    Dumevska, Biljana; Chami, Omar; McKernan, Robert; Goel, Divya; Schmidt, Uli

    2016-03-01

    The Genea042 human embryonic stem cell line was derived from a donated, fully commercially consented ART blastocyst, through ICM outgrowth on inactivated human feeders. The line showed pluripotent cell morphology and genomic analysis verified a 46, XX karyotype and female allele pattern through traditional karyotyping, CGH and STR analysis. Pluripotency of Genea042 was demonstrated with 81% of cells expressing Nanog, 95% Oct4, 53% Tra1-60 and 97% SSEA4, a PluriTest Pluripotency score of 30.06, Novelty score of 1.24 and Alkaline Phosphatase activity. The cell line was negative for Mycoplasma and any visible contamination. PMID:27345994

  9. Derivation of Genea002 human embryonic stem cell line.

    PubMed

    Dumevska, Biljana; Bosman, Alexis; McKernan, Robert; Goel, Divya; Peura, Teija; Schmidt, Uli

    2016-01-01

    The Genea002 human embryonic stem cell line was derived from a donated, fully commercially consented ART blastocyst, through ICM outgrowth on inactivated feeders. The line showed pluripotent cell morphology and genomic analysis verified a 46, XY karyotype by CGH and male Allele pattern through STR analysis. Pluripotency of Genea002 was demonstrated with 75% of cells expressing Nanog, 93% Oct4, 83% Tra1-60 and 98% SSEA4, a Pluritest pluripotency score of 24.55, Novelty score of 1.39, teratomas with tissues from all embryonic germ layers and Alkaline Phosphatase activity. The cell line was negative for Mycoplasma and any visible contamination. PMID:27345802

  10. Derivation of Genea052 human embryonic stem cell line.

    PubMed

    Dumevska, Biljana; Chami, Omar; McKernan, Robert; Goel, Divya; Schmidt, Uli

    2016-03-01

    The Genea052 human embryonic stem cell line was derived from a donated, fully commercially consented ART blastocyst, through ICM outgrowth on inactivated human feeders. The line showed pluripotent cell morphology and genomic analysis verified a 46, XY karyotype and male allele pattern through CGH and STR analysis. Pluripotency of Genea052 was demonstrated with 85% of cells expressing Nanog, 87% Oct4, 60% Tra1-60 and 97% SSEA4, a PluriTest Pluripotency score of 27.21, Novelty score of 1.2 and tri-lineage teratoma formation. The cell line was negative for Mycoplasma and any visible contamination. PMID:27345996

  11. Derivation of human embryonic stem cell line Genea023.

    PubMed

    Dumevska, Biljana; Bosman, Alexis; McKernan, Robert; Goel, Divya; Schmidt, Uli; Peura, Teija

    2016-03-01

    The Genea023 human embryonic stem cell line was derived from a donated, fully commercially consented ART blastocyst, through ICM outgrowth on inactivated feeders. The line showed pluripotent cell morphology and genomic analysis verified a 46, XY karyotype and male allele pattern through CGH and STR analysis. Pluripotency of Genea023 was demonstrated with 85% of cells expressed Nanog, 98% Oct4, 55% Tra1-60 and 98% SSEA4, gave a Pluritest Pluripotency score of 42.76, Novelty of 1.23, demonstrated Alkaline Phosphatase activity and tri-lineage teratoma formation. The cell line was negative for Mycoplasma and visible contamination. PMID:27346015

  12. Derivation of Genea015 human embryonic stem cell line.

    PubMed

    Dumevska, Biljana; Chami, Omar; McKernan, Robert; Goel, Divya; Schmidt, Uli

    2016-03-01

    The Genea015 human embryonic stem cell line was derived from a donated, fully commercially consented ART blastocyst, through ICM outgrowth on inactivated human feeders. The line showed pluripotent cell morphology and genomic analysis verified a 46, XY karyotype and male Allele pattern through traditional karyotyping, CGH and STR analysis. Pluripotency of Genea015 was demonstrated with 80% of cells expressing Nanog, 97% Oct4, 75% Tra1-60 and 98% SSEA4, a PluriTest Pluripotency score of 29.52, Novelty score of 1.3 and Alkaline Phosphatase activity. The cell line was negative for Mycoplasma and any visible contamination. PMID:27346028

  13. Derivation of human embryonic stem cell line Genea022.

    PubMed

    Dumevska, Biljana; Bosman, Alexis; McKernan, Robert; Schmidt, Uli; Peura, Teija

    2016-03-01

    The Genea022 human embryonic stem cell line was derived from a donated, fully commercially consented ART blastocyst, through ICM outgrowth on inactivated feeders. The line showed pluripotent cell morphology and genomic analysis verified a 46, XY karyotype and male allele pattern through CGH and STR analysis. Pluripotency of Genea022 was demonstrated with 84% of cells expressed Nanog, 98% Oct4, 55% Tra1-60 and 97% SSEA4, gave a Pluritest Pluripotency score of 42.95, Novelty of 1.23, demonstrated Alkaline Phosphatase activity and tri-lineage teratoma formation. The cell line was negative for Mycoplasma and visible contamination. PMID:27346017

  14. Derivation of Genea047 human embryonic stem cell line.

    PubMed

    Dumevska, Biljana; Chami, Omar; McKernan, Robert; Goel, Divya; Schmidt, Uli

    2016-03-01

    The Genea047 human embryonic stem cell line was derived from a donated, fully commercially consented ART blastocyst, through ICM outgrowth on inactivated human feeders. The line showed pluripotent cell morphology and genomic analysis verified a 46, XX karyotype and female allele pattern through traditional karyotyping, CGH and STR analysis. Pluripotency of Genea047 was demonstrated with 88% of cells expressing Nanog, 95% Oct4, 59% Tra1-60 and 99% SSEA4, a PluriTest Pluripotency score of 30.86, Novelty score of 1.23 and tri-lineage teratoma formation. The cell line was negative for Mycoplasma and any visible contamination. PMID:27345995

  15. Derivation of Genea043 human embryonic stem cell line.

    PubMed

    Dumevska, Biljana; Chami, Omar; McKernan, Robert; Goel, Divya; Schmidt, Uli

    2016-01-01

    The Genea043 human embryonic stem cell line was derived from a donated, fully commercially consented ART blastocyst, through ICM outgrowth on inactivated human feeders. The line showed pluripotent cell morphology and genomic analysis verified a 46, XY karyotype and male allele pattern through traditional karyotyping, CGH and STR analysis. Pluripotency of Genea043 was demonstrated with 92% of cells expressing Nanog, 95% Oct4, 61% Tra1-60 and 99% SSEA4, a PluriTest Pluripotency score of 31.74, Novelty score of 1.2 and Alkaline Phosphatase activity. The cell line was negative for Mycoplasma and any visible contamination. PMID:27345801

  16. Derivation of Genea016 human embryonic stem cell line.

    PubMed

    Dumevska, Biljana; Chami, Omar; McKernan, Robert; Goel, Divya; Peura, Teija; Schmidt, Uli

    2016-01-01

    The Genea016 human embryonic stem cell line was derived from a donated, fully commercially consented ART blastocyst, through ICM outgrowth on inactivated human feeders. The line showed pluripotent cell morphology and genomic analysis verified a 46, XX karyotype and female Allele pattern through traditional karyotyping, CGH and STR analysis. Pluripotency of Genea016 was demonstrated with 77% of cells expressing Nanog, 95% Oct4, 53% Tra1-60 and 98% SSEA4, a PluriTest Pluripotency score of 28.4, Novelty score of 1.37 and Alkaline Phosphatase activity. The cell line was negative for Mycoplasma and any visible contamination. PMID:27345780

  17. Differential effects of bisphosphonates on breast cancer cell lines.

    PubMed

    Verdijk, R; Franke, H R; Wolbers, F; Vermes, I

    2007-02-01

    Bisphosphonates may induce direct anti-tumor effects in breast cancer cells in vitro. In this study, six bisphosphonates were administered to three breast cancer cell lines. Cell proliferation was measured by quantification of the expression of Cyclin D1 mRNA. Apoptosis was determined by flow cytometry of a DNA fragmentation assay. We demonstrated that bisphosphonates have direct effects on cell proliferation and apoptosis in different breast cancer cell lines. However, not all bisphosphonates act equally on breast cancer cells in vitro. Zoledronate seems to be the most potent of the six bisphosphonates. This in vitro study showed that bisphosphonates possess promising anti-tumor potential.

  18. Recombinant protein production from stable mammalian cell lines and pools.

    PubMed

    Hacker, David L; Balasubramanian, Sowmya

    2016-06-01

    We highlight recent developments for the production of recombinant proteins from suspension-adapted mammalian cell lines. We discuss the generation of stable cell lines using transposons and lentivirus vectors (non-targeted transgene integration) and site-specific recombinases (targeted transgene integration). Each of these methods results in the generation of cell lines with protein yields that are generally superior to those achievable through classical plasmid transfection that depends on the integration of the transfected DNA by non-homologous DNA end-joining. This is the main reason why these techniques can also be used for the generation of stable cell pools, heterogenous populations of recombinant cells generated by gene delivery and genetic selection without resorting to single cell cloning. This allows the time line from gene transfer to protein production to be reduced.

  19. Recombinant protein production from stable mammalian cell lines and pools.

    PubMed

    Hacker, David L; Balasubramanian, Sowmya

    2016-06-01

    We highlight recent developments for the production of recombinant proteins from suspension-adapted mammalian cell lines. We discuss the generation of stable cell lines using transposons and lentivirus vectors (non-targeted transgene integration) and site-specific recombinases (targeted transgene integration). Each of these methods results in the generation of cell lines with protein yields that are generally superior to those achievable through classical plasmid transfection that depends on the integration of the transfected DNA by non-homologous DNA end-joining. This is the main reason why these techniques can also be used for the generation of stable cell pools, heterogenous populations of recombinant cells generated by gene delivery and genetic selection without resorting to single cell cloning. This allows the time line from gene transfer to protein production to be reduced. PMID:27322762

  20. Deriving cell lines from zebrafish embryos and tumors.

    PubMed

    Choorapoikayil, Suma; Overvoorde, John; den Hertog, Jeroen

    2013-09-01

    Over the last two decades the zebrafish has emerged as a powerful model organism in science. The experimental accessibility, the broad range of zebrafish mutants, and the highly conserved genetic and biochemical pathways between zebrafish and mammals lifted zebrafish to become one of the most attractive vertebrate models to study gene function and to model human diseases. Zebrafish cell lines are highly attractive to investigate cell biology and zebrafish cell lines complement the experimental tools that are available already. We established a straightforward method to culture cells from a single zebrafish embryo or a single tumor. Here we describe the generation of fibroblast-like cell lines from wild-type and ptenb(-/-) embryos and an endothelial-like cell line from a tumor of an adult ptena(+/-)ptenb(-/-) zebrafish. This protocol can easily be adapted to establish stable cell lines from any mutant or transgenic zebrafish line and the average time to obtain a pro-stable cell line is 3-5 months.

  1. Effects of multiwalled carbon nanotubes and triclocarban on several eukaryotic cell lines: elucidating cytotoxicity, endocrine disruption, and reactive oxygen species generation

    NASA Astrophysics Data System (ADS)

    Simon, Anne; Maletz, Sibylle X.; Hollert, Henner; Schäffer, Andreas; Maes, Hanna M.

    2014-08-01

    To date, only a few reports about studies on toxic effects of carbon nanotubes (CNT) are available, and their results are often controversial. Three different cell lines (rainbow trout liver cells (RTL-W1), human adrenocortical carcinoma cells (T47Dluc), and human adrenocarcinoma cells (H295R)) were exposed to multiwalled carbon nanotubes, the antimicrobial agent triclocarban (TCC) as well as the mixture of both substances in a concentration range of 3.13 to 50 mg CNT/L, 31.25 to 500 μg TCC/L, and 3.13 to 50 mg CNT/L + 1% TCC (percentage relative to carbon nanotubes concentration), respectively. Triclocarban is a high-production volume chemical that is widely used as an antimicrobial compound and is known for its toxicity, hydrophobicity, endocrine disruption, bioaccumulation potential, and environmental persistence. Carbon nanotubes are known to interact with hydrophobic organic compounds. Therefore, triclocarban was selected as a model substance to examine mixture toxicity in this study. The influence of multiwalled carbon nanotubes and triclocarban on various toxicological endpoints was specified: neither cytotoxicity nor endocrine disruption could be observed after exposure of the three cell lines to carbon nanotubes, but the nanomaterial caused intracellular generation of reactive oxygen species in all cell types. For TCC on the other hand, cell vitality of 80% could be observed at a concentration of 2.1 mg/L for treated RTL-W1 cells. A decrease of luciferase activity in the ER Calux assay at a triclocarban concentration of 125 μg/L and higher was observed. This effect was less pronounced when multiwalled carbon nanotubes were present in the medium. Taken together, these results demonstrate that multiwalled carbon nanotubes induce the production of reactive oxygen species in RTL-W1, T47Dluc, and H295R cells, reveal no cytotoxicity, and reduce the bioavailability and toxicity of the biocide triclocarban.

  2. Effects of multiwalled carbon nanotubes and triclocarban on several eukaryotic cell lines: elucidating cytotoxicity, endocrine disruption, and reactive oxygen species generation.

    PubMed

    Simon, Anne; Maletz, Sibylle X; Hollert, Henner; Schäffer, Andreas; Maes, Hanna M

    2014-01-01

    To date, only a few reports about studies on toxic effects of carbon nanotubes (CNT) are available, and their results are often controversial. Three different cell lines (rainbow trout liver cells (RTL-W1), human adrenocortical carcinoma cells (T47Dluc), and human adrenocarcinoma cells (H295R)) were exposed to multiwalled carbon nanotubes, the antimicrobial agent triclocarban (TCC) as well as the mixture of both substances in a concentration range of 3.13 to 50 mg CNT/L, 31.25 to 500 μg TCC/L, and 3.13 to 50 mg CNT/L + 1% TCC (percentage relative to carbon nanotubes concentration), respectively. Triclocarban is a high-production volume chemical that is widely used as an antimicrobial compound and is known for its toxicity, hydrophobicity, endocrine disruption, bioaccumulation potential, and environmental persistence. Carbon nanotubes are known to interact with hydrophobic organic compounds. Therefore, triclocarban was selected as a model substance to examine mixture toxicity in this study. The influence of multiwalled carbon nanotubes and triclocarban on various toxicological endpoints was specified: neither cytotoxicity nor endocrine disruption could be observed after exposure of the three cell lines to carbon nanotubes, but the nanomaterial caused intracellular generation of reactive oxygen species in all cell types. For TCC on the other hand, cell vitality of 80% could be observed at a concentration of 2.1 mg/L for treated RTL-W1 cells. A decrease of luciferase activity in the ER Calux assay at a triclocarban concentration of 125 μg/L and higher was observed. This effect was less pronounced when multiwalled carbon nanotubes were present in the medium. Taken together, these results demonstrate that multiwalled carbon nanotubes induce the production of reactive oxygen species in RTL-W1, T47Dluc, and H295R cells, reveal no cytotoxicity, and reduce the bioavailability and toxicity of the biocide triclocarban.

  3. Eternity and functionality - rational access to physiologically relevant cell lines.

    PubMed

    Lipps, Christoph; May, Tobias; Hauser, Hansjörg; Wirth, Dagmar

    2013-12-01

    In the first 50 years of cell culture, the development of new cell lines was mainly based on trial and error. Due to the understanding of the molecular networks of aging, senescence, proliferation, and adaption by mutation, the generation of new cell lines with physiologic properties has become more systematic. This endeavor has been supported by the availability of new technological achievements and increasing knowledge about the biology of cell differentiation and cell-cell communication. Here, we review some promising developments that are contributing toward this goal. These include molecular tools frequently used for the immortalization process. In addition to these broadly acting immortalization regimens, we focus on the developments of cell type-specific immortalization and on the methodologies of how to control the growth of newly established cell lines.

  4. Application of DNA fingerprints for cell-line individualization.

    PubMed Central

    Gilbert, D A; Reid, Y A; Gail, M H; Pee, D; White, C; Hay, R J; O'Brien, S J

    1990-01-01

    DNA fingerprints of 46 human cell lines were derived using minisatellite probes for hypervariable genetic loci. The incidence of 121 HaeIII DNA fragments among 33 cell lines derived from unrelated individuals was used to estimate allelic and genotypic frequencies for each fragment and for composite individual DNA fingerprints. We present a quantitative estimate of the extent of genetic difference between individuals, an estimate based on the percentage of restriction fragments at which they differ. The average percent difference (APD) among pairwise combinations from the population of 33 unrelated cell lines was 76.9%, compared with the APD in band sharing among cell lines derived from the same individual (less than or equal to 1.2%). Included in this survey were nine additional cell lines previously implicated as HeLa cell derivatives, and these lines were clearly confirmed as such by DNA fingerprints (APD less than or equal to 0.6%). On the basis of fragment frequencies in the tested cell line population, a simple genetic model was developed to estimate the frequencies of each DNA fingerprint in the population. The median incidence was 2.9 X 10(-17), and the range was 2.4 X 10(-21) to 6.6 X 10(-15). This value approximates the probability that a second cell line selected at random from unrelated individuals will match a given DNA fingerprint. Related calculations address the chance that any two DNA fingerprints would be identical among a large group of cell lines. This estimate is still very slight; for example, the chance of two or more common DNA fingerprints among 1 million distinct individuals is less than .001. The procedure provides a straightforward, easily interpreted, and statistically robust method for identification and individualization of human cells. Images p[504]-a PMID:1975479

  5. Network analysis reveals potential markers for pediatric adrenocortical carcinoma

    PubMed Central

    Kulshrestha, Anurag; Suman, Shikha; Ranjan, Rakesh

    2016-01-01

    Pediatric adrenocortical carcinoma (ACC) is a rare malignancy with a poor outcome. Molecular mechanisms of pediatric ACC oncogenesis and advancement are not well understood. Accurate and timely diagnosis of the disease requires identification of new markers for pediatric ACC. Differentially expressed genes (DEGs) were identified from the gene expression profile of pediatric ACC and obtained from Gene Expression Omnibus. Gene Ontology functional and pathway enrichment analysis was implemented to recognize the functions of DEGs. A protein–protein interaction (PPI) and gene–gene functional interaction (GGI) network of DEGs was constructed. Hub gene detection and enrichment analysis of functional modules were performed. Furthermore, a gene regulatory network incorporating DEGs–microRNAs–transcription factors was constructed and analyzed. A total of 431 DEGs including 228 upregulated and 203 downregulated DEGs were screened. These genes were largely involved in cell cycle, steroid biosynthesis, and p53 signaling pathways. Upregulated genes, CDK1, CCNB1, CDC20, and BUB1B, were identified as the common hubs of PPI and GGI networks. All the four common hub genes were also part of modules of the PPI network. Moreover, all the four genes were also present in the largest module of GGI network. A gene regulatory network consisting of 82 microRNAs and 100 transcription factors was also constructed. CDK1, CCNB1, CDC20, and BUB1B may serve as potential biomarker of pediatric ACC and as potential targets for therapeutic approach, although experimental studies are required to authenticate our findings. PMID:27555782

  6. [DNA fingerprinting analysis of silkworm embryo cell lines].

    PubMed

    Pan, Min Hui; Feng, Zhen Yue; Tian, Zhi Qiang; Liu, Min; Lu, Cheng

    2006-12-01

    DNA extraction and the polymerase chain reaction (PCR) were used on DNA genomes study of cell lines of Bombyx mori. DNA polymorphic marker analysis was conducted and DNA fingerprint of cell lines of Bombyx mori. was carried out using ISSR and RAPD. Primers that can reliably find polymorphic bands were screened out. 26 ISSR primers were selected from them any available, and 797 polymorphic bands were abtained through PCR amplification in 9 samples, including 3 embryo cell lines of Bombyx mori (BmE-SWU1, BmE-SWU2, BmE-SWU3), 5 passage cell lines (BmE, BmN, Sf9, Sf21, Hi5) and the embryos from which BmE-SWU1 originated. The ration of polymorphic bands was 89.9%. 43 RAPD primers were selected out through PCR amplification, and 1205 polymorphic bands were obtained in 9 samples. The ration of polymorphic bands was 76.6%. There were many DNA polymorphic bands differences in the cell lines of Bombyx mori. The special DNA markers of the 3 embryo cell lines were found respectively. The similarity index Nei and genetic distance of the 9 samples were calculated and the phylogeny tree of 9 samples was constructed by UPGMA. Results showed that 2 groups were divided,one group including the 3 embryo cell lines and the embryo of XQ has close relative. Another group constructed by five insect cell lines came from different species, their genetic distance was closer than the 3 embryo cell lines.

  7. Radiosensitivity of hepatoma cell lines and human normal liver cell lines exposed to 12C6+ ions

    NASA Astrophysics Data System (ADS)

    Jing, X.; Yang, J.; Li, W.; Guo, C.; Dang, B.; Wang, J.; Zhou, L.; Wei, W.; Gao, Q.

    AIM To investigate the radiosensitivity of hepatoma cell lines and human normal liver cell lines METHODS Accelerated carbon ions by heavy ion research facility in Lanzhou HIRFL have high LET We employed it to study the radiosensitivity of hepatoma cell lines SMMC-7721 and human normal liver cell lines L02 using premature chromosome condensation technique PCC Cell survive was documented by a colony assay Chromatid breaks were measured by counting the number of chromatid breaks and isochromatid breaks immediately after prematurely chromosome condensed by Calyculin-A RESULTS The survival curve of the two cell lines presented a good linear relationship and the survival fraction of L02 is higher than that of SMMC-7721 Additionally the two types of G 2 phase chromosome breaks chromatid breaks and isochromatid breaks of L02 are lower than that of SMMC-7721 CONCLUSION Human normal liver cell line have high radioresistance than that of hepatoma cell line It imply that it is less damage to normal organs when radiotherapy to hepatoma

  8. Development of a cell line from Echinococcus granulosus germinal layer.

    PubMed

    Albani, Clara María; Cumino, Andrea Carina; Elissondo, María Celina; Denegri, Guillermo María

    2013-10-01

    In vitro culture of parasitic helminths provides an important tool to study cell regeneration and physiology, as well as for molecular biology and genetic engineering studies. In the present study, we established in vitro propagation of cells from Echinococcus granulosus germinal cyst layer. E. granulosus germinal cells grew beyond 100 passages and showed no signs of reduced proliferation capacity. Microscopic analysis revealed that cells grew both attached to the substrate and in suspension, forming three-dimensional structures like mammalian stem cell aggregates. Examination of the chromosome number of attached germinal cells showed a high degree of heteroploidy, suggesting the occurrence of transformation during culture. Monolayer cells survived cryopreservation and were able to proliferate after thawing. Based on the characteristics displayed by E. granulosus germinal cells, we establish a cell line from the E. granulosus germinal layer. Furthermore, we propose that this cell line could be useful for drug screening and for obtaining parasite material.

  9. Vaccine production: upstream processing with adherent or suspension cell lines.

    PubMed

    Genzel, Yvonne; Rödig, Jana; Rapp, Erdmann; Reichl, Udo

    2014-01-01

    The production of viral vaccines in cell culture can be accomplished with primary, diploid, or continuous (transformed) cell lines. Each cell line, each virus type, and each vaccine preparation require the specific design of upstream and downstream processing. Media have to be selected as well as production vessels, cultivation conditions, and modes of operation. Many viruses only replicate to high titers in adherently growing cells, but similar to processes established for recombinant protein production, an increasing number of suspension cell lines is being evaluated for future use. Here, we describe key issues to be considered for the establishment of large-scale virus production in bioreactors. As an example upstream processing of cell culture-derived influenza virus production is described in more detail for adherently growing and for suspension cells. In particular, use of serum-containing, serum-free, and chemically defined media as well as choice of cultivation vessel are considered. PMID:24297427

  10. [Decontamination of continual cell lines spontaneously infected with mycoplasmas].

    PubMed

    Machatková, M; Jurmanová, K; Snejdar, V

    1986-07-01

    The continual cell lines of bovine kidneys MDBK and AUBEK, and porcine kidneys RPD and IBRS, spontaneously infected with Mycoplasma arginini and Acholeplasma laidlawii, were decontaminated by the method of selective elimination. Two elimination procedures were modified to be used for the decontamination: one based on the reduction of infection by the light treatment of the cultures, the other based on the selection of mycoplasma-free cell population through cell clonation. On the basis of a long-continued control of the cell clones a methodical procedure of the preparation of mycoplasma-free cell lines was worked out. PMID:3090766

  11. Development and characterization of a largemouth bass cell line.

    PubMed

    Getchell, Rodman G; Groocock, Geoffrey H; Cornwell, Emily R; Schumacher, Vanessa L; Glasner, Lindsay I; Baker, Barry J; Frattini, Stephen A; Wooster, Gregory A; Bowser, Paul R

    2014-09-01

    Abstract The development and characterization of a new cell line, derived from the ovary of Largemouth Bass Micropterus salmoides, is described. Gonad tissue was collected from Largemouth Bass that were electrofished from Oneida Lake, New York. The tissue was processed and grown in culture flasks at approximately 22°C for more than 118 passages during an 8-year period from 2004 to 2011. The identity of these cells as Largemouth Bass origin was confirmed by sequencing a portion of the cytochrome b gene. Growth rate at three different temperatures was documented. The cell line was susceptible to Largemouth Bass virus (LMBV) and its replication was compared with that of Bluegill Lepomis macrochirus fry (BF-2), one of the cell lines recommended for LMBV isolation by the American Fisheries Society Fish Health Section Blue Book. Quantitative PCR results from the replication trial showed the BF-2 cell line produced approximately 10-fold more LMBV copies per cell than the new Largemouth Bass cell line after 6 d, while the titration assay showed similar quantities in each cell line after 1 week. Received February 18, 2014; accepted April 16, 2014. PMID:25229492

  12. Development and characterization of a largemouth bass cell line.

    PubMed

    Getchell, Rodman G; Groocock, Geoffrey H; Cornwell, Emily R; Schumacher, Vanessa L; Glasner, Lindsay I; Baker, Barry J; Frattini, Stephen A; Wooster, Gregory A; Bowser, Paul R

    2014-09-01

    Abstract The development and characterization of a new cell line, derived from the ovary of Largemouth Bass Micropterus salmoides, is described. Gonad tissue was collected from Largemouth Bass that were electrofished from Oneida Lake, New York. The tissue was processed and grown in culture flasks at approximately 22°C for more than 118 passages during an 8-year period from 2004 to 2011. The identity of these cells as Largemouth Bass origin was confirmed by sequencing a portion of the cytochrome b gene. Growth rate at three different temperatures was documented. The cell line was susceptible to Largemouth Bass virus (LMBV) and its replication was compared with that of Bluegill Lepomis macrochirus fry (BF-2), one of the cell lines recommended for LMBV isolation by the American Fisheries Society Fish Health Section Blue Book. Quantitative PCR results from the replication trial showed the BF-2 cell line produced approximately 10-fold more LMBV copies per cell than the new Largemouth Bass cell line after 6 d, while the titration assay showed similar quantities in each cell line after 1 week. Received February 18, 2014; accepted April 16, 2014.

  13. Apoptotic effect of noscapine in breast cancer cell lines.

    PubMed

    Quisbert-Valenzuela, Edwin O; Calaf, Gloria M

    2016-06-01

    Cancer is a public health problem in the world and breast cancer is the most frequently cancer in women. Approximately 15% of the breast cancers are triple-negative. Apoptosis regulates normal growth, homeostasis, development, embryogenesis and appropriate strategy to treat cancer. Bax is a protein pro-apoptotic enhancer of apoptosis in contrast to Bcl-2 with antiapoptotic properties. Initiator caspase-9 and caspase-8 are features of intrinsic and extrinsic apoptosis pathway, respectively. NF-κB is a transcription factor known to be involved in the initiation and progression of breast cancer. Noscapine, an alkaloid derived from opium is used as antitussive and showed antitumor properties that induced apoptosis in cancer cell lines. The aim of the present study was to determine the apoptotic effect of noscapine in breast cancer cell lines compared to breast normal cell line. Three cell lines were used: i) a control breast cell line MCF-10F; ii) a luminal-like adenocarcinoma triple-positive breast cell line MCF-7; iii) breast cancer triple-negative cell line MDA-MB-231. Our results showed that noscapine had lower toxicity in normal cells and was an effective anticancer agent that induced apoptosis in breast cancer cells because it increases Bax gene and protein expression in three cell lines, while decreases Bcl-xL gene expression, and Bcl-2 protein expression decreased in breast cancer cell lines. Therefore, Bax/Bcl-2 ratio increased in the three cell lines. This drug increased caspase-9 gene expression in breast cancer cell lines and caspase-8 gene expression increased in MCF-10F and MDA-MB-231. Furthermore, it increased cleavage of caspase-8, suggesting that noscapine-induced apoptosis is probably due to the involvement of extrinsic and intrinsic apoptosis pathways. Antiapoptotic gene and protein expression diminished and proapoptotic gene and protein expression increased noscapine-induced expression, probably due to decrease in NF-κB gene and protein expression

  14. Characterization of an epithelial cell line from bovine mammary gland.

    PubMed

    German, Tania; Barash, Itamar

    2002-05-01

    Elucidation of the bovine mammary gland's unique characteristics depends on obtaining an authentic cell line that will reproduce its function in vitro. Representative clones from bovine mammary cell populations, differing in their attachment capabilities, were cultured. L-1 cells showed strong attachment to the plate, whereas H-7 cells detached easily. Cultures established from these clones were nontumorigenic upon transplantation to an immunodeficient host; they exhibited the epithelial cell characteristics of positive cytokeratin but not smooth muscle actin staining. Both cell lines depended on fetal calf serum for proliferation. They exhibited distinct levels of differentiation on Matrigel in serum-free, insulin-supplemented medium on the basis of their organization and beta-lactoglobulin (BLG) secretion. H-7 cells organized into mammospheres, whereas L-1 cells arrested in a duct-like morphology. In both cell lines, prolactin activated phosphorylation of the signal transducer and activator of transcription, Stat5-a regulator of milk protein gene transcription, and of PHAS-I-an inhibitor of translation initiation in its nonphosphorylated form. De novo synthesis and secretion of BLG were detected in differentiated cultures: in L-1 cells, BLG was dependent on lactogenic hormones for maximal induction but was less stringently controlled than was beta-casein in the mouse CID-9 cell line. L-1 cells also encompassed a near-diploid chromosomal karyotype and may serve as a tool for studying functional characteristics of the bovine mammary gland.

  15. Molecular profiling reveals primary mesothelioma cell lines recapitulate human disease.

    PubMed

    Chernova, T; Sun, X M; Powley, I R; Galavotti, S; Grosso, S; Murphy, F A; Miles, G J; Cresswell, L; Antonov, A V; Bennett, J; Nakas, A; Dinsdale, D; Cain, K; Bushell, M; Willis, A E; MacFarlane, M

    2016-07-01

    Malignant mesothelioma (MM) is an aggressive, fatal tumor strongly associated with asbestos exposure. There is an urgent need to improve MM patient outcomes and this requires functionally validated pre-clinical models. Mesothelioma-derived cell lines provide an essential and relatively robust tool and remain among the most widely used systems for candidate drug evaluation. Although a number of cell lines are commercially available, a detailed comparison of these commercial lines with freshly derived primary tumor cells to validate their suitability as pre-clinical models is lacking. To address this, patient-derived primary mesothelioma cell lines were established and characterized using complementary multidisciplinary approaches and bioinformatic analysis. Clinical markers of mesothelioma, transcriptional and metabolic profiles, as well as the status of p53 and the tumor suppressor genes CDKN2A and NF2, were examined in primary cell lines and in two widely used commercial lines. Expression of MM-associated markers, as well as the status of CDKN2A, NF2, the 'gatekeeper' in MM development, and their products demonstrated that primary cell lines are more representative of the tumor close to its native state and show a degree of molecular diversity, thus capturing the disease heterogeneity in a patient cohort. Molecular profiling revealed a significantly different transcriptome and marked metabolic shift towards a greater glycolytic phenotype in commercial compared with primary cell lines. Our results highlight that multiple, appropriately characterised, patient-derived tumor cell lines are required to enable concurrent evaluation of molecular profiles versus drug response. Furthermore, application of this approach to other difficult-to-treat tumors would generate improved cellular models for pre-clinical evaluation of novel targeted therapies.

  16. Molecular profiling reveals primary mesothelioma cell lines recapitulate human disease

    PubMed Central

    Chernova, T; Sun, X M; Powley, I R; Galavotti, S; Grosso, S; Murphy, F A; Miles, G J; Cresswell, L; Antonov, A V; Bennett, J; Nakas, A; Dinsdale, D; Cain, K; Bushell, M; Willis, A E; MacFarlane, M

    2016-01-01

    Malignant mesothelioma (MM) is an aggressive, fatal tumor strongly associated with asbestos exposure. There is an urgent need to improve MM patient outcomes and this requires functionally validated pre-clinical models. Mesothelioma-derived cell lines provide an essential and relatively robust tool and remain among the most widely used systems for candidate drug evaluation. Although a number of cell lines are commercially available, a detailed comparison of these commercial lines with freshly derived primary tumor cells to validate their suitability as pre-clinical models is lacking. To address this, patient-derived primary mesothelioma cell lines were established and characterized using complementary multidisciplinary approaches and bioinformatic analysis. Clinical markers of mesothelioma, transcriptional and metabolic profiles, as well as the status of p53 and the tumor suppressor genes CDKN2A and NF2, were examined in primary cell lines and in two widely used commercial lines. Expression of MM-associated markers, as well as the status of CDKN2A, NF2, the ‘gatekeeper' in MM development, and their products demonstrated that primary cell lines are more representative of the tumor close to its native state and show a degree of molecular diversity, thus capturing the disease heterogeneity in a patient cohort. Molecular profiling revealed a significantly different transcriptome and marked metabolic shift towards a greater glycolytic phenotype in commercial compared with primary cell lines. Our results highlight that multiple, appropriately characterised, patient-derived tumor cell lines are required to enable concurrent evaluation of molecular profiles versus drug response. Furthermore, application of this approach to other difficult-to-treat tumors would generate improved cellular models for pre-clinical evaluation of novel targeted therapies. PMID:26891694

  17. Mercury specifically induces LINE-1 activity in a human neuroblastoma cell line.

    PubMed

    Habibi, Laleh; Shokrgozar, Mohammad Ali; Tabrizi, Mina; Modarressi, Mohammad Hossein; Akrami, Seyed Mohammad

    2014-01-01

    L1 retro-elements comprise 17% of the human genome. Approximately 100 copies of these autonomous mobile elements are active in our DNA and can cause mutations, gene disruptions, and genomic instability. Therefore, human cells control the activities of L1 elements, in order to prevent their deleterious effects through different mechanisms. However, some toxic agents increase the retrotransposition activity of L1 elements in somatic cells. In order to identify specific effects of neurotoxic metals on L1 activity in neuronal cells, we studied the effects of mercury and cobalt on L1-retroelement activity by measuring levels of cellular transcription, protein expression, and genomic retrotransposition in a neuroblastoma cell line compared with the effects in three non-neuronal cell lines. Our results show that mercury increased the expression of L1 RNA, the activity of the L1 5'UTR, and L1 retrotransposition exclusively in the neuroblastoma cell line but not in non-neuronal cell lines. However, cobalt increased the expression of L1 RNA in neuroblastoma cells, HeLa cells, and wild-type human fibroblasts, and also increased the activity of the L1 5'UTR as well as the SV40 promoter in HeLa cells but not in neuroblastoma cells. Exposure to cobalt did not result in increased retrotransposition activity in HeLa cells or neuroblastoma cells. We conclude that non-toxic levels of the neurotoxic agent mercury could influence DNA by increasing L1 activities, specifically in neuronal cells, and may make these cells susceptible to neurodegeneration over time.

  18. Pathway-specific differences between tumor cell lines and normal and tumor tissue cells

    PubMed Central

    Ertel, Adam; Verghese, Arun; Byers, Stephen W; Ochs, Michael; Tozeren, Aydin

    2006-01-01

    Background Cell lines are used in experimental investigation of cancer but their capacity to represent tumor cells has yet to be quantified. The aim of the study was to identify significant alterations in pathway usage in cell lines in comparison with normal and tumor tissue. Methods This study utilized a pathway-specific enrichment analysis of publicly accessible microarray data and quantified the gene expression differences between cell lines, tumor, and normal tissue cells for six different tissue types. KEGG pathways that are significantly different between cell lines and tumors, cell lines and normal tissues and tumor and normal tissue were identified through enrichment tests on gene lists obtained using Significance Analysis of Microarrays (SAM). Results Cellular pathways that were significantly upregulated in cell lines compared to tumor cells and normal cells of the same tissue type included ATP synthesis, cell communication, cell cycle, oxidative phosphorylation, purine, pyrimidine and pyruvate metabolism, and proteasome. Results on metabolic pathways suggested an increase in the velocity nucleotide metabolism and RNA production. Pathways that were downregulated in cell lines compared to tumor and normal tissue included cell communication, cell adhesion molecules (CAMs), and ECM-receptor interaction. Only a fraction of the significantly altered genes in tumor-to-normal comparison had similar expressions in cancer cell lines and tumor cells. These genes were tissue-specific and were distributed sparsely among multiple pathways. Conclusion Significantly altered genes in tumors compared to normal tissue were largely tissue specific. Among these genes downregulation was a major trend. In contrast, cell lines contained large sets of significantly upregulated genes that were common to multiple tissue types. Pathway upregulation in cell lines was most pronounced over metabolic pathways including cell nucleotide metabolism and oxidative phosphorylation. Signaling

  19. Hypothalamic-pituitary-adrenocortical axis: neuropsychiatric aspects.

    PubMed

    Jacobson, Lauren

    2014-04-01

    Evidence of aberrant hypothalamic-pituitary-adrenocortical (HPA) activity in many psychiatric disorders, although not universal, has sparked long-standing interest in HPA hormones as biomarkers of disease or treatment response. HPA activity may be chronically elevated in melancholic depression, panic disorder, obsessive-compulsive disorder, and schizophrenia. The HPA axis may be more reactive to stress in social anxiety disorder and autism spectrum disorders. In contrast, HPA activity is more likely to be low in PTSD and atypical depression. Antidepressants are widely considered to inhibit HPA activity, although inhibition is not unanimously reported in the literature. There is evidence, also uneven, that the mood stabilizers lithium and carbamazepine have the potential to augment HPA measures, while benzodiazepines, atypical antipsychotics, and to some extent, typical antipsychotics have the potential to inhibit HPA activity. Currently, the most reliable use of HPA measures in most disorders is to predict the likelihood of relapse, although changes in HPA activity have also been proposed to play a role in the clinical benefits of psychiatric treatments. Greater attention to patient heterogeneity and more consistent approaches to assessing treatment effects on HPA function may solidify the value of HPA measures in predicting treatment response or developing novel strategies to manage psychiatric disease.

  20. Global Conservation of Protein Status between Cell Lines and Xenografts.

    PubMed

    Biau, Julian; Chautard, Emmanuel; Court, Frank; Pereira, Bruno; Verrelle, Pierre; Devun, Flavien; De Koning, Leanne; Dutreix, Marie

    2016-08-01

    Common preclinical models for testing anticancer treatment include cultured human tumor cell lines in monolayer, and xenografts derived from these cell lines in immunodeficient mice. Our goal was to determine how similar the xenografts are compared with their original cell line and to determine whether it is possible to predict the stability of a xenograft model beforehand. We studied a selection of 89 protein markers of interest in 14 human cell cultures and respective subcutaneous xenografts using the reverse-phase protein array technology. We specifically focused on proteins and posttranslational modifications involved in DNA repair, PI3K pathway, apoptosis, tyrosine kinase signaling, stress, cell cycle, MAPK/ERK signaling, SAPK/JNK signaling, NFκB signaling, and adhesion/cytoskeleton. Using hierarchical clustering, most cell culture-xenograft pairs cluster together, suggesting a global conservation of protein signature. Particularly, Akt, NFkB, EGFR, and Vimentin showed very stable protein expression and phosphorylation levels highlighting that 4 of 10 pathways were highly correlated whatever the model. Other proteins were heterogeneously conserved depending on the cell line. Finally, cell line models with low Akt pathway activation and low levels of Vimentin gave rise to more reliable xenograft models. These results may be useful for the extrapolation of cell culture experiments to in vivo models in novel targeted drug discovery. PMID:27567954

  1. Guanylate-Binding Protein-1 protects ovarian cancer cell lines but not breast cancer cell lines from killing by paclitaxel.

    PubMed

    Tipton, Aaron R; Nyabuto, Geoffrey O; Trendel, Jill A; Mazur, Travis M; Wilson, John P; Wadi, Suzan; Justinger, Jacob S; Moore, Garret L; Nguyen, Peter T; Vestal, Deborah J

    2016-09-30

    Forced expression of the cytokine-induced large GTPase, human Guanylate-Binding Protein-1 (hGBP-1), in ovarian cancer cell lines increases resistance to paclitaxel. Elevated hGBP-1 RNA in ovarian tumors correlates with shorter recurrence-free survival. In contract, hGBP-1 is part of a gene signature predicting improved prognosis in all subtypes of breast cancers. hGBP-1 does not confer paclitaxel resistance on MCF-7 and TMX2-28 breast cancer cells. Expression of the isotype of the hGBP-1-interacting protein, PIM1, which may contribute to paclitaxel resistance when associated with hGBP-1, is different in breast and ovarian cancer cell lines. Breast cancer cell lines express the 44 kDa isoform of PIM-1, and ovarian cancer cell lines express the 33 kDa isoform. PMID:27590579

  2. MORPHOMETRIC SUBTYPING FOR A PANEL OF BREAST CANCER CELL LINES

    SciTech Connect

    Han, Ju; Chang, Hang; Fontenay, Gerald; Wang, Nicholas J.; Gray, Joe W.; Parvin, Bahram

    2009-05-08

    A panel of cell lines of diverse molecular background offers an improved model system for high-content screening, comparative analysis, and cell systems biology. A computational pipeline has been developed to collect images from cell-based assays, segment individual cells and colonies, represent segmented objects in a multidimensional space, and cluster them for identifying distinct subpopulations. While each segmentation strategy can vary for different imaging assays, representation and subpopulation analysis share a common thread. Application of this pipeline to a library of 41 breast cancer cell lines is demonstrated. These cell lines are grown in 2D and imaged through immunofluorescence microscopy. Subpopulations in this panel are identified and shown to correlate with previous subtyping literature that was derived from transcript data.

  3. Effects of ethanol on an intestinal epithelial cell line

    SciTech Connect

    Nano, J.L.; Cefai, D.; Rampal, P. )

    1990-02-01

    The effect of exposure of an intestinal epithelial cell line to various concentrations of ethanol (217 mM (1%) to 652 mM (3%)) during 24, 48, and 72 hr was investigated in vitro using a rat intestinal epithelial cell line (IRD 98). Incubation of these cells in the presence of ethanol significantly decreased cell growth. This inhibition was accompanied by a strong increase in cellular protein. Stimulation of specific disaccharidases, gamma-glutamyl transferase, and aminopeptidase activities by ethanol was dose- and time-dependent. Ethanol induces a change in the relative proportions of the different lipid classes synthesized; triglycerides, fatty acids, and cholesterol esters were preferentially synthethysed. Our findings show that cell lines are good models for investigation of the effects of ethanol, and that alcohol considerably modifies the functions of intestinal epithelial cells.

  4. Derivation of human embryonic stem cell lines, towards clinical quality.

    PubMed

    Hovatta, Outi

    2006-01-01

    Human embryonic stem (hES) cells offer an excellent source of cells for transplantation in the treatment of severe diseases. To be clinically safe, the lines have to be derived using strict quality criteria and good manufacturing practice. Animal proteins are immunogenic and may contain microbes, and they should not be used in establishing or propagating hES cells. Derivation systems have been improved towards clinical quality by establishing all 25 hES cell lines using human skin fibroblasts as feeder cells instead of mouse fibroblasts. A further 21 cell lines have been established using synthetic serum instead of fetal calf serum in the medium. In the five latest derivations, the inner cell mass was isolated mechanically instead of by immunosurgery (animal antibodies). Feeder-free derivation would be optimal, but it is not yet considered safe. Clinical-quality lines can be derived by establishing the skin fibroblast feeders in the good manufacturing practice laboratory with human serum in the medium, and by establishing the hES cells on such feeders. In this process, a serum replacement that contains only human protein can be used, the inner cell mass has to be isolated mechanically, and the colonies have to be split mechanically for passaging. Somatic cell nuclear transfer would help to overcome rejection of transplanted cells. PMID:17147930

  5. DNA fingerprinting of the NCI-60 cell line panel.

    PubMed

    Lorenzi, Philip L; Reinhold, William C; Varma, Sudhir; Hutchinson, Amy A; Pommier, Yves; Chanock, Stephen J; Weinstein, John N

    2009-04-01

    The National Cancer Institute's NCI-60 cell line panel, the most extensively characterized set of cells in existence and a public resource, is frequently used as a screening tool for drug discovery. Because many laboratories around the world rely on data from the NCI-60 cells, confirmation of their genetic identities represents an essential step in validating results from them. Given the consequences of cell line contamination or misidentification, quality control measures should routinely include DNA fingerprinting. We have, therefore, used standard DNA microsatellite short tandem repeats to profile the NCI-60, and the resulting DNA fingerprints are provided here as a reference. Consistent with previous reports, the fingerprints suggest that several NCI-60 lines have common origins: the melanoma lines MDA-MB-435, MDA-N, and M14; the central nervous system lines U251 and SNB-19; the ovarian lines OVCAR-8 and OVCAR-8/ADR (also called NCI/ADR); and the prostate lines DU-145, DU-145 (ATCC), and RC0.1. Those lines also show that the ability to connect two fingerprints to the same origin is not affected by stable transfection or by the development of multidrug resistance. As expected, DNA fingerprints were not able to distinguish different tissues-of-origin. The fingerprints serve principally as a barcodes.

  6. Aging effects on oxidative phosphorylation in rat adrenocortical mitochondria.

    PubMed

    Solinas, Paola; Fujioka, Hisashi; Radivoyevitch, Tomas; Tandler, Bernard; Hoppel, Charles L

    2014-06-01

    Does aging in itself lead to alteration in adrenocortical mitochondrial oxidative phosphorylation? Mitochondria from Fischer 344 (F344) rats (6 and 24 months old), Brown Norway rats (6 and 32 months old) and F344-Brown Norway hybrid rats (6 and 30 months old) were compared. Mitochondria were isolated from extirpated adrenal cortex. The yields of mitochondria were quantitatively similar in all rat strains irrespective of age. In order to assess the activity of each mitochondrial complex, several different substrates were tested and the rate of oxidative phosphorylation measured. Aging does not affect mitochondrial activity except in the F344 rat adrenal cortex where the maximal ADP-stimulated oxidative phosphorylation decreased with age. We hypothesize that impaired synthesis of steroid hormones by the adrenal cortex with age in F344 rats might be due to decreased adrenocortical mitochondrial oxidative phosphorylation. We conclude that aging results in adrenocortical mitochondria effects that are non-uniform across different rat strains.

  7. Antiproliferative Effect of Solanum nigrum on Human Leukemic Cell Lines

    PubMed Central

    Gabrani, Reema; Jain, Ramya; Sharma, Anjali; Sarethy, Indira P.; Dang, Shweta; Gupta, S.

