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Sample records for intranuclear p27kip1 protein

  1. [Prognostic value of the inmunohïstochemical expression of protein p27KIP1 in laryngeal cancer].

    PubMed

    García Lozano, M C; Orradre Romero, J L; Caro García, M; Martínez Alvarez, A; Lasso Luis, O; Piris Pinilla, M A

    2006-01-01

    In this paper we carried out an immunohistochemical study of protein p27KIP1 expression in a series of 195 patients with laryngeal carcinoma that were diagnosticated, treated and followed at the Department of Otolaryngology at "Virgen de la Salud" Hospital (Toledo, Spain). In the cases with lymph node metastasis we also studied p27KIP1 expression at this level. Furthermore we have analysed the value of protein p27KIP1 expression as a prognostic factor (tumor recurrence, deads due to cancer and survival) and we evaluate the relationship between p27KIP1 expression and other clinic and pathologic parameters. PMID:16566195

  2. Cell cycle regulatory protein p27KIP1 is a substrate and interacts with the protein kinase CK2.

    PubMed

    Tapia, Julio C; Bolanos-Garcia, Victor M; Sayed, Muhammed; Allende, Catherine C; Allende, Jorge E

    2004-04-01

    The protein kinase CK2 is constituted by two catalytic (alpha and/or alpha') and two regulatory (beta) subunits. CK2 phosphorylates more than 300 proteins with important functions in the cell cycle. This study has looked at the relation between CK2 and p27(KIP1), which is a regulator of the cell cycle and a known inhibitor of cyclin-dependent kinases (Cdk). We demonstrated that in vitro recombinant Xenopus laevis CK2 can phosphorylate recombinant human p27(KIP1), but this phosphorylation occurs only in the presence of the regulatory beta subunit. The principal site of phosphorylation is serine-83. Analysis using pull down and surface plasmon resonance (SPR) techniques showed that p27(KIP1) interacts with the beta subunit through two domains present in the amino and carboxyl ends, while CD spectra showed that p27(KIP1) phosphorylation by CK2 affects its secondary structure. Altogether, these results suggest that p27(KIP1) phosphorylation by CK2 probably involves a docking event mediated by the CK2beta subunit. The phosphorylation of p27(KIP1) by CK2 may affect its biological activity. PMID:15034923

  3. The Relevance of Women's Diseases, Jun Activation-domain Binding Protein 1 (JAB1) and p27kip1

    PubMed Central

    Kim, Mijin; Lee, Hae-Hyeog

    2016-01-01

    The Jun activation-domain binding protein 1 (Jab1) recognize a potential coactivator of activator protein 1 (AP-1) such as c-fos, c-jun transcription factor and the fifth subunit of the COP9 signalosome complex. Also, Jab1 activate the c-jun gene resulted cell proliferation. Not only a powerful tumor suppressor but also regulator of apoptosis negative cdk inhibitor p27kip1 are involved in the cell cycle. This is Jab1 and p27kip1 interact with each other, Jab1 accelerate p27kip1 from nuclear to cytoplasm through ubiquitin/proteasome pathway. However, information about the relationship between Jab1 and p27kip1 is not known much. Taken together, the results of this study identify function and structure of Jab1 and p27kip1 were described in a recent article on the basis of relevant. Besides Jab1 and p27kip1 will organize the relationship between the disease and women. PMID:27152307

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

    DOE PAGESBeta

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

    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

  5. 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

  6. Cryptic sequence features within the disordered protein p27Kip1 regulate cell cycle signaling

    PubMed Central

    Das, Rahul K.; Huang, Yongqi; Phillips, Aaron H.; Kriwacki, Richard W.; Pappu, Rohit V.

    2016-01-01

    Peptide motifs embedded within intrinsically disordered regions (IDRs) of proteins are often the sites of posttranslational modifications that control cell-signaling pathways. How do IDR sequences modulate the functionalities of motifs? We answer this question using the polyampholytic C-terminal IDR of the cell cycle inhibitory protein p27Kip1 (p27). Phosphorylation of Thr-187 (T187) within the p27 IDR controls entry into S phase of the cell division cycle. Additionally, the conformational properties of polyampholytic sequences are predicted to be influenced by the linear patterning of oppositely charged residues. Therefore, we designed sequence variants of the p27 IDR to alter charge patterning outside the primary substrate motif containing T187. Computer simulations and biophysical measurements confirm predictions regarding the impact of charge patterning on the global dimensions of IDRs. Through functional studies, we uncover cryptic sequence features within the p27 IDR that influence the efficiency of T187 phosphorylation. Specifically, we find a positive correlation between T187 phosphorylation efficiency and the weighted net charge per residue of an auxiliary motif. We also find that accumulation of positive charges within the auxiliary motif can diminish the efficiency of T187 phosphorylation because this increases the likelihood of long-range intra-IDR interactions that involve both the primary and auxiliary motifs and inhibit their contributions to function. Importantly, our findings suggest that the cryptic sequence features of the WT p27 IDR negatively regulate T187 phosphorylation signaling. Our approaches provide a generalizable strategy for uncovering the influence of sequence contexts on the functionalities of primary motifs in other IDRs. PMID:27140628

  7. Cryptic sequence features within the disordered protein p27Kip1 regulate cell cycle signaling.

    PubMed

    Das, Rahul K; Huang, Yongqi; Phillips, Aaron H; Kriwacki, Richard W; Pappu, Rohit V

    2016-05-17

    Peptide motifs embedded within intrinsically disordered regions (IDRs) of proteins are often the sites of posttranslational modifications that control cell-signaling pathways. How do IDR sequences modulate the functionalities of motifs? We answer this question using the polyampholytic C-terminal IDR of the cell cycle inhibitory protein p27(Kip1) (p27). Phosphorylation of Thr-187 (T187) within the p27 IDR controls entry into S phase of the cell division cycle. Additionally, the conformational properties of polyampholytic sequences are predicted to be influenced by the linear patterning of oppositely charged residues. Therefore, we designed sequence variants of the p27 IDR to alter charge patterning outside the primary substrate motif containing T187. Computer simulations and biophysical measurements confirm predictions regarding the impact of charge patterning on the global dimensions of IDRs. Through functional studies, we uncover cryptic sequence features within the p27 IDR that influence the efficiency of T187 phosphorylation. Specifically, we find a positive correlation between T187 phosphorylation efficiency and the weighted net charge per residue of an auxiliary motif. We also find that accumulation of positive charges within the auxiliary motif can diminish the efficiency of T187 phosphorylation because this increases the likelihood of long-range intra-IDR interactions that involve both the primary and auxiliary motifs and inhibit their contributions to function. Importantly, our findings suggest that the cryptic sequence features of the WT p27 IDR negatively regulate T187 phosphorylation signaling. Our approaches provide a generalizable strategy for uncovering the influence of sequence contexts on the functionalities of primary motifs in other IDRs. PMID:27140628

  8. Homeodomain-interacting protein kinase-2 stabilizes p27(kip1) by its phosphorylation at serine 10 and contributes to cell motility.

    PubMed

    Pierantoni, Giovanna Maria; Esposito, Francesco; Tornincasa, Mara; Rinaldo, Cinzia; Viglietto, Giuseppe; Soddu, Silvia; Fusco, Alfredo

    2011-08-19

    HIPK2 is a serine/threonine kinase that acts as a coregulator of an increasing number of factors involved in cell survival and proliferation during development and in response to different types of stress. Here we report on a novel target of HIPK2, the cyclin-dependent kinase inhibitor p27(kip1). HIPK2 phosphorylates p27(kip1) in vitro and in vivo at serine 10, an event that accounts for 80% of the total p27(kip1) phosphorylation and plays a crucial role in the stability of the protein. Indeed, HIPK2 depletion by transient or stable RNA interference in tumor cells of different origin was consistently associated with strong reduction of p27(kip1) phosphorylation at serine 10 and of p27(kip1) stability. An initial evaluation of the functional relevance of this HIPK2-mediated regulation of p27(kip1) revealed a contribution to cell motility, rather than to cell proliferation, but only in cells that do not express wild-type p53. PMID:21715331

  9. Rat Protein Tyrosine Phosphatase η Suppresses the Neoplastic Phenotype of Retrovirally Transformed Thyroid Cells through the Stabilization of p27Kip1

    PubMed Central

    Trapasso, Francesco; Iuliano, Rodolfo; Boccia, Angelo; Stella, Antonella; Visconti, Roberta; Bruni, Paola; Baldassarre, Gustavo; Santoro, Massimo; Viglietto, Giuseppe; Fusco, Alfredo

    2000-01-01

    The r-PTPη gene encodes a rat receptor-type protein tyrosine phosphatase whose expression is negatively regulated by neoplastic cell transformation. Here we first demonstrate a dramatic reduction in DEP-1/HPTPη (the human homolog of r-PTPη) expression in a panel of human thyroid carcinomas. Subsequently, we show that the reexpression of the r-PTPη gene in highly malignant rat thyroid cells transformed by retroviruses carrying the v-mos and v-ras-Ki oncogenes suppresses their malignant phenotype. Cell cycle analysis demonstrated that r-PTPη caused G1 growth arrest and increased the cyclin-dependent kinase inhibitor p27Kip1 protein level by reducing the proteasome-dependent degradation rate. We propose that the r-PTPη tumor suppressor activity is mediated by p27Kip1 protein stabilization, because suppression of p27Kip1 protein synthesis using p27-specific antisense oligonucleotides blocked the growth-inhibitory effect induced by r-PTPη. Furthermore, we provide evidence that in v-mos- or v-ras-Ki-transformed thyroid cells, the p27Kip1 protein level was regulated by the mitogen-activated protein (MAP) kinase pathway and that r-PTPη regulated p27Kip1 stability by preventing v-mos- or v-ras-Ki-induced MAP kinase activation. PMID:11094075

  10. Prognostic implications of expression of the cell cycle inhibitor protein p27Kip1.

    PubMed

    Cariou, S; Catzavelos, C; Slingerland, J M

    1998-01-01

    Mitogenic and growth inhibitory signals influence the activity of a family of cyclin dependent kinases (cdks). p27 is an important cdk inhibitor, acting in G1 to inhibit cyclin-cdks. As negative growth regulators, the cdk inhibitors may function as tumor suppressors. While the p16 gene plays a tumor suppressor role in cancers, p27 gene mutations have been identified only rarely. While high levels of p27 protein are expressed in normal human mammary epithelium, loss of p27 is frequent and is of independent prognostic significance in breast cancers. Low p27 is also a poor prognostic factor in colon, gastric, esophageal, lung, and prostate carcinomas, and enhanced proteasomal degradation may underlie loss of p27 in tumor cells. Loss of p27 has not been significantly correlated with tumor proliferation in a number of studies and may reflect alterations in differentiation and adhesion-dependent growth regulation germane to oncogenesis and tumor progression. Efforts to confirm the prognostic value of p27 are under way in a number of large breast cancer studies. These studies may also indicate whether loss of p27 in association with other traditional or novel markers has greater prognostic potential than each factor alone. p27 immunostaining is inexpensive and reliable and may become part of the routine histopathologic processing of tumors in the near future. Widespread application of p27 in prognostic testing will require greater uniformity in scoring techniques and determination of the cut off levels which distinguish individuals at high and low risk of cancer recurrence and death. Finally, the greatest utility of p27 may lie in the information it sheds on the biology of aberrant growth regulation in breast cancer and the potential to use this in the generation of novel therapeutic strategies. PMID:10066070

  11. 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.

  12. The Down syndrome-related protein kinase DYRK1A phosphorylates p27Kip1 and Cyclin D1 and induces cell cycle exit and neuronal differentiation

    PubMed Central

    Soppa, Ulf; Schumacher, Julian; Florencio Ortiz, Victoria; Pasqualon, Tobias; Tejedor, Francisco J; Becker, Walter

    2014-01-01

    A fundamental question in neurobiology is how the balance between proliferation and differentiation of neuronal precursors is maintained to ensure that the proper number of brain neurons is generated. Substantial evidence implicates DYRK1A (dual specificity tyrosine-phosphorylation-regulated kinase 1A) as a candidate gene responsible for altered neuronal development and brain abnormalities in Down syndrome. Recent findings support the hypothesis that DYRK1A is involved in cell cycle control. Nonetheless, how DYRK1A contributes to neuronal cell cycle regulation and thereby affects neurogenesis remains poorly understood. In the present study we have investigated the mechanisms by which DYRK1A affects cell cycle regulation and neuronal differentiation in a human cell model, mouse neurons, and mouse brain. Dependent on its kinase activity and correlated with the dosage of overexpression, DYRK1A blocked proliferation of SH-SY5Y neuroblastoma cells within 24 h and arrested the cells in G1 phase. Sustained overexpression of DYRK1A induced G0 cell cycle exit and neuronal differentiation. Furthermore, we provide evidence that DYRK1A modulated protein stability of cell cycle-regulatory proteins. DYRK1A reduced cellular Cyclin D1 levels by phosphorylation on Thr286, which is known to induce proteasomal degradation. In addition, DYRK1A phosphorylated p27Kip1 on Ser10, resulting in protein stabilization. Inhibition of DYRK1A kinase activity reduced p27Kip1 Ser10 phosphorylation in cultured hippocampal neurons and in embryonic mouse brain. In aggregate, these results suggest a novel mechanism by which overexpression of DYRK1A may promote premature neuronal differentiation and contribute to altered brain development in Down syndrome. PMID:24806449

  13. Cyclin dependent kinase inhibitor p27Kip1 expression in normal and neoplastic cervical epithelium.

    PubMed Central

    Troncone, G; Vetrani, A; de Rosa, G; Gerbasio, D; Palombini, L

    1999-01-01

    AIM: To investigate whether there is loss of the p27Kip1 protein in developing cervical cancer and whether p27Kip1 immunoreactivity has any relation to the proliferative indicator Ki-67. METHODS: The expression of p27Kip1 and Ki-67 was assessed by immunohistochemistry in serial sections from normal epithelium (13), low grade (27) and high grade (19) squamous intraepithelial lesions (LSIL, HSIL), and invasive cervical cancer (23). In the SIL cases the presence of human papillomavirus (HPV) genomic sequences was assessed by in situ hybridisation. The results were evaluated by image analysis, and reported as mean score of the percentage of p27Kip1 and of Ki-67 positive cells in each histological group. RESULTS: In general, p27Kip1 immunostaining was related to squamous differentation, and was intense in normal epithelium (47%), while it was reduced in SIL lesions as an effect of the decreased number of differentiating cells. However, decrease in the p27Kip1 expression was more evident in LSIL (36%) than in HSIL (39%); in the latter, p27Kip1 had a different intraepithelial distribution in that the staining extended to the basal cells. The average levels of p27Kip1 were similar in SIL lesions associated to low, intermediate, and high risk HPV types. Compared with normal epithelium and dysplasia, invasive cancer showed significantly lower p27Kip1 levels (23%). There was no relation between p27Kip1 and Ki-67 labelling indices in any of the histological groups examined. CONCLUSIONS: A reduction in p27Kip1 protein occurs in cervical cancer independently of the proliferative status. The changes in p27Kip1 expression may be related to the unregulated kinetics of developing cervical cancer. Images PMID:10711250

  14. Cdc6 Protein Activates p27KIP1-bound Cdk2 Protein Only after the Bound p27 Protein Undergoes C-terminal Phosphorylation*

    PubMed Central

    Uranbileg, Baasanjav; Yamamoto, Hanako; Park, Jung-ha; Mohanty, Atish R.; Arakawa-Takeuchi, Shiho; Jinno, Shigeki; Okayama, Hiroto

    2012-01-01

    In mammalian cells Cdk2 activity during the G1-S transition is mainly controlled by p27KIP1. Although the amount and subcellular localization of p27 influence Cdk2 activity, how Cdk2 activity is regulated during this phase transition still remains virtually unknown. Here we report an entirely new mechanism for this regulation. Cdc6 the AAA+ ATPase, known to assemble prereplicative complexes on chromosomal replication origins and activate p21CIP1-bound Cdk2, also activated p27-bound Cdk2 in its ATPase and cyclin binding motif-dependent manner but only after the p27 bound to the Cdk2 was phosphorylated at the C terminus. ROCK, which mediates a signal for cell anchorage to the extracellular matrix and activates the mTORC1 cascade as well as controls cytoskeleton assembly, was partly responsible for C-terminal phosphorylation of the p27. In vitro reconstitution demonstrated ROCK (Rho-associated kinase)-mediated phosphorylation of Cdk2-bound p27 at the C terminus and subsequent activation of the Cdk2 by Cdc6. PMID:22223646

  15. Brk/Protein tyrosine kinase 6 phosphorylates p27KIP1, regulating the activity of cyclin D-cyclin-dependent kinase 4.

    PubMed

    Patel, Priyank; Asbach, Benedikt; Shteyn, Elina; Gomez, Cindy; Coltoff, Alexander; Bhuyan, Sadia; Tyner, Angela L; Wagner, Ralf; Blain, Stacy W

    2015-05-01

    Cyclin D and cyclin-dependent kinase 4 (cdk4) are overexpressed in a variety of tumors, but their levels are not accurate indicators of oncogenic activity because an accessory factor such as p27(Kip1) is required to assemble this unstable dimer. Additionally, tyrosine (Y) phosphorylation of p27 (pY88) is required to activate cdk4, acting as an "on/off switch." We identified two SH3 recruitment domains within p27 that modulate pY88, thereby modulating cdk4 activity. Via an SH3-PXXP interaction screen, we identified Brk (breast tumor-related kinase) as a high-affinity p27 kinase. Modulation of Brk in breast cancer cells modulates pY88 and increases resistance to the cdk4 inhibitor PD 0332991. An alternatively spliced form of Brk (Alt Brk) which contains its SH3 domain blocks pY88 and acts as an endogenous cdk4 inhibitor, identifying a potentially targetable regulatory region within p27. Brk is overexpressed in 60% of breast carcinomas, suggesting that this facilitates cell cycle progression by modulating cdk4 through p27 Y phosphorylation. p27 has been considered a tumor suppressor, but our data strengthen the idea that it should also be considered an oncoprotein, responsible for cyclin D-cdk4 activity. PMID:25733683

  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. PMID:26902224

  17. O-GlcNAc glycosylation of p27(kip1) promotes astrocyte migration and functional recovery after spinal cord contusion.

    PubMed

    Mao, Xingxing; Zhang, Dongmei; Tao, Tao; Liu, Xiaojuan; Sun, Xiaolei; Wang, Youhua; Shen, Aiguo

    2015-12-10

    Glial scar formation derived from astrocyte proliferation and migration influences the functional recovery after spinal cord injury. Cyclin-dependent kinase inhibitor p27(kip1), whose activity is closely related to its phosphorylation state, reportedly regulates astrocyte proliferation and migration. In this study, we reported that p27(Kip1) undergoes O-GlcNAc modification at Ser 2, Ser 110 and Thr 197. Inhibiting O-GlcNAcylation on Ser 2 by gene mutation (S2A) attenuated the phosphorylation of Ser 10, and vice versa. Interestingly, compared with wild type p27(Kip1), S2A p27(Kip1) displayed a decreased interaction with CRM1 and reduced nuclear export following serum starvation and release. In addition, the interaction between stathmin and S2A p27(Kip1) was also decreased. Cytoskeletal proteins microtubules appeared high density in astrocytes transfected with S2A p27(Kip1) especially at the leading edge of the scratch wound. Accordingly, scratch-wound assay revealed that the motility of astrocytes transfected with S2A p27(Kip1) was faster than that of control. Finally, we injected lentiviral vectors immediately after spinal cord contusion, and found the lesion volume of the rat injected with S2A p27(Kip1) was smaller than that of rat injected with wild type p27(Kip1). Besides, the BBB and CBS behavioral tests showed greater functional recovery in S2A p27(Kip1) treated rats. Taken together, our findings revealed a novel function of O-GlcNAc modification of p27(Kip1) in mediating astrocytes migration and functional recovery after spinal cord contusion. PMID:26562163

  18. Structural Basis of the Cks1-Dependent Recognition of P27Kip1 by the SCF skp2 Ubiquitin Ligase

    SciTech Connect

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

    2005-01-01

    The ubiquitin-mediated proteolysis of the Cdk2 inhibitor p27(Kip1) plays a central role in cell cycle progression, and enhanced degradation of p27(Kip1) is associated with many common cancers. Proteolysis of p27(Kip1) is triggered by Thr187 phosphorylation, which leads to the binding of the SCF(Skp2) (Skp1-Cul1-Rbx1-Skp2) ubiquitin ligase complex. Unlike other known SCF substrates, p27(Kip1) ubiquitination also requires the accessory protein Cks1. The crystal structure of the Skp1-Skp2-Cks1 complex bound to a p27(Kip1) phosphopeptide shows that Cks1 binds to the leucine-rich repeat (LRR) domain and C-terminal tail of Skp2, whereas p27(Kip1) binds to both Cks1 and Skp2. The phosphorylated Thr187 side chain of p27(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(Kip1) to the SCF(Skp2)-Cks1 complex.

  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. CacyBP/SIP nuclear translocation regulates p27Kip1 stability in gastric cancer cells

    PubMed Central

    Niu, Ying-Lin; Li, Ya-Jun; Wang, Jing-Bo; Lu, Yuan-Yuan; Liu, Zhen-Xiong; Feng, Shan-Shan; Hu, Jian-Guo; Zhai, Hui-Hong

    2016-01-01

    AIM: To investigate the mechanism of calcyclin binding protein/Siah-1 interacting protein (CacyBP/SIP) nuclear translocation in promoting the proliferation of gastric cancer (GC) cells. METHODS: The effect of CacyBP/SIP nuclear translocation on cell cycle was investigated by cell cycle analysis. Western blot analysis was used to assess the change in expression of cell cycle regulatory proteins and proteasome-mediated degradation of p27Kip1. Co-immunoprecipitation (co-IP) analysis was performed to examine the binding of CacyBP/SIP with Skp1. A CacyBP/SIP truncation mutant which lacked the Skp1 binding site was constructed and fused to a fluorescent protein. Subsequently, the effect on Skp1 binding with the fusion protein was examined by co-IP, while localization of fluorescent fusion protein observed by confocal laser microscopy, and change in p27Kip1 protein expression assessed by Western blot analysis. RESULTS: CacyBP/SIP nuclear translocation induced by gastrin promoted progression of GC cells from G1 phase. However, while CacyBP/SIP nuclear translocation was inhibited using siRNA to suppress CacyBP/SIP expression, cell cycle was clearly inhibited. CacyBP/SIP nuclear translocation significantly decreased the level of cell cycle inhibitor p27Kip1, increased Cyclin E protein expression whereas the levels of Skp1, Skp2, and CDK2 were not affected. Upon inhibition of CacyBP/SIP nuclear translocation, there were no changes in protein levels of p27Kip1 and Cyclin E, while p27Kip1 decrease could be prevented by the proteasome inhibitor MG132. Moreover, CacyBP/SIP was found to bind to Skp1 by immunoprecipitation, an event that was abolished by mutant CacyBP/SIP, which also failed to stimulate p27Kip1 degradation, even though the mutant could still translocate into the nucleus. CONCLUSION: CacyBP/SIP nuclear translocation contributes to the proliferation of GC cells, and CacyBP/SIP exerts this effect, at least in part, by stimulating ubiquitin-mediated degradation of p27

  2. Expression of p27Kip1, a cell cycle repressor protein, is inversely associated with potential carcinogenic risk in the genetic rodent models of obesity and long-lived Ames dwarf mice

    PubMed Central

    Eto, Isao

    2014-01-01

    Introduction The association of genetic rodent models of obesity and cancer still remains a controversial issue. Although this controversy has largely been resolved in recent years for homozygous leptin receptor-deficient obese Zucker rats and homozygous long-lived Ames dwarf mice, it is still unresolved for homozygous leptin-deficient obese ob/ob mice. Objective The objective of the present study described below was to investigate whether the expression of the cell cycle repressor protein p27(Kip1) is (a) down-regulated in the tumor-free homozygous leptin receptor-deficient obese Zucker rats as well as tumor-free homozygous leptin-deficient obese ob/ob mice and (b) up-regulated in the tumor-free homozygous long-lived Ames dwarf mice. Methods To achieve this objective, we first performed western immunoblot analysis of the hepatic expression of p27. We then performed western immunoblot analysis and proteomic analysis of the hepatic expression of the proteins involved in the upstream molecular signaling pathways for the expression of p27. Lastly, we analyzed the serum levels of glucose, insulin, and branched-chain amino acids, all of which have been shown to regulate, causally and inversely, the expression of p27. Results/Conclusions The results indicated that the hepatic expression of p27 was down-regulated in the homozygous leptin receptor-deficient obese Zucker rats and up-regulated in the homozygous long-lived Ames dwarf mice as expected. We also found that the hepatic expression of p27 was down-regulated in the homozygous leptin-deficient obese ob/ob mice. This last observation was not completely consistent with all of the results of the published studies where homozygous leptin-deficient obese ob/ob mice were used. PMID:23357529

  3. p27(Kip1) participates in the regulation of endoreplication in differentiating chick retinal ganglion cells.

    PubMed

    Ovejero-Benito, María C; Frade, José M

    2015-01-01

    Nuclear DNA duplication in the absence of cell division (i.e. endoreplication) leads to somatic polyploidy in eukaryotic cells. In contrast to some invertebrate neurons, whose nuclei may contain up to 200,000-fold the normal haploid DNA amount (C), polyploid neurons in higher vertebrates show only 4C DNA content. To explore the mechanism that prevents extra rounds of DNA synthesis in these latter cells we focused on the chick retina, where a population of tetraploid retinal ganglion cells (RGCs) has been described. We show that differentiating chick RGCs that express the neurotrophic receptors p75 and TrkB while lacking retinoblastoma protein, a feature of tetraploid RGCs, also express p27(Kip1). Two different short hairpin RNAs (shRNA) that significantly downregulate p27(Kip1) expression facilitated DNA synthesis and increased ploidy in isolated chick RGCs. Moreover, this forced DNA synthesis could not be prevented by Cdk4/6 inhibition, thus suggesting that it is triggered by a mechanism similar to endoreplication. In contrast, p27(Kip1) deficiency in mouse RGCs does not lead to increased ploidy despite previous observations have shown ectopic DNA synthesis in RGCs from p27(Kip1-/-) mice. This suggests that a differential mechanism is used for the regulation of neuronal endoreplication in mammalian versus avian RGCs. PMID:25946375

  4. Huntingtin with an expanded polyglutamine repeat affects the Jab1-p27(Kip1) pathway.

    PubMed

    Cong, S Y; Pepers, B A; Zhou, T T; Kerkdijk, H; Roos, R A; van Ommen, G J; Dorsman, J C

    2012-06-01

    Expansion of polyglutamine repeats is the cause of at least nine inherited human neurodegenerative disorders, including Huntington's disease (HD). It is widely accepted that deregulation of the transcriptional coactivator CBP by expanded huntingtin (htt) plays an important role in HD molecular pathogenesis. In this study, we report on a novel target of expanded polyglutamine stretches, the transcriptional coactivator Jun activation domain-binding protein 1 (Jab1), which shares DNA-sequence-specific transcription factor targets with CBP. Jab1 also plays a major role in the degradation of the cyclin-dependent-kinase inhibitor and putative transcription cofactor p27(Kip1). We found that Jab1 accumulates in aggregates when co-expressed with either expanded polyglutamine stretches or N-terminal fragments of mutant htt. In addition, the coactivator function of Jab1 was suppressed both by aggregated expanded polyglutamine solely and by mutant htt. Inhibition by mutant htt even preceded the appearance of microscopic aggregation. In an exon 1 HD cell model, we found that endogenous Jab1 could be recruited into aggregates and that this was accompanied by the accumulation of p27(Kip1). Accumulation of p27(Kip1) was also found in brains derived from HD patients. The repression of Jab1 by various mechanisms coupled with an increase of p27(Kip1) at late stages may have important transcriptional effects. In addition, the interference with the Jab1-p27(Kip1) pathway may contribute to the observed lower incidence of cancer in HD patients and may also be relevant for the understanding of the molecular pathogenesis of polyglutamine disorders in general. PMID:22426400

  5. Epidermal growth factor upregulates Skp2/Cks1 and p27kip1 in human extrahepatic cholangiocarcinoma cells

    PubMed Central

    Kim, Ja-yeon; Kim, Hong Joo; Park, Jung Ho; Park, Dong Il; Cho, Yong Kyun; Sohn, Chong Il; Jeon, Woo Kyu; Kim, Byung Ik; Kim, Dong Hoon; Chae, Seoung Wan; Sohn, Jin Hee

    2014-01-01

    AIM: To evaluate the expression status of S-phase kinase-associated protein 2 (Skp2)/cyclin-dependent kinases regulatory subunit 1 (Cks1) and p27kip1, and assess the prognostic significance of Skp2/Cks1 expression with p27kip1 in patients with extrahepatic cholangiocarcinoma. METHODS: Seventy-six patients who underwent curative resection for histologically confirmed extrahepatic cholangiocarcinoma at our institution from December 1994 to March 2008 were enrolled. Immunohistochemical staining for Skp2, Cks1, p27kip1, and Ki67, along with other relevant molecular biologic experiments, were performed. RESULTS: By Cox regression analyses, advanced age (> 65 years), advanced AJCC tumor stage, poorly differentiated histology, and higher immunostaining intensity of Skp2 were identified as independent prognostic factors in patients with extrahepatic cholangiocarcinoma. Exogenous epidermal growth factor (EGF, especially 0.1-10 ng/mL) significantly increased the proliferation indices by MTT assay and the mRNA levels of Skp2/Cks1 and p27kip1 in SNU-1196, SNU-1079, and SNU-245 cells. The protein levels of Skp2/Cks1 (from nuclear lysates) and p27kip1 (from cytosolic lysate) were also significantly increased in these cells. There were significant reductions in the protein levels of Skp2/Cks1 and p27kip1 (from nuclear lysate) after the treatment of LY294002. By chromatin immunoprecipitation assay, we found that E2F1 transcription factor directly binds to the promoter site of Skp2. CONCLUSION: Higher immunostaining intensity of Skp2/Cks1 was an independent prognostic factor for patients with extrahepatic cholangiocarcinoma. EGF upregulates the mRNA and protein levels of Skp2/Cks1 and p27kip1 via the PI3K/Akt pathway and direct binding of E2F1 transcription factor with the Skp2 promoter. PMID:24574749

  6. 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. PMID:27279267

  7. 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

  8. Histone Deacetylase Inhibitors Increase p27Kip1 by Affecting Its Ubiquitin-Dependent Degradation through Skp2 Downregulation

    PubMed Central

    Borriello, Adriana; Naviglio, Silvio; Bencivenga, Debora; Caldarelli, Ilaria; Tramontano, Annunziata; Speranza, Maria Carmela; Stampone, Emanuela; Sapio, Luigi; Negri, Aide; Oliva, Adriana; Sinisi, Antonio Agostino; Spina, Annamaria; Della Ragione, Fulvio

    2016-01-01

    Histone deacetylase inhibitors (HDACIs) represent an intriguing class of pharmacologically active compounds. Currently, some HDACIs are FDA approved for cancer therapy and many others are in clinical trials, showing important clinical activities at well tolerated doses. HDACIs also interfere with the aging process and are involved in the control of inflammation and oxidative stress. In vitro, HDACIs induce different cellular responses including growth arrest, differentiation, and apoptosis. Here, we evaluated the effects of HDACIs on p27Kip1, a key cyclin-dependent kinase inhibitor (CKI). We observed that HDACI-dependent antiproliferative activity is associated with p27Kip1 accumulation due to a reduced protein degradation. p27Kip1 removal requires a preliminary ubiquitination step due to the Skp2-SCF E3 ligase complex. We demonstrated that HDACIs increase p27Kip1 stability through downregulation of Skp2 protein levels. Skp2 decline is only partially due to a reduced Skp2 gene expression. Conversely, the protein decrease is more profound and enduring compared to the changes of Skp2 transcript. This argues for HDACIs effects on Skp2 protein posttranslational modifications and/or on its removal. In summary, we demonstrate that HDACIs increase p27Kip1 by hampering its nuclear ubiquitination/degradation. The findings might be of relevance in the phenotypic effects of these compounds, including their anticancer and aging-modulating activities. PMID:26682002

  9. 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

  10. 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

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

    PubMed

    Del Debbio, Carolina Beltrame; Mir, Qulsum; 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

  12. Reduced levels of the cell-cycle inhibitor p27Kip1 in epithelial dysplasia and carcinoma of the oral cavity.

    PubMed Central

    Jordan, R. C.; Bradley, G.; Slingerland, J.

    1998-01-01

    Recent studies have shown that the cyclin-dependent kinase (cdk) inhibitors play important roles in cell cycle progression in normal cells. Alterations in the cdk inhibitors also appear to be important in cancer development in a number of human tumors. p27Kip1 is a member of the CIP/KIP family of cdk inhibitors that negatively regulates cyclin-cdk complexes. Reduced levels of p27Kip1 protein have been identified in a number of human cancers, and in some cases reduced p27Kip1 is associated with an increase in proliferative fraction. In the present study, we examined p27Kip1 protein by immunohistochemistry in 10 normal and 36 dysplastic epithelia and in 8 squamous cell carcinomas from one anatomical site within the oral cavity, the floor of the mouth. Proliferative activity was assessed in serial sections by determining the expression of the cell cycle proteins Ki-67 and cyclin A. p27kip1 protein was significantly reduced in oral dysplasias and carcinomas compared with that in normal epithelial controls. In addition, there was a significant reduction in p27Kip1 protein between low- and high-grade dysplasias, suggesting that changes in p27Kip1 expression may be an early event in oral carcinogenesis. There was increasing expression of Ki-67 and cyclin A proteins with increasingly severe grades of dysplasia compared with normal controls. Although there was a strong correlation between Ki-67 and cyclin A scores (r2= 0.61) for all categories of disease, there was a weak negative correlation between Ki-67 and p27Kip1 levels (r2 = 0.29) and between cyclin A and p27Kip1 levels (r2 = 0.25). In conclusion, this study has found that a reduction in the proportion of cells expressing p27Kip1 protein is frequently associated with oral dysplasia and carcinoma from the floor of the mouth. Furthermore, reductions in p27Kip1 levels are associated with increased cell proliferation, although other changes likely contribute to altered cell kinetics during carcinogenesis at this site

  13. Depletion of p18/LAMTOR1 promotes cell survival via activation of p27(kip1) -dependent autophagy under starvation.

    PubMed

    Zada, Sahib; Noh, Hae Sook; Baek, Seon Mi; Ha, Ji Hye; Hahm, Jong Ryeal; Kim, Deok Ryong

    2015-11-01

    The MAPK and mTOR signal pathways in endosomes or lysosomes play a crucial role in cell survival and death. They are also closely associated with autophagy, a catabolic process highly regulated under various cellular stress or nutrient deprivation. Recently we have isolated a protein, named p18/LAMTOR1, that specifically regulates the ERK or mTOR pathway in lysosomes. p18/LAMTOR1 also interacts with p27(kip1) . Here we examined how p18/LAMTOR1 plays a role in autophagy under nutrient deprivation. The p18(+/+) MEF cells were more susceptible to cell death under starvation or in the presence of AICAR in comparison with p18(-/-) MEF cells. Cleavage of caspase-3 was increased in p18(+/+) MEF cells under starvation, and phosphorylation at the threonine 198 of p27(kip1) was highly elevated in starved p18(-/-) MEF cells. Furthermore, LC3-II formation and other autophagy-associated proteins were largely increased in p18-deficient cells, and suppression of p27(kip1) expression in p18(-/-) MEF cells mitigated starvation-induced cell death. These data suggest that ablation of p18/LAMTOR1 suppresses starvation-induced cell death by stimulating autophagy through modulation of p27(kip1) activity. PMID:26032166

  14. CPEB1 restrains proliferation of Glioblastoma cells through the regulation of p27Kip1 mRNA translation

    PubMed Central

    Galardi, Silvia; Petretich, Massimo; Pinna, Guillaume; D’Amico, Silvia; Loreni, Fabrizio; Michienzi, Alessandro; Groisman, Irina; Ciafrè, Silvia Anna

    2016-01-01

    The cytoplasmic element binding protein 1 (CPEB1) regulates many important biological processes ranging from cell cycle control to learning and memory formation, by controlling mRNA translation efficiency via 3′ untranslated regions (3′UTR). In the present study, we show that CPEB1 is significantly downregulated in human Glioblastoma Multiforme (GBM) tissues and that the restoration of its expression impairs glioma cell lines growth. We demonstrate that CPEB1 promotes the expression of the cell cycle inhibitor p27Kip1 by specifically targeting its 3′UTR, and competes with miR-221/222 binding at an overlapping site in the 3′UTR, thus impairing miR-221/222 inhibitory activity. Upon binding to p27Kip1 3′UTR, CPEB1 promotes elongation of poly-A tail and the subsequent translation of p27Kip1 mRNA. This leads to higher levels of p27Kip1 in the cell, in turn significantly inhibiting cell proliferation, and confers to CPEB1 a potential value as a tumor suppressor in Glioblastoma. PMID:27142352

  15. CPEB1 restrains proliferation of Glioblastoma cells through the regulation of p27(Kip1) mRNA translation.

    PubMed

    Galardi, Silvia; Petretich, Massimo; Pinna, Guillaume; D'Amico, Silvia; Loreni, Fabrizio; Michienzi, Alessandro; Groisman, Irina; Ciafrè, Silvia Anna

    2016-01-01

    The cytoplasmic element binding protein 1 (CPEB1) regulates many important biological processes ranging from cell cycle control to learning and memory formation, by controlling mRNA translation efficiency via 3' untranslated regions (3'UTR). In the present study, we show that CPEB1 is significantly downregulated in human Glioblastoma Multiforme (GBM) tissues and that the restoration of its expression impairs glioma cell lines growth. We demonstrate that CPEB1 promotes the expression of the cell cycle inhibitor p27(Kip1) by specifically targeting its 3'UTR, and competes with miR-221/222 binding at an overlapping site in the 3'UTR, thus impairing miR-221/222 inhibitory activity. Upon binding to p27(Kip1) 3'UTR, CPEB1 promotes elongation of poly-A tail and the subsequent translation of p27(Kip1) mRNA. This leads to higher levels of p27(Kip1) in the cell, in turn significantly inhibiting cell proliferation, and confers to CPEB1 a potential value as a tumor suppressor in Glioblastoma. PMID:27142352

  16. Embryonic Lethal Abnormal Vision-like HuR-dependent mRNA Stability Regulates Post-transcriptional Expression of Cyclin-dependent Kinase Inhibitor p27Kip1

    PubMed Central

    Ziegeler, Gudrun; Ming, Jie; Koseki, Jana C.; Sevinc, Sema; Chen, Ting; Ergun, Suleyman; Qin, Xuebin; Aktas, Bertal H.

    2010-01-01

    The cyclin-dependent kinase inhibitor p27Kip1 plays a critical role in regulating entry into and exit from the cell cycle. Post-transcriptional regulation of p27Kip1 expression is of significant interest. The embryonic lethal abnormal vision (ELAV)-like RNA-binding protein HuR is thought be important for the translation of p27Kip1, however, different reports attributed diametrically opposite roles to HuR. We report here an alternative mechanism wherein HuR regulates stability of the p27Kip1 mRNA. Specifically, human and mouse p27Kip1 mRNAs interact with HuR protein through multiple U-rich elements in both 5′ and 3′ untranslated regions (UTR). These interactions, which occur in vitro and in vivo, stabilize p27Kip1 mRNA and play a critical role in its accumulation. Deleting HuR binding sites or knocking down HuR expression destabilizes p27Kip1 mRNA and reduces its accumulation. We also identified a CT repeat in the 5′ UTR of full-length p27Kip1 mRNA isoforms that interact with a ∼41-kDa protein and represses p27Kip1 expression. This CT-rich element and diffuse elements in the 3′ UTR regulate post-transcriptional expression of p27Kip1 at the level of translation. This is the first demonstration that HuR-dependent mRNA stability and HuR-independent mRNA translation plays a critical role in the regulation of post-transcriptional p27Kip1 expression. PMID:20332085

  17. 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

  18. 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

  19. Regulation of the p27Kip1 tumor suppressor by miR-221 and miR-222 promotes cancer cell proliferation

    PubMed Central

    le Sage, Carlos; Nagel, Remco; Egan, David A; Schrier, Mariette; Mesman, Elly; Mangiola, Annunziato; Anile, Corrado; Maira, Giulio; Mercatelli, Neri; Ciafrè, Silvia Anna; Farace, Maria Giulia; Agami, Reuven

    2007-01-01

    MicroRNAs (miRNAs) are potent post-transcriptional regulators of protein coding genes. Patterns of misexpression of miRNAs in cancer suggest key functions of miRNAs in tumorigenesis. However, current bioinformatics tools do not entirely support the identification and characterization of the mode of action of such miRNAs. Here, we used a novel functional genetic approach and identified miR-221 and miR-222 (miR-221&222) as potent regulators of p27Kip1, a cell cycle inhibitor and tumor suppressor. Using miRNA inhibitors, we demonstrate that certain cancer cell lines require high activity of miR-221&222 to maintain low p27Kip1 levels and continuous proliferation. Interestingly, high levels of miR-221&222 appear in glioblastomas and correlate with low levels of p27Kip1 protein. Thus, deregulated expression of miR-221&222 promotes cancerous growth by inhibiting the expression of p27Kip1. PMID:17627278

  20. Involvement of p27KIP1 in G1 arrest mediated by an anti-epidermal growth factor receptor monoclonal antibody.

    PubMed

    Wu, X; Rubin, M; Fan, Z; DeBlasio, T; Soos, T; Koff, A; Mendelsohn, J

    1996-04-01

    Activation of the cyclin dependent kinases (CDK4/CDK6 and CDK2) is required for G1 phase progression and entry into S-phase. The activation of these kinases is regulated by checkpoints that monitor environmental and intracellular conditions. Progression into S-phase is controlled, in part, by the availability of growth factors, and we have investigated the relationship between growth factor availability and the activation of the CDK kinases. Blocking activation of epidermal growth factor (EGF) receptor tyrosine kinase with anti-EGF receptor monoclonal antibody (mAb) 225 induces G1 phase cell cycle arrest in DiFi human colon adenocarcinoma cells. When DiFi cells are treated with mAb 225 for 24 h, we observe marked decreases in the activities of CDK2 kinase and cyclin E-associated CDK kinase which are not accompanied by reduced levels of cyclin E and CDK2 proteins. However, the amount of cyclin/CDK kinase inhibitor p27KIP1 increases in the mAb-treated cells and p27KIP1 is bound to CDK2 in increasing amounts. Immunodepletion of p27KIP1 removes an inhibitory activity from lysates of mAb-treated cells: the immunodepleted and heated lysates lose the capacity to inhibit cyclin E/CDK2 activity in an in vitro assay. The results suggest that G1 arrest in the cell cycle induced by EGF receptor blockade involves p27KIP1. PMID:8622855

  1. 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. PMID:19264608

  2. PRMT5 Is Upregulated in Malignant and Metastatic Melanoma and Regulates Expression of MITF and p27Kip1

    PubMed Central

    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 p27Kip1. These results are the first to report elevated PRMT5 expression in human melanoma specimens and indicate this protein may regulate MITF and p27Kip1 expression in human melanoma cells. PMID:24098663

  3. A prostatic intraepithelial neoplasia-dependent p27kip1 checkpoint induces senescence, inhibits cell proliferation and cancer progression

    PubMed Central

    Majumder, Pradip K.; Grisanzio, Chiara; O’Connell, Fionnuala; Barry, Marc; Brito, Joseph M.; Xu, Qing; Guney, Isil; Berger, Raanan; Herman, Paula; Bikoff, Rachel; Fedele, Giuseppe; Baek, Won-Ki; Wang, Shunyou; Ellwood-Yen, Katharine; Wu, Hong; Sawyers, Charles L.; Signoretti, Sabina; Hahn, William C.; Loda, Massimo; Sellers, William R.

