RESUMO
Members of the Aurora/Ipl1p family of mitotically regulated serine/threonine kinases are emerging as key regulators of chromosome segregation and cytokinesis. Proper chromosome segregation and cytokinesis ensure that each daughter cell receives the full complement of genetic material. Defects in these processes can lead to aneuploidy and the propagation of genetic abnormalities. This review discusses the Aurora/Ipl1p kinases in terms of their protein structure and proposed function in mitotic cells and also the potential role of aurora2 in human cancer.
Assuntos
Divisão Celular , Segregação de Cromossomos , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Animais , Aurora Quinases , Divisão Celular/genética , Regulação da Expressão Gênica no Desenvolvimento , Humanos , RatosRESUMO
The complete amino acid sequence of amphiregulin, a bifunctional cell growth modulator, was determined. The truncated form contains 78 amino acids, whereas a larger form of amphiregulin contains six additional amino acids at the amino-terminal end. The amino-terminal half of amphiregulin is extremely hydrophilic and contains unusually high numbers of lysine, arginine, and asparagine residues. The carboxyl-terminal half of amphiregulin (residues 46 to 84) exhibits striking homology to the epidermal growth factor (EGF) family of proteins. Amphiregulin binds to the EGF receptor but not as well as EGF does. Amphiregulin fully supplants the requirement for EGF or transforming growth factor-alpha in murine keratinocyte growth, but it is a much weaker growth stimulator in other cell systems.
Assuntos
Glicoproteínas/fisiologia , Substâncias de Crescimento/fisiologia , Peptídeos e Proteínas de Sinalização Intercelular , Sequência de Aminoácidos , Anfirregulina , Animais , Ligação Competitiva , Divisão Celular , Família de Proteínas EGF , Células Epidérmicas , Fator de Crescimento Epidérmico/fisiologia , Receptores ErbB/metabolismo , Humanos , Queratinas/metabolismo , Camundongos , Dados de Sequência Molecular , Ensaio Radioligante , Homologia de Sequência do Ácido Nucleico , Fatores de Crescimento Transformadores/fisiologiaRESUMO
The completion of the budding yeast genome sequencing project has made it possible to determine not only the total number of genes, but also the exact number of genes of a particular type 1-3. As a consequence, we now know exactly how many protein kinases are encoded by the yeast genome, a number of considerable interest because of the importance of protein phosphorylation in the control of so many cellular processes.
Assuntos
Genes Fúngicos , Genoma Fúngico , Família Multigênica , Proteínas Quinases/genética , Saccharomyces cerevisiae/genéticaRESUMO
Amphiregulin (AR) is a heparin-binding, heparin-inhibited member of the epidermal growth factor (EGF) family and an autocrine growth factor for human keratinocytes. Previous studies have shown that AR expression is increased in psoriatic epidermis. To test the hypothesis that aberrant AR expression is central to the development of psoriatic lesions, we constructed a transgene (K14-ARGE) encoding a human keratin 14 promoter-driven AR gene. Our results indicate that transgene integration and subsequent expression of AR in basal keratinocytes correlated with a psoriasis-like skin phenotype. Afflicted mice demonstrated shortened life spans, prominent scaling and erythematous skin with alopecia, and occasional papillomatous epidermal growths. Histologic examination revealed extensive areas of marked hyperkeratosis with focal parakeratosis, acanthosis, dermal and epidermal lymphocytic and neutrophilic infiltration, and dilated blood vessels within the papillary dermis. Our results reveal that AR exerts activity in the skin that is distinct from that of transgenic transforming growth factor-alpha or other cytokines, and induces skin pathology with striking similarities to psoriasis. Our observations also link the keratinocyte EGF receptor-ligand system to psoriatic inflammation, and suggest that aberrant expression of AR in the epidermis may represent a critical step in the development or propagation of psoriatic lesions.
