RESUMO
DNA replication stress promotes genome instability in cancer. However, the contribution of the replication stress response to the development of malignancies remains unresolved. The DNA replication stress response protein SMARCAL1 stabilizes DNA replication forks and prevents replication fork collapse, a cause of DNA breaks and apoptosis. While the fork regression/remodeling functions of SMARCAL1 have been investigated, its in vivo functions in replication stress and cancer are unclear. Using a gamma radiation (IR)-induced replication stress T-cell lymphoma mouse model, we observed a significant inhibition of lymphomagenesis in mice lacking one or both alleles of Smarcal1. Notably, a quarter of the Smarcal1-deficient mice did not develop tumors. Moreover, hematopoietic stem/progenitor cells (HSPCs) and developing thymocytes in Smarcal1-deficient mice showed increased DNA damage and apoptosis during the proliferation burst following IR and an impaired ability to repopulate the thymus after IR. Additionally, mice lacking Smarcal1 showed significant HSPC defects when challenged to respond to other replication stress stimuli. Thus, our data reveal the critical function of the DNA replication stress response and, specifically, Smarcal1 in hematopoietic cell survival and tumor development. Our results also provide important insight into the immunodeficiency observed in individuals with mutations in SMARCAL1 by suggesting that it is an HSPC defect.
Assuntos
DNA Helicases/genética , Instabilidade Genômica/genética , Linfoma de Células T/genética , Animais , Apoptose/genética , Proliferação de Células/genética , Proliferação de Células/efeitos da radiação , Dano ao DNA/genética , Dano ao DNA/efeitos da radiação , Replicação do DNA/genética , Replicação do DNA/efeitos da radiação , DNA de Cadeia Simples/efeitos da radiação , Modelos Animais de Doenças , Raios gama , Instabilidade Genômica/efeitos da radiação , Células-Tronco Hematopoéticas/patologia , Humanos , Linfócitos/patologia , Linfócitos/efeitos da radiação , Linfoma de Células T/patologia , Camundongos , MutaçãoRESUMO
The oncogene Mdmx is overexpressed in many human malignancies, and together with Mdm2, negatively regulates the p53 tumor suppressor. However, a p53-independent function of Mdmx that impacts genome stability has been described, but this function is not well understood. In the present study, we determined that of the 13 different cancer types evaluated, 6-90% of those that had elevated levels of Mdmx had concurrent inactivation (mutated or deleted) of p53. We show elevated levels of Mdmx-inhibited double-strand DNA break repair and induced chromosome and chromatid breaks independent of p53, leading to genome instability. Mdmx impaired early DNA damage-response signaling, such as phosphorylation of the serine/threonine-glutamine motif, mediated by the ATM kinase. Moreover, we identified Mdmx associated with Nbs1 of the Mre11-Rad50-Nbs1 (MRN) DNA repair complex, and this association increased upon DNA damage and was detected at chromatin. Elevated Mdmx levels also increased cellular transformation in a p53-independent manner. Unexpectedly, all Mdmx-mediated phenotypes also occurred in cells lacking Mdm2 and were independent of the Mdm2-binding domain (RING) of Mdmx. Therefore, Mdmx-mediated inhibition of the DNA damage response resulted in delayed DNA repair and increased genome instability and transformation independent of p53 and Mdm2. Our results reveal a novel p53- and Mdm2-independent oncogenic function of Mdmx that provides new insight into the many cancers that overexpress Mdmx.