    2008-01-01

    SUMMARY Transgenic expression of activated AKT1 in the murine prostate induces Prostatic Intraepithelial Neoplasia (PIN) that does not progress to invasive prostate cancer (CaP). In luminal epithelial cells of Akt-driven PIN we show the concomitant induction of p27kip1 and senescence. Genetic ablation of p27Kip1 led to down regulation of senescence markers and progression to cancer. In humans, p27Kip1 and senescence markers were elevated in PIN not associated with CaP, but were decreased and absent, respectively in cancer-associated PIN and in CaP. Importantly, p27Kip1 up-regulation in mouse and human in situ lesions did not depend upon mTOR or Akt activation but was instead specifically associated with alterations in cellular polarity, architecture and adhesion molecules. These data suggest that a p27Kip1-driven checkpoint limits progression of PIN to CaP. PMID:18691549

  4. Rheb promotes cancer cell survival through p27Kip1-dependent activation of autophagy.

    PubMed

    Campos, Tania; Ziehe, Javiera; Palma, Mario; Escobar, David; Tapia, Julio C; Pincheira, Roxana; Castro, Ariel F

    2016-02-01

    We previously found that the small GTPase Rheb regulates the cell-cycle inhibitor p27KIP1 (p27) in colon cancer cells by a mTORC1-independent mechanism. However, the biological function of the Rheb/p27 axis in cancer cells remains unknown. Here, we show that siRNA-mediated depletion of Rheb decreases survival of human colon cancer cells under serum deprivation. As autophagy can support cell survival, we analyzed the effect of Rheb on this process by detecting the modification of the autophagy marker protein LC3 by western blot and imunofluorescence. We found that Rheb promotes autophagy in several human cancer cell lines under serum deprivation. Accordingly, blocking autophagy inhibited the pro-survival effect of Rheb in colon cancer cells. We then analyzed whether p27 was involved in the biological effect of Rheb. Depletion of p27 inhibited colon cancer cell survival, and Rheb induction of autophagy. These results suggest that p27 has an essential role in the effect of Rheb in response to serum deprivation. In addition, we demonstrated that the role of p27 in autophagy stands on the N-terminal portion of the protein, where the CDK-inhibitory domain is located. Our results indicate that a Rheb/p27 axis accounts for the activation of autophagy that supports cancer cell survival. Our work therefore highlights a biological function of Rheb and prompts the need for future studies to address whether the mTORC1-independent Rheb/p27 axis could contribute to tumorigenesis and/or resistance to mTOR inhibitors. PMID:25594310

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

    PubMed

    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

  6. Differential modulation of paclitaxel-mediated apoptosis by p21Waf1 and p27Kip1.

    PubMed

    Schmidt, M; Lu, Y; Liu, B; Fang, M; Mendelsohn, J; Fan, Z

    2000-05-11

    The impact of the cyclin dependent kinase (CDK) inhibitors p21Waf1 and p27Kip1 on paclitaxel-mediated cytotoxicity was investigated in RKO human colon adenocarcinoma cells with the ecdysone-inducible expression of p21Waf1 or p27Kip1. Ectopic expression of p27Kip1 arrested cells at G1 phase, whereas p21Waf1 expression arrested cells at G1 and G2. Expression of p21Waf1 after paclitaxel treatment produced much greater resistance to paclitaxel than did expression of p27Kip1. We attributed this difference to the additional block at G2 induced by p21Waf1, which prevented cells from entering M phase and becoming paclitaxel susceptible. Expression of p21Waf1 inhibited p34cdc2 activity and markedly reduced paclitaxel-mediated mitotic arrest, from 87.5 to 23%. In contrast, p27Kip1 expression also inhibited p34cdc2 but reduced mitotic arrest only slightly, from 87. 4 to 74.5%. We concluded that the G2 block produced by p21Waf1, but not by p27Kip1, contributed to their unequal modulation of sensitivity to paclitaxel-mediated apoptosis in RKO cells, and there is no causal relationship between paclitaxel-mediated cytotoxicity and elevation of p34cdc2 activity. PMID:10828884

  7. p27Kip1 expression as a prognostic marker for squamous cell carcinoma of the head and neck

    PubMed Central

    DE ALMEIDA, MIGUEL REIS; PÉREZ-SAYÁNS, MARIO; SUÁREZ-PEÑARANDA, JOSÉ MANUEL; SOMOZA-MARTÍN, JOSÉ MANUEL; GARCÍA-GARCÍA, ABEL

    2015-01-01

    Regulation of the cell cycle is essential for carcinogenesis. The cell cycle is controlled by cyclin-dependent kinases (CDKs), which are upregulated by cyclins and downregulated by CDK inhibitors (CDKIs). Decreased p27Kip1 expression has been associated with survival rate, tumor size, histological differentiation and the presence of lymph node metastasis in patients with various types of cancer. The aim of the current study is to provide a literature review on the association between p27Kip1 expression and the clinical and pathological aspects of head and neck squamous cell carcinoma (HNSCC), and the expression of other CDKIs of the Cip/Kip family and cyclins. Throughout the literature, different methodologies were used to determine the immunohistochemical expression of p27Kip1; thus, results concerning p27Kip1 expression in HNSCC vary widely. However, it has now been confirmed that p27Kip1 is underexpressed in SCC cells. p27 may be a promising marker for determining the prognosis of HNSCC, despite the marked variability of the results obtained. An association between p27 expression and survival rate, time to recurrence and tumor stage has been observed. Based on the information currently available, it is premature to recommend the analysis of p27Kip1 expression in guiding HNSCC treatment planning. However, although relatively unstudied, the correlation between p27Kip1 expression and other tumor suppressor genes may turn out to be important in determining the prognosis of HNSCC. Further prospective studies utilizing standardized laboratory methodologies and statistics that facilitate meta-analyses are required to confirm this proposal. PMID:26722226

  8. 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. PMID:25498792

  9. NSun2 delays replicative senescence by repressing p27 (KIP1) translation and elevating CDK1 translation

    PubMed Central

    Xing, Junyue; Liu, Zhenyun; Jiang, Bin; Dou, Yali; Gorospe, Myriam; Wang, Wengong

    2015-01-01

    A rise in the levels of the cyclin-dependent kinase (CDK) inhibitor p27KIP1 is important for the growth arrest of senescent cells, but the mechanisms responsible for this increase are poorly understood. Here, we show that the tRNA methyltransferase NSun2 represses the expression of p27 in replicative senescence. NSun2 methylated the 5′-untranslated region (UTR) of p27 mRNA at cytosine C64 in vitro and in cells, thereby repressing the translation of p27. During replicative senescence, increased p27 protein levels were accompanied by decreased NSun2 protein levels. Knockdown of NSun2 in human diploid fibroblasts (HDFs) elevated p27 levels and reduced the expression of CDK1 (encoded by CDK1 mRNA, a previously reported target of NSun2), which in turn further repressed cell proliferation and accelerated replicative senescence, while overexpression of NSun2 exerted the opposite effect. Ectopic overexpression of the p27 5′UTR fragment rescued the effect of NSun2 overexpression in lowering p27, increasing CDK1, promoting cell proliferation, and delaying replicative senescence. Our findings indicate that NSun2-mediated mRNA methylation regulates p27 and CDK1 levels during replicative senescence. PMID:26687548

  10. A new tumour suppression mechanism by p27Kip1: EGFR down-regulation mediated by JNK/c-Jun pathway inhibition

    PubMed Central

    Fang, Yong; Wang, Yihong; Wang, Yulei; Meng, Yan; Zhu, Junlan; Jin, Honglei; Li, Jingxia; Zhang, Dongyun; Yu, Yonghui; Wu, Xue-Ru; Huang, Chuanshu

    2014-01-01

    p27Kip1 is a potent inhibitor of cyclin-dependent kinases that drive G1-to-S cell-cycle transition. Reduced p27Kip1 expression is prevalent in a wide range of human tumours; however, the exact mechanism(s) of p27Kip1-mediated tumour suppression remains obscure. In the present study, we identified a close inverse relationship between p27Kip1 and EGFR (epidermal growth factor receptor) expression: the parental T24 human bladder cancer cells had high p27Kip1 expression but low EGFR expression and, in striking contrast, the metastatic derivative of T24 (T24T) had low p27Kip1 expression but high EGFR expression. This relationship was also found in various human cancer tissues, and was not only just correlative but also causal; depletion of p27Kip1 in MEF (mouse embryonic fibroblast) cells resulted in markedly elevated EGFR expression, a result reproducible with an Egfr promoter-luciferase reporter in both T24 and MEF cells, suggesting transcriptional repression of EGFR by p27Kip1. Indeed, p27Kip1 was found to regulate EGFR expression via the JNK (c-Jun N-terminal kinase)/c-Jun transcription factor: p27Kip1 deficiency activated JNK/c-Jun, whereas inhibition of JNK/c-Jun by dominant-negative mutants dramatically repressed Egfr transcription. Furthermore, the proximal promoter of the Egfr gene was crucial for its transcription, where the recruiting activity of c-Jun was much greater in p27Kip1−/− cells than in p27Kip1+/+ cells. Introduction of GFP–p27Kip1 into T24T cells suppressed JNK/c-Jun activation, EGFR expression and anchorage-independent growth. The results of the present study demonstrate that p27Kip1 suppresses JNK/c-Jun activation and EGFR expression in MEFs and human bladder cancer cells, and the results obtained are consistent with those from human cancer specimens. The present study provides new insights into p27Kip1 suppression of cancer cell growth, migration and metastasis. PMID:25121353

  11. MYC antagonizes the differentiation induced by imatinib in chronic myeloid leukemia cells through downregulation of p27(KIP1.).

    PubMed

    Gómez-Casares, M T; García-Alegria, E; López-Jorge, C E; Ferrándiz, N; Blanco, R; Alvarez, S; Vaqué, J P; Bretones, G; Caraballo, J M; Sánchez-Bailón, P; Delgado, M D; Martín-Perez, J; Cigudosa, J C; León, J

    2013-04-25

    Chronic myeloid leukemia (CML) progresses from a chronic to a blastic phase where the leukemic cells are proliferative and undifferentiated. The CML is nowadays successfully treated with BCR-ABL kinase inhibitors as imatinib and dasatinib. In the CML-derived K562 cell line, low concentrations of imatinib induce proliferative arrest and erythroid differentiation. We found that imatinib upregulated the cell cycle inhibitor p27(KIP1) (p27) in a time- and -concentration dependent manner, and that the extent of imatinib-mediated differentiation was severely decreased in cells with depleted p27. MYC (c-Myc) is a transcription factor frequently deregulated in human cancer. MYC is overexpressed in untreated CML and is associated to poor response to imatinib. Using K562 sublines with conditional MYC expression (induced by Zn(2+) or activated by 4-hydroxy-tamoxifen) we show that MYC prevented the erythroid differentiation induced by imatinib and dasatinib. The differentiation inhibition is not due to increased proliferation of MYC-expressing clones or enhanced apoptosis of differentiated cells. As p27 overexpression is reported to induce erythroid differentiation in K562, we explored the effect of MYC on imatinib-dependent induction of p27. We show that MYC abrogated the imatinib-induced upregulation of p27 concomitantly with the differentiation inhibition, suggesting that MYC inhibits differentiation by antagonizing the imatinib-mediated upregulation of p27. This effect occurs mainly by p27 protein destabilization. This was in part due to MYC-dependent induction of SKP2, a component of the ubiquitin ligase complex that targets p27 for degradation. The results suggest that, although MYC deregulation does not directly confer resistance to imatinib, it might be a factor that contributes to progression of CML through the inhibition of differentiation. PMID:22710719

  12. eIF3a improve cisplatin sensitivity in ovarian cancer by regulating XPC and p27Kip1 translation

    PubMed Central

    Zhang, Yu; Yu, Jing-Jing; Tian, Yan; Li, Zheng-Zheng; Zhang, Cai-Yi; Zhang, Shu-Fen; Cao, Lan-Qin; Zhang, Yi; Qian, Chen-Yue; Zhang, Wei; Zhou, Hong-Hao; Yin, Ji-Ye; Liu, Zhao-Qian

    2015-01-01

    The eukaryotic translation initiation factor 3a (eIF3a) is one of the core subunits of the translation initiation complex eIF3, responsible for ribosomal subunit joining and mRNA recruitment to the ribosome. Our previous study identified that it was correlated with platinum response in lung cancer. The current study aims to test the hypothesis that eIF3a may affect the drug response and prognosis of ovarian cancer patients receiving platinum-based chemotherapy by regulating xeroderma pigmentosum complementation group C (XPC) and p27Kip1. Immunohistochemistry and western blot was used to determine the expression of eIF3a in 126 human ovarian cancer tissues followed by association analysis of eIF3a expression with patient's response and survival. Ectopic over-expression and RNA interference knockdown of eIF3a were carried out in A2780/cisplatin (DDP) and its parental A2780 cells, respectively, to determine the effect of altered eIF3a expression on cellular response to cisplatin by employing MTT assay. Western Blot analyses were also carried out to determine the regulation of eIF3a on XPC and p27Kip1. eIF3a expression was associated with response of ovarian cancer patients to DDP-based chemotherapy and their survival. Overexpression and knockdown of eIF3a increased and decreased the cellular response to cisplatin in A2780/DDP and A2780 cells, respectively. In addition, XPC and p27Kip1 were down regulated by eIF3a. eIF3a improves ovarian cancer patients' response to DDP-based chemotherapy via down regulating XPC and p27Kip1. PMID:26213845

  13. A single-nucleotide polymorphism in the human p27kip1 gene (-838C>A) affects basal promoter activity and the risk of myocardial infarction

    PubMed Central

    González, Pelayo; Díez-Juan, Antonio; Coto, Eliecer; Álvarez, Victoria; Reguero, Julian R; Batalla, Alberto; Andrés, Vicente

    2004-01-01

    Background Excessive proliferation of vascular smooth muscle cells and leukocytes within the artery wall is a major event in the development of atherosclerosis. The growth suppressor p27kip1 associates with several cyclin-dependent kinase/cyclin complexes, thereby abrogating their capacity to induce progression through the cell cycle. Recent studies have implicated p27kip1 in the control of neointimal hyperplasia. For instance, p27kip1 ablation in apolipoprotein-E-null mice enhanced arterial cell proliferation and accelerated atherogenesis induced by dietary cholesterol. Therefore, p27kip1 is a candidate gene to modify the risk of developing atherosclerosis and associated ischaemic events (i.e., myocardial infarction and stroke). Results In this study we found three common single-nucleotide polymorphisms in the human p27kip1 gene (+326T>G [V109G], -79C>T, and -838C>A). The frequency of -838A carriers was significantly increased in myocardial infarction patients compared to healthy controls (odds ratio [OR] = 1.73, 95% confidence interval [95%CI] = 1.12–2.70). In addition, luciferase reporter constructs driven by the human p27kip1 gene promoter containing A at position -838 had decreased basal transcriptional activity when transiently transfected in Jurkat cells, compared with constructs bearing C in -838 (P = 0.04). Conclusions These data suggest that -838A is associated with reduced p27kip1 promoter activity and increased risk of myocardial infarction. PMID:15061869

  14. Differential roles of p21(Waf1) and p27(Kip1) in modulating chemosensitivity and their possible application in drug discovery studies.

    PubMed

    Schmidt, M; Lu, Y; Parant, J M; Lozano, G; Bacher, G; Beckers, T; Fan, Z

    2001-11-01

    In this study, the differential role of the cyclin-dependent kinase (CDK) inhibitors p21(Waf1) and p27(Kip1) in cell cycle regulation was proposed for use in screening natural or synthetic compounds for cell cycle-dependent (particularly M phase-dependent) antineoplastic activity. p21(Waf1) or p27(Kip1) was ectopically expressed with an ecdysone-inducible mammalian expression system in a human colon adenocarcinoma cell line. Induction of p21(Waf1) or p27(Kip1) expression inhibited the activities of CDK2 and completely arrested cells at G(1) phase of the cell cycle by p27(Kip1) and at G(1) and G(2) phases by p21(Waf1). We examined the sensitivity of these cells to several antineoplastic agents known to be cell cycle-dependent or -independent. Substantially increased resistance to cell cycle-dependent antineoplastic agents was found in the cells when the expression of p21(Waf1) or p27(Kip1) was induced. In contrast, only a desensitization to cell cycle-independent antineoplastic agents was found in the cells arrested by p21(Waf1) or p27(Kip1). Because p21(Waf1) induces an additional block at G(2) phase that inhibits cell entry into M phase, we further examined the difference between p21(Waf1)- and p27(Kip1)-induced cells in their sensitivity to D-24851, a novel M phase-dependent compound. We found that induction of p21(Waf1) after exposure of the cells to D-24851 conferred stronger resistance than did induction of p27(Kip1). Taken together, our results suggest that the differential effect of p21(Waf1) and p27(Kip1) on cell cycle regulation may be advantageous for screening chemical libraries for novel antineoplastic candidates that are cell cycle-dependent, and M phase-dependent in particular. PMID:11641417

  15. Knockdown of CRM1 inhibits the nuclear export of p27(Kip1) phosphorylated at serine 10 and plays a role in the pathogenesis of epithelial ovarian cancer.

    PubMed

    Wang, You; Wang, Yingying; Xiang, Jingying; Ji, Fang; Deng, Yan; Tang, Chunhui; Yang, Shuyun; Xi, Qinghua; Liu, Rong; Di, Wen

    2014-02-01

    In a previous study, the nuclear export protein chromosomal region maintenance (CRM1) was correlated with p27(Kip1) in glioma. The aims of the present study were to investigate the expression of CRM1 and pSer10p27 and their functional roles in epithelial ovarian cancer (EOC) tissues. Using immunohistochemical analysis, CRM1 and pSer10p27 expression levels were shown to be associated with histologic stage and grade (P<0.05). High CRM1 and pSer10p27 expression levels were prognostic indicators of overall survival (P<0.05). Knockdown of CRM1 and pSer10p27 expression arrested cell cycle progression and inhibited the proliferation of SKOV3 cells both in vitro and in vivo. These data support the idea that pSer10p27 and CRM1 play cooperative roles in EOC. PMID:24018641

  16. Ras activity late in G1 phase required for p27kip1 downregulation, passage through the restriction point, and entry into S phase in growth factor-stimulated NIH 3T3 fibroblasts.

    PubMed Central

    Takuwa, N; Takuwa, Y

    1997-01-01

    It is well documented that Ras functions as a molecular switch for reentry into the cell cycle at the border between G0 and G1 by transducing extracellular growth stimuli into early G1 mitogenic signals. In the present study, we investigated the role of Ras during the late stage of the G1 phase by using NIH 3T3 (M17) fibroblasts in which the expression of a dominant negative Ras mutant, p21(Ha-Ras[Asn17]), is induced in response to dexamethasone treatment. We found that delaying the expression of Ras(Asn17) until late in the G1 phase by introducing dexamethasone 3 h after the addition of epidermal growth factor (EGF) abolished the downregulation of the p27kip1 cyclin-dependent kinase (CDK) inhibitor which normally occurred during this period, with resultant suppression of cyclin Ds/CDK4 and cyclin E/CDK2 and G1 arrest. The immunodepletion of p27kip1 completely eliminated the CDK inhibitor activity from EGF-stimulated, dexamethasone-treated cell lysate. The failure of p27kip1 downregulation and G1 arrest was also observed in cells in which Ras(Asn17) was induced after growth stimulation with a phorbol ester or alpha-thrombin and was mimicked by the addition late in the G1 phase of inhibitors for phosphatidylinositol-3-kinase. Ras-mediated downregulation of p27kip1 involved both the suppression of synthesis and the stimulation of the degradation of the protein. Unlike the earlier expression of Ras(Asn17) at the border between G0 and G1, its delayed expression did not compromise the EGF-stimulated transient activation of extracellular signal-regulated kinases or inhibit the stimulated expression of a principal D-type cyclin, cyclin D1, until close to the border between G1 and S. We conclude that Ras plays temporally distinct, phase-specific roles throughout the G1 phase and that Ras function late in G1 is required for p27kip1 downregulation and passage through the restriction point, a prerequisite for entry into the S phase. PMID:9271412

  17. 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 Central

    Su, Liang-Cheng; Jiang, Shih Sheng; Chan, Tzu-Min; Chang, Chung-Ho; Chen, Li-Tzong; Kung, Hsing-Jien; Wang, Horng-Dar; Chuu, Chih-Pin

    2015-01-01

    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. PMID:25788262

  18. Cell cycle arrest in Metformin treated breast cancer cells involves activation of AMPK, downregulation of cyclin D1, and requires p27Kip1 or p21Cip1

    PubMed Central

    Zhuang, Yongxian; Miskimins, W Keith

    2008-01-01

    Background The antihyperglycemic drug metformin may have beneficial effects on the prevention and treatment of cancer. Metformin is known to activate AMP-activated protein kinase (AMPK). It has also been shown to inhibit cyclin D1 expression and proliferation of some cultured cancer cells. However, the mechanisms of action by which metformin mediates cell cycle arrest are not completely understood. Results In this study, metformin was found to inhibit proliferation of most cultured breast cancer cell lines. This was independent of estrogen receptor, HER2, or p53 status. Inhibition of cell proliferation was associated with arrest within G0/G1 phase of the cell cycle. As in previous studies, metformin treatment led to activation of (AMPK) and downregulation of cyclin D1. However, these events were not sufficient for cell cycle arrest because they were also observed in the MDA-MB-231 cell line, which is not sensitive to growth arrest by metformin. In sensitive breast cancer lines, the reduction in cyclin D1 led to release of sequestered CDK inhibitors, p27Kip1 and p21Cip1, and association of these inhibitors with cyclin E/CDK2 complexes. The metformin-resistant cell line MDA-MB-231 expresses significantly lower levels of p27Kip1 and p21Cip1 than the metformin-sensitive cell line, MCF7. When p27Kip1 or p21Cip1 were overexpressed in MDA-MB-231, the cells became sensitive to cell cycle arrest in response to metformin. Conclusion Cell cycle arrest in response to metformin requires CDK inhibitors in addition to AMPK activation and cyclin D1 downregulation. This is of interest because many cancers are associated with loss or downregulation of CDK inhibitors and the results may be relevant to the development of anti-tumor reagents that target the AMPK pathway. PMID:19046439

  19. FGFR1 signaling stimulates proliferation of human mesenchymal stem cells by inhibiting the cyclin-dependent kinase inhibitors p21(Waf1) and p27(Kip1).

    PubMed

    Dombrowski, Christian; Helledie, Torben; Ling, Ling; Grünert, Martin; Canning, Claire A; Jones, C Michael; Hui, James H; Nurcombe, Victor; van Wijnen, Andre J; Cool, Simon M

    2013-12-01

    Signaling through fibroblast growth factor receptor one (FGFR1) is a known inducer of proliferation in both embryonic and human adult mesenchymal stem cells (hMSCs) and positively regulates maintenance of stem cell viability. Leveraging the mitogenic potential of FGF2/FGFR1 signaling in stem cells for therapeutic applications necessitates a mechanistic understanding of how this receptor stimulates cell cycle progression. Using small interfering RNA (siRNA) depletion, antibody-inhibition, and small molecule inhibition, we establish that FGFR1 activity is rate limiting for self-renewal of hMSCs. We show that FGFR1 promotes stem cell proliferation through multiple mechanisms that unite to antagonize cyclin-dependent kinase (CDK) inhibitors. FGFR1 not only stimulates c-Myc to suppress transcription of the CDK inhibitors p21(Waf1) and p27(Kip1), thus promoting cell cycle progression but also increases the activity of protein kinase B (AKT) and the level of S-phase kinase-associated protein 2 (Skp2), resulting in the nuclear exclusion and reduction of p21(Waf1). The in vivo importance of FGFR1 signaling for the control of proliferation in mesenchymal progenitor populations is underscored by defects in ventral mesoderm formation during development upon inhibition of its signaling. Collectively, these studies demonstrate that FGFR1 signaling mediates the continuation of MSC growth and establishes a receptor target for enhancing the expansion of mesenchymal progenitors while maintaining their multilineage potential. PMID:23939995

  20. 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

  1. HBx-upregulated lncRNA UCA1 promotes cell growth and tumorigenesis by recruiting EZH2 and repressing p27Kip1/CDK2 signaling

    PubMed Central

    Hu, Jiao-Jiao; Song, Wei; Zhang, Shao-Dan; Shen, Xiao-Hui; Qiu, Xue-Mei; Wu, Hua-Zhang; Gong, Pi-Hai; Lu, Sen; Zhao, Zhu-Jiang; He, Ming-Liang; Fan, Hong

    2016-01-01

    It is well accepted that HBx plays the major role in hepatocarcinogenesis associated with hepatitis B virus (HBV) infections. However, little was known about its role in regulating long noncoding RNAs (lncRNAs), a large group of transcripts regulating a variety of biological processes including carcinogenesis in mammalian cells. Here we report that HBx upregulates UCA1 genes and downregulates p27 genes in hepatic LO2 cells. Further studies show that the upregulated UCA1 promotes cell growth by facilitating G1/S transition through CDK2 in both hepatic and hepatoma cells. Knock down of UCA1 in HBx-expressing hepatic and hepatoma cells resulted in markedly increased apoptotic cells by elevating the cleaved caspase-3 and caspase-8. More importantly, UCA1 is found to be physically associated with enhancer of zeste homolog 2 (EZH2), which suppresses p27Kip1 through histone methylation (H3K27me3) on p27Kip1 promoter. We also show that knockdown of UCA1 in hepatoma cells inhibits tumorigenesis in nude mice. In a clinic study, UCA1 is found to be frequently up-regulated in HBx positive group tissues in comparison with the HBx negative group, and exhibits an inverse correlation between UCA1 and p27Kip1 levels. Our findings demonstrate an important mechanism of hepatocarcinogenesis through the signaling of HBx-UCA1/EZH2-p27Kip1 axis, and a potential target of HCC. PMID:27009634

  2. HBx-upregulated lncRNA UCA1 promotes cell growth and tumorigenesis by recruiting EZH2 and repressing p27Kip1/CDK2 signaling.

    PubMed

    Hu, Jiao-Jiao; Song, Wei; Zhang, Shao-Dan; Shen, Xiao-Hui; Qiu, Xue-Mei; Wu, Hua-Zhang; Gong, Pi-Hai; Lu, Sen; Zhao, Zhu-Jiang; He, Ming-Liang; Fan, Hong

    2016-01-01

    It is well accepted that HBx plays the major role in hepatocarcinogenesis associated with hepatitis B virus (HBV) infections. However, little was known about its role in regulating long noncoding RNAs (lncRNAs), a large group of transcripts regulating a variety of biological processes including carcinogenesis in mammalian cells. Here we report that HBx upregulates UCA1 genes and downregulates p27 genes in hepatic LO2 cells. Further studies show that the upregulated UCA1 promotes cell growth by facilitating G1/S transition through CDK2 in both hepatic and hepatoma cells. Knock down of UCA1 in HBx-expressing hepatic and hepatoma cells resulted in markedly increased apoptotic cells by elevating the cleaved caspase-3 and caspase-8. More importantly, UCA1 is found to be physically associated with enhancer of zeste homolog 2 (EZH2), which suppresses p27Kip1 through histone methylation (H3K27me3) on p27Kip1 promoter. We also show that knockdown of UCA1 in hepatoma cells inhibits tumorigenesis in nude mice. In a clinic study, UCA1 is found to be frequently up-regulated in HBx positive group tissues in comparison with the HBx negative group, and exhibits an inverse correlation between UCA1 and p27Kip1 levels. Our findings demonstrate an important mechanism of hepatocarcinogenesis through the signaling of HBx-UCA1/EZH2-p27Kip1 axis, and a potential target of HCC. PMID:27009634

  3. Upregulation of p27Kip1 by demethylation sensitizes cisplatin-resistant human ovarian cancer SKOV3 cells.

    PubMed

    Zhao, Yan; Li, Qiaoyan; Wu, Xiaoying; Chen, Puxiang

    2016-08-01

    Ovarian cancer has a poor prognosis due to its chemoresistance, and p27Kip1 (p27) has been implicated in tumor prognosis and drug-resistance. However, the regulatory mechanisms of p27 in drug‑resistance in ovarian cancer remain unknown. The current study successfully established chemoresistant cell lines using paclitaxel (TAX), cisplatin (DDP) and carboplatin (CBP) in SKOV3 ovarian cancer cells. The results indicated that the expression levels of p27 were dramatically downregulated in chemoresistant cells. However, 5-aza-2'-deoxycytidine (5-aza) treatment restored p27 expression in DDP-resistant cells, and increased their sensitivity to DDP. In addition, it was observed that the methylation of DDP‑resistant cells, which was downregulated by 5‑aza treatment, was significantly higher compared with SKOV3 cells. Additionally, the overexpression of p27 arrested the cell cycle in S phase and promoted an apoptotic response to DDP. In conclusion, p27 was involved in chemoresistance of SKOV3 cells. Upregulated p27 expression induced by demethylation may enhance sensitivity to DDP through the regulation of the cell cycle. PMID:27314502

  4. The MLL fusion gene, MLL-AF4, regulates cyclin-dependent kinase inhibitor CDKN1B (p27kip1) expression

    PubMed Central

    Xia, Zhen-Biao; Popovic, Relja; Chen, Jing; Theisler, Catherine; Stuart, Tara; Santillan, Donna A.; Erfurth, Frank; Diaz, Manuel O.; Zeleznik-Le, Nancy J.

    2005-01-01

    MLL, involved in many chromosomal translocations associated with acute myeloid and lymphoid leukemia, has >50 known partner genes with which it is able to form in-frame fusions. Characterizing important downstream target genes of MLL and of MLL fusion proteins may provide rational therapeutic strategies for the treatment of MLL-associated leukemia. We explored downstream target genes of the most prevalent MLL fusion protein, MLL-AF4. To this end, we developed inducible MLL-AF4 fusion cell lines in different backgrounds. Overexpression of MLL-AF4 does not lead to increased proliferation in either cell line, but rather, cell growth was slowed compared with similar cell lines inducibly expressing truncated MLL. We found that in the MLL-AF4-induced cell lines, the expression of the cyclin-dependent kinase inhibitor gene CDKN1B was dramatically changed at both the RNA and protein (p27kip1) levels. In contrast, the expression levels of CDKN1A (p21) and CDKN2A (p16) were unchanged. To explore whether CDKN1B might be a direct target of MLL and of MLL-AF4, we used chromatin immunoprecipitation (ChIP) assays and luciferase reporter gene assays. MLL-AF4 binds to the CDKN1B promoter in vivo and regulates CDKN1B promoter activity. Further, we confirmed CDKN1B promoter binding by ChIP in MLL-AF4 as well as in MLL-AF9 leukemia cell lines. Our results suggest that CDKN1B is a downstream target of MLL and of MLL-AF4, and that, depending on the background cell type, MLL-AF4 inhibits or activates CDKN1B expression. This finding may have implications in terms of leukemia stem cell resistance to chemotherapy in MLL-AF4 leukemias. PMID:16169901

  5. 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

  6. SKP2 oncogene is a direct MYC target gene and MYC down-regulates p27(KIP1) through SKP2 in human leukemia cells.

    PubMed

    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

    2011-03-18

    SKP2 is the ubiquitin ligase subunit that targets p27(KIP1) (p27) for degradation. SKP2 is induced in the G(1)-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

  7. Down-regulation of both p21/Cip1 and p27/Kip1 produces a more aggressive prostate cancer phenotype

    PubMed Central

    Roy, Srirupa; Singh, Rana P.; Agarwal, Chapla; Siriwardana, Sunitha; Sclafani, Robert; Agarwal, Rajesh

    2009-01-01

    Roles of cyclin dependent kinase inhibitors, p21/Cip1 (p21) and p27/Kip1 (p27) in prostate cancer (PCa) progression is still not clear. Lower p27 protein expression in PCa tissues is often associated with poor prognosis, but prognostic significance of p21 is still controversial. Herein, we investigated the role of these molecules in determining PCa growth characteristics. We generated human PCa DU145 cell variants with knocked down levels of p21 (DU-p21) or p27 (DU-p27), or both (DU-p21+p27) via retroviral transduction of respective shRNAs and compared their various characteristics with empty vector-transduced DU145 (DU-EV) cells in vitro as well as in vivo. Knocking down either p21 or p27 did not show any significant change in doubling time, clonogenicity and cell cycle progression in DU145 cells, but simultaneous knock-down of both p21 and p27 significantly enhanced these parameters. In athymic mice, DU-p21+p27 tumors showed higher growth rate than the comparable growth of DU-EV, DU-p21 and DU-p27 tumors. Concurrently, DU-p21+p27 tumors had significantly higher proliferation rate, showing 54% and 48% increase in proliferating cell nuclear antigen (PCNA) and Ki-67-positive cells, respectively, compared to DU-EV tumors. DU-p21+p27 tumors also showed higher microvessel density and increased expression of vascular endothelial growth factor (VEGF). Proliferation and angiogenic status of DU-p21 and DU-p27 tumors was comparable to DU-EV tumors. Both in vitro and in vivo results implicate that p21 and p27 have compensatory roles in advanced prostate cancer cells, and ablation or down-modulation of both these molecules essentially enhances the aggressive prostate carcinoma phenotype. PMID:18583941

  8. 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

    2016-04-01

    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. PMID:26963853

  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. PMID:27282281

  10. A novel miRNA-mediated STOP sign in lung cancer: miR-340 inhibits the proliferation of lung cancer cells through p27KIP1

    PubMed Central

    Fernandez, Serena; Risolino, Maurizio; Verde, Pasquale

    2015-01-01

    Oncosuppressor miRNAs inhibit cancer cell proliferation by targeting key components of the cell cycle machinery. In our recent report we showed that miR-340 is a novel tumor suppressor in non-small cell lung cancer. miR-340 inhibits neoplastic cell proliferation and induces p27KIP1 by targeting multiple translational and post-translational regulators of this cyclin-dependent kinase inhibitor. PMID:27308439

  11. Cooperation of p27Kip1 and p18INK4c in Progestin-Mediated Cell Cycle Arrest in T-47D Breast Cancer Cells

    PubMed Central

    Swarbrick, Alexander; Lee, Christine S. L.; Sutherland, Robert L.; Musgrove, Elizabeth A.

    2000-01-01

    The steroid hormone progesterone regulates proliferation and differentiation in the mammary gland and uterus by cell cycle phase-specific actions. The long-term effect of progestins on T-47D breast cancer cells is inhibition of cellular proliferation. This is accompanied by decreased G1 cyclin-dependent kinase (CDK) activities, redistribution of the CDK inhibitor p27Kip1 among these CDK complexes, and alterations in the elution profile of cyclin E-Cdk2 upon gel filtration chromatography, such that high-molecular-weight complexes predominate. This study aimed to determine the relative contribution of CDK inhibitors to these events. Following progestin treatment, the majority of cyclin E- and D-CDK complexes were bound to p27Kip1 and few were bound to p21Cip1. In vitro, recombinant His6-p27 could quantitatively reproduce the effects on cyclin E-Cdk2 kinase activity and the shift in molecular weight observed following progestin treatment. In contrast, cyclin D-Cdk4 was not inhibited by His6-p27 in vitro or p27Kip1 in vivo. However, an increase in the expression of the Cdk4/6 inhibitor p18INK4c and its extensive association with Cdk4 and Cdk6 were apparent following progestin treatment. Recombinant p18INK4c led to the reassortment of cyclin-CDK-CDK inhibitor complexes in vitro, with consequent decrease in cyclin E-Cdk2 activity. These results suggest a concerted model of progestin action whereby p27Kip1 and p18INK4c cooperate to inhibit cyclin E-Cdk2 and Cdk4. Since similar models have been developed for growth inhibition by transforming growth factor β and during adipogenesis, interaction between the Cip/Kip and INK4 families of inhibitors may be a common theme in physiological growth arrest and differentiation. PMID:10713180

  12. Cell-permeable Carboxyl-terminal p27Kip1 Peptide Exhibits Anti-tumor Activity by Inhibiting Pim-1 Kinase*

    PubMed Central

    Morishita, Daisuke; Takami, Miho; Yoshikawa, Seiko; Katayama, Ryohei; Sato, Shigeo; Kukimoto-Niino, Mutsuko; Umehara, Takashi; Shirouzu, Mikako; Sekimizu, Kazuhisa; Yokoyama, Shigeyuki; Fujita, Naoya

    2011-01-01

    The incidence and death rate of prostate cancer is increasing rapidly. In addition, the low sensitivity of prostate cancer to chemotherapy makes it difficult to treat this condition. The serine/threonine kinase Pim-1 plays an important role in cell cycle progression and apoptosis inhibition, resulting in prostate tumorigenesis. Therefore, Pim-1 inhibition has been expected to be an attractive target for developing new anti-cancer drugs. However, no small compounds targeting Pim-1 have progressed to clinical use because of their lack of specificity. Here, we have reported a new cell-permeable Pim-1 inhibitory p27Kip1 peptide that could interfere with the binding of Pim-1 to its substrates and act as an anti-cancer drug. The peptide could bind to Pim-1 and inhibit phosphorylation of endogenous p27Kip1 and Bad by Pim-1. Treatment of prostate cancer with the peptide induces G1 arrest and subsequently apoptosis in vitro. However, the peptide showed almost no growth inhibitory or apoptosis-inducing effects in normal cells. The peptide could inhibit tumor growth in in vivo prostate cancer xenograft models. Moreover, the peptide treatment could overcome resistance to taxol, one of the first line chemotherapeutic agents for prostate cancer, and a combination of the peptide with taxol synergistically inhibited prostate cancer growth in vivo. These results indicate that a Pim-1 inhibitory p27Kip1 peptide could be developed as an anti-cancer drug against prostate cancer. PMID:21062737

  13. Augmentation of antitumor activity of 5-fluorouracil by interferon alpha is associated with up-regulation of p27Kip1 in human hepatocellular carcinoma cells.

