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Impaired Immune Responses and B-Cell Proliferation in Mice Lacking the Id3 Gene

Pan, Lihua; Sato, Shinichi; Frederick, Joshua P.; Sun, Xiao-Hong; Zhuang, Yuan
Fonte: American Society for Microbiology Publicador: American Society for Microbiology
Tipo: Artigo de Revista Científica
Publicado em /09/1999 Português
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B-lymphocyte activation and proliferation induced by the B-cell receptor (BCR) signals are important steps in the initiation of humoral immune responses. How the BCR signals are translated by nuclear transcription factors into cell cycle progression is poorly understood. Id3 is an immediate-early gene responding to growth and mitogenic signals in many cell types including B cells. The primary function of the Id3 protein has been defined as that of inhibitor of basic-helix-loop-helix (bHLH) transcription factors. The interaction between Id3 and bHLH proteins, many of which are essential for cellular differentiation, has been proposed as a key regulatory event leading to cellular proliferation instead of differentiation. To further investigate the role of Id3 in tissue and embryo development and the mechanism of Id3-mediated growth regulation, we generated and analyzed Id3-deficient mice. While these mice display no overt abnormality in tissue and embryo development, their humoral immunity is compromised. The amounts of immunoglobulins produced in Id3-deficient mice immunized with a T-cell-dependent antigen and a type 2 T-cell-independent antigen are attenuated and severely impaired, respectively. Further analysis of lymphocytes isolated from Id3-deficient mice reveals a B-cell defect in their proliferation response to BCR cross-linking but not to lipopolysaccharide or a combination of BCR cross-linking and interleukin-4. Analyses of cultured lymphocytes also suggest involvement of Id3 in cytokine production in T cells and isotype switching in B cells. Finally...

Elevated Cyclin E Levels, Inactive Retinoblastoma Protein, and Suppression of the p27KIP1 Inhibitor Characterize Early Development of Promyeloid Cells into Macrophages

Liu, Qiang; VanHoy, Roger W.; Zhou, J. H.; Dantzer, Robert; Freund, Gregory G.; Kelley, Keith W.
Fonte: American Society for Microbiology Publicador: American Society for Microbiology
Tipo: Artigo de Revista Científica
Publicado em /09/1999 Português
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772.245%
Cyclin-dependent kinase inhibitors such as p27KIP1 have recently been shown to lead to cellular differentiation by causing cell cycle arrest, but it is unknown whether similar events occur in differentiating promyeloid cells. Hematopoietic progenitor cells undergo lineage-restricted differentiation, which is accompanied by expression of distinct maturation markers. Here we show that the classical growth factor insulin-like growth factor I (IGF-I) potently promotes vitamin D3-induced macrophage differentiation of promyeloid cells, as assessed by measurement of a coordinate increase in expression of the integrin α subunit CD11b, the CD14 lipopolysaccharide receptor, and the macrophage-specific esterase, α-naphthyl acetate esterase, as early as 24 h following initiation of terminal differentiation. Addition of IGF-I to cells undergoing vitamin D3-induced differentiation also leads to an early increase in expression of cyclin E, phosphorylation of the retinoblastoma tumor suppressor protein, and a doubling of the cell number. Early expression of CD11b (24 h) is simultaneously accompanied by inhibition in the expression of p27KIP1. Cell cycle analysis with propidium iodide revealed that CD11b expression at 24 h following initiation of differentiation occurs at all phases of the cell cycle instead of only those cells arrested in G0/G1. Similarly...