Assuntos
Glicoproteínas/genética , Substâncias de Crescimento/genética , Peptídeos e Proteínas de Sinalização Intercelular , Psoríase/genética , Anfirregulina , Animais , Complexo CD3/metabolismo , Família de Proteínas EGF , Epiderme/fisiologia , Regulação da Expressão Gênica , Humanos , Queratinas/genética , Antígeno Ki-67/metabolismo , Camundongos , Camundongos Transgênicos , Psoríase/patologiaRESUMO
The five members of the human epidermal growth factor (EGF) family (EGF, transforming growth factor alpha [TGF-alpha], heparin-binding EGF-like growth factor [HB-EGF], betacellulin, and amphiregulin [AR]) are synthesized as transmembrane proteins whose extracellular domains are proteolytically processed to release the biologically active mature growth factors. These factors all activate the EGF receptor, but in contrast to EGF and TGF-alpha, the mature forms of HB-EGF and AR are also glycosylated, heparin-binding proteins. We have constructed a series of mutants to examine the influence of the distinct precursor domains in the biosynthesis of AR. The transmembrane and cytoplasmic domains of the precursor are not required for secretion of bioactive AR from either COS or mammary epithelium-derived cells, although proteolytic removal of the N-terminal pro-region is less efficient in the absence of the membrane anchor. Deletion of the N-terminal pro-region, however, results in rapid intracellular degradation of the molecule with no detectable secretion of active growth factor. AR secretion is preserved by replacing the native pro-region with the corresponding domain of the HB-EGF precursor but not with that of the TGF-alpha precursor. In the absence of any N-terminal pro-region, secretion of the molecule is restored by deleting the N-terminal heparin-binding domain of mature AR. Both EGF and TGF-alpha, in contrast, can be secreted without their pro-regions. However, if the protein is fused with the AR heparin-binding domain, TGF-alpha secretion is inhibited unless the AR pro-region is also present. We propose that the heparin-binding domain of mature AR necessitates the presence of a specific structural motif in an N-terminal pro-region to permit proper folding, and thus secretion, of a bioactive molecule.
Assuntos
Glicoproteínas/metabolismo , Substâncias de Crescimento/metabolismo , Peptídeos e Proteínas de Sinalização Intercelular , Precursores de Proteínas/metabolismo , Sequência de Aminoácidos , Anfirregulina , Família de Proteínas EGF , Receptores ErbB/metabolismo , Heparina/metabolismo , Hexosaminidases/metabolismo , Humanos , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Processamento de Proteína Pós-Traducional , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Recombinantes/metabolismo , Deleção de Sequência , Relação Estrutura-Atividade , Transfecção , Fator de Crescimento Transformador alfa/metabolismoRESUMO
Deregulated signaling by the four members of the epidermal growth factor receptor tyrosine kinase family (erbB family) is implicated in the genesis or progression of human cancers. However, efforts to analyze signaling by these receptors have been hampered by the diversity of ligands and extensive interreceptor cross talk. We have expressed the four human erbB family receptors, singly and in pairwise combinations, in a pro-B-lymphocyte cell line (Ba/F3) and investigated the range of interactions activated by the epidermal growth factor homology domain of the agonist neuregulin beta. The results provide the first comprehensive analysis of the response of this receptor family to a single peptide agonist. This peptide induced complex patterns of receptor tyrosine phosphorylation and regulation of Ba/F3 cell survival and proliferation. These data demonstrate the existence of several previously undocumented receptor interactions driven by neuregulin.
Assuntos
Receptores ErbB/metabolismo , Glicoproteínas/farmacologia , Transdução de Sinais/efeitos dos fármacos , Linfócitos B , Contagem de Células/efeitos dos fármacos , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Ativação Enzimática , Receptores ErbB/agonistas , Receptores ErbB/biossíntese , Receptores ErbB/genética , Glicoproteínas/síntese química , Humanos , Interleucina-3/farmacologia , Neurregulinas , Peptídeos/síntese química , FosforilaçãoRESUMO
We have isolated the gene for a novel growth regulator, amphiregulin (AR), that is evolutionarily related to epidermal growth factor (EGF) and transforming growth factor alpha (TGF-alpha). AR is a bifunctional growth modulator: it interacts with the EGF/TGF-alpha receptor to promote the growth of normal epithelial cells and inhibits the growth of certain aggressive carcinoma cell lines. The 84-amino-acid mature protein is embedded within a 252-amino-acid transmembrane precursor, an organization similar to that of the TGF-alpha precursor. Human placenta and ovaries were found to express significant amounts of the 1.4-kilobase AR transcript, implicating AR in the regulation of normal cell growth. In addition, the AR gene was localized to chromosomal region 4q13-4q21, a common breakpoint for acute lymphoblastic leukemia.