Assuntos
Instabilidade Genômica , Neoplasias/metabolismo , Proteínas Nucleares/metabolismo , Proteínas Proto-Oncogênicas c-mdm2/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Transportadores de Cassetes de Ligação de ATP/genética , Transportadores de Cassetes de Ligação de ATP/metabolismo , Hidrolases Anidrido Ácido , Animais , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Dano ao DNA/genética , Reparo do DNA/genética , Enzimas Reparadoras do DNA/genética , Enzimas Reparadoras do DNA/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Humanos , Proteína Homóloga a MRE11 , Camundongos , Camundongos Knockout , Neoplasias/genética , Neoplasias/patologia , Proteínas Nucleares/genética , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas c-mdm2/genética , Transdução de Sinais/genética , Proteína Supressora de Tumor p53/genéticaRESUMO
Mdm2 binding protein (MTBP) has been implicated in cell-cycle arrest and the Mdm2/p53 tumor suppressor pathway through its interaction with Mdm2. To determine the function of MTBP in tumorigenesis and its potential role in the Mdm2/p53 pathway, we crossed Mtbp-deficient mice to Emu-myc transgenic mice, in which overexpression of the oncogene c-Myc induces B-cell lymphomas primarily through inactivation of the Mdm2/p53 pathway. We report that Myc-induced B-cell lymphoma development in Mtbp heterozygous mice was profoundly delayed. Surprisingly, reduced levels of Mtbp did not lead to an increase in B-cell apoptosis or affect Mdm2. Instead, an Mtbp deficiency inhibited Myc-induced proliferation and the upregulation of Myc target genes necessary for cell growth. Consistent with a role in proliferation, Mtbp expression was induced by Myc and other factors that promote cell-cycle progression and was elevated in lymphomas from humans and mice. Therefore, Mtbp functioned independent of Mdm2 and was a limiting factor for the proliferative and transforming functions of Myc. Thus, Mtbp is a previously unrecognized regulator of Myc-induced tumorigenesis.
Assuntos
Linfócitos B/patologia , Proteínas de Transporte/metabolismo , Genes myc , Linfoma de Células B/metabolismo , Animais , Apoptose/genética , Linfócitos B/metabolismo , Processos de Crescimento Celular/fisiologia , Células Cultivadas , Feminino , Genes Supressores de Tumor , Humanos , Linfoma de Células B/genética , Linfoma de Células B/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Proteínas Proto-Oncogênicas c-mdm2/metabolismo , Proteína Supressora de Tumor p53/metabolismoRESUMO
We have identified a third member of the junctional adhesion molecule (JAM) family. At the protein level JAM3 displays 36 and 32% identity to JAM2 and JAM1, respectively. The coding region is distributed over 9 exons and maps to chromosome 11q25. The gene shows widespread tissue expression with higher levels apparent in the kidney, brain, and placenta. At the cellular level we show expression of JAM3 transcript within endothelial cells. Our major finding is that JAM3 and JAM2 are binding partners. Thus, JAM3 ectodomain binds firmly to JAM2-Fc. This heterotypic interaction is maintained when JAM3-Fc is used to capture Chinese hamster ovary cells expressing full-length JAM2. In static adhesion assays we show that JAM3 is unable to bind to leukocyte cell lines. This is consistent with the lack of JAM2 expression. However, using JAM2-Fc pull-down experiments in combination with polyclonal anti-JAM3 serum, we demonstrate that JAM3 is the previously uncharacterized 43-kDa counter-receptor that mediates JAM2 adhesion to T cells. Most significantly we demonstrate up-regulation of JAM3 protein on peripheral blood lymphocytes following activation. Finally we show the utility of JAM3 ectodomain as an inhibitor of JAM2 adhesion.
Assuntos
Moléculas de Adesão Celular , Imunoglobulinas/genética , Proteínas de Membrana/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Células CHO , Mapeamento Cromossômico , Cromossomos Humanos Par 11 , Clonagem Molecular , Cricetinae , Primers do DNA , DNA Complementar , Bases de Dados de Ácidos Nucleicos , Etiquetas de Sequências Expressas , Humanos , Imunoglobulinas/química , Imunoglobulinas/metabolismo , Proteínas de Membrana/química , Proteínas de Membrana/metabolismo , Dados de Sequência Molecular , RNA Mensageiro/genética , Homologia de Sequência de AminoácidosRESUMO
We have cloned a novel cDNA belonging to the Ig superfamily that shows 44% similarity to the junctional adhesion molecule (JAM) and maps to chromosome 21q21.2. The open reading frame of JAM2 predicts a 34-kDa type I integral membrane protein that features two Ig-like folds and three N-linked glycosylation sites in the extracellular domain. A single protein kinase C phosphorylation consensus site and a PDZ-binding motif are present in the short intracellular tail. Heterologous expression of JAM2 in Chinese hamster ovary cells defined a 48-kDa protein that localizes predominantly to the intercellular borders. Northern blot analysis showed that JAM2 is preferentially expressed in the heart. JAM2 homotypic interactions were demonstrated by the ability of JAM2-Fc to capture JAM2-expressing Chinese hamster ovary cells. We further showed that JAM2, but not JAM1, is capable of adhering to the HSB and HPB-ALL lymphocyte cell lines. Neutralizing mouse anti-JAM2 polyclonal antibodies provided evidence against homotypic interactions in this assay. Biotinylation of HSB cell membranes revealed a 43-kDa counter-receptor that precipitates specifically with JAM2-Fc. These characteristics of JAM2 led us to hypothesize a role for this novel protein in adhesion events associated with cardiac inflammatory conditions.
