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At least 1,315 records · Page 73Linked to original sources
PubMed1998

Both Rb/p16INK4a inactivation and telomerase activity are required to immortalize human epithelial cells.

Normal human cells undergo a limited number of divisions in culture and enter a non-dividing state called replicative senescence. Senescence is accompanied by several changes, including an increase in inhibitors of cyclin-dependent kinases and telomere shortening. The mechanisms by which viral oncogenes reverse these processes are not fully understood, although a general requirement for oncoproteins such as human papillomavirus E6 and E7 has suggested that the p53 and Rb pathways are targeted. Expression of the catalytic component of telomerase, hTERT, alone significantly extends the lifespan of human fibroblasts. Here we show that telomerase activity is not sufficient for immortalization of human keratinocyte or mammary epithelial cells: we find that neither addition of hTERT nor induction of telomerase activity by E6, both of which are active in maintaining telomere length, results in immortalization. Inactivation of the Rb/p16 pathway by E7 or downregulation of p16 expression, in combination with telomerase activity, however, is able to immortalize epithelial cells efficiently. Elimination of p53 and of the DNA-damage-induced G1 checkpoint is not necessary for immortalization, neither is elimination of p19ARF.

Breast↗
PubMed2000

Familial dyserythropoietic anaemia and thrombocytopenia due to an inherited mutation in GATA1.

Haematopoietic development is regulated by nuclear protein complexes that coordinate lineage-specific patterns of gene expression. Targeted mutagenesis in embryonic stem cells and mice has revealed roles for the X-linked gene Gata1 in erythrocyte and megakaryocyte differentiation. GATA-1 is the founding member of a family of DNA-binding proteins that recognize the motif WGATAR through a conserved multifunctional domain consisting of two C4-type zinc fingers. Here we describe a family with X-linked dyserythropoietic anaemia and thrombocytopenia due to a substitution of methionine for valine at amino acid 205 of GATA-1. This highly conserved valine is necessary for interaction of the amino-terminal zinc finger of GATA-1 with its essential cofactor, FOG-1 (for friend of GATA-1; refs 9-12). We show that the V205M mutation abrogates the interaction between Gata-1 and Fog-1, inhibiting the ability of Gata-1 to rescue erythroid differentiation in an erythroid cell line deficient for Gata-1 (G1E). Our findings underscore the importance of FOG-1:Gata-1 associations in both megakaryocyte and erythroid development, and suggest that other X-linked anaemias or thrombocytopenias may be caused by defects in GATA1.

Adult↗
PubMed1997

Immunoconjugates made of an anti-EGF receptor monoclonal antibody and type 1 ribosome-inactivating proteins from Saponaria ocymoides or Vaccaria pyramidata.

The present paper describes two immunoconjugates consisting of an anti-epidermal growth factor receptor (EGFR) monoclonal antibody (MAb), named Mint5, covalently linked to the type 1 ribosome-inactivating proteins (RIPs) ocymoidine (Ocy) and pyramidatine (Pyra) from Saponaria ocymoides and Vaccaria pyramidata respectively. Both antibody and toxins are shown to retain their respective biological properties upon chemical conjugation. The immunoconjugates exert specific inhibition of EGFR expressing target cell proliferation and protein synthesis in in vitro assays and also inhibit the growth of grafted human tumour cells in nude mice.

Animals↗
PubMed2002

The F-box protein slimb controls the levels of clock proteins period and timeless.

The Drosophila circadian clock is driven by daily fluctuations of the proteins Period and Timeless, which associate in a complex and negatively regulate the transcription of their own genes. Protein phosphorylation has a central role in this feedback loop, by controlling Per stability in both cytoplasmic and nuclear compartments as well as Per/Tim nuclear transfer. However, the pathways regulating degradation of phosphorylated Per and Tim are unknown. Here we show that the product of the slimb (slmb) gene--a member of the F-box/WD40 protein family of the ubiquitin ligase SCF complex that targets phosphorylated proteins for degradation--is an essential component of the Drosophila circadian clock. slmb mutants are behaviourally arrhythmic, and can be rescued by targeted expression of Slmb in the clock neurons. In constant darkness, highly phosphorylated forms of the Per and Tim proteins are constitutively present in the mutants, indicating that the control of their cyclic degradation is impaired. Because levels of Per and Tim oscillate in slmb mutants maintained in light:dark conditions, light- and clock-controlled degradation of Per and Tim do not rely on the same mechanisms.

