RESUMO
To identify the site of stimulation of sucrase by a sucrose diet, changes in sucrase-specific activity of jejunal mucosa were studied after introduction of sucrose diet to carbohydrate-deprived rats. Results were correlated with simultaneous changes in villus gradients of sucrase-specific activity. Simultaneous with the introduction of sucrose diet, [(3)H]thymidine (100 muCi) was administered intravenously, and rates of cell migration measured during adaptation to the new diet. After a 72-h fast, rats fed sucrose diet for 6, 12, or 18 h showed no change in sucrase-specific activity in either whole mucosa or villus gradients. However, within 18-24 h after starting a sucrose diet, there was a marked rise in whole mucosal sucrase-specific activity above fasting values (99 +/- 14 vs. 38 +/- 4 muM glucose/min per g protein, P < 0.001) in association with the development of a region of increased activity at the lower villus (154 +/- 22 vs. 60 +/- 9 muM glucose/min per g protein, P < 0.02, but with no change in villus tip activity (56 +/- 5 vs. 46 +/- 8 muM glucose/min per g protein). Similar changes were seen in animals fed 24 h of sucrose diet after a 72-h carbohydratefree diet. Fasted animals fed sucrose diet for 36 h had increased sucrase-specific activity at the villus tip (144 +/- 11 muM glucose/min per g protein) as well as at the lower villus region, and this pattern persisted at 1 wk of sucrose diet. Maximal activity patterns for isomaltase and maltase paralleled those for sucrase, but the villus gradients for lactase were unaffected by sucrose diet. The region of maximal sucrase-specific activity always coincided with or followed the leading edge of radioactivity as determined by liquid scintillation counting. Therefore, sucrose-mediated changes in sucrase activity of the jejunal mucosa in the rat appear to be initiated at the level of the crypt epithelial cell and are expressed after a latent period of 18-24 h during which these cells mature and migrate toward the villus tip.
Assuntos
Mucosa Intestinal/enzimologia , Jejuno/enzimologia , Sacarase/metabolismo , Sacarose/metabolismo , Animais , Movimento Celular , Carboidratos da Dieta/administração & dosagem , Mucosa Intestinal/citologia , Jejuno/citologia , Masculino , Ratos , Fatores de TempoRESUMO
Adenyl cyclase activity in intestinal membranes has been studied during development in the rabbit fetus from fetal day 17 to 10 days postnatally and in the human fetus from the 10th to the 17th wk of gestation. In the rabbit, the enzyme was already present by fetal day 17 and showed a fourfold peak rise in specific activity by 22 days. By 28 days, the specific activity had fallen toward adult levels and remained constant throughout gestation and the 1st wk of life. Fluoridestimulated activity showed a similar curve, and was 2.5-5 times the basal values. Activities in jejunum and ileum were comparable at all time points studied. Phosphodiesterase activity did not change during gestation. When fetal intestinal segments were incubated in vitro with purified cholera enterotoxin, adenyl cyclase activity in subsequently prepared membranes was increased two- to threefold. This level was not regularly further elevated by fluoride ion. Lithium ion inhibited both the basal and fluoride-stimulated enzyme activity in membranes prepared from rabbit fetuses at term. Lactase activity (reflecting the development of the microvilli) in either whole intestinal homogenates or in the membrane fractions showed a differnet pattern of development, with a rise beginning on fetal day 24 and a plateau just after birth. In intestinal membranes prepared from human fetuses, the activity of both basal and fluoride-stimulated adenyl cyclase tripled from the 10th to the 17th wk of gestation. The data both in the rabbit and in man show that intestinal adenyl cyclase is capable of responding to cholera enterotoxin quite early in gestation. In the rabbit, this occurs before the time of appearance or ville or of an enzyme marker (lactase) for microville. The results support the concept that adenyl cyclase is present in plasma membrane other than the brush border.