    PubMed

    Eguchi, H; Nagano, H; Yamamoto, H; Miyamoto, A; Kondo, M; Dono, K; Nakamori, S; Umeshita, K; Sakon, M; Monden, M

    2000-07-01

    Several clinical trials have demonstrated the effectiveness of combination therapy with 5-fluorouracil (5-FU) and IFN-alpha in colon cancer, hepatocellular carcinoma (HCC), and other malignancies. In our preliminary clinical studies, we have observed outstanding effects with this combination therapy in patients with advanced HCC. However, the underlying mechanism by which IFN-alpha modulates the effects of 5-FU is unknown. We, therefore, conducted a mechanistic study using two HCC cell lines, PLC/PRF/5 and HuH7. IFN-alpha significantly enhanced the growth inhibitory effect of 5-FU in PLC/PRF/5 cells but not in HuH7 cells, and the isobolographic analysis indicated that this effect was synergistic. Flow cytometric analysis showed a delay in the progression of G0-G1 to S phase in PLC/PRF/5, and a sustained, induction of the cyclin-dependent kinase inhibitor p27-Kip1 and down-regulation of cyclin D1 was observed. Moreover, increased expression of p27Kip1 was associated with reduced CDK-2-associated kinase activity. Another difference in the two cell types was that PLC/PRF/5 expressed abundant IFN receptors, but HuH7 did not. Apoptosis assays were not helpful in explaining the mechanism. Our results suggest that the synergistic effects of 5-FU and IFN-alpha may in part be attributable to alterations in cell cycle progression via up-regulation of p27Kip1. PMID:10914738

  14. p27kip1 Maintains a Subset of Leukemia Stem Cells in the Quiescent State in Murine MLL-Leukemia

    PubMed Central

    Zhang, Jun; Seet, Christopher; Sun, Clare; Li, Jing; You, Dewen; Volk, Andrew; Breslin, Peter; Li, Xingyu; Wei, Wei; Qian, Zhijian; Zeleznik-Le, Nancy J.; Zhang, Zhou; Zhang, Jiwang

    2013-01-01

    MLL (mixed-lineage leukemia)-fusion genes induce the development of leukemia through deregulation of normal MLL target genes, such as HOXA9 and MEIS1. Both HOXA9 and MEIS1 are required for MLL-fusion gene-induced leukemogenesis. Co-expression of HOXA9 and MEIS1 induces acute myeloid leukemia (AML) similar to that seen in mice in which MLL-fusion genes are over-expressed. p27kip1 (p27 hereafter), a negative regulator of the cell cycle, has also been defined as an MLL target, the expression of which is up-regulated in MLL leukemic cells (LCs). To investigate whether p27 plays a role in the pathogenesis of MLL-leukemia, we examined the effects of p27 deletion (p27-/-) on MLL-AF9 (MA9)-induced murine AML development. HOXA9/MEIS1 (H/M)-induced, p27 wild-type (p27+/+) and p27-/- AML were studied in parallel as controls. We found that LCs from both MA9-AML and H/M-AML can be separated into three fractions, a CD117-CD11bhi differentiated fraction as well as CD117+CD11bhi and CD117+CD11blo, two less differentiated fractions. The CD117+CD11blo fraction, comprising only 1-3% of total LCs, expresses higher levels of early hematopoietic progenitor markers but lower levels of mature myeloid cell markers compared to other populations of LCs. p27 is expressed and is required for maintaining the quiescent and drug-resistant states of the CD117+CD11blo fraction of MA9-LCs but not of H/M-LCs. p27 deletion significantly compromises the leukemogenic capacity of CD117+CD11blo MA9-LCs by reducing the frequency of leukemic stem cells (LSCs) but does not do so in H/M-LCs. In addition, we found that p27 is highly expressed and required for cell cycle arrest in the CD117-CD11bhi fraction in both types of LCs. Furthermore, we found that c-Myc expression is required for maintaining LCs in an undifferentiated state independently of proliferation. We concluded that p27 represses the proliferation of LCs, which is specifically required for maintaining the quiescent and drug-resistant states of a

  15. A vitamin D3 analog induces a G1-phase arrest in CaCo-2 cells by inhibiting cdk2 and cdk6: roles of cyclin E, p21Waf1, and p27Kip1.

    PubMed

    Scaglione-Sewell, B A; Bissonnette, M; Skarosi, S; Abraham, C; Brasitus, T A

    2000-11-01

    Previous studies by our laboratory have shown that a noncalcemic fluorinated analog of 1alpha,25-dihydroxyvitamin D3, 1alpha,25-dihydroxy-16-ene-23-yne-26,27-hexafluorocholcal ciferol (F6-D3), significantly reduced the frequency of colonic adenomas and completely abolished the development of colonic adenocarcinomas in rats treated with azoxymethane. The mechanisms involved in this analog's chemopreventive actions, however, remain unclear. In the present study, we now show that although both 1alpha,25-dihydroxyvitamin D3 and F6-D3 inhibited the proliferation of CaCo-2 cells, a human colonic adenocarcinoma cell line, by increasing their doubling times, only F6-D3 caused an arrest of these cells in the G1 phase of their cell cycle. This arrest was accompanied by an increase in the expression of the cyclin-dependent kinase (cdk) inhibitor proteins, p2Waf1 and p27Kip1, which served to decrease the activity of cyclin-dependent kinase 2 and cyclin-dependent kinase 6, whereas the expression and phosphorylation of pRB were unchanged. In contrast to the increased expression of these cdk inhibitors, the expression of cyclin E was decreased, which further inhibited the activity of cyclin-dependent kinase 2. Collectively, the inhibition of these cyclin-dependent kinases served to arrest the CaCo-2 cells, independent of changes in pRB. Furthermore, antibody neutralization studies suggest that transforming growth factor-beta may mediate the coassociations between cdk2 and p27Kip1 and cyclin E induced by F6-D3. These data indicate that cell cycle arrest may, at least in part, underlie the chemopreventive actions of F6-D3 observed in the azoxymethane model of colon cancer. Furthermore, if the antiproliferative action observed in CaCo-2 cells also occurs in human colonic epithelium, F6-D3 may have chemopreventive potential against human colon cancer, as well. PMID:11089522

  16. Thrombin Induces Tumor Cell Cycle Activation and Spontaneous Growth by Down-regulation of p27Kip1, in Association with the Up-regulation of Skp2 and MiR-222

    PubMed Central

    Hu, Liang; Ibrahim, Sherif; Liu, Cynthia; Skaar, Jeffrey; Pagano, Michele; Karpatkin, Simon

    2009-01-01

    The effect of thrombin on tumor cell cycle activation and spontaneous growth was examined in synchronized serum-starved tumor cell lines and a model of spontaneous prostate cancer development in TRAMP mice. BrdUrd incorporation and propidium iodide staining of prostate LNCaP cells arrested in G0 and treated with thrombin or serum revealed a 48- and 29-fold increase in S phase cells, respectively, at 8 hours. Similar results were obtained with TRAMP cells and a glioblastoma cell line, T98G. Cell cycle kinases and inhibitors in synchronized tumor cells revealed high levels of p27Kip1 and low levels of Skp2 and cyclins D1 and A. Addition of thrombin, TFLLRN, or serum down-regulated p27Kip1 with concomitant induction of Skp2, Cyclin D1, and Cyclin A with similar kinetics. LNCaP p27Kip1-transfected cells or Skp2 knockdown cells were refractory to thrombin-induced cell cycle activation. MicroRNA 222, an inhibitor of p27Kip1, was robustly up-regulated by thrombin. The in vitro observations were tested in vivo with transgenic TRAMP mice. Repetitive thrombin injection enhanced prostate tumor volume 6- to 8-fold (P < 0.04). Repetitive hirudin, a specific potent antithrombin, decreased tumor volume 13- to 24-fold (P < 0.04). Thus, thrombin stimulates tumor cell growth in vivo by down-regulation of p27Kip1. PMID:19351827

  17. Ganglioside GM3 modulates tumor suppressor PTEN-mediated cell cycle progression--transcriptional induction of p21(WAF1) and p27(kip1) by inhibition of PI-3K/AKT pathway.

    PubMed

    Choi, Hee-Jung; Chung, Tae-Wook; Kang, Sung-Koo; Lee, Young-Choon; Ko, Jeong-Heon; Kim, Jong-Guk; Kim, Cheorl-Ho

    2006-07-01

    The simple ganglioside GM3 has been shown to have anti-proliferative effects in several in vitro and in vivo cancer models. Although the exogenous ganglioside GM3 has an inhibitory effect on cancer cell proliferation, the exact mechanism by which it prevents cell proliferation remains unclear. Previous studies showed that MDM2 is an oncoprotein that controls tumorigenesis through both p53-dependent and p53-independent mechanisms, and tumor suppressor phosphatase and tensin homolog deleted on chromosome 10 (PTEN), a dual-specificity phosphatase that antagonizes phosphatidylinositol 3-kinase (PI-3K)/AKT signaling, is capable of blocking MDM2 nuclear translocation and destabilizing the MDM2 protein. Results from our current study show that GM3 treatment dramatically increases cyclin-dependent kinase (CDK) inhibitor (CKI) p21(WAF1) expression through the accumulation of p53 protein by the PTEN-mediated inhibition of the PI-3K/AKT/MDM2 survival signaling in HCT116 colon cancer cells. Moreover, the data herein clearly show that ganglioside GM3 induces p53-dependent transcriptional activity of p21(WAF1), as evidenced by the p21(WAF1) promoter-driven luciferase reporter plasmid (full-length p21(WAF1) promoter and a construct lacking the p53-binding sites). Additionally, ganglioside GM3 enhances expression of CKI p27(kip1) through the PTEN-mediated inhibition of the PI-3K/AKT signaling. Furthermore, the down-regulation of the cyclin E and CDK2 was clearly observed in GM3-treated HCT116 cells, but the down-regulation of cyclin D1 and CDK4 was not. On the contrary, suppression of PTEN levels by RNA interference restores the enhanced expression of p53-dependent p21(WAF1) and p53-independent p27(kip1) through inactivating the effect of PTEN on PI-3K/AKT signaling modulated by ganglioside GM3. These results suggest that ganglioside GM3-stimulated PTEN expression modulates cell cycle regulatory proteins, thus inhibiting cell growth. We conclude that ganglioside GM3 represents a

  18. Molecular cloning and functional characterization of a cell-permeable superoxide dismutase targeted to lung adenocarcinoma cells. Inhibition cell proliferation through the Akt/p27kip1 pathway.

    PubMed

    Lu, Min; Gong, Xingguo; Lu, Yuwen; Guo, Jianjun; Wang, Chenhui; Pan, Yuanjiang

    2006-05-12

    In clinical oncology, many trials with superoxide dismutase (SOD) have failed to demonstrate antitumor ability and in many cases even caused deleterious effects because of low tumor-targeting ability. In the current research, the Nostoc commune Fe-SOD coding sequence was amplified from genomic DNA. In addition, the single chain variable fragment (ScFv) was constructed from the cDNA of an LC-1 hybridoma cell line secreting anti-lung adenocarcinoma monoclonal antibody. After modification, the SOD and ScFv were fused and co-expressed, and the resulting fusion protein produced SOD and LC-1 antibody activity. Tracing SOD-ScFv by fluorescein isothiocyanate and superoxide anions (O2*-) in SPC-A-1 cells showed that the fusion protein could recognize and enter SPC-A-1 cells to eliminate O2*-. The lower oxidative stress resulting from the decrease in cellular O2*- delayed the cell cycle at G1 and significantly slowed SPC-A-1 cell growth in association with the dephosphorylation of the serine-threonine protein kinase Akt and expression of p27kip1. The tumor-targeting fusion protein resulting from this research overcomes two disadvantages of SODs previously used in the clinical setting, the inability to target tumor cells or permeate the cell membrane. These findings lay the groundwork for development of an efficient antitumor drug targeted by the ScFv. PMID:16551617

  19. 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

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

    PubMed

    Qiu, Mei-Ting; Fan, Qiong; Zhu, Zhu; 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

    2015-10-13

    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

  1. Targeting the cyclin-binding groove site to inhibit the catalytic activity of CDK2/cyclin A complex using p27(KIP1)-derived peptidomimetic inhibitors.

    PubMed

    Karthiga, Arumugasamy; Tripathi, Sunil Kumar; Shanmugam, Ramasamy; Suryanarayanan, Venkatesan; Singh, Sanjeev Kumar

    2015-01-01

    Functionally activated cyclin-dependent kinase 2 (CDK2)/cyclin A complex has been validated as an interesting therapeutic target to develop the efficient antineoplastic drug based on the cell cycle arrest. Cyclin A binds to CDK2 and activates the kinases as well as recruits the substrate and inhibitors using a hydrophobic cyclin-binding groove (CBG). Blocking the cyclin substrate recruitment on CBG is an alternative approach to override the specificity hurdle of the currently available ATP site targeting CDK2 inhibitors. Greater understanding of the interaction of CDK2/cyclin A complex with p27 (negative regulator) reveals that the Leu-Phe-Gly (LFG) motif region of p27 binds with the CBG site of cyclin A to arrest the malignant cell proliferation that induces apoptosis. In the present study, Replacement with Partial Ligand Alternatives through Computational Enrichment (REPLACE) drug design strategies have been applied to acquire LFG peptide-derived peptidomimetics library. The peptidomimetics function is equivalent with respect to substrate p27 protein fashion but does not act as an ATP antagonist. The combined approach of molecular docking, molecular dynamics (MD), and molecular electrostatic potential and ADME/T prediction were carried out to evaluate the peptidomimetics. Resultant interaction and electrostatic potential maps suggested that smaller substituent is desirable at the position of phenyl ring to interact with Trp217, Arg250, and Gln254 residues in the active site. The best docked poses were refined by the MD simulations which resulted in conformational changes. After equilibration, the structure of the peptidomimetic and receptor complex was stable. The results revealed that the various substrate protein-derived peptidomimetics could serve as perfect leads against CDK2 protein. PMID:25584078

  2. 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.

  3. CRM1/Ran-mediated nuclear export of p27(Kip1) involves a nuclear export signal and links p27 export and proteolysis.

    PubMed

    Connor, Michael K; Kotchetkov, Rouslan; Cariou, Sandrine; Resch, Ansgar; Lupetti, Rafaella; Beniston, Richard G; Melchior, Frauke; Hengst, Ludger; Slingerland, Joyce M

    2003-01-01

    We show that p27 localization is cell cycle regulated and we suggest that active CRM1/RanGTP-mediated nuclear export of p27 may be linked to cytoplasmic p27 proteolysis in early G1. p27 is nuclear in G0 and early G1 and appears transiently in the cytoplasm at the G1/S transition. Association of p27 with the exportin CRM1 was minimal in G0 and increased markedly during G1-to-S phase progression. Proteasome inhibition in mid-G1 did not impair nuclear import of p27, but led to accumulation of p27 in the cytoplasm, suggesting that export precedes degradation for at least part of the cellular p27 pool. p27-CRM1 binding and nuclear export were inhibited by S10A mutation but not by T187A mutation. A putative nuclear export sequence in p27 is identified whose mutation reduced p27-CRM1 interaction, nuclear export, and p27 degradation. Leptomycin B (LMB) did not inhibit p27-CRM1 binding, nor did it prevent p27 export in vitro or in heterokaryon assays. Prebinding of CRM1 to the HIV-1 Rev nuclear export sequence did not inhibit p27-CRM1 interaction, suggesting that p27 binds CRM1 at a non-LMB-sensitive motif. LMB increased total cellular p27 and may do so indirectly, through effects on other p27 regulatory proteins. These data suggest a model in which p27 undergoes active, CRM1-dependent nuclear export and cytoplasmic degradation in early G1. This would permit the incremental activation of cyclin E-Cdk2 leading to cyclin E-Cdk2-mediated T187 phosphorylation and p27 proteolysis in late G1 and S phase. PMID:12529437

  4. KLRG1 impairs CD4+ T cell responses via p16ink4a and p27kip1 pathways: role in hepatitis B vaccine failure in individuals with hepatitis C virus infection.

    PubMed

    Shi, Lei; Wang, Jia M; Ren, Jun P; Cheng, Yong Q; Ying, Ruo S; Wu, Xiao Y; Lin, Shu M; Griffin, Jeddidiah W D; Li, Guang Y; Moorman, Jonathan P; Yao, Zhi Q

    2014-01-15

    Coinfection of hepatitis B virus (HBV) with hepatitis C virus (HCV) is quite common, leading to an increase in morbidity and mortality. As such, HBV vaccination is recommended in HCV-infected individuals. However, HBV vaccine responses in HCV-infected individuals are often blunted compared with uninfected populations. The mechanism for this failure of vaccine response in HCV-infected subjects remains unclear. In this study, we investigated the expression and function of an inhibitory receptor, killer cell lectin-like receptor subfamily G member 1 (KLRG1), in the regulation of CD4(+) T cells and HBV vaccine responses during HCV infection. We demonstrated that KLRG1 was overexpressed on CD4(+) T cells from HCV-infected, HBV vaccine nonresponders compared with HBV vaccine responders. The capacity of CD4(+) T cells to proliferate and secrete IL-2 cytokine was inversely associated with the level of KLRG1 expression. Importantly, blocking KLRG1 signaling resulted in a significant improvement in CD4(+) T cell proliferation and IL-2 production in HCV-infected, HBV vaccine nonresponders in response to TCR stimulation. Moreover, blockade of KLRG1 increased the phosphorylation of Akt (Ser(473)) and decreased the expression of cell cycle inhibitors p16(ink4a) and p27(kip1), which subsequently enhanced the expression of cyclin-dependent kinase 2 and cyclin E. These results suggest that the KLRG1 pathway impairs CD4(+) T cell responses to neoantigen and induces a state of immune senescence in individuals with HCV infection, raising the possibility that blocking this negative-signaling pathway might improve HBV vaccine responses in the setting of chronic viral infection. PMID:24337749

  5. KLRG1 impairs CD4+ T cell responses via p16ink4a and p27kip1 pathways - Role in hepatitis B vaccine failure in individuals with hepatitis C virus infection

    PubMed Central

    Shi, Lei; Wang, Jia M.; Ren, Jun P.; Cheng, Yong Q.; Ying, Ruo S.; Wu, Xiao Y.; Lin, Shu M.; Griffin, Jeddidiah WD; Li, Guang Y.; Moorman, Jonathan P.; Yao, Zhi Q.

    2014-01-01

    Co-infection of hepatitis B virus (HBV) with hepatitis C virus (HCV) is quite common, leading to an increase in morbidity and mortality. As such, HBV vaccination is recommended in HCV-infected individuals. HBV vaccine responses in HCV-infected individuals, however, are often blunted when compared to uninfected populations. The mechanism for this failure of vaccine response in HCV-infected subjects remains unclear. In this study, we investigated the expression and function of an inhibitory receptor, killer cell lectin-like receptor subfamily G member 1 (KLRG1), in regulation of CD4+ T cells and HBV vaccine responses during HCV infection. We demonstrated that KLRG1 was over-expressed on CD4+ T cells from HCV-infected, HBV vaccine non-responders (HBV-NR) compared to those responders (HBV-R). The capacity of CD4+ T cell to proliferate and secrete IL-2 cytokine was inversely associated with the level of KLRG1 expression. Importantly, blocking KLRG1 signaling resulted in a significant improvement of CD4+ T cell proliferation and IL-2 production in HCV-infected, HBV-NR in response to T cell receptor (TCR) stimulation. Moreover, blockade of KLRG1 increased the phosphorylation of Akt (Ser473) and decreased the expression of cell cycle inhibitors p16ink4a and p27kip1, which subsequently enhanced CDK 2 and cyclin E expressions. These results suggest that the KLRG1 pathway impairs CD4+ T cell responses to neo-antigen and induces a state of immune senescence in individuals with HCV infection, raising the possibility that blocking this negative signaling pathway might improve HBV vaccine responses in the setting of chronic viral infection. PMID:24337749

  6. Intranuclear localization and UV response of ERCC5/XPG protein

    SciTech Connect

    Park, M.S.; Marrone, B.L.; MacInnes, M.A.

    1995-11-01

    The human ERCC5/XPG protein is defective in the hereditary genetic disorder xeroderma pigmentosum, group-G. The XPG gene encodes a single-strand DNA endonuclease which is essential for the incision step of nucleotide excision repair for a wide variety of DNA damages. We have shown previously by indirect immunofluorescence and biochemical fractionation that the XPG protein is localized in the nucleus, in discrete foci, and probably associated with the nuclear matrix. However, the intranuclear localization of XPG is markedly altered for a short time after UV irradiation. Here, we report the identification of XPG protein regions involved in the UV response, and its putative nuclear localization signals (NLS) using a B-galactosidase (B-gal) reporter gene system. Control and fusion reporter genes were expressed in Hela S3 cells after CaPO{sub 4} transfection. B-gal protein was detected by indirect immuno-fluorescence using an anti B-gal monoclonal antibody and FITC-labeled goat anti-mouse antiserum. Two NLS peptides of the XPG carboxy-terminal region (AA 1029-1069 and 1146-1186 term) were shown to independently localize B-gal fusion proteins to the nucleus (>90%). The C-terminus peptide was observed to further localize B-gal into nuclear foci and the perinucleolar regions. When B-gal was fused with two copies of the C-terminal NLS, in tandem, B-gal was extensively sublocalized to the perinucleolar regions. Shortly after cell UV irradiation (5 J/m{sup 2}) this B-gal fusion protein became dissociated from the perinucleolar regions whereupon it was distributed throughout the nucleus. Within 6 hours post-irradiation, the fusion protein reassociated again with the perinucleolar regions. These observations confirm and extend a similar UV response of endogenous XPG protein in UV-irradiation human cells. The involvement of XPG protein and its UV responses will be discussed in context of models nuclear matrix and preferential DNA repair in actively transcribed genes.

  7. Compartmentalization and Functionality of Nuclear Disorder: Intrinsic Disorder and Protein-Protein Interactions in Intra-Nuclear Compartments

    PubMed Central

    Meng, Fanchi; Na, Insung; Kurgan, Lukasz; Uversky, Vladimir N.

    2015-01-01

    The cell nucleus contains a number of membrane-less organelles or intra-nuclear compartments. These compartments are dynamic structures representing liquid-droplet phases which are only slightly denser than the bulk intra-nuclear fluid. They possess different functions, have diverse morphologies, and are typically composed of RNA (or, in some cases, DNA) and proteins. We analyzed 3005 mouse proteins localized in specific intra-nuclear organelles, such as nucleolus, chromatin, Cajal bodies, nuclear speckles, promyelocytic leukemia (PML) nuclear bodies, nuclear lamina, nuclear pores, and perinuclear compartment and compared them with ~29,863 non-nuclear proteins from mouse proteome. Our analysis revealed that intrinsic disorder is enriched in the majority of intra-nuclear compartments, except for the nuclear pore and lamina. These compartments are depleted in proteins that lack disordered domains and enriched in proteins that have multiple disordered domains. Moonlighting proteins found in multiple intra-nuclear compartments are more likely to have multiple disordered domains. Protein-protein interaction networks in the intra-nuclear compartments are denser and include more hubs compared to the non-nuclear proteins. Hubs in the intra-nuclear compartments (except for the nuclear pore) are enriched in disorder compared with non-nuclear hubs and non-nuclear proteins. Therefore, our work provides support to the idea of the functional importance of intrinsic disorder in the cell nucleus and shows that many proteins associated with sub-nuclear organelles in nuclei of mouse cells are enriched in disorder. This high level of disorder in the mouse nuclear proteins defines their ability to serve as very promiscuous binders, possessing both large quantities of potential disorder-based interaction sites and the ability of a single such site to be involved in a large number of interactions. PMID:26712748

  8. The intranuclear mobility of messenger RNA binding proteins is ATP dependent and temperature sensitive

    PubMed Central

    Calapez, Alexandre; Pereira, Henrique M.; Calado, Angelo; Braga, José; Rino, José; Carvalho, Célia; Tavanez, João Paulo; Wahle, Elmar; Rosa, Agostinho C.; Carmo-Fonseca, Maria

    2002-01-01

    fAter being released from transcription sites, messenger ribonucleoprotein particles (mRNPs) must reach the nuclear pore complexes in order to be translocated to the cytoplasm. Whether the intranuclear movement of mRNPs results largely from Brownian motion or involves molecular motors remains unknown. Here we have used quantitative photobleaching techniques to monitor the intranuclear mobility of protein components of mRNPs tagged with GFP. The results show that the diffusion coefficients of the poly(A)-binding protein II (PABP2) and the export factor TAP are significantly reduced when these proteins are bound to mRNP complexes, as compared with nonbound proteins. The data further show that the mobility of wild-type PABP2 and TAP, but not of a point mutant variant of PABP2 that fails to bind to RNA, is significantly reduced when cells are ATP depleted or incubated at 22°C. Energy depletion has only minor effects on the intranuclear mobility of a 2,000-kD dextran (which corresponds approximately in size to 40S mRNP particles), suggesting that the reduced mobility of PABP2 and TAP is not caused by a general alteration of the nuclear environment. Taken together, the data suggest that the mobility of mRNPs in the living cell nucleus involves a combination of passive diffusion and ATP-dependent processes. PMID:12473688

  9. Protein complex formation and intranuclear dynamics of NAC1 in cancer cells.

    PubMed

    Nakayama, Naomi; Kato, Hiroaki; Sakashita, Gyosuke; Nariai, Yuko; Nakayama, Kentaro; Kyo, Satoru; Urano, Takeshi

    2016-09-15

    Nucleus accumbens-associated protein 1 (NAC1) is a cancer-related transcription regulator protein that is also involved in the pluripotency and differentiation of embryonic stem cells. NAC1 is overexpressed in various carcinomas including ovarian, cervical, breast, and pancreatic carcinomas. NAC1 knock-down was previously shown to result in the apoptosis of ovarian cancer cell lines and to rescue their sensitivity to chemotherapy, suggesting that NAC1 may be a potential therapeutic target, but protein complex formation and the dynamics of intranuclear NAC1 in cancer cells remain poorly understood. In this study, analysis of HeLa cell lysates by fast protein liquid chromatography (FPLC) on a sizing column showed that the NAC1 peak corresponded to an apparent molecular mass of 300-500 kDa, which is larger than the estimated molecular mass (58 kDa) of the protein. Furthermore, live cell photobleaching analyses with green fluorescent protein (GFP)-fused NAC1 proteins revealed the intranuclear dynamics of NAC1. Collectively our results demonstrate that NAC1 forms a protein complex to function as a transcriptional regulator in cancer cells. PMID:27424155

  10. Autophagy-related intrinsically disordered proteins in intra-nuclear compartments.

    PubMed

    Na, Insung; Meng, Fanchi; Kurgan, Lukasz; Uversky, Vladimir N

    2016-08-16

    Recent analyses indicated that autophagy can be regulated via some nuclear transcriptional networks and many important players in the autophagy and other forms of programmed cell death are known to be intrinsically disordered. To this end, we analyzed similarities and differences in the intrinsic disorder distribution of nuclear and non-nuclear proteins related to autophagy. We also looked at the peculiarities of the distribution of the intrinsically disordered autophagy-related proteins in various intra-nuclear organelles, such as the nucleolus, chromatin, Cajal bodies, nuclear speckles, promyelocytic leukemia (PML) nuclear bodies, nuclear lamina, nuclear pores, and perinucleolar compartment. This analysis revealed that the autophagy-related proteins constitute about 2.5% of the non-nuclear proteins and 3.3% of the nuclear proteins, which corresponds to a substantial enrichment by about 32% in the nucleus. Curiously, although, in general, the autophagy-related proteins share similar characteristics of disorder with a generic set of all non-nuclear proteins, chromatin and nuclear speckles are enriched in the intrinsically disordered autophagy proteins (29 and 37% of these proteins are disordered, respectively) and have high disorder content at 0.24 and 0.27, respectively. Therefore, our data suggest that some of the nuclear disordered proteins may play important roles in autophagy. PMID:27377881

  11. Valosin-containing protein immunoreactivity in tauopathies, synucleinopathies, polyglutamine diseases and intranuclear inclusion body disease.

    PubMed

    Mori, Fumiaki; Tanji, Kunikazu; Toyoshima, Yasuko; Sasaki, Hidenao; Yoshida, Mari; Kakita, Akiyoshi; Takahashi, Hitoshi; Wakabayashi, Koichi

    2013-12-01

    Valosin-containing protein (VCP) is associated with multiple cellular functions, including ubiquitin-dependent protein degradation. Mutations in VCP are known to cause inclusion body myopathy with Paget's disease and frontotemporal dementia and familial amyotrophic lateral sclerosis (fALS; ALS14), both of which are characterized by trans-activation response DNA protein 43 (TDP-43)-positive neuronal cytoplasmic and nuclear inclusions. Recently, immunoreactivity for fALS-associated proteins (TDP-43, fused in sarcoma (FUS), optineurin and ubiquilin-2) were reported to be present in cytoplasmic and nuclear inclusions in various neurodegenerative diseases. However, the extent and frequency of VCP-immunoreactive structures in these neurodegenerative diseases are uncertain. We immunohistochemically examined the brains of 72 cases with neurodegenerative diseases and five control cases. VCP immunoreactivity was present in Lewy bodies in Parkinson's disease and dementia with Lewy bodies, and neuronal nuclear inclusions in five polyglutamine diseases and intranuclear inclusion body disease, as well as in Marinesco bodies in aged control subjects. However, other neuronal and glial cytoplasmic inclusions in tauopathies and TDP-43 proteinopathies were unstained. These findings suggest that VCP may have common mechanisms in the formation or degradation of cytoplasmic and nuclear inclusions of neurons, but not of glial cells, in several neurodegenerative conditions. PMID:23782134

  12. Nuclear translocation and regulation of intranuclear distribution of cytoplasmic poly(A)-binding protein are distinct processes mediated by two Epstein Barr virus proteins.

    PubMed

    Park, Richard; El-Guindy, Ayman; Heston, Lee; Lin, Su-Fang; Yu, Kuan-Ping; Nagy, Mate; Borah, Sumit; Delecluse, Henri-Jacques; Steitz, Joan; Miller, George

    2014-01-01

    Many viruses target cytoplasmic polyA binding protein (PABPC) to effect widespread inhibition of host gene expression, a process termed viral host-shutoff (vhs). During lytic replication of Epstein Barr Virus (EBV) we observed that PABPC was efficiently translocated from the cytoplasm to the nucleus. Translocated PABPC was diffusely distributed but was excluded from viral replication compartments. Vhs during EBV infection is regulated by the viral alkaline nuclease, BGLF5. Transfection of BGLF5 alone into BGLF5-KO cells or uninfected 293 cells promoted translocation of PAPBC that was distributed in clumps in the nucleus. ZEBRA, a viral bZIP protein, performs essential functions in the lytic program of EBV, including activation or repression of downstream viral genes. ZEBRA is also an essential replication protein that binds to viral oriLyt and interacts with other viral replication proteins. We report that ZEBRA also functions as a regulator of vhs. ZEBRA translocated PABPC to the nucleus, controlled the intranuclear distribution of PABPC, and caused global shutoff of host gene expression. Transfection of ZEBRA alone into 293 cells caused nuclear translocation of PABPC in the majority of cells in which ZEBRA was expressed. Co-transfection of ZEBRA with BGLF5 into BGLF5-KO cells or uninfected 293 cells rescued the diffuse intranuclear pattern of PABPC seen during lytic replication. ZEBRA mutants defective for DNA-binding were capable of regulating the intranuclear distribution of PABPC, and caused PABPC to co-localize with ZEBRA. One ZEBRA mutant, Z(S186E), was deficient in translocation yet was capable of altering the intranuclear distribution of PABPC. Therefore ZEBRA-mediated nuclear translocation of PABPC and regulation of intranuclear PABPC distribution are distinct events. Using a click chemistry-based assay for new protein synthesis, we show that ZEBRA and BGLF5 each function as viral host shutoff factors. PMID:24705134

  13. Orchid Fleck Virus Structural Proteins N and P Form Intranuclear Viroplasm-Like Structures in the Absence of Viral Infection

    PubMed Central

    Chiba, Sotaro; Andika, Ida Bagus; Maruyama, Kazuyuki; Tamada, Tetsuo; Suzuki, Nobuhiro

    2013-01-01

    Orchid fleck virus (OFV) has a unique two-segmented negative-sense RNA genome that resembles that of plant nucleorhabdoviruses. In infected plant cells, OFV and nucleorhabdoviruses induce an intranuclear electron-lucent viroplasm that is believed to be the site for virus replication. In this study, we investigated the molecular mechanism by which OFV viroplasms are produced in vivo. Among OFV-encoded proteins, the nucleocapsid protein (N) and the putative phosphoprotein (P) were present in nuclear fractions of OFV-infected Nicotiana benthamiana plants. Transient coexpression of N and P, in the absence of virus infection, was shown to be sufficient for formation of an intranuclear viroplasm-like structure in plant cells. When expressed independently as a fluorescent protein fusion product in uninfected plant cells, N protein accumulated throughout the cell, while P protein accumulated in the nucleus. However, the N protein, when coexpressed with P, was recruited to a subnuclear region to induce a large viroplasm-like focus. Deletion and substitution mutagenesis demonstrated that the P protein contains a nuclear localization signal (NLS). Artificial nuclear targeting of the N-protein mutant was insufficient for formation of viroplasm-like structures in the absence of P. A bimolecular fluorescence complementation assay confirmed interactions between the N and P proteins within subnuclear viroplasm-like foci and interactions of two of the N. benthamiana importin-α homologues with the P protein but not with the N protein. Taken together, our results suggest that viroplasm formation by OFV requires nuclear accumulation of both the N and P proteins, which is mediated by P-NLS, unlike nucleorhabdovirus viroplasm utilizing the NLS on protein N. PMID:23616651

  14. Intranuclear Actin Regulates Osteogenesis

    PubMed Central

    Sen, Buer; Xie, Zhihui; Uzer, Gunes; Thompson, William R.; Styner, Maya; Wu, Xin; Rubin, Janet

    2016-01-01

    Depolymerization of the actin cytoskeleton induces nuclear trafficking of regulatory proteins and global effects on gene transcription. We here show that in mesenchymal stem cells (MSCs), cytochalasin D treatment causes rapid cofilin-/importin-9-dependent transfer of G-actin into the nucleus. The continued presence of intranuclear actin, which forms rod-like structures that stain with phalloidin, is associated with induction of robust expression of the osteogenic genes osterix and osteocalcin in a Runx2-dependent manner, and leads to acquisition of osteogenic phenotype. Adipogenic differentiation also occurs, but to a lesser degree. Intranuclear actin leads to nuclear export of Yes-associated protein (YAP); maintenance of nuclear YAP inhibits Runx2 initiation of osteogenesis. Injection of cytochalasin into the tibial marrow space of live mice results in abundant bone formation within the space of 1 week. In sum, increased intranuclear actin forces MSC into osteogenic lineage through controlling Runx2 activity; this process may be useful for clinical objectives of forming bone. PMID:26140478

  15. Intranuclear Delivery of a Novel Antibody-Derived Radiosensitizer Targeting the DNA-Dependent Protein Kinase Catalytic Subunit

    SciTech Connect

    Xiong Hairong; Lee, Robert J.; Haura, Eric B.; Edwards, John G.; Dynan, William S.; Li Shuyi

    2012-07-01

    Purpose: To inhibit DNA double-strand break repair in tumor cells by delivery of a single-chain antibody variable region fragment (ScFv 18-2) to the cell nucleus. ScFv 18-2 binds to a regulatory region of the DNA-dependent protein kinase (DNA-PK), an essential enzyme in the nonhomologous end-joining pathway, and inhibits DNA end-joining in a cell-free system and when microinjected into single cells. Development as a radiosensitizer has been limited by the lack of a method for intranuclear delivery to target cells. We investigated a delivery method based on folate receptor-mediated endocytosis. Methods and Materials: A recombinant ScFv 18-2 derivative was conjugated to folate via a scissile disulfide linker. Folate-ScFv 18-2 was characterized for its ability to be internalized by tumor cells and to influence the behavior of ionizing radiation-induced repair foci. Radiosensitization was measured in a clonogenic survival assay. Survival curves were fitted to a linear-quadratic model, and between-group differences were evaluated by an F test. Sensitization ratios were determined based on mean inhibitory dose. Results: Human KB and NCI-H292 lung cancer cells treated with folate-conjugated ScFv 18-2 showed significant radiosensitization (p < 0.001). Sensitization enhancement ratios were 1.92 {+-} 0.42 for KB cells and 1.63 {+-} 0.13 for NCI-H292 cells. Studies suggest that treatment inhibits repair of radiation-induced DSBs, as evidenced by the persistence of {gamma}-H2AX-stained foci and by inhibition of staining with anti-DNA-PKcs phosphoserine 2056. Conclusions: Folate-mediated endocytosis is an effective method for intranuclear delivery of an antibody-derived DNA repair inhibitor.

  16. Studies of a nuclear matrix protein restricted to normal brain cells and lead-induced intranuclear inclusion bodies of kidney

    SciTech Connect

    Shelton, K.; Egle, P.; Redford, K.; Bigbee, J.

    1986-05-01

    A nuclear matrix protein, p32/6.3, with an unusual tissue distribution, has been identified. Protein from 21 tissues was surveyed by immunoprobing Western blots. In normal adult rats p32/6.3 is found only in grey matter from the cerebrum and the cerebellum, occurring in both neurons and astrocytes. Other brain cell types have not been examined. The protein appears to be developmentally regulated. It is detectable in the brain within a few days after birth and reaches adult levels within one to two weeks. Brain p32/6.3 has been found in all animals tested including rat, mouse, dog, cow, pig, chicken and human. This conservation indicates a fundamental role for p32/6.3 in the nucleus of brain cells. Possible functions for p32/6.3 may be indicated by a second novel occurrence. Chronic lead poisoning characteristically induces intranuclear inclusion bodies in the cells lining kidney proximal tubules. p32/6.3 is a major constituent of these inclusion bodies. They are also rich in lead and other metals including calcium, iron, zinc, copper and cadmium. These diverse observations suggest that p32/6.3 may have a role in metal homeostasis in the brain of normal animals.

  17. Intranuclear location of the adenovirus type 5 E1B 55-kilodalton protein.

    PubMed Central

    Smiley, J K; Young, M A; Flint, S J

    1990-01-01

    The intracellular location of the adenovirus type 5 E1B 55-kilodalton (kDa) protein, particularly the question of whether it is associated with nuclear pore complexes, was examined. Fractionation of adenovirus type 5-infected HeLa cell nuclei by an established procedure (N. Dwyer and G. Blobel, J. Cell. Biol. 70:581-591, 1976) yielded one population of E1B 55-kDa protein molecules released by digestion of nuclei with RNase A and a second population recovered in the pore complex-lamina fraction. Free and E1B 55-kDa protein-bound forms of the E4 34-kDa protein (P. Sarnow, C. A. Sullivan, and A. J. Levine, Virology 120:387-394, 1982) were largely recovered in the pore complex-lamina fraction. Nevertheless, the association of E1B 55-kDa protein molecules with this nuclear envelope fraction did not depend on interaction of the E1B 55-kDa protein with the E4 34-kDa protein. Comparison of the immunofluorescence patterns observed with antibodies recognizing the E1B 55-kDa protein or cellular pore complex proteins and of the behavior of these viral and cellular proteins during in situ fractionation suggests that the E1B 55-kDa protein does not become intimately or stably associated with pore complexes in adenovirus-infected cells. Images PMID:2143545

  18. Modulation of VEGF-induced endothelial cell cycle protein expression through cyclic AMP hydrolysis by PDE2 and PDE4.

    PubMed

    Favot, Laure; Keravis, Thérèse; Lugnier, Claire

    2004-09-01

    Endothelial cell proliferation in response to VEGF plays an important role in physiological and pathological angiogenesis. The role of PDE2 and PDE4 in VEGF-induced proliferation in HUVEC was investigated: 1) VEGF increased cAMP-hydrolytic activity by up-regulating the expression of PDE2 and PDE4 isozymes; 2) VEGF increased progression in cell cycle with an increase in p42/p44 MAP kinase, cyclin A and cyclin D1 expressions and with a decrease in p21 waf1/cip1 and p27 kip1 expressions; 3) EHNA (20 micro M), a selective PDE2 inhibitor, RP73401 (10 micro M), a selective PDE4 inhibitor blocked the VEGF-induced increase in p42/p44 MAP kinase expression; 4) RP73401, but not EHNA, blocked the VEGF-induced increase in cyclin A and decrease in p27 kip1 expressions; 5) EHNA, contrary to RP73401, enhanced the VEGF-induced increase of cyclin A and decrease of p27 kip1. 6) EHNA and RP73401 together blocked the VEGF-induced increase in cyclin D1 and decrease in p21 waf1/cip1 expressions; 7) Inhibition of VEGF-upregulated PDE2 and PDE4 reversed the VEGF-induced alterations in cell cycle protein expression, bringing back endothelial cells to a non-proliferating status. Consequently, PDE2 and PDE4 inhibitions were able to inhibit VEGF-induced endothelial cell proliferation by restoring cell cycle key protein expression, and might thus be useful in excessive angiogenesis. Furthermore, the differences between PDE2 and PDE4 effects may suggest compartmentalized effects. PMID:15351862

  19. Serum insensitive, intranuclear protein delivery by the multipurpose cationic lipid SAINT-2.

    PubMed

    van der Gun, Bernardina T F; Monami, Amélie; Laarmann, Sven; Raskó, Tamás; Slaska-Kiss, Krystyna; Weinhold, Elmar; Wasserkort, Reinhold; de Leij, Lou F M H; Ruiters, Marcel H J; Kiss, Antal; McLaughlin, Pamela M J

    2007-11-20

    Cationic liposomal compounds are widely used to introduce DNA and siRNA into viable cells, but none of these compounds are also capable of introducing proteins. Here we describe the use of a cationic amphiphilic lipid SAINT-2:DOPE for the efficient delivery of proteins into cells (profection). Labeling studies demonstrated equal delivery efficiency for protein as for DNA and siRNA. Moreover, proteins complexed with Saint-2:DOPE were successfully delivered, irrespective of the presence of serum, and the profection efficiency was not influenced by the size or the charge of the protein:cationic liposomal complex. Using beta-galactosidase as a reporter protein, enzymatic activity was detected in up to 98% of the adherent cells, up to 83% of the suspension cells and up to 70% of the primary cells after profection. A delivered antibody was detected in the cytoplasm for up to 7 days after profection. Delivery of the methyltransferase M.SssI resulted in DNA methylation, leading to a decrease in E-cadherin expression. The lipid-mediated multipurpose transport system reported here can introduce proteins into the cell with an equal delivery efficiency as for nucleotides. Delivery is irrespective of the presence of serum, and the protein can exert its function both in the cytoplasm and in the nucleus. Furthermore, DNA methylation by M.SssI delivery as a novel tool for gene silencing has potential applications in basic research and therapy. PMID:17884225

  20. NF-κB hyper-activation by HTLV-1 tax induces cellular senescence, but can be alleviated by the viral anti-sense protein HBZ.