Assuntos
Glicoproteínas/genética , Substâncias de Crescimento/genética , Peptídeos e Proteínas de Sinalização Intercelular , Sequência de Aminoácidos , Anfirregulina , Sequência de Bases , Northern Blotting , Cromossomos Humanos Par 4 , Clonagem Molecular , DNA/genética , Família de Proteínas EGF , Fator de Crescimento Epidérmico/genética , Genes , Humanos , Dados de Sequência Molecular , Conformação Proteica , Precursores de Proteínas/genética , RNA Mensageiro/genética , Mapeamento por Restrição , Distribuição Tecidual , Fatores de Crescimento Transformadores/genéticaRESUMO
A novel human keratinocyte-derived autocrine factor (KAF) was purified from conditioned medium by using heparin affinity chromatography as the first step. Purified KAF stimulated the growth of normal human keratinocytes, mouse AKR-2B cells, and a mouse keratinocyte cell line (BALB/MK). Heparin sulfate inhibited KAF mitogenic activity on all cell types tested and inhibited the ability of KAF to compete with epidermal growth factor for cell surface binding. Interestingly, KAF stimulated the growth of BALB/MK cells at high cell density but failed to stimulate these cells at clonal density. Protein microsequencing of the first 20 NH2-terminal amino acid residues of purified KAF revealed identity to the NH2 terminus of human amphiregulin (AR). Northern (RNA) blot analysis with AR-specific cRNA demonstrated that human keratinocytes, as well as mammary epithelial cell cultures, expressed high levels of AR mRNA. In contrast, AR mRNA was not detected in normal human fibroblasts or melanocytes and was present at reduced levels in several mammary tumor cell lines. The mitogenic activity of purified AR was also shown to be inhibited by heparin sulfate, and an AR-specific enzyme-linked immunosorbent assay (ELISA) revealed that KAF and AR are antigenically related. We have previously shown that human keratinocytes can grow in an autocrine manner. Our present study demonstrates that one of the growth factors responsible for this autocrine growth (KAF) is similar or identical to AR and that KAF and AR bioactivity can be negatively regulated by heparin sulfate.
Assuntos
Glicoproteínas/genética , Substâncias de Crescimento/genética , Heparitina Sulfato/farmacologia , Peptídeos e Proteínas de Sinalização Intercelular , Queratinócitos/fisiologia , Sequência de Aminoácidos , Anfirregulina , Animais , Sequência de Bases , Ligação Competitiva , Divisão Celular/efeitos dos fármacos , Linhagem Celular , Células Cultivadas , Replicação do DNA/efeitos dos fármacos , Família de Proteínas EGF , Fator de Crescimento Epidérmico/metabolismo , Glicoproteínas/isolamento & purificação , Glicoproteínas/farmacologia , Substâncias de Crescimento/isolamento & purificação , Substâncias de Crescimento/farmacologia , Humanos , Queratinócitos/efeitos dos fármacos , Masculino , Camundongos , Dados de Sequência Molecular , Sondas de Oligonucleotídeos , RNA Mensageiro/genética , Homologia de Sequência do Ácido Nucleico , Pele/citologiaRESUMO
c-erbB-3 is a member of the type I family of growth factor receptors which includes the epidermal growth factor (EGF) receptor and c-erbB-2. Whereas for EGF receptor and c-erbB-2 the expression patterns in normal tissues are well documented, there is currently little information available about the sites of c-erbB-3 expression. In order to examine the normal tissue distribution of c-erbB-3, polyclonal antibodies were raised against eight synthetic peptides corresponding to distinct sites on the intracellular domain of c-erbB-3. Of these, three produced antibodies which reacted with a 160-kDa protein on immunoblots of human embryonal cells (293 cells) transfected with the cDNA encoding c-erbB-3, and two of the three antibodies immunoprecipitated a protein of similar size from the same cells. These antibodies were used for immunochemical staining of a wide variety of normal human adult and fetal tissues employing formalin-fixed, paraffin-embedded material. The c-erbB-3 protein was identified in cells of the gastrointestinal, reproductive, respiratory and urinary tracts as well as the skin, endocrine and nervous system in a distribution distinctly different from that observed for EGF receptor and c-erbB-2. The level of expression of the mRNA for c-erbB-3 was also examined in extracts of a selection of fetal tissues. In general the sites of mRNA and protein expression were concordant.