Assuntos
Adenilil Ciclases/metabolismo , Cólera , Enterotoxinas/farmacologia , Íleo/enzimologia , Jejuno/enzimologia , Animais , Animais Recém-Nascidos , Feminino , Feto/metabolismo , Fluoretos/farmacologia , Galactosidases/metabolismo , Idade Gestacional , Humanos , Íleo/efeitos dos fármacos , Técnicas In Vitro , Mucosa Intestinal/crescimento & desenvolvimento , Mucosa Intestinal/metabolismo , Jejuno/efeitos dos fármacos , Lítio/farmacologia , Diester Fosfórico Hidrolases/metabolismo , Gravidez , Coelhos , Estimulação QuímicaRESUMO
The molecular basis of adult human "lactase deficiency" has long been a subject of controversy. To address this issue, small intestinal biopsies from orienta, black, and white patients were analyzed. Adjacent samples were assayed for lactase and sucrase specific activities and the sucrase/lactase ratio (high ratio signifies lactase deficiency), and the results were compared to lactase steady-state mRNA levels detected in Northern blots probed with a human lactase mDNA. All oriental patients had high ratios and no detectable lactase mRNA. Four black patients had a similar pattern; two with low ratios had detectable mRNA. The group of white patients displayed a range of findings, from high ratio/no mRNA to low ratio/considerable mRNA. Elevated levels of lactase mRNA always correlated with the presence of elevated levels of lactase enzyme activity, suggesting that the difference in levels of adult human intestinal lactase activity among racial groups may be regulated at the level of gene transcription.
Assuntos
beta-Galactosidase/genética , Adulto , Sequência de Bases , Humanos , Lactase , Dados de Sequência Molecular , RNA Mensageiro/análise , Transcrição Gênica , beta-Galactosidase/análiseRESUMO
Four transformed cell lines were established from cultures of human embryo kidney (HEK) cells microinjected or transfected with cloned adenovirus 12 (Ad12) EcoRI-C DNA (0 through 16.5 map units of the left-hand end of the viral genome). Each cell line showed a different growth pattern. Southern blotting demonstrated that all of the cell lines contained Ad12-specific DNA sequences, but in the microinjected isolates these were at a much lower copy number than in the transfected isolate. Two cell lines (Ad12 HEK 1 and 3) appeared to contain tandemly repeated Ad12 EcoRI-C DNA fragments. Immunoprecipitation and Western blotting confirmed that Ad12 early region 1 (E1) proteins were being expressed by all four of the transformed cell lines, but indicated that E1A polypeptide expression was considerably less than E1B polypeptide expression. All of the Ad12-transformed HEK cell lines were tumorigenic when inoculated intracranially into athymic nude mice.
Assuntos
Adenovírus Humanos , Transformação Celular Viral , Animais , Sequência de Bases , Divisão Celular , Linhagem Celular , DNA Viral/isolamento & purificação , Embrião de Mamíferos , Humanos , Rim , Camundongos , Neoplasias Experimentais/etiologia , Proteínas Virais/isolamento & purificaçãoRESUMO
In this paper, we describe for the first time the transformation of normal rat cells by DNA equivalent to adenovirus type 12 Early Region 1 (E1A). This DNA was 30-fold less efficient at transformation than DNA encoding the entire E1 region. Those established lines expressing a full complement of adenovirus type 12 E1 proteins were phenotypically indistinguishable from adenovirus type 12 virus-transformed cell lines. However, cell lines produced by plasmids carrying subgenomic fragments of E1 DNA and therefore not expressing E1B Mr 52,000 protein took longer to establish and produced tumors only after a protracted latent period. A Giemsa-banding study showed that adenovirus transformation can occur without disruption of the normal rat karyotype.
