RESUMO
We used the serial analysis of gene expression (SAGE) method to systematically analyze transcripts present in non-small cell lung cancer. Over 226,000 SAGE tags were sequence analyzed from two independent primary lung cancers and two normal human bronchial/tracheal epithelial cell cultures. A total of 226,000 SAGE tags were sequence identified, representing 43,254 unique transcripts. Comparison of the tags present in the tumor with those identified in the normal tissue revealed 175 transcript tags that were overrepresented in the normal tissue and 142 tags that were overexpressed in the tumor by 10-fold or more. Northern hybridization was performed on 15 of the most abundantly expressed tags identified in the tumors. These tags were derived from either a known gene or a matched expressed sequence tag clone. The transcripts for 3 of the 15 genes, PGP 9.5, B-myb, and human mutT, were abundantly expressed in primary lung cancers (10 of 18, 15 of 18, and 6 of 12 tumors, respectively). In contrast, the presence of PGP9.5 and B-myb was much less frequent in primary tumors derived from other tissue origins. These results suggest that at least a portion of the transcripts identified by SAGE are frequently associated with lung cancer, and that their overexpression may contribute to lung tumorigenesis. The identification and further characterization of genes generated by SAGE should provide potential new targets for the diagnosis, prognosis, and therapy of lung cancer.
Assuntos
Carcinoma Pulmonar de Células não Pequenas/genética , Carcinoma de Células Escamosas/genética , Expressão Gênica , Técnicas Genéticas , Neoplasias Pulmonares/genética , Brônquios , Células Cultivadas , Humanos , RNA Mensageiro/análise , Sitios de Sequências Rotuladas , TraqueiaRESUMO
Serial analysis of gene expression (SAGE) allows for a quantitative, representative, and comprehensive profile of gene expression. We have utilized SAGE technology to contrast the differential gene expression profile in rat embryo fibroblast cells producing temperature-sensitive p53 tumor suppressor protein at permissive or non-permissive temperatures. Analysis of approximately 15,000 genes revealed that the expression of 14 genes (P < 0.001, > or = 0.03% abundance) was dependent on functional p53 protein, whereas the expression of three genes was significantly higher in cells producing non-functional p53 protein. Those genes whose expression was increased by functional p53 include RAS, U6 snRNA, cyclin G, EGR-1, and several novel genes. The expression of actin, tubulin, and HSP70 genes was elevated at the non-permissive temperature for p53 function. Interestingly, the expression of several genes was dependent on a non-temperature-sensitive mutant p53 suggesting altered transcription profiles dependent on specific p53 mutant proteins. These results demonstrate the utility of SAGE for rapidly and reproducibly evaluating global transcriptional responses within different cell populations.
Assuntos
Clonagem Molecular/métodos , Regulação da Expressão Gênica , Genes p53 , Transcrição Gênica , Animais , Divisão Celular/genética , Linhagem Celular , Embrião de Mamíferos , Fibroblastos/citologia , Fibroblastos/metabolismo , Ratos , Reprodutibilidade dos Testes , TemperaturaRESUMO
Transcriptionally regulated growth-response genes play a pivotal role in the determination of the fate of a cell. p53 is known to transcriptionally regulate genes important in regulating cell growth potential. Using differential reverse transcription-PCR analysis of rat embryo fibroblast cells containing a temperature-sensitive p53 allele, we were able to isolate several transcripts up-regulated specifically in cells harboring functional p53 protein. Two of these genes, SM20 and microsomal epoxide hydrolase (mEH), are previously described genes. Two previously uncharacterized cDNAs, cell growth regulatory (CGR) genes CGR11 and CGR19, were isolated. The predicted amino acid sequence of these novel proteins contain known motifs; EF-hand domains (CGR11) and a ring-finger domain (CGR19), suggestive of function. CGR11 and CGR19 appear to be primary response genes expressed to moderate levels in functional p53 cells. Both CGR11 and CGR19 are able to inhibit the growth of several cell lines.
