RESUMEN
Recent studies have shown that hyperinsulinemia may increase the cancer risk. Moreover, many tumors demonstrate an increased activation of IR signaling pathways. Phosphatidylinositol 3-kinase (PI3K) is necessary for insulin action. In epithelial cells, which do not express GLUT4 and gluconeogenic enzymes, insulin-mediated PI3K activation regulates cell survival, growth, and motility. Although the involvement of the regulatory subunit of PI3K (p85α (PI3K)) in insulin signal transduction has been extensively studied, the function of its N-terminus remains elusive. It has been identified as a serine (S83) in the p85α (PI3K) that is phosphorylated by protein kinase A (PKA). To determine the molecular mechanism linking PKA to insulin-mediated PI3K activation, we used p85α (PI3K) mutated forms to prevent phosphorylation (p85A) or to mimic the phosphorylated residue (p85D). We demonstrated that phosphorylation of p85α (PI3K)S83 modulates the formation of the p85α (PI3K)/IRS-1 complex and its subcellular localization influencing the kinetics of the insulin signaling both on MAPK-ERK and AKT pathways. Furthermore, the p85α (PI3K)S83 phosphorylation plays a central role in the control of insulin-mediated cell proliferation, cell migration, and adhesion. This study highlights the p85α (PI3K)S83 role as a key regulator of cell proliferation and motility induced by insulin in MCF-7 cells breast cancer model.
Asunto(s)
Movimiento Celular , Fosfatidilinositol 3-Quinasa Clase Ia/metabolismo , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Insulina/metabolismo , Movimiento Celular/efectos de los fármacos , Proliferación Celular , Supervivencia Celular , Humanos , Insulina/farmacología , Proteínas Sustrato del Receptor de Insulina/metabolismo , Espacio Intracelular/metabolismo , Células MCF-7 , Fosforilación , Unión Proteica , Transporte de Proteínas , Transducción de Señal/efectos de los fármacosRESUMEN
Insulin-like growth factor II (IGF-II) is a mitogen for many cell types and an important modulator of muscle growth and differentiation. IGF-II gene is prevalently expressed during prenatal development and its gene activity is regulated by genomic imprinting, in that the allele inherited from the father is active and the allele inherited from the mother is inactive in most normal tissues. IGF-II expression is activated in several types of human neoplasms and an alteration of IGF-II imprinting has been described in Beckwith-Wiedemann syndrome and Wilms' tumor. Here we show that monoallelic expression of IGF-II gene is conserved in normal adult muscle tissue whereas two or more copies of active IGF-II alleles, arising by either relaxation of imprinting or duplication of the active allele, are found in 9 out of 11 (82%) rhabdomyosarcomas retaining heterozygosity at 11p15, regardless of the histological subtype. Since IGF-II has been indicated as an autocrine growth factor for rhabdomyosarcoma cells, these findings strongly suggest that acquisition of a double dosage of active IGF-II gene is an important step for the initiation or progression of rhabdomyosarcoma tumorigenesis. Among different types of muscle tumors, relaxation of imprinting seems to arise prevalently in rhabdomyosarcomas, since we have detected only one case of partial reactivation of the maternal IGF-II allele out of 7 leiomyosarcomas tested.
Asunto(s)
Alelos , Regulación Neoplásica de la Expresión Génica , Impresión Genómica , Factor II del Crecimiento Similar a la Insulina/genética , Proteínas Musculares/genética , Músculos/metabolismo , Proteínas de Neoplasias/genética , Rabdomiosarcoma/genética , Neoplasias de los Tejidos Blandos/genética , Adulto , Secuencia de Bases , Niño , Femenino , Genes , Humanos , Factor II del Crecimiento Similar a la Insulina/biosíntesis , Masculino , Datos de Secuencia Molecular , Proteínas Musculares/biosíntesis , Proteínas de Neoplasias/biosíntesisRESUMEN
Rat thyroid cells (FRTL5), transfected with the sequence coding for rat insulin-like growth factor II (IGF-II) presented mRNA specific for the transfected IGF-II in most of the clones obtained (Tr clones). Tr7 and Tr12 cells maintained their ability to respond to the mitogenic effect of thyrotropin (TSH), while either exogenous IGF-I or IGF-II or insulin failed to stimulate their proliferation. In the absence of exogenous mitogens the Tr7 and Tr12 clones vigorously incorporated [3H]thymidine into DNA. This activity was significantly inhibited by sm1.2, a monoclonal antibody against rat IGF-II. Tr7 and Tr12 clones possess type I IGF receptors, known to mediate the mitogenic effect of IGF-II, with affinity similar to those present on the membrane of the parental cells but with reduced capacity. Finally, media conditioned by Tr7 and Tr12 increase basal thymidine incorporation in quiescent FRTL5 cells and amplify that induced by TSH. Endogenous IGFs may play an important role in the regulation of thyroid cell proliferation by modulating the mitogenic effect of TSH and by supporting TSH-independent growth.
