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Sample records for 3-methylcholanthrene induces differential

  1. Prevention of 3-methylcholanthrene-induced fibrosarcomas in rats pre-inoculated with endogenous rat retrovirus.

    Fish, D C; Demarais, J T; Djurickovic, D B; Huebner, R J

    1981-01-01

    Weanling Fischer 344 rats received a single intraperitoneal injection of a 1000-fold concentrated preparation of endogenous nontransforming rat retrovirus. Ten days later, the rats were each given a single subcutaneous injection of 3-methylcholanthrene. The rats inoculated with the endogenous rat retrovirus were significantly protected against the development of cancer, whereas uninoculated rats and rats given one of several murine retroviruses or baboon retrovirus were not protected.

  2. Analysis of Ki-ras Exon 2 Gene Mutations in 3-Methylcholanthrene and Butylated Hydroxytoluene-Induced Rat Lung Tissues

    POLAT, Fikriye; ÖZDEMİR, Öztürk; ELAGÖZ, Şahende

    2008-01-01

    3-Methylcholanthrene (MCA) is a polycyclic aromatic hydrocarbon and potent carcinogenic agent that is often used in experimental cancer studies. Butylated hydroxytoluene (BHT) has been widely used for many years as an antioxidant to preserve and stabilize the freshness, nutritional value, flavor, and color of foods. The aim of the present study was to investigate the role of the application of MCA and BHT in the development of lung cancer, and to detect any mutation in the Ki-ras gene exon 2....

  3. CD8+ T cells are crucial for the ability of congenic normal mice to reject highly immunogenic sarcomas induced in nude mice with 3-methylcholanthrene

    Boesen, M; Svane, I M; Engel, A M; Rygaard, J; Thomsen, Allan Randrup; Werdelin, O

    2000-01-01

    An attempt was made to identify the selection pressures put upon a growing tumour by CD8+ T cells. To this end tumours induced with 3-methylcholanthrene in T cell-deficient nude mice and in congenic T cell-competent nu/+ mice were transplanted to nu/+ recipients. The rejection rate of the sarcomas...... from nude mice was almost twice that of the sarcomas from nu/+ mice. Depletion of CD8+ T cells from nu/+ recipients prior to transplantation made them accept nude tumours that were consistently rejected by untreated nu/+ recipients. These findings suggest that a methylcholanthrene sarcoma during its...

  4. Transformation of SV40-immortalized human uroepithelial cells by 3-methylcholanthrene increases IFN- and Large T Antigen-induced transcripts

    Easton Marilyn J

    2010-02-01

    Full Text Available Abstract Background Simian Virus 40 (SV40 immortalization followed by treatment of cells with 3-methylcholanthrene (3-MC has been used to elicit tumors in athymic mice. 3-MC carcinogenesis has been thoroughly studied, however gene-level interactions between 3-MC and SV40 that could have produced the observed tumors have not been explored. The commercially-available human uroepithelial cell lines were either SV40-immortalized (HUC or SV40-immortalized and then 3-MC-transformed (HUC-TC. Results To characterize the SV40 - 3MC interaction, we compared human gene expression in these cell lines using a human cancer array and confirmed selected changes by RT-PCR. Many viral Large T Antigen (Tag expression-related changes occurred in HUC-TC, and it is concluded that SV40 and 3-MC may act synergistically to transform cells. Changes noted in IFP 9-27, 2'-5' OAS, IF 56, MxA and MxAB were typical of those that occur in response to viral exposure and are part of the innate immune response. Because interferon is crucial to innate immune host defenses and many gene changes were interferon-related, we explored cellular growth responses to exogenous IFN-γ and found that treatment impeded growth in tumor, but not immortalized HUC on days 4 - 7. Cellular metabolism however, was inhibited in both cell types. We conclude that IFN-γ metabolic responses were functional in both cell lines, but IFN-γ anti-proliferative responses functioned only in tumor cells. Conclusions Synergism of SV40 with 3-MC or other environmental carcinogens may be of concern as SV40 is now endemic in 2-5.9% of the U.S. population. In addition, SV40-immortalization is a generally-accepted method used in many research materials, but the possibility of off-target effects in studies carried out using these cells has not been considered. We hope that our work will stimulate further study of this important phenomenon.

  5. Activation of liver X receptor inhibits the development of pulmonary carcinomas induced by 3-methylcholanthrene and butylated hydroxytoluene in BALB/c mice.

    Wang, Qixue; Sun, Lei; Yang, Xiaoxiao; Ma, Xingzhe; Li, Qi; Chen, Yuanli; Liu, Ying; Zhang, Di; Li, Xiaoju; Xiang, Rong; Wei, Yuquan; Han, Jihong; Duan, Yajun

    2016-01-01

    We previously reported that LXR ligand, T0901317, inhibited the growth of inoculated Lewis lung carcinoma in C57BL/6 mice by activating IFN-γ production. However, the effects of T0901317 on carcinogen-induced pulmonary carcinomas remain unknown. In this study, we initially conducted a statistical analysis on the data of human lung cancer samples extracted from the TCGA database, and determined that survival rate/time of lung cancer patients and grade of lung adenocarcinoma were positively and negatively related to lung IFN-γ levels, respectively. We then determined the inhibitory effects of T0901317 on mouse pulmonary carcinomas induced by 3-methylcholanthrene (MCA) and butylated hydroxytoluene (BHT) or urethane. We found that T0901317 reduced morbidity and mortality in MCA/BHT-injected BALB/c mice by inhibiting lung adenocarcinoma. T0901317 also protected C57BL/6 mice, but not IFN-γ deficient (IFN-γ(-/-), C57BL/6 background) mice, against MCA/BHT-induced lung hyperplasia/inflammation. In addition, we determined that T0901317 inhibited urethane-induced lung tumors in BABL/c mice. Furthermore, we determined that T0901317 prevented metastasis of 4T1 breast cancer cells in BALB/c mice. Administration of T0901317 substantially increased serum IFN-γ levels and lung IFN-γ expression in BABL/c and C57BL/6 mice. Taken together, our study demonstrates that LXR inhibits MCA/BHT-induced pulmonary carcinomas in BABL/c mice and the inhibition is associated with induction of IFN-γ production. PMID:27250582

  6. Activation of liver X receptor inhibits the development of pulmonary carcinomas induced by 3-methylcholanthrene and butylated hydroxytoluene in BALB/c mice

    Wang, Qixue; Sun, Lei; Yang, Xiaoxiao; Ma, Xingzhe; Li, Qi; Chen, Yuanli; Liu, Ying; Zhang, Di; Li, Xiaoju; Xiang, Rong; Wei, Yuquan; Han, Jihong; Duan, Yajun

    2016-01-01

    We previously reported that LXR ligand, T0901317, inhibited the growth of inoculated Lewis lung carcinoma in C57BL/6 mice by activating IFN-γ production. However, the effects of T0901317 on carcinogen-induced pulmonary carcinomas remain unknown. In this study, we initially conducted a statistical analysis on the data of human lung cancer samples extracted from the TCGA database, and determined that survival rate/time of lung cancer patients and grade of lung adenocarcinoma were positively and negatively related to lung IFN-γ levels, respectively. We then determined the inhibitory effects of T0901317 on mouse pulmonary carcinomas induced by 3-methylcholanthrene (MCA) and butylated hydroxytoluene (BHT) or urethane. We found that T0901317 reduced morbidity and mortality in MCA/BHT-injected BALB/c mice by inhibiting lung adenocarcinoma. T0901317 also protected C57BL/6 mice, but not IFN-γ deficient (IFN-γ−/−, C57BL/6 background) mice, against MCA/BHT-induced lung hyperplasia/inflammation. In addition, we determined that T0901317 inhibited urethane-induced lung tumors in BABL/c mice. Furthermore, we determined that T0901317 prevented metastasis of 4T1 breast cancer cells in BALB/c mice. Administration of T0901317 substantially increased serum IFN-γ levels and lung IFN-γ expression in BABL/c and C57BL/6 mice. Taken together, our study demonstrates that LXR inhibits MCA/BHT-induced pulmonary carcinomas in BABL/c mice and the inhibition is associated with induction of IFN-γ production. PMID:27250582

  7. Developmental regulation of the 3-methylcholanthrene- and dioxin-inducible CYP1A5 gene in chick embryo liver in vivo.

    Bentivegna, C S; Ihnat, M A; Baptiste, N S; Hamilton, J W

    1998-07-01

    The cDNA sequences for two dioxin-inducible cytochrome P450s in chicken, CYP1A4 and CYP1A5, have recently been reported which correspond to two dioxin-inducible forms of P450 previously designated as TCDDAHH and TCDDAA, respectively. The developmental expression of CYP1A4-associated aryl hydrocarbon (benzo[a]pyrene) hydroxylase (AHH) activity and its association with expression of the Ah receptor had previously been characterized in chick embryo liver. The purpose of this study was to examine the developmental regulation of the second dioxin-inducible P450 gene, CYP1A5, in chick embryo liver. A partial gene sequence for CYP1A5 indicated that the intron/exon organization of this gene was identical to that of the CYP1A1 and CYP1A2 mammalian genes and was present in a single copy in the genome. CYP1A5 mRNA was expressed basally in chick embryo liver and was highly inducible by the Ah receptor ligands, 3-methylcholanthrene, beta-naphthoflavone, and 3,4,3', 4'-tetrachlorobiphenyl (TCB), but not by the phenobarbital analog, glutethimide. CYP1A5 mRNA levels were increased 40- to 50-fold within 5 h after a single TCB treatment, corresponding to a 30- to 40-fold increase in the transcription rate of the CYP1A5 gene at this time point. In contrast to a previous report that CYP1A5 mRNA expression was inducible by estradiol, we observed no effects of estradiol or dexamethasone on CYP1A5 mRNA expression, either alone or in combination with TCB. Basal and TCB-inducible CYP1A5 mRNA expression was maximal in liver at 8 days of development and remained high throughout the remainder of embryonic development. Thus, CYP1A5 appears to be regulated in a very similar manner to CYP1A4 in chick embryo liver. PMID:9705900

  8. 3-methylcholanthrene induces differential recruitment of aryl hydrocarbon receptor to human promoters

    Pansoy, Andrea; Ahmed, Shaimaa; Valen, Eivind;

    2010-01-01

    responsive element. Conventional ChIP confirmed recruitment of AHR to 26 regions with target gene responses to 3MC varying from activation to inhibition to having no effect. A comparison of identified AHR-3MC bound regions with AHR-TCDD bound regions in from our previous study (Ahmed, S., Valen, E., Sandelin...... periodicity between 1.5 to 2 h. Re-treatment of cells with 3MC failed to alter the oscillatory binding profiles of AHR or ARNT. Cells became responsive to 3MC but not TCDD after 24 h of exposure to 3MC, highlighting important differences in AHR responsiveness between the two ligands. Our results reveal a...

  9. Aberrant methylation accounts for cell adhesion-related gene silencing during 3-methylcholanthrene and diethylnitrosamine induced multistep rat lung carcinogenesis associated with overexpression of DNA methyltransferases 1 and 3a

    To evaluate the significance of alterations in cell adhesion-related genes methylation during lung multistep carcinogenesis induced by the genotoxic carcinogens 3-methylcholanthrene (MCA) and diethylnitrosamine (DEN), tissue samples microdissected from MCA/DEN-induced rat lung carcinogenesis model were subjected to methylation-specific PCR to evaluate the DNA methylation status of CADM1, TIMP3, E-cadherin and N-cadherin. Immunohistochemistry was used to determine protein expression of CADM1, TIMP3, N-cadherin and the DNA methyltransferases (DNMTs) 1, 3a and 3b. E-cadherin hypermethylation was not detected in any tissue. CADM1, TIMP3 and N-cadherin hypermethylation was correlated with the loss of their protein expression during the progression of pathologic lesions. The prevalence of DNA methylation of at least one gene and the average number of methylated genes increased with the histological progression. DNMT1 and DNMT3a protein expression increased progressively during the stages of lung carcinogenesis, whereas DNMT3b overexpression was only found in several samples. Furthermore, DNMT1 protein expression levels were correlated with CADM1 methylation, and DNMT3a protein expression levels were correlated with CADM1, TIMP3 and N-cadherin methylation. The average number of methylated genes during carcinogenesis was significantly correlated with DNMT1 and DNMT3a protein expression levels. Moreover, mRNA expression of CADM1 significantly increased after treatment with DNMT inhibitor 5-aza-2'-deoxycytidine in CADM1-methylated primary tumor cell lines. Our findings suggest that an accumulation of hypermethylation accounts for cell adhesion-related gene silencing is associated with dynamic changes in the progression of MCA/DEN-induced rat lung carcinogenesis. We suggest that DNMT1 and DNMT3a protein overexpression may be responsible for this aberrant DNA methylation.

  10. Activation of liver X receptor inhibits the development of pulmonary carcinomas induced by 3-methylcholanthrene and butylated hydroxytoluene in BALB/c mice

    Qixue Wang; Lei Sun; Xiaoxiao Yang; Xingzhe Ma; Qi Li; Yuanli Chen; Ying Liu; Di Zhang; Xiaoju Li; Rong Xiang; Yuquan Wei; Jihong Han; Yajun Duan

    2016-01-01

    We previously reported that LXR ligand, T0901317, inhibited the growth of inoculated Lewis lung carcinoma in C57BL/6 mice by activating IFN-γ production. However, the effects of T0901317 on carcinogen-induced pulmonary carcinomas remain unknown. In this study, we initially conducted a statistical analysis on the data of human lung cancer samples extracted from the TCGA database, and determined that survival rate/time of lung cancer patients and grade of lung adenocarcinoma were positively and...

  11. Protective effect of Ocimum sanctum on 3-methylcholanthrene, 7,12-dimethylbenz(a)anthracene and aflatoxin B1 induced skin tumorigenesis in mice

    A study on the protective effect of alcoholic extract of the leaves of Ocimum sanctum on 3-mthylcholanthrene (MCA), 7,12-dimethylbenzanthracene (DMBA) and aflatoxin B1 (AFB1) induced skin tumorigenesis in a mouse model has been investigated. The study involved pretreatment of mice with the leaf extract prior to either MCA application or tetradecanoyl phorbol acetate (TPA) treatment in a two-stage tumor protocol viz a viz, DMBA/TPA and AFB1/TPA. The results of the present study indicate that the pretreatment with alcoholic extract of the leaves of O. sanctum decreased the number of tumors in MCA, DMBA/TPA and AFB1/TPA treated mice. The skin tumor induced animals pretreated with alcoholic extract led to a decrease in the expression of cutaneous γ-glutamyl transpeptidase (GGT) and glutathione-S-transferase-P (GST-P) protein. The histopathological examination of skin tumors treated with leaf extract showed increased infiltration of polymorphonuclear, mononuclear and lymphocytic cells, decreased ornithine decarboxylase activity with concomitant enhancement of interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α) in the serum, implying the in vivo antiproliferative and immunomodulatory activity of leaf extract. The decrease in cutaneous phase I enzymes and elevation of phase II enzymes in response to topical application of leaf extract prior to MCA, AFB1, DMBA/TPA and AFB1/TPA treatment indicate the possibility of impairment in reactive metabolite(s) formation and thereby reducing skin carcinogenicity. Furthermore, pretreatment of leaf extract in the carcinogen induced animals resulted in elevation of glutathione levels and decrease in lipid peroxidation along with heat shock protein expression, indicating a scavenging or antioxidant potential of the extract during chemical carcinogenesis. Thus it can be concluded that leaf extract of O. sanctum provides protection against chemical carcinogenesis in one or more of the following mechanisms: (i) by acting as an

  12. Effect of interleukin 12 on tumor induction by 3-methylcholanthrene.

    Noguchi, Y; Jungbluth, A; Richards, E C; Old, L J

    1996-01-01

    Interleukin (IL)-12 has strong antitumor activity in transplantable tumor systems in the mouse. The present study was designed to determine whether tumor induction by 3-methylcholanthrene (3-MC), a carcinogenic hydrocarbon, can be inhibited by IL-12. BALB/cBy mice were injected subcutaneously with 25 micrograms or 100 micrograms of 3-MC and treated with 100 ng, 10 ng, or 1 ng of IL-12 for 5 days a week for 18 weeks, with a schedule of 3 weeks on and 1 week off. In mice injected with 25 microg...

  13. Expression of CYP2A3 mRNA and its regulation by 3-methylcholanthrene, pyrazole, and ß-ionone in rat tissues

    A.B. Robottom-Ferreira

    2003-07-01

    Full Text Available Cytochrome P450 (CYP 2A enzymes are involved in the metabolism of numerous drugs and hormones and activate different carcinogens. Human CYP2A6, mouse CYP2A5 and rat CYP2A3 are orthologous enzymes that present high similarity in their amino acid sequence and share substrate specificities. However, different from the human and mouse enzyme, CYP2A3 is not expressed in the rat liver. There are limited data about expression of CYP2A3 in extrahepatic tissues and its regulation by typical CYP inducers. Therefore, the objective of the present study was to analyze CYP2A3 mRNA expression in different rat tissues by RT-PCR, and to study the influence of 3-methylcholanthrene, pyrazole and ß-ionone treatment on its expression. Male Wistar rats were divided into four groups of 5 rats each, and were treated ip for 4 days with 3-methylcholanthrene (25 mg/kg body weight, pyrazole (150 mg/kg body weight, ß-ionone (1 g/kg body weight, or vehicle. Total RNA was extracted from tissues and CYP2A3 mRNA levels were analyzed by semiquantitative RT-PCR. CYP2A3 mRNA was constitutively expressed in the esophagus, lung and nasal epithelium, but not along the intestine, liver, or kidney. CYP2A3 mRNA levels were increased in the esophagus by treatment with 3-methylcholanthrene and pyrazole (17- and 7-fold, respectively, in lung by pyrazole and ß-ionone (3- and 4-fold, respectively, although not statistically significant, in the distal part of the intestine and kidney by 3-methylcholanthrene and pyrazole, and in the proximal part of the intestine by pyrazole. CYP2A3 mRNA was not induced in nasal epithelium, liver or in the middle part of the intestine. These data show that, in the rat, CYP2A3 is constitutively expressed in several extrahepatic tissues and its regulation occurs through a complex mechanism that is essentially tissue specific.

  14. Consequences of 3-methylcholanthrene-type induction for the metabolism of 4-aminobiphenyl in isolated rat hepatocytes.

    Orzechowski, A; Schrenk, D; Schut, H A; Bock, K W

    1994-03-01

    Carcinogenic aromatic amines such as 4-aminobiphenyl (4-ABP) are extensively metabolized by both oxidative and conjugation reactions. Thus the burden of genotoxic metabolites of 4-ABP in a target organ is probably influenced by the balance of N-hydroxylation and alternative metabolic pathways in the hepatocyte. In freshly isolated rat hepatocytes, 4-ABP (at a substrate concentration of 10 microM) was mainly N-acetylated (54% of total metabolites), while 2% N-hydroxy-4-ABP-N-glucuronide and 21% of unconjugated N-hydroxylated metabolites were detectable. Ring-hydroxylated metabolites and the primary N-glucuronide of 4-ABP accounted for 8% and 4%, respectively. Pretreatment of rats with 3-methylcholanthrene (MC), a dioxin-type inducer of CYP1A isozymes and phenol UDP-glucuronosyltransferase (UGT1A1), led to a dramatic decrease of N-acetylated (2% of total metabolites) and an increase of N-hydroxylated (54% as free and glucuronidated compound) and ring-hydroxylated (35%) metabolites. Essentially similar effects were seen at a substrate concentration of 50 microM. Consistently, MC-type induction with beta-naphthoflavone resulted in a significant increase in the formation of DNA adducts of 4-ABP, detected by 32P-postlabeling of hepatocellular DNA. The results suggest that, similar to a previous study with 2-naphthylamine (2-NA), MC treatment leads to a marked shift from conjugation to N-oxidation. However, N-hydroxy-4-ABP (in contrast to N-hydroxy-2-NA) is mostly released from hepatocytes in the unconjugated form. PMID:8118934

  15. The effect of 3-methylcholanthrene and butylated hydroxytoluene on glycogen levels of liver, muscle, testis, and tumor tissues of rats

    POLAT, Fikriye; DERE, Egemen; GÜL, Eylem; YELKUVAN, İzzet; ÖZDEMİR, Öztürk; BİNGÖL, Günsel

    2013-01-01

    This study examined the effects of separate and combined applications of 3-methylcholanthrene, a polycyclic aromatic hydrocarbon and potent carcinogenic agent, and butylated hydroxytoluene, the antioxidant food additive, on the glycogen levels of liver, muscle, testis, and tumor tissues in rats. Adult male Wistar albino rats weighing 100-110 g at 8 weeks of age were used in this study. This study consisted of a control group (n = 9) and 3 different experiment groups in which rats were chronic...

  16. Influences of 3-methylcholanthrene, phenobarbital and dexamethasone on xenobiotic metabolizing-related cytochrome P450 enzymes and steroidogenesis in human fetal adrenal cortical cells

    Hui WANG; Min HUANG; Ren-xiu PENG; Jiang LE

    2006-01-01

    Aim: To explore the influence and possible mechanism of xenobiotics on adrenal steroidogenesis during fetal development. Methods: Primary human fetal adrenal cortical cells were prepared, cultured and treated with 3-methylcholanthrene, phenobarbital and dexamethasone. The activities of 7-ethoxyresorufin 0-dealkylase, benzphetamine, aminopyrine and erythromycin N-demethylases were measured by enzyme assays. At the same time, quantitative analysis of steroid hormones cortisol, aldosterone, testosterone and progesterone were carried out in cultural medium by radioimmunoassays. Results: The activities of benzphetamine and aminopyrine Ar-demethylase were increased in the cultural fetal adrenal cells treated with phenobarbital (0.25-1 mmol/L) for 24 h. Dexamethasone (25-100 μmol/L) also increased the activity of erythromycin W-demethylase. The activity of 7-ethoxyresorufin 0-dealkylase was undetected in the cells treated without and with 3-methylcholanthrene (0.5-2 μmol/L). Meanwhile, the contents of medium cortisol, aldosterone and progesterone were decreased after treatment with 3-methylcholanthrene. Cortisol, aldosterone and progesterone concentrations were also slightly decreased with phenobarbital. Dexamethasone enhanced the productions of cortisol and progesterone remarkably. The trend of testosterone concentration was uncertain after 3-methylcholanthrene, phenobarbital or dexamethasone treatment. Conclusion: 3-Methylcholanthrene, phenobarbital or dexamethasone could interfere with the synthesis of cortisol, aldosterone and progesterone in primary human fetal adrenal cortical cells, which likely act through xenobiotic metabolizing-related cytochrome P450 isoform activation.

  17. Induction of Cyp1a1 and Cyp1b1 and formation of DNA adducts in C57BL/6, Balb/c, and F1 mice following in utero exposure to 3-methylcholanthrene

    Fetal mice are more sensitive to chemical carcinogens than are adults. Previous studies from our laboratory demonstrated differences in the mutational spectrum induced in the Ki-ras gene from lung tumors isolated from [D2 x B6D2F1]F2 mice and Balb/c mice treated in utero with 3-methylcholanthrene (MC). We thus determined if differences in metabolism, adduct formation, or adduct repair influence strain-specific responses to transplacental MC exposure in C57BL/6 (B6), Balb/c (BC), and reciprocal F1 crosses between these two strains of mice. The induction of Cyp1a1 and Cyp1b1 in fetal lung and liver tissue was determined by quantitative fluorescent real-time PCR. MC treatment caused maximal induction of Cyp1a1 and Cyp1b1 RNA 2-8 h after injection in both organs. RNA levels for both genes then declined in both fetal organs, but a small biphasic, secondary increase in Cyp1a1 was observed specifically in the fetal lung 24-48 h after MC exposure in all four strains. Cyp1a1 induction by MC at 4 h was 2-5 times greater in fetal liver (7000- to 16,000-fold) than fetal lung (2000- to 6000-fold). Cyp1b1 induction in both fetal lung and liver was similar and much lower than that observed for Cyp1a1, with induction ratios of 8- to 18-fold in fetal lung and 10- to 20-fold in fetal liver. The overall kinetics and patterns of induction were thus very similar across the four strains of mice. The only significant strain-specific effect appeared to be the relatively poor induction of Cyp1b1 in the parental strain of B6 mice, especially in fetal lung tissue. We also measured the levels of MC adducts and their disappearance from lung tissue by the P32 post-labeling assay on gestation days 18 and 19 and postnatal days 1, 4, 11, and 18. Few differences were seen between the different strains of mice; the parental strain of B6 mice had nominally higher levels of DNA adducts 2 (gestation day 19) and 4 (postnatal day 1) days after injection, although this was not statistically significant

  18. Transcription factor oscillations induce differential gene expressions.

    Wee, Keng Boon; Yio, Wee Kheng; Surana, Uttam; Chiam, Keng Hwee

    2012-06-01

    Intracellular protein levels of diverse transcription factors (TFs) vary periodically with time. However, the effects of TF oscillations on gene expression, the primary role of TFs, are poorly understood. In this study, we determined these effects by comparing gene expression levels induced in the presence and in the absence of TF oscillations under same mean intracellular protein level of TF. For all the nonlinear TF transcription kinetics studied, an oscillatory TF is predicted to induce gene expression levels that are distinct from a nonoscillatory TF. The conditions dictating whether TF oscillations induce either higher or lower average gene expression levels were elucidated. Subsequently, the predicted effects from an oscillatory TF, which follows sigmoid transcription kinetics, were applied to demonstrate how oscillatory dynamics provide a mechanism for differential target gene transactivation. Generally, the mean TF concentration at which oscillations occur relative to the promoter binding affinity of a target gene determines whether the gene is up- or downregulated whereas the oscillation amplitude amplifies the magnitude of the differential regulation. Notably, the predicted trends of differential gene expressions induced by oscillatory NF-κB and glucocorticoid receptor match the reported experimental observations. Furthermore, the biological function of p53 oscillations is predicted to prime the cell for death upon DNA damage via differential upregulation of apoptotic genes. Lastly, given N target genes, an oscillatory TF can generate between (N-1) and (2N-1) distinct patterns of differential transactivation. This study provides insights into the mechanism for TF oscillations to induce differential gene expressions, and underscores the importance of TF oscillations in biological regulations. PMID:22713556

  19. Impact-induced differentiation in icy bodies

    Tonks, W Brian; Melosh, H Jay

    2016-01-01

    By the time icy objects grow to about the mass of Europa and silicate bodies grow to approximately lunar mass, a large high-speed impact can generate an intact melt region that allows the dense material to gravitationally segregate, forming a large density anomaly. If this anomaly generates sufficient differential stress, it rapidly segregates to the object's center, triggering whole body differentiation. We used an impact melting model based on the Hugoniot equations, the linear shock-particle velocity relationship, and the empirical relationship between shock pressure and distance coupled with a Monte Carlo simulation of the late accretion process to determine conditions under which large impacts trigger differentiation in icy bodies. In a gas-free environment, impacts of projectiles in the satellite's accretion zone have a small probability of triggering differentiation in surviving proto-satellites as small as Triton. The probability increases to 100% by the time surviving proto-satellites grow to the mas...

  20. Differential effects of mercury, lead and copper on the constitutive and inducible expression of aryl hydrocarbon receptor (AHR)-regulated genes in cultured hepatoma Hepa 1c1c7 cells.

    Korashy, Hesham M; El-Kadi, Ayman O S

    2004-09-01

    Both simultaneous and sequential exposure to heavy metals and aryl hydrocarbon receptor (AHR)-ligands potentially occur in human populations, yet there have been relatively few studies of combined effects of heavy metals and AHR-ligands on AHR-regulated genes. To investigate the effects of heavy metals on AHR-regulated genes; cytochrome P450 1a1 (cyp1a1), NAD(P)H:quinone oxidoreductase (QOR) and glutathione S-transferase Ya (GST Ya), murine hepatoma Hepa 1c1c7 cells were incubated with increasing concentrations of Hg2+ (2.5-10 microM), Pb2+ (10-100 microM), and Cu2+ (1-100 microM) alone or with the AHR-ligand, 2,3,7,8-tetrachlorodibenzo-p-dioxin (0.1 nM), 3-methylcholanthrene (0.25 microM), beta-naphthoflavone (10 microM), or benzo[a]pyrene (1 microM). The results clearly showed that metals alone did not significantly alter the cyp1a1 activity and protein levels but increased its mRNA expression, whereas a significant reduction in AHR ligand-mediated induction of cyp1a1 activity was observed by all metals. The decrease in cyp1a1 activity was associated with an increase, no change, or decrease in cyp1a1 mRNA and protein levels by Hg2+, Pb2+ and Cu2+ respectively, suggesting pre- and post-transcription mechanisms are involved. With respect to QOR, the activity and mRNA levels were increased by all metals in the absence or presence of an AHR-ligand, with the exception of Cu2+ which significantly decreased the induction of QOR. Differently, GST Ya activity was significantly increased by Cu2+ and Pb2+ and inhibited by Hg2+, while its mRNA was increased by Hg2+ and Pb2+ and decreased by Cu2+. All metals significantly increased the expression of heme oxygenase-1, which coincided with the changes in the phase I and phase II enzyme activities. These results demonstrate that heavy metals differentially modulate the constitutive and the inducible expression of AHR-regulated genes. PMID:15297030

  1. On Volatility Induced Stationarity for Stochastic Differential Equations

    Albin, J.M.P.; Astrup Jensen, Bjarne; Muszta, Anders;

    2006-01-01

    This article deals with stochastic differential equations with volatility induced stationarity. We study of theoretical properties of such equations, as well as numerical aspects, together with a detailed study of three examples.......This article deals with stochastic differential equations with volatility induced stationarity. We study of theoretical properties of such equations, as well as numerical aspects, together with a detailed study of three examples....

  2. Transcription Factor Oscillations Induce Differential Gene Expressions

    Wee, Keng Boon; Yio, Wee Kheng; Surana, Uttam; Chiam, Keng Hwee

    2012-01-01

    Intracellular protein levels of diverse transcription factors (TFs) vary periodically with time. However, the effects of TF oscillations on gene expression, the primary role of TFs, are poorly understood. In this study, we determined these effects by comparing gene expression levels induced in the presence and in the absence of TF oscillations under same mean intracellular protein level of TF. For all the nonlinear TF transcription kinetics studied, an oscillatory TF is predicted to induce ge...

  3. Induced Differentiation of Adipose-derived Stromal Cells into Myoblasts

    吴桂珠; 郑秀; 江忠清; 王金华; 宋岩峰

    2010-01-01

    This study aimed to induce the differentiation of isolated and purified adipose-derived stromal cells(ADSCs) into myoblasts,which may provide a new strategy for tissue engineering in patients with stress urinary incontinence(SUI).ADSCs,isolated and cultured ex vivo,were identified by flow cytometry and induced to differentiate into myoblasts in the presence of an induction solution consisting of DMEM supplemented with 5-azacytidine(5-aza),5% FBS,and 5% horse serum.Cellular morphology was observed under an i...

  4. Mechanisms that uncouple growth and differentiation in myeloid leukemia cells: restoration of requirement for normal growth-inducing protein without restoring induction of differentiation-inducing protein.

    Lotem, J; Sachs, L

    1982-01-01

    There are different macrophage- and granulocyte-inducing (MGI) proteins. Normal myeloid precursors are induced to multiply by one form (MGI-1) and to differentiate by another form (MGI-2). There are clones of myeloid leukemia cells that no longer require MGI-1 for growth but can still be induced to differentiate by MGI-2. After induction of differentiation in these leukemia cells by adding MCI-2 or inducing endogenous production of MGI-2 by lipopolysaccharide, the differentiating leukemia cel...

  5. Development of basal and induced aryl hydrocarbon (benzo[a]pyrene) hydroxylase activity in the chicken embryo in ovo.

    Hamilton, J W; Denison, M S; Bloom, S E

    1983-06-01

    The development of the hepatic microsomal mixed-function oxidase system was studied to determine the basal level of embryonic enzyme activity and the inducibility of this system throughout growth and differentiation. Chicken embryo livers were assayed for basal and inducible hepatic aryl hydrocarbon hydroxylase (AHHase; designated elsewhere as AHH) activity from the first appearance of the liver as a discrete organ at 5 days of incubation (DI) through day 10 after hatching. In addition, whole-embryo and viscera preparations were assayed at 3 and 4 DI. Basal AHHase activity was equal to or greater than adult levels from 3 DI through hatching in all preparations (approximately 0.3-0.5 nmol/min per mg). A 3-fold increase in basal activity above adult values occurred at hatching. The onset of inducibility in chicken embryo liver between 5 and 6 DI was concomitant with hepatocyte differentiation. A developmental profile of 24-hr 3,4,3', 4'-tetrachlorobiphenyl-induced AHHase activity showed 15- to 30-fold induction over controls from 7 DI through day 10 after hatching, with a maximum of 15 nmol/min per mg at 14 DI and day 1 after hatching, a specific activity greater than 50% greater than maximal induction in the adult. Embryonic AHHase activity was also induced by 2,3,7,8-tetrachlorodibenzo-p-dioxin, 3-methylcholanthrene, beta-naphthoflavone, and sodium phenobarbital. Induction kinetics throughout embryonic development were similar to those reported for the adult chicken and other animals. These findings demonstrate development of a mixed-function oxidase system in very early embryogenesis and then in the liver as it differentiates. Liver AHHase activity is inducible throughout development and perinatally but such activity is under strict developmental regulation. The chicken embryo has adult levels of AHHase activity which would be sufficient to achieve metabolic activation of promutagens/carcinogens before and after hepatocyte differentiation. PMID:6407011

  6. Globular adiponectin induces differentiation and fusion of skeletal muscle cells

    Tania Fiaschi; Domenico Cirelli; Giuseppina Comito; Stefania Gelmini; Giampietro Ramponi; Maria Serio; Paola Chiarugi

    2009-01-01

    The growing interest in skeletal muscle regeneration is associated with the opening of new therapeutic strategies for muscle injury after trauma, as well as several muscular degenerative pathologies, including dystrophies, muscu-lar atrophy, and cachexia. Studies focused on the ability of extracellular factors to promote myogenesis are therefore highly promising. We now report that an adipocyte-derived factor, globular adiponectin (gAd), is able to induce mus-cle gene expression and cell differentiation, gAd, besides its well-known ability to regulate several metabolic func-tions in muscle, including glucose uptake and consumption and fatty acid catabolism, is able to block cell cycle entry of myoblasts, to induce the expression of specific skeletal muscle markers such as myosin heavy chain or eaveolin-3, as well as to provoke cell fusion into multinucleated syneytia and, finally, muscle fibre formation, gAd exerts its pro-differentiative activity through redox-dependent activation of p38, Akt and 5'-AMP-activated protein kinase path-ways. Interestingly, differentiating myoblasts are autocrine for adiponectiu, and the mimicking of pro-inflammatory settings or exposure to oxidative stress strongly increases the production of the hormone from differentiating cells. These data suggest a novel function of adiponectin, directly coordinating the myogenic differentiation program and serving an autocrine function during skeletal myogenesis.

  7. Testicular acid phosphatase induces odontoblast differentiation and mineralization.

    Choi, Hwajung; Kim, Tak-Heun; Yun, Chi-Young; Kim, Jung-Wook; Cho, Eui-Sic

    2016-04-01

    Odontoblasts differentiate from dental mesenchyme during dentin formation and mineralization. However, the molecular mechanisms controlling odontoblast differentiation remain poorly understood. Here, we show that expression of testicular acid phosphatase (ACPT) is restricted in the early stage of odontoblast differentiation in proliferating dental mesenchymal cells and secretory odontoblasts. ACPT is expressed earlier than tissue-nonspecific alkaline phosphatase (TNAP) and partly overlaps with TNAP in differentiating odontoblasts. In MDPC-23 odontoblastic cells, expression of ACPT appears simultaneously with a decrease in β-catenin activity and is abolished with the expression of Phex and Dsp. Knockdown of ACPT in MDPC-23 cells stimulates cell proliferation together with an increase in active β-catenin and cyclin D1. In contrast, the overexpression of ACPT suppresses cell proliferation with a decrease in active β-catenin and cyclin D1. Expression of TNAP, Osx, Phex and Dsp is reduced by knockdown of ACPT but is enhanced by ACPT overexpression. When ACPT is blocked with IgG, alkaline phosphatase activity is inhibited but cell proliferation is unchanged regardless of ACPT expression. These findings suggest that ACPT inhibits cell proliferation through β-catenin-mediated signaling in dental mesenchyme but elicits odontoblast differentiation and mineralization by supplying phosphate during dentin formation. Thus, ACPT might be a novel candidate for inducing odontoblast differentiation and mineralization for dentin regeneration. PMID:26547858

  8. Radiation-Induced Differentiation in Human Lung Fibroblast

    Park, Sa-Rah; Ahn, Ji-Yeon; Han, Young-Soo; Shim, Jie-Young; Yun, Yeon-Sook; Song, Jie-Young [Korea Institute of Radiological and Medical Sciences, Seoul (Korea, Republic of)

    2007-10-15

    One of the most common tumors in many countries is lung cancer and patients with lung cancer may take radiotherapy. Although radiotherapy may have its own advantages, it can also induce serious problems such as acute radiation pneumonitis and pulmonary fibrosis. Pulmonary fibrosis is characterized by excessive production of {alpha}-SMA and accumulation of extracellular matrix (ECM) such as collagen and fibronectin. There has been a great amount of research about fibrosis but the exact mechanism causing the reaction is not elucidated especially in radiation-induced fibrosis. Until now it has been known that several factors such as transforming growth factor (TGF-{beta}), tumor necrosis factor (TNF), interleukin (IL)-1, IL-6, platelet-derived growth factor (PDGF) and fibroblast growth factor (FGF) are related to fibrosis. Among them TGF-{beta} with Smad signaling is known to be the main stream and other signaling molecules such as MAPK, ERK and JNK (3) also participates in the process. In addition to those above factors, it is thought that more diverse and complicate mechanisms may involve in the radiationinduced fibrosis. Therefore, to investigate the underlying mechanisms in radiation induced fibrosis, first of all, we confirmed whether radiation induces trans differentiation in human normal lung fibroblasts. Here, we suggest that not only TGF-{beta} but also radiation can induce trans differentiation in human lung fibroblast WI-38 and IMR-90.

  9. Insulin Cannot Induce Adipogenic Differentiation in Primary Cardiac Cultures.

    Parameswaran, Sreejit; Sharma, Rajendra K

    2016-09-01

    Cardiac tissue contains a heterogeneous population of cardiomyocytes and nonmyocyte population especially fibroblasts. Fibroblast differentiation into adipogenic lineage is important for fat accumulation around the heart which is important in cardiac pathology. The differentiation in fibroblast has been observed both spontaneously and due to increased insulin stimulation. The present study aims to observe the effect of insulin in adipogenic differentiation of cardiac cells present in primary murine cardiomyocyte cultures. Oil Red O (ORO) staining has been used for observing the lipid accumulations formed due to adipogenic differentiation in murine cardiomyocyte cultures. The accumulated lipids were quantified by ORO assay and normalized using protein estimation. The lipid accumulation in cardiac cultures did not increase in presence of insulin. However, addition of other growth factors like insulin-like growth factor 1 and epidermal growth factor promoted adipogenic differentiation even in the presence of insulin and other inhibitory molecules such as vitamins. Lipid accumulation also increased in cells grown in media without insulin after an initial exposure to insulin-containing growth media. The current study adds to the existing knowledge that the insulin by itself cannot induce adipogenic induction in the cardiac cultures. The data have significance in the understanding of cardiovascular health especially in diabetic patients. PMID:27574386

  10. Acidic environments induce differentiation of Proteus mirabilis into swarmer morphotypes.

    Fujihara, Masatoshi; Obara, Hisato; Watanabe, Yusaku; Ono, Hisaya K; Sasaki, Jun; Goryo, Masanobu; Harasawa, Ryô

    2011-07-01

    Although swarmer morphotypes of Proteus mirabilis have long been considered to result from surfaced-induced differentiation, the present findings show that, in broth medium containing urea, acidic conditions transform some swimmer cells into elongated swarmer cells. This study has also demonstrates that P. mirabilis cells grown in acidic broth medium containing urea enhance virulence factors such as flagella production and cytotoxicity to human bladder carcinoma cell line T24, though no significant difference in urease activity under different pH conditions was found. Since there is little published data on the behavior of P. mirabilis at various hydrogen-ion concentrations, the present study may clarify aspects of cellular differentiation of P. mirabilis in patients at risk of struvite formation due to infection with urease-producing bacteria, as well as in some animals with acidic or alkaline urine. PMID:21707738

  11. ROS Mediates Radiation-Induced Differentiation in Human Lung Fibroblast

    Park, Sa Rah; Ahn, Ji Yeon; Kim, Mi Hyeung; Lim, Min Jin; Yun, Yeon Sook; Song, Jie Young [Korea Institute of Radiological and Medical Sciences, Seoul (Korea, Republic of)

    2009-05-15

    One of the most common tumors worldwide is lung cancer and the number of patients with lung cancer received radiotherapy is increasing rapidly. Although radiotherapy may have lots of advantages, it can also induce serious adverse effects such as acute radiation pneumonitis and pulmonary fibrosis. Pulmonary fibrosis is characterized by excessive production of smooth muscle actin-alpha (a-SMA) and accumulation of extracellular matrix (ECM) such as collagen and fibronectin. There has been a great amount of research about fibrosis but the exact mechanism causing the reaction is not elucidated especially in radiation-induced fibrosis. Until now it has been known that several factors such as transforming growth factor (TGF-b), tumor necrosis factor (TNF), IL-6, platelet-derived growth factor (PDGF) and reactive oxygen species are related to fibrosis. It is also reported that reactive oxygen species (ROS) can be induced by radiation and can act as a second messenger in various signaling pathways. Therefore we focused on the role of ROS in radiation induced fibrosis. Here, we suggest that irradiation generate ROS mainly through NOX4, result in differentiation of lung fibroblast into myofibroblast.

  12. Decreased Intracellular pH Induced by Cariporide Differentially Contributes to Human Umbilical Cord-Derived Mesenchymal Stem Cells Differentiation

    Wei Gao

    2014-01-01

    Full Text Available Background/Aims: Na+/H+ exchanger 1 (NHE1 is an important regulator of intracellular pH (pHi. High pHi is required for cell proliferation and differentiation. Our previous study has proven that the pHi of mesenchymal stem cells is higher than that of normal differentiated cells and similar to tumor cells. NHE1 is highly expressed in both mesenchymal stem cells and tumor cells. Targeted inhibition of NHE1 could induce differentiation of K562 leukemia cells. In the present paper we explored whether inhibition of NHE1 could induce differentiation of mesenchymal stem cells. Methods: MSCs were obtained from human umbilical cord and both the surface phenotype and functional characteristics were analyzed. Selective NHE1 inhibitor cariporide was used to treat human umbilical cord-derived mesenchymal stem cells (hUC-MSCs. The pHi and the differentiation of hUC-MSCs were compared upon cariporide treatment. The putative signaling pathway involved was also explored. Results: The pHi of hUC-MSCs was decreased upon cariporide treatment. Cariporide up-regulated the osteogenic differentiation of hUC-MSCs while the adipogenic differentiation was not affected. For osteogenic differentiation, β-catenin expression was up-regulated upon cariporide treatment. Conclusion: Decreased pHi induced by cariporide differentially contributes to hUC-MSCs differentiation.

  13. Pathologic endoplasmic reticulum stress induced by glucotoxic insults inhibits adipocyte differentiation and induces an inflammatory phenotype.