Amino Acid Motifs↗
PubMed2004

Reading the Hedgehog morphogen gradient by measuring the ratio of bound to unbound Patched protein.

Morphogens are 'form-generating' substances that spread from localized sites of production and specify distinct cellular outcomes at different concentrations. A cell's perception of morphogen concentration is thought to be determined by the number of active receptors, with inactive receptors making little if any contribution. Patched (Ptc), the receptor for the morphogen Hedgehog (Hh), is active in the absence of ligand and blocks the expression of target genes by inhibiting Smoothened (Smo), an essential transducer of the Hh signal. Hh binding to Ptc abrogates the ability of Ptc to inhibit Smo, thereby unleashing Smo activity and inducing target gene expression. Here, we show that a cell's measure of ambient Hh concentration is not determined solely by the number of active (unliganded) Ptc molecules. Instead, we find that Hh-bound Ptc can titrate the inhibitory action of unbound Ptc. Furthermore, we demonstrate that this effect is sufficient to allow normal reading of the Hh gradient in the presence of a form of Ptc that cannot bind the ligand but retains its ability to inhibit Smo. These results support a model in which the ratio of bound to unbound Ptc molecules determines the cellular response to Hh.

Animals↗
PubMed1997

Transgenic pigs produce functional human factor VIII in milk.

Deficiency or abnormality of coagulation factor VIII (FVIII) causes a bleeding disorder called hemophilia A. Treatment involves FVIII concentrates prepared from pooled human plasma or recombinant FVIII (rFVIII) prepared from mammalian cell culture. The cost of highly purified FVIII or rFVIII is a major factor in hemophilia therapy and restricts prophylaxis. We have sought to generate a new source of rFVIII by targeting expression of the human FVIII cDNA to the mammary gland of transgenic pigs using the regulatory sequences of the mouse whey acidic protein gene. The identity of processed heterodimeric rFVIII was confirmed using specific antibodies, by thrombin digestion and activity assays. The secretion of as much as 2.7 micrograms/ml of rFVIII in milk was over tenfold higher than in normal plasma. Up to 0.62 U/ml of rFVIII was detected in an assay in which rFVIII restored normal clotting activity to FVIII-deficient human plasma.

Animals↗
PubMed2005

Root gravitropism requires lateral root cap and epidermal cells for transport and response to a mobile auxin signal.

Re-orientation of Arabidopsis seedlings induces a rapid, asymmetric release of the growth regulator auxin from gravity-sensing columella cells at the root apex. The resulting lateral auxin gradient is hypothesized to drive differential cell expansion in elongation-zone tissues. We mapped those root tissues that function to transport or respond to auxin during a gravitropic response. Targeted expression of the auxin influx facilitator AUX1 demonstrated that root gravitropism requires auxin to be transported via the lateral root cap to all elongating epidermal cells. A three-dimensional model of the root elongation zone predicted that AUX1 causes the majority of auxin to accumulate in the epidermis. Selectively disrupting the auxin responsiveness of expanding epidermal cells by expressing a mutant form of the AUX/IAA17 protein, axr3-1, abolished root gravitropism. We conclude that gravitropic curvature in Arabidopsis roots is primarily driven by the differential expansion of epidermal cells in response to an influx-carrier-dependent auxin gradient.

Arabidopsis↗
PubMed2005

Competing waves of oligodendrocytes in the forebrain and postnatal elimination of an embryonic lineage.

The developmental origin of oligodendrocyte progenitors (OLPs) in the forebrain has been controversial. We now show, by Cre-lox fate mapping in transgenic mice, that the first OLPs originate in the medial ganglionic eminence (MGE) and anterior entopeduncular area (AEP) in the ventral forebrain. From there, they populate the entire embryonic telencephalon including the cerebral cortex before being joined by a second wave of OLPs from the lateral and/or caudal ganglionic eminences (LGE and CGE). Finally, a third wave arises within the postnatal cortex. When any one population is destroyed at source by the targeted expression of diphtheria toxin, the remaining cells take over and the mice survive and behave normally, with a normal complement of oligodendrocytes and myelin. Thus, functionally redundant populations of OLPs compete for space in the developing brain. Notably, the embryonic MGE- and AEP-derived population is eliminated during postnatal life, raising questions about the nature and purpose of the competition.