    PubMed

    Zhi, Huijun; Yang, Liangpeng; Kuo, Yu-Liang; Ho, Yik-Khuan; Shih, Hsiu-Ming; Giam, Chou-Zen

    2011-04-01

    Activation of I-κB kinases (IKKs) and NF-κB by the human T lymphotropic virus type 1 (HTLV-1) trans-activator/oncoprotein, Tax, is thought to promote cell proliferation and transformation. Paradoxically, expression of Tax in most cells leads to drastic up-regulation of cyclin-dependent kinase inhibitors, p21(CIP1/WAF1) and p27(KIP1), which cause p53-/pRb-independent cellular senescence. Here we demonstrate that p21(CIP1/WAF1)-/p27(KIP1)-mediated senescence constitutes a checkpoint against IKK/NF-κB hyper-activation. Senescence induced by Tax in HeLa cells is attenuated by mutations in Tax that reduce IKK/NF-κB activation and prevented by blocking NF-κB using a degradation-resistant mutant of I-κBα despite constitutive IKK activation. Small hairpin RNA-mediated knockdown indicates that RelA induces this senescence program by acting upstream of the anaphase promoting complex and RelB to stabilize p27(KIP1) protein and p21(CIP1/WAF1) mRNA respectively. Finally, we show that down-regulation of NF-κB by the HTLV-1 anti-sense protein, HBZ, delay or prevent the onset of Tax-induced senescence. We propose that the balance between Tax and HBZ expression determines the outcome of HTLV-1 infection. Robust HTLV-1 replication and elevated Tax expression drive IKK/NF-κB hyper-activation and trigger senescence. HBZ, however, modulates Tax-mediated viral replication and NF-κB activation, thus allowing HTLV-1-infected cells to proliferate, persist, and evolve. Finally, inactivation of the senescence checkpoint can facilitate persistent NF-κB activation and leukemogenesis. PMID:21552325

  1. Organ differences in the impact of p27(kip1) deficiency on carcinogenesis induced by N-methyl-N-nitrosourea.

    PubMed

    Ogawa, Kumiko; Murasaki, Toshiya; Sugiura, Satoshi; Nakanishi, Makoto; Shirai, Tomoyuki

    2013-06-01

    To evaluate the impact of p27 on carcinogenesis in various organs, N-methyl-N-nitrosourea (MNU), a direct-acting alkylating agent, was given to p27 knock-out mice. Groups of 20-40 male and female mice with null, hetero- or wild-type p27 alleles were given drinking water containing 240 ppm MNU or distilled water every other week for five cycles. The incidence and multiplicity of the induced proliferative lesions were then histologically evaluated at weeks 14 and 20. MNU treatment induced various lesions including squamous hyperplasia and squamous cell carcinoma in the forestomach, atypical hyperplasia and adenocarcinomas in the fundic and pyloric glands, adenomas and adenocarcinomas in the duodenum, malignant lymphomas in the thymus, liver, kidney and spleen and alveolar hyperplasia, adenomas, adenocarcinomas and malignant lymphomas in the lung. Although the incidences of the lesions in the forestomach, fundic and pyloric glands did not differ among the p27 genotypes, those of alveolar hyperplasia of the lung and malignant lymphoma of the thymus were significantly increased in p27-null males as compared with both wild- and hetero-type animals. Moreover, in both p27(+/+) and p27(+/-) cases, the rates for p27-positive cells were obviously increased in proliferative lesions of the pyloric gland and the lung. However, an increased rate of p27-positive cells was not observed in malignant lymphoma of the thymus. These findings suggest that p27 does not control the cell cycle equally in all organs affected by MNU-induced carcinogenesis. PMID:22183835

  2. A pathway in quiescent cells that controls p27Kip1 stability, subcellular localization, and tumor suppression

    PubMed Central

    Besson, Arnaud; Gurian-West, Mark; Chen, Xueyan; Kelly-Spratt, Karen S.; Kemp, Christopher J.; Roberts, James M.

    2006-01-01

    We have created two knock-in mouse models to study the mechanisms that regulate p27 in normal cells and cause misregulation of p27 in tumors: p27S10A, in which Ser10 is mutated to Ala; and p27CK–, in which point mutations abrogate the ability of p27 to bind cyclins and CDKs. These two mutant alleles identify steps in a pathway that controls the proteasomal degradation of p27 uniquely in quiescent cells: Dephosphorylation of p27 on Ser10 inhibits p27 nuclear export and promotes its assembly into cyclin–CDK complexes, which is, in turn, necessary for p27 turnover. We further show that Ras-dependent lung tumorigenesis is associated with increased phosphorylation on Ser10 and cytoplasmic mislocalization of p27. Indeed, we find that p27S10A is refractory to Ras-induced cytoplasmic translocation and that p27S10A mice are tumor resistant. Thus, phosphorylation of p27 on Ser10 is an important event in the regulation of the tumor suppressor function of p27. PMID:16391232

  3. Herpes simplex virus type 1 tegument proteins VP1/2 and UL37 are associated with intranuclear capsids

    SciTech Connect

    Bucks, Michelle A.; O'Regan, Kevin J.; Murphy, Michael A.; Wills, John W.; Courtney, Richard J. . E-mail: rcourtney@psu.edu

    2007-05-10

    The assembly of the tegument of herpes simplex virus type 1 (HSV-1) is a complex process that involves a number of events at various sites within virus-infected cells. Our studies focused on determining whether tegument proteins, VP1/2 and UL37, are added to capsids located within the nucleus. Capsids were isolated from the nuclear fraction of HSV-1-infected cells and purified by rate-zonal centrifugation to separate B capsids (containing the scaffold proteins and no viral DNA) and C capsids (containing DNA and no scaffold proteins). Western blot analyses of these capsids indicated that VP1/2 associated primarily with C capsids and UL37 associated with B and C capsids. The results demonstrate that at least two of the tegument proteins of HSV-1 are associated with capsids isolated from the nuclear fraction, and these capsid-tegument protein interactions may represent initial events of the tegumentation process.

  4. Enhancement of gene targeting in human cells by intranuclear permeation of the Saccharomyces cerevisiae Rad52 protein

    PubMed Central

    Kalvala, Arjun; Rainaldi, Giuseppe; Di Primio, Cristina; Liverani, Vania; Falaschi, Arturo; Galli, Alvaro

    2010-01-01

    The introduction of exogenous DNA in human somatic cells results in a frequency of random integration at least 100-fold higher than gene targeting (GT), posing a seemingly insurmountable limitation for gene therapy applications. We previously reported that, in human cells, the stable over-expression of the Saccharomyces cerevisiae Rad52 gene (yRAD52), which plays the major role in yeast homologous recombination (HR), caused an up to 37-fold increase in the frequency of GT, indicating that yRAD52 interacts with the double-strand break repair pathway(s) of human cells favoring homologous integration. In the present study, we tested the effect of the yRad52 protein by delivering it directly to the human cells. To this purpose, we fused the yRAD52 cDNA to the arginine-rich domain of the TAT protein of HIV (tat11) that is known to permeate the cell membranes. We observed that a recombinant yRad52tat11 fusion protein produced in Escherichia coli, which maintains its ability to bind single-stranded DNA (ssDNA), enters the cells and the nuclei, where it is able to increase both intrachromosomal recombination and GT up to 63- and 50-fold, respectively. Moreover, the non-homologous plasmid DNA integration decreased by 4-fold. yRAD52tat11 proteins carrying point mutations in the ssDNA binding domain caused a lower or nil increase in recombination proficiency. Thus, the yRad52tat11 could be instrumental to increase GT in human cells and a ‘protein delivery approach’ offers a new tool for developing novel strategies for genome modification and gene therapy applications. PMID:20519199

  5. Effects of Deletion and Overexpression of the Autographa californica Nuclear Polyhedrosis Virus FP25K Gene on Synthesis of Two Occlusion-Derived Virus Envelope Proteins and Their Transport into Virus-Induced Intranuclear Membranes

    PubMed Central

    Rosas-Acosta, Germán; Braunagel, Sharon C.; Summers, Max D.

    2001-01-01

    Partial deletions within Autographa californica open reading frame 61 (FP25K) alter the expression and accumulation profile of several viral proteins and the transport of occlusion-derived virus (ODV)-E66 to intranuclear membranes during infection (S. C. Braunagel et al., J. Virol. 73:8559–8570, 1999). Here we show the effects of a full deletion and overexpression of FP25K on the transport and expression of two ODV envelope proteins, ODV-E66 (E66) and ODV-E25 (E25). Deletion and overexpression of FP25K substantially altered the levels of expression of E66 during infection. Compared with cells infected with wild-type (wt) virus, the levels of E66 were reduced fivefold in cells infected with a viral mutant lacking FP25K (ΔFP25K) and were slightly increased in cells infected with a viral mutant overexpressing FP25K (FP25Kpolh). In contrast, no significant changes were observed in the levels of E25 among wt-, ΔFP25K-, and FP25Kpolh-infected cells. The changes observed in the levels of E66 among the different viral mutants were not accompanied by changes in either the time of synthesis, membrane association, protein turnover, or steady-state transcript abundance. Deletion of FP25K also substantially altered the transport and localization of E66 during infection. In cells infected with the ΔFP25K mutant virus, E66 accumulated in localized regions at the nuclear periphery and the outer nuclear membrane and did not traffic to intranuclear membranes. In contrast, in cells infected with the FP25Kpolh mutant virus E66 trafficked to intranuclear membranes. For comparison, E25 was normally transported to intranuclear membranes in both ΔFP25K- and FP25Kpolh-infected cells. Altogether these studies suggest that FP25K affects the synthesis of E66 at a posttranscriptional level, probably by altering the translation of E66; additionally, the block in transport of E66 at the nuclear envelope in ΔFP25K-infected cells suggests that the pathway of E66 trafficking to the inner

  6. The interplay between TEAD4 and KLF5 promotes breast cancer partially through inhibiting the transcription of p27Kip1

    PubMed Central

    Zhou, Zhongmei; Zhang, Hailin; Liu, Rong; Wu, Jing; Qin, Junying; Ma, Yun; Chen, Liang; Li, Shumo; Chen, Wenlin; Li, Fubing; Shi, Peiguo; Wu, Yingying; Shen, Jian; Chen, Ceshi

    2015-01-01

    Growing evidence suggests that YAP/TAZ are mediators of the Hippo pathway and promote breast cancer. However, the roles of YAP/TAZ transcription factor partners TEADs in breast cancer remain unclear. Here we found that TEAD4 was expressed in breast cancer cell lines, especially in triple negative breast cancers (TNBC) cell lines. TEAD4 binds to KLF5. Knockdown of either TEAD4 or KLF5 in HCC1937 and HCC1806 cells induced the expression of CDK inhibitor p27. Depletion of either TEAD4 or KLF5 activated the p27 gene promoter and increased the p27 mRNA levels. Depletion of p27 partially prevents growth inhibition caused by TEAD4 and KLF5 knockdown. TEAD4 overexpression stimulated proliferation in vitro and tumor growth in mice, while stable knockdown of TEAD4 inhibited proliferation in vitro and tumor growth in mice. Thus TEAD4 and KLF5, in collaboration, promoted TNBC cell proliferation and tumor growth in part by inhibiting p27 gene transcription. TEAD4 is a potential target and biomarker for the development of novel therapeutics for breast cancer. PMID:25970772

  7. The STAR protein QKI-7 recruits PAPD4 to regulate post-transcriptional polyadenylation of target mRNAs

    PubMed Central

    Yamagishi, Ryota; Tsusaka, Takeshi; Mitsunaga, Hiroko; Maehata, Takaharu; Hoshino, Shin-ichi

    2016-01-01

    Emerging evidence has demonstrated that regulating the length of the poly(A) tail on an mRNA is an efficient means of controlling gene expression at the post-transcriptional level. In early development, transcription is silenced and gene expression is primarily regulated by cytoplasmic polyadenylation. In somatic cells, considerable progress has been made toward understanding the mechanisms of negative regulation by deadenylation. However, positive regulation through elongation of the poly(A) tail has not been widely studied due to the difficulty in distinguishing whether any observed increase in length is due to the synthesis of new mRNA, reduced deadenylation or cytoplasmic polyadenylation. Here, we overcame this barrier by developing a method for transcriptional pulse-chase analysis under conditions where deadenylases are suppressed. This strategy was used to show that a member of the Star family of RNA binding proteins, QKI, promotes polyadenylation when tethered to a reporter mRNA. Although multiple RNA binding proteins have been implicated in cytoplasmic polyadenylation during early development, previously only CPEB was known to function in this capacity in somatic cells. Importantly, we show that only the cytoplasmic isoform QKI-7 promotes poly(A) tail extension, and that it does so by recruiting the non-canonical poly(A) polymerase PAPD4 through its unique carboxyl-terminal region. We further show that QKI-7 specifically promotes polyadenylation and translation of three natural target mRNAs (hnRNPA1, p27kip1 and β-catenin) in a manner that is dependent on the QKI response element. An anti-mitogenic signal that induces cell cycle arrest at G1 phase elicits polyadenylation and translation of p27kip1 mRNA via QKI and PAPD4. Taken together, our findings provide significant new insight into a general mechanism for positive regulation of gene expression by post-transcriptional polyadenylation in somatic cells. PMID:26926106

  8. Hyperinvasive Meningococci Induce Intra-nuclear Cleavage of the NF-κB Protein p65/RelA by Meningococcal IgA Protease

    PubMed Central

    Besbes, Anissa; Le Goff, Salomé; Antunes, Ana; Terrade, Aude; Hong, Eva; Giorgini, Dario; Taha, Muhamed-Kheir; Deghmane, Ala-Eddine

    2015-01-01

    Differential modulation of NF-κB during meningococcal infection is critical in innate immune response to meningococcal disease. Non-invasive isolates of Neisseria meningitidis provoke a sustained NF-κB activation in epithelial cells. However, the hyperinvasive isolates of the ST-11 clonal complex (ST-11) only induce an early NF-κB activation followed by a sustained activation of JNK and apoptosis. We show that this temporal activation of NF-κB was caused by specific cleavage at the C-terminal region of NF-κB p65/RelA component within the nucleus of infected cells. This cleavage was mediated by the secreted 150 kDa meningococcal ST-11 IgA protease carrying nuclear localisation signals (NLS) in its α-peptide moiety that allowed efficient intra-nuclear transport. In a collection of non-ST-11 healthy carriage isolates lacking NLS in the α-peptide, secreted IgA protease was devoid of intra-nuclear transport. This part of iga polymorphism allows non-invasive isolates lacking NLS, unlike hyperinvasive ST-11 isolates of N. meningitides habouring NLS in their α-peptide, to be carried asymptomatically in the human nasopharynx through selective eradication of their ability to induce apoptosis in infected epithelial cells. PMID:26241037

  9. Neuronal intranuclear inclusion disease in identical twins.

    PubMed

    Haltia, M; Somer, H; Palo, J; Johnson, W G

    1984-04-01

    A pair of female identical twins exhibited slurred speech, nystagmus, and oculogyral spasms starting at age 11. The patients then had episodic rage, extrapyramidal and lower motor neuron abnormalities, and grand mal seizures, but retained largely normal intelligence, until death at age 21. Severe loss of nigral and craniospinal motor neurons was noted postmortem. Round, eosinophilic, autofluorescent inclusion bodies, 3 to 10 microns in diameter, were observed in the nuclei of most nerve cell types of the central and peripheral nervous systems and retina. Ultrastructurally the inclusions appeared as masses of filaments without a limiting membrane, the constituent filaments having a diameter of 8.5 to 9.5 nm. Histochemical results suggested the presence of proteins with a high content of tryptophan. Four similar cases have been reported previously under various designations. We propose the name neuronal intranuclear inclusion disease for the disorder. PMID:6331275

  10. A novel partner for D-type cyclins: protein kinase A-anchoring protein AKAP95.

    PubMed Central

    Arsenijevic, Tatjana; Degraef, Chantal; Dumont, Jacques E; Roger, Pierre P; Pirson, Isabelle

    2004-01-01

    Using a yeast interaction screen to search for proteins that interact with cyclin D3 in thyroid gland, we identified the cAMP-dependent AKAP95 (protein kinase A-anchoring protein 95). AKAP95 is a scaffolding protein that primarily co-fractionates with the nuclear matrix, whereas a minor fraction associates with chromatin in interphase cells. In co-transfected Chinese-hamster ovary cells, AKAP95 strongly interacted with the three D-type cyclins, but not with CDK4 (cyclin-dependent kinase 4) or with p27kip1. CDK4 displaced the interaction between cyclin D3 and AKAP95, suggesting that AKAP95 could not be the elusive bridging adaptor between D-type cyclins and CDK4 or play a role in the regulation of cyclin D3-CDK4 activity. Interaction between endogenous AKAP95 and cyclin D3 or cyclin D1 was detected in canine thyrocytes, human fibroblasts and NIH-3T3 cells. As both AKAP95 and cyclins D were recently reported to associate with minichromosome maintenance proteins [Eide, Tasken, Carlson, Williams, Jahnsen, Tasken and Collas (2003) J. Biol. Chem. 278, 26750-26756; Gladden and Diehl (2003) J. Biol. Chem. 278, 9754-9760], we hypothesize that the interaction between AKAP95 and D-type cyclins might serve to facilitate the emerging regulatory role of cyclin D-CDK4 in the formation of the prereplication complex at the DNA replication origins. PMID:14641107

  11. DNA polymerase alpha associated protein P1, a murine homolog of yeast MCM3, changes its intranuclear distribution during the DNA synthetic period.

    PubMed Central

    Kimura, H; Nozaki, N; Sugimoto, K

    1994-01-01

    We isolated a murine gene for the DNA polymerase alpha associated protein P1, which shares high homology with the budding yeast MCM3 protein, which is a member of a protein family involved in the early event of DNA replication having a putative DNA-dependent ATPase motif. Using a polyclonal anti-P1 antibody raised against a beta-galactosidase-P1 fusion protein, we identified at least two forms of P1 protein in the nucleus of a mouse cell line, an underphosphorylated form that was associated with a particular nuclear structure and a hyperphosphorylated form loosely bound to the nucleus. During progression through S phase, P1 disappeared, first from the euchromatic region, then from the heterochromatic region, apparently in parallel with temporally ordered DNA replication. Thus, it is likely that the underphosphorylated P1 is dissociated from the nuclear structure after DNA replication by cell cycle-dependent phosphorylation. This is the first direct observation of a protein whose behavior is consistent with that of a hypothetical factor which restricts the chromatin to replicate once per cell cycle in higher eukaryotes. Images PMID:7925275

  12. Spatial Localization of Genes Determined by Intranuclear DNA Fragmentation with the Fusion Proteins Lamin KRED and Histone KRED und Visible Light

    PubMed Central

    Waldeck, Waldemar; Mueller, Gabriele; Glatting, Karl-Heinz; Hotz-Wagenblatt, Agnes; Diessl, Nicolle; Chotewutmonti, Sasithorn; Langowski, Jörg; Semmler, Wolfhard; Wiessler, Manfred; Braun, Klaus

    2013-01-01

    The highly organized DNA architecture inside of the nuclei of cells is accepted in the scientific world. In the human genome about 3 billion nucleotides are organized as chromatin in the cell nucleus. In general, they are involved in gene regulation and transcription by histone modification. Small chromosomes are localized in a central nuclear position whereas the large chromosomes are peripherally positioned. In our experiments we inserted fusion proteins consisting of a component of the nuclear lamina (lamin B1) and also histone H2A, both combined with the light inducible fluorescence protein KillerRed (KRED). After activation, KRED generates reactive oxygen species (ROS) producing toxic effects and may cause cell death. We analyzed the spatial damage distribution in the chromatin after illumination of the cells with visible light. The extent of DNA damage was strongly dependent on its localization inside of nuclei. The ROS activity allowed to gain information about the location of genes and their functions via sequencing and data base analysis of the double strand breaks of the isolated DNA. A connection between the damaged gene sequences and some diseases was found. PMID:23869190

  13. Ultrastructural Studies of H-1 Parvovirus Replication VI. Simultaneous Autoradiographic and Immunochemical Intranuclear Localization of Viral DNA Synthesis and Protein Accumulation

    PubMed Central

    Singer, Irwin I.; Rhode, Solon L.

    1978-01-01

    The localization of H-1 viral replicative-form double-stranded DNA and progeny single-stranded DNA replication in parasynchronously infected, simian virus 40-transformed newborn human kidney cells was studied with high-resolution electron microscope autoradiography (80-nm silver grains). We analyzed wild-type H-1 and ts1 H-1 (a conditional mutant defective in progeny single-stranded DNA synthesis). The proportion of the total DNA synthesis that was viral was estimated to be >90% by comparing the amount of [3H]thymidine uptake in cultures infected with wild-type H-1 versus ts14 (an H-1 mutant defective in DNA replication). Simultaneous staining with cytochrome c-conjugated anti-H-1 immunoglobulin G was performed to ensure that cells incorporating [3H]thymidine (2- to 60-min pulses) were H-1 infected. The sites of H-1 replicative-form (in ts1-infected cells) and progeny (in wild-type-infected cells) DNA synthesis were identical. Immunospecifically labeled nuclei at the earliest stages of infection exhibited dense clusters of silver grains over material extruded from nucleolar fibrillar centers. These foci became larger with increasing cellular damage, forming a limited number of H-1 DNA synthetic centers in the euchromatin. Each island-like focus was surrounded by tufts of heterochromatin containing high concentrations of unassembled H-1 capsid proteins. In late phases of infection, the heterochromatin became completely marginated, and the nucleoplasm contained only euchromatin that exhibited randomly distributed sites of H-1 DNA replication. This indicates that H-1 DNA synthesis begins at localized euchromatic or nucleolar sites and then spreads outward. Immunostained heterochromatin and nucleolar chromatin never incorporated [3H]thymidine. Our results suggest that H-1 proteins and cellular cofactors associated with the fibrillar component of the nucleolus and the euchromatin may play a role in the regulation of H-1 DNA synthesis. Images PMID:340710

  14. An unusual degenerative disorder of neurons associated with a novel intranuclear hyaline inclusion (neuronal intranuclear hyaline inclusion disease). A clinicopathological study of a case.

    PubMed

    Sung, J H; Ramirez-Lassepas, M; Mastri, A R; Larkin, S M

    1980-03-01

    A 21-year-old woman with an unusual, progressive, degenerative neurological disorder is described. The disorder is characterized clinically by behavioral abnormality, peculiar involuntary movements, and ataxia starting in early childhood and subsequent development of dementia, choreoathetosis, rectal and bladder incontinence, bulbar and spinal muscular weakness, pes cavus, kyphoscoliosis, and generalized seizures. The clinical manifestations are correlated, with widespread pathological changes affecting almost all neuronal systems. The pathological changes are discussed in relation to the wide spectrum of "multisystem atrophies." Particular attention is directed to the ubiquitous occurrence of a novel intranuclear, eosinophilic, hyaline inclusion in almost all types of central, peripheral, and autonomic neurons. The ubiquitous neuronal involvement seems to explain the diffuse multiple system degeneration. The pathogenesis of the neuronal inclusions is unknown, but it is speculated that the disorder may represent a metabolic abnormality affecting the nuclear protein of neurons, rather than a viral infection. The pathological features, consisting of the neuronal intranuclear hyaline inclusions associated with multiple system atrophy, have not hitherto been described, and "neuronal intranuclear hyaline inclusion disease" is proposed as a name for the disorder. Rectal biopsy demonstrating the intranuclear hyaline inclusions in ganglion cells of the hyenteric plexuses may serve as a diagnostic procedure for the disorder. PMID:6154779

  15. Neuronal intranuclear inclusion disease and juvenile parkinsonism.

    PubMed

    O'Sullivan, J D; Hanagasi, H A; Daniel, S E; Tidswell, P; Davies, S W; Lees, A J

    2000-09-01

    Juvenile parkinsonism (onset age <20 yrs) is uncommon and few cases with neuropathologic confirmation have been reported. We present the case of a 17-year-old boy who presented with asymmetric arm tremor and bulbar symptoms. His paternal great aunt had parkinsonism with onset at age 22 years. Examination revealed parkinsonism in the absence of additional neurologic signs except for delayed pupillary responses to light. He responded well to levodopa but developed motor fluctuations and disabling dyskinesias after 3 years of treatment. Following attempted withdrawal of levodopa at age 24 years, he developed severe aspiration pneumonia complicated by cardiorepiratory arrests and he died 6 months later. At autopsy, the dominant histologic feature was wide-spread neuronal hyaline intranuclear inclusions. Neuronal depletion was observed in the substantia nigra, locus ceruleus, and, to a lesser extent, in the frontal cortex, and inclusions were particularly prominent in these areas. Inclusions were immunoreactive for ubiquitin and were typical of those seen in neuronal intranuclear inclusion disease (NIID), a rare, multisytem neurodegenerative disease. NIID should be considered in the differential diagnosis of juvenile parkinsonism. A link between NIID and hereditary neurodegenerative disorders characterized by expanded polyglutamine tracts is supported by the similar appearance of intranuclear inclusions in both conditions and by a family history in some cases of NIID. PMID:11009211

  16. Heat Shock Protein B1-Deficient Mice Display Impaired Wound Healing

    PubMed Central

    McNamee, Kay; Przybycien, Paulina M.; Lu, Xin; Williams, Richard O.; Bou-Gharios, George; Saklatvala, Jeremy; Dean, Jonathan L. E.

    2013-01-01

    There is large literature describing in vitro experiments on heat shock protein (hsp)B1 but understanding of its function in vivo is limited to studies in mice overexpressing human hspB1 protein. Experiments in cells have shown that hspB1 has chaperone activity, a cytoprotective role, regulates inflammatory gene expression, and drives cell proliferation. To investigate the function of the protein in vivo we generated hspB1-deficient mice. HspB1-deficient fibroblasts display increased expression of the pro-inflammatory cytokine, interleukin-6, compared to wild-type cells, but reduced proliferation. HspB1-deficient fibroblasts exhibit reduced entry into S phase and increased expression of cyclin-dependent kinase inhibitors p27kip1 and p21waf1. The expression of hspB1 protein and mRNA is also controlled by the cell cycle. To investigate the physiological function of hspB1 in regulating inflammation and cell proliferation we used an excisional cutaneous wound healing model. There was a significant impairment in the rate of healing of wounds in hspB1-deficient mice, characterised by reduced re-epithelialisation and collagen deposition but also increased inflammation. HspB1 deficiency augments neutrophil infiltration in wounds, driven by increased chemokine (C-X-C motif) ligand 1 expression. This appears to be a general mechanism as similar results were obtained in the air-pouch and peritonitis models of acute inflammation. PMID:24143227

  17. Protein Kinase C alpha (PKCα) dependent signaling mediates endometrial cancer cell growth and tumorigenesis

    PubMed Central

    Haughian, James M.; Reno, Elaine M.; Thorne, Alicia M.; Bradford, Andrew P.

    2009-01-01

    Endometrial cancer is the most common invasive gynecologic malignancy, yet molecular mechanisms and signaling pathways underlying its etiology and pathophysiology remain poorly characterized. We sought to define a functional role for the protein kinase C (PKC) isoform, PKCα, in an established cell model of endometrial adenocarcinoma. Ishikawa cells depleted of PKCα protein grew slower, formed fewer colonies in anchorage-independent growth assays and exhibited impaired xenograft tumor formation in nude mice. Consistent with impaired growth, PKCα knockdown increased levels of the cyclin dependent kinase (CDK) inhibitors p21Cip1/WAF1 (p21) and p27Kip1 (p27). Despite the absence of functional phosphatase and tensin homologue (PTEN) protein in Ishikawa cells, PKCα knockdown reduced Akt phosphorylation at serine 473 and concomitantly inhibited phosphorylation of the Akt target, glycogen synthase kinase-3β (GSK-3β). PKCα knockdown also resulted in decreased basal ERK phosphorylation and attenuated ERK activation following EGF stimulation. p21 and p27 expression was not increased by treatment of Ishikawa cells with ERK and Akt inhibitors, suggesting PKCα regulates CDK expression independently of Akt and ERK. Immunohistochemical analysis of grade 1 endometrioid adenocarcinoma revealed aberrant PKCα expression, with foci of elevated PKCα staining, not observed in normal endometrium. These studies demonstrate a critical role for PKCα signaling in endometrial tumorigenesis by regulating expression of CDK inhibitors p21 and p27 and activation of Akt and ERK dependent proliferative pathways. Thus, targeting PKCα may provide novel therapeutic options in endometrial tumors. PMID:19672862

  18. Protein Kinase C Signaling Mediates a Program of Cell Cycle Withdrawal in the Intestinal Epithelium

    PubMed Central

    Frey, Mark R.; Clark, Jennifer A.; Leontieva, Olga; Uronis, Joshua M.; Black, Adrian R.; Black, Jennifer D.

    2000-01-01

    Members of the protein kinase C (PKC) family of signal transduction molecules have been widely implicated in regulation of cell growth and differentiation, although the underlying molecular mechanisms involved remain poorly defined. Using combined in vitro and in vivo intestinal epithelial model systems, we demonstrate that PKC signaling can trigger a coordinated program of molecular events leading to cell cycle withdrawal into G0. PKC activation in the IEC-18 intestinal crypt cell line resulted in rapid downregulation of D-type cyclins and differential induction of p21waf1/cip1 and p27kip1, thus targeting all of the major G1/S cyclin-dependent kinase complexes. These events were associated with coordinated alterations in expression and phosphorylation of the pocket proteins p107, pRb, and p130 that drive cells to exit the cell cycle into G0 as indicated by concomitant downregulation of the DNA licensing factor cdc6. Manipulation of PKC isozyme levels in IEC-18 cells demonstrated that PKCα alone can trigger hallmark events of cell cycle withdrawal in intestinal epithelial cells. Notably, analysis of the developmental control of cell cycle regulatory molecules along the crypt–villus axis revealed that PKCα activation is appropriately positioned within intestinal crypts to trigger this program of cell cycle exit–specific events in situ. Together, these data point to PKCα as a key regulator of cell cycle withdrawal in the intestinal epithelium. PMID:11076962

  19. Ultrastructural cytochemical analysis of intranuclear arsenic inclusions

    SciTech Connect

    Sorensen, E.M.B.

    1987-01-01

    To establish the chemical composition of the arsenic inclusion, freshly isolated preparations of inclusions and epon-embedded thin sections of inclusions were subjected to ultrastructural cytochemical analysis. Intranuclear inclusions are composed of amorphous, arsenic-containing subunits aligned linearly to form a coiled complex. Lipase, ribonuclease, deoxyribonuclease, trypsin, pepsin, protease, amylase, or ethylenediaminetetraacetic acid (EDTA) was used to digest or chelate these inclusions. Following enzymatic digestion or chelation, the electron opacity of inclusions was compared with that of control sections exposed for equal times to equivalent solutions lacking the enzymes. Exposure to amylase caused a consistent reduction in the electron opacity of thin sections of inclusions and almost complete digestion of the freshly isolated preparations of inclusions. This was indicative of the presence of a carbohydrate moiety within arsenic inclusions. Incubation of inclusions with EDTA resulted in solubilization of freshly isolated and thin-sectioned embedded material. These data indicated that the intranuclear arsenic inclusion is composed of both metallic and carbohydrate moieties, confirming earlier studies which identified arsenic within inclusions using instrumental neutron activation analysis and x-ray microprobe analysis.

  20. The p53 tumor suppressor protein protects against chemotherapeutic stress and apoptosis in human medulloblastoma cells

    PubMed Central

    Parasido, Erika; Tricoli, Lucas; Sivakumar, Angiela; Mikhaiel, John P.; Yenugonda, Venkata; Rodriguez, Olga C.; Karam, Sana D.; Rood, Brian R.; Avantaggiati, Maria Laura; Albanese, Chris

    2015-01-01

    Medulloblastoma (MB), a primitive neuroectodermal tumor, is the most common malignant childhood brain tumor and remains incurable in about a third of patients. Currently, survivors carry a significant burden of late treatment effects. The p53 tumor suppressor protein plays a crucial role in influencing cell survival in response to cellular stress and while the p53 pathway is considered a key determinant of anti-tumor responses in many tumors, its role in cell survival in MB is much less well defined. Herein, we report that the experimental drug VMY-1-103 acts through induction of a partial DNA damage-like response as well induction of non-survival autophagy. Surprisingly, the genetic or chemical silencing of p53 significantly enhanced the cytotoxic effects of both VMY and the DNA damaging drug, doxorubicin. The inhibition of p53 in the presence of VMY revealed increased late stage apoptosis, increased DNA fragmentation and increased expression of genes involved in apoptosis, including CAPN12 and TRPM8, p63, p73, BIK, EndoG, CIDEB, P27Kip1 and P21cip1. These data provide the groundwork for additional studies on VMY as a therapeutic drug and support further investigations into the intriguing possibility that targeting p53 function may be an effective means of enhancing clinical outcomes in MB. PMID:26540407

  1. The soybean-derived peptide lunasin inhibits non-small cell lung cancer cell proliferation by suppressing phosphorylation of the retinoblastoma protein

    PubMed Central

    McConnell, Elizabeth J.; Devapatla, Bharat; Yaddanapudi, Kavitha; Davis, Keith R.

    2015-01-01

    Lunasin, a soybean bioactive peptide, has both chemopreventive and chemotherapeutic activities. The aim of this study was to determine the chemotherapeutic potential of lunasin against human lung cancer. Treatment of non-small cell lung cancer (NSCLC) cells with highly purified soybean-derived lunasin caused limited, cell-line specific anti-proliferative effects on anchorage-dependent growth whereas two normal bronchial epithelial cell lines were unaffected. Lunasin's antiproliferative effects were potentiated upon utilization of anchorage-independent conditions. Furthermore, NSCLC cell lines that were unaffected by lunasin in anchorage-dependent assays exhibited a dose-dependent inhibition in colony formation or colony size. Mouse xenograft studies revealed that 30 mg lunasin/kg body weight per day decreased NSCLC H1299 tumor volume by 63.0% at day 32. Mechanistic studies using cultured NSCLC H661 cells showed that lunasin inhibited cell cycle progression at the G1/S phase interface without inducing apoptosis. Immunoblot analyses of key cell-cycle proteins demonstrated that lunasin altered the expression of the G1 specific cyclin-dependent kinase complex components, increased levels of p27Kip1, reduced levels of phosphorylated Akt, and ultimately inhibited the sequential phosphorylation of the retinoblastoma protein (RB). These results establish for the first time that lunasin can inhibit NSCLC proliferation by suppressing cell-cycle dependent phosphorylation of RB. PMID:25609198

  2. RNF135, RING finger protein, promotes the proliferation of human glioblastoma cells in vivo and in vitro via the ERK pathway

    PubMed Central

    Liu, Yongjian; Wang, Feng; Liu, Yongsheng; Yao, Yiqun; Lv, Xiupeng; Dong, Bin; Li, Jun; Ren, Siyang; Yao, Yiwen; Xu, Yinghui

    2016-01-01

    Ring finger protein 135 (RNF135), located on chromosome 17q11.2, is a RING finger domain-containing E3 ubiquitin ligase that was identified as a bio-marker and therapy target of glioblastoma. In our study, we confirmed that RNF135 was up-regulated in glioblastoma tissues compared with normal brain (NB) tissues, and that RNF135 knockdown inhibited proliferation and migration and led to cell cycle arrest in the G0/G1 phase in vivo. By lowering RNF135 expression, phosphorylated Erk and cell cycle protein CDK4 were down-regulated, while p27Kip1 and p21Waf1/Cip1 were up-regulated in U87 and U251 cells in vitro. In addition, using the immunofluorescence double labelling method, we found that RNF135 and P-Erk were co-localized in the cytoplasm and were highly expressed in glioblastoma samples compared with NB tissues. Moreover, the growth of U87 cell-transplanted tumours in nude mice was inhibited while transduced with Lv-shRNF135. Taken together, our findings demonstrate the biological effects of RNF135 in glioblastoma cell proliferation, migration and cell cycle, and its role in the progression of glioblastoma may be associated with the ERK signal transduction pathway. PMID:26856755

  3. The cyclopentenone 15-deoxy-delta(12,14)-prostaglandin J2 inhibits G1/S transition and retinoblastoma protein phosphorylation in immortalized lymphocytes from Alzheimer's disease patients.

    PubMed

    Muñoz, Ursula; de Las Cuevas, Natividad; Bartolomé, Fernando; Hermida, Ofelia G; Bermejo, Félix; Martín-Requero, Angeles

    2005-10-01

    Epidemiologic studies indicated that non-steroidal anti-inflammatory drugs (NSAIDs) might prevent or delay the clinical features of Alzheimer disease (AD). The pharmacological activity of NSAIDs is generally attributed to inhibition of cyclooxygenase and peroxisome proliferator-activated receptor gamma (PPARgamma) activation. Based on the antineoplastic and apoptotic effects of PPARgamma activation in a number of cell types, we hypothesized that NSAIDs could protect neurons by controlling the regulation of cell cycle. Recent work suggests that uncoordinated expression of cell cycle molecules and perturbation of cell cycle checkpoints may be one of the mechanisms by which post-mitotic neurons die. Since cell cycle dysfunction is not restricted to neurons in AD, we found it interesting to study the role of PPARgamma activation on cell proliferation in immortalized lymphocytes from AD patients. We report here that 15-deoxy-delta(12,14)-prostaglandin J2 (15d-PGJ2), but not NSAIDs or thiazolidinediones inhibited the serum-mediated enhancement of cell proliferation in AD by blocking the events critical for G1/S transition. The cyclopentenone induced a partial inhibition of retinoblastoma protein phosphorylation and increased levels of the CDK inhibitor p27kip1. PMID:16061222

  4. Nuclear export of proteins and drug resistance in cancer

    PubMed Central

    Turner, Joel G.; Dawson, Jana; Sullivan, Daniel M.

    2015-01-01

    The intracellular location of a protein is crucial to its normal functioning in a cell. Cancer cells utilize the normal processes of nuclear-cytoplasmic transport through the nuclear pore complex of a cell to effectively evade anti-neoplastic mechanisms. CRM1-mediated export is increased in various cancers. Proteins that are exported in cancer include tumor-suppressive proteins such as retinoblastoma, APC, p53, BRAC1, FOXO proteins, INI1/hSNF5, galectin-3, Bok, nucleophosmin, RASSF2, Merlin, p21CIP, p27KIP1, N-WASP/FAK, estradiol receptor and Tob, drug targets topoisomerase I and IIα and BCR-ABL, and the molecular chaperone protein Hsp90. Here, we review in detail the current processes and known structures involved in the export of a protein through the nuclear pore complex. We also discuss the export receptor molecule CRM1 and its binding to the leucine-rich nuclear export signal of the cargo protein and the formation of a nuclear export trimer with RanGTP. The therapeutic potential of various CRM1 inhibitors will be addressed, including leptomycin B, ratjadone, KOS-2464, and specific small molecule inhibitors of CRM1, N-azolylacrylate analogs, FOXO export inhibitors, valtrate, acetoxychavicol acetate, CBS9106, and SINE inhibitors. We will also discuss examples of how drug resistance may be reversed by targeting the exported proteins topoisomerase IIα, BCR-ABL, and galectin-3. As effective and less toxic CRM1 export inhibitors become available, they may be used as both single agents and in combination with current chemotherapeutic drugs. We believe that the future development of low-toxicity, small-molecule CRM1 inhibitors may provide a new approach to treating cancer. PMID:22209898

  5. INTRANUCLEAR MATRIX METALLOPROTEINASES PROMOTE DNA DAMAGE AND APOPTOSIS INDUCED BY OXYGEN–GLUCOSE DEPRIVATION IN NEURONS

    PubMed Central

    HILL, J. W.; PODDAR, R.; THOMPSON, J. F.; ROSENBERG, G. A.; YANG, Y.

    2016-01-01

    Degradation of the extracellular matrix by elevated matrix metalloproteinase (MMP) activity following ischemia/reperfusion is implicated in blood–brain barrier disruption and neuronal death. In contrast to their characterized extracellular roles, we previously reported that elevated intranuclear MMP-2 and -9 (gelatinase) activity degrades nuclear DNA repair proteins and promotes accumulation of oxidative DNA damage in neurons in rat brain at 3-h reperfusion after ischemic stroke. Here, we report that treatment with a broad-spectrum MMP inhibitor significantly reduced neuronal apoptosis in rat ischemic hemispheres at 48-h reperfusion after a 90-min middle cerebral artery occlusion (MCAO). Since extracellular gelatinases in brain tissue are known to be neurotoxic during acute stroke, the contribution of intranuclear MMP-2 and -9 activities in neurons to neuronal apoptosis has been unclear. To confirm and extend our in vivo observations, oxygen–glucose deprivation (OGD), an in vitro model of ischemia/reperfusion, was employed. Primary cortical neurons were subjected to 2-h OGD with reoxygenation. Increased intranuclear gelatinase activity was detected immediately after reoxygenation onset and was maximal at 24 h, while extracellular gelatinase levels remained unchanged. We detected elevated levels of both MMP-2 and -9 in neuronal nuclear extracts and gelatinase activity in neurons co-localized primarily with MMP-2. We found a marked decrease in PARP1, XRCC1, and OGG1, and decreased PARP1 activity. Pretreatment of neurons with selective MMP-2/9 inhibitor II significantly decreased gelatinase activity and downregulation of DNA repair enzymes, decreased accumulation of oxidative DNA damage, and promoted neuronal survival after OGD. Our results confirm the nuclear localization of gelatinases and their nuclear substrates observed in an animal stroke model, further supporting a novel role for intranuclear gelatinase activity in an intrinsic apoptotic pathway in neurons

  6. Intranuclear bacteria: inside the cellular control center of eukaryotes.

    PubMed

    Schulz, Frederik; Horn, Matthias

    2015-06-01

    Intracellular bacteria including major pathogens live in the cytoplasm or in cytoplasmic vacuoles within their host cell. However, some can invade more unusual intracellular niches such as the eukaryotic nucleus. Phylogenetically diverse intranuclear bacteria have been discovered in various protist, arthropod, marine invertebrate, and mammalian hosts. Although targeting the same cellular compartment, they have apparently developed fundamentally-different infection strategies. The nucleus provides a rich pool of nutrients and protection against host cytoplasmic defense mechanisms; intranuclear bacteria can directly manipulate the host by interfering with nuclear processes. The impact on their host cells ranges from stable associations with a neutral or beneficial effect on host fitness to rapid host lysis. The analysis of the intranuclear lifestyle will extend our current framework for understanding host-pathogen interactions. PMID:25680230

  7. Constitutive photomorphogenesis protein 1 (COP1) and COP9 signalosome, evolutionarily conserved photomorphogenic proteins as possible targets of melatonin.