Assuntos
Moléculas de Adesão Celular , Imunoglobulinas/metabolismo , Leucócitos/metabolismo , Proteínas de Membrana/metabolismo , Sequência de Aminoácidos , Animais , Sequência de Bases , Células CHO , Adesão Celular , Mapeamento Cromossômico , Cromossomos Humanos Par 21 , Clonagem Molecular , Cricetinae , DNA Complementar , Humanos , Imunoglobulinas/química , Imunoglobulinas/genética , Proteínas de Membrana/química , Proteínas de Membrana/genética , Dados de Sequência MolecularRESUMO
The kinase insert domain-containing receptor (KDR) tyrosine kinase mediates calcium mobilization in endothelial cells and plays a key role during physiological and pathological angiogenesis. To provide a detailed understanding of how KDR is activated, we analyzed the kinetics of ligand-receptor interaction using BIAcore. Both predimerized (KDR-Fc) and monomeric (KDR-cbu) receptors were examined with vascular endothelial cell growth factor (VEGF) homodimers and VEGF/placental growth factor (PlGF) heterodimers. VEGF binds to KDR-Fc with ka = 3.6 +/- 0.07e6, kd = 1.34 +/- 0.19e-4, and KD = 37.1 +/- 4.9 pM. These values are similar to those displayed by monomeric KDR where ka = 5.23 +/- 1.4e6, kd = 2.74 +/- 0.76e-4, and KD = 51.7 +/- 5.8 pM were apparent. In contrast, VEGF/PlGF bound to KDR-Fc with ka = 7.3 +/- 1.6e4, kd = 4.4 +/- 1. 2e-4, and KD = 6 +/- 1.2 nM. Thus, the heterodimer displays a 160-fold reduced KD for binding to predimerized KDR, which is mainly a consequence of a 50-fold reduction in ka. We were unable to detect association between VEGF/PlGF and monomeric KDR. However, nanomolar concentrations of VEGF/PlGF were able to elicit weak calcium mobilization in endothelial cells. This latter observation may indicate partial predimerization of KDR on the cell surface or facilitation of binding due to accessory receptors.
Assuntos
Fatores de Crescimento Endotelial/metabolismo , Linfocinas/metabolismo , Receptores Proteína Tirosina Quinases/metabolismo , Sítios de Ligação , Técnicas Biossensoriais , Linhagem Celular , Dimerização , Endotélio Vascular/metabolismo , Humanos , Cinética , Fator de Crescimento Placentário , Proteínas da Gravidez/metabolismo , Ligação Proteica , Receptores de Fatores de Crescimento/metabolismo , Receptores de Fatores de Crescimento do Endotélio Vascular , Fator A de Crescimento do Endotélio Vascular , Fatores de Crescimento do Endotélio VascularRESUMO
We have prepared a polyclonal mouse antibody directed against the first three immunoglobulin-like domains of the kinase insert domain-containing receptor (KDR) tyrosine kinase. It possesses the ability to inhibit binding of the 165-amino acid splice variant of vascular endothelial cell growth factor (VEGF165) to recombinant KDR in vitro as well as to reduce VEGF165 binding to human umbilical vein endothelial cells (HUVEC). These results confirm that the first three immunoglobulin-like domains of KDR are involved in VEGF165 interactions. The anti-KDR antibody is able to completely block VEGF165-mediated intracellular Ca2+ mobilization in HUVEC. Therefore, it appears that binding of VEGF165 to the fms-like tyrosine kinase (Flt-1) in these cells does not translate into a Ca2+ response. This is further exemplified by the lack of response to placental growth factor (PlGF), an Flt-1-specific ligand. Additionally, PlGF is unable to potentiate the effects of submaximal concentrations of VEGF165. Surprisingly, the VEGF-PlGF heterodimer was also very inefficient at eliciting a Ca2+ signaling event in HUVEC. We conclude that KDR activation is crucial for mobilization of intracellular Ca2+ in HUVEC in response to VEGF165.
