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Sample records for inhibitor p27kip1 expression

  1. High-Throughput Screening Reveals Alsterpaullone, 2-Cyanoethyl as a Potent p27Kip1 Transcriptional Inhibitor

    PubMed Central

    Walters, Brandon J.; Lin, Wenwei; Diao, Shiyong; Brimble, Mark; Iconaru, Luigi I.; Dearman, Jennifer; Goktug, Asli; Chen, Taosheng; Zuo, Jian

    2014-01-01

    p27Kip1 is a cell cycle inhibitor that prevents cyclin dependent kinase (CDK)/cyclin complexes from phosphorylating their targets. p27Kip1 is a known tumor suppressor, as the germline loss of p27Kip1 results in sporadic pituitary formation in aged rodents, and its presence in human cancers is indicative of a poor prognosis. In addition to its role in cancer, loss of p27Kip1 results in regenerative phenotypes in some tissues and maintenance of stem cell pluripotency, suggesting that p27Kip1 inhibitors could be beneficial for tissue regeneration. Because p27Kip1 is an intrinsically disordered protein, identifying direct inhibitors of the p27Kip1 protein is difficult. Therefore, we pursued a high-throughput screening strategy to identify novel p27Kip1 transcriptional inhibitors. We utilized a luciferase reporter plasmid driven by the p27Kip1 promoter to transiently transfect HeLa cells and used cyclohexamide as a positive control for non-specific inhibition. We screened a “bioactive” library consisting of 8,904 (4,359 unique) compounds, of which 830 are Food and Drug Administration (FDA) approved. From this screen, we successfully identified 111 primary hits with inhibitory effect against the promoter of p27Kip1. These hits were further refined using a battery of secondary screens. Here we report four novel p27Kip1 transcriptional inhibitors, and further demonstrate that our most potent hit compound (IC50 = 200 nM) Alsterpaullone 2-cyanoethyl, inhibits p27Kip1 transcription by preventing FoxO3a from binding to the p27Kip1 promoter. This screen represents one of the first attempts to identify inhibitors of p27Kip1 and may prove useful for future tissue regeneration studies. PMID:24646893

  2. Oligodendrocyte Regeneration after Neonatal Hypoxia Requires FoxO1-Mediated p27Kip1 Expression

    PubMed Central

    Jablonska, Beata; Scafidi, Joseph; Aguirre, Adan; Vaccarino, Flora; Nguyen, Vien; Borok, Erzsebet; Horvath, Tamas L.; Rowitch, David H.; Gallo, Vittorio

    2012-01-01

    Diffuse white matter injury (DWMI) caused by hypoxia is associated with permanent neurodevelopmental disabilities in preterm infants. The cellular and molecular mechanisms producing DWMI are poorly defined. Using a mouse model of neonatal hypoxia, we demonstrate a biphasic effect on oligodendrocyte development, resulting in hypomyelination. Oligodendrocyte death and oligodendrocyte progenitor cell (OPC) proliferation during the week after hypoxia were followed by delayed oligodendrocyte differentiation and abnormal myelination, as demonstrated by electron microscopy. Cdk2 activation was essential for the regenerative OPC response after hypoxia and was accompanied by reduced FoxO1-dependent p27 Kip1 expression. p27 Kip1 was also reduced in OPCs in human infant white matter lesions after hypoxia. The negative effects of hypoxia on oligodendrogenesis and myelination were more pronounced in p27 Kip1-null mice; conversely, overexpression of FoxO1 or p27 Kip1 in OPCs after hypoxia promoted oligodendrogenesis. Our studies demonstrate for the first time that neonatal hypoxia affects the Foxo1/p27 Kip1 pathway during white matter development. We also show that molecular manipulation of this pathway enhances oligodendrocyte regeneration during a critical developmental time window after DWMI. Thus, FoxO1 and p27 Kip1 may serve as promising target molecules for promoting timely oligodendrogenesis in neonatal DWMI. PMID:23077062

  3. p27KIP1 is abnormally expressed in Diffuse Large B-Cell Lymphomas and is associated with an adverse clinical outcome

    PubMed Central

    Sáez, Al; Sánchez, E; Sánchez-Beato, M; Cruz, M A; Chacón, I; Muñoz, E; Camacho, F I; Martínez-Montero, J C; Mollejo, M; Garcia, J F; Piris, M A

    1999-01-01

    Cell cycle progression is regulated by the combined action of cyclins, cyclin-dependent kinases (CDKs), and CDK-inhibitors (CDKi), which are negative cell cycle regulators. p27KIP1 is a CDKi key in cell cycle regulation, whose degradation is required for G1/S transition. In spite of the absence of p27KIP1 expression in proliferating lymphocytes, some aggressive B-cell lymphomas have been reported to show an anomalous p27KIP1 staining. We analysed p27KIP1 expression in a series of Diffuse Large B-cell Lymphoma (DLBCL), correlating it with the proliferative index and clinical outcome, to characterize the implications of this anomalous staining in lymphomagenesis in greater depth. For the above mentioned purposes, an immunohistochemical technique in paraffin-embedded tissues was employed, using commercially available antibodies, in a series of 133 patients with known clinical outcomes. Statistical analysis was performed in order to ascertain which clinical and molecular variables may influence outcome, in terms of disease-free survival (DFS) and overall survival (OS). The relationships between p27KIP1 and MIB-1 (Ki-67) were also tested. An abnormally high expression of p27KIP1 was found in lymphomas of this type. The overall correlation between p27KIP1 and MIB-1 showed there to be no significant relationship between these two parameters, this differing from observations in reactive lymphoid and other tissues. Analysis of the clinical relevance of these findings showed that a high level of p27KIP1 expression in this type of tumour is an adverse prognostic marker, in both univariate and multivariate analysis. These results show that there is abnormal p27KIP1 expression in DLBCL, with adverse clinical significance, suggesting that this anomalous p27KIP1 protein may be rendered non-functional through interaction with other cell cycle regulator proteins. © 1999 Cancer Research Campaign PMID:10424746

  4. Expression of p21(Cip1/Waf1/Sdi1) and p27(Kip1) cyclin-dependent kinase inhibitors during human hematopoiesis.

    PubMed

    Taniguchi, T; Endo, H; Chikatsu, N; Uchimaru, K; Asano, S; Fujita, T; Nakahata, T; Motokura, T

    1999-06-15

    Expression of p21 and p27 cyclin-dependent kinase inhibitors is associated with induced differentiation and cell-cycle arrest in some hematopoietic cell lines. However, it is not clear how these inhibitors are expressed during normal hematopoiesis. We examined various human hematopoietic colonies derived from cord blood CD34(+) cells, bone marrow, and peripheral blood cells using a quantitative reverse transcription-polymerase chain reaction assay, immunochemistry, and/or Western blot analysis. p21 mRNA was expressed increasingly over time in all of the colonies examined (granulocytes, macrophages, megakaryocytes, and erythroblasts), whereas p27 mRNA levels remained low, except for erythroid bursts. Erythroid bursts expressed both p21 and p27 mRNAs with differentiation but expressed neither protein, whereas both proteins were expressed in megakaryocytes and peripheral blood monocytes. In bone marrow, p21 was immunostained almost exclusively in a subset of megakaryocytes and p27 protein was present in megakaryocytes, plasma cells, and endothelial cells. In megakaryocytes, reciprocal expression of p27 to Ki-67 was evident and an inverse relationship between p21 and Ki-67 positivities was also present, albeit less obvious. These observations suggest that a complex lineage-specific regulation is involved in p21 and p27 expression and that these inhibitors are involved in cell-cycle exit in megakaryocytes.

  5. Nanog induces suppression of senescence through downregulation of p27KIP1 expression

    PubMed Central

    Münst, Bernhard; Thier, Marc Christian; Winnemöller, Dirk; Helfen, Martina; Thummer, Rajkumar P.; Edenhofer, Frank

    2016-01-01

    ABSTRACT A comprehensive analysis of the molecular network of cellular factors establishing and maintaining pluripotency as well as self renewal of pluripotent stem cells is key for further progress in understanding basic stem cell biology. Nanog is necessary for the natural induction of pluripotency in early mammalian development but dispensable for both its maintenance and its artificial induction. To gain further insight into the molecular activity of Nanog, we analyzed the outcomes of Nanog gain-of-function in various cell models employing a recently developed biologically active recombinant cell-permeant protein, Nanog-TAT. We found that Nanog enhances the proliferation of both NIH 3T3 and primary fibroblast cells. Nanog transduction into primary fibroblasts results in suppression of senescence-associated β-galactosidase activity. Investigation of cell cycle factors revealed that transient activation of Nanog correlates with consistent downregulation of the cell cycle inhibitor p27KIP1 (also known as CDKN1B). By performing chromatin immunoprecipitation analysis, we confirmed bona fide Nanog-binding sites upstream of the p27KIP1 gene, establishing a direct link between physical occupancy and functional regulation. Our data demonstrates that Nanog enhances proliferation of fibroblasts through transcriptional regulation of cell cycle inhibitor p27 gene. PMID:26795560

  6. Induction of anergy in Th1 cells associated with increased levels of cyclin-dependent kinase inhibitors p21Cip1 and p27Kip1.

    PubMed

    Jackson, S K; DeLoose, A; Gilbert, K M

    2001-01-15

    Th1 cells exposed to Ag and the G(1) blocker n-butyrate in primary cultures lose their ability to proliferate in Ag-stimulated secondary cultures. The ability of n-butyrate to induce anergy in Ag-stimulated, but not resting, Th1 cells was shown here to be blocked by cycloheximide. Subsequent experiments to delineate the nature of the protein apparently required for n-butyrate-induced Th1 cell anergy focused on the role of cyclin-dependent kinase (cdk) inhibitors p21(Cip1) and p27(Kip1). Normally, entry into S phase by Th1 cells occurs around 24 h after Ag stimulation and corresponds with relatively low levels of both p21(Cip1) and p27(Kip1). However, unlike control Th1 cells, anergic Th1 cells contained high levels of both p21(Cip1) and p27(Kip1) when examined 24 h after Ag stimulation. The increase in p21(Cip1) observed in Ag-stimulated anergic Th1 cells appeared to be initiated in primary cultures. In contrast, the increase in p27(Kip1) observed in these anergic Th1 cells appears to represent a re-expression of the protein much earlier than control cells following Ag stimulation in secondary cultures. The anergic Th1 cells contained functionally active cdk inhibitors capable of inhibiting the activity of both endogenous and exogenous cdks. Consequently, it appears that n-butyrate-induced anergy in Th1 cells correlated with the up-regulation of p21(Cip1) and perhaps the downstream failure to maintain low levels of p27(Kip1). Increased levels of both p21(Cip1) and p27(Kip1) at the end of G(1) could prevent cdk-mediated entry into S phase, and thus help maintain the proliferative unresponsiveness found in the anergic Th1 cells.

  7. Cip2a promotes cell cycle progression in triple-negative breast cancer cells by regulating the expression and nuclear export of p27Kip1.

    PubMed

    Liu, H; Qiu, H; Song, Y; Liu, Y; Wang, H; Lu, M; Deng, M; Gu, Y; Yin, J; Luo, K; Zhang, Z; Jia, X; Zheng, G; He, Z

    2016-10-03

    Triple-negative breast cancer (TNBC) is very aggressive and currently has no specific therapeutic targets; as a consequence, TNBC exhibits poor clinical outcome. In this study, we showed that cancerous inhibitor of protein phosphatase 2A (Cip2a) represents a promising target in TNBC because Cip2a was highly expressed in TNBC cells and tumor tissues, and its expression showed an inverse correlation with overall survival in patients with TNBC. We found that inhibition of Cip2a in TNBC cells induced cell cycle arrest at the G2/M phase, inhibited cell proliferation and delayed tumor growth in the xenograft model. Moreover, Cip2a markedly decreased the expression and nuclear localization of p27Kip1 and this is critical for the ability of Cip2a to promote TNBC progression. Mechanistically, our studies showed that Cip2a promoted p27Kip1 phosphoration at Ser10 via inhibiting Akt-associated PP2A activity, which seems to relocalize p27Kip1 to the cytoplasm in TNBC cells. On the other hand, Cip2a also recruited c-myc to mediate the transcriptional inhibition of p27Kip1. Notably, we observed negative correlation between Cip2a and p27Kip1 expression in TNBC specimens. In addition, our data showed that Cip2a depletion could sensitize TNBC to PARP inhibition. Collectively, these data suggested that Cip2a effectively promotes TNBC cell cycle progression and tumor growth via regulation of PP2A/c-myc/p27Kip1 signaling, which could serve as a potential therapeutic target for TNBC patients.Oncogene advance online publication, 3 October 2016; doi:10.1038/onc.2016.355.

  8. Growth arrest by the cyclin-dependent kinase inhibitor p27Kip1 is abrogated by c-Myc.

    PubMed Central

    Vlach, J; Hennecke, S; Alevizopoulos, K; Conti, D; Amati, B

    1996-01-01

    We show here that c-Myc antagonizes the cyclin-dependent kinase (CDK) inhibitor p27Kip1. p27 expressed from recombinant retroviruses in Rat1 cells associated with and inhibited cyclin E/CDK2 complexes, induced accumulation of the pRb and p130 proteins in their hypophosphorylated forms, and arrested cells in G1. Prior expression of c-Myc prevented inactivation of cyclin E/CDK2 as well as dephosphorylation of pRb and p130, and allowed continuous cell proliferation in the presence of p27. This effect did not require ubiquitin-mediated degradation of p27. Myc altered neither the susceptibility of cyclin E/CDK2 to inhibition by p27, nor the intrinsic CDK-inhibitory activity of p27, but induced sequestration of p27 in a form unable to bind cyclin E/CDK2. Neither Myc itself nor other G1-cyclin/CDK complexes were directly responsible for p27 sequestration. Retroviral expression of G1 cyclins (D1-3, E or A) or of the Cdc25A phosphatase did not overcome p27-induced arrest. Growth rescue by Myc required dimerization with Max, DNA binding and an intact transcriptional activation domain, as previously shown for cellular transformation. We propose that this activity is mediated by the product of an as yet unknown Myc-Max target gene(s) and represents an essential aspect of Myc's mitogenic and oncogenic functions. Images PMID:8978686

  9. miR-222 confers the resistance of breast cancer cells to Adriamycin through suppression of p27(kip1) expression.

    PubMed

    Wang, Dan-Dan; Li, Jian; Sha, Huan-Huan; Chen, Xiu; Yang, Su-Jin; Shen, Hong-Yu; Zhong, Shan-Liang; Zhao, Jian-Hua; Tang, Jin-Hai

    2016-09-15

    Adriamycin (Adr) is a potent chemotherapeutic agent for chemotherapy of breast cancer patients. Despite impressive initial clinical responses, some developed drug resistance to Adr-based therapy and the mechanisms underlying breast cancer cells resistance to Adr are not well known. In our previous study, in vitro, we verified that miR-222 was upregulated in Adr-resistant breast cancer cells (MCF-7/Adr) compared with the sensitive parental cells (MCF-7/S). Here, miR-222 inhibitors or mimics were transfected into MCF-7 cell lines. RT-qPCR and western blot were used to detect the expression of p27(kip1). Immunofluorescence showed that miR-222 altered the subcellular location of p27(kip1) in nucleus. MTT was employed to verify the sensitivity of breast cancer cell lines to Adr. Flow cytometry showed the apoptosis and cell cycles of the cells after adding Adr. The results showed that downregulation of miR-222 in MCF-7/Adr increased sensitivity to Adr and Adr-induced apoptosis, and arrested the cells in G1 phase, accompanied by more expressions of p27(kip1), especially in nucleus. Furthermore, overexpressed miR-222 in MCF-7/S had the inverse results. Taken together, the results found that miR-222 induced Adr-resistance at least in part via suppressing p27(kip1) expression and altering its subcellular localization, and miR-222 inhibitors could reverse Adr-resistance of breast cancer cells. These results disclosed that the future holds much promise for the targeted therapeutic in the treatment of Adr-resistant breast cancer.

  10. Rho/ROCK pathway inhibition by the CDK inhibitor p27(kip1) participates in the onset of macrophage 3D-mesenchymal migration.

    PubMed

    Gui, Philippe; Labrousse, Arnaud; Van Goethem, Emeline; Besson, Arnaud; Maridonneau-Parini, Isabelle; Le Cabec, Véronique

    2014-09-15

    Infiltration of macrophages into tissue can promote tumour development. Depending on the extracellular matrix architecture, macrophages can adopt two migration modes: amoeboid migration--common to all leukocytes, and mesenchymal migration--restricted to macrophages and certain tumour cells. Here, we investigate the initiating mechanisms involved in macrophage mesenchymal migration. We show that a single macrophage is able to use both migration modes. Macrophage mesenchymal migration is correlated with decreased activity of Rho/Rho-associated protein kinase (ROCK) and is potentiated when ROCK is inhibited, suggesting that amoeboid inhibition participates in mechanisms that initiate mesenchymal migration. We identify the cyclin-dependent kinase (CDK) inhibitor p27(kip1) (also known as CDKN1B) as a new effector of macrophage 3D-migration. By using p27(kip1) mutant mice and small interfering RNA targeting p27(kip1), we show that p27(kip1) promotes mesenchymal migration and hinders amoeboid migration upstream of the Rho/ROCK pathway, a process associated with a relocation of the protein from the nucleus to the cytoplasm. Finally, we observe that cytoplasmic p27(kip1) is required for in vivo infiltration of macrophages within induced tumours in mice. This study provides the first evidence that silencing of amoeboid migration through inhibition of the Rho/ROCK pathway by p27(kip1) participates in the onset of macrophage mesenchymal migration.

  11. PRMT5 is upregulated in malignant and metastatic melanoma and regulates expression of MITF and p27(Kip1.).

    PubMed

    Nicholas, Courtney; Yang, Jennifer; Peters, Sara B; Bill, Matthew A; Baiocchi, Robert A; Yan, Fengting; Sïf, Saïd; Tae, Sookil; Gaudio, Eugenio; Wu, Xin; Grever, Michael R; Young, Gregory S; Lesinski, Gregory B

    2013-01-01

    Protein arginine methyltransferase-5 (PRMT5) is a Type II arginine methyltransferase that regulates various cellular functions. We hypothesized that PRMT5 plays a role in regulating the growth of human melanoma cells. Immunohistochemical analysis indicated significant upregulation of PRMT5 in human melanocytic nevi, malignant melanomas and metastatic melanomas as compared to normal epidermis. Furthermore, nuclear PRMT5 was significantly decreased in metastatic melanomas as compared to primary cutaneous melanomas. In human metastatic melanoma cell lines, PRMT5 was predominantly cytoplasmic, and associated with its enzymatic cofactor Mep50, but not STAT3 or cyclin D1. However, histologic examination of tumor xenografts from athymic mice revealed heterogeneous nuclear and cytoplasmic PRMT5 expression. Depletion of PRMT5 via siRNA inhibited proliferation in a subset of melanoma cell lines, while it accelerated growth of others. Loss of PRMT5 also led to reduced expression of MITF (microphthalmia-associated transcription factor), a melanocyte-lineage specific oncogene, and increased expression of the cell cycle regulator p27(Kip1). These results are the first to report elevated PRMT5 expression in human melanoma specimens and indicate this protein may regulate MITF and p27(Kip1) expression in human melanoma cells.

  12. Expression patterns of cyclins D1, E and cyclin-dependent kinase inhibitors p21waf1/cip1, p27kip1 in colorectal carcinoma: correlation with other cell cycle regulators (pRb, p53 and Ki-67 and PCNA) and clinicopathological features.

    PubMed

    Ioachim, E

    2008-11-01

    Aberrations in the cell cycle regulators are common features of many tumours and several have been shown to have prognostic significant in colorectal cancer. The expression patterns of cyclins D1 and E as well as cyclin-dependent kinase (CDK) inhibitors p21waf1/cip1 and p27kip1 and their interrelationship with other cell cycle checkpoint proteins [p53, pRb, Ki-67 and proliferative cell nuclear antigen (PCNA)] were investigated in colorectal cancer in order to ascertain coregulation and influence on tumour behaviour or survival. These molecular markers were localisated immunohistochemically using the monoclonal antibodies anticyclin D1 (DCS-6), anticyclin E (13A3), anti-p21 (4D10), anti-p27 (1B4), anti-p53 (DO7), anti-Rb (AB-5), MIB1 and PC10 in colorectal cancer tissue from 97 patients. Data were analysed statistically using the spss software program. Overexpression of cyclin D1, cyclin E and p21waf1/cip1 proteins (>5% positive neoplastic cells) was observed in 5.9%, 30% and 7.2% of the cases respectively. Increased levels of cyclin D1 (p = 0.0001) and p21waf1/cip1 protein (p = 0.03) in tumours with mucous differentiation were observed. Overexpression of cyclin D1 was correlated with tumour stage (p = 0.03), the lymph node involvement (p = 0.02), as well as p21waf1/cip1 protein expression (p < 0.0001). Cyclin E was positively correlated with p21waf1/cip1 (p = 0.014), as well as with the cell proliferation as measured by PCNA-labelling index (p = 0.011) and Ki-67 score (p = 0.007). A positive relationship of p21waf1/cip1 expression with the proliferative-associated index Ki-67 was noted (p = 0.005). Downregulation of p27kip1 was observed in 47.4% of the cases and was correlated with downregulation of pRb (p = 0.002) and PCNA score (p = 0.004). The prognostic significance of cyclins D1, E and CDK inhibitors p21waf1/cip1, p27kip1 in determining the risk of recurrence and overall survival with both univariate (long-rang test) and multivariate (Cox regression) methods

  13. p27kip1 controls H-Ras/MAPK activation and cell cycle entry via modulation of MT stability

    PubMed Central

    Fabris, Linda; Berton, Stefania; Pellizzari, Ilenia; Segatto, Ilenia; D’Andrea, Sara; Armenia, Joshua; Bomben, Riccardo; Schiappacassi, Monica; Gattei, Valter; Philips, Mark R.; Vecchione, Andrea; Belletti, Barbara; Baldassarre, Gustavo

    2015-01-01

    The cyclin-dependent kinase (CDK) inhibitor p27kip1 is a critical regulator of the G1/S-phase transition of the cell cycle and also regulates microtubule (MT) stability. This latter function is exerted by modulating the activity of stathmin, an MT-destabilizing protein, and by direct binding to MTs. We recently demonstrated that increased proliferation in p27kip1-null mice is reverted by concomitant deletion of stathmin in p27kip1/stathmin double-KO mice, suggesting that a CDK-independent function of p27kip1 contributes to the control of cell proliferation. Whether the regulation of MT stability by p27kip1 impinges on signaling pathway activation and contributes to the decision to enter the cell cycle is largely unknown. Here, we report that faster cell cycle entry of p27kip1-null cells was impaired by the concomitant deletion of stathmin. Using gene expression profiling coupled with bioinformatic analyses, we show that p27kip1 and stathmin conjunctly control activation of the MAPK pathway. From a molecular point of view, we observed that p27kip1, by controlling MT stability, impinges on H-Ras trafficking and ubiquitination levels, eventually restraining its full activation. Our study identifies a regulatory axis controlling the G1/S-phase transition, relying on the regulation of MT stability by p27kip1 and finely controlling the spatiotemporal activation of the Ras-MAPK signaling pathway. PMID:26512117

  14. p27kip1 controls H-Ras/MAPK activation and cell cycle entry via modulation of MT stability.

    PubMed

    Fabris, Linda; Berton, Stefania; Pellizzari, Ilenia; Segatto, Ilenia; D'Andrea, Sara; Armenia, Joshua; Bomben, Riccardo; Schiappacassi, Monica; Gattei, Valter; Philips, Mark R; Vecchione, Andrea; Belletti, Barbara; Baldassarre, Gustavo

    2015-11-10

    The cyclin-dependent kinase (CDK) inhibitor p27(kip1) is a critical regulator of the G1/S-phase transition of the cell cycle and also regulates microtubule (MT) stability. This latter function is exerted by modulating the activity of stathmin, an MT-destabilizing protein, and by direct binding to MTs. We recently demonstrated that increased proliferation in p27(kip1)-null mice is reverted by concomitant deletion of stathmin in p27(kip1)/stathmin double-KO mice, suggesting that a CDK-independent function of p27(kip1) contributes to the control of cell proliferation. Whether the regulation of MT stability by p27(kip1) impinges on signaling pathway activation and contributes to the decision to enter the cell cycle is largely unknown. Here, we report that faster cell cycle entry of p27(kip1)-null cells was impaired by the concomitant deletion of stathmin. Using gene expression profiling coupled with bioinformatic analyses, we show that p27(kip1) and stathmin conjunctly control activation of the MAPK pathway. From a molecular point of view, we observed that p27(kip1), by controlling MT stability, impinges on H-Ras trafficking and ubiquitination levels, eventually restraining its full activation. Our study identifies a regulatory axis controlling the G1/S-phase transition, relying on the regulation of MT stability by p27(kip1) and finely controlling the spatiotemporal activation of the Ras-MAPK signaling pathway.

  15. Cooperation between the Cdk inhibitors p27KIP1 and p57KIP2 in the control of tissue growth and development

    PubMed Central

    Zhang, Pumin; Wong, Calvin; DePinho, Ronald A.; Harper, J. Wade; Elledge, Stephen J.

    1998-01-01

    Cell cycle exit is required for terminal differentiation of many cell types. The retinoblastoma protein Rb has been implicated both in cell cycle exit and differentiation in several tissues. Rb is negatively regulated by cyclin-dependent kinases (Cdks). The main effectors that down-regulate Cdk activity to activate Rb are not known in the lens or other tissues. In this study, using multiple mutant mice, we show that the Cdk inhibitors p27KIP1 and p57KIP2 function redundantly to control cell cycle exit and differentiation of lens fiber cells and placental trophoblasts. These studies demonstrate that p27KIP1 and p57KIP2 are critical terminal effectors of signal transduction pathways that control cell differentiation. PMID:9784491

  16. Genetic characterization of p27kip1 and stathmin in controlling cell proliferation in vivo

    PubMed Central

    Berton, Stefania; Pellizzari, Ilenia; Fabris, Linda; D'Andrea, Sara; Segatto, Ilenia; Canzonieri, Vincenzo; Marconi, Daniela; Schiappacassi, Monica; Benevol, Sara; Gattei, Valter; Colombatti, Alfonso; Belletti, Barbara; Baldassarre, Gustavo

    2014-01-01

    The CDK inhibitor p27kip1 is a critical regulator of cell cycle progression, but the mechanisms by which p27kip1 controls cell proliferation in vivo are still not fully elucidated. We recently demonstrated that the microtubule destabilizing protein stathmin is a relevant p27kip1 binding partner. To get more insights into the in vivo significance of this interaction, we generated p27kip1 and stathmin double knock-out (DKO) mice. Interestingly, thorough characterization of DKO mice demonstrated that most of the phenotypes of p27kip1 null mice linked to the hyper-proliferative behavior, such as the increased body and organ weight, the outgrowth of the retina basal layer and the development of pituitary adenomas, were reverted by co-ablation of stathmin. In vivo analyses showed a reduced proliferation rate in DKO compared to p27kip1 null mice, linked, at molecular level, to decreased kinase activity of CDK4/6, rather than of CDK1 and CDK2. Gene expression profiling of mouse thymuses confirmed the phenotypes observed in vivo, showing that DKO clustered with WT more than with p27 knock-out tissue. Taken together, our results demonstrate that stathmin cooperates with p27kip1 to control the early phase of G1 to S phase transition and that this function may be of particular relevance in the context of tumor progression. PMID:25486569

  17. Genetic characterization of p27(kip1) and stathmin in controlling cell proliferation in vivo.

    PubMed

    Berton, Stefania; Pellizzari, Ilenia; Fabris, Linda; D'Andrea, Sara; Segatto, Ilenia; Canzonieri, Vincenzo; Marconi, Daniela; Schiappacassi, Monica; Benevol, Sara; Gattei, Valter; Colombatti, Alfonso; Belletti, Barbara; Baldassarre, Gustavo

    2014-01-01

    The CDK inhibitor p27(kip1) is a critical regulator of cell cycle progression, but the mechanisms by which p27(kip1) controls cell proliferation in vivo are still not fully elucidated. We recently demonstrated that the microtubule destabilizing protein stathmin is a relevant p27(kip1) binding partner. To get more insights into the in vivo significance of this interaction, we generated p27(kip1) and stathmin double knock-out (DKO) mice. Interestingly, thorough characterization of DKO mice demonstrated that most of the phenotypes of p27(kip1) null mice linked to the hyper-proliferative behavior, such as the increased body and organ weight, the outgrowth of the retina basal layer and the development of pituitary adenomas, were reverted by co-ablation of stathmin. In vivo analyses showed a reduced proliferation rate in DKO compared to p27(kip1) null mice, linked, at molecular level, to decreased kinase activity of CDK4/6, rather than of CDK1 and CDK2. Gene expression profiling of mouse thymuses confirmed the phenotypes observed in vivo, showing that DKO clustered with WT more than with p27 knock-out tissue. Taken together, our results demonstrate that stathmin cooperates with p27(kip1) to control the early phase of G1 to S phase transition and that this function may be of particular relevance in the context of tumor progression.

  18. SKP2 siRNA inhibits the degradation of P27kip1 and down-regulates the expression of MRP in HL-60/A cells.

    PubMed

    Xiao, Jie; Yin, Songmei; Li, Yiqing; Xie, Shuangfeng; Nie, Danian; Ma, Liping; Wang, Xiuju; Wu, Yudan; Feng, Jianhong

    2009-08-01

    S-phase kinase-associated protein 2 (SKP2) gene is a tumor suppressor gene, and is involved in the ubiquitin-mediated degradation of P27kip1. SKP2 and P27kip1 affect the proceeding and prognosis of leukemia through regulating the proliferation, apoptosis and differentiation of leukemia cells. In this study, we explored the mechanism of reversing of HL-60/A drug resistance through SKP2 down-regulation. HL-60/A cells were nucleofected by Amaxa Nucleofector System with SKP2 siRNA. The gene and protein expression levels of Skp2, P27kip1, and multi-drug resistance associated protein (MRP) were determined by reverse transcription-polymerase chain reaction and western blot analysis, respectively. The cell cycle was analyzed by flow cytometry. The 50% inhibitory concentration value was calculated using cytotoxic analysis according to the death rate of these two kinds of cells under different concentrations of chemotherapeutics to compare the sensitivity of the cells. HL-60/A cells showed multi-drug resistance phenotype characteristic by cross-resistance to adriamycin, daunorubicin, and arabinosylcytosine, due to the expression of MRP. We found that the expression of SKP2 was higher in HL-60/A cells than in HL-60 cells, but the expression of P27kip1 was lower. The expression of SKP2 in HL-60/A cells nucleofected by SKP2 siRNA was down-regulated whereas the protein level of P27kip1 was up-regulated. Compared with the MRP expression level in the control group (nucleofected by control siRNA), the mRNA and protein expression levels of MRP in HL-60/A cells nucleofected by SKP2 siRNA were lower, and the latter cells were more sensitive to adriamycin, daunorubicin, and arabinosylcytosine. Down-regulating the SKP2 expression and arresting cells in the G0/G1 phase improve drug sensitivity of leukemia cells with down-regulated MRP expression.

  19. c-Myc represses FOXO3a-mediated transcription of the gene encoding the p27(Kip1) cyclin dependent kinase inhibitor.

    PubMed

    Chandramohan, Vidyalakshmi; Mineva, Nora D; Burke, Brian; Jeay, Sébastien; Wu, Min; Shen, Jian; Yang, William; Hann, Stephen R; Sonenshein, Gail E

    2008-08-15

    The p27(Kip1) (p27) cyclin-dependent kinase inhibitor and c-Myc oncoprotein play essential roles in control of cell cycle progression and apoptosis. Induction of p27 (CDKN1B) gene transcription by Forkhead box O proteins such as FOXO3a leads to growth arrest and apoptosis. Previously, we observed that B cell receptor (surface IgM) engagement of WEHI 231 immature B lymphoma cells with an anti-IgM antibody results in activation of FOXO3a, growth arrest and apoptosis. As ectopic c-Myc expression in these cells prevented anti-IgM induction of p27 and cell death, we hypothesized that c-Myc represses FOXO3a-mediated transcription. Here we show that c-Myc inhibits FOXO3a-mediated activation of the p27 promoter in multiple cell lines. The mechanism of this repression was explored using a combination of co-immunoprecipitation, oligonucleotide precipitation, and chromatin immunoprecipitation experiments. The studies demonstrate a functional association of FOXO3a and c-Myc on a proximal Forkhead binding element in the p27 promoter. This association involves the Myc box II domain of c-Myc and the N-terminal DNA-binding portion of FOXO3a. Analysis of publicly available microarray datasets showed an inverse pattern of c-MYC and p27 RNA expression in primary acute myeloid leukemia, prostate cancer and tongue squamous cell carcinoma samples. The inhibition of FOXO3a-mediated activation of the p27 gene by the high aberrant expression of c-Myc in many tumor cells likely contributes to their uncontrolled proliferation and invasive phenotype.

  20. pRb2/p130, vascular endothelial growth factor, p27(KIP1), and proliferating cell nuclear antigen expression in hepatocellular carcinoma: their clinical significance.

    PubMed

    Claudio, Pier Paolo; Russo, Giuseppe; Kumar, Christine A C Y; Minimo, Corrado; Farina, Antonio; Tutton, Steve; Nuzzo, Gennaro; Giuliante, Felice; Angeloni, Giulia; Maria, Vellone; Vecchio, Fabio Maria; Campli, Cristiana Di; Giordano, Antonio

    2004-05-15

    Hepatocarcinoma (HCC) is the fifth most common cancer, with more than one million fatalities occurring annually worldwide. Multiple risk factors are associated with HCC disease etiology, the highest incidence being in patients with chronic hepatitis B virus and hepatitis C virus, although other factors such as genetic makeup and environmental exposure are involved. Multiple genetic alterations including the activation of oncogenes and inactivation of tumor suppressor genes are required for malignancy in human cancers and are correlated with increased stages of carcinogenesis and further tumor progression. In this study of 21 HCC patients, we analyzed pRb2/p130, vascular endothelial growth factor (VEGF), p27((KIP1)), and proliferating cell nuclear antigen as potential HCC molecular biomarkers. In our sample set, we found that p27((KIP1)) was absent. Univariate survival analysis showed that proliferating cell nuclear antigen expression (diffuse staining >50% of positive cells in tumor) was confirmed as a significant HCC prognostic biomarker for determining patient survival agreeing with previous studies (P = 0.0126, log-rank test). Lower pRb2/p130 expression was associated to a borderline P value of inverse correlation with tumor malignancy and to a positive correlation with respect to the time from HCC diagnosis (Spearman coefficient = 0.568; P < 0.05). Conversely, higher VEGF expression was associated with a poor survival (P = 0.0257, log-rank test). We demonstrate for the first time that pRb2/p130 is inversely correlated with VEGF expression and tumor aggressiveness (P < 0.05) in p27((KIP1))-negative HCC patients. pRb2/p130 and VEGF expression are independent from tumor staging, suggesting their possible role as independent prognostic molecular biomarkers in HCC. Furthermore, we have evidence that VEGF together with pRb2/p130 may act as new HCC biomarkers in a p27((KIP1))-independent manner. Additional studies with larger numbers of patient data would allow the

  1. p27kip1 overexpression promotes paclitaxel-induced apoptosis in pRb-defective SaOs-2 cells.

    PubMed

    Gabellini, Chiara; Pucci, Bruna; Valdivieso, Paola; D'Andrilli, Giuseppina; Tafani, Marco; De Luca, Antonio; Masciullo, Valeria

    2006-08-15

    p27kip1 is a cyclin-dependent kinase (CDK) inhibitor, which controls several cellular processes in strict collaboration with pRb. We evaluated the role of p27kip1 in paclitaxel-induced apoptosis in the pRb-defective SaOs-2 cells. Following 48 h of exposure of SaOs-2 cells to 100 nM paclitaxel, we observed an increase in p27kip1 expression caused by the decrease of the ubiquitin-proteasome activity. Such increase was not observed in SaOs-2 cells treated with the caspase inhibitors Z-VAD-FMK, suggesting that p27kip1 enhancement at 48 h is strictly related to apoptosis. Finally, we demonstrated that SaOs-2 cells transiently overexpressing the p27kip1 protein are more susceptible to paclitaxel-induced apoptosis than SaOs-2 cells transiently transfected with the empty vector. Indeed, after 48 h of paclitaxel treatment, 41.8% of SaOs-2 cells transiently transfected with a pcDNA3-p27kip1 construct were Annexin V-positive compared to 30.6% of SaOs-2 cells transfected with the empty vector (P < 0.05). In conclusion, we demonstrated that transfection of the pRb-defective SaOs-2 cells with the p27kip1 gene via plasmid increases their susceptibility to paclitaxel-induced apoptosis. The promoting effect of p27kip1 overexpression on apoptosis makes p27kip1 and proteasomal inhibitors interesting tools for therapy in patients with pRb-defective cancers.

  2. Effect of hypoxia and Beraprost sodium on human pulmonary arterial smooth muscle cell proliferation: the role of p27kip1

    PubMed Central

    Kadowaki, Maiko; Mizuno, Shiro; Demura, Yoshiki; Ameshima, Shingo; Miyamori, Isamu; Ishizaki, Takeshi

    2007-01-01

    Background Hypoxia induces the proliferation of pulmonary arterial smooth muscle cell (PASMC) in vivo and in vitro, and prostacyclin analogues are thought to inhibit the growth of PASMC. Previous studies suggest that p27kip1, a kind of cyclin-dependent kinase inhibitor, play an important role in the smooth muscle cell proliferation. However, the mechanism of hypoxia and the subcellular interactions between p27kip1 and prostacyclin analogues in human pulmonary arterial smooth muscle cell (HPASMC) are not fully understood. Methods We investigated the role of p27kip1 in the ability of Beraprost sodium (BPS; a stable prostacyclin analogue) to inhibit the proliferation of HPASMC during hypoxia. To clarify the biological effects of hypoxic air exposure and BPS on HPASMC, the cells were cultured in a hypoxic chamber under various oxygen concentrations (0.1–21%). Thereafter, DNA synthesis was measured as bromodeoxyuridine (BrdU) incorporation, the cell cycle was analyzed by flow cytometry with propidium iodide staining. The p27kip1 mRNA and protein expression and it's stability was measured by real-time RT-PCR and Western blotting. Further, we assessed the role of p27kip1 in HPASMC proliferation using p27kip1 gene knockdown using small interfering RNA (siRNA) transfection. Results Although severe hypoxia (0.1% oxygen) suppressed the proliferation of serum-stimulated HPASMC, moderate hypoxia (2% oxygen) enhanced proliferation in accordance with enhanced p27kip1 protein degradation, whereas BPS suppressed HPASMC proliferation under both hypoxic and normoxic conditions by suppressing p27kip1 degradation with intracellular cAMP-elevation. The 8-bromo-cyclic adenosine monophosphate (8-Br-cAMP), a cAMP analogue, had similar action as BPS in the regulation of p27kip1. Moderate hypoxia did not affect the stability of p27kip1 protein expression, but PDGF, known as major hypoxia-induced growth factors, significantly decreased p27kip1 protein stability. We also demonstrated that

  3. p27kip1 expression limits H-Ras-driven transformation and tumorigenesis by both canonical and non-canonical mechanisms

    PubMed Central

    Segatto, Ilenia; Citron, Francesca; D'Andrea, Sara; Cusan, Martina; Benevol, Sara; Perin, Tiziana; Massarut, Samuele; Canzonieri, Vincenzo; Schiappacassi, Monica; Belletti, Barbara; Baldassarre, Gustavo

    2016-01-01

    The tumor suppressor protein p27Kip1 plays a pivotal role in the control of cell growth and metastasis formation. Several studies pointed to different roles for p27Kip1 in the control of Ras induced transformation, although no explanation has been provided to elucidate these differences. We recently demonstrated that p27kip1 regulates H-Ras activity via its interaction with stathmin. Here, using in vitro and in vivo models, we show that p27kip1 is an important regulator of Ras induced transformation. In H-RasV12 transformed cells, p27kip1 suppressed cell proliferation and tumor growth via two distinct mechanisms: 1) inhibition of CDK activity and 2) impairment of MT-destabilizing activity of stathmin. Conversely, in K-Ras4BV12 transformed cells, p27kip1 acted mainly in a CDK-dependent but stathmin-independent manner. Using human cancer-derived cell lines and primary breast and sarcoma samples, we confirmed in human models what we observed in mice. Overall, we highlight a pathway, conserved from mouse to human, important in the regulation of H-Ras oncogenic activity that could have therapeutic and diagnostic implication in patients that may benefit from anti-H-Ras therapies. PMID:27579539

  4. p27kip1 expression limits H-Ras-driven transformation and tumorigenesis by both canonical and non-canonical mechanisms.

    PubMed

    Pellizzari, Ilenia; Fabris, Linda; Berton, Stefania; Segatto, Ilenia; Citron, Francesca; D'Andrea, Sara; Cusan, Martina; Benevol, Sara; Perin, Tiziana; Massarut, Samuele; Canzonieri, Vincenzo; Schiappacassi, Monica; Belletti, Barbara; Baldassarre, Gustavo

    2016-10-04

    The tumor suppressor protein p27Kip1 plays a pivotal role in the control of cell growth and metastasis formation.Several studies pointed to different roles for p27Kip1 in the control of Ras induced transformation, although no explanation has been provided to elucidate these differences. We recently demonstrated that p27kip1 regulates H-Ras activity via its interaction with stathmin.Here, using in vitro and in vivo models, we show that p27kip1 is an important regulator of Ras induced transformation. In H-RasV12 transformed cells, p27kip1 suppressed cell proliferation and tumor growth via two distinct mechanisms: 1) inhibition of CDK activity and 2) impairment of MT-destabilizing activity of stathmin. Conversely, in K-Ras4BV12 transformed cells, p27kip1 acted mainly in a CDK-dependent but stathmin-independent manner.Using human cancer-derived cell lines and primary breast and sarcoma samples, we confirmed in human models what we observed in mice.Overall, we highlight a pathway, conserved from mouse to human, important in the regulation of H-Ras oncogenic activity that could have therapeutic and diagnostic implication in patients that may benefit from anti-H-Ras therapies.

  5. Accumulation of the cyclin-dependent kinase inhibitor p27/Kip1 and the timing of oligodendrocyte differentiation.

    PubMed Central

    Durand, B; Gao, F B; Raff, M

    1997-01-01

    Many types of vertebrate precursor cells divide a limited number of times before they stop and terminally differentiate. In no case is it known what causes them to stop dividing. We have been studying this problem in the proliferating precursor cells that give rise to postmitotic oligodendrocytes, the cells that make myelin in the central nervous system. We show here that two components of the cell cycle control system, cyclin D1 and the Cdc2 kinase, are present in the proliferating precursor cells but not in differentiated oligodendrocytes, suggesting that the control system is dismantled in the oligodendrocytes. More importantly, we show that the cyclin-dependent kinase (Cdk) inhibitor p27 progressively accumulates in the precursor cells as they proliferate and is present at high levels in oligodendrocytes. Our findings are consistent with the possibility that the accumulation of p27 is part of both the intrinsic counting mechanism that determines when precursor cell proliferation stops and differentiation begins and the effector mechanism that arrests the cell cycle when the counting mechanism indicates it is time. The recent findings of others that p27-deficient mice have an increased number of cells in all of the organs examined suggest that this function of p27 is not restricted to the oligodendrocyte cell lineage. PMID:9029151

  6. Effects of adenovirus-mediated expression of p27Kip1, p21Waf1 and p16INK4A in cell lines derived from t(2;5) anaplastic large cell lymphoma and Hodgkin's disease.

    PubMed

    Turturro, Franceso; Arnold, Marilyn D; Frist, Audrey Y; Seth, Prem

    2002-06-01

    We investigated the response of SUDHL-1 and L428 cells, derived from t(2;5)-anaplastic large cell lymphoma (ALCL) and Hodgkin's disease (HD), respectively, to recombinant adenoviruses expressing cyclin-dependent kinase inhibitors (CDKIs) p27Kip1 (Adp27), p21Waf1 (Adp21) and p16INK4A (Adp16). Cell cycle analysis of SUDHL-1 cells after 24 h of infection with 200 multiplicity of infection (MOI) of Adp27, Adp21, and Adp16, showed very high levels of cell debris in the subG1 area. The magnitude of cell debris-events was Adp27/Adp21 > Adp16. Cell cycle analysis of L428 cells revealed absence of cell debris and increased G2 phase in all the groups of cells tested as compared to the controls (mock and AdNull). A minimal increase in G1 phase was also evident in cells infected with Adp27 (52%) compared to uninfected cells (43%), AdNull (45%) and to cells infected with Adp21 (37%) and Adp16 (31%). The presence of significant levels of Coxsackie-adenovirus receptor (CAR) on the cell surface of L428 cells excluded the cell membrane-barrier as responsible for the differences in cell observed in response to the recombinant adenovirus-mediated CDKIs expression as compared to SUDHL-1. We also showed that the recombinant adenovirus-mediated cytotoxicity measured as apoptosis was MOI- and vector-dependent in SUDHL-1 cells at lower MOI (100). In conclusion, the therapeutic effect induced by recombinant adenoviruses expressing p27Kip1, p21Waf1 and p16INK4A is cell-dependent in cells derived from selected lymphoid malignancies. Biochemical cellular differences more than cell surface barriers seem to be responsible for differences in response to recombinant adenovirus-mediated expression of cytotoxic genes. Moreover, the cytotoxicity of recombinant adenoviruses expressing p27Kip1, p21Waf1 and p16INK4A may be further explored as a tool for gene therapy of t(2;5)-derived ALCL.

  7. Notch Signaling Activates Stem Cell Properties of Müller Glia through Transcriptional Regulation and Skp2-mediated Degradation of p27Kip1

    PubMed Central

    Parameswaran, Sowmya; Mathews, Saumi; Xia, Xiaohuan; Zheng, Li; Neville, Andrew J.; Ahmad, Iqbal

    2016-01-01

    Müller glia (MG), the sole glial cells generated by retinal progenitors, have emerged as a viable cellular target for therapeutic regeneration in degenerative blinding diseases, as they possess dormant stem cell properties. However, the mammalian MG does not display the neurogenic potential of their lower vertebrate counterparts, precluding their practical clinical use. The answer to this barrier may be found in two interlinked processes underlying the neurogenic potential, i.e., the activation of the dormant stem cell properties of MG and their differentiation along the neuronal lineage. Here, we have focused on the former and examined Notch signaling-mediated activation of MG. We demonstrate that one of the targets of Notch signaling is the cyclin-dependent kinase inhibitor (CKI), p27Kip1, which is highly expressed in quiescent MG. Notch signaling facilitates the activation of MG by inhibiting p27Kip1 expression. This is likely achieved through the Notch- p27Kip1 and Notch-Skp2-p27Kip1 axes, the former inhibiting the expression of p27Kip1 transcripts and the latter levels of p27Kip1 proteins by Skp2-mediated proteasomal degradation. Thus, Notch signaling may facilitate re-entry of MG into the cell cycle by inhibiting p27Kip1 expression both transcriptionally and post-translationally. PMID:27011052

  8. CRM-1 knockdown inhibits extrahepatic cholangiocarcinoma tumor growth by blocking the nuclear export of p27Kip1

    PubMed Central

    Luo, Jian; Chen, Yongjun; Li, Qiang; Wang, Bing; Zhou, Yanqiong; Lan, Hongzhen

    2016-01-01

    Cholangiocarcinoma is a deadly disease which responds poorly to surgery and conventional chemotherapy or radiotherapy. Early diagnosis is difficult due to the anatomical and biological characteristics of cholangiocarcinoma. Cyclin-dependent kinase inhibitor 1B (p27Kip1) is a cyclin-dependent kinase inhibitor and in the present study, we found that p27Kip1 expression was suppressed in the nucleus and increased in the cytoplasm in 53 samples of cholangiocarcinoma from patients with highly malignant tumors (poorly-differentiated and tumor-node-metastsis (TNM) stage III–IV) compared with that in samples from 10 patients with chronic cholangitis. The expression of phosphorylated (p-)p27Kip1 (Ser10), one of the phosphorylated forms of p27Kip1, was increased in the patient samples with increasing malignancy and clinical stage. Coincidentally, chromosome region maintenance 1 (CRM-1; also referred to as exportin 1 or Xpo1), a critical protein responsible for protein translocation from the nucleus to the cytoplasm, was also overexpressed in the tumor samples which were poorly differentiated and of a higher clinical stage. Through specific short hairpin RNA (shRNA)-mediated knockdown of CRM-1 in the cholangiocarcinoma cell line QBC939, we identified an elevation of cytoplasmic p27Kip1 and a decrease of nuclear p27Kip1. Furthermore, the viability and colony formation ability of QBC939 cells was largely reduced with G1 arrest. Consistent with the findings of the in vitro experiments, in a xenograft mouse model, the tumors formed in the CRM-1 knockdown group were markedly smaller and weighed less than those in the control group in vivo. Taken together, these findings demonstrated that the interplay between CRM-1 and p27Kip1 may provide potentially potent biomarkers and functional targets for the development of future cholangiocarcinoma treatments. PMID:27279267

  9. CRM-1 knockdown inhibits extrahepatic cholangiocarcinoma tumor growth by blocking the nuclear export of p27Kip1.

    PubMed

    Luo, Jian; Chen, Yongjun; Li, Qiang; Wang, Bing; Zhou, Yanqiong; Lan, Hongzhen

    2016-08-01

    Cholangiocarcinoma is a deadly disease which responds poorly to surgery and conventional chemotherapy or radiotherapy. Early diagnosis is difficult due to the anatomical and biological characteristics of cholangiocarcinoma. Cyclin-dependent kinase inhibitor 1B (p27Kip1) is a cyclin‑dependent kinase inhibitor and in the present study, we found that p27Kip1 expression was suppressed in the nucleus and increased in the cytoplasm in 53 samples of cholangiocarcinoma from patients with highly malignant tumors (poorly-differentiated and tumor-node-metastsis (TNM) stage III-IV) compared with that in samples from 10 patients with chronic cholangitis. The expression of phosphorylated (p-)p27Kip1 (Ser10), one of the phosphorylated forms of p27Kip1, was increased in the patient samples with increasing malignancy and clinical stage. Coincidentally, chromosome region maintenance 1 (CRM-1; also referred to as exportin 1 or Xpo1), a critical protein responsible for protein translocation from the nucleus to the cytoplasm, was also overexpressed in the tumor samples which were poorly differentiated and of a higher clinical stage. Through specific short hairpin RNA (shRNA)-mediated knockdown of CRM-1 in the cholangiocarcinoma cell line QBC939, we identified an elevation of cytoplasmic p27Kip1 and a decrease of nuclear p27Kip1. Furthermore, the viability and colony formation ability of QBC939 cells was largely reduced with G1 arrest. Consistent with the findings of the in vitro experiments, in a xenograft mouse model, the tumors formed in the CRM-1 knockdown group were markedly smaller and weighed less than those in the control group in vivo. Taken together, these findings demonstrated that the interplay between CRM-1 and p27Kip1 may provide potentially potent biomarkers and functional targets for the development of future cholangiocarcinoma treatments.

  10. MARCKS Signaling Differentially Regulates Vascular Smooth Muscle and Endothelial Cell Proliferation through a KIS-, p27kip1- Dependent Mechanism

    PubMed Central

    Yu, Dan; Makkar, George; Dong, Tuo; Strickland, Dudley K.; Sarkar, Rajabrata; Monahan, Thomas Stacey

    2015-01-01

    Background Overexpression of the myristolated alanine-rich C kinase substrate (MARCKS) occurs in vascular proliferative diseases such as restenosis after bypass surgery. MARCKS knockdown results in arrest of vascular smooth muscle cell (VSMC) proliferation with little effect on endothelial cell (EC) proliferation. We sought to identify the mechanism of differential regulation by MARCKS of VSMC and EC proliferation in vitro and in vivo. Methods and Results siRNA-mediated MARCKS knockdown in VSMCs inhibited proliferation and prevented progression from phase G0/G1 to S. Protein expression of the cyclin-dependent kinase inhibitor p27kip1, but not p21cip1 was increased by MARCKS knockdown. MARCKS knockdown did not affect proliferation in VSMCs derived from p27kip1-/- mice indicating that the effect of MARCKS is p27kip1-dependent. MARCKS knockdown resulted in decreased phosphorylation of p27kip1 at threonine 187 and serine 10 as well as, kinase interacting with stathmin (KIS), cyclin D1, and Skp2 expression. Phosphorylation of p27kip1 at serine 10 by KIS is required for nuclear export and degradation of p27kip1. MARCKS knockdown caused nuclear trapping of p27kip1. Both p27kip1 nuclear trapping and cell cycle arrest were released by overexpression of KIS, but not catalytically inactive KIS. In ECs, MARCKS knockdown paradoxically increased KIS expression and cell proliferation. MARCKS knockdown in a murine aortic injury model resulted in decreased VSMC proliferation determined by bromodeoxyuridine (BrdU) integration assay, and inhibition of vascular wall thickening. MARCKS knockdown increased the rate of re-endothelialization. Conclusions MARCKS knockdown arrested VSMC cell cycle by decreasing KIS expression. Decreased KIS expression resulted in nuclear trapping of p27kip1 in VSMCs. MARCKS knockdown paradoxically increased KIS expression in ECs resulting in increased EC proliferation. MARCKS knockdown significantly attenuated the VSMC proliferative response to vascular

  11. Transforming growth factor-beta, transforming growth factor-beta receptor II, and p27Kip1 expression in nontumorous and neoplastic human pituitaries.

    PubMed Central

    Jin, L.; Qian, X.; Kulig, E.; Sanno, N.; Scheithauer, B. W.; Kovacs, K.; Young, W. F.; Lloyd, R. V.

    1997-01-01

    Transforming growth factor (TGF)-beta has been implicated in the regulation of normal and neoplastic anterior pituitary cell function. TGF-beta regulates the expression of various proteins, including p27Kip1 (p27), a cell cycle inhibitory protein. We examined TGF-beta, TGF-beta type II receptor (TGF-beta-RII), and p27 expression in normal pituitaries, pituitary adenomas, and carcinomas to analyze the possible roles of these proteins in pituitary tumorigenesis. Normal pituitary, pituitary adenomas, and pituitary carcinomas all expressed TGF-beta and TGF-beta-RII immunoreactivity. Reverse transcription polymerase chain reaction analysis showed TGF-beta 1, -beta 2, and -beta 3 isoforms and TGF-beta-RII in normal pituitaries and pituitary adenomas. Pituitary adenomas cells cultured for 7 days in defined media showed a biphasic response to TGF-beta with significant inhibition of follicle-stimulating hormone secretion at higher concentrations (10(-9) mol/L) and stimulation of follicle-stimulating hormone secretion at lower concentrations (10(-13) mol/L) of TGF-beta 1 in gonadotroph adenomas. Immunohistochemical analysis for p27 protein expression showed the highest levels in nontumorous pituitaries with decreased immunoreactivity in adenomas and carcinomas. When nontumorous pituitaries and various adenomas were analyzed for p27 and specific hormone production, growth hormone, luteinizing hormone, and thyroid-stimulating hormone cells and tumors had the highest percentages of cells expressing p27, whereas adrenocorticotrophic hormone cells and tumors had the lowest percentages. Immunoblotting analysis showed that adrenocorticotrophic hormone adenomas also had the lowest levels of p27 protein. Semiquantitative reverse transcription polymerase chain reaction and Northern hybridization analysis did not show significant differences in p27 mRNA expression in the various types of adenomas or in nontumorous pituitaries. In situ hybridization for p27 mRNA showed similar

  12. Contact inhibition modulates intracellular levels of miR-223 in a p27kip1-dependent manner

    PubMed Central

    Armenia, Joshua; Fabris, Linda; Lovat, Francesca; Berton, Stefania; Segatto, Ilenia; D'Andrea, Sara; Ivan, Cristina; Cascione, Luciano; Calin, George A.; Croce, Carlo M.; Colombatti, Alfonso; Vecchione, Andrea; Belletti, Barbara; Baldassarre, Gustavo

    2014-01-01

    MicroRNAs (miRs) are a large class of small regulatory RNAs that function as nodes of signaling networks. This implicates that miRs expression has to be finely tuned, as observed during cell cycle progression. Here, using an expression profiling approach, we provide evidence that the CDK inhibitor p27Kip1 regulates miRs expression following cell cycle exit. By using wild type and p27KO cells harvested in different phases of the cell cycle we identified several miRs regulated by p27Kip1 during the G1 to S phase transition. Among these miRs, we identified miR-223 as a miR specifically upregulated by p27Kip1 in G1 arrested cells. Our data demonstrate that p27Kip1 regulated the expression of miR-223, via two distinct mechanisms. p27Kip1 directly stabilized mature miR-223 expression, acting as a RNA binding protein and it controlled E2F1 expression that, in turn, regulated miR-223 promoter activity. The resulting elevated miR-223 levels ultimately participated to arresting cell cycle progression following contact inhibition. Importantly, this mechanism of growth control was conserved in human cells and deranged in breast cancers. Here, we identify a novel and conserved function of p27Kip1 that, by modulating miR-223 expression, contributes to proper regulation of cell cycle exit following contact inhibition. Thus we propose a new role for miR-223 in the regulation of breast cancer progression. PMID:24727437

  13. Contact inhibition modulates intracellular levels of miR-223 in a p27kip1-dependent manner.

    PubMed

    Armenia, Joshua; Fabris, Linda; Lovat, Francesca; Berton, Stefania; Segatto, Ilenia; D'Andrea, Sara; Ivan, Cristina; Cascione, Luciano; Calin, George A; Croce, Carlo M; Colombatti, Alfonso; Vecchione, Andrea; Belletti, Barbara; Baldassarre, Gustavo

    2014-03-15

    MicroRNAs (miRs) are a large class of small regulatory RNAs that function as nodes of signaling networks. This implicates that miRs expression has to be finely tuned, as observed during cell cycle progression. Here, using an expression profiling approach, we provide evidence that the CDK inhibitor p27Kip1 regulates miRs expression following cell cycle exit. By using wild type and p27KO cells harvested in different phases of the cell cycle we identified several miRs regulated by p27Kip1 during the G1 to S phase transition. Among these miRs, we identified miR-223 as a miR specifically upregulated by p27Kip1 in G1 arrested cells. Our data demonstrate that p27Kip1 regulated the expression of miR-223, via two distinct mechanisms. p27Kip1 directly stabilized mature miR-223 expression, acting as a RNA binding protein and it controlled E2F1 expression that, in turn, regulated miR-223 promoter activity. The resulting elevated miR-223 levels ultimately participated to arresting cell cycle progression following contact inhibition. Importantly, this mechanism of growth control was conserved in human cells and deranged in breast cancers. Here, we identify a novel and conserved function of p27Kip1 that, by modulating miR-223 expression, contributes to proper regulation of cell cycle exit following contact inhibition. Thus we propose a new role for miR-223 in the regulation of breast cancer progression.

  14. Loss of Cell Cycle Regulators p27Kip1 and Cyclin E in Transitional Cell Carcinoma of the Bladder Correlates with Tumor Grade and Patient Survival

    PubMed Central

    Del Pizzo, Joseph J.; Borkowski, Andrew; Jacobs, Stephen C.; Kyprianou, Natasha

    1999-01-01

    The cyclin-dependent kinase inhibitor p27Kip1 is a powerful molecular determinant of cell cycle progression. Loss of expression of p27Kip1 has been shown to be predictive of disease progression in several human malignancies. In this study we investigated the expression of two key cell cycle regulators, p27Kip1 and cyclin E, in the progression of transitional cell carcinoma of the bladder. An immunohistochemical analysis was conducted in a series of 50 bladder tumor specimens, including 3 metastatic lymph nodes, and 7 normal bladder specimens, using specific antibodies against the two regulators of the cell cycle, p27Kip1 and cyclin E. The degree of immunoreactivity was correlated with the pathological tumor grade, stage, and patient survival. A uniformly intense immunoreactivity for p27Kip1 and cyclin E was observed in epithelial cells of normal bladder tissue. Malignant bladder tissue demonstrated a heterogeneous pattern of significantly reduced p27Kip1 and cyclin E immunoreactivity, compared with normal urothelium (P < 0.01). In addition, there was progressive loss of expression of both cell cycle proteins with increasing tumor grade and pathological stage. Expression of p27Kip1 was significantly lower in the poorly differentiated tumors (grades III) compared to well and moderately differentiated (grades I and II) tumors (P = 0.004). Moreover, the expression of cyclin E was lower in grade III tumors compared to grade I and II lesions, although this difference failed to reach statistical significance. Most significantly, Kaplan-Meier plots of patient survival show increased mortality risk associated with low levels of p27Kip1 (P = 0.001) and cyclin E (P = 0.002) expression. This is the first evidence that loss of expression of p27Kip1 and cyclin E in human bladder transitional cell carcinoma cells correlates with advancing histological aggressiveness and poor patient survival. These results have clinical importance, because they support a role for p27Kip1 and

  15. Alterations in TP53, cyclin D2, c-Myc, p21WAF1/CIP1 and p27KIP1 expression associated with progression in B-CLL.

    PubMed

    Halina, Antosz; Artur, Paterski; Barbara, Marzec-Kotarska; Joanna, Sajewicz; Anna, Dmoszyńska

    2010-12-01

    B-cell chronic lymphocytic leukaemia (B-CLL) originates from B lymphocytes that may differ in the activation level, maturation state or cellular subgroups in peripheral blood. Tumour progression in CLL B cells seems to result in gradual accumulation of the clone of resting B lymphocytes in the early phases (G0/G1) of the cell cycle. The G1 phase is impaired in B-CLL. We investigated the gene expression of five key cell cycle regulators: TP 53, c-Myc, cyclin D2, p21WAF1/CIP1 and p27KIP1, which primarily regulate the G1 phase of the cell cycle, or S-phase entry and ultimately control the proliferation and cell growth as well as their role in B-CLL progression. The study was conducted in peripheral blood CLL lymphocytes of 40 previously untreated patients. Statistical analysis of correlations of TP53, cyclin D2, c-Myc, p21WAF1/CIP1 and p27KIP1 expressions in B-CLL patients with different Rai stages demonstrated that the progression of disease was accompanied by increases in p53, cyclin D2 and c-Myc mRNA expression. The expression of p27KIP1 was nearly statistically significant whereas that of p21 WAF1/CIP1 showed no such correlation. Moreover, high expression levels of TP53 and c-Myc genes were found to be closely associated with more aggressive forms of the disease requiring earlier therapy.

  16. Fad24, a Positive Regulator of Adipogenesis, Is Required for S Phase Re-entry of C2C12 Myoblasts Arrested in G0 Phase and Involved in p27(Kip1) Expression at the Protein Level.

    PubMed

    Ochiai, Natsuki; Nishizuka, Makoto; Osada, Shigehiro; Imagawa, Masayoshi

    2016-05-01

    Factor for adipocyte differentiation 24 (fad24) is a positive regulator of adipogenesis. We previously found that human fad24 is abundantly expressed in skeletal muscle. However, the function of fad24 in skeletal muscle remains largely unknown. Because skeletal muscle is a highly regenerative tissue, we focused on the function of fad24 in skeletal muscle regeneration. In this paper, we investigated the role of fad24 in the cell cycle re-entry of quiescent C2C12 myoblasts-mimicked satellite cells. The expression levels of fad24 and histone acetyltransferase binding to ORC1 (hbo1), a FAD24-interacting factor, were elevated at the early phase of the regeneration process in response to cardiotoxin-induced muscle injury. The knockdown of fad24 inhibited the proliferation of quiescent myoblasts, whereas fad24 knockdown did not affect differentiation. S phase entry following serum activation is abrogated by fad24 knockdown in quiescent cells. Furthermore, fad24 knockdown cells show a marked accumulation of p27(Kip1) protein. These results suggest that fad24 may have an important role in the S phase re-entry of quiescent C2C12 cells through the regulation of p27(Kip1) at the protein level.

  17. CBP and p27KIP1 in Prostate Carcinogenesis

    DTIC Science & Technology

    2008-02-01

    acid ( SAHA ), are effective for the treatment of prostate tumors developed in CBP/p27KIP1 mice. As scheduled in our proposal, we have generated CBP...on tumors developed in CBP/p27KIP1 double knockout mice. 6. References Fero ML et al., A syndrome of multiorgan hyperplasia with features of

  18. Nkx3.1 and p27(KIP1) cooperate in proliferation inhibition and apoptosis induction in human androgen-independent prostate cancer cells.

    PubMed

    Wang, Ping; Ma, Qi; Luo, JinDan; Liu, Ben; Tan, FuQing; Zhang, ZhiGen; Chen, ZhaoDian

    2009-05-01

    Prostate cancer (PC), which responds well to androgen ablation initially, invariably progresses to treatment resistance. The so-called androgen-independent PC is also a concern, since there is no effective therapy so far. Nkx3.1 is a putative prostate tumor suppressor that is expressed exclusively in the prostate under the regulation of androgen, and p27(KIP1) functions as a cell proliferation inhibitor and apoptosis trigger by disrupting the cyclin-dependent kinase (CDK)-cyclin complex. Lack of expressions of Nkx3.1 and/or p27(KIP1) have been detected in most advanced PC and is associated with poor clinical progression. Here, we show that endogenous expressions of both Nkx3.1 and p27(KIP1) are lost in the androgen-independent PC3 PC cells, while remaining intact in LNCaP PC cells, which contain functional androgen receptor (AR) and are hormone-responsive. Ectopic restoration of either Nkx3.1 or p27(KIP1) in PC3 cells results in reduced cell proliferation, and increased cell death. Both effects are synergistically enhanced when the two molecules are coexpressed. p27(KIP1) overexpression in PC3 results in increased cell population ceased at the G0/G1 phase, and this cell-cycle-arresting effect is significantly enhanced by the coexpression of Nkx3.1. Flow cytometry further revealed that Nkx3.1 and p27(KIP1) also cooperatively render more PC3 cells undergoing apoptosis. Consistently, the coexpression of Nkx3.1 and p27(KIP1) leads to the decreased expression of Bcl-2 oncogene and a concomitantly upregulated Bax expression. It also activates caspase 3 and leads to increased cleavage of PARP. Our findings thus reveal the crucial relevance of the combined antiproliferative and proapoptotic activities of Nkx3.1 and p27(KIP1) in androgen-independent PC cells, and further suggest that a combined, rather than single gene manipulation may be of clinical value for hormone-refractory PC.

  19. Phosphorylation and subcellular localization of p27Kip1 regulated by hydrogen peroxide modulation in cancer cells.

    PubMed

    Ibañez, Irene L; Bracalente, Candelaria; Notcovich, Cintia; Tropper, Ivanna; Molinari, Beatriz L; Policastro, Lucía L; Durán, Hebe

    2012-01-01

    The Cyclin-dependent kinase inhibitor 1B (p27Kip1) is a key protein in the decision between proliferation and cell cycle exit. Quiescent cells show nuclear p27Kip1, but this protein is exported to the cytoplasm in response to proliferating signals. We recently reported that catalase treatment increases the levels of p27Kip1 in vitro and in vivo in a murine model. In order to characterize and broaden these findings, we evaluated the regulation of p27Kip1 by hydrogen peroxide (H(2)O(2)) in human melanoma cells and melanocytes. We observed a high percentage of p27Kip1 positive nuclei in melanoma cells overexpressing or treated with exogenous catalase, while non-treated controls showed a cytoplasmic localization of p27Kip1. Then we studied the levels of p27Kip1 phosphorylated (p27p) at serine 10 (S10) and at threonine 198 (T198) because phosphorylation at these sites enables nuclear exportation of this protein, leading to accumulation and stabilization of p27pT198 in the cytoplasm. We demonstrated by western blot a decrease in p27pS10 and p27pT198 levels in response to H(2)O(2) removal in melanoma cells, associated with nuclear p27Kip1. Melanocytes also exhibited nuclear p27Kip1 and lower levels of p27pS10 and p27pT198 than melanoma cells, which showed cytoplasmic p27Kip1. We also showed that the addition of H(2)O(2) (0.1 µM) to melanoma cells arrested in G1 by serum starvation induces proliferation and increases the levels of p27pS10 and p27pT198 leading to cytoplasmic localization of p27Kip1. Nuclear localization and post-translational modifications of p27Kip1 were also demonstrated by catalase treatment of colorectal carcinoma and neuroblastoma cells, extending our findings to these other human cancer types. In conclusion, we showed in the present work that H(2)O(2) scavenging prevents nuclear exportation of p27Kip1, allowing cell cycle arrest, suggesting that cancer cells take advantage of their intrinsic pro-oxidant state to favor cytoplasmic localization of p27

  20. Phosphorylation and Subcellular Localization of p27Kip1 Regulated by Hydrogen Peroxide Modulation in Cancer Cells

    PubMed Central

    Ibañez, Irene L.; Bracalente, Candelaria; Notcovich, Cintia; Tropper, Ivanna; Molinari, Beatriz L.; Policastro, Lucía L.; Durán, Hebe

    2012-01-01

    The Cyclin-dependent kinase inhibitor 1B (p27Kip1) is a key protein in the decision between proliferation and cell cycle exit. Quiescent cells show nuclear p27Kip1, but this protein is exported to the cytoplasm in response to proliferating signals. We recently reported that catalase treatment increases the levels of p27Kip1 in vitro and in vivo in a murine model. In order to characterize and broaden these findings, we evaluated the regulation of p27Kip1 by hydrogen peroxide (H2O2) in human melanoma cells and melanocytes. We observed a high percentage of p27Kip1 positive nuclei in melanoma cells overexpressing or treated with exogenous catalase, while non-treated controls showed a cytoplasmic localization of p27Kip1. Then we studied the levels of p27Kip1 phosphorylated (p27p) at serine 10 (S10) and at threonine 198 (T198) because phosphorylation at these sites enables nuclear exportation of this protein, leading to accumulation and stabilization of p27pT198 in the cytoplasm. We demonstrated by western blot a decrease in p27pS10 and p27pT198 levels in response to H2O2 removal in melanoma cells, associated with nuclear p27Kip1. Melanocytes also exhibited nuclear p27Kip1 and lower levels of p27pS10 and p27pT198 than melanoma cells, which showed cytoplasmic p27Kip1. We also showed that the addition of H2O2 (0.1 µM) to melanoma cells arrested in G1 by serum starvation induces proliferation and increases the levels of p27pS10 and p27pT198 leading to cytoplasmic localization of p27Kip1. Nuclear localization and post-translational modifications of p27Kip1 were also demonstrated by catalase treatment of colorectal carcinoma and neuroblastoma cells, extending our findings to these other human cancer types. In conclusion, we showed in the present work that H2O2 scavenging prevents nuclear exportation of p27Kip1, allowing cell cycle arrest, suggesting that cancer cells take advantage of their intrinsic pro-oxidant state to favor cytoplasmic localization of p27Kip1. PMID

  1. Autophagy regulates T lymphocyte proliferation through selective degradation of the cell-cycle inhibitor CDKN1B/p27Kip1.

    PubMed

    Jia, Wei; He, Ming-Xiao; McLeod, Ian X; Guo, Jian; Ji, Dong; He, You-Wen

    2015-01-01

    The highly conserved cellular degradation pathway, macroautophagy, regulates the homeostasis of organelles and promotes the survival of T lymphocytes. Previous results indicate that Atg3-, Atg5-, or Pik3c3/Vps34-deficient T cells cannot proliferate efficiently. Here we demonstrate that the proliferation of Atg7-deficient T cells is defective. By using an adoptive transfer and Listeria monocytogenes (LM) mouse infection model, we found that the primary immune response against LM is intrinsically impaired in autophagy-deficient CD8(+) T cells because the cell population cannot expand after infection. Autophagy-deficient T cells fail to enter into S-phase after TCR stimulation. The major negative regulator of the cell cycle in T lymphocytes, CDKN1B, is accumulated in autophagy-deficient naïve T cells and CDKN1B cannot be degraded after TCR stimulation. Furthermore, our results indicate that genetic deletion of one allele of CDKN1B in autophagy-deficient T cells restores proliferative capability and the cells can enter into S-phase after TCR stimulation. Finally, we found that natural CDKN1B forms polymers and is physiologically associated with the autophagy receptor protein SQSTM1/p62 (sequestosome 1). Collectively, autophagy is required for maintaining the expression level of CDKN1B in naïve T cells and selectively degrades CDKN1B after TCR stimulation.

  2. miR-222 induces Adriamycin resistance in breast cancer through PTEN/Akt/p27(kip1) pathway.

    PubMed

    Wang, Dan-Dan; Yang, Su-Jin; Chen, Xiu; Shen, Hong-Yu; Luo, Long-Ji; Zhang, Xiao-Hui; Zhong, Shan-Liang; Zhao, Jian-Hua; Tang, Jin-Hai

    2016-11-01

    The high resistant rate of Adriamycin (Adr) is associated with a poor prognosis of breast cancer in women worldwide. Since miR-222 might contribute to chemoresistance in many cancer types, in this study, we aimed to investigate its efficacy in breast cancer through PTEN/Akt/p27 (kip1) pathway. Firstly, in vivo, we verified that miR-222 was upregulated in chemoresistant tissues after surgery compared with the paired preneoadjuvant samples of 21 breast cancer patients. Then, human breast cancer Adr-resistant cell line (MCF-7/Adr) was constructed to validate the pathway from the parental sensitive cell line (MCF-7/S). MCF-7/Adr and MCF-7/S were transfected with miR-222 mimics, miR-222 inhibitors, or their negative controls, respectively. The results showed that inhibition of miR-222 in MCF-7/Adr significantly increased the expressions of PTEN and p27 (kip1) and decreased phospho-Akt (p-Akt) both in mRNA and protein levels (p < 0.05) by using quantitative real-time PCR (qRT-PCR) and western blot. MTT and flow cytometry suggested that lower expressed miR-222 enhanced apoptosis and decreased the IC50 of MCF-7/Adr cells. Additionally, immunofluorescence demonstrated that the subcellular location of p27 (kip1) was dislocated resulting from the alteration of miR-222. Conversely, in MCF-7/S transfected with miR-222 mimics, upregulation of miR-222 is associated with decreasing PTEN and p27 (kip1) and increasing Akt accompanied by less apoptosis and higher IC50. Importantly, Adr resistance induced by miR-222 overexpression through PTEN/Akt/p27 was completely blocked by LY294002, an Akt inhibitor. Taken together, these data firstly elucidated that miR-222 could reduce the sensitivity of breast cancer cells to Adr through PTEN/Akt/p27 (kip1) signaling pathway, which provided a potential target to increase the sensitivity to Adr in breast cancer treatment and further improved the prognosis of breast cancer patients.

  3. MicroRNAs 221 and 222 target p27Kip1 in Marek's disease virus-transformed tumour cell line MSB-1.

    PubMed

    Lambeth, Luke S; Yao, Yongxiu; Smith, Lorraine P; Zhao, Yuguang; Nair, Venugopal

    2009-05-01

    MicroRNAs (miRNAs) are a class of short RNAs that function as post-transcriptional suppressors of protein expression and are involved in a variety of biological processes, including oncogenesis. Several recent studies have implicated the involvement of miR-221 and miR-222 in tumorigenesis as these miRNAs are upregulated in a number of cancers and affect the expression of cell cycle regulatory proteins such as the cyclin-dependent kinase (cdk) inhibitor p27(Kip1). Marek's disease virus (MDV) is a highly oncogenic herpesvirus that affects poultry, causing acute neoplastic disease with lymphomatous lesions in several organs. MDV-encoded oncogenes such as Meq are directly implicated in the neoplastic transformation of T cells and have been well studied. More recently, however, the involvement of both host and virus-encoded miRNAs in the induction of MD lymphomas is being increasingly recognized. We analysed the miRNA expression profiles in the MDV-transformed lymphoblastoid cell line MSB-1 and found that endogenous miRNAs miR-221 and miR-222 were significantly upregulated. Demonstration of the conserved binding sites for these miRNAs in the chicken p27(Kip1) 3'-untranslated region sequence and the repression of luciferase activity of reporter constructs indicated that miR-221 and miR-222 target p27(Kip1) in these cells. We also found that overexpression of miR-221 and miR-222 decreased p27(Kip1) levels and that treatment with retrovirally expressed antagomiRs partially alleviated this suppression. These data show that an oncogenic herpesvirus, as in the case of many cancers, can exploit the miRNA machinery for suppressing cell cycle regulatory molecules such as p27(Kip1) in the induction and progression of T-cell lymphomas.

  4. Attenuated expression of menin and p27 (Kip1) in an aggressive case of multiple endocrine neoplasia type 1 (MEN1) associated with an atypical prolactinoma and a malignant pancreatic endocrine tumor.

    PubMed

    Ishida, Emi; Yamada, Masanobu; Horiguchi, Kazuhiko; Taguchi, Ryo; Ozawa, Atsushi; Shibusawa, Nobuyuki; Hashimoto, Koshi; Satoh, Tetsuro; Yoshida, Sachiko; Tanaka, Yoshiki; Yokota, Machiko; Tosaka, Masahiko; Hirato, Junko; Yamada, Shozo; Yoshimoto, Yuhei; Mori, Masatomo

    2011-01-01

    Tumors in multiple endocrine neoplasia type 1 (MEN1) are generally benign. Since information on the pathogenesis of MEN1 in malignant cases is limited, we conducted genetic analysis and compared the expression of menin, p27(Kip1)(p27)/CDKN1B and p18(Ink4C)(p18)/CDKN2C with levels in benign cases. We describe the case of a 56 year-old male with an atypical prolactinoma and malignant pancreatic neuroenocrine tumor. At age 50, he had undergone transsphenoidal surgery to remove a prolactinoma. However, the tumor relapsed twice. Histological analysis of the recurrent prolactinoma revealed the presence of prolactin, a high MIB-1 index (32.1 %), p53-positive cells (0.2%), and an unusual association with FSH-positive cells. A few years later, he was also found to have a non-functioning pancreatic tumor with probable metastasis to the extradullar region. The metastatic region tested positive for chromogranin and CD56, and negative for prolactin, with 1.2 % of cells p53-positive. Although genetic analyses of the MEN1, p27, and p18 genes demonstrated no mutation, numbers of menin, p27 and p18 immuno-positive cells were significantly down-regulated in the recurrent prolactinoma, but that of p18 was intact in the metastatic region. Furthermore, MEN1 and p27 mRNA levels of the recurrent prolactinoma were down-regulated, particularly the MEN1 mRNA level, compared to levels in 10 cases of benign prolactinoma, while the p18 mRNA level was similar to that of normal pituitary. The tumor in this case may be a subtype of MEN1 showing more aggressive and malignant features probably induced by low levels of menin and p27.

  5. Gfer inhibits Jab1-mediated degradation of p27kip1 to restrict proliferation of hematopoietic stem cells

    PubMed Central

    Teng, Ellen C.; Todd, Lance R.; Ribar, Thomas J.; Lento, William; Dimascio, Leah; Means, Anthony R.; Sankar, Uma

    2011-01-01

    Growth factor erv1-like (Gfer) is an evolutionarily conserved sulfhydryl oxidase that is enriched in embryonic and adult stem cells and plays an essential prosurvival role in pluripotent embryonic stem cells. Here we show that knockdown (KD) of Gfer in hematopoietic stem cells (HSCs) compromises their in vivo engraftment potential and triggers a hyper-proliferative response that leads to their exhaustion. KD of Gfer in HSCs does not elicit a significant alteration of mitochondrial morphology or loss of cell viability. However, these cells possess significantly reduced levels of the cyclin-dependent kinase inhibitor p27kip1. In contrast, overexpression of Gfer in HSCs results in significantly elevated total and nuclear p27kip1. KD of Gfer results in enhanced binding of p27kip1 to its inhibitor, the COP9 signalosome subunit jun activation-domain binding protein 1 (Jab1), leading to its down-regulation. Conversely, overexpression of Gfer results in its enhanced binding to Jab1 and inhibition of the Jab1-p27kip1 interaction. Furthermore, normalization of p27kip1 in Gfer-KD HSCs rescues their in vitro proliferation deficits. Taken together, our data demonstrate the presence of a novel Gfer-Jab1-p27kip1 pathway in HSCs that functions to restrict abnormal proliferation. PMID:21346186

  6. p27Kip1 Is Required to Mediate a G1 Cell Cycle Arrest Downstream of ATM following Genotoxic Stress

    PubMed Central

    Cassimere, Erica K.; Mauvais, Claire; Denicourt, Catherine

    2016-01-01

    The DNA damage response (DDR) is a coordinated signaling network that ensures the maintenance of genome stability under DNA damaging stress. In response to DNA lesions, activation of the DDR leads to the establishment of cell cycle checkpoints that delay cell-cycle progression and allow repair of the defects. The tumor suppressor p27Kip1 is a cyclin-CDK inhibitor that plays an important role in regulating quiescence in a variety of tissues. Several studies have suggested that p27Kip1 also plays a role in the maintenance of genomic integrity. Here we demonstrate that p27Kip1 is essential for the establishment of a G1 checkpoint arrest after DNA damage. We also uncovered that ATM phosphorylates p27Kip1 on a previously uncharacterized residue (Ser-140), which leads to its stabilization after induction of DNA double-strand breaks. Inhibition of this stabilization by replacing endogenous p27Kip1 with a Ser-140 phospho-mutant (S140A) significantly sensitized cells to IR treatments. Our findings reveal a novel role for p27Kip1 in the DNA damage response pathway and suggest that part of its tumor suppressing functions relies in its ability to mediate a G1 arrest after the induction of DNA double strand breaks. PMID:27611996

  7. Differential Roles of Hath1, MUC2 and P27Kip1 in Relation with Gamma-Secretase Inhibition in Human Colonic Carcinomas: A Translational Study

    PubMed Central

    Souazé, Frédérique; Bou-Hanna, Chantal; Kandel, Christine; Leclair, François; Devallière, Julie; Charreau, Béatrice; Bézieau, Stéphane; Mosnier, Jean-François; Laboisse, Christian L.