    2012-01-01

    Solanum nigrum is used in various traditional medical systems for antiproliferative, antiinflammatory, antiseizure and hepatoprotective activities. We have evaluated organic solvent and aqueous extracts obtained from berries of Solanum nigrum for antiproliferative activity on leukemic cell lines, Jurkat and HL-60 (Human promyelocytic leukemia cells). The cell viability after the treatment with Solanum nigrum extract was measured by MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide) assay. Results indicated increased cytotoxicity with increasing extract concentrations. Comparative analysis indicated that 50% inhibitory concentration value of methanol extract is the lowest on both cell lines. PMID:23716874

  8. Measles virus persistence in an immortalized murine macrophage cell line.

    PubMed

    Goldman, M B; Buckthal, D J; Picciotto, S; O'Bryan, T A; Goldman, J N

    1995-02-20

    Persistent infection with the Edmonston strain of measles virus (MV) has been established in IC-21 cells, an immortalized murine macrophage cell line. Persistence was established immediately without syncytia formation or cytopathic effects. MV was expressed in the majority of the cells as evidenced by immunofluorescence microscopy, flow cytometry, infectious centers assays, and limiting dilution analysis. Hemagglutinin (H) and phosphoprotein expressed in persistently infected IC-21 cells had retarded migration in SDS-PAGE gels when compared to these proteins expressed in Vero cells. H protein differences were also found between freshly infected IC-21 cells and persistently infected IC-21 cells passaged for over 2 years. Six sublines of IC-21 cells, infected at different times, have maintained these characteristics for 2 years of passage. During this time period the intensity of immunofluorescence and the number of infectious virus particles recoverable fluctuated in five of the six cell lines. In one cell line virus expression remained at a consistent high level. The ability to establish a persistent MV infection in murine macrophages allows studies using a cell important in disseminating the infection. It facilitates experiments on immunological aspects of viral immunity by enabling cell mixing experiments with histocompatible cell populations and by making available the wide array of cellular and humoral reagents in the mouse. PMID:7871720

  9. Metronidazole Decreases Viability of DLD-1 Colorectal Cancer Cell Line

    PubMed Central

    Sadowska, Anna; Krętowski, Rafał; Szynaka, Beata; Cechowska-Pasko, Marzanna

    2013-01-01

    Abstract The aim of our study was to evaluate the impact of metronidazole (MTZ) on DLD-1 colorectal cancer cell (CRC) line. Toxicity of MTZ was determined by MTT test. Cells were incubated with MTZ used in different concentrations for 24, 48, and 72 hours. The effect of MTZ on DNA synthesis was measured as [3H]-thymidine incorporation. The morphological changes in human DLD-1 cell line were defined by transmission electron microscope OPTON 900. The influence of MTZ on the apoptosis of DLD-1 cell lines was detected by flow cytometry and fluorescence microscopy, while cell concentration, volume, and diameter were displayed by Scepter Cell Counter from Millipore. Our results show that cell viability was diminished in all experimental groups in comparison with the control, and the differences were statistically significant. We did not find any significant differences in [3H]-thymidine incorporation in all experimental groups and times of observation. Cytofluorimetric assays demonstrated a statistically significant increase of apoptotic rate in MTZ concentrations 10 and 50 μg/mL after 24 hours; 0.1, 10, 50, and 250 μg/mL after 48 hours; and in all concentrations after 72 hours compared with control groups. In the ultrastructural studies, necrotic or apoptotic cells were occasionally seen. In conclusion, MTZ affects human CRC cell line viability. The reduction of cell viability was consistent with the apoptotic test. PMID:23777253

  10. Inducible human immunodeficiency virus type 1 packaging cell lines.

    PubMed Central

    Yu, H; Rabson, A B; Kaul, M; Ron, Y; Dougherty, J P

    1996-01-01

    Packaging cell lines are important tools for transferring genes into eukaryotic cells. Human immunodeficiency virus type 1 (HIV-1)-based packaging cell lines are difficult to obtain, in part owing to the problem that some HIV-1 proteins are cytotoxic in a variety of cells. To overcome this, we have developed an HIV-1-based packaging cell line which has an inducible expression system. The tetracycline-inducible expression system was utilized to control the expression of the Rev regulatory protein, which in turn controls the expression of the late proteins including Gag, Pol, and Env. Western blotting (immunoblotting) demonstrated that the expression of p24gag and gp120env from the packaging cells peaked on days 6 and 7 postinduction. Reverse transcriptase activity could be detected by day 4 after induction and also peaked on days 6 and 7. Defective vector virus could be propagated, yielding titers as high as 7 x 10(3) CFU/ml, while replication-competent virus was not detectable at any time. Thus, the cell line should enable the transfer of specific genes into CD4+ cells and should be a useful tool for studying the biology of HIV-1. We have also established an inducible HIV-1 Env-expressing cell line which could be used to propagate HIV-1 vectors that require only Env in trans. The env-minus vector virus titer produced from the Env-expressing cells reached 2 x 10(4) CFU/ml. The inducible HIV-1 Env-expressing cell line should be a useful tool for the study of HIV-1 Env as well. PMID:8676479

  11. Chronic effects of mercuric chloride ingestion on rat adrenocortical function

    SciTech Connect

    Agrawal, R.; Chansouria, J.P.N. )

    1989-09-01

    Mercurial contamination of environment has increased. Mercury accumulates in various organs and adversely affects their functions. Some of the most prominent toxic effects of inorganic mercury compounds include neurotoxicity, hepatotoxicity and nephrotoxicity. Besides this, mercury has also been reported to affect various endocrine glands like pituitary, thyroid, gonadal and adrenal glands. There have been no reports on the toxic effects of chronic oral administration of varying doses of mercuric chloride on adrenocortical function in albino rats. The present work was undertaken to study the adrenocortical response to chronic oral administration of mercuric chloride of varying dose and duration in albino rats.

  12. Regeneration of lateral line and inner ear vestibular cells.

    PubMed

    Jørgensen, J M

    1991-01-01

    Labelling experiments with [3H]thymidine demonstrate a continuous production of cells in the mechanoreceptive lateral line organs of the eel (Anguilla anguilla) and butterfly fish (Pantodon buchholzi) as well as in the electroreceptive ampullary organ of the transparent catfish (Kryptopterus bicirrhus). Shortly after [3H]thymidine injection many cells are labelled in the middle and basal parts of the sensory organ and after a few days' survival sensory cells are also labelled. The vestibular sensory organs of selected species of fishes, amphibians, reptiles and birds also show a continuous production of cells. In the budgerigar (Melopsittacus undulatus) labelled cells are found in the basal and middle layer of the sensory epithelium a few hours after injection with [3H]thymidine. A few days after the injection labelled cells are found in non-calyceal hair cells. After one month the calyceal cells are also labelled. Similar experiments with the bat Pipistrellus nathusii and with normal and gentamicin-treated mice (Mus musculus) show no labelled cells in the inner ear sensory epithelia. The lateral line organs and vestibular epithelia of non-mammalian vertebrates all contain a small number of dark cells with the characteristics of apoptotic cells. Macrophages and inclusions in some cells, thought to be remnants of apoptotic cells, are occasionally seen. Fixation at different osmolarities has little effect on the number of dark cells. It is suggested that the continually produced cells replace apoptotic dying cells.

  13. Experimental Adaptation of Rotaviruses to Tumor Cell Lines

    PubMed Central

    Guerrero, Carlos A.; Guerrero, Rafael A.; Silva, Elver; Acosta, Orlando; Barreto, Emiliano

    2016-01-01

    A number of viruses show a naturally extended tropism for tumor cells whereas other viruses have been genetically modified or adapted to infect tumor cells. Oncolytic viruses have become a promising tool for treating some cancers by inducing cell lysis or immune response to tumor cells. In the present work, rotavirus strains TRF-41 (G5) (porcine), RRV (G3) (simian), UK (G6-P5) (bovine), Ym (G11-P9) (porcine), ECwt (murine), Wa (G1-P8), Wi61 (G9) and M69 (G8) (human), and five wild-type human rotavirus isolates were passaged multiple times in different human tumor cell lines and then combined in five different ways before additional multiple passages in tumor cell lines. Cell death caused by the tumor cell-adapted isolates was characterized using Hoechst, propidium iodide, 7-AAD, Annexin V, TUNEL, and anti-poly-(ADP ribose) polymerase (PARP) and -phospho-histone H2A.X antibodies. Multiple passages of the combined rotaviruses in tumor cell lines led to a successful infection of these cells, suggesting a gain-of-function by the acquisition of greater infectious capacity as compared with that of the parental rotaviruses. The electropherotype profiles suggest that unique tumor cell-adapted isolates were derived from reassortment of parental rotaviruses. Infection produced by such rotavirus isolates induced chromatin modifications compatible with apoptotic cell death. PMID:26828934

  14. Exometabolom analysis of breast cancer cell lines: Metabolic signature

    PubMed Central

    Willmann, Lucas; Erbes, Thalia; Halbach, Sebastian; Brummer, Tilman; Jäger, Markus; Hirschfeld, Marc; Fehm, Tanja; Neubauer, Hans; Stickeler, Elmar; Kammerer, Bernd

    2015-01-01

    Cancer cells show characteristic effects on cellular turnover and DNA/RNA modifications leading to elevated levels of excreted modified nucleosides. We investigated the molecular signature of different subtypes of breast cancer cell lines and the breast epithelial cell line MCF-10A. Prepurification of cell culture supernatants was performed by cis-diol specific affinity chromatography using boronate-derivatized polyacrylamide gel. Samples were analyzed by application of reversed phase chromatography coupled to a triple quadrupole mass spectrometer. Collectively, we determined 23 compounds from RNA metabolism, two from purine metabolism, five from polyamine/methionine cycle, one from histidine metabolism and two from nicotinate and nicotinamide metabolism. We observed major differences of metabolite excretion pattern between the breast cancer cell lines and MCF-10A, just as well as between the different breast cancer cell lines themselves. Differences in metabolite excretion resulting from cancerous metabolism can be integrated into altered processes on the cellular level. Modified nucleosides have great potential as biomarkers in due consideration of the heterogeneity of breast cancer that is reflected by the different molecular subtypes of breast cancer. Our data suggests that the metabolic signature of breast cancer cell lines might be a more subtype-specific tool to predict breast cancer, rather than a universal approach. PMID:26293811

  15. A clinical and immunological study of adrenocortical insufficiency (Addison's disease)

    PubMed Central

    Irvine, W. J.; Stewart, A. G.; Scarth, Laura

    1967-01-01

    Fifty-one patients with adrenocortical insufficiency were subdivided into three groups according to the nature of their adrenal disease; twelve patients with idiopathic, twenty-three patients with probable idiopathic and sixteen patients with tuberculous adrenal insufficiency. The importance of objective confirmation of a clinical diagnosis of adrenal insufficiency is stressed and the difficulties of classification of many patients with adult onset adrenal insufficiency are discussed. Idiopathic and probable idiopathic adrenal insufficiency had a sex ratio that was predominantly female (2·5:1) with a mean age of onset of 33 years. Antibodies to adrenal cortex were detected by the methods of immunofluorescence and complement fixation. They were detected in the serum of 80% (20:25) of the females with idiopathic or probable idiopathic adrenal insufficiency and in only 10% (1:10) of the males. The titre of the adrenal antibody was low (≤32) as tested either by immunofluorescence or complement fixation. The serum of only one patient with tuberculous adrenal insufficiency reacted with adrenal tissue in the complement fixation test but the immunofluorescence method showed that this serum reacted with the vascular endothelium and not the secretory cells. No correlation was observed between the duration of the clinical illness and the presence, or absence, or titre of the adrenal antibody. Adrenal antibody was not detected in the sera of fifty-one control subjects matched for age and sex. Four of sixty-nine patients with lymphadenoid goitre, one out of ninety-three patients with diabetes mellitus and none of 230 patients with thyrotoxicosis, primary hypothyroidism or pernicious anaemia had antibody in the serum specific for adrenocortical secretory cells. There is a clinical and immunological overlap between idiopathic adrenal insufficiency and other diseases associated with autoimmune phenomena— thyroid disease, atrophic gastritis and hypoparathyroidism. It is

  16. Generation of stable Drosophila cell lines using multicistronic vectors.

    PubMed

    González, Monika; Martín-Ruíz, Itziar; Jiménez, Silvia; Pirone, Lucia; Barrio, Rosa; Sutherland, James D

    2011-01-01

    Insect cell culture is becoming increasingly important for applications including recombinant protein production and cell-based screening with chemical or RNAi libraries. While stable mammalian cell lines expressing a protein of interest can be efficiently prepared using IRES-based vectors or viral-based approaches, options for stable insect cell lines are more limited. Here, we describe pAc5-STABLEs, new vectors for use in Drosophila cell culture to facilitate stable transformation. We show that viral-derived 2A-like (or "CHYSEL") peptides function in Drosophila cells and can mediate the multicistronic expression of two or three proteins of interest under control of the Actin5C constitutive promoter. The current vectors allow mCherry and/or GFP fusions to be generated for positive selection by G418 resistance in cells and should serve as a flexible platform for future applications. PMID:22355594

  17. Three-dimensional cultured glioma cell lines

    NASA Technical Reports Server (NTRS)

    Gonda, Steve R. (Inventor); Marley, Garry M. (Inventor)

    1991-01-01

    Three-dimensional glioma spheroids were produced in vitro with size and histological differentiation previously unattained. The spheroids were grown in liquid media suspension in a Johnson Space Center (JSC) Rotating Wall Bioreactor without using support matrices such as microcarrier beads. Spheroid volumes of greater than 3.5 cu mm and diameters of 2.5 mm were achieved with a viable external layer or rim of proliferating cells, a transitional layer beneath the external layer with histological differentiation, and a degenerative central region with a hypoxic necrotic core. Cell debris was evident in the degenerative central region. The necrotics centers of some of the spheroids had hyaline droplets. Granular bodies were detected predominantly in the necrotic center.

  18. Solid Oxide Fuel Cell Systems PVL Line

    SciTech Connect

    Susan Shearer - Stark State College; Gregory Rush - Rolls-Royce Fuel Cell Systems

    2012-05-01

    In July 2010, Stark State College (SSC), received Grant DE-EE0003229 from the U.S. Department of Energy (DOE), Golden Field Office, for the development of the electrical and control systems, and mechanical commissioning of a unique 20kW scale high-pressure, high temperature, natural gas fueled Stack Block Test System (SBTS). SSC worked closely with subcontractor, Rolls-Royce Fuel Cell Systems (US) Inc. (RRFCS) over a 13 month period to successfully complete the project activities. This system will be utilized by RRFCS for pre-commercial technology development and training of SSC student interns. In the longer term, when RRFCS is producing commercial products, SSC will utilize the equipment for workforce training. In addition to DOE Hydrogen, Fuel Cells, and Infrastructure Technologies program funding, RRFCS internal funds, funds from the state of Ohio, and funding from the DOE Solid State Energy Conversion Alliance (SECA) program have been utilized to design, develop and commission this equipment. Construction of the SBTS (mechanical components) was performed under a Grant from the State of Ohio through Ohio's Third Frontier program (Grant TECH 08-053). This Ohio program supported development of a system that uses natural gas as a fuel. Funding was provided under the Department of Energy (DOE) Solid-state Energy Conversion Alliance (SECA) program for modifications required to test on coal synthesis gas. The subject DOE program provided funding for the electrical build, control system development and mechanical commissioning. Performance testing, which includes electrical commissioning, was subsequently performed under the DOE SECA program. Rolls-Royce Fuel Cell Systems is developing a megawatt-scale solid oxide fuel cell (SOFC) stationary power generation system. This system, based on RRFCS proprietary technology, is fueled with natural gas, and operates at elevated pressure. A critical success factor for development of the full scale system is the capability to

  19. Molecular dissection of AKT activation in lung cancer cell lines

    PubMed Central

    Guo, Yanan; Du, Jinyan; Kwiatkowski, David J

    2013-01-01

    AKT is a critical signaling node downstream of PI3K, which is often activated in cancer. We analyzed the state of activation of AKT in 80 human non-small cell lung cancer cell lines under serum starvation conditions. We identified 13 lines which showed persistent AKT activation in the absence of serum. In 12 of the 13 lines, AKT activation could be attributed to loss of PTEN, activating mutation in EGFR or PIK3CA, or amplification of ERBB2. HCC2429 was the only cell line that had no alterations in those genes, but had high phospho-AKT(Ser473) levels under serum starvation conditions. However, the activation of AKT in HCC2429 was PI3K- and mTORC2-dependent based upon use of specific inhibitors. Kinome tyrosine phosphorylation profiling showed that both Notch and SRC were highly activated in this cell line. Despite the activation of Notch, AKT activation and cell survival were not affected by Notch inhibitors DAPT or Compound E. In contrast, SRC inhibitors PP2 and dasatinib both significantly decreased pAKT(Ser473) levels and reduced cell survival by inducing apoptosis. Further, a combination of SRC and mTOR inhibition synergistically blocked activation of AKT and induced apoptosis. Over-expression of SRC has been identified previously in human lung cancers, and these results suggest that a combination of SRC and mTOR inhibitors may have unique therapeutic benefit for a subset of lung cancers with these molecular features. PMID:23319332

  20. Heterozygous Embryonic Stem Cell Lines Derived from Nonhuman Primate Parthenotes

    PubMed Central

    Dighe, Vikas; Clepper, Lisa; Pedersen, Darlene; Byrne, James; Ferguson, Betsy; Gokhale, Sumita; Penedo, M. Cecilia T.; Wolf, Don; Mitalipov, Shoukhrat

    2009-01-01

    Monoparental parthenotes represent a potential source of histocompatible stem cells that should be isogenic with the oocyte donor and therefore suitable for use in cell or tissue replacement therapy. We generated five rhesus monkey parthenogenetic embryonic stem cell (PESC) lines with stable, diploid female karyotypes that were morphologically indistinguishable from biparental controls, expressed key pluripotent markers, and generated cell derivatives representative of all three germ layers following in vivo and in vitro differentiation. Interestingly, high levels of heterozygosity were observed at the majority of loci that were polymorphic in the oocyte donors. Some PESC lines were also heterozygous in the major histocompatibility complex region, carrying haplotypes identical to those of the egg donor females. Expression analysis revealed transcripts from some imprinted genes that are normally expressed from only the paternal allele. These results indicate that limitations accompanying the potential use of PESC-derived phenotypes in regenerative medicine, including aberrant genomic imprinting and high levels of homozygosity, are cell line-dependent and not always present. PESC lines were derived in high enough yields to be practicable, and their derivatives are suitable for autologous transplantation into oocyte donors or could be used to establish a bank of histocompatible cell lines for a broad spectrum of patients. PMID:18192229

  1. Heterozygous embryonic stem cell lines derived from nonhuman primate parthenotes.

    PubMed

    Dighe, Vikas; Clepper, Lisa; Pedersen, Darlene; Byrne, James; Ferguson, Betsy; Gokhale, Sumita; Penedo, M Cecilia T; Wolf, Don; Mitalipov, Shoukhrat

    2008-03-01

    Monoparental parthenotes represent a potential source of histocompatible stem cells that should be isogenic with the oocyte donor and therefore suitable for use in cell or tissue replacement therapy. We generated five rhesus monkey parthenogenetic embryonic stem cell (PESC) lines with stable, diploid female karyotypes that were morphologically indistinguishable from biparental controls, expressed key pluripotent markers, and generated cell derivatives representative of all three germ layers following in vivo and in vitro differentiation. Interestingly, high levels of heterozygosity were observed at the majority of loci that were polymorphic in the oocyte donors. Some PESC lines were also heterozygous in the major histocompatibility complex region, carrying haplotypes identical to those of the egg donor females. Expression analysis revealed transcripts from some imprinted genes that are normally expressed from only the paternal allele. These results indicate that limitations accompanying the potential use of PESC-derived phenotypes in regenerative medicine, including aberrant genomic imprinting and high levels of homozygosity, are cell line-dependent and not always present. PESC lines were derived in high enough yields to be practicable, and their derivatives are suitable for autologous transplantation into oocyte donors or could be used to establish a bank of histocompatible cell lines for a broad spectrum of patients.

  2. Novel cell lines derived from transgenic mice expressing recombinant human proteins. Transgenic hepatoma-derived cell lines.

    PubMed

    Perraud, F; Dalemans, W; Ali-Hadji, D; Pavirani, A

    1992-01-01

    We have used transgenic mouse technology to establish immortalized hepatoma cell lines stably secreting heterologous proteins, such as human alpha 1-antitrypsin and human factor IX. Hepatocyte-specific regulatory DNA sequences were used to target both the expression of an onc gene and the gene coding for the human protein to the liver of transgenic mice which eventually developed hepatocellular carcinomas. Tumour cells were subsequently established as permanent cell lines, which maintained a differentiated phenotype under specific culture conditions, being capable of producing biologically active and correctly processed human alpha 1-antitrypsin and factor IX. Moreover, a preliminary analysis has shown that certain cell lines express elevated total cytochrome P450 activity. These cells could therefore represent a useful alternative to the use of animals or primary cultures in drug safety testing. PMID:1369183

  3. Germ line development: lessons learned from pluripotent stem cells.

    PubMed

    Martínez-Arroyo, Ana M; Medrano, Jose V; Remohí, José; Simón, Carlos

    2014-10-01

    Current knowledge about mammalian germ line development is mainly based on the mouse model and little is known about how this fundamental process occurs in humans. This review summarizes our current knowledge of genetic and epigenetic germ line development in mammals, mainly focusing on primordial germ cell (PGC) specification events, comparing the differences between mouse and human models. We also emphasize the knowledge derived from the most successful strategies used to generate germ cell-like cells in vitro in both models and major obstacles to obtaining bona fide in vitro-derived gametes are considered. PMID:25461452

  4. Guidelines for the use of cell lines in biomedical research

    PubMed Central

    Geraghty, R J; Capes-Davis, A; Davis, J M; Downward, J; Freshney, R I; Knezevic, I; Lovell-Badge, R; Masters, J R W; Meredith, J; Stacey, G N; Thraves, P; Vias, M

    2014-01-01

    Cell-line misidentification and contamination with microorganisms, such as mycoplasma, together with instability, both genetic and phenotypic, are among the problems that continue to affect cell culture. Many of these problems are avoidable with the necessary foresight, and these Guidelines have been prepared to provide those new to the field and others engaged in teaching and instruction with the information necessary to increase their awareness of the problems and to enable them to deal with them effectively. The Guidelines cover areas such as development, acquisition, authentication, cryopreservation, transfer of cell lines between laboratories, microbial contamination, characterisation, instability and misidentification. Advice is also given on complying with current legal and ethical requirements when deriving cell lines from human and animal tissues, the selection and maintenance of equipment and how to deal with problems that may arise. PMID:25117809

  5. Guidelines for the use of cell lines in biomedical research.

    PubMed

    Geraghty, R J; Capes-Davis, A; Davis, J M; Downward, J; Freshney, R I; Knezevic, I; Lovell-Badge, R; Masters, J R W; Meredith, J; Stacey, G N; Thraves, P; Vias, M

    2014-09-01

    Cell-line misidentification and contamination with microorganisms, such as mycoplasma, together with instability, both genetic and phenotypic, are among the problems that continue to affect cell culture. Many of these problems are avoidable with the necessary foresight, and these Guidelines have been prepared to provide those new to the field and others engaged in teaching and instruction with the information necessary to increase their awareness of the problems and to enable them to deal with them effectively. The Guidelines cover areas such as development, acquisition, authentication, cryopreservation, transfer of cell lines between laboratories, microbial contamination, characterisation, instability and misidentification. Advice is also given on complying with current legal and ethical requirements when deriving cell lines from human and animal tissues, the selection and maintenance of equipment and how to deal with problems that may arise. PMID:25117809

  6. Derivation of human embryonic stem cell line Genea019.

    PubMed

    Dumevska, Biljana; Peura, Teija; McKernan, Robert; Goel, Divya; Schmidt, Uli

    2016-03-01

    The Genea019 human embryonic stem cell line was derived from a donated, fully commercially consented ART blastocyst, through ICM outgrowth on inactivated feeders. The line showed pluripotent cell morphology and genomic analysis verified a 46, XX karyotype, female Allele pattern and unaffected Htt CAG repeat length, compared to HD affected sibling Genea020. Pluripotency of Genea019 was demonstrated with 75% of cells expressing Nanog, 89% Oct4, 48% Tra1-60 and 85% SSEA4, a Pluritest Pluripotency score of 22.97, Novelty score of 1.42, tri-lineage teratoma formation and Alkaline Phosphatase activity. The cell line was negative for Mycoplasma and any visible contamination. PMID:27346002

  7. Tools for Targeted Genome Engineering of Established Drosophila Cell Lines

    PubMed Central

    Cherbas, Lucy; Hackney, Jennifer; Gong, Lei; Salzer, Claire; Mauser, Eric; Zhang, Dayu; Cherbas, Peter

    2015-01-01

    We describe an adaptation of φC31 integrase–mediated targeted cassette exchange for use in Drosophila cell lines. Single copies of an attP-bounded docking platform carrying a GFP-expression marker, with or without insulator elements flanking the attP sites, were inserted by P-element transformation into the Kc167 and Sg4 cell lines; each of the resulting docking-site lines carries a single mapped copy of one of the docking platforms. Vectors for targeted substitution contain a cloning cassette flanked by attB sites. Targeted substitution occurs by integrase-mediated substitution between the attP sites (integrated) and the attB sites (vector). We describe procedures for isolating cells carrying the substitutions and for eliminating the products of secondary off-target events. We demonstrate the technology by integrating a cassette containing a Cu2+-inducible mCherry marker, and we report the expression properties of those lines. When compared with clonal lines made by traditional transformation methods, which lead to the illegitimate insertion of tandem arrays, targeted insertion lines give more uniform expression, lower basal expression, and higher induction ratios. Targeted substitution, though intricate, affords results that should greatly improve comparative expression assays—a major emphasis of cell-based studies. PMID:26450921

  8. Comparative antibiotic eradication of mycoplasma infections from continuous cell lines.

    PubMed

    Uphoff, Cord C; Drexler, Hans G

    2002-02-01

    Accumulating data implicate mycoplasma contamination as the single biggest problem in the culture of continuous cell lines. Mycoplasma infection can affect virtually every parameter and functional activity of the eukaryotic cells. A successful alternative to discarding infected cultures is to attempt to eliminate the contaminants by treatment with specific and efficient antimycoplasma antibiotics. The addition of antibiotics to the culture medium during a limited period of time (1-3 wk) is a simple, inexpensive, and very practical approach for decontaminating continuous cell lines. Here, we examined the effectiveness of several antibiotic treatment protocols that we have employed routinely in our cell lines bank. On an aggregate, 673 cultures from 236 chronically mycoplasma-positive cell lines were exposed to one of the following five antibiotic regimens: mycoplasma removal agent (quinolone; a 1-wk treatment), enrofloxacin (quinolone; 1 wk), sparfloxacin (quinolone; 1 wk), ciprofloxacin (quinolone; 2 wk), and BM-Cyclin (alternating tiamulin and minocycline; 3 wk). The mycoplasma infection was permanently (as determined by three solid mycoplasma detection assays) eliminated by the various antibiotics in 66-85% of the cultures treated. Mycoplasma resistance was seen in 7-21%, and loss of the culture as a result of cytotoxically caused cell death occurred in 3-11% of the cultures treated. Overall, 223 of the 236 mycoplasma-positive cell lines could be cured in a first round of antibiotic treatment with at least one regimen. Taken together, 95% of the mycoplasma-infected cell lines were permanently cleansed of the contaminants by antibiotic treatment, which validates this approach as an efficient and technically simple mycoplasma eradication method.

  9. Comparative antibiotic eradication of mycoplasma infections from continuous cell lines.

    PubMed

    Uphoff, Cord C; Drexler, Hans G

    2002-02-01

    Accumulating data implicate mycoplasma contamination as the single biggest problem in the culture of continuous cell lines. Mycoplasma infection can affect virtually every parameter and functional activity of the eukaryotic cells. A successful alternative to discarding infected cultures is to attempt to eliminate the contaminants by treatment with specific and efficient antimycoplasma antibiotics. The addition of antibiotics to the culture medium during a limited period of time (1-3 wk) is a simple, inexpensive, and very practical approach for decontaminating continuous cell lines. Here, we examined the effectiveness of several antibiotic treatment protocols that we have employed routinely in our cell lines bank. On an aggregate, 673 cultures from 236 chronically mycoplasma-positive cell lines were exposed to one of the following five antibiotic regimens: mycoplasma removal agent (quinolone; a 1-wk treatment), enrofloxacin (quinolone; 1 wk), sparfloxacin (quinolone; 1 wk), ciprofloxacin (quinolone; 2 wk), and BM-Cyclin (alternating tiamulin and minocycline; 3 wk). The mycoplasma infection was permanently (as determined by three solid mycoplasma detection assays) eliminated by the various antibiotics in 66-85% of the cultures treated. Mycoplasma resistance was seen in 7-21%, and loss of the culture as a result of cytotoxically caused cell death occurred in 3-11% of the cultures treated. Overall, 223 of the 236 mycoplasma-positive cell lines could be cured in a first round of antibiotic treatment with at least one regimen. Taken together, 95% of the mycoplasma-infected cell lines were permanently cleansed of the contaminants by antibiotic treatment, which validates this approach as an efficient and technically simple mycoplasma eradication method. PMID:11929000

  10. Establishment, Immortalisation and Characterisation of Pteropid Bat Cell Lines

    PubMed Central

    Crameri, Gary; Todd, Shawn; Grimley, Samantha; McEachern, Jennifer A.; Marsh, Glenn A.; Smith, Craig; Tachedjian, Mary; De Jong, Carol; Virtue, Elena R.; Yu, Meng; Bulach, Dieter; Liu, Jun-Ping; Michalski, Wojtek P.; Middleton, Deborah; Field, Hume E.; Wang, Lin-Fa

    2009-01-01

    Background Bats are the suspected natural reservoir hosts for a number of new and emerging zoonotic viruses including Nipah virus, Hendra virus, severe acute respiratory syndrome coronavirus and Ebola virus. Since the discovery of SARS-like coronaviruses in Chinese horseshoe bats, attempts to isolate a SL-CoV from bats have failed and attempts to isolate other bat-borne viruses in various mammalian cell lines have been similarly unsuccessful. New stable bat cell lines are needed to help with these investigations and as tools to assist in the study of bat immunology and virus-host interactions. Methodology/Findings Black flying foxes (Pteropus alecto) were captured from the wild and transported live to the laboratory for primary cell culture preparation using a variety of different methods and culture media. Primary cells were successfully cultured from 20 different organs. Cell immortalisation can occur spontaneously, however we used a retroviral system to immortalise cells via the transfer and stable production of the Simian virus 40 Large T antigen and the human telomerase reverse transcriptase protein. Initial infection experiments with both cloned and uncloned cell lines using Hendra and Nipah viruses demonstrated varying degrees of infection efficiency between the different cell lines, although it was possible to infect cells in all tissue types. Conclusions/Significance The approaches developed and optimised in this study should be applicable to bats of other species. We are in the process of generating further cell lines from a number of different bat species using the methodology established in this study. PMID:20011515

  11. Monoclonal antibodies against the human leukemia cell line K 562.

    PubMed

    Böttger, V; Hering, S; Jantscheff, P; Micheel, B

    1985-01-01

    Three monoclonal antibodies raised against K 562, a cell line originally established from a patient with chronic myeloid leukemia (CML) in terminal blast crisis, were selected according to their distinct reaction pattern. Whereas two antibodies (ZIK-C1-A/C5 and ZIK-C1-A/H5 also designated C and H) recognized antigens, present on K 562 cells and other immature and mature hematopoietic cells (cell lines and normal blood and bone marrow cells), antibody ZIK-C1-A/D9 also designated Y showed an exclusive binding to K 562 cells. The results obtained (here and in the following paper) indicate, that antibody ZIK-C1-A/D9 defines an early differentiation antigen of hematopoiesis or a leukemia-associated antigen.

  12. JNK Inhibition Inhibits Lateral Line Neuromast Hair Cell Development

    PubMed Central

    Cai, Chengfu; Lin, Jinchao; Sun, Shaoyang; He, Yingzi

    2016-01-01

    JNK signaling is known to play a role in regulating cell behaviors such as cell cycle progression, cell proliferation, and apoptosis, and recent studies have suggested important roles for JNK signaling in embryonic development. However, the precise function of JNK signaling in hair cell development remains poorly studied. In this study, we used the small molecule JNK inhibitor SP600125 to examine the effect of JNK signaling abrogation on the development of hair cells in the zebrafish lateral line neuromast. Our results showed that SP600125 reduced the numbers of both hair cells and supporting cells in neuromasts during larval development in a dose-dependent manner. Additionally, JNK inhibition strongly inhibited the proliferation of neuromast cells, which likely explains the decrease in the number of differentiated hair cells in inhibitor-treated larvae. Furthermore, western blot and in situ analysis showed that JNK inhibition induced cell cycle arrest through induction of p21 expression. We also showed that SP600125 induced cell death in developing neuromasts as measured by cleaved caspase-3 immunohistochemistry, and this was accompanied with an induction of p53 gene expression. Together these results indicate that JNK might be an important regulator in the development of hair cells in the lateral line in zebrafish by controlling both cell cycle progression and apoptosis. PMID:26903805

  13. Non-targeted radiation effects in vertebrate cell lines

    NASA Astrophysics Data System (ADS)

    Ryan, Lorna

    Radiation effects, such as bystander effects, hyper radiosensitivity/induced radioresistance (HRS/IRR) and adaptive response that are not related to direct DNA damage are now accepted. However the inter-relationship between them and the possible impact on the scientific basis for radiation protection are highly controversial. This thesis attempts to elucidate the mechanisms of some of these well known but little understood effects. Each paper examines some aspect of bystander effects, adaptive responses and HRS/IRR in an effort to understand how they vary with cell type, dose and time of exposure to single or multiple doses. All the effects involve non-linear dose effect curves and are mainly evident following low doses. Overall findings of the thesis include (1) A clear difference was observed between radioresistant, tumorigenic cell lines with mutant p53 gene expression, and radiosensitive, more normal, cell lines with wild type p53. In general death inducing bystander responses are induced in normal cell populations exposed to low doses of radiation while survival inducing IRR and adaptive responses are seen in the radioresistant tumorigenic cell lines. (2) A cohort of fish cell lines which demonstrated survival promoting bystander effects, also did not show a protective adaptive responses. (3) Adaptive responses traditionally occur when a large challenge dose is given 4--6hrs following low (10--100mGy) priming doses but this thesis shows that for the epithelial cell lines tested, the size of the priming dose (range 0.1--2Gy) does not appear to alter the size of the recovery response. Additionally increased survival could be detected in some cases when the challenge dose was given within one hour of the priming dose. The overall conclusion is that cell lines induce either a bystander response or a protective/adaptive response depending on genetic background and other factors. Care is needed in the interpretation of data generated from only one or two cell lines

  14. SENSORY HAIR CELL REGENERATION IN THE ZEBRAFISH LATERAL LINE

    PubMed Central

    Lush, Mark E.; Piotrowski, Tatjana

    2014-01-01

    Damage or destruction of sensory hair cells in the inner ear leads to hearing or balance deficits that can be debilitating, especially in older adults. Unfortunately, the damage is permanent, as regeneration of the inner ear sensory epithelia does not occur in mammals. Zebrafish and other non-mammalian vertebrates have the remarkable ability to regenerate sensory hair cells and understanding the molecular and cellular basis for this regenerative ability will hopefully aid us in designing therapies to induce regeneration in mammals. Zebrafish not only possess hair cells in the ear but also in the sensory lateral line system. Hair cells in both organs are functionally analogous to hair cells in the inner ear of mammals. The lateral line is a mechanosensory system found in most aquatic vertebrates that detects water motion and aids in predator avoidance, prey capture, schooling and mating. Although hair cell regeneration occurs in both the ear and lateral line, most research to date has focused on the lateral line due to its relatively simple structure and accessibility. Here we review the recent discoveries made during the characterization of hair cell regeneration in zebrafish. PMID:25045019

  15. Comparative Metabolic Flux Profiling of Melanoma Cell Lines

    PubMed Central

    Scott, David A.; Richardson, Adam D.; Filipp, Fabian V.; Knutzen, Christine A.; Chiang, Gary G.; Ronai, Ze'ev A.; Osterman, Andrei L.; Smith, Jeffrey W.

    2011-01-01

    Metabolic rewiring is an established hallmark of cancer, but the details of this rewiring at a systems level are not well characterized. Here we acquire this insight in a melanoma cell line panel by tracking metabolic flux using isotopically labeled nutrients. Metabolic profiling and flux balance analysis were used to compare normal melanocytes to melanoma cell lines in both normoxic and hypoxic conditions. All melanoma cells exhibited the Warburg phenomenon; they used more glucose and produced more lactate than melanocytes. Other changes were observed in melanoma cells that are not described by the Warburg phenomenon. Hypoxic conditions increased fermentation of glucose to lactate in both melanocytes and melanoma cells (the Pasteur effect). However, metabolism was not strictly glycolytic, as the tricarboxylic acid (TCA) cycle was functional in all melanoma lines, even under hypoxia. Furthermore, glutamine was also a key nutrient providing a substantial anaplerotic contribution to the TCA cycle. In the WM35 melanoma line glutamine was metabolized in the “reverse” (reductive) direction in the TCA cycle, particularly under hypoxia. This reverse flux allowed the melanoma cells to synthesize fatty acids from glutamine while glucose was primarily converted to lactate. Altogether, this study, which is the first comprehensive comparative analysis of metabolism in melanoma cells, provides a foundation for targeting metabolism for therapeutic benefit in melanoma. PMID:21998308

  16. Morphology and growth of murine cell lines on model biomaterials.

    PubMed

    Godek, Marisha L; Duchsherer, Nichole L; McElwee, Quinn; Grainger, David W

    2004-01-01

    All biomaterial implants are assaulted by the host "foreign body" immune response. Understanding the complex, dynamic relationship between cells, biomaterials and milieu is an important first step towards controlling this reaction. Material surface chemistry dictates protein adsorption, and thus subsequent cell interactions. The cell-implant is a microenvironment involving 1) proteins that coat the surface and 2) cells that interact with these proteins. Macrophages and fibroblasts are two cell types that interact with proteins on biomaterials surfaces and play different related, but equally important, roles in biomaterials rejection and implant failure. Growth characteristics of four murine cell lines on model biomaterials surfaces were examined. Murine monocyte-macrophages (RAW 264.7 and J774A.1), murine macrophage (IC-21) and murine fibroblast (NIH 3T3) cell lines were tested to determine whether differences exist in adhesion, proliferation, differentiation, spreading, and fusion (macrophage lineages only) on these surfaces. Differences were observed in the ability of cells to adhere to and subsequently proliferate on polymer surfaces. (Monocyte-) macrophages grew well on all surfaces tested and growth rates were measured on three representative polymer biomaterials surfaces: tissue culture polystyrene (TCPS), polystyrene, and Teflon-AF. J774A.1 cultures grown on TCPS and treated with exogenous cytokines IL-4 and GM-CSF were observed to contain multinucleate cells with unusual morphologies. Thus, (monocyte-) macrophage cell lines were found to effectively attach to and interrogate each surface presented, with evidence of extensive spreading on Teflon-AF surfaces, particularly in the IC-21 cultures. The J774A.1 line was able to proliferate and/or differentiate to more specialized cell types (multinucleate/dendritic-like cells) in the presence of soluble chemokine cues. PMID:15133927

  17. Establishment of a rat nasal epithelial tumor cell line.

    PubMed

    Hood, A T; Currie, D; Garte, S J

    1987-04-01

    A new cell line designated NAS 2BL has been established from a rat nasal tumor induced by inhalation of the direct-acting carcinogen methylmethane sulfonate. The cells are epithelial in morphology, have a generation time of 34 h, require 10% fetal bovine serum for optimal growth, and exhibit keratinization at confluence. The karyotype is aneuploid, with several marker chromosomes, and the cells are transformed by the criterion of nude mouse tumorigenicity.

  18. Artificial islets from hybrid spheroids of three pancreatic cell lines.

    PubMed

    Jo, Y H; Jang, I J; Nemeno, J G; Lee, S; Kim, B Y; Nam, B M; Yang, W; Lee, K M; Kim, H; Takebe, T; Kim, Y S; Lee, J I

    2014-05-01

    Pancreatic islets have been the focus of recent studies exploring the pathologic mechanisms of diabetes mellitus as well as more effective and radical treatments for this disease. Islet transplantation is a promising therapeutic strategy; however, isolation of pancreatic islets for this purpose has been challenging, because the technique is time consuming and technically difficult, and tissue handling can be variable. Pseudo-islets can be used as an alternative to naïve islets, but require cellular sources or artificial materials. In this study, pancreas-derived cells were used to generate pseudo-islets. Because the pancreas is composed of a variety of cell types, namely α cells, β cells, δ cells, and other pancreatic cells that perform different functions, we used 3 different cell lines-NIT-1 (a β-cell line), α TC1 clone 6 (an α-cell line), and TGP52 (a pancreatic epithelial-like cell line)-which we cocultured in nonadhesive culture plates to produce hybrid cellular spheroids. These pseudo-islets had an oval shape and were morphologically similar to naïve islets; additionally, they expressed and secreted the pancreatic hormones insulin, glucagon, and somatostatin, as confirmed by reverse-transcription polymerase chain reaction and enzyme-linked immunosorbent assay. The results demonstrate that pseudo-islets that mimic naïve islets can be successfully generated by a coculture method. These artificial islets can potentially be used for in vitro tests related to diabetes mellitus, specifically, in drug discovery or for investigating pathology. Moreover, they can be useful for examining basic questions pertaining to cell-cell interactions and tissue development. PMID:24815150

  19. Metabolic reprogramming: a new relevant pathway in adult adrenocortical tumors

    PubMed Central

    Longatto-Filho, Adhemar; Faria, André M.; Fragoso, Maria C. B. V.; Lovisolo, Silvana M.; Lerário, Antonio M.; Almeida, Madson Q.

    2015-01-01

    Adrenocortical carcinomas (ACCs) are complex neoplasias that may present unexpected clinical behavior, being imperative to identify new biological markers that can predict patient prognosis and provide new therapeutic options. The main aim of the present study was to evaluate the prognostic value of metabolism-related key proteins in adrenocortical carcinoma. The immunohistochemical expression of MCT1, MCT2, MCT4, CD147, CD44, GLUT1 and CAIX was evaluated in a series of 154 adult patients with adrenocortical neoplasia and associated with patients' clinicopathological parameters. A significant increase in was found for membranous expression of MCT4, GLUT1 and CAIX in carcinomas, when compared to adenomas. Importantly MCT1, GLUT1 and CAIX expressions were significantly associated with poor prognostic variables, including high nuclear grade, high mitotic index, advanced tumor staging, presence of metastasis, as well as shorter overall and disease free survival. In opposition, MCT2 membranous expression was associated with favorable prognostic parameters. Importantly, cytoplasmic expression of CD147 was identified as an independent predictor of longer overall survival and cytoplasmic expression of CAIX as an independent predictor of longer disease-free survival. We provide evidence for a metabolic reprogramming in adrenocortical malignant tumors towards the hyperglycolytic and acid-resistant phenotype, which was associated with poor prognosis. PMID:26587828

  20. The Role of gsp Mutations on the Development of Adrenocortical Tumors and Adrenal Hyperplasia.

    PubMed

    Villares Fragoso, Maria Candida Barisson; Wanichi, Ingrid Quevedo; Cavalcante, Isadora Pontes; Mariani, Beatriz Marinho de Paula

    2016-01-01

    Somatic GNAS point mutations, commonly known as gsp mutations, are involved in the pathogenesis of McCune-Albright syndrome (MAS) and have also been described in autonomous hormone-producing tumors, such as somatotropinoma, corticotrophoma, thyroid cancer, ovarian and testicular Leydig cell tumors, and primary macronodular adrenocortical hyperplasia (PMAH) (1-3). The involvement of gsp mutations in adrenal tumors was first described by Lyons et al. Since then, several studies have detected the presence of gsp mutations in adrenal tumors, but none of them could explain its presence along or the mechanism that leads to tumor formation and hormone hypersecretion. As a result, the molecular pathogenesis of the majority of sporadic adrenocortical tumors remains unclear (3). PMAH has also been reported with gsp somatic mutations in a few cases. Fragoso et al. identified two distinct gsp somatic mutations affecting arginine residues on codon 201 of GNAS in a few patients with PMAH who lacked any features or manifestations of MAS. Followed by this discovery, other studies have continued looking for gsp mutations based on strong prior evidence demonstrating that increased cAMP signaling is sufficient for cell proliferation and cortisol production (2, 4). With consideration for the previously reported findings, we conjecture that although somatic activating mutations in GNAS are a rare molecular event, these mutations could probably be sufficient to induce the development of macronodule hyperplasia and variable cortisol secretion. In this manuscript, we revised the presence of gsp mutations associated with adrenal cortical tumors and hyperplasia. PMID:27512387

  1. The Role of gsp Mutations on the Development of Adrenocortical Tumors and Adrenal Hyperplasia

    PubMed Central

    Villares Fragoso, Maria Candida Barisson; Wanichi, Ingrid Quevedo; Cavalcante, Isadora Pontes; Mariani, Beatriz Marinho de Paula

    2016-01-01

    Somatic GNAS point mutations, commonly known as gsp mutations, are involved in the pathogenesis of McCune–Albright syndrome (MAS) and have also been described in autonomous hormone-producing tumors, such as somatotropinoma, corticotrophoma, thyroid cancer, ovarian and testicular Leydig cell tumors, and primary macronodular adrenocortical hyperplasia (PMAH) (1–3). The involvement of gsp mutations in adrenal tumors was first described by Lyons et al. Since then, several studies have detected the presence of gsp mutations in adrenal tumors, but none of them could explain its presence along or the mechanism that leads to tumor formation and hormone hypersecretion. As a result, the molecular pathogenesis of the majority of sporadic adrenocortical tumors remains unclear (3). PMAH has also been reported with gsp somatic mutations in a few cases. Fragoso et al. identified two distinct gsp somatic mutations affecting arginine residues on codon 201 of GNAS in a few patients with PMAH who lacked any features or manifestations of MAS. Followed by this discovery, other studies have continued looking for gsp mutations based on strong prior evidence demonstrating that increased cAMP signaling is sufficient for cell proliferation and cortisol production (2, 4). With consideration for the previously reported findings, we conjecture that although somatic activating mutations in GNAS are a rare molecular event, these mutations could probably be sufficient to induce the development of macronodule hyperplasia and variable cortisol secretion. In this manuscript, we revised the presence of gsp mutations associated with adrenal cortical tumors and hyperplasia. PMID:27512387

  2. [Adrenocortical zonation in human growth hormone gene (hGH) transgenic mice].

    PubMed

    Tanaka, S; Tojo, H; Matsuzawa, A

    1993-04-01

    Adrenocortical zonation was histologically examined in 2 infertile female transgenic (Tg) mice which carried human growth hormone (hGH) gene and had a high circulating level of hGH. The adrenal cortices of Tg mice were characterized by the appearance of hypertrophied zona fasciculata cells with the cytoplasm filled with many lipid droplets even in size, the absence of the distinctive z. reticularis and the persistence of the X zone with nodules in one and without them in the other. The last finding might be explained by the reflection of the strain differences used in Tg mouse construction. It was, however, suggested that the X zone degeneration might not be associated with the adenohypophysis. The changes observed in z. fasciculata cells were interpreted as the effects of ACTH-like function of circulating hGH in addition to the known PRL-like function. The absence of the z. reticularis may be explained by its morphological similarity to z. fasciculata under the effects of the circulating hGH at a high level. In addition to genetic analysis of various inbred strains, Tg mice with ectopic hGH production provide a new methodology in analyzing the mechanism of adrenocortical zone formation.

  3. Comparative proteomic profiling of Hodgkin lymphoma cell lines.

    PubMed

    Vergara, D; Simeone, P; De Matteis, S; Carloni, S; Lanuti, P; Marchisio, M; Miscia, S; Rizzello, A; Napolitano, R; Agostinelli, C; Maffia, M

    2016-01-01

    Classical Hodgkin lymphoma (cHL) is a malignancy with complex pathogenesis. The hallmark of the disease is the presence of large mononucleated Hodgkin and bi- or multinucleated Reed/Sternberg (H/RS) cells. The origin of HRS cells in cHL is controversial as these cells show the coexpression of markers of several lineages. Using a proteomic approach, we compared the protein expression profile of cHL models of T- and B-cell derivation to find proteins differentially expressed in these cell lines. A total of 67 proteins were found differentially expressed between the two cell lines including metabolic proteins and proteins involved in the regulation of the cytoskeleton and/or cell migration, which were further validated by western blotting. Additionally, the expression of selected B- and T-cell antigens was also assessed by flow cytometry to reveal significant differences in the expression of different surface markers. Bioinformatics analysis was then applied to our dataset to find enriched pathways and networks, and to identify possible key regulators. In the present study, a proteomic approach was used to compare the protein expression profiles of two cHL cell lines. The identified proteins and/or networks, many of which not previously related to cHL, may be important to better define the pathogenesis of the disease, to identify novel diagnostic markers, and to design new therapeutic strategies. PMID:26588820

  4. Implantation of Vascular Grafts Lined with Genetically Modified Endothelial Cells

    NASA Astrophysics Data System (ADS)

    Wilson, James M.; Birinyi, Louis K.; Salomon, Robert N.; Libby, Peter; Callow, Allan D.; Mulligan, Richard C.