    Longo, Michele; Spinelli, Rosa; D'Esposito, Vittoria; Zatterale, Federica; Fiory, Francesca; Nigro, Cecilia; Raciti, Gregory A; Miele, Claudia; Formisano, Pietro; Beguinot, Francesco; Di Jeso, Bruno

    2016-06-01

    Adipocyte differentiation is critical in obesity. By controlling new adipocyte recruitment, adipogenesis contrasts adipocyte hypertrophy and its adverse consequences, such as insulin resistance. Contrasting data are present in literature on the effect of endoplasmic reticulum (ER) stress and subsequent unfolded protein response (UPR) on adipocyte differentiation, being reported to be either necessary or inhibitory. In this study, we sought to clarify the effect of ER stress and UPR on adipocyte differentiation. We have used two different cell lines, the widely used pre-adipocyte 3T3-L1 cells and a murine multipotent mesenchymal cell line, W20-17 cells. A strong ER stress activator, thapsigargin, and a pathologically relevant inducer of ER stress, glucosamine (GlcN), induced ER stress and UPR above those occurring in the absence of perturbation and inhibited adipocyte differentiation. Very low concentrations of 4-phenyl butyric acid (PBA, a chemical chaperone) inhibited only the overactivation of ER stress and UPR elicited by GlcN, leaving unaltered the part physiologically activated during differentiation, and reversed the inhibitory effect of GlcN on differentiation. In addition, GlcN stimulated proinflammatory cytokine release and PBA prevented these effects. An inhibitor of NF-kB also reversed the effects of GlcN on cytokine release. These results indicate that while ER stress and UPR activation is "physiologically" activated during adipocyte differentiation, the "pathologic" part of ER stress activation, secondary to a glucotoxic insult, inhibits differentiation. In addition, such a metabolic insult, causes a shift of the preadipocyte/adipocyte population towards a proinflammatory phenotype. PMID:26940722

  14. Laboratory-induced hyperventilation differentiates female sexual arousal disorder subtypes.

    Brotto, Lori A; Klein, Carolin; Gorzalka, Boris B

    2009-08-01

    The effects of heightened sympathetic nervous system (SNS) activity via laboratory-induced hyperventilation (LIH) on subjective and physiological sexual arousal were examined in a heterogeneous group of women with Sexual Arousal Disorder (SAD; n = 60), as well as across subtypes of SAD, in comparison to a control group of women without sexual difficulties (n = 42). Participants took part in 2 min of rapid breathing, a technique previously found to increase SNS activity, immediately prior to viewing erotic stimuli. Physiological arousal (i.e., vaginal pulse amplitude; VPA) was measured via the vaginal photoplethysmograph and subjective arousal was measured via self-report questionnaires. LIH differentiated women with SAD from those in the control group, with LIH increasing VPA in the latter, but having no significant effect in the heterogeneous SAD group. However, among subtypes of SAD, LIH differentiated women with genital (n = 16) and subjective (n = 16) subtypes of SAD from women with combined SAD (n = 28) and women without sexual difficulties. Specifically, women in the control group and those with combined SAD had a significant increase in VPA whereas women with genital or subjective SAD had a significant decrease in VPA following LIH. There was no significant effect of LIH on any self-report measure of sexual arousal following erotic stimuli. Implications of the results for the conceptualization, diagnosis, and treatment of SAD are discussed. PMID:18343989

  15. Optimal time point for the transplantation of neural stem cells induced to differentiate with retinoic acid

    Shuxin Wang; Dengji Pan; Na Liu; Yongming Liu; Juan Chen; Houjie Ni; Zhouping Tang

    2011-01-01

    Previous studies have demonstrated that differentiated neural stem cells (NSCs) are more suitable for transplantation than non-differentiated NSCs. In this study, NSCs were expanded in vitro for two passages, induced with retinoic acid to differentiate, and harvested between 1-6 days later. They were subsequently cultured in artificial cerebrospinal fluid for an additional 3 days, during which their growth and morphology was monitored. NSCs induced for 4 days exhibited a peak rate of cells differentiating into neurons and robust growth. Our results indicate that the optimal time point for transplanting NSCs is following a 4-day period of induced differentiation.

  16. Static and Dynamic Experiment Evaluations of a Displacement Differential Self-Induced Magnetorheological Damper

    Guoliang Hu; Wei Zhou; Mingke Liao; Weihua Li

    2015-01-01

    This paper presents the development of a novel magnetorheological damper (MRD) which has a self-induced ability. In this study, a linear variable differential sensor (LVDS) based on the electromagnetic induction mechanism was integrated with a conventional MRD. The structure of the displacement differential self-induced magnetorheological damper (DDSMRD) was developed, and the theory of displacement differential self-induced performance was deduced. The static experiments of the DDSMRD under ...

  17. Derivation, characterization and retinal differentiation of induced pluripotent stem cells

    Subba Rao Mekala; Vasundhara Vauhini; Usha Nagarajan; Savitri Maddileti; Subhash Gaddipati; Indumathi Mariappan

    2013-03-01

    Millions of people world over suffer visual disability due to retinal dystrophies which can be age-related or a genetic disorder resulting in gradual degeneration of the retinal pigmented epithelial (RPE) cells and photoreceptors. Therefore, cell replacement therapy offers a great promise in treating such diseases. Since the adult retina does not harbour any stem cells, alternative stem cell sources like the embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) offer a great promise for generating different cell types of the retina. Here, we report the derivation of four iPSC lines from mouse embryonic fibroblasts (MEFs) using a cocktail of recombinant retroviruses carrying the genes for Oct4, Sox2, Klf4 and cMyc. The iPS clone MEF-4F3 was further characterized for stemness marker expression and stable reprogramming by immunocytochemistry, FACS and RT-PCR analysis. Methylation analysis of the nanog promoter confirmed the reprogrammed epigenetic state. Pluripotency was confirmed by embryoid body (EB) formation and lineage-specific marker expression. Also, upon retinal differentiation, patches of pigmented cells with typical cobble-stone phenotype similar to RPE cells are generated within 6 weeks and they expressed ZO-1 (tight junction protein), RPE65 and bestrophin (mature RPE markers) and showed phagocytic activity by the uptake of fluorescent latex beads.

  18. Embryonic carcinoma cells show specific dielectric resistance profiles during induced differentiation.

    Simin Öz

    Full Text Available Induction of differentiation in cancer stem cells by drug treatment represents an important approach for cancer therapy. The understanding of the mechanisms that regulate such a forced exit from malignant pluripotency is fundamental to enhance our knowledge of tumour stability. Certain nucleoside analogues, such as 2'-deoxy-5-azacytidine and 1β-arabinofuranosylcytosine, can induce the differentiation of the embryonic cancer stem cell line NTERA 2 D1 (NT2. Such induced differentiation is associated with drug-dependent DNA-damage, cellular stress and the proteolytic depletion of stem cell factors. In order to further elucidate the mode of action of these nucleoside drugs, we monitored differentiation-specific changes of the dielectric properties of growing NT2 cultures using electric cell-substrate impedance sensing (ECIS. We measured resistance values of untreated and retinoic acid treated NT2 cells in real-time and compared their impedance profiles to those of cell populations triggered to differentiate with several established substances, including nucleoside drugs. Here we show that treatment with retinoic acid and differentiation-inducing drugs can trigger specific, concentration-dependent changes in dielectric resistance of NT2 cultures, which can be observed as early as 24 hours after treatment. Further, low concentrations of nucleoside drugs induce differentiation-dependent impedance values comparable to those obtained after retinoic acid treatment, whereas higher concentrations induce proliferation defects. Finally, we show that impedance profiles of substance-induced NT2 cells and those triggered to differentiate by depletion of the stem cell factor OCT4 are very similar, suggesting that reduction of OCT4 levels has a dominant function for differentiation induced by nucleoside drugs and retinoic acid. The data presented show that NT2 cells have specific dielectric properties, which allow the early identification of differentiating

  19. Phenazopyridine induces and synchronizes neuronal differentiation of embryonic stem cells.

    Suter, David M; Preynat-Seauve, Olivier; Tirefort, Diderik; Feki, Anis; Krause, Karl-Heinz

    2009-09-01

    Embryonic stem (ES) cells are powerful tools to understand mechanisms of neuronal differentiation and to engineer neurons for in vitro studies and cell therapy. We developed a screening approach to identify small organic molecules driving neuronal differentiation of ES cells. For this purpose, we used a lentivector carrying a dual luciferase reporter system to engineer an ES cell line which allowed us to screen for small organic molecules enhancing neuronal differentiation. One of them, phenazopyridine, was further analysed in human ES cells. Phenazopyridine: (i) enhanced neuronal differentiation, (ii) increased cell survival, (iii) decreased the amount of non-neuronal and undifferentiated cells and (iv) synchronized the cellular differentiation state. Phenazopyridine allowed the development of a differentiation protocol compatible with the generation of clinical grade neural precursors, which were able differentiate into different neuronal subtypes, astrocytes and oligodendrocytes. In summary, we describe a powerful approach to identify small molecules directing stem cell differentiation. This led to the establishment of a new application for an old drug and the development of a novel clinical grade protocol for neuronal differentiation of ES cells. PMID:20196783

  20. PI3K/AKT and ERK regulate retinoic acid-induced neuroblastoma cellular differentiation

    Qiao, Jingbo [Department of Pediatric Surgery, Vanderbilt University Medical Center, Nashville, TN 37232 (United States); Paul, Pritha; Lee, Sora [Department of Pediatric Surgery, Vanderbilt University Medical Center, Nashville, TN 37232 (United States); Department of Cancer Biology, Vanderbilt University Medical Center, Nashville, TN 37232 (United States); Qiao, Lan; Josifi, Erlena; Tiao, Joshua R. [Department of Pediatric Surgery, Vanderbilt University Medical Center, Nashville, TN 37232 (United States); Chung, Dai H., E-mail: dai.chung@vanderbilt.edu [Department of Pediatric Surgery, Vanderbilt University Medical Center, Nashville, TN 37232 (United States); Department of Cancer Biology, Vanderbilt University Medical Center, Nashville, TN 37232 (United States)

    2012-08-03

    Highlights: Black-Right-Pointing-Pointer Retinoic acid (RA) induces neuroblastoma cells differentiation, which is accompanied by G0/G1 cell cycle arrest. Black-Right-Pointing-Pointer RA resulted in neuroblastoma cell survival and inhibition of DNA fragmentation; this is regulated by PI3K pathway. Black-Right-Pointing-Pointer RA activates PI3K and ERK1/2 pathway; PI3K pathway mediates RA-induced neuroblastoma cell differentiation. Black-Right-Pointing-Pointer Upregulation of p21 is necessary for RA-induced neuroblastoma cell differentiation. -- Abstract: Neuroblastoma, the most common extra-cranial solid tumor in infants and children, is characterized by a high rate of spontaneous remissions in infancy. Retinoic acid (RA) has been known to induce neuroblastoma differentiation; however, the molecular mechanisms and signaling pathways that are responsible for RA-mediated neuroblastoma cell differentiation remain unclear. Here, we sought to determine the cell signaling processes involved in RA-induced cellular differentiation. Upon RA administration, human neuroblastoma cell lines, SK-N-SH and BE(2)-C, demonstrated neurite extensions, which is an indicator of neuronal cell differentiation. Moreover, cell cycle arrest occurred in G1/G0 phase. The protein levels of cyclin-dependent kinase inhibitors, p21 and p27{sup Kip}, which inhibit cell proliferation by blocking cell cycle progression at G1/S phase, increased after RA treatment. Interestingly, RA promoted cell survival during the differentiation process, hence suggesting a potential mechanism for neuroblastoma resistance to RA therapy. Importantly, we found that the PI3K/AKT pathway is required for RA-induced neuroblastoma cell differentiation. Our results elucidated the molecular mechanism of RA-induced neuroblastoma cellular differentiation, which may be important for developing novel therapeutic strategy against poorly differentiated neuroblastoma.

  1. Differentiation-inducing factor-1 induces cyclin D1 degradation through the phosphorylation of Thr286 in squamous cell carcinoma

    Differentiation-inducing factors (DIFs) are morphogens which induce cell differentiation in Dictyostelium. We reported that DIF-1 and DIF-3 inhibit proliferation and induce differentiation in mammalian cells. In this study, we investigated the effect of DIF-1 on oral squamous cell carcinoma cell lines NA and SAS, well differentiated and poorly differentiated cell lines, respectively. Although DIF-1 did not induce the expression of cell differentiation makers in these cell lines, it inhibited the proliferation of NA and SAS in a dose-dependent manner by restricting the cell cycle in the G0/G1 phase. DIF-1 induced cyclin D1 degradation, but this effect was prevented by treatment with lithium chloride and SB216763, the inhibitors of glycogen synthase kinase-3β (GSK-3β). Depletion of endogenous GSK-3β by RNA interference also attenuated the effect of DIF-1 on cyclin D1 degradation. Therefore, we investigated the effect of DIF-1 on GSK-3β and found that DIF-1 dephosphorylated GSK-3β on Ser9 and induced the nuclear translocation of GSK-3β, suggesting that DIF-1 activated GSK-3β. Then, we examined the effect of DIF-1 on cyclin D1 mutants (Thr286Ala, Thr288Ala, and Thr286/288Ala). We revealed that Thr286Ala and Thr286/288Ala mutants were highly resistant to DIF-1-induced degradation compared with wild-type cyclin D1, indicating that the phosphorylation of Thr286 was critical for cyclin D1 degradation induced by DIF-1. These results suggest that DIF-1 induces degradation of cyclin D1 through the GSK-3β-mediated phosphorylation of Thr286

  2. BMP-2 Induced Expression of Alx3 That Is a Positive Regulator of Osteoblast Differentiation.

    Takashi Matsumoto

    Full Text Available Bone morphogenetic proteins (BMPs regulate many aspects of skeletal development, including osteoblast and chondrocyte differentiation, cartilage and bone formation, and cranial and limb development. Among them, BMP-2, one of the most potent osteogenic signaling molecules, stimulates osteoblast differentiation, while it inhibits myogenic differentiation in C2C12 cells. To evaluate genes involved in BMP-2-induced osteoblast differentiation, we performed cDNA microarray analyses to compare BMP-2-treated and -untreated C2C12 cells. We focused on Alx3 (aristaless-like homeobox 3 which was clearly induced during osteoblast differentiation. Alx3, a homeobox gene related to the Drosophilaaristaless gene, has been linked to developmental functions in craniofacial structures and limb development. However, little is known about its direct relationship with bone formation. In the present study, we focused on the mechanisms of Alx3 gene expression and function during osteoblast differentiation induced by BMP-2. In C2C12 cells, BMP-2 induced increase of Alx3 gene expression in both time- and dose-dependent manners through the BMP receptors-mediated SMAD signaling pathway. In addition, silencing of Alx3 by siRNA inhibited osteoblast differentiation induced by BMP-2, as showed by the expressions of alkaline phosphatase (Alp, Osteocalcin, and Osterix, while over-expression of Alx3 enhanced osteoblast differentiation induced by BMP-2. These results indicate that Alx3 expression is enhanced by BMP-2 via the BMP receptors mediated-Smad signaling and that Alx3 is a positive regulator of osteoblast differentiation induced by BMP-2.

  3. Wnt signaling pathway participates in valproic acid-induced neuronal differentiation of neural stem cells

    Wang, Li; Liu, Yuan; Li, Sen; Zai-yun LONG; Wu, Ya-min

    2015-01-01

    Neural stem cells (NSCs) are multipotent cells that have the capacity for differentiation into the major cell types of the nervous system, i.e. neurons, astrocytes and oligodendrocytes. Valproic acid (VPA) is a widely prescribed drug for seizures and bipolar disorder in clinic. Previously, a number of researches have been shown that VPA has differential effects on growth, proliferation and differentiation in many types of cells. However, whether VPA can induce NSCs from embryonic cerebral cor...

  4. Inducing endoderm differentiation by modulating mechanical properties of soft substrates.

    Jaramillo, Maria; Singh, Satish S; Velankar, Sachin; Kumta, Prashant N; Banerjee, Ipsita

    2015-01-01

    Early embryonic stem cell (ESC) differentiation is marked by the formation of three germ layers from which all tissues types arise. Conventionally, ESCs are differentiated by altering their chemical microenvironment. Recently however, it was established that a mechanical microenvironment can also contribute towards cellular phenotype commitment. In this study, we report how the cellular mechanical microenvironment of soft substrates affects the differentiation and phenotypic commitment of ESCs. Mouse ESCs were cultured in a fibrin hydrogel matrix in 2D and 3D cultures. The gelation characteristics of the substrates were modulated by systematically altering the fibrinogen concentration and the fibrinogen-thrombin crosslinking ratio. Analysis of the ESCs cultured on different substrate conditions clearly illustrated the strong influence that substrate physical characteristics assert on cellular behaviours. Specifically, it was found that ESCs had a higher proliferation rate in gels of lower stiffness. Early differentiation events were studied by analyzing the gene and protein expression levels of early germ layer markers. Our results revealed that lower substrate stiffness elicited stronger upregulation of endoderm related genes Sox17, Afp and Hnf4 compared to stiffer substrates. While both 2D and 3D cultures showed a similar response, the effects were much stronger in 3D culture. These results suggest that physical cues can be used to modulate ESC differentiation into clinically relevant tissues such as liver and pancreas. PMID:23008262

  5. Lignin Induces ES Cells to Differentiate into Neuroectodermal Cells through Mediation of the Wnt Signaling Pathway

    Inoue, Yu; Hasegawa, Seiji; Yamada, Takaaki; Date, Yasushi; MIZUTANI, Hiroshi; Nakata, Satoru; Akamatsu, Hirohiko

    2013-01-01

    Embryonic stem cells (ES cells) are characterized by their pluripotency and infinite proliferation potential. Ever since ES cells were first established in 1981, there have been a growing number of studies aimed at clinical applications of ES cells. In recent years, various types of differentiation inducement systems using ES cells have been established. Further studies have been conducted to utilize differentiation inducement systems in the field of regenerative medicine. For cellular treatm...

  6. Tinospora cordifolia Induces Differentiation and Senescence Pathways in Neuroblastoma Cells.

    Mishra, Rachana; Kaur, Gurcharan

    2015-08-01

    Children diagnosed with neuroblastomas often suffer from severe side as well as late effects of conventional treatments like chemotherapy and radiotherapy. Recent advances in understanding of molecular pathways involved in cellular differentiation and apoptosis have helped in the development of new therapeutic approach based on differentiation-based therapy of malignant tumours. Natural medicines with their holistic therapeutic approach are known to selectively eliminate cancer cells thus provide a better substitute for the conventional treatment modes. The current study was aimed to investigate the anti-cancer potential of aqueous ethanolic extract of Tinospora cordifolia (TCE) using IMR-32 human neuroblastoma cell line as a model system. TCE is highly recommended in Ayurveda for its general body and metal health-promoting properties. TCE treatment was seen to arrest the majority of cells in G0/G1 phase and modulated the expression of DNA clamp sliding protein (PCNA) and cyclin D1. Further, TCE-treated cells showed differentiation as revealed by their morphology and the expression of neuronal cell specific differentiation markers NF200, MAP-2 and NeuN in neuroblastoma cells. The differentiated phenotype was associated with induction of senescence and pro-apoptosis pathways by enhancing expression of senescence marker mortalin and Rel A subunit of nuclear factor kappa beta (NFkB) along with decreased expression of anti-apoptotic marker, Bcl-xl. TCE exhibited anti-metastatic activity and significantly reduced cell migration in the scratched area along with downregulation of neural cell adhesion molecule (NCAM) polysialylation and secretion of matrix metalloproteinases (MMPs). Our data suggest that crude extract or active phytochemicals from this plant may be a potential candidate for differentiation-based therapy of malignant neuroblastoma cells. PMID:25280667

  7. Graphene induces spontaneous cardiac differentiation in embryoid bodies

    Ahadian, Samad; Zhou, Yuanshu; Yamada, Shukuyo; Estili, Mehdi; Liang, Xiaobin; Nakajima, Ken; Shiku, Hitoshi; Matsue, Tomokazu

    2016-03-01

    Graphene was embedded into the structure of mouse embryoid bodies (EBs) using the hanging drop technique. The inclusion of 0.2 mg per mL graphene in the EBs did not affect the viability of the stem cells. However, the graphene decreased the stem cell proliferation, probably by accelerating cell differentiation. The graphene also enhanced the mechanical properties and electrical conductivity of the EBs. Interestingly, the cardiac differentiation of the EB-graphene was significantly greater than that of the EBs at day 5 of culture, as confirmed by high-throughput gene analysis. Electrical stimulation (voltage, 4 V; frequency, 1 Hz; and duration, 10 ms for 2 continuous days) further enhanced the cardiac differentiation of the EBs, as demonstrated by analyses of the cardiac protein and gene expression and the beating activity of the EBs. Taken together, the results demonstrated that graphene played a major role in directing the cardiac differentiation of EBs, which has potential cell therapy and tissue regeneration applications.Graphene was embedded into the structure of mouse embryoid bodies (EBs) using the hanging drop technique. The inclusion of 0.2 mg per mL graphene in the EBs did not affect the viability of the stem cells. However, the graphene decreased the stem cell proliferation, probably by accelerating cell differentiation. The graphene also enhanced the mechanical properties and electrical conductivity of the EBs. Interestingly, the cardiac differentiation of the EB-graphene was significantly greater than that of the EBs at day 5 of culture, as confirmed by high-throughput gene analysis. Electrical stimulation (voltage, 4 V; frequency, 1 Hz; and duration, 10 ms for 2 continuous days) further enhanced the cardiac differentiation of the EBs, as demonstrated by analyses of the cardiac protein and gene expression and the beating activity of the EBs. Taken together, the results demonstrated that graphene played a major role in directing the cardiac

  8. Activation of PPARγ is not involved in butyrate-induced epithelial cell differentiation

    Histone deacetylase-inhibitors affect growth and differentiation of intestinal epithelial cells by inducing expression of several transcription factors, e.g. Peroxisome proliferator-activated receptor γ (PPARγ) or vitamin D receptor (VDR). While activation of VDR by butyrate mainly seems to be responsible for cellular differentiation, the activation of PPARγ in intestinal cells remains to be elucidated. The aim of this study was to determine the role of PPARγ in butyrate-induced cell growth inhibition and differentiation induction in Caco-2 cells. Treatment with PPARγ ligands ciglitazone and BADGE (bisphenol A diglycidyl) enhanced butyrate-induced cell growth inhibition in a dose- and time-dependent manner, whereas cell differentiation was unaffected after treatment with PPARγ ligands rosiglitazone and MCC-555. Experiments were further performed in dominant-negative PPARγ mutant cells leading to an increase in cell growth whereas butyrate-induced cell differentiation was again unaffected. The present study clearly demonstrated that PPARγ is involved in butyrate-induced inhibition of cell growth, but seems not to play an essential role in butyrate-induced cell differentiation

  9. The Preliminary Experimental Study of Induced Differentiation of Embryonic Stem Cells into Corneal Epithelial Cells

    Ling Yu; Jian Ge; Zhichong Wang; Bing Huang; Keming Yu; Chongde Long; Xigu Chen

    2001-01-01

    Purpose:To study preliminarily induced differentiation of embryonic stem cells intocorneal epithelial cells in vitro.Methods: Murine embryonic stem cells were co-cultured with Rabbit limbal cornealepithelial cells in Transwell system to induce differentiation. Mophological andimmunohistochemical examination were implemented.Results: The induced cells from embryonic stem cells have an epithelial appearance.The cells formed a network and were confluent into film gradually after beingco-cultured with rabbit limbal corneal epithelial cells for 24 ~ 96 hours. The cells rangedmosaic structure and localized together with clear rim. Most of the cells showedpolygonal appearance. Transmission electron microscope showed lots of microvilli on thesurface of induced cells and tight junctions between them. These epithelial-like cellsexpressed the corneal epithelial cell specific marker cytokeratin3/cytokeratinl2.Conclusion: The potential mechanism of the differentiation of murine embryonic stemcells into corneal epithelial cells induced by limbal corneal epithelial cell-derivedinducing activity is to be further verified.

  10. Natural Product Vibsanin A Induces Differentiation of Myeloid Leukemia Cells through PKC Activation.

    Yu, Zu-Yin; Xiao, He; Wang, Li-Mei; Shen, Xing; Jing, Yu; Wang, Lin; Sun, Wen-Feng; Zhang, Yan-Feng; Cui, Yu; Shan, Ya-Jun; Zhou, Wen-Bing; Xing, Shuang; Xiong, Guo-Lin; Liu, Xiao-Lan; Dong, Bo; Feng, Jian-Nan; Wang, Li-Sheng; Luo, Qing-Liang; Zhao, Qin-Shi; Cong, Yu-Wen

    2016-05-01

    All-trans retinoic acid (ATRA)-based cell differentiation therapy has been successful in treating acute promyelocytic leukemia, a unique subtype of acute myeloid leukemia (AML). However, other subtypes of AML display resistance to ATRA-based treatment. In this study, we screened natural, plant-derived vibsane-type diterpenoids for their ability to induce differentiation of myeloid leukemia cells, discovering that vibsanin A potently induced differentiation of AML cell lines and primary blasts. The differentiation-inducing activity of vibsanin A was mediated through direct interaction with and activation of protein kinase C (PKC). Consistent with these findings, pharmacological blockade of PKC activity suppressed vibsanin A-induced differentiation. Mechanistically, vibsanin A-mediated activation of PKC led to induction of the ERK pathway and decreased c-Myc expression. In mouse xenograft models of AML, vibsanin A administration prolonged host survival and inhibited PKC-mediated inflammatory responses correlated with promotion of skin tumors in mice. Collectively, our results offer a preclinical proof of concept for vibsanin A as a myeloid differentiation-inducing compound, with potential application as an antileukemic agent. Cancer Res; 76(9); 2698-709. ©2016 AACR. PMID:26984756

  11. SKW 6.4 cell differentiation induced by interleukin 6 is stimulated by butyrate.

    Kawamoto, T; Gohda, E; Iji, H; Fujiwara, M; Yamamoto, I

    1998-08-01

    We investigated if sodium butyrate (NaBu), an inhibitor of histone deacetylase, and its analogs modulate cytokine-induced differentiation of the human B cell line SKW 6.4 transformed by the Epstein-Barr virus. NaBu markedly enhanced interleukin (IL)-6-induced IgM production with an accompanying increase in the level of histone H4 acetylation and augmented IgM production induced by IL-4 and phorbol 12-myristate 13-acetate. From both the enhancing effect of cell differentiation and the effect of inducing histone hyperacetylation in SKW 6.4 cells, other histone deacetylase inhibitors and NaBu analogs were divided into three groups: those that increased both IL-6-induced antibody production and histone acetylation, those that caused histone hyperacetylation, but failed to induce the differentiation, and those that were ineffective at inducing either activity. No agent that enhanced IgM production without inducing histone hyperacetylation was found among the inhibitors and analogs we tested. These results suggest that the increase in the histone acetylation is necessary, but it is insufficient to augment differentiation of SKW 6.4 cells. Thus another activity of NaBu in addition to the inhibition of histone deacetylase may be involved in promoting IL-6-induced differentiation. Our results also suggest that fatty acids that have a straight chain of four carbon atoms or are branched with four and five carbon atoms, which contain no hydrophilic substituents, or those with similar structures, show this other activity. PMID:9826026

  12. Forced expression of Hnf4a induces hepatic gene activation through directed differentiation.

    Yahoo, Neda; Pournasr, Behshad; Rostamzadeh, Jalal; Fathi, Fardin

    2016-08-01

    Embryonic stem (ES) cells are capable of unlimited self-renewal and have a diverse differentiation potential. These unique features make ES cells as an attractive source for developmental biology studies. Having the mature hepatocyte in the lab with functional activities is valuable in drug discovery studies. Overexpression of hepatocyte lineage-specific transcription factors (TFs) becomes a promising approach in pluripotent cell differentiation toward liver cells. Many studies generate transgenic ES cell lines to examine the effects of specific TFs overexpression in cell differentiation. In the present report, we have addressed whether a suspension or adherent model of differentiation is an appropriate way to study the role of Hnf4a overexpression. We generated ES cells that carried a doxycycline (Dox)-inducible Hnf4a using lentiviral vectors. The transduced cells were subjected to induced Hnf4a overexpression through both spontaneous and directed differentiation methods. Gene expression analysis showed substantially increased expression of hepatic gene markers, particularly Ttr and endogenous Hnf4a, in transduced cells differentiated by the directed approach. These results demonstrated that forced expression of TFs during directed differentiation would be an appropriate way to study relevant gene activation and the effects of overexpression in the context of hepatic differentiation. PMID:27233607

  13. Myogenic differentiation of FSHD patient specific induced pluripotent stem cells

    Bosnakovski, Darko

    2012-01-01

    Human induced pluripotent stem (IPS) cells overcome several disadvantages of human embryonic stem cells, including host specificity and ethical issues. Patient-specific IPS cells can be generated from every donor by using different cell types, making them a suitable tool for autologous cell therapy and tissue engineering. IPS cells generated from patients with genetic disorders capture the disease genotype in the cell, making them a good model for studying the pathology of the diseas...

  14. Differentiation of murine embryonic stem and induced pluripotent stem cells to renal lineage in vitro

    Embryonic stem (ES) cells which have the unlimited proliferative capacity and extensive differentiation potency can be an attractive source for kidney regeneration therapies. Recent breakthroughs in the generation of induced pluripotent stem (iPS) cells have provided with another potential source for the artificially-generated kidney. The purpose of this study is to know how to differentiate mouse ES and iPS cells into renal lineage. We used iPS cells from mouse fibroblasts by transfection of four transcription factors, namely Oct4, Sox2, c-Myc and Klf4. Real-time PCR showed that renal lineage markers were expressed in both ES and iPS cells after the induction of differentiation. It also showed that a tubular specific marker, KSP progressively increased to day 18, although the differentiation of iPS cells was slower than ES cells. The results indicated that renal lineage cells can be differentiated from both murine ES and iPS cells. Several inducing factors were tested whether they influenced on cell differentiation. In ES cells, both of GDNF and BMP7 enhanced the differentiation to metanephric mesenchyme, and Activin enhanced the differentiation of ES cells to tubular cells. Activin also enhanced the differentiation of iPS cells to tubular cells, although the enhancement was lower than in ES cells. ES and iPS cells have a potential to differentiate to renal lineage cells, and they will be an attractive resource of kidney regeneration therapy. This differentiation is enhanced by Activin in both ES and iPS cells.

  15. Differentiation of murine embryonic stem and induced pluripotent stem cells to renal lineage in vitro

    Morizane, Ryuji [Department of Internal Medicine, Keio University School of Medicine, Tokyo (Japan); Monkawa, Toshiaki, E-mail: monkawa@sc.itc.keio.ac.jp [Department of Internal Medicine, Keio University School of Medicine, Tokyo (Japan); Itoh, Hiroshi [Department of Internal Medicine, Keio University School of Medicine, Tokyo (Japan)

    2009-12-25

    Embryonic stem (ES) cells which have the unlimited proliferative capacity and extensive differentiation potency can be an attractive source for kidney regeneration therapies. Recent breakthroughs in the generation of induced pluripotent stem (iPS) cells have provided with another potential source for the artificially-generated kidney. The purpose of this study is to know how to differentiate mouse ES and iPS cells into renal lineage. We used iPS cells from mouse fibroblasts by transfection of four transcription factors, namely Oct4, Sox2, c-Myc and Klf4. Real-time PCR showed that renal lineage markers were expressed in both ES and iPS cells after the induction of differentiation. It also showed that a tubular specific marker, KSP progressively increased to day 18, although the differentiation of iPS cells was slower than ES cells. The results indicated that renal lineage cells can be differentiated from both murine ES and iPS cells. Several inducing factors were tested whether they influenced on cell differentiation. In ES cells, both of GDNF and BMP7 enhanced the differentiation to metanephric mesenchyme, and Activin enhanced the differentiation of ES cells to tubular cells. Activin also enhanced the differentiation of iPS cells to tubular cells, although the enhancement was lower than in ES cells. ES and iPS cells have a potential to differentiate to renal lineage cells, and they will be an attractive resource of kidney regeneration therapy. This differentiation is enhanced by Activin in both ES and iPS cells.

  16. Metformin induces differentiation in acute promyelocytic leukemia by activating the MEK/ERK signaling pathway

    Huai, Lei; Wang, Cuicui; Zhang, Cuiping; Li, Qihui; Chen, Yirui; Jia, Yujiao; Li, Yan; Xing, Haiyan; Tian, Zheng; Rao, Qing; Wang, Min [State Key Laboratory of Experimental Hematology, Institute of Hematology and Blood Diseases Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College, Tianjin 300020 (China); Wang, Jianxiang, E-mail: wangjx@ihcams.ac.cn [State Key Laboratory of Experimental Hematology, Institute of Hematology and Blood Diseases Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College, Tianjin 300020 (China)

    2012-06-08

    Highlights: Black-Right-Pointing-Pointer Metformin induces differentiation in NB4 and primary APL cells. Black-Right-Pointing-Pointer Metformin induces activation of the MEK/ERK signaling pathway in APL cells. Black-Right-Pointing-Pointer Metformin synergizes with ATRA to trigger maturation of NB4 and primary APL cells. Black-Right-Pointing-Pointer Metformin induces the relocalization and degradation of the PML-RAR{alpha} fusion protein. Black-Right-Pointing-Pointer The study may be applicable for new differentiation therapy in cancer treatment. -- Abstract: Recent studies have shown that metformin, a widely used antidiabetic agent, may reduce the risk of cancer development. In this study, we investigated the antitumoral effect of metformin on both acute myeloid leukemia (AML) and acute promyelocytic leukemia (APL) cells. Metformin induced apoptosis with partial differentiation in an APL cell line, NB4, but only displayed a proapoptotic effect on several non-M3 AML cell lines. Further analysis revealed that a strong synergistic effect existed between metformin and all-trans retinoic acid (ATRA) during APL cell maturation and that metformin induced the hyperphosphorylation of extracellular signal-regulated kinase (ERK) in APL cells. U0126, a specific MEK/ERK activation inhibitor, abrogated metformin-induced differentiation. Finally, we found that metformin induced the degradation of the oncoproteins PML-RAR{alpha} and c-Myc and activated caspase-3. In conclusion, these results suggest that metformin treatment may contribute to the enhancement of ATRA-induced differentiation in APL, which may deepen the understanding of APL maturation and thus provide insight for new therapy strategies.

  17. Metformin induces differentiation in acute promyelocytic leukemia by activating the MEK/ERK signaling pathway

    Highlights: ► Metformin induces differentiation in NB4 and primary APL cells. ► Metformin induces activation of the MEK/ERK signaling pathway in APL cells. ► Metformin synergizes with ATRA to trigger maturation of NB4 and primary APL cells. ► Metformin induces the relocalization and degradation of the PML-RARα fusion protein. ► The study may be applicable for new differentiation therapy in cancer treatment. -- Abstract: Recent studies have shown that metformin, a widely used antidiabetic agent, may reduce the risk of cancer development. In this study, we investigated the antitumoral effect of metformin on both acute myeloid leukemia (AML) and acute promyelocytic leukemia (APL) cells. Metformin induced apoptosis with partial differentiation in an APL cell line, NB4, but only displayed a proapoptotic effect on several non-M3 AML cell lines. Further analysis revealed that a strong synergistic effect existed between metformin and all-trans retinoic acid (ATRA) during APL cell maturation and that metformin induced the hyperphosphorylation of extracellular signal-regulated kinase (ERK) in APL cells. U0126, a specific MEK/ERK activation inhibitor, abrogated metformin-induced differentiation. Finally, we found that metformin induced the degradation of the oncoproteins PML-RARα and c-Myc and activated caspase-3. In conclusion, these results suggest that metformin treatment may contribute to the enhancement of ATRA-induced differentiation in APL, which may deepen the understanding of APL maturation and thus provide insight for new therapy strategies.

  18. A cost-effective system for differentiation of intestinal epithelium from human induced pluripotent stem cells

    Soichiro Ogaki; Mayu Morooka; Kaito Otera; Shoen Kume

    2015-01-01

    The human intestinal epithelium is a useful model for pharmacological studies of absorption, metabolism, drug interactions, and toxicology, as well as for studies of developmental biology. We established a rapid and cost effective system for differentiation of human induced pluripotent stem (iPS) cells into definitive endoderm (DE) cells. In the presence of dimethyl sulfoxide (DMSO), a low concentration of Activin at 6.25 ng/ml is sufficient to give a similar differentiation efficiency with t...

  19. STRUCTURAL REMODELING OF PROTEOGLYCANS UPON RETINOIC ACID-INDUCED DIFFERENTIATION OF NCCIT CELLS*

    Gasimli, Leyla; Stansfield, Hope E.; Nairn, Alison V.; Liu, Haiying; Janet L. Paluh; Yang, Bo; Dordick, Jonathan S.; Moremen, Kelley W.; Linhardt, Robert J.

    2012-01-01

    Pluripotent and multipotent cells become increasingly lineage restricted through differentiation. Alterations to the cellular proteoglycan composition and structure should accompany these changes to influence cell proliferation, delineation of tissues and acquisition of cell migration capabilities. Retinoic acid plays an important role in pre-patterning of the early embryo. Retinoic acid can be used in vitro to induce differentiation, causing pluripotent and multipotent cells to become increa...

  20. Src transduces erythropoietin-induced differentiation signals through phosphatidylinositol 3-kinase

    Kubota, Yoshitsugu; Tanaka, Terukazu; Kitanaka, Akira; Ohnishi, Hiroaki; Okutani, Yuichi; Waki, Masato; Ishida, Toshihiko; Kamano, Hiroshi

    2001-01-01

    In this study, we examined the molecular mechanism of erythropoietin-initiated signal transduction of erythroid differentiation through Src and phosphatidylinositol 3-kinase (PI3-kinase). Antisense oligonucleotides against src but not lyn inhibited the formation of erythropoietin-dependent colonies derived from human bone marrow cells and erythropoietin-induced differentiation of K562 human erythroleukaemia cells. Antisense p85α oligonucleotide or LY294002, a selective inhibitor of PI3-kinase...

  1. Electrical Stimulation Promotes Cardiac Differentiation of Human Induced Pluripotent Stem Cells

    Damián Hernández; Rodney Millard; Priyadharshini Sivakumaran; Wong, Raymond C. B.; Crombie, Duncan E.; Hewitt, Alex W.; Helena Liang; Hung, Sandy S. C.; Alice Pébay; Shepherd, Robert K.; Gregory J Dusting; Lim, Shiang Y

    2016-01-01

    Background. Human induced pluripotent stem cells (iPSCs) are an attractive source of cardiomyocytes for cardiac repair and regeneration. In this study, we aim to determine whether acute electrical stimulation of human iPSCs can promote their differentiation to cardiomyocytes. Methods. Human iPSCs were differentiated to cardiac cells by forming embryoid bodies (EBs) for 5 days. EBs were then subjected to brief electrical stimulation and plated down for 14 days. Results. In iPS(Foreskin)-2 cell...

  2. Effects and mechanisms of melatonin on neural differentiation of induced pluripotent stem cells.

    Shu, Tao; Wu, Tao; Pang, Mao; Liu, Chang; Wang, Xuan; Wang, Juan; Liu, Bin; Rong, Limin

    2016-06-01

    Melatonin, a lipophilic molecule mainly synthesized in the pineal gland, has properties of antioxidation, anti-inflammation, and antiapoptosis to improve neuroprotective functions. Here, we investigate effects and mechanisms of melatonin on neural differentiation of induced pluripotent stem cells (iPSCs). iPSCs were induced into neural stem cells (NSCs), then further differentiated into neurons in medium with or without melatonin, melatonin receptor antagonist (Luzindole) or Phosphatidylinositide 3 kinase (PI3K) inhibitor (LY294002). Melatonin significantly promoted the number of neurospheres and cell viability. In addition, Melatonin markedly up-regulated gene and protein expression of Nestin and MAP2. However, Luzindole or LY294002 attenuated these increase. The expression of pAKT/AKT were increased by Melatonin, while Luzindole or LY294002 declined these melatonin-induced increase. These results suggest that melatonin significantly increased neural differentiation of iPSCs via activating PI3K/AKT signaling pathway through melatonin receptor. PMID:27130826

  3. Direct hepatic differentiation of mouse embryonic stem cells induced by valproic acid and cytokines

    Xue-Jun Dong; Guo-Rong Zhang; Qing-Jun Zhou; Ruo-Lang Pan; Ye Chen; Li-Xin Xiang; Jian-Zhong Shao

    2009-01-01

    AIM: To develop a protocol for direct hepatic lineage differentiation from early developmental progenitors to a population of mature hepatocytes. METHODS: Hepatic progenitor cells and then mature hepatocytes from mouse embryonic stem (ES) cells were obtained in a sequential manner, induced by valproic acid (VPA) and cytokines (hepatocyte growth factor, epidermal growth factor and insulin). Morphological changes of the differentiated cells were examined by phase-contrast microscopy and electron microscopy. Reverse transcription polymerase chain reaction and immunocytochemical analyses were used to evaluate the gene expression profiles of the VPA-induced hepatic progenitors and the hepatic progenitor-derived hepatocytes. Glycogen storage, cytochrome P450 activity, transplantation assay, differentiation of bile duct-like structures and tumorigenic analyses were performed for the functional identification of the differentiated cells. Furthermore, FACS and electron microscopy were used for the analyses of cell cycle profile and apoptosis in VPA-induced hepatic differentiated cells. RESULTS: Based on the combination of VPA and cytokines, mouse ES cells differentiated into a uniform and homogeneous cell population of hepatic progenitor cells and then matured into functional hepatocytes. The progenitor population shared several characteristics with ES cells and hepatic stem/progenitor cells, and represented a novel progenitor cell between ES and hepatic oval cells in embryonic development. The differentiated hepatocytes from progenitor cells shared typical characteristics with mature hepatocytes, including the patterns of gene expression, immunological markers, in vitro hepatocyte functions and in vivo capacity to restore acute-damaged liver function. In addition, the differentiation of hepatic progenitor cells from ES cells was accompanied by significant cell cycle arrest and selective survival of differentiating cells towards hepatic lineages. CONCLUSION: Hepatic cells

  4. Autocrine fibroblast growth factor 18 mediates dexamethasone-induced osteogenic differentiation of murine mesenchymal stem cells.

    Hamidouche, Zahia; Fromigué, Olivia; Nuber, Ulrike; Vaudin, Pascal; Pages, Jean-Christophe; Ebert, Regina; Jakob, Franz; Miraoui, Hichem; Marie, Pierre J

    2010-08-01

    The potential of mesenchymal stem cells (MSC) to differentiate into functional bone forming cells provides an important tool for bone regeneration. The identification of factors capable of promoting osteoblast differentiation in MSCs is therefore critical to enhance the osteogenic potential of MSCs. Using microarray analysis combined with biochemical and molecular approach, we found that FGF18, a member of the FGF family, is upregulated during osteoblast differentiation induced by dexamethasone in murine MSCs. We showed that overexpression of FGF18 by lentiviral (LV) infection, or treatment of MSCs with recombinant human (rh)FGF18 increased the expression of the osteoblast specific transcription factor Runx2, and enhanced osteoblast phenotypic marker gene expression and in vitro osteogenesis. Molecular silencing using lentiviral shRNA demonstrated that downregulation of FGFR1 or FGFR2 abrogated osteoblast gene expression induced by either LV-FGF18 or rhFGF18, indicating that FGF18 enhances osteoblast differentiation in MSCs via activation of FGFR1 or FGFR2 signaling. Biochemical and pharmacological analyses showed that the induction of phenotypic osteoblast markers by LV-FGF18 is mediated by activation of ERK1/2-MAPKs and PI3K signaling in MSCs. These results reveal that FGF18 is an essential autocrine positive regulator of the osteogenic differentiation program in murine MSCs and indicate that osteogenic differentiation induced by FGF18 in MSCs is triggered by FGFR1/FGFR2-mediated ERK1/2-MAPKs and PI3K signaling. PMID:20432451

  5. Synaptic network activity induces neuronal differentiation of adult hippocampal precursor cells through BDNF signaling

    HarishBabu

    2009-09-01

    Full Text Available Adult hippocampal neurogenesis is regulated by activity. But how do neural precursor cells in the hippocampus respond to surrounding network activity and translate increased neural activity into a developmental program? Here we show that long-term potential (LTP-like synaptic activity within a cellular network of mature hippocampal neurons promotes neuronal differentiation of newly generated cells. In co-cultures of precursor cells with primary hippocampal neurons, LTP-like synaptic plasticity induced by addition of glycine in Mg2+-free media for 5 min, produced synchronous network activity and subsequently increased synaptic strength between neurons. Furthermore, this synchronous network activity led to a significant increase in neuronal differentiation from the co-cultured neural precursor cells. When applied directly to precursor cells, glycine and Mg2+-free solution did not induce neuronal differentiation. Synaptic plasticity-induced neuronal differentiation of precursor cells was observed in the presence of GABAergic neurotransmission blockers but was dependent on NMDA-mediated Ca2+ influx. Most importantly, neuronal differentiation required the release of brain-derived neurotrophic factor (BDNF from the underlying substrate hippocampal neurons as well as TrkB receptor phosphorylation in precursor cells. This suggests that activity-dependent stem cell differentiation within the hippocampal network is mediated via synaptically evoked BDNF signaling.