    2013-01-01

    Hath1, a bHLH transcription factor negatively regulated by the γ-secretase-dependent Notch pathway, is required for intestinal secretory cell differentiation. Our aim was fourfold: 1) determine whether Hath1 is able to alter the phenotype of colon cancer cells that are committed to a differentiated phenotype, 2) determine whether the Hath1-dependent alteration of differentiation is coupled to a restriction of anchorage-dependent growth, 3) decipher the respective roles of three putative tumor suppressor genes Hath1, MUC2 and P27kip1 in this coupling and, 4) examine how our findings translate to primary tumors. Human colon carcinoma cell lines that differentiate along a mucin secreting (MUC2/MUC5AC) and/or enterocytic (DPPIV) lineages were maintained on inserts with or without a γ-secretase inhibitor (DBZ). Then the cells were detached and their ability to survive/proliferate in the absence of substratum was assessed. γ-secretase inhibition led to a Hath1-mediated preferential induction of MUC2 over MUC5AC, without DPPIV modification, in association with a decrease in anchorage-independent growth. While P27kip1 silencing relieved the cells from the Hath1-induced decrease of anchorage-independent growth, MUC2 silencing did not modify this parameter. Hath1 ectopic expression in the Hath1 negative enterocytic Caco2 cells led to a decreased anchorage-independent growth in a P27kip1-independent manner. In cultured primary human colon carcinomas, Hath1 was up-regulated in 7 out of 10 tumors upon DBZ treatment. Parallel MUC2 up-regulation occurred in 4 (4/7) and P27kip1 in only 2 (2/7) tumors. Interestingly, the response patterns of primary tumors to DBZ fitted with the hierarchical model of divergent signalling derived from our findings on cell lines. PMID:23409082

  8. Ras/ERK1 pathway regulation of p27KIP1-mediated G1-phase cell-cycle arrest in cordycepin-induced inhibition of the proliferation of vascular smooth muscle cells.

    PubMed

    Jung, Su-Mi; Park, Sung-Soo; Kim, Wun-Jae; Moon, Sung-Kwon

    2012-04-15

    Cordycepin, the main constituent of Cordyceps militaris, demonstrated an anti-atherogenic effect in experimental animals. However, the effects of cordycepin on cell-cycle regulation and the signaling pathway in vascular smooth muscle cells (VSMC) remain largely unknown; therefore, unexpected roles of cordycepin-induced inhibition in VSMC growth were investigated. Mechanisms in cordycepin-treated VSMC were examined via an MTT assay, a thymidine uptake experiment, FACS analysis, immunoblot analysis, kinase assay, immunoprecipitation assay, and transient transfection assays. Cordycepin inhibited cell growth, induced G1-phase cell-cycle arrest, down-regulated cyclins and cyclin-dependent kinase (CDK) expression, and up-regulated p27KIP1 expression in VSMC. Cordycepin induced activation of JNK, p38MAPK and ERK1/2. Blocking of the ERK function using either ERK1/2-specific inhibitor U0126 or a small interfering RNA (si-ERK1) reversed p27KIP1 expression, inhibition of cell growth, and decreased cell-cycle proteins in cordycepin-treated VSMC. Ras activation was increased by cordycepin. Transfection of cells with dominant negative Ras (RasN17) mutant genes rescued cordycepin-induced ERK1/2 activity, increased p27KIP1 expression, inhibited cell proliferation, and reduced cell cycle proteins. In conclusion, our findings indicate that Ras/ERK1 pathways participate in p27KIP1-mediated G1-phase cell-cycle arrest induced by cordycepin via a decrease in cyclin/CDK complexes in VSMC.

  9. Damage-specific DNA binding protein 1 (DDB1) is involved in ubiquitin-mediated proteolysis of p27Kip1 in response to UV irradiation.

    PubMed

    Iovine, Barbara; Iannella, Maria Luigia; Bevilacqua, Maria Assunta

    2011-05-01

    Damage-specific DNA binding protein 1 (DDB1) is a conserved protein component of the damaged DNA binding protein complex (DDB) that recognizes UV-induced DNA lesions and initiates the nucleotide excision repair process. DDB1 is also part of an E3 ubiquitin-ligase complex that targets a variety of substrates for proteolysis including the cyclin-dependent kinase inhibitor p27(Kip1). The mechanism regulating the trafficking of DDB1 and its relationship with UV irradiation is not known, although cell cycle progression is implicated in the molecular machinery driving DDB1 into the nucleus. We evaluated the involvement of DDB1 in ubiquitination of the cdk inhibitor p27(Kip1) in response to UV irradiation. First, we observed that low and high doses of UV irradiation exert different effects on p27(Kip1) protein levels. Indeed, low but not high UV doses induced p27(Kip1) protein proteolysis in several human cell lines and UV-dependent degradation is dominant over other genotoxic agents such as cisplatin. We also demonstrate that p27(Kip1) reduction is not due to transcriptional regulation and that the proteasome inhibitor MG132 affects p27(Kip1) degradation. We observed that at low UV doses the decrease in p27(Kip1) nuclear protein related with DDB1 translocation into the nucleus; conversely, high doses of UV-induced p27(Kip1) accumulation and unchanged level of DDB1. The knockdown of DDB1 or Skp2 prevents UV-induced degradation of p27(Kip1) suggesting that DDB1 is essential to regulation of p27(kip1) turnover after a mild DNA damage. Our findings support the concept that DDB1 contributes to the activation of DNA repair mechanisms and could be a key factor in regulating the cell cycle in response to UV-induced DNA damage. Although the temporal order with which DDB1 contributes to ubiquitination of p27(Kip1) or initiates the nucleotide excision repair process remains to be established, our results represent a major step towards clarifying these issues.

  10. miR-222 is upregulated in epithelial ovarian cancer and promotes cell proliferation by downregulating P27(kip1.)

    PubMed

    Sun, Chaoyang; Li, Na; Zhou, Bo; Yang, Zongyuan; Ding, Dong; Weng, Danhui; Meng, Li; Wang, Shixuan; Zhou, Jianfeng; Ma, Ding; Chen, Gang

    2013-08-01

    Epithelial ovarian cancer (EOC) is the leading cause of female reproductive system cancer mortality in females. The majority of cases of ovarian carcinomas are not identified until a late stage. Identifying the molecular changes that occur during the development and progression of ovarian cancer is an urgent requirement. MicroRNAs (miRNAs) have been identified as gene expression regulators that induce mRNA degradation or translation blockade through pairing to the 3' untranslated region (3-'UTR) of the target mRNAs. In the present study, miR-222 was observed to be frequently upregulated in ovarian cancer. miR-222 upregulation induced an enhancement of ovarian cancer cell proliferation potential, possibly by downregulating its target, P27(Kip1). A bioinformatic analysis showed that the 3'-UTR of the P27(Kip1) mRNA contained a highly-conserved putative miR-222 binding site. Luciferase reporter assays demonstrated that P27(Kip1) was a direct target of miR-222. Consistently, there was an inverse correlation between the P27(Kip1) and miR-222 expression levels in the ovarian cancer cell lines and tissues. Overall, the present results suggest that miR-222 upregulation in human ovarian cancer may promote ovarian cancer cell proliferation during ovarian carcinogenesis.

  11. p27kip1 in Intestinal Tumorigenesis and Chemoprevention in the Mouse

    PubMed Central

    Yang, WanCai; Bancroft, Laura; Liang, Jiao; Zhuang, Min; Augenlicht, Leonard H.

    2010-01-01

    Targeted inactivation of p27kip1 was sufficient for intestinal tumor formation in mice, but this was strictly a function of diet: tumors formed in p27+/− or p27−/− mice fed control AIN-76A diet and were increased by a western-style diet but did not develop in mice fed standard chow diet. When crossed with the Apc1638N+/− mouse, Apc+/−,p27+/− or Apc+/−,p27−/− mice not only formed twice as many tumors than the sum of the tumors from mutation at either locus alone, but on AIN76A diet also developed intestinal intussusception, a tumor-associated pathology in patients leading to intestinal blockage that has not been reported for intestinal cancer in mouse models. Moreover, the frequency of intussusception was increased when the compound mutant mice were maintained on the western diet, leading to early death. Despite this more aggressive tumor phenotype generated by inactivation of p27 than by inactivation of another cyclin-dependent kinase inhibitor, p21WAF1/cip1, the nonsteroidal anti-inflammatory drug sulindac was still effective in inhibiting intestinal tumor formation in Apc+/−,p27+/− or Apc+/−,p27−/− mice, which contrasts with the abrogation of the effects of sulindac in Apc+/−,p21+/− or Apc+/−,p21−/− mice, indicating that p27 is not necessary for tumor inhibition by sulindac. Furthermore, tumor inhibition by sulindac was linked to the induction of p21 expression by the drug, regardless of p27 status, leading to suppression of cell proliferation and promotion of cell differentiation and apoptosis in the intestinal mucosa. PMID:16230399

  12. Tuberous sclerosis complex suppression in cerebellar development and medulloblastoma: separate regulation of mTOR activity and p27Kip1 localization

    PubMed Central

    Bhatia, Bobby; Northcott, Paul A.; Hambardzumyan, Dolores; Govindarajan, Baskaran; Brat, Daniel J.; Arbiser, Jack L.; Holland, Eric C.; Taylor, Michael D.; Kenney, Anna Marie

    2009-01-01

    During development, proliferation of cerebellar granule neuron precursors (CGNPs), candidate cells-of-origin for the pediatric brain tumor medulloblastoma, requires signaling by Sonic hedgehog (Shh) and insulin-like growth factor (IGF), whose pathways are also implicated in medulloblastoma. One of the consequences of IGF signaling is inactivation of the mTOR-suppressing Tuberous Sclerosis Complex (TSC), comprised of TSC1 and TSC2, leading to increased mRNA translation. We show that mice in which TSC function is impaired display increased mTOR pathway activation, enhanced CGNP proliferation, GSK-3α/β inactivation, and cytoplasmic localization of the cyclin-dependent kinase (cdk) inhibitor p27Kip1, which has been proposed to cause its inactivation or gain of oncogenic functions. We observed the same characteristics in wild-type primary cultures of CGNPs in which TSC1 and/or TSC2 were knocked down, and in mouse medulloblastomas induced by ectopic Shh pathway activation. Moreover, Shh-induced mouse medulloblastomas manifested Akt-mediated TSC2 inactivation, and the mutant TSC2 allele synergized with aberrant Shh signaling to increase medulloblastoma incidence in mice. Driving exogenous TSC2 expression in Shh-induced medulloblastoma cells corrected p27Kip1 localization and reduced proliferation. GSK-3α/β inactivation in the tumors in vivo and in primary CGNP cultures was mTOR-dependent, whereas p27Kip1 cytoplasmic localization was regulated upstream of mTOR, by TSC2. These results indicate that a balance between Shh mitogenic signaling and TSC function regulating new protein synthesis and cdk inhibition is essential for normal development and prevention of tumor formation or expansion. PMID:19738049

  13. Siah1/SIP regulates p27(kip1) stability and cell migration under metabolic stress.

    PubMed

    Nagano, Yoshito; Fukushima, Toru; Okemoto, Kazuo; Tanaka, Keiichiro; Bowtell, David D L; Ronai, Ze'ev; Reed, John C; Matsuzawa, Shu-ichi

    2011-08-01

    p27(kip1) has been implicated in cell cycle regulation, functioning as an inhibitor of cyclin-dependent kinase activity. In addition, p27 was also shown to affect cell migration, with accumulation of cytoplasmic p27 associated with tumor invasiveness. However, the mechanism underlying p27 regulation as a cytoplasmic protein is poorly understood. Here we show that glucose starvation induces proteasome-dependent degradation of cytoplasmic p27, accompanied by a decrease in cell motility. We also show that the glucose limitation-induced p27 degradation is regulated through an ubiquitin E3 ligase complex involving Siah1 and SIP/CacyBP. SIP (-/-) embryonic fibroblasts have increased levels of cytosolic p27 and exhibit increased cell motility compared to wild-type cells. These observations suggest that the Siah1/SIP E3 ligase complex regulates cell motility through degradation of p27.

  14. A p27Kip1 mutant that does not inhibit CDK activity promotes centrosome amplification and micronucleation.

    PubMed

    Sharma, S S; Ma, L; Bagui, T K; Forinash, K D; Pledger, W J

    2012-08-30

    Mitotic catastrophe occurs when cells enter mitosis with damaged DNA or excess centrosomes. Cells overexpressing the centrosome protein CP110 or depleted of cyclin F, which targets CP110 for destruction, have more than two centrosomes and undergo mitotic catastrophe. Our studies show centrosome reduplication and mitotic catastrophe in osteosarcoma cells inducibly expressing a p27Kip1 mutant (termed p27K) that binds cyclins but not cyclin-dependent kinases (CDKs). p27K inhibited cell proliferation but not CDK activity or cell cycle progression. It did not induce apoptosis; however, cells expressing p27K had more than two centrosomes and, indicative of mitotic catastrophe, irregularly shaped nuclei or multiple micronuclei. p27K interacted with cyclin F in vivo (as did endogenous p27Kip1) and displaced cyclin F from CP110. Depletion of CP110 rescued p27K-expressing cells from centrosome reduplication and mitotic catastrophe. Collectively, our data show that p27Kip1 can perturb mitosis and suggest that it does so by sequestering cyclin F, which prevents its interaction with and the subsequent degradation of CP110, ultimately resulting in centrosome reduplication, mitotic catastrophe and abrogation of cell proliferation.

  15. CKS1B, overexpressed in aggressive disease, regulates multiple myeloma growth and survival through SKP2- and p27Kip1-dependent and -independent mechanisms

    PubMed Central

    Colla, Simona; Wu, Xiaosong; Chen, Bangzheng; Stewart, James P.; Kuehl, W. Michael; Barlogie, Bart

    2007-01-01

    Overexpression of CKS1B, a gene mapping within a minimally amplified region between 153 to 154 Mb of chromosome 1q21, is linked to a poor prognosis in multiple myeloma (MM). CKS1B binds to and activates cyclin-dependent kinases and also interacts with SKP2 to promote the ubiquitination and proteasomal degradation of p27Kip1. Overexpression of CKS1B or SKP2 contributes to increased p27Kip1 turnover, cell proliferation, and a poor prognosis in many tumor types. Using 4 MM cell lines harboring MAF-, FGFR3/MMSET-, or CCND1-activating translocations, we show that lentiviral delivery of shRNA directed against CKS1B resulted in ablation of CKS1B mRNA and protein with concomitant stabilization of p27Kip1, cell cycle arrest, and apoptosis. Although shRNA-mediated knockdown of SKP2 and forced expression of a nondegradable form of p27Kip1 (p27T187A) led to cell cycle arrest, apoptosis was modest. Of importance, while knockdown of SKP2 or overexpression of p27T187A induced cell cycle arrest in KMS28PE, an MM cell line with biallelic deletion of CDKN1B/p27Kip1, CKS1B ablation induced strong apoptosis. These data suggest that CKS1B influences myeloma cell growth and survival through SKP2- and p27Kip1-dependent and -independent mechanisms and that therapeutic strategies aimed at abolishing CKS1B function may hold promise for the treatment of high-risk disease for which effective therapies are currently lacking. PMID:17303695

  16. Butein inhibits cell proliferation and induces cell cycle arrest in acute lymphoblastic leukemia via FOXO3a/p27kip1 pathway

    PubMed Central

    Wang, Li-Na; Tian, Yun; Shi, Dingbo; Wang, Jingshu; Qin, Ge; Li, Anchuan; Liang, Yan-Ni; Zhou, Huan-Juan; Ke, Zhi-Yong; Huang, Wenlin; Deng, Wuguo; Luo, Xue-Qun

    2016-01-01

    Acute lymphoblastic leukemia (ALL) is a common hematological malignancy characterized by the uncontrolled proliferation of leukemia cells in children. Discovering and developing effective chemotherapeutic drugs are needed for ALL. In this study, we investigated the anti-leukemic activity of butein and its action mechanisms in ALL. Butein was found to significantly suppress the cellular proliferation of ALL cell lines and primary ALL blasts in a dose-dependent manner. It also induced cell cycle arrest by decreasing the expression of cyclin E and CDK2. We also found that butein promoted nuclear Forkhead Class box O3a (FOXO3a) localization, enhanced the binding of FOXO3a on the p27kip1 gene promoter and then increased the expression of p27kip1. Moreover, we showed that FOXO3a knockdown significantly decreased the proliferation inhibition by butein, whereas overexpression of FOXO3a enhanced the butein-mediated proliferation inhibition. However, overexpression of FOXO3a mutation (C-terminally truncated FOXO3a DNA-binding domain) decreased the proliferation inhibition by butein through decreasing the expression of p27kip1. Our results therefore demonstrate the therapeutic potential of butein for ALL via FOXO3a/p27kip1 pathway. PMID:26919107

  17. Interferon-γ-induced p27KIP1 binds to and targets MYC for proteasome-mediated degradation

    PubMed Central

    Zakaria, Siti Mariam; Frings, Oliver; Fahlén, Sara; Nilsson, Helén; Goodwin, Jacob; von der Lehr, Natalie; Su, Yingtao; Lüscher, Bernhard; Castell, Alina; Larsson, Lars-Gunnar

    2016-01-01

    The Myc oncoprotein is tightly regulated at multiple levels including ubiquitin-mediated protein turnover. We recently demonstrated that inhibition of Cdk2-mediated phosphorylation of Myc at Ser-62 pharmacologically or through interferon (IFN)-γ-induced expression of p27Kip1 (p27) repressed Myc's activity to suppress cellular senescence and differentiation. In this study we identified an additional activity of p27 to interfere with Myc independent of Ser-62 phosphorylation. p27 is required and sufficient for IFN-γ-induced turnover of Myc. p27 interacted with Myc in the nucleus involving the C-termini of the two proteins, including Myc box 4 of Myc. The C-terminus but not the Cdk2 binding fragment of p27 was sufficient for inducing Myc degradation. Protein expression data of The Cancer Genome Atlas breast invasive carcinoma set revealed significantly lower Myc protein levels in tumors with highly expressed p27 lacking phosphorylation at Thr-157 - a marker for active p27 localized in the nucleus. Further, these conditions correlated with favorable tumor stage and patient outcome. This novel regulation of Myc by IFN-γ/p27KIP1 potentially offers new possibilities for therapeutic intervention in tumors with deregulated Myc. PMID:26701207

  18. Caffeic acid phenethyl ester induced cell cycle arrest and growth inhibition in androgen-independent prostate cancer cells via regulation of Skp2, p53, p21Cip1 and p27Kip1.

    PubMed

    Lin, Hui-Ping; Lin, Ching-Yu; Huo, Chieh; Hsiao, Ping-Hsuan; Su, Liang-Cheng; Jiang, Shih Sheng; Chan, Tzu-Min; Chang, Chung-Ho; Chen, Li-Tzong; Kung, Hsing-Jien; Wang, Horng-Dar; Chuu, Chih-Pin

    2015-03-30

    Prostate cancer (PCa) patients receiving the androgen ablation therapy ultimately develop recurrent castration-resistant prostate cancer (CRPC) within 1-3 years. Treatment with caffeic acid phenethyl ester (CAPE) suppressed cell survival and proliferation via induction of G1 or G2/M cell cycle arrest in LNCaP 104-R1, DU-145, 22Rv1, and C4-2 CRPC cells. CAPE treatment also inhibited soft agar colony formation and retarded nude mice xenograft growth of LNCaP 104-R1 cells. We identified that CAPE treatment significantly reduced protein abundance of Skp2, Cdk2, Cdk4, Cdk7, Rb, phospho-Rb S807/811, cyclin A, cyclin D1, cyclin H, E2F1, c-Myc, SGK, phospho-p70S6kinase T421/S424, phospho-mTOR Ser2481, phospho-GSK3α Ser21, but induced p21Cip1, p27Kip1, ATF4, cyclin E, p53, TRIB3, phospho-p53 (Ser6, Ser33, Ser46, Ser392), phospho-p38 MAPK Thr180/Tyr182, Chk1, Chk2, phospho-ATM S1981, phospho-ATR S428, and phospho-p90RSK Ser380. CAPE treatment decreased Skp2 and Akt1 protein expression in LNCaP 104-R1 tumors as compared to control group. Overexpression of Skp2, or siRNA knockdown of p21Cip1, p27Kip1, or p53 blocked suppressive effect of CAPE treatment. Co-treatment of CAPE with PI3K inhibitor LY294002 or Bcl-2 inhibitor ABT737 showed synergistic suppressive effects. Our finding suggested that CAPE treatment induced cell cycle arrest and growth inhibition in CRPC cells via regulation of Skp2, p53, p21Cip1, and p27Kip1.

  19. Regulation of p27Kip1 phosphorylation and G1 cell cycle progression by protein phosphatase PPM1G

    PubMed Central

    Sun, Chuang; Wang, Gaohang; Wrighton, Katharine H; Lin, Han; Songyang, Zhou; Feng, Xin-Hua; Lin, Xia

    2016-01-01

    The cell cycle, an essential process leading to the cell division, is stringently controlled by the key cell cycle regulators, cyclin-CDK complexes, whose activity is further regulated by a variety of mechanisms. p27Kip1 is a cyclin-CDK inhibitor that arrests the cell cycle at the G1 phase by blocking the activation of cyclin E-CDK2 complex, preventing the improper entry to the cell cycle. Dysfunction of p27 has been frequently observed in many types of human cancers, resulting from p27 protein degradation and cytoplasmic mislocalization, which are highly regulated by the phosphorylation status of p27. Although the kinases that phosphorylate p27 have been extensively studied, phosphatases that dephosphorylate p27 remain to be elucidated. By using genomic phosphatase screening, we identified a PPM family phosphatase, PPM1G, which could reduce p27 phosphorylation at T198. We further confirmed that PPM1G is a novel p27 phosphatase by demonstrating that PPM1G can interact with and dephosphorylate p27 in cells and in vitro. Functionally, ectopic expression of PPM1G enhanced p27 protein stability and delayed cell cycle progression from G1 to S phase. In accordance, knockdown of PPM1G accelerated p27 degradation during G1 phase and rendered cells resistant to the cell cycle arrest induced by serum deprivation. Mechanistically, PPM1G inhibited the interaction of p27 to 14-3-3θ, a chaperone protein that facilitates p27 nuclear export. Knockdown of PPM1G promoted the cytoplasmic localization of p27. Taken together, our studies identified PPM1G as a novel regulator of p27 that dephosphorylates p27 at T198 site and, together with p27 kinases, PPM1G controls cell cycle progression by maintaining the proper level of p27 protein. PMID:27822412

  20. High-Sensitivity IHC Detection of Phosphorylated p27/Kip1 in Human Tissues Using Secondary Antibody Conjugated to Polymer-HRP.

    PubMed

    Grahek, Michael; Ptak, Ana; Kalyuzhny, Alexander E

    2017-01-01

    A complex composed of goat anti-rabbit secondary antibody conjugated to a polymer coated with horseradish peroxidase (HRP) molecules was used to develop rapid and highly sensitive immunostaining protocol for the detection of phosphorylated p27/Kip1 (T157) in human tissues. This polymer-HRP complex produced much better sensitivity detection compared to conventional biotin-streptavidin-HRP chemistry. Using polymer-HRP made it possible to reduce primary antibody concentration, eliminate some incubation steps such as avidin-biotin blocking and incubation with separate biotinylated secondary antibodies, and shorten the incubation time with primary antibody. Specificity of the detection was confirmed by eliminating labeling after treating tissues with lambda phosphatase to remove phosphate groups from p27/Kip1. Secondary antibodies conjugated to polymer-HRP is a reagent of choice in both research and diagnostic pathology allowing detecting low abundant and weakly expressed tissue targets.

  1. Role of RhoA, mDia, and ROCK in cell shape-dependent control of the Skp2-p27kip1 pathway and the G1/S transition.

    PubMed

    Mammoto, Akiko; Huang, Sui; Moore, Kimberly; Oh, Philmo; Ingber, Donald E

    2004-06-18

    Cell shape-dependent control of cell-cycle progression underlies the spatial differentials of growth that drive tissue morphogenesis, yet little is known about how cell distortion impacts the biochemical signaling machinery that is responsible for growth control. Here we show that the Rho family GTPase, RhoA, conveys the "cell shape signal" to the cell-cycle machinery in human capillary endothelial cells. Cells accumulating p27(kip1) and arrested in mid G(1) phase when spreading were inhibited by restricted extracellular matrix adhesion, whereas constitutively active RhoA increased expression of the F-box protein Skp2 required for ubiquitination-dependent degradation of p27(kip1) and restored G(1) progression in these cells. Studies with dominant-negative and constitutively active forms of mDia1, a downstream effector of RhoA, and with a pharmacological inhibitor of ROCK, another RhoA target, revealed that RhoA promoted G(1) progression by altering the balance of activities between these two downstream effectors. These data indicate that signaling proteins such as mDia1 and ROCK, which are thought to be involved primarily in cytoskeletal remodeling, also mediate cell growth regulation by coupling cell shape to the cell-cycle machinery at the level of signal transduction.

  2. Insect peptide CopA3-induced protein degradation of p27Kip1 stimulates proliferation and protects neuronal cells from apoptosis

    SciTech Connect

    Nam, Seung Taek; Kim, Dae Hong; Lee, Min Bum; Nam, Hyo Jung; Kang, Jin Ku; Park, Mi Jung; Lee, Ik Hwan; Seok, Heon; Lee, Dong Gun; Hwang, Jae Sam; Kim, Ho

    2013-07-19

    Highlights: •CopA3 peptide isolated from the Korean dung beetle has antimicrobial activity. •Our study reported that CopA3 has anticancer and immunosuppressive effects. •We here demonstrated that CopA3 has neurotropic and neuroprotective effects. •CopA3 degrades p27Kip1 protein and this mediates effects of CopA3 on neuronal cells. -- Abstract: We recently demonstrated that the antibacterial peptide, CopA3 (a D-type disulfide dimer peptide, LLCIALRKK), inhibits LPS-induced macrophage activation and also has anticancer activity in leukemia cells. Here, we examined whether CopA3 could affect neuronal cell proliferation. We found that CopA3 time-dependently increased cell proliferation by up to 31 ± 2% in human neuroblastoma SH-SY5Y cells, and up to 29 ± 2% in neural stem cells isolated from neonatal mouse brains. In both cell types, CopA3 also significantly inhibited the apoptosis and viability losses caused by 6-hydroxy dopamine (a Parkinson disease-mimicking agent) and okadaic acid (an Alzheimer’s disease-mimicking agent). Immunoblotting revealed that the p27Kip1 protein (a negative regulator of cell cycle progression) was markedly degraded in CopA3-treated SH-SY5Y cells. Conversely, an adenovirus expressing p27Kip1 significantly inhibited the antiapoptotic effects of CopA3 against 6-hydroxy dopamine- and okadaic acid-induced apoptosis, and decreased the neurotropic effects of CopA3. These results collectively suggest that CopA3-mediated protein degradation of p27Kip1 may be the main mechanism through which CopA3 exerts neuroprotective and neurotropic effects.

  3. Insect peptide CopA3-induced protein degradation of p27Kip1 stimulates proliferation and protects neuronal cells from apoptosis.

    PubMed

    Nam, Seung Taek; Kim, Dae Hong; Lee, Min Bum; Nam, Hyo Jung; Kang, Jin Ku; Park, Mi Jung; Lee, Ik Hwan; Seok, Heon; Lee, Dong Gun; Hwang, Jae Sam; Kim, Ho

    2013-07-19

    We recently demonstrated that the antibacterial peptide, CopA3 (a D-type disulfide dimer peptide, LLCIALRKK), inhibits LPS-induced macrophage activation and also has anticancer activity in leukemia cells. Here, we examined whether CopA3 could affect neuronal cell proliferation. We found that CopA3 time-dependently increased cell proliferation by up to 31 ± 2% in human neuroblastoma SH-SY5Y cells, and up to 29 ± 2% in neural stem cells isolated from neonatal mouse brains. In both cell types, CopA3 also significantly inhibited the apoptosis and viability losses caused by 6-hydroxy dopamine (a Parkinson disease-mimicking agent) and okadaic acid (an Alzheimer's disease-mimicking agent). Immunoblotting revealed that the p27Kip1 protein (a negative regulator of cell cycle progression) was markedly degraded in CopA3-treated SH-SY5Y cells. Conversely, an adenovirus expressing p27Kip1 significantly inhibited the antiapoptotic effects of CopA3 against 6-hydroxy dopamine- and okadaic acid-induced apoptosis, and decreased the neurotropic effects of CopA3. These results collectively suggest that CopA3-mediated protein degradation of p27Kip1 may be the main mechanism through which CopA3 exerts neuroprotective and neurotropic effects.

  4. SIRT1-mediated downregulation of p27Kip1 is essential for overcoming contact inhibition of Kaposi's sarcoma-associated herpesvirus transformed cells.

    PubMed

    He, Meilan; Yuan, Hongfeng; Tan, Brandon; Bai, Rosemary; Kim, Heon Seok; Bae, Sangsu; Che, Lu; Kim, Jin-Soo; Gao, Shou-Jiang

    2016-11-15

    Kaposi's sarcoma-associated herpesvirus (KSHV) is an oncogenic virus associated with Kaposi's sarcoma (KS), a malignancy commonly found in AIDS patients. Despite intensive studies in the last two decades, the mechanism of KSHV-induced cellular transformation and tumorigenesis remains unclear. In this study, we found that the expression of SIRT1, a metabolic sensor, was upregulated in a variety of KSHV-infected cells. In a model of KSHV-induced cellular transformation, SIRT1 knockdown with shRNAs or knockout by CRISPR/Cas9 gene editing dramatically suppressed cell proliferation and colony formation in soft agar of KSHV-transformed cells by inducing cell cycle arrest and contact inhibition. SIRT1 knockdown or knockout induced the expression of cyclin-dependent kinase inhibitor 1B (p27Kip1). Consequently, p27 knockdown rescued the inhibitory effect of SIRT1 knockdown or knockout on cell proliferation and colony formation. Furthermore, treatment of KSHV-transformed cells with a SIRT1 inhibitor, nicotinamide (NAM), had the same effect as SIRT1 knockdown and knockout. NAM significantly inhibited cell proliferation in culture and colony formation in soft agar, and induced cell cycle arrest. Significantly, NAM inhibited the progression of tumors and extended the survival of mice in a KSHV-induced tumor model. Collectively, these results demonstrate that SIRT1 suppression of p27 is required for KSHV-induced tumorigenesis and identify a potential therapeutic target for KS.

  5. SIRT1-mediated downregulation of p27Kip1 is essential for overcoming contact inhibition of Kaposi's sarcoma-associated herpesvirus transformed cells

    PubMed Central

    Tan, Brandon; Bai, Rosemary; Kim, Heon Seok; Bae, Sangsu; Che, Lu; Kim, Jin-Soo; Gao, Shou-Jiang

    2016-01-01

    Kaposi's sarcoma-associated herpesvirus (KSHV) is an oncogenic virus associated with Kaposi's sarcoma (KS), a malignancy commonly found in AIDS patients. Despite intensive studies in the last two decades, the mechanism of KSHV-induced cellular transformation and tumorigenesis remains unclear. In this study, we found that the expression of SIRT1, a metabolic sensor, was upregulated in a variety of KSHV-infected cells. In a model of KSHV-induced cellular transformation, SIRT1 knockdown with shRNAs or knockout by CRISPR/Cas9 gene editing dramatically suppressed cell proliferation and colony formation in soft agar of KSHV-transformed cells by inducing cell cycle arrest and contact inhibition. SIRT1 knockdown or knockout induced the expression of cyclin-dependent kinase inhibitor 1B (p27Kip1). Consequently, p27 knockdown rescued the inhibitory effect of SIRT1 knockdown or knockout on cell proliferation and colony formation. Furthermore, treatment of KSHV-transformed cells with a SIRT1 inhibitor, nicotinamide (NAM), had the same effect as SIRT1 knockdown and knockout. NAM significantly inhibited cell proliferation in culture and colony formation in soft agar, and induced cell cycle arrest. Significantly, NAM inhibited the progression of tumors and extended the survival of mice in a KSHV-induced tumor model. Collectively, these results demonstrate that SIRT1 suppression of p27 is required for KSHV-induced tumorigenesis and identify a potential therapeutic target for KS. PMID:27708228

  6. Cellular mechanism through which parathyroid hormone-related protein induces proliferation in arterial smooth muscle cells: definition of an arterial smooth muscle PTHrP/p27kip1 pathway.

    PubMed

    Fiaschi-Taesch, Nathalie; Sicari, Brian M; Ubriani, Kiran; Bigatel, Todd; Takane, Karen K; Cozar-Castellano, Irene; Bisello, Alessandro; Law, Brian; Stewart, Andrew F

    2006-10-27

    Parathyroid hormone-related protein (PTHrP) is present in vascular smooth muscle (VSM), is markedly upregulated in response to arterial injury, is essential for normal VSM proliferation, and also markedly accentuates neointima formation following rat carotid angioplasty. PTHrP contains a nuclear localization signal (NLS) through which it enters the nucleus and leads to marked increases in retinoblastoma protein (pRb) phosphorylation and cell cycle progression. Our goal was to define key cell cycle molecules upstream of pRb that mediate cell cycle acceleration induced by PTHrP. The cyclin D/cdk-4,-6 system and its upstream regulators, the inhibitory kinases (INKs), are not appreciably influenced by PTHrP. In striking contrast, cyclin E/cdk-2 kinase activity is markedly increased by PTHrP, and this is a result of a specific, marked, PTHrP-induced proteasomal degradation of p27(kip1). Adenoviral restoration of p27(kip1) fully reverses PTHrP-induced cell cycle progression, indicating that PTHrP mediates its cell cycle acceleration in VSM via p27(kip1). In confirmation, adenoviral delivery of PTHrP to murine primary vascular smooth muscle cells (VSMCs) significantly decreases p27(kip1) expression and accelerates cell cycle progression. p27(kip1) is well known to be a central cell cycle regulatory molecule involved in both normal and pathological VSM proliferation and is a target of widely used drug-eluting stents. The current observations define a novel "PTHrP/p27(kip1) pathway" in the arterial wall and suggest that this pathway is important in normal arterial biology and a potential target for therapeutic manipulation of the arterial response to injury.

  7. Contact with fibrillar collagen inhibits melanoma cell proliferation by up-regulating p27KIP1

    PubMed Central

    Henriet, Patrick; Zhong, Zhi-Duan; Brooks, Peter C.; Weinberg, Kenneth I.; DeClerck, Yves A.

    2000-01-01

    It is known that the extracellular matrix regulates normal cell proliferation, and it is assumed that anchorage-independent malignant cells escape this regulatory function. Here we demonstrate that human M24met melanoma cells remain responsive to growth regulatory signals that result from contact with type I collagen and that the effect on proliferation depends on the physical structure of the collagen. On polymerized fibrillar collagen, M24met cells are growth arrested at the G1/S checkpoint and maintain high levels of p27KIP1 mRNA and protein. In contrast, on nonfibrillar (denatured) collagen, the cells enter the cell cycle, and p27KIP1 is down-regulated. These growth regulatory effects involve contact between type I collagen and the collagen-binding integrin α2β1, which appears restricted in the presence of fibrillar collagen. Thus melanoma cells remain sensitive to negative growth regulatory signals originating from fibrillar collagen, and the proteolytic degradation of fibrils is a mechanism allowing tumor cells to escape these restrictive signals. PMID:10944199

  8. Cooperative role between p21cip1/waf1 and p27kip1 in premature senescence in glandular proliferative lesions in mice.

    PubMed

    García-Fernández, R A; García-Palencia, P; Suarez, C; Sánchez, M A; Gil-Gómez, G; Sánchez, B; Rollán, E; Martín-Caballero, J; Flores, J M

    2014-03-01

    Cellular senescence has been considered a novel target for cancer therapy. It has also been pointed out that p21(cip1/waf1) and p27(kip1) cyclin-dependent kinase inhibitors (CKIs) play a role in cellular senescence in some tumor types. Therefore, in order to address the possibility of a cooperative role between p21 and p27 proteins in senescence in vivo we analyzed cellular senescence in spontaneous glandular proliferative lesions (adrenal, thyroid and pituitary glands) in a double-KO mice model, using γH2AX, p53, p16, PTEN and Ki67 as senescence markers. The results obtained showed that p21p27 double-null mice had the lowest number of γH2AX positive cells in glandular hyperplasias and benign tumors. Also, in this group, Ki67 proliferation index correlated with a lower immunohistochemical expression of γH2AX and p53. The expression of p16 and PTEN do not seem to cause synergism of senescence in the benign lesions analyzed in p21p27 double-KO mice. These observations suggest an intrinsic cooperation between p21 and p27 CKIs in the activation of stress-induced cellular senescence and tumor progression in vivo, which would be a physiological mechanism to prevent tumor cell proliferation.

  9. The B56γ3 regulatory subunit-containing protein phosphatase 2A outcompetes Akt to regulate p27KIP1 subcellular localization by selectively dephosphorylating phospho-Thr157 of p27KIP1

    PubMed Central

    Lai, Tai-Yu; Yang, Yu-San; Hong, Wei-Fu; Chiang, Chi-Wu

    2016-01-01

    The B56γ-containing protein phosphatase 2A (PP2A-B56γ) has been postulated to have tumor suppressive functions. Here, we report regulation of p27KIP1 subcellular localization by PP2A-B56γ3. B56γ3 overexpression enhanced nuclear localization of p27KIP1, whereas knockdown of B56γ3 decreased p27KIP1 nuclear localization. B56γ3 overexpression decreased phosphorylation at Thr157 (phospho-Thr157), whose phosphorylation promotes cytoplasmic localization of p27KIP1, whereas B56γ3 knockdown significantly increased the level of phospho-Thr157. In vitro, PP2A-B56γ3 catalyzed dephosphorylation of phospho-Thr157 in a dose-dependent and okadaic acid-sensitive manner. B56γ3 did not increase p27KIP1 nuclear localization by down-regulating the upstream kinase Akt activity and outcompeted a myristoylated constitutively active Akt (Aktca) in regulating Thr157 phosphorylation and subcellular localization of p27KIP1. In addition, results of interaction domain mapping revealed that both the N-terminal and C-terminal domains of p27 and a domain at the C-terminus of B56γ3 are required for interaction between p27 and B56γ3. Furthermore, we demonstrated that p27KIP1 levels are positively correlated with B56γ levels in both non-tumor and tumor parts of a set of human colon tissue specimens. However, positive correlation between nuclear p27KIP1 levels and B56γ levels was found only in the non-tumor parts, but not in tumor parts of these tissues, implicating a dysregulation in PP2A-B56γ3-regulated p27KIP1 nuclear localization in these tumor tissues. Altogether, this study provides a new mechanism by which the PP2A-B56γ3 holoenzyme plays its tumor suppressor role. PMID:26684356

  10. Auditory Hair Cell-Specific Deletion of p27Kip1 in Postnatal Mice Promotes Cell-Autonomous Generation of New Hair Cells and Normal Hearing

    PubMed Central

    Walters, Bradley J.; Liu, Zhiyong; Crabtree, Mark; Coak, Emily; Cox, Brandon C.

    2014-01-01

    Hearing in mammals relies upon the transduction of sound by hair cells (HCs) in the organ of Corti within the cochlea of the inner ear. Sensorineural hearing loss is a widespread and permanent disability due largely to a lack of HC regeneration in mammals. Recent studies suggest that targeting the retinoblastoma (Rb)/E2F pathway can elicit proliferation of auditory HCs. However, previous attempts to induce HC proliferation in this manner have resulted in abnormal cochlear morphology, HC death, and hearing loss. Here we show that cochlear HCs readily proliferate and survive following neonatal, HC-specific, conditional knock-out of p27Kip1 (p27CKO), a tumor suppressor upstream of Rb. Indeed, HC-specific p27CKO results in proliferation of these cells without the upregulation of the supporting cell or progenitor cell proteins, Prox1 or Sox2, suggesting that they remain HCs. Furthermore, p27CKO leads to a significant addition of postnatally derived HCs that express characteristic synaptic and stereociliary markers and survive to adulthood, although a portion of the newly derived inner HCs exhibit cytocauds and lack VGlut3 expression. Despite this, p27CKO mice exhibit normal hearing as measured by evoked auditory brainstem responses, which suggests that the newly generated HCs may contribute to, or at least do not greatly detract from, function. These results show that p27Kip1 actively maintains HC quiescence in postnatal mice, and suggest that inhibition of p27Kip1 in residual HCs represents a potential strategy for cell-autonomous auditory HC regeneration. PMID:25411503

  11. Auditory hair cell-specific deletion of p27Kip1 in postnatal mice promotes cell-autonomous generation of new hair cells and normal hearing.

    PubMed

    Walters, Bradley J; Liu, Zhiyong; Crabtree, Mark; Coak, Emily; Cox, Brandon C; Zuo, Jian

    2014-11-19

    Hearing in mammals relies upon the transduction of sound by hair cells (HCs) in the organ of Corti within the cochlea of the inner ear. Sensorineural hearing loss is a widespread and permanent disability due largely to a lack of HC regeneration in mammals. Recent studies suggest that targeting the retinoblastoma (Rb)/E2F pathway can elicit proliferation of auditory HCs. However, previous attempts to induce HC proliferation in this manner have resulted in abnormal cochlear morphology, HC death, and hearing loss. Here we show that cochlear HCs readily proliferate and survive following neonatal, HC-specific, conditional knock-out of p27(Kip1) (p27CKO), a tumor suppressor upstream of Rb. Indeed, HC-specific p27CKO results in proliferation of these cells without the upregulation of the supporting cell or progenitor cell proteins, Prox1 or Sox2, suggesting that they remain HCs. Furthermore, p27CKO leads to a significant addition of postnatally derived HCs that express characteristic synaptic and stereociliary markers and survive to adulthood, although a portion of the newly derived inner HCs exhibit cytocauds and lack VGlut3 expression. Despite this, p27CKO mice exhibit normal hearing as measured by evoked auditory brainstem responses, which suggests that the newly generated HCs may contribute to, or at least do not greatly detract from, function. These results show that p27(Kip1) actively maintains HC quiescence in postnatal mice, and suggest that inhibition of p27(Kip1) in residual HCs represents a potential strategy for cell-autonomous auditory HC regeneration.

  12. The Rho GTPase effector ROCK regulates cyclin A, cyclin D1, and p27Kip1 levels by distinct mechanisms.

    PubMed

    Croft, Daniel R; Olson, Michael F

    2006-06-01

    The members of the Rho GTPase family are well known for their regulation of actin cytoskeletal structures. In addition, they influence progression through the cell cycle. The RhoA and RhoC proteins regulate numerous effector proteins, with a central and vital signaling role mediated by the ROCK I and ROCK II serine/threonine kinases. The requirement for ROCK function in the proliferation of numerous cell types has been revealed by studies utilizing ROCK-selective inhibitors such as Y-27632. However, the mechanisms by which ROCK signaling promotes cell cycle progression have not been thoroughly characterized. Using a conditionally activated ROCK-estrogen receptor fusion protein, we found that ROCK activation is sufficient to stimulate G1/S cell cycle progression in NIH 3T3 mouse fibroblasts. Further analysis revealed that ROCK acts via independent pathways to alter the levels of cell cycle regulatory proteins: cyclin D1 and p21(Cip1) elevation via Ras and the mitogen-activated protein kinase pathway, increased cyclin A via LIM kinase 2, and reduction of p27(Kip1) protein levels. Therefore, the influence of ROCK on cell cycle regulatory proteins occurs by multiple independent mechanisms.

  13. Downregulation of the KIP family members p27(KIP1) and p57(KIP2) by SKP2 and the role of methylation in p57(KIP2) inactivation in nonsmall cell lung cancer.

    PubMed

    Pateras, Ioannis S; Apostolopoulou, Kalliopi; Koutsami, Marilena; Evangelou, Kostas; Tsantoulis, Petros; Liloglou, Triantafillos; Nikolaidis, Giorgos; Sigala, Fragiska; Kittas, Christos; Field, John K; Kotsinas, Athanassios; Gorgoulis, Vassilis G

    2006-12-01

    Knowing the status of molecules involved in cell cycle control in cancer is vital for therapeutic approaches aiming at their restoration. The p27(KIP1) and p57(KIP2) cyclin-dependent kinase inhibitors are nodal factors controlling normal cell cycle. Their expression in normal lung raises the question whether they have a mutual exclusive or redundant role in nonsmall cell lung cancer (NSCLC). A comparative comprehensive analysis was performed in a series of 70 NSCLCs. The majority of cases showed significantly reduced expression of both members compared to normal counterparts. Low KIP protein levels correlated with increased proliferation, which seems to be histological subtype preponderant. At mechanistic level, degradation by SKP2 was demonstrated, in vivo and in vitro, by siRNA-methodology, to be the most important downregulating mechanism of both KIPs in NSCLC. Decreased p57(KIP) (2)-transcription complements the above procedure in lowering p57(KIP2)-protein levels. Methylation was the main cause of decreased p57(KIP) (2)-mRNA levels. Allelic loss and imprinting from LIT1 mRNA contribute also to decreased p57(KIP2) transcription. In vitro recapitulation of the in vivo findings, in A549 lung cells (INK4A-B((-/-))), suggested that inhibition of the SKP2-degradation mechanism restores p27(KIP1) and p57(KIP2) expression. Double siRNA treatments demonstrated that each KIP is independently capable of restraining cell growth. An additional demethylation step is required for complete reconstitution of p57(KIP2) expression in NSCLC.

  14. The inhibition of activated hepatic stellate cells proliferation by arctigenin through G0/G1 phase cell cycle arrest: persistent p27(Kip1) induction by interfering with PI3K/Akt/FOXO3a signaling pathway.

    PubMed

    Li, Ao; Wang, Jun; Wu, Mingjun; Zhang, Xiaoxun; Zhang, Hongzhi

    2015-01-15

    Proliferation of hepatic stellate cells (HSCs) is vital for the development of fibrosis during liver injury. In this study, we describe that arctigenin (ATG), a major bioactive component of Fructus Arctii, exhibited selective cytotoxic activity via inhibiting platelet-derived growth factor-BB (PDGF-BB)-activated HSCs proliferation and arrested cell cycle at G0/G1 phase, which could not be observed in normal human hepatocytes in vitro. The cyclin-dependent kinase (CDK) 4/6 activities could be strongly inhibited by ATG through down-regulation of cyclin D1 and CDK4/6 expression in early G1 phase arrest. In the ATG-treated HSCs, the expression level of p27(Kip1) and the formation of CDK2-p27(Kip1) complex were also increased. p27(Kip1) silencing significantly attenuated the effect of ATG, including cell cycle arrest and suppression of proliferation in activated HSCs. We also found that ATG suppressed PDGF-BB-induced phosphorylation of Akt and its downstream transcription factor Forkhead box O 3a (FOXO3a), decreased binding of FOXO3a to 14-3-3 protein, and stimulated nuclear translocation of FOXO3a in activated HSCs. Furthermore, knockdown of FOXO3a expression by FOXO3a siRNA attenuated ATG-induced up-regulation of p27(Kip1) in activated HSCs. All the above findings suggested that ATG could increase the levels of p27(Kip1) protein through inhibition of Akt and improvement of FOXO3a activity, in turn inhibited the CDK2 kinase activity, and eventually caused an overall inhibition of HSCs proliferation.

  15. HBx-dependent cell cycle deregulation involves interaction with cyclin E/A-cdk2 complex and destabilization of p27Kip1.

    PubMed

    Mukherji, Atish; Janbandhu, Vaibhao C; Kumar, Vijay

    2007-01-01

    The HBx (X protein of hepatitis B virus) is a promiscuous transactivator implicated to play a key role in hepatocellular carcinoma. However, HBx-regulated molecular events leading to deregulation of cell cycle or establishment of a permissive environment for hepatocarcinogenesis are not fully understood. Our cell culture-based studies suggested that HBx had a profound effect on cell cycle progression even in the absence of serum. HBx presence led to an early and sustained level of cyclin-cdk2 complex during the cell cycle combined with increased protein kinase activity of cdk2 heralding an early proliferative signal. The increased cdk2 activity also led to an early proteasomal degradation of p27(Kip1) that could be reversed by HBx-specific RNA interference and blocked by a chemical inhibitor of cdk2 or the T187A mutant of p27. Further, our co-immunoprecipitation and in vitro binding studies with recombinant proteins suggested a direct interaction between HBx and the cyclin E/A-cdk2 complex. Interference with different signalling cascades known to be activated by HBx suggested a constitutive requirement of Src kinases for the association of HBx with these complexes. Notably, the HBx mutant that did not interact with cyclin E/A failed to destabilize p27(Kip1) or deregulate the cell cycle. Thus HBx appears to deregulate the cell cycle by interacting with the key cell cycle regulators independent of its well-established role in transactivation.

  16. Discovery of Small Molecules that Inhibit the Disordered Protein, p27Kip1

    PubMed Central

    Iconaru, Luigi I.; Ban, David; Bharatham, Kavitha; Ramanathan, Arvind; Zhang, Weixing; Shelat, Anang A.; Zuo, Jian; Kriwacki, Richard W.

    2015-01-01

    Disordered proteins are highly prevalent in biological systems, they control myriad signaling and regulatory processes, and their levels and/or cellular localization are often altered in human disease. In contrast to folded proteins, disordered proteins, due to conformational heterogeneity and dynamics, are not considered viable drug targets. We challenged this paradigm by identifying through NMR-based screening small molecules that bound specifically, albeit weakly, to the disordered cell cycle regulator, p27Kip1 (p27). Two groups of molecules bound to sites created by transient clusters of aromatic residues within p27. Conserved chemical features within these two groups of small molecules exhibited complementarity to their binding sites within p27, establishing structure-activity relationships for small molecule:disordered protein interactions. Finally, one compound counteracted the Cdk2/cyclin A inhibitory function of p27 in vitro, providing proof-of-principle that small molecules can inhibit the function of a disordered protein (p27) through sequestration in a conformation incapable of folding and binding to a natural regulatory target (Cdk2/cyclin A). PMID:26507530

  17. Discovery of Small Molecules that Inhibit the Disordered Protein, p27Kip1

    DOE PAGES

    Iconaru, Luigi I.; Ban, David; Bharatham, Kavitha; ...

    2015-10-28

    In disordered proteins we see that they are highly prevalent in biological systems. They control myriad signaling and regulatory processes, and their levels and/or cellular localization are often altered in human disease. In contrast to folded proteins, disordered proteins, due to conformational heterogeneity and dynamics, are not considered viable drug targets. We challenged this paradigm by identifying through NMR-based screening small molecules that bound specifically, albeit weakly, to the disordered cell cycle regulator, p27Kip1 (p27). Moreover, two groups of molecules bound to sites created by transient clusters of aromatic residues within p27. Conserved chemical features within these two groups ofmore » small molecules exhibited complementarity to their binding sites within p27, establishing structure-activity relationships for small molecule: disordered protein interactions. Finally, one compound counteracted the Cdk2/cyclin A inhibitory function of p27 in vitro, providing proof-of- principle that small molecules can inhibit the function of a disordered protein (p27) through sequestration in a conformation incapable of folding and binding to a natural regulatory target (Cdk2/cyclin A).« less

  18. p27KIP1 loss promotes proliferation and phagocytosis but prevents epithelial–mesenchymal transition in RPE cells after photoreceptor damage

    PubMed Central

    ul Quraish, Reeshan; Sudou, Norihiro; Nomura-Komoike, Kaori; Sato, Fumi

    2016-01-01

    Purpose p27KIP1 (p27), originally identified as a cell cycle inhibitor, is now known to have multifaceted roles beyond cell cycle regulation. p27 is required for the normal histogenesis of the RPE, but the role of p27 in the mature RPE remains elusive. To define the role of p27 in the maintenance and function of the RPE, we investigated the effects of p27 deletion on the responses of the RPE after photoreceptor damage. Methods Photoreceptor damage was induced in wild-type (WT) and p27 knockout (KO) mice with N-methyl-N-nitrosourea (MNU) treatment. Damage-induced responses of the RPE were investigated with bromodeoxyuridine (BrdU) incorporation assays, immunofluorescence, and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assays at different stages after MNU treatment. Subcellular localization of p27 in the WT RPE was also analyzed in vivo and in vitro. Results MNU treatment induced photoreceptor-specific degeneration in the WT and KO retinas. BrdU incorporation assays revealed virtually no proliferation of RPE cells in the WT retinas while, in the KO retinas, approximately 16% of the RPE cells incorporated BrdU at day 2 after MNU treatment. The RPE in the KO retinas developed aberrant protrusions into the outer nuclear layer in response to photoreceptor damage and engulfed outer segment debris, as well as TUNEL-positive photoreceptor cells. Increased phosphorylation of myosin light chains and their association with rhodopsin-positive phagosomes were observed in the mutant RPE, suggesting possible deregulation of cytoskeletal dynamics. In addition, WT RPE cells exhibited evidence of the epithelial–mesenchymal transition (EMT), including morphological changes, induction of α-smooth muscle actin expression, and attenuated expression of tight junction protein ZO-1 while these changes were absent in the KO retinas. In the normal WT retinas, p27 was localized to the nuclei of RPE cells while nuclear and cytoplasmic p27 was detected in RPE cells

  19. [P27(Kip1), cyclin E and endogenous TGF-beta1 changes in apoptosis of NB4 cells induced by As(2)O(3) and/or TGF-beta1 and their significance].

    PubMed

    Liang, Ying; Li, Yan; Wang, Yue; Li, Xia; Wang, Ping-Ping; Wang, Bai-Xun

    2009-02-01

    This study was aimed to investigate the effects of arsenic trioxide (As(2)O(3)) and/or transforming growth factor-beta1 (TGF-beta1)on cell apoptosis and the changes of P27(Kip1), cyclin E and endogenous TGF-beta1 mRNA levels in NB4 cells. As(2)O(3) cytotoxicity to NB4 cells and the IC(50) were assayed with MTT, the apoptotic morphological changes were observed by Wright-Giemsa staining; the cell cycle and apoptosis were detected with flow cytometry. Semiquantitative RT-PCR was used to examine P27(Kip1), cyclin E and endogenous TGF-beta1 mRNA levels. The results showed that the As(2)O(3) and TGF-beta1 significantly suppressed the growth of NB4 cells, and promoted the apoptosis of these cells. The growth inhibition and apoptosis of NB4 cells treated with As(2)O(3) were in dose-and time-dependent manners. IC(50) were about 12 micromol/L for 24 hours, about 5 micromol/L for 48 hours, and about 3 micromol/L for 72 hours respectively. Cell cycle arrest in NB4 cells was induced by As(2)O(3) and/or TGF-beta1. The arrest of NB4 cells treated by 5 micromol/L As(2)O(3) was in G(2)/M phase, and 5 ng/ml TGF-beta1 in G(1) phase. However, the arrest of NB4 cells caused by combination of As(2)O(3) and TGF-beta1 was in S phase. After treating with As(2)O(3), P27(Kip1) and endogenous TGF-beta1 mRNA expressions of NB4 cells were up-regulated, and cyclin E mRNA expression was down-regulated. When NB4 cells were treated with TGF-beta1 alone, P27(Kip1) and cyclin E mRNA expressions were the same as that treated by As(2)O(3). Exogenous TGF-beta1 enhanced the above effects of As(2)O(3) in combination group. It is concluded that As(2)O(3) and TGF-beta1 are able to induce apoptosis and cell cycle abnormal distribution in NB4 cells. As(2)O(3) and exogenous TGF-beta1 may up-regulate endogenous TGF-beta1, which induce apoptosis of NB4 cells through consequently high expression of P27(Kip1). TGF-beta1 may lead to cell cycle arrest by inhibiting the expression of cyclin E directly, or by the

  20. p27 kip1 haplo-insufficiency improves cardiac function in early-stages of myocardial infarction by protecting myocardium and increasing angiogenesis by promoting IKK activation.

    PubMed

    Zhou, Ningtian; Fu, Yuxuan; Wang, Yunle; Chen, Pengsheng; Meng, Haoyu; Guo, Shouyu; Zhang, Min; Yang, Zhijian; Ge, Yingbin

    2014-08-07

    p27(kip1) (p27) is widely known as a potent cell cycle inhibitor in several organs, especially in the heart. However, its role has not been fully defined during the early phase of myocardial infarction (MI). In this study, we investigated the relationships between p27, vascular endothelial growth factor/hepatocyte growth factor (VEGF/HGF) and NF-κB in post-MI cardiac function repair both in vivo and in the hypoxia/ischemia-induced rat myocardiocyte model. In vivo, haplo-insufficiency of p27 improved cardiac function, diminished the infarct zone, protected myocardiocytes and increased angiogenesis by enhancing the production of VEGF/HGF. In vitro, the presence of conditioned medium from hypoxia/ischemia-induced p27 knockdown myocardiocytes reduced the injury caused by hypoxia/ischemia in myocardiocytes, and this effect was reversed by VEGF/HGF neutralizing antibodies, consistent with the cardioprotection being due to VEGF/HGF secretion. We also observed that p27 bound to IKK and that p27 haplo-insufficiency promoted IKK/p65 activation both in vivo and in vitro, thereby inducing the NF-κB downstream regulator, VEGF/HGF. Furthermore, IKKi and IKK inhibitor negated the effect of VEGF/HGF. Therefore, we conclude that p27 haplo-insufficiency protects against heart injury by VEGF/HGF mediated cardioprotection and increased angiogenesis through promoting IKK activation.

  1. KDM4B and KDM4A promote endometrial cancer progression by regulating androgen receptor, c-myc, and p27kip1

    PubMed Central

    Kwan, Suet-Ying; Chen, Limo; Chen, Jin-Hong; Ying, Zuo-Lin; Zhou, Ye; Gu, Wei; Wang, Li-Hua; Cheng, Wei-Wei; Zeng, Jianfang; Wan, Xiao-Ping; Mok, Samuel C.; Wong, Kwong-Kwok; Bao, Wei

    2015-01-01

    Epidemiological evidence suggests that elevated androgen levels and genetic variation related to the androgen receptor (AR) increase the risk of endometrial cancer (EC). However, the role of AR in EC is poorly understood. We report that two members of the histone demethylase KDM4 family act as major regulators of AR transcriptional activityin EC. In the MFE-296 cell line, KDM4B and AR upregulate c-myc expression, while in AN3CA cells KDM4A and AR downregulate p27kip1. Additionally, KDM4B expression is positively correlated with AR expression in EC cell lines with high baseline AR expression, while KDM4A and AR expression are positively correlated in low-AR cell lines. In clinical specimens, both KDM4B and KDM4A expression are significantly higher in EC tissues than that in normal endometrium. Finally, patients with alterations in AR, KDM4B, KDM4A, and c-myc have poor overall and disease-free survival rates. Together, these findings demonstrate that KDM4B and KDM4A promote EC progression by regulating AR activity. PMID:26397136

  2. Accumulation of p27(kip1) is associated with cyclin D3 overexpression in the oxyphilic (Hurthle cell) variant of follicular thyroid carcinoma

    PubMed Central

    Troncone, G; Iaccarino, A; Russo, M; Palmieri, E A; Volante, M; Papotti, M; Viglietto, G; Palombini, L

    2007-01-01

    Background The down regulation of protein p27kip1 (p27) in most cases of thyroid cancer has relevant diagnostic and prognostic implications. However, the oxyphilic (Hurthle cell) variant of follicular thyroid carcinoma expresses more p27 than benign oxyphilic lesions do. Aim To evaluate the mechanism underlying this difference in expression of p27. Methods Because high levels of cyclin D3 lead to p27 accumulation in cell lines and clinical samples of thyroid cancer, the immunocytochemical pattern of cyclin D3 in oxyphilic (n = 47) and non‐oxyphilic (n = 70) thyroid neoplasms was investigated. Results In the whole study sample, there was a significant correlation between p27 and cyclin D3 expression (Spearman's r: 0.64; p<0.001). The expression of cyclin D3 and p27 was significantly higher in the oxyphilic variant of follicular carcinomas than in non‐oxyphilic carcinomas (p<0.001). In the former, cyclin D3 overexpression and p27 accumulation were observed in a median of 75% and 55% of cells, respectively. In co‐immunoprecipitation experiments, the level of p27‐bound cyclin D3 was much higher in oxyphilic neoplasias than in normal thyroids and other thyroid tumours. Conclusion These results show that increased p27 expression in the oxyphilic (Hurthle cell) variant of follicular thyroid carcinoma results from cyclin D3 overexpression. PMID:16798934

  3. Knockdown of AMPKα2 Promotes Pulmonary Arterial Smooth Muscle Cells Proliferation via mTOR/Skp2/p27(Kip1) Signaling Pathway.

    PubMed

    Ke, Rui; Liu, Lu; Zhu, Yanting; Li, Shaojun; Xie, Xinming; Li, Fangwei; Song, Yang; Yang, Lan; Gao, Li; Li, Manxiang

    2016-05-31

    It has been shown that activation of adenosine monophosphate-activated protein kinase (AMPK) suppresses proliferation of a variety of tumor cells as well as nonmalignant cells. In this study, we used post-transcriptional gene silencing with small interfering RNA (siRNA) to specifically examine the effect of AMPK on pulmonary arterial smooth muscle cells (PASMCs) proliferation and to further elucidate its underlying molecular mechanisms. Our results showed that knockdown of AMPKα2 promoted primary cultured PASMCs proliferation; this was accompanied with the elevation of phosphorylation of mammalian target of rapamycin (mTOR) and S-phase kinase-associated protein 2 (Skp2) protein level and reduction of p27(Kip1). Importantly, prior silencing of mTOR with siRNA abolished AMPKα2 knockdown-induced Skp2 upregulation, p27(Kip1) reduction as well as PASMCs proliferation. Furthermore, pre-depletion of Skp2 by siRNA also eliminated p27(Kip1) downregulation and PASMCs proliferation caused by AMPKα2 knockdown. Taken together, our study indicates that AMPKα2 isoform plays an important role in regulation of PASMCs proliferation by modulating mTOR/Skp2/p27(Kip1) axis, and suggests that activation of AMPKα2 might have potential value in the prevention and treatment of pulmonary arterial hypertension.

  4. Knockdown of AMPKα2 Promotes Pulmonary Arterial Smooth Muscle Cells Proliferation via mTOR/Skp2/p27Kip1 Signaling Pathway

    PubMed Central

    Ke, Rui; Liu, Lu; Zhu, Yanting; Li, Shaojun; Xie, Xinming; Li, Fangwei; Song, Yang; Yang, Lan; Gao, Li; Li, Manxiang

    2016-01-01

    It has been shown that activation of adenosine monophosphate-activated protein kinase (AMPK) suppresses proliferation of a variety of tumor cells as well as nonmalignant cells. In this study, we used post-transcriptional gene silencing with small interfering RNA (siRNA) to specifically examine the effect of AMPK on pulmonary arterial smooth muscle cells (PASMCs) proliferation and to further elucidate its underlying molecular mechanisms. Our results showed that knockdown of AMPKα2 promoted primary cultured PASMCs proliferation; this was accompanied with the elevation of phosphorylation of mammalian target of rapamycin (mTOR) and S-phase kinase-associated protein 2 (Skp2) protein level and reduction of p27Kip1. Importantly, prior silencing of mTOR with siRNA abolished AMPKα2 knockdown-induced Skp2 upregulation, p27Kip1 reduction as well as PASMCs proliferation. Furthermore, pre-depletion of Skp2 by siRNA also eliminated p27Kip1 downregulation and PASMCs proliferation caused by AMPKα2 knockdown. Taken together, our study indicates that AMPKα2 isoform plays an important role in regulation of PASMCs proliferation by modulating mTOR/Skp2/p27Kip1 axis, and suggests that activation of AMPKα2 might have potential value in the prevention and treatment of pulmonary arterial hypertension. PMID:27258250

  5. p27kip1 stabilization is essential for the maintenance of cell cycle arrest in response to DNA damage

    PubMed Central

    Cuadrado, Myriam; Gutierrez-Martinez, Paula; Swat, Aneta; Nebreda, Angel R.; Fernandez-Capetillo, Oscar

    2013-01-01

    One of the current models of cancer proposes that oncogenes activate a DNA damage response (DDR), which would limit the growth of the tumor in its earliest stages. In this context, and in contrast to studies focused on the acute responses to a one-time genotoxic insult, understanding how cells respond to a persistent source of DNA damage might become critical for future studies in the field. We here report the discovery of a novel damage-responsive pathway, which involves p27kip1 and retinoblastoma tumour suppressors (TS), and which is only implemented after a persistent exposure to clastogens. In agreement with its late activation, we show that this pathway is critical for the maintenance –but not the initiation- of the cell cycle arrest triggered by DNA damage. Interestingly, this late response is independent of the canonical ATM- and ATR-dependent DDR, but downstream of p38 MAPK. Our results might help to reconcile the oncogene-induced DNA damage model with the clinical evidence that points to non-DDR members as the most important TSs in human cancer. PMID:19843869

  6. Role of Intrinsic Flexibility in Signal Transduction Mediated by the Cell Cycle Regulator, p27Kip1

    PubMed Central

    Galea, Charles A.; Nourse, Amanda; Wang, Yuefeng; Sivakolundu, Sivashankar G.; Heller, William T.; Kriwacki, Richard W.

    2008-01-01

    Summary p27Kip1 (p27), which controls eukaryotic cell division through interactions with cyclin-dependent kinases (Cdks), integrates and transduces pro-mitogenic signals from various non-receptor tyrosine kinases (NRTKs) by orchestrating its own phosphorylation, ubiquitination and degradation. Intrinsic flexibility allows p27 to act as a “conduit” for sequential signaling mediated by tyrosine and threonine phosphorylation and ubiquitination. While the structural features of the Cdk/cyclin-binding domain of p27 are understood, how the C-terminal regulatory domain coordinates multi-step signaling leading to p27 degradation is poorly understood. We show that the 100-residue p27 C-terminal domain is extended and flexible when p27 is bound to Cdk2/cyclin A. We propose that the intrinsic flexibility of p27 provides a molecular basis for the sequential signal transduction conduit that regulates p27 degradation and cell division. Other intrinsically unstructured proteins possessing multiple sites of post-translational modification may participate in similar signaling conduits. PMID:18177895

  7. Substituting Threonine 187 with Alanine in p27Kip1 Prevents Pituitary Tumorigenesis by Two-Hit Loss of Rb1 and Enhances Humoral Immunity in Old Age*

    PubMed Central

    Zhao, Hongling; Bauzon, Frederick; Bi, Enguang; Yu, J. Jessica; Fu, Hao; Lu, Zhonglei; Cui, Jinhua; Jeon, Hyungjun; Zang, Xingxing; Ye, B. Hilda; Zhu, Liang

    2015-01-01

    p27Kip1 (p27) is an inhibitor of cyclin-dependent kinases. Inhibiting p27 protein degradation is an actively developing cancer therapy strategy. One focus has been to identify small molecule inhibitors to block recruitment of Thr-187-phosphorylated p27 (p27T187p) to SCFSkp2/Cks1 ubiquitin ligase. Since phosphorylation of Thr-187 is required for this recruitment, p27T187A knockin (KI) mice were generated to determine the effects of systemically blocking interaction between p27 and Skp2/Cks1 on tumor susceptibility and other proliferation related mouse physiology. Rb1+/− mice develop pituitary tumors with full penetrance and the tumors are invariably Rb1−/−, modeling tumorigenesis by two-hit loss of RB1 in humans. Immunization induced humoral immunity depends on rapid B cell proliferation and clonal selection in germinal centers (GCs) and declines with age in mice and humans. Here, we show that p27T187A KI prevented pituitary tumorigenesis in Rb1+/− mice and corrected decline in humoral immunity in older mice following immunization with sheep red blood cells (SRBC). These findings reveal physiological contexts that depend on p27 ubiquitination by SCFSkp2-Cks1 ubiquitin ligase and therefore help forecast clinical potentials of Skp2/Cks1-p27T187p interaction inhibitors. We further show that GC B cells and T cells use different mechanisms to regulate their p27 protein levels, and propose a T helper cell exhaustion model resembling that of stem cell exhaustion to understand decline in T cell-dependent humoral immunity in older age. PMID:25583987

  8. Substituting threonine 187 with alanine in p27Kip1 prevents pituitary tumorigenesis by two-hit loss of Rb1 and enhances humoral immunity in old age.

    PubMed

    Zhao, Hongling; Bauzon, Frederick; Bi, Enguang; Yu, J Jessica; Fu, Hao; Lu, Zhonglei; Cui, Jinhua; Jeon, Hyungjun; Zang, Xingxing; Ye, B Hilda; Zhu, Liang

    2015-02-27

    p27Kip1 (p27) is an inhibitor of cyclin-dependent kinases. Inhibiting p27 protein degradation is an actively developing cancer therapy strategy. One focus has been to identify small molecule inhibitors to block recruitment of Thr-187-phosphorylated p27 (p27T187p) to SCF(Skp2/Cks1) ubiquitin ligase. Since phosphorylation of Thr-187 is required for this recruitment, p27T187A knockin (KI) mice were generated to determine the effects of systemically blocking interaction between p27 and Skp2/Cks1 on tumor susceptibility and other proliferation related mouse physiology. Rb1(+/-) mice develop pituitary tumors with full penetrance and the tumors are invariably Rb1(-/-), modeling tumorigenesis by two-hit loss of RB1 in humans. Immunization induced humoral immunity depends on rapid B cell proliferation and clonal selection in germinal centers (GCs) and declines with age in mice and humans. Here, we show that p27T187A KI prevented pituitary tumorigenesis in Rb1(+/-) mice and corrected decline in humoral immunity in older mice following immunization with sheep red blood cells (SRBC). These findings reveal physiological contexts that depend on p27 ubiquitination by SCF(Skp2-Cks1) ubiquitin ligase and therefore help forecast clinical potentials of Skp2/Cks1-p27T187p interaction inhibitors. We further show that GC B cells and T cells use different mechanisms to regulate their p27 protein levels, and propose a T helper cell exhaustion model resembling that of stem cell exhaustion to understand decline in T cell-dependent humoral immunity in older age.

  9. The role of p21(waf1/cip1) and p27(Kip1) in HDACi-mediated tumor cell death and cell cycle arrest in the Eμ-myc model of B-cell lymphoma.

    PubMed

    Newbold, A; Salmon, J M; Martin, B P; Stanley, K; Johnstone, R W

    2014-11-20

    Following the establishment of histone deacetylases (HDACs) as promising therapeutic targets for the reversal of aberrant epigenetic states associated with cancer, the development of HDAC inhibitors (HDACi) and their underlying mechanisms of action has been a significant area of scientific interest. HDACi induce diverse biological responses including the inhibition of cell proliferation by blocking progression through the G1 or G2/M phases of the cell cycle. As a putative tumor-suppressor protein, p21(waf1/cip1) influences cell proliferation by inhibiting the activity of cyclin-cyclin-dependent kinase (CDK) complexes at the G1/S and G2/M cell cycle checkpoints. HDACi transcriptionally activate CDKN1A, and it has been proposed that induction of p21(waf1/cip1) can determine if a cell undergoes apoptosis or cell cycle arrest following HDACi treatment. In the Eμ-myc transgenic mouse model of B-cell lymphoma, knockout of cdkn1a had no effect on disease latency, indicating that p21(waf1/cip1) did not function as a tumor suppressor in this system. Although HDACi robustly induced expression of p21(waf1/cip1) in wild-type Eμ-myc lymphomas, deletion of cdkn1a did not sensitize the lymphoma cells to HDACi-induced apoptosis and HDACi-induced cell cycle arrest still occurred. However, knockdown of cdkn1b in cdkn1a knockout lymphomas resulted in defective vorinostat-mediated arrest at G1/S indicating an essential role of p27(Kip1) in mediating this biological response to vorinostat. These data demonstrate that induction of cdkn1a does not regulate HDACi-mediated tumor cell apoptosis and refute the notion that p21(waf1/cip1) is an obligate mediator of HDACi-induced cell cycle arrest.

  10. SKP2 Oncogene Is a Direct MYC Target Gene and MYC Down-regulates p27KIP1 through SKP2 in Human Leukemia Cells*

    PubMed Central

    Bretones, Gabriel; Acosta, Juan C.; Caraballo, Juan M.; Ferrándiz, Nuria; Gómez-Casares, M. Teresa; Albajar, Marta; Blanco, Rosa; Ruiz, Paula; Hung, Wen-Chun; Albero, M. Pilar; Perez-Roger, Ignacio; León, Javier

    2011-01-01

    SKP2 is the ubiquitin ligase subunit that targets p27KIP1 (p27) for degradation. SKP2 is induced in the G1-S transit of the cell cycle, is frequently overexpressed in human cancer, and displays transformation activity in experimental models. Here we show that MYC induces SKP2 expression at the mRNA and protein levels in human myeloid leukemia K562 cells with conditional MYC expression. Importantly, in these systems, induction of MYC did not activate cell proliferation, ruling out SKP2 up-regulation as a consequence of cell cycle entry. MYC-dependent SKP2 expression was also detected in other cell types such as lymphoid, fibroblastic, and epithelial cell lines. MYC induced SKP2 mRNA expression in the absence of protein synthesis and activated the SKP2 promoter in luciferase reporter assays. With chromatin immunoprecipitation assays, MYC was detected bound to a region of human SKP2 gene promoter that includes E-boxes. The K562 cell line derives from human chronic myeloid leukemia. In a cohort of chronic myeloid leukemia bone marrow samples, we found a correlation between MYC and SKP2 mRNA levels. Analysis of cancer expression databases also indicated a correlation between MYC and SKP2 expression in lymphoma. Finally, MYC-induced SKP2 expression resulted in a decrease in p27 protein in K562 cells. Moreover, silencing of SKP2 abrogated the MYC-mediated down-regulation of p27. Our data show that SKP2 is a direct MYC target gene and that MYC-mediated SKP2 induction leads to reduced p27 levels. The results suggest the induction of SKP2 oncogene as a new mechanism for MYC-dependent transformation. PMID:21245140

  11. A Cytostatic Ruthenium(II)-Platinum(II) Bis(terpyridyl) Anticancer Complex That Blocks Entry into S Phase by Up-regulating p27(KIP1).

    PubMed

    Ramu, Vadde; Gill, Martin R; Jarman, Paul J; Turton, David; Thomas, Jim A; Das, Amitava; Smythe, Carl

    2015-06-15

    Cytostatic agents that interfere with specific cellular components to prevent cancer cell growth offer an attractive alternative, or complement, to traditional cytotoxic chemotherapy. Here, we describe the synthesis and characterization of a new binuclear Ru(II) -Pt(II) complex [Ru(tpy)(tpypma)Pt(Cl)(DMSO)](3+) (tpy=2,2':6',2''-terpyridine and tpypma=4-([2,2':6',2''-terpyridine]-4'-yl)-N-(pyridin-2-ylmethyl)aniline), VR54, which employs the extended terpyridine tpypma ligand to link the two metal centres. In cell-free conditions, VR54 binds DNA by non-intercalative reversible mechanisms (Kb =1.3×10(5)  M(-1) ) and does not irreversibly bind guanosine. Cellular studies reveal that VR54 suppresses proliferation of A2780 ovarian cancer cells with no cross-resistance in the A2780CIS cisplatin-resistant cell line. Through the preparation of mononuclear Ru(II) and Pt(II) structural derivatives it was determined that both metal centres are required for this anti-proliferative activity. In stark contrast to cisplatin, VR54 neither activates the DNA-damage response network nor induces significant levels of cell death. Instead, VR54 is cytostatic and inhibits cell proliferation by up-regulating the cyclin-dependent kinase inhibitor p27(KIP1) and inhibiting retinoblastoma protein phosphorylation, which blocks entry into S phase and results in G1 cell cycle arrest. Thus, VR54 inhibits cancer cell growth by a gain of function at the G1 restriction point. This is the first metal-coordination compound to demonstrate such activity.

  12. Cell adhesion induces p27Kip1-associated cell-cycle arrest through down-regulation of the SCFSkp2 ubiquitin ligase pathway in mantle-cell and other non-Hodgkin B-cell lymphomas

    PubMed Central

    Lwin, Tint; Hazlehurst, Lori A.; Dessureault, Sophie; Lai, Raymond; Bai, Wenlong; Sotomayor, Eduardo; Moscinski, Lynn C.; Dalton, William S.

    2007-01-01

    Mounting evidence suggests that dynamic interactions between a tumor and its microenvironment play a critical role in tumor development, cell-cycle progression, and response to therapy. In this study, we used mantle cell lymphoma (MCL) as a model to characterize the mechanisms by which stroma regulate cell-cycle progression. We demonstrated that adhesion of MCL and other non-Hodgkin lymphoma (NHL) cells to bone marrow stromal cells resulted in a reversible G1 arrest associated with elevated p27Kip1 and p21 (WAF1) proteins. The adhesion-mediated p27Kip1 and p21 increases were posttranslationally regulated via the down-regulation of Skp2, a subunit of SCFSkp2 ubiquitin ligase. Overexpression of Skp2 in MCL decreased p27Kip1, whereas inhibition of Skp2 by siRNA increased p27Kip1 and p21 levels. Furthermore, we found cell adhesion up-regulated Cdh1 (an activating subunit of anaphase-promoting complex [APC] ubiquitin ligase), and reduction of Cdh1 by siRNA induced Skp2 accumulation and hence p27Kip1 degradation, thus implicating Cdh1 as an upstream effector of the Skp2/p27Kip1 signaling pathway. Overall, this report, for the first time, demonstrates that cell-cell contact controls the tumor cell cycle via ubiquitin-proteasome proteolytic pathways in MCL and other NHLs. The understanding of this novel molecular pathway may prove valuable in designing new therapeutic approaches for modifying tumor cell growth and response to therapy. PMID:17502456

  13. In vivo antitumor effects of 4,7-dimethoxy-5-methyl-1,3-benzodioxole isolated from the fruiting body of Antrodia camphorata through activation of the p53-mediated p27/Kip1 signaling pathway.

    PubMed

    Tu, Shih-Hsin; Wu, Chih-Hsiung; Chen, Li-Ching; Huang, Ching-Shui; Chang, Hui-Wen; Chang, Chien-Hsi; Lien, Hsiu-Man; Ho, Yuan-Soon

    2012-04-11

    In this study, 4,7-dimethoxy-5-methyl-1,3-benzodioxole (SY-1) was isolated from three different sources of dried Antrodia camphorata (AC) fruiting bodies. AC is a medicinal mushroom that grows on the inner heartwood wall of Cinnamomum kanehirai Hay (Lauraceae), which is an endemic species that is used in Chinese medicine for its antitumor properties. We demonstrated that SY-1 [given as a 1-30 mg/kg body weight intraperitoneal (ip) injection three times per week] profoundly decreased the growth of COLO-205 human colon cancer cell tumor xenografts in an athymic nude mouse model. We further demonstrated that significant AC extract-mediated antitumor effects were observed at the highest concentration (5 g/kg body weight/day). No gross toxicity signs were observed (i.e., body weight changes, general appearance, or individual organ effects). Frozen COLO-205 xenograft tumors were pulverized in liquid N(2), and the expression of cell cycle regulatory proteins was detected by immunoblotting. We found that the p53-mediated p27/Kip1 protein was significantly induced in the low-dose (1 mg/kg body weight) SY-1-treated tumors, whereas the p21/Cip1 protein levels did not change. The G0/G1 phase cell cycle regulators induced by SY-1 were also associated with a significant decrease in cyclins D1, D3, and A. These results provide further evidence that SY-1 may have significance for cancer chemotherapy.

  14. Nrdp1-mediated ErbB3 degradation inhibits glioma cell migration and invasion by reducing cytoplasmic localization of p27(Kip1).

    PubMed

    Shi, Hengliang; Gong, Hui; Cao, Kuan; Zou, Shenshan; Zhu, Bingxin; Bao, Hanmo; Wu, Yuxuan; Gao, Yong; Tang, Yuan; Yu, Rutong

    2015-09-01

    We previously reported that loss of Nrdp1 contributes to human glioma progression by reducing apoptosis. However, the role of Nrdp1 in glioma migration and invasion has not been investigated. Here, we report that ErbB3, a substrate of Nrdp1, is undetectable in normal brain tissues and grade II/III glioma tissues, but is abundant in a certain percentage of grade IV glioma tissues and is associated with the loss of Nrdp1. This suggests that Nrdp1 may be involved in glioma migration and invasion by regulating ErbB3. Thus, the role of Nrdp1/ErbB3 signaling in glioma cell migration and invasion was investigated using Nrdp1 loss- and gain-of-function. The results show that down-regulation of Nrdp1 by use of short hairpin RNA promoted glioma cell migration and invasion. In contrast, overexpression of Nrdp1 significantly inhibited glioma cell migration and invasion. Further investigation on molecular targets revealed that Nrdp1 decreased the level of ErbB3, which resulted in decreasing p-AKT thereby reducing cytoplasmic p27(Kip1). Taken together, these findings suggest that Nrdp1-mediated ErbB3 degradation suppresses glioma migration and invasion and that loss of Nrdp1 may amplify ErbB3 signaling to contribute to glioma migration and invasion. These findings suggest that Nrdp1 may be a target for glioma therapy.

  15. TGF-β activates APC through Cdh1 binding for Cks1 and Skp2 proteasomal destruction stabilizing p27kip1 for normal endometrial growth.

    PubMed

    Pavlides, Savvas C; Lecanda, Jon; Daubriac, Julien; Pandya, Unnati M; Gama, Patricia; Blank, Stephanie; Mittal, Khushbakhat; Shukla, Pratibha; Gold, Leslie I

    2016-01-01

    We previously reported that aberrant TGF-β/Smad2/3 signaling in endometrial cancer (ECA) leads to continuous ubiquitylation of p27(kip1)(p27) by the E3 ligase SCF-Skp2/Cks1 causing its degradation, as a putative mechanism involved in the pathogenesis of this cancer. In contrast, normal intact TGF-β signaling prevents degradation of nuclear p27 by SCF-Skp2/Cks1 thereby accumulating p27 to block Cdk2 for growth arrest. Here we show that in ECA cell lines and normal primary endometrial epithelial cells, TGF-β increases Cdh1 and its binding to APC/C to form the E3 ligase complex that ubiquitylates Cks1 and Skp2 prompting their proteasomal degradation and thus, leaving p27 intact. Knocking-down Cdh1 in ECA cell lines increased Skp2/Cks1 E3 ligase activity, completely diminished nuclear and cytoplasmic p27, and obviated TGF-β-mediated inhibition of proliferation. Protein synthesis was not required for TGF-β-induced increase in nuclear p27 and decrease in Cks1 and Skp2. Moreover, half-lives of Cks1 and Skp2 were extended in the Cdh1-depleted cells. These results suggest that the levels of p27, Skp2 and Cks1 are strongly or solely regulated by proteasomal degradation. Finally, an inverse relationship of low p27 and high Cks1 in the nucleus was shown in patients in normal proliferative endometrium and grade I-III ECAs whereas differentiated secretory endometrium showed the reverse. These studies implicate Cdh1 as the master regulator of TGF-β-induced preservation of p27 tumor suppressor activity. Thus, Cdh1 is a potential therapeutic target for ECA and other human cancers showing an inverse relationship between Cks1/Skp2 and p27 and/or dysregulated TGF-β signaling.