Species-Specific Elements in the Large T-Antigen J Domain Are Required for Cellular Transformation and DNA Replication by Simian Virus 40

Sullivan, Christopher S.; Tremblay, James D.; Fewell, Sheara W.; Lewis, John A.; Brodsky, Jeffrey L.; Pipas, James M.
Fonte: American Society for Microbiology Publicador: American Society for Microbiology
Tipo: Artigo de Revista Científica
Publicado em /08/2000 Português
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The J domain of simian virus 40 (SV40) large T antigen is required for efficient DNA replication and transformation. Despite previous reports demonstrating the promiscuity of J domains in heterologous systems, results presented here show the requirement for specific J-domain sequences in SV40 large-T-antigen-mediated activities. In particular, chimeric-T-antigen constructs in which the SV40 T-antigen J domain was replaced with that from the yeast Ydj1p or Escherichia coli DnaJ proteins failed to replicate in BSC40 cells and did not transform REF52 cells. However, T antigen containing the JC virus J domain was functional in these assays, although it was less efficient than the wild type. The inability of some large-T-antigen chimeras to promote DNA replication and elicit cellular transformation was not due to a failure to interact with hsc70, since a nonfunctional chimera, containing the DnaJ J domain, bound hsc70. However, this nonfunctional chimeric T antigen was reduced in its ability to stimulate hsc70 ATPase activity and unable to liberate E2F from p130, indicating that transcriptional activation of factors required for cell growth and DNA replication may be compromised. Our data suggest that the T-antigen J domain harbors species-specific elements required for viral activities in vivo.

Forkhead Transcription Factors Are Critical Effectors of Cell Death and Cell Cycle Arrest Downstream of PTEN

Nakamura, Noriaki; Ramaswamy, Shivapriya; Vazquez, Francisca; Signoretti, Sabina; Loda, Massimo; Sellers, William R.
Fonte: American Society for Microbiology Publicador: American Society for Microbiology
Tipo: Artigo de Revista Científica
Publicado em /12/2000 Português
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PTEN acts as a tumor suppressor, at least in part, by antagonizing phosphoinositide 3-kinase (PI3K)/Akt signaling. Here we show that Forkhead transcription factors FKHRL1 and FKHR, substrates of the Akt kinase, are aberrantly localized to the cytoplasm and cannot activate transcription in PTEN-deficient cells. Restoration of PTEN function restores FKHR to the nucleus and restores transcriptional activation. Expression of a constitutively active form of FKHR that cannot be phosphorylated by Akt produces the same effect as reconstitution of PTEN on PTEN-deficient tumor cells. Specifically, activated FKHR induces apoptosis in cells that undergo PTEN-mediated cell death and induces G1 arrest in cells that undergo PTEN-mediated cell cycle arrest. Furthermore, both PTEN and constitutively active FKHR induce p27KIP1 protein but not p21. These data suggest that Forkhead transcription factors are critical effectors of PTEN-mediated tumor suppression.

Rfg1, a Protein Related to the Saccharomyces cerevisiae Hypoxic Regulator Rox1, Controls Filamentous Growth and Virulence in Candida albicans

Kadosh, David; Johnson, Alexander D.
Fonte: American Society for Microbiology Publicador: American Society for Microbiology
Tipo: Artigo de Revista Científica
Publicado em /04/2001 Português
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855.618%
Candida albicans, the major fungal pathogen in humans, can undergo a reversible transition from ellipsoidal single cells (blastospores) to filaments composed of elongated cells attached end to end. This transition is thought to allow for rapid colonization of host tissues, facilitating the spread of infection. Here, we report the identification of Rfg1, a transcriptional regulator that controls filamentous growth of C. albicans in an environment-dependent manner. Rfg1 is important for virulence of C. albicans in a mouse model and is shown to control a number of genes that have been implicated in this process. The closest relative to Rfg1 in Saccharomyces cerevisiae is Rox1, a key repressor of hypoxic genes. However, Rfg1 does not appear to play a role in the regulation of hypoxic genes in C. albicans. These results demonstrate that a regulatory protein that controls the hypoxic response in S. cerevisiae controls filamentous growth and virulence in C. albicans. The observations described in this paper raise new and intriguing questions about the evolutionary relationship between these processes.