Assuntos
Proteínas de Ligação ao Cálcio/genética , Divisão Celular/genética , Regulação da Expressão Gênica , Genes Reguladores , Inibidores do Crescimento/genética , Proteínas/genética , Proteína Supressora de Tumor p53/fisiologia , Sequência de Aminoácidos , Animais , Proteínas de Ligação ao Cálcio/biossíntese , Proteínas de Ligação ao Cálcio/química , DNA Complementar/genética , Fibroblastos/citologia , Fibroblastos/metabolismo , Genes p53 , Inibidores do Crescimento/biossíntese , Inibidores do Crescimento/química , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Dados de Sequência Molecular , Biossíntese de Proteínas , Estrutura Terciária de Proteína , Proteínas/química , RNA Mensageiro/genética , RNA Mensageiro/isolamento & purificação , Ratos , Proteínas Recombinantes/metabolismo , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Especificidade da Espécie , Técnica de Subtração , TemperaturaRESUMO
The p53 tumor suppressor gene is a logical target for cancer therapy. Several therapeutic strategies can be envisioned based upon recent advances concerning structure and function of the p53 protein, its interaction with cellular and viral proteins and its roles in repairing DNA, regulating cell division and promoting apoptosis.
Assuntos
Genes p53/efeitos dos fármacos , Neoplasias/genética , Neoplasias/terapia , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Regulação Neoplásica da Expressão Gênica , Terapia Genética , Vetores Genéticos/uso terapêutico , Humanos , Mutação , Neoplasias/imunologia , Proteína Supressora de Tumor p53/química , Proteína Supressora de Tumor p53/fisiologiaRESUMO
CD4-IgG2 is a novel fusion protein comprising human IgG2 in which the Fv portions of both heavy and light chains have been replaced by the V1 and V2 domains of human CD4. This tetrameric protein is being developed as an immunoprophylactic agent to reduce the probability of infection following HIV-1 exposure, in settings such as occupational or perinatal exposure to the virus. CD4-IgG2 has been expressed in Chinese hamster ovary cells and is secreted as a fully assembled heterotetramer. The protein binds with nanomolar affinity to purified gp120 from both a laboratory-adapted strain and a primary isolate of HIV-1. Pharmacokinetic studies in rabbits demonstrated that CD4-IgG2 has a plasma terminal half-life greater than 1 day, compared with 15 min for soluble CD4 (sCD4). CD4-IgG2 does not bind to Fc receptors on the surface of U937 monocyte/macrophage cells. Compared to molecules that incorporate the Fc portion of IgG1, CD4-IgG2 has less potential to mediate functions such as antibody-dependent enhancement of infection or transplacental transmission of HIV-1. When tested in a virus-free HIV-1 envelope glycoprotein-mediated cell fusion assay, the tetrameric CD4-IgG2 molecule inhibited syncytium formation more effectively than monomeric sCD4 or a dimeric CD4-gamma 2 fusion protein. This suggests the protein will block cell-to-cell transmission of HIV-1. Moreover, CD4-IgG2 effectively neutralized a panel of laboratory-adapted strains and primary isolates of HIV-1, including strains with different tropisms and isolated from different stages of the disease, at concentrations that should be readily achieved in vivo.
Assuntos
Antivirais/farmacologia , Imunoadesinas CD4/farmacologia , Infecções por HIV/prevenção & controle , HIV-1/efeitos dos fármacos , Animais , Antivirais/farmacocinética , Células CHO , Cricetinae , Células Gigantes/virologia , Humanos , Testes de Neutralização , Coelhos , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/farmacocinética , Proteínas Recombinantes de Fusão/farmacologiaRESUMO
CD4-based molecules were tested in combination with HIV-1-neutralizing antibodies directed against the V3 loop of gp120 or against gp41, for inhibition of HIV-1 envelope-mediated cell fusion. A virus-free cell fusion assay was developed, using Chinese hamster ovary cells that stably express HIV-1 gp120/gp41. These cells were incubated with dilutions of CD4-based molecules, antibodies, or mixtures of both, then overlaid with C8166 CD4+ T cells. Syncytia were counted and the degree of inhibition of cell fusion was determined. Synergy, additivity, or antagonism was calculated by the combination index (CI) method. The CD4-based molecules included soluble human CD4 as well as fusion proteins composed of CD4 linked to human immunoglobulin gamma 1 or gamma 2 heavy chains. Combinations of CD4-based molecules and monoclonal or polyclonal anti-V3 loop antibodies were synergistic in blocking HIV-1 envelope-mediated cell fusion (CI = 0.21-0.91 at 95% inhibition). Synergy was also observed with combinations of the CD4-based molecules and a broadly neutralizing anti-gp41 monoclonal antibody (2F5) (CI = 0.29-0.65 at 95% inhibition). These results demonstrate that molecules inhibiting HIV attachment act synergistically with molecules inhibiting HIV-1 fusion. The results suggest that CD4-based therapeutics would be more effective in patients with naturally occurring anti-V3 loop or anti-gp41 antibodies. In addition, there may be an advantage in coadministering CD4-based molecules and antibodies that block fusion, especially broadly neutralizing anti-gp41 antibodies, as a combination therapy for HIV-1 infections.