  16. The Cell Cycle Inhibitor p27KIP1: A Key Mediator of G1 Arrest by Androgen Ablation an dby Vitamin D3 Analog

    DTIC Science & Technology

    2000-02-01

    prostatic epithelium. We demonstrated higher appeared fully reversible on removal of the high dose PSA production in conditioned media from cells DHT at 48 h...34endorsement or approval of the products or services of these organizations. In conducting research using animals, the investigator(s) * red to the "Guide...fell from 20% to 2%, while the G1 fraction rose from 74% to 90% by 24 hours. Loss of phosphorylation of the retinoblastoma protein was noted and cdk4

  17. Genetic association between the cyclin-dependent kinase inhibitor gene p27/Kip1 polymorphism (rs34330) and cancer susceptibility: a meta-analysis

    PubMed Central

    Cheng, Xiao-Ke; Wang, Xue-Jun; Li, Xiao-Dong; Ren, Xue-Qun

    2017-01-01

    The p27 rs34330 (-79C/T) polymorphism has been widely studied for human cancer susceptibility. The current findings, however, still remained controversial. Therefore, we performed the meta-analysis to provide a more accurate result. Eligible studies were identified from PubMed database up to June 2015. The association of p27 rs34330 polymorphism and cancer susceptibility was estimated with odds ratios and corresponding 95% confidence intervals. The meta-analysis was performed with Stata 12. A total of ten studies with 11,214 cases and more than 8,776 controls were included in the meta-analysis (including breast, lung, thyroid, endometrial, and hepatocellular cancer). In pooled analysis, p27 gene rs34330 polymorphism significantly increased the cancer susceptibility. Subgroup analysis indicated that the elevated risk was observed under all the genetic models for Asians and under three genetic models for Caucasians. Results of sensitivity analysis were similar to the overall results. The results suggested that the p27 rs34330 polymorphism increased the cancer susceptibility, especially in Asians. Further well-designed and large sample size studies are warranted to verify the conclusion. PMID:28317869

  18. The Cell Cycle Inhibitor p27KIP1: A Key of G1 Arrest by Androgen Ablation and by Vitamin D3 Analog

    DTIC Science & Technology

    2001-02-01

    EB 1089 . Work during final year of the grant period has addressed how processes regulating p27 are altered during prostate cancer progression...Effects of androgens and vDR activation by EB 1089 on p27 function were assayed. We demonstrated that physiologic concentrations of DHT and EB 1089 have...DHT and EB 1089 in pre-clinical trials using LNCaP xenografts in immunodeficient mice. Our preliminary data analysis of these in vivo studies in

  19. PKCeta enhances cell cycle progression, the expression of G1 cyclins and p21 in MCF-7 cells.

    PubMed

    Fima, E; Shtutman, M; Libros, P; Missel, A; Shahaf, G; Kahana, G; Livneh, E

    2001-10-11

    Protein kinase C encodes a family of enzymes implicated in cellular differentiation, growth control and tumor promotion. However, not much is known with respect to the molecular mechanisms that link protein kinase C to cell cycle control. Here we report that the expression of PKCeta in MCF-7 cells, under the control of a tetracycline-responsive inducible promoter, enhanced cell growth and affected the cell cycle at several points. The induced expression of another PKC isoform, PKCdelta, in MCF-7 cells had opposite effects and inhibited their growth. PKCeta expression activated cellular pathways in these cells that resulted in the increased expression of the G1 phase cyclins, cyclin D and cyclin E. Expression of the cyclin-dependent kinase inhibitor p21(WAF1) was also specifically elevated in PKCeta expressing cells, but its overall effects were not inhibitory. Although, the protein levels of the cyclin-dependent kinase inhibitor p27(KIP1) were not altered by the induced expression of PKCeta, the cyclin E associated Cdk2 kinase activity was in correlation with the p27(KIP1) bound to the cyclin E complex and not by p21(WAF1) binding. PKCeta expression enhanced the removal of p27(KIP1) from this complex, and its re-association with the cyclin D/Cdk4 complex. Reduced binding of p27(KIP1) to the cyclin D/Cdk4 complex at early time points of the cell cycle also enhanced the activity of this complex, while at later time points the decrease in bound p21(WAF1) correlated with its increased activity in PKCeta-expressing cells. Thus, PKCeta induces altered expression of several cell cycle functions, which may contribute to its ability to affect cell growth.

  20. Over-expression of Skp2 is associated with resistance to preoperative doxorubicin-based chemotherapy in primary breast cancer

    PubMed Central

    Davidovich, Shirly; Ben-Izhak, Ofer; Shapira, Ma'anit; Futerman, Boris; Hershko, Dan D

    2008-01-01

    Introduction Preoperative chemotherapy is often used in patients with locally advanced breast cancer. However, commonly used clinical and pathological parameters are poor predictors of response to this type of therapy. Recent studies have suggested that altered regulation of the cell cycle in cancer may be involved in resistance to chemotherapy. Over-expression of the ubiquitin ligase Skp2 results in loss of the cell cycle inhibitor p27Kip1 and is associated with poor prognosis in early breast cancer. The purpose of the present study was to examine the role of these proteins as predictors of clinical outcome and response to chemotherapy in locally advanced breast cancer. Methods The expression levels of Skp2 and p27Kip1 were determined by immunohistochemistry both before and after preoperative chemotherapy in 40 patients with locally advanced breast cancer. All patients were treated with cyclophosphamide/doxorubicin (adriamycin)/5-fluorouracil (CAF) and some patients received additional treatment with docetaxel. Expression data were compared with patients' clinical and pathological features, clinical outcome, and response to chemotherapy. Results Skp2 expression before preoperative chemotherapy was inversely related to p27Kip1 levels, tumor grade, and expression of estrogen and progesterone receptors. Both Skp2 and p27Kip1 were found to be accurate prognostic markers for disease-free and overall survival. High preoperative expression of Skp2 was associated with resistance to CAF therapy in 94% of patients (P < 0.0001) but not with resistance to docetaxel. Conclusion Skp2 expression may be a useful marker for predicting response to doxorubicin-based preoperative chemotherapy and clinical outcome in patients with locally advanced breast cancer. PMID:18644126

  1. Structural basis of the Cks1-dependent recognition of p27(Kip1) by the SCF(Skp2) ubiquitin ligase

    SciTech Connect

    Hao, B.; Zheng, N.; Schulman, B.A.; Wu, G.; Miller, J.J.; Pagano, M.; Pavletich, N.P.

    2010-07-19

    The ubiquitin-mediated proteolysis of the Cdk2 inhibitor p27{sup Kip1} plays a central role in cell cycle progression, and enhanced degradation of p27{sup Kip1} is associated with many common cancers. Proteolysis of p27{sup Kip1} is triggered by Thr187 phosphorylation, which leads to the binding of the SCF{sup Skp2} (Skp1-Cul1-Rbx1-Skp2) ubiquitin ligase complex. Unlike other known SCF substrates, p27{sup Kip1} ubiquitination also requires the accessory protein Cks1. The crystal structure of the Skp1-Skp2-Cks1 complex bound to a p27{sup Kip1} phosphopeptide shows that Cks1 binds to the leucine-rich repeat (LRR) domain and C-terminal tail of Skp2, whereas p27{sup Kip1} binds to both Cks1 and Skp2. The phosphorylated Thr187 side chain of p27{sup Kip1} is recognized by a Cks1 phosphate binding site, whereas the side chain of an invariant Glu185 inserts into the interface between Skp2 and Cks1, interacting with both. The structure and biochemical data support the proposed model that Cdk2-cyclin A contributes to the recruitment of p27{sup Kip1} to the SCF{sup Skp2}-Cks1 complex.

  2. Identification of Genes, including the Gene Encoding p27Kip1, Regulated by Serine 276 Phosphorylation of the p65 Subunit of NF-κB

    PubMed Central

    Prasad, Ratna Chakraborty; Wang, Xiaohui L.; Law, Brian K.; Davis, Bradley; Green, Gail; Boone, Braden; Sims, Lauren; Law, Mary

    2009-01-01

    Phosphorylation of the p65 subunit of NF-κB is required for its transcriptional activity. Recent reports show that phosphorylation of p65 at serine 276 regulates only a subset of genes, such as those encoding IL-6, IL-8, Gro-β, and ICAM-1. In order to identify additional genes regulated by serine 276 phosphorylation, HepG2 hepatoma cells were infected with adenoviruses encoding either wild-type p65 or the S276A mutant of p65, followed by DNA microarray analysis. The results show that mutation of serine 276 affected the expression of several genes that encode proteins involved in cell cycle regulation, signal transduction, transcription, and metabolism. Notably, expression of S276A increased the mRNA and protein level of p27, a cell cycle inhibitory protein, which led to an increased association of p27 with cdk2, and inhibition of cdk2 activity. Furthermore, while wild-type NF-κB is known to increase cell proliferation in a number of different cancer cell lines, our data show that S276A inhibits cell proliferation. Evidence is mounting that NF-κB plays a pivotal role in oncogenesis. Therapeutic agents that regulate the phosphorylation of serine 276 and p27 gene expression, therefore, may be useful as anti-cancer agents in the future. PMID:19038492

  3. Aortic endothelial cells regulate proliferation of human monocytes in vitro via a mechanism synergistic with macrophage colony-stimulating factor. Convergence at the cyclin E/p27(Kip1) regulatory checkpoint.

    PubMed

    Antonov, A S; Munn, D H; Kolodgie, F D; Virmani, R; Gerrity, R G

    1997-06-15

    Monocyte-derived macrophages (Mphis) are pivotal participants in the pathogenesis of atherosclerosis. Evidence from both animal and human plaques indicates that local proliferation may contribute to accumulation of lesion Mphis, and the major Mphi growth factor, macrophage colony stimulating factor (MCSF), is present in atherosclerotic plaques. However, most in vitro studies have failed to demonstrate that human monocytes/Mphis possess significant proliferative capacity. We now report that, although human monocytes cultured in isolation showed only limited MCSF-induced proliferation, monocytes cocultured with aortic endothelial cells at identical MCSF concentrations underwent enhanced (up to 40-fold) and prolonged (21 d) proliferation. In contrast with monocytes in isolation, this was optimal at low seeding densities, required endothelial cell contact, and could not be reproduced by coculture with smooth muscle cells. Intimal Mphi isolated from human aortas likewise showed endothelial cell contact-dependent, MCSF-induced proliferation. Consistent with a two-signal mechanism governing Mphi proliferation, the cell cycle regulatory protein, cyclin E, was rapidly upregulated by endothelial cell contact in an MCSFindependent fashion, but MCSF was required for successful downregulation of the cell cycle inhibitory protein p27(Kip1) before cell cycling. Thus endothelial cells and MCSF differentially and synergistically regulate two Mphi genes critical for progression through the cell cycle.

  4. The ability of antigen, but not interleukin-2, to promote n-butyrate-induced T helper 1 cell anergy is associated with increased expression and altered association patterns of cyclin-dependent kinase inhibitors

    PubMed Central

    Jackson, Stephanie K; DeLoose, Annick; Gilbert, Kathleen M

    2002-01-01

    The ability of the cell cycle inhibitor n-butyrate to induce T helper 1 (Th1) cell anergy is dependent upon its ability to block the cell cycle progression of activated Th1 cells in G1. Results reported here show that although both interleukin (IL)-2 and antigen (Ag) push Th1 cells into G1 where they are blocked by n-butyrate, only the Ag-activated Th1 cells demonstrate functional anergy once the n-butyrate has been removed from the culture. Because n-butyrate-induced Th1 cell anergy has been linked to increased expression of the cyclin-dependent kinase inhibitors p21Cip1 and p27Kip1, mechanistic experiments focused on the role of these inhibitors. It was found that when Th1 cells were reincubated in Ag-stimulated secondary cultures, the Th1 cells previously exposed to Ag and n-butyrate (anergic Th1 cells) demonstrated a cumulative increase in p21Cip1 and p27Kip1 when compared with Th1 cells previously exposed to recombinant (r)IL-2 and n-butyrate (non-anergic Th1 cells). p27Kip1 in the anergic Th1 cells from the secondary cultures was associated with cyclin-dependent kinases (cdks). In contrast, p21Cip1 in the anergic Th1 cells, although present at high levels, did not associate significantly with cdks, suggesting that p21Cip1 may target some other protein in the anergic Th1 cells. Taken together, these findings suggest that Th1 cell exposure to Ag and n-butyrate, rather than IL-2 and n-butyrate, is needed to induce the cumulative increase in p21Cip1 and p27Kip1 that is associated with the proliferative unresponsiveness in anergic Th1 cells. In addition, p21Cip1 may inhibit proliferation in the anergic Th1 cells by some mechanism other than suppression of cdks that is unique to the induction of Th1 cell anergy. PMID:12153511

  5. The ability of antigen, but not interleukin-2, to promote n-butyrate-induced T helper 1 cell anergy is associated with increased expression and altered association patterns of cyclin-dependent kinase inhibitors.

    PubMed

    Jackson, Stephanie K; DeLoose, Annick; Gilbert, Kathleen M

    2002-08-01

    The ability of the cell cycle inhibitor n-butyrate to induce T helper 1 (Th1) cell anergy is dependent upon its ability to block the cell cycle progression of activated Th1 cells in G1. Results reported here show that although both interleukin (IL)-2 and antigen (Ag) push Th1 cells into G1 where they are blocked by n-butyrate, only the Ag-activated Th1 cells demonstrate functional anergy once the n-butyrate has been removed from the culture. Because n-butyrate-induced Th1 cell anergy has been linked to increased expression of the cyclin-dependent kinase inhibitors p21Cip1 and p27Kip1, mechanistic experiments focused on the role of these inhibitors. It was found that when Th1 cells were reincubated in Ag-stimulated secondary cultures, the Th1 cells previously exposed to Ag and n-butyrate (anergic Th1 cells) demonstrated a cumulative increase in p21Cip1 and p27Kip1 when compared with Th1 cells previously exposed to recombinant (r)IL-2 and n-butyrate (non-anergic Th1 cells). p27Kip1 in the anergic Th1 cells from the secondary cultures was associated with cyclin-dependent kinases (cdks). In contrast, p21Cip1 in the anergic Th1 cells, although present at high levels, did not associate significantly with cdks, suggesting that p21Cip1 may target some other protein in the anergic Th1 cells. Taken together, these findings suggest that Th1 cell exposure to Ag and n-butyrate, rather than IL-2 and n-butyrate, is needed to induce the cumulative increase in p21Cip1 and p27Kip1 that is associated with the proliferative unresponsiveness in anergic Th1 cells. In addition, p21Cip1 may inhibit proliferation in the anergic Th1 cells by some mechanism other than suppression of cdks that is unique to the induction of Th1 cell anergy.

  6. An essential role for Ink4 and Cip/Kip cell-cycle inhibitors in preventing replicative stress

    PubMed Central

    Quereda, V; Porlan, E; Cañamero, M; Dubus, P; Malumbres, M

    2016-01-01

    Cell-cycle inhibitors of the Ink4 and Cip/Kip families are involved in cellular senescence and tumor suppression. These inhibitors are individually dispensable for the cell cycle and inactivation of specific family members results in increased proliferation and enhanced susceptibility to tumor development. We have now analyzed the consequences of eliminating a substantial part of the cell-cycle inhibitory activity in the cell by generating a mouse model, which combines the absence of both p21Cip1 and p27Kip1 proteins with the endogenous expression of a Cdk4 R24C mutant insensitive to Ink4 inhibitors. Pairwise combination of Cdk4 R24C, p21-null and p27-null alleles results in frequent hyperplasias and tumors, mainly in cells of endocrine origin such as pituitary cells and in mesenchymal tissues. Interestingly, complete abrogation of p21Cip1 and p27Kip1 in Cdk4 R24C mutant mice results in a different phenotype characterized by perinatal death accompanied by general hypoplasia in most tissues. This phenotype correlates with increased replicative stress in developing tissues such as the nervous system and subsequent apoptotic cell death. Partial inhibition of Cdk4/6 rescues replicative stress signaling as well as p53 induction in the absence of cell-cycle inhibitors. We conclude that one of the major physiological activities of cell-cycle inhibitors is to prevent replicative stress during development. PMID:26292757

  7. Resveratrol induces cell cycle arrest and apoptosis with docetaxel in prostate cancer cells via a p53/ p21WAF1/CIP1 and p27KIP1 pathway.

    PubMed

    Singh, Santosh Kumar; Banerjee, Saswati; Acosta, Edward P; Lillard, James W; Singh, Rajesh

    2017-02-13

    Resveratrol (RES) is the most effective natural products used for the treatment of a variety of cancers. In this study, we tested the effect of RES in enhancing the efficacy of docetaxel (DTX) treatment in prostate cancer (PCa) cells. The C4-2B and DU-145 cell lines were treated with RES, DTX and combination followed by evaluating the apoptosis and cell cycle progression. The combined drug treatment up-regulates the pro-apoptotic genes (BAX, BID, and BAK), cleaved PARP and down regulates the anti-apoptotic genes (MCL-1, BCL-2, BCL-XL) promoting apoptosis. In C4-2B cells the combination up regulated the expression of p53, and cell cycle inhibitors (p21WAF1/CIP1, p27KIP), which, in turn, inhibited the expression of CDK4, cyclin D1, cyclin E1 and induced hypo-phosphorylation of Rb thus blocking the transition of cells in the G0/G1 to S phase. In contrast, the synergistic effect was not profound in DU145 due to its lesser sensitivity to DTX. The suppression of cyclin B1 and CDK1 expression in both cell lines inhibits the further progression of cells in G2/M phase. The current study demonstrates that combination treatment blocks the cell cycle arrest by modulation of key regulators and promotes apoptosis via p53 dependent and independent mechanism in PCa.

  8. Nuclear factor of activated T cells (NFAT) signaling regulates PTEN expression and intestinal cell differentiation

    PubMed Central

    Wang, Qingding; Zhou, Yuning; Jackson, Lindsey N.; Johnson, Sara M.; Chow, Chi-Wing; Evers, B. Mark

    2011-01-01

    The nuclear factor of activated T cell (NFAT) proteins are a family of transcription factors (NFATc1–c4) involved in the regulation of cell differentiation and adaptation. Previously we demonstrated that inhibition of phosphatidylinositol 3-kinase or overexpression of PTEN enhanced intestinal cell differentiation. Here we show that treatment of intestinal-derived cells with the differentiating agent sodium butyrate (NaBT) increased PTEN expression, NFAT binding activity, and NFAT mRNA expression, whereas pretreatment with the NFAT signaling inhibitor cyclosporine A (CsA) blocked NaBT-mediated PTEN induction. Moreover, knockdown of NFATc1 or NFATc4, but not NFATc2 or NFATc3, attenuated NaBT-induced PTEN expression. Knockdown of NFATc1 decreased PTEN expression and increased the phosphorylation levels of Akt and downstream targets Foxo1 and GSK-3α/β. Furthermore, overexpression of NFATc1 or the NFATc4 active mutant increased PTEN and p27kip1 expression and decreased Akt phosphorylation. In addition, pretreatment with CsA blocked NaBT-mediated induction of intestinal alkaline phosphatase (IAP) activity and villin and p27kip1 expression; knockdown of either NFATc1 or NFATc4 attenuated NaBT-induced IAP activity. We provide evidence showing that NFATc1 and NFATc4 are regulators of PTEN expression. Importantly, our results suggest that NFATc1 and NFATc4 regulation of intestinal cell differentiation may be through PTEN regulation. PMID:21148296

  9. Susceptibility of p27 kip1 knockout mice to urinary bladder carcinogenesis induced by N-butyl-N-(4-hydroxybutyl)nitrosamine may not simply be due to enhanced proliferation.

    PubMed

    Hikosaka, Atsuya; Ogawa, Kumiko; Sugiura, Satoshi; Asamoto, Makoto; Takeshita, Fumitaka; Sato, Shin-Ya; Nakanishi, Makoto; Kohri, Kenjiro; Shirai, Tomoyuki

    2008-03-15

    Deregulated proliferation is one of the fundamental characteristics of carcinogenesis. p27 is one of the most well characterized negative cell cycle regulator. In our previous study, expression of p27 protein was found to be dramatically suppressed on stimulation of cell proliferation by calculi in the rodent urinary bladder, withdrawal of the insult resulting in re-expression of p27 and regression of urothelial hyperplastic lesions. In the present study, to evaluate how loss of function impacts on urinary bladder carcinogenesis, N-butyl-N-(4-hydroxybutyl)nitrosamine (BBN), a bladder carcinogen was given to p27 knockout mice. Males and females with either null, hetero or wild-type p27 alleles were divided into 2 groups, one given drinking water containing 0.05% BBN for 10 weeks and the other receiving distilled water, then, killed at week 20. The experiment was repeated for confirmation of the outcome. In the second experiment, performed with a larger number of animals, the incidence of urinary bladder carcinomas was significantly higher in female p27-null mice than in their wild-type counterparts. p27 deficiency also resulted in their increase of relative weights of urinary bladders and section areas of carcinomas in BBN-treated mice. Interestingly, while BrdU labeling indices generally increased with progression of mucosal proliferative lesions, from normal epithelium, through hyperplasia to carcinoma, there was no significant variation with the p27 genotype, in tumors as well as normal urothelium. These findings suggest that p27 deficient mice have elevated susceptibility to BBN-induction of urinary bladder carcinogenesis through a mechanism which might be independent of acceleration of cell cycling.

  10. A-62176, a potent topoisomerase inhibitor, inhibits the expression of human epidermal growth factor receptor 2.

    PubMed

    Kim, Hye-Lin; Jeon, Kyung-Hwa; Jun, Kyu-Yeon; Choi, Yongmun; Kim, Dae-Kee; Na, Younghwa; Kwon, Youngjoo

    2012-12-01

    HER2 overexpression is observed in ∼6-35% of all gastric cancers, while co-amplification of topoisomerase IIα occurs in ∼32-90% of all cancers with HER2 amplification. The present study reports that HER2 expression is down-regulated by A-62176, a fluoroquinophenoxazine derivative that we previously demonstrated to inhibit topoisomerase I and IIα. The results suggest that A-62176 inhibits the interaction between the ESX, an ets transcription factor, and its co-activator Sur2, leading to the attenuation of HER2-mediated phosphorylation of MAPK/Akt. A-62176 arrests the cell cycle in the G1 phase via the down-regulation of cyclin D1 and the up-regulation of p27(Kip1) in NCI-N87 gastric cancer cells. The combination of A-62176 with doxorubicin provides a strong synergistic activity. We propose that A-62176 is a dual inhibitor that impairs the expression of HER2 and restrains the activity of topoisomerase IIα. Our results may lead to the rational design of anticancer molecules targeting a subgroup of gastric cancer cells overexpressing both HER2 and topoisomerase IIα.

  11. Potentiated antitumor effects of a combination therapy with a farnesyltransferase inhibitor L-744,832 and butyrate in vitro.

    PubMed

    Kopec, Maciej; Strusinska, Katarzyna; Legat, Magdalena; Makowski, Marcin; Jakobisiak, Marek; Golab, Jakub

    2004-05-01

    Farnesyltransferase inhibitors, butyrate and butyric acid derivatives have previously been reported to exert anti-tumor activity in experimental models in vitro and in vivo and have recently gained acceptance as potential anticancer agents. In our study, we examined antitumor effects of a combination of a farnesyltransferase inhibitor L-744,832 and butyrate in vitro against MDA-MB-231 and MIA PaCa-2 human cancer cells. This combination therapy showed synergistic antitumor activity against MDA-MB-231 cells, which was at least in part due to induction of p27KIP1 expression. Both drugs increased intracellular levels of p53 as well but there was no significant difference between the groups treated with single drugs and the group treated with their combination. In MIA PaCa-2 cells, the combination therapy exerted additive antitumor activity. Our results illustrate possible application of the farnesyltransferase inhibitor L-744,832 and butyrate as a combination therapy of cancer.

  12. In vitro and in vivo anti-tumor activity of miR-221/222 inhibitors in multiple myeloma.

    PubMed

    Di Martino, Maria Teresa; Gullà, Annamaria; Cantafio, Maria Eugenia Gallo; Lionetti, Marta; Leone, Emanuela; Amodio, Nicola; Guzzi, Pietro Hiram; Foresta, Umberto; Conforti, Francesco; Cannataro, Mario; Neri, Antonino; Giordano, Antonio; Tagliaferri, Pierosandro; Tassone, Pierfrancesco

    2013-02-01

    A rising body of evidence suggests that silencing microRNAs (miRNAs) with oncogenic potential may represent a successful therapeutic strategy for human cancer. We investigated the therapeutic activity of miR-221/222 inhibitors against human multiple myeloma (MM) cells. Enforced expression of miR-221/222 inhibitors triggered in vitro anti-proliferative effects and up-regulation of canonic miR-221/222 targets, including p27Kip1, PUMA, PTEN and p57Kip2, in MM cells highly expressing miR-221/222. Conversely, transfection of miR-221/222 mimics increased S-phase and down-regulated p27Kip1 protein expression in MM with low basal miR-221/222 levels. The effects of miR-221/222 inhibitors was also evaluated in MM xenografts in SCID/ NOD mice. Significant anti-tumor activity was achieved in xenografted mice by the treatment with miR-221/222 inhibitors, together with up-regulation of canonic protein targets in tumors retrieved from animals. These findings provide proof of principle that silencing the miR-221/222 cluster exerts significant therapeutic activity in MM cells with high miR-221/222 level of expression, which mostly occurs in TC2 and TC4 MM groups. These findings suggest that MM genotyping may predict the therapeutic response. All together our results support a framework for clinical development of miR-221/222 inhibitors-based therapeutic strategy in this still incurable disease.

  13. The expression of AIP-related molecules in elucidation of cellular pathways in pituitary adenomas.

    PubMed

    Heliövaara, Elina; Raitila, Anniina; Launonen, Virpi; Paetau, Anders; Arola, Johanna; Lehtonen, Heli; Sane, Timo; Weil, Robert J; Vierimaa, Outi; Salmela, Pasi; Tuppurainen, Karoliina; Mäkinen, Markus; Aaltonen, Lauri A; Karhu, Auli

    2009-12-01

    Germline mutations in the aryl hydrocarbon receptor interacting protein (AIP) gene predispose to the development of pituitary adenomas. Here, we characterized AIP mutation positive (AIPmut+) and AIP mutation negative (AIPmut-) pituitary adenomas by immunohistochemistry. The expressions of the AIP-related proteins aryl hydrocarbon receptor (AHR), AHR nuclear translocator (ARNT), cyclin-dependent kinase inhibitor 1B encoding p27(Kip1), and hypoxia-inducible factor 1-alpha were examined in 14 AIPmut+ and 53 AIPmut- pituitary adenomas to detect possible expression differences. In addition, the expression of CD34, an endothelial and hematopoietic stem cell marker, was analyzed. We found ARNT to be less frequently expressed in AIPmut+ pituitary adenomas (P = 0.001), suggesting that AIP regulates the ARNT levels. AIP small interfering RNA-treated HeLa, HEK293, or Aip-null mouse embryonic fibroblast cells did not show lowered expression of ARNT. Instead, in the pituitary adenoma cell line GH3, Aip silencing caused a partial reduction of Arnt and a clear increase in cell proliferation. We also observed a trend for increased expression of nuclear AHR in AIPmut+ samples, although the difference was not statistically significant (P = 0.06). The expressions of p27(Kip1), hypoxia-inducible factor 1-alpha, or CD34 did not differ between tumor types. The present study shows that the expression of ARNT protein is significantly reduced in AIPmut+ tumors. We suggest that the down-regulation of ARNT may be connected to an imbalance in AHR/ARNT complex formation arising from aberrant cAMP signaling.

  14. A retroviral expression system based on tetracycline-regulated tricistronic transactivator/repressor vectors for functional analyses of antiproliferative and toxic genes.

    PubMed

    Ausserlechner, Michael J; Obexer, Petra; Deutschmann, Andrea; Geiger, Kathrin; Kofler, Reinhard

    2006-08-01

    Establishment of stably transfected mammalian cells with conditional expression of antiproliferative or proapoptotic proteins is often hampered by varying expression within bulk-selected cells and high background in the absence of the inducing drug. To overcome such limitations, we designed a gene expression system that transcribes the tetracycline-dependent rtTA2-M2-activator, TRSID-silencer, and selection marker as a tricistronic mRNA from a single retroviral vector. More than 92% of bulk-selected cells expressed enhanced green fluorescent protein or luciferase over more than three orders of magnitude in an almost linear, dose-dependent manner. To functionally test this system, we studied how dose-dependent expression of p27(Kip1) affects proliferation and viability of SH-EP neuroblastoma cells. Low to moderate p27(Kip1) expression caused transient G(0)-G(1) accumulation without reduced viability, whereas high p27(Kip1) levels induced significant apoptosis after 72 hours. This proves that this expression system allows concentration-dependent analysis of gene function and implicates p27(Kip1) as a critical regulator of both proliferation and apoptosis in SH-EP neuroblastoma cells.

  15. The expression of the ubiquitin ligase subunit Cks1 in human breast cancer

    PubMed Central

    Slotky, Merav; Shapira, Ma'anit; Ben-Izhak, Ofer; Linn, Shai; Futerman, Boris; Tsalic, Medy; Hershko, Dan D

    2005-01-01

    Introduction Loss of the cell-cycle inhibitory protein p27Kip1 is associated with a poor prognosis in breast cancer. The decrease in the levels of this protein is the result of increased proteasome-dependent degradation, mediated and rate-limited by its specific ubiquitin ligase subunits S-phase kinase protein 2 (Skp2) and cyclin-dependent kinase subunit 1 (Cks1). Skp2 was recently found to be overexpressed in breast cancers, but the role of Cks1 in these cancers is unknown. The present study was undertaken to examine the role of Cks1 expression in breast cancer and its relation to p27Kip1 and Skp2 expression and to tumor aggressiveness. Methods The expressions of Cks1, Skp2, and p27Kip1 were examined immunohistochemically on formalin-fixed, paraffin-wax-embedded tissue sections from 50 patients with breast cancer and by immunoblot analysis on breast cancer cell lines. The relation between Cks1 levels and patients' clinical and histological parameters were examined by Cox regression and the Kaplan–Meier method. Results The expression of Cks1 was strongly associated with Skp2 expression (r = 0.477; P = 0.001) and inversely with p27Kip1 (r = -0.726; P < 0.0001). Overexpression of Cks1 was associated with loss of tumor differentiation, young age, lack of expression of estrogen receptors and of progesterone receptors, and decreased disease-free (P = 0.0007) and overall (P = 0.041) survival. In addition, Cks1 and Skp2 expression were increased by estradiol in estrogen-dependent cell lines but were down-regulated by tamoxifen. Conclusion These results suggest that Cks1 is involved in p27Kip1 down-regulation and may have an important role in the development of aggressive tumor behavior in breast cancer. PMID:16168119

  16. Structure-based repurposing of FDA-approved drugs as inhibitors of NEDD8-activating enzyme.

    PubMed

    Zhong, Hai-Jing; Liu, Li-Juan; Chan, Daniel Shiu-Hin; Wang, Hui-Min; Chan, Philip Wai Hong; Ma, Dik-Lung; Leung, Chung-Hang

    2014-07-01

    We report the discovery of an inhibitor of NEDD8-activating enzyme (NAE) by an integrated virtual screening approach. Piperacillin 1 inhibited NAE activity in cell-free and cell-based systems with high selectivity. Furthermore, piperacillin 1 was able to inhibit the degradation of the NAE downstream protein substrate p27(kip1). Our molecular modeling and kinetic studies suggested that this compound may act as a non-covalent ATP-competitive inhibitor of NAE.

  17. A functional link between Wnt signaling and SKP2-independent p27 turnover in mammary tumors

    PubMed Central

    Miranda-Carboni, Gustavo A.; Krum, Susan A.; Yee, Kathleen; Nava, Miguel; Deng, Qiming E.; Pervin, Shehla; Collado-Hidalgo, Alicia; Galić, Zoran; Zack, Jerome A.; Nakayama, Keiko; Nakayama, Keiichi I.; Lane, Timothy F.

    2008-01-01

    Loss of the CDK inhibitor p27KIP1 is widely linked with poor prognosis in human cancer. In Wnt10b-expressing mammary tumors, levels of p27KIP1 were extremely low; conversely, Wnt10b-null mammary cells expressed high levels of this protein, suggesting Wnt-dependent regulation of p27KIP1. Interestingly we found that Wnt-induced turnover of p27KIP1 was independent from classical SCFSKP2-mediated degradation in both mouse and human cells. Instead, turnover required Cullin 4A and Cullin 4B, components of an alternative E3 ubiquitin ligase induced in response to active Wnt signaling. We found that CUL4A was a novel Wnt target gene in both mouse and human cells and that CUL4A physically interacted with p27KIP1 in Wnt-responding cells. We further demonstrated that both Cul4A and Cul4B were required for Wnt-induced p27KIP1 degradation and S-phase progression. CUL4A and CUL4B are therefore components of a conserved Wnt-induced proteasome targeting (WIPT) complex that regulates p27KIP1 levels and cell cycle progression in mammalian cells. PMID:19056892

  18. The p27Kip1 Tumor Suppressor and Multi-Step Tumorigenesis

    DTIC Science & Technology

    2001-08-01

    completion of the Celera mouse genome . Sequenced IPCR clones were searched against the Celera database and clones that fell within the same Celera ...in all of the lymphomas containing XPC-1 insertions. There is significant sequence conservation between the murine XPC-1 locus and the syntenic human ...Xq26 region, and sequences homologous to A1464896 and the cloned insertion sites are present in the human Xq26 region with spacing quite similar to

  19. Bortezomib induces apoptosis and growth suppression in human medulloblastoma cells, associated with inhibition of AKT and NF-ĸB signaling, and synergizes with an ERK inhibitor.

    PubMed

    Yang, Fan; Jove, Veronica; Chang, Shirley; Hedvat, Michael; Liu, Lucy; Buettner, Ralf; Tian, Yan; Scuto, Anna; Wen, Wei; Yip, M L Richard; Van Meter, Timothy; Yen, Yun; Jove, Richard

    2012-04-01

    Medulloblastoma is the most common brain tumor in children. Here, we report that bortezomib, a proteasome inhibitor, induced apoptosis and inhibited cell proliferation in two established cell lines and a primary culture of human medulloblastomas. Bortezomib increased the release of cytochrome c to cytosol and activated caspase-9 and caspase-3, resulting in cleavage of PARP. Caspase inhibitor (Z-VAD-FMK) could rescue medulloblastoma cells from the cytotoxicity of bortezomib. Phosphorylation of AKT and its upstream regulator mTOR were reduced by bortezomib treatment in medulloblastoma cells. Bortezomib increased the expression of Bad and Bak, pro-apoptotic proteins, and p21Cip1 and p27Kip1, negative regulators of cell cycle progression, which are associated with the growth suppression and induction of apoptosis in these tumor cells. Bortezomib also increased the accumulation of phosphorylated IĸBα, and decreased nuclear translocation of NF-ĸB. Thus, NF-ĸB signaling and activation of its downstream targets are suppressed. Moreover, ERK inhibitors or downregulating ERK with ERK siRNA synergized with bortezomib on anticancer effects in medulloblastoma cells. Bortezomib also inhibited the growth of human medulloblastoma cells in a mouse xenograft model. These findings suggest that proteasome inhibitors are potentially promising drugs for treatment of pediatric medulloblastomas.

  20. Constitutively expressed COX-2 in osteoblasts positively regulates Akt signal transduction via suppression of PTEN activity.

    PubMed

    Li, Ching-Ju; Chang, Je-Ken; Wang, Gwo-Jaw; Ho, Mei-Ling

    2011-02-01

    Cyclooxygenase-2 (COX-2) is thought to be an inducible enzyme, but increasing reports indicate that COX-2 is constitutively expressed in several organs. The status of COX-2 expression in bone and its physiological role remains undefined. Non-selective non-steroidal anti-inflammatory drugs (NSAIDs) and selective COX-2 inhibitors, which commonly suppress COX-2 activity, were reported to suppress osteoblast proliferation via Akt/FOXO3a/p27(Kip1) signaling, suggesting that COX-2 may be the key factor of the suppressive effects of NSAIDs on proliferation. Although Akt activation correlates with PTEN deficiency and cell viability, the role of COX-2 on PTEN/Akt regulation remains unclear. In this study, we hypothesized that COX-2 may be constitutively expressed in osteoblasts and regulate PTEN/Akt-related proliferation. We examined the localization and co-expression of COX-2 and p-Akt in normal mouse femurs and in cultured mouse (mOBs) and human osteoblasts (hOBs). Our results showed that osteoblasts adjacent to the trabeculae, periosteum and endosteum in mouse femurs constitutively expressed COX-2, while COX-2 co-expressed with p-Akt in osteoblasts sitting adjacent to trabeculae in vivo, and in mOBs and hOBs in vitro. We further used COX-2 siRNA to test the role of COX-2 in Akt signaling in hOBs; COX-2 silencing significantly inhibited PTEN phosphorylation, enhanced PTEN activity, and suppressed p-Akt level and proliferation. However, replenishment of the COX-2 enzymatic product, PGE2, failed to reverse COX-2-dependent Akt phosphorylation. Furthermore, transfection with recombinant human COX-2 (rhCOX-2) significantly reversed COX-2 siRNA-suppressed PTEN phosphorylation, but this effect was reduced when the enzymatic activity of rhCOX-2 was blocked. This finding indicated that the effect of COX-2 on PTEN/Akt signaling is not related to PGE2 but still dependent on COX-2 enzymatic activity. Conversely, COX-1 silencing did not affect PTEN/Akt signaling. Our findings provide

  1. FoxF1 and FoxF2 transcription factors synergistically promote Rhabdomyosarcoma carcinogenesis by repressing transcription of p21Cip1 CDK inhibitor

    PubMed Central

    Cai, Yuqi; Le, Tien; Turpin, Brian; Kalinichenko, Vladimir V.; Kalin, Tanya V.

    2016-01-01

    The role of Forkhead Box F1 (FoxF1) transcription factor in carcinogenesis is not well characterized. Depending on tissue and histological type of cancer, FoxF1 was shown to be either oncogene or tumor suppressor. Alveolar rhabdomyosarcoma (RMS) is the most aggressive pediatric soft tissue sarcoma. While FoxF1 is highly expressed in alveolar RMS, the functional role of FoxF1 in RMS is unknown. The present study demonstrates that expression of FoxF1 and its closely related transcription factor FoxF2 are essential for rhabdomyosarcoma tumor growth. Depletion of FoxF1 or FoxF2 in rhabdomyosarcoma cells decreased tumor growth in orthotopic mouse models of RMS. The decreased tumorigenesis was associated with the reduced tumor cell proliferation. Cell cycle regulatory proteins Cdk2, Cdk4/6, Cyclin D1 and Cyclin E2 were decreased in FoxF1- and FoxF2-deficient RMS tumors. Depletion of either FoxF1 or FoxF2 delayed G1-S cell cycle progression, decreased levels of phosphorylated Rb and increased protein levels of the CDK inhibitors, p21Cip1 and p27Kip1. Depletion of both FoxF1 and FoxF2 in tumor cells completely abrogated RMS tumor growth in mice. Overexpression of either FoxF1 or FoxF2 in tumor cells was sufficient to increase carcinogenesis in orthotopic RMS mouse model. FoxF1 and FoxF2 directly bound to and repressed transcriptional activity of p21Cip1 promoter through −556/−545 bp region, but did not affect p27Kip1 transcription. Knockdown of p21Cip1 restored cell cycle progression in the FoxF1- or FoxF2-deficient tumor cells. Altogether, FoxF1 and FoxF2 promoted RMS tumorigenesis by inducing tumor cell proliferation via transcriptional repression of p21Cip1 gene promoter. Due to robust oncogenic activity in RMS tumors, FoxF1 and FoxF2 may represent promising targets for anti-tumor therapy. PMID:27425595

  2. Oncogenic signals of HER-2/neu in regulating the stability of the cyclin-dependent kinase inhibitor p27.

    PubMed

    Yang, H Y; Zhou, B P; Hung, M C; Lee, M H

    2000-08-11

    Overexpression and activation of HER-2/neu, a proto-oncogene, play a pivotal role in cancer formation. Strong expression of HER-2/neu in cancers has been associated with poor prognosis. Reduced expression of p27(Kip1), a cyclin-dependent kinase inhibitor, correlates with poor clinical outcome in many types of carcinomas. Because many cancers with the overexpression of HER-2/neu overlap with those affected by reduced p27 expression, we studied the link between HER-2/neu oncogenic signals and p27 regulation. We found that down-regulation of p27 correlates with HER-2/neu overexpression. To address the molecular mechanism of this inverse correlation, we found that reduction of p27 is caused by enhanced ubiquitin-mediated degradation, and the HER-2/Grb2/MAPK pathway is involved in the decrease of p27 stability. Also, HER-2/neu activity causes mislocation of p27 and Jun activation domain-binding protein 1 (JAB1), an exporter of p27, into the cytoplasm, thereby facilitating p27 degradation. These results reveal that HER-2/neu signals reduce p27 stability and thus present potential points for therapeutic intervention in HER-2/neu-associated cancers.

  3. Hepatocyte growth factor (HGF) inhibits skeletal muscle cell differentiation: a role for the bHLH protein twist and the cdk inhibitor p27.

    PubMed

    Leshem, Y; Spicer, D B; Gal-Levi, R; Halevy, O

    2000-07-01

    Hepatocyte growth factor (HGF) plays a crucial role in regulating the differentiation of both fetal and adult skeletal myoblasts. This study aimed at defining the intracellular factors that mediate the effect of HGF on adult myoblast differentiation. HGF increased Twist expression while decreasing p27(kip1) protein levels and not affecting the induction of p21(Cip1/Waf1) in satellite cells. Like HGF, overexpression of Twist did not affect p21 expression while inhibiting muscle-specific proteins. Both ectopic Twist-antisense (Twist-AS) and p27 partially rescued the effects of HGF on bromodeoxyuridine (BrdU) incorporation and myosin heavy chain (MHC) expression in muscle satellite cells; the two plasmids together effected full rescue, suggesting that HGF independently regulates these two factors to mediate its effects. Ectopic p27 promoted differentiation in the presence of HGF by blocking the induction of Twist. Using Twist-AS to lower Twist levels restored the HGF-dependent reduction of p27 and MHC. In the presence of ectopic HGF, satellite cells formed thin mononuclear myotubes. Neither ectopic p27, Twist-AS, or their combination reversed this change in cell morphology, suggesting that HGF acts through additional mediators to inhibit downstream events during myogenesis. Taken together, the results suggest that the effects of HGF on muscle cell proliferation and differentiation are mediated through changes in the expression levels of the myogenic-inhibitory basic helix-loop-helix (bHLH) protein Twist and the cell-cycle inhibitor p27.

  4. Effects of the combined blockade of EGFR and ErbB-2 on signal transduction and regulation of cell cycle regulatory proteins in breast cancer cells.

    PubMed

    D'Alessio, Amelia; De Luca, Antonella; Maiello, Monica R; Lamura, Luana; Rachiglio, Anna Maria; Napolitano, Maria; Gallo, Marianna; Normanno, Nicola

    2010-09-01

    Treatment of breast cancer cells with a combination of the EGFR-tyrosine kinase inhibitor (EGFR-TKI) gefitinib and the anti-ErbB-2 monoclonal antibody trastuzumab results in a synergistic antitumor effect. In this study, we addressed the mechanisms involved in this phenomenon. The activation of signaling pathways and the expression of cell cycle regulatory proteins were studied in SK-Br-3 and BT-474 breast cancer cells, following treatment with EGFR and/or ErbB-2 inhibitors. Treatment with the gefitinib/trastuzumab combination produced, as compared with a single agent, a more prolonged blockade of AKT and MAPK activation, a more pronounced accumulation of cells in the G0/G1 phase of the cell cycle, a more significant increase in the levels of p27(kip1) and of hypophosphorylated pRb2, and a decrease in the levels of Cyclin D1 and survivin. Similar findings were observed with the EGFR/ErbB-2 inhibitor lapatinib. Gefitinib, trastuzumab, and their combination increased the stability of p27(kip1), with the combination showing the highest effects. Blockade of both receptors with gefitinib/trastuzumab or lapatinib induced a significant increase in the levels of p27(kip1) mRNA and in the nuclear levels of the p27(kip1) transcription factor FKHRL-1. Inhibition of PI3K signaling also produced a significant raise in p27(kip1) mRNA. Finally, down-modulation of FKHRL-1 with siRNAs prevented the lapatinib-induced increase of p27(kip1) mRNA. The synergism deriving from EGFR and ErbB-2 blockade is mediated by several different alterations in the activation of signaling proteins and in the expression of cell cycle regulatory proteins, including transcriptional and posttranscriptional regulation of p27(kip1) expression.

  5. Minibrain drives the Dacapo-dependent cell cycle exit of neurons in the Drosophila brain by promoting asense and prospero expression.

    PubMed

    Shaikh, Mirja N; Gutierrez-Aviño, Francisco; Colonques, Jordi; Ceron, Julian; Hämmerle, Barbara; Tejedor, Francisco J

    2016-09-01

    A key aim of neurodevelopmental research is to understand how precursor cells decide to stop dividing and commence their terminal differentiation at the correct time and place. Here, we show that minibrain (mnb), the Drosophila ortholog of the Down syndrome candidate gene DYRK1A, is transiently expressed in newborn neuronal precursors known as ganglion cells (GCs). Mnb promotes the cell cycle exit of GCs through a dual mechanism that regulates the expression of the cyclin-dependent kinase inhibitor Dacapo, the homolog of vertebrate p27(Kip1) (Cdkn1b). Mnb upregulates the expression of the proneural transcription factor (TF) Asense, which promotes Dacapo expression. Mnb also induces the expression of Prospero, a homeodomain TF that in turn inhibits the expression of Deadpan, a pan-neural TF that represses dacapo In addition to its effects on Asense and Prospero, Mnb also promotes the expression of the neuronal-specific RNA regulator Elav, strongly suggesting that Mnb facilitates neuronal differentiation. These actions of Mnb ensure the precise timing of neuronal birth, coupling the mechanisms that regulate neurogenesis, cell cycle control and terminal differentiation of neurons.

  6. WIF1, a Wnt pathway inhibitor, regulates SKP2 and c-myc expression leading to G1 arrest and growth inhibition ofhuman invasive urinary bladder cancer cells

    PubMed Central

    Tang, Yaxiong; Simoneau, Anne R.; Liao, Wu-xiang; Yi, Guo; Hope, Christopher; Liu, Feng; Li, Shunqiang; Xie, Jun; Holcombe, Randall F.; Jurnak, Frances A.; Mercola, Dan; Hoang, Bang H.; Zi, Xiaolin

    2009-01-01

    Epigenetic silencing of secreted wingless-type (Wnt) antagonists through hypermethylation is associated with tobacco smoking and with invasive bladder cancer. The secreted Wnt inhibitory factor-1 (WIF1) has shown consistent growth-inhibitory effect on various cancer cell lines. Therefore,we assessed the mechanisms of action of WIF1 by either restoring WIF1 expression in invasive bladder cancer cell lines (T24 and TSU-PR1) or using a recombinant protein containing functional WIF1 domain. Both ectopic expression of WIF1 and treatment with WIF1 domain protein resulted in cell growth inhibition via G1 arrest. The G1 arrest induced by WIF1 is associated with down-regulation of SKP2 and c-myc and up-regulation of p21/WAF1 and p27/Kip1. Conversely, reexpression of SKP2 in WIF1-overexpressing TSU-PR1 cells attenuated the WIF1-induced G1 arrest. Furthermore, inhibition of nuclear Wnt signaling by either dominant-negative LEF1 or short hairpin RNA of TCF4 also reduced SKP2 expression. The human SKP2 gene contains two TCF/LEF1 consensus binding sites within the promoter. Chromatin immunoprecipitation/real-time PCR analysis revealed that both WIF1 and dominant-negative LEF1 expression decreased the in vivo binding of TCF4 and β-catenin to the SKP2 promoter. Together,our results suggest that mechanisms of WIF1-induced G1 arrest include (a) SKP2 down-regulation leading to p27/Kip1 accumulation and (b) c-myc down-regulation releasing p21/WAF1 transcription. Additionally,we show that WIF1 inhibits in vivo bladder tumor growth in nude mice. These observations suggest a mechanism for transformation of bladder epithelium on loss of WIF1 function and provide new targets such as SKP2 for intervention in WIF1-deficient bladder cancer. PMID:19174556

  7. EPO gene expression promotes proliferation, migration and invasion via the p38MAPK/AP-1/MMP-9 pathway by p21WAF1 expression in vascular smooth muscle cells.

    PubMed

    Park, Sung Lyea; Won, Se Yeon; Song, Jun-Hui; Kambe, Taiho; Nagao, Masaya; Kim, Wun-Jae; Moon, Sung-Kwon

    2015-03-01

    The use of recombinant human erythropoietin (rHuEpo) can lead to hypertrophy and hyperplasia, and has induced the proliferation of vascular smooth muscle cells (VSMCs). The effect of the EPO gene in the migration and invasion of VSMCs remains unclear. In this study, overexpression of the EPO gene increased the DNA synthesis and phosphorylation of ERK1/2 and p38MAPK in VSMCs. In addition, EPO gene expression induced the migration and invasion of VSMCs via the expression of MMP-9 by the activation of NF-κB and AP-1 binding. A blockade of p38MAPK by specific p38MAPK inhibitor SB203580 led to a suppression of the increased DNA synthesis, migration, and invasion of VSMCs that was induced by the EPO gene. SB203580 treatment blocked the increased expression of MMP-9 through the binding activity of AP-1. Transfection of the EPO gene with VSMCs was associated with the up-regulation of cyclin D1/CDK4, cyclin E/CDK2, and p21WAF1, and with the down-regulation of p27KIP1. The specific suppression of p21WAF1 expression by siRNA rescued the enhancement of DNA synthesis via the phosphorylation of p38MAPK and the increase in migration and invasion through AP-1-mediated MMP-9 expression in EPO gene transfectants. These novel findings demonstrate that p21WAF1 regulates the proliferation, migration and invasion of VSMC induced by EPO gene.

  8. Reduced Expression of the Retinoblastoma Protein Shows That the Related Signaling Pathway Is Essential for Mediating the Antineoplastic Activity of Erufosine

    PubMed Central

    Zaharieva, Maya M.; Kirilov, Milen; Chai, Minquang; Berger, Stefan M.; Konstantinov, Spiro; Berger, Martin R.

    2014-01-01

    Erufosine is a new antineoplastic agent of the group of alkylphosphocholines, which interferes with signal transduction and induces apoptosis in various leukemic and tumor cell lines. The present study was designed to examine for the first time the mechanism of resistance to erufosine in malignant cells with permanently reduced expression of the retinoblastoma (Rb) protein. Bearing in mind the high number of malignancies with reduced level of this tumor-suppressor, this investigation was deemed important for using erufosine, alone or in combination, in patients with compromised RB1 gene expression. For this purpose, clones of the leukemic T-cell line SKW-3 were used, which had been engineered to constantly express differently low Rb levels. The alkylphosphocholine induced apoptosis, stimulated the expression of the cyclin dependent kinase inhibitor p27Kip1 and inhibited the synthesis of cyclin D3, thereby causing a G2 phase cell cycle arrest and death of cells with wild type Rb expression. In contrast, Rb-deficiency impeded the changes induced by eru-fosine in the expression of these proteins and abrogated the induction of G2 arrest, which was correlated with reduced antiproliferative and anticlonogenic activities of the compound. In conclusion, analysis of our results showed for the first time that the Rb signaling pathway is essential for mediating the antineoplastic activity of erufosine and its efficacy in patients with malignant diseases may be predicted by determining the Rb status. PMID:24987858

  9. Down-regulation of dihydrofolate reductase inhibits the growth of endothelial EA.hy926 cell through induction of G1 cell cycle arrest via up-regulating p53 and p21(waf1/cip1) expression.

    PubMed

    Fei, Zhewei; Gao, Yong; Qiu, Mingke; Qi, Xianqin; Dai, Yuxin; Wang, Shuqing; Quan, Zhiwei; Liu, Yingbin; Ou, Jingmin

    2016-03-01

    Folic acid supplementation may meliorate cardiovascular disease risk by improving vascular endothelial structure and function. However, the underlying mechanisms are still lack of a global understanding. To be used, folic acid must be converted to 7,8-dihydrofolate by dihydrofolate reductase to generate one-carbon derivatives serving as important cellular cofactors in the synthesis of nucleotides and amino acids required for cell growth. Therefore, this study explored the effect of dihydrofolate reductase knockdown on endothelial EA.hy926 cell growth and the mechanism involved. We found that down-regulation of dihydrofolate reductase inhibited EA.hy926 cell proliferation, and induced G1 phase arrest. Meanwhile, the expression of regulators necessary for G1/S phase transition, such as cyclin-dependent kinases CDK2, CDK4 and CDK6, were remarkably down-regulated; by contrast, the cell cycle inhibitors p21(waf/cip1), p27(Kip1) and p53 were significantly up-regulated after dihydrofolate reductase knockdown. Furthermore, supplementation of 5-methyltetrahydrofolate to the dihydrofolate reductase knockdown cells could weaken the inhibitory effect of dihydrofolate reductase knockdown on cell proliferation, simultaneously, inducing the expression of p53 and p21(waf/cip1) falling back moderately. Our findings suggest that attenuating dihydrofolate reductase may cause imbalanced expression of cell cycle regulators, especially up-regulation of p53-p21(waf/cip1) pathway, leading to G1 cell cycle arrest, thereby inhibiting the growth of endothelial EA.hy926 cells.

  10. Gfer is a critical regulator of HSC proliferation.

    PubMed

    Sankar, Uma; Means, Anthony R

    2011-07-15

    Hematopoietic stem cells (HSC) are a relatively quiescent pool of cells that perform the arduous task of replacing the short-lived mature cells of the peripheral blood. While a rapid expansion of HSCs under periods of hematological stress is warranted, their enhanced proliferation during homeostasis leads to loss of function. We recently reported that in HSCs, the evolutionarily conserved growth factor erv1-like (Gfer) acts to counter jun activation domain-binding protein 1 (Jab1)-mediated nuclear export and destabilization of the cell cycle inhibitor, p27kip1, by directly binding to and sequestering the COP9 signalosome (CSN) subunit. Through this mechanism, Gfer promotes quiescence and maintains the functional integrity of HSCs. Here, we extend our study to demonstrate an association between Gfer and Ca2+/calmodulin-dependent protein kinase IV (CaMKIV) in the regulation of HSC proliferation. Highly proliferative and functionally deficient Camk4-/- HSCs possess significantly lower levels of Gfer and p27kip1. Ectopic expression of Gfer restores quiescence and elevates p27kip1 expression in Camk4-/- HSCs. These results further substantiate a critical role for Gfer in the restriction of unwarranted proliferation in HSCs through the inhibition of Jab1 and subsequent stabilization and nuclear retention of p27kip1. This Gfer-mediated pro-quiescence mechanism could be therapeutically exploited in the treatment of hematological malignancies associated with elevated Jab1 and reduced p27kip1.

  11. Gfer is a critical regulator of HSC proliferation

    PubMed Central

    Means, Anthony R

    2011-01-01

    Hematopoietic stem cells (HSC) are a relatively quiescent pool of cells that perform the arduous task of replacing the short-lived mature cells of the peripheral blood. While a rapid expansion of HSCs under periods of hematological stress is warranted, their enhanced proliferation during homeostasis leads to loss of function. We recently reported that in HSCs, the evolutionarily conserved growth factor erv1-like (Gfer) acts to counter jun activation domain-binding protein 1 (Jab1)-mediated nuclear export and destabilization of the cell cycle inhibitor, p27kip1, by directly binding to and sequestering the COP9 signalosome (CSN) subunit. Through this mechanism, Gfer promotes quiescence and maintains the functional integrity of HSCs. Here, we extend our study to demonstrate an association between Gfer and Ca2+/calmodulin-dependent protein kinase IV (CaMKIV) in the regulation of HSC proliferation. Highly proliferative and functionally deficient Camk4-/- HSCs possess significantly lower levels of Gfer and p27kip1. Ectopic expression of Gfer restores quiescence and elevates p27kip1 expression in Camk4-/- HSCs. These results further substantiate a critical role for Gfer in the restriction of unwarranted proliferation in HSCs through the inhibition of Jab1 and subsequent stabilization and nuclear retention of p27kip1. This Gfer-mediated pro-quiescence mechanism could be therapeutically exploited in the treatment of hematological malignancies associated with elevated Jab1 and reduced p27kip1. PMID:21636978

  12. Effects of apicidin, a histone deacetylase inhibitor, on the regulation of apoptosis in H-ras-transformed breast epithelial cells.

    PubMed

    Park, Hyeyoung; Im, Ji Young; Kim, Jeonga; Choi, Wahn Soo; Kim, Hyung Sik

    2008-03-01

    The cellular susceptibility of cancer cells to histone deacetylase (HDAC) inhibitors is increased by the etopic expression of oncogenic Ras. However, the ability of HDAC inhibitors to regulate the apoptotic pathway in human breast cancer cells is still not completely understood. In this study, the anti-proliferative effects of apicidin were compared in H-ras-transformed human breast epithelial (MCF10A-ras) and non-transformed epithelial (MCF10A) cells. MCF10A-ras cells showed a significantly higher growth rate than MCF10A cells. Apicidin significantly increased the levels of acetylated histone H3 and H4 in both cell lines. Western blot analysis and flow cytometry were used to determine if the anti-proliferative effects of apicidin in MCF10A and MCF10A-ras cells could be mediated by modulating the cell cycle. Apicidin attenuated the expression of cyclin E and CDK2 in MCF10A cells, decreased cyclin D1 and cyclin E levels in MCF10A-ras cells, and increased the levels of CDK inhibitors, p21WAF1/Cip1 and p27Kip1, in both cell lines. Notably, the levels of hyperphosphorylation of the Rb protein levels were lower in the MCF10A-ras cells after apicidin treatment. Studies on the regulation of apoptosis showed that apicidin induces the up-regulation of p53 and the downstream activation of ERK in MCF10A-ras cells. The up-regulation of p53 promoted Bax expression leading to activation of caspases-9 and -6, and eventually to apoptosis in MCF10A-ras cells. In addition, apicidin significantly increased the levels of ERK1/2 phosphorylation in MCF10A-ras cells. Therefore, the apicidin-mediated ERK pathway appears to play an important role in modulating the pro-apoptotic pathway in MCF10A-ras cells.

  13. The Role of Intrinsic Flexibility in Signal Transduction Mediated by the Cell Cycle Regulator, p27Kip1

    SciTech Connect

    Galea, Charles A.; Nourse, Amanda; Wang, Yuefeng; Sivakolundu, Sivashankar G.; Heller, William T; Kriwacki, Richard W

    2008-02-01

    p27{sup Kip1} (p27), which controls eukaryotic cell division through interactions with cyclin-dependent kinases (Cdks), integrates and transduces promitogenic signals from various nonreceptor tyrosine kinases by orchestrating its own phosphorylation, ubiquitination and degradation. Intrinsic flexibility allows p27 to act as a 'conduit' for sequential signaling mediated by tyrosine and threonine phosphorylation and ubiquitination. While the structural features of the Cdk/cyclin-binding domain of p27 are understood, how the C-terminal regulatory domain coordinates multistep signaling leading to p27 degradation is poorly understood. We show that the 100-residue p27 C-terminal domain is extended and flexible when p27 is bound to Cdk2/cyclin A. We propose that the intrinsic flexibility of p27 provides a molecular basis for the sequential signal transduction conduit that regulates p27 degradation and cell division. Other intrinsically unstructured proteins possessing multiple sites of posttranslational modification may participate in similar signaling conduits.

  14. A novel mutation in the upstream open reading frame of the CDKN1B gene causes a MEN4 phenotype.

    PubMed

    Occhi, Gianluca; Regazzo, Daniela; Trivellin, Giampaolo; Boaretto, Francesca; Ciato, Denis; Bobisse, Sara; Ferasin, Sergio; Cetani, Filomena; Pardi, Elena; Korbonits, Márta; Pellegata, Natalia S; Sidarovich, Viktoryia; Quattrone, Alessandro; Opocher, Giuseppe; Mantero, Franco; Scaroni, Carla

    2013-03-01

    The CDKN1B gene encodes the cyclin-dependent kinase inhibitor p27(KIP1), an atypical tumor suppressor playing a key role in cell cycle regulation, cell proliferation, and differentiation. Impaired p27(KIP1) expression and/or localization are often observed in tumor cells, further confirming its central role in regulating the cell cycle. Recently, germline mutations in CDKN1B have been associated with the inherited multiple endocrine neoplasia syndrome type 4, an autosomal dominant syndrome characterized by varying combinations of tumors affecting at least two endocrine organs. In this study we identified a 4-bp deletion in a highly conserved regulatory upstream ORF (uORF) in the 5'UTR of the CDKN1B gene in a patient with a pituitary adenoma and a well-differentiated pancreatic neoplasm. This deletion causes the shift of the uORF termination codon with the consequent lengthening of the uORF-encoded peptide and the drastic shortening of the intercistronic space. Our data on the immunohistochemical analysis of the patient's pancreatic lesion, functional studies based on dual-luciferase assays, site-directed mutagenesis, and on polysome profiling show a negative influence of this deletion on the translation reinitiation at the CDKN1B starting site, with a consequent reduction in p27(KIP1) expression. Our findings demonstrate that, in addition to the previously described mechanisms leading to reduced p27(KIP1) activity, such as degradation via the ubiquitin/proteasome pathway or non-covalent sequestration, p27(KIP1) activity can also be modulated by an uORF and mutations affecting uORF could change p27(KIP1) expression. This study adds the CDKN1B gene to the short list of genes for which mutations that either create, delete, or severely modify their regulatory uORFs have been associated with human diseases.

  15. A Novel Mutation in the Upstream Open Reading Frame of the CDKN1B Gene Causes a MEN4 Phenotype

    PubMed Central

    Occhi, Gianluca; Regazzo, Daniela; Trivellin, Giampaolo; Boaretto, Francesca; Ciato, Denis; Bobisse, Sara; Ferasin, Sergio; Cetani, Filomena; Pardi, Elena; Korbonits, Márta; Pellegata, Natalia S.; Sidarovich, Viktoryia; Quattrone, Alessandro; Opocher, Giuseppe; Mantero, Franco; Scaroni, Carla

    2013-01-01

    The CDKN1B gene encodes the cyclin-dependent kinase inhibitor p27KIP1, an atypical tumor suppressor playing a key role in cell cycle regulation, cell proliferation, and differentiation. Impaired p27KIP1 expression and/or localization are often observed in tumor cells, further confirming its central role in regulating the cell cycle. Recently, germline mutations in CDKN1B have been associated with the inherited multiple endocrine neoplasia syndrome type 4, an autosomal dominant syndrome characterized by varying combinations of tumors affecting at least two endocrine organs. In this study we identified a 4-bp deletion in a highly conserved regulatory upstream ORF (uORF) in the 5′UTR of the CDKN1B gene in a patient with a pituitary adenoma and a well-differentiated pancreatic neoplasm. This deletion causes the shift of the uORF termination codon with the consequent lengthening of the uORF–encoded peptide and the drastic shortening of the intercistronic space. Our data on the immunohistochemical analysis of the patient's pancreatic lesion, functional studies based on dual-luciferase assays, site-directed mutagenesis, and on polysome profiling show a negative influence of this deletion on the translation reinitiation at the CDKN1B starting site, with a consequent reduction in p27KIP1 expression. Our findings demonstrate that, in addition to the previously described mechanisms leading to reduced p27KIP1 activity, such as degradation via the ubiquitin/proteasome pathway or non-covalent sequestration, p27KIP1 activity can also be modulated by an uORF and mutations affecting uORF could change p27KIP1 expression. This study adds the CDKN1B gene to the short list of genes for which mutations that either create, delete, or severely modify their regulatory uORFs have been associated with human diseases. PMID:23555276

  16. Hair cell regeneration or the expression of related factors that regulate the fate specification of supporting cells in the cochlear ducts of embryonic and posthatch chickens.

    PubMed

    Jiang, Lingling; Jin, Ran; Xu, Jincao; Ji, Yubin; Zhang, Meiguang; Zhang, Xuebo; Zhang, Xinwen; Han, Zhongming; Zeng, Shaoju

    2016-02-01

    Hair cells in posthatch chickens regenerate spontaneously through mitosis or the transdifferentiation of supporting cells in response to antibiotic injury. However, how embryonic chicken cochleae respond to antibiotic treatment remains unknown. This study is the first to indicate that unlike hair cells in posthatch chickens, the auditory epithelium was free from antibiotic injury (25-250 mg gentamicin/kg) in embryonic chickens, although FITC-conjugated gentamicin actually reached embryonic hair cells. Next, we examined and counted the cells and performed labeling for BrdU, Sox2, Atoh1/Math1, PV or p27(kip1) (triple or double labeling) in the injured cochlea ducts after gentamicin treatment at 2 h (h), 15 h, 24 h, 2 days (d), 3 d and 7 d after BrdU treatment in posthatch chickens. Our results indicated that following gentamicin administration, proliferating cells (BrdU+) were labeled for Atoh1/Math1 in the damaged areas 3d after gentamicin administration, whereas hair cells (PV+) renewed through mitosis (BrdU+) or direct transdifferentiation (BrdU-) were evident only after 5 d of gentamicin administration. In addition, Sox2 expression was up-regulated in triggered supporting cells at an early stage of regeneration, but stopped at the advent of mature hair cells. Our study also indicated that p27(kip1) was expressed in both hair cells and supporting cells but was down-regulated in a subgroup of the supporting cells that gave rise to hair cells. These data and the obtained dynamic changes of the cells labeled for BrdU, Sox2, Atoh1/Math1, PV or p27(kip1) are useful for understanding supporting cell behaviors and their fate specification during hair cell regeneration.

  17. Altered expression of G1/S regulatory genes occurs early and frequently in lung carcinogenesis in transforming growth factor-beta1 heterozygous mice.

    PubMed

    Kang, Yang; Ozbun, Laurent L; Angdisen, Jerry; Moody, Terry W; Prentice, Margaret; Diwan, Bhalchandra A; Jakowlew, Sonia B

    2002-07-01

    We developed the AJBL6 transforming growth factor-beta 1 (TGF-beta1) heterozygous (HT) mouse by mating A/J mice with C57BL/6 TGF-beta1 HT mice that shows increased carcinogen-induced lung lesions with decreased latency to examine progressive events in lung tumorigenesis. Mouse cDNA macroarrays were used to identify cell cycle genes that are differentially regulated in ethyl carbamate-induced lung adenocarcinomas compared with normal lung tissue in AJBL6 TGF-beta1 HT mice using probes that were generated from tissues isolated using laser capture microdissection. While expression of the genes for cyclin D1, CDK4, and E2F1 increased in lung adenocarcinomas relative to normal lung, expression of p15(Ink4b), p16(Ink4a), p21(Cip1), p27(Kip1), p57(Kip2), and pRb genes decreased in comparison. Competitive RT-PCR showed that the levels of cyclin D1 and CDK4 mRNAs were 2- and 3-fold higher, respectively, in lung adenocarcinomas than in normal lung, while the mRNAs for p15(Ink4b), p16(Ink4a), p21(Cip1), p27(Kip1), and pRb were 3- to 4-fold lower in adenocarcinomas than in normal lung, thus validating the macroarray findings. Competitive RT-PCR of microdissected lesions also showed that the levels of cyclin D1 and CDK4 mRNAs increased significantly, while the mRNAs for p15(Ink4b) and p27(Kip1) decreased significantly as lung tumorigenesis progressed. Immunohistochemical staining for cyclin D1 and CDK4 showed staining in >80% of nuclei in adenocarcinomas compared with fewer than 20% of nuclei staining positively in normal lung. In contrast, while >60% of normal lung cells showed immunostaining for p15(Ink4b), p16(Ink4a), p21(Cip1), p27(Kip1), and pRb, staining for these proteins decreased in hyperplasias, adenomas, and adenocarcinomas. These data show that multiple components of the cyclin D1/CDK4/p16(Ink4a)/pRb signaling pathway are frequently altered early in lung lesions of AJBL6 TGF-beta1 HT mice that are induced by ethyl carbamate as a function of progressive lung

  18. Casticin induced apoptotic cell death and altered associated gene expression in human colon cancer colo 205 cells.

    PubMed

    Shang, Hung-Sheng; Liu, Jia-You; Lu, Hsu-Feng; Chiang, Han-Sun; Lin, Chia-Hain; Chen, Ann; Lin, Yuh-Feng; Chung, Jing-Gung

    2016-11-14

    Casticin, a polymethoxyflavone, derived from natural plant Fructus Viticis exhibits biological activities including anti-cancer characteristics. The anti-cancer and alter gene expression of casticin on human colon cancer cells and the underlying mechanisms were investigated. Flow cytometric assay was used to measure viable cell, cell cycle and sub-G1 phase, reactive oxygen species (ROS) and Ca(2+) productions, level of mitochondria membrane potential (ΔΨm ) and caspase activity. Western blotting assay was used to detect expression of protein level associated with cell death. Casticin induced cell morphological changes, decreased cell viability and induced G2/M phase arrest in colo 205 cells. Casticin increased ROS production but decreased the levels of ΔΨm , and Ca(2+) , increased caspase-3, -8, and -9 activities. The cDNA microarray indicated that some of the cell cycle associated genes were down-regulated such as cyclin-dependent kinase inhibitor 1A (CDKN1A) (p21, Cip1) and p21 protein (Cdc42/Rac)-activated kinase 3 (PAK3). TNF receptor-associated protein 1 (TRAP1), CREB1 (cAMP responsive element binding protein 1) and cyclin-dependent kinase inhibitor 1B (CDKN1B) (p27, Kip1) genes were increased but matrix metallopeptidase 2 (MMP-2), toll-like receptor 4 (TLR4), PRKAR2B (protein kinase, cAMP-dependent, regulatory, type II, bet), and CaMK4 (calcium/calmodulin-dependent protein kinase IV) genes were inhibited. Results suggest that casticin induced cell apoptosis via the activation of the caspase- and/or mitochondria-dependent signaling cascade, the accumulation of ROS and altered associated gene expressions in colo 205 human colon cancer cells.

  19. 19-nor vitamin-D analogs: a new class of potent inhibitors of proliferation and inducers of differentiation of human myeloid leukemia cell lines.

    PubMed

    Asou, H; Koike, M; Elstner, E; Cambell, M; Le, J; Uskokovic, M R; Kamada, N; Koeffler, H P

    1998-10-01

    We have studied the in vitro biological activities and mechanisms of action of 1,25-dihydroxyvitamin D3 (1,25D3) and nine potent 1,25D3 analogs on proliferation and differentiation of myeloid leukemia cell lines (HL-60, retinoic acid-resistant HL-60 [RA-res HL-60], NB4 and Kasumi-1). The common novel structural motiff for almost all the analogs included removal of C-19 (19-nor); each also had unsaturation of the side chain. All the compounds were potent; for example, the concentration of analogs producing a 50% clonal inhibition (ED50) ranged between 1 x 10(-9) to 4 x 10(-11) mol/L when using the HL-60 cell line. The most active compound [1, 25(OH)2-16,23E-diene-26-trifluoro-19-nor-cholecalciferol (Ro 25-9716)] had an ED50 of 4 x 10(-11) mol/L; in contrast, the 1,25D3 produced an ED50 of 10(-9) mol/L with the HL-60 target cells. Ro 25-9716 (10(-9) mol/L, 3 days) was a strong inducer of myeloid differentiation because it caused 92% of the HL-60 cells to express CD11b and 75% of these cells to reduce nitroblue tetrazolium (NBT). This compound (10(-8) mol/L, 4 days) also caused HL-60 cells to arrest in the G1 phase of the cell cycle (88% cells in G1 v 48% of the untreated control cells). The p27(kip-1), a cyclin-dependent kinase inhibitor which is important in blocking the cell cycle, was induced more quickly and potently by Ro 25-9716 (10(-7) mol/L, 0 to 5 days) than by 1,25D3, suggesting a possible mechanism by which these analogs inhibit proliferation of leukemic growth. The NB4 promyelocytic leukemia cells cultured with the Ro 25-9716 were also inhibited in their clonal proliferation (ED50, 5 x 10(-11) mol/L) and their expression of CD11b was enhanced (80% positive [10(-9) mol/L, 4 days] v 27% untreated NB4 cells). Moreover, the combination of Ro 25-9716 (10(-9) mol/L) and all-trans retinoic acid (ATRA, 10(-7) mol/L) induced 92% of the NB4 cells to reduce NBT, whereas only 26% of the cells became NBT positive after a similar exposure to the combination of 1,25D3

  20. Placental estrogen suppresses cyclin D1 expression in the nonhuman primate fetal adrenal cortex.

    PubMed

    Dumitrescu, Adina; Aberdeen, Graham W; Pepe, Gerald J; Albrecht, Eugene D

    2014-12-01

    We have previously shown that estrogen selectively suppresses growth of the fetal zone of the baboon fetal adrenal cortex, which produces the C19-steroid precursors, eg, dehydroepiandrosterone sulfate, which are aromatized to estrogen within the placenta. In the present study, we determined whether fetal adrenal expression of cell cycle regulators are altered by estrogen and thus provide a mechanism by which estrogen regulates fetal adrenocortical development. Cyclin D1 mRNA levels in the whole fetal adrenal were increased 50% (P < .05), and the number of cells in the fetal adrenal definitive zone expressing cyclin D1 protein was increased 2.5-fold (P < .05), whereas the total number of cells in the fetal zone and fetal serum dehydroepiandrosterone sulfate levels were elevated 2-fold (P < .05) near term in baboons in which fetal serum estradiol levels were decreased by 95% (P < .05) after maternal administration of the aromatase inhibitor letrozole and restored to normal by concomitant administration of letrozole plus estradiol throughout second half of gestation. However, fetal adrenocortical expression of cyclin D2, the cyclin-dependent kinase (Cdk)-2, Cdk4, and Cdk6, and Cdk regulatory proteins p27(Kip1) and p57(Kip2) were not changed by letrozole or letrozole plus estradiol administration. We suggest that estrogen controls the growth of the fetal zone of the fetal adrenal by down-regulating cyclin D1 expression and thus proliferation of progenitor cells within the definitive zone that migrate to the fetal zone. We propose that estrogen restrains growth and function of the fetal zone via cyclin D1 to maintain estrogen levels in a physiological range during primate pregnancy.

  1. Placental Estrogen Suppresses Cyclin D1 Expression in the Nonhuman Primate Fetal Adrenal Cortex*

    PubMed Central

    Dumitrescu, Adina; Aberdeen, Graham W.; Pepe, Gerald J.

    2014-01-01

    We have previously shown that estrogen selectively suppresses growth of the fetal zone of the baboon fetal adrenal cortex, which produces the C19-steroid precursors, eg, dehydroepiandrosterone sulfate, which are aromatized to estrogen within the placenta. In the present study, we determined whether fetal adrenal expression of cell cycle regulators are altered by estrogen and thus provide a mechanism by which estrogen regulates fetal adrenocortical development. Cyclin D1 mRNA levels in the whole fetal adrenal were increased 50% (P < .05), and the number of cells in the fetal adrenal definitive zone expressing cyclin D1 protein was increased 2.5-fold (P < .05), whereas the total number of cells in the fetal zone and fetal serum dehydroepiandrosterone sulfate levels were elevated 2-fold (P < .05) near term in baboons in which fetal serum estradiol levels were decreased by 95% (P < .05) after maternal administration of the aromatase inhibitor letrozole and restored to normal by concomitant administration of letrozole plus estradiol throughout second half of gestation. However, fetal adrenocortical expression of cyclin D2, the cyclin-dependent kinase (Cdk)-2, Cdk4, and Cdk6, and Cdk regulatory proteins p27Kip1 and p57Kip2 were not changed by letrozole or letrozole plus estradiol administration. We suggest that estrogen controls the growth of the fetal zone of the fetal adrenal by down-regulating cyclin D1 expression and thus proliferation of progenitor cells within the definitive zone that migrate to the fetal zone. We propose that estrogen restrains growth and function of the fetal zone via cyclin D1 to maintain estrogen levels in a physiological range during primate pregnancy. PMID:25247468

  2. Cell Cycle Regulatory Proteins p27(kip), Cyclins Dl and E and Proliferative Activity in Oncocytic (Hurthle Cell) Lesions of the Thyroid.

    PubMed

    Maynes, Lincoln J.; Hutzler, Michael J.; Patwardhan, Nilima A.; Wang, Songtao; Khan, Ashraf

    2000-01-01

    Cyclins are prime cell-cycle regulators central to the control of cell proliferation in eukaryotic cells. The formation of cyclin/cyclin-dependent kinases (CDK) complexes activates the kinases and initiates a cascade of events, which directs cells through the cell cycle. CDK inhibitors (CDKIs) such as p27(kip1) inhibit cyclln-CDK complexes and function as negative regulators of the cell cycle. Previous studies have shown that p27(kip1) is decreased In malignant relative to benign thyroid tumors, but its role and Interaction with other cell cycle regulatory proteins have not been well established In oncocytic lesions of the thyroid. We studied the expression of p27(kip1), cyclins D1 and E, and Ki67 In 20 cases of oncocytic adenoma (AD). 6 cases of oncocytic carcinoma (CA). 8 cases of Hashimoto's thyroiditis (HT). and 9 cases of nodular goiter with oncocytic change (NG) by Immunohistochemlstry. In the latter two lesions only oncocytic cells were evaluated. The positive staining was stratified Into four groups. Statistical analysis was done using the Kruslcal-Wallis one-way analysis of variance test, and, when significant the Dunn multiple-comparisons procedure was used to determine pairwise differences. AllI 20 AD were p27(kip1) posItive, 10 were 4+, 2 were 3+, and the remaining 8 were 1+. In contrast all 6 CA showed 4+ p27(kip1) staining, of the 8 HT 2 were 4+, two 3+, three1+, and I was negative.All 9 NG were p27 positive, 7 showed 4+, one 3+, and one 1+ staining. On pairwise comparison differences in p27(kip1) staining between AD and CA and between HT and CA were statistically significant (p=0.0243 and p=0.0142, respectively). In all but one case Ki67 expression was either very low (<3%) or negative. No significant differences were seen in the expression of cyclin D1 or cyclin E among the groups observed. In conclusion, the increased p27(kip1) expression in malignant oncocytlc tumors relative to benign oncocytic lesions is unlike any other malignant progression

  3. ARTD1 regulates cyclin E expression and consequently cell-cycle re-entry and G1/S progression in T24 bladder carcinoma cells.

    PubMed

    Léger, Karolin; Hopp, Ann-Katrin; Fey, Monika; Hottiger, Michael O

    2016-08-02

    ADP-ribosylation is involved in a variety of biological processes, many of which are chromatin-dependent and linked to important functions during the cell cycle. However, any study on ADP-ribosylation and the cell cycle faces the problem that synchronization with chemical agents or by serum starvation and subsequent growth factor addition already activates ADP-ribosylation by itself. Here, we investigated the functional contribution of ARTD1 in cell cycle re-entry and G1/S cell cycle progression using T24 urinary bladder carcinoma cells, which synchronously re-enter the cell cycle after splitting without any additional stimuli. In synchronized cells, ARTD1 knockdown, but not inhibition of its enzymatic activity, caused specific down-regulation of cyclin E during cell cycle re-entry and G1/S progression through alterations of the chromatin composition and histone acetylation, but not of other E2F-1 target genes. Although Cdk2 formed a functional complex with the residual cyclin E, p27(Kip 1) protein levels increased in G1 upon ARTD1 knockdown most likely due to inappropriate cyclin E-Cdk2-induced phosphorylation-dependent degradation, leading to decelerated G1/S progression. These results provide evidence that ARTD1 regulates cell cycle re-entry and G1/S progression via cyclin E expression and p27(Kip 1) stability independently of its enzymatic activity, uncovering a novel cell cycle regulatory mechanism.

  4. Aberrant expression of cyclin D1 in cancer

    PubMed Central

    Inoue, Kazushi; Fry, Elizabeth A.

    2016-01-01

    Cyclin D1 binds and activates cyclin-dependent kinases 4/6 (Cdk4/6) to phosphorylate the retinoblastoma (RB) family proteins, relieving E2F/DPs from the negative restraint of RB proteins and histone deacetylases. The cyclin D-Cdk4/6 complexes activate cyclin E/Cdk2 through titration of the Cdk inhibitors p21Cip1/p27Kip1. Cyclin E/Cdk2 further phosphorylates RBs, thereby activating E2F/DPs, and cells enter the S phase of the cell cycle. Cyclin D-Cdk4/6 also phosphorylates MEP50 subunit of the protein arginine methyltransferase 5 (PRMT5), which cooperates with cyclin D1 to drive lymphomagenesis in vivo. Activated PRMPT5 causes arginine methylation of p53 to suppress expression of pro-apoptotic and anti-proliferative target genes, explaining the molecular mechanism for tumorigenesis. Cyclin D1 physically interacts with transcription factors such as estrogen receptor, androgen receptor, and Myb family proteins to regulate gene expression in Cdk-independent fashion. Dmp1 is a Myb-like protein that quenches the oncogenic signals from activated Ras or HER2 by inducing Arf/p53-dependent cell cycle arrest. Cyclin D1 binds to Dmp1α to activate both Arf and Ink4a promoters to induce cell cycle arrest or apoptosis in non-transformed cells to prevent them from neoplastic transformation. Dmp1-deficiency significantly accelerates mouse mammary tumorigenesis with reduced apoptosis and increased metastasis. Cyclin D1 interferes with ligand activation of PPARγ involved in cellular differentiation; it also physically interacts with histone deacetylases (HDACs) and p300 to repress gene expression. It has also been shown that cyclin D1 accelerates tumorigenesis through transcriptional activation of miR-17/20 and Dicer1 which, in turn, represses cyclin D1 expression. Identification of cyclin D1-binding proteins/promoters will be essential for further clarification of its biological activities. PMID:28090171

  5. 1α,25 dihydroxi-vitamin D₃ modulates CDK4 and CDK6 expression and localization.

    PubMed

    Irazoqui, Ana P; Heim, Nadia B; Boland, Ricardo L; Buitrago, Claudia G

    2015-03-27

    We recently reported that the vitamin D receptor (VDR) and p38 MAPK participate in pro-differentiation events triggered by 1α,25(OH)₂-vitamin D₃ [1,25D] in skeletal muscle cells. Specifically, our studies demonstrated that 1,25D promotes G0/G1 arrest of cells inducing cyclin D3 and cyclin dependent kinases inhibitors (CKIs) p21(Waf1/Cip1) and p27(Kip1) expression in a VDR and p38 MAPK dependent manner. In this work we present data indicating that cyclin-dependent kinases (CDKs) 4 and 6 also play a role in the mechanism by which 1,25D stimulates myogenesis. To investigate VDR involvement in hormone regulation of CDKs 4 and 6, we significantly reduced its expression by the use of a shRNA against mouse VDR, generating the skeletal muscle cell line C2C12-VDR. Investigation of changes in cellular cycle regulating proteins by immunoblotting showed that the VDR is involved in the 1,25D -induced CDKs 4 and 6 protein levels at 6 h of hormone treatment. CDK4 levels remains high during S phase peak and G0/G1 arrest while CDK6 expression decreases at 12 h and increases again al 24 h. The up-regulation of CDKs 4 and 6 by 1,25D (6 h) was abolished in C2C12 cells pre-treated with the ERK1/2 inhibitor, UO126. Moreover, CDKs 4 and 6 expression induced by the hormone nor was detected when α and β isoforms of p38 MAPK were inhibited by compound SB203580. Confocal images show that there is not co-localization between VDR and CDKs at 6 h of hormone treatment, however CDK4 and VDR co-localizates in nucleus after 12 h of 1,25D exposure. Of relevance, at this time 1,25D promotes CDK6 localization in a peri-nuclear ring. Our data demonstrate that the VDR, ERK1/2 and p38 MAPK are involved in the control of CDKs 4 and 6 by 1,25D in skeletal muscle cells sustaining the operation of a VDR and MAPKs -dependent mechanism in hormone modulation of myogenesis.