Growth Suppression by an E2F-Binding-Defective Retinoblastoma Protein (RB): Contribution from the RB C Pocket†

Whitaker, Laura L.; Su, Heyun; Baskaran, Rajasekaran; Knudsen, Erik S.; Wang, Jean Y. J.
Fonte: American Society for Microbiology Publicador: American Society for Microbiology
Tipo: Artigo de Revista Científica
Publicado em /07/1998 Português
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Growth suppression by the retinoblastoma protein (RB) is dependent on its ability to form complexes with transcription regulators. At least three distinct protein-binding activities have been identified in RB: the large A/B pocket binds E2F, the A/B pocket binds the LXCXE peptide motif, and the C pocket binds the nuclear c-Abl tyrosine kinase. Substitution of Trp for Arg 661 in the B region of RB (mutant 661) inactivates both E2F and LXCXE binding. The tumor suppression function of mutant 661 is not abolished, because this allele predisposes its carriers to retinoblastoma development with a low penetrance. In cell-based assays, 661 is shown to inhibit G1/S progression. This low-penetrance mutant also induces terminal growth arrest with reduced but detectable activity. We have constructed mutations that disrupt C pocket activity. When overproduced, the RB C-terminal fragment did not induce terminal growth arrest but could inhibit G1/S progression, and this activity was abolished by the C-pocket mutations. In full-length RB, the C-pocket mutations reduced but did not abolish RB function. Interestingly, combination of the C-pocket and 661 mutations completely abolished RB’s ability to cause an increase in the percentage of cells in G1 and to induce terminal growth arrest. These results suggest that the A/B or C region can induce a prolongation of G1 through mechanisms that are independent of each other. In contrast...

A Novel Pathway for Mammary Epithelial Cell Invasion Induced by the Helix-Loop-Helix Protein Id-1

Desprez, Pierre-Yves; Lin, Claudia Qiao; Thomasset, Nicole; Sympson, Carolyn J.; Bissell, Mina J.; Campisi, Judith
Fonte: American Society for Microbiology Publicador: American Society for Microbiology
Tipo: Artigo de Revista Científica
Publicado em /08/1998 Português
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Mammary epithelial cells undergo changes in growth, invasion, and differentiation throughout much of adulthood, and most strikingly during pregnancy, lactation, and involution. Although the pathways of milk protein expression are being elucidated, little is known, at a molecular level, about control of mammary epithelial cell phenotypes during normal tissue morphogenesis and evolution of aggressive breast cancer. We developed a murine mammary epithelial cell line, SCp2, that arrests growth and functionally differentiates in response to a basement membrane and lactogenic hormones. In these cells, expression of Id-1, an inhibitor of basic helix-loop-helix transcription factors, declines prior to differentiation, and constitutive Id-1 expression blocks differentiation. Here, we show that SCp2 cells that constitutively express Id-1 slowly invade the basement membrane but remain anchorage dependent for growth and do not form tumors in nude mice. Cells expressing Id-1 secreted a ∼120-kDa gelatinase. From inhibitor studies, this gelatinase appeared to be a metalloproteinase, and it was the only metalloproteinase detectable in conditioned medium from these cells. A nontoxic inhibitor diminished the activity of this metalloproteinase in vitro and repressed the invasive phenotype of Id-1-expressing cells in culture. The implications of these findings for normal mammary-gland development and human breast cancer were investigated. A gelatinase of ∼120 kDa was expressed by the mammary gland during involution...

Tissue Hyperplasia and Enhanced T-Cell Signalling via ZAP-70 in c-Cbl-Deficient Mice