Assuntos
Antígenos CD4/farmacologia , Imunoadesinas CD4/farmacologia , Produtos do Gene env/imunologia , Células Gigantes , Anticorpos Anti-HIV/imunologia , HIV-1/patogenicidade , Sequência de Aminoácidos , Animais , Células CHO , Fusão Celular , Cricetinae , Sinergismo Farmacológico , Proteína gp120 do Envelope de HIV/imunologia , Proteína gp41 do Envelope de HIV/imunologia , HIV-1/imunologia , Células HeLa , Humanos , Dados de Sequência Molecular , Testes de Neutralização , Fragmentos de Peptídeos/imunologia , Proteínas Recombinantes de Fusão/farmacologia , Proteínas Recombinantes/farmacologiaRESUMO
Peptidylglycine alpha-amidating enzyme (alpha-AE) catalyzes the conversion of glycine-extended prohormones to their biologically active alpha-amidated forms. We have derived a clonal Chinese hamster ovary cell line that secretes significant quantities of active alpha-AE. Enzyme production was increased by selection for methotrexate-resistant cells expressing a dicistronic message. Amplification of the alpha-AE gene was monitored by Southern blot analysis, enzyme activity, and immunoreactive protein throughout the selection process. The soluble enzyme is bifunctional as determined by the ability to convert either the glycine-extended substrate, dansyl-Tyr--Val--Gly, or the intermediate, dansyl-Tyr--Val--alpha-hydroxyglycine, to the dansyl-Tyr--Val--NH2 product. The recombinant alpha-AE was purified by a simple two-step chromatographic process. The purified enzyme is partially glycosylated and the glycosylated and nonglycosylated forms of the enzyme were separated on a Con A-Sepharose column. The kinetic constants for dansyl-Tyr--Val--Gly, dansyl-Tyr--Val--alpha-hydroxyglycine, ascorbate, and catechol were the same for both forms of alpha-AE. In addition, mimosine is competitive vs ascorbate with K(is) = 3.5 microM for the nonglycosylated alpha-AE and K(is) = 4.2 microM for the glycosylated alpha-AE. Therefore, the presence or absence of asparagine-linked oligosaccharide does not affect the catalytic efficiency of the enzyme. Overexpression of the recombinant enzyme in CHO cells greatly enhances expression of the endogenous gene, implicating a feedback mechanism on the alpha-AE gene.
Assuntos
Oxigenases de Função Mista/metabolismo , Complexos Multienzimáticos , Sequência de Aminoácidos , Animais , Northern Blotting , Southern Blotting , Western Blotting , Células CHO , Cromatografia por Troca Iônica , Células Clonais , Cricetinae , DNA/genética , DNA/isolamento & purificação , Eletroforese em Gel de Poliacrilamida , Cinética , Oxigenases de Função Mista/genética , Oxigenases de Função Mista/isolamento & purificação , Dados de Sequência Molecular , Plasmídeos , RNA/genética , RNA/isolamento & purificação , Ratos , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Especificidade por Substrato , Tetra-Hidrofolato Desidrogenase/genética , Tetra-Hidrofolato Desidrogenase/metabolismo , TransfecçãoRESUMO
A rat alpha-amidating enzyme (alpha-AE) cDNA has been expressed in mouse C127 cells using a bovine papilloma virus vector in which transcription was regulated by the mouse metallothionein 1 promoter. The cDNA encoding the full length alpha-AE protein was modified to terminate translation at a site preceding the transmembrane and cytoplasmic domains, thereby enabling functional enzyme to be secreted into the medium. Purification of recombinant alpha-AE to homogeneity indicated that the enzyme was synthesized and secreted as two proteins of 75-77 kDA. The observed heterogeneity was due to inefficient glycosylation at Asn660, as demonstrated by glycopeptidase F digestion. Using the synthetic peptide, dansyl-Tyr-Val-Gly, the specific activity of the recombinant enzyme at pH 7.0 was found to be 1.4 mumol/min/mg and the Km of the enzyme was determined to be 3 microM. The purified recombinant enzyme has maximal activity at pH 4.5-5.5; however, a rapid inactivation of the enzyme occurs in acidic solutions in vitro. This inactivation is diminished when activity is measured at pH 7.0-10.0. The availability of large amounts of readily purified, active recombinant alpha-AE should allow detailed probing of reaction mechanism, copper coordination chemistry, and turn-over-based inactivation events.