Animals↗
PubMed2001

Ex vivo generation of cytotoxic T lymphocytes specific for one or two distinct viruses for the prophylaxis of patients receiving an allogeneic bone marrow transplant.

Human adenovirus (AdV) infection and EBV-lymphoproliferative disease (LPD) are serious complications following allogeneic stem cell transplantation. In the healthy individual these viruses cause minor, self-limiting diseases but in the immunocompromised patient they are responsible for significant morbidity and mortality. The limitations of anti-viral drugs and a better understanding of the cellular immune response to viral pathogens have prompted interest in developing adoptive immunotherapy for transplant patients. Ex vivo expanded cytotoxic T lymphocytes (CTLs) specific for EBV have been used effectively both as prophylaxis against EBV-LPD and as treatment of established EBV+ lymphoma. To generate CTLs specific for AdV, we infected immature dendritic cells with virus, in the presence of lipid, and subsequently used these cells to stimulate PBMNCs. Cytotoxicity assays showed that the resulting CTLs specifically lysed AdV-expressing targets and that this was mediated predominantly by CD4+ T cells. To generate CTLs specific for both AdV and EBV, we developed a CD40 ligand co-culture system to infect B-lymphoblastoid cell lines (LCLs) with high efficiency. PBMNCs from healthy AdV-seropositive donors were stimulated weekly with autologous AdV+-LCLs. Chromium release assays demonstrated that the resultant CTLs had specificity against both EBV and AdV and that this was mediated by both CD4+ and CD8+ T cells. Our findings have potential implications for post-transplant AdV and EBV immunotherapy in recipients of allogeneic stem cell transplants.

Adenoviridae↗
PubMed2000

Surface expression of phosphatidylserine on macrophages is required for phagocytosis of apoptotic thymocytes.

Cells generally maintain an asymmetric distribution of phospholipids across the plasma membrane bilayer, restricting the phospholipid, phosphatidylserine (PS), to the inner leaflet of the plasma membrane. When cells undergo apoptosis, this asymmetric transbilayer distribution is lost, bringing PS to the surface where it acts as a signal for engulfment by phagocytes. The fluorescent dye merocyanine 540 specifically stains the plasma membrane of apoptotic cells which have lost their asymmetric distribution of phospholipids. However, it also stains non-apoptotic macrophages, suggesting that phospholipid asymmetry may not be maintained in these cells, and thus that they may express PS on their surface. Here, the PS-binding protein, annexin V, was used to show that in fact normal macrophages do express PS on their surface. Furthermore, pre-treating macrophages with annexin V was found to inhibit phagocytosis of apoptotic thymocytes and thymocytes on which PS expression was artificially induced, but did not inhibit phagocytosis of latex beads or Fc receptor-mediated phagocytosis of opsonized erythrocytes. These results indicate that PS is constitutively expressed on the surface of macrophages and is functionally significant for the phagocytosis of PS-expressing target cells.

Animals↗
PubMed2000

Drosophila models of human neurodegenerative disease.

Drosophila has provided a powerful genetic system in which to elucidate fundamental cellular pathways in the context of a developing and functioning nervous system. Recently, Drosophila has been applied toward elucidating mechanisms of human neurodegenerative disease, including Alzheimer's, Parkinson's and Huntington's diseases. Drosophila allows study of the normal function of disease proteins, as well as study of effects of familial mutations upon targeted expression of human mutant forms in the fly. These studies have revealed new insight into the normal functions of such disease proteins, as well as provided models in Drosophila that will allow genetic approaches to be applied toward elucidating ways to prevent or delay toxic effects of such disease proteins. These, and studies to come that follow from the recently completed sequence of the Drosophila genome, underscore the contributions that Drosophila as a model genetic system stands to contribute toward the understanding of human neurodegenerative disease.

Amyloid beta-Protein Precursor↗
PubMed1999

Dominant effects of the bcr-abl oncogene on Drosophila morphogenesis.