    1989-06-01

    The possibility of using the vascular endothelial cell as a target for gene replacement therapy was explored. Recombinant retroviruses were used to transduce the lacZ gene into endothelial cells harvested from mongrel dogs. Prosthetic vascular grafts seeded with the genetically modified cells were implanted as carotid interposition grafts into the dogs from which the original cells were harvested. Analysis of the graft 5 weeks after implantation revealed genetically modified endothelial cells lining the luminal surface of the graft. This technology could be used in the treatment of atherosclerosis disease and the design of new drug delivery systems.

  5. Characterization of a Novel Radiation-Induced Sarcoma Cell Line

    PubMed Central

    Lang, J.E.; Zhu, W.; Nokes, B.T.; Sheth, G.R.; Novak, P.; Fuchs, L.; Watts, G.S.; Futscher, B.W.; Mineyev, N.; Ring, A.; LeBeau, L.; Nagle, R.; Cranmer, L.D.

    2014-01-01

    Background Radiation-induced sarcoma (RIS) is a potential complication of cancer treatment. No widely available cell line models exist to facilitate studies of RIS. Methods We derived a spontaneously immortalized primary human cell line, UACC-SARC1, from a RIS. Results Short tandem repeat (STR) profiling of UACC-SARC1 was virtually identical to its parental tumor. Immunohistochemistry (IHC) analysis of the tumor and immunocytochemistry (ICC) analysis of UACC-SARC1 revealed shared expression of vimentin, osteonectin, CD68, Ki67 and PTEN but tumor-restricted expression of the histiocyte markers α1-antitrypsin and α1-antichymotrypsin. Karyotyping of the tumor demonstrated aneuploidy. Comparative genomic hybridization (CGH) provided direct genetic comparison between the tumor and UACC-SARC1. Sequencing of 740 mutation hotspots revealed no mutations in UACC-SARC1 nor in the tumor. NOD/SCID gamma mouse xenografts demonstrated tumor formation and metastasis. Clonogenicity assays demonstrated that 90% of single cells produced viable colonies. NOD/SCID gamma mice produced useful patient-derived xenografts for orthotopic or metastatic models. Conclusion Our novel RIS strain constitutes a useful tool for pre-clinical studies of this rare, aggressive disease. UACC-SARC1 is an aneuploid cell line with complex genomics lacking common oncogenes or tumor suppressor genes as drivers of its biology. The UACC-SARC1 cell line will enable further studies of the drivers of RIS. Synopsis We derived a spontaneously immortalized primary human cell line, UACC-SARC1, from a radiation-induced sarcoma (RIS). Our novel RIS cell line constitutes a useful tool for pre-clinical studies of this rare, aggressive disease. PMID:25644184

  6. Casein gene expression in mouse mammary epithelial cell lines: Dependence upon extracellular matrix and cell type

    SciTech Connect

    Medina, D.; Oborn, C.J. ); Li, M.L.; Bissell, M.J. )

    1987-09-01

    The COMMA-D mammary cell line exhibits mammary-specific functional differentiation under appropriate conditions in cell culture. The cytologically heterogeneous COMMA-D parental line and the clonal lines DB-1, TA-5, and FA-1 derived from the COMMA-D parent were examined for similar properties of functional differentiation. In monolayer cell culture, the cell lines DB-1, TA-5, FA-1, and MA-4 were examined for expression of mammary-specific and epithelial-specific proteins by an indirect immunofluorescence assay. The clonal cell lines were relatively homogeneous in their respective staining properties and seemed to represent three subpopulations found in the heterogeneous parental COMMA-D lines. None of the four clonal lines appeared to represent myoepithelial cells. The cell lines were examined for expression of {beta}-casein mRNA in the presence or absence of prolactin. The inducibility of {beta}-casein in the COMMA-D cell line was further enhanced by a reconstituted basement membrane preparation enriched in laminin, collagen IV, and proteoglycans. These results support the hypothesis that the functional response of inducible mammary cell populations is a result of interaction among hormones, multiple extracellular matrix components, and specific cell types.

  7. Human cell lines: A promising alternative for recombinant FIX production.

    PubMed

    de Sousa Bomfim, Aline; Cristina Corrêa de Freitas, Marcela; Picanço-Castro, Virgínia; de Abreu Soares Neto, Mário; Swiech, Kamilla; Tadeu Covas, Dimas; Maria de Sousa Russo, Elisa

    2016-05-01

    Factor IX (FIX) is a vitamin K-dependent protein, and it has become a valuable pharmaceutical in the Hemophilia B treatment. We evaluated the potential of recombinant human FIX (rhFIX) expression in 293T and SK-Hep-1 human cell lines. SK-Hep-1-FIX cells produced higher levels of biologically active protein. The growth profile of 293T-FIX cells was not influenced by lentiviral integration number into the cellular genome. SK-Hep-1-FIX cells showed a significantly lower growth rate than SK-Hep-1 cells. γ-carboxylation process is significant to FIX biological activity, thus we performed a expression analysis of genes involved in this process. The 293T gene expression suggests that this cell line could efficiently carboxylate FIX, however only 28% of the total secreted protein is active. SK-Hep-1 cells did not express high amounts of VKORC1 and carboxylase, but this cell line secreted large amounts of active protein. Enrichment of culture medium with Ca(+2) and Mg(+2) ions did not affect positively rhFIX expression in SK-Hep-1 cells. In 293T cells, the addition of 0.5 mM Ca(+2) and 1 mM Mg(+2) resulted in higher rhFIX concentration. SK-Hep-1 cell line proved to be very effective in rhFIX production, and it can be used as a novel biotechnological platform for the production of recombinant proteins.

  8. Phase transitions in tumor growth: II prostate cancer cell lines

    NASA Astrophysics Data System (ADS)

    Llanos-Pérez, J. A.; Betancourt-Mar, A.; De Miguel, M. P.; Izquierdo-Kulich, E.; Royuela-García, M.; Tejera, E.; Nieto-Villar, J. M.

    2015-05-01

    We propose a mechanism for prostate cancer cell lines growth, LNCaP and PC3 based on a Gompertz dynamics. This growth exhibits a multifractal behavior and a "second order" phase transition. Finally, it was found that the cellular line PC3 exhibits a higher value of entropy production rate compared to LNCaP, which is indicative of the robustness of PC3, over to LNCaP and may be a quantitative index of metastatic potential tumors.

  9. Adrenocortical cancer (ACC) - literature overview and own experience.

    PubMed

    Dworakowska, Dorota; Drabarek, Agata; Wenzel, Ingrid; Babińska, Anna; Świątkowska-Stodulska, Renata; Sworczak, Krzysztof

    2014-01-01

    Adrenocortical carcinoma (ACC) is a malignant endocrine tumour. The rarity of the disease has stymied therapeutic development. Age distribution shows two peaks: the first and fifth decades of life, with children and women more frequently affected. Although 60-70% of ACCs are biochemically found to overproduce hormones, it is not clinically apparent in many cases. If present, endocrine symptoms include signs of hypercortisolaemia, virilisation or gynaecomastia. ACC carries a poor prognosis, and a cure can be achieved only by complete surgical resection. Mitotane is used both as an adjuvant treatment and also in non-operative patients. The role of radio- and chemotherapy is still controversial. The post-operative disease free survival is low and oscillates around 30% due to high tumour recurrence rate. The diagnosis is based on tumour histological assessment with the use of the Weiss score, however urinary steroid profiling (if available) can serve to differentiate between ACC and other adrenal tumours. Conventional prognostic markers in ACC include stage and grade of disease, and, as currently reported, the presence of hypercortisolaemia. Molecular analysis has had a significant impact on the understanding of the pathogenetic mechanism of ACC development and the evaluation of prognostic and predictive markers, among which alterations of the IGF system, the Wnt pathway, p53 and molecules involved in cancer cell invasion properties and angiogenesis seem to be very promising. We here summarise our own experience related to the management of ACC and present a literature overview. We have not aimed to include a detailed summary of the molecular alterations biology described in ACC, as this has already been addressed in other papers. PMID:25554619

  10. Psychiatric implications of altered limbic-hypothalamic-pituitary-adrenocortical activity.

    PubMed

    Holsboer, F

    1989-01-01

    Hormones of the limbic-hypothalamic-pituitary-adrenocortical (LHPA) system are much involved in central nervous system regulation. The major LHPA neuropeptides, corticotropin-releasing hormone (CRH), vasopressin (AVP) and corticotropin (ACTH) do not only coordinate the neuroendocrine response to stress, but also induce behavioral adaptation. Transcription and post-translational processing of these neuropeptides is regulated by corticosteroids secreted from the adrenal cortex after stimulation by ACTH and other proopiomelanocortin derived peptides. These steroids play a key role as regulators of cell development, homeostatic maintenance and adaptation to environmental challenges. They execute vitally important actions through genomic effects resulting in altered gene expression and nongenomic effects leading to altered neuronal excitability. Since excessive secretory activity of this particular neuroendocrine system is part of an acute stress response or depressive symptom pattern, there is good reason to suspect that central actions of these steroids and peptides are involved in pathophysiology determining the clinical phenotype, drug response and relapse liability. This overview summarizes the clinical neuroendocrine investigations of the author and his collaborators, while they worked at the Department of Psychiatry in Mainz. The major conclusions from this work were: (1) aberrant hormonal responses to challenges with dexamethasone, ACTH or CRH are reflecting altered brain physiology in affective illness and related disorders; (2) hormones of the LHPA axis influence also nonendocrine behavioral systems such as sleep EEG; (3) physiologically significant interactions exist between LHPA hormones, the thyroid, growth hormone, gonadal and other neuroendocrine systems; (4) hormones of the LHPA axis constitute a bidirectional link between immunoregulation and brain activity; and (5) future psychiatric research topics such as molecular genetics of affective disorders

  11. DNA Methylation Profiling Identifies Global Methylation Differences and Markers of Adrenocortical Tumors

    PubMed Central

    Rechache, Nesrin S.; Wang, Yonghong; Stevenson, Holly S.; Killian, J. Keith; Edelman, Daniel C.; Merino, Maria; Zhang, Lisa; Nilubol, Naris; Stratakis, Constantine A.; Meltzer, Paul S.

    2012-01-01

    Context: It is not known whether there are any DNA methylation alterations in adrenocortical tumors. Objective: The objective of the study was to determine the methylation profile of normal adrenal cortex and benign and malignant adrenocortical tumors. Methods: Genome-wide methylation status of CpG regions were determined in normal (n = 19), benign (n = 48), primary malignant (n = 8), and metastatic malignant (n = 12) adrenocortical tissue samples. An integrated analysis of genome-wide methylation and mRNA expression in benign vs. malignant adrenocortical tissue samples was also performed. Results: Methylation profiling revealed the following: 1) that methylation patterns were distinctly different and could distinguish normal, benign, primary malignant, and metastatic tissue samples; 2) that malignant samples have global hypomethylation; and 3) that the methylation of CpG regions are different in benign adrenocortical tumors by functional status. Normal compared with benign samples had the least amount of methylation differences, whereas normal compared with primary and metastatic adrenocortical carcinoma samples had the greatest variability in methylation (adjusted P ≤ 0.01). Of 215 down-regulated genes (≥2-fold, adjusted P ≤ 0.05) in malignant primary adrenocortical tumor samples, 52 of these genes were also hypermethylated. Conclusions: Malignant adrenocortical tumors are globally hypomethylated as compared with normal and benign tumors. Methylation profile differences may accurately distinguish between primary benign and malignant adrenocortical tumors. Several differentially methylated sites are associated with genes known to be dysregulated in malignant adrenocortical tumors. PMID:22472567

  12. Analysis of LINE-1 expression in human pluripotent cells.

    PubMed

    Muñoz-Lopez, Martin; Garcia-Cañadas, Marta; Macia, Angela; Morell, Santiago; Garcia-Perez, Jose L

    2012-01-01

    Half of the human genome is composed of repeated DNA, and some types are mobile within our genome (transposons and retrotransposons). Despite their abundance, only a small fraction of them are currently active in our genome (Long Interspersed Element-1 (LINE-1), Alu, and SVA elements). LINE-1 or L1 elements are a family of active non-LTR retrotransposons, the ongoing mobilization of which still impacts our genome. As selfish DNA elements, L1 activity is more prominent in early human development, where new insertions would be transmitted to the progeny. Here, we describe the conventional methods aimed to determine the expression level of LINE-1 elements in pluripotent human cells.

  13. Registration of Human Embryonic Stem Cell Lines: Korea, 2010

    PubMed Central

    Lee, Ji-Yoon; Lee, Dae-Yeon; Choi, Young-Sil; Lee, Kyoung-Jae; Kim, Yong-Ou

    2011-01-01

    In an effort to increase the credibility of human embryonic stem cell (hESC) lines established in Korea, obligatory registration was introduced by the Bioethics and Safety Act 2008, effective as of January 1, 2010. The DNA fingerprint, chromosome stability, expression of pluripotency markers, and contamination of mycoplasma of the submitted lines were analyzed by Korea Centers for Disease Control and Prevention (KCDC). The characterization data and ethical aspects, such as informed consent for donation of surplus embryos, were reviewed by a 10-member advisory review committee for stem cell registry. A total of 55 domestic hESC lines were submitted for registration in 2010; among them 51 were registered. Among these submitted lines, 26 were additionally characterized by KCDC, while 25 lines previously characterized by the Ministry of Education, Science and Technology were not additionally analyzed by KCDC. Registration completed an oversight system for embryo research by registering the products of licensed embryo research projects, making embryo research more transparent in Korea. Information about hESC lines is available at the website of the Korea Stem Cell Registry (kscr.nih.go.kr). PMID:24159464

  14. Isolation of dengue virus with a human promonocyte cell line.

    PubMed

    Liu, W T; Chen, C L; Lee, S S; Chan, C C; Lo, F L; Ko, Y C

    1991-05-01

    In October-November, 1988 there was an outbreak of dengue fever in the Kaoshiung area of southern Taiwan. We collected 100 serum samples from 96 patients at the onset of their fever for virus cultures and identification. A human promonocyte cell line (HL-CZ) established in our laboratory was used and proved to be susceptible for dengue virus propagation. Type 1 dengue virus in the HL-CZ cell culture was identified by immunofluorescence tests using monoclonal antibodies, and also by hemagglutination tests with goose red blood cells. The density of the virus particles, as measured by sucrose gradient ultracentrifugation, ranged from 1.186 to 1.224 g/ml. The virus yield from this cell culture is comparable with that from the C6/36 mosquito cell line. There was a significant correlation between the antibody responses tested with Western dot blots and hemagglutination inhibition techniques.

  15. Effect of Predatory Bacteria on Human Cell Lines

    PubMed Central

    Gupta, Shilpi; Tang, Chi; Tran, Michael; Kadouri, Daniel E.

    2016-01-01

    Predatory bacteria are Gram-negative bacteria that prey on other Gram-negative bacteria and have been considered as potential therapeutic agents against multi-drug resistant pathogens. In vivo animal models have demonstrated that predatory bacteria are non-toxic and non-immunogenic in rodents. In order to consider the use of predatory bacteria as live antibiotics, it is important to investigate their effect on human cells. The aim of this study was to determine the effect of Bdellovibrio bacteriovorus strains 109J and HD100, and Micavibrio aeruginosavorus strain ARL-13 on cell viability and inflammatory responses of five human cell lines, representative of clinically relevant tissues. We found that the predators were not cytotoxic to any of the human cell lines tested. Microscopic imaging showed no signs of cell detachment, as compared to predator-free cells. In comparison to an E. coli control, exposure to higher concentrations of the predators did not trigger a significant elevation of pro-inflammatory cytokines in four of the five human cell lines tested. Our work underlines the non-pathogenic attributes of predatory bacteria on human cells and highlights their potential use as live antibiotics against human pathogens. PMID:27579919

  16. Modeling adenovirus latency in human lymphocyte cell lines.

    PubMed

    Zhang, Yange; Huang, Wen; Ornelles, David A; Gooding, Linda R

    2010-09-01

    Species C adenovirus establishes a latent infection in lymphocytes of the tonsils and adenoids. To understand how this lytic virus is maintained in these cells, four human lymphocytic cell lines that support the entire virus life cycle were examined. The T-cell line Jurkat ceased proliferation and died shortly after virus infection. BJAB, Ramos (B cells), and KE37 (T cells) continued to divide at nearly normal rates while replicating the virus genome. Viral genome numbers peaked and then declined in BJAB cells below one genome per cell at 130 to 150 days postinfection. Ramos and KE37 cells maintained the virus genome at over 100 copies per cell over a comparable period of time. BJAB cells maintained the viral DNA as a monomeric episome. All three persistently infected cells lost expression of the cell surface coxsackie and adenovirus receptor (CAR) within 24 h postinfection, and CAR expression remained low for at least 340 days postinfection. CAR loss proceeded via a two-stage process. First, an initial loss of cell surface staining for CAR required virus late gene expression and a CAR-binding fiber protein even while CAR protein and mRNA levels remained high. Second, CAR mRNA disappeared at around 30 days postinfection and remained low even after virus DNA was lost from the cells. At late times postinfection (day 180), BJAB cells could not be reinfected with adenovirus, even when CAR was reintroduced to the cells via retroviral transduction, suggesting that the expression of multiple genes had been stably altered in these cells following infection. PMID:20573817

  17. Derivation of a Homozygous Human Androgenetic Embryonic Stem Cell Line.

    PubMed

    Ding, Chenhui; Huang, Sunxing; Qi, Quan; Fu, Rui; Zhu, Wanwan; Cai, Bing; Hong, Pingping; Liu, Zhengxin; Gu, Tiantian; Zeng, Yanhong; Wang, Jing; Xu, Yanwen; Zhao, Xiaoyang; Zhou, Qi; Zhou, Canquan

    2015-10-01

    Human embryonic stem cells (hESCs) have long been considered as a promising source for cell replacement therapy. However, one major obstacle for the use of these cells is immune compatibility. Histocompatible human parthenogenetic ESCs have been reported as a new method for generating human leukocyte antigen (HLA)-matched hESCs. To further investigate the possibility of obtaining histocompatible stem cells from uniparental embryos, we tried to produce androgenetic haploid human embryos by injecting a single spermatozoon into enucleated human oocyte, and establish human androgenetic embryonic stem (hAGES) cell lines from androgenetic embryos. In the present study, a diploid hAGES cell line has been established, which exhibits typical features of human ESCs, including the expression of pluripotency markers, having differentiation potential in vitro and in vivo, and stable propagation in an undifferentiated state (>P40). Bisulfite sequencing of the H19, Snrpn, Meg3, and Kv imprinting control regions suggested that hAGES cells maintained to a certain extent a sperm methylation pattern. Genome-wide single nucleotide polymorphism, short tandem repeat, and HLA analyses revealed that the hAGES cell genome was highly homozygous. These results suggest that hAGES cells from spermatozoon could serve as a useful tool for studying the mechanisms underlying genomic imprinting in humans. It might also be used as a potential resource for cell replacement therapy as parthenogenetic stem cells.

  18. Oral bioavailability of glyphosate: studies using two intestinal cell lines.

    PubMed

    Vasiluk, Luba; Pinto, Linda J; Moore, Margo M

    2005-01-01

    Glyphosate is a commonly used nonselective herbicide that inhibits plant growth through interference with the production of essential aromatic amino acids. In vivo studies in mammals with radiolabeled glyphosate have shown that 34% of radioactivity was associated with intestinal tissue 2 h after oral administration. The aim of our research was to investigate the transport, binding, and toxicity of glyphosate to the cultured human intestinal epithelial cell line, Caco-2, and the rat small intestinal crypt-derived cell line, ileum epithelial cells-18 (IEC-18). An in vitro analysis of the transport kinetics of [14C]-glyphosate showed that 4 h after exposure, approximately 8% of radiolabeled glyphosate moved through the Caco-2 monolayer in a dose-dependent manner. Binding of glyphosate to cells was saturable and approximately 4 x 10(11) binding sites/cell were estimated from bound [14C]. Exposure of Caco-2 cells to > or =10 mg/ml glyphosate reduced transmembrane electrical resistance (TEER) by 82 to 96% and increased permeability to [3H]-mannitol, indicating that paracellular permeability increased in glyphosate-treated cells. At 10-mg/ml glyphosate, both IEC-18 and Caco-2 cells showed disruption in the actin cytoskeleton. In Caco-2 cells, significant lactate dehydrogenase leakage was observed when cells were exposed to 15 mg/ml of glyphosate. These data indicate that at doses >10 mg/ml, glyphosate significantly disrupts the barrier properties of cultured intestinal cells.

  19. Characterization of cloned cells from an immortalized fetal pulmonary type II cell line

    SciTech Connect

    Henderson, R.F.; Waide, J.J.; Lechner, J.F.

    1995-12-01

    A cultured cell line that maintained expression of pulmonary type II cell markers of differentiation would be advantageous to generate a large number of homogenous cells in which to study the biochemical functions of type II cells. Type II epithelial cells are the source of pulmonary surfactant and a cell of origin for pulmonary adenomas. Last year our laboratory reported the induction of expression of two phenotypic markers of pulmonary type II cells (alkaline phosphatase activity and surfactant lipid synthesis) in cultured fetal rat lung epithelial (FRLE) cells, a spontaneously immortalized cell line of fetal rat lung type II cell origin. Subsequently, the induction of the ability to synthesize surfactant lipid became difficult to repeat. We hypothesized that the cell line was heterogenuous and some cells were more like type II cells than others. The purpose of this study was to test this hypothesis and to obtain a cultured cell line with type II cell phenotypic markers by cloning several FRLE cells and characterizing them for phenotypic markers of type II cells (alkaline phosphatase activity and presence of surfactant lipids). Thirty cloned cell lines were analyzed for induced alkaline phosphatase activity (on x-axis) and for percent of phospholipids that were disaturated (i.e., surfactant).

  20. Immunological features of idiopathic Addison's disease: characterization of the adrenocortical antigens

    PubMed Central

    Goudie, R. B.; McDonald, E.; Anderson, J. R.; Gray, Kathleen

    1968-01-01

    The antibodies in the sera of two patients with idiopathic Addison's disease, one reacting specifically with adrenocortical cells, the other with the various cell types which produce steroid hormones, have been found to be predominently IgG. The behaviour of the two antibodies in complement-fixation tests is in agreement with their reactivity, reported in the previous paper, in immunofluorescence tests. Cell fractionation studies suggest that both adrenal antigens are associated with the microsomal fraction. The biochemical properties of the antigens are consistent with the view that they are lipoproteins, possibly with associated carbohydrate. While the properties of the adrenal antigens are generally similar to those of the thyroid and gastric parietal cell organ-specific autoantigens, they differ from these, and from each other, in their susceptibility to destruction by certain enzymes and chemical treatments. ImagesFig. 1Fig. 3 PMID:4171048

  1. Innervation regulates synaptic ribbons in lateral line mechanosensory hair cells.

    PubMed

    Suli, Arminda; Pujol, Remy; Cunningham, Dale E; Hailey, Dale W; Prendergast, Andrew; Rubel, Edwin W; Raible, David W

    2016-06-01

    Failure to form proper synapses in mechanosensory hair cells, the sensory cells responsible for hearing and balance, leads to deafness and balance disorders. Ribbons are electron-dense structures that tether synaptic vesicles to the presynaptic zone of mechanosensory hair cells where they are juxtaposed with the post-synaptic endings of afferent fibers. They are initially formed throughout the cytoplasm, and, as cells mature, ribbons translocate to the basolateral membrane of hair cells to form functional synapses. We have examined the effect of post-synaptic elements on ribbon formation and maintenance in the zebrafish lateral line system by observing mutants that lack hair cell innervation, wild-type larvae whose nerves have been transected and ribbons in regenerating hair cells. Our results demonstrate that innervation is not required for initial ribbon formation but suggest that it is crucial for regulating the number, size and localization of ribbons in maturing hair cells, and for ribbon maintenance at the mature synapse.

  2. Innervation regulates synaptic ribbons in lateral line mechanosensory hair cells.

    PubMed

    Suli, Arminda; Pujol, Remy; Cunningham, Dale E; Hailey, Dale W; Prendergast, Andrew; Rubel, Edwin W; Raible, David W

    2016-06-01

    Failure to form proper synapses in mechanosensory hair cells, the sensory cells responsible for hearing and balance, leads to deafness and balance disorders. Ribbons are electron-dense structures that tether synaptic vesicles to the presynaptic zone of mechanosensory hair cells where they are juxtaposed with the post-synaptic endings of afferent fibers. They are initially formed throughout the cytoplasm, and, as cells mature, ribbons translocate to the basolateral membrane of hair cells to form functional synapses. We have examined the effect of post-synaptic elements on ribbon formation and maintenance in the zebrafish lateral line system by observing mutants that lack hair cell innervation, wild-type larvae whose nerves have been transected and ribbons in regenerating hair cells. Our results demonstrate that innervation is not required for initial ribbon formation but suggest that it is crucial for regulating the number, size and localization of ribbons in maturing hair cells, and for ribbon maintenance at the mature synapse. PMID:27103160

  3. LINEing germ and embryonic stem cells' silencing of retrotransposons.

    PubMed

    Ishiuchi, Takashi; Torres-Padilla, Maria-Elena

    2014-07-01

    Almost half of our genome is occupied by transposable elements. Although most of them are inactive, one type of non-long terminal repeat (LTR) retrotransposon, long interspersed nuclear element 1 (LINE1), is capable of retrotransposition. Two studies in this issue, Pezic and colleagues (pp. 1410-1428) and Castro-Diaz and colleagues (pp. 1397-1409), provide novel insight into the regulation of LINE1s in human embryonic stem cells and mouse germ cells and shed new light on the conservation of complex mechanisms to ensure silencing of transposable elements in mammals.

  4. Argininosuccinate synthetase 1 suppression and arginine restriction inhibit cell migration in gastric cancer cell lines.

    PubMed

    Shan, Yan-Shen; Hsu, Hui-Ping; Lai, Ming-Derg; Yen, Meng-Chi; Chen, Wei-Ching; Fang, Jung-Hua; Weng, Tzu-Yang; Chen, Yi-Ling

    2015-01-01

    Gastric cancer metastasis remains a major cause of cancer-related deaths. There is an urgent need to develop new therapeutic approaches targeting metastatic gastric cancer. Argininosuccinate synthetase 1 (ASS1) expression is increased in gastric cancer. We detected the protein expression of ASS1 in human gastric cancer cell lines (AGS, NCI-N87, and MKN45) and in murine gastric cancer cell lines (3I and 3IB2). We used vector-mediated short hairpin RNA (shRNA) expression to silence ASS1 expression in the MKN45 and 3IB2 cell lines, and analyzed the effects of this protein on cell migration and metastasis. We demonstrated that ASS1 silencing suppressed cell migration in the MKN45 and 3IB2 cell lines. ASS1 knockdown significantly reduced liver metastasis in mice after the intrasplenic implantation of 3IB2 cancer cell clones. To determine whether arginine restriction may represent a therapeutic approach to treat gastric cancer, the sensitivity of tumor cells to arginine depletion was determined in gastric cancer cells. Arginine depletion significantly inhibited cell migration in the gastric cancer cell line. The silencing of ASS1 expression in MKN45 and 3IB2 gastric cancer cells markedly decreased STAT3 protein expression. In conclusion, our results indicate that the ASS1 protein is required for cell migration in gastric cancer cell lines. PMID:25928182

  5. Mouse DRG Cell Line with Properties of Nociceptors.

    PubMed

    Doran, Ciara; Chetrit, Jonathan; Holley, Matthew C; Grundy, David; Nassar, Mohammed A

    2015-01-01

    In vitro cell lines from DRG neurons aid drug discovery because they can be used for early stage, high-throughput screens for drugs targeting pain pathways, with minimal dependence on animals. We have established a conditionally immortal DRG cell line from the Immortomouse. Using immunocytochemistry, RT-PCR and calcium microfluorimetry, we demonstrate that the cell line MED17.11 expresses markers of cells committed to the sensory neuron lineage. Within a few hours under differentiating conditions, MED17.11 cells extend processes and following seven days of differentiation, express markers of more mature DRG neurons, such as NaV1.7 and Piezo2. However, at least at this time-point, the nociceptive marker NaV1.8 is not expressed, but the cells respond to compounds known to excite nociceptors, including the TRPV1 agonist capsaicin, the purinergic receptor agonist ATP and the voltage gated sodium channel agonist, veratridine. Robust calcium transients are observed in the presence of the inflammatory mediators bradykinin, histamine and norepinephrine. MED17.11 cells have the potential to replace or reduce the use of primary DRG culture in sensory, pain and developmental research by providing a simple model to study acute nociception, neurite outgrowth and the developmental specification of DRG neurons. PMID:26053673

  6. Mouse DRG Cell Line with Properties of Nociceptors

    PubMed Central

    Doran, Ciara; Chetrit, Jonathan; Holley, Matthew C.; Grundy, David; Nassar, Mohammed A.

    2015-01-01

    In vitro cell lines from DRG neurons aid drug discovery because they can be used for early stage, high-throughput screens for drugs targeting pain pathways, with minimal dependence on animals. We have established a conditionally immortal DRG cell line from the Immortomouse. Using immunocytochemistry, RT-PCR and calcium microfluorimetry, we demonstrate that the cell line MED17.11 expresses markers of cells committed to the sensory neuron lineage. Within a few hours under differentiating conditions, MED17.11 cells extend processes and following seven days of differentiation, express markers of more mature DRG neurons, such as NaV1.7 and Piezo2. However, at least at this time-point, the nociceptive marker NaV1.8 is not expressed, but the cells respond to compounds known to excite nociceptors, including the TRPV1 agonist capsaicin, the purinergic receptor agonist ATP and the voltage gated sodium channel agonist, veratridine. Robust calcium transients are observed in the presence of the inflammatory mediators bradykinin, histamine and norepinephrine. MED17.11 cells have the potential to replace or reduce the use of primary DRG culture in sensory, pain and developmental research by providing a simple model to study acute nociception, neurite outgrowth and the developmental specification of DRG neurons. PMID:26053673

  7. Combined Use of Etomidate and Dexmedetomidine Produces an Additive Effect in Inhibiting the Secretion of Human Adrenocortical Hormones

    PubMed Central

    Gu, Hongbin; Zhang, Mazhong; Cai, Meihua; Liu, Jinfen

    2015-01-01

    Background The direct effects of etomidate were investigated on the secretion of cortisol and its precursors by dispersed cells from the adrenal cortex of human of animals. Dexmedetomidine (DEX) is an anesthetic agent that may interfere with cortisol secretion via an unknown mechanism, such as involving inhibition of 11β-hydroxylase and the cholesterol side-chain cleavage enzyme system. The aim of this study was to determine whether dexmedetomidine (DEX) has a similar inhibitory effect on adrenocortical function, and whether combined use of etomidate (ETO) and DEX could produce a synergistic action in inhibiting the secretion of human adrenocortical hormones. Material/Methods Human adrenocortical cells were exposed to different concentrations of ETO and DEX. The dose-effect model between the ETO concentration and the mean secretion of cortisone (CORT) and aldosterone (ALDO) per hour was estimated. Results Hill’s equation well-described the dose-effect correlation between the ETO concentration and the amount of ALDO and CORT secretion. When the DEX concentration was introduced into the model by using E0 (basal secretion) as the covariate, the goodness of fit of the ETO-CORT dose-effect model was improved significantly and the objective function value was reduced by 4.55 points (P<0.05). The parameters of the final ETO-ALDO pharmacodynamics model were EC50=9.74, Emax=1.20, E0=1.33, and γ=18.5; the parameters of the final ETO-CORT pharmacodynamics model were EC50=9.49, Emax=8.16, E0=8.57, and γ=37.0. In the presence of DEX, E0 was 8.57–0.0247×(CDEX–4.6), and the other parameters remained unchanged. All parameters but γ were natural logarithm conversion values. Conclusions Combined use of DEX and ETO reduced ETO’s inhibitory E0 (basal secretion) of CORT from human adrenocortical cells in a dose-dependent manner, suggesting that combined use of ETO and DEX produced an additive effect in inhibiting the secretion of human adrenocortical hormones. PMID

  8. Dengue-2 virus infection of human mononuclear cell lines and establishment of persistent infections.

    PubMed

    Kurane, I; Kontny, U; Janus, J; Ennis, F A

    1990-01-01

    Twenty three human mononuclear cell lines including ten myelomonocytic cell lines, eight B cell lines and five T cell lines, were examined to determine whether they could be infected with dengue-2 virus. All the cell lines were infected with dengue-2 virus as determined by immunofluorescent staining and by virus titration of culture supernatant fluids. K562, Jiyoye and Jurkat, respectively, showed the highest percentage of infected cells of these myelomonocytic, B and T cell lines. Antibody to dengue-2 virus at subneutralizing concentrations augmented dengue-2 virus infection of myelomonocytic cell lines, but not of B cell lines or of T cell lines. Persistent dengue-2 virus infection was established using a myelomonocytic cell line (K562), a B cell line (Raji), and a T cell line (HSB-2). These cell lines maintained a high percentage (more than 70%) of dengue-2 virus antigen-positive cells for at least 25 weeks. Very low titers of infectious dengue-2 virus were detected in the culture supernatant fluids of the persistently infected cells. Dengue-2 virus antigen-positive Raji cell clones were established from persistently-infected Raji cells using limiting dilutions and all of the cells in these clones were dengue-2 virus antigen-positive. These findings demonstrate that a variety of human mononuclear cell lines can be infected with dengue-2 virus and may be useful as models for the analysis of dengue virus-human cell interactions in dengue virus infections.

  9. Cytolytic activity against tumor cells by macrophage cell lines and augmentation by macrophage stimulants.

    PubMed

    Taniyama, T; Holden, H T

    1980-07-15

    Previous studies have shown that macrophage cell lines retained the ability to phagocytize, to secrete lysosomal enzymes, and to function as effector cells in antibody-dependent cellular cytoxicity. In this paper, the cytolytic activity of murine macrophage cell lines against tumor target cells was assessed using an 18-h 51Cr release assay. Of the macrophage cell lines tested, RAW 264, PU5-1.8 and IC-21 had intermediate to high levels of spontaneous cytolytic activity, P388D, and J774 had low to intermediate levels, while /WEHI-3 showed little or no cytolytic activity against RBL-5, MBL-2 and TU-5 target cells. Tumor-cell killing by macrophage cell lines could be augmented by the addition of macrophage stimulants, such as bacterial lipopolysaccharide and poly I:C, indicating that the activation of macrophages by these stimulants does not require the participation of other cell types. Treatment with interferon also augmented the tumor-cell killing by macrophage cell lines. Although the mechanism by which these cell lines exert their spontaneous or boosted cytotoxic activity is not clear, it does not appear to be due to depletion of nutrients since cell lines with high metabolic and proliferative activities, such as WEHI-3 and RBL-5, showed little or no cytotoxicity and supernatants from the macrophage cell lines did not exert any cytotoxic effects in their essay. Thus, it appears that the different macrophage cell lines represent different levels of activation and/or differentiation and may be useful for studying the development of these processes as well as providing a useful tool for analyzing the mechanisms of macrophage-mediated cytolysis. PMID:6165690

  10. Characterization of hepatocellular carcinoma cell lines based on cell adhesion molecules.

    PubMed

    Jung, Cheol Woong; Song, Tae-Jin; Lee, Kun-Ok; Choi, Sae Byeol; Kim, Wan Bae; Suh, Sung Ock; Kim, Young Chul; Choi, Sang Yong

    2012-06-01

    Many studies which focus on the molecules and mechanisms related to the characteristics of the cancer have been performed. In particular, cell adhesion molecules (CAMs) are known to play a central role in the adhesion of cancer cells to vascular endothelial cells. In this study, the expression of CAMs in hepatocellular carcinoma (HCC) cell lines was analyzed and correlated with the characteristics of various HCC cell lines. Eight human HCC cell lines were used in this study. We analyzed the expression of ICAM-1, E-selectin and the integrin subunits of HCC cell lines by western blot analysis and ELISA kit. We estimated the expression of integrin-α5 using western blot analysis and RT-PCR to compare the expression at the gene level with the protein level. In addition, we determined the expression of TGF-β1, as one of the markers for the cellular activity compared to the levels of expression with the expression of integrin-α3 and -α5. ICAM-1 was highly expressed in all of the cell lines except SNU398 and Hep3B, which exhibit a more aggressive nature among the studied HCC cell lines. E-selectin and integrin subunits varied in all HCC cell lines. In particular, integrin-β2 was highly expressed on all HCC cell lines. In conclusion, the levels of expression of the CAMs may not affect cellular activity, morphology or tumorigenicity. However, most HCC cell lines show various expressions of CAMs, suggesting that HCC cell lines expressing the major CAMs remain candidates for molecular targeted therapy, which may need to be patient-tailored for therapy according to the molecular profile.

  11. Hypoxic cell turnover in different solid tumor lines

    SciTech Connect

    Ljungkvist, Anna S.E. . E-mail: a.ljungkvist@rther.umcn.nl; Bussink, Johan; Kaanders, Johannes H.A.M.; Rijken, Paulus F.J.W.; Begg, Adrian C.; Raleigh, James A.; Kogel, Albert J. van der

    2005-07-15

    Purpose: Most solid tumors contain hypoxic cells, and the amount of tumor hypoxia has been shown to have a negative impact on the outcome of radiotherapy. The efficacy of combined modality treatments depends both on the sequence and timing of the treatments. Hypoxic cell turnover in tumors may be important for optimal scheduling of combined modality treatments, especially when hypoxic cell targeting is involved. Methods and Materials: Previously we have shown that a double bioreductive hypoxic marker assay could be used to detect changes of tumor hypoxia in relation to the tumor vasculature after carbogen and hydralazine treatments. This assay was used in the current study to establish the turnover rate of hypoxic cells in three different tumor models. The first hypoxic marker, pimonidazole, was administered at variable times before tumor harvest, and the second hypoxic marker, CCI-103F, was injected at a fixed time before harvest. Hypoxic cell turnover was defined as loss of pimonidazole (first marker) relative to CCI-103F (second marker). Results: The half-life of hypoxic cell turnover was 17 h in the murine C38 colon carcinoma line, 23 h and 49 h in the human xenograft lines MEC82 and SCCNij3, respectively. Within 24 h, loss of pimonidazole-stained areas in C38 and MEC82 occurred concurrent with the appearance of pimonidazole positive cell debris in necrotic regions. In C38 and MEC82, most of the hypoxic cells had disappeared after 48 h, whereas in SCCNij3, viable cells that had been labeled with pimonidazole were still observed after 5 days. Conclusions: The present study demonstrates that the double hypoxia marker assay can be used to study changes in both the proportion of hypoxic tumor cells and their lifespan at the same time. The present study shows that large differences in hypoxic cell turnover rates may exist among tumor lines, with half-lives ranging from 17-49 h.

  12. Cell membrane fatty acid composition differs between normal and malignant cell lines.

    PubMed

    Meng, Xialong; Riordan, Neil H; Riordan, Hugh D; Mikirova, Nina; Jackson, James; González, Michael J; Miranda-Massari, Jorge R; Mora, Edna; Trinidad Castillo, Waleska

    2004-06-01

    Twenty-eight fatty acids (C8:0 to C24:l n-9) were measured by gas chromatography in four normal cell lines (C3H / 10T1 / 2, CCD-18Co, CCD-25SK and CCD-37Lu) and seven cancer cell lines (C-41, Caov-3, LS-180, PC-3, SK-MEL-28, SK-MES-1 and U-87 MG). Results show differences in the content and proportions of fatty acids when comparing cancer cell lines with their normal counterparts. Cancer cell lines showed lower C20: 4 n-6, C24:1 n-9, polyunsaturated fatty acids (PUFA's) and ratios of C20:4 n-6 to C20:5 n-3 and C16:0 to C18:1 n-9 and stearic to oleic (SA/OA) than their normal counterparts. All cancer cell lines had SA/OA ratios lower than 7.0 while normal cell lines had ratios greater than 0.7 (p<0.05). In addition, the ratios of total saturated fatty acids (SFA) to PUFA'S and the concentration of C18:1 n-9, C18:2 n-6, C20:5 n-3 were higher in cancer cell lines as compared to normal cell lines. A positive correlation was detected between C16:0 and longer SFA'S (r = +0.511, p<0.05) in normal cell lines whereas a negative correlation (r=0.608, p<0.05) was obtained for malignant cell lines. Moreover, cancerous cell lines exhibited a particular desaturation defect and an abnormal incorporation of C18:2 n-6 and C20-4 n-6 fatty acids. PMID:15377057

  13. UOK 268 Cell Line for Hereditary Leiomyomatosis and Renal Cell Carcinoma | NCI Technology Transfer Center | TTC

    Cancer.gov

    The National Cancer Institute’s Urologic Oncology Branch seeks parties to co-develop the UOK 262 immortalized cell line as research tool to study aggressive hereditary leiomyomatosis and renal cell carcinoma (HLRCC)-associated recurring kidney cancer.

  14. Comparative proteome analysis across non-small cell lung cancer cell lines.

    PubMed

    Grundner-Culemann, Kathrin; Dybowski, J Nikolaj; Klammer, Martin; Tebbe, Andreas; Schaab, Christoph; Daub, Henrik

    2016-01-01

    Non-small cell lung cancer (NSCLC) cell lines are widely used model systems to study molecular aspects of lung cancer. Comparative and in-depth proteome expression data across many NSCLC cell lines has not been generated yet, but would be of utility for the investigation of candidate targets and markers in oncogenesis. We employed a SILAC reference approach to perform replicate proteome quantifications across 23 distinct NSCLC cell lines. On average, close to 4000 distinct proteins were identified and quantified per cell line. These included many known targets and diagnostic markers, indicating that our proteome expression data represents a useful resource for NSCLC pre-clinical research. To assess proteome diversity within the NSCLC cell line panel, we performed hierarchical clustering and principal component analysis of proteome expression data. Our results indicate that general proteome diversity among NSCLC cell lines supersedes potential effects common to K-Ras or epidermal growth factor receptor (EGFR) oncoprotein expression. However, we observed partial segregation of EGFR or KRAS mutant cell lines for certain principal components, which reflected biological differences according to gene ontology enrichment analyses. Moreover, statistical analysis revealed several proteins that were significantly overexpressed in KRAS or EGFR mutant cell lines. PMID:26361996

  15. YPEL4 modulates HAC15 adrenal cell proliferation and is associated with tumor diameter.

    PubMed

    Oki, Kenji; Plonczynski, Maria W; Gomez-Sanchez, Elise P; Gomez-Sanchez, Celso E

    2016-10-15

    Yippee-like (YPEL) proteins are thought to be related to cell proliferation because of their structure and location in the cell. The aim of this study was to clarify the effects of YPEL4 on aldosterone production and cell proliferation in the human adrenocortical cell line (HAC15) and aldosterone producing adenoma (APA). Basal aldosterone levels in HAC15 cells over-expressing YPEL4 was higher than those of control HAC15 cells. The positive effects of YPEL4 on cell proliferation were detected by XTT assay and crystal violet staining. YPEL4 levels in 39 human APA were 2.4-fold higher compared to those in 12 non-functional adrenocortical adenomas, and there was a positive relationship between YPEL4 levels and APA diameter (r = 0.316, P < 0.05). In summary, we have demonstrated that YPEL4 stimulates human adrenal cortical cell proliferation, increasing aldosterone production as a consequence. These results in human adrenocortical cells are consistent with the clinical observations with APA in humans. PMID:27333825

  16. 76 FR 16609 - Proposed Information Collection; Comment Request; Identification of Human Cell Lines Project

    Federal Register 2010, 2011, 2012, 2013, 2014

    2011-03-24

    ...; Identification of Human Cell Lines Project AGENCY: National Institute of Standards and Technology (NIST...) profiling up to 1500 human cell line samples as part of the Identification of Human Cell Lines Project. All... for Biotechnology Information (NCBI) and will be used to differentiate among cell lines, as...

  17. Cryopreservation of specialized chicken lines using cultured primordial germ cells.

    PubMed

    Nandi, S; Whyte, J; Taylor, L; Sherman, A; Nair, V; Kaiser, P; McGrew, M J

    2016-08-01

    Biosecurity and sustainability in poultry production requires reliable germplasm conservation. Germplasm conservation in poultry is more challenging in comparison to other livestock species. Embryo cryopreservation is not feasible for egg-laying animals, and chicken semen conservation has variable success for different chicken breeds. A potential solution is the cryopreservation of the committed diploid stem cell precursors to the gametes, the primordial germ cells ( PGCS: ). Primordial germ cells are the lineage-restricted cells found at early embryonic stages in birds and form the sperm and eggs. We demonstrate here, using flocks of partially inbred, lower-fertility, major histocompatibility complex- ( MHC-: ) restricted lines of chicken, that we can easily derive and cryopreserve a sufficient number of independent lines of male and female PGCs that would be sufficient to reconstitute a poultry breed. We demonstrate that germ-line transmission can be attained from these PGCs using a commercial layer line of chickens as a surrogate host. This research is a major step in developing and demonstrating that cryopreserved PGCs could be used for the biobanking of specialized flocks of birds used in research settings. The prospective application of this technology to poultry production will further increase sustainability to meet current and future production needs. PMID:27099306

  18. Whole-genome sequencing of nine esophageal adenocarcinoma cell lines.

    PubMed

    Contino, Gianmarco; Eldridge, Matthew D; Secrier, Maria; Bower, Lawrence; Fels Elliott, Rachael; Weaver, Jamie; Lynch, Andy G; Edwards, Paul A W; Fitzgerald, Rebecca C

    2016-01-01

    Esophageal adenocarcinoma (EAC) is highly mutated and molecularly heterogeneous. The number of cell lines available for study is limited and their genome has been only partially characterized. The availability of an accurate annotation of their mutational landscape is crucial for accurate experimental design and correct interpretation of genotype-phenotype findings. We performed high coverage, paired end whole genome sequencing on eight EAC cell lines-ESO26, ESO51, FLO-1, JH-EsoAd1, OACM5.1 C, OACP4 C, OE33, SK-GT-4-all verified against original patient material, and one esophageal high grade dysplasia cell line, CP-D. We have made available the aligned sequence data and report single nucleotide variants (SNVs), small insertions and deletions (indels), and copy number alterations, identified by comparison with the human reference genome and known single nucleotide polymorphisms (SNPs). We compare these putative mutations to mutations found in primary tissue EAC samples, to inform the use of these cell lines as a model of EAC. PMID:27594985

  19. Cryopreservation of specialized chicken lines using cultured primordial germ cells

    PubMed Central

    Nandi, S.; Whyte, J.; Taylor, L.; Sherman, A.; Nair, V.; Kaiser, P.; McGrew, M. J.

    2016-01-01

    Biosecurity and sustainability in poultry production requires reliable germplasm conservation. Germplasm conservation in poultry is more challenging in comparison to other livestock species. Embryo cryopreservation is not feasible for egg-laying animals, and chicken semen conservation has variable success for different chicken breeds. A potential solution is the cryopreservation of the committed diploid stem cell precursors to the gametes, the primordial germ cells (PGCs). Primordial germ cells are the lineage-restricted cells found at early embryonic stages in birds and form the sperm and eggs. We demonstrate here, using flocks of partially inbred, lower-fertility, major histocompatibility complex- (MHC-) restricted lines of chicken, that we can easily derive and cryopreserve a sufficient number of independent lines of male and female PGCs that would be sufficient to reconstitute a poultry breed. We demonstrate that germ-line transmission can be attained from these PGCs using a commercial layer line of chickens as a surrogate host. This research is a major step in developing and demonstrating that cryopreserved PGCs could be used for the biobanking of specialized flocks of birds used in research settings. The prospective application of this technology to poultry production will further increase sustainability to meet current and future production needs. PMID:27099306

  20. DIVERSITY OF ARSENIC METABOLISM IN CULTURED HUMAN CANCER CELL LINES

    EPA Science Inventory

    Diversity of arsenic metabolism in cultured human cancer cell lines.

    Arsenic has been known to cause a variety of malignancies in human. Pentavalent As (As 5+) is reduced to trivalent As (As3+) which is further methylated by arsenic methyltransferase(s) to monomethylarson...

  1. METHYLATION OF ARSENITE BY SOME MAMMALIAN CELL LINES

    EPA Science Inventory

    THIS ABSTRACT WAS SUBMITTED ELECTRONICALLY;. SPACE CONSTRAINTS WERE SEVERE)

    Methylation of Arsenite by Some Mammalian Cell Lines.