  6. Blocking p55PIK signaling inhibits proliferation and induces differentiation of leukemia cells.

    Wang, G; Deng, Y; Cao, X; Lai, S; Tong, Y; Luo, X; Feng, Y; Xia, X; Gong, J; Hu, J

    2012-11-01

    p55PIK, a regulatory subunit of phosphatidylinositol 3-kinases, promotes cell cycle progression by interacting with cell cycle modulators such as retinoblastoma protein (Rb) via its unique amino-terminal 24 amino-acid residue (N24). Overexpression of N24 specifically inhibits these interactions and leads to cell cycle arrest. Herein, we describe the generation of a fusion protein (Tat transactivator protein (TAT)-N24) that contains the protein transduction domain and N24, and examined its effects on the proliferation and differentiation of leukemia cells. TAT-N24 not only blocks cell proliferation but remarkably induces differentiation of leukemia cells in vitro and in vivo. Systemically administered TAT-N24 also significantly decreases growth of leukemia cell tumors in animal models. Furthermore, overexpression of p55PIK in leukemia cells leads to increased proliferation; however, TAT-N24 blocks this effect and concomitantly induces differentiation. There is significant upregulation of p55PIK mRNA and protein expression in leukemia cells from patients. TAT-N24 inhibits cell cycle progression and induces differentiation of bone marrow cells derived from patients with several different types of leukemia. These results show that cell-permeable N24 peptide induces leukemia cell differentiation and suggest that p55PIK may be a novel drug target for the treatment of hematopoetic malignancies. PMID:22722333

  7. Silver nanoparticles impede phorbol myristate acetate-induced monocyte-macrophage differentiation and autophagy

    Xu, Yingying; Wang, Liming; Bai, Ru; Zhang, Tianlu; Chen, Chunying

    2015-09-01

    Monocytes/macrophages are important constituents of the innate immune system. Monocyte-macrophage differentiation is not only crucial for innate immune responses, but is also related to some cardiovascular diseases. Silver nanoparticles (AgNPs) are one of the most widely used nanomaterials because of their broad-spectrum antimicrobial properties. However, the effect of AgNPs on the functions of blood monocytes is scarcely reported. Here, we report the impedance effect of AgNPs on THP-1 monocyte differentiation, and that this effect was mediated by autophagy blockade and lysosomal impairment. Firstly, AgNPs inhibit phorbol 12-myristate 13-acetate (PMA)-induced monocyte differentiation by down-regulating both expression of surface marker CD11b and response to lipopolysaccharide (LPS) stimulation. Secondly, autophagy is activated during PMA-induced THP-1 monocyte differentiation, and the autophagy inhibitor chloroquine (CQ) can inhibit this process. Thirdly, AgNPs block the degradation of the autophagy substrate p62 and induce autophagosome accumulation, which demonstrates the blockade of autophagic flux. Fourthly, lysosomal impairments including alkalization and decrease of lysosomal membrane stability were observed in AgNP-treated THP-1 cells. In conclusion, we demonstrate that the impedance of monocyte-macrophage differentiation by AgNPs is mediated by autophagy blockade and lysosomal dysfunction. Our results suggest that crosstalk exists in different biological effects induced by AgNPs.

  8. A localized differentiation-inducing-factor sink in the front of the Dictyostelium slug.

    Kay, R R; Large, S.; Traynor, D.; Nayler, O

    1993-01-01

    Differentiation-inducing factor 1 [DIF-1; 1-(3,5-dichloro-2,6-dihydroxy-4-methoxyphenyl)-hexan-1-one] induces stalk cell differentiation during Dictyostelium development. It is present as a gradient in the multicellular slug, its lowest concentration being in the anterior. Here we demonstrate the existence of a localized sink for DIF-1, also in the anterior of the slug, which could be responsible for generating the DIF-1 gradient. DIF-1 is metabolized extensively by developing cells, initiall...

  9. 5-Azacytidine Induces Cardiac Differentiation of Human Umbilical Cord-Derived Mesenchymal Stem Cells by Activating Extracellular Regulated Kinase

    Qian, Qian; QIAN, HUI; Zhang, Xu; Zhu, Wei; Yan, Yongmin; Ye, Shengqin; Peng, Xiujuan; Li, Wei; Xu, Zhe; Sun, Lingyun; Xu, Wenrong

    2011-01-01

    5-Azacytidine (5-Aza) induces differentiation of mesenchymal stem cells (MSCs) into cardiomyocytes. However, the underlying mechanisms are not well understood. Our previous work showed that 5-Aza induces human bone marrow-derived MSCs to differentiate into cardiomyocytes. Here, we demonstrated that 5-Aza induced cardiac differentiation of human umbilical cord-derived MSCs (hucMSCs) and explored the potential signaling pathway. Our results showed that hucMSCs had cardiomyocyte phenotypes after...

  10. Mannosylerythritol lipid induces granulocytic differentiation and inhibits the tyrosine phosphorylation of human myelogenous leukemia cell line K562

    Isoda, Hiroko; Nakahara, Tadaatsu

    1997-01-01

    Mannosylerythritol lipid (MEL), which induced granulocytic differentiation of human promyelocytic leukemia cell line HL60, also induced differentiation of human myelogenous leukemia cell line K562. MEL inhibited insulin-dependent cell proliferation and induced leukocyte esterase activity of K562 cells. MEL markedly increased the differentiation-associated characteristics in granulocytes, such as nitroblue tetrazolium (NBT) reducing ability, expression of Fc receptors, and phagocytic activity ...

  11. Undifferentiated spleen colonies: an attempt to induce differentiation into eosinophils

    Repeated injections of casein caused a 4-fold increase in the overall number of colonies in the spleens of sublethally irradiated, polycythemic mice. Chronic (12 injections) casein treatment considerably increased the number of eosinophil and of undifferentiated colonies, but not that of granuloid ones. A reduction in number and size of eosinophil colonies took place between the sixth and the ninth day after irradiation. No such reduction occurred with undifferentiated colonies. Stimulation with casein for only 3 (consecutive) days before irradiation caused a similar increase in eosinophil colonies, only a moderate increase in undifferentiated ones and a very marked increase in granuloid colonies. Evidence for a change of undifferentiated colonies into eosinophil ones under conditions of eosinopoiesis was looked for, but not detected. No correlation was found between the amyloid-inducing capacity of caseins and their eosinopoietic potency

  12. Activin A induces Langerhans cell differentiation in vitro and in human skin explants.

    Tiziana Musso

    Full Text Available Langerhans cells (LC represent a well characterized subset of dendritic cells located in the epidermis of skin and mucosae. In vivo, they originate from resident and blood-borne precursors in the presence of keratinocyte-derived TGFbeta. In vitro, LC can be generated from monocytes in the presence of GM-CSF, IL-4 and TGFbeta. However, the signals that induce LC during an inflammatory reaction are not fully investigated. Here we report that Activin A, a TGFbeta family member induced by pro-inflammatory cytokines and involved in skin morphogenesis and wound healing, induces the differentiation of human monocytes into LC in the absence of TGFbeta. Activin A-induced LC are Langerin+, Birbeck granules+, E-cadherin+, CLA+ and CCR6+ and possess typical APC functions. In human skin explants, intradermal injection of Activin A increased the number of CD1a+ and Langerin+ cells in both the epidermis and dermis by promoting the differentiation of resident precursor cells. High levels of Activin A were present in the upper epidermal layers and in the dermis of Lichen Planus biopsies in association with a marked infiltration of CD1a+ and Langerin+ cells. This study reports that Activin A induces the differentiation of circulating CD14+ cells into LC. Since Activin A is abundantly produced during inflammatory conditions which are also characterized by increased numbers of LC, we propose that this cytokine represents a new pathway, alternative to TGFbeta, responsible for LC differentiation during inflammatory/autoimmune conditions.

  13. Differential effects of energy balance on experimentally-induced colitis

    Sarah J McCaskey; Elizabeth A Rondini; Ingeborg M Langohr; Jenifer I Fenton

    2012-01-01

    AIM:To characterize the influence of diet-induced changes in body fat on colitis severity in SMAD3-/-mice.METHODS:SMAD3-/-mice (6-8 wk of age) were randomly assigned to receive a calorie restricted (30%of control; CR),control (CON),or high fat (HF) diet for 20 wk and were gavaged with sterile broth or with Helicobacter hepaticus (H.hepaticus) to induce colitis.Four weeks after infection,mice were sacrificed and the cecum and colons were processed for histological evaluation.RESULTS:Dietary treatment significantly influenced body composition prior to infection (P < 0.05),with CR mice having less (14% ± 2%) and HF-fed mice more body fat (32% ± 7%) compared to controls (22% ±4%).Differences in body composition were associated with alterations in plasma levels of leptin (HF > CON > CR) and adiponectin (CON > HF ≥ CR) (P < 0.05).There were no significant differences in colitis scores between CON and HF-fed mice 4 wk post-infection.Consistent with this,differences in proliferation and inflammation markers (COX-2,iNOS),and infiltrating cell types (CD3+ T lymphocytes,macrophages) were not observed.Unexpectedly,only 40% of CR mice survived infection with H.hepaticus,with mortality observed as early as 1 wk following induction of colitis.CONCLUSION:Increased adiposity does not influence colitis severity in SMAD3-/-mice.Importantly,caloric restriction negatively impacts survival following pathogen challenge,potentially due to an impaired immune response.

  14. Hypoxia induces differential translation of enolase/MBP-1

    Hypoxic microenvironments in tumors contribute to transformation, which may alter metabolism, growth, and therapeutic responsiveness. The α-enolase gene encodes both a glycolytic enzyme (α-enolase) and a DNA-binding tumor suppressor protein, c-myc binding protein (MBP-1). These divergent α-enolase gene products play central roles in glucose metabolism and growth regulation and their differential regulation may be critical for tumor adaptation to hypoxia. We have previously shown that MBP-1 and its binding to the c-myc P2 promoter regulates the metabolic and cellular growth changes that occur in response to altered exogenous glucose concentrations. To examine the regulation of α-enolase and MBP-1 by a hypoxic microenvironment in breast cancer, MCF-7 cells were grown in low, physiologic, or high glucose under 1% oxygen. Our results demonstrate that adaptation to hypoxia involves attenuation of MBP-1 translation and loss of MBP-1-mediated regulation of c-myc transcription, evidenced by decreased MBP-1 binding to the c-myc P2 promoter. This allows for a robust increase in c-myc expression, 'early c-myc response', which stimulates aerobic glycolysis resulting in tumor acclimation to oxidative stress. Increased α-enolase mRNA and preferential translation/post-translational modification may also allow for acclimatization to low oxygen, particularly under low glucose concentrations. These results demonstrate that malignant cells adapt to hypoxia by modulating α-enolase/MBP-1 levels and suggest a mechanism for tumor cell induction of the hyperglycolytic state. This important 'feedback' mechanism may help transformed cells to escape the apoptotic cascade, allowing for survival during limited glucose and oxygen availability

  15. Differential effect of baicalein on ionizing radiation induced cell death in normal lymphocytes and lymphoma cells

    Baicalein (5,6,7-trihydroxy-2-phenyl-4H-1-benzopyran-4-one), a naturally occurring flavone, present in Indian and Chinese medicinal plants has been reported to possess potent antioxidant activity. Previous reports from our laboratory have elucidated the radical scavenging and radioprotective potential of this compound in cell free system. To investigate potential of baicalein as a radioprotector, we have studied its effect on normal lymphocytes and lymphoma cells (EL-4 cells) in presence of radiation. Baicalein protected murine splenic lymphocytes against radiation (4Gy) induced apoptosis as assessed by propidium iodide staining. It inhibited background cell death in lymphocytes whereas, baicalein induced concentration dependent cell death in EL-4 cells and did not protect against radiation induced apoptosis. Interestingly, baicalein scavenged radiation derived ROS (reactive oxygen species) in both the cell types suggesting that, it is not exhibiting differential antioxidant action. Despite scavenging radiation derived ROS, which are principal mediators of radiation induced cell death, baicalein induced cell death in EL-4 cells. To investigate the reason for this differential behavior, we investigated the effect of baicalein on pro-survival molecules viz. ERK and NF-kB. Baicalein induced phosphorylation of ERK in normal lymphocytes in a time dependent manner, but, it did not alter pERK levels in EL-4 cells. Baicalein treatment per se induced degradation of IkBα and increased nuclear accumulation of NF-kB in normal lymphocytes. Whereas, baicalein pre-treatment reduced basal NF-kB levels in EL-4 cells and it also suppressed TNF-α induced nuclear accumulation of NF-kB. This study suggests that, differential regulation of pro-survival transcription factor NF-kB may be playing a role in differential effect of baicalein in normal lymphocytes and lymphoma cells. (author)

  16. Reversible structural alterations of undifferentiated and differentiated human neuroblastoma cells induced by phorbol ester.

    Tint, I S; Bonder, E. M.; Feder, H. H.; Reboulleau, C P; Vasiliev, J M; Gelfand, I M

    1992-01-01

    Morphological alterations in the structure of undifferentiated and morphologically differentiated human neuroblastoma cells induced by phorbol 12-myristate 13-acetate (PMA), an activator of protein kinase C, were examined by video microscopy and immunomorphology. In undifferentiated cells, PMA induced the formation of motile actin-rich lamellas and of stable cylindrical processes rich in microtubules. Formation of stable processes resulted either from the collapse of lamellas or the movement ...

  17. Ameloblasts serum-free conditioned medium: bone morphogenic protein 4-induced odontogenic differentiation of mouse induced pluripotent stem cells.

    Liu, Li; Liu, Ying-Feng; Zhang, Jing; Duan, Yin-Zhong; Jin, Yan

    2016-06-01

    Induced pluripotent stem (iPS) cells possess the ability of self-renewal and can differentiate into cells of the three germ layers, both in vitro and in vivo. Here we report a new method to efficiently induce differentiation of mouse iPS cells into the odontogenic lineage. Using ameloblasts serum-free conditioned medium (ASF-CM), we successfully generated ameloblast-like cells from mouse iPS cells. Importantly, culturing mouse iPS cells in ASF-CM supplemented with BMP4 (ASF-BMP4) promoted odontogenic differentiation, which was evident by the upregulation of ameloblast-specific as well as odontoblast-specific genes. On the other hand, culturing mouse iPS cells in ASF-CM supplemented with noggin (ASF-noggin), an inhibitor of BMP4, abrogated this effect. These results suggest that mouse iPS cells can be induced by ASF-BMP4 to differentiate into ameloblast-like and odontoblast-like cells. The results of our study raise the possibility of using patient-specific iPS cells for tooth regeneration in the future. Copyright © 2016 John Wiley & Sons, Ltd. PMID:23606575

  18. Wnt signaling induces epithelial differentiation during cutaneous wound healing

    Hocking Anne

    2006-01-01

    Full Text Available Abstract Background Cutaneous wound repair in adult mammals does not regenerate the original epithelial architecture and results in altered skin function. We propose that lack of regeneration may be due to the absence of appropriate molecular signals to promote regeneration. In this study, we investigated the regulation of Wnt signaling during cutaneous wound healing and the consequence of activating either the beta-catenin-dependent or beta-catenin-independent Wnt signaling on epidermal architecture during wound repair. Results We determined that the expression of Wnt ligands that typically signal via the beta-catenin-independent pathway is up-regulated in the wound while the beta-catenin-dependent Wnt signaling is activated in the hair follicles adjacent to the wound edge. Ectopic activation of beta-catenin-dependent Wnt signaling with lithium chloride in the wound resulted in epithelial cysts and occasional rudimentary hair follicle structures within the epidermis. In contrast, forced expression of Wnt-5a in the deeper wound induced changes in the interfollicular epithelium mimicking regeneration, including formation of epithelia-lined cysts in the wound dermis, rudimentary hair follicles and sebaceous glands, without formation of tumors. Conclusion These findings suggest that adult interfollicular epithelium is capable of responding to Wnt morphogenic signals necessary for restoring epithelial tissue patterning in the skin during wound repair.

  19. Static and Dynamic Experiment Evaluations of a Displacement Differential Self-Induced Magnetorheological Damper

    Guoliang Hu

    2015-01-01

    Full Text Available This paper presents the development of a novel magnetorheological damper (MRD which has a self-induced ability. In this study, a linear variable differential sensor (LVDS based on the electromagnetic induction mechanism was integrated with a conventional MRD. The structure of the displacement differential self-induced magnetorheological damper (DDSMRD was developed, and the theory of displacement differential self-induced performance was deduced. The static experiments of the DDSMRD under different displacement positions were carried out by applying sine excitation signals to the excitation coils, and the experimental results show that the self-induced voltage is proportional to the damper piston displacement. Meanwhile, the dynamic experiments were also carried out using the fatigue test machine to investigate the change of the self-induced voltage under the typical direct current inputs and the different piston rod displacements; the experimental results also show that the self-induced voltage is proportional to the damper piston displacements. Additionally, the dynamic mechanical performance of the DDSMRD was evaluated. The theory deduction and the experimental results indicate that the proposed DDSMRD has the ability of the integrated displacement sensor in addition to the output controllable damping force.

  20. Lignin Induces ES Cells to Differentiate into Neuroectodermal Cells through Mediation of the Wnt Signaling Pathway

    Inoue, Yu; Hasegawa, Seiji; Yamada, Takaaki; Date, Yasushi; Mizutani, Hiroshi; Nakata, Satoru; Akamatsu, Hirohiko

    2013-01-01

    Embryonic stem cells (ES cells) are characterized by their pluripotency and infinite proliferation potential. Ever since ES cells were first established in 1981, there have been a growing number of studies aimed at clinical applications of ES cells. In recent years, various types of differentiation inducement systems using ES cells have been established. Further studies have been conducted to utilize differentiation inducement systems in the field of regenerative medicine. For cellular treatments using stem cells including ES cells, differentiation induction should be performed in a sufficient manner to obtain the intended cell lineages. Lignin is a high-molecular amorphous material that forms plants together with cellulose and hemicelluloses, in which phenylpropane fundamental units are complexly condensed. Lignin derivatives have been shown to have several bioactive functions. In spite of these findings, few studies have focused on the effects of lignin on stem cells. Our study aimed to develop a novel technology using lignin to effectively induce ES cells to differentiate into neuroectodermal cells including ocular cells and neural cells. Since lignin can be produced at a relatively low cost in large volumes, its utilization is expected for more convenient differentiation induction technologies and in the field of regenerative medicine in the future. PMID:23805217

  1. Murine bone cell lines as models for spaceflight induced effects on differentiation and gene expression

    Lau, P.; Hellweg, C. E.; Baumstark-Khan, C.; Reitz, G.

    Critical health factors for space crews especially on long-term missions are radiation exposure and the absence of gravity DNA double strand breaks DSB are presumed to be the most deleterious DNA lesions after radiation as they disrupt both DNA strands in close proximity Besides radiation risk the absence of gravity influences the complex skeletal apparatus concerning muscle and especially bone remodelling which results from mechanical forces exerting on the body Bone is a dynamic tissue which is life-long remodelled by cells from the osteoblast and osteoclast lineage Any imbalance of this system leads to pathological conditions such as osteoporosis or osteopetrosis Osteoblastic cells play a crucial role in bone matrix synthesis and differentiate either into bone-lining cells or into osteocytes Premature terminal differentiation has been reported to be induced by a number of DNA damaging or cell stress inducing agents including ionising and ultraviolet radiation as well as treatment with mitomycin C In the present study we compare the effects of sequential differentiation by adding osteoinductive substances ss -glycerophosphate and ascorbic acid Radiation-induced premature differentiation was investigated regarding the biosynthesis of specific osteogenic marker molecules and the differentiation dependent expression of marker genes The bone cell model established in our laboratory consists of the osteocyte cell line MLO-Y4 the osteoblast cell line OCT-1 and the subclones 4 and 24 of the osteoblast cell line MC3T3-E1 expressing several

  2. Cytokine-Regulated GADD45G Induces Differentiation and Lineage Selection in Hematopoietic Stem Cells

    Frederic B. Thalheimer

    2014-07-01

    Full Text Available The balance of self-renewal and differentiation in long-term repopulating hematopoietic stem cells (LT-HSC must be strictly controlled to maintain blood homeostasis and to prevent leukemogenesis. Hematopoietic cytokines can induce differentiation in LT-HSCs; however, the molecular mechanism orchestrating this delicate balance requires further elucidation. We identified the tumor suppressor GADD45G as an instructor of LT-HSC differentiation under the control of differentiation-promoting cytokine receptor signaling. GADD45G immediately induces and accelerates differentiation in LT-HSCs and overrides the self-renewal program by specifically activating MAP3K4-mediated MAPK p38. Conversely, the absence of GADD45G enhances the self-renewal potential of LT-HSCs. Videomicroscopy-based tracking of single LT-HSCs revealed that, once GADD45G is expressed, the development of LT-HSCs into lineage-committed progeny occurred within 36 hr and uncovered a selective lineage choice with a severe reduction in megakaryocytic-erythroid cells. Here, we report an unrecognized role of GADD45G as a central molecular linker of extrinsic cytokine differentiation and lineage choice control in hematopoiesis.

  3. Nicotinamide induces differentiation of embryonic stem cells into insulin-secreting cells

    The poly(ADP-ribose) polymerase (PARP) inhibitor, nicotinamide, induces differentiation and maturation of fetal pancreatic cells. In addition, we have previously reported evidence that nicotinamide increases the insulin content of cells differentiated from embryonic stem (ES) cells, but the possibility of nicotinamide acting as a differentiating agent on its own has never been completely explored. Islet cell differentiation was studied by: (i) X-gal staining after neomycin selection; (ii) BrdU studies; (iii) single and double immunohistochemistry for insulin, C-peptide and Glut-2; (iv) insulin and C-peptide content and secretion assays; and (v) transplantation of differentiated cells, under the kidney capsule, into streptozotocin (STZ)-diabetic mice. Here we show that undifferentiated mouse ES cells treated with nicotinamide: (i) showed an 80% decrease in cell proliferation; (ii) co-expressed insulin, C-peptide and Glut-2; (iii) had values of insulin and C-peptide corresponding to 10% of normal mouse islets; (iv) released insulin and C-peptide in response to stimulatory glucose concentrations; and (v) after transplantation into diabetic mice, normalized blood glucose levels over 7 weeks. Our data indicate that nicotinamide decreases ES cell proliferation and induces differentiation into insulin-secreting cells. Both aspects are very important when thinking about cell therapy for the treatment of diabetes based on ES cells

  4. Differentiation induced by physiological and pharmacological stimuli leads to increased antigenicity of human neuroblastoma cells

    Lena-Maria Carlson; Sven P(a)hlman; Anna De Geer; Per Kogner; Jelena Levitskaya

    2008-01-01

    Sympathetic neuronal differentiation is associated with favorable prognosis of neuroblastoma (NB), the most common extra-cranial solid tumor of early childhood. Differentiation agents have proved useful in clinical protocols of NB treatment, but using them as a sole treatment is not sufficient to induce tumor elimination in patients. Therefore, complementary approaches, such as immunotherapy, are warranted. Here we demonstrate that differentiation of NB cell lines and ex vivo isolated tumor cells in response to physiological or pharmacological stimuli is associated with acquisition of increased antigenicity. This manifests as increased expression of surface major histocompatibility class I complexes and ICAM-1 molecules and translates into increased sensitivity of NB cells to lysis by cytotoxic T lymphocytes (CTLs) and natural killer (NK) cells. The latter is paralleled by enhanced ability of differentiated cells to form immune conjugates and bind increased amounts of granzyme B to the cell surface. We demonstrate, for the first time, that, regardless of the stimulus applied, the differentiation state in NBs is associated with increased tumor antigenicity that enables more efficient elimination of tumor cells by cytotoxic lymphocytes and paves the way for combined application of differentiation-inducing agents and immunotherapy as an auxiliary approach in NB patients.

  5. Retinoic acid induces HL-60 cell differentiation via the upregulation of miR-663

    Zhuan Zhou

    2011-04-01

    Full Text Available Abstract Background Differentiation of the acute myeloid leukemia (AML cell line HL-60 can be induced by all trans-retinoic acid (ATRA; however, the mechanism regulating this process has not been fully characterized. Methods Using bioinformatics and in vitro experiments, we identified the microRNA gene expression profile of HL-60 cells during ATRA induced granulocytic differentiation. Results Six microRNAs were upregulated by ATRA treatment, miR-663, miR-494, miR-145, miR-22, miR-363* and miR-223; and three microRNAs were downregulated, miR-10a, miR-181 and miR-612. Additionally, miR-663 expression was regulated by ATRA. We used a lentivirus (LV backbone incorporating the spleen focus forming virus (SFFV-F promoter to drive miR-663 expression, as the CMV (Cytomegalovirus promoter is ineffective in some lymphocyte cells. Transfection of LV-miR-663 induced significant HL-60 cell differentiation in vitro. Conclusions Our results show miR-663 may play an important role in ATRA induced HL-60 cell differentiation. Lentivirus delivery of miR-663 could potentially be used directly as an anticancer treatment in hematological malignancies

  6. Strain Gauges Indicate Differential-CTE-Induced Failures

    Harris, Brian

    2007-01-01

    A method of detecting mechanical failure induced by variation in temperature at an adhesive bond between two materials that have different coefficients of thermal expansion (CTEs) involves monitoring of strain-gauge readings. This method can be regarded as an exploitation of the prior observation that the readings of strain gauges commonly used in tensile and compressive testing of material specimens include features indicative of incremental failures in the specimens. In this method, one or more strain gauges are bonded to either or both of the two materials near the bond between the materials. (The adhesive used to bond the strain gauges would not ordinarily be the same as the one used to bond the two materials). Then strain-gauge readings are recorded as the temperature of the materials is varied through a range of interest. Any significant discontinuity in the slope of the resulting strain-versus-temperature curve(s) is taken to be a qualitative indication of a failure of the bond between the two materials and/or a failure within one of the materials in the vicinity of the bond. The method has been demonstrated in experiments on specimens consisting of polyacrylonitrile-fiber/epoxy-matrix laminated composite plates bonded by epoxy to smaller plates made, variously, of aluminum, titanium, and a low-CTE nickel/iron alloy. In preparation for each experiment, strain gauges were bonded, by use of cryogenic-rated adhesives, to the composite plate near the corners of the metal plate (see Figure 1). In each experiment, strain-gauge and temperature readings were taken as the specimen was cooled from room temperature to 20 K. The specimens were then returned to room temperature and ultrasonically inspected for damage in the bond region. No failure events were detectable in the strain-gauge readings from the composite/ titanium and composite/low-thermalexpansion- alloy specimens, and ultrasonic inspection of these specimens revealed no damage. However, failure events were

  7. Smurf1 plays a role in EGF inhibition of BMP2-induced osteogenic differentiation

    Lee, Hye-Lim; Park, Hyun-Jung; Kwon, Arang [Department of Molecular Genetics, School of Dentistry and Dental Research Institute, Seoul National University, Seoul 110-749 (Korea, Republic of); Baek, Kyunghwa [Department of Pharmacology, College of Dentistry and Research Institute of Oral Science, Gangneung-Wonju National University, Gangneung 210-702, Gangwondo (Korea, Republic of); Woo, Kyung Mi; Ryoo, Hyun-Mo; Kim, Gwan-Shik [Department of Molecular Genetics, School of Dentistry and Dental Research Institute, Seoul National University, Seoul 110-749 (Korea, Republic of); Baek, Jeong-Hwa, E-mail: baekjh@snu.ac.kr [Department of Molecular Genetics, School of Dentistry and Dental Research Institute, Seoul National University, Seoul 110-749 (Korea, Republic of)

    2014-05-01

    It has been demonstrated that epidermal growth factor (EGF) plays a role in supporting the proliferation of bone marrow stromal cells in bone but inhibits their osteogenic differentiation. However, the mechanism underlying EGF inhibition of osteoblast differentiation remains unclear. Smurf1 is an E3 ubiquitin ligase that targets Smad1/5 and Runx2, which are critical transcription factors for bone morphogenetic protein 2 (BMP2)-induced osteoblast differentiation. In this study, we investigated the effect of EGF on the expression of Smurf1, and the role of Smurf1 in EGF inhibition of osteogenic differentiation using C2C12 cells, a murine myoblast cell line. EGF increased Smurf1 expression, which was blocked by inhibiting the activity of either JNK or ERK. Chromatin immunoprecipitation and Smurf1 promoter assays demonstrated that c-Jun and Runx2 play roles in the EGF induction of Smurf1 transcription. EGF suppressed BMP2-induced expression of osteogenic marker genes, which were rescued by Smurf1 knockdown. EGF downregulated the protein levels of Runx2 and Smad1 in a proteasome-dependent manner. EGF decreased the transcriptional activity of Runx2 and Smurf1, which was partially rescued by Smurf1 silencing. Taken together, these results suggest that EGF increases Smurf1 expression via the activation of JNK and ERK and the subsequent binding of c-Jun and Runx2 to the Smurf1 promoter and that Smurf1 mediates the inhibitory effect of EGF on BMP2-induced osteoblast differentiation. - Highlights: • EGF increases the expression level of Smurf1 in mesenchymal precursor cells. • EGF reduces the protein levels and transcriptional activity of Runx2 and Smad1. • EGF suppresses BMP2-induced osteogenic differentiation, which is rescued by Smurf1 knockdown.

  8. Smurf1 plays a role in EGF inhibition of BMP2-induced osteogenic differentiation

    It has been demonstrated that epidermal growth factor (EGF) plays a role in supporting the proliferation of bone marrow stromal cells in bone but inhibits their osteogenic differentiation. However, the mechanism underlying EGF inhibition of osteoblast differentiation remains unclear. Smurf1 is an E3 ubiquitin ligase that targets Smad1/5 and Runx2, which are critical transcription factors for bone morphogenetic protein 2 (BMP2)-induced osteoblast differentiation. In this study, we investigated the effect of EGF on the expression of Smurf1, and the role of Smurf1 in EGF inhibition of osteogenic differentiation using C2C12 cells, a murine myoblast cell line. EGF increased Smurf1 expression, which was blocked by inhibiting the activity of either JNK or ERK. Chromatin immunoprecipitation and Smurf1 promoter assays demonstrated that c-Jun and Runx2 play roles in the EGF induction of Smurf1 transcription. EGF suppressed BMP2-induced expression of osteogenic marker genes, which were rescued by Smurf1 knockdown. EGF downregulated the protein levels of Runx2 and Smad1 in a proteasome-dependent manner. EGF decreased the transcriptional activity of Runx2 and Smurf1, which was partially rescued by Smurf1 silencing. Taken together, these results suggest that EGF increases Smurf1 expression via the activation of JNK and ERK and the subsequent binding of c-Jun and Runx2 to the Smurf1 promoter and that Smurf1 mediates the inhibitory effect of EGF on BMP2-induced osteoblast differentiation. - Highlights: • EGF increases the expression level of Smurf1 in mesenchymal precursor cells. • EGF reduces the protein levels and transcriptional activity of Runx2 and Smad1. • EGF suppresses BMP2-induced osteogenic differentiation, which is rescued by Smurf1 knockdown

  9. Tryptanthrin induces growth inhibition and neuronal differentiation in the human neuroblastoma LA-N-1 cells.

    Liao, Xuemei; Leung, Kwok Nam

    2013-04-25

    Neuroblastoma is one of the most common extracranial solid cancers found in young children. The prognosis of neuroblastoma patients in advanced stages having N-myc amplification remains poor despite intensive multimodal therapy. Agents that trigger neuroblastoma cells to undergo cellular differentiation and thereby stop proliferation have attracted considerable interest as an alternative therapy. Tryptanthrin (12-dihydro-6,12-dioxoindolo-(2,1-b)-quinazoline) is a weakly basic alkaloid isolated from the dried roots of medicinal indigo plants known as Banlangen. It has been shown to possess various biological activities, such as anti-microbial, anti-inflammatory and anti-tumor activities. However, its effects and mechanism(s) of action on human neuroblastoma cells remain poorly understood. Therefore, the objective of this study is to investigate the effects of tryptanthrin on the growth and differentiation of human neuroblastoma LA-N-1 cells with N-myc amplification. Our results show that tryptanthrin inhibited the growth of the human neuroblastoma cells in a dose- and time-dependent manner. Mechanistic studies indicated that tryptanthrin induced cell cycle arrest of the human neuroblastoma LA-N-1 cells at the G0/G1 phase. Tryptanthrin also induced neuronal differentiation of LA-N-1 cells, as assessed by morphological criteria, enhancement of acetylcholine esterase activity and up-regulation of various differentiation markers. Moreover, tryptanthrin treatment led to the significant reduction of N-myc expression in LA-N-1 cells while siRNA directed against N-myc induced morphological differentiation of LA-N-1 cells. These results, when taken together, suggest that tryptanthrin suppressed the growth and induced neuronal differentiation in the human neuroblastoma LA-N-1 cells and might be exploited as a potential therapeutic candidate for the treatment of high-risk neuroblastomas with N-myc-amplification. PMID:23500671

  10. Quantitative glycomics monitoring of induced pluripotent- and embryonic stem cells during neuronal differentiation

    Michiyo Terashima

    2014-11-01

    Full Text Available Alterations in the structure of cell surface glycoforms occurring during the stages of stem cell differentiation remain unclear. We describe a rapid glycoblotting-based cellular glycomics method for quantitatively evaluating changes in glycoform expression and structure during neuronal differentiation of murine induced pluripotent stem cells (iPSCs and embryonic stem cells (ESCs. Our results show that changes in the expression of cellular N-glycans are comparable during the differentiation of iPSCs and ESCs. The expression of bisect-type N-glycans was significantly up-regulated in neurons that differentiated from both iPSCs and ESCs. From a glycobiological standpoint, iPSCs are an alternative neural cell source in addition to ESCs.

  11. Differentiation patterns of mouse embryonic stem cells and induced pluripotent stem cells into neurons.

    Nakamura, Mai; Kamishibahara, Yu; Kitazawa, Ayako; Kawaguchi, Hideo; Shimizu, Norio

    2016-05-01

    Mouse embryonic stem (ES) cells and induced pluripotent stem (iPS) cells have the ability to differentiate in vitro into various cell lineages including neurons. The differentiation of these cells into neurons has potential applications in regenerative medicine. Previously, we reported that a chick dorsal root ganglion (DRG)-conditioned medium (CM) promoted the differentiation of mouse ES and iPS cells into neurons. Here, we used real-time PCR to investigate the differentiation patterns of ES and iPS cells into neurons when DRG-CM was added. DRG-CM promoted the expression levels of βIII-tubulin gene (a marker of postmitotic neurons) in ES and iPS cells. ES cells differentiated into neurons faster than iPS cells, and the maximum peaks of gene expression involved in motor, sensory, and dopaminergic neurons were different. Rho kinase (ROCK) inhibitors could be very valuable at numerous stages in the production and use of stem cells in basic research and eventual cell-based therapies. Thus, we investigated whether the addition of a ROCK inhibitor Y-27632 and DRG-CM on the basis of the differentiation patterns promotes the neuronal differentiation of ES cells. When the ROCK inhibitor was added to the culture medium at the initial stages of cultivation, it stimulated the neuronal differentiation of ES cells more strongly than that stimulated by DRG-CM. Moreover, the combination of the ROCK inhibitor and DRG-CM promoted the neuronal differentiation of ES cells when the ROCK inhibitor was added to the culture medium at day 3. The ROCK inhibitor may be useful for promoting neuronal differentiation of ES cells. PMID:25354731

  12. Glucocorticoid-induced differentiation of fetal rat calvarial osteoblasts is mediated by bone morphogenetic protein-6.

    Boden, S D; Hair, G; Titus, L; Racine, M; McCuaig, K; Wozney, J M; Nanes, M S

    1997-07-01

    Glucocorticoids (GCs) at physiological concentrations promote osteoblast differentiation from fetal calvarial cells, calvarial organ cultures, and bone marrow stromal cells; however, the cellular pathways involved are not known. Bone morphogenetic proteins (BMPs) are recognized as important mediators of osteoblast differentiation. Specific roles for individual BMPs during postembryonic membranous bone formation have yet to be determined. We recently reported that GC potentiated the osteoblast differentiation effects of BMP-2 and BMP-4, but not of BMP-6, which, by itself, was the most potent of the three. In the present study, we used fetal rat secondary calvarial cultures to study the role of BMP-6 during early osteoblast differentiation. Treatment with the GC triamcinolone (10(-9) M) resulted in a 5- to 8-fold increase in BMP-6 steady-state messenger RNA levels, peaking at 12 h. In contrast, BMPs -2, -4, -5, -7, and transforming growth factor (TGF)-beta1 messenger RNA levels increased by less than 2-fold, after GC treatment, compared with untreated control cultures at 24 h. BMP-6 protein secretion increased 6- to 7-fold by 12 h and 12-fold (from 7.5 to 90 ng/ml) by 24 h, as measured by quantitative Western analysis. Treatment of cells with oligodeoxynucleotides antisense to BMP-6 diminished secretion of BMP-6 protein and significantly inhibited the GC-induced differentiation, as determined by a 10-fold decrease in the number of mineralized bone nodules, compared with controls that were treated with sense oligonucleotides or no oligonucleotides (ANOVA, P < 0.05). The antisense oligonucleotide inhibition of differentiation was rescued by treatment with exogenous recombinant human BMP-6. We conclude that GC-induced differentiation of osteoblasts from the pluripotent precursors is mediated, in part, by BMP-6. These results suggest that BMP-6 has an important and unique role during early osteoblast differentiation. PMID:9202223

  13. Genistein as an inducer of tumor cell differentiation : possible mechanisms of action.

    Constantinou, A.; Huberman, E.; Center for Mechanistic Biology and Biotechnology; Univ. of Illinois at Chicago

    1995-01-01

    Decreased activity of either topoisomerases or tyrosine kinases has been implicated in the differentiation of a number of cell types. It is therefore conceivable that genistein, because of its reported ability to inhibit these activities in vitro, may be an inducer of cellular differentiation. We investigated this possibility in human promyelocytic HL-60 and erythroid K-562 leukemia cells and in human SK-MEL-131 melanoma cells. Our results indicated that genistein, in a dose-dependent manner, inhibited cell multiplication and induced cell differentiation. The maturing HL-60 cells acquired granulocytic and monocytic markers. The differentiating K-562 cells stained positively with benzidine, which indicates the production of hemoglobin, an erythroid marker. Following genistein treatment, maturing SK-MEL-131 melanoma cells formed dendrite-like structures and exhibited increased tyrosinase activity and melanin content. Experiments were designed to identify the molecular mechanism of genistein's action. Data from our laboratory suggest that this isoflavone triggers the pathway that leads to cellular differentiation by stabilizing protein-linked DNA strand breakage. Other possible mechanisms reported in the literature are discussed.

  14. Choline kinase alpha expression during RA-induced neuronal differentiation: role of C/EBPβ.

    Domizi, Pablo; Aoyama, Chieko; Banchio, Claudia

    2014-04-01

    Neuronal differentiation is a complex process characterized by a halt in proliferation and extension of neurites from the cell body. This process is accompanied by changes in gene expression that mediate the redirection leading to neurite formation and function. Acceleration of membrane phospholipids synthesis is associated with neurite elongation, and phosphatidylcholine (PtdCho) is the major membrane phospholipid in mammalian cells. The transcription of two genes in particular encoding key enzymes in the CDP-choline pathway for PtdCho biosynthesis are stimulated; the Chka gene for choline kinase (CK) alpha isoform and the Pcyt1a gene for the CTP:phosphocholine cytidylyltransferase (CCT) alpha isoform. We report that the stimulation of CKα expression during retinoic acid (RA) induced differentiation depends on a promoter region that contains two CCAAT/Enhancer-binding Protein-β (C/EBPβ) sites. We demonstrate that during neuronal differentiation of Neuro-2a cells, RA induces Chka expression by a mechanism that involves ERK1/2 activation which triggers C/EBPβ expression. Elevated levels of C/EBPβ bind to the Chka proximal promoter (Box1) inducing CKα expression. In addition we identified a downstream sequence named Box2 which together with Box1 is required for the promoter to reach the full induction. This is the first elucidation of the mechanism by which the expression of Chka is coordinately regulated during neuronal differentiation. PMID:24440820

  15. The benzophenanthridine alkaloid fagaronine induces erythroleukemic cell differentiation by gene activation.

    Dupont, Claude; Couillerot, Eric; Gillet, Reynald; Caron, Catherine; Zeches-Hanrot, Monique; Riou, Jean-François; Trentesaux, Chantal

    2005-06-01

    Fagaronine, a benzophenanthridine alkaloid from Fagara zanthoxyloides Lam. (Rutaceae), has been tested on the erythroleukemic cell line K562 in order to explain some previous results on cell differentiation. In this study we showed that fagaronine induces a significant hemoglobinization of the human erythroleukemic cell line K562. This hemoglobin synthesis was accompanied by a strong increase of erythroid mRNA expression such as gamma- and alpha-globin, and PBGD, an enzyme of heme synthesis. In addition, the Epo-R transcripts were also stimulated indicating that cells are engaged in a maturation process. Both transcription factors GATA-1 and NF-E2, which play an important role in the regulation of genes involved in the erythroid differentiation, were also transcriptionally up-regulated. To elucidate the possible role of GATA-1 in the FAG-induced differentiation of K562 cells, we transfected reporter constructs containing regulatory regions of erythroid genes encompassing GATA-1 binding sites. After 48 hours of treatment, FAG stimulated the EPO-R and gamma-globin promoters by 2- to 3-fold and the promoter/enhancer region of GATA-1 gene by 3.2-fold. A mutation within the GATA-1 binding sites strongly decreased the promoter activation induced by FAG. Taken together, our results represent a demonstration that FAG exerts its differentiating activity by a specific activation of the regulating GATA-1 regions of genes involved in the erythroid phenotype expression. PMID:15971117

  16. Identification of Centella asiatica’s Effective Ingredients for Inducing the Neuronal Differentiation

    Hui Jiang

    2016-01-01

    Full Text Available Centella asiatica, commonly known as Gotu kola, has been widely used as a traditional herb for decades. Yet, the study on which compounds or compound combinations actually lead to its brain benefits remains scarce. To study the neuroprotection effects of Centella asiatica, neuronal differentiation of PC12 cells was applied. In our pilot study, we isolated 45 Centella asiatica fractions and tested their abilities for inducing neuronal differentiation on PC12 cells. The most effective fraction showed robust induction in neurite outgrowth and neurofilament expression. LC-MS fingerprint analysis of this fraction revealed asiatic acid and madecassic acid as the dominant components. A further investigation on the pure combination of these two compounds indicated that the combination of these two compounds extensively promoted nerve differentiation in vitro. Application of PD98059, a protein MEK inhibitor, attenuated combination-induced neurofilament expression, indicating the combination-induced nerve differentiation through activation of MEK signaling pathway. Our results support the use of combination of asiatic acid and madecassic acid as an effective mean to intervene neurodegenerative diseases in which neurotrophin deficiency is involved.

  17. Titanium With Nanotopography Induces Osteoblast Differentiation by Regulating Endogenous Bone Morphogenetic Protein Expression and Signaling Pathway.

    M S Castro-Raucci, Larissa; S Francischini, Marcelo; N Teixeira, Lucas; P Ferraz, Emanuela; B Lopes, Helena; T de Oliveira, Paulo; Hassan, Mohammad Q; Losa, Adalberto L; Beloti, Marcio M

    2016-07-01

    We aimed at evaluating the effect of titanium (Ti) with nanotopography (Nano) on the endogenous expression of BMP-2 and BMP-4 and the relevance of this process to the nanotopography-induced osteoblast differentiation. MC3T3-E1 cells were grown on Nano and machined (Machined) Ti surfaces and the endogenous BMP-2/4 expression and the effect of BMP receptor BMPR1A silencing in both osteoblast differentiation and expression of genes related to TGF-β/BMP signaling were evaluated. Nano supported higher BMP-2 gene and protein expression and upregulated the osteoblast differentiation compared with Machined Ti surface. The BMPR1A silencing inhibited the osteogenic potential induced by Nano Ti surface as indicated by reduced alkaline phosphatase (ALP), osteocalcin and RUNX2 gene expression, RUNX2 protein expression and ALP activity. In addition, the expression of genes related to TGF-β/BMP signaling was deeply affected by BMPR1A-silenced cells grown on Nano Ti surface. In conclusion, we have demonstrated for the first time that nanotopography induces osteoblast differentiation, at least in part, by upregulating the endogenous production of BMP-2 and modulating BMP signaling pathway. J. Cell. Biochem. 117: 1718-1726, 2016. © 2015 Wiley Periodicals, Inc. PMID:26681207

  18. A Receptor Tyrosine Kinase Inhibitor, Dovitinib (TKI-258), Enhances BMP-2-Induced Osteoblast Differentiation In Vitro.