  6. Fibroblast growth factor signaling regulates Dach1 expression during skeletal development.

    PubMed

    Horner, A; Shum, L; Ayres, J A; Nonaka, K; Nuckolls, G H

    2002-09-01

    Dach1 is a mouse homologue of the Drosophila dachshund gene, which is a key regulator of cell fate determination during eye, leg, and brain development in the fly. We have investigated the expression and growth factor regulation of Dach1 during pre- and postnatal skeletal development in the mouse limb to understand better the function of Dach1. Dach1 was expressed in the distal mesenchyme of the early embryonic mouse limb bud and subsequently became restricted to the tips of digital cartilages. Dach1 protein was localized to postmitotic, prehypertrophic, and early hypertrophic chondrocytes during the initiation of ossification centers, but Dach1 was not expressed in growth plates that exhibited extensive ossification. Dach1 colocalized with Runx2/Cbfa1 in chondrocytes but not in the forming bone collar or primary spongiosa. Dach1 also colocalized with cyclin-dependent kinase inhibitors p27 (Kip1) and p57 (Kip2) in chondrocytes of the growth plate and in the epiphysis before the formation of the secondary ossification center. Because fibroblast growth factors (FGF), bone morphogenetic proteins (BMP), and hedgehog molecules (Hh) regulate skeletal patterning of the limb bud and chondrocyte maturation in developing endochondral bones, we investigated the regulation of Dach1 by these growth and differentiation factors. Expression of Dach1 in 11 days postcoitus mouse limb buds in organ culture was up-regulated by implanting beads soaked in FGF1, 2, 8, or 9 but not FGF10. BMP4-soaked beads down-regulated Dach1 expression, whereas Shh and bovine serum albumin had no effect. Furthermore, FGF4 or 8 could substitute for the apical ectodermal ridge in maintaining Dach1 expression in the limb buds. Immunolocalization of FGFR2 and FGFR3 revealed overlap with Dach1 expression during skeletal patterning and chondrocyte maturation. We conclude that Dach1 is a target gene of FGF signaling during limb skeletal development, and Dach1 may function as an intermediary in the FGF

  7. Danusertib, a potent pan-Aurora kinase and ABL kinase inhibitor, induces cell cycle arrest and programmed cell death and inhibits epithelial to mesenchymal transition involving the PI3K/Akt/mTOR-mediated signaling pathway in human gastric cancer AGS and NCI-N78 cells.

    PubMed

    Yuan, Chun-Xiu; Zhou, Zhi-Wei; Yang, Yin-Xue; He, Zhi-Xu; Zhang, Xueji; Wang, Dong; Yang, Tianxing; Pan, Si-Yuan; Chen, Xiao-Wu; Zhou, Shu-Feng

    2015-01-01

    Gastric cancer is the second leading cause of cancer-related death worldwide, with a poor response to current chemotherapy. Danusertib is a pan-inhibitor of the Aurora kinases and a third-generation Bcr-Abl tyrosine kinase inhibitor with potent anticancer effects, but its antitumor effect and underlying mechanisms in the treatment of human gastric cancer are unknown. This study aimed to investigate the effects of danusertib on cell growth, apoptosis, autophagy, and epithelial to mesenchymal transition and the molecular mechanisms involved in human gastric cancer AGS and NCI-N78 cells. The results showed that danusertib had potent growth-inhibitory, apoptosis-inducing, and autophagy-inducing effects on AGS and NCI-N78 cells. Danusertib arrested AGS and NCI-N78 cells in G2/M phase, with downregulation of expression of cyclin B1 and cyclin-dependent kinase 1 and upregulation of expression of p21 Waf1/Cip1, p27 Kip1, and p53. Danusertib induced mitochondria-mediated apoptosis, with an increase in expression of proapoptotic protein and a decrease in antiapoptotic proteins in both cell lines. Danusertib induced release of cytochrome c from the mitochondria to the cytosol and triggered activation of caspase 9 and caspase 3 in AGS and NCI-N78 cells. Further, danusertib induced autophagy, with an increase in expression of beclin 1 and conversion of microtubule-associated protein 1A/1B-light chain 3 (LC3-I) to LC3-II in both cell lines. Inhibition of phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt)/mammalian target of rapamycin (mTOR) and p38 mitogen-activated protein kinase pathways as well as activation of 5' AMP-activated protein kinase contributed to the proautophagic effect of danusertib in AGS and NCI-N78 cells. SB202191 and wortmannin enhanced the autophagy-inducing effect of danusertib in AGS and NCI-N78 cells. In addition, danusertib inhibited epithelial to mesenchymal transition with an increase in expression of E-cadherin and a decrease in expression of

  8. Danusertib, a potent pan-Aurora kinase and ABL kinase inhibitor, induces cell cycle arrest and programmed cell death and inhibits epithelial to mesenchymal transition involving the PI3K/Akt/mTOR-mediated signaling pathway in human gastric cancer AGS and NCI-N78 cells

    PubMed Central

    Yuan, Chun-Xiu; Zhou, Zhi-Wei; Yang, Yin-Xue; He, Zhi-Xu; Zhang, Xueji; Wang, Dong; Yang, Tianxing; Pan, Si-Yuan; Chen, Xiao-Wu; Zhou, Shu-Feng

    2015-01-01

    Gastric cancer is the second leading cause of cancer-related death worldwide, with a poor response to current chemotherapy. Danusertib is a pan-inhibitor of the Aurora kinases and a third-generation Bcr-Abl tyrosine kinase inhibitor with potent anticancer effects, but its antitumor effect and underlying mechanisms in the treatment of human gastric cancer are unknown. This study aimed to investigate the effects of danusertib on cell growth, apoptosis, autophagy, and epithelial to mesenchymal transition and the molecular mechanisms involved in human gastric cancer AGS and NCI-N78 cells. The results showed that danusertib had potent growth-inhibitory, apoptosis-inducing, and autophagy-inducing effects on AGS and NCI-N78 cells. Danusertib arrested AGS and NCI-N78 cells in G2/M phase, with downregulation of expression of cyclin B1 and cyclin-dependent kinase 1 and upregulation of expression of p21 Waf1/Cip1, p27 Kip1, and p53. Danusertib induced mitochondria-mediated apoptosis, with an increase in expression of proapoptotic protein and a decrease in antiapoptotic proteins in both cell lines. Danusertib induced release of cytochrome c from the mitochondria to the cytosol and triggered activation of caspase 9 and caspase 3 in AGS and NCI-N78 cells. Further, danusertib induced autophagy, with an increase in expression of beclin 1 and conversion of microtubule-associated protein 1A/1B-light chain 3 (LC3-I) to LC3-II in both cell lines. Inhibition of phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt)/mammalian target of rapamycin (mTOR) and p38 mitogen-activated protein kinase pathways as well as activation of 5′ AMP-activated protein kinase contributed to the proautophagic effect of danusertib in AGS and NCI-N78 cells. SB202191 and wortmannin enhanced the autophagy-inducing effect of danusertib in AGS and NCI-N78 cells. In addition, danusertib inhibited epithelial to mesenchymal transition with an increase in expression of E-cadherin and a decrease in expression

  9. The investigational Aurora kinase A inhibitor alisertib (MLN8237) induces cell cycle G2/M arrest, apoptosis, and autophagy via p38 MAPK and Akt/mTOR signaling pathways in human breast cancer cells

    PubMed Central

    Li, Jin-Ping; Yang, Yin-Xue; Liu, Qi-Lun; Pan, Shu-Ting; He, Zhi-Xu; Zhang, Xueji; Yang, Tianxin; Chen, Xiao-Wu; Wang, Dong; Qiu, Jia-Xuan; Zhou, Shu-Feng

    2015-01-01

    Alisertib (ALS) is an investigational potent Aurora A kinase inhibitor currently undergoing clinical trials for the treatment of hematological and non-hematological malignancies. However, its antitumor activity has not been tested in human breast cancer. This study aimed to investigate the effect of ALS on the growth, apoptosis, and autophagy, and the underlying mechanisms in human breast cancer MCF7 and MDA-MB-231 cells. In the current study, we identified that ALS had potent growth-inhibitory, pro-apoptotic, and pro-autophagic effects in MCF7 and MDA-MB-231 cells. ALS arrested the cells in G2/M phase in MCF7 and MDA-MB-231 cells which was accompanied by the downregulation of cyclin-dependent kinase (CDK)1/cell division cycle (CDC) 2, CDK2, and cyclin B1 and upregulation of p21 Waf1/Cip1, p27 Kip1, and p53, suggesting that ALS induces G2/M arrest through modulation of p53/p21/CDC2/cyclin B1 pathways. ALS induced mitochondria-mediated apoptosis in MCF7 and MDA-MB-231 cells; ALS significantly decreased the expression of B-cell lymphoma 2 (Bcl-2), but increased the expression of B-cell lymphoma 2-associated X protein (Bax) and p53-upregulated modulator of apoptosis (PUMA), and increased the expression of cleaved caspases 3 and 9. ALS significantly increased the expression level of membrane-bound microtubule-associated protein 1 light chain 3 (LC3)-II and beclin 1 and induced inhibition of phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt)/mammalian target of rapamycin (mTOR) and p38 mitogen-activated protein kinase (MAPK) pathways in MCF7 and MDA-MB-231 cells as indicated by their altered phosphorylation, contributing to the pro-autophagic activities of ALS. Furthermore, treatment with wortmannin markedly downregulated ALS-induced p38 MAPK activation and LC3 conversion. In addition, knockdown of the p38 MAPK gene by ribonucleic acid interference upregulated Akt activation and resulted in LC3-II accumulation. These findings indicate that ALS promotes cellular

  10. The investigational Aurora kinase A inhibitor alisertib (MLN8237) induces cell cycle G2/M arrest, apoptosis, and autophagy via p38 MAPK and Akt/mTOR signaling pathways in human breast cancer cells.

    PubMed

    Li, Jin-Ping; Yang, Yin-Xue; Liu, Qi-Lun; Pan, Shu-Ting; He, Zhi-Xu; Zhang, Xueji; Yang, Tianxin; Chen, Xiao-Wu; Wang, Dong; Qiu, Jia-Xuan; Zhou, Shu-Feng

    2015-01-01

    Alisertib (ALS) is an investigational potent Aurora A kinase inhibitor currently undergoing clinical trials for the treatment of hematological and non-hematological malignancies. However, its antitumor activity has not been tested in human breast cancer. This study aimed to investigate the effect of ALS on the growth, apoptosis, and autophagy, and the underlying mechanisms in human breast cancer MCF7 and MDA-MB-231 cells. In the current study, we identified that ALS had potent growth-inhibitory, pro-apoptotic, and pro-autophagic effects in MCF7 and MDA-MB-231 cells. ALS arrested the cells in G2/M phase in MCF7 and MDA-MB-231 cells which was accompanied by the downregulation of cyclin-dependent kinase (CDK)1/cell division cycle (CDC) 2, CDK2, and cyclin B1 and upregulation of p21 Waf1/Cip1, p27 Kip1, and p53, suggesting that ALS induces G2/M arrest through modulation of p53/p21/CDC2/cyclin B1 pathways. ALS induced mitochondria-mediated apoptosis in MCF7 and MDA-MB-231 cells; ALS significantly decreased the expression of B-cell lymphoma 2 (Bcl-2), but increased the expression of B-cell lymphoma 2-associated X protein (Bax) and p53-upregulated modulator of apoptosis (PUMA), and increased the expression of cleaved caspases 3 and 9. ALS significantly increased the expression level of membrane-bound microtubule-associated protein 1 light chain 3 (LC3)-II and beclin 1 and induced inhibition of phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt)/mammalian target of rapamycin (mTOR) and p38 mitogen-activated protein kinase (MAPK) pathways in MCF7 and MDA-MB-231 cells as indicated by their altered phosphorylation, contributing to the pro-autophagic activities of ALS. Furthermore, treatment with wortmannin markedly downregulated ALS-induced p38 MAPK activation and LC3 conversion. In addition, knockdown of the p38 MAPK gene by ribonucleic acid interference upregulated Akt activation and resulted in LC3-II accumulation. These findings indicate that ALS promotes cellular

  11. CXCR4-independent rescue of the myeloproliferative defect of the Gata1low myelofibrosis mouse model by Aplidin.

    PubMed

    Verrucci, Maria; Pancrazzi, Alessandro; Aracil, Miguel; Martelli, Fabrizio; Guglielmelli, Paola; Zingariello, Maria; Ghinassi, Barbara; D'Amore, Emanuela; Jimeno, José; Vannucchi, Alessandro M; Migliaccio, Anna Rita

    2010-11-01

    The discovery of JAK2 mutations in Philadelphia-negative myeloproliferative neoplasms has prompted investigators to evaluate mutation-targeted treatments to restore hematopoietic cell functions in these diseases. However, the results of the first clinical trials with JAK2 inhibitors are not as promising as expected, prompting a search for additional drugable targets to treat these disorders. In this paper, we used the hypomorphic Gata1(low) mouse model of primary myelofibrosis (PMF), the most severe of these neoplasms, to test the hypothesis that defective marrow hemopoiesis and development of extramedullary hematopoiesis in myelofibrosis is due to insufficient p27(Kip1) activity and is treatable by Aplidin, a cyclic depsipeptide that activates p27(Kip1) in several cancer cells. Aplidin restored expression of Gata1 and p27(Kip1) in Gata1(low) hematopoietic cells, proliferation of marrow progenitor cells in vitro and maturation of megakaryocytes in vivo (reducing TGF-beta/VEGF levels released in the microenvironment by immature Gata1(low) megakaryocytes). Microvessel density, fibrosis, bone growth, and marrow cellularity were normal in Aplidin-treated mice and extramedullary hematopoiesis did not develop in liver although CXCR4 expression in Gata1(low) progenitor cells remained low. These results indicate that Aplidin effectively alters the natural history of myelofibrosis in Gata1(low) mice and suggest this drug as candidate for clinical evaluation in PMF.

  12. CacyBP/SIP promotes the proliferation of colon cancer cells.

    PubMed

    Zhai, Huihong; Shi, Yongquan; Chen, Xiong; Wang, Jun; Lu, Yuanyuan; Zhang, Faming; Liu, Zhengxiong; Lei, Ting; Fan, Daiming

    2017-01-01

    CacyBP/SIP is a component of the ubiquitin pathway and is overexpressed in several transformed tumor tissues, including colon cancer, which is one of the most common cancers worldwide. It is unknown whether CacyBP/SIP promotes the proliferation of colon cancer cells. This study examined the expression level, subcellular localization, and binding activity of CacyBP/SIP in human colon cancer cells in the presence and absence of the hormone gastrin. We found that CacyBP/SIP was expressed in a high percentage of colon cancer cells, but not in normal colonic surface epithelium. CacyBP/SIP promoted the cell proliferation of colon cancer cells under both basal and gastrin stimulated conditions as shown by knockdown studies. Gastrin stimulation triggered the translocation of CacyBP/SIP to the nucleus, and enhanced interaction between CacyBP/SIP and SKP1, a key component of ubiquitination pathway which further mediated the proteasome-dependent degradation of p27kip1 protein. The gastrin induced reduction in p27kip1 was prevented when cells were treated with the proteasome inhibitor MG132. These results suggest that CacyBP/SIP may be promoting growth of colon cancer cells by enhancing ubiquitin-mediated degradation of p27kip1.

  13. CacyBP/SIP promotes the proliferation of colon cancer cells

    PubMed Central

    Chen, Xiong; Wang, Jun; Lu, Yuanyuan; Zhang, Faming; Liu, Zhengxiong; Lei, Ting; Fan, Daiming

    2017-01-01

    CacyBP/SIP is a component of the ubiquitin pathway and is overexpressed in several transformed tumor tissues, including colon cancer, which is one of the most common cancers worldwide. It is unknown whether CacyBP/SIP promotes the proliferation of colon cancer cells. This study examined the expression level, subcellular localization, and binding activity of CacyBP/SIP in human colon cancer cells in the presence and absence of the hormone gastrin. We found that CacyBP/SIP was expressed in a high percentage of colon cancer cells, but not in normal colonic surface epithelium. CacyBP/SIP promoted the cell proliferation of colon cancer cells under both basal and gastrin stimulated conditions as shown by knockdown studies. Gastrin stimulation triggered the translocation of CacyBP/SIP to the nucleus, and enhanced interaction between CacyBP/SIP and SKP1, a key component of ubiquitination pathway which further mediated the proteasome-dependent degradation of p27kip1 protein. The gastrin induced reduction in p27kip1 was prevented when cells were treated with the proteasome inhibitor MG132. These results suggest that CacyBP/SIP may be promoting growth of colon cancer cells by enhancing ubiquitin-mediated degradation of p27kip1. PMID:28196083

  14. 2′,4′-dihydroxychalcone, a flavonoid isolated from Herba oxytropis, suppresses PC-3 human prostate cancer cell growth by induction of apoptosis

    PubMed Central

    SHENG, YUQING; ZOU, MINGCHANG; WANG, YAN; LI, QIHENG

    2015-01-01

    Natural products are a promising source for the development of novel cancer therapies, due to their potential effectiveness and low toxicity profiles. As a main component of Herba oxytropis, 2′,4′-dihydroxychalcone (TFC) is known to demonstrate anti-tumor activity in vitro. In the present study, TFC was found to potently inhibit proliferation and induce apoptosis in PC-3 human prostate cancer cells in a dose-dependent manner. The results demonstrated that the induction of apoptosis is associated with cell cycle arrest at the G0/G1 phase and activation of caspase-3/-7. Additional mechanistic studies of two biomarkers, phosphatase and tensin homolog (PTEN) and cyclin-dependent kinase inhibitor 1B (p27Kip1), in prostate cancer revealed that TFC treatment significantly upregulated the expression of PTEN and p27Kip1. The findings of the present study indicate that TFC-induced apoptosis in PC-3 cells via upregulation of PTEN and p27Kip1, which results in cell cycle arrest in G0/G1 phase, activation of caspase-3/-7 and induction of apoptosis. Therefore, TFC may be a potential compound for human prostate cancer therapy. PMID:26788200

  15. Proteolysis of Xenopus Cip-type CDK inhibitor, p16Xic2, is regulated by PCNA binding and CDK2 phosphorylation

    PubMed Central

    2013-01-01

    Background Cell division is positively regulated by cyclin-dependent kinases (CDKs) partnered with cyclins and negatively regulated by CDK inhibitors. In the frog, Xenopus laevis, three types of CDK inhibitors have been described: p27Xic1 (Xic1) which shares sequence homology with both p21Cip1 and p27Kip1 from mammals, p16Xic2 (Xic2) which shares sequence homology with p21Cip1, and p17Xic3 (Xic3) which shares sequence homology with p27Kip1. While past studies have demonstrated that during DNA polymerase switching, Xic1 is targeted for protein turnover dependent upon DNA, Proliferating Cell Nuclear Antigen (PCNA), and the ubiquitin ligase CRL4Cdt2, little is known about the processes that regulate Xic2 or Xic3. Methods We used the Xenopus interphase egg extract as a model system to examine the regulation of Xic2 by proteolysis and phosphorylation. Results Our studies indicated that following primer synthesis during the initiation of DNA replication, Xic2 is targeted for DNA- and PCNA-dependent ubiquitin-mediated proteolysis and that Cdt2 can promote Xic2 turnover. Additionally, during interphase, Xic2 is phosphorylated by CDK2 at Ser-98 and Ser-131 in a DNA-independent manner, inhibiting Xic2 turnover. In the presence of double-stranded DNA ends, Xic2 is also phosphorylated at Ser-78 and Ser-81 by a caffeine-sensitive kinase, but this phosphorylation does not alter Xic2 turnover. Conversely, in the presence or absence of DNA, Xic3 was stable in the Xenopus interphase egg extract and did not exhibit a shift indicative of phosphorylation. Conclusions During interphase, Xic2 is targeted for DNA- and PCNA-dependent proteolysis that is negatively regulated by CDK2 phosphorylation. During a response to DNA damage, Xic2 may be alternatively regulated by phosphorylation by a caffeine-sensitive kinase. Our studies suggest that the three types of Xenopus CDK inhibitors, Xic1, Xic2, and Xic3 appear to be uniquely regulated which may reflect their specialized roles during cell

  16. The pan-inhibitor of Aurora kinases danusertib induces apoptosis and autophagy and suppresses epithelial-to-mesenchymal transition in human breast cancer cells.

    PubMed

    Li, Jin-Ping; Yang, Yin-Xue; Liu, Qi-Lun; Zhou, Zhi-Wei; Pan, Shu-Ting; He, Zhi-Xu; Zhang, Xueji; Yang, Tianxin; Pan, Si-Yuan; Duan, Wei; He, Shu-Ming; Chen, Xiao-Wu; Qiu, Jia-Xuan; Zhou, Shu-Feng

    2015-01-01

    Danusertib (Danu) is a pan-inhibitor of Aurora kinases and a third-generation breakpoint cluster region-Abelson murine leukemia viral oncogene homolog 1 (Bcr-Abl) tyrosine kinase inhibitor, but its antitumor effect and underlying mechanisms in the treatment of human breast cancer remain elusive. This study aimed to investigate the effects of Danu on the growth, apoptosis, autophagy, and epithelial-to-mesenchymal transition (EMT) and the molecular mechanisms in human breast cancer MCF7 and MDA-MB-231 cells. The results demonstrated that Danu remarkably inhibited cell proliferation, induced apoptosis and autophagy, and suppressed EMT in both breast cancer cell lines. Danu arrested MCF7 and MDA-MB-231 cells in G2/M phase, accompanied by the downregulation of cyclin-dependent kinase 1 and cyclin B1 and upregulation of p21 Waf1/Cip1, p27 Kip1, and p53. Danu significantly decreased the expression of B-cell lymphoma-extra-large (Bcl-xl) and B-cell lymphoma 2 (Bcl-2), but increased the expression of Bcl-2-associated X protein (Bax) and p53-upregulated modulator of apoptosis (PUMA), and promoted the cleavage of caspases 3 and 9. Furthermore, Danu significantly increased the expression levels of the membrane-bound microtubule-associated protein 1A/1B-light chain 3 (LC3-II) and beclin 1 in breast cancer cells, two markers for autophagy. Danu induced the activation of p38 mitogen-activated protein kinase (MAPK) and extracellular signal-regulated kinases 1 and 2 (Erk1/2) and inhibited the activation of protein kinase B (Akt)/mammalian target of rapamycin (mTOR) signaling pathways in breast cancer cells. Treatment with wortmannin (a phosphatidylinositol 3-kinase inhibitor) markedly inhibited Danu-induced activation of p38 MAPK and conversion of cytosolic LC3-I to membrane-bound LC3-II. Pharmacological inhibition and small interfering RNA-mediated knockdown of p38 MAPK suppressed Akt activation, resulting in LC3-II accumulation and enhanced autophagy. Pharmacological inhibition

  17. The pan-inhibitor of Aurora kinases danusertib induces apoptosis and autophagy and suppresses epithelial-to-mesenchymal transition in human breast cancer cells

    PubMed Central

    Li, Jin-Ping; Yang, Yin-Xue; Liu, Qi-Lun; Zhou, Zhi-Wei; Pan, Shu-Ting; He, Zhi-Xu; Zhang, Xueji; Yang, Tianxin; Pan, Si-Yuan; Duan, Wei; He, Shu-Ming; Chen, Xiao-Wu; Qiu, Jia-Xuan; Zhou, Shu-Feng

    2015-01-01

    Danusertib (Danu) is a pan-inhibitor of Aurora kinases and a third-generation breakpoint cluster region-Abelson murine leukemia viral oncogene homolog 1 (Bcr-Abl) tyrosine kinase inhibitor, but its antitumor effect and underlying mechanisms in the treatment of human breast cancer remain elusive. This study aimed to investigate the effects of Danu on the growth, apoptosis, autophagy, and epithelial-to-mesenchymal transition (EMT) and the molecular mechanisms in human breast cancer MCF7 and MDA-MB-231 cells. The results demonstrated that Danu remarkably inhibited cell proliferation, induced apoptosis and autophagy, and suppressed EMT in both breast cancer cell lines. Danu arrested MCF7 and MDA-MB-231 cells in G2/M phase, accompanied by the downregulation of cyclin-dependent kinase 1 and cyclin B1 and upregulation of p21 Waf1/Cip1, p27 Kip1, and p53. Danu significantly decreased the expression of B-cell lymphoma-extra-large (Bcl-xl) and B-cell lymphoma 2 (Bcl-2), but increased the expression of Bcl-2-associated X protein (Bax) and p53-upregulated modulator of apoptosis (PUMA), and promoted the cleavage of caspases 3 and 9. Furthermore, Danu significantly increased the expression levels of the membrane-bound microtubule-associated protein 1A/1B-light chain 3 (LC3-II) and beclin 1 in breast cancer cells, two markers for autophagy. Danu induced the activation of p38 mitogen-activated protein kinase (MAPK) and extracellular signal-regulated kinases 1 and 2 (Erk1/2) and inhibited the activation of protein kinase B (Akt)/mammalian target of rapamycin (mTOR) signaling pathways in breast cancer cells. Treatment with wortmannin (a phosphatidylinositol 3-kinase inhibitor) markedly inhibited Danu-induced activation of p38 MAPK and conversion of cytosolic LC3-I to membrane-bound LC3-II. Pharmacological inhibition and small interfering RNA-mediated knockdown of p38 MAPK suppressed Akt activation, resulting in LC3-II accumulation and enhanced autophagy. Pharmacological inhibition

  18. Meta-Analysis of Differential Connectivity in Gene Co-Expression Networks in Multiple Sclerosis

    PubMed Central

    Creanza, Teresa Maria; Liguori, Maria; Liuni, Sabino; Nuzziello, Nicoletta; Ancona, Nicola

    2016-01-01

    Differential gene expression analyses to investigate multiple sclerosis (MS) molecular pathogenesis cannot detect genes harboring genetic and/or epigenetic modifications that change the gene functions without affecting their expression. Differential co-expression network approaches may capture changes in functional interactions resulting from these alterations. We re-analyzed 595 mRNA arrays from publicly available datasets by studying changes in gene co-expression networks in MS and in response to interferon (IFN)-β treatment. Interestingly, MS networks show a reduced connectivity relative to the healthy condition, and the treatment activates the transcription of genes and increases their connectivity in MS patients. Importantly, the analysis of changes in gene connectivity in MS patients provides new evidence of association for genes already implicated in MS by single-nucleotide polymorphism studies and that do not show differential expression. This is the case of amiloride-sensitive cation channel 1 neuronal (ACCN1) that shows a reduced number of interacting partners in MS networks, and it is known for its role in synaptic transmission and central nervous system (CNS) development. Furthermore, our study confirms a deregulation of the vitamin D system: among the transcription factors that potentially regulate the deregulated genes, we find TCF3 and SP1 that are both involved in vitamin D3-induced p27Kip1 expression. Unveiling differential network properties allows us to gain systems-level insights into disease mechanisms and may suggest putative targets for the treatment. PMID:27314336

  19. Downregulation of Toll-Like Receptor 9 Expression by Beta Human Papillomavirus 38 and Implications for Cell Cycle Control

    PubMed Central

    Pacini, Laura; Savini, Claudia; Ghittoni, Raffaella; Saidj, Djamel; Lamartine, Jerome; Hasan, Uzma A.; Accardi, Rosita

    2015-01-01

    ABSTRACT Innate immunity is the first line of host defense against infections. Many oncogenic viruses can deregulate several immune-related pathways to guarantee the persistence of the infection. Here, we show that the cutaneous human papillomavirus 38 (HPV38) E6 and E7 oncoproteins suppress the expression of the double-stranded DNA sensor Toll-like receptor 9 (TLR9) in human foreskin keratinocytes (HFK), a key mediator of the antiviral innate immune host response. In particular, HPV38 E7 induces TLR9 mRNA downregulation by promoting accumulation of ΔNp73α, an antagonist of p53 and p73. Inhibition of ΔNp73α expression by antisense oligonucleotide in HPV38 E6/E7 HFK strongly rescues mRNA levels of TLR9, highlighting a key role of ΔNp73α in this event. Chromatin immunoprecipitation experiments showed that ΔNp73α is part of a negative transcriptional regulatory complex with IκB kinase beta (IKKβ) that binds to a NF-κB responsive element within the TLR9 promoter. In addition, the Polycomb protein enhancer of zeste homolog 2 (EZH2), responsible for gene expression silencing, is also recruited into the complex, leading to histone 3 trimethylation at lysine 27 (H3K27me3) in the same region of the TLR9 promoter. Ectopic expression of TLR9 in HPV38 E6/E7 cells resulted in an accumulation of the cell cycle inhibitors p21WAF1 and p27Kip1, decreased CDK2-associated kinase activity, and inhibition of cellular proliferation. In summary, our data show that HPV38, similarly to other viruses with well-known oncogenic activity, can downregulate TLR9 expression. In addition, they highlight a new role for TLR9 in cell cycle regulation. IMPORTANCE The mucosal high-risk HPV types have been clearly associated with human carcinogenesis. Emerging lines of evidence suggest the involvement of certain cutaneous HPV types in development of skin squamous cell carcinoma, although this association is still under debate. Oncogenic viruses have evolved different strategies to hijack the

  20. Transgenic expression of cyclin-dependent kinase 4 results in epidermal hyperplasia, hypertrophy, and severe dermal fibrosis.

    PubMed

    Miliani de Marval, P L; Gimenez-Conti, I B; LaCava, M; Martinez, L A; Conti, C J; Rodriguez-Puebla, M L

    2001-07-01

    In a previous report we have described the effects of expression of D-type cyclins in epithelial tissues of transgenic mice. To study the involvement of the D-type cyclin partner cyclin-dependent kinase 4 (CDK4) in epithelial growth and differentiation, transgenic mice were generated carrying the CDK4 gene under the control of a keratin 5 promoter. As expected, transgenic mice showed expression of CDK4 in the epidermal basal-cell layer. Epidermal proliferation increased dramatically and basal cell hyperplasia and hypertrophy were observed. The hyperproliferative phenotype of these transgenic mice was independent of D-type cyclin expression because no overexpression of these proteins was detected. CDK4 and CDK2 kinase activities increased in transgenic animals and were associated with elevated binding of p27(Kip1) to CDK4. Expression of CDK4 in the epidermis results in an increased spinous layer compared with normal epidermis, and a mild hyperkeratosis in the cornified layer. In addition to epidermal changes, severe dermal fibrosis was observed and part of the subcutaneous adipose tissue was replaced by connective tissue. Also, abnormal expression of keratin 6 associated with the hyperproliferative phenotype was observed in transgenic epidermis. This model provides in vivo evidence for the role of CDK4 as a mediator of proliferation in epithelial cells independent of D-type cyclin expression.

  1. Chalcone-based small-molecule inhibitors attenuate malignant phenotype via targeting deubiquitinating enzymes.

    PubMed

    Issaenko, Olga A; Amerik, Alexander Yu

    2012-05-01

    The ubiquitin-proteasome system (UPS) is usurped by many if not all cancers to regulate their survival, proliferation, invasion, angiogenesis and metastasis. Bioflavonoids curcumin and chalcones exhibit anti-neoplastic selectivity through inhibition of the 26S proteasome-activity within the UPS. Here, we provide evidence for a novel mechanism of action of chalcone-based derivatives AM146, RA-9 and RA-14, which exert anticancer activity by targeting deubiquitinating enzymes (DUB) without affecting 20S proteasome catalytic-core activity. The presence of the α,β-unsaturated carbonyl group susceptible to nucleophilic attack from the sulfhydryl of cysteines in the active sites of DUB determines the capacity of novel small-molecules to act as cell-permeable, partly selective DUB inhibitors and induce rapid accumulation of polyubiquitinated proteins and deplete the pool of free ubiquitin. These chalcone-derivatives directly suppress activity of DUB UCH-L1, UCH-L3, USP2, USP5 and USP8, which are known to regulate the turnover and stability of key regulators of cell survival and proliferation. Inhibition of DUB-activity mediated by these compounds downregulates cell-cycle promoters, e.g., cyclin D1 and upregulates tumor suppressors p53, p27(Kip1) and p16(Ink4A). These changes are associated with arrest in S-G 2/M, abrogated anchorage-dependent growth and onset of apoptosis in breast, ovarian and cervical cancer cells without noticeable alterations in primary human cells. Altogether, this work provides evidence of antitumor activity of novel chalcone-based derivatives mediated by their DUB-targeting capacity; supports the development of pharmaceuticals to directly target DUB as a most efficient strategy compared with proteasome inhibition and also provides a clear rationale for the clinical evaluation of these novel small-molecule DUB inhibitors.

  2. Profiling of differentially expressed genes in haemophilia A with inhibitor.

    PubMed

    Hwang, S H; Lim, J A; Kim, M J; Kim, H C; Lee, H W; Yoo, K Y; You, C W; Lee, K S; Kim, H S

    2012-05-01

    Inhibitor development is the most significant complication in the therapy of haemophilia A (HA) patients. In spite of many studies, not much is known regarding the mechanism underlying inhibitor development. To understand the mechanism, we analysed profiles of differentially expressed genes (DEGs) between inhibitor and non-inhibitor HA via a microarray technique. Twenty unrelated Korean HAs were studied: 11 were non-inhibitor and nine were HA with inhibitor (≥5 BU mL(-1)). Microarray analysis was conducted using a Human Ref-8 expression Beadchip system (Illumina) and the data were analysed using Beadstudio software. We identified 545 DEGs in inhibitor HA as compared with the non-inhibitor patients; 384 genes were up-regulated and 161 genes were down-regulated. Among them, 75 genes whose expressions were altered by at least two-fold (>+2 or <-2) were selected and classified via the PANTHER classification method. The expressions of signal transduction and immunity-related genes differed significantly in the two groups. For validation of the DEGs, semi-quantitative RT-PCR (semi-qRT-PCR) was conducted with the six selected DEGs. The results corresponded to the microarray data, with the exception of one gene. We also examined the expression of the genes associated with the antigen presentation process via real-time PCR. The average levels of IL10, CTLA4 and TNFα slightly reduced, whereas that of IFNγ increased in the inhibitor HA group. We are currently unable to explain whether this phenomenon is a function of the inhibitor-inducing factor or is an epiphenomenon of antibody production. Nevertheless, our results provide a possible explanation for inhibitor development.

  3. Small gastrointestinal stromal tumor in the stomach: identification of precursor for clinical gastrointestinal stromal tumor using c-kit and α-smooth muscle actin expression.

    PubMed

    Mikami, Tetuo; Nemoto, Yuta; Numata, Yoshiko; Hana, Kiyomi; Nakada, Norihiro; Ichinoe, Masaaki; Murakumo, Yoshiki; Okayasu, Isao

    2013-12-01

    Gastrointestinal stromal tumors (GISTs) are the most common mesenchymal tumors of the digestive tract. To find precursors for clinical GISTs of the stomach, small gastric stromal tumors of less than 3 cm were collected and examined immunohistochemically with analysis of the KIT mutation. Sixty-eight of 74 lesions were classified into 4 representative groups according to the expression of c-kit and α-smooth muscle actin (αSMA): group A, c-kit diffusely positive and αSMA negative (18 cases); group B, c-kit diffusely positive and αSMA focally positive (13); group C, c-kit focally positive and αSMA diffusely positive (27); and group D, c-kit negative and αSMA diffusely positive (10). Of the 4 groups, groups A and B of c-kit diffuse expression showed higher cellularity and labeling indices of p27(Kip1) and Ki-67 than did groups C and D of diffuse αSMA expression. Incidence of KIT exon 11 mutation in groups A and B was 86% (25/29), whereas that in groups C and D was 0% (0/20). Small gastric stromal tumors with c-kit diffuse expression were considered precursors for clinical GIST because they were significantly different from c-kit focally positive or negative tumors. The mutation of KIT is considered as an early event in tumorigenesis of GIST.

  4. The molecular responses of skeletal muscle satellite cells to continuous expression of IGF-1: implications for the rescue of induced muscular atrophy in aged rats

    NASA Technical Reports Server (NTRS)

    Chakravarthy, M. V.; Booth, F. W.; Spangenburg, E. E.

    2001-01-01

    Approximately 50% of humans older than 85 years have physical frailty due to weak skeletal muscles. This indicates a need for determining mechanisms to combat this problem. A critical cellular factor for postnatal muscle growth is a population of myogenic precursor cells called satellite cells. Given the complex process of sarcopenia, it has been postulated that, at some point in this process, a limited satellite cell proliferation potential could become rate-limiting to the regrowth of old muscles. It is conceivable that if satellite cell proliferative capacity can be maintained or enhanced with advanced age, sarcopenia could potentially be delayed or prevented. Therefore, the purposes of this paper are to describe whether IGF-I can prevent muscular atrophy induced by repeated cycles of hindlimb immobilization, increase the in vitro proliferation in satellite cells from these muscles and, if so, the molecular mechanisms by which IGF-I mediates this increased proliferation. Our results provide evidence that IGF-I can enhance aged muscle regrowth possibly through increased satellite cell proliferation. The results also suggest that IGF-I enhances satellite cell proliferation by decreasing the cell cycle inhibitor, p27Kip1, through the PI3'-K/Akt pathway. These data provide molecular evidence for IGF-I's rescue effect upon aging-associated skeletal muscle atrophy.

  5. UCH-L1 induces podocyte hypertrophy in membranous nephropathy by protein accumulation.

    PubMed

    Lohmann, Frithjof; Sachs, Marlies; Meyer, Tobias N; Sievert, Henning; Lindenmeyer, Maja T; Wiech, Thorsten; Cohen, Clemens D; Balabanov, Stefan; Stahl, R A K; Meyer-Schwesinger, Catherine

    2014-07-01

    Podocytes are terminally differentiated cells of the glomerular filtration barrier that react with hypertrophy in the course of injury such as in membranous nephropathy (MGN). The neuronal deubiquitinase ubiquitin C-terminal hydrolase L1 (UCH-L1) is expressed and activated in podocytes of human and rodent MGN. UCH-L1 regulates the mono-ubiquitin pool and induces accumulation of poly-ubiquitinated proteins in affected podocytes. Here, we investigated the role of UCH-L1 in podocyte hypertrophy and in the homeostasis of the hypertrophy associated "model protein" p27(Kip1). A better understanding of the basic mechanisms leading to podocyte hypertrophy is crucial for the development of specific therapies in MGN. In human and rat MGN, hypertrophic podocytes exhibited a simultaneous up-regulation of UCH-L1 and of cytoplasmic p27(Kip1) content. Functionally, inhibition of UCH-L1 activity and knockdown or inhibition of UCH-L1 attenuated podocyte hypertrophy by decreasing the total protein content in isolated glomeruli and in cultured podocytes. In contrast, UCH-L1 levels and activity increased podocyte hypertrophy and total protein content in culture, specifically of cytoplasmic p27(Kip1). UCH-L1 enhanced cytoplasmic p27(Kip1) levels by nuclear export and decreased poly-ubiquitination and proteasomal degradation of p27(Kip1). In parallel, UCH-L1 increased podocyte turnover, migration and cytoskeletal rearrangement, which are associated with known oncogenic functions of cytoplasmic p27(Kip1) in cancer. We propose that UCH-L1 induces podocyte hypertrophy in MGN by increasing the total protein content through altered degradation and accumulation of proteins such as p27(Kip1) in the cytoplasm of podocytes. Modification of both UCH-L1 activity and levels could be a new therapeutic avenue to podocyte hypertrophy in MGN.

  6. Suppression of caspase-11 expression by histone deacetylase inhibitors

    SciTech Connect

    Heo, Hyejung; Yoo, Lang; Shin, Ki Soon; Kang, Shin Jung

    2009-01-02

    It has been well documented that histone deacetylase inhibitors suppress inflammatory gene expression. Therefore, we investigated whether histone deacetylase inhibitors modulate the expression of caspase-11 that is known as an inducible caspase regulating both inflammation and apoptosis. In the present study, we show that sodium butyrate and trichostatin A, two structurally unrelated inhibitors of histone deacetylase (HDAC), effectively suppressed the induction of caspase-11 in mouse embryonic fibroblasts stimulated with lipopolysaccharides. Sodium butyrate inhibited the activation of upstream signaling events for the caspase-11 induction such as activation of p38 mitogen-activated protein kinase and c-Jun N-terminal kinase, degradation of inhibitor of {kappa}B, and activation of nuclear factor-{kappa}B. These results suggest that the HDAC inhibitor suppressed cytosolic signaling events for the induction of caspase-11 by inhibiting the deacetylation of non-histone proteins.

  7. Long-term insulin-like growth factor-I expression in skeletal muscles attenuates the enhanced in vitro proliferation ability of the resident satellite cells in transgenic mice

    NASA Technical Reports Server (NTRS)

    Chakravarthy, M. V.; Fiorotto, M. L.; Schwartz, R. J.; Booth, F. W.

    2001-01-01

    Insulin-like growth factor-I (IGF-I) overexpression for 1-month in mouse skeletal muscle increases satellite cell proliferation potential. However, it is unknown whether this beneficial enhancement by IGF-I expression would persist over a longer-term duration in aged mice. This is an important issue to address if a prolonged course of IGF-I is to be used clinically in muscle-wasting conditions where satellite cells may become limiting. Using the IGF-I transgenic (IGF-I Tg) mouse that selectively expresses the IGF-I transgene in striated muscles, we found that 18-months of continuous IGF-I overexpression led to a loss in the enhanced in vitro proliferative capacity of satellite cells from Tg skeletal muscles. Also 18-month-old IGF-I Tg satellite cells lost the enhanced BrdU incorporation, greater pRb and Akt phosphorylations, and decreased p27(Kip1) levels initially observed in cells from 1-month-old IGF-I Tg mice. The levels of those biochemical markers reverted to similar values seen in the 18-months WT littermates. These findings, therefore, suggest that there is no further beneficial effect on enhancing satellite cell proliferation ability with persistent long-term expression of IGF-I in skeletal muscles of these transgenic mice.

  8. Antiviral cytokines induce hepatic expression of the granzyme B inhibitors, proteinase inhibitor 9 and serine proteinase inhibitor 6.

    PubMed

    Barrie, Mahmoud B; Stout, Heather W; Abougergi, Marwan S; Miller, Bonnie C; Thiele, Dwain L

    2004-05-15

    Expression of the granzyme B inhibitors, human proteinase inhibitor 9 (PI-9), or the murine orthologue, serine proteinase inhibitor 6 (SPI-6), confers resistance to CTL or NK killing by perforin- and granzyme-dependent effector mechanisms. In light of prior studies indicating that virally infected hepatocytes are selectively resistant to this CTL effector mechanism, the present studies investigated PI-9 and SPI-6 expression in hepatocytes and hepatoma cells in response to adenoviral infection and to cytokines produced during antiviral immune responses. Neither PI-9 nor SPI-6 expression was detected by immunoblotting in uninfected murine or human hepatocytes. Similarly, human Huh-7 hepatoma cells were found to express only very low levels of PI-9 relative to levels detected in perforin- and granzyme-resistant CTL or lymphokine-activated killer cells. Following in vivo adenoviral infection or in vitro culture with IFN-alphabeta or IFN-gamma, SPI-6 expression was induced in murine hepatocytes. Similarly, after culture with IFN-alpha, induction of PI-9 mRNA and protein expression was observed in human hepatocytes and Huh-7 cells. IFN-gamma and TNF-alpha also induced 4- to 10-fold higher levels of PI-9 mRNA expression in Huh-7 cells, whereas levels of mRNA encoding a related serine proteinase inhibitor, proteinase inhibitor 8, were unaffected by culture of Huh-7 cells with IFN-alpha, IFN-gamma, or TNF-alpha. These findings indicate that cytokines that promote antiviral cytopathic responses also regulate expression of the cytoprotective molecules, PI-9 and SPI-6, in hepatocytes that are potential targets of CTL and NK effector mechanisms.

  9. Causal co-expression method with module analysis to screen drugs with specific target.

    PubMed

    Yu, Shuhao; Zheng, Lulu; Li, Yixue; Li, Chunyan; Ma, Chenchen; Yu, Yang; Li, Xuan; Hao, Pei

    2013-04-10

    The considerable increase of investment in research and development by the pharmaceutical industry over the past three decades has not added the number of approved new drugs. An important issue ignored by drug discovery practice is the multi-dimensional interaction network between drugs and their targets. Thus, it is essential to view drug actions through the lens of network biology. In the current study, based on the co-expression network of transcription factors and their downstream genes, we proposed a novel approach, called causal co-expression method with module analysis, to screen drugs with specific target and fewer side effects. We presented a causal co-expression method with module analysis and it could be used in analyzing the microarray data of different drug candidates. At first, the differential wiring value (DW) was calculated to find some causal transcription factors (TFs) by combining with differential expression genes in the regulated networks. After the discovery of the causal TFs, co-expression module analysis method was applied to mine molecular pharmacology pathways around these causal TFs at molecular level. We applied our methods to two drug candidates, Argyrin A and Bortezomib, both with anti-cancer activities. We first obtained some differentially expressed transcription factors of cells treated with Argyrin A or Bortezomib. Nearly all these transcription factors are associated with the tumor suppressor protein p27kip1. Furthermore, module analysis showed that Bortezomib inhibited tumor growth not specifically by cell cycle and cell proliferation pathway, but through many basic metabolic processes which result in cell toxicity. In contrast, Argyrin A had influence on cell cycle, and was involved in DNA damage repair at the same time, showing that Argyrin A was a more suitable drug for anti-cancer treatment. Our study revealed that the causal co-expression method with module analysis was effective and can be used as a tool to evaluate drug

  10. Apoptosis, cell proliferation and modulation of cyclin-dependent kinase inhibitor p21(cip1) in vascular remodelling during vein arterialization in the rat.

    PubMed

    Borin, Thaiz Ferraz; Miyakawa, Ayumi Aurea; Cardoso, Leandro; de Figueiredo Borges, Luciano; Gonçalves, Giovana Aparecida; Krieger, Jose Eduardo

    2009-06-01

    Neo-intima development and atherosclerosis limit long-term vein graft use for revascularization of ischaemic tissues. Using a rat model, which is technically less challenging than smaller rodents, we provide evidence that the temporal morphological, cellular, and key molecular events during vein arterialization resemble the human vein graft adaptation. Right jugular vein was surgically connected to carotid artery and observed up to 90 days. Morphometry demonstrated gradual thickening of the medial layer and important formation of neo-intima with deposition of smooth muscle cells (SMC) in the subendothelial layer from day 7 onwards. Transmission electron microscopy showed that SMCs switch from the contractile to synthetic phenotype on day 3 and new elastic lamellae formation occurs from day 7 onwards. Apoptosis markedly increased on day 1, while alpha-actin immunostaining for SMC almost disappeared by day 3. On day 7, cell proliferation reached the highest level and cellular density gradually increased until day 90. The relative magnitude of cellular changes was higher in the intima vs. the media layer (100 vs. 2 times respectively). Cyclin-dependent kinase inhibitors (CDKIs) p27(Kip1) and p16(INKA) remained unchanged, whereas p21(Cip1) was gradually downregulated, reaching the lowest levels by day 7 until day 90. Taken together, these data indicate for the first time that p21(Cip1) is the main CDKI protein modulated during the arterialization process the rat model of vein arterialization that may be useful to identify and validate new targets and interventions to improve the long-term patency of vein grafts.

  11. Germline and somatic mutations in cyclin-dependent kinase inhibitor genes CDKN1A, CDKN2B, and CDKN2C in sporadic parathyroid adenomas.

    PubMed

    Costa-Guda, Jessica; Soong, Chen-Pang; Parekh, Vaishali I; Agarwal, Sunita K; Arnold, Andrew

    2013-10-01

    The molecular pathogenesis of sporadic parathyroid adenomas is incompletely understood. The possible role of cyclin-dependent kinase inhibitor (CDKI) genes was raised by recognition of cyclin D1 as a parathyroid oncogene, identification of rare germline mutations in CDKI genes in patients with multiple endocrine neoplasia type 1; that in rodents, mutation in Cdkn1b caused parathyroid tumors; and subsequently through identification of rare predisposing germline sequence variants and somatic mutation of CDKN1B, encoding p27(kip1), in sporadic human parathyroid adenoma. We therefore sought to determine whether mutations/variants in the other six CDKI genes CDKN1A, CDKN1C, CDKN2A, CDKN2B, CDKN2C, and CDKN2D, encoding p21, p57, p14(ARF)/p16, p15, p18, and p19, respectively, contribute to the development of typical parathyroid adenomas. In a series of 85 sporadic parathyroid adenomas, direct DNA sequencing identified alterations in five adenomas (6 %): Two contained distinct heterozygous changes in CDKN1A, one germline and one of undetermined germline status; one had a CDKN2B germline alteration, accompanied by loss of the normal allele in the tumor (LOH); two had variants of CDKN2C, one somatic and one germline with LOH. Abnormalities of three of the mutant proteins were readily demonstrable in vitro. Thus, germline mutations/rare variants in CDKN1A, CDKN2B, and CDKN2C likely contribute to the development of a significant subgroup of common sporadic parathyroid adenomas, and somatic mutation in CDKN2C further suggests a direct role for CDKI alteration in conferring a selective growth advantage to parathyroid cells, providing novel support for the concept that multiple CDKIs can play primary roles in human neoplasia.

  12. The expression of growth-arrest genes in the liver and kidney of the protein-restricted rat fetus.

    PubMed

    Maloney, Christopher A; Lilley, Christina; Cruickshank, Morven; McKinnon, Caroline; Hay, Susan M; Rees, William D

    2005-07-01

    During fetal life, there are periods of rapid cell proliferation, which are uniquely sensitive to nutritional perturbation. Feeding the pregnant rat a protein-restricted diet alters the growth trajectory of major fetal organs such as the kidney. By day 21 of gestation, the ratio of kidney weight to total body weight is reduced in the fetuses of dams fed a protein-deficient diet. In contrast, the ratio of fetal liver weight to total body weight is unchanged. To investigate the mechanisms underlying this disproportionate change in organ growth in the low-protein group, cell proliferation and differentiation have been assessed in the liver and kidney. The steady-state levels of mRNA for the growth-arrest and DNA-damage gene gadd153/CHOP-10, CCAAT enhancer-binding proteins alpha and beta were unaffected by maternal diet in both fetal liver and kidney. The mRNA for alpha-fetoprotein, albumin and hepatic glucokinase were unchanged in the liver, suggesting that maternal protein deficiency does not alter the state of differentiation. The steady-state levels of the mRNA coding for the cyclin-dependent protein kinase inhibitors (p15(INK4a), p19(INK4d), p21(CIP1), p27(KIP1) and p57(KIP2)) were unchanged in the fetal livers but were significantly increased in the kidneys of fetuses from dams fed the low-protein diet. These results show that the asymmetrical growth of the kidney is associated with increases in mRNA for the Cip/Kip cyclin-dependent kinase inhibitors and that these may reflect specific lesions in organ development.

  13. Induction of p21(Waf1/Cip1) by garcinol via downregulation of p38-MAPK signaling in p53-independent H1299 lung cancer.

    PubMed

    Yu, Sheng-Yung; Liao, Chiung-Ho; Chien, Ming-Hsien; Tsai, Tsung-Yu; Lin, Jen-Kun; Weng, Meng-Shih

    2014-03-05

    Garcinol, a polyisoprenylated benzophenone, from Garcinia indica fruit rind has possessed anti-inflammatory, antioxidant, antiproliferation, and anticancer activities. However, the anticancer mechanisms of garcinol in lung cancer were still unclear. Therefore, we examine the effects of garcinol on antiproliferation in human lung cancer cells. Treatments with garcinol for 24 h exhibited morphological changes and inhibited the proliferation of H460 (p53-wild type) and H1299 (p53-null) cells in dose- and time-dependent manners. Furthermore, a significant G1 cell cycle arrest was observed in a dose-dependent treatment after H1299 cells were exposed in garcinol, whereas garcinol induced apoptosis rather than cell cycle arrest in H460 cells. Moreover, cyclin-dependent kinase 2 (CDK2), cyclin-dependent kinase 4 (CDK4), cyclin D1, and cyclin D3 were decreased, although cyclin E and cyclin-dependent kinase 6 (CDK6) were increased in garcinol-treated H1299 cells. Meanwhile, the protein levels of CDK inhibitors p21(Waf1/Cip1) and p27(KIP1) also exhibited upregulation after garcinol treatments. The enhanced protein-associated level between p21(Waf1/Cip1) and CDK4/2 rather than p27(KIP1) and CDK4/2 was demonstrated in garcinol-treated cells. Additionally, knock-down p21(Waf1/Cip1) by specific siRNA competently prevented garcinol-induced G1 arrest. Besides, garcinol also inhibited ERK and p38-MAPK activations in time-dependent mode. The pretreatment with p38-MAPK inhibitor but not ERK inhibitor raised garcinol-induced G1 population cells. Co-treatment with p38-MAPK inhibitor and garcinol synergistically elevated cyclin E, p21(Waf1/Cip1), and p27(Kip1) expressions. Meanwhile, overexpression dominant negative p38-MAPK also enhanced garcinol-induced p21(Waf1/Cip1) expression in H1299 cells. Accordingly, our data suggested that garcinol induced G1 cell cycle arrest and apoptosis in lung cancer cells under different p53 statuses. The p53-independent G1 cell cycle arrest induced by

  14. Progression in Cutaneous Malignant Melanoma Is Associated with Distinct Expression Profiles

    PubMed Central

    Alonso, Soledad R.; Ortiz, Pablo; Pollán, Marina; Pérez-Gómez, Beatriz; Sánchez, Lydia; Acuña, Ma Jesús; Pajares, Raquel; Martínez-Tello, Francisco J.; Hortelano, Carlos M.; Piris, Miguel A.; Rodríguez-Peralto, José L.

    2004-01-01

    Cutaneous malignant melanoma remains the leading cause of skin cancer death in industrialized countries. Clinical and histological variables that predict survival, such as Breslow’s index, tumor size, ulceration, or vascular invasion have been identified in malignant melanoma. Nevertheless, the potential relevance of biological variables still awaits an in-depth exploration. Using tissue microarrays (TMAs), we retrospectively analyzed 165 malignant melanoma samples from 88 patients corresponding to distinct histological progression phases, radial, vertical, and metastases. A panel of 39 different antibodies for cell cycle, apoptosis, melanoma antigens, transcription factors, DNA mismatch repair, and other proteins was used. Integrating the information, the study has identified expression profiles distinguishing specific melanoma progression stages. Most of the detected alterations were linked to the control of cell cycle G1/S transition; cyclin D1 was expressed in radial cases 48% (12 of 25) with significant lost of expression in vertical cases 14% (9 of 65), P = 0.002; whereas p16INK4a (89% in vertical versus 71% in metastatic cases, P = 0.009) and p27KIP1 (76% in radial versus 45% in vertical cases, P = 0.010) were diminished in advanced stages. The study also defines a combination of biological markers associated with shorter overall survival in patients with vertical growth phase melanoma, that provided a predictor model with four antibodies (Ki67, p16INK4a, p21CIP1, and Bcl-6). This predictor model was validated using an independent series of 72 vertical growth phase melanoma patients. PMID:14695333

  15. N-myc downstream regulated 1 (NDRG1) is regulated by eukaryotic initiation factor 3a (eIF3a) during cellular stress caused by iron depletion.

    PubMed

    Lane, Darius J R; Saletta, Federica; Suryo Rahmanto, Yohan; Kovacevic, Zaklina; Richardson, Des R

    2013-01-01

    Iron is critical for cellular proliferation and its depletion leads to a suppression of both DNA synthesis and global translation. These observations suggest that iron depletion may trigger a cellular "stress response". A canonical response of cells to stress is the formation of stress granules, which are dynamic cytoplasmic aggregates containing stalled pre-initiation complexes that function as mRNA triage centers. By differentially prioritizing mRNA translation, stress granules allow for the continued and selective translation of stress response proteins. Although the multi-subunit eukaryotic initiation factor 3 (eIF3) is required for translation initiation, its largest subunit, eIF3a, may not be essential for this activity. Instead, eIF3a is a vital constituent of stress granules and appears to act, in part, by differentially regulating specific mRNAs during iron depletion. Considering this, we investigated eIF3a's role in modulating iron-regulated genes/proteins that are critically involved in proliferation and metastasis. In this study, eIF3a was down-regulated and recruited into stress granules by iron depletion as well as by the classical stress-inducers, hypoxia and tunicamycin. Iron depletion also increased expression of the metastasis suppressor, N-myc downstream regulated gene-1 (NDRG1), and a known downstream repressed target of eIF3a, namely the cyclin-dependent kinase inhibitor, p27(kip1). To determine if eIF3a regulates NDRG1 expression, eIF3a was inducibly over-expressed or ablated. Importantly, eIF3a positively regulated NDRG1 expression and negatively regulated p27(kip1) expression during iron depletion. This activity of eIF3a could be due to its recruitment to stress granules and/or its ability to differentially regulate mRNA translation during cellular stress. Additionally, eIF3a positively regulated proliferation, but negatively regulated cell motility and invasion, which may be due to the eIF3a-dependent changes in expression of NDRG1 and p27

  16. Protein inhibitor of activated STAT3 inhibits adipogenic gene expression

    SciTech Connect

    Deng Jianbei; Hua Kunjie; Caveney, Erica J.; Takahashi, Nobuyuki; Harp, Joyce B. . E-mail: jharp@unc.edu

    2006-01-20

    Protein inhibitor of activated STAT3 (PIAS3), a cytokine-induced repressor of signal transducer and activator of transcription 3 (STAT3) and a modulator of a broad array of nuclear proteins, is expressed in white adipose tissue, but its role in adipogenesis is not known. Here, we determined that PIAS3 was constitutively expressed in 3T3-L1 cells at all stages of adipogenesis. However, it translocated from the nucleus to the cytoplasm 4 days after induction of differentiation by isobutylmethylxanthine, dexamethasone, and insulin (MDI). In ob/ob mice, PIAS3 expression was increased in white adipose tissue depots compared to lean mice and was found in the cytoplasm of adipocytes. Overexpression of PIAS3 in differentiating preadipocytes, which localized primarily to the nucleus, inhibited mRNA level gene expression of adipogenic transcription factors C/EBP{alpha} and PPAR{gamma}, as well as their downstream target genes aP2 and adiponectin. PIAS3 also inhibited C/EBP{alpha} promoter activation mediated specifically by insulin, but not dexamethasone or isobutylmethylxanthine. Taken together, these data suggest that PIAS3 may play an inhibitory role in adipogenesis by modulating insulin-activated transcriptional activation events. Increased PIAS3 expression in adipose tissue may play a role in the metabolic disturbances of obesity.

  17. Cross talk between cell death and cell cycle progression: BCL-2 regulates NFAT-mediated activation.

    PubMed Central

    Linette, G P; Li, Y; Roth, K; Korsmeyer, S J

    1996-01-01

    BCL-2-deficient T cells demonstrate accelerated cell cycle progression and increased apoptosis following activation. Increasing the levels of BCL-2 retarded the G0-->S transition, sustained the levels of cyclin-dependent kinase inhibitor p27Kip1, and repressed postactivation death. Proximal signal transduction events and immediate early gene transcription were unaffected. However, the transcription and synthesis of interleukin 2 and other delayed early cytokines were markedly attenuated by BCL-2. In contrast, a cysteine protease inhibitor that also blocks apoptosis had no substantial affect upon cytokine production. InterleUkin 2 expression requires several transcription factors of which nuclear translocation of NFAT (nuclear factor of activated T cells) and NFAT-mediated transactivation were impaired by BCL-2. Thus, select genetic aberrations in the apoptotic pathway reveal a cell autonomous coregulation of activation. Images Fig. 3 Fig. 4 Fig. 7 PMID:8790367

  18. Expression of matrix metalloproteinase and its tissue inhibitor in haemangioma.

    PubMed

    Zhong, Shan; Yang, Guohua; Xia, Cong; Duanlian, Zhang; Shan, Shengguo

    2009-10-01

    The action mechanism of matrix metalloproteinases-2 (MMP-2) and tissue inhibitor of metalloproteinases-2 (TIMP-2) in the genesis, development and degeneration of haemangioma was investigated by detecting their expression in the tissue of haemangioma in different phases by using the immunohistochemistry. Fifty paraffin-embedded specimens of skin capillary haemangioma were collected, which were documented in the Department of Pathology, Renmin Hospital of Wuhan University from 2000 to 2006. All samples were stained by regular HE method, and proliferative cell nuclear antigen (PCNA) was tested by immunohistochemical S-P method. The samples were classified according to the Mulliken criteria and the expression pattern of PCNA. Immunohistochemical S-P method was applied to detect the expression of MMP-2 and TIMP-2 in proliferative and degenerative phases of cutaneous capillary haemangioma, and in normal skin tissues. In combination with the detection of the expression of factor VIII-related antigen, it was verified that in haemangioma tissues, the cells expressing MMP-2 and TIMP-2 were vascular endothelial cells. The MMP-2 and TIMP-2 expression was quantitatively analyzed by image analysis system (HPIAS-1000), and one-way ANOVA(107) and SNK(q) test were done to analyze average absorbance (A) and positive area rate of immunohistochemically positive particles by using SPSS11.5. The results showed: (1) Among 50 samples of haemangioma, there were 26 proliferative haemangiomas, and 24 degenerative haemangiomas, respectively; (2) The expression of MMP-2 was weak in normal vascular endothelial cells, cytoplasm of connective tissues and extracellular matrix around blood vessels. The expression of MMP-2 in proliferative group was significantly higher than in degenerative group and control group (normal skin) (P<0.05), but there was no statistically significant difference between the latter two groups; (3) TIMP-2 was highly expressed in normal tissues, degenerative vascular

  19. Effects of tobacco genetically modified to express protease inhibitor bovine spleen trypsin inhibitor on non-target soil organisms.

    PubMed

    O'Callaghan, Maureen; Brownbridge, Michael; Stilwell, Wendy B; Gerard, Emily M; Burgess, Elisabeth P J; Barraclough, Emma I; Christeller, John T

    2007-01-01

    Effects of tobacco genetically modified to express the protease inhibitor bovine spleen trypsin inhibitor (BSTI) were examined in laboratory assays against three earthworm and one collembolan species. BSTI is a serine protease inhibitor that can bind to the digestive trypsins of insects feeding on modified plants, resulting in reduced growth and survival. Protease inhibitors are active against a broad range of insects, so may have a large impact on non-target organisms. Survival and fecundity of the collembolan Folsomia candida were unaffected by consumption of artificial diet containing BSTI-expressing tobacco leaf or powdered freeze-dried BSTI-expressing tobacco leaf that was added to soil. Similarly, mortality and growth of earthworms Aporrectodea caliginosa and Lumbricus rubellus did not differ significantly between soil augmented with BSTI-expressing tobacco leaves or unmodified control leaves. The redworm Eisenia fetida gained less weight when provided with BSTI-expressing leaves in one assay, but when the experiment was repeated, there was no significant difference between treatments. BSTI-expressing tobacco and unmodified control leaves decomposed at the same rate, indicating that the inhibitor had no effect on the overall function of the decomposer community of micro-flora and fauna in soil.

  20. Screening of cell cycle fusion proteins to identify kinase signaling networks.

    PubMed

    Trojanowsky, Michelle; Vidovic, Dusica; Simanski, Scott; Penas, Clara; Schurer, Stephan; Ayad, Nagi G

    2015-01-01

    Kinase signaling networks are well-established mediators of cell cycle transitions. However, how kinases interact with the ubiquitin proteasome system (UPS) to elicit protein turnover is not fully understood. We sought a means of identifying kinase-substrate interactions to better understand signaling pathways controlling protein degradation. Our prior studies used a luciferase fusion protein to uncover kinase networks controlling protein turnover. In this study, we utilized a similar approach to identify pathways controlling the cell cycle protein p27(Kip1). We generated a p27(Kip1)-luciferase fusion and expressed it in cells incubated with compounds from a library of pharmacologically active compounds. We then compared the relative effects of the compounds on p27(Kip1)-luciferase fusion stabilization. This was combined with in silico kinome profiling to identify potential kinases inhibited by each compound. This approach effectively uncovered known kinases regulating p27(Kip1) turnover. Collectively, our studies suggest that this parallel screening approach is robust and can be applied to fully understand kinase-ubiquitin pathway interactions.

  1. Corneal Antifibrotic Switch Identified in Genetic and Pharmacological Deficiency of Vimentin*

    PubMed Central

    Bargagna-Mohan, Paola; Paranthan, Riya R.; Hamza, Adel; Zhan, Chang-Guo; Lee, Do-Min; Kim, Kyung Bo; Lau, Daniel L.; Srinivasan, Cidambi; Nakayama, Keiko; Nakayama, Keiichi I.; Herrmann, Harald; Mohan, Royce

    2012-01-01

    The type III intermediate filaments (IFs) are essential cytoskeletal elements of mechanosignal transduction and serve critical roles in tissue repair. Mice genetically deficient for the IF protein vimentin (Vim−/−) have impaired wound healing from deficits in myofibroblast development. We report a surprising finding made in Vim−/− mice that corneas are protected from fibrosis and instead promote regenerative healing after traumatic alkali injury. This reparative phenotype in Vim−/− corneas is strikingly recapitulated by the pharmacological agent withaferin A (WFA), a small molecule that binds to vimentin and down-regulates its injury-induced expression. Attenuation of corneal fibrosis by WFA is mediated by down-regulation of ubiquitin-conjugating E3 ligase Skp2 and up-regulation of cyclin-dependent kinase inhibitors p27Kip1 and p21Cip1. In cell culture models, WFA exerts G2/M cell cycle arrest in a p27Kip1- and Skp2-dependent manner. Finally, by developing a highly sensitive imaging method to measure corneal opacity, we identify a novel role for desmin overexpression in corneal haze. We demonstrate that desmin down-regulation by WFA via targeting the conserved WFA-ligand binding site shared among type III IFs promotes further improvement of corneal transparency without affecting cyclin-dependent kinase inhibitor levels in Vim−/− mice. This dissociates a direct role for desmin in corneal cell proliferation. Taken together, our findings illuminate a previously unappreciated pathogenic role for type III IF overexpression in corneal fibrotic conditions and also validate WFA as a powerful drug lead toward anti-fibrosis therapeutic development. PMID:22117063

  2. The Human Ubiquitin Conjugating Enzyme, UBE2E3, Is Required for Proliferation of Retinal Pigment Epithelial Cells

    PubMed Central

    Plafker, Kendra S.; Farjo, Krysten M.; Wiechmann, Allan F.; Plafker, Scott M.

    2008-01-01

    Purpose Cell cycle progression is governed by the coordinated activities of kinases, phosphatases, and the ubiquitin system. The entire complement of ubiquitin pathway components that mediate this process in retinal pigment epithelial (RPE) cells remains to be identified. This study was undertaken to determine whether the human ubiquitin-conjugating enzyme, UBE2E3, is essential for RPE cell proliferation. Methods UBE2E3 expression and localization in telomerase-immortalized, human RPE cells was determined with a UBE2E3-specific antibody. The necessity for UBE2E3 in RPE proliferation was determined using small interfering (si)RNA to target the expression of the enzyme. Cell counts and immunolabeling for the proliferation marker Ki-67 and the cyclin-dependent kinase inhibitor p27Kip1 were performed to assess the consequences of UBE2E3 depletion. A mouse strain harboring a disrupted allele of UbcM2 (the mouse counterpart of UBE2E3) with the coding sequence for β-galactosidase was used to track the developmental expression of the enzyme in murine RPE cells. Results UBE2E3 localized in the nucleus of the immortalized RPE cells. Depletion of the enzyme by siRNA resulted in a cell-cycle exit accompanied by a loss of Ki-67, an increase in p27Kip1, and a doubling in cell area. Rescue experiments confirmed the specificity of the RNA interference. In vivo, UbcM2 was transcriptionally downregulated during RPE development in the mouse. Conclusions UBE2E3 is essential for the proliferation of RPE-1 cells and is downregulated during RPE layer maturation in the developing mouse eye. These findings indicate that UBE2E3 is a major enzyme in modulating the balance between RPE cell proliferation and differentiation. PMID:18614808

  3. Rapamycin ameliorates IgA nephropathy via cell cycle-dependent mechanisms.

    PubMed

    Tian, Jihua; Wang, Yanhong; Liu, Xinyan; Zhou, Xiaoshuang; Li, Rongshan

    2015-07-01

    IgA nephropathy is the most frequent type of glomerulonephritis worldwide. The role of cell cycle regulation in the pathogenesis of IgA nephropathy has been studied. The present study was designed to explore whether rapamycin ameliorates IgA nephropathy via cell cycle-dependent mechanisms. After establishing an IgA nephropathy model, rats were randomly divided into four groups. Coomassie Brilliant Blue was used to measure the 24-h urinary protein levels. Renal function was determined using an autoanalyzer. Proliferation was assayed via Proliferating Cell Nuclear Antigen (PCNA) immunohistochemistry. Rat mesangial cells were cultured and divided into the six groups. Methylthiazolyldiphenyl-tetrazolium bromide (MTT) and flow cytometry were used to detect cell proliferation and the cell cycle phase. Western blotting was performed to determine cyclin E, cyclin-dependent kinase 2, p27(Kip1), p70S6K/p-p70S6K, and extracellular signal-regulated kinase 1/2/p- extracellular signal-regulated kinase 1/2 protein expression. A low dose of the mammalian target of rapamycin (mTOR) inhibitor rapamycin prevented an additional increase in proteinuria, protected kidney function, and reduced IgA deposition in a model of IgA nephropathy. Rapamycin inhibited mesangial cell proliferation and arrested the cell cycle in the G1 phase. Rapamycin did not affect the expression of cyclin E and cyclin-dependent kinase 2. However, rapamycin upregulated p27(Kip1) at least in part via AKT (also known as protein kinase B)/mTOR. In conclusion, rapamycin can affect cell cycle regulation to inhibit mesangial cell proliferation, thereby reduce IgA deposition, and slow the progression of IgAN.

  4. microRNA-200b and microRNA-200c promote colorectal cancer cell proliferation via targeting the reversion-inducing cysteine-rich protein with Kazal motifs.

    PubMed

    Pan, Yi; Liang, Hongwei; Chen, Weixu; Zhang, Hongjie; Wang, Nan; Wang, Feng; Zhang, Suyang; Liu, Yanqing; Zhao, Chihao; Yan, Xin; Zhang, Junfeng; Zhang, Chen-Yu; Gu, Hongwei; Zen, Ke; Chen, Xi

    2015-01-01

    MicroRNA-200b and microRNA-200c (miR-200b/c) are 2 of the most frequently upregulated oncomiRs in colorectal cancer cells. The role of miR-200b/c during colorectal tumorigenesis, however, remains unclear. In the present study, we report that miR-200b/c can promote colorectal cancer cell proliferation via targeting the reversion-inducing cysteine-rich protein with Kazal motifs (RECK). Firstly, bioinformatics analysis predicted RECK as a conserved target of miR-200b/c. By overexpressing or knocking down miR-200b/c in colorectal cancer cells, we experimentally validated that miR-200b/c are direct regulators of RECK. Secondly, an inverse correlation between the levels of miR-200b/c and RECK protein was found in human colorectal cancer tissues and cell lines. Thirdly, we demonstrated that repression of RECK by miR-200b/c consequently triggered SKP2 (S-phase kinase-associated protein 2) elevation and p27(Kip1) (also known as cyclin-dependent kinase inhibitor 1B) degradation in colorectal cancer cells, which eventually promotes cancer cell proliferation. Finally, promoting tumor cell growth by miR-200b/c-targeting RECK was also observed in the xenograft mouse model. Taken together, our results demonstrate that miR-200b/c play a critical role in promoting colorectal tumorigenesis through inhibiting RECK expression and subsequently triggering SKP2 elevation and p27(Kip1) degradation.

  5. Gene expression abnormalities and oligodendrocyte deficits in the internal capsule in schizophrenia.

    PubMed

    Kerns, David; Vong, Ghe S; Barley, Kevin; Dracheva, Stella; Katsel, Pavel; Casaccia, Patrizia; Haroutunian, Vahram; Byne, William

    2010-07-01

    Deficits in the expression of oligodendrocyte (Ol) and myelin genes have been described in numerous brain regions in schizophrenia (SZ) in association with abnormalities of cell cycle markers. We have previously reported a SZ-associated decrease in the expression of genes expressed after, but not prior to, the terminal differentiation of Ols in the posterior limb of the internal capsule (ICp). This pattern of deficits could reflect a failure of Ol precursors to exit the cell cycle and differentiate to meet the demands imposed by the high rate of apoptosis among myelinating Ols. Here we explore this hypothesis using quantitative real time PCR to examine the mRNA expression of additional genes in the ICp of the previously examined sample of 14 subjects with SZ and 15 normal controls (NCs). The genes examined in the present study were chosen because they are associated with particular phases of the cell cycle (CCND1, CCND2, p21(Cip1), p27(Kip1), and p57(Kip2)), with DNA replication and repair (PCNA), apoptosis (CASP3), or the Notch signaling pathway (JAG1, HES1, HES5, andDTX1). The Notch pathway influences whether Ol precursors continue to proliferate or exit the cell cycle. We also determined the densities of Ols in the ICp. Genes associated with maintenance of the cell cycle tended to exhibit increased expression levels in SZ relative to NCs and to be negatively correlated with the expression levels of the previously assessed mature Ol genes. In contrast, genes associated with cell cycle arrest tended to show the opposite pattern (decreased expression in SZ and positive correlations with mature Ol genes). CASP3 and PCNA expression levels were significantly decreased in SZ and positively correlated with mature Ol genes, suggesting that myelinating Ols may turnover more rapidly in normal controls than in subjects with SZ. JAG1 expression was significantly increased in SZ and exhibited positive correlations with mediators of the canonical Notch pathway but negative

  6. A secretory protease inhibitor requires androgens for its expression in male sex accessory tissues but is expressed constitutively in pancreas.

    PubMed Central

    Mills, J S; Needham, M; Parker, M G

    1987-01-01

    A full length cDNA clone encoding a mouse prostatic secretory glycoprotein (p12) whose synthesis is dependent upon testicular androgens has been cloned and characterized. The predicted amino acid sequence of p12 shares extensive homology with several members of the Kazal family of secretory protease inhibitors, in particular the pancreatic secretory trypsin inhibitors. In agreement with sequence data, prostatic secretory p12, purified from mouse ventral prostate secretion, exhibits anti-trypsin activity. Steady-state levels of protease inhibitor mRNA in ventral prostate are reduced from approximately 0.06% in normal mice to undetectable after androgen withdrawal but are inducible within 4 h by re-administration of testosterone. Androgen-dependent expression of the secretory protease inhibitor mRNA was also observed in coagulating gland and seminal vesicle. In seminal vesicle, a tissue of different embryonic origin to the prostate, the kinetics of secretory protease inhibitor mRNA loss after castration are not as rapid as in the ventral prostate and coagulating gland. Low-level androgen independent expression was also observed in the pancreas. There appears to be a single gene for this secretory protease inhibitor and yet expression is markedly stimulated by testosterone in the sex accessory tissues and unaffected by this hormone in the pancreas. Images Fig. 3. Fig. 4. Fig. 5. Fig. 6. Fig. 7. PMID:3428272

  7. Differential regulation of Bdnf expression in cortical neurons by class-selective histone deacetylase inhibitors.

    PubMed

    Koppel, Indrek; Timmusk, Tõnis

    2013-12-01

    Histone deactylase (HDAC) inhibitors show promise as therapeutics for neurodegenerative and psychiatric diseases. Increased expression of brain-derived neurotrophic factor (BDNF) has been associated with memory-enhancing and neuroprotective properties of these drugs, but the mechanism of BDNF induction is not well understood. Here, we compared the effects of a class I/IIb selective HDAC inhibitor SAHA, a class I selective inhibitor MS-275, a class II selective inhibitor MC1568 and a HDAC6 selective inhibitor tubacin on Bdnf mRNA expression in rat primary neurons. We show that inhibition of class II HDACs resulted in rapid upregulation of Bdnf mRNA levels, whereas class I HDAC inhibition produced a markedly delayed Bdnf induction. In contrast to relatively slow upregulation of Bdnf transcripts, histone acetylation at BDNF promoters I and IV was rapidly induced by SAHA. Bdnf induction by SAHA and MS-275 at 24 h was sensitive to protein synthesis inhibition, suggesting that delayed Bdnf induction by HDAC inhibitors is secondary to changed expression of its regulators. HDAC4 and HDAC5 repressed Bdnf promoter IV activity, supporting the role of class II HDACs in regulation of Bdnf expression. In addition, we show a critical role for the cAMP/Ca2+ response element (CRE) in induction of Bdnf promoter IV by MS-275, MC1568, SAHA and sodium valproate. In contrast, MEF2-binding CaRE1 element was not necessary for promoter IV induction by HDAC inhibition. Finally, we show that similarly to Bdnf, the studied HDAC inhibitors differentially induced expression of neuronal activity-regulated genes c-fos and Arc. Together, our findings implicate class II HDACs in transcriptional regulation of Bdnf and indicate that class II selective HDAC inhibitors may have potential as therapeutics for nervous system disorders.

  8. Assay for isolation of inhibitors of her2-kinase expression.

    PubMed

    Chiosis, Gabriela; Keeton, Adam B

    2009-01-01

    Her2 (ErbB2) protein is overexpressed in breast and other solid tumors, and its expression is associated with progressive disease. Current therapies directed toward Her2 either block dimerization of the receptor or inhibit tyrosine kinase activity to disrupt intracellular signaling. However, little is known about alternative mechanisms for suppressing Her2 expression, possibly by inducing degradation or blocking synthesis. Here, we describe a hybrid western-blotting and enzyme-linked immunosorbent assay (ELISA) designed to identify in low- to medium-throughput format noncytotoxic compounds that reduce expression of Her2 protein.

  9. Adenosine Attenuates Human Coronary Artery Smooth Muscle Cell Proliferation by Inhibiting Multiple Signaling Pathways That Converge on Cyclin D.

    PubMed

    Dubey, Raghvendra K; Fingerle, Jürgen; Gillespie, Delbert G; Mi, Zaichuan; Rosselli, Marinella; Imthurn, Bruno; Jackson, Edwin K

    2015-12-01

    The goal of this study was to determine whether and how adenosine affects the proliferation of human coronary artery smooth muscle cells (HCASMCs). In HCASMCs, 2-chloroadenosine (stable adenosine analogue), but not N(6)-cyclopentyladenosine, CGS21680, or N(6)-(3-iodobenzyl)-adenosine-5'-N-methyluronamide, inhibited HCASMC proliferation (A2B receptor profile). 2-Chloroadenosine increased cAMP, reduced phosphorylation (activation) of ERK and Akt (protein kinases known to increase cyclin D expression and activity, respectively), and reduced levels of cyclin D1 (cyclin that promotes cell-cycle progression in G1). Moreover, 2-chloroadenosine inhibited expression of S-phase kinase-associated protein-2 (Skp2; promotes proteolysis of p27(Kip1)) and upregulated levels of p27(Kip1) (cell-cycle regulator that impairs cyclin D function). 2-Chloroadenosine also inhibited signaling downstream of cyclin D, including hyperphosphorylation of retinoblastoma protein and expression of cyclin A (S phase cyclin). Knockdown of A2B receptors prevented the effects of 2-chloroadenosine on ERK1/2, Akt, Skp2, p27(Kip1), cyclin D1, cyclin A, and proliferation. Likewise, inhibition of adenylyl cyclase and protein kinase A abrogated 2-chloroadenosine's inhibitory effects on Skp2 and stimulatory effects on p27(Kip1) and rescued HCASMCs from 2-chloroadenosine-mediated inhibition. Knockdown of p27(Kip1) also reversed the inhibitory effects of 2-chloroadenosine on HCASMC proliferation. In vivo, peri-arterial (rat carotid artery) 2-chloroadenosine (20 μmol/L for 7 days) downregulated vascular expression of Skp2, upregulated vascular expression of p27(Kip1), and reduced neointima hyperplasia by 71% (P<0.05; neointimal thickness: control, 37 424±18 371 pixels; treated, 10 352±2824 pixels). In conclusion, the adenosine/A2B receptor/cAMP/protein kinase A axis inhibits HCASMC proliferation by blocking multiple signaling pathways (ERK1/2, Akt, and Skp2) that converge at cyclin D, a key G1 cyclin

  10. Ectopic Expression of a Neospora caninum Kazal Type Inhibitor Triggers Developmental Defects in Toxoplasma and Plasmodium

    PubMed Central

    Goulielmaki, Evi; Kaforou, Sofia; Kim, Kami; Waters, Andrew P.; Carruthers, Vern B.; Siden-Kiamos, Inga; Koussis, Konstantinos

    2015-01-01

    Regulated proteolysis is known to control a variety of vital processes in apicomplexan parasites including invasion and egress of host cells. Serine proteases have been proposed as targets for drug development based upon inhibitor studies that show parasite attenuation and transmission blockage. Genetic studies suggest that serine proteases, such as subtilisin and rhomboid proteases, are essential but functional studies have proved challenging as active proteases are difficult to express. Proteinaceous Protease Inhibitors (PPIs) provide an alternative way to address the role of serine proteases in apicomplexan biology. To validate such an approach, a Neospora caninum Kazal inhibitor (NcPI-S) was expressed ectopically in two apicomplexan species, Toxoplasma gondii tachyzoites and Plasmodium berghei ookinetes, with the aim to disrupt proteolytic processes taking place within the secretory pathway. NcPI-S negatively affected proliferation of Toxoplasma tachyzoites, while it had no effect on invasion and egress. Expression of the inhibitor in P. berghei zygotes blocked their development into mature and invasive ookinetes. Moreover, ultra-structural studies indicated that expression of NcPI-S interfered with normal formation of micronemes, which was also confirmed by the lack of expression of the micronemal protein SOAP in these parasites. Our results suggest that NcPI-S could be a useful tool to investigate the function of proteases in processes fundamental for parasite survival, contributing to the effort to identify targets for parasite attenuation and transmission blockage. PMID:25803874

  11. Ectopic expression of a Neospora caninum Kazal type inhibitor triggers developmental defects in Toxoplasma and Plasmodium.