Assuntos
Cálcio/metabolismo , Fatores de Crescimento Endotelial/fisiologia , Endotélio Vascular/metabolismo , Linfocinas/fisiologia , Receptores Proteína Tirosina Quinases/metabolismo , Receptores de Fatores de Crescimento/metabolismo , Veias Umbilicais/metabolismo , Animais , Transporte Biológico/fisiologia , Células Cultivadas , Endotélio Vascular/citologia , Ativação Enzimática/fisiologia , Humanos , Técnicas Imunológicas , Camundongos , Receptores de Fatores de Crescimento do Endotélio Vascular , Proteínas Recombinantes , Veias Umbilicais/citologia , Fator A de Crescimento do Endotélio Vascular , Fatores de Crescimento do Endotélio VascularRESUMO
Vascular endothelial cell growth factor interacts with the receptor tyrosine kinases Flt-1 and KDR/Flk-1. We report that both receptors bind to PLC gamma and display specificity for the N-SH2 over the C-SH2 domain. Extensive site-directed mutagenesis of Flt-1 reveals that the juxta-membrane Y794, and the carboxyl terminal Y1169, are two major sites of interaction. Amino acids in the +1, +2 and +3 positions following these tyrosines are LSI and IPI, respectively. Peptide maps generated from wild type and mutant Flt-1 confirms that these residues are autophosphorylated. As predicted, mutagenesis of the analogous amino acids in KDR, positions Y801F and Y1175F, which lie in contexts YLSI and YIVL, respectively, reduced interactions of PLC gamma with this receptor. We conclude that both Flt-1 and KDR have the potential to signal through PLC gamma via phosphotyrosine residues located in juxta-membrane and carboxyl tail regions.
Assuntos
Fatores de Crescimento Endotelial/metabolismo , Isoenzimas/metabolismo , Linfocinas/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Receptores Proteína Tirosina Quinases/metabolismo , Receptores de Fatores de Crescimento/metabolismo , Fosfolipases Tipo C/metabolismo , Sequência de Aminoácidos , Sítios de Ligação , Western Blotting , Cromatografia Líquida de Alta Pressão , Análise Mutacional de DNA , Isoenzimas/química , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Oligodesoxirribonucleotídeos/química , Mapeamento de Peptídeos , Fosfolipase C gama , Fosforilação , Fosfotirosina/metabolismo , Ligação Proteica , Proteínas Proto-Oncogênicas/química , Proteínas Proto-Oncogênicas/genética , Receptores Proteína Tirosina Quinases/química , Receptores Proteína Tirosina Quinases/genética , Receptores de Fatores de Crescimento/genética , Receptores de Fatores de Crescimento do Endotélio Vascular , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Tripsina/metabolismo , Fosfolipases Tipo C/química , Fator A de Crescimento do Endotélio Vascular , Receptor 1 de Fatores de Crescimento do Endotélio Vascular , Fatores de Crescimento do Endotélio Vascular , Domínios de Homologia de srcRESUMO
Vascular Endothelial Growth Factor (VEGF) mediates its actions through the Flt-1 and KDR(Flk-1) receptor tyrosine kinases. To localize the extracellular region of Flt-1 that is involved in ligand interactions, we prepared secreted fusion proteins between various combinations of its seven extracellular IgG-like folds. Ligand binding studies show that in combination, domains one and two (amino acids 1-234) are sufficient to achieve VEGF165 interactions. Either domain alone is insufficient to achieve this effect. However, Scatchard analysis reveals that despite the binding capabilities of this construct, the Kd is five fold lower than ligand binding to the full extracellular domain. We find that addition of domain three to this minimal site restores high affinity receptor binding. Further, we show that domains one and two are sufficient to achieve interactions of Flt-1 with Placental Growth Factor (PIGF-1).