    Lee, Yura; Bae, Kyoung Jun; Chon, Hae Jung; Kim, Seong Hwan; Kim, Soon Ae; Kim, Jiyeon

    2016-05-31

    Dovitinib (TKI258) is a small molecule multi-kinase inhibitor currently in clinical phase I/II/III development for the treatment of various types of cancers. This drug has a safe and effective pharmacokinetic/pharmacodynamic profile. Although dovitinib can bind several kinases at nanomolar concentrations, there are no reports relating to osteoporosis or osteoblast differentiation. Herein, we investigated the effect of dovitinib on human recombinant bone morphogenetic protein (BMP)-2-induced osteoblast differentiation in a cell culture model. Dovitinib enhanced the BMP-2-induced alkaline phosphatase (ALP) induction, which is a representative marker of osteoblast differentiation. Dovitinib also stimulated the translocation of phosphorylated Smad1/5/8 into the nucleus and phosphorylation of mitogen-activated protein kinases, including ERK1/2 and p38. In addition, the mRNA expression of BMP-4, BMP-7, ALP, and OCN increased with dovitinib treatment. Our results suggest that dovitinib has a potent stimulating effect on BMP-2-induced osteoblast differentiation and this existing drug has potential for repositioning in the treatment of bone-related disorders. PMID:27025387

  19. Differentiation of human B lymphocyte subpopulations induced by an alloreactive helper T-cell clone

    We have used cloned alloreactive helper T cells to determine if direct T cell-B cell interaction can induce differentiation of human peripheral blood B cells which do not respond to pokeweed mitogen (PWM). T-cell clone 2F8 was derived from a one-way mixed lymphocyte reaction. 2F8 cells are T3+T4+T8-IL-2R+ and proliferate in response to irradiated stimulator cells, but not autologous cells, in the absence of exogenous interleukin-2. 2F8 cells provide allospecific help for polyclonal proliferation and differentiation of B cells in the absence of any other stimulus. The magnitude of this response is comparable to that of the response of the same B cells to PWM and fresh autologous T cells. 2F8 cells could also provide nonspecific help for unrelated donor B cells in the presence of PWM, with no requirement for costimulation by irradiated stimulator cells. Allospecific stimulation of B cells was completely inhibited by antibodies to class II major histocompatibility complex (MHC) framework determinants and was abrogated by 1000-rad irradiation. Cloned 2F8 T cells stimulated differentiation of both small, high-density B cells and larger B cells, generating up to 30% plasma cells with either fraction. B cells forming rosettes with mouse erythrocytes were also induced to differentiate by the helper T cell clone. As found previously, neither small, high-density B cells nor mouse rosette+ B cells responded well to PWM. Direct interaction with allospecific T cells induces differentiation of a broader spectrum of B cells than soluble growth and differentiation factors in conjunction with polyclonal activators such as PWM and protein A containing staphylococci

  20. Expression of the X-CGD gene during induced differentiation of myeloid leukemia cell line HL-60.

    Barker, K A; Orkin, S H; Newburger, P E

    1988-01-01

    The expression of the X-CGD gene, which encodes the heavy-chain subunit of the phagocyte cytochrome b, was studied during induced myeloid differentiation of HL-60 cells. Incubation of the cells with a combined regimen of retinoic acid and dimethyl formamide resulted in granulocytic morphological differentiation and acquisition of nitroblue tetrazolium reduction, a measure of superoxide generation. During the 5-day course of induced differentiation, the levels of X-CGD mRNA transcripts rose 13...

  1. Cisplatin induces resistance by triggering differentiation of testicular embryonal carcinoma cells.

    Paolo B Abada

    Full Text Available Although testicular germ cell tumors are generally quite responsive to treatment with cisplatin, a small fraction of them acquire resistance during therapy. Even when cisplatin treatment is successful the patient is often left with a residual teratoma at the site of the primary tumor suggesting that cisplatin may trigger differentiation in some tumors. Using the human embryonal carcinoma cell line NTera2/D1, we confirmed that exposure to the differentiating agent retinoic acid produced a reduction in pluripotency markers NANOG and POU5F1 (Oct3/4 and an acute concentration-dependent increase in resistance to both cisplatin and paclitaxel that reached as high as 18-fold for cisplatin and 61-fold for paclitaxel within four days. A two day exposure to cisplatin also produced a concentration-dependent decrease in the expression of the NANOG and POU5F1 and increased expression of three markers whose levels increase with differentiation including Nestin, SCG10 and Fibronectin. In parallel, exposure to cisplatin induced up to 6.2-fold resistance to itself and 104-fold resistance to paclitaxel. Paclitaxel did not induce differentiation or resistance to either itself or cisplatin. Neither retinoic acid nor cisplatin induced resistance in cervical or prostate cancer cell lines or other germ cell tumor lines in which they failed to alter the expression of NANOG and POU5F1. Forced expression of NANOG prevented the induction of resistance to cisplatin by retinoic acid. We conclude that cisplatin can acutely induce resistance to itself and paclitaxel by triggering a differentiation response in pluripotent germ cell tumor cells.

  2. Cisplatin induces resistance by triggering differentiation of testicular embryonal carcinoma cells.

    Abada, Paolo B; Howell, Stephen B

    2014-01-01

    Although testicular germ cell tumors are generally quite responsive to treatment with cisplatin, a small fraction of them acquire resistance during therapy. Even when cisplatin treatment is successful the patient is often left with a residual teratoma at the site of the primary tumor suggesting that cisplatin may trigger differentiation in some tumors. Using the human embryonal carcinoma cell line NTera2/D1, we confirmed that exposure to the differentiating agent retinoic acid produced a reduction in pluripotency markers NANOG and POU5F1 (Oct3/4) and an acute concentration-dependent increase in resistance to both cisplatin and paclitaxel that reached as high as 18-fold for cisplatin and 61-fold for paclitaxel within four days. A two day exposure to cisplatin also produced a concentration-dependent decrease in the expression of the NANOG and POU5F1 and increased expression of three markers whose levels increase with differentiation including Nestin, SCG10 and Fibronectin. In parallel, exposure to cisplatin induced up to 6.2-fold resistance to itself and 104-fold resistance to paclitaxel. Paclitaxel did not induce differentiation or resistance to either itself or cisplatin. Neither retinoic acid nor cisplatin induced resistance in cervical or prostate cancer cell lines or other germ cell tumor lines in which they failed to alter the expression of NANOG and POU5F1. Forced expression of NANOG prevented the induction of resistance to cisplatin by retinoic acid. We conclude that cisplatin can acutely induce resistance to itself and paclitaxel by triggering a differentiation response in pluripotent germ cell tumor cells. PMID:24475288

  3. Induced differentiation of cancer cells: second generation potent hybrid polar compounds target cell cycle regulators

    Hybrid polar compounds are potent inducers of differentiation of a wide variety of cancer transformed cells. Hexamethylene bisacetamide (HMBA) has been used as a prototype of these compounds to investigate their mechanism of action. Employing murine erythroleukemia (MEL) cells as a model, three characteristics of inducer-mediated commitment to terminal differentiation were demonstrated: (I) induced commitment was stochastic, requiring up to 5 cell cycles to recruit essentially all cells to commit to growth arrest in G1; (II) inducers caused a prolongation of the initial G1; and (III) the hybrid polar compounds induced a wide variety of transformed cells to terminal differentiation. These findings suggested that the rate limiting factor or factors for induction by these agents may be at the level of protein(s) regulating G1-to-S progression, which are common to most eukaryotic cells. It was found that HMBA induced a profound suppression of cyclin dependent kinase, cdk4, which reflected a marked decrease in stability of the protein, and is a critical change in the pathway of induced differentiation. HMBA also induced an increase in pRB and in the active, underphosphorylated form of this protein, an increase in the pRB related protein, p107, and an increase in the cyclin dependent kinase inhibitor, p21. Further, the free form of the transcription factor, E2F, was markedly decreased within hours of exposure of transformed cells to HMBA and found to complex with p107 and cdk 2. A phase II clinical trial was conducted using HMBA to treat patients with myelodysplastic syndrome (MDS) or acute myelogenous leukemia. Of 28 patients, 9 patients achieved a complete or partial remission lasting from 1 to 16 months. These clinical studies also provided direct evidence that HMBA induces differentiation of transformed cells in patients. In four separate courses of treatment with HMBA, a patient with MDS and the monosomy 7 karyotype marking the malignant clone of bone marrow blast

  4. Inhibition of the NAD-dependent protein deacetylase SIRT2 induces granulocytic differentiation in human leukemia cells.

    Yoshitaka Sunami

    Full Text Available Sirtuins, NAD-dependent protein deacetylases, play important roles in cellular functions such as metabolism and differentiation. Whether sirtuins function in tumorigenesis is still controversial, but sirtuins are aberrantly expressed in tumors, which may keep cancerous cells undifferentiated. Therefore, we investigated whether the inhibition of sirtuin family proteins induces cellular differentiation in leukemic cells. The sirtuin inhibitors tenovin-6 and BML-266 induce granulocytic differentiation in the acute promyelocytic leukemia (APL cell line NB4. This differentiation is likely caused by an inhibition of SIRT2 deacetylase activity, judging from the accumulation of acetylated α-tubulin, a major SIRT2 substrate. Unlike the clinically used differentiation inducer all-trans retinoic acid, tenovin-6 shows limited effects on promyelocytic leukemia-retinoic acid receptor α (PML-RAR-α stability and promyelocytic leukemia nuclear body formation in NB4 cells, suggesting that tenovin-6 does not directly target PML-RAR-α activity. In agreement with this, tenovin-6 induces cellular differentiation in the non-APL cell line HL-60, where PML-RAR-α does not exist. Knocking down SIRT2 by shRNA induces granulocytic differentiation in NB4 cells, which demonstrates that the inhibition of SIRT2 activity is sufficient to induce cell differentiation in NB4 cells. The overexpression of SIRT2 in NB4 cells decreases the level of granulocytic differentiation induced by tenovin-6, which indicates that tenovin-6 induces granulocytic differentiation by inhibiting SIRT2 activity. Taken together, our data suggest that targeting SIRT2 is a viable strategy to induce leukemic cell differentiation.

  5. Sphingosine 1-phosphate receptor activation enhances BMP-2-induced osteoblast differentiation

    Sato, Chieri [Division of Rheumatology, Department of Internal Medicine, Hyogo College of Medicine, 1-1 Mukogawa-cho, Nishinomiya, Hyogo 663-8501 (Japan); Iwasaki, Tsuyoshi, E-mail: tsuyo-i@huhs.ac.jp [Division of Pharmacotherapy, Department of Pharmacy, School of Pharmacy, Hyogo University of Health Sciences, 1-3-6 Minatojima, Chuo-ku, Kobe 650-8530 (Japan); Kitano, Sachie; Tsunemi, Sachi; Sano, Hajime [Division of Rheumatology, Department of Internal Medicine, Hyogo College of Medicine, 1-1 Mukogawa-cho, Nishinomiya, Hyogo 663-8501 (Japan)

    2012-06-22

    Highlights: Black-Right-Pointing-Pointer We investigated the role of S1P signaling for osteoblast differentiation. Black-Right-Pointing-Pointer Both S1P and FTY enhanced BMP-2-stimulated osteoblast differentiation by C2C12 cells. Black-Right-Pointing-Pointer S1P signaling enhanced BMP-2-stimulated Smad and ERK phosphorylation by C2C12 cells. Black-Right-Pointing-Pointer MEK/ERK signaling is a pathway underlying S1P signaling for osteoblast differentiation. -- Abstract: We previously demonstrated that sphingosine 1-phosphate (S1P) receptor-mediated signaling induced proliferation and prostaglandin productions by synovial cells from rheumatoid arthritis (RA) patients. In the present study we investigated the role of S1P receptor-mediated signaling for osteoblast differentiation. We investigated osteoblast differentiation using C2C12 myoblasts, a cell line derived from murine satellite cells. Osteoblast differentiation was induced by the treatment of bone morphogenic protein (BMP)-2 in the presence or absence of either S1P or FTY720 (FTY), a high-affinity agonist of S1P receptors. Osteoblast differentiation was determined by osteoblast-specific transcription factor, Runx2 mRNA expression, alkaline phosphatase (ALP) activity and osteocalcin production by the cells. Smad1/5/8 and extracellular signal-regulated kinase (ERK) 1/2 phosphorylation was examined by Western blotting. Osteocalcin production by C2C12 cells were determined by ELISA. Runx2 expression and ALP activity by BMP-2-stimulated C2C12 cells were enhanced by addition of either S1P or FTY. Both S1P and FTY enhanced BMP-2-induced ERK1/2 and Smad1/5/8 phosphorylation. The effect of FTY was stronger than that of S1P. S1P receptor-mediated signaling on osteoblast differentiation was inhibited by addition of mitogen-activated protein kinase/ERK kinase (MEK) 1/2 inhibitor, indicating that the S1P receptor-mediated MEK1/2-ERK1/2 signaling pathway enhanced BMP-2-Smad signaling. These results indicate that S1P

  6. Sphingosine 1-phosphate receptor activation enhances BMP-2-induced osteoblast differentiation

    Highlights: ► We investigated the role of S1P signaling for osteoblast differentiation. ► Both S1P and FTY enhanced BMP-2-stimulated osteoblast differentiation by C2C12 cells. ► S1P signaling enhanced BMP-2-stimulated Smad and ERK phosphorylation by C2C12 cells. ► MEK/ERK signaling is a pathway underlying S1P signaling for osteoblast differentiation. -- Abstract: We previously demonstrated that sphingosine 1-phosphate (S1P) receptor-mediated signaling induced proliferation and prostaglandin productions by synovial cells from rheumatoid arthritis (RA) patients. In the present study we investigated the role of S1P receptor-mediated signaling for osteoblast differentiation. We investigated osteoblast differentiation using C2C12 myoblasts, a cell line derived from murine satellite cells. Osteoblast differentiation was induced by the treatment of bone morphogenic protein (BMP)-2 in the presence or absence of either S1P or FTY720 (FTY), a high-affinity agonist of S1P receptors. Osteoblast differentiation was determined by osteoblast-specific transcription factor, Runx2 mRNA expression, alkaline phosphatase (ALP) activity and osteocalcin production by the cells. Smad1/5/8 and extracellular signal-regulated kinase (ERK) 1/2 phosphorylation was examined by Western blotting. Osteocalcin production by C2C12 cells were determined by ELISA. Runx2 expression and ALP activity by BMP-2-stimulated C2C12 cells were enhanced by addition of either S1P or FTY. Both S1P and FTY enhanced BMP-2-induced ERK1/2 and Smad1/5/8 phosphorylation. The effect of FTY was stronger than that of S1P. S1P receptor-mediated signaling on osteoblast differentiation was inhibited by addition of mitogen-activated protein kinase/ERK kinase (MEK) 1/2 inhibitor, indicating that the S1P receptor-mediated MEK1/2-ERK1/2 signaling pathway enhanced BMP-2-Smad signaling. These results indicate that S1P receptor-mediated signaling plays a crucial role for osteoblast differentiation.

  7. A cost-effective system for differentiation of intestinal epithelium from human induced pluripotent stem cells.

    Ogaki, Soichiro; Morooka, Mayu; Otera, Kaito; Kume, Shoen

    2015-01-01

    The human intestinal epithelium is a useful model for pharmacological studies of absorption, metabolism, drug interactions, and toxicology, as well as for studies of developmental biology. We established a rapid and cost effective system for differentiation of human induced pluripotent stem (iPS) cells into definitive endoderm (DE) cells. In the presence of dimethyl sulfoxide (DMSO), a low concentration of Activin at 6.25 ng/ml is sufficient to give a similar differentiation efficiency with that using Activin at 100 ng/ml at the presence of Wnt activator. In the presence of DMSO, Activin at low concentration triggered hiPS cells to undergo differentiation through G1 arrest, reduce apoptosis, and potentiate activation of downstream targets, such as SMAD2 phosphorylation and SOX17 expression. This increased differentiation into CDX2 + SOX17 + DE cells. The present differentiation procedure therefore permits rapid and efficient derivation of DE cells, capable of differentiating into intestinal epithelium upon BIO and DAPT treatment and of giving rise to functional cells, such as enterocytes. PMID:26616277

  8. Retinoic acid induces nuclear accumulation of Raf1 during differentiation of HL-60 cells

    Smith, James; Bunaciu, Rodica P.; Reiterer, Gudrun [Department of Biomedical Sciences, T4-008 VRT, Cornell University, Ithaca, NY 14853 (United States); Coder, David; George, Thaddeus [Amnis Corporation, Seattle, Washington (United States); Asaly, Michael [Department of Biomedical Sciences, T4-008 VRT, Cornell University, Ithaca, NY 14853 (United States); Yen, Andrew, E-mail: ay13@cornell.edu [Department of Biomedical Sciences, T4-008 VRT, Cornell University, Ithaca, NY 14853 (United States)

    2009-08-01

    All trans-retinoic acid (RA) is a standard therapeutic agent used in differentiation induction therapy treatment of acute promyelocytic leukemia (APL). RA and its metabolites use a diverse set of signal transduction pathways during the differentiation program. In addition to the direct transcriptional targets of the nuclear RAR and RXR receptors, signals derived from membrane receptors and the Raf-MEK-ERK pathway are required. Raf1 phosphorylation and the prolonged activation of Raf1 persisting during the entire differentiation process are required for RA-dependent differentiation of HL-60 cells. Here we identify a nuclear redistribution of Raf1 during the RA-induced differentiation of HL-60 cells. In addition, the nuclear accumulation of Raf1 correlates with an increase in Raf1 phosphorylated at serine 621. The serine 621 phosphorylated Raf1 is predominantly localized in the nucleus. The RA-dependent nuclear accumulation of Raf1 suggests a novel nuclear role for Raf1 during the differentiation process.

  9. Effect of anthocyanidins on myogenic differentiation in induced and non-induced primary myoblasts from rainbow trout (Oncorhynchus mykiss).

    Villasante, Alejandro; Powell, Madison S; Murdoch, Gordon K; Overturf, Ken; Cain, Kenneth; Wacyk, Jurij; Hardy, Ronald W

    2016-01-01

    A study was conducted to test whether an anthocyanidin mixture (peonidin, cyanidin and pelargonidin chloride) modulates myogenesis in both induced and non-induced myogenic cells from juvenile rainbow trout (Oncorhynchus mykiss). We evaluated three different anthocyanidin concentrations (1×, 2.5× and 10×) at two sampling times (24 and 36h). To test for treatment effects, we analyzed the expression of myoD and pax7 as well as two target genes of the Notch signaling pathway, hey2 and her6. In induced myogenic cells, the lowest and middle anthocyanidin doses caused significantly greater expression of myoD after 24h of treatment compared to control. A significantly higher expression of pax7 in cells exposed to either anthocyanidin treatment during 36h compared was observed. Similarly, the pax7/myoD ratio was significantly lower in cells exposed to the lowest anthocyanidin doses during 24h compared to control. No significant effect of anthocyanidin treatments on the expression of hey2 and her6 at either sampling point was detected. In non-induced cells, we observed no effect of anthocyanidins on myoD expression and significant down-regulation on pax7 expression in cells exposed to either anthocyanidin mixture concentrations after 24 and 36h of treatment compared to control. Further, the pax7/myoD ratio was significantly lower in cells exposed to either anthocyanidin doses at both sampling time. In non-induced cells, the highest anthocyanidin dose provoked significantly greater expression of hey2 after 24h of treatment compared to control. We detected no such effect in non-induced cells exposed to the lowest and middle anthocyanidin doses during 24h of treatment. The expression of her6 was unaffected by anthocyanidin treatments at either sampling time or doses compared to control. Collectively, these findings provide evidence that anthocyanidins modulate specific components of the myogenic programming in fish, thereby potentially affecting somatic growth in fish fed

  10. Experimental study of millimeter wave-induced differentiation of bone marrow mesenchymal stem cells into chondrocytes.

    Wu, Guang-Wen; Liu, Xian-Xiang; Wu, Ming-Xia; Zhao, Jin-Yan; Chen, Wen-Lie; Lin, Ru-Hui; Lin, Jiu-Mao

    2009-04-01

    Low power millimeter wave irradiation is widely used in clinical medicine. We describe the effects of this treatment on cultured mesenchymal stem cells (MSCs) and attempted to identify the underlying mechanism. Cells cultured using the whole marrow attachment culture method proliferated dispersedly or in clones. Flow cytometric analyses showed that the MSCs were CD90 positive, but negative for CD45. The negative control group (A) did not express detectable levels of Cbfa1 or Sox9 mRNA at any time point, while cells in the millimeter wave-induced groups (B and C) increasingly expressed both genes after the fourth day post-induction. Statistical analysis showed that starting on the fourth day post-induction, there were very significant differences in the expression of Cbfa1 and Sox9 mRNA between groups A and B as well as A and C at any given time point, between treated groups B and C after identical periods of induction, and within each treated group at different induction times. Transition electron microscopy analysis showed that the rough endoplasmic reticulum of cells in the induced groups was richer and more developed than in cells of the negative control group, and that the shape of cells shifted from long-spindle to near ellipse. Toluidine blue staining revealed heterochromia in the cytoplasm and extracellular matrix of cells in the induced groups, whereas no obvious heterochromia was observed in negative control cells. Induced cells also exhibited positive immunohistochemical staining of collagen II, in contrast to the negative controls. These results show that millimeter wave treatment successfully induced MSCs to differentiate as chondrocytes and the extent of differentiation increased with treatment duration. Our findings suggest that millimeter wave irradiation can be employed as a novel non-drug inducing method for the differentiation of MSCs into chondrocytes. PMID:19288021

  11. Differentiation-inducing factor-1 and -2 function also as modulators for Dictyostelium chemotaxis.

    Hidekazu Kuwayama

    Full Text Available BACKGROUND: In the early stages of development of the cellular slime mold Dictyostelium discoideum, chemotaxis toward cAMP plays a pivotal role in organizing discrete cells into a multicellular structure. In this process, a series of signaling molecules, such as G-protein-coupled cell surface receptors for cAMP, phosphatidylinositol metabolites, and cyclic nucleotides, function as the signal transducers for controlling dynamics of cytoskeleton. Differentiation-inducing factor-1 and -2 (DIF-1 and DIF-2 were originally identified as the factors (chlorinated alkylphenones that induce Dictyostelium stalk cell differentiation, but it remained unknown whether the DIFs had any other physiologic functions. METHODOLOGY/PRINCIPAL FINDINGS: To further elucidate the functions of DIFs, in the present study we investigated their effects on chemotaxis under various conditions. Quite interestingly, in shallow cAMP gradients, DIF-1 suppressed chemotaxis whereas DIF-2 promoted it greatly. Analyses with various mutants revealed that DIF-1 may inhibit chemotaxis, at least in part, via GbpB (a phosphodiesterase and a decrease in the intracellular cGMP concentration ([cGMP](i. DIF-2, by contrast, may enhance chemotaxis, at least in part, via RegA (another phosphodiesterase and an increase in [cGMP](i. Using null mutants for DimA and DimB, the transcription factors that are required for DIF-dependent prestalk differentiation, we also showed that the mechanisms for the modulation of chemotaxis by DIFs differ from those for the induction of cell differentiation by DIFs, at least in part. CONCLUSIONS/SIGNIFICANCE: Our findings indicate that DIF-1 and DIF-2 function as negative and positive modulators for Dictyostelium chemotaxis, respectively. To our knowledge, this is the first report in any organism of physiologic modulators (small molecules for chemotaxis having differentiation-inducing activity.

  12. B-Raf inhibitors induce epithelial differentiation in BRAF-mutant colorectal cancer cells.

    Herr, Ricarda; Köhler, Martin; Andrlová, Hana; Weinberg, Florian; Möller, Yvonne; Halbach, Sebastian; Lutz, Lisa; Mastroianni, Justin; Klose, Martin; Bittermann, Nicola; Kowar, Silke; Zeiser, Robert; Olayioye, Monilola A; Lassmann, Silke; Busch, Hauke; Boerries, Melanie; Brummer, Tilman

    2015-01-01

    BRAF mutations are associated with aggressive, less-differentiated and therapy-resistant colorectal carcinoma. However, the underlying mechanisms for these correlations remain unknown. To understand how oncogenic B-Raf contributes to carcinogenesis, in particular to aspects other than cellular proliferation and survival, we generated three isogenic human colorectal carcinoma cell line models in which we can dynamically modulate the expression of the B-Raf(V600E) oncoprotein. Doxycyclin-inducible knockdown of endogenous B-Raf(V600E) decreases cellular motility and invasion in conventional and three-dimensional (3D) culture, whereas it promotes cell-cell contacts and induces various hallmarks of differentiated epithelia. Importantly, all these effects are recapitulated by B-Raf (PLX4720, vemurafenib, and dabrafenib) or MEK inhibitors (trametinib). Surprisingly, loss of B-Raf(V600E) in HT29 xenografts does not only stall tumor growth, but also induces glandular structures with marked expression of CDX2, a tumor-suppressor and master transcription factor of intestinal differentiation. By performing the first transcriptome profiles of PLX4720-treated 3D cultures of HT29 and Colo-205 cells, we identify several upregulated genes linked to epithelial differentiation and effector functions, such as claudin-1, a Cdx-2 target gene encoding a critical tight junction component. Thereby, we provide a mechanism for the clinically observed correlation between mutant BRAF and the loss of Cdx-2 and claudin-1. PLX4720 also suppressed several metastasis-associated transcripts that have not been implicated as targets, effectors or potential biomarkers of oncogenic B-Raf signaling so far. Together, we identify a novel facet of clinically applied B-Raf or MEK inhibitors by showing that they promote cellular adhesion and differentiation of colorectal carcinoma cells. PMID:25381152

  13. Differential Costs of Two Distinct Resistance Mechanisms Induced by Different Herbivore Species in Arabidopsis.

    Onkokesung, Nawaporn; Reichelt, Michael; van Doorn, Arjen; Schuurink, Robert C; Dicke, Marcel

    2016-02-01

    Plants respond to herbivory with the induction of resistance, mediated by distinct phytohormonal signaling pathways and their interactions. Phloem feeders are known to induce plant resistance via the salicylic acid pathway, whereas biting-chewing herbivores induce plant resistance mainly via the jasmonate pathway. Here, we show that a specialist caterpillar (biting-chewing herbivore) and a specialist aphid (phloem feeder) differentially induce resistance against Pieris brassicae caterpillars in Arabidopsis (Arabidopsis thaliana) plants. Caterpillar feeding induces resistance through the jasmonate signaling pathway that is associated with the induction of kaempferol 3,7-dirhamnoside, whereas aphid feeding induces resistance via a novel mechanism involving sinapoyl malate. The role of sinapoyl malate is confirmed through the use of a mutant compromised in the biosynthesis of this compound. Caterpillar-induced resistance is associated with a lower cost in terms of plant growth reduction than aphid-induced resistance. A strong constitutive resistance against P. brassicae caterpillars in combination with a strong growth attenuation in plants of a transfer DNA (T-DNA) insertion mutant of WRKY70 (wrky70) suggest that the WRKY70 transcription factor, a regulator of downstream responses mediated by jasmonate-salicylic acid signaling cross talk, is involved in the negative regulation of caterpillar resistance and in the tradeoff between growth and defense. In conclusion, different mechanisms of herbivore-induced resistance come with different costs, and a functional WRKY70 transcription factor is required for the induction of low-cost resistance. PMID:26603653

  14. Myostatin acts as an autocrine/paracrine negative regulator in myoblast differentiation from human induced pluripotent stem cells

    Highlights: ► iPS-derived cells express myostatin and its receptor upon myoblast differentiation. ► Myostatin inhibits myoblast differentiation by inhibiting MyoD and Myo5a induction. ► Silencing of myostatin promotes differentiation of human iPS cells into myoblasts. -- Abstract: Myostatin, also known as growth differentiation factor (GDF-8), regulates proliferation of muscle satellite cells, and suppresses differentiation of myoblasts into myotubes via down-regulation of key myogenic differentiation factors including MyoD. Recent advances in stem cell biology have enabled generation of myoblasts from pluripotent stem cells, but it remains to be clarified whether myostatin is also involved in regulation of artificial differentiation of myoblasts from pluripotent stem cells. Here we show that the human induced pluripotent stem (iPS) cell-derived cells that were induced to differentiate into myoblasts expressed myostatin and its receptor during the differentiation. An addition of recombinant human myostatin (rhMyostatin) suppressed induction of MyoD and Myo5a, resulting in significant suppression of myoblast differentiation. The rhMyostatin treatment also inhibited proliferation of the cells at a later phase of differentiation. RNAi-mediated silencing of myostatin promoted differentiation of human iPS-derived embryoid body (EB) cells into myoblasts. These results strongly suggest that myostatin plays an important role in regulation of myoblast differentiation from iPS cells of human origin. The present findings also have significant implications for potential regenerative medicine for muscular diseases

  15. Intercellular Communication between Keratinocytes and Fibroblasts Induces Local Osteoclast Differentiation: a Mechanism Underlying Cholesteatoma-Induced Bone Destruction.

    Iwamoto, Yoriko; Nishikawa, Keizo; Imai, Ryusuke; Furuya, Masayuki; Uenaka, Maki; Ohta, Yumi; Morihana, Tetsuo; Itoi-Ochi, Saori; Penninger, Josef M; Katayama, Ichiro; Inohara, Hidenori; Ishii, Masaru

    2016-06-01

    Bone homeostasis is maintained by a balance in activity between bone-resorbing osteoclasts and bone-forming osteoblasts. Shifting the balance toward bone resorption causes osteolytic bone diseases such as rheumatoid arthritis and periodontitis. Osteoclast differentiation is regulated by receptor activator of nuclear factor κB ligand (RANKL), which, under some pathological conditions, is produced by T and B lymphocytes and synoviocytes. However, the mechanism underlying bone destruction in other diseases is little understood. Bone destruction caused by cholesteatoma, an epidermal cyst in the middle ear resulting from hyperproliferation of keratinizing squamous epithelium, can lead to lethal complications. In this study, we succeeded in generating a model for cholesteatoma, epidermal cyst-like tissue, which has the potential for inducing osteoclastogenesis in mice. Furthermore, an in vitro coculture system composed of keratinocytes, fibroblasts, and osteoclast precursors was used to demonstrate that keratinocytes stimulate osteoclast differentiation through the induction of RANKL in fibroblasts. Thus, this study demonstrates that intercellular communication between keratinocytes and fibroblasts is involved in the differentiation and function of osteoclasts, which may provide the molecular basis of a new therapeutic strategy for cholesteatoma-induced bone destruction. PMID:27001307

  16. Introduction of v-Ha-ras oncogene induces differentiation of cultured human medullary thyroid carcinoma cells

    Medullary thyroid carcinoma (MTC) is an endocrine tumor of the thyroid C cells that expresses high levels of the neuroendocrine peptide hormone calcitonin. During tumor progression in the host, there is an apparent loss of differentiation in MTC cells that involves a consistent decrease in calcitonin content of the tumor cells associated with decreased expression of the calcitonin gene and/or changes in a mRNA alternative-processing pattern away from that characteristic of the parent thyroid C cell. The authors now report that introduction of the viral Harvey ras (v-Ha-ras) oncogene into cultured human MTC cells can reverse such changes in gene expression and can induce endocrine differentiation of the tumor cells. The expression of v-Ha-ras is associated with decreased cellular proliferation and DNA synthesis. There is a marked increase in the number of cytoplasmic secretory granules that are a classic feature of differentiated thyroid C cells. v-Ha-ras expression induces increased expression of the calcitonin gene and the processing of the primary gene transcript is shifted to favor calcitonin mRNA rather than calcitonin-gene-related peptide (CGRP) mRNA production. These studies with cultured human MTC cells provide a model system to study the role of Ha-ras and related genes in neuroendocrine differentiation. The findings suggest an important approach for identifying genes in solid tumors whose altered expression may play a role in the impaired maturational capacity characteristic of cancer cells during tumor progression

  17. Chronic heat-shock treatment driven differentiation induces apoptosis in Leishmania donovani.

    Raina, Puneet; Kaur, Sukhbir

    2006-09-01

    The present study investigates the role of apoptosis in the regulation of cell numbers of Leishmania donovani during the in vitro differentiation of promastigote stage to amastigote stage in axenic conditions. We report that apoptosis is induced in Leishmania donovani due to chronic heat-shock treatment of 37 ( degrees )C that also mediates the differentiation of promastigotes to amastigotes. This is characterized by the fragmentation of DNA, blebbing in the parasite cell membrane, nuclear condensation, formation of preapoptotic bodies and involvement of Ca(++) in the apoptotic process. The flowcytometric analysis shows an early and steep rise in percentage apoptotic nuclei till 48-hour stage of differentiation and then a gradual decline, suggesting synergistic action of Ca(++) ATPase and probably Hsp70. Hsp70 might be rescuing cells from apoptosis in the death signaling pathway. Incubation of the culture with Ca(++) chelator EGTA (1 mM) brings down the percentage of apoptotic nuclei considerably showing thereby that calcium is needed for the process of cell death here that occurs by apoptosis. The survival of the infective individuals appears to be decided by the parasite in the early stages of its differentiation. Our studies show the potential of the physiological temperature of 37 ( degrees )C in inducing apoptosis in Leishmania donovani and the therapeutic use it can be put to. PMID:16718376

  18. Differential diagnosis of particular interest - Osteoarthropathy induced by haemochromatosis; Die interessante Differentialdiagnose - die Haemochromatose-Osteoarthropathie

    Kittner, T. [Universitaetsklinikum ``Carl Gustaf Carus``, TU Dresden, Inst. und Poliklinik fuer Radiologische Diagnostik (Germany); Nitzsche, H. [Universitaetsklinikum ``Carl Gustaf Carus``, TU Dresden, Inst. und Poliklinik fuer Radiologische Diagnostik (Germany); Klengel, S. [Universitaetsklinikum ``Carl Gustaf Carus``, TU Dresden, Inst. und Poliklinik fuer Radiologische Diagnostik (Germany)

    1996-01-01

    Two cases have been selected to explain the efficiency of X-radiography and MRI and the relevant diagnostic images leading to the diagnosis of haemochromatosis-induced osteoarthropathy of the hand joints, and to possible differential diagnoses. Clinical as well as paraclinical aspects are included in the report. (orig.) [Deutsch] Anhand zweier Fallbeispiele werden das konventionell-radiologische sowie kernspintomographische Erscheinungsbild der Haemochromatose-Osteoarthropathie der Handgelenke sowie moegliche Differentialdiagnosen beschrieben. Klinische sowie paraklinische Aspekte finden gleichfalls Beachtung. (orig.)

  19. Sad but true? - How induced emotional states differentially bias self-rated Big Five personality traits

    Querengässer, Jan; Schindler, Sebastian

    2014-01-01

    Background The Big Five are seen as stable personality traits. This study hypothesized that their measurement via self-ratings is differentially biased by participants’ emotions. The relationship between habitual emotions and personality should be mirrored in a patterned influence of emotional states upon personality scores. Methods We experimentally induced emotional states and compared baseline Big Five scores of ninety-eight German participants (67 female; mean age 22.2) to thei...

  20. Selective differentiation and proliferation of hematopoietic cells induced by recombinant human interleukins.

    Saito, H; Hatake, K.; Dvorak, A. M.; Leiferman, K M; Donnenberg, A D; Arai, N.; Ishizaka, K; Ishizaka, T

    1988-01-01

    Effects of recombinant human interleukins on hematopoiesis were explored by using suspension cultures of mononuclear cells of human umbilical-cord blood and bone marrow. The results showed that interleukin 5 induced the selective differentiation and proliferation of eosinophils. After 3 weeks in culture with interleukin 5, essentially all nonadherent cells in both bone marrow and cord blood cell cultures became eosinophilic myelocytes. Culture of the same cells with interleukin 4 resulted in ...

  1. Latexin is involved in bone morphogenetic protein-2-induced chondrocyte differentiation

    Latexin is the only known carboxypeptidase A inhibitor in mammals. We previously demonstrated that BMP-2 significantly induced latexin expression in Runx2-deficient mesenchymal cells (RD-C6 cells), during chondrocyte and osteoblast differentiation. In this study, we investigated latexin expression in the skeleton and its role in chondrocyte differentiation. Immunohistochemical studies revealed that proliferating and prehypertrophic chondrocytes expressed latexin during skeletogenesis and bone fracture repair. In the early phase of bone fracture, latexin mRNA expression was dramatically upregulated. BMP-2 upregulated the expression of the mRNAs of latexin, Col2a1, and the gene encoding aggrecan (Agc1) in a micromass culture of C3H10T1/2 cells. Overexpression of latexin additively stimulated the BMP-2-induced expression of the mRNAs of Col2a, Agc1, and Col10a1. BMP-2 treatment upregulated Sox9 expression, and Sox9 stimulated the promoter activity of latexin. These results indicate that latexin is involved in BMP-2-induced chondrocyte differentiation and plays an important role in skeletogenesis and skeletal regeneration.

  2. Mutated recombinant human glucagon-like peptide-1 induces differentiation of PC12 cells

    Jin Wu; Lan Zhang; Zhongwei Sun; Gang Huang; Jing Huang; Bing Mei

    2011-01-01

    "Glucagon-like peptide-1 (GLP-1) and its long-acting analogues have neuroprotective and neurotrophic properties and are emerging as potential treatments for neurodegenerative diseases. Its short half-life has limited the application of GLP-1 in the clinic. We generated a mutated form of human GLP-1 (mGLP-1) using site-directed mutagenesis and gene recombination techniques, and found that these modifications significantly prolonged the biological half-life of GLP-1 compared with native GLP-1 (nGLP-1). This study investigated the role of mGLP-1 on inducing PC12 cell differentiation. mGLP-1 induced PC12 cell differentiation with neurite outgrowth and increased the expression of growth-associated protein-43 and neuronal class III ?-tubulin, and significantly increased cyclic adenosine monophosphate level. No significant difference was found between mGLP-1 and nGLP-1. The results indicate that mGLP-1 activates the GLP-1 receptor, induces PC12 cell differentiation, and has neurotrophic effects.

  3. Differentiation of bone mesenchymal stem cells into hepatocyte-like cells induced by liver tissue homogenate.

    Xing, X K; Feng, H G; Yuan, Z Q

    2016-01-01

    This study investigated the efficacy and feasibility of inducing the differentiation of bone marrow-derived mesenchymal stem cells (BMSCs) into hepatocyte-like cells in vitro using Sprague Dawley rats, as a model of hepatocyte generation for cell transplantation. BMSCs were isolated and grown using the adherent method and exposed to 5 or 10% liver tissue homogenate, before being collected for analysis after 0, 7, 14, and 21 days. Immunofluorescence and western blotting were employed to detect the liver-specific markers a-fetoprotein (AFP) and albumin (ALB). Supernatant urea content was also measured to verify that differentiation had been induced. After 7 days in the presence of 10% liver tissue homogenate, BMSCs demonstrated hepatocyte-like morphological characteristics, and with prolonged culture time, liver-specific markers were gradually produced at levels indicating cell maturation. AFP expression peaked at 14 days then began to decrease, while both urea and ALB levels increased with induction time. Overall, marker expression in the 5% homogenate group was less than or equal to the 10% group at each time point. Thus, in a rat model, liver tissue homogenate obtained from partial hepatectomy can induce the differentiation of BMSCs into hepatocyte-like cells. This method is simple, feasible, and has remarkable real-world application potential. PMID:27525848

  4. Differential interferometry for measurement of density fluctuations and fluctuation-induced transport (invited)

    Differential interferometry employs two parallel laser beams with a small spatial offset (less than beam width) and frequency difference (1-2 MHz) using common optics and a single mixer for a heterodyne detection. The differential approach allows measurement of the electron density gradient, its fluctuations, as well as the equilibrium density distribution. This novel interferometry technique is immune to fringe skip errors and is particularly useful in harsh plasma environments. Accurate calibration of the beam spatial offset, accomplished by use of a rotating dielectric wedge, is required to enable broad application of this approach. Differential interferometry has been successfully used on the Madison Symmetric Torus reversed-field pinch plasma to directly measure fluctuation-induced transport along with equilibrium density profile evolution during pellet injection. In addition, by combining differential and conventional interferometry, both linear and nonlinear terms of the electron density fluctuation energy equation can be determined, thereby allowing quantitative investigation of the origin of the density fluctuations. The concept, calibration, and application of differential interferometry are presented.

  5. Differential effects of chronic alcohol administration to rats on the activation of aromatic amines to mutagens in the Ames test.

    Steele, C M; Ioannides, C

    1986-05-01

    Male Wistar albino rats were maintained on alcohol-containing liquid diets for 4 weeks. Hepatic post-mitochondrial preparations derived from these animals were more efficient than control in activating 4-aminobiphenyl and 2-aminofluorene to mutagens in the Ames test. The alcohol-induced enhancement in mutagenicity was not inhibited by dimethylsulphoxide indicating that the generation of hydroxyl radicals is not involved. The activation of 2-naphthylamine was not affected by the treatment with alcohol but the mutagenicities of 2-aminoanthracene, benzo[a]pyrene and 3-methylcholanthrene were inhibited. The same treatment markedly increased hepatic microsomal aniline p-hydroxylase and ethoxyresorufin O-de-ethylase activities and to a lesser extent benzphetamine N-demethylase and microsomal levels of total cytochromes P-450. It is concluded that chronic alcohol administration to rats modulates the metabolic activation of pre-carcinogens to their reactive intermediates presumably by causing the redistribution of cytochrome P-450 isozymes. PMID:3009048

  6. Ultraviolet radiation B induces differentiation and protein kinase C in normal human epidermal keratinocytes

    Mid-wave ultraviolet radiation (UVB, 280-320 nm) is highly efficient at inducing erythema, pyrimidine dimers in DNA, oncogene expression and initiation of cutaneous tumors. These UVB-induced responses of epidermal cells have been correlated with the direct effects of UVB on DNA. However, UVB has also been shown to have biologic effects at the cellular level that appear to mimic some of the membrane-associated effects produced by phorbol ester tumor promoters such as 12-O-tetradecanoyl phorbol-13-acetate (TPA). For example, we have previously shown that both UVB irradiation and TPA treatment are followed by release of arachidonic acid and a rapid, dose-dependent inhibition of epidermal growth factor (EGF) binding. TPA generates cellular responses through activation of a phospholipid-dependent, calcium-sensitive protein kinase, protein kinase C (PKC). The primary goal of the studies described here was to compare the cellular effects of TPA with those of UVB with special regard to PKC and keratinocyte growth control, using normal human epidermal keratinocytes. The results obtained showed that both TPA and UVB radiation induced differentiation in normal human keratinocytes. UVB radiation, however, increased both cytosolic and membrane-associated levels of PKC, in contrast to TPA, which increased PKC primarily in the membrane fraction. PCK is probably not the initial chromophore or target molecule of UVB, but because activation of PKC has been shown to be essential for keratinocyte differentiation, differentiation induced by UVB may be caused by activation of PKC by UVB-induced release of diacylglycerol or arachidonic acid. (au) 25 refs

  7. Uremic Toxins Enhance Statin-Induced Cytotoxicity in Differentiated Human Rhabdomyosarcoma Cells

    Hitoshi Uchiyama

    2014-09-01

    Full Text Available The risk of myopathy and rhabdomyolysis is considerably increased in statin users with end-stage renal failure (ESRF. Uremic toxins, which accumulate in patients with ESRF, exert cytotoxic effects that are mediated by various mechanisms. Therefore, accumulation of uremic toxins might increase statin-induced cytotoxicity. The purpose of this study was to determine the effect of four uremic toxins—hippuric acid, 3-carboxy-4-methyl-5-propyl-2-furanpropionate, indole-3-acetic acid, and 3-indoxyl sulfate—on statin-induced myopathy. Differentiated rhabdomyosarcoma cells were pre-treated with the uremic toxins for seven days, and then the cells were treated with pravastatin or simvastatin. Cell viability and apoptosis were assessed by viability assays and flow cytometry. Pre-treatment with uremic toxins increased statin- but not cisplatin-induced cytotoxicity (p < 0.05 vs. untreated. In addition, the pre-treatment increased statin-induced apoptosis, which is one of the cytotoxic factors (p < 0.05 vs. untreated. However, mevalonate, farnesol, and geranylgeraniol reversed the effects of uremic toxins and lowered statin-induced cytotoxicity (p < 0.05 vs. untreated. These results demonstrate that uremic toxins enhance statin-induced apoptosis and cytotoxicity. The mechanism underlying this effect might be associated with small G-protein geranylgeranylation. In conclusion, the increased severity of statin-induced rhabdomyolysis in patients with ESRF is likely due to the accumulation of uremic toxins.