    PubMed

    Tampaki, Zoi; Mwakubambanya, Ramadhan S; Goulielmaki, Evi; Kaforou, Sofia; Kim, Kami; Waters, Andrew P; Carruthers, Vern B; Siden-Kiamos, Inga; Loukeris, Thanasis G; Koussis, Konstantinos

    2015-01-01

    Regulated proteolysis is known to control a variety of vital processes in apicomplexan parasites including invasion and egress of host cells. Serine proteases have been proposed as targets for drug development based upon inhibitor studies that show parasite attenuation and transmission blockage. Genetic studies suggest that serine proteases, such as subtilisin and rhomboid proteases, are essential but functional studies have proved challenging as active proteases are difficult to express. Proteinaceous Protease Inhibitors (PPIs) provide an alternative way to address the role of serine proteases in apicomplexan biology. To validate such an approach, a Neospora caninum Kazal inhibitor (NcPI-S) was expressed ectopically in two apicomplexan species, Toxoplasma gondii tachyzoites and Plasmodium berghei ookinetes, with the aim to disrupt proteolytic processes taking place within the secretory pathway. NcPI-S negatively affected proliferation of Toxoplasma tachyzoites, while it had no effect on invasion and egress. Expression of the inhibitor in P. berghei zygotes blocked their development into mature and invasive ookinetes. Moreover, ultra-structural studies indicated that expression of NcPI-S interfered with normal formation of micronemes, which was also confirmed by the lack of expression of the micronemal protein SOAP in these parasites. Our results suggest that NcPI-S could be a useful tool to investigate the function of proteases in processes fundamental for parasite survival, contributing to the effort to identify targets for parasite attenuation and transmission blockage.

  12. SMARCE1 suppresses EGFR expression and controls responses to MET and ALK inhibitors in lung cancer.

    PubMed

    Papadakis, Andreas I; Sun, Chong; Knijnenburg, Theo A; Xue, Yibo; Grernrum, Wipawadee; Hölzel, Michael; Nijkamp, Wouter; Wessels, Lodewyk F A; Beijersbergen, Roderick L; Bernards, Rene; Huang, Sidong

    2015-04-01

    Recurrent inactivating mutations in components of SWI/SNF chromatin-remodeling complexes have been identified across cancer types, supporting their roles as tumor suppressors in modulating oncogenic signaling pathways. We report here that SMARCE1 loss induces EGFR expression and confers resistance to MET and ALK inhibitors in non-small cell lung cancers (NSCLCs). We found that SMARCE1 binds to regulatory regions of the EGFR locus and suppresses EGFR transcription in part through regulating expression of Polycomb Repressive Complex component CBX2. Addition of the EGFR inhibitor gefitinib restores the sensitivity of SMARCE1-knockdown cells to MET and ALK inhibitors in NSCLCs. Our findings link SMARCE1 to EGFR oncogenic signaling and suggest targeted treatment options for SMARCE1-deficient tumors.

  13. Insect and wound induced GUS gene expression from a Beta vulgaris proteinase inhibitor gene promoter

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Inducible gene promoters that are specifically activated by pathogen invasion or insect pest attack are needed for effective expression of resistance genes to control plant diseases. In the present study, a promoter from a serine proteinase inhibitor gene (BvSTI) shown to be up-regulated in resist...

  14. Structural basis for decreased induction of class IB PI3-kinases expression by MIF inhibitors.

    PubMed

    Singh, Abhay Kumar; Pantouris, Georgios; Borosch, Sebastian; Rojanasthien, Siripong; Cho, Thomas Yoonsang

    2017-01-01

    Macrophage migration inhibitory factor (MIF) is a master regulator of proinflammatory cytokines and plays pathological roles when not properly regulated in rheumatoid arthritis, lupus, atherosclerosis, asthma and cancer. Unlike canonical cytokines, MIF has vestigial keto-enol tautomerase activity. Most of the current MIF inhibitors were screened for the inhibition of this enzymatic activity. However, only some of the enzymatic inhibitors inhibit receptor-mediated biological functions of MIF, such as cell recruitment, through an unknown molecular mechanism. The goal of this study was to understand the molecular basis underlying the pharmacological inhibition of biological functions of MIF. Here, we demonstrate how the structural changes caused upon inhibitor binding translate into the alteration of MIF-induced downstream signalling. Macrophage migration inhibitory factor activates phosphoinositide 3-kinases (PI3Ks) that play a pivotal role in immune cell recruitment in health and disease. There are several different PI3K isoforms, but little is known about how they respond to MIF. We demonstrate that MIF up-regulates the expression of Class IB PI3Ks in leucocytes. We also demonstrate that MIF tautomerase active site inhibitors down-regulate the expression of Class IB PI3Ks as well as leucocyte recruitment in vitro and in vivo. Finally, based on our MIF:inhibitor complex crystal structures, we hypothesize that the reduction in Class IB PI3K expression occurs because of the displacement of Pro1 towards the second loop of MIF upon inhibitor binding, which results in increased flexibility of the loop 2 and sub-optimal MIF binding to its receptors. These results will provide molecular insights for fine-tuning the biological functions of MIF.

  15. Inhibitors of angiotensin-converting enzyme modulate mitosis and gene expression in pancreatic cancer cells

    SciTech Connect

    Reddy, M.K.; Baskaran, K.; Molteni, A.

    1995-12-01

    The angiotensin-converting enzyme (ACE) inhibitor captopril inhibits mitosis in several cell types that contain ACE and renin activity. In the present study, we evaluated the effect of the ACE inhibitors captopril and CGS 13945 (10{sup {minus}8} to 10{sup {minus}2}M) on proliferation and gene expression in hamster pancreatic duct carcinoma cells in culture. These cells lack renin and ACE activity. Both ACE inhibitors produced a dose-dependent reduction in tumor cell proliferation within 24 hr. Captopril at a concentration of 0.36 mM and CGS 13945 at 150 {mu}M decreased cellular growth rate to approximately half that of the control. Neither drug influenced the viability or the cell cycle distribution of the tumor cells. Slot blot analysis of mRNA for four genes, proliferation associated cell nuclear antigen (PCNA), K-ras, protein kinase C-{Beta} (PKC-{Beta}) and carbonic anhydrase II (CA II) was performed. Both ACE inhibitors increased K-ras expression by a factor of 2, and had no effect on CA II mRNA levels. Captopril also lowered PCNA by 40% and CGS 13945 lowered PKC-{Beta} gene expression to 30% of the control level. The data demonstrate that ACE inhibitors exhibit antimitotic activity and differential gene modulation in hamster pancreatic duct carcinoma cells. The absence of renin and ACE activity in these cells suggests that the antimitotic action of captopril and CGS 13945 is independent of renin-angiotensin regulation. The growth inhibition may occur through downregulation of growth-related gene expression. 27 refs., 5 figs.

  16. Withaferin A Effectively Targets Soluble Vimentin in the Glaucoma Filtration Surgical Model of Fibrosis

    PubMed Central

    Bargagna-Mohan, Paola; Deokule, Sunil P.; Thompson, Kyle; Wizeman, John; Srinivasan, Cidambi; Vooturi, Sunil; Kompella, Uday B.; Mohan, Royce

    2013-01-01

    Withaferin A (WFA) is a natural product that binds to soluble forms of the type III intermediate filament (IF) vimentin. Currently, it is unknown under what pathophysiological contexts vimentin is druggable, as cytoskeltal vimentin-IFs are abundantly expressed. To investigate druggability of vimentin, we exploited rabbit Tenon's capsule fibroblast (RbTCF) cell cultures and the rabbit glaucoma filtration surgical (GFS) model of fibrosis. WFA potently caused G0/G1 cell cycle inhibition (IC50 25 nM) in RbTCFs, downregulating ubiquitin E3 ligase skp2 and inducing p27Kip1 expression. Transforming growth factor (TGF)-ß-induced myofibroblast transformation caused development of cell spheroids with numerous elongated invadopodia, which WFA blocked potently by downregulating soluble vimentin and α-smooth muscle actin (SMA) expression. In the pilot proof-of-concept study using the GFS model, subconjunctival injections of a low WFA dose reduced skp2 expression in Tenon's capsule and increased p27Kip1 expression without significant alteration to vimentin-IFs. This treatment maintains significant nanomolar WFA concentrations in anterior segment tissues that correspond to WFA's cell cycle targeting activity. A ten-fold higher WFA dose caused potent downregulation of soluble vimentin and skp2 expression, but as found in cell cultures, no further increase in p27Kip1 expression was observed. Instead, this high WFA dose potently induced vimentin-IF disruption and downregulated α-SMA expression that mimicked WFA activity in TGF-ß-treated RbTCFs that blocked cell contractile activity at submicromolar concentrations. These findings illuminate that localized WFA injection to ocular tissues exerts pharmacological control over the skp2-p27Kip1 pathway by targeting of soluble vimentin in a model of surgical fibrosis. PMID:23667686

  17. Gene expression profiling in response to the histone deacetylase inhibitor BL1521 in neuroblastoma

    SciTech Connect

    Ruijter, Annemieke J.M. de; Kemp, Stephan . E-mail: a.b.vankuilenburg@amc.uva.nl

    2005-10-01

    Neuroblastoma is a childhood tumor with a poor survival in advanced stage disease despite intensive chemotherapeutic regimes. The new histone deacetylase (HDAC) inhibitor BL1521 has shown promising results in neuroblastoma. Inhibition of HDAC resulted in a decrease in proliferation and metabolic activity, induction of apoptosis and differentiation of neuroblastoma cells. In order to elucidate the mechanism mediating the effects of BL1521 on neuroblastoma cells, we investigated the gene expression profile of an MYCN single copy (SKNAS) and an MYCN amplified (IMR32) neuroblastoma cell line after treatment with BL1521 using the Affymetrix oligonucleotide array U133A. An altered expression of 255 genes was observed in both neuroblastoma cell lines. The majority of these genes were involved in gene expression, cellular metabolism, and cell signaling. We observed changes in the expression of vital genes belonging to the cell cycle (cyclin D1 and CDK4) and apoptosis (BNIP3, BID, and BCL2) pathway in response to BL1521. The expression of 37 genes was altered by both BL1521 and Trichostatin A, which could indicate a common gene set regulated by different HDAC inhibitors. BL1521 treatment changed the expression of a number of MYCN-associated genes. Several genes in the Wnt and the Delta/Notch pathways were changed in response to BL1521 treatment, suggesting that BL1521 is able to induce the differentiation of neuroblastoma cells into a more mature phenotype.

  18. Expression of BMP and Actin Membrane Bound Inhibitor Is Increased during Terminal Differentiation of MSCs

    PubMed Central

    Karl, Alexandra; Berner, Arne; Schmitz, Paul; Koch, Matthias; Nerlich, Michael; Mueller, Michael B.

    2016-01-01

    Chondrogenic differentiating mesenchymal stem cells (MSCs) are mimicking embryonal endochondral ossification and become hypertrophic. BMP (bone morphogenetic protein) and Activin Membrane Bound Inhibitor (BAMBI) is a pseudoreceptor that regulates the activity of transforming growth factor-β (TGF-β) and BMP signalling during chondrogenesis. Both TGF-β and BMP signalling are regulators of chondrogenic cell differentiation. Human bone marrow derived MSCs were chondrogenically predifferentiated in aggregate culture for 14 days. Thereafter, one group was subjected to hypertrophy enhancing media conditions while controls were kept in chondrogenic medium until day 28. Histological evaluation, gene expression by PCR, and Western blot analysis were carried out at days 1, 3, 7, 14, 17, 21, and 28. A subset of cultures was treated with the BMP inhibitor Noggin to test for BMP dependent expression of BAMBI. Hypertrophic differentiated pellets showed larger cells with increased collagen 10 and alkaline phosphatase staining. There was significantly increased expression of BAMBI on gene expression and protein level in hypertrophic cultures compared to the chondrogenic control and increased BMP4 gene expression. Immunohistochemistry showed intense staining of BAMBI in hypertrophic cells. BAMBI expression was dose-dependently downregulated by Noggin. The pseudoreceptor BAMBI is upregulated upon enhancement of hypertrophy in MSC chondrogenic differentiation by a BMP dependent mechanism. PMID:27843458

  19. Hypoxia inducible factor 1α expression and effects of its inhibitors in canine lymphoma

    PubMed Central

    KAMBAYASHI, Satoshi; IGASE, Masaya; KOBAYASHI, Kosuke; KIMURA, Ayana; SHIMOKAWA MIYAMA, Takako; BABA, Kenji; NOGUCHI, Shunsuke; MIZUNO, Takuya; OKUDA, Masaru

    2015-01-01

    Hypoxic conditions in various cancers are believed to relate with their malignancy, and hypoxia inducible factor-1α (HIF-1α) has been shown to be a major regulator of the response to low oxygen. In this study, we examined HIF-1α expression in canine lymphoma using cell lines and clinical samples and found that these cells expressed HIF-1α. Moreover, the HIF-1α inhibitors, echinomycin, YC-1 and 2-methoxyestradiol, suppressed the proliferation of canine lymphoma cell lines. In a xenograft model using NOD/scid mice, echinomycin treatment resulted in a dose-dependent regression of the tumor. Our results suggest that HIF-1α contributes to the proliferation and/or survival of canine lymphoma cells. Therefore, HIF-1α inhibitors may be potential agents to treat canine lymphoma. PMID:26050843

  20. Natural haemozoin induces expression and release of human monocyte tissue inhibitor of metalloproteinase-1.

    PubMed

    Polimeni, Manuela; Valente, Elena; Ulliers, Daniela; Opdenakker, Ghislain; Van den Steen, Philippe E; Giribaldi, Giuliana; Prato, Mauro

    2013-01-01

    Recently matrix metalloproteinase-9 (MMP-9) and its endogenous inhibitor (tissue inhibitor of metalloproteinase-1, TIMP-1) have been implicated in complicated malaria. In vivo, mice with cerebral malaria (CM) display high levels of both MMP-9 and TIMP-1, and in human patients TIMP-1 serum levels directly correlate with disease severity. In vitro, natural haemozoin (nHZ, malarial pigment) enhances monocyte MMP-9 expression and release. The present study analyses the effects of nHZ on TIMP-1 regulation in human adherent monocytes. nHZ induced TIMP-1 mRNA expression and protein release, and promoted TNF-α, IL-1β, and MIP-1α/CCL3 production. Blocking antibodies or recombinant cytokines abrogated or mimicked nHZ effects on TIMP-1, respectively. p38 MAPK and NF-κB inhibitors blocked all nHZ effects on TIMP-1 and pro-inflammatory molecules. Still, total gelatinolytic activity was enhanced by nHZ despite TIMP-1 induction. Collectively, these data indicate that nHZ induces inflammation-mediated expression and release of human monocyte TIMP-1 through p38 MAPK- and NF-κB-dependent mechanisms. However, TIMP-1 induction is not sufficient to counterbalance nHZ-dependent MMP-9 enhancement. Future investigation on proteinase-independent functions of TIMP-1 (i.e. cell survival promotion and growth/differentiation inhibition) is needed to clarify the role of TIMP-1 in malaria pathogenesis.

  1. Identification and expression analysis of BMP signaling inhibitors genes of the DAN family in amphioxus.

    PubMed

    Le Petillon, Yann; Oulion, Silvan; Escande, Marie-Line; Escriva, Hector; Bertrand, Stephanie

    2013-12-01

    Bone morphogenetic proteins (BMPs) are members of the Transforming Growth Factor-β (TGF-β) family implicated in many developmental processes in metazoans such as embryo axes specification. Their wide variety of actions is in part controlled by inhibitors that impede the interaction of BMPs with their specific receptors. Here, we focused our attention on the Differential screening-selected gene Aberrative in Neuroblastoma (DAN) family of inhibitors. Although they are well-characterized in vertebrates, few data are available for this family in other metazoan species. In order to understand the evolution of potential developmental roles of these inhibitors in chordates, we identified the members of this family in the cephalochordate amphioxus, and characterized their expression patterns during embryonic development. Our data suggest that the function of Cerberus/Dand5 subfamily genes is conserved among chordates, whereas Gremlin1/2 and NBL1 subfamily genes seem to have acquired divergent expression patterns in each chordate lineage. On the other hand, the expression of Gremlin in the amphioxus neural plate border during early neurulation strengthens the hypothesis of a conserved neural plate border gene network in chordates.

  2. Effects of the kava chalcone flavokawain A differ in bladder cancer cells with wild-type versus mutant p53.

    PubMed

    Tang, Yaxiong; Simoneau, Anne R; Xie, Jun; Shahandeh, Babbak; Zi, Xiaolin

    2008-11-01

    Flavokawain A is the predominant chalcone from kava extract. We have assessed the mechanisms of flavokawain A's action on cell cycle regulation. In a p53 wild-type, low-grade, and papillary bladder cancer cell line (RT4), flavokawain A increased p21/WAF1 and p27/KIP1, which resulted in a decrease in cyclin-dependent kinase-2 (CDK2) kinase activity and subsequent G(1) arrest. The increase of p21/WAF1 protein corresponded to an increased mRNA level, whereas p27/KIP1 accumulation was associated with the down-regulation of SKP2, which then increased the stability of the p27/KIP1 protein. The accumulation of p21/WAF1 and p27/KIP1 was independent of cell cycle position and thus not a result of the cell cycle arrest. In contrast, flavokawain A induced a G(2)-M arrest in six p53 mutant-type, high-grade bladder cancer cell lines (T24, UMUC3, TCCSUP, 5637, HT1376, and HT1197). Flavokawain A significantly reduced the expression of CDK1-inhibitory kinases, Myt1 and Wee1, and caused cyclin B1 protein accumulation leading to CDK1 activation in T24 cells. Suppression of p53 expression by small interfering RNA in RT4 cells restored Cdc25C expression and down-regulated p21/WAF1 expression, which allowed Cdc25C and CDK1 activation, which then led to a G(2)-M arrest and an enhanced growth-inhibitory effect by flavokawain A. Consistently, flavokawain A also caused a pronounced CDK1 activation and G(2)-M arrest in p53 knockout but not in p53 wild-type HCT116 cells. This selectivity of flavokawain A for inducing a G(2)-M arrest in p53-defective cells deserves further investigation as a new mechanism for the prevention and treatment of bladder cancer.

  3. Skp2 Regulates G2/M Progression in a p53-dependent Manner

    PubMed Central

    Hu, Rong

    2008-01-01

    Targeted proteasomal degradation mediated by E3 ubiquitin ligases controls cell cycle progression, and alterations in their activities likely contribute to malignant cell proliferation. S phase kinase-associated protein 2 (Skp2) is the F-box component of an E3 ubiquitin ligase complex that targets p27Kip1 and cyclin E1 to the proteasome. In human melanoma, Skp2 is highly expressed, regulated by mutant B-RAF, and required for cell growth. We show that Skp2 depletion in melanoma cells resulted in a tetraploid cell cycle arrest. Surprisingly, co-knockdown of p27Kip1 or cyclin E1 failed to prevent the tetraploid arrest induced by Skp2 knockdown. Enhanced Aurora A phosphorylation and repression of G2/M regulators cyclin B1, cyclin-dependent kinase 1, and cyclin A indicated a G2/early M phase arrest in Skp2-depleted cells. Furthermore, expression of nuclear localized cyclin B1 prevented tetraploid accumulation after Skp2 knockdown. The p53 status is most frequently wild type in melanoma, and the tetraploid arrest and down-regulation of G2/M regulatory genes were strongly dependent on wild-type p53 expression. In mutant p53 melanoma lines, Skp2 depletion did not induce cell cycle arrest despite up-regulation of p27Kip1. These data indicate that elevated Skp2 expression may overcome p53-dependent cell cycle checkpoints in melanoma cells and highlight Skp2 actions that are independent of p27Kip1 degradation. PMID:18716061

  4. Effects of the Kava Chalcone Flavokawain A Differ in Bladder Cancer Cells with Wild-type versus Mutant p53

    PubMed Central

    Tang, Yaxiong; Simoneau, Anne R.; Xie, Jun; Shahandeh, Babbak; Zi, Xiaolin

    2010-01-01

    Flavokawain A is the predominant chalcone from kava extract. We have assessed the mechanisms of flavokawain A's action on cell cycle regulation. In a p53 wild-type, low-grade, and papillary bladder cancer cell line (RT4), flavokawain A increased p21/WAF1 and p27/KIP1, which resulted in a decrease in cyclin-dependent kinase-2 (CDK2) kinase activity and subsequent G1 arrest. The increase of p21/WAF1 protein corresponded to an increased mRNA level, whereas p27/KIP1 accumulation was associated with the down-regulation of SKP2 and then increased the stability of the p27/KIP1 protein. The accumulation of p21/WAF1 and p27/KIP1 was independent of cell cycle position and thus not a result of the cell cycle arrest. In contrast, flavokawain A induced a G2-M arrest in six p53 mutant-type, high-grade bladder cancer cell lines (T24, UMUC3, TCCSUP, 5637, HT1376, and HT1197). Flavokawain A significantly reduced the expression of CDK1-inhibitory kinases, Myt1 and Wee1, and caused cyclin B1 protein accumulation leading to CDK1 activation in T24 cells. Suppression of p53 expression by small interfering RNA in RT4 cells restored Cdc25C expression and down-regulated p21/WAF1 expression, which allowed Cdc25C and CDK1 activation and then led to a G2-M arrest and an enhanced growth-inhibitory effect by flavokawain A. Consistently, flavokawain A also caused a pronounced CDK1 activation and G2-M arrest in p53 knockout but not in p53 wild-type HCT116 cells. This selectivity of flavokawain A for inducing a G2-M arrest in p53-defective cells deserves further investigation as a new mechanism for the prevention and treatment of bladder cancer. PMID:19138991

  5. A colostrum trypsin inhibitor gene expressed in the Cape fur seal mammary gland during lactation.

    PubMed

    Pharo, Elizabeth A; Cane, Kylie N; McCoey, Julia; Buckle, Ashley M; Oosthuizen, W H; Guinet, Christophe; Arnould, John P Y

    2016-03-01

    The colostrum trypsin inhibitor (CTI) gene and transcript were cloned from the Cape fur seal mammary gland and CTI identified by in silico analysis of the Pacific walrus and polar bear genomes (Order Carnivora), and in marine and terrestrial mammals of the Orders Cetartiodactyla (yak, whales, camel) and Perissodactyla (white rhinoceros). Unexpectedly, Weddell seal CTI was predicted to be a pseudogene. Cape fur seal CTI was expressed in the mammary gland of a pregnant multiparous seal, but not in a seal in its first pregnancy. While bovine CTI is expressed for 24-48 h postpartum (pp) and secreted in colostrum only, Cape fur seal CTI was detected for at least 2-3 months pp while the mother was suckling its young on-shore. Furthermore, CTI was expressed in the mammary gland of only one of the lactating seals that was foraging at-sea. The expression of β-casein (CSN2) and β-lactoglobulin II (LGB2), but not CTI in the second lactating seal foraging at-sea suggested that CTI may be intermittently expressed during lactation. Cape fur seal and walrus CTI encode putative small, secreted, N-glycosylated proteins with a single Kunitz/bovine pancreatic trypsin inhibitor (BPTI) domain indicative of serine protease inhibition. Mature Cape fur seal CTI shares 92% sequence identity with Pacific walrus CTI, but only 35% identity with BPTI. Structural homology modelling of Cape fur seal CTI and Pacific walrus trypsin based on the model of the second Kunitz domain of human tissue factor pathway inhibitor (TFPI) and porcine trypsin (Protein Data Bank: 1TFX) confirmed that CTI inhibits trypsin in a canonical fashion. Therefore, pinniped CTI may be critical for preventing the proteolytic degradation of immunoglobulins that are passively transferred from mother to young via colostrum and milk.

  6. CT-2576, an inhibitor of phospholipid signaling, suppresses constitutive and induced expression of human immunodeficiency virus.

    PubMed Central

    Leung, D W; Peterson, P K; Weeks, R; Gekker, G; Chao, C C; Kaplan, A H; Balantac, N; Tompkins, C; Underiner, G E; Bursten, S

    1995-01-01

    Viruses such as human immunodeficiency virus (HIV) require cellular activation for expression. Cellular activation in lymphoid cells is associated with augmented accumulation of certain phosphatidic acid (PA) species derived from the hydrolysis of glycan phosphatidylinositol (GPI). This suggests that activation of a phospholipid pathway may play a role in initiation of viral replication. To test this hypothesis, we examined the effect of tat gene expression on the production of cellular PA species, as the Tat protein is essential for HIV expression and has been implicated in activating the expression of multiple host cellular genes. Expression of tat increased the expression of PA. We then tested whether synthetic inhibitors of PA metabolism would inhibit activation of the HIV long terminal repeat by Tat and tumor necrosis factor alpha (TNF-alpha). CT-2576 suppressed both PA generation induced by Tat and HIV long terminal repeat-directed gene expression in response to Tat or TNF-alpha at a posttranscriptional step. CT-2576 also inhibited constitutive as well as TNF-alpha- and interleukin 6-induced expression of HIV p24 antigen in chronically infected U1 cells and in peripheral blood lymphocytes acutely infected with a clinical isolate of HIV. Pharmacological inhibition of synthesis of selected PA species may therefore provide a therapeutic approach to suppression of HIV replication. Images Fig. 1 Fig. 3 Fig. 5 PMID:7761405

  7. Role of the phosphatidylinositol 3-kinase/Akt and mTOR/P70S6-kinase pathways in the proliferation and apoptosis in multiple myeloma.

    PubMed

    Pene, Frédéric; Claessens, Yann-Erick; Muller, Odile; Viguié, Franck; Mayeux, Patrick; Dreyfus, François; Lacombe, Catherine; Bouscary, Didier

    2002-09-26

    Multiple myeloma (MM) is a plasma cell malignancy preliminary localized in the bone marrow and characterized by its capacity to disseminate. IL-6 and IGF-1 have been shown to mediate proliferative and anti-apoptotic signals in plasmocytes. However, in primary plasma-cell leukemia (PCL) and in end-stage aggressive extramedullar disease, the cytokine requirement for both effects may be not mandatory. This suggests that constitutive activation of signaling pathways occurs. One of the signaling pathways whose deregulation may play an oncogenic role in MM is the phosphatidylinositol 3-kinase (PI 3-K) pathway. In human growth factor-independent MM cell lines OPM2 and RPMI8226, we show that the PI 3-K inhibitors LY294002 and Wortmannin strongly inhibited cell proliferation, whereas inhibition of the mammalian Target Of Rapamycin (mTOR)/P70-S6-kinase (P70(S6K)) pathway with rapamycin or of the Mitogen-Activated Protein Kinase (MAPK) pathway with PD98059 had minimal effect on proliferation. In both cell lines, constitutive activation of the PI 3-K/Akt/FKHRL-1, mTOR/P70(S6K) and MAPK pathways was detected. LY294002 inhibited phosphorylation of Akt, FKHRL-1 and P70(S6K) but had no effect on ERK1/2 phosphorylation, indicating that the PI 3-K and MAPK pathways are independent. IGF-1 but not IL-6 increased phosphorylation of Akt, FKHRL-1 and P70(S6K). Purified plasmocytes from four patients with MM and two patients with primary PCL were studied. In three of them including the two patients with PCL, constitutive phosphorylation of Akt, FKHRL-1 and P70(S6K) was present, inhibited by LY294002 and enhanced by IGF-1. In these patients with constitutive Akt activation, normal PTEN expression was detected. PI 3-K inhibition induced caspase-dependent apoptosis as confirmed by inhibition with the large spectrum caspase inhibitor Z-VAD-FMK and cleavage of pro-caspase-3. Both cell lines spontaneously expressed Skp2 and cyclin D1 proteins at high levels but no p27(Kip1) protein. In the

  8. The novel ATP-competitive MEK/Aurora kinase inhibitor BI-847325 overcomes acquired BRAF inhibitor resistance through suppression of Mcl-1 and MEK expression

    PubMed Central

    Phadke, Manali S.; Sini, Patrizia; Smalley, Keiran S. M.

    2015-01-01

    Resistance to BRAF inhibitors is a major clinical problem. Here we evaluate BI-847325, an ATP-competitive inhibitor of MEK and Aurora kinases, in treatment-naïve and drug-resistant BRAF-mutant melanoma models. BI-847325 potently inhibited growth and survival of melanoma cell lines that were both BRAF inhibitor naïve and resistant in 2D culture, 3D cell culture conditions and in colony formation assays. Western blot studies showed BI-847325 to reduce expression of phospho-ERK and phospho-histone 3 in multiple models of vemurafenib resistance. Mechanistically, BI-847325 decreased the expression of MEK and Mcl-1 while increasing the expression of the pro-apoptotic protein BIM. Strong suppression of MEK expression was observed after 48 h of treatment, with no recovery following >72 h of washout. siRNA mediated knockdown of Mcl-1 enhanced the effects of BI-847325, whereas Mcl-1 overexpression reversed this in both 2D cell culture and 3D spheroid melanoma models. In vivo, once weekly BI-847325 (70 mg/kg) led to durable regression of BRAF-inhibitor naive xenografts with no regrowth seen (>65 days of treatment). In contrast, treatment with the vemurafenib analog PLX4720 was associated with tumor relapse at >30 days. BI-847325 also suppressed the long-term growth of xenografts with acquired PLX4720 resistance. Analysis of tumor samples revealed BI-847325 to induce apoptosis associated with suppression of phospho-ERK, total MEK, phospho-Histone3 and Mcl-1 expression. Our studies indicate that BI-847325 is effective in overcoming BRAF inhibitor resistance and has long-term inhibitory effects upon BRAF-mutant melanoma in vivo, through a mechanism associated with the decreased expression of both MEK and Mcl-1. PMID:25873592

  9. Recombinant expression, purification, and kinetic and inhibitor characterisation of human site-1-protease.

    PubMed

    Bodvard, Kristofer; Mohlin, Johanna; Knecht, Wolfgang

    2007-02-01

    Human site-1-protease (S1P, MEROPS S08.8063), also widely known as subtilisin/kexin isozyme 1 (SKI-1), is a membrane bound subtilisin-related serine protease, that belongs to a group of nine mammalian proprotein convertases. Among these proteases, S1P displays unique substrate specificity, by showing preferred cleavage after non-basic amino acids. S1P plays a key role in a proteolytic pathway that controls the cholesterol content of membranes, cells and blood. S1P also participates in the activation of viral coat glycoproteins of the lassa virus, the lympocytic choriomeningitis virus and the crimean congo hemorrhagic fever virus. We expressed recombinant human S1P using the baculovirus expression vector system and characterized the highly purified enzyme. Featuring a new chromogenic substrate (Acetyl-Arg-Arg-Leu-Leu-p-nitroanilide) we show that the enzymatic activity of S1P is not calcium dependent, but can be modulated by a variety of mono- and divalent cations. S1P displayed pronounced positive cooperativity with a substrate derived from the viral coat glycoprotein of the lassa virus. The screening of a limited number of protease inhibitors showed that S1P was not inhibited by specific inhibitors of other proprotein convertases or by Pefabloc SC (4-(2-aminoethyl) benzene sulphonyl fluoride, AEBSF). We found 3,4-dichloroisocoumarin (DCI) to be a potent slow binding inhibitor of human S1P, with a K(iapp) = 6.8 microM, thus representing a new small molecule inhibitor of S1P. These findings show that S1P differs significantly from other proprotein convertases with respect to kinetics, co-factor requirement and inhibition.

  10. Melatonin as a negative mitogenic hormonal regulator of human prostate epithelial cell growth: potential mechanisms and clinical significance.

    PubMed

    Tam, Chun W; Chan, Kwok W; Liu, Vincent W S; Pang, Bo; Yao, Kwok-Ming; Shiu, Stephen Y W

    2008-11-01

    Circannual variation in the human serum levels of prostate-specific antigen, a growth marker of the prostate gland, has been reported recently. The present study was conducted to investigate the role of the photoperiodic hormone melatonin (MLT) and its membrane receptors in the modulation of human prostate growth. Expression of MT(1) and MT(2) receptors was detected in benign human prostatic epithelial tissues and RWPE-1 cells. MLT and 2-iodomelatonin inhibited RWPE-1 cell proliferation and up-regulated p27(Kip1) gene and protein expression in the cells. The effects of MLT were blocked by the nonselective MT(1)/MT(2) receptor antagonist luzindole, but were not affected by the selective MT(2) receptor antagonist 4-phenyl-2-propionamidotetraline. Of note, the antiproliferative action of MLT on benign prostate epithelial RWPE-1 cells was effected via increased p27(Kip1) gene transcription through MT(1) receptor-mediated activation of protein kinase A (PKA) and protein kinase C (PKC) in parallel, a signaling process which has previously been demonstrated in 22Rv1 prostate cancer cells. Taken together, the demonstration of the MT(1)/PKA+PKC/p27(Kip1) antiproliferative pathway in benign and malignant prostate epithelial cell lines indicated the potential importance of this MLT receptor-mediated signaling mechanism in growth regulation of the human prostate gland in health and disease. Collectively, our data support the hypothesis that MLT may function as a negative mitogenic hormonal regulator of human prostate epithelial cell growth.

  11. Insulin-like growth factor-I extends in vitro replicative life span of skeletal muscle satellite cells by enhancing G1/S cell cycle progression via the activation of phosphatidylinositol 3'-kinase/Akt signaling pathway

    NASA Technical Reports Server (NTRS)

    Chakravarthy, M. V.; Abraha, T. W.; Schwartz, R. J.; Fiorotto, M. L.; Booth, F. W.

    2000-01-01

    Interest is growing in methods to extend replicative life span of non-immortalized stem cells. Using the insulin-like growth factor I (IGF-I) transgenic mouse in which the IGF-I transgene is expressed during skeletal muscle development and maturation prior to isolation and during culture of satellite cells (the myogenic stem cells of mature skeletal muscle fibers) as a model system, we elucidated the underlying molecular mechanisms of IGF-I-mediated enhancement of proliferative potential of these cells. Satellite cells from IGF-I transgenic muscles achieved at least five additional population doublings above the maximum that was attained by wild type satellite cells. This IGF-I-induced increase in proliferative potential was mediated via activation of the phosphatidylinositol 3'-kinase/Akt pathway, independent of mitogen-activated protein kinase activity, facilitating G(1)/S cell cycle progression via a down-regulation of p27(Kip1). Adenovirally mediated ectopic overexpression of p27(Kip1) in exponentially growing IGF-I transgenic satellite cells reversed the increase in cyclin E-cdk2 kinase activity, pRb phosphorylation, and cyclin A protein abundance, thereby implicating an important role for p27(Kip1) in promoting satellite cell senescence. These observations provide a more complete dissection of molecular events by which increased local expression of a growth factor in mature skeletal muscle fibers extends replicative life span of primary stem cells than previously known.

  12. Expression of ADAMs and their inhibitors in sputum from patients with asthma.

    PubMed

    Paulissen, Geneviève; Rocks, Natacha; Quesada-Calvo, Florence; Gosset, Philippe; Foidart, Jean-Michel; Noel, Agnès; Louis, Renaud; Cataldo, Didier D

    2006-01-01

    ADAMs (a disintegrin and metalloprotease) constitute a family of cell surface proteins containing disintegrin and metalloprotease domains which associate features of adhesion molecules and proteases. ADAMTSs (a disintegrin and metalloprotease with thrombospondin motifs) bear thrombospondin type I motifs in C-terminal extremity, and most of them are secreted proteins. Because genetic studies have shown that ADAM-33 gene polymorphisms are associated with asthma, we designed this study to assess mRNA expression profile of several ADAM and ADAMTS proteases in sputum from patients with asthma and to investigate the relationship between expression of these proteases and asthma-associated inflammation and airway obstruction. mRNA expression profile of selected ADAM and ADAMTS proteinases (ADAM-8, -9, -10, -12, -15, -17, and -33; ADAMTS-1, -2, -15, -16, -17, -18, and -19), their physiological inhibitors TIMP-1 and TIMP-3, and RECK, a membrane-anchored MMP activity regulator, was obtained by RT-PCR analysis performed on cells collected by sputum induction from 21 patients with mild to moderate asthma and 17 healthy individuals. mRNA levels of ADAM-8, ADAM-9, ADAM-12, TIMP-1, and TIMP-3 were significantly increased, whereas mRNA levels coding for ADAMTS-1, ADAMTS-15, and RECK were significantly decreased in patients with asthma compared with control patients. ADAM-8 expression was negatively correlated with the forced expiratory volume at the first second (FEV(1)) (r = -0.57, P < 0.01), whereas ADAMTS-1 and RECK expressions were positively correlated to FEV(1) (r = 0.45, P < 0.05, and r = 0.55, P = 0.01, respectively). We conclude that expression of ADAMs and ADAMTSs and their inhibitors is modulated in airways from patients with asthma and that these molecules may play a role in the pathogenesis of asthma.

  13. Salinomycin induces cell death via inactivation of Stat3 and downregulation of Skp2.

    PubMed

    Koo, K H; Kim, H; Bae, Y-K; Kim, K; Park, B-K; Lee, C-H; Kim, Y-N

    2013-06-27

    Salinomycin has been shown to control breast cancer stem cells, although the mechanisms underlying its anticancer effects are not clear. Deregulation of cell cycle regulators play critical roles in tumorigenesis, and they have been considered as anticancer targets. In this study, we investigated salinomycin effect on cell cycle progression using OVCAR-8 ovarian cancer cell line and multidrug-resistant NCI/ADR-RES and DXR cell lines that are derived from OVCAR-8. Parental OVCAR-8 cells are sensitive to several anticancer drugs, but NCI/ADR-RES and DXR cells are resistant to several anticancer drugs. However, salinomycin caused cell growth inhibition and apoptosis via cell cycle arrest at G1 in all three cell lines. Salinomycin inhibited signal transducer and activator of transcription 3 (Stat3) activity and thus decreased expression of Stat3-target genes, including cyclin D1, Skp2, and survivin. Salinomycin induced degradation of Skp2 and thus accumulated p27Kip1. Knockdown of Skp2 further increased salinomycin-induced G1 arrest, but knockdown of p27Kip1 attenuated salinomycin effect on G1 arrest. Cdh1, an E3 ligase for Skp2, was shifted to nuclear fractions upon salinomycin treatment. Cdh1 knockdown by siRNA reversed salinomycin-induced Skp2 downregulation and p27Kip1 upregulation, indicating that salinomycin activates the APC(Cdh1)-Skp2-p27Kip1 pathway. Concomitantly, si-Cdh1 inhibited salinomycin-induced G1 arrest. Taken together, our data indicate that salinomycin induces cell cycle arrest and apoptosis via downregulation or inactivation of cell cycle-associated oncogenes, such as Stat3, cyclin D1, and Skp2, regardless of multidrug resistance.

  14. Altered expression of metalloproteinase-2 and tissue inhibitor of metalloproteinase-2 in cervical disc herniation patients.

    PubMed

    Zhuang, H M; Xu, G T; Wen, S F; Guo, Y Y; Huang, Q

    2016-04-26

    The aim of the current study was to examine matrix metalloproteinase-2 (MMP-2) and tissue inhibitor of metalloproteinase-2 (TIMP-2) expression in patients with cervical disc herniation (CDH). A total of 127 specimens from CDH patients undergoing posterior spinal surgery were obtained for the case group, which was divided into three subgroups: lateral protrusion (N = 102), median protrusion (N = 18), and paramedian protrusion (N = 7). Another 55 specimens from subjects who had cervical spine trauma and underwent spinal canal decompression were obtained for the control group. Routine hematoxylin and eosin staining was performed for pathological diagnosis. Immunohistochemical (IHC) analysis was used to determine MMP-2 and TIMP-2 expression. Under light microscopy, MMP-2-positive cells presented brown-yellow or dark brown staining in the cell membrane or cytoplasm. MMP-2 expression in the case group was significantly higher than that in controls (P < 0.05). Furthermore, MMP-2 expression in the lateral and median protrusion groups was significantly higher compared to that in the paramedian protrusion group (both P < 0.05), while there was no apparent difference in MMP-2 expression between the lateral and median protrusion groups (P > 0.05). IHC results showed that TIMP-2 expression in cases was significantly lower than that in controls (P < 0.05). Spearman correlation analysis indicated that MMP- 2 was negatively correlated with TIMP-2 expression (r = -0.418, P < 0.001). In conclusion, MMP-2 expression increased, whereas TIMP- 2 expression decreased in CDH patients, suggesting that MMP-2 and TIMP-2 expression may contribute to CDH development.

  15. Tissue inhibitor of metalloproteinase-1 and -2 RNA expression in rat and human liver fibrosis.

    PubMed Central

    Herbst, H.; Wege, T.; Milani, S.; Pellegrini, G.; Orzechowski, H. D.; Bechstein, W. O.; Neuhaus, P.; Gressner, A. M.; Schuppan, D.

    1997-01-01

    The remodeling of extracellular matrix during chronic liver disease may partially be attributed to altered activity of matrix metalloproteinases and their tissue inhibitors (TIMPs). Expression of TIMP-1 and -2 was studied by in situ hybridization combined with immunohistochemistry in rat (acute and chronic carbon tetrachloride intoxication and secondary biliary fibrosis) and human livers and on isolated rat hepatic stellate cells. TIMP-1 and -2 transcripts appeared in rat livers within 1 to 3 hours after intoxication, pointing to a role in the protection against accidental activation of matrix metalloproteinases, and were present at high levels in all fibrotic rat and human livers predominantly in stellate cells. TIMP-2 RNA distribution largely matched with previously reported patterns of matrix metalloproteinase-2 (72-kd gelatinase) expression, suggesting generation of a TIMP-2/matrix metalloproteinase-2 complex (large inhibitor of metalloproteinases). Isolated stellate cells expressed TIMP-1 and -2 RNA. Addition of transforming growth factor-beta 1 enhanced TIMP-1 and matrix metalloproteinase-2 RNA levels in vitro, whereas TIMP-2-specific signals were reduced, likely to result in a stoichiometric excess of matrix-metalloproteinase-2 over TIMP-2. In the context of previous demonstrations of transforming growth factor-beta 1 and matrix metalloproteinase-2 in vivo, these patterns suggest an intrahepatic environment permitting only limited matrix degradation, ultimately resulting in redistribution of extracellular matrix with relative accumulation of collagen type 1. Images Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 PMID:9137090

  16. Proton pump inhibitor induced collagen expression in colonocytes is associated with collagenous colitis

    PubMed Central

    Mori, Shiori; Kadochi, Yui; Luo, Yi; Fujiwara-Tani, Rina; Nishiguchi, Yukiko; Kishi, Shingo; Fujii, Kiyomu; Ohmori, Hitoshi; Kuniyasu, Hiroki

    2017-01-01

    AIM To elucidate the role of proton pump inhibitors (PPIs) in collagenous disease, direct effect of PPI on colonocytes was examined. METHODS Collagenous colitis is a common cause of non-bloody, watery diarrhea. Recently, there has been increasing focus on the use of proton PPIs as a risk factor for developing collagenous colitis. Mouse CT26 colonic cells were treated with PPI and/or PPI-induced alkaline media. Expression of fibrosis-associated genes was examined by RT-PCR. In human materials, collagen expression was examined by immunohistochemistry. RESULTS CT26 cells expressed a Na+-H+ exchanger gene (solute carrier family 9, member A2). Treatment with PPI and/or PPI-induced alkaline media caused growth inhibition and oxidative stress in CT26 cells. The treatment increased expression of fibrosis inducing factors, transforming growth factor β and fibroblast growth factor 2. The treatment also decreased expression of a negative regulator of collagen production, replication factor C1, resulting in increased expression of collagen types III and IV in association with lipid peroxide. In biopsy specimens from patients with collagenous colitis, type III and IV collagen were increased. Increase of type III collagen was more pronounced in PPI-associated collagenous colitis than in non-PPI-associated disease. CONCLUSION From these findings, the reaction of colonocytes to PPI might participate in pathogenesis of collagenous colitis. PMID:28321159

  17. Expression of X-linked Inhibitor of Apoptosis Protein in Neoplastic Thyroid Disorder

    PubMed Central

    Yim, Ji Hye; Kim, Sun A; Kim, Won Gu; Jeon, Min Ji; Han, Ji Min; Sung, Tae Yon; Kim, Tae Yong; Kim, Won Bae; Hong, Suck Joon; Shong, Young Kee; Gong, Gyungyub

    2011-01-01

    X-linked inhibitor of apoptosis protein (XIAP) is associated with tumor genesis, growth, progression and metastasis, and acts by blocking caspase-mediated apoptosis. In the present study, we sought to evaluate the expression patterns of XIAP in various neoplastic thyroid disorders and determine the association between XIAP expression and clinicopathologic factors. Expression of XIAP was evaluated with immunohistochemical staining using monoclonal anti-XIAP in 164 specimens of conventional papillary thyroid carcinoma (PTC) and 53 specimens of other malignant or benign thyroid tumors. XIAP positivity was observed in 128 (78%) of the 164 conventional PTC specimens. Positive rates of XIAP expression in follicular variant PTC, follicular, medullary, poorly differentiated, and anaplastic thyroid carcinoma specimens were 20%, 25%, 38%, 67%, and 38%, respectively. Six nodular hyperplasia specimens were negative and 1 of 7 follicular adenomas (8%) was positive for XIAP. Lateral neck lymph node metastases were more frequent in patients negative for XIAP expression (P = 0.01). Immunohistochemical staining for XIAP as a novel molecular marker may thus be helpful in the differential diagnosis of thyroid cancer. Moreover, high XIAP expression in conventional PTC is strongly associated with reduced risk of lateral neck lymph node metastasis. PMID:21935275

  18. Modulators of inhibitor of growth (ING) family expression in development and disease.

    PubMed

    Maher, Stacey K; Helbing, Caren C

    2009-05-01

    The inhibitor of growth (ING) gene family proteins regulate many critical cellular processes such as cell proliferation and growth, apoptosis, DNA repair, senescence, angiogenesis, and drug resistance. Their transcripts and proteins are differentially expressed in health and disease and there is evidence for developmental regulation. The vast majority of studies have characterized ING levels in the context of cancer. However, relatively little attention has been paid to the expression of ING family members in other contexts. This review summarizes the findings from human and animal model systems that provide insight into the factors influencing the expression of these important proteins. We examine the influence of cell cycle and aging as well as genotoxic stress on ING expression levels and evaluate several emerging areas of inquiry demonstrating that ING gene activity may be modulated by factors such as the p53 tumor suppressor, DNA methylation, and ING proteins themselves with external factors such as hormones, reactive oxygen species, TGFbeta signalling, and other proteins of pathological significance also influencing ING levels. We then briefly discuss the influence of post-translational modification and changes in subcellular localization as it pertains to modulation of ING expression. Understanding how ING expression is modulated represents a vital aspect of effective drug targeting strategies.

  19. Gastrin stimulates expression of plasminogen activator inhibitor-1 in gastric epithelial cells.

    PubMed

    Nørsett, Kristin G; Steele, Islay; Duval, Cedric; Sammut, Stephen J; Murugesan, Senthil V M; Kenny, Susan; Rainbow, Lucille; Dimaline, Rod; Dockray, Graham J; Pritchard, D Mark; Varro, Andrea

    2011-09-01

    Plasminogen activator inhibitor (PAI)-1 is associated with cancer progression, fibrosis and thrombosis. It is expressed in the stomach but the mechanisms controlling its expression there, and its biological role, are uncertain. We sought to define the role of gastrin in regulating PAI-1 expression and to determine the relevance for gastrin-stimulated cell migration and invasion. In gastric biopsies from subjects with elevated plasma gastrin, the abundances of PAI-1, urokinase plasminogen activator (uPA), and uPA receptor (uPAR) mRNAs measured by quantitative PCR were increased compared with subjects with plasma concentrations in the reference range. In patients with hypergastrinemia due to autoimmune chronic atrophic gastritis, there was increased abundance of PAI-1, uPA, and uPAR mRNAs that was reduced by octreotide or antrectomy. Immunohistochemistry revealed localization of PAI-1 to parietal cells and enterochromaffin-like cells in micronodular neuroendocrine tumors in hypergastrinemic subjects. Transcriptional mechanisms were studied by using a PAI-1-luciferase promoter-reporter construct transfected into AGS-G(R) cells. There was time- and concentration-dependent increase of PAI-1-luciferase expression in response to gastrin that was reversed by inhibitors of the PKC and MAPK pathways. In Boyden chamber assays, recombinant PAI-1 inhibited gastrin-stimulated AGS-G(R) cell migration and invasion, and small interfering RNA treatment increased responses to gastrin. We conclude that elevated plasma gastrin concentrations are associated with increased expression of gastric PAI-1, which may act to restrain gastrin-stimulated cell migration and invasion.

  20. Neurotropic and neuroprotective activities of the earthworm peptide Lumbricusin.

    PubMed

    Kim, Dae Hong; Lee, Ik Hwan; Nam, Seung Taek; Hong, Ji; Zhang, Peng; Hwang, Jae Sam; Seok, Heon; Choi, Hyemin; Lee, Dong Gun; Kim, Jae Il; Kim, Ho

    2014-06-06

    We recently isolated a polypeptide from the earthworm Lumbricus terrestris that is structurally similar to defensin, a well-known antibacterial peptide. An 11-mer antibacterial peptide (NH2-RNRRWCIDQQA), designated Lumbricusin, was synthesized based on the amino acid sequence of the isolated polypeptide. Since we previously reported that CopA3, a dung beetle peptide, enhanced neuronal cell proliferation, we here examined whether Lumbricusin exerted neurotropic and/or neuroprotective effects. Lumbricusin treatment induced a time-dependent increase (∼51%) in the proliferation of human neuroblastoma SH-SY5Y cells. Lumbricusin also significantly inhibited the apoptosis and decreased viability induced by treatment with 6-hydroxy dopamine, a Parkinson's disease-mimicking agent. Immunoblot analyses revealed that Lumbricusin treatment increased ubiquitination of p27(Kip1) protein, a negative regulator of cell-cycle progression, in SH-SY5Y cells, and markedly promoted its degradation. Notably, adenoviral-mediated over-expression of p27(Kip1) significantly blocked the antiapoptotic effect of Lumbricusin in 6-hydroxy dopamine-treated SH-SY5Y cells. These results suggest that promotion of p27(Kip1) degradation may be the main mechanism underlying the neuroprotective and neurotropic effects of Lumbricusin.

  1. The Calpain Inhibitor MDL28170 Induces the Expression of Apoptotic Markers in Leishmania amazonensis Promastigotes

    PubMed Central

    Marinho, Fernanda A.; Gonçalves, Keyla C. S.; Oliveira, Simone S. C.; Gonçalves, Diego S.; Matteoli, Filipe P.; Seabra, Sergio H.; Oliveira, Ana Carolina S.; Bellio, Maria; Oliveira, Selma S.; Souto-Padrón, Thaïs; d'Avila-Levy, Claudia M.; Santos, André L. S.; Branquinha, Marta H.

    2014-01-01

    Background Human cutaneous leishmaniasis is caused by distinct species, including Leishmania amazonensis. Treatment of cutaneous leishmaniasis is far from satisfactory due to increases in drug resistance and relapses, and toxicity of compounds to the host. As a consequence for this situation, the development of new leishmanicidal drugs and the search of new targets in the parasite biology are important goals. Methodology/Principal Findings In this study, we investigated the mechanism of death pathway induced by the calpain inhibitor MDL28170 on Leishmania amazonensis promastigote forms. The combined use of different techniques was applied to contemplate this goal. MDL28170 treatment with IC50 (15 µM) and two times the IC50 doses induced loss of parasite viability, as verified by resazurin assay, as well as depolarization of the mitochondrial membrane, which was quantified by JC-1 staining. Scanning and transmission electron microscopic images revealed drastic alterations on the parasite morphology, some of them resembling apoptotic-like death, including cell shrinking, surface membrane blebs and altered chromatin condensation pattern. The lipid rearrangement of the plasma membrane was detected by Annexin-V labeling. The inhibitor also induced a significant increase in the proportion of cells in the sub-G0/G1 phase, as quantified by propidium iodide staining, as well as genomic DNA fragmentation, detected by TUNEL assay. In cells treated with MDL28170 at two times the IC50 dose, it was also possible to observe an oligonucleossomal DNA fragmentation by agarose gel electrophoresis. Conclusions/Significance The data presented in the current study suggest that MDL28170 induces apoptotic marker expression in promastigotes of L. amazonensis. Altogether, the results described in the present work not only provide a rationale for further exploration of the mechanism of action of calpain inhibitors against trypanosomatids, but may also widen the investigation of the

  2. PD-1 inhibits T cell proliferation by upregulating p27 and p15 and suppressing Cdc25A.

    PubMed

    Patsoukis, Nikolaos; Sari, Duygu; Boussiotis, Vassiliki A

    2012-12-01

    The programmed cell death-1 (PD)-1 receptor (CD279) is a potent T cell inhibitor with a critical role in peripheral tolerance, but it can also compromise anti-viral and antitumor T cell responses. The effects of PD-1 on the cell cycle leading to inhibition of T cell expansion are poorly understood. Recently, we examined the effects of PD-1 on the molecular control of the cell cycle machinery and on TCR-activated signaling pathways that regulate these downstream outcomes. Our studies showed that PD-1 blocks cell cycle progression in the G 1 phase. PD-1 did not alter the expression of G 1 phase cyclins or cyclin-dependent kinases (Cdks) but, instead, suppressed the transcription of SKP2, the substrate recognition component of the SCF (Skp2) ubiquitin ligase that leads p27 (kip1) to degradation and resulted in accumulation of p27 (kip1) . Subsequently, T cells receiving PD-1 signals displayed impaired Cdk2 activation and failed to phosphorylate two critical Cdk2 substrates, the retinoblastoma gene product (Rb) and the TGFβ-specific transcription factor Smad3, leading to suppression of E2F target genes but enhanced Smad3 transactivation. These events resulted in upregulation of the Cdk4/6 inhibitor p15 (INK4B) and repression of the Cdk-activating phosphatase Cdc25A. The suppressive effect of PD-1 on Skp2 expression was mediated by inhibition of both PI3K/Akt and Ras/MEK/Erk pathways and was only partially reversed by IL-2, which restored activation of MEK/Erk but not Akt. Thus, PD-1 targets Ras and PI3K/Akt signaling to inhibit transcription of Skp2 and to activate Smad3 as an integral component of a pathway that regulates blockade of cell cycle progression in T lymphocytes. Here, we discuss the detailed sequence of these signaling events and their implications in mediating cell-intrinsic and -extrinsic mechanisms that inhibit proliferation of T effector cells in response to PD-1-mediated signaling.

  3. Tigutcystatin, a cysteine protease inhibitor from Triatoma infestans midgut expressed in response to Trypanosoma cruzi

    SciTech Connect

    Buarque, Diego S.; Spindola, Leticia M.N.; Martins, Rafael M.; Braz, Gloria R.C.; Tanaka, Aparecida S.

    2011-09-23

    Highlights: {yields} Tigutcystatin inhibits Trypanosoma cruzi cysteine proteases with high specificity. {yields} Tigutcystatin expression is up-regulated in response to T. cruzi infection. {yields} It is the first cysteine proteases inhibitor characterized from a triatomine insect. -- Abstract: The insect Triatoma infestans is a vector of Trypanosoma cruzi, the etiological agent of Chagas disease. A cDNA library was constructed from T. infestans anterior midgut, and 244 clones were sequenced. Among the EST sequences, an open reading frame (ORF) with homology to a cystatin type 2 precursor was identified. Then, a 288-bp cDNA fragment encoding mature cystatin (lacking signal peptide) named Tigutcystatin was cloned fused to a N-terminal His tag in pET-14b vector, and the protein expressed in Escherichia coli strain Rosetta gami. Tigutcystatin purified and cleaved by thrombin to remove His tag presented molecular mass of 11 kDa and 10,137 Da by SDS-PAGE and MALDI-TOF mass spectrometry, respectively. Purified Tigutcystatin was shown to be a tight inhibitor towards cruzain, a T. cruzi cathepsin L-like enzyme (K{sub i} = 3.29 nM) and human cathepsin L (K{sub i} = 3.78 nM). Tissue specific expression analysis showed that Tigutcystatin was mostly expressed in anterior midgut, although amplification in small intestine was also detected by semi quantitative RT-PCR. qReal time PCR confirmed that Tigutcystatin mRNA is significantly up-regulated in anterior midgut when T. infestans is infected with T. cruzi. Together, these results indicate that Tigutcystatin may be involved in modulation of T. cruzi in intestinal tract by inhibiting parasite cysteine proteases, which represent the virulence factors of this protozoan.

  4. Expression and localization of Inter-alpha Inhibitors in rodent brain.

    PubMed

    Chen, X; Rivard, L; Naqvi, S; Nakada, S; Padbury, J F; Sanchez-Esteban, J; Stopa, E G; Lim, Y-P; Stonestreet, B S

    2016-06-02

    Inter-alpha Inhibitor Proteins (IAIPs) are a family of related serine protease inhibitors. IAIPs are important components of the systemic innate immune system. We have identified endogenous IAIPs in the central nervous system (CNS) of sheep during development and shown that treatment with IAIPs reduces neuronal cell death and improves behavioral outcomes in neonatal rats after hypoxic-ischemic brain injury. The presence of IAIPs in CNS along with their exogenous neuroprotective properties suggests that endogenous IAIPs could be part of the innate immune system in CNS. The purpose of this study was to characterize expression and localization of IAIPs in CNS. We examined cellular expressions of IAIPs in vitro in cultured cortical mouse neurons, in cultured rat neurons, microglia, and astrocytes, and in vivo on brain sections by immunohistochemistry from embryonic (E) day 18 mice and postnatal (P) day 10 rats. Cultured cortical mouse neurons expressed the light chain gene Ambp and heavy chain genes Itih-1, 2, 3, 4, and 5 mRNA transcripts and IAIP proteins. IAIP proteins were detected by immunohistochemistry in cultured cells as well as brain sections from E18 mice and P10 rats. Immunoreactivity was found in neurons, microglia, astrocytes and oligodendroglia in multiple brain regions including cortex and hippocampus, as well as within both the ependyma and choroid plexus. Our findings suggest that IAIPs are endogenous proteins expressed in a wide variety of cell types and regions both in vitro and in vivo in rodent CNS. We speculate that endogenous IAIPs may represent endogenous neuroprotective immunomodulatory proteins within the CNS.

  5. Cloning, expression and characterization of Bauhinia variegata trypsin inhibitor BvTI.

    PubMed

    de Souza, Adriana F; Torquato, Ricardo J S; Tanaka, Aparecida S; Sampaio, Claudio A M

    2005-11-01

    A Bauhinia variegata trypsin inhibitor (BvTI) cDNA fragment was cloned into the pCANTAB5E phagemid. The clone pAS 1.1.3 presented a cDNA fragment of 733 bp, including the coding region for a mature BvTI protein comprising 175 amino acid residues. The deduced amino acid sequence for BvTI confirmed it as a member of the Kunitz-type plant serine proteinase inhibitor family. The BvTI cDNA fragment encoding the mature form was cloned into the expression vector, pET-14b, and ex-pressed in E. coli BL21 (DE3) pLysS in an active form. In addition, a BvTI mutant form, r(mut)BvTI, with a Pro residue as the fifth amino acid in place of Leu, was produced. The recombinant proteins, rBvTI and r(mut)BvTI, were purified on a trypsin-Sepharose column, yielding 29 and 1.44 mg/l of active protein, respectively, and showed protein bands of approximately 21.5 kDa by SDS-PAGE. Trypsin inhibition activity was comparable for rBvTI (Ki=4 nM) and r(mut)BvTI (Ki=6 nM). Our data suggest that the Leu to Pro substitution at the fifth amino-terminal residue was not crucial for proteinase inhibition.

  6. Transcriptional upregulation of the human MRP2 gene expression by serine/threonine protein kinase inhibitors.

    PubMed

    Pułaski, L; Szemraj, J; Uchiumi, T; Kuwano, M; Bartosz, G

    2005-01-01

    Transcriptional regulation by cellular signalling pathways of multidrug resistance proteins that pump anticancer drugs out of cells is one of key issues in the development of the multidrug resistance phenotype. In our study, we have used the reporter gene approach as well as determination of mRNA levels in two cancer cell lines of human origin, MCF-7 and A549, to study the regulation of multidrug resistance proteins 2 and 3 (MRP2 AND MRP3) by serine/threonine protein kinases. Since a prototypic PKC inducer, PMA, caused a marked upregulation of transcription from both human MRP2 and MRP3 promoters, a role for PKC isoforms in positive control of expression of these proteins could be postulated. Interestingly, broad-spectrum serine-threonine protein kinase inhibitors which also inhibit PKC, staurosporine and H-7, stimulated expression from the MRP2 promoter instead of inhibiting it. This effect was not seen for MRP3. MRP2 induction by staurosporine and H-7 was shown to have phenotypic consequences in whole cells, rendering them more resistant to etoposide and increasing their ability to export calcein through the plasma membrane. These results point to the involvement of serine/threonine protein kinases in negative regulation of the human MRP2 gene and to the necessity of testing novel anti-cancer drugs acting as protein kinase inhibitors with regard to their potential ability to induce multidrug resistance.

  7. Human intestine luminal ACE2 and amino acid transporter expression increased by ACE-inhibitors.

    PubMed

    Vuille-dit-Bille, Raphael N; Camargo, Simone M; Emmenegger, Luca; Sasse, Tom; Kummer, Eva; Jando, Julia; Hamie, Qeumars M; Meier, Chantal F; Hunziker, Schirin; Forras-Kaufmann, Zsofia; Kuyumcu, Sena; Fox, Mark; Schwizer, Werner; Fried, Michael; Lindenmeyer, Maja; Götze, Oliver; Verrey, François

    2015-04-01

    Sodium-dependent neutral amino acid transporter B(0)AT1 (SLC6A19) and imino acid (proline) transporter SIT1 (SLC6A20) are expressed at the luminal membrane of small intestine enterocytes and proximal tubule kidney cells where they exert key functions for amino acid (re)absorption as documented by their role in Hartnup disorder and iminoglycinuria, respectively. Expression of B(0)AT1 was shown in rodent intestine to depend on the presence of the carboxypeptidase angiotensin-converting enzyme 2 (ACE2). This enzyme belongs to the renin-angiotensin system and its expression is induced by treatment with ACE-inhibitors (ACEIs) or angiotensin II AT1 receptor blockers (ARBs) in many rodent tissues. We show here in the Xenopus laevis oocyte expression system that human ACE2 also functionally interacts with SIT1. To investigate in human intestine the potential effect of ACEIs or ARBs on ACE2, we analysed intestinal biopsies taken during routine gastroduodenoscopy and ileocolonoscopy from 46 patients of which 9 were under ACEI and 13 ARB treatment. Analysis of transcript expression by real-time PCR and of proteins by immunofluorescence showed a co-localization of SIT1 and B(0)AT1 with ACE2 in the brush-border membrane of human small intestine enterocytes and a distinct axial expression pattern of the tested gene products along the intestine. Patients treated with ACEIs displayed in comparison with untreated controls increased intestinal mRNA levels of ACE2, peptide transporter PEPT1 (SLC15A1) and AA transporters B(0)AT1 and PAT1 (SLC36A1). This study unravels in human intestine the localization and distribution of intestinal transporters involved in amino acid absorption and suggests that ACEIs impact on their expression.

  8. PML nuclear bodies contribute to the basal expression of the mTOR inhibitor DDIT4

    PubMed Central

    Salsman, Jayme; Stathakis, Alex; Parker, Ellen; Chung, Dudley; Anthes, Livia E.; Koskowich, Kara L.; Lahsaee, Sara; Gaston, Daniel; Kukurba, Kimberly R.; Smith, Kevin S.; Chute, Ian C.; Léger, Daniel; Frost, Laura D.; Montgomery, Stephen B.; Lewis, Stephen M.; Eskiw, Christopher; Dellaire, Graham

    2017-01-01

    The promyelocytic leukemia (PML) protein is an essential component of PML nuclear bodies (PML NBs) frequently lost in cancer. PML NBs coordinate chromosomal regions via modification of nuclear proteins that in turn may regulate genes in the vicinity of these bodies. However, few PML NB-associated genes have been identified. PML and PML NBs can also regulate mTOR and cell fate decisions in response to cellular stresses. We now demonstrate that PML depletion in U2OS cells or TERT-immortalized normal human diploid fibroblasts results in decreased expression of the mTOR inhibitor DDIT4 (REDD1). DNA and RNA immuno-FISH reveal that PML NBs are closely associated with actively transcribed DDIT4 loci, implicating these bodies in regulation of basal DDIT4 expression. Although PML silencing did reduce the sensitivity of U2OS cells to metabolic stress induced by metformin, PML loss did not inhibit the upregulation of DDIT4 in response to metformin, hypoxia-like (CoCl2) or genotoxic stress. Analysis of publicly available cancer data also revealed a significant correlation between PML and DDIT4 expression in several cancer types (e.g. lung, breast, prostate). Thus, these findings uncover a novel mechanism by which PML loss may contribute to mTOR activation and cancer progression via dysregulation of basal DDIT4 gene expression. PMID:28332630

  9. Molecular mechanism of hepatitis B virus (HBV) on suppression of raf kinase inhibitor protein (RKIP) expression

    PubMed Central

    Cheng, Xiao-Ke; Yu, Guo-Zheng; Li, Xiao-Dong; Ren, Xue-Qun

    2017-01-01

    Raf kinase inhibitor protein (RKIP) has been shown to be a suppressor of the mitogen-activated protein kinase pathway and is reported to be involved in human malignancy. However, the molecular mechanism of hepatitis B virus (HBV) in regulating RKIP expression is not yet clarified. In this study, we compared RKIP expression in 107 pairs of matched liver cancer and adjacent non-cancerous liver tissues. Among seven HBV-encoded proteins, we found HBV X (HBX) protein could significantly inhibit the expression level of RKIP, indicating that HBV could suppress RKIP expression through regulating HBX. To further elucidate the mechanism, analyses on transcriptional regulation and promoter methylation inhibition were conducted in Huh7 cells. Our results showed that HBX can interact with AP1 protein to inhibit the RKIP transcription. Moreover, we observed that the promoter methylation level of RKIP could be enhanced by HBV. In conclusion, our study revealed that RKIP could act as a molecular marker for HBV-infected liver cancer, but had no tumor-suppressing effect. PMID:27902472

  10. PML nuclear bodies contribute to the basal expression of the mTOR inhibitor DDIT4.

    PubMed

    Salsman, Jayme; Stathakis, Alex; Parker, Ellen; Chung, Dudley; Anthes, Livia E; Koskowich, Kara L; Lahsaee, Sara; Gaston, Daniel; Kukurba, Kimberly R; Smith, Kevin S; Chute, Ian C; Léger, Daniel; Frost, Laura D; Montgomery, Stephen B; Lewis, Stephen M; Eskiw, Christopher; Dellaire, Graham

    2017-03-23

    The promyelocytic leukemia (PML) protein is an essential component of PML nuclear bodies (PML NBs) frequently lost in cancer. PML NBs coordinate chromosomal regions via modification of nuclear proteins that in turn may regulate genes in the vicinity of these bodies. However, few PML NB-associated genes have been identified. PML and PML NBs can also regulate mTOR and cell fate decisions in response to cellular stresses. We now demonstrate that PML depletion in U2OS cells or TERT-immortalized normal human diploid fibroblasts results in decreased expression of the mTOR inhibitor DDIT4 (REDD1). DNA and RNA immuno-FISH reveal that PML NBs are closely associated with actively transcribed DDIT4 loci, implicating these bodies in regulation of basal DDIT4 expression. Although PML silencing did reduce the sensitivity of U2OS cells to metabolic stress induced by metformin, PML loss did not inhibit the upregulation of DDIT4 in response to metformin, hypoxia-like (CoCl2) or genotoxic stress. Analysis of publicly available cancer data also revealed a significant correlation between PML and DDIT4 expression in several cancer types (e.g. lung, breast, prostate). Thus, these findings uncover a novel mechanism by which PML loss may contribute to mTOR activation and cancer progression via dysregulation of basal DDIT4 gene expression.

  11. A cotton gene encoding a polygalacturonase inhibitor-like protein is specifically expressed in petals.

    PubMed

    Shi, Haiyan; Zhu, Li; Zhou, Ying; Li, Gang; Chen, Liang; Li, Xuebao

    2009-04-01

    A cDNA encoding a polygalacturonase-inhibitor-like protein (PGIP) was isolated from cotton flower cDNA library. The cDNA, designated GhPS1 (GenBank accession No. ABO47744), encodes a protein with 370 amino acids that shares high similarity with the known plant PGIPs. Fluorescent microscopy indicated that GhPS1 protein localizes on the cell membranes as well as in cytoplasm. Real-time quantitative RT-PCR and Northern blot analyses showed that GhPS1 was specifically expressed in cotton petals. Furthermore, the GhPS1 expression was gradually up-regulated in petal development, and its transcripts were accumulated to the highest level in the petals at anthesis. However, its expression level was declined rapidly in senesced petals after flowering. At low temperature, the GhPS1 gene expression was gradually decreased to very low level in petals. Collectively, our results suggest that GhPS1 gene might be involved in cotton petal development and senescence, and in response to cold stress.

  12. Mammary serine protease inhibitor and CD138 immunohistochemical expression in ovarian serous and clear cell carcinomas.

    PubMed

    Hasby, Eiman Adel

    2016-04-01

    This study aims to investigate the immunohistochemical expression of mammary serine protease inhibitor (maspin) and CD138 in primary ovarian high-grade serous carcinomas (HGSC) as compared to low-grade serous carcinomas (LGSC) and clear cell carcinomas and investigate if the studied markers have a correlation to International Federation of Gynaecology and Obstetrics (FIGO) stage, Ki67 proliferation index, and to each other. Maspin cellular location varied significantly between studied groups with only nuclear expression seen in 46.7 % of LGSC group, mixed nuclear and cytoplasmic in 13.3, 28.6, and 20 % of LGSC, HGSC, and clear cell carcinoma, respectively, and was only cytoplasmic in 26.7, 71.4, and 80 % of LGSC, HGSC, and clear cell carcinoma, respectively. Mean maspin and CD138 counts were significantly higher in HGSC and clear cell carcinoma compared to LGSC. Both maspin and CD138 scores varied significantly between studied groups and were positively correlated with adverse prognostic factors in studied carcinomas including FIGO stage and Ki67 proliferation index. Besides, both maspin and CD138 had significant correlation to each other. These findings suggest that epithelial cytoplasmic expression of maspin and CD138 may have a significant role in tumorigenesis in ovarian high-grade serous carcinomas and clear cell carcinomas; these markers may regulate tumor cell proliferation, and their significant correlation to each other may suggest that CD138 probably induces maspin expression to protect tumor growth factors from being lysed by proteolytic enzymes.

  13. Altered gene expression in rat mesenteric tissue following in vivo exposure to a phosphodiesterase 4 inhibitor

    SciTech Connect

    Dagues, Nicolas . E-mail: nicolas.dagues@pfizer.com; Pawlowski, Valerie; Guigon, Ghislaine; Ledieu, David; Sobry, Cecile; Hanton, Gilles; Freslon, Jean-Louis; Chevalier, Stephan

    2007-01-01

    Vascular injury is a relatively common finding during the pre-clinical toxicity testing of drugs. The mechanisms of the injury are poorly understood and in turn, sensitive and specific biomarkers for pre-clinical and clinical monitoring do not exist. The present study was undertaken to investigate the molecular mechanisms of drug-induced vascular injury in mesenteric tissue of rats treated with the selective phosphodiesterase 4 (PDE4) inhibitor CI-1044. In a time-course study, male Sprague Dawley rats were given daily doses of 40 or 80 mg/kg for 1, 2 or 3 successive days and were euthanized the following day. Gene expression profiles in mesenteric tissue were determined using Affymetrix RG{sub U}34A microarrays and fibrinogen and cytokine measurements were performed in blood samples. Hierarchical clustering analysis produced a clear pattern separation of the animals with inflammation, animal with inflammation and necrosis and animals without any lesion. Genes associated with inflammation, procoagulation, extracellular matrix remodeling were up-regulated. An altered expression of genes involved in vascular tone regulation, lipid and glucose metabolism was also observed. Selected genes expression changes were confirmed by TaqMan real-time RT-PCR. The inflammatory process was also detected in the bloodstream at the protein level since fibrinogen, IL6 and IL1{beta} concentrations were increased in treated animals. Overall, the present study reveals several molecular changes supporting the hypothesis by which PDE4 inhibitor-induced vascular lesions in rats are triggered by an inflammatory mechanism and/or a vascular tone dysregulation.

  14. Shear stress-induced Ets-1 modulates protease inhibitor expression in microvascular endothelial cells.

    PubMed

    Milkiewicz, Malgorzata; Uchida, Cassandra; Gee, Eric; Fudalewski, Tomasz; Haas, Tara L

    2008-11-01

    Elevated shear stress within the skeletal muscle microvasculature is implicated in the induction of a longitudinal splitting form of angiogenesis, which is characterized by the lack of basement membrane breakage. We investigated whether the transcriptional regulator, Ets-1, is responsive to changes in hemodynamic forces and if so, whether Ets-1 controls microvascular endothelial cell integrity by inducing the expression of inhibitors of matrix degrading proteases. Rats were treated with prazosin for 2, 4, and 7 days to increase in microvascular shear stress in hindlimb skeletal muscles. In complimentary in vitro experiments, rat microvascular skeletal muscle endothelial cells were exposed to laminar shear stress (15 dyne/cm(2)) for 0.5, 2, and 24 h. TaqMan PCR analysis of laser microdissected capillaries isolated from EDL muscles demonstrated transient (after 2 days) induction of Ets-1 gene expression. In cultured cells, a transient up-regulation of Ets-1 mRNA was observed after 2 h shear stimulation, accompanied by increased phosphorylation of Ets-1 and enhanced Ets-1 DNA binding activity. This response was modulated by ERK1/2 and p38 MAP kinases, but was not dependent on NOS or COX-2 activity. PAI-1, TIMP-1 and TIMP-3 mRNA were elevated significantly in prazosin treated EDL, and in response to shear stimulation in vitro. In cultured endothelial cells, Ets-1 RNA interference abolished the shear-induced increases in Ets-1, PAI-1, TIMP-1, and TIMP-3 mRNA expression. These results suggest that enhanced laminar shear stress may act to preserve the integrity of microvascular walls in part through Ets-1-dependent induction of protease inhibitors.

  15. GABA maintains the proliferation of progenitors in the developing chick ciliary marginal zone and non-pigmented ciliary epithelium.

    PubMed

    Ring, Henrik; Mendu, Suresh Kumar; Shirazi-Fard, Shahrzad; Birnir, Bryndis; Hallböök, Finn

    2012-01-01

    GABA is more than the main inhibitory neurotransmitter found in the adult CNS. Several studies have shown that GABA regulates the proliferation of progenitor and stem cells. This work examined the effects of the GABA(A) receptor system on the proliferation of retinal progenitors and non-pigmented ciliary epithelial (NPE) cells. qRT-PCR and whole-cell patch-clamp electrophysiology were used to characterize the GABA(A) receptor system. To quantify the effects on proliferation by GABA(A) receptor agonists and antagonists, incorporation of thymidine analogues was used. The results showed that the NPE cells express functional extrasynaptic GABA(A) receptors with tonic properties and that low concentration of GABA is required for a baseline level of proliferation. Antagonists of the GABA(A) receptors decreased the proliferation of dissociated E12 NPE cells. Bicuculline also had effects on progenitor cell proliferation in intact E8 and E12 developing retina. The NPE cells had low levels of the Cl-transporter KCC2 compared to the mature retina, suggesting a depolarising role for the GABA(A) receptors. Treatment with KCl, which is known to depolarise membranes, prevented some of the decreased proliferation caused by inhibition of the GABA(A) receptors. This supported the depolarising role for the GABA(A) receptors. Inhibition of L-type voltage-gated Ca(2+) channels (VGCCs) reduced the proliferation in the same way as inhibition of the GABA(A) receptors. Inhibition of the channels increased the expression of the cyclin-dependent kinase inhibitor p27(KIP1), along with the reduced proliferation. These results are consistent with that when the membrane potential indirectly regulates cell proliferation with hyperpolarisation of the membrane potential resulting in decreased cell division. The increased expression of p27(KIP1) after inhibition of either the GABA(A) receptors or the L-type VGCCs suggests a link between the GABA(A) receptors, membrane potential, and intracellular Ca

  16. Alisertib induces G2/M arrest, apoptosis, and autophagy via PI3K/Akt/mTOR- and p38 MAPK-mediated pathways in human glioblastoma cells

    PubMed Central

    Liu, Zheng; Wang, Feng; Zhou, Zhi-Wei; Xia, He-Chun; Wang, Xin-Yu; Yang, Yin-Xue; He, Zhi-Xu; Sun, Tao; Zhou, Shu-Feng

    2017-01-01

    Glioblastoma (GBM) is the most common brain tumor with poor response to current therapeutics. Alisertib (ALS), a second-generation selective Aurora kinase A (AURKA) inhibitor, has shown potent anticancer effects on solid tumors in animal studies. This study aimed to investigate the killing effect of ALS on GBM cell line DAOY and the possible underlying mechanisms using both bioinformatic and cell-based approaches. Our molecular docking showed that ALS preferentially bound AURKA over AURKB via hydrogen bond formation, charge interaction, and π-π stacking. ALS also bound key regulating proteins of cell cycle, apoptosis and autophagy, such as cyclin-dependent kinase 1 (CDK1/CDC2), CDK2, cyclin B1, p27 Kip1, p53, cytochrome C, cleaved caspase 3, Bax, Bcl-2, Bcl-xl, phosphatidylinositol 3-kinase (PI3K), protein kinase B (Akt), mammalian target of rapamycin (mTOR), 5’-adenosine monophosphate-activated protein kinase (AMPK), p38 mitogen-activated protein kinase (MAPK), beclin 1, phosphatase and tensin homolog (PTEN), and microtubule-associated protein light chain 3 (LC3). ALS exhibited potent growth-inhibitory, pro-apoptotic, and pro-autophagic effects on DAOY cells in a concentration-dependent manner. Notably, ALS remarkably induced G2/M arrest mainlyvia regulating the expression of CDK1/CDC2, CDK2, cyclin B1, p27 Kip1, and p53 in DAOY cells. ALS significantly induced the expression of mitochondria-mediated pro-apoptotic proteins such as Baxbut inhibited the expression of anti-apoptotic proteins such as Bcl-2 and Bcl-xl, with a significant increase in the release of cytochrome C and the activation of caspases 3 and 9. ALS also induced PI3K/Akt/mTOR and p38 MAPK signaling pathways while activating the AMPK signaling pathway. Taken together, these findings indicate that ALS exerts a potent inhibitory effect on cell proliferation and induces mitochondria-dependent apoptosis and autophagy with the involvement of PI3K/Akt/mTOR- and p38 MAPK-mediated signaling pathways in

  17. Cannabidiol as a novel inhibitor of Id-1 gene expression in aggressive breast cancer cells.

    PubMed

    McAllister, Sean D; Christian, Rigel T; Horowitz, Maxx P; Garcia, Amaia; Desprez, Pierre-Yves

    2007-11-01

    Invasion and metastasis of aggressive breast cancer cells is the final and fatal step during cancer progression, and is the least understood genetically. Clinically, there are still limited therapeutic interventions for aggressive and metastatic breast cancers available. Clearly, effective and nontoxic therapies are urgently required. Id-1, an inhibitor of basic helix-loop-helix transcription factors, has recently been shown to be a key regulator of the metastatic potential of breast and additional cancers. Using a mouse model, we previously determined that metastatic breast cancer cells became significantly less invasive in vitro and less metastatic in vivo when Id-1 was down-regulated by stable transduction with antisense Id-1. It is not possible at this point, however, to use antisense technology to reduce Id-1 expression in patients with metastatic breast cancer. Here, we report that cannabidiol (CBD), a cannabinoid with a low-toxicity profile, could down-regulate Id-1 expression in aggressive human breast cancer cells. The CBD concentrations effective at inhibiting Id-1 expression correlated with those used to inhibit the proliferative and invasive phenotype of breast cancer cells. CBD was able to inhibit Id-1 expression at the mRNA and protein level in a concentration-dependent fashion. These effects seemed to occur as the result of an inhibition of the Id-1 gene at the promoter level. Importantly, CBD did not inhibit invasiveness in cells that ectopically expressed Id-1. In conclusion, CBD represents the first nontoxic exogenous agent that can significantly decrease Id-1 expression in metastatic breast cancer cells leading to the down-regulation of tumor aggressiveness.

  18. Transgenic expression of pectin methylesterase inhibitors limits tobamovirus spread in tobacco and Arabidopsis.

    PubMed

    Lionetti, Vincenzo; Raiola, Alessandro; Cervone, Felice; Bellincampi, Daniela

    2014-04-01

    Plant infection by a virus is a complex process influenced by virus-encoded factors and host components which support replication and movement. Critical factors for a successful tobamovirus infection are the viral movement protein (MP) and the host pectin methylesterase (PME), an important plant counterpart that cooperates with MP to sustain viral spread. The activity of PME is modulated by endogenous protein inhibitors (pectin methylesterase inhibitors, PMEIs). PMEIs are targeted to the extracellular matrix and typically inhibit plant PMEs by forming a specific and stable stoichiometric 1:1 complex. PMEIs counteract the action of plant PMEs and therefore may affect plant susceptibility to virus. To test this hypothesis, we overexpressed genes encoding two well-characterized PMEIs in tobacco and Arabidopsis plants. Here, we report that, in tobacco plants constitutively expressing a PMEI from Actinidia chinensis (AcPMEI), systemic movement of Tobacco mosaic virus (TMV) is limited and viral symptoms are reduced. A delayed movement of Turnip vein clearing virus (TVCV) and a reduced susceptibility to the virus were also observed in Arabidopsis plants overexpressing AtPMEI-2. Our results provide evidence that PMEIs are able to limit tobamovirus movement and to reduce plant susceptibility to the virus.

  19. Expression of Arabidopsis Bax Inhibitor-1 in transgenic sugarcane confers drought tolerance.

    PubMed

    Ramiro, Daniel Alves; Melotto-Passarin, Danila Montewka; Barbosa, Mariana de Almeida; Santos, Flavio Dos; Gomez, Sergio Gregorio Perez; Massola Júnior, Nelson Sidnei; Lam, Eric; Carrer, Helaine

    2016-09-01

    The sustainability of global crop production is critically dependent on improving tolerance of crop plants to various types of environmental stress. Thus, identification of genes that confer stress tolerance in crops has become a top priority especially in view of expected changes in global climatic patterns. Drought stress is one of the abiotic stresses that can result in dramatic loss of crop productivity. In this work, we show that transgenic expression of a highly conserved cell death suppressor, Bax Inhibitor-1 from Arabidopsis thaliana (AtBI-1), can confer increased tolerance of sugarcane plants to long-term (>20 days) water stress conditions. This robust trait is correlated with an increased tolerance of the transgenic sugarcane plants, especially in the roots, to induction of endoplasmic reticulum (ER) stress by the protein glycosylation inhibitor tunicamycin. Our findings suggest that suppression of ER stress in C4 grasses, which include important crops such as sorghum and maize, can be an effective means of conferring improved tolerance to long-term water deficit. This result could potentially lead to improved resilience and yield of major crops in the world.