We targeted expression of human/fly chimeric Bcr-Abl proteins to the developing central nervous system (CNS) and eye imaginal disc of Drosophila melanogaster. Neural expression of human/fly chimeric P210 Bcr-Abl or P185 Bcr-Abl rescued abl mutant flies from pupal lethality, indicating that P210 and P185 Bcr-Abl can substitute functionally for Drosophila Abl during axonogenesis. However, increased levels of neurally expressed P210 or P185 Bcr-Abl but not Drosophila Abl produced CNS defects and lethality. Expression of P210 or P185 in the eye imaginal disc produced a dominant rough eye phenotype that was dependent on dosage of the transgene. Drosophila Enabled, previously identified as a suppressor of the abl mutant phenotype and substrate for Drosophila Abl kinase, had markedly increased phosphotyrosine levels in Bcr-Abl expressing Drosophila, indicating that it is a substrate for Bcr-Abl as well. Drosophila, therefore, is a suitable model system to identify Bcr-Abl interactions important for signal transduction and oncogenesis.

Animals↗
PubMed1999

Early cellular abnormalities induced by RET/PTC1 oncogene in thyroid-targeted transgenic mice.

The RET/PTC1 oncogene, a rearranged form of the RET proto-oncogene, has been reported to be associated with human papillary thyroid carcinomas. We have shown that targeted expression of RET/PTC1 in the thyroid gland leads to the development of thyroid carcinomas in transgenic mice with histologic and cytologic similarities to human papillary thyroid carcinoma. To further investigate how RET/PTC1 expression contributes to the pathogenesis of papillary thyroid tumor, the time of tumor onset and the early phenotypic consequences of RET/PTC1 expression in thyrocytes were determined. All high copy transgenic mice developed bilateral thyroid tumors as early as 4 days of age. At embryological days 16-18, increased proliferation rate, distorted thyroid follicle formation and reduced radioiodide concentrating activity were identified in transgenic embryos. The reduced radioiodide concentrating activity was attributed to decreased expression of the sodium-iodide symporter. Our study showed that RET/PTC1 not only increased proliferation of thyrocytes, it also altered morphogenesis and differentiation. These findings provide a model for the role of RET/PTC1 in the formation of abnormal follicles with reduced iodide uptake ability observed in human papillary thyroid carcinoma.

Age of Onset↗
PubMed2002

Loss of p19ARF enhances the defects of Mdm2 overexpression in the mammary gland.

The protein encoded by the murine double minute 2 (Mdm2) gene inactivates the function of the tumor suppressor p53. The targeted expression of the mdm2 transgene (BLGmdm2) to the mammary epithelium disrupts the cell cycle, causing multiple rounds of DNA synthesis without proper cell division and consequently poor mammary gland development. These phenotypes in the mammary epithelia of the transgenic mice are not dependent on either p53 or the transcription factor E2F1, as mice null for these genes carrying the BLGmdm2 transgene exhibit similar defects to mice carrying the BLGmdm2 transgene alone. p19ARF, an alternative splice product of the INK4a/ARF locus, has been shown to interact directly with MDM2. Therefore, BLGmdm2 transgenic mice null for p19ARF were created to gain insight into the mechanism by which mdm2 overexpression disrupts the cell cycle. The BLGmdm2 phenotype in the absence of p19ARF was worse than BLGmdm2 mice and visible as early as day 15 of pregnancy. By day 5 of lactation the phenotype was very pronounced. Histological analysis of the mammary gland showed a decrease in ductal branching, smaller and fewer lobuloalveolar structures, and a decrease in luminal secretions. Multinucleated and enlarged cells were present due to continued replication in the absence of cytokinesis. Thus, the absence of p19ARF in this in vivo system enhanced the defect caused by mdm2 overexpression.

Animals↗
PubMed2002

Inhibition of Rel/Nuclear Factor-kappaB signaling in skin results in defective DNA damage-induced cell cycle arrest and Ha-ras- and p53-independent tumor development.