Assuntos
Adenovírus Humanos/genética , Transformação Celular Neoplásica , Transformação Celular Viral , DNA Viral/análise , Genes Virais , Plasmídeos , Animais , Sequência de Bases , Encéfalo/patologia , Linhagem Celular , Rim/patologia , Camundongos , Ratos , Proteínas Virais/análiseRESUMO
Using radioimmunoassays we have confirmed earlier reports that the adenovirus 12 E1B 19K protein is expressed on the surface of transformed and infected human cells. Immunoprecipitation and radioimmunoassay of Ad 12 infected primary human embryo kidney cells demonstrated a considerable time lag in the translocation of the 19K protein to the external membrane after its expression within the cell. We have used antisera raised against synthetic octapeptides to detect and map the orientation of the polypeptide in the plasma membrane. The data obtained suggest that the C-terminus of the protein is exposed on the outside of the cell whereas the N-terminus is free in the cytoplasm. The predicted secondary structure of the 19K protein demonstrates a hydrophobic region which is highly conserved amongst Ad2, 5, 7 and 12 (amino acids 83-100 in Ad12) and we conclude that this forms the transmembrane domain. These observations are discussed in relation to the known functions of the Ad12 E1B 19K protein.
Assuntos
Infecções por Adenoviridae/metabolismo , Transformação Celular Viral , Proteínas Oncogênicas Virais/análise , Acilação , Proteínas Precoces de Adenovirus , Linhagem Celular Transformada , Membrana Celular/análise , Humanos , Proteínas Oncogênicas Virais/imunologia , Conformação Proteica , Linfócitos T Citotóxicos/imunologia , Tripsina/farmacologiaRESUMO
One dimensional [1H] and [31P] nuclear magnetic resonance (NMR) studies have been carried out on purified wild type and mutant (Gly-12----Asp) N-ras protein expressed at high level in E. coli. Both proteins were isolated as stable 1:1 molar complexes with GDP with the upper limit for the first order rate constant for nucleotide dissociation 3 x 10(-4)s-1. From observation of the [31P] NMR spectrum after the addition of GTP it was concluded that the rate of nucleotide hydrolysis is appreciably greater than that of nucleotide exchange. Differences in the [31P] spectra of mutant and wild type proteins suggest that the mutation has a direct influence on the catalytic step. [1H] NMR spectra obtained for both mutant and wild type p21 were consistent with proteins of considerable stability and the addition of urea to concentrations of 4M appeared to cause little disruption in secondary structure. Additionally, the protein environment of the bound nucleotide remained well defined in the presence of a number of added reagents and over the pH range 5.8-9.5. The data are discussed in the light of the known crystal structure for H-ras p21 and indicate that the transforming mutation of aspartate for glycine-12 results in structural perturbations near the nucleotide binding site.
Assuntos
Nucleotídeos de Guanina/metabolismo , Guanosina Difosfato/metabolismo , Guanosina Trifosfato/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Sítios de Ligação , GTP Fosfo-Hidrolases/metabolismo , Histidina , Concentração de Íons de Hidrogênio , Espectroscopia de Ressonância Magnética , Ligação Proteica , Conformação Proteica , Proteínas Proto-Oncogênicas p21(ras) , Relação Estrutura-AtividadeRESUMO
Using a transformed human embryo retinal cell line (Ad 2 E1A + N-ras HER 313A), which expresses activated N-ras p21 at a high level, we have examined various properties of the protein. Immunoprecipitated p21 has covalently bound lipid attached to it through an alkali-labile thioester bond. This incorporation of fatty acid into the protein proceeds in vitro and probably in vivo via a palmitoyl-CoA intermediate and is catalysed by a crude microsomal preparation. A novel purification procedure has been developed for the protein based on its solubility in high concentrations of ethanol. Residual protein impurities were removed by gel filtration in the presence of detergent. Using a membrane preparation from Ad 2 E1A + N-ras HER 313A cells, we have shown that N-ras p21 is firmly anchored in the cell membrane and is not removed by extraction with salts, chelating agents or reducing agents, but is only solubilised by detergents at high concentrations. Exposure of cell membrane preparations and purified N-ras p21 to proteolytic enzymes gives rise to similar degradation patterns. Based on these observations and the known amino acid sequence of p21, it is concluded that attachment to the cell membrane is through the lipid at the C-terminus and not through the incorporation of the polypeptide chain into the lipid bilayer. These results are discussed in relation to the hypothesis that the mode of action of p21 is analogous to that of G proteins.