  20. CXCL12-mediated induction of plasminogen activator inhibitor-1 expression in human CXCR4 positive astroglioma cells.

    PubMed

    Oh, Jae-Wook; Olman, Mitchell; Benveniste, Etty Nadia

    2009-04-01

    Glioblastoma is the most malignant and common brain tumor. To promote their growth, these glioma cells secrete a variety of soluble factors including plasminogen activator inhibitor-1 (PAI-1), which functions as an inhibitor of plasminogen activators. We report here with the basis of microarray gene expression analysis that CXCR4 expressing glioma cells are capable of expressing PAI-1 mRNA and protein upon CXCL12 stimulation. Pretreatment with U0126, an inhibitor of mitogen activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) kinase (MEK) 1/2, abrogated CXCL12-induced PAI-1 expression. Pertussis toxin (PTX), an inhibitor of Galpha(i) proteins, also had inhibitory effects, indicating that the activation of Galpha(i) and ERK MAPK are required for this response. Interestingly, CXCL12 showed additive effects with another PAI-1 inducers, tumor necrosis factor (TNF)-alpha and/or tumor growth factor (TGF)-beta1, in increasing PAI-1 expression. These results indicate that CXCL12/CXCR4 signaling in glioma cells may be another mechanism for these cells to express PAI-1, which may be involved in angiogenesis and tumor invasion in brain tumors.

  1. Restoration of SHIP activity in a human leukemia cell line downregulates constitutively activated phosphatidylinositol 3-kinase/Akt/GSK-3beta signaling and leads to an increased transit time through the G1 phase of the cell cycle.

    PubMed

    Horn, S; Endl, E; Fehse, B; Weck, M M; Mayr, G W; Jücker, M

    2004-11-01

    The inositol 5-phosphatase SHIP (SHIP-1) is a negative regulator of signal transduction in hematopoietic cells and targeted disruption of SHIP in mice leads to a myeloproliferative disorder. We analyzed the effects of SHIP on the human leukemia cell line Jurkat in which expression of endogenous SHIP protein is not detectable. Restoration of SHIP expression in Jurkat cells with an inducible expression system caused a 69% reduction of phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P(3)) and a 65% reduction of Akt kinase activity, which was associated with reduced phosphorylation of glycogen synthase kinase 3beta (GSK-3beta) (Ser-9) without changing the phosphorylation of Bad (Ser-136), FKHR (Ser-256) or MAPK (Thr-202/Tyr-204). SHIP-expressing Jurkat cells showed an increased transit time through the G1 phase of the cell cycle, but SHIP did not cause a complete cell cycle arrest or apoptosis. Extension of the G1 phase was associated with an increased stability of the cell cycle inhibitor p27(Kip1) and reduced phosphorylation of the retinoblastoma protein Rb at serine residue 780. Our data indicate that restoration of SHIP activity in a human leukemia cell line, which has lost expression of endogenous SHIP, downregulates constitutively activated phosphatidylinositol 3-kinase/Akt/GSK-3beta signaling and leads to an increased transit time through the G1 phase of the cell cycle.

  2. Pediatric Primitive Neuroectodermal Tumors of the Central Nervous System Differentially Express Granzyme Inhibitors

    PubMed Central

    Vermeulen, Jeroen F.; van Hecke, Wim; Spliet, Wim G. M.; Villacorta Hidalgo, José; Fisch, Paul; Broekhuizen, Roel; Bovenschen, Niels

    2016-01-01

    Background Central nervous system (CNS) primitive neuroectodermal tumors (PNETs) are malignant primary brain tumors that occur in young infants. Using current standard therapy, up to 80% of the children still dies from recurrent disease. Cellular immunotherapy might be key to improve overall survival. To achieve efficient killing of tumor cells, however, immunotherapy has to overcome cancer-associated strategies to evade the cytotoxic immune response. Whether CNS-PNETs can evade the immune response remains unknown. Methods We examined by immunohistochemistry the immune response and immune evasion strategies in pediatric CNS-PNETs. Results Here, we show that CD4+, CD8+, γδ-T-cells, and Tregs can infiltrate pediatric CNS-PNETs, although the activation status of cytotoxic cells is variable. Pediatric CNS-PNETs evade immune recognition by downregulating cell surface MHC-I and CD1d expression. Intriguingly, expression of SERPINB9, SERPINB1, and SERPINB4 is acquired during tumorigenesis in 29%, 29%, and 57% of the tumors, respectively. Conclusion We show for the first time that brain tumors express direct granzyme inhibitors (serpins) as a potential mechanism to overcome cellular cytotoxicity, which may have consequences for cellular immunotherapy. PMID:26963506

  3. Clinically Viable Gene Expression Assays with Potential for Predicting Benefit from MEK Inhibitors.

    PubMed

    Brant, Roz; Sharpe, Alan; Liptrot, Tom; Dry, Jonathan R; Harrington, Elizabeth A; Barrett, J Carl; Whalley, Nicky; Womack, Christopher; Smith, Paul; Hodgson, Darren R

    2017-03-15

    Purpose: To develop a clinically viable gene expression assay to measure RAS/RAF/MEK/ERK (RAS-ERK) pathway output suitable for hypothesis testing in non-small cell lung cancer (NSCLC) clinical studies.Experimental Design: A published MEK functional activation signature (MEK signature) that measures RAS-ERK functional output was optimized for NSCLC in silico NanoString assays were developed for the NSCLC optimized MEK signature and the 147-gene RAS signature. First, platform transfer from Affymetrix to NanoString, and signature modulation following treatment with KRAS siRNA and MEK inhibitor, were investigated in cell lines. Second, the association of the signatures with KRAS mutation status, dynamic range, technical reproducibility, and spatial and temporal variation was investigated in NSCLC formalin-fixed paraffin-embedded tissue (FFPET) samples.Results: We observed a strong cross-platform correlation and modulation of signatures in vitro Technical and biological replicates showed consistent signature scores that were robust to variation in input total RNA; conservation of scores between primary and metastatic tumor was statistically significant. There were statistically significant associations between high MEK (P = 0.028) and RAS (P = 0.003) signature scores and KRAS mutation in 50 NSCLC samples. The signatures identify overlapping but distinct candidate patient populations from each other and from KRAS mutation testing.Conclusions: We developed a technically and biologically robust NanoString gene expression assay of MEK pathway output, compatible with the quantities of FFPET routinely available. The gene signatures identified a different patient population for MEK inhibitor treatment compared with KRAS mutation testing. The predictive power of the MEK signature should be studied further in clinical trials. Clin Cancer Res; 23(6); 1471-80. ©2016 AACRSee related commentary by Xue and Lito, p. 1365.

  4. Differential effects of histone deacetylase inhibitors on phorbol ester- and TGF-beta1 induced murine tissue inhibitor of metalloproteinases-1 gene expression.

    PubMed

    Young, David A; Billingham, Olivia; Sampieri, Clara L; Edwards, Dylan R; Clark, Ian M

    2005-04-01

    Expression of the tissue inhibitor of metalloproteinases-1 (Timp-1) gene can be induced by either phorbol myristate acetate (PMA) or transforming growth factor beta1 (TGF-beta1), although the signalling pathways involved are not clearly defined. Canonically, histone deacetylase inhibitors (HDACi) such as trichostatin A (TSA) or sodium butyrate (NaB) increase total cellular histone acetylation and activate expression of susceptible genes. Remarkably, PMA and TGF-beta1 stimulation of Timp-1 show a differential response to TSA or NaB. TSA or NaB potentiate PMA-induced Timp-1 expression but repress TGF-beta1-induced Timp-1 expression. The repression of TGF-beta1-induced Timp-1 by TSA was maximal at 5 ng.mL(-1), while for the superinduction of PMA-induced Timp-1 expression, the maximal dose is > 500 ng x mL(-1) TSA. A further HDACi, valproic acid, did not block TGF-beta1-induced Timp-1 expression, demonstrating that different HDACs impact on the induction of Timp-1. For either PMA or TGF-beta1 to induce Timp-1 expression, new protein synthesis is required, and the induction of AP-1 factors closely precedes that of Timp-1. The effects of the HDACi can be reiterated in transient transfection using Timp-1 promoter constructs. Mutation or deletion of the AP-1 motif (-59/-53) in the Timp-1 promoter diminishes PMA-induction of reporter constructs, however, the further addition of TSA still superinduces the reporter. In c-Jun-/- cells, PMA still stimulates Timp-1 expression, but TSA superinduction is lost. Transfection of a series of Timp-1 promoter constructs identified three regions through which TSA superinduces PMA-induced Timp-1 and we have demonstrated specific protein binding to two of these regions which contain either an avian erythroblastosis virus E26 (v-ets) oncogene homologue (Ets) or Sp1 binding motif.

  5. Plants with increased expression of ent-kaurene oxidase are resistant to chemical inhibitors of this gibberellin biosynthesis enzyme.

    PubMed

    Swain, Stephen M; Singh, Davinder P; Helliwell, Chris A; Poole, Andrew T

    2005-02-01

    The gibberellin (GA) biosynthetic pathway includes the three-step oxidation of ent-kaurene to ent-kaurenoic acid, catalyzed by the enzyme ent-kaurene oxidase (KO). Arabidopsis plants overexpressing the KO cDNA under the control of the cauliflower mosaic virus 35S promoter, with or without a translational fusion to a modified green fluorescent protein (GFP), are very similar to wild-type (WT) plants under normal growth conditions. In contrast, when WT and 35S:KO (or 35S:KO-GFP) seeds, seedlings or pollen tubes are grown in the presence of chemical inhibitors of KO, such as paclobutrazol and uniconazole, plants with increased KO expression are partially resistant to the effects of these inhibitors. In combination with the observation that decreased KO levels increase the sensitivity to KO inhibitors, the 35S:KO phenotypes demonstrate that the modification of KO enzyme levels could be used to create transgenic crop plants with altered KO inhibitor response. These results also suggest that the KO gene could be used as a selectable marker for plant regeneration based on resistance to KO inhibitors. Finally, the observation that pollen tubes expressing 35S:KO or 35S:KO-GFP have decreased sensitivity to KO inhibitors provides further evidence for a physiological role for GAs in pollen tube elongation.

  6. Berberine attenuates high glucose-induced proliferation and extracellular matrix accumulation in mesangial cells: involvement of suppression of cell cycle progression and NF-κB/AP-1 pathways.

    PubMed

    Lan, Tian; Wu, Teng; Chen, Cheng; Chen, Xiaolan; Hao, Jie; Huang, Junying; Wang, Lijing; Huang, Heqing

    2014-03-25

    Berberine has been shown to have renoprotective effects on diabetes through attenuating TGF-β1 and fibronectin (FN) expression. However, how berberine regulates TGF-β1 and FN is not fully clear. Here we investigated whether berberine inhibited TGF-β1 and FN expression in high glucose-cultured mesangial cells. Berberine significantly inhibited mesangial cell proliferation and hypertrophy by increasing the cell population in G1-phase and reducing that in S-phase. In addition, berberine reversed high glucose-induced down-regulation of cyclin-dependent kinase inhibitor p21(Waf1)/(Cip1) and p27(Kip1). Berberine inhibited p65 translocation to the nucleus and c-jun phosphorylation induced by high glucose. Furthermore, berberine attenuated high glucose-induced expression of TGF-β1 and FN. Using a luciferase reporter assay, we found that high glucose-induced transcription activity of NF-κB and AP-1 was blocked by berberine. Electrophoretic mobility shift assay showed that high glucose increased that NF-κB and AP-1 DNA binding activity. These data indicate that berberine inhibited mesangial cell proliferation and hypertrophy by modulating cell cycle progress. In addition, berberine suppressed high glucose-induced TGF-β1 and FN expression by blocking NF-κB/AP-1 pathways.

  7. The highly virulent variola and monkeypox viruses express secreted inhibitors of type I interferon.

    PubMed

    Fernández de Marco, María del Mar; Alejo, Alí; Hudson, Paul; Damon, Inger K; Alcami, Antonio

    2010-05-01

    Variola virus (VARV) caused smallpox, one of the most devastating human diseases and the first to be eradicated, but its deliberate release represents a dangerous threat. Virulent orthopoxviruses infecting humans, such as monkeypox virus (MPXV), could fill the niche left by smallpox eradication and the cessation of vaccination. However, immunomodulatory activities and virulence determinants of VARV and MPXV remain largely unexplored. We report the molecular characterization of the VARV- and MPXV-secreted type I interferon-binding proteins, which interact with the cell surface after secretion and prevent type I interferon responses. The proteins expressed in the baculovirus system have been purified, and their interferon-binding properties characterized by surface plasmon resonance. The ability of these proteins to inhibit a broad range of interferons was investigated to identify potential adaptation to the human immune system. Furthermore, we demonstrate by Western blot and activity assays the expression of the type I interferon inhibitor during VARV and MPXV infections. These findings are relevant for the design of new vaccines and therapeutics to smallpox and emergent virulent orthopoxviruses because the type I interferon-binding protein is a major virulence factor in animal models, vaccination with this protein induces protective immunity, and its neutralization prevents disease progression.

  8. Development and bioassay of transgenic Chinese cabbage expressing potato proteinase inhibitor II gene

    PubMed Central

    Zhang, Junjie; Liu, Fan; Yao, Lei; Luo, Chen; Yin, Yue; Wang, Guixiang; Huang, Yubi

    2012-01-01

    Lepidopteran larvae are the most injurious pests of Chinese cabbage production. We attempted the development of transgenic Chinese cabbage expressing the potato proteinase inhibitor II gene (pinII) and bioassayed the pest-repelling ability of these transgenic plants. Cotyledons with petioles from aseptic seedlings were used as explants for Agrobacterium-mediated in vitro transformation. Agrobacterium tumefaciens C58 contained the binary vector pBBBasta-pinII-bar comprising pinII and bar genes. Plants showing vigorous PPT resistance were obtained by a series concentration selection for PPT resistance and subsequent regeneration of leaf explants dissected from the putative chimera. Transgenic plants were confirmed by PCR and genomic Southern blotting, which showed that the bar and pinII genes were integrated into the plant genome. Double haploid homozygous transgenic plants were obtained by microspore culture. The pinII expression was detected using quantitative real time polymerase chain reaction (qRT-PCR) and detection of PINII protein content in the transgenic homozygous lines. Insect-feeding trials using the larvae of cabbage worm (Pieris rapae) and the larvae of the diamondback moth (Plutella xylostella) showed higher larval mortality, stunted larval development, and lower pupal weights, pupation rates, and eclosion rates in most of the transgenic lines in comparison with the corresponding values in the non-transformed wild-type line. PMID:23136521

  9. Glycol-split nonanticoagulant heparins are inhibitors of hepcidin expression in vitro and in vivo

    PubMed Central

    Poli, Maura; Asperti, Michela; Naggi, Annamaria; Campostrini, Natascia; Girelli, Domenico; Corbella, Michela; Benzi, Marina; Besson-Fournier, Celine; Coppin, Helene; Maccarinelli, Federica; Finazzi, Dario

    2014-01-01

    Hepcidin controls systemic iron availability, and its excess contributes to the anemia of chronic diseases, the most prevalent anemia in hospitalized patients. We previously reported that heparins are efficient hepcidin inhibitors both in vitro and in vivo, but their anticoagulant activity limits therapeutic use. We studied nonanticoagulant heparins produced by N-acetylation and oxidation/reduction (glycol-split) that lost antithrombin-binding affinity. Four nonanticoagulant heparins inhibited hepcidin expression in hepatic HepG2 cells and primary hepatocytes. The 2 most potent ones used in mice suppressed liver hepcidin expression and serum hepcidin in 6 hours, with a significant decrease of spleen iron. This occurred also in lipopolysaccharide (LPS)-treated animals that mimic inflammation, as well as after chronic 1-week treatments, without evident adverse effects on coagulation. Heparin injections increased iron mobilization and facilitated the recovery from the anemia induced by heat-killed Brucella abortus, a model of inflammatory anemia. The heparins were used also in Bmp6−/− mice. A single dose of heparin reduced the already low level of hepcidin of these mice and prevented its induction by LPS. These nonanticoagulant compounds impair bone morphogenetic protein /sons of mothers against decapentaplegic signaling with no evident adverse effect in vivo, even when administered chronically. They may offer a strategy for the treatment of diseases with high hepcidin levels. PMID:24398330

  10. Cardiac glycosides are potent inhibitors of interferon-β gene expression

    PubMed Central

    Ye, Junqiang; Chen, Shuibing; Maniatis, Tom

    2011-01-01

    We report that bufalin and other cardiac glycoside inhibitors of the sodium-potassium ATPase (sodium pump) potently inhibit the induction of the interferon-β (IFNβ) gene by virus, dsRNA or dsDNA. Cardiac glycosides increase the intracellular sodium concentration, which appears to inhibit the ATPase activity of the RNA sensor RIG-I, an essential and early component in the IFNβ activation pathway. This, in turn, prevents the activation of the critical transcription factors IRF3 and NFκB. Bufalin inhibition can be overcome by expressing a drug-resistant variant of the sodium pump, and knocking down the pump by shRNA inhibits IFNβ expression. Thus, bufalin acts exclusively through the sodium pump. We also show that bufalin inhibits tumor necrosis factor (TNF) signaling, at least in part by interfering with the nuclear translocation of NFκB. These findings suggest that bufalin could be used to treat inflammatory and autoimmune diseases where IFN or TNF are hyperactivated. PMID:21076398

  11. A Dynamical Framework for the All-or-None G1/S Transition

    PubMed Central

    Barr, Alexis R.; Heldt, Frank S.; Zhang, Tongli; Bakal, Chris; Novák, Béla

    2016-01-01

    Summary The transition from G1 into DNA replication (S phase) is an emergent behavior resulting from dynamic and complex interactions between cyclin-dependent kinases (Cdks), Cdk inhibitors (CKIs), and the anaphase-promoting complex/cyclosome (APC/C). Understanding the cellular decision to commit to S phase requires a quantitative description of these interactions. We apply quantitative imaging of single human cells to track the expression of G1/S regulators and use these data to parametrize a stochastic mathematical model of the G1/S transition. We show that a rapid, proteolytic, double-negative feedback loop between Cdk2:Cyclin and the Cdk inhibitor p27Kip1 drives a switch-like entry into S phase. Furthermore, our model predicts that increasing Emi1 levels throughout S phase are critical in maintaining irreversibility of the G1/S transition, which we validate using Emi1 knockdown and live imaging of G1/S reporters. This work provides insight into the general design principles of the signaling networks governing the temporally abrupt transitions between cell-cycle phases. PMID:27136687

  12. Fibroblast growth factor receptor signaling is essential for lens fiber cell differentiation.

    PubMed

    Zhao, Haotian; Yang, Tianyu; Madakashira, Bhavani P; Thiels, Cornelius A; Bechtle, Chad A; Garcia, Claudia M; Zhang, Huiming; Yu, Kai; Ornitz, David M; Beebe, David C; Robinson, Michael L

    2008-06-15

    The vertebrate lens provides an excellent model to study the mechanisms that regulate terminal differentiation. Although fibroblast growth factors (FGFs) are thought to be important for lens cell differentiation, it is unclear which FGF receptors mediate these processes during different stages of lens development. Deletion of three FGF receptors (Fgfr1-3) early in lens development demonstrated that expression of only a single allele of Fgfr2 or Fgfr3 was sufficient for grossly normal lens development, while mice possessing only a single Fgfr1 allele developed cataracts and microphthalmia. Profound defects were observed in lenses lacking all three Fgfrs. These included lack of fiber cell elongation, abnormal proliferation in prospective lens fiber cells, reduced expression of the cell cycle inhibitors p27(kip1) and p57(kip2), increased apoptosis and aberrant or reduced expression of Prox1, Pax6, c-Maf, E-cadherin and alpha-, beta- and gamma-crystallins. Therefore, while signaling by FGF receptors is essential for lens fiber differentiation, different FGF receptors function redundantly.

  13. [Effect of proteasome inhibitor bortezomib on proliferation, apoptosis and SHIP gene expression in K562 cells].

    PubMed

    Jia, Zhi-Qiang; Wei, Yu-Tao; Li, Ai-Ming; Cheng, Zhi-Yong

    2013-08-01

    This study was aimed to investigate the effects of proteasome inhibitor bortezomib on proliferation, apoptosis and the SHIP expression of K562 cells. K562 cells were treated with bortezomib of different concentrations. Cell proliferation was analyzed by MTT assay, cell apoptosis was detected by flow cytometry and SHIP mRNA expression was assayed by RT-PCR.The results showed that after being treated with 10, 20, 50 and 100 nmol/L bortezomib for 24 h, the inhibitory rates of K562 cells were (5.76 ± 1.47)%, (10.55 ± 1.59)%, (17.14 ± 2.05)% and (27.69 ± 3.57)% respectively, and were higher than that in control (1.30 ± 0.10); when K562 cells were treated with 20 nmol/L bortezomib for 24, 48 and 72 h, the inhibitory rates of cell proliferation were (10.55 ± 1.59)%, (16.33 ± 2.53)% and (19.78 ± 1.56)% respectively, there was statistic difference of cell proliferation rate between 24 h group and 48 h group (P < 0.05). After being treated with 10,20,50,100 nmol/L bortezomib for 24 h, the apoptotic rates of K562 cells were (12.7 ± 0.6)%, (26.9 ± 0.9)%, (32.6 ± 1.2)% and (72.5 ± 1.5)% respectively,and all higher than that in control (1.0 ± 0.5)% (P < 0.05). According to results of RT-PCR detection, the expression level of SHIP mRNA was obviously up-regulated after treatment with bortezomib, and showed statistical difference in comparison with control. It is concluded that bortezomib inhibits proliferation of K562 cells in time and concentration-dependent manner and induces apoptosis through up-regulation of SHIP gene.

  14. Anti-Cancer Effect of IN-2001 in T47D Human Breast Cancer.

    PubMed

    Joung, Ki Eun; Min, Kyung Nan; Kim, Dae-Kee; Sheen, Yhun Yhong

    2012-01-01

    Histone deacetylases (HDACs) are enzymes involved in the remodelling of chromatin, and have a key role in the epigenetic regulation of gene expression. Histone deacetylase (HDAC) inhibitors are emerging as an exciting new class of potential anti-cancer agents. In recent years, a number of structurally diverse HDAC inhibitors have been identified and these HDAC inhibitors induce growth arrest, differentiation and/or apoptosis of cancer cells in vitro and in vivo. However, the underlying molecular mechanisms remain unclear. This study aimed at investigating the anti-tumor activity of various HDAC inhibitors, IN-2001, using T47D human breast cancer cells. Moreover, the possible mechanism by which HDAC inhibitors exhibit anti-tumor activity was also explored. In estrogen receptor positive T47D cells, IN-2001, HDAC inhibitor showed anti-proliferative effects in dose-and time-dependent manner. In T47D human breast cancer cells showed anti-tumor activity of IN-2001 and the growth inhibitory effects of IN-2001 were related to the cell cycle arrest and induction of apoptosis. Flow cytometry studies revealed that IN-2001 showed accumulation of cells at G2/M phase. At the same time, IN-2001 treatment time-dependently increased sub-G1 population, representing apoptotic cells. IN-2001-mediated cell cycle arrest was associated with induction of cdk inhibitor expression. In T47D cells, IN-2001 as well as other HDAC inhibitors treatment significantly increased p21(WAF1) and p27(KIP1) expression. In addition, thymidylate synthase, an essential enzyme for DNA replication and repair, was down-regulated by IN-2001 and other HDAC inhibitors in the T47D human breast cancer cells. In summary, IN-2001 with a higher potency than other HDAC inhibitors induced growth inhibition, cell cycle arrest, and eventual apoptosis in human breast cancer possibly through modulation of cell cycle and apoptosis regulatory proteins, such as cdk inhibitors, cyclins, and thymidylate synthase.

  15. Differential Expression of Kunitz and Bowman-Birk Soybean Proteinase Inhibitors in Plant and Callus Tissue 1

    PubMed Central

    Tan-Wilson, Anna L.; Hartl, Philippe M.; Delfel, Norman E.; Wilson, Karl A.

    1985-01-01

    Bowman-Birk soybean trypsin inhibitor (BBSTI) but not Kunitz soybean trypsin inhibitor (KSTI) was found in samples of undifferentiated and partially differentiated Amsoy 71 tissue culture callus. This suggests the differential metabolism of these two classes of proteinase inhibitors, whether the difference be in synthesis, in rates of degradation, or both. The differential metabolism of the proteinase inhibitors is also seen in the plant. Both BBSTI and KSTI were found in the hypocotyl, root, and epicotyl of the Amsoy 71 soybean seedling in addition to their expected presence in the cotyledons. Whereas the ratio of KSTI to BBSTI in the cotyledon was higher, the ratio of BBSTI to KSTI was higher in the extracotyledonary tissues of the seedling. The levels of both classes of proteinase inhibitors declined during seedling growth, except in the epicotyl and the proximal root. In both of these tissues, an increase in BBSTI, but not in KSTI content, expressed as milligrams inhibitor per plant part, occurred. Images Fig. 1 Fig. 4 PMID:16664236

  16. Flavokawain A induces deNEDDylation and Skp2 degradation leading to inhibition of tumorigenesis and cancer progression in the TRAMP transgenic mouse model

    PubMed Central

    Li, Xuesen; Yokoyama, Noriko N.; Zhang, Saiyang; Ding, Lina; Liu, Hong-min; Lilly, Michael B.; Mercola, Dan; Zi, Xiaolin

    2015-01-01

    S phase kinase-associated protein 2 (Skp2) has been shown to be required for spontaneous tumor development that occurs in the retinoblastoma protein (pRb) deficient mice. Here we have demonstrated that flavokawain A (FKA), a novel chalcone from the kava plant, selectively inhibited the growth of pRb deficient cell lines and resulted in a proteasome-dependent and ubiquitination-mediated Skp2 degradation. Degradation of Skp2 by FKA was found to be involved in a functional Cullin1, but independent of Cdh1 expression. Further studies have demonstrated that FKA docked into the ATP binding pocket of the precursor cell-expressed developmentally down-regulated 8 (NEDD8)-activating enzyme (NAE) complex, inhibited NEDD8 conjugations to both Cullin1 and Ubc12 in PC3 cells and Ubc12 NEDDylation in an in vitro assay. Finally, dietary feeding of the autochthonous transgenic adenocarcinoma of the mouse prostate (TRAMP) mice with FKA inhibited the formation of high-grade prostatic intra-epithelial neoplasia lesions (HG-PIN) and prostate adenocarcinomas, reduced the tumor burden and completely abolished distant organ metastasis. Immunohistochemistry studies revealed that dietary FKA feeding resulted in marked anti-proliferative and apoptotic effects via down-regulation of Skp2 and NEDD8 and up-regulation of p27/Kip1 in the prostate of TRAMP mice. Our findings therefore provide evidence that FKA is a promising NEDDylation inhibitor for targeting Skp2 degradation in prostate cancer prevention and treatment. PMID:26497688

  17. Flavokawain A induces deNEDDylation and Skp2 degradation leading to inhibition of tumorigenesis and cancer progression in the TRAMP transgenic mouse model.

    PubMed

    Li, Xuesen; Yokoyama, Noriko N; Zhang, Saiyang; Ding, Lina; Liu, Hong-min; Lilly, Michael B; Mercola, Dan; Zi, Xiaolin

    2015-12-08

    S phase kinase-associated protein 2 (Skp2) has been shown to be required for spontaneous tumor development that occurs in the retinoblastoma protein (pRb) deficient mice. Here we have demonstrated that flavokawain A (FKA), a novel chalcone from the kava plant, selectively inhibited the growth of pRb deficient cell lines and resulted in a proteasome-dependent and ubiquitination-mediated Skp2 degradation. Degradation of Skp2 by FKA was found to be involved in a functional Cullin1, but independent of Cdh1 expression. Further studies have demonstrated that FKA docked into the ATP binding pocket of the precursor cell-expressed developmentally down-regulated 8 (NEDD8)-activating enzyme (NAE) complex, inhibited NEDD8 conjugations to both Cullin1 and Ubc12 in PC3 cells and Ubc12 NEDDylation in an in vitro assay. Finally, dietary feeding of the autochthonous transgenic adenocarcinoma of the mouse prostate (TRAMP) mice with FKA inhibited the formation of high-grade prostatic intra-epithelial neoplasia lesions (HG-PIN) and prostate adenocarcinomas, reduced the tumor burden and completely abolished distant organ metastasis. Immunohistochemistry studies revealed that dietary FKA feeding resulted in marked anti-proliferative and apoptotic effects via down-regulation of Skp2 and NEDD8 and up-regulation of p27/Kip1 in the prostate of TRAMP mice. Our findings therefore provide evidence that FKA is a promising NEDDylation inhibitor for targeting Skp2 degradation in prostate cancer prevention and treatment.

  18. High fat diet triggers cell cycle arrest and excessive apoptosis of granulosa cells during the follicular development.

    PubMed

    Wu, Yanqing; Zhang, Zhenghong; Liao, Xinghui; Wang, Zhengchao

    2015-10-23

    The regulatory mechanism of granulosa cells (GCs) proliferation during the follicular development is complicated and multifactorial, which is essential for the oocyte growth and normal ovarian functions. To investigate the role of high fat diet (HFD) on the proliferation of GCs, 4-week old female mice were fed with HFD or normal control diet (NC) for 15 weeks or 20 weeks and then detected the expression level of some regulatory molecules of cell cycle and apoptosis. The abnormal ovarian morphology was observed at 20 weeks. Further mechanistic studies indicated that HFD induced-obesity caused elevated apoptotic levels in GCs of the ovaries in a time-dependent manner. Moreover, cell cycle progress was also impacted after HFD fed. The cell cycle inhibitors, p27(Kip1) and p21(Cip1), were significantly induced in the ovaries from the mice in HFD group when compared with that in the ovaries from the mice in NC group. Subsequently, the expression levels of Cyclin D1, D3 and CDK4 were also significantly influenced in the ovaries from the mice fed with HFD in a time-dependent manner. The present results suggested that HFD induced-obesity may trigger cell cycle arrest and excessive apoptosis of GCs, causing the abnormal follicular development and ovarian function failure.

  19. Induction of cell cycle arrest in prostate cancer cells by the dietary compound isoliquiritigenin.

    PubMed

    Lee, Yeo Myeong; Lim, Do Young; Choi, Hyun Ju; Jung, Jae In; Chung, Won-Yoon; Park, Jung Han Yoon

    2009-02-01

    Isoliquiritigenin (ISL), a flavonoid chalcone that is present in licorice, shallot, and bean sprouts, is known to have antitumorigenic activities. The present study examined whether ISL alters prostate cancer cell cycle progression. DU145 human and MatLyLu (MLL) rat prostate cancer cells were cultured with various concentrations of ISL. In both DU145 and MLL cells treated with ISL, the percentage of cells in the G1 phase increased, and the incorporation of [(3)H]thymidine decreased. ISL decreased the protein levels of cyclin D1, cyclin E, and cyclin-dependent kinase (CDK) 4, whereas cyclin A and CDK2 expressions were unaltered in cells treated with ISL. The expression of the CDK inhibitor p27(KIP1) was increased in cells treated with 20 micromol/L ISL. In addition, treatment of cells with 20 micromol/L ISL for 24 hours led to G2/M cell cycle arrest. Cell division control (CDC) 2 protein levels remained unchanged. The protein levels of phospho-CDC2 (Tyr15) and cyclin B1 were increased, and the CDC25C level was decreased by ISL dose-dependently. We demonstrate that ISL promotes cell cycle arrest in DU145 and MLL cells, thereby providing insights into the mechanisms underlying its antitumorigenic activities.

  20. Dachshund homologues play a conserved role in islet cell development

    PubMed Central

    Kalousova, Anna; Mavropoulos, Anastasia; Adams, Bruce A.; Nekrep, Nada; Li, Zhongmei; Krauss, Stephan; Stainier, Didier Y.; German, Michael S.

    2010-01-01

    All metazoans use insulin to control energy metabolism, but they secrete it from different cells: neurons in the central nervous system in invertebrates and endocrine cells in the gut or pancreas in vertebrates. Despite their origins in different germ layers, all of these insulin-producing cells share common functional features and gene expression patterns. In this study, we tested the role in insulin-producing cells of the vertebrate homologues of Dachshund, a transcriptional regulator that marks the earliest committed progenitors of the neural insulin-producing cells in Drosophila. Both zebrafish and mice expressed a single dominant Dachshund homologue in the pancreatic endocrine lineage, and in both species loss of this homologue reduced the numbers of all islet cell types including the insulin-producing β-cells. In mice, Dach1 gene deletion left pancreatic progenitor cells unaltered, but blocked the perinatal burst of proliferation of differentiated β-cells that normally generates most of the β-cell mass. In β-cells, Dach1 bound to the promoter of the cell cycle inhibitor p27Kip1, which constrains β-cell proliferation. Taken together, these data demonstrate a conserved role for Dachshund homologues in the production of insulin-producing cells. PMID:20869363

  1. Enhanced malignant tumorigenesis in Cdk4 transgenic mice.

    PubMed

    Miliani de Marval, Paula L; Macias, Everardo; Conti, Claudio J; Rodriguez-Puebla, Marcelo L

    2004-03-11

    In a previous study, we reported that overexpression of cyclin-dependent kinase-4 (CDK4) in mouse epidermis results in epidermal hyperplasia, hypertrophy and severe dermal fibrosis. In this study, we have investigated the susceptibility to skin tumor formation by forced expression of CDK4. Skin tumors from transgenic mice showed a dramatic increase in the rate of malignant progression to squamous cell carcinomas (SCC) in an initiation-promotion protocol. Histopathological analysis of papillomas from transgenic mice showed an elevated number of premalignant lesions characterized by dysplasia and marked atypia. Interestingly, transgenic mice also developed tumors in initiated but not promoted skin, demonstrating that CDK4 replaced the action of tumor promoters. These results suggest that expression of cyclin D1 upon ras activation synergizes with CDK4 overexpression. However, cyclin D1 transgenic mice and double transgenic mice for cyclin D1 and CDK4 did not show increased malignant progression in comparison to CDK4 transgenic mice. Biochemical analysis of tumors showed that CDK4 sequesters the CDK2 inhibitors p27Kip1 and p21Cip1, suggesting that indirect activation of CDK2 plays an important role in tumor development. These results indicate that, contrary to the general assumption, the catalytic subunit, CDK4, has higher oncogenic activity than cyclin D1, revealing a potential use of CDK4 as therapeutic target.

  2. Rho-associated kinase (ROCK) inhibition reverses low cell activity on hydrophobic surfaces.

    PubMed

    Tian, Yu Shun; Kim, Hyun Jung; Kim, Hyun-Man

    2009-08-28

    Hydrophobic polymers do not offer an adequate scaffold surface for cells to attach, migrate, proliferate, and differentiate. Thus, hydrophobic scaffolds for tissue engineering have traditionally been physicochemically modified to enhance cellular activity. However, modifying the surface by chemical or physical treatment requires supplementary engineering procedures. In the present study, regulation of a cell signal transduction pathway reversed the low cellular activity on a hydrophobic surface without surface modification. Inhibition of Rho-associated kinase (ROCK) by Y-27632 markedly enhanced adhesion, migration, and proliferation of osteoblastic cells cultured on a hydrophobic polystyrene surface. ROCK inhibition regulated cell-cycle-related molecules on the hydrophobic surface. This inhibition also decreased expression of the inhibitors of cyclin-dependent kinases such as p21(cip1) and p27(kip1) and increased expression of cyclin A and D. These results indicate that defective cellular activity on the hydrophobic surface can be reversed by the control of a cell signal transduction pathway without physicochemical surface modification.

  3. CBP loss cooperates with PTEN haploinsufficiency to drive prostate cancer: implications for epigenetic therapy

    PubMed Central

    Ding, Liya; Chen, Shuai; Liu, Ping; Pan, Yunqian; Zhong, Jian; Regan, Kevin M.; Wang, Liguo; Yu, Chunrong; Rizzardi, Tony; Cheng, Liang; Zhang, Jun; Schmechel, Stephen C.; Cheville, John C.; van Deursen, Jan; Tindall, Donald J.; Huang, Haojie

    2014-01-01

    Despite the high incidence and mortality of prostate cancer, the etiology of this disease is not fully understood. In this study, we develop functional evidence for CBP and PTEN interaction in prostate cancer based on findings of their correlate expression in the human disease. Cbppc−/−;Ptenpc+/− mice exhibited higher cell proliferation in the prostate and an early onset of high-grade prostatic intraepithelial neoplasia. Levels of EZH2 methyltransferase were increased along with its Thr350 phosphorylation in both mouse Cbp−/−;Pten+/− and human prostate cancer cells. CBP loss and PTEN deficiency cooperated to trigger a switch from K27-acetylated histone H3 to K27-trimethylated bulk histones, in a manner associated with decreased expression of the growth inhibitory EZH2 target genes DAB2IP, p27KIP1 and p21CIP1. Conversely, treatment with the histone deacetylase inhibitor panobinostat reversed this switch, in a manner associated with tumor suppression in Cbppc−/−;Ptenpc+/− mice. Our findings show how CBP and PTEN interact to mediate tumor suppression in the prostate, establishing a central role for histone modification in the etiology of prostate cancer and providing a rationale for clinical evaluation of epigenetic targeted therapy in prostate cancer patients. PMID:24491799

  4. Transgenic cowpea (Vigna unguiculata) seeds expressing a bean alpha-amylase inhibitor 1 confer resistance to storage pests, bruchid beetles.

    PubMed

    Solleti, Siva Kumar; Bakshi, Souvika; Purkayastha, Jubilee; Panda, Sanjib Kumar; Sahoo, Lingaraj

    2008-12-01

    Cowpea is one of the important grain legumes. Storage pests, Callosobruchus maculatus and C. chinensis cause severe damage to the cowpea seeds during storage. We employ a highly efficient Agrobacterium-mediated cowpea transformation method for introduction of the bean (Phaseolus vulgaris) alpha-amylase inhibitor-1 (alphaAI-1) gene into a commercially important Indian cowpea cultivar, Pusa Komal and generated fertile transgenic plants. The use of constitutive expression of additional vir genes in resident pSB1 vector in Agrobacterium strain LBA4404, thiol compounds during cocultivation and a geneticin based selection system resulted in twofold increase in stable transformation frequency. Expression of alphaAI-1 gene under bean phytohemagglutinin promoter results in accumulation of alphaAI-1 in transgenic seeds. The transgenic protein was active as an inhibitor of porcine alpha-amylase in vitro. Transgenic cowpeas expressing alphaAI-1 strongly inhibited the development of C. maculatus and C. chinensis in insect bioassays.

  5. Inducible expression of a fusion gene encoding two proteinase inhibitors leads to insect and pathogen resistance in transgenic rice.

    PubMed

    Quilis, Jordi; López-García, Belén; Meynard, Donaldo; Guiderdoni, Emmanuel; San Segundo, Blanca

    2014-04-01

    Plant proteinase inhibitors (PIs) are considered as candidates for increased insect resistance in transgenic plants. Insect adaptation to PI ingestion might, however, compromise the benefits received by transgenic expression of PIs. In this study, the maize proteinase inhibitor (MPI), an inhibitor of insect serine proteinases, and the potato carboxypeptidase inhibitor (PCI) were fused into a single open reading frame and introduced into rice plants. The two PIs were linked using either the processing site of the Bacillus thuringiensis Cry1B precursor protein or the 2A sequence from the foot-and-mouth disease virus (FMDV). Expression of each fusion gene was driven by the wound- and pathogen-inducible mpi promoter. The mpi-pci fusion gene was stably inherited for at least three generations with no penalty on plant phenotype. An important reduction in larval weight of Chilo suppressalis fed on mpi-pci rice, compared with larvae fed on wild-type plants, was observed. Expression of the mpi-pci fusion gene confers resistance to C. suppressalis (striped stem borer), one of the most important insect pest of rice. The mpi-pci expression systems described may represent a suitable strategy for insect pest control, better than strategies based on the use of single PI genes, by preventing insect adaptive responses. The rice plants expressing the mpi-pci fusion gene also showed enhanced resistance to infection by the fungus Magnaporthe oryzae, the causal agent of the rice blast disease. Our results illustrate the usefulness of the inducible expression of the mpi-pci fusion gene for dual resistance against insects and pathogens in rice plants.

  6. Synergistic Activation of Latent HIV-1 Expression by Novel Histone Deacetylase Inhibitors and Bryostatin-1

    PubMed Central

    Martínez-Bonet, Marta; Isabel Clemente, Maria; Jesús Serramía, Maria; Muñoz, Eduardo; Moreno, Santiago; Ángeles Muñoz-Fernández, Maria

    2015-01-01

    Viral reactivation from latently infected cells has become a promising therapeutic approach to eradicate HIV. Due to the complexity of the viral latency, combinations of efficient and available drugs targeting different pathways of latency are needed. In this work, we evaluated the effect of various combinations of bryostatin-1 (BRY) and novel histone deacetylase inhibitors (HDACIs) on HIV-reactivation and on cellular phenotype. The lymphocyte (J89GFP) or monocyte/macrophage (THP89GFP) latently infected cell lines were treated with BRY, panobinostat (PNB) and romidepsin (RMD) either alone or in combination. Thus, the effect on the viral reactivation was evaluated. We calculated the combination index for each drug combination; the BRY/HDACIs showed a synergistic HIV-reactivation profile in the majority of the combinations tested, whereas non-synergistic effects were observed when PNB was mixed with RMD. Indeed, the 75% effective concentrations of BRY, PNB and RMD were reduced in these combinations. Moreover, primary CD4 T cells treated with such drug combinations presented similar activation and proliferation profiles in comparison with single drug treated cells. Summing up, combinations between BRY, PNB and/or RMD presented a synergistic profile by inducing virus expression in HIV-latently infected cells, rendering these combinations an attractive novel and safe option for future clinical trials. PMID:26563568

  7. NANOG Expression as a Responsive Biomarker during Treatment with Hedgehog Signal Inhibitor in Acute Myeloid Leukemia

    PubMed Central

    Kakiuchi, Seiji; Minami, Yosuke; Miyata, Yoshiharu; Mizutani, Yu; Goto, Hideaki; Kawamoto, Shinichiro; Yakushijin, Kimikazu; Kurata, Keiji; Matsuoka, Hiroshi; Minami, Hironobu

    2017-01-01

    Aberrant activation of the Hedgehog (Hh) signaling pathway is involved in the maintenance of leukemic stem cell (LSCs) populations. PF-0444913 (PF-913) is a novel inhibitor that selectively targets Smoothened (SMO), which regulates the Hh pathway. Treatment with PF-913 has shown promising results in an early phase study of acute myeloid leukemia (AML). However, a detailed mode of action for PF-913 and relevant biomarkers remain to be elucidated. In this study, we examined bone marrow samples derived from AML patients under PF-913 monotherapy. Gene set enrichment analysis (GSEA) revealed that PF-913 treatment affected the self-renewal signature and cell-cycle regulation associated with LSC-like properties. We then focused on the expression of a pluripotency factor, NANOG, because previous reports showed that a downstream effector in the Hh pathway, GLI, directly binds to the NANOG promoter and that the GLI-NANOG axis promotes stemness and growth in several cancers. In this study, we found that a change in NANOG transcripts was closely associated with GLI-target genes and NANOG transcripts can be a responsive biomarker during PF-913 therapy. Additionally, the treatment of AML with PF-913 holds promise, possibly through inducing quiescent leukemia stem cells toward cell cycling. PMID:28245563

  8. HDAC inhibitor AR-42 decreases CD44 expression and sensitizes myeloma cells to lenalidomide.

    PubMed

    Canella, Alessandro; Cordero Nieves, Hector; Sborov, Douglas W; Cascione, Luciano; Radomska, Hanna S; Smith, Emily; Stiff, Andrew; Consiglio, Jessica; Caserta, Enrico; Rizzotto, Lara; Zanesi, Nicola; Stefano, Volinia; Kaur, Balveen; Mo, Xiaokui; Byrd, John C; Efebera, Yvonne A; Hofmeister, Craig C; Pichiorri, Flavia

    2015-10-13

    Multiple myeloma (MM) is a hematological malignancy of plasma cells in the bone marrow. Despite multiple treatment options, MM is inevitably associated with drug resistance and poor outcomes. Histone deacetylase inhibitors (HDACi's) are promising novel chemotherapeutics undergoing evaluation in clinical trials for the potential treatment of patients with MM. Although in preclinical studies HDACi's have proven anti-myeloma activity, but in the clinic single-agent HDACi treatments have been limited due to low tolerability. Improved clinical outcomes were reported only when HDACi's were combined with other drugs. Here, we show that a novel pan-HDACi AR-42 downregulates CD44, a glycoprotein that has been associated with lenalidomide and dexamethasone resistance in myeloma both in vitro and in vivo. We also show that this CD44 downregulation is in part mediated by miR-9-5p, targeting insulin-like growth factor 2 mRNA binding protein 3 (IGF2BP3), which directly binds to CD44 mRNA and increases its stability. Importantly, we also demonstrate that AR-42 enhances anti-myeloma activity of lenalidomide in primary MM cells isolated from lenalidomide resistant patients and in in vivo MM mouse model. Thus, our findings shed light on potential novel combinatorial therapeutic approaches modulating CD44 expression, which may help overcome lenalidomide resistance in myeloma patients.

  9. HDAC inhibitor AR-42 decreases CD44 expression and sensitizes myeloma cells to lenalidomide

    PubMed Central

    Sborov, Douglas W.; Cascione, Luciano; Radomska, Hanna S.; Smith, Emily; Stiff, Andrew; Consiglio, Jessica; Caserta, Enrico; Rizzotto, Lara; Zanesi, Nicola; Stefano, Volinia; Kaur, Balveen; Mo, Xiaokui; Byrd, John C.; Efebera, Yvonne A.

    2015-01-01

    Multiple myeloma (MM) is a hematological malignancy of plasma cells in the bone marrow. Despite multiple treatment options, MM is inevitably associated with drug resistance and poor outcomes. Histone deacetylase inhibitors (HDACi's) are promising novel chemotherapeutics undergoing evaluation in clinical trials for the potential treatment of patients with MM. Although in preclinical studies HDACi's have proven anti-myeloma activity, but in the clinic single-agent HDACi treatments have been limited due to low tolerability. Improved clinical outcomes were reported only when HDACi's were combined with other drugs. Here, we show that a novel pan-HDACi AR-42 downregulates CD44, a glycoprotein that has been associated with lenalidomide and dexamethasone resistance in myeloma both in vitro and in vivo. We also show that this CD44 downregulation is in part mediated by miR-9–5p, targeting insulin-like growth factor 2 mRNA binding protein 3 (IGF2BP3), which directly binds to CD44 mRNA and increases its stability. Importantly, we also demonstrate that AR-42 enhances anti-myeloma activity of lenalidomide in primary MM cells isolated from lenalidomide resistant patients and in in vivo MM mouse model. Thus, our findings shed light on potential novel combinatorial therapeutic approaches modulating CD44 expression, which may help overcome lenalidomide resistance in myeloma patients. PMID:26429859

  10. Stimulus-Dependent Inhibitor of Apoptosis Protein Expression Prolongs the Duration of B Cell Signalling

    PubMed Central

    Shinohara, Hisaaki; Inoue, Kentaro; Yumoto, Noriko; Nagashima, Takeshi; Okada-Hatakeyama, Mariko

    2016-01-01

    Different dynamic behaviours of signalling activity can induce distinct biological responses in a variety of cells. However, the molecular mechanisms that determine the dynamics of kinase activities in immune cells are not well understood. In this study, we showed that the duration of both IκB kinase (IKK) and extracellular signal-regulated kinase (ERK) activities in B cell receptor (BCR)- and CD40-signalling pathways in B cells were regulated by transcriptional feedback loops. We conducted a time-course transcriptome analysis after BCR or CD40 stimulation and identified the following four candidate genes as feedback regulators for IKK and ERK: inhibitor of apoptosis protein (IAP), TNF alpha-induced protein 3, dual-specificity phosphatase 5, and sprouty homolog 2. Quantitative experiments and mathematical modelling suggested that IAP inhibition shortened the duration of IKK and ERK activity following both BCR and CD40 pathway stimulation, indicating a positive role for IAP in B cell signalling. Furthermore, transient kinase activities induced by IAP blockage reduced the levels of delayed expression genes. Together, our findings suggest that IKK and ERK activity durations can be fine-tuned by the coordinated regulation of positive and negative transcriptional feedback and that these network properties determine the biological output of B cells. PMID:27277891

  11. Diazepam binding inhibitor gene expression: Location in brain and peripheral tissues of rate

    SciTech Connect

    Alho, H.; Fremeau, R.T. Jr.; Tiedge, H.; Wilcox, J.; Bovolin, P.; Brosius, J.; Roberts, J.L.; Costa, E.

    1988-09-01

    Diazepam binding inhibitor (DBI), an endogenous 10-kDa polypeptide was isolated from rat and human brain by monitoring displacement of radioactive diazepam bound to specific recognition sites in brain synaptic and mitochondrial membranes. The cellular location of DBI mRNA was studied in rat brain and selected peripheral tissues by in situ hybridization histochemistry with a /sup 35/S-labeled single-stranded complementary RNA probe. DBI mRNA was heterogeneously distributed in rat brain, with particularly high levels in the area postrema, the cerebellar cortex, and ependyma of the third ventricle. Intermediate levels were found in the olfactory bulb, pontine nuclei, inferior colliculi, arcuate nucleus, and pineal gland. Relatively low but significant levels of silver grains were observed overlying many mesencephalic and telencephalic areas that have previously been shown to contain numerous DBI-immunoreactive neurons and a high density of central benzodiazepine receptors. In situ hybridizations also revealed high levels of DBI mRNA in the posterior lobe of the pituitary gland, liver, and germinal center of the white pulp of spleen, all tissues that are rich in peripheral benzodiazepine binding sites. The tissue-specific pattern of DBI gene expression described here could be exploited to further understand the physiological function of DBI in the brain and periphery.

  12. Maintenance of the Extracellular Matrix in Rat Anterior Pituitary Gland: Identification of Cells Expressing Tissue Inhibitors of Metalloproteinases.

    PubMed

    Azuma, Morio; Tofrizal, Alimuddin; Maliza, Rita; Batchuluun, Khongorzul; Ramadhani, Dini; Syaidah, Rahimi; Tsukada, Takehiro; Fujiwara, Ken; Kikuchi, Motoshi; Horiguchi, Kotaro; Yashiro, Takashi

    2015-12-25

    The extracellular matrix (ECM) is important in creating cellular environments in tissues. Recent studies have demonstrated that ECM components are localized in anterior pituitary cells and affect cell activity. Thus, clarifying the mechanism responsible for ECM maintenance would improve understanding of gland function. Tissue inhibitors of metalloproteinases (TIMPs) are endogenous inhibitors of matrix metalloproteinases and participate in ECM degradation. In this study, we investigated whether cells expressing TIMPs are present in rat anterior pituitary gland. Reverse transcription polymerase chain reaction was used to analyze expression of the TIMP family (TIMP1-4), and cells producing TIMPs in the gland were identified by using in situ hybridization. Expression of TIMP1, TIMP2, and TIMP3 mRNAs was detected, and the TIMP-expressing cells were located in the gland. The TIMP-expressing cells were also investigated by means of double-staining with in situ hybridization and immunohistochemical techniques. Double-staining revealed that TIMP1 mRNA was expressed in folliculostellate cells. TIMP2 mRNA was detected in folliculostellate cells, prolactin cells, and thyroid-stimulating hormone cells. TIMP3 mRNA was identified in endothelial cells, pericytes, novel desmin-immunopositive perivascular cells, and folliculostellate cells. These findings indicate that TIMP1-, TIMP2-, and TIMP3-expressing cells are present in rat anterior pituitary gland and that they are involved in maintaining ECM components.

  13. Effects of Histone Deacetylase Inhibitor (HDACi); Trichostatin-A (TSA) on the expression of housekeeping genes.

    PubMed

    Mogal, Ashish; Abdulkadir, Sarki A

    2006-04-01

    In quantitative RT-PCR (qRT-PCR), analysis of gene expression is dependent on normalization using housekeeping genes such as 18S rRNA, GAPDH and beta actin. However, variability in their expression has been reported to be caused by factors like drug treatment, pathological states and cell-cycle phase. An emerging area of cancer research focuses on identifying the role of epigenetic alterations such as histone modifications and DNA methylation in the initiation and progression of cancer. Histone acetylation is the best studied modification so far and has been probed through the use of histone deacetylase inhibitors (HDACi). Further, modulation of histone acetylation is currently being explored as a therapeutic strategy in the treatment of cancer and HDACis have shown promise in inhibiting tumorigenesis and metastasis. Trichostatin-A (TSA) is the most widely used HDACi. Therefore, we were driven to identify a suitable internal control for RT-PCR following TSA treatment. We performed quantitative RT-PCR analysis using mouse prostate tissue explants, human prostate cancer (LNCaP) cells and human breast cancer (T-47D and ZR-75-1) cells following TSA treatment. Expression of housekeeping genes including 18S rRNA, beta actin, GAPDH and ribosomal highly-basic 23-kDa protein (rb 23-kDa, RPL13A) were compared in vehicle versus TSA treated samples. Our results showed marked variations in 18S rRNA, beta actin mRNA and GAPDH mRNA levels in mouse prostate explants and a human prostate cancer (LNCaP) cell line following TSA treatment. Furthermore, in two human breast cancer cell lines (T-47D and ZR-75-1) 18S rRNA, beta actin mRNA and GAPDH mRNA levels varied significantly. However, RPL13A mRNA levels remained constant in all the conditions tested. Therefore, we recommend use of RPL13A as a standard for normalization during TSA treatment.

  14. Phosphorylation of ezrin-radixin-moesin-binding phosphoprotein 50 (EBP50) by Akt promotes stability and mitogenic function of S-phase kinase-associated protein-2 (Skp2).

    PubMed

    Song, Gyun Jee; Leslie, Kristen L; Barrick, Stacey; Mamonova, Tatyana; Fitzpatrick, Jeremy M; Drombosky, Kenneth W; Peyser, Noah; Wang, Bin; Pellegrini, Maria; Bauer, Philip M; Friedman, Peter A; Mierke, Dale F; Bisello, Alessandro

    2015-01-30

    The regulation of the cell cycle by the ubiquitin-proteasome system is dependent on the activity of E3 ligases. Skp2 (S-phase kinase associated protein-2) is the substrate recognition subunit of the E3 ligase that ubiquitylates the cell cycle inhibitors p21(cip1) and p27(kip1) thus promoting cell cycle progression. Increased expression of Skp2 is frequently observed in diseases characterized by excessive cell proliferation, such as cancer and neointima hyperplasia. The stability and cellular localization of Skp2 are regulated by Akt, but the molecular mechanisms underlying these effects remain only partly understood. The scaffolding protein Ezrin-Binding Phosphoprotein of 50 kDa (EBP50) contains two PDZ domains and plays a critical role in the development of neointimal hyperplasia. Here we report that EBP50 directly binds Skp2 via its first PDZ domain. Moreover, EBP50 is phosphorylated by Akt on Thr-156 within the second PDZ domain, an event that allosterically promotes binding to Skp2. The interaction with EBP50 causes cytoplasmic localization of Skp2, increases Skp2 stability and promotes proliferation of primary vascular smooth muscle cells. Collectively, these studies define a novel regulatory mechanism contributing to aberrant cell growth and highlight the importance of scaffolding function of EBP50 in Akt-dependent cell proliferation.

  15. Zebrafish phenotypic screen identifies novel Notch antagonists.

    PubMed

    Velaithan, Vithya; Okuda, Kazuhide Shaun; Ng, Mei Fong; Samat, Norazwana; Leong, Sze Wei; Faudzi, Siti Munirah Mohd; Abas, Faridah; Shaari, Khozirah; Cheong, Sok Ching; Tan, Pei Jean; Patel, Vyomesh

    2017-04-01

    Zebrafish represents a powerful in vivo model for phenotype-based drug discovery to identify clinically relevant small molecules. By utilizing this model, we evaluated natural product derived compounds that could potentially modulate Notch signaling that is important in both zebrafish embryogenesis and pathogenic in human cancers. A total of 234 compounds were screened using zebrafish embryos and 3 were identified to be conferring phenotypic alterations similar to embryos treated with known Notch inhibitors. Subsequent secondary screens using HEK293T cells overexpressing truncated Notch1 (HEK293TΔE) identified 2 compounds, EDD3 and 3H4MB, to be potential Notch antagonists. Both compounds reduced protein expression of NOTCH1, Notch intracellular domain (NICD) and hairy and enhancer of split-1 (HES1) in HEK293TΔE and downregulated Notch target genes. Importantly, EDD3 treatment of human oral cancer cell lines demonstrated reduction of Notch target proteins and genes. EDD3 also inhibited proliferation and induced G0/G1 cell cycle arrest of ORL-150 cells through inducing p27(KIP1). Our data demonstrates the utility of the zebrafish phenotypic screen and identifying EDD3 as a promising Notch antagonist for further development as a novel therapeutic agent.

  16. 17β-Estradiol Induces Overproliferation in Adenomyotic Human Uterine Smooth Muscle Cells of the Junctional Zone Through Hyperactivation of the Estrogen Receptor-Enhanced RhoA/ROCK Signaling Pathway.

    PubMed

    Sun, Fu-Qing; Duan, Hua; Wang, Sha; Li, Jin-Jiao

    2015-11-01

    Adenomyosis (ADS) is a common estrogen-dependent gynecological disease with unknown etiology. Recent models favor abnormal thickening of the junctional zone (JZ) may be the causative factor in the development of ADS. RhoA, a small guanosine triphosphatase which controls multiple cellular processes, is involved in the control of cell proliferation. Here we demonstrate that treatment of human uterine smooth muscle cells (SMCs) of the JZ with 17β-estradiol (E2) increased expression of RhoA and its downstream effectors (-associated coiled coil containing protein kinase [ROCK] 1 and ROCK2). Compared with non-ADS cells, RhoA, ROCK1, and ROCK2 were overexpressed and hyperactivated in ADS cells. These effects were suppressed in the presence of ICI 182,780, supporting an estrogen receptor (ER)-dependent mechanism. Hyperactivation of ER-enhanced RhoA/ROCK signaling was associated with overproliferation in ADS human uterine SMCs of the JZ. Moreover, E2-induced overproliferation was accompanied by downregulation of cyclin-dependent kinases inhibitors (CKIs; p21(Waf1/Cip1) and p27(Kip1)) and upregulation of cyclin-dependent kinases (CDKs) and cyclins (cyclin D1, cyclin E1, CDK2, CDK4, and CDK6).

  17. Anti-proliferation effect on human breast cancer cells via inhibition of pRb phosphorylation by taiwanin E isolated from Eleutherococcus trifoliatus.

    PubMed

    Wang, Hui-Chun; Tseng, Yen-Hsueh; Wu, Hui-Rong; Chu, Fang-Hua; Kuo, Yueh-Hsiung; Wang, Sheng-Yang

    2014-09-01

    Eleutherococcus trifoliatus has been used as a folk medicine since ancient times, especially as refreshing qi medicines. In our current study, taiwanin E, which possesses strong cytotoxicity, was isolated from the branches of E. trifoliatus by using a bioactivity guided fractionation procedure. Taiwanin E presented a potent anti-proliferation activity on the growth of a human breast adenocarcinoma cell line (MCF-7), with an IC50 value for cytotoxicity of 1.47 μM. Cell cycle analysis revealed that the proportion of cells in the G0/G1 phase increased in a dose-dependent manner (from 79.4% to 90.2%) after 48 h exposure to taiwanin E at a dosage range from 0.5 to 4μM. After treatment with taiwanin E, phosphorylation of retinoblastoma protein (pRb) in MCF-7 cells was inhibited, accompanied by a decrease in the levels of cyclin D1, cyclin D3 and cyclin-dependent kinase 4 (cdk4) and cdk6; in addition, there was an increase in the expression of cyclin-dependent kinase inhibitors p21(WAF-1/Cip) and p27(Kip1). The results suggest that taiwanin E inhibits cell cycle progression of MCF-7 at the G0/G1 transition.

  18. Isolation, expression and characterization of a novel dual serine protease inhibitor, OH-TCI, from king cobra venom.

    PubMed

    He, Ying-Ying; Liu, Shu-Bai; Lee, Wen-Hui; Qian, Jin-Qiao; Zhang, Yun

    2008-10-01

    Snake venom Kunitz/BPTI members are good tools for understanding of structure-functional relationship between serine proteases and their inhibitors. A novel dual Kunitz/BPTI serine proteinase inhibitor named OH-TCI (trypsin- and chymotrypsin-dual inhibitor from Ophiophagus hannah) was isolated from king cobra venom by three chromatographic steps of gel filtration, trypsin affinity and reverse phase HPLC. OH-TCI is composed of 58 amino acid residues with a molecular mass of 6339Da. Successful expression of OH-TCI was performed as the maltose-binding fusion protein in E. coli DH5alpha. Much different from Oh11-1, the purified native and recombinant OH-TCI both had strong inhibitory activities against trypsin and chymotrypsin although the sequence identity (74.1%) between them is very high. The inhibitor constants (K(i)) of recombinant OH-TCI were 3.91 x 10(-7) and 8.46 x10(-8)M for trypsin and chymotrypsin, respectively. To our knowledge, it was the first report of Kunitz/BPTI serine proteinase inhibitor from snake venom that had equivalent trypsin and chymotrypsin inhibitory activities.

  19. Renin-angiotensin-aldosterone system inhibitors improve membrane stability and change gene-expression profiles in dystrophic skeletal muscles.

    PubMed

    Chadwick, Jessica A; Bhattacharya, Sayak; Lowe, Jeovanna; Weisleder, Noah; Rafael-Fortney, Jill A

    2017-02-01

    Angiotensin-converting enzyme inhibitors (ACEi) and mineralocorticoid receptor (MR) antagonists are FDA-approved drugs that inhibit the renin-angiotensin-aldosterone system (RAAS) and are used to treat heart failure. Combined treatment with the ACEi lisinopril and the nonspecific MR antagonist spironolactone surprisingly improves skeletal muscle, in addition to heart function and pathology in a Duchenne muscular dystrophy (DMD) mouse model. We recently demonstrated that MR is present in all limb and respiratory muscles and functions as a steroid hormone receptor in differentiated normal human skeletal muscle fibers. The goals of the current study were to begin to define cellular and molecular mechanisms mediating the skeletal muscle efficacy of RAAS inhibitor treatment. We also compared molecular changes resulting from RAAS inhibition with those resulting from the current DMD standard-of-care glucocorticoid treatment. Direct assessment of muscle membrane integrity demonstrated improvement in dystrophic mice treated with lisinopril and spironolactone compared with untreated mice. Short-term treatments of dystrophic mice with specific and nonspecific MR antagonists combined with lisinopril led to overlapping gene-expression profiles with beneficial regulation of metabolic processes and decreased inflammatory gene expression. Glucocorticoids increased apoptotic, proteolytic, and chemokine gene expression that was not changed by RAAS inhibitors in dystrophic mice. Microarray data identified potential genes that may underlie RAAS inhibitor treatment efficacy and the side effects of glucocorticoids. Direct effects of RAAS inhibitors on membrane integrity also contribute to improved pathology of dystrophic muscles. Together, these data will inform clinical development of MR antagonists for treating skeletal muscles in DMD.

  20. Effects of protein tyrosine kinase inhibitors on cytokine-induced adhesion molecule expression by human umbilical vein endothelial cells.

    PubMed Central

    May, M. J.; Wheeler-Jones, C. P.; Pearson, J. D.

    1996-01-01

    1. Endothelial cells can be stimulated by the pro-inflammatory cytokines interleukin (IL)-1 alpha and tumour necrosis factor (TNF) alpha to express the leukocyte adhesion molecules E-selectin, vascular cell adhesion molecule (VCAM)-1 and intercellular adhesion molecule (ICAM)-1 but the intracellular signalling mechanisms leading to this expression are incompletely understood. We have investigated the role of protein tyrosine kinases (PTK) in adhesion molecule expression by cytokine-activated human umbilical vein endothelial cells (HUVEC) using the PTK inhibitors genistein and herbimycin A, and the protein tyrosine phosphatase (PTP) inhibitor sodium orthovanadate. 2. Maximal E-selectin expression induced by incubation of HUVEC for 4 h with IL-1 alpha (100 u ml-1) and TNF alpha (100 u ml-1) was dose-dependently inhibited by genistein and herbimycin A. Although similar effects were seen on phorbol 12-myristate, 13-acetate (PMA)-induced expression, this was not due to inhibition of protein kinase C (PKC) activity as the selective inhibitors of PKC, bisindolylmaleimide (BIM), Ro31-7549 or Ro31-8220 did not affect IL-1 alpha- or TNF alpha-induced E-selectin expression at concentrations which maximally inhibited PMA-induced expression. 3. Genistein inhibited VCAM-1 expression induced by incubation of HUVEC for 24 h with TNF alpha or IL-1 alpha whereas it did not affect ICAM-1 expression induced by 24 h incubation with either of these cytokines. Herbimycin A inhibited both VCAM-1 and ICAM-1 expression induced by TNF alpha. 4. Basal expression of E-selectin, VCAM-1 and ICAM-1 was dose-dependently enhanced by sodium orthovanadate. In contrast, vanadate differentially affected TNF alpha-induced expression of these molecules with maximal E-selectin and ICAM-1 expression being slightly enhanced and VCAM-1 expression dose-dependently reduced. 5. We also studied the effects of PTK and PTP inhibitors on adhesion of the human pre-myeloid cell line U937 to TNF alpha-stimulated HUVEC

  1. Class-Specific Histone Deacetylase Inhibitors Promote 11-Beta Hydroxysteroid Dehydrogenase Type 2 Expression in JEG-3 Cells

    PubMed Central

    Togher, Katie L.; Kenny, Louise C.

    2017-01-01

    Exposure to maternal cortisol plays a crucial role in fetal organogenesis. However, fetal overexposure to cortisol has been linked to a range of short- and long-term adverse outcomes. Normally, this is prevented by the expression of an enzyme in the placenta called 11-beta hydroxysteroid dehydrogenase type 2 (11β-HSD2) which converts active cortisol to its inactive metabolite cortisone. Placental 11β-HSD2 is known to be reduced in a number of adverse pregnancy complications, possibly through an epigenetic mechanism. As a result, a number of pan-HDAC inhibitors have been examined for their ability to promote 11β-HSD2 expression. However, it is not known if the effects of pan-HDAC inhibition are a general phenomenon or if the effects are dependent upon a specific class of HDACs. Here, we examined the ability of pan- and class-specific HDAC inhibitors to regulate 11β-HSD2 expression in JEG3 cells. We find that pan-, class I, or class IIa HDAC inhibition promoted 11β-HSD2 expression and prevented cortisol or interleukin-1β-induced decrease in its expression. These results demonstrate that targeting a specific class of HDACs can promote 11β-HSD2 expression in JEG3 cells. This adds to the growing body of evidence suggesting that HDACs may be crucial in maintaining normal fetal development. PMID:28321257

  2. Src family tyrosine kinase inhibitors suppress Nav1.1 expression in cultured rat spiral ganglion neurons.

    PubMed

    Chen, Huiying; Zeng, Qingjiao; Yao, Chen; Cai, Zheng; Wei, Tingjia; Huang, Zhihui; Su, Jiping

    2016-03-01

    Src family kinases regulate neuronal voltage-gated Na(+) channels, which generate action potentials. The mechanisms of action, however, remain poorly understood. The aim of the present study was to further elucidate the effects of Src family kinases on Nav1.1 mRNA and protein expression in spiral ganglion neurons. Immunofluorescence staining techniques detected Nav1.1 expression in the spiral ganglion neurons. Additionally, quantitative PCR and western blot techniques were used to analyze Nav1.1 mRNA and protein expression, respectively, in spiral ganglion neurons following exposure to Src family kinase inhibitors PP2 (1 and 10 μM) and SU6656 (0.1 and 1 μM) for different lengths of time (6 and 24 h). In the spiral ganglion neurons, Nav1.1 protein expression was detected in the somas and axons. The Src family kinase inhibitors PP2 and SU6665 significantly decreased Nav1.1 mRNA and protein expression (p < 0.05), respectively, in the spiral ganglion neurons, and changes in expression were not dependent on time or dose (p > 0.05).

  3. A functional pectin methylesterase inhibitor protein (SolyPMEI) is expressed during tomato fruit ripening and interacts with PME-1.

    PubMed

    Reca, Ida Barbara; Lionetti, Vincenzo; Camardella, Laura; D'Avino, Rossana; Giardina, Thierry; Cervone, Felice; Bellincampi, Daniela

    2012-07-01

    A pectin methylesterase inhibitor (SolyPMEI) from tomato has been identified and characterised by a functional genomics approach. SolyPMEI is a cell wall protein sharing high similarity with Actinidia deliciosa PMEI (AdPMEI), the best characterised inhibitor from kiwi. It typically affects the activity of plant pectin methylesterases (PMEs) and is inactive against a microbial PME. SolyPMEI transcripts were mainly expressed in flower, pollen and ripe fruit where the protein accumulated at breaker and turning stages of ripening. The expression of SolyPMEI correlated during ripening with that of PME-1, the major fruit specific PME isoform. The interaction of SolyPMEI with PME-1 was demonstrated in ripe fruit by gel filtration and by immunoaffinity chromatography. The analysis of the zonal distribution of PME activity and the co-localization of SolyPMEI with high esterified pectins suggest that SolyPMEI regulates the spatial patterning of distribution of esterified pectins in fruit.

  4. Expression of an S phase-stabilized version of the CDK inhibitor Dacapo can alter endoreplication

    PubMed Central

    Swanson, Christina I.; Meserve, Joy H.; McCarter, Patrick C.; Thieme, Alexis; Mathew, Tony; Elston, Timothy C.; Duronio, Robert J.

    2015-01-01

    In developing organisms, divergence from the canonical cell division cycle is often necessary to ensure the proper growth, differentiation, and physiological function of a variety of tissues. An important example is endoreplication, in which endocycling cells alternate between G and S phase without intervening mitosis or cytokinesis, resulting in polyploidy. Although significantly different from the canonical cell cycle, endocycles use regulatory pathways that also function in diploid cells, particularly those involved in S phase entry and progression. A key S phase regulator is the Cyclin E-Cdk2 kinase, which must alternate between periods of high (S phase) and low (G phase) activity in order for endocycling cells to achieve repeated rounds of S phase and polyploidy. The mechanisms that drive these oscillations of Cyclin E-Cdk2 activity are not fully understood. Here, we show that the Drosophila Cyclin E-Cdk2 inhibitor Dacapo (Dap) is targeted for destruction during S phase via a PIP degron, contributing to oscillations of Dap protein accumulation during both mitotic cycles and endocycles. Expression of a PIP degron mutant Dap attenuates endocycle progression but does not obviously affect proliferating diploid cells. A mathematical model of the endocycle predicts that the rate of destruction of Dap during S phase modulates the endocycle by regulating the length of G phase. We propose from this model and our in vivo data that endo S phase-coupled destruction of Dap reduces the threshold of Cyclin E-Cdk2 activity necessary to trigger the subsequent G-S transition, thereby influencing endocycle oscillation frequency and the extent of polyploidy. PMID:26493402

  5. Discovery of novel heteroaryl-substituted chalcones as inhibitors of TNF-alpha-induced VCAM-1 expression.

    PubMed

    Meng, Charles Q; Zheng, X Sharon; Ni, Liming; Ye, Zhihong; Simpson, Jacob E; Worsencroft, Kimberly J; Hotema, Martha R; Weingarten, M David; Skudlarek, Jason W; Gilmore, Joshua M; Hoong, Lee K; Hill, Russell R; Marino, Elaine M; Suen, Ki-Ling; Kunsch, Charles; Wasserman, Martin A; Sikorski, James A

    2004-03-22

    Novel chalcone derivatives have been discovered as potent inhibitors of TNF-alpha-induced VCAM-1 expression. Thienyl or benzothienyl substitution at the meta-position of ring B helps boost potency while large substitution at the para-position on ring B is detrimental. Various substitutions are tolerated on ring A. A lipophilicity-potency relationship has been observed in several sub-series of compounds.

  6. Gene and protein expression in pituitary corticotroph adenomas: a systematic review of the literature.

    PubMed

    Seltzer, Justin; Ashton, Charles E; Scotton, Thomas C; Pangal, Dhiraj; Carmichael, John D; Zada, Gabriel

    2015-02-01

    , and gene underexpression in 58 genes and 15 proteins was reported. Immunohistochemistry was used in 39 of the studies, and reverse transcriptase polymerase chain reaction was used in 26 of the studies, primarily, and as validation for 4 others. Thirteen studies used both immunohistochemistry and reverse transcriptase polymerase chain reaction. Other methods used included microarray, in situ hybridization, Northern blot analysis, and Western blot analysis. Expression of prioritized genes emphasized in multiple studies were often validated on both the gene and protein levels. Genes/proteins found to be overexpressed in ACTH-PAs relative to the normal pituitary gland included hPTTG1/securin, NEUROD1/NeuroD1 (Beta2), HSD11B2/11β-hydroxysteroid dehydrogenase 2, AKT/Akt, protein kinase B, and CCND1/cyclin D1. Candidate genes/proteins found to be underexpressed in ACTH-PAs relative to the normal pituitary gland included CDKN1B/p27(Kip1), CDKN2A/p16, KISS1/kisspeptin, ACTHR/ACTH-R, and miR-493. CONCLUSIONS On the basis of the authors' systematic review, many significant gene and protein targets that may contribute to tumorigenesis, invasion, and hormone production/secretion of ACTH have been identified and validated in ACTH-PAs. Many of these potential targets have not been fully analyzed for their therapeutic and diagnostic potential but may represent candidate molecular targets for biomarker development and drug targeting. This review may help catalyze additional research efforts using modern profiling and sequencing techniques and alteration of gene expression.

  7. Tension Force Downregulates Matrix Metalloproteinase Expression and Upregulates the Expression of Their Inhibitors through MAPK Signaling Pathways in MC3T3-E1 cells

    PubMed Central

    Karasawa, Yoko; Tanaka, Hideki; Nakai, Kumiko; Tanabe, Natsuko; Kawato, Takayuki; Maeno, Masao; Shimizu, Noriyoshi

    2015-01-01

    Objective: Matrix metalloproteinases (MMPs), produced by osteoblasts, catalyze the turnover of extracellular matrix (ECM) molecules in osteoid, and the regulation of MMP activity depends on interactions between MMPs and tissue inhibitors of metalloproteinases (TIMPs). We focused on the degradation process of ECM in osteoid that was exposed to mechanical strain, and conducted an in vitro study using MC3T3-E1 osteoblastic cells to examine the effects of tension force (TF) on the expression of MMPs and TIMPs, and activation of mitogen-activated protein kinase (MAPK) pathways. Design: Cells were incubated on flexible-bottomed culture plates and stimulated with or without cyclic TF for 24 hours. The expression of MMPs and TIMPs was examined at mRNA and protein levels by real-time RT-PCR and Western blotting, respectively. The phosphorylation of extracellular signal-regulated kinase (ERK) 1/2, p38 MAPK, and stress-activated protein kinases/c-jun N-terminal kinases (SAPK/JNK) were examined by Western blotting. Results: TF decreased the expression of MMP-1, -3, -13 and phosphorylated ERK1/2. In contrast, TF increased the expression of TIMP-2, -3 and phosphorylated SAPK/JNK. The expression of MMP-2, -14, TIMP-1, -4 and phosphorylated p38 MAPK was unaffected by TF. MMP-1, -3 and -13 expression decreased in cells treated with the ERK inhibitor PD98059 compared with untreated control cells. The JNK inhibitor SP600125 inhibited the TF-induced upregulation of TIMP-2 and -3. Conclusions: The results suggest that TF suppresses the degradation process that occurs during ECM turnover in osteoid via decreased production of MMP-1, -3 and -13, and increased production of TIMP-2 and -3 through the MAPK signaling pathways in osteoblasts. PMID:26640410

  8. Expression of matrix metalloproteinases, their tissue inhibitors, and osteopontin in the wall of thoracic and abdominal aortas with dilatative pathology.

    PubMed

    Lesauskaite, Vaiva; Epistolato, Maria Carmela; Castagnini, Marta; Urbonavicius, Sigitas; Tanganelli, Piero

    2006-08-01

    Matrix metalloproteinases (MMPs) degrade extracellular matrix and may play a central role in the pathogenesis of aortic aneurysms. We studied 2 groups of patients: 15 with dilatative pathology of the ascending thoracic aorta and 17 with aneurysm of the abdominal aortic wall (AAA). We compared the expression of MMPs, tissue inhibitors of matrix metalloproteinases (TIMPs), and osteopontin in the wall of thoracic and abdominal aneurysms. In AAA, MMP-9 and TIMP-1 expression in inflammatory cells was higher than in smooth muscle cells (SMCs) (median score: 3.5 versus 1, P < .0001; 2 versus 1, P < .04, respectively), whereas MMP-2 demonstrated higher expression in SMCs than in inflammatory cells (median score: 0 versus 4, P < .0001). In ATA, MMP-2, MMP-9, TIMP-1, TIMP-2, TIMP-3, and osteopontin expression in SMCs was higher than in inflammatory cells (median score: 3 versus 0, P < .0001; 4 versus 1, P < .0005; 2 versus 0, P < .001; 5 versus 2, P < .0001; 2 versus 0, P < .005; and 5 versus 1.5, P < .0001, respectively), when both inflammatory cells of the media and the adventitia were considered together. The cellular expression of MMP-9 and their tissue inhibitors TIMP-1, TIMP-2, and TIMP-3 differs in the dilatative pathology of abdominal and thoracic aortas, so the hypothetical model of morphogenesis of AAA cannot completely explain the formation of dilatative pathology of the ascending thoracic aorta.

  9. Novel inhibitors of urokinase-type plasminogen activator and matrix metalloproteinase expression in metastatic cancer cell lines.

    PubMed

    Cakarovski, Kristina; Leung, Jenny Y; Restall, Christina; Carin-Carlson, Anna; Yang, Eunice; Perlmutter, Patrick; Anderson, Robin; Medcalf, Robert; Dear, Anthony E

    2004-07-01

    The plasminogen-activating (PA) and matrix metalloproteinase (MMP) enzyme systems are implicated in proteolytic turnover of the extracellular matrix (ECM) associated with biologic processes including wound healing, inflammation and angiogenesis. Aberrant expression of components of the PA and MMP enzyme systems occurs in the pathogenesis of metastatic cancer. Oxamflatin (Ox), a novel hydroxamic acid derivative, inhibits u-PA mRNA expression and proteolytic activity while simultaneously upregulating the expression of the natural inhibitor of u-PA, plasminogen activator inhibitor type 2 (PAI-2) in metastatic cancer cells. We have characterized the effects of Ox and a novel derivative, Metacept-1 (MCT-1), on PA and MMP-mediated proteolysis and invasion in several metastatic tumor lines. Both compounds are able to inhibit u-PA-, MMP-2- and MMP-9-mediated gene expression at low micromolar concentrations as well as u-PA- and MMP-mediated proteolysis as assessed by zymography, with MCT-1 being the more effective of the 2 agents in some assays. Cellular invasion assays correlate with gene expression and zymography experiments identifying both Ox and MCT-1 as able to inhibit invasion of metastatic cancer cell lines through matrigel at nanomolar concentrations, with MCT-1 more effective than Ox in 2 of the 3 cancer cell lines assessed.

  10. Cell-free expression of human glucosamine 6-phosphate N-acetyltransferase (HsGNA1) for inhibitor screening.

    PubMed

    Ma, Yi; Ghoshdastider, Umesh; Wang, Jufang; Ye, Wei; Dötsch, Volker; Filipek, Slawomir; Bernhard, Frank; Wang, Xiaoning

    2012-12-01

    Glucosamine 6-phosphate N-acetyltransferase (GNA1; EC 2.3.1.4) is required for the de novo synthesis of N-acetyl-d-glucosamine-6-phosphate (GlcNAc-6P), which is an essential precursor in Uridine diphosphate N-acetylglucosamine (UDP-GlcNAc) biosynthesis pathway. Therefore, GNA1 is indispensable for the viability of organisms. Here, a novel cell-free expression strategy was developed to efficiently produce large amounts of human GNA1(HsGNA1) and HsGNA1-sGFP for throughput inhibitor screening. The binding site of inhibitor glucose-6-phosphate (G6P) to hGNA was identified by simulated annealing. Subtle differences to the binding site of Aspergillius GNA1(AfGNA1) can be harnessed for inhibitor design. HsGNA1 may be also useful as an antimicrobial and chemotherapeutic target against cancer. Additionally HsGNA1 inhibitors/modulators can possibly be administered with other drugs in the next generation of personalized medicine.

  11. Resistance through inhibition: ectopic expression of serine protease inhibitor offers stress tolerance via delayed senescence in yeast cell.

    PubMed

    Joshi, Rakesh S; Tanpure, Rahul S; Singh, Rajan Kumar; Gupta, Vidya S; Giri, Ashok P

    2014-09-26

    Protease inhibitors have been known to confer multiple stress tolerance in transgenic plants. We have assessed growth of yeast (Pichia pastoris GS115) strains expressing inhibitory repeat domains (PpIRD(+)) of previously characterized Capsicum annuum protease inhibitors under high salt, heavy metal and oxidative stress. PpIRD(+) strains exhibited multiple stress tolerance and showed differential molecular responses at transcriptional and translational level on exposure to stress inducing agents like heavy metal, high salt and H2O2. PpIRD(+) strains display significant reduction in metacaspase (Yca1) activity, the key enzyme in apoptosis, indicates the possibility of cross reactivity of IRDs (serine protease inhibitor) with cysteine proteases. PpIRD(+) and Saccharomyces cerevisiae knockout with Yca1 (ΔYca1) strain showed similar growth characteristics under stress, which indicated the delayed senescence due to cellular metacaspase inhibition. Molecular docking study showed a close proximity of IRDs reactive site and the active site of metacaspase in the complex that signified their strong interactions. Maintenance of GAPDH activity, primary target of metacaspase, in PpIRD(+) strain evidenced the inhibition of metacaspase activity and survival of these cells under stress. This report demonstrates a potential molecular mechanism of protease inhibitor-based multiple stress tolerance in yeast strains.