    PubMed

    Sanchez-Barcelo, Emilio J; Mediavilla, Maria D; Vriend, Jerry; Reiter, Russel J

    2016-08-01

    The ubiquitin proteasome system has been proposed as a possible mechanism involved in the multiple actions of melatonin. COP1 (constitutive photomorphogenesis protein 1), a RING finger-type ubiquitin E3 ligase formerly identified in Arabidopsis, is a central switch for the transition from plant growth underground in darkness (etiolation) to growth under light exposure (photomorphogenesis). In darkness, COP1 binds to photomorphogenic transcription factors driving its degradation via the 26S proteasome; blue light, detected by cryptochromes, and red and far-red light detected by phytochromes, negatively regulate COP1. Homologues of plant COP1 containing all the structural features present in Arabidopsis as well as E3 ubiquitin ligase activity have been identified in mice and humans. Substrates for mammalian (m) COP1 include p53, AP-1 and c-Jun, p27(Kip1) , ETV1, MVP, 14-3-3σ, C/EBPα, MTA1, PEA3, ACC, TORC2 and FOXO1. This mCOP1 target suggests functions related to tumorigenesis, gluconeogenesis, and lipid metabolism. The role of mCOP1 in tumorigenesis (either as a tumor promoter or tumor suppressor), as well as in glucose metabolism (inhibition of gluconeogenesis) and lipid metabolism (inhibition of fatty acid synthesis), has been previously demonstrated. COP1, along with numerous other ubiquitin ligases, is regulated by the COP9 signalosome; this protein complex is associated with the oxidative stress sensor Keap1 and the deubiquitinase USP15. The objective of this review was to provide new information on the possible role of COP1 and COP9 as melatonin targets. The hypothesis is based on common functional aspects of melatonin and COP1 and COP9, including their dependence on light, regulation of the metabolism, and their control of tumor growth. PMID:27121162

  8. Nucleon removal from unstable nuclei investigated via intranuclear cascade

    SciTech Connect

    Louchart, C.; Obertelli, A.; Boudard, A.; Flavigny, F.

    2011-01-15

    An intranuclear cascade model for one-nucleon removal cross sections at intermediate energies beyond the sudden and inert-core assumptions is presented. Indirect processes are taken into account in the reaction mechanism via a Monte Carlo-based approach. The formalism is applied to a set of weakly bound nuclei for which data are available. Results are in correct agreement with experiment including the case of deeply bound nucleon removal. A comparison to eikonal predictions is performed.

  9. MIF4G domain containing protein regulates cell cycle and hepatic carcinogenesis by antagonizing CDK2-dependent p27 stability.

    PubMed

    Wan, C; Hou, S; Ni, R; Lv, L; Ding, Z; Huang, X; Hang, Q; He, S; Wang, Y; Cheng, C; Gu, X X; Xu, G; Shen, A

    2015-01-01

    The CDK inhibitor p27(kip1) plays crucial roles in cell cycle regulation and cancer progression. Through yeast two-hybrid screening, we identified MIF4G domain containing protein (MIF4GD) as a novel binding partner for p27. The association of MIF4GD and p27 was verified using immunoprecipitation and glutathione S-transferase (GST) pull-down assays. Interaction with MIF4GD led to the stabilization of p27 both in the nucleus and in the cytoplasm in hepatocellular carcinoma (HCC) cells as a result of suppressed phosphorylation of p27 by CDK2 at threonine187. Serum stimulation decreased the levels of MIF4GD and p27 simultaneously. In addition, MIF4GD overexpression resulted in increased p27 levels and reduced cell proliferation, while knockdown of MIF4GD promoted cell cycle progression with decreased p27 levels in cells. Furthermore, overexpression of MIF4GD reduced colony formation and inhibited xenograft tumor growth in nude mice. Finally, we found that both MIF4GD and p27 were expressed at low levels in HCC tissues compared to non-cancerous tissues, and that low expression levels of MIF4GD and p27 were associated with significantly worse prognosis in HCC patients. Our results suggest that MIF4GD is a potential regulator of p27-dependent cell proliferation in HCC. These findings provide a rational framework for the development of potential HCC therapy by targeting the MIF4GD-p27 interaction. PMID:24336329

  10. A model for mammalian cochlear hair cell differentiation in vitro: effects of retinoic acid on cytoskeletal proteins and potassium conductances.

    PubMed

    Helyer, R; Cacciabue-Rivolta, D; Davies, D; Rivolta, M N; Kros, C J; Holley, M C

    2007-02-01

    We have established a model for the in-vitro differentiation of mouse cochlear hair cells and have used it to explore the influence of retinoic acid on proliferation, cytoskeletal proteins and voltage-gated potassium conductances. The model is based on the conditionally immortal cell line University of Sheffield/ventral otocyst-epithelial cell line clone 36 (US/VOT-E36), derived from ventral otic epithelial cells of the mouse at embryonic day 10.5 and transfected with a reporter for myosin VIIa. Retinoic acid did not increase cell proliferation but led to up-regulation of myosin VIIa and formation of prominent actin rings that gave rise to numerous large, linear actin bundles. Cells expressing myosin VIIa had larger potassium conductances and did not express the cyclin-dependent kinase inhibitor p27(kip1). US/VOT-E36 endogenously expressed the voltage-gated potassium channel alpha-subunits Kv1.3 and Kv2.1, which we subsequently identified in embryonic and neonatal hair cells in both auditory and vestibular sensory epithelia in vivo. These subunits could underlie the embryonic and neonatal delayed-rectifiers recorded in nascent hair cells in vivo. Kv2.1 was particularly prominent on the basolateral membrane of cochlear inner hair cells. Kv1.3 was distributed throughout all hair cells but tended to be localized to the cuticular plates. US/VOT-E36 recapitulates a coherent pattern of cell differentiation under the influence of retinoic acid and will provide a convenient model for screening the effects of other extrinsic factors on the differentiation of cochlear epithelial cell types in vitro. PMID:17331193

  11. Intranuclear dynamics of the Nup107-160 complex

    PubMed Central

    Morchoisne-Bolhy, Stéphanie; Geoffroy, Marie-Claude; Bouhlel, Imène B.; Alves, Annabelle; Audugé, Nicolas; Baudin, Xavier; Van Bortle, Kevin; Powers, Maureen A.; Doye, Valérie

    2015-01-01

    Nup98 is a glycine-leucine-phenylalanine-glycine (GLFG) repeat–containing nucleoporin that, in addition to nuclear transport, contributes to multiple aspects of gene regulation. Previous studies revealed its dynamic localization within intranuclear structures known as GLFG bodies. Here we show that the mammalian Nup107-160 complex (Y-complex), a major scaffold module of the nuclear pore, together with its partner Elys, colocalizes with Nup98 in GLFG bodies. The frequency and size of GLFG bodies vary among HeLa sublines, and we find that an increased level of Nup98 is associated with the presence of bodies. Recruitment of the Y-complex and Elys into GLFG bodies requires the C-terminal domain of Nup98. During cell division, Y-Nup–containing GLFG bodies are disassembled in mitotic prophase, significantly ahead of nuclear pore disassembly. FRAP studies revealed that, unlike at nuclear pores, the Y-complex shuttles into and out of GLFG bodies. Finally, we show that within the nucleoplasm, a fraction of Nup107, a key component of the Y-complex, displays reduced mobility, suggesting interaction with other nuclear components. Together our data uncover a previously neglected intranuclear pool of the Y-complex that may underscore a yet-uncharacterized function of these nucleoporins inside the nucleus, even in cells that contain no detectable GLFG bodies. PMID:25904327

  12. 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

  13. 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) induces expression of p27(kip¹) and FoxO3a in female rat cerebral cortex and PC12 cells.

    PubMed

    Xu, Guangfei; Liu, Jiao; Yoshimoto, Katsuhiko; Chen, Gang; Iwata, Takeo; Mizusawa, Noriko; Duan, Zhiqing; Wan, Chunhua; Jiang, Junkang

    2014-05-01

    2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is a potent toxin that alters normal brain development, producing cognitive disability and motor dysfunction. Previous studies in rats have proved that female rats are more sensitive to TCDD lethality than male ones. Recent studies have shown that TCDD induces cell cycle arrest and apoptosis, but the regulatory proteins involved in these processes have yet to be elucidated. In this study, we constructed an acute TCDD injury female rat model, and investigated the effects of TCDD on apoptosis and expression of cell cycle regulators, forkhead box class O 3a (FoxO3a) and p27(kip1), in the central nervous system (CNS). Increased levels of active caspase-3 were observed in the cerebral cortex of female rats treated with TCDD, suggesting that TCDD-induced apoptosis occurs in the CNS. The terminal deoxynucleotidyl transferase-mediated biotinylated-dUTP nick-end labeling assay showed that apoptosis primarily occurred in neurons. Furthermore, Western blot analysis, reverse transcription-polymerase chain reaction, and immunohistochemistry showed a significant up-regulation of FoxO3a and p27(kip1) in the cerebral cortex. Immunofluorescent labeling indicated that FoxO3a and p27(kip1) were predominantly localized in apoptotic neurons, but not in astrocytes. In vitro experiments using PC12, a rat neuron-like pheochromocytoma cell line, also revealed that TCDD induced apoptosis and an increase in FoxO3a and p27(kip1) expression. Furthermore, knockdown of FoxO3a expression inhibited p27(kip1) transcription and TCDD-induced apoptosis. Based on our data, induction of FoxO3a may play an important role in TCDD-induced neurotoxicity. PMID:24594276

  14. Distribution and frequency of intranuclear inclusions in female CGG KI mice modeling the fragile X premutation☆

    PubMed Central

    Schluter, Erik W.; Hunsaker, Michael R.; Greco, Claudia M.; Willemsen, Rob; Berman, Robert F.

    2012-01-01

    The fragile X-associated tremor/ataxia syndrome (FXTAS) is an adult-onset neurodegenerative disorder caused by CGG trinucleotide repeat expansions in the fragile X mental retardation 1 (FMR1) gene. The neuropathological hallmark of FXTAS is the presence of ubiquitin-positive intranuclear inclusions in neurons and in astroglia. Intranuclear inclusions have also been reported in the neurons of male CGG KI mice carrying an expanded CGG trinucleotide repeat and used to model FXTAS, but no study has been carried out quantifying inclusions in female CGG KI mice heterozygous for the fragile X premutation. We used histologic and immunocytochemical methods to determine the pathological features of intranuclear inclusions in astroglia and neurons. In female CGG KI mice, ubiquitin-positive intranuclear inclusions were found in neurons and astroglia throughout the brain in cortical and subcortical regions. These inclusions increased in number and became larger with advanced age and increasing CGG repeat length, supporting hypotheses that these pathologic features are progressive across the lifespan. The number of inclusions in neurons was reduced by ~25% in female CGG KI mice compared to male CGG KI mice, but not so low as the 50% predicted. These data emphasize the need to evaluate the neurocognitive and pathological features in female carriers of the fragile X premutation with and without FXTAS symptomatology is warranted, as this population shows similar neuropathological features present in male FXTAS patients. PMID:22796595

  15. Nucleosome-like structural subunits of intranuclear parental adenovirus type 2 DNA.

    PubMed Central

    Sergeant, A; Tigges, M A; Raskas, H J

    1979-01-01

    The intranuclear structure of parental adenovirus 2 DNA was studied using digestion with micrococcal nuclease as a probe. When cultures were infected with 32P-labeled virions, at a multiplicity of 3,000 particles per cell, 14 to 21% of parental DNA penetrated the cell and reached the nucleus. Of this parental DNA, 60% could be solubilized by extensive digestion with micrococcal nuclease. The nuclease-resistant fraction contained viral deoxyribonucleoprotein monomers and oligomers. These nucleosome-like structures contained DNA fragments which are integral multiples of a unit-length DNA of approximately 185 base pairs. The monomeric DNA is similar in length to the unit-length DNA contained in cellular nucleosomes. However, the viral oligomers are slightly smaller than their cellular counterparts. DNA-DNA hybridization demonstrated that all segments of the viral genome, including those expressed as mRNA only at late times, are represented in the nucleosomal viral DNA. The amount of early intranuclear viral chromatin was proportional to multiplicity of infection up to multiplicities of 4,000 particles per cell. However, viral transcriptional activity did not increase in direct proportion to the amount of viral chromatin. Maximum accumulation of intranuclear viral chromatin was achieved by 3 h after infection. The intranuclear parental viral chromatin remained resistant to nuclease digestion even at late times in infection, after viral DNA replication had begun. Images PMID:448800

  16. 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. PMID:11709714

  17. Disruption of NAD+ binding site in glyceraldehyde 3-phosphate dehydrogenase affects its intranuclear interactions

    PubMed Central

    Phadke, Manali; Krynetskaia, Natalia; Mishra, Anurag; Barrero, Carlos; Merali, Salim; Gothe, Scott A; Krynetskiy, Evgeny

    2015-01-01

    AIM: To characterize phosphorylation of human glyceraldehyde 3-phosphate dehydrogenase (GAPDH), and mobility of GAPDH in cancer cells treated with chemotherapeutic agents. METHODS: We used proteomics analysis to detect and characterize phosphorylation sites within human GAPDH. Site-specific mutagenesis and alanine scanning was then performed to evaluate functional significance of phosphorylation sites in the GAPDH polypeptide chain. Enzymatic properties of mutated GAPDH variants were assessed using kinetic studies. Intranuclear dynamics parameters (diffusion coefficient and the immobile fraction) were estimated using fluorescence recovery after photobleaching (FRAP) experiments and confocal microscopy. Molecular modeling experiments were performed to estimate the effects of mutations on NAD+ cofactor binding. RESULTS: Using MALDI-TOF analysis, we identified novel phosphorylation sites within the NAD+ binding center of GAPDH at Y94, S98, and T99. Using polyclonal antibody specific to phospho-T99-containing peptide within GAPDH, we demonstrated accumulation of phospho-T99-GAPDH in the nuclear fractions of A549, HCT116, and SW48 cancer cells after cytotoxic stress. We performed site-mutagenesis, and estimated enzymatic properties, intranuclear distribution, and intranuclear mobility of GAPDH mutated variants. Site-mutagenesis at positions S98 and T99 in the NAD+ binding center reduced enzymatic activity of GAPDH due to decreased affinity to NAD+ (Km = 741 ± 257 μmol/L in T99I vs 57 ± 11.1 µmol/L in wild type GAPDH. Molecular modeling experiments revealed the effect of mutations on NAD+ binding with GAPDH. FRAP (fluorescence recovery after photo bleaching) analysis showed that mutations in NAD+ binding center of GAPDH abrogated its intranuclear interactions. CONCLUSION: Our results suggest an important functional role of phosphorylated amino acids in the NAD+ binding center in GAPDH interactions with its intranuclear partners. PMID:26629320

  18. 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.

  19. 14-3-3ζ coordinates adipogenesis of visceral fat

    PubMed Central

    Lim, Gareth E.; Albrecht, Tobias; Piske, Micah; Sarai, Karnjit; Lee, Jason T. C; Ramshaw, Hayley S.; Sinha, Sunita; Guthridge, Mark A.; Acker-Palmer, Amparo; Lopez, Angel F.; Clee, Susanne M.; Nislow, Corey; Johnson, James D.

    2015-01-01

    The proteins that coordinate complex adipogenic transcriptional networks are poorly understood. 14-3-3ζ is a molecular adaptor protein that regulates insulin signalling and transcription factor networks. Here we report that 14-3-3ζ-knockout mice are strikingly lean from birth with specific reductions in visceral fat depots. Conversely, transgenic 14-3-3ζ overexpression potentiates obesity, without exacerbating metabolic complications. Only the 14-3-3ζ isoform is essential for adipogenesis based on isoform-specific RNAi. Mechanistic studies show that 14-3-3ζ depletion promotes autophagy-dependent degradation of C/EBP-δ, preventing induction of the master adipogenic factors, Pparγ and C/EBP-α. Transcriptomic data indicate that 14-3-3ζ acts upstream of hedgehog signalling-dependent upregulation of Cdkn1b/p27Kip1. Indeed, concomitant knockdown of p27Kip1 or Gli3 rescues the early block in adipogenesis induced by 14-3-3ζ knockdown in vitro. Adipocyte precursors in 14-3-3ζKO embryos also appear to have greater Gli3 and p27Kip1 abundance. Together, our in vivo and in vitro findings demonstrate that 14-3-3ζ is a critical upstream driver of adipogenesis. PMID:26220403

  20. Nucleospora cyclopteri n. sp., an intranuclear microsporidian infecting wild lumpfish, Cyclopterus lumpus L., in Icelandic waters

    PubMed Central

    2013-01-01

    Background Commercial fisheries of lumpfish Cyclopterus lumpus have been carried out in Iceland for centuries. Traditionally the most valuable part is the eggs which are harvested for use as a caviar substitute. Previously reported parasitic infections from lumpfish include an undescribed intranuclear microsporidian associated with abnormal kidneys and mortalities in captive lumpfish in Canada. During Icelandic lumpfish fisheries in spring 2011, extensive enlargements to the kidneys were observed in some fish during processing. The aim of this study was to identify the pathogen responsible for these abnormalities. Methods Lumpfish from the Icelandic coast were examined for the causative agent of kidney enlargement. Fish were dissected and used in histological and molecular studies. Results Lumpfish, with various grades of clinical signs, were observed at 12 of the 43 sites sampled around Iceland. From a total of 77 fish examined, 18 had clear clinical signs, the most prominent of which was an extensive enlargement and pallor of the kidneys. The histopathology of the most severely affected fish consisted of extensive degeneration and necrosis of kidney tubules and vacuolar degeneration of the haematopoietic tissue. Intranuclear microsporidians were detected in all organs examined in fish with prominent clinical signs and most organs of apparently healthy fish using the new PCR and histological examination. One or multiple uniformly oval shaped spores measuring 3.12 ± 0.15 × 1.30 ± 0.12 μm were observed in the nucleus of affected lymphocytes and lymphocyte precursor cells. DNA sequencing provided a ribosomal DNA sequence that was strongly supported in phylogenetic analyses in a clade containing other microsporidian parasites from the Enterocytozoonidae, showing highest similarity to the intranuclear microsporidian Nucleospora salmonis. Conclusions Intranuclear microsporidian infections are common in wild caught lumpfish from around the Icelandic

  1. 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

    2014-06-01

    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. PMID:24796676

  2. Induction of intranuclear membranes by overproduction of Opi1p and Scs2p, regulators for yeast phospholipid biosynthesis, suggests a mechanism for Opi1p nuclear translocation.

    PubMed

    Masuda, Miki; Oshima, Ayaka; Noguchi, Tetsuko; Kagiwada, Satoshi

    2016-03-01

    In the yeast Saccharomyces cerevisiae, the expression of phospholipid biosynthetic genes is suppressed by the Opi1p negative regulator. Opi1p enters into the nucleoplasm from the nuclear membrane to suppress the gene expression under repressing conditions. The binding of Opi1p to the nuclear membrane requires an integral membrane protein, Scs2p and phosphatidic acid (PA). Although it is demonstrated that the association of Opi1p with membranes is affected by PA levels, how Opi1p dissociates from Scs2p is unknown. Here, we found that fluorescently labelled Opi1p accumulated on a perinuclear region in an Scs2p-dependent manner. Electron microscopic analyses indicated that the perinuclear region consists of intranuclear membranes, which may be formed by the invagination of the nuclear membrane due to the accumulation of Opi1p and Scs2p in a restricted area. As expected, localization of Opi1p and Scs2p in the intranuclear membranes was detected by immunoelectron microscopy. Biochemical analysis showed that Opi1p recovered in the membrane fraction was detergent insoluble while Scs2p was soluble, implying that Opi1p behaves differently from Scs2p in the fraction. We hypothesize that Opi1p dissociates from Scs2p after targeting to the nuclear membrane, making it possible to be released from the membrane quickly when PA levels decrease. PMID:26590299

  3. Widespread occurrence of an intranuclear bacterial parasite in vent and seep bathymodiolin mussels.

    PubMed

    Zielinski, Frank U; Pernthaler, Annelie; Duperron, Sébastien; Raggi, Luciana; Giere, Olav; Borowski, Christian; Dubilier, Nicole

    2009-05-01

    Many parasitic bacteria live in the cytoplasm of multicellular animals, but only a few are known to regularly invade their nuclei. In this study, we describe the novel bacterial parasite "Candidatus Endonucleobacter bathymodioli" that invades the nuclei of deep-sea bathymodiolin mussels from hydrothermal vents and cold seeps. Bathymodiolin mussels are well known for their symbiotic associations with sulfur- and methane-oxidizing bacteria. In contrast, the parasitic bacteria of vent and seep animals have received little attention despite their potential importance for deep-sea ecosystems. We first discovered the intranuclear parasite "Ca. E. bathymodioli" in Bathymodiolus puteoserpentis from the Logatchev hydrothermal vent field on the Mid-Atlantic Ridge. Using primers and probes specific to "Ca. E. bathymodioli" we found this intranuclear parasite in at least six other bathymodiolin species from vents and seeps around the world. Fluorescence in situ hybridization and transmission electron microscopy analyses of the developmental cycle of "Ca. E. bathymodioli" showed that the infection of a nucleus begins with a single rod-shaped bacterium which grows to an unseptated filament of up to 20 microm length and then divides repeatedly until the nucleus is filled with up to 80,000 bacteria. The greatly swollen nucleus destroys its host cell and the bacteria are released after the nuclear membrane bursts. Intriguingly, the only nuclei that were never infected by "Ca. E. bathymodioli" were those of the gill bacteriocytes. These cells contain the symbiotic sulfur- and methane-oxidizing bacteria, suggesting that the mussel symbionts can protect their host nuclei against the parasite. Phylogenetic analyses showed that the "Ca. E. bathymodioli" belongs to a monophyletic clade of Gammaproteobacteria associated with marine metazoans as diverse as sponges, corals, bivalves, gastropods, echinoderms, ascidians and fish. We hypothesize that many of the sequences from this clade

  4. Acid-fast intranuclear inclusion bodies in the kidneys of mallards fed lead shot

    USGS Publications Warehouse

    Locke, L.N.; Bagley, G.E.; Irby, H.D.

    1966-01-01

    Acid-fast intranuclear inclusion bodies were found in the cells of the proximal convoluted tubules of the kidneys of mallards fed one, two, three or eight number 6 lead shot and maintained on cracked or whole corn and on grain-duck pellet diets. No acid-fast inclusion bodies were found in mallards fed one or three lead shot but maintained on a duck pellet ration. Dietary factors may be responsible for the failure of mallards fed a duck pellet ration to develop lead Inclusion bodies when treated with one or three lead shot. The authors suggest these inclusion bodies can be used as presumptive evidence for lead intoxication in mallards.

  5. Neuronal intranuclear inclusions are ultrastructurally and immunologically distinct from cytoplasmic inclusions of neuronal intermediate filament inclusion disease.

    PubMed

    Mosaheb, Sabrina; Thorpe, Julian R; Hashemzadeh-Bonehi, Lida; Bigio, Eileen H; Gearing, Marla; Cairns, Nigel J

    2005-10-01

    Abnormal neuronal cytoplasmic inclusions (NCIs) containing aggregates of alpha-internexin and the neurofilament (NF) subunits, NF-H, NF-M, and NF-L, are the signature lesions of neuronal intermediate filament (IF) inclusion disease (NIFID). The disease has a clinically heterogeneous phenotype, including frontotemporal dementia, pyramidal and extrapyramidal signs presenting at a young age. NCIs are variably ubiquitinated and about half of cases also have neuronal intranuclear inclusions (NIIs), which are also ubiquitinated. NIIs have been described in polyglutamine-repeat expansion diseases, where they are strongly ubiquitin immunoreactive. The fine structure of NIIs of NIFID has not previously been described. Therefore, to determine the ultrastructure of NIIs, immunoelectron microscopy was undertaken on NIFID cases and normal aged control brains. Our results indicate that the NIIs of NIFID are strongly ubiquitin immunoreactive. However, unlike NCIs which contain ubiquitin, alpha-internexin and NF epitopes, NIIs contain neither epitopes of alpha-internexin nor NF subunits. Neither NIIs nor NCIs were recognised by antibodies to expanded polyglutamine repeats. The NII of NIFID lacks a limiting membrane and contains straight filaments of 20 nm mean width (range 11-35 nm), while NCIs contain filaments with a mean width of 10 nm (range 5-18 nm; t-test, P<0.001). Biochemistry revealed no differences in neuronal IF protein mobilities between NIFID and normal brain tissue. Therefore, NIIs of NIFID contain filaments morphologically and immunologically distinct from those of NCIs, and both types of inclusion lack expanded polyglutamine tracts of the triplet-repeat expansion diseases. These observations indicate that abnormal protein aggregation follows separate pathways in different neuronal compartments of NIFID. PMID:16025283

  6. Extended Intranuclear Cascade model for pickup reactions induced by 50-MeV-range protons

    NASA Astrophysics Data System (ADS)

    Uozumi, Yusuke; Mori, Taiki; Sonoda, Akifumi; Nakano, Masahiro

    2016-06-01

    The intranuclear cascade model was investigated to explain (p, dx) and (p, ax) reactions at incident energies of around 50 MeV. Since these reactions are governed mainly by the direct pickup process, the model was expanded to include exclusive pickup processes leading to hole-state-excitations. The energy of the outgoing clusters is determined with single-particle energies of transferred nucleons, the reaction Q-value, and the recoil of the residual nucleus. The rescattering of the produced cluster inside the nucleus is treated within the intranuclear cascade model. The emission angle is given by the sum of momentum vectors of transferred nucleons in addition to the deflection at the nuclear surface, which was introduced to explain angular distributions of elastic scattering. Double differential cross sections of reactions were calculated and compared with experimental data. The proposed model showed a high predictive power over the wide range of emission energies and angles. The treatment ofthe cluster transport inside the nucleus was also verified.

  7. Hypoxic ischemic encephalopathy in a case of intranuclear rod myopathy without any prenatal sentinel event.

    PubMed

    Kawase, Koya; Nishino, Ichizo; Sugimoto, Mari; Kouwaki, Masanori; Koyama, Norihisa; Yokochi, Kenji

    2015-02-01

    Intranuclear rod myopathy (IRM), a variant of nemaline myopathy, is characterized by the presence of nemaline bodies in myonuclei. We report a case of IRM presenting with hypoxic ischemic encephalopathy (HIE). There were no prenatal complications caused by fetal brain injury. Although no nemaline bodies were observed in the cytoplasm, intranuclear rods were observed in some fibers under light and electron microscopy. Molecular analysis identified a heterozygous variant, c.449C>T (p.Thr150Ile), in ACTA1. On magnetic resonance imaging at 9days of age, injuries to the basal ganglia, thalamus, and brainstem consistent with perinatal HIE were seen. Respiratory insufficiency at birth was strongly suspected to be the cause of HIE. Our case highlights that a patient with a congenital neuromuscular disorder who presents with severe respiratory dysfunction requiring substantial resuscitative efforts at birth can be complicated by HIE without any prenatal sentinel event. Prenatal detection of neuromuscular disorders, careful management of delivery, and neonatal resuscitation and adequate respiratory management are important in preventing irreversible brain injury in these patients. PMID:24787270

  8. Intranuclear Pseudo-inclusions and Grooves in Fine Needle Aspiration Cytology of Pulmonary Carcinoid Tumor.

    PubMed

    Mokhtari, Maral; Kumar, Perikala Vijayananda

    2016-02-01

    Cytologic findings of pulmonary carcinoid have been well described. We report new cytological findings in a case of carcinoid tumor. The patient is a 36-year-old man presenting with hemoptysis of about six months in duration. Chest CT scans showed a well-defined round polypoid lesion measuring 1 × 1 cm within the right upper lobe of the bronchus with hyperinflation of the right upper lobe. Trans-bronchial fine needle aspiration and biopsy were done. Cytologic smears showed isolated and loose clusters of uniform round to spindle shape cells with round centrally located nuclei, fine granular (salt and pepper) chromatin and pale cytoplasm. Intranuclear pseudo-inclusions and grooves were seen in some tumor cells. No mitotic figures or necrosis were evident. A cytological diagnosis of carcinoid tumor was made and histopathologic examination and subsequent immunohistochemical study confirmed the diagnosis. Carcinoid tumor may be reliably diagnosed on fine needle aspiration cytology smears. Intranuclear pseudo-inclusions and grooves may be evident in tumor cells. PMID:26838088

  9. Accessing the tonotopic organization of the ventral cochlear nucleus by intranuclear microstimulation.

    PubMed

    McCreery, D B; Shannon, R V; Moore, J K; Chatterjee, M

    1998-12-01

    This study is part of a program to develop an auditory prosthesis for the profoundly deaf, based on multichannel microstimulation in the cochlear nucleus. The functionality of such a device is dependent on its ability to access the tonotopic axis of the human ventral cochlear nucleus in an orderly fashion. In these studies, we utilized the homologies between the human and feline ventral cochlear nuclei and the known tonotopic organization of the central nucleus of the inferior colliculus (IC). In anesthetized cats, stimuli were delivered to three or four locations along the dorsal-to-ventral axis of the posteroventral cochlear nucleus (PVCN), and for each stimulus location, we recorded the multiunit neuronal activity and the field potentials at 20 or more locations along the dorsolateral-ventromedial (tonotopic) axis of the IC. The current source-sink density (CSD), which delimits regions of neuronal activity, was computed from the sequence of field potentials recorded along this axis. The multiunit activity and the CSD analysis both showed that the tonotopic organization of the PVCN can be accessed in an orderly manner by intranuclear microstimulation in several regions of the PVCN, using the range of stimulus pulse amplitudes that have been shown in previous studies to be noninjurious during prolonged intranuclear microstimulation via chronically implanted microelectrodes. We discuss the applicability of these findings to the design of clinical auditory prostheses for implantation into the human cochlear nucleus. PMID:9865886

  10. Memory Loss and Frontal Cognitive Dysfunction in a Patient with Adult-onset Neuronal Intranuclear Inclusion Disease.

    PubMed

    Araki, Kunihiko; Sone, Jun; Fujioka, Yusuke; Masuda, Michihito; Ohdake, Reiko; Tanaka, Yasuhiro; Nakamura, Tomohiko; Watanabe, Hirohisa; Sobue, Gen

    2016-01-01

    Neuronal intranuclear inclusion disease (NIID) is an uncommon progressive neurodegenerative disorder. Adult-onset NIID can result in prominent dementia. We herein describe the case of a 74-year-old man who presented with dementia, cerebellar ataxia, neuropathy, and autonomic dysfunction. Diffusion-weighted imaging showed hyperintensity of the corticomedullary junction. Fluid-attenuated inversion recovery images showed frontal-dominant white matter hyperintensity. NIID was diagnosed from the presence of intranuclear inclusions in a skin biopsy sample. Neuropsychological testing revealed memory loss and frontal cognitive dysfunction, especially in relation to language and executive functions. We were therefore able to confirm the association of NIID with cognitive dysfunction. PMID:27523009

  11. Intranuclear bundles of microfilaments and microtubules in chromaffin cells of the auricle of the heart of a lungfish, Protopterus aethiopicus.

    PubMed

    Scheuermann, D W; Adriaensen, D; De Groodt-Lasseel, M H

    1988-01-01

    Intranuclear microtubular-microfilamentous rod-like inclusions were investigated in chromaffin cells of the auricle of the heart of lungfishes. In conventional electron microscopy, these inclusions reveal a wide variety in appearance, depending on their orientation to the plane of sectioning. Whereas originally they were merely interpreted as a bundle of microfilaments, application of a goniometer stage showed the rod- or spindle-shaped intranuclear inclusions to have a basic substructure of parallel arranged microtubules among microfilaments, which are clearly connected to chromatin granules, occasionally penetrating dense areas of chromatin. The chemical nature and biological significance of these structures, which so far remain enigmatic, are discussed. PMID:3227775

  12. Protein kinase C delta inhibits Caco-2 cell proliferation by selective changes in cell cycle and cell death regulators.

    PubMed

    Cerda, S R; Mustafi, R; Little, H; Cohen, G; Khare, S; Moore, C; Majumder, P; Bissonnette, M

    2006-05-25

    PKC-delta is a serine/threonine kinase that mediates diverse signal transduction pathways. We previously demonstrated that overexpression of PKC-delta slowed the G1 progression of Caco-2 colon cancer cells, accelerated apoptosis, and induced cellular differentiation. In this study, we further characterized the PKC-delta dependent signaling pathways involved in these tumor suppressor actions in Caco-2 cells overexpressing PKC-delta using a Zn2+ inducible expression vector. Consistent with a G1 arrest, increased expression of PKC-delta caused rapid and significant downregulation of cyclin D1 and cyclin E proteins (50% decreases, P<0.05), while mRNA levels remained unchanged. The PKC agonist, phorbol 12-myristate 13-acetate (TPA, 100 nM, 4 h), induced two-fold higher protein and mRNA levels of p21(Waf1), a cyclin-dependent kinase (cdk) inhibitor in PKC-delta transfectants compared with empty vector (EV) transfected cells, whereas the PKC-delta specific inhibitor rottlerin (3 microM) or knockdown of this isoenzyme with specific siRNA oligonucleotides blocked p21(Waf1) expression. Concomitantly, compared to EV control cells, PKC-delta upregulation decreased cyclin D1 and cyclin E proteins co-immunoprecipitating with cdk6 and cdk2, respectively. In addition, overexpression of PKC-delta increased binding of cdk inhibitor p27(Kip1) to cdk4. These alterations in cyclin-cdks and their inhibitors are predicted to decrease G1 cyclin kinase activity. As an independent confirmation of the direct role PKC-delta plays in cell growth and cell cycle regulation, we knocked down PKC-delta using specific siRNA oligonucleotides. PKC-delta specific siRNA oligonucleotides, but not irrelevant control oligonucleotides, inhibited PKC-delta protein by more than 80% in Caco-2 cells. Moreover, PKC-delta knockdown enhanced cell proliferation ( approximately 1.4-2-fold, P<0.05) and concomitantly increased cyclin D1 and cyclin E expression ( approximately 1.7-fold, P<0.05). This was a specific

  13. Intranuclear interactomic inhibition of NF-κB suppresses LPS-induced severe sepsis

    SciTech Connect

    Park, Sung-Dong; Cheon, So Yeong; Park, Tae-Yoon; Shin, Bo-Young; Oh, Hyunju; Ghosh, Sankar; Koo, Bon-Nyeo; Lee, Sang-Kyou

    2015-08-28

    Suppression of nuclear factor-κB (NF-κB) activation, which is best known as a major regulator of innate and adaptive immune responses, is a potent strategy for the treatment of endotoxic sepsis. To inhibit NF-κB functions, we designed the intra-nuclear transducible form of transcription modulation domain (TMD) of RelA (p65), called nt-p65-TMD, which can be delivered effectively into the nucleus without influencing the cell viability, and work as interactomic inhibitors via disruption of the endogenous p65-mediated transcription complex. nt-p65-TMD effectively inhibited the secretion of pro-inflammatory cytokines, including TNF-α, IL-1β, or IL-6 from BV2 microglia cells stimulated by lipopolysaccharide (LPS). nt-p65-TMD did not inhibit tyrosine phosphorylation of signaling mediators such as ZAP-70, p38, JNK, or ERK involved in T cell activation, but was capable of suppressing the transcriptional activity of NF-κB without the functional effect on that of NFAT upon T-cell receptor (TCR) stimulation. The transduced nt-p65-TMD in T cell did not affect the expression of CD69, however significantly inhibited the secretion of T cell-specific cytokines such as IL-2, IFN-γ, IL-4, IL-17A, or IL-10. Systemic administration of nt-p65-TMD showed a significant therapeutic effect on LPS-induced sepsis model by inhibiting pro-inflammatory cytokines secretion. Therefore, nt-p65-TMD can be a novel therapeutics for the treatment of various inflammatory diseases, including sepsis, where a transcription factor has a key role in pathogenesis, and further allows us to discover new functions of p65 under normal physiological condition without genetic alteration. - Highlights: • The nt-p65-TMD is intra-nuclear interactomic inhibitor of endogenous p65. • The nt-p65-TMD effectively inhibited the secretion of pro-inflammatory cytokines. • The excellent therapeutic potential of nt-p65-TMD was confirmed in sepsis model.

  14. A novel intranuclear RNA vector system for long-term stem cell modification

    PubMed Central

    Ikeda, Yasuhiro; Makino, Akiko; Matchett, William E.; Holditch, Sara J.; Lu, Brian; Dietz, Allan B.; Tomonaga, Keizo

    2015-01-01

    Genetically modified stem and progenitor cells have emerged as a promising regenerative platform in the treatment of genetic and degenerative disorders, highlighted by their successful therapeutic use in inherent immunodeficiencies. However, biosafety concerns over insertional mutagenesis resulting from integrating recombinant viral vectors have overshadowed the widespread clinical applications of genetically modified stem cells. Here, we report an RNA-based episomal vector system, amenable for long-term transgene expression in stem cells. Specifically, we used a unique intranuclear RNA virus, Borna disease virus (BDV), as the gene transfer vehicle, capable of persistent infections in various cell types. BDV-based vectors allowed for long-term transgene expression in mesenchymal stem cells (MSCs) without affecting cellular morphology, cell surface CD105 expression, or the adipogenicity of MSCs. Similarly, replication-defective BDV vectors achieved long-term transduction of human induced pluripotent stem cells (iPSCs), while maintaining the ability to differentiate into three embryonic germ layers. Thus, the BDV-based vectors offer a genomic modification-free, episomal RNA delivery system for sustained stem cell transduction. PMID:26632671

  15. A novel intranuclear RNA vector system for long-term stem cell modification.

    PubMed

    Ikeda, Y; Makino, A; Matchett, W E; Holditch, S J; Lu, B; Dietz, A B; Tomonaga, K

    2016-03-01

    Genetically modified stem and progenitor cells have emerged as a promising regenerative platform in the treatment of genetic and degenerative disorders, highlighted by their successful therapeutic use in inherent immunodeficiencies. However, biosafety concerns over insertional mutagenesis resulting from integrating recombinant viral vectors have overshadowed the widespread clinical applications of genetically modified stem cells. Here, we report an RNA-based episomal vector system, amenable for long-term transgene expression in stem cells. Specifically, we used a unique intranuclear RNA virus, borna disease virus (BDV), as the gene transfer vehicle, capable of persistent infections in various cell types. BDV-based vectors allowed for long-term transgene expression in mesenchymal stem cells (MSCs) without affecting cellular morphology, cell surface CD105 expression or the adipogenicity of MSCs. Similarly, replication-defective BDV vectors achieved long-term transduction of human induced pluripotent stem cells, while maintaining the ability to differentiate into three embryonic germ layers. Thus, the BDV-based vectors offer a genomic modification-free, episomal RNA delivery system for sustained stem cell transduction. PMID:26632671

  16. Processes involving few degrees of freedom in the frame of Intranuclear Cascade approaches

    NASA Astrophysics Data System (ADS)

    Cugnon, J.; Boudard, A.; David, J.-C.; Leray, S.; Mancusi, D.

    2016-05-01

    This article focuses on spallation reactions, i.e. interactions of energetic nucleons, basically with a kinetic energy in the 100MeV to a few GeV range, with a target nucleus. These processes are described rather successfully by the so-called Intranuclear Cascade (INC) plus evaporation models. They can be viewed as a first stage of nucleon-nucleon collisions, ejecting fast particles, followed by evaporation of slow particles from the target remnant. These cascade + evaporation models have, now, globally reached a high level of predictive power, owing in particular to successive research programs. The present work, which is an outcome of one of these programs, the recent European Union ANDES research program, deals with a set of reactions (or observable quantities), which can be due to a single collision, such as the one-nucleon removal reactions or the quasi-elastic elastic process. A survey of the experimental data is presented, which allows to clearly point out that, often, the INC models are unsatisfactory for the description of these peculiar events, whereas they are rather successful for the rest of the experimental data. This paradoxical situation is tentatively related to quasi-particle effects which are neglected in INC models.

  17. Intranuclear cascade with emission of light fragment code implemented in the transport code system PHITS

    NASA Astrophysics Data System (ADS)

    Sawada, Y.; Uozumi, Y.; Nogamine, S.; Yamada, T.; Iwamoto, Y.; Sato, T.; Niita, K.