Assuntos
Genes ras , Proteínas de Membrana/isolamento & purificação , Proteínas Proto-Oncogênicas/isolamento & purificação , Acilação , Compartimento Celular , Linhagem Celular , Detergentes , Proteínas de Ligação ao GTP/isolamento & purificação , Proteínas de Ligação ao GTP/metabolismo , Humanos , Peso Molecular , Peptídeo Hidrolases/metabolismo , Processamento de Proteína Pós-Traducional , Proteínas Proto-Oncogênicas/metabolismoRESUMO
The efficiency of transformation of primary human embryo retinal (HER) cells by the adenovirus E1 region (E1A + E1B) depended on the virus serotype whereas transformation by E1A alone was a rare event regardless of serotype. Activated human c-Ha-ras and N-ras genes co-operated differentially with different E1As for HER transformation but were ineffective without E1A. Ras + E1A co-transformants containing Ad 12 E1A established directly from foci, in contrast to those containing Ad 2 or Ad 5 E1A. A spectrum of activated ras gene expression was found in stable co-transformants with mRNA and protein levels being lower in Ad 12 E1A + N-ras than Ad 2 E1A + N-ras cell lines. Down regulation of E1A transcription in the absence of E1B was found in Ad 2 E1A + Ha-ras transformants only but E1A protein levels were similar to those in Ad 2 E1A + N-ras or Ad 5 E1A + E1B cell lines. HER cell transformants which contained Ad 12 E1A were more tumourigenic than those which contained the Ad 2 or Ad 5 E1A. This unique transformation system shows that stable malignant transformation of primary human cells in vitro is a complex process requiring the combined activities of two or more types of genes.
Assuntos
Adenovírus Humanos/genética , Transformação Celular Viral , Genes ras , Proteínas Oncogênicas Virais/genética , Proteínas Proto-Oncogênicas/genética , Proteínas Precoces de Adenovirus , Adenovírus Humanos/imunologia , Células Cultivadas , Regulação da Expressão Gênica , Humanos , Proteínas Oncogênicas Virais/imunologia , Retina/citologia , Sorotipagem , Transcrição GênicaRESUMO
Chromosomal translocation and subsequent de-regulation of the c-myc proto-oncogene are considered to be critical events in the multi-stage evolution of Burkitt lymphoma (BL). It is widely accepted that Myc protein functions as a competence factor for proliferation. However, recent studies indicate that it can also act in some cell types as a regulator of apoptosis. BL cell populations display a high frequency of apoptosis in vivo, a property which is also readily demonstrable in vitro in group I BL cell lines. Such lines are known to retain the cell surface marker characteristics of the parental tumour cells and, in the case of Epstein-Barr virus-positive tumours, their restricted viral protein expression. We have shown previously that apoptosis in a group I BL cell line is inhibited by interferon (IFN)-alpha. Here we show that IFN-alpha-mediated suppression of apoptosis in group I BL cells corresponds temporally with inhibition of Myc protein levels. Furthermore, inhibition of Myc expression following treatment with c-myc anti-sense oligonucleotides markedly enhanced survival of group I BL cells. These results indicate that, whilst c-myc may facilitate cycling of tumour cells in which it is de-regulated, it also stimulates their apoptosis.