  12. Histone deacetylase inhibitors up-regulate LL-37 expression independent of toll-like receptor mediated signalling in airway epithelial cells.

    PubMed

    Liu, Quan; Liu, Juan; Roschmann, Kristina Irene Lisolette; van Egmond, Danielle; Golebski, Korneliusz; Fokkens, Wytske Johanna; Wang, Dehui; van Drunen, Cornelis Maria

    2013-04-11

    HDAC inhibitors have been proposed as anticancer agents. However, their roles in innate genes expression remain not well known. Cathelicidin LL-37 is one of the few human bactericidal peptides, but the regulation of histone acetylation on LL-37 expression in airway epithelium remains largely unknown. Therefore, we investigated the effects of two non-selective HDACi, trichostatin A (TSA) and sodium butyrate (SB), on the expression of the cathelicidin LL-37 in human airway epithelial cells. LL37 in human NCI-H292 airway epithelial cells and the primary cultures of normal nasal epithelial cells(PNEC) in response to HDAC inhibitors with or without poly (I:C) stimulation was assessed using real-time PCR and western blot. In parallel, IL-6 expression was evaluated by ELISA. Our results showed that HDAC inhibitors up-regulated LL-37 gene expression independent of poly (I:C) stimulation in PNEC as well as in NCI-H292 cells. HDAC inhibitors increased LL37 protein expression in NCI-H292 cells but not in PNEC. In addition, HDAC inhibitors significantly inhibited poly (I:C)-induced IL-6 production in both of the epithelial cells. In conclusion, HDAC inhibitors directly up-regulated LL-37 gene expression in human airway epithelial cells.

  13. Histone deacetylase inhibitors up-regulate LL-37 expression independent of toll-like receptor mediated signalling in airway epithelial cells

    PubMed Central

    2013-01-01

    HDAC inhibitors have been proposed as anticancer agents. However, their roles in innate genes expression remain not well known. Cathelicidin LL-37 is one of the few human bactericidal peptides, but the regulation of histone acetylation on LL-37 expression in airway epithelium remains largely unknown. Therefore, we investigated the effects of two non-selective HDACi, trichostatin A (TSA) and sodium butyrate (SB), on the expression of the cathelicidin LL-37 in human airway epithelial cells. LL37 in human NCI-H292 airway epithelial cells and the primary cultures of normal nasal epithelial cells(PNEC) in response to HDAC inhibitors with or without poly (I:C) stimulation was assessed using real-time PCR and western blot. In parallel, IL-6 expression was evaluated by ELISA. Our results showed that HDAC inhibitors up-regulated LL-37 gene expression independent of poly (I:C) stimulation in PNEC as well as in NCI-H292 cells. HDAC inhibitors increased LL37 protein expression in NCI-H292 cells but not in PNEC. In addition, HDAC inhibitors significantly inhibited poly (I:C)-induced IL-6 production in both of the epithelial cells. In conclusion, HDAC inhibitors directly up-regulated LL-37 gene expression in human airway epithelial cells. PMID:23577829

  14. Loss of expression of LyGDI (ARHGDIB), a rho GDP-dissociation inhibitor, in Hodgkin lymphoma.

    PubMed

    Ma, Liya; Xu, Gaixiang; Sotnikova, Anna; Szczepanowski, Monika; Giefing, Maciej; Krause, Kristina; Krams, Matthias; Siebert, Reiner; Jin, Jie; Klapper, Wolfram

    2007-10-01

    The guanosine triphosphatase (GTPase) inhibitor LyGDI (ARHGDIB, Ly/D4-GDI, RhoGDIb or RhoGDI 2) is abundantly expressed in haematopoetic cells and possibly plays a role in the onset of apoptosis. Gene expression profiling of Hodgkin cell lines revealed that LyGDI expression was downregulated in these cell lines. The present study evaluated the expression of LyGDI in Hodgkin cells in vivo and studied the function of LyGDI in Hodgkin cell lines in vitro. Our results showed that virtually all Hodgkin and Reed-Sternberg cells in classical Hodgkin lymphoma lacked LyGDI protein expression. On the other hand, almost all non-Hodgkin lymphomas, except for anaplastic large cell lymphomas, expressed LyGDI protein. Transfection of the classical Hodgkin cell line L428 with a vector containing full-length LyGDI-induced apoptosis in a subset of cells. However, the majority of Hodgkin cells with transgenic expression of LyGDI escaped apoptosis. Our data show that lack of LyGDI expression is a frequent feature of cHL but that it is not of vital importance for the growth and survival of these cells.

  15. Histone deacetylase inhibitors decrease Toll-like receptor-mediated activation of proinflammatory gene expression by impairing transcription factor recruitment

    PubMed Central

    Bode, Konrad A; Schroder, Kate; Hume, David A; Ravasi, Timothy; Heeg, Klaus; Sweet, Matthew J; Dalpke, Alexander H

    2007-01-01

    Post-translational modifications of histone proteins are major mechanisms that modify chromatin structure and regulate gene expression in eukaryotes. Activation of histone acetyltransferases or inhibition of histone deacetylases (HDACs) is generally believed to allow chromatin to assume a more open state, permitting transcriptional activity. We report here the surprising observation that treatment of murine dendritic cells with the HDAC inhibitors trichostatin A (TSA) or suberoylanilide hydroxamic acid (SAHA) in non-apoptotic concentrations strongly inhibited induction of both interleukin-12 protein p40 (IL-12p40) mRNA and protein upon stimulation of Toll-like receptors (TLRs). Moreover, TLR-mediated up-regulation of costimulatory molecules was also inhibited. Up-regulation of tumour necrosis factor-α mRNA and protein in response to TLR agonists was only affected upon prolonged exposure to HDAC inhibitors and regulation of IL-1β was not affected. Similar effects were apparent in murine and human macrophages. Regarding the mode of action, HDAC inhibition increased the acetylation status at the IL-12p40 locus. Nevertheless, IL-12p40 chromatin remodelling, binding of Rel-A and IRF1 to the IL-12p40 promoter and transcriptional activation were abrogated. In contrast, HDAC inhibitors had no effects on upstream nuclear factor-κB and mitogen-activated protein kinase activation. Thus HDACs positively regulate the expression of a subset of cytokine genes by enabling transcription factor recruitment. PMID:17635610

  16. Effect of phosphodiesterase 4 inhibitors on NFAT-dependent cyclooxygenase-2 expression in human T lymphocytes.

    PubMed

    Jimenez, José L; Iñiguez, Miguel A; Muñoz-Fernández, M Angeles; Fresno, Manuel

    2004-12-01

    Transcriptional induction of cyclooxygenase-2 (COX-2) occurs early after T cell receptor triggering and has functional implications in inflammation. Here, we show that phosphodiesterase (PDE)-4 inhibitors block COX-2 induction and prostaglandin synthesis in activated T cells. COX-2 inhibition by PDE4 inhibitors occurs mainly at the transcriptional level. Two response elements for the nuclear factor of activated T cells (NFAT) in the COX-2 promoter were required for inhibition by these drugs. PDE4 inhibitors did not affect NFAT nuclear translocation upon T cell activation; rather they prevented NFAT binding to DNA and induction of the transactivation function of GAL4-NFAT. These effects seem to be cAMP/PKA independent as they were not mimicked by the permeable analog dBcAMP or by forskolin, neither can be reverted by the PKA inhibitors H89 or KT-5720. These results may explain some of the anti-inflammatory properties of PDE4 inhibitors through the blockade of NFAT-mediated transactivation of pro-inflammatory genes such as COX-2.

  17. Antizyme inhibitor 2 hypomorphic mice. New patterns of expression in pancreas and adrenal glands suggest a role in secretory processes.

    PubMed

    López-Garcia, Carlos; Ramos-Molina, Bruno; Lambertos, Ana; López-Contreras, Andrés J; Cremades, Asunción; Peñafiel, Rafael

    2013-01-01

    The intracellular levels of polyamines, polycations implicated in proliferation, differentiation and cell survival, are regulated by controlling their biosynthesis, catabolism and transport. Antizymes and antizyme inhibitors are key regulatory proteins of polyamine levels by affecting ornithine decarboxylase, the rate-limiting biosynthetic enzyme, and polyamine uptake. We recently described the molecular function of a novel antizyme inhibitor (AZIN2). However, the physiological function of AZIN2 in mammals is mostly unknown. To gain insight on the tissue expression profile of AZIN2 and to find its possible physiological role, we have generated, transgenic mice with severe Azin2 hypomorphism. This mouse model expresses transgenic bacterial β-D-galactosidase as a reporter gene, under the control of the Azin2 endogenous promoter, what allows a very sensitive and specific detection of the expression of the gene in the different tissues of transgenic mice. The biochemical and histochemical analyses of β-D-galactosidase together with the quantification of Azin2 mRNA levels, corroborated that AZIN2 is mainly expressed in testis and brain, and showed for the first time that AZIN2 is also expressed in the adrenal glands and pancreas. In these tissues, AZIN2 was not expressed in all type of cells, but rather in specific type of cells. Thus, AZIN2 was mainly found in the haploid germinal cells of the testis and in different brain regions such as hippocampus and cerebellum, particularly in specific type of neurons. In the adrenal glands and pancreas, the expression was restricted to the adrenal medulla and to the Langerhans islets, respectively. Interestingly, plasma insulin levels were significantly reduced in the transgenic mice. These results support the idea that AZIN2 may have a role in the modulation of reproductory and secretory functions and that this mouse model might be an interesting tool for the progress of our understanding on the role of AZIN2 and polyamines in

  18. Histone Deacetylase Inhibitors Reduce the Number of Herpes Simplex Virus-1 Genomes Initiating Expression in Individual Cells

    PubMed Central

    Shapira, Lev; Ralph, Maya; Tomer, Enosh; Cohen, Shai; Kobiler, Oren

    2016-01-01

    Although many viral particles can enter a single cell, the number of viral genomes per cell that establish infection is limited. However, mechanisms underlying this restriction were not explored in depth. For herpesviruses, one of the possible mechanisms suggested is chromatinization and silencing of the incoming genomes. To test this hypothesis, we followed infection with three herpes simplex virus 1 (HSV-1) fluorescence expressing recombinants in the presence or absence of histone deacetylases inhibitors (HDACi’s). Unexpectedly, a lower number of viral genomes initiated expression in the presence of these inhibitors. This phenomenon was observed using several HDACi: Trichostatin A (TSA), Suberohydroxamic Acid, Valporic Acid, and Suberoylanilide Hydroxamic Acid. We found that HDACi presence did not change the progeny outcome from the infected cells but did alter the kinetic of the gene expression from the viral genomes. Different cell types (HFF, Vero, and U2OS), which vary in their capability to activate intrinsic and innate immunity, show a cell specific basal average number of viral genomes establishing infection. Importantly, in all cell types, treatment with TSA reduced the number of viral genomes. ND10 nuclear bodies are known to interact with the incoming herpes genomes and repress viral replication. The viral immediate early protein, ICP0, is known to disassemble the ND10 bodies and to induce degradation of some of the host proteins in these domains. HDACi treated cells expressed higher levels of some of the host ND10 proteins (promyelocytic leukemia and ATRX), which may explain the lower number of viral genomes initiating expression per cell. Corroborating this hypothesis, infection with three HSV-1 recombinants carrying a deletion in the gene coding for ICP0, show a reduction in the number of genomes being expressed in U2OS cells. We suggest that alterations in the levels of host proteins involved in intrinsic antiviral defense may result in

  19. Frequent expression loss of Inter-alpha-trypsin inhibitor heavy chain (ITIH) genes in multiple human solid tumors: A systematic expression analysis

    PubMed Central

    Hamm, Alexander; Veeck, Juergen; Bektas, Nuran; Wild, Peter J; Hartmann, Arndt; Heindrichs, Uwe; Kristiansen, Glen; Werbowetski-Ogilvie, Tamra; Del Maestro, Rolando; Knuechel, Ruth; Dahl, Edgar

    2008-01-01

    Background The inter-alpha-trypsin inhibitors (ITI) are a family of plasma protease inhibitors, assembled from a light chain – bikunin, encoded by AMBP – and five homologous heavy chains (encoded by ITIH1, ITIH2, ITIH3, ITIH4, and ITIH5), contributing to extracellular matrix stability by covalent linkage to hyaluronan. So far, ITIH molecules have been shown to play a particularly important role in inflammation and carcinogenesis. Methods We systematically investigated differential gene expression of the ITIH gene family, as well as AMBP and the interacting partner TNFAIP6 in 13 different human tumor entities (of breast, endometrium, ovary, cervix, stomach, small intestine, colon, rectum, lung, thyroid, prostate, kidney, and pancreas) using cDNA dot blot analysis (Cancer Profiling Array, CPA), semiquantitative RT-PCR and immunohistochemistry. Results We found that ITIH genes are clearly downregulated in multiple human solid tumors, including breast, colon and lung cancer. Thus, ITIH genes may represent a family of putative tumor suppressor genes that should be analyzed in greater detail in the future. For an initial detailed analysis we chose ITIH2 expression in human breast cancer. Loss of ITIH2 expression in 70% of cases (n = 50, CPA) could be confirmed by real-time PCR in an additional set of breast cancers (n = 36). Next we studied ITIH2 expression on the protein level by analyzing a comprehensive tissue micro array including 185 invasive breast cancer specimens. We found a strong correlation (p < 0.001) between ITIH2 expression and estrogen receptor (ER) expression indicating that ER may be involved in the regulation of this ECM molecule. Conclusion Altogether, this is the first systematic analysis on the differential expression of ITIH genes in human cancer, showing frequent downregulation that may be associated with initiation and/or progression of these malignancies. PMID:18226209

  20. Expression of phosphorylated raf kinase inhibitor protein (pRKIP) is a predictor of lung cancer survival

    PubMed Central

    2011-01-01

    Background Raf-1 kinase inhibitor protein (RKIP) has been reported to negatively regulate signal kinases of major survival pathways. RKIP activity is modulated in part by phosphorylation on Serine 153 by protein kinase C, which leads to dissociation of RKIP from Raf-1. RKIP expression is low in many human cancers and represents an indicator of poor prognosis and/or induction of metastasis. The prognostic power has typically been based on total RKIP expression and has not considered the significance of phospho-RKIP. Methods The present study examined the expression levels of both RKIP and phospho-RKIP in human lung cancer tissue microarray proteomics technology. Results Total RKIP and phospho-RKIP expression levels were similar in normal and cancerous tissues. phospho-RKIP levels slightly decreased in metastatic lesions. However, the expression levels of phospho-RKIP, in contrast to total RKIP, displayed significant predictive power for outcome with normal expression of phospho-RKIP predicting a more favorable survival compared to lower levels (P = 0.0118); this was even more pronounced in more senior individuals and in those with early stage lung cancer. Conclusions This study examines for the first time, the expression profile of RKIP and phospho-RKIP in lung cancer. Significantly, we found that phospho-RKIP was a predictive indicator of survival. PMID:21689459

  1. Peroxisome proliferator-activated receptorα agonists differentially regulate inhibitor of DNA binding expression in rodents and human cells.

    PubMed

    González, María Del Carmen; Corton, J Christopher; Acero, Nuria; Muñoz-Mingarro, Dolores; Quirós, Yolanda; Alvarez-Millán, Juan José; Herrera, Emilio; Bocos, Carlos

    2012-01-01

    Inhibitor of DNA binding (Id2) is a helix-loop-helix (HLH) transcription factor that participates in cell differentiation and proliferation. Id2 has been linked to the development of cardiovascular diseases since thiazolidinediones, antidiabetic agents and peroxisome proliferator-activated receptor (PPAR) gamma agonists, have been reported to diminish Id2 expression in human cells. We hypothesized that PPARα activators may also alter Id2 expression. Fenofibrate diminished hepatic Id2 expression in both late pregnant and unmated rats. In 24 hour fasted rats, Id2 expression was decreased under conditions known to activate PPARα. In order to determine whether the fibrate effects were mediated by PPARα, wild-type mice and PPARα-null mice were treated with Wy-14,643 (WY). WY reduced Id2 expression in wild-type mice without an effect in PPARα-null mice. In contrast, fenofibrate induced Id2 expression after 24 hours of treatment in human hepatocarcinoma cells (HepG2). MK-886, a PPARα antagonist, did not block fenofibrate-induced activation of Id2 expression, suggesting a PPARα-independent effect was involved. These findings confirm that Id2 is a gene responsive to PPARα agonists. Like other genes (apolipoprotein A-I, apolipoprotein A-V), the opposite directional transcriptional effect in rodents and a human cell line further emphasizes that PPARα agonists have different effects in rodents and humans.

  2. Peroxisome Proliferator-Activated Receptorα Agonists Differentially Regulate Inhibitor of DNA Binding Expression in Rodents and Human Cells

    PubMed Central

    González, María del Carmen; Corton, J. Christopher; Acero, Nuria; Muñoz-Mingarro, Dolores; Quirós, Yolanda; Álvarez-Millán, Juan José; Herrera, Emilio; Bocos, Carlos

    2012-01-01

    Inhibitor of DNA binding (Id2) is a helix-loop-helix (HLH) transcription factor that participates in cell differentiation and proliferation. Id2 has been linked to the development of cardiovascular diseases since thiazolidinediones, antidiabetic agents and peroxisome proliferator-activated receptor (PPAR) gamma agonists, have been reported to diminish Id2 expression in human cells. We hypothesized that PPARα activators may also alter Id2 expression. Fenofibrate diminished hepatic Id2 expression in both late pregnant and unmated rats. In 24 hour fasted rats, Id2 expression was decreased under conditions known to activate PPARα. In order to determine whether the fibrate effects were mediated by PPARα, wild-type mice and PPARα-null mice were treated with Wy-14,643 (WY). WY reduced Id2 expression in wild-type mice without an effect in PPARα-null mice. In contrast, fenofibrate induced Id2 expression after 24 hours of treatment in human hepatocarcinoma cells (HepG2). MK-886, a PPARα antagonist, did not block fenofibrate-induced activation of Id2 expression, suggesting a PPARα-independent effect was involved. These findings confirm that Id2 is a gene responsive to PPARα agonists. Like other genes (apolipoprotein A-I, apolipoprotein A-V), the opposite directional transcriptional effect in rodents and a human cell line further emphasizes that PPARα agonists have different effects in rodents and humans. PMID:22701468

  3. Gene expression patterns of invertase gene families and modulation of the inhibitor gene in tomato sucrose metabolism.

    PubMed

    Zhang, Y L; Zhang, A H; Jiang, J

    2013-01-24

    Patterns of gene expression in the different types of sucrose metabolism in the tomato are highly variable and heritable. This genetic variation causes considerable functional differences. We examined the patterns of expression of invertase (Inv) gene families and an invertase inhibitor (INH) gene involved in elongating roots, hypocotyls, and fruit of the tomato (Lycopersicon esculentum cv. Micro-Tom and L. chmielewskii) through a real-time quantitative PCR analysis. We found that the Lin6 gene plays an important role in the vegetative growth stage. Lin5 and Lin7 did not express in Micro-Tom, but did express in L. chmielewskii. Overall relative expression levels of sucrose Inv gene families were significantly lower in L. chmielewskii during the reproductive growth stage than in Micro-Tom, being up to hundreds of times lower. It was not expressed in the dissepiment in L. chmielewskii. We suggest that differences in sucrose accumulation in tomato fruit is mainly due to differentially expressed invertase gene families at the later fruit growth stages.

  4. Alpha-1 proteinase inhibitor M358R reduces thrombin generation when displayed on the surface of cells expressing tissue factor.

    PubMed

    Gierczak, Richard F; Pepler, Laura; Bhagirath, Vinai; Liaw, Patricia C; Sheffield, William P

    2014-11-01

    The M358R variant of alpha-1-proteinase inhibitor (API) is a potent soluble inhibitor of thrombin. Previously we engineered AR-API M358R, a membrane-bound form of this protein and showed that it inhibited exogenous thrombin when expressed on transfected cells lacking tissue factor (TF). To determine the suitability of AR-API M358R for gene transfer to vascular cells to limit thrombogenicity, we tested the ability of AR-API M358R to inhibit endogenous thrombin generated in plasma via co-expression co-expressing it on the surface of cells expressing TF. Transfected AR-API M358R formed inhibitory complexes with thrombin following exposure of recalcified, defibrinated plasma to TF on T24/83 cells, but discontinuously monitored thrombin generation was unaffected. Similarly, AR-API M358R expression did not reduce continuously monitored thrombin generation by T24/83 cell suspensions exposed to recalcified normal plasma in a Thrombogram-Thrombinoscope-type thrombin generation assay (TGA); in contrast, 1 μM hirudin variant 3 or soluble API M358R abolished thrombin generation. Gene transfer of TF to HEK 293 conferred the ability to support TF-dependent thrombin generation on HEK 293 cells. Co-transfection of HEK 293 cells with a 9:1 excess of DNA encoding AR-API M358R to that encoding TF reduced peak thrombin generation approximately 3-fold compared to controls. These in vitro results suggest that surface display of API M358R inhibits thrombin generation when the tethered serpin is expressed in excess of TF, and suggest its potential to limit thrombosis in appropriate vascular beds in animal models.

  5. Physalis angulata induced G2/M phase arrest in human breast cancer cells.

    PubMed

    Hsieh, Wen-Tsong; Huang, Kuan-Yuh; Lin, Hui-Yi; Chung, Jing-Gung

    2006-07-01

    Physalis angulata (PA) is employed in herbal medicine around the world. It is used to treat diabetes, hepatitis, asthma and malaria in Taiwan. We have evaluated PA as a cancer chemopreventive agent in vitro by studying the role of PA in regulation of proliferation, cell cycle and apoptosis in human breast cancer cell lines. PA inhibited cell proliferation and induced G2/M arrest and apoptosis in human breast cancer MAD-MB 231 and MCF-7 cell lines. In this study, under treatment with various concentrations of PA in MDA-MB 231 cell line, we checked mRNA levels for cyclin A and cyclin B1 and the protein levels of cyclin A and cyclin B1, Cdc2 (cyclin-dependent kinases), p21(waf1/cip1) and P27(Kip1) (cyclin-dependent kinase inhibitors), Cdc25C, Chk2 and Wee1 kinase (cyclin-dependent kinase relative factors) in cell cycle G2/M phase. From those results, we determined that PA arrests MDA-MB 231 cells at the G2/M phase by (i) inhibiting synthesis or stability of mRNA and their downstream protein levels of cyclin A and cyclin B1, (ii) increasing p21(waf1/cip1) and P27(kip1) levels, (iii) increasing Chk2, thus causing an increase in Cdc25C phosphorylation/inactivation and inducing a decrease in Cdc2 levels and an increase in Wee1 level. According to the results obtained, PA appears to possess anticarcinogenic properties; these results suggest that the effect of PA on the levels of phosphorylated/inactivated Cdc25C are mediated by Chk2 activation, at least in part, via p21(waf1/cip1) and P27(kip1) cyclin-dependent kinase inhibitors pathway to arrest cells at G2/M phase in breast cancer carcinoma cells.

  6. The ectopic expression of a pectin methyl esterase inhibitor increases pectin methyl esterification and limits fungal diseases in wheat.

    PubMed

    Volpi, Chiara; Janni, Michela; Lionetti, Vincenzo; Bellincampi, Daniela; Favaron, Francesco; D'Ovidio, Renato

    2011-09-01

    Cell wall pectin methyl esterification can influence plant resistance because highly methyl-esterified pectin can be less susceptible to the hydrolysis by pectic enzymes such as fungal endopolygalacturonases (PG). Pectin is secreted into the cell wall in a highly methyl-esterified form and, here, is de-methyl esterified by pectin methyl esterase (PME). The activity of PME is controlled by specific protein inhibitors called PMEI; consequently, an increased inhibition of PME by PMEI might modify the pectin methyl esterification. In order to test the possibility of improving wheat resistance by modifying the methyl esterification of pectin cell wall, we have produced durum wheat transgenic lines expressing the PMEI from Actinidia chinensis (AcPMEI). The expression of AcPMEI endows wheat with a reduced endogenous PME activity, and transgenic lines expressing a high level of the inhibitor showed a significant increase in the degree of methyl esterification. These lines showed a significant reduction of disease symptoms caused by the fungal pathogens Bipolaris sorokiniana or Fusarium graminearum. This increased resistance was related to the impaired ability of these fungal pathogens to grow on methyl-esterified pectin and to a reduced activity of the fungal PG to hydrolyze methyl-esterified pectin. In addition to their importance for wheat improvement, these results highlight the primary role of pectin despite its low content in the wheat cell wall.

  7. PDE5 expression in human thyroid tumors and effects of PDE5 inhibitors on growth and migration of cancer cells.

    PubMed

    Sponziello, Marialuisa; Verrienti, Antonella; Rosignolo, Francesca; De Rose, Roberta Francesca; Pecce, Valeria; Maggisano, Valentina; Durante, Cosimo; Bulotta, Stefania; Damante, Giuseppe; Giacomelli, Laura; Di Gioia, Cira Rosaria Tiziana; Filetti, Sebastiano; Russo, Diego; Celano, Marilena

    2015-11-01

    Recent studies have revealed in normal thyroid tissue the presence of the transcript of several phosphodiesterases (PDEs), enzymes responsible for the hydrolysis of cyclic nucleotides. In this work, we analyzed the expression of PDE5 in a series of human papillary thyroid carcinomas (PTCs) presenting or not BRAF V600E mutation and classified according to ATA risk criteria. Furthermore, we tested the effects of two PDE5 inhibitors (sildenafil, tadalafil) against human thyroid cancer cells. PDE5 gene and protein expression were analyzed in two different cohorts of PTCs by real-time PCR using a TaqMan micro-fluid card system and Western blot. MTT and migration assay were used to evaluate the effects of PDE5 inhibitors on proliferation and migration of TPC-1, BCPAP, and 8505C cells. In a first series of 36 PTCs, we found higher expression levels of PDE5A in tumors versus non-tumor (normal) tissues. PTCs with BRAF mutation showed higher levels of mRNA compared with those without mutation. No significant differences were detected between subgroups with low and intermediate ATA risk. Upregulation of PDE5 was also detected in tumor tissue proteins. Similar results were obtained analyzing the second cohort of 50 PTCs. Moreover, all tumor tissues with high PDE5 levels showed reduction of Thyroglobulin, TSH receptor, Thyroperoxidase, and NIS transcripts. In thyroid cancer cells in vitro, sildenafil and tadalafil determined a reduction of proliferation and cellular migration. Our findings demonstrate for the first time an overexpression of PDE5 in PTCs, and the ability of PDE5 inhibitors to block the proliferation of thyroid cancer cells in culture, therefore, suggesting that specific inhibition of PDE5 may be proposed for the treatment of these tumors.

  8. Reduced Expression of the ROCK Inhibitor Rnd3 Is Associated with Increased Invasiveness and Metastatic Potential in Mesenchymal Tumor Cells

    PubMed Central

    Belgiovine, Cristina; Frapolli, Roberta; Bonezzi, Katiuscia; Chiodi, Ilaria; Favero, Francesco; Mello-Grand, Maurizia; Dei Tos, Angelo P.; Giulotto, Elena; Taraboletti, Giulia; D'Incalci, Maurizio; Mondello, Chiara

    2010-01-01

    Background Mesenchymal and amoeboid movements are two important mechanisms adopted by cancer cells to invade the surrounding environment. Mesenchymal movement depends on extracellular matrix protease activity, amoeboid movement on the RhoA-dependent kinase ROCK. Cancer cells can switch from one mechanism to the other in response to different stimuli, limiting the efficacy of antimetastatic therapies. Methodology and Principal Findings We investigated the acquisition and molecular regulation of the invasion capacity of neoplastically transformed human fibroblasts, which were able to induce sarcomas and metastases when injected into immunocompromised mice. We found that neoplastic transformation was associated with a change in cell morphology (from fibroblastic to polygonal), a reorganization of the actin cytoskeleton, a decrease in the expression of several matrix metalloproteases and increases in cell motility and invasiveness. In a three-dimensional environment, sarcomagenic cells showed a spherical morphology with cortical actin rings, suggesting a switch from mesenchymal to amoeboid movement. Accordingly, cell invasion decreased after treatment with the ROCK inhibitor Y27632, but not with the matrix protease inhibitor Ro 28-2653. The increased invasiveness of tumorigenic cells was associated with reduced expression of Rnd3 (also known as RhoE), a cellular inhibitor of ROCK. Indeed, ectopic Rnd3 expression reduced their invasive ability in vitro and their metastatic potential in vivo. Conclusions These results indicate that, during neoplastic transformation, cells of mesenchymal origin can switch from a mesenchymal mode of movement to an amoeboid one. In addition, they point to Rnd3 as a possible regulator of mesenchymal tumor cell invasion and to ROCK as a potential therapeutic target for sarcomas. PMID:21209796

  9. Differential gene expression profiling of mouse skin after sulfur mustard exposure: Extended time response and inhibitor effect

    SciTech Connect

    Gerecke, Donald R. Chen Minjun; Isukapalli, Sastry S.; Gordon, Marion K.; Chang, Y.-C.; Tong Weida; Androulakis, Ioannis P.; Georgopoulos, Panos G.

    2009-01-15

    Sulfur mustard (HD, SM), is a chemical warfare agent that within hours causes extensive blistering at the dermal-epidermal junction of skin. To better understand the progression of SM-induced blistering, gene expression profiling for mouse skin was performed after a single high dose of SM exposure. Punch biopsies of mouse ears were collected at both early and late time periods following SM exposure (previous studies only considered early time periods). The biopsies were examined for pathological disturbances and the samples further assayed for gene expression profiling using the Affymetrix microarray analysis system. Principal component analysis and hierarchical cluster analysis of the differently expressed genes, performed with ArrayTrack showed clear separation of the various groups. Pathway analysis employing the KEGG library and Ingenuity Pathway Analysis (IPA) indicated that cytokine-cytokine receptor interaction, cell adhesion molecules (CAMs), and hematopoietic cell lineage are common pathways affected at different time points. Gene ontology analysis identified the most significantly altered biological processes as the immune response, inflammatory response, and chemotaxis; these findings are consistent with other reported results for shorter time periods. Selected genes were chosen for RT-PCR verification and showed correlations in the general trends for the microarrays. Interleukin 1 beta was checked for biological analysis to confirm the presence of protein correlated to the corresponding microarray data. The impact of a matrix metalloproteinase inhibitor, MMP-2/MMP-9 inhibitor I, against SM exposure was assessed. These results can help in understanding the molecular mechanism of SM-induced blistering, as well as to test the efficacy of different inhibitors.

  10. Pectinesterase Inhibitor from Jelly Fig (Ficus awkeotsang Makino) Achene Inhibits Surface Antigen Expression by Human Hepatitis B Virus.

    PubMed

    Huang, Yu-Chuen; Jiang, Chii-Ming; Chen, Yu-Jen; Chen, Yu-Yawn

    2013-01-01

    Pectinesterase inhibitor (PEI) isolated from jelly fig (Ficus awkeotsang Makino) is an edible component of a popular drink consumed in Asia. Hepatitis B virus (HBV) infection is prevalent in Asia, and current treatments for HBV infection need improvement. This study aimed to evaluate the effect of PEI on the surface antigen expression by HBV (HBsAg). Human hepatoma cell lines Hep3B and Huh7 served as in vitro models for assessing the cytotoxicity and HBsAg expression. A culture of primary hepatocytes cultured from mice served as the normal counterpart. Cell viability was measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) colorimetric assay. HBsAg expression was evaluated by measuring HBsAg secretion into the culture medium using an enzyme-linked immunosorbent assay. The results showed that PEI did not affect the viability of the human hepatoma cell lines or primary mouse hepatocytes. PEI inhibited the expression of HBsAg in hepatoma cell lines harboring endogenous (Hep3B) and integrated (Huh7) HBV genomes in a concentration- and time-dependent manner, thus implicating a universal activity against HBV gene expression. In conclusion, it suggests that PEI from jelly fig inhibits the expression of human HBsAg in host cells without toxic effects on normal primary hepatocytes.

  11. Small-molecule inhibitors suppress the expression of both type III secretion and amylovoran biosynthesis genes in Erwinia amylovora.

    PubMed

    Yang, Fan; Korban, Schuyler S; Pusey, P Lawrence; Elofsson, Michael; Sundin, George W; Zhao, Youfu

    2014-01-01

    The type III secretion system (T3SS) and exopolysaccharide (EPS) amylovoran are two essential pathogenicity factors in Erwinia amylovora, the causal agent of the serious bacterial disease fire blight. In this study, small molecules that inhibit T3SS gene expression in E. amylovora under hrp (hypersensitive response and pathogenicity)-inducing conditions were identified and characterized using green fluorescent protein (GFP) as a reporter. These compounds belong to salicylidene acylhydrazides and also inhibit amylovoran production. Microarray analysis of E. amylovora treated with compounds 3 and 9 identified a total of 588 significantly differentially expressed genes. Among them, 95 and 78 genes were activated and suppressed by both compounds, respectively, when compared with the dimethylsulphoxide (DMSO) control. The expression of the majority of T3SS genes in E. amylovora, including hrpL and the avrRpt2 effector gene, was suppressed by both compounds. Compound 3 also suppressed the expression of amylovoran precursor and biosynthesis genes. However, both compounds induced significantly the expression of glycogen biosynthesis genes and siderophore biosynthesis, regulatory and transport genes. Furthermore, many membrane, lipoprotein and exported protein-encoding genes were also activated by both compounds. Similar expression patterns were observed for compounds 1, 2 and 4. Using crab apple flower as a model, compound 3 was capable of reducing disease development in pistils. These results suggest a common inhibition mechanism shared by salicylidene acylhydrazides and indicate that small-molecule inhibitors that disable T3SS function could be explored to control fire blight disease.

  12. Auranofin Suppresses Plasminogen Activator Inhibitor-2 Expression through Annexin A5 Induction in Human Prostate Cancer Cells

    PubMed Central

    Shin, Dong-Won; Kwon, Yeo-Jung; Ye, Dong-Jin; Baek, Hyoung-Seok; Lee, Joo-Eun; Chun, Young-Jin

    2017-01-01

    Auranofin has been developed as antirheumatic drugs, which is currently under clinical development for the treatment of chronic lymphocytic leukemia. Previous report showed that auranofin induced apoptosis by enhancement of annexin A5 expression in PC-3 cells. To understand the role of annexin A5 in auranofin-mediated apoptosis, we performed microarray data analysis to study annexin A5-controlled gene expression in annexin A5 knockdown PC-3 cells. Of differentially expressed genes, plasminogen activator inhibitor (PAI)-2 was increased by annexin A5 siRNA confirmed by qRT-PCR and western blot. Treatment with auranofin decreased PAI-2 and increased annexin A5 expression as well as promoting apoptosis. Furthermore, auranofin-induced apoptosis was recovered by annexin A5 siRNA but it was promoted by PAI-2 siRNA. Interestingly, knockdown of annexin A5 rescued PAI-2 expression suppressed by auranofin. Taken together, our study suggests that induction of annexin A5 by auranofin may enhance apoptosis through suppression of PAI-2 expression in PC-3 cells. PMID:27956714

  13. Expression screening of bacterial libraries of recombinant alpha-1 proteinase inhibitor variants for candidates with thrombin inhibitory capacity.

    PubMed

    Bhakta, Varsha; Gierczak, Richard F; Sheffield, William P

    2013-12-01

    Exhaustive mutagenesis studies of the reactive centre loop (RCL), a key structural component of proteins belonging to the serpin superfamily of protease inhibitors, are complicated by the size of the RCL, serpin conformational complexity, and, for most serpins, the lack of a serpin-dependent phenotype of expressing cells. Here, we describe a thrombin capture assay that distinguished thrombin-inhibitory recombinant human alpha-1 proteinase inhibitor (API M358R) from non-inhibitory API variants in Escherichia coli lysates prepared from either single clones or pools. Binding of API proteins in the lysates to thrombin immobilized on microtiter plate wells was quantified via colour generated by a peroxidase-coupled anti-API antibody. Bacterial expression plasmids encoding inhibitory API M358R were mixed 1:99 with plasmids encoding non-inhibitory API T345R/M358R and the resulting library screened in pools of 10. All above-background signals arising from pools or subsequently re-probed single clones were linked to the presence of plasmids encoding API M358R. Screening of a portion of another expression library encoding hypervariable API with all possibilities at codons 352-358 also yielded only novel, thrombin-inhibitory variants. Probing a smaller library expressing all possible codons at Ala347 yielded the wild type, 6 different functional variants, one partially active variant, and two variants with no thrombin-inhibitory activity. API antigen levels varied considerably less among Ala347 variants than activity levels, and comparison of rate constants of inhibition of purified API variants to their corresponding thrombin capture assay lysate values was used to establish the sensitivity and specificity of the assay. The results indicate that the approach is sufficiently robust to correctly identify functional versus non-functional candidates in API expression libraries, and could be of value in systematically probing structure/function relationships not only in the API

  14. Histone deacetylase inhibitors suppress TF-kappaB-dependent agonist-driven tissue factor expression in endothelial cells and monocytes.

    PubMed

    Wang, Jianguo; Mahmud, Shawn A; Bitterman, Peter B; Huo, Yuqing; Slungaard, Arne

    2007-09-28

    Histone deacetylase inhibitors (HDACi), such as trichostatin A (TSA), can regulate gene expression by promoting acetylation of histones and transcription factors. Human tissue factor (TF) expression is partly governed by a unique, NF-kappaB-related "TF-kappaB" promoter binding site. We find that TSA and four other HDACi (apicidin, MS-275, sodium butyrate, and valproic acid) all inhibit by approximately 90% TF activity and protein level induction in human umbilical vein endothelial cells stimulated by the physiologic agonists tumor necrosis factor (TNF)-alpha, interleukin-1beta, lipopolysaccharide, and HOSCN without affecting expression of the NF-kappaB-regulated adhesion molecules ICAM-1 and E-selectin. TSA and butyrate also blunt TF induction approximately 50% in vitro in peripheral blood mononuclear cells and in vivo in thioglycolate-elicited murine peritoneal macrophages. In human umbilical vein endothelial cells, TSA attenuates by approximately 70% TNF-alpha stimulation of TF mRNA transcription without affecting that of ICAM-1. By electrophoretic mobility shift assay analyses, TNF-alpha and lipopolysaccharide induce strong p65/p50 and p65/c-Rel heterodimer binding to both NF-kappaB and TF-kappaB probes. TSA nearly abolishes TF-kappaB binding without affecting NF-kappaB binding. A chromatin immunoprecipitation assay and a promoter-luciferase reporter system confirm that TSA inhibits TF-kappaB but not NF-kappaB activation. Chromatin immunoprecipitation and small interfering RNA inhibitor studies demonstrate that HDAC3 plays a significant role in TNF-alpha-mediated TF induction. Thus, HDACi transcriptionally inhibit agonist-induced TF expression in endothelial cells and monocytes by a TF-kappaB- and HDAC3-dependent mechanism. We conclude that histone deacetylases, particularly HDAC3, play a hitherto unsuspected role in regulating TF expression and raise the possibility that HDACi might be a novel therapy for thrombotic disorders.

  15. Effect of selective serotonin reuptake inhibitors on expression of 5-HT1AR and neurotransmitters in rats with vascular dementia.

    PubMed

    Guo, K; Yin, G; Zi, X H; Zhu, H X; Pan, Q

    2016-12-02

    5-hydroxytryptamine receptor 1A (5-HT1AR) is closely associated with cognitive functions. Selective serotonin reuptake inhibitors (SSRIs) can protect individuals from brain damage following ischemia/hypoxia. To investigate the function of SSRIs in vascular dementia (VD), we established a rat model of VD, and observed the effect of SSRIs on the expression of 5-HT1AR mRNA and neurotransmitters. Male SD rats (6 months) were randomly assigned into sham, model, and SSRI groups (N = 30). VD was achieved by permanent ligation of the bilateral common carotid artery. Escitalopram, a highly selective 5-HT reabsorption inhibitor, was ip injected into the rats for three consecutive weeks. The Morris water-maze was used to test learning and memory. H&E staining for neuronal injury was conducted on cortical and hippocampal tissues. HPLC was used to determine the levels of dopamine (DA), 5-HT, and norepinephrine (NE). RT-PCR was used to determine expression of 5-HT1AR mRNA. As compared to control rats, model animals demonstrated elongated escape latency, lower platform crossing times, and significant injuries to hippocampal CA1 neurons. This was accompanied by reductions in DA, 5-HT, and NE levels in hippocampal tissues, as well as reduced cortical 5-HT and decreased 5-HT1AR mRNA expression (P < 0.05). Escitalopram treatments reduced escape latency, elevated platform crossing times, improved CA1 neuronal damage, increased DA and 5-HT levels in hippocampal and cortical neurons, as well as elevated expression of 5-HT1AR mRNA (P < 0.05). Therefore, SSRIs may improve cognitive dysfunction of VD rats, possibly by stimulating expression of neurotransmitters and protecting neurons.

  16. Spatial and temporal expression patterns of diverse Pin-II proteinase inhibitor genes in Capsicum annuum Linn.

    PubMed

    Tamhane, Vaijayanti A; Giri, Ashok P; Kumar, Pavan; Gupta, Vidya S

    2009-08-01

    Pin-II type proteinase inhibitor (PI) genes were cloned from fruit and stem tissues of Capsicum annuum L. var Phule Jyoti using primers designed from reported CanPI gene sequence (AF039398). In total, 21 novel CanPIs, members of the Pin-II PI family, were identified in the study, with three isoforms of 1-inhibitory repeat domain (IRD), eight isoforms of 2-IRD, three isoforms of 3-IRD, five isoforms of 4-IRD and two partial CanPI sequences. Most of the sequences showed variation (2 to 20%) in the deduced AA sequences which were pronounced close to the reactive site loop. Expression patterns of CanPIs in the fruit and stem tissues of mature C. annuum plants were shown to vary qualitatively and quantitatively using semi-quantitative RT-PCR expression analysis. In the fruit tissue, CanPIs with different IRDs (from 1 to 4) were expressed simultaneously. In stem tissue, 1- and 2-IRD CanPIs were strongly expressed along moderate expression of 3- and 4-IRD genes. Analysis of CanPI protein activity showed a range of active forms across the tissues. CanPI expression was differentially up-regulated upon wounding and insect attack. Although infestation by aphids (Myzus persicae) and lepidopteran pests (Spodoptera litura) specifically induced 4-IRD CanPIs, virus-infected leaves did not affect CanPI expression. Analysis of CanPI protein activity indicated that the up-regulation in CanPI expression was not always correlated with increase in PI activity. Our results demonstrated that CanPI expression is regulated spatially, temporally as well as qualitatively and quantitatively.

  17. The Drosophila melanogaster seminal fluid protein Acp62F is a protease inhibitor that is toxic upon ectopic expression.

    PubMed Central

    Lung, Oliver; Tram, Uyen; Finnerty, Casey M; Eipper-Mains, Marcie A; Kalb, John M; Wolfner, Mariana F

    2002-01-01

    Drosophila melanogaster seminal fluid proteins stimulate sperm storage and egg laying in the mated female but also cause a reduction in her life span. We report here that of eight Drosophila seminal fluid proteins (Acps) and one non-Acp tested, only Acp62F is toxic when ectopically expressed. Toxicity to preadult male or female Drosophila occurs upon one exposure, whereas multiple exposures are needed for toxicity to adult female flies. Of the Acp62F received by females during mating, approximately 10% enters the circulatory system while approximately 90% remains in the reproductive tract. We show that in the reproductive tract, Acp62F localizes to the lumen of the uterus and the female's sperm storage organs. Analysis of Acp62F's sequence, and biochemical assays, reveals that it encodes a trypsin inhibitor with sequence and structural similarities to extracellular serine protease inhibitors from the nematode Ascaris. In light of previous results demonstrating entry of Acp62F into the mated female's hemolymph, we propose that Acp62F is a candidate for a molecule to contribute to the Acp-dependent decrease in female life span. We propose that Acp62F's protease inhibitor activity exerts positive protective functions in the mated female's reproductive tract but that entry of a small amount of this protein into the female's hemolymph could contribute to the cost of mating. PMID:11805057

  18. Two variants of the major serine protease inhibitor from the sea anemone Stichodactyla helianthus, expressed in Pichia pastoris.

    PubMed

    García-Fernández, Rossana; Ziegelmüller, Patrick; González, Lidice; Mansur, Manuel; Machado, Yoan; Redecke, Lars; Hahn, Ulrich; Betzel, Christian; Chávez, María de Los Ángeles

    2016-07-01

    The major protease inhibitor from the sea anemone Stichodactyla helianthus (ShPI-1) is a non-specific inhibitor that binds trypsin and other trypsin-like enzymes, as well as chymotrypsin, and human neutrophil elastase. We performed site-directed mutagenesis of ShPI-1 to produce two variants (rShPI-1/K13L and rShPI/Y15S) that were expressed in Pichia pastoris, purified, and characterized. After a single purification step, 65 mg and 15 mg of protein per liter of culture supernatant were obtained for rShPI-1/K13L and rShPI/Y15S, respectively. Functional studies demonstrated a 100-fold decreased trypsin inhibitory activity as result of the K13L substitution at the reactive (P1) site. This protein variant has a novel tight-binding inhibitor activity of pancreatic elastase and increased activity toward neutrophil elastase in comparison to rShPI-1A. In contrast, the substitution Y15S at P2' site did not affect the Ki value against trypsin, but did reduce activity 10-fold against chymotrypsin and neutrophil elastase. Our results provide two new ShPI-1 variants with modified inhibitory activities, one of them with increased biomedical potential. This study also offers new insight into the functional impact of the P1 and P2' sites on ShPI-1 specificity.

  19. NF-kappaB inhibitor dehydroxymethylepoxyquinomicin suppresses osteoclastogenesis and expression of NFATc1 in mouse arthritis without affecting expression of RANKL, osteoprotegerin or macrophage colony-stimulating factor.

    PubMed

    Kubota, Tetsuo; Hoshino, Machiko; Aoki, Kazuhiro; Ohya, Keiichi; Komano, Yukiko; Nanki, Toshihiro; Miyasaka, Nobuyuki; Umezawa, Kazuo

    2007-01-01

    Inhibition of NF-kappaB is known to be effective in reducing both inflammation and bone destruction in animal models of arthritis. Our previous study demonstrated that a small cell-permeable NF-kappaB inhibitor, dehydroxymethylepoxyquinomicin (DHMEQ), suppresses expression of proinflammatory cytokines and ameliorates mouse arthritis. It remained unclear, however, whether DHMEQ directly affects osteoclast precursor cells to suppress their differentiation to mature osteoclasts in vivo. The effect of DHMEQ on human osteoclastogenesis also remained elusive. In the present study, we therefore examined the effect of DHMEQ on osteoclastogenesis using a mouse collagen-induced arthritis model, and using culture systems of fibroblast-like synovial cells obtained from patients with rheumatoid arthritis, and of osteoclast precursor cells from peripheral blood of healthy volunteers. DHMEQ significantly suppressed formation of osteoclasts in arthritic joints, and also suppressed expression of NFATc1 along the inner surfaces of bone lacunae and the eroded bone surface, while serum levels of soluble receptor activator of NF-kappaB ligand (RANKL), osteoprotegerin and macrophage colony-stimulating factor were not affected by the treatment. DHMEQ also did not suppress spontaneous expression of RANKL nor of macrophage colony-stimulating factor in culture of fibroblast-like synovial cells obtained from patients with rheumatoid arthritis. These results suggest that DHMEQ suppresses osteoclastogenesis in vivo, through downregulation of NFATc1 expression, without significantly affecting expression of upstream molecules of the RANKL/receptor activator of NF-kappaB/osteoprotegerin cascade, at least in our experimental condition. Furthermore, in the presence of RANKL and macrophage colony-stimulating factor, differentiation and activation of human osteoclasts were also suppressed by DHMEQ, suggesting the possibility of future application of NF-kappaB inhibitors to rheumatoid arthritis

  20. Transgenic expression of a myostatin inhibitor derived from follistatin increases skeletal muscle mass and ameliorates dystrophic pathology in mdx mice.

    PubMed

    Nakatani, Masashi; Takehara, Yuka; Sugino, Hiromu; Matsumoto, Mitsuru; Hashimoto, Osamu; Hasegawa, Yoshihisa; Murakami, Tatsuya; Uezumi, Akiyoshi; Takeda, Shin'ichi; Noji, Sumihare; Sunada, Yoshihide; Tsuchida, Kunihiro

    2008-02-01

    Myostatin is a potent negative regulator of skeletal muscle growth. Therefore, myostatin inhibition offers a novel therapeutic strategy for muscular dystrophy by restoring skeletal muscle mass and suppressing the progression of muscle degeneration. The known myostatin inhibitors include myostatin propeptide, follistatin, follistatin-related proteins, and myostatin antibodies. Although follistatin shows potent myostatin-inhibiting activities, it also acts as an efficient inhibitor of activins. Because activins are involved in multiple functions in various organs, their blockade by follistatin would affect multiple tissues other than skeletal muscles. In the present study, we report the characterization of a myostatin inhibitor derived from follistatin, which does not affect activin signaling. The dissociation constants (K(d)) of follistatin to activin and myostatin are 1.72 nM and 12.3 nM, respectively. By contrast, the dissociation constants (K(d)) of a follistatin-derived myostatin inhibitor, designated FS I-I, to activin and myostatin are 64.3 microM and 46.8 nM, respectively. Transgenic mice expressing FS I-I, under the control of a skeletal muscle-specific promoter showed increased skeletal muscle mass and strength. Hyperplasia and hypertrophy were both observed. We crossed FS I-I transgenic mice with mdx mice, a model for Duchenne muscular dystrophy. Notably, the skeletal muscles in the mdx/FS I-I mice showed enlargement and reduced cell infiltration. Muscle strength is also recovered in the mdx/FS I-I mice. These results indicate that myostatin blockade by FS I-I has a therapeutic potential for muscular dystrophy.

  1. Signal transduction of receptor-mediated antiproliferative action of melatonin on human prostate epithelial cells involves dual activation of Gα(s) and Gα(q) proteins.

    PubMed

    Shiu, Stephen Y W; Pang, Bo; Tam, Chun W; Yao, Kwok-Ming

    2010-10-01

    Melatonin has been shown to inhibit the proliferation of malignant and transformed human prostate epithelial cells by transcriptional up-regulation of p27(Kip1) expression via MTNR1A receptor-mediated activation of protein kinase A (PKA) and protein kinase C (PKC) in parallel. Given that melatonin MTNR1A receptor is a G protein-coupled receptor, this study was conducted to identify the specific G proteins that mediate the antiproliferative action of melatonin on human prostate epithelial cells. In 22Rv1 and RWPE-1 cells, knockdown of either Gα(s) or Gα(q) , but not Gα(i2) expression by RNA interference, abrogated the effects of melatonin on p27(Kip1) and cell proliferation. Conversely, cellular overexpression of activated mutants of Gα(s) and Gα(q) in 22Rv1 and RWPE-1 cells mimicked the effects of melatonin on prostate epithelial cell antiproliferation by increasing p27(Kip1) expression through downstream activation of PKA and PKC in parallel. Moreover, melatonin or 2-iodomelatonin induced elevation of adenosine-3',5'-cyclic monophosphate (cAMP) in 22Rv1 and RWPE-1 cells. The effects of 2-iodomelatonin on cAMP were blocked by the nonselective MTNR1A/MTNR1B receptor antagonist luzindole but were not affected by the selective MTNR1B receptor antagonist 4-phenyl-2-propionamidotetraline (4-P-PDOT). Furthermore, knockdown of Gα(s) mitigated the stimulatory effects of 2-iodomelatonin on cAMP. Collectively, the data demonstrated, for the first time, functional coupling of MTNR1A receptor to Gα(s) in cancerous or transformed human cells expressing endogenous melatonin receptors. Our results also showed that dual activation of Gα(s) and Gα(q) proteins is involved in the signal transduction of MTNR1A receptor-mediated antiproliferative action of melatonin on human prostate epithelial cells.

  2. Substrates and inhibitors display different sensitivity to expression level of the dopamine transporter in heterologously expressing cells.

    PubMed

    Chen, Nianhang; Reith, Maarten E A

    2007-04-01

    The use of heterologous expression systems for studying dopamine (DA) transporter (DAT) function has provided important information corroborating and complementing in situ obtained knowledge. Preliminary experiments with human embryonic kidney cells (HEK293) heterologously expressing varying amounts of DAT suggested fluctuations in the potency of cocaine in inhibiting DA uptake and led to the present systematic assessment of the impact of the density of DAT on its function. Transiently expressing intact HEK293 cells, transfected with increasing amounts of DAT cDNA, displayed increasing levels of surface DAT, binding of the cocaine analog [(3)H]2beta-carbomethoxy-3beta-(4-fluorophenyl)tropane ([(3)H]CFT), and uptake of [(3)H]DA, [(3)H]N-methyl-4-phenylpyridinium ([(3)H]MPP(+)), [(3)H]norepinephrine, and [(3)H]serotonin. However, the amount of DAT cDNA and the DAT expression level required to produce 50% of maximal activity was threefold higher for CFT binding than for DA uptake. Increased DAT expression was accompanied by weakened potency in inhibiting [(3)H]DA uptake for cocaine, CFT, benztropine, and its analog JHW025, GBR 12909 and mazindol; their potency in inhibiting [(3)H]CFT binding was unaffected. Inhibition of uptake by the substrates DA, m-tyramine, d-amphetamine, or MPP(+) was also unaffected. Increasing DAT in stably expressing HEK293 cells by stimulation of gene expression with sodium butyrate also decreased the uptake inhibitory potency of a number of the above blockers without affecting the interaction between substrates and DAT. The present results prompt discussion of models explaining how factors regulating DAT expression at the plasma membrane can regulate DAT function and pharmacology.

  3. The FAAH inhibitor URB597 efficiently reduces tyrosine hydroxylase expression through CB1- and FAAH-independent mechanisms

    PubMed Central

    Bosier, Barbara; Muccioli, Giulio G; Lambert, Didier M

    2013-01-01

    Background Anandamide and 2-arachidonoylglycerol are neuromodulatory lipids interacting with cannabinoid receptors, whose availability is regulated by the balance between ‘on demand’ generation and enzymatic degradation [by fatty acid amide hydrolase (FAAH)/monoacylglycerol lipase]. Given the reported effects of anandamide on dopamine transmission, we investigated the influence of endocannabinoids and URB597, a well-known FAAH inhibitor, on the expression of tyrosine hydroxylase (TH), the rate-limiting enzyme in dopamine synthesis. Experimental Approach We investigated TH expression in N1E115 neuroblastoma using a reporter gene assay, as well as mRNA and protein quantifications. FAAH inhibition was confirmed by measuring radiolabelled substrate hydrolysis and endogenous endocannabinoids. Key Results Anandamide decreased TH promoter activity in N1E115 cells through CB1 receptor activation. Unexpectedly, URB597 reduced TH expression (pEC50 = 8.7 ± 0.2) through FAAH-independent mechanisms. Indeed, four structurally unrelated inhibitors of FAAH had no influence on TH expression, although all the inhibitors increased endocannabinoid levels. At variance with the endocannabinoid responses, the use of selective antagonists indicated that the URB597-mediated decrease in TH expression was not directed by the CB1 receptor, but rather by abnormal-cannabidiol-sensitive receptors and PPARs. Further supporting the physiological relevance of these in vitro data, URB597 administration resulted in reduced TH mRNA levels in mice brain. Conclusions While confirming the implication of endocannabinoids on the modulation of TH, we provide strong evidence for additional physiologically relevant off-target effects of URB597. In light of the numerous preclinical studies involving URB597, particularly in anxiety and depression, the existence of non-CB1 and non-FAAH mediated influences of URB597 on key enzymes of the catecholaminergic transmission system should be taken into account when

  4. The metastasis suppressor RARRES3 as an endogenous inhibitor of the immunoproteasome expression in breast cancer cells

    NASA Astrophysics Data System (ADS)

    Anderson, Alison M.; Kalimutho, Murugan; Harten, Sarah; Nanayakkara, Devathri M.; Khanna, Kum Kum; Ragan, Mark A.

    2017-01-01

    In breast cancer metastasis, the dynamic continuum involving pro- and anti-inflammatory regulators can become compromised. Over 600 genes have been implicated in metastasis to bone, lung or brain but how these genes might contribute to perturbation of immune function is poorly understood. To gain insight, we adopted a gene co-expression network approach that draws on the functional parallels between naturally occurring bone marrow-derived mesenchymal stem cells (BM-MSCs) and cancer stem cells (CSCs). Our network analyses indicate a key role for metastasis suppressor RARRES3, including potential to regulate the immunoproteasome (IP), a specialized proteasome induced under inflammatory conditions. Knockdown of RARRES3 in near-normal mammary epithelial and breast cancer cell lines increases overall transcript and protein levels of the IP subunits, but not of their constitutively expressed counterparts. RARRES3 mRNA expression is controlled by interferon regulatory factor IRF1, an inducer of the IP, and is sensitive to depletion of the retinoid-related receptor RORA that regulates various physiological processes including immunity through modulation of gene expression. Collectively, these findings identify a novel regulatory role for RARRES3 as an endogenous inhibitor of IP expression, and contribute to our evolving understanding of potential pathways underlying breast cancer driven immune modulation.

  5. The metastasis suppressor RARRES3 as an endogenous inhibitor of the immunoproteasome expression in breast cancer cells

    PubMed Central

    Anderson, Alison M.; Kalimutho, Murugan; Harten, Sarah; Nanayakkara, Devathri M.; Khanna, Kum Kum; Ragan, Mark A.

    2017-01-01

    In breast cancer metastasis, the dynamic continuum involving pro- and anti-inflammatory regulators can become compromised. Over 600 genes have been implicated in metastasis to bone, lung or brain but how these genes might contribute to perturbation of immune function is poorly understood. To gain insight, we adopted a gene co-expression network approach that draws on the functional parallels between naturally occurring bone marrow-derived mesenchymal stem cells (BM-MSCs) and cancer stem cells (CSCs). Our network analyses indicate a key role for metastasis suppressor RARRES3, including potential to regulate the immunoproteasome (IP), a specialized proteasome induced under inflammatory conditions. Knockdown of RARRES3 in near-normal mammary epithelial and breast cancer cell lines increases overall transcript and protein levels of the IP subunits, but not of their constitutively expressed counterparts. RARRES3 mRNA expression is controlled by interferon regulatory factor IRF1, an inducer of the IP, and is sensitive to depletion of the retinoid-related receptor RORA that regulates various physiological processes including immunity through modulation of gene expression. Collectively, these findings identify a novel regulatory role for RARRES3 as an endogenous inhibitor of IP expression, and contribute to our evolving understanding of potential pathways underlying breast cancer driven immune modulation. PMID:28051153

  6. Tissue inhibitor of matrix metalloproteinase-1 expression in colorectal cancer liver metastases is associated with vascular structures.

    PubMed

    Illemann, Martin; Eefsen, Rikke Helene Løvendahl; Bird, Nigel Charles; Majeed, Ali; Osterlind, Kell; Laerum, Ole Didrik; Alpízar-Alpízar, Warner; Lund, Ida Katrine; Høyer-Hansen, Gunilla

    2016-02-01

    Metastatic growth by colorectal cancer cells in the liver requires the ability of the cancer cells to interact with the new microenvironment. This interaction results in three histological growth patterns of liver metastases: desmoplastic, pushing, and replacement. In primary colorectal cancer several proteases, involved in the degradation of extracellular matrix components, are up-regulated. In liver metastases, their expression is growth pattern dependent. Tissue inhibitor of matrix metalloproteinase-1 (TIMP-1) is a strong prognostic marker in plasma from colorectal cancer patients, with significant higher levels in patients with metastatic disease. We therefore wanted to determine the expression pattern of TIMP-1 in primary colorectal cancers and their matching liver metastases. TIMP-1 mRNA was primarily seen in α-smooth-muscle actin (α-SMA)-positive cells. In all primary tumors and liver metastases with desmoplastic growth pattern, TIMP-1 mRNA was primarily found in α-SMA-positive myofibroblasts located at the invasive front. Some α-SMA-positive cells with TIMP-1 mRNA were located adjacent to CD34-positive endothelial cells, identifying them as pericytes. This indicates that TIMP-1 in primary tumors and liver metastases with desmoplastic growth pattern has dual functions; being an MMP-inhibitor at the cancer periphery and involved in tumor-induced angiogenesis in the pericytes. In the liver metastases with pushing or replacement growth patterns, TIMP-1 was primarily expressed by activated hepatic stellate cells at the metastasis/liver parenchyma interface. These cells were located adjacent to CD34-positive endothelial cells, suggesting a function in tumor-induced angiogenesis. We therefore conclude that TIMP-1 expression is growth pattern dependent in colorectal cancer liver metastases.

  7. Selective Inducible Nitric Oxide Synthase Inhibitor Reversed Zinc Chloride-Induced Spatial Memory Impairment via Increasing Cholinergic Marker Expression.

    PubMed

    Tabrizian, Kaveh; Azami, Kian; Belaran, Maryam; Soodi, Maliheh; Abdi, Khosrou; Fanoudi, Sahar; Sanati, Mehdi; Mottaghi Dastjerdi, Negar; Soltany Rezaee-Rad, Mohammad; Sharifzadeh, Mohammad

    2016-10-01

    Zinc, an essential micronutrient and biochemical element of the human body, plays structural, catalytic, and regulatory roles in numerous physiological functions. In the current study, the effects of a pretraining oral administration of zinc chloride (10, 25, and 50 mg/kg) for 14 consecutive days and post-training bilateral intra-hippocampal infusion of 1400W as a selective inducible nitric oxide synthase (iNOS) inhibitor (10, 50, and 100 μM/side), alone and in combination, on the spatial memory retention in Morris water maze (MWM) were investigated. Animals were trained for 4 days and tested 48 h after completion of training. Also, the molecular effects of these compounds on the expression of choline acetyltransferase (ChAT), as a cholinergic marker in the CA1 region of the hippocampus and medial septal area (MSA), were evaluated. Behavioral and molecular findings of this study showed that a 2-week oral administration of zinc chloride (50 mg/kg) impaired spatial memory retention in MWM and decreased ChAT expression. Immunohistochemical analysis of post-training bilateral intra-hippocampal infusion of 1400W revealed a significant increase in ChAT immunoreactivity. Furthermore, post-training bilateral intra-hippocampal infusion of 1400W into the CA1 region of the hippocampus reversed zinc chloride-induced spatial memory impairment in MWM and significantly increased ChAT expression in comparison with zinc chloride-treated animals. Taken together, these results emphasize the role of selective iNOS inhibitors in reversing zinc chloride-induced spatial memory deficits via modulation of cholinergic marker expression.

  8. Human and rodent carboxylesterases: immunorelatedness, overlapping substrate specificity, differential sensitivity to serine enzyme inhibitors, and tumor-related expression.

    PubMed

    Xie, Mingxing; Yang, Dongfang; Liu, Lan; Xue, Bob; Yan, Bingfang

    2002-05-01

    Carboxylesterases hydrolyze numerous endogenous and foreign compounds with diverse structures. Humans and rodents express multiple forms of carboxylesterases, which share a high degree of sequence identity (approximately 70%). Alignment analyses locate in carboxylesterases several functional subsites such the catalytic triad as seen in acetylcholinesterase. The aim of this study was to determine among human and rodent carboxylesterases the immunorelatedness, overlapping substrate specificity, differential sensitivity to serine enzyme inhibitors, tissue distribution, and tumor-related expression. Six antibodies against whole carboxylesterases or synthetic peptides were tested for their reactivity toward 11 human or rodent recombinant carboxylesterases. The antibodies against whole proteins generally exhibited a broader cross-reactivity than the anti-peptide antibodies. All carboxylesterases hydrolyzed para-nitrophenylacetate and para-nitrophenylbutyrate. However, the relative activity varied markedly from enzyme to enzyme (>20-fold), and some carboxylesterases showed a clear substrate preference. Carboxylesterases with the same functional subsites had a similar profile on substrate specificity and sensitivity toward phenylmethylsulfonyl fluoride (PMSF) and paraoxon, suggesting that these subsites play determinant roles in the recognition of substrates and inhibitors. Among three human carboxylesterases, HCE-1 hydrolyzed both substrates to a similar extent, whereas HCE-2 and HCE-3 showed an opposite substrate preference. All three enzymes were inhibited by PMSF and paraoxon, but they showed a marked difference in relative sensitivities. Based on immunoblotting analyses, HCE-1 was present in all tissues examined, whereas HCE-2 and HCE-3 were expressed in a tissue-restricted pattern. Colon carcinomas expressed slightly higher levels of HCE-1 and HCE-2 than the adjacent normal tissues, whereas the opposite was true with HCE-3.

  9. Decreased Expression of Inhibitor of Caspase-Activated DNase (ICAD) in Renal Cell Carcinoma – Tissue Microarray of Human Samples

    PubMed Central

    Rajandram, Retnagowri; Razack, Azad H. A.; Ng, Keng Lim

    2016-01-01

    Although primary localised tumours of renal cell carcinoma (RCC) can be treated relatively successfully with surgery, metastatic RCC has poor prognosis because of late diagnosis and resistance to therapies. In the present study, we were interested in profiling the protein expression of “inhibitor of caspase-activated DNase” (ICAD), an apoptosis inhibitor, in kidney cancer and its paired normal kidney. Immunohistochemistry with automated batch staining and morphometry using digital pathology were used to compare ICAD in 121 RCC specimens with their paired normal kidney tissue. Tissue microarray of formalin-fixed, paraffin-embedded archival tissue was used. Intensity and localisation of ICAD were compared between normal and cancer samples, and against grading within the cancers. The results demonstrated that, in this cohort, ICAD was highly expressed in the proximal tubular epithelium of normal kidney, and significantly decreased in clear cell RCC tissue (p < 0.05) as well as other subtypes of RCC (p < 0.01) compared with normal kidney. There was a tendency towards nuclear localisation of ICAD in clear cell RCC, but not in other subtypes of RCC. No significant association was found between ICAD intensity and grade of RCC. In summary, down-regulation of ICAD occurs in RCC. ICAD normally inhibits DNA fragmentation and apoptosis; thus, its down-regulation was unexpected in a cancer known for its resistance to apoptosis. However, these RCC samples were from primary, not metastatic, RCC sites, and down-regulated ICAD may be part of a progressive pathway that promotes RCC metastasis.

  10. Functional expression of recombinant human ribonuclease/angiogenin inhibitor in stably transformed Drosophila melanogaster S2 cells.

    PubMed

    Park, Jong-Hwa; Hwang, In-Sook; Kim, Kyung-Il; Lee, Jong-Min; Park, Young-Min; Park, Chang-Ho; Chung, In Sik

    2008-05-01

    A recombinant plasmid harboring heterologous genes coding human ribonuclease/angiogenin inhibitor (RAI) was expressed in stably transformed Drosophila melanogaster Schneider 2 (S2) cells. Stably transformed polyclonal cell populations expressing RAI were isolated after 4 weeks of selection with hygromycin B. Recombinant RAI with a molecular weight of 50 kDa was detected in the intracellular (cell) and extracellular (medium) fractions of S2 cells. Recombinant RAI was purified from the extracellular fraction using a two-step purification scheme comprised of Ni-NTA and ion-exchange chromatography. Purified RAI migrated on SDS-PAGE as a single band in the elution fraction containing 300 mM NaCl. The ribonuclease inhibitor activity of purified RAI was measured using yeast tRNA and RNase A. Purified RAI exhibited an activity of approximately 8 U mug(-1) for the inhibition of RNA degradation by RNase A. Cultivation of stably transformed S2 cells using HyQ((R))SFX-insect MP medium increased cell growth by 79% and approximately doubled the production of recombinant RAI.

  11. Latex of Euphorbia antiquorum-induced S-phase arrest via active ATM kinase and MAPK pathways in human cervical cancer HeLa cells.

    PubMed

    Hsieh, Wen-Tsong; Lin, Hui-Yi; Chen, Jou-Hsuan; Lin, Wen-Chung; Kuo, Yueh-Hsiung; Wood, W Gibson; Lu, Hsu-Feng; Chung, Jing-Gung

    2015-09-01

    Latex of Euphorbia antiquorum (EA) has demonstrated great chemotherapeutic potential for cancer. However, the mechanisms of anti-proliferation of EA on cancer cell remain to be further investigated. The purpose of this study was to explore the influence of EA in human cervical cancer cells. Here, the cell cycle distribution by flow cytometry was examined and the protein expression by the western blotting methods was analyzed. From the cytometric results it was shown that EA-induced S-phase arrest in a concentration manner both in human cervical cancer HeLa and CaSki cells. According the western blot results it was illustrated that EA could downregulate early cyclin E1-Cdk2; and cyclin A-Cdc2 provides a significant additional quantity of S-phase promotion, that in turn promoted the expression of p21(waf1/cip1) and p27(kip1) which were the inhibitors in the complex of cyclin A and Cdc2 that led to cell cycle arrest. Moreover, EA promoted the activation of ataxia telangiectasia mutated (ATM) and check-point kinase-2 (Chk2); however, it negatively regulated the expression of Topoisomerases I and II, Cdc25A, and Cdc25C signaling. Caffeine, an ATM/ATR inhibitor significantly reversed EA downregulation in the levels of Cdc25A. Furthermore, JNK inhibitor SP600125 and p38 MAPK inhibitor SB203580 both could reverse the EA upregulation of the protein of Chk2 level, significantly. This study, therefore, revealed that EA could downregulate topoisomerase, and activate ATM kinase, which then induce parallel Chk 1/2 and MAPK signaling pathways to promote the degradation of Cdc25A to induced S-phase arrest in human cervical cancer HeLa cells.

  12. From microRNA functions to microRNA therapeutics: novel targets and novel drugs in breast cancer research and treatment (Review).

    PubMed

    Piva, Roberta; Spandidos, Demetrios A; Gambari, Roberto

    2013-10-01

    MicroRNAs (miRNAs or miRs) are a family of small non‑coding RNAs that regulate gene expression by the sequence-selective targeting of mRNAs, leading to translational repression or mRNA degradation, depending on the degree of complementarity with target mRNA sequences. miRNAs play a crucial role in cancer. In the case of breast tumors, several studies have demonstrated a correlation between: i) the expression profile of oncogenic miRNAs (oncomiRs) and tumor suppressor miRNAs; and ii) the tumorigenic potential of triple-negative [estrogen receptor (ER), progesterone receptor (PR) and Her2/neu] primary breast cancers. Among the miRNAs involved in breast cancer, miR-221 plays a crucial role for the following reasons: i) miR-221 is significantly overexpressed in triple-negative primary breast cancer; ii) the oncosuppressor p27Kip1, a validated miR-221 target is downregulated in aggressive cancer cell lines; and iii) the upregulation of a key transcription factor, Slug, appears to be crucial, since it binds to the miR-221/miR-222 promoter and is responsible for the high expression of the miR-221/miR-222 cluster in breast cancer cells. A Slug/miR-221 network has been suggested, linking miR-221 activity with the downregulation of a Slug repressor, leading to Slug/miR-221 upregulation and p27Kip1 downregulation. Interference with this process can be achieved using antisense miRNA (antagomiR) molecules targeting miR-221, inducing the downregulation of Slug and the upregulation of p27Kip1.

  13. Co-active receptor tyrosine kinases mitigate the effect of FGFR inhibitors in FGFR1-amplified lung cancers with low FGFR1 protein expression.

    PubMed

    Kotani, H; Ebi, H; Kitai, H; Nanjo, S; Kita, K; Huynh, T G; Ooi, A; Faber, A C; Mino-Kenudson, M; Yano, S

    2016-07-07

    Targeted therapies are effective in subsets of lung cancers with EGFR mutations and anaplastic lymphoma kinase (ALK) translocations. Large-scale genomics have recently expanded the lung cancer landscape with FGFR1 amplification found in 10-20% of squamous cell carcinomas (SCCs). However, the response rates have been low for biomarker-directed fibroblast growth factor receptor (FGFR) inhibitor therapy in SCC, which contrasts to the relatively high rates of response seen in EGFR mutant and ALK-translocated lung cancers treated with epidermal growth factor receptor (EGFR) inhibitors and ALK inhibitors, respectively. In order to better understand the low response rates of FGFR1-amplified lung cancers to FGFR inhibitors, relationships between gene copy number, mRNA expression and protein expression of FGFR1 were assessed in cell lines, tumor specimens and data from The Cancer Genome Atlas. The importance of these factors for the sensitivity to FGFR inhibitors was determined by analyzing drug screen data and conducting in vitro and in vivo experiments. We report that there was a discrepancy between FGFR1 amplification level and FGFR1 protein expression in a number of these cell lines, and the cancers with unexpectedly low FGFR1 expression were uniformly resistant to the different FGFR inhibitors. Further interrogation of the receptor tyrosine kinase activity in these discordant cell lines revealed co-activation of HER2 and platelet-derived growth factor receptor-α (PDGFRα) caused by gene amplification or ligand overexpression maintained phosphoinositide 3-kinase (PI3K) and MEK/ERK signaling even in the presence of FGFR inhibitor. Accordingly, co-inhibition of FGFR1 and HER2 or PDGFRα led to enhanced drug responses. In contrast, FGFR1-amplified high FGFR1 protein-expressing lung cancers are sensitive to FGFR inhibitor monotherapy by downregulating ERK signaling. Addition of a PI3K inhibitor to these high FGFR1 protein-expressing cancers further sensitized them to FGFR

  14. cDNA cloning and heterologous expression of a wheat proteinase inhibitor of subtilisin and chymotrypsin (WSCI) that interferes with digestive enzymes of insect pests.

    PubMed

    Di Gennaro, Simone; Ficca, Anna G; Panichi, Daniela; Poerio, Elia

    2005-04-01

    A cDNA encoding the proteinase inhibitor WSCI (wheat subtilisin/chymotrypsin inhibitor) was isolated by RT-PCR. Degenerate oligonucleotide primers were designed based on the amino acid sequence of WSCI and on the nucleotide sequence of the two homologous inhibitors (CI-2A and CI-2B) isolated from barley. For large-scale production, wsci cDNA was cloned into the E. coli vector pGEX-2T. The fusion protein GST-WSCI was efficiently produced in the bacterial expression system and, as the native inhibitor, was capable of inhibiting bacterial subtilisin, mammalian chymotrypsins and chymotrypsin-like activities present in crude extracts of a number of insect larvae ( Helicoverpa armigera , Plodia interpunctella and Tenebrio molitor ). The recombinant protein produced was also able to interfere with chymotrypsin-like activity isolated from immature wheat caryopses. These findings support a physiological role for this inhibitor during grain maturation.

  15. Characterization of Plasmodium phosphatidylserine decarboxylase expressed in yeast and application for inhibitor screening.

    PubMed

    Choi, Jae-Yeon; Kumar, Vidya; Pachikara, Niseema; Garg, Aprajita; Lawres, Lauren; Toh, Justin Y; Voelker, Dennis R; Ben Mamoun, Choukri

    2016-03-01

    Phospholipid biosynthesis is critical for the development, differentiation and pathogenesis of several eukaryotic pathogens. Genetic studies have validated the pathway for phosphatidylethanolamine synthesis from phosphatidylserine catalyzed by phosphatidylserine decarboxylase enzymes (PSD) as a suitable target for development of antimicrobials; however no inhibitors of this class of enzymes have been discovered. We show that the Plasmodium falciparum PSD can restore the essential function of the yeast gene in strains requiring PSD for growth. Genetic, biochemical and metabolic analyses demonstrate that amino acids between positions 40 and 70 of the parasite enzyme are critical for proenzyme processing and decarboxylase activity. We used the essential role of Plasmodium PSD in yeast as a tool for screening a library of anti-malarials. One of these compounds is 7-chloro-N-(4-ethoxyphenyl)-4-quinolinamine, an inhibitor with potent activity against P. falciparum, and low toxicity toward mammalian cells. We synthesized an analog of this compound and showed that it inhibits PfPSD activity and eliminates Plasmodium yoelii infection in mice. These results highlight the importance of 4-quinolinamines as a novel class of drugs targeting membrane biogenesis via inhibition of PSD activity.

  16. Characterization of Plasmodium phosphatidylserine decarboxylase expressed in yeast and application for inhibitor screening

    PubMed Central

    Choi, Jae-Yeon; Lawres, Lauren; Toh, Justin Y.; Voelker, Dennis R.; Ben Mamoun, Choukri

    2016-01-01

    Summary Phospholipid biosynthesis is critical for the development, differentiation and pathogenesis of several eukaryotic pathogens. Genetic studies have validated the pathway for phosphatidylethanolamine synthesis from phosphatidylserine catalyzed by phosphatidylserine decarboxylase enzymes (PSD) as a suitable target for development of antimicrobials; however no inhibitors of this class of enzymes have been discovered. We show that the Plasmodium falciparum PSD can restore the essential function of the yeast gene in strains requiring PSD for growth. Genetic, biochemical and metabolic analyses demonstrate that amino acids between positions 40 and 70 of the parasite enzyme are critical for proenzyme processing and decarboxylase activity. We used the essential role of Plasmodium PSD in yeast as a tool for screening a library of anti-malarials. One of these compounds is 7-chloro-N-(4-ethoxyphenyl)-4-quinolinamine, an inhibitor with potent activity against P. falciparum, and low toxicity toward mammalian cells. We synthesized an analog of this compound and showed that it inhibits PfPSD activity and eliminates Plasmodium yoelii infection in mice. These results highlight the importance of 4-quinolinamines as a novel class of drugs targeting membrane biogenesis via inhibition of PSD activity PMID:26585333

  17. Peroxisome Proliferator-Activated Receptor γ Induces the Expression of Tissue Factor Pathway Inhibitor-1 (TFPI-1) in Human Macrophages

    PubMed Central

    Copin, C.; Derudas, B.; Marx, N.

    2016-01-01

    Tissue factor (TF) is the initiator of the blood coagulation cascade after interaction with the activated factor VII (FVIIa). Moreover, the TF/FVIIa complex also activates intracellular signalling pathways leading to the production of inflammatory cytokines. The TF/FVIIa complex is inhibited by the tissue factor pathway inhibitor-1 (TFPI-1). Peroxisome proliferator-activated receptor gamma (PPARγ) is a transcription factor that, together with PPARα and PPARβ/δ, controls macrophage functions. However, whether PPARγ activation modulates the expression of TFP1-1 in human macrophages is not known. Here we report that PPARγ activation increases the expression of TFPI-1 in human macrophages in vitro as well as in vivo in circulating peripheral blood mononuclear cells. The induction of TFPI-1 expression by PPARγ ligands, an effect shared by the activation of PPARα and PPARβ/δ, occurs also in proinflammatory M1 and in anti-inflammatory M2 polarized macrophages. As a functional consequence, treatment with PPARγ ligands significantly reduces the inflammatory response induced by FVIIa, as measured by variations in the IL-8, MMP-2, and MCP-1 expression. These data identify a novel role for PPARγ in the control of TF the pathway. PMID:28115923

  18. The DNA methylation inhibitor induces telomere dysfunction and apoptosis of leukemia cells that is attenuated by telomerase over-expression.

    PubMed

    Zhang, Xiaolu; Li, Bingnan; de Jonge, Nick; Björkholm, Magnus; Xu, Dawei

    2015-03-10

    DNA methyltransferase inhibitors (DNMTIs) such as 5-azacytidine (5-AZA) have been used for treatment of acute myeloid leukemia (AML) and other malignancies. Although inhibiting global/gene-specific DNA methylation is widely accepted as a key mechanism behind DNMTI anti-tumor activity, other mechanisms are likely involved in DNMTI's action. Because telomerase reverse transcriptase (TERT) plays key roles in cancer through telomere elongation and telomere lengthening-independent activities, and TERT has been shown to confer chemo- or radio-resistance to cancer cells, we determine whether DNMTIs affect telomere function and whether TERT/telomerase interferes with their anti-cancer efficacy. We showed that 5-AZA induced DNA damage and telomere dysfunction in AML cell lines by demonstrating the presence of 53-BP1 foci and the co-localization of 53-BP1 foci with telomere signals, respectively. Telomere dysfunction was coupled with diminished TERT expression, shorter telomere and apoptosis in 5-AZA-treated cells. However, 5-AZA treatment did not lead to changes in the methylation status of subtelomere regions. Down-regulation of TERT expression similarly occurred in primary leukemic cells derived from AML patients exposed to 5-AZA. TERT over-expression significantly attenuated 5-AZA-mediated DNA damage, telomere dysfunction and apoptosis of AML cells. Collectively, 5-AZA mediates the down-regulation of TERT expression, and induces telomere dysfunction, which consequently exerts an anti-tumor activity.

  19. NAD(P)H:Quinone Oxidoreductase-1 Expression Sensitizes Malignant Melanoma Cells to the HSP90 Inhibitor 17-AAG

    PubMed Central

    Kasai, Shuya; Arakawa, Nobuyuki; Okubo, Ayaka; Shigeeda, Wataru; Yasuhira, Shinji; Masuda, Tomoyuki; Akasaka, Toshihide; Shibazaki, Masahiko; Maesawa, Chihaya

    2016-01-01

    The KEAP1-NRF2 pathway regulates cellular redox homeostasis by transcriptional induction of genes associated with antioxidant synthesis and detoxification in response to oxidative stress. Previously, we reported that KEAP1 mutation elicits constitutive NRF2 activation and resistance to cisplatin (CDDP) and dacarbazine (DTIC) in human melanomas. The present study was conducted to clarify whether an HSP90 inhibitor, 17-AAG, efficiently eliminates melanoma with KEAP1 mutation, as the NRF2 target gene, NQO1, is a key enzyme in 17-AAG bioactivation. In melanoma and non-small cell lung carcinoma cell lines with or without KEAP1 mutations, NQO1 expression and 17-AAG sensitivity are inversely correlated. NQO1 is highly expressed in normal melanocytes and in several melanoma cell lines despite the presence of wild-type KEAP1, and the NQO1 expression is dependent on NRF2 activation. Because either CDDP or DTIC produces reactive oxygen species that activate NRF2, we determined whether these agents would sensitize NQO1-low melanoma cells to 17-AAG. Synergistic cytotoxicity of the 17-AAG and CDDP combination was detected in four out of five NQO1-low cell lines, but not in the cell line with KEAP1 mutation. These data indicate that 17-AAG could be a potential chemotherapeutic agent for melanoma with KEAP1 mutation or NQO1 expression. PMID:27045471

  20. Broad 4-Hydroxyphenylpyruvate Dioxygenase Inhibitor Herbicide Tolerance in Soybean with an Optimized Enzyme and Expression Cassette[W][OPEN

    PubMed Central

    Siehl, Daniel L.; Tao, Yumin; Albert, Henrik; Dong, Yuxia; Heckert, Matthew; Madrigal, Alfredo; Lincoln-Cabatu, Brishette; Lu, Jian; Fenwick, Tamara; Bermudez, Ericka; Sandoval, Marian; Horn, Caroline; Green, Jerry M.; Hale, Theresa; Pagano, Peggy; Clark, Jenna; Udranszky, Ingrid A.; Rizzo, Nancy; Bourett, Timothy; Howard, Richard J.; Johnson, David H.; Vogt, Mark; Akinsola, Goke; Castle, Linda A.