    2012-11-01

    The Intranuclear Cascade with Emission of Light Fragment (INC-ELF) code has been developed and implemented in the Particle and Heavy Ion Transport code System (PHITS). The INC-ELF code explicitly includes nucleon correlations within the framework of the INC model to describe light fragment emissions from nuclear spallation reactions by using the model in Phys. Rev. C 84, (2011) 064617. In addition to the degrees of freedom of nucleons, the developed code also accounts for pions, Δs, and N∗s, and can cover energy ranges up to 3 GeV. The predictive capabilities of the ELF/PHITS system have been verified through comparison with a diverse set of experimental observations. In particular, the verification was conducted with abundant double-differential cross-section data covering a wide range of reactions (e.g., (p, p'x), (p, nx), (p, dx), (p, 3Hex), (p, αx) and (p, πx) reactions) over a wide energy range (between 400 MeV and 1.5 GeV). As a result, our ELF/PHITS code has demonstrated strong predictive capability for all of these data, although areas requiring future study remain due to the lack of experimental data on high-energy cluster production.

  18. Balbiani Ring mRNPs Diffuse through and Bind to Clusters of Large Intranuclear Molecular Structures

    PubMed Central

    Veith, Roman; Sorkalla, Thomas; Baumgart, Eugen; Anzt, Johannes; Häberlein, Hanns; Tyagi, Sanjay; Siebrasse, Jan Peter; Kubitscheck, Ulrich

    2010-01-01

    A detailed conception of intranuclear messenger ribonucleoprotein particle (mRNP) dynamics is required for the understanding of mRNP processing and gene expression outcome. We used complementary state-of-the-art fluorescence techniques to quantify native mRNP mobility at the single particle level in living salivary gland cell nuclei. Molecular beacons and fluorescent oligonucleotides were used to specifically label BR2.1 mRNPs by an in vivo fluorescence in situ hybridization approach. We characterized two major mobility components of the BR2.1 mRNPs. These components with diffusion coefficients of 0.3 ± 0.02 μm2/s and 0.73 ± 0.03 μm2/s were observed independently of the staining method and measurement technique used. The mobility analysis of inert tracer molecules revealed that the gland cell nuclei contain large molecular nonchromatin structures, which hinder the mobility of large molecules and particles. The mRNPs are not only hindered by these mobility barriers, but in addition also interact presumably with these structures, what further reduces their mobility and effectively leads to the occurrence of the two diffusion coefficients. In addition, we provide evidence that the remarkably high mobility of the large, 50 nm-sized BR2.1 mRNPs was due to the absence of retarding chromatin. PMID:20959109

  19. Topochemistry of Internuclear and Intranuclear Interneurons of the Vasomotor Area in the Medulla Oblongata of Hypertensive Rats.

    PubMed

    Chertok, V M; Kotsyuba, A E; Startseva, M S

    2016-01-01

    Immunohistochemical examination with the antiserum against neuronal NO synthase and cystathionine β-synthase was used to study the following two pools of interneurons in Wistar rats at various periods after the development of renovascular hypertension: intranuclear interneurons (lying in the projection of the solitary nucleus, reticular gigantocellular nucleus, and parvocellular nucleus) and 2 groups of internuclear interneurons (small interneurons, area 50-300 μ(2); and large interneurons, area above 350 μ(2)). Intranuclear and internuclear interneurons probably play a role in the central mechanisms of hemodynamics regulation. These interneurons differ by not only in topochemical parameters, but also functional properties (different resistances to BP changes). Intranuclear interneurons are characterized by high sensitivity of the gas transmitter systems to a continuous increase in BP, which results in remodeling and dysfunction of the bulbar part of the cardiovascular center. Large internuclear interneurons demonstrate a strong reaction to BP rise, which confirms their involvement into hemodynamics regulation. By contrast, small internuclear interneurons retain their characteristics in arterial hypertension and probably perform an integrative function. PMID:26746841

  20. Nitro-linoleic acid inhibits vascular smooth muscle cell proliferation via the Keap1/Nrf2 signaling pathway

    PubMed Central

    Villacorta, Luis; Zhang, Jifeng; Garcia-Barrio, Minerva T.; Chen, Xi-lin; Freeman, Bruce A.; Chen, Yuqing E.; Cui, Taixing

    2007-01-01

    Nitroalkenes, the nitration products of unsaturated fatty acids formed via NO-dependent oxidative reactions, have been demonstrated to exert strong biological actions in endothelial cells and monocytes/macrophages; however, little is known about their effects on vascular smooth muscle cells (VSMCs). The present study examined the role of nitro-linoleic acid (LNO2) in the regulation of VSMC proliferation. We observed that LNO2 inhibited VSMC proliferation in a dose-dependent manner. In addition, LNO2 induced growth arrest of VSMCs in the G1/S phase of the cell cycle with an upregulation of the cyclin-dependent kinase inhibitor p27kip1. Furthermore, LNO2 triggered nuclear factor-erythroid 2-related factor 2 (Nrf2) nuclear translocation and activation of the antioxidant-responsive element-driven transcriptional activity via impairing Kelch-like ECH-associating protein 1 (Keap1)-mediated negative control of Nrf2 activity in VSMCs. LNO2 upregulated the expression of Nrf2 protein levels, but not mRNA levels, in VSMCs. A forced activation of Nrf2 led to an upregulation of p27kip1 and growth inhibition of VSMCs. In contrast, knock down of Nrf2 using an Nrf2 siRNA approach reversed the LNO2-induced upregulation of p27kip1 and inhibition of cellular proliferation in VSMCs. These studies provide the first evidence that nitroalkene LNO2 inhibits VSMC proliferation through activation of the Keap1/Nrf2 signaling pathway, suggesting an important role of nitroalkenes in vascular biology. PMID:17468336

  1. Intranuclear verrucomicrobial symbionts and evidence of lateral gene transfer to the host protist in the termite gut.

    PubMed

    Sato, Tomoyuki; Kuwahara, Hirokazu; Fujita, Kazuma; Noda, Satoko; Kihara, Kumiko; Yamada, Akinori; Ohkuma, Moriya; Hongoh, Yuichi

    2014-05-01

    In 1944, Harold Kirby described microorganisms living within nuclei of the protists Trichonympha in guts of termites; however, their taxonomic assignment remains to be accomplished. Here, we identified intranuclear symbionts of Trichonympha agilis in the gut of the termite Reticulitermes speratus. We isolated single nuclei of T. agilis, performed whole-genome amplification, and obtained bacterial 16S rRNA genes by PCR. Unexpectedly, however, all of the analyzed clones were from pseudogenes of 16S rRNA with large deletions and numerous sequence variations even within a single-nucleus sample. Authentic 16S rRNA gene sequences were finally recovered by digesting the nuclear DNA; these pseudogenes were present on the host Trichonympha genome. The authentic sequences represented two distinct bacterial species belonging to the phylum Verrucomicrobia, and the pseudogenes have originated from each of the two species. Fluorescence in situ hybridization confirmed that both species are specifically localized, and occasionally co-localized, within nuclei of T. agilis. Transmission electron microscopy revealed that they are distorted cocci with characteristic electron-dense and lucent regions, which resemble the intranuclear symbionts illustrated by Kirby. For these symbionts, we propose a novel genus and species, 'Candidatus Nucleococcus trichonymphae' and 'Candidatus Nucleococcus kirbyi'. These formed a termite-specific cluster with database sequences, other members of which were also detected within nuclei of various gut protists, including both parabasalids and oxymonads. We suggest that this group is widely distributed as intranuclear symbionts of diverse protists in termite guts and that they might have affected the evolution of the host genome through lateral gene transfer. PMID:24335826

  2. Intranuclear verrucomicrobial symbionts and evidence of lateral gene transfer to the host protist in the termite gut

    PubMed Central

    Sato, Tomoyuki; Kuwahara, Hirokazu; Fujita, Kazuma; Noda, Satoko; Kihara, Kumiko; Yamada, Akinori; Ohkuma, Moriya; Hongoh, Yuichi

    2014-01-01

    In 1944, Harold Kirby described microorganisms living within nuclei of the protists Trichonympha in guts of termites; however, their taxonomic assignment remains to be accomplished. Here, we identified intranuclear symbionts of Trichonympha agilis in the gut of the termite Reticulitermes speratus. We isolated single nuclei of T. agilis, performed whole-genome amplification, and obtained bacterial 16S rRNA genes by PCR. Unexpectedly, however, all of the analyzed clones were from pseudogenes of 16S rRNA with large deletions and numerous sequence variations even within a single-nucleus sample. Authentic 16S rRNA gene sequences were finally recovered by digesting the nuclear DNA; these pseudogenes were present on the host Trichonympha genome. The authentic sequences represented two distinct bacterial species belonging to the phylum Verrucomicrobia, and the pseudogenes have originated from each of the two species. Fluorescence in situ hybridization confirmed that both species are specifically localized, and occasionally co-localized, within nuclei of T. agilis. Transmission electron microscopy revealed that they are distorted cocci with characteristic electron-dense and lucent regions, which resemble the intranuclear symbionts illustrated by Kirby. For these symbionts, we propose a novel genus and species, ‘Candidatus Nucleococcus trichonymphae' and ‘Candidatus Nucleococcus kirbyi'. These formed a termite-specific cluster with database sequences, other members of which were also detected within nuclei of various gut protists, including both parabasalids and oxymonads. We suggest that this group is widely distributed as intranuclear symbionts of diverse protists in termite guts and that they might have affected the evolution of the host genome through lateral gene transfer. PMID:24335826

  3. Human T-cell leukemia virus type 1 infection leads to arrest in the G1 phase of the cell cycle.

    PubMed

    Liu, Meihong; Yang, Liangpeng; Zhang, Ling; Liu, Baoying; Merling, Randall; Xia, Zheng; Giam, Chou-Zen

    2008-09-01

    Infection by the human T-cell leukemia virus type 1 (HTLV-1) is thought to cause dysregulated T-cell proliferation, which in turn leads to adult T-cell leukemia/lymphoma. Early cellular changes after HTLV-1 infection have been difficult to study due to the poorly infectious nature of HTLV-1 and the need for cell-to-cell contact for HTLV-1 transmission. Using a series of reporter systems, we show that HeLa cells cease proliferation within one or two division cycles after infection by HTLV-1 or transduction of the HTLV-1 tax gene. HTLV-1-infected HeLa cells, like their tax-transduced counterparts, expressed high levels of p21(CIP1/WAF1) and p27(KIP1), developed mitotic abnormalities, and became arrested in G(1) in senescence. In contrast, cells of a human osteosarcoma lineage (HOS) continued to divide after HTLV-1 infection or Tax expression, albeit at a reduced growth rate and with mitotic aberrations. Unique to HOS cells is the dramatic reduction of p21(CIP1/WAF1) and p27(KIP1) expression, which is in part associated with the constitutive activation of the phosphatidylinositol-3-kinase (PI3K)-protein kinase B (Akt) pathway. The loss of p21(CIP1/WAF1) and p27(KIP1) in HOS cells apparently allows HTLV-1- and Tax-induced G(1) arrest to be bypassed. Finally, HTLV-1 infection and Tax expression also cause human SupT1 T cells to arrest in the G(1) phase of the cell cycle. These results suggest that productive HTLV-1 infection ordinarily leads to Tax-mediated G(1) arrest. However, T cells containing somatic mutations that inactivate p21(CIP1/WAF1) and p27(KIP1) may continue to proliferate after HTLV-1 infection and Tax expression. These infected cells can expand clonally, accumulate additional chromosomal abnormalities, and progress to cancer. PMID:18596104

  4. A honey bee hexamerin, HEX 70a, is likely to play an intranuclear role in developing and mature ovarioles and testioles.

    PubMed

    Martins, Juliana R; Anhezini, Lucas; Dallacqua, Rodrigo P; Simões, Zilá L P; Bitondi, Márcia M G

    2011-01-01

    Insect hexamerins have long been known as storage proteins that are massively synthesized by the larval fat body and secreted into hemolymph. Following the larval-to-pupal molt, hexamerins are sequestered by the fat body via receptor-mediated endocytosis, broken up, and used as amino acid resources for metamorphosis. In the honey bee, the transcript and protein subunit of a hexamerin, HEX 70a, were also detected in ovaries and testes. Aiming to identify the subcellular localization of HEX 70a in the female and male gonads, we used a specific antibody in whole mount preparations of ovaries and testes for analysis by confocal laser-scanning microscopy. Intranuclear HEX 70a foci were evidenced in germ and somatic cells of ovarioles and testioles of pharate-adult workers and drones, suggesting a regulatory or structural role. Following injection of the thymidine analog EdU we observed co-labeling with HEX 70a in ovariole cell nuclei, inferring possible HEX 70a involvement in cell proliferation. Further support to this hypothesis came from an injection of anti-HEX 70a into newly ecdysed queen pupae where it had a negative effect on ovariole thickening. HEX 70a foci were also detected in ovarioles of egg laying queens, particularly in the nuclei of the highly polyploid nurse cells and in proliferating follicle cells. Additional roles for this storage protein are indicated by the detection of nuclear HEX 70a foci in post-meiotic spermatids and spermatozoa. Taken together, these results imply undescribed roles for HEX 70a in the developing gonads of the honey bee and raise the possibility that other hexamerins may also have tissue specific functions. PMID:22205988

  5. A Honey Bee Hexamerin, HEX 70a, Is Likely to Play an Intranuclear Role in Developing and Mature Ovarioles and Testioles

    PubMed Central

    Martins, Juliana R.; Anhezini, Lucas; Dallacqua, Rodrigo P.; Simões, Zilá L. P.; Bitondi, Márcia M. G.

    2011-01-01

    Insect hexamerins have long been known as storage proteins that are massively synthesized by the larval fat body and secreted into hemolymph. Following the larval-to-pupal molt, hexamerins are sequestered by the fat body via receptor-mediated endocytosis, broken up, and used as amino acid resources for metamorphosis. In the honey bee, the transcript and protein subunit of a hexamerin, HEX 70a, were also detected in ovaries and testes. Aiming to identify the subcellular localization of HEX 70a in the female and male gonads, we used a specific antibody in whole mount preparations of ovaries and testes for analysis by confocal laser-scanning microscopy. Intranuclear HEX 70a foci were evidenced in germ and somatic cells of ovarioles and testioles of pharate-adult workers and drones, suggesting a regulatory or structural role. Following injection of the thymidine analog EdU we observed co-labeling with HEX 70a in ovariole cell nuclei, inferring possible HEX 70a involvement in cell proliferation. Further support to this hypothesis came from an injection of anti-HEX 70a into newly ecdysed queen pupae where it had a negative effect on ovariole thickening. HEX 70a foci were also detected in ovarioles of egg laying queens, particularly in the nuclei of the highly polyploid nurse cells and in proliferating follicle cells. Additional roles for this storage protein are indicated by the detection of nuclear HEX 70a foci in post-meiotic spermatids and spermatozoa. Taken together, these results imply undescribed roles for HEX 70a in the developing gonads of the honey bee and raise the possibility that other hexamerins may also have tissue specific functions. PMID:22205988

  6. Draft Genome Sequences of Two Novel Amoeba-Resistant Intranuclear Bacteria, “Candidatus Berkiella cookevillensis” and “Candidatus Berkiella aquae”

    PubMed Central

    Mehari, Yohannes T.; Arivett, Brock A.; Farone, Anthony L.; Gunderson, John H.

    2016-01-01

    “Candidatus Berkiella cookevillensis” and “Candidatus Berkiella aquae” are obligate intranuclear endosymbionts of freshwater amoebae. Here, we present the draft genome sequences of these two bacteria, with total sizes of 2,990,361 bp and 3,626,027 bp, respectively. PMID:26893424

  7. Cross sections of proton- and neutron-induced reactions by the Liège intranuclear cascade model

    NASA Astrophysics Data System (ADS)

    Chen, Jian; Dong, Tiekuang; Ren, Zhongzhou

    2016-06-01

    The purpose of the paper is mainly to test the validity of the Liège intranuclear cascade (INCL) model in calculating the cross sections of proton-induced reactions for cosmogenic nuclei using the newly compiled database of proton cross sections. The model calculations of 3He display the rising tendency of cross sections with the increase of energy, in accordance with the experimental data. Meanwhile, the differences between the theoretical results and experimental data of production cross sections (10Be and 26Al) are generally within a factor of 3, meaning that the INCL model works quite well for the proton-induced reactions. Based on the good agreement, we predict the production cross sections of 26Al from reactions n + 27Al, n + 28Si, and n + 40Ca and those of 10Be from reactions n + 16O and n + 28Si. The results also show a good agreement with a posteriori excitation functions.

  8. Antimicrobial Peptide, Lumbricusin, Ameliorates Motor Dysfunction and Dopaminergic Neurodegeneration in a Mouse Model of Parkinson's Disease.

    PubMed

    Kim, Dae Hong; Lee, Ik Hwan; Nam, Seung Taek; Hong, Ji; Zhang, Peng; Lu, Li Fang; Hwang, Jae Sam; Park, Ki Cheol; Kim, Ho

    2015-10-01

    We recently reported that the antimicrobial peptide Lumbricusin (NH2-RNRRWCIDQQA), isolated from the earthworm, increases cell proliferation in neuroblastoma SH-SY5Y cells. Here, we investigated whether Lumbricusin has neurotropic activity in mouse neural stem cells (MNSCs) and a protective effect in a mouse model of Parkinson's disease (PD). In MNSCs isolated from mouse brains, Lumbricusin treatment significantly increased cell proliferation (up to 12%) and reduced the protein expression of p27(Kip1) through proteasomal protein degradation but not transcriptional regulation. Lumbricusin inhibited the 6-OHDA-induced apoptosis of MNSCs, and also showed neuroprotective effects in a mouse PD model, ameliorating the motor impairments seen in the pole, elevated body swing, and rotation tests. These results suggest that the Lumbricusin-induced promotion of neural cell proliferation via p27(Kip1) degradation has a protective effect in an experimental PD model. Thus, the antimicrobial peptide Lumbricusin could possibly be developed as a potential therapeutic agent for the treatment of PD. PMID:26215270

  9. CDK2 and mTOR are direct molecular targets of isoangustone A in the suppression of human prostate cancer cell growth.

    PubMed

    Lee, Eunjung; Son, Joe Eun; Byun, Sanguine; Lee, Seung Joon; Kim, Yeong A; Liu, Kangdong; Kim, Jiyoung; Lim, Soon Sung; Park, Jung Han Yoon; Dong, Zigang; Lee, Ki Won; Lee, Hyong Joo

    2013-10-01

    Licorice extract which is used as a natural sweetener has been shown to possess inhibitory effects against prostate cancer, but the mechanisms responsible are poorly understood. Here, we report a compound, isoangustone A (IAA) in licorice that potently suppresses the growth of aggressive prostate cancer and sought to clarify its mechanism of action. We analyzed its inhibitory effects on the growth of PTEN-deleted human prostate cancer cells, in vitro and in vivo. Administration of IAA significantly attenuated the growth of prostate cancer cell cultures and xenograft tumors. These effects were found to be attributable to inhibition of the G1/S phase cell cycle transition and the accumulation of p27(kip1). The elevated p27(kip1) expression levels were concurrent with the decrease of its phosphorylation at threonine 187 through suppression of CDK2 kinase activity and the reduced phosphorylation of Akt at Serine 473 by diminishing the kinase activity of the mammalian target of rapamycin (mTOR). Further analysis using recombinant proteins and immunoprecipitated cell lysates determined that IAA exerts suppressive effects against CDK2 and mTOR kinase activity by direct binding with both proteins. These findings suggested that the licorice compound IAA is a potent molecular inhibitor of CDK2 and mTOR, with strong implications for the treatment of prostate cancer. Thus, licorice-derived extracts with high IAA content warrant further clinical investigation for nutritional sources for prostate cancer patients. PMID:23707764

  10. Pfaffosidic Fraction from Hebanthe paniculata Induces Cell Cycle Arrest and Caspase-3-Induced Apoptosis in HepG2 Cells

    PubMed Central

    da Silva, Tereza Cristina; Cogliati, Bruno; Latorre, Andréia Oliveira; Akisue, Gokithi; Nagamine, Márcia Kazumi; Haraguchi, Mitsue; Hansen, Daiane; Sanches, Daniel Soares; Dagli, Maria Lúcia Zaidan

    2015-01-01

    Hebanthe paniculata roots (formerly Pfaffia paniculata and popularly known as Brazilian ginseng) show antineoplastic, chemopreventive, and antiproliferative properties. Functional properties of these roots and their extracts are usually attributed to the pfaffosidic fraction, which is composed mainly by pfaffosides A–F. However, the therapeutic potential of this fraction in cancer cells is not yet entirely understood. This study aimed to analyze the antitumoral effects of the purified pfaffosidic fraction or saponinic fraction on the human hepatocellular carcinoma HepG2 cell line. Cellular viability, proliferation, and apoptosis were evaluated, respectively, by MTT assay, BrdU incorporation, activated caspase-3 immunocytochemistry, and DNA fragmentation assay. Cell cycle was analyzed by flow cytometry and the cell cycle-related proteins were analyzed by quantitative PCR and Western blot. The cells exposed to pfaffosidic fraction had reduced viability and cellular growth, induced G2/M at 48 h or S at 72 h arrest, and increased sub-G1 cell population via cyclin E downregulation, p27KIP1 overexpression, and caspase-3-induced apoptosis, without affecting the DNA integrity. Antitumoral effects of pfaffosidic fraction from H. paniculata in HepG2 cells originated by multimechanisms of action might be associated with cell cycle arrest in the S phase, by CDK2 and cyclin E downregulation and p27KIP1 overexpression, besides induction of apoptosis through caspase-3 activation. PMID:26075002

  11. 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

    2016-08-01

    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. PMID:27295004

  12. Cell-cycle arrest induced by the bacterial adenylate cyclase toxins from Bacillus anthracis and Bordetella pertussis

    PubMed Central

    Gray, Mary C.; Hewlett, Erik L.

    2014-01-01

    Summary Bacillus anthracis Edema Toxin (ET) and Bordetella pertussis Adenylate Cyclase Toxin (ACT) enter host cells and produce cAMP. To understand the cellular consequences, we exposed J774 cells to these toxins at ng/ml (pM) concentrations, then followed cell number and changes in cell signaling pathways. Under these conditions, both toxins produce a concentration-dependent inhibition of cell proliferation without cytotoxicity. ET and ACT increase the proportion of cells in G1/G0 and reduce S-phase, such that a single addition of ET or ACT inhibits cell division for 3 to 6 days. Treatment with ET or ACT produces striking changes in proteins controlling cell cycle, including virtual elimination of phosphorylated ERK 1/2 and Cyclin D1 and increases in phospho-CREB and p27Kip1. Importantly, PD98059, a MEK inhibitor, elicits a comparable reduction in Cyclin D1 to that produced by the toxins and blocks proliferation. These data show that non-lethal concentrations of ET and ACT impose a prolonged block on the proliferation of J774 cells by impairment of the progression from G1/G0 to S-phase in a process involving cAMP-mediated increases in phospho-CREB and p27Kip1 and reductions in phospho-ERK 1/2 and Cyclin D1. This phenomenon represents a new mechanism by which these toxins affect host cells. PMID:20946259

  13. Combination treatment with proteasome inhibitors and antiestrogens has a synergistic effect mediated by p21WAF1 in estrogen receptor-positive breast cancer.

    PubMed

    Maynadier, Marie; Basile, Ilaria; Gallud, Audrey; Gary-Bobo, Magali; Garcia, Marcel

    2016-08-01

    Although antiestrogens significantly improve the survival of patients with ER-positive breast cancer, therapeutic resistance remains a major limitation. The combinatorial use of antiestrogen with other therapies was proposed to increase their efficiency and more importantly, to prevent or delay the resistance phenomenon. In the present study, we addressed their combined effects with proteasome inhibitors (PIs). The effects of antiestrogens (hydroxyl-tamoxifen, raloxifen and fulvestrant) currently used in endocrine therapy were tested in combination with PIs, bortezomib or MG132, on the growth of three ER-positive breast cancer cell lines and in two cellular models of acquired antiestrogen resistance. When compared to single treatments, these combined treatments were significantly more effective in preventing the growth of the cell lines. The regulation of key cell cycle proteins, the cyclin-dependent kinase inhibitors, p21WAF1 and p27KIP1, were also studied. Bortezomib and MG132 drastically increased p21WAF1 expression through elevation of its mRNA concentration. Notably, p27KIP1 regulation was quite different from that of p21WAF1. Furthermore, the effect of bortezomib in combination with antiestrogen was evaluated on antiestrogen-resistant cell lines. The growth of two antiestrogen-resistant cell lines appeared responsive to proteasome inhibition and was strongly decreased by a combined therapy with an antiestrogen. Collectively, these findings provide new perspectives for the use of PIs in combination with endocrine therapies for breast cancer and possibly to overcome acquired hormonal resistance. PMID:27373750

  14. In vivo optical detection of intranuclear cancer biomarkers using gold nanoparticles

    NASA Astrophysics Data System (ADS)

    Kumar, Sonia; Sokolov, Konstantin; Richards-Kortum, Rebecca

    2006-02-01

    Specific genotypes of human papillomavirus (HPV) are well correlated with cervical oncogenesis. The major transforming and immortalizing protein in high risk HPVs, namely HPV16, is E7 protein. E7 protein functions by deregulating the cell cycle and promoting S-phase reentry in differentiated keratinocytes. Currently, clinical diagnosis of cervical cancer is based on phenotypic changes observed in a screening Papanicolaou smear. Although screening has been effective in reducing the occurrence of cervical cancer, the low specificity of the Pap smear results in resources wasted on the evaluation of low-grade lesions not likely to progress to cervical cancer. Molecular characterization of active HPV infections using molecular specific contrast agents are combined with in-vivo optical imaging is proposed to be a cost-effective, non-invasive technique for the detection of cervical pre-cancers. Contrast is achieved by exploiting the peak absorbance and scattering shift in aggregated gold nanoparticles over isolated ones and molecular specificity is achieved via recognition moieties with high affinities for E7. Conjugates of gold nanoparticles and HPV16 anti-E7 antibodies are delivered into the nucleus of living cells and imaged with reflectance confocal microscopy. These contrast agents have been used to successfully enhance contrast in HPV16+ cervical cancer cells over HPV- cells by a factor of 2.5. Further characterization and development of these contrast agents will provide a robust, low cost screening tool for the detection of cervical pre-cancers.

  15. Atypical sporadic CJD-MM phenotype with white matter kuru plaques associated with intranuclear inclusion body and argyrophilic grain disease.

    PubMed

    Berghoff, Anna S; Trummert, Anita; Unterberger, Ursula; Ströbel, Thomas; Hortobágyi, Tibor; Kovacs, Gabor G

    2015-08-01

    We describe an atypical neuropathological phenotype of sporadic Creutzfeldt-Jakob disease in a 76-year-old man. The clinical symptoms were characterized by progressive dementia, gait ataxia, rigidity and urinary incontinence. The disease duration was 6 weeks. MRI did not show prominent atrophy or hyperintensities in cortical areas, striatum or thalamus. Biomarker examination of the cerebrospinal fluid deviated from that seen in pure Alzheimer's disease. Triphasic waves in the EEG were detected only later in the disease course, while 14-3-3 assay was positive. PRNP genotyping revealed methionine homozygosity (MM) at codon 129. Neuropathology showed classical CJD changes corresponding to the MM type 1 cases. However, a striking feature was the presence of abundant kuru-type plaques in the white matter. This rare morphology was associated with neuropathological signs of intranuclear inclusion body disease and advanced stage of argyrophilic grain disease. These alterations did not show correlation with each other, thus seemed to develop independently. This case further highlights the complexity of neuropathological alterations in the ageing brain. PMID:25783686

  16. Inclusion of correlations in the empherical selection of intranuclear cascade nucleons from high energy hadron-nucleus collisions

    NASA Astrophysics Data System (ADS)

    Alsmiller, F. S.; Alsmiller, R. G.

    1989-06-01

    The very high energy (5 GeV to 20 Tev) hadron-nucleus differential particle production model found in the Monte Carlo transport code FLUKA87 has been adapted for inclusion in the transport code HETC88. The empirical selection of intranuclear cascade nucleons has been modified to provide simple correlations with the randomly selected number of hadron-nucleon collisions. A standard method of calculating the extension energy of the compound nucleus preceding an added evaporation step by assuming the particles are produced in a one-dimensional nuclear well is applied. This method, coupled with the above correlations, lead to improved correlations of the excitation energy with the A and Z of the compound nucleus, and then to greatly improved distributions of the residual nuclei following evaporation. The frequency distribution of low enegy ( β < 0.7) charged particles show good agreement with experiment for 200 GeV protons incident on emulsions. Average multiplicities of showe and grey particles after evaporation for protons and pions incident on several elements are also compared with experiment.

  17. A quantitative investigation of neuronal cytoplasmic and intranuclear inclusions in the pontine and inferior olivary nuclei in multiple system atrophy.

    PubMed

    Nishie, M; Mori, F; Yoshimoto, M; Takahashi, H; Wakabayashi, K

    2004-10-01

    Multiple system atrophy (MSA) is a sporadic neurodegenerative disease characterized by the presence of neuronal and oligodendroglial alpha-synuclein aggregates. To investigate the relationship between the occurrence of neuronal cytoplasmic and intranuclear inclusions (NCIs and NNIs, respectively) and the progression of neuronal degeneration, we performed a quantitative analysis of the pontine and inferior olivary nuclei based on 14 cases of MSA. alpha-Synuclein immunohistochemistry revealed that NCIs and NNIs were present in both brain nuclei in all the cases. The average incidence of NCIs in the pontine and inferior olivary nuclei was 9.1% and 25.8%, respectively, and that of NNIs was 9.2% and 9.0%, respectively. The number of NNIs was strongly correlated with that of neurones in the pontine and inferior olivary nuclei. Although the number of NCIs was not correlated with the neuronal population in both nuclei, the NCI count in patients with moderate MSA was higher than in patients with mild MSA. The NNI count was much higher than the NCI count in the pontine nucleus in four patients, and was the same in the olivary nucleus in three of the four patients. Moreover, the neuronal population in the NNI-predominant cases was significantly higher than in the NCI-predominant cases. These findings suggest that NCI formation is accelerated by the progression of the disease process, and that in MSA, NNI formation is an earlier phenomenon than NCI formation. PMID:15488031

  18. Neuronal ubiquitinated intranuclear inclusions in familial and non-familial frontotemporal dementia of the motor neuron disease type associated with amyotrophic lateral sclerosis.

    PubMed

    Bigio, Eileen H; Johnson, Nancy A; Rademaker, Alfred W; Fung, Bing B; Mesulam, M-Marsel; Siddique, Nailah; Dellefave, Lisa; Caliendo, Janice; Freeman, Stefanie; Siddique, Teepu

    2004-08-01

    Ubiquitinated cytoplasmic inclusions (Ub-CIs) in superficial frontal cortex and dentate gyrus neurons are the hallmark of frontotemporal degeneration of the motor neuron disease-type (FTD-MND-type). To date, 2 reports have described intranuclear ubiquitinated inclusions (Ub-INIs) in 9 cases of familial FTD-MND-type (without clinical or pathologic motor neuron disease, MND). In the current study we found an additional 11 cases with Ub-INIs. We have identified for the first time among these cases 2 with a negative family history and 3 that have concomitant amyotrophic lateral sclerosis (ALS). The results of the present study i) confirm a previous report of significantly lower average brain weight and longer duration in cases with Ub-INIs, ii) reveal significantly greater striatal neuronal loss and gliosis in cases with intranuclear inclusions, and iii) demonstrate that intranuclear inclusions correlate with cytoplasmic inclusions and dystrophic neurites in frontal cortex and striatum but not in dentate gyrus. In addition, the current study confirms that Ub-INIs are found in familial FTD-MND-type, but also extends the presence of Ub-INIs to familial FTD-MND (with concomitant ALS), and probably also to non-familial FTD-MND-type. PMID:15330335

  19. Efficient intranuclear gene delivery by CdSe aqueous quantum dots electrostatically-coated with polyethyleneimine

    NASA Astrophysics Data System (ADS)

    Au, Giang H. T.; Y Shih, Wan; Shih, Wei-Heng

    2015-01-01

    Quantum dots (QDs) are semiconducting nanoparticles with photoluminescence properties that do not photobleach. Due to these advantages, using QDs for non-viral gene delivery has the additional benefit of being able to track the delivery of the genes in real time as it happens. We investigate the efficacy of mercaptopropionic acid (MPA)-capped CdSe aqueous quantum dots (AQDs) electrostatically complexed with branched polyethyleneimine (PEI) both as a non-viral gene delivery vector and as a fluorescent probe for tracking the delivery of genes into nuclei. The MPA-capped CdSe AQDs that were completely synthesized in water were the model AQDs. A nominal MPA:Cd:Se = 4:3:1 was chosen for optimal photoluminescence and zeta potential. The gene delivery study was carried out in vitro using a human colon cancer cell line, HT29 (ATCC). The model gene was a plasmid DNA (pDNA) that can express red fluorescent protein (RFP). Positively charged branched PEI was employed to provide a proton buffer to the AQDs to allow for endosomal escape. It is shown that by using a PEI-AQD complex with a PEI/AQD molar ratio of 300 and a nominal pDNA/PEI-AQD ratio of 6, we can achieve 75 ± 2.6% RFP expression efficiency with cell vitality remaining at 78 ± 4% of the control.

  20. Design of a microfluidic device to quantify dynamic intra-nuclear deformation during cell migration through confining environments

    PubMed Central

    Davidson, Patricia M.; Sliz, Josiah; Isermann, Philipp; Denais, Celine; Lammerding, Jan

    2015-01-01

    The ability of cells to migrate through tissues and interstitial space is an essential factor during development and tissue homeostasis, immune cell mobility, and in various human diseases. Deformation of the nucleus and its associated lamina during 3-D migration is gathering increasing interest in the context of cancer metastasis, with the underlying hypothesis that a softer nucleus, resulting from reduced levels of lamin A/C, may aid tumour spreading. However, current methods to study the migration of cells in confining three dimensional (3-D) environments are limited by their imprecise control over the confinement, physiological relevance, and/or compatibility with high resolution imaging techniques. We describe the design of a polydimethylsiloxane (PDMS) microfluidic device composed of channels with precisely-defined constrictions mimicking physiological environments that enable high resolution imaging of live and fixed cells. The device promotes easy cell loading and rapid, yet long-lasting (>24 hours) chemotactic gradient formation without the need for continuous perfusion. Using this device, we obtained detailed, quantitative measurements of dynamic nuclear deformation as cells migrate through tight spaces, revealing distinct phases of nuclear translocation through the constriction, buckling of the nuclear lamina, and severe intranuclear strain. Furthermore, we found that lamin A/C-deficient cells exhibited increased and more plastic nuclear deformations compared to wild-type cells but only minimal changes in nuclear volume, implying that low lamin A/C levels facilitate migration through constrictions by increasing nuclear deformability rather than compressibility. The integration of our migration devices with high resolution time-lapse imaging provides a powerful new approach to study intracellular mechanics and dynamics in a variety of physiologically-relevant applications, ranging from cancer cell invasion to immune cell recruitment. PMID:26549481

  1. Evidence that FOXO3a is involved in oocyte apoptosis in the neonatal rat ovary.

    PubMed

    Sui, Xu-Xia; Luo, Li-Li; Xu, Jin-Jie; Fu, Yu-Cai

    2010-08-01

    Previous studies have proposed that the forkhead transcription factor FOXO3a is involved in cell cycle arrest and apoptosis and that it may also repress follicular development by inducing cell cycle arrest in ovaries. We have recently demonstrated that FOXO3a induces oocyte apoptosis of neonatal rat ovaries under in vitro conditions. In the present study, we evaluated the role of FOXO3a in oocyte apoptosis under in vivo conditions. Ovaries from rats were obtained from newborns on postnatal day (PD) 1, 2, 3, and 4. TUNEL assay results showed that oocyte apoptosis occurred mainly on PD 1 and 2. Immunohistochemical staining of FOXO3a, Bim, Fas ligand (FasL), p27KIP1, caspase-8, and caspase-3 showed that they were all expressed mainly in naked oocytes on PD 1 and 2. The percentage of positive FOXO3a staining of oocytes reached peak levels in the ovaries of 2-day-old rats, which was consistent with the rate of the apoptotic profiles determined by TUNEL. The percentage between TUNEL-positive and FOXO3a-positive oocytes in the nucleus showed no statistical differences within the 4-day-old rat ovaries. Furthermore, the positive oocyte percentage of the target factors of FOXO3a (Bim, p27KIP1, and FasL) and pro-apoptotic proteins (caspase-3 and caspase-8) also reached peak levels in the ovaries of 2-day-old rats, which was similar to the rate of FOXO3a-positive oocytes. These results suggest that FOXO3a in the oocyte nucleus is involved in oocyte apoptosis; that is, FOXO3a-positive oocytes may be the apoptotic cells. To verify this, rat oocytes were subjected to TUNEL and immunofluorescent double-labeling assays. We found that TUNEL-positive cells were also FOXO3a-, Bim-, or FasL-positive. To identify the downstream target of FOXO3a, double immunofluorescent staining with antibodies to Bim and FasL was performed. We found that FOXO3a-positive cells were also Bim- and FasL-positive. We conclude that the overexpression of FOXO3a in the oocyte nucleus of neonatal rat ovaries

  2. Bax alpha perturbs T cell development and affects cell cycle entry of T cells.

    PubMed Central

    Brady, H J; Gil-Gómez, G; Kirberg, J; Berns, A J

    1996-01-01

    Bax alpha can heterodimerize with Bcl-2 and Bcl-X(L), countering their effects, as well as promoting apoptosis on overexpression. We show that bax alpha transgenic mice have greatly reduced numbers of mature T cells, which results from an impaired positive selection in the thymus. This perturbation in positive selection is accompanied by an increase in the number of cycling thymocytes. Further to this, mature T cells overexpressing Bax alpha have lower levels of p27Kip1 and enter S phase more rapidly in response to interleukin-2 stimulation than do control T cells, while the converse is true of bcl-2 transgenic T cells. These data indicate that apoptotic regulatory proteins can modulate the level of cell cycle-controlling proteins and thereby directly impact on the cell cycle. Images PMID:9003775

  3. p62 positive, TDP-43 negative, neuronal cytoplasmic and intranuclear inclusions in the cerebellum and hippocampus define the pathology of C9orf72-linked FTLD and MND/ALS.

    PubMed

    Al-Sarraj, Safa; King, Andrew; Troakes, Claire; Smith, Bradley; Maekawa, Satomi; Bodi, Istvan; Rogelj, Boris; Al-Chalabi, Ammar; Hortobágyi, Tibor; Shaw, Christopher E

    2011-12-01

    Neuronal cytoplasmic inclusions (NCIs) containing phosphorylated TDP-43 (p-TDP-43) are the pathological hallmarks of motor neuron disease/amyotrophic lateral sclerosis (MND/ALS) and FTLD-TDP. The vast majority of NCIs in the brain and spinal cord also label for ubiquitin and p62, however, we have previously reported a subset of TDP-43 proteinopathy patients who have unusual and abundant p62 positive, TDP-43 negative inclusions in the cerebellum and hippocampus. Here we sought to determine whether these cases carry the hexanucleotide repeat expansion in C9orf72. Repeat primer PCR was performed in 36 MND/ALS, FTLD-MND/ALS and FTLD-TDP cases and four controls. Fourteen individuals with the repeat expansion were detected. In all the 14 expansion mutation cases there were abundant globular and star-shaped p62 positive NCIs in the pyramidal cell layer of the hippocampus, the vast majority of which were p-TDP-43 negative. p62 positive NCIs were also abundant in the cerebellar granular and molecular layers in all cases and in Purkinje cells in 12/14 cases but they were only positive for p-TDP-43 in the granular layer of one case. Abundant p62 positive, p-TDP-43 negative neuronal intranuclear inclusions (NIIs) were seen in 12/14 cases in the pyramidal cell layer of the hippocampus and in 6/14 cases in the cerebellar granular layer. This unusual combination of inclusions appears pathognomonic for C9orf72 repeat expansion positive MND/ALS and FTLD-TDP which we believe form a pathologically distinct subset of TDP-43 proteinopathies. Our results suggest that proteins other than TDP-43 are binding p62 and aggregating in response to the mutation which may play a mechanistic role in neurodegeneration. PMID:22101323

  4. G1-checkpoint function including a cyclin-dependent kinase 2 regulatory pathway as potential determinant of 7-hydroxystaurosporine (UCN-01)-induced apoptosis and G1-phase accumulation.