    Methylation of arsenite is thought to play an important role in the carcinogenicity of arsenic.
    Aim 1: Determine if there is diffe...

  2. Analysis of tumour cell composition in tumours composed of paired mixtures of mammary tumour cell lines.

    PubMed Central

    Miller, B. E.; Miller, F. R.; Wilburn, D. J.; Heppner, G. H.

    1987-01-01

    In order to quantitate the effects of tumour subpopulation interactions, we have devised a method to determine the subpopulation composition of tumours by using paired tumour cell lines able to grow in different selective media. Line 4T07 forms colonies in thioguanine but not in HAT and line 168 forms colonies in HAT but not in thioguanine. An independent technique of determining tumour cell content was used to validate this method: line 168 and 4T07 cells are distinguishable by flow cytometry after staining with propidium iodide for DNA content. Mixtures of cell suspensions prepared from each unmixed tumour, as well as from tumours arising from mixtures of these lines, were analysed by both the colony formation assay and by the DNA content assay. The colony formation assay yielded values in good agreement with the DNA content assay, but was considerably more sensitive in that it was able to quantitate minority subpopulations that constituted less than 10% of the tumour. Both methods revealed that in tumours arising from mixtures, the tumour cells were almost entirely line 4T07, even when the inoculum had contained a high proportion of 168 cells. Since line 168 cells are very tumorigenic per se, these results suggest that line 4T07 cells are capable of interfering with 168 proliferation in mixed tumours, either directly or through a host-mediated mechanism. PMID:3426919

  3. 9-{beta}-arabinofuranosyladenine preferentially sensitizes radioresistant squamous cell carcinoma cell lines to x-rays

    SciTech Connect

    Heaton, D.; Mustafi, R.; Schwartz, J.L. |

    1992-06-01

    The effect of 9-{beta}-arabinofuranosyladenine (ara-A) on sensitivity to the deleterious effects of x-rays was studied in six squamous cell carcinoma cell lines. Three lines were relatively radioresistant, having D{sub 0} values of 2.31 to 2.89 Gy, and the other three lines were relatively radiosensitive, having D{sub 0} values of between 1.07 and 1.45 Gy. Ara-A (50 or 500 {mu}M) was added to cultures 30 min prior to irradiation and removed 30 min after irradiation, and sensitivity was measured in terms of cell survival. The radiosensitizing effect of ara-A was very dependent on the inherent radiosensitivity of the tumor cell line. Fifty micromolar concentrations of ara-A sensitized only the two most radioresistant lines, SCC-12B.2 and JSQ-3. Five hundred micromolar concentrations of ara-A sensitized the more sensitive cell lines, SQ-20B and SQ-9G, but failed to have any effect on the radiation response of the two most sensitive cell lines, SQ-38 and SCC-61. Concentrations of ara-A as low as 10 {mu}M were equally efficient in inhibiting DNA synthesis in all six cell lines. These results suggest that the target for the radiosensitizing effect of ara-A is probably related to the factor controlling the inherent radiosensitivity of human tumor cells. Therefore, ara-A might be useful in overcoming radiation resistance in vivo.

  4. Cytotoxic effects of Euterpe oleracea Mart. in malignant cell lines

    PubMed Central

    2014-01-01

    Background Euterpe oleracea Mart., a plant from the Amazon region, is commonly known as açaí or juçara; it has high nutritional value and elevated levels of lipids, proteins, and minerals. Açaí is an abundant and much consumed fruit by the Amazon local population, and studies have demonstrated that it is rich in phytochemicals with antioxidant, anti-inflammatory, and anticancer activities. Therefore, the aim of this study was to test this plant for anticancer activity in different human malignant cell lines. Methods Cell lines derived from breast and colorectal adenocarcinomas were treated with 10, 20, and 40 μg/mL of bark, seed, and total açaí fruit hydroalcoholic extracts for 24 and 48 h. After treatment, cell viability was measured using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays, and cell morphological features were observed by light and transmission electron microscopy. The type of cell death was also evaluated. The data were analyzed statistically by one-way analysis of variance (ANOVA), followed by Dunnett’s or Tukey’s post hoc tests, as appropriate. Results We observed that of all the cell lines tested, MCF-7 was the only line that responded to açaí treatment. The extracts caused significant reduction (p < 0.01) in cell viability and altered cell morphological features by inducing the appearance of autophagic vacuoles, as observed by transmission electron microscopy. Furthermore, increased expression of LC3BII, a protein marker of autophagosome formation, was observed by western blotting. Caspase Glo™ assays and morphologic observations by DAPI nuclear staining and transmission electron microscopy did not indicate any apoptotic events. Conclusions The present study demonstrated that açaí possesses antitumorigenic potential in the MCF-7 cell line. Further studies are needed to identify the compound (s) responsible for this cytotoxic activity and the molecular target in the cell. This discovery of the

  5. Mitochondrial DNA determines androgen dependence in prostate cancer cell lines

    PubMed Central

    Higuchi, M; Kudo, T; Suzuki, S; Evans, TT; Sasaki, R; Wada, Y; Shirakawa, T; Sawyer, JR; Gotoh, A

    2008-01-01

    Prostate cancer progresses from an androgen-dependent to androgen-independent stage after androgen ablation therapy. Mitochondrial DNA plays a role in cell death and metastatic competence. Further, heteroplasmic large-deletion mitochondrial DNA is verycommon in prostate cancer. To investigate the role of mitochondrial DNA in androgen dependence of prostate cancers, we tested the changes of normal and deleted mitochondrial DNA in accordance with the progression of prostate cancer. We demonstrated that the androgen-independent cell line C4-2, established byinoculation of the androgen-dependent LNCaP cell line into castrated mice, has a greatlyreduced amount of normal mitochondrial DNA and an accumulation of large-deletion DNA. Strikingly, the depletion of mitochondrial DNA from androgen-dependent LNCaP resulted in a loss of androgen dependence. Reconstitution of normal mitochondrial DNA to the mitochondrial DNA-depleted clone restored androgen dependence. These results indicate that mitochondrial DNA determines androgen dependence of prostate cancer cell lines. Further, mitochondrial DNA-deficient cells formed tumors in castrated athymic mice, whereas LNCaP did not. The accumulation of large deletion and depletion of mitochondrial DNA maythus playa role in the development of androgen independence, leading to progression of prostate cancers. PMID:16278679

  6. Cytotoxic effect of Plantago spp. on cancer cell lines.

    PubMed

    Gálvez, Marina; Martín-Cordero, Carmen; López-Lázaro, Miguel; Cortés, Felipe; Ayuso, María Jesús

    2003-10-01

    Methanolic extracts from seven Plantago species used in traditional medicine for the treatment of cancer, were evaluated for cytotoxic activity against three human cancer cell lines recommended by the National Cancer Institute (NCI, USA). The results showed that Plantago species exhibited cytotoxic activity, showing a certain degree of selectivity against the tested cells in culture. Since the flavonoids are able to strongly inhibit the proliferation of human cancer cell lines, we have identified luteolin-7-O-beta-glucoside as major flavonoid present in most of the Plantago species. Also, we have evaluated this compound and its aglycon, luteolin, for their cytotoxic and DNA topoisomerase I poisons activities. These results could justify the traditional use of the Plantago species and topoisomerase-mediated DNA damage might be a possible mechanism by which flavonoids of Plantago exert their cytotoxicity potential.

  7. Plasmids and packaging cell lines for use in phage display

    DOEpatents

    Bradbury, Andrew M.

    2012-07-24

    The invention relates to a novel phagemid display system for packaging phagemid DNA into phagemid particles which completely avoids the use of helper phage. The system of the invention incorporates the use of bacterial packaging cell lines which have been transformed with helper plasmids containing all required phage proteins but not the packaging signals. The absence of packaging signals in these helper plasmids prevents their DNA from being packaged in the bacterial cell, which provides a number of significant advantages over the use of both standard and modified helper phage. Packaged phagemids expressing a protein or peptide of interest, in fusion with a phage coat protein such as g3p, are generated simply by transfecting phagemid into the packaging cell line.

  8. Targeted genetic modification of cell lines for recombinant protein production

    PubMed Central

    Piskareva, Olga; Muniyappa, Mohan

    2007-01-01

    Considerable increases in productivity have been achieved in biopharmaceutical production processes over the last two decades. Much of this has been a result of improvements in media formulation and process development. Though advances have been made in cell line development, there remains considerable opportunity for improvement in this area. The wealth of transcriptional and proteomic data being generated currently hold the promise of specific molecular interventions to improve the performance of production cell lines in the bioreactor. Achieving this—particularly for multi-gene modification—will require specific, targeted and controlled genetic manipulation of these cells. This review considers some of the current and potential future techniques that might be employed to realise this goal. PMID:19003191

  9. Patient sues UCLA over patent on cell line.

    PubMed

    Culliton, B J

    1984-09-28

    A patient's lawsuit against the University of California is raising questions about an individual's rights of ownership in relation to body tissues that have been turned over for biomedical research but are subsequently used commercially. In 1976, John Moore had his spleen removed at the University of California, Los Angeles, in connection with his leukemia treatment. The university and researchers David Golde and Shirley Quan recently received a patent on the biologically interesting Mo cell line, which was derived from Moore's spleen cells. Moore's suit claims that the university misappropriated his tissues and that the researchers failed to obtain a valid informed consent because they did not formally tell him about the potential commercial applications of the cell line.

  10. Cytotoxic effect of Plantago spp. on cancer cell lines.

    PubMed

    Gálvez, Marina; Martín-Cordero, Carmen; López-Lázaro, Miguel; Cortés, Felipe; Ayuso, María Jesús

    2003-10-01

    Methanolic extracts from seven Plantago species used in traditional medicine for the treatment of cancer, were evaluated for cytotoxic activity against three human cancer cell lines recommended by the National Cancer Institute (NCI, USA). The results showed that Plantago species exhibited cytotoxic activity, showing a certain degree of selectivity against the tested cells in culture. Since the flavonoids are able to strongly inhibit the proliferation of human cancer cell lines, we have identified luteolin-7-O-beta-glucoside as major flavonoid present in most of the Plantago species. Also, we have evaluated this compound and its aglycon, luteolin, for their cytotoxic and DNA topoisomerase I poisons activities. These results could justify the traditional use of the Plantago species and topoisomerase-mediated DNA damage might be a possible mechanism by which flavonoids of Plantago exert their cytotoxicity potential. PMID:12963131

  11. Over-expression of secreted proteins from mammalian cell lines

    PubMed Central

    Dalton, Annamarie C; Barton, William A

    2014-01-01

    Secreted mammalian proteins require the development of robust protein over-expression systems for crystallographic and biophysical studies of protein function. Due to complex disulfide bonds and distinct glycosylation patterns preventing folding and expression in prokaryotic expression hosts, many secreted proteins necessitate production in more complex eukaryotic expression systems. Here, we elaborate on the methods used to obtain high yields of purified secreted proteins from transiently or stably transfected mammalian cell lines. Among the issues discussed are the selection of appropriate expression vectors, choice of signal sequences for protein secretion, availability of fusion tags for enhancing protein stability and purification, choice of cell line, and the large-scale growth of cells in a variety of formats. PMID:24510886

  12. Derivation of Human Skin Fibroblast Lines for Feeder Cells of Human Embryonic Stem Cells.

    PubMed

    Unger, Christian; Felldin, Ulrika; Rodin, Sergey; Nordenskjöld, Agneta; Dilber, Sirac; Hovatta, Outi

    2016-01-01

    After the first derivations of human embryonic stem cell (hESC) lines on fetal mouse feeder cell layers, the idea of using human cells instead of mouse cells as feeder cells soon arose. Mouse cells bear a risk of microbial contamination, and nonhuman immunogenic proteins are absorbed from the feeders to hESCs. Human skin fibroblasts can be effectively used as feeder cells for hESCs. The same primary cell line, which can be safely used for up to 15 passages after stock preparations, can be expanded and used for large numbers of hESC derivations and cultures. These cells are relatively easy to handle and maintain. No animal facilities or animal work is needed. Here, we describe the derivation, culture, and cryopreservation procedures for research-grade human skin fibroblast lines. We also describe how to make feeder layers for hESCs using these fibroblasts. PMID:26840224

  13. Ellipticine cytotoxicity to cancer cell lines — a comparative study

    PubMed Central

    Stiborová, Marie; Poljaková, Jitka; Martínková, Eva; Bořek-Dohalská, Lucie; Eckschlager, Tomáš; Kizek, Rene; Frei, Eva

    2011-01-01

    Ellipticine is a potent antineoplastic agent exhibiting multiple mechanisms of action. This anticancer agent should be considered a pro-drug, whose pharmacological efficiency and/or genotoxic side effects are dependent on its cytochrome P450 (CYP)- and/or peroxidase-mediated activation to species forming covalent DNA adducts. Ellipticine can also act as an inhibitor or inducer of biotransformation enzymes, thereby modulating its own metabolism leading to its genotoxic and pharmacological effects. Here, a comparison of the toxicity of ellipticine to human breast adenocarcinoma MCF-7 cells, leukemia HL-60 and CCRF-CEM cells, neuroblastoma IMR-32, UKF-NB-3 and UKF-NB-4 cells and U87MG glioblastoma cells and mechanisms of its action to these cells were evaluated. Treatment of all cells tested with ellipticine resulted in inhibition of cell growth and proliferation. This effect was associated with formation of two covalent ellipticine-derived DNA adducts, identical to those formed by 13-hydroxy- and 12-hydroxyellipticine, the ellipticine metabolites generated by CYP and peroxidase enzymes, in MCF-7, HL-60, CCRF-CEM, UKF-NB-3, UKF-NB-4 and U87MG cells, but not in neuroblastoma UKF-NB-3 cells. Therefore, DNA adduct formation in most cancer cell lines tested in this comparative study might be the predominant cause of their sensitivity to ellipticine treatment, whereas other mechanisms of ellipticine action also contribute to its cytotoxicity to neuroblastoma UKF-NB-3 cells. PMID:21753906

  14. Highly efficient site-specific transgenesis in cancer cell lines

    PubMed Central

    2012-01-01

    Background Transgenes introduced into cancer cell lines serve as powerful tools for identification of genes involved in cancer. However, the random nature of genomic integration site of a transgene highly influences the fidelity, reliability and level of its expression. In order to alleviate this bottleneck, we characterized the potential utility of a novel PhiC31 integrase-mediated site-specific insertion system (PhiC31-IMSI) for introduction of transgenes into a pre-inserted docking site in the genome of cancer cells. Methods According to this system, a “docking-site” was first randomly inserted into human cancer cell lines and clones with a single copy were selected. Subsequently, an “incoming” vector containing the gene of interest was specifically inserted in the docking-site using PhiC31. Results Using the Pc-3 and SKOV-3 cancer cell lines, we showed that transgene insertion is reproducible and reliable. Furthermore, the selection system ensured that all surviving stable transgenic lines harbored the correct integration site. We demonstrated that the expression levels of reporter genes, such as green fluorescent protein and luciferase, from the same locus were comparable among sister, isogenic clones. Using in vivo xenograft studies, we showed that the genetically altered cancer cell lines retain the properties of the parental line. To achieve temporal control of transgene expression, we coupled our insertion strategy with the doxycycline inducible system and demonstrated tight regulation of the expression of the antiangiogenic molecule sFlt-1-Fc in Pc-3 cells. Furthermore, we introduced the luciferase gene into the insertion cassette allowing for possible live imaging of cancer cells in transplantation assays. We also generated a series of Gateway cloning-compatible intermediate cassettes ready for high-throughput cloning of transgenes and demonstrated that PhiC31-IMSI can be achieved in a high throughput 96-well plate format. Conclusions The novel

  15. Carbon nanoparticles for gene transfection in eukaryotic cell lines.

    PubMed

    Zanin, H; Hollanda, L M; Ceragioli, H J; Ferreira, M S; Machado, D; Lancellotti, M; Catharino, R R; Baranauskas, V; Lobo, A O

    2014-06-01

    For the first time, oxygen terminated cellulose carbon nanoparticles (CCN) was synthesised and applied in gene transfection of pIRES plasmid. The CCN was prepared from catalytic of polyaniline by chemical vapour deposition techniques. This plasmid contains one gene that encodes the green fluorescent protein (GFP) in eukaryotic cells, making them fluorescent. This new nanomaterial and pIRES plasmid formed π-stacking when dispersed in water by magnetic stirring. The frequencies shift in zeta potential confirmed the plasmid strongly connects to the nanomaterial. In vitro tests found that this conjugation was phagocytised by NG97, NIH-3T3 and A549 cell lines making them fluorescent, which was visualised by fluorescent microscopy. Before the transfection test, we studied CCN in cell viability. Both MTT and Neutral Red uptake tests were carried out using NG97, NIH-3T3 and A549 cell lines. Further, we use metabolomics to verify if small amounts of nanomaterial would be enough to cause some cellular damage in NG97 cells. We showed two mechanisms of action by CCN-DNA complex, producing an exogenous protein by the transfected cell and metabolomic changes that contributed by better understanding of glioblastoma, being the major finding of this work. Our results suggested that this nanomaterial has great potential as a gene carrier agent in non-viral based therapy, with low cytotoxicity, good transfection efficiency, and low cell damage in small amounts of nanomaterials in metabolomic tests.

  16. Survival of different cell lines in alginate-agarose microcapsules.

    PubMed

    Orive, G; Hernández, R M; Gascón, A R; Igartua, M; Pedraz, J L

    2003-01-01

    Cell microencapsulation has emerged as a promising therapeutic strategy to treat a wide range of diseases. The optimisation of this technology depends on several critical issues such as the careful selection of the cell line, the controlled manufacture of microcapsules and the suitable adaptation of the construct design to the selected cell line. In this work, we studied the behavior of hybridoma cells once enclosed in solid and liquefied core alginate-agarose beads. Results show that hybridoma cells presented a better growing pattern and improved their viability and antibody production within liquefied beads. However, when these beads were evaluated with a compression resistance study, they were found to be mechanically more fragile than solid ones. To address this problem, we entrapped non-autologous cells (BHK fibroblast and C2C12 myoblast) in solid alginate-agarose beads and observed that they showed an improved growing profile and prolonged their viability up to 70 days in comparison to the 15 days seen for the hybridoma cells.

  17. Characterization of a transformed rat retinal ganglion cell line.

    PubMed

    Krishnamoorthy, R R; Agarwal, P; Prasanna, G; Vopat, K; Lambert, W; Sheedlo, H J; Pang, I H; Shade, D; Wordinger, R J; Yorio, T; Clark, A F; Agarwal, N

    2001-01-31

    The purpose of the present study was to establish a rat retinal ganglion cell line by transformation of rat retinal cells. For this investigation, retinal cells were isolated from postnatal day 1 (PN1) rats and transformed with the psi2 E1A virus. In order to isolate retinal ganglion cells (RGC), single cell clones were chosen at random from the transformed cells. Expression of Thy-1 (a marker for RGC), glial fibrillary acidic protein (GFAP, a positive marker for Muller cells), HPC-1/syntaxin (a marker for amacrine cells), 8A1 (a marker for horizontal and ganglion cells) and neurotrophins was studied using reverse transcriptase-polymerase chain reaction (RT-PCR), immunoblotting and immunocytochemistry. One of the retinal cell clones, designated RGC-5, was positive for Thy-1, Brn-3C, Neuritin, NMDA receptor, GABA-B receptor, and synaptophysin expression and negative for GFAP, HPC-1, and 8A1, suggesting that it represented a putative RGC clone. The results of RT-PCR analysis were confirmed by immunocytochemistry for Thy-1 and GFAP. Upon further characterization by immunoblotting, the RGC-5 clone was positive for Thy-1, negative for GFAP, 8A1 and syntaxin. RGC 5 cells were also positive for the expression of neurotrophins and their cognate receptors. To establish the physiological relevance of RGC-5, the effects of serum/trophic factor deprivation and glutamate toxicity were analyzed to determine if these cells would undergo apoptosis. The protective effects of neurotrophins on RGC-5 after serum deprivation was also investigated. Apoptosis was studied by terminal deoxynucleotidyl transferase-mediated fluoresceinated dUTP nick end labeling (TUNEL). Serum deprivation resulted in apoptosis and supplementation with both BDNF and NT-4 in the growth media, protected the RGC-5 cells from undergoing apoptosis. On differentiation with succinyl concanavalin A (sConA), RGC-5 cells became sensitive to glutamate toxicity, which could be reversed by inclusion of ciplizone (MK801

  18. Pubertal outcome in a female with virilizing adrenocortical carcinoma

    PubMed Central

    Breidbart, Emily; Cameo, Tamara; Garvin, James H.; Hibshoosh, Hanina

    2016-01-01

    Adrenocortical tumors are neoplasms that rarely occur in pediatric patients. Adrenocortical carcinoma (ACC) is even more uncommon, and is an aggressive malignancy with 5-year survival of 55% in a registry series. There is a lack of information on long-term endocrine outcome in survivors. We describe a 10-year follow-up in a patient who presented at 3 years 5 months with a 1-year history of axillary odor and 6 months’ history of pubic hair development with an increased clitoral size. Androgen levels were increased and a pelvic sonogram revealed a suprarenal mass of the left kidney. The tumor was successfully removed. At 6 years 11 months, androgen levels increased again. Workup for tumor recurrence was negative and the findings likely represented early adrenarche. The patient had menarche at an appropriate time and attained a height appropriate for her family. PMID:26812773

  19. Gallium-67 uptake by a benign adrenocortical adenoma

    SciTech Connect

    Jackson, J.A.; Naul, L.G.; Montgomery, J.L.; Carpentier, W.R.; Roberts, J.W.

    1988-08-01

    A 55-yr-old man presented with an atypical relapsing meningitis and was found to have intense unilateral adrenal uptake by /sup 67/Ga imaging. Computed tomography showed a 4-cm right adrenal mass which was hypointense on the T1-weighted images and mildly hyperintense on the T2-weighted images of a magnetic resonance (MR) scan. At surgery, a coincidental benign adrenocortical adenoma was found. Because /sup 67/Ga uptake is usually associated with inflammatory or malignant lesions and malignant adrenal lesions are hyperintense on T2-weighted MR images, these findings contributed to diagnostic uncertainty in this patient. Thus, a nonhyperfunctional adrenocortical adenoma may be associated with abnormal /sup 67/Ga uptake and atypical MR findings.

  20. Cecal perforation and adrenocortical adenoma in a dog.

    PubMed

    Moore, M P; Robinette, J D

    1987-07-01

    Cecal perforation was diagnosed in a dog with a history of acute vomiting. The dog also had an adrenocortical adenoma. Intestinal perforation can be a serious complication of cortico-steroid treatment in the dog, but has not been attributable to hyperadrenocorticism. Fever and an inflammatory CBC were not observed, which could have been secondary to adrenal-dependent hyperadrenocorticism. The acute abdominal crisis associated with peritonitis required quick resolution in an attempt to save the dog, but also precluded any further diagnostic procedures for possible hyperadrenocorticism. The signs that suggested hyper-adrenocorticism in this dog included alopecia, lymphopenia, eosinopenia, high liver enzyme activities, hypercholesterolemia, and one large and one small adrenal gland. This latter finding presumably indicated negative feedback suppression and atrophy attributable to a functional adrenocortical adenoma.

  1. Bioenergetic analysis of ovarian cancer cell lines: profiling of histological subtypes and identification of a mitochondria-defective cell line.

    PubMed

    Dier, Usawadee; Shin, Dong-Hui; Hemachandra, L P Madhubhani P; Uusitalo, Larissa M; Hempel, Nadine

    2014-01-01

    Epithelial ovarian cancer (EOC) is the most lethal of all gynecological cancers, and encompasses distinct histological subtypes that have specific genetic and tissues-of-origin differences. Ovarian clear cell carcinoma (OCCC) represents approximately 10% of cases and has been termed a stress responsive cancer. OCCC is characterized by increased expression of oxidative stress and glycolysis-related genes. In the present study, we hypothesized that bioenergetic profiling might uniquely distinguish OCCC from other EOC histological subtypes. Using an extracellular flux analyzer, OCCC lines (ES-2, TOV-21-G) were shown to be highly metabolically active, with high oxygen consumption rate (OCR) and high extracellular acidification rate (ECAR), indicative of enhanced mitochondrial oxidative phosphorylation and glycolytic rate, respectively. A high bioenergetics profile was associated with the cell lines' ability to form anchorage independent spheroids. Given their high glycolytic and mitochondrial activity, OCCC cells displayed strong sensitivity to 2-deoxy-D-glucose and Rotenone growth inhibition, although this chemosensitivity profile was not specific to only OCCC cells. Bioenergetic profiling also identified a non-OCCC cell line, OVCA420, to have severely compromised mitochondrial function, based on low OCR and a lack of stimulation of maximal respiration following application of the uncoupler FCCP. This was accompanied by mitochondrial morphology changes indicative of enhanced fission, increased expression of the mitochondrial fission protein Drp1, a loss of mitochondrial membrane potential and dependence on glycolysis. Importantly, this loss of mitochondrial function was accompanied by the inability of OVCA420 cells to cope with hypoxic stress, and a compromised ability to stabilize HIF-1α in response to 1% O2 hypoxia. This knowledge may be imperative for researchers planning to utilize this cell line for further studies of metabolism and hypoxia, and suggests that

  2. Apoptosis inhibitor of macrophage (AIM) reduces cell number in canine histiocytic sarcoma cell lines

    PubMed Central

    UCHIDA, Mona; SAEKI, Kohei; MAEDA, Shingo; TAMAHARA, Satoshi; YONEZAWA, Tomohiro; MATSUKI, Naoaki

    2016-01-01

    Apoptosis inhibitor of macrophage (AIM) is initially reported to protect macrophages from apoptosis. In this study, we determined the effect of AIM on the macrophage-derived tumor, histiocytic sarcoma cell lines (HS) of dogs. Five HS and five other tumor cell lines were used. When recombinant canine AIM was applied to non-serum culture media, cell numbers of all the HS and two of other tumor cell lines decreased dose-dependently. The DNA fragmentation, TUNEL staining and flow cytometry tests revealed that AIM induced both of apoptosis and cell cycle arrest in the HS. Although AIM is known as an apoptosis inhibitor, these results suggest that a high dose of AIM could have an opposite function in HS and some tumor cell lines. PMID:27246397

  3. Plurihormonal Cosecretion by a Case of Adrenocortical Oncocytic Neoplasm.

    PubMed

    Corrales, J J; Robles-Lázaro, C; Sánchez-Marcos, A I; González-Sánchez, M C; Antúnez-Plaza, P; Miralles, J M

    2016-01-01

    Adrenocortical oncocytic neoplasms (oncocytomas) are extremely rare; only approximately 159 cases have been described so far. The majority are nonfunctional and benign. We describe an unusual case of a functional oncocytoma secreting an excess of glucocorticoids (cortisol) and androgens (androstenedione and DHEAS), a pattern of plurihormonal cosecretion previously not reported in men, presenting with endocrine manifestations of Cushing's syndrome. The neoplasm was considered to be of uncertain malignant potential (borderline) according to the Lin-Weiss-Bisceglia criteria. PMID:27413559

  4. Plurihormonal Cosecretion by a Case of Adrenocortical Oncocytic Neoplasm

    PubMed Central

    Corrales, J. J.; Robles-Lázaro, C.; Sánchez-Marcos, A. I.; González-Sánchez, M. C.; Antúnez-Plaza, P.; Miralles, J. M.

    2016-01-01

    Adrenocortical oncocytic neoplasms (oncocytomas) are extremely rare; only approximately 159 cases have been described so far. The majority are nonfunctional and benign. We describe an unusual case of a functional oncocytoma secreting an excess of glucocorticoids (cortisol) and androgens (androstenedione and DHEAS), a pattern of plurihormonal cosecretion previously not reported in men, presenting with endocrine manifestations of Cushing's syndrome. The neoplasm was considered to be of uncertain malignant potential (borderline) according to the Lin-Weiss-Bisceglia criteria. PMID:27413559

  5. Beta-cell gene expression and functional characterisation of the human insulinoma cell line CM.

    PubMed

    Baroni, M G; Cavallo, M G; Mark, M; Monetini, L; Stoehrer, B; Pozzilli, P

    1999-04-01

    Animal insulinoma cell lines are widely used to study physiological and pathophysiological mechanisms involved in glucose metabolism and to establish in vitro models for studies on beta-cells. In contrast, human insulinoma cell lines are rarely used because of difficulties in obtaining and culturing them for long periods. The aim of our study was to investigate, under different experimental conditions, the capacity of the human insulinoma cell line CM to retain beta-cell function, particularly the expression of constitutive beta-cell genes (insulin, the glucose transporters GLUT1 and GLUT2, glucokinase), intracellular and secreted insulin, beta-cell granules, and cAMP content. Results showed that CM cells from an early-passage express specific beta-cell genes in response to glucose stimulation, in particular the insulin and GLUT genes. Such capacity is lost at later passages when cells are cultured at standard glucose concentrations. However, if cultured at lower glucose concentration (0.8 mM) for a longer time, CM cells re-acquire the capacity to respond to glucose stimulation, as shown by the increased expression of beta-cell genes (insulin, GLUT2, glucokinase). Nonetheless, insulin secretion could not be restored under such experimental conditions despite the presence of intracellular insulin, although cAMP response to a potent activator of adenylate cyclase, forskolin, was present indicating a viable system. In conclusion, these data show that the human insulinoma cell line CM, at both early-passage and late-passage, posseses a functional glucose-signalling pathway and insulin mRNA expression similar to normal beta-cells, representing, therefore, a good model for studies concerning the signalling and expression of beta-cells. Furthermore, we have previously shown that it is also a good model for immunological studies. In this respect it is important to note that the CM cell line is one of the very few existing human beta-cell lines in long-term culture.

  6. Recycling of 5'-nucleotidase in a rat hepatoma cell line.

    PubMed Central

    van den Bosch, R A; du Maine, A P; Geuze, H J; van der Ende, A; Strous, G J

    1988-01-01

    Intracellular movement of cell surface 5'-nucleotidase was studied in H4S cells, a rat hepatoma cell line. Surface labelled cells were incubated for various periods at 37 degrees C and treated with neuraminidase at 0 degrees C. Removal of sialic acid residues from glycoproteins results in a change of their isoelectric points. Analysis with isoelectric focusing was then used to distinguish between cell surface and intracellular 5'-nucleotidase. Incubation of 125I-surface-labelled cells at 37 degrees C resulted in a gradual decrease of labelled 5'-nucleotidase at the plasma membrane until, at 60 to 90 min, a steady state was reached with 52% of the label on the cell surface and 48% intracellular. Pretreatment of the cells with the weak base primaquine had no influence on this distribution while at the same time uptake of iron via the transferrin receptor was inhibited. Using immunoelectron microscopy 5'-nucleotidase was found on the cell surface, in multivesicular endosomes and the Golgi complex. Preincubation of the cells in the presence of cycloheximide caused a reduction of labelling in the Golgi complex, whereas the label in the other compartments was retained. These results lead to the conclusion that 5'-nucleotidase does not recycle through the Golgi complex and that in contrast to the transferrin receptor the recycling of 5'-nucleotidase is not inhibited by primaquine. Images PMID:2850162

  7. Whole-genome sequencing of nine esophageal adenocarcinoma cell lines

    PubMed Central

    Contino, Gianmarco; Eldridge, Matthew D.; Secrier, Maria; Bower, Lawrence; Fels Elliott, Rachael; Weaver, Jamie; Lynch, Andy G.; Edwards, Paul A.W.; Fitzgerald, Rebecca C.

    2016-01-01

    Esophageal adenocarcinoma (EAC) is highly mutated and molecularly heterogeneous. The number of cell lines available for study is limited and their genome has been only partially characterized. The availability of an accurate annotation of their mutational landscape is crucial for accurate experimental design and correct interpretation of genotype-phenotype findings. We performed high coverage, paired end whole genome sequencing on eight EAC cell lines—ESO26, ESO51, FLO-1, JH-EsoAd1, OACM5.1 C, OACP4 C, OE33, SK-GT-4—all verified against original patient material, and one esophageal high grade dysplasia cell line, CP-D. We have made available the aligned sequence data and report single nucleotide variants (SNVs), small insertions and deletions (indels), and copy number alterations, identified by comparison with the human reference genome and known single nucleotide polymorphisms (SNPs). We compare these putative mutations to mutations found in primary tissue EAC samples, to inform the use of these cell lines as a model of EAC. PMID:27594985

  8. Whole-genome sequencing of nine esophageal adenocarcinoma cell lines

    PubMed Central

    Contino, Gianmarco; Eldridge, Matthew D.; Secrier, Maria; Bower, Lawrence; Fels Elliott, Rachael; Weaver, Jamie; Lynch, Andy G.; Edwards, Paul A.W.; Fitzgerald, Rebecca C.

    2016-01-01

    Esophageal adenocarcinoma (EAC) is highly mutated and molecularly heterogeneous. The number of cell lines available for study is limited and their genome has been only partially characterized. The availability of an accurate annotation of their mutational landscape is crucial for accurate experimental design and correct interpretation of genotype-phenotype findings. We performed high coverage, paired end whole genome sequencing on eight EAC cell lines—ESO26, ESO51, FLO-1, JH-EsoAd1, OACM5.1 C, OACP4 C, OE33, SK-GT-4—all verified against original patient material, and one esophageal high grade dysplasia cell line, CP-D. We have made available the aligned sequence data and report single nucleotide variants (SNVs), small insertions and deletions (indels), and copy number alterations, identified by comparison with the human reference genome and known single nucleotide polymorphisms (SNPs). We compare these putative mutations to mutations found in primary tissue EAC samples, to inform the use of these cell lines as a model of EAC.

  9. Fibronectin synthesized by a human hepatoma cell line

    SciTech Connect

    Glasgow, J.E.; Colman, R.W.

    1984-07-01

    Fibronectin is a family of immunologically similar glycoproteins which mediate a variety of cell-cell and cell-substratum interactions. It is a constituent of the extracellular matrix of connective tissue and circulates in plasma. When suspension and adherent cultures of a human hepatoma cell line (SK-HEP-1) were incubated in serum-free medium, the resulting conditioned medium contained material which was specifically immunoprecipitated by antisera to human plasma fibronectin. By double immunodiffusion, a component in the conditioned culture medium was shown to form a line of identity with fibronectin in human plasma and to migrate as an alpha 2- to beta-globulin during immunoelectrophoresis. Human fibronectin was quantified in conditioned medium by electroimmunodiffusion, and was found to increase for at least three days at about 0.1 micrograms/10(6) cells/day. Adherent cultures of SK-HEP-1 cells were incubated with L-(/sup 35/S)methionine to label newly synthesized proteins. Labeled fibronectin in conditioned medium or in cell extracts comigrated with fibronectin in human plasma as shown by autoradiography following crossed-immunoelectrophoresis. Fibronectin was demonstrated in the extra-cellular matrix of adherent SK-HEP-1 cultures by immunofluorescence. It was shown previously that SK-HEP-1 cells synthesize alpha 1-protease inhibitor, one of the products of normal hepatocytes. The finding that these hepatoma cells also synthesize fibronectin supports the concept that the hepatocyte may be one source of circulating fibronectin, a possibility consistent with the established role of this cell type in blood plasma protein synthesis.

  10. Cysteine modified polyaniline films improve biocompatibility for two cell lines.

    PubMed

    Yslas, Edith I; Cavallo, Pablo; Acevedo, Diego F; Barbero, César A; Rivarola, Viviana A

    2015-06-01

    This work focuses on one of the most exciting application areas of conjugated conducting polymers, which is cell culture and tissue engineering. To improve the biocompatibility of conducting polymers we present an easy method that involves the modification of the polymer backbone using l-cysteine. In this publication, we show the synthesis of polyaniline (PANI) films supported onto Polyethylene terephthalate (PET) films, and modified using cysteine (PANI-Cys) in order to generate a biocompatible substrate for cell culture. The PANI-Cys films are characterized by Fourier Transform infrared and UV-visible spectroscopy. The changes in the hydrophilicity of the polymer films after and before the modification were tested using contact angle measurements. After modification the contact angle changes from 86°±1 to 90°±1, suggesting a more hydrophylic surface. The adhesion properties of LM2 and HaCaT cell lines on the surface of PANI-Cys films in comparison with tissue culture plastic (TCP) are studied. The PANI-Cys film shows better biocompatibility than PANI film for both cell lines. The cell morphologies on the TCP and PANI-Cys film were examined by florescence and Atomic Force Microscopy (AFM). Microscopic observations show normal cellular behavior when PANI-Cys is used as a substrate of both cell lines (HaCaT and LM2) as when they are cultured on TCP. The ability of these PANI-Cys films to support cell attachment and growth indicates their potential use as biocompatible surfaces and in tissue engineering.

  11. Mouse Models Recapitulating Human Adrenocortical Tumors: What Is Lacking?

    PubMed

    Leccia, Felicia; Batisse-Lignier, Marie; Sahut-Barnola, Isabelle; Val, Pierre; Lefrançois-Martinez, A-Marie; Martinez, Antoine

    2016-01-01

    Adrenal cortex tumors are divided into benign forms, such as primary hyperplasias and adrenocortical adenomas (ACAs), and malignant forms or adrenocortical carcinomas (ACCs). Primary hyperplasias are rare causes of adrenocorticotropin hormone-independent hypercortisolism. ACAs are the most common type of adrenal gland tumors and they are rarely "functional," i.e., producing steroids. When functional, adenomas result in endocrine disorders, such as Cushing's syndrome (hypercortisolism) or Conn's syndrome (hyperaldosteronism). By contrast, ACCs are extremely rare but highly aggressive tumors that may also lead to hypersecreting syndromes. Genetic analyses of patients with sporadic or familial forms of adrenocortical tumors (ACTs) led to the identification of potentially causative genes, most of them being involved in protein kinase A (PKA), Wnt/β-catenin, and P53 signaling pathways. Development of mouse models is a crucial step to firmly establish the functional significance of candidate genes, to dissect mechanisms leading to tumors and endocrine disorders, and in fine to provide in vivo tools for therapeutic screens. In this article, we will provide an overview on the existing mouse models (xenografted and genetically engineered) of ACTs by focusing on the role of PKA and Wnt/β-catenin pathways in this context. We will discuss the advantages and limitations of models that have been developed heretofore and we will point out necessary improvements in the development of next generation mouse models of adrenal diseases. PMID:27471492

  12. Mouse Models Recapitulating Human Adrenocortical Tumors: What Is Lacking?

    PubMed Central

    Leccia, Felicia; Batisse-Lignier, Marie; Sahut-Barnola, Isabelle; Val, Pierre; Lefrançois-Martinez, A-Marie; Martinez, Antoine

    2016-01-01

    Adrenal cortex tumors are divided into benign forms, such as primary hyperplasias and adrenocortical adenomas (ACAs), and malignant forms or adrenocortical carcinomas (ACCs). Primary hyperplasias are rare causes of adrenocorticotropin hormone-independent hypercortisolism. ACAs are the most common type of adrenal gland tumors and they are rarely “functional,” i.e., producing steroids. When functional, adenomas result in endocrine disorders, such as Cushing’s syndrome (hypercortisolism) or Conn’s syndrome (hyperaldosteronism). By contrast, ACCs are extremely rare but highly aggressive tumors that may also lead to hypersecreting syndromes. Genetic analyses of patients with sporadic or familial forms of adrenocortical tumors (ACTs) led to the identification of potentially causative genes, most of them being involved in protein kinase A (PKA), Wnt/β-catenin, and P53 signaling pathways. Development of mouse models is a crucial step to firmly establish the functional significance of candidate genes, to dissect mechanisms leading to tumors and endocrine disorders, and in fine to provide in vivo tools for therapeutic screens. In this article, we will provide an overview on the existing mouse models (xenografted and genetically engineered) of ACTs by focusing on the role of PKA and Wnt/β-catenin pathways in this context. We will discuss the advantages and limitations of models that have been developed heretofore and we will point out necessary improvements in the development of next generation mouse models of adrenal diseases. PMID:27471492

  13. Design of a stable cell line producing a recombinant monoclonal anti-TNFα antibody based on a CHO cell line.

    PubMed

    Voronina, E V; Seregin, Y A; Litvinova, N A; Shvets, V I; Shukurov, R R

    2016-01-01

    Recombinant monoclonal antibodies (mAbs) against tumor necrosis factor alpha are widely used in the biopharmaceutical therapy of autoimmune diseases. Currently, a large number of drugs based on these antibodies are available. Accordingly, the development of these products for the Russian market is an important goal. The aim of the current study is to describe the development of one such technology. CHO-DG44-derived cell lines producing mAb were developed using two strategies, one based on individual clones and the other based on cell pools. To obtain recombinant cell lines with highly amplified genes of interest, the clones underwent dihydrofolate reductase-mediated gene amplification. Using the best strategy for the selection and amplification of mAb-producing clones, we achieved the production of more than 1 g/L in small scale, non-optimized conditions. PMID:27652157

  14. Bovine viral diarrhea virus (BVDV) in cell lines used for somatic cell cloning.

    PubMed

    Stringfellow, David A; Riddell, Kay P; Givens, M Daniel; Galik, Patricia K; Sullivan, Eddie; Dykstra, Christine C; Robl, James; Kasinathan, Poothapillai

    2005-03-01

    Culture of cell lines from fetuses or postnatal animals is an essential part of somatic cell cloning. Fetal bovine serum (FBS) is commonly used in media for propagation of these cells. Unfortunately, bovine fetuses and postnatal animals as well as FBS are all possible sources of non-cytopathic bovine viral diarrhea virus (BVDV) which is widely distributed among cattle. This study was prompted when screening of samples sent to veterinary diagnostic labs revealed that 15 of 39 fetal fibroblast cell lines used in cloning research were positive for BVDV as determined by various assays including reverse transcription-polymerase chain reaction (RT-PCR). Goals of the research were to use both virus isolation and reverse transcription-nested polymerase chain reaction (RT-nPCR) to confirm which of the cell lines were actually infected with BVDV and to assay samples of media, FBS and the earliest available passages of each cell line in an attempt to determine the source of the viral infections. Sequence analysis of amplified cDNA from all isolates was performed to provide a definitive link between possible sources of virus and infected cell lines. Only 5 of the 39 cell lines were actually infected with BVDV. Three of these five lines were not infected at the earliest cryopreserved passage, leading to the conclusion that they likely became infected after culture in media containing contaminated FBS. In fact, sequence comparison of the amplified cDNA from one lot of FBS confirmed that it was the source of infection for one of these cell lines. Since BVDV was isolated from the remaining two cell lines at the earliest available passage, the fetuses from which they were established could not be ruled out as the source of the virus.

  15. Interaction of a mouse macrophage cell line with homologous erythrocytes.

    PubMed

    Singer, J A; Walker, W S; Morrison, M

    1982-06-01

    The interaction of the IC-21 murine macrophage cell line and homologous red blood cells (RBC) was assessed in the absence of exogenous opsonins. These results were used to evaluate this system as a potential model for macrophage-mediated clearance of old or damaged RBC. The binding and ingestion of density-separated and unseparated RBC by IC-21 cells were quantitated in assays that involved both 51Cr-labeled RBC and direct microscopy. The number of unseparated RBC that bound to IC-21 macrophages depended on the number of RBC added. Macrophages phagocytized an appreciable proportion of RBC within 3 hours with the ratio of RBC:macrophage of 10, a point at which the RBC-binding was not rate limiting. The mouse RBC were separated into dense- and less-dense fractions which are presumably enriched for old and young cells, respectively. When these RBC fractions were incubated with the IC-21 macrophage, significantly more of these dense cells were phagocytized. These results show that IC-21 macrophage cell line is a useful model for defining the processes whereby aged or damaged RBC are recognized and removed from circulation by macrophages. PMID:7120230

  16. Toxicity of Calcium Hydroxide Nanoparticles on Murine Fibroblast Cell Line

    PubMed Central

    Dianat, Omid; Azadnia, Sina; Mozayeni, Mohammad Ali

    2015-01-01

    Introduction: One of the major contributing factors, which may cause failure of endodontic treatment, is the presence of residual microorganisms in the root canal system. For years, most dentists have been using calcium hydroxide (CH) as the intracanal medicament between treatment sessions to eliminate remnant microorganisms. Reducing the size of CH particles into nanoparticles enhances the penetration of this medicament into dentinal tubules and increases their antimicrobial efficacy. This in vitro study aimed to compare the cytotoxicity of CH nanoparticles and conventional CH on fibroblast cell line using the Mosmann’s Tetrazolium Toxicity (MTT) assay. Methods and Materials: This study was conducted on L929 murine fibroblast cell line by cell culture and evaluation of the direct effect of materials on the cultured cells. Materials were evaluated in two groups of 10 samples each at 24, 48 and 72 h. At each time point, 10 samples along with 5 positive and 5 negative controls were evaluated. The samples were transferred into tubes and exposed to fibroblast cells. The viability of cells was then evaluated. The Two-way ANOVA was used for statistical analysis and the level of significance was set at 0.05. Results: Cytotoxicity of both materials decreased over time and for conventional CH was lower than that of nanoparticles. However, this difference was not statistically significant (P>0.05). Conclusion: The cytotoxicity of CH nanoparticles was similar to that of conventional CH. PMID:25598810

  17. Effects of prolonged sodium restriction on the morphology and function of rat adrenocortical autotransplants.

    PubMed

    Belloni, A S; Neri, G; Andreis, P G; Musajo, F G; Boscaro, M; Mazzocchi, G; Nussdorfer, G G

    1991-07-01

    Regenerated adrenocortical nodules were obtained by implanting fragments of the capsular tissue of excised adrenal glands into the musculus gracilis of rats (Belloni et al. 1990). Five months after the operation, operated rats showed a normal basal blood level of corticosterone, but a very low concentration of circulating aldosterone associated with a slightly increased plasma renin activity (PRA). Regenerated nodules were well encapsulated and some septa extended into the parenchyma from the connective-tissue capsule. The majority of parenchymal cells were similar to those of the zonae fasciculata and reticularis of the normal adrenal gland, while zona glomerulosa-like cells were exclusively located around septa (juxta-septal zone; JZ). In vitro studies demonstrated that nodules were functioning as far as glucocorticoid production was concerned, while mineralocorticoid yield was very low. Prolonged sodium restriction significantly increased PRA and plasma aldosterone concentration, and provoked a marked hypertrophy of JZ, which was due to increases in both the number and average volume of JZ cells. Accordingly, the in vitro basal production of aldosterone and other 18-hydroxylated steroids was notably enhanced. The plasma level of corticosterone, as well as zona fasciculata/reticularis-like cells and in vitro production of glucocorticoids by regenerated nodules were not affected. These findings, indicating that autotransplanted adrenocortical nodules respond to a prolonged sodium restriction similar to the normal adrenal glands, suggest that the relative deficit in mineralocorticoid production is not due to an intrinsic defect of the zona glomerulosa-like JZ, but is probably caused by the impairment of its adequate stimulation under basal conditions.(ABSTRACT TRUNCATED AT 250 WORDS)

  18. Methylprednisolone Pulse Treatment of Graves' Ophthalmopathy Is Not Associated with Secondary Adrenocortical Insufficiency

    PubMed Central

    Jespersen, Sofie; Nygaard, Birte; Kristensen, Lars Østergaard

    2015-01-01

    Objective Graves' ophthalmopathy (GO) is an inflammatory disease in the orbital region. The first-line medical treatment is glucocorticoids. An important potential side effect of glucocorticoid treatment is suppression of the hypothalamic-pituitary-adrenal (HPA) axis with impairment of endogenous cortisol production, implicating symptoms of adrenocortical insufficiency, especially in the period after cessation of therapy with possible risks in cases of intercurrent illness. The aim of this study was to evaluate HPA axis function before and after methylprednisolone pulse treatment of GO. Study Design HPA axis function was evaluated by measurements of plasma ACTH and an ACTH stimulation test with plasma cortisol measurements at 0 and 30 min after an intravenous bolus of synthetic ACTH (Synacthen® 250 µg). This was done in 12 patients with GO before and at cessation of methylprednisolone pulse treatment (500 mg i.v. per week for 6 weeks followed by 250 mg i.v. per week for an additional 6 weeks). Results All patients included fulfilled the criteria of intact HPA axis function before and at cessation of methylprednisolone pulse treatment. Data are given as medians (with ranges). Before glucocorticoid treatment basal plasma cortisol was 290 nM (196-579) and 786 nM (612-1,050) after ACTH stimulation. At cessation of therapy the corresponding values were 309 nM (88-718) and 852 nM (524-1,011), respectively. Thus, all patients passed a 30-min stimulated plasma cortisol of 500 nM. Before treatment plasma ACTH was 4.2 pmol/l (4-16) and at cessation of therapy the corresponding value was 4.8 pmol/l (2-9; p = 0.27). Conclusion Transient suppression of the HPA axis with secondary adrenocortical insufficiency does not seem to be a common phenomenon after intravenous methylprednisolone pulse therapy for GO. Therefore, routine precautions are not necessary. However, our results do not exclude that transient secondary adrenocortical insufficiency might occur occasionally. PMID

  19. Bisphosphonates induce apoptosis in human breast cancer cell lines

    PubMed Central

    Senaratne, S G; Pirianov, G; Mansi, J L; Arnett, T R; Colston, K W

    2000-01-01

    Breast cancer has a prodigious capacity to metastasize to bone. In women with advanced breast cancer and bone metastases, bisphosphonates reduce the incidence of hypercalcaemia and skeletal morbidity. Recent clinical findings suggest that some bisphosphonates reduce the tumour burden in bone with a consequent increase in survival, raising the possibility that bisphosphonates may have a direct effect on breast cancer cells. We have investigated the in vitro effects of bisphosphonates zoledronate, pamidronate, clodronate and EB 1053 on growth, viability and induction of apoptosis in three human breast cancer cell lines (MDA-MB-231, Hs 578T and MCF-7). Cell growth was monitored by crystal violet dye assay, and cell viability was quantitated by MTS dye reduction. Induction of apoptosis was determined by identification of morphological features of apoptosis using time-lapse videomicroscopy, identifying morphological changes in nucleis using Hoechst staining, quantitation of DNA fragmentation, level of expression of bcl-2 and bax proteins and identification of the proteolytic cleavage of Poly (ADP)-ribose polymerase (PARP). All four bisphosphonates significantly reduced cell viability in all three cell lines. Zoledronate was the most potent bisphosphonate with IC50values of 15, 20 and 3 μM respectively in MDA-MB-231, MCF-7 and Hs 578T cells. Corresponding values for pamidronate were 40, 35 and 25 μM, whereas clodronate and EB 1053 were more than two orders of magnitude less potent. An increase in the proportion of cells having morphological features characteristic of apoptosis, characteristic apoptotic changes in the nucleus, time-dependent increase in the percentage of fragmented chromosomal DNA, down-regulation in bcl-2 protein and proteolytic cleavage of PARP, all indicate that bisphosphonates have direct anti-tumour effects on human breast cancer cells. © 2000 Cancer Research Campaign PMID:10780527

  20. Identification and Characterization of CD133pos Subpopulation Cells From a Human Laryngeal Cancer Cell Line

    PubMed Central

    Qiu, Hai-ou; Wang, Huifang; Che, Na; Li, Dong; Mao, Yong; Zeng, Qiao; Ge, Rongming

    2016-01-01

    Background Recent research indicates that CD133 are expressed in several kinds of stem cells, among which, its high expression in laryngeal carcinoma has caused wide concern. To further explore efficaciously targeting drugs to laryngeal carcinoma stem cells (CSCs), we transplanted a solid tumor from CSCs into abdominal subcutaneous tissue of nude mice, and then compared the biological characteristics of laryngeal solid tumors with or without cisplatin intervention. Material/Methods In this study, the expression of CD133 was detected in the Hep-2 cell line by flow cytometry. By applying magnetic cell sorting (MACS) technology, we reported the results of purifying CD133-positive cells from a Hep-2 cell line. Cell proliferation, colony formation, and tumor-forming ability were examined in vitro and in vivo to identify the marker of CSCs in Hep-2 cell line. Results Upon flow cytometry analysis, CD133 was expressed constantly on 40.12±1.32% in Hep-2 cell line. Cell proliferation and colony formation ability were higher in CD133-positive cells compared to CD133-negative cells, and the in vivo tumorigenesis experiment showed the same results as in vitro assay. The 2 subpopulations cells were both sensitive to DDP, among which, the effect of DPP on proliferation ability and tumor-forming ability of CD133-positive cells was obviously greater than that of CD133-negative cells. Conclusions Above all, our study revealed that CD133-positive cells have properties of higher proliferation, colony formation, and tumorigenesis in Hep-2 cell line, indicating that CD133 could be a marker to characterize laryngeal cancer stem cells. PMID:27049928

  1. Designing of promiscuous inhibitors against pancreatic cancer cell lines

    NASA Astrophysics Data System (ADS)

    Kumar, Rahul; Chaudhary, Kumardeep; Singla, Deepak; Gautam, Ankur; Raghava, Gajendra P. S.