Asunto(s)
Factor II del Crecimiento Similar a la Insulina/farmacología , Glándula Tiroides/efectos de los fármacos , Animales , División Celular/efectos de los fármacos , Células Cultivadas , Medios de Cultivo/farmacología , Replicación del ADN/efectos de los fármacos , Sinergismo Farmacológico , Insulina/farmacología , Factor I del Crecimiento Similar a la Insulina/farmacología , Factor II del Crecimiento Similar a la Insulina/genética , Mitosis/efectos de los fármacos , Ratas , Proteínas Recombinantes/farmacología , Glándula Tiroides/citología , Tirotropina/farmacología , TransfecciónRESUMEN
A new member of the ras genes superfamily was isolated from a cDNA library derived from a rat liver cell line (BRL-3A). The predicted 201 amino acids ras-like protein shows 30-35% homology with other members of the ras and ras-related gene products so far described. Conserved features include the GTP-binding and hydrolysis domains and the carboxyl terminal cysteine residues. A protein of the expected size (Mr 23,000) was synthesized in an in vitro transcription-translation system. The BRL-ras gene is present in single copy in the rat genome and is ubiquitously expressed at high levels in all tissues and cell lines examined.
Asunto(s)
Genes ras , Proteínas Proto-Oncogénicas/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Línea Celular , Clonación Molecular , Hígado , Datos de Secuencia Molecular , Conformación Proteica , Proteínas Proto-Oncogénicas p21(ras) , Ratas , Homología de Secuencia de Ácido NucleicoRESUMEN
The rat insulin-like growth factor II (rIGF-II) gene, which exists as a single copy in the genome, is expressed as a multitranscript family of mRNA molecules ranging in size from 4.6 to 1 kilobases. Part of this heterogeneity can be ascribed to the presence of two different promoters, each transcribing alternative 5'-noncoding regions which are spliced to common coding exons. In the present study we use a combination of DNA sequence analysis of the gene, mapping of the mRNA molecules by Northern analysis and ribonuclease protection experiments, and DNA sequence analysis of cDNA clones complementary to different regions of the genome to establish the structure of several rIGF-II mRNA species. These results indicate that RNA heterogeneity also arises from the use of different polyadenylation sites. In addition, a variant 2 kilobases RNA was observed that was colinear with the distal 1700 base pairs of the 3147 base pair long exon 3, and may arise by alternative RNA splicing. These posttranscriptional modifications of RNAs arising from the rIGF-II transcription unit may generate molecules with different functional potential.
Asunto(s)
Factor II del Crecimiento Similar a la Insulina/genética , Empalme del ARN , Somatomedinas/genética , Animales , Secuencia de Bases , Northern Blotting , Factor II del Crecimiento Similar a la Insulina/análisis , Datos de Secuencia Molecular , Regiones Promotoras Genéticas , Ratas , Ratas Endogámicas , Ribonucleasas/análisis , Ribonucleasas/genética , Transcripción GenéticaRESUMEN
Insulin-like growth factor II (IGF-II) is a mitogenic polypeptide present in rat plasma at high levels during fetal and early postnatal life and is believed to play an important, although as yet undefined, role in fetal development. Both in humans and rats, expression of the IGF-II gene results in the appearance of several mRNA species. In the present study, cDNA and synthetic oligonucleotide probes were used to isolate and characterize the rat IGF-II gene from genomic libraries. The rat IGF-II gene extends over 12 kilobase pairs and contains two 5'-noncoding exons and three protein-coding exons. The two 5' exons represent alternative 5' regions of different mRNA molecules and are expressed from two distinct promoters. The two promoters are transcribed with different efficiencies but exhibit similar tissue-specific expression and regulation with developmental age.