Murphy, Maria A.; Schnall, Ralf G.; Venter, Deon J.; Barnett, Louise; Bertoncello, Ivan; Thien, Christine B. F.; Langdon, Wallace Y.; Bowtell, David D. L.
Fonte: American Society for Microbiology Publicador: American Society for Microbiology
Tipo: Artigo de Revista Científica
Publicado em /08/1998 Português
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The c-Cbl protein is tyrosine phosphorylated and forms complexes with a wide range of signalling partners in response to various growth factors. How c-Cbl interacts with proteins, such as Grb2, phosphatidylinositol 3-kinase, and phosphorylated receptors, is well understood, but its role in these complexes is unclear. Recently, the Caenorhabditis elegans Cbl homolog, Sli-1, was shown to act as a negative regulator of epidermal growth factor receptor signalling. This finding forced a reassessment of the role of Cbl proteins and highlighted the desirability of testing genetically whether c-Cbl acts as a negative regulator of mammalian signalling. Here we investigate the role of c-Cbl in development and homeostasis in mice by targeted disruption of the c-Cbl locus. c-Cbl-deficient mice were viable, fertile, and outwardly normal in appearance. Bone development and remodelling also appeared normal in c-Cbl mutants, despite a previously reported requirement for c-Cbl in osteoclast function. However, consistent with a high level of expression of c-Cbl in the hemopoietic compartment, c-Cbl-deficient mice displayed marked changes in their hemopoietic profiles, including altered T-cell receptor expression, lymphoid hyperplasia, and primary splenic extramedullary hemopoiesis. The mammary fat pads of mutant female mice also showed increased ductal density and branching compared to those of their wild-type littermates...

Requirement of PKR Dimerization Mediated by Specific Hydrophobic Residues for Its Activation by Double-Stranded RNA and Its Antigrowth Effects in Yeast

Patel, Rekha C.; Sen, Ganes C.
Fonte: American Society for Microbiology Publicador: American Society for Microbiology
Tipo: Artigo de Revista Científica
Publicado em /12/1998 Português
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855.47305%
The roles of protein dimerization and double-stranded RNA (dsRNA) binding in the biochemical and cellular activities of PKR, the dsRNA-dependent protein kinase, were investigated. We have previously shown that both properties of the protein are mediated by the same domain. Here we show that dimerization is mediated by hydrophobic residues present on one side of an amphipathic α-helical structure within this domain. Appropriate substitution mutations of residues on that side produced mutants with increased or decreased dimerization activities. Using these mutants, we demonstrated that dimerization is not essential for dsRNA binding. However, enhancing dimerization artificially, by providing an extraneous dimerization domain, increased dsRNA binding of both wild-type and mutant proteins. In vitro, the dimerization-defective mutants could not be activated by dsRNA but were activated normally by heparin. In Saccharomyces cerevisiae, unlike wild-type PKR, these mutants could not inhibit cell growth and the dsRNA-binding domain of the dimerization-defective mutants could not prevent the antigrowth effect of wild-type PKR. These results demonstrate the biological importance of the dimerization properties of PKR.

Ras Signals to the Cell Cycle Machinery via Multiple Pathways To Induce Anchorage-Independent Growth

Yang, Jaw-Ji; Kang, Jong-Sun; Krauss, Robert S.
Fonte: American Society for Microbiology Publicador: American Society for Microbiology
Tipo: Artigo de Revista Científica
Publicado em /05/1998 Português
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771.4416%
Several specific cell cycle activities are dependent on cell-substratum adhesion in nontransformed cells, and the ability of the Ras oncoprotein to induce anchorage-independent growth is linked to its ability to abrogate this adhesion requirement. Ras signals via multiple downstream effector proteins, a synergistic combination of which may be required for the highly altered phenotype of fully transformed cells. We describe here studies on cell cycle regulation of anchorage-independent growth that utilize Ras effector loop mutants in NIH 3T3 and Rat 6 cells. Stable expression of activated H-Ras (12V) induced soft agar colony formation by both cell types, but each of three effector loop mutants (12V,35S, 12V,37G, and 12V,40C) was defective in producing this response. Expression of all three possible pairwise combinations of these mutants synergized to induce anchorage-independent growth of NIH 3T3 cells, but only the 12V,35S-12V,37G and 12V,37G-12V,40C combinations were complementary in Rat 6 cells. Each individual effector loop mutant partially relieved adhesion dependence of pRB phosphorylation, cyclin E-dependent kinase activity, and expression of cyclin A in NIH 3T3, but not Rat 6, cells. The pairwise combinations of effector loop mutants that were synergistic in producing anchorage-independent growth in Rat 6 cells also led to synergistic abrogation of the adhesion requirement for these cell cycle activities. The relationship between complementation in producing anchorage-independent growth and enhancement of cell cycle activities was not as clear in NIH 3T3 cells that expressed pairs of mutants...