Assuntos
Oxigenases de Função Mista/isolamento & purificação , Complexos Multienzimáticos , Sequência de Aminoácidos , Animais , Sequência de Bases , Linhagem Celular , Vetores Genéticos , Cinética , Camundongos , Oxigenases de Função Mista/genética , Oxigenases de Função Mista/metabolismo , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Sondas de Oligonucleotídeos , Ratos , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , TransfecçãoRESUMO
The alpha-amidating enzyme activity in rat medullary thyroid carcinoma (MTC) consists of multiple, active enzymes that can be resolved by ion-exchange chromatography. Amino acid sequences from one form of purified rat MTC alpha-amidating enzyme have been utilized to design oligonucleotide probes for isolating cDNAs encoding this protein. Sequence analysis of multiple cDNA clones indicates that there are at least two types of cDNA in rat tissues. These cDNAs differ primarily by the absence (type A) or the presence (type B) of a 315-base internal sequence. Additional heterogeneity in the 3' coding regions of the different mRNAs has also been found. Both types of cDNA predict primary translation products that are preproenzymes which must be post-translationally processed at both their amino and carboxyl termini. Sequence analysis of the purified peak III protein from rat MTC demonstrates that the type A mRNA encodes this 75-kDa protein. This analysis also provides support for the assignment of the C-terminal processing site. In addition, data are presented which demonstrate that type B mRNA is also functional. The implications of the internal and carboxyl-end heterogeneity are discussed.
Assuntos
Carcinoma/genética , DNA/análise , Oxigenases de Função Mista , Complexos Multienzimáticos , Oxirredutases atuantes sobre Doadores de Grupo CH-NH/genética , RNA Mensageiro/metabolismo , Neoplasias da Glândula Tireoide/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Carcinoma/enzimologia , Clonagem Molecular , Dados de Sequência Molecular , Peso Molecular , Splicing de RNA , Ratos , Mapeamento por Restrição , Neoplasias da Glândula Tireoide/enzimologia , Células Tumorais CultivadasRESUMO
The biosynthesis and secretion of alpha amidating enzymes from CA-77 cells has been investigated to determine the relationship among the various forms of alpha amidating enzyme seen after purification of alpha amidating enzyme activity from conditioned cell culture media. Initially 2 proteins of 104 kD and 94 kD are synthesized. With time the 104 kD precursor is processed to 41 kD and 43 kD, and the 94 kD precursor is processed to 75 kD. The 41 kD, 43 kD, and 75 kD proteins are secreted into the medium as functional enzymes. In comparing these data with known cDNA sequence for alpha amidating enzyme we conclude that the 104 kD and 94 kD precursors are membrane bound proteins which are posttranslationally processed to generate secreted alpha amidating enzyme.
Assuntos
Precursores Enzimáticos/metabolismo , Oxigenases de Função Mista , Complexos Multienzimáticos , Oxirredutases atuantes sobre Doadores de Grupo CH-NH/metabolismo , Processamento de Proteína Pós-Traducional , Animais , Linhagem Celular , Membrana Celular/metabolismo , Eletroforese em Gel de Poliacrilamida , Glicosilação , Hidrólise , Microssomos/metabolismo , RNA Mensageiro/metabolismo , Ratos , Espectrometria de FluorescênciaRESUMO
Stimulation of Madin-Darby canine kidney cells by 12-O-tetradecanoyl-phorbol-13-acetate (TPA) results in an increase in prostaglandin synthesis. We have measured the specific activity of the cyclooxygenase system in a cell-free assay and demonstrate that a fourfold induction occurs by 6 h poststimulation. This induction could be prevented by continuous treatment with either cycloheximide or actinomycin D. When cycloheximide was added to cells 6 h post-TPA stimulation we found only a 50% reduction in prostaglandin synthesis over the ensuing 6 h in vitro; this decrease in activity was not observed when actinomycin was used. On the other hand, cells stimulated with TPA for 6 h and subsequently treated with cycloheximide or actinomycin D ceased prostaglandin synthesis completely within 1 h. These results suggest that TPA stimulation of prostaglandin synthesis requires both transcriptional and translational events but that a factor(s) in addition to or in place of the cyclooxygenase might be crucial.