    2014-04-01

    Pancreatic cancer remains the most devastating disease with worst prognosis. There is a pressing need to accelerate the drug discovery process to identify new effective drug candidates against pancreatic cancer. We have developed QSAR models for predicting promiscuous inhibitors using the pharmacological data. Our models achieved maximum Pearson correlation coefficient of 0.86, when evaluated on 10-fold cross-validation. Our models have also successfully validated the drug-to-oncogene relationship and further we used these models to screen FDA approved drugs and tested them in vitro. We have integrated these models in a webserver named as DiPCell, which will be useful for screening and designing novel promiscuous drug molecules. We have also identified the most and least effective drugs for pancreatic cancer cell lines. On the other side, we have identified resistant pancreatic cancer cell lines, which need investigative scanner on them to put light on resistant mechanism in pancreatic cancer.

  2. Regulation of alkaline phosphatase expression in human choriocarcinoma cell lines.

    PubMed Central

    Hamilton, T A; Tin, A W; Sussman, H H

    1979-01-01

    The coincident expression of two structurally distinct isoenzymes of human alkaline phosphatase was demonstrated in two independently derived gestational choriocarcinoma cell lines. These proteins were shown to have enzymatic, antigenic, and physical-chemical properties resembling those of isoenzymes from term placenta and adult liver. The regulation of these isoenzymes has been studied during the exposure of both cell lines to 5-bromodeoxyuridine and dibutyryl cyclic AMP. The responses of the alkaline phosphatase isoenzymes to these agents have also been compared with the response of another protein phenotypic to placenta, the alpha subunit of chorionic gonadotropin. The results show that (i) the separate structural genes coding for placental and liver alkaline phosphatases are regulated in a noncoordinate fashion; (ii) both alkaline phosphatase genes respond independently of the alpha subunit; and (iii) the induction of the placental type isoenzyme occurs via at least two independent pathways. Images PMID:218197

  3. Reversal of diabetes following transplantation of an insulin-secreting human liver cell line: Melligen cells.

    PubMed

    Lawandi, Janet; Tao, Chang; Ren, Binhai; Williams, Paul; Ling, Dora; Swan, M Anne; Nassif, Najah T; Torpy, Fraser R; O'Brien, Bronwyn A; Simpson, Ann M

    2015-01-01

    As an alternative to the transplantation of islets, a human liver cell line has been genetically engineered to reverse type 1 diabetes (TID). The initial liver cell line (Huh7ins) commenced secretion of insulin in response to a glucose concentration of 2.5 mmol/l. After transfection of the Huh7ins cells with human islet glucokinase, the resultant Melligen cells secreted insulin in response to glucose within the physiological range; commencing at 4.25 mmol/l. Melligen cells exhibited increased glucokinase enzymatic activity in response to physiological glucose concentrations, as compared with Huh7ins cells. When transplanted into diabetic immunoincompetent mice, Melligen cells restored normoglycemia. Quantitative real-time polymerase chain reaction (qRT-PCR) revealed that both cell lines expressed a range of β-cell transcription factors and pancreatic hormones. Exposure of Melligen and Huh7ins cells to proinflammatory cytokines (TNF-α, IL-1β, and IFN-γ) affected neither their viability nor their ability to secrete insulin to glucose. Gene expression (microarray and qRT-PCR) analyses indicated the survival of Melligen cells in the presence of known β-cell cytotoxins was associated with the expression of NF-κB and antiapoptotic genes (such as BIRC3). This study describes the successful generation of an artificial β-cell line, which, if encapsulated to avoid allograft rejection, may offer a clinically applicable cure for T1D. PMID:26029722

  4. Restoration of WNT4 inhibits cell growth in leukemia-derived cell lines

    PubMed Central

    2013-01-01

    Background WNT signaling pathways are significantly altered during cancer development. Vertebrates possess two classes of WNT signaling pathways: the “canonical” WNT/β-catenin signaling pathway, and the “non-canonical” pathways including WNT/Ca2+ and WNT/Planar cell polarity [PCP] signaling. WNT4 influences hematopoietic progenitor cell expansion and survival; however, WNT4 function in cancer development and the resulting implications for oncogenesis are poorly understood. The aim of this study was twofold: first, to determine the expression of WNT4 in mature peripheral blood cells and diverse leukemia-derived cells including cell lines from hematopoietic neoplasms and cells from patients with leukemia; second, to identify the effect of this ligand on the proliferation and apoptosis of the blast-derived cell lines BJAB, Jurkat, CEM, K562, and HL60. Methods We determined WNT4 expression by quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR) in peripheral blood mononuclear cells (PBMCs) and T- and B-lymphocytes from healthy individuals, as well as from five leukemia-derived cell lines and blasts derived from patients with leukemia. To analyze the effect of WNT4 on cell proliferation, PBMCs and cell lines were exposed to a commercially available WNT4 recombinant human protein. Furthermore, WNT4 expression was restored in BJAB cells using an inducible lentiviral expression system. Cell viability and proliferation were measured by the addition of WST-1 to cell cultures and counting cells; in addition, the progression of the cell cycle and the amount of apoptosis were analyzed in the absence or presence of WNT4. Finally, the expression of WNT-pathway target genes was measured by qRT-PCR. Results WNT4 expression was severely reduced in leukemia-derived cell lines and blasts derived from patients with leukemia. The exposure of cell lines to WNT4 recombinant protein significantly inhibited cell proliferation; inducing WNT4 expression in BJAB

  5. Effect of human procathepsin D on proliferation of human cell lines.

    PubMed

    Vĕtvicka, V; Vĕktvicková, J; Fusek, M

    1994-05-16

    We have used human procathepsin D isolated from supernatant of human breast cancer cell line ZR-75-1 to test its mitogenic activity for a broad spectrum of human-derived cell lines. These cell lines included: breast cancer cell lines ZR-75-1, MDA-MB-436, MBA-MD-483 and MDA-MB-231, B lymphoblastoid cell line Raji, the monocytoid cell line U937, T lymphoblastoid cell line 8402, epitheloid carcinoma cell line HELA, hepatocellular carcinoma cell line Hep G2, breast milk epithelial cell line HBL-100 and angiosarcoma cell line HAEND-1. We have tested the level of proliferation of these cell lines depending on the presence of procathepsin D in the medium. In parallel we have also measured the effect of insulin-like growth factor II under the same experimental conditions. We have found a significant difference between the influence of IGF II and that of procathepsin D. While IGF II promoted in practically the same way the proliferation of all cell lines tested, procathepsin D had a very pronounced effect on breast cancer cell lines only. This finding might help to explain some contradictory results of prognostic significance of procathepsin D in human breast cancer.

  6. Perhexiline maleate toxicity on human liver cell lines.

    PubMed Central

    Le Gall, J Y; Guillouzo, A; Glaise, D; Deugnier, Y; Messner, M; Bourel, M

    1980-01-01

    When added to the culture medium of human liver cell lines, perhexiline maleate induced formation of numerous myeloid bodies containing unicentric or multicentric smooth membranes within a few days. The nine lysosomal enzyme activities studied, except for beta-galactosidase which decreased, remained unchanged. These results indicate that on cultured human liver cells perhexiline maleate has an effect similar to that described on hepatocytes of some patients treated with this drug and suggest that myeloid body formation is not due to impairment of lysosomal enzyme activities. Images Fig. 2 Fig. 3 Fig. 4 Fig. 5 Fig. 6 Fig. 7 Fig. 8 Fig. 9 PMID:7192674

  7. Pseudoislet of hybrid cellular spheroids from commercial cell lines.

    PubMed

    Jo, Y H; Nam, B M; Kim, B Y; Nemeno, J G; Lee, S; Yeo, J E; Yang, W; Park, S H; Kim, Y S; Lee, J I

    2013-10-01

    Investigators conducting diabetes-related research have focused on islet transplantation as a radical therapy for type 1 diabetes mellitus. Pancreatic islet isolation, an essential process, is a very demanding work because of the proteolytic enzymes, species, treatment time, and individual difference. Replacement of primary isolated pancreatic islets must be carried out continuously for various in vitro tests, making primary isolated islets a useful tool for cell transplantation research. Hence, we sought to develop pseudoislets from commercial pancreas-derived cell lines. In this study, we used RIN-5F and RIN-m cells, which secrete insulin, somatostatin, or glucagon. To manufacture hybrid cellular spheroids, the cells were cultured under hanging drop plate and nonadhesive plate methods. We observed that hybrid cellular pseudoislets exhibited an oval shape, with sizes ranging from 590 to 1200 μm. Their morphology was similar to naïve islets. Cell line pseudoislets secreted and expressed insulin, glucagon, and somatostatin, as confirmed by reverse transcriptase polymerase chain reaction, enzyme-linked immunosorbent assay, and immunohistochemistry analyses. Thus, the current artificially manufactured biomimetic pseudoislets resembled pancreatic islets of the endocrine system, appearing as cellular aggregates that secreted insulin, glucagon, and somatostatin. Enhanced immunoisolation techniques may lead to the development of new islet sources for pancreatic transplantation through this pseudoislet strategy.

  8. Impairment of cell cycle progression by aflatoxin B1 in human cell lines.

    PubMed

    Ricordy, R; Gensabella, G; Cacci, E; Augusti-Tocco, G

    2002-05-01

    Aflatoxin B1 is a mycotoxin produced by Aspergillus flavus and Aspergillus parasiticum, which may be present as a food contaminant. It is known to cause acute toxic effects and act as a carcinogenic agent. The carcinogenic action has been related to its ability to form unstable adducts with DNA, which represent possible mutagenic sites. On the other hand, the primary cellular target responsible for its toxic action has not yet been clearly identified. Previous data suggested a possible correlation between cell proliferation and responsiveness to aflatoxin toxicity. These observations led us to investigate the effect of the toxin on cell cycle progression of three human cell lines (HepG2, SK-N-MC and SK-N-SH derived from liver and nervous tissue tumours); they were shown to display different responses to toxin exposure and have different growth kinetics. We performed analysis of the cell cycle, DNA synthesis and expression of p21 and p53 in the presence and absence of the toxin in all cell lines exposed. The results of cell cycle cytofluorometric analysis show significant alterations of cell cycle progression as a result of toxin treatment. In all cell lines exposure to a 24 h toxin treatment causes a dose-dependent accumulation in S phase, however, the ability to recover from impairment to traverse S phase varies in the cell lines under study. SK-N-MC cells appear more prone to resume DNA synthesis when the toxin is removed, while the other two cell lines maintain a significant inhibition of DNA synthesis, as indicated by cytofluorimetry and [(3)H]dTR incorporation. The level of p53 and p21 expression in the three cell lines was examined by western blot analysis and significant differences were detected. The ready resumption of DNA synthesis displayed by SK-N-MC cells could possibly be related to the absence of p53 control of cell cycle progression.

  9. MicroRNA profiles in various hepatocellular carcinoma cell lines

    PubMed Central

    Morishita, Asahiro; Iwama, Hisakazu; Fujihara, Shintaro; Sakamoto, Teppei; Fujita, Koji; Tani, Joji; Miyoshi, Hisaaki; Yoneyama, Hirohito; Himoto, Takashi; Masaki, Tsutomu

    2016-01-01

    Hepatocellular carcinoma (HCC) is one of the most common causes of cancer-associated mortality worldwide. Although surgery is considered the most effective treatment for patients with HCC, its indication is restricted by limited criteria and a high relapse rate following surgery; therefore, systemic chemotherapy is required for patients with advanced-stage HCC to prolong their survival. MicroRNAs (miRNAs) are endogenous non-coding RNAs of 18–22 nucleotides in length. It has been reported that aberrant expression of miRNAs is a feature shared by various types of human cancer. Previous studies have indicated that the modulation of non-coding RNAs, particularly miRNAs, may be a valuable therapeutic target for HCC. The aim of the present study was to elucidate the miRNA profiles associated with differentiation and hepatitis B virus (HBV) infection observed in HCC cell lines. The human Alex, Hep3B, HepG2, HuH1, HuH7, JHH1, JHH2, JHH5, JHH6, HLE, HLF and Li-7 HCC cell lines were used for an miRNA array. Replicate data were analyzed following their classification into: i) Poorly- and well-differentiated human HCC cells and ii) HBV-positive and -negative human HCC cells. Out of the 1,719 miRNAs, 4 were found to be significantly upregulated and 52 significantly downregulated in the poorly-differentiated cells, as compared with the well-differentiated cells. Conversely, in the HBV-positive cells 125 miRNAs were found to be upregulated and 2 downregulated, as compared with the HBV-negative cells. Unsupervised hierarchical clustering analysis with Pearson's correlation revealed that the miRNA expression levels were clustered both together and separately in each group. In conclusion, miRNA profile characterization based on various parameters may be a novel approach to determine the etiology of HCC. PMID:27588118

  10. Responding to hypoxia: lessons from a model cell line.

    PubMed

    Seta, K A; Spicer, Z; Yuan, Y; Lu, G; Millhorn, D E

    2002-08-20

    Mammalian cells require a constant supply of oxygen to maintain adequate energy production, which is essential for maintaining normal function and for ensuring cell survival. Sustained hypoxia can result in cell death. It is, therefore, not surprising that sophisticated mechanisms have evolved that allow cells to adapt to hypoxia. "Oxygen-sensing" is a special phenotype that functions to detect changes in oxygen tension and to transduce this signal into organ system functions that enhance the delivery of oxygen to tissue in various organisms. Oxygen-sensing cells can be segregated into two distinct cell types: those that functionally depolarize (excitable) and those that do not functionally depolarize (nonexcitable) in response to reduced oxygen. Theoretically, excitable cells have all the same signaling capabilities as the nonexcitable cells, but the nonexcitable cells cannot have all the signaling capabilities as excitable cells. A number of signaling pathways have been identified that regulate gene expression during hypoxia. These include the Ca2+-calmodulin pathway, the 3'-5' adenosine monophosphate (cAMP)-protein kinase A (PKA) pathway, the p42 and p44 mitogen-activated protein kinase [(MAPK); also known as the extracellular signal-related kinase (ERK) for ERK1 and ERK2] pathway, the stress-activated protein kinase (SAPK; also known as p38 kinase) pathway, and the phosphatidylinositol 3-kinase (PI3K)-Akt pathway. In this review, we describe hypoxia-induced signaling in the model O2-sensing rat pheochromocytoma (PC12) cell line, the current level of understanding of the major signaling events that are activated by reduced O2, and how these signaling events lead to altered gene expression in both excitable and nonexcitable oxygen-sensing cells. PMID:12189251

  11. Boldine: a potential new antiproliferative drug against glioma cell lines.

    PubMed

    Gerhardt, Daniéli; Horn, Ana Paula; Gaelzer, Mariana Maier; Frozza, Rudimar Luiz; Delgado-Cañedo, Andrés; Pelegrini, Alessandra Luiza; Henriques, Amélia T; Lenz, Guido; Salbego, Christianne

    2009-12-01

    Malignant gliomas are the most common and devastating primary tumors of the central nervous system. Currently no efficient treatment is available. This study evaluated the effect and underlying mechanisms of boldine, an aporphine alkaloid of Peumus boldus, on glioma proliferation and cell death. Boldine decreased the cell number of U138-MG, U87-MG and C6 glioma lines at concentrations of 80, 250 and 500 muM. We observed that cell death caused by boldine was cell-type specific and dose-dependent. Exposure to boldine for 24 h did not activate key mediators of apoptosis. However, it induced alterations in the cell cycle suggesting a G(2)/M arrest in U138-MG cells. Boldine had no toxic effect on non-tumor cells when used at the same concentrations as those used on tumor cells. Based on these results, we speculate that boldine may be a promising compound for evaluation as an anti-cancer agent. PMID:19050827

  12. Rapid micropatterning of cell lines and human pluripotent stem cells on elastomeric membranes.

    PubMed

    Paik, Isha; Scurr, David J; Morris, Bryan; Hall, Graham; Denning, Chris; Alexander, Morgan R; Shakesheff, Kevin M; Dixon, James E

    2012-10-01

    Tissue function during development and in regenerative medicine completely relies on correct cell organization and patterning at micro and macro scales. We describe a rapid method for patterning mammalian cells including human embryonic stem cells (HESCs) and induced pluripotent stem cells (iPSCs) on elastomeric membranes such that micron-scale control of cell position can be achieved over centimeter-length scales. Our method employs surface engineering of hydrophobic polydimethylsiloxane (PDMS) membranes by plasma polymerization of allylamine. Deposition of plasma polymerized allylamine (ppAAm) using our methods may be spatially restricted using a micro-stencil leaving faithful hydrophilic ppAAm patterns. We employed airbrushing to create aerosols which deposit extracellular matrix (ECM) proteins (such as fibronectin and Matrigel™) onto the same patterned ppAAm rich regions. Cell patterns were created with a variety of well characterized cell lines (e.g., NIH-3T3, C2C12, HL1, BJ6, HESC line HUES7, and HiPSC line IPS2). Individual and multiple cell line patterning were also achieved. Patterning remains faithful for several days and cells are viable and proliferate. To demonstrate the utility of our technique we have patterned cells in a variety of configurations. The ability to rapidly pattern cells at high resolution over macro scales should aid future tissue engineering efforts for regenerative medicine applications and in creating in vitro stem cell niches. PMID:22511037

  13. Establishment and characterization of feeder-cell-dependent bovine fetal liver cell lines

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The establishment and initial characterization of bovine fetal liver cell lines is described. Bovine fetal hepatocytes were cultured from the liver of a 34-day bovine fetus by physical disruption of the liver tissue. Released liver cells and clumps of cells were plated on STO feeder layers and wer...

  14. Rapid micropatterning of cell lines and human pluripotent stem cells on elastomeric membranes.

    PubMed

    Paik, Isha; Scurr, David J; Morris, Bryan; Hall, Graham; Denning, Chris; Alexander, Morgan R; Shakesheff, Kevin M; Dixon, James E

    2012-10-01

    Tissue function during development and in regenerative medicine completely relies on correct cell organization and patterning at micro and macro scales. We describe a rapid method for patterning mammalian cells including human embryonic stem cells (HESCs) and induced pluripotent stem cells (iPSCs) on elastomeric membranes such that micron-scale control of cell position can be achieved over centimeter-length scales. Our method employs surface engineering of hydrophobic polydimethylsiloxane (PDMS) membranes by plasma polymerization of allylamine. Deposition of plasma polymerized allylamine (ppAAm) using our methods may be spatially restricted using a micro-stencil leaving faithful hydrophilic ppAAm patterns. We employed airbrushing to create aerosols which deposit extracellular matrix (ECM) proteins (such as fibronectin and Matrigel™) onto the same patterned ppAAm rich regions. Cell patterns were created with a variety of well characterized cell lines (e.g., NIH-3T3, C2C12, HL1, BJ6, HESC line HUES7, and HiPSC line IPS2). Individual and multiple cell line patterning were also achieved. Patterning remains faithful for several days and cells are viable and proliferate. To demonstrate the utility of our technique we have patterned cells in a variety of configurations. The ability to rapidly pattern cells at high resolution over macro scales should aid future tissue engineering efforts for regenerative medicine applications and in creating in vitro stem cell niches.

  15. Preparation of cell lines for single-cell analysis of transcriptional activation dynamics.

    PubMed

    Rafalska-Metcalf, Ilona U; Janicki, Susan M

    2013-01-01

    Imaging molecularly defined regions of chromatin in single living cells during transcriptional activation has the potential to provide new insight into gene regulatory mechanisms. Here, we describe a method for isolating cell lines with multi-copy arrays of reporter transgenes, which can be used for real-time high-resolution imaging of transcriptional activation dynamics in single cells.

  16. Primed Pluripotent Cell Lines Derived from Various Embryonic Origins and Somatic Cells in Pig

    PubMed Central

    Park, Jin-Kyu; Kim, Hye-Sun; Uh, Kyung-Jun; Choi, Kwang-Hwan; Kim, Hyeong-Min; Lee, Taeheon; Yang, Byung-Chul; Kim, Hyun-Jong; Ka, Hak-Hyun; Kim, Heebal; Lee, Chang-Kyu

    2013-01-01

    Since pluripotent embryonic stem cell (ESC) lines were first derived from the mouse, tremendous efforts have been made to establish ESC lines in several domestic species including the pig; however, authentic porcine ESCs have not yet been established. It has proven difficult to maintain an ESC-like state in pluripotent porcine cell lines due to the frequent occurrence of spontaneous differentiation into an epiblast stem cell (EpiSC)-like state during culture. We have been able to derive EpiSC-like porcine ESC (pESC) lines from blastocyst stage porcine embryos of various origins, including in vitro fertilized (IVF), in vivo derived, IVF aggregated, and parthenogenetic embryos. In addition, we have generated induced pluripotent stem cells (piPSCs) via plasmid transfection of reprogramming factors (Oct4, Sox2, Klf4, and c-Myc) into porcine fibroblast cells. In this study, we analyzed characteristics such as marker expression, pluripotency and the X chromosome inactivation status in female of our EpiSC-like pESC lines along with our piPSC line. Our results show that these cell lines demonstrate the expression of genes associated with the Activin/Nodal and FGF2 pathways along with the expression of pluripotent markers Oct4, Sox2, Nanog, SSEA4, TRA 1–60 and TRA 1–81. Furthermore all of these cell lines showed in vitro differentiation potential, the X chromosome inactivation in female and a normal karyotype. Here we suggest that the porcine species undergoes reprogramming into a primed state during the establishment of pluripotent stem cell lines. PMID:23326334

  17. Human Fucci Pancreatic Beta Cell Lines: New Tools to Study Beta Cell Cycle and Terminal Differentiation

    PubMed Central

    Carlier, Géraldine; Maugein, Alicia; Cordier, Corinne; Pechberty, Séverine; Garfa-Traoré, Meriem; Martin, Patrick; Scharfmann, Raphaël; Albagli, Olivier

    2014-01-01

    Regulation of cell cycle in beta cells is poorly understood, especially in humans. We exploited here the recently described human pancreatic beta cell line EndoC-βH2 to set up experimental systems for cell cycle studies. We derived 2 populations from EndoC-βH2 cells that stably harbor the 2 genes encoding the Fucci fluorescent indicators of cell cycle, either from two vectors, or from a unique bicistronic vector. In proliferating non-synchronized cells, the 2 Fucci indicators revealed cells in the expected phases of cell cycle, with orange and green cells being in G1 and S/G2/M cells, respectively, and allowed the sorting of cells in different substeps of G1. The Fucci indicators also faithfully red out alterations in human beta cell proliferative activity since a mitogen-rich medium decreased the proportion of orange cells and inflated the green population, while reciprocal changes were observed when cells were induced to cease proliferation and increased expression of some beta cell genes. In the last situation, acquisition of a more differentiated beta cell phenotype correlates with an increased intensity in orange fluorescence. Hence Fucci beta cell lines provide new tools to address important questions regarding human beta cell cycle and differentiation. PMID:25259951

  18. Cytotoxic Activity of New Acetoxycoumarin Derivatives in Cancer Cell Lines

    PubMed Central

    Musa, Musiliyu A.; Badisa, Veera L. D.; Latinwo, Lekan M.; Cooperwood, John; Sinclair, Andre; Abdullah, Ahkinyala

    2012-01-01

    Background Coumarin and their derivatives are important and useful compounds with diverse pharmacological properties. In the present study, we evaluated the in vitro cytotoxic activity of new acetoxycoumarin derivatives: 4-(7-methoxy-4-methyl-2-oxo-2H-chromen-3-yl)phenyl acetate (1), 4-(1-methyl-3-oxo-3H-benzo[f]chromen-2-yl)phenyl acetate (2), 4-(6-propionamido-4-methyl-2-oxo-2H-chromen-3-yl)phenyl acetate (3), 4-(7-acetoxy-2-oxo-4-phenyl-2H-chromen-3-yl)phenyl acetate (4), 4-(2-oxo-4-phenyl-2H-chromen-3-yl)phenyl acetate (5), 4-(6-bromo-2-oxo-4-phenyl-2H-chromen-3-yl)phenyl acetate (6), 4-(7-(diethylamino)-4-methyl-2-oxo-2H-chromen-3-yl)phenyl acetate (7), 4-(6,8-dibromo-4-methyl-2-oxo-2H-chromen-3-yl)phenyl acetate (8) against A549 human lung cancer, CRL 1548 rat liver cancer and CRL 1439 normal rat liver cells. Materials and Methods The cytotoxic activity was evaluated by crystal violet dye-binding assay. The effect of compounds 5 and 7 on different phases of the cell cycle was determined using flow cytometry. Results In the A549 lung cancer cell line, the 50% lethal dose (LD50) values for compounds 1–4, 6 and 8 were found to be >100 μM while those for 5 and 7 were 89.3 and 48.1 μM, respectively after 48 h treatment. In the CRL 1548 liver cancer cell line, only compound 7 showed toxicity, with an LD50 of 45.1 μM. Compounds 5 and 7 caused different cell phase arrest in lung and liver cancer cell lines. Conclusion The results indicate that 4-(7-(diethylamino)-4-methyl-2-oxo-2H-chromen-3-yl)phenyl acetate (7) had the highest cytotoxic activity in all of the examined cell lines. PMID:21737617

  19. PRKACA: the catalytic subunit of protein kinase A and adrenocortical tumors.

    PubMed

    Berthon, Annabel S; Szarek, Eva; Stratakis, Constantine A

    2015-01-01

    Cyclic-AMP (cAMP)-dependent protein kinase (PKA) is the main effector of cAMP signaling in all tissues. Inactivating mutations of the PRKAR1A gene, coding for the type 1A regulatory subunit of PKA, are responsible for Carney complex and primary pigmented nodular adrenocortical disease (PPNAD). PRKAR1A inactivation and PKA dysregulation have been implicated in various types of adrenocortical pathologies associated with ACTH-independent Cushing syndrome (AICS) from PPNAD to adrenocortical adenomas and cancer, and other forms of bilateral adrenocortical hyperplasias (BAH). More recently, mutations of PRKACA, the gene coding for the catalytic subunit C alpha (Cα), were also identified in the pathogenesis of adrenocortical tumors. PRKACA copy number gain was found in the germline of several patients with cortisol-producing BAH, whereas the somatic Leu206Arg (c.617A>C) recurrent PRKACA mutation was found in as many as half of all adrenocortical adenomas associated with AICS. In vitro analysis demonstrated that this mutation led to constitutive Cα activity, unregulated by its main partners, the PKA regulatory subunits. In this review, we summarize the current understanding of the involvement of PRKACA in adrenocortical tumorigenesis, and our understanding of PKA's role in adrenocortical lesions. We also discuss potential therapeutic advances that can be made through targeting of PRKACA and the PKA pathway. PMID:26042218

  20. PRKACA: the catalytic subunit of protein kinase A and adrenocortical tumors

    PubMed Central

    Berthon, Annabel S.; Szarek, Eva; Stratakis, Constantine A.

    2015-01-01

    Cyclic-AMP (cAMP)-dependent protein kinase (PKA) is the main effector of cAMP signaling in all tissues. Inactivating mutations of the PRKAR1A gene, coding for the type 1A regulatory subunit of PKA, are responsible for Carney complex and primary pigmented nodular adrenocortical disease (PPNAD). PRKAR1A inactivation and PKA dysregulation have been implicated in various types of adrenocortical pathologies associated with ACTH-independent Cushing syndrome (AICS) from PPNAD to adrenocortical adenomas and cancer, and other forms of bilateral adrenocortical hyperplasias (BAH). More recently, mutations of PRKACA, the gene coding for the catalytic subunit C alpha (Cα), were also identified in the pathogenesis of adrenocortical tumors. PRKACA copy number gain was found in the germline of several patients with cortisol-producing BAH, whereas the somatic Leu206Arg (c.617A>C) recurrent PRKACA mutation was found in as many as half of all adrenocortical adenomas associated with AICS. In vitro analysis demonstrated that this mutation led to constitutive Cα activity, unregulated by its main partners, the PKA regulatory subunits. In this review, we summarize the current understanding of the involvement of PRKACA in adrenocortical tumorigenesis, and our understanding of PKA's role in adrenocortical lesions. We also discuss potential therapeutic advances that can be made through targeting of PRKACA and the PKA pathway. PMID:26042218

  1. Adrenocortical response to low-dose ACTH test in female patients with rheumatoid arthritis.

    PubMed

    Radikova, Zofia; Rovensky, Jozef; Vlcek, Miroslav; Penesova, Adela; Kerlik, Jana; Vigas, Milan; Imrich, Richard

    2008-12-01

    Alterations in adrenal steroid production have been suggested in females with rheumatoid arthritis (RA). The aim of the present study was to assess adrenocortical function in RA females. We examined 11 female RA patients (RA: age 30 +/- 2 years, BMI 21.0 +/- 0.7 kg/m(2)) and 10 matched healthy controls (C: age 31 +/- 1 years, BMI 21.6 +/- 0.6 kg/m(2)). Low-dose adrenocorticotropic hormone (ACTH) test (i.v. bolus of 1 microg synthetic ACTH) was performed at 10.00 h with blood sampling every 15 min for 90 min. Cortisol, 17-OH-progesterone (17OHP), androstenedione (ASD), and dehydroepiandrosterone (DHEA) were assayed in plasma. Baseline cortisol levels were higher in RA patients (RA: 385 +/- 38 versus C: 229 +/- 28 nmol/L, P= 0.007). In both study groups, ACTH administration increased all the four steroids measured (P < 0.001). Cortisol response to ACTH administration was diminished in RA patients when compared to controls (Delta(max): 284 +/- 24 in RA versus 424 +/- 31 nmol/L in C, P= 0.002). ACTH-induced maximal rise in plasma DHEA was significantly lower in RA patients when compared to controls (Delta(max): 2.59 +/- 0.68 in RA versus 5.57 +/- 1.25 ng/mL in C, P= 0.015). No significant between-groups differences were found in responses of ASD or 17OHP. The molar ratio of ASD:cortisol was significantly lower (P < 0.05) in RA patients at base line, but did not differ during ACTH test. After ACTH bolus, the cortisol:17OHP ratio decreased significantly in the RA group (P < 0.001), whereas there was no change in the control group. The present results show decreased secretion of cortisol and DHEA in RA patients in response to ACTH, suggesting a subtle HPA hypofunction at the adrenocortical level. PMID:19120158

  2. Establishment of a novel human medulloblastoma cell line characterized by highly aggressive stem-like cells.

    PubMed

    Silva, Patrícia Benites Gonçalves da; Rodini, Carolina Oliveira; Kaid, Carolini; Nakahata, Adriana Miti; Pereira, Márcia Cristina Leite; Matushita, Hamilton; Costa, Silvia Souza da; Okamoto, Oswaldo Keith

    2016-08-01

    Medulloblastoma is a highly aggressive brain tumor and one of the leading causes of morbidity and mortality related to childhood cancer. These tumors display differential ability to metastasize and respond to treatment, which reflects their high degree of heterogeneity at the genetic and molecular levels. Such heterogeneity of medulloblastoma brings an additional challenge to the understanding of its physiopathology and impacts the development of new therapeutic strategies. This translational effort has been the focus of most pre-clinical studies which invariably employ experimental models using human tumor cell lines. Nonetheless, compared to other cancers, relatively few cell lines of human medulloblastoma are available in central repositories, partly due to the rarity of these tumors and to the intrinsic difficulties in establishing continuous cell lines from pediatric brain tumors. Here, we report the establishment of a new human medulloblastoma cell line which, in comparison with the commonly used and well-established cell line Daoy, is characterized by enhanced proliferation and invasion capabilities, stem cell properties, increased chemoresistance, tumorigenicity in an orthotopic metastatic model, replication of original medulloblastoma behavior in vivo, strong chromosome structural instability and deregulation of genes involved in neural development. These features are advantageous for designing biologically relevant experimental models in clinically oriented studies, making this novel cell line, named USP-13-Med, instrumental for the study of medulloblastoma biology and treatment.

  3. Hepatocellular carcinoma cell lines retain the genomic and transcriptomic landscapes of primary human cancers

    PubMed Central

    Qiu, Zhixin; Zou, Keke; Zhuang, Liping; Qin, Jianjie; Li, Hong; Li, Chao; Zhang, Zhengtao; Chen, Xiaotao; Cen, Jin; Meng, Zhiqiang; Zhang, Haibin; Li, Yixue; Hui, Lijian

    2016-01-01

    Hepatocellular carcinoma (HCC) cell lines are useful in vitro models for the study of primary HCCs. Because cell lines acquire additional mutations in culture, it is important to understand to what extent HCC cell lines retain the genetic landscapes of primary HCCs. Most HCC cell lines were established during the last century, precluding comparison between cell lines and primary cancers. In this study, 9 Chinese HCC cell lines with matched patient-derived cells at low passages (PDCs) were established in the defined culture condition. Whole genome analyses of 4 HCC cell lines showed that genomic mutation landscapes, including mutations, copy number alterations (CNAs) and HBV integrations, were highly stable during cell line establishment. Importantly, genetic alterations in cancer drivers and druggable genes were reserved in cell lines. HCC cell lines also retained gene expression patterns of primary HCCs during in vitro culture. Finally, sequential analysis of HCC cell lines and PDCs at different passages revealed their comparable and stable genomic and transcriptomic levels if maintained within proper passages. These results show that HCC cell lines largely retain the genomic and transcriptomic landscapes of primary HCCs, thus laying the rationale for testing HCC cell lines as preclinical models in precision medicine. PMID:27273737

  4. Hepatocellular carcinoma cell lines retain the genomic and transcriptomic landscapes of primary human cancers.

    PubMed

    Qiu, Zhixin; Zou, Keke; Zhuang, Liping; Qin, Jianjie; Li, Hong; Li, Chao; Zhang, Zhengtao; Chen, Xiaotao; Cen, Jin; Meng, Zhiqiang; Zhang, Haibin; Li, Yixue; Hui, Lijian

    2016-06-07

    Hepatocellular carcinoma (HCC) cell lines are useful in vitro models for the study of primary HCCs. Because cell lines acquire additional mutations in culture, it is important to understand to what extent HCC cell lines retain the genetic landscapes of primary HCCs. Most HCC cell lines were established during the last century, precluding comparison between cell lines and primary cancers. In this study, 9 Chinese HCC cell lines with matched patient-derived cells at low passages (PDCs) were established in the defined culture condition. Whole genome analyses of 4 HCC cell lines showed that genomic mutation landscapes, including mutations, copy number alterations (CNAs) and HBV integrations, were highly stable during cell line establishment. Importantly, genetic alterations in cancer drivers and druggable genes were reserved in cell lines. HCC cell lines also retained gene expression patterns of primary HCCs during in vitro culture. Finally, sequential analysis of HCC cell lines and PDCs at different passages revealed their comparable and stable genomic and transcriptomic levels if maintained within proper passages. These results show that HCC cell lines largely retain the genomic and transcriptomic landscapes of primary HCCs, thus laying the rationale for testing HCC cell lines as preclinical models in precision medicine.

  5. PKA catalytic subunit mutations in adrenocortical Cushing's adenoma impair association with the regulatory subunit.

    PubMed

    Calebiro, Davide; Hannawacker, Annette; Lyga, Sandra; Bathon, Kerstin; Zabel, Ulrike; Ronchi, Cristina; Beuschlein, Felix; Reincke, Martin; Lorenz, Kristina; Allolio, Bruno; Kisker, Caroline; Fassnacht, Martin; Lohse, Martin J

    2014-01-01

    We recently identified a high prevalence of mutations affecting the catalytic (Cα) subunit of protein kinase A (PKA) in cortisol-secreting adrenocortical adenomas. The two identified mutations (Leu206Arg and Leu199_Cys200insTrp) are associated with increased PKA catalytic activity, but the underlying mechanisms are highly controversial. Here we utilize a combination of biochemical and optical assays, including fluorescence resonance energy transfer in living cells, to analyze the consequences of the two mutations with respect to the formation of the PKA holoenzyme and its regulation by cAMP. Our results indicate that neither mutant can form a stable PKA complex, due to the location of the mutations at the interface between the catalytic and the regulatory subunits. We conclude that the two mutations cause high basal catalytic activity and lack of regulation by cAMP through interference of complex formation between the regulatory and the catalytic subunits of PKA. PMID:25477193

  6. Adrenocortical carcinoma: An extremely uncommon entity and the role of Immunohistochemistry in its diagnosis.

    PubMed

    Gogoi, G; Baruah, Manash P; Borah, P; Borgohain, M

    2012-12-01

    Adrenocortcal carcinoma is an extremely uncommon entity with an incidence of two in one millionth population. Here we present a 60 year gentleman with pain in abdomen, nausea, and backache, and weight loss. Contrast enhanced computed tomography (CECT) abdomen revealed a heterogenous well defined mass measuring (15 × 10.3 × 13) cm(3) on the left suprarenal region with central necrosis which extended medially up to the midline. Locally, the growth infiltrated the upper pole of left kidney. Initially, the differential diagnosis included that of renal cell carcinoma arising from upper pole of left kidney involving adrenal gland. The patient underwent left radical nephrectomy and left adrenalectomy. Histological evaluation could not differentiate it from of malignant pheochromocytoma, but immunohistochemistry confirmed it as adrenocortical carcinoma. This case highlights the crucial role of immunohistochemistry in establishing the diagnosis like tumors. PMID:23565434

  7. KLN205--a murine lung carcinoma cell line.

    PubMed

    Kaneko, T; LePage, G A; Shnitka, T K

    1980-10-01

    KLN205 cells, a cloned cell line established from the Nettesheim lung carcinoma, grow in various synthetic media such as MEM, Fisher's or Roswell Park Memorial Institute Medium (RPMI) with the addition of 5 to 20% fetal bovine serum (FBS), calf-serum (CS) or horse serum (HS). They grow optimally in minimum Eagle's medium plus nonessential amino acids (NEAA) plus 5 to 10% FBS or HS. The cells are transplantable to DBA/2, BDF1, AKD2F1, and BALB/c, but not to C3H/He or ICR mice. The growth curves, plating efficiency, ultrastructural characteristics, modal number of chromosomes and transplantability to mice of various strains are almost the same for early and late passage of cells passaged in vitro. These parameters for 16th and 36th passages were: doubling time, 31 and 33 hr; plating efficiency, 12.4 +/- 1.2 and 14.6 +/- 2.6%; modal number of chromosomes, 73 and 76; lung colony formation in DBA/2, 50 and 45.9/mouse; and subcutaneous tumor diameter 24.5 and 27.4 mm, respectively. Only the numbers of lung colonies formed in BDF1 mice were different: 24.4/mouse with 16th passage cells, and 10.2/mouse with 36th passage cells. The results suggest that KLN205 is a relatively stable cultured cell line through 36 passages. As was expected, immunosuppression by higher concentrations of triaminolone acetonide (TA) enhanced lung colony formation in BDF1 mice. On the other hand, a low concentration of TA inhibited lung colony formation in DBA/2 mice, which was unexpected. These results suggest that KLN205 offers a model for investigations on metastases to lungs as well as chemotherapy for lung carcinoma.

  8. Single-walled carbon nanohorn (SWNH) aggregates inhibited proliferation of human liver cell lines and promoted apoptosis, especially for hepatoma cell lines

    PubMed Central

    Zhang, Jinqian; Sun, Qiang; Bo, Jian; Huang, Rui; Zhang, Mengran; Xia, Zhenglin; Ju, Lili; Xiang, Guoan

    2014-01-01

    Single-walled carbon nanohorns (SWNHs) may be useful as carriers for anticancer drugs due to their particular structure. However, the interactions between the material itself and cancerous or normal cells have seldom been studied. To address this problem, the effects of raw SWNH material on the biological functions of human liver cell lines were studied. Our results showed that unmodified SWNHs inhibited mitotic entry, growth, and proliferation of human liver cell lines and promoted their apoptosis, especially in hepatoma cell lines. Individual spherical SWNH particles were found inside the nuclei of human hepatoma HepG2 cells and the lysosomes of normal human liver L02 cells, implying that SWNH particles could penetrate into human liver cells_and the different interacted mechanisms on human normal cell lines compared to hepatoma cell lines. Further research on the mechanisms and application in treatment of hepatocellular carcinoma with SWNHs is needed. PMID:24523586

  9. Astaxanthin Inhibits Proliferation of Human Gastric Cancer Cell Lines by Interrupting Cell Cycle Progression

    PubMed Central

    Kim, Jung Ha; Park, Jong-Jae; Lee, Beom Jae; Joo, Moon Kyung; Chun, Hoon Jai; Lee, Sang Woo; Bak, Young-Tae

    2016-01-01

    Background/Aims Astaxanthin is a carotenoid pigment that has antioxidant, antitumoral, and anti-inflammatory properties. In this in vitro study, we investigated the mechanism of anticancer effects of astaxanthin in gastric carcinoma cell lines. Methods The human gastric adenocarcinoma cell lines AGS, KATO-III, MKN-45, and SNU-1 were treated with various concentrations of astaxanthin. A cell viability test, cell cycle analysis, and immunoblotting were performed. Results The viability of each cancer cell line was suppressed by astaxanthin in a dose-dependent manner with significantly decreased proliferation in KATO-III and SNU-1 cells. Astaxanthin increased the number of cells in the G0/G1 phase but reduced the proportion of S phase KATO-III and SNU-1 cells. Phosphorylated extracellular signal-regulated kinase (ERK) was decreased in an inverse dose-dependent correlation with astaxanthin concentration, and the expression of p27kip-1 increased the KATO-III and SNU-1 cell lines in an astaxanthin dose-dependent manner. Conclusions Astaxanthin inhibits proliferation by interrupting cell cycle progression in KATO-III and SNU-1 gastric cancer cells. This may be caused by the inhibition of the phosphorylation of ERK and the enhanced expression of p27kip-1. PMID:26470770

  10. The comparison of glycosphingolipids isolated from an epithelial ovarian cancer cell line and a nontumorigenic epithelial ovarian cell line using MALDI-MS and MALDI-MS/MS.

    PubMed

    Rajanayake, Krishani K; Taylor, William R; Isailovic, Dragan

    2016-08-01

    Glycosphingolipids (GSLs) are important biomolecules, which are linked to many diseases such as GSL storage disorders and cancer. Consequently, the expression of GSLs may be altered in ovarian cancer cell lines in comparison to apparently healthy cell lines. Here, differential expressions of GSLs in an epithelial ovarian cancer cell line SKOV3 and a nontumorigenic epithelial ovarian cell line T29 were studied using matrix-assisted laser desorption/ionization-mass spectrometry (MALDI-MS) and MALDI-MS/MS. The isolation of GSLs from SKOV3 and T29 cell lines was carried out using Folch partition. GSLs were successfully detected by MALDI-MS, and structurally assigned by a comparison of their MALDI-MS/MS fragmentation patterns with MS/MS data found in SimLipid database. Additionally, LIPID MAPS was used to assign GSL ion masses in MALDI-MS spectra. Seventeen neutral GSLs were identified in Folch partition lower (chloroform/methanol) phases originating from both cell lines, while five globo series neutral GSLs were identified only in the Folch partition lower phase of SKOV3 cell line. Several different sialylated GSLs were detected in Folch partition upper (water/methanol) phases of SKOV3 and T29 cell lines. Overall, this study demonstrates the alteration and increased glycosylation of GSLs in an epithelial ovarian cancer cell line in comparison to a nontumorigenic epithelial ovarian cell line. PMID:27267063

  11. Interactions of Streptococcus iniae with phagocytic cell line.

    PubMed

    El Aamri, Fatima; Remuzgo-Martínez, S; Acosta, Félix; Real, Fernando; Ramos-Vivas, José; Icardo, José M; Padilla, Daniel

    2015-04-01

    Streptococcus iniae has become one of the most serious aquatic pathogens in the last decade, causing large losses in wild and farmed fish worldwide. There is clear evidence that this pathogen is capable not only of causing serious disease in fish but also of being transferred to and infecting humans. In this study, we investigate the interaction of S. iniae with two murine macrophage cell lines, J774-A1 and RAW 264.7. Cytotoxicity assay demonstrated significant differences between live and UV-light killed IUSA-1 strains. The burst respiratory activity decreased to baseline after 1 and 4 h of exposure for J774-A1 and RAW 264.7, respectively. Immunofluorescent and ultrastructural study of infected cells confirmed the intracellular localization of bacteria at 1 h and 24 h post-infection. Using qRT-PCR arrays, we investigated the changes in the gene expression of immune relevant genes associated with macrophage activation. In this screening, we identified 11 of 84 genes up-regulated, we observed over-expression of pro-inflammatory response as IL-1α, IL-1β, and TNF-α, without a good anti-inflammatory response. Present findings suggest a capacity of S. iniae to modulate a mammalian macrophages cell lines to their survival and replication intracellular, which makes this cell type as a reservoir for continued infection.