Asunto(s)
Genes , Factor I del Crecimiento Similar a la Insulina/genética , Regiones Promotoras Genéticas , ARN Mensajero/genética , Somatomedinas/genética , Transcripción Genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Clonación Molecular , ADN/metabolismo , Enzimas de Restricción del ADN , Hibridación de Ácido Nucleico , Ratas , Distribución TisularRESUMEN
Insulin-like growth factor II (IGF-II) is a mitogenic polypeptide that is thought to play a role in fetal growth and development. To study the hormonal and developmental regulation of IGF-II gene expression, we have isolated a cDNA clone for rat IGF-II (rIGF-II) from a 12S [1.2-kilobase-pair (kbp)] fraction of mRNA from a rat liver cell line (BRL-3A) that directs the cell-free synthesis of pre-pro-rIGF-II. In the present study, the rIGF-II probe was used to determine the size of IGF-II RNA. Surprisingly, in BRL-3A cells and in neonatal liver, the probe hybridized under stringent conditions 10-20 times more strongly to a larger (4 kbp) RNA than to 1.2-kbp RNA. The 4-kbp RNA is almost exclusively cytoplasmic and is colinear with a 551-base fragment of the rIGF-II cDNA insert containing coding and 3' noncoding regions. The 4-kbp and 1.2-kbp RNA species are regulated coordinately with developmental age, being high in liver from neonatal rats but not detectable in liver from older animals, suggesting that both IGF-II mRNA species arise from a single primary transcript by alternative RNA processing. Although oligodeoxynucleotide hybridization and S1 nuclease protection experiments suggest that the 4-kbp RNA contains an intact protein-coding region, fractions enriched in 4-kbp RNA do not direct the translation of pre-pro-rIGF-II in vitro. This may indicate that the 4-kbp RNA specifies an altered protein product that has not yet been recognized, or alternatively that it contains a normal protein-coding region but requires further RNA processing to be activated for translation.
Asunto(s)
Factor II del Crecimiento Similar a la Insulina/genética , Somatomedinas/genética , Animales , Compartimento Celular , Línea Celular , Sistema Libre de Células , Clonación Molecular , ADN/genética , Regulación de la Expresión Génica , Hígado/embriología , Hígado/fisiología , Peso Molecular , Hibridación de Ácido Nucleico , Biosíntesis de Proteínas , ARN Mensajero/genética , Ratas , Transcripción GenéticaRESUMEN
We present evidence that the fos oncogene encodes a transcriptional trans-activation function. trans-activation was assayed by cotransfection into NIH 3T3 mouse fibroblasts of v-fos DNA containing plasmids together with a plasmid containing a test promoter. Three v-fos DNAs were used: (i) pFBR-1, a plasmid containing the FBR proviral sequences; (ii) pFBJ-2, a plasmid harboring the FBJ proviral sequences; (iii) pMF-J, a plasmid containing the FBJ fos sequences linked to a mouse metallothionein promoter. Each of the three v-fos DNA plasmids stimulated the expression of a cotransfected chimeric gene consisting of a promoter segment of the mouse alpha 1(III) collagen gene linked to the gene for chloramphenicol transacetylase. In similar experiments the v-fos gene also stimulated the long terminal repeat promoter of Rous sarcoma virus (RSV) but neither the early promoter of simian virus 40 nor the beta-actin promoter. Evidence that the trans-activation function is specified by the v-fos coding sequences comes from the fact that a frameshift mutation in the v-fos coding sequence inhibits the trans-activation. Two mutations that map around nucleotide -100 in the RSV promoter do not respond to cotransfection with v-fos, whereas other mutations respond like the wild-type RSV promoter. These experiments suggest that the v-fos gene either encodes or induces an activator of transcription that recognizes specific sequences in promoters.
Asunto(s)
Virus del Sarcoma Aviar/genética , Colágeno/genética , Oncogenes , Regiones Promotoras Genéticas , Factores de Transcripción/genética , Transcripción Genética , Animales , Línea Celular , Clonación Molecular , Regulación de la Expresión Génica , Ratones , Proteínas Oncogénicas Virales/genética , Secuencias Repetitivas de Ácidos NucleicosRESUMEN
Insulin-like growth factor-I (IGF-I) and IGF-II are mitogenic polypeptides of relative molecular mass (Mr) approximately 7,500 isolated from human plasma each containing four peptide domains in a single chain and identical at more than 60% of their amino acid loci. The B- and A-domains of the IGFs are approximately 40% identical to the B- and A-chains of human insulin. IGF-I and IGF-II have similar in vitro biological activities and receptor reactivity, but are immunologically distinct. IGF-I appears to mediate the effects of growth hormone on cartilage to promote skeletal growth whereas IGF-II may have a special role in fetal development and in the central nervous system. To investigate the in vivo role of IGF-II, we have studied IGF-II biosynthesis in the BRL-3A rat liver cell line. BRL-3A cells synthesize and secrete a 7,484 Mr protein 93% identical to human IGF-II and representing rat IGF-II (rIGF-II). Rat IGF-II is synthesized as a approximately 22,000 Mr prepro-rIGF-II (ref. 12) from 12 S poly(A)+mRNA. In addition, approximately 20,000 Mr pro-rIGF-II has been identified in lysates of biosynthetically labelled intact BRL-3A cells. We report here the isolation of an almost complete cDNA clone for rIGF-II. Our results indicate that pro-rIGF-II is synthesized as a 156 amino acid peptide precursor (17,619 Mr) containing mature rIGF-II 1-67 at its amino-terminus and an 89-residue carboxy-terminal peptide extension.