  8. Corticosteroids reverse cytokine-induced block of survival and differentiation of oligodendrocyte progenitor cells from rats

    Marx Romy

    2008-09-01

    Full Text Available Abstract Background Periventricular leukomalacia (PVL is a frequent complication of preterm delivery. Proinflammatory cytokines, such as interferon-γ (IFN-γ and tumor necrosis factor α (TNF-α released from astrocytes and microglia activated by infection or ischemia have previously been shown to impair survival and maturation of oligodendrocyte progenitors and could thus be considered as potential factors contributing to the generation of this disease. The first goal of the present study was to investigate whether exposure of oligodendrocyte precursors to these cytokines arrests the maturation of ion currents in parallel to its effects on myelin proteins and morphological maturation. Secondly, in the search for agents, that can protect differentiating oligodendrocyte precursor cells from cytokine-induced damage we investigated effects of coapplications of corticosteroids with proinflammatory cytokines on the subsequent survival and differentiation of oligodendrocyte progenitor cells. Methods To exclude influences from factors released from other cell types purified cultures of oligodendrocyte precursors were exposed to cytokines and/or steroids and allowed to differentiate for further 6 days in culture. Changes in membrane surface were investigated with capacitance recordings and Scanning Ion Conductance Microscopy. Na+- and K+- currents were investigated using whole cell patch clamp recordings. The expression of myelin specific proteins was investigated using western blots and the precursor cells were identified using immunostaining with A2B5 antibodies. Results Surviving IFN-γ and TNF-α treated cells continued to maintain voltage-activated Na+- and K+ currents characteristic for the immature cells after 6 days in differentiation medium. Corticosterone, dihydrocorticosterone and, most prominently dexamethasone, counteracted the deleterious effects of IFN-γ and TNF-α on cell survival, A2B5-immunostaining and expression of myelin basic

  9. Activation of JNKs is essential for BMP9-induced osteogenic differentiation of mesenchymal stem cells

    Yan-fang Zhao

    2013-08-01

    Full Text Available Although BMP9 is highly capable of promoting osteogenicdifferentiation of mesenchymal stem cell (MSCs, the molecularmechanism involved remains to be fully elucidated. Here, weexplore the possible involvement and detail role of JNKs (c-JunN-terminal kinases in BMP9-induced osteogenic differentiationof MSCs. It was found that BMP9 stimulated the activation ofJNKs in MSCs. BMP9-induced osteogenic differentiation ofMSCs was dramatically inhibited by JNKs inhibitor SP600125.Moreover, BMP9-activated Smads signaling was decreased bySP600125 treatment in MSCs. The effects of inhibitor arereproduced with adenoviruses expressing siRNA targeted JNKs.Taken together, our results revealed that JNKs was activated inBMP9-induced osteogenic differentiation of MSCs. What ismost noteworthy, however, is that inhibition of JNKs activityresulted in reduction of BMP9-induced osteogenic differentiationof MSCs, implying that activation of JNKs is essential forBMP9 osteoinductive activity. [BMB Reports 2013; 46(8:422-427

  10. Dendritic cells enhance UHMWPE wear particle-induced osteoclast differentiation of macrophages.

    Cang, Dingwei; Guo, Kaijin; Zhao, Fengchao

    2015-10-01

    Ultra-high molecular weight polyethylene (UHMWPE) has been widely used in large joint replacement. Osteolysis induced by the UHMWPE wear particles is one of the main causes of replacement failure. This study aims to elucidate whether dendritic cells play a role in UHMWPE particle-induced osteolysis. An in vitro Raw 264.7 and DC 2.4 coculture system was employed to examine the effects of dendritic cells on the inflammatory and osteoclastogenic responses of Raw 264.7 toward UHMWPE particles. The expression of cytokines, NF-κB, and osteoclast marker genes was analyzed by ELISA, western blot, or quantitative PCR. The osteoclast differentiation was measured by TRAP staining and flow cytometry. UHMWPE particles induced Raw 264.7 cells to differentiate into osteoclasts, which was enhanced by coculturing with DC 2.4 cells. DC 2.4 cells augmented UHMWPE particle-elicited activation of NF-κB signaling, higher levels of TNF-α and MCP-1, and an increased expression of MMP-9, Calcr, and Ctsk, though DC 2.4 coculture alone did not significantly cause the aforementioned changes. These results suggest that dendritic cells, among other immune cells recruited by UHMWPE particle induced inflammation, could further exacerbate inflammation and osteolysis. PMID:25808788

  11. Scoparone attenuates RANKL-induced osteoclastic differentiation through controlling reactive oxygen species production and scavenging

    Lee, Sang-Hyun; Jang, Hae-Dong, E-mail: haedong@hnu.kr

    2015-02-15

    Scoparone, one of the bioactive components of Artemisia capillaris Thunb, has various biological properties including immunosuppressive, hepatoprotective, anti-allergic, anti-inflammatory, and antioxidant effects. This study aims at evaluating the anti-osteoporotic effect of scoparone and its underlying mechanism in vitro. Scoparone demonstrated potent cellular antioxidant capacity. It was also found that scoparone inhibited the receptor activator of nuclear factor-κB ligand (RANKL)-induced osteoclast differentiation and suppressed cathepsin K and tartrate-resistant acid phosphatase (TRAP) expression via c-jun N-terminal kinase (JNK)/extracellular signal-regulated kinase (ERK)/p38-mediated c-Fos–nuclear factor of activated T cells, cytoplasmic 1 (NFATc1) signaling pathway. During osteoclast differentiation, the production of general reactive oxygen species (ROS) and superoxide anions was dose-dependently attenuated by scoparone. In addition, scoparone diminished NADPH (nicotinamide adenine dinucleotide phosphate) oxidase 1 (Nox1) expression and activation via the tumor necrosis factor receptor-associated factor 6 (TRAF6)–cSrc–phosphatidylinositol 3-kinase (PI3k) signaling pathway and prevented the disruption of mitochondrial electron transport chain system. Furthermore, scoparone augmented the expression of superoxide dismutase 1 (SOD1) and catalase (CAT). The overall results indicate that the inhibitory effect of scoparone on RANKL-induced osteoclast differentiation is attributed to the suppressive effect on ROS and superoxide anion production by inhibiting Nox1 expression and activation and protecting the mitochondrial electron transport chain system and the scavenging effect of ROS resulting from elevated SOD1 and CAT expression. - Highlights: • Scoparone dose-dependently inhibited RANKL-induced osteoclast differentiation. • Scoparone diminished general ROS and superoxide anions in a dose-dependent manner. • Scoparone inhibited Nox1 expression and

  12. Dopamine inhibits proliferation, induces differentiation and apoptosis of K562 leukaemia cells

    HE Qun; YUAN Lin-bo

    2007-01-01

    Background Dopamine exerts its effects mainly in nervous system through D1, D2 or D3 receptors. There are few reports dealing with the effects of dopamine on leukaemia cells. However, some dopamine agonists or antagonists do show biological effects on some types of leukaemia cells. Here, we report the effects of dopamine on the proliferation,differentiation and apoptosis of K562 leukaemia cells.Methods Proliferation was determined by MTT assay and cell counting both in liquid and semisolid cultures.Differentiation was verified by morphology, benzidine staining and flow cytometry. Apoptosis was checked by Hoechst 33258 staining and flow cytometry. The two groups were untreated group and treated group (dopamine 10-9 mol/L-10-4mol/L).Results In liquid culture, MTT assay and colony assay, dopamine inhibited proliferation of K562 cells. Inhibition rate was 29.28% at 10-6 mol/L and 36.10% at 10-5 mol/L after culture for 5 days in MTT assay. In benzidine staining and CD71 expression, dopamine induced K562 cells toward erythroid differentiation by increased 155% at 10-6 mol/L and by 171% at 10-5 mol/L after culture for 5 days in benzidine staining. In Hoechst 33258 staining and flow cytometry,dopamine induced K562 cells toward apoptosis. The sub G1 peak stained by PI was 14.23% at 10-4 mol/L dopamine after culture for 3 days compared with the control (0.81%) in flow cytometry.Conclusion Dopamine inhibites proliferation and induces both differentiation and apoptosis of K562 leukaemia cells.

  13. Scoparone attenuates RANKL-induced osteoclastic differentiation through controlling reactive oxygen species production and scavenging

    Scoparone, one of the bioactive components of Artemisia capillaris Thunb, has various biological properties including immunosuppressive, hepatoprotective, anti-allergic, anti-inflammatory, and antioxidant effects. This study aims at evaluating the anti-osteoporotic effect of scoparone and its underlying mechanism in vitro. Scoparone demonstrated potent cellular antioxidant capacity. It was also found that scoparone inhibited the receptor activator of nuclear factor-κB ligand (RANKL)-induced osteoclast differentiation and suppressed cathepsin K and tartrate-resistant acid phosphatase (TRAP) expression via c-jun N-terminal kinase (JNK)/extracellular signal-regulated kinase (ERK)/p38-mediated c-Fos–nuclear factor of activated T cells, cytoplasmic 1 (NFATc1) signaling pathway. During osteoclast differentiation, the production of general reactive oxygen species (ROS) and superoxide anions was dose-dependently attenuated by scoparone. In addition, scoparone diminished NADPH (nicotinamide adenine dinucleotide phosphate) oxidase 1 (Nox1) expression and activation via the tumor necrosis factor receptor-associated factor 6 (TRAF6)–cSrc–phosphatidylinositol 3-kinase (PI3k) signaling pathway and prevented the disruption of mitochondrial electron transport chain system. Furthermore, scoparone augmented the expression of superoxide dismutase 1 (SOD1) and catalase (CAT). The overall results indicate that the inhibitory effect of scoparone on RANKL-induced osteoclast differentiation is attributed to the suppressive effect on ROS and superoxide anion production by inhibiting Nox1 expression and activation and protecting the mitochondrial electron transport chain system and the scavenging effect of ROS resulting from elevated SOD1 and CAT expression. - Highlights: • Scoparone dose-dependently inhibited RANKL-induced osteoclast differentiation. • Scoparone diminished general ROS and superoxide anions in a dose-dependent manner. • Scoparone inhibited Nox1 expression and

  14. Isolation, Culture and Induced Differentiation of Fetal Porcine Islet Derived Pancreatic Stem Cell

    FENG Ruo-peng; ZHANG Hui-ru; WANG Yun; QIAO Hai; ZHAO Ting; SHEN Wen-zheng; DOU Zhong-ying

    2007-01-01

    To isolate and culture the porcine pancreatic stem cells and investigate their function, the fetal porcine pancreatic stem cells were isolated by the method of suspending plus adhering culture. The isolated cells were then identified by irnmunohistochemical staining, and their culture viability measured through the MTT method in vitro. This induced them to differentiate into endocrine cells and detect their function. The isolated IPSCS did not express nestin, but expressed CK-19, a marker of ductal epithelia cells and oc-actin, a smooth muscle marker, demonstrating the growth characteristics of ES-like cells, and strong proliferative ability, after 18 passages. They could excrete insulin, and showed ultrastructure changes after being induced. Porcine pancreatic stem cells can be isolated by this method, induced to form islet-like clusters, and can secret insulin.

  15. ESAT6-induced IFNgamma and CXCL9 can differentiate severity of tuberculosis.

    Zahra Hasan

    Full Text Available BACKGROUND: Protective responses against Mycobacterium tuberculosis are dependent on appropriate T cell and macrophage activation. Mycobacterial antigen six kDa early secreted antigenic target (ESAT6 and culture filtrate protein 10 (CFP10 can detect M. tuberculosis specific IFNgamma responses. However, most studies have been performed in non-endemic regions and to study pulmonary tuberculosis (PTB. We have studied ESAT6 and CFP10 induced cytokine and chemokines responses in PTB and extrapulmonary (EPul TB. METHODOLOGY: IFNgamma, IL10, CXCL9 and CCL2 responses were determined using an ex vivo whole blood assay system in PTB (n = 30 and EPulTB patients with limited (LNTB, n = 24 or severe (SevTB, n = 22 disease, and in healthy endemic controls (ECs. Responses to bacterial LPS were also determined. PRINCIPAL FINDINGS: ESAT6- and CFP10-induced IFNgamma was comparable between ECs and TB patients. Both ESAT6- and CFP10-induced IFNgamma secretion was greater in LNTB than PTB. ESAT6-induced CXCL9 was greater in EPulTB as compared with PTB, with an increase in SevTB as compared with LNTB. CFP10-induced CCL2 was higher in PTB than LNTB patients. LPS-stimulated CXCL9 was greatest in SevTB and LPS-induced CCL2 was increased in PTB as compared with LNTB patients. A positive correlation between ESAT6-induced IFNgamma and CXCL9 was present in all TB patients, but IFNgamma and CCL2 was only correlated in LNTB. ESAT-induced CCL2 and CXCL9 were significantly associated in LNTB while correlation in response to LPS was only present in SevTB. CONCLUSIONS: ESAT6 induced IFNgamma and CXCL9 can differentiate between limited and severe TB infections.

  16. Differential microRNA expression in aristolochic acid-induced upper urothelial tract cancers ex vivo.

    Tao, Le; Zeng, Yigang; Wang, Jun; Liu, Zhihong; Shen, Bing; Ge, Jifu; Liu, Yong; Guo, Yifeng; Qiu, Jianxin

    2015-11-01

    Aristolochic acid (AA) is a carcinogenic, mutagenic and nephrotoxic compound commonly isolated from members of the plant family of Aristolochiaceae (such as Aristolochia and Asarum) and used in Chinese herbal medicine. Use of AA and AA‑containing plants causes chronic kidney disease (CKD) and upper urinary tract carcinoma (UUC); however, the underlying mechanism remains to be defined. miRNAs regulate a number of biological processes, including cell proliferation, differentiation and metabolism. This study explored differentially expressed miRNAs between AA‑induced upper urothelial tract cancer (AAN‑UUC) and non‑AAN‑UUC tissues. Patients with AAN‑UUC and non‑AAN‑UUC (n=20/group) were recruited in the present study. Five tissue samples from each group were used for miRNA microarray profiling and the rest of the tissue samples were subjected to reverse transcription-quantitative polymerase chain reaction analysis including seven selected miRNAs for confirmation. A total of 29 miRNAs were differentially expressed between AAN‑UUC and non‑AAN‑UUC tissues (Pontology analyses predicted the functions and targeted genes of these differentially expressed miRNAs, i.e. Akt3, FGFR3, PSEN1, VEGFa and AR. Subsequently, expression of the selected differentially expressed miRNAs (Hsa‑miR‑4795‑5p, Hsa‑miR‑488, Hsa‑miR‑4784, Hsa‑miR‑330, Hsa‑miR‑3916, Hsa‑miR‑4274 and Hsa‑miR‑181c) was validated in another set of tissue samples. A total of 29 miRNAs were identified to be differentially expressed between AAN‑UUC and non‑AAN‑UUC tissues and these miRNA target genes in FGFR3 and Akt pathways, which regulate cell growth and tumor progression, respectively. PMID:26397152

  17. A hybrid microfluidic system for regulation of neural differentiation in induced pluripotent stem cells.

    Hesari, Zahra; Soleimani, Massoud; Atyabi, Fatemeh; Sharifdini, Meysam; Nadri, Samad; Warkiani, Majid Ebrahimi; Zare, Mehrak; Dinarvand, Rassoul

    2016-06-01

    Controlling cellular orientation, proliferation, and differentiation is valuable in designing organ replacements and directing tissue regeneration. In the present study, we developed a hybrid microfluidic system to produce a dynamic microenvironment by placing aligned PDMS microgrooves on surface of biodegradable polymers as physical guidance cues for controlling the neural differentiation of human induced pluripotent stem cells (hiPSCs). The neuronal differentiation capacity of cultured hiPSCs in the microfluidic system and other control groups was investigated using quantitative real time PCR (qPCR) and immunocytochemistry. The functionally of differentiated hiPSCs inside hybrid system's scaffolds was also evaluated on the rat hemisected spinal cord in acute phase. Implanted cell's fate was examined using tissue freeze section and the functional recovery was evaluated according to the Basso, Beattie, and Bresnahan (BBB) locomotor rating scale. Our results confirmed the differentiation of hiPSCs to neuronal cells on the microfluidic device where the expression of neuronal-specific genes was significantly higher compared to those cultured on the other systems such as plain tissue culture dishes and scaffolds without fluidic channels. Although survival and integration of implanted hiPSCs did not lead to a significant functional recovery, we believe that combination of fluidic channels with nanofiber scaffolds provides a great microenvironment for neural tissue engineering, and can be used as a powerful tool for in situ monitoring of differentiation potential of various kinds of stem cells. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 104A: 1534-1543, 2016. PMID:26914600

  18. Effect of amorphous silica nanoparticles on in vitro RANKL-induced osteoclast differentiation in murine macrophages

    Nagano Kazuya

    2011-01-01

    Full Text Available Abstract Amorphous silica nanoparticles (nSP have been used as a polishing agent and/or as a remineralization promoter for teeth in the oral care field. The present study investigates the effects of nSP on osteoclast differentiation and the relationship between particle size and these effects. Our results revealed that nSP exerted higher cytotoxicity in macrophage cells compared with submicron-sized silica particles. However, tartrate-resistant acid phosphatase (TRAP activity and the number of osteoclast cells (TRAP-positive multinucleated cells were not changed by nSP treatment in the presence of receptor activator of nuclear factor κB ligand (RANKL at doses that did not induce cytotoxicity by silica particles. These results indicated that nSP did not cause differentiation of osteoclasts. Collectively, the results suggested that nanosilica exerts no effect on RANKL-induced osteoclast differentiation of RAW264.7 cells, although a detailed mechanistic examination of the nSP70-mediated cytotoxic effect is needed.

  19. TBTC induces adipocyte differentiation in human bone marrow long term culture

    Organotins are widely used in agriculture and the chemical industry, causing persistent and widespread pollution. Organotins may affect the brain, liver and immune system and eventually human health. Recently, it has been shown that tri-butyltin (TBT) interacts with nuclear receptors PPARγ (peroxisome proliferator-activated receptor γ) and RXR (retinoid x receptor) leading to adipocyte differentiation in the 3T3 cell line. Since adipocytes are known to influence haematopoiesis, for instance through the expression of cytokines and adhesion molecules, it was considered of interest to further study the adipocyte-stimulating effect of TBTC in human bone marrow cultures. Nile Red spectrofluorimetric analysis showed a significant increase of adipocytes in TBTC-treated cultures after 14 days of long term culture. Real-time PCR and Western blot analysis confirmed the high expression of the specific adipocyte differentiation marker aP2 (adipocyte-specific fatty acid binding protein). PPARγ, but not RXR, mRNA was increased after 24 h and 48 h exposure. TBTC also induced a decrease in a number of chemokines, interleukins, and growth factors. Also the expression of leptin, a hormone involved in haematopoiesis, was down regulated by TBTC treatment. It therefore appears that TBTC induced adipocyte differentiation, whilst reducing a number of haematopoietic factors. This study indicates that TBTC may interfere in the haematopoietic process through an alteration of the stromal layer and cytokine homeostasis

  20. Statins activate GATA-6 and induce differentiated vascular smooth muscle cells

    The beneficial effects of 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors (statins) beyond cholesterol lowering involve their direct actions on vascular smooth muscle cells (VSMCs). However, the effects of statins on phenotypic modulation of VSMCs are unknown. We herein show that simvastatin (Sm) and atorvastatin (At) inhibited DNA synthesis in human aortic VSMCs dose-dependently, while cell toxicity was not observed below the concentration of 1 μM of Sm or 100 nM of At. Stimulating proliferative VSMCs with Sm or At induced the expression of SM-α-actin and SM-MHC, highly specific markers of differentiated phenotype. Sm up-regulated the binding activity of GATA-6 to SM-MHC GATA site and activated the transfected SM-MHC promoter in proliferative VSMCs, while mutating the GATA-6 binding site abolished this activation. Geranylgeranylpyrophosphate (10 μM), an inhibitor of Rho family proteins, abolished the statin-mediated induction of the differentiated phenotype in VSMCs. These findings suggest that statins activate GATA-6 and induce differentiated VSMCs

  1. Paracrine sonic hedgehog signalling by prostate cancer cells induces osteoblast differentiation

    Iannaccone Philip

    2009-03-01

    Full Text Available Abstract Background Sonic hedgehog (Shh and components of its signalling pathway have been identified in human prostate carcinoma and increased levels of their expression appear to correlate with disease progression and metastasis. The mechanism through which Shh signalling could promote metastasis in bone, the most common site for prostate carcinoma metastasis, has not yet been investigated. The present study determined the effect of Shh signalling between prostate cancer cells and pre-osteoblasts on osteoblast differentiation, a requisite process for new bone formation that characterizes prostate carcinoma metastasis. Results LNCaP human prostate cancer cells modified to overexpress Shh (designated LNShh cells and MC3T3 mouse pre-osteoblasts were maintained as mixed populations within the same culture chamber. In this non-conventional mixed culture system, LNShh cells upregulated the expression of Shh target genes Gli1 and Patched 1 (Ptc1 in MC3T3 cells and this was inhibited by cyclopamine, a specific chemical inhibitor of hedgehog signalling. Concomitantly, MC3T3 cells exhibited time-dependent decreased cell proliferation, upregulated alkaline phosphatase Akp2 gene expression, and increased alkaline phosphatase activity indicative of early phase osteoblast differentiation. LNShh cell-induced differentiation was inhibited in MC3T3 cells stably transfected with a dominant negative form of Gli1, a transcription factor that mediates Shh signalling. Interestingly, LNShh cells did not significantly increase the endogenous expression of the osteoblast differentiation transcription factor Runx2 and its target genes osteocalcin and osteopontin. Consistent with these results, exogenous Shh peptide did not upregulate Runx2 expression in MC3T3 cells. However, Runx2 levels were increased in MC3T3 cells by ascorbic acid, a known stimulator of osteoblast differentiation. Conclusion Altogether, these data demonstrate that Shh-expressing prostate cancer

  2. Structurally distinct polycyclic aromatic hydrocarbons induce differential transcriptional responses in developing zebrafish

    Polycyclic aromatic hydrocarbons (PAHs) are ubiquitous in the environment as components of fossil fuels and by-products of combustion. These multi-ring chemicals differentially activate the aryl hydrocarbon receptor (AHR) in a structurally dependent manner, and induce toxicity via both AHR-dependent and -independent mechanisms. PAH exposure is known to induce developmental malformations in zebrafish embryos, and recent studies have shown cardiac toxicity induced by compounds with low AHR affinity. Unraveling the potentially diverse molecular mechanisms of PAH toxicity is essential for understanding the hazard posed by complex PAH mixtures present in the environment. We analyzed transcriptional responses to PAH exposure in zebrafish embryos exposed to benz(a)anthracene (BAA), dibenzothiophene (DBT) and pyrene (PYR) at concentrations that induced developmental malformations by 120 h post-fertilization (hpf). Whole genome microarray analysis of mRNA expression at 24 and 48 hpf identified genes that were differentially regulated over time and in response to the three PAH structures. PAH body burdens were analyzed at both time points using GC–MS, and demonstrated differences in PAH uptake into the embryos. This was important for discerning dose-related differences from those that represented unique molecular mechanisms. While BAA misregulated the least number of transcripts, it caused strong induction of cyp1a and other genes known to be downstream of the AHR, which were not induced by the other two PAHs. Analysis of functional roles of misregulated genes and their predicted regulatory transcription factors also distinguished the BAA response from regulatory networks disrupted by DBT and PYR exposure. These results indicate that systems approaches can be used to classify the toxicity of PAHs based on the networks perturbed following exposure, and may provide a path for unraveling the toxicity of complex PAH mixtures. - Highlights: • Defined global mRNA expression

  3. Structurally distinct polycyclic aromatic hydrocarbons induce differential transcriptional responses in developing zebrafish

    Goodale, Britton C. [Department of Environmental and Molecular Toxicology, The Environmental Health Sciences Center, Oregon State University, Corvallis, OR (United States); Tilton, Susan C. [Computational Biology and Bioinformatics, Pacific Northwest National Laboratory (United States); Corvi, Margaret M.; Wilson, Glenn R. [Department of Environmental and Molecular Toxicology, The Environmental Health Sciences Center, Oregon State University, Corvallis, OR (United States); Janszen, Derek B. [Computational Biology and Bioinformatics, Pacific Northwest National Laboratory (United States); Anderson, Kim A. [Department of Environmental and Molecular Toxicology, The Environmental Health Sciences Center, Oregon State University, Corvallis, OR (United States); Waters, Katrina M. [Computational Biology and Bioinformatics, Pacific Northwest National Laboratory (United States); Tanguay, Robert L., E-mail: tanguay.robert@oregonstate.edu [Department of Environmental and Molecular Toxicology, The Environmental Health Sciences Center, Oregon State University, Corvallis, OR (United States)

    2013-11-01

    Polycyclic aromatic hydrocarbons (PAHs) are ubiquitous in the environment as components of fossil fuels and by-products of combustion. These multi-ring chemicals differentially activate the aryl hydrocarbon receptor (AHR) in a structurally dependent manner, and induce toxicity via both AHR-dependent and -independent mechanisms. PAH exposure is known to induce developmental malformations in zebrafish embryos, and recent studies have shown cardiac toxicity induced by compounds with low AHR affinity. Unraveling the potentially diverse molecular mechanisms of PAH toxicity is essential for understanding the hazard posed by complex PAH mixtures present in the environment. We analyzed transcriptional responses to PAH exposure in zebrafish embryos exposed to benz(a)anthracene (BAA), dibenzothiophene (DBT) and pyrene (PYR) at concentrations that induced developmental malformations by 120 h post-fertilization (hpf). Whole genome microarray analysis of mRNA expression at 24 and 48 hpf identified genes that were differentially regulated over time and in response to the three PAH structures. PAH body burdens were analyzed at both time points using GC–MS, and demonstrated differences in PAH uptake into the embryos. This was important for discerning dose-related differences from those that represented unique molecular mechanisms. While BAA misregulated the least number of transcripts, it caused strong induction of cyp1a and other genes known to be downstream of the AHR, which were not induced by the other two PAHs. Analysis of functional roles of misregulated genes and their predicted regulatory transcription factors also distinguished the BAA response from regulatory networks disrupted by DBT and PYR exposure. These results indicate that systems approaches can be used to classify the toxicity of PAHs based on the networks perturbed following exposure, and may provide a path for unraveling the toxicity of complex PAH mixtures. - Highlights: • Defined global mRNA expression

  4. Inhibition of protein kinase C induces differentiation in Neuro-2a cells

    1-(5-Isoquinolinylsulfonyl)-2-methylpiperazine (H7), a potent inhibitor of protein kinase C, induced neuritogenesis in Neuro-2a cells, whereas N-(2-guanidinoethyl)-5-isoquinolinesulfonamide (HA 1004), which inhibits more efficiently cAMP- and cGMP-dependent protein kinases, did not. The effect, noticeable after 3 hr, was maximum (13-fold increase at 500 μM H7) between 1 and 3 days and was maintained over 2 months. In controls, 90% of the cells were undifferentiated, whereas after 3 hr with 500 μM H7 only 25% of the cells remained undifferentiated. DNA synthesis decreased as the number of differentiated cells increased. Differentiation is also functional since acetylcholinesterase activity increased ∼7-fold after 48 hr with 500 μM H7. Phorbol 12-myristate 13-acetate, a specific activator of protein kinase C, prevented or reversed the induction of neuritogenesis and the inhibition of DNA synthesis by H7. There is a good correlation between the level of protein kinase C and the percentage of differentiated cells. The results indicate that protein kinase C may play a key role in the control of differentiation of neural cells. Some possible clinical implications are briefly discussed

  5. Bystander-induced apoptosis and premature differentiation in primary urothelial explants after charged particle microbeam irradiation

    The ureter primary explant technique was developed to study bystander effects under in vivo like conditions where stem and differentiated cells are present. Irradiation was performed with a 3He2+ charged particle microbeam available at the Gray Cancer Institute, with high (∼2 μm) precision. Tissue sections from porcine ureters were pre-irradiated with the microbeam at a single location with 103He2+ particles (5 MeV; LET 70 keV.μm-1). After irradiation, the tissue section was incubated for 7 days, thus allowing the explant outgrowth to form. Total cellular damage (total fraction of micronucleated and apoptotic cells) was measured according to morphological criteria. Apoptosis was also assessed using a 3'-OH DNA end-labelling technique. Premature differentiation was estimated using antibodies to uroplakin III, a specific marker of terminal urothelial differentiation. Results of our experiments demonstrated a significant bystander-induced differentiation and a less significant increase in apoptotic and micronucleated cells. A hypothesis based on the protective nature of the bystander effect is proposed. (author)

  6. Inhibition of protein kinase C induces differentiation in Neuro-2a cells.

    Miñana, M D; Felipo, V; Grisolía, S

    1990-01-01

    1-(5-Isoquinolinylsulfonyl)-2-methylpiperazine (H7), a potent inhibitor of protein kinase C, induced neuritogenesis in Neuro-2a cells, whereas N-(2-guanidinoethyl)-5-isoquinolinesulfonamide (HA 1004), which inhibits more efficiently cAMP- and cGMP-dependent protein kinases, did not. The effect, noticeable after 3 hr, was maximum (13-fold increase at 500 microM H7) between 1 and 3 days and was maintained over 2 months. In controls, 90% of the cells were undifferentiated, whereas after 3 hr with 500 microM H7 only 25% of the cells remained undifferentiated. DNA synthesis decreased as the number of differentiated cells increased. Differentiation is also functional since acetylcholinesterase activity increased approximately 7-fold after 48 hr with 500 microM H7. Phorbol 12-myristate 13-acetate, a specific activator of protein kinase C, prevented or reversed the induction of neuritogenesis and the inhibition of DNA synthesis by H7. There is a good correlation between the level of protein kinase C and the percentage of differentiated cells. The results indicate that protein kinase C may play a key role in the control of differentiation of neural cells. Some possible clinical implications are briefly discussed. Images PMID:1693437

  7. Inhibition of protein kinase C induces differentiation in Neuro-2a cells

    Minana, M.D.; Felipo, V.; Grisolia, S. (Instituto de Investigaciones Citologicas de la Caja de Ahorros de Valencia (Spain))

    1990-06-01

    1-(5-Isoquinolinylsulfonyl)-2-methylpiperazine (H7), a potent inhibitor of protein kinase C, induced neuritogenesis in Neuro-2a cells, whereas N-(2-guanidinoethyl)-5-isoquinolinesulfonamide (HA 1004), which inhibits more efficiently cAMP- and cGMP-dependent protein kinases, did not. The effect, noticeable after 3 hr, was maximum (13-fold increase at 500 {mu}M H7) between 1 and 3 days and was maintained over 2 months. In controls, 90% of the cells were undifferentiated, whereas after 3 hr with 500 {mu}M H7 only 25% of the cells remained undifferentiated. DNA synthesis decreased as the number of differentiated cells increased. Differentiation is also functional since acetylcholinesterase activity increased {approx}7-fold after 48 hr with 500 {mu}M H7. Phorbol 12-myristate 13-acetate, a specific activator of protein kinase C, prevented or reversed the induction of neuritogenesis and the inhibition of DNA synthesis by H7. There is a good correlation between the level of protein kinase C and the percentage of differentiated cells. The results indicate that protein kinase C may play a key role in the control of differentiation of neural cells. Some possible clinical implications are briefly discussed.

  8. Influence of suppressor gene p16 on retinoic acid inducing cancer cell A549 differentiation

    2001-01-01

    Objective To investigate the role of suppressor gene p16 in the process of differential regulation of retinoic acid (RA) on the A549 lung cancer cells.Methods Tumor suppressor gene p16 was transferred into A549 cells and the cells were treated with all-trans retinoic acid (ATR) at the dosage of 5×10-6 mol/L for 4 d. After that, the proliferation and differentiation of A549 cells were examined by growth curve and cytometry analysis, the change of lung lineage-specific marker MUC1 was tested by immunohistochemical staining. Meanwhile, Western blot was used to observe the change of p16 protein expression in A549 cells treated with ATRA.Results ATRA could obviously inhibit the growth and induce the differentiation of A549 Cells that were transferred with p16 gene. There were more cells arrested in G1/G0 phase and the expression of MUG1 was markedly down-regulated than in control cells. The expression of p16 protein was up-regulated in A549 cells treated with ATRA.Conclusion Suppressor gene p16 could enhance the effects of RA and proliferated suppression and differential induction of A549 cells.

  9. Algal Toxin Azaspiracid-1 Induces Early Neuronal Differentiation and Alters Peripherin Isoform Stoichiometry

    Linda V. Hjørnevik

    2015-12-01

    Full Text Available Azaspiracid-1 is an algal toxin that accumulates in edible mussels, and ingestion may result in human illness as manifested by vomiting and diarrhoea. When injected into mice, it causes neurotoxicological symptoms and death. Although it is well known that azaspiracid-1 is toxic to most cells and cell lines, little is known about its biological target(s. A rat PC12 cell line, commonly used as a model for the peripheral nervous system, was used to study the neurotoxicological effects of azaspiracid-1. Azaspiracid-1 induced differentiation-related morphological changes followed by a latter cell death. The differentiated phenotype showed peripherin-labelled neurite-like processes simultaneously as a specific isoform of peripherin was down-regulated. The precise mechanism behind this down-regulation remains uncertain. However, this study provides new insights into the neurological effects of azaspiracid-1 and into the biological significance of specific isoforms of peripherin.

  10. The nucleus of differentiated root plant cells: modifications induced by arbuscular mycorrhizal fungi

    G Lingua

    2009-12-01

    Full Text Available The nuclei of plant cells show marked differences in chromatin organisation, related to their DNA content, which ranges from the type with large strands of condensed chromatin (reticulate or chromonematic nuclei to one with mostly decondensed chromatin (chromocentric or diffuse nuclei. A loosening of the chromatin structure generally occurs in actively metabolising cells, such as differentiating and secretory cells, in relation to their high transcriptional activity. Endoreduplication may occur, especially in plants with a small genome, which increases the availability of nuclear templates, the synthesis of DNA, and probably regulates gene expression. Here we describe structural and quantitative changes of the chromatin and their relationship with transcription that occur in differentiated cells following an increase of their metabolism. The nuclei of root cortical cells of three plants with different 2C DNA content (Allium porrum, Pisum sativum and Lycopersicon esculentm and their modifications induced by arbuscular mycorrhization, which strongly increase the metabolic activity of colonised cells, are taken as examples.

  11. Advances of imaging on differential diagnosis between recurrence of glioma and radiation-induced brain injury

    Differentiating recurrence of glioma from radiation-induced brain injury is a central challenge in neuro-oncology. The 2 very different outcomes after brain tumor treatment often appear similar on traditional imaging studies. They may even manifest with similar clinical symptoms. Distinguishing treatment injury from tumor recurrence is crucial for diagnosis and treatment planning. In this article, we reviewed the latest developments and key findings from research studies exploring the efficacy of structural and functional imaging modalities in differentiating treatment injury from tumor recurrence with DWI, MRS, DCE-MR, DSC-MR, PET, and SPECT. And we discussed the advantages and disadvantages of each approach to provide useful information for making proper diagnosis and treatment planning. (authors)

  12. Metabolism of arachidonic acid in phorbol ester, interferon and dimethyl sulfoxide differentiation induced U937 cells

    U937, a human macrophage cell line can metabolize arachidonic acid to a prostaglandin E2-like substance, and an unidentified lipoxygenase product. This metabolism occurs at very low levels however since these cells have low lipase and fatty acid oxygenase activities. The investigated the appearance of these enzyme activities during differentiation induced by phorbol-12-myristate-13-acetate (PMA), human gamma interferon (INF), and dimethyl sulfoxide (DMSO) on days 1,3 and 5 of stimulation using 3H-arachidonic acid (3H-AA). Culture supernatants were analyzed for free 3H-AA and 3H metabolites by radio-thin layer chromatography (3H-MET). The increasing percentage of 3H-AA release suggests the appearance of phospholipase activity during differentiation

  13. The prelamin A pre-peptide induces cardiac and skeletal myoblast differentiation

    Prelamin A processing is unique amongst mammalian proteins and results in the production of a farnesylated and carboxymethylated peptide. We examined the effect of pathogenic LMNA mutations on prelamin A processing, and of the covalently modified peptide on cardiac and skeletal myoblast differentiation. Here we report a mutation associated with dilated cardiomyopathy prevents prelamin A peptide production. In addition, topical application of the covalently modified C-terminal peptide to proliferating skeletal and cardiac myoblasts induced myotube and striated tissue formation, respectively. Western blot analysis revealed that skeletal and cardiac myoblasts are the first cell lines examined to contain unprocessed prelamin A, and immunostaining of peptide-treated cells revealed a previously unidentified role for prelamin A in cytoskeleton formation and intercellular organization. These results demonstrate a direct role for prelamin A in myoblast differentiation and indicate the prelamin A peptide may have therapeutic potential

  14. Preadipocyte factor 1 induces pancreatic ductal cell differentiation into insulin-producing cells.

    Rhee, Marie; Lee, Seung-Hwan; Kim, Ji-Won; Ham, Dong-Sik; Park, Heon-Seok; Yang, Hae Kyung; Shin, Ju-Young; Cho, Jae-Hyoung; Kim, Young-Bum; Youn, Byung-Soo; Sul, Hei Sook; Yoon, Kun-Ho

    2016-01-01

    The preadipocyte factor 1 (Pref-1) is involved in the proliferation and differentiation of various precursor cells. However, the intracellular signaling pathways that control these processes and the role of Pref-1 in the pancreas remain poorly understood. Here, we showed that Pref-1 induces insulin synthesis and secretion via two independent pathways. The overexpression of Pref-1 activated MAPK signaling, which induced nucleocytoplasmic translocation of FOXO1 and PDX1 and led to the differentiation of human pancreatic ductal cells into β-like cells and an increase in insulin synthesis. Concurrently, Pref-1 activated Akt signaling and facilitated insulin secretion. A proteomics analysis identified the Rab43 GTPase-activating protein as a downstream target of Akt. A serial activation of both proteins induced various granular protein syntheses which led to enhanced glucose-stimulated insulin secretion. In a pancreatectomised diabetic animal model, exogenous Pref-1 improved glucose homeostasis by accelerating pancreatic ductal and β-cell regeneration after injury. These data establish a novel role for Pref-1, opening the possibility of applying this molecule to the treatment of diabetes. PMID:27044861

  15. STRUCTURAL REMODELING OF PROTEOGLYCANS UPON RETINOIC ACID-INDUCED DIFFERENTIATION OF NCCIT CELLS*

    Gasimli, Leyla; Stansfield, Hope E.; Nairn, Alison V.; Liu, Haiying; Paluh, Janet L.; Yang, Bo; Dordick, Jonathan S.; Moremen, Kelley W.; Linhardt, Robert J.

    2012-01-01

    Pluripotent and multipotent cells become increasingly lineage restricted through differentiation. Alterations to the cellular proteoglycan composition and structure should accompany these changes to influence cell proliferation, delineation of tissues and acquisition of cell migration capabilities. Retinoic acid plays an important role in pre-patterning of the early embryo. Retinoic acid can be used in vitro to induce differentiation, causing pluripotent and multipotent cells to become increasingly lineage restricted. We examined retinoic acid-induced changes in the cellular proteoglycan composition of the well-characterized teratocarcinoma line NCCIT. Our analysis revealed changes in the abundance of transcripts for genes encoding core proteins, enzymes that are responsible for early and late linkage region biosynthesis, as well as enzymes for GAG chain extension and modification. Transcript levels for genes encoding core proteins used as backbones for polysaccharide synthesis revealed highly significant increases in expression of lumican and decorin, 1500-fold and 2800-fold, respectively. Similarly, glypican 3, glypican 5, versican and glypican 6 showed increases between 5 and 70-fold. Significant decreases in biglycan, serglycin, glypican 4, aggrecan, neurocan, CD74 and glypican 1 were observed. Disaccharide analysis of the glycans in heparin/heparan sulfate and chondroitin/dermatan sulfate revealed retinoic acid-induced changes restricted to chondroitin/dermatan sulfate glycans. Our study provides the first detailed analysis of changes in the glycosaminoglycan profile of human pluripotent cells upon treatment with the retinoic acid morphogen. PMID:23053635

  16. Structural remodeling of proteoglycans upon retinoic acid-induced differentiation of NCCIT cells.

    Gasimli, Leyla; Stansfield, Hope E; Nairn, Alison V; Liu, Haiying; Paluh, Janet L; Yang, Bo; Dordick, Jonathan S; Moremen, Kelley W; Linhardt, Robert J

    2013-07-01

    Pluripotent and multipotent cells become increasingly lineage restricted through differentiation. Alterations to the cellular proteoglycan composition and structure should accompany these changes to influence cell proliferation, delineation of tissues and acquisition of cell migration capabilities. Retinoic acid plays an important role in pre-patterning of the early embryo. Retinoic acid can be used in vitro to induce differentiation, causing pluripotent and multipotent cells to become increasingly lineage restricted. We examined retinoic acid-induced changes in the cellular proteoglycan composition of the well-characterized teratocarcinoma line NCCIT. Our analysis revealed changes in the abundance of transcripts for genes encoding core proteins, enzymes that are responsible for early and late linkage region biosynthesis, as well as enzymes for GAG chain extension and modification. Transcript levels for genes encoding core proteins used as backbones for polysaccharide synthesis revealed highly significant increases in expression of lumican and decorin, 1,500-fold and 2,800-fold, respectively. Similarly, glypican 3, glypican 5, versican and glypican 6 showed increases between 5 and 70-fold. Significant decreases in biglycan, serglycin, glypican 4, aggrecan, neurocan, CD74 and glypican 1 were observed. Disaccharide analysis of the glycans in heparin/heparan sulfate and chondroitin/dermatan sulfate revealed retinoic acid-induced changes restricted to chondroitin/dermatan sulfate glycans. Our study provides the first detailed analysis of changes in the glycosaminoglycan profile of human pluripotent cells upon treatment with the retinoic acid morphogen. PMID:23053635

  17. Proteomics unveil corticoid-induced S100A11 shuttling in keratinocyte differentiation

    Unlike classical protein extraction techniques, proteomic mapping using a selective subcellular extraction kit revealed S100A11 as a new member of the S100 protein family modulated by glucocorticoids in keratinocytes. Glucocorticoids (GC)-induced S100A11 redistribution in the 'organelles and membranes' compartment. Microscopic examination indicated that glucocorticoids specifically routed cytoplasmic S100A11 toward perinuclear compartment. Calcium, a key component of skin terminal differentiation, directed S100A11 to the plasma membrane as previously reported. When calcium was added to glucocorticoids, minor change was observed at the proteomic level while confocal microscopy revealed a rapid and dramatic translocation of S100A11 toward plasma membrane. This effect was accompanied by strong nuclear condensation, loss of mitochondrial potential and DNA content, and increased high molecular weight S100A11 immunoreactivity, suggesting corticoids accelerate calcium-induced terminal differentiation. Finally, our results suggest GC-induced S100A11 relocalization could be a key step in both keratinocyte homeostasis and glucocorticoids side effects in human epidermis

  18. Using delay differential equations to induce alternans in a model of cardiac electrophysiology.

    Eastman, Justin; Sass, Julian; Gomes, Johnny M; Dos Santos, Rodrigo Weber; Cherry, Elizabeth M

    2016-09-01

    Cardiac electrical alternans is a period-2 dynamical behavior with alternating long and short action potential durations (APD) that often precedes dangerous arrhythmias associated with cardiac arrest. Despite the importance of alternans, many current ordinary differential equations models of cardiac electrophysiology do not produce alternans, thereby limiting the use of these models for studying the mechanisms that underlie this condition. Because delay differential equations (DDEs) commonly induce complex dynamics in other biological systems, we investigate whether incorporating DDEs can lead to alternans development in cardiac models by studying the Fox et al. canine ventricular action potential model. After suppressing the alternans in the original model, we show that alternans can be obtained by introducing DDEs in the model gating variables, and we quantitatively compare the DDE-induced alternans with the alternans present in the original model. We analyze the behavior of the voltage, currents, and gating variables of the model to study the effects of the delays and to determine how alternans develops in that setting, and we discuss the mathematical and physiological implications of our findings. In future work, we aim to apply our approach to induce alternans in models that do not naturally exhibit such dynamics. PMID:27302910

  19. Structurally Distinct Polycyclic Aromatic Hydrocarbons Induce Differential Transcriptional Responses in Developing Zebrafish

    Goodale, Britton; Tilton, Susan C.; Corvi, Margaret M.; Wilson, Glenn V.; Janszen, Derek B.; Anderson, Kim A.; Waters, Katrina M.; Tanguay, Robert

    2013-11-01

    Polycyclic aromatic hydrocarbons (PAHs) are ubiquitous in the environment as components of fossil fuels and by-products of combustion. These multi-ring chemicals differentially activate the aryl hydrocarbon receptor (AHR) in a structurally dependent manner, and induce toxicity via both AHR-dependent and -independent mechanisms. PAH exposure is known to induce developmental malformations in zebrafish embryos, and recent studies have shown cardiac toxicity induced by compounds with low AHR affinity. Unraveling the potentially diverse molecular mechanisms of PAH toxicity is essential for understanding the hazard posed by complex PAH mixtures present in the environment. We analyzed transcriptional responses to PAH exposure in zebrafish embryos exposed to benz(a)anthracene (BAA), dibenzothiophene (DBT) and pyrene (PYR) at concentrations that induced developmental malformations by 120 h post-fertilization (hpf). Whole genome microarray analysis of mRNA expression at 24 and 48 hpf identified genes that were differentially regulated over time and in response to the three PAH structures. PAH body burdens were analyzed at both time points using GC-MS, and demonstrated differences in PAH uptake into the embryos. This was important for discerning dose-related differences from those that represented unique molecular mechanisms. While BAA misregulated the least number of transcripts, it caused strong induction of cyp1a and other genes known to be downstream of the AHR, which were not induced by the other two PAHs. Analysis of functional roles of misregulated genes and their predicted regulatory transcription factors also distinguished the BAA response from regulatory networks disrupted by DBT and PYR exposure. These results indicate that systems approaches can be used to classify the toxicity of PAHs based on the networks perturbed following exposure, and may provide a path for unraveling the toxicity of complex PAH mixtures.