    PubMed

    Akiyama, T; Sugiyama, K; Shimizu, M; Tamaoki, T; Akinaga, S

    1999-12-01

    7-Hydroxystaurosporine (UCN-01), which was originally identified as a protein kinase C selective inhibitor, is currently in clinical trials as an anti-cancer drug. We previously showed that UCN-01 induced preferential G1-phase accumulation in tumor cells and this effect was associated with the retinoblastoma (Rb) protein and its regulatory factors, such as cyclin-dependent kinase 2 (CDK2) and CDK inhibitors p21Cip1/WAF1 and p27Kip1. We demonstrate here that G1-phase accumulation was induced by UCN-01 in Rb-proficient cell lines (WiDr and HCT116 human colon carcinomas and WI-38 human lung fibroblast), and it was accompanied by dephosphorylation of Rb. In addition, UCN-01-induced G1-phase accumulation was also demonstrated in a Rb-defective cell line (Saos-2 human osteosarcoma), but not in a simian virus 40 (SV40)-transformed cell line (WI-38 VA13). Apoptosis was induced by UCN-01 in the two Rb-deficient cell lines, but not in the other Rb-proficient cell lines. These observations suggest that G1-checkpoint function might be important for cell survival during UCN-01 treatment. In addition, there may be a UCN-01-responsive factor in the G1-checkpoint machinery other than Rb which is targeted by SV40. Further studies revealed a correlation between UCN-01-induced G1-phase accumulation and reduction of cellular CDK2 kinase activity. This reduction was strictly dependent on down-regulation of the Thr160-phosphorylated form of CDK2 protein, and coincided in part with up-regulation of p27Kip1, but it was independent of the level of the p21Cip1/WAF1 protein. These results suggest that G1-checkpoint function, including a CDK2-regulatory pathway, may be a significant determinant of the sensitivity of tumor cells to UCN-01. PMID:10665655

  5. Extension of the Liège intranuclear cascade model at incident energies between 2 and 12 GeV. Aspects of pion production

    NASA Astrophysics Data System (ADS)

    Pedoux, Sophie; Cugnon, Joseph

    2011-09-01

    The validity of the standard version of the Liège Intra-Nuclear Cascade (INCL4) model, which has been shown to be quite successful for the description of spallation reactions, is limited to an upper incident energy of ˜2 GeV, because inelastic elementary processes are restricted to the excitation and de-excitation of the Delta resonance. In this paper, the INCL4 model is extended to higher incident energy by including other inelastic elementary collisions. However, excitation of heavier baryonic resonances is replaced by direct multipion production in elementary nucleon-nucleon and pion-nucleon collisions. The predictions of the modified model for production of charged pions by proton and pion beams off nuclei are compared with experimental data of the HARP Collaboration for beam energies between 2 and 12 GeV. The apparent duality between the approach based on excitation of numerous baryonic resonances and our approach is briefly discussed.

  6. Kick-starting the cell cycle: From growth-factor stimulation to initiation of DNA replication

    NASA Astrophysics Data System (ADS)

    Aguda, Baltazar D.

    2001-03-01

    The essential genes, proteins and associated regulatory networks involved in the entry into the mammalian cell cycle are identified, from activation of growth-factor receptors to intracellular signal transduction pathways that impinge on the cell cycle machinery and ultimately on the initiation of DNA replication. Signaling pathways mediated by the oncoproteins Ras and Myc induce the activation of cyclin-dependent kinases CDK4 and CDK2, and the assembly and firing of pre-replication complexes require a collaboration among E2F, CDK2, and Cdc7 kinase. A proposed core mechanism of the restriction point, the major checkpoint prior to commitment to DNA synthesis, involves cyclin E/CDK2, the phosphatase Cdc25A, and the CDK inhibitor p27Kip1.

  7. Proliferation and differentiation characteristics of neural stem cells during course of cerebral cortical histogenesis.

    PubMed

    Mitsuhashi, Takayuki; Takahashi, Takao

    2016-01-01

    Recent advancements in the research field of stem cell biology have enabled the realization of regenerative medicine in various systems of the body, including the central nervous system. However, fundamental knowledge regarding how neural stem cells divide and generate young neurons in mammals, especially in vivo, is still inadequate. In this article, we shall summarize the concept of cell cycle/division of neural stem cells that generate projection neurons in the murine cerebral cortex. We shall also review the molecular mechanisms that modulate the critical parameters related to the cell cycle regulatory mechanisms, with special reference to the cell cycle regulatory protein p27(Kip1) , an inhibitor of progression of the cell cycle at the G1 phase. A better understanding of the mechanisms controlling cell cycle progression is expected to contribute to the development of novel strategies to increase the efficiency of neural cell/tissue production, both in vivo and in vitro. PMID:26058879

  8. Development and molecular characterization of polymeric micro-nanofibrous scaffold of a defined 3-D niche for in vitro chemosensitivity analysis against acute myeloid leukemia cells

    PubMed Central

    Nair, Maya S; Mony, Ullas; Menon, Deepthy; Koyakutty, Manzoor; Sidharthan, Neeraj; Pavithran, Keechilat; Nair, Shantikumar V; Menon, Krishnakumar N

    2015-01-01

    Standard in vitro drug testing employs 2-D tissue culture plate systems to test anti-leukemic drugs against cell adhesion-mediated drug-resistant leukemic cells that harbor in 3-D bone marrow microenvironments. This drawback necessitates the fabrication of 3-D scaffolds that have cell adhesion-mediated drug-resistant properties similar to in vivo niches. We therefore aimed at exploiting the known property of polyurethane (PU)/poly-l-lactic acid (PLLA) in forming a micro-nanofibrous structure to fabricate unique, not presented before, as far as we are aware, 3-D micro-nanofibrous scaffold composites using a thermally induced phase separation technique. Among the different combinations of PU/PLLA composites generated, the unique PU/PLLA 60:40 composite displayed micro-nanofibrous morphology similar to decellularized bone marrow with increased protein and fibronectin adsorption. Culturing of acute myeloid leukemia (AML) KG1a cells in FN-coated PU/PLLA 60:40 shows increased cell adhesion and cell adhesion-mediated drug resistance to the drugs cytarabine and daunorubicin without changing the original CD34+/CD38−/CD33− phenotype for 168 hours compared to fibronectin tissue culture plate systems. Molecularly, as seen in vivo, increased chemoresistance is associated with the upregulation of anti-apoptotic Bcl2 and the cell cycle regulatory protein p27Kip1 leading to cell growth arrest. Abrogation of Bcl2 activity by the Bcl2-specific inhibitor ABT 737 led to cell death in the presence of both cytarabine and daunorubicin, demonstrating that the cell adhesion-mediated drug resistance induced by Bcl2 and p27Kip1 in the scaffold was similar to that seen in vivo. These results thus show the utility of a platform technology, wherein drug testing can be performed before administering to patients without the necessity for stromal cells. PMID:26028971

  9. In Vitro and in Vivo Anti-tumor Activity of miR-221/222 Inhibitors in Multiple Myeloma

    PubMed Central

    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

    2013-01-01

    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. PMID:23479461

  10. 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. PMID:23479461

  11. Chimeric DNA-RNA hammerhead ribozyme targeting transforming growth factor-beta 1 mRNA inhibits neointima formation in rat carotid artery after balloon injury.

    PubMed

    Ando, Hideyuki; Fukuda, Noboru; Kotani, Motoko; Yokoyama, Shin ichiro; Kunimoto, Satoshi; Matsumoto, Koichi; Saito, Satoshi; Kanmatsuse, Katsuo; Mugishima, Hideo

    2004-01-12

    We designed and synthesized a chimeric DNA-RNA hammerhead ribozyme targeting transforming growth factor (TGF)-beta 1 mRNA and found that this ribozyme effectively and specifically inhibited growth of vascular smooth muscle cells. We examined the effects of the chimeric DNA-RNA hammerhead ribozyme targeting TGF-beta 1 mRNA on neointima formation and investigated the underlying mechanism to develop a possible gene therapy for coronary artery restenosis after percutaneous transluminal coronary angioplasty. Expression of mRNAs encoding TGF-beta 1, p27kip1, and connective tissue growth factor (CTGF) in carotid artery increased after balloon injury. Fluorescein-isothiocyanate (FITC)-labeled ribozyme was taken up into the midlayer smooth muscle of the injured carotid artery. Both 2 and 5 mg of ribozyme reduced neointima formation by 65% compared to that of controls. Ribozyme markedly decreased expression of TGF-beta 1 mRNA and protein in injured vessel. Mismatch ribozyme had no effect on expression of TGF-beta 1 mRNA protein in injured vessel. Ribozyme markedly decreased expression of fibronectin, p27kip1, and CTGF mRNAs in injured vessel, whereas a mismatch ribozyme had no effect on these mRNAs. These findings indicate that the chimeric DNA-RNA hammerhead ribozyme targeting TGF-beta 1 mRNA inhibits neointima formation in rat carotid artery after balloon injury with suppression of TGF-beta 1 and inhibition of extracellular matrix and CTGF. In conclusion, the hammerhead ribozyme against TGF-beta 1 may have promise as a therapy for coronary artery restenosis after percutaneous transluminal coronary angioplasty. PMID:14729108

  12. Retinoic acid inhibits the proliferative response induced by CD40 activation and interleukin-4 in mantle cell lymphoma.

    PubMed

    Guidoboni, Massimo; Zancai, Paola; Cariati, Roberta; Rizzo, Silvana; Dal Col, Jessica; Pavan, Alessandro; Gloghini, Annunziata; Spina, Michele; Cuneo, Antonio; Pomponi, Fabrizio; Bononi, Antonio; Doglioni, Claudio; Maestro, Roberta; Carbone, Antonino; Boiocchi, Mauro; Dolcetti, Riccardo

    2005-01-15

    Mantle cell lymphoma (MCL) is an aggressive B-cell non-Hodgkin's lymphoma with poor response to therapy and unfavorable prognosis. Here, we show that retinoic acid (RA) isomers significantly inhibit the proliferation of both primary MCL cultures (n = 7) and established cell lines (Granta 519 and SP-53) as shown by [(3)H]thymidine uptake and carboxyfluorescein diacetate succinimidyl ester labeling coupled with cyclin D1 staining. RA induces cell accumulation in G(0)-G(1) together with a marked up-regulation of p27(Kip1) by inhibiting ubiquitination and proteasome-dependent degradation of the protein. The p21(Cip1) inhibitor was also up-regulated by RA in Granta 519 cells, whereas the expression of cyclin D1 is unaffected. Most of RA-induced p27(Kip1) was bound to cyclin D1/cyclin-dependent kinase 4 complexes, probably contributing to the decreased cyclin-dependent kinase 4 kinase activity and pRb hypophosphorylation observed in RA-treated cells. Experiments with receptor-selective ligands indicate that RA receptor alpha cooperates with retinoid X receptors in mediating RA-dependent MCL cell growth inhibition. Notably, RA isomers, and particularly 9-cis-RA, also inhibited the growth-promoting effect induced in primary MCL cells by CD40 activation alone or in combination with interleukin-4. Immunohistochemical analysis showed that significant numbers of CD40L-expressing lymphoid cells are present in lymph node biopsies of MCL patients. These results therefore further strengthen the possibility that triggering of CD40 by infiltrating CD40L+ cells may continuously promote the growth of MCL cells in vivo. On these grounds, our findings that RA inhibits basal MCL proliferation as well as MCL growth-promoting effects exerted by microenvironmental factors make these compounds highly attractive in terms of potential clinical efficacy in this setting. PMID:15695403

  13. Hadronic event generation for hadron cascade calculations and detector simulation, Part IV: The application of the intranuclear cascade model to reactions of pions, nucleons, kaons, and their antiparticles with nuclei below 6 GeV/c

    SciTech Connect

    Haenbssgen, K.

    1987-02-01

    An extension of the intranuclear cascade model is described. The primary hadrons may be pions, kaons, nucleons, and their antiparticles. Secondary particles produced include hyperons or antihyperons. A large amount of experimental data is described by the model. The model is constructed via the Monte Carlo generation of complete events, based on a model of the nucleus structure and the hadron/nucleon interaction inside the nucleus. Calculated average multiplicities and single and double differential cross sections are compared with experimental data.

  14. PICA95: An intranuclear-cascade code for 25-MeV to 3.5-GeV photon-induced nuclear reactions

    SciTech Connect

    Fu, C.Y.; Gabriel, T.A.; Lillie, R.A.

    1997-05-01

    PICA95, an intranuclear-cascade code for calculating photon-induced nuclear reactions for incident photon energies up to 3.5 GeV, is an extension of the original PICA code package that works for incident photon energies up to 400 MeV. The original code includes the quasi-deuteron breakup and single-pion production channels. The extension to an incident photon energy of 3.5 GeV requires the addition of multiple-pion production channels capable of emitting up to five pions. Relativistic phase-space relations are used to conserve energy and momentum in multi-body breakups. Fermi motion of the struck nucleon is included in the phase-space calculations as well as secondary nuclear collisions of the produced particles. Calculated doubly differential cross sections for the productions of protons, neutrons, {pi}{sup +}, {pi}{sup 0}, and {pi}{sup {minus}} for incident photon energies of 500 MeV, 1 GeV, and 2 GeV are compared with predictions by other codes. Due to the sparsity of experimental data, more experiments are needed in order to refine the gamma nuclear collision model.

  15. Hydroxyurea-induced replication stress causes poly(ADP-ribose) polymerase-2 accumulation and changes its intranuclear location in root meristems of Vicia faba.

    PubMed

    Rybaczek, Dorota

    2016-07-01

    Replication stress induced by 24 and 48h exposure to 2.5mM hydroxyurea (HU) increased the activity of poly(ADP-ribose) polymerase-2 (PARP-2; EC 2.4.2.30) in root meristem cells of Vicia faba. An increase in the number of PARP-2 foci was accompanied by their delocalization from peripheral areas to the interior of the nucleus. Our results indicate that the increase in PARP-2 was connected with an increase in S139-phosphorylated H2AX histones. The findings suggest the possible role of PARP-2 in replication stress. We also confirm that the intranuclear location of PARP-2 depends on the duration of HU-induced replication stress, confirming the role of PARP-2 as an indicator of stress intensity. Finally, we conclude that the more intense the HU-mediated replication stress, the greater the probability of PARP-2 activation or H2AXS139 phosphorylation, but also the greater the chance of increasing the efficiency of repair processes and a return to normal cell cycle progression. PMID:27155387

  16. Intra-Nuclear Single-Particle Tracking (I-SPT) to Reveal the Functional Architecture of Chromosomes.

    PubMed

    Récamier, Vincent

    2016-01-01

    Chromosome architecture needs to be investigated in relation with the chemical function of DNA. The kinetics of gene expression, DNA replication, and repair are driven by the mechanisms by which a functional nuclear protein finds its substrate in the nucleus. Single-particle tracking (SPT) is a method to quantify fluorescent molecules dynamics from the tracks of the single molecules recorded by high-resolution microscopes. SPT offers direct observation of the movement and single-molecule resolution. Usually SPT is performed on membranes because of higher contrast. Here, we introduce a novel method to record the trajectories of weakly fluorescent molecules in the nucleus of living cells. I-SPT uses some specific detection and analysis tools to enable the computation of reliable statistics on nuclear particle movement. PMID:27283314

  17. Polyreactive anti-DNA monoclonal antibodies and a derived peptide as vectors for the intracytoplasmic and intranuclear translocation of macromolecules.

    PubMed

    Avrameas, A; Ternynck, T; Nato, F; Buttin, G; Avrameas, S

    1998-05-12

    Naturally occurring polyreactive anti-DNA mAbs derived from a nonimmunized (NZB x NZW)F1 mouse with spontaneous lupus erythematosus penetrated and accumulated in the nuclei of a variety of cultured cells. These mAbs and their F(ab')2 and Fab' fragments, covalently coupled to fluorescein, peroxidase, or a 15-mer polynucleotide, also translocated to the cell nuclei. A 30-amino acid peptide corresponding to the combined sequences of the complementary-determining regions 2 and 3 of the heavy chain variable region of one mAb was able to penetrate into the cytoplasm and nucleus of cells of several lines. This peptide recognized DNA and was strongly polyreactive. Streptavidin-peroxidase conjugates complexed with the N-biotinylated peptide were rapidly translocated into cells. Similarly, peroxidase or anti-peroxidase polyclonal antibodies covalently coupled to the N-cysteinylated peptide through an heterobifunctional maleimide cross-linker were also rapidly internalized and frequently accumulated in nuclei. The peptide carrying 19 lysine residues at its N-terminal was highly effective in transfecting 3T3 cells with a plasmid containing the luciferase gene. Thus, penetrating mAbs and derived peptides are versatile vectors for the intracellular delivery of proteins and genes. PMID:9576929

  18. Proteins.

    ERIC Educational Resources Information Center

    Doolittle, Russell F.

    1985-01-01

    Examines proteins which give rise to structure and, by virtue of selective binding to other molecules, make genes. Binding sites, amino acids, protein evolution, and molecular paleontology are discussed. Work with encoding segments of deoxyribonucleic acid (exons) and noncoding stretches (introns) provides new information for hypotheses. (DH)

  19. Protein

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Proteins are the major structural and functional components of all cells in the body. They are macromolecules that comprise 1 or more chains of amino acids that vary in their sequence and length and are folded into specific 3-dimensional structures. The sizes and conformations of proteins, therefor...

  20. Hsp105 reduces the protein aggregation and cytotoxicity by expanded-polyglutamine proteins through the induction of Hsp70

    SciTech Connect

    Yamagishi, Nobuyuki; Goto, Kazumasa; Nakagawa, Satomi; Saito, Youhei; Hatayama, Takumi

    2010-09-10

    Hsp105{alpha} and Hsp105{beta} are major heat shock proteins in mammalian cells and belong to the HSP105/110 family. Hsp105{alpha} is expressed constitutively in the cytoplasm of cells, while Hsp105{beta}, an alternatively spliced form of Hsp105{alpha}, is expressed specifically in the nucleus of cells during mild heat shock. Here, we show that not only Hsp105{beta} but also Hsp105{alpha} accumulated in the nucleus of cells following the expression of enhanced green fluorescent protein with a pathological length polyQ tract (EGFP-polyQ97) and suppressed the intranuclear aggregation of polyQ proteins and apoptosis induced by EGFP-polyQ97. Mutants of Hsp105{alpha} and Hsp105{beta} with changes in the nuclear localization signal sequences, which localized exclusively in the cytoplasm with or without the expression of EGFP-polyQ97, did not suppress the intranuclear aggregation of polyQ proteins and apoptosis induced by EGFP-polyQ97. Furthermore, Hsp70 was induced by the co-expression of Hsp105{alpha} and EGFP-polyQ97, and the knockdown of Hsp70 reduced the inhibitory effect of Hsp105{alpha} and Hsp105{beta} on the intranuclear aggregation of polyQ proteins and apoptosis induced by EGFP-polyQ97. These observations suggested that Hsp105{alpha} and Hsp105{beta} suppressed the expanded polyQ tract-induced protein aggregation and apoptosis through the induction of Hsp70.

  1. The slow Wallerian degeneration protein, WldS, binds directly to VCP/p97 and partially redistributes it within the nucleus.

    PubMed

    Laser, Heike; Conforti, Laura; Morreale, Giacomo; Mack, Till G M; Heyer, Molly; Haley, Jane E; Wishart, Thomas M; Beirowski, Bogdan; Walker, Simon A; Haase, Georg; Celik, Arzu; Adalbert, Robert; Wagner, Diana; Grumme, Daniela; Ribchester, Richard R; Plomann, Markus; Coleman, Michael P

    2006-03-01

    Slow Wallerian degeneration (Wld(S)) mutant mice express a chimeric nuclear protein that protects sick or injured axons from degeneration. The C-terminal region, derived from NAD(+) synthesizing enzyme Nmnat1, is reported to confer neuroprotection in vitro. However, an additional role for the N-terminal 70 amino acids (N70), derived from multiubiquitination factor Ube4b, has not been excluded. In wild-type Ube4b, N70 is part of a sequence essential for ubiquitination activity but its role is not understood. We report direct binding of N70 to valosin-containing protein (VCP; p97/Cdc48), a protein with diverse cellular roles including a pivotal role in the ubiquitin proteasome system. Interaction with Wld(S) targets VCP to discrete intranuclear foci where ubiquitin epitopes can also accumulate. Wld(S) lacking its N-terminal 16 amino acids (N16) neither binds nor redistributes VCP, but continues to accumulate in intranuclear foci, targeting its intrinsic NAD(+) synthesis activity to these same foci. Wild-type Ube4b also requires N16 to bind VCP, despite a more C-terminal binding site in invertebrate orthologues. We conclude that N-terminal sequences of Wld(S) protein influence the intranuclear location of both ubiquitin proteasome and NAD(+) synthesis machinery and that an evolutionary recent sequence mediates binding of mammalian Ube4b to VCP. PMID:16371511

  2. The Slow Wallerian Degeneration Protein, WldS, Binds Directly to VCP/p97 and Partially Redistributes It within the NucleusD⃞

    PubMed Central

    Laser, Heike; Conforti, Laura; Morreale, Giacomo; Mack, Till G.M.; Heyer, Molly; Haley, Jane E.; Wishart, Thomas M.; Beirowski, Bogdan; Walker, Simon A.; Haase, Georg; Celik, Arzu; Adalbert, Robert; Wagner, Diana; Grumme, Daniela; Ribchester, Richard R.; Plomann, Markus; Coleman, Michael P.

    2006-01-01

    Slow Wallerian degeneration (WldS) mutant mice express a chimeric nuclear protein that protects sick or injured axons from degeneration. The C-terminal region, derived from NAD+ synthesizing enzyme Nmnat1, is reported to confer neuroprotection in vitro. However, an additional role for the N-terminal 70 amino acids (N70), derived from multiubiquitination factor Ube4b, has not been excluded. In wild-type Ube4b, N70 is part of a sequence essential for ubiquitination activity but its role is not understood. We report direct binding of N70 to valosin-containing protein (VCP; p97/Cdc48), a protein with diverse cellular roles including a pivotal role in the ubiquitin proteasome system. Interaction with WldS targets VCP to discrete intranuclear foci where ubiquitin epitopes can also accumulate. WldS lacking its N-terminal 16 amino acids (N16) neither binds nor redistributes VCP, but continues to accumulate in intranuclear foci, targeting its intrinsic NAD+ synthesis activity to these same foci. Wild-type Ube4b also requires N16 to bind VCP, despite a more C-terminal binding site in invertebrate orthologues. We conclude that N-terminal sequences of WldS protein influence the intranuclear location of both ubiquitin proteasome and NAD+ synthesis machinery and that an evolutionary recent sequence mediates binding of mammalian Ube4b to VCP. PMID:16371511

  3. Molecular control of brain size: Regulators of neural stem cell life, death and beyond

    SciTech Connect

    Joseph, Bertrand; Hermanson, Ola

    2010-05-01

    The proper development of the brain and other organs depends on multiple parameters, including strictly controlled expansion of specific progenitor pools. The regulation of such expansion events includes enzymatic activities that govern the correct number of specific cells to be generated via an orchestrated control of cell proliferation, cell cycle exit, differentiation, cell death etc. Certain proteins in turn exert direct control of these enzymatic activities and thus progenitor pool expansion and organ size. The members of the Cip/Kip family (p21Cip1/p27Kip1/p57Kip2) are well-known regulators of cell cycle exit that interact with and inhibit the activity of cyclin-CDK complexes, whereas members of the p53/p63/p73 family are traditionally associated with regulation of cell death. It has however become clear that the roles for these proteins are not as clear-cut as initially thought. In this review, we discuss the roles for proteins of the Cip/Kip and p53/p63/p73 families in the regulation of cell cycle control, differentiation, and death of neural stem cells. We suggest that these proteins act as molecular interfaces, or 'pilots', to assure the correct assembly of protein complexes with enzymatic activities at the right place at the right time, thereby regulating essential decisions in multiple cellular events.

  4. New approach to description of (d,xn) spectra at energies below 50 MeV in Monte Carlo simulation by intra-nuclear cascade code with Distorted Wave Born Approximation

    NASA Astrophysics Data System (ADS)

    Hashimoto, S.; Iwamoto, Y.; Sato, T.; Niita, K.; Boudard, A.; Cugnon, J.; David, J.-C.; Leray, S.; Mancusi, D.

    2014-08-01

    A new approach to describing neutron spectra of deuteron-induced reactions in the Monte Carlo simulation for particle transport has been developed by combining the Intra-Nuclear Cascade of Liège (INCL) and the Distorted Wave Born Approximation (DWBA) calculation. We incorporated this combined method into the Particle and Heavy Ion Transport code System (PHITS) and applied it to estimate (d,xn) spectra on natLi, 9Be, and natC targets at incident energies ranging from 10 to 40 MeV. Double differential cross sections obtained by INCL and DWBA successfully reproduced broad peaks and discrete peaks, respectively, at the same energies as those observed in experimental data. Furthermore, an excellent agreement was observed between experimental data and PHITS-derived results using the combined method in thick target neutron yields over a wide range of neutron emission angles in the reactions. We also applied the new method to estimate (d,xp) spectra in the reactions, and discussed the validity for the proton emission spectra.

  5. 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

  6. YSY01A, a Novel Proteasome Inhibitor, Induces Cell Cycle Arrest on G2 Phase in MCF-7 Cells via ERα and PI3K/Akt Pathways

    PubMed Central

    Xue, Bingjie; Huang, Wei; Yuan, Xia; Xu, Bo; Lou, Yaxin; Zhou, Quan; Ran, Fuxiang; Ge, Zemei; Li, Runtao; Cui, Jingrong

    2015-01-01

    Given that the proteasome is essential for multiple cellular processes by degrading diverse regulatory proteins, inhibition of the proteasome has emerged as an attractive target for anti-cancer therapy. YSY01A is a novel small molecule compound targeting the proteasome. The compound was found to suppress viability of MCF-7 cells and cause limited cell membrane damage as determined by sulforhodamine B assay (SRB) and CytoTox 96® non-radioactive cytotoxicity assay. High-content screening (HCS) further shows that YSY01A treatment induces cell cycle arrest on G2 phase within 24 hrs. Label-free quantitative proteomics (LFQP), which allows extensive comparison of cellular responses following YSY01A treatment, suggests that various regulatory proteins including cell cycle associated proteins and PI3K/Akt pathway may be affected. Furthermore, YSY01A increases p-CDC-2, p-FOXO3a, p53, p21Cip1 and p27Kip1 but decreases p-Akt, p-ERα as confirmed by Western blotting. Therefore, YSY01A represents a potential therapeutic for breast cancer MCF-7 by inducing G2 phase arrest via ERα and PI3K/Akt pathways. PMID:25767601

  7. Calpain Genetic Disruption and HSP90 Inhibition Combine To Attenuate Mammary Tumorigenesis.

    PubMed

    Grieve, Stacy; Gao, Yan; Hall, Christine; Hu, Jing; Greer, Peter A

    2016-08-01

    Calpain is an intracellular Ca(2+)-regulated protease system whose substrates include proteins involved in proliferation, survival, migration, invasion, and sensitivity to therapeutic drugs. Genetic disruption of calpain attenuated the tumorigenic potential of breast cancer cells and hypersensitized cells to 17AAG, an inhibitor of the molecular chaperone HSP90. Calpain-1 or -2 overexpression rendered cells resistant to 17AAG, whereas downregulation or inhibition of calpain-1/2 led to increased cell death in multiple breast cancer cell lines, including models of HER2(+) (SKBR3) and triple-negative basal-cell-like (MDA-MB-231) breast cancer. In an MDA-MB-231 orthotopic xenograft model, calpain knockdown or 17AAG treatment independently attenuated tumor growth and metastasis, while the combination was most effective. Calpain knockdown was associated with increased 17AAG-induced degradation of the HSP90 clients cyclin D1 and AKT and multidrug resistance protein 2, which correlated with increased expression of antimitogenic p27(KIP1) and proapoptotic BIM proteins. Like other therapeutics, 17AAG can be effluxed by specific ABC transporters. Calpain expression positively correlated with the expression of P glycoprotein in mouse embryonic fibroblasts. Importantly, we show that calpain affects ABC transporter function and efflux of clinically relevant doxorubicin. These observations provide a compelling rationale for exploring the combination of calpain inhibition with new or existing cancer therapeutics. PMID:27215381

  8. Euphol from Euphorbia tirucalli selectively inhibits human gastric cancer cell growth through the induction of ERK1/2-mediated apoptosis.

    PubMed

    Lin, Ming-Wei; Lin, An-Shen; Wu, Deng-Chyang; Wang, Sophie S W; Chang, Fang-Rong; Wu, Yang-Chang; Huang, Yaw-Bin

    2012-12-01

    Gastric cancer is one of the most common malignancies worldwide, and the main cause of cancer-related death in Asia. The present study assessed the anticancer effects of euphol, a triterpene alcohol with anti-inflammatory and antiviral activities on human gastric cancer cells. Euphol showed higher cytotoxicity activity against human gastric CS12 cancer cells than against noncancer CSN cells. In addition, it up-regulated the pro-apoptotic protein BAX and down-regulated the prosurvival protein Bcl-2, causing mitochondrial dysfunction, possibly by caspase-3 activation. The anti-proliferative effects of euphol were associated with the increased p27(kip1) levels and decreased cyclin B1 levels. Inhibition of ERK1/2 activation by PD98059 reversed euphol-induced pro-apoptotic protein expression and cell death. Taken together, these findings suggest that euphol selectively induced gastric cancer cells apoptosis by modulation of ERK signaling, and could thus be of value for cancer therapy. PMID:22634261

  9. Dendrotoxin-κ suppresses tumor growth induced by human lung adenocarcinoma A549 cells in nude mice

    PubMed Central

    Jang, Soo Hwa; Ryu, Pan Dong

    2011-01-01

    Voltage-gated K+ (Kv) channels have been considered to be a regulator of membrane potential and neuronal excitability. Recently, accumulated evidence has indicated that several Kv channel subtypes contribute to the control of cell proliferation in various types of cells and are worth noting as potential emerging molecular targets of cancer therapy. In the present study, we investigated the effects of the Kv1.1-specific blocker, dendrotoxin-κ (DTX-κ), on tumor formation induced by the human lung adenocarcinoma cell line A549 in a xenograft model. Kv1.1 mRNA and protein was expressed in A549 cells and the blockade of Kv1.1 by DTX-κ, reduced tumor formation in nude mice. Furthermore, treatment with DTX-κ significantly increased protein expression of p21Waf1/Cip1, p27Kip1, and p15INK4B and significantly decreased protein expression of cyclin D3 in tumor tissues compared to the control. These results suggest that DTX-κ has anti-tumor effects in A549 cells through the pathway governing G1-S transition. PMID:21368561

  10. 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. PMID:19298190

  11. REGULATION OF PTEN EXPRESSION IN INTESTINAL EPITHELIAL CELLS BY JNK ACTIVATION AND NF-κB INHIBITION

    PubMed Central

    Wang, QingDing; Zhou, Yuning; Wang, Xiaofu; Chung, Dai H.; Evers, B. Mark

    2008-01-01

    The tumor suppressor protein PTEN plays an important role in intestinal cell proliferation and differentiation and tumor suppression by antagonizing phosphatidylinositol 3-kinase (PI3K). Despite its importance, the molecular mechanisms regulating PTEN expression are largely undefined. Here, we show that treatment of the colon cancer cell line, HT29, with the differentiating agent sodium butyrate (NaBT) increased PTEN protein and mRNA expression and induced JNK activation. Inhibition of c-Jun-NH2-terminal kinase (JNK) by chemical or genetic methods attenuated NaBT-induced PTEN expression. In addition, our findings demonstrated a cross-talk between NF-κB and JNK with respect to PTEN regulation. Overexpression of the NF-κB superrepressor increased PTEN expression and JNK activity, whereas overexpression of the p65 NF-κB subunit reduced both basal and NaBT-mediated JNK activation and PTEN expression. Moreover, we showed that overexpression of PTEN or treatment with NaBT increased expression of the cyclin dependent kinase inhibitor p27kip1 in HT29 cells; this induction was attenuated by inhibition of PTEN or JNK expression or overexpression of p65. Finally, we demonstrate a role for PTEN in NaBT-mediated cell death and differentiation. Our findings suggest that the NF-κB/JNK/PTEN pathway plays a critical role in normal intestinal homeostasis and colon carcinogenesis. PMID:17699782

  12. Integrin signaling at the M/G1 transition induces expression of cyclin E.

    PubMed

    Hulleman, E; Bijvelt, J J; Verkleij, A J; Verrips, C T; Boonstra, J

    1999-12-15

    The activities of the mammalian G1 cyclins, cyclin D and cyclin E, during cell cycle progression (G1/S) are believed to be regulated by cell attachment and the presence of growth factors. In order to study the importance of cell attachment and concomitant integrin signaling on the expression of G1 cyclins during the natural adhesion process from mitosis to interphase, protein expression was monitored in cells that were synchronized by mitotic shake off. Here we show that in Chinese hamster ovary (CHO) and neuroblastoma (N2A) cells, expression of cyclin E at the M/G1 transition is regulated by both growth factors and cell attachment, while expression of cyclin D seems to be entirely dependent on the presence of serum. Expression of cyclin E appears to be correlated with the phosphorylation of the retinoblastoma protein, suggesting a link with the activity of the cyclin D/cdk4 complex. Expression of the cdk inhibitors p21(cip1/Waf1) and p27(Kip1) is not changed upon serum depletion or detachment of cells during early G1, suggesting no direct role for these CKIs in the regulation of cyclin activity. Although inhibition of cyclin E/cdk2 kinase activity has been reported previously, this is the first time that cyclin E expression is shown to be dependent on cell attachment. PMID:10585265

  13. Novel dichlorophenyl urea compounds inhibit proliferation of human leukemia HL-60 cells by inducing cell cycle arrest, differentiation and apoptosis.

    PubMed

    Figarola, James L; Weng, Yehua; Lincoln, Christopher; Horne, David; Rahbar, Samuel

    2012-08-01

    Two novel dichlorophenyl urea compounds, SR4 and SR9, were synthesized in our laboratory and evaluated for anti-cancer activities. Specifically, we investigated the antiproliferative properties of these new compounds on promyelocytic HL-60 leukemia cells by analyzing their effects on cell differentiation, cell cycle progression and apoptosis. SR4 and SR9 were both cytotoxic to HL-60 cells in a dose-and time-dependent manner, with IC(50) of 1.2 μM and 2.2 μM, respectively, after 72 h treatment. Both compounds strongly suppressed growth of HL-60 cells by promoting cell cycle arrest at the G0/G1 transition, with concomitant decrease in protein levels of cyclins D1 and E2 and cyclin-dependent kinases (CDK 2 and CDK 4), and increased protein expression of CDK inhibitors p21(WAF1/Cip1) and p27(Kip1). In addition, either compounds induce cell differentiation as detected by increased NBT staining and expression of CD11b and CD14. Treatment with SR compounds also promoted mitochondrial-dependent apoptosis as confirmed by Annexin V-FITC double staining, DNA fragmentation, increased expression of caspase 3, 7 and 9, cytochrome c release, PARP degradation, and collapse in mitochondrial membrane potential (ΔΨ(MT)). Collectively, these results provide evidence that SR4 and SR9 have the potential for the treatment of human leukemia and merit further investigation as therapeutic agents against other types of cancer. PMID:21728022

  14. KSHV vCyclin Counters the Senescence/G1 Arrest Response Triggered by NF-κB Hyper-activation

    PubMed Central

    Zhi, Huijun; Zahoor, Muhammad Atif; Druck Shudofsky, Abigail M.; Giam, Chou-Zen

    2014-01-01

    Many oncogenic viruses activate NF-κB as a part of their replicative cycles. We have shown recently that persistent and potentially oncogenic activation of NF-κB by the human T-lymphotropic virus 1 (HTLV-1) oncoprotein Tax immediately triggers a host senescence response mediated by cyclin-dependent kinase inhibitors: p21CIP1/WAF1 (p21) and p27Kip1 (p27) Here we demonstrate that RelA/NF-κB activation by Kaposi sarcoma herpesvirus (KSHV) latency protein vFLIP also leads to p21/p27 up-regulation and G1 cell cycle arrest. Remarkably, KSHV vCyclin, another latency protein co-expressed with vFLIP from a bicistronic latency-specific mRNA, was found to prevent the senescence and G1 arrest induced by HTLV-1 Tax and vFLIP respectively. This is due to the known ability of vCyclin/CDK6 complex to resist p21 and p27 inhibition and cause p27 degradation23. In KSHV-transformed BCBL-1 cells, sustained vFLIP expression with shRNA-mediated vCyclin depletion resulted in G1 arrest. The functional interdependence of vFLIP and vCyclin explains why they are co-translated from the same viral mRNA. Importantly, deregulation of the G1 cyclin-dependent kinase can facilitate chronic IKK/NF-κB activation. PMID:24469036

  15. Fusarochromanone Induces G1 Cell Cycle Arrest and Apoptosis in COS7 and HEK293 Cells

    PubMed Central

    Gu, Ying; Chen, Xin; Shang, Chaowei; Singh, Karnika; Barzegar, Mansoureh; Mahdavian, Elahe; Salvatore, Brian A.; Jiang, Shanxiang; Huang, Shile

    2014-01-01

    Fusarochromanone (FC101), a mycotoxin produced by the fungus Fusarium equiseti, is frequently observed in the contaminated grains and feedstuffs, which is toxic to animals and humans. However, the underlying molecular mechanism remains to be defined. In this study, we found that FC101 inhibited cell proliferation and induced cell death in COS7 and HEK293 cells in a concentration-dependent manner. Flow cytometric analysis showed that FC101 induced G1 cell cycle arrest and apoptosis in the cells. Concurrently, FC101 downregulated protein expression of cyclin D1, cyclin-dependent kinases (CDK4 and CDK6), and Cdc25A, and upregulated expression of the CDK inhibitors (p21Cip1 and p27Kip1), resulting in hypophosphorylation of Rb. FC101 also inhibited protein expression of Bcl-2, Bcl-xL, Mcl-1 and survivin, and induced expression of BAD, leading to activation of caspase 3 and cleavage of PARP, indicating caspase-dependent apoptosis. However, Z-VAD-FMK, a pan-caspase inhibitor, only partially prevented FC101-induced cell death, implying that FC101 may induce cell death through both caspase-dependent and -independent mechanisms. Our results support the notion that FC101 executes its toxicity at least by inhibiting cell proliferation and inducing cell death. PMID:25384025

  16. Non-CDK-bound p27 (p27{sup NCDK}) is a marker for cell stress and is regulated through the Akt/PKB and AMPK-kinase pathways

    SciTech Connect

    Bjoerklund, Mia A.; Vaahtomeri, Kari; Peltonen, Karita; Viollet, Benoit; Maekelae, Tomi P.; Band, Arja M.; Laiho, Marikki

    2010-03-10

    p27Kip1 (p27) tumour suppressor protein is regulated by multiple mechanisms including its turnover, localization and complex formation with its key targets, cyclin-dependent kinases (CDK) and cyclins. We have earlier shown that p27 exists in cells in a form that lacks cyclin/CDK interactions (hence non-CDK, p27{sup NCDK}) but the nature of p27{sup NCDK} has remained unresolved. Here we demonstrate that the epitope recognized by the p27{sup NCDK}-specific antibody resides in the p27 CDK-interaction domain and that p27{sup NCDK} is regulated by the balance of CDK inhibitors and cyclin-CDK complexes. We find that signalling by cellular growth promoting pathways, like phosphoinositol 3-kinase (PI3K) and specifically Akt/PKB kinase, inversely correlates with p27{sup NCDK} levels whereas total p27 levels are unaffected. p27{sup NCDK}, but not total p27, is increased by cellular perturbations such as hyperosmotic and metabolic stress and activation of AMP-activated protein kinase (AMPK). By using AMPK catalytic subunit proficient and deficient cells we further demonstrate that the AMPK pathway governs p27{sup NCDK} responses to metabolic stress and PI3K inhibition. These results indicate that p27{sup NCDK} is a sensitive marker for both cell stress and proliferation over and above p27 and is regulated by Akt/PKB and AMPK pathways.

  17. Overexpression of the human ZNF300 gene enhances growth and metastasis of cancer cells through activating NF-kB pathway

    PubMed Central

    Wang, Tao; Wang, Xian-guo; Xu, Jun-hua; Wu, Xiang-Peng; Qiu, Hong-ling; Yi, Hong; Li, Wen-Xin

    2012-01-01

    Abstract Zinc finger proteins (ZNF) play important roles in various physiological processes. Here we report that ZNF300, a novel zinc finger protein, identified specifically in humans, promotes tumour development by modulating the NF-κB pathway. Inflammatory factors were found to induce ZNF300 expression in HeLa cell line, and ZNF300 expression further enhanced NF-κB signalling by activating TRAF2 and physically interacting with IKKβ. Furthermore, ZNF300 overexpression increased ERK1/2 phosphorylation and the expression of c-myc, IL-6, and IL-8 but decreased the expression of p21waf-1 and p27Kip1; whose down-regulation led to the opposite effect. Most importantly, ZNF300 overexpression stimulated cancer cell proliferation in vitro and significantly enhanced tumour development and metastasis in mouse xenograft model, while knocking down ZNF300 led to the opposite effects. We have identified a novel function for ZNF300 in tumour development that may uniquely link inflammation and NF-κB to tumourigenesis in humans but not in mice. PMID:21777376

  18. 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

  19. 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

    2015-12-01

    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

  20. G1/S Cell Cycle Arrest Provides Anoikis Resistance through Erk-Mediated Bim Suppression†

    PubMed Central

    Collins, Nicole L.; Reginato, Maurico J.; Paulus, Jessica K.; Sgroi, Dennis C.; LaBaer, Joshua; Brugge, Joan S.