Asunto(s)
Sustancias de Crecimiento/genética , Insulina/genética , Péptidos/genética , Somatomedinas/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Clonación Molecular , ADN/genética , Peso Molecular , Precursores de Proteínas/genética , ARN Mensajero/genética , RatasRESUMEN
The DNA of the attenuator region of the histidine operon of Escherichia coli has been transcribed in a purified in vitro system and found to synthesize two major RNA transcripts. The first one, 180 nucleotides long, has been identified as the histidine-specific leader RNA. It contains the coding sequence for the leader peptide [Di Nocera, P. P., Blasi, F., Di Lauro, R., Frunzio, R. & Bruni, C. B. (1978) Proc. Natl. Acad. Sci. USA 75, 4276-4280] and is terminated at the attenuator site. Termination of transcription at this site is extremely efficient in the in vitro system. The leader RNA also has been detected in vivo in a minicell producer strain transformed with plasmids harboring the regulatory region of the histidine operon of E. coli. A second RNA molecule is synthesized in the in vitro system. It has a divergent direction of transcription with respect to the histidine leader RNA, but its role, if any, in the regulation of the histidine operon remains to be ascertained. The existence of the histidine leader RNA lends support to the regulatory mechanism which postulates that regulation of the histidine operon is dependent on the alternative secondary structures that the leader RNA may assume, depending on whether or not the histidine-rich leader peptide is translated.
Asunto(s)
Escherichia coli/genética , Histidina/genética , Operón , ARN Bacteriano/genética , Transcripción Genética , Secuencia de Bases , Enzimas de Restricción del ADN , Ribonucleasa T1RESUMEN
A restriction fragment has been isolated and its nucleotide sequence determined. This fragment contains sites for RNA polymerase binding, initiation and termination of transcription of the Escherichia coli histidine operon. In vitro transcription of plasmids containing this region generates one single histidine-specific, attenuated, small RNA: the leader RNA. This RNA is more efficiently transcribed when the template DNA is supercoiled. Another promoter was identified on the same fragment of deoxyribonucleic acid by in vitro transcription, DNA sequencing and RNA polymerase binding. Both promoters, transcribing in opposite direction, are very A-T rich and are separated by a G-C rich region containing a palyndromic structure.
Asunto(s)
Escherichia coli/genética , Histidina/genética , Operón , Transcripción Genética , Secuencia de Bases , Enzimas de Restricción del ADN , ADN Superhelicoidal/metabolismo , ARN Polimerasas Dirigidas por ADN/metabolismo , Plásmidos , ARN Bacteriano/biosíntesisRESUMEN
A fragment of deoxyribonucleic acid 5,300 base paris long and containing the promoter-proximal portion of the histidine operon of Escherichia coli K-12, has been cloned in plasmid pBR313 (plasmids pCB2 and pCB3). Restriction mapping, partial nucleotide sequencing, and studies on functional expression in vivo and on protein synthesis in minicells have shown that the fragment contains the regulatory region of the operon, the hisG, hisD genes, and part of the hisC gene. Another plasmid (pCB5) contained the hisG gene and part of the hisD gene. Expression of the hisG gene in the latter plasmid was under control of the tetracycline promoter of the pBR313 plasmid. The in vivo expression of the two groups of plasmids described above, as well as their effect on the expression of the histidine genes not carried by the plasmids but present on the host chromosome, has been studied. The presence of multiple copies of pCB2 or pCB3, but not of pCB5, prevented derepression of the chromosomal histidine operon. Possible interpretations of this phenomenon are discussed.
Asunto(s)
Escherichia coli/genética , Histidina/biosíntesis , Operón , Plásmidos , Secuencia de Bases , Clonación Molecular , Enzimas de Restricción del ADN , ADN Bacteriano/análisis , ADN Bacteriano/genética , Genes , Genes Reguladores , Vectores GenéticosRESUMEN
The attenuator region of the histidine operon of Escherichia coli K-12 has a potential coding capacity for two peptides, one of 16 amino acids and another of 30 amino acids. This region is followed by a perfect palindrome of 14 base pairs separated by five nucleotides. A G+C-rich region precedes and follows a possible transcription termination sequence. These features are compatible with a model in which active translation of a leader mRNA interferes with transcription termination, thus causing derepression of the histidine operon. The sequence of the region coding for the hypothetical 16-amino acid peptide is of particular relevance because it indicates the site and a possible mechanism of action of histidyl-tRNAhis in regulating histidine gene expression. Seven contiguous histidine codons are present within this sequence: : formula: (see text)