  20. Creating an Animal Model of Tendinopathy by Inducing Chondrogenic Differentiation with Kartogenin.

    Ting Yuan

    Full Text Available Previous animal studies have shown that long term rat treadmill running induces over-use tendinopathy, which manifests as proteoglycan accumulation and chondrocytes-like cells within the affected tendons. Creating this animal model of tendinopathy by long term treadmill running is however time-consuming, costly and may vary among animals. In this study, we used a new approach to develop an animal model of tendinopathy using kartogenin (KGN, a bio-compound that can stimulate endogenous stem/progenitor cells to differentiate into chondrocytes. KGN-beads were fabricated and implanted into rat Achilles tendons. Five weeks after implantation, chondrocytes and proteoglycan accumulation were found at the KGN implanted site. Vascularity as well as disorganization in collagen fibers were also present in the same site along with increased expression of the chondrocyte specific marker, collagen type II (Col. II. In vitro studies confirmed that KGN was released continuously from KGN-alginate in vivo beads and induced chondrogenic differentiation of tendon stem/progenitor cells (TSCs suggesting that chondrogenesis after KGN-bead implantation into the rat tendons is likely due to the aberrant differentiation of TSCs into chondrocytes. Taken together, our results showed that KGN-alginate beads can be used to create a rat model of tendinopathy, which, at least in part, reproduces the features of over-use tendinopathy model created by long term treadmill running. This model is mechanistic (stem cell differentiation, highly reproducible and precise in creating localized tendinopathic lesions. It is expected that this model will be useful to evaluate the effects of various topical treatments such as NSAIDs and platelet-rich plasma (PRP for the treatment of tendinopathy.

  1. Creating an Animal Model of Tendinopathy by Inducing Chondrogenic Differentiation with Kartogenin.

    Yuan, Ting; Zhang, Jianying; Zhao, Guangyi; Zhou, Yiqin; Zhang, Chang-Qing; Wang, James H-C

    2016-01-01

    Previous animal studies have shown that long term rat treadmill running induces over-use tendinopathy, which manifests as proteoglycan accumulation and chondrocytes-like cells within the affected tendons. Creating this animal model of tendinopathy by long term treadmill running is however time-consuming, costly and may vary among animals. In this study, we used a new approach to develop an animal model of tendinopathy using kartogenin (KGN), a bio-compound that can stimulate endogenous stem/progenitor cells to differentiate into chondrocytes. KGN-beads were fabricated and implanted into rat Achilles tendons. Five weeks after implantation, chondrocytes and proteoglycan accumulation were found at the KGN implanted site. Vascularity as well as disorganization in collagen fibers were also present in the same site along with increased expression of the chondrocyte specific marker, collagen type II (Col. II). In vitro studies confirmed that KGN was released continuously from KGN-alginate in vivo beads and induced chondrogenic differentiation of tendon stem/progenitor cells (TSCs) suggesting that chondrogenesis after KGN-bead implantation into the rat tendons is likely due to the aberrant differentiation of TSCs into chondrocytes. Taken together, our results showed that KGN-alginate beads can be used to create a rat model of tendinopathy, which, at least in part, reproduces the features of over-use tendinopathy model created by long term treadmill running. This model is mechanistic (stem cell differentiation), highly reproducible and precise in creating localized tendinopathic lesions. It is expected that this model will be useful to evaluate the effects of various topical treatments such as NSAIDs and platelet-rich plasma (PRP) for the treatment of tendinopathy. PMID:26848746

  2. Neurogenic differentiation factor NeuroD confers protection against radiation-induced intestinal injury in mice.

    Li, Ming; Du, Aonan; Xu, Jing; Ma, Yanchao; Cao, Han; Yang, Chao; Yang, Xiao-Dong; Xing, Chun-Gen; Chen, Ming; Zhu, Wei; Zhang, Shuyu; Cao, Jianping

    2016-01-01

    The gastrointestinal tract, especially the small intestine, is particularly sensitive to radiation, and is prone to radiation-induced injury as a result. Neurogenic differentiation factor (NeuroD) is an evolutionarily-conserved basic helix-loop-helix (bHLH) transcription factor. NeuroD contains a protein transduction domain (PTD), which allows it to be exogenously delivered across the membrane of mammalian cells, whereupon its transcription activity can be unleashed. Whether NeuroD has therapeutic effects for radiation-induced injury remains unclear. In the present study, we prepared a NeuroD-EGFP recombinant protein, and explored its protective effects on the survival and intestinal damage induced by ionizing radiation. Our results showed that NeuroD-EGFP could be transduced into small intestine epithelial cells and tissues. NeuroD-EGFP administration significantly increased overall survival of mice exposed to lethal total body irradiation (TBI). This recombinant NeuroD also reduced radiation-induced intestinal mucosal injury and apoptosis, and improved crypt survival. Expression profiling of NeuroD-EGFP-treated mice revealed upregulation of tissue inhibitor of metalloproteinase 1 (TIMP-1), a known inhibitor of apoptosis in mammalian cells. In conclusion, NeuroD confers protection against radiation-induced intestinal injury, and provides a novel therapeutic clinical option for the prevention of intestinal side effects of radiotherapy and the treatment of victims of incidental exposure. PMID:27436572

  3. The endocrine disruptor diethylstilbestrol induces adipocyte differentiation and promotes obesity in mice

    Epidemiology studies indicate that exposure to endocrine disruptors during developmental “window” contributes to adipogenesis and the development of obesity. Implication of endocrine disruptor such as diethylstilbestrol (DES) on adipose tissue development has been poorly investigated. Here we evaluated the effects of DES on adipocyte differentiation in vitro and in vivo, and explored potential mechanism involved in its action. DES induced 3T3-L1 preadipocyte differentiation in a dose-dependent manner, and activated the expression of estrogen receptor (ER) and peroxisome proliferator-acivated receptor (PPAR) γ as well as its target genes required for adipogenesis in vitro. ER mediated the enhancement of DES-induced PPARγ activity. Moreover, DES perturbed key regulators of adipogenesis and lipogenic pathway in vivo. In utero exposure to low dose of DES significantly increased body weight, liver weight and fat mass in female offspring at postnatal day (PND) 60. In addition, serum triglyceride and glucose levels were also significantly elevated. These results suggest that perinatal exposure to DES may be expected to increase the incidence of obesity in a sex-dependent manner and can act as a potential chemical stressor for obesity and obesity-related disorders. -- Highlights: ► DES induced adipocyte differentiation in a dose-dependent manner in 3T3-L1 cells. ► DES activated adipogenic critical regulators and markers in vitro and in vivo. ► Perinatal exposure to DES led to the obese phenotype in female offspring. ► DES might be a potential chemical stressor for obesity and obesity-related disorders.

  4. The endocrine disruptor diethylstilbestrol induces adipocyte differentiation and promotes obesity in mice

    Hao, Chan-Juan; Cheng, Xue-Jia; Xia, Hong-Fei, E-mail: hongfeixia@yahoo.com.cn; Ma, Xu

    2012-08-15

    Epidemiology studies indicate that exposure to endocrine disruptors during developmental “window” contributes to adipogenesis and the development of obesity. Implication of endocrine disruptor such as diethylstilbestrol (DES) on adipose tissue development has been poorly investigated. Here we evaluated the effects of DES on adipocyte differentiation in vitro and in vivo, and explored potential mechanism involved in its action. DES induced 3T3-L1 preadipocyte differentiation in a dose-dependent manner, and activated the expression of estrogen receptor (ER) and peroxisome proliferator-acivated receptor (PPAR) γ as well as its target genes required for adipogenesis in vitro. ER mediated the enhancement of DES-induced PPARγ activity. Moreover, DES perturbed key regulators of adipogenesis and lipogenic pathway in vivo. In utero exposure to low dose of DES significantly increased body weight, liver weight and fat mass in female offspring at postnatal day (PND) 60. In addition, serum triglyceride and glucose levels were also significantly elevated. These results suggest that perinatal exposure to DES may be expected to increase the incidence of obesity in a sex-dependent manner and can act as a potential chemical stressor for obesity and obesity-related disorders. -- Highlights: ► DES induced adipocyte differentiation in a dose-dependent manner in 3T3-L1 cells. ► DES activated adipogenic critical regulators and markers in vitro and in vivo. ► Perinatal exposure to DES led to the obese phenotype in female offspring. ► DES might be a potential chemical stressor for obesity and obesity-related disorders.

  5. Leishmania donovani infection induces anemia in hamsters by differentially altering erythropoiesis in bone marrow and spleen.

    William P Lafuse

    Full Text Available Leishmania donovani is a parasite that causes visceral leishmaniasis by infecting and replicating in macrophages of the bone marrow, spleen, and liver. Severe anemia and leucopenia is associated with the disease. Although immune defense mechanisms against the parasite have been studied, we have a limited understanding of how L. donovani alters hematopoiesis. In this study, we used Syrian golden hamsters to investigate effects of L. donovani infection on erythropoiesis. Infection resulted in severe anemia and leucopenia by 8 weeks post-infection. Anemia was associated with increased levels of serum erythropoietin, which indicates the hamsters respond to the anemia by producing erythropoietin. We found that infection also increased numbers of BFU-E and CFU-E progenitor populations in the spleen and bone marrow and differentially altered erythroid gene expression in these organs. In the bone marrow, the mRNA expression of erythroid differentiation genes (α-globin, β-globin, ALAS2 were inhibited by 50%, but mRNA levels of erythroid receptor (c-kit, EpoR and transcription factors (GATA1, GATA2, FOG1 were not affected by the infection. This suggests that infection has a negative effect on differentiation of erythroblasts. In the spleen, erythroid gene expression was enhanced by infection, indicating that the anemia activates a stress erythropoiesis response in the spleen. Analysis of cytokine mRNA levels in spleen and bone marrow found that IFN-γ mRNA is highly increased by L. donovani infection. Expression of the IFN-γ inducible cytokine, TNF-related apoptosis-inducing ligand (TRAIL, was also up-regulated. Since TRAIL induces erythroblasts apoptosis, apoptosis of bone marrow erythroblasts from infected hamsters was examined by flow cytometry. Percentage of erythroblasts that were apoptotic was significantly increased by L. donovani infection. Together, our results suggest that L. donovani infection inhibits erythropoiesis in the bone marrow by

  6. Using the laser-induced fluorescence spectroscopy in the differentiation between normal and neoplastichuman breast tissue.

    Hage, R; Galhanone, P R; Zângaro, R A; Rodrigues, K C; Pacheco, M T T; Martin, A A; Netto, M M; Soares, F A; da Cunha, I W

    2003-01-01

    This article reports results of the in vitro study for potential evaluation of the laser-induced fluorescence spectroscopy in the differentiation between normal and neoplastic human breast tissue. A coumarine dye laser pumped by nitrogen laser generated an excitation light centered at 458 nm. In order to collect the fluorescence signal was used an optical fiber catheter coupled to a spectrometer and CCD detector. Fluorescence spectra were recorded from normal and neoplastic (benign and malignant) human breast tissue, adding up 94 different areas. The discrimination between normal and neoplasm groups reach a sensitivity and specificity of 100%. PMID:14505202

  7. Changes in binding of staphylococcal leukocidin to HL-60 cells during differentiation induced by dimethyl sulfoxide.

    Morinaga, N; Nagamori, M; Kato, I.

    1988-01-01

    The susceptibility of HL-60 cells to the cytotoxic activity of leukocidin increased depending on the degree of differentiation induced by dimethyl sulfoxide (DMSO). To compare binding characteristics of two components (S and F) of leukocidin to HL-60 and DMSO-treated HL-60 cells, the S and F components were labeled with 125I. Scatchard analysis of the binding curve of the 125I-labeled S component to HL-60 cells showed two classes of binding sites. The binding sites with higher affinity had a ...

  8. Δn89β-Catenin Induces Precocious Development, Differentiation, and Neoplasia in Mammary Gland

    Imbert, Alexandra; Eelkema, Rachel; Jordan, Sara; Feiner, Helen; Cowin, Pamela

    2001-01-01

    To investigate the role of β-catenin in mammary gland development and neoplasia, we expressed a stabilized, transcriptionally active form of β-catenin lacking the NH2-terminal 89 amino acids (ΔN89β-catenin) under the control of the mouse mammary tumor virus long terminal repeat. Our results show that ΔN89β-catenin induces precocious lobuloalveolar development and differentiation in the mammary glands of both male and female mice. Virgin ΔN89β-catenin mammary glands resemble those found in wil...

  9. Ganoderma lucidum Polysaccharides Induce Macrophage-Like Differentiation in Human Leukemia THP-1 Cells via Caspase and p53 Activation

    Jia-Wei Hsu

    2011-01-01

    Full Text Available Differentiation therapy by induction of tumor cells is an important method in the treatment of hematological cancers such as leukemia. Tumor cell differentiation ends cancer cells' immortality, thus stopping cell growth and proliferation. In our previous study, we found that fucose-containing polysaccharide fraction F3 extracted from Ganoderma lucidum can bring about cytokine secretion and cell death in human leukemia THP-1 cells. This prompted us to further investigate on how F3 induces the differentiation in human leukemia cells. We integrated time-course microarray analysis and network modeling to study the F3-induced effects on THP-1 cells. In addition, we determined the differentiation effect using Liu's staining, nitroblue tetrazolium (NBT reduction assay, flow cytometer, western blotting and Q-PCR. We also examined the modulation and regulation by F3 during the differentiation process. Dynamic gene expression profiles showed that cell differentiation was induced in F3-treated THP-1 cells. Furthermore, F3-treated THP-1 cells exhibited enhanced macrophage differentiation, as demonstrated by changes in cell adherence, cell cycle arrest, NBT reduction and expression of differentiation markers including CD11b, CD14, CD68, matrix metalloproteinase-9 and myeloperoxidase. In addition, caspase cleavage and p53 activation were found to be significantly enhanced in F3-treated THP-1 cells. We unraveled the role of caspases and p53 in F3-induced THP-1 cells differentiation into macrophages. Our results provide a molecular explanation for the differentiation effect of F3 on human leukemia THP-1 cells and offer a prospect for a potential leukemia differentiation therapy.

  10. Non-coding RNAs change their expression profile after Retinoid induced differentiation of the promyelocytic cell line NB4

    Caporaso Maria G

    2010-01-01

    Full Text Available Abstract Background The importance of non-coding RNAs (ncRNAs as fine regulators of eukaryotic gene expression has emerged by several studies focusing on microRNAs (miRNAs. miRNAs represent a newly discovered family of non coding-RNAs. They are thought to be crucial players of human hematopoiesis and related tumorigenesis and to represent a potential tool to detect the early stages of cancer. More recently, the expression regulation of numerous long ncRNAs has been linked to cell growth, differentiation and cancer although the molecular mechanism of their function is still unknown. NB4 cells are promyelocytic cells that can be induced to differentiation upon retinoic acid (ATRA treatment and represent a feasible model to study changes of non coding RNAs expression between cancer cells and their terminally differentiated counterpart. Findings we screened, by microarray analysis, the expression of 243 miRNAs and 492 human genes transcribing for putative long ncRNAs different from miRNAs in NB4 cells before and after ATRA induced differentiation. Our data show that 8 miRNAs, and 58 long ncRNAs were deregulated by ATRA induced NB4 differentiation. Conclusion our data suggest that ATRA-induced differentiation lead to deregulation of a large number of the ncRNAs that can play regulatory roles in both tumorigenesis and differentiation.

  11. Comparative Neuronal Differentiation of Self-Renewing Neural Progenitor Cell Lines Obtained from Human Induced Pluripotent Stem Cells

    Chiara Verpelli

    2013-10-01

    Full Text Available Most human neuronal disorders are associated with genetic alterations that cause defects in neuronal development and induce precocious neurodegeneration. In order to fully characterize the molecular mechanisms underlying the onset of these devastating diseases, it is important to establish in vitro models able to recapitulate the human pathology as closely as possible. Here we compared three different differentiation protocols for obtaining functional neurons from human induced pluripotent stem cells (hiPSCs: human neural progenitors (hNPs obtained from hiPSCs were differentiated by co-culturing them with rat primary neurons, glial cells or simply by culturing them on matrigel in neuronal differentiation medium, and the differentiation level was compared using immunofluorescence, biochemical and electrophysiological methods.We show that the differentiated neurons displayed distinct maturation properties depending on the protocol used and the faster morphological and functional maturation was obtained when hNPs were co-cultured with rat primary neurons.

  12. Orphan nuclear receptor Nur77 is required for the differentiation of C6 glioma cells induced by cholera toxin

    Dong XU; Yi-jun HUANG; Yan LI; Wei YIN; Guang-mei YAN

    2009-01-01

    Aim: To investigate a possible regulator gene involved in the cholera toxin-induced differentiation of rat C6 glioma cells. Methods: The global changes in the mRNA expression pattern induced by cholera toxin were analyzed using gene chip microarray. The selected gene was then silenced by RNA interference or overexpressed with an ORF plasmid to determine its necessity in this process. Results: Nur77, a member of the orphan nuclear receptor family (NR4A), was markedly up-regulated during the process of differentiation. Furthermore, RNAi of nur77 attenuated the induction effect of cholera toxin on C6 cells, whereas overexpression of nur77 led to similarly differentiated behavior, including morphologic and biomarker changes, as well as cell cycle arrest. Conclusion: Nur77 participated actively and essentially as an important regulator in the cholera toxin-induced differentiation of C6 cells.

  13. Wnt signaling induces differentiation of progenitor cells in organotypic keratinocyte cultures

    Liu Bob Y

    2007-02-01

    Full Text Available Abstract Background Interfollicular skin develops normally only when the activity of the progenitor cells in the basal layer is counterbalanced by the exit of cells into the suprabasal layers, where they differentiate and cornify to establish barrier function. Distinct stem and progenitor compartments have been demonstrated in hair follicles and sebaceous glands, but there are few data to describe the control of interfollicular progenitor cell activity. Wnt signaling has been shown to be an important growth-inducer of stem cell compartments in skin and many other tissues. Results Here, we test the effect of ectopic Wnt1 expression on the behavior of interfollicular progenitor cells in an organotypic culture model, and find that Wnt1 signaling inhibits their growth and promotes terminal differentiation. Conclusion These results are consistent with the phenotypes reported for transgenic mice engineered to have gain or loss of function of Wnt signaling in skin, which would recommend our culture model as an accurate one for molecular analysis. Since it is known that canonical ligands are expressed in skin, it is likely that this pathway normally regulates the balance of growth and differentiation, and suggests it could be important to pathogenesis.

  14. Mechanisms of all-trans retinoic acid-induced differentiation of acute promyelocytic leukemia cells

    Ji-Wang Zhang; Jian Gu; Zhen-Yi Wang; Sai-Juan Chen; Zhu Chen

    2000-09-01

    Retinoic acids (RA) play a key role in myeloid differentiation through their agonistic nuclear receptors (RAR/RXR) to modulate the expression of target genes. In acute promyelocytic leukemia (APL) cells with rearrangement of retinoic acid receptor (RAR) (including: PML-RAR, PLZF-RAR, NPM-RAR, NuMA-RAR or STAT5b-RAR) as a result of chromosomal translocations, the RA signal pathway is disrupted and myeloid differentiation is arrested at the promyelocytic stage. Pharmacologic dosage of all-trans retinoic acid (ATRA) directly modulates PML-RAR and its interaction with the nuclear receptor co-repressor complex, which restores the wild-type RAR/RXR regulatory pathway and induces the transcriptional expression of downstream genes. Analysing gene expression profiles in APL cells before and after ATRA treatment represents a useful approach to identify genes whose functions are involved in this new cancer treatment. A chronologically well coordinated modulation of ATRA-regulated genes has thus been revealed which seems to constitute a balanced functional network underlying decreased cellular proliferation, initiation and progression of maturation, and maintenance of cell survival before terminal differentiation.

  15. Centrifuge model test on earthquake-induced differential settlement of foundation on cohesive ground

    SHAMOTO; Yasuhiro; HOTTA; Hiroyuki

    2009-01-01

    Dynamic centrifuge model test was conducted to study the earthquake-induced differential settlement of foundation on cohesive ground, and the influence of asymmetry of building was investigated. During the experiment, the overconsolidated kaolin clay ground with a three-dimensional asymmetrical structure model was shaken by a basically balanced input motion, and bender elements were used to measure shear wave velocities of model ground to reveal the soil fabric evolution during and after shaking. The test results show that, the total seismic settlement of foundation is composed of instantaneous and long-term post-earthquake settlements, and most of the differential settlement occurs immediately after the earthquake while the post-earthquake settlement is relatively uniform despite its large amplitude. The asymmetry of building affects the settlement behavior considerably. Compared with 1-or 2-dimensional structures, more evident differential settlement occurs under threedimensional asymmetrical building during shaking, which accounts for one-half of the total seismic settlements and results in complex spatial tilting effects of foundation.

  16. Glyphosate Inhibits PPAR Gamma Induction and Differentiation of Preadipocytes and is able to Induce Oxidative Stress.

    Martini, Claudia N; Gabrielli, Matías; Brandani, Javier N; Vila, María Del C

    2016-08-01

    Glyphosate-based herbicides (GF) are extensively used for weed control. Thus, it is important to investigate their putative toxic effects. We have reported that GF at subagriculture concentrations inhibits proliferation and differentiation to adipocytes of 3T3-L1 fibroblasts. In this investigation, we evaluated the effect of GF on genes upregulated during adipogenesis. GF was able to inhibit the induction of PPAR gamma, the master gene in adipogenesis but not C/EBP beta, which precedes PPAR gamma activation. GF also inhibited differentiation and proliferation of another model of preadipocyte: mouse embryonic fibroblasts. In exponentially growing 3T3-L1 cells, GF increased lipid peroxidation and the activity of the antioxidant enzyme, superoxide dismutase. We also found that proliferation was inhibited with lower concentrations of GF when time of exposure was extended. Thus, GF was able to inhibit proliferation and differentiation of preadipocytes and to induce oxidative stress, which is indicative of its ability to alter cellular physiology. PMID:27044015

  17. Donor Dependent Variations in Hematopoietic Differentiation among Embryonic and Induced Pluripotent Stem Cell Lines

    Féraud, Olivier; Valogne, Yannick; Melkus, Michael W.; Zhang, Yanyan; Oudrhiri, Noufissa; Haddad, Rima; Daury, Aurélie; Rocher, Corinne; Larbi, Aniya; Duquesnoy, Philippe; Divers, Dominique; Gobbo, Emilie; Brunet de la Grange, Philippe; Louache, Fawzia; Bennaceur-Griscelli, Annelise; Mitjavila-Garcia, Maria Teresa

    2016-01-01

    Hematopoiesis generated from human embryonic stem cells (ES) and induced pluripotent stem cells (iPS) are unprecedented resources for cell therapy. We compared hematopoietic differentiation potentials from ES and iPS cell lines originated from various donors and derived them using integrative and non-integrative vectors. Significant differences in differentiation toward hematopoietic lineage were observed among ES and iPS. The ability of engraftment of iPS or ES-derived cells in NOG mice varied among the lines with low levels of chimerism. iPS generated from ES cell-derived mesenchymal stem cells (MSC) reproduce a similar hematopoietic outcome compared to their parental ES cell line. We were not able to identify any specific hematopoietic transcription factors that allow to distinguish between good versus poor hematopoiesis in undifferentiated ES or iPS cell lines. There is a relatively unpredictable variation in hematopoietic differentiation between ES and iPS cell lines that could not be predicted based on phenotype or gene expression of the undifferentiated cells. These results demonstrate the influence of genetic background in variation of hematopoietic potential rather than the reprogramming process. PMID:26938212

  18. Differential protein expression in the midgut of Culex quinquefasciatus mosquitoes induced by the insecticide temephos.

    Games, P D; Alves, S N; Katz, B B; Tomich, J M; Serrão, J E

    2016-09-01

    Mosquitoes are vectors for pathogens of malaria, lymphatic filariasis, dengue, chikungunya, yellow fever and Japanese encephalitis. Culex quinquefasciatus Say, 1823 (Diptera: Culicidae) is a known vector of lymphatic filariasis. Its control in Brazil has been managed using the organophosphate temephos. Studies examining the proteins of Cx. quinquefasciatus that are differentially expressed in response to temephos further understanding of the modes of action of the insecticide and may potentially identify resistance factors in the mosquito. In the present study, a comparative proteomic analysis, using 2-dimensional electrophoresis coupled with matrix-assisted laser desorption/ionization (MALDI) time of flight (TOF)/TOF mass spectrometry, and bioinformatics analyses were performed to identify midgut proteins in Cx. quinquefasciatus larvae that were differentially expressed in response to exposure to temephos relative to those in untreated controls. A total of 91 protein spots were differentially expressed; 40 were upregulated and 51 were downregulated by temephos. A total of 22 proteins, predominantly upregulated, were identified as known to play a role in the immune response, whereas the downregulated proteins were involved in energy and protein catabolism. This is the first proteome study of the midgut of Cx. quinquefasciatus and it provides insights into the molecular mechanisms of insecticide-induced responses in the mosquito. PMID:27072633

  19. Potato suberin induces differentiation and secondary metabolism in the genus Streptomyces.

    Lerat, Sylvain; Forest, Martin; Lauzier, Annie; Grondin, Gilles; Lacelle, Serge; Beaulieu, Carole

    2012-01-01

    Bacteria of the genus Streptomyces are soil microorganisms with a saprophytic life cycle. Previous studies have revealed that the phytopathogenic agent S. scabiei undergoes metabolic and morphological modifications in the presence of suberin, a complex plant polymer. This paper investigates morphological changes induced by the presence of potato suberin in five species of the genus Streptomyces, with emphasis on S. scabiei. Streptomyces scabiei, S. acidiscabies, S. avermitilis, S. coelicolor and S. melanosporofaciens were grown both in the presence and absence of suberin. In all species tested, the presence of the plant polymer induced the production of aerial hyphae and enhanced resistance to mechanical lysis. The presence of suberin in liquid minimal medium also induced the synthesis of typical secondary metabolites in S. scabiei and S. acidiscabies (thaxtomin A), S. coelicolor (actinorhodin) and S. melanosporofaciens (geldanamycin). In S. scabiei, the presence of suberin modified the fatty acid composition of the bacterial membrane, which translated into higher membrane fluidity. Moreover, suberin also induced thickening of the bacterial cell wall. The present data indicate that suberin hastens cellular differentiation and triggers the onset of secondary metabolism in the genus Streptomyces. PMID:22129602

  20. The inhibitory effect of vitamin K on RANKL-induced osteoclast differentiation and bone resorption.

    Wu, Wei-Jie; Kim, Min Seuk; Ahn, Byung-Yong

    2015-10-01

    To further understand the correlation between vitamin K and bone metabolism, the effects of vitamins K1, menaquinone-4 (MK-4), and menaquinone-7 (MK-7) on RANKL-induced osteoclast differentiation and bone resorption were comparatively investigated. Vitamin K2 groups (MK-4 and MK-7) were found to significantly inhibit RANKL-medicated osteoclast cell formation of bone marrow macrophages (BMMs) in a dose-dependent manner, without any evidence of cytotoxicity. The mRNA expression of specific osteoclast differentiation markers, such as c-Fos, NFATc1, OSCAR, and TRAP, as well as NFATc1 protein expression and TRAP activity in RANKL-treated BMMs were inhibited by vitamin K2, although MK-4 exhibited a significantly greater efficiency compared to MK-7. In contrast, the same dose of vitamin K1 had no inhibitory effect on RANKL-induced osteoclast cell formation, but increased the expression of major osteoclastogenic genes. Interestingly, vitamins K1, MK-4 and MK-7 all strongly inhibited osteoclastic bone resorption (p vitamins K1, MK-4 and MK-7 have anti-osteoporotic properties, while their regulation effects on osteoclastogenesis are somewhat different. PMID:26267519

  1. Preparation of an antibody that recognizes and neutralizes Dictyostelium differentiation-inducing factor-1.

    Kubohara, Yuzuru; Kikuchi, Haruhisa; Nakamura, Koji; Matsuo, Yusuke; Oshima, Yoshiteru

    2010-05-28

    In the development of the cellular slime mold Dictyostelium discoideum, the differentiation-inducing factor-1 (DIF-1; 1-(3,5-dichloro-2,6-dihydroxy-4-methoxyphenyl)hexan-1-one) plays an important role in the regulation of cell differentiation and chemotaxis; however, the cellular signaling systems involving DIF-1 remain to be elucidated. To obtain a probe for DIF-1, we synthesized a DIF derivative (DIF-1-NH(2); 6-amino-1-(3,5-dichloro-2,6-dihydroxy-4-methoxyphenyl)hexan-1-one), and prepared an anti-DIF-1 antibody using a DIF-1-NH(2)-conjugated macromolecule as the immunogen. A 100-fold dilution of the antibody bound to DIF-1-NH(2)-conjugated resin, and this binding was inhibited by co-addition of 20 microM DIF-1 or DIF-1-NH(2). In a monolayer culture of HM44 cells, a DIF-deficient D. discoideum strain, 0.5 nM exogenous DIF-1 induced stalk cell formation in approximately 60% of the cells; this induction was dose-dependently inhibited by the antibody (diluted 12.5- or 25-fold). Furthermore, this inhibition by the antibody was recovered by co-addition of 2.5 or10 nM DIF-1. The results indicate that the anti-DIF-1 antibody recognizes DIF-1 and neutralizes its function. PMID:20416278

  2. A Sphingolipid Inhibitor Induces a Cytokinesis Arrest and Blocks Stage Differentiation in Giardia lamblia▿

    Sonda, Sabrina; Štefanić, Saša; Hehl, Adrian B.

    2008-01-01

    Sphingolipid biosynthesis pathways have recently emerged as a promising target for therapeutic intervention against pathogens, including parasites. A key step in the synthesis of complex sphingolipids is the glucosylation of ceramide, mediated by glucosylceramide (GlcCer) synthase, whose activity can be inhibited by PPMP (1-phenyl-2-palmitoylamino-3-morpholino-1-propanol). In this study, we investigated whether PPMP inhibits the proliferation and differentiation of the pathogenic parasite Giardia lamblia, the major cause of parasite-induced diarrhea worldwide. PPMP was found to block in vitro parasite replication in a dose-dependent manner, with a 50% inhibitory concentration of 3.5 μM. The inhibition of parasite replication was irreversible at 10 μM PPMP, a concentration that did not affect mammalian cell metabolism. Importantly, PPMP inhibited the completion of cell division at a specific stage in late cytokinesis. Microscopic analysis of cells incubated with PPMP revealed the aberrant accumulation of cellular membranes belonging to the endoplasmic reticulum network in the caudal area of the parasites. Finally, PPMP induced a 90% reduction in G. lamblia differentiation into cysts, the parasite stage responsible for the transmission of the disease. These results show that PPMP is a powerful inhibitor of G. lamblia in vitro and that as-yet-uncharacterized sphingolipid biosynthetic pathways are potential targets for the development of anti-G. lamblia agents. PMID:18086854

  3. A sphingolipid inhibitor induces a cytokinesis arrest and blocks stage differentiation in Giardia lamblia.

    Sonda, Sabrina; Stefanic, Sasa; Hehl, Adrian B

    2008-02-01

    Sphingolipid biosynthesis pathways have recently emerged as a promising target for therapeutic intervention against pathogens, including parasites. A key step in the synthesis of complex sphingolipids is the glucosylation of ceramide, mediated by glucosylceramide (GlcCer) synthase, whose activity can be inhibited by PPMP (1-phenyl-2-palmitoylamino-3-morpholino-1-propanol). In this study, we investigated whether PPMP inhibits the proliferation and differentiation of the pathogenic parasite Giardia lamblia, the major cause of parasite-induced diarrhea worldwide. PPMP was found to block in vitro parasite replication in a dose-dependent manner, with a 50% inhibitory concentration of 3.5 muM. The inhibition of parasite replication was irreversible at 10 muM PPMP, a concentration that did not affect mammalian cell metabolism. Importantly, PPMP inhibited the completion of cell division at a specific stage in late cytokinesis. Microscopic analysis of cells incubated with PPMP revealed the aberrant accumulation of cellular membranes belonging to the endoplasmic reticulum network in the caudal area of the parasites. Finally, PPMP induced a 90% reduction in G. lamblia differentiation into cysts, the parasite stage responsible for the transmission of the disease. These results show that PPMP is a powerful inhibitor of G. lamblia in vitro and that as-yet-uncharacterized sphingolipid biosynthetic pathways are potential targets for the development of anti-G. lamblia agents. PMID:18086854

  4. Neural stem cells was induced to differentiate into cholinergic neurons in vitro

    The cholinergic-inducing effect of BMP4 on isolated and cultivated rat's cerebral neural stem cells (NSCs) was examined. NSCs which were isolated from two month's old rat's brain region like hippocampus and striatum were cultivated in a medium containing EGF and bFGF, and were identified with morphological character by microscope and nestin immunocytochemistry test. After 24 hours, half NSCs were cultivated with a BMP4-added medium as a experimental group instead of the primary medium, while the an other half NSCs being cultivated with the primary medium as a control group. After 8 days the expression of choline acetyltransferase (ChAT) of the cultivated cells was observated by indirect immunofluorescence test. Results showed that more positive cells were found in the experimental group, and the fluorescence intensity were stronger; while less positive cells were found in the control group, and the fluorescence intensity was weaker. The differentiational efficiency of the NSCs was examined by FITC-labelled Flow Cytometry. The results showed that about 16% cells of the experimental group appeared ChAT-positive, while that of control group only 7%. So BMP4 may have the function of inducing NSCs to differentiate into neurons with cholinergic characteristic. (authors)

  5. Spirulina phycocyanin induces differential protein expression and apoptosis in SKOV-3 cells.

    Pan, Ruowang; Lu, Rongmao; Zhang, Ying; Zhu, Mei; Zhu, Wen; Yang, Rongrong; Zhang, Enyong; Ying, Jun; Xu, Teng; Yi, Huiguang; Li, Jinsong; Shi, Mengru; Zhou, Li; Xu, Zuyuan; Li, Peizhen; Bao, Qiyu

    2015-11-01

    The present study was designed to determine the effects of phycocyanin (PC) on Human ovarian cancer SKOV-3 cells and the underlying molecular mechanisms of action. The inhibitory effects of PC on the cell proliferation were detected by MTT assay. The IC50 values of PC were 182.0μM and 133.6μM for 24h and 48h exposure, respectively. PC induced apoptosis in SKOV-3 cells was observed by electron microscopy and flow cytometry. The apoptosis rate was increased from 1.6% to 19.8% after PC exposure. The fluorescence intensity of ROS and the activities of Caspase-3, Caspase-8, and Caspase-9 were increased. Differentiated expression protein spots were selected and identified using proteomic techniques. There were 698±73 and 683±79 protein spots resolved in untreated and PC-treated cells, respectively. Forty five differential protein spots were analyzed by MALDI-TOF-MS, including mtSSB, PSME3, and nucleolin. The mRNA expression profiles determined by RT-PCR were consistent with that of the two-dimensional electrophoresis. The decreased proteins such as HSP60, nucleolin, PPase, peroxiredoxin-4 and the increased protein (mtSSB) were identified in SKOV-3 cells after PC treatment, indicating that the effects of PC on tumor cell apoptosis may be relate to multiple target proteins. And the mitochondrial pathway may be the main pathway for PC-induced apoptosis. PMID:26410814

  6. Current-induced forces: a new mechanism to induce negative differential resistance and current-switching effect in molecular junctions

    Gu, Lei; Fu, Hua-Hua

    2015-12-01

    Current-induced forces can excite molecules, polymers and other low-dimensional materials, which in turn leads to an effective gate voltage through Holstein interaction. Here, by taking a short asymmetric DNA junction as an example, and using the Langevin approach, we find that when suppression of charge transport by the effective gate voltage surpasses the current increase from an elevated voltage bias, the current-voltage (I-V) curves display strong negative differential resistance (NDR) and perfect current-switching characteristics. The asymmetric DNA chain differs in mechanical stability under inverse voltages and the I-V curve is asymmetric about inverse biases, which can be used to understand recent transport experiments on DNA chains, and meanwhile provides a new strategy to realize NDR in molecular junctions and other low-dimensional quantum systems.

  7. Role of SP-1 in SDS-Induced Adipose Differentiation Related Protein Synthesis in Human Keratinocytes

    Emanuela Corsini

    2007-01-01

    Full Text Available Skin irritation is a complex phenomenon, and keratinocytes play an important role in it. We have recently characterized the expression and protective role of adipose differentiation related protein (ADRP in skin irritation. In particular, ADRP expression is induced to recover functional cell membrane following the cell damage caused by skin irritants. The purpose of this study was to characterize in a human keratinocyte cells line (NCTC 2544 the biochemical events that lead to ADRP expression following SDS treatment, and in particular, to investigate the role of transcription factor SP-1. Analysis of ADRP promoter region revealed the presence of a potential binding site for the transcription factor SP-1 close to the start site. Evaluated by measuring the DNA binding activity, we found that SDS induced a dose and time related SP-1 activation, which was correlated with SDS-induced ADRP mRNA expression. Furthermore, SDS-induced SP-1 activation, ADRP mRNA expression and lipid droplets accumulation could be modulated by mithramycin A, an antibiotic that selectively binds to the GC box preventing SP-1 binding and gene expression. This demonstrated that SDS-induced ADRP expression was mediated in part through the transcription factor SP-1. In addition, SDS-induced SP-1 activation and ADRP expression could be modulated by the calcium chelator BAPTA, indicating a role of calcium in ADRP-induction. Thus, every time an irritant perturbs the membrane barrier, it renders the membrane leaky and allows extracellular calcium to enter the cells, an event that provides the upstream mechanisms initiating the signaling cascade that triggers the activation of SP-1 and culminates in the enhancement of ADRP expression, which helps to restore the normal homeostasis and ultimately repairs the to membrane.

  8. High and Low LET Radiation Differentially Induce Normal Tissue Damage Signals

    Purpose: Radiotherapy using high linear energy transfer (LET) radiation is aimed at efficiently killing tumor cells while minimizing dose (biological effective) to normal tissues to prevent toxicity. It is well established that high LET radiation results in lower cell survival per absorbed dose than low LET radiation. However, whether various mechanisms involved in the development of normal tissue damage may be regulated differentially is not known. Therefore the aim of this study was to investigate whether two actions related to normal tissue toxicity, p53-induced apoptosis and expression of the profibrotic gene PAI-1 (plasminogen activator inhibitor 1), are differentially induced by high and low LET radiation. Methods and Materials: Cells were irradiated with high LET carbon ions or low LET photons. Cell survival assays were performed, profibrotic PAI-1 expression was monitored by quantitative polymerase chain reaction, and apoptosis was assayed by annexin V staining. Activation of p53 by phosphorylation at serine 315 and serine 37 was monitored by Western blotting. Transfections of plasmids expressing p53 mutated at serines 315 and 37 were used to test the requirement of these residues for apoptosis and expression of PAI-1. Results: As expected, cell survival was lower and induction of apoptosis was higher in high -LET irradiated cells. Interestingly, induction of the profibrotic PAI-1 gene was similar with high and low LET radiation. In agreement with this finding, phosphorylation of p53 at serine 315 involved in PAI-1 expression was similar with high and low LET radiation, whereas phosphorylation of p53 at serine 37, involved in apoptosis induction, was much higher after high LET irradiation. Conclusions: Our results indicate that diverse mechanisms involved in the development of normal tissue damage may be differentially affected by high and low LET radiation. This may have consequences for the development and manifestation of normal tissue damage.

  9. BMP7 transfection induces in-vitro osteogenic differentiation of dental pulp mesenchymal stem cells

    Ka Po John Yau

    2013-01-01

    Full Text Available Objective: To assess whether in-vitro osteogenic differentiation of human dental pulp mesenchymal stem cells can be induced by transient transfection with the gene encoding human bone morphogenic protein 7 (BMP7. Materials and Methods: A mesenchymal stem cell population was isolated from the dental pulp of two extracted permanent premolars, expanded and characterized. The human BMP7 gene, as a recombinant pcDNA3.1/V5-His-TOPO-BMP7 plasmid, was transfected into the cells. Three negative controls were used: No plasmid, empty vector, and an unrelated vector encoding green fluorescent protein. After the interval of 24 and 48 h, mRNA levels of alkaline phosphatase and osteocalcin as markers of in-vitro osteogenic differentiation were measured by real-time polymerase chain reaction and standardized against β-actin mRNA levels. Results: The level of alkaline phosphatase mRNA was significantly higher for the BMP7 group than for all three negative controls 48 h after transfection (706.9 vs. 11.24 for untransfected cells, 78.05 for empty vector, and 73.10 for green fluorescent protein vector. The level of osteocalcin mRNA was significantly higher for the BMP7 group than for all three negative controls 24 h after transfection (1.0, however, decreased after another 24 h. Conclusions: In-vitro osteoblastic differentiation of human dental pulp mesenchymal stem cells, as indicated by expression of alkaline phosphatase and osteocalcin, can be induced by transient transfection with the BMP7 gene.

  10. DNA-damage response gene GADD45A induces differentiation in hematopoietic stem cells without inhibiting cell cycle or survival.

    Wingert, Susanne; Thalheimer, Frederic B; Haetscher, Nadine; Rehage, Maike; Schroeder, Timm; Rieger, Michael A

    2016-03-01

    Hematopoietic stem cells (HSCs) maintain blood cell production life-long by their unique abilities of self-renewal and differentiation into all blood cell lineages. Growth arrest and DNA-damage-inducible 45 alpha (GADD45A) is induced by genotoxic stress in HSCs. GADD45A has been implicated in cell cycle control, cell death and senescence, as well as in DNA-damage repair. In general, GADD45A provides cellular stability by either arresting the cell cycle progression until DNA damage is repaired or, in cases of fatal damage, by inducing apoptosis. However, the function of GADD45A in hematopoiesis remains controversial. We revealed the changes in murine HSC fate control orchestrated by the expression of GADD45A at single cell resolution. In contrast to other cellular systems, GADD45A expression did not cause a cell cycle arrest or an alteration in the decision between cell survival and apoptosis in HSCs. Strikingly, GADD45A strongly induced and accelerated the differentiation program in HSCs. Continuous tracking of individual HSCs and their progeny via time-lapse microscopy elucidated that once GADD45A was expressed, HSCs differentiate into committed progenitors within 29 hours. GADD45A-expressing HSCs failed to long-term reconstitute the blood of recipients by inducing multilineage differentiation in vivo. Importantly, γ-irradiation of HSCs induced their differentiation by upregulating endogenous GADD45A. The differentiation induction by GADD45A was transmitted by activating p38 Mitogen-activated protein kinase (MAPK) signaling and allowed the generation of megakaryocytic-erythroid, myeloid, and lymphoid lineages. These data indicate that genotoxic stress-induced GADD45A expression in HSCs prevents their fatal transformation by directing them into differentiation and thereby clearing them from the system. Stem Cells 2016;34:699-710. PMID:26731607

  11. Extended passaging increases the efficiency of neural differentiation from induced pluripotent stem cells

    Koehler Karl R

    2011-08-01

    Full Text Available Abstract Background The use of induced pluripotent stem cells (iPSCs for the functional replacement of damaged neurons and in vitro disease modeling is of great clinical relevance. Unfortunately, the capacity of iPSC lines to differentiate into neurons is highly variable, prompting the need for a reliable means of assessing the differentiation capacity of newly derived iPSC cell lines. Extended passaging is emerging as a method of ensuring faithful reprogramming. We adapted an established and efficient embryonic stem cell (ESC neural induction protocol to test whether iPSCs (1 have the competence to give rise to functional neurons with similar efficiency as ESCs and (2 whether the extent of neural differentiation could be altered or enhanced by increased passaging. Results Our gene expression and morphological analyses revealed that neural conversion was temporally delayed in iPSC lines and some iPSC lines did not properly form embryoid bodies during the first stage of differentiation. Notably, these deficits were corrected by continual passaging in an iPSC clone. iPSCs with greater than 20 passages (late-passage iPSCs expressed higher expression levels of pluripotency markers and formed larger embryoid bodies than iPSCs with fewer than 10 passages (early-passage iPSCs. Moreover, late-passage iPSCs started to express neural marker genes sooner than early-passage iPSCs after the initiation of neural induction. Furthermore, late-passage iPSC-derived neurons exhibited notably greater excitability and larger voltage-gated currents than early-passage iPSC-derived neurons, although these cells were morphologically indistinguishable. Conclusions These findings strongly suggest that the efficiency neuronal conversion depends on the complete reprogramming of iPSCs via extensive passaging.