Assuntos
Proteínas Proto-Oncogênicas/biossíntese , Adulto , Sequência de Aminoácidos , Anticorpos , Epitopos , Feminino , Feto/metabolismo , Expressão Gênica , Humanos , Imuno-Histoquímica , Masculino , Dados de Sequência Molecular , Especificidade de Órgãos , Peptídeos , Proteínas Proto-Oncogênicas/imunologia , RNA Mensageiro/biossíntese , Receptor ErbB-3RESUMO
Complementary DNA (cDNA) encoding a novel member of the receptor tyrosine kinase (RTK) family has been isolated from colon carcinoma tissue. Colon carcinoma kinase 4 (CCK-4) mRNA is highly expressed in human lung tissue and at lower levels in the thyroid gland and ovary. While no mRNA was found in human adult colon tissues, expression varied remarkably in colon carcinoma-derived cell lines. CCK-4 cDNA encodes a chicken KLG-related, 1071 amino acid-long transmembrane glycoprotein containing several genetic alterations within the RTK consensus sequences. These define CCK-4 as a catalytically inactive member of the RTK family of proteins and, in analogy to HER3, suggest a potentially tumor-characteristic role as a signal amplifier or modulator for an as yet unidentified kinase-competent partner.
Assuntos
Moléculas de Adesão Celular , Neoplasias do Colo/enzimologia , Proteínas de Neoplasias/genética , Receptores Proteína Tirosina Quinases/genética , Adulto , Sequência de Aminoácidos , Sequência de Bases , Neoplasias do Colo/genética , DNA de Neoplasias/análise , DNA de Neoplasias/genética , DNA de Cadeia Simples/análise , DNA de Cadeia Simples/genética , Expressão Gênica , Humanos , Dados de Sequência Molecular , Proteínas de Neoplasias/análise , Reação em Cadeia da Polimerase , RNA Mensageiro/análise , RNA Mensageiro/genética , Receptores Proteína Tirosina Quinases/análise , Receptores Proteína Tirosina Quinases/classificação , TransfecçãoRESUMO
Betacellulin is a member of the epidermal growth factor (EGF) family. These soluble proteins are ligands for one or more of the four receptor tyrosine kinases encoded by the erbB gene family (erbB-1/epidermal growth factor receptor (EGFR), neu/erbB-2/HER2, erbB-3/HER3 and erbB-4/HER4). While evidence suggests that betacellulin is a ligand for the EGFR, the ability of betacellulin to regulate other erbB family receptors has not been analysed. Previously we engineered derivatives of the mouse Ba/F3 hematopoietic cell line to ectopically express erbB family receptors, singly and in pairwise combinations. We have stimulated this panel of cell lines with betacellulin and two other EGF family members, EGF itself and neuregulin-beta (NRG-beta). In the cell lines expressing a single erbB family receptor, betacellulin not only stimulated EGFR tyrosine phosphorylation, but it activated erbB-4 as well. Furthermore, in the double recombinant Ba/F3 derivatives, betacellulin stimulated a complex pattern of receptor phosphorylation distinct from the patterns activated by NRG-beta and EGF. Moreover, betacellulin stimulated a complex pattern of interleukin-3 independence in the Ba/F3 derivatives distinct from those activated by NRG-beta and EGF. These data identify a novel receptor for betacellulin and establish that different EGF family ligands activate distinct patterns of receptor phosphorylation and coupling to cellular signaling pathways.
Assuntos
Fator de Crescimento Epidérmico/farmacologia , Receptores ErbB/efeitos dos fármacos , Glicoproteínas/farmacologia , Substâncias de Crescimento/farmacologia , Peptídeos e Proteínas de Sinalização Intercelular , Sequência de Aminoácidos , Betacelulina , Linhagem Celular , Humanos , Interleucina-3/farmacologia , Dados de Sequência Molecular , Neurregulinas , Fosforilação , Receptor ErbB-4RESUMO
Activation of erbB-1 receptors by glial TGFalpha has been shown to be a component of the developmental program by which the neuroendocrine brain controls mammalian sexual development. The participation of other members of the erbB family may be required, however, for full signaling capacity. Here, we show that activation of astrocytic erbB-2/erbB-4 receptors plays a significant role in the process by which the hypothalamus controls the advent of mammalian sexual maturation. Hypothalamic astrocytes express both the erbB-2 and erbB-4 genes, but no erbB-3, and respond to neuregulins (NRGs) by releasing prostaglandin E(2) (PGE(2)), which acts on neurosecretory neurons to stimulate secretion of luteinizing hormone-releasing hormone (LHRH), the neuropeptide controlling sexual development. The actions of TGFalpha and NRGs in glia are synergistic and involve recruitment of erbB-2 as a coreceptor, via erbB-1 and erbB-4, respectively. Hypothalamic expression of both erbB-2 and erbB-4 increases first in a gonad-independent manner before the onset of puberty, and then, at the time of puberty, in a sex steroid-dependent manner. Disruption of erbB-2 synthesis in hypothalamic astrocytes by treatment with an antisense oligodeoxynucleotide inhibited the astrocytic response to NRGs and, to a lesser extent, that to TGFalpha and blocked the erbB-dependent, glia-mediated, stimulation of LHRH release. Intracerebral administration of the oligodeoxynucleotide to developing animals delayed the initiation of puberty. Thus, activation of the erbB-2-erbB-4 receptor complex appears to be a critical component of the signaling process by which astrocytes facilitate the acquisition of female reproductive capacity in mammals.