    2005-01-01

    Proper attachment to the extracellular matrix is essential for cell survival. Detachment from the extracellular matrix results in an apoptotic process termed anoikis. Anoikis induction in MCF-10A mammary epithelial cells is due not only to loss of survival signals following integrin disengagement, but also to consequent downregulation of epidermal growth factor (EGFR) and loss of EGFR-induced survival signals. Here we demonstrate that G1/S arrest by overexpression of the cyclin-dependent kinase inhibitors p16INK4a, p21Cip1, or p27Kip1 or by treatment with mimosine or aphidicolin confers anoikis resistance in MCF-10A cells. G1/S arrest-mediated anoikis resistance involves suppression of the BH3-only protein Bim. Furthermore, in G1/S-arrested cells, Erk phosphorylation is maintained in suspension and is necessary for Bim suppression. Following G1/S arrest, known proteins upstream of Erk, including Raf and Mek, are not activated. However, retained Erk activation under conditions in which Raf and Mek activation is lost is observed, suggesting that G1/S arrest acts at the level of Erk dephosphorylation. Thus, anoikis resistance by G1/S arrest is mediated by a mechanism involving Bim suppression through maintenance of Erk activation. These results provide a novel link between cell cycle arrest and survival, and this mechanism could contribute to the survival of nonreplicating, dormant tumor cells that avert apoptosis during early stages of metastasis. PMID:15923641

  1. Direct interactions with both p27 and Cdk2 regulate Spy1-mediated proliferation in vivo and in vitro.

    PubMed

    Al Sorkhy, Mohammad; Fifield, Bre-Anne; Myers, Dorothy; Porter, Lisa A

    2016-01-01

    Families of cyclin-like proteins have emerged that bind and activate cyclin dependent kinases (Cdk)s, directing the phosphorylation of noncanonical Cdk substrates. One of these proteins, Spy1, has demonstrated the unique ability to directly bind and activate both Cdk1 and Cdk2, as well as binding and promoting the degradation of at least one Cdk inhibitor, p27(Kip1). Spy1 accelerates somatic cell growth and proliferation and is implicated in a number of human cancers including the breast, brain and liver. Herein we isolate key residues mediating the direct interaction with p27. We use mutants of Spy1 to determine the physiological role of direct interactions with distinct binding partners Cdk2 and p27. We demonstrate that disrupting the direct interaction with either Spy1 binding partner decreased endogenous activity of Cdk2, as well as Spy1-mediated proliferation. However, only the direct interaction with p27 was essential for Spy1-mediated effects on p27 stability. In vivo neither mutation completely prevented tumorigenesis, although each mutation slowed the rate of Spy1-mediated tumorigenesis and decreased overall tumor volumes. This work supports the conclusion that direct interaction with both p27 and Cdk2 contribute to Spy1-mediated effects on cell growth. It is important to elucidate the dynamics of these interactions and to consider these data when assessing functional outcomes. PMID:26771716

  2. Intranuclear binding in space and time of exon junction complex and NXF1 to premRNPs/mRNPs in vivo.

    PubMed

    Björk, Petra; Persson, Jan-Olov; Wieslander, Lars

    2015-10-12

    Eukaryotic gene expression requires the ordered association of numerous factors with precursor messenger RNAs (premRNAs)/messenger RNAs (mRNAs) to achieve efficiency and regulation. Here, we use the Balbiani ring (BR) genes to demonstrate the temporal and spatial association of the exon junction complex (EJC) core with gene-specific endogenous premRNAs and mRNAs. The EJC core components bind cotranscriptionally to BR premRNAs during or very rapidly after splicing. The EJC core does not recruit the nonsense-mediated decay mediaters UPF2 and UPF3 until the BR messenger RNA protein complexes (mRNPs) enter the interchromatin. Even though several known adapters for the export factor NXF1 become part of BR mRNPs already at the gene, NXF1 binds to BR mRNPs only in the interchromatin. In steady state, a subset of the BR mRNPs in the interchromatin binds NXF1, UPF2, and UPF3. This binding appears to occur stochastically, and the efficiency approximately equals synthesis and export of the BR mRNPs. Our data provide unique in vivo information on how export competent eukaryotic mRNPs are formed. PMID:26459599

  3. Intranuclear binding in space and time of exon junction complex and NXF1 to premRNPs/mRNPs in vivo

    PubMed Central

    Björk, Petra; Persson, Jan-Olov

    2015-01-01

    Eukaryotic gene expression requires the ordered association of numerous factors with precursor messenger RNAs (premRNAs)/messenger RNAs (mRNAs) to achieve efficiency and regulation. Here, we use the Balbiani ring (BR) genes to demonstrate the temporal and spatial association of the exon junction complex (EJC) core with gene-specific endogenous premRNAs and mRNAs. The EJC core components bind cotranscriptionally to BR premRNAs during or very rapidly after splicing. The EJC core does not recruit the nonsense-mediated decay mediaters UPF2 and UPF3 until the BR messenger RNA protein complexes (mRNPs) enter the interchromatin. Even though several known adapters for the export factor NXF1 become part of BR mRNPs already at the gene, NXF1 binds to BR mRNPs only in the interchromatin. In steady state, a subset of the BR mRNPs in the interchromatin binds NXF1, UPF2, and UPF3. This binding appears to occur stochastically, and the efficiency approximately equals synthesis and export of the BR mRNPs. Our data provide unique in vivo information on how export competent eukaryotic mRNPs are formed. PMID:26459599

  4. Upregulation of sex-determining region Y-box 9 (SOX9) promotes cell proliferation and tumorigenicity in esophageal squamous cell carcinoma

    PubMed Central

    Hong, Yingcai; Chen, Wen; Du, Xiaojun; Ning, Huiwen; Chen, Huaisheng; Shi, Ruiqing; Lin, Shaolin; Xu, Rongyu; Zhu, Jinrong; Wu, Shu; Zhou, Haiyu

    2015-01-01

    Sex-determining region Y-box 9 (SOX9), a vital transcription factor, play important roles in numerous biological and pathological processes. However, the clinical significance and biological role of SOX9 expression has not been characterized in human esophageal squamous cell cancer (ESCC). Herein, we found that SOX9 was markedly upregulated, at both mRNA and protein level, in ESCC cell lines and ESCC tissues and that SOX9 expression was significantly correlated with tumor clinical stage, T classification, N classification, M classification, pathological differentiation, and shorter overall survival. The proliferation and tumorigenicity of ESCC cells were dramatically induced by SOX9 overexpression but were inhibited by SOX9 knockdown both in vitro and in vivo. Moreover, we demonstrated that upregulation of SOX9 increased the expression of phosphorylated Akt, the cyclin-dependent kinase (CDK) regulator cyclin D1, phosphorylated forkhead box O (FOXO)1, and phosphorylated FOXO3, but SOX9 downregulation decreased their expression, whereas the levels of the CDK inhibitors p21Cip1 and p27Kip1 were attenuated in SOX9-transduced cells. Taken together, our results suggest that SOX9 plays an important role in promoting the proliferation and tumorigenesis of ESCC and may represent a novel prognostic marker for the disease. PMID:26384302

  5. Inhibition of liver trans-sulphuration pathway by propargylglycine mimics gene expression changes found in the mammary gland of weaned lactating rats: role of glutathione.

    PubMed Central

    Zaragozá, Rosa; García, Concha; Rus, A Diana; Pallardó, Federico V; Barber, Teresa; Torres, Luis; Miralles, Vicente J; Viña, Juan R

    2003-01-01

    In the lactating mammary gland, weaning produces mitochondrial cytochrome c release and nuclear DNA fragmentation, as determined by gel electrophoresis. This is followed by a significant decrease in lactation. Weaning for 2 h produces an early induction of the tumour suppressor/transcription factor p53, whereas the oncoprotein c-Jun and c-Jun N-terminal kinase are elevated after 24 h of weaning when compared with controls. The expression of p21(cip1) and p27(kip1), cyclin-dependent kinase inhibitors, was significantly higher in weaned rats when compared with control lactating rats. All the changes mentioned above also happen in the lactating mammary gland when propargylglycine, an inhibitor of the liver trans-sulphuration pathway, is administered. This effect is partially reversed by N -acetylcysteine administration. The administration of buthionine sulphoximine, an irreversible inhibitor of gamma-glutamylcysteine synthetase, to lactating rats produces a decrease in GSH levels and changes in protein concentrations and gene transcripts similar to those in rats with impaired trans-sulphuration pathway. These data suggest that the inter-tissue flux of GSH is an important mechanism of L-cysteine delivery to the lactating mammary gland and emphasize the importance of this physiological event in maintaining the gene expression required to sustain lactation. PMID:12723969

  6. Myc-induced SUMOylation is a therapeutic vulnerability for B-cell lymphoma

    PubMed Central

    Hoellein, Alexander; Fallahi, Mohammad; Schoeffmann, Stephanie; Steidle, Sabine; Schaub, Franz X.; Rudelius, Martina; Laitinen, Iina; Nilsson, Lisa; Goga, Andrei; Peschel, Christian; Nilsson, Jonas A.; Cleveland, John L.

    2014-01-01

    Myc oncogenic transcription factors (c-Myc, N-Myc, and L-Myc) coordinate the control of cell growth, division, and metabolism. In cancer, Myc overexpression is often associated with aggressive disease, which is in part due to the destruction of select targets by the ubiquitin-proteasome system (eg, SCFSkp2-directed destruction of the Cdk inhibitor p27Kip1). We reasoned that Myc would also regulate SUMOylation, a related means of posttranslational modification of proteins, and that this circuit would play essential roles in Myc-dependent tumorigenesis. Here, we report marked increases in the expression of genes that encode regulators and components of the SUMOylation machinery in mouse and human Myc-driven lymphomas, resulting in hyper-SUMOylation in these tumors. Further, inhibition of SUMOylation by genetic means disables Myc-induced proliferation, triggering G2/M cell-cycle arrest, polyploidy, and apoptosis. Using genetically defined cell models and conditional expression systems, this response was shown to be Myc specific. Finally, in vivo loss-of-function and pharmacologic studies demonstrated that inhibition of SUMOylation provokes rapid regression of Myc-driven lymphoma. Thus, targeting SUMOylation represents an attractive therapeutic option for lymphomas with MYC involvement. PMID:25143484

  7. Laminin α5 influences the architecture of the mouse small intestinal mucosa

    PubMed Central

    Mahoney, Zhen X.; Stappenbeck, Thaddeus S.; Miner, Jeffrey H.

    2008-01-01

    Summary The mammalian intestine displays two distinct patterns of mucosal organization. The small intestine contains mucosal epithelial invaginations called crypts of Lieberkühn that are continuous with evaginations into the lumen called villi. The colon also contains crypts, but its epithelial surface is lined by flat surface cuffs. The epithelial cells of both organs communicate with the underlying mesenchyme through a basement membrane that is composed of a variety of extracellular matrix proteins, including members of the laminin family. The basement membranes of the small intestine and colon contain distinct laminin subtypes; notably, the villus basement membrane is rich in laminin α5. Here we show that diminution of laminin α5 in a mouse model led to a compensatory deposition of colonic laminins that resulted in a transformation from a small intestinal to a colonic mucosal architecture. The alteration in mucosal architecture was associated with reduced levels of nuclear p27Kip1, a cell cycle regulator, and altered intestinal epithelial cell proliferation, migration, and differentiation. Our results suggest that laminin α5 plays a crucial role in establishing and maintaining the specific mucosal pattern of the mouse small intestine. PMID:18628307

  8. Age-specific functional epigenetic changes in p21 and p16 in injury-activated satellite cells.

    PubMed

    Li, Ju; Han, Suhyoun; Cousin, Wendy; Conboy, Irina M

    2015-03-01

    The regenerative capacity of muscle dramatically decreases with age because old muscle stem cells fail to proliferate in response to tissue damage. Here, we uncover key age-specific differences underlying this proliferative decline: namely, the genetic loci of cyclin/cyclin-dependent kinase (CDK) inhibitors (CDKIs) p21 and p16 are more epigenetically silenced in young muscle stem cells, as compared to old, both in quiescent cells and those responding to tissue injury. Interestingly, phosphorylated ERK (pERK) induced in these cells by ectopic FGF2 is found in association with p21 and p16 promoters, and moreover, only in the old cells. Importantly, in the old satellite cells, FGF2/pERK silences p21 epigenetically and transcriptionally, which leads to reduced p21 protein levels and enhanced cell proliferation. In agreement with the epigenetic silencing of the loci, young muscle stem cells do not depend as much as old on ectopic FGF/pERK for their myogenic proliferation. In addition, other CDKIs, such asp15(INK4B) and p27(KIP1) , become elevated in satellite cells with age, confirming and explaining the profound regenerative defect of old muscle. This work enhances our understanding of tissue aging, promoting strategies for combating age-imposed tissue degeneration. PMID:25447026

  9. Age-specific functional epigenetic changes in p21 and p16 in injury-activated satellite cells

    PubMed Central

    Li, Ju; Han, Suhyoun; Cousin, Wendy; Conboy, Irina M.

    2014-01-01

    The regenerative capacity of muscle dramatically decreases with age because old muscle stem cells fail to proliferate in response to tissue damage. Here we uncover key age-specific differences underlying this proliferative decline: namely, the genetic loci of CDK inhibitors (CDKI) p21 and p16 are more epigenetically silenced in young muscle stem cells, as compared to old, both in quiescent cells and those responding to tissue injury. Interestingly, phosphorylated ERK (pERK) induced in these cells by ectopic FGF-2 is found in association with p21 and p16 promoters, and moreover, only in the old cells. Importantly, in the old satellite cells FGF-2/pERK silences p21 epigenetically and transcriptionally, which leads to reduced p21 protein levels and enhanced cell proliferation. In agreement with the epigenetic silencing of the loci, young muscle stem cells do not depend as much as old on ectopic FGF/pERK for their myogenic proliferation. In addition, other CDKIs, such asp15INK4B and p27KIP1, become elevated in satellite cells with age, confirming and explaining the profound regenerative defect of old muscle. This work enhances our understanding of tissue aging, promoting strategies for combating age-imposed tissue degeneration. PMID:25447026

  10. Novel functions of core cell cycle regulators in neuronal migration.

    PubMed

    Godin, Juliette D; Nguyen, Laurent

    2014-01-01

    The cerebral cortex is one of the most intricate regions of the brain, which required elaborated cell migration patterns for its development. Experimental observations show that projection neurons migrate radially within the cortical wall, whereas interneurons migrate along multiple tangential paths to reach the developing cortex. Tight regulation of the cell migration processes ensures proper positioning and functional integration of neurons to specific cerebral cortical circuits. Disruption of neuronal migration often lead to cortical dysfunction and/or malformation associated with neurological disorders. Unveiling the molecular control of neuronal migration is thus fundamental to understand the physiological or pathological development of the cerebral cortex. Generation of functional cortical neurons is a complex and stratified process that relies on decision of neural progenitors to leave the cell cycle and generate neurons that migrate and differentiate to reach their final position in the cortical wall. Although accumulating work shed some light on the molecular control of neuronal migration, we currently do not have a comprehensive understanding of how cell cycle exit and migration/differentiation are coordinated at the molecular level. The current chapter tends to lift the veil on this issue by discussing how core cell cycle regulators, and in particular p27(Kip1) acts as a multifunctional protein to control critical steps of neuronal migration through activities that go far beyond cell cycle regulation. PMID:24243100

  11. Maduramicin Inhibits Proliferation and Induces Apoptosis in Myoblast Cells

    PubMed Central

    Chen, Xin; Gu, Ying; Singh, Karnika; Shang, Chaowei; Barzegar, Mansoureh; Jiang, Shanxiang; Huang, Shile

    2014-01-01

    Maduramicin, a polyether ionophore antibiotic derived from the bacterium Actinomadura yumaensis, is currently used as a feed additive against coccidiosis in poultry worldwide. It has been clinically observed that maduramicin can cause skeletal muscle and heart cell damage, resulting in skeletal muscle degeneration, heart failure, and even death in animals and humans, if improperly used. However, the mechanism of its toxic action in myoblasts is not well understood. Using mouse myoblasts (C2C12) and human rhabdomyosarcoma (RD and Rh30) cells as an experimental model for myoblasts, here we found that maduramicin inhibited cell proliferation and induced cell death in a concentration-dependent manner. Further studies revealed that maduramicin induced accumulation of the cells at G0/G1 phase of the cell cycle, and induced apoptosis in the cells. Concurrently, maduramicin downregulated protein expression of cyclin D1, cyclin-dependent kinases (CDK4 and CDK6), and CDC25A, and upregulated expression of the CDK inhibitors (p21Cip1 and p27Kip1), resulting in decreased phosphorylation of Rb. Maduramicin also induced expression of BAK, BAD, DR4, TRADD and TRAIL, leading to activation of caspases 8, 9 and 3 as well as cleavage of poly ADP ribose polymerase (PARP). Taken together, our results suggest that maduramicin executes its toxicity in myoblasts at least by inhibiting cell proliferation and inducing apoptotic cell death. PMID:25531367

  12. Transgenic Expression of Cyclin-Dependent Kinase 4 Results in Epidermal Hyperplasia, Hypertrophy, and Severe Dermal Fibrosis

    PubMed Central

    Miliani de Marval, Paula L.; Gimenez-Conti, Irma B.; LaCava, Margaret; Martinez, Luis A.; Conti, Claudio J.; Rodriguez-Puebla, Marcelo L.

    2001-01-01

    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 p27Kip1 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. PMID:11438484

  13. Lysophosphatidic acid induces YAP-promoted proliferation of human corneal endothelial cells via PI3K and ROCK pathways

    PubMed Central

    Hsueh, Yi-Jen; Chen, Hung-Chi; Wu, Sung-En; Wang, Tze-Kai; Chen, Jan-Kan; Ma, David Hui-Kang

    2015-01-01

    The first two authors contributed equally to this work.Silence of p120-catenin has shown promise in inducing proliferation in human corneal endothelial cells (HCECs), but there is concern regarding off-target effects in potential clinical applications. We aimed to develop ex vivo expansion of HCECs using natural compounds, and we hypothesized that lysophosphatidic acid (LPA) can unlock the mitotic block in contact-inhibited HCECs via enhancing nuclear translocation of yes-associated protein (YAP). Firstly, we verified that exogenous YAP could induce cell proliferation in contact-inhibited HCEC monolayers and postconfluent B4G12 cells. In B4G12 cells, enhanced cyclin D1 expression, reduced p27KIP1/p21CIP1 levels, and the G1/S transition were detected upon transfection with YAP. Secondly, we confirmed that LPA induced nuclear expression of YAP and promoted cell proliferation. Moreover, PI3K and ROCK, but not ERK or p38, were required for LPA-induced YAP nuclear translocation. Finally, cells treated with LPA or transfected with YAP remained hexagonal in shape, in addition to unchanged expression of ZO-1, Na/K-ATPase, and smooth muscle actin (SMA), suggestive of a preserved phenotype, without endothelial–mesenchymal transition. Collectively, our findings indicate an innovative strategy for ex vivo cultivation of HCECs for transplantation and cell therapy. PMID:26029725

  14. Testing the importance of p27 degradation by the SCFskp2 pathway in murine models of lung and colon cancer.

    PubMed

    Timmerbeul, Inke; Garrett-Engele, Carrie M; Kossatz, Uta; Chen, Xueyan; Firpo, Eduardo; Grünwald, Viktor; Kamino, Kenji; Wilkens, Ludwig; Lehmann, Ulrich; Buer, Jan; Geffers, Robert; Kubicka, Stefan; Manns, Michael P; Porter, Peggy L; Roberts, James M; Malek, Nisar P

    2006-09-19

    Decreased expression of the CDK inhibitor p27kip1 in human tumors directly correlates with increased resistance to chemotherapies, increased rates of metastasis, and an overall increased rate of patient mortality. It is thought that decreased p27 expression in tumors is caused by increased proteasomal turnover, in particular activation of the pathway governed by the SCFskp2 E3 ubiquitin protein ligase. We have directly tested the importance of the SCFskp-mediated degradation of p27 in tumorigenesis by analyzing the tumor susceptibility of mice that express a form of p27 that cannot be ubiquitinated and degraded by this pathway (p27T187A). In mouse models of both lung and colon cancer down-regulation of p27 promotes tumorigenesis. However, we found that preventing p27 degradation by the SCFskp2 pathway had no impact on tumor incidence or overall survival in either tumor model. Our study unveiled a previously unrecognized role for the control of p27 mRNA abundance in the development of non-small cell lung cancers. In the colon cancer model, the frequency of intestinal adenomas was similarly unaffected by the p27T187A mutation, but, unexpectedly, we found that it inhibited progression of intestinal adenomas to carcinomas. These studies may guide the choice of clinical settings in which pharmacologic inhibitors of the Skp2 pathway might be of therapeutic value. PMID:16966613

  15. A Dihydroxy-pentamethoxyflavone from Gardenia obtusifolia suppresses proliferation and promotes apoptosis of tumor cells through modulation of multiple cell signaling pathways.

    PubMed

    Phromnoi, Kanokkarn; Reuter, Simone; Sung, Bokyung; Limtrakul, Pornngarm; Aggarwal, Bharat B

    2010-09-01

    We sought to determine the molecular basis for the anticancer activities of 5,3'-dihydroxy-3,6,7,8,4'-pentamethoxyflavone (DH-PMF), isolated from Gardenia obtusifolia traditionally used in Thailand for a variety of ailments. As little as 1 μM DH-PMF inhibited the proliferation of prostate, colon, kidney, lung, head and neck, pancreas, breast, leukemia, and myeloma cancer cell lines. DH-PMF also suppressed the colony-forming ability of tumor cells, with 50% inhibition occurring at a dose less than 10 nM. DH-PMF induced G(2)/M and subG(1) cell cycle arrest, increased the levels of p21(WAF1/CIP1) and p27(KIP1), and reduced the expression of cyclin D1, CDC2, and c-MYC. Furthermore, DH-PMF inhibited AKT and glycogen synthase kinase 3 beta (GSK3β) activation, reduced cell survival proteins, and induced apoptosis, as indicated by annexin V staining, TUNEL assay, and activation of caspase-8, -9 and -3. Overall, our results demonstrate that DH-PMF induces suppression of cell proliferation through modulation of AKT-GSK3β pathways and induction of cyclin-dependent kinase (CDK) inhibitors. PMID:20944143

  16. Cycle Inhibiting Factors (CIFs) Are a Growing Family of Functional Cyclomodulins Present in Invertebrate and Mammal Bacterial Pathogens

    PubMed Central

    Jubelin, Grégory; Chavez, Carolina Varela; Taieb, Frédéric; Banfield, Mark J.; Samba-Louaka, Ascel; Nobe, Rika; Nougayrède, Jean-Philippe; Zumbihl, Robert; Givaudan, Alain; Escoubas, Jean-Michel; Oswald, Eric

    2009-01-01

    The cycle inhibiting factor (Cif) produced by enteropathogenic and enterohemorrhagic Escherichia coli was the first cyclomodulin to be identified that is injected into host cells via the type III secretion machinery. Cif provokes cytopathic effects characterized by G1 and G2 cell cycle arrests, accumulation of the cyclin-dependent kinase inhibitors (CKIs) p21waf1/cip1 and p27kip1 and formation of actin stress fibres. The X-ray crystal structure of Cif revealed it to be a divergent member of a superfamily of enzymes including cysteine proteases and acetyltransferases that share a conserved catalytic triad. Here we report the discovery and characterization of four Cif homologs encoded by different pathogenic or symbiotic bacteria isolated from vertebrates or invertebrates. Cif homologs from the enterobacteria Yersinia pseudotuberculosis, Photorhabdus luminescens, Photorhabdus asymbiotica and the β-proteobacterium Burkholderia pseudomallei all induce cytopathic effects identical to those observed with Cif from pathogenic E. coli. Although these Cif homologs are remarkably divergent in primary sequence, the catalytic triad is strictly conserved and was shown to be crucial for cell cycle arrest, cytoskeleton reorganization and CKIs accumulation. These results reveal that Cif proteins form a growing family of cyclomodulins in bacteria that interact with very distinct hosts including insects, nematodes and humans. PMID:19308257

  17. Biological mechanisms of action of novel C-10 non-acetal trioxane dimers in prostate cancer cell lines.

    PubMed

    Alagbala, Adebusola A; McRiner, Andrew J; Borstnik, Kristina; Labonte, Tanzina; Chang, Wonsuk; D'Angelo, John G; Posner, Gary H; Foster, Barbara A

    2006-12-28

    The mechanisms of action of three C-10 non-acetal trioxane dimers (TDs) were examined in human (LNCaP) and mouse (TRAMP-C1A and -C2H) prostate cancer cell lines. 1 (AJM3/23), 2 (GHP-TM-III-07w), and 3 (GHP-KB-06) inhibited cell growth with 3 being the most potent in C1A (GI50 = 18.0 nM), C2H (GI50 = 17.0 nM), and LNCaP (GI50 = 17.9 nM) cells. In comparison to a standard cytotoxic agent such as doxorubicin (GI50 = 45.3 nM), 3 (GI50 = 17.9 nM) inhibited LNCaP cell growth more potently. TDs induced G0/G1 cell cycle arrest in LNCaP cells and decreased cells in the S phase. These changes correlated with modulation of G1 phase cell cycle proteins including decreased cyclin D1, cyclin E, and cdk2 and increased p21waf1 and p27Kip1. TDs also promoted apoptosis in LNCaP cells with increased expression of proapoptotic bax. These results demonstrate that TDs are potentially useful agents that warrant further preclinical development for treatment of prostate cancer. PMID:17181166

  18. Casticin induces ovarian cancer cell apoptosis by repressing FoxM1 through the activation of FOXO3a

    PubMed Central

    JIANG, LING; CAO, XIAO-CHENG; CAO, JIAN-GUO; LIU, FEI; QUAN, MEI-FANG; SHENG, XI-FENG; REN, KAI-QUN

    2013-01-01

    Casticin, a polymethoxyflavone, is reported to have anticancer activities. The aim of the present study was to examine the molecular mechanisms by which casticin induces apoptosis in ovarian cancer cells. The human ovarian cancer cell lines SKOV3 and A2780 were cultured in vitro. Various molecular techniques, including histone/DNA enzyme-linked immunosorbent assay (ELISA), reverse transcription polymerase chain reaction (RT-PCR), western blot analysis and gene transfection, were used to assess the expression of FOXO3a and forkhead box protein M1 (FoxM1) in casticin-treated ovarian cancer cell lines. Casticin-induced apoptotic cell death was accompanied by the activation of transcription factor FOXO3a, with a concomitant decrease in the expression levels of FoxM1 and its downstream target factors, namely survivin and polo-like kinase 1 (PLK1), and an increase in p27KIP1. A small inhibitory RNA (siRNA) knockout of FoxM1 potentiated casticin-induced apoptosis in ovarian cancer cells. Silencing FOXO3a expression using siRNA increased FoxM1 expression levels and clearly attenuated the induction of apoptosis by casticin treatment. These results show that casticin-induced apoptosis in ovarian cancer may be caused by the activation of FOXO3a, leading to FoxM1 inhibition. PMID:23761826

  19. Pterostilbine, an active component of blueberries, sensitizes colon cancer cells to 5-fluorouracil cytotoxicity.

    PubMed

    Tolba, Mai F; Abdel-Rahman, Sherif Z

    2015-01-01

    Although colorectal cancer (CRC) treatment with 5-fluorouracil (5-FU) is the first line of therapy for this debilitating disease, treatment effectiveness is often hampered by the development of drug resistance and toxicity at high doses. ER-β can play an important role in CRC development and possibly in its response to therapy. Pterostilbene (PT) possesses antioxidant and anticancer effects that are mediated by ER-β. In the current study, we test the hypothesis that PT sensitizes colon cancer cells to 5-FU and we examine the underlying mechanism(s) by which PT exerts its cytotoxic effects in CRC cells. Our data indicate that PT exhibited a more potent cytotoxic effect in Caco-2 compared to HCT-116 cells. PT/5-FU co-treatment was more effective in Caco-2 cells. Our data indicate that ER-β is expressed at higher levels in Caco-2 cells and its levels are further boosted with PT treatment. PT significantly suppressed Akt and ERK phosphorylations, and enhanced FOXO-1 and p27(kip1) levels in Caco-2 cells. PT also induced a significant increase in Caco-2 cells at pre-G phase coupled with increased Bax/Bcl-2 ratio and PARP cleavage. These results provide a rationale for novel combination treatment strategies, especially for patients with 5-FU-resistant tumors expressing ER-β protein. PMID:26472352

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

    PubMed

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

    2015-08-01

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

  1. FGFR1-Frs2/3 Signalling Maintains Sensory Progenitors during Inner Ear Hair Cell Formation

    PubMed Central

    Ono, Kazuya; Kita, Tomoko; Sato, Shigeru; O'Neill, Paul; Mak, Siu-Shan; Paschaki, Marie; Ito, Masataka; Gotoh, Noriko; Kawakami, Kiyoshi; Sasai, Yoshiki; Ladher, Raj K.

    2014-01-01

    Inner ear mechanosensory hair cells transduce sound and balance information. Auditory hair cells emerge from a Sox2-positive sensory patch in the inner ear epithelium, which is progressively restricted during development. This restriction depends on the action of signaling molecules. Fibroblast growth factor (FGF) signalling is important during sensory specification: attenuation of Fgfr1 disrupts cochlear hair cell formation; however, the underlying mechanisms remain unknown. Here we report that in the absence of FGFR1 signaling, the expression of Sox2 within the sensory patch is not maintained. Despite the down-regulation of the prosensory domain markers, p27Kip1, Hey2, and Hes5, progenitors can still exit the cell cycle to form the zone of non-proliferating cells (ZNPC), however the number of cells that form sensory cells is reduced. Analysis of a mutant Fgfr1 allele, unable to bind to the adaptor protein, Frs2/3, indicates that Sox2 maintenance can be regulated by MAP kinase. We suggest that FGF signaling, through the activation of MAP kinase, is necessary for the maintenance of sensory progenitors and commits precursors to sensory cell differentiation in the mammalian cochlea. PMID:24465223

  2. FGFR1-Frs2/3 signalling maintains sensory progenitors during inner ear hair cell formation.

    PubMed

    Ono, Kazuya; Kita, Tomoko; Sato, Shigeru; O'Neill, Paul; Mak, Siu-Shan; Paschaki, Marie; Ito, Masataka; Gotoh, Noriko; Kawakami, Kiyoshi; Sasai, Yoshiki; Ladher, Raj K

    2014-01-01

    Inner ear mechanosensory hair cells transduce sound and balance information. Auditory hair cells emerge from a Sox2-positive sensory patch in the inner ear epithelium, which is progressively restricted during development. This restriction depends on the action of signaling molecules. Fibroblast growth factor (FGF) signalling is important during sensory specification: attenuation of Fgfr1 disrupts cochlear hair cell formation; however, the underlying mechanisms remain unknown. Here we report that in the absence of FGFR1 signaling, the expression of Sox2 within the sensory patch is not maintained. Despite the down-regulation of the prosensory domain markers, p27(Kip1), Hey2, and Hes5, progenitors can still exit the cell cycle to form the zone of non-proliferating cells (ZNPC), however the number of cells that form sensory cells is reduced. Analysis of a mutant Fgfr1 allele, unable to bind to the adaptor protein, Frs2/3, indicates that Sox2 maintenance can be regulated by MAP kinase. We suggest that FGF signaling, through the activation of MAP kinase, is necessary for the maintenance of sensory progenitors and commits precursors to sensory cell differentiation in the mammalian cochlea. PMID:24465223

  3. Pterostilbine, an active component of blueberries, sensitizes colon cancer cells to 5-fluorouracil cytotoxicity

    PubMed Central

    Tolba, Mai F.; Abdel-Rahman, Sherif Z.

    2015-01-01

    Although colorectal cancer (CRC) treatment with 5-fluorouracil (5-FU) is the first line of therapy for this debilitating disease, treatment effectiveness is often hampered by the development of drug resistance and toxicity at high doses. ER-β can play an important role in CRC development and possibly in its response to therapy. Pterostilbene (PT) possesses antioxidant and anticancer effects that are mediated by ER-β. In the current study, we test the hypothesis that PT sensitizes colon cancer cells to 5-FU and we examine the underlying mechanism(s) by which PT exerts its cytotoxic effects in CRC cells. Our data indicate that PT exhibited a more potent cytotoxic effect in Caco-2 compared to HCT-116 cells. PT/5-FU co-treatment was more effective in Caco-2 cells. Our data indicate that ER-β is expressed at higher levels in Caco-2 cells and its levels are further boosted with PT treatment. PT significantly suppressed Akt and ERK phosphorylations, and enhanced FOXO-1 and p27kip1 levels in Caco-2 cells. PT also induced a significant increase in Caco-2 cells at pre-G phase coupled with increased Bax/Bcl-2 ratio and PARP cleavage. These results provide a rationale for novel combination treatment strategies, especially for patients with 5-FU-resistant tumors expressing ER-β protein. PMID:26472352

  4. 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 Central

    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

    2012-01-01

    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. PMID:22313636

  5. CK2 regulates in vitro the activity of the yeast cyclin-dependent kinase inhibitor Sic1.

    PubMed

    Barberis, Matteo; Pagano, Mario A; Gioia, Luca De; Marin, Oriano; Vanoni, Marco; Pinna, Lorenzo A; Alberghina, Lilia

    2005-11-01

    We have previously demonstrated that the cyclin-dependent kinase inhibitor (Cki) Sic1 of Saccharomyces cerevisiae is phosphorylated in vitro by the CK2 kinase on Ser(201) residue. Moreover, we have collected evidence showing that Sic1 is functionally and structurally related to mammalian Cki p27(Kip1) and binds to the mammalian Cdk2/cyclin A complex with a similar mode of inhibition. In this paper, we use SPR analysis to investigate the binding of Sic1 to the catatytic and regulatory subunits of CK2. Evidence is presented showing that phosphorylation of Sic1 at the CK2 consensus site QES(201)EDEED increases the binding of a Sic1-derived peptide to the Cdk2/cyclin A complex, a functional homologue of the yeast Cdk1/Clb5,6. Moreover, Sic1 fully phosphorylated in vitro on Ser(201) by CK2 is shown to be a stronger inhibitor of the Cdk/cyclin complexes than the unphosphorylated protein. Taken together, these data disclose the possibility that CK2 plays a role in the regulation of Sic1 activity. PMID:16168390

  6. Growth-Inhibitory and Antiangiogenic Activity of the MEK Inhibitor PD0325901 in Malignant Melanoma with or without BRAF Mutations12

    PubMed Central

    Ciuffreda, Ludovica; Del Bufalo, Donatella; Desideri, Marianna; Di Sanza, Cristina; Stoppacciaro, Antonella; Ricciardi, Maria Rosaria; Chiaretti, Sabina; Tavolaro, Simona; Benassi, Barbara; Bellacosa, Alfonso; Foà, Robin; Tafuri, Agostino; Cognetti, Francesco; Anichini, Andrea; Zupi, Gabriella; Milella, Michele

    2009-01-01

    The Raf/MEK/ERK pathway is an important mediator of tumor cell proliferation and angiogenesis. Here, we investigated the growth-inhibitory and antiangiogenic properties of PD0325901, a novel MEK inhibitor, in human melanoma cells. PD0325901 effects were determined in a panel of melanoma cell lines with different genetic aberrations. PD0325901 markedly inhibited ERK phosphorylation and growth of both BRAF mutant and wild-type melanoma cell lines, with IC50 in the nanomolar range even in the least responsive models. Growth inhibition was observed both in vitro and in vivo in xenograft models, regardless of BRAF mutation status, and was due to G1-phase cell cycle arrest and subsequent induction of apoptosis. Cell cycle (cyclin D1, c-Myc, and p27KIP1) and apoptosis (Bcl-2 and survivin) regulators were modulated by PD0325901 at the protein level. Gene expression profiling revealed profound modulation of several genes involved in the negative control of MAPK signaling and melanoma cell differentiation, suggesting alternative, potentially relevant mechanisms of action. Finally, PD0325901 inhibited the production of the proangiogenic factors vascular endothelial growth factor and interleukin 8 at a transcriptional level. In conclusion, PD0325901 exerts potent growth-inhibitory, proapoptotic, and antiangiogenic activity in melanoma lines, regardless of their BRAF mutation status. Deeper understanding of the molecular mechanisms of action of MEK inhibitors will likely translate into more effective treatment strategies for patients experiencing malignant melanoma. PMID:19649202

  7. Encorafenib (LGX818), a potent BRAF inhibitor, induces senescence accompanied by autophagy in BRAFV600E melanoma cells.

    PubMed

    Li, Zhen; Jiang, Ke; Zhu, Xiaofang; Lin, Guibin; Song, Fei; Zhao, Yongfu; Piao, Yongjun; Liu, Jiwei; Cheng, Wei; Bi, Xiaolin; Gong, Peng; Song, Zhiqi; Meng, Songshu

    2016-01-28

    Encorafenib (LGX818) is a new-generation BRAF inhibitor that is under evaluation in clinical trials. However, the underlying mechanism remains to be elucidated. Here we show that LGX818 potently decreased ERK phosphorylation and inhibited proliferation in BRAFV600E melanoma cell lines. Moreover, LGX818 downregulated CyclinD1 in a glycogen synthase kinase 3β-independent manner and induced cell cycle arrest in the G1 phase, Surprisingly, LGX818 triggered cellular senescence in BRAFV600E melanoma cells, as evidenced by increased β-galactosidase staining, while no appreciable induction of apoptosis was detected, as determined by Annexin V and propidium iodide staining and immunoblot analysis of caspase-3 processing and poly (ADP-ribose) polymerase cleavage. Increased p27KIP1 expression and retinoblastoma protein activation were detected during LGX818-induced senescence. Additionally, inhibition of dual-specificity tyrosine phosphorylation-regulated kinase 1B by AZ191 reversed LGX818-induced CyclinD1 turnover and senescence. Interestingly, autophagy is triggered through inhibition of the mTOR/70S6K pathway during LGX818-induced senescence. Moreover, autophagy inhibition by pharmacological and genetic regulation attenuates LGX818-induced senescence. Notably, combining LGX818 with autophagy modulators has anti-proliferative effect in LGX818-resistant BRAF mutant melanoma cells. Altogether, we uncovered a mechanism by which LGX818 exerts its anti-tumor activity in BRAFV600E melanoma cells. PMID:26586345

  8. Fangchinoline induces G1 arrest in breast cancer cells through cell-cycle regulation.

    PubMed

    Xing, Zhibo; Zhang, Youxue; Zhang, Xianyu; Yang, Yanmei; Ma, Yuyan; Pang, Da

    2013-12-01

    Fangchinoline, an alkaloid derived from the dry roots of Stephaniae tetrandrine S. Moore (Menispermaceae), has been shown to possess cytotoxic, anti-inflammatory, and antioxidant properties. In this study, we used Fangchinoline to inhibit breast cancer cell proliferation and to investigate its underlying molecular mechanisms. Human breast cancer cell lines, MCF-7 and MDA-MB-231, were both used in this study. We found that Fangchinoline significantly decreased cell proliferation in a dose-dependent manner and induced G1-phase arrest in both cell lines. In addition, upon analysis of expression of cell cycle-related proteins, we found that Fangchinoline reduced expression of cyclin D1, cyclin D3, and cyclin E, and increased expression of the cyclin-dependent kinase (CDK) inhibitors, p21/WAF1, and p27/KIP1. Moreover, Fangchinoline also inhibited the kinase activities of CDK2, CDK4, and CDK6. These results suggest that Fangchinoline can inhibit human breast cancer cell proliferation and thus may have potential applications in cancer therapy. PMID:23401195

  9. A Dihydroxy-pentamethoxyflavone from Gardenia obtusifolia Suppresses Proliferation and Promotes Apoptosis of Tumor Cells Through Modulation of Multiple Cell Signaling Pathways

    PubMed Central

    PHROMNOI, KANOKKARN; REUTER, SIMONE; SUNG, BOKYUNG; LIMTRAKUL, PORNNGARM; AGGARWAL, BHARAT B.

    2011-01-01

    We sought to determine the molecular basis for the anticancer activities of 5,3′-dihydroxy-3,6,7,8,4′-pentamethoxyflavone (DH-PMF), isolated from Gardenia obtusifolia traditionally used in Thailand for a variety of ailments. As little as 1 μM DH-PMF inhibited the proliferation of prostate, colon, kidney, lung, head and neck, pancreas, breast, leukemia, and myeloma cancer cell lines. DH-PMF also suppressed the colony-forming ability of tumor cells, with 50% inhibition occurring at a dose less than 10 nM. DH-PMF induced G2/M and subG1 cell cycle arrest, increased the levels of p21WAF1/CIP1 and p27KIP1, and reduced the expression of cyclin D1, CDC2, and c-MYC. Furthermore, DH-PMF inhibited AKT and glycogen synthase kinase 3 beta (GSK3β) activation, reduced cell survival proteins, and induced apoptosis, as indicated by annexin V staining, TUNEL assay, and activation of caspase-8, -9 and -3. Overall, our results demonstrate that DH-PMF induces suppression of cell proliferation through modulation of AKT-GSK3β pathways and induction of cyclin-dependent kinase (CDK) inhibitors. PMID:20944143

  10. Synthesis, Characterization and Anti-Cancer Activity of Hydrazide Derivatives Incorporating a Quinoline Moiety.

    PubMed

    Bingul, Murat; Tan, Owen; Gardner, Christopher R; Sutton, Selina K; Arndt, Greg M; Marshall, Glenn M; Cheung, Belamy B; Kumar, Naresh; Black, David StC

    2016-01-01

    Identification of the novel (E)-N'-((2-chloro-7-methoxyquinolin-3-yl)methylene)-3-(phenylthio)propanehydrazide scaffold 18 has led to the development of a new series of biologically active hydrazide compounds. The parent compound 18 and new quinoline derivatives 19-26 were prepared from the corresponding quinoline hydrazones and substituted carboxylic acids using EDC-mediated peptide coupling reactions. Further modification of the parent compound 18 was achieved by replacement of the quinoline moiety with other aromatic systems. All the newly synthesized compounds were evaluated for their anti-cancer activity against the SH-SY5Y and Kelly neuroblastoma cell lines, as well as the MDA-MB-231 and MCF-7 breast adenocarcinoma cell lines. Analogues 19 and 22 significantly reduced the cell viability of neuroblastoma cancer cells with micromolar potency and significant selectivity over normal cells. The quinoline hydrazide 22 also induced G₁ cell cycle arrest, as well as upregulation of the p27(kip1) cell cycle regulating protein. PMID:27428941