RESUMO
Usher syndrome (USH) is an autosomal recessive disorder characterized by retinitis pigmentosa and hearing loss. USH type 2 (USH2) is the most common type of USH and is frequently caused by mutations in USH2A, which accounts for 74-90% of USH2 cases. This is the first study reporting the results of scanning for USH2A mutations in Japanese patients with USH2. In 8 of 10 unrelated patients, we identified 14 different mutations. Of these mutations, 11 were novel. Although the mutation spectrum that we identified differed from that for Caucasians, the incidence of mutations in USH2A was 80% for all patients tested, which is consistent with previous findings. Further, c.8559-2A>G was identified in four patients and accounted for 26.7% of mutated alleles; it is thus a frequent mutation in Japanese patients. Hence, mutation screening for c.8559-2A>G in USH2A may prove very effective for the early diagnosis of USH2.
Assuntos
Proteínas da Matriz Extracelular/genética , Predisposição Genética para Doença/genética , Mutação/genética , Síndromes de Usher/genética , Adulto , Povo Asiático , Análise Mutacional de DNA , Primers do DNA/genética , Feminino , Testes Genéticos , Humanos , Japão , Masculino , Pessoa de Meia-IdadeRESUMO
The RCC1 gene, a regulator for the onset of chromosome condensation was found to encode a protein with a molecular mass of 45 kD, determined using the antibody against the synthetic peptides prepared according to the amino acid sequence of the putative RCC1 protein. The p45 located in the nuclei was released from the isolated nuclei, either by DNase I digestion or by treatment with 0.3 M NaCl. Consistently, p45 bound to the DNA-cellulose column was eluted with 0.3 M NaCl. After sequential treatment with DNase I and 2 M NaCl, almost all of the RCC1 protein were released from the nuclei. Thus, RCC1 protein locates on the chromatin and is not a component of the nuclear matrix. In mitotic cells, p45 is dispersed into the cytoplasm. Presumably, RCC1 protein plays a role in regulating the onset of chromosome condensation, at the level of transcription or of mRNA maturation.
Assuntos
Proteínas de Ciclo Celular , Núcleo Celular/ultraestrutura , Cromossomos/ultraestrutura , Proteínas de Ligação a DNA/genética , Genes Reguladores , Fatores de Troca do Nucleotídeo Guanina , Proteínas Nucleares , Sequência de Aminoácidos , Animais , Anticorpos , Linhagem Celular , Cromossomos Humanos/ultraestrutura , Imunofluorescência , Células HeLa/citologia , Humanos , Mitose , Dados de Sequência Molecular , Peso Molecular , Peptídeos/síntese química , Peptídeos/imunologiaRESUMO
Growth factors and cell anchorage jointly regulate transit through G1 in almost all cell types, but the cell cycle basis for this combined requirement remains largely uncharacterized. We show here that cell adhesion and growth factors jointly regulate the cyclin D1- and E-dependent kinases. Adhesion to substratum regulates both the induction and translation of cyclin D1 mRNA. Nonadherent cells fail to phosphorylate the retinoblastoma protein (Rb), and enforced expression of cyclin D1 rescues Rb phosphorylation and entry into S phase when G1 cells are cultured in the absence of substratum. Nonadherent cells also fail to activate the cyclin E-associated kinase, and this effect can be linked to an increased association of the cdk inhibitors, p21 and p27. These data describe a striking convergence in the cell cycle controls used by the two major signal transduction systems responsible for normal and abnormal cell growth. Taken together with our previous studies showing adhesion-dependent expression of cyclin A, they also establish the cell cycle basis for explaining the combined requirement for growth factors and the extracellular matrix in transit through the Rb checkpoint, entry into S phase, and anchorage-dependent growth.