Assuntos
Astrócitos/fisiologia , Córtex Cerebral/fisiologia , Receptores ErbB/fisiologia , Hipotálamo/fisiologia , Neurregulinas/fisiologia , Receptor ErbB-2/fisiologia , Maturidade Sexual/fisiologia , Animais , Neoplasias da Mama , Córtex Cerebral/crescimento & desenvolvimento , Ventrículos Cerebrais/efeitos dos fármacos , Ventrículos Cerebrais/fisiologia , Dinoprostona/sangue , Receptores ErbB/genética , Estradiol/farmacologia , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Genes erbB-1 , Humanos , Hipotálamo/crescimento & desenvolvimento , Neurregulinas/genética , Oligodesoxirribonucleotídeos Antissenso/administração & dosagem , Oligodesoxirribonucleotídeos Antissenso/farmacologia , Proteínas Oncogênicas v-erbB , Ovariectomia , Fosforilação , Fosfotirosina/metabolismo , Gravidez , Progesterona/farmacologia , Ratos , Ratos Sprague-Dawley , Receptor ErbB-2/genética , Receptor ErbB-4 , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais , Transfecção , Células Tumorais CultivadasRESUMO
Advances in our understanding of the signal transduction pathways involved in cellular growth control have provided several new strategies for cancer therapy. Recent advances now make it possible to develop selective inhibitors targeting genomic instability, the growth, survival, and invasion of the tumor, and its nourishment through the growth of new blood vessels.
Assuntos
Antineoplásicos/síntese química , Desenho de Fármacos , Neoplasias/tratamento farmacológico , Animais , Antineoplásicos/uso terapêutico , Antineoplásicos/toxicidade , Apoptose , Divisão Celular , Sobrevivência Celular , Humanos , Invasividade Neoplásica , Neoplasias/irrigação sanguínea , Neoplasias/genética , Neoplasias/patologia , Neovascularização PatológicaRESUMO
The actions of 17 beta-estradiol (E2) and protein kinase C (PKC) appear to converge in the regulation of expression of certain growth modulatory genes, such as the growth factor amphiregulin (AR). AR is known to modulate cell growth by binding to the epidermal growth factor receptor. In the current report we established the mechanisms of the PKC-activating phorbol ester tumor promoter, 12-O-tetradecanoylphorbol-13-acetate (TPA) and the steroid hormone E2 on the induction of AR expression in human breast carcinoma cell lines. TPA (100 nM) and E2 (1 nM) induce AR messenger RNA (mRNA) expression by 6- to 8-fold and 3- to 6-fold, respectively, in a time- and dose-dependent manner. In addition, immunoreactive AR protein is induced by both TPA and E2 by 6- to 8-fold and 2- to 4-fold, respectively. The PKC-modulating drugs, bryostatin and H-7, and antiestrogens (ICI 164,384 and 4-hydroxytamoxifen) interfere with AR induction by TPA and estrogen, respectively. The effects of TPA and E2 on the induction of AR mRNA were both closely associated with enhanced transcription of the AR gene. However, TPA had an additional effect at the posttranscriptional level by stabilizing the AR mRNA. The protein synthesis inhibitor, cycloheximide, prevented AR induction by TPA, suggesting that a component of the TPA induction of AR is indirect and dependent upon protein synthesis. Conversely, the E2 induction of AR transcription was found to be a direct response, independent of protein synthesis. The results presented herein thus demonstrate that TPA and E2 are able to stimulate AR gene transcription by two separate mechanisms.