  12. Preliminary Study on in Vitro Induced Differentiation of Embryonic Stem Cells into Neurons

    JianGe; ShunongLi; 等

    2002-01-01

    Purpose;To study preliminarily in vitro induced differentiation of embryonic stem cells into neurons for further investigation of an alternative for the treatment of glaumatous neuropathy.Materials and methods:Supernatant of cultured Buffalo rat liver cells (buffalorat liver cell-conditioned medium,BRL-CM)was used for culturing embryonic stem cells(ES-D3 cell line)..Morphological features of undifferentiated ES cells were studied by HE staining and electron microscopy.Based on the methods used by Bain et al,we modified the methods and used retinoic,acid(RA) as an inducer to differentiate ES-D3 cells and cytosine arabinoside(Ara-C) as inhibitor of proliferative cells.The growth of the cells was observed under phase contrast microscope.Reuslts:ES-D3 cells cultured by BRL-CM grew in aggregates and remained undifferentiated.Electromicroscopy showed large nucleus and a large amount of mitochondria in undifferentiated ES cells and many processes on the surfaces.In the first day after the adding of retinoic acid,some neuron-like cells with one,two or more processes were present.In the second day after adding RA and the first day after the plus of 10μm Ara-C,a large amount of neuron-like cells appeared,with the formation of neuron-like networks.Conclusions:Combined use of RA and Ara-C can induce ES cells to different into neuron-like cells.Our present preliminary study might provide insights into an alternative for the treatment of glaucomatous neuropathy by the transplantation of embryonic stem cells.Eye Science 2000;16:1-6.

  13. Simvastatin induces differentiation of rabbit articular chondrocytes via the ERK-1/2 and p38 kinase pathways.

    Han, Yohan; Kim, Song Ja

    2016-08-15

    Statins are competitive inhibitors of hydroxy-methyl-glutaryl Coenzyme A (HMG-CoA) reductase, a key enzyme involved in the conversion of HMG-CoA to the cholesterol precursor mevalonate. Some statins, such as simvastatin (simvastatin), have been shown to have anti-cancer and anti-inflammatory effects, reducing cartilage degradation in osteoarthritic rabbits in vivo. However, the regulatory mechanisms undergirding simvastatin mediated chondrocyte differentiation have not been well elucidated. Thus, we investigated the action and mechanism of simvastatin on differentiation of rabbit articular chondrocytes through western blot analyses, RT-PCR, and immunohistochemical (IHC) and immunofluorescence (IF) staining. Simvastatin treatment was found to induce type II collagen expression and sulfated-proteoglycan synthesis in a dose- and time-dependent manner. Indeed, RT-PCR revealed increased expression of type II collagen on treatment with simvastatin. Both IHC and IF staining indicated differentiation of chondrocytes. Simvastatin treatment reduced activation of ERK-1/2 and stimulated activation of p38 kinase. Inhibition of ERK-1/2 with PD98059 enhanced simvastatin induced differentiation, whereas inhibition of p38 kinase with SB203580 inhibited simvastatin induced differentiation. Simvastatin treatment also inhibits loss of type II collagen in serial monolayer culture. Collectively, our results indicate that ERK-1/2 and p38 kinase regulate simvastatin-induced differentiation of chondrocytes in opposing manners. Thus, these findings suggest that simvastatin may be a potential therapeutic drug for osteoarthritis. PMID:27475840

  14. NGF induces adult stem Leydig cells to proliferate and differentiate during Leydig cell regeneration

    Highlights: •Nerve growth factor has shown significant changes on mRNA levels during Adult Leydig cells regeneration. •We established the organ culture model of rat seminiferous tubules with ethane dimethyl sulphonate (EDS) treatment. •Nerve growth factor has shown proliferation and differentiation-promoting effects on Adult stem Leydig cells. •Nerve growth factor induces progenitor Leydig cells to proliferate and differentiate and immature Leydig cells to proliferate. -- Abstract: Nerve growth factor (NGF) has been reported to be involved in male reproductive physiology. However, few reports have described the activity of NGF during Leydig cell development. The objective of the present study was to examine the role of NGF during stem-Leydig-cell (SLC) regeneration. We investigated the effects of NGF on Leydig-cell (LC) regeneration by measuring mRNA levels in the adult rat testis after ethane dimethanesulfonate (EDS) treatment. Furthermore, we used the established organ culture model of rat seminiferous tubules to examine the regulation of NGF during SLC proliferation and differentiation using EdU staining, real-time PCR and western blotting. Progenitor Leydig cells (PLCs) and immature Leydig cells (ILCs) were also used to investigate the effects of NGF on LCs at different developmental stages. NGF mRNA levels changed significantly during Leydig-cell regeneration in vivo. In vitro, NGF significantly promoted the proliferation of stem Leydig cells and also induced steroidogenic enzyme gene expression and 3β-HSD protein expression. The data from PLCs and ILCs showed that NGF could increase Cyclin D1 and Hsd 17b3 mRNA levels in PLCs and Cyclin D1 mRNA levels in ILCs. These results indicate that NGF may play an important role during LC regeneration by regulating the proliferation and differentiation of LCs at different developmental stages, from SLCs to PLCs and from PLCs to ILCs. The discovery of this effect of NGF on Leydig cells will provide useful

  15. NGF induces adult stem Leydig cells to proliferate and differentiate during Leydig cell regeneration

    Zhang, Lei [Department of Cell Biology, College of Life Science and Technology, Jinan University, 510632 Guangzhou (China); Wang, Huaxi [Southern Medical University, 510515 Guangzhou (China); Yang, Yan [College of Pharmacy, Jinan University, 510632 Guangzhou (China); Liu, Hui [Department of Cell Biology, College of Life Science and Technology, Jinan University, 510632 Guangzhou (China); Zhang, Qihao; Xiang, Qi [Department of Cell Biology, College of Life Science and Technology, Jinan University, 510632 Guangzhou (China); National Engineering Research Center of Genetic Medicine, 510632 Guangzhou (China); Ge, Renshan [Population Council, Rockefeller University, 10065 New York (United States); Su, Zhijian, E-mail: tjnuszj@jnu.edu.cn [Department of Cell Biology, College of Life Science and Technology, Jinan University, 510632 Guangzhou (China); Huang, Yadong, E-mail: tydhuang@jnu.edu.cn [Department of Cell Biology, College of Life Science and Technology, Jinan University, 510632 Guangzhou (China); National Engineering Research Center of Genetic Medicine, 510632 Guangzhou (China)

    2013-06-28

    Highlights: •Nerve growth factor has shown significant changes on mRNA levels during Adult Leydig cells regeneration. •We established the organ culture model of rat seminiferous tubules with ethane dimethyl sulphonate (EDS) treatment. •Nerve growth factor has shown proliferation and differentiation-promoting effects on Adult stem Leydig cells. •Nerve growth factor induces progenitor Leydig cells to proliferate and differentiate and immature Leydig cells to proliferate. -- Abstract: Nerve growth factor (NGF) has been reported to be involved in male reproductive physiology. However, few reports have described the activity of NGF during Leydig cell development. The objective of the present study was to examine the role of NGF during stem-Leydig-cell (SLC) regeneration. We investigated the effects of NGF on Leydig-cell (LC) regeneration by measuring mRNA levels in the adult rat testis after ethane dimethanesulfonate (EDS) treatment. Furthermore, we used the established organ culture model of rat seminiferous tubules to examine the regulation of NGF during SLC proliferation and differentiation using EdU staining, real-time PCR and western blotting. Progenitor Leydig cells (PLCs) and immature Leydig cells (ILCs) were also used to investigate the effects of NGF on LCs at different developmental stages. NGF mRNA levels changed significantly during Leydig-cell regeneration in vivo. In vitro, NGF significantly promoted the proliferation of stem Leydig cells and also induced steroidogenic enzyme gene expression and 3β-HSD protein expression. The data from PLCs and ILCs showed that NGF could increase Cyclin D1 and Hsd 17b3 mRNA levels in PLCs and Cyclin D1 mRNA levels in ILCs. These results indicate that NGF may play an important role during LC regeneration by regulating the proliferation and differentiation of LCs at different developmental stages, from SLCs to PLCs and from PLCs to ILCs. The discovery of this effect of NGF on Leydig cells will provide useful

  16. Pristane-induced effects on cytochrome P-4501A, ornithine decarboxylase and putrescine in rats.

    Harper, C M; Soni, M G; Mehendale, H M; Cuchens, M A

    1995-08-16

    The effects of pristane (2,6,10,14-tetramethylpentadecane) on cytochrome P-4501A (cP4501A) activity in microsomes, as well as on ornithine decarboxylase (ODC) activity and concomitant putrescine levels were examined in Copenhagen rats. In general, pristane treatment led to increased cP4501A levels when compared to basal levels, while co-treatment with 3-methylcholanthrene (3-MC) and pristane elicited augmented cP4501A responses when compared to responses induced by 3-MC alone. Increases in both ODC activity and putrescine levels were also observed in pristane treated rats. Collectively, these results indicate that pristane influences cP4501A activity and elicits promoter-like responses as reflected in elevated ODC activity and increased amount of putrescine. PMID:7656217

  17. Bladder cancer cell in co-culture induces human stem cell differentiation to urothelial cells through paracrine FGF10 signaling

    Chung, Seyung S.; Koh, Chester J.

    2013-01-01

    FGF10 is required for embryonic epidermal morphogenesis including brain development, lung morphogenesis, and initiation of limb bud formation. In this study, we investigated the role of FGF10 as a lead induction factor for stem cell differentiation toward urothelial cell. To this end, human multi-potent stem cell in vitro system was employed. Human amniotic fluid stem cells were co-cultured with immortalized bladder cancer lines to induce directed differentiation into urothelial cells. Urothe...

  18. Multilineage-differentiating stress-enduring (Muse) cells are a primary source of induced pluripotent stem cells in human fibroblasts

    Wakao, Shohei; Kitada, Masaaki; Kuroda, Yasumasa; Shigemoto, Taeko; Matsuse, Dai; Akashi, Hideo; Tanimura, Yukihiro; Tsuchiyama, Kenichiro; Kikuchi, Tomohiko; Goda, Makoto; Nakahata, Tatsutoshi; Fujiyoshi, Yoshinori; Dezawa, Mari

    2011-01-01

    The stochastic and elite models have been proposed for the mechanism of induced pluripotent stem (iPS) cell generation. In this study we report a system that supports the elite model. We previously identified multilineage-differentiating stress-enduring (Muse) cells in human dermal fibroblasts that are characterized by stress tolerance, expression of pluripotency markers, self-renewal, and the ability to differentiate into endodermal-, mesodermal-, and ectodermal-lineage cells from a single c...

  19. Neuronal Differentiation of Induced Pluripotent Stem Cells on Surfactant Templated Chitosan Hydrogels.

    Worthington, Kristan S; Green, Brian J; Rethwisch, Mary; Wiley, Luke A; Tucker, Budd A; Guymon, C Allan; Salem, Aliasger K

    2016-05-01

    The development of effective tissue engineering materials requires careful consideration of several properties beyond biocompatibility, including permeability and mechanical stiffness. While surfactant templating has been used for over a decade to control the physical properties of photopolymer materials, the potential benefit of this technique with regard to biomaterials has yet to be fully explored. Herein we demonstrate that surfactant templating can be used to tune the water uptake and compressive modulus of photo-cross-linked chitosan hydrogels. Interestingly, templating with quaternary ammonium surfactants also hedges against property fluctuations that occur with changing pH. Further, we demonstrate that, after adequate surfactant removal, these materials are nontoxic, support the attachment of induced pluripotent stem cells and facilitate stem cell differentiation to neuronal phenotypes. These results demonstrate the utility of surfactant templating for optimizing the properties of biomaterials intended for a variety of applications, including retinal regeneration. PMID:27008004

  20. Differential gene expression induced by high LET charged particles in normal human fibroblasts

    We investigated differential gene expression of normal human skin HSF42 fibroblasts induced by heavy ions using cDNA microarray technology. Irradiation with 3 types of heavy ions was performed at Heavy Ion Medical Accelerator in Chiba (HIMAC) facility. Out of 7458 genes, we found 61 significant genes (40 up-regulated and 21 down-regulated) that distinguished between human skin fibroblast HSF42 cells non-irradiated and irradiated with 1 Gy of neon particles and 62 significant genes (48 up-regulated and 14 down-regulated) that distinguished between HSF42 cells non-irradiated and irradiated with 1 Gy of silicon particles. Furthermore, we are going to analyze profiles of HSF42 cells exposed to carbon particles and compare those profiles between different types of beams. (author)

  1. On the algebra of deformed differential operators, and induced integrable Toda field theory

    We build in this paper the algebra of q-deformed pseudo-differential operators shown to be an essential step towards setting a q-deformed integrability program. In fact, using the results of this q-deformed algebra, we derive the q-analogues of the generalised KdV hierarchy. We focus in particular the first leading orders of this q-deformed hierarchy namely the q-KdV and q-Boussinesq integrable systems. We also present the q-generalisation of the conformal transformations of the currents un, n ≥ 2 and discuss the primary condition of the fields wn, n ≥ 2 by using the Volterra gauge group transformations for the q-covariant Lax operators. An induced su(n)-Toda(su(2)-Liouville) field theory construction is discussed and other important features are presented. (author)

  2. Development of a system of measuring double-differential cross sections for proton-induced reactions

    Harada, M.; Watanabe, Y.; Sato, K. [Kyushu Univ., Fukuoka (Japan); Meigo, S.

    1997-03-01

    We report the present status of a counter telescope and a data acquisition system which are being developed for the measurement of double-differential cross sections of all light-charged particles emitted from proton-induced reactions on {sup 12}C at incident energies less than 90 MeV. The counter telescope consists of an active collimator made of a plastic scintillator, two thin silicon {Delta}E-detectors and a CsI(Tl) E-detectors with photo-diode readout. Signals from each detector are processed using the data acquisition system consisting of the front-end electronics (CAMAC) and two computers connected with the ethernet LAN: a personal computer as the data collector and server, and a UNIX workstation as the monitor and analyzer. (author)

  3. 5-Azacytidine Induces Cardiac Differentiation of Human Umbilical Cord-Derived Mesenchymal Stem Cells by Activating Extracellular Regulated Kinase

    Qian, Qian; Qian, Hui; Zhang, Xu; Zhu, Wei; Yan, Yongmin; Ye, Shengqin; Peng, Xiujuan; Li, Wei; Xu, Zhe; Sun, Lingyun

    2012-01-01

    5-Azacytidine (5-Aza) induces differentiation of mesenchymal stem cells (MSCs) into cardiomyocytes. However, the underlying mechanisms are not well understood. Our previous work showed that 5-Aza induces human bone marrow-derived MSCs to differentiate into cardiomyocytes. Here, we demonstrated that 5-Aza induced cardiac differentiation of human umbilical cord-derived MSCs (hucMSCs) and explored the potential signaling pathway. Our results showed that hucMSCs had cardiomyocyte phenotypes after 5-Aza treatment. In addition, myogenic cells differentiated from hucMSCs were positive for mRNA and protein of desmin, β-myosin heavy chain, cardiac troponin T, A-type natriuretic peptide, and Nkx2.5. Human diploid lung fibroblasts treated with 5-Aza expressed no cardiac-specific genes. 5-Aza did not induce hucMSCs to differentiate into osteoblasts. Further study revealed that 5-Aza treatment activated extracellular signal related kinases (ERK) in hucMSCs, but protein kinase C showed no response to 5-Aza administration. U0126, a specific inhibitor of ERK, could inhibit 5-Aza-induced expression of cardiac-specific genes and proteins in hucMSCs. Increased phosphorylation of signal transducers and activators of transcription 3, and up-regulation of myocyte enhancer-binding factor-2c and myogenic differentiation antigen in 5-Aza-treated hucMSCs were also suppressed by U0126. Taken together, these results suggested that sustained activation of ERK by 5-Aza contributed to the induction of the differentiation of hucMSCs into cardiomyocytes in vitro. PMID:21476855

  4. Differentiation inducing factor-1 (DIF-1) induces gene and protein expression of the Dictyostelium nuclear calmodulin-binding protein nucleomorphin.

    O'Day, Danton H; Poloz, Yekaterina; Myre, Michael A

    2009-02-01

    The nucleomorphin gene numA1 from Dictyostelium codes for a multi-domain, calmodulin binding protein that regulates nuclear number. To gain insight into the regulation of numA, we assessed the effects of the stalk cell differentiation inducing factor-1 (DIF-1), an extracellular signalling molecule, on the expression of numA1 RNA and protein. For comparison, the extracellular signalling molecules cAMP (mediates chemotaxis, prestalk and prespore differentiation) and ammonia (NH(3)/NH(4)(+); antagonizes DIF) were also studied. Starvation, which is a signal for multicellular development, results in a greater than 80% decrease in numA1 mRNA expression within 4 h. Treatment with ammonium chloride led to a greater than 90% inhibition of numA1 RNA expression within 2 h. In contrast, the addition of DIF-1 completely blocked the decrease in numA1 gene expression caused by starvation. Treatment of vegetative cells with cAMP led to decreases in numA1 RNA expression that were equivalent to those seen with starvation. Western blotting after various morphogen treatments showed that the maintenance of vegetative levels of numA1 RNA by DIF-1 in starved cells was reflected in significantly increased numA1 protein levels. Treatment with cAMP and/or ammonia led to decreased protein expression and each of these morphogens suppressed the stimulatory effects of DIF-1. Protein expression levels of CBP4a, a calcium-dependent binding partner of numA1, were regulated in the same manner as numA1 suggesting this potential co-regulation may be related to their functional relationship. NumA1 is the first calmodulin binding protein shown to be regulated by developmental morphogens in Dictyostelium being upregulated by DIF-1 and down-regulated by cAMP and ammonia. PMID:19000924

  5. Differential gene expression and lipid metabolism in fatty liver induced by acute ethanol treatment in mice

    Ethanol induces cumulative liver damage including steatosis, steatohepatitis and cirrhosis. The aim of this study is to investigate the global intrahepatic gene expression profile in the mouse liver treated with ethanol. A single oral dose of 0.5 or 5 g/kg ethanol was administered to male ICR mice, and liver samples were obtained after 6, 24 and 72 h. Histopathological evaluation showed typical fatty livers in the high-dose group at 24 h. Microarray analysis identified 28 genes as being ethanol responsive (two-way ANOVA; p < 0.05), after adjustment by the Benjamini-Hochberg multiple testing correction; these genes displayed ≥ 2-fold induction or repression. The expression of genes that are known to be involved in fatty acid synthesis was examined. The transcript for lipogenic transcription factor, sterol regulatory element (SRE)-binding factor 1 (Srebf1), was upregulated by acute ethanol exposure. Of the genes known to contain SRE or SRE-like sequences and to be regulated by SRE-binding protein 1 (SREBP1), those encoding malic enzyme (Mod1), ATP-citrate lyase (Acly), fatty acid synthase (Fasn) and stearyl-CoA desaturase (Scd1) were induced by ethanol. Quantitative real-time PCR confirmed the changes in the expression levels of the selected genes. The change in the Srebf1 mRNA level correlates well with that of the SREBP1 protein expression as well as its binding to the promoters of the target genes. The present study identifies differentially expressed genes that can be applied to the biomarkers for alcohol-binge-induced fatty liver. These results support the hypothesis by which ethanol-induced steatosis in mice is mediated by the fatty acid synthetic pathway regulated by SREBP1

  6. D609 induces vascular endothelial cells and marrow stromal cells differentiation into neuron-like cells

    Nan WANG; Chun-qing DU; Shao-shan WANG; Kun XIE; Shang-li ZHANG; Jun-ying MIAO

    2004-01-01

    AIM: To investigate the effect of tricyclodecane-9-yl-xanthogenate (D609) on cell differentiation in vascular endothelial cells (VECs) and marrow stromal cells (MSCs). METHODS: Morphological changes were observed under phase contrast microscope. Electron microscope and immunostaining were used for VECs identification. The expressions of neuron-specific enolase (NSE) and glial fibrillary acidic protein (GFAP) were examined by immunohistochemistry. RESULTS: After 6 h of induction with D609, some VECs showed morphological changes characteristic of neurones. 9 h later, more VECs became neuron-like cells. About 30.8 % of VECs displayed positive NSE (P<0.01), while the expression of GFAP was negative. When MSCs were exposed to D609, the cells displayed neuronal morphologies, such as pyramidal cell bodies and processes formed extensive networks at 3 h. 6 h later, almost all of the cells exhibited a typical neuronal appearance, and 85.6 % of MSCs displayed intensive positive NSE, but GFAP did not express. CONCLUSION: D609 induces VECs and MSCs differentiation into neuron-like cells.

  7. Oligonucleotide microarray identifies genes differentially expressed during tumorigenesis of DMBA-induced pancreatic cancer in rats.

    Jun-Chao Guo

    Full Text Available The extremely dismal prognosis of pancreatic cancer (PC is attributed, at least in part, to lack of early diagnosis. Therefore, identifying differentially expressed genes in multiple steps of tumorigenesis of PC is of great interest. In the present study, a 7,12-dimethylbenzanthraene (DMBA-induced PC model was established in male Sprague-Dawley rats. The gene expression profile was screened using an oligonucleotide microarray, followed by real-time quantitative polymerase chain reaction (qRT-PCR and immunohistochemical staining validation. A total of 661 differentially expressed genes were identified in stages of pancreatic carcinogenesis. According to GO classification, these genes were involved in multiple molecular pathways. Using two-way hierarchical clustering analysis, normal pancreas, acute and chronic pancreatitis, PanIN, early and advanced pancreatic cancer were completely discriminated. Furthermore, 11 upregulated and 142 downregulated genes (probes were found by Mann-Kendall trend Monotone test, indicating homologous genes of rat and human. The qRT-PCR and immunohistochemistry analysis of CXCR7 and UBe2c, two of the identified genes, confirmed the microarray results. In human PC cell lines, knockdown of CXCR7 resulted in decreased migration and invasion. Collectively, our data identified several promising markers and therapeutic targets of PC based on a comprehensive screening and systemic validation.

  8. Transcriptional Elongation Factor Elongin A Regulates Retinoic Acid-Induced Gene Expression during Neuronal Differentiation

    Takashi Yasukawa

    2012-11-01

    Full Text Available Elongin A increases the rate of RNA polymerase II (pol II transcript elongation by suppressing transient pausing by the enzyme. Elongin A also acts as a component of a cullin-RING ligase that can target stalled pol II for ubiquitylation and proteasome-dependent degradation. It is not known whether these activities of Elongin A are functionally interdependent in vivo. Here, we demonstrate that Elongin A-deficient (Elongin A−/− embryos exhibit abnormalities in the formation of both cranial and spinal nerves and that Elongin A−/− embryonic stem cells (ESCs show a markedly decreased capacity to differentiate into neurons. Moreover, we identify Elongin A mutations that selectively inactivate one or the other of the aforementioned activities and show that mutants that retain the elongation stimulatory, but not pol II ubiquitylation, activity of Elongin A rescue neuronal differentiation and support retinoic acid-induced upregulation of a subset of neurogenesis-related genes in Elongin A−/− ESCs.

  9. Understanding Cell Shape Phenotypes Associated with Stem Cell Differentiation Induced by Topographical Cues of Nanofiber Microenvironment

    Chen, Desu; Sarkar, Sumona; Losert, Wolfgang

    It is increasingly important to understand cell responses to bioinspired material structures and topographies designed to guide cell functional alterations. In this study, we investigated association between early stage cell morphological response and osteogenic differentiation of human bone marrow stromal cells (hBMSCs) induced by poly(ɛ-caprolactone) (PCL) nanofiber scaffolds (PCL-NF). Accounting for both multi-parametric complexity and biological heterogeneity, we developed an analysis framework based on support vector machines and a multi-cell level averaging method (supercell) to determine the most pronounced cell shape features describing shape phenotypes of cells in PCL-NF compared to cells on flat PCL films. We found that smaller size and more dendritic shape were the major morphological responses of hBMSCs to PCL-NF on day 1 of cell culture. Further, we investigated the shape phenotypes of hBMSCs in PCL-NF of different fiber densities to monitor the transition between 2-D and 3-D topographies. We tracked the genotypic, phenotypic and morphological responses of hBMSCs to different fiber densities at multiple time points to identify correlations between hBMSCs differentiation and early stage morphology in PCL-NF scaffolds.

  10. [Solarium-induced pseudoporphyria and variegate porphyria as rare differential diagnoses of porphyria cutanea tarda].

    Kochs, C; Mühlenstädt, E; Neumann, N J; Hanneken, S

    2009-10-01

    Three patients presented with typical porphyria cutanea tarda-like vesicles, erosions and scars as well as increased fragility, primarily on the back of the hands. In two of the three, porphyrin workup was normal. Skin biopsy was compatible with porphyria cutanea tarda (PCT) or pseudoporphyria. The common aspect in the patients' history was the frequent use of solaria for many years, so that UV-induced pseudoporphyria was diagnosed. Treatment was strict abstention from UV radiation and regular dermatologic controls for signs of skin damage. Porphyrin analysis in the third patient showed normal excretion of total urine porphyrins and precursors; however, fecal porphyrins were elevated with dominating coproporphyrins in HPLC and the plasma fluorescence scan yielded a peak at 625 nm. Subsequent mutation analysis showed a mutation in the protoporphyrinogen oxidase gene, thereby confirming the diagnosis of variegate porphyria. Five months after the initial diagnosis the patient presented with the first acute attack. Further investigations revealed a metastasized carcinoma of the colon, which probably triggered the acute attack. Our cases show rare differential diagnoses in patients presenting with typical PCT-like skin lesions. The discrimination between porphyria cutanea tarda and its differential diagnoses is very important since it has an important impact not only on the treatment modality but also on the course and the prognosis of the disease. PMID:19756434

  11. The BRPF2/BRD1-MOZ complex is involved in retinoic acid-induced differentiation of embryonic stem cells.

    Cho, Hye In; Kim, Min Seong; Jang, Yeun Kyu

    2016-08-01

    The scaffold protein BRPF2 (also called BRD1), a key component of histone acetyltransferase complexes, plays an important role in embryonic development, but its function in the differentiation of embryonic stem cells (ESCs) remains unknown. In the present study, we investigated whether BRPF2 is involved in mouse ESC differentiation. BRPF2 depletion resulted in abnormal formation of embryoid bodies, downregulation of differentiation-associated genes, and persistent maintenance of alkaline phosphatase activity even after retinoic acid-induced differentiation, indicating impaired differentiation of BRPF2-depleted ESCs. We also found reduced global acetylation of histone H3 lysine 14 (H3K14) in BRPF2-depleted ESCs, irrespective of differentiation status. Further, co-immunoprecipitation analysis revealed a physical association between BRPF2 and the histone acetyltransferase MOZ in differentiated ESCs, suggesting the role of BRPF2-MOZ complexes in ESC differentiation. Together, these results suggest that BRPF2-MOZ complexes play an important role in the differentiation of ESCs via H3K14 acetylation. PMID:27256846

  12. Transforming growth factor-β1 induces intestinal myofibroblast differentiation and modulates their migration

    Julia Brenmoehl; Sandra Nicole Miller; Claudia Hofmann; Daniela Vogl; Werner Falk; Jürgen Scholmerich; Gerhard Rogler

    2009-01-01

    AIM:To investigate the effects of transforming growth factor β1 (TGF-β1) on the differentiation of colonic lamina propria fibroblasts (CLPF) into myofibroblasts in vitro. METHODS:Primary CLPF cultures were incubated with TGF-β1 and analyzed for production of α-smooth muscle actin (α-SMA), fibronectin (FN) and FN isoforms. Migration assays were performed in a modified 48-well Boyden chamber. Levels of total and phosphorylated focal adhesion kinase (FAK) in CLPF were analyzed after induction of migration. RESULTS:Incubation of CLPF with TGF-β1 for 2 d did not change α-SMA levels, while TGF-β1 treatment for 6 d significantly increased α-SMA production. Short term incubation (6 h) with TGF-β1 enhanced CLPF migration, while long term treatment (6 d) of CLPF with TGF-β1 reduced migration to 15%-37% compared to untreated cells. FN and FN isoform mRNA expression were increased after short term incubation with TGF-β1 (2 d) in contrast to long term incubation with TGF-β1 for 6 d. After induction of migration, TGF-β1-preincubated CLPF showed higher amounts of FN and its isoforms and lower levels of total and phosphorylated FAK than untreated cells. CONCLUSION:Long term incubation of CLPF with TGF-β1 induced differentiation into myofibroblasts with enhanced α-SMA, reduced migratory potential and FAK phosphorylation, and increased FN production. In contrast, short term contact (6 h) of fibroblasts with TGF-β1 induced a dose-dependent increase of cell migration and FAK phosphorylation without induction of α-SMA production.

  13. Effects of the phytoestrogen coumestrol on RANK-ligand-induced differentiation of osteoclasts

    Phytoestrogens, which have structural similarity to 17β-estradiol, have been reported to act as agonists/antagonists of estrogen in animals and humans. Estrogen is known to have an important role in maintaining bone mass, because the concentration of serum estrogen decreases after menopause and the estrogen deficiency causes bone loss. In this study, we investigated the effects of coumestrol and other phytoestrogens on osteoclast differentiation using estrogen receptor α-transfected RAW264.7 (RAW264.7-ERα) cells. When the cells were cultured with the receptor activator of nuclear factor kappa B-ligand (RANKL), both formation of tartrate-resistant acid phosphatase (TRAP) positive multinucleated cells and TRAP activity were increased compared with control cells that were cultured in the absence of RANKL. Coumestrol decreased RANKL-induced formation of TRAP-positive multinucleated cells and TRAP activity dose-dependently. RANKL-stimulated RAW264.7-ERα cells formed resorption pits on calcium phosphate films and the pit formation was inhibited by coumestrol in a dose-dependent manner. RT-PCR analyses revealed that coumestrol (10 μM) decreased mRNA levels of calcitonin receptor (CTR) and matrix metalloproteinase-9 (MMP9) in RANKL-treated cells. In addition, pretreatment of coumestrol decreased RANKL-induced phosphorylation of extracellular signal-regulated kinases/p44/42 (ERK1/2). These results suggest that coumestrol has an inhibitory effect on the differentiation of osteoclasts, at least partially via ERK1/2 pathway

  14. Multiple Differential Networks Strategy Reveals Carboplatin and Melphalan-Induced Dynamic Module Changes in Retinoblastoma.

    Chen, Cui; Ma, Feng-Wei; Du, Cui-Yun; Wang, Ping

    2016-01-01

    BACKGROUND Retinoblastoma (RB) is the most common malignant tumor of the eye in childhood. The objective of this paper was to investigate carboplatin (CAR)- and melphalan (MEL)-induced dynamic module changes in RB based on multiple (M) differential networks, and to generate systems-level insights into RB progression. MATERIAL AND METHODS To achieve this goal, we constructed M-differential co-expression networks (DCNs), assigned a weight to each edge, and identified seed genes in M DCNs by ranking genes based on their topological features. Starting with seed genes, a module search was performed to explore candidate modules in CAR and MEL condition. M-DMs were detected according to significance evaluations of M-modules, which originated from refinement of candidate modules. Further, we revealed dynamic changes in M-DM activity and connectivity on the basis of significance of Module Connectivity Dynamic Score (MCDS). RESULTS In the present study, M=2, a total of 21 seed genes were obtained. By assessing module search, refinement, and evaluation, we gained 18 2-DMs. Moreover, 3 significant 2-DMs (Module 1, Module 2, and Module 3) with dynamic changes across CAR and MEL condition were determined, and we denoted them as dynamic modules. Module 1 had 27 nodes of which 6 were seed genes and 56 edges. Module 2 was composed of 28 nodes and 54 edges. A total of 28 nodes interacted with 45 edges presented in Module 3. CONCLUSIONS We have identified 3 dynamic modules with changes induced by CAR and MEL in RB, which might give insights in revealing molecular mechanism for RB therapy. PMID:27144687

  15. Coal mining induced land subsidence monitoring using multiband spaceborne differential interferometric synthetic aperture radar data

    Yue, Huanyin; Liu, Guang; Guo, Huadong; Li, Xinwu; Kang, Zhizhong; Wang, Runfeng; Zhong, Xuelian

    2011-01-01

    The differential interferometric synthetic aperture radar (SAR)(DInSAR) technique has been applied to the earth surface deformation monitoring in many areas. In this paper, the DInSAR technique is used to process the spaceborne SAR data including C band ENVISAT ASAR, L band JERS SAR, and ALOS PALSAR data to derive the temporal land subsidence information in the Fengfeng coal mine area, Hebei province in China. Since JERS and ALOS do not have precise orbit, an orbit adjustment must be accomplished before the DInSAR interferogram was formed. Twenty-three differential interferograms are derived to show the temporal change of the land subsidence range and position. At the acquisition time of ENVISAT ASAR, the leveling in the Dashucun coal mine in Fengfeng area was carried, the historical excavation data in 8 coal mines in Fengfeng area from 1992 to 2007 were collected as well. In our analysis, the DInSAR results are compared with leveling data and historical excavation data. The comparison results show the DInSAR subsidence results are consistent with the leveling results and the historical excavation data, and the L band DInSAR shows more advantages than C band in the coal mining induced subsidence monitoring in a rural area. The feasibility and limitations in coal mining induced subsidence monitoring with DInSAR are analyzed, and the possibility of underground mining activity monitoring by spaceborne InSAR data is evaluated. The experimental results show that both C and L band can accomplish monitoring mining area subsidence, but C band has more restricted conditions of its perpendicular baseline. In order to get a satisfactory outcome in mining area subsidence by the DInSAR method, the time series of SAR images of every visit and SAR deformation interferograms should be archived.

  16. Differentiation of bone marrow derived Thy-1+β2M-cells into hepatocytes induced by coculture with transgenic CFSCs

    WANG Yunfang; NAN Xue; ZHANG Rui; LI Yanhua; YUE Wen; YAN Fang; PEI Xuetao

    2004-01-01

    Studies of transplantation in vivo indicted that bone marrow derived stem cells had a potential to differentiate into mature hepatocytes. However, there are lots of doubts and uncertainties in the influencing factors and control agents of effectively inducing stem cell differentiation in vitro, the efficiency of stem cells' differentiation into hepatocytes and differentiated cells' life-span and functional state,etc. In this study, rat bone marrow derived Thy-1+β2M- cells (BDTCs) were induced to differentiate into hepatocytes by co-culturing with CFSC/HGF feeder layers which expressed hHGF efficiently and stably. RT-PCR and immunofluorescent texts proved induced BDTCs expressed infant and adult hepatocyte specific genes. Further more, these cells displayed functions of indocyanine green (ICG) uptake, ammonium metabolism and albumin production. It was shown that growth factors together with hepatic nonparenchyma cells provided a feasible microenvironment for differentiation of bone marrow stem cells into hepatocytes. The studies not only provided a significant biological model for going deep into the mechanism of stem cell plasticity, but also offered a theoretical and technical foundation of gene and stem cell engineering-based regenerative medicine for end-stage liver diseases.

  17. An ester extract of Cochinchina momordica seeds induces differentiation of melanoma B16 F1 cells via MAPKs signaling.

    Zhao, Lian-Mei; Han, Li-Na; Ren, Feng-Zhi; Chen, Shu-Hong; Liu, Li-Hua; Wang, Ming-Xia; Sang, Mei-Xiang; Shan, Bao-En

    2012-01-01

    Cochinchina momordica seeds (CMS) have been widely used due to antitumor activity by Mongolian tribes of China. However, the details of the underlying mechanisms remain unknown. In the present study, we found that an EtOAc (ethyl ester) extract of CMS (CMSEE) induced differentiation and caused growth inhibition of melanoma B16 F1 cells. CMSEE at the concentration of 5-200 μg/ml exhibited strongest anti-proliferative effects on B16 F1 cells among other CMS fractions (water or petroleum ether). Moreover, CMSEE induced melanoma B16 F1 cell differentiation, characterized by dendrite-like outgrowth, increasing melanogenesis production, as well as enhancing tyrosinase activity. Western blot analysis showed that sustained phosphorylation of p38 MAP accompanied by decrease in ERK1/2 and JNK dephosphorylation were involved in CMSEE-induced B16 F1 cell differentiation. Notably, 6 compounds that were isolated and identified may be responsible for inducing differentiation of CMSEE. These results indicated that CMSEE contributes to the differentiation of B16 F1 cells through modulating MAPKs activity, which may throw some light on the development of potentially therapeutic strategies for melanoma treatment. PMID:23098473

  18. Ectopic expression of neurogenin 2 alone is sufficient to induce differentiation of embryonic stem cells into mature neurons.

    Eva C Thoma

    Full Text Available Recent studies show that combinations of defined key developmental transcription factors (TFs can reprogram somatic cells to pluripotency or induce cell conversion of one somatic cell type to another. However, it is not clear if single genes can define a cell̀s identity and if the cell fate defining potential of TFs is also operative in pluripotent stem cells in vitro. Here, we show that ectopic expression of the neural TF Neurogenin2 (Ngn2 is sufficient to induce rapid and efficient differentiation of embryonic stem cells (ESCs into mature glutamatergic neurons. Ngn2-induced neuronal differentiation did not require any additional external or internal factors and occurred even under pluripotency-promoting conditions. Differentiated cells displayed neuron-specific morphology, protein expression, and functional features, most importantly the generation of action potentials and contacts with hippocampal neurons. Gene expression analyses revealed that Ngn2-induced in vitro differentiation partially resembled neurogenesis in vivo, as it included specific activation of Ngn2 target genes and interaction partners. These findings demonstrate that a single gene is sufficient to determine cell fate decisions of uncommitted stem cells thus giving insights into the role of key developmental genes during lineage commitment. Furthermore, we present a promising tool to improve directed differentiation strategies for applications in both stem cell research and regenerative medicine.

  19. Purple Sweet Potato Leaf Extract Induces Apoptosis and Reduces Inflammatory Adipokine Expression in 3T3-L1 Differentiated Adipocytes

    Shou-Lun Lee

    2015-01-01

    Full Text Available Background. Purple sweet potato leaves (PSPL are widely grown and are considered a healthy vegetable in Taiwan. PSPL contain a high content of flavonoids, and the boiling water-extracted PSPL (PSPLE is believed to prevent metabolic syndrome. However, its efficacy has not yet been verified. Therefore, we investigated the effect of PSPLE on adipocytes. Methods. The differentiated 3T3-L1 cells used in this study were derived from preadipocytes that were differentiated into adipocytes using an adipogenic agent (insulin, dexamethasone, and 3-isobutyl-1-methylxanthine; approximately 90% of the cells were differentiated using this method. Results. Treating the differentiated 3T3-L1 cells with PSPLE caused a dose-dependent decrease in the number of adipocytes rather than preadipocytes. In addition, treatment with PSPLE resulted in apoptosis of the differentiated 3T3-L1 cells as determined by DAPI analysis and flow cytometry. PSPLE also increased the expression of cleaved caspase-3 and poly ADP-ribose polymerase (PARP. Furthermore, PSPLE induced downregulation of interleukin-6 (IL-6 and tumor necrosis factor-α (TNF-α gene expression in the differentiated 3T3-L1 cells. Conclusions. These results suggest that PSPLE not only induced apoptosis but also downregulated inflammation-associated genes in the differentiated 3T3-L1 cells.

  20. Soluble Jagged 1/Fc chimera protein induces the differentiation and maturation of bone marrow-derived dendritic cells

    XING FeiYue; LIU Jing; YU Zhe; JI YuHua

    2008-01-01

    A soluble Jagged 1/Fc chimera protein (Jagged 1/Fc) was directly used to induce differentiation and maturation of bone marrow-derived dendritic cells (DCs) in mice in vitro. A model of inducing and am-plifying DCs in vitro was established. The effect of Jagged 1/Fc on morphology of DCs induced by both rmGM-CSF and rmlL-4 was observed under a confocal microscope. A fluorescein-labeled monoclonal antibody staining combined with flow cytometry was applied to detect the effect of Jagged 1/Fc on the expression of CD11c, MHC-Ⅱ, CD86, CD80 and CD40 molecules on the surface of DCs. The results showed that Jagged 1/Fc did not affect the morphological properties of DC differentiation induced by both rmGM-CSF and rmlL-4. But it could promote the differentiation and maturation of DCs induced by both. The effect of it was strikingly different in the expression profile of co-stimulating molecules and the morphologic properties of DCs from lipopolysaccharide (LPS). The levels of MHC-Ⅱ and CD40 molecule expression on the surface of DCs stimulated by Jagged 1/Fc were significantly lower than those stimulated by LPS, and the level of CD80 expression on the surface of DCs induced by Jagged 1/Fc was near to that induced by LPS. Jagged 1/Fc had no influence on the expression of CD86 mole-cule on the surface of DCs. Jagged 1/Fc when used alone could not maintain the growth, differentiation and maturation of DCs. All the findings indicate that Jagged 1/Fc influences the differentiation and maturation of DCs, which is not markedly similar to LPS, providing important evidence for its devel-opment and application as a novel immunosuppressant.

  1. Modulation of the retinoic acid-induced cell apoptosis and differentiation by the human TR4 orphan nuclear receptor

    In our previous studies, the TR4 orphan nuclear receptor (TR4) has been demonstrated to suppress retinoic acid (RA)-induced transactivation via a negative feedback control mechanism and in situ analysis showed that TR4 is extensively expressed in mouse brain, especially in regions where the cells are proliferating. To further study the potential roles of TR4 during cell differentiation, a tetracycline-inducible system with anti-sense TR4 in teratocarcinoma P19 cell lines was generated to analyze the retinoic acid-induced differentiation of these cells. The results indicated that the expression of TR4 reduced by doxycycline anti-sense TR4 would alter the retinoic acid-induced differentiation pathway that results in the changes of cell morphology and cell cycle profile. Unexpectedly, our data further indicated that the RA-induced apoptosis, judging by DNA fragmentation, could also be altered by the induction of anti-sense TR4. Together, these findings provide the first in vivo evidence that an orphan nuclear receptor, such as TR4, may play major roles in the RA-mediated apoptosis or differentiation in P19 cells

  2. Lipopolysaccharide-activated microglial-induced neuroglial cell differentiation in bone marrow mesenchymal stem cells

    Xiaoguang Luo; Chunlin Ge; Yan Ren; Hongmei Yu; Zhe Wu; Qiushuang Wang; Chaodong Zhang

    2008-01-01

    BACKGROUND: Microglia are very sensitive to environmental changes, often becoming activated by pathological conditions. Activated microglia can exert a dual role in injury and repair in various diseases of the central nervous system, including cerebral ischemia, Parkinson's disease, and Alzheimer's disease. OBJECTIVE: An immortal microglial cell line, BV2, was treated with varying concentrations of lipopolysaccharide (LPS) to induce a pathological situation. Supernatant was harvested and incubated with bone marrow mesenchymal stem cells and, concomitantly, bone marrow mesenchymal stem cell differentiation was observed. DESIGN: A controlled observation, in vitro experiment. SETTING: Department of Neurology, First Affiliated Hospital of China Medical University. MATERIALS: Five male 2-3-week-old Sprague Dawley rats were purchased from Animal Laboratory Center of China Medical University and included in this study. The protocol was performed in accordance with ethical guidelines for the use and care of animals. The microglial cell line BV2 was produced by Cell Research Institute of Chinese Academy of Sciences. LPS was produced by Sigma Company, USA. METHODS: This study was performed in the Central Laboratory of China Medical University from September 2006 to March 2007. Rat femoral and tibial bone marrow was collected for separation and primary culture of bone marrow mesenchymal stem cells. Bone marrow mesenchymal stem cell cultures were divided into 5 groups: control group, non-activated group, as well as low-, medium-, and high-dose LPS groups. In the control group, bone marrow mesenchymal stem cells were cultured with Dulbecco's modified Eagle's medium (DMEM) supplemented with fetal bovine serum (volume fraction 0.1). In the non-activated group, bone marrow mesenchymal stem cells were incubated with non-activated BV2 supernatant. In the low-, medium-, and high-dose LPS groups, bone marrow mesenchymal stem cells were incubated with LPS (0.01, 0.1 and 1

  3. Identification of Differentially Expressed Genes Induced by Ammonium Nitrogen in Rice Using mRNA Differential Display

    ZHU Guo-hui; HUANG Zhuo-lie

    2008-01-01

    RNAs isolated from ammonium- and nitrate-treated rice leaves were used to screen differentially expressed genes through mRNA differential display. A total of 72 bands appeared significant differences and some of them were further confirmed by reverse Northern and Northem blot. The results showed that two genes, A-02 (Oryza sativa drought stress related mRNA) and A-03 (Zea mays partial mRNA for TFIIB-related protein) were highly up-regulated in the ammonium-fed rice leaves. The enzyme assays showed that the activities of the two anti-oxidative enzymes, catalase and peroxidase, and the content of a non-enzymic antioxidant, glutathione, were significantly higher in the ammonium-fed rice leaves than those in the nitrate-fed ones, indicating that the ammonium nutrition might be beneficial for rice plants to improve the stress resistance during growth and development.