Assuntos
Apoptose/fisiologia , Linfoma de Burkitt/patologia , Proteínas Proto-Oncogênicas c-myc/fisiologia , Sequência de Bases , Linfoma de Burkitt/fisiopatologia , Divisão Celular/fisiologia , Humanos , Interferon-alfa/farmacologia , Ionomicina/farmacologia , Dados de Sequência Molecular , Oligonucleotídeos Antissenso , Proto-Oncogene Mas , Proteínas Proto-Oncogênicas/farmacologia , Proteínas Proto-Oncogênicas c-bcl-2 , Células Tumorais CultivadasRESUMO
The adenovirus early region 1B (Ad E1B) genes have no transforming capability of their own but markedly increase the transformation frequency of Ad E1A following co-transfection into mammalian cells. The larger E1B proteins of both Ad2/5 and Ad12 bind to p53 and inhibit its ability to transcriptionally activate other genes. We have previously demonstrated that synthetic peptides identical to the binding sites for p53 on both the Ad2 and Ad12 E1B proteins will disrupt the interaction in vivo and in vitro. In the work presented here we have examined the effects of complex dissociation on Ad E1-transformed human cells. It has been shown, using confocal microscopy, that when the peptide identical to the p53 binding site was added to Ad5 E1-transformed cells it initally located in the cytoplasmic dense bodies where it caused disruption of the p53/E1B complex. Peptide and p53 then translocated to the nucleus. In Ad12 E1-transformed cells the peptide localized in the nucleus directly and there caused a reorganization of p53 staining from a highly organized, 'flecked' distribution to one in which nuclear staining was homogeneous and diffuse. Peptides added to either Ad5 E1 or Ad12 E1 transformed cells resulted in the release of transcriptionally active p53. Interestingly, the level of p53 then fell presumably as a result of proteasomal action - this was probably a reflection of the short half-life of 'free' (i.e. dissociated) p53 compared to that of the bound protein. Free p53 did not cause apoptosis in target cells probably due to the presence of the smaller (19K) E1B proteins. However, addition of peptide leads to a significant reduction in cell growth rate. We have further demonstrated that a significant proportion of those cells which had taken up peptide had ceased DNA synthesis, probably due to a p53-induced cell cycle arrest. The role of the larger EIB protein during transformation is considered in view of these data.
Assuntos
Adenoviridae/fisiologia , Proteínas E1B de Adenovirus/fisiologia , Transformação Celular Neoplásica , Proteína Supressora de Tumor p53/fisiologia , Sequência de Aminoácidos , Animais , Apoptose , Divisão Celular , Linhagem Celular , Humanos , Dados de Sequência MolecularRESUMO
Mutation of the retinoblastoma (Rb) gene is found frequently in human sarcomas, lung, bladder and breast carcinomas and is the molecular basis for hereditary predisposition to retinoblastoma. The Rb protein is a nuclear phosphoprotein that is differentially phosphorylated during the cell cycle. Its precise function is unknown but it has been suggested that it may act as a transcriptional regulator or as a regulator of cellular DNA synthesis. The Rb protein forms specific complexes with the oncogenes of three different groups of DNA tumour viruses. We have prepared a new monoclonal antibody to the Rb protein and used it to establish sensitive immunoassays for Rb complexed to T antigen. In SV40-infected and transformed cells these assays showed that Rb enters a trimolecular complex containing p53, Rb and T. A large panel of human tumour cell lines was tested for expression, cellular localization and T-binding activity of Rb using the new antibody.
Assuntos
Anticorpos Monoclonais/isolamento & purificação , Antígenos Transformantes de Poliomavirus/metabolismo , Proteína do Retinoblastoma/biossíntese , Proteína Supressora de Tumor p53/metabolismo , Antígenos Transformantes de Poliomavirus/análise , Linhagem Celular Transformada , Núcleo Celular/química , Hibridomas/metabolismo , Imunoensaio/métodos , Testes de Precipitina , Proteína do Retinoblastoma/análise , Proteína Supressora de Tumor p53/análiseRESUMO
Plasma membrane-enriched fractions were prepared from human embryonic retinal cells transformed with either adenovirus E1A and oncogenic ras DNA, or E1A and E1B DNA. Ras comprised 5-10% of the membrane protein from the E1A/ras transformed cells, whereas the membranes from E1A/E1B transformed cells did not overexpress Ras. The membranes from E1A/ras cells contained MAP kinase kinase kinase (MAPKKK) activity, even after washing in 0.5 M NaCl, whereas the membranes from E1A/E1B cells did not. Neither membrane fraction contained MAP kinase kinase or MAP kinase activity after washing with 0.5M NaCl. Immunoblotting experiments revealed about 10-fold more c-Raf in the membranes from E1A/ras cells than from E1A/E1B cells, and 50-60% of the MAPKKK activity in Triton X100-solubilised membranes from E1A/ras cells was immunoprecipitated with anti-Raf antibodies. A striking enrichment of c-Raf in the plasma membranes of E1A/ras cells was also demonstrated by immunocytochemistry, where it was co-localized with Ras. The MAPKKK activity in E1A/ras membranes was unaffected by incubation with protein phosphatases or by inclusion of protein phosphatase inhibitors during isolation, nor was it activated by GTP-Ras or inhibited by GDP-Ras. The results support the view that Ras and c-Raf interact with one another, but that neither c-Raf phosphorylation nor its interaction with GTP-Ras are alone sufficient for activation. The identification of MAPKKK activity in the membranes of ras-transformed cells may prove useful in elucidating the mechanism by which Raf is activated by Ras.