Assuntos
Neoplasias da Mama/química , Estrogênios/farmacologia , Glicoproteínas/análise , Glicoproteínas/genética , Substâncias de Crescimento/análise , Substâncias de Crescimento/genética , Peptídeos e Proteínas de Sinalização Intercelular , Acetato de Tetradecanoilforbol/farmacologia , 1-(5-Isoquinolinasulfonil)-2-Metilpiperazina , Anfirregulina , Antineoplásicos/farmacologia , Western Blotting , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Briostatinas , Meios de Cultivo Condicionados , Cicloeximida/farmacologia , Relação Dose-Resposta a Droga , Família de Proteínas EGF , Estradiol/análogos & derivados , Estradiol/farmacologia , Antagonistas de Estrogênios/farmacologia , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Isoquinolinas/farmacologia , Lactonas/farmacologia , Macrolídeos , Piperazinas/farmacologia , Alcamidas Poli-Insaturadas , Proteína Quinase C/antagonistas & inibidores , Proteína Quinase C/fisiologia , RNA Mensageiro/análise , RNA Mensageiro/genética , Receptores de Estrogênio/agonistas , Tamoxifeno/análogos & derivados , Tamoxifeno/farmacologia , Células Tumorais CultivadasRESUMO
Transforming growth factor-beta (TGF-beta) is capable of affecting the proliferation of many cell types. To identify novel genes whose protein products may mediate cellular responses to this factor, a cDNA library was made from mRNA isolated from a human lung adenocarcinoma cell line (A549) that had been treated for 3 days with TGF-beta. The library was screened by differential hybridization and a cDNA clone, beta ig-h3, was isolated. This gene was induced up to 20-fold in A549 cells after 2 days of treatment with TGF-beta 1. It was also induced in several other cell lines, including PC-3 and H2981. DNA sequence analysis of beta ig-h3 indicated that it encoded a novel protein, beta IG-H3, of 683 amino acids, which contained an amino-terminal secretory sequence and a carboxy-terminal Arg-Gly-Asp (RGD) sequence that can serve as a ligand recognition site for several integrins. beta IG-H3 also contained short amino acid regions homologous to similar regions in Drosophila fasciclin-I and four homologous internal domains, which can be folded into a potential bivalent structure and could act as a bridge between cells expressing the appropriate ligand. beta ig-h3 RNA was detected in several cell lines and tissues. COS cells transfected with plasmids encoding beta IG-H3 secreted a major 68-kD protein that was detected by immunoblotting using antipeptide antibodies. Since beta ig-h3 is induced in several cell lines whose proliferation is affected by TGF-beta 1, it may be involved in mediating some of the signals of this multifunctional growth modulator.
Assuntos
Proteínas da Matriz Extracelular , Proteínas de Neoplasias/genética , Proteínas/genética , Fator de Crescimento Transformador beta/farmacologia , Adenocarcinoma , Sequência de Aminoácidos , Sequência de Bases , Diferenciação Celular/genética , Divisão Celular/genética , Linhagem Celular , Clonagem Molecular , DNA , Humanos , Immunoblotting , Cinética , Dados de Sequência Molecular , Proteínas/fisiologia , Homologia de Sequência , Transcrição Gênica , Transfecção , Células Tumorais CultivadasAssuntos
Glicoproteínas/isolamento & purificação , Substâncias de Crescimento/isolamento & purificação , Peptídeos e Proteínas de Sinalização Intercelular , Acetato de Tetradecanoilforbol/farmacologia , Anfirregulina , Divisão Celular/efeitos dos fármacos , Linhagem Celular , Cromatografia em Gel/métodos , Cromatografia Líquida de Alta Pressão/métodos , Meios de Cultura , Replicação do DNA/efeitos dos fármacos , Família de Proteínas EGF , Eletroforese em Gel de Poliacrilamida , Glicoproteínas/farmacologia , Substâncias de Crescimento/farmacologia , Humanos , Peso MolecularRESUMO
A variety of sexually transmitted diseases frequently accompany infection with human papillomavirus and stimulate inflammation of the cervical mucosa. Inflammation and cell injury cause release of proinflammatory cytokines, which in turn might regulate growth of human papillomavirus-infected cells. This study compared the interaction of the proinflammatory cytokine, interleukin-6 (IL-6), and its soluble receptor with normal ecto- and endocervical cells, human papillomavirus-immortalized ectocervical cells, and squamous carcinoma-derived cell lines. Proliferation of normal cervical cells was enhanced by IL-6 but inhibited by its soluble receptor. However, both IL-6 and its soluble receptor significantly stimulated growth of the three immortal and four cervical carcinoma-derived cell lines analyzed. Stimulation by IL-6 was dose dependent and was blocked by an antibody that neutralized IL-6 activity. IL-6-mediated proliferation was accompanied by increased expression of RNAs encoding transforming growth factor-alpha and amphiregulin, two epidermal growth factor receptor ligands. Furthermore, growth stimulation by IL-6 was significantly inhibited by antibodies that either blocked signal transduction by the epidermal growth factor receptor or that neutralized transforming growth factor-alpha or amphiregulin activity. Thus, IL-6 stimulates proliferation of human papillomavirus-immortalized cervical cells via an epidermal growth factor receptor-dependent pathway involving autocrine stimulation by transforming growth factor-alpha and amphiregulin.