Control of asgE Expression during Growth and Development of Myxococcus xanthus

Garza, Anthony G.; Harris, Baruch Z.; Greenberg, Brandon M.; Singer, Mitchell
Fonte: American Society for Microbiology Publicador: American Society for Microbiology
Tipo: Artigo de Revista Científica
Publicado em /12/2000 Português
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771.6528%
One of the earliest events in the Myxococcus xanthus developmental cycle is production of an extracellular cell density signal called A-signal (or A-factor). Previously, we showed that cells carrying an insertion in the asgE gene fail to produce normal levels of this cell-cell signal. In this study we found that expression of asgE is growth phase regulated and developmentally regulated. Several lines of evidence indicate that asgE is cotranscribed with an upstream gene during development. Using primer extension analyses, we identified two 5′ ends for this developmental transcript. The DNA sequence upstream of one 5′ end has similarity to the promoter regions of several genes that are A-signal dependent, whereas sequences located upstream of the second 5′ end show similarity to promoter elements identified for genes that are C-signal dependent. Consistent with this result is our finding that mutants failing to produce A-signal or C-signal are defective for developmental expression of asgE. In contrast to developing cells, the large majority of the asgE transcript found in vegetative cells appears to be monocistronic. This finding suggests that asgE uses different promoters for expression during vegetative growth and development. Growth phase regulation of asgE is abolished in a relA mutant...

Regulation of Transforming Growth Factor α Expression in a Growth Factor-Independent Cell Line

Howell, Gillian M.; Humphrey, Lisa E.; Ziober, Barry L.; Awwad, Rana; Periyasamy, Basker; Koterba, Alan; Li, Wenhui; Willson, James K. V.; Coleman, Kevin; Carboni, Joan; Lynch, Mark; Brattain, Michael G.
Fonte: American Society for Microbiology Publicador: American Society for Microbiology
Tipo: Artigo de Revista Científica
Publicado em /01/1998 Português
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Aberrant transcriptional regulation of transforming growth factor α (TGFα) appears to be an important contributor to the malignant phenotype and the growth factor independence with which malignancy is frequently associated. However, little is known about the molecular mechanisms responsible for dysregulation of TGFα expression in the malignant phenotype. In this paper, we report on TGFα promoter regulation in the highly malignant growth factor-independent cell line HCT116. The HCT116 cell line expresses TGFα and the epidermal growth factor receptor (EGFR) but is not growth inhibited by antibodies to EGFR or TGFα. However, constitutive expression of TGFα antisense RNA in the HCT116 cell line resulted in the isolation of clones with markedly reduced TGFα mRNA and which were dependent on exogenous growth factors for proliferation. We hypothesized that if TGFα autocrine activation is the major stimulator of TGFα expression in this cell line, TGFα promoter activity should be reduced in the antisense TGFα clones in the absence of exogenous growth factor. This was the case. Moreover, transcriptional activation of the TGFα promoter was restored in an antisense-TGFα-mRNA-expressing clone which had reverted to a growth factor-independent phenotype. Using this model system...

Src Catalytic but Not Scaffolding Function Is Needed for Integrin-Regulated Tyrosine Phosphorylation, Cell Migration, and Cell Spreading

Cary, Leslie A.; Klinghoffer, Richard A.; Sachsenmaier, Christoph; Cooper, Jonathan A.
Fonte: American Society for Microbiology Publicador: American Society for Microbiology
Tipo: Artigo de Revista Científica
Publicado em /04/2002 Português
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855.6323%
Src family kinases (SFKs) are crucial for signaling through a variety of cell surface receptors, including integrins. There is evidence that integrin activation induces focal adhesion kinase (FAK) autophosphorylation at Y397 and that Src binds to and is activated by FAK to carry out subsequent phosphorylation events. However, it has also been suggested that Src functions as a scaffolding molecule through its SH2 and SH3 domains and that its kinase activity is not necessary. To examine the role of SFKs in integrin signaling, we have expressed various Src molecules in fibroblasts lacking other SFKs. In cells plated on fibronectin, FAK could indeed autophosphorylate at Y397 independently of Src but with lower efficiency than when Src was present. This step was promoted by kinase-inactive Src, but Src kinase activity was required for full rescue. Src kinase activity was also required for phosphorylation of additional sites on FAK and for other integrin-directed functions, including cell migration and spreading on fibronectin. In contrast, Src mutations in the SH2 or SH3 domain greatly reduced binding to FAK, Cas, and paxillin but had little effect on tyrosine phosphorylation or biological assays. Furthermore, our indirect evidence indicates that Src kinase activity does not need to be regulated to promote cell migration and FAK phosphorylation. Although Src clearly plays important roles in integrin signaling...