    2014-01-01

    With an optimized expression cassette consisting of the soybean (Glycine max) native promoter modified for enhanced expression driving a chimeric gene coding for the soybean native amino-terminal 86 amino acids fused to an insensitive shuffled variant of maize (Zea mays) 4-hydroxyphenylpyruvate dioxygenase (HPPD), we achieved field tolerance in transgenic soybean plants to the HPPD-inhibiting herbicides mesotrione, isoxaflutole, and tembotrione. Directed evolution of maize HPPD was accomplished by progressively incorporating amino acids from naturally occurring diversity and novel substitutions identified by saturation mutagenesis, combined at random through shuffling. Localization of heterologously expressed HPPD mimicked that of the native enzyme, which was shown to be dually targeted to chloroplasts and the cytosol. Analysis of the native soybean HPPD gene revealed two transcription start sites, leading to transcripts encoding two HPPD polypeptides. The N-terminal region of the longer encoded peptide directs proteins to the chloroplast, while the short form remains in the cytosol. In contrast, maize HPPD was found almost exclusively in chloroplasts. Evolved HPPD enzymes showed insensitivity to five inhibitor herbicides. In 2013 field trials, transgenic soybean events made with optimized promoter and HPPD variant expression cassettes were tested with three herbicides and showed tolerance to four times the labeled rates of mesotrione and isoxaflutole and two times the labeled rates of tembotrione. PMID:25192697

  1. Fatty acid synthase regulates estrogen receptor-α signaling in breast cancer cells

    PubMed Central

    Menendez, J A; Lupu, R

    2017-01-01

    Fatty acid synthase (FASN), the key enzyme for endogenous synthesis of fatty acids, is overexpressed and hyperactivated in a biologically aggressive subset of sex steroid-related tumors, including breast carcinomas. Using pharmacological and genetic approaches, we assessed the molecular relationship between FASN signaling and estrogen receptor alpha (ERα) signaling in breast cancer. The small compound C75, a synthetic slow-binding inhibitor of FASN activity, induced a dramatic augmentation of estradiol (E2)-stimulated, ERα-driven transcription. FASN and ERα were both necessary for the synergistic activation of ERα transcriptional activity that occurred following co-exposure to C75 and E2: first, knockdown of FASN expression using RNAi (RNA interference) drastically lowered (>100 fold) the amount of E2 required for optimal activation of ERα-mediated transcriptional activity; second, FASN blockade synergistically increased E2-stimulated ERα-mediated transcriptional activity in ERα-negative breast cancer cells stably transfected with ERα, but not in ERα-negative parental cells. Non-genomic, E2-regulated cross-talk between the ERα and MAPK pathways participated in these phenomena. Thus, treatment with the pure antiestrogen ICI 182 780 or the potent and specific inhibitor of MEK/ERK, U0126, was sufficient to abolish the synergistic nature of the interaction between FASN blockade and E2-stimulated ERα transactivation. FASN inhibition suppressed E2-stimulated breast cancer cell proliferation and anchorage-independent colony formation while promoting the reduction of ERα protein. FASN blockade resulted in the increased expression and nuclear accumulation of the cyclin-dependent kinase inhibitors p21WAF1/CIP1 and p27Kip1, two critical mediators of the therapeutic effects of antiestrogen in breast cancer, while inactivating AKT, a key mediator of E2-promoted anchorage-independent growth. The ability of FASN to regulate E2/ERα signaling may represent a

  2. Further characterization of the GlyT-1 inhibitor Org25935: anti-alcohol, neurobehavioral, and gene expression effects.

    PubMed

    Lidö, Helga Höifödt; Jonsson, Susanne; Hyytiä, Petri; Ericson, Mia; Söderpalm, Bo

    2017-02-04

    The glycine transporter-1 inhibitor Org25935 is a promising candidate in a treatment concept for alcohol use disorder targeting the glycine system. Org25935 inhibits ethanol-induced dopamine elevation in brain reward regions and reduces ethanol intake in Wistar rats. This study aimed to further characterise the compound and used ethanol consumption, behavioral measures, and gene expression as parameters to investigate the effects in Wistar rats and, as pharmacogenetic comparison, Alko-Alcohol (AA) rats. Animals were provided limited access to ethanol in a two-bottle free-choice paradigm with daily drug administration. Acute effects of Org25935 were estimated using locomotor activity and neurobehavioral status. Effects on gene expression in Wistar rats were measured with qPCR. The higher but not the lower dose of Org25935 reduced alcohol intake in Wistar rats. Unexpectedly, Org25935 reduced both ethanol and water intake and induced strong CNS-depressive effects in AA-rats (withdrawn from further studies). Neurobehavioral effects by Org25935 differed between the strains (AA-rats towards sedation). Org25935 did not affect gene expression at the mRNA level in the glycine system of Wistar rats. The data indicate a small therapeutic range for the anti-alcohol properties of Org25935, a finding that may guide further evaluations of the clinical utility of GlyT-1 inhibitors. The results point to the importance of pharmacogenetic considerations when developing drugs for alcohol-related medical concerns. Despite the lack of successful clinical outcomes, to date, the heterogeneity of drug action of Org25935 and similar agents and the unmet medical need justify further studies of glycinergic compounds in alcohol use disorder.

  3. Epigenetic bivalent marking is permissive to the synergy of HDAC and PARP inhibitors on TXNIP expression in breast cancer cells.

    PubMed

    Baldan, Federica; Mio, Catia; Lavarone, Elisa; Di Loreto, Carla; Puglisi, Fabio; Damante, Giuseppe; Puppin, Cinzia

    2015-05-01

    Studies on stem cell differentiation led to the identification of paused genes, characterized by the contemporary presence of both activator and repressor epigenetic markers (bivalent marking). TXNIP is an oncosuppressor gene the expression of which was reduced in breast cancer. In the present study, we evaluated whether the concept of epigenetic bivalent marking can be applied to TXNIP gene in breast cancer cells. Using chromatin immunoprecipitation (ChIP), three histone modifications were investigated: two associated with transcriptional activation, lysines 9-14 acetylation of H3 histone (H3K9K14ac) and lysine 4 trimethylation of H3 histone (H3K4me3), and one associated with transcriptional silencing, lysine 27 trimethylation of H3 histone (H3K27me3). According to the bivalent marking model, TXNIP gene appears to be paused in MDA157 cells (markers of active and repressed transcription are present), but are definitively silenced in MDA468 cells (presence of only markers of transcription repression). This was proven by evaluating TXNIP mRNA and protein levels after the treatment of cell lines with a histone deacetylase inhibitor (SAHA) and a poly-ADP-ribose polymerases inhibitor (PJ34). In MDA157 cells, SAHA and PJ34 showed a synergistic effect: a large increment was observed in TXNIP mRNA and protein levels. By contrast, in MDA468 cells, synergy between the two compounds was not observed. Therefore, the pausing epigenetic signature was permissive for synergy between SAHA and PJ34 on TXNIP gene expression. The synergy between SAHA and PJ34 on TXNIP expression was associated with variation in cell viability and apoptosis. In MDA157 cells, but not in MDA468 cells, combined treatment of SAHA and PJ34 induced a decrease in cell viability and an increase of apoptosis. Thus, our data support the hypothesis that TXNIP is an effective target for the treatment of breast cancer.

  4. The expression and phylogenetics of the Inhibitor Cysteine Knot peptide OCLP1 in the honey bee Apis mellifera.

    PubMed

    Bloch, Guy; Cohen, Mira

    2014-06-01

    Small cysteine-rich peptides have diverse functions in insects including antimicrobial defense, phenoloxidase activity regulation, and toxic inhibition of ion channels of prey or predator. We combined bioinformatics and measurements of transcript abundance to start characterizing AmOCLP1, a recently discovered Inhibitor Cysteine Knot peptide in the honey bee Apis mellifera. We found that the genomes of ants, bees, and the wasp Nasonia vitripennis encode orthologous sequences indicating that OCLP1 is a conserved peptide and not unique to the honey bee. Search of available EST libraries and quantitative real time PCR analyses indicate that the transcript of AmOCLP1 is ubiquitous with expression in life stages ranging from embryos to adults and in all tested tissues. In worker honey bees AmOCLP1 expression was not associated with age or task and did not show clear enrichment in any of the tested tissues. There was however a consistent trend toward higher transcript levels in the abdomen of foragers relative to levels in the head or thorax, and compared to levels in the abdomen of younger worker bees. By contrast, in drones AmOCLP1 transcript levels appeared higher in the head relative to the abdomen. Finer analyses of the head and abdomen indicated that the AmOCLP1 transcript is not enriched in the stinger and the associated venom sac or in cephalic exocrine glands. The evolutionary conservation in the Hymenoptera, the ubiquitous expression, and the lack of enrichment in the venom gland, stinger, exocrine glands, and the brain are not consistent with the hypotheses that OCLP1 is a secreted honeybee toxin or an endotoxin acting in the central nervous system. Rather we hypothesize that OCLP1 is a conserved antimicrobial or phenoloxidase inhibitor peptide.

  5. Molecular Cloning and Functional Studies of Two Kazal-Type Serine Protease Inhibitors Specifically Expressed by Nasonia vitripennis Venom Apparatus

    PubMed Central

    Qian, Cen; Fang, Qi; Wang, Lei; Ye, Gong-Yin

    2015-01-01

    Two cDNA sequences of Kazal-type serine protease inhibitors (KSPIs) in Nasonia vitripennis, NvKSPI-1 and NvKSPI-2, were characterized and their open reading frames (ORFs) were 198 and 264 bp, respectively. Both NvKSPI-1 and NvKSPI-2 contained a typical Kazal-type domain. Real-time quantitative PCR (RT-qPCR) results revealed that NvKSPI-1 and NvKSPI-2 mRNAs were mostly detected specifically in the venom apparatus, while they were expressed at lower levels in the ovary and much lower levels in other tissues tested. In the venom apparatus, both NvKSPI-1 and NvKSPI-2 transcripts were highly expressed on the fourth day post eclosion and then declined gradually. The NvKSPI-1 and NvKSPI-2 genes were recombinantly expressed utilizing a pGEX-4T-2 vector, and the recombinant products fused with glutathione S-transferase were purified. Inhibition of recombinant GST-NvKSPI-1 and GST-NvKSPI-2 to three serine protease inhibitors (trypsin, chymotrypsin, and proteinase K) were tested and results showed that only NvKSPI-1 could inhibit the activity of trypsin. Meanwhile, we evaluated the influence of the recombinant GST-NvKSPI-1 and GST-NvKSPI-2 on the phenoloxidase (PO) activity and prophenoloxidase (PPO) activation of hemolymph from a host pupa, Musca domestica. Results showed PPO activation in host hemolymph was inhibited by both recombinant proteins; however, there was no significant inhibition on the PO activity. Our results suggested that NvKSPI-1 and NvKSPI-2 could inhibit PPO activation in host hemolymph and trypsin activity in vitro. PMID:26248077

  6. The pan-neural bHLH proteins DEADPAN and ASENSE regulate mitotic activity and cdk inhibitor dacapo expression in the Drosophila larval optic lobes.

    PubMed

    Wallace, K; Liu, T H; Vaessin, H

    2000-01-01

    Developmental regulators and cell cycle regulators have to interface in order to ensure appropriate cell proliferation during organogenesis. Our analysis of the roles of the pan-neural genes deadpan and asense defines critical roles for these genes in regulation of mitotic activities in the larval optic lobes. Loss of deadpan results in reduced cell proliferation, while ectopic deadpan expression causes over-proliferation. In contrast, loss of asense results in increased proliferation, while ectopic asense expression causes reduced proliferation. Consistent with these observations endogenous Deadpan is expressed in mitotic areas of the optic lobes, and endogenous Asense is expressed in cells that will become quiescent. Altered Deadpan or Asense expression results in altered expression of the cyclin dependent kinase inhibitor gene dacapo. Thus, regulation of mitotic activity during optic lobe development may, at least in part, involve deadpan and asense mediated regulation of the cyclin dependent kinase inhibitor gene dacapo. genesis 26:77-85, 2000.

  7. PI3K inhibitors LY294002 and IC87114 reduce inflammation in carrageenan-induced paw oedema and down-regulate inflammatory gene expression in activated macrophages.

    PubMed

    Eräsalo, Heikki; Laavola, Mirka; Hämäläinen, Mari; Leppänen, Tiina; Nieminen, Riina; Moilanen, Eeva

    2015-01-01

    PI3K/Akt pathway is a well-characterized pathway controlling cellular processes such as proliferation, migration and survival, and its role in cancer is vastly studied. There is also evidence to suggest the involvement of this pathway in the regulation of inflammatory responses. In this study, an attempt was made to investigate the role of PI3Ks in acute inflammation in vivo using pharmacological inhibitors against PI3Ks in the carrageenan-induced paw oedema model. A non-selective PI3K inhibitor LY294002 and a PI3Kδ-selective inhibitor IC87114 were used. Both of these inhibitors reduced inflammatory oedema upon carrageenan challenge in the mouse paw. To explain this result, the effects of the two inhibitors on inflammatory gene expression were investigated in activated macrophages. LY294002 and IC87114 prevented Akt phosphorylation as expected and down-regulated the expression of inflammatory factors IL-6, MCP-1,TNFα and iNOS. These findings suggest that PI3K inhibitors could be used to attenuate inflammatory responses and that the mechanism of action behind this effect is the down-regulation of inflammatory gene expression.

  8. Altered Expression of Brain Proteinase-Activated Receptor-2, Trypsin-2 and Serpin Proteinase Inhibitors in Parkinson's Disease.

    PubMed

    Hurley, Michael J; Durrenberger, Pascal F; Gentleman, Steve M; Walls, Andrew F; Dexter, David T

    2015-09-01

    Neuroinflammation is thought to contribute to cell death in neurodegenerative disorders, but the factors involved in the inflammatory process are not completely understood. Proteinase-activated receptor-2 (PAR2) expression in brain is increased in Alzheimer's disease and multiple sclerosis, but the status of PAR2 in Parkinson's disease is unknown. This study examined expression of PAR2 and endogenous proteinase activators (trypsin-2, mast cell tryptase) and proteinase inhibitors (serpin-A5, serpin-A13) in areas vulnerable and resistant to neurodegeneration in Parkinson's disease at different Braak α-synuclein stages of the disease in post-mortem brain. In normal aged brain, expression of PAR-2, trypsin-2, and serpin-A5 and serpin-A13 was found in neurons and microglia, and alterations in the amount of immunoreactivity for these proteins were found in some brain regions. Namely, there was a decrease in neurons positive for serpin-A5 in the dorsal motor nucleus, and serpin-A13 expression was reduced in the locus coeruleus and primary motor cortex, while expression of PAR2, trypsin-2 and both serpins was reduced in neurons within the substantia nigra. There was an increased number of microglia that expressed serpin-A5 in the dorsal motor nucleus of vagus and elevated numbers of microglia that expressed serpin-A13 in the substantia nigra of late Parkinson's disease cases. The number of microglia that expressed trypsin-2 increased in primary motor cortex of incidental Lewy body disease cases. Analysis of Parkinson's disease cases alone indicated that serpin-A5 and serpin-A13, and trypsin-2 expression in midbrain and cerebral cortex was different in cases with a high incidence of L-DOPA-induced dyskinesia and psychosis compared to those with low levels of these treatment-induced side effects. This study showed that there was altered expression in brain of PAR2 and some proteins that can control its function in Parkinson's disease. Given the role of PAR2 in

  9. Expression of a TGF-{beta} regulated cyclin-dependent kinase inhibitor in normal and immortalized airway epithelial cells

    SciTech Connect

    Tierney, L.A.; Bloomfield, C.; Johnson, N.F.

    1995-12-01

    Tumors arising from epithelial cells, including lung cancers are frequently resistant to factors that regulate growth and differentiation in normal in normal cells. Once such factor is transforming growth factor-{Beta} (TGF-{Beta}). Escape from the growth-inhibitory effects of TGF-{Beta} is thought to be a key step in the transformation of airway epithelial cells. most lung cancer cell lines require serum for growth. In contrast, normal human bronchial epithelial (NHBE) cells are exquisitely sensitive to growth-inhibitory and differentiating effects of TGF-{Beta}. The recent identification of a novel cyclin-dependent kinase inhibitor, p15{sup INK4B}, which is regulated by TGF-{Beta}, suggests a mechanism by which TGF-{Beta} mediates growth arrest in NHBE cells. The purpose of this study was two-fold: (1) to determine if p15{sup INK4B} is induced by TGF-{Beta} in NHBE cells or immortalized bronchial epithelial (R.1) cells and if that induction corresponds to a G1/S cell-cycle arrest; (2) to determine the temporal relationship between p15{sup INK4B} induction, cell-cycle arrest, and the phosphorylation state of the pRB because it is thought that p15{sup INK4B} acts indirectly by preventing phosphorylation of the RB gene product. In this study, expression of p15{sup INK4B} was examined in NHBE cells and R.1 cells at different time intervals following TGF-{Beta} treatment. The expression of this kinase inhibitor and its relationship to the cell and the pRb phosphorylation state were examined in cells that were both sensitive (NHBE) and resistant (R.1) to the effects of TGF-{Beta}. These results suggest that the cyclin-dependent kinase inhibitor, p15{sup INK4B}, is involved in airway epithelial cell differentiation and that loss or reduction of expression plays a role in the resistance of transformed or neoplastic cells to the growth-inhibitory effects of TGF-{Beta}.

  10. Expression of Raf kinase inhibitor protein is downregulated in response to Newcastle disease virus infection to promote viral replication.

    PubMed

    Yin, Renfu; Liu, Xinxin; Bi, Yuhai; Xie, Guangyao; Zhang, Pingze; Meng, Xin; Ai, Lili; Xu, Rongyi; Sun, Yuzhang; Stoeger, Tobias; Ding, Zhuang

    2015-09-01

    Newcastle disease virus (NDV) causes a severe and economically significant disease affecting almost the entire poultry industry worldwide. However, factors that affect NDV replication in host cells are poorly understood. Raf kinase inhibitory protein (RKIP) is a physiological inhibitor of c-RAF kinase and NF-κB signalling, known for their functions in the control of immune response as well as tumour invasion and metastasis. In the present study, we investigated the consequences of overexpression of host RKIP during viral infection. We demonstrate that NDV infection represses RKIP expression thereby promoting virus replication. Experimental upregulation of RKIP in turn acts as a potential antiviral defence mechanism in host cells that restricts NDV replication by repressing the activation of Raf/MEK/ERK and IκBα/NF-κB signalling pathways. Our results not only extend the concept of linking NDV-host interactions, but also reveal RKIP as a new class of protein-kinase-inhibitor protein that affects NDV replication with therapeutic potential.

  11. Reversible linkage of two distinct small molecule inhibitors of Myc generates a dimeric inhibitor with improved potency that is active in myc over-expressing cancer cell lines.

    PubMed

    Wanner, Jutta; Romashko, Darlene; Werner, Douglas S; May, Earl W; Peng, Yue; Schulz, Ryan; Foreman, Kenneth W; Russo, Suzanne; Arnold, Lee D; Pingle, Maneesh; Bergstrom, Donald E; Barany, Francis; Thomson, Stuart

    2015-01-01

    We describe the successful application of a novel approach for generating dimeric Myc inhibitors by modifying and reversibly linking two previously described small molecules. We synthesized two directed libraries of monomers, each comprised of a ligand, a connector, and a bioorthogonal linker element, to identify the optimal dimer configuration required to inhibit Myc. We identified combinations of monomers, termed self-assembling dimeric inhibitors, which displayed synergistic inhibition of Myc-dependent cell growth. We confirmed that these dimeric inhibitors directly bind to Myc blocking its interaction with Max and affect transcription of MYC dependent genes. Control combinations that are unable to form a dimer do not show any synergistic effects in these assays. Collectively, these data validate our new approach to generate more potent and selective inhibitors of Myc by self-assembly from smaller, lower affinity components. This approach provides an opportunity for developing novel therapeutics against Myc and other challenging protein:protein interaction (PPI) target classes.

  12. Reversible Linkage of Two Distinct Small Molecule Inhibitors of Myc Generates a Dimeric Inhibitor with Improved Potency That Is Active in Myc Over-Expressing Cancer Cell Lines

    PubMed Central

    Wanner, Jutta; Romashko, Darlene; Werner, Douglas S.; May, Earl W.; Peng, Yue; Schulz, Ryan; Foreman, Kenneth W.; Russo, Suzanne; Arnold, Lee D.; Pingle, Maneesh; Bergstrom, Donald E.; Barany, Francis; Thomson, Stuart

    2015-01-01

    We describe the successful application of a novel approach for generating dimeric Myc inhibitors by modifying and reversibly linking two previously described small molecules. We synthesized two directed libraries of monomers, each comprised of a ligand, a connector, and a bioorthogonal linker element, to identify the optimal dimer configuration required to inhibit Myc. We identified combinations of monomers, termed self-assembling dimeric inhibitors, which displayed synergistic inhibition of Myc-dependent cell growth. We confirmed that these dimeric inhibitors directly bind to Myc blocking its interaction with Max and affect transcription of MYC dependent genes. Control combinations that are unable to form a dimer do not show any synergistic effects in these assays. Collectively, these data validate our new approach to generate more potent and selective inhibitors of Myc by self-assembly from smaller, lower affinity components. This approach provides an opportunity for developing novel therapeutics against Myc and other challenging protein:protein interaction (PPI) target classes. PMID:25875098

  13. Altered expression of urokinase-type plasminogen activator and plasminogen activator inhibitor in high-risk soft tissue sarcomas.

    PubMed

    Benassi, M S; Ponticelli, F; Azzoni, E; Gamberi, G; Pazzaglia, L; Chiechi, A; Conti, A; Spessotto, P; Scapolan, M; Pignotti, E; Bacchini, P; Picci, P

    2007-09-01

    In recent years, classification of soft-tissue sarcomas (STS) has improved with cytogenetic analyses, but their clinical behavior is still not easily predictable. The aim of this study was to detect alterations in the urokinase-type plasminogen system, involved in tumor growth and invasion, by comparing mRNA levels of its components with those of paired normal tissues, and relating them with patient clinical course. Real-time PCR was performed on human STS cell lines and tissues from highly malignant STS, including leiomyosarcomas and malignant fibrous histiocytomas, to evaluate the expression of urokinase-type plasminogen activator (uPA), uPA receptor (uPAR) and plasminogen activator inhibitor-1 (PAI-1). Immunohistochemistry of gene products was also performed. Median mRNA values of all genes studied were higher in tumors than in paired normal tissues. In agreement with data on STS cell lines, significant up-regulation for uPA and PAI-1 genes compared to reference values was seen. Moreover, different levels of expression were related to histotype and metastatic phenotype. There was accordance between uPA mRNA and protein expression, while immunodetection of PAI-1 product was weak and scattered. Clearly, the controversial role of PAI-1 protein requires further biological analyses, but evident involvement of uPA/PAI-1 gene overexpression in STS malignancy may highlight a molecular defect useful in discriminating STS high-risk patients.

  14. Extracellular regulated kinase 1/2 signaling is a critical regulator of interleukin-1β-mediated astrocyte tissue inhibitor of metalloproteinase-1 expression.

    PubMed

    Fields, Jerel; Cisneros, Irma E; Borgmann, Kathleen; Ghorpade, Anuja

    2013-01-01

    Astrocytes are essential for proper central nervous system (CNS) function and are intricately involved in neuroinflammation. Despite evidence that immune-activated astrocytes contribute to many CNS pathologies, little is known about the inflammatory pathways controlling gene expression. Our laboratory identified altered levels of tissue inhibitor of metalloproteinase (TIMP)-1 in brain lysates from human immunodeficiency virus (HIV)-1 infected patients, compared to age-matched controls, and interleukin (IL)-1β as a key regulator of astrocyte TIMP-1. Additionally, CCAAT enhancer binding protein (C/EBP)β levels are elevated in brain specimens from HIV-1 patients and the transcription factor contributes to astrocyte TIMP-1 expression. In this report we sought to identify key signaling pathways necessary for IL-1β-mediated astrocyte TIMP-1 expression and their interaction with C/EBPβ. Primary human astrocytes were cultured and treated with mitogen activated protein kinase-selective small molecule inhibitors, and IL-1β. TIMP-1 and C/EBPβ mRNA and protein expression were evaluated at 12 and 24 h post-treatment, respectively. TIMP-1 promoter-driven luciferase plasmids were used to evaluate TIMP-1 promoter activity in inhibitor-treated astrocytes. These data show that extracellular regulated kinase (ERK) 1/2-selective inhibitors block IL-1β-induced astrocyte TIMP-1 expression, but did not decrease C/EBPβ expression in parallel. The p38 kinase (p38K) inhibitors partially blocked both IL-1β-induced astrocyte TIMP-1 expression and C/EBPβ expression. The ERK1/2-selective inhibitor abrogated IL-1β-mediated increases in TIMP-1 promoter activity. Our data demonstrate that ERK1/2 activation is critical for IL-1β-mediated astrocyte TIMP-1 expression. ERK1/2-selective inhibition may elicit a compensatory response in the form of enhanced IL-1β-mediated astrocyte C/EBPβ expression, or, alternatively, ERK1/2 signaling may function to moderate IL-1β-mediated astrocyte C

  15. Expression of matrix metalloproteinases-2, -8, -13, -26, and tissue inhibitors of metalloproteinase-1 in human osteosarcoma.

    PubMed

    Korpi, Jarkko T; Hagström, Jaana; Lehtonen, Niko; Parkkinen, Jyrki; Sorsa, Timo; Salo, Tuula; Laitinen, Minna

    2011-03-01

    Osteosarcoma (OS) is among most common malignant tumour of bone. Matrix metalloproteinases (MMPs) are predominantly associated with poor prognosis of several cancers, although some of them, like MMP-8, seem to have a protective role in some cancers. We analyzed the distribution patterns of MMP-2, -8, -13, -26, and tissue inhibitor of matrix metalloproteinase (TIMP)-1 in 25 OS patients. MMP-2, -8, -13, -26 and TIMP-1 were mostly detected in sarcoma cells. Response to chemotherapy affected the amount of MMP-2, -8, and -13 in resection sections when compared to biopsies: patients with excellent or good response had less positivity to MMP-2 in chemotherapy samples than those with moderate or poor response. We conclude that MMP-2, -8, -13, -26, and TIMP-1 are expressed in OS tissue, and all, except protective MMP-8, were also found in metastases indicating that MMPs and TIMP-1 can participate in the OS progression.

  16. Killing of cancer cells through the use of eukaryotic expression vectors harbouring genes encoding nucleases and ribonuclease inhibitor.

    PubMed

    Glinka, Elena M

    2015-05-01

    Cancer gene therapy vectors are promising tools for killing cancer cells with the purpose of eradicating malignant tumours entirely. Different delivery methods of vectors into the cancer cells, including both non-viral and viral, as well as promoters for the targeted expression of genes encoding anticancer proteins were developed for effective and selective killing of cancer cells without harming healthy cells. Many vectors have been created to kill cancer cells, and some vectors suppress malignant tumours with high efficiency. This review is focused on vectors bearing genes for nucleases such as deoxyribonucleases (caspase-activated DNase, deoxyribonuclease I-like 3, endonuclease G) and ribonucleases (human polynucleotide phosphorylase, ribonuclease L, α-sarcin, barnase), as well as vectors harbouring gene encoding ribonuclease inhibitor. The data concerning the functionality and the efficacy of such vectors are presented.

  17. The Proteasome Inhibitor Bortezomib Is a Potent Inducer of Zinc Finger AN1-type Domain 2a Gene Expression

    PubMed Central

    Rossi, Antonio; Riccio, Anna; Coccia, Marta; Trotta, Edoardo; La Frazia, Simone; Santoro, M. Gabriella

    2014-01-01

    The zinc finger AN1-type domain 2a gene, also known as arsenite-inducible RNA-associated protein (AIRAP), was recently identified as a novel human canonical heat shock gene strictly controlled by heat shock factor (HSF) 1. Little is known about AIRAP gene regulation in human cells. Here we report that bortezomib, a proteasome inhibitor with anticancer and antiangiogenic properties used in the clinic for treatment of multiple myeloma, is a potent inducer of AIRAP expression in human cells. Using endothelial cells as a model, we unraveled the molecular mechanism regulating AIRAP expression during proteasome inhibition. Bortezomib induces AIRAP expression at the transcriptional level early after treatment, concomitantly with polyubiquitinated protein accumulation and HSF activation. AIRAP protein is detected at high levels for at least 48 h after bortezomib exposure, together with the accumulation of HSF2, a factor implicated in differentiation and development regulation. Different from heat-mediated induction, in bortezomib-treated cells, HSF1 and HSF2 interact directly, forming HSF1-HSF2 heterotrimeric complexes recruited to a specific heat shock element in the AIRAP promoter. Interestingly, whereas HSF1 has been confirmed to be critical for AIRAP gene transcription, HSF2 was found to negatively regulate AIRAP expression after bortezomib treatment, further emphasizing an important modulatory role of this transcription factor under stress conditions. AIRAP function is still not defined. However, the fact that AIRAP is expressed abundantly in primary human cells at bortezomib concentrations comparable with plasma levels in treated patients suggests that AIRAP may participate in the regulatory network controlling proteotoxic stress during bortezomib treatment. PMID:24619424

  18. Histone deacetylase inhibitors promote eNOS expression in vascular smooth muscle cells and suppress hypoxia-induced cell growth.

    PubMed

    Tan, Xiaoling; Feng, Lan; Huang, Xiaoyong; Yang, Yidong; Yang, Chengzhong; Gao, Yuqi

    2017-03-07

    Hypoxia stimulates excessive growth of vascular smooth muscle cells (VSMCs) contributing to vascular remodelling. Recent studies have shown that histone deacetylase inhibitors (HDIs) suppress VSMC proliferation and activate eNOS expression. However, the effects of HDI on hypoxia-induced VSMC growth and the role of activated eNOS in VSMCs are unclear. Using an EdU incorporation assay and flow cytometry analysis, we found that the HDIs, butyrate (Bur) and suberoylanilide hydroxamic acid (SAHA) significantly suppressed the proliferation of hypoxic VSMC lines and induced apoptosis. Remarkable induction of cleaved caspase 3, p21 expression and reduction of PCNA expression were also observed. Increased eNOS expression and enhanced NO secretion by hypoxic VSMC lines were detected using Bur or SAHA treatment. Knockdown of eNOS by siRNA transfection or exposure of hypoxic VSMCs to NO scavengers weakened the effects of Bur and SAHA on the growth of hypoxic VSMCs. In animal experiments, administration of Bur to Wistar rats exposed to hypobaric hypoxia for 28 days ameliorated the thickness and collagen deposition in pulmonary artery walls. Although the mean pulmonary arterial pressure (mPAP) was not obviously decreased with Bur in hypoxic rats, right ventricle hypertrophy index (RVHI) was decreased and the oxygen partial pressure of arterial blood was elevated. Furthermore, cell viability was decreased and eNOS and cleaved caspase 3 were induced in HDI-treated rat pulmonary arterial SMCs. These findings imply that HDIs prevent hypoxia-induced VSMC growth, in correlation with activated eNOS expression and activity in hypoxic VSMCs.

  19. Beta vulgaris L. serine proteinase inhibitor gene expression correlates to insect pest resistance in sugar beet

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Analyzing genes that can be used for improving sugar beet resistance to the sugar beet root maggot (SBRM, Tetanops myopaeformis Roder), one of the most destructive insect pests of sugar beet in North America, was a major goal in our investigation. We report on the expression patterns of a sugar beet...

  20. A Gene Expression Profile of BRCAness that Predicts for Responsiveness to Platinum and PARP Inhibitors

    DTIC Science & Technology

    2014-08-01

    allylamino-17-demethoxygeldanamycin) downregulated HR, ATM and Fanconi Anemia pathways. In HR- proficient EOC cells, 17-AAG suppressed HR as assessed...downregulated HR (pɘ.005), ATM (p=0.015) and Fanconi Anemia (pɘ.005) pathways, and downregulated the expression levels of several genes of these

  1. Gene expression of cyclin-dependent kinase inhibitors and effect of heparin on their expression in mice with hypoxia-induced pulmonary hypertension

    SciTech Connect

    Yu Lunyin; Quinn, Deborah A.; Garg, Hari G.; Hales, Charles A. . E-mail: chales@partners.org

    2006-07-14

    The balance between cell proliferation and cell quiescence is regulated delicately by a variety of mediators, in which cyclin-dependent kinases (CDK) and CDK inhibitors (CDKI) play a very important role. Heparin which inhibits pulmonary artery smooth muscle cell (PASMC) proliferation increases the levels of two CDKIs, p21 and p27, although only p27 is important in inhibition of PASMC growth in vitro and in vivo. In the present study we investigated the expression profile of all the cell cycle regulating genes, including all seven CDKIs (p21, p27, p57, p15, p16, p18, and p19), in the lungs of mice with hypoxia-induced pulmonary hypertension. A cell cycle pathway specific gene microarray was used to profile the 96 genes involved in cell cycle regulation. We also observed the effect of heparin on gene expression. We found that (a) hypoxic exposure for two weeks significantly inhibited p27 expression and stimulated p18 activity, showing a 98% decrease in p27 and 81% increase in p18; (b) other CDKIs, p21, p57, p15, p16, and p19 were not affected significantly in response to hypoxia; (c) heparin treatment restored p27 expression, but did not influence p18; (d) ERK1/2 and p38 were mediators in heparin upregulation of p27. This study provides an expression profile of cell cycle regulating genes under hypoxia in mice with hypoxia-induced pulmonary hypertension and strengthens the previous finding that p27 is the only CDKI involved in heparin regulation of PASMC proliferation and hypoxia-induced pulmonary hypertension.

  2. Combined treatment by octreotide and everolimus: Octreotide enhances inhibitory effect of everolimus in aggressive meningiomas.

    PubMed

    Graillon, Thomas; Defilles, Céline; Mohamed, Amira; Lisbonis, Christophe; Germanetti, Anne-Laure; Chinot, Olivier; Figarella-Branger, Dominique; Roche, Pierre-Hugues; Adetchessi, Tarek; Fuentes, Stéphane; Metellus, Philippe; Dufour, Henry; Enjalbert, Alain; Barlier, Anne

    2015-08-01

    Treatment for recurrent and aggressive meningiomas remains an unmet medical need in neuro-oncology, and chemotherapy exhibits limited clinical activity, if any. Merlin expression, encoded by the NF2 gene, is lost in a majority of meningiomas, and merlin is a negative regulator of mTORC1. The sst2 somatostatin receptor, targeted by octreotide, is highly expressed in meningiomas. To investigate new therapeutic strategies, we evaluated the activity of everolimus (mTOR inhibitor), BKM-120 and BEZ-235 (new Pi3K/Akt/mTOR inhibitors), octreotide and a combined treatment (octreotide plus everolimus), on cell proliferation, signaling pathways, and cell cycle proteins, respectively. The in vitro study was conducted on human meningioma primary cells extracted from fresh tumors, allowing the assessment of somatostatin analogs at the concentration levels used in patients. The results were correlated to WHO grades. Further, everolimus decreased cell viability of human meningiomas, but concomitantly, induced Akt activation, reducing the antiproliferative effect of the drug. The new Pi3K inhibitors were not more active than everolimus alone, limiting their clinical relevance. In contrast, a clear cooperative inhibitory effect of octreotide and everolimus was observed on cell proliferation in all tested meningiomas, including WHO grades II-III. Octreotide not only reversed everolimus-induced Akt phosphorylation but also displayed additive and complementary effects with everolimus on downstream proteins involved in translation (4EB-P1), and controlling cell cycle (p27Kip1 and cyclin D1). We have demonstrated a co-operative action between everolimus and octreotide on cell proliferation in human meningiomas, including aggressive ones, establishing the basis for a clinical trial.

  3. Disulfiram Suppresses Growth of the Malignant Pleural Mesothelioma Cells in Part by Inducing Apoptosis

    PubMed Central

    Muthu, Magesh; Jamal, Shazia; Chen, Di; Yang, Huanjie; Polin, Lisa A.; Tarca, Adi L.; Pass, Harvey I.; Dou, Q. Ping; Sharma, Sunita; Wali, Anil; Rishi, Arun K.

    2014-01-01

    Dithiocarbamate compound Disulfiram (DSF) that binds with copper and functions as an inhibitor of aldehyde dehydrogenase is a Food and Drug Administration approved agent for treatment of alcoholism. Copper complexed DSF (DSF-Cu) also possesses anti-tumor and chemosensitizing properties; however, its molecular mechanisms of action remain unclear. Here we investigated malignant pleural mesothelioma (MPM) suppressive effects of DSF-Cu and the molecular mechanisms involved. DSF-Cu inhibited growth of the murine as well as human MPM cells in part by increasing levels of ubiquitinated proteins. DSF-Cu exposure stimulated apoptosis in MPM cells that involved activation of stress-activated protein kinases (SAPKs) p38 and JNK1/2, caspase-3, and cleavage of poly-(ADP-ribose)-polymerase, as well as increased expression of sulfatase 1 and apoptosis transducing CARP-1/CCAR1 protein. Gene-array based analyses revealed that DSF-Cu suppressed cell growth and metastasis-promoting genes including matrix metallopeptidase 3 and 10. DSF inhibited MPM cell growth and survival by upregulating cell cycle inhibitor p27Kip1, IGFBP7, and inhibitors of NF-κB such as ABIN 1 and 2 and Inhibitory κB (IκB)α and β proteins. DSF-Cu promoted cleavage of vimentin, as well as serine-phosphorylation and lysine-63 linked ubiquitination of podoplanin. Administration of 50 mg/kg DSF-Cu by daily i.p injections inhibited growth of murine MPM cell-derived tumors in vivo. Although podoplanin expression often correlates with metastatic disease and poor prognosis, phosphorylation of serines in cytoplasmic domain of podoplanin has recently been shown to interfere with cellular motility and migration signaling. Post-translational modification of podoplanin and cleavage of vimentin by DSF-Cu underscore a metastasis inhibitory property of this agent and together with our in vivo studies underscore its potential as an anti-MPM agent. PMID:24690739

  4. Ectopic expression of a tobacco vacuolar invertase inhibitor in guard cells confers drought tolerance in Arabidopsis.

    PubMed

    Chen, Su-Fen; Liang, Ke; Yin, Dong-Mei; Ni, Di-An; Zhang, Zhi-Guo; Ruan, Yong-Ling

    2016-12-01

    There are several hypotheses that explain stomatal behavior. These include the concept of osmoregulation mediated by potassium and its counterions malate and chlorine and the more recent starch-sugar hypothesis. We have previously reported that the activity of the sucrose cleavage enzyme, vacuolar invertase (VIN), is significantly higher in guard cells than in other leaf epidermal cells and its activity is correlated with stomatal aperture. Here, we examined whether VIN indeed controls stomatal movement under normal and drought conditions by transforming Arabidopsis with a tobacco vacuolar invertase inhibitor homolog (Nt-inhh) under the control of an abscisic acid-sensitive and guard cell-specific promoter (AtRab18). The data obtained showed that guard cells of transgenic Arabidopsis plants had lower VIN activity, stomatal aperture and conductance than that of wild-type plants. Moreover, the transgenic plants also displayed higher drought tolerance than wild-type plants. The data indicate that VIN is a promising target for manipulating stomatal function to increase drought tolerance.

  5. Isolation and characterization of a proteinaceous α-amylase inhibitor AAI-CC5 from Streptomyces sp. CC5, and its gene cloning and expression.

    PubMed

    Sun, Zhibin; Lu, Weihao; Liu, Pingping; Wang, Hui; Huang, Yan; Zhao, Yuguo; Kong, Yi; Cui, Zhongli

    2015-02-01

    An α-amylase inhibitor producing Streptomyces sp. strain CC5 was isolated from soil. A proteinaceous α-amylase inhibitor AAI-CC5 was purified from strain CC5. AAI-CC5 specifically inhibited mammalian α-amylases. The molecular weight of the inhibitor was determined to be 8,212 Da by MALDI-TOF Mass Spectrum. The N-terminal 15 amino acid residues of the purified AAI-CC5 were DTGSPAPECVEYFQS, which is dissimilar to other reported proteinaceous α-amylase inhibitors. AAI-CC5 is a pH insensitive and heat-stable protein, and cannot be hydrolysed by trypsin. AAI-CC5 was cloned and expressed in Escherichia coli BL21 (DE3) with a hexa-histidine tag on the C terminal. AAI-CC5 shared 82 % identity with Parvulustat. The recombinant α-amylase inhibitor was purified to homogeneity by one-step affinity chromatography using Ni(2+)-NTA resin with molecular mass of 9,404 Da. Steady state kinetics studies of α-amylase and the inhibitor revealed an irreversible, non-competitive inhibition mechanism with IC50 and Ki value of 6.43 ×1 10(-11) and 4.45 × 10(-11) M respectively. These results suggest this novel α-amylase inhibitor possessed powerful inhibitory activity for α-amylase, and it may be a candidate in research of diabetes therapy and obesity treatment.

  6. Gene Expression Profiling of Rat Hippocampus Following Exposure to the Acetylcholinesterase Inhibitor Soman

    DTIC Science & Technology

    2009-01-01

    from seizure-related damage , and thus, neurodegeneration of soman- sensitive brain areas is a potential postexposure outcoVle. We performed gene...2009. 14. ABSTRACT See reprint. 15. SUBJECT TERMS Acetylcholinesterase, nerve agents, soman, neurodegeneration , gene expression profiling...Kniffin,:j: Christina P. Tompkins,:j: Tracey A. Hamilton,:j: and Robert K. Kan:j: Cell and Molecular Biology Branch, and Comparative Pathology

  7. Inhibition of Wild-Type p53-Expressing AML by the Novel Small Molecule HDM2 Inhibitor CGM097.

    PubMed

    Weisberg, Ellen; Halilovic, Ensar; Cooke, Vesselina G; Nonami, Atsushi; Ren, Tao; Sanda, Takaomi; Simkin, Irene; Yuan, Jing; Antonakos, Brandon; Barys, Louise; Ito, Moriko; Stone, Richard; Galinsky, Ilene; Cowens, Kristen; Nelson, Erik; Sattler, Martin; Jeay, Sebastien; Wuerthner, Jens U; McDonough, Sean M; Wiesmann, Marion; Griffin, James D

    2015-10-01

    The tumor suppressor p53 is a key regulator of apoptosis and functions upstream in the apoptotic cascade by both indirectly and directly regulating Bcl-2 family proteins. In cells expressing wild-type (WT) p53, the HDM2 protein binds to p53 and blocks its activity. Inhibition of HDM2:p53 interaction activates p53 and causes apoptosis or cell-cycle arrest. Here, we investigated the ability of the novel HDM2 inhibitor CGM097 to potently and selectively kill WT p53-expressing AML cells. The antileukemic effects of CGM097 were studied using cell-based proliferation assays (human AML cell lines, primary AML patient cells, and normal bone marrow samples), apoptosis, and cell-cycle assays, ELISA, immunoblotting, and an AML patient-derived in vivo mouse model. CGM097 potently and selectively inhibited the proliferation of human AML cell lines and the majority of primary AML cells expressing WT p53, but not mutant p53, in a target-specific manner. Several patient samples that harbored mutant p53 were comparatively unresponsive to CGM097. Synergy was observed when CGM097 was combined with FLT3 inhibition against oncogenic FLT3-expressing cells cultured both in the absence as well as the presence of cytoprotective stromal-secreted cytokines, as well as when combined with MEK inhibition in cells with activated MAPK signaling. Finally, CGM097 was effective in reducing leukemia burden in vivo. These data suggest that CGM097 is a promising treatment for AML characterized as harboring WT p53 as a single agent, as well as in combination with other therapies targeting oncogene-activated pathways that drive AML.

  8. A phenotype from tumor stroma based on the expression of metalloproteases and their inhibitors, associated with prognosis in breast cancer

    PubMed Central

    Eiró, Noemí; Fernandez-Garcia, Belen; Vázquez, Julio; del Casar, José M; González, Luis O; Vizoso, Francisco J

    2015-01-01

    The objective of the present work was to evaluate the impact of the phenotype of both mononuclear inflammatory cells (MICs) and cancer-associated fibroblast (CAFs) in early breast cancer patients, specifically assessed as to their expression of MMP/TIMP relative to their position within the tumor (i.e., localization at the tumor center or invasive front) and the occurrence of distant metastases.. An immunohistochemical study was performed using tissue arrays and specific antibodies against matrix metalloproteinase (MMP)−1, −2, −7, −9, −11, −13 and −14, tissue inhibitors of metalloproteinase (TIMP)−1, −2 and −3, both at tumor center and at invasive front, in 107 patients with primary ductal invasive breast tumors. Data were analyzed by unsupervised hierarchical clustering analysis. Our results indicated that MMP-11 expression by MICs, and TIMP-2 expression by CAFs at either the tumor center or the invasive front, were the most potent independent prognostic factors for predicting the clinical outcome of patients. Using the unsupervised hierarchical clustering analysis, we found well-defined clusters of cases identifying subgroups of tumors showing a high molecular profile of MMPs/TIMPs expression by stromal cells (CAFs and MICs), both at the tumor center and at the invasive front, which were strongly associated with a higher prevalence of distant metastasis. In addition, we found combinations of these clusters defining subpopulations of breast carcinomas differing widely in their clinical outcome. The results presented here identify biologic markers useful to categorize patients into different subgroups based on their tumor stroma, which may contribute to improved understanding of the prognosis of breast cancer patients. PMID:26140253

  9. Expression Profiles and RNAi Silencing of Inhibitor of Apoptosis Transcripts in Aedes, Anopheles, and Culex Mosquitoes (Diptera: Culicidae).

    PubMed

    Puglise, Jason M; Estep, Alden S; Becnel, James J

    2016-03-01

    Effective mosquito control is vital to curtail the devastating health effects of many vectored diseases. RNA interference (RNAi)-mediated control of mosquitoes is an attractive alternative to conventional chemical pesticides. Previous studies have suggested that transcripts for inhibitors of apoptosis (IAPs) may be good RNAi targets. To revisit and extend previous reports, we examined the expression of Aedes aegypti (L.) IAPs (AaeIAPs) 1, 2, 5, 6, 9, and a viral IAP-associated factor (vIAF) as well as Anopheles quadrimaculatus Say and Culex quinquefasciatus Say IAP1 homologs (AquIAP1 and CquIAP1) in adult females. Expression profiles of IAPs suggested that some older female mosquitoes had significantly higher IAP mRNA levels when compared to the youngest ones. Minor differences in expression of AaeIAPs were observed in mosquitoes that imbibed a bloodmeal, but the majority of the time points (up to 48 h) were not significantly different. Although in vitro experiments with the Ae. aegypti Aag-2 cell line demonstrated that the various AaeIAPs could be effectively knocked down within one day after dsRNA treatment, only Aag-2 cells treated with dsIAP1 displayed apoptotic morphology. Gene silencing and mortality were also evaluated after topical application and microinjection of the same dsRNAs into female Ae. aegypti. In contrast to previous reports, topical administration of dsRNA against AaeIAP1 did not yield a significant reduction in gene expression or increased mortality. Knockdown of IAP1 and other IAPs by microinjection did not result in significant mortality. In toto, our findings suggest that IAPs may not be suitable RNAi targets for controlling adult mosquito populations.

  10. Inhibitor of apoptosis protein expression in glioblastomas and their in vitro and in vivo targeting by SMAC mimetic GDC-0152

    PubMed Central

    Tchoghandjian, A; Soubéran, A; Tabouret, E; Colin, C; Denicolaï, E; Jiguet-Jiglaire, C; El-Battari, A; Villard, C; Baeza-Kallee, N; Figarella-Branger, D

    2016-01-01

    Glioblastomas (GBMs) are the most aggressive primary brain tumors in adult and remain a therapeutic challenge. Targeting key apoptosis regulators with the ultimate aim to restore apoptosis in tumor cells could be an interesting therapeutic strategy. The inhibitors of apoptosis proteins (IAPs) are regulators of cell death and represent attractive targets, especially because they can be antagonized by SMAC mimetics. In this study, we first investigated the expression of cIAP1, cIAP2, XIAP and ML-IAP in human GBM samples and in four different cell lines. We showed that all GBM samples and GBM cell lines expressed all these IAPs, although the expression of each IAP varied from one case to another. We then showed that high level of ML-IAP predicted worse progression-free survival and overall survival in both univariate and multivariate analyses in two independent cohorts of 58 and 43 primary human GBMs. We then used GDC-0152, a SMAC mimetic that antagonizes these IAPs and confirmed that GDC-0152 treatment in vitro decreased IAPs in all the cell lines studied. It affected cell line viability and triggered apoptosis, although the effect was higher in U87MG and GL261 than in GBM6 and GBM9 cell lines. In vivo, GDC-0152 effect on U87MG orthotopic xenografts was dose dependent; it postponed tumor formation and slowed down tumor growth, significantly improving survival of GBM-bearing mice. This study revealed for the first time that ML-IAP protein expression correlates with GBM patient survival and that its antagonist GDC-0152 improves outcome in xenografted mouse. PMID:27490930

  11. Variola virus immune evasion design: expression of a highly efficient inhibitor of human complement.

    PubMed

    Rosengard, Ariella M; Liu, Yu; Nie, Zhiping; Jimenez, Robert

    2002-06-25

    Variola virus, the most virulent member of the genus Orthopoxvirus, specifically infects humans and has no other animal reservoir. Variola causes the contagious disease smallpox, which has a 30-40% mortality rate. Conversely, the prototype orthopoxvirus, vaccinia, causes no disease in immunocompetent humans and was used in the global eradication of smallpox, which ended in 1977. However, the threat of smallpox persists because clandestine stockpiles of variola still exist. Although variola and vaccinia share remarkable DNA homology, the strict human tropism of variola suggests that its proteins are better suited than those of vaccinia to overcome the human immune response. Here, we demonstrate the functional advantage of a variola complement regulatory protein over that of its vaccinia homologue. Because authentic variola proteins are not available for study, we molecularly engineered and characterized the smallpox inhibitor of complement enzymes (SPICE), a homologue of a vaccinia virulence factor, vaccinia virus complement control protein (VCP). SPICE is nearly 100-fold more potent than VCP at inactivating human C3b and 6-fold more potent at inactivating C4b. SPICE is also more human complement-specific than is VCP. By inactivating complement components, SPICE serves to inhibit the formation of the C3/C5 convertases necessary for complement-mediated viral clearance. SPICE provides the first evidence that variola proteins are particularly adept at overcoming human immunity, and the decreased function of VCP suggests one reason why the vaccinia virus vaccine was associated with relatively low mortality. Disabling SPICE may be therapeutically useful if smallpox reemerges.

  12. Abnormal expression of plasminogen activator inhibitors in patients with gestational trophoblastic disease.

    PubMed Central

    Estellés, A.; Grancha, S.; Gilabert, J.; Thinnes, T.; Chirivella, M.; España, F.; Aznar, J.; Loskutoff, D. J.

    1996-01-01

    We previously reported significantly elevated levels of plasminogen activator inhibitor type 1 (PAI-1) in plasma and placenta from pregnant women with severe pre-eclampsia, and pre-eclampsia is a frequent problem in molar pregnancies. As increases in PAI-1 may contribute to the placental alterations that occur in pre-eclampsia, we have begun to investigate changes in PAI-1 as well as PAI-2 and several other components of the fibrinolytic system in patients with trophoblastic disease. Significant increases in plasma PAI-1 and decreases in plasma PAI-2 levels were observed in molar pregnancies when compared with the levels in normal pregnant women of similar gestational age. PAI-1 antigen levels also were increased, and PAI-2 levels were decreased in placenta from women with molar pregnancies compared with placenta obtained by spontaneous abortion. Immunohistochemical analysis revealed strong positive and specific staining of PAI-1 in trophoblastic epithelium in molar pregnancies and relatively weak staining of PAI-2. No association between the distribution of PAI-1 and vitronectin was found, and no specific signal for tissue type PA, urokinase type PA, tumor necrosis factor-alpha, or interleukin-1 was detected. In situ hybridization revealed an increase in PAI-1 but not PAI-2 mRNAs in placenta from molar pregnancies in comparison with placenta from abortions. These results demonstrate increased PAI-1 protein and mRNA in trophoblastic disease and suggest that localized elevated levels of PAI-1 may contribute to the hemostatic problems associated with this disorder. Images Figure 1 Figure 2 Figure 3 PMID:8863672

  13. The tumor suppressor ING1b is a novel corepressor for the androgen receptor and induces cellular senescence in prostate cancer cells.

    PubMed

    Esmaeili, Mohsen; Jennek, Susanne; Ludwig, Susann; Klitzsch, Alexandra; Kraft, Florian; Melle, Christian; Baniahmad, Aria

    2016-06-01

    The androgen receptor (AR) signaling is critical for prostate cancer (PCa) progression to the castration-resistant stage with poor clinical outcome. Altered function of AR-interacting factors may contribute to castration-resistant PCa (CRPCa). Inhibitor of growth 1 (ING1) is a tumor suppressor that regulates various cellular processes including cell proliferation. Interestingly, ING1 expression is upregulated in senescent primary human prostate cells; however, its role in AR signaling in PCa was unknown. Using a proteomic approach by surface-enhanced laser desorption ionization-mass spectrometry (SELDI-MS) combined with immunological techniques, we provide here evidence that ING1b interacts in vivo with the AR. The interaction was confirmed by co-immunoprecipitation, in vitro GST-pull-down, and quantitative intracellular colocalization analyses. Functionally, ING1b inhibits AR-responsive promoters and endogenous key AR target genes in the human PCa LNCaP cells. Conversely, ING1b knockout (KO) mouse embryonic fibroblasts (MEFs) exhibit enhanced AR activity, suggesting that the interaction with ING1b represses the AR-mediated transcription. Also, data suggest that ING1b expression is downregulated in CRPCa cells compared with androgen-dependent LNCaP cells. Interestingly, its ectopic expression induces cellular senescence and reduces cell migration in both androgen-dependent and CRPCa cells. Intriguingly, ING1b can also inhibit androgen-induced growth in LNCaP cells in a similar manner as AR antagonists. Moreover, ING1b upregulates different cell cycle inhibitors including p27(KIP1), which is a novel target for ING1b. Taken together, our findings reveal a novel corepressor function of ING1b on various AR functions, thereby inhibiting PCa cell growth.

  14. Inhibitor of DASH proteases affects expression of adhesion molecules in osteoclasts and reduces myeloma growth and bone disease.

    PubMed

    Pennisi, Angela; Li, Xin; Ling, Wen; Khan, Sharmin; Gaddy, Dana; Suva, Larry J; Barlogie, Bart; Shaughnessy, John D; Aziz, Nazneen; Yaccoby, Shmuel

    2009-06-01

    Dipeptidyl peptidase (DPP) IV activity and/or structure homologues (DASH) are serine proteases implicated in tumourigenesis. We previously found that a DASH protease, fibroblast activation protein (FAP), was involved in osteoclast-induced myeloma growth. Here we further demonstrated expression of various adhesion molecules in osteoclasts cultured alone or cocultured with myeloma cells, and tested the effects of DASH inhibitor, PT-100, on myeloma cell growth, bone disease, osteoclast differentiation and activity, and expression of adhesion molecules in osteoclasts. PT-100 had no direct effects on viability of myeloma cells or mature osteoclasts, but significantly reduced survival of myeloma cells cocultured with osteoclasts. Real-time PCR array for 85 adhesion molecules revealed upregulation of 17 genes in osteoclasts after coculture with myeloma cells. Treatment of myeloma/osteoclast cocultures with PT-100 significantly downregulated 18 of 85 tested genes in osteoclasts, some of which are known to play roles in tumourigenesis and osteoclastogenesis. PT-100 also inhibited osteoclast differentiation and subsequent pit formation. Resorption activity of mature osteoclasts and differentiation of osteoblasts were not affected by PT-100. In primary myelomatous severe combined immunodeficient (SCID)-hu mice PT-100 reduced osteoclast activity, bone resorption and tumour burden. These data demonstrated that DASH proteases are involved in myeloma bone disease and tumour growth.

  15. Pentoxifylline Regulates Plasminogen Activator Inhibitor-1 Expression and Protein Kinase A Phosphorylation in Radiation-Induced Lung Fibrosis

    PubMed Central

    Bae, Chang-Hwan; Jin, Young-Woo; Lee, Seung-Sook

    2017-01-01

    Purpose. Radiation-induced lung fibrosis (RILF) is a serious late complication of radiotherapy. In vitro studies have demonstrated that pentoxifylline (PTX) has suppressing effects in extracellular matrix production in fibroblasts, while the antifibrotic action of PTX alone using clinical dose is yet unexplored. Materials and Methods. We used micro-computed tomography (micro-CT) and histopathological analysis to evaluate the antifibrotic effects of PTX in a rat model of RILF. Results. Micro-CT findings showed that lung density, volume loss, and mediastinal shift are significantly increased at 16 weeks after irradiation. Simultaneously, histological analysis demonstrated thickening of alveolar walls, destruction of alveolar structures, and excessive collagen deposition in the irradiated lung. PTX treatment effectively attenuated the fibrotic changes based on both micro-CT and histopathological analyses. Western analysis also revealed increased levels of plasminogen activator inhibitor- (PAI-) 1 and fibronectin (FN) and PTX treatment reduced expression of PAI-1 and FN by restoring protein kinase A (PKA) phosphorylation but not TGF-β/Smad in both irradiated lung tissues and epithelial cells. Conclusions. Our results demonstrate the antifibrotic effect of PTX on radiation-induced lung fibrosis and its effect on modulation of PKA and PAI-1 expression as possible antifibrotic mechanisms. PMID:28337441

  16. Insulin continues to induce plasminogen activator inhibitor 1 gene expression in insulin-resistant mice and adipocytes.

    PubMed Central

    Samad, F.; Pandey, M.; Bell, P. A.; Loskutoff, D. J.

    2000-01-01

    BACKGROUND: Although the association between insulin resistance and cardiovascular risk is well established, the underlying molecular mechanisms are poorly understood. The antifibrinolytic molecule plasminogen activator inhibitor 1 (PAI-1) is a cardiovascular risk factor that is consistently elevated in insulin-resistant states such as obesity and non-insulin-dependent diabetes mellitus (NIDDM). The strong positive correlation between this elevated PAI-1 and the degree of hyperinsulinemia not only implicates insulin itself in this increase, but also suggests that PAI-1 is regulated by a pathway that does not become insulin resistant. The data in this report supports this hypothesis. MATERIALS AND METHODS: We show that insulin stimulates PAI-1 gene expression in metabolically insulin-resistant ob/ob mice and in insulin-resistant 3T3-L1 adipocytes. Moreover, we provide evidence that glucose transport and PAI-1 gene expression are mediated by different insulin signaling pathways. These observations suggest that the compensatory hyperinsulinemia that is frequently associated with insulin-resistant states, directly contribute to the elevated PAI-1. CONCLUSIONS: These results provide a potential mechanism for the abnormal increases in cardiovascular risk genes in obesity, NIDDM, and polycystic ovary disease. PMID:11055587

  17. Largazole, a class I histone deacetylase inhibitor, enhances TNF-α-induced ICAM-1 and VCAM-1 expression in rheumatoid arthritis synovial fibroblasts

    SciTech Connect

    Ahmed, Salahuddin; Riegsecker, Sharayah; Beamer, Maria; Rahman, Ayesha; Bellini, Joseph V.; Bhansali, Pravin; Tillekeratne, L.M. Viranga

    2013-07-15

    In the present study, we evaluated the effect of largazole (LAR), a marine-derived class I HDAC inhibitor, on tumor necrosis factor-α (TNF-α)-induced expression of intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1), and matrix metalloproteinase-2 (MMP-2) activity. LAR (1–5 μM) had no adverse effect on the viability of RA synovial fibroblasts. Among the different class I HDACs screened, LAR (0.5–5 μM) inhibited the constitutive expression of HDAC1 (0–30%). Surprisingly, LAR increased class II HDAC [HDAC6] by ∼ 220% with a concomitant decrease in HDAC5 [30–58%] expression in RA synovial fibroblasts. SAHA (5 μM), a pan-HDAC inhibitor, also induced HDAC6 expression in RA synovial fibroblasts. Pretreatment of RA synovial fibroblasts with LAR further enhanced TNF-α-induced ICAM-1 and VCAM-1 expression. However, LAR inhibited TNF-α-induced MMP-2 activity in RA synovial fibroblasts by 35% when compared to the TNF-α-treated group. Further, the addition of HDAC6 specific inhibitor Tubastatin A with LAR suppressed TNF-α + LAR-induced ICAM-1 and VCAM-1 expression and completely blocked MMP-2 activity, suggesting a role of HDAC6 in LAR-induced ICAM-1 and VCAM-1 expression. LAR also enhanced TNF-α-induced phospho-p38 and phospho-AKT expression, but inhibited the expression of phospho-JNK and nuclear translocation of NF-κBp65 in RA synovial fibroblasts. These results suggest that LAR activates p38 and Akt pathways and influences class II HDACs, in particular HDAC6, to enhance some of the detrimental effects of TNF-α in RA synovial fibroblasts. Understanding the exact role of different HDAC isoenzymes in RA pathogenesis is extremely important in order to develop highly effective HDAC inhibitors for the treatment of RA. - Highlights: • Largazole enhances TNF-α-induced ICAM-1 and VCAM-1. • Largazole upregulates class II HDAC (HDAC6) in RA synovial fibroblasts. • Largazole also induces the expression of phospho-p38

  18. Angiotensin-converting enzyme (ACE) inhibitors modulate cellular retinol-binding protein 1 and adiponectin expression in adipocytes via the ACE-dependent signaling cascade.

    PubMed

    Kohlstedt, Karin; Gershome, Cynthia; Trouvain, Caroline; Hofmann, Wolf-Karsten; Fichtlscherer, Stephan; Fleming, Ingrid

    2009-03-01

    Inhibitors of the angiotensin-converting enzyme (ACE) decrease angiotensin II production and activate an intracellular signaling cascade that affects gene expression in endothelial cells. Because ACE inhibitors have been reported to delay the onset of type 2 diabetes, we determined ACE signaling-modulated gene expression in endothelial cells and adipocytes. Using differential gene expression analysis, several genes were identified that were 3-fold up- or down-regulated by ramiprilat in cells expressing wild-type ACE versus cells expressing a signaling-dead ACE mutant. One up-regulated gene was the cellular retinol-binding protein 1 (CRBP1). In adipocytes, the overexpression of CRBP1 enhanced (4- to 5-fold) the activity of promoters containing response elements for retinol-dependent nuclear receptors [retinoic acid receptor (RAR) and retinoid X receptor (RXR)] or peroxisome proliferator-activated receptors (PPAR). CRBP1 overexpression also enhanced the promoter activity (by 470 +/- 40%) and expression/release of the anti-inflammatory and antiatherogenic adipokine adiponectin (cellular adiponectin by 196 +/- 24%, soluble adiponectin by 228 +/- 74%). Significantly increased adiponectin secretion was also observed after ACE inhibitor treatment of human preadipocytes, an effect prevented by small interfering RNA against CRBP1. Furthermore, in ob/ob mice, ramipril markedly potentiated both the basal (approximately 2-fold) and rosiglitazonestimulated circulating levels of adiponectin. In patients with coronary artery disease or type 2 diabetes, ACE inhibition also significantly increased plasma adiponectin levels (1.6- or 2.1-fold, respectively). In summary, ACE inhibitors affect adipocyte homeostasis via CRBP1 through the activation of RAR/RXR-PPAR signaling and up-regulation of adiponectin. The latter may contribute to the beneficial effects of ACE inhibitors on the development of type 2 diabetes in patients with an activated renin-angiotensin system.

  19. miR-17 inhibitor suppressed osteosarcoma tumor growth and metastasis via increasing PTEN expression

    SciTech Connect

    Gao, Yong; Luo, Ling-hui; Li, Shuai; Yang, Cao

    2014-02-07

    Highlights: • miR-17 was increased in OS tissues and cell lines. • Inhibition of miR-17 suppressed OS cell proliferation. • Inhibition of miR-17 suppressed OS cell migration and invasion. • PTEN was a target of miR-17. • miR-17 was negatively correlated with PTEN in OS tissues. - Abstract: MicroRNAs (miRNAs) play essential roles in cancer development and progression. Here, we investigated the role of miR-17 in the progression and metastasis of osteosarcoma (OS). miR-17 was frequently increased in OS tissues and cell lines. Inhibition of miR-17 in OS cell lines substantially suppressed cell proliferation, migration, and invasion. Phosphatase and tensin homolog (PTEN) was identified as a target of miR-17, and ectopic expression of miR-17 inhibited PTEN by direct binding to its 3′-untranslated region (3′-UTR). Expression of miR-17 was negatively correlated with PTEN in OS tissues. Together, these findings indicate that miR-17 acts as an oncogenic miRNA and may contribute to the progression and metastasis of OS, suggesting miR-17 as a potential novel diagnostic and therapeutic target of OS.

  20. Apigenin: Selective CK2 inhibitor increases Ikaros expression and improves T cell homeostasis and function in murine pancreatic cancer

    PubMed Central

    Nelson, Nadine; Szekeres, Karoly; Iclozan, Cristina; Rivera, Ivannie Ortiz; McGill, Andrew; Johnson, Gbemisola; Nwogu, Onyekachi

    2017-01-01

    Pancreatic cancer (PC) evades immune destruction by favoring the development of regulatory T cells (Tregs) that inhibit effector T cells. The transcription factor Ikaros is critical for lymphocyte development, especially T cells. We have previously shown that downregulation of Ikaros occurs as a result of its protein degradation by the ubiquitin-proteasome system in our Panc02 tumor-bearing (TB) mouse model. Mechanistically, we observed a deregulation in the balance between Casein Kinase II (CK2) and protein phosphatase 1 (PP1), which suggested that increased CK2 activity is responsible for regulating Ikaros’ stability in our model. We also showed that this loss of Ikaros expression is associated with a significant decrease in CD4+ and CD8+ T cell percentages but increased CD4+CD25+ Tregs in TB mice. In this study, we evaluated the effects of the dietary flavonoid apigenin (API), on Ikaros expression and T cell immune responses. Treatment of splenocytes from naïve mice with (API) stabilized Ikaros expression and prevented Ikaros downregulation in the presence of murine Panc02 cells in vitro, similar to the proteasome inhibitor MG132. In vivo treatment of TB mice with apigenin (TB-API) improved survival, reduced tumor weights and prevented splenomegaly. API treatment also restored protein expression of some Ikaros isoforms, which may be attributed to its moderate inhibition of CK2 activity from splenocytes of TB-API mice. This partial restoration of Ikaros expression was accompanied by a significant increase in CD4+ and CD8+ T cell percentages and a reduction in Treg percentages in TB-API mice. In addition, CD8+ T cells from TB-API mice produced more IFN-γ and their splenocytes were better able to prime allogeneic CD8+ T cell responses compared to TB mice. These results provide further evidence that Ikaros is regulated by CK2 in our pancreatic cancer model. More importantly, our findings suggest that API may be a possible therapeutic agent for stabilizing Ikaros

  1. Global gene expression in larval zebrafish (Danio rerio) exposed to selective serotonin reuptake inhibitors (fluoxetine and sertraline) reveals unique expression profiles and potential biomarkers of exposure.

    PubMed

    Park, June-Woo; Heah, Tze Ping; Gouffon, Julia S; Henry, Theodore B; Sayler, Gary S

    2012-08-01

    Larval zebrafish (Danio rerio) were exposed (96 h) to selective serotonin reuptake inhibitors (SSRIs) fluoxetine and sertraline and changes in transcriptomes analyzed by Affymetrix GeneChip Zebrafish Array were evaluated to enhance understanding of biochemical pathways and differences between these SSRIs. The number of genes differentially expressed after fluoxetine exposure was 288 at 25 μg/L and 131 at 250 μg/L; and after sertraline exposure was 33 at 25 μg/L and 52 at 250 μg/L. Same five genes were differentially regulated in both SSRIs indicating shared molecular pathways. Among these, the gene coding for FK506 binding protein 5, annotated to stress response regulation, was highly down-regulated in all treatments (results confirmed by qRT-PCR). Gene ontology analysis indicated at the gene expression level that regulation of stress response and cholinesterase activities were influenced by these SSRIs, and suggested that changes in transcription of these genes could be used as biomarkers of SSRI exposure.

  2. PULMONARY LOCALIZATION AND EXPRESSION OF PLASMINOGEN ACTIVATOR INHIBITOR-1 (PAI-1) IN HEALTHY OR HYPERTENSIVE RATS EXPOSED TO PARTICULATE MATTER (PM)

    EPA Science Inventory

    PULMONARY LOCALIZATION AND EXPRESSION OF PLASMINOGEN ACTIVATOR INHIBITOR-1 (PAI-1) IN HEALTHY OR HYPERTENSIVE RATS EXPOSED TO PARTICULATE MATTER (PM). GS Backus1, R Vincent2, UP Kodavanti2, 1Curriculum in Toxicology, UNC, Chapel Hill; 2NHEERL, ORD, US EPA, Research Triangle Park,...

  3. Purification, characterization, and specificity of dextranase inhibitor (Dei) expressed from Streptococcus sobrinus UAB108 gene cloned in Escherichia coli.

    PubMed Central

    Sun, J W; Wanda, S Y; Curtiss, R

    1995-01-01

    The dextranase inhibitor gene (dei) from Streptococcus sobrinus UAB108 was previously cloned, expressed, and sequenced. Its gene product (Dei) has now been purified as a single band with apparent molecular mass of 43 kDa, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The specific activity of Dei increased 121-fold upon purification. Most Dei activity (91.2%) was located in the periplasmic fraction from recombinant Escherichia coli cells. Dei competitively inhibits dextranase (Dex). This competitive inhibition mechanism has been further shown by detection and recovery of the intermediate enzyme-inhibitor (Dex-Dei) complex by gel filtration technology using fast protein liquid chromatography. Calibration of their molecular masses indicated that native Dei exists as a tetramer, Dex exists as dimer, and the Dex-Dei complex consists of two Dex molecules with two Dei molecules. Deletion analysis indicates that the intact Dei molecule is essential for Dei activity but not for glucan binding and immune cross-reaction. Dei is a special kind of glucan-binding protein with ability to inhibit Dex with high specificity. It can inhibit endogenous Dex, which can make more branches in glucan with the cooperation of the glucosyltransferase GTF-I. This inhibition cause the accumulation of water-soluble glucan. The latter reaction product can inhibit plaque formation and adherence of the mutans group of streptococcal cells. Dei derived from S. sobrinus UAB108 can inhibit only Dex from S. sobrinus (serotypes d and g), S. downei (previously S. sobrinus, serotype h), and S. macacae (serotype h). This finding suggests that Dei is another important protein existing in some serotypes of the mutans group of streptococci which participates in sucrose metabolism through its interaction with Dex. PMID:7896691

  4. Selective serotonin reuptake inhibitors, fluoxetine and paroxetine, attenuate the expression of the established behavioral sensitization induced by methamphetamine.

    PubMed

    Kaneko, Yujiro; Kashiwa, Atsushi; Ito, Takashi; Ishii, Sumikazu; Umino, Asami; Nishikawa, Toru

    2007-03-01

    To obtain an insight into the development of a new pharmacotherapy that prevents the treatment-resistant relapse of psychostimulant-induced psychosis and schizophrenia, we have investigated in the mouse the effects of selective serotonin reuptake inhibitors (SSRI), fluoxetine (FLX) and paroxetine (PRX), on the established sensitization induced by methamphetamine (MAP), a model of the relapse of these psychoses, because the modifications of the brain serotonergic transmission have been reported to antagonize the sensitization phenomenon. In agreement with previous reports, repeated MAP treatment (1.0 mg/kg a day, subcutaneously (s.c.)) for 10 days induced a long-lasting enhancement of the increasing effects of a challenge dose of MAP (0.24 mg/kg, s.c.) on motor activity on day 12 or 29 of withdrawal. The daily injection of FLX (10 mg/kg, s.c.) or PRX (8 mg/kg, s.c.) from 12 to 16 days of withdrawal of repeated MAP administration markedly attenuated the ability of the MAP pretreatment to augment the motor responses to the challenge dose of the stimulant 13 days after the SSRI injection. The repeated treatment with FLX or PRX alone failed to affect the motor stimulation following the challenge of saline and MAP 13 days later. These results suggest that the intermittent and repetitive elevation of serotonergic tone may inhibit the expression of the motor sensitization induced by pretreatment with MAP. It is proposed that clinically available serotonin reuptake inhibitors could be useful for preventing the recurrence of hallucinatory-paranoid state in drug-induced psychosis and schizophrenia.

  5. Involvement of ERK1/2, p38 MAPK, and PI3K/Akt signaling pathways in the regulation of cell cycle progression by PTHrP in colon adenocarcinoma cells.

    PubMed

    Calvo, Natalia; Martín, María Julia; de Boland, Ana Russo; Gentili, Claudia

    2014-08-01

    Parathyroid hormone-related peptide (PTHrP) is distributed in most fetal and adult tissues, and its expression correlates with the severity of colon carcinoma. Recently we obtained evidence that in Caco-2 cells, a cell line from human colorectal adenocarcinoma, exogenous PTHrP increases the number of live cells, via ERK1/2, p38 MAPK, and PI3-kinase and induces the expression of cyclin D1, a cell cycle regulatory protein. In this study, we further investigated the role of PTHrP in the regulation of the cell cycle progression in these intestinal cells. Flow cytometry analysis revealed that PTHrP treatment diminishes the number of cells in the G0/G1 phase and increases the number in both S and G2/M phases. The hormone increases the expression of CDK6 and diminishes the amount of negative cell cycle regulators p27Kip1, p15INK4B, and p53. However, PTHrP does not modify the expression of cyclin D3, CDK4, and p16INK4A. In addition, inhibitors of ERK1/2 (PD98059), p38 MAPK (SB203580), and PI3Kinase (LY294002) reversed PTHrP response in Caco-2 cells. Taken together, our results suggest that PTHrP positively modulates cell cycle progression and changes the expression of proteins involved in cell cycle regulation via ERK1/2, p38 MAPK, and PI3K signaling pathways in Caco-2 cells.

  6. Iroquois homeobox transcription factor (Irx5) promotes G1/S-phase transition in vascular smooth muscle cells by CDK2-dependent activation

    PubMed Central

    Liu, Dong; Pattabiraman, Vaishnavi; Bacanamwo, Methode

    2016-01-01

    The Iroquois homeobox (Irx5) gene is essential in embryonic development and cardiac electrophysiology. Although recent studies have reported that IRX5 protein is involved in regulation of the cell cycle and apoptosis in prostate cancer cells, little is known about the role of IRX5 in the adult vasculature. Here we report novel observations on the role of IRX5 in adult vascular smooth muscle cells (VSMCs) during proliferation in vitro and in vivo. Comparative studies using primary human endothelial cells, VSMCs, and intact carotid arteries to determine relative expression of Irx5 in the peripheral vasculature demonstrate significantly higher expression in VSMCs. Sprague-Dawley rat carotid arteries were subjected to balloon catherization, and the presence of IRX5 was examined by immunohistochemistry after 2 wk. Results indicate markedly elevated IRX5 signal at 14 days compared with uninjured controls. Total RNA was isolated from injured and uninjured arteries, and Irx5 expression was measured by RT-PCR. Results demonstrate a significant increase in Irx5 expression at 3–14 days postinjury compared with controls. Irx5 genetic gain- and loss-of-function studies using thymidine and 5-bromo-2′-deoxyuridine incorporation assays resulted in modulation of DNA synthesis in primary rat aortic VSMCs. Quantitative RT-PCR results revealed modulation of cyclin-dependent kinase inhibitor 1B (p27kip1), E2F transcription factor 1 (E2f1), and proliferating cell nuclear antigen (Pcna) expression in Irx5-transduced VSMCs compared with controls. Subsequently, apoptosis was observed and confirmed by morphological observation, caspase-3 cleavage, and enzymatic activation compared with control conditions. Taken together, these results indicate that Irx5 plays an important role in VSMC G1/S-phase cell cycle checkpoint control and apoptosis. PMID:27170637

  7. Cell cycle inhibitor p57 expression in normal and diabetic rat placentas during some stages of pregnancy.

    PubMed

    Acar, N; Korgun, E T; Ustunel, I

    2012-01-01

    Placentomegaly, an abnormal increase in the size of the placenta, is commonly seen in human diabetic pregnancies and diabetic animal experimental models. Proper placental development depends on the proliferation and differentiation of trophoblasts. However, our knowledge about the mitotic regulators that play key roles in synchronizing these events is limited. p57 is a cyclin-dependent kinase (CDK) inhibitor acting in the G1/S transition of the cell cycle. There is no data regarding p57 expression in either rat or human diabetic placentas. The purpose of this study was to investigate p57 expression in control and diabetic rat placentas at different stages of pregnancy. Diabetes was induced by streptozotocin on the first day of pregnancy, and placentas were taken on days 11, 13, 17, and 21 of pregnancy. Our results showed that on day 11, p57 immunostaining intensity was stronger in control group placentas compared to the diabetic group. On day 13, p57 immunostaining intensity increased in both groups, but increased more in the diabetic group. On day 17, p57 immunostaining intensity decreased in both the control and diabetic groups compared to day 13, yet the intensity remained higher in control placentas compared to diabetic placentas. On day 21 of pregnancy, p57 immunostaining intensity increased in the control group and it decreased from the day 17 level in the diabetic group. Western blot results showed consistency with immunohistochemistry results. Our study shows different expression patterns of p57 between control and diabetic rat placentas, which indicate p57 may play a role in abnormal placental formation resulting in placentomegaly arising from diabetes.

  8. The histone deacetylase inhibitor valproic acid inhibits NKG2D expression in natural killer cells through suppression of STAT3 and HDAC3

    PubMed Central

    Ni, Lulu; Wang, Lixin; Yao, Chao; Ni, Zhongya; Liu, Fei; Gong, Chenyuan; Zhu, Xiaowen; Yan, Xuewei; Watowich, Stephanie S.; Lee, Dean A.; Zhu, Shiguo

    2017-01-01

    NKG2D is a major activating receptor of NK cells and plays a critical role in tumor immunosurveillance. NKG2D expression in NK cells is inhibited by the histone deacetylase (HDAC) inhibitor valproic acid (VPA) and enhanced by the narrow-spectrum HDAC inhibitor entinostat. We previously demonstrated that entinostat enhanced NKG2D transcription by increasing acetylation of Histones H3 and H4. However, the mechanism by which VPA reduces NKG2D expression in NK cells is not known. We have also shown that NKG2D transcription is regulated by STAT3 phosphorylation. In this study, we investigated regulation of NKG2D expression in NK cells by VPA and entinostat by assessing protein expression, phosphorylation, and interaction of HDACs and STAT3. We find that VPA selectively inhibits STAT3 tyrosine705 phosphorylation, but entinostat does not. STAT3 complexes with HDAC3, and HDAC3 inhibition represses STAT3 phosphorylation and therefore NKG2D expression. NK cells from STAT3 wild-type mice downregulate NKG2D in response to VPA, but not NK cells from STAT3 knockout mice. These results show that VPA is a potent inhibitor of STAT3 phosphorylation and demonstrate that histone acetylation and STAT3 tyrosine705 phosphorylation cooperate in regulating NKG2D expression in NK cells. PMID:28338101

  9. Expression and localization of inhibitor of differentiation (ID) proteins during tissue and vascular remodelling in the human corpus luteum.

    PubMed

    Nio-Kobayashi, Junko; Narayanan, Rachna; Giakoumelou, Sevasti; Boswell, Lyndsey; Hogg, Kirsten; Duncan, W Colin

    2013-02-01

    Members of the transforming growth factor-β (TGF-β) superfamily are likely to have major roles in the regulation of tissue and vascular remodelling in the corpus luteum (CL). There are four inhibitor-of-differentiation (ID1-4) genes that are regulated by members of the TGF-β superfamily and are involved in the transcriptional regulation of cell growth and differentiation. We studied their expression, localization and regulation in dated human corpora lutea from across the luteal phase (n = 22) and after human chorionic gonadotrophin (hCG) administration in vivo (n = 5), and in luteinized granulosa cells (LGCs), using immunohistochemistry and quantitative RT-PCR. ID1-4 can be localized to multiple cell types in the CL across the luteal phase. Endothelial cell ID3 (P < 0.05) and ID4 (P < 0.05) immunostaining intensities peak at the time of angiogenesis but overall ID1 (P < 0.05) and ID3 (P < 0.05) expression peaks at the time of luteolysis, and luteal ID3 expression is inhibited by hCG in vivo (P < 0.01). In LGC cultures in vitro, hCG had no effect on ID1, down-regulated ID3 (P < 0.001), and up-regulated ID2 (P < 0.001) and ID4 (P < 0.01). Bone morphogenic proteins (BMPs) had no effect on ID4 expression but up-regulated ID1 (P < 0.01 to P < 0.005). BMP up-regulation of ID2 (P < 0.05) was additive to the hCG up-regulation of ID2 expression (P < 0.001), while BMP cancelled out the down regulative effect of hCG on ID3 regulation. As well as documenting regulation patterns specific for ID1, ID2, ID3 and ID4, we have shown that IDs are located and differentially regulated in the human CL, suggesting a role in the transcriptional regulation of luteal cells during tissue and vascular remodelling.

  10. Amplification and Overexpression of SKP2 Are Associated with Metastasis of Non-Small-Cell Lung Cancers to Lymph Nodes

    PubMed Central

    Yokoi, Sana; Yasui, Kohichiroh; Mori, Miki; Iizasa, Toshihiko; Fujisawa, Takehiko; Inazawa, Johji

    2004-01-01

    SKP2, an F-box protein constituting the substrate recognition subunit of the SCFSKP2 ubiquitin ligase complex, is implicated in ubiquitin-mediated degradation of the cyclin-dependent kinase inhibitor p27KIP1. Our earlier studies revealed SKP2 as a target gene within the 5p13 amplicon that is often seen in small-cell lung cancers. In the present study we examined amplification status and expression levels of SKP2 in non-small-cell lung cancer (NSCLC) and investigated its clinicopathological significance in this type of tumor because amplification of DNA at 5p13 is observed frequently in NSCLCs as well as in small-cell lung cancers. SKP2 exhibited amplification in 5 (20%) of 25 cell lines derived from NSCLC, and the transcript was overexpressed in 11 (44%) of the 25 lines. Moreover, expression of SKP2 was up-regulated significantly in 60 primary NSCLC tumors as compared to nontumorous lung tissues (P < 0.0001). Elevated expressio