  12. Hypoxia induces adipogenic differentitation of myoblastic cell lines

    SciTech Connect

    Itoigawa, Yoshiaki; Kishimoto, Koshi N.; Okuno, Hiroshi; Sano, Hirotaka; Kaneko, Kazuo; Itoi, Eiji

    2010-09-03

    Research highlights: {yields} C2C12 and G8 myogenic cell lines treated by hypoxia differentiate into adipocytes. {yields} The expression of C/EBP{beta}, {alpha} and PPAR{gamma} were increased under hypoxia. {yields} Myogenic differentiation of C2C12 was inhibited under hypoxia. -- Abstract: Muscle atrophy usually accompanies fat accumulation in the muscle. In such atrophic conditions as back muscles of kyphotic spine and the rotator cuff muscles with torn tendons, blood flow might be diminished. It is known that hypoxia causes trans-differentiation of mesenchymal stem cells derived from bone marrow into adipocytes. However, it has not been elucidated yet if hypoxia turned myoblasts into adipocytes. We investigated adipogenesis in C2C12 and G8 murine myogenic cell line treated by hypoxia. Cells were also treated with the cocktail of insulin, dexamethasone and IBMX (MDI), which has been known to inhibit Wnt signaling and promote adipogenesis. Adipogenic differentiation was seen in both hypoxia and MDI. Adipogenic marker gene expression was assessed in C2C12. CCAAT/enhancer-binding protein (C/EBP) {beta}, {alpha} and peroxisome proliferator activating receptor (PPAR) {gamma} were increased by both hypoxia and MDI. The expression profile of Wnt10b was different between hypoxia and MDI. The mechanism for adipogenesis of myoblasts in hypoxia might be regulated by different mechanism than the modification of Wnt signaling.

  13. Mechanisms of prodigiosin cytotoxicity in human neuroblastoma cell lines.

    PubMed

    Francisco, Roser; Pérez-Tomás, Ricardo; Gimènez-Bonafé, Pepita; Soto-Cerrato, Vanessa; Giménez-Xavier, Pol; Ambrosio, Santiago

    2007-10-31

    Prodigiosin is a bacterial red pigment with cytotoxic properties and potential antitumor activity that has been tested against different cancerous cells. In this study we report the effect and mechanisms of action of prodigiosin against different human neuroblastoma cell lines: SH-SY5Y, LAN-1, IMR-32 (N-type) and SK-N-AS (S-type). We compare the anticancerous effect of prodigiosin with that of cisplatin at different concentrations during 24 h of exposure. Prodigiosin is more potent, with IC50 values lower than 1.5 microM in N-type neuroblastoma cells and around 7 microM in the S-type neuroblastoma cell line. We describe prodigiosin as a proton sequestering agent that destroys the intracellular pH gradient, and propose that its main cytotoxic effect could be related to its action on mitochondria, where it exerts an uncoupling effect on the electronic chain transport of protons to mitochondrial ATP synthase. As a result of this action, ATP production is reduced but without decreasing in oxygen consumption. This mechanism of action differs from those induced by conventional chemotherapeutic drugs, suggesting a possible role for prodigiosin to enhance the effect of antitumor agents in the treatment of neuroblastoma.

  14. Characterization of cell lines stably transfected with rubella virus replicons

    SciTech Connect

    Tzeng, Wen-Pin; Xu, Jie; Frey, Teryl K.

    2012-07-20

    Rubella virus (RUBV) replicons expressing a drug resistance gene and a gene of interest were used to select cell lines uniformly harboring the replicon. Replicons expressing GFP and a virus capsid protein GFP fusion (C-GFP) were compared. Vero or BHK cells transfected with either replicon survived drug selection and grew into a monolayer. However, survival was {approx}9-fold greater following transfection with the C-GFP-replicon than with the GFP-expressing replicon and while the C-GFP-replicon cells grew similarly to non-transfected cells, the GFP-replicon cells grew slower. Neither was due to the ability of the CP to enhance RNA synthesis but survival during drug selection was correlated with the ability of CP to inhibit apoptosis. Additionally, C-GFP-replicon cells were not cured of the replicon in the absence of drug selection. Interferon-alpha suppressed replicon RNA and protein synthesis, but did not cure the cells, explaining in part the ability of RUBV to establish persistent infections.

  15. Airway goblet cells: responsive and adaptable front-line defenders.

    PubMed

    Rogers, D F

    1994-09-01

    development of a hypersecretory epithelium include excessive discharge of mucus and increased expression of airway mucin messenger ribonucleic acid (mRNA). Cessation of chronic airway stress rapidly reverses the increased number of goblet cells. Irritant-induced increases in number of goblet cells can be inhibited by a variety of drugs with anti-inflammatory and mucoregulatory properties, and the reversal to normal numbers after cessation of the irritation is speeded by these drugs. The ability of goblet cells to be progenitors of ciliated cells, to rapidly produce vast quantities of mucus in response to acute airway insult, and to change in number according to variations in chronic insult indicates that these cells are vitally important responsive and adaptable front-line defenders of the airways. PMID:7995400

  16. Genetically-defined novel oral squamous cell carcinoma cell lines for the development of molecular therapies

    PubMed Central

    Fadlullah, Muhammad Zaki Hidayatullah; Chiang, Ivy Kim-Ni; Dionne, Kalen R.; Yee, Pei San; Gan, Chai Phei; Sam, Kin Kit; Tiong, Kai Hung; Ng, Adrian Kwok Wen; Martin, Daniel; Lim, Kue Peng; Kallarakkal, Thomas George; Mustafa, Wan Mahadzir Wan; Lau, Shin Hin; Abraham, Mannil Thomas; Zain, Rosnah Binti; Rahman, Zainal Ariff Abdul; Molinolo, Alfredo; Patel, Vyomesh; Gutkind, J. Silvio; Tan, Aik Choon; Cheong, Sok Ching

    2016-01-01

    Emerging biological and translational insights from large sequencing efforts underscore the need for genetically-relevant cell lines to study the relationships between genomic alterations of tumors, and therapeutic dependencies. Here, we report a detailed characterization of a novel panel of clinically annotated oral squamous cell carcinoma (OSCC) cell lines, derived from patients with diverse ethnicity and risk habits. Molecular analysis by RNAseq and copy number alterations (CNA) identified that the cell lines harbour CNA that have been previously reported in OSCC, for example focal amplications in 3q, 7p, 8q, 11q, 20q and deletions in 3p, 5q, 8p, 18q. Similarly, our analysis identified the same cohort of frequently mutated genes previously reported in OSCC including TP53, CDKN2A, EPHA2, FAT1, NOTCH1, CASP8 and PIK3CA. Notably, we identified mutations (MLL4, USP9X, ARID2) in cell lines derived from betel quid users that may be associated with this specific risk factor. Gene expression profiles of the ORL lines also aligned with those reported for OSCC. By focusing on those gene expression signatures that are predictive of chemotherapeutic response, we observed that the ORL lines broadly clustered into three groups (cell cycle, xenobiotic metabolism, others). The ORL lines noted to be enriched in cell cycle genes responded preferentially to the CDK1 inhibitor RO3306, by MTT cell viability assay. Overall, our in-depth characterization of clinically annotated ORL lines provides new insight into the molecular alterations synonymous with OSCC, which can facilitate in the identification of biomarkers that can be used to guide diagnosis, prognosis, and treatment of OSCC. PMID:27050151

  17. LINE-1 induces hTERT and ensures telomere maintenance in tumour cell lines.

    PubMed

    Aschacher, T; Wolf, B; Enzmann, F; Kienzl, P; Messner, B; Sampl, S; Svoboda, M; Mechtcheriakova, D; Holzmann, K; Bergmann, M

    2016-01-01

    A hallmark of cancer cells is an activated telomere maintenance mechanism, which allows prolonged survival of the malignant cells. In more than 80% of tumours, telomeres are elongated by the enzyme telomerase, which adds de novo telomere repeats to the ends of chromosomes. Cancer cells are also characterized by expression of active LINE-1 elements (L1s, long interspersed nuclear elements-1). L1 elements are abundant retrotransposons in the eukaryotic genome that are primarily known for facilitating aberrant recombination. Using L1-knockdown (KD), we show for the first time that L1 is critical for telomere maintenance in telomerase-positive tumour cells. The reduced length of telomeres in the L1-KD-treated cells correlated with an increased rate of telomere dysfunction foci, a reduced expression of shelterin proteins and an increased rate of anaphase bridges. The decreased telomere length was associated with a decreased telomerase activity and decreased telomerase mRNA level; the latter was increased upon L1 overexpression. L1-KD also led to a decrease in mRNA and protein expression of cMyc and KLF-4, two main transcription factors of telomerase and altered mRNA levels of other stem-cell-associated proteins such as CD44 and hMyb, as well as a corresponding reduced growth of spheroids. The KD of KLF-4 or cMyc decreased the level of L1-ORF1 mRNA, suggesting a specific reciprocal regulation with L1. Thus, our findings contribute to the understanding of L1 as a pathogenicity factor in cancer cells. As L1 is only expressed in pathophysiological conditions, L1 now appears to be target in the rational treatment of telomerase-positive cancer.

  18. Molecular signatures in response to Isoliquiritigenin in lymphoblastoid cell lines

    SciTech Connect

    Lee, Jae-Eun; Hong, Eun-Jung; Nam, Hye-Young; Hwang, Meeyul; Kim, Ji-Hyun; Han, Bok-Ghee; Jeon, Jae-Pil

    2012-10-19

    Highlights: Black-Right-Pointing-Pointer We identified the inhibitory effect of ISL on cell proliferation of LCLs. Black-Right-Pointing-Pointer We found ISL-induced genes and miRNAs through microarray approach. Black-Right-Pointing-Pointer ISL-treated LCLs represented gene expression changes in cell cycle and p53 pathway. Black-Right-Pointing-Pointer We revealed 12 putative mRNA-miRNA functional pairs associated with ISL effect. -- Abstract: Isoliquiritigenin (ISL) has been known to induce cell cycle arrest and apoptosis of various cancer cells. However, genetic factors regulating ISL effects remain unclear. The aim of this study was to identify the molecular signatures involved in ISL-induced cell death of EBV-transformed lymphoblastoid cell lines (LCLs) using microarray analyses. For gene expression and microRNA (miRNA) microarray experiments, each of 12 LCL strains was independently treated with ISL or DMSO as a vehicle control for a day prior to total RNA extraction. ISL treatment inhibited cell proliferation of LCLs in a dose-dependent manner. Microarray analysis showed that ISL-treated LCLs represented gene expression changes in cell cycle and p53 signaling pathway, having a potential as regulators in LCL survival and sensitivity to ISL-induced cytotoxicity. In addition, 36 miRNAs including five miRNAs with unknown functions were differentially expressed in ISL-treated LCLs. The integrative analysis of miRNA and gene expression profiles revealed 12 putative mRNA-miRNA functional pairs. Among them, miR-1207-5p and miR-575 were negatively correlated with p53 pathway- and cell cycle-associated genes, respectively. In conclusion, our study suggests that miRNAs play an important role in ISL-induced cytotoxicity in LCLs by targeting signaling pathways including p53 pathway and cell cycle.

  19. Integrative proteomic profiling of ovarian cancer cell lines reveals precursor cell associated proteins and functional status

    PubMed Central

    Coscia, F.; Watters, K. M.; Curtis, M.; Eckert, M. A.; Chiang, C. Y.; Tyanova, S.; Montag, A.; Lastra, R. R.; Lengyel, E.; Mann, M.

    2016-01-01

    A cell line representative of human high-grade serous ovarian cancer (HGSOC) should not only resemble its tumour of origin at the molecular level, but also demonstrate functional utility in pre-clinical investigations. Here, we report the integrated proteomic analysis of 26 ovarian cancer cell lines, HGSOC tumours, immortalized ovarian surface epithelial cells and fallopian tube epithelial cells via a single-run mass spectrometric workflow. The in-depth quantification of >10,000 proteins results in three distinct cell line categories: epithelial (group I), clear cell (group II) and mesenchymal (group III). We identify a 67-protein cell line signature, which separates our entire proteomic data set, as well as a confirmatory publicly available CPTAC/TCGA tumour proteome data set, into a predominantly epithelial and mesenchymal HGSOC tumour cluster. This proteomics-based epithelial/mesenchymal stratification of cell lines and human tumours indicates a possible origin of HGSOC either from the fallopian tube or from the ovarian surface epithelium. PMID:27561551

  20. Creation and characterization of a cell-death reporter cell line for hepatitis C virus infection

    PubMed Central

    Chen, Zhilei; Simeon, Rudo; Chockalingam, Karuppiah; Rice, Charles M.

    2010-01-01

    The present study describes the creation and characterization of a hepatoma cell line, n4mBid, that supports all stages of the hepatitis C virus (HCV) life cycle and strongly reports HCV infection by a cell-death phenotype. The n4mBid cell line is derived from the highly HCV-permissive Huh-7.5 hepatoma cell line and contains a modified Bid protein (mBid) that is cleaved and activated by the HCV serine protease NS3-4A. N4mBid exhibited a 10–20 fold difference in cell viability between the HCV-infected and mock-infected states, while the parental Huh-7.5 cells showed <2 fold difference under the same conditions. The pronounced difference in n4mBid cell viability between the HCV- and mock-infected states in a 96-well plate format points to its usefulness in cell survival-based high-throughput screens for anti-HCV molecules. The degree of cell death was found to be proportional to the intracellular load of HCV. HCV-low n4mBid cells, expressing an anti-HCV short hairpin RNA, showed a significant growth advantage over naïve cells and could be rapidly enriched after HCV infection, suggesting the possibility of using n4mBid cells for the cell survival-based selection of genetic anti-HCV factors. PMID:20188762

  1. Characterization of hybrids between bovine (MDBK) and mouse (L-cell) cell lines.

    PubMed

    Chinchar, V G; Floyd, A D; Chinchar, G D; Taylor, M W

    1979-02-01

    Hypoxanthine-guanine phosphoribosyltransferase (HGPRT)-deficient mutants of a bovine kidney cell line (MDBK) were selected following mutagenesis with ethylmethane sulfonate or ICR-170G. MDBK mutants were hybridized to thymidine kinase-deficient L cells and selected in HAT medium. Parental and hybrid cells were characterized for isozyme patterns of lactic dehydrogenase malate dehydrogenase, glucose-6-phosphate dehydrogenase, and glutamate oxalate transaminase. Chromosomes of MDBK can be distinguished from mouse L cells by configuration and by fluorescent staining with Hoechst 33-258 stain. Hybrid cells contained both MDBK and L-cell chromosomes and had elevated DNA content. MDBK cells are normally restrictive for mengovirus replication. Both permissive and restrictive hybrids were found. Our data indicate that there was preferential loss of MDBK chromosomes in the hybrid cell lines.

  2. Internalization of cystatin C in human cell lines.

    PubMed

    Ekström, Ulf; Wallin, Hanna; Lorenzo, Julia; Holmqvist, Bo; Abrahamson, Magnus; Avilés, Francesc X

    2008-09-01

    Altered protease activity is considered important for tumour invasion and metastasis, processes in which the cysteine proteases cathepsin B and L are involved. Their natural inhibitor cystatin C is a secreted protein, suggesting that it functions to control extracellular protease activity. Because cystatins added to cell cultures can inhibit polio, herpes simplex and coronavirus replication, which are intracellular processes, the internalization and intracellular regulation of cysteine proteases by cystatin C should be considered. The extension, mechanism and biological importance of this hypothetical process are unknown. We investigated whether internalization of cystatin C occurs in a set of human cell lines. Demonstrated by flow cytometry and confocal microscopy, A-431, MCF-7, MDA-MB-453, MDA-MB-468 and Capan-1 cells internalized fluorophore-conjugated cystatin C when exposed to physiological concentrations (1 microm). During cystatin C incubation, intracellular cystatin C increased after 5 min and accumulated for at least 6 h, reaching four to six times the baseline level. Western blotting showed that the internalized inhibitor was not degraded. It was functionally intact and extracts of cells exposed to cystatin C showed a higher capacity to inhibit papain and cathepsin B than control cells (decrease in enzyme activity of 34% and 37%, respectively). The uptake of labelled cystatin C was inhibited by unlabelled inhibitor, suggesting a specific pathway for the internalization. We conclude that the cysteine protease inhibitor cystatin C is internalized in significant quantities in various cancer cell lines. This is a potentially important physiological phenomenon not previously described for this group of inhibitors.

  3. Melatonin decreases cell proliferation, impairs myogenic differentiation and triggers apoptotic cell death in rhabdomyosarcoma cell lines.

    PubMed

    Codenotti, Silvia; Battistelli, Michela; Burattini, Sabrina; Salucci, Sara; Falcieri, Elisabetta; Rezzani, Rita; Faggi, Fiorella; Colombi, Marina; Monti, Eugenio; Fanzani, Alessandro

    2015-07-01

    Melatonin is a small indole produced by the pineal gland and other tissues, and has numerous functions that aid in the maintenance of the whole body homeostasis, ranging from the regulation of circadian rhythms and sleep to protection from oxidative stress. Melatonin has also been reported to counteract cell growth and chemoresistance in different types of cancer. In the present study, we investigated the effects of exogenous melatonin administration on different human cell lines and primary mouse tumor cultures of rhabdomyosarcoma (RMS), the most frequent soft tissue sarcoma affecting childhood. The results showed that melatonin significantly affected the behavior of RMS cells, leading to inhibition of cell proliferation and impairment of myogenic differentiation followed by increased apoptotic cell death, as observed by immunoblotting analysis of apoptosis-related markers including Bax, Bcl-2 and caspase-3. Similar findings were observed using a combination of microscopy techniques, including scanning/transmission electron and confocal microscopy. Furthermore, melatonin in combination with doxorubicin or cisplatin, two compounds commonly used for the treatment of solid tumors, increased the sensitivity of RMS cells to apoptosis. These data indicated that melatonin may be effective in counteracting RMS tumor growth and chemoresistance.

  4. Proteomic patterns of cervical cancer cell lines, a network perspective

    PubMed Central

    2011-01-01

    Background Cervical cancer is a major mortality factor in the female population. This neoplastic is an excellent model for studying the mechanisms involved in cancer maintenance, because the Human Papilloma Virus (HPV) is the etiology factor in most cases. With the purpose of characterizing the effects of malignant transformation in cellular activity, proteomic studies constitute a reliable way to monitor the biological alterations induced by this disease. In this contextual scheme, a systemic description that enables the identification of the common events between cell lines of different origins, is required to distinguish the essence of carcinogenesis. Results With this study, we sought to achieve a systemic perspective of the common proteomic profile of six cervical cancer cell lines, both positive and negative for HPV, and which differ from the profile corresponding to the non-tumourgenic cell line, HaCaT. Our objectives were to identify common cellular events participating in cancer maintenance, as well as the establishment of a pipeline to work with proteomic-derived results. We analyzed by means of 2D SDS-PAGE and MALDI-TOF mass spectrometry the protein extracts of six cervical cancer cell lines, from which we identified a consensus of 66 proteins. We call this group of proteins, the "central core of cervical cancer". Starting from this core set of proteins, we acquired a PPI network that pointed, through topological analysis, to some proteins that may well be playing a central role in the neoplastic process, such as 14-3-3ζ. In silico overrepresentation analysis of transcription factors pointed to the overexpression of c-Myc, Max and E2F1 as key transcription factors involved in orchestrating the neoplastic phenotype. Conclusions Our findings show that there is a "central core of cervical cancer" protein expression pattern, and suggest that 14-3-3ζ is key to determine if the cell proliferates or dies. In addition, our bioinformatics analysis suggests that

  5. Heparanase augments inflammatory chemokine production from colorectal carcinoma cell lines.

    PubMed

    Tsunekawa, Naoki; Higashi, Nobuaki; Kogane, Yusuke; Waki, Michihiko; Shida, Hiroaki; Nishimura, Yoshio; Adachi, Hayamitsu; Nakajima, Motowo; Irimura, Tatsuro

    2016-01-22

    To explore possible roles of heparanase in cancer-host crosstalk, we examined whether heparanase influences expression of inflammatory chemokines in colorectal cancer cells. Murine colorectal carcinoma cells incubated with heparanase upregulated MCP-1, KC, and RANTES genes and released MCP-1 and KC proteins. Heparanase-dependent production of IL-8 was detected in two human colorectal carcinoma cell lines. Addition of a heparanase inhibitor Heparastatin (SF4) did not influence MCP-1 production, while both latent and mature forms of heparanase augmented MCP-1 release, suggesting that heparanase catalytic activity was dispensable for MCP-1 production. In contrast, addition of heparin to the medium suppressed MCP-1 release in a dose-dependent manner. Similarly, targeted suppression of Ext1 by RNAi significantly suppressed cell surface expression of heparan sulfate and MCP-1 production in colon 26 cells. Taken together, it is concluded that colon 26 cells transduce the heparanase-mediated signal through heparan sulfate binding. We propose a novel function for heparanase independent of its endoglycosidase activity, namely as a stimulant for chemokine production. PMID:26713365

  6. Complementation analysis of the murine scid cell line

    SciTech Connect

    Zdzienicka, M.Z. |; Priestly, A.; Jeggo, P.A.

    1995-09-01

    It has been shown that several X-ray-sensitive Chinese hamster cell mutants defective in repair of DNA double-strand breaks (DSBs) are also impaired in the process of V(D)J recombination. The hamster mutants with this phenotype represent three distinct complementation groups, represented by the xrs series, XR-1 and V-3. The murine scid cell line also shows the same phenotype, and therefore we examined whether the scid mutant represents a new complementation group or belongs to one of the existing groups. Scid cells were fused with hamster cell mutants representing the three complementation groups. Hybrids between V-3 and scid cells were only partially complemented for X-ray sensitivity, whereas hybrids derived from fusions with the other mutants were resistant to X rays. These results suggest that V-3 and scid cells are defective in the same gene. To confirm this finding, a single human chromosome 8, which is known to carry the scid gene, was introduced into V-3 cells by microcell-mediated chromosome transfer. Nine hybrid clones derived from V-3 and carrying human chromosome 8 were obtained, and seven were found to be partially complemented for X-ray sensitivity. When human chromosome 8 was introduced into scid cells, seven of eight hybrid clones became resistant to X rays. The results indicate that the defective genes in V-3 and scid are both localized on human chromosome 8. This supports the results from the fusion analysis that V-3 and scid cells are defective in the same gene. 53 refs., 4 figs., 1 tab.

  7. Cycle reset in a melanoma cell line caused by cooling.

    PubMed

    Dewey, D L

    1987-11-01

    When cells in culture are released from G0 into cycle by diluting into fresh medium there is a delay of many hours before they re-enter the cycle and start DNA synthesis. A mouse melanoma cell line designated HP2 has been used to investigate the effects of non-standard temperatures between the time of plating and DNA synthesis. When the cells were incubated in a 5% CO2 box at 8 degrees C for periods during the G0-G1 transition there was an extra delay before the start of S, approximately equal to the time that the cells were held at 8 degrees C and independent of the time when the cold pulse was administered. When the cells were cooled to 25 degrees C the delay was longer than the time for which the cells had been kept at 25 degrees C, and this extra delay was also dependent on the point in G0-G1 when the cells were cooled, as though the cells could be reset to an earlier time by this treatment. It is suggested that a labile substance required for progression is destroyed faster than it is made at 25 degrees C but at 8 degrees C the rate of destruction is very low. Another phenomenon noted during these cooling experiments was that the peak height of the S phase profile, as measured by frequent pulse-thymidine incorporation experiments, was substantially higher for cells which had been cooled at a later stage in the G0-G1 transition, even though the overall times at 37 degrees C and at the colder temperature were identical. By varying the temperature of the cold pulse it was possible to separate the change in the peak height and the delay as separate entities. PMID:3502929

  8. Establishment of Immortalized Human Erythroid Progenitor Cell Lines Able to Produce Enucleated Red Blood Cells

    PubMed Central

    Kurita, Ryo; Suda, Noriko; Sudo, Kazuhiro; Miharada, Kenichi; Hiroyama, Takashi; Miyoshi, Hiroyuki; Tani, Kenzaburo; Nakamura, Yukio

    2013-01-01

    Transfusion of red blood cells (RBCs) is a standard and indispensable therapy in current clinical practice. In vitro production of RBCs offers a potential means to overcome a shortage of transfusable RBCs in some clinical situations and also to provide a source of cells free from possible infection or contamination by microorganisms. Thus, in vitro production of RBCs may become a standard procedure in the future. We previously reported the successful establishment of immortalized mouse erythroid progenitor cell lines that were able to produce mature RBCs very efficiently. Here, we have developed a reliable protocol for establishing immortalized human erythroid progenitor cell lines that are able to produce enucleated RBCs. These immortalized cell lines produce functional hemoglobin and express erythroid-specific markers, and these markers are upregulated following induction of differentiation in vitro. Most importantly, these immortalized cell lines all produce enucleated RBCs after induction of differentiation in vitro, although the efficiency of producing enucleated RBCs remains to be improved further. To the best of our knowledge, this is the first demonstration of the feasibility of using immortalized human erythroid progenitor cell lines as an ex vivo source for production of enucleated RBCs. PMID:23533656

  9. Sourcing human embryos for embryonic stem cell lines: Problems & perspectives

    PubMed Central

    Mehta, Rajvi H.

    2014-01-01

    The ability to successfully derive human embryonic stem cells (hESC) lines from human embryos following in vitro fertilization (IVF) opened up a plethora of potential applications of this technique. These cell lines could have been successfully used to increase our understanding of human developmental biology, transplantation medicine and the emerging science of regenerative medicine. The main source for human embryos has been ‘discarded’ or ‘spare’ fresh or frozen human embryos following IVF. It is a common practice to stimulate the ovaries of women undergoing any of the assisted reproductive technologies (ART) and retrieve multiple oocytes which subsequently lead to multiple embryos. Of these, only two or maximum of three embryos are transferred while the rest are cryopreserved as per the decision of the couple. In case a couple does not desire to ‘cryopreserve’ their embryos then all the embryos remaining following embryo transfer can be considered ‘spare’ or if a couple is no longer in need of the ‘cryopreserved’ embryos then these also can be considered as ‘spare’. But, the question raised by the ethicists is, “what about ‘slightly’ over-stimulating a woman to get a few extra eggs and embryos? The decision becomes more difficult when it comes to ‘discarded’ embryos. As of today, the quality of the embryos is primarily assessed based on morphology and the rate of development mainly judged by single point assessment. Despite many criteria described in the literature, the quality assessment is purely subjective. The question that arises is on the decision of ‘discarding’ embryos. What would be the criteria for discarding embryos and the potential ‘use’ of ESC derived from the ‘abnormal appearing’ embryos? This paper discusses some of the newer methods to procure embryos for the derivation of embryonic stem cell lines which will respect the ethical concerns but still provide the source material. PMID:25673530

  10. ULTRASTRUCTURE, STEROIDOGENIC POTENTIAL, AND ENERGY METABOLISM OF THE SNELL ADRENOCORTICAL CARCINOMA 494

    PubMed Central

    Kimmel, G. L.; Péron, F. G.; Haksar, A.; Bedigian, E.; Robidoux, W. F.; Lin, M. T.

    1974-01-01

    Electron microscope studies were carried out with the adrenocortical carcinoma 494 and normal adrenal cortex tissue. The mitochondria of the tumor cells showed marked differences when compared with mitochondria from fasciculata cells of the normal adrenal cortex. These differences were primarily related to mitochondrial number and crista structure. Corticosterone production in isolated tumor cells was extremely low and neither ACTH nor dibutyryl cyclic AMP had any stimulatory effect. Normal adrenal cells showed at least a tenfold increase under identical conditions. In the presence of corticosteroid precursors the amount of corticosterone produced by the tumor cells was much less than that produced by normal cells. The results indicate a reduced capacity for 11β-hydroxylation in the tumor mitochondria and a possible reduced capacity for biosynthetic steps before the 11β-hydroxylation reaction. Glycolysis in isolated tumor cells was also lower than in normal cells. Isolated tumor mitochondria oxidized succinate normally with a good degree of coupling with phosphorylation. However, unlike normal adrenal mitochondria, the tumor mitochondria showed little or no oxygen uptake with other Krebs cycle substrates. These data suggest that the tumor mitochondria may be lacking in the flavoprotein dehydrogenases responsible for the oxidation of NADH and NADPH, although other components of the respiratory chain may be intact. PMID:4366105

  11. Development of buffalo (Bubalus bubalis) embryonic stem cell lines from somatic cell nuclear transferred blastocysts.

    PubMed

    Shah, Syed Mohmad; Saini, Neha; Ashraf, Syma; Singh, Manoj K; Manik, Radheysham; Singla, Suresh K; Palta, Prabhat; Chauhan, Manmohan Singh

    2015-11-01

    We developed buffalo embryonic stem cell lines from somatic cell nuclear transfer derived blastocysts, produced by hand-guided cloning technique. The inner cell mass of the blastocyst was cut mechanically using a Microblade and cultured onto feeder cells in buffalo embryonic stem (ES) cell culture medium at 38 °C in a 5% CO2 incubator. The stem cell colonies were characterized for alkaline phosphatase activity, karyotype, pluripotency and self-renewal markers like OCT4, NANOG, SOX2, c-Myc, FOXD3, SSEA-1, SSEA-4, TRA-1-60, TRA-1-81 and CD90. The cell lines also possessed the capability to differentiate across all the three germ layers under spontaneous differentiation conditions. PMID:26987926

  12. Development and Characterization of Six New Human Papillary Thyroid Carcinoma Cell Lines

    PubMed Central

    Henderson, Ying C.; Ahn, Soon-Hyun; Ryu, Junsun; Chen, Yunyun; Williams, Michelle D.; El-Naggar, Adel K.; Gagea, Mihai; Schweppe, Rebecca E.; Haugen, Bryan R.; Lai, Stephen Y.

    2015-01-01

    Context: Cell lines are a widely used tool in cancer research. However, despite the relatively high incidence of papillary thyroid carcinoma (PTC), there are only four PTC cell lines available for international research audience. Objective: The objective of this study was to establish and characterize new PTC cell lines that represent primary tumor biology. Surgical specimens were obtained to generate PTC cell lines. Short tandem repeat profiling was used to confirm the uniqueness of the cell lines against databases of known cell lines and mutations were assessed using Sequenom. The expression of thyroid-specific genes was examined using real-time PCR. Tumorigenicity was determined using an orthotopic thyroid xenograft tumor mouse model. Results: Six PTC cell lines (five conventional PTCs and one follicular variant of PTC) were generated and found to be unique when compared by short tandem repeat profiling against databases of all existing cell lines. The five conventional PTC cell lines carry the BRAF V600E mutation and the follicular variant of PTC cell line had an NRAS mutation. Five of the six cell lines had a mutation in the promoter of the human telomerase reverse transcriptase gene. None of the cell lines have RET/PTC rearrangements. Three cell lines were tumorigenic in the orthotopic thyroid xenograft tumor mouse model. Conclusions: These five characterized conventional PTC cell lines and the unique follicular variant of PTC cell line should be valuable reagents for thyroid cancer research. The three tumorigenic cell lines can be used for in vivo testing of targeted therapeutic and novel agents. PMID:25427145

  13. Erythropoietin modulates intracellular calcium in a human neuroblastoma cell line

    PubMed Central

    Assandri, Roberta; Egger, Marcel; Gassmann, Max; Niggli, Ernst; Bauer, Christian; Forster, Ian; Görlach, Agnes

    1999-01-01

    Recent investigations have shown that the glycoprotein erythropoietin (Epo) and its specific receptor (EpoR) are present in the mammalian brain including human, monkey and mouse. These findings suggest a local action of Epo in the nervous system. The aim of this study was to elucidate a possible functional interaction of Epo with neuronal cells. To examine the influence of externally applied Epo on Ca2+ homeostasis the human neuroblastoma cell line SK-N-MC was chosen as a suitable in vitro model for undifferentiated neuronal cells. Expression of the EpoR in SK-N-MC cells was detected by reverse transcription-PCR, Western blot and immunofluorescence analysis. Patch-clamp studies of SK-N-MC cells confirmed the expression of T-type Ca2+ channels, whose peak macroscopic current was increased by the addition of recombinant human Epo (rhEpo) to the bathing medium. Confocal laser scanning microscopy analysis of SK-N-MC cells confirmed a transient increase in intracellular free [Ca2+] in response to externally applied rhEpo. The transient response to Epo was dependent on external Ca2+ and remained even after depletion of internal Ca2+ stores by caffeine or thapsigargin. However, after depletion the response to Epo was absent when cells were superfused with the T-type Ca2+ channel blocker flunarizine. This study demonstrates that Epo can interact with neuronal cells by affecting Ca2+ homeostasis through an increase in Ca2+ influx via plasma membrane T-type voltage-dependent Ca2+ channels. PMID:10087335

  14. Comparative proteomic phenotyping of cell lines and primary cells to assess preservation of cell type-specific functions.

    PubMed

    Pan, Cuiping; Kumar, Chanchal; Bohl, Sebastian; Klingmueller, Ursula; Mann, Matthias

    2009-03-01

    Biological experiments are most often performed with immortalized cell lines because they are readily available and can be expanded without limitation. However, cell lines may differ from the in vivo situation in important aspects. Here we introduce a straightforward methodology to compare cell lines to their cognate primary cells and to derive a comparative functional phenotype. We used SILAC (stable isotope labeling by amino acids in cell culture) for quantitative, mass spectrometry-based comparison of the hepatoma cell line Hepa1-6 with primary hepatocytes. The resulting quantitative proteome of 4,063 proteins had an asymmetric distribution, with many proteins down-regulated in the cell line. Bioinformatic analysis of the quantitative proteomics phenotypes revealed that Hepa1-6 cells were deficient in mitochondria, reflecting re-arrangement of metabolic pathways, drastically up-regulate cell cycle-associated functions and largely shut down drug metabolizing enzymes characteristic for the liver. This quantitative knowledge of changes provides an important basis to adapt cell lines to more closely resemble physiological conditions.

  15. Novel antiproliferative flavonoids induce cell cycle arrest in human prostate cancer cell lines.

    PubMed

    Haddad, A Q; Venkateswaran, V; Viswanathan, L; Teahan, S J; Fleshner, N E; Klotz, L H

    2006-01-01

    Epidemiologic studies have demonstrated an inverse association between flavonoid intake and prostate cancer (PCa) risk. The East Asian diet is very high in flavonoids and, correspondingly, men in China and Japan have the lowest incidence of PCa worldwide. There are thousands of different naturally occurring and synthetic flavonoids. However, only a few have been studied in PCa. Our aim was to identify novel flavonoids with antiproliferative effect in PCa cell lines, as well as determine their effects on cell cycle. We have screened a representative subgroup of 26 flavonoids for antiproliferative effect on the human PCa (LNCaP and PC3), breast cancer (MCF-7), and normal prostate stromal cell lines (PrSC). Using a fluorescence-based cell proliferation assay (Cyquant), we have identified five flavonoids, including the novel compounds 2,2'-dihydroxychalcone and fisetin, with antiproliferative and cell cycle arresting properties in human PCa in vitro. Most of the flavonoids tested exerted antiproliferative effect at lower doses in the PCa cell lines compared to the non-PCa cells. Flow cytometry was used as a means to determine the effects on cell cycle. PC3 cells were arrested in G2/M phase by flavonoids. LNCaP cells demonstrated different cell cycle profiles. Further studies are warranted to determine the molecular mechanism of action of 2,2'-DHC and fisetin in PCa, and to establish their effectiveness in vivo.

  16. Characterization of UV radiation sensitive frog cell lines

    SciTech Connect

    Smith-Stein, A.C.

    1983-01-01

    Twenty-one subclones of nine frog cell isolates were tested for sensitivity to a panel of DNA damaging agents. Two clones were identified which had a greater than wild type level of sensitivity to UV radiation but had a wild type level of sensitivity to the other agents. These clones were the haploid RRP602-7 and the diploid RRP802-1. RRP802-1 was found to be unstable with respect to UV sensitivity. The line was cloned in order to isolate stable sensitive and wild type derivatives. RRP802-1-16, a UV sensitive clone and RRP802-1-13, a clone with a wild type level of sensitivity to UV radiation, were isolated. The UV radiation sensitivity of RRP602-7, RRP802-1 and RRP802-1-16 did not correlate with cell size, cell shape, cell cycle distribution or ploidy. The cell cycle distribution after UV irradiation, the rate of DNA synthesis after UV-irradiation, the DNA polymerase ..cap alpha.. activity and the sister chromatid exchange frequency were all measured in RRP602-7, RRP802-1 and RRP802-1-16 in order to examine the DNA repair capacity. The presence of DNA repair pathways was examined directly in RRP602-7, RRP802-1 and RRP802-1-16. All were found to be proficient in photo-reactivation repair and postreplication repair of UV elicited DNA damage.

  17. Amelioration of vanadium-induced testicular toxicity and adrenocortical hyperactivity by vitamin E acetate in rats.

    PubMed

    Chandra, Amar K; Ghosh, Rituparna; Chatterjee, Aparajita; Sarkar, Mahitosh

    2007-12-01

    Vanadium toxicity is a challenging problem to the health professionals and a cutting-edge medical problem. Vanadium has been recognized as industrial hazards that adversely affect human and animal reproductive health. Since testicular function is exquisitely susceptible to reactive-oxygen species, the present study elucidates the possible involvement of oxidative stress in vanadium-induced testicular toxicity and the prophylactic effects of vitamin E acetate against such adverse effects of vanadium. The study also characterizes the effects of vanadium on rat adrenal steroidogenesis and determines the underlying mechanisms of testicular and adrenal interactions in response to vanadium exposure. Significantly reduced sperm count associated with decreased serum testosterone and gonadotropins level in the vanadium-injected group of rats compared to control substantially proves the ongoing damaging effects of vanadium-induced ROS on developing germ cells. This is in turn reflected in the appreciable increase in testicular lipid peroxidation level and decline in the activities of steroidogenic and antioxidant enzymes. However, oral administration of vitamin E acetate could protect testes from the toxic effects of vanadium. Vanadium also results in adrenocortical hyperactivity, as evidenced by the elevated secretion of glucocorticoids, adrenal gland hypertrophy and increased activity of adrenal Delta(5)3beta-HSD. However, reversibility of these alterations in adrenocortical activities was vividly reflected after vitamin E acetate supplementation. All these studies reveal that oxidative stress is the major mechanism of health deterioration and that vanadium can act as a stressor metal causing chronic stress effects through excitation of hypothalamo-pituitary-adrenal axis. However antioxidant support by vitamin E acetate may provide significant protection.

  18. DAX1 Overexpression in Pediatric Adrenocortical Tumors: A Synergic Role with SF1 in Tumorigenesis.

    PubMed

    de Sousa, G R V; Soares, I C; Faria, A M; Domingues, V B; Wakamatsu, A; Lerario, A M; Alves, V A F; Zerbini, M C N; Mendonca, B B; Fragoso, M C B V; Latronico, A C; Almeida, M Q

    2015-08-01

    DAX1 transcription factor is a key determinant of adrenogonadal development, acting as a repressor of SF1 targets in steroidogenesis. It was recently demonstrated that DAX1 regulates pluripotency and differentiation in murine embryonic stem cells. In this study, we investigated DAX1 expression in adrenocortical tumors (ACTs) and correlated it with SF1 expression and clinical parameters. DAX1 and SF1 protein expression were assessed in 104 ACTs from 34 children (25 clinically benign and 9 malignant) and 70 adults (40 adenomas and 30 carcinomas). DAX1 gene expression was studied in 49 ACTs by quantitative real-time PCR. A strong DAX1 protein expression was demonstrated in 74% (25 out of 34) and 24% (17 out of 70) of pediatric and adult ACTs, respectively (χ(2)=10.1, p=0.002). In the pediatric group, ACTs with a strong DAX1 expression were diagnosed at earlier ages than ACTs with weak expression [median 1.2 (range, 0.5-4.5) vs. 2.2 (0.9-9.4), p=0.038]. DAX1 expression was not associated with functional status in ACTs. Interestingly, a positive correlation was observed between DAX1 and SF1 protein expression in both pediatric and adult ACTs (r=0.55 for each group separately; p<0.0001). In addition, DAX1 gene expression was significantly correlated with SF1 gene expression (p<0.0001, r=0.54). In conclusion, DAX1 strong protein expression was more frequent in pediatric than in adult ACTs. Additionally, DAX1 and SF1 expression positively correlated in ACTs, suggesting that these transcription factors might cooperate in adrenocortical tumorigenesis. PMID:25985323

  19. Conventional chemotherapy and emerging targeted therapy for advanced adrenocortical carcinoma.

    PubMed

    Xu, Yun-Ze; Zhu, Yu

    2013-02-01

    Adrenocortical carcinoma (ACC) is a rare but typically aggressive malignancy. Radical surgery remains the potentially curative option. However, about one third of patients initially present with distant metastases. Regarding to chemotherapy, mitotane alone or in combination with cytotoxic drugs should be the first selection. Meanwhile, a phase lll clinical trial of etoposide, doxorubicin, cisplatin plus mitotane or streptozotocin plus mitotane is currently undergoing worldwide. The study on molecular pathogenesis of ACC is progressing. A lot of targeted therapies are also enrolled in preclinical investigations and clinical trials, including small-molecule tyrosine kinase inhibitors, antiangiogenic compounds. This article introduced the conventional chemotherapy, newly developed targeted therapy for advanced ACC.

  20. Effects of centrifugation on gonadal and adrenocortical steroids in rats

    NASA Technical Reports Server (NTRS)

    Kakihana, R.; Butte, J. C.

    1980-01-01

    Many endocrine systems are sensitive to external changes in the environment. Both the pituitary adrenal and pituitary gonadal systems are affected by stress including centrifugation stress. The effect of centrifugation on the pituitary gonadal and pituitary adrenocortical systems was examined by measuring the gonadal and adrenal steroids in the plasma and brain following different duration and intensity of centrifugation stress in rats. Two studies were completed and the results are presented. The second study was carried out to describe the developmental changes of brain, plasma and testicular testosterone and dihydrotestosterone in Sprague Dawley rats so that the effect of centrifugation stress on the pituitary gonadal syatem could be better evaluated in future studies.

  1. Response of a mouse hybridoma cell line to heat shock, agitation, and sparging

    NASA Technical Reports Server (NTRS)

    Passini, Cheryl A.; Goochee, Charles F.

    1989-01-01

    A mouse hybridoma cell line is used as a model system for studying the effect of environmental stress on attachment-independent mammalian cells. The full time course of recovery for a mouse hybridoma cell line from both a mild and intermediate heat shock is examined. The pattern of intracellular synthesis is compared for actively growing, log phase cells and nondividing, stationary phase cells.

  2. 9 CFR 113.52 - Requirements for cell lines used for production of biologics.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... 9 Animals and Animal Products 1 2011-01-01 2011-01-01 false Requirements for cell lines used for... STANDARD REQUIREMENTS Ingredient Requirements § 113.52 Requirements for cell lines used for production of... cell line used to prepare a biological product shall be tested as prescribed in this section. A...

  3. 9 CFR 113.52 - Requirements for cell lines used for production of biologics.

    Code of Federal Regulations, 2012 CFR

    2012-01-01

    ... 9 Animals and Animal Products 1 2012-01-01 2012-01-01 false Requirements for cell lines used for... STANDARD REQUIREMENTS Ingredient Requirements § 113.52 Requirements for cell lines used for production of... cell line used to prepare a biological product shall be tested as prescribed in this section. A...

  4. 9 CFR 113.52 - Requirements for cell lines used for production of biologics.

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    ... 9 Animals and Animal Products 1 2013-01-01 2013-01-01 false Requirements for cell lines used for... STANDARD REQUIREMENTS Ingredient Requirements § 113.52 Requirements for cell lines used for production of... cell line used to prepare a biological product shall be tested as prescribed in this section. A...

  5. 9 CFR 113.52 - Requirements for cell lines used for production of biologics.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... 9 Animals and Animal Products 1 2010-01-01 2010-01-01 false Requirements for cell lines used for... STANDARD REQUIREMENTS Ingredient Requirements § 113.52 Requirements for cell lines used for production of... cell line used to prepare a biological product shall be tested as prescribed in this section. A...

  6. 9 CFR 113.52 - Requirements for cell lines used for production of biologics.

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ... 9 Animals and Animal Products 1 2014-01-01 2014-01-01 false Requirements for cell lines used for... STANDARD REQUIREMENTS Ingredient Requirements § 113.52 Requirements for cell lines used for production of... cell line used to prepare a biological product shall be tested as prescribed in this section. A...

  7. Establishment of an ASPL-TFE3 renal cell carcinoma cell line (S-TFE).

    PubMed

    Hirobe, Megumi; Masumori, Naoya; Tanaka, Toshiaki; Kitamura, Hiroshi; Tsukamoto, Taiji

    2013-06-01

    Xp11 translocation renal cell carcinoma is a rare disease diagnosed in children and adolescents in the advanced stage with an aggressive clinical course. Various gene fusions including the transcription factor E3 (TFE3) gene located on chromosome X cause the tumor. We established an Xp11 translocation renal cell carcinoma cell line from a renal tumor in a 18-y-old Japanese female and named it "S-TFE." The cell line and its xenograft demonstrated definite gene fusion including TFE3. They showed strong nuclear staining for TFE3 in immunohistochemistry, TFE3 gene rearrangement in dual-color, break-apart FISH analysis and ASPL-TFE3 type 1 fusion transcripts detected by RT-PCR and direct DNA sequencing. Although many renal cell carcinoma cell lines have been established and investigated, only a few cell lines are recognized as Xp11.2 translocation carcinoma. S-TFE will be useful to examine the characteristics and drug susceptibility of Xp11 translocation renal cell carcinoma. PMID:23760492

  8. 3-Bromopyruvate induces necrotic cell death in sensitive melanoma cell lines

    SciTech Connect

    Qin, J.-Z.; Xin, H.; Nickoloff, B.J.

    2010-05-28

    Clinicians successfully utilize high uptake of radiolabeled glucose via PET scanning to localize metastases in melanoma patients. To take advantage of this altered metabolome, 3-bromopyruvate (BrPA) was used to overcome the notorious resistance of melanoma to cell death. Using four melanoma cell lines, BrPA triggered caspase independent necrosis in two lines, whilst the other two lines were resistant to killing. Mechanistically, sensitive cells differed from resistant cells by; constitutively lower levels of glutathione, reduction of glutathione by BrPA only in sensitive cells; increased superoxide anion reactive oxygen species, loss of outer mitochondrial membrane permeability, and rapid ATP depletion. Sensitive cell killing was blocked by N-acetylcysteine or glutathione. When glutathione levels were reduced in resistant cell lines, they became sensitive to killing by BrPA. Taken together, these results identify a metabolic-based Achilles' heel in melanoma cells to be exploited by use of BrPA. Future pre-clinical and clinical trials are warranted to translate these results into improved patient care for individuals suffering from metastatic melanoma.

  9. Development of a cell line from the American eel brain expressing endothelial cell properties.

    PubMed

    Bloch, Sophia R; Vo, Nguyen T K; Walsh, Sarah K; Chen, Cici; Lee, Lucy E J; Hodson, Peter V; Bols, Niels C

    2016-04-01

    A cell line (eelB) was developed from the outgrowth of adherent cells from brain explants of the American eel, Anguilla rostrata (Lesueur). EelB cells have been grown routinely in L-15 with 10% fetal bovine serum (FBS), undergone over 100 passages, and cryopreserved successfully. The cells from late-passage cultures (>45) were polygonal, formed capillary-like structures (CLS) on Matrigel, and stained immunocytochemically for von Willebrand factor (vWF) and for three tight junction proteins, zonula occludens-1 (ZO-1), claudin 3, and claudin 5. These results suggest that eelB is an endothelial cell line, one of the few from fish and the first from the brain. Despite this, eelB did not respond to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) with the induction of CYP1A protein. The cells from early-passage cultures (<20) had more varied shapes and did not form CLS on Matrigel. Only cells from early-passage cultures formed in suspension three-dimensional aggregates that had some cells expressing alkaline phosphatase and nestin. These cells are thought to be neural stem cells and the aggregates neurospheres. The emergence of endothelial-like cells upon the continued subcultivation of cells from early-passage cultures that had neural stem cells has been described previously for mammals, but this is a first for teleosts. Remarkably, cells from all passage levels were stained strongly for senescence-associated β-galactosidase (SA β-Gal) activity.