Assuntos
Ácidos Araquidônicos/metabolismo , Rim/metabolismo , Forbóis/farmacologia , Prostaglandina-Endoperóxido Sintases/biossíntese , Acetato de Tetradecanoilforbol/farmacologia , Animais , Ácido Araquidônico , Células Cultivadas , Cicloeximida/farmacologia , Dactinomicina/farmacologia , Cães , Indução Enzimática/efeitos dos fármacos , Rim/enzimologia , CinéticaRESUMO
Challenge of Madin-Darby canine kidney (MDCK) cells with the divalent cation ionophore A23187 caused a marked increase in the deacylation of [3H]arachidonic acid but not of [14C]palmitic acid. When the cells were treated with 12-O-tetradecanoyl-phorbol-13-acetate (TPA) and A23187, there was an additional increase in the deacylation of [3H]arachidonic acid compared to that observed with either agent alone. In contrast to deacylation, the stimulation of prostaglandin production by A23187 was small compared to the stimulation by TPA. Cycloheximide inhibited synthesis of prostaglandins in TPA-treated cells, but did not block the stimulated deacylation caused by either TPA or A23187. These data indicate that, while both TPA and A23187 stimulated the deacylation of [3H]arachidonic acid, TPA had an additional, cycloheximide-sensitive effect that was required for efficient conversion of the release fatty acids to prostaglandins. Thus, although required, deacylation appeared to be independent of and insufficient to stimulate maximum prostaglandin synthesis in these cells.
Assuntos
Ácidos Araquidônicos/metabolismo , Calcimicina/farmacologia , Rim/metabolismo , Forbóis/farmacologia , Acetato de Tetradecanoilforbol/farmacologia , Animais , Ácido Araquidônico , Cicloeximida/farmacologia , Dinoprostona , Cães , Rim/efeitos dos fármacos , Prostaglandinas E/biossínteseRESUMO
The tumor-promoting phorbol diester, 12-O-tetradecanoyl-phorbol-13-acetate (TPA), stimulates Madin-Darby canine (MDCK) cells to deacylate cellular phospholipid and to produce prostaglandins. We have used this system to characterize the kinetics of deacylation of [3H]arachidonate and the further metabolism of arachidonate by the cyclooxygenase system. Stimulation of the appearance of [3H]arachidonic acid in extracellular fluids was found to be maximal 2 h after treatment with TPA and its subsequent removal. The production of prostaglandins then followed for up to 24 h. Phospholipase activity was not inhibited by indomethacin over the range of 0.01-100 micrograms/ml. In contrast, prostaglandin synthesis was inhibited at 1 microgram/ml indomethacin. Further, there was a significant stimulation of deacylation within 15 min in the presence of TPA that increased to nearly 30% of the total radioactivity within 1 h. Likewise, stimulation of prostaglandin production was detected within 15 min, but, unlike the deacylation process, did not increase significantly during TPA treatment. The source of arachidonic acid in the early stimulation period was found to be primarily phosphatidylethanolamine, but phosphatidylcholine and phosphatidylinositol were also deacylated. The results presented here argue that the phospholipase and cyclooxygenase are not tightly coupled in this system. Furthermore, we conclude that the earliest effect of TPA with regard to increased prostaglandin production in the MDCK cell is the direct stimulation of phospholipase activity.
Assuntos
Rim/metabolismo , Forbóis/farmacologia , Fosfolipídeos/metabolismo , Acetato de Tetradecanoilforbol/farmacologia , Animais , Ácido Araquidônico , Ácidos Araquidônicos/metabolismo , Linhagem Celular , Cães , Indometacina/farmacologia , Rim/efeitos dos fármacos , Cinética , Prostaglandinas/biossínteseRESUMO
Madin-Darby canine kidney cells deacylate arachidonic acid from cellular phospholipid in response to 12-O-tetradecanoyl-phorbol-13-acetate (TPA) and convert the free arachidonic acid to prostaglandins. We have used this system to characterize the acyl specificity of deacylation. Cells were labeled with either [14C]linoleic, [14C]eicosatrienoic (delta 8,11,14 or delta 5,8,11), or [14C]arachidonic acid and stimulated with 10 nM TPA. We found that TPA stimulated the deacylation of all four acids, primarily from phosphatidylethanolamine and phosphatidylcholine.l Only products from linoleic (presumably through chain elongation and desaturation), eicosatrienoic (delta 8,11,14), and arachidonic acids produced prostaglandins. Those produced from linoleic and eicosatrienoic acid (delta 8,11,14)-labeled cells were determined to be primarily of the 1-series, while arachidonic acid-labeled cells produced prostaglandins of the 2-series. Together these results indicate that the stimulated deacylation of phospholipids is not specific for arachidonic acid and that the membrane acyl composition controls the particular series of prostaglandin which is produced.