Etk/Bmx as a Tumor Necrosis Factor Receptor Type 2-Specific Kinase: Role in Endothelial Cell Migration and Angiogenesis

Pan, Shi; An, Ping; Zhang, Rong; He, Xiangrong; Yin, Guoyong; Min, Wang
Fonte: American Society for Microbiology Publicador: American Society for Microbiology
Tipo: Artigo de Revista Científica
Publicado em /11/2002 Português
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853.968%
Tumor necrosis factor (TNF) is a cytokine that mediates many pathophysiologial processes, including angiogenesis. However, the molecular signaling involved in TNF-induced angiogenesis has not been determined. In this study, we examined the role of Etk/Bmx, an endothelial/epithelial tyrosine kinase involved in cell adhesion, migration, and survival in TNF-induced angiogenesis. We show that TNF activates Etk specifically through TNF receptor type 2 (TNFR2) as demonstrated by studies using a specific agonist to TNFR2 and TNFR2-deficient cells. Etk forms a preexisting complex with TNFR2 in a ligand-independent manner, and the association is through multiple domains (pleckstrin homology domain, TEC homology domain, and SH2 domain) of Etk and the C-terminal domain of TNFR2. The C-terminal 16-amino-acid residues of TNFR2 are critical for Etk association and activation, and this Etk-binding and activating motif in TNFR2 is not overlapped with the TNFR-associated factor type 2 (TRAF2)-binding sequence. Thus, TRAF2 is not involved in TNF-induced Etk activation, suggesting a novel mechanism for Etk activation by cytokine receptors. Moreover, a constitutively active form of Etk enhanced, whereas a dominant-negative Etk blocked, TNF-induced endothelial cell migration and tube formation. While most TNF actions have been attributed to TNFR1...

Growth Suppression of Pre-T Acute Lymphoblastic Leukemia Cells by Inhibition of Notch Signaling

Weng, Andrew P.; Nam, Yunsun; Wolfe, Michael S.; Pear, Warren S.; Griffin, James D.; Blacklow, Stephen C.; Aster, Jon C.
Fonte: American Society for Microbiology Publicador: American Society for Microbiology
Tipo: Artigo de Revista Científica
Publicado em /01/2003 Português
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772.2854%
Constitutive NOTCH signaling in lymphoid progenitors promotes the development of immature T-cell lymphoblastic neoplasms (T-ALLs). Although it is clear that Notch signaling can initiate leukemogenesis, it has not previously been established whether continued NOTCH signaling is required to maintain T-ALL growth. We demonstrate here that the blockade of Notch signaling at two independent steps suppresses the growth and survival of NOTCH1-transformed T-ALL cells. First, inhibitors of presenilin specifically induce growth suppression and apoptosis of a murine T-ALL cell line that requires presenilin-dependent proteolysis of the Notch receptor in order for its intracellular domain to translocate to the nucleus. Second, a 62-amino-acid peptide derived from a NOTCH coactivator, Mastermind-like-1 (MAML1), forms a transcriptionally inert nuclear complex with NOTCH1 and CSL and specifically inhibits the growth of both murine and human NOTCH1-transformed T-ALLs. These studies show that continued growth and survival of NOTCH1-transformed lymphoid cell lines require nuclear access and transcriptional coactivator recruitment by NOTCH1 and identify at least two steps in the Notch signaling pathway as potential targets for chemotherapeutic intervention.