  10. Identification of a mitotic death signature in cancer cell lines.

    PubMed

    Sakurikar, Nandini; Eichhorn, Joshua M; Alford, Sarah E; Chambers, Timothy C

    2014-02-28

    This study examined the molecular mechanism of action of anti-mitotic drugs. The hypothesis was tested that death in mitosis occurs through sustained mitotic arrest with robust Cdk1 signaling causing complete phosphorylation of Mcl-1 and Bcl-xL, and conversely, that mitotic slippage is associated with incomplete phosphorylation of Mcl-1/Bcl-xL. The results, obtained from studying six different cancer cell lines, strongly support the hypothesis and identify for the first time a unique molecular signature for mitotic death. The findings represent an important advance in understanding anti-mitotic drug action and provide insight into cancer cell susceptibility to such drugs which has important clinical implications. PMID:24099917

  11. Identification of a mitotic death signature in cancer cell lines.

    PubMed

    Sakurikar, Nandini; Eichhorn, Joshua M; Alford, Sarah E; Chambers, Timothy C

    2014-02-28

    This study examined the molecular mechanism of action of anti-mitotic drugs. The hypothesis was tested that death in mitosis occurs through sustained mitotic arrest with robust Cdk1 signaling causing complete phosphorylation of Mcl-1 and Bcl-xL, and conversely, that mitotic slippage is associated with incomplete phosphorylation of Mcl-1/Bcl-xL. The results, obtained from studying six different cancer cell lines, strongly support the hypothesis and identify for the first time a unique molecular signature for mitotic death. The findings represent an important advance in understanding anti-mitotic drug action and provide insight into cancer cell susceptibility to such drugs which has important clinical implications.

  12. Control of Differentiation of a Mammary Cell Line by Lipids

    NASA Astrophysics Data System (ADS)

    Dulbecco, Renato; Bologna, Mauro; Unger, Michael

    1980-03-01

    A rat mammary cell line (LA7) undergoes spontaneous differentiation into domes due to production of specific inducers by the cells. Some of these inducers may be lipids, and we show that lipids regulate this differentiation as both inducers and inhibitors. One inhibitor is the tumor promoter tetradecanoyl-13 phorbol 12-acetate. The inducers are saturated fatty acids of two groups: butyric acid and acids with chain lengths from C13 to C16, especially myristic acid (C14). Other inducers are myristoyl and palmitoyl lysolecithins, myristic acid methyl ester, and two cationic detergents with a tetradecenyl chain. We propose that the lipids with a C14-C16 alkyl chain affect differentiation by recognizing specific receptors through their alkyl chains and that the effects obtained depend on the head groups. These lipids may be physiological regulators in the mammary gland.

  13. In vitro generation of hematopoietic stem cells from an embryonic stem cell line.

    PubMed Central

    Palacios, R; Golunski, E; Samaridis, J

    1995-01-01

    Hematopoietic stem cells (HSC) are unique in that they give rise both to new stem cells (self-renewal) and to all blood cell types. The cellular and molecular events responsible for the formation of HSC remain unknown mainly because no system exists to study it. Embryonic stem (ES) cells were induced to differentiate by coculture with the stromal cell line RP010 and the combination of interleukin (IL) 3, IL-6, and F (cell-free supernatants from cultures of the FLS4.1 fetal liver stromal cell line). Cell cytometry analysis of the mononuclear cells produced in the cultures was consistent with the presence of PgP-1+ Lin- early hematopoietic (B-220- Mac-1- JORO 75- TER 119-) cells and of fewer B-220+ IgM- B-cell progenitors and JORO 75+ T-lymphocyte progenitors. The cell-sorter-purified PgP-1+ Lin- cells produced by induced ES cells could repopulate the lymphoid, myeloid, and erythroid lineages of irradiated mice. The ES-derived PgP-1+ Lin- cells must possess extensive self-renewal potential, as they were able to produce hematopoietic repopulation of secondary mice recipients. Indeed, marrow cells from irradiated mice reconstituted (15-18 weeks before) with PgP-1+ Lin- cell-sorter-purified cells generated by induced ES cells repopulated the lymphoid, myeloid, and erythroid lineages of secondary mouse recipients assessed 16-20 weeks after their transfer into irradiated secondary mice. The results show that the culture conditions described here support differentiation of ES cells into hematopoietic cells with functional properties of HSC. It should now be possible to unravel the molecular events leading to the formation of HSC. Images Fig. 3 PMID:7638225

  14. Characterisation and manipulation of docetaxel resistant prostate cancer cell lines

    PubMed Central

    2011-01-01

    Background There is no effective treatment strategy for advanced castration-resistant prostate cancer. Although Docetaxel (Taxotere®) represents the most active chemotherapeutic agent it only gives a modest survival advantage with most patients eventually progressing because of inherent or acquired drug resistance. The aims of this study were to further investigate the mechanisms of resistance to Docetaxel. Three Docetaxel resistant sub-lines were generated and confirmed to be resistant to the apoptotic and anti-proliferative effects of increasing concentrations of Docetaxel. Results The resistant DU-145 R and 22RV1 R had expression of P-glycoprotein and its inhibition with Elacridar partially and totally reversed the resistant phenotype in the two cell lines respectively, which was not seen in the PC-3 resistant sublines. Resistance was also not mediated in the PC-3 cells by cellular senescence or autophagy but multiple changes in pro- and anti-apoptotic genes and proteins were demonstrated. Even though there were lower basal levels of NF-κB activity in the PC-3 D12 cells compared to the Parental PC-3, docetaxel induced higher NF-κB activity and IκB phosphorylation at 3 and 6 hours with only minor changes in the DU-145 cells. Inhibition of NF-κB with the BAY 11-7082 inhibitor reversed the resistance to Docetaxel. Conclusion This study confirms that multiple mechanisms contribute to Docetaxel resistance and the central transcription factor NF-κB plays an immensely important role in determining docetaxel-resistance which may represent an appropriate therapeutic target. PMID:21982118

  15. First Case Report of a Sporadic Adrenocortical Carcinoma With Gastric Metastasis and a Synchronous Gastrointestinal Stromal Tumor of the Stomach.

    PubMed

    Kovecsi, Attila; Jung, Ioan; Bara, Tivadar; Bara, Tivadar; Azamfirei, Leonard; Kovacs, Zsolt; Gurzu, Simona

    2015-09-01

    Adrenocortical carcinoma is a rare tumor with high aggresivity that can associate systemic metastases. A 71-year-old man was hospitalized for gastric cancer. The abdominal computed tomography also revealed a tumor above the right kidney. Total gastrectomy and right adrenalectomy were performed. The encapsulated tumor of the adrenal gland weighed 560 grams and presented diffuse tumor architecture under microscope, with capsular, sinusoidal, and vascular invasion. The large tumor cells had a polygonal shape, with slight basophilic, eosinophilic, or vacuolated cytoplasm, pleomorphic nuclei, and a high mitotic rate. In the stomach, the protruded tumor was covered by normal mucosa; under microscope, the tumor cells were observed only in the submucosal layer. In primary adrenal tumor and gastric metastasis the tumor cells were marked by vimentin, inhibin, synaptophysin, neuron-specific enolase, and calretinin. Based on these criteria, the diagnosis of adrenocortical carcinoma (ACC) with gastric metastasis and no lymph node metastases was established. A synchronous 10 × 10-mm-sized gastrointestinal stromal tumor (GIST) of the stomach, without mitoses, was also identified. So far, as we know, this is the 15th case of ever reported synchronous/metachronous sporadic ACCs; the ACC-related gastric metastases either synchronous ACC and GIST, has not been reported in the literature previously.

  16. Oxidative stress induces hypomethylation of LINE-1 and hypermethylation of the RUNX3 promoter in a bladder cancer cell line.

    PubMed

    Wongpaiboonwattana, Wikrom; Tosukhowong, Piyaratana; Dissayabutra, Thasinas; Mutirangura, Apiwat; Boonla, Chanchai

    2013-01-01

    Increased oxidative stress and changes in DNA methylation are frequently detected in bladder cancer patients. We previously demonstrated a relationship between increased oxidative stress and hypomethylation of the transposable long-interspersed nuclear element-1 (LINE-1). Promoter hypermethylation of a tumor suppressor gene, runt-related transcription factor 3 (RUNX3), may also be associated with bladder cancer genesis. In this study, we investigated changes of DNA methylation in LINE-1 and RUNX3 promoter in a bladder cancer cell (UM-UC-3) under oxidative stress conditions, stimulated by challenge with H2O2 for 72 h. Cells were pretreated with an antioxidant, tocopheryl acetate for 1 h to attenuate oxidative stress. Methylation levels of LINE-1 and RUNX3 promoter were measured by combined bisulfite restriction analysis PCR and methylation-specific PCR, respectively. Levels of LINE-1 methylation were significantly decreased in H2O2-treated cells, and reestablished after pretreated with tocopheryl acetate. Methylation of RUNX3 promoter was significantly increased in cells exposed to H2O2. In tocopheryl acetate pretreated cells, it was markedly decreased. In conclusion, hypomethylation of LINE-1 and hypermethylation of RUNX3 promoter in bladder cancer cell line was experimentally induced by reactive oxygen species (ROS). The present findings support the hypothesis that oxidative stress promotes urothelial cell carcinogenesis through modulation of DNA methylation. Our data also imply that mechanistic pathways of ROS-induced alteration of DNA methylation in a repetitive DNA element and a gene promoter might differ.

  17. The MDCK epithelial cell line expresses a cell surface antigen of the kidney distal tubule

    PubMed Central

    1982-01-01

    Monoclonal antibodies were prepared against the Madin-Darby canine kidney (MDCK) cell line to identify epithelial cell surface macromolecules involved in renal function. Lymphocyte hybrids were generated by fusing P3U-1 myeloma cells with spleen cells from a C3H mouse immunized with MDCK cells. Hybridomas secreting anti-MDCK antibodies were obtained and clonal lines isolated in soft agarose. We are reporting on one hybridoma line that secretes a monoclonal antibody that binds to MDCK cells at levels 20-fold greater than background binding. Indirect immunofluorescence microscopy was utilized to study the distribution of antibody binding on MDCK cells and on frozen sections of dog kidney and several nonrenal tissues. In the kidney the fluorescence staining pattern demonstrates that the antibody recognizes an antigenic determinant that is expressed only on the epithelial cells of the thick ascending limb of Henle's loops and the distal convoluted tubule and appears to be localized on the basolateral plasma membrane. This antigen also has a unique distribution in non-renal tissues and can only be detected on cells known to be active in transepithelial ion movements. These results indicate the probable distal tubule origin of MDCK and suggest that the monoclonal antibody recognizes a cell surface antigen involved in physiological functions unique to the kidney distal tubule and transporting epithelia of nonrenal tissues. PMID:6178742

  18. Epigenetic alterations differ in phenotypically distinct human neuroblastoma cell lines

    PubMed Central

    2010-01-01

    Background Epigenetic aberrations and a CpG island methylator phenotype have been shown to be associated with poor outcomes in children with neuroblastoma (NB). Seven cancer related genes (THBS-1, CASP8, HIN-1, TIG-1, BLU, SPARC, and HIC-1) that have been shown to have epigenetic changes in adult cancers and play important roles in the regulation of angiogenesis, tumor growth, and apoptosis were analyzed to investigate the role epigenetic alterations play in determining NB phenotype. Methods Two NB cell lines (tumorigenic LA1-55n and non-tumorigenic LA1-5s) that differ in their ability to form colonies in soft agar and tumors in nude mice were used. Quantitative RNA expression analyses were performed on seven genes in LA1-5s, LA1-55n and 5-Aza-dC treated LA1-55n NB cell lines. The methylation status around THBS-1, HIN-1, TIG-1 and CASP8 promoters was examined using methylation specific PCR. Chromatin immunoprecipitation assay was used to examine histone modifications along the THBS-1 promoter. Luciferase assay was used to determine THBS-1 promoter activity. Cell proliferation assay was used to examine the effect of 5-Aza-dC on NB cell growth. The soft agar assay was used to determine the tumorigenicity. Results Promoter methylation values for THBS-1, HIN-1, TIG-1, and CASP8 were higher in LA1-55n cells compared to LA1-5s cells. Consistent with the promoter methylation status, lower levels of gene expression were detected in the LA1-55n cells. Histone marks associated with repressive chromatin states (H3K9Me3, H3K27Me3, and H3K4Me3) were identified in the THBS-1 promoter region in the LA1-55n cells, but not the LA1-5s cells. In contrast, the three histone codes associated with an active chromatin state (acetyl H3, acetyl H4, and H3K4Me3) were present in the THBS-1 promoter region in LA1-5s cells, but not the LA1-55n cells, suggesting that an accessible chromatin structure is important for THBS-1 expression. We also show that 5-Aza-dC treatment of LA1-55n cells

  19. Origin of the U87MG glioma cell line: Good news and bad news.

    PubMed

    Allen, Marie; Bjerke, Mia; Edlund, Hanna; Nelander, Sven; Westermark, Bengt

    2016-08-31

    Human tumor-derived cell lines are indispensable tools for basic and translational oncology. They have an infinite life span and are easy to handle and scalable, and results can be obtained with high reproducibility. However, a tumor-derived cell line may not be authentic to the tumor of origin. Two major questions emerge: Have the identity of the donor and the actual tumor origin of the cell line been accurately determined? To what extent does the cell line reflect the phenotype of the tumor type of origin? The importance of these questions is greatest in translational research. We have examined these questions using genetic profiling and transcriptome analysis in human glioma cell lines. We find that the DNA profile of the widely used glioma cell line U87MG is different from that of the original cells and that it is likely to be a bona fide human glioblastoma cell line of unknown origin.

  20. Origin of the U87MG glioma cell line: Good news and bad news.

    PubMed

    Allen, Marie; Bjerke, Mia; Edlund, Hanna; Nelander, Sven; Westermark, Bengt

    2016-08-31

    Human tumor-derived cell lines are indispensable tools for basic and translational oncology. They have an infinite life span and are easy to handle and scalable, and results can be obtained with high reproducibility. However, a tumor-derived cell line may not be authentic to the tumor of origin. Two major questions emerge: Have the identity of the donor and the actual tumor origin of the cell line been accurately determined? To what extent does the cell line reflect the phenotype of the tumor type of origin? The importance of these questions is greatest in translational research. We have examined these questions using genetic profiling and transcriptome analysis in human glioma cell lines. We find that the DNA profile of the widely used glioma cell line U87MG is different from that of the original cells and that it is likely to be a bona fide human glioblastoma cell line of unknown origin. PMID:27582061

  1. Human embryonic stem cell lines derived from single blastomeres of two 4-cell stage embryos

    PubMed Central

    Geens, Mieke; Mateizel, Ileana; Sermon, Karen; De Rycke, Martine; Spits, Claudia; Cauffman, Greet; Devroey, Paul; Tournaye, Herman; Liebaers, Inge; Van de Velde, Hilde

    2009-01-01

    BACKGROUND Recently, we demonstrated that single blastomeres of a 4-cell stage human embryo are able to develop into blastocysts with inner cell mass and trophectoderm. To further investigate potency at the 4-cell stage, we aimed to derive pluripotent human embryonic stem cells (hESC) from single blastomeres. METHODS Four 4-cell stage embryos were split on Day 2 of preimplantation development and the 16 blastomeres were individually cultured in sequential medium. On Day 3 or 4, the blastomere-derived embryos were plated on inactivated mouse embryonic fibroblasts (MEFs). RESULTS Ten out of sixteen blastomere-derived morulae attached to the MEFs, and two produced an outgrowth. They were mechanically passaged onto fresh MEFs as described for blastocyst ICM-derived hESC, and shown to express the typical stemness markers by immunocytochemistry and/or RT–PCR. In vivo pluripotency was confirmed by the presence of all three germ layers in the teratoma obtained after injection in immunodeficient mice. The first hESC line displays a mosaic normal/abnormal 46, XX, dup(7)(q33qter), del(18)(q23qter) karyotype. The second hESC line displays a normal 46, XY karyotype. CONCLUSION We report the successful derivation and characterization of two hESC lines from single blastomeres of four split 4-cell stage human embryos. These two hESC lines were derived from distinct embryos, proving that at least one of the 4-cell stage blastomeres is pluripotent. PMID:19633307

  2. Characterization of a mantle cell lymphoma cell line resistant to the Chk1 inhibitor PF-00477736.

    PubMed

    Restelli, Valentina; Chilà, Rosaria; Lupi, Monica; Rinaldi, Andrea; Kwee, Ivo; Bertoni, Francesco; Damia, Giovanna; Carrassa, Laura

    2015-11-10

    Mantle cell lymphoma (MCL) is an aggressive B-cell lymphoma characterized by the chromosomal translocation t(11;14) that leads to constitutive expression of cyclin D1, a master regulator of the G1-S phase. Chk1 inhibitors have been recently shown to be strongly effective as single agents in MCL. To investigate molecular mechanisms at the basis of Chk1 inhibitor activity, a MCL cell line resistant to the Chk1 inhibitor PF-00477736 (JEKO-1 R) was obtained and characterized. The JEKO-1 R cell line was cross resistant to another Chk1 inhibitor (AZD-7762) and to the Wee1 inhibitor MK-1775. It displayed a shorter doubling time than parental cell line, likely due to a faster S phase. Cyclin D1 expression levels were decreased in resistant cell line and its re-overexpression partially re-established PF-00477736 sensitivity. Gene expression profiling showed an enrichment in gene sets involved in pro-survival pathways in JEKO-1 R. Dasatinib treatment partly restored PF-00477736 sensitivity in resistant cells suggesting that the pharmacological interference of pro-survival pathways can overcome the resistance to Chk1 inhibitors. These data further corroborate the involvement of the t(11;14) in cellular sensitivity to Chk1 inhibitors, fostering the clinical testing of Chk1 inhibitors as single agents in MCL. PMID:26439697

  3. Characterization of a mantle cell lymphoma cell line resistant to the Chk1 inhibitor PF-00477736

    PubMed Central

    Restelli, Valentina; Chilà, Rosaria; Lupi, Monica; Rinaldi, Andrea; Kwee, Ivo; Bertoni, Francesco; Damia, Giovanna; Carrassa, Laura

    2015-01-01

    Mantle cell lymphoma (MCL) is an aggressive B-cell lymphoma characterized by the chromosomal translocation t(11;14) that leads to constitutive expression of cyclin D1, a master regulator of the G1-S phase. Chk1 inhibitors have been recently shown to be strongly effective as single agents in MCL. To investigate molecular mechanisms at the basis of Chk1 inhibitor activity, a MCL cell line resistant to the Chk1 inhibitor PF-00477736 (JEKO-1 R) was obtained and characterized. The JEKO-1 R cell line was cross resistant to another Chk1 inhibitor (AZD-7762) and to the Wee1 inhibitor MK-1775. It displayed a shorter doubling time than parental cell line, likely due to a faster S phase. Cyclin D1 expression levels were decreased in resistant cell line and its re-overexpression partially re-established PF-00477736 sensitivity. Gene expression profiling showed an enrichment in gene sets involved in pro-survival pathways in JEKO-1 R. Dasatinib treatment partly restored PF-00477736 sensitivity in resistant cells suggesting that the pharmacological interference of pro-survival pathways can overcome the resistance to Chk1 inhibitors. These data further corroborate the involvement of the t(11;14) in cellular sensitivity to Chk1 inhibitors, fostering the clinical testing of Chk1 inhibitors as single agents in MCL. PMID:26439697

  4. "Fluorescent Cell Chip" for immunotoxicity testing: development of the c-fos expression reporter cell lines.

    PubMed

    Trzaska, Dominika; Zembek, Patrycja; Olszewski, Maciej; Adamczewska, Violetta; Ullerås, Erik; Dastych, Jarosław

    2005-09-01

    The Fluorescent Cell Chip for in vitro immunotoxicity testing employs cell lines derived from lymphocytes, mast cells, and monocytes-macrophages transfected with various EGFP cytokine reporter gene constructs. While cytokine expression is a valid endpoint for in vitro immunotoxicity screening, additional marker for the immediate-early response gene expression level could be of interest for further development and refinement of the Fluorescent Cell Chip. We have used BW.5147.3 murine thymoma transfected with c-fos reporter constructs to obtain reporter cell lines expressing ECFP under the control of murine c-fos promoter. These cells upon serum withdrawal and readdition and incubation with heavy metal compounds showed paralleled induction of c-Fos expression as evidenced by Real-Time PCR and ECFP fluorescence as evidenced by computer-supported fluorescence microscopy. In conclusion, we developed fluorescent reporter cell lines that could be employed in a simple and time-efficient screening assay for possible action of chemicals on c-Fos expression in lymphocytes. The evaluation of usefulness of these cells for the Fluorescent Cell Chip-based detection of immunotoxicity will require additional testing with a larger number of chemicals.

  5. PACAP protects against TNFα-induced cell death in olfactory epithelium and olfactory placodal cell lines

    PubMed Central

    Kanekar, Shami; Gandham, Mahendra; Lucero, Mary T

    2010-01-01

    In mouse olfactory epithelium (OE), pituitary adenylate cyclase activating peptide (PACAP) protects against axotomy-induced apoptosis. We used mouse OE to determine whether PACAP protects neurons during exposure to the inflammatory cytokine TNFα. Live slices of neonatal mouse OE were treated with 40 ng/ml TNFα ± 40 nM PACAP for 6 hours and dying cells were live-labeled with 0.5% propidium iodide. TNFα significantly increased the percentage of dying cells while co-incubation with PACAP prevented cell death. PACAP also prevented TNFα-mediated cell death in the olfactory placodal (OP) cell lines, OP6 and OP27. Although OP cell lines express all three PACAP receptors (PAC1, VPAC1,VPAC2), PACAP’s protection of these cells from TNFα was mimicked by the specific PAC1 receptor agonist maxadilan and abolished by the PAC1 antagonist PACAP6–38. Treatment of OP cell lines with blockers or activators of the PLC and AC/MAPKK pathways revealed that PACAP-mediated protection from TNFα involved both pathways. PACAP may therefore function through PAC1 receptors to protect neurons from cell death during inflammatory cytokine release in vivo as would occur upon viral infection or allergic rhinitis-associated injury. PMID:20654718

  6. Different toxic effects of YTX in tumor K-562 and lymphoblastoid cell lines

    PubMed Central

    Fernández-Araujo, Andrea; Sánchez, Jon A.; Alfonso, Amparo; Vieytes, Mercedes R.; Botana, Luis M.

    2015-01-01

    Yessotoxin (YTX) modulates cellular phosphodiesterases (PDEs). In this regard, opposite effects had been described in the tumor model K-562 cell line and fresh human lymphocytes in terms of cell viability, cyclic adenosine 3',5'-cyclic monophosphate (cAMP) production and protein expression after YTX treatment. Studies in depth of the pathways activated by YTX in K-562 cell line, have demonstrated the activation of two different cell death types, apoptosis, and autophagy after 24 and 48 h of treatment, respectively. Furthermore, the key role of type 4A PDE (PDE4A) in both pathways activated by YTX was demonstrated. Therefore, taking into account the differences between cellular lines and fresh cells, a study of cell death pathways activated by YTX in a non-tumor cell line with mitotic activity, was performed. The cellular model used was the lymphoblastoid cell line that represents a non-tumor model with normal apoptotic and mitotic machinery. In this context, cell viability and cell proliferation, expression of proteins involved in cell death activated by YTX and mitochondrial mass, were studied after the incubation with the toxin. Opposite to the tumor model, no cell death activation was observed in lymphoblastoid cell line in the presence of YTX. In this sense, variations in apoptosis hallmarks were not detected in the lymphoblastoid cell line after YTX incubation, whereas this type I of programmed cell death was observed in K-562 cells. On the other hand, autophagy cell death was triggered in this cellular line, while other autophagic process is suggested in lymphoblastoid cells. These YTX effects are related to PDE4A in both cellular lines. In addition, while cell death is triggered in K-562 cells after YTX treatment, in lymphoblastoid cells the toxin stops cellular proliferation. These results point to YTX as a specific toxic compound of tumor cells, since in the non-tumor lymphoblastoid cell line, no cell death hallmarks are observed. PMID:26136685

  7. The Cancer-Related Transcription Factor Runx2 Modulates Cell Proliferation in Human Osteosarcoma Cell Lines

    PubMed Central

    Lucero, Claudia M.J.; Vega, Oscar A.; Osorio, Mariana M.; Tapia, Julio C.; Antonelli, Marcelo; Stein, Gary S.; Van Wijnen, Andre J.; Galindo, Mario A.

    2013-01-01

    Runx2 regulates osteogenic differentiation and bone formation, but also suppresses pre-osteoblast proliferation by affecting cell cycle progression in the G1 phase. The growth suppressive potential of Runx2 is normally inactivated in part by protein destabilization, which permits cell cycle progression beyond the G1/S phase transition, and Runx2 is again up-regulated after mitosis. Runx2 expression also correlates with metastasis and poor chemotherapy response in osteosarcoma. Here we show that six human osteosarcoma cell lines (SaOS, MG63, U2OS, HOS, G292, and 143B) have different growth rates, which is consistent with differences in the lengths of the cell cycle. Runx2 protein levels are cell cycle-regulated with respect to the G1/S phase transition in U2OS, HOS, G292, and 143B cells. In contrast, Runx2 protein levels are constitutively expressed during the cell cycle in SaOS and MG63 cells. Forced expression of Runx2 suppresses growth in all cell lines indicating that accumulation of Runx2 in excess of its pre-established levels in a given cell type triggers one or more anti-proliferative pathways in osteosarcoma cells. Thus, regulatory mechanisms controlling Runx2 expression in osteosarcoma cells must balance Runx2 protein levels to promote its putative oncogenic functions, while avoiding suppression of bone tumor growth. PMID:22949168

  8. Gene expression profiling analysis of osteosarcoma cell lines

    PubMed Central

    SUN, LU; LI, JIE; YAN, BING

    2015-01-01

    Osteosarcoma (OS) is the most common type of primary bone malignancy and has a poor prognosis. To investigate the mechanisms of osteosarcoma, the present analyzed the GSE28424 microarray. GSE28424 was downloaded from the Gene Expression Omnibus, and included a collective of 19 OS cell lines and four normal bone cell lines, which were used as controls. Subsequently, the differentially expressed genes (DEGs) were screened using the Limma package in Bioconductor. Gene Ontology (GO) and pathway enrichment analysis of the DEGs was performed using the Database for Annotation, Visualization and Integrated Discovery, interactions between the proteins encoded by the DEGs were identified using STRING, and the protein-protein interaction (PPI) network was visualized using Cytoscape. In addition, modular analysis of the PPI network was performed using the Clique Percolation Method (CPM) in CFinder. A total of 1,170 DEGs were screened, including 530 upreguated and 640 downregulated genes. The enriched functions included organelle fission, immune response and response to wounding. In addition, RPL8 was observed to be involved with the ribosomal pathway in module A of the PPI network of the DEGs. PLCG1, SYK and PLCG2 were also involved in the B-cell receptor signaling pathway in module B and the Fc-epsilon RI signaling pathway in module C. In addition, AURKA (degree=39), MAD2L1 (degree=38), CDCA8 (degree=38), BUB1 (degree=37) and MELK (degree=37) exhibited higher degrees of connectivity in module F. The results of the present study suggested that the RPL8, PLCG1, PLCG2, SYK, MAD2L1, AURKA, CDCA8, BUB1 and MELK genes may be involved in OS. PMID:26096802

  9. Postnatal foraging demands alter adrenocortical activity and psychosocial development.

    PubMed

    Lyons, D M; Kim, S; Schatzberg, A F; Levine, S

    1998-05-01

    Mother squirrel monkeys stop carrying infants at earlier ages in high-demand (HD) conditions where food is difficult to find relative to low-demand (LD) conditions. To characterize these transitions in psychosocial development, from 10- to 21-weeks postpartum we collected measures of behavior, adrenocortical activity, and social transactions coded for initiator (mother or infant), goal (make-contact or break-contact), and outcome (success or failure). Make-contact attempts were most often initiated by HD infants, but mothers often opposed these attempts and less than 50% were successful. Break-contact attempts were most often initiated by LD infants, but mothers often opposed these attempts and fewer LD than HD infant break-contact attempts were successful. Plasma levels of cortisol were significantly higher in HD than LD mothers, but differences in adrenocortical activity were less consistent in their infants. HD and LD infants also spent similar amounts of time nursing on their mothers and feeding on solid foods. By rescheduling some transitions in development (carry-->self-transport), and not others (nursing-->self-feeding), mothers may have partially protected infants from the immediate impact of an otherwise stressful foraging task. PMID:9589217

  10. Adrenocortical adaptation to chronic intermittent stress in hemispherectomized pigeon.

    PubMed

    Ramade, F; Bayle, J D

    1984-07-01

    Hemispherectomized pigeons were exposed daily to electrical footshocks delivered for 15 sec, at the same hour, for 8 weeks. Serial blood samples were obtained through a chronic vascular catheter. The adrenocortical response to chronic intermittent stress was measured kinetically at one week intervals. The initial response including several successive peaks of plasma corticosterone progressively adapted: Late peaks disappeared and only the first one subsisted 12-14 min after stressor application; this first peak diminished in magnitude; furthermore, an anticipatory peak occurred, starting 14 min before stress. In pigeons lesioned in the anterior dorsomedial thalamus, the only response to the stressor was of the single peak (12-14 min) type without any development of anticipatory conditioned response. This phenomenon was consistant all over the experimental period. Thalamic-hypothalamic interrelationships may be suggested to provide neuronal loops that underlie the long lasting, pulsatile repetitive components of the adrenocortical response to acute stress and also the adaptative process of such a response to chronic intermittent stress, including a conditioned, anticipatory endocrine activation. PMID:6505055

  11. Mechanisms of lymphocyte adhesion to endothelial cells: studies using a LFA-1-deficient cell line.

    PubMed Central

    Haskard, D O; Strobel, S; Thornhill, M; Pitzalis, C; Levinsky, R J

    1989-01-01

    In order to investigate the role of lymphocyte function-associated antigen 1 (LFA-1) in lymphocyte adhesion to endothelial cells (EC), we have studied the adhesion of a LFA-1-deficient lymphoblastoid cell line, ICH-KM, which has < 10% of the cell surface LFA-1 expressed on a normal lymphoblastoid cell line, ICH-BJ. The adhesion of ICH-KM cells to unstimulated EC was 49.9 +/- 8.6% (mean +/- SD) that of ICH-BJ cells. Moreover, phorbol ester-stimulated ICH-KM cells showed a considerably weaker increase in adhesion to unstimulated EC compared with ICH-BJ cells (mean +/- SD increase in percentage adhesion, 3.8 +/- 2.3 compared with 18.5 +/- 8.0; P<0.025). In contrast, there was no significant difference between the enhanced adhesion of ICH-KM cells and ICH-BJ cells to interleukin-1 (IL-1)-stimulated EC. Thus ICH-KM cells showed a 22.7 +/- 11.0 (mean +/- SD) increase in percentage adhesion to IL-1-stimulated EC compared with the 24.8 +/- 8.5 increase in percentage adhesion of ICH-BJ cells. Anti-LFA-1 monoclonal antibodies had no effect on the enhanced adhesion of ICH-KM and ICH-BJ cells to IL-1-stimulated EC but abolished the differences in adhesion between the two cell lines. The study therefore indicates that although a major part of unstimulated and phorbol ester-stimulated lymphocyte-EC adhesion is dependent upon LFA-1, the enhanced adhesion due to stimulation of EC with IL-1 is not dependent upon this molecule. The data therefore supports the existence of cytokine-inducible LFA-1-independent adhesion molecules for lymphocytes on EC. PMID:15493272

  12. Standardized orthotopic xenografts in zebrafish reveal glioma cell-line-specific characteristics and tumor cell heterogeneity

    PubMed Central

    Welker, Alessandra M.; Jaros, Brian D.; Puduvalli, Vinay K.; Imitola, Jaime; Kaur, Balveen; Beattie, Christine E.

    2016-01-01

    ABSTRACT Glioblastoma (GBM) is a deadly brain cancer, for which few effective drug treatments are available. Several studies have used zebrafish models to study GBM, but a standardized approach to modeling GBM in zebrafish was lacking to date, preventing comparison of data across studies. Here, we describe a new, standardized orthotopic xenotransplant model of GBM in zebrafish. Dose-response survival assays were used to define the optimal number of cells for tumor formation. Techniques to measure tumor burden and cell spread within the brain over real time were optimized using mouse neural stem cells as control transplants. Applying this standardized approach, we transplanted two patient-derived GBM cell lines, serum-grown adherent cells and neurospheres, into the midbrain region of embryonic zebrafish and analyzed transplanted larvae over time. Progressive brain tumor growth and premature larval death were observed using both cell lines; however, fewer transplanted neurosphere cells were needed for tumor growth and lethality. Tumors were heterogeneous, containing both cells expressing stem cell markers and cells expressing markers of differentiation. A small proportion of transplanted neurosphere cells expressed glial fibrillary acidic protein (GFAP) or vimentin, markers of more differentiated cells, but this number increased significantly during tumor growth, indicating that these cells undergo differentiation in vivo. By contrast, most serum-grown adherent cells expressed GFAP and vimentin at the earliest times examined post-transplant. Both cell types produced brain tumors that contained Sox2+ cells, indicative of tumor stem cells. Transplanted larvae were treated with currently used GBM therapeutics, temozolomide or bortezomib, and this resulted in a reduction in tumor volume in vivo and an increase in survival. The standardized model reported here facilitates robust and reproducible analysis of glioblastoma tumor cells in real time and provides a platform for

  13. Establishment of a highly differentiated thyroid cancer cell line of Hürthle cell origin.

    PubMed

    Zielke, A; Tezelman, S; Jossart, G H; Wong, M; Siperstein, A E; Duh, Q Y; Clark, O H

    1998-06-01

    Hürthle cell carcinomas (HCC) of the thyroid are a variant of follicular thyroid tumors. In contrast to follicular thyroid carcinoma, HCC rarely take up radioiodine and frequently metastasize to the lymph nodes. Histologically they are indistinguishable from Hürthle cell adenomas except for evidence of invasion and metastasis. How these carcinomas develop and why they behave differently than other follicular tumors is not known. Although some differentiated thyroid cancer cell lines exist, none are from Hürthle cell tumors. We have established a well-differentiated thyroid cancer cell line from a metastasis of a HCC, designated XTC.UC1. In vitro, XTC cells display epitheloid morphology, grow with a population doubling time of 4.3 +/- 0.3 days, migrate, and invade through reconstituted basement membranes. The cells are immunoreactive for and release thyroglobulin, respond to thyrotropin (TSH) with increase of intracellular cyclic adenosine monophosphate (cAMP), proliferation, and invasion of reconstituted basement membrane, thus exhibiting characteristics of well-differentiated thyroid carcinoma. In vivo, xenografted XTC cells grow with a doubling time of 9.8 +/- 0.8 days. Tumors spontaneously metastasize to the lymph nodes and less frequently to the lungs and the liver. The cells retained their differentiated function in vivo as assessed by human thyroglobulin (hTG) secretion and immunohistochemistry. This is a first report of the establishment of a unique, highly differentiated thyroid carcinoma cell line derived from an HCC. Based on the ability to invade through reconstituted basement membrane in vitro and the potential to metastasize in vivo, this cell line may provide a unique model to study invasion and metastazation of well-differentiated thyroid cancer.

  14. Natural Killer Cells for Immunotherapy – Advantages of the NK-92 Cell Line over Blood NK Cells

    PubMed Central

    Klingemann, Hans; Boissel, Laurent; Toneguzzo, Frances

    2016-01-01

    Natural killer (NK) cells are potent cytotoxic effector cells for cancer therapy and potentially for severe viral infections. However, there are technical challenges to obtain sufficient numbers of functionally active NK cells from a patient’s blood since they represent only 10% of the lymphocytes and are often dysfunctional. The alternative is to obtain cells from a healthy donor, which requires depletion of the allogeneic T cells to prevent graft-versus-host reactions. Cytotoxic cell lines have been established from patients with clonal NK-cell lymphoma. Those cells can be expanded in culture in the presence of IL-2. Except for the NK-92 cell line, though, none of the other six known NK cell lines has consistently and reproducibly shown high antitumor cytotoxicity. Only NK-92 cells can easily be genetically manipulated to recognize specific tumor antigens or to augment monoclonal antibody activity through antibody-dependent cellular cytotoxicity. NK-92 is also the only cell line product that has been infused into patients with advanced cancer with clinical benefit and minimal side effects. PMID:27014270

  15. Side population cells isolated from KATO III human gastric cancer cell line have cancer stem cell-like characteristics

    PubMed Central

    She, Jun-Jun; Zhang, Peng-Ge; Wang, Xuan; Che, Xiang-Ming; Wang, Zi-Ming

    2012-01-01

    AIM: To investigate whether the side population (SP) cells possess cancer stem cell-like characteristics in vitro and the role of SP cells in tumorigenic process in gastric cancer. METHODS: We analyzed the presence of SP cells in different human gastric carcinoma cell lines, and then isolated and identified the SP cells from the KATO III human gastric cancer cell line by flow cytometry. The clonogenic ability and self-renewal were evaluated by clone and sphere formation assays. The related genes were determined by reverse transcription polymerase chain reaction. To compare tumorigenic ability, SP and non-side population (NSP) cells from the KATO III human gastric cancer cell line were subcutaneously injected into nude mice. RESULTS: SP cells from the total population accounted for 0.57% in KATO III, 1.04% in Hs-746T, and 0.02% in AGS (CRL-1739). SP cells could grow clonally and have self-renewal capability in conditioned media. The expression of ABCG2, MDRI, Bmi-1 and Oct-4 was different between SP and NSP cells. However, there was no apparent difference between SP and NSP cells when they were injected into nude mice. CONCLUSION: SP cells have some cancer stem cell-like characteristics in vitro and can be used for studying the tumorigenic process in gastric cancer. PMID:22969237

  16. Identification of Replication Competent Murine Gammaretroviruses in Commonly Used Prostate Cancer Cell Lines

    PubMed Central

    Sfanos, Karen Sandell; Aloia, Amanda L.; Hicks, Jessica L.; Esopi, David M.; Steranka, Jared P.; Shao, Wei; Sanchez-Martinez, Silvia; Yegnasubramanian, Srinivasan; Burns, Kathleen H.; Rein, Alan; De Marzo, Angelo M.

    2011-01-01

    A newly discovered gammaretrovirus, termed XMRV, was recently reported to be present in the prostate cancer cell line CWR22Rv1. Using a combination of both immunohistochemistry with broadly-reactive murine leukemia virus (MLV) anti-sera and PCR, we determined if additional prostate cancer or other cell lines contain XMRV or MLV-related viruses. Our study included a total of 72 cell lines, which included 58 of the 60 human cancer cell lines used in anticancer drug screens and maintained at the NCI-Frederick (NCI-60). We have identified gammaretroviruses in two additional prostate cancer cell lines: LAPC4 and VCaP, and show that these viruses are replication competent. Viral genome sequencing identified the virus in LAPC4 and VCaP as nearly identical to another known xenotropic MLV, Bxv-1. We also identified a gammaretrovirus in the non-small-cell lung carcinoma cell line EKVX. Prostate cancer cell lines appear to have a propensity for infection with murine gammaretroviruses, and we propose that this may be in part due to cell line establishment by xenograft passage in immunocompromised mice. It is unclear if infection with these viruses is necessary for cell line establishment, or what confounding role they may play in experiments performed with these commonly used lines. Importantly, our results suggest a need for regular screening of cancer cell lines for retroviral “contamination”, much like routine mycoplasma testing. PMID:21698104

  17. Cell cycle analysis and cytotoxic potential of Ruta graveolens against human tumor cell lines.

    PubMed

    Varamini, P; Soltani, M; Ghaderi, A

    2009-01-01

    There are reports on the presence of various compounds exerting different biological activities in Ruta graveolens, a plant of Rutaceae family. The aim of the present study was to evaluate in vitro cytotoxicity of the total extract of R. graveolens against tumor cell lines of different origin. Aerial parts of the plant was extracted with 70% ethanol by sonication method and cytotoxic activity was examined on RAJI, RAMOS, RPMI8866, U937, Jurkat, MDA-MB-453, MCF-7, LNCap-FGC-10, 5637, HeLa, SK-OV-3, A549, Mehr-80 and also peripheral blood mononuclear cells (PBMC) by the use of WST-1 assay. Results were expressed as IC(50) values. R. graveolens extract showed high cytotoxic activity against RAJI and RAMOS, two Burkitt's lymphoma cell lines, with an IC(50) equal to 24.3 microg/ml and 35.2 microg/ml respectively and LNCap-FGC-10, a prostate adenocarcinoma cell line with an IC(50) equal to 27.6 microg/ml as well as Mehr-80, a newly established Large Cell Lung Carcinoma (IC(50)=46.2 microg/ml). No significant anti-proliferative activity was observed on other cell lines including MCF-7, MDA-MB-453, SK-OV-3, HeLa, 5637, JURKAT and RPMI8866. Adverse cytotoxic effect of R. graveolens was investigated against PBMCs and a significantly lower effect of this extract (IC(50)=104 microg/ml) was seen on normal cells compared with RAJI and RAMOS, two haematopoietic cell lines.

  18. A Human Cell Line Model for Interferon-α Driven Dendritic Cell Differentiation

    PubMed Central

    Ruben, Jurjen M.; Visser, Lindy L.; Heinhuis, Kimberley M.; O’Toole, Tom; Bontkes, Hetty J.; Westers, Theresia M.; Ossenkoppele, Gert J.; de Gruijl, Tanja D.; van de Loosdrecht, Arjan A.

    2015-01-01

    The CD34+ MUTZ-3 acute myeloid leukemia cell line has been used as a dendritic cell (DC) differentiation model. This cell line can be cultured into Langerhans cell (LC) or interstitial DC-like cells using the same cytokine cocktails used for the differentiation of their primary counterparts. Currently, there is an increasing interest in the study and clinical application of DC generated in the presence of IFNα, as these IFNα-DC produce high levels of inflammatory cytokines and have been suggested to be more potent in their ability to cross-present protein antigens, as compared to the more commonly used IL-4-DC. Here, we report on the generation of IFNα-induced MUTZ-DC. We show that IFNα MUTZ-DC morphologically and phenotypically display characteristic DC features and are functionally equivalent to “classic” IL-4 MUTZ-DC. IFNα MUTZ-DC ingest exogenous antigens and can subsequently cross-present HLA class-I restricted epitopes to specific CD8+ T cells. Importantly, mature IFNα MUTZ-DC express CCR7, migrate in response to CCL21, and are capable of priming naïve antigen-specific CD8+ T cells. In conclusion, we show that the MUTZ-3 cell line offers a viable and sustainable model system to study IFNα driven DC development and functionality. PMID:26252775

  19. A cell-permeable fluorescent polymeric thermometer for intracellular temperature mapping in mammalian cell lines.

    PubMed

    Hayashi, Teruyuki; Fukuda, Nanaho; Uchiyama, Seiichi; Inada, Noriko

    2015-01-01

    Changes in intracellular temperatures reflect the activity of the cell. Thus, the tool to measure intracellular temperatures could provide valuable information about cellular status. We previously reported a method to analyze the intracellular temperature distribution using a fluorescent polymeric thermometer (FPT) in combination with fluorescence lifetime imaging microscopy (FLIM). Intracellular delivery of the FPT used in the previous study required microinjection. We now report a novel FPT that is cell permeable and highly photostable, and we describe the application of this FPT to the imaging of intracellular temperature distributions in various types of mammalian cell lines. This cell-permeable FPT displayed a temperature resolution of 0.05°C to 0.54°C within the range from 28°C to 38°C in HeLa cell extracts. Using our optimized protocol, this cell-permeable FPT spontaneously diffused into HeLa cells within 10 min of incubation and exhibited minimal toxicity over several hours of observation. FLIM analysis confirmed a temperature difference between the nucleus and the cytoplasm and heat production near the mitochondria, which were also detected previously using the microinjected FPT. We also showed that this cell-permeable FPT protocol can be applied to other mammalian cell lines, COS7 and NIH/3T3 cells. Thus, this cell-permeable FPT represents a promising tool to study cellular states and functions with respect to temperature.

  20. Establishment and characterization of a clear cell carcinoma cell line, designated NOCC, derived from human ovary.

    PubMed

    Ohyama, Akihiro; Toyomura, Junko; Tachibana, Toshiaki; Isonishi, Seiji; Takahashi, Haruka; Ishikawa, Hiroshi

    2016-10-01

    A cell line, designated NOCC, was established from the ascites of a patient with clear cell adenocarcinoma of the ovary. The cell line has been grown without interruption and continuously propagated by serial passaging (more than 76 times) over 7 years. The cells are spherical to polygonal-shaped, display neoplastic, and pleomorphic features, and grow in a jigsaw puzzle-like pattern while forming monolayers without contact inhibition. The cells proliferate rapidly, but are easily floated as a cell sheet. The population doubling time is about 29 h. The number of chromosomes ranges from 60 to 83. The modal number of chromosomes is 70-74 at the 30th passage. NOCC cells secreted 750.5 ng/ml of VEGF over 3 days of culture. Hypoxia inducible factor-1α (HIF-1α) is a primary regulator of VEGF under hypoxic conditions. NOCC cells were not sensitive to the anticancer drugs BEV, DOX, GEM, ETP, CDDP, or TXT. The graft of NOCC cells to a scid mouse displayed similar histological aspects to the original tumor. Both the NOCC cells and the graft of the NOCC cells gave a positive PAS reaction. PMID:27541369

  1. Analysis of differential protein expression in normal and neoplastic human breast epithelial cell lines

    SciTech Connect

    Williams, K.; Chubb, C.; Huberman, E.; Giometti, C.S.

    1997-07-01

    High resolution two dimensional get electrophoresis (2DE) and database analysis was used to establish protein expression patterns for cultured normal human mammary epithelial cells and thirteen breast cancer cell lines. The Human Breast Epithelial Cell database contains the 2DE protein patterns, including relative protein abundances, for each cell line, plus a composite pattern that contains all the common and specifically expressed proteins from all the cell lines. Significant differences in protein expression, both qualitative and quantitative, were observed not only between normal cells and tumor cells, but also among the tumor cell lines. Eight percent of the consistently detected proteins were found in significantly (P < 0.001) variable levels among the cell lines. Using a combination of immunostaining, comigration with purified protein, subcellular fractionation, and amino-terminal protein sequencing, we identified a subset of the differentially expressed proteins. These identified proteins include the cytoskeletal proteins actin, tubulin, vimentin, and cytokeratins. The cell lines can be classified into four distinct groups based on their intermediate filament protein profile. We also identified heat shock proteins; hsp27, hsp60, and hsp70 varied in abundance and in some cases in the relative phosphorylation levels among the cell lines. Finally, we identified IMP dehydrogenase in each of the cell lines, and found the levels of this enzyme in the tumor cell lines elevated 2- to 20-fold relative to the levels in normal cells.

  2. In vitro invasion of small-cell lung cancer cell lines correlates with expression of epidermal growth factor receptor.

    PubMed Central

    Damstrup, L.; Rude Voldborg, B.; Spang-Thomsen, M.; Brünner, N.; Skovgaard Poulsen, H.