Assuntos
Fatores de Crescimento Endotelial/fisiologia , Linfocinas/fisiologia , Proteínas Proto-Oncogênicas/química , Receptores Proteína Tirosina Quinases/química , Animais , Humanos , Imunoglobulina G/química , Camundongos , Ligação Proteica , Receptores de Fatores de Crescimento/fisiologia , Proteínas Recombinantes de Fusão , Transdução de Sinais , Relação Estrutura-Atividade , Fator A de Crescimento do Endotélio Vascular , Receptor 1 de Fatores de Crescimento do Endotélio Vascular , Fatores de Crescimento do Endotélio VascularRESUMO
We have isolated and cloned a novel epithelial Cl- channel protein from a bovine tracheal cDNA expression library using an antibody probe. The antibody (alpha p38) was raised against a 38-kDa component of a homopolymeric protein that behaves as a Ca2+/calmodulin kinase II-, DIDS-, and dithiothreitol (DTT)-sensitive, anion-selective channel when incorporated into planar lipid bilayers. The full-length cDNA is 3001 base pairs long and codes for a 903-amino acid protein. The clone does not show any significant homology to any other previously reported Cl- channel sequence. Northern analysis of bovine tracheal mRNA with a cDNA probe corresponding to the cloned sequence revealed a band at 3.1 kilobases, suggesting that close to the full-length sequence has been cloned. The full-length open reading frame (2712 base pairs) has been expressed in Xenopus oocytes and in mammalian COS-7 cells. In oocytes, expression of the clone was associated with the appearance of a novel DIDS-, and DTT-sensitive, anion-selective conductance that was outwardly rectified and exhibited a reversal potential close to 0 mV. Whole-cell patch clamp studies in COS-7 cells transfected with the clone identified an ionomycin-, and DTT-sensitive chloride conductance that was not apparent in mock-transfected or control cells. In vitro translation studies have shown that the primary transcript codes for a protein migrating at 140 kDa under reduced conditions, significantly larger than the polypeptide recognized by alpha p38. We therefore suggest that either the 140-kDa translated product is a prepro form of the 38-kDa subunit of the previously identified bovine tracheal anion channel and that the primary transcript is post-translationally cleaved to yield the final product, or that the cloned channel and the previously identified bovine tracheal anion channel protein share an epitope that is recognized by the alpha p38 antibody.
Assuntos
Canais de Cloreto/genética , Traqueia/metabolismo , Sequência de Aminoácidos , Animais , Sequência de Bases , Western Blotting , Bovinos , Linhagem Celular , Clonagem Molecular , DNA Complementar , Epitélio/metabolismo , Bicamadas Lipídicas , Potenciais da Membrana , Dados de Sequência Molecular , Biossíntese de Proteínas , Transcrição Gênica , XenopusRESUMO
A bovine homologue of the rat and human epithelial Na+ channel subunits, alpha-rENaC and alpha-hENaC, was cloned. The cDNA clone, termed alpha-bENaC, was isolated from a bovine renal papillary collecting duct cDNA expression library. The bovine cDNA is 3,584 base pairs (bp) long, has an open reading frame of 2,094 bp encoding a 697-amino acid protein, and is 75-85% homologous to its rat and human counterparts. In vitro translation of the transcribed cRNA yields an 80-kDa polypeptide and one at 92 kDa in the presence of pancreatic microsomes. The clone exhibits consensus sequences for N-linked glycosylation and for phosphorylation by protein kinase C, but not for protein kinase A. After expression in Xenopus laevis oocytes, a small amiloride-sensitive Na+ conductance that exhibited inward rectification and a reversal potential greater than +30 mV, consistent with the predicted equilibrium potential for Na+, was identified. The expressed alpha-bENaC-associated Na+ current was not responsive to elevations in adenosine 3',5'-cyclic monophosphate but could be stimulated by phorbol 12-myristate 13-acetate, an activator of protein kinase C. alpha-bENaC also formed amiloride-sensitive chimeric channels when coexpressed with the rat beta- and gamma-ENaC subunits in Xenopus oocytes. alpha-bENaC therefore represents a novel isoform of a growing family of epithelial Na+ channels.