T-Cadherin-Mediated Cell Growth Regulation Involves G2 Phase Arrest and Requires p21CIP1/WAF1 Expression

Huang, Zhi-yong; Wu, YanLi; Hedrick, Nicolé; Gutmann, David H.
Fonte: American Society for Microbiology Publicador: American Society for Microbiology
Tipo: Artigo de Revista Científica
Publicado em /01/2003 Português
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771.7238%
Members of the cadherin family have been implicated as growth regulators in multiple tumor types. Based on recent studies from our laboratory implicating T-cadherin expression in mouse brain tumorigenesis, we examined the role of T-cadherin in astrocytoma growth regulation. In this report, we show that T-cadherin expression increased during primary astrocyte physiologic growth arrest in response to contact inhibition and serum starvation in vitro, suggesting a function for T-cadherin in astrocyte growth regulation. We further demonstrate that transient and stable reexpression of T-cadherin in deficient C6 glioma cell lines results in growth suppression. In addition, T-cadherin-expressing C6 cell lines demonstrated increased homophilic cell aggregation, increased cell attachment to fibronectin, and decreased cell motility. Cell cycle flow cytometry demonstrated that T-cadherin reexpression resulted in G2 phase arrest, which was confirmed by mitotic index analysis. This growth arrest was p53 independent, as T-cadherin could still mediate growth suppression in p53−/− mouse embryonic fibroblasts. T-cadherin-expressing C6 cell lines exhibited increased p21CIP1/WAF1, but not p27Kip1, expression. Lastly, T-cadherin-mediated growth arrest was dependent on p21CIP1/WAF1 expression and was eliminated in p21CIP1/WAF1-deficient fibroblasts. Collectively...

Direct Involvement of HERV-W Env Glycoprotein in Human Trophoblast Cell Fusion and Differentiation

Frendo, Jean-Louis; Olivier, Delphine; Cheynet, Valérie; Blond, Jean-Luc; Bouton, Olivier; Vidaud, Michel; Rabreau, Michèle; Evain-Brion, Danièle; Mallet, François
Fonte: American Society for Microbiology Publicador: American Society for Microbiology
Tipo: Artigo de Revista Científica
Publicado em /05/2003 Português
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854.8594%
We recently demonstrated that the product of the HERV-W env gene, a retroviral envelope protein also dubbed syncytin, is a highly fusogenic membrane glycoprotein inducing the formation of syncytia on interaction with the type D mammalian retrovirus receptor. In addition, the detection of HERV-W Env protein (Env-W) expression in placental tissue sections led us to propose a role for this fusogenic glycoprotein in placenta formation. To evaluate this hypothesis, we analyzed the involvement of Env-W in the differentiation of primary cultures of human villous cytotrophoblasts that spontaneously differentiate by cell fusion into syncytiotrophoblasts in vitro. First, we observed that HERV-W env mRNA and glycoprotein expression are colinear with primary cytotrophoblast differentiation and with expression of human chorionic gonadotropin (hCG), a marker of syncytiotrophoblast formation. Second, we observed that in vitro stimulation of trophoblast cell fusion and differentiation by cyclic AMP is also associated with a concomitant increase in HERV-W env and hCG mRNA and protein expression. Finally, by using specific antisense oligonucleotides, we demonstrated that inhibition of Env-W protein expression leads to a decrease of trophoblast fusion and differentiation...

Activation of the Phosphatidylinositol 3-Kinase/Protein Kinase Akt Pathway Mediates Nitric Oxide-Induced Endothelial Cell Migration and Angiogenesis