In recent years a growth inhibitory role in skin for the Rel/NF-kappaB transcription factors has been established, and the block of Rel/NF-kappaB signaling results in rapid development of spontaneous skin cancer. The molecular mechanism underlying tumor development is however unknown. In the present study, we show that inhibition of NF-kappaB signaling in mouse skin by targeted expression of degradation resistant IkappaB-alpha generates transgenic keratinocytes unable to arrest the cell cycle in response to DNA damage induced by gamma-radiation. The results indicate that transgenic keratinocytes have a defect at the G1-S checkpoint whereas the G2-M checkpoint response was found to be intact. However, transgenic keratinocytes still respond by induction of the cyclin dependent kinase inhibitor p21(Cip1/Waf) after exposure to gamma-radiation. In the spontaneous skin tumors that develop in transgenic mice no mutations were found in the Ha-ras or p53 gene, suggesting that inhibition of NF-kappaB signaling in skin can induce cancer development independently of initiating mutations in the Ha-ras gene or additional mutations in the p53 gene. These findings demonstrate an involvement of NF-kappaB signaling in the DNA damage response and cell cycle checkpoint control in the skin.

Animals↗
PubMed2003

Fibroblast growth factor receptor signalling is crucial for liver homeostasis and regeneration.

Several growth factors have been suggested to play a crucial role in liver regeneration, but a functional proof is still missing. Since fibroblast growth factors are important for the initiation of mammalian liver development, we determined the roles of these mitogens in liver repair by targeted expression of a dominant-negative fibroblast growth factor receptor (FGFR) in hepatocytes of transgenic mice. The liver of young animals appeared histologically normal, and liver function was not obviously impaired. In aged transgenic mice, the frequency of fatty liver development was strongly increased compared to control animals. Following partial hepatectomy, transgenic mice showed markedly reduced hepatocyte proliferation because of an arrest in the late G(1) phase of the cell cycle. These data demonstrate a key role of FGFR signalling in repair after liver injury.

Animals↗
PubMed2006

The role of the MKK6/p38 MAPK pathway in Wip1-dependent regulation of ErbB2-driven mammary gland tumorigenesis.

There is increasing evidence for the role of wild-type p53 induced phosphatase 1 (Wip1) phosphatase in the regulation of tumorigenesis. To evaluate Wip1 as a breast cancer oncogene, we generated a mouse strain with targeted expression of Wip1 to the breast epithelium. We found that these mice are prone to cancer when intercrossed with transgenics expressing the ErbB2 oncogene but not conditional knockouts for Brca2. This tumor-prone phenotype of Wip1 is fully eliminated through attenuation of proliferation by activating the MKK6/p38 mitogen-activated protein kinases (MAPK) cascade in mice bearing a constitutively active form of MKK6. We propose that Wip1 phosphatase operates within the MKK6/p38 MAPK signaling pathway to promote ErbB2-driven mammary gland tumorigenesis.

Animals↗
PubMed2000

Improvement of receptor-mediated gene delivery to HepG2 cells using an amphiphilic gelling agent.

Gene transfer was performed using asialo-oroso-mucoid-polylysine (ASOR-PL) conjugates to allow targeted expression of the gene in cells of hepatic origin. In a gel-electrophoretic analysis, the ASOR-PL conjugate produced a complete DNA retardation effect at the optimal ratio of 222:1 (ASOR-PL conjugate/pCMV beta-gal plasmid). The gene-transfer efficiency of the ASOR-PL conjugate was evaluated in HepG2 cells that express asialoglycoprotein receptor and NIH 3T3 cells that do not. The expression was assayed by 5-bromo-4-chloroindol-3-yl beta-D-galactopyranoside ('X-Gal') staining and Chlorophenol Red beta-D-galactopyranoside. When an expression vector for the tumour-suppressor gene p53, pCMVp53, complexed to ASOR-PL conjugate, was transfected into HepG2 cells, the exogenously provided p53 gene was detected in the HepG2 cells by PCR. To improve the efficiency of DNA delivery and expression of the therapeutic proteins poloxamer 407, a fusogenic peptide, influenza-virus haemagglutinin HA2 and chloroquine were individually incorporated into the system. The expression level of beta-galactosidase in HepG2 cells was increased by about four times by the presence of poloxamer 407, whereas the fusogenic peptide HA2 and chloroquine had no effects. When HepG2 cells were transfected with pCMVp53 in the presence of poloxamer 407, the mRNA of transfected p53 could be detected by reverse transcriptase PCR. The current findings open the possibility that a receptor-mediated gene-delivery system for hepatic gene therapy using ASOR-PL conjugate in combination with poloxamer 407 may be developed in the future.

3T3 Cells↗