Assuntos
Clonagem Molecular , Rim/metabolismo , Canais de Sódio/genética , Canais de Sódio/metabolismo , Sequência de Aminoácidos , Animais , Elementos Antissenso (Genética)/genética , Sequência de Bases , Northern Blotting , Bovinos , Epitélio/metabolismo , Humanos , Immunoblotting , Isomerismo , Sondas Moleculares/genética , Dados de Sequência Molecular , Oócitos/metabolismo , Biossíntese de Proteínas , Ratos , Homologia de Sequência de Aminoácidos , Canais de Sódio/química , Xenopus laevisRESUMO
Zn(II) is an essential trace element. In spermatozoa, Zn(II) modulates metabolism and chromatin condensation. The mechanisms of uptake and distribution of this ion in sperm cells have not been explored. In rat spermatids, our results indicate that 1) 65Zn(II) binds with fast kinetics to a labile, presumably extracellular, compartment. This binding is temperature insensitive and not modified by metabolic inhibitors. 2) Entry of 65Zn(II) in the absence of externally added proteins occurs through a mediated transport system that allows exchange to reach steady state in approximately 15 min at 34 degrees C. 3) Upon entering the cells, 65Zn(II) binds tightly to cellular organelles. 4) Exchangeable Zn(II) bound to cytoplasmic proteins plus free intracellular Zn(II) appears to be < 20% of total exchangeable Zn(II). 5) The intracellular exchangeable Zn(II) compartment is decreased by metabolic inhibitors, showing a direct or indirect link between energy metabolism and cellular Zn(II) levels. 6) 65Zn(II) efflux from rat spermatids is a process with a rate constant of 0.16 +/- 0.13 min-1 at 34 degrees C. This exit rate of Zn(II) is likely to be affected by Zn(II) release from cytoplasmic binding sites or organelles.
Assuntos
Espermátides/metabolismo , Zinco/farmacocinética , Animais , Antimicina A/análogos & derivados , Antimicina A/farmacologia , Transporte Biológico , Calcimicina/farmacologia , Desoxiglucose/farmacologia , Troca Iônica , Masculino , Concentração Osmolar , Ratos , Temperatura , Distribuição Tecidual , Zinco/antagonistas & inibidoresRESUMO
We characterized the electrophysiological properties of a chloride channel protein isolated from bovine trachea after incorporation into planar lipid bilayers, and studied the effects of thiol-modulating agents on channel regulation both in bilayers and vesicular iodide uptake studies. Our experiments showed that this protein formed perfectly anion-selective channels in the bilayer, with an anion permeability sequence of I- (2.1) > NO3- (1.7) > Br- (1.2) > Cl- (1.0). The conductance of this channel was 25-30 picosiemens in 150 mM Cl-, and saturated with increasing chloride concentration. This channel could be completely inhibited by 4,4'-bis(isothiocyano)-2,2'-stilbenedisulfonate. Immunoblot analysis, using polyclonal antibodies (anti-p38), revealed one major band at 140 kDa. Upon reduction with dithiothreitol, 64- and 38-kDa polypeptides were observed. Functional experiments showed that reduction was accompanied by loss of 125I- uptake and single-channel activity. In the presence of dithiothreitol, only the low molecular mass protein forms (64 and 38 kDa) were detected by anti-p38 antibodies on Western blots. Cross-linking of S-S bonds with Cu(2+)-o-phenanthroline led to activation of chloride channels in vesicles and bilayers. Over-aggregation of chloride channels by this S-S cross-linking reagent caused inhibition of 125I- uptake by 80-100% and the abolishment of single-channel activity. We propose that the native chloride channel from bovine trachea can exist in vivo in different structural and functional forms depending upon its thiol-disulfide oxidation reduction status. The oxidized form has a molecular mass of 140 kDa and represents a fully active chloride channel. Inactivation of this channel might occur by over-aggregation of protein subunits, or by dissociation of the 140-kDa subunit by disulfide bond reduction.