    1998-01-01

    Formation of metastasis is a multistep process involving attachment to the basement membrane, local proteolysis and migration into surrounding tissues, lymph or bloodstream. In the present study, we have analysed the correlation between in vitro invasion and presence of the epidermal growth factor receptor (EGFR) in a panel of 21 small-cell lung cancer (SCLC) cell lines. We have previously reported that ten of these cell lines expressed EGFR protein detected by radioreceptor and affinity labelling assays. In 11 small-cell lung cancer (SCLC) cell lines, EGFR mRNA was detected by Northern blot analysis. In vitro invasion in a Boyden chamber assay was found in all EGFR-positive cell lines, whereas no invasion was detected in the EGFR-negative cell lines. Quantification of the in vitro invasion in 12 selected SCLC cell lines demonstrated that, in the EGFR-positive cell lines, between 5% and 16% of the cells added to the upper chamber were able to traverse the Matrigel membrane. Expression of several matrix metalloproteases (MMP), of tissue inhibitor of MMP (TIMP) and of cathepsin B was evaluated by immunoprecipitation, Western blot analysis and reverse transcriptase polymerase chain reaction (RT-PCR). However, in vitro invasive SCLC cell lines could not be distinguished from non-invasive cell lines based on the expression pattern of these molecules. In six SCLC cell lines, in vitro invasion was also determined in the presence of the EGFR-neutralizing monoclonal antibody mAb528. The addition of this antibody resulted in a significant reduction of the in vitro invasion in three selected EGFR-positive cell lines. Our results show that only EGFR-positive SCLC cell lines had the in vitro invasive phenotype, and it is therefore suggested that the EGFR might play an important role for the invasion potential of SCLC cell lines. Images Figure 1 Figure 3 Figure 4 PMID:9744504

  3. Diverse Hormone Response Networks in 41 Independent Drosophila Cell Lines

    DOE PAGES

    Stoiber, Marcus; Celniker, Susan; Cherbas, Lucy; Brown, Ben; Cherbas, Peter

    2016-01-15

    Steroid hormones induce cascades of gene activation and repression with transformative effects on cell fate . Steroid transduction plays a major role in the development and physiology of nearly all metazoan species, and in the progression of the most common forms of cancer. Despite the paramount importance of steroids in developmental and translational biology, a complete map of transcriptional response has not been developed for any hormone . In the case of 20-hydroxyecdysone (ecdysone) in Drosophila melanogaster, these trajectories range from apoptosis to immortalization. We mapped the ecdysone transduction network in a cohort of 41 cell lines, the largest suchmore » atlas yet assembled. We found that the early transcriptional response mirrors the distinctiveness of physiological origins: genes respond in restricted patterns, conditional on the expression levels of dozens of transcription factors. Only a small cohort of genes is constitutively modulated independent of initial cell state. Ecdysone-responsive genes tend to organize into directional same-stranded units, with consecutive genes induced from the same strand. Here, we identify half of the ecdysone receptor heterodimer as the primary rate-limiting step in the response, and find that initial receptor isoform levels modulate the activated cohort of target transcription factors. In conclusion, this atlas of steroid response reveals organizing principles of gene regulation by a model type II nuclear receptor and lays the foundation for comprehensive and predictive understanding of the ecdysone transduction network in the fruit fly.« less

  4. Comparison of antibody molecules produced from two cell lines with contrasting productivities and aggregate contents.

    PubMed

    Ishii, Yoichi; Imamoto, Yasufumi; Yamamoto, Rie; Tsukahara, Masayoshi; Wakamatsu, Kaori

    2015-01-01

    Cell culture processes that produce therapeutic antibodies with high productivity (titer) and low aggregate content reduce the risk of adverse effects and expense to patients. To elucidate the mechanism of aggregate formation, we compared trastuzumab samples produced from two contrasting cell lines: cell line A, which exhibits high titer and low aggregate content, and cell line B, which exhibits low titer and high aggregate content. Cell line B produced significantly fewer (approximately 1/3) antibodies compared with cell line A and contained higher (approximately 3-fold) percentages of aggregates. The aggregates of antibodies found in the protein A-purified samples of cell line B were associated mostly with noncovalent interactions. Cell line B exhibited a low content of monomers/dimers of light chains in the medium and within cells. Because light chains are essential for the correct folding of heavy chains and secretion of mature antibodies, the characteristics of cell line B may be attributed to low levels of light chain production. In addition, protein A-purified antibodies from cell line B (but not those from cell line A) contained fragments that are expected to expose the hydrophobic CH3 domain, which may serve as nuclei for aggregation. PMID:25501618

  5. Lipid analysis of eight human breast cancer cell lines with ToF-SIMS

    PubMed Central

    Robinson, Michael A.; Graham, Daniel J.; Morrish, Fionnuala; Hockenbery, David; Gamble, Lara J.

    2015-01-01

    In this work, four triple negative (TN) cell lines, three ER+ and PR+ receptor positive (RP) cell lines, and one ER+, PR+, and HER2+ cell line were chemically distinguished from one another using time-of-flight secondary ion mass spectrometry (ToF-SIMS) and principal component analysis (PCA). PCA scores separation was observed between the individual cell lines within a given classification (TN and RP) and there were distinctly different trends found in the fatty acid and lipid compositions of the two different classifications. These trends indicated that the RP cell lines separated out based on the carbon chain length of the lipids while the TN cell lines showed separation based on cholesterol-related peaks (in the positive ion data). Both cell types separated out by trends in fatty acid chain length and saturation in the negative ions. These chemical differences may be manifestations of unique metabolic processes within each of the different cell lines. Additionally, the HER2+ cell line was distinguished from three other RP cell types as having a unique distribution of fatty acids including anticorrelation to 18-carbon chain fatty acids. As these cell lines could not be grown in the same growth media, a combination of chemical fixation, rinsing, C60+ presputtering, and selection of cellular regions-of-interest is also presented as a successful method to acquire ToF-SIMS data from cell lines grown in different media. PMID:26319020

  6. Evaluation of cytokine gene expression after avian influenza virus infection in avian cell lines and primary cell cultures

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The innate immune responses elicited by avian influenza virus (AIV) infection has been studied by measuring cytokine gene expression by relative real time PCR (rRT-PCR) in vitro, using both cell lines and primary cell cultures. Continuous cell lines offer advantages over the use of primary cell cult...

  7. Derivation and characterization of matched cell lines from primary and recurrent serous ovarian cancer

    PubMed Central

    2012-01-01

    Background Cell line models have proven to be effective tools to investigate a variety of ovarian cancer features. Due to the limited number of cell lines, particularly of the serous subtype, the heterogeneity of the disease, and the lack of cell lines that model disease progression, there is a need to further develop cell line resources available for research. This study describes nine cell lines derived from three ovarian cancer cases that were established at initial diagnosis and at subsequent relapse after chemotherapy. Methods The cell lines from three women diagnosed with high-grade serous ovarian cancer (1369, 2295 and 3133) were derived from solid tumor (TOV) and ascites (OV), at specific time points at diagnosis and relapse (R). Primary treatment was a combination of paclitaxel/carboplatin (1369, 3133), or cisplatin/topotecan (2295). Second line treatment included doxorubicin, gemcitabine and topotecan. In addition to molecular characterization (p53, HER2), the cell lines were characterized based on cell growth characteristics including spheroid growth, migration potential, and anchorage independence. The in vivo tumorigenicity potential of the cell lines was measured. Response to paclitaxel and carboplatin was assessed using a clonogenic assay. Results All cell lines had either a nonsense or missense TP53 mutations. The ability to form compact spheroids or aggregates was observed in six of nine cell lines. Limited ability for migration and anchorage independence was observed. The OV3133(R) cell line, formed tumors at subcutaneous sites in SCID mice. Based on IC50 values and dose response curves, there was clear evidence of acquired resistance to carboplatin for TOV2295(R) and OV2295(R2) cell lines. Conclusion The study identified nine new high-grade serous ovarian cancer cell lines, derived before and after chemotherapy that provides a unique resource for investigating the evolution of this common histopathological subtype of ovarian cancer. PMID:22931248

  8. Detection of antigens specific for B-lymphoid cultured cell lines with human alloantisera.

    PubMed

    Mann, D L; Abelson, L; Harris, S; Amos, D B

    1975-07-01

    Human sera were tested for cytotoxicity to pairs of long-term tissue-cultured cell lines. Each pair had been derived from the same individual and one of the pairs possessed the characteristics of either "T" or "B" cells. The alloantisera used were HL-A-typing reagents or sera obtained from Amish multiparas. Selected cytotoxicity was found against the B-cell lines by direct testing. Cytotoxicity was abolished by absorption with B-cell line but not by absorption with the T-cell lines. The results suggest that a group of allotypic antigens may be expressed exculsively on human B cells. PMID:1080182

  9. DNA Methylation Heterogeneity Patterns in Breast Cancer Cell Lines.

    PubMed

    Tian, Sunny; Bertelsmann, Karina; Yu, Linda; Sun, Shuying

    2016-01-01

    Heterogeneous DNA methylation patterns are linked to tumor growth. In order to study DNA methylation heterogeneity patterns for breast cancer cell lines, we comparatively study four metrics: variance, I (2) statistic, entropy, and methylation state. Using the categorical metric methylation state, we select the two most heterogeneous states to identify genes that directly affect tumor suppressor genes and high- or moderate-risk breast cancer genes. Utilizing the Gene Set Enrichment Analysis software and the ConsensusPath Database visualization tool, we generate integrated gene networks to study biological relations of heterogeneous genes. This analysis has allowed us to contribute 19 potential breast cancer biomarker genes to cancer databases by locating "hub genes" - heterogeneous genes of significant biological interactions, selected from numerous cancer modules. We have discovered a considerable relationship between these hub genes and heterogeneously methylated oncogenes. Our results have many implications for further heterogeneity analyses of methylation patterns and early detection of breast cancer susceptibility. PMID:27688708

  10. DNA Methylation Heterogeneity Patterns in Breast Cancer Cell Lines

    PubMed Central

    Tian, Sunny; Bertelsmann, Karina; Yu, Linda; Sun, Shuying

    2016-01-01

    Heterogeneous DNA methylation patterns are linked to tumor growth. In order to study DNA methylation heterogeneity patterns for breast cancer cell lines, we comparatively study four metrics: variance, I2 statistic, entropy, and methylation state. Using the categorical metric methylation state, we select the two most heterogeneous states to identify genes that directly affect tumor suppressor genes and high- or moderate-risk breast cancer genes. Utilizing the Gene Set Enrichment Analysis software and the ConsensusPath Database visualization tool, we generate integrated gene networks to study biological relations of heterogeneous genes. This analysis has allowed us to contribute 19 potential breast cancer biomarker genes to cancer databases by locating “hub genes” – heterogeneous genes of significant biological interactions, selected from numerous cancer modules. We have discovered a considerable relationship between these hub genes and heterogeneously methylated oncogenes. Our results have many implications for further heterogeneity analyses of methylation patterns and early detection of breast cancer susceptibility.

  11. DNA Methylation Heterogeneity Patterns in Breast Cancer Cell Lines

    PubMed Central

    Tian, Sunny; Bertelsmann, Karina; Yu, Linda; Sun, Shuying

    2016-01-01

    Heterogeneous DNA methylation patterns are linked to tumor growth. In order to study DNA methylation heterogeneity patterns for breast cancer cell lines, we comparatively study four metrics: variance, I2 statistic, entropy, and methylation state. Using the categorical metric methylation state, we select the two most heterogeneous states to identify genes that directly affect tumor suppressor genes and high- or moderate-risk breast cancer genes. Utilizing the Gene Set Enrichment Analysis software and the ConsensusPath Database visualization tool, we generate integrated gene networks to study biological relations of heterogeneous genes. This analysis has allowed us to contribute 19 potential breast cancer biomarker genes to cancer databases by locating “hub genes” – heterogeneous genes of significant biological interactions, selected from numerous cancer modules. We have discovered a considerable relationship between these hub genes and heterogeneously methylated oncogenes. Our results have many implications for further heterogeneity analyses of methylation patterns and early detection of breast cancer susceptibility. PMID:27688708

  12. Effects of feeder cells (human cancer cell lines) on the development of mouse embryos by co-culture.

    PubMed

    Ishiwata, I; Tokieda, Y; Ishiwata, C; Okane, N; Iguchi, M; Sato, K; Ishikawa, H

    1997-12-01

    In order to establish the best co-culture system on embryogenesis such as egg fertilization, egg cleavage, blastocyst formation, hatching and implantation etc., several kinds of cell lines as a feeder cell and mouse fertilized eggs (zygotes) were co-cultured in the organ culture dish, and embryotrophic effects of feeder cells were investigated. Best feeder cell on the embryogenesis was SKG-II cell line derived from squamous cell carcinoma of human uterine cervix which was chosen from 10 of the human tumor cell lines. Furthermore, in order to isolate and determinate embryotrophic factors produced by feeder cells, we established a SKG-II SF subline which was grown in serum free medium derived from SKG-II cell line. The SKG-II SF cell line secreted an epidermal growth factor (EGF) into the medium. Also, cleavaged egg produced and secreted interleukin (IL)-1 alpha into the medium. PMID:9573483

  13. Enhancement of Radiation Response in Osteosarcoma and Rhabomyosarcoma Cell Lines by Histone Deacetylase Inhibition

    SciTech Connect

    Blattmann, Claudia; Oertel, Susanne; Ehemann, Volker

    2010-09-01

    Purpose: Histone deacetylase inhibitors (HDACIs) can enhance the sensitivity of cells to photon radiation treatment (XRT) by altering numerous molecular pathways. We investigated the effect of pan-HDACIs such as suberoylanilide hydroxamic acid (SAHA) on radiation response in two osteosarcoma (OS) and two rhabdomyosarcoma (RMS) cell lines. Methods and Materials: Clonogenic survival, cell cycle analysis, and apoptosis were examined in OS (KHOS-24OS, SAOS2) and RMS (A-204, RD) cell lines treated with HDACI and HDACI plus XRT, respectively. Protein expression was investigated via immunoblot analysis, and cell cycle analysis and measurement of apoptosis were performed using flow cytometry. Results: SAHA induced an inhibition of cell proliferation and clonogenic survival in OS and RMS cell lines and led to a significant radiosensitization of all tumor cell lines. Other HDACI such as M344 and valproate showed similar effects as investigated in one OS cell line. Furthermore, SAHA significantly increased radiation-induced apoptosis in the OS cell lines, whereas in the RMS cell lines radiation-induced apoptosis was insignificant with and without SAHA. In all investigated sarcoma cell lines, SAHA attenuated radiation-induced DNA repair protein expression (Rad51, Ku80). Conclusion: Our results show that HDACIs enhance radiation action in OS and RMS cell lines. Inhibition of DNA repair, as well as increased apoptosis induction after exposure to HDACIs, can be mechanisms of radiosensitization by HDACIs.

  14. Photodynamic therapy-induced programmed cell death in carcinoma cell lines

    NASA Astrophysics Data System (ADS)

    He, Xiao-Yan; Sikes, Robert A.; Thomsen, Sharon L.; Chung, L.; Jacques, Steven L.

    1993-06-01

    The mode of cell death following photodynamic therapy (PDT) was investigated from the perspective of programmed cell death (apoptosis). Human prostate carcinoma cells (PC3), human non-small cell lung carcinoma (H322a), and rat mammary carcinoma (MTF7) were treated by PDT following sensitization with dihematoporphyrin ether (DHE). The response of these carcinoma cell lines to PDT was variable. An examination of extracted cellular DNA by gel electrophoresis showed the characteristic DNA ladder pattern indicative of internucleosomal cleavage of DNA during apoptosis. MTF7 and PC3 responded to PDT by inducing apoptosis while H322a had no apoptotic response. The magnitude of the response and the PDT dosage required to induce the effect were different in PC3 and MTF7. MTF7 cells responded with rapid apoptosis at the dose of light and drug that yielded 50% cell death (LD50). In contrast, PC3 showed only marginal apoptosis at the LD50 but had a marked response at the LD85. Furthermore, the onset of apoptosis followed slower kinetics in PC3 (2 hr - 4 hr) than in MTF7 (< 1 hr). H322a cells were killed by PDT but failed to exhibit any apoptotic response. This study indicates that apoptosis may occur during PDT induced cell death, but this pathway is not universal for all cancer cell lines.

  15. Quality Check in Oral Cell Lines: The Need for Molecular Characterization.

    PubMed

    Patil, Shankargouda; Rao, Roopa S; Raj, A Thirumal

    2015-11-01

    Oral cell lines have provided valuable insights into the various molecular pathways in oral carcinogenesis. Several landmark studies in oral oncology have utilized commercially available normal, dysplastic and cancer cell lines to decode the genetic alterations leading to the development of oral cancer. Most of these studies have shown a significant degree of variation in their mutation landscapes. These variations were thought to represent the heterogeneity of oral cancer.(1) But in a recent study, Dickman et al have shown that normal and dysplastic cell lines carry specific genetic alterations within the parent cell line, thus questioning the authenticity of several published mutation profiles. These genetic alterations in the commercial cell lines have been attributed to several factors, the most common being immortalization. Normal and dysplastic cell lines unlike cancer cell lines attain senescence following limited number of replication. Immortalization of the normal and dysplastic cell lines would aid the researcher in maintaining a viable population of cells for further studies. Ideally, the immortalized cell line must possess potential for indefinite replication and must retain the genetic makeup of its parent cell line.(2). PMID:26718303

  16. Establishment and characterization of 7 novel hepatocellular carcinoma cell lines from patient-derived tumor xenografts.

    PubMed

    Xin, Hong; Wang, Ke; Hu, Gang; Xie, Fubo; Ouyang, Kedong; Tang, Xuzhen; Wang, Minjun; Wen, Danyi; Zhu, Yizhun; Qin, Xiaoran

    2014-01-01

    Hepatocellular carcinoma (HCC) is a common cancer with poor prognosis worldwide and the molecular mechanism is not well understood. This study aimed to establish a collection of human HCC cell lines from patient-derived xenograft (PDX) models. From the 20 surgical HCC sample collections, 7 tumors were successfully developed in immunodeficient mice and further established 7 novel HCC cell lines (LIXC002, LIXC003, LIXC004, LIXC006, LIXC011, LIXC012 and CPL0903) by primary culture. The characterization of cell lines was defined by morphology, growth kinetics, cell cycle, chromosome analysis, short tandem repeat (STR) analysis, molecular profile, and tumorigenicity. Additionally, response to clinical chemotherapeutics was validated both in vitro and in vivo. STR analysis indicated that all cell lines were unique cells different from known cell lines and free of contamination by bacteria or mycoplasma. The other findings were quite heterogeneous between individual lines. Chromosome aberration could be found in all cell lines. Alpha-fetoprotein was overexpressed only in 3 out of 7 cell lines. 4 cell lines expressed high level of vimentin. Ki67 was strongly stained in all cell lines. mRNA level of retinoic acid induced protein 3 (RAI3) was decreased in all cell lines. The 7 novel cell lines showed variable sensitivity to 8 tested compounds. LIXC011 and CPL0903 possessed multiple drug resistance property. Sorafenib inhibited xenograft tumor growth of LIXC006, but not of LIXC012. Our results indicated that the 7 novel cell lines with low passage maintaining their clinical and pathological characters could be good tools for further exploring the molecular mechanism of HCC and anti-cancer drug screening.

  17. Biological characteristics of a novel giant cell tumor cell line derived from spine.

    PubMed

    Zhou, Zhenhua; Li, Yan; Xu, Leqin; Wang, Xudong; Chen, Su; Yang, Cheng; Xiao, Jianru

    2016-07-01

    Giant cell tumor of bone(GCTB) is a special bone tumor for it consists of various cell types, and its biological characteristics is different from common benign or malignant neoplasm. In the present study, we report the biological features of a primary Asian GCTB cell line named GCTB28. We analyzed extensive properties of the GCTB28 cells including morphological observations, growth, cell cycle, karyotype, proliferation, proteins expression, surface biomarker verification, and tumorigenicity in nude mice. We found that the stromal cells of GCTB were endowed with self-renewal capacity and played dominant roles in GCTB development. Moreover, we confirmed that GCTB cells can be CD33(-)CD14(-) phenotype which was not in accord with previous study. This study provides an in vitro model system to investigate pathogenic mechanisms and molecular characteristics of GCTB and also provides a useful tool for researching the therapeutic targeting of GCTB.

  18. Hepatocyte growth factor/cMET pathway activation enhances cancer hallmarks in adrenocortical carcinoma

    PubMed Central

    Phan, Liem M.; Fuentes-Mattei, Enrique; Wu, Weixin; Velazquez-Torres, Guermarie; Sircar, Kanishka; Wood, Christopher G.; Hai, Tao; Jimenez, Camilo; Cote, Gilbert J.; Ozsari, Levent; Hofmann, Marie-Claude; Zheng, Siyuan; Verhaak, Roeland; Pagliaro, Lance; Cortez, Maria Angelica; Lee, Mong-Hong; Yeung, Sai-Ching J.; Habra, Mouhammed Amir

    2015-01-01

    Adrenocortical carcinoma (ACC) is a rare malignancy with poor prognosis and limited response to chemotherapy. Hepatocyte growth factor (HGF) and its receptor cMET augment cancer growth and resistance to chemotherapy, but their role in ACC has not been examined. In this study, we investigated the association between HGF/cMET expression and cancer hallmarks of ACC. Transcriptomic and immunohistochemical analyses indicated that increased HGF/cMET expression in human ACC samples was positively associated with cancer-related biological processes including proliferation and angiogenesis, and negatively correlated with apoptosis. Accordingly, treatment of ACC cells with exogenous HCG resulted in increased cell proliferation in vitro and in vivo while short hairpin RNA-mediated knockdown or pharmacological inhibition of cMET suppressed cell proliferation and tumor growth. Moreover, exposure of cells to mitotane, cisplatin, or radiation rapidly induced pro-cMET expression and was associated with an enrichment of genes (e.g., CYP450 family) related to therapy resistance further implicating cMET in the anticancer drug response. Together, these data suggest an important role for HGF/cMET signaling in ACC growth and resistance to commonly used treatments. Targeting cMET, alone or in combination with other drugs, could provide a breakthrough in the management of this aggressive cancer. PMID:26282167

  19. Macrophage cell lines derived from major histocompatibility complex II-negative mice

    NASA Technical Reports Server (NTRS)

    Beharka, A. A.; Armstrong, J. W.; Chapes, S. K.; Spooner, B. S. (Principal Investigator)

    1998-01-01

    Two bone-marrow-derived macrophage cell lines, C2D and C2Dt, were isolated from major histocompatibility class II negative knock-out mice. The C2D cell line was stabilized by continuous culture in colony-stimulating factor-1 and the C2Dt cell line was transformed with SV40 virus large T antigen. These cells exhibited phenotypic properties of macrophages including morphology and expression of Mac 1 and Mac 2 cell surface molecules. These cells also had comparable growth to the bone-marrow-derived macrophage cell line B6MP102. These new cell lines were not spontaneously cytotoxic and were only capable of modest killing of F5b tumor cells when stimulated with LPS and interferon-gamma, but not when stimulated with LPS alone or with staphylococcal exotoxin. C2D and C2Dt cells phagocytosed labeled Staphylococcus aureus similarly to B6MP102 cells but less well than C2D peritoneal macrophages. These cell lines secreted interleukin-6, but not tumor necrosis factor or nitric oxide in response to LPS or staphlococcal enterotoxins A or B C2D(t) cells were tumorigenic in C2D and C57BL/6J mice but C2D cells were not. These data suggest that macrophage cell lines can be established from bone marrow cells of major histocompatibility complex II-negative mice.

  20. A bioassay for metals utilizing a human cell line.

    PubMed

    Shea, J; Moran, T; Dehn, P F

    2008-06-01

    The purpose of this study was to assess the ability of the HepG2 cell line to function as a bioassay for metal contamination in sediments, using metallothionein (MT) as a biomarker of exposure. Sediments were collected from the eastern and western ends of Lake Erie, extracted using EPA method 200.7, and analyzed for cadmium (Cd), mercury (Hg) and lead (Pb) levels using ICP-AES. Sediment extracts were neutralized then used at a 2.5% final concentration in the exposure medium. MT levels were measured using the cadmium-hemoglobin affinity assay after a 48 h exposure. Fortified blanks from the ICP protocol served as positive controls. Also, HepG2 cells were exposed to Cd, Pb or combinations of Cd and Pb to determine whether or not induction of MT observed in cells exposed to sediment extracts was due to a single metal, combinations of metals, pH, or some other factor. Additionally, cells were exposed to a range of Cd concentrations approximating the levels found in the extracts (0.0005-0.1mg/L) to determine if a concentration-response occurred. Total metal levels ranged from 527 to 33.5mg/kg with lead the predominant metal, accounting for 100-88.9% of the total quantifiable metals in the sediments. The biomarker response (MT induction) was strongly correlated (r2=0.9919, r2=0.990) with total metal and lead levels in the sediments, respectively, which supports recent field studies indicating the biomarker can discern differences in the strength of the inducing agent. Statistically significant MT induction was associated with sediments which contained measurable Cd concentrations and no significant differences were observed when comparing Cd only and Cd+Pb exposed cells indicating no interactions between Cd and Pb were occurring and supporting our finding that Cd was the main inducing agent in sediment extracts. MT levels also increased significantly in a concentration-dependent manner when cells were exposed only to Cd. Results suggest this human bioassay and the MT

  1. Immune suppressor factor confers stromal cell line with enhanced supporting activity for hematopoietic stem cells

    SciTech Connect

    Nakajima, Hideaki . E-mail: hnakajim@ims.u-tokyo.ac.jp; Shibata, Fumi; Fukuchi, Yumi; Goto-Koshino, Yuko; Ito, Miyuki; Urano, Atsushi; Nakahata, Tatsutoshi; Aburatani, Hiroyuki; Kitamura, Toshio

    2006-02-03

    Immune suppressor factor (ISF) is a subunit of the vacuolar ATPase proton pump. We earlier identified a short form of ISF (ShIF) as a stroma-derived factor that supports cytokine-independent growth of mutant Ba/F3 cells. Here, we report that ISF/ShIF supports self-renewal and expansion of primary hematopoietic stem cells (HSCs). Co-culture of murine bone marrow cells with a stromal cell line overexpressing ISF or ShIF (MS10/ISF or MS10/ShIF) not only enhanced their colony-forming activity and the numbers of long-term culture initiating cells, but also maintained the competitive repopulating activity of HSC. This stem cell supporting activity depended on the proton-transfer function of ISF/ShIF. Gene expression analysis of ISF/ShIF-transfected cell lines revealed down-regulation of secreted frizzled-related protein-1 and tissue inhibitor of metalloproteinase-3, and the restoration of their expressions in MS10/ISF cells partially reversed its enhanced LTC-IC supporting activity to a normal level. These results suggest that ISF/ShIF confers stromal cells with enhanced supporting activities for HSCs by modulating Wnt-activity and the extracellular matrix.

  2. HIG-82: an established cell line from rabbit periarticular soft tissue, which retains the "activatable" phenotype.

    PubMed

    Georgescu, H I; Mendelow, D; Evans, C H

    1988-10-01

    We have isolated a continuous cell line from soft tissue lining the knee joints of rabbits. Designated HIG-82, this line was produced by spontaneous establishment of an aging, late-passage culture of primary cells. Like unpassaged, primary cells, HIG-82 cells can be activated by a number of stimuli, including phorbol myristate acetate (PMA), interleukin-1 (IL-1), and the endocytosis of latex beads. Activated cells secrete collagenase, gelatinase, caseinase (stromelysin), and prostaglandin E2 (PGE2) into their culture medium. Pseudodiploid, HIG-82 cells combine a high plating efficiency with a doubling time of approximately 24 h. As primary tissue of this origin is difficult to obtain in large quantities and shows cellular heterogeneity, the HIG-82 cell line should facilitate research into the biology and biochemistry of the fibroblastic cells that line the diarthrodial joints of mammals. Such cells are likely to be important in the pathophysiology of various arthritides.

  3. Structure of retroviral RNAs produced by cell lines derived from spontaneous lymphomas of AKR mice.

    PubMed Central

    Pedersen, F S; Crowther, R L; Hays, E F; Nowinski, R C; Haseltine, W A

    1982-01-01

    The retrovirus expression of eight independent lymphoid cell lines derived from spontaneous thymomas of AKR mice was investigated. The RNase T1 fingerprints of viral 70S RNA produced by these cell lines were compared with genome structures of the non-leukemogenic Akv virus and with two types of cloned leukemogenic viruses derived from one of the thymoma cell lines. Viral RNAs from three cell lines, SL3, 4, and 7, were indistinguishable from one another. The fingerprint patterns indicated that these cell lines produce equal amounts of two prototype, leukomogenic SL viruses that were previously isolated from the SL3 cell line. Viral RNA produced by the SL1 and SL2 cell lines contained similar components, but at a different ratio. Two other cell lines (SL5 and SL11) produced viral RNAs that resemble those of AKR mink cell focus-forming viruses. One additional line, SL9, produced viral RNA of a novel structure. The complex pattern of viral RNA expression observed for these lymphoid cell lines can be interpreted in terms of recombination among three types of endogenous viral sequences: the Akv virus, a xenotropic virus, and an SL (for spontaneous leukemia) virus. Images PMID:7086955

  4. Characterization of novel low passage primary and metastatic colorectal cancer cell lines

    PubMed Central

    Boot, Arnoud; van Eendenburg, Jaap; Crobach, Stijn; Ruano, Dina; Speetjens, Frank; Calame, Jan; Oosting, Jan; Morreau, Hans; van Wezel, Tom

    2016-01-01

    In vitro models are essential to understanding the molecular characteristics of colorectal cancer (CRC) and the testing of therapies for CRC. Many efforts to establish and characterize primary CRC cell lines have been published, most describing a small number of novel cell lines. However, there remains a lack of a large panel of uniformly established and characterized cell lines. To this end we established 20 novel CRC cell lines, of which six were derived from liver metastases. Genetic, genomic and transcriptomic profiling was performed in order to characterize these new cell lines. All data are made publically available upon publication. By combining mutation profiles with CNA and gene expression profiles, we generated an overall profile of the alterations in the major CRC-related signaling pathways. The combination of mutation profiles with genome, transcriptome and methylome data means that these low passage cell lines are among the best characterized of all CRC cell lines. This will allow researchers to select model cell lines appropriate to specific experiments, facilitating the optimal use of these cell lines as in vitro models for CRC. All cell lines are available for further research. PMID:26894854

  5. Selective migration of neuralized embryonic stem cells to stem cell factor and media conditioned by glioma cell lines

    PubMed Central

    Serfozo, Peter; Schlarman, Maggie S; Pierret, Chris; Maria, Bernard L; Kirk, Mark D

    2006-01-01

    Background Pluripotent mouse embryonic stem (ES) cells can be induced in vitro to become neural progenitors. Upon transplantation, neural progenitors migrate toward areas of damage and inflammation in the CNS. We tested whether undifferentiated and neuralized mouse ES cells migrate toward media conditioned by glioma cell lines (C6, U87 & N1321) or Stem Cell Factor (SCF). Results Cell migration assays revealed selective migration by neuralized ES cells to conditioned media as well as to synthetic SCF. Migration of undifferentiated ES cells was extensive, but not significantly different from that of controls (Unconditioned Medium). RT-PCR analysis revealed that all the three tumor cell lines tested synthesized SCF and that both undifferentiated and neuralized ES cells expressed c-kit, the receptor for SCF. Conclusion Our results demonstrate that undifferentiated ES cells are highly mobile and that neural progenitors derived from ES cells are selectively attracted toward factors produced by gliomas. Given that the glioma cell lines synthesize SCF, SCF may be one of several factors that contribute to the selective migration observed. PMID:16436212

  6. Integromic analysis of the NCI-60 cancer cell lines.

    PubMed

    Weinstein, John N

    2004-01-01

    Microarray-based transcript profiling has become exceedingly popular, particularly for breast cancer. However, other 'omic' profiling technologies at the DNA, RNA, protein, functional, and pharmacological levels are also becoming increasingly practical. We define 'integromics' as the melding of such diverse types of data from different experimental platforms. The whole can sometimes be more than the sum of its parts. We describe here a set of integromic studies in which we have profiled the 60 human cancer cell lines (the NCI-60) used by the National Cancer Institute to screen >100,000 chemical compounds over the last 13 years. Patterns of potency in the screen can be mapped into molecular structures of the compounds or into molecular characteristics of the cells. Here we discuss conceptual and experimental aspects of the profiling, as well as a number of bioinformatic computer programs (CIMminer, MedMiner, MatchMiner, and GoMiner) that we have developed for biological interpretation of the profiles. As briefly reviewed here, we have used the combination of NCI-60 data types to identify markers for distinguishing tumor types and to obtain pharmacogenomic clues for possible individualization of a cancer therapy. PMID:15687693

  7. Opioid binding site in EL-4 thymoma cell line

    SciTech Connect

    Fiorica, E.; Spector, S.

    1988-01-01

    Using EL-4 thymoma cell-line we found a binding site similar to the k opioid receptor of the nervous system. The Scatchard analysis of the binding of (/sup 3/H) bremazocine indicated a single site with a K/sub D/ = 60 +/- 17 nM and Bmax = 2.7 +/- 0.8 pmols/10/sup 6/ cells. To characterize this binding site, competition studies were performed using selective compounds for the various opioid receptors. The k agonist U-50,488H was the most potent displacer of (/sup 3/H) bremazocine with an IC/sub 50/ value = 0.57..mu..M. The two steroisomers levorphanol and dextrorphan showed the same affinity for this site. While morphine, (D-Pen/sup 2/, D-Pen/sup 5/) enkephalin and ..beta..-endorphin failed to displace, except at very high concentrations, codeine demonstrated a IC/sub 50/ = 60..mu..M, that was similar to naloxone. 32 references, 3 figures, 2 tables.

  8. The endocannabinoid system in the human granulosa cell line KGN.

    PubMed

    Ernst, Jana; Grabiec, Urszula; Greither, Thomas; Fischer, Bernd; Dehghani, Faramarz

    2016-03-01

    Ovarian steroidogenesis is embedded in a sensitive network of regulatory mechanisms crucial for human fertility. The endocannabinoid system (ECS) represents an intrinsic modulating system involved in the regulation of endocrine functions. In the present study we characterized the ECS in the human granulosa cell line KGN and its impact on gonadotropin sensitivity and steroid hormone synthesis under basal and FSH-stimulated conditions. Expression studies were performed and estradiol was measured. CB1, CB2, DAGL, FAAH, GPR55, MAGL, NAPE-PLD and TRPV1 were expressed without FSH-dependent effects. Treatment with selective cannabinoid receptor agonists reduced basal but not FSH-stimulated estradiol and CYP19. Progesterone was not altered by ECS manipulation. CB1 agonist changed the expression of miRNAs associated with granulosa cell function, e.g. miR-23a, miR-24, miR-181a and miR-320a. Present data indicate a modulating role of the intrinsic ovarian ECS in the regulation of estradiol synthesis. PMID:26773729

  9. Comparison of betanodavirus replication efficiency in ten Indian fish cell lines.

    PubMed

    Sarath Babu, V; Abdul Majeed, S; Nambi, K S N; Taju, G; Madan, N; Sundar Raj, N; Sahul Hameed, A S

    2013-06-01

    Ten cell lines established from Indian marine, brackishwater and freshwater fish were tested for their susceptibility to fish nodavirus. In addition, the efficiency of betanodavirus replication was tested in these cell lines. Multiple vacuolation, a typical cytopathic effect for virus infection, was observed in infected SISK, SISS, SIGE and ICF cells. Infection of the different fish cell lines was confirmed by RT-PCR, immunodot blot assay and indirect ELISA. The virus concentration in culture supernatant collected from infected sea bass and grouper cell lines increased progressively from 10(3) at day 1 postinfection to 10(8) TCID50 ml(-1) at day 9. The amount of virus in different cell lines was also quantified by real-time PCR. These results indicate the suitability of the SISK, SISS, and SIGE cell lines for fish nodavirus propagation for developing viral diagnostics and vaccines. PMID:23392632

  10. 188Rhenium-induced cell death and apoptosis in a panel of tumor cell lines

    NASA Astrophysics Data System (ADS)

    Antoccia, Antonio; Banzato, Alessandra; Bello, Michele; Bollini, Dante; De Notaristefani, Francesco; Giron, Cecilia; Mazzi, Ulderico; Alafort, Laura Melendez; Moschini, Giuliano; Nadali, Anna; Navarria, Francesco; Perrotta, Andrea; Rosato, Antonio; Tanzarella, Caterina; Uzunov, Nikolay

    2007-02-01

    Assessment of "in vitro" tumor growth inhibition and radiobiological effects, such as apoptosis, have been evaluated in human neoplastic cells of different histotypes (H460 lung cancer cells, U87 glioblastoma, LnCaP prostate tumor cells) treated using solutions of 188Rhenium-perrhenate. The MTT assay, which measures mitochondrial metabolism in the entire cell culture is a recognized test for cytotoxicity and was used in cells exposed 48-72 h to specific activities ranged from 37 to 148 GBq/l. Whereas H460 and LnCaP were particularly sensitive to treatment, U87 glioblastoma cells behaved as radioresistant ones. However, evaluation of 188Re-induced apoptosis indicated that this kind of cell death contributed only marginally to the reduction in cell viability of H460 and LNCaP lines, suggesting the existence of protective mechanisms against apoptosis. In this respect, the membrane receptor, CD44, whose expression is dysregulated in most malignant cell types has proven to alter the response of cancer cells to apoptotic stimuli, including ionizing radiation. Cell samples decorated with a FITC-labelled CD44 antibody indicated, that in H460 and U87 cells the CD44(+) correlated well with an apoptosis-resistant response. Conversely, LnCap cells proven as CD44(-) did not display however sensitivity to radio-induced apoptosis.

  11. Acrylamide affects proliferation and differentiation of the neural progenitor cell line C17.2 and the neuroblastoma cell line SH-SY5Y.

    PubMed

    Attoff, K; Kertika, D; Lundqvist, J; Oredsson, S; Forsby, A

    2016-09-01

    Acrylamide is a well-known neurotoxic compound and people get exposed to the compound by food consumption and environmental pollutants. Since acrylamide crosses the placenta barrier, the fetus is also being exposed resulting in a risk for developmental neurotoxicity. In this study, the neural progenitor cell line C17.2 and the neuroblastoma cell line SH-SY5Y were used to study proliferation and differentiation as alerting indicators for developmental neurotoxicity. For both cell lines, acrylamide reduced the number of viable cells by reducing proliferation and inducing cell death in undifferentiated cells. Acrylamide concentrations starting at 10fM attenuated the differentiation process in SH-SY5Y cells by sustaining cell proliferation and neurite outgrowth was reduced at concentrations from 10pM. Acrylamide significantly reduced the number of neurons starting at 1μM and altered the ratio between the different phenotypes in differentiating C17.2 cell cultures. Ten micromolar of acrylamide also reduced the expression of the neuronal and astrocyte biomarkers. Although the neurotoxic concentrations in the femtomolar range seem to be specific for the SH-SY5Y cell line, the fact that micromolar concentrations of acrylamide seem to attenuate the differentiation process in both cell lines raises the interest to further investigations on the possible developmental neurotoxicity of acrylamide. PMID:27241584

  12. Acrylamide affects proliferation and differentiation of the neural progenitor cell line C17.2 and the neuroblastoma cell line SH-SY5Y.

    PubMed

    Attoff, K; Kertika, D; Lundqvist, J; Oredsson, S; Forsby, A

    2016-09-01

    Acrylamide is a well-known neurotoxic compound and people get exposed to the compound by food consumption and environmental pollutants. Since acrylamide crosses the placenta barrier, the fetus is also being exposed resulting in a risk for developmental neurotoxicity. In this study, the neural progenitor cell line C17.2 and the neuroblastoma cell line SH-SY5Y were used to study proliferation and differentiation as alerting indicators for developmental neurotoxicity. For both cell lines, acrylamide reduced the number of viable cells by reducing proliferation and inducing cell death in undifferentiated cells. Acrylamide concentrations starting at 10fM attenuated the differentiation process in SH-SY5Y cells by sustaining cell proliferation and neurite outgrowth was reduced at concentrations from 10pM. Acrylamide significantly reduced the number of neurons starting at 1μM and altered the ratio between the different phenotypes in differentiating C17.2 cell cultures. Ten micromolar of acrylamide also reduced the expression of the neuronal and astrocyte biomarkers. Although the neurotoxic concentrations in the femtomolar range seem to be specific for the SH-SY5Y cell line, the fact that micromolar concentrations of acrylamide seem to attenuate the differentiation process in both cell lines raises the interest to further investigations on the possible developmental neurotoxicity of acrylamide.

  13. Isolation of a pluripotent cell line from early mouse embryos cultured in medium conditioned by teratocarcinoma stem cells.

    PubMed

    Martin, G R

    1981-12-01

    This report describes the establishment directly from normal preimplantation mouse embryos of a cell line that forms teratocarcinomas when injected into mice. The pluripotency of these embryonic stem cells was demonstrated conclusively by the observation that subclonal cultures, derived from isolated single cells, can differentiate into a wide variety of cell types. Such embryonic stem cells were isolated from inner cell masses of late blastocysts cultured in medium conditioned by an established teratocarcinoma stem cell line. This suggests that such conditioned medium might contain a growth factor that stimulates the proliferation or inhibits the differentiation of normal pluripotent embryonic cells, or both. This method of obtaining embryonic stem cells makes feasible the isolation of pluripotent cells lines from various types of noninbred embryo, including those carrying mutant genes. The availability of such cell lines should made possible new approaches to the study of early mammalian development.

  14. Absence of keratins 8 and 18 expression in rodent epithelial cell lines associates with keratin gene mutation and DNA methylation: cell line selective effects on cell invasion

    PubMed Central

    Omary, M. Bishr

    2016-01-01

    Epithelial-mesenchymal transition (EMT) in carcinoma is associated with dramatic up-regulation of vimentin and down-regulation of the simple-type keratins 8 and 18 (K8/K18), but the mechanisms of these changes are poorly understood. We demonstrate that two commonly-studied murine (CT26) and rat (IEC-6) intestinal cell lines have negligible K8/K18 but high vimentin protein expression. Proteasome inhibition led to a limited increase in K18 but not K8 stabilization, thereby indicating that K8/K18 absence is not due, in large part, to increased protein turnover. CT26 and IEC-6 cells had <10% of normal K8/K18 mRNA and exhibited decreased mRNA stability, with K8 being higher in IEC-6 versus CT26 and K18 being higher in CT26 versus IEC-6 cells. Keratin gene sequencing showed that KRT8 in CT26 cells had a 21-nucleotide deletion while K18 in IEC-6 cells had a 9-amino acid in-frame insertion. Furthermore, the KRT8 promoter in CT26 and the KRT18 promoter in IEC-6 are hypermethylated. Inhibition of DNA methylation using 5-azacytidine increased K8 or K18 in some but all the tested rodent epithelial cell lines. Restoring K8 and K18 by lentiviral transduction reduced CT26 but not IEC-6 cell matrigel invasion. K8/K18 re-introduction also decreased E-cadherin expression in IEC-6 but not CT26 cells, suggesting that the effect of keratin expression on epithelial to mesenchymal transition is cell-line dependent. Therefore, some commonly utilized rodent epithelial cell lines, unexpectedly, manifest barely detectable keratin expression but have high levels of vimentin. In the CT26 and IEC-6 intestinal cell lines, keratin expression correlates with keratin gene insertion or deletion and with promoter methylation, which likely suppress keratin transcription or mRNA stability. PMID:25882495

  15. Derivation and Osmotolerance Characterization of Three Immortalized Tilapia (Oreochromis mossambicus) Cell Lines

    PubMed Central

    Gardell, Alison M.; Qin, Qin; Rice, Robert H.; Li, Johnathan; Kültz, Dietmar

    2014-01-01

    Fish cell cultures are becoming more widely used models for investigating molecular mechanisms of physiological response to environmental challenge. In this study, we derived two immortalized Mozambique tilapia (Oreochromis mossambicus) cell lines from brain (OmB) and lip epithelium (OmL), and compared them to a previously immortalized bulbus arteriosus (TmB) cell line. The OmB and OmL cell lines were generated without or with Rho-associated kinase (ROCK) inhibitor/3T3 feeder layer supplementation. Although both approaches were successful, ROCK inhibitor/feeder layer supplementation was found to offer the advantages of selecting for epithelial-like cell type and decreasing time to immortalization. After immortalization (≥ passage 5), we characterized the proteomes of the newly derived cell lines (OmB and OmL) using LCMS and identified several unique cell markers for each line. Subsequently, osmotolerance for each of the three cell lines following acute exposure to elevated sodium chloride was evaluated. The acute maximum osmotolerance of these tilapia cell lines (>700 mOsm/kg) was markedly higher than that of any other known vertebrate cell line, but was significantly higher in the epithelial-like OmL cell line. To validate the physiological relevance of these tilapia cell lines, we quantified the effects of acute hyperosmotic challenge (450 mOsm/kg and 700 mOsm/kg) on the transcriptional regulation of two enzymes involved in biosynthesis of the compatible organic osmolyte, myo-inositol. Both enzymes were found to be robustly upregulated in all three tilapia cell lines. Therefore, the newly established tilapia cells lines represent valuable tools for studying molecular mechanisms involved in the osmotic stress response of euryhaline fish. PMID:24797371

  16. Lines

    ERIC Educational Resources Information Center

    Mires, Peter B.

    2006-01-01

    National Geography Standards for the middle school years generally stress the teaching of latitude and longitude. There are many creative ways to explain the great grid that encircles our planet, but the author has found that students in his college-level geography courses especially enjoy human-interest stories associated with lines of latitude…

  17. Network signatures of cellular immortalization in human lymphoblastoid cell lines

    SciTech Connect

    Shim, Sung-Mi; Jung, So-Young; Nam, Hye-Young; Kim, Hye-Ryun; Lee, Mee-Hee; Kim, Jun-Woo; Han, Bok-Ghee; Jeon, Jae-Pil

    2013-11-15

    Highlights: •We identified network signatures of LCL immortalization from transcriptomic profiles. •More than 41% of DEGs are possibly regulated by miRNAs in LCLs. •MicroRNA target genes in LCLs are involved in apoptosis and immune-related functions. •This approach is useful to find functional miRNA targets in specific cell conditions. -- Abstract: Human lymphoblastoid cell line (LCL) has been used as an in vitro cell model in genetic and pharmacogenomic studies, as well as a good model for studying gene expression regulatory machinery using integrated genomic analyses. In this study, we aimed to identify biological networks of LCL immortalization from transcriptomic profiles of microRNAs and their target genes in LCLs. We first selected differentially expressed genes (DEGs) and microRNAs (DEmiRs) between early passage LCLs (eLCLs) and terminally differentiated late passage LCLs (tLCLs). The in silico and correlation analysis of these DEGs and DEmiRs revealed that 1098 DEG–DEmiR pairs were found to be positively (n = 591 pairs) or negatively (n = 507 pairs) correlated with each other. More than 41% of DEGs are possibly regulated by miRNAs in LCL immortalizations. The target DEGs of DEmiRs were enriched for cellular functions associated with apoptosis, immune response, cell death, JAK–STAT cascade and lymphocyte activation while non-miRNA target DEGs were over-represented for basic cell metabolisms. The target DEGs correlated negatively with miR-548a-3p and miR-219-5p were significantly associated with protein kinase cascade, and the lymphocyte proliferation and apoptosis, respectively. In addition, the miR-106a and miR-424 clusters located in the X chromosome were enriched in DEmiR–mRNA pairs for LCL immortalization. In this study, the integrated transcriptomic analysis of LCLs could identify functional networks of biologically active microRNAs and their target genes involved in LCL immortalization.

  18. Evaluation of effect of triterpenes and limonoids on cell growth, cell cycle and apoptosis in human tumor cell line.

    PubMed

    Cazal, Cristiane M; Choosang, Kantima; Severino, Vanessa Gisele P; Soares, Marcio S; Sarria, Andre Lucio F; Fernandes, Joao B; Silva, Maria Fatima G F; Vieira, Paulo Cezar; Pakkong, Pannee; Almeida, Gabriela M; Vasconcelos, M Helena; Nascimento, Maria S J; Pinto, Madalena M M

    2010-12-01

    Six triterpenes and eight limonoids were evaluated for their capacity to inhibit the growth of three human tumour cell lines, breast adenocarcinoma (MCF-7), non-small cell lung cancer (NCI-H460) and melanoma (A375-C5). The mechanisms involved in the observed cell growth arrest of the four most potent compounds were carried out by studying their effect in cell cycle profile and programmed cell death. The results showed that one triterpene (odoratol) and two limonoids (gedunin and cedrelone) caused cell cycle arrest while only the limonoids gedunin and cedrelone were found to be very potent inducers of apoptosis. PMID:21269253

  19. Retrovirus-mediated conditional immortalization and analysis of established cell lines of osteoclast precursor cells