Kawasaki, Koh; Smith, Robert S.; Hsieh, Chung-Ming; Sun, Jianxin; Chao, Julie; Liao, James K.
Fonte: American Society for Microbiology Publicador: American Society for Microbiology
Tipo: Artigo de Revista Científica
Publicado em /08/2003 Português
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855.6787%
To test the hypothesis that the phosphatidylinositol 3-kinase (PI3 kinase)/protein kinase Akt signaling pathway is involved in nitric oxide (NO)-induced endothelial cell migration and angiogenesis, we treated human and bovine endothelial cells with NO donors, S-nitroso-l-glutathione (GSNO) and S-nitroso-N-penicillamine (SNAP). Both GSNO and SNAP increased Akt phosphorylation and activity, which were blocked by cotreatment with the PI3 kinase inhibitor wortmannin. The mechanism was due to the activation of soluble guanylyl cyclase because 8-bromo-cyclic GMP activated PI3 kinase and the soluble guanylyl cyclase inhibitor 1H-[1,2,4]oxadiazolo[4,3-α]quinoxalin-1-one (ODQ) blocked NO-induced PI3 kinase activity. Indeed, transfection with adenovirus containing endothelial cell NO synthase (eNOS) or protein kinase G (PKG) increased endothelial cell migration, which was inhibited by cotransfection with a dominant-negative mutant of PI3 kinase (dnPI3 kinase). In a rat model of hind limb ischemia, adenovirus-mediated delivery of human eNOS cDNA in adductor muscles resulted in time-dependent expression of recombinant eNOS, which was accompanied by significant increases in regional blood perfusion and capillary density. Coinjection of adenovirus carrying dnPI3 kinase abolished neovascularization in ischemic hind limb induced by eNOS gene transfer. These findings indicate that NO promotes endothelial cell migration and neovascularization via cGMP-dependent activation of PI3 kinase and suggest that this pathway is important in mediating NO-induced angiogenesis.

A Transforming Growth Factor β-Induced Smad3/Smad4 Complex Directly Activates Protein Kinase A

Zhang, Lizhi; Duan, Chao Jun; Binkley, Charles; Li, Gangyong; Uhler, Michael D.; Logsdon, Craig D.; Simeone, Diane M.
Fonte: American Society for Microbiology Publicador: American Society for Microbiology
Tipo: Artigo de Revista Científica
Publicado em /03/2004 Português
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Transforming growth factor β (TGFβ) interacts with cell surface receptors to initiate a signaling cascade critical in regulating growth, differentiation, and development of many cell types. TGFβ signaling involves activation of Smad proteins which directly regulate target gene expression. Here we show that Smad proteins also regulate gene expression by using a previously unrecognized pathway involving direct interaction with protein kinase A (PKA). PKA has numerous effects on growth, differentiation, and apoptosis, and activation of PKA is generally initiated by increased cellular cyclic AMP (cAMP). However, we found that TGFβ activates PKA independent of increased cAMP, and our observations support the conclusion that there is formation of a complex between Smad proteins and the regulatory subunit of PKA, with release of the catalytic subunit from the PKA holoenzyme. We also found that the activation of PKA was required for TGFβ activation of CREB, induction of p21Cip1, and inhibition of cell growth. Taken together, these data indicate an important and previously unrecognized interaction between the TGFβ and PKA signaling pathways.

Hematopoietic Cell Fate and the Initiation of Leukemic Properties in Primitive Primary Human Cells Are Influenced by Ras Activity and Farnesyltransferase Inhibition

Dorrell, Craig; Takenaka, Katsuto; Minden, Mark D.; Hawley, Robert G.; Dick, John E.
Fonte: American Society for Microbiology Publicador: American Society for Microbiology
Tipo: Artigo de Revista Científica
Publicado em /08/2004 Português
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The Ras pathway transduces divergent signals determining normal cell fate and is frequently activated in hematopoietic malignancies, but the manner in which activation contributes to human leukemia is poorly understood. We report that a high level of activated H-Ras signaling in transduced primary human hematopoietic progenitors reduced their proliferation and enhanced monocyte/macrophage differentiation. However, the exposure of these cells to a farnesyltransferase inhibitor and establishment of a moderate level of Ras activity showed increased proliferation, an elevated frequency of primitive blast-like cells, and progenitors with enhanced self-renewal capacity. These results suggest that the amplitude of Ras pathway signaling is a determinant of myeloid cell fate and that moderate Ras activation in primitive hematopoietic cells can be an early event in leukemogenesis.