  4. Differential responses in pear and quince genotypes induced by Fe deficiency and bicarbonate.

    Donnini, Silvia; Castagna, Antonella; Ranieri, Annamaria; Zocchi, Graziano

    2009-07-15

    Most of the studies carried out on Fe deficiency condition in arboreous plants have been performed, with the exception of those carried out on plants grown in the field, in hydroponic culture utilizing a total iron depletion growth condition. This can cause great stress to plants. By introducing Fe deficiency induced by the presence of bicarbonate, we found significant differences between Pyrus communis L. cv. Conference and Cydonia oblonga Mill. BA29 and MA clones, characterized by different levels of tolerance to chlorosis. Pigment content and the main protein-pigment complexes were investigated by HPLC and protein gel blot analysis, respectively. While similar changes in the structural organization of photosystems (PSs) were observed in both species under Fe deficiency, a different reorganization of the photosynthetic apparatus was found in the presence of bicarbonate between tolerant and susceptible genotypes, in agreement with the photosynthetic electron transport rate measured in isolated thylakoids. In order to characterize the intrinsic factors determining the efficiency of iron uptake in a tolerant genotype, the main mechanisms induced by Fe deficiency in Strategy I species, such as Fe3+-chelate reductase (EC 1.16.1.7) and H+-ATPase (EC 3.6.3.6) activities, were also investigated. We demonstrate that physiological and biochemical root responses in quince and pear are differentially affected by iron starvation and bicarbonate supply, and we show a high correlation between tolerance and Strategy I activation. PMID:19269060

  5. Novel analogs targeting histone deacetylase suppress aggressive thyroid cancer cell growth and induce re-differentiation.

    Jang, S; Yu, X-M; Odorico, S; Clark, M; Jaskula-Sztul, R; Schienebeck, C M; Kupcho, K R; Harrison, A D; Winston-McPherson, G N; Tang, W; Chen, H

    2015-08-01

    To develop novel therapies for aggressive thyroid cancers, we have synthesized a collection of histone deacetylase (HDAC) inhibitor analogs named AB1 to AB13, which have different linkers between a metal chelating group and a hydrophobic cap. The purpose of this study was to screen out the most effective compounds and evaluate the therapeutic efficacy. AB2, AB3 and AB10 demonstrated the lowest half-maximal inhibitory concentration (IC50) values in one metastatic follicular and two anaplastic thyroid cancer cell lines. Treatment with each of the three ABs resulted in an increase in apoptosis markers, including cleaved poly adenosine diphosphate ribose polymerase (PARP) and cleaved caspase 3. Additionally, the expression of cell-cycle regulatory proteins p21(WAF1) and p27(Kip1) increased with the treatment of ABs while cyclin D1 decreased. Furthermore, AB2, AB3 and AB10 were able to induce thyrocyte-specific genes in the three thyroid cancer cell lines indicated by increased expression levels of sodium iodide symporter, paired box gene 8, thyroid transcription factor 1 (TTF1), TTF2 and thyroid-stimulating hormone receptors. AB2, AB3 and AB10 suppress thyroid cancer cell growth via cell-cycle arrest and apoptosis. They also induce cell re-differentiation, which could make aggressive cancer cells more susceptible to radioactive iodine therapy. PMID:26251030

  6. Nanopit-induced osteoprogenitor cell differentiation: The effect of nanopit depth.

    Davison, Martin J; McMurray, Rebecca J; Smith, Carol-Anne; Dalby, Matthew J; Meek, Rm Dominic

    2016-01-01

    We aimed to assess osteogenesis in osteoprogenitor cells by nanopits and to assess optimal feature depth. Topographies of depth 80, 220 and 333 nm were embossed onto polycaprolactone discs. Bone marrow-derived mesenchymal stromal cells were seeded onto polycaprolactone discs, suspended in media and incubated. Samples were fixed after 3 and 28 days. Cells were stained for the adhesion molecule vinculin and the osteogenic transcription factor RUNX2 after 3 days. Adhesion was lowest on planar controls and it was the shallowest, and 80-nm-deep pits supported optimal adhesion formation. Deep pits (80 and 220 nm) induced most RUNX2 accumulation. After 28 days, osteocalcin and osteopontin expression were used as markers of osteoblastic differentiation. Deep pits (220 nm) produced cells with the highest concentrations of osteopontin and osteocalcin. All topographies induced higher expression levels than controls. We demonstrated stimulation of osteogenesis in a heterogeneous population of mesenchymal stromal cells. All nanopit depths gave promising results with an optimum depth of 220 nm after 28 days. Nanoscale modification of implant surfaces could optimise fracture union or osteointegration. PMID:27298716

  7. Differential Effects of Olanzapine and Haloperidol on MK-801-induced Memory Impairment in Mice

    Song, Jae Chun; Seo, Mi Kyoung; Park, Sung Woo; Lee, Jung Goo; Kim, Young Hoon

    2016-01-01

    Objective We investigated the differential effects of the antipsychotic drugs olanzapine and haloperidol on MK-801-induced memory impairment and neurogenesis in mice. Methods MK-801 (0.1 mg/kg) was administered 20 minutes prior to behavioral testing over 9 days. Beginning on the sixth day of MK-801 treatment, either olanzapine (0.05 mg/kg) or haloperidol (0.05 mg/kg) was administered 40 minutes prior to MK-801 for the final 4 days. Spatial memory performance was measured using a Morris water maze (MWM) test for 9 days (four trials/day). Immunohistochemistry with bromodeoxyuridine (BrdU) was used to identify newborn cells labeled in tissue sections from the dentate gyrus of the hippocampus. Results MK-801 administration over 9 days significantly impaired memory performance in the MWM test compared to untreated controls (p<0.05) and these deficits were blocked by treatment with olanzapine (p<0.05) but not haloperidol. The administration of MK-801 also resulted in a decrease in the number of BrdU-labeled cells in the dentate gyrus (28.6%; p<0.01), which was prevented by treatment with olanzapine (p<0.05) but not haloperidol. Conclusion These results suggest that olanzapine has a protective effect against cognitive impairments induced by MK-801 in mice via the stimulating effects of neurogenesis. PMID:27489382

  8. Co-expression analysis of differentially expressed genes in hepatitis C virus-induced hepatocellular carcinoma.

    Song, Qingfeng; Zhao, Chang; Ou, Shengqiu; Meng, Zhibin; Kang, Ping; Fan, Liwei; Qi, Feng; Ma, Yilong

    2015-01-01

    The aim of the current study was to investigate the molecular mechanisms underlying hepatitis C virus (HCV)-induced hepatocellular carcinoma (HCC) using the expression profiles of HCV-infected Huh7 cells at different time points. The differentially expressed genes (DEGs) were identified with the Samr package in R software once the data were normalized. Functional and pathway enrichment analysis of the identified DEGs was also performed. Subsequently, MCODE in Cytoscape software was applied to conduct module analysis of the constructed co-expression networks. A total of 1,100 DEGs were identified between the HCV-infected and control samples at 12, 18, 24 and 48 h post-infection. DEGs at 24 and 48 h were involved in the same signaling pathways and biological processes, including sterol biosynthetic processes and tRNA amino-acylation. There were 22 time series genes which were clustered into 3 expression patterns, and the demarcation point of the 2 expression patterns that 401 overlapping DEGs at 24 and 48 h clustered into was 24 h post-infection. tRNA synthesis-related biological processes emerged at 24 and 48 h. Replication and assembly of HCV in HCV-infected Huh7 cells occurred mainly at 24 h post-infection. In view of this, the screened time series genes have the potential to become candidate target molecules for monitoring, diagnosing and treating HCV-induced HCC. PMID:25339452

  9. Anticancer chemotherapy-induced intratumoral recruitment and differentiation of antigen-presenting cells.

    Ma, Yuting; Adjemian, Sandy; Mattarollo, Stephen R; Yamazaki, Takahiro; Aymeric, Laetitia; Yang, Heng; Portela Catani, João Paulo; Hannani, Dalil; Duret, Helene; Steegh, Kim; Martins, Isabelle; Schlemmer, Frederic; Michaud, Mickaël; Kepp, Oliver; Sukkurwala, Abdul Qader; Menger, Laurie; Vacchelli, Erika; Droin, Nathalie; Galluzzi, Lorenzo; Krzysiek, Roman; Gordon, Siamon; Taylor, Philip R; Van Endert, Peter; Solary, Eric; Smyth, Mark J; Zitvogel, Laurence; Kroemer, Guido

    2013-04-18

    The therapeutic efficacy of anthracyclines relies on antitumor immune responses elicited by dying cancer cells. How chemotherapy-induced cell death leads to efficient antigen presentation to T cells, however, remains a conundrum. We found that intratumoral CD11c(+)CD11b(+)Ly6C(hi) cells, which displayed some characteristics of inflammatory dendritic cells and included granulomonocytic precursors, were crucial for anthracycline-induced anticancer immune responses. ATP released by dying cancer cells recruited myeloid cells into tumors and stimulated the local differentiation of CD11c(+)CD11b(+)Ly6C(hi) cells. Such cells efficiently engulfed tumor antigens in situ and presented them to T lymphocytes, thus vaccinating mice, upon adoptive transfer, against a challenge with cancer cells. Manipulations preventing tumor infiltration by CD11c(+)CD11b(+)Ly6C(hi) cells, such as the local overexpression of ectonucleotidases, the blockade of purinergic receptors, or the neutralization of CD11b, abolished the immune system-dependent antitumor activity of anthracyclines. Our results identify a subset of tumor-infiltrating leukocytes as therapy-relevant antigen-presenting cells. PMID:23562161

  10. Ram ion scattering caused by space shuttle v x B induced differential charging

    Observations of secondary, high-inclination ions streams have been reported in the literature. The authors of these previous papers attributed the source of the secondary ions to a disturbed region in the plasma about 10 m form the space shuttle Orbiter. A new theory has been developed which shows how v x B induces differential charging on the plasma diagnostics package (PDP) can scatter the ram ion flux. Some of these ions are reflected back to the PDP and may be the source of the observed ion distributions. The effect is unique to large spacecraft; it occurs only when the magnitude of the induced v x B potentials are much larger than the electron thermal energy and of the order of the ion ram energy. That the ion streams observed at large angles must have been reflected from the PDP surface is demonstrated with three-dimensional sheath and particle trajectory calculations using the low earth orbit version of the NASA Charging Analyzer Program (NASCAP/LEO)

  11. UV-induced DNA excision repair in rat fibroblasts during immortalization and terminal differentiation in vitro

    UV-induced DNA excision repair was studied as DNA repair synthesis and dimer removal in rat fibroblast cultures, initiated from either dense or sparse inocula of primary cells grown from skin biopsies. During passaging in vitro an initial increase in DNA repair synthesis, determined both autoradiographically as unscheduled DNA synthesis (UDS) and by means of the BrdU photolysis assay as the number and average size of repair patches, was found to be associated with a morphological shift from small spindle-shaped to large pleiomorphic cells observed over the first twenty generations. In cell populations in growth crisis, a situation exclusively associated with thin-inoculum cultures in which the population predominantly consisted of large pleiomorphic cells, UDS was found to occur at a low level. After development of secondary cultures into immortal cell lines, both repair synthesis and morphology appeared to be the same as in the original primary spindle-shaped cells. At all passages the capacity to remove UV-induced pyrimidine dimers was found to be low, as indicated by the persistence of Micrococcus luteus UV endonuclease-sensitive sites. These results are discussed in the context of terminal differentiation and immortalization of rat fibroblasts upon establishment in vitro

  12. Lapatinib induces autophagic cell death and differentiation in acute myeloblastic leukemia

    Chen YJ

    2016-07-01

    Full Text Available Yu-Jen Chen,1–4 Li-Wen Fang,5 Wen-Chi Su,6,7 Wen-Yi Hsu,1 Kai-Chien Yang,1 Huey-Lan Huang8 1Department of Medical Research, 2Department of Radiation Oncology, Mackay Memorial Hospital, 3Institute of Traditional Medicine, School of Medicine, National Yang-Ming University, 4Institute of Pharmacology, Taipei Medical University, Taipei, 5Department of Nutrition, I-Shou University, Kaohsiung, 6Research Center for Emerging Viruses, China Medical University Hospital, 7Graduate Institute of Clinical Medical Science, China Medical University, Taichung, 8Department of Bioscience Technology, College of Health Science, Chang Jung Christian University, Tainan, Taiwan, Republic of China Abstract: Lapatinib is an oral-form dual tyrosine kinase inhibitor of epidermal growth factor receptor (EGFR or ErbB/Her superfamily members with anticancer activity. In this study, we examined the effects and mechanism of action of lapatinib on several human leukemia cells lines, including acute myeloid leukemia (AML, chronic myeloid leukemia (CML, and acute lymphoblastic leukemia (ALL cells. We found that lapatinib inhibited the growth of human AML U937, HL-60, NB4, CML KU812, MEG-01, and ALL Jurkat T cells. Among these leukemia cell lines, lapatinib induced apoptosis in HL-60, NB4, and Jurkat cells, but induced nonapoptotic cell death in U937, K562, and MEG-01 cells. Moreover, lapatinib treatment caused autophagic cell death as shown by positive acridine orange staining, the massive formation of vacuoles as seen by electronic microscopy, and the upregulation of LC3-II, ATG5, and ATG7 in AML U937 cells. Furthermore, autophagy inhibitor 3-methyladenine and knockdown of ATG5, ATG7, and Beclin-1 using short hairpin RNA (shRNA partially rescued lapatinib-induced cell death. In addition, the induction of phagocytosis and ROS production as well as the upregulation of surface markers CD14 and CD68 was detected in lapatinib-treated U937 cells, suggesting the induction of

  13. Copper induced immunotoxicity promote differential apoptotic pathways in spleen and thymus

    proliferated. Apart from the role of inflammation, our findings also have identified the role of other partially responsible apoptotic molecules like p27, p73, p62, poly (ADP-ribose) polymerase (PARP) that differentially changed due to copper treatment in spleen and thymus of Swiss albino mice. Present study firstly demonstrates how apoptotic pathways differentially regulate copper induced immunosuppression

  14. Differentiation between glioma recurrence and radiation-induced brain injuries using perfusion-weighted MR imaging

    Objective: To evaluate the feasibility of perfusion weighted imaging (PWI) in the differentiation of recurrent glioma and radiation-induced brain injuries. Methods: Fifteen patients with previously resected and irradiated glioma, presenting newly developed abnormal enhancement, were included in the study. The final diagnosis was determined either histologically or clinicoradiologically. PWI was obtained with a gradient echo echo-planar-imaging (GRE-EPI) sequence. The normalized rCBV ratio [CBV (abnormal enhancement)/CBV (contralateral tissue)], rCBF ratio [CBF (abnormal enhancement)/CBF (contralateral tissue)] and rMTT ratio [(MTT abnormal enhancement)/MTT (contralateral tissue)] were calculated, respectively. The regions of interest (ROIs) consisting of 20-40 mm2 were placed in the abnormal enhanced areas on postcontrast T1-weighted images. Ten to fifteen ROIs measurements were performed in each lesion and the mean value was obtained. Mann-Whitney test was used to determine whether there was a difference in the rCBV/rCBF/MTT ratios between glioma recurrence and radiated injuries. Results: Nine of the 15 patients were proved recurrent glioma, 6 were proved radiation-induced brain injuries. The mean rCBV ratio [2.87 (0.70-4.91)] in glioma recurrence was markedly higher than that [0.70 (0.12-1.62)] in radiation injuries (Z=-2.55, P<0.05). The mean rCBF ratio [1.89 (0.64-3.96)] in glioma recurrence was markedly higher than that [0.56 (0.12-2.08)] in radiation injuries (Z=-2.08, P<0.05). The areas under rCBV and rCBF ROC curve were 0.893 and 0.821. If the rCBV ratio ≤ 0.77, the diagnosis sensitivity of radiation-induced brain injuries was 100.0%; If ≥ 2.44, the diagnosis specificity of recurrent glioma was 100.0%. Conclusion: PWI was an effective technique in distinguishing glioma recurrence from radiation injuries and rCBV and rCBF ratios were of great value in the differentiation. (authors)

  15. The tankyrase-specific inhibitor JW74 affects cell cycle progression and induces apoptosis and differentiation in osteosarcoma cell lines

    Wnt/β-catenin is a major regulator of stem cell self-renewal and differentiation and this signaling pathway is aberrantly activated in a several cancers, including osteosarcoma (OS). Attenuation of Wnt/β-catenin activity by tankyrase inhibitors is an appealing strategy in treatment of OS. The efficacy of the tankyrase inhibitor JW74 was evaluated in three OS cell lines (KPD, U2OS, and SaOS-2) both at the molecular and functional level. At the molecular level, JW74 induces stabilization of AXIN2, a key component of the β-catenin destruction complex, resulting in reduced levels of nuclear β-catenin. At the functional level, JW74 induces reduced cell growth in all three tested cell lines, in part due to a delay in cell cycle progression and in part due to an induction of caspase-3-mediated apoptosis. Furthermore, JW74 induces differentiation in U2OS cells, which under standard conditions are resistant to osteogenic differentiation. JW74 also enhances differentiation of OS cell lines, which do not harbor a differentiation block. Interestingly, microRNAs (miRNAs) of the let-7 family, which are known tumor suppressors and inducers of differentiation, are significantly upregulated following treatment with JW74. We demonstrate for the first time that tankyrase inhibition triggers reduced cell growth and differentiation of OS cells. This may in part be due to an induction of let-7 miRNA. The presented data open for novel therapeutic strategies in the treatment of malignant OS

  16. Ascorbic acid enhances the cardiac differentiation of induced pluripotent stem cells through promoting the proliferation of cardiac progenitor cells

    Nan Cao; Bin Wei; Liu Wang; Ying Jin; Huang-Tian Yang; Zumei Liu; Zhongyan Chen; Jia Wang; Taotao Chen; Xiaoyang Zhao; Yu Ma; Lianju Qin; Jiuhong Kang

    2012-01-01

    Generation of induced pluripotent stem cells (iPSCs) has opened new avenues for the investigation of heart diseases,drug screening and potential autologous cardiac regeneration.However,their application is hampered by inefficient cardiac differentiation,high interline variability,and poor maturation of iPSC-derived cardiomyoeytes (iPS-CMs).To identify efficient inducers for cardiac differentiation and maturation of iPSCs and elucidate the mechanisms,we systematically screened sixteen cardiomyocyte inducers on various murine (m) iPSCs and found that only ascorbic acid (AA) consistently and robustly enhanced the cardiac differentiation of eleven lines including eight without spontaneous cardiogenic potential.We then optimized the treatment conditions and demonstrated that differentiation day 2-6,a period for the specification of cardiac progenitor cells (CPCs),was a critical time for AA to take effect.This was further confirmed by the fact that AA increased the expression of cardiovascular but not mesodermal markers.Noteworthily,AA treatment led to approximately 7.3-fold (miPSCs) and 30.2-fold (human iPSCs) augment in the yield of iPS-CMs.Such effect was attributed to a specific increase in the proliferation of CPCs via the MEK-ERK1/2 pathway by promoting collagen synthesis.In addition,AA-induced cardiomyocytes showed better sareomerie organization and enhanced responses of action potentials and calcium transients to β-adrenergic and muscarinic stimulations.These findings demonstrate that AA is a suitable cardiomyocyte inducer for iPSCs to improve cardiac differentiation and maturation simply,universally,and efficiently.These findings also highlight the importance of stimulating CPC proliferation by manipulating extracellular microenvironment in guiding cardiac differentiation of the pluripotent stem cells.

  17. ERβ induces the differentiation of cultured osteoblasts by both Wnt/β-catenin signaling pathway and estrogen signaling pathways

    Yin, Xinhua [Department of Spine Surgery, Xiangya Hospital of Central South University, Changsha (China); Wang, Xiaoyuan [Department of Nephrology, Xi An Honghui Hospital, Xi an (China); Hu, Xiongke; Chen, Yong; Zeng, Kefeng [Department of Spine Surgery, Xiangya Hospital of Central South University, Changsha (China); Zhang, Hongqi, E-mail: zhq9699@126.com [Department of Spine Surgery, Xiangya Hospital of Central South University, Changsha (China)

    2015-07-01

    Although 17β-estradial (E2) is known to stimulate bone formation, the underlying mechanisms are not fully understood. Recent studies have implicated the Wnt/β-catenin pathway as a major signaling cascade in bone biology. The interactions between Wnt/β-catenin signaling pathway and estrogen signaling pathways have been reported in many tissues. In this study, E2 significantly increased the expression of β-catenin by inducing phosphorylations of GSK3β at serine 9. ERβ siRNAs were transfected into MC3T3-E1 cells and revealed that ERβ involved E2-induced osteoblasts proliferation and differentiation via Wnt/β-catenin signaling. The osteoblast differentiation genes (BGP, ALP and OPN) and proliferation related gene (cyclin D1) expression were significantly induced by E2-mediated ERβ. Furthermore immunofluorescence and immunoprecipitation analysis demonstrated that E2 induced the accumulation of β-catenin protein in the nucleus which leads to interaction with T-cell-specific transcription factor/lymphoid enhancer binding factor (TCF/LEF) transcription factors. Taken together, these findings suggest that E2 promotes osteoblastic proliferation and differentiation by inducing proliferation-related and differentiation-related gene expression via ERβ/GSK-3β-dependent Wnt/β-catenin signaling pathway. Our findings provide novel insights into the mechanisms of action of E2 in osteoblastogenesis. - Highlights: • 17β-estradial (E2) promotes GSK3-β phosphorylation. • E2 activates the Wnt/β-catenin signaling pathway. • The Wnt/β-catenin signaling pathway interacts with estrogen signaling pathways. • E2-mediated ER induced osteoblast differentiation and proliferation related genes expression.

  18. ERβ induces the differentiation of cultured osteoblasts by both Wnt/β-catenin signaling pathway and estrogen signaling pathways

    Although 17β-estradial (E2) is known to stimulate bone formation, the underlying mechanisms are not fully understood. Recent studies have implicated the Wnt/β-catenin pathway as a major signaling cascade in bone biology. The interactions between Wnt/β-catenin signaling pathway and estrogen signaling pathways have been reported in many tissues. In this study, E2 significantly increased the expression of β-catenin by inducing phosphorylations of GSK3β at serine 9. ERβ siRNAs were transfected into MC3T3-E1 cells and revealed that ERβ involved E2-induced osteoblasts proliferation and differentiation via Wnt/β-catenin signaling. The osteoblast differentiation genes (BGP, ALP and OPN) and proliferation related gene (cyclin D1) expression were significantly induced by E2-mediated ERβ. Furthermore immunofluorescence and immunoprecipitation analysis demonstrated that E2 induced the accumulation of β-catenin protein in the nucleus which leads to interaction with T-cell-specific transcription factor/lymphoid enhancer binding factor (TCF/LEF) transcription factors. Taken together, these findings suggest that E2 promotes osteoblastic proliferation and differentiation by inducing proliferation-related and differentiation-related gene expression via ERβ/GSK-3β-dependent Wnt/β-catenin signaling pathway. Our findings provide novel insights into the mechanisms of action of E2 in osteoblastogenesis. - Highlights: • 17β-estradial (E2) promotes GSK3-β phosphorylation. • E2 activates the Wnt/β-catenin signaling pathway. • The Wnt/β-catenin signaling pathway interacts with estrogen signaling pathways. • E2-mediated ER induced osteoblast differentiation and proliferation related genes expression

  19. Variation of radiation sensitivity of Friend Erythroleukemia cells cultured in the presence of the differentiation inducer DMSO

    Differentiation of Friend erythroleukemia cells (FELC) was induced with 1.5% dimethyl sulfoxide (DMSO) in the culture medium. Cell growth, erythroid differentiation, and radiosensitivity of the proliferative capacity of the cells were measured and compared to a noninduced control culture of identical age. Induced cells first appeared on Day 2 after DMSO addition, and increased to a maximum of 80 to 90% of the cell population on Day 5, whereas in the control culture, induction was less than 2% of the cells. Radiosensitivity of the cells in the induced culture relative to that of cells in the control culture, showed an age-dependent variation. On days 1 and 2 after DMSO addition, the cells in the induced culture were less radiosensitive than those in the control culture. At later times, this relationship was reversed, and between days 3 and 5 the clonable cells in the induced culture were less radiosensitive than those in the control culture. These results suggest that the metabolic events associated with commitment of FELC to differentiate affect their ability to cope with the radiation-induced lesions underlying the loss of division capacity

  20. Cholinergic activation enhances retinoic acid-induced differentiation in the human NB-4 acute promyelocytic leukemia cell line.

    Chotirat, Sadudee; Suriyo, Tawit; Hokland, Marianne; Hokland, Peter; Satayavivad, Jutamaad; Auewarakul, Chirayu U

    2016-07-01

    The non-neuronal cholinergic system (NNCS) has been shown to play a role in regulating hematopoietic differentiation. We determined the expression of cholinergic components in leukemic cell lines by Western blotting and in normal leukocyte subsets by flow cytometry and found a heterogeneous expression of choline acetyltransferase (ChAT), acetylcholinesterase (AChE), choline transporter (CHT), M3 muscarinic acetylcholine receptor (M3-mAChR) and α7 nicotinic acetylcholine receptor (α7-nAChR). We then evaluated NNCS role in differentiation of human NB-4 acute promyelocytic leukemia cell line and discovered a dramatic induction of M3-mAChR after all-trans retinoic acid (ATRA) treatment (p<0.0001). Adding carbachol which is a cholinergic agonist to the ATRA treatment resulted in an increase of a granulocytic differentiation marker (CD11b) as compared with ATRA treatment alone (p<0.05), indicating that cholinergic activation enhanced ATRA in inducing NB-4 maturation. The combination of carbachol and ATRA treatment for 72h also resulted in decreased viability and increased cleaved caspase-3 expression when compared with ATRA treatment alone (p<0.05). However, this combination did not cause poly (ADP-ribose) polymerase (PARP) cleavage. Overall, we have shown that NB-4 cells expressed M3-mAChR in a differentiation-dependent manner and cholinergic stimulation induced maturation and death of ATRA-induced differentiated NB-4 cells. PMID:27282572

  1. Development of a rapid culture method to induce adipocyte differentiation of human bone marrow-derived mesenchymal stem cells

    Ninomiya, Yuichi [Translational Research Center, Saitama International Medical, Saitama Medical University, 1397-1 Yamane, Hidaka, Saitama 350-1298 (Japan); Sugahara-Yamashita, Yzumi; Nakachi, Yutaka; Tokuzawa, Yoshimi; Okazaki, Yasushi [Division of Functional Genomics and Systems Medicine, Research Center for Genomic Medicine, Saitama Medical University, Saitama 350-1241 (Japan); Nishiyama, Masahiko, E-mail: yamacho@saitama-med.ac.jp [Translational Research Center, Saitama International Medical, Saitama Medical University, 1397-1 Yamane, Hidaka, Saitama 350-1298 (Japan)

    2010-04-02

    Human mesenchymal stem cells (hMSCs) derived from bone marrow are multipotent stem cells that can regenerate mesenchymal tissues such as adipose, bone or muscle. It is thought that hMSCs can be utilized as a cell resource for tissue engineering and as human models to study cell differentiation mechanisms, such as adipogenesis, osteoblastogenesis and so on. Since it takes 2-3 weeks for hMSCs to differentiate into adipocytes using conventional culture methods, the development of methods to induce faster differentiation into adipocytes is required. In this study we optimized the culture conditions for adipocyte induction to achieve a shorter cultivation time for the induction of adipocyte differentiation in bone marrow-derived hMSCs. Briefly, we used a cocktail of dexamethasone, insulin, methylisobutylxanthine (DIM) plus a peroxisome proliferator-activated receptor {gamma} agonist, rosiglitazone (DIMRo) as a new adipogenic differentiation medium. We successfully shortened the period of cultivation to 7-8 days from 2-3 weeks. We also found that rosiglitazone alone was unable to induce adipocyte differentiation from hMSCs in vitro. However, rosiglitazone appears to enhance hMSC adipogenesis in the presence of other hormones and/or compounds, such as DIM. Furthermore, the inhibitory activity of TGF-{beta}1 on adipogenesis could be investigated using DIMRo-treated hMSCs. We conclude that our rapid new culture method is very useful in measuring the effect of molecules that affect adipogenesis in hMSCs.

  2. Inhibitory effect of leptin on rosiglitazone-induced differentiation of primary adipocytes prepared from TallyHO/Jng mice

    Research highlights: → In this study, we investigated the effects of leptin on adipocyte differentiation prepared from subcutaneous fat of TallyHo mice. → Leptin inhibited the adipocytes differentiation at physiological concentration via inhibition of PPARγ expression. → Inhibitors of ERK and STAT1 restored the leptin's inhibitory activity both in vitro and in vivo. -- Abstract: The effects of leptin on rosiglitazone-induced adipocyte differentiation were investigated in the primary adipocytes prepared from subcutaneous fat of TallyHO/Jng (TallyHO) mouse, a recently developed model animal for type 2 diabetes mellitus (T2DM). The treatment of leptin inhibited the rosiglitazone-induced adipocyte differentiation with a decreased expression of peroxisome proliferator-activated receptor γ (PPARγ) a key adipogenic transcription factor, both in mRNA and protein levels. Leptin (10 nM) was sufficient to inhibit the adipocyte differentiation, which seemed to come from increased expression of leptin receptor genes in the fat of TallyHO mice. The inhibition of adipogenesis by leptin was restored by the treatment of inhibitors for extracellular-signal-regulated kinase (ERK) (PD98059) and signal transducer and activator of transcription-1 (STAT1) (fludarabine). Furthermore, in vivo intraperitoneal administration of PD98059 and fludarabine increased the PPARγ expression in the subcutaneous fat of TallyHO mice. These data suggest that leptin could inhibit the PPARγ expression and adipocyte differentiation in its physiological concentration in TallyHO mice.

  3. A comparison of the chemical and liver extract-induced hepatic differentiation of adipose derived stem cells.

    Nhung, Truong Hai; Nam, Nguyen Hai; Nguyen, Nguyen Thi Kim; Nghia, Huynh; Van Thanh, Nguyen; Ngoc, Phan Kim; Van Pham, Phuc

    2015-11-01

    Adipose-derived stem cells (ADSCs) have been put forward as promising therapeutics for end-stage liver disease (ESLD). In the present study, we compared the effects of defined chemicals and liver extract on the hepatic differentiation of ADSCs. ADSCs were isolated according to the method described in our previously published study. Subsequently, the differentiation of ADSCs was induced separately by chemicals (including hepatic growth factor (HGF), fibroblast growth factor (FGF), and oncostatin M (OSM)) and liver extract (30 μg/ml) in a total period of 21 d. The efficiency of hepatic differentiation was evaluated by changes in the cell morphology, gene expression, and cellular function. The results showed that the liver extract promoted the hepatic differentiation of ADSCs to a significantly greater extent than the chemicals. In the group of ADSCs treated with liver extract, changes in the cell morphology began sooner, and the expression of alpha-FP and albumin genes was higher than that in the chemically treated group. The ADSCs in both the groups stained positive for anti-alpha trypsin (AAT) and albumin markers. The cells also exhibited glycogen storage capacity. Therefore, we concluded that the liver extract could efficiently induce the differentiation of ADSCs into hepatocyte-like cells. This study reveals the potential of mesenchymal stem cell differentiation in the liver extract, which supports further preclinical and clinical research on the application of ADSCs in ESLD treatment. PMID:26275888

  4. Inhibitory effect of leptin on rosiglitazone-induced differentiation of primary adipocytes prepared from TallyHO/Jng mice

    Kim, Ki Young; Kim, Joo Young; Sung, Yoon-Young; Jung, Won Hoon; Kim, Hee-Youn; Park, Ji Seon; Cheon, Hyae Gyeong [Medicinal Science Division, Korea Research Institute of Chemical Technology, 100 Jang-dong, Yuseong, 305-600 Daejon (Korea, Republic of); Rhee, Sang Dal, E-mail: sdrhee@krict.re.kr [Medicinal Science Division, Korea Research Institute of Chemical Technology, 100 Jang-dong, Yuseong, 305-600 Daejon (Korea, Republic of)

    2011-03-25

    Research highlights: {yields} In this study, we investigated the effects of leptin on adipocyte differentiation prepared from subcutaneous fat of TallyHo mice. {yields} Leptin inhibited the adipocytes differentiation at physiological concentration via inhibition of PPAR{gamma} expression. {yields} Inhibitors of ERK and STAT1 restored the leptin's inhibitory activity both in vitro and in vivo. -- Abstract: The effects of leptin on rosiglitazone-induced adipocyte differentiation were investigated in the primary adipocytes prepared from subcutaneous fat of TallyHO/Jng (TallyHO) mouse, a recently developed model animal for type 2 diabetes mellitus (T2DM). The treatment of leptin inhibited the rosiglitazone-induced adipocyte differentiation with a decreased expression of peroxisome proliferator-activated receptor {gamma} (PPAR{gamma}) a key adipogenic transcription factor, both in mRNA and protein levels. Leptin (10 nM) was sufficient to inhibit the adipocyte differentiation, which seemed to come from increased expression of leptin receptor genes in the fat of TallyHO mice. The inhibition of adipogenesis by leptin was restored by the treatment of inhibitors for extracellular-signal-regulated kinase (ERK) (PD98059) and signal transducer and activator of transcription-1 (STAT1) (fludarabine). Furthermore, in vivo intraperitoneal administration of PD98059 and fludarabine increased the PPAR{gamma} expression in the subcutaneous fat of TallyHO mice. These data suggest that leptin could inhibit the PPAR{gamma} expression and adipocyte differentiation in its physiological concentration in TallyHO mice.

  5. Inhibition of glycogen synthase kinase-3β attenuates glucocorticoid-induced suppression of myogenic differentiation in vitro.

    Zhenyu Ma

    Full Text Available Glucocorticoids are the only therapy that has been demonstrated to alter the progress of Duchenne muscular dystrophy (DMD, the most common muscular dystrophy in children. However, glucocorticoids disturb skeletal muscle metabolism and hamper myogenesis and muscle regeneration. The mechanisms involved in the glucocorticoid-mediated suppression of myogenic differentiation are not fully understood. Glycogen synthase kinase-3β (GSK-3β is considered to play a central role as a negative regulator in myogenic differentiation. Here, we showed that glucocorticoid treatment during the first 48 h in differentiation medium decreased the level of phosphorylated Ser9-GSK-3β, an inactive form of GSK-3β, suggesting that glucocorticoids affect GSK-3β activity. We then investigated whether GSK-3β inhibition could regulate glucocorticoid-mediated suppression of myogenic differentiation in vitro. Two methods were employed to inhibit GSK-3β: pharmacological inhibition with LiCl and GSK-3β gene knockdown. We found that both methods resulted in enhanced myotube formation and increased levels of muscle regulatory factors and muscle-specific protein expression. Importantly, GSK-3β inhibition attenuated glucocorticoid-induced suppression of myogenic differentiation. Collectively, these data suggest the involvement of GSK-3β in the glucocorticoid-mediated impairment of myogenic differentiation. Therefore, the inhibition of GSK-3β may be a strategy for preventing glucocorticoid-induced muscle degeneration.

  6. Development of a rapid culture method to induce adipocyte differentiation of human bone marrow-derived mesenchymal stem cells

    Human mesenchymal stem cells (hMSCs) derived from bone marrow are multipotent stem cells that can regenerate mesenchymal tissues such as adipose, bone or muscle. It is thought that hMSCs can be utilized as a cell resource for tissue engineering and as human models to study cell differentiation mechanisms, such as adipogenesis, osteoblastogenesis and so on. Since it takes 2-3 weeks for hMSCs to differentiate into adipocytes using conventional culture methods, the development of methods to induce faster differentiation into adipocytes is required. In this study we optimized the culture conditions for adipocyte induction to achieve a shorter cultivation time for the induction of adipocyte differentiation in bone marrow-derived hMSCs. Briefly, we used a cocktail of dexamethasone, insulin, methylisobutylxanthine (DIM) plus a peroxisome proliferator-activated receptor γ agonist, rosiglitazone (DIMRo) as a new adipogenic differentiation medium. We successfully shortened the period of cultivation to 7-8 days from 2-3 weeks. We also found that rosiglitazone alone was unable to induce adipocyte differentiation from hMSCs in vitro. However, rosiglitazone appears to enhance hMSC adipogenesis in the presence of other hormones and/or compounds, such as DIM. Furthermore, the inhibitory activity of TGF-β1 on adipogenesis could be investigated using DIMRo-treated hMSCs. We conclude that our rapid new culture method is very useful in measuring the effect of molecules that affect adipogenesis in hMSCs.

  7. Dexamethasone Induces Cardiomyocyte Terminal Differentiation via Epigenetic Repression of Cyclin D2 Gene.

    Gay, Maresha S; Dasgupta, Chiranjib; Li, Yong; Kanna, Angela; Zhang, Lubo

    2016-08-01

    Dexamethasone treatment of newborn rats inhibited cardiomyocyte proliferation and stimulated premature terminal differentiation of cardiomyocytes in the developing heart. Yet mechanisms remain undetermined. The present study tested the hypothesis that the direct effect of glucocorticoid receptor-mediated epigenetic repression of cyclin D2 gene in the cardiomyocyte plays a key role in the dexamethasone-mediated effects in the developing heart. Cardiomyocytes were isolated from 2-day-old rats. Cells were stained with a cardiomyocyte marker α-actinin and a proliferation marker Ki67. Cyclin D2 expression was evaluated by Western blot and quantitative real-time polymerase chain reaction. Promoter methylation of CcnD2 was determined by methylated DNA immunoprecipitation (MeDIP). Overexpression of Cyclin D2 was conducted by transfection of FlexiCcnD2 (+CcnD2) construct. Treatment of cardiomyocytes isolated from newborn rats with dexamethasone for 48 hours significantly inhibited cardiomyocyte proliferation with increased binucleation and decreased cyclin D2 protein abundance. These effects were blocked with Ru486 (mifepristone). In addition, the dexamethasone treatment significantly increased cyclin D2 gene promoter methylation in newborn rat cardiomyocytes. 5-Aza-2'-deoxycytidine inhibited dexamethasone-mediated promoter methylation, recovered dexamethasone-induced cyclin D2 gene repression, and blocked the dexamethasone-elicited effects on cardiomyocyte proliferation and binucleation. In addition, the overexpression of cyclin D2 restored the dexamethasone-mediated inhibition of proliferation and increase in binucleation in newborn rat cardiomyocytes. The results demonstrate that dexamethasone acting on glucocorticoid receptors has a direct effect and inhibits proliferation and stimulates premature terminal differentiation of cardiomyocytes in the developing heart via epigenetic repression of cyclin D2 gene. PMID:27302109

  8. Vorinostat induces apoptosis and differentiation in myeloid malignancies: genetic and molecular mechanisms.

    Gabriela Silva

    Full Text Available BACKGROUND: Aberrant epigenetic patterns are central in the pathogenesis of haematopoietic diseases such as myelodysplastic syndromes (MDS and acute myeloid leukaemia (AML. Vorinostat is a HDACi which has produced responses in these disorders. The purpose of this study was to address the functional effects of vorinostat in leukemic cell lines and primary AML and MDS myeloid cells and to dissect the genetic and molecular mechanisms by which it exerts its action. METHODOLOGY/PRINCIPAL FINDINGS: Functional assays showed vorinostat promoted cell cycle arrest, inhibited growth, and induced apoptosis and differentiation of K562, HL60 and THP-1 and of CD33(+ cells from AML and MDS patients. To explore the genetic mechanism for these effects, we quantified gene expression modulation by vorinostat in these cells. Vorinostat increased expression of genes down-regulated in MDS and/or AML (cFOS, COX2, IER3, p15, RAI3 and suppressed expression of genes over-expressed in these malignancies (AXL, c-MYC, Cyclin D1 and modulated cell cycle and apoptosis genes in a manner which would favor cell cycle arrest, differentiation, and apoptosis of neoplastic cells, consistent with the functional assays. Reporter assays showed transcriptional effect of vorinostat on some of these genes was mediated by proximal promoter elements in GC-rich regions. Vorinostat-modulated expression of some genes was potentiated by mithramycin A, a compound that interferes with SP1 binding to GC-rich DNA sequences, and siRNA-mediated SP1 reduction. ChIP assays revealed vorinostat inhibited DNA binding of SP1 to the proximal promoter regions of these genes. These results suggest vorinostat transcriptional action in some genes is regulated by proximal promoter GC-rich DNA sequences and by SP1. CONCLUSION: This study sheds light on the effects of vorinostat in AML and MDS and supports the implementation of clinical trials to explore the use of vorinostat in the treatment of these diseases.

  9. Social defeat during adolescence and adulthood differentially induce BDNF-regulated immediate early genes

    Caroline M. Coppens

    2011-11-01

    Full Text Available Stressful life events generally enhance the vulnerability for the development of human psychopathologies such as anxiety disorders and depression. The incidence rates of adult mental disorders steeply rises during adolescence in parallel with a structural and functional reorganization of the neural circuitry underlying stress reactivity. However, the mechanisms underlying susceptibility to stress and manifestation of mental disorders during adolescence are little understood. We hypothesized that heightened sensitivity to stress during adolescence reflects age-dependent differences in the expression of activity-dependent genes involved in synaptic plasticity. Therefore, we compared the effect of social stress during adolescence with social stress in adulthood on the expression of a panel of genes linked to induction of long-term potentiation (LTP and brain-derived neurotrophic factor (BDNF signaling. We show that social defeat during adolescence and adulthood differentially regulates expression of the immediate early genes BDNF, Arc, Carp, and Tieg1, as measured by qPCR in tissue lysates from prefrontal cortex, nucleus accumbens, and hippocampus. In the hippocampus, mRNA levels for all four genes were robustly elevated following social defeat in adolescence, whereas none were induced by defeat in adulthood. The relationship to coping style was also examined using adult reactive and proactive coping rats. Gene expression levels of reactive and proactive animals were similar in the prefrontal cortex and hippocampus. However, a trend toward a differential expression of BDNF and Arc mRNA in the nucleus accumbens was detected. BDNF mRNA was increased in the nucleus accumbens of proactive defeated animals, whereas the expression level in reactive defeated animals was comparable to control animals. The results demonstrate striking differences in immediate early gene expression in response to social defeat in adolescent and adult rats.

  10. Adipogenic human adenovirus Ad-36 induces commitment, differentiation, and lipid accumulation in human adipose-derived stem cells

    Pasarica, Magdalena; Mashtalir, Nazar; McAllister, Emily J;

    2008-01-01

    Human adenovirus Ad-36 is causatively and correlatively linked with animal and human obesity, respectively. Ad-36 enhances differentiation of rodent preadipocytes, but its effect on adipogenesis in humans is unknown. To indirectly assess the role of Ad-36-induced adipogenesis in human obesity, the...