Assuntos
Divisão Celular/fisiologia , Colo do Útero/citologia , Interleucina-6/fisiologia , Receptores de Interleucina/fisiologia , Neoplasias do Colo do Útero/patologia , Linhagem Celular Transformada , Colo do Útero/patologia , Colo do Útero/virologia , Ensaio de Imunoadsorção Enzimática , Células Epiteliais , Epitélio/patologia , Epitélio/virologia , Feminino , Humanos , Interleucina-6/biossíntese , Papillomaviridae , RNA Mensageiro/biossíntese , Receptores de Interleucina/biossíntese , Receptores de Interleucina-6 , Células Tumorais CultivadasRESUMO
With the availability of the nearly complete genomic sequence of C. elegans, the first multicellular organism to be sequenced, molecular biology has definitely entered the postgenomic era. Annotation of the genomic sequence, which refers to identifying the genes and other biologically relevant sections of the genome, is an important and nontrivial next step. A first-pass annotation will be necessarily incomplete but will drive further biological experiments, which in turn will help to annotate the genome better. Given the scale of the genome sequence analysis, it is clear that the annotation should be automated as much as possible without sacrificing the quality of analysis. In this work, we outline our approach to identifying the protein kinases of C. elegans from the genomic sequence. We describe new tools we have developed for analysis, management and visualization of genomic data. By developing modular and scalable solutions, this study has provided a framework for future analysis of the Drosophila and human genomes.
Assuntos
Caenorhabditis elegans/enzimologia , Biologia Computacional , Proteínas Quinases/genética , Animais , Sistemas de Gerenciamento de Base de Dados , SoftwareRESUMO
Caenorhabditis elegans should soon be the first multicellular organism whose complete genomic sequence has been determined. This achievement provides a unique opportunity for a comprehensive assessment of the signal transduction molecules required for the existence of a multicellular animal. Although the worm C. elegans may not much resemble humans, the molecules that regulate signal transduction in these two organisms prove to be quite similar. We focus here on the content and diversity of protein kinases present in worms, together with an assessment of other classes of proteins that regulate protein phosphorylation. By systematic analysis of the 19,099 predicted C. elegans proteins, and thorough analysis of the finished and unfinished genomic sequences, we have identified 411 full length protein kinases and 21 partial kinase fragments. We also describe 82 additional proteins that are predicted to be structurally similar to conventional protein kinases even though they share minimal primary sequence identity. Finally, the richness of phosphorylation-dependent signaling pathways in worms is further supported with the identification of 185 protein phosphatases and 128 phosphoprotein-binding domains (SH2, PTB, STYX, SBF, 14-3-3, FHA, and WW) in the worm genome.
Assuntos
Caenorhabditis elegans/enzimologia , Modelos Biológicos , Proteínas Quinases/metabolismo , Transdução de Sinais , Animais , Proteínas Fúngicas , Humanos , Proteínas Quinases/classificação , Saccharomyces cerevisiae/enzimologia , Especificidade da EspécieRESUMO
We have isolated a cDNA clone coding for the simian TGF-beta precursor from a cDNA library made from an African green monkey cell line, BSC-40. The deduced amino acid sequence of the mature simian TGF-beta shows 100% homology with that of the human TGF-beta: Strong sequence homology was found between the precursor regions of the human and simian proteins with only five amino acid changes out of 278. The simian (and murine) precursor sequence was found to code for one less amino acid residue than the human. The implications of these results with respect to the isolation of a growth inhibitor from these cells functionally related to the TGF-beta molecule are discussed.