Assuntos
Transformação Celular Neoplásica , Genes ras , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas/análise , Retina/enzimologia , Sequência de Aminoácidos , Linhagem Celular Transformada , Membrana Celular/enzimologia , Humanos , MAP Quinase Quinase Quinases , Dados de Sequência Molecular , Proteínas Proto-Oncogênicas c-rafRESUMO
Bcl-2 can inhibit apoptosis induced by a variety of stimuli, including radiation and its presence in tumour cells would be expected to indicate poor prognosis. Bcl-2-expressing tumours, however, are often low-grade and highly responsive to therapy. To investigate this apparent paradox, we analysed in vitro the responses of Burkitt lymphoma (BL) cells to gamma-irradiation in the presence and absence of Bcl-2. High-level expression of Bcl-2 was shown to promote BL cell survival following irradiation. However, a significant proportion of Bcl-2-rescued cells subsequently underwent apoptosis after an extended period in culture. In addition, in different BL lines, Bcl-2 was found either to promote or to inhibit long-term proliferative activity following gamma-irradiation. This differential regulation of proliferation correlated both with differential effects of Bcl-2 on the cell cycle and with differences in p53 status. Thus, by one week after irradiation, BL cells expressing only wild-type p53 (wt/wt) had arrested in G1, whereas those with a mutant allele (wt/mu) were arrested in all phases of the cell cycle. The proportion of Bcl-2-rescued cells that subsequently underwent apoptosis was reduced by ligation of CD40 at the time of irradiation in wt/wt BL cells, but not in wt/mu cells. CD40-ligation reduced both G1-arrest and apoptosis in parallel. These results indicate that, whilst Bcl-2 can delay apoptosis in BL cells following gamma-irradiation, the protein can also cause growth-arrest and thereby promote apoptosis. Long-term survival following Bcl-2-mediated rescue of gamma-irradiated cells may depend on p53 status and require additional death-repressing or growth-promoting signals.
Assuntos
Linfoma de Células B/patologia , Proteínas Proto-Oncogênicas c-bcl-2/fisiologia , Antígenos CD40/metabolismo , Divisão Celular/fisiologia , Divisão Celular/efeitos da radiação , Sobrevivência Celular/fisiologia , Sobrevivência Celular/efeitos da radiação , Raios gama , Humanos , Ligação Proteica , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Células Tumorais CultivadasRESUMO
Previous studies have established that adenovirus 2/5 early region 1B (Ad E1B) 58K protein binds p53 strongly and co-localizes with it to cytoplasmic dense bodies whilst the homologous Ad12E1B54K protein binds only weakly and co-localizes primarily to the nucleus in Ad12E1 transformed cells. We have used these properties of the E1B proteins from different viral serotypes to map the p53 binding site on the Ad2/5 protein. A set of chimaeric genes was constructed containing different proportions of the Ad12 and Ad2E1B DNA. These, together with Ad12E1A and E1B19K DNA, were transfected into baby rat kidney cells and transformed lines isolated. From an examination of the properties of these Ad12/Ad2E1B fusion proteins in co-immunoprecipitation and subcellular localization experiments it has been concluded that the p53 binding site on Ad2E1B58K protein lies between amino acids 216 and 235 and that the homologous region on Ad12E1B54K protein also binds p53. In addition, a unique nuclear localization signal is located on Ad12E1B54K between residues 228 and 239. We suggest that primary structure differences in these regions of the Ad2 and Ad12E1B proteins are responsible for the different subcellular localizations in AdE1 transformants.
Assuntos
Proteínas E1B de Adenovirus/química , Núcleo Celular/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Proteínas Virais de Fusão/química , Adenoviridae/química , Adenoviridae/genética , Proteínas E1B de Adenovirus/metabolismo , Sequência de Aminoácidos , Animais , Sítios de Ligação , Linhagem Celular , Humanos , Dados de Sequência Molecular , Fragmentos de Peptídeos/metabolismo , Ratos , Transfecção , Proteínas Virais de Fusão/metabolismoRESUMO
Using a Burkitt lymphoma cell line to model human B-cell apoptosis in vitro, we observed that crosslinking, by antibody, of cell surface immunoglobulin induced G1 growth-arrest followed by apoptosis. By contrast, cells treated with the Ca(2+)-ionophore, ionomycin, generated apoptotic signals in G2/M as well as in G1. Both ionomycin and anti-immunoglobulin treatment induced rapid dephosphorylation of Rb prior to apoptosis. Apoptosis was repressed following exposure to CD40-ligand and was accompanied by hyperphosphorylation of Rb and cell-cycle progression but not Bcl-2 expression. Expression of Bcl-2 protein in stable bcl-2-transfectants, also resulted in repression of apoptosis and anti-immunoglobulin-treated cells no longer underwent growth-arrest. In Bcl-2-expressing cells in which apoptosis was repressed, Rb remained hyperphosphorylated, even during G1-arrest induced by ionomycin. TGF beta treatment of Bcl-2-expressing cells induced G1-arrest, de-phosphorylation of Rb and apoptosis. These results suggest that the functional activity of Bcl-2 in B-lymphoma cells is dependent upon, or leads to, sustained hyperphosphorylation of Rb and that Rb hyperphosphorylation can be uncoupled from cell-cycle progression.
Assuntos
Apoptose/efeitos dos fármacos , Apoptose/fisiologia , Linfócitos B/patologia , Linfoma de Burkitt/patologia , Glicoproteínas de Membrana/farmacologia , Proteínas Proto-Oncogênicas/farmacologia , Proteína do Retinoblastoma/fisiologia , Anticorpos Anti-Idiotípicos/farmacologia , Ligante de CD40 , Ciclo Celular/fisiologia , Fase G1/efeitos dos fármacos , Fase G1/fisiologia , Humanos , Ionomicina/farmacologia , Ionóforos/farmacologia , Fosforilação , Proteínas Proto-Oncogênicas/fisiologia , Proteínas Proto-Oncogênicas c-bcl-2 , Proteína do Retinoblastoma/metabolismo , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologia , Células Tumorais CultivadasRESUMO
Polyclonal antibodies to the human retinoblastoma gene product (Rb1) have been produced in rats by immunisation with a fusion protein comprising part of Rb1 together with the E. coli beta-Gal sequence. We have used these antibodies in Western blotting studies to screen a number of human foetal tissues and organs and found approximately similar levels of expression of Rb1 in all of them. The protein seems to be somewhat more abundant in some cell lines produced by transfection of human embryo retinal (HER) cells with adenovirus 12 early region 1 (Ad 12 E1), Ad 5 E1, Ad 2 E1A + mutant N-ras or SV40 DNA. Using co-immunoprecipitation followed by Western blotting we have shown that the Rb1 protein binds to Ad 12 E1A 266 and 235 amino acid proteins. This interaction is ionic strength dependent but is unaffected by non-ionic detergent up to a concentration of at least 1%. In Ad 12 infected human cells it appears that less E1A is bound to Rb1 than in the transformants. These results are discussed in view of the known similarities and differences between the amino acid sequences of Ad 12 and Ad 5 E1A proteins.
Assuntos
Adenovírus Humanos/genética , Transformação Celular Neoplásica , Neoplasias Oculares/genética , Expressão Gênica , Oncogenes , Fosfoproteínas/genética , Retinoblastoma/genética , Sequência de Aminoácidos , Animais , Western Blotting , Linhagem Celular , Núcleo Celular/metabolismo , Núcleo Celular/ultraestrutura , Feto , Imunofluorescência , Humanos , Dados de Sequência Molecular , Fosfoproteínas/análise , Proteínas Recombinantes de Fusão/análise , Proteína do Retinoblastoma , Homologia de Sequência do Ácido NucleicoRESUMO
Measurement of colony forming ability following exposure to gamma-rays has been performed on human retinoblasts transformed with either adenovirus 5 or 12 early region 1 DNA, adenovirus early region 1A plus activated N- or H-ras DNA or SV40 DNA. In contrast to recently reported results (M.D. Sklar, 1988, Science, 239, 645-647), we found no general correlation between transformation with activated ras and increased radiation resistance. Similarly, there was no correlation between D0 values and the level of expression of ras p21 in transformed human retinoblasts as determined by liquid competition assay. Indeed, cell lines with very similar D0 values had ras contents varying by up to one hundred fold. Cell lines transformed with SV40 DNA were generally less sensitive to ionising radiation than adenovirus and/or ras transformants, but even so the variation in sensitivity within these encompassed the whole spectrum of values obtained for the ras transformants. It may be interesting to note, however, that two out of the three ras transformants which were least sensitive to gamma-rays were cell lines expressing the highest levels of p21.
Assuntos
Transformação Celular Neoplásica , Genes ras , Tolerância a Radiação , Proteínas Precoces de Adenovirus , Sobrevivência Celular/efeitos da radiação , Raios gama , Humanos , Mutação , Proteínas Oncogênicas Virais/genética , Vírus 40 dos Símios/genéticaRESUMO
Adenovirus early region 1A (Ad E1A) is a multifunctional protein which is essential for adenovirus-mediated transformation and oncogenesis. Whilst E1A is generally considered to exert its influence on recipient cells through regulation of transcription it also increases the level of cellular p53 by increasing the protein half-life. With this in view, we have investigated the relationship of Ad E1A to the proteasome, which is normally responsible for degradation of p53. Here we have shown that both Ad5 and Ad12 E1A 12S and 13S proteins can be co-immunoprecipitated with proteasomes and that the larger Ad12 E1A protein binds strongly to at least three components of the 26S but not 20S proteasome. One of these interacting species has been identified as mammalian SUGI, a proteasome regulatory component which also plays a role in the cell as a mediator of transcription. In vitro assays have demonstrated a direct interaction between Ad12 E1A 13S protein and mouse SUGI. Following infection of human cells with Ad5 wt and Ad5 mutants with lesions in the E1A gene it has been shown that human SUG1 can be co-immunoprecipitated with full-length E1A and with E1A carrying a deletion in conserved region 1 which is the region considered to be responsible for increased expression of p53. We have concluded therefore that Ad EIA binds strongly to SUGI but that this interaction is not responsible for inhibition of proteasome activity. This is consistent with the observation that purified Ad12 E1A inhibits the activity of the purified 20S but not 26S proteasomes. We have also demonstrated that SUGI can be co-immunoprecipitated with SV40 T and therefore we suggest that this may represent a common interaction of transforming proteins of DNA tumour viruses.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal , Proteínas E1A de Adenovirus/metabolismo , Proteínas de Transporte/metabolismo , Cisteína Endopeptidases/metabolismo , Complexos Multienzimáticos/metabolismo , Fatores de Transcrição , ATPases Associadas a Diversas Atividades Celulares , Antígenos Transformantes de Poliomavirus/metabolismo , Linhagem Celular Transformada , Regulação da Expressão Gênica , Genes p53 , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Proteínas com Domínio LIM , Complexo de Endopeptidases do Proteassoma , Ligação Proteica , Células Tumorais CultivadasRESUMO
Sucrase-isomaltase immunoprecipitated from brush border of an intestinal transplant lacking pancreatic proteases was found to be a single, high molecular weight protein. Elastase digestion converted this protein into two subunits which co-migrated on electrophoresis with those normally found on the microvillus membrane. The high molecular weight form had full sucrase and isomaltase activities.