Assuntos
Quinases relacionadas a CDC2 e CDC28 , Proteínas de Ciclo Celular , Ciclo Celular/fisiologia , Quinases Ciclina-Dependentes/metabolismo , Ciclinas/biossíntese , Ciclinas/metabolismo , Proteínas Oncogênicas/biossíntese , Proteínas Serina-Treonina Quinases/metabolismo , Proteína do Retinoblastoma/metabolismo , Proteínas Supressoras de Tumor , Células 3T3 , Animais , Adesão Celular/fisiologia , Ciclina D1 , Quinase 2 Dependente de Ciclina , Inibidor de Quinase Dependente de Ciclina p21 , Inibidor de Quinase Dependente de Ciclina p27 , Quinases Ciclina-Dependentes/análise , Quinases Ciclina-Dependentes/antagonistas & inibidores , Ciclinas/análise , Ciclinas/genética , Cicloeximida/farmacologia , Inibidores Enzimáticos , Fibroblastos/citologia , Expressão Gênica , Humanos , Camundongos , Proteínas Associadas aos Microtúbulos/análise , Proteínas Associadas aos Microtúbulos/metabolismo , Mitógenos/farmacologia , Proteínas Oncogênicas/genética , Fosforilação , Proteínas Serina-Treonina Quinases/análise , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Inibidores da Síntese de Proteínas/farmacologia , Proteínas , RNA Mensageiro/análiseRESUMO
Eukaryotic cells become committed to proliferate during the G1 phase of the cell cycle. In budding yeast, commitment occurs when the catalytic subunit of a protein kinase, encoded by the CDC28 gene (the homolog of the fission yeast cdc2+ gene), binds to a positively acting regulatory subunit, a cyclin. Related kinases are also required for progression through the G1 phase in higher eukaryotes. The role of cyclins in controlling G1 progression in mammalian cells was tested by construction of fibroblasts that constitutively overexpress human cyclin E. This was found to shorten the duration of G1, decrease cell size, and diminish the serum requirement for the transition from G1 to S phase. These observations show that cyclin levels can be rate-limiting for G1 progression in mammalian cells and suggest that cyclin synthesis may be the target of physiological signals that control cell proliferation.
Assuntos
Ciclinas/fisiologia , Fibroblastos/citologia , Fase G1/fisiologia , Animais , Divisão Celular/fisiologia , Linhagem Celular , Clonagem Molecular , Ciclinas/genética , Fibroblastos/metabolismo , Citometria de Fluxo , Expressão Gênica , Vetores Genéticos , Humanos , Canamicina Quinase , Masculino , Fosfotransferases/genética , Ratos , Proteínas Recombinantes de Fusão/metabolismo , Retroviridae/genética , Fase S/fisiologia , Fatores de Tempo , TransfecçãoRESUMO
Cell adhesion has an essential role in regulating proliferation during the G1 phase of the cell cycle, and loss of this adhesion requirement is a classic feature of oncogenic transformation. The appearance of cyclin A messenger RNA and protein in late G1 was dependent on cell adhesion in both NRK and NIH 3T3 fibroblasts. In contrast, the expression of Cdc2, Cdk2, cyclin D1, and cyclin E was independent of adhesion in both cell lines. Transfection of NRK cells with a cyclin A complementary DNA resulted in adhesion-independent accumulation of cyclin A protein and cyclin A-associated kinase activity. These transfected cells also entered S phase and complete multiple rounds of cell division in the absence of cell adhesion. Thus, cyclin A is a target of the adhesion-dependent signals that control cell proliferation.
Assuntos
Quinases relacionadas a CDC2 e CDC28 , Adesão Celular/fisiologia , Ciclo Celular/fisiologia , Quinases Ciclina-Dependentes , Ciclinas/biossíntese , Proteínas Serina-Treonina Quinases , Células 3T3 , Animais , Proteína Quinase CDC2/biossíntese , Linhagem Celular , Quinase 2 Dependente de Ciclina , Fibroblastos/citologia , Fibroblastos/metabolismo , Regulação da Expressão Gênica , Humanos , Camundongos , Proteínas Quinases/biossíntese , Ratos , TransfecçãoRESUMO
OBJECTIVE: Severe congenital neutropenia (SCN), also known as Kostmann syndrome (SCN3, OMIM 610738), includes a variety of haematological disorders caused by different genetic abnormalities. Mutations in ELA2 are most often the cause in autosomal dominant or sporadic forms. Recently, mutations in HAX1 have been identified as the cause of some autosomal recessive forms of SCN, including those present in the original pedigree first reported by Kostmann. We sought to determine the relationship between HAX1 gene mutations and the clinical characteristics of Japanese cases of SCN. METHODS: The genes implicated in SCN (ELA2, HAX1, Gfi-1, WAS, and P14) were analysed in 18 Japanese patients with SCN. The clinical features of these patients were obtained from medical records. Immunoblotting of HAX1 was performed on cell extracts from peripheral blood leucocytes from patients and/or their parents. RESULTS: We found five patients with HAX1 deficiency and 11 patients with mutations in the ELA2 gene. In HAX1 deficiency, a homozygous single base pair substitution (256C>T), which causes the nonsense change R86X, was identified in three affected individuals. Two sibling patients showed a compound heterozygous mutation consisting of a single base pair substitution (256C>T) and a 59 bp deletion at nucleotides 376-434. There was no detectable phenotype in any heterozygous carrier. All patients with HAX1 deficiency had experienced developmental delay. Three patients carrying R86X also suffered from epileptic seizures. In contrast, no SCN patient with heterozygous mutations in the ELA2 gene suffered from any neurodevelopmental abnormality. CONCLUSIONS: These findings suggest that the R86X mutation in the HAX1 gene is an abnormality in Japanese SCN patients with HAX1 deficiency and may lead to neurodevelopmental abnormalities and severe myelopoietic defects.
Assuntos
Deficiências do Desenvolvimento/genética , Mutação , Neutropenia/congênito , Neutropenia/genética , Proteínas/genética , Proteínas Adaptadoras de Transdução de Sinal , Sequência de Bases , Feminino , Homozigoto , Humanos , Lactente , Masculino , Dados de Sequência Molecular , LinhagemRESUMO
Four bentonites from various sources were exposed to batch adsorption testing, selective sequential extraction and consolidation tests to investigate their metal retention capacity and hydraulic conductivity for geoenvironmental application such as in clay barrier materials. The Japanese bentonites (JB1-JB3) contain approximately 2-4% of carbonate and trace amount of zeolite (JB2 and JB3), whereas the US bentonite has < 1% carbonate and no zeolite. The rank of smectite content in the bentonites are USB > JB1 > JB3 > JB2. The materials ranked as JB2 approximately JB3 > JB1 > USB, according to retention capacity, while after the removal of carbonate the retention capacity order was JB1 approximately JB2 approximately JB3 > USB. SSE results indicate that carbonate plays a major role at low Pb solution concentration and precipitate as PbCO3. Once the carbonate is exhausted, the clay composition dominates the sorption process. The hydraulic conductivity of the bentonite mixtures (basalt + 10% bentonite) using water was kUSB < kJB1 < kJB3 < kJB2, consistent with the smectite content and swelling power, with USB having the highest proportion of smectite. Among the Japanese bentonites studied, JB1 is the best candidate for barrier material, comparable to the widely used USB.
Assuntos
Bentonita/química , Chumbo/química , Adsorção , Carbonatos/análise , Engenharia/métodos , Concentração de Íons de Hidrogênio , Tamanho da Partícula , Gravidade Específica , Propriedades de Superfície , Difração de Raios XRESUMO
The tsBN2 cell line, a temperature-sensitive (ts) mutant of baby hamster kidney cell line BHK21/13, seems to possess a mutation in the gene that controls initiation of chromosome condensation. At the nonpermissive temperature (39.5 degrees C), the chromatin of tsBN2 cells is prematurely condensed, and the cells die. Using tsBN2 cells as a recipient of DNA-mediated gene transfer, we investigated a human gene that is responsible for regulation of chromosome condensation and cell proliferation. We found that the human gene complementing the tsBN2 mutation resides in the area of the 40- to 50-kilobase HindIII fragment, derived from HeLa cells. Based on this finding, we initiated cloning of a human gene complementing the tsBN2 mutation. From lambda and cosmid libraries carrying partial digests of DNA from the secondary transformants, the 41.8-kilobase HindIII fragment containing the human DNA was isolated. The cloned human DNA was conserved in ts+ transformants through primary and secondary transfections. Two cosmid clones convert the ts- phenotype of tsBN2 cells to ts+ with more than 100 times a higher efficiency, compared with cases of transfection with total human DNA. Thus, the cloned DNA fragments contain an active human gene that complements the tsBN2 mutation.
Assuntos
Ciclo Celular , Cromatina/ultraestrutura , Clonagem Molecular , Genes , Teste de Complementação Genética , Células HeLa , Humanos , Mitose , Mutação , Transformação GenéticaRESUMO
At the nonpermissive temperature, premature chromosome condensation (PCC) occurs in tsBN2 cells derived from the BHK cell line, which can be converted to the Ts+ phenotype by the human RCC1 gene. To prove that the RCC1 gene is the mutant gene in tsBN2 cells, which have RCC1 mRNA and protein of the same sizes as those of BHK cells, RCC1 cDNAs were isolated from BHK and tsBN2 cells and sequenced to search for mutations. The hamster (BHK) RCC1 cDNA encodes a protein of 421 amino acids homologous to the human RCC1 protein. In a comparison of the base sequences of BHK and BN2 RCC1 cDNAs, a single base change, cytosine to thymine (serine to phenylalanine), was found in the 256th codon of BN2 RCC1 cDNA. The same transition was verified in the RCC1 genomic DNA by the polymerase chain reaction method. BHK RCC1 cDNA, but not tsBN2 RCC1 cDNA, complemented the tsBN2 mutation, although both have the same amino acid sequence except for one amino acid at the 256th codon. This amino acid change, serine to phenylalanine, was estimated to cause a profound structural change in the RCC1 protein.
Assuntos
Proteínas de Ciclo Celular , Ciclo Celular , Cromossomos/fisiologia , Proteínas de Ligação a DNA/genética , Genes , Fatores de Troca do Nucleotídeo Guanina , Mutação , Proteínas Nucleares , Sequência de Aminoácidos , Animais , Sequência de Bases , Linhagem Celular , Clonagem Molecular , Cricetinae , DNA/genética , Biblioteca Gênica , Humanos , Rim , Dados de Sequência Molecular , Hibridização de Ácido Nucleico , Sondas de Oligonucleotídeos , Fenótipo , Reação em Cadeia da Polimerase , Conformação Proteica , Mapeamento por Restrição , Homologia de Sequência do Ácido Nucleico , TemperaturaRESUMO
Cyclin E was first identified by screening human cDNA libraries for genes that would complement G1 cyclin mutations in Saccharomyces cerevisiae and has subsequently been found to have specific biochemical and physiological properties that are consistent with it performing a G1 function in mammalian cells. Most significantly, the cyclin E-Cdk2 complex is maximally active at the G1/S transition, and overexpression of cyclin E decreases the time it takes the cell to complete G1 and enter S phase. We have now found that mammalian cells express two forms of cyclin E protein which differ from each other by the presence or absence of a 15-amino-acid amino-terminal domain. These proteins are encoded by alternatively spliced mRNAs and are localized to the nucleus during late G1 and early S phase. Fibroblasts engineered to constitutively overexpress either form of cyclin E showed elevated cyclin E-dependent kinase activity and a shortened G1 phase of the cell cycle. The overexpressed cyclin E protein was detected in the nucleus during all cell cycle phases, including G0. Although the cyclin E protein could be overexpressed in quiescent cells, the cyclin E-Cdk2 complex was inactive. It was not activated until 6 to 8 h after readdition of serum, 4 h earlier than the endogenous cyclin E-Cdk2. This premature activation of cyclin E-Cdk2 was consistent with the extent of G1 shortening caused by cyclin E overexpression. Microinjection of affinity-purified anti-cyclin E antibodies during G1 inhibited entry into S phase, whereas microinjection performed near the G1/S transition was ineffective. These results demonstrate that cyclin E is necessary for entry into S phase. Moreover, we found that cyclin E, in contrast to cyclin D1, was required for the G1/S transition even in cells lacking retinoblastoma protein function. Therefore, cyclins E and D1 control two different transitions within the human cell cycle.
Assuntos
Ciclinas/fisiologia , Fase G1/fisiologia , Proteínas Nucleares/fisiologia , Fase S/fisiologia , Processamento Alternativo , Sequência de Aminoácidos , Animais , Anticorpos , Sequência de Bases , Linhagem Celular , Ciclina D1 , Ciclinas/antagonistas & inibidores , Ciclinas/genética , Primers do DNA/genética , Replicação do DNA , DNA Complementar/genética , Expressão Gênica , Humanos , Dados de Sequência Molecular , Proteínas Nucleares/genética , Proteínas Oncogênicas/fisiologia , RNA Mensageiro/genética , Proteína do Retinoblastoma/fisiologiaRESUMO
The KMDB/MutationView is a graphical database of mutations in human disease-causing genes and its current version consists of nine category-based sub-databases including diseases of eye, heart, ear, brain, cancer, syndrome, autoimmunity, muscle and blood. The KMDB/MutationView stores mutation data of 97 genes involved in 87 different disease and is accessible through http://mutview.dmb.med. keio.ac.jp.
Assuntos
Bases de Dados Factuais , Doença , Genes/genética , Mutação , Doença/etiologia , Humanos , InternetRESUMO
A431 cells hyperproduce EGF receptors and possess inactive p53 proteins. It has been suggested that a cyclin-dependent kinase (CDK) inhibitor p21/WAF1 plays a crucial role in the EGF-induced cell-cycle arrest of A431 cells. Here, we investigated the role of WAF1 gene transcription in the EGF-induced cell-cycle arrest by transfecting the 18-mer antisense oligonucleotide which corresponds to the 5' region of WAF1 gene (AS/WAF1). When A431 cells were treated with EGF, a cascade of responses were observed, including immediate hyperphosphorylation of EGF receptor on tyrosine residues, accumulation of WAF1 mRNA and p21/WAF1 protein, dephosphorylation of RB protein which is a substrate of CDK-cyclin, and cell-cycle arrest. In the presence of AS/WAF1, EGF induced the tyrosine-phosphorylation of EGF receptor, but WAF1 mRNA was reduced to a half; accumulation of p21/WAF1 protein and its downstream responses were no longer observed; A431 cells grew continuously. Thus, the transfection of antisense efficiently prevented A431 cells from the EGF-induced arrest. These observations suggest that p21/WAF1 protein is a major effector molecule of the EGF-mediated cell-cycle arrest of A431 cells.
Assuntos
Quinases Ciclina-Dependentes/antagonistas & inibidores , Ciclinas/metabolismo , Inibidores Enzimáticos/metabolismo , Fator de Crescimento Epidérmico/farmacologia , Inibidores do Crescimento/farmacologia , Oligonucleotídeos Antissenso/farmacologia , Ciclo Celular/efeitos dos fármacos , Divisão Celular/efeitos dos fármacos , Inibidor de Quinase Dependente de Ciclina p21 , Ciclinas/biossíntese , Ciclinas/genética , Fator de Crescimento Epidérmico/antagonistas & inibidores , Humanos , RNA Mensageiro/biossíntese , Transfecção , Células Tumorais CultivadasRESUMO
TRAIL has gained much attention for its specific induction of apoptosis in cancer cells but not in normal cells. This phenomenon has been explained thus: that cancer cells dominantly express death receptors while normal cells express decoy receptors. However, recent reports have shown that some cancer cell lines are resistant to TRAIL-induced apoptosis despite the absence of decoy receptors and the presence of death receptors. This suggested the existance of an inhibitory factor. We herein showed that NF-kappaB is a key molecule underlying the TRAIL-resistant mechanism in renal cell carcinoma (RCC) cell lines. We observed that NF-kappaB is constitutively activated in resistant cell lines. Forced expression of antisense cDNA of IkappaBalpha, a specific inhibitor of NF-kappaB, in TRAIL-sensitive cell lines with a low NF-kappaB activity result in constitutive activation of NF-kappaB and resistance to TRAIL-induced apoptosis. Adenoviral expression of a stable form of IkappaBalpha in the TRAIL-resistant cell lines induced apoptosis. These data suggest that RCC can be classified into two subsets: TRAIL-sensitive RCC with a low NF-kappaB activity and TRAIL-resistant RCC with constitutively activated NF-kappaB. In the former group TRAIL can be a treatment option, while in the latter group a molecular approach targeting NF-kappaB appears to be a promising therapy.
Assuntos
Antineoplásicos/metabolismo , Apoptose , Carcinoma de Células Renais/metabolismo , Proteínas I-kappa B , Neoplasias Renais/metabolismo , Glicoproteínas de Membrana/metabolismo , NF-kappa B/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , Antineoplásicos/farmacologia , Proteínas Reguladoras de Apoptose , Carcinoma de Células Renais/tratamento farmacológico , Caspase 8 , Caspase 9 , Caspases/genética , Caspases/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Ativação Enzimática , Proteínas Ligadas por GPI , Humanos , Neoplasias Renais/tratamento farmacológico , Ligantes , Glicoproteínas de Membrana/farmacologia , Inibidor de NF-kappaB alfa , Receptores do Ligante Indutor de Apoptose Relacionado a TNF , Receptores do Fator de Necrose Tumoral/genética , Membro 10c de Receptores do Fator de Necrose Tumoral , Ligante Indutor de Apoptose Relacionado a TNF , Células Tumorais Cultivadas , Receptores Chamariz do Fator de Necrose Tumoral , Fator de Necrose Tumoral alfa/farmacologiaRESUMO
Previously we cloned a novel adaptor protein, APS (adaptor molecules containing PH and SH2 domains) which was tyrosine phosphorylated in response to c-kit or B cell receptor stimulation. Here we report that APS was expressed in some human osteosarcoma cell lines, markedly so in SaOS-2 cells, and was tyrosine-phosphorylated in response to several growth factors, including platelet derived growth factor (PDGF), insulin-like growth factor (IGF), and granulocyte-macrophage colony stimulating factor (GM-CSF). Ectopic expression of the wild type APS, but not C-terminal truncated APS, in NIH3T3 fibroblasts suppressed PDGF-induced MAP kinase (Erk2) activation, c-fos and c-myc induction as well as cell proliferation. In vitro binding experiments suggest that APS bound to the beta type PDGF receptor, mainly via phosphotyrosine 1021 (pY1021). Indeed, tyrosine phosphorylation of PLC-gamma, which has been demonstrated to bind to pY1021, but not that of PI3 kinase and associated proteins, was reduced in APS transformants. PDGF induced phosphorylation of the tyrosine residue of APS close to the C-terminal end. In vitro and in vivo binding experiments indicate that the tyrosine phosphorylated C-terminal region of APS bound to c-Cbl, which has been shown to be a negative regulator of tyrosine kinases. Since coexpression of c-Cbl with wild type APS, but not C-terminal truncated APS, synergistically inhibited PDGF-induced c-fos promoter activation, c-Cbl could be a mechanism of inhibitory action of APS on PDGF receptor signaling.
Assuntos
Proteínas Adaptadoras de Transporte Vesicular , Proteínas Sanguíneas , Proteínas/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Receptores do Fator de Crescimento Derivado de Plaquetas/metabolismo , Ubiquitina-Proteína Ligases , Domínios de Homologia de src , Proteínas Adaptadoras de Transdução de Sinal , Divisão Celular , Expressão Gênica , Humanos , Mitógenos , Osteossarcoma , Fosfoproteínas/metabolismo , Fosforilação , Fator de Crescimento Derivado de Plaquetas/metabolismo , Proteínas Proto-Oncogênicas c-cbl , Células Tumorais Cultivadas , Tirosina/metabolismoRESUMO
Point mutations of Asp-376 of the alpha-subunit of Torpedo californica Na+/K(+)-ATPase (the site of phosphorylation during the catalytic cycle) to Asn, Glu or Thr led to virtual abolishment of Na+/K(+)-ATPase activity and ouabain-binding capacity. Replacement of Lys-507 of the same subunit (the putative ATP-binding site) by Met resulted in decreases in Na+/K(+)-ATPase activity and ouabain-binding capacity. These results are in agreement with those reported for rabbit sarcoplasmic reticulum Ca2(+)-ATPase (Maruyama, K. and MacLennan, D.H. (1988) Proc. Natl. Acad. Sci. USA 85, 3314-3318).
Assuntos
Ácido Aspártico , Lisina , Mutação , ATPase Trocadora de Sódio-Potássio/genética , Trifosfato de Adenosina/metabolismo , Animais , Sequência de Bases , Sítios de Ligação , Feminino , Expressão Gênica , Cinética , Substâncias Macromoleculares , Dados de Sequência Molecular , Oligodesoxirribonucleotídeos/isolamento & purificação , Oócitos/enzimologia , Fosforilação , Mapeamento por Restrição , ATPase Trocadora de Sódio-Potássio/metabolismo , Torpedo , XenopusRESUMO
The "Fab immunogene" is a novel gene transfer vehicle in which the Fab fragment of anti-human epidermal growth factor (EGF) receptor antibody B4G7 is conjugated with poly-L-lysine to form an affinity complex with DNA. It was developed to target delivery of therapeutic genes into EGF receptor-hyperproducing tumor cells. Various characteristic features of the immunogene have been documented (Chen et al., 1998). Here we add further evidence to prove that in vitro transfer of beta-galactosidase/Fab immunogene is exclusively to EGF receptor-positive cells and that the herpes simplex virus thymidine kinase (TK)/Fab immunogene induces substantial suicide effects on A431 tumor cells when treated together with ganciclovir. The in vivo specificity of the immunogene transfer was examined using A431 tumor-bearing nude mice. When these nude mice were injected intraperitoneally with the chloramphenicol acetyltransferase (CAT)/Fab immunogene, CAT DNA was detected in the tumors as well as in liver and kidney but not brain, whereas CAT mRNA and enzyme activity were detected only in the tumors. Local and intraperitoneal injection of the TK/Fab immunogene and subsequent administration of ganciclovir effectively suppressed the growth of A431 tumors transplanted on the backs of nude mice. These observations suggest a possible application of the Fab immunogene system in cancer gene therapy.
Assuntos
Carcinoma de Células Escamosas/terapia , Técnicas de Transferência de Genes , Terapia Genética , Fragmentos Fab das Imunoglobulinas/genética , Animais , Cloranfenicol O-Acetiltransferase/genética , Cloranfenicol O-Acetiltransferase/metabolismo , Sistemas de Liberação de Medicamentos , Receptores ErbB/imunologia , Ganciclovir/uso terapêutico , Herpesvirus Humano 1/enzimologia , Herpesvirus Humano 1/genética , Camundongos , Camundongos Nus , Timidina Quinase/genética , beta-Galactosidase/genéticaRESUMO
The argG gene, encoding argininosuccinate synthetase, was cloned from Streptomyces lavendulae KCCS0055 by colony hybridization using the argG-carrying 2.1-kb fragment of S. coelicolor DNA as a probe. The restriction map of the cloned DNA fragment was very similar to that of S. coelicolor. This DNA fragment could complement the argG mutation of both S. lividans 1326 I10 and Escherichia coli K-12 JE5694, suggesting that the fragment contained a promoter for both E. coli and S. lividans. The subcloning experiment using E. coli K-12 JE5694 as a host has indicated that the essential region for argG is contained in the 2.5-kb DNA fragment. The translational product was identified as a 56-kDa kDa protein in minicells and by conventional gel electrophoresis. Determination of the nucleotide (nt) sequence of the 2.5-kb DNA fragment revealed one open reading frame of 1449 bp. The amino acid (aa) sequence analysis showed that the N-terminus was Ser, and 9 aa from the N terminus were completely identical with those deduced from the nt sequence. Nuclease S1 mapping indicated that the transcription start point is located near the start codon.
Assuntos
Argininossuccinato Sintase/genética , Streptomyces/enzimologia , Sequência de Aminoácidos , Argininossuccinato Sintase/biossíntese , Sequência de Bases , Clonagem Molecular , Escherichia coli , Expressão Gênica , Dados de Sequência Molecular , Biossíntese de Proteínas , Proteínas Recombinantes/biossíntese , Endonucleases Específicas para DNA e RNA de Cadeia Simples , Transcrição GênicaRESUMO
The N-terminus of MDM2 proto-oncoprotein interacts with p53 and down modulates p53 activity by inhibiting transcriptional activity and promoting p53 degradation. MDMX is structurally related to MDM2 and also binds to p53. However, the function of MDMX has not been clarified yet. We found that MDM2 hetero-oligomerized with MDMX through their C-terminal RING finger domains. Yeast two-hybrid analysis revealed that the hetero-oligomerization between MDMX and MDM2 was more stable than the homo-oligomerization of each protein. MDM2 has been shown to be degraded by the ubiquitin-proteasome pathway, while MDMX was a stable protein. Interaction of MDMX with MDM2 through the C-terminal RING finger domains resulted in inhibiting degradation of MDM2. These data indicate that MDMX functions as a regulator of MDM2.
Assuntos
Proteínas Nucleares , Proteínas Proto-Oncogênicas/metabolismo , Dedos de Zinco , Sítios de Ligação , Clonagem Molecular , Ligação Proteica , Conformação Proteica , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas c-mdm2 , Proteínas Recombinantes/metabolismo , Saccharomyces cerevisiae/genética , Proteína Supressora de Tumor p53/metabolismoRESUMO
We previously developed the "immunogene" approach toward cancer gene therapy using epidermal growth factor receptor (EGFR)-mediated endocytosis. Here, we describe an improved immunogene system, in which the antigen-binding (Fab) fragments of the monoclonal antibody (Ab) B4G7 against the human EGFR were conjugated with poly-L-lysine to form a gene delivery vehicle (designated Fab "immunoporter"). Within 12 hours, the beta-galactosidase beta-gal) gene was transferred via the Fab immunoporter to virtually all of the nuclei of human squamous carcinoma A431 cells that overproduce the EGFR, and the beta-gal enzyme activity was detected within 24 hours and retained for more than 3 days. The beta-gal gene was not transferred into human and mouse cells that were deficient in EGFRs, but it was delivered if those mouse cells were transformed with human EGFR genes. Beta-gal gene transfer via the Fab immunoporter was inhibited by pretreatment with excess amounts of the Fab fragment. The transfer efficiency of the beta-gal gene to A431 cells via the Fab immunoporter was approximately 2%, which is as high as the lipofection method and 20- to 100-fold higher than the whole Ab immunoporter. The transfer of the herpes simplex virus thymidine kinase gene into A431 tumor cells as a form of the thymidine kinase/Fab immunogene was successful, and subsequent treatment with ganciclovir induced remarkable suicide effects which conferred 1000-fold higher drug sensitivity. Thus, the Fab immunogene was substantially improved with regard to the whole Ab immunogene and could be used as a potent gene transfer vehicle for the in vivo targeting of EGFR-hyperproducing tumor cells.
Assuntos
Receptores ErbB/imunologia , Marcação de Genes/métodos , Técnicas de Transferência de Genes , Fragmentos Fab das Imunoglobulinas , Animais , Anticorpos Monoclonais , Núcleo Celular/química , Ganciclovir/farmacologia , Genes Reporter , Humanos , Camundongos , Proteínas Tirosina Quinases/genética , Simplexvirus/genética , Fatores de Tempo , Células Tumorais Cultivadas/enzimologia , beta-Galactosidase/genética , beta-Galactosidase/metabolismoRESUMO
This study indicates that skeletal muscle metabolism may affect the results of maximal bicycle and treadmill exercise differently, and that maximal bicycle exercise was limited by quadriceps muscle metabolism rather than by cardiopulmonary capacity. In contrast, maximal treadmill exercise was not limited, eliciting more cardiopulmonary reserve and attaining greater peak oxygen uptake than maximal bicycle exercise.