RESUMO
The p53 tumour suppressor promotes cell-cycle arrest or apoptosis in response to cellular stress, such as DNA damage and oncogenesis. This role of p53 is important for its tumour-suppression function and depends, at least in part, on its ability to bind to specific DNA sequences and activate the transcription of target genes. The pathway through which p53 promotes apoptosis is not fully understood. Here we describe a new gene regulated by p53 that encodes a predicted protein of 915 amino acids in mice (910 amino acids in humans), which we have named Pidd. The mouse Pidd cDNA contains a p53 consensus DNA binding sequence upstream of the Pidd-coding region. This sequence element bound to p53 and conferred p53-dependent inducibility on a heterologous reporter gene. Moreover, Pidd RNA was induced by ionizing radiation in a p53-dependent manner and the basal level of Pidd RNA was dependent on Trp53 status. Overexpression of Pidd inhibited cell growth in a p53-like manner by inducing apoptosis. Antisense inhibition of Pidd expression attenuated p53-mediated apoptosis. Our data suggest that Pidd is an effector of p53-dependent apoptosis.
Assuntos
Apoptose , Proteínas de Transporte/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Sequência de Aminoácidos , Animais , Northern Blotting , Proteínas de Transporte/química , Linhagem Celular , Clonagem Molecular , DNA Complementar/metabolismo , Proteínas Adaptadoras de Sinalização de Receptores de Domínio de Morte , Relação Dose-Resposta à Radiação , Etiquetas de Sequências Expressas , Vírus da Leucemia Murina de Friend/genética , Genes Reporter , Humanos , Células K562 , Cinética , Luciferases/metabolismo , Camundongos , Dados de Sequência Molecular , Oligonucleotídeos Antissenso/metabolismo , Plasmídeos/metabolismo , Ligação Proteica , Estrutura Terciária de Proteína , Homologia de Sequência de Aminoácidos , Temperatura , Fatores de Tempo , Distribuição Tecidual , Transfecção , Transformação GenéticaRESUMO
We have investigated whether the p53 oncogene is expressed in the blast cells of patients with acute myeloblastic leukemia. p53 protein was detected in the blast cells of 19 out of 34 patients, but not in normal myelopoietic cells. We find a highly significant correlation between p53 protein synthesis in leukemic blast cells and the secondary plating efficiency of these cells (p = 0.0001). The latter provides an estimate for the self renewal capacity of progenitor cells in the blast population. These data indicate that p53 may be involved in leukemic stem cell renewal.
Assuntos
Leucemia Mieloide Aguda/metabolismo , Proteínas de Neoplasias/genética , Oncogenes , Fosfoproteínas/genética , Divisão Celular , Epitopos/análise , Humanos , Leucemia Mieloide Aguda/patologia , Proteínas de Neoplasias/análise , Proteínas de Neoplasias/biossíntese , Fosfoproteínas/análise , Fosfoproteínas/biossíntese , Fosforilação , Proteína Supressora de Tumor p53RESUMO
BACKGROUND: Oral epithelial dysplasia (OED) is a histologically detectable lesion that may progress to carcinoma but there are no accurate markers that predict progression. This study examined the development of carcinoma from oral dysplastic lesions, and the association between abnormal DNA content and progression to carcinoma. METHODS: Epithelial dysplasias from the Oral Pathology Diagnostic Service were matched against the Ontario Cancer Registry database to identify cases that progressed to carcinoma. A case-control study was conducted to compare DNA image cytometry of dysplasias that progressed with those that have not progressed. For a subset of the progressed dysplasias, DNA content of the carcinoma was also analysed. RESULTS: A total of 8% of epithelial dysplasias progressed to carcinoma after 6-131 months. In all, 28 of 99 dysplasias showed abnormal DNA content by image cytometry. In multivariate analysis of time to progression, abnormal DNA content was a significant predictor with hazard ratio of 3.3 (95% confidence interval: 1.5-7.4) corrected for site and grade of dysplasia. Analysis of sequential samples of dysplasia and carcinoma suggested that epithelial cell populations with grossly abnormal DNA content were transient intermediates during oral cancer development. CONCLUSIONS: Abnormal DNA content is a significant biomarker of a subset of OED that progress to carcinoma.
Assuntos
DNA de Neoplasias/ultraestrutura , Progressão da Doença , Mucosa Bucal/patologia , Neoplasias Bucais/genética , Lesões Pré-Cancerosas/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Carcinoma/genética , Feminino , Humanos , Citometria por Imagem , Processamento de Imagem Assistida por Computador , Masculino , Pessoa de Meia-Idade , Mucosa Bucal/metabolismo , RiscoRESUMO
BACKGROUND: Tumour necrosis factor (TNF) is capable of activating the cell death pathway, and has been implicated in killing transformed cells. However, TNF also activates survival signals, including NF-kappaB activation and the subsequent expression of anti-apoptotic genes, leading to protection against TNF toxicity. METHODS: In this study, we show that, although untransformed mouse embryonic fibroblasts (MEFs) were resistant to TNF killing, E1A/Ras-transformed MEFs were susceptible to extensive apoptosis induced by TNF. The key factors for determining TNF sensitivity were explored by comparing wild-type and E1A/Ras-transformed MEFs. RESULTS: TNF signalling to NF-kappaB and to its target genes such as IkappaBalpha seemed to be mostly intact in E1A/Ras-transformed cells. Instead, the induction of A20 was completely abolished in E1A/Ras-transformed MEFs, although A20 is known to be NF-kappaB dependent. Reintroduction of A20 into E1A/Ras-transformed MEFs rescued these cells from TNF-induced death and reduced the formation of the FADD/caspase-8 complex. This impaired A20 induction in E1A/Ras MEFs was not because of the stabilisation of p53 or a defective TNF-induced p38 and Jun N-terminal kinase (JNK) signalling. Consistently, we found a reduced A20 promoter activity but normal NF-kappaB activity in TNF-treated E1A/Ras MEFs. However, Bcl-3 seemed to have a role in the transactivation of the A20 promoter in E1A/Ras cells. CONCLUSIONS: Our results suggest that specific inhibition of certain survival factors, such as A20, may determine the sensitivity to TNF-induced apoptosis in transformed cells such as E1A/Ras MEFs.
Assuntos
Proteínas E1A de Adenovirus/genética , Apoptose/efeitos dos fármacos , Cisteína Endopeptidases/fisiologia , Genes ras , Peptídeos e Proteínas de Sinalização Intracelular/fisiologia , NF-kappa B/fisiologia , Fator de Necrose Tumoral alfa/farmacologia , Animais , Proteína 3 do Linfoma de Células B , Proteína Reguladora de Apoptosis Semelhante a CASP8 e FADD/análise , Caspase 8/metabolismo , Linhagem Celular Transformada , Células Cultivadas , Cisteína Endopeptidases/genética , Regulação da Expressão Gênica , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Proteínas Quinases JNK Ativadas por Mitógeno/fisiologia , Camundongos , Proteínas Proto-Oncogênicas/fisiologia , Fatores de Transcrição/fisiologia , Proteína 3 Induzida por Fator de Necrose Tumoral alfa , Proteína Supressora de Tumor p53/fisiologia , Proteínas Quinases p38 Ativadas por Mitógeno/fisiologiaRESUMO
The localization and characterization of carbohydrates in adrenal medullary cells were studied by histochemical and cytochemical methods. Adrenaline (A)-and noradrenaline (N)-storing granules were argentaphobic when ultrathin sections of Araldite-embedded medullae were stained according to the periodic acid-thiocarbohydrazide-silver proteinate technique of Thiery. A small amount of glycogen in the form of single beta-particles as well as lysosomes were, however, visualized by this technique. The entire core of the A granules was markedly positive after ultrathin sections of glutaraldehyde-fixed, glycol methacrylate (GMA)-embedded medullae were stained with phosphotungstic acid (PTA) at low pH (0.3). The N granules, in contrast, were mostly unreactive. In the A cells, PTA stained a large part of the Golgi complex, whereas in the N cells the Golgi complex was mostly unstained. In both cell types, the cell coat, lysosomes, and multivesticular bodies reacted to PTA. The periodic acid-Schiff (PAS) technique showed A but not N granules in semithin sections of GMA- or Araldite-embedded medullae. The PTA and PAS stains were abolished by acetylation, restored by saponification, unchanged by methylation, and greatly diminished by sulfation. In ultrathin sections of GMA- or Araldite-embedded medullae incubated with colloidal iron according to various techniques, the cell coat and lysosomes of both cell types were stained, unlike all the other cytoplasmic organelles. These results indicate that A granules and the Golgi complex of A cells, unlike the same structures in N cells, are rich in glycoproteins which are probably not acidic.
Assuntos
Medula Suprarrenal/análise , Carboidratos/isolamento & purificação , Medula Suprarrenal/ultraestrutura , Animais , Parede Celular/análise , Cricetinae , Grânulos Citoplasmáticos/análise , Grânulos Citoplasmáticos/ultraestrutura , Epinefrina/isolamento & purificação , Feminino , Glicogênio/isolamento & purificação , Glicoproteínas/isolamento & purificação , Complexo de Golgi/análise , Complexo de Golgi/ultraestrutura , Histocitoquímica , Lisossomos/análise , Microscopia Eletrônica , Norepinefrina/isolamento & purificação , Organoides/análise , Ácido Fosfotúngstico , Ratos , Coloração e RotulagemRESUMO
Normal somatic cells have a finite life span [1] and lose telomeric DNA, present at the ends of chromosomes, each time they divide as a function of age in vivo or in culture [2-4]. In contrast, many cancer cells and cell lines established from tumours maintain their telomere length by activation of an RNA-protein complex called telomerase, an enzyme originally discovered in Tetrahymena [5], that synthesizes telomeric repeats [6-8]. These findings have led to the formation of the 'telomere hypothesis', which proposes that critical shortening of telomeric DNA due to the end-replication problem [9] is the signal for the initiation of cellular senescence [10,11]. In yeast, the EST2 gene product, the catalytic subunit of telomerase, is essential for telomere maintenance in vivo [12-14]. The recent cloning of the cDNA encoding the catalytic subunit of human telomerase (hTERT) [15,16] makes it possible to test the telomere hypothesis. In this study, we expressed hTERT in normal human diploid fibroblasts, which lack telomerase activity, to determine whether telomerase activity could be reconstituted leading to extension of replicative life span. Our results show that retroviral-mediated expression of hTERT resulted in functional telomerase activity in normal aging human cells. Moreover, reconstitution of telomerase activity in vivo led to an increase in the length of telomeric DNA and to extension of cellular life span. These findings provide direct evidence in support of the telomere hypothesis, indicating that telomere length is one factor that can determine the replicative life span of human cells.
Assuntos
Replicação do DNA , RNA , Telomerase/metabolismo , Telômero/metabolismo , Animais , Catálise , Células Cultivadas , Senescência Celular , Proteínas de Ligação a DNA , Fibroblastos/enzimologia , Humanos , Conformação Proteica , Proteínas/metabolismo , Retroviridae , TetrahymenaRESUMO
Murine erythroleukemia cells that lack endogenous p53 expression were transfected with a temperature-sensitive p53 allele. The temperature-sensitive p53 protein behaves as a mutant polypeptide at 37 degrees C and as a wild-type polypeptide at 32 degrees C. Three independent clones expressing the temperature-sensitive p53 protein were characterized with respect to p53-mediated G1 cell cycle arrest, apoptosis, and differentiation. Clone ts5.203 responded to p53 activation at 32 degrees C by undergoing G1 arrest, apoptosis, and differentiation. Apoptosis was seen in cells representative of all phases of the cell cycle and was not restricted to cells arrested in G1. The addition of a cytokine (erythropoietin, c-kit ligand, or interleukin-3) to the culture medium of ts5.203 cells blocked p53-mediated apoptosis and differentiation but not p53-mediated G1 arrest. These observations indicate that apoptosis and G1 arrest can be effectively uncoupled through the action of cytokines acting as survival factors and are consistent with the idea that apoptosis and G1 arrest represent separate functions of p53. Clones ts15.15 and tsCB3.4 responded to p53 activation at 32 degrees C by undergoing G1 arrest but not apoptosis. We demonstrate that tsCB3.4 secretes a factor with erythropoietin-like activity and that ts15.15 secretes a factor with interleukin-3 activity and suggest that autocrine secretion of these cytokines blocks p53-mediated apoptosis. These data provide a framework in which to understand the variable responses of cells to p53 overexpression.
Assuntos
Apoptose , Citocinas/fisiologia , Fase G1 , Proteína Supressora de Tumor p53/fisiologia , Animais , Sequência de Bases , Diferenciação Celular , Núcleo Celular/metabolismo , Proteínas de Ligação a DNA/metabolismo , Eritropoetina/farmacologia , Interleucina-3/farmacologia , Camundongos , Dados de Sequência Molecular , Oligodesoxirribonucleotídeos/química , Conformação Proteica/efeitos dos fármacos , Fator de Células-Tronco/farmacologia , Células Tumorais CultivadasRESUMO
An amphotropic retroviral vector containing the bacterial neomycin phosphotransferase gene (neo) was used to infect blast cells from patients with acute myeloblastic leukemia. The infected cells acquired a G418-resistant phenotype that was stable as measured in a clonogenic assay and in long-term suspension culture. Thus, gene transfer into stem cells was accomplished by this procedure. This approach for manipulating gene expression in blast stem cells provides a means to assess the roles of a variety of genes in self-renewal, differentiation, and leukemogenesis.
Assuntos
Crise Blástica/genética , Escherichia coli/genética , Genes Bacterianos , Genes , Leucemia Mieloide Aguda/genética , Fosfotransferases/genética , Transfecção , DNA de Neoplasias/genética , DNA de Neoplasias/isolamento & purificação , Escherichia coli/enzimologia , Humanos , Canamicina Quinase , Células Tumorais Cultivadas/enzimologiaRESUMO
The murine allele temperature-sensitive (ts) p53Val-135 encodes a ts p53 protein that behaves as a mutant polypeptide at 37 degrees C and as a wild-type polypeptide at 32 degrees C. This ts allele was introduced into the p53 nonproducer Friend erythroleukemia cell line DP16-1. The DP16-1 cell line was derived from the spleen cells of a mouse infected with the polycythemia strain of Friend virus, and like other erythroleukemia cell lines transformed by this virus, it grows independently of erythropoietin, likely because of expression of the viral gp55 protein which binds to and activates the erythropoietin receptor. When incubated at 32 degrees C, DP16-1 cells expressing ts p53Val-135 protein, arrested in the G0/G1 phase of the cell cycle, rapidly lost viability and expressed hemoglobin, a marker of erythroid differentiation. Erythropoietin had a striking effect on p53Val-135-expressing cells at 32 degrees C by prolonging their survival and diminishing the extent of hemoglobin production. This response to erythropoietin was not accompanied by down-regulation of viral gp55 protein.
Assuntos
Eritropoetina/farmacologia , Vírus da Leucemia Murina de Friend/genética , Hemoglobinas/biossíntese , Leucemia Eritroblástica Aguda/metabolismo , Proteína Supressora de Tumor p53/genética , Animais , Ciclo Celular , Diferenciação Celular , Divisão Celular , Linhagem Celular Transformada , Transformação Celular Viral , Leucemia Eritroblástica Aguda/genética , Camundongos , Camundongos Endogâmicos DBA , Mutação , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Baço/citologia , Vírus Formadores de Foco no Baço/genética , Proteínas do Envelope Viral/genéticaRESUMO
The Friend erythroleukemia virus complex contains no cell-derived oncogene. Transformation by this virus may therefore involve mutations affecting cellular gene expression. We provide evidence that inactivating mutations of the cellular p53 gene are a common feature in Friend virus-induced malignancy, consistent with an antioncogene role for p53 in this disease. We have shown that frequent rearrangements of the p53 gene cause loss of expression or synthesis of truncated proteins, whereas overexpression of p53 protein is seen in other Friend cell lines. We now demonstrate that p53 expression in the latter cells is also abnormal, as a result of missense mutations in regions encoding highly conserved amino acids. Three of these aberrant alleles obtained from cells from different mice were cloned and found to function as dominant oncogenes in gene transfer assays, supporting the view that certain naturally occurring missense mutations in p53 confer a dominant negative phenotype on the encoded protein.
Assuntos
Transformação Celular Neoplásica , Vírus da Leucemia Murina de Friend/genética , Genes Dominantes , Proteínas Nucleares/genética , Proteínas Oncogênicas/genética , Fosfoproteínas/genética , Alelos , Sequência de Aminoácidos , Animais , Sequência de Bases , Células Cultivadas , Clonagem Molecular , Embrião de Mamíferos , Epitopos/análise , Amplificação de Genes , Leucemia Eritroblástica Aguda/microbiologia , Leucemia Experimental/microbiologia , Dados de Sequência Molecular , Sondas de Oligonucleotídeos , Proteínas Oncogênicas/análise , Proteínas Oncogênicas/imunologia , Fosfoproteínas/análise , Fosfoproteínas/imunologia , RNA/genética , RNA Antissenso , RNA Mensageiro/antagonistas & inibidores , Ratos , Ratos Endogâmicos F344 , Ribonucleases , Proteína Supressora de Tumor p53RESUMO
Inactivation of the cellular p53 gene is a common feature of Friend virus-induced murine erythroleukemia cell lines and may represent a necessary step in the progression of this disease. As well, frequent loss or mutation of p53 alleles in diverse human tumors is consistent with the view of p53 as a tumor suppressor gene. To examine the significance of p53 gene inactivation in tumorigenesis, we have attempted to express transfected wild-type p53 in three p53-negative tumor cell lines: murine DP16-1 Friend erythroleukemia cells, human K562 cells, and SKOV-3 cells. We found that aberrant p53 proteins, which differ from wild-type p53 by a single amino acid substitution, were expressed stably in these cells, whereas wild-type p53 expression was not tolerated. The inability of p53-negative tumor cell lines to support long-term expression of wild-type p53 protein is consistent with the view that p53 is a tumor suppressor gene.
Assuntos
Transformação Celular Neoplásica/genética , Genes Supressores de Tumor , Proteína Supressora de Tumor p53/fisiologia , Animais , Sequência de Bases , Northern Blotting , Southern Blotting , Divisão Celular , DNA de Neoplasias/genética , Expressão Gênica , Camundongos , Dados de Sequência Molecular , Oligonucleotídeos , RNA Neoplásico/genética , Transfecção , Células Tumorais Cultivadas , Proteína Supressora de Tumor p53/genéticaRESUMO
Carcinoembryonic antigen (CEA) expression is perhaps the most prevalent of phenotypic changes observed in human cancer cells. The molecular genetic basis of this phenomenon, however, is completely unknown. Twenty-seven CEA cDNA clones were isolated from a human colon adenocarcinoma cell line. Most of these clones are full length and consist of a number (usually three) of surprisingly similar long (534 base pairs) repeats between a 5' end of 520 base pairs and a 3' end with three different termination points. The predicted translation product of these clones consists of a processed signal sequence of 34 amino acids, an amino-terminal sequence of 107 amino acids, which includes the known terminal amino acid sequence of CEA, three repeated domains of 178 amino acids each, and a membrane-anchoring domain of 27 amino acids, giving a total of 702 amino acids and a molecular weight of 72,813 for the mature protein. The repeated domains have conserved features, including the first 67 amino acids at their N termini and the presence of four cysteine residues. Comparisons with the amino acid sequences of other proteins reveals homology of the repeats with various members of the immunoglobulin supergene family, particularly the human T-cell receptor gamma chain. CEA cDNA clones in the SP-65 vector were shown to produce transcripts in vitro which could be translated in vitro to yield a protein of molecular weight 73,000 which in turn could be precipitated with CEA-specific antibodies. CEA cDNA clones were also inserted into an animal cell expression vector and introduced by transfection into mammalian cell lines. These transfectants produced a CEA-immunoprecipitable glycoprotein which could be visualized by immunofluorescence on the cell surface.
Assuntos
Antígeno Carcinoembrionário/genética , Adenocarcinoma/genética , Sequência de Aminoácidos , Sequência de Bases , Clonagem Molecular , Neoplasias do Colo/genética , DNA/genética , Humanos , Imunoglobulinas/genética , Dados de Sequência Molecular , Família Multigênica , RNA Mensageiro/genética , Células Tumorais CultivadasRESUMO
The p53 gene is rearranged in an erythroleukemic cell line (DP15-2) transformed by Friend retrovirus. Here, we characterize the mutation and identify a deletion of approximately equal to 3.0 kilobases that removes exon 2 coding sequences. The gene is expressed in DP15-2 cells and results in synthesis of a 44,000-dalton protein that is missing the N-terminal amino acid residues of p53. The truncated protein is unusually stable and accumulates to high levels intracellularly. Moreover, it appears to have undergone a change in conformation as revealed by epitope mapping studies. This study represents the first description of an altered p53 gene product arising by mutation during neoplastic progression and identifies a region in the p53 protein molecule that plays a role in determining p53 stability in vivo.
Assuntos
Leucemia Eritroblástica Aguda/genética , Proteínas de Neoplasias/genética , Fosfoproteínas/genética , Proto-Oncogenes , Animais , Anticorpos Monoclonais , Sequência de Bases , Deleção Cromossômica , DNA de Neoplasias/genética , Vírus da Leucemia Murina de Friend , Regulação da Expressão Gênica , Camundongos , Peso Molecular , Proteínas de Neoplasias/imunologia , Fosfoproteínas/imunologia , Proteína Supressora de Tumor p53RESUMO
Rat embryo fibroblast clones transformed with the human papillomavirus type 16 E7 gene and the H-ras oncogene (ER clones) fall into two groups on the basis of endogenous p53 genotype, wild type or mutant. We have compared these clones with the aim of indentifying physiological differences that could be attributed to p53 protein function. We show that all ER clones, regardless of p53 gene status, are tumorigenic and metastatic in severe combined immunodeficiency mice. We demonstrate that only the wild-type p53 protein expressed in ER clones is functional on the basis of its site-specific double-stranded DNA-binding activity and its ability to confer a G1 delay on cells following treatment with ionizing radiation. These data indicate that disruption of the p53 growth-regulatory pathway is not a prerequisite for the malignant conversion of rat embryo fibroblasts expressing the E7 gene and mutant ras. Differences in phenotype that were correlated with loss of p53 protein function included the following: serum-independent growth of ER clones in culture, decreased tumor doubling time in vivo, and increased radioresistance. In addition, we demonstrate the p53-dependent G1 checkpoint alone does not determine radiosensitivity.
Assuntos
Transformação Celular Neoplásica , Fase G1/fisiologia , Genes p53 , Genes ras , Proteínas Oncogênicas Virais/genética , Papillomaviridae/genética , Animais , Sequência de Bases , Sítios de Ligação , Ciclo Celular/efeitos da radiação , Linhagem Celular Transformada , Sobrevivência Celular/efeitos da radiação , Células Clonais , Sequência Consenso , Embrião de Mamíferos , Fibroblastos , Citometria de Fluxo , Raios gama , Camundongos , Camundongos SCID , Dados de Sequência Molecular , Metástase Neoplásica/patologia , Transplante de Neoplasias , Oligodesoxirribonucleotídeos , Proteínas E7 de Papillomavirus , Ratos , Fatores de Transcrição/genética , Transplante Heterólogo , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismoRESUMO
Life span determination in normal human cells may be regulated by nucleoprotein structures called telomeres, the physical ends of eukaryotic chromosomes. Telomeres have been shown to be essential for chromosome stability and function and to shorten with each cell division in normal human cells in culture and with age in vivo. Reversal of telomere shortening by the forced expression of telomerase in normal cells has been shown to elongate telomeres and extend the replicative life span (H. Vaziri and S. Benchimol, Curr. Biol. 8:279-282, 1998; A. G. Bodnar et al., Science 279:349-352, 1998). Extension of the life span as a consequence of the functional inactivation of p53 is frequently associated with loss of genomic stability. Analysis of telomerase-induced extended-life-span fibroblast (TIELF) cells by G banding and spectral karyotyping indicated that forced extension of the life span by telomerase led to the transient formation of aberrant structures, which were subsequently resolved in higher passages. However, the p53-dependent G1 checkpoint was intact as assessed by functional activation of p53 protein in response to ionizing radiation and subsequent p53-mediated induction of p21(Waf1/Cip1/Sdi1). TIELF cells were not tumorigenic and had a normal DNA strand break rejoining activity and normal radiosensitivity in response to ionizing radiation.
Assuntos
Fibroblastos/fisiologia , Fase G1 , Genoma Humano , Telomerase/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Sobrevivência Celular , Senescência Celular/genética , Senescência Celular/fisiologia , DNA , Dano ao DNA , Reparo do DNA , Humanos , Hibridização in Situ Fluorescente , Transdução de Sinais , TelômeroRESUMO
Normal and diseased human tissues were analyzed for the transcription of genes of the carcinoembryonic (CEA) family. Epithelial tissues of colonic origin, whether malignant or normal, all express two closely related mRNA species of 3.0- and 3.5-kilobase mRNA which code for CEA. Only tissues of colonic origin were found to express these CEA-specific transcripts. Colon carcinomas consistently express a 2.6-kilobase mRNA species as well which codes for nonspecific cross-reacting antigen. Nonneoplastic colon mucosas, on the other hand, express lower or nondetectable levels of this transcript. Most breast carcinomas produce only the nonspecific cross-reacting antigen mRNA, whereas leukocytes of chronic myelogeneous leukemia express both nonspecific cross-reacting antigen mRNA and a 2.3-kilobase mRNA corresponding to a yet undefined gene of the CEA family. Thus the multiple CEA-like products reported to be produced by these tissues correspond to only four different mRNA species coding for three different peptides. These data suggest a less complex organization of the CEA family than was previously suspected and point to posttranscriptional modifications, such as variable patterns of glycosylation, as the likely reason for much of the observed complexity in CEA-like glycoproteins.
Assuntos
Antígeno Carcinoembrionário/genética , Genes , Neoplasias/imunologia , Transcrição Gênica , DNA/genética , Enzimas de Restrição do DNA , DNA de Neoplasias/genética , Feminino , Humanos , Hibridização de Ácido Nucleico , RNA Mensageiro/genética , Valores de ReferênciaRESUMO
Embryonic fibroblast cell lines were established from mice deficient, heterozygous, or proficient for Msh2, one of the three known DNA mismatch repair genes involved in hereditary nonpolyposis colon cancer (HNPCC). Cell lines were established by transfection of primary mouse embryo fibroblasts with E7 and Ras oncogenes or mutant p53. Spontaneously immortalized cells derived from the primary cultures were also studied. To determine whether these cells developed a mutator phenotype similar to that found in colon cancer cells deficient in mismatch repair, we measured mutation rates, microsatellite instability, and sensitivities to a range of DNA-damaging agents. The mutator phenotype detected in the E7 and Ras or mutant p53-immortalized Msh2-/- mouse cells was similar to that found in human mismatch repair-deficient colorectal carcinoma cell lines. Mutation rates to ouabain resistance were increased 8-12-fold relative to lines from Msh2+/+ mice, and microsatellite instability was detectable in 12-18% of subclones derived from the Msh2-/- line but was undetectable in subclones developed from the Msh2+/+ line. Furthermore, E7 and Ras or spontaneously immortalized Msh2-/- cells were significantly more resistant to the cytotoxic effects of 6-thioguanine relative to Msh2+/+ cells. In contrast, these lines showed various responses to UV light and cis-platinum, suggesting that mismatch repair deficiency was not the sole determinant for sensitivity to these DNA-damaging agents. Particular attention was paid to the properties of cells heterozygous for the Msh2 mutant gene, which would mimic the situation of an HNPCC carrier. However, our studies failed to reveal any properties of these cells that might provide a growth advantage or predispose them for the acquisition of further mutations. This observation is consistent with the model that inactivation of the wild-type Msh2 allele is a critical step for tumorigenesis in HNPCC patients.
Assuntos
Cinamatos , Neoplasias Colorretais Hereditárias sem Polipose/genética , Reparo do DNA/genética , Proteínas de Ligação a DNA/genética , Fibroblastos , Mutagênese/genética , Proteínas Proto-Oncogênicas/genética , Animais , Antibacterianos , Antineoplásicos/farmacologia , Linhagem Celular , Cisplatino/farmacologia , Proteínas de Ligação a DNA/metabolismo , Resistência a Medicamentos/genética , Fibroblastos/efeitos dos fármacos , Fibroblastos/patologia , Fibroblastos/efeitos da radiação , Genes p53/genética , Genes ras/genética , Humanos , Higromicina B/análogos & derivados , Camundongos , Camundongos Endogâmicos C57BL , Repetições de Microssatélites/genética , Proteína 2 Homóloga a MutS , Fenótipo , Proteínas Proto-Oncogênicas/deficiência , Proteínas Proto-Oncogênicas/metabolismo , Radiossensibilizantes/farmacologia , Transfecção , Ensaio Tumoral de Célula-TroncoRESUMO
Intact and rearranged p53 genes from Friend virus-induced erythroleukemic cell lines which code for proteins of 53- (p53) and 44-kDa (p44), respectively, were cloned into pUC18 and tested for their ability to immortalize rat embryo fibroblasts. The functional p53 gene from normal Balb/c mouse liver was also tested for immortalizing activity. Immortal cells were obtained with the three genes although the efficiency of immortalization by p44 was lower than that by p53. Expression of murine p53 and p44 in the established rat cell lines was confirmed by metabolic labeling and Western Blotting. Our results demonstrate that elevated expression of the mouse p53 gene, driven by its natural promoter and in the absence of strong heterologous promoters and/or enhancers, can efficiently immortalize early-passage rat embryo fibroblasts. p53-immortalized cells but not p44-immortalized cells could be morphologically transformed by secondary transfection with activated Ha-ras. Thus 5'-coding sequences of the p53 gene appear necessary for ras complementation but not for immortalization.
Assuntos
Transformação Celular Neoplásica/genética , Proteínas de Neoplasias/fisiologia , Fosfoproteínas/fisiologia , Proteínas Proto-Oncogênicas/fisiologia , Animais , Ciclo Celular , Linhagem Celular , Células Cultivadas , DNA de Neoplasias/genética , Regulação da Expressão Gênica , Genes , Leucemia Eritroblástica Aguda/genética , Camundongos , Peso Molecular , Neoplasias Experimentais/genética , Ratos , Relação Estrutura-Atividade , Transfecção , Proteína Supressora de Tumor p53RESUMO
Telomere shortening may be one of several factors that contribute to the onset of senescence in human cells. The p53 and pRb pathways are involved in the regulation of cell cycle progression from G1 into S phase and inactivation of these pathways leads to extension of life span. Short dysfunctional telomeres may be perceived as damaged DNA and may activate these pathways, leading to prolonged arrest in G1, typical of cells in senescence. Inactivation of the p53 and pRb pathways, however, does not lead to cell immortalization. Cells that overcome senescence and have an extended life span continue to lose telomeric DNA and subsequently enter a second phase of growth arrest termed 'crisis'. Forced expression of telomerase in human cells leads to the elongation of telomeres and immortalization. The development of human cancer is frequently associated with the inactivation of the pRb and p53 pathways, attesting to the importance of senescence in restricting the tumor-forming ability of human cells. Cancer cells must also maintain telomere length and, in the majority of cases, this is associated with expression of telomerase activity.
Assuntos
Senescência Celular , Proteína do Retinoblastoma/metabolismo , Telomerase/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Animais , Ciclo Celular , Dano ao DNA/genética , Genoma , Humanos , Neoplasias/enzimologia , Neoplasias/genética , Neoplasias/patologia , Transdução de Sinais , Telomerase/antagonistas & inibidores , Telômero/química , Telômero/genética , Telômero/metabolismoRESUMO
The 3' untranslated region of human p53 mRNA represses translation both in vitro and in vivo. Here, we identify a cis-acting 66-nucleotide U-rich sequence in the human p53 mRNA 3' untranslated region that mediates translational repression. Using UV cross-linking, we detect a 40 kDa protein that interacts specifically with the p53 3'UTR containing the repressor element. Enhanced translation of p53 mRNA contributes to the accumulation of p53 protein in cells exposed to gamma-radiation and could be a consequence of relieving the inhibition